Determination of Region-Specific Roles of the M3 Muscarinic Acetylcholine Receptor in Gastrointestinal Motility.

BACKGROUND: The specific role of the M3 muscarinic acetylcholine receptor in gastrointestinal motility under physiological conditions is unclear, due to a lack of subtype-selective compounds. AIMS: The objective of this study was to determine the region-specific role of the M3 receptor in gastrointestinal motility. METHODS: We developed a novel positive allosteric modulator (PAM) for the M3 receptor, PAM-369. The effects of PAM-369 on the carbachol-induced contractile response of porcine esophageal smooth muscle and mouse colonic smooth muscle (ex vivo) and on the transit in mouse small intestine and rat colon (in vivo) were examined. RESULTS: PAM-369 selectively potentiated the M3 receptor under the stimulation of its orthosteric ligands without agonistic or antagonistic activity. Half-maximal effective concentrations of PAM activity for human, mouse, and rat M3 receptors were 0.253, 0.345, and 0.127 µM, respectively. PAM-369 enhanced carbachol-induced contraction in porcine esophageal smooth muscle and mouse colonic smooth muscle without causing any contractile responses by itself. The oral administration of 30 mg/kg PAM-369 increased the small intestinal transit in both normal motility and loperamide-induced intestinal dysmotility mice but had no effects on the colonic transit, although the M3 receptor mRNA expression is higher in the colon than in the small intestine. CONCLUSIONS: This study provided the first direct evidence that the M3 receptor has different region-specific roles in the motility function between the small intestine and colon in physiological and pathophysiological contexts. Selective PAMs designed for targeted subtypes of muscarinic receptors are useful for elucidating the subtype-specific function.

Pivotal role of Rho-associated kinase 2 in generating the intrinsic circadian rhythm of vascular contractility.

BACKGROUND: The circadian variation in the incidence of cardiovascular events may be attributable to the circadian changes in vascular contractility. The circadian rhythm of vascular contractility is determined by the interplay between the central and peripheral clocks. However, the molecular mechanism of the vascular intrinsic clock that generates the circadian rhythm of vascular contractility still remains largely unknown. METHODS AND RESULTS: The agonist-induced phosphorylation of myosin light chain in cultured smooth muscle cells synchronized by dexamethasone pulse treatment exhibited an apparent circadian oscillation, with a 25.4-hour cycle length. The pharmacological inhibition and knockdown of Rho-associated kinase 2 (ROCK2) abolished the circadian rhythm of myosin light chain phosphorylation. The expression and activity of ROCK2 exhibited a circadian rhythm in phase with that of myosin light chain phosphorylation. A clock gene, RORα, activated the promoter of the ROCK2 gene, whereas its knockdown abolished the rhythmic expression of ROCK2. In the mouse aorta, ROCK2 expression exhibited the circadian oscillation, with a peak at Zeitgeber time 0/24 and a nadir at Zeitgeber time 12. The myofilament Ca(2+) sensitization induced by GTPγS and U46619, a thromboxane A2 analog, at Zeitgeber time 0/24 was greater than that seen at Zeitgeber time 12. The circadian rhythm of ROCK2 expression and myofilament Ca(2+) sensitivity was abolished in staggerer mutant mice, which lack a functional RORα. CONCLUSIONS: ROCK2 plays a pivotal role in generating the intrinsic circadian rhythm of vascular contractility by receiving a cue from RORα. The ROCK2-mediated intrinsic rhythm of vascular contractility may underlie the diurnal variation of the incidence of cardiovascular diseases.

Therapeutic Effect of Proteinase-Activated Receptor-1 Antagonist on Colitis-Associated Carcinogenesis.

BACKGROUND & AIMS: Inflammatory bowel disease is associated with carcinogenesis, which limits the prognosis of the patients. The local expression of proteinases and proteinase-activated receptor 1 (PAR1) increases in inflammatory bowel disease. The present study investigated the therapeutic effects of PAR1 antagonism on colitis-associated carcinogenesis. METHODS: A colitis-associated carcinogenesis model was prepared in mice by treatment with azoxymethane (AOM) and dextran sulfate sodium (DSS). PAR1 antagonist E5555 was administered in long- and short-term protocol, starting on the day of AOM injection and 1 week after completing AOM/DSS treatment, respectively. The fecal samples were collected for metagenome analysis of gut microbiota. The intestinal myofibroblasts of the Crohn's disease patients were used to elucidate underlying cellular mechanisms. Caco-2 cells were used to investigate a possible source of PAR1 agonist proteinases. RESULTS: AOM/DSS model showed weight loss, diarrhea, tumor development, inflammation, fibrosis, and increased production of inflammatory cytokines. The ß-diversity, but not α-diversity, of microbiota significantly differed between AOM/DSS and control mice. E5555 alleviated these pathological changes and altered the microbiota ß-diversity in AOM/DSS mice. The thrombin expression was up-regulated in tumor and non-tumor areas, whereas PAR1 mRNA expression was higher in tumor areas compared with non-tumor areas. E5555 inhibited thrombin-triggered elevation of cytosolic Ca2+ concentration and ERK1/2 phosphorylation, as well as IL6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in intestinal myofibroblasts. Caco-2 cell-conditioned medium contained immunoreactive thrombin, which cleaved the recombinant protein containing the extracellular domain of PAR1 at the thrombin cleavage site. CONCLUSIONS: PAR1 antagonism is proposed to be a novel therapeutic strategy for treatment of inflammatory bowel disease and its associated carcinogenesis.

Cilostazol suppresses angiotensin II-induced vasoconstriction via protein kinase A-mediated phosphorylation of the transient receptor potential canonical 6 channel.

OBJECTIVE: The goal of this study was to determine whether inhibition of transient receptor potential canonical (TRPC) channels underlies attenuation of angiotensin II (Ang II)-induced vasoconstriction by phosphodiesterase (PDE) 3 inhibition. METHODS AND RESULTS: Pretreatment of rat thoracic aorta with cilostazol, a selective PDE3 inhibitor, suppressed vasoconstriction induced by Ang II but not that induced by KCl. The Ang II-induced contraction was largely dependent on Ca(2+) influx via receptor-operated cation channels. Cilostazol specifically suppressed diacylglycerol-activated TRPC channels (TRPC3/TRPC6/TRPC7) through protein kinase A (PKA)-dependent phosphorylation of TRPC channels in HEK293 cells. In contrast, we found that phosphorylation of TRPC6 at Thr69 was essential for the suppression of Ang II-induced Ca(2+) influx by PDE3 inhibition in rat aortic smooth muscle cells (RAoSMCs). Cilostazol specifically induced phosphorylation of endogenous TRPC6 at Thr69. The endogenous TRPC6, but not TRPC3, formed a ternary complex with PDE3 and PKA in RAoSMCs, suggesting the specificity of TRPC6 phosphorylation by PDE3 inhibition. Furthermore, inhibition of PDE3 suppressed the Ang II-induced contraction of reconstituted ring with RAoSMCs, which were abolished by the expression of a phosphorylation-deficient mutant of TRPC6. CONCLUSIONS: PKA-mediated phosphorylation of TRPC6 at Thr69 is essential for the vasorelaxant effects of PDE3 inhibition against the vasoconstrictive actions of Ang II.

Critical role of Rho proteins in myosin light chain di-phosphorylation during early phase of endothelial barrier disruption.

We previously reported the Rho-associated coiled-coil containing protein kinase (ROCK)-mediated di-phosphorylation of myosin light chain (MLC) and actin bundle formation at the cell periphery as early events of the endothelial barrier disruption. We herein examined the role of RhoA during early events of barrier disruption. Treatment of cultured porcine aortic endothelial cells with simvastatin prevented the decrease in trans-endothelial electrical resistance, MLC di-phosphorylation and peripheral actin bundle formation seen 3 min after thrombin stimulation. Co-treatment with geranylgeranyl pyrophosphate rescued the thrombin-induced events. Thrombin increased a GTP-bound form of RhoA and phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at the ROCK site. The intracellular introduction of the inhibitory protein of RhoA inhibited the thrombin-induced di-phosphorylation of MLC. However, knockdown of either one of RhoA, RhoB or RhoC failed to inhibit thrombin-induced MLC di-phosphorylation. The findings suggest that Rho proteins play a critical role during early events of thrombin-induced barrier disruption.

Involvement of reactive oxygen species in thrombin-induced pulmonary vasoconstriction.

RATIONALE: Pulmonary vascular thrombosis and thrombotic arteriopathy are common pathological findings in pulmonary arterial hypertension. Thrombin may thus play an important role in the pathogenesis and pathophysiology of pulmonary arterial hypertension. OBJECTIVES: The present study aimed to elucidate the contractile effect of thrombin in the pulmonary artery and clarify its underlying mechanisms. METHODS: The changes in cytosolic Ca²(+) concentrations ([Ca²(+)](i)), 20-kD myosin light chain (MLC20) phosphorylation, and contraction were monitored in the isolated porcine pulmonary artery. The production of reactive oxygen species (ROS) was evaluated by fluorescence imaging. MEASUREMENTS AND MAIN RESULTS: In the presence of extracellular Ca²(+), thrombin induced a sustained contraction accompanied by an increase in [Ca²(+)](i) and the phosphorylation of MLC20. In the absence of extracellular Ca²(+), thrombin induced a contraction without either [Ca²(+)](i) elevation or MLC20 phosphorylation. This Ca²(+)- and MLC20 phosphorylation-independent contraction was mimicked by hydrogen peroxide and inhibited by N-acetyl cysteine. Fluorescence imaging revealed thrombin to induce the production of ROS. A Rho-kinase inhibitor, Y27632, inhibited not only the thrombin-induced Ca²(+)- and MLC20 phosphorylation-dependent contraction, but also the Ca²(+)- and MLC20 phosphorylation-independent contraction and the ROS production. These effects of thrombin were mimicked by a proteinase-activated receptor 1 (PAR1)-activating peptide. CONCLUSIONS: This study elucidated the Ca²(+)- and MLC20 phosphorylation-independent ROS-mediated noncanonical mechanism as well as Ca²(+)- and MLC20 phosphorylation-dependent canonical mechanism that are involved in the thrombin-induced PAR1-mediated pulmonary vasoconstriction. Rho-kinase was suggested to play multiple roles in the development of thrombin-induced pulmonary vasoconstriction.

Chronic Inhibition of Toll-Like Receptor 9 Ameliorates Pulmonary Hypertension in Rats.

Background Recent accumulating evidence suggests that toll-like receptor 9 (TLR9) is involved in the pathogenesis of cardiovascular diseases. However, its role in pulmonary hypertension remains uncertain. We hypothesized that TLR9 is involved in the development of pulmonary hypertension. Methods and Results A rat model of monocrotaline-induced pulmonary hypertension was used to investigate the effects of TLR9 on hemodynamic parameters, vascular remodeling, and survival. Monocrotaline-exposed rats significantly showed increases in plasma levels of mitochondrial DNA markers, which are recognized by TLR9, TLR9 activation in the lung, and interleukin-6 mRNA level in the lung on day 14 after monocrotaline injection. Meanwhile, monocrotaline-exposed rats showed elevated right ventricular systolic pressure, total pulmonary vascular resistance index and vascular remodeling, together with macrophage accumulation on day 21. In the preventive protocol, administration (days -3 to 21 after monocrotaline injection) of selective (E6446) or nonselective TLR9 inhibitor (chloroquine) significantly ameliorated the elevations of right ventricular systolic pressure and total pulmonary vascular resistance index as well as vascular remodeling and macrophage accumulation on day 21. These inhibitors also significantly reduced NF-κB activation and interleukin-6 mRNA levels to a similar extent. In the short-term reversal protocol, E646 treatment (days 14-17 after monocrotaline injection) almost normalized NF-κB activation and interleukin-6 mRNA level, and reduced macrophage accumulation. In the prolonged reversal protocol, E6446 treatment (days 14-24 after monocrotaline injection) reversed total pulmonary vascular resistance index and vascular remodeling, and improved survival in monocrotaline-exposed rats. Conclusions TLR9 is involved in the development of pulmonary hypertension concomitant via activation of the NF-κB‒IL-6 pathway. Inhibition of TLR9 may be a novel therapeutic strategy for pulmonary hypertension.

Increased Lung Uric Acid Deteriorates Pulmonary Arterial Hypertension.

Background Recent studies have demonstrated that uric acid (UA) enhances arginase activity, resulting in decreased NO in endothelial cells. However, the role of lung UA in pulmonary arterial hypertension (PAH) remains uncertain. We hypothesized that increased lung UA level contributes to the progression of PAH. Methods and Results In cultured human pulmonary arterial endothelial cells, voltage-driven urate transporter 1 (URATv1) gene expression was detected, and treatment with UA increased arginase activity. In perfused lung preparations of VEGF receptor blocker (SU5416)/hypoxia/normoxia-induced PAH model rats, addition of UA induced a greater pressure response than that seen in the control and decreased lung cGMP level. UA-induced pressor responses were abolished by benzbromarone, a UA transporter inhibitor, or L-norvaline, an arginase inhibitor. In PAH model rats, induction of hyperuricemia by administering 2% oxonic acid significantly increased lung UA level and induced greater elevation of right ventricular systolic pressure with exacerbation of occlusive neointimal lesions in small pulmonary arteries, compared with nonhyperuricemic PAH rats. Administration of benzbromarone to hyperuricemic PAH rats significantly reduced lung UA levels without changing XOR (xanthine oxidoreductase) activity, and attenuated right ventricular systolic pressure increase and occlusive lesion development. Topiroxostat, a XOR inhibitor, significantly reduced lung XOR activity in PAH rats, with no effects on increase in right ventricular systolic pressure, arterial elastance, and occlusive lesions. XOR-knockout had no effects on right ventricular systolic pressure increase and arteriolar muscularization in hypoxia-exposed mice. Conclusions Increased lung UA per se deteriorated PAH, whereas XOR had little impact. The mechanism of increased lung UA may be a novel therapeutic target for PAH complicated with hyperuricemia.

Upregulation of proteinase-activated receptor-2 and increased response to trypsin in endothelial cells after exposure to oxidative stress in rat aortas.

BACKGROUND/AIMS: The effects of oxidative stress on the vascular responsiveness to the agonists of proteinase-activated receptors (PARs) were investigated. METHODS: Serum-free incubation was utilized to impose oxidative stress to isolated rat aortas. Spontaneously hypertensive rats (SHR) were investigated as a model of in vivo oxidative stress. RESULTS: Thrombin, trypsin, PAR1-activating peptide (PAR1-AP), PAR2-AP and PAR4-AP induced little or no effect in the aortas of female Wistar-Kyoto rats (WKY). Serum-free incubation induced endothelium-dependent relaxant responses to PAR2 agonists, but not PAR1 or PAR4 agonists, in a manner sensitive to diphenyleneiodonium or ascorbic acid. In male aortas, trypsin and PAR2-AP induced a transient endothelium-dependent relaxation without serum-free incubation. The acetylcholine-induced endothelium-dependent relaxation and the sodium nitroprusside-induced endothelium-independent relaxation remained unchanged. Immunoblot analyses revealed the upregulation of PAR2 in endothelial cells, which was abolished by either diphenyleneiodonium or ascorbic acid. Aortas of female SHR expressed a higher level of PAR2 than WKY and responded to trypsin without serum-free incubation. Treatment with ascorbic acid attenuated the trypsin-induced relaxation and the PAR2 expression in SHR. CONCLUSION: This study provides the first evidence that oxidative stress upregulates PAR2 in endothelial cells, thereby enhancing the endothelium-dependent relaxant response to PAR2 agonists in rat aortas.

Current perspective on the role of the thrombin receptor in cerebral vasospasm after subarachnoid hemorrhage.

Cerebral vasospasm is a persistent arterial narrowing typically observed during the 3 - 14 days after subarachnoid hemorrhage (SAH). Vasospasm is frequently associated with ischemic neurological deficits or even death, resulting in a poor prognosis for patients with SAH. However, the mechanism underlying cerebral vasospasm remains elusive, and no effective therapeutic strategies have been established. A large amount of thrombin is produced during SAH. Recent investigations have uncovered a key role of the thrombin receptor in the pathogenesis of cerebral vasospasm. Thrombin has little contractile effect in the normal cerebral artery, but it induces an enhanced and prolonged contraction after SAH, owing to the up-regulation of thrombin receptor PAR(1) (proteinase-activated receptor 1) and the impairment of receptor desensitization in arterial smooth muscle. Thrombin-mediated activation of PAR(1) is an irreversible process, as it is initiated by the proteolytic removal of the N-terminal region. Since the mechanism of receptor desensitization is impaired after SAH, the thrombin-induced contraction irreversibly persists even after terminating thrombin stimulation. Intrathecal administration of a PAR(1) antagonist prevents the PAR(1) up-regulation and the increased reactivity to thrombin. PAR(1) is suggested to play a key role in cerebral vasospasm and may be useful as a therapeutic target for prevention and treatment of cerebral vasospasm.

Enhanced contractile response of the basilar artery to platelet-derived growth factor in subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: The level of platelet-derived growth factor (PDGF) in cerebrospinal fluid is elevated in subarachnoid hemorrhage (SAH). Therefore, the contractile effect of PDGF on the basilar artery was examined in SAH. METHODS: A rabbit double-hemorrhage SAH model was used. In the medial layers of the control basilar artery, PDGF had no effect on contraction up to 1 nmol/L, whereas 3 nmol/L PDGF induced slight contraction. In SAH, PDGF induced an enhanced contraction with an increase in [Ca(2+)](i) at 1 nmol/L and higher concentrations. The levels of [Ca(2+)](i) and tension induced by 1 nmol/L PDGF in SAH were 17% and 20%, respectively, of those obtained with 118 mmol/L K(+) depolarization. The PDGF-induced elevation of [Ca(2+)](i) and contraction seen in SAH were abolished in the absence of extracellular Ca(2+). In alpha-toxin-permeabilized strips of SAH animals, PDGF induced no further development of tension during contraction induced by 300 nmol/L Ca(2+), suggesting no direct effect on myofilament Ca(2+) sensitivity. Genistein at 10 micromol/L completely inhibited the tension induced by 1 nmol/L PDGF. The level of myosin light-chain phosphorylation was significantly increased by 1 nmol/L PDGF. CONCLUSIONS: These results show that the contractile response to PDGF of the basilar artery was enhanced in SAH. The PDGF-induced contraction depended mostly on tyrosine phosphorylation and Ca(2+)-dependent myosin light-chain phosphorylation. The enhancement of the responsiveness to PDGF may therefore contribute to the development of cerebral vasospasm after SAH.

Involvement of STIM1 in the proteinase-activated receptor 1-mediated Ca2+ influx in vascular endothelial cells.

Thrombin increases the cytosolic Ca(2+) concentrations and induces NO production by activating proteinase-activated receptor 1 (PAR(1)) in vascular endothelial cells. The store-operated Ca(2+) influx is a major Ca(2+) influx pathway in non-excitable cells including endothelial cells and it has been reported to play a role in the thrombin-induced Ca(2+) signaling in endothelial cells. Recent studies have identified stromal interaction molecule 1 (STIM1) to function as a sensor of the store site Ca(2+) content, thereby regulating the store-operated Ca(2+) influx. However, the functional role of STIM1 in the thrombin-induced Ca(2+) influx and NO production in endothelial cells still remains to be elucidated. Fura-2 and diaminorhodamine-4M fluorometry was utilized to evaluate the thrombin-induced changes in cytosolic Ca(2+) concentrations and NO production, respectively, in porcine aortic endothelial cells transfected with small interfering RNA (siRNA) targeted to STIM1. STIM1-targeted siRNA suppressed the STIM1 expression and the thapsigargin-induced Ca(2+) influx. The degree of suppression of the STIM1 expression correlated well to the degree of suppression of the Ca(2+) influx. The knockdown of STIM1 was associated with a substantial inhibition of the Ca(2+) influx and a partial reduction of the NO production induced by thrombin. The thrombin-induced Ca(2+) influx exhibited the similar sensitivity toward the Ca(2+) influx inhibitors to that seen with the thapsigargin-induced Ca(2+) influx. The present study provides the first evidence that STIM1 plays a critical role in the PAR(1)-mediated Ca(2+) influx and Ca(2+)-dependent component of the NO production in endothelial cells.

Proteinase-activated receptor 1 antagonism ameliorates experimental pulmonary hypertension.

AIMS: Pulmonary hypertension (PH) is characterized by progressive increases in pulmonary vascular resistance (PVR). Thrombotic lesions are common pathological findings. The pulmonary artery has a unique property regarding the vasoconstrictive response to thrombin, which is mediated by proteinase-activated receptor 1 (PAR1). We aim to elucidate the role of PAR1 in the development and progression of PH. METHODS AND RESULTS: A rat model of monocrotaline-induced PH and a mouse model of hypoxia (Hx)-induced PH were used to investigate the effects of atopaxar (a PAR1 antagonist) and PAR1 knockout on haemodynamic parameters, right ventricular hypertrophy (RVH), vascular remodelling and survival. In perfused lung preparations, the pressor response to PAR1 agonist was significantly augmented in monocrotaline-induced PH. Both the preventive and therapeutic administration of atopaxar significantly inhibited the increase in PVR and the development of RVH and prolonged survival. A real-time PCR revealed that the level of PAR1 mRNA in the pulmonary artery was significantly higher than that in any of the systemic arteries examined in control rats, and the level was significantly up-regulated in monocrotaline-induced PH. PAR1 gene knockout significantly attenuated the haemodynamic and histological findings in the mouse model of Hx-induced PH. CONCLUSION: The specific expression of PAR1 in the pulmonary artery and its up-regulation were suggested to play a critical role in the development and progression of experimental PH in murine models. PAR1 is a potential therapeutic target for the treatment of PH.

Involvement of different receptor subtypes in prostaglandin E2-induced contraction and relaxation in the lower esophageal sphincter and esophageal body.

Prostaglandin E2 (PGE2) plays a role in the pathogenesis of gastro-esophageal reflux disease (GERD). There are 4 subtypes of PGE2, PGE2 receptor 1, 2, 3 and 4 (EP 1-4). In GERD patents, PGE2, EP2 and EP4 are upregulated. However, the effects of PGE2 on esophageal motility remain elusive. We examined how PGE2 regulates motility in the porcine circular smooth muscle of the lower esophageal sphincter (LES), and the circular and longitudinal smooth muscle of the esophagus body in organ bath. PGE2 induced tonic relaxation in the LES and circular smooth muscle, but transient contraction in longitudinal smooth muscle. The relaxation of the LES and circular smooth muscle was similar in pattern and mechanism, but was much larger in the LES. The relaxation was completely blocked by a voltage-gated K+ channel blocker or 40 mM K+ depolarization, indicating the involvement of K+ channel. Longitudinal smooth muscle contraction was completely blocked by an L-type Ca2+ channel blocker, showing the contribution of Ca2+ movement. The involvement of the EP receptor in motility was examined with selective receptor agonists and antagonists. Activation of EP2 and EP4 caused relaxation in the LES and circular smooth muscle. Compatible with PGE2, EP2 and EP4 agonists caused more significant relaxation in the LES than in circular smooth muscle. EP1 contributed to the longitudinal smooth muscle contraction. The different effects of PGE2 in the LES, circular and longitudinal smooth muscle contributes to esophageal motility, their impairment might increase the amount and frequency of esophageal reflux.

Rac1-dependent transcriptional up-regulation of p27Kip1 by homophilic cell-cell contact in vascular endothelial cells.

The mechanism for the transcriptional up-regulation of p27Kip1 due to the formation of the cell-cell contact was investigated in vascular endothelial cells. The induction of the cell-cell contact by adding an extra number of endothelial cells activated Rac1, up-regulated p27Kip1 mRNA and protein, and also facilitated the cell cycle arrest. Transduction of the Rac1 inhibitor protein using the cell-penetrating peptide or treatment with a Rac1 inhibitor NSC23766 inhibited the p27Kip1 up-regulation and delayed the cell cycle arrest. Rac1 was therefore suggested to mediate the contact-induced transcriptional up-regulation of p27Kip1. The role of Rac1 in the regulation of the p27Kip1 promoter activity was next examined with a luciferase reporter assay. The promoter activity was increased by inducing the cell-cell contact, which was significantly inhibited by the Rac1 inhibitory protein and NSC23766. The evaluation of various truncated promoter regions determined region -620 to -573 nucleotides from the initiation codon to be responsible for the contact-induced, Rac1-dependent activation of the p27Kip1 promoter. The present study thus demonstrated for the first time that the activation of Rac1 due to the cell-cell contact plays a critical role in the transcriptional up-regulation of p27Kip1 in vascular endothelial cells.

Plasmin induces endothelium-dependent nitric oxide-mediated relaxation in the porcine coronary artery.

OBJECTIVE: Plasmin is a key enzyme in fibrinolysis. We attempted to determine the possible role of plasmin in the regulation of vascular tone, while also investigating the mechanism of plasmin-induced vasorelaxation. METHODS AND RESULTS: In porcine coronary artery, plasmin induced an endothelium-dependent relaxation. This relaxing effect was mostly abolished by a proteinase inhibitor, a plasmin inhibitor, or a nitric oxide (NO) synthase inhibitor. The preceding stimulation with plasmin significantly inhibited the subsequent relaxation induced by thrombin but not that induced by proteinase-activated receptor-1-activating peptide. The relaxation induced by trypsin and substance P remained unaffected by the preceding plasmin stimulation. The pretreatment with plasmin, thrombin, or trypsin significantly attenuated the plasmin-induced relaxation. In porcine coronary artery endothelial cells (PCAECs) and human umbilical vein endothelial cells (HUVECs), plasmin induced a transient elevation in the cytosolic Ca2+ concentrations ([Ca2+]i). The preceding stimulation with plasmin inhibited the subsequent [Ca2+]i elevation induced by thrombin but not that induced by trypsin. In PCAECs, plasmin concentration-dependently induced NO production. CONCLUSIONS: The present study demonstrated, for the first time, that plasmin induced an endothelium-dependent NO-mediated relaxation in the porcine coronary artery, while also showing plasmin to specifically inactivate the thrombin receptor.

Endogenous Hydrogen Sulfide Contributes to Tone Generation in Porcine Lower Esophageal Sphincter Via Na+/Ca2+ Exchanger.

BACKGROUND AND AIMS: Hydrogen sulfide (H2S) is a major physiologic gastrotransmitter. Its role in the regulation of the lower esophageal sphincter (LES) function remains unknown. The present study addresses this question. METHODS: Isometric contraction was monitored in circular smooth muscle strips of porcine LES. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored in fura-2-loaded strips with front-surface fluorometry. The contribution of endogenous H2S to LES contractility was investigated by examining the effects of inhibitors of H2S-generating enzymes, including cystathionine-ß-synthase, cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase, on the LES function. RESULTS: Porcine LES strips myogenically maintained a tetrodotoxin-resistant basal tone. Application of AOA (cystathionine-ß-synthase inhibitor) or L-aspartic acid (L-Asp; 3-mercaptopyruvate sulfurtransferase inhibitor) but not DL-PAG (cystathionine-γ-lyase inhibitor), decreased this basal tone. The relaxant effects of AOA and L-Asp were additive. Maximum relaxation was obtained by combination of 1 mM AOA and 3 mM L-Asp. Immunohistochemical analyses revealed that cystathionine-ß-synthase and 3-mercaptopyruvate sulfurtransferase, but not cystathionine-γ-lyase, were expressed in porcine LES. AOA+L-Asp-induced relaxation was accompanied by a decrease in [Ca2+]i and inversely correlated with the extracellular Na+ concentration ([Na+]o) (25-137.4 mM), indicating involvement of an Na+/Ca2+ exchanger. The reduction in the basal [Ca2+]i level by AOA was significantly augmented in the antral smooth muscle sheets of Na+/Ca2+ exchanger transgenic mice compared with wild-type mice. CONCLUSIONS: Endogenous H2S regulates the LES myogenic tone by maintaining the basal [Ca2+]i via Na+/Ca2+ exchanger. H2S-generating enzymes may be a potential therapeutic target for esophageal motility disorders, such as achalasia.

Long-term inhibition of RhoA attenuates vascular contractility by enhancing endothelial NO production in an intact rabbit mesenteric artery.

RhoA plays a critical role in regulating NO production in cultured endothelial cells. To determine its role in in situ endothelial cells, we investigated the effects of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors and a RhoA-binding domain of Rho-kinase (RB) on vascular contractility in the isolated rabbit mesenteric artery. Ex vivo treatment of the strips with 3x10(-5) mol/L simvastatin and fluvastatin for approximately 24 to 30 hours significantly attenuated the contractile response to phenylephrine and high K+ in the presence of endothelium. The addition of N(omega)-nitro-L-arginine methyl ester and the removal of endothelium abolished the attenuation of the contractile response. The cotreatment with geranylgeranyl pyrophosphate prevented the statin-induced attenuation of the contractile response, whereas geranylgeranyl transferase inhibitor mimicked the effect of simvastatin. Treatment with simvastatin enhanced the bradykinin-induced endothelium-dependent relaxation in the mesenteric artery, whereas it had no effect on the bradykinin-induced [Ca2+]i elevation in endothelial cells of the aortic valves. Introduction of RB to the strips using a cell-penetrating peptide of Tat protein (TATHA-RB) attenuated the contractile responses in a NO-dependent manner. However, a Rac1/Cdc42-binding fragment of p21-activated protein kinase, RB without Tat peptide or TATHA-protein A had no effect. The in vivo treatment of rabbit with simvastatin and TATHA-RB attenuated the contractility in a NO-dependent manner. Simvastatin and TATHA-RB significantly upregulated eNOS in the rabbit mesenteric artery. The present study provides the first evidence that RhoA plays a physiological role in suppressing NO production in in situ endothelial cells.

Trypsin induces biphasic muscle contraction and relaxation via transient receptor potential vanilloid 1 and neurokinin receptors 1/2 in porcine esophageal body.

Duodenal reflux of fluids containing trypsin relates to refractory gastroesophageal reflux disease (GERD). Esophageal peristalsis and clearance are important factors in GERD pathogenesis. However, the function of trypsin in esophageal body contractility is not fully understood. In this study, effects of trypsin on circular smooth muscle (CSM) and longitudinal smooth muscle (LSM) of the porcine esophageal body were examined. Trypsin elicited a concentration dependent biphasic response, a major contraction and a subsequent relaxation only in CSM. In CSM, contraction occurred at trypsin concentrations of 100nM and relaxation at 1µM. A proteinase-activated receptor (PAR)2 activating peptide, SLIGKV-NH2 (1mM), induced a monophasic contraction. Those responses were unaffected by tetrodotoxin though abolished by the gap junction uncouplers carbenoxolone and octanol. They were also partially inhibited by a transient receptor potential vanilloid type 1 (TRPV1) antagonist and abolished by combination of neurokinin receptor 1 (NK1) and NK2 antagonists, but not by an NK3 antagonist, suggesting a PAR2-TRPV1-substance P pathway in sensory neurons. Substance P (100nM), an agonist for various NK receptors (NK1, NK2 and NK3) with differing affinities, induced significant contraction in CSM, but not in LSM. The contraction was also blocked by the combination of NK1 and NK2 antagonists, but not by the NK3 antagonist. Moreover, substance P-induced contractions were unaffected by the TRPV1 antagonist, but inhibited by a gap junction uncoupler. In conclusion, trypsin induced a biphasic response only in CSM and this was mediated by PAR2, TRPV1 and NK1/2. Gap junctions were indispensable in this tachykinin-induced response.

Rac1 regulation of surface expression of protease-activated receptor-1 and responsiveness to thrombin in vascular smooth muscle cells.

OBJECTIVE: Protease-activated receptor-1 (PAR1) mediates the thrombin-induced proliferation and hypertrophy of vascular smooth muscle cells. A role of Rac1 in the regulation of PAR1 expression was investigated. METHODS AND RESULTS: Treatment with simvastatin, a hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitor, for 24 hours attenuated the transient [Ca2+]i elevation induced by thrombin. Immunofluorescence staining revealed that simvastatin decreased the surface expression of PAR1 in a manner dependent on protein geranylgeranylation. Introduction of a Rac1/Cdc42 inhibitory fragment but not a RhoA inhibitory fragment using a cell-penetrating peptide also attenuated the response to thrombin and decreased the surface expression of PAR1. Finally, downregulation of Rac1, but not RhoA, using an RNA interference technique attenuated the thrombin-induced [Ca2+]i elevation. However, the level of PAR1 mRNA and the total amount of PAR1 protein remained unchanged. CONCLUSIONS: Here, we provide for the first time 3 lines of evidence that Rac1 plays a critical role in maintaining the surface expression of PAR1 and the responsiveness to thrombin in vascular smooth muscle cells. Rac1 is suggested to regulate the constitutive trafficking of PAR1 and thereby regulate the surface expression of PAR1.