vascular smooth muscle contraction is activated by an increase in cytosolic free 
 ca concentration ( [ ca]i ) as a result of ca 
 entry from the extracellular space and/or ca release from intracellular stores , 
 primarily the sarcoplasmic reticulum ( 1 ) . 

ca diffuses to the contractile machinery where it binds to calmodulin ( cam ) 
 ( 2 ) .
the ( ca)4-cam complex 
 induces a conformational change in myosin light chain kinase ( mlck ) , which involves removal of 
 the autoinhibitory domain from the active site , thereby converting the kinase from an inactive 
 to an active state ( 3 ) .
mlck is physically bound through 
 its n - terminus to actin filaments and , upon activation , phosphorylates nearby 
 myosin molecules ( 4 ) .
myosin ii filaments are composed 
 of hexameric myosin molecules , each consisting of two heavy chains and two pairs of light 
 chains ( 17-kda essential light chains ( lc17 ) and 20-kda regulatory light chains 
 ( lc20 ) ) located in the neck region of the myosin molecule ( fig .
smooth muscle myosin ii is a 
 hexameric protein composed of two heavy chains ( 205 kda each ) and two pairs of light 
 chains : the 17-kda essential light chains ( lc17 ) and the 20-kda regulatory light chains 
 ( lc20 ) .
the n - termini of the heavy chains make up most of the head domains ( pink ) and 
 the c - termini of the heavy chains account for the complete coiled - coil rod domain ( which 
 is responsible for assembly of myosin filaments ) and the terminal unstructured tail 
 ( black ) .
each globular head includes an actin - binding site ( blue ) and an atp - binding 
 site ( green ) .
the light chains , lc17 ( yellow ) and lc20 ( red ) , are associated with the 
 neck region .
vascular smooth muscle myosin light chain diphosphorylation : 
 mechanism , function and pathological implications .
activated mlck phosphorylates ser19 of lc20 and this simple 
 post - translational modification induces a conformational change that is transmitted to the 
 myosin heads , resulting in actin interaction and a marked increase in the actin - activated 
 mgatpase activity of myosin ( 5 ) .
the energy derived from 
 the hydrolysis of atp then drives cross - bridge cycling and the development of force or 
 contraction of the muscle .
relaxation follows the removal of ca from the cytosol , 
 primarily by caatpases , which pump ca out of the cell and back into the 
 sarcoplasmic reticulum ( 6 ) .
mlck is inactivated as 
 ca dissociates from cam and the autoinhibitory domain of mlck blocks the active 
 site .
phosphorylated myosin is then dephosphorylated by myosin light chain phosphatase ( mlcp ) , 
 a type 1 protein serine / threonine phosphatase ( 7 ) .  
smooth muscle myosin ii is a 
 hexameric protein composed of two heavy chains ( 205 kda each ) and two pairs of light 
 chains : the 17-kda essential light chains ( lc17 ) and the 20-kda regulatory light chains 
 ( lc20 ) .
the n - termini of the heavy chains make up most of the head domains ( pink ) and 
 the c - termini of the heavy chains account for the complete coiled - coil rod domain ( which 
 is responsible for assembly of myosin filaments ) and the terminal unstructured tail 
 ( black ) .
each globular head includes an actin - binding site ( blue ) and an atp - binding 
 site ( green ) .
the light chains , lc17 ( yellow ) and lc20 ( red ) , are associated with the 
 neck region .
walsh mp . vascular smooth muscle myosin light chain diphosphorylation : 
 mechanism , function and pathological implications .
mlcp is a trimeric phosphatase with a 38-kda catalytic subunit ( pp1c ) , a 130-kda 
 regulatory subunit ( mypt1 ) and a 21-kda subunit of uncertain function .
mlcp is inhibited by both 
 protein kinase c ( pkc ) and rhoa / rho - associated kinase ( rok ) pathways .
phosphorylation of the 
 cytosolic phosphatase inhibitory protein of 17-kda ( cpi-17 ) at thr38 by pkc converts cpi-17 
 to a potent inhibitor of mlcp , which is achieved by direct interaction of phosphorylated 
 cpi-17 with pp1c ( 8) .
in addition , and 
 apparently of greater physiological significance , rok phosphorylates mypt1 to induce 
 inhibition of mlcp activity .
rok phosphorylates mypt1 at thr697 and thr855 ( rat numbering ) 
 in vitro and both phosphorylation events result in phosphatase inhibition 
 ( 10 ) .
however , it appears that rok predominantly 
 phosphorylates thr855 in intact tissues ( 11 ) . 

activation of pkc and rok pathways , therefore , can result in decreased mlcp activity , 
 resulting in an increase in the ratio of mlck : mlcp activity and an increase in force .
since 
 rok and novel pkc isoforms are ca - independent , this results in ca 
 sensitization of contraction , i.e. an increase in force without an increase in 
 [ ca]i .
the possibility that lc20 may also be phosphorylated at thr18 was originally 
 demonstrated in vitro when it was shown that high concentrations of mlck 
 phosphorylate thr18 in addition to ser19 ( 12 ) .
it is 
 clear , however , that mlck does not phosphorylate thr18 of lc20 in intact smooth 
 muscle tissues , and most contractile stimuli are associated with lc20 
 phosphorylation exclusively at ser19 , which can be attributed to mlck ( 13 ) .
interest in thr18 phosphorylation was revived , however , when it was 
 shown that treatment of smooth muscle tissues with membrane - permeant phosphatase inhibitors 
 ( such as calyculin - a ) induced ca - independent contractions that correlated with 
 diphosphorylation of lc20 at thr18 and ser19 ( 14 ) .
two ca - independent kinases ( integrin - linked kinase ( ilk ) and 
 zipper - interacting protein kinase ( zipk ) ) were shown to be the most likely kinases 
 responsible for lc20 diphosphorylation ( 15,16,17 ) .
phosphorylation of lc20 at thr18 and ser19 has been observed in several smooth 
 muscle tissues , e.g. bovine tracheal smooth muscle in response to neural stimulation or 
 carbachol ( 18 , 19 ) , rabbit thoracic aorta treated with prostaglandin - f2 ( 20 , 21 ) and renal 
 afferent arterioles stimulated with endothelin-1 ( 22 ) .
lc20 diphosphorylation has frequently been associated with 
 pathophysiological conditions involving hypercontractility , including cerebral vasospasm 
 ( 23 ) , coronary ( 24 , 25 ) and femoral arterial vasospasm 
 ( 26 ) , intimal hyperplasia ( 27 ) and hypertension ( 28 ) .
early studies comparing the properties of smooth muscle myosin phosphorylated at ser19 ( by 
 low concentrations of mlck ) and at both thr18 and ser19 ( by high concentrations of mlck ) 
 indicated that the additional phosphorylation at thr18 increased the actomyosin mgatpase 
 activity some two- to three - fold ( 12 , 29,30,31 )
. however , the velocity of movement of myosin - coated 
 beads along actin cables ( 32 ) or of actin filaments 
 over immobilized myosin in the in vitro motility assay ( 31 ) was similar whether lc20 was 
 phosphorylated at ser19 alone or at both thr18 and ser19 .
we recently addressed the 
 hypothesis that phosphorylation at thr18 may enhance the level of steady - state force 
 achieved with ser19 phosphorylation ( 13 ) . to test 
 this hypothesis
the 
 objective was to achieve stoichiometric phosphorylation of lc20 at ser19 , then 
 elicit phosphorylation at thr18 and observe whether or not there was a further increase in 
 steady - state force .
the challenge was to achieve stoichiometric phosphorylation at ser19 
 since this can not be done in intact tissue due to the competing actions of mlck and mlcp , 
 which results in stable phosphorylation of lc20 at steady - state of  0.5  mol 
 pi / mol lc20 .
furthermore , this problem can not be overcome by 
 inhibition of phosphatase activity since phosphatase inhibitors such as calyculin - a and 
 okadaic acid unmask the basal activities of ilk and zipk , which phosphorylate both thr18 and 
 ser19 of lc20 .
we took advantage of the fact that protein kinases generally , 
 including mlck , can utilize adenosine 5-o-(3-thiotriphosphate ) ( atps ) as 
 a substrate to thiophosphorylate their protein substrates ( 33 ) , but the thiophosphorylated protein ( lc20 in this case ) is a very 
 poor phosphatase substrate ( 34 ) .
it was necessary to 
 use triton - skinned tissue for this experiment since the plasma membrane is impermeant to 
 atps .
stoichiometric thiophosphorylation of lc20 at ser19 in triton - skinned rat caudal 
 arterial smooth muscle .
( a ) the viability of triton - skinned rat caudal arterial smooth 
 muscle strips was initially verified by transfer from relaxing solution ( pca 9 ) to 
 high [ ca ] ( pca 4.5 ) solution containing atp and an atp regenerating system ( rs ) , 
 which induced a contractile response .
tissues were then relaxed by 3 washes in pca 9 
 solution containing atp and rs .
atp was then removed by 6 washes in pca 9 solution 
 without atp or rs .
tissues were then incubated in pca 4.5 solution containing atps 
 ( 4  mm ) in the absence of atp and rs .
excess atps was then removed by washing twice 
 with pca 9 solution without atp or rs .
once steady - state force was established , microcystin 
 ( 1 m ) was added in pca 9 solution containing atp and rs .
tissues were harvested at 
 the indicated times during this protocol for phos - tag sds - page and western blotting 
 with anti - pan lc20 ( b ) , as shown by the arrows in ( a ) ( the numbers correspond to the 
 lanes in ( b ) ) : ( i ) lanes 1 and 8 , tissue incubated at pca 9 showing exclusively 
 unphosphorylated lc20 ; ( ii ) lanes 2 and 3 , pca 4.5 + atps in the absence of atp and 
 rs ; ( iii ) lane 4 , pca 9 in the absence of atp and rs following thiophosphorylation ; 
 ( iv ) lane 5 , at the plateau of force development following transfer to pca 9 solution 
 containing atp and rs ; ( v ) lanes 6 and 7 , following treatment with microcystin at pca 
 9 in the presence of atp and rs .
an additional control is included in lane 9 : 
 triton - skinned tissue treated with microcystin at pca 9 for 60  min to identify 
 unphosphorylated ( 0p ) , monophosphorylated ( 1p ) , and diphosphorylated ( 2p ) lc20 bands . 

thiophosphorylated forms of lc20 are indicated as follows : 1sp , monothiophosphorylated 
 lc20 ; 2sp , dithiophosphorylated lc20 ; 1sp1p , lc20 thiophosphorylated at one site and 

myosin regulatory light chain diphosphorylation slows 
 relaxation of arterial smooth muscle . 2012 ; 287(29 ) : 2406476 .
 the american society for biochemistry and molecular 
 biology . shows the experimental protocol and corresponding force measurements ( fig .
2a ) and analysis of lc20 
 ( thio)phosphorylation by phos - tag sds - page ( see below for a description of this technique , 
 which enables the separation of unphosphorylated and phosphorylated forms of 
 lc20 ) ( fig .
2b ) . at resting tension 
 ( pca 9 ) , lc20 is unphosphorylated ( lanes 1 and 8 , fig .
a control contraction was elicited by increasing 
 [ ca ] to pca 4.5 in the presence of atp and relaxation followed removal of 
 ca ( fig .
when basal force was 
 restored , the tissue was washed several times in pca 9 solution without atp to remove all 
 atp , following which atps was added at pca 4.5 to elicit close - to - stoichiometric 
 lc20 thiophosphorylation ( fig . 2b , 
 lanes 2 and 3 ) .
contraction did not occur under these conditions since atps is not 
 hydrolysed by the actin - activated myosin mgatpase and , therefore , atps can not support 
 cross - bridge cycling ( 35,36,37 ) .
following lc20 
 thiophosphorylation , atps was washed out by several washes with pca 9 solution ( fig .
atp was then added at pca 9 , whereupon the tissue contracted rapidly due to the 
 fact that lc20 was previously thiophosphorylated .
the level of force achieved during this treatment was comparable to 
 that elicited initially by addition of atp at pca 4.5 ( fig .
finally , the phosphatase inhibitor microcystin was added at pca 9 in the 
 presence of atp , whereupon ser19-thiophosphorylated lc20 was phosphorylated at 
 thr18 ( 1s1p in lanes 6 and 7 of fig .
small 
 amounts of monophosphorylated ( 1p ) and diphosphorylated ( 2p ) lc20 were also 
 detected due to the presence ( fig .
2b , lane 5 ) of 
 a small amount of unphosphorylated lc20 prior to the addition of microcystin . 

2b , lane 9 shows a control triton - skinned 
 tissue treated with microcystin and atp at pca 9 to indicate the migration of 
 unphosphorylated ( 0p ) , monophosphorylated ( 1p ) and diphosphorylated ( 2p ) lc20 , as 
 previously established ( 13 ) .
the key finding from 
 this experiment was that phosphorylation of lc20 at thr18 on top of 
 close - to - stoichiometric thiophosphorylation of lc20 at ser19 did not elicit an 
 increase in force .  
stoichiometric thiophosphorylation of lc20 at ser19 in triton - skinned rat caudal 
 arterial smooth muscle .
( a ) the viability of triton - skinned rat caudal arterial smooth 
 muscle strips was initially verified by transfer from relaxing solution ( pca 9 ) to 
 high [ ca ] ( pca 4.5 ) solution containing atp and an atp regenerating system ( rs ) , 
 which induced a contractile response .
tissues were then relaxed by 3 washes in pca 9 
 solution containing atp and rs .
atp was then removed by 6 washes in pca 9 solution 
 without atp or rs .
tissues were then incubated in pca 4.5 solution containing atps 
 ( 4  mm ) in the absence of atp and rs .
excess atps was then removed by washing twice 
 with pca 9 solution without atp or rs .
once steady - state force was established , microcystin 
 ( 1 m ) was added in pca 9 solution containing atp and rs .
tissues were harvested at 
 the indicated times during this protocol for phos - tag sds - page and western blotting 
 with anti - pan lc20 ( b ) , as shown by the arrows in ( a ) ( the numbers correspond to the 
 lanes in ( b ) ) : ( i ) lanes 1 and 8 , tissue incubated at pca 9 showing exclusively 
 unphosphorylated lc20 ; ( ii ) lanes 2 and 3 , pca 4.5 + atps in the absence of atp and 
 rs ; ( iii ) lane 4 , pca 9 in the absence of atp and rs following thiophosphorylation ; 
 ( iv ) lane 5 , at the plateau of force development following transfer to pca 9 solution 
 containing atp and rs ; ( v ) lanes 6 and 7 , following treatment with microcystin at pca 
 9 in the presence of atp and rs .
an additional control is included in lane 9 : 
 triton - skinned tissue treated with microcystin at pca 9 for 60  min to identify 
 unphosphorylated ( 0p ) , monophosphorylated ( 1p ) , and diphosphorylated ( 2p ) lc20 bands . 

thiophosphorylated forms of lc20 are indicated as follows : 1sp , monothiophosphorylated 
 lc20 ; 2sp , dithiophosphorylated lc20 ; 1sp1p , lc20 thiophosphorylated at one site and 
 phosphorylated at the other .
we next addressed the hypothesis that phosphorylation of lc20 at both thr18 and 
 ser19 reduces the rate of dephosphorylation and relaxation compared to phosphorylation at 
 ser19 alone . to test this hypothesis ,
lc20 was phosphorylated at ser19 only or at 
 both thr18 and ser19 to comparable stoichiometry .
this was achieved by treatment of 
 triton - skinned rat caudal arterial smooth muscle strips with atp at pca 4.5 ( for 
 monophosphorylation at ser19 ) or with atp and the phosphatase inhibitor okadaic acid at pca 
 9 ( for diphosphorylation at thr18 and ser19 ) . the time - courses of dephosphorylation and 
 relaxation were then followed to determine if there was a reduction in the rates of 
 dephosphorylation and relaxation when lc20 was diphosphorylated at thr18 and 
 ser19 compared to monophosphorylated at ser19 . as shown in fig .
comparison of the time courses of relaxation and lc20 dephosphorylation in 
 triton - skinned rat caudal arterial smooth muscle following contraction with ca or 
 okadaic acid in the absence of ca .
triton - skinned tissues that had been contracted 
 with ca ( open circles ) or okadaic acid ( 20 m ) at pca 9 ( closed circles ) were 
 transferred to pca 9 solution and the time courses of dephosphorylation ( a ) and 
 relaxation ( b ) were followed .
tissues were harvested at 10 , 20 , 30 , 40 , 50 , 75 , and 
 100% relaxation and lc20 phosphorylation levels were quantified by phos - tag sds - page 
 and western blotting with anti - pan lc20 .
 the american society for 
 biochemistry and molecular biology . , both treatments induced lc20 phosphorylation to 0.5  mol 
 pi / mol lc20 . at pca 4.5 ,
this was exclusively ser19 phosphorylation , 
 whereas in response to okadaic acid at pca 9 , phosphorylation occurred at both thr18 and 
 ser19 ( fig .
3b ) clearly shows that both 
 dephosphorylation and relaxation rates were significantly slower when lc20 was 
 diphosphorylated at thr18 and ser19 compared to monophosphorylated at ser19 .
this finding 
 suggests a functional effect of lc20 diphosphorylation and raises the possibility 
 that pathological situations of hypercontractility may result from impaired relaxation due 
 to this dephosphorylation event .
mlcp has been shown to dephosphorylate thr18 in addition to 
 ser19 ( 38 ) suggesting that the same phosphatase 
 dephosphorylates mono- and diphosphorylated lc20 in situ .  
comparison of the time courses of relaxation and lc20 dephosphorylation in 
 triton - skinned rat caudal arterial smooth muscle following contraction with ca or 
 okadaic acid in the absence of ca .
triton - skinned tissues that had been contracted 
 with ca ( open circles ) or okadaic acid ( 20 m ) at pca 9 ( closed circles ) were 
 transferred to pca 9 solution and the time courses of dephosphorylation ( a ) and 
 relaxation ( b ) were followed .
tissues were harvested at 10 , 20 , 30 , 40 , 50 , 75 , and 
 100% relaxation and lc20 phosphorylation levels were quantified by phos - tag sds - page 
 and western blotting with anti - pan lc20 .
4 . schematic diagram illustrating the anatomical relationship of the afferent 
 arteriole , glomerulus , and the efferent arteriole . the afferent arteriole controls the 
 glomerular inflow resistance . this vessel must constrict rapidly in response to 
 fluctuations in blood pressure to prevent pressure elevations from being transmitted 
 to the downstream glomerular capillaries .
when renal 
 perfusion pressure is compromised , a sustained increase in efferent arteriolar tone 
 maintains adequate filtration pressure within the upstream glomerular capillaries , 
 thereby preserving renal function .
provides a schematic representation of the renal microcirculation , which consists of 
 the afferent arteriole conveying blood from the interlobular artery to the glomerular 
 capillaries where it is filtered prior to returning to the systemic circulation via the 
 efferent arteriole .
the afferent arteriole plays a key role in regulating glomerular inflow 
 resistance and must be able to respond very rapidly to sudden changes in systemic blood 
 pressure in order to protect the fragile glomeruli from pressure - induced damage ( 39 ) .
angiotensin ii ( ang ii ) is a renal - selective 
 vasoconstrictor that contributes to renal vascular resistance under normal physiological 
 conditions and thereby plays an important role in modulating renal hemodynamics ( 40 ) .
endothelin-1 ( et-1 ) , on the other hand , is a renal 
 vasoconstrictor that does not contribute to renal vascular resistance under normal 
 physiological conditions , but is implicated in abnormal renal vasoconstriction and reduced 
 glomerular filtration in pathological states such as diabetes and chronic kidney disease 
 ( 41,42,43,44,45,46,47 ) .  
schematic diagram illustrating the anatomical relationship of the afferent 
 arteriole , glomerulus , and the efferent arteriole .
this vessel must constrict rapidly in response to 
 fluctuations in blood pressure to prevent pressure elevations from being transmitted 
 to the downstream glomerular capillaries .
when renal 
 perfusion pressure is compromised , a sustained increase in efferent arteriolar tone 
 maintains adequate filtration pressure within the upstream glomerular capillaries , 
 thereby preserving renal function .
( 52 ) with permission . based on our studies of the effects of lc20 diphosphorylation described above
, 
 we developed a hypothesis that pathological situations of hypercontractility may result from 
 impaired relaxation due to diphosphorylation of lc20 at thr18 and ser19 .
we have 
 tested this hypothesis by studying the effects of two contractile stimuli , one physiological 
 ( ang ii ) and one pathophysiological ( et-1 ) , on renal afferent arteriolar constriction and 
 lc20 phosphorylation .
we compared the patterns of phosphorylation of 
 lc20 in the afferent arteriole to determine whether or not lc20 
 diphosphorylation is associated with the pathophysiological stimulus et-1 and not the 
 physiological stimulus ang ii .
this proved to be a challenging proposition due largely to 
 the very small size of the afferent arteriole : a single afferent arteriole has a diameter of 
 1520 m , contains < 100 smooth muscle cells and is 1/10 the size of a 
 human eyelash .
this necessitated developing a technique to isolate individual afferent 
 arterioles and enhance the sensitivity of detection and quantification of lc20 
 phosphorylation .
perfusion of the renal artery with molten agarose followed by cooling to 
 solidify the agarose enabled dissection and recovery of intact afferent arterioles ( 48 ) .
phos - tag sds - page ( 49 ) proved to be a suitable technique for rapid and efficient separation of 
 unphosphorylated and phosphorylated forms of lc20 ( 50 ) . in this technique ,
tissue proteins are separated in laemmli sds gels 
 in which a phosphate - binding ligand ( phos - tag reagent ) is immobilized in the running gel . in 
 the presence of mn ions , the migration of proteins containing phosphorylated 
 serine , threonine and/or tyrosine residues is retarded due to binding to the ligand .
the 
 higher the stoichiometry of phosphorylation , the slower the migration rate through the gel . 
 the protein of interest ( lc20 in this instance ) is then detected by western 
 blotting with an antibody that recognizes all forms of the protein , phosphorylated and 
 unphosphorylated .
the effectiveness of the separation of the various lc20 species 
 by phos - tag sds - page can be clearly seen in figs .
quantification of the different 
 lc20 species by densitometric scanning enables determination of the 
 stoichiometry of phosphorylation , and the individual phosphorylation sites can be identified 
 by using phosphorylation site - specific antibodies in parallel western blotting experiments . 

we were able to increase the sensitivity of detection of lc20 > 4,000-fold 
 over existing methods ( 50 ) .
this was achieved by : ( i ) 
 the use of biotinylated secondary antibodies in conjunction with streptavidin - conjugated 
 horseradish peroxidase , combined with enhanced chemiluminescence detection of 
 lc20 species , ( ii ) fixing the lc20 on the pvdf membrane by treatment 
 with glutaraldehyde , and ( iii ) incorporating cangetsignal ( toyobo , japan ) into 
 the protocol .
utilization of a minimum number of steps in the protocol with the least 
 possible number of sample transfers maximized lc20 yield .
the limit of detection 
 of lc20 was thereby increased from 200 fmol ( 4 ng ) to 0.05 fmol ( 1 pg ) ; we 
 estimate that a single afferent arteriole contains 2.5 fmol ( 50 pg ) of lc20 . using this approach , we succeeded in quantifying lc20 phosphorylation levels in 
 single isolated afferent arterioles and observed that ang ii induced exclusively 
 monophosphorylation of lc20 whereas et-1 induced diphosphorylation , both in a 
 time- and concentration - dependent manner ( 22 ) . 

et-1-induced diphosphorylation was confirmed to occur at thr18 and ser19 using 
 phosphorylation site - specific antibodies .
et-1-induced lc20 diphosphorylation was 
 confirmed by the proximity ligation assay ( 51 ) . 

furthermore , afferent arteriolar vasodilation ( relaxation ) occurred more slowly following 
 washout of et-1 than ang ii ( 22 ) .
these findings are , 
 therefore , consistent with the hypothesis that pathophysiological signals such as et-1 that 
 are associated with prolonged vasoconstrictor responses involve lc20 
 diphosphorylation , whereas physiological signals such as ang ii induce lc20 
 phosphorylation exclusively at ser19 .
the additional phosphorylation at thr18 induced by 
 et-1 is , therefore , proposed to account , at least in part , for the sustained contractile 
 response of the afferent arteriole to et-1 compared to ang ii .
lc20 is phosphorylated at thr18 and ser19 in a ca - independent 
 manner by ilk and/or zipk , which are associated with the contractile machinery in vascular 
 smooth muscle .
this occurs in concert with inhibition of mlcp by rok - catalysed 
 phosphorylation of mypt1 , the regulatory and targeting subunit of the phosphatase . 

diphosphorylation of lc20 occurs in response to et-1 ( pathological stimulus ) but 
 not ang ii ( physiological stimulus ) in renal afferent arterioles , and diphosphorylation of 
 lc20 is associated with decreased rates of lc20 dephosphorylation 
 and relaxation .
ilk and zipk are , therefore , potential therapeutic targets for the treatment 
 of diseases associated with hypercontractility , such as hypertension , cerebral vasospasm 
 following subarachnoid hemorrhage , coronary arterial vasospasm , intimal hyperplasia , acute 
 renal insufficiency and chronic kidney disease .
 smooth muscle contraction is activated primarily by phosphorylation at ser19 of the 
 regulatory light chain subunits ( lc20 ) of myosin ii , catalysed by 
 ca2+/calmodulin - dependent myosin light chain kinase . 
 
 ca2 + -independent contraction can be induced by inhibition of myosin light chain 
 phosphatase , which correlates with diphosphorylation of lc20 at ser19 and 
 thr18 , catalysed by integrin - linked kinase ( ilk ) and zipper - interacting protein kinase 
 ( zipk ) . lc20 
 diphosphorylation at ser19 and thr18 has been detected in 
 mammalian vascular smooth muscle tissues in response to specific contractile stimuli ( e.g. 
 endothelin-1 stimulation of rat renal afferent arterioles ) and in pathophysiological 
 situations associated with hypercontractility ( e.g. cerebral vasospasm following 
 subarachnoid hemorrhage ) . 
 comparison of the effects of lc20 monophosphorylation 
 at ser19 and diphosphorylation at ser19 and thr18 on contraction and relaxation of 
 triton - skinned rat caudal arterial smooth muscle revealed that phosphorylation at thr18 
 has no effect on steady - state force induced by ser19 phosphorylation . on the other hand , 
 
 the rates of dephosphorylation and relaxation are significantly slower following 
 diphosphorylation at thr18 and ser19 compared to monophosphorylation at ser19 . 
 we propose 
 that this diphosphorylation mechanism underlies the prolonged contractile response of 
 particular vascular smooth muscle tissues to specific stimuli , e.g. endothelin-1 
 stimulation of renal afferent arterioles , and the vasospastic behavior observed in 
 pathological conditions such as cerebral vasospasm following subarachnoid hemorrhage and 
 coronary arterial vasospasm . 
 ilk and zipk may , therefore , be useful therapeutic targets 
 for the treatment of such conditions . 
The central role of myosin regulatory light chain phosphorylation in the activation of smooth
muscle contraction
Ca
Myosin regulatory light chain diphosphorylation
Evidence of myosin regulatory light chain diphosphorylation in intact smooth muscle
tissues
The functional effects of diphosphorylation of myosin regulatory light chains
The renal microvasculature
Conclusions
Conflict of interest