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Background Thromboxane A2 (TXA2 ) participates in many pathophysiological processes of coronary artery disease. However, its mechanism of TXA2 -induced contraction in the coronary artery remains to be clarified. Methods Multi Myograph System was used to measure the isometric tension of the mouse coronary arteries and identify the effect and pathway of TXA2 analogues U46619. Confocal laser scanning microscopy was used to measure the intracellular calcium concentration ([Ca2+ ]i ) in mouse coronary artery smooth muscle cells. Results Results from the experiment had shown that contraction in coronary artery was generated by U46619 in a concentration- dependent manner, which was completely abolished by a specific TXA2 receptor blocker, GR32191. PI-PLC inhibitors U73122 and D609 and Rho-Kinase inhibitor Y-27632 can block the U46619 elicited coronary artery contraction in a dose-dependent manner. Then, the vasoconstriction response to U46619 was obviously inhibited by two pan-PKC inhibitors chelerythrine or Gӧ6983, and a selective PKCδ inhibitor rottlerin, but wasn't blocked by a selective PKCζ inhibitor PKC-PS or a selective PKCβ inhibitor hispidin. Meanwhile, the PKC activator PDBu-induced vasoconstriction was significantly inhibited by 1 μM nifedipine,then mostly inhibited by 100 μM 2-APB and 10 μM Y27632. We further found that the response to U46619 was inhibited respectively by three calcium channel blockers nifedipine, SKF96356 or 2-APB in a concentration-dependent manner. Although Store-operated Ca2+ (SOC) channels generated the increase of [Ca2+ ]i in mouse coronary artery smooth muscle cells, SOC channels didn't contribute to the vasoconstriction in mouse coronary arteries. Caffeine-induced sarcoplasmic reticulum (SR) Ca2+ release could obviously induce coronal vasoconstriction. In addition, NPPB, a cell membrane Ca2+ activated C1- channel blocker, could obviously inhibit the U46619-induced vasoconstriction. Conclusions The U46619-induced mouse coronary artery contraction was involved in the increase in [Ca2+ ]i mediated by Cav1.2, TRPC channels and SR release through the activation of G-protein-coupled TP receptors and the kinases signaling pathway in TP downstream proteins, while SOC channels didn't participate in the vasoconstriction.

It is well-established that long-term exposure of the vasculature to metabolic disturbances leads to abnormal vascular tone, while the physiological regulation of vascular tone upon acute metabolic challenge remains unknown. Here, we found that acute glucose challenge induced transient increases in blood pressure and vascular constriction in humans and mice. Ex vivo study in isolated thoracic aortas from mice showed that glucose-induced vascular constriction is dependent on glucose oxidation in vascular smooth muscle cells. Specifically, mitochondrial membrane potential (ΔΨm), an essential component in glucose oxidation, was increased along with glucose influx and positively regulated vascular smooth muscle tone. Mechanistically, mitochondrial hyperpolarization inhibited the activity of myosin light chain phosphatase (MLCP) in a Ca2+-independent manner through activation of Rho-associated kinase, leading to cell contraction. However, ΔΨm regulated smooth muscle tone independently of the small G protein RhoA, a major regulator of Rho-associated kinase signaling. Furthermore, myosin phosphatase target subunit 1 (MYPT1) was found to be a key molecule in mediating MLCP activity regulated by ΔΨm. ΔΨm positively phosphorylated MYPT1, and either knockdown or knockout of MYPT1 abolished the effects of glucose in stimulating smooth muscle contraction. In addition, smooth muscle-specific Mypt1 knockout mice displayed blunted response to glucose challenge in blood pressure and vascular constriction and impaired clearance rate of circulating metabolites. These results suggested that glucose influx stimulates vascular smooth muscle contraction via mitochondrial hyperpolarization-inactivated myosin phosphatase, which represents a novel mechanism underlying vascular constriction and circulating metabolite clearance.

Class II phosphatidylinositol 3-kinases (PI3K), PI3K-C2α and PI3K-C2β, are involved in cellular processes including endocytosis, cilia formation and autophagy. However, the role of PI3K-C2α and PI3K-C2β at the organismal level is not well understood. We found that double knockout (KO) mice with both smooth muscle-specific KO of PI3K-C2α and global PI3K-C2β KO, but not single KO mice of either PI3K-C2α or PI3K-C2β, exhibited reductions in arterial blood pressure and substantial attenuation of contractile responses of isolated aortic rings. In wild-type vascular smooth muscle cells, double knockdown of PI3K-C2α and PI3K-C2β but not single knockdown of either PI3K markedly inhibited contraction with reduced phosphorylation of 20-kDa myosin light chain and MYPT1 and Rho activation, but without inhibition of the intracellular Ca2+ mobilization. These data indicate that PI3K-C2α and PI3K-C2β play the redundant but essential role for vascular smooth muscle contraction and blood pressure regulation mainly through their involvement in Rho activation.

Scutellaria baicalensis Georgi is an extensively used medicinal herb for the treatment of hypertension in traditional Chinese medicine. Baicalin, is an important flavonoid in Scutellaria baicalensis Georgi extracts, which exhibits therapeutic effects on anti-hypertension, but its underlying mechanisms remain to be further explored. Therefore, we investigated the effects and molecular mechanisms of Baicalin on anti-hypertension. In vivo studies revealed that Baicalin treatment significantly attenuated the elevation in blood pressure, the pulse propagation and thickening of the abdominal aortic wall in C57BL/6 mice infused with Angiotensin II (Ang II). Moreover, RNA-sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified 537 differentially expressed transcripts and multiple enriched signaling pathways (including vascular smooth muscle contraction and calcium signaling pathway). Consistently, we found that Baicalin pretreatment significantly alleviated the Ang II induced constriction of abdominal aortic ring, while promoted NE pre-contracted vasodilation of abdominal aortic ring at least partly dependent on L-type calcium channel. In addition, Ang II stimulation significantly increased cell viability and PCNA expression, while were attenuated after Baicalin treatment. Moreover, Baicalin pretreatment attenuated Ang II-induced intracellular Ca2+ release, Angiotensin II type 1 receptor (AT1R) expression and activation of MLCK/p-MLC pathway in vascular smooth muscle cells (VSMCs). The present work further addressed the pharmacological and mechanistic insights on anti-hypertension of Baicalin, which may help better understand the therapeutic effect of Scutellaria baicalensis Georgi on anti-hypertension.

Pulmonary hypertension (PH) is a serious disorder with high morbidity and mortality rate. We analyzed the right-ventricular systolic pressure (RVSP), right-ventricular hypertrophy (RVH), lung histology, and transcriptomes of six-week-old male rats with PH induced by (1) hypoxia (HO), (2) administration of monocrotaline (CM), or (3) administration of monocrotaline and exposure to hypoxia (HM). The results in PH rats were compared to those in control rats (CO). After four weeks exposure, increased RVSP and RVH, pulmonary arterial wall thickening, and alteration of the lung transcriptome were observed in all PH groups. The HM group exhibited the largest alterations, as well as neointimal lesions and obliteration of the lumen in small arteries. We found that PH increased the expression of caveolin1, matrix metallopeptidase 2, and numerous inflammatory and cell proliferation genes. The cell cycle, vascular smooth muscle contraction, and oxidative phosphorylation pathways, as well as their interplay, were largely perturbed. Our results also suggest that the upregulated Rhoa (Ras homolog family member A) mediates its action through expression coordination with several ATPases. The upregulation of antioxidant genes and the extensive mitochondrial damage observed, especially in the HM group, indicate metabolic shift toward aerobic glycolysis.

Thoracic aortic dissection (TAD) is a catastrophic disease worldwide, but the pathogenic genes and pathways are largely unclear. This study aims at integrating two gene expression profile datasets and verifying hub genes and pathways involved in TAD as well as exploring potential molecular mechanisms. We will combine our mRNAs expression profile (6 TAD tissues versus 6 non-TAD tissues) and GSE52093 downloaded from the Gene Expression Omnibus (GEO) database. The two mRNAs expression profiles contained 13 TAD aortic tissues and 11 non-TAD tissues. The two expression profile datasets were integrated and we found out coexpression of differentially expressed genes (DEGs) using bioinformatics methods. The gene ontology and pathway enrichment of DEGs were performed by DAVID and Kyoto Encyclopedia of Genes and Genomes online analyses, respectively. The protein-protein interaction networks of the DEGs were constructed according to the data from the STRING database. Cytohubber calculating result shows the top 10 hub genes with CDC20, AURKA, RFC4, MCM4, TYMS, MCM2, DLGAP5, FANCI, BIRC5, and POLE2. Module analysis revealed that TAD was associated with significant pathways including cell cycle, vascular smooth muscle contraction, and adrenergic signaling in cardiomyocytes. The qRT-PCR result showed that the expression levels of all the hub genes were significantly increased in OA samples (p < 0.05), and these candidate genes could be used as potential diagnostic biomarkers and therapeutic targets of TAD.

Depolarization of the plasma membrane is a key mechanism of activation of contraction of vascular smooth muscle. This is commonly achieved in isolated, de-endothelialized vascular smooth muscle strips by increasing extracellular [K(+)] (replacing Na(+) by K(+)) and leads to a rapid phasic contraction followed by a sustained tonic contraction. The initial phasic contractile response is due to opening of voltage-gated Ca(2+) channels and entry of extracellular Ca(2+), which binds to calmodulin, leading to activation of myosin light chain kinase, phosphorylation of the regulatory light chains of myosin II at Ser19 and cross-bridge cycling. The subsequent tonic contractile response involves, in addition to myosin light chain kinase activation, Ca(2+)-induced Ca(2+) sensitization whereby Ca(2+) entry activates the RhoA/Rho-associated kinase pathway leading to phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase) and inhibition of the phosphatase. Investigations into the mechanism of activation of RhoA by Ca(2+) have implicated a genistein-sensitive tyrosine kinase, and recent evidence indicates this to be the Ca(2+)-dependent tyrosine kinase, Pyk2.

金沢大学医薬保健研究域医学系, Class II phosphoinositide 3-kinases (PI3Ks), PI3K-C2α and PI3K-C2β, are highly homologous and distinct from class I and class III PI3Ks in catalytic products and domain structures. In contrast to class I and class III PI3Ks, physiological roles of PI3K-C2α and PI3K-C2β are not fully understood. Because we previously demonstrated that PI3K-C2α is involved in vascular smooth muscle contraction, we studied the phenotypes of smooth muscle-specific knockout (KO) mice of PI3K-C2α and PI3K-C2β. The pup numbers born from single PI3K-C2α-KO and single PI3K-C2β-KO mothers were similar to those of control mothers, but those from double KO (DKO) mothers were smaller compared with control mice. However, the number of intrauterine fetuses in pregnant DKO mothers was similar to that in control mice. Both spontaneous and oxytocin-induced contraction of isolated uterine smooth muscle (USM) strips was diminished in DKO mice but not in either of the single KO mice, compared with control mice. Furthermore, contraction of USM of DKO mice was less sensitive to a Rho kinase inhibitor. Mechanistically, the extent of oxytocin-induced myosin light chain phosphorylation was greatly reduced in USM from DKO mice compared with control mice. The oxytocin-induced rise in the intracellular Ca2+ concentration in USM was similar in DKO and control mice. However, Rho activation in the intracellular compartment was substantially attenuated in DKO mice compared with control mice, as evaluated by fluorescence resonance energy transfer imaging technique. These data indicate that both PI3K-C2α and PI3K-C2β are required for normal USM contraction and parturition mainly through their involvement in Rho activation., Embargo Period 12 months

Vascular smooth muscle (VSM) plays an important role in the regulation of vascular function. Identifying the mechanisms of VSM contraction has been a major research goal in order to determine the causes of vascular dysfunction and exaggerated vasoconstriction in vascular disease. Major discoveries over several decades have helped to better understand the mechanisms of VSM contraction. Ca(2+) has been established as a major regulator of VSM contraction, and its sources, cytosolic levels, homeostatic mechanisms and subcellular distribution have been defined. Biochemical studies have also suggested that stimulation of Gq protein-coupled membrane receptors activates phospholipase C and promotes the hydrolysis of membrane phospholipids into inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG). IP(3) stimulates initial Ca(2+) release from the sarcoplasmic reticulum, and is buttressed by Ca(2+) influx through voltage-dependent, receptor-operated, transient receptor potential and store-operated channels. In order to prevent large increases in cytosolic Ca(2+) concentration ([Ca(2+)](c)), Ca(2+) removal mechanisms promote Ca(2+) extrusion via the plasmalemmal Ca(2+) pump and Na(+)/Ca(2+) exchanger, and Ca(2+) uptake by the sarcoplasmic reticulum and mitochondria, and the coordinated activities of these Ca(2+) handling mechanisms help to create subplasmalemmal Ca(2+) domains. Threshold increases in [Ca(2+)](c) form a Ca(2+)-calmodulin complex, which activates myosin light chain (MLC) kinase, and causes MLC phosphorylation, actin–myosin interaction, and VSM contraction. Dissociations in the relationships between [Ca(2+)](c), MLC phosphorylation, and force have suggested additional Ca(2+) sensitization mechanisms. DAG activates protein kinase C (PKC) isoforms, which directly or indirectly via mitogen-activated protein kinase phosphorylate the actin-binding proteins calponin and caldesmon and thereby enhance the myofilaments force sensitivity to Ca(2+). PKC-mediated phosphorylation of PKC-potentiated phosphatase inhibitor protein-17 (CPI-17), and RhoA-mediated activation of Rho-kinase (ROCK) inhibit MLC phosphatase and in turn increase MLC phosphorylation and VSM contraction. Abnormalities in the Ca(2+) handling mechanisms and PKC and ROCK activity have been associated with vascular dysfunction in multiple vascular disorders. Modulators of [Ca(2+)](c), PKC and ROCK activity could be useful in mitigating the increased vasoconstriction associated with vascular disease.

There is accumulating evidence that makes the link between the circadian variation in blood pressure and circadian variations in vascular contraction. The importance of vascular endothelium-derived redox-active and redox-derived species in the signalling pathways involved in controlling vascular smooth muscle contraction are well known, and when linked to the circadian variations in the processes involved in generating these species, suggests a cellular mechanism for the circadian variations in blood pressure that links directly to the peripheral circadian clock. Relaxation of vascular smooth muscle cells involves endothelial-derived relaxing factor (EDRF) which is nitric oxide (NO) produced by endothelial NO synthase (eNOS), and endothelial-derived hyperpolarising factor (EDHF) which includes hydrogen peroxide (H2O2) produced by NADPH oxidase (Nox). Both of these enzymes appear to be under the direct control of the circadian clock mechanism in the endothelial cells, and disruption to the clock results in endothelial and vascular dysfunction. In this review, we focus on EDRF and EDHF and summarise the recent findings on the influence of the peripheral circadian clock mechanism on processes involved in generating the redox species involved and how this influences vascular contractility, which may account for some of the circadian variations in blood pressure and peripheral resistance. Moreover, the direct link between the peripheral circadian clock and redox-signalling pathways in the vasculature, has a bearing on vascular endothelial dysfunction in disease and aging, which are both known to lead to dysfunction of the circadian clock.

Introduction It is well known that moderate to high doses of ionizing radiation have a toxic effect on the organism. However, there are few experimental studies on the mechanisms of LDR ionizing radiation on nerve regeneration after peripheral nerve injury. Methods We established the rats’ peripheral nerve injury model via repaired Peripheral nerve injury nerve, vascular endothelial growth factor a and Growth associated protein-43 were detected from different treatment groups. We performed transcriptome sequencing focusing on investigating the differentially expressed genes and gene functions between the control group and 1 Gy group. Sequencing was done by using high-throughput RNA-sequencing (RNA-seq) technologies. Results The results showed the 1 Gy group to be the most effective promoting repair. RNA-sequencing identified 619 differently expressed genes between control and treated groups. A Gene Ontology analysis of the differentially expressed genes revealed enrichment in the functional pathways. Among them, candidate genes associated with nerve repair were identified. Discussion Pathways involved in cell-substrate adhesion, vascular smooth muscle contraction and cell adhesion molecule signaling may be involved in recovery from peripheral nerve injury.

The purpose of this study was to determine the effects of resveratrol (RSV) and the molecular mechanisms by which it regulates vascular smooth muscle contraction and blood pressure in mice. In cultured human vascular smooth muscle cells (VSMCs), we found that the activation of AMP-activated protein kinase (AMPK) by RSV inhibited angiotensin II (AngII)-induced phosphorylation of myosin phosphatase-targeting subunit 1 (MYPT1) and myosin light chain (MLC). Inversely, AMPK inhibition with RNA interference and compound C, an AMPK inhibitor, abolished the inhibitory effect of RSV on AngII-induced MYPT1 and MLC phosphorylation. Thiazovivin, a Rho-associated kinase (ROCK) inhibitor, reversed AngII-induced MYPT1 and MLC phosphorylation, suggesting that ROCK functions as an upstream kinase for MYPT1/MLC. RSV reversed AngII-induced Ras homolog gene family member A (RhoA) and ROCK activity, whereas AMPK inhibition via pharmacological or genetic means abolished this effect. In addition, gene silencing of p190-guanosine triphosphatase-activating protein blocked the effects of RSV-induced AMPK activation on MLC, MYPT1 and RhoA in VSMCs. Ex vivo analyses demonstrated that AngII-induced aorta contractions were dramatically inhibited by RSV, and this effect was abolished by AMPK inhibition. Finally, daily chronic administration of RSVl alleviated hypertension in the experimental model of AngII-induced hypertensive mice, and these effects were accompanied by the activation of AMPK, significantly decreased RhoA activity and phosphorylation levels of MYPT1 and MLC in AngII-treated murine aortic VSMCs. More importantly, administration of compound C significantly abolished the effects of RSV. In conclusion, AMPK suppression of the p190-GAP-dependent RhoA/ROCK/MYPT1/MLC pathway contributes to the hypotensive effect of RSV in AngII-treated mice.

Myosin regulatory light chain (LC20) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates LC20 (its only known substrate) exclusively at S19. Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 at T18 and S19 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par-4 (prostate-apoptosis response-4). Phosphorylation of MYPT1 at T696 and T853 inhibits MLCP activity whereas phosphorylation of Par-4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of MLCK, ROCK and ZIPK in these phosphorylation events, we investigated the time courses of phosphorylation of LC20, MYPT1 and Par-4 in serum-stimulated human vascular smooth muscle cells (from coronary and umbilical arteries), and examined the effects of siRNA-mediated MLCK, ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T853, and Par-4 at T163, peaking within 30-120 s. MLCK knockdown or inhibition, or Ca2+ chelation with EGTA, had no effect on serum-induced LC20 phosphorylation. ROCK knockdown decreased the levels of phosphorylation of LC20 at T18 and S19, of MYPT1 at T696 and T853, and of Par-4 at T163, whereas ZIPK knockdown decreased LC20 diphosphorylation, but increased phosphorylation of MYPT1 at T696 and T853 and of Par-4 at T163. ROCK inhibition with GSK429286A reduced serum-induced phosphorylation of LC20 at T18 and S19, MYPT1 at T853 and Par-4 at T163, while ZIPK inhibition by HS38 reduced only LC20 diphosphorylation. We also demonstrated that serum stimulation induced phosphorylation (activation) of ZIPK, which was inhibited by ROCK and ZIPK down-regulation and inhibition. Finally, basal phosphorylation of LC20 in the absence of serum stimulation was unaffected by MLCK, ROCK or ZIPK knockdown or inhibition. We conclude that: (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1, Par-4 and ZIPK, in contrast to the slower phosphorylation of kinases and other proteins involved in other signaling pathways (Akt, ERK1/2, p38 MAPK and HSP27), (ii) ROCK and ZIPK, but not MLCK, are involved in serum-induced phosphorylation of LC20, (iii) ROCK, but not ZIPK, directly phosphorylates MYPT1 at T853 and Par-4 at T163 in response to serum stimulation, (iv) ZIPK phosphorylation is enhanced by serum stimulation and involves phosphorylation by ROCK and autophosphorylation, and (v) basal phosphorylation of LC20 under serum-free conditions is not attributable to MLCK, ROCK or ZIPK.

Introduction Phosphorylation of myosin light chains is a biochemical readout of smooth muscle cell contraction. α2-Adrenoceptor agonists and antagonists may have important applications in cardiovascular drug development. To assess α2-adrenoceptor-mediated drug effects on vascular smooth muscle contraction, we developed a cell-based assay for the quantitative determination of myosin light chain phosphorylation (pMLC20) in cultured A7r5 smooth muscle cells from rat aorta, transfected to express the human α2B-adrenoceptor (A7r5-α2B cell line). Methods: In a 96-well format, confluent and serum-starved cells (+/− inhibitor preincubation) were treated with receptor ligands for 5–120 s and the evoked pMLC20 response was monitored with a quantitative in-cell immunoassay, employing time-resolved fluorescence technology. Western blotting, immunofluorescent labelling and intracellular calcium concentration measurements were used for assay validation. Results: The α2-adrenoceptor agonist dexmedetomidine induced rapid, transient and dose-dependent (EC50 30–65 nM) myosin light chain phosphorylation, peaking at 20–45 s with an Emax value of approximately 60% over vehicle control. The endogenous agonist arginine vasopressin produced responses that were comparable to those evoked by dexmedetomidine. Blockers of α2-adrenoceptors, myosin light chain kinase, Gi-proteins, Gβγ subunits, L-type calcium channels and phospholipase C antagonized the dexmedetomidine-evoked myosin light chain phosphorylation, whereas blockers of protein kinase C and protein kinase A potentiated the response to dexmedetomidine. Discussion: The novel method is suitable as a ligand profiling tool to assess the capacity of ligands to evoke or inhibit vascular smooth muscle cell contraction and for investigating the intracellular pathways involved in this process. The assay now allows the quantitative determination of pMLC20 signal induction or inhibition in vascular smooth muscle cells and is superior to conventional Western blotting due to the reduced number of cells required and the potential for measurement of detailed time curves, multiple treatments and replicates on each plate.

It is well established that the release of vasodilators and vasoconstrictors from vascular endothelium regulates vascular smooth muscle contraction. In this report, we investigate the role of the endothelium in the development and maintenance of constitutive vascular contractility. For that purpose, contractile activity of cultured bovine aortic smooth muscle cells (BASMCs) embedded in collagen gels was monitored by changes in gel diameter. After culturing for 5 days, ATP- and high KCl solution-induced contractions were significantly enhanced in the gels that were overlaid with bovine aortic endothelial cells (BAECs) or were cultured with conditioned medium of cultured BAECs. ATP-induced Ca(2+) transients, recorded in BASMCs cultured with conditioned medium of BAECs, were markedly augmented, but high KCl-induced Ca(2+) transients were not affected. BASMCs in control gels were spindle shaped, and those in endothelium-treated gels were more elongated and interconnected. The endothelial conditioned medium also strongly affected the intracellular distribution of actin fibers. Conditioned medium of BAECs contained TGFβ1 and TGFβ2. The TGFβ receptor antagonist SB431542 as well as simultaneous treatment with TGFβ1 and TGFβ2 neutralizing antibodies completely reversed the above effects of endothelial conditioned medium on BASMCs. BAECs medium induced phosphorylation of Smad2 and increased ATP-induced phosphorylation of myosin light chain in BASMCs. The present results indicate that the release of TGFβ1 and TGFβ2 from vascular endothelium affects the contractility of vascular smooth muscle cells by altering their morphology and agonist-induced Ca(2+) mobilization.

APS12-2 and APS3 are synthetic analogues of polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai. The aim of the present study was to determine the possible direct contractile effects of these two synthetic molecules on coronary arteries, in order partly to explain hemodynamic and cardiotoxic effects of APS12-2 previously observed in in vivo studies and to reveal possible adverse effects on the organism in the case of their clinical use. In contrast to APS3, APS12-2 caused a concentration-dependent vascular smooth muscle contraction of isolated porcine coronary ring preparations in a concentration-range from 1.36 to 13.60 μM. Lanthanum chloride (5 mM) and verapamil (10 μM) completely abolished the APS12-2 evoked contraction of the coronary rings. Pre-incubation with indomethacin (10 μM) had no effect on the contractile responses of coronary ring preparations. These results indicate that APS12-2 contracts vascular smooth muscle in a concentration-dependent manner, due to an increase of Ca2+ influx through the voltage-gated Ca2+ channels. Our data show for the first time that APS12-2 induces concentration-dependent contraction of coronary ring preparations, which may contribute to the cardiotoxic effects of APS12-2, in addition to hyperkalemia.

Potassium channels importantly contribute to the regulation of vascular smooth muscle (VSM) contraction and growth. They are the dominant ion conductance of the VSM cell membrane and importantly determine and regulate membrane potential. Membrane potential, in turn, regulates the open-state probability of voltage-gated Ca2+ channels (VGCC), Ca2+ influx through VGCC, intracellular Ca2+ and VSM contraction. Membrane potential also affects release of Ca2+ from internal stores and the Ca2+ sensitivity of the contractile machinery such that K+ channels participate in all aspects of regulation of VSM contraction. Potassium channels also regulate proliferation of VSM cells through membrane potential-dependent and membrane potential-independent mechanisms. Vascular smooth muscle cells express multiple isoforms of at least five classes of K+ channels contribute to the regulation of contraction and cell proliferation (growth). This review will examine the structure, expression and function of large-conductance, Ca2+-activated K+ (BKCa) channels, intermediate-conductance Ca2+-activated K+ (KCa3.1) channels, multiple isoforms of voltage-gated K+ (KV) channels, ATP-sensitive K+ (KATP) channels, and inward-rectifier K+ (KIR) channels in both contractile and proliferating VSM cells.

The smooth muscle cell directly drives the contraction of the vascular wall and hence regulates the size of the blood vessel lumen. We review here the current understanding of the molecular mechanisms by which agonists, therapeutics, and diseases regulate contractility of the vascular smooth muscle cell and we place this within the context of whole body function. We also discuss the implications for personalized medicine and highlight specific potential target molecules that may provide opportunities for the future development of new therapeutics to regulate vascular function.

Vascular smooth muscle contraction occurs following an initial response to an increase in intracellular calcium concentration and a sustained response following increases in the sensitivity of contractile proteins to calcium (calcium sensitization). This latter process is regulated by the rhoA/rho kinase pathway and activated by serotonin. In multiple cell types, signaling molecules compartmentalize within caveolae to regulate their activation. We hypothesized that serotonin differentially compartmentalizes rhoA within caveolar versus noncaveolar lipid rafts to regulate sustained vascular contractions. To test this hypothesis, we measured aortic contractions in response to serotonin in wild-type (WT) and cav-1-deficient mice (cav-1 KO). RhoA-dependent contractions in response to serotonin were markedly augmented in arteries from cav-1 KO mice despite a modest reduction in rhoA expression compared with WT. We found that under basal conditions, rhoA in WT arteries was primarily localized within high-density sucrose gradient fractions but temporally shifted to low-density fractions in response to serotonin. In contrast, rhoA in cav-1 KO arteries was primarily in low-density fractions and shifted to high-density fractions in a similar timeframe as that seen in WT mice. We conclude that localization of rhoA to caveolar versus noncaveolar lipid rafts differentially regulates its activation and contractions to rhoA-dependent agonists with greater activation associated with its localization to noncaveolar rafts. Disruption of rhoA localization within caveolae may contribute to increased activation and enhanced vascular contractions in cardiovascular disease.

The aim of the present study is to determine the effects and molecular mechanisms by which activation of LKB1-AMP-activated protein kinase (AMPK) by metformin regulates vascular smooth muscle contraction. The essential ability of vascular smooth muscle cells (VSMCs) to contract and relax in response to an elevation and reduction in intravascular pressure is necessary for appropriate blood flow regulation. Thus, vessel contraction is a critical mechanism for systemic blood flow regulation. In cultured rat VSMCs, AMPK activation through LKB1 by metformin-inhibited phenylephrine-mediated myosin light chain kinase (MLCK) and myosin light chain phosphorylation (p-MLC). Conversely, inhibition of AMPK and LKB1 reversed phenylephrine-induced MLCK and p-MLC phosphorylation. Measurement of the tension trace in rat aortic rings also showed that the effect of AMPK activation by metformin decreased phenylephrine-induced contraction. Metformin inhibited PE-induced p-MLC and α-smooth muscle actin co-localization. Our results suggest that activation of AMPK by LKB1 decreases VSMC contraction by inhibiting MLCK and p-MLC, indicating that induction by the AMPK-LKB1 pathway may be a new therapeutic target to lower high blood pressure.

We reported previously that ATP2B1 was one of the genes for hypertension receptivity in a large-scale Japanese population, which has been replicated recently in Europeans and Koreans. ATP2B1 encodes the plasma membrane calcium ATPase isoform 1, which plays a critical role in intracellular calcium homeostasis. In addition, it is suggested that ATP2B1 plays a major role in vascular smooth muscle contraction. Because the ATP2B1 knockout (KO) mouse is embryo-lethal, we generated mice with vascular smooth muscle cell-specific KO of ATP2B1 using the Cre-loxP system to clarify the relationship between ATP2B1 and hypertension. The KO mice expressed significantly lower levels of ATP2B1 mRNA and protein in the aorta compared with control mice. KO mice showed significantly higher systolic blood pressure as measured by tail-cuff method and radiotelemetric method. Similar to ATP2B1, the expression of the Na + -Ca 2+ exchanger isoform 1 mRNA was decreased in vascular smooth muscle cells of KO mice. However, ATP2B4 expression was increased in KO mice. The cultured vascular smooth muscle cells of KO mice showed increased intracellular calcium concentration not only in basal condition but also in phenylephrine-stimulated condition. Furthermore, phenylephrine-induced vasoconstriction was significantly increased in vascular rings of the femoral artery of KO mice. These results suggest that ATP2B1 plays important roles in the regulation of blood pressure through alteration of calcium handling and vasoconstriction in vascular smooth muscle cells.

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway and a major source of nicotinamide adenine dinucleotide phosphate reduced (NADPH), which regulates numerous enzymatic (including glutathione reductase and NADPH oxidase that, respectively, generates reduced glutathione and reactive oxygen species) reactions involved in various cellular actions, yet its physiological function is seldom investigated. We, however, recently showed that inhibiting G6PD causes precontracted coronary artery (CA) to relax in an endothelium-derived relaxing factor- and second messenger-independent manner. Here we assessed the role of G6PD in regulating CA contractility. Treating bovine CAs for 20 min with potassium chloride (KCl; 30 mM), amphotericin B (50 μM), or U46619 (100 nM) significantly (p

In this study, we compared the potencies of diosgenin, a plant-derived sapogenin structurally similar to estrogen and progesterone, on vascular smooth muscle functions ranging from contraction and migration to apoptosis. The effects of diosgenin on vascular smooth muscle cell viability and migration were measured using a primary mouse aortic smooth muscle cell culture. The effects of diosgenin on smooth muscle cell contraction and calcium signaling were investigated in the isolated mouse aorta using wire myography and confocal microscopy, respectively. Here, we report that in cultured cells diosgenin (≥ 25 μM) induces apoptosis as measured by the number of annexin V-positive cells and caspase-3 cleavage, while decreasing cell viability as indicated by protein kinase B/Akt phosphorylation. In addition, diosgenin blocks smooth muscle cell migration in a transwell Boyden chamber in response to serum treatment and response to injury in a cell culture system. Diosgenin (≥ 25 μM) also significantly blocks receptor-mediated calcium signals and smooth muscle contraction in the isolated aorta. There is no difference in the inhibitory effects of diosgenin on vascular smooth muscle contraction between the endothelium-intact and endothelium-denuded aortic segments, indicating that they are caused by altered smooth muscle activity. Our findings suggest that over the concentration range of 10 to 15 μM diosgenin may provide overall beneficial effects on diseased vascular smooth muscle cells by blocking migration and contraction without any significant cytopathic effects, implying a potential therapeutic value for diosgenin in vascular disorders.

Cardiovascular disease remains as the leading cause of death in the developed world. However, there is limited knowledge about how cardiovascular risk factors actually cause vascular disease. Traditional cardiovascular risk factors include increased circulating levels of low-density lipoproteins, cigarette smoking and hypertension (both strongly related to arterial wall injury), inflammation and atherosclerosis. The vascular endothelin receptors are a protein family that belongs to the larger family of G-protein coupled receptors. They mediate vascular smooth muscle contraction, proliferation and apoptosis, which are important events in the pathogenesis of atherosclerotic vascular disease. Recent investigations into intracellular signaling mechanisms suggest that the above risk factors increase the expression of endothelin receptors in vascular smooth muscle cells by activating intracellular mitogen-activated protein kinase pathways and downstream transcription factors such as nuclear factor-kappaB. Understanding the mechanisms involved in vascular endothelin receptor upregulation during cardiovascular disease may provide novel therapeutic approaches.

Abnormal vascular smooth muscle cell (VSMC) contraction plays an important role in vascular diseases. The RhoA/ROCK signaling pathway is now well recognized to mediate vascular smooth muscle contraction in response to vasoconstrictors by inhibiting myosin phosphatase (MLCP) activity and increasing myosin light chain phosphorylation. Two ROCK isoforms, ROCK1 and ROCK2, are expressed in many tissues, yet the isoform-specific roles of ROCK1 and ROCK2 in vascular smooth muscle and the mechanism of ROCK-mediated regulation of MLCP are not well understood. In this study, ROCK2, but not ROCK1, bound directly to the myosin binding subunit of MLCP, yet both ROCK isoforms regulated MLCP and myosin light chain phosphorylation. Despite that both ROCK1 and ROCK2 regulated MLCP, the ROCK isoforms had distinct and opposing effects on VSMC morphology and ROCK2, but not ROCK1, had a predominant role in VSMC contractility. These data support that although the ROCK isoforms both regulate MLCP and myosin light chain phosphorylation through different mechanisms, they have distinct roles in VSMC function.

To explore the possible role of heat shock protein 20 (HSP20) -linked regulation of actin-myosin interaction in living vascular smooth muscle contraction, we studied the effects of HSP20p and TnIp, synthetic peptides originating from an actin tropomyosin binding region of human heat shock protein 20 [residues 110-121; GFVAREFHRRYR] and that of rabbit cardiac troponin I [residues 136-147; GKFKRPTLRRVR], respectively, on the active stress and phosphorylation level of myosin regulatory light chain (MLC(20)) during relaxation of skinned (cell membrane permeabilized) preparations from "tonic" carotid artery and "phasic" taenia cecum from guinea pig. Active stress of the skinned preparations, resulting from actin-myosin interaction, biphasically decayed following Ca(2+) removal (relaxation). Decay of MLC(20) phosphorylation level by Ca(2+) removal was much faster than active stress in an exponential manner. In skinned carotid artery, HSP20p did neither affect relaxation time course nor MLC(20) dephosphorylation, whereas, in skinned taenia cecum, the peptide slowed relaxation time course through inhibition of MLC(20) dephosphorylation and slowing "latch"-bridge dissociation. On the other hand, TnIp accelerated relaxation time course without affecting MLC(20) dephosphorylation in both skinned carotid artery and skinned taenia cecum. Our present results suggest that, HSP20p slows the relaxation processes through intracellular regulatory mechanisms such as Rho A/Rho-kinase mediated pathways, which are known to be dominant in "phasic" smooth muscles but to be recessive in "tonic" smooth muscles.

Previously, we reported that hyposmotic swelling evoked transient vascular smooth muscle cell (SMC) contraction that was completely abolished by L-type Ca(2+) channel blockers. In contrast, sustained contraction revealed in hyper- and isoosmotically-shrunken SMCs was insensitive to L-type channel blockers and was diminished in Ca(2+)-free medium by only 30-50%. Several research groups reported cell volume-dependent cytoskeleton network rearrangements. This study examines the role of cytoskeleton proteins in cell volume-dependent contraction of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat thoracic aorta. Hyperosmotic shrinkage and hyposmotic swelling were triggered by modulation of medium osmolality; isosmotic shrinkage was induced by VSMR transfer from hypo- to isosmotic medium. The relative content of globular (G) and fibrillar (F) actin was estimated by fluorescence microscopy. Hyperosmotic shrinkage and hyposmotic swelling led to elevation of the F-actin/G-actin ratio by 2.5- and 1.8-fold respectively. Contraction of shrunken and swollen VSMR was insensitive to modulators of microtubules such as vinblastine, colchicine and docetaxel. Microfilament disassembly by cytochalasin B resulted in dramatic attenuation of the maximal amplitude of contraction of hyperosmotically-shrunken and hyposmotically-swollen VSMR, and almost completely abolished the contraction triggered by isosmotic shrinkage. These data suggest that both L-type Ca(2+) channel-mediated contraction of swollen vascular SMC and Ca(2+)(o)-insensitive contractions of shrunken cells are triggered by reorganization of the microfilament network caused by elevation of the F-actin/G-actin ratio.

We here review existing models of vascular smooth muscle cell, endothelial cell and cell-cell communication, which have been developed to better understand vascular tone and blood flow autoregulation. In particular, we discuss models that intended to explain modulation of myogenic tone by intraluminal pressure in resistance arterioles. Modeling efforts in the recent past have witnessed a shift from empirical models to models with mechanistic details that underscore different physical aspects of vascular regulation. Future models should synthesize mechanistic interactions in a hierarchy, from molecular regulation of ion channels to whole organ blood flow control.

The perivascular adipose tissue (PVAT) releases a variety of factors that affect vascular function. PVAT in the thoracic aorta shares characteristics with the brown adipose tissue, including a large amount of mitochondria. PVAT-derived factors influence both endothelial and smooth muscle function via several signaling mechanisms including the release/generation of reactive nitrogen and oxygen species. Considering the importance of reactive oxygen species (ROS) on vascular function and that mitochondria are an important source of ROS, we hypothesized that mitochondria-derived ROS in the PVAT modulates vascular reactivity. Vascular reactivity to norephinephrine (NE) was evaluated in thoracic aortic rings, with or without endothelium and/or PVAT, from male Wistar rats. Mitochondrial uncoupling, as well as hydrogen peroxide (H2O2) removal, increased the contraction in vessels surrounded by PVAT. PVAT stimulated with NE exhibited increased protein expression, determined by Western blot analysis, of manganese superoxide dismutase (Mn-SOD) and decreased protein expression of catalase. Ultimately, NE increased superoxide anion (O2(-)) generation in PVAT via increases in intracellular calcium. These results clearly demonstrate that mitochondrial electron transport chain (mETC) in PVAT contributes to modulation of aortic muscle contraction by generating higher amounts of O2(-) that is, in turn, dismutated to hydrogen peroxide, which then acts as a pivotal signaling molecule regulating vascular smooth muscle contraction.

血管平滑筋の異常収縮は，虚血性心疾患やくも膜下出血後の脳血管攣縮などの疾患の病態生理に重要な役割を果たしている．この異常収縮の分子機構として，RhoキナーゼによるCa2+-sensitizationが重要である事が知られているが，我々はこれまでに，Rhoキナーゼの上流の，血管平滑筋Ca2+-sensitizationのシグナル分子として，スフィンゴシルホスホリルコリン（SPC）を同定し，更に，SPCが，Srcファミリーチロシンキナーゼ（Src-TK）を介して，Rhoキナーゼを活性化する事を明らかにした．我々は，Src-TKの中で，血管平滑筋組織および培養血管平滑筋細胞において，Fynおよびc-Srcが発現している事を確認し，どちらが血管平滑筋Ca2+-sensitizationのシグナル伝達に関与するかを検討した．培養血管平滑筋細胞において，SPC刺激により，Fynは細胞内から形質膜へ移動したが，c-Srcの細胞内局在は変化しなかった．そこで，1）siRNAによるFynの特異的ノックダウン，2）β-escinスキンド血管平滑筋標本における組み換えFynタンパクの導入，の2つの方法を用いて検討し，血管平滑筋Ca2+-sensitizationのシグナル伝達におけるFynの関与を直接的に証明しうる所見を得た．また，ヒトおよびコレステロール負荷家兎の血管平滑筋のSPC反応性と血清コレステロール濃度が相関する事を見出した．β-サイクロデキストリン処理により，細胞膜から選択的にコレステロールを除去すると，血管平滑筋組織において，SPC収縮は特異的に抑制され，培養平滑筋細胞では，ラフトのマーカーであるカベオリン-1による標識が著しく抑制され，SPCによるFynおよびRhoキナーゼの細胞内から形質膜への移動は抑制された．以上の所見より，血管平滑筋Ca2+-sensitizationのシグナル伝達において，コレステロールおよび細胞膜ラフトが，重要な役割を果たす事が示唆された．

The chemical compound [4-(4-bromo-2-cyano-phenylcarbamoyl)-benzyl]-phosphonic acid diethyl ester (NO-1886) is a lipoprotein lipase activator having beneficial effects on both diabetes control and the cardiovascular system. Preventing accumulation of lipids in the cell wall, in addition to improving insulin actions on vasculature, may indirectly contribute to the reducing effect of NO-1886 on vascular resistance. However, the direct effect of NO-1886 on vascular resistance, i.e., whether NO-1886 directly modulates the function of vascular endothelium and/or smooth muscle cells has not been investigated. In this study we therefore investigated the direct effect of NO-1886 on vascular contractility using rat aortic rings and cultured smooth muscle cell-line A10. The results show that administration of NO-1886 attenuated aortic contraction induced by phenylephrine and/or a high K(+) environment, in both the presence and absence of aortic endothelium. 1-(5-Chloronaphthalene-1-sulfonyl)homopiperazine hydrochloride (ML-9), a myosin light chain kinase (MLCK) inhibitor, blocked this inhibitory effect of NO-1886, whereas inhibitors of other signaling molecules such as calmodulin, protein kinase C and Rho-kinase had no effect. The vasorelaxant effect of NO-1886 was blocked in the absence of extracellular Ca(2+), or in the presence of the Ca(2+) channel inhibitor, verapamil. NO-1886 attenuated smooth muscle contraction induced by the cumulative addition of CaCl(2). In A10 cells, NO-1886 inhibited the membrane depolarization-induced initial peak of [Ca(2+)](i) in the presence of extracellular Ca(2+). This inhibition did not occur in the absence of extracellular Ca(2+). Taken together these results demonstrate that NO-1886 attenuates smooth muscle contraction and causes vasorelaxation by an extracellular Ca(2+)- and MLCK-dependent mechanism.

A novel inhibitor of zipper-interacting protein kinase (ZIPK) was used to examine the involvement of ZIPK in the regulation of smooth muscle contraction. Pretreatment of de-endothelialized rat caudal arterial smooth muscle strips with the pyrazolo[3,4-d]pyrimidinone inhibitor 2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo [3,4-d]-pyrimidin-6-yl)thio)propanamide (HS38) decreased the velocity of contraction (time to reach half-maximal force) induced by the phosphatase inhibitor calyculin A in the presence of Ca(2+) without affecting maximal force development. This effect was reversed following washout of HS38 and correlated with a reduction in the rate of phosphorylation of myosin 20-kDa regulatory light chains (LC20) but not of protein kinase C-potentiated inhibitory protein for myosin phosphatase of 17 kDa (CPI-17), prostate apoptosis response-4, or myosin phosphatase-targeting subunit 1 (MYPT1), all of which have been implicated in the regulation of vascular contractility. A structural analog of HS38, with inhibitory activity toward proviral integrations of Moloney (PIM) virus 3 kinase but not ZIPK, had no effect on calyculin A-induced contraction or protein phosphorylations. We conclude that a pool of constitutively active ZIPK is involved in regulation of vascular smooth muscle contraction through direct phosphorylation of LC20 upon inhibition of myosin light chain phosphatase activity. HS38 also significantly attenuated both phasic and tonic contractile responses elicited by phenylephrine, angiotensin II, endothelin-1, U46619, and K(+)-induced membrane depolarization in the presence of Ca(2+), which correlated with inhibition of phosphorylation of LC20, MYPT1, and CPI-17. These effects of HS38 suggest that ZIPK also lies downstream from G protein-coupled receptors that signal through both Gα12/13 and Gαq/11.

Rho kinase (ROCK1) mediates vascular smooth muscle contraction and is a potential target for the treatment of hypertension and related disorders. Indazole amide 3 was identified as a potent and selective ROCK1 inhibitor but possessed poor oral bioavailability. Optimization of this lead resulted in the discovery of a series of dihydropyridones, exemplified by 13, with improved pharmacokinetic parameters relative to the initial lead. Indazole substitution played a critical role in decreasing clearance and improving oral bioavailability.

We have recently demonstrated in vascular smooth muscle (VSM) that membrane depolarization by high KCl induces Ca(2+)-dependent Rho activation and myosin phosphatase (MLCP) inhibition (Ca(2+)-induced Ca(2+)-sensitization) through the mechanisms involving phosphorylation of myosin-targeting protein 1 (MYPT1) and 17-kDa protein kinase C (PKC)-potentiated inhibitory protein of PP1 (CPI-17). In the present study, we investigated whether and how cAMP affected Ca(2+)-dependent MLCP inhibition by examining the effects of forskolin, cell-permeable dibutyryl cAMP (dbcAMP), and isoproterenol. Forskolin, but not its inactive analog 1,9-dideoxyforskolin, inhibited KCl-induced contraction and the 20-kDa myosin light chain (MLC) phosphorylation without inhibiting Ca(2+) mobilization in rabbit aortic VSM. dbcAMP mimicked these forskolin effects. We recently suggested that Ca(2+)-mediated Rho activation is dependent on class II alpha-isoform of phosphoinositide 3-kinase (PI3K-C2alpha). Forskolin inhibited KCl-induced stimulation of PI3K-C2alpha activity. KCl-induced membrane depolarization stimulated Rho in a manner dependent on a PI3K but not PKC and stimulated phosphorylation of MYPT1 at Thr(850) and CPI-17 at Thr(38) in manners dependent on both PI3K and Rho kinase, but not PKC. Forskolin, dbcAMP, and isoproterenol inhibited KCl-induced Rho activation and phosphorylation of MYPT1 and CPI-17. Consistent with these data, forskolin, isoproterenol, a PI3K inhibitor, or a Rho kinase inhibitor, but not a PKC inhibitor, abolished KCl-induced diphosphorylation of MLC. These observations indicate that cAMP inhibits Ca(2+)-mediated activation of the MLCP-regulating signaling pathway comprising PI3K-C2alpha, Rho, and Rho kinase in a manner independent of Ca(2+) and point to the novel mechanism of the cAMP actions in the regulation of vascular smooth muscle contraction.

Vascular smooth muscle cells (VSMCs) are not terminally differentiated and, owing to their remarkable plasticity, can change to a dedifferentiated state in response to vascular injury. Our understanding of the contractility of VSMCs is mainly based on the data obtained from normal adult animals. However, to obtain a better understanding of the abnormal contractility seen in the vascular diseases such as hypertension and vasospasm superimposed on atherosclerosis, it is important to also know the contractility of proliferating dedifferentiated VSMCs. To this end, we studied the contractility of cultured VSMCs that undergo dedifferentiation similar to that induced by vascular injury. There are only a few reports in which the contractility of cultured VSMCs has been extensively studied. We established a method to investigate the contractility of the cultured VSMCs and determined that their contraction is dramatically changed to be more dependent on the Rho-Rho kinase system but less dependent on the PKC-CPI-17 (protein kinase C-potentiated protein phosphatase 1 inhibitory protein)-mediated pathway. In this review, we focus on the contractility of the cultured VSMCs as a model of the proliferating dedifferentiated VSMCs.

Vascular smooth muscle contraction is mediated by activation of extracellular signal-regulated kinase (ERK) 1/2, an isoform of mitogen-activated protein kinase (MAPK). However, the role of stress-activated protein kinase/c-Jun N-terminal kinase (JNK) in vascular smooth muscle contraction has not been defined. We investigated the role of JNK in the contractile response to norepinephrine (NE) in rat aortic smooth muscle. NE evoked contraction in a dose-dependent manner, and this effect was inhibited by the JNK inhibitor SP600125. NE increased the phosphorylation of JNK, which was greater in aortic smooth muscle from hypertensive rats than from normotensive rats. NE-induced JNK phosphorylation was significantly inhibited by SP600125 and the conventional-type PKC (cPKC) inhibitor Gö6976, but not by the Rho kinase inhibitor Y27632 or the phosphatidylinositol 3-kinase inhibitor LY294002. Thymeleatoxin, a selective activator of cPKC, increased JNK phosphorylation, which was inhibited by Gö6976. SP600125 attenuated the phosphorylation of caldesmon, an actin-binding protein whose phosphorylation is increased by NE. These results show that JNK contributes to NE-mediated contraction through phosphorylation of caldesmon in rat aortic smooth muscle, and that this effect is regulated by the PKC pathway, especially cPKC. Keywords:: c-Jun N-terminal kinase, mitogen-activated protein kinase, vascular smooth muscle contraction, caldesmon, hypertension

Clinically metamizol (MZ) has been related to alteration on haemodynamic parameters and modifications on blood pressure in humans when administered intravenously. These effects have been observed at MZ therapeutic doses. Experimentally, MZ is able to induce relaxation on several types of vascular smooth muscles and modulates the contraction induced by phenylephrine. However, the mechanism underlying the MZ effects on vascular reactivity is not clear. Potassium channels (K) present on vascular smooth muscle cells closely regulate the vascular reactivity and membrane potential. There are four described types of K in vascular tissue: K voltage sensitive (K(V)), K calcium sensitive (K(Ca)2+), K ATP sensitive (K(ATP) and K inward rectification (K(IR), voltage sensitive). The aim of this work was to investigate MZ effects on angiotensin II (AT II) and noradrenaline (NA) induced contraction and to evaluate the K participation on MZ modulating effect on vascular smooth muscle contraction, using isometric and patch clamp techniques. MZ induces relaxation in a concentration dependent manner. Furthermore, MZ strongly inhibits in a concentration dependent fashion the contraction induced by AT II. However, MZ inhibition on NA induced contraction was moderated compared with that observed on AT II. MZ effects on AT II induced contraction was blocked by glybenclamide (a specific K(ATP) blocker, 3 microM, *p < 0.01). In patch clamp experiments, MZ (3 mM) induces an increase on potassium current (K+) mediated by K(ATP) in similar way as diazoxide (a specific K(ATP) opener, 3 microM). Our results suggest that MZ induces relaxation and inhibits contraction induced by AT II acting as a K(ATP) opener.

UNLABELLED Sevoflurane dilates blood vessels and reduces arterial blood pressure in a dose-dependent manner. Angiotensin II (Ang II) is one of the primary regulators of vascular tension and arterial blood pressure, and the p44/42 mitogen-activated protein kinases (p44/42 MAPK) are involved in Ang II-mediated vascular smooth muscle contraction. We designed this study to examine the effects of sevoflurane on Ang II-induced, p44/42 MAPK-mediated contraction of rat aortic smooth muscle. The effects of the p44/42 MAPK kinase (MEK1/2) inhibitor, PD 098059 (10(-5) molar [M], 5 x 10(-5) M and 10(-4) M), and sevoflurane (1.7%, 3.4%, and 5.1%) on Ang II-induced contraction and p44/42 MAPK phosphorylation were tested in rat aortic smooth muscle, using isometric force measurement and Western blot analysis, respectively. Ang II induced both a transient contractile response and phosphorylation of p44/42 MAPK, which were significantly attenuated by PD 098059 (P < 0.05-0.01). Sevoflurane inhibited Ang II-induced contractile response in a dose-dependent manner (P < 0.05 and 0.01 in response to 3.4% and 5.1% sevoflurane, respectively). Sevoflurane also dose-dependently depressed Ang II-elicited p44/42 MAPK phosphorylation (P < 0.01 in response to 3.4% and 5.1% sevoflurane). These results suggest that the inhibitory effect of sevoflurane on Ang II-induced vasoconstriction is, at least in part, caused by the inhibition of the p44/42 MAPK-mediated signaling pathway. IMPLICATIONS The present study demonstrates that sevoflurane can dose-dependently inhibit both angiotensin II (Ang II)-induced contraction and p44/42 MAPK phosphorylation of rat aortic smooth muscle. These data suggest that sevoflurane-produced inhibition of Ang II-induced vasoconstriction is, at least in part, caused by depression of the p44/42 MAPK-mediated signaling pathway.

Background: Isoflurane induces greater effects on vasodilation and decreasing blood pressure than sevoflurane. Tyrosine kinase-catalyzed protein tyrosine phosphorylation plays an important role in regulating vascular smooth muscle contraction. The aim of the present study was to compare the effects of isoflurane and sevoflurane on tyrosine phosphorylation-mediated vascular constriction, by assessing the degree of sodium orthovanadate (Na 3 VO 4 , tyrosine phosphatase inhibitor)-induced contraction and protein tyrosine phosphorylation of rat aortic smooth muscle. Methods: Na 3 VO 4 -induced contraction and protein tyrosine phosphorylation of rat aortic smooth muscle were measured in the presence of genistein, a tyrosine kinase inhibitor, and different concentrations of isoflurane and sevoflurane, using isometric force measurement and Western blot, respectively. Results: Na 3 VO 4 (10 4 M) induced sustained contraction and tyrosine phosphorylation of substrates that were both markedly attenuated in the presence of genistein (5 × 10 5 M). Isoflurane and sevoflurane dose-dependently (1, 2, 3 MAC) attenuated the Na 3 VO 4 -induced contraction (P < 0.05-0.005, n = 8), with a greater degree of inhibition by isoflurane than sevoflurane at 2 MAC (P < 0.01) and 3 MAC (P < 0.05). Both anesthetics also attenuated the total band density of the Na 3 VO 4 -induced, tyrosine-phosphorylated substrates in a concentration-dependent manner (P < 0.05-0.005, n = 4), with much greater attenuation by isoflurane than sevoflurane at 1 and 2 MAC (P < 0.05), respectively. Conclusion: The results of the present study demonstrate that isoflurane exhibits a greater degree of inhibition on the Na 3 VO 4 -stimulated contraction and protein tyrosine phosphorylation of rat aortic smooth muscle compared with sevoflurane. These findings suggest that isoflurane depresses the protein tyrosine phosphorylation-mediated contraction of vascular smooth muscle to a greater degree than sevoflurane.

A model is presented that highlights the principal factors determining the form and extent of contraction in arteries upon stimulation of their sympathetic nerve supply. This model incorporates a previous quantitative model of the process of noradrenaline (NAd) diffusion into the vascular media and reuptake into sympathetic varicosities during nerve stimulation (J. Theor. Biol. 226 (2004) 359). It is also dependent on a model of how the subsequent activation of metabotropic receptors initiates a G-protein cascade, resulting in the production of inositol trisphosphate (IP3) and an increase in intracellular calcium concentration, [Ca2+]i, in the smooth muscle cells (J. Theor. Biol. 223 (2003) 93). In the present work we couple this rise in [Ca2+]i to the increase in phosphorylated myosin bound to actin in the cells and hence determine the force development in arteries due to nerve stimulation. The model accounts for force development as a function of [Ca2+]i and for the rate of change of force as a function of the rate of change of [Ca2+]i in single smooth muscle cells. It also accounts for the characteristic time course of the force developed by the media of the rat-tail artery upon nerve stimulation. This consists of a rapid rise to a transient peak followed by a sustained plateau of contraction during the stimulation period, after which the contraction slowly decays back to baseline at a rate dependent on the strength of the stimulation. The model indicates that the transient peak is primarily due to the partial block of the IP3 receptor by the rise in [Ca2+]i and that the main determinant of the equilibrium condition indicated by the plateau phase is the rate of pumping of calcium into the sarcoplasmic reticulum. The relatively slow decline of contraction at the end of nerve stimulation is primarily a consequence of the slow rates of removal of NAd from the media by diffusion and reuptake into the sympathetic varicosities. The model thus provides a quantitative account of vascular smooth muscle contraction upon sympathetic nerve stimulation.

1. Airway smooth muscle (ASM) has recently been termed the 'frustrated' cell of the lung given that contraction of ASM has no proven useful physiological function in adults and yet is indelibly associated with pathological conditions by virtue of its unwanted airflow-limiting actions in asthma. In contrast, pulmonary vascular smooth muscle contraction plays an essential role in the control of blood flow through the lung. 2. Little is known of the differences in phenotype between human ASM and pulmonary vascular smooth muscle (VSM) tissues, but differences in contractile protein and transcription factor expression and regulation of contractile protein promoter activity have been documented. Similarly, the embryological signals in mice required for differentiation of ASM versus pulmonary VSM are distinct. 3. Bronchoconstriction in asthma is currently treated with beta2-adrenoceptor agonists, which relax contracted ASM cells. An additional approach may be to use gene therapy to render ASM unable to contract (via disruption of their contractile apparatus organization). 4. Application of ASM-specific gene therapies would rely on minimal actions on other lung smooth muscle tissues, including pulmonary and bronchial vascular smooth muscle. The combination of mRNA analysis of laser-captured microdissected tissue with in situ immunohistochemical staining for protein should be very useful in terms of being able to characterize definitively the differences in mRNA and protein expression between the smooth muscle species of the lung. Any discovery of an ASM-selective target could provide a novel lead for ASM-directed anti-asthma therapy.

ObjectiveAbdominal aortic aneurysm (AAA) is a common disease with as yet unclear cause. Increased matrix metalloproteinase (MMP) levels in the plasma and aorta are a consistent finding in AAA. Although the role of MMPs in AAA has largely been attributed to degradation of the extracellular matrix proteins, the effects of MMPs on the mechanisms of aortic contraction are unclear. The purpose of this study was to test the hypothesis that MMPs promote aortic dilation by inhibiting the Ca2+ mobilization mechanisms of smooth muscle contraction.MethodsIsometric contraction and 45Ca2+ influx were measured in aortic strips isolated from male Sprague-Dawley rats treated or not treated with MMP-2 and MMP-9.ResultsIn normal Krebs solution (2.5 mmol/L Ca2+) phenylephrine (10−5 mol/L) caused contraction of the aortic strips, which was significantly inhibited (P < .05) by MMP-2 (maximum, 48.9% ± 5.0%) and to a greater extent by MMP-9 (maximum, 69.8% ± 6.2%). The MMP-induced inhibition of phenylephrine contraction depended on concentration and time. The inhibitory effects of MMPs on phenylephrine contraction were reversible. In Ca2+-free (2 mmol/L ethylene glycol bis[β-aminoethyl ether]-N,N,N′,N′-tetraacetic acid) Krebs solution phenylephrine caused a small contraction that was not inhibited by MMP-2 or MMP-9, which suggests that MMPs do not inhibit Ca2+ release from the intracellular stores. Membrane depolarization with 96 mmol/L of potassium chloride, which stimulates Ca2+ entry from the extracellular space, caused a time-dependent and reversible contraction, which was inhibited by MMP-2 and MMP-9. Histologic studies of MMP-treated tissues stained with hematoxylin-eosin or Verhoeff stain for elastin confirmed the absence of degradation of the extracellular matrix. MMP-2 and MMP-9 also caused significant inhibition of 45Ca2+ influx induced by phenylephrine and potassium chloride.ConclusionsThese data suggest that MMP-2 and MMP-9 promote aortic dilation by inhibiting the Ca2+ entry mechanism of vascular smooth muscle contraction.Clinical relevanceAbdominal aortic aneurysm (AAA) is a slow and progressive disease. The late stages of AAA are characterized by degenerative changes in the extracellular matrix and smooth muscle components of the aortic wall. The present study describes novel inhibitory effects of matrix metalloproteinase (MMP) on the Ca2+ entry mechanisms of aortic smooth muscle contraction, even in the absence of extracellular matrix degradation. The MMP-induced inhibition of aortic contraction may further explain the role of increased MMP activity particularly during the early development of AAA. Chronic exposure to MMPs may lead to protracted inhibition of aortic contraction, progressive aortic dilation, and aneurysm formation. MMP-9 is a more potent inhibitor of aortic contraction than MMP-2, consistent with a more dominant role in AAA. Restoration and preservation of smooth muscle contractile function by specific inhibitors of MMPs may represent a new strategy in preventing the progression of small AAA.

This study elucidates the role of cell volume in contractions of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat aorta. We observed that hyposmotic swelling as well as hyper- and isosmotic shrinkage led to VSMR contractions. Swelling-induced contractions were accompanied by activation of Ca2+ influx and were abolished by nifedipine and verapamil. In contrast, contractions of shrunken cells were insensitive to the presence of L-type channel inhibitors and occurred in the absence of Ca2+ o. Thirty minutes preincubation with bumetanide, a potent Na+, K+, CI- cotransport (NKCC) inhibitor, decreased Cl(-)i content, nifedipine-sensitive 45Ca uptake and contractions triggered by modest depolarization ([K+]o = 36 mM). Elevation of [K+]o to 66 mM completely abolished the effect of bumetanide on these parameters. Bumetanide almost completely abrogated phenylephrine-induced contraction, partially suppressed contractions triggered by hyperosmotic shrinkage, but potentiated contractions of isosmotically shrunken VSMR. Our results suggest that bumetanide suppresses contraction of modestly depolarized cells via NKCC inhibition and Cl(-)i-mediated membrane hyperpolarization, whereas augmented contraction of isosmotically shrunken VSMR by bumetanide is a consequence of suppression of NKCC-mediated regulatory volume increase. The mechanism of bumetanide inhibition of contraction of phenylephrine-treated and hyperosmotically shrunken VSMR should be examined further.

Nicotinamide has been shown to be an effective tumor oxygenator in preclinical studies and is part of a promising clinical protocol for the treatment of cancer of the larynx. It has been known for some time that nicotinamide sensitizes tumors, at least in part, by modulating vascular smooth muscle contraction; however, the cellular target within the smooth muscle cell has yet to be identified. Our previous studies have eliminated targets within several agonist and antagonist signaling pathways in smooth muscle, suggesting that it must act at a point close to the contractile machinery of the cell. The present study investigated the effect of nicotinamide on four key steps responsible for force generation via actin/myosin interaction in the smooth muscle cell: calcium binding to calmodulin, calcium-calmodulin binding to smooth muscle myosin light chain kinase (MLCK) inhibitor peptide 480-501 (smMLCIP), modulation of MLCK-dependent signaling, and MLCK-induced phosphorylation of the regulatory myosin light chain, MLC20. Nicotinamide abolished the phosphorylation of MLC20, but had no significant effect on the other endpoints tested. We conclude that the vasorelaxant effects of nicotinamide are mediated mainly through inhibition of MLC20 phosphorylation, and that this could be a promising target for the development of novel tumor oxygenators to enhance radio- and chemotherapy.

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 μM), an inhibitor of p42/p44 MAPK kinase, partially attenuated the ET-1-induced contraction in WKY and SHR. However, SB203580 (10 μM), 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 μM) and SB203580 (10 μM) 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 μM) 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. Keywords:: p38 mitogen-activated protein kinase, endothelin, contraction, spontaneously hypertensive rat

A pool of actin monomers is induced to polymerize into actin filaments during contractile stimulation of smooth muscle. The inhibition of actin dynamics by actin polymerization inhibitors depresses active force generation in smooth muscle. In this study, we hypothesized that Crk-associated substrate plays a role in the regulation of contraction and actin dynamics in vascular smooth muscle. Antisense or sense oligodeoxynucleotides for Crk-associated substrate were introduced into carotid smooth muscle tissues by chemical loading. The treatment of smooth muscle strips with antisense oligodeoxynucleotides inhibited the expression of Crk-associated substrates; it did not influence the expression of actin, myosin heavy chain, and paxillin. Sense oligodeoxynucleotides did not affect the expression of these proteins in smooth muscle tissues. Force generation in response to stimulation with norepinephrine or KCl was significantly lower in antisense-treated muscle strips than in sense-treated strips or in muscle strips not treated with oligodeoxynucleotides. The downregulation of Crk-associated substrate did not attenuate increases in phosphorylation of the 20-kDa regulatory light chain of myosin in response to stimulation with norepinephrine. The increase in F-actin/G-actin ratio during contractile stimulation was significantly inhibited in antisense-treated smooth muscle strips. Contractile activation of smooth muscle increased the association of profilin with actin monomers; the depletion of Crk-associated substrate inhibited the increases in the profilin-actin complex in response to contractile stimulation. These results suggest that Crk-associated substrate is a necessary molecule of signaling cascades that regulate active force generation in smooth muscle. This molecule may regulate actin dynamics in smooth muscle in response to contractile stimulation.

Background Volatile anesthetics inhibit vascular smooth muscle contraction, but the mechanisms responsible are uncertain. In this study, the effects of halothane on Ca2+ signaling and Ca2+ activation of contractile proteins were examined in high K+-depolarized smooth muscle from rat mesenteric resistance arteries. Methods Vessels were cannulated and held at a constant transmural pressure (40 mmHg). Image analysis and microfluorimetry were used to simultaneously measure vessel diameter and smooth muscle intracellular [Ca2+] concentration ([Ca2+]i). Myosin light chain (MLC) phosphorylation was measured using the Western blotting technique. Results Step increases in extracellular [Ca2+] concentration (0-10 mM) in high K+ (40 mM)-depolarized smooth muscle produced incremental increases in [Ca2+]i, MLC phosphorylation, and contraction. Halothane (0.5-4.5%) inhibited contraction in a concentration-dependent manner, but the decrease in [Ca2+]i was small, and there was a marked shift in the [Ca2+]i-contraction relationship to the right, indicating an important Ca2+ desensitizing effect. Halothane (0.5-4.5%) did not affect MLC phosphorylation or the [Ca2+]-MLC phosphorylation relationship, but the MLC phosphorylation-contraction relationship was also shifted rightward, indicating an "MLC phosphorylation" desensitizing effect. In contrast, control relaxations produced by the Ca2+ channel blocker nifedipine were accompanied by decreases in both [Ca2+]i and MLC phosphorylation, and nifedipine had no affect on the [Ca2+]i-contraction, [Ca2+]i-MLC phosphorylation, and MLC phosphorylation-contraction relationships. Conclusions In high K+-depolarized vascular smooth muscle, halothane relaxation is largely mediated by a Ca2+ and MLC phosphorylation desensitizing effect. These results suggest that the relaxing action of halothane is independent of the classic Ca2+-induced myosin phosphorylation contraction mechanism.

Vascular smooth muscle contraction plays a defining role in the regulation and maintenance of blood pressure, and its deregulation is associated with many clinical syndromes including hypertension, coronary vasospasm and congestive heart failure. Over the past 20 years, there has been a growing understanding of the regulation of 20 kDa myosin light chain phosphorylation by myosin light chain kinase and myosin light chain phosphatase, the role of splice-variant isoforms of both the myosin heavy chain and the essential myosin light chain, as well as the signaling pathways involved in smooth muscle contraction under normal and pathophysiological conditions. This review will attempt to recapitulate the data in the field, primarily focusing on the contractile response of smooth muscle, and the molecular determinants responsible for the regulation of vascular tone.