Yasuyuki Tokinaga

Vasodilation mediated by inward rectifier K+ channels in cerebral microvessels of hypertensive and normotensive rats

Although inward rectifier K+ channels contribute to the regulation of cerebral circulation, dilation of cerebral microvasculature mediated by these channels has not been demonstrated in chronic hypertension. We designed the present study to examine the roles of inward rectifier K+ channels in the vasodilation produced by increased levels of extracellular K+ in cerebral parenchymal arterioles from hypertensive and normotensive rats. During constriction to prostaglandin F2&agr; (5 × 10−7 M), the arterioles within brain slices were evaluated using computer-assisted microscopy. Potassium chloride (KCl) induced vasodilation in cerebral arterioles from normotensive (5–10 mM) and hypertensive (5–15 mM) rats, whereas an inward rectifier K+ channel antagonist barium chloride (BaCl2; 10−5 M) completely abolished the vasodilation in both strains. In arterioles of hypertensive rats, vasodilator responses to KCl were augmented compared with those in normotensive rats. In contrast, the vasodilator responses induced by sodium nitroprusside (3 × 10−8 to 3 × 10−6 M) in these two strains were similar. These results suggest that in cerebral cortex parenchymal microvessels, inward rectifier K+ channels play a crucial role in vasodilation produced by extracellular K+ and that the dilation of cerebral arterioles via these channels is augmented in chronic hypertension.

Involvement of Ca2+Sensitization in Ropivacaine-induced Contraction of Rat Aortic Smooth Muscle

Background:The mechanisms of amino-amide local anesthetic agent-induced vasoconstriction remain unclear. The current study was designed to examine the roles of the protein kinase C (PKC), Rho kinase, and p44/42 mitogen-activated protein kinase (p44/42 MAPK) signaling pathways in calcium (Ca2+)–sensitization mechanisms in ropivacaine-induced vascular contraction. Methods:Endothelium-denuded rat aortic rings, segments, and strips were prepared. The cumulative dose–response relations of contraction and intracellular Ca2+ concentration to ropivacaine were tested, using isometric force transducers and a fluorometer, respectively. The dose-dependent ropivacaine-induced phosphorylation of PKC and p44/42 MAPK and the membrane translocation of Rho kinase were also detected using Western blotting. Results:Ropivacaine induced a dose-dependent biphasic contractile response and an increase in intracellular Ca2+ concentration of rat aortic rings, increasing at concentrations of 3 × 10−5 m to 3 × 10−4 m and decreasing from 10−3 m to 3 × 10−3 m, with a greater tension/intracellular Ca2+ concentration ratio than that induced with potassium chloride. The contraction was attenuated in a dose-dependent manner, by the PKC inhibitors bisindolylmaleimide I and calphostin C, the Rho-kinase inhibitor Y 27632, and the p44/42 MAPK inhibitor PD 098059. Ropivacaine also induced an increase in phosphorylation of PKC and p44/42 MAPK, and membrane translocation of Rho kinase in accordance with the contractile responses, which were also significantly inhibited by bisindolylmaleimide I and calphostin C, Y 27632, and PD 098059, correspondingly. Conclusion:These findings demonstrated that PKC-, Rho kinase–, and p44/42 MAPK–mediated Ca2+-sensitization mechanisms are involved in the ropivacaine-induced biphasic contraction of rat aortic smooth muscle.

The vascular relaxing effects of sevoflurane and isoflurane are more important in hypertensive than in normotensive rats.

PurposeThe vascular response to anesthetics is altered in hypertensive patients since the functional and structural integrities of vascular smooth muscle and endothelium are deranged. The effects of anesthetics on angiotensin II (Ang ll)-induced changes in vascular tone are not well understood. We investigated the effects of sevoflurane and isoflurane on Ang II-induced vasoconstriction in spontaneously hypertensive rats (SHR).MethodsThe dose-dependent effects of sevoflurane and isoflurane on the Ang II-induced contraction of aortic rings, in the presence and absence of an intact endothelium, were investigated in normotensive Wistar-Kyoto rats (WKY) and SHR and compared using isometric force transducers.ResultsAng II (10−9-10−6 M) induced a similar transient phasic contraction of endothelium-intact rings from the two rat strains in a dose-dependent manner. Removal of the endothelium augmented the Ang II-elicited phasic contraction, to a greater extent in the SHR group than in the WKY group. Sevoflurane and isoflurane (1–3 minimum alveolar concentration) concentration-dependently inhibited the Ang II-induced contraction of endothelium-intact rings from WKY; an effect that was greatly enhanced following removal of the endothelium. Agreater degree of attenuation of the Ang II-induced contraction of both endothelium-intact and -denuded rings by the two anesthetics was observed in the SHR group. The inhibitory effects of isoflurane on the Ang II-induced contraction of aortic rings from both strains appeared to be stronger than that of sevoflurane at equipotent concentrations.ConclusionOur finding that the inhibitory effects of isoflurane and sevoflurane on Ang II-induced vasoconstriction are enhanced in SHR may, at least in part, account for the anesthesia-induced systemic hypotension frequently seen in hypertensive patients.RésuméObjectifLa réponse vasculaire aux anesthésiques est modifiée chez les patients hypertendus, car l’intégrité fonctionnelle et structurale des muscles lisses vasculaires et de l’endothélium est perturbée. Les effets des anesthésiques sur les changements de tonus vasculaire induits par l’angiotensine II (Ang II) ne sont pas très bien compris. Nous avons exploré les effets du sévoflurane et de l’isoflurane sur la vasoconstriction induite par l’Ang II chez des rats spontanément hypertendus (PSH).MéthodeLes effets reliés à la dose du sévoflurane et de l’isoflurane sur la contraction d’anneaux aortiques induite par l’Ang II, en présence et en l’absence d’un endothélium intact, ont été examinés chez des rats Wistar-Kyoto (WKY) normotendus et RSH et comparés en utilisant des transducteurs de force isométrique.RésultatsL’Ang II (10−9-10−6 M) a induit une contraction phasique transitoire, dépendante de la dose, des anneaux intacts d’endothélium prélevés chez les deux souches de rats. Le retrait de l’endothélium a augmenté la contraction phasique induite par l’Ang II, davantage dans le groupe RSH que dans le groupe WKY. Le sévoflurane et l’isoflurane (de concentration alvéolaire minimale de 1–3) ont inhibé la contraction induite pas l’Ang II des anneaux intacts d’endothélium de WKY en fonction de la concentration. C’est un effet qui a été grandement accru à la suite du retrait de l’endothélium. Une plus importante atténuation de la contraction induite par l’Ang II des anneaux dénudés et des anneaux intacts d’endothélium par les deux anesthésiques a été observée dans le groupe RSH. Les effets inhibiteurs de l’isoflurane sur la contraction des anneaux aortiques des deux souches induite par l’Ang II sont apparus plus prononcés que ceux du sévoflurane pour des concentrations équivalentes.ConclusionNotre résultat voulant que les effets inhibiteurs de l’isoflurane et du sévoflurane sur la vasoconstriction induite par l’Ang II soient accrus chez les RSH peut, au moins en partie, expliquer l’hypotension générale induite par l’anesthésie observée chez les patients hypertendus.

Sevoflurane inhibits angiotensin II-induced, protein kinase c-mediated but not Ca2+-elicited contraction of rat aortic smooth muscle.

BackgroundWhether volatile anesthetics attenuate angiotensin II–mediated vascular tone has not been determined. The current study was designed to investigate the effects of sevoflurane on the angiotensin II–stimulated, Ca2+- and protein kinase C (PKC)–mediated contraction of rat aortic smooth muscle. MethodsThe dose-dependent effects of sevoflurane on angiotensin II (10−7 m)–induced contraction, the increase in intracellular Ca2+ concentration, and PKC phosphorylation of rat aortic smooth muscle were measured using an isometric force transducer, a fluorometer, and Western blotting, respectively. ResultsAngiotensin II induced a transient increase in intracellular Ca2+ concentration, phosphorylation of Ca2+-dependent PKC (cPKC)-&agr;, and consequently, a transient contraction of rat aortic smooth muscle. Phosphorylation of the Ca2+-independent PKC-&egr; was not detected. The angiotensin II–induced contraction was almost completely abolished by removing extracellular Ca2+ and was significantly inhibited by the selective cPKC inhibitor Gö 6976 (10−5 m) but was not inhibited by the nonselective PKC inhibitor Ro 31-8425 (10−5 m). Sevoflurane dose-dependently inhibited the angiotensin II–induced contraction, with reductions of 14.2 ± 5.2% (P > 0.05), 26.7 ± 8.9% (P < 0.05), and 38.5 ± 12.8% (P < 0.01) (n = 10) in response to 1.7, 3.4, and 5.1% sevoflurane, respectively. The angiotensin II–elicited increase in intracellular Ca2+ concentration was not significantly influenced by 3.4, 5.1, or 8.5% sevoflurane. However, cPKC-&agr; phosphorylation induced by angiotensin II was inhibited dose dependently by 1.7, 3.4, and 5.1% sevoflurane, with depressions of 20.5 ± 14.2% (P > 0.05), 37.0 ± 17.8% (P < 0.05), and 62.5 ± 12.2% (P < 0.01) (n = 4), respectively. ConclusionThe current study indicates that Ca2+ and cPKC-&agr; are involved in angiotensin II–induced vascular contraction. Sevoflurane dose-dependently inhibited the angiotensin II–stimulated, cPKC-mediated but not Ca2+-elicited contraction of rat aortic smooth muscle.

Sevoflurane Inhibits Guanosine 5′-[γ-thio]triphosphate–stimulated, Rho/Rho-kinase–mediated Contraction of Isolated Rat Aortic Smooth Muscle

Background The Rho/Rho-kinase signaling pathway plays an important role in mediating Ca2+ sensitization of vascular smooth muscle. The effect of anesthetics on Rho/Rho-kinase–mediated vasoconstriction has not been determined to date. This study is designed to examine the possible inhibitory effects of sevoflurane on the Rho/Rho-kinase pathway by measuring guanosine 5′-[&ggr;-thio]triphosphate (GTP&ggr;S)-stimulated contraction and translocation of RhoA (one of the three Rho subtypes) and Rock-2 (one of the two Rho-kinase subtypes) from the cytosol to the membrane in rat aortic smooth muscle. Methods GTP&ggr;S-induced contraction of rat aortic endothelium-denuded rings was measured using an isometric force transducer, and GTP&ggr;S-stimulated membrane translocation of RhoA and Rock-2 in smooth muscle cells was detected with Western blotting in the presence and absence of sevoflurane. Results GTP&ggr;S (10−4 m) induced a sustained contraction, which was significantly inhibited by the Rho-kinase inhibitor, Y27632 (3 × 10−6 m). Before treatment with GTP&ggr;S, RhoA and Rock-2 were detected primarily in the cytosolic fraction. GTP&ggr;S (10−4 m) stimulated the translocation of RhoA and Rock-2 from the cytosol to the membrane, which was sustained for more than 60 min. Sevoflurane (1.7, 3.4, and 5.1%) concentration dependently inhibited the GTP&ggr;S-induced constriction of rat aortic smooth muscle with a reduction of constriction of 52–75% (P < 0.01, n = 8), and attenuated the translocation of RhoA and Rock-2 by 31–66% and 34–78%, respectively (P < 0.05–0.01, respectively; n = 4). Conclusion The current findings show that sevoflurane depresses the GTP&ggr;S-stimulated contraction and translocation of both Rho and Rho-kinase from the cytosol in a concentration-dependent manner, indicating that sevoflurane is able to inhibit vasoconstriction mediated by the Rho/Rho-kinase pathway in rat aortic smooth muscle.

Mechanism of the ropivacaine-induced increase in intracellular Ca2+ concentration in rat aortic smooth muscle

Background:  Ropivacaine is a long‐acting local anesthetic with low cardiac toxicity that induces vasoconstriction in vitro and in vivo. Vascular smooth muscle tone is regulated by changes in both intracellular Ca2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity. Therefore, the aim of this study was to examine the mechanism underlying the increase in [Ca2+]i in ropivacaine‐induced vascular contraction.

The mechanism behind the inhibitory effect of isoflurane on angiotensin II-induced vascular contraction is different from that of sevoflurane.

BACKGROUND: Angiotensin II (Ang II)-induced vascular contraction is mediated both by a Ca2+-mediated signaling pathway and a Ca2+ sensitization mechanism. We recently demonstrated that sevoflurane inhibits the contractile response to Ang II, mainly by inhibiting protein kinase C (PKC) phosphorylation that regulates myofilament Ca2+ sensitivity, without significant alteration of intracellular Ca2+ concentration ([Ca2+]i) in rat aortic smooth muscle. The current study was designed to determine the mechanisms by which isoflurane inhibits Ang II-induced contraction of rat aortic smooth muscle. METHODS: The effects of isoflurane on vasoconstriction, increase in [Ca2+]i, and phosphorylation of PKC in response to Ang II (10−7 M) were investigated, using an isometric force transducer, a fluorometer, and Western blotting, respectively. RESULTS: Ang II elicited a transient contraction of rat aortic smooth muscle that was associated with an increase in [Ca2+]i and PKC phosphorylation. Isoflurane (1.2%–3.5%) inhibited Ang II-induced contraction of rat aortic smooth muscle in a concentration-dependent manner (P < 0.05 at 1.2%, P < 0.01 at 2.3% and 3.5% isoflurane, n = 6). Isoflurane also inhibited elevation of [Ca2+]i in response to Ang II (P < 0.01 at 2.3% and 3.5% isoflurane, n = 6), but failed to affect Ang II-induced phosphorylation of PKC at concentrations up to 3.5% (n = 7). CONCLUSION: These results suggest that, unlike sevoflurane, the inhibitory effect of isoflurane on Ang II-induced contraction is mainly mediated by attenuation of the Ca2+-mediated signaling pathway.

Volatile anesthetics inhibit angiotensin II-induced vascular contraction by modulating myosin light chain phosphatase inhibiting protein, CPI-17 and regulatory subunit, MYPT1 phosphorylation.

BACKGROUND: Vascular contraction is regulated by myosin light chain (MLC) phosphorylation. Inhibition of MLC phosphatase (MLCP) increases MLC phosphorylation for a given Ca2+ concentration, and results in promoting myofilament Ca2+ sensitivity. MLCP activity is mainly determined by protein kinase C (PKC) and Rho kinase through the phosphorylation of both PKC-potentiated inhibitory protein (CPI-17) and myosin phosphatase target subunit (MYPT1). We have previously demonstrated that sevoflurane inhibits PKC phosphorylation and membrane translocation of Rho kinase. This study was designed to investigate the effects of sevoflurane and isoflurane on CPI-17, MYPT1, and MLC phosphorylation in response to angiotensin II (Ang II) in rat aortic smooth muscle. METHODS: The effects of sevoflurane or isoflurane (1–3 minimum alveolar concentration) on the vasoconstriction and phosphorylation of MLC, CPI-17, MYPT1 at Thr853 and MYPT1 at Thr696 in response to Ang II were investigated using isometric force transducer and Western blotting, respectively. RESULTS: Ang II (10−7 M) elicited a transient contraction of rat aortic smooth muscle that was inhibited by both sevoflurane and isoflurane in a concentration-dependent manner. Ang II also induced an increase in the phosphorylation of MLC, CPI-17, MYPT1/Thr853 and MYPT1/Thr696. Sevoflurane inhibited the phosphorylation of MLC, CPI-17, and MYPT1/Thr853 in response to Ang II in a concentration-dependent manner. Isoflurane also inhibited MLC phosphorylation in response to Ang II, which was associated with decreases in MYPT1/Thr853, but not in CPI-17. Neither sevoflurane nor isoflurane affected the Ang II-induced phosphorylation of MYPT1/Thr696. CONCLUSION: Although both volatile anesthetics inhibited Ang II-induced vasoconstriction and MLC phosphorylation to similar extent, the mechanisms behind the inhibitory effects of each anesthetic on MLCP activity appear to differ.

The Inhibitory Effects of Isoflurane on Protein Tyrosine Phosphorylation–modulated Contraction of Rat Aortic Smooth Muscle

Background:Tyrosine kinase–catalyzed protein tyrosine phosphorylation plays an important role in initiating and modulating vascular smooth muscle contraction. The aim of the current study was to examine the effects of isoflurane on sodium orthovanadate (Na3VO4), a potent protein tyrosine phosphatase inhibitor–induced, tyrosine phosphorylation–mediated contraction of rat aortic smooth muscle. Methods:The Na3VO4-induced contraction of rat aortic smooth muscle and tyrosine phosphorylation of proteins including phospholipase Cγ-1 (PLCγ-1) and p44/p42 mitogen-activated protein kinase (MAPK) were assessed in the presence of different concentrations of isoflurane, using isometric force measurement and Western blotting methods, respectively. Results:Na3VO4 (10−4 m) induced a gradually sustained contraction and significant increase in protein tyrosine phosphorylation of a set of substrates including PLCγ-1 and p42MAPK, all of which were markedly inhibited by genistein (5 × 10−5 m), a tyrosine kinase inhibitor. Isoflurane (1.2–3.5%) dose-dependently depressed the Na3VO4-induced contraction (P < 0.05–0.005; n = 8). Isoflurane also attenuated the total density of the Na3VO4-induced, tyrosine-phosphorylated substrate bands and the density of tyrosine-phosphorylated PLCγ-1 band and p42MAPK band (P < 0.05–0.005; n = 4) in a concentration-dependent manner. Conclusion:The findings of the current study, that isoflurane dose-dependently inhibits both the Na3VO4-stimulated contraction and tyrosine phosphorylation of a set of proteins including PLCγ-1 and p42MAPK in rat aortic smooth muscle, suggest that isoflurane depresses protein tyrosine phosphorylation–modulated contraction of vascular smooth muscle, especially that mediated by the tyrosine-phosphorylated PLCγ-1 and MAPK signaling pathways.

The inhibitory effects of sevoflurane on angiotensin II- induced, p44/42 mitogen-activated protein kinase-mediated contraction of rat aortic smooth muscle.

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 × 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.