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.

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.

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.

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.

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.

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.

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.

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.

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.

Myosin di-phosphorylation and peripheral actin bundle formation as initial events during endothelial barrier disruption.

The phosphorylation of the 20-kD myosin light chain (MLC) and actin filament formation play a key role in endothelial barrier disruption. MLC is either mono- or di-phosphorylated (pMLC and ppMLC) at T18 or S19. The present study investigated whether there are any distinct roles of pMLC and ppMLC in barrier disruption induced by thrombin. Thrombin induced a modest bi-phasic increase in pMLC and a robust mono-phasic increase in ppMLC. pMLC localized in the perinuclear cytoplasm during the initial phase, while ppMLC localized in the cell periphery, where actin bundles were formed. Later, the actin bundles were rearranged into stress fibers, where pMLC co-localized. Rho-kinase inhibitors inhibited thrombin-induced barrier disruption and peripheral localization of ppMLC and actin bundles. The double, but not single, mutation of phosphorylation sites abolished the formation of peripheral actin bundles and the barrier disruption, indicating that mono-phosphorylation of MLC at either T18 or S19 is functionally sufficient for barrier disruption. Namely, the peripheral localization, but not the degree of phosphorylation, is suggested to be essential for the functional effect of ppMLC. These results suggest that MLC phosphorylation and actin bundle formation in cell periphery are initial events during barrier disruption.

Experimental realization of spatially separated entanglement with continuous variables using laser pulse trains.

Spatially separated entanglement is demonstrated by interfering two high-repetition squeezed pulse trains. The entanglement correlation of the quadrature amplitudes between individual pulses is interrogated. It is characterized in terms of the sufficient inseparability criterion with an optimum result of in the frequency domain and in the time domain. The quantum correlation is also observed when the two measurement stations are separated by a physical distance of 4.5 m, which is sufficiently large to demonstrate the space-like separation, after accounting for the measurement time.

Trypsin-induced biphasic regulation of tone in the porcine lower esophageal sphincter.

The lower esophageal sphincter (LES) plays an important role in coordinated esophageal motility. The present study aimed to elucidate how trypsin affects LES contractility. Porcine LES circular smooth muscle strips were prepared. Contractile responses to trypsin were assessed. Trypsin (300nM) induced a transient contraction. At concentrations of 1µM or higher, trypsin induced biphasic responses, consisting of a transient contraction followed by a transient relaxation. Pretreatment with either 1µM tetrodotoxin or carbenoxolone had no effect on these responses. In contrast, trypsin-induced responses were completely blocked by pretreatment with the serine protease inhibitor. Pretreatment with 10µM FSLLRY-NH2, a PAR2 antagonist, significantly inhibited trypsin-induced biphasic responses. Trypsin (1µM)-induced contractions were partially inhibited by pretreatment with 10µM Y-27632. In addition, trypsin (10µM)-induced relaxation was partially inhibited by pretreatment with 10µM Y-27632, 10µM PD98059 or 10µM SB203580. Trypsin-induced relaxation was abolished by increasing the extracellular K(+) concentration to 40mM, but not by pretreatment with l-arginine methyl ester. Furthermore, trypsin-induced relaxation was partially inhibited by pretreatment with 10µM glibenclamide or 1µM 4-aminopyridine. Trypsin causes biphasic regulation of LES tone by directly acting on smooth muscle. Rho-associated protein kinase (ROK) is involved in trypsin-induced contraction, whereas ROK, ERK1/2, p38MAPK, and membrane hyperpolarization are involved in relaxation. The regulation of LES tone by trypsin may play a role in esophageal motility.

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.

Mechanisms underlying potentiation of endothelin-1-induced myofilament Ca(2+) sensitization after subarachnoid hemorrhage.

Increased vascular smooth muscle contractility has an important role in the development of cerebral vasospasm after subarachnoid hemorrhage (SAH). Myofilament Ca(2+) sensitivity is a major determinant of smooth muscle contractility. We investigated changes in the Ca(2+)-sensitizing effect of endothelin-1 (ET-1) and the mechanisms underlying ET-1-induced Ca(2+) sensitization after SAH using a rabbit SAH model. After SAH, the contractile response to ET-1 was enhanced, and the ET(A) receptor expression was upregulated in the basilar artery. In α-toxin-permeabilized preparations, ET-1 induced enhanced and prolonged contraction after SAH, suggesting that ET-1-induced Ca(2+) sensitization is potentiated after SAH. Endothelin-1-induced Ca(2+) sensitization became less sensitive to inhibitors of Rho-associated coiled-coil protein kinase (ROCK) and protein kinase C (PKC) after SAH. The expression of PKCα, ROCK2, PKC-potentiated phosphatase inhibitor of 17 kDa (CPI-17) and myosin phosphatase target subunit 1 (MYPT1) was upregulated, and the level of phosphorylation of CPI-17 and MYPT1 was elevated after SAH. This study demonstrated for the first time that the Ca(2+)-sensitizing effect of ET-1 on myofilaments is potentiated after SAH. The increased expression and activity of PKCα, ROCK2, CPI-17, and MYPT1, as well as the upregulation of ET(A) receptor expression are suggested to underlie the enhanced and prolonged Ca(2+) sensitization induced by ET-1.

Combined argatroban and anti-oxidative agents prevents increased vascular contractility to thrombin and other ligands after subarachnoid haemorrhage.

BACKGROUND AND PURPOSE: Increased vascular contractility plays a fundamental role in cerebral vasospasm in subarachnoid haemorrhage (SAH). We investigated the role of thrombin and its receptor, proteinase-activated receptor 1 (PAR1), and other G protein-coupled receptors in the increased contractility, and examined the preventive effects of the thrombin inhibitor, argatroban, and anti-oxidative agents, vitamin C and tempol. EXPERIMENTAL APPROACH: A rabbit model of SAH was utilized. Contractile responses of the isolated basilar artery and the level of oxidative stress of brain tissues were evaluated. KEY RESULTS: Contractile responses to thrombin and PAR1-activating peptide (PAR1-AP) were enhanced and prolonged after SAH. The thrombin-induced contraction persisted even after terminating thrombin stimulation. When sequentially stimulated with PAR1-AP, the second response was maintained in SAH, while it was substantially attenuated in the control. Only a combination of argatroban with vitamin C or tempol prevented both the enhancement and prolongation of the contractile response to PAR1-AP and restored the reversibility of the thrombin-induced contraction. The responses to angiotensin II, vasopressin and PGF(2α) were enhanced and prolonged after SAH to varying degrees, and responded differently to the treatment. The response to vasopressin exhibited a similar phenomenon to that seen with PAR1-AP. Oxidative stress was increased in SAH, and normalized by the treatment with argatroban, vitamin C or their combination. CONCLUSIONS AND IMPLICATIONS: Increased vascular reactivity to agonists in SAH was attributable to the enhancement and prolongation of the contractile response. A combination of argatroban and anti-oxidative agents was required to prevent both the enhancement and prolongation of the contractile response.

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.

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.

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.

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.