**Figure 10.** Effects of exogenous NO on contraction window in mouse aortic segments following repolarization of Vm to VK with 1 µM levcromakalim. eNOS was active (blue circles) or inhibited (red circles) and in this situation exogenous NO could be applied (100 nM DEANO, purple circles). The fitted (sigmoidal concentration-response curves with variable slope) curves in A were differentiated in B to show the voltage-dependence of the change of force development per mV change of Vclamp. Data: mean±SEM; n=4; \*, \*\*, \*\*\*: P<0.05, 0.01, 0.001 eNOS inactive versus eNOS active; #, ##, ###: P<0.05, 0.01, 0.001 +100 nM DEANO versus eNOS inactive.

The shift of the contraction window by eNOS inhibition could be completely reversed by adding exogenous NO (100 nM DEANO) (figure 10). The addition of exogenous NO to segments, in which eNOS activity was inhibited, caused a rightward (depolarising) shift of the voltage-dependency of contraction. Also in this situation, effects of eNOS inhibition or exogenous NO addition on Vm were avoided by performing the experiments in the presence of 1 µM levcromakalim. Results confirmed that NO altered the voltage-dependency of the Ltype Ca2+ channel-mediated contraction, probably by direct effects of NO on the channel´s gating properties. It should be noted that for strong depolarization above -20 mV (90 and 124 mM K+), addition of exogenous NO caused significant relaxation of the pre-constricted segments. Hence, the window contraction in the presence of NO is narrower than in the absence of NO (figure 10B). Whether these results can also be explained by NO-dependent changes of the voltage dependence of activation of the L-type Ca2+ channels needs further investigation.

The above data may provide an explanation for the absence of a decrease of intracellular VSMC Ca2+ with relaxation upon addition of NO to segments depolarized with 50 mM K+, whereas similar experiments in phenylephrine-constricted segments displayed relaxation with an abrupt decrease of intracellular Ca2+ upon addition of NO (see figure 2). Firstly, depolarization with elevated K+ did not empty the SR Ca2+ stores, and, hence, did not stimulate SOCE via NO-sensitive non-selective cation channels. Secondly, at 50 mM K+ Vm of the VSMCs was clamped at depolarized potentials and NO cannot elicit hyperpolarisation of Vm. Thirdly, at 50 mM K+, the window Ca2+ influx and concomitant contraction were maximal and addition of NO will only cause small relaxations, probably via effects on Ca2+ sensitivity and not on intracellular Ca2+ concentrations.

#### **6. NO efficacy and VSMC L-type Ca2+ channels along the thoracic aorta**

Current Basic and Pathological Approaches to

0.001 +100 nM DEANO versus eNOS inactive.

sensitivity and not on intracellular Ca2+ concentrations.

investigation.

82 the Function of Muscle Cells and Tissues – From Molecules to Humans

**Figure 10.** Effects of exogenous NO on contraction window in mouse aortic segments following repolarization of Vm to VK with 1 µM levcromakalim. eNOS was active (blue circles) or inhibited (red circles) and in this situation exogenous NO could be applied (100 nM DEANO, purple circles). The fitted (sigmoidal concentration-response curves with variable slope) curves in A were differentiated in B to show the voltage-dependence of the change of force development per mV change of Vclamp. Data: mean±SEM; n=4; \*, \*\*, \*\*\*: P<0.05, 0.01, 0.001 eNOS inactive versus eNOS active; #, ##, ###: P<0.05, 0.01,

The shift of the contraction window by eNOS inhibition could be completely reversed by adding exogenous NO (100 nM DEANO) (figure 10). The addition of exogenous NO to segments, in which eNOS activity was inhibited, caused a rightward (depolarising) shift of the voltage-dependency of contraction. Also in this situation, effects of eNOS inhibition or exogenous NO addition on Vm were avoided by performing the experiments in the presence of 1 µM levcromakalim. Results confirmed that NO altered the voltage-dependency of the Ltype Ca2+ channel-mediated contraction, probably by direct effects of NO on the channel´s gating properties. It should be noted that for strong depolarization above -20 mV (90 and 124 mM K+), addition of exogenous NO caused significant relaxation of the pre-constricted segments. Hence, the window contraction in the presence of NO is narrower than in the absence of NO (figure 10B). Whether these results can also be explained by NO-dependent changes of the voltage dependence of activation of the L-type Ca2+ channels needs further

The above data may provide an explanation for the absence of a decrease of intracellular VSMC Ca2+ with relaxation upon addition of NO to segments depolarized with 50 mM K+, whereas similar experiments in phenylephrine-constricted segments displayed relaxation with an abrupt decrease of intracellular Ca2+ upon addition of NO (see figure 2). Firstly, depolarization with elevated K+ did not empty the SR Ca2+ stores, and, hence, did not stimulate SOCE via NO-sensitive non-selective cation channels. Secondly, at 50 mM K+ Vm of the VSMCs was clamped at depolarized potentials and NO cannot elicit hyperpolarisation of Vm. Thirdly, at 50 mM K+, the window Ca2+ influx and concomitant contraction were maximal and addition of NO will only cause small relaxations, probably via effects on Ca2+-

Increased stiffness of elastic arteries represents an early risk factor for cardiovascular diseases (O'Rourke & Mancia, 1999) and, therefore, the assessment of mechanical properties of the aorta is important to understand the mechanisms of cardiovascular disease. It has been shown that in the aorta of C57Bl6 mice, the circumferential modulus is greatest (most rigid) near the diaphragm, and that about 85% of volume compliance is in the thoracic compared with abdominal aorta (Guo & Kassab, 2003). Because as well NO as CaBs have been described to de-stiffen large arteries (Fitch et al., 2006; Safar et al., 1989; Safar et al., 2011), we wondered whether NO release (basal and stimulated) and L-type Ca2+ channel activity differed along the length of the thoracic aorta.

**Figure 11.** Dissection of the mouse thoracic aorta in five 2 mm wide segments. The mouse aorta was removed from the animal, stripped of adherent tissue and dissected systematically. Starting at the diaphragm, the ascending thoracic aorta was cut in segments of 2 mm width (TA5 up to TA1).

In this part of the chapter, we show some preliminary data on aortic segment differences with respect to NO and L-type Ca2+ channels. In order to test whether the release or efficacy of NO differs between different locations along the thoracic aorta of C57Bl6 mice, the aorta was divided into 5 segments (figure 11) and in each segment endogenous and exogenous NO efficacy was determined. Basal release of NO and its efficacy to counteract contraction was measured in each aortic segment by measuring isometric contractions to the α1 adrenoceptor agonist phenylephrine before and after inhibition of eNOS with 300 µM LNAME/LNNA. The increase of force (Δ force) and the shift of logEC50 (Δ logEC50) by eNOS inhibition are an index of the basal release of endogenous NO in each segment (figures 12A and B).

Although not significant, the effects of eNOS inhibition on phenylephrine-induced contractions were smaller in the atherosclerosis-resistant segments TA2, TA3 and TA4 as compared with the atherosclerosis-prone segments TA1 and TA5 (figure 12A). In accordance, inhibition of eNOS caused a significantly larger shift of the segment´s sensitivity to phenylephrine in the atherosclerosis-prone segment TA1 compared with the other segments (figure 12B). Results may point to a higher basal eNOS activity or release of basal NO in the atherosclerosis-prone segments TA1 and TA5. Subsequently, endogenous release of NO was evoked by adding increasing concentrations of ACh to segments pre-contracted with 1 µM phenylephrine. Figures 12C and D show maximal relaxation evoked by and sensitivity (logEC50) for ACh in each segment. Although TA1 seemed to have the highest NO efficacy without endothelial stimulation (basal NO, figures A and B), this atherosclerosis-prone segment caused only relaxations of about 65% and displayed the lowest sensitivity for ACh-induced relaxations. This was due to endothelial dysfunction because the effects of exogenous NO (DEANO) were not significantly different between the different aortic segments (figures 12 E and F). Results of these experiments show that endothelial function (basal and stimulated release of NO) may differ between atherosclerosis-prone and –resistant segments along the thoracic aorta of wild-type mice, which may have important consequences for the tonus and compliance of the thoracic aorta.

Contraction by Ca2+ Influx via the L-Type Ca2+ Channel

Voltage Window in Mouse Aortic Segments is Modulated by Nitric Oxide 85

When the impact of ACh-stimulated eNOS activity on L-type Ca2+ channels was investigated further (see figure 6), but now in atherosclerosis-prone and -resistant segments, figures 13 A and C show that as well in the presence of external Ca2+ as after eliciting contraction by readdition of Ca2+ to zero Ca2+ (SOCE contraction), relaxation in prone segments in comparison with resistant segments was attenuated. When SOCE via cation-selective channels was inhibited with 50 µM 2-APB, the SOCE contraction by phenylephrine was smaller in the resistant than in the prone segments (figure 13B, values at -9 logM ACh). Because this SOCEcontraction by phenylephrine is due to influx of Ca2+ via L-type Ca2+ channels only (see figure 5), this suggests that the L-type Ca2+ channel-mediated SOCE contraction in prone segments is larger than in resistant segments. This L-type Ca2+ channel-mediated contraction by phenylephrine in prone segments displayed attenuated relaxation to ACh (figure 13D), again

pointing to the lower capacity of NO to relax contractions evoked by L-type Ca2+ influx.

**Figure 13.** Absolute (mN, A, B) and relative (%, C, D) ACh-induced relaxation of atherosclerosis-prone (TA1,white symbols) and atherosclerosis-resistant (TA2/TA3, black symbols) segments constricted with 2 µM phenylephrine in the presence of extracellular Ca2+ (control conditions, circles) and after eliciting contraction by re-addition of external Ca2+ to zero Ca2+ (SOCE, squares). Relaxation curves were fitted with sigmoidal concentration-response equations with variable slope and significantly differed between prone and resistant segments. After inhibition of SOCE via non-selective cation channels with 50 µM 2- APB (triangles), the SOCE contraction, which is mediated through L-type Ca2+ influx only, was larger in prone than in resistant segments. Relaxation of these phenylephrine-induced SOCE contractions by

**Figure 12.** Basal and agonist-stimulated NO efficacy for different aortic segments. A and B show maximal increase of force development (Δforce) and the shift of the logEC50 (ΔlogEC50) for phenylephrine after inhibition of eNOS with the 300 µM LNAME/LNNA combination (measure of basal NO release). C and D show % relaxation and logEC50 for ACh-induced relaxation of the contraction elicited with 1 µM phenylephrine (stimulated endogenous NO), hence in the absence of LNAME/LNNA. E and F show % relaxation and logEC50 for DEANO-induced relaxation of the contraction elicited with 1 µM phenylephrine (exogenous NO) after inhibition of eNOS with 300 µM LNAME/LNNA. Data: mean ±SEM; n=6 or more; \*, \*\*, \*\*\*: P<0.05, 0.01, 0.001 versus TA1.

When the impact of ACh-stimulated eNOS activity on L-type Ca2+ channels was investigated further (see figure 6), but now in atherosclerosis-prone and -resistant segments, figures 13 A and C show that as well in the presence of external Ca2+ as after eliciting contraction by readdition of Ca2+ to zero Ca2+ (SOCE contraction), relaxation in prone segments in comparison with resistant segments was attenuated. When SOCE via cation-selective channels was inhibited with 50 µM 2-APB, the SOCE contraction by phenylephrine was smaller in the resistant than in the prone segments (figure 13B, values at -9 logM ACh). Because this SOCEcontraction by phenylephrine is due to influx of Ca2+ via L-type Ca2+ channels only (see figure 5), this suggests that the L-type Ca2+ channel-mediated SOCE contraction in prone segments is larger than in resistant segments. This L-type Ca2+ channel-mediated contraction by phenylephrine in prone segments displayed attenuated relaxation to ACh (figure 13D), again pointing to the lower capacity of NO to relax contractions evoked by L-type Ca2+ influx.

Current Basic and Pathological Approaches to

compliance of the thoracic aorta.

84 the Function of Muscle Cells and Tissues – From Molecules to Humans

for ACh in each segment. Although TA1 seemed to have the highest NO efficacy without endothelial stimulation (basal NO, figures A and B), this atherosclerosis-prone segment caused only relaxations of about 65% and displayed the lowest sensitivity for ACh-induced relaxations. This was due to endothelial dysfunction because the effects of exogenous NO (DEANO) were not significantly different between the different aortic segments (figures 12 E and F). Results of these experiments show that endothelial function (basal and stimulated release of NO) may differ between atherosclerosis-prone and –resistant segments along the thoracic aorta of wild-type mice, which may have important consequences for the tonus and

**Figure 12.** Basal and agonist-stimulated NO efficacy for different aortic segments. A and B show maximal increase of force development (Δforce) and the shift of the logEC50 (ΔlogEC50) for phenylephrine after inhibition of eNOS with the 300 µM LNAME/LNNA combination (measure of basal NO release). C and D show % relaxation and logEC50 for ACh-induced relaxation of the contraction elicited with 1 µM phenylephrine (stimulated endogenous NO), hence in the absence of LNAME/LNNA. E and F show % relaxation and logEC50 for DEANO-induced relaxation of the contraction elicited with 1 µM phenylephrine (exogenous NO) after inhibition of eNOS with 300 µM LNAME/LNNA. Data: mean ±SEM;

n=6 or more; \*, \*\*, \*\*\*: P<0.05, 0.01, 0.001 versus TA1.

**Figure 13.** Absolute (mN, A, B) and relative (%, C, D) ACh-induced relaxation of atherosclerosis-prone (TA1,white symbols) and atherosclerosis-resistant (TA2/TA3, black symbols) segments constricted with 2 µM phenylephrine in the presence of extracellular Ca2+ (control conditions, circles) and after eliciting contraction by re-addition of external Ca2+ to zero Ca2+ (SOCE, squares). Relaxation curves were fitted with sigmoidal concentration-response equations with variable slope and significantly differed between prone and resistant segments. After inhibition of SOCE via non-selective cation channels with 50 µM 2- APB (triangles), the SOCE contraction, which is mediated through L-type Ca2+ influx only, was larger in prone than in resistant segments. Relaxation of these phenylephrine-induced SOCE contractions by

ACh were compromised more in prone than in resistant segments and could not be fitted with sigmoidal concentration-response equations (B). Data: mean±SEM; n=7; \*, \*\*, \*\*\*: P<0.05, 0.01, 0.001 prone versus resistant; #, ###: 2-APB versus SOCE.

Contraction by Ca2+ Influx via the L-Type Ca2+ Channel

Voltage Window in Mouse Aortic Segments is Modulated by Nitric Oxide 87

benzothiazepines such as diltiazem and dihydropyridines such as nifedipine). They reduce blood pressure more effectively in hypertensive than in normotensive subjects (Leonetti et al., 1982) and they inhibit L-type Ca2+ influx more effectively in vascular tissue than in heart (Godfraind et al., 1984; Godfraind, 2005; Striessnig et al., 1998). Several attempts have been made to explain these unique features. In hypertension, there is an increase of reactivity to vasoconstrictors, which is not only due to a higher number of L-type Ca2+ channels (Godfraind, 2005; Pesic et al., 2004; Pratt et al., 2002), but also to depolarization of the resting potential of hypertensive VSMCs (Morel & Godfraind, 1994; Pesic et al., 2004). Depolarization leads to an increased proportion L-type Ca2+ channels in the inactivated state, which according to the "modulated receptor theory" may have higher affinity to CaBs than channels in the resting state (Bean et al., 1986; Godfraind, 2005; Morel & Godfraind, 1987). Cardiac muscle cells are hyperpolarized with respect to VSMC and hence less susceptible to block by CaBs. Another specific feature of L-type Ca2+ channels is that their population is not homogeneous because of the occurrence of alternatively spliced isoforms (Koch et al., 1990). Among the 55 known human Ltype Ca2+ channel exons 19 undergo alternative splicing and display differences in tissue distribution, physiology, pharmacology and disease-related up- and/or down-regulation (Liao et al., 2005; Tang et al., 2007; Tiwari et al., 2006). Some of these isoforms were dominant in aorta (> 50%) and less abundant in heart (<5%). Moreover, the VSM-specific splice variant of the L-type Ca2+ channel displayed hyperpolarised window current at voltages where there is overlap between activation and inactivation curves and enhanced inhibition by nifedipine in comparison with the predominant cardiac isoform (Liao et al., 2007). The selective affinity of CaBs for the different L-type Ca2+ channel subpopulations may further contribute to the different susceptibility of VSMC or cardiac cells to CaBs. Because the L-type Ca2+ channel window in VSMCs may be responsible for "time-independent" baseline Ca2+ influx at normal resting or slightly depolarized membrane potentials (Fleischmann et al., 1994; Poburko et al., 2004), depolarization of the resting potential by hypertension not only favours the inactivated state of the L-type Ca2+ channel, but is also expected to increase L-type Ca2+ influx via the L-type Ca2+ channel window and

Preliminary results of this chapter, finally, indicate that NO efficacy and L-type Ca2+ channel distribution may differ along the length of the thoracic aorta. Basal and stimulated eNOS activation or NO release occurred differently along the thoracic aorta. Close to the aortic arch, segments released more basal NO but less stimulated NO than segments close to the diaphragm. Similarly, L-type Ca2+ channel distribution and related window contraction were higher in atherosclerosis-prone than in -resistant segments. These preliminary data need to be further explored, but may have important consequences for the compliance of the different aortic segments and their susceptibility to the development of atherosclerosis.

to evoke inhibition by CaBs (Fleischmann et al., 1994).

*Laboratory of Physiopharmacology, University of Antwerp, Wilrijk, Belgium*

*Laboratory of Pharmacology, University of Antwerp, Wilrijk, Belgium*

Cor E. Van Hove, Johanna van Langen and Hidde Bult

**Author details**

Paul Fransen

#### **7. Conclusions**

The present chapter investigated the relaxing efficacy of NO for contractions induced by α1 adrenoceptor stimulation with phenylephrine or by depolarization with elevated extracellular K+ and confirmed previous observations that relaxation by NO is attenuated when the contraction was due to L-type Ca2+ influx (Van Hove et al., 2009). Contractions, which were initiated by Ca2+ release from the SR as with phenylephrine were very sensitive to endogenous or exogenous NO. Here, NO caused relaxation by inhibiting the IP3-mediated contraction, by stimulating Ca2+ re-uptake to the SR via stimulation of SERCA, by inhibiting SOCE via cation channels and by reducing SOCE via L-type Ca2+ channels. Although phenylephrine has been described to cause depolarization of the VSMCs, Vm is not clamped at depolarized values and addition of NO is expected to cause hyperpolarization, thereby reducing Ca2+ influx via L-type Ca2+ channels. By clamping Vm to depolarized values with elevation of K+, we were able to show contractions due to Ca2+ influx via L-type Ca2+ channels only and more specifically, to Ca2+ influx via non-inactivating Ca2+ channels (window contraction). This window contraction could be decreased by repolarizing Vm of VSMCs with levcromakalim (an opener of ATP-dependent K+ channels) or by adding exogenous NO as long as the extracellular K+ concentration was below 50 mM. Because NO also shifted the voltage-dependence of the window contraction in the presence of levcromakalim, it was hypothesized that NO, but not levcromakalim exerted a direct effect on the gating properties of the L-type Ca2+ channel. Although it has been described that NO affects L-type Ca2+ channels directly via S-nitrosylation (Almanza et al., 2007), an effect on the voltagedependent contraction has not been directly demonstrated yet. Hence, it is hypothesized that in mouse aortic segments NO changed the voltage-dependence of the contraction mediated by L-type Ca2+ influx, probably via S-nitrosylation of the cysteine residues located in the gating part of the channel´s α1C-subunit.

An interplay between NO and L-type Ca2+ channels was also suggested by a number of other observations: a) When treated with the CaB, lacidipine, Western-type diet-evoked hypertension and atherosclerosis development in apoE-deficient mice was reduced, whereas endothelial function was preserved (Kyselovic et al., 2005), b) after partly inhibiting L-type Ca2+ influx induced by high K+ with the CaB nifedipine, relaxation by exogenous NO was ameliorated (Van Hove et al., 2009) and c) ApoE-/- mice, which spontaneously develop atherosclerotic lesions at the age of 6 months when fed a normal diet, displayed altered Ca2+ homeostasis at the age of 4 months, hence, before development of plaques. In comparison with wild-type C57Bl6 mice, they showed higher baseline intracellular Ca2+ in the SMCs (Van Assche et al., 2007), lower baseline Ca2+ in endothelial cells and decreased basal but normal or even enhanced agonist-evoked NO release and efficacy (Fransen et al., 2008).

The present study might also provide an explanation for the unique features of the antihypertensive classes of CaBs with different chemical structure (phenylalkylamines such as verapamil, benzothiazepines such as diltiazem and dihydropyridines such as nifedipine). They reduce blood pressure more effectively in hypertensive than in normotensive subjects (Leonetti et al., 1982) and they inhibit L-type Ca2+ influx more effectively in vascular tissue than in heart (Godfraind et al., 1984; Godfraind, 2005; Striessnig et al., 1998). Several attempts have been made to explain these unique features. In hypertension, there is an increase of reactivity to vasoconstrictors, which is not only due to a higher number of L-type Ca2+ channels (Godfraind, 2005; Pesic et al., 2004; Pratt et al., 2002), but also to depolarization of the resting potential of hypertensive VSMCs (Morel & Godfraind, 1994; Pesic et al., 2004). Depolarization leads to an increased proportion L-type Ca2+ channels in the inactivated state, which according to the "modulated receptor theory" may have higher affinity to CaBs than channels in the resting state (Bean et al., 1986; Godfraind, 2005; Morel & Godfraind, 1987). Cardiac muscle cells are hyperpolarized with respect to VSMC and hence less susceptible to block by CaBs. Another specific feature of L-type Ca2+ channels is that their population is not homogeneous because of the occurrence of alternatively spliced isoforms (Koch et al., 1990). Among the 55 known human Ltype Ca2+ channel exons 19 undergo alternative splicing and display differences in tissue distribution, physiology, pharmacology and disease-related up- and/or down-regulation (Liao et al., 2005; Tang et al., 2007; Tiwari et al., 2006). Some of these isoforms were dominant in aorta (> 50%) and less abundant in heart (<5%). Moreover, the VSM-specific splice variant of the L-type Ca2+ channel displayed hyperpolarised window current at voltages where there is overlap between activation and inactivation curves and enhanced inhibition by nifedipine in comparison with the predominant cardiac isoform (Liao et al., 2007). The selective affinity of CaBs for the different L-type Ca2+ channel subpopulations may further contribute to the different susceptibility of VSMC or cardiac cells to CaBs. Because the L-type Ca2+ channel window in VSMCs may be responsible for "time-independent" baseline Ca2+ influx at normal resting or slightly depolarized membrane potentials (Fleischmann et al., 1994; Poburko et al., 2004), depolarization of the resting potential by hypertension not only favours the inactivated state of the L-type Ca2+ channel, but is also expected to increase L-type Ca2+ influx via the L-type Ca2+ channel window and to evoke inhibition by CaBs (Fleischmann et al., 1994).

Preliminary results of this chapter, finally, indicate that NO efficacy and L-type Ca2+ channel distribution may differ along the length of the thoracic aorta. Basal and stimulated eNOS activation or NO release occurred differently along the thoracic aorta. Close to the aortic arch, segments released more basal NO but less stimulated NO than segments close to the diaphragm. Similarly, L-type Ca2+ channel distribution and related window contraction were higher in atherosclerosis-prone than in -resistant segments. These preliminary data need to be further explored, but may have important consequences for the compliance of the different aortic segments and their susceptibility to the development of atherosclerosis.

#### **Author details**

Current Basic and Pathological Approaches to

versus resistant; #, ###: 2-APB versus SOCE.

the gating part of the channel´s α1C-subunit.

**7. Conclusions**

86 the Function of Muscle Cells and Tissues – From Molecules to Humans

ACh were compromised more in prone than in resistant segments and could not be fitted with sigmoidal concentration-response equations (B). Data: mean±SEM; n=7; \*, \*\*, \*\*\*: P<0.05, 0.01, 0.001 prone

The present chapter investigated the relaxing efficacy of NO for contractions induced by α1 adrenoceptor stimulation with phenylephrine or by depolarization with elevated extracellular K+ and confirmed previous observations that relaxation by NO is attenuated when the contraction was due to L-type Ca2+ influx (Van Hove et al., 2009). Contractions, which were initiated by Ca2+ release from the SR as with phenylephrine were very sensitive to endogenous or exogenous NO. Here, NO caused relaxation by inhibiting the IP3-mediated contraction, by stimulating Ca2+ re-uptake to the SR via stimulation of SERCA, by inhibiting SOCE via cation channels and by reducing SOCE via L-type Ca2+ channels. Although phenylephrine has been described to cause depolarization of the VSMCs, Vm is not clamped at depolarized values and addition of NO is expected to cause hyperpolarization, thereby reducing Ca2+ influx via L-type Ca2+ channels. By clamping Vm to depolarized values with elevation of K+, we were able to show contractions due to Ca2+ influx via L-type Ca2+ channels only and more specifically, to Ca2+ influx via non-inactivating Ca2+ channels (window contraction). This window contraction could be decreased by repolarizing Vm of VSMCs with levcromakalim (an opener of ATP-dependent K+ channels) or by adding exogenous NO as long as the extracellular K+ concentration was below 50 mM. Because NO also shifted the voltage-dependence of the window contraction in the presence of levcromakalim, it was hypothesized that NO, but not levcromakalim exerted a direct effect on the gating properties of the L-type Ca2+ channel. Although it has been described that NO affects L-type Ca2+ channels directly via S-nitrosylation (Almanza et al., 2007), an effect on the voltagedependent contraction has not been directly demonstrated yet. Hence, it is hypothesized that in mouse aortic segments NO changed the voltage-dependence of the contraction mediated by L-type Ca2+ influx, probably via S-nitrosylation of the cysteine residues located in

An interplay between NO and L-type Ca2+ channels was also suggested by a number of other observations: a) When treated with the CaB, lacidipine, Western-type diet-evoked hypertension and atherosclerosis development in apoE-deficient mice was reduced, whereas endothelial function was preserved (Kyselovic et al., 2005), b) after partly inhibiting L-type Ca2+ influx induced by high K+ with the CaB nifedipine, relaxation by exogenous NO was ameliorated (Van Hove et al., 2009) and c) ApoE-/- mice, which spontaneously develop atherosclerotic lesions at the age of 6 months when fed a normal diet, displayed altered Ca2+ homeostasis at the age of 4 months, hence, before development of plaques. In comparison with wild-type C57Bl6 mice, they showed higher baseline intracellular Ca2+ in the SMCs (Van Assche et al., 2007), lower baseline Ca2+ in endothelial cells and decreased basal but normal or even enhanced agonist-evoked NO release and efficacy (Fransen et al., 2008).

The present study might also provide an explanation for the unique features of the antihypertensive classes of CaBs with different chemical structure (phenylalkylamines such as verapamil, Paul Fransen *Laboratory of Physiopharmacology, University of Antwerp, Wilrijk, Belgium*

Cor E. Van Hove, Johanna van Langen and Hidde Bult *Laboratory of Pharmacology, University of Antwerp, Wilrijk, Belgium*

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Contraction by Ca2+ Influx via the L-Type Ca2+ Channel

Voltage Window in Mouse Aortic Segments is Modulated by Nitric Oxide 91

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Current Basic and Pathological Approaches to

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35133-35142

90 the Function of Muscle Cells and Tissues – From Molecules to Humans

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Current Basic and Pathological Approaches to

92 the Function of Muscle Cells and Tissues – From Molecules to Humans

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**Chapter 5**

© 2012 Deng et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

© 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**MAP Kinase-Mediated and**

Maoxian Deng, Lixia Deng and Yarong Xue

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/47732

**1. Introduction**

**MLC20 in Smooth Muscle Cells**

**MLCK-Independent Phosphorylation of**

Smooth muscle cells constitute the walls of various organs and tubes in the body, including the blood vessels, gastrointestinal tract, respiratory tract, bladder, and reproductive tracts. The primary function of smooth muscle contraction is to generate force, which is utilized to perform many physiological processes such as blood flow and blood pressure maintenance, gastrointestinal motility, bronchial diameter regulation, bladder evacuation, and fetus expulsion. Smooth muscle contraction is caused by the sliding of myosin and actin filaments over each other. Movement of the two types of filaments happens when the globular myosin heads protruding from myosin filaments attach and interact with actin filaments to form crossbridges. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with a new ATP. The myosin interacts with the actin to convert chemical energy, in the form of ATP, to mechanical energy. The coordinated regulation of contraction is a key property of the smooth muscle. When the smooth muscle functions normally, it contributes to general health and wellness. Contractile abnormalities of the smooth muscle are considered to underlie many diseases and disorders, including hypertension, vasospasm, diabetes-associated microvascular abnormalities, bronchial asthma, preterm labor, urinary incontinence, megacolon, and irritable bowel syndrome. Not surprisingly, inadequate contraction and relaxation of smooth muscle may cause the dysfunction of these hollow organs, which is usually associated with morbidity and mortality. Hence, the precise regulation of smooth muscle contraction is much more important in smooth muscles than in striated muscle. The muscle cells can respond to physiological and pathological signals from the environment to adapt to the environmental demands. This

**Chapter 5**

### **MAP Kinase-Mediated and MLCK-Independent Phosphorylation of MLC20 in Smooth Muscle Cells**

Maoxian Deng, Lixia Deng and Yarong Xue

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/47732

#### **1. Introduction**

Current Basic and Pathological Approaches to

92 the Function of Muscle Cells and Tissues – From Molecules to Humans

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Tsai, E. J. & Kass, D. A. (2009) Cyclic GMP signaling in cardiovascular pathophysiology and

Van Assche, T.; Fransen, P.; Guns, P. J.; Herman, A. G.; & Bult, H. (2007) Altered Ca2+ handling of smooth muscle cells in aorta of apolipoprotein E-deficient mice before

Van Hove, C. E.; Van der Donckt, C.; Herman, A. G.; Bult, H.; & Fransen, P. (2009) Vasodilator efficacy of nitric oxide depends on mechanisms of intracellular calcium mobilization in

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Westerhof, N.; Lankhaar, J. W.; & Westerhof, B. E. (2009) The arterial Windkessel*.*

Weston, A. H.; Richards, G. R.; Burnham, M. P.; Feletou, M.; Vanhoutte, P. M.; & Edwards, G. (2002) K+-induced hyperpolarization in rat mesenteric artery: identification, localization and role of Na+/K+-ATPases*. Br.J.Pharmacol.*,Vol.136, No.6, pp. 918-926 White, D. G. & Martin, W. (1989) Differential control and calcium-dependence of production of endothelium-derived relaxing factor and prostacyclin by pig aortic endothelial cells*.*

Yuan, X. J.; Tod, M. L.; Rubin, L. J.; & Blaustein, M. P. (1996) NO hyperpolarizes pulmonary artery smooth muscle cells and decreases the intracellular Ca2+ concentration by activating

Zhou, Z. H.; Wang, J.; Xiao, H.; Chen, Z. J.; Wang, M.; Cheng, X.; & Liao, Y. H. (2008) A novel autoantibody in patients with primary hypertension: antibody against L-type Ca2+

voltage-gated K+ channels*. Proc.Natl.Acad.Sci.U.S.A*,Vol.93, No.19, pp. 10489-10494 Zhang, D. X.; Mendoza, S. A.; Bubolz, A. H.; Mizuno, A.; Ge, Z. D.; Li, R.; Warltier, D. C.; Suzuki, M.; & Gutterman, D. D. (2009) Transient receptor potential vanilloid type 4 deficient mice exhibit impaired endothelium-dependent relaxation induced by

acetylcholine in vitro and in vivo*. Hypertension*,Vol.53, No.3, pp. 532-538

channel*. Chin Med.J.(Engl.)*,Vol.121, No.16, pp. 1513-1517

development of atherosclerotic lesions*. Cell Calcium*,Vol.41, No.3, pp. 295-302 Van Assche, T.; Hendrickx, J.; Crauwels, H. M.; Guns, P. J.; Martinet, W.; Fransen, P.; Raes, M.; & Bult, H. (2011) Transcription profiles of aortic smooth muscle cells from atherosclerosis-prone and -resistant regions in young apolipoprotein E-deficient mice

mouse aortic smooth muscle cells*. Br.J.Pharmacol.*,Vol.158, No.3, pp. 920-930

waveform*. Am.J.Physiol Heart Circ.Physiol*,Vol.301, No.2, pp. H382-H390

Smooth muscle cells constitute the walls of various organs and tubes in the body, including the blood vessels, gastrointestinal tract, respiratory tract, bladder, and reproductive tracts. The primary function of smooth muscle contraction is to generate force, which is utilized to perform many physiological processes such as blood flow and blood pressure maintenance, gastrointestinal motility, bronchial diameter regulation, bladder evacuation, and fetus expulsion. Smooth muscle contraction is caused by the sliding of myosin and actin filaments over each other. Movement of the two types of filaments happens when the globular myosin heads protruding from myosin filaments attach and interact with actin filaments to form crossbridges. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with a new ATP. The myosin interacts with the actin to convert chemical energy, in the form of ATP, to mechanical energy. The coordinated regulation of contraction is a key property of the smooth muscle. When the smooth muscle functions normally, it contributes to general health and wellness. Contractile abnormalities of the smooth muscle are considered to underlie many diseases and disorders, including hypertension, vasospasm, diabetes-associated microvascular abnormalities, bronchial asthma, preterm labor, urinary incontinence, megacolon, and irritable bowel syndrome. Not surprisingly, inadequate contraction and relaxation of smooth muscle may cause the dysfunction of these hollow organs, which is usually associated with morbidity and mortality. Hence, the precise regulation of smooth muscle contraction is much more important in smooth muscles than in striated muscle. The muscle cells can respond to physiological and pathological signals from the environment to adapt to the environmental demands. This

© 2012 Deng et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

adaptation is accomplished through signal transduction, which activates factors that signal pathways and ultimately lead to muscle contraction or relaxation. The regulation of smooth muscle contraction has two main mechanisms: neuromuscular and myogenic. In the neuromuscular mechanism, the smooth muscle receives principally neural innervation from the autonomic nervous system inside the same tissue, even though the central nervous system may be involved in the regulation of smooth muscle contraction. The myogenic mechanism, on the other hand, plays a more important role in the regulation of smooth muscle contraction. Under this mechanism, smooth muscle cell contraction is regulated principally by the mechanical (stretch) activation of the contractile proteins myosin and the actin in the intact body. A change in membrane potential, brought by the firing of action potentials or by the activation of stretchdependent ion channels in the plasma membrane, can also trigger contraction. These smooth muscle cells also develop tonic and phasic contractions in response to changes in load or length. In addition, the contractile state of the smooth muscle is controlled by the hormones, autocrine/paracrine agents, and other local chemical signals. Regardless of the stimulus, the smooth muscle cells use crossbridge cycling between the actin and myosin to develop force and calcium ions (Ca2+) that serve to initiate contraction in light of the current view. The deep mechanism for both myogenic and neuromuscular regulations is involved in cell signaling. Because current therapies for disorders and diseases of smooth muscles are costly and primarily palliative and do not target the cause of the disease, attention is being focused on the detailed understanding of the molecular basis of smooth muscle function and regulation and identification of abnormalities (dysfunctional proteins and signaling pathways) that lead to contractile pathologies and the development of strategies to reverse such abnormalities. The muscle contraction regulation mediated by signaling pathways is the key event. In recent years, a variety of signaling pathways has been implicated in the regulation of muscle contraction. A growing body of evidence shows a fine vista for the research of pathway-mediated regulation of the smooth muscle. One of the widely accepted notions is that the increase in Ca2+ concentration activates the Ca2+-calmodulin (CaM)–myosin light chain kinase (MLCK) pathway and stimulates the 20-kDa myosin light chain (MLC20) phosphorylation. The MLC20 phosphorylation at Ser19 causes a conformational change that increases the angle in the neck domain of the myosin heavy chain which mobilizes the crossbridges and causes the actin thin filament to slide along the myosin thick filament. Through an unknown mechanism, this interaction between the myosin and actin activates the ATPase activity of the myosin head region and leads to the development of a contractile force. Therefore, in theory, the factors that cause the inhibition of the MLCK activity and expression, such as calcium deprivation and calmodulin inactivation, could abolish consequentially MLC20 phosphorylation and smooth muscle cell contraction. However, increasing evidence supports Ca2+ independent contraction of smooth muscle (Deng et al., 2001; Harnett & Biancani, 2003; Ratz et al., 2009). Here, we will describe an MLCK-independent and mitogen-activated protein (MAP) kinase-mediated phosphorylation of MLC20.

MAP Kinase-Mediated and MLCK-Independent Phosphorylation of MLC20 in Smooth Muscle Cells 95

**2. Expression and function of MLC20 and MLCK in smooth muscle**

in regulating muscle contraction.

the activities of the MLCK and MLCP.

smooth muscle MLCK (smMLCK).

The myosin II (also called conventional myosin), a hexameric protein complex, is composed of two identical 200-kD heavy chains and two sets of light chains: the 17-kD light chain (MLC17, also known as ELC) and the 20-kD regulatory light chain (MLC20). The exact function of the MLC17 is unclear but it may contribute to the structural stability of the myosin head along with the MLC20. The MLC20 is a small ring around the neck region of heavy chains of myosin and is also known as the regulatory light chain (also called RLC20, RLC2, and LC2). It is believed to participate actively in muscle contraction. The MLC20 is expressed not only in smooth muscle, but also in cardiac, skeletal, and nonmuscle cells. Only the MLC20 in the smooth muscle appears to play a unique role in increasing the actinactivated ATPase activity. The MLC20 is activated by phosphorylation at multiple serine and threonine residues. The phosphorylation of the MLC20 is thought to play a pivotal role

Up to now, the MLC20 is the only known physiological substrate of the MLCK. Protein kinase C (PKC), CaM-kinase II, Rho-kinase, p21-activated kinase, and p34cdc2 kinase also phosphorylate three residues on the N-terminus of the MLC20, Ser-1, Ser-2, and Thr-9. In addition, the integrin-linked kinase and ZIP-kinase are reported to phosphorylate the MLC20 in the absence of Ca2+, at Ser19 and Thr18, thus activating the myosin activity (Niiro & Ikebe, 2001). *In vivo* phosphorylation of the MLC20 isoforms is accomplished by raising free intracellular Ca2+ and subsequent activation of the Ca2+-calmodulin-dependent MLCK. Ca2+/calmodulin-dependent MLCK is considered the primary regulator of MLC20 phosphorylation among potential regulators of MLC20 phosphorylation. The dephosphorylation of phosphorylated MLC20 is catalyzed by the myosin light chain phosphatase (MLCP), which counters the MLCK that promotes contraction by phosphorylating MLC20. The degree of phosphorylation of the RLC depends on the ratio of

The myosin light chain kinase (MLCK or MYLK) is a Ca2+/CaM-activated kinase found in smooth, cardiac, and skeletal muscles as well as in many mammalian nonmuscle cells. It is a serine/threonine-specific protein kinase that phosphorylates the MLC20 of myosin II. There are three isoforms of the MLCK, i.e., smooth muscle (smMLCK, ~130-kDa), skeletal muscle (skMLCK, ~220-kDa), and cardiac muscle (cMLCK). The short MLCK (smMLCK, ~130kDa) is best known as the conventional smooth muscle MLCK. The smMLCK is encoded by the mylk1 gene, which expresses three transcripts in a cell-specific manner due to the alternate promoters, long MLCK (210- to 220-kDa), short MLCK (130-kDa), and the noncatalytic gene product, called telokin. The short smooth muscle MLCK (130-kDa) is ubiquitous in all adult tissues with the highest amounts in smooth muscle tissues. Since both the short and long MLCK are found in smooth muscles or cultured/embryonic smooth muscle tissues, they are called smooth muscle MLCK (smMLCK). The two smMLCKs have been extensively described, and their function is to regulate the activity of the nonmuscle and smooth muscle myosin II. Here we will deal only with the short

#### **2. Expression and function of MLC20 and MLCK in smooth muscle**

Current Basic and Pathological Approaches to

94 the Function of Muscle Cells and Tissues – From Molecules to Humans

protein (MAP) kinase-mediated phosphorylation of MLC20.

adaptation is accomplished through signal transduction, which activates factors that signal pathways and ultimately lead to muscle contraction or relaxation. The regulation of smooth muscle contraction has two main mechanisms: neuromuscular and myogenic. In the neuromuscular mechanism, the smooth muscle receives principally neural innervation from the autonomic nervous system inside the same tissue, even though the central nervous system may be involved in the regulation of smooth muscle contraction. The myogenic mechanism, on the other hand, plays a more important role in the regulation of smooth muscle contraction. Under this mechanism, smooth muscle cell contraction is regulated principally by the mechanical (stretch) activation of the contractile proteins myosin and the actin in the intact body. A change in membrane potential, brought by the firing of action potentials or by the activation of stretchdependent ion channels in the plasma membrane, can also trigger contraction. These smooth muscle cells also develop tonic and phasic contractions in response to changes in load or length. In addition, the contractile state of the smooth muscle is controlled by the hormones, autocrine/paracrine agents, and other local chemical signals. Regardless of the stimulus, the smooth muscle cells use crossbridge cycling between the actin and myosin to develop force and calcium ions (Ca2+) that serve to initiate contraction in light of the current view. The deep mechanism for both myogenic and neuromuscular regulations is involved in cell signaling. Because current therapies for disorders and diseases of smooth muscles are costly and primarily palliative and do not target the cause of the disease, attention is being focused on the detailed understanding of the molecular basis of smooth muscle function and regulation and identification of abnormalities (dysfunctional proteins and signaling pathways) that lead to contractile pathologies and the development of strategies to reverse such abnormalities. The muscle contraction regulation mediated by signaling pathways is the key event. In recent years, a variety of signaling pathways has been implicated in the regulation of muscle contraction. A growing body of evidence shows a fine vista for the research of pathway-mediated regulation of the smooth muscle. One of the widely accepted notions is that the increase in Ca2+ concentration activates the Ca2+-calmodulin (CaM)–myosin light chain kinase (MLCK) pathway and stimulates the 20-kDa myosin light chain (MLC20) phosphorylation. The MLC20 phosphorylation at Ser19 causes a conformational change that increases the angle in the neck domain of the myosin heavy chain which mobilizes the crossbridges and causes the actin thin filament to slide along the myosin thick filament. Through an unknown mechanism, this interaction between the myosin and actin activates the ATPase activity of the myosin head region and leads to the development of a contractile force. Therefore, in theory, the factors that cause the inhibition of the MLCK activity and expression, such as calcium deprivation and calmodulin inactivation, could abolish consequentially MLC20 phosphorylation and smooth muscle cell contraction. However, increasing evidence supports Ca2+ independent contraction of smooth muscle (Deng et al., 2001; Harnett & Biancani, 2003; Ratz et al., 2009). Here, we will describe an MLCK-independent and mitogen-activated

The myosin II (also called conventional myosin), a hexameric protein complex, is composed of two identical 200-kD heavy chains and two sets of light chains: the 17-kD light chain (MLC17, also known as ELC) and the 20-kD regulatory light chain (MLC20). The exact function of the MLC17 is unclear but it may contribute to the structural stability of the myosin head along with the MLC20. The MLC20 is a small ring around the neck region of heavy chains of myosin and is also known as the regulatory light chain (also called RLC20, RLC2, and LC2). It is believed to participate actively in muscle contraction. The MLC20 is expressed not only in smooth muscle, but also in cardiac, skeletal, and nonmuscle cells. Only the MLC20 in the smooth muscle appears to play a unique role in increasing the actinactivated ATPase activity. The MLC20 is activated by phosphorylation at multiple serine and threonine residues. The phosphorylation of the MLC20 is thought to play a pivotal role in regulating muscle contraction.

Up to now, the MLC20 is the only known physiological substrate of the MLCK. Protein kinase C (PKC), CaM-kinase II, Rho-kinase, p21-activated kinase, and p34cdc2 kinase also phosphorylate three residues on the N-terminus of the MLC20, Ser-1, Ser-2, and Thr-9. In addition, the integrin-linked kinase and ZIP-kinase are reported to phosphorylate the MLC20 in the absence of Ca2+, at Ser19 and Thr18, thus activating the myosin activity (Niiro & Ikebe, 2001). *In vivo* phosphorylation of the MLC20 isoforms is accomplished by raising free intracellular Ca2+ and subsequent activation of the Ca2+-calmodulin-dependent MLCK. Ca2+/calmodulin-dependent MLCK is considered the primary regulator of MLC20 phosphorylation among potential regulators of MLC20 phosphorylation. The dephosphorylation of phosphorylated MLC20 is catalyzed by the myosin light chain phosphatase (MLCP), which counters the MLCK that promotes contraction by phosphorylating MLC20. The degree of phosphorylation of the RLC depends on the ratio of the activities of the MLCK and MLCP.

The myosin light chain kinase (MLCK or MYLK) is a Ca2+/CaM-activated kinase found in smooth, cardiac, and skeletal muscles as well as in many mammalian nonmuscle cells. It is a serine/threonine-specific protein kinase that phosphorylates the MLC20 of myosin II. There are three isoforms of the MLCK, i.e., smooth muscle (smMLCK, ~130-kDa), skeletal muscle (skMLCK, ~220-kDa), and cardiac muscle (cMLCK). The short MLCK (smMLCK, ~130kDa) is best known as the conventional smooth muscle MLCK. The smMLCK is encoded by the mylk1 gene, which expresses three transcripts in a cell-specific manner due to the alternate promoters, long MLCK (210- to 220-kDa), short MLCK (130-kDa), and the noncatalytic gene product, called telokin. The short smooth muscle MLCK (130-kDa) is ubiquitous in all adult tissues with the highest amounts in smooth muscle tissues. Since both the short and long MLCK are found in smooth muscles or cultured/embryonic smooth muscle tissues, they are called smooth muscle MLCK (smMLCK). The two smMLCKs have been extensively described, and their function is to regulate the activity of the nonmuscle and smooth muscle myosin II. Here we will deal only with the short smooth muscle MLCK (smMLCK).

The activation of myosin motors by the MLCK modulates a variety of contractile processes, including smooth muscle contraction, cell adhesion, migration, and proliferation. The dysregulation of these processes contribute to a number of diseases. It is widely accepted that the MLCK phosphorylates the MLC20 of smooth and nonmuscle myosin II in the presence of Ca2+ and calmodulin. The phosphorylation of the MLC20 facilitates myosin interaction with actin filaments. Once there is an influx of calcium cations into the muscle, either from the sarcoplasmic reticulum or, more important, from the extracellular space, the contraction of smooth muscle fibers may begin. First, the elevation of the Ca2+ concentration in smooth muscles causes the Ca2+ to bind to the calmodulin (CaM). Then, the complex of the Ca2+ and CaM (Ca2+/CaM) activates the MLCK, which would phosphorylate the myosin light chain (MLC20) at serine residue 19 to generate phospho-MLC20. The phosphorylation of the MLC20 enables the myosin crossbridge to bind to the actin filament and allows contraction to begin (through the crossbridge cycle). The Ca2+-CaM activates the MLCK by reversal of an auto-inhibited state. In contrast, reducing intracellular calcium concentration inactivates MLCK but does not stop smooth muscle contraction since the MLC20 has been physically modified through phosphorylation. This regulatory model of myosin phosphorylation is widely accepted as the intracellular path for the induction of smooth muscle contraction.

MAP Kinase-Mediated and MLCK-Independent Phosphorylation of MLC20 in Smooth Muscle Cells 97

**Figure 1.** Inconsistence between MLC20 phosphorylation and MLCK expression in smooth muscle

alternative regulation system may play an active role in the MLC20 phosphorylation.

In the experiments, fresh smooth muscle tissues were homogenized on ice with pre-cooled small glass homogenizers within ice-cold lysis buffer (50 mM Tris, 300 mM NaCl, 3 mM EGTA, 0.1 mM sodium orthovanadate, 10% glycerol v/v, 1% NP-40 v/v, 0.3% SDS w/v, protease and phosphatase inhibitor cocktails from Sigma Co., Germany, pH 7.6). Twenty micrograms of protein were loaded onto 10% SDS-PAGE gels that were subjected to electrophoresis in running buffer (BioRad Co., Hercules, CA). The proteins were then transferred to the Immobilon-P Transfer Membrane (Millipore Co., MA), followed by blocking it with 5% fat-free milk in TBST buffer. The MLCK was detected with a specific antibody. After it was washed with 1PBS for three times, the membranes were re-probed with antibody anti-phospho MLC20. The results show that the levels of MLCK expression and phosphorylated MLC20 in different smooth muscles from the same mouse are markedly different (Figure 1). For instance, the large intestine of mouse 1 contains a high level of phosphor MLC20 but the MLCK expression is low. In contrast, the small intestine has low phosphor MLC20 but the MLCK expression is high. Besides, the MLCK expression and phosphor MLC20 in the same type of smooth muscle tissue are inconsistent among different individuals. For example, the large intestine of mouse 1 contains a low expression of MLCK and high phosphor MLC20. Conversely, mice 2 and 5 express high levels of MLCK in their large intestines but the MLC20 phosphorylation is low. The conspicuous inconsistence between the MLC20 phosphorylation and MLCK expression was subsequently verified by Western blotting in independent experiments. These interesting results cannot be explained by the current regulatory mode. Obviously, they suggest that an

tissues. \*in, intestine; †, MLCK expression; ‡, phosphor MLC20.

However, to a certain extent, this viewpoint about the Ca2+-, MLCK- and MLC20-dependent regulatory mechanism is founded on a presumption. Several observations of smooth muscle contraction cannot be explained by the mode of phosphorylation (Kohama K & K., 1995). For example, when the uterine smooth muscle is subjected to prolonged incubation in a Ca2+-free medium, the oxytocin induces the contraction of the muscle without any signs of MLC20 phosphorylation (Oishi et al., 1991). Another example is the MLC20 phosphorylated in rat embryo fibroblasts, which contains no detectable MLCK (Emmert et al., 2004). In addition, these are increasing evidence to support the calcium- and MLCK-independent mechanism of MLC20 phosphorylation (McFawn et al., 2003; El-Toukhy et al., 2006; Cho et al., 2011). According to the current mode, the expression of the MLCK in tissues/cells is necessarily consistent with the MLC20 phosphorylation. However, there has not been any report about the consistence between the MLCK expression and MLC20 phosphorylation. Herein, we first investigated the consistency between the MLCK expression and phosphor MLC20. In the following section we will describe a recent finding, the significant inconsistence between MLCK expression, and MLC20 phosphorylation in multiple smooth muscles in mice.

#### **3. Inconsistence between MLCK expression and MLC20 phosphorylation**

According to the current view, the MLCK expression must co-localize with the MLC20 phosphorylation in cells or tissues. However, by a series of experiments, we recently found that phosphorylation (Ser19) of the MLC20 is inconsistent with 130-kDa MLCK expression in mouse aorta, bladder, large/small intestines, stomach, and uterus (Deng et al., 2011) (Figure.1A and B).

Current Basic and Pathological Approaches to

muscle contraction.

muscles in mice.

(Figure.1A and B).

96 the Function of Muscle Cells and Tissues – From Molecules to Humans

The activation of myosin motors by the MLCK modulates a variety of contractile processes, including smooth muscle contraction, cell adhesion, migration, and proliferation. The dysregulation of these processes contribute to a number of diseases. It is widely accepted that the MLCK phosphorylates the MLC20 of smooth and nonmuscle myosin II in the presence of Ca2+ and calmodulin. The phosphorylation of the MLC20 facilitates myosin interaction with actin filaments. Once there is an influx of calcium cations into the muscle, either from the sarcoplasmic reticulum or, more important, from the extracellular space, the contraction of smooth muscle fibers may begin. First, the elevation of the Ca2+ concentration in smooth muscles causes the Ca2+ to bind to the calmodulin (CaM). Then, the complex of the Ca2+ and CaM (Ca2+/CaM) activates the MLCK, which would phosphorylate the myosin light chain (MLC20) at serine residue 19 to generate phospho-MLC20. The phosphorylation of the MLC20 enables the myosin crossbridge to bind to the actin filament and allows contraction to begin (through the crossbridge cycle). The Ca2+-CaM activates the MLCK by reversal of an auto-inhibited state. In contrast, reducing intracellular calcium concentration inactivates MLCK but does not stop smooth muscle contraction since the MLC20 has been physically modified through phosphorylation. This regulatory model of myosin phosphorylation is widely accepted as the intracellular path for the induction of smooth

However, to a certain extent, this viewpoint about the Ca2+-, MLCK- and MLC20-dependent regulatory mechanism is founded on a presumption. Several observations of smooth muscle contraction cannot be explained by the mode of phosphorylation (Kohama K & K., 1995). For example, when the uterine smooth muscle is subjected to prolonged incubation in a Ca2+-free medium, the oxytocin induces the contraction of the muscle without any signs of MLC20 phosphorylation (Oishi et al., 1991). Another example is the MLC20 phosphorylated in rat embryo fibroblasts, which contains no detectable MLCK (Emmert et al., 2004). In addition, these are increasing evidence to support the calcium- and MLCK-independent mechanism of MLC20 phosphorylation (McFawn et al., 2003; El-Toukhy et al., 2006; Cho et al., 2011). According to the current mode, the expression of the MLCK in tissues/cells is necessarily consistent with the MLC20 phosphorylation. However, there has not been any report about the consistence between the MLCK expression and MLC20 phosphorylation. Herein, we first investigated the consistency between the MLCK expression and phosphor MLC20. In the following section we will describe a recent finding, the significant inconsistence between MLCK expression, and MLC20 phosphorylation in multiple smooth

**3. Inconsistence between MLCK expression and MLC20 phosphorylation**

According to the current view, the MLCK expression must co-localize with the MLC20 phosphorylation in cells or tissues. However, by a series of experiments, we recently found that phosphorylation (Ser19) of the MLC20 is inconsistent with 130-kDa MLCK expression in mouse aorta, bladder, large/small intestines, stomach, and uterus (Deng et al., 2011)

**Figure 1.** Inconsistence between MLC20 phosphorylation and MLCK expression in smooth muscle tissues. \*in, intestine; †, MLCK expression; ‡, phosphor MLC20.

In the experiments, fresh smooth muscle tissues were homogenized on ice with pre-cooled small glass homogenizers within ice-cold lysis buffer (50 mM Tris, 300 mM NaCl, 3 mM EGTA, 0.1 mM sodium orthovanadate, 10% glycerol v/v, 1% NP-40 v/v, 0.3% SDS w/v, protease and phosphatase inhibitor cocktails from Sigma Co., Germany, pH 7.6). Twenty micrograms of protein were loaded onto 10% SDS-PAGE gels that were subjected to electrophoresis in running buffer (BioRad Co., Hercules, CA). The proteins were then transferred to the Immobilon-P Transfer Membrane (Millipore Co., MA), followed by blocking it with 5% fat-free milk in TBST buffer. The MLCK was detected with a specific antibody. After it was washed with 1PBS for three times, the membranes were re-probed with antibody anti-phospho MLC20. The results show that the levels of MLCK expression and phosphorylated MLC20 in different smooth muscles from the same mouse are markedly different (Figure 1). For instance, the large intestine of mouse 1 contains a high level of phosphor MLC20 but the MLCK expression is low. In contrast, the small intestine has low phosphor MLC20 but the MLCK expression is high. Besides, the MLCK expression and phosphor MLC20 in the same type of smooth muscle tissue are inconsistent among different individuals. For example, the large intestine of mouse 1 contains a low expression of MLCK and high phosphor MLC20. Conversely, mice 2 and 5 express high levels of MLCK in their large intestines but the MLC20 phosphorylation is low. The conspicuous inconsistence between the MLC20 phosphorylation and MLCK expression was subsequently verified by Western blotting in independent experiments. These interesting results cannot be explained by the current regulatory mode. Obviously, they suggest that an alternative regulation system may play an active role in the MLC20 phosphorylation.

#### **4. Unknown substance(s) other than MLCK phosphorylates MLC20**

Even though some of the smooth muscle does not show phosphor MLC20 bands (Figure 1), those tissues still contain the MLC20 because we noticed in subsequent experiments that all these frozen lysates had similar levels of phosphor MLC20 after storage in -80C and several cycles of freezing and thawing. The MLC20 in some tissues was not visualized by any specific antibody against the phosphor MLC20 only because it was not phosphorylated. The MLC20 phosphorylation in MLCK-free tissues suggests that unknown molecule(s) may phosphorylate the MLC20 in phosphor MLC20-containing tissues.

MAP Kinase-Mediated and MLCK-Independent Phosphorylation of MLC20 in Smooth Muscle Cells 99

phosphorylate MLC20, while the extract without detectable MLCK phosphorylated MLC20 of extract 1 contains no detectable MLCK (line 3). Several independent experiments were conducted to verify the results. These experiments suggest that MLC20 can be phosphorylated *in vitro* and that non-MLCK substance(s) may trigger the MLC20

**Figure 2.** Unknown substance(s) in smooth muscle tissue but not immunoprecipitated MLCK induced MLC20 phosphorylation. (A) Small amount of extract 2 without MLCK induced MLC20 phosphorylation of MLC20 in extract 1 (lines 2 and 3). (B) Precipitated MLCK did not induce MLC20 phosphorylation (line 2). The extract 2 containing no detectable MLCK increased significantly the phosphorylation of MLC 20 in extract 1(line 3). MLCK\*, MLCK freshly isolated by co-immunoprecipitation from smooth

**5. Inhibition of MLCK expression and activity do not affect MLC20**

and MLCK, and 3) depriving the calcium ion of cultured hBSM cells.

According to the current regulatory model, intracellular calcium ion is the trigger of smooth muscle contraction. The contractile initiation, maintenance, and strength are dependent on the control of intracellular free Ca2+ level. The canonical excitation-contraction coupling pathway is triggered by neural, hormonal or myogenic stimulation to elicit the influx of extracellular or intracellular Ca2+ (from the sarcoplasmic reticulum) into the cytosol. The Ca2+ binds to calmodulin to form the Ca2+/CaM complex. At an increased level of Ca2+, rapid binding of the Ca2+ to CaM occurs and triggers MLCK activation. Once activated, the MLCK phosphorylates MLC20 leads to crossbridges cycling and generation of the contractile force. Therefore, several factors may affect the MLC20 phosphorylation, including expression of the MLCK gene, MLCK activity, free Ca2+ concentration, and CaM activity. The following experiments were designed to access the effects of these factors on the role of the MLCK in MLC20 phosphorylation: 1) using siRNA to knockdown the MLCK expression in human bladder smooth muscle (hBSM) cells, 2) inactivating the MLCK by inhibitors of calmodulin

We first investigated the effect of alteration in MLCK expression on the MLC20 phosphorylation. To inhibit the MLCK expression, the siRNA against MLCK (from Santa Cruz Biotechnology Inc.) was transfected with Lipofectamine 2000 into cultured hBSM cells at a cell density of 40-50% confluence, which were isolated from the peri-cancer tissue of a bladder cancer patient. Only lower than seventh passage primary cells were used for experiments. Two days after transfection, cells were harvested for Western blotting. MLCK,

phosphorylation.

muscle tissue; *p*-MLC20, phosphor MLC20.

**phosphorylation**

Thus, we proposed a hypothesis that something else other than the MLCK phosphorylates MLC20 in the smooth muscle tissues, which contain no detectable MLCK. Even though we did not know what kind of substance(s) the phosphorylated MLC20 is/are in the phosphor MLC20-containing and MLCK-free tissue, it may mobilize the phosphorylative process of the MLC20. To test our hypothesis, we designed an unconventional experiment, and added a small amount of tissue extract which was freshly prepared from the phosphor MLC20 containing tissues without any detectable MLCK (extract 2), to another extract that contained the unphosphorylated but had no phosphorylated MLC20 and MLCK (extract 1). In other words, we theorized that extract 2 is a catalyst and extract 1 is a substrate of extract 2. By rapid Western blotting assay (with one-hour incubation at room temperature), we chose the tissue extract containing the high phosphor MLC20 but no detectable MLCK as a catalyst (extract 2), and the one containing no phosphor MLC20 and detectable MLCK as the substrate (extract 1). Then, a small amount of exact 2 was added directly into extract 1, as shown in Figure 2A. After the one-hour 37C incubation in water, as expected, the added extract 2 phosphorylated strongly MLC20 of the extract 1 (lines 2 and 3 in Figure 2A). This suggests that high phosphor MLC20 tissue content may have non-MLCK substance(s) that induce/s MLC20 phosphorylation.

To evaluate the further roles of the MLCK and the possible non-MLCK substance(s) in the MLC20 phosphorylation, we assayed the effects of the isolated MLCK on MLC20 phosphorylation (Figure 2B). Firstly, the MLCK was isolated from the fresh smooth muscle tissue by the anti-MLCK monoclonal antibody from Sigma Co., following conventional immunoprecipitation protocol. Secondly, the isolated MLCK was immediately added to the freshly prepared extract 1, which contains unphosphorylated MLC20 but no detectable MLCK (line 2 in Figure 2B). In the meantime, a small amount of extract 2 that contains a high level phosphor MLC20 but no detectable MLCK was added into the same 'substrate' extract 1 as a control (line 3). The mixtures were incubated in 37C water for one hour, followed by Western blotting assay. Extracts 1, 2 and isolated MLCK were loaded to lines 1, 5 and 4, respectively, for assays of its own MLCK and phosphor MLC20. No protease and phosphatase inhibitor were added into the reaction system. After the membrane was washed with PBS for three times, the actin and phospho-MLC20 were simultaneously probed by mixture solution of the antibodies against actin and phospho-MLC20 (Membrane was not striped by striping buffer because the sizes of the three targeting proteins were much different). The results show that the immunoprecipitated MLCK failed to phosphorylate MLC20, while the extract without detectable MLCK phosphorylated MLC20 of extract 1 contains no detectable MLCK (line 3). Several independent experiments were conducted to verify the results. These experiments suggest that MLC20 can be phosphorylated *in vitro* and that non-MLCK substance(s) may trigger the MLC20 phosphorylation.

Current Basic and Pathological Approaches to

induce/s MLC20 phosphorylation.

98 the Function of Muscle Cells and Tissues – From Molecules to Humans

phosphorylate the MLC20 in phosphor MLC20-containing tissues.

**4. Unknown substance(s) other than MLCK phosphorylates MLC20**

Even though some of the smooth muscle does not show phosphor MLC20 bands (Figure 1), those tissues still contain the MLC20 because we noticed in subsequent experiments that all these frozen lysates had similar levels of phosphor MLC20 after storage in -80C and several cycles of freezing and thawing. The MLC20 in some tissues was not visualized by any specific antibody against the phosphor MLC20 only because it was not phosphorylated. The MLC20 phosphorylation in MLCK-free tissues suggests that unknown molecule(s) may

Thus, we proposed a hypothesis that something else other than the MLCK phosphorylates MLC20 in the smooth muscle tissues, which contain no detectable MLCK. Even though we did not know what kind of substance(s) the phosphorylated MLC20 is/are in the phosphor MLC20-containing and MLCK-free tissue, it may mobilize the phosphorylative process of the MLC20. To test our hypothesis, we designed an unconventional experiment, and added a small amount of tissue extract which was freshly prepared from the phosphor MLC20 containing tissues without any detectable MLCK (extract 2), to another extract that contained the unphosphorylated but had no phosphorylated MLC20 and MLCK (extract 1). In other words, we theorized that extract 2 is a catalyst and extract 1 is a substrate of extract 2. By rapid Western blotting assay (with one-hour incubation at room temperature), we chose the tissue extract containing the high phosphor MLC20 but no detectable MLCK as a catalyst (extract 2), and the one containing no phosphor MLC20 and detectable MLCK as the substrate (extract 1). Then, a small amount of exact 2 was added directly into extract 1, as shown in Figure 2A. After the one-hour 37C incubation in water, as expected, the added extract 2 phosphorylated strongly MLC20 of the extract 1 (lines 2 and 3 in Figure 2A). This suggests that high phosphor MLC20 tissue content may have non-MLCK substance(s) that

To evaluate the further roles of the MLCK and the possible non-MLCK substance(s) in the MLC20 phosphorylation, we assayed the effects of the isolated MLCK on MLC20 phosphorylation (Figure 2B). Firstly, the MLCK was isolated from the fresh smooth muscle tissue by the anti-MLCK monoclonal antibody from Sigma Co., following conventional immunoprecipitation protocol. Secondly, the isolated MLCK was immediately added to the freshly prepared extract 1, which contains unphosphorylated MLC20 but no detectable MLCK (line 2 in Figure 2B). In the meantime, a small amount of extract 2 that contains a high level phosphor MLC20 but no detectable MLCK was added into the same 'substrate' extract 1 as a control (line 3). The mixtures were incubated in 37C water for one hour, followed by Western blotting assay. Extracts 1, 2 and isolated MLCK were loaded to lines 1, 5 and 4, respectively, for assays of its own MLCK and phosphor MLC20. No protease and phosphatase inhibitor were added into the reaction system. After the membrane was washed with PBS for three times, the actin and phospho-MLC20 were simultaneously probed by mixture solution of the antibodies against actin and phospho-MLC20 (Membrane was not striped by striping buffer because the sizes of the three targeting proteins were much different). The results show that the immunoprecipitated MLCK failed to

**Figure 2.** Unknown substance(s) in smooth muscle tissue but not immunoprecipitated MLCK induced MLC20 phosphorylation. (A) Small amount of extract 2 without MLCK induced MLC20 phosphorylation of MLC20 in extract 1 (lines 2 and 3). (B) Precipitated MLCK did not induce MLC20 phosphorylation (line 2). The extract 2 containing no detectable MLCK increased significantly the phosphorylation of MLC 20 in extract 1(line 3). MLCK\*, MLCK freshly isolated by co-immunoprecipitation from smooth muscle tissue; *p*-MLC20, phosphor MLC20.

#### **5. Inhibition of MLCK expression and activity do not affect MLC20 phosphorylation**

According to the current regulatory model, intracellular calcium ion is the trigger of smooth muscle contraction. The contractile initiation, maintenance, and strength are dependent on the control of intracellular free Ca2+ level. The canonical excitation-contraction coupling pathway is triggered by neural, hormonal or myogenic stimulation to elicit the influx of extracellular or intracellular Ca2+ (from the sarcoplasmic reticulum) into the cytosol. The Ca2+ binds to calmodulin to form the Ca2+/CaM complex. At an increased level of Ca2+, rapid binding of the Ca2+ to CaM occurs and triggers MLCK activation. Once activated, the MLCK phosphorylates MLC20 leads to crossbridges cycling and generation of the contractile force. Therefore, several factors may affect the MLC20 phosphorylation, including expression of the MLCK gene, MLCK activity, free Ca2+ concentration, and CaM activity. The following experiments were designed to access the effects of these factors on the role of the MLCK in MLC20 phosphorylation: 1) using siRNA to knockdown the MLCK expression in human bladder smooth muscle (hBSM) cells, 2) inactivating the MLCK by inhibitors of calmodulin and MLCK, and 3) depriving the calcium ion of cultured hBSM cells.

We first investigated the effect of alteration in MLCK expression on the MLC20 phosphorylation. To inhibit the MLCK expression, the siRNA against MLCK (from Santa Cruz Biotechnology Inc.) was transfected with Lipofectamine 2000 into cultured hBSM cells at a cell density of 40-50% confluence, which were isolated from the peri-cancer tissue of a bladder cancer patient. Only lower than seventh passage primary cells were used for experiments. Two days after transfection, cells were harvested for Western blotting. MLCK, *pan*-actin and phosphor MLC20 were detected by specific antibodies. Western blotting analysis showed that no significant alteration of MLC20 phosphorylation level was observed while the MLCK expression was knocked down (Figure 3A). Together with the data about the expression of MLCK and MLC20 phosphorylation from mice, this experiment provides further proof that MLC20 phosphorylation is independent of MLCK gene expression.

MAP Kinase-Mediated and MLCK-Independent Phosphorylation of MLC20 in Smooth Muscle Cells 101

Putting the data together, we firmly believe that an alternative mechanism plays an important part in the regulation of MLC20 phosphorylation. This potential regulatory

**Figure 4.** Two main pathways involved in regulation of smooth muscle cell contraction and migration. The contractile response is initiated by a rapid and transient rise in intracellular Ca2+, followed by a Ca2+-calmodulin interaction, the MLCK activation to initiate phosphorylation of the MLC20 and therefore contraction and cell migration (the blue). Agonists (such as ET-1) bind to serpentine receptors on the membrane of the smooth muscle cells to activate the RhoA/Rho kinase signaling pathway, which inactivates the MLC phosphatase (MLCP) by phosphorylation, and resulted in increased unphosphorylated MLCP and sustained smooth muscle contraction (the green). GAP, GTPase activating protein;

After we verified the MLCK-independent regulation of MLC phosphorylation, we were interested in searching for the non-MLCK mechanism that may function as the trigger of MLC20 phosphorylation. The cell signaling pathways would be most likely to address this intractable issue. In recent years, a multitude of signaling pathways has been suggested to regulate the smooth muscle contractility. However, these pathways can be broken down into two major pathways (Figure 4): the calcium–calmodulin signalling pathway and Rho/Rho-kinase pathway. The former, the Ca2+/CaM pathway, regulates smooth muscle contraction by the binding of increased intracellular calcium ion with CaM, which activates the MLCK. The MLCK phosphorylates the MLC20 at the neck of the myosin heavy chains. The phosphorylation increases the ATPase activity and thereby produces contraction. In the Rho/Rho-kinase pathway, the Rho A-GTP, activated by multiple stimuli, activates the Rho kinase (also known as Rok or Rock). The activated kinase phosphorylates the myosin

**6. MAP kinase pathways and functions in smooth muscle cells**

phosphatase and therefore modulates the MLC20 phosphorylation.

mechanism is MLCK-independent.

GEF, GDP/GTP exchange factor.

Equally vital to the MLCK gene expression, the MLCK activity or activation may contribute to its functions. Then, the subsequent experiments were designed to study the effects of the MLCK activity on the MLC20 phosphorylation. The MLCK and its upstream molecule, the CaM, were directly inactivated by the corresponding commercial inhibitory peptides (EMD Chemicals Inc., NJ) in both permeabilized or unpermeabilized hBSM cells (Figure 3B, C) and mouse smooth muscle extracts (Figure 3D). The MLC20 phosphorylation was examined by Western blotting. In addition, ethylene glycol tetraacetic acid (EGTA), a Ca2+ chelating agent, was added into the medium to prevent the formation of Ca2+/CaM complex in cultured hBSM cells (Figure 3B and C). CaM and MLCK inhibitory peptides were added into mixtures of smooth muscle extracts 1 and 2, in which small amount of extract 2 was able to phosphorylate MLC20 of extract 1 (shown in figure 2). After one-hour incubation at 37C, *pan*-actin and phosphor MLC20 were assayed by Western blotting. Likewise, none of the inhibitors of the MLCK and CaM, and EGTA inhibit the MLC20 phosphorylation in both cultured hBSM cells and tissue extracts.

**Figure 3.** Inhibition of MLCK expression and activity does not block MLC20 phosphorylation. (A) siRNA-mediated MLCK knockdown did not inhibit MLC20 phosphorylation. (B and C) Inhibitors of CaM and MLCK and EGTA did not suppress MLC20 phosphorylation in permeabilized and unpermeabilized cells. (D) Neither CaM nor MLCK inhibitors blocked MLC20 phosphorylation in smooth muscle extracts.

Putting the data together, we firmly believe that an alternative mechanism plays an important part in the regulation of MLC20 phosphorylation. This potential regulatory mechanism is MLCK-independent.

Current Basic and Pathological Approaches to

cultured hBSM cells and tissue extracts.

cle extracts.

100 the Function of Muscle Cells and Tissues – From Molecules to Humans

*pan*-actin and phosphor MLC20 were detected by specific antibodies. Western blotting analysis showed that no significant alteration of MLC20 phosphorylation level was observed while the MLCK expression was knocked down (Figure 3A). Together with the data about the expression of MLCK and MLC20 phosphorylation from mice, this experiment provides

Equally vital to the MLCK gene expression, the MLCK activity or activation may contribute to its functions. Then, the subsequent experiments were designed to study the effects of the MLCK activity on the MLC20 phosphorylation. The MLCK and its upstream molecule, the CaM, were directly inactivated by the corresponding commercial inhibitory peptides (EMD Chemicals Inc., NJ) in both permeabilized or unpermeabilized hBSM cells (Figure 3B, C) and mouse smooth muscle extracts (Figure 3D). The MLC20 phosphorylation was examined by Western blotting. In addition, ethylene glycol tetraacetic acid (EGTA), a Ca2+ chelating agent, was added into the medium to prevent the formation of Ca2+/CaM complex in cultured hBSM cells (Figure 3B and C). CaM and MLCK inhibitory peptides were added into mixtures of smooth muscle extracts 1 and 2, in which small amount of extract 2 was able to phosphorylate MLC20 of extract 1 (shown in figure 2). After one-hour incubation at 37C, *pan*-actin and phosphor MLC20 were assayed by Western blotting. Likewise, none of the inhibitors of the MLCK and CaM, and EGTA inhibit the MLC20 phosphorylation in both

further proof that MLC20 phosphorylation is independent of MLCK gene expression.

**Figure 3.** Inhibition of MLCK expression and activity does not block MLC20 phosphorylation. (A) siRNA-mediated MLCK knockdown did not inhibit MLC20 phosphorylation. (B and C) Inhibitors of CaM and MLCK and EGTA did not suppress MLC20 phosphorylation in permeabilized and unpermeabilized cells. (D) Neither CaM nor MLCK inhibitors blocked MLC20 phosphorylation in smooth mus-

**Figure 4.** Two main pathways involved in regulation of smooth muscle cell contraction and migration. The contractile response is initiated by a rapid and transient rise in intracellular Ca2+, followed by a Ca2+-calmodulin interaction, the MLCK activation to initiate phosphorylation of the MLC20 and therefore contraction and cell migration (the blue). Agonists (such as ET-1) bind to serpentine receptors on the membrane of the smooth muscle cells to activate the RhoA/Rho kinase signaling pathway, which inactivates the MLC phosphatase (MLCP) by phosphorylation, and resulted in increased unphosphorylated MLCP and sustained smooth muscle contraction (the green). GAP, GTPase activating protein; GEF, GDP/GTP exchange factor.

#### **6. MAP kinase pathways and functions in smooth muscle cells**

After we verified the MLCK-independent regulation of MLC phosphorylation, we were interested in searching for the non-MLCK mechanism that may function as the trigger of MLC20 phosphorylation. The cell signaling pathways would be most likely to address this intractable issue. In recent years, a multitude of signaling pathways has been suggested to regulate the smooth muscle contractility. However, these pathways can be broken down into two major pathways (Figure 4): the calcium–calmodulin signalling pathway and Rho/Rho-kinase pathway. The former, the Ca2+/CaM pathway, regulates smooth muscle contraction by the binding of increased intracellular calcium ion with CaM, which activates the MLCK. The MLCK phosphorylates the MLC20 at the neck of the myosin heavy chains. The phosphorylation increases the ATPase activity and thereby produces contraction. In the Rho/Rho-kinase pathway, the Rho A-GTP, activated by multiple stimuli, activates the Rho kinase (also known as Rok or Rock). The activated kinase phosphorylates the myosin phosphatase and therefore modulates the MLC20 phosphorylation.

Current Basic and Pathological Approaches to 102 the Function of Muscle Cells and Tissues – From Molecules to Humans

In the following sections, we will describe a MAP kinase-mediated phosphorylative mechanism of MLC20.

MAP Kinase-Mediated and MLCK-Independent Phosphorylation of MLC20 in Smooth Muscle Cells 103

(MEK1/2). The ERK is activated by a large variety of diverse extracellular stimuli, including growth factors, cytokines, virus infection, transforming agents, and carcinogens. Activated ERK1/2 phosphorylate numerous substrates, including various membrane proteins (CD120a, Syk, and calnexin), nuclear substrates (SRC-1, Pax6, Elk-1, MEF2, c-Fos, c-Myc, and STAT3), cytoskeletal proteins, and several MAPK-activated protein kinases (MKs), and MAPK-interacting kinases(MNKs). As a consequence of the activation, ERK pathway regulates many distinct and even opposing cellular processing, including proliferation,

ERK pathway may be involved in regulation of contraction, migration and proliferation of smooth muscle cells. PD-098059, a specific inhibitor of MAP kinase kinase (MEK1/2, the upstream of ERK1/2), reduced vascular smooth muscle contraction and increase in blood pressure induced by Ang II and phenylephrine, perhaps by inhibiting ERK1/2 activation (Escano *et al.*, 2008) and airway smooth muscle contraction by isoprenaline (Lelliott *et al.*, 2012). Direct inhibition of ERK1/2 by U-0126 attenuates Ang II- and isoprenaline-induced contraction of bronchial smooth muscle (Sakai *et al.*, 2010; Lelliott *et al.*, 2012). Similar role of ERK1/2 are also observed in other smooth muscle (cells) (Jeong *et al.*, 2011). However, some reports show that the inhibitors of ERK and its upstream do not affect vascular smooth muscle contraction (Do *et al.*, 2009; Bauer *et al.*, 2011; Sathish *et al.*, 2011). Thus, this role of ERK in regulation of smooth muscle contraction is not universal, or even controversial.

The biological process of cell migration is similar to that of muscle cell contraction. The functional role of ERK in the regulation of smooth muscle cell migration is reported by a large number of articles (Aitken & Bagl, 2001; Kavurma & Khachigian, 2003). Many reports about the regulatory model of ERK1/2 in smooth muscle cell migration are consistent, even though it is not entirely clear how ERK activation promotes smooth muscle cell motility. For example, inhibition of ERK by inhibitor and antisense oligonucleotide blocks smooth muscle migration (Graf *et al.*, 1997; Gerthoffer, 2007). Our study confirmed role of ERK1/2 in smooth muscle cell migration by blocking movement by PD-098059(Deng *et al.*, 2011). In addition, Raf-1 kinase, the upstream molecule of MEK1/2 that activates ERK1/2, may be involved in

The p38 cascade is composed of MEKK3, MKK3/4/6, p38 and several MAPKAPKs (EIk-1, CHOP, ATF2 and MEF2A). The p38 family includes four splice variants: p38α, p38, p38γand p38δ. Like the rest of MAP kinases, p38 are ubiquitously expressed in various types of tissues, including various smooth muscle cells. Two main MAPKKs, MEK3 and MEK6, function as the upstream molecules of p38, which are known to activate p38. In addition, MEK4, an upstream kinase of JNK, can aid in the activation of p38α and p38δ in specific cell types. The p38 MAPK cascade is activated by numerous promigratory stimuli, including platelet-derived growth factor (PDGF), Ang II, S1P, and thrombin. This kinase is also responsive to a wide range of environmental stresses, such as ultraviolet irradiation, heat shock, osmotic shock, as well as response to inflammatory cytokines, but less by serum

differentiation, metabolism, morphology, survival and even apoptosis.

regulation of smooth muscle contraction (Sathishkumar *et al.*, 2010).

**6.2. p38 pathway**

**Figure 5.** The main components of the three MAP kinase pathways. MAP kinase cascade is typically composed of four kinases that establish a sequential activation pathway comprising a MAP kinase kinase kinase kinase (MAP4K), MAP kinase kinase kinase (MAP3K), MAP kinase kinase (MAP2K), and MAP kinase (MAPK). Extracellular/intracellular stimuli activate MAP4K, MAP3K, MAP2K and MAPK sequentially, leading to the multiple biological effects.

MAP kinases (MAPKs) comprise a family of serine threonine kinases, which include three major sets of kinases: extracellular signal-regulated kinases (ERK1 and ERK2), c-Jun aminoterminal kinase/stress-activated protein kinases (JNKs), and p38 MAPKs (Figure 5). These kinases constitute three major discrete cascades and serve as focal points in response to a variety of extracellular stimuli. Members of all three MAPK families, the ERK, JNK, and the p38 MAPKs, are expressed in various types of tissues and cells, including smooth muscles.

#### **6.1. ERK pathway**

The best-characterized members of the MAPK family are ERK1 and ERK2, also known as p44 MAPK and p42 MAPK, respectively. The canonical ERK cascade comprises Ras, Rafs, MEK1/2, ERK1/2 and several MAPK-activating protein kinases (MAPKAPK, such as EIk-1, Sap1a and c-Fos). ERK1 and ERK2 are expressed to various extents in all tissues, including various types of smooth muscle. ERK1/2 is distributed throughout quiescent cells, but upon stimulation, a significant population of ERK1/2 accumulates in the nucleus. Immediate upstream kinases that activate ERK cascade are mitogen-activated protein kinase kinase 1/2 (MEK1/2). The ERK is activated by a large variety of diverse extracellular stimuli, including growth factors, cytokines, virus infection, transforming agents, and carcinogens. Activated ERK1/2 phosphorylate numerous substrates, including various membrane proteins (CD120a, Syk, and calnexin), nuclear substrates (SRC-1, Pax6, Elk-1, MEF2, c-Fos, c-Myc, and STAT3), cytoskeletal proteins, and several MAPK-activated protein kinases (MKs), and MAPK-interacting kinases(MNKs). As a consequence of the activation, ERK pathway regulates many distinct and even opposing cellular processing, including proliferation, differentiation, metabolism, morphology, survival and even apoptosis.

ERK pathway may be involved in regulation of contraction, migration and proliferation of smooth muscle cells. PD-098059, a specific inhibitor of MAP kinase kinase (MEK1/2, the upstream of ERK1/2), reduced vascular smooth muscle contraction and increase in blood pressure induced by Ang II and phenylephrine, perhaps by inhibiting ERK1/2 activation (Escano *et al.*, 2008) and airway smooth muscle contraction by isoprenaline (Lelliott *et al.*, 2012). Direct inhibition of ERK1/2 by U-0126 attenuates Ang II- and isoprenaline-induced contraction of bronchial smooth muscle (Sakai *et al.*, 2010; Lelliott *et al.*, 2012). Similar role of ERK1/2 are also observed in other smooth muscle (cells) (Jeong *et al.*, 2011). However, some reports show that the inhibitors of ERK and its upstream do not affect vascular smooth muscle contraction (Do *et al.*, 2009; Bauer *et al.*, 2011; Sathish *et al.*, 2011). Thus, this role of ERK in regulation of smooth muscle contraction is not universal, or even controversial.

The biological process of cell migration is similar to that of muscle cell contraction. The functional role of ERK in the regulation of smooth muscle cell migration is reported by a large number of articles (Aitken & Bagl, 2001; Kavurma & Khachigian, 2003). Many reports about the regulatory model of ERK1/2 in smooth muscle cell migration are consistent, even though it is not entirely clear how ERK activation promotes smooth muscle cell motility. For example, inhibition of ERK by inhibitor and antisense oligonucleotide blocks smooth muscle migration (Graf *et al.*, 1997; Gerthoffer, 2007). Our study confirmed role of ERK1/2 in smooth muscle cell migration by blocking movement by PD-098059(Deng *et al.*, 2011). In addition, Raf-1 kinase, the upstream molecule of MEK1/2 that activates ERK1/2, may be involved in regulation of smooth muscle contraction (Sathishkumar *et al.*, 2010).

#### **6.2. p38 pathway**

Current Basic and Pathological Approaches to

mechanism of MLC20.

102 the Function of Muscle Cells and Tissues – From Molecules to Humans

sequentially, leading to the multiple biological effects.

**6.1. ERK pathway**

In the following sections, we will describe a MAP kinase-mediated phosphorylative

**Figure 5.** The main components of the three MAP kinase pathways. MAP kinase cascade is typically composed of four kinases that establish a sequential activation pathway comprising a MAP kinase kinase kinase kinase (MAP4K), MAP kinase kinase kinase (MAP3K), MAP kinase kinase (MAP2K), and MAP kinase (MAPK). Extracellular/intracellular stimuli activate MAP4K, MAP3K, MAP2K and MAPK

MAP kinases (MAPKs) comprise a family of serine threonine kinases, which include three major sets of kinases: extracellular signal-regulated kinases (ERK1 and ERK2), c-Jun aminoterminal kinase/stress-activated protein kinases (JNKs), and p38 MAPKs (Figure 5). These kinases constitute three major discrete cascades and serve as focal points in response to a variety of extracellular stimuli. Members of all three MAPK families, the ERK, JNK, and the p38 MAPKs, are expressed in various types of tissues and cells, including smooth muscles.

The best-characterized members of the MAPK family are ERK1 and ERK2, also known as p44 MAPK and p42 MAPK, respectively. The canonical ERK cascade comprises Ras, Rafs, MEK1/2, ERK1/2 and several MAPK-activating protein kinases (MAPKAPK, such as EIk-1, Sap1a and c-Fos). ERK1 and ERK2 are expressed to various extents in all tissues, including various types of smooth muscle. ERK1/2 is distributed throughout quiescent cells, but upon stimulation, a significant population of ERK1/2 accumulates in the nucleus. Immediate upstream kinases that activate ERK cascade are mitogen-activated protein kinase kinase 1/2 The p38 cascade is composed of MEKK3, MKK3/4/6, p38 and several MAPKAPKs (EIk-1, CHOP, ATF2 and MEF2A). The p38 family includes four splice variants: p38α, p38, p38γand p38δ. Like the rest of MAP kinases, p38 are ubiquitously expressed in various types of tissues, including various smooth muscle cells. Two main MAPKKs, MEK3 and MEK6, function as the upstream molecules of p38, which are known to activate p38. In addition, MEK4, an upstream kinase of JNK, can aid in the activation of p38α and p38δ in specific cell types. The p38 MAPK cascade is activated by numerous promigratory stimuli, including platelet-derived growth factor (PDGF), Ang II, S1P, and thrombin. This kinase is also responsive to a wide range of environmental stresses, such as ultraviolet irradiation, heat shock, osmotic shock, as well as response to inflammatory cytokines, but less by serum and growth factors. Substrates of p38 *in vivo* include transcription factors (ATF2, MEF2C), the RNA binding protein tristetraprolin, and several protein kinases, such as MAPKAP kinases 2, 3, and 5. The p38 signaling has been implicated in cellular responses including inflammation, cell cycle, cell death, development, cell differentiation, senescence, and tumorigenesis. Because p38 MAPKs are key regulators of inflammatory cytokine expression, they appear to be involved in many human diseases such as asthma and autoimmunity.

MAP Kinase-Mediated and MLCK-Independent Phosphorylation of MLC20 in Smooth Muscle Cells 105

phenylephrine (Lee *et al.*, 2006; Strittmatter *et al.*, 2012). In addition, overexpression of an inactive mutant of JNK partially inhibits migration induced by PDGF-BB and Ang II (Ohtsu *et al.*, 2005). The mutant may attenuate natural JNK activity by interfering its binding to the up- and down-stream molecules. Besides, SP600125 inhibits smooth muscle migration

By using the MAPK inhibitors and adenoviral overexpression, we found a MAP kinase

Research on the role of the ERK, p38 MAPK, and JNK is facilitated by effective and relatively selective small molecule inhibitors. We first used the p38, JNK, and ERK inhibitors to access the effects of MAP kinase signaling on MLC20 phosphorylation in cultured hBSM

The SB203580, BIRB 796, SB 202190, and VX-702 have inhibitory effects for the p38 kinase but only the SB 203580, a specific inhibitor of p38α and p38β isoforms, is widely used for the p38 function-related studies. The half maximal inhibitory concentration (IC50) values recommended by the manufacturer are 50 and 500 nM for the p38αand p38β, respectively. The concentration ranges used in published reports are 10–50 M (Kramer et al., 1996; Fatima et al., 2001; Kim et al., 2002). The JNK inhibitors include the SP600125, JNK inhibitor I (a peptide), JNK inhibitor IX, and JNK inhibitor VIII. The SP600125, a JNK inhibitor for three isoforms of JNKs, is often used to inhibit JNKs in most studies. The recommended IC50 values are 40 nM for JNK-1 and JNK-2, and 90 nM for JNK-3. The concentration ranges used by most previous researchers are 10–30 µM (Renlund et al., 2008). The recently used concentration is 20 µM (Takahashi et al., 2011). The ERK inhibitors include the PD98059, ERK Inhibitor II (C18H13N7), U0126, and inhibitory peptide. It is a 13-amino acid peptide that corresponds to the N-terminus of MEK1 and functions as a specific inhibitor of ERK activation. The IC50 recommended by the manufacturer is 2.5 µM. Different concentrations such as 10, 20, 40, and 50 µM were used in previous researches (Mathur et al., 2004; Monick et al., 2008). The concentration of 60 µM was used in our studies. Considering the difficulty to transport a peptide across a cell membrane, we permeabilized cells before the ERK

**7. Regulation of MAP kinase pathways on MLC20 phosphorylation**

**7.1. Inhibition of MAP kinase pathways increases phosphorylation level of**

pathway-mediated regulatory mechanism of MLC20 phosphorylation.

inhibitor treatment, even though the inhibitor peptide is cell-permeable.

In this study, the cultured hBSM cells were exposed to 30 M of SB203580 (a p38 inhibitor), SP600125 (a JNK inhibitor), and 60 M of ERK inhibitory peptide for 40 min. Cells for ERK inhibition were previously permeabilized (Deng *et al.*, 2011). Proteins of cell lysates were separated by SDS-PAGE electrophoresis and then transferred to PVDF membranes. Actin, phosphor MLC20 and MLCK on the same membrane were detected. Inhibition of MAP kinase activity increased MLC20 phosphorylation (Figure 6A). To minimize the effects of the

(Kavurma & Khachigian, 2003).

**MLC20**

cells (Figure 6A).

The p38 MAPKs may participate in multiple processes in smooth muscle cells including contraction, oxidative stress signaling, and cytokine synthesis . Contraction induced by some but not all agonists depends on p38 activity. SB203580, the specific inhibitor of p38, blocks spontaneous and agent-induced smooth muscle contraction (Lee *et al.*, 2007; Barona *et al.*, 2011). Besides, by using SB203580 and siRNA, researchers have demonstrated that p38 is involved in regulation of smooth muscle cell migration (Mugabe *et al.*, 2010).

#### **6.3. JNK pathway**

The JNK cascade comprises generally MAP-ERK kinase kinase-1 (MEKK1), MKK4/7, JNKs and MAPK-activated C-Jun, JunB and ATF2. JNK family, encoded by three genes, jnk1, jnk2, jnk3, includes three main spliced forms, JNK1, -2, and -3, also known as SAPK, SAPK, and SAPK, respectively. JNK1 and JNK2, are believed to be expressed in every cell and tissue type, whereas the JNK3 protein is found primarily in brain (Bode & Dong, 2007). JNK activation is much more complex than that of ERK1/ERK2 owing to inputs by a greater number of MAP4Ks and MAP3Ks. This diversity of upstream messages allows a wide range of stimuli to activate this MAPK pathway, including UV and -irradiation, protein synthesis inhibitors (anisomycin), hyperosmolarity, toxins, ischemia/reperfusion injury in heart attacks, heat shock, anticancer drugs (cisplatinum, adriamycin, or etoposide), ceramide, peroxide, and inflammatory cytokines such as TNF. The activated JNKs translocate to the nucleus where they phosphorylate the effector molecules. A well-known substrate for JNKs is the transcription factor c-Jun.  Some non-transcription factors can also be regulated by JNKs, such as Bcl-2, paxillin, and Bcl-xL. The dual role of JNK in both apoptotic and survival signaling pathways indicates that the functional role of JNK is complex. JNKs are involved in various physiological and pathological processes, such as apoptosis, neurodegeneration, neural development, cell differentiation and proliferation, inflammation, cytokine production and regulation of responses to stimuli.

Compared with ERK and p38 MAPKs, less is known about the role of JNK MAPKs in contraction and migration of smooth muscle cells. The chemical inhibitors of JNK family members are less selective than MEK (then ERK1/2) and p38 MAPK inhibitors and therefore somewhat less useful for definitive studies of kinase action in cells. However, recent evidences support an important role for JNK in regulation of airway smooth muscle contraction (Lei *et al.*, 2011). SP600125, a JNK inhibitor, attenuates vascular smooth muscle contraction and human prostate smooth muscle induced by norepinephrine and phenylephrine (Lee *et al.*, 2006; Strittmatter *et al.*, 2012). In addition, overexpression of an inactive mutant of JNK partially inhibits migration induced by PDGF-BB and Ang II (Ohtsu *et al.*, 2005). The mutant may attenuate natural JNK activity by interfering its binding to the up- and down-stream molecules. Besides, SP600125 inhibits smooth muscle migration (Kavurma & Khachigian, 2003).

#### **7. Regulation of MAP kinase pathways on MLC20 phosphorylation**

Current Basic and Pathological Approaches to

**6.3. JNK pathway**

104 the Function of Muscle Cells and Tissues – From Molecules to Humans

production and regulation of responses to stimuli.

and growth factors. Substrates of p38 *in vivo* include transcription factors (ATF2, MEF2C), the RNA binding protein tristetraprolin, and several protein kinases, such as MAPKAP kinases 2, 3, and 5. The p38 signaling has been implicated in cellular responses including inflammation, cell cycle, cell death, development, cell differentiation, senescence, and tumorigenesis. Because p38 MAPKs are key regulators of inflammatory cytokine expression, they appear to be involved in many human diseases such as asthma and autoimmunity.

The p38 MAPKs may participate in multiple processes in smooth muscle cells including contraction, oxidative stress signaling, and cytokine synthesis . Contraction induced by some but not all agonists depends on p38 activity. SB203580, the specific inhibitor of p38, blocks spontaneous and agent-induced smooth muscle contraction (Lee *et al.*, 2007; Barona *et al.*, 2011). Besides, by using SB203580 and siRNA, researchers have demonstrated that p38 is

The JNK cascade comprises generally MAP-ERK kinase kinase-1 (MEKK1), MKK4/7, JNKs and MAPK-activated C-Jun, JunB and ATF2. JNK family, encoded by three genes, jnk1, jnk2, jnk3, includes three main spliced forms, JNK1, -2, and -3, also known as SAPK, SAPK, and SAPK, respectively. JNK1 and JNK2, are believed to be expressed in every cell and tissue type, whereas the JNK3 protein is found primarily in brain (Bode & Dong, 2007). JNK activation is much more complex than that of ERK1/ERK2 owing to inputs by a greater number of MAP4Ks and MAP3Ks. This diversity of upstream messages allows a wide range of stimuli to activate this MAPK pathway, including UV and -irradiation, protein synthesis inhibitors (anisomycin), hyperosmolarity, toxins, ischemia/reperfusion injury in heart attacks, heat shock, anticancer drugs (cisplatinum, adriamycin, or etoposide), ceramide, peroxide, and inflammatory cytokines such as TNF. The activated JNKs translocate to the nucleus where they phosphorylate the effector molecules. A well-known substrate for JNKs is the transcription factor c-Jun.  Some non-transcription factors can also be regulated by JNKs, such as Bcl-2, paxillin, and Bcl-xL. The dual role of JNK in both apoptotic and survival signaling pathways indicates that the functional role of JNK is complex. JNKs are involved in various physiological and pathological processes, such as apoptosis, neurodegeneration, neural development, cell differentiation and proliferation, inflammation, cytokine

Compared with ERK and p38 MAPKs, less is known about the role of JNK MAPKs in contraction and migration of smooth muscle cells. The chemical inhibitors of JNK family members are less selective than MEK (then ERK1/2) and p38 MAPK inhibitors and therefore somewhat less useful for definitive studies of kinase action in cells. However, recent evidences support an important role for JNK in regulation of airway smooth muscle contraction (Lei *et al.*, 2011). SP600125, a JNK inhibitor, attenuates vascular smooth muscle contraction and human prostate smooth muscle induced by norepinephrine and

involved in regulation of smooth muscle cell migration (Mugabe *et al.*, 2010).

By using the MAPK inhibitors and adenoviral overexpression, we found a MAP kinase pathway-mediated regulatory mechanism of MLC20 phosphorylation.

#### **7.1. Inhibition of MAP kinase pathways increases phosphorylation level of MLC20**

Research on the role of the ERK, p38 MAPK, and JNK is facilitated by effective and relatively selective small molecule inhibitors. We first used the p38, JNK, and ERK inhibitors to access the effects of MAP kinase signaling on MLC20 phosphorylation in cultured hBSM cells (Figure 6A).

The SB203580, BIRB 796, SB 202190, and VX-702 have inhibitory effects for the p38 kinase but only the SB 203580, a specific inhibitor of p38α and p38β isoforms, is widely used for the p38 function-related studies. The half maximal inhibitory concentration (IC50) values recommended by the manufacturer are 50 and 500 nM for the p38αand p38β, respectively. The concentration ranges used in published reports are 10–50 M (Kramer et al., 1996; Fatima et al., 2001; Kim et al., 2002). The JNK inhibitors include the SP600125, JNK inhibitor I (a peptide), JNK inhibitor IX, and JNK inhibitor VIII. The SP600125, a JNK inhibitor for three isoforms of JNKs, is often used to inhibit JNKs in most studies. The recommended IC50 values are 40 nM for JNK-1 and JNK-2, and 90 nM for JNK-3. The concentration ranges used by most previous researchers are 10–30 µM (Renlund et al., 2008). The recently used concentration is 20 µM (Takahashi et al., 2011). The ERK inhibitors include the PD98059, ERK Inhibitor II (C18H13N7), U0126, and inhibitory peptide. It is a 13-amino acid peptide that corresponds to the N-terminus of MEK1 and functions as a specific inhibitor of ERK activation. The IC50 recommended by the manufacturer is 2.5 µM. Different concentrations such as 10, 20, 40, and 50 µM were used in previous researches (Mathur et al., 2004; Monick et al., 2008). The concentration of 60 µM was used in our studies. Considering the difficulty to transport a peptide across a cell membrane, we permeabilized cells before the ERK inhibitor treatment, even though the inhibitor peptide is cell-permeable.

In this study, the cultured hBSM cells were exposed to 30 M of SB203580 (a p38 inhibitor), SP600125 (a JNK inhibitor), and 60 M of ERK inhibitory peptide for 40 min. Cells for ERK inhibition were previously permeabilized (Deng *et al.*, 2011). Proteins of cell lysates were separated by SDS-PAGE electrophoresis and then transferred to PVDF membranes. Actin, phosphor MLC20 and MLCK on the same membrane were detected. Inhibition of MAP kinase activity increased MLC20 phosphorylation (Figure 6A). To minimize the effects of the quantitative difference in protein loading on experimental judgment in Western blotting assay, we always incubated membranes with mixed primary antibodies against *pan*-actin and phosphor MLC20, followed by incubation with corresponding and different second antibody after PBS washing. The actin gene is a housekeeping gene, which is expressed invariably in various cells/tissues usually at different treatments. The *pan*-actin bands can show us how much protein is loaded in the wells of SDS-PAGE gels. Then, the membrane was re-probed with specific antibody against the MLCK. Unexpectedly, all of the three MAP kinase inhibitors appear to increase the phosphor MLC20 level (Figure 6A), especially treatments by the SB 203580 and SP600125 in lines 2–3. The MLCK expression was not altered when the phosphor MLC20 level increased due to the MAP kinase inhibitor treatment.

MAP Kinase-Mediated and MLCK-Independent Phosphorylation of MLC20 in Smooth Muscle Cells 107

**7.2. Activation of MAP kinase pathways inhibits MLC20 phosphorylation**

in hBSM cells.

molecules.

were titrated by the agarose overlay plaque assay.

Contrary to the MAP kinase-inhibition experiment (Figure 6A), the activity of the MAP kinases was to be increased in this experiment. The activity of the MAP kinase in quiescent cells is relatively low (Graf et al., 1997; Pizon & Baldacci, 2000; Hatton et al., 2003; Kuramochi et al., 2004; Mackeigan et al., 2005), and we observed low activation of MAP kinases in the hBSM cells used in this study. It is difficult to observe the alteration of the MAP kinase activity during a change of MLC20 phosphorylation. Thus, we increased the intracellular levels of the phosphor MAP kinases by adenovirus-mediated overexpression of active human MAP-ERK kinase kinase-1 (MEKK1), the upstream molecule of MAP kinases

The generation of MEKK1-containing adenovirus is described in brief as follows. To overexpress MEKK1, an HA-tagged 4.9-kb cDNA for active human wild-type MEKK1 was cloned at the downstream of cytomegalovirus (CMV) promoter of pAdtrack, a shuttle vector that transports target gene MEKK1 to the viral plasmid. To prepare the MEKK1-containing viral DNA, the shuttle vector was recombined with the adenoviral DNA in bacteria. Then, the recombinant adenoviral plasmid was used to produce the adenovirus in packaging cells following conventional protocol for adenovirus preparation. Two control adenoviruses were prepared in the same procedure but human MEKK1 was replaced by the -galactosidase (LacZ) or green fluorescent protein (GFP). The control virus helped us to distinguish the MEKK1 effects from that of non-MEKK1 virus infection. After the amplification, the viruses