Sensitization Of Smooth Muscle Contraction Research Articles

Investigating the effects of the Rho-kinase enzyme inhibitors AS1892802 and fasudil hydrochloride on the contractions of isolated pregnant rat myometrium

ObjectivesRho-kinases (ROCKs), are one of the dynamic structures of the actin cytoskeleton and they mediate different biological processes, including regulation of calcium sensitivity of smooth muscle contraction. The activation of Rho A/ROCK system is thought to be effective on the termination time of the pregnancy process. The aim of this study, was to investigate in vitro effects of the ROCK enzyme inhibitors, clinically available fasudil hydrochloride, and a new promising inhibitor AS1892802, on the contractions of isolated pregnant rat myometrium. Study designTerm pregnant Wistar albino rats (n=12), weighing 200–220g, were used in this study. Myometrial tissues obtained from rats were dissected into four full-thickness longitudinal muscle strips and then myometrial tension was recorded isometrically. The inhibitory effects of cumulative concentrations of AS1892802 and of fasudil hydrochloride in the presence and absence of ODQ (guanylate cyclase inhibitor), l-NAME (nitric oxide synthase inhibitor) and l-NNA (endothelial nitric oxide synthase inhibitor) on oxytocin-induced myometrial contractions were measured, and values for −log10EC50 (pD2) and mean maximal inhibition (Emax) were compared. ResultsBoth ROCK inhibitors, AS1892802 and fasudil hydrochloride starting from the concentrations of 10−6M reached statistical significance on contraction amplitude and frequency of myometrial strips (p<0.05). The inhibition of the amplitude and frequency of myometrial contractions was antagonized with ODQ (10−5M; only amplitude), l-NAME (3×10−5M) and l-NNA (10−5M) (p<0.05). ConclusionThese results suggest that fasudil hydrochloride and AS1892802 may contribute to the development of new tocolytic drugs. We conclude that AS1892802 and fasudil hydrochloride perform this inhibitory effect partially through ROCK inhibition and the NO/cGMP pathway.

Trophic action of sympathetic nerves reduces arterial smooth muscle Ca(2+) sensitivity during early post-natal development in rats.

A decrease in the Ca(2+) sensitivity of smooth muscle contraction is a hallmark of functional remodelling of blood vessels during development. However, the responsible factors are largely unknown. Here, we tested the hypothesis that the post-natal decline of arterial Ca(2+) sensitivity is the result of trophic effects of sympathetic nerves. Contractile responses, intracellular Ca(2+) levels and protein expression profiles were compared in saphenous arteries from young (1- and 2-week-old) and adult rats using wire myography, Ca(2+) fluorimetry and Western blotting respectively. We observed a lower Ca(2+) sensitivity of contractions induced by methoxamine, an agonist of α1 -adrenoceptors, and U46619, an agonist of thromboxane A2 receptors, in arteries from adult as compared to young animals. Post-natal maturation was associated with stronger expression of regulatory proteins mediating Ca(2+) -dependent contraction (myosin light chain kinase (MLCK), myosin targeting subunit (MYPT1) and h-caldesmon) and weaker expression of proteins regulating Ca(2+) -independent contraction (Rho kinase, extracellular-regulated kinases (ERK1/2) and mitogen-activated protein kinases p38 MAPK) in vessels from adult rats. To eliminate the trophic action of sympathetic nerves, we performed lumbar sympathectomy in adult rats. This resulted in higher Ca(2+) sensitivity of agonist-induced contractions in denervated as compared to control arteries. Furthermore, denervated arteries contained less MLCK, MYPT1 and h-caldesmon and more ERK1/2 and p38 MAPK. Sympathetic denervation reverses developmental changes both in Ca(2+) sensitivity and in the expression of regulatory proteins back to the early post-natal phenotype in the rat saphenous artery. We conclude that trophic effects of sympathetic nerves govern functional remodelling of arteries during early post-natal development.

GATA-6 and NF-κB Activate CPI-17 Gene Transcription and Regulate Ca2+ Sensitization of Smooth Muscle Contraction

Protein kinase C (PKC)-potentiated inhibitory protein of 17 kDa (CPI-17) inhibits myosin light chain phosphatase, altering the levels of myosin light chain phosphorylation and Ca(2+) sensitivity in smooth muscle. In this study, we characterized the CPI-17 promoter and identified binding sites for GATA-6 and nuclear factor kappa B (NF-κB). GATA-6 and NF-κB upregulated CPI-17 expression in cultured human and mouse bladder smooth muscle (BSM) cells in an additive manner. CPI-17 expression was decreased upon GATA-6 silencing in cultured BSM cells and in BSM from NF-κB knockout (KO) mice. Moreover, force maintenance by BSM strips from KO mice was decreased compared with the force maintenance of BSM strips from wild-type mice. GATA-6 and NF-κB overexpression was associated with CPI-17 overexpression in BSM from men with benign prostatic hyperplasia (BPH)-induced bladder hypertrophy and in a mouse model of bladder outlet obstruction. Thus, aberrant expression of NF-κB and GATA-6 deregulates CPI-17 expression and the contractile function of smooth muscle. Our data provide insight into how GATA-6 and NF-κB mediate CPI-17 transcription, PKC-mediated signaling, and BSM remodeling associated with lower urinary tract symptoms in patients with BPH.

Cross-talk between Rho-associated Kinase and Cyclic Nucleotide-dependent Kinase Signaling Pathways in the Regulation of Smooth Muscle Myosin Light Chain Phosphatase

Ca(2+) sensitization of smooth muscle contraction depends upon the activities of protein kinases, including Rho-associated kinase, that phosphorylate the myosin phosphatase targeting subunit (MYPT1) at Thr(697) and/or Thr(855) (rat sequence numbering) to inhibit phosphatase activity and increase contractile force. Both Thr residues are preceded by the sequence RRS, and it has been suggested that phosphorylation at Ser(696) prevents phosphorylation at Thr(697). However, the effects of Ser(854) and dual Ser(696)-Thr(697) and Ser(854)-Thr(855) phosphorylations on myosin phosphatase activity and contraction are unknown. We characterized a suite of MYPT1 proteins and phosphospecific antibodies for specificity toward monophosphorylation events (Ser(696), Thr(697), Ser(854), and Thr(855)), Ser phosphorylation events (Ser(696)/Ser(854)) and dual Ser/Thr phosphorylation events (Ser(696)-Thr(697) and Ser(854)-Thr(855)). Dual phosphorylation at Ser(696)-Thr(697) and Ser(854)-Thr(855) by cyclic nucleotide-dependent protein kinases had no effect on myosin phosphatase activity, whereas phosphorylation at Thr(697) and Thr(855) by Rho-associated kinase inhibited phosphatase activity and prevented phosphorylation by cAMP-dependent protein kinase at the neighboring Ser residues. Forskolin induced phosphorylation at Ser(696), Thr(697), Ser(854), and Thr(855) in rat caudal artery, whereas U46619 induced Thr(697) and Thr(855) phosphorylation and prevented the Ser phosphorylation induced by forskolin. Furthermore, pretreatment with forskolin prevented U46619-induced Thr phosphorylations. We conclude that cross-talk between cyclic nucleotide and RhoA signaling pathways dictates the phosphorylation status of the Ser(696)-Thr(697) and Ser(854)-Thr(855) inhibitory regions of MYPT1 in situ, thereby regulating the activity of myosin phosphatase and contraction.

Excitatory nerve stimulation and agonist stimulation induce gastric fundus smooth muscle contraction via stimulus dependent Ca2+ sensitization pathways‐not via myosin light chain phosphorylation

Ca2+ sensitization of smooth muscle contraction occurs by myosin light chain phosphatase (MLCP) inhibition. CPI‐17 phosphorylation or MYPT‐1 phosphorylation by Rho kinase (ROK) inhibits MLCP. Studies have shown that bath application of acetylcholine (Ach) induces Ca2+ sensitization in the gastrointestinal smooth muscles. We addressed a question; does excitatory nerve stimulation also cause Ca2+ sensitization in fundus smooth muscle? We stimulated fundus smooth muscle at a series of frequencies and determined the phosphorylated levels of MYPT‐1 at Thr 696 and 853 (pT696 and pT853), CPI‐17 (pT38) and LC‐20 at Ser 19 (pS19) by western blotting at different phases of contractions. Nerve stimulation robustly increased levels of pT38 but not pT696, pT853, and pS19. In contrast, bath application of Ach increased phosphorylation of pT696, pT853, and pT38, but not pS19. The L‐type Ca2+ channel blocker nicardipine completely blocked Ach induced contraction with a partial block in the increase of pT696 (but not pT853) and pT38 levels. The ROK inhibitor SAR1x partially blocked Ach induced contraction with a complete block of the increase in pT853 (but not pT696) and a partial block of the increase in pT38. At present, we conclude that excitatory nerve stimulation induces Ca2+ sensitization in gastric fundus smooth muscle by a different pathway than bath application of Ach. Supported by NIH grant RR018751 and DK40569.

Ca2+ sensitization contributes to basal contractions of smooth muscles from different regions of a murine gastrointestinal tract

Ca2+ sensitization of smooth muscle contraction occurs by myosin light chain phosphatase (MLCP) inhibition. CPI-17 phosphorylation or MYPT-1 phosphorylation by Rho kinase inhibits MLCP. We employed western blotting to examine the levels of proteins involved in Ca2+ sensitization to address a question; is basal Ca2+ sensitization different among smooth muscles from antrum, fundus and proximal colon (PC) in a murine gastrointestinal (GI) tract? Fundus and PC smooth muscles showed higher levels of Ca2+ sensitization proteins than antrum. Myosin light chain (MLC) and pSer19-MLC levels were lower in fundus and PC compared to that in antrum. Rho kinase inhibitorY-27632, reduced contractions and MYPT-1 pThr853 levels, perhaps with different sensitivities, in all three smooth muscles. Y-27632 reduced MYPT-1 pThr696 levels in fundus, but not in antrum and PC smooth muscles. L-type Ca2+ channel blocker, nicardipine, reduced antrum and PC smooth muscle contractions without affecting MYPT-1 pThr853 or pThr696 levels. Whereas, nicardipine did not affect fundus smooth muscle contraction but still reduced MYPT-1 phosphorylation. pSer19-MLC levels were reduced by Y-27632 or nicardipine in fundus and PC, but not in antrum smooth muscles. These findings suggest that both Ca2+ dependent mechanisms and MLCP inhibition by MYPT-1 phosphorylation contribute to basal contractile properties of GI smooth muscles. Supported by NIH Grant RR018751.

The effects of the small GTPase RhoA on the muscarinic contraction of airway smooth muscle result from its role in regulating actin polymerization

The small GTPase RhoA increases the Ca(2+) sensitivity of smooth muscle contraction and myosin light chain (MLC) phosphorylation by inhibiting the activity of MLC phosphatase. RhoA is also a known regulator of cytoskeletal dynamics and actin polymerization in many cell types. In airway smooth muscle (ASM), contractile stimulation induces MLC phosphorylation and actin polymerization, which are both required for active tension generation. The objective of this study was to evaluate the primary mechanism by which RhoA regulates active tension generation in intact ASM during stimulation with acetylcholine (ACh). RhoA activity was inhibited in canine tracheal smooth muscle tissues by expressing the inactive RhoA mutant, RhoA T19N, in the intact tissues or by treating them with the cell-permeant RhoA inhibitor, exoenzyme C3 transferase. RhoA inactivation reduced ACh-induced contractile force by approximately 60% and completely inhibited ACh-induced actin polymerization but inhibited ACh-induced MLC phosphorylation by only approximately 20%. Inactivation of MLC phosphatase with calyculin A reversed the reduction in MLC phosphorylation caused by RhoA inactivation, but calyculin A did not reverse the depression of active tension and actin polymerization caused by RhoA inactivation. The MLC kinase inhibitor, ML-7, inhibited ACh-induced MLC phosphorylation by approximately 80% and depressed active force by approximately 70% but did not affect ACh-induced actin polymerization, demonstrating that ACh-stimulated actin polymerization occurs independently of MLC phosphorylation. We conclude that the RhoA-mediated regulation of ACh-induced contractile tension in ASM results from its role in mediating actin polymerization rather than from effects on MLC phosphatase or MLC phosphorylation.

Role of Calcium-independent Phospholipase A2β in High Glucose-induced Activation of RhoA, Rho Kinase, and CPI-17 in Cultured Vascular Smooth Muscle Cells and Vascular Smooth Muscle Hypercontractility in Diabetic Animals

Previous studies suggest that high glucose-induced RhoA/Rho kinase/CPI-17 activation is involved in diabetes-associated vascular smooth muscle hypercontractility. However, the upstream signaling that links high glucose and RhoA/Rho kinase/CPI-17 activation is unknown. Here we report that calcium-independent phospholipase A(2)beta (iPLA(2)beta) is required for high glucose-induced RhoA/Rho kinase/CPI-17 activation and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility. We demonstrate that high glucose increases iPLA(2)beta mRNA, protein, and iPLA(2) activity in a time-dependent manner. Protein kinase C is involved in high glucose-induced iPLA(2)beta protein up-regulation. Inhibiting iPLA(2)beta activity with bromoenol lactone or preventing its expression by genetic deletion abolishes high glucose-induced RhoA/Rho kinase/CPI-17 activation, and restoring expression of iPLA(2)beta in iPLA(2)beta-deficient cells also restores high glucose-induced CPI-17 phosphorylation. Pharmacological and genetic inhibition of 12/15-lipoxygenases has effects on high glucose-induced CPI-17 phosphorylation similar to iPLA(2)beta inhibition. Moreover, increases in iPLA(2) activity and iPLA(2)beta protein expression are also observed in both type 1 and type 2 diabetic vasculature. Pharmacological and genetic inhibition of iPLA(2)beta, but not iPLA(2)gamma, diminishes diabetes-associated vascular smooth muscle hypercontractility. In summary, our results reveal a novel mechanism by which high glucose-induced, protein kinase C-mediated iPLA(2)beta up-regulation activates the RhoA/Rho kinase/CPI-17 via 12/15-lipoxygenases and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility.

Participation of Rho-associated kinase in electrical stimulated and acetylcholine-induced contraction of feline esophageal smooth muscle

The RhoA/Rho-associated kinase (ROCK) signaling pathway has been known to play a critical role in Ca 2+-sensitization of smooth muscle contraction. In this study, we investigated the role of ROCK in feline esophageal body smooth muscle contraction induced by electrical field stimulation and exogenous acetylcholine in vitro. Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-(4-pyridyl) cyclohexanecarboxamide dihydrochloride], ROCK inhibitor, and specific antibodies to ROCK1 and ROCK2 proteins, which are two isoforms of ROCK, were used. Electrical field stimulation induced off-contraction and on-contraction in the presence of N G-nitro-L-arginine methylester, originating from the cholinergic nerve. Y-27632 inhibited both excitatory contractions in a concentration-dependent manner. Exogenous acetylcholine concentration-dependently induced two types of contractions: an initial contraction which occurred immediately after the addition of acetylcholine during short periods, and a sustained contraction which sluggishly continued after the initial contraction. Maximal initial and sustained contractions were reached at 10 − 5 M acetylcholine. Y-27632 significantly inhibited both acetylcholine-induced contractions in a concentration-dependent manner. Western blot analysis revealed that acetylcholine maximally increased the level of phosphorylation in the 20 kDa regulatory light chain of myosin II (MLC 20) at Ser 19 from 0.25 min to 1 min, and then declined after 2 min. The level changes of MLC 20 phosphorylation during the 5 min paralleled with those of acetylcholine-induced contractions. The expression of ROCK1 and ROCK2 in membrane fractions of muscle was increased by acetylcholine; more specifically, ROCK2 continually expressed up to 5 min. Taken together, ROCK may be involved in neural-evoked and acetylcholine-induced contraction via translocation to the membrane in feline esophageal smooth muscle.

A Novel STAT6 Inhibitor AS1517499 Ameliorates Antigen-Induced Bronchial Hypercontractility in Mice

Interleukin-13 (IL-13) is one of the central mediators for development of airway hyperresponsiveness in asthma. The signal transducer and activation of transcription 6 (STAT6) is one of the major signal transducers activated by IL-13, and a possible involvement of IL-13/STAT6 pathway in the augmented bronchial smooth muscle (BSM) contraction has been suggested. In the present study, the effect of a novel STAT6 inhibitor, AS1517499, on the development of antigen-induced BSM hyperresponsiveness was investigated. In cultured human BSM cells, IL-13 (100 ng/ml) caused a phosphorylation of STAT6 and an up-regulation of RhoA, a monomeric GTPase responsible for Ca2+ sensitization of smooth muscle contraction: both events were inhibited by co-incubation with AS1517499 (100 nM). In BALB/c mice that were actively sensitized and repeatedly challenged with ovalbumin antigen, an increased IL-13 level in bronchoalveolar lavage fluids and a phosphorylation of STAT6 in bronchial tissues were observed after the last antigen challenge. These mice had an augmented BSM contractility to acetylcholine together with an up-regulation of RhoA in bronchial tissues. Intraperitoneal injections of AS1517499 (10 mg/kg) 1 hour before each ovalbumin exposure inhibited both the antigen-induced up-regulation of RhoA and BSM hyperresponsiveness, almost completely. A partial but significant inhibition of antigen-induced production of IL-13 was also found. These findings suggest that the inhibitory effects of STAT6 inhibitory agents, such as AS1517499, both on RhoA and IL-13 up-regulations might be useful for asthma treatment.

Molecular mechanisms of Ca2+-sensitization of smooth muscle contraction

Molecular mechanisms of Ca2+-sensitization of smooth muscle contraction

2P229 血管平滑筋のカルシウム非依存性収縮におけるアクトミオシン運動の解析(細胞生物的課題(接着・運動・骨格・伝達・膜),ポスター発表,第45回日本生物物理学会年会)

2P229 血管平滑筋のカルシウム非依存性収縮におけるアクトミオシン運動の解析(細胞生物的課題(接着・運動・骨格・伝達・膜),ポスター発表,第45回日本生物物理学会年会)

Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus

KCl has long been used as a convenient stimulus to bypass G protein-coupled receptors (GPCR) and activate smooth muscle by a highly reproducible and relatively "simple" mechanism involving activation of voltage-operated Ca2+ channels that leads to increases in cytosolic free Ca2+ ([Ca2+]i), Ca2+-calmodulin-dependent myosin light chain (MLC) kinase activation, MLC phosphorylation and contraction. This KCl-induced stimulus-response coupling mechanism is a standard tool-set used in comparative studies to explore more complex mechanisms generated by activation of GPCRs. One area where this approach has been especially productive is in studies designed to understand Ca2+ sensitization, the relationship between [Ca2+]i and force produced by GPCR agonists. Studies done in the late 1980s demonstrated that a unique relationship between stimulus-induced [Ca2+]i and force does not exist: for a given increase in [Ca2+]i, GPCR activation can produce greater force than KCl, and relaxant agents can produce the opposite effect to cause Ca2+ desensitization. Such changes in Ca2+ sensitivity are now known to involve multiple cell signaling strategies, including translocation of proteins from cytosol to plasma membrane, and activation of enzymes, including RhoA kinase and protein kinase C. However, recent studies show that KCl can also cause Ca2+ sensitization involving translocation and activation of RhoA kinase. Rather than complicating the Ca2+ sensitivity story, this surprising finding is already providing novel insights into mechanisms regulating Ca2+ sensitivity of smooth muscle contraction. KCl as a "simple" stimulus promises to remain a standard tool for smooth muscle cell physiologists, whose focus is to understand mechanisms regulating Ca2+ sensitivity.

Involvement of RhoA-mediated Ca2+ sensitization in antigen-induced bronchial smooth muscle hyperresponsiveness in mice

BackgroundIt has recently been suggested that RhoA plays an important role in the enhancement of the Ca2+ sensitization of smooth muscle contraction. In the present study, a participation of RhoA-mediated Ca2+ sensitization in the augmented bronchial smooth muscle (BSM) contraction in a murine model of allergic asthma was examined.MethodsOvalbumin (OA)-sensitized BALB/c mice were repeatedly challenged with aerosolized OA and sacrificed 24 hours after the last antigen challenge. The contractility and RhoA protein expression of BSMs were measured by organ-bath technique and immunoblotting, respectively.ResultsRepeated OA challenge to sensitized mice caused a BSM hyperresponsiveness to acetylcholine (ACh), but not to high K+-depolarization. In α-toxin-permeabilized BSMs, ACh induced a Ca2+ sensitization of contraction, which is sensitive to Clostridium botulinum C3 exoenzyme, indicating that RhoA is implicated in this Ca2+ sensitization. Interestingly, the ACh-induced, RhoA-mediated Ca2+ sensitization was significantly augmented in permeabilized BSMs of OA-challenged mice. Moreover, protein expression of RhoA was significantly increased in the hyperresponsive BSMs.ConclusionThese findings suggest that the augmentation of Ca2+ sensitizing effect, probably via an up-regulation of RhoA protein, might be involved in the enhanced BSM contraction in antigen-induced airway hyperresponsiveness.

Extracellular signal-regulated kinase1/2 in contraction of vascular smooth muscle

Smooth muscle contractility is regulated by both intracellular Ca 2+ concentration ([Ca 2+] i) and Ca 2+ sensitivity of the contractile apparatus. Extracellular signal-regulated kinases1/2 (ERK1/2) have been implicated in modulating Ca 2+ sensitivity of smooth muscle contraction but mechanisms of action remain elusive. This study investigated the roles of ERK1/2 in modulating [Ca 2+] i, calcium sensitivity and the 20-kDa myosin light chain (MLC 20) phosphorylation during contraction activated by α 1-adrenoceptor agonist phenylephrine and thromboxane A 2 mimetic U46619 in rat tail artery strips. A specific inhibitor for ERK1/2 activation, U0126, inhibited phenylephrine- and U46619-induced contraction, shifting both concentration-response curves rightward. During phenylephrine-stimulated contraction, U0126 exhibited concentration-dependent inhibition towards force but significant decreases in [Ca 2+] i were detected only at higher concentration. Both phenylephrine and U46619 induced a transient activation of ERK1/2 which was abolished by U0126 but unaffected by a general tyrosine kinase inhibitor genistein or Rho kinase inhibitor Y27632 at concentrations inhibiting more than 50% force. Interestingly, U0126 had no effect on steady-state MLC 20 phosphorylation levels stimulated by both receptor agonists. These results indicated that during contraction of rat tail artery smooth muscle activated by α 1-adrenoceptor agonist or thromboxane A 2 analogue, ERK1/2 increase Ca 2+ sensitivity that does not involve the modulation of MLC 20 phosphorylation.

Actions Downstream of Cyclic GMP/Protein Kinase G Can Reverse Protein Kinase C-mediated Phosphorylation of CPI-17 and Ca2+Sensitization in Smooth Muscle

Ca(2+) sensitivity of smooth muscle contraction is modulated by several systems converging on myosin light chain phosphatase (MLCP). Rho-Rho kinase is considered to inhibit MLCP via phosphorylation, whereas protein kinase C (PKC) induced sensitization has been shown to be dependent on phosphorylation of the inhibitory protein CPI-17. We have explored the interaction of cGMP-dependent protein kinase (PKG) with Ca(2+) sensitization pathways using permeabilized mouse smooth muscle. Three conditions giving approximately 50% of maximal active force were compared in small intestinal preparations: 1). Ca(2+)-activated unsensitized muscle (pCa 5.9 with Rho kinase inhibitor Y27632); 2). Rho-Rho kinase-sensitized muscle (pCa 6.1 with guanosine 5'-3-O-(thio)triphosphate); and 3). PKC-sensitized muscle (pCa 6.0 with Y27632 and PKC activator phorbol 12,13-dibutyrate). 8-Br-cGMP relaxed the sensitized muscles but had marginal effects on unsensitized preparations, showing that PKG reverses both PKC and Rho-mediated Ca(2+) sensitization. CPI-17 was present in permeabilized intestinal tissue. In PKC-sensitized preparations, CPI-17 phosphorylation decreased in response to 8-Br-cGMP. The rate of PKC-mediated phosphorylation in the presence of the MLCP inhibitor microcystin-LR was not influenced by 8-Br-cGMP. PKC-induced Ca(2+) sensitization also was reversed in vascular smooth muscle tissues (portal vein and femoral artery). We conclude that actions downstream of cGMP/PKG can reverse PKC-mediated phosphorylation of CPI-17 and Ca(2+) sensitization in smooth muscle.

Commentary

commentary

Identification of the endogenous smooth muscle myosin phosphatase-associated kinase.

Ca(2+) sensitization of smooth muscle contraction involves inhibition of myosin light chain phosphatase (SMPP-1M) and enhanced myosin light chain phosphorylation. Inhibition of SMPP-1M is modulated through phosphorylation of the myosin targeting subunit (MYPT1) by either Rho-associated kinase (ROK) or an unknown SMPP-1M-associated kinase. Activated ROK is predominantly membrane-associated and its putative substrate, SMPP-1M, is mainly myofibrillar-associated. This raises a conundrum about the mechanism of interaction between these enzymes. We present ZIP-like kinase, identified by "mixed-peptide" Edman sequencing after affinity purification, as the previously unidentified SMPP-1M-associated kinase. ZIP-like kinase was shown to associate with MYPT1 and phosphorylate the inhibitory site in intact smooth muscle. Phosphorylation of ZIP-like kinase was associated with an increase in kinase activity during carbachol stimulation, suggesting that the enzyme may be a terminal member of a Ca(2+) sensitizing kinase cascade.

Arachidonic acid-induced Ca2+ sensitization of smooth muscle contraction through activation of Rho-kinase.

Arachidonic acid activates isolated Rho-kinase and contracts permeabilized smooth muscle fibres. Various assays were carried out to examine the mechanism of this activation. Native Rho-kinase was activated 5-6 times by arachidonic acid but an N-terminal, constitutively-active fragment of Rho-kinase, expressed as a glutathione-S-transferase (GST) fusion protein and including the catalytic subunit (GST-Rho-kinase-CAT), was not. GST-Rho-kinase-CAT was inhibited by a C-terminal fragment of Rho-kinase and arachidonic acid removed this inhibition. These results suggest that the C-terminal part of Rho-kinase, containing the RhoA binding site and the pleckstrin homology domain, acts as an autoinhibitor. It is suggested further that activation by arachidonic acid is due to its binding to the autoinhibitory region and subsequent release from the catalytic site. Arachidonic acid, at concentrations greater than 30 microM, increases force in alpha-toxin-permeabilized femoral artery but not in Triton X-100-skinned fibres. The content of Rho-kinase in the latter was lower than in alpha-toxin-treated or intact fibres. The arachidonic acid-induced contraction was not observed at a pCa above 8.0 and was inhibited by Y-27632 and wortmannin, inhibitors of Rho-kinase and myosin light-chain kinase (MLCK), respectively. The activation of Rho-kinase and subsequent phosphorylation of the myosin phosphatase target subunit inhibits myosin phosphatase and increases myosin phosphorylation.

Phosphorylation of CPI-17, an Inhibitor of Myosin Phosphatase, by Protein Kinase N

CPI-17 is a phosphorylation-dependent inhibitory protein for smooth muscle myosin phosphate. Phosphorylation at Thr38, in vitro, by protein kinase C or Rho-kinase enhances the inhibitory potency toward myosin phosphatase. Phosphorylation of CPI-17 by protein kinase N (PKN), a fatty acid- and Rho-activated serine/threonine kinase, and its effect on smooth muscle myosin phosphatase activity were investigated. CPI-17 was phosphorylated by GST-PKN-CAT, a constitutively active GST-fusion fragment of PKN, to 1.46 mol of P/mol of CPI-17, in vitro. The Km value of CPI-17 for PKN was 0.96 μM. Phosphorylation of PKN dramatically increased the inhibitory effect of CPI-17 on myosin phosphatase activity. The major and inhibitory phosphorylation site was identified as Thr38 using a point mutant of CPI-17 and a phosphorylation-state specific antibody. Thus, CPI-17 is a substrate of PKN and might be involved in the Ca2+ sensitization of smooth muscle contraction as a downstream effector of Rho and/or arachidonic acid.