The viral infection efficiency in the cultured hBSM cells was examined by observing the percentage of the GFP-positive living cells under a fluorescence microscope. The viral expressive efficiency of MEKK1 was assayed in an independent experiment by Western blotting using an antibody against the HA-Tag (Deng et al., 2011). The exogenous MEKK1 activity expressed by the adenovirus was estimated by its capacity to activate the downstream molecules, including the JNK, ERK, and p38. These results show that the infection efficiency of the adenoviruses in the cultured hBSM cells reached up to 99% (the cell density was 70% when infected). The MEKK1 expressed by the adenovirus activated strikingly JNK1 and JNK2. In the meantime, it also activated the ERK1 and p38 kinase to a lower extent. Then, the MEKK1 adenovirus was used for the evaluation of the roles of the MAP kinase on MLC20 phosphorylation. When the cell density was between 60–70%, the hBSM cells in 6-well plates were infected with the MEKK1 and the control viruses at different doses (Deng et al., 2011). Three days after infection, the cells were harvested for Western blotting. The experimental results show that the MEKK1 adenovirus infection inhibited the MLC20 phosphorylation significantly (line 3 in Figure 6B). The inhibition of the MAP kinase pathway for the MLC20 phosphorylation is already hinted in Figure 6A but the extent of inhibition is beyond our expectation. Apparently, the inhibitory efficiency depends on viral infection efficiency, expressive capacity of MEKK1 adenovirus, and activating ability of MEKK1 for the downstream

**Figure 6.** Negative regulation of MAP kinase pathway on MLC20 phosphorylation. (A) All of p38, JNK and ERK inhibitors raised phosphorylation level of MLC20. (B)Activation of MAP kinases by adenovirus-mediated overexpression of MEKK1 inhibited significantly MLC20 phosphorylation (line 3). MAPK inhibitors counteracted inhibition of MEKK1 on MLC20 phosphorylation (line4~6). (C, D) The level of MLC20 phosphorylation increased with JNK and p38 inhibitors in hBSM cells. (E, F) MLC phosphorylation decreased with MEKK1-viral doses. *P*-MLC20, phosphor MLC20; SB, SB203580; SP, SP600125; adv, adenovirus.

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

Current Basic and Pathological Approaches to

adenovirus.

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

MLC20 level increased due to the MAP kinase inhibitor treatment.

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

**Figure 6.** Negative regulation of MAP kinase pathway on MLC20 phosphorylation. (A) All of p38, JNK and ERK inhibitors raised phosphorylation level of MLC20. (B)Activation of MAP kinases by adenovirus-mediated overexpression of MEKK1 inhibited significantly MLC20 phosphorylation (line 3). MAPK inhibitors counteracted inhibition of MEKK1 on MLC20 phosphorylation (line4~6). (C, D) The level of MLC20 phosphorylation increased with JNK and p38 inhibitors in hBSM cells. (E, F) MLC phosphorylation decreased with MEKK1-viral doses. *P*-MLC20, phosphor MLC20; SB, SB203580; SP, SP600125; adv,

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 in hBSM cells.

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 were titrated by the agarose overlay plaque assay.

The viral infection efficiency in the cultured hBSM cells was examined by observing the percentage of the GFP-positive living cells under a fluorescence microscope. The viral expressive efficiency of MEKK1 was assayed in an independent experiment by Western blotting using an antibody against the HA-Tag (Deng et al., 2011). The exogenous MEKK1 activity expressed by the adenovirus was estimated by its capacity to activate the downstream molecules, including the JNK, ERK, and p38. These results show that the infection efficiency of the adenoviruses in the cultured hBSM cells reached up to 99% (the cell density was 70% when infected). The MEKK1 expressed by the adenovirus activated strikingly JNK1 and JNK2. In the meantime, it also activated the ERK1 and p38 kinase to a lower extent. Then, the MEKK1 adenovirus was used for the evaluation of the roles of the MAP kinase on MLC20 phosphorylation. When the cell density was between 60–70%, the hBSM cells in 6-well plates were infected with the MEKK1 and the control viruses at different doses (Deng et al., 2011). Three days after infection, the cells were harvested for Western blotting. The experimental results show that the MEKK1 adenovirus infection inhibited the MLC20 phosphorylation significantly (line 3 in Figure 6B). The inhibition of the MAP kinase pathway for the MLC20 phosphorylation is already hinted in Figure 6A but the extent of inhibition is beyond our expectation. Apparently, the inhibitory efficiency depends on viral infection efficiency, expressive capacity of MEKK1 adenovirus, and activating ability of MEKK1 for the downstream molecules.

In addition, we set up other treatments, using MAP kinase inhibitors to counteract the activation of the MEKK1 for the MAP kinases so that we could evaluate more accurately the role of the MEKK1-activated MAP kinase pathway in the regulation of the MLC20 phosphorylation. If it is true that the MEKK1 regulates negatively the MLC20 phosphorylation, the MAP kinase inhibitors would augment the MLC20 phosphorylation through suppression of the activity of the MEKK1 downstream molecules. As expected, the MAP kinase inhibitors neutralized the inhibition of the MEKK1 adenovirus on the MLC20 phosphorylation (lines 4–6 in Figure 6B). Likewise, the actin bands are detected to show a similar amount of loaded proteins. Obviously, the experimental results and even conclusions depend on the balance between the viral doses and inhibitor concentrations. A relatively low-concentration inhibitor may not affect the role of a relatively high-dose infection of MEKK1 adenovirus in MLC20 phosphorylation, and vice versa.

MAP Kinase-Mediated and MLCK-Independent Phosphorylation of MLC20 in Smooth Muscle Cells 109

In addition, when laboratory animals are killed, the muscle is usually relaxed, or rapidly relaxed in seconds (except for some smooth muscles). Because muscle contraction and relaxation are rapid, the MLC20 phosphorylation and dephosphorylation are also very fast, especially in the skeletal muscle. Even for smooth muscles, the muscle contraction and relaxation, such as the bladder emptying by detrusor and relaxation of tracheal/bronchial smooth muscle elicited by agents, may be fast. In one word, the MLC20 is phosphorylated in contracting muscle but unphosphorylated in relaxed muscle, and the transformation between the phosphorylation and nonphosphorylation is rapid. The muscle samples used in a lot of published articles for the MLC20 phosphorylation are almost in non–contracting status. However, those reports show that the phosphorylated MLC20 in various types of muscles are actually in relaxed state. Besides, a lot of data from different laboratories demonstrate that the MLC20 is phosphorylated strongly in cultured cells. As we all know, cells in culture dishes stop moving and are considered quiescent cells when they reach a high density like 90%. This suggests that the MLC20 is heavily phosphorylated in nonmigrating cells. Up to now, there is no direct evidence to support that the MLC20

*Department of Animal Biology, School of Animal Husbandry and Veterinary Medicine, Jiangsu Polytechnic College of Agriculture and Forestry, Jurong, Jiangsu,China*

*West China Center of Medical Sciences, Sichuan University, Chengdu, Sichuan, China*

Aitken K & Bagl DJ. (2001). Stretch-induced bladder smooth muscle cell (SMC) proliferation is mediated by RHAMM-dependent extracellular-regulated kinase (erk) signaling.

Barona I, Fagundes DS, Gonzalo S, Grasa L, Arruebo MP, Plaza MA & Murillo MD. (2011). Role of TLR4 and MAPK in the local effect of LPS on intestinal contractility. *The Journal*

Bauer RM, Strittmatter F, Gratzke C, Gottinger J, Schlenker B, Reich O, Stief CG, Hedlund P, Andersson KE & Hennenberg M. (2011). Coupling of alpha1-adrenoceptors to ERK1/2

Bode AM & Dong Z. (2007). The functional contrariety of JNK. *Molecular carcinogenesis* 46,

*School of Life Sciences, Nanjing University, Nanjing Jiangsu, China*

in the human prostate. *Urologia internationalis* 86, 427-433.

*of pharmacy and pharmacology* 63, 657-662.

phosphorylation triggers muscle contraction.

**Author details**

Maoxian Deng

Lixia Deng

Yarong Xue

**9. References**

591-598.

*Urology* 57, 109.

The dose-response relationship is a mathematic relationship between the dose and the organism's reaction to the dose. It describes the change in reaction or effect on an organism caused by differing levels of doses. Herein, we used dose-gradient methods to provide additional evidence that the MLC20 phosphorylation is regulated by the MAP kinase pathways. In the studies, the hBSM cells were exposed to different concentrations of the MAP kinase inhibitors for 40 minutes, followed by Western blotting assays for the phosphor MLC20. The results show that phosphor MLC20 level increases with the doses of inhibitors (Figure 6C and D, ERK inhibitor not shown) (Deng et al., 2011). A similar method was used to examine the effects of the MEKK1 at different infection doses (i.e., pfu/cell) on the MLC20 phosphorylation. The phosphor MLC20 decreased with doses of the MEKK1 adenovirus (Figure 6 E and F).

At this point, our experiments have demonstrated the negative regulation of MAP kinase pathway on the MLC20 phosphorylation.

#### **8. Suggestions for the future investigation**

The role of the MLC20 phosphorylation in contraction needs to be reassessed, even though it is accepted widely as a trigger of muscle contraction. A number of experiments have already verified the role of the MAP kinase pathway in the migration of different cell types and smooth muscle contraction. Therefore, according to current views, it is reasonable to assume that the MAP kinases would phosphorylate the MLC20, either via one or more enzymes. However, a series of experiments above showed the negative regulation of the MAP kinase pathways on the MLC20 phosphorylation. Because of conflict between our conclusion and current views about the mechanism of the MLC20 phosphorylation, we subsequently carried out a preliminary experiment to explore the synchrony between the cell migration and MLC20 phosphorylation. The migrating cells were fixed immediately and the phosphor MLC20 inside cells was immunostained with a specific antibody. However, we did not see a difference in the fluorescence intensity of the phosphor MLC20 between the migrating and quiescent cells (Deng et al., 2011).

In addition, when laboratory animals are killed, the muscle is usually relaxed, or rapidly relaxed in seconds (except for some smooth muscles). Because muscle contraction and relaxation are rapid, the MLC20 phosphorylation and dephosphorylation are also very fast, especially in the skeletal muscle. Even for smooth muscles, the muscle contraction and relaxation, such as the bladder emptying by detrusor and relaxation of tracheal/bronchial smooth muscle elicited by agents, may be fast. In one word, the MLC20 is phosphorylated in contracting muscle but unphosphorylated in relaxed muscle, and the transformation between the phosphorylation and nonphosphorylation is rapid. The muscle samples used in a lot of published articles for the MLC20 phosphorylation are almost in non–contracting status. However, those reports show that the phosphorylated MLC20 in various types of muscles are actually in relaxed state. Besides, a lot of data from different laboratories demonstrate that the MLC20 is phosphorylated strongly in cultured cells. As we all know, cells in culture dishes stop moving and are considered quiescent cells when they reach a high density like 90%. This suggests that the MLC20 is heavily phosphorylated in nonmigrating cells. Up to now, there is no direct evidence to support that the MLC20 phosphorylation triggers muscle contraction.

#### **Author details**

Current Basic and Pathological Approaches to

vice versa.

(Figure 6 E and F).

pathway on the MLC20 phosphorylation.

quiescent cells (Deng et al., 2011).

**8. Suggestions for the future investigation**

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

In addition, we set up other treatments, using MAP kinase inhibitors to counteract the activation of the MEKK1 for the MAP kinases so that we could evaluate more accurately the role of the MEKK1-activated MAP kinase pathway in the regulation of the MLC20 phosphorylation. If it is true that the MEKK1 regulates negatively the MLC20 phosphorylation, the MAP kinase inhibitors would augment the MLC20 phosphorylation through suppression of the activity of the MEKK1 downstream molecules. As expected, the MAP kinase inhibitors neutralized the inhibition of the MEKK1 adenovirus on the MLC20 phosphorylation (lines 4–6 in Figure 6B). Likewise, the actin bands are detected to show a similar amount of loaded proteins. Obviously, the experimental results and even conclusions depend on the balance between the viral doses and inhibitor concentrations. A relatively low-concentration inhibitor may not affect the role of a relatively high-dose infection of MEKK1 adenovirus in MLC20 phosphorylation, and

The dose-response relationship is a mathematic relationship between the dose and the organism's reaction to the dose. It describes the change in reaction or effect on an organism caused by differing levels of doses. Herein, we used dose-gradient methods to provide additional evidence that the MLC20 phosphorylation is regulated by the MAP kinase pathways. In the studies, the hBSM cells were exposed to different concentrations of the MAP kinase inhibitors for 40 minutes, followed by Western blotting assays for the phosphor MLC20. The results show that phosphor MLC20 level increases with the doses of inhibitors (Figure 6C and D, ERK inhibitor not shown) (Deng et al., 2011). A similar method was used to examine the effects of the MEKK1 at different infection doses (i.e., pfu/cell) on the MLC20 phosphorylation. The phosphor MLC20 decreased with doses of the MEKK1 adenovirus

At this point, our experiments have demonstrated the negative regulation of MAP kinase

The role of the MLC20 phosphorylation in contraction needs to be reassessed, even though it is accepted widely as a trigger of muscle contraction. A number of experiments have already verified the role of the MAP kinase pathway in the migration of different cell types and smooth muscle contraction. Therefore, according to current views, it is reasonable to assume that the MAP kinases would phosphorylate the MLC20, either via one or more enzymes. However, a series of experiments above showed the negative regulation of the MAP kinase pathways on the MLC20 phosphorylation. Because of conflict between our conclusion and current views about the mechanism of the MLC20 phosphorylation, we subsequently carried out a preliminary experiment to explore the synchrony between the cell migration and MLC20 phosphorylation. The migrating cells were fixed immediately and the phosphor MLC20 inside cells was immunostained with a specific antibody. However, we did not see a difference in the fluorescence intensity of the phosphor MLC20 between the migrating and Maoxian Deng *Department of Animal Biology, School of Animal Husbandry and Veterinary Medicine, Jiangsu Polytechnic College of Agriculture and Forestry, Jurong, Jiangsu,China*

Lixia Deng *West China Center of Medical Sciences, Sichuan University, Chengdu, Sichuan, China*

Yarong Xue

*School of Life Sciences, Nanjing University, Nanjing Jiangsu, China*

#### **9. References**

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**Chapter 6**

© 2012 Alfonzo 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,

**Two Guanylylcyclases Regulate the Muscarinic**

Adolfo Borges, Ramona González de Alfonzo and Itala Lippo de Becemberg

Muscarinic activation of Airway smooth muscle (ASM) is of major importance to the physiological and patho-physiological actions of acetylcholine, which induces bronchoconstriction, airway smooth muscle thickening, and the modulation of cytokine and chemokine production by these cells as described in [1]. The parasympathetic nervous system is the dominant neuronal pathway in the control of airway smooth muscle tone. Stimulation of cholinergic nerves causes bronchoconstriction, and mucus secretion as in [2]. The human airways are innervated via efferent and afferent autonomic nerves, which regulate many aspects of airway function. It has been suggested that neural control of the airways may be abnormal in asthmatic patients, and that neurogenic mechanisms may contribute to the pathogenesis and pathophysiology of asthma. Although abnormalities of the cholinergic innervation have been suggested in asthma, thus far the evidence for cholinergic dysfunction

Acetylcholine (Ach) is the predominant parasympathetic neurotransmitter, acting as an autocrine or paracrine hormone in the airways and its role in the regulation of bronchomotor tone and mucus secretion from airway submucosal glands in the respiratory tract is well established as described in [2]. More recent findings suggest that acetylcholine regulates additional functions in the respiratory tract, may promoting airway inflammation and remodelling, including airway smooth muscle thickening in inflammatory lung diseases as reported in references [3-5]. Another source of ACh is the non-neuronal cells and tissues, particularly inflammatory cells and the airway epithelium as described in references [6-8].

Collectively, these findings indicate that acetylcholine, derived from the vagal nerve and from non-neuronal origins such as the airway epithelium, may induce cell responses associated with airway wall remodelling and trigger proinflammatory Cytokines as IL-6 and

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

**Activation of Airway Smooth Muscle**

Marcelo J. Alfonzo, Fabiola Placeres-Uray, Walid Hassan-Soto,

in asthmatic subjects is not convincing as mentioned in references [1,2].

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/48684

**1. Introduction**

### **Two Guanylylcyclases Regulate the Muscarinic Activation of Airway Smooth Muscle**

Marcelo J. Alfonzo, Fabiola Placeres-Uray, Walid Hassan-Soto, Adolfo Borges, Ramona González de Alfonzo and Itala Lippo de Becemberg

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/48684

#### **1. Introduction**

Current Basic and Pathological Approaches to

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Sakai H, Nishizawa Y, Nishimura A, Chiba Y, Goto K, Hanazaki M & Misawa M. (2010). Angiotensin II induces hyperresponsiveness of bronchial smooth muscle via an

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Takahashi S, Ebihara A, Kajiho H, Kontani K, Nishina H & Katada T. (2011). RASSF7 negatively regulates pro-apoptotic JNK signaling by inhibiting the activity of

type II receptor-mediated signal transduction. *Endocrinology* 149, 108-115.

Muscarinic activation of Airway smooth muscle (ASM) is of major importance to the physiological and patho-physiological actions of acetylcholine, which induces bronchoconstriction, airway smooth muscle thickening, and the modulation of cytokine and chemokine production by these cells as described in [1]. The parasympathetic nervous system is the dominant neuronal pathway in the control of airway smooth muscle tone. Stimulation of cholinergic nerves causes bronchoconstriction, and mucus secretion as in [2]. The human airways are innervated via efferent and afferent autonomic nerves, which regulate many aspects of airway function. It has been suggested that neural control of the airways may be abnormal in asthmatic patients, and that neurogenic mechanisms may contribute to the pathogenesis and pathophysiology of asthma. Although abnormalities of the cholinergic innervation have been suggested in asthma, thus far the evidence for cholinergic dysfunction in asthmatic subjects is not convincing as mentioned in references [1,2].

Acetylcholine (Ach) is the predominant parasympathetic neurotransmitter, acting as an autocrine or paracrine hormone in the airways and its role in the regulation of bronchomotor tone and mucus secretion from airway submucosal glands in the respiratory tract is well established as described in [2]. More recent findings suggest that acetylcholine regulates additional functions in the respiratory tract, may promoting airway inflammation and remodelling, including airway smooth muscle thickening in inflammatory lung diseases as reported in references [3-5]. Another source of ACh is the non-neuronal cells and tissues, particularly inflammatory cells and the airway epithelium as described in references [6-8].

Collectively, these findings indicate that acetylcholine, derived from the vagal nerve and from non-neuronal origins such as the airway epithelium, may induce cell responses associated with airway wall remodelling and trigger proinflammatory Cytokines as IL-6 and

© 2012 Alfonzo 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.

IL-8 release in [9] by structural cells of the airways, including the airway smooth muscle itself. In addition, muscarinic receptors regulate proliferative and proinflammatory functions of the airway smooth muscle as mentioned in references [9,10].

Two Guanylylcyclases Regulate the Muscarinic Activation of Airway Smooth Muscle 115

More recently, we showed that muscarinic agonists, via M2AChR induced a massive and selective α1β1-NOsGC migration from cytoplasm to plasma membranes in a dosedependent manner. Such migration was blocked by PTX, suggesting the involvement of

Some of the signal cascades activated by mAChRs at TSM are linked to the generation of second messengers such as cyclic nucleotides: cAMP and cGMP as described in reference [32]. In this review, we address the cGMP generation as a product of the muscarinic activation of ASM. It is well known that in mammalian cells, cGMP is produced by the action of two distinctive guanylyl cyclases, named the NO-sensitive soluble guanylylcyclases (NO-sGC) as reported in [33] and the single membrane-spanning guanylyl

The muscarinic activation of tracheal smooth muscle (TSM) fragments associated with smooth muscle contraction, involves the generation of two cGMP signals, at 20-s and 60-s as

Effect of ODQ on the time course of cGMP signals induced by agonist muscarinic carbachol (1x10-5 M) in TSM. Tracheal smooth muscle strips were pre-incubated for 30 min with ODQ (1H-[1, 2,4 ] Oxadiazolo[4,3-a]quinoxalin-1 one) (■) (100 nM) selective inhibitor of NO-sGC, following the Procedure 2 as described in reference 36. Control experiments (□) without drug were run simultaneously under the same conditions. Each value is the mean ± SE of 4 different tracheas and the cGMP determinations were carried out by triplicate as described in reference [36].

**Figure 1.** Time course of muscarinic agonist action on cGMP levels from TSM in the presence of ODQ.

**2. Body: Cyclic GMP signals during muscarinic activation**

described in reference [36] and a kinetic behavior is shown in Figure 1.

Go/Gi proteins in [31].

Taken from reference [36].

cyclases as published in references [33-35].

Acetylcholine acting on muscarinic receptors (mAChRs) anchored at the airway smooth muscle sarcolemma are involved in the generation of a number of signal transducing cascades allowing the activation of the smooth muscle machinery as described in [11]. Airway smooth muscle muscarinic activation is mediated through mAChRs, which are members of the so called G protein-coupled receptors (GPCR) family, which are cell surface receptors, that activate intracellular responses by coupling G proteins as described in [12] to specific effectors in [13]. Molecular cloning studies have revealed the existence of five mammalian subtypes of muscarinic receptors (m1-m5) as described in [14]. In trachea, smooth muscle expresses mRNAs coding for both m2 and m3 receptors as reported in [15]. Airways smooth muscarinic receptors have been identified as a mixed population of M2 and M3 subtypes roughly in a 4:1 ratio using pharmacological ligand binding studies in [16,17] being the M2 subtype, the most abundant muscarinic receptor in tracheal plasma membranes as reported in references [16,18,19]. In addition, a muscarinic antagonist heterogeneity associated with the M3AchR subtype present in plasma membrane fractions from tracheal smooth muscle has been described in [20].

It has been claimed that M3AChR represents a primary target of acetylcholine in the airways, involved in the regulation of bronchoconstriction as stated in references [17,18,21,22]. Classically, M3AChRs in ASM are coupled to phospholipase C (PLC)/protein kinase C (PKC) pathway via pertussis toxin (PTX)-insensitive G proteins of the Gq/11 family. The contractile response evoked by M3AChRs stimulation is attributed to the formation of inositol trisphosphate (IP3), the subsequent release of Ca2+ from intracellular stores, the additional influx of extracellular calcium, and the Ca2+-sensitizing effect of PKC as mentioned in [21,23,24].

On the other hand, the stimulation of M2 muscarinic receptors (M2AChRs) in ASM inhibits adenylyl cyclase via activation of PTX-sensitive G proteins of the Gi/o family in [25,26] and therefore M2AChRs are thought to counteract relaxation as reported in [27]. Experimental evidence has been provided that M2AChRs participate directly in ASM contraction but the molecular mechanisms by which the M2AChRs in ASM induce contraction is, still unknown.

Recently, it has been shown that M2AChRs stimulate Gi/o proteins to released βγ dimer, which inhibit the Large Conductance Ca2+-activated K+ Channel Activity (BK channels) as described in [28]. The inhibition of BK channel activity favors contraction of ASM and these BK channels are opposed to the M2AChR-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges as mentioned in [29].

In addition, the influence of M2AChRs to modulate the relaxant effects of atrial natriuretic peptide (ANP) has been reported. Thus, the stimulation of M2AChRs suppresses ANPinduced activation of particulate guanylyl cyclase via a PTX-sensitive G protein as reported in [30].

More recently, we showed that muscarinic agonists, via M2AChR induced a massive and selective α1β1-NOsGC migration from cytoplasm to plasma membranes in a dosedependent manner. Such migration was blocked by PTX, suggesting the involvement of Go/Gi proteins in [31].

Some of the signal cascades activated by mAChRs at TSM are linked to the generation of second messengers such as cyclic nucleotides: cAMP and cGMP as described in reference [32]. In this review, we address the cGMP generation as a product of the muscarinic activation of ASM. It is well known that in mammalian cells, cGMP is produced by the action of two distinctive guanylyl cyclases, named the NO-sensitive soluble guanylylcyclases (NO-sGC) as reported in [33] and the single membrane-spanning guanylyl cyclases as published in references [33-35].

#### **2. Body: Cyclic GMP signals during muscarinic activation**

Current Basic and Pathological Approaches to

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

from tracheal smooth muscle has been described in [20].

mentioned in [29].

in [30].

IL-8 release in [9] by structural cells of the airways, including the airway smooth muscle itself. In addition, muscarinic receptors regulate proliferative and proinflammatory

Acetylcholine acting on muscarinic receptors (mAChRs) anchored at the airway smooth muscle sarcolemma are involved in the generation of a number of signal transducing cascades allowing the activation of the smooth muscle machinery as described in [11]. Airway smooth muscle muscarinic activation is mediated through mAChRs, which are members of the so called G protein-coupled receptors (GPCR) family, which are cell surface receptors, that activate intracellular responses by coupling G proteins as described in [12] to specific effectors in [13]. Molecular cloning studies have revealed the existence of five mammalian subtypes of muscarinic receptors (m1-m5) as described in [14]. In trachea, smooth muscle expresses mRNAs coding for both m2 and m3 receptors as reported in [15]. Airways smooth muscarinic receptors have been identified as a mixed population of M2 and M3 subtypes roughly in a 4:1 ratio using pharmacological ligand binding studies in [16,17] being the M2 subtype, the most abundant muscarinic receptor in tracheal plasma membranes as reported in references [16,18,19]. In addition, a muscarinic antagonist heterogeneity associated with the M3AchR subtype present in plasma membrane fractions

It has been claimed that M3AChR represents a primary target of acetylcholine in the airways, involved in the regulation of bronchoconstriction as stated in references [17,18,21,22]. Classically, M3AChRs in ASM are coupled to phospholipase C (PLC)/protein kinase C (PKC) pathway via pertussis toxin (PTX)-insensitive G proteins of the Gq/11 family. The contractile response evoked by M3AChRs stimulation is attributed to the formation of inositol trisphosphate (IP3), the subsequent release of Ca2+ from intracellular stores, the additional influx of extracellular calcium, and the Ca2+-sensitizing effect of PKC as mentioned in [21,23,24].

On the other hand, the stimulation of M2 muscarinic receptors (M2AChRs) in ASM inhibits adenylyl cyclase via activation of PTX-sensitive G proteins of the Gi/o family in [25,26] and therefore M2AChRs are thought to counteract relaxation as reported in [27]. Experimental evidence has been provided that M2AChRs participate directly in ASM contraction but the molecular mechanisms by which the M2AChRs in ASM induce contraction is, still unknown. Recently, it has been shown that M2AChRs stimulate Gi/o proteins to released βγ dimer, which inhibit the Large Conductance Ca2+-activated K+ Channel Activity (BK channels) as described in [28]. The inhibition of BK channel activity favors contraction of ASM and these BK channels are opposed to the M2AChR-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges as

In addition, the influence of M2AChRs to modulate the relaxant effects of atrial natriuretic peptide (ANP) has been reported. Thus, the stimulation of M2AChRs suppresses ANPinduced activation of particulate guanylyl cyclase via a PTX-sensitive G protein as reported

functions of the airway smooth muscle as mentioned in references [9,10].

The muscarinic activation of tracheal smooth muscle (TSM) fragments associated with smooth muscle contraction, involves the generation of two cGMP signals, at 20-s and 60-s as described in reference [36] and a kinetic behavior is shown in Figure 1.

Effect of ODQ on the time course of cGMP signals induced by agonist muscarinic carbachol (1x10-5 M) in TSM. Tracheal smooth muscle strips were pre-incubated for 30 min with ODQ (1H-[1, 2,4 ] Oxadiazolo[4,3-a]quinoxalin-1 one) (■) (100 nM) selective inhibitor of NO-sGC, following the Procedure 2 as described in reference 36. Control experiments (□) without drug were run simultaneously under the same conditions. Each value is the mean ± SE of 4 different tracheas and the cGMP determinations were carried out by triplicate as described in reference [36].

**Figure 1.** Time course of muscarinic agonist action on cGMP levels from TSM in the presence of ODQ. Taken from reference [36].

Interestingly, the 20-s signal is linked to the onset and the 60s signal is related to the plateau of the smooth muscle contraction as described in [36]. The 20-s signal is associated with the activity of a Soluble guanylyl cyclases, which are nitric oxide stimulated guanylyl cyclase (NO-sGC), which are described in [33] and the second, the 60-s signal, is linked to membrane-bound natriuretic peptide receptor guanylyl cyclase (NPR-GC) in references [33- 35], which has been previously characterized at TSM in [37,38].

Two Guanylylcyclases Regulate the Muscarinic Activation of Airway Smooth Muscle 117

Isolated BTSM strips were incubated in KRB at 37C in the presence and absence of muscarinic agonist Cch (1 x 10−5 M) for 70 sec in [36]. After each 10 s, the BTSM strips were removed and immediately frozen in liquid N2 and processed to prepare crude membranes fraction as described in [54]. In these membrane sediments, GC assays were performed in duplicate as described previously in [53]. Empty symbols represent GC assays performed by duplicate in the presence of 3 mM MnCl2, 0.2M GTP, 5 mM creatine phosphate, and 10 IU phospho-creatine kinase in 0.01% defatted BSA. Full symbols represent GC activity in the presence of 100 µM SNP. Basal (□), muscarinic agonist CC 1 x 10−5 M (○), basal

**Figure 2.** Time-course of guanylyl cyclase activity in crude plasma membrane fractions isolated from

**Figure 3.** Western blotting of α1β1 NO-sGC-heterodimer from plasma membranes of BTSM under

plus SNP (▲), and CC+SNP (■). Each value is the mean of three different BTSM strips.

BTSM strips under muscarinic agonist action. Taken from reference [31].

muscarinic activation as described in reference [31].

The details of the activation of an NO-sGC, generating the early 20-s cGMP signal, presents in the plasma membrane of BTSM, has been previously established in reference [31].

Soluble guanylyl cyclases are nitric oxide stimulated guanylyl cyclase (NO-sGC) due to the fact that the primary and best-studied endogenous activator is nitric oxide (NO). NO-sGC is a heterodimeric hemoprotein formed by two different subunits, α- and β-subunits, which exist in four types (α1, α2, β1, and β2), each the product of a separate gene as described in [39,40]. Structurally, each subunit consists of N-terminal H-NOX domain, a central domain related to the dimerization, and a C-terminal consensus nucleotide catalytic cyclase domain as described in [41,42]. For the formation of a catalytically active enzyme, both α- and βsubunits are required as reported in [43]. Although the α1β1 isoform is ubiquitous, the α2β1 isoform is less broadly distributed described in [39-43].

The best-characterized heterodimers are the α1/β1 and the α2/β1 isoforms as mentioned in [44] being the first ones, relevant in our studies as in [31]. At molecular level, His-105 at the amino terminus of the β1 subunit of NO-sGC is the axial ligand of the pentacoordinated reduced iron center of heme, which is required for NO activation of the enzyme. Thus, NO activates sGC by binding to the sixth position of the heme ring, which breaks the bond between the axial histidine and iron to form a 5-coordinated ring with NO in the fifth position as reported in [45].

By using several experimental approaches as biochemical, pharmacological and molecular biology methods, we established that the first 20-s signal is a product of NO-sGC being sensitive to ODQ, as shown in Figure 2, which is translocated from cytoplasm to the inner face of the airway smooth muscle sarcolemma under muscarinic activation.

In addition, there is a coupling mechanism between M2AChRs and NO-sGC involving a Go/Gi heterotrimeric proteins as previously demonstrated in [31]. Thus, in intact smooth muscle fragments and isolated plasma membranes from bovine tracheal smooth muscle, we showed that the heterodimer of NO-sGC involved in such translocation is the α1β1sGC. The experimental evidence using Western blotting with specific antibodies against all subunits of NO-sGC demonstrate that under muscarinic activation, the α1β1NO-sGC-heterodimer isoform is translocated from cytoplasm to plasma membranes of BTSM. These experimental data are shown in Figure 3.

Since the capability of this α1β1-sGC to migrate to plasma membranes under muscarinic activation, a purification procedure and further identification of this α1β1-sGC heterodimer was also performed. Such NO-sGC translocation involves a M2AChR subtype as previously described in [31].

Current Basic and Pathological Approaches to

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

35], which has been previously characterized at TSM in [37,38].

isoform is less broadly distributed described in [39-43].

position as reported in [45].

data are shown in Figure 3.

described in [31].

Interestingly, the 20-s signal is linked to the onset and the 60s signal is related to the plateau of the smooth muscle contraction as described in [36]. The 20-s signal is associated with the activity of a Soluble guanylyl cyclases, which are nitric oxide stimulated guanylyl cyclase (NO-sGC), which are described in [33] and the second, the 60-s signal, is linked to membrane-bound natriuretic peptide receptor guanylyl cyclase (NPR-GC) in references [33-

The details of the activation of an NO-sGC, generating the early 20-s cGMP signal, presents

Soluble guanylyl cyclases are nitric oxide stimulated guanylyl cyclase (NO-sGC) due to the fact that the primary and best-studied endogenous activator is nitric oxide (NO). NO-sGC is a heterodimeric hemoprotein formed by two different subunits, α- and β-subunits, which exist in four types (α1, α2, β1, and β2), each the product of a separate gene as described in [39,40]. Structurally, each subunit consists of N-terminal H-NOX domain, a central domain related to the dimerization, and a C-terminal consensus nucleotide catalytic cyclase domain as described in [41,42]. For the formation of a catalytically active enzyme, both α- and βsubunits are required as reported in [43]. Although the α1β1 isoform is ubiquitous, the α2β1

The best-characterized heterodimers are the α1/β1 and the α2/β1 isoforms as mentioned in [44] being the first ones, relevant in our studies as in [31]. At molecular level, His-105 at the amino terminus of the β1 subunit of NO-sGC is the axial ligand of the pentacoordinated reduced iron center of heme, which is required for NO activation of the enzyme. Thus, NO activates sGC by binding to the sixth position of the heme ring, which breaks the bond between the axial histidine and iron to form a 5-coordinated ring with NO in the fifth

By using several experimental approaches as biochemical, pharmacological and molecular biology methods, we established that the first 20-s signal is a product of NO-sGC being sensitive to ODQ, as shown in Figure 2, which is translocated from cytoplasm to the inner

In addition, there is a coupling mechanism between M2AChRs and NO-sGC involving a Go/Gi heterotrimeric proteins as previously demonstrated in [31]. Thus, in intact smooth muscle fragments and isolated plasma membranes from bovine tracheal smooth muscle, we showed that the heterodimer of NO-sGC involved in such translocation is the α1β1sGC. The experimental evidence using Western blotting with specific antibodies against all subunits of NO-sGC demonstrate that under muscarinic activation, the α1β1NO-sGC-heterodimer isoform is translocated from cytoplasm to plasma membranes of BTSM. These experimental

Since the capability of this α1β1-sGC to migrate to plasma membranes under muscarinic activation, a purification procedure and further identification of this α1β1-sGC heterodimer was also performed. Such NO-sGC translocation involves a M2AChR subtype as previously

face of the airway smooth muscle sarcolemma under muscarinic activation.

in the plasma membrane of BTSM, has been previously established in reference [31].

Isolated BTSM strips were incubated in KRB at 37C in the presence and absence of muscarinic agonist Cch (1 x 10−5 M) for 70 sec in [36]. After each 10 s, the BTSM strips were removed and immediately frozen in liquid N2 and processed to prepare crude membranes fraction as described in [54]. In these membrane sediments, GC assays were performed in duplicate as described previously in [53]. Empty symbols represent GC assays performed by duplicate in the presence of 3 mM MnCl2, 0.2M GTP, 5 mM creatine phosphate, and 10 IU phospho-creatine kinase in 0.01% defatted BSA. Full symbols represent GC activity in the presence of 100 µM SNP. Basal (□), muscarinic agonist CC 1 x 10−5 M (○), basal plus SNP (▲), and CC+SNP (■). Each value is the mean of three different BTSM strips.

**Figure 2.** Time-course of guanylyl cyclase activity in crude plasma membrane fractions isolated from BTSM strips under muscarinic agonist action. Taken from reference [31].

**Figure 3.** Western blotting of α1β1 NO-sGC-heterodimer from plasma membranes of BTSM under muscarinic activation as described in reference [31].

Identification of α1/β1 sGC heterodimer in membrane fractions isolated from BTSM strips under muscarinic agonist exposure. Isolated BTSM were incubated in KRB at 37°C in the presence and absence of muscarinic agonist CC (1 x10−5 M) during 0, 20 s and 60 s. The samples were immediately frozen and pulverized with a mortar in liquid nitrogen, following the protocols described to isolate crude plasma membranes fraction as described in [31,54]. The final membranes sediments were electrophoresed in 12% PAGE-SDS, transferred to nitrocellulose membranes, and probed with specific antibodies against the α1, α2, (A) β1 and β2 (B) of NO-sGC subunits. The specific antibodies against α2 and β2 produced negative results. This experiment was performed three times with similar results.

Two Guanylylcyclases Regulate the Muscarinic Activation of Airway Smooth Muscle 119

In the other hand, the 60-s cGMP signal is a product of a Natriuretic Peptide Receptor Guanylylcyclase-B (NPR-GC-B), which was previously identified at TSM, using biochemical as reported in [38] and molecular biology approaches as described in [37]. The NPR-GC-B (GC-B) is a membrane-spanning homodimer form, which contains an extracellular ligandbinding, trans-membrane, kinase homology, dimerization and carboxyl-terminal catalytic domains that was published in references [46,47]. NPR-GC-B, which is also called NPR-B or NPR2, is activated by CNP, which exists in 22 and 53 amino acid forms that are structurally

Furthermore, this GC-B from TSM is a novel G-protein coupled guanylyl cyclase presents in isolated plasma membranes fractions from this smooth muscle subtype as described in [53]. This G-protein-coupled NPR-GG-B showed complex kinetics and regulation, which is summarized in a proposed model as shown in Figure 5 as illustrated in reference [38]. This NPR-B was activated by Natriuretic Peptides (CNP-53>CNP-22>ANP-28) at the Ligand Extracellular Domain, stimulated by Gq-protein activators such as mastoparan, and inhibited by a chloride sensitive-Gi/o, interacting at the Juxtamembrane Domain. The Kinase Homology Domain was evaluated by the ATP inhibition of Mn2+-activated-NPR-B, which was partially reversed by mastoparan. The Catalytic Domain was studied by its kinetics of Mn2+/Mg2+ and GTP, and the catalytic effect with GTP analogs with modifications of the βγ phosphates and ribose moieties. Most NPR-B biochemical properties remained after detergent-solubilization but the mastoparan-activation and chloride-inhibition of NPR-B disappeared. This NPR-GC-B is a highly regulated nano-machinery with domains acting at crosstalk points with other signal transducing cascades initiated by G-Protein Coupled Receptors (GPCR) and affected by intracellular ligands such as chloride, Mn2+, Mg2+, ATP and GTP. In addition, this model contains a novel GPRM domain, a G-protein-regulatory site, which was identified and char-

acterized as the binding domain for the G proteins subunits as described in [38].

GC-B is abundantly expressed in brain, lung, bone, heart and ovary tissue as reported in [50]. GC-B contains three intramolecular disulfide bonds and is highly glycosylated on asparagine residues. Moreover, GC-B is highly phosphorylated and dephosphorylation is associated with receptor inhibition as described in [51]. ATP increases the enzymatic activity of GC-B by reducing the Michaelis-Menten constant for GTP, an order of magnitude and this nucleotide seems to be essential for maximal activity as demonstrated in refer-

On the other hand, this Natriuretic Peptide Receptor Guanylylcyclase–B (NPR-GC-B), which has been previously identified as the predominant functional TSM membrane-bound GC subtype by using molecular biology in [37] and biochemical approaches as described in [38]. Nucleotide sequence of membrane-bound GC transcripts retrieved by RT-PCR correspond to the bovine GC-B isoform, indicating the predominance of this NPR-sensitive GC-B over GC-A and GC-C, which are also expressed in TSM in reference [37]. Both ANP and CNP stimulates the TSM membrane-bound GC activity in a concentration-dependent manner, but the CNP effect (10-8 to 10-5 M) was significantly higher than that exerted by ANP, whereas guanylin (a GC-C activator) showed no effect. Given that CNP specifically activates GC-B

similar to ANP and BNP as reported in references [48,49].

ence [52].

Thus, we demonstrate that the first 20-s cGMP signal is a product of a novel signaling cascade involving M2AChR coupled to Go/i proteins, which facilitates the α1β1-sGC isoform migration from cytoplasm to the BTSM sarcolemma. These experimental evidences support a model for the M2AChR, Gi/o, and heterodimer of α1β1 of NO-sGC novel signal transducing cascade as illustrated in Figure 4.

**Figure 4.** Model for the M2AChR, Gi/o, and heterodimer of α1β1 of NO-sGC novel signal transducing cascade in mammalian cells taken from reference [31].

Proposed model for a novel signal transducing cascade in mammalian cells involving three distinct molecular entities: M2AChR, Gi/o protein, and heterodimer of α1β1 of NO-sGC. (A) Basal condition: The hetero-dimer (α1β1NO-sGC) is located in the cytoplasm and the M2AChR and Gi/o proteins are macromolecules spanning and associated respectively with the plasma membrane bilayer. (B) Under muscarinic exposure, the agonist (Cch) binds at the extracellular domains of M2AChR causing the activation of M2AChR. This induces conformational changes at the M2AChR cytoplasm domains stimulating a PTX-sensitive G protein (Gi/o), which may induce a migration and further activation of the α1β1 heterodimer NOsGC from cytoplasm to the plasma membrane resulting in a fast rise in cGMP production, which is related to the 20-s cGMP signal generated during the muscarinic activation of tracheal smooth muscle cell.

In the other hand, the 60-s cGMP signal is a product of a Natriuretic Peptide Receptor Guanylylcyclase-B (NPR-GC-B), which was previously identified at TSM, using biochemical as reported in [38] and molecular biology approaches as described in [37]. The NPR-GC-B (GC-B) is a membrane-spanning homodimer form, which contains an extracellular ligandbinding, trans-membrane, kinase homology, dimerization and carboxyl-terminal catalytic domains that was published in references [46,47]. NPR-GC-B, which is also called NPR-B or NPR2, is activated by CNP, which exists in 22 and 53 amino acid forms that are structurally similar to ANP and BNP as reported in references [48,49].

Current Basic and Pathological Approaches to

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

transducing cascade as illustrated in Figure 4.

cascade in mammalian cells taken from reference [31].

cheal smooth muscle cell.

Identification of α1/β1 sGC heterodimer in membrane fractions isolated from BTSM strips under muscarinic agonist exposure. Isolated BTSM were incubated in KRB at 37°C in the presence and absence of muscarinic agonist CC (1 x10−5 M) during 0, 20 s and 60 s. The samples were immediately frozen and pulverized with a mortar in liquid nitrogen, following the protocols described to isolate crude plasma membranes fraction as described in [31,54]. The final membranes sediments were electrophoresed in 12% PAGE-SDS, transferred to nitrocellulose membranes, and probed with specific antibodies against the α1, α2, (A) β1 and β2 (B) of NO-sGC subunits. The specific antibodies against α2 and β2 produced negative results. This experiment was performed three times with similar results. Thus, we demonstrate that the first 20-s cGMP signal is a product of a novel signaling cascade involving M2AChR coupled to Go/i proteins, which facilitates the α1β1-sGC isoform migration from cytoplasm to the BTSM sarcolemma. These experimental evidences support a model for the M2AChR, Gi/o, and heterodimer of α1β1 of NO-sGC novel signal

**Figure 4.** Model for the M2AChR, Gi/o, and heterodimer of α1β1 of NO-sGC novel signal transducing

Proposed model for a novel signal transducing cascade in mammalian cells involving three distinct molecular entities: M2AChR, Gi/o protein, and heterodimer of α1β1 of NO-sGC. (A) Basal condition: The hetero-dimer (α1β1NO-sGC) is located in the cytoplasm and the M2AChR and Gi/o proteins are macromolecules spanning and associated respectively with the plasma membrane bilayer. (B) Under muscarinic exposure, the agonist (Cch) binds at the extracellular domains of M2AChR causing the activation of M2AChR. This induces conformational changes at the M2AChR cytoplasm domains stimulating a PTX-sensitive G protein (Gi/o), which may induce a migration and further activation of the α1β1 heterodimer NOsGC from cytoplasm to the plasma membrane resulting in a fast rise in cGMP production, which is related to the 20-s cGMP signal generated during the muscarinic activation of traFurthermore, this GC-B from TSM is a novel G-protein coupled guanylyl cyclase presents in isolated plasma membranes fractions from this smooth muscle subtype as described in [53]. This G-protein-coupled NPR-GG-B showed complex kinetics and regulation, which is summarized in a proposed model as shown in Figure 5 as illustrated in reference [38]. This NPR-B was activated by Natriuretic Peptides (CNP-53>CNP-22>ANP-28) at the Ligand Extracellular Domain, stimulated by Gq-protein activators such as mastoparan, and inhibited by a chloride sensitive-Gi/o, interacting at the Juxtamembrane Domain. The Kinase Homology Domain was evaluated by the ATP inhibition of Mn2+-activated-NPR-B, which was partially reversed by mastoparan. The Catalytic Domain was studied by its kinetics of Mn2+/Mg2+ and GTP, and the catalytic effect with GTP analogs with modifications of the βγ phosphates and ribose moieties. Most NPR-B biochemical properties remained after detergent-solubilization but the mastoparan-activation and chloride-inhibition of NPR-B disappeared. This NPR-GC-B is a highly regulated nano-machinery with domains acting at crosstalk points with other signal transducing cascades initiated by G-Protein Coupled Receptors (GPCR) and affected by intracellular ligands such as chloride, Mn2+, Mg2+, ATP and GTP. In addition, this model contains a novel GPRM domain, a G-protein-regulatory site, which was identified and characterized as the binding domain for the G proteins subunits as described in [38].

GC-B is abundantly expressed in brain, lung, bone, heart and ovary tissue as reported in [50]. GC-B contains three intramolecular disulfide bonds and is highly glycosylated on asparagine residues. Moreover, GC-B is highly phosphorylated and dephosphorylation is associated with receptor inhibition as described in [51]. ATP increases the enzymatic activity of GC-B by reducing the Michaelis-Menten constant for GTP, an order of magnitude and this nucleotide seems to be essential for maximal activity as demonstrated in reference [52].

On the other hand, this Natriuretic Peptide Receptor Guanylylcyclase–B (NPR-GC-B), which has been previously identified as the predominant functional TSM membrane-bound GC subtype by using molecular biology in [37] and biochemical approaches as described in [38]. Nucleotide sequence of membrane-bound GC transcripts retrieved by RT-PCR correspond to the bovine GC-B isoform, indicating the predominance of this NPR-sensitive GC-B over GC-A and GC-C, which are also expressed in TSM in reference [37]. Both ANP and CNP stimulates the TSM membrane-bound GC activity in a concentration-dependent manner, but the CNP effect (10-8 to 10-5 M) was significantly higher than that exerted by ANP, whereas guanylin (a GC-C activator) showed no effect. Given that CNP specifically activates GC-B receptors and that it activates GC-A receptors only marginally, the significant CNP effect on TSM cGMP production indicates that NPR-GC-B is the most abundantly expressed tracheal membrane-spanning GC isoform, further confirming the RT-PCR results in [37].

Two Guanylylcyclases Regulate the Muscarinic Activation of Airway Smooth Muscle 121

**Figure 6.** Schematic model of signal cascade involving m3AChR/Gq16/NPR-GC-B at BTSM in [56].

Gqα16ßγ protein as indicated in the scheme.

A model for coupling M3AChR, via Gqα16ßγ to activate NPR-GC-B in plasma membranes from BTSM. This model is composed of three separate and different molecular entities, M3AChR, a GPCR seven transmembrane receptor, a heterotrimeric G protein and homodimeric NPR-GC (cGMP producing enzyme) as the effector. The drawings do not take into account the actual structural biology (molecular mass) of these entities; it is a scheme to suggest the ow of information in this novel signal transducing cascade, which is indicated by the dashed lines. Thus, a muscarinic agonist (ACh) binds at extracellullar domains of M3AChR inducing conformational changes at the cytoplasmic i3M3AChR domain, which stimulates the Gqα16ßγ, to release its active subunits that interact with NPR-GC. Mastoparan and its active analogues may act at the interactions between i3M3AChR domain and the

Mastoparans are tetradecapeptides isolated from wasp venom as reported in [57,58] and they are well known as heterotrimeric G-protein activators as described in [59,60]. Moreover, mastoparan is able to activate the NPR-GC-B associated with plasma membranes fractions from TSM in [56]. Following these results, we evaluate the effect of these peptides on TSM contraction. It was found that mastoparan and analogs were able to inhibit in a selective manner the BTSM contraction induced by muscarinic agonist as carbachol (CC) as shown in Figure 7. Thus, our original findings indicated that mastoparans in the nM range decreased the contractile maximal responses induced by CC without changing its EC50. One explanation for the decrement on the contractile maximal responses by mastoparans may be related to the ability of these tetradecapeptides to disturb the function and the contractile machinery of the BTSM type through cytotoxic mechanisms, which have been described in other biological models, specifically in the µM concentration range as described in [61,62].

This assumption is not supported by our results on the effects of classic TSM spasmogens as 5-HT in as described in reference [63], which produced potent contractions even in the presence of mastoparan (nM) as shown in Figure 8. These findings can be explained since this bioactive amine has been claimed to exert their physiological effects on TSM through

**Figure 5.** Proposed model of the G-protein coupled NPR-GC-B as illustrated in reference [38].

This NPR-GC-B is regulated in an opposite way by two MAChRs signaling cascades, acting the M2AChR as an inhibitor and the M3AChRs as an activator of this NPR-GC-B as mentioned in [55].

This novel signal transducing system is illustrated as a model in Figure 6. The relevant feature of these two opposite signal cascades that regulate this NPR-GC-B is the coupling of two MAChRs and distinctive G proteins. Thus, this activation system coupled a M3AChR to a Gq16 being stimulated by mastoparans (tetradecapeptides from wasp venoms), whereas, on the inhibitor cascade of NPR-GC-B, via M2AChR coupled to a Go/Gi protein has been previously described in references [35,38,56].

Current Basic and Pathological Approaches to

mentioned in [55].

previously described in references [35,38,56].

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

receptors and that it activates GC-A receptors only marginally, the significant CNP effect on TSM cGMP production indicates that NPR-GC-B is the most abundantly expressed tracheal

membrane-spanning GC isoform, further confirming the RT-PCR results in [37].

**Figure 5.** Proposed model of the G-protein coupled NPR-GC-B as illustrated in reference [38].

This NPR-GC-B is regulated in an opposite way by two MAChRs signaling cascades, acting the M2AChR as an inhibitor and the M3AChRs as an activator of this NPR-GC-B as

This novel signal transducing system is illustrated as a model in Figure 6. The relevant feature of these two opposite signal cascades that regulate this NPR-GC-B is the coupling of two MAChRs and distinctive G proteins. Thus, this activation system coupled a M3AChR to a Gq16 being stimulated by mastoparans (tetradecapeptides from wasp venoms), whereas, on the inhibitor cascade of NPR-GC-B, via M2AChR coupled to a Go/Gi protein has been

**Figure 6.** Schematic model of signal cascade involving m3AChR/Gq16/NPR-GC-B at BTSM in [56].

A model for coupling M3AChR, via Gqα16ßγ to activate NPR-GC-B in plasma membranes from BTSM. This model is composed of three separate and different molecular entities, M3AChR, a GPCR seven transmembrane receptor, a heterotrimeric G protein and homodimeric NPR-GC (cGMP producing enzyme) as the effector. The drawings do not take into account the actual structural biology (molecular mass) of these entities; it is a scheme to suggest the ow of information in this novel signal transducing cascade, which is indicated by the dashed lines. Thus, a muscarinic agonist (ACh) binds at extracellullar domains of M3AChR inducing conformational changes at the cytoplasmic i3M3AChR domain, which stimulates the Gqα16ßγ, to release its active subunits that interact with NPR-GC. Mastoparan and its active analogues may act at the interactions between i3M3AChR domain and the Gqα16ßγ protein as indicated in the scheme.

Mastoparans are tetradecapeptides isolated from wasp venom as reported in [57,58] and they are well known as heterotrimeric G-protein activators as described in [59,60]. Moreover, mastoparan is able to activate the NPR-GC-B associated with plasma membranes fractions from TSM in [56]. Following these results, we evaluate the effect of these peptides on TSM contraction. It was found that mastoparan and analogs were able to inhibit in a selective manner the BTSM contraction induced by muscarinic agonist as carbachol (CC) as shown in Figure 7. Thus, our original findings indicated that mastoparans in the nM range decreased the contractile maximal responses induced by CC without changing its EC50. One explanation for the decrement on the contractile maximal responses by mastoparans may be related to the ability of these tetradecapeptides to disturb the function and the contractile machinery of the BTSM type through cytotoxic mechanisms, which have been described in other biological models, specifically in the µM concentration range as described in [61,62].

This assumption is not supported by our results on the effects of classic TSM spasmogens as 5-HT in as described in reference [63], which produced potent contractions even in the presence of mastoparan (nM) as shown in Figure 8. These findings can be explained since this bioactive amine has been claimed to exert their physiological effects on TSM through

#### Current Basic and Pathological Approaches to 122 the Function of Muscle Cells and Tissues – From Molecules to Humans

specific GPCRs. These receptors are the 5-HT2A, which induced activation of the Gq/11 protein and its downstream effector phospholipase C (PLC) leading to intracellular phosphatidylinositol turnover and Ca2+mobilization as reported in [64]. These Gq/11 proteins are mastoparan-insensitive ones. The latter facts can explain the mastoparaninsensitivity of the serotoninergic transducing cascades at TSM. Furthermore, our results demonstrated that mastoparan inhibits selectively the muscarinic activation without altering other spasmogens transducing cascades at TSM.

Two Guanylylcyclases Regulate the Muscarinic Activation of Airway Smooth Muscle 123

The disappearance of the second signal of cGMP (60-s) correlates well with a significant reduction on the contractile maximal responses as here described in Figure 7. This 60-s cGMP signal is a product of NPR-GC-B as above mentioned in references [35,54,55]. Recently, we further recognized that muscarinic agonist and mastoparan activations of NPR-GC-B in isolated BTSM plasma membranes fraction involve this mastoparan-sensitive-Gq16 protein in [54]. Thus, the pre-exposure of TSM strips to mastoparan stimulated the Gq16 protein, disrupting this signal cascade inducing the failure of this muscarinic-dependent TSM contraction. However, the serotoninergic-dependent TSM contraction remained functionally

BTSM fragments were stabilized for 1 h using a procedure described previously in [36]. Later, mastoparan (50 nM) was added for 10 min, followed by carbachol (CC) (1x 10-4 M), serotonin or 5-hydroxytryptamine (5-HT) (1x 10-4 M) and Atropine (AT) (1x10-4M) was added. Values between parentheses are the final concentration of drugs in the incubation

Until now, the BTSM muscarinic activation is unique biological system that involves two cGMP signals as second messengers in references [35,36]. In addition, this activation is a highly regulated biological process, which starts with M2/M3AChRs coupled to two different heterotrimeric G proteins, leading to a fine time regulation and stimulation of two distinctive guanylyl cyclases that accomplish the generation of these two (20-s and 60-s) cGMP

Based in our results, it can be postulated that the first cGMP signal (20-s) is a product of the activation of one M2AChR subtype coupled to a PTX-sensitive-Gi/o protein that leads to the translocation and further activation of the heterodimer α1β1 NO-sGC isoform, anchored to plasma membrane. Recently, it has been suggested that a plasma membrane-bound GC, may provide a localized pool of cGMP in [68], which seems to be in a similar trend suggested by our work. The second signal peak is a product of a the activation of M3AChR coupled to the stimulation of mastoparan-sensitive Gq16 as reported in reference [54] to turn on a

media. This trace is representative of 3 experiments performed with 3 different tracheas fragments. **Figure 8.** Serotonin (5HT) induced contractile responses in mastoparan-treated BTSM strips.

transmembrane-homodimer as NPR-GC-B as described in [35,55].

signal peaks.

active indicating that the smooth muscle machinery remains fully active.

The BTSM strips contractile activity was measured using Procedure 1 as previously described in [36]. Carbachol cumulative responses using concentrations from 1x10–9 M to 1x10–4 M were measured in recording period until 3 min. Mastoparan was pre-incubated for 10 min before the muscarinic agonist (carbachol) addition. Experimental conditions: (■) Control, (▲) 0.5nM (▼) 1 nM (○) 5 nM (□) 10 nM (●) 50nM. The maximal contractile activity was considered as 100% (3.2 ± 0.2 g) and this value was used to estimate other contractile responses. Each value is the mean ± SEM of three different tracheas assayed in duplicate.

**Figure 7.** Carbachol cumulative concentration curves responses from BTSM, pre-treated with mastoparan.

From the data above mentioned, mastoparan can affect specifically the signal cascades associated with the muscarinic activation at TSM sarcolemma, which are initiated with a mAChRs coupled to heterotrimeric G-proteins in reference [65]. These results indicate that the most like candidates implicated in mastoparan effects are the heterotrimeric G-proteins coupled to these mAChRs. G protein involvement on the mastoparan inhibition on TSM muscarinic activation is supported by the ability of this G-protein activator to alter the generation of the two GMPc signals at 20-s and 60-s as previously described in [36]. Mastoparan (50 nM) induced a potent inhibition of these cGMP signals, as shown in Figure 8. After mastoparan pre-incubation, the kinetics of cGMP intracellular levels at TSM was evaluated following the muscarinic agonist exposure. Interestingly, the first cGMP signal (20-s) decreased in more than 60% and the second signal peak (60-s) completely vanished.

The disappearance of the second signal of cGMP (60-s) correlates well with a significant reduction on the contractile maximal responses as here described in Figure 7. This 60-s cGMP signal is a product of NPR-GC-B as above mentioned in references [35,54,55]. Recently, we further recognized that muscarinic agonist and mastoparan activations of NPR-GC-B in isolated BTSM plasma membranes fraction involve this mastoparan-sensitive-Gq16 protein in [54]. Thus, the pre-exposure of TSM strips to mastoparan stimulated the Gq16 protein, disrupting this signal cascade inducing the failure of this muscarinic-dependent TSM contraction. However, the serotoninergic-dependent TSM contraction remained functionally active indicating that the smooth muscle machinery remains fully active.

Current Basic and Pathological Approaches to

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

other spasmogens transducing cascades at TSM.

three different tracheas assayed in duplicate.

completely vanished.

paran.

specific GPCRs. These receptors are the 5-HT2A, which induced activation of the Gq/11 protein and its downstream effector phospholipase C (PLC) leading to intracellular phosphatidylinositol turnover and Ca2+mobilization as reported in [64]. These Gq/11 proteins are mastoparan-insensitive ones. The latter facts can explain the mastoparaninsensitivity of the serotoninergic transducing cascades at TSM. Furthermore, our results demonstrated that mastoparan inhibits selectively the muscarinic activation without altering

The BTSM strips contractile activity was measured using Procedure 1 as previously described in [36]. Carbachol cumulative responses using concentrations from 1x10–9 M to 1x10–4 M were measured in recording period until 3 min. Mastoparan was pre-incubated for 10 min before the muscarinic agonist (carbachol) addition. Experimental conditions: (■) Control, (▲) 0.5nM (▼) 1 nM (○) 5 nM (□) 10 nM (●) 50nM. The maximal contractile activity was considered as 100% (3.2 ± 0.2 g) and this value was used to estimate other contractile responses. Each value is the mean ± SEM of

**Figure 7.** Carbachol cumulative concentration curves responses from BTSM, pre-treated with masto-

From the data above mentioned, mastoparan can affect specifically the signal cascades associated with the muscarinic activation at TSM sarcolemma, which are initiated with a mAChRs coupled to heterotrimeric G-proteins in reference [65]. These results indicate that the most like candidates implicated in mastoparan effects are the heterotrimeric G-proteins coupled to these mAChRs. G protein involvement on the mastoparan inhibition on TSM muscarinic activation is supported by the ability of this G-protein activator to alter the generation of the two GMPc signals at 20-s and 60-s as previously described in [36]. Mastoparan (50 nM) induced a potent inhibition of these cGMP signals, as shown in Figure 8. After mastoparan pre-incubation, the kinetics of cGMP intracellular levels at TSM was evaluated following the muscarinic agonist exposure. Interestingly, the first cGMP signal (20-s) decreased in more than 60% and the second signal peak (60-s)

**Figure 8.** Serotonin (5HT) induced contractile responses in mastoparan-treated BTSM strips.

Until now, the BTSM muscarinic activation is unique biological system that involves two cGMP signals as second messengers in references [35,36]. In addition, this activation is a highly regulated biological process, which starts with M2/M3AChRs coupled to two different heterotrimeric G proteins, leading to a fine time regulation and stimulation of two distinctive guanylyl cyclases that accomplish the generation of these two (20-s and 60-s) cGMP signal peaks.

Based in our results, it can be postulated that the first cGMP signal (20-s) is a product of the activation of one M2AChR subtype coupled to a PTX-sensitive-Gi/o protein that leads to the translocation and further activation of the heterodimer α1β1 NO-sGC isoform, anchored to plasma membrane. Recently, it has been suggested that a plasma membrane-bound GC, may provide a localized pool of cGMP in [68], which seems to be in a similar trend suggested by our work. The second signal peak is a product of a the activation of M3AChR coupled to the stimulation of mastoparan-sensitive Gq16 as reported in reference [54] to turn on a transmembrane-homodimer as NPR-GC-B as described in [35,55].

Current Basic and Pathological Approaches to

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

Two Guanylylcyclases Regulate the Muscarinic Activation of Airway Smooth Muscle 125

Cyclic GMP (cGMP) production in airway smooth muscle cells (ASMC) from CONTROL and OVA- sensitized rats. The ASMC were incubated for 15 min in the presence of IBMX as described in reference [70]. Cyclic GMP production was determined in the presence of Cch, SNP and ODQ as described in Methods. The cGMP produced was estimated by duplicate using a radioimmunoassay kit from Amersham as described in [70]. Each value is the mean + of 5 different experiments. In both groups, the amounts of cell cultures plates were obtained for a pool of 5 rats. The

**Figure 10.** The effect of NO-sGC activators and inhibitors on total GC activity from Control and OVA

Trying to understand the role of the NPR-GC-B in the ASMC, we studied the effect of several activators of this NPR-GC such as natriuretic peptides (ANP and CNP) and mastoparans as previously described in BTSM in references [37,38,71]. Thus, ASMC from Control and OVA-exposed rats were cultured and exposed to these NPR-GC-B activators. Thus, in Figure 11, the total GC activity at OVA-ASMC was stimulated by CNP, ANP and muscarinic agonist (Cch) indicating that NPR-GC-B was present in Control ASMC, which are similar results described in intact BTSM strips and isolated plasma membranes from BTSM in references [37,38,53,55]. In addition, the OVA-ASMC showed a more significant stimulation by these NPR-GC activators. Interestinly, the combination of CC plus ANP and CNP increased in more than 6 times the GC activity. As expected, mastoparan, an activator of NPR-GC-B, via Gq16 dramatically increased in 5 times, the GC activity as reported in [72]. These data showed for the first time that there is an hyperstimulation of M3AChR/Gq16/NPR-GC-B

Taking together all these results indicate that the ASMC from OVA-sensitized rats express a reduced NO-sGC activity and an increased in the NPR-GC-B activity. This imbalance between these two guanylyl cyclases can contribute to airway hyperreactivity and might be implicated in the hyperplastic smooth muscle responses and remodeling present in asthma.

stadistical significance between CONTROL vs OVA was established as *p*<0.05 (\*) and *p*< 0.001 (\*\*).

ASMC. Taken from reference [70].

cascade in ASMC from a experimental asthma model.

BTSM strips were assayed using following Procedure 2 as described in [36]. Pre-incubation for 10 min with mastoparan (□) (50nM) was performed. Control experiments (▲) without drugs were run simultaneously under the same conditions. Each value is the mean X ± SEM of four different tracheas, and the cGMP determinations were carried out in triplicate as described in references [36,53]. Statistically significant difference between the Control with respect to mastoparan as indicated with asterisk (\**p*<0.05).

**Figure 9.** Mastoparan effect on cGMP signal peaks induced by muscarinic agonist (carbachol) in TSM.

Thus, a dysfunction of these M2/M3AChR signal transducing cascades has been implied in the pathophysiological mechanisms of bronchial asthma as mentioned in references [1,3,4] and Chronic Obstructive Pulmonary Disease (COPD) in [3]. In this sense, we attempted to evaluate these novel signal transducing cascades involving guanylyl cyclase activities above discussed, in an experimental asthma model in rats as described in reference [70]. In addition, it has been claimed that excessive NO production that occurs in asthma induces a down-regulation of NO-sGC as reported in [69].

Following these rationale, we used cultured Airway Smooth Muscle Cells (ASMC) from Control and an experimental asthma model (Ovoalbumin exposed rats or OVA-ASMC), which were sensitized to Ovoalbumin using a procedure described in [70,71]. In these ASMC, we evaluate the cGMP production by NPR-GC-B stimulated by muscarinic agonists, mastoparans, natriuretic peptides (ANP, CNP) and for NO-sGC, a classic NO donor as Sodiun NitroPrusside (SNP) and a selective inhibitor for the NO-sGC as 1H-[1, 2,4] Oxadiazolo[ 4,3-a]quinoxalin-1-one (ODQ) were used.

All ASMC exposed to a NO-donor compound as SNP and muscarinic agonist as CC increased cGMP intracellular levels, which were inhibited by ODQ, suggesting that NO-sGC is present and this activity is partially responsible for cGMP production in these ASMC. However, OVA-ASMC showed low basal cGMP production compared to CONTROL ASMC in reference [71] as shown in Figure 10 possibly due to substantial reduced NO-sGC expression reflected in decrease the steady state levels of NO-sGC subunit mRNAs and protein level expression, as described in intact lung tissue from OVA-sensitized mice described elsewhere in reference [69].

Current Basic and Pathological Approaches to

to mastoparan as indicated with asterisk (\**p*<0.05).

down-regulation of NO-sGC as reported in [69].

zolo[ 4,3-a]quinoxalin-1-one (ODQ) were used.

elsewhere in reference [69].

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

BTSM strips were assayed using following Procedure 2 as described in [36]. Pre-incubation for 10 min with mastoparan (□) (50nM) was performed. Control experiments (▲) without drugs were run simultaneously under the same conditions. Each value is the mean X ± SEM of four different tracheas, and the cGMP determinations were carried out in triplicate as described in references [36,53]. Statistically significant difference between the Control with respect

**Figure 9.** Mastoparan effect on cGMP signal peaks induced by muscarinic agonist (carbachol) in TSM.

Thus, a dysfunction of these M2/M3AChR signal transducing cascades has been implied in the pathophysiological mechanisms of bronchial asthma as mentioned in references [1,3,4] and Chronic Obstructive Pulmonary Disease (COPD) in [3]. In this sense, we attempted to evaluate these novel signal transducing cascades involving guanylyl cyclase activities above discussed, in an experimental asthma model in rats as described in reference [70]. In addition, it has been claimed that excessive NO production that occurs in asthma induces a

Following these rationale, we used cultured Airway Smooth Muscle Cells (ASMC) from Control and an experimental asthma model (Ovoalbumin exposed rats or OVA-ASMC), which were sensitized to Ovoalbumin using a procedure described in [70,71]. In these ASMC, we evaluate the cGMP production by NPR-GC-B stimulated by muscarinic agonists, mastoparans, natriuretic peptides (ANP, CNP) and for NO-sGC, a classic NO donor as Sodiun NitroPrusside (SNP) and a selective inhibitor for the NO-sGC as 1H-[1, 2,4] Oxadia-

All ASMC exposed to a NO-donor compound as SNP and muscarinic agonist as CC increased cGMP intracellular levels, which were inhibited by ODQ, suggesting that NO-sGC is present and this activity is partially responsible for cGMP production in these ASMC. However, OVA-ASMC showed low basal cGMP production compared to CONTROL ASMC in reference [71] as shown in Figure 10 possibly due to substantial reduced NO-sGC expression reflected in decrease the steady state levels of NO-sGC subunit mRNAs and protein level expression, as described in intact lung tissue from OVA-sensitized mice described

Cyclic GMP (cGMP) production in airway smooth muscle cells (ASMC) from CONTROL and OVA- sensitized rats. The ASMC were incubated for 15 min in the presence of IBMX as described in reference [70]. Cyclic GMP production was determined in the presence of Cch, SNP and ODQ as described in Methods. The cGMP produced was estimated by duplicate using a radioimmunoassay kit from Amersham as described in [70]. Each value is the mean + of 5 different experiments. In both groups, the amounts of cell cultures plates were obtained for a pool of 5 rats. The stadistical significance between CONTROL vs OVA was established as *p*<0.05 (\*) and *p*< 0.001 (\*\*).

**Figure 10.** The effect of NO-sGC activators and inhibitors on total GC activity from Control and OVA ASMC. Taken from reference [70].

Trying to understand the role of the NPR-GC-B in the ASMC, we studied the effect of several activators of this NPR-GC such as natriuretic peptides (ANP and CNP) and mastoparans as previously described in BTSM in references [37,38,71]. Thus, ASMC from Control and OVA-exposed rats were cultured and exposed to these NPR-GC-B activators. Thus, in Figure 11, the total GC activity at OVA-ASMC was stimulated by CNP, ANP and muscarinic agonist (Cch) indicating that NPR-GC-B was present in Control ASMC, which are similar results described in intact BTSM strips and isolated plasma membranes from BTSM in references [37,38,53,55]. In addition, the OVA-ASMC showed a more significant stimulation by these NPR-GC activators. Interestinly, the combination of CC plus ANP and CNP increased in more than 6 times the GC activity. As expected, mastoparan, an activator of NPR-GC-B, via Gq16 dramatically increased in 5 times, the GC activity as reported in [72]. These data showed for the first time that there is an hyperstimulation of M3AChR/Gq16/NPR-GC-B cascade in ASMC from a experimental asthma model.

Taking together all these results indicate that the ASMC from OVA-sensitized rats express a reduced NO-sGC activity and an increased in the NPR-GC-B activity. This imbalance between these two guanylyl cyclases can contribute to airway hyperreactivity and might be implicated in the hyperplastic smooth muscle responses and remodeling present in asthma. All these previous experimental data unravels some of complex molecular mechanisms associated with the muscarinic activation of ASM. This muscarinic activation is the most physiological, pathophysiological and pharmacological relevant mechanisms because Ach is the neurotransmitter-linked stimulation of ASM in asthma and COPD. Thus, this works opens new trends for the pathophysiological and pharmacological mechanisms, which may lead to new therapeutic approaches for the treatment of these chronic respiratory diseases, such as asthma and COPD, in which, the ASM is involved.

Two Guanylylcyclases Regulate the Muscarinic Activation of Airway Smooth Muscle 127

Natriuretic Peptide Receptor Guanylyl Cyclase-B (NPR-GC-B). The 20-s cGMP signal is linked to the activation of a M2AChR coupled to Go/Gi proteins inducing a massive and transient α1β1-NO-sGC translocation from cytoplasm to plasma membranes of ASM as

The 60-s cGMP signal is associated with a NPR-GC-B in [37], a novel G-protein coupled NPR-GC-B as described in references [38,53,55], which is nano-machine regulated by GPCR and also modulated, in an opposite way, by an activator M3AChRs coupled to Gq16 to activate NPR-GC-B (M3AChR/Gq16/NPR-GC-B cascade) that is stimulated by mastoparan as reported in [56] and an inhibitor M2AChR signal cascade that was partially char-

Mastoparan inhibited in a selective manner the muscarinic-dependent ASM contraction and affected the two cGMP signals. The ASM muscarinic activation is unique biological system involving two cGMP signals and a dysfunction of these M2/M3AChR cascades has been

We used another mammalian model as ASM from rats, which are more prone to develop experimental asthma using Ovoalbumin leading to the OVA-sensitized rats model as described in [70,71]. In isolated and cultured Airway Smooth Muscle Cells (ASMC) from Control and OVA, we evaluated the cGMP production in these ASM cultured cells. All ASMC showed cGMP increments by a classic NO donor as SNP being ODQ-sensitive, indicating that NO-sGC is present, but OVA-ASMC showed low basal cGMP production compared to Control ASMC described in [71], which confirmed the molecular biology results reported elsewhere in reference [69]. Moreover, NPR-GC-B is present in Control ASMC but OVA ASMC displayed an hyperstimulation of M3AChR/Gq16/NPR-GC-B cascade, which is an

These results indicate that the OVA-ASMC express a reduced NO-sGC and increased NPR-GC-B activities. This imbalance between these two GCs can contribute to airway hyperreactivity and might be implicated in the abnormal ASM responses present in asthma and

This work was supported by grants from CDCH-UCV # PG -09-7401-2008/2 (RGA) and CDCH-UCV # PI -09-7726.2009/2 (ILB) and financial support for the publication of this book chapter. WHS is a Graduate student at Ph-D program of Curso de Postgrado en Ciencias

Marcelo J. Alfonzo, Fabiola Placeres-Uray, Walid Hassan-Soto, Adolfo Borges,

*Sección de Biomembranas, Instituto de Medicina Experimental Facultad de Medicina, Universidad Central de Venezuela, Apdo, Sabana Grande, Caracas, Venezuela*

Ramona González de Alfonzo and Itala Lippo de Becemberg

implied in asthma and COPD as published in references [1-6].

original experimental findings as reported in [72].

reported in [31].

acterized.

COPD.

**Author details**

**Acknowledgement**

The effect of NPR-GC activators on total GC activity in Control and OVA ASMC. The OVA and Control ASMC were incubated for 15 min in the presence of 100 µM IBMX (non-selective PDEs inhibitor). Cyclic GMP production was determined in the presence of Cch (1x10-5 M), Mastoparan (1x10-7M), CNP (1x10-7M) and ANP (1x10-7M). The reaction was stopped by removing the medium immediately and freezing with liquid N2 was later 500 µl of TCA 6%, mixed vigorously and centrifuged for 1,500 g x 15 min. The acid extract was treated 2 times with ether saturated with water and the water phase was then lyophilized and suspended in 0.150 ml of water. The cGMP produced by these cells were determined using a radioimmunoassay kit as previously described in reference [70].

**Figure 11.** The effect of NPR-GC activators on total GC activity in Control and OVA ASMC.

#### **3. Conclusion**

In this review, we exposed the recent experimental evidences, in relation to the generation of cGMP, on the muscarinic activation of ASM, which is the essential element in the bronchoconstriction presents in asthma.

Moreover, this muscarinic activation seems to be involved in the remodelling and functional changes of ASM described in asthma and COPD as described in references [1-6,73]. We discussed the existence of two cGMP signals, at 20-s and 60-s in [36], which are products of two distinctive guanylyl cyclases, the NO-sensitive soluble guanylyl cyclases (NO-sGC) and Natriuretic Peptide Receptor Guanylyl Cyclase-B (NPR-GC-B). The 20-s cGMP signal is linked to the activation of a M2AChR coupled to Go/Gi proteins inducing a massive and transient α1β1-NO-sGC translocation from cytoplasm to plasma membranes of ASM as reported in [31].

The 60-s cGMP signal is associated with a NPR-GC-B in [37], a novel G-protein coupled NPR-GC-B as described in references [38,53,55], which is nano-machine regulated by GPCR and also modulated, in an opposite way, by an activator M3AChRs coupled to Gq16 to activate NPR-GC-B (M3AChR/Gq16/NPR-GC-B cascade) that is stimulated by mastoparan as reported in [56] and an inhibitor M2AChR signal cascade that was partially characterized.

Mastoparan inhibited in a selective manner the muscarinic-dependent ASM contraction and affected the two cGMP signals. The ASM muscarinic activation is unique biological system involving two cGMP signals and a dysfunction of these M2/M3AChR cascades has been implied in asthma and COPD as published in references [1-6].

We used another mammalian model as ASM from rats, which are more prone to develop experimental asthma using Ovoalbumin leading to the OVA-sensitized rats model as described in [70,71]. In isolated and cultured Airway Smooth Muscle Cells (ASMC) from Control and OVA, we evaluated the cGMP production in these ASM cultured cells. All ASMC showed cGMP increments by a classic NO donor as SNP being ODQ-sensitive, indicating that NO-sGC is present, but OVA-ASMC showed low basal cGMP production compared to Control ASMC described in [71], which confirmed the molecular biology results reported elsewhere in reference [69]. Moreover, NPR-GC-B is present in Control ASMC but OVA ASMC displayed an hyperstimulation of M3AChR/Gq16/NPR-GC-B cascade, which is an original experimental findings as reported in [72].

These results indicate that the OVA-ASMC express a reduced NO-sGC and increased NPR-GC-B activities. This imbalance between these two GCs can contribute to airway hyperreactivity and might be implicated in the abnormal ASM responses present in asthma and COPD.

#### **Author details**

Current Basic and Pathological Approaches to

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

such as asthma and COPD, in which, the ASM is involved.

All these previous experimental data unravels some of complex molecular mechanisms associated with the muscarinic activation of ASM. This muscarinic activation is the most physiological, pathophysiological and pharmacological relevant mechanisms because Ach is the neurotransmitter-linked stimulation of ASM in asthma and COPD. Thus, this works opens new trends for the pathophysiological and pharmacological mechanisms, which may lead to new therapeutic approaches for the treatment of these chronic respiratory diseases,

The effect of NPR-GC activators on total GC activity in Control and OVA ASMC. The OVA and Control ASMC were incubated for 15 min in the presence of 100 µM IBMX (non-selective PDEs inhibitor). Cyclic GMP production was determined in the presence of Cch (1x10-5 M), Mastoparan (1x10-7M), CNP (1x10-7M) and ANP (1x10-7M). The reaction was stopped by removing the medium immediately and freezing with liquid N2 was later 500 µl of TCA 6%, mixed vigorously and centrifuged for 1,500 g x 15 min. The acid extract was treated 2 times with ether saturated with water and the water phase was then lyophilized and suspended in 0.150 ml of water. The cGMP produced by these cells

In this review, we exposed the recent experimental evidences, in relation to the generation of cGMP, on the muscarinic activation of ASM, which is the essential element in the bron-

Moreover, this muscarinic activation seems to be involved in the remodelling and functional changes of ASM described in asthma and COPD as described in references [1-6,73]. We discussed the existence of two cGMP signals, at 20-s and 60-s in [36], which are products of two distinctive guanylyl cyclases, the NO-sensitive soluble guanylyl cyclases (NO-sGC) and

were determined using a radioimmunoassay kit as previously described in reference [70].

**3. Conclusion**

choconstriction presents in asthma.

**Figure 11.** The effect of NPR-GC activators on total GC activity in Control and OVA ASMC.

Marcelo J. Alfonzo, Fabiola Placeres-Uray, Walid Hassan-Soto, Adolfo Borges, Ramona González de Alfonzo and Itala Lippo de Becemberg *Sección de Biomembranas, Instituto de Medicina Experimental Facultad de Medicina, Universidad Central de Venezuela, Apdo, Sabana Grande, Caracas, Venezuela*

#### **Acknowledgement**

This work was supported by grants from CDCH-UCV # PG -09-7401-2008/2 (RGA) and CDCH-UCV # PI -09-7726.2009/2 (ILB) and financial support for the publication of this book chapter. WHS is a Graduate student at Ph-D program of Curso de Postgrado en Ciencias Fisiológicas. Facultad de Medicina, Universidad Central de Venezuela (UCV). The authors thank to Dr. Marcelo Alfonzo-González for the editing process of this manuscript.

Two Guanylylcyclases Regulate the Muscarinic Activation of Airway Smooth Muscle 129

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[19] Misle AJ, Bécemberg IL, Alfonzo RG, Alfonzo MJ. Methoctramine binding sites sensitive to alkylation on muscarinic receptors from tracheal smooth muscle. Biochem Phar-

[20] Misle A, Bruges G, Herrera VN, Alfonzo MJ, Becemberg IL, Alfonzo, RG. G-proteindependent antagonists binding in M3 AChR from tracheal smooth muscle. Arch Venez

[21] Meurs H, Roffel AF, Postema JB, Timmermans A, Elzinga, CRS, Kauffman, HF, Zaagsma J. Evidence for a direct relationship between phosphoinositide metabolism and airway smooth muscle contraction induced by muscarinic agonists. Eur J Pharmacol.1988;

[22] Roffel AF, Elzinga CR, Zaagsma J. Muscarinic M3 receptors mediate contraction of human central and peripheral airway smooth muscle. Pulm Pharmacol. 1990; 3: 47–51 [23] Grandordy BM, Cuss FM, Sampson AS, Palmer JB, Barnes PJ. Phosphatidylinositol response to cholinergic agonists in airway smooth muscle: relationship to contraction

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**Chapter 7**

© 2012 Salim and Ali, 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,

**Melanophores: Smooth Muscle Cells in Disguise**

Melanophores are specialized cells derived from the neural crest that contain membrane bound vesicles called melanosomes. Melanosomes are filled with melanin, a dark, nonfluorescent pigment that plays a principal role in physiological color adaptation of animals. Melanophores regulate melanosome trafficking on cytoskeletal filaments to generate a range of striking chromatic patterns. The mechanism of physiological color change by these melanophores encompasses both physical and biochemical aspects of melanosome dynamics. Melanophores aggregate or disperse their melanosomes when the host requires changing its color in response to the environmental cues (e.g., social interactions or camouflage). Interestingly, morphological, embryological and physiological evidence has revealed that melanophores of fish, amphibian and reptiles are functionally modified smooth muscle cells [1]. Moreover, during contraction of melanophore there is an aggregation of melanin granules which is to be considered the visible counterpart of an aggregation of colloidal particles that occur during smooth muscle contraction. The biochemical events underlying melanosome dispersion is analogous to those for smooth muscle relaxation: both processes result from increase in cAMP levels and require the

Ligands like neurotransmitters and hormones bind to specific receptors to activate contraction in smooth muscle. Subsequent to this binding there is an increase in cellular phospholipase C activity via coupling to a G protein resulting into the activation of a membrane phospholipid phosphatidylinositol 4, 5-biphosphate (PIP2). Phospholipase C (PLC) produces two potent second messengers from PIP2: Diacylglycerol (DAG) and inositol 1, 4, 5-triphosphate (IP3). IP3 binds to specific receptors, causing release of calcium (Ca2+). Later in the cascade DAG activates PKC, which further phosphorylates specific target proteins. In smooth muscles PKC promotes phosphorylation of Ca2+

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

Saima Salim and Sharique A. Ali

http://dx.doi.org/10.5772/48256

**1. Introduction**

Additional information is available at the end of the chapter

presence of extracellular Ca2+ for their action.

**1.1. Regulation of smooth muscle contraction**

### **Melanophores: Smooth Muscle Cells in Disguise**

Saima Salim and Sharique A. Ali

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/48256

#### **1. Introduction**

Current Basic and Pathological Approaches to

cytes. Cardiov Res. 2005; 68:65-74.

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human primates. Cell Tissue Res. 2006; 326:707-714.

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21324.

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

[61] Jones S, Howl J. Charge delocalization and the design of novel mastoparan analogues: enhanced cytotoxicity and secretory efficacy of [Lys5, Lys8, Aib10]MP. Regul Pept.

[62] Sugama J, Yu JZ, Rasenick MM, Nakahata N. Mastoparan inhibits betaadrenoceptor-G(s) signaling by changing the localization of Galpha(s) in lipid rafts. Cell Signal 2007;

[63] Shi J, Damjanoska KJ, Singh RK, Carrasco GA, Garcia F, Grippo AJ, Landry M, Sullivan NR, Battaglia G, Muma NA. Agonist induced-phosphorylation of Galpha11 protein re-

[65] Murthy KS, Makhlouf GM. Differential coupling of muscarinic m2 and m3 receptors to adenylyl cyclases V/VI in smooth muscle. Concurrent M2-mediated inhibition via Galphai3 and m3-mediated stimulation via Gbetagammaq. J Biol Chem. 1997; 272: 21317-

[66] Agulló L, Garcia-Dorado D, Escalona N, Ruiz-Meana M, Mirabet M, Inserte J, Soler-Soler J. Membrane association of nitric oxide-sensitive guanylyl cyclase in cardiomyo-

[67] Bidmon HJ, Mohlberg H, Habermann G, Buse E, Zilles K, Behrends S. Cerebellar localization of the NO-receptive soluble guanylyl cyclase subunits-alpha(2)/beta (1) in non-

[68] Belligham M, Evans TJ. The α2β2 isoform of guanylyl cyclase mediates plasma membrane localized nitric oxide signaling. Cellular Signalling 2007; 19: 2183-2193. [69] Papapetropoulos A, Simoes DC, Xanthou G, Roussos C, Gratziou C. Soluble guanylyl cyclase expression is reduced in allergic asthma. Am J Physiol Lung Cell Mol Physiol.

[70] Hjoberg J, Shore S, Kobzik L, Okinaga S, Hallock A, Vallone J, Subramaniam V, De Sanctis GT, Elias JA, Drazen JM, Silverman ES. Expression of nitric oxide synthase-2 in the lungs decreases airway resistance and responsiveness. J Appl Physiol. 2004; 97: 249-

[71] Placeres-Uray F, de Alfonzo RG, de Becemberg IL, Alfonzo MJ. Soluble guanylyl cyclase is reduced in airway smooth muscle cells from a murine model of allergic asth-

[72] [Placeres-Uray F, de Alfonzo RG, Alfonzo MJ, de Becemberg IL. Hypersensitivity of the M3AChR/ Gq16 protein/NPR-GC-B coupling mechanism associated to muscarinic activation of airway smooth muscle cells in a rat asthma model. World Allergy Organiza-

[73] Kolahian S, Gosens R. Cholinergic Regulation of Airway Inflammation and Remodel-

duces coupling to 5-HT2A receptors. J Pharmacol Exp Ther. 2007; 323:248-56. [64] Shi J, Zemaitaitis B, Muma NA. Phosphorylation of Gq11 Protein Contributes to Agonist-Induced Desensitization of 5-HT2A Receptor Signaling. Mol Pharmacol. 2007;

> Melanophores are specialized cells derived from the neural crest that contain membrane bound vesicles called melanosomes. Melanosomes are filled with melanin, a dark, nonfluorescent pigment that plays a principal role in physiological color adaptation of animals. Melanophores regulate melanosome trafficking on cytoskeletal filaments to generate a range of striking chromatic patterns. The mechanism of physiological color change by these melanophores encompasses both physical and biochemical aspects of melanosome dynamics. Melanophores aggregate or disperse their melanosomes when the host requires changing its color in response to the environmental cues (e.g., social interactions or camouflage). Interestingly, morphological, embryological and physiological evidence has revealed that melanophores of fish, amphibian and reptiles are functionally modified smooth muscle cells [1]. Moreover, during contraction of melanophore there is an aggregation of melanin granules which is to be considered the visible counterpart of an aggregation of colloidal particles that occur during smooth muscle contraction. The biochemical events underlying melanosome dispersion is analogous to those for smooth muscle relaxation: both processes result from increase in cAMP levels and require the presence of extracellular Ca2+ for their action.

#### **1.1. Regulation of smooth muscle contraction**

Ligands like neurotransmitters and hormones bind to specific receptors to activate contraction in smooth muscle. Subsequent to this binding there is an increase in cellular phospholipase C activity via coupling to a G protein resulting into the activation of a membrane phospholipid phosphatidylinositol 4, 5-biphosphate (PIP2). Phospholipase C (PLC) produces two potent second messengers from PIP2: Diacylglycerol (DAG) and inositol 1, 4, 5-triphosphate (IP3). IP3 binds to specific receptors, causing release of calcium (Ca2+). Later in the cascade DAG activates PKC, which further phosphorylates specific target proteins. In smooth muscles PKC promotes phosphorylation of Ca2+

channels that regulates cross-bridge cycling. Ca2+ binds to a calcium modulated protein called calmodulin, resulting into the activation of MLC kinase, which is a myosin light chain protein [2]. This step initiates the shortening of the muscle cell together with actin via cross bridge cycling. The state of contraction is maintained by a Ca2+ sensitizing mechanism that is brought about by another protein, Rho kinase also known as ROCK. ROCK increases the activity of the motor protein myosin II by two different mechanisms: Firstly, phosphorylation of the myosin light chain (MLC) increases the myosin II ATPase activity. Thus several bundled and active myosins, which are asynchronously active on several actin filaments, move actin filaments against each other resulting in the net shortening of actin fibres. Secondly by inactivating MLC phosphatase, leading to increased levels of phosphorylated MLC [2, 3, 100]. (Figure 1)

Melanophores: Smooth Muscle Cells in Disguise 135

**1.2. Regulation of smooth muscle relaxation**

process of smooth muscle relaxation [2,100] (Figure 1).

resulting into relaxation [2].

**reference to smooth muscles**

The process of relaxation requires a decreased intracellular Ca2+ concentration and increased myosin light chain (MLC) phosphatase activity. The plasma membrane contain Ca Mg-ATPases along with Na+/ Ca2+ exchangers that remove Ca2+ from the cytosol. Also the voltage operated Ca2+ channels in the plasma membrane close to the entry of Ca2+ in the cell

In short the contractile activity in smooth muscle is initiated by a Ca2+-calmodulin interaction to stimulate phosphorylation of the light chain of myosin. A Ca2+ sensitization of the contractile proteins is signaled by the RhoA/Rho kinase pathway to inhibit the dephosphorylation of the light chain by myosin phosphatase, maintaining force generation. Removal of Ca2+from the cytosol and stimulation of myosin phosphatase initiate the

The comparison of melanophores to smooth muscle cells dates back as early as 1917 when Spaeth provided evidence for the similarity of melanophores with smooth muscle cells. Rhythmical pulsations in the isolated scale melanophores of *Fundulus heteroclitus* were first observed by Spaeth [1]. The observation followed by the in depth analysis of the behavior and responses of melanophores to various stimuli and their parallelism to smooth muscle cells led to the conceptualization of melanophore as a modified derivative of smooth muscle. Rhythmical movements are, of course, intimately associated with this latter tissue and their persistence for considerable periods of time in isolated strips of the esophagus and stomach of frogs and other vertebrates is well known. The rhythmical movements in isolated scale melanophores are comparable and perhaps similar to these movements in smooth muscle. This discovery opens a new field for research that might contribute in

The biochemical events underlying the melanosome dispersion are analogous to smooth muscle relaxation since both these processes result from an increase in cyclic AMP (cAMP) levels [102]. Unlike cardiac muscle, increased cAMP in smooth muscle causes relaxation. The reason for this is that cAMP normally inhibits myosin light chain kinase (MLCK), the enzyme that is responsible for phosphorylating smooth muscle myosin and causing contraction. It is known that the increase in cAMP initiates the influx of Ca2+ from the cytoplasm to the endoplasmic reticulum and/or mitochondria or out of the smooth muscle cell. Whereas the melanosome dispersion progresses in the absence of Ca2+ and presence of extracellular Ca2+ (from the action of MSH) [4]. The crucial role of Ca2+ and MSH in the bidirectional movement of melanophores has been shown in the figure 2. Extracellular stimuli (either hormonal; adrenaline or mechanical stimulus) could result in inward movement of Ca2+ from the extracellular matrix. This is followed by activation of phospholipase C (PLC) promoting 1) inositol trisphosphate (IP3)-dependent release of Ca2+

**2. Historical and comparative perspective of melanophores with**

understanding the ramifications of inter and intra-cellular dynamics.

**Figure 1.** Regulation of Smooth Muscle Contraction and Relaxation. Contraction is triggered by the influx of calcium through the transmembrane channels (ligand-gated (noradrenaline) and the voltagegated Ca2+-channels). The calcium combines with calmodulin to form a complex that converts myosin light chain kinase to its active form (MLCK\*). The latter phosphorylates the myosin light chains (MLC), thereby initiating the interaction of myosin with actin. Activation of RhoA leads to the activation of Rho-kinase (ROCK), which in turn phosphorylates the regulatory myosin-binding subunit of myosin phosphatase (MLCP), resulting into the inhibition of the enzyme. Substances that increase the cAMP may cause relaxation in smooth muscle by accelerating the inactivation of MLCK and facilitating the expulsion of calcium from the cell. (Reproduced with permission from Macmillan Publishers Ltd: [100] Internat. Journ. of Impot. Res. 16: 459–469, Copyright © 2004, Nature Publishing Group.

#### **1.2. Regulation of smooth muscle relaxation**

Current Basic and Pathological Approaches to

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

increased levels of phosphorylated MLC [2, 3, 100]. (Figure 1)