Shosuke Takahashi

Long-Term Inhibition of Rho-Kinase Suppresses Angiotensin II–Induced Cardiovascular Hypertrophy in Rats In Vivo Effect on Endothelial NAD(P)H Oxidase System

Abstract— Intracellular signaling pathway mediated by small GTPase Rho and its effector Rho-kinase plays an important role in regulation of vascular smooth muscle contraction and other cellular functions. We have recently demonstrated that Rho-kinase is substantially involved in angiotensin II–induced gene expressions and various cellular responses in vitro. However, it remains to be examined whether Rho-kinase is involved in the angiotensin II–induced cardiovascular hypertrophy in vivo and, if so, what mechanisms are involved. Long-term infusion of angiotensin II for 4 weeks caused hypertrophic changes of vascular smooth muscle and cardiomyocytes in rats. Both changes were significantly suppressed by concomitant oral treatment with fasudil, which is metabolized to a specific Rho-kinase inhibitor, hydroxyfasudil, after oral administration. Angiotensin II caused a perivascular accumulation of macrophages and Rho-kinase activation, both of which were also significantly suppressed by fasudil. Vascular NAD(P)H oxidase expression (nox1, nox4, gp91phox, and p22phox) and endothelial production of superoxide anions were markedly increased by angiotensin II, both of which were also significantly suppressed by fasudil. Thus, fasudil ameliorated the impaired endothelium-dependent relaxations caused by angiotensin II without affecting vasodilator function of vascular smooth muscle. These results provide evidence that Rho-kinase is substantially involved in the angiotensin II–induced cardiovascular hypertrophy in rats in vivo. The suppression of endothelial NAD(P)H oxidase upregulation and resultant superoxide production and the amelioration of endothelial vasodilator function may be involved in this process.

Inhibition of Myosin Phosphatase by Upregulated Rho-Kinase Plays a Key Role for Coronary Artery Spasm in a Porcine Model With Interleukin-1β

BACKGROUND We recently demonstrated that the Rho-kinase-mediated pathway plays an important role for coronary artery spasm in our porcine model with interleukin-1beta (IL-1beta). In this study, we examined whether or not Rho-kinase is upregulated at the spastic site and if so, how it induces vascular smooth muscle hypercontraction. METHODS AND RESULTS Segments of the left porcine coronary artery were chronically treated from the adventitia with IL-1beta-bound microbeads. Two weeks after the operation, as reported previously, intracoronary serotonin repeatedly induced coronary hypercontractions at the IL-1beta-treated site both in vivo and in vitro, which were markedly inhibited by Y-27632, one of the specific inhibitors of Rho-kinase. Reverse transcription-polymerase chain reaction analysis demonstrated that the expression of Rho-kinase mRNA was significantly increased in the spastic compared with the control segment. Western blot analysis showed that during the serotonin-induced contractions, the extent of phosphorylation of the myosin-binding subunit of myosin phosphatase (MBS), one of the major substrates of Rho-kinase, was significantly greater in the spastic than in the control segment and that the increase in MBS phosphorylations was also markedly inhibited by Y-27632. There was a highly significant correlation between the extent of MBS phosphorylations and that of contractions. CONCLUSIONS These results indicate that Rho-kinase is upregulated at the spastic site and plays a key role in inducing vascular smooth muscle hypercontraction by inhibiting myosin phosphatase through the phosphorylation of MBS in our porcine model.

Inhibition of Superoxide Production and Ca2+ Mobilization in Human Neutrophils by Halothane, Enflurane, and Isoflurane

The inhibitory effects of three inhalation anesthetics, i.e., halothane, enflurane, and isoflurane, on superoxide production and the intracellular mobilization of calcium in human neutrophils were studied. The superoxide production induced by N-formyl-methionylleucyl-phcnylalanine (FMLP) was inhibited by the anesthetics, but the binding of FML|3H|P to the cells and the superoxide-forming NADPH oxidasc of the phagocytic vesicles were not inhibited. The inhibition of the cellular superoxide production was partially reversed by the addition of a calcium ionophore, A23187. The increase in intracellular free calcium monitored by a calcium-sensitive fluorescent probe, quin-2 and the release of calcium from hydrophobic environment monitored by chlortetracycline were inhibited dose dependently by the anesthetics. These observations suggest that decreased mobilization of intracellular Ca2+ is one of the mechanisms by which the anesthetics inhibited the superoxide production of human neutrophils stimulated by FMLP.

Evidence for Protein Kinase C-Mediated Activation of Rho- Kinase in a Porcine Model of Coronary Artery Spasm

Objective—We have recently demonstrated that protein kinase C (PKC) and Rho-kinase play important roles in coronary vasospasm in a porcine model. However, it remains to be examined whether there is an interaction between the two molecules to cause the spasm. Methods and Results—A segment of left porcine coronary artery was chronically treated with IL-1&bgr;–bound microbeads in vivo. Two weeks after the operation, phorbol ester caused coronary spasm in vivo and coronary hypercontractions in vitro at the IL-1&bgr;–treated segment; both were significantly inhibited by hydroxyfasudil, a specific Rho-kinase inhibitor. Guanosine 5′-[&ggr;-thio]triphosphate (GTP&ggr;S), which activates Rho with a resultant activation of Rho-kinase, enhanced Ca2+ sensitization of permeabilized vascular smooth muscle cells, which were resistant to the blockade of PKC by calphostin C. The GTP&ggr;S-induced Ca2+ sensitization was greater in the spastic segment than in the control segment. Western blot analysis revealed that only PKC&dgr; isoform was activated during the hypercontraction. Conclusions—These results demonstrate that PKC and Rho-kinase coexist on the same intracellular signaling pathway, with PKC located upstream on Rho-kinase, and that among the PKC isoforms, only PKC&dgr; may be involved. Thus, the strategy to inhibit Rho-kinase rather than PKC may be a more specific and useful treatment for coronary spasm.

Effects of Lidocaine on Intracellular Ca2+ and Tension in Airway Smooth Muscle

Background:Many studies have demonstrated that lidocaine directly relaxes airway smooth muscle. The underlying mechanisms, especially in relation to Ca2+mobilization, remain to be elucidated. Methods:Using front-surface fluorometry and fura-2-loaded porcine tracheal smooth muscle strips, intracellular Ca2+ concentration ([Ca2+]1) and isometric tension were simultaneously measured. Results:In cases of 40 mM K+-induced contraction and 1 µM acetylcholine (ACh)-induced contraction, the cumulative application of lidocaine (10-6˜3 X 10-3 M) caused a concentration-dependent decrease in [Ca2+]1 and tension, and almost complete relaxation. To examine the effect of lidocaine on Ca2+ sensitivity of the contractile apparatus, the [Ca2+]1-tension relationship was determined by changing the extracellular Ca2+ concentration during 40 mM K+ induced depolarization, with and without treatment with lidocaine. Although treatment with 1 mM lidocaine inhibited increases in both [Ca2+]1 and tension induced by extracellular Ca2+, it had little effect on the [Ca2+]1-tension relationship. In the presence of 1 µM ACh, the [Ca2+]1-tension relationship shifted markedly to the left, thereby indicating an increase in Ca2+ sensitivity of the contractile apparatus; this shift was inhibited by 1 mM lidocaine. In the absence of extracellular Ca2+, 1 mM lidocaine inhibited the release of stored Ca2+ induced by 1 µM ACh, but not that by 20 mM caffeine. Conclusions:Lidocaine directly relaxes airway smooth muscle by decreasing [Ca2+]1. In addition, lidocaine inhibits the ACh-induced increase of Ca2+ sensitivity of the contractile apparatus, although it has little effect on Ca2+ sensitivity during high K+ depolarization. The decrease in [Ca2+]1 is attributed to inhibition of the influx of extracellular Ca2+, as induced by high K+ depolarization and by ACh, and to the inhibition of the ACh-induced release of stored Ca2+.

Effects of volatile anesthetics on acetylcholine-induced relaxation in the rabbit mesenteric resistance artery

Background Vascular endothelium plays an important role in the regulation of vascular tone. Volatile anesthetics have been shown to attenuate endothelium‐mediated relaxation in conductance arteries, such as aorta. However, significant differences in volatile anesthetic pharmacology between these large vessels and the small vessels that regulate systemic vascular resistance and blood flow have been documented, yet little is known about volatile anesthetic action on endothelial function in resistance arteries. Furthermore, endothelium‐dependent relaxation mediated by factors other than endothelium‐derived relaxing factor (EDRF) has recently been recognized, and there is no information available regarding volatile anesthetic action on non‐EDRF‐mediated endothelium‐dependent relaxation. Methods Employing isometric tension recording and microelectrode methods, the authors first characterized the endothelium‐dependent relaxing and hyperpolarizing actions of acetylcholine (ACh) in rabbit small mesenteric arteries, and tested the sensitivities of these actions to EDRF pathway inhibitors and Potassium sup + channel blockers. They then examined the effects of the volatile anesthetics isoflurane, enflurane, and sevoflurane on ACh‐induced endothelium‐dependent relaxation that was sensitive to EDRF inhibitors and that which was resistant to the EDRF inhibitors but sensitive to blockers of ACh‐induced hyperpolarization. The effects of the volatile anesthetics on endothelium‐independent sodium nitroprusside (SNP)‐induced relaxation were also studied. Results Acetylcholine concentration‐dependently caused both endothelium‐dependent relaxation and hyperpolarization of vascular smooth muscle. The relaxation elicited by low concentrations of ACh (less or equal to 0.1 micro Meter) was almost completely abolished by the EDRF inhibitors NG ‐nitro L‐arginine (LNNA), oxyhemoglobin (HbO sub 2), and methylene blue (MB). The relaxation elicited by higher concentrations of ACh (greater or equal to 0.3 micro Meter) was only attenuated by the EDRF inhibitors. The remaining relaxation, as well as the ACh‐induced hyperpolarization that was also resistant to EDRF inhibitors, were both specifically blocked by tetraethylammonium (TEA greater or equal to 10 mM). Sodium nitroprusside, a NO donor, produced dose‐dependent relaxation, but not hyperpolarization, in the endothelium‐denuded (E[‐]) strips, and the relaxation was inhibited by MB and HbO2, but not TEA (greater or equal to 10 mM). One MAC isoflurane, enflurane, and sevoflurane inhibited both ACh relaxation that was sensitive to the EDRF inhibitors and the ACh relaxation resistant to the EDRF inhibitors and sensitive to TEA, but not SNP relaxation (in the E[‐] strips). An additional finding was that the anesthetics all significantly inhibited norepinephrine (NE) contractions in the presence and absence of the endothelium or after exposure to the EDRF inhibitors. Conclusions The results confirm that ACh has a hyperpolarizing action in rabbit small mesenteric resistance arteries that is independent of EDRF inhibitors but blocked by the Potassium sup + channel blocker TEA. The ACh relaxation in these resistance arteries thus appears to consist of distinct EDRF‐mediated and hyperpolarization‐mediated components. Isoflurane, enflurane, and sevoflurane inhibited both components of the ACh‐induced relaxation in these small arteries, indicating a more global depression of endothelial function or ACh signaling in endothelial cells, rather than a specific effect on the EDRF pathway. All these anesthetics exerted vasodilating action in the presence of NE, the primary neurotransmitter of the sympathetic nervous system, which plays a major role in maintaining vasomotor tone in vivo. This strongly indicates that the vasodilating action of these anesthetics probably dominates over their inhibitory action on the EDRF pathway and, presumably, contributes to their known hypotensive effects in vivo. Finally, the vasodilating action of these anesthetics is, at least in part, independent from endothelium.

Mechanisms of Direct Inhibitory Action of Isoflurane on Vascular Smooth Muscle of Mesenteric Resistance Arteries

Background Isoflurane has been shown to directly inhibit vascular reactivity. However, less information is available regarding its underlying mechanisms in systemic resistance arteries. Methods Endothelium-denuded smooth muscle strips were prepared from rat mesenteric resistance arteries. Isometric force and intracellular Ca2+ concentration ([Ca2+]i) were measured simultaneously in the fura-2-loaded strips, whereas only the force was measured in the &bgr;-escin membrane-permeabilized strips. Results Isoflurane (3–5%) inhibited the increases in both [Ca2+]i and force induced by either norepinephrine (0.5 &mgr;m) or KCl (40 mm). These inhibitions were similarly observed after depletion of intracellular Ca2+ stores by ryanodine. Regardless of the presence of ryanodine, after washout of isoflurane, its inhibition of the norepinephrine response (both [Ca2+]i and force) was significantly prolonged, whereas that of the KCl response was quickly restored. In the ryanodine-treated strips, the norepinephrine- and KCl-induced increases in [Ca2+]i were both eliminated by nifedipine, a voltage-gated Ca2+ channel blocker, whereas only the former was inhibited by niflumic acid, a Ca2+-activated Cl− channel blocker. Isoflurane caused a rightward shift of the Ca2+-force relation only in the fura-2-loaded strips but not in the &bgr;-escin-permeabilized strips. Conclusions In mesenteric resistance arteries, isoflurane depresses vascular smooth muscle reactivity by directly inhibiting both Ca2+ mobilization and myofilament Ca2+ sensitivity. Isoflurane inhibits both norepinephrine- and KCl-induced voltage-gated Ca2+ influx. During stimulation with norepinephrine, isoflurane may prevent activation of Ca2+-activated Cl− channels and thereby inhibit voltage-gated Ca2+ influx in a prolonged manner. The presence of the plasma membrane appears essential for its inhibition of the myofilament Ca2+ sensitivity.

Propofol-induced Increase in Vascular Capacitance Is Due to Inhibition of Sympathetic Vasoconstrictive Activity

Background Venodilation is thought to be one of the mechanisms underlying propofol‐induced hypotension. The purpose of this study is to test two hypotheses: (1) propofol increases systemic vascular capacitance, and (2) the capacitance change produced by propofol is a result of an inhibition of sympathetic vasoconstrictor activity. Methods In 33 Wistar rats previously anesthetized with urethane and ketamine, vascular capacitance was examined before and after propofol infusion by measuring mean circulatory filling pressure (Pmcf). The P (mcf) was measured during a brief period of circulatory arrest produced by inflating an indwelling balloon in the right atrium. Rats were assigned into four groups: an intact group, a sympathetic nervous system (SNS)‐block group produced by hexamethonium infusion, a SNS‐block + noradrenaline (NA) group, and a hypovolemic group. The Pmcf was measured at a control state and 2 min after a bolus administration of 2, 10, and 20 mg/kg of propofol. Results The mean arterial pressure (MAP) was decreased by propofol dose‐dependently in intact, hypovolemic, and SNS‐block groups, but the decrease in MAP was less in the SNS‐block group (‐25%) than in the intact (‐50%) and hypovolemic (‐61%) groups. In the SNS‐block + NA group, MAP decreased only at 20 mg/kg of propofol (‐18%). The Pmcf decreased in intact and hypovolemic groups in a dose‐dependent fashion but was unchanged in the SNS‐block and SNS‐block + NA groups. Conclusions The results have provided two principal findings: (1) propofol decreases Pmcf dose‐dependently, and (2) the decrease in Pmcf by propofol is elicited only when the sympathetic nervous system is intact, suggesting that propofol increases systemic vascular capacitance as a result of an inhibition of sympathetic nervous system.

Influence of diabetes mellitus, hypercholesterolemia, and their combination on EDHF-mediated responses in mice

The endothelium synthesizes and releases several vasodilator substances, including vasodilator prostaglandins, NO, and EDHF. NO-mediated relaxations are reduced by various risk factors, such as diabetes mellitus and hypercholesterolemia. However, it remains to be elucidated whether EDHF-mediated relaxations also are reduced by those factors and their combination. In this study, we addressed this point in mice. We used small mesenteric arteries from control, diabetic (streptozotocin-induced), apolipoprotein-E-deficient (ApoE−/−), and diabetic ApoE−/− mice. In control mice, endothelium-dependent relaxations to acetylcholine were largely mediated by EDHF. This EDHF-mediated component was slightly reduced in diabetic mice, preserved in ApoE−/− mice, and markedly reduced in diabetic ApoE−/− mice with an increase in NO-mediated component and a negative contribution of indomethacin-sensitive endothelium-derived contracting factor (EDCF). Endothelium-independent relaxations to sodium nitroprusside or NS1619, a direct opener of calcium-activated K channels, were attenuated in ApoE−/− and diabetic ApoE−/− mice. Endothelium-dependent hyperpolarizations were significantly reduced in diabetic mice, preserved in ApoE−/− mice, and again markedly reduced in diabetic ApoE−/− mice. These results indicate that hypercholesterolemia alone minimally affects the EDHF-mediated relaxations, and diabetes mellitus significantly attenuated the responses, whereas their combination markedly attenuates the responses with a compensatory involvement of NO and a negative contribution of EDCF.

Effects of Sevoflurane and Isoflurane on Electrocorticographic Activities in Patients With Temporal Lobe Epilepsy

To compare the neuroexcitatory effects of sevoflurane and isoflurane, we recorded electrocorticograms (ECoG) during wakefulness and during sevoflurane and isoflurane anesthesia in six patients with temporal lobe epilepsy (TLE). These patients had subdural grid electrodes chronically implanted in the temporal region. During sevoflurane anesthesia at 1.5 minimum alveolar concentration (MAC) of the combination with 67% nitrous oxide (N2O), a marked increase in interictal paroxysmal activities was observed in four patients. Two patients had a slight increase in paroxysmal activities. Activated areas were widely distributed, not being confined to the ictal onset zone of spontaneous seizures. However, isoflurane anesthesia at 1.5 MAC of the combination with 67% N2O was associated with less increased paroxysmal activity. While the neuroexcitatory properties of sevoflurane proved greater than those of isoflurane, the widespread irritative response to sevoflurane administration was not helpful in localizing the epileptogenic area.