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MUSCLE CELL BIOLOGY AND CELL MOTILITY
Departments of 1Biochemistry, 2Pediatrics, and 3Emergency Medicine, Virginia Commonwealth University School of Medicine, and 4Reanimation Engineering Shock Center, Virginia Commonwealth University, Richmond, Virginia
Submitted 21 October 2005 ; accepted in final form 17 January 2006
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ABSTRACT |
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-escin-permeabilized muscle, FSK and 8b-cGMP accelerated the relaxation rate when tissues were returned to pCa 9, suggesting that both agents can elevate MLCP activity. However, a component of the Ca2+ desensitization attributed to PKG activation in intact tissues appeared to involve a MLC phosphorylation-independent component. Inhibition of KCl-induced tonic force by the ROK inhibitor, Y-27632, and by PE pretreatment, were synergistically potentiated by 8b-cGMP, but not FSK. FSK and PE pretreatment, but not 8b-cGMP, inhibited the KCl-induced increase in site-specific myosin phosphatase target protein-1 phosphorylation at Thr853. These data support the hypothesis that PKA and PE pretreatment converge on a common Ca2+-desensitization pathway, but that PKG can act by a mechanism different from that activated by PKA and PE pretreatment. vascular smooth muscle; Ca2+ sensitization; RhoA kinase; signal transduction
Ca2+ sensitization of contraction can be caused by activation of ROK and PKC, and both systems appear to operate to varying degrees when VSM is stimulated by GPCRs (3) and reviewed by (6, 40, 45, 50). Because of the complexity inherent in this integrative cell signaling network, the precise mechanism(s) by which relaxant agents and contractile GPCR-induced negative feedback (i.e., arterial memory) control the degree of overall Ca2+ sensitization remains to be determined. The present study was designed to examine and compare Ca2+ desensitization causing relaxation that is induced by activation of PKA, PKG, and arterial memory, and that specifically involves alterations in the ROK signaling pathway. To achieve this end, we examined force, [Ca2+]i, MLC phosphorylation, and myosin phosphatase target protein-1 (MYPT1) site-specific phosphorylation produced by K+ depolarization (KCl) in rabbit artery. KCl has been used extensively as a smooth muscle contractile stimulus to bypasses GPCR activation and cause force development and maintenance by stimulating only increases in [Ca2+]i (see Ref. 10 for review). However, recent studies (20, 41, 42, 48) indicate that KCl also causes Ca2+ sensitization solely by activation of ROK, not by activation of both ROK and PKC like many GPCR stimuli [3, and reviewed by Ratz et al. (37)]. Results from this study will provide information that can be used in the formation of a more comprehensive and integrative model of the regulation of smooth muscle contraction.
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METHODS |
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distilled and deionized, was used throughout. The endothelium of each artery was removed by gently rubbing the intimal surface with a metal rod. Isometric force. Contractile force (F) was measured as previously described (31). Tissues were cut into 3- to 4-mm-wide artery rings and each muscle ring was secured in a tissue bath (Radnoti Glass Technology, Monrovia, CA) between stainless steel wires attached to a micrometer and isometric force transducer for, respectively, length adjustments and isometric force measurements (model 52, Harvard Apparatus, S. Natick, MA). Tissues were allowed to equilibrate for 1 h at 37°C in physiological saline solution with aeration. The muscle length for which active force was maximum (Lo) was determined for each tissue using an abbreviated length-tension curve and KCl as the stimulus (7, 35). Voltage signals from force transducers were digitized (CIO-DAS16F, Computer Boards, Middleboro, MA) and visualized on a computer screen as force (g). Data acquisition and analyses were accomplished using DasyLab (DasyTec, Amherst, NH) and Excel (Microsoft) software. For each preloaded tissue, the degree of steady-state force (F) produced at Lo by incubation in KCl for 5–10 min was equal to the optimal force (Fo) for muscle contraction, and subsequent contractions were calculated as F/Fo. No further length changes were imposed once Lo was established.
[Ca2+]i. [Ca2+]i was measured as previously described (31). Tissues at Lo in an aerated muscle chamber designed for microscopic imaging (Danish Myo Technology) were placed on the stage of an inverted microscope (Olympus IX71) and loaded for 2.5 h with 7.5 µM fura 2-PE3 (AM) and 0.01% (wt/vol) Pluronic F-127 (TefLabs, Austin, TX) to enhance solubility. Fluorescence emission at 510 nm was collected by a photomultiplier tube for excitations at 340 and 380 nm (DeltaRam V, Photon Technology International, Lawrenceville, NJ) and emission intensities were expressed as 340 nm/380 nm ratios using Felix software (Photon Technology International) to measure changes in [Ca2+]i. Background fluorescence, determined by incubating tissues in 4 mM MnCl2 plus 30 µM ionomycin, was subtracted from all 340 nm and 380 nm signals before calculating the 340 nm/380 nm fluorescence ratios.
MLC phosphorylation. Two-dimensional (isoelectric focusing/SDS-PAGE) was performed as previously described (31, 48) to measure the degree of MLC phosphorylation. Isoelectric variants of the 20-kDa light chains were detected by colloidal gold stain (Amersham) and quantified after digitization by Scion Image Software.
Western blot analysis. Phosphorylation of MYPT1 was measured by Western blot analysis using phospho-specific antibodies as described previously (34). Artery rings were quick-frozen in an acetone-dry ice slurry, thawed, homogenized in 1% SDS, 10% glycerol, 20 mM dithiothreitol, 25 mM Tris·HCl (pH 6.8), 5 mM EGTA, 1 mM EDTA, 50 mM NaF, 1 mM sodium orthovanadate, 20 mg/ml leupeptin, 2 mg/ml aprotinin, and 20 mg/ml 4-(amidino-phenyl)-methanesulfonyl fluoride, heated 10 min at 100°C, clarified by centrifugation at 5,000 g for 10 min, and stored at –70°C. Thawed homogenates were assayed for protein concentration and loaded into gel wells at 10 µg for total MYPT1 and 50 µg for phospho-MYPT1 assays. Proteins were separated by one-dimensional SDS-PAGE on 12% gels, followed by Western blot analysis onto Immobilon-P membranes (Millipore; Bedford, MA). Phosphorylated MYPT1 was identified using anti-MYPT1-p853 and anti-MYPT1-p696 antibodies (Upstate) and total MYPT1 (BD Transduction Labs) was assessed to quantify loading accuracy. Antibodies were detected using horseradish peroxidase-labeled secondary antibody (Santa Cruz) and enhanced chemiluminescence (ECL) and ECL film (Amersham). Quantification of visualized bands was obtained by digital image analysis (Scion Image software). To compensate for gel-to-gel variability and efficiencies of Western blot analysis, antibody labeling, ECL reaction, and film development, a control basal sample, was included in one lane of each gel, and band intensities from other lanes were reported as the degree of change from control basal.
Tissue permeabilization.
Artery rings at Lo were depleted of sarcoplasmic reticular Ca2+ by contracting three times with 10 µM phenylephrine (PE) in a Ca2+-free solution. The tissues were then permeabilized with 40 µM -escin at 5°C for 45 min and continued for 60 min at 30°C. The initial treatment with -escin at a low temperature helps the slow penetration and/or binding of -escin to the surface membrane of the smooth muscle cells (18). -Escin was dissolved in a "relaxing solution" contained 74.1 mM potassium methanesulfonate, 4.0 mM magnesium methanesulphonate, 4 mM Na2ATP, 4 mM EGTA, 5 mM creatine phosphate, and 30 mM PIPES, neutralized with 1 M KOH to pH 7.1 at 20°C. Ionic strength was kept constant at 0.18 M by adjusting the concentration of potassium methanesulfonate. To activate muscle contraction at Ca2+-clamped levels of 1 µM (pCa = 6) and 10 µM (pCa = 5) free Ca2+, a "contracting solution" was made by including the appropriate volume of a 1 M CaCl2 stock (Fluka), as determined using WEBMAXC (27). Calmodulin (1 µM) was added to the solutions throughout each experiment to compensate for its loss during permeabilization. To induce relaxation for relaxation velocity measurements, tissues precontracted with pCa = 6 contracting solution were rapidly washed in the relaxing solution (pCa = 9) containing 3 µM wortmannin to inhibit MLC kinase activity.
Drugs. 8-bromo-cGMP (8br-cGMP), dibutyryl cAMP (db-cAMP), nifedipine, and phentolamine were from Sigma. Forskolin (FSK), ionomycin, S-nitroso-N-acetylpenicillamine (SNAP) and Y-27632 were from Calbiochem. Fura 2-PE3 (AM) and Pluronic F-127 were from Tef Labs (Austin, TX). Nifedipine and ionomycin were dissolved in ethanol; FSK was dissolved in dimethylsulfoxide (DMSO). Ethanol and DMSO were added at a final concentration no >0.1%, a concentration that had no effect on KCl-induced contraction.
Statistics. The null hypothesis was examined using Student's t-test (when 2 groups were compared) or with one-way ANOVA. To determine differences between groups following ANOVA, the Student-Neuman-Keuls post hoc test was used. In all cases, the null hypothesis was rejected at P < 0.05. For each study described, the n value was equal to the number of rabbits from which arteries were taken.
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RESULTS |
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20–30%), whereas 10 µM FSK caused a strong inhibition of 60% (Fig. 1B). In this study and all subsequent studies, relaxant agents were added at least 20 min before and during stimulation with KCl (except for those agents shown in Figs. 2 and 7). KCl-induced peak force, however, was not inhibited even by 10 µM FSK (Fig. 1, A–C). To assess the ability of FSK and 8b-cGMP to inhibit KCl-induced increases in [Ca2+]i, tissues were contracted twice with KCl, a first time in the absence (control, C), and a second time in the presence (+relaxant, R), of FSK or 8b-cGMP (see Fig. 1D for example of protocol). Neither 10 µM FSK (Fig. 1E) nor 100 µM 8br-cGMP (Fig. 1F) produced a reduction in KCl-induced peak or tonic increases in [Ca2+]i. Likewise, the NO donor and activator of soluble guanylyl cyclase, SNAP (51), did not cause a decrease in KCl-induced tonic [Ca2+]i, even at 100 µM (Fig. 2A, example of n = 2). By comparison, and as expected, a Ca2+ channel blocker (1 µM nifedipine) caused an immediate reduction in KCl-induced tonic [Ca2+]i (Fig. 2B) and force (data not shown) in fura-loaded tissues.
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60%) reductions in KCl-induced increases in tonic (10 min) MLC phosphorylation and force (Fig. 3; open triangle). A lower FSK concentration (1 µM) also significantly reduced both KCl-induced tonic increases in MLC phosphorylation (Fig. 3, inset; F) and force, but by a lesser degree (Fig. 3, solid triangle). The effect of FSK was most likely due to increases in cAMP levels causing PKA activation because 1 mM db-cAMP, a cell-permeable activator of PKA, likewise reduced KCl-induced tonic increases in MLC phosphorylation (Fig. 3, inset; cA) and force (Fig. 3, inverted triangle). 8b-cGMP (100 µM) did not significantly reduce basal MLC phosphorylation or force (data not shown), but did reduce the average KCl-induced steady-state MLC phosphorylation level. This reduction was not significantly different than control despite the finding that the average value was close to that produced by 1 µM FSK (Fig. 3, inset; cG). When taking the reduction by 8b-cGMP in average basal MLC phosphorylation into account, 8bc-GMP reduced the average value for a KCl-stimulated tonic increase in MLC phosphorylation by only 1% compared with control. Moreover, 100 µM 8b-cGMP + 10 µM FSK (Fig. 3, solid octagon) significantly reduced the level of tonic force produced by KCl compared with that produced by 10 µM FSK alone (Fig. 3, solid triangle with center hole), but MLC phosphorylation was not further inhibited. Also, 30 µM SNAP appeared to reduce KCl-induced tonic force without reducing MLC phosphorylation (n = 2). In general, activation of PKA produced nearly equivalent reductions in MLC phosphorylation and force, and activation of PKG to a level that inhibited force by 20–30% appeared to do so, in part, through a MLC phosphorylation-independent mechanism. However, high concentrations of NO invoked by 100 µM SNAP that caused strong inhibition of force may additionally cause a reduction in MLC phosphorylation similar to that seen by activators of PKA.
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15–20 min), relaxed by addition of 3 µM Y-27632 (Fig. 4A). Y-27632 alone caused 50% relaxation of KCl-contracted artery (Fig. 4B, Con), and caused 60% relaxation of tissues contracted with KCl in the presence of FSK (Fig. 4B). This small difference in the average values of percent relaxation was not statistically significant. However, Y-27632 produced a nearly complete relaxation (>80%) of tissues contracted with KCl in the presence of 8b-cGMP (Fig. 4B; 8br-cG) that was significantly greater than the 50–60% relaxation produced by control tissues and by tissues exposed also to FSK. Moreover, relaxation in the presence of the NO donor, SNAP, was comparable to that produced in the presence of 8b-cGMP (30 µM SNAP: 76 ± 3%, 100 µM SNAP: 81 ± 2%, n = 2), while relaxation in the presence of db-cAMP was comparable to that produced in the presence of FSK (1 mM db-cAMP: 55 ± 1%, n = 2). These data support the hypothesis that PKA and Y-27632 caused Ca2+ desensitization by the same mechanism, namely, inhibition of ROK, whereas PKG can act by a pathway distinct from ROK inhibition.
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FSK (10 µM) reduced, 8b-cGMP (100 µM) had no effect on, and PE pretreatment increased, the degree of basal MYPT1-p853 (Fig. 6A), whereas only 8b-cGMP caused an elevation in the level of basal MYPT1-p696 (Fig. 6B). At 2 min of KCl-induced contraction, MYPT1-p853 was elevated above the basal level in control tissues and in tissues exposed to 8b-cGMP, but not in tissues exposed to FSK or in tissues that had been pretreated with PE (Fig. 6A). KCl did not cause an increase above the basal level in MYPT1-p696, and FSK, 8b-cGMP and PE pretreatment also exerted no effect on KCl-induced MYPT1-p696 (Fig. 6B). In summary, the pattern of MYPT1 phosphorylation was different when comparing the effects of FSK, 8b-cGMP, and PE pretreatment. However, it was apparent that both PE pretreatment and FSK, but not 8b-cGMP, prevented the KCl-induced increase of MYPT1-p853 observed at 2 min. These data are consistent with the hypothesis that PE pretreatment and PKA, but not PKG, caused Ca2+ desensitization by allowing MLC phosphatase activation through prevention of KCl-activated, MYPT1-p853-induced MLC phosphatase inhibition.
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30% (Fig. 7, B, C, and E), and compared with control (Fig. 7A), 8b-cGMP (Fig. 7B) and FSK (Fig. 7C) increased the rate of relaxation produced when tissues precontracted with pCa 6 were exposed to relaxing solution (Fig. 7, D and F). These data suggest that both FSK and 8b-cGMP caused relaxation of Ca2+activated permeabilized muscle by enhancing MLC phosphatase activity. Together, the results from this study suggest that permeabilized and intact muscle display differences in responsiveness to FSK and 8b-cGMP. |
DISCUSSION |
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/PKC
-induced Ca2+ sensitization involving CPI-17 (3). During KCl-induced contraction of rabbit artery, both FSK and 8b-cGMP caused relaxation solely by causing Ca2+ desensitization, because neither FSK nor 8b-cGMP caused reductions in stimulated [Ca2+]i, but both agents caused reductions in tonic force. Both FSK and 8b-cGMP appeared to elevate MLCP activity in permeabilized artery because both agents increased the rate of relaxation. Moreover, activation of PKA by FSK and db-cAMP, and strong activation of PKG by 100 µM SNAP, caused concomitant reductions in KCl-induced tonic increases in MLC phosphorylation and force in intact tissues. However, weaker activation of PKG by 30 µM SNAP or 100 µM 8b-cGMP, and especially by 100 µM 8b-cGMP in the presence of 10 µM FSK, caused 20–30% inhibition of KCl-induced tonic force, whereas inducing either no reduction, or less of a reduction, in MLC phosphorylation. Together, these data support studies indicating that both FSK and 8b-cGMP can cause relaxation by acting at the level of MLCP regulation (2, 24, 43, 53, 54) but introduce the idea that in intact artery stimulated with KCl, whereas FSK activates this mechanism even at 1 µM, weak activation of PKG by 8b-cGMP may cause relaxation also by acting downstream from MLCP regulation to dissociate MLC phosphorylation from tonic force. These results provide strong support for the hypothesis that, despite the potential for extensive cross-talk (see Refs. 16 and 28 for review). PKA and PKG do not necessarily converge on identical signaling pathways to cause relaxation. Although the precise mechanisms causing dissociation of MLC phosphorylation from contraction remain to be determined, both thin-filament regulatory proteins and heat shock protein-20 may play a role (22, 39, 52).
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-adrenergic receptor agonist PE) appeared to be the same mechanism used by the relaxant, FSK, and not 8b-cGMP (Fig. 8). A surprising but enlightening consequence of this convergence of Ca2+-desensitizing signals was that the delayed Ca2+ desensitization invoked in tissues by pretreatment with PE was actually reversed by simultaneous exposure of tissues to PE and the activator of PKA, FSK (Fig. 8). That is, PE pretreatment in the presence of FSK did not lead to Ca2+ desensitization during a subsequent KCl-induced contraction, as it does in the absence of FSK (see Fig. 5). Thus perhaps the most novel aspect of the present study is the revelation that VSM retains a "memory" of prior exposure to GPCR agonists and relaxant agents such that the degree of Ca2+ sensitivity produced subsequently can yield nearly the full range of contractile responses from very weak to very strong (see Fig. 5A). That is, the degree of Ca2+ sensitivity produced at any given instance reflects, for some time, the relative activity levels of prior stimulation with GPCR agonists and relaxant agents activating PKA and PKG. One implication is that, not only do the signaling systems involved in regulating Ca2+ sensitivity exist in a complex spatiotemporal organization (see Ref. 5 for review), but the temporal duration of influence on contraction is not restricted solely to the duration of exposure to the stimuli activating the mechanisms. Rather, the influence of GPCR agonists and of activators of PKA and PKG can last for some time after complete cessation of the stimulation period, when tissues are otherwise in their basal state. The consequence is an ability of VSM to produce highly variable degrees of Ca2+ sensitivity, and therefore, force (see Fig. 5A for example). Additional studies are required to identify how Ca2+ desensitizing signaling systems induced by PKA and PE pretreatment interact spatially and temporally at the subcellular level.
PKA and PKG potentially can cause reductions in ROK-induced Ca2+ sensitization (2, 13, 24, 43, 53, 54). Moreover, the present study supports this contention because FSK and 8b-cGMP did appear to increase MLCP activity in permeabilized artery. Thus, it was somewhat surprising that in KCl-stimulated intact tissues, PKG activation was found to cause relaxation, in part, by a mechanism that appeared to be independent of reductions in MLC phosphorylation levels, at least when PKG was stimulated with 100 µM 8b-cGMP or 30 µM SNAP, concentrations that induced inhibition of tonic force by 30%. However, nitrovasodilators uncouple force from MLC phosphorylation in intact swine carotid artery stimulated with histamine (19). Moreover, in support of these data, we also found that 8b-cGMP did not reduce the increase in MYPT1-p853 produced by KCl, whereas both FSK and PE pretreatment did. Although PKG (1) may cause Ca2+ desensitization by inhibiting CPI-17 phosphorylation, KCl does not elevate CPI-17 phosphorylation in rabbit femoral artery (11). Interestingly, 8b-cGMP alone elevated the basal level of MYPT1-p696, but this increase was not evident during KCl stimulation. Because phosphorylation at both MYPT1-p853 and MYPT1-p696 have been linked to MLCP inhibition (4, 8, 11, 26, 49), the significance of this increased basal MYPT1-p696 phosphorylation was unclear. One possible explanation is that the phospho-specific antibody used could not clearly discriminate between MYPT1-p696 and MYPT1-p695, and that 8b-cGMP actually caused an increase in MYPT1-p695, not MYPT1-p696. PKG does not directly inhibit MLCP activity despite phosphorylation of MYPT1, but rather, causes reductions in MLCP-plasma membrane binding (25). A recent study (53) has revealed that phosphorylation by PKA and PKG of sites adjacent to those involved in regulation of MLCP activity has no direct effect on MLCP activity, but inhibits the ability of ROK to phosphorylate the MYPT1 regulatory sites. Moreover, PKG directly binds MYPT1, and uncoupling of this interaction decreases the ability of PKG to phosphorylate MYPT1 (46). In summary, 8b-cGMP caused an increase in MYPT1-p696 (or possibly MYPT1-p695) before muscle stimulation with KCl, but this phosphorylation, as well as MYPT1-p853, was not elevated above the basal level during KCl-induced contraction in the presence of 8b-cGMP. Together, this information supports a speculative model that PKG and MYPT1 were associated before stimulation with KCl, but not during the KCl-induced contraction, and that during a KCl-induced contraction, PKA (but not PKG) can access MYPT1 to prevent MYPT1-p853, permitting MLCP activation. In any event, it is clear that a complete understanding of in situ regulation of MLCP activity will likely require a great deal of spatiotemporal information about phosphorylation status and location(s) of both catalytic and regulatory subunits and their binding partners.
In conclusion, a growing body of literature implicates altered Ca2+ sensitization and desensitization pathways in many disorders involving dysregulation of vascular smooth muscle contraction, including hypertension (47), vasospasm (17, 44), and hemorrhagic shock (55). The degree of Ca2+ sensitivity of contraction is not only dependent on the integration of signals from various contractile and relaxant stimuli, but also, as revealed by the present study, on the history of VSM stimulation. To fully understand the implications of dysregulation of Ca2+ sensitivity and its relation to certain vascular disorders, what will be required is a more complete understanding not only of direct, or immediate, stimulus-response coupling mechanisms, but also of mechanisms permitting VSM to retain information about prior stimulation, and of how such information is integrated into the spatiotemporal signaling network controlling Ca2+ sensitivity.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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) and at 10 min of a KCl-induced contraction (
), 10 µM FSK (
), 100 µM 8-bromo-cGMP (8b-cGMP; 
), 30 µM SNAP (
), 100 µM SNAP (
P < 0.05, compared with PE.
