LY333531 attenuates contraction of tumor necrosis factor-α-sensitized human airway smooth muscle cells.

OBJECTIVE: Protein kinase C (PKC) has been implicated in the increased contraction of human airway smooth muscle cells (HASMCs) in asthma. Using the three-dimensional collagen gel contraction system, the study aimed to determine the effects of LY333531, a specific inhibitor of the PKC-ß isoform, on the contraction of tumor necrosis factor (TNF)-α-sensitized HASMCs. METHODS: Cultured HASMCs were divided into five groups: the control group received no treatment, and the cells in the TNF-α group were sensitized with 10 ng/mL TNF-α for 48 h, while TNF-α was administered to sensitize HASMCs in the presence of 0.1, 0.2, and 0.5 µM LY333531 for 48 h in the 0.1LY, 0.2LY, and 0.5LY groups, respectively. Following this, HASMCs contraction was stimulated with 1 mM acetylcholine (ACh) for up to 24 h in each group and assessed using a three-dimensional collagen gel contraction assay. Furthermore, western blot and immunofluorescence analysis were performed. RESULTS: The collagen gel contraction assay revealed that TNF-α increased the protein expression of phosphorylated PKC-ß2, CPI-17, and MLC while exacerbating ACh-induced HASMCs contraction. LY333531 significantly attenuated HASMCs contraction and downregulated the protein expression of both p-CPI-17 and p-MLC. CONCLUSIONS: At least in part by regulating CPI-17 and MLC phosphorylation, LY333531 attenuates augmented contraction of TNF-α-sensitized HASMCs in a collagen gel contraction system.

Phosphorylated CPI-17 and MLC2 as Biomarkers of Coronary Artery Spasm-Induced Sudden Cardiac Death.

Coronary artery spasm (CAS) plays an important role in the pathogeneses of various ischemic heart diseases and has gradually become a common cause of life-threatening arrhythmia. The specific molecular mechanism of CAS has not been fully elucidated, nor are there any specific diagnostic markers for the condition. Therefore, this study aimed to examine the specific molecular mechanism underlying CAS, and screen for potential diagnostic markers. To this end, we successfully constructed a rat CAS model and achieved in vitro culture of a human coronary-artery smooth-muscle cell (hCASMC) contraction model. Possible molecular mechanisms by which protein kinase C (PKC) regulated CAS through the C kinase-potentiated protein phosphatase 1 inhibitor of 17 kDa (CPI-17)/myosin II regulatory light chain (MLC2) pathway were studied in vivo and in vitro to screen for potential molecular markers of CAS. We performed hematoxylin and eosin staining, myocardial zymogram, and transmission electron microscopy to determine myocardial and coronary artery injury in CAS rats. Then, using immunohistochemical staining, immunofluorescence staining, and Western blotting, we further demonstrated a potential molecular mechanism by which PKC regulated CAS via the CPI-17/MLC2 pathway. The results showed that membrane translocation of PKCα occurred in the coronary arteries of CAS rats. CPI-17/MLC2 signaling was observably activated in coronary arteries undergoing CAS. In addition, in vitro treatment of hCASMCs with angiotensin II (Ang II) increased PKCα membrane translocation while consistently activating CPI-17/MLC2 signaling. Conversely, GF-109203X and calphostin C, specific inhibitors of PKC, inactivated CPI-17/MLC2 signaling. We also collected the coronary artery tissues from deceased subjects suspected to have died of CAS and measured their levels of phosphorylated CPI-17 (p-CPI-17) and MLC2 (p-MLC2). Immunohistochemical staining was positive for p-CPI-17 and p-MLC2 in the tissues of these subjects. These findings suggest that PKCα induced CAS through the CPI-17/MLC2 pathway; therefore, p-CPI-17 and p-MLC2 could be used as potential markers for CAS. Our data provide novel evidence that therapeutic strategies against PKC or CPI-17/MLC2 signaling might be promising in the treatment of CAS.

The Evolution of Duplicated Genes of the Cpi-17/Phi-1 (ppp1r14) Family of Protein Phosphatase 1 Inhibitors in Teleosts.

The Cpi-17 (ppp1r14) gene family is an evolutionarily conserved, vertebrate specific group of protein phosphatase 1 (PP1) inhibitors. When phosphorylated, Cpi-17 is a potent inhibitor of myosin phosphatase (MP), a holoenzyme complex of the regulatory subunit Mypt1 and the catalytic subunit PP1. Myosin phosphatase dephosphorylates the regulatory myosin light chain (Mlc2) and promotes actomyosin relaxation, which in turn, regulates numerous cellular processes including smooth muscle contraction, cytokinesis, cell motility, and tumor cell invasion. We analyzed zebrafish homologs of the Cpi-17 family, to better understand the mechanisms of myosin phosphatase regulation. We found single homologs of both Kepi (ppp1r14c) and Gbpi (ppp1r14d) in silico, but we detected no expression of these genes during early embryonic development. Cpi-17 (ppp1r14a) and Phi-1 (ppp1r14b) each had two duplicate paralogs, (ppp1r14aa and ppp1r14ab) and (ppp1r14ba and ppp1r14bb), which were each expressed during early development. The spatial expression pattern of these genes has diverged, with ppp1r14aa and ppp1r14bb expressed primarily in smooth muscle and skeletal muscle, respectively, while ppp1r14ab and ppp1r14ba are primarily expressed in neural tissue. We observed that, in in vitro and heterologous cellular systems, the Cpi-17 paralogs both acted as potent myosin phosphatase inhibitors, and were indistinguishable from one another. In contrast, the two Phi-1 paralogs displayed weak myosin phosphatase inhibitory activity in vitro, and did not alter myosin phosphorylation in cells. Through deletion and chimeric analysis, we identified that the difference in specificity for myosin phosphatase between Cpi-17 and Phi-1 was encoded by the highly conserved PHIN (phosphatase holoenzyme inhibitory) domain, and not the more divergent N- and C- termini. We also showed that either Cpi-17 paralog can rescue the knockdown phenotype, but neither Phi-1 paralog could do so. Thus, we provide new evidence about the biochemical and developmental distinctions of the zebrafish Cpi-17 protein family.

Relationship between uterine smooth muscular CPI-17-signal pathway-mediated Ca2+ sensitivity changes and uterine atony-induced postpartum haemorrhage.

This study aimed to investigate the changes of protein kinase C (PKC)-potentiated phosphatase inhibitor of 17 ku (CPI-17) expression, PKC activity and Rho kinase activity in the maternal uterine smooth muscle (USM), and their roles in the occurrence of uterine atony-induced postpartum haemorrhage (UAI-PPH). Sixty primiparaes who had a caesarean section performed were divided into the case group (with UAI-PPH) and the control group (the uterine contraction was good, without the PPH). The USM-p-CPI-17 (Thr38) protein levels, the activities of PKC and Rho kinase in the case group and the control group were 0.43 ± 0.20, 4.30 ± 0.91, 10.85 ± 1.70 and 0.67 ± 0.32, 0.099 ± 0.028, 0.20 ± 0.071, respectively (p < .05). The down-regulated expression of CPI-17 phosphorylated proteins might be one of the important factors of UAI-PPH, while the activity reduction of PKC and Rho kinase might be the reason that led to the phosphorylation level reduction of USM-CPI-17 in UAI-PPH. Impact Statement What is already known on this subject? The studies have shown that in the late pregnancy period, the total protein and phosphorylated protein of myometrial CPI-17 are significantly higher than in the non-pregnancy state, and they were all involved in regulating and enhancing the Ca2+ sensitivity of USMC during the pregnancy. The data regarding the CPI-17-signal pathway-mediated Ca2+ sensitivity in UAI-PPH is sparse. What do the results of this study add? We have shown that the down-regulated expression of CPI-17 phosphorylated proteins might be one of the important factors of UAI-PPH, while the activity reduction of PKC and Rho kinase might be the reason that led to the phosphorylation level reduction of USM-CPI-17 in UAI-PPH. What are the implications of these findings for clinical practice and/or further research? Further studies are needed to confirm the pathogenesis of CPI-17-signal pathway-mediated Ca2+ sensitivity in UAI-PPH.

The functional role of protease-activated receptors on contractile responses by activation of Ca2+ sensitization pathways in simian colonic muscles.

It has been known that activation of protease-activated receptors (PARs) affects gastrointestinal motility. In this study, we tested the effects of PAR agonists on electrical and contractile responses and Ca2+ sensitization pathways in simian colonic muscles. The Simian colonic muscle was initially hyperpolarized by PAR agonists. After the transient hyperpolarization, simian colonic muscle repolarized to the control resting membrane potential (RMP) without a delayed depolarization. Apamin significantly reduced the initial hyperpolarization, suggesting that activation of small conductance Ca2+-activated K+ (SK) channels is involved in the initial hyperpolarization. In contractile experiments, PAR agonists caused an initial relaxation followed by an increase in contractions. These delayed contractile responses were not matched with the electrical responses that showed no after depolarization of the RMP. To investigate the possible involvement of Rho-associated protein kinase 2 (ROCK) pathways in the PAR effects, muscle strips were treated with ROCK inhibitors, which significantly reduced the PAR agonist-induced contractions. Furthermore, PAR agonists increased MYPT1 phosphorylation, and ROCK inhibitors completely blocked MYPT1 phosphorylation. PAR agonists alone had no effect on CPI-17 phosphorylation. In the presence of apamin, PAR agonists significantly increased CPI-17 phosphorylation, which was blocked by protein kinase C (PKC) inhibitors suggesting that Ca2+ influx is increased by apamin and is activating PKC. In conclusion, these studies show that PAR activators induce biphasic responses in simian colonic muscles. The initial inhibitory responses by PAR agonists are mainly mediated by activation of SK channels and delayed contractile responses are mainly mediated by the CPI-17 and ROCK Ca2+ sensitization pathways in simian colonic muscles. NEW & NOTEWORTHY In the present study, we found that the contractile responses of simian colonic muscles to protease-activated receptor (PAR) agonists are different from the previously reported contractile responses of murine colonic muscles. Ca2+ sensitization pathways mediate the contractile responses of simian colonic muscles to PAR agonists without affecting the membrane potential. These findings emphasize novel mechanisms of PAR agonist-induced contractions possibly related to colonic dysmotility in inflammatory bowel disease.

Prolonged bed rest impairs rapid CPI-17 phosphorylation and contraction in rat mesenteric resistance arteries to cause orthostatic hypotension.

Prolonged bed rest (PBR) causes orthostatic hypotension (OH). Rapid constriction of splanchnic resistance arteries in response to a sudden increase in sympathetic tone contributes to the recovery of orthostatic arterial pressure upon standing. However, the molecular mechanism of PBR-induced dysfunction in arterial constriction is not fully understood. Previously, we showed that CPI-17, a regulatory protein for myosin phosphatase, mediates α1A-adrenergic receptor-induced rapid contraction of small mesenteric arteries. Here, we tested whether PBR associated with OH affects the α1-adrenergic receptor-induced CPI-17 signaling pathway in mesenteric arteries using rats treated by head-down tail-suspension hindlimb unloading (HDU), an experimental OH model. In normal anesthetized rats, mean arterial pressure (MAP) rapidly reduced upon 90° head-up tilt from supine position and then immediately recovered without change in heart rate, suggesting a rapid arterial constriction. On the other hand, after a 4-week HDU treatment, the fast orthostatic MAP recovery failed for 1 min. Alpha1A subtype-specific antagonist suppressed the orthostatic MAP recovery with a small decrease in basal blood pressure, whereas non-specific α1-antagonist prazosin strongly reduced both basal MAP and orthostatic recovery. The HDU treatment resulted in 68% reduction in contraction in parallel with 83% reduction in CPI-17 phosphorylation in denuded mesenteric arteries 10 s after α1-agonist stimulation. The treatment with either Ca2+-release channel opener or PKC inhibitor mimicked the deficiency in HDU arteries. These results suggest that an impairment of the rapid PKC/CPI-17 signaling pathway downstream of α1A-adrenoceptors in peripheral arterial constriction, as an end organ of orthostatic blood pressure reflex, is associated with OH in prolonged bed rest patients.

High fat diet confers vascular hyper-contractility against angiotensin II through upregulation of MLCK and CPI-17.

Obesity is a critical risk factor for the hypertension. Although angiotensin II (Ang II) in obese individuals is known to be upregulated in obesity-induced hypertension, direct evidence that explains the underlying mechanism for increased vascular tone and consequent increase in blood pressure (BP) is largely unknown. The purpose of this study is to investigate the novel mechanism underlying Ang II-induced hyper-contractility and hypertension in obese rats. Eight-week old male Sprague-Dawley rats were fed with 60% fat diet or normal diet for 4 months. Body weight, plasma lipid profile, plasma Ang II level, BP, Ang II-induced vascular contraction, and expression of regulatory proteins modulating vascular contraction with/without Ang II stimulation were measured. As a result, high fat diet (HFD) accelerated age-dependent body weight gaining along with increased plasma Ang II concentration. It also increased BP and Ang II-induced aortic contraction. Basal expression of p-CPI-17 and myosin light chain (MLC) kinase was increased by HFD along with increased phosphorylation of MLC. Ang II-induced phosphorylation of CPI-17 and MLC were also higher in HFD group than control group. In conclusion HFD-induced hypertension is through at least in part by increased vascular contractility via increased expression and activation of contractile proteins and subsequent MLC phosphorylation induced by increased Ang II.

Regulation of mitogen-activated protein kinase by protein kinase C and mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle.

Vascular smooth muscle contraction is primarily regulated by phosphorylation of myosin light chain. There are also modulatory pathways that control the final level of force development. We tested the hypothesis that protein kinase C (PKC) and mitogen-activated protein (MAP) kinase modulate vascular smooth muscle activity via effects on MAP kinase phosphatase-1 (MKP-1). Swine carotid arteries were mounted for isometric force recording and subjected to histamine stimulation in the presence and absence of inhibitors of PKC [bisindolylmaleimide-1 (Bis)], MAP kinase kinase (MEK) (U0126), and MKP-1 (sanguinarine) and flash frozen for measurement of MAP kinase, PKC-potentiated myosin phosphatase inhibitor 17 (CPI-17), and caldesmon phosphorylation levels. CPI-17 was phosphorylated in response to histamine and was inhibited in the presence of Bis. Caldesmon phosphorylation levels increased in response to histamine stimulation and were decreased in response to MEK inhibition but were not affected by the addition of Bis. Inhibition of PKC significantly increased p42 MAP kinase, but not p44 MAP kinase. Inhibition of MEK with U0126 inhibited both p42 and p44 MAP kinase activity. Inhibition of MKP-1 with sanguinarine blocked the Bis-dependent increase of MAP kinase activity. Sanguinarine alone increased MAP kinase activity due to its effects on MKP-1. Sanguinarine increased MKP-1 phosphorylation, which was inhibited by inhibition of MAP kinase. This suggests that MAP kinase has a negative feedback role in inhibiting MKP-1 activity. Therefore, PKC catalyzes MKP-1 phosphorylation, which is reversed by MAP kinase. Thus the fine tuning of vascular contraction is due to the concerted effort of PKC, MAP kinase, and MKP-1.

Resolvin E1 normalizes contractility, Ca2+ sensitivity and smooth muscle cell migration rate in TNF-α- and IL-6-pretreated human pulmonary arteries.

Pulmonary hypertension (PH) is a rare disease in which pathophysiology is characterized by an increase in proinflammatory mediators, chronic endothelial dysfunctions, and a high migration rate of smooth muscle cells (SMC). Over the course of the last decade, various treatments have been proposed to relax the pulmonary arteries, none of which have been effective in resolving PH. Our hypothesis is that artery-relaxing drugs are not the long-term solution, but rather the inhibition of tissue inflammation, which underlies human pulmonary artery (HPA) dysfunctions that lead to abnormal vasoconstriction. The goal of the present study was to assess the anti-inflammatory effects of resolvin E1 (RvE1) with concomitant effects on SMC migration and on HPA reactivity. The role and mode of action of RvE1 and its precursor, monoacylglyceride eicosapentaenoic acid were assessed on HPA under proinflammatory conditions, involving a combined pretreatment with 10 ng/ml TNF-α and 10 ng/ml IL-6. Our results show that TNF-α and IL-6 treatment induced hyperreactivity and Ca(2+) hypersensitivity in response to pharmaco-mechanical stimuli, including 80 mM KCl, 1 µM phorbol 12-13-dibutyrate, and 30 nM U-46619. Furthermore, the proinflammatory treatment increased the migration rate of SMC isolated from HPA. The phosphorylation level of regulatory contractile proteins (CPI-17, MYPT-1), and proinflammatory signaling pathways (c-Fos, c-Jun, NF-κB) were also significantly increased compared with control conditions. Conversely, 300 nM RvE1 was able to normalize all of the above abnormal events triggered by proinflammation. In conclusion, RvE1 can resolve human arterial hyperreactivity via the resolution of inflammatory markers.

Reversal of IL-13-induced inflammation and Ca(2+) sensitivity by resolvin and MAG-DHA in association with ASA in human bronchi.

The aim of this study was to investigate the effects of resolvin D1 (RvD1), as well as the combined treatment of docosahexaenoic acid monoglyceride (MAG-DHA) and acetylsalicylic acid (ASA), on the resolution of inflammation markers and Ca(2+) sensitivity in IL-13-pretreated human bronchi (HB). Tension measurements performed with 300 nM RvD1 largely abolished (50%) the over-reactivity triggered by 10 ng/ml IL-13 pretreatment and reversed hyper Ca(2+) sensitivity. Addition of 300 nM 17(S)-HpDoHE, the metabolic intermediate between DHA and RvD1, displayed similar effects. In the presence of 100 µM ASA (a COX inhibitor), the inhibitory effect of 1 µM MAG-DHA on muscarinic tone was further amplified, but not in the presence of Ibuprofen. Western blot analysis revealed that the combined treatment of MAG-DHA and ASA upregulated GPR-32 expression and downregulated cytosolic TNFα detection, hence preventing IκBα degradation and p65-NFκB phosphorylation. The Ca(2+) sensitivity of HB was also quantified on ß-escin permeabilized preparations. The presence of ASA potentiated the inhibitory effects of MAG-DHA in reducing the Ca(2+) hypersensitivity triggered by IL-13 by decreasing the phosphorylation levels of the PKC-potentiated inhibitor protein-17 regulatory protein (CPI-17). In summary, MAG-DHA combined with ASA, as well as exogenously added RvD1, may represent valuable assets against critical AHR disorder.

Resolvin D1 reverses reactivity and Ca2+ sensitivity induced by ET-1, TNF-α, and IL-6 in the human pulmonary artery.

Pulmonary hypertension (PH) is a rare and progressive disease characterized by an inflammatory status and vessel wall remodeling, resulting in increased pulmonary artery resistance. During the last decade, treatments have been proposed; most of them target the endothelial pathways that stimulate smooth muscle cell relaxation. However, PH remains associated with significant morbidity. We hypothesized that inflammation plays a crucial role in the severity of the abnormal vasoconstriction in PH. The goal of this study was to assess the effects of resolvin D1 (RvD1), a potent anti-inflammatory agent, on the pharmacological reactivity of human pulmonary arteries (HPAs) via an in vitro model of induced hyperreactivity. The effects of RvD1 and monoacylglyceride compounds were measured on contractile activity and Ca(2+) sensitivity developed by HPAs that had been pretreated (or not) under proinflammatory conditions with either 10 ng/ml TNF-α or 10 ng/ml IL-6 or under hyperreactive conditions with 5 nM endothelin-1. The results demonstrated that, compared with controls, 24-h pretreatment with TNF-α, IL-6, or endothelin-1 increased reactivity and Ca(2+) sensitivity of HPAs as revealed by agonist challenges with 80 mM KCl, 1 µM serotonin (5-hydroxytryptamine), 30 nM U-46619, and 1 µM phorbol 12,13-dibutyrate. However, 300 nM RvD1 as well as 1 µM monoacylglyceride-docosapentaenoic acid monoglyceride strongly reversed the overresponsiveness induced by both proinflammatory and hyperreactive treatments. In pretreated pulmonary artery smooth muscle cells, Western blot analyses revealed that RvD1 treatment decreased the phosphorylation level of CPI-17 and expression of transmembrane protein member 16A while increasing the detection of G protein-coupled receptor 32. The present data demonstrate that RvD1, a trihydroxylated docosahexaenoic acid derivative, decreases induced overreactivity in HPAs via a reduction in CPI-17 phosphorylation and transmembrane protein member 16A expression.

Myosin phosphatase isoforms as determinants of smooth muscle contractile function and calcium sensitivity of force production.

The dephosphorylation of myosin by the MP causes smooth muscle relaxation. MP is also a key target of signals that regulate vascular tone and thus blood flow and pressure. Here, we review studies from the past two decades that support the hypothesis that the regulated expression of MP subunits is a critical determinant of smooth muscle responses to constrictor and dilator signals. In particular, the highly regulated splicing of the regulatory subunit Mypt1 Exon 24 is proposed to tune sensitivity to NO/cGMP-mediated relaxation. The regulated transcription of the MP inhibitory subunit CPI-17 is proposed to determine sensitivity to agonist-mediated constriction. The expression of these subunits is specific in the microcirculation and varies in developmental and disease contexts. To date, the relationship between MP subunit expression and vascular function in these different contexts is correlative; confirmation of the hypothesis will require the generation of genetically engineered mice to test the role of MP subunits and their isoforms in the specificity of vascular smooth muscle responses to constrictor and dilator signals.

A bioinformatic and computational study of myosin phosphatase subunit diversity.

Variability in myosin phosphatase (MP) subunits may provide specificity in signaling pathways that regulate muscle tone. We utilized public databases and computational algorithms to investigate the phylogenetic diversity of MP regulatory (PPP1R12A-C) and inhibitory (PPP1R14A-D) subunits. The comparison of exonic coding sequences and expression data confirmed or refuted the existence of isoforms and their tissue-specific expression in different model organisms. The comparison of intronic and exonic sequences identified potential expressional regulatory elements. As examples, smooth muscle MP regulatory subunit (PPP1R12A) is highly conserved through evolution. Its alternative exon E24 is present in fish through mammals with two invariant features: 1) a reading frame shift generating a premature termination codon and 2) a hexanucleotide sequence adjacent to the 3' splice site hypothesized to be a novel suppressor of exon splicing. A characteristic of the striated muscle MP regulatory subunit (PPP1R12B) locus is numerous and phylogenetically variable transcriptional start sites. In fish this locus only codes for the small (M21) subunit, suggesting the primordial function of this gene. Inhibitory subunits show little intragenic variability; their diversity is thought to have arisen by expansion and tissue-specific expression of different gene family members. We demonstrate differences in the regulatory landscape between smooth muscle enriched (PPP1R14A) and more ubiquitously expressed (PPP1R14B) family members and identify deeply conserved intronic sequence and predicted transcriptional cis-regulatory elements. This bioinformatic and computational study has uncovered a number of attributes of MP subunits that supports selection of ideal model organisms and testing of hypotheses regarding their physiological significance and regulated expression.

Effect of Kaempferol on Modulation of Vascular Contractility Mainly through PKC and CPI-17 Inactivation.

In this study, we investigated the efficacy of kaempferol (a flavonoid found in plants and plant-derived foods such as kale, beans, tea, spinach and broccoli) on vascular contractibility and aimed to clarify the detailed mechanism underlying the relaxation. Isometric contractions of divested muscles were stored and linked with western blot analysis which was carried out to estimate the phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and phosphorylation-dependent inhibitory protein for myosin phosphatase (CPI-17) and to estimate the effect of kaempferol on the RhoA/ROCK/CPI-17 pathway. Kaempferol conspicuously impeded phorbol ester-, fluoride- and a thromboxane mimetic-derived contractions regardless of endothelial nitric oxide synthesis, indicating its direct effect on smooth muscles. It also conspicuously impeded the fluoride-derived elevation in phospho-MYPT1 rather than phospho-CPI-17 levels and phorbol 12,13-dibutyrate-derived increase in phospho-CPI-17 and phospho-ERK1/2 levels, suggesting the depression of PKC and MEK activities and subsequent phosphorylation of CPI-17 and ERK1/2. Taken together, these outcomes suggest that kaempferol-derived relaxation incorporates myosin phosphatase retrieval and calcium desensitization, which appear to be modulated by CPI-17 dephosphorylation mainly through PKC inactivation.

The Effect of Luteolin on the Modulation of Vascular Contractility via ROCK and CPI-17 Inactivation.

In this investigation, we made a study of the efficacy of luteolin (a flavonoid found in plants such as vegetables, herbs and fruits) on vascular contractibility and to elucidate the mechanism underlying the relaxation. Isometric contractions of denuded muscles were stored and combined with western blot analysis which was conducted to assess the phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and phosphorylation-dependent inhibitory protein for myosin phosphatase (CPI-17) and to examine the effect of luteolin on the RhoA/ROCK/CPI-17 pathway. Luteolin significantly alleviated phorbol ester-, fluoride- and thromboxane mimetic-elicited contractions regardless of endothelial nitric oxide synthesis, implying its direct effect on smooth muscle. It also significantly alleviated the fluoride-elicited elevation in pCPI-17 and pMYPT1 levels and phorbol 12,13-dibutyrate-elicited increase in pERK1/2 level, suggesting depression of ROCK and PKC/MEK activity and ensuing phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that luteolin-elicited relaxation includes myosin phosphatase reactivation and calcium desensitization, which seems to be arbitrated by CPI-17 dephosphorylation via ROCK/PKC inhibition.

The Effect of Galangin on the Regulation of Vascular Contractility via the Holoenzyme Reactivation Suppressing ROCK/CPI-17 rather than PKC/CPI-17.

In this study, we investigated the influence of galangin on vascular contractibility and to determine the mechanism underlying the relaxation. Isometric contractions of denuded aortic muscles were recorded and combined with western blot analysis which was performed to measure the phosphorylation of phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) and to evaluate the effect of galangin on the RhoA/ROCK/CPI-17 pathway. Galangin significantly inhibited phorbol ester-, fluoride- and thromboxane mimetic-induced vasoconstrictions regardless of endothelial nitric oxide synthesis, suggesting its direct effect on vascular smooth muscle. Galangin significantly inhibited the fluoride-dependent increase in pMYPT1 and pCPI-17 levels and phorbol 12,13-dibutyrate-dependent increase in pERK1/2 level, suggesting repression of ROCK and MEK activity and subsequent phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that galangin-induced relaxation involves myosin phosphatase reactivation and calcium desensitization, which appears to be mediated by CPI-17 dephosphorylation via not PKC but ROCK inactivation.

Characterization of Contractile Machinery of Vascular Smooth Muscles in Hypertension.

Hypertension is a key risk factor for cardiovascular disease and it is a growing public health problem worldwide. The pathophysiological mechanisms of vascular smooth muscle (VSM) contraction contribute to the development of hypertension. Calcium (Ca2+)-dependent and -independent signaling mechanisms regulate the balance of the myosin light chain kinase and myosin light chain phosphatase to induce myosin phosphorylation, which activates VSM contraction to control blood pressure (BP). Here, we discuss the mechanism of the contractile machinery in VSM, especially RhoA/Rho kinase and PKC/CPI-17 of Ca2+ sensitization pathway in hypertension. The two signaling pathways affect BP in physiological and pathophysiological conditions and are highlighted in pulmonary, pregnancy, and salt-sensitive hypertension.

CPI-17-mediated contraction of vascular smooth muscle is essential for the development of hypertension in obese mice.

Several factors have been implicated in obesity-related hypertension, but the genesis of the hypertension is largely unknown. In this study, we found a significantly upregulated expression of CPI-17 (C-kinase-potentiated protein phosphatase 1 inhibitor of 17 kDa) and protein kinase C (PKC) isoforms in the vascular smooth muscles of high-fat diet (HFD)-fed obese mice. The obese wild-type mice showed a significant elevation of blood pressure and enhanced calcium-sensitized contraction of vascular smooth muscles. However, the obese CPI-17-deficient mice showed a normotensive blood pressure, and the calcium-sensitized contraction was consistently reduced. In addition, the mutant muscle displayed an abolished responsive force to a PKC activator and a 30%-50% reduction in both the initial peak force and sustained force in response to various G protein-coupled receptor (GPCR) agonists. Our observations showed that CPI-17-mediated calcium sensitization is mediated through a GPCR/PKC/CPI-17/MLCP/RLC signaling pathway. We therefore propose that the upregulation of CPI-17-mediated calcium-sensitized vasocontraction by obesity contributes to the development of obesity-related hypertension.

Levobupivacaine-induced vasoconstriction involves caldesmon phosphorylation mediated by tyrosine kinase-induced ERK phosphorylation.

The goals of this study were to examine the cellular signaling pathways associated with the phosphorylation of caldesmon, the phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17), and the 20-kDa regulatory light chain of myosin (MLC20) induced by levobupivacaine in isolated rat aortas. The effects of genistein, tyrphostin 23, GF109203X, PD98059, Y-27632, 1-butanol, and ML-7 HCl on levobupivacaine-induced contraction were assessed. The effect of genistein on the simultaneous calcium-tension curves induced by levobupivacaine was examined. The effects of GF109203X, genistein, PD98059 and extracellular signal-regulated kinase (ERK) siRNA on levobupivacaine-induced caldesmon phosphorylation were investigated. The effect of genistein on the ERK and tyrosine phosphorylation induced by levobupivacaine was examined. The effect of GF109203X, PD98059, Y-27632, SP600125, and ML-7 HCl on the levobupivacaine-induced phosphorylation of CPI-17 and MLC20 were investigated. Genistein, tyrphostin 23, GF109203X, PD98059, Y-27632, ML-7 HCl, and 1-butanol attenuated levobupivacaine-induced contraction. Genistein caused a right downward shift of the calcium-tension curves induced by levobupivacaine. Genistein attenuated levobupivacaine-induced phosphorylation of protein tyrosine, ERK and caldesmon. PD98059, ERK siRNA and GF109203X attenuated levobupivacaine-induced caldesmon phosphorylation. GF109203X, Y-27632, SP600125, ML-7 HCl and PD98059 attenuated CPI-17 phosphorylation and MLC20 phosphorylation induced by levobupivacaine. These results suggest that levobupivacaine-induced caldesmon phosphorylation contributing to levobupivacaine-induced contraction is mediated by a pathway involving ERK, which is activated by tyrosine kinase or protein kinase C (PKC). The phosphorylation of CPI-17 and MLC20 induced by levobupivacaine is mediated by cellular signaling pathways involving PKC, Rho-kinase, and c-Jun NH2-terminal kinase or PKC, Rho-kinase, ERK, and myosin light chain kinase.

Differential Regulation of Myosin Regulatory Light Chain Phosphorylation by Protein Kinase C Isozymes in Human Uterine Myocytes.

BACKGROUND: Preterm birth is the most common cause of neonatal morbidity and mortality and a common precedent to lifelong disability. Current treatment has minimal efficacy. OBJECTIVE: We assessed the role of isozymes of the protein kinase C (PKC) family in regulating the phosphorylation of myosin regulatory light chains (RLCs), which regulate uterine contractility. We also explored the mechanisms through which these isozymes function. STUDY DESIGN: We used a previously characterized and validated quantitative in-cell Western (ICW) assay to measure site-specific phosphorylations on myosin RLC and CPI-17. Cultures of human uterine myocytes (hUM) were treated with the potent contractile stimulant oxytocin to induce uterine contractility or a pharmacological mimic of diacyl-glycerol to stimulate the conventional and novel isozymes of the PKC family. Combinations of isozyme-selective inhibitors were used to determine the effects of the conventional and novel classes of isozymes. RESULTS: Stimulation of PKC using phospho-dibutyrate caused immediate, concentration-dependent inhibition of uterine activity ex vivo. Using the ICW assay with hUM, the oxytocin-stimulated increase in the pro-contractile phosphorylations of myosin RLCs at serine19 and threonine18 was completely inhibited by prior treatment with phorbol-12-myristate-13-acetate, which stimulates both convention and novel classes of isozymes. Our results suggest that the conventional class of isozymes cause a reduction in phosphorylations at serine19 and threonine18 by reducing activity of myosin light chain kinase. The novel class of isozymes has 2 mechanisms of action: the first is activation of CPI-17 through phosphorylation at threonine38, which results in reduced activity of myosin light chain phosphatase and increased levels of activated myosin RLC; the second is increased phosphorylation of the N-terminal region of myosin RLC. CONCLUSIONS: Specific agonists for the conventional isozymes or inhibitors of the novel isozymes of the PKC family could be useful pharmacological agents for regulation of uterine activity.