Mayumi Hirano

Protein kinase network in the regulation of phosphorylation and dephosphorylation of smooth muscle myosin light chain

The contraction of smooth muscle is regulated primarily by intracellular Ca2+ signal. It is well established that the elevation of the cytosolic Ca2+ level activates myosin light chain kinase, which phosphorylates 20 kDa regulatory myosin light chain and activates myosin ATPase. The simultaneous measurement of cytosolic Ca2+ concentration and force development revealed that the alteration of the Ca2+-sensitivity of the contractile apparatus as well as the Ca2+ signal plays a critical role in the regulation of smooth muscle contraction. The fluctuation of an extent of myosin phosphorylation for a given change in Ca2+ concentration is considered to contribute to the major mechanisms regulating the Ca2+-sensitivity. The level of myosin phosphorylation is determined by the balance between phosphorylation and dephosphorylation. The phosphorylation level for a given Ca2+ elevation is increased either by Ca2+-independent activation of phosphorylation process or inhibition of dephosphorylation. In the last decade, the isolation and cloning of myosin phosphatase facilitated the understanding of regulatory mechanism of dephosphorylation process at the molecular level. The inhibition of myosin phosphatase can be achieved by (1) alteration of hetrotrimeric structure, (2) phosphorylation of 110 kDa regulatory subunit MYPT1 at the specific site and (3) inhibitory protein CPI-17 upon its phosphorylation. Rho-kinase was first identified to phosphorylate MYPT1, and later many kinases were found to phosphorylate MYPT1 and inhibit dephosphorylation of myosin. Similarly, the phosphorylation of CPI-17 can be catalysed by multiple kinases. Moreover, the myosin light chain can be phosphorylated by not only authentic myosin light chain kinase in a Ca2+-dependent manner but also by multiple kinases in a Ca2+-independent manner, thus adding a novel mechanism to the regulation of the Ca2+-sensitivity by regulating the phosphorylation process. It is now clarified that the protein kinase network is involved in the regulation of myosin phosphorylation and dephosphorylation. However, the physiological role of each component remains to be determined. One approach to accomplish this purpose is to investigate the effects of the dominant negative mutants of the signalling molecule on the smooth muscle contraction. In this regards, a protein transduction technique utilizing the cell-penetrating peptides would provide a useful tool. In the preliminary study, we succeeded in introducing a fragment of MYPT1 into the arterial strips, and found enhancement of contraction.

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 3×10-5 mol/L simvastatin and fluvastatin for ≈24 to 30 hours significantly attenuated the contractile response to phenylephrine and high K+ in the presence of endothelium. The addition of N&ohgr;-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.

Distinct Ca2+ Requirement for NO Production between Proteinase-Activated Receptor 1 and 4 (PAR1 and PAR4) in Vascular Endothelial Cells

Proteinase-activated receptors 1 and 4 (PAR1 and PAR4) are the major receptors mediating thrombin-induced NO production in endothelial cells. The intracellular signaling following their activation still remains to be elucidated. The present study provides the first evidence for the distinct Ca2+ requirement for the NO production between PAR1 and PAR4. The activation of PAR1 by the activating peptide (PAR1-AP) elevated cytosolic Ca2+ concentrations ([Ca2+]i) and activated NO production in porcine aortic and human umbilical vein endothelial cells, whereas it had little effect on bovine aortic endothelial cells. PAR4 activation by PAR4-AP consistently induced NO production without an appreciable [Ca2+]i elevation in three types of endothelial cells. The PAR1-mediated NO production was significantly inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), whereas the PAR4-mediated NO production was resistant. NO production following the PAR1 and PAR4 activation was significantly inhibited by pertussis toxin, but it was resistant to a Gαq/11 inhibitor, YM254890 [(1R)-1-{(3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-[(1R)-1-methoxyethyl]-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl}-2-methylpropyl rel-(2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate]. However, YM254890 abrogated the PAR1-mediated Ca2+ signal. PAR4-mediated NO production was substantially inhibited by the inhibitors of phosphotidylinositol-3 kinase (PI3K) and Akt, as well as by the dominant negative mutant of Akt. The PAR1-mediated NO production was relatively resistant to inhibitors of PI3K. An immunoblot analysis revealed a transient increase in the phosphorylation of Akt and endothelial NO synthase following the PAR4 stimulation. In conclusion, PAR1 and PAR4 engage distinct signal transduction mechanisms to activate NO production in vascular endothelial cells. PAR4 preferably activates Gαi/o and induced NO production in a manner mostly independent of Ca2+ but dependent on the PI3K/Akt pathway, whereas PAR1 activates both the Ca2+-dependent and -independent mechanisms.

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, ROR&agr;, 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 Ca2+ sensitization induced by GTP&ggr;S 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 Ca2+ sensitivity was abolished in staggerer mutant mice, which lack a functional ROR&agr;. Conclusions— ROCK2 plays a pivotal role in generating the intrinsic circadian rhythm of vascular contractility by receiving a cue from ROR&agr;. The ROCK2-mediated intrinsic rhythm of vascular contractility may underlie the diurnal variation of the incidence of cardiovascular diseases.

Up‐regulation of proteinase‐activated receptor 1 and increased contractile responses to thrombin after subarachnoid haemorrhage

The mechanism for the development of post‐haemorrhagic cerebral vasospasm after subarachnoid haemorrhage (SAH) still remains unknown.

Impaired feedback regulation of the receptor activity and the myofilament Ca2+ sensitivity contributes to increased vascular reactiveness after subarachnoid hemorrhage.

Cerebral vasospasm determines the prognosis of subarachnoid hemorrhage (SAH). The increased vascular reactiveness has an important role in the development of cerebral vasospasm. This study analyzed the roles of the receptor-mediated signaling and the myofilament Ca2+ sensitivity in the increased vascular reactiveness in SAH, using the basilar artery of a rabbit SAH model. Endothelin-1, thrombin, and phenylephrine induced transient increases in [Ca2+]i, myosin light chain phosphorylation, and contraction in the controls. All these responses were not only enhanced but also became sustained in SAH. In the sequential stimulation of thrombin receptor or α1-adrenoceptor, the second response was substantially attenuated in the controls, whereas it was maintained in SAH. The thrombin-induced contraction in SAH irreversibly persisted even after terminating the thrombin stimulation. This contraction was completely reversed by trypsin and a Gαq inhibitor YM254890, thus suggesting the sustained receptor activity during the sustained contraction. YM254890 also inhibited the endothelin-1- and phenylephrine-induced sustained contraction. Furthermore, the GTPγS-induced transient contraction in the control α-toxin-permeabilized strips was converted to a sustained contraction in SAH. The results provide the first evidence that the feedback inactivation of the receptor activity and the myofilament Ca2+ sensitivity was impaired in SAH, thus contributing to the increased vascular reactiveness.

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 Ca2+ 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 Ca2+ 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. Conclusion—PKA-mediated phosphorylation of TRPC6 at Thr69 is essential for the vasorelaxant effects of PDE3 inhibition against the vasoconstrictive actions of Ang II.

Mechanism of down-regulation of L-type Ca2+ channel in the proliferating smooth muscle cells of rat aorta

The mechanism of down‐regulation of L‐type Ca2+ channel (L‐VOC) was investigated in rat aortic smooth muscle cells in primary culture. On culture days 3–5, the cells actively incorporated the 5‐bromo‐2′‐deoxy‐uridine (BrdU), and did not respond to K+ depolarization nor express α1C subunit of L‐VOC. At confluence on day 8, BrdU incorporation decreased, and the cells up‐regulated α1C subunit mRNA, expressed α1C subunit protein at cell periphery, and responded to K+ depolarization. Treating the proliferating cells on day 3 with serum‐free media or 10 μM PD98059, a MAP kinase kinase inhibitor, for 2 days induced the expression of α1C subunit protein and the responsiveness to K+ depolarization. However, the serum starvation, but not PD98059, decreased the BrdU incorporation and increased the α1C subunit mRNA. It is concluded that the expression of L‐VOC is substantially suppressed in the proliferating cells due to two mechanisms; a MAP kinase‐mediated post‐transcriptional down‐regulation and the transcriptional down‐regulation by additional mitogenic signals. J. Cell. Biochem. 87: 242–251, 2002.

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.

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 and Results— 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 [Ca2+]i at 1 nmol/L and higher concentrations. The levels of [Ca2+]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 [Ca2+]i and contraction seen in SAH were abolished in the absence of extracellular Ca2+. In α-toxin–permeabilized strips of SAH animals, PDGF induced no further development of tension during contraction induced by 300 nmol/L Ca2+, suggesting no direct effect on myofilament Ca2+ sensitivity. Genistein at 10 &mgr;mol/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 Ca2+-dependent myosin light-chain phosphorylation. The enhancement of the responsiveness to PDGF may therefore contribute to the development of cerebral vasospasm after SAH.