Cellular regulation of basal tone in internal anal sphincter smooth muscle by RhoA/ROCK

Sustained contractions of smooth muscle cells (SMC) maintain basal tone in the internal anal sphincter (IAS). To examine the molecular bases for the myogenic tone in the IAS, the present studies focused on the role of RhoA/ROCK in the SMC isolated from the IAS vs. the adjoining phasic tissues of the rectal smooth muscle (RSM) and anococcygeus smooth muscle (ASM) of rat. We also compared cellular distribution of RhoA/ROCK, levels of RhoA-GTP, RhoA-Rho guanine nucleotide dissociation inhibitor (GDI) complex formation, levels of p(Thr696)-MYPT1, and SMC relaxation caused by RhoA inhibition. Levels of RhoA/ROCK were higher at the cell membrane in the IAS SMC compared with those from the RSM and ASM. C3 exoenzyme (RhoA inhibitor) and Y 27632 (ROCK inhibitor) caused a concentration-dependent relaxation of the IAS SMC. In addition, active ROCK-II (primary isoform of ROCK in SMC) caused further shortening in the IAS SMC. C3 exoenzyme increased RhoA-RhoGDI binding and reduced the levels of RhoA-GTP and p(Thr696)-MYPT1. ROCK inhibitor attenuated PKC-induced contractions in IAS SMC. Conversely, a PKC inhibitor (G? 6850, which causes partial relaxation of the SMC) had no significant effect on ROCK-II-induced contractions. Further experiments showed the highest levels of RhoA, active form of RhoA (RhoA-GTP), ROCK-II, 20-kDa myosin regulatory light chain (MLC(20)), phospho-MYPT1, and phospho-MLC(20) in the IAS vs. RSM and ASM SMC. However, the trend was the reverse with the levels of inactive RhoA (GDP-RhoA-RhoGDI complex) and MYPT1. We conclude that RhoA/ROCK play a critical role in maintenance of spontaneous tone in the IAS SMC via inhibition of myosin light chain phosphatase.     

RhoA/ROCK pathway is the major molecular determinant of basal tone in intact human internal anal sphincter

The knowledge of molecular control mechanisms underlying the basal tone in the intact human internal anal sphincter (IAS) is critical for the pathophysiology and rational therapy for a number of debilitating rectoanal motility disorders. We determined the role of RhoA/ROCK and PKC pathways by comparing the effects of ROCK- and PKC-selective inhibitors Y 27632 and G? 6850 (10(-8) to 10(-4) M), respectively, on the basal tone in the IAS vs. the rectal smooth muscle (RSM). Western blot studies were performed to determine the levels of RhoA/ROCK II, PKC-α, MYPT1, CPI-17, and MLC(20) in the unphosphorylated and phosphorylated forms, in the IAS vs. RSM. Confocal microscopic studies validated the membrane distribution of ROCK II. Finally, to confirm a direct relationship, we examined the enzymatic activities and changes in the basal IAS tone and p-MYPT1, p-CPI-17, and p-MLC(20), before and after Y 27632 and G? 6850. Data show higher levels of RhoA/ROCK II and related downstream signal transduction proteins in the IAS vs. RSM. In addition, data show a significant correlation between the active RhoA/ROCK levels, ROCK enzymatic activity, downstream proteins, and basal IAS tone, before and after ROCK inhibitor. From these data we conclude 1) RhoA/ROCK and downstream signaling are constitutively active in the IAS, and this pathway (in contrast with PKC) is the critical determinant of the basal tone in intact human IAS; and 2) RhoA and ROCK are potential therapeutic targets for a number of rectoanal motility disorders for which currently there is no satisfactory treatment.     

RhoA- and PKC-alpha-mediated phosphorylation of MYPT and its association with HSP27 in colonic smooth muscle cells

Agonist-induced activation of the RhoA/Rho kinase (ROCK) pathway results in inhibition of myosin phosphatase and maintenance of myosin light chain (MLC20) phosphorylation. We have shown that RhoA/ROCKII translocates and associates with heat shock protein (HSP)27 in the particulate fraction. We hypothesize that inhibition of the 130-kDa regulatory myosin-binding subunit (MYPT) requires its association with HSP27 in the particulate fraction. Furthermore, it is not certain whether regulation of MYPT by CPI-17 or by ROCKII is due to cross talk between RhoA and PKC-alpha. Presently, we examined the cross talk between RhoA and PKC-alpha in the regulation of MYPT phosphorylation in rabbit colon smooth muscle cells. Acetylcholine induced 1) sustained phosphorylation of PKC-alpha, CPI-17, and MYPT; 2) an increase in the association of phospho-MYPT with HSP27 in the particulate fraction; 3) a decrease in myosin phosphatase activity (66.21+/-3.52 and 42.19+/-3.85% nM/ml lysate at 30 s and 4 min); and 4) an increase in PKC activity (298.12+/-46.60% and 290.59+/-22.07% at 30 s and 4 min). Inhibition of RhoA/ROCKII by Y-27632 inhibited phosphorylation of MYPT and its association with HSP27. Both Y27632 and a negative dominant construct of RhoA inhibited phosphorylation of MYPT and CPI-17. Inhibition of PKCs or calphostin C or selective inhibition of PKC-alpha by negative dominant constructs inhibited phosphorylation of MYPT and CPI-17. The results suggest that 1) acetylcholine induces activation of both RhoA and/or PKC-alpha pathways, suggesting cross talk between RhoA and PKC-alpha resulting in phosphorylation of MYPT, inhibition of myosin phosphatase activity, and maintenance of MLC phosphorylation; and 2) phosphorylated MYPT is associated with HSP27 and translocated to the particulate fraction, suggesting a scaffolding role for HSP27 in mediating the association of the complex MYPT/RhoA-ROCKII. Thus both pathways (PKC and RhoA) converge on the regulation of myosin phosphatase activities and modulate sustained phosphorylation of MLC20.     

Involvement of rho and rho-associated kinase in sphincteric smooth muscle contraction by angiotensin II

The tonic smooth muscles of lower esophageal sphincter (LES) and internal anal sphincter (IAS) are subject to modulation by the neurohumoral agents. We report that angiotensin (Ang) II-induced contraction of rat IAS and LES smooth muscle cells (SMC) was inhibited by Clostridium botulinum C3 exozyme, HA 1077 and Y 27632, suggesting a role for Rho kinase and a Rho-associated kinase (ROK). Ang II-induced contraction of the SMC was also attenuated by genistein, antibodies to the pp60(c-src), p(190) RhoGTPase-activating protein (p190 RhoGAP), carboxyl terminus of Galpha13, carboxyl terminus peptide, and ADP ribosylation factor (ARF) antibody. Ang II-induced increase in p(190) RhoGAP tyrosine phosphorylation was attenuated by genistein. Furthermore, Ang II-induced increase in smooth muscle tone and phosphorylation of myosin light chain (MLC; 20 kDa; MLC20-P) were attenuated by Y 27632 and genistein. The results suggest an important role for Galpha13 and pp60(c-src) in the intracellular events responsible for the activation of RhoA/ROK in Ang II-induced contraction of LES and IAS SMC.     

Ectopic expression of caveolin-1 restores physiological contractile response of aged colonic smooth muscle

Reduced colonic motility has been observed in aged rats with a parallel reduction in acetylcholine (ACh)-induced myosin light chain (MLC(20)) phosphorylation. MLC(20) phosphorylation during smooth muscle contraction is maintained by a coordinated signal transduction cascade requiring both PKC-alpha and RhoA. Caveolae are membrane microdomains that permit rapid and efficient coordination of different signal transduction cascades leading to sustained smooth muscle contraction of the colon. Here, we show that normal physiological contraction can be reinstated in aged colonic smooth muscle cells (CSMCs) upon transfection with wild-type caveolin-1 through the activation of both the RhoA/Rho kinase and PKC pathways. Our data demonstrate that impaired contraction in aging is an outcome of altered membrane translocation of PKC-alpha and RhoA with a concomitant reduction in the association of these molecules with the caveolae-specific protein caveolin-1, resulting in a parallel decrease in the myosin phosphatase-targeting subunit (MYPT) and CPI-17 phosphorylation. Decreased MYPT and CPI-17 phosphorylation activates MLC phosphatase activity, resulting in MLC(20) dephosphorylation, which may be responsible for decreased colonic motility in aged rats. Importantly, transfection of CSMCs from aged rats with wild-type yellow fluorescent protein-caveolin-1 cDNA restored translocation of RhoA and PKC-alpha and phosphorylation of MYPT, CPI-17, and MLC(20), thereby restoring the contractile response to levels comparable with young adult rats. Thus, we propose that caveolin-1 gene transfer may represent a promising therapeutic treatment to correct the age-related decline in colonic smooth muscle motility.     

Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo

Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca(2+) concentration ([Ca(2+)](i)). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca(2+)](i) with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rho-kinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.     

G(q)-dependent signalling by the lysophosphatidic acid receptor LPA(3) in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA

The present study characterized the signalling pathways initiated by the bioactive lipid, LPA (lysophosphatidic acid) in smooth muscle. Expression of LPA(3) receptors, but not LPA(1) and LPA(2), receptors was demonstrated by Western blot analysis. LPA stimulated phosphoinositide hydrolysis, PKC (protein kinase C) and Rho kinase (Rho-associated kinase) activities: stimulation of all three enzymes was inhibited by expression of the G(alphaq), but not the G(alphai), minigene. Initial contraction and MLC(20) (20 kDa regulatory light chain of myosin II) phosphorylation induced by LPA were abolished by inhibitors of PLC (phospholipase C)-beta (U73122) or MLCK (myosin light-chain kinase; ML-9), but were not affected by inhibitors of PKC (bisindolylmaleimide) or Rho kinase (Y27632). In contrast, sustained contraction, and phosphorylation of MLC(20) and CPI-17 (PKC-potentiated inhibitor 17 kDa protein) induced by LPA were abolished selectively by bisindolylmaleimide. LPA-induced activation of IKK2 {IkappaB [inhibitor of NF-kappaB (nuclear factor kappaB)] kinase 2} and PKA (protein kinase A; cAMP-dependent protein kinase), and degradation of IkappaBalpha were blocked by the RhoA inhibitor (C3 exoenzyme) and in cells expressing dominant-negative mutants of IKK2(K44A) or RhoA(N19RhoA). Phosphorylation by Rho kinase of MYPT1 (myosin phosphatase targeting subunit 1) at Thr(696) was masked by phosphorylation of MYPT1 at Ser(695) by PKA derived from IkappaB degradation via RhoA, but unmasked in the presence of PKI (PKA inhibitor) or C3 exoenzyme and in cells expressing IKK2(K44A). We conclude that LPA induces initial contraction which involves activation of PLC-beta and MLCK and phosphorylation of MLC(20), and sustained contraction which involves activation of PKC and phosphorylation of CPI-17 and MLC(20). Although Rho kinase was activated, phosphorylation of MYPT1 at Thr(696) by Rho kinase was masked by phosphorylation of MYPT1 at Ser(695) via cAMP-independent PKA derived from the NF-kappaB pathway.     

Signaling pathways mediating gastrointestinal smooth muscle contraction and MLC20 phosphorylation by motilin receptors

The signaling cascades initiated by motilin receptors in gastric and intestinal smooth muscle cells were characterized. Motilin bound with high affinity (IC(50) 0.7 +/- 0.2 nM) to receptors on smooth muscle cells; the receptors were rapidly internalized via G protein-coupled receptor kinase 2 (GRK2). Motilin selectively activated G(q) and G(13), stimulated G alpha(q)-dependent phosphoinositide (PI) hydrolysis and 1,4,5-trisphosphate (IP(3))-dependent Ca(2+) release, and increased cytosolic free Ca(2+). PI hydrolysis was blocked by expression of G alpha(q) minigene and augmented by overexpression of dominant negative RGS4(N88S) or GRK2(K220R). Motilin induced a biphasic, concentration-dependent contraction (EC(50) = 1.0 +/- 0.2 nM), consisting of an initial peak followed by a sustained contraction. The initial Ca(2+)-dependent contraction and myosin light-chain (MLC)(20) phosphorylation were inhibited by the PLC inhibitor U-73122 and the MLC kinase inhibitor ML-9 but were not affected by the Rho kinase inhibitor Y27632 or the PKC inhibitor bisindolylmaleimide. Sustained contraction and MLC(20) phosphorylation were RhoA dependent and mediated by two downstream messengers: PKC and Rho kinase. The latter was partly inhibited by expression of G alpha(q) or G alpha(13) minigene and abolished by coexpression of both minigenes. Sustained contraction and MLC(20) phosphorylation were partly inhibited by Y27632 and bisindolylmaleimide and abolished by a combination of both inhibitors. The inhibition reflected phosphorylation of two MLC phosphatase inhibitors: CPI-17 via PKC and MYPT1 via Rho kinase. We conclude that motilin initiates a G alpha(q)-mediated cascade involving Ca(2+)/calmodulin activation of MLC kinase and transient MLC(20) phosphorylation and contraction as well as a sustained G alpha(q)- and G alpha(13)-mediated, RhoA-dependent cascade involving phosphorylation of CPI-17 by PKC and MYPT1 by Rho kinase, leading to inhibition of MLC phosphatase and sustained MLC(20) phosphorylation and contraction.     

Bioengineered human IAS reconstructs with functional and molecular properties similar to intact IAS

Because of its critical importance in rectoanal incontinence, we determined the feasibility to reconstruct internal anal sphincter (IAS) from human IAS smooth muscle cells (SMCs) with functional and molecular attributes similar to the intact sphincter. The reconstructs were developed using SMCs from the circular smooth muscle layer of the human IAS, grown in smooth muscle differentiation media under sterile conditions in Sylgard-coated tissue culture plates with central Sylgard posts. The basal tone in the reconstructs and its changes were recorded following 0 Ca(2+), KCl, bethanechol, isoproterenol, protein kinase C (PKC) activator phorbol 12,13-dibutyrate, and Rho kinase (ROCK) and PKC inhibitors Y-27632 and G?-6850, respectively. Western blot (WB), immunofluorescence (IF), and immunocytochemical (IC) analyses were also performed. The reconstructs developed spontaneous tone (0.68 ± 0.26 mN). Bethanechol (a muscarinic agonist) and K(+) depolarization produced contraction, whereas isoproterenol (β-adrenoceptor agonist) and Y-27632 produced a concentration-dependent decrease in the tone. Maximal decrease in basal tone with Y-27632 and G?-6850 (each 10(-5) M) was 80.45 ± 3.29 and 17.76 ± 3.50%, respectively. WB data with the IAS constructs' SMCs revealed higher levels of RhoA/ROCK, protein kinase C-potentiated inhibitor or inhibitory phosphoprotein for myosin phosphatase (CPI-17), phospho-CPI-17, MYPT1, and 20-kDa myosin light chain vs. rectal smooth muscle. WB, IF, and IC studies of original SMCs and redispersed from the reconstructs for the relative distribution of different signal transduction proteins confirmed the feasibility of reconstruction of IAS with functional properties similar to intact IAS and demonstrated the development of myogenic tone with critical dependence on RhoA/ROCK. We conclude that it is feasible to bioengineer IAS constructs using human IAS SMCs that behave like intact IAS.     

Uncoupling of GPCR and RhoA-induced Ca2+-sensitization of chicken amnion smooth muscle lacking CPI-17

Ca2+-sensitization of smooth muscle occurs through inhibition of myosin light chain phosphatase (MLCP) leading to an increase in the MLCK:MLCP activity ratio. MLCP is inhibited through phosphorylation of its regulatory subunit (MYPT-1) following activation of the RhoA/Rho kinase (ROK) pathway or through phosphorylation of the PP1c inhibitory protein, CPI-17, by PKC delta or ROK. Here, we explore the crosstalk between these two modes of MLCP inhibition in a smooth muscle of a natural CPI-17 knockout, chicken amnion. GTPgammaS elicited Ca2+-sensitized force which was relaxed by GDI or Y-27632, however, U46619, carbachol and phorbol ester failed to induce Ca2+-sensitized force, but were rescued by recombinant CPI-17, and were sensitive to Y-27632 inhibition. In the presence, but not absence, of CPI-17, U46619 also significantly increased GTP.RhoA. There was no affect on MYPT-1 phosphorylation at T695, however, T850 phosphorylation increased in response to GTPgammaS stimulation. Together, these data suggest a role for CPI-17 upstream of RhoA activation possibly through activation of another PP1 family member targeted by CPI-17.     

Increased contractility of hepatic stellate cells in cirrhosis is mediated by enhanced Ca2+-dependent and Ca2+-sensitization pathways

Activation of hepatic stellate cells (HSCs) results in cirrhosis and portal hypertension due to intrahepatic resistance. Activated HSCs increase their contraction after receptor agonist stimulation; however, the signaling pathways for the regulation of contraction are not fully understood. The aim of this study was to elucidate the change in contractile mechanisms of HSCs after cirrhotic activation. The expression pattern of contractile regulatory proteins was analyzed with quantitative RT-PCR and Western blotting. The phosphorylation levels of myosin light chain (MLC), 17-kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17), and MLC phosphatase targeting subunit 1 (MYPT1) after endothelin-1 (ET-1) stimulation in culture-activated HSCs were measured using phosphorylation-specific antibodies. In vivo-activated HSCs were isolated from rats subjected to bile duct ligation and repeated dimethylnitrosoamine injections. HSCs showed increased expression of not only α-smooth muscle actin, but also the contractile regulatory proteins MLC kinase (MLCK), Rho kinase 2 (ROCK2), and CPI-17 during HSC activation in vitro. In culture-activated HSCs, ET-1 increased phosphorylation of CPI-17 at Thr18, which was markedly inhibited by the PKC inhibitor Ro-31-8425. ET-1 induced phosphorylation of MYPT1 at Thr853, which was suppressed by the ROCK inhibitor Y-27632. ET-1 induced sustained phosphorylation of MLC at Thr18/Ser19, which was inhibited by both Ro-31-8425 and Y-27632. Consistent with the data obtained from the in vitro study, HSCs isolated from cirrhotic rats showed increased expression of α-smooth muscle actin, MLCK, CPI-17, and ROCK2 compared with HSCs from nontreated rats. Furthermore, MLC phosphorylation in in vivo-activated HSCs was increased, according to enhanced phosphorylation of CPI-17 and MYPT1 in the presence of ET-1. These results suggest that activated HSCs may participate in constriction of hepatic sinusoids in the cirrhotic liver through both Ca(2+)-dependent (MLCK pathway) and Ca(2+)-sensitization mechanism (CPI-17 and MYPT1 pathways).     

RhoA-Rho kinase pathway mediates thrombin- and U-46619-induced phosphorylation of a myosin phosphatase inhibitor, CPI-17, in vascular smooth muscle cells

Protein kinase C-potentiated phosphatase inhibitor of 17 kDa (CPI-17) mediates some agonist-induced smooth muscle contraction by suppressing the myosin phosphatase in a phosphorylation-dependent manner. The physiologically relevant kinases that phosphorylate CPI-17 remain to be identified. Several previous studies have shown that some agonist-induced CPI-17 phosphorylation in smooth muscle tissues was attenuated by the Rho kinase (ROCK) inhibitor Y-27632, suggesting that ROCK is involved in agonist-induced CPI-17 phosphorylation. However, Y-27632 has recently been found to inhibit protein kinase C (PKC)-, a well-recognized CPI-17 kinase. Thus the role of ROCK in agonist-induced CPI-17 phosphorylation remains uncertain. The present study was designed to address this important issue. We selectively activated the RhoA pathway using inducible adenovirus-mediated expression of a constitutively active mutant RhoA (V14RhoA) in primary cultured rabbit aortic vascular smooth muscle cells (VSMCs). V14RhoA caused expression level-dependent CPI-17 phosphorylation at Thr38 as well as myosin phosphatase phosphorylation at Thr853. Importantly, we have shown that V14RhoA-induced CPI-17 phosphorylation was not affected by the PKC inhibitor GF109203X but was abolished by Y-27632, suggesting that ROCK but not PKC was involved. Furthermore, we have shown that the contractile agonists thrombin and U-46619 induced CPI-17 phosphorylation in VSMCs. Similarly to V14RhoA-induced CPI-17 phosphorylation, thrombin-induced CPI-17 phosphorylation was not affected by inhibition of PKC with GF109203X, but it was blocked by inhibition of RhoA with adenovirus-mediated expression of exoenzyme C3 as well as by Y-27632. Taken together, our present data provide the first clear evidence indicating that ROCK is responsible for thrombin- and U-46619-induced CPI-17 phosphorylation in primary cultured VSMCs. protein kinase C; signal transduction; adenovirus     

PHI-1 induced enhancement of myosin phosphorylation in chicken smooth muscle

Herein, we provide evidence that in chicken smooth muscle, G-protein stimulation by a Rho-kinase pathway leads to an increase in myosin light chain phosphorylation. Additionally, G-protein stimulation did not increase MYPT1 phosphorylation at Thr695 or Thr850, and CPI-17, was not expressed in chicken smooth muscle. However, PHI-1 was present in chicken smooth muscle tissues. Both agonist and GTP(gamma)S stimulation result in an increase in PHI-1 phosphorylation, which is inhibited by inhibitors to both Rho-kinase (Y-27632) and (PKC) GF109203x. These data suggest that PHI-1 may act as a CPI-17 analog in chicken smooth muscle and inhibit myosin phosphatase activity during G-protein stimulation to produce Ca2+ sensitization.     

Acetylcholine-induced phosphorylation of CPI-17 in rat bronchial smooth muscle: the roles of Rho-kinase and protein kinase C

It has been demonstrated that CPI-17 provokes an inhibition of myosin light chain phosphatase to increase myosin light chain phosphorylaton and Ca(2+) sensitivity during contraction of vascular smooth muscle. However, expression and agonist-mediated regulation of CPI-17 in bronchial smooth muscle have not been documented. Thus, expression and phosphorylation of CPI-17 mediated by PKC and ROCK were investigated using rat bronchial preparations. Acetylcholine (ACh)-induced contraction and Ca(2+) sensitization were both attenuated by 10(-6) mol Y-27632 /L, a ROCK inhibitor, 10(-6) mol calphostin C/L, a PKC inhibitor, and their combination. A PKC activator, PDBu, induced a Ca(2+) sensitization in alpha-toxin-permeabilized bronchial smooth muscle. In this case, the Ca(2+) sensitizing effect was significantly inhibited by caphostin C but not by Y-27632. An immunoblot study demonstrated CPI-17 expression in the rat bronchial smooth muscle. Acetylcholine induced a phosphorylation of CPI-17 in a concentration-dependent manner, which was significantly inhibited by Y-27632 and calphostin C. In conclusion, these data suggest that both PKC and ROCK are involved in force development, Ca(2+) sensitization, and CPI-17 phosphorylation induced by ACh stimulation in rat bronchial smooth muscle. As such, RhoA/ROCK, PKC/CPI-17, and RhoA/ROCK/CPI pathways may play important roles in the ACh-induced Ca(2+) sensitization of bronchial smooth muscle contraction.     

Selectivity of ROCK inhibitors in the spontaneously tonic smooth muscle

The selectivity of different Rho kinase (ROCK) inhibitors in the spontaneously tonic smooth muscle has not been investigated. We examined this issue using Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarbox anecarboxamide, 2HCl], H-1152 [(S)-(+)-(2-methyl-5-isoquinolinyl) sulfonylhomopiperazine, 2HCl], HA-1077 [(5 isoquinolinesulfonyl) homopiperazine, 2HCl], and ROCK inhibitor II [N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea]. We compared these inhibitors in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). ROCK, protein kinase C (PKC), and myosin light chain kinase (MLCK) activities were determined in the IAS, before and after different ROCK inhibitors. Y-27632 and H-1152 were approximately 30-fold more potent in the IAS (IC(50): 4.4 x 10(-7) and 7.9 x 10(-8) M, respectively) vs. the phasic rectal smooth muscle (RSM) (IC(50): 1.3 x 10(-5) and 2.5 x 10(-6) M, respectively). HA-1077 and ROCK inhibitor II were equipotent in the IAS vs. RSM. In the IAS, H-1152 was the most potent whereas ROCK inhibitor II is the least. Y-27632 and H-1152 caused concentration-dependent decrease in the IAS tone that correlates directly with the decreases in ROCK activity, without significant effect in the PKC and MLCK activities. This specifically selective correlation between ROCK activity and decrease in the IAS tone was absent in the case of HA-1077 and ROCK inhibitor II, which also inhibited PKC and MLCK. We conclude that the IAS tone is critically dependent on ROCK activity, and H-1152 and Y-27632 are the most selective and potent ROCK inhibitors in the IAS.     

Length-dependent regulation of basal myosin phosphorylation and force in detrusor smooth muscle

Urinary bladder (detrusor) smooth muscle is active in the absence of an external stimulus. Tone occurs even "at rest" during the filling phase, and it is elevated in patients with overactive bladder. This study examined the role of muscle length on tone and the level of basal myosin light chain phosphorylation (MLC(20P)). MLC(20P) was 23.9 +/- 1% (n = 58) at short lengths (zero preload; L(z)). An increase in length from L(z) to the optimal length for contraction (L(o)) caused a reduction in MLC(20P) to 15.8 +/- 1% (n = 49). Whereas 10 microM staurosporine reduced MLC(20P) at L(z), 1 microM staurosporine, a Ca(2+)-free solution, and inhibitors of MLC kinase, protein kinase C (PKC) and RhoA kinase (ROK) did not. However, 1 microM staurosporine and inhibitors of ROK inhibited MLC(20P) and tone at L(o). These data support the hypothesis that a Ca(2+)-independent kinase, possibly ZIP-like kinase, regulates MLC(20P) at L(z), whereas in detrusor stretched to L(o), additional kinases, such as ROK, participate.     

Gq/G13 signaling by ET-1 in smooth muscle: MYPT1 phosphorylation via ETA and CPI-17 dephosphorylation via ETB

We analyzed the signaling pathways initiated by endothelin receptors ET_A and ET_B in intestinal circular and longitudinal smooth muscle cells. The response to endothelin-1 (ET-1) consisted of two phases in both cell types. The initial, transient phase of contraction and phosphorylation of 20-kDa myosin light chain (MLC₂₀) was mediated additively by ET_A and ET_B receptors and initiated by G_q-, Ca²⁺/calmodulin-dependent activation of MLC kinase. In contrast, the sustained phase was mediated selectively by ET_A receptors via a pathway involving sequential activation of G₁₃, RhoA, and Rho kinase, resulting in phosphorylation of MYPT1 at Thr⁶⁹⁶ and phosphorylation of MLC₂₀. Although PKC was activated, CPI-17 was not phosphorylated and hence did not contribute to inhibition of MLC phosphatase. The absence of CPI-17 phosphorylation by PKC reflected active dephosphorylation of CPI-17 by protein phosphatase 2A (PP2A). PP2A was activated via a pathway involving ET_B-dependent stimulation of p38 MAPK activity. CPI-17 phosphorylation was unmasked in the presence of the ET_B antagonist BQ-788, but not the ET_A antagonist BQ-123, and in the presence of a low concentration of okadaic acid, which selectively inactivates PP2A. The resultant phosphorylation of CPI-17 was blocked by bisindolylmaleimide, providing direct confirmation that it was PKC dependent. We conclude that the two phases of the intestinal smooth muscle response to ET-1 involve distinct receptors, G proteins, and signaling pathways. The sustained response is mediated via selective ET_A-dependent phosphorylation of MYPT1. In contrast, ET_B initiates an inhibitory pathway involving p38 MAPK-dependent activation of PP2A that causes dephosphorylation of CPI-17. endothelin receptor type A; endothelin receptor type B; myosin phosphatase targeting subunit     

The importance of Rho-associated kinase-induced Ca2+ sensitization as a component of electromechanical and pharmacomechanical coupling in rat ureteric smooth muscle

Ureteric peristalsis, which occurs via alternating contraction and relaxation of ureteric smooth muscle, ensures the unidirectional flow of urine from the kidney to the bladder. Understanding of the molecular mechanisms underlying ureteric excitation–contraction coupling, however, is limited. To address these knowledge deficits, and in particular to test the hypothesis that Ca²⁺ sensitization via activation of the RhoA/Rho-associated kinase (ROK) pathway plays an important role in ureteric smooth muscle contraction, we carried out a thorough characterization of the electrical activity, Ca²⁺ signaling, MYPT1 (myosin targeting subunit of myosin light chain phosphatase, MLCP) and myosin regulatory light chain (LC₂₀) phosphorylation, and force responses to membrane depolarization induced by KCl (electromechanical coupling) and carbachol (CCh) (pharmacomechanical coupling). The effects of ROK inhibition on these parameters were investigated. We conclude that the tonic, but not the phasic component of KCl- or CCh-induced ureteric smooth muscle contraction is highly dependent on ROK-catalyzed phosphorylation of MYPT1 at T855, leading to inhibition of MLCP and increased LC₂₀ phosphorylation.     

Role of phospholipase A2 (group I secreted) in the genesis of basal tone in the internal anal sphincter smooth muscle

The role of phospholipase A(2) (PLA(2)) in the genesis of basal tone in the internal anal sphincter (IAS) is not known. We determined the effects of PLA(2) and inhibitors on the basal tone and intraluminal pressures (IASP) in the rat IAS vs. rectal smooth muscles (RSM). In addition, we determined the correlations between the IAS tone, PLA(2) levels, and the actual enzymatic activity. Inhibition of PLA(2) by 4-bromophenacyl bromide (universal inhibitor of PLA(2)) and MJ33 [selective inhibitor of secreted isoform of PLA(2) (sPLA(2))] caused concentration-dependent decrease in the IAS tone and in the IASP. Maximal decreases in the IAS tone and IASP by 4-bromophenacyl bromide and MJ33 were 58.8 +/- 6.9 and 51.5 +/- 6.3%, and 66.7 +/- 5.1 and 79.8 +/- 8.2%, respectively. The sPLA(2) inhibitors were approximately 100 times more potent in decreasing the IASP than the mean blood pressure. Conversely, the selective inhibitors of the cytosolic and calcium-independent PLA(2) arachidonyl trifluoromethyl ketone and bromoenol lactone, respectively, produced no significant effect. The IAS had characteristically higher levels of sPLA(2) activity (26.5 +/- 4.9 micromol.min(-1).ml(-1)) vs. the RSM (3.2 +/- 0.4 micromol.min(-1).ml(-1)), and higher levels of sPLA(2) as shown by Western blot and RT-PCR. Interestingly, administration of sPLA(2) transformed RSM into the tonic smooth muscle like that of the IAS: it developed basal tone and relaxed in response to the electrical field stimulation. From the present data, we conclude that sPLA(2) plays a critical role in the genesis of tone in the IAS. PLA(2) inhibitors may provide potential therapeutic target for treating anorectal motility disorders.     

Identification and characterization of the unique guanine nucleotide exchange factor, SmgGDS, in vascular smooth muscle cells

The guanine nucleotide exchange factor (GEF), SmgGDS, promotes nucleotide exchange by several GTPases in both the Ras and Rho families, especially by RhoA. Because RhoA plays an important role in regulating the contraction of vascular smooth muscle cells (VSMC), we examined the expression and function of SmgGDS in VSMC. SmgGDS is expressed in primary rat aortic smooth muscle (ASM) cells, primary bovine coronary artery smooth muscle (BCASM) cells, and the immortalized A7r5 line of rat ASM cells. Down regulation of SmgGDS expression by siRNA transfection resulted in a decrease of RhoA-GTP levels, enhanced cell spreading, and loss of the characteristic elongated morphology of VSMC. A similar morphology was also observed following treatment with the Rho-kinase inhibitor, Y27632. In contrast, cells with reduced RhoA expression exhibit an elongated shape. Subsequent immunofluorescent staining revealed a disruption of the myosin filament organization in the cells with reduced SmgGDS expression. Further studies analyzed the effect of SmgGDS siRNA transfection on the contraction of A7r5 cells and BCASM cells, which is also a Rho-regulated pathway. Transfection of SmgGDS siRNA or RhoA siRNA resulted in an impaired ability of the A7r5 and BCASM cells to undergo contraction in a collagen gel matrix. However, phosphorylation of the myosin-binding subunit of myosin phosphatase (MYPT1) or the light chain of myosin II (MLC) was not altered by downregulating expression of either SmgGDS or RhoA GTPase. Taken together these results identify SmgGDS as a novel regulator of myosin organization and contraction in VSMC.