Possible involvement of CPI-17 in augmented bronchial smooth muscle contraction in antigen-induced airway hyper-responsive rats

Airway hyper-responsiveness (AHR) associated with heightened airway resistance and inflammation is a characteristic feature of asthma. It has been demonstrated that contractile responsiveness and Ca(2+) sensitization to acetylcholine (ACh) in repeated antigen challenge-induced airway hyper-responsive bronchial preparation were significantly increased. The CPI-17 (PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa) is activated by protein kinase C and acts on a myosin light-chain phosphatase-specific target. The aim of the present study was to explore the role of CPI-17 in hyper-responsiveness of bronchial smooth muscle in antigen-induced AHR rats. In immunoblotting, the levels of expression of CPI-17 mRNA and protein were significantly increased in bronchus from rats that were repeatedly challenged with antigen. ACh-induced CPI-17 phosphorylation and translocation to membrane fraction were also significantly increased in bronchus from antigen-challenged rats. In conclusion, we suggest that augmented expression and activation of CPI-17 observed in the hyper-responsive bronchial smooth muscle might be responsible for the enhanced ACh-induced Ca(2+) sensitization of bronchial smooth muscle contraction associated with AHR.     

Augmented acetylcholine-induced translocation of RhoA in bronchial smooth muscle from antigen-induced airway hyperresponsive rats

Acetylcholine (ACh)-induced translocation of RhoA in bronchial smooth muscle of repeatedly antigen-challenged rats that have a marked airway hyperresponsiveness (AHR) was examined. ACh induced time- and concentration-dependent translocation of RhoA to the plasma membrane, indicating an activation of RhoA in bronchial smooth muscle. The level of ACh-induced RhoA translocation was further increased markedly in the AHR group as compared to that in the control group. It is suggested that the augmented activation of RhoA observed in the hyperresponsive bronchial smooth muscle might be responsible for the enhanced ACh-induced Ca(2+) sensitization of bronchial smooth muscle contraction associated with AHR.     

A role for Rho-kinase in Ca-independent contractions induced by phorbol-12,13-dibutyrate

1. Phorbol-12,13-dibutyrate (PDBu) is an activator of protein kinase C (PKC) that causes contractions in both physiological salt solutions and Ca(2+)-depleted solutions. In the present study, we tested the hypothesis that Rho-kinase plays a role in Ca(2+)-independent contractions induced by PDBu in vascular smooth muscles. 2. In Ca(2+)-free solution, 0.1 and 1 micromol/L PDBu induced contraction and myosin light chain (MLC(20)) phosphorylation, both of which were approximately 40% of responses obtained in normal Krebs' solution. Hydroxyfasudil (H1152; 1 micromol/L), an inhibitor of Rho-kinase, but not ML7 (10 micromol/L), an inhibitor of myosin light chain kinase, inhibited Ca(2+)-independent contractions induced by PDBu. 3. In Ca(2+)-free solution, PDBu increased phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and CPI-17 (PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa). This action was inhibited by H1152, with the phosphorylation of CPI-17 almost completely abolished by 1 micromol/L Ro31-8220, an inhibitor of PKC. 4. In Ca(2+)-free solution, PDBu increased the amount of GTP-RhoA (an activated form of RhoA). This increase was blocked by the PKC inhibitor Ro31-8220, but not by the Rho kinase inhibitor H1152. 5. In conclusion, RhoA/Rho-kinase plays an important role in Ca(2+)-independent contractions induced by PDBu in vascular smooth muscles. The results of the present study suggest that PDBu induces Ca(2+)-independent contractions by inhibiting myosin light chain phospatase (MLCP) through activation of GTP-RhoA and subsequent phosphorylation of MYPT1 and CPI-17.     

Probable involvement of the augmented agonist-induced Ca2+ sensitization of airway smooth muscle contraction in the pathogenesis of airway hyperresponsiveness]

Nonspecific airway hyperresponsiveness (AHR) is a common feature of allergic bronchial asthmatics, but the underlying mechanism (s) of AHR have yet to be elucidated. The importance of AHR in the pathogenesis of asthma has been suggested by its relevance to the severity of this disease. There is thus a need to understand the underlying mechanisms of AHR for the sake of asthma therapy. In the present minireview, we discussed the involvement of the augmented agonist-induced Ca2+ sensitization of airway smooth muscle contraction in the pathogenesis of AHR. Treatment with acetylcholine (ACh) of a beta-escin-permeabilized intrapulmonary bronchial smooth muscle of the rat induced a stronger contractile force even when the Ca2+ concentration was clamped at 1 microM. The ACh-induced Ca2+ sensitization of myofilaments was found to be significantly greater in antigen-induced airway hyperresponsive rats than in control rats. The ACh-induced Ca2+ sensitization was completely blocked by treatment with Clostridium botulinum C3 exoenzyme, an inactivator of the Rho family proteins. Moreover, the protein level of RhoA in the intrapulmonary bronchi was demonstrated to be significantly increased in the airway hyperresponsive rats. Thus, the increased airway smooth muscle contractility observed in asthmatics may be related to the augmented agonist-induced, Rho-mediated Ca2+ sensitization of myofilaments.     

Leukotriene C(4) enhances the contraction of porcine tracheal smooth muscle through the activation of Y-27632, a rho kinase inhibitor, sensitive pathway

1. An unsaturated fatty acid, leukotriene C(4) (LTC(4)), has a potent contractile effect on human airway smooth muscle, and has been implicated in the pathogenesis of human asthma. Using front-surface fluorometry with fura-PE3, the effect of LTC(4) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension were investigated in porcine tracheal smooth muscle strips. 2. The application of LTC(4) induced little or no contraction despite a small and transient increase in [Ca(2+)](i). In the presence of LTC(4), however, the contractions evoked by high K(+) depolarization or a low concentration of carbachol (CCh) were markedly enhanced without inducing any changes in the [Ca(2+)](i) levels, thus indicating that LTC(4) increases the Ca(2+) responsiveness of the contractile apparatus. This LTC(4)-induced increase in Ca(2+) responsiveness could partly be reproduced in the permeabilized preparation of tracheal smooth muscle strips. 3. The LTC(4)-induced enhancement of contraction was accompanied by an increase in myosin light chain (MLC) phosphorylation and was blocked by a rho kinase inhibitor (Y-27632), but not by either a PKC inhibitor (calphostin C) or a tyrosine kinase inhibitor (genistein). 4. These results indicated that, in porcine tracheal smooth muscle, LTC(4) enhances the contraction by increasing the Ca(2+) responsiveness of the contractile apparatus in a MLC phosphorylation dependent manner, possibly through the activation of the rho-rho kinase pathway.     

Tumor necrosis factor-alpha-induced activation of RhoA in airway smooth muscle cells: role in the Ca2+ sensitization of myosin light chain20 phosphorylation

Tumor necrosis factor-alpha (TNF), an inflammatory cytokine, has a potentially important role in the pathogenesis of bronchial asthma and may contribute to airway hyper-responsiveness. Recent evidence has revealed that TNF can increase the Ca(2+) sensitivity of agonist-stimulated myosin light chain(20) (MLC(20)) phosphorylation and contractility in guinea pig airway smooth muscle (ASM). In the present study, the potential intracellular pathways responsible for this TNF-induced Ca(2+) sensitization were investigated. In permeabilized cultured guinea pig ASM cells, recombinant human TNF stimulated an increase in Ca(2+)-activated MLC(20) phosphorylation under Ca(2+) "clamp" conditions. This increased MLC(20) phosphorylation was inhibited by preincubation with the Rho-kinase inhibitor Y27632. TNF also increased the proportion of GTP-bound RhoA, as measured using rhotekin Rho-binding domain, in a time course compatible with a role in the TNF-induced Ca(2+) sensitization. In cultured human ASM cells, recombinant human TNF also activated RhoA with a similar time course. In addition, TNF stimulated phosphorylation of the regulatory subunit of the myosin phosphatase, which was inhibited by Y27632. Although human ASM cells expressed both receptor subtypes, TNF-R1 and TNF-R2, the activation of RhoA was predominantly via stimulation of the TNF-R1, although RhoA did not immunoprecipitate with the TNF-R1. In conclusion, the TNF-induced increase in the Ca(2+) sensitivity of MLC(20) phosphorylation is through stimulation of the TNF-R1 receptor and via a RhoA/Rho-kinase pathway leading to inhibition of the myosin light chain phosphatase. This intracellular mechanism may contribute to TNF-induced airway hyper-responsiveness.     

Pharmacomechanical coupling in vascular smooth muscle cells--an overview

In vascular smooth muscles, neurotransmitters or autacoids produce contraction through activation of Ca2(+)-influx and release of Ca2+ from intracellular store sites. These agonists appear to activate Ca2(+)-influxes in both voltage-dependent and voltage-independent manners. The release of Ca2+ is though to be linked to the action of inositol 1,4,5-trisphosphate. The phosphorylation of myosin light chain may be the mechanism for the Ca2(-+)-induced contraction in smooth muscles. Some agonists only transiently increase cellular Ca2+ and the phosphorylation of myosin, but they produce a sustained contraction in various vascular tissues. Hence, additional high Ca2(+)-sensitive mechanisms are no doubt involved in the contraction of vascular smooth muscle. In the present article, attention will be directed to the mechanisms and agonist-induced contraction in arterial smooth muscle.     

Calcium-independent activation of the contractile apparatus in smooth muscle of mouse aorta by protein phosphatase inhibition

Smooth muscle contraction is primarily regulated by reversible phosphorylation of the 20-kDa light chains of myosin (MLC(20)) involving Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK) and serine/threonine protein phosphatases (PP).The aim of this study was to investigate the effects of the protein phosphatases (PP) type 1 (PP1) and type 2A (PP2A) inhibitor cantharidin (Cant), its structural analogue endothall (ETA) and microcystin LR (MC) on force of contraction and MLC(20)-phosphorylation in arterial smooth muscle of mouse aorta. Cant increased force of contraction and MLC(20)-phosphorylation in intact arterial rings of mouse aorta in the presence of Ca(2+) whereas ETA and MC were ineffective under the same experimental conditions. In contrast, all compounds induced contraction and led to enhanced MLC(20)-phosphorylation in nominally Ca(2+)-free solution in fibers of mouse aorta permeabilised (skinned) with Triton X-100.In addition, Western blot analysis revealed that skinning of mouse aorta did not result in a loss of PP1 and PP2A compared to intact rings. Thus, both PP must be tightly bound to structural proteins, e.g. myosin. The findings indicate a Ca(2+)-independent mechanism of smooth muscle contraction involving inhibition of PP1- and/or PP2A-activities leading to enhanced force and MLC(20)-phosphorylation of arterial smooth muscle.     

Direct inhibition of contractile apparatus by analogues of amiloride in the smooth muscle of guinea-pig taenia caecum and chicken gizzard

The relaxant effects of amiloride and its analogues, benzamil, 5-(N,N-diethyl)-amiloride (DEAM) and 5-(N-ethyl-N-isopropyl)-amiloride (EIAM), were investigated using smooth muscle of guinea-pig taenia caeci and chicken gizzard. High K+-induced contractions of intact taenia and gizzard were inhibited by these compounds (1-100 microM) with the order of potency; benzamil greater than or equal to EIAM greater than DEAM greater than amiloride. Contractions of permealized taenia and gizzard were also inhibited by these compounds at concentrations 8-35 times higher than those needed to inhibit the contractions of intact tissues. These compounds inhibited 20 K myosin light chain (MLC) phosphorylation at the concentrations needed to inhibit the contraction in the permealized muscles. Calmodulin (CaM) activity, as monitored by erythrocyte membrane (Ca2+ + Mg2+)-ATPase and phosphodiesterase activities, was inhibited by DEAM and EIAM at similar concentrations as those to inhibit the MLC phosphorylation. Benzamil also inhibited CaM activity at concentrations 4-8 times higher than those required to inhibit MLC phosphorylation. However, amiloride failed to inhibit CaM activity. Among these compounds, amiloride and benzamil inhibited Ca2+/CaM-independent MLC phosphorylation due to trypsin-treated MLC kinase. Taenia tissue gradually accumulated these compounds and the tissue/medium ratio exceeded 3.5-17 after a 3-hr incubation period. These results indicate that amiloride and its analogues inhibit smooth muscle contraction mainly by the direct inhibition of MLC phosphorylation. The inhibitory effect of amiloride may be attributable to the inhibition of MLC kinase, whereas the inhibitory effect of DEAM and EIAM may largely be attributable to the inhibition of CaM. Benzamil may inhibit contraction by the inhibition of both MLC kinase and CaM. Differences in the drug-sensitivity between intact and permealized tissues may be attributable to the difference in drug accumulation by the cell.     

Characterization of capsaicin-induced, capsazepine-insensitive relaxation of ileal smooth muscle of rats

The mechanisms underlying the capsaicin-induced relaxation of the acetylcholine- as well as KCl-contraction were studied by measuring isometric force and phosphorylation of 20-kDa regulatory light chain subunit of myosin (MLC(20)) in ileal longitudinal smooth muscles of rats. Capsaicin relaxed acetylcholine- and KCl-stimulated preparations in a concentration-dependent manner; the former was less sensitive to capsaicin than the latter and maximum responses to capsaicin (a percentage of papaverine-induced relaxation) were 70.6+/-7.5%, n=10 and 97.1+/-0.9%, n=13, P<0.05, respectively. The response showed no desensitization. Like nifedipine, capsaicin relaxed the tissue precontracted with an agonist of L-type Ca(2+) channels as well. The relaxant effect of capsaicin was not inhibited by capsazepine (a selective antagonist of vanilloid VR1 receptors), nitro-l-arginine, indomethacin, guanethidine, nor by inhibitors of soluble guanylate cyclase. Capsaicin inhibited acetylcholine-induced transient contraction in a Ca(2+)-free, EGTA solution. Phosphorylation of MLC(20) (a percentage of phosphorylated to total MLC(20)) was increased 1 min after application of 10 microM acetylcholine (7.8+/-2.0%, n=6 vs. 22.6+/-3.2%, n=6) and of 65.9 mM KCl (2.2+/-0.3%, n=8 vs. 10.7+/-1.7%, n=12). Capsaicin reduced the KCl-induced increase more markedly than acetylcholine-induced increase in MLC(20) phosphorylation. When the tissue was contracted for 20 min with acetylcholine, MLC(20) phosphorylation was increased, and capsaicin reduced markedly the contraction and abolished MLC(20) phosphorylation both elicited by acetylcholine. It is suggested that capsaicin relaxes the rat ileum via its direct action on smooth muscle, and that capsaicin inhibits contractile mechanisms involving extracellular Ca(2+) influx via non-L-type Ca(2+) channels, possibly via store-operated Ca(2+) channels and Ca(2+) release from intracellular storage sites. The effects of capsaicin on acetylcholine- and KCl-induced contraction could be explained by a decrease in MLC(20) phosphorylation.     

Flavone inhibits vascular contraction by decreasing phosphorylation of the myosin phosphatase target subunit

1. Flavonoids modulate vascular tone through an endothelium-dependent or -independent mechanism. Although a few mechanisms for endothelium-independent relaxation have been suggested, such as interference with protein kinase C or cAMP or cGMP phosphodiesterase, the inhibition of Ca(2+) release from intracellular stores or Ca(2+) influx from extracellular fluids, the mode of action of flavonoids remains elusive. 2. We hypothesized that treatment with flavone inhibits vascular smooth muscle contraction by decreasing the phosphorylation of the myosin phosphatase target subunit (MYPT1). 3. Rat aortic rings were denuded of endothelium, mounted in organ baths and contracted with U46619, a thromboxane A(2) analogue. 4. Flavone dose-dependently inhibited the U46619-induced contractile response and myosin light chain (MLC(20)) phosphorylation. At 10(-7) mol/L, U46619 induced vascular contraction with the concomitant phosphorylation of MYPT1 at Thr855, but not at Thr697. Incubation with flavone (100 or 300 micromol/L) for 30 min attenuated the phosphorylation of MYPT1(Thr855), but not MYPT1(Thr697). 5. It is concluded that treatment with flavone inhibits vascular smooth muscle contraction by decreasing the phosphorylation of the MYPT1. These results suggest that flavone causes endothelium-independent relaxation through, at least in part, the inhibition of p160 Rho-associated coiled-coil-containing protein kinase (ROCK) signalling.     

Glucocorticoids ameliorate antigen-induced bronchial smooth muscle hyperresponsiveness by inhibiting upregulation of RhoA in rats

To determine the mechanism(s) of the inhibitory effect of glucocorticoids on airway hyperresponsiveness in allergic bronchial asthma, the effects of systemic treatment with glucocorticoids on bronchial smooth muscle hyperresponsiveness and RhoA upregulation were investigated in rats with allergic bronchial asthma. Rats were sensitized and repeatedly challenged with 2,4-dinitrophenylated Ascaris suum antigen. Animals were also treated with prednisolone or beclomethasone (each 10 mg/kg, i.p.) once a day during the antigen inhalation period. Repeated antigen inhalation caused a marked bronchial smooth muscle hyperresponsiveness to acetylcholine with an upregulation of RhoA. Augmented acetylcholine-induced activation of RhoA and phosphorylation of myosin light chain were observed in bronchial smooth muscles of the antigen-exposed animals. Systemic treatment with either glucocorticoid used inhibited the bronchial smooth muscle hypercontraction until the level of the sensitized control rats that received saline inhalation instead of antigen challenge. Interestingly, both glucocorticoids also inhibited the upregulation of RhoA and augmented acetylcholine-induced activation of RhoA and phosphorylation of myosin light chain. In conclusion, glucocorticoids ameliorated the augmented bronchial smooth muscle contraction by inhibiting upregulation of RhoA. These effects of glucocorticoids may account for, in part, their beneficial effects in the treatment of asthma.     

Effect of a peptide inhibitor of protein kinase C on G-protein-mediated increase in myofilament Ca(2+)-sensitivity in rabbit arterial skinned muscle.

1. To investigate the role of protein kinase C in the increase mediated by guanosine 5'-triphosphate (GTP)-binding proteins (G-proteins) in the sensitivity of the contractile proteins to Ca2+ in vascular smooth muscle, the effect of a novel peptide inhibitor of protein kinase C (PKC19-36) on Ca(2+)-induced contraction and myosin light chain (MLC) phosphorylation was studied in the presence and absence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in beta-escin-skinned smooth muscle strips of rabbit mesenteric artery. For comparison, the effects were also observed of PKC19-36 on the action of phorbol 12,13-dibutylate (PDBu, an activator of PKC) on the two Ca(2+)-induced responses. 2. In beta-escin-skinned strips treated with ionomycin, Ca2+ (0.1-3 microM) concentration-dependently produced contraction in parallel with an increase in MLC-phosphorylation. GTP gamma S (10 microM) and PDBu (0.1 microM) each shifted both the Ca(2+)-force and Ca(2+)-MLC-phosphorylation relationships to the left without a significant change in either maximum response. The relationship between force and MLC-phosphorylation was not modified by either GTP gamma S or PDBu, indicating that the sensitivity of MLC-phosphorylation to Ca2+ is enhanced by both GTP gamma S and PDBu. 3. PKC19-36 itself modified neither the contraction nor MLC-phosphorylation induced by Ca2+ but it did block the PDBu-induced enhancement of these two Ca(2+)-induced responses. By contrast, PKC19-36 did not modify the GTP gamma S-induced enhancement of the two Ca(2+)-induced responses.(ABSTRACT TRUNCATED AT 250 WORDS)     

p38 Mitogen-activated protein kinase regulates vasoconstriction in spontaneously hypertensive rats

We investigated whether p42/p44 mitogen-activated protein kinase (MAPK) and/or p38 MAPK participates in the regulation of vascular smooth muscle contraction by endothelin-1 (ET-1) in Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR). ET-1 (10 nM) induced a sustained contraction in WKY and SHR aortas. PD98059 (100 microM), an inhibitor of p42/p44 MAPK kinase, partially attenuated the ET-1-induced contraction in WKY and SHR. However, SB203580 (10 microM), an inhibitor of p38 MAPK, relaxed the ET-1-induced contraction to the resting levels in SHR, but not in WKY. ET-1 (10 nM) increased phosphorylation of both p42/p44 MAPK and p38 MAPK in WKY and SHR. However, in SHR, p38 MAPK phosphorylation in response to ET-1 stimulation was increased more than in WKY. PD98059 (100 microM) and SB203580 (10 microM) abolished the phosphorylation of p42/p44 MAPK and p38 MAPK in response to ET-1 stimulation in WKY and SHR, respectively. On the other hand, SB203580 (10 microM) did not affect myosin light chain (MLC) phosphorylation in response to ET-1 (10 nM) stimulation in WKY and SHR. From these results, it is concluded that p42/p44 MAPK and/or p38 MAPK partially regulates the ET-1-induced vasoconstriction in WKY. However, p38 MAPK, rather than p42/p44 MAPK, activation plays an important role for the maintenance of ET-1-induced vasoconstriction in SHR through a MLC phosphorylation-independent pathway.     

Continuous elevation of intracellular Ca2+ is essential for the development of cerebral vasospasm

Subarachnoid hemorrhage (SAH)-induced cerebral vasospasm causes serious neurological morbidity and mortality mainly because of the absence of effective treatment. Therefore, we reviewed the molecular mechanisms involved in the development of the cerebral vasospasm based on the experimental data in the two-hemorrhage canine model. The characteristic feature of vasospasm is a continuous elevation of intracellular Ca2+ levels in the cerebral artery, as indicated by the continuous activation of mu-calpain and Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylation of the myosin light chain. In contrast, KCl- or serotonin-induced vasocontraction displays a transient increase in Ca2+ concentration. The elevation of intracellular Ca2+ levels in vasospasm is induced through enhanced Ca2+ release from the sarcoplasmic reticulum and influx from the extracellular space by the activation of tyrosine kinase pathway and also probably by the proteolysis of Ca2+ channel by mu-calpain. Topical application of L-type Ca2+ channel blockers, ethylene-glycol-bis(beta-aminoethylether)N,N'-tetraacetic acid, genistein, calpeptin (a selective inhibitor of calpain), or ML-9 (a selective inhibitor of MLCK) induces the reversal of vasospasm probably as a result of a decrease in intracellular Ca2+ levels mainly due to a reduction of Ca2+ influx by these three inhibitors. Rho-kinase is also activated during vasospasm. It inhibits myosin phosphatase through phosphorylation at the myosin phosphatase target subunit 1 and also probably through phosphorylation of the 17-kDa smooth muscle-specific myosin phosphatase inhibitor (CPI-17) to bring about Ca2+-independent vasospasm. This interpretation is supported by the reversal of vasospasm with Y-27632, a specific inhibitor of Rho-kinase. Arachidonic acid produced during vasospasm might inhibit myosin phosphatase probably directly and via activation of Rho-kinase or atypical protein kinase C (PKC). PKC activated during vasospasm may inhibit myosin phosphates directly and by phosphorylating CPI-17. The protein levels of thin filament-associated proteins, calponin and caldesmon, are decreased in vasospasm, whereas their phosphorylation levels are increased. Both changes probably contribute to the enhancement of vascular smooth muscle contractility. Furthermore, contractile and cytoskeletal proteins appear to be degraded in vasospasm probably by proteolysis with mu-calpain, suggesting that degradation of the structural and functional mechanisms related to smooth muscle contraction occurs. Thus, the mechanisms responsible for the development of cerebral vasospasm are complicated, but the prevention of intracellular Ca2+ elevation induced by SAH may not activate MLCK, calpain and PKC to largely suppressing the development of vasospasm.     

Regulatory light chains of striated muscle myosin. Structure,function and malfunction

Striated (skeletal and cardiac) muscle is activated by the binding of Ca(2+) to troponin C and is regulated by the thin filament proteins, tropomyosin and troponin. Unlike in molluscan or smooth muscles, the myosin regulatory light chains (RLC) of striated muscles do not play a major regulatory role and their function is still not well understood. The N-terminal domain of RLC contains a 'Ca(2+)-Mg(2+)'-binding site and, analogous to that of smooth muscle myosin, also contains a phosphorylation site. During muscle contraction, the increase in Ca(2+) concentration activates the Ca(2+)/calmodulin-dependent myosin light chain kinase and leads to phosphorylation of the RLC. In agreement with other laboratories we have demonstrated that phosphorylation and Ca(2+) binding to the RLC play an important modulatory role in striated muscle contraction. Furthermore, the ventricular isoform of human cardiac RLC has been shown to be one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular hypertrophy, myofibrillar disarray and sudden cardiac death. Our recent studies have demonstrated that phosphorylation and Ca(2+) binding to human ventricular RLC are significantly altered by the FHC mutations and that their detrimental effects depend upon the specific position of the missense mutation, whether located in the proximity of the RLC 'Ca(2+)-Mg(2+)'-binding site or the phosphorylation site (Serine 15). We have also shown that there is a functional coupling between Ca(2+) and/or Mg(2+) binding to the RLC and phosphorylation and that the FHC mutations can affect this relationship. Further in vivo studies are necessary to investigate the mechanisms involved in the pathogenesis of RLC-linked FHC.     

Essential role for class II phosphoinositide 3-kinase alpha-isoform in Ca2+-induced, Rho- and Rho kinase-dependent regulation of myosin phosphatase and contraction in isolated vascular smooth muscle cells

The laser confocal fluorescent microscope-based observation of contractile responses in green fluorescent protein-expressing differentiated vascular smooth muscle cells, combined with the RNA interference-mediated gene-silencing technique, allowed us to determine the role of phosphoinositide 3-kinase (PI3K) class II alpha-isoform (PI3K-C2alpha) as a novel, Ca2+-dependent regulator of myosin light-chain phosphatase (MLCP) and contraction. The Ca2+-ionophore ionomycin induced a robust contractile response with an increase in the intracellular free Ca2+ concentration ([Ca2+]i). The PI3K-C2alpha-specific short interfering RNA (siRNA) induced a selective and marked reduction in PI3K-C2alpha protein expression. The siRNA-mediated knockdown of PI3K-C2alpha, but not class I PI3K p110alpha, suppressed ionomycin-induced contraction without altering Ca2+-mobilization. PI3K-C2alpha is uniquely less sensitive to the PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) than the other PI3K members, including p110alpha. Ionomycin-induced contraction was inhibited only by a relatively high concentration of LY294002. Consistent with our previous observations showing that ionomycin and membrane depolarization induced Rho activation in vascular smooth muscle tissues in a Ca2+-dependent manner, ionomycin-induced contraction was dependent on Rho and Rho-kinase. Ionomycin induced phosphorylation of the MLCP-regulatory subunit myosin targeting protein 1(MYPT1) at Thr850 and the 20-kDa myosin light chain (MLC) in a Rho kinase-dependent manner. Knockdown of PI3K-C2alpha suppressed phosphorylation of both MYPT1 and MLC. The receptor agonist noradrenaline, which induced a rapid increase in the [Ca2+]i and Ca2+-dependent contraction, stimulated phosphorylation of MYPT1 and MLC, which was also dependent on Ca2+, PI3K-C2alpha, and Rho-kinase. These observations indicate that PI3K-C2alpha is necessary for Ca2+-induced Rho- and Rho kinase-dependent negative regulation of MLCP and consequently MLC phosphorylation and contraction.     

Effects of Y-27632 on acetylcholine-induced contraction of intact and permeabilized intrapulmonary bronchial smooth muscles in rats.

In the present study, the effects of a selective Rho-associated coiled-coil forming protein kinase (ROCK) inhibitor, Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] on acetylcholine-induced contraction and Ca(2+) sensitization of rat bronchial smooth muscle were examined. Intact and beta-escin-permeabilized muscles of the third branch of intrapulmonary bronchi were used. In intact muscles, Y-27632 (10(-6)-10(-4) M) concentration-dependently inhibited acetylcholine-induced contractile responses. In acetylcholine (10(-3) M)-precontracted intact muscles, the maximal relaxation (about 50% inhibition of contraction) was obtained by a concentration of 10(-4) M Y-27632, which had no effect on the resting tone. In beta-escin-permeabilized muscles, addition of acetylcholine (10(-5)-10(-3) M) plus GTP (100 microM) induced a further contraction, i.e., Ca(2+) sensitization at a constant Ca(2+) concentration of pCa=6.0. The acetylcholine-induced Ca(2+) sensitization was completely blocked in the presence of 10(-4) M Y-27632, whereas the Ca(2+)-induced contraction itself was not affected by Y-27632. Immunoblot study revealed the expression of ROCK-I and ROCK-II proteins in the intrapulmonary bronchi of rats. These findings suggest that Y-27632 dilates acetylcholine-mediated contraction of rat bronchial smooth muscle by inhibiting RhoA/ROCK-mediated Ca(2+) sensitization.     

Rho-kinase inhibitor inhibits both myosin phosphorylation-dependent and -independent enhancement of myofilament Ca2+ sensitivity in the bovine middle cerebral artery

The role of Rho kinase in Ca2+ sensitization of the contractile apparatus in smooth muscle was investigated in the bovine middle cerebral artery. U46619, a thromboxane A2 analog, induced a greater sustained contraction with a smaller [Ca2+]i elevation than that seen with 118 mm K+. The level of myosin light chain (MLC) phosphorylation obtained in the initial phase of the contraction was higher than that seen with 118 mm K+; thereafter, it gradually declined to a comparable level in the late phase. During the steady state of the U46619-induced contraction, Y27632 (10 microM), a Rho-kinase inhibitor, partially inhibited [Ca2+]i, although it substantially inhibited tension and MLC phosphorylation. Wortmannin (10 microM), an MLC kinase inhibitor, had no significant effect on [Ca2+]i, but it completely inhibited MLC phosphorylation and partially inhibited tension. The wortmannin-resistant tension development was thus not associated with MLC phosphorylation, and this component was completely inhibited by Y27632. In conclusion, U46619 enhanced Ca2+ sensitivity in a manner both dependent and independent of MLC phosphorylation in the bovine middle cerebral artery. Both mechanisms of Ca2+ sensitization can be inhibited by the Rho-kinase inhibitor.     

Inhibition of high K+-induced contraction by the ROCKs inhibitor Y-27632 in vascular smooth muscle: possible involvement of ROCKs in a signal transduction pathway

In the isolated rat aorta, a ROCKs (rhoA-dependent coiled coil serine/threonine kinases) inhibitor, Y-27632, inhibited the contractions induced not only by receptor agonists but also by high K(+) with the similar IC(50) values (0.8 - 4.9 microM). However, Y-27632 did not inhibit the increment of cytosolic Ca(2+) concentration ([Ca(2+)](i)) due to these stimulants. The Y-27632-induced inhibition of contraction was accompanied by an inhibition of myocin light chain (MLC) phosphorylation, although inhibition of contraction was stronger than that of MLC phosphorylation during the initial phase of contraction. Y-27632 had no effect on the myocin light chain kinase (MLCK) activity. This inhibitor also did not directly change the phosphatase activity. These results suggest that Y-27632 is a selective inhibitor of ROCKs with no direct inhibitory effect on [Ca(2+)](i), calmodulin, MLCK, or phosphatase. Y-27632 disrupted the actin filament network and decreased the filamentous actin, implying that the stronger inhibition by Y-27632 on early phase of contraction than MLC phosphorylation may be explained by this effect. These results suggest that the high K(+)-induced MLC phosphorylation and contraction are mediated not only by the classical Ca(2+)/calmodulin-dependent MLCK system but also by a novel MLC phosphorylation pathway involving ROCKs. One of the possibilities is that high K(+) activates ROCKs to inhibit myosin phosphatase resulting in an augmentation of MLC phosphorylation and contraction.