Nitric Oxide-Induced Decrease in Calcium Sensitivity of Resistance Arteries Is Attributable to Activation of the Myosin Light Chain Phosphatase and Antagonized by the RhoA/Rho Kinase Pathway
Background— NO-induced dilations in resistance arteries (RAs) are not associated with decreases in vascular smooth muscle cell Ca2+. We tested whether a cGMP-dependent activation of the smooth muscle myosin light chain phosphatase (MLCP) resulting in a Ca2+ desensitization of the contractile apparatus was the underlying mechanism and whether it could be antagonized by the RhoA pathway.
Methods and Results— The Ca2+ sensitivity of RA was assessed as the relation between changes in diameter and [Ca2+]i in depolarized RA (120 mol/L K+) exposed to stepwise increases in Ca2+ex (0 to 3 mmol/L). Effects of 10 μmol/L sodium nitroprusside (SNP) on Ca2+ sensitivity were determined before and after application of the soluble guanylate cyclase inhibitor ODQ (1 μmol/L) and the MLCP inhibitor calyculin A (120 nmol/L) and in presence of the RhoA-activating phospholipid sphingosine-1-phosphate (S1P, 12 nmol/L). SNP-induced dilations were also studied in controls and in RAs pretreated with the Rho kinase inhibitor Y27632 or transfected with a dominant-negative RhoA mutant (N19RhoA). Constrictions elicited by increasing Ca2+ex were significantly attenuated by SNP, which, however, left associated increases in [Ca2+]i unaffected. This NO-induced attenuation was blocked by ODQ, calyculin A, and S1P. The S1P-induced translocation of RhoA indicating activation of the GTPase was not reversed by SNP. Inhibition of RhoA/Rho kinase by N19RhoA or Y27632 significantly augmented SNP-induced dilations.
Conclusions— NO dilates RA by activating the MLCP in a cGMP-dependent manner, thereby reducing the apparent Ca2+ sensitivity of the contractile apparatus. MLCP inactivation via the RhoA/Rho kinase pathway antagonizes this Ca2+-desensitizing effect that, in turn, can be restored using RhoA/Rho kinase inhibitors.
Received January 13, 2003; revision received February 27, 2003; accepted March 12, 2003.
Smooth muscle relaxation results from a decrease in intracellular free Ca2+ ([Ca2+]i) or a decrease in myofilament Ca2+ sensitivity. Most studies in large arteries and cultured vascular smooth muscle cells (VSMCs) demonstrated that NO exerted its dilatory effect by a cGMP-mediated reduction of [Ca2+]i, leading to a decreased phosphorylation of the myosin light chain (MLC20).1 However, this mechanism seems not to play a predominant role in resistance arteries (RAs). We have previously reported NO-induced dilations of hamster skeletal muscle RAs that occurred without changes in [Ca2+]i.2 In permeabilized rabbit ileum smooth muscle, Wu et al3 demonstrated dilations at constant [Ca2+]i that resulted from activation of the myosin light chain phosphatase (MLCP) and subsequently reduced MLC20 phosphorylation. Because the state of MLC20 phosphorylation controls actin-myosin interaction, any increase in MLCP activity results in a rightward shift of the [Ca2+]i constriction curve reflecting a reduction of the apparent myofilament Ca2+ sensitivity. In the aforementioned study, MLCP was activated by cGMP, the intracellular second messenger of NO. More recent studies have shown that cGMP/cGMP-dependent kinase (cGKIα)-dependent activation of MLCP also occurs in cultured mesangial,4 VSMCs,5 and intact arterial smooth muscle,6 suggesting that the NO-induced reduction of Ca2+ sensitivity observed in RA could indeed result from an increase in MLCP activity. Additional evidence for a potential role of MLCP in controlling microvascular tone comes from an earlier study in which we showed that oxidized low-density lipoproteins (oxLDLs) induced contraction by increasing the myofilament Ca2+ sensitivity via a RhoA/Rho kinase-dependent mechanism.7 Rho kinase inactivates MLCP by phosphorylation of the myosin-binding subunit (MYPT1), a process that seems to involve a ZIP-like MLCP-associated kinase.8
In this study, we investigated whether NO exerts its dilator effect in RA via an activation of MLCP and whether this effect was dependent on cGMP. We additionally studied whether a RhoA/Rho kinase-dependent inactivation of MLCP could modulate this effect of NO. Thus, in addition to the MLCP inhibitor calyculin A, the RhoA/Rho kinase-activating sphingolipid mediator sphingosine-1-phosphat (S1P) was used. To investigate the RhoA pathway, we not only used appropriate pharmacological inhibitors but also inhibited steps of the RhoA signaling cascade by transfecting the vessels with plasmids coding for the specific RhoA inhibitor C3 transferase or targeted mutations of RhoA.
We found that the Ca2+-desensitizing effect of NO was dependent on cGMP and a functionally intact MLCP. It was antagonized by S1P-induced RhoA/Rho kinase-dependent inhibition of MLCP, suggesting a critical balance in the control of MLCP between NO and RhoA/Rho kinase. The NO-antagonizing effects of RhoA/Rho kinase may contribute to the pathogenesis of hypertension and the functional impairment of endothelium-dependent dilatation.
MOPS-buffered salt solution contained (in mmol/L) NaCl 145, KCl 4.7, CaCl2 1.5, MgSO4 1.17, NaH2PO4 1.2, pyruvate 2.0, EDTA 0.02, MOPS 3.0, and glucose 5.0. In depolarizing solution with 120 mmol/L KCl, NaCl was compensatorily reduced to 29.7 mmol/L. Fura 2-AM was purchased from Molecular Probes, and norepinephrine (NE), acetylcholine (ACh), NS1619, and sodium nitroprusside (SNP) were from Sigma Chemicals. Y27632 was from Welfide Corporation. C3 transferase and N19RhoA plasmids were kindly provided by Dr Alan Hall, University College London, UK. Effectene was from Qiagen, and Trans LT was from Mobitec. Concentrations given in the text refer to final bath concentrations.
Preparation of Small RA and [Ca2+]i and Diameter Measurements
The care of the animals and the experimental procedures were in accordance with German animal protection laws. The preparation of the vessels and the technique of Ca2+ (fura 2) and diameter measurements were previously described.2,9 Briefly, RAs (maximal outer diameter, 180 to 250 μm) from gracilis muscle of female hamsters were cannulated with micropipettes and studied at 45 mm Hg transmural pressure. Fura 2 was alternately excited at 340 or 380 nm. The ratio F340 nm/F380 nm at 510 nm was calculated after subtraction of the background fluorescence (obtained after fura 2-quenching with 8 mmol/L MnCl2). Diameters were simultaneously recorded by videomicroscopy at wavelengths >610 nm to avoid interference with fura 2-measurements.
Transfection of Intact RA
To transfect plasmids containing C3 transferase or the respective mutated RhoA sequences (N19RhoA, RhoAAla-188) into VSMCs, arteries were incubated for 18 to 21 hours in an artery culture system10 with culture medium containing antibiotics, the transfectant Effectene (16 μL/mL), and 5 μg of the respective plasmid. Unspecific effects of the transfection procedure were assessed by comparing vascular responses of nontransfected RA and arteries transfected with green fluorescent protein (GFP). In arteries transfected with RhoA-GFP fusion protein, all VSMCs per microscopic field showed GFP-related fluorescence (confocal microscopy, excitation 488 nm, emission 525 to 565 nm; Figure 5b). The technique to transfect intact C3 transferase protein using trans LT was previously described.7
Immunofluorescence and Digital Imaging
Arteries were fixed with 3.7% formaldehyde, permeabilized with 0.3% Triton X-100, blocked with 1% BSA, and incubated with the primary antibody (MLCP: rabbit anti-mouse, 1:200, Covance; RhoA: mouse monoclonal, 1:200, Santa Cruz Biotechnology). FITC-labeled goat anti-rabbit or donkey anti-mouse (1:200 each) were used as secondary antibodies. Images were obtained using a Zeiss LSM410 confocal microscope equipped with a Kr/Ar laser and a 40x/1.2W water immersion objective.
Tissue samples of hamster aorta were quick-frozen in liquid nitrogen and homogenized. Cytosolic and particulate fractions were separated by centrifugation of the homogenate at 100 000g (Beckman Coulter, Optima Max-E). Pellets were resuspended in lysis buffer plus 1% Triton-X 100. Protein-matched samples were electrophoresed by SDS-PAGE (7%), transferred to nitrocellulose membranes (Amersham), and subjected to immunostaining using a polyclonal primary antibody (rabbit anti-mouse, 1:500). An HRP-labeled secondary antibody (goat anti-rabbit, 1:10 000, Santa Cruz) was used with ECLplus (Amersham) to visualize the signal.
Changes in diameter and [Ca2+]i were continuously recorded in 70 vessels from 41 animals. All vessels studied developed spontaneous tone (9.6±1% of maximal diameter). The viability of each vessel was assessed by its constriction to NE (0.3 μmol/L) and a dilation >80% in response to 1 μmol/L ACh.
The apparent Ca2+ sensitivity of the arteries was assessed by stepwise increasing the extracellular Ca2+ concentration (Ca2+ex, 0 to 3 mmol/L) around the arteries kept in depolarizing solution (120 mmol/L K+). Depolarization-dependent opening of voltage-gated calcium channels allowed increases in Ca2+ex to be reproducibly followed by increases in VSMC [Ca2+]i.7 The Ca2+ sensitivity was assessed under control conditions, in the presence of SNP and in the combined presence of SNP and the respective modulating substance or protein (ODQ, calyculin A, S1P, RhoAAla-188).
Additionally, dose-response curves for SNP were obtained in arteries preconstricted by 0.3 μmol/L NE under control conditions, in the presence of the Rho kinase inhibitor Y27632 (1 μmol/L), or in N19RhoA-transfected arteries.
Dilations are expressed as the following: percent of maximum dilation=[(diaVD−diaNE)/(diamax−diaNE)]×100, with diaVD and diaNE representing steady-state diameters 2 minutes after administration of NE or the respective vasodilator and diamax being the maximal diameter obtained in Ca2+-free 1 mmol/L EGTA-containing MOPS buffer.
Because of methodological uncertainties in calculating exact values for [Ca2+]i in intact vessels,11 fluorescence ratios (F340 nm/ F380 nm) are presented instead. Calibration curves obtained in a cell-free system indicated that the range of ratios observed here (0.4 to 6.3) fitted into the linear range of the curve that comprises physiological intracellular Ca2+ concentrations (42.2 to 1520 nmol/L).
Steady-state values from different groups were compared with ANOVA followed by post hoc analysis of the means. Data are presented as mean±SEM. Differences were considered significant at P<0.05.
Curves were compared using a nonlinear regression analysis applied first to every individual curve and then to the pooled data. Curves were considered to be different if the F-test indicated a significantly smaller sum of squares for the deviations in each individual fit compared with the deviation in the fit to the pooled data.12
NO-Induced Desensitization of the Contractile Apparatus Is Dependent on cGMP
Stepwise constrictions of K+-depolarized arteries occurring in parallel to increases in Ca2+ex were significantly attenuated in the presence of 10 μmol/L SNP (P<0.05, n=7, Figure 1). Increases in [Ca2+]i were virtually identical in control and SNP-treated arteries for any given concentration of Ca2+ex (Figure 2), suggesting that NO decreased the myofilament Ca2+ sensitivity. This NO effect was entirely mediated by cGMP because it was blocked after inhibition of the soluble guanylate cyclase by ODQ (1 μmol/L, P<0.05, n=7, Figure 1). [Ca2+]i was not significantly different in control, SNP-treated RA, or SNP/ODQ-treated RA.
MLCP Mediates the Ca2+-Desensitizing Effect of NO
The potential involvement of the MLCP in NO-induced Ca2+ desensitization was assessed in RA pretreated with the MLCP inhibitor calyculin A at a concentration (120 nmol/L) considered to be specific for the MLCP.13 Calyculin A almost abolished the desensitizing effect of NO (P<0.05, n=7, Figure 2), suggesting that this effect requires a fully functional MLCP. None of the myofilament Ca2+ sensitivity-modulating treatments affected VSMC [Ca2+]i (Figure 2).
Activation of RhoA/Rho Kinase Antagonizes NO-Induced Desensitization and Dilations
At concentrations <1 μmol/L, the sphingolipid mediator S1P induced constrictions of RA that were abolished after treatment with the RhoA inhibitor C3 transferase (n=7) or the Rho kinase inhibitor Y27632 (n=7, Table). S1P-induced activation of RhoA/Rho kinase induced a translocation of the MLCP subunit MYPT1 to the VSMC plasma membrane (Figure 3), an effect that has recently been linked to inhibition of MLCP.14 S1P-induced translocation was absent in arteries transfected with the dominant-negative RhoA mutant N19RhoA and those pretreated with Y27632 (1 μmol/L, Figure 3).
S1P (10 nmol/L, n=11), which per se increased the Ca2+ sensitivity only in a medium concentration range of Ca2+ex (0.25 to 0.75 mmol/L), abolished the NO-induced Ca2+ desensitization over the whole range of Ca2+ex (Figure 4a).
RhoA showing a cytosolic localization under resting conditions was translocated to the membrane after stimulation with 100 nmol/L S1P (Figures 4b and 4c). This translocation was not affected by subsequent addition of SNP (10 μmol/L, 3 minutes, Figure 4d).
SNP(1 μmol/L)-induced dilations after preconstriction with 1 μmol/L S1P were significantly smaller (by 64±8%, n=4) than those after preconstriction with 0.3 μmol/L NE despite virtually identical preconstriction levels (130±10 vs 128±2 μm, n=4).
Transfection of RhoAAla-188 Does Not Affect NO-Induced Ca2+-Desensitizing Effects
Recently, Sauzeau et al16 showed that in permeabilized aortic rings, SNP directly inactivated RhoA through a cGK I-mediated translocation of activated RhoA back to the cytosol. To test the involvement of this mechanism in desensitizing effects of NO in RA, intact RAs were transfected with RhoAAla-188 (n=6) that cannot be phosphorylated by cGK I. Desensitizing effects of NO were fully maintained in RhoAAla-188- transfected RA (Figure 4e). VSMC [Ca2+]i was not affected by SNP.
Inactivation of RhoA/Rho Kinase Augments Dilations Induced by NO
The Ca2+/diameter curve in C3 transferase-transfected RA was significantly shifted to the right (Figure 5a), suggesting a high basal activity of RhoA/Rho kinase.15 To test whether this high basal activity antagonized Ca2+-desensitizing and dilatory effects of NO under resting conditions, NO-induced dilations were studied in arteries treated with Y27632 (n=5) or transfected with N19RhoA (n=6). These inhibitions of RhoA/Rho kinase significantly augmented NO-induced dilations (Figure 5c).
Genetic inhibition of RhoA by transfection with N19RhoA did not affect Ca2+-dependent dilations induced by ACh (0.01 to 1 μmol/L in the presence of L-NA/indomethacin, 30 μmol/L, each, n=4, Figure 5d) or the KCa channel opener NS1619 (1 to 100 μmol/L, n=4, Figure 5d). Dilations by ACh (+L-NA/indomethacin) and NS1619 were abolished in the presence of the KCa channel inhibitor charybdotoxin (n=4 each, P<0.001).
This study demonstrates that NO decreases the myofilament Ca2+ sensitivity in VSMCs of hamster skeletal muscle RA. This effect was attributable to an activation of smooth muscle MLCP, presumably resulting in a subsequent decrease in MLC20 phosphorylation and hence a vasodilation that did not require a decrease in [Ca2+]i. Activation of the RhoA/Rho kinase pathway that has recently been shown to mediate Ca2+-sensitizing effects of a variety of agonists via inhibition of MLCP17 opposed the NO-induced desensitization. Both pathways, the Ca2+-desensitizing NO pathway as well as the Ca2+-sensitizing RhoA/Rho kinase pathway, terminate in modulation of MLCP activity,5,7 suggesting that its level of activity is a major determinant of microvascular tone.
The finding that dilations in response to NO occur independently of changes in VSMC [Ca2+]i is at variance with several studies demonstrating NO-induced decreases in [Ca2+]i. The latter were cGMP-mediated and involved various mechanisms such as activation of Ca2+-ATPases located either at the plasma membrane or intracellular Ca2+ stores, inhibition of phospholipase C or IP3 receptors, and activation of Ca2+-activated K+ channels.18 None of these mechanisms seems to be effective in the microvessels studied here, because NO-induced dilations were not associated with decreases in VSMC [Ca2+]i.2 Recent studies in other preparations also challenged the classical model of action for NO.4,6,19,20 However, the cellular mechanism of NO-induced, Ca2+-independent dilations in microvessels remains unclear. Wu et al3 demonstrated Ca2+-independent relaxations by 8-Br-cGMP in skinned rabbit ileum smooth muscle that were based on activation of MLCP. Accordingly, cGMP-dependent relaxations of mesangial cells were also shown to depend on MLCP activation.4 Recently, Surks et al5 provided a possible molecular basis demonstrating that cGKIα, which finally mediates the effects of NO and cGMP, is targeted to the contractile apparatus by a leucine zipper interaction with the MLCP myosin-binding subunit.
The results of the present study strongly suggest that the Ca2+-desensitizing effect of NO in RA was also mediated by a cGMP-dependent activation of MLCP, because inhibition of the cGMP-generating enzyme soluble guanylate cyclase by ODQ as well as MLCP inhibition by calyculin A used in a concentration shown to be specific for the SMPP-1 abolished the NO effect.
We additionally hypothesized that RhoA/Rho kinase that inhibit the MLCP via phosphorylation of MYPT1 antagonize the NO-induced Ca2+ desensitization. The phospholipid S1P known to stimulate RhoA/Rho kinase-dependent processes such as proliferation, migration, matrix reassembly, and angiogenesis 21–23 was used to activate RhoA/Rho kinase. S1P induced strong constrictions in RA that were abolished by C3 transferase or the Rho kinase inhibitor Y27632, indicating that they were mediated by RhoA/Rho kinase. Furthermore, S1P induced a translocation of MYPT1 to the plasma membrane, an effect that was absent in arteries transfected with N19RhoA or pretreated with Y27632 and, therefore, RhoA/Rho kinase dependent. The translocation of MYPT1 has recently been suggested to underlie the inhibition of MLCP.14 Indeed, NO-induced Ca2+ desensitization that depends on a functional MLCP was completely prevented in the presence of S1P.
Accordingly, the amplitude of NO-induced dilations mediated by MLCP activation was modulated by the respective constricting stimulus with significantly smaller dilations in S1P-preconstricted (activation of RhoA) than NE-preconstricted (increase in VSMC Ca2+) arteries. ACh-induced dilations (in the presence of L-NA/indomethacin EDHF-mediated and strictly Ca2+ dependent)2 that do not require an activated MLCP remained almost unaffected in S1P-preconstricted arteries. The fact that these Ca2+-dependent dilations were affected at all, albeit to a lesser extent, presumably reflects the reduced activity of MLCP in S1P-constricted arteries.
Inhibition of basal RhoA/Rho kinase activity significantly augmented NO-induced dilations. However, it did not affect Ca2+-dependent and exclusively EDHF-mediated dilations after ACh2 or dilations induced by the KCa channel opener NS1619. This suggests that even in resting VSMC, part of the desensitizing NO effect is physiologically and specifically antagonized by RhoA. In fact, there seems to be a high RhoA activity in unstimulated arteries because C3 transferase substantially decreased myofilament Ca2+ sensitivity. Virtually identical constrictions at maximal Ca2+, however, suggest that the number of potentially recruitable actin-myosin interactions were similar in control and C3 transferase-transfected arteries, arguing against unspecific cytoskeletal effects of the RhoA inhibition. Unaffected responses to NE and ACh in N19RhoA-, C3 transferase-, or Y27632-treated arteries add to this interpretation.
A high basal activity of RhoA/Rho kinase resulting in a low MLCP activity in unstimulated arteries may explain the weak effect of calyculin A on microvascular resting tone and the confinement of S1P-induced Ca2+-sensitizing effects to low [Ca2+]i concentrations. A moderately active MLCP could only antagonize the MLCK at low Ca2+ but would become increasingly ineffective with increasing [Ca2+]i and hence high MLCK activity. At this point any additional inhibition of MLCP as induced by S1P becomes irrelevant for the regulation of microvascular tone.
A physiological antagonism between NO and RhoA/Rho kinase in the microvasculature may be pathophysiologically important, because several cardiovascular diseases are associated with activation of RhoA/Rho kinase.24 Interestingly, both pathways terminate in phosphorylating MYPT15,8,17 suggesting that this protein subunit is the molecular substrate for this antagonistic crosstalk.
In contrast to our results pointing to a direct activation of MLCP underlying the NO-induced Ca2+ desensitization in RA, Sauzeau et al16 recently demonstrated direct inactivation of RhoA through NO-induced cGK1-mediated phosphorylation in large arteries. However, we found no differences in NO-induced Ca2+-desensitizing effect between control and arteries that overexpressed RhoAAla-188, a mutant that cannot be phosphorylated by cGK I, suggesting that a direct inactivation of RhoA is not effective in RA.
Interestingly, the slope of the control curve of RhoAAla-188-transfected arteries differed from that of nontransfected arteries. Unspecific effects of the transfection method are unlikely, because overexpression of GFP did not affect vascular function. A problem with mutants like RhoAAla-188 is that they function by replacing the respective native protein and overtaking its function. The point mutation in RhoAAla-188 may have decreased its biological activity because of changes in its quaternary structure. However, this does not comprise the conclusion that the desensitizing effect of NO is maintained despite overexpression of RhoAAla-188. Furthermore, immunostainings of RhoA showed S1P-induced translocation from the cytosol to the plasma membrane that could not be reversed by NO, additionally suggesting that a direct inactivation of RhoA is not mandatory for the Ca2+ desensitization in microvessels.
In summary, we demonstrated that NO decreases the Ca2+ sensitivity of the contractile apparatus in RA. This effect is dependent on cGMP and a functionally intact MLCP and can be antagonized by RhoA/Rho kinase-mediated inhibition of the MLCP. Accordingly, inhibition of RhoA/Rho kinase augments NO-induced dilations in RA. Future studies are necessary to show whether a pharmacologically reduced activity of the RhoA system can help to restore attenuated responses to NO in the microvasculature, allowing new pharmacological approaches to enhance the efficacy of NO under pathophysiological conditions.
The authors thank Sabine D’Avis for expert technical assistance and Eberhard Scholz and Johannes Veit for their enthusiastic help in performing part of the experiments during their laboratory training.
Cornwell TL, Lincoln TM. Regulation of intracellular Ca2+ levels in cultured vascular smooth muscle cells: reduction of Ca2+ by atriopeptin and 8-bromo-cyclic GMP is mediated by cyclic GMP-dependent protein kinase. J Biol Chem. 1989; 264: 1146–1155.
Torrecillas G, Diez-Marques ML, Garcia-Escribano C, et al. Mechanisms of cGMP-dependent mesangial-cell relaxation: a role for myosin light-chain phosphatase activation. Biochem J. 2000; 346 (pt 1): 217–222.
Surks HK, Mochizuki N, Kasai Y, et al. Regulation of myosin phosphatase by a specific interaction with cGMP- dependent protein kinase Ialpha. Science. 1999; 286: 1583–1587.
Etter EF, Eto M, Wardle RL, et al. Activation of myosin light chain phosphatase in intact arterial smooth muscle during nitric oxide-induced relaxation. J Biol Chem. 2001; 276: 34681–34685.
Bolz SS, Galle J, Derwand R, et al. Oxidized LDL increases the sensitivity of the contractile apparatus in isolated resistance arteries for Ca2+ via a rho- and rho kinase-dependent mechanism. Circulation. 2000; 102: 2402–2410.
MacDonald JA, Borman MA, Muranyi A, et al. Identification of the endogenous smooth muscle myosin phosphatase-associated kinase. Proc Natl Acad Sci U S A. 2001; 98: 2419–2424.
Bolz SS, Fisslthaler B, Pieperhoff S, et al. Antisense oligonucleotides against cytochrome P450 2C8 attenuate EDHF-mediated Ca(2+) changes and dilation in isolated resistance arteries. FASEB J. 2000; 14: 255–260.
Bolz SS, Pieperhoff S, de Wit C, et al. Intact endothelial and smooth muscle function in small resistance arteries after 48 h in vessel culture. Am J Physiol Heart Circ Physiol. 2000; 279: H1434–H1439.
Motulsky HJ, Ransnas LA. Fitting curves to data using nonlinear regression: a practical and nonmathematical review. FASEB J. 1987; 1: 365–374.
Shin HM, Je HD, Gallant C, et al. Differential association and localization of myosin phosphatase subunits during agonist-induced signal transduction in smooth muscle. Circ Res. 2002; 90: 546–553.
Gong MC, Fujihara H, Somlyo AV, et al. Translocation of rhoA associated with Ca2+ sensitization of smooth muscle. J Biol Chem. 1997; 272: 10704–10709.
Sauzeau V, Le JH, Cario-Toumaniantz C, et al. Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle. J Biol Chem. 2000; 275: 21722–21729.
Kimura K, Ito M, Amano M, et al. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science. 1996; 273: 245–248.
Pabelick CM, Warner DO, Perkins WJ, et al. S-nitrosoglutathione-induced decrease in calcium sensitivity of airway smooth muscle. Am J Physiol Lung Cell Mol Physiol. 2000; 278: L521–L527.
Wang F, Van BJ, Hobson JP, et al. Sphingosine 1-phosphate stimulates cell migration through a G(i)-coupled cell surface receptor: potential involvement in angiogenesis. J Biol Chem. 1999; 274: 35343–35350.