Yoshinobu Tomiyama

Molecular mechanisms of the inhibitory effects of propofol and thiamylal on sarcolemmal adenosine triphosphate-sensitive potassium channels

BackgroundBoth propofol and thiamylal inhibit adenosine triphosphate–sensitive potassium (KATP) channels. In the current study, the authors investigated the effects of these anesthetics on the activity of recombinant sarcolemmal KATP channels encoded by inwardly rectifying potassium channel (Kir6.1 or Kir6.2) genes and sulfonylurea receptor (SUR1, SUR2A, or SUR2B) genes. MethodsThe authors used inside-out patch clamp configurations to investigate the effects of propofol and thiamylal on the activity of recombinant KATP channels using COS-7 cells transfected with various types of KATP channel subunits. ResultsPropofol inhibited the activities of the SUR1/Kir6.2 (EC50 = 77 &mgr;m), SUR2A/Kir6.2 (EC50 = 72 &mgr;m), and SUR2B/Kir6.2 (EC50 = 71 &mgr;m) channels but had no significant effects on the SUR2B/Kir6.1 channels. Propofol inhibited the truncated isoform of Kir6.2 (Kir6.2&Dgr;C36) channels (EC50 = 78 &mgr;m) that can form functional KATP channels in the absence of SUR molecules. Furthermore, the authors identified two distinct mutations R31E (arginine residue at position 31 to glutamic acid) and K185Q (lysine residue at position 185 to glutamine) of the Kir6.2&Dgr;C36 channel that significantly reduce the inhibition of propofol. In contrast, thiamylal inhibited the SUR1/Kir6.2 (EC50 = 541 &mgr;m), SUR2A/Kir6.2 (EC50 = 248 &mgr;m), SUR2B/Kir6.2 (EC50 = 183 &mgr;m), SUR2B/Kir6.1 (EC50 = 170 &mgr;m), and Kir6.2&Dgr;C36 channels (EC50 = 719 &mgr;m). None of the mutants significantly affects the sensitivity of thiamylal. ConclusionsThese results suggest that the major effects of both propofol and thiamylal on KATP channel activity are mediated via the Kir6.2 subunit. Site-directed mutagenesis study suggests that propofol and thiamylal may influence Kir6.2 activity by different molecular mechanisms; in thiamylal, the SUR subunit seems to modulate anesthetic sensitivity.

Cerebral Blood Flow During Hemodilution and Hypoxia in Rats Role of ATP-Sensitive Potassium Channels

BACKGROUND AND PURPOSE Hypoxia and hemodilution both reduce arterial oxygen content (CaO(2)) and increase cerebral blood flow (CBF), but the mechanisms by which hemodilution increases CBF are largely unknown. ATP-sensitive potassium (K(ATP)) channels are activated by intravascular hypoxia, and contribute to hypoxia-mediated cerebrovasodilatation. Although CaO(2) can be reduced to equal levels by hypoxia or hemodilution, intravascular PO(2) is reduced only during hypoxia. We therefore tested the hypothesis that K(ATP) channels would be unlikely to contribute to cerebrovasodilatation during hemodilution. METHODS Glibenclamide (19.8 microg) or vehicle was injected into the cisterna magna of barbiturate-anesthetized rats. The dose of glibenclamide was chosen to yield an estimated CSF concentration of 10(-4) M. Thirty minutes later, some animals underwent either progressive isovolumic hemodilution or hypoxia (over 30 minutes) to achieve a CaO(2) of approximately 7.5 mL O(2)/dL. Other animals did not undergo hypoxia or hemodilution and served as controls. Six groups of animals were studied: control/vehicle (n=4), control/glibenclamide (n=4), hemodilution/vehicle (n=10), hemodilution/glibenclamide (n=10), hypoxia/vehicle (n=10), and hypoxia/glibenclamide (n=10). CBF was then measured with (3)H-nicotine in the forebrain, cerebellum, and brain stem. RESULTS In control/vehicle rats, CBF ranged from 72 mL. 100 g(-1). min(-1) in forebrain to 88 mL. 100 g(-1) x min(-1) in the brain stem. Glibenclamide treatment of control animals did not influence CBF in any brain area. Hemodilution increased CBF in all brain areas, with flows ranging from 128 mL. 100 g(-1) x min(-1) in forebrain to 169 mL. 100 g(-1) x min(-1) in the brain stem. Glibenclamide treatment of hemodiluted animals did not affect CBF in any brain area. Hypoxia resulted in a greater CBF than did hemodilution, ranging from 172 mL. 100 g(-1) x min(-1) in forebrain to 259 mL. 100 g(-1) x min(-1) in the brain stem. Glibenclamide treatment of hypoxic animals significantly reduced CBF in all brain areas (P<0.05). CONCLUSIONS Both hypoxia and hemodilution increased CBF. Glibenclamide treatment significantly attenuated the CBF increase during hypoxia but not after hemodilution. This finding supports our hypothesis that K(ATP) channels do not contribute to increasing CBF during hemodilution. Because intravascular PO(2) is normal during hemodilution, this finding supports the hypothesis that intravascular PO(2) is an important regulator of cerebral vascular tone and exerts its effect in part by activation of K(ATP) channels in the cerebral circulation.

Molecular mechanisms underlying ketamine-mediated inhibition of sarcolemmal adenosine triphosphate-sensitive potassium channels

Background: Ketamine inhibits adenosine triphosphate-sensitive potassium (KATP) channels, which results in the blocking of ischemic preconditioning in the heart and inhibition of vasorelaxation induced by KATP channel openers. In the current study, the authors investigated the molecular mechanisms of ketamine’s actions on sarcolemmal KATP channels that are reassociated by expressed subunits, inwardly rectifying potassium channels (Kir6.1 or Kir6.2) and sulfonylurea receptors (SUR1, SUR2A, or SUR2B). Methods: The authors used inside-out patch clamp configurations to investigate the effects of ketamine on the activities of reassociated Kir6.0/SUR channels containing wild-type, mutant, or chimeric SURs expressed in COS-7 cells. Results: Ketamine racemate inhibited the activities of the reassociated KATP channels in a SUR subtype-dependent manner: SUR2A/Kir6.2 (IC50 = 83 &mgr;m), SUR2B/Kir6.1 (IC50 = 77 &mgr;m), SUR2B/Kir6.2 (IC50 = 89 &mgr;m), and SUR1/Kir6.2 (IC50 = 1487 &mgr;m). S-(+)-ketamine was significantly less potent than ketamine racemate in blocking all types of reassociated KATP channels. The ketamine racemate and S-(+)-ketamine both inhibited channel currents of the truncated isoform of Kir6.2 (Kir6.2&Dgr;C36) with very low affinity. Application of 100 &mgr;m magnesium adenosine diphosphate significantly enhanced the inhibitory potency of ketamine racemate. The last transmembrane domain of SUR2 was essential for the full inhibitory effect of ketamine racemate. Conclusions: These results suggest that ketamine-induced inhibition of sarcolemmal KATP channels is mediated by the SUR subunit. These inhibitory effects of ketamine exhibit specificity for cardiovascular KATP channels, at least some degree of stereoselectivity, and interaction with intracellular magnesium adenosine diphosphate.

Hemodilution, cerebral O2 delivery, and cerebral blood flow: A study using hyperbaric oxygenation

Hemodilution reduces blood viscosity and O2content ([Formula: see text]) and increases cerebral blood flow (CBF). Viscosity and [Formula: see text] may contribute to increasing CBF after hemodilution. However, because hematocrit is the major contributor to blood viscosity and[Formula: see text], it has been difficult to assess their relative importance. By varying blood viscosity without changing[Formula: see text], prior investigation in hemodiluted animals has suggested that both factors play roughly equal roles. To further investigate the relationship of hemodilution, blood viscosity,[Formula: see text], and CBF, we took the opposite approach in hemodiluted animals, i.e., we varied[Formula: see text] without changing blood viscosity. Hyperbaric O2 was used to restore[Formula: see text] to normal after hemodilution. Pentobarbital sodium-anesthetized rats underwent isovolumic hemodilution with 6% hetastarch, and forebrain CBF was measured with [3H]nicotine. One group of animals did not undergo hemodilution and served as controls (Con). In the three experimental groups, hematocrit was reduced from 44% to 17-19%. Con and hemodiluted (HDil) groups were ventilated with 40% O2 at 101 kPa (1 atmosphere absolute), which resulted in[Formula: see text] values of 19.7 ± 1.3 and 8.1 ± 0.7 (SD) ml O2/dl, respectively. A second group of hemodiluted animals (HBar) was ventilated with 100% O2 at 506 kPa (5 atmospheres absolute) in a hyperbaric chamber, which restored[Formula: see text] to an estimated 18.5 ± 0.5 ml O2/dl by increasing dissolved O2. A fourth group of hemodiluted animals (HCon) served as hyperbaric controls and were ventilated with 10% O2 at 506 kPa, resulting in[Formula: see text] of 9.1 ± 0.6 ml O2/dl. CBF was 79 ± 19 ml ⋅ 100 g-1 ⋅ min-1in the Con group and significantly increased to 123 ± 9 ml ⋅ 100 g-1 ⋅ min-1in the HDil group. When[Formula: see text] was restored to baseline with dissolved O2 in the HBar group, CBF decreased to 104 ± 20 ml ⋅ 100 g-1 ⋅ min-1. When normoxia was maintained during hyperbaric exposure in the HCon group, CBF was 125 ± 18 ml ⋅ 100 g-1 ⋅ min-1, a value indistinguishable from that in normobaric HDil animals. Our data demonstrate that the reduction in [Formula: see text] after hemodilution is responsible for 40-60% of the increase in CBF.

Molecular mechanisms of the inhibitory effects of bupivacaine, levobupivacaine, and ropivacaine on sarcolemmal adenosine triphosphate-sensitive potassium channels in the cardiovascular system

Background:Sarcolemmal adenosine triphosphate–sensitive potassium (KATP) channels in the cardiovascular system may be involved in bupivacaine-induced cardiovascular toxicity. The authors investigated the effects of local anesthetics on the activity of reconstituted KATP channels encoded by inwardly rectifying potassium channel (Kir6.0) and sulfonylurea receptor (SUR) subunits. Methods:The authors used an inside-out patch clamp configuration to investigate the effects of bupivacaine, levobupivacaine, and ropivacaine on the activity of reconstituted KATP channels expressed in COS-7 cells and containing wild-type, mutant, or chimeric SURs. Results:Bupivacaine inhibited the activities of cardiac KATP channels (IC50 = 52 &mgr;m) stereoselectively (levobupivacaine, IC50 = 168 &mgr;m; ropivacaine, IC50 = 249 &mgr;m). Local anesthetics also inhibited the activities of channels formed by the truncated isoform of Kir6.2 (Kir6.2&Dgr;C36) stereoselectively. Mutations in the cytosolic end of the second transmembrane domain of Kir6.2 markedly decreased both the local anesthetics’ affinity and stereoselectivity. The local anesthetics blocked cardiac KATP channels with approximately eightfold higher potency than vascular KATP channels; the potency depended on the SUR subtype. The 42 amino acid residues at the C-terminal tail of SUR2A, but not SUR1 or SUR2B, enhanced the inhibitory effect of bupivacaine on the Kir6.0 subunit. Conclusions:Inhibitory effects of local anesthetics on KATP channels in the cardiovascular system are (1) stereoselective: bupivacaine was more potent than levobupivacaine and ropivacaine; and (2) tissue specific: local anesthetics blocked cardiac KATP channels more potently than vascular KATP channels, via the intracellular pore mouth of the Kir6.0 subunit and the 42 amino acids at the C-terminal tail of the SUR2A subunit, respectively.

Clinically relevant concentrations of propofol have no effect on adenosine triphosphate-sensitive potassium channels in rat ventricular myocytes

Background Activation of adenosine triphosphate–sensitive potassium (KATP) channels produces cardioprotective effects during ischemia. Because propofol is often used in patients who have coronary artery disease undergoing a wide variety of surgical procedures, it is important to evaluate the direct effects of propofol on KATP channel activities in ventricular myocardium during ischemia. Methods The effects of propofol (0.4–60.1 &mgr;g/ml) on both sarcolemmal and mitochondrial KATP channel activities were investigated in single, quiescent rat ventricular myocytes. Membrane currents were recorded using cell-attached and inside-out patch clamp configurations. Flavoprotein fluorescence was measured to evaluate mitochondrial oxidation mediated by mitochondrial KATP channels. Results In the cell-attached configuration, open probability of KATP channels was reduced by propofol in a concentration-dependent manner (EC50 = 14.2 &mgr;g/ml). In the inside-out configurations, propofol inhibited KATP channel activities without changing the single-channel conductance (EC50 = 11.4 &mgr;g/ml). Propofol reduced mitochondrial oxidation in a concentration-dependent manner with an EC50 of 14.6 &mgr;g/ml. Conclusions Propofol had no effect on the sarcolemmal KATP channel activities in patch clamp configurations and the mitochondrial flavoprotein fluorescence induced by diazoxide at clinically relevant concentrations (< 2 &mgr;m), whereas it significantly inhibited both KATP channel activities at very high, nonclinical concentrations (> 5.6 &mgr;g/ml; 31 &mgr;m).

Lidocaine and mexiletine inhibit mitochondrial oxidation in rat ventricular myocytes

BackgroundAccumulating evidence suggests that mitochondrial rather than sarcolemmal adenosine triphosphate–sensitive K+ (KATP) channels may have an important role in the protection of myocardium during ischemia. Because both lidocaine and mexiletine are frequently used antiarrhythmic drugs during myocardial ischemia, it is important to investigate whether they affect mitochondrial KATP channel activities. MethodsMale Wistar rats were anesthetized with ether. Single, quiescent ventricular myocytes were dispersed enzymatically. The authors measured flavoprotein fluorescence to evaluate mitochondrial redox state. Lidocaine or mexiletine was applied after administration of diazoxide (25 &mgr;m), a selective mitochondrial KATP channel opener. The redox signal was normalized to the baseline flavoprotein fluorescence obtained during exposure to 2,4-dinitrophenol, a protonophore that uncouples respiration from ATP synthesis and collapses the mitochondrial potential. ResultsDiazoxide-induced oxidation of flavoproteins and the redox changes were inhibited by 5-hydroxydecanoic acid, a selective mitochondrial KATP channel blocker, suggesting that flavoprotein fluorescence can be used as an index of mitochondrial oxidation mediated by mitochondrial KATP channels. Lidocaine (10−3 to 10 mm) and mexiletine (10−3 to 10 mm) reduced oxidation of the mitochondrial matrix in a dose-dependent manner with an EC50 of 98 ± 63 &mgr;m for lidocaine and 107 ± 89 &mgr;m for mexiletine. ConclusionsBoth lidocaine and mexiletine reduced flavoprotein fluorescence induced by diazoxide in rat ventricular myocytes, indicating that these antiarrhythmic drugs may produce impairment of mitochondrial oxidation mediated by mitochondrial KATP channels.

Phenylephrine increases pulmonary blood flow in children with tetralogy of Fallot

PurposeAlthough it has been reported that the increase in blood pressure improves arterial oxygen saturation (SaO2) in children with tetralogy of Fallot, no prospective study has demonstrated that an increase in blood pressure induces an increase in pulmonary blood flow in these patients. The purpose of this study was to see whether a phenylephrine-induced increase in systemic blood pressure increased pulmonary blood flow, resulting in improved arterial oxygénation in tetralogy of Fallot.MethodsIn 14 consecutive children with tetralogy of Fallot (2–32 months old), transesophageal pulsed Doppler signals of left upper pulmonary venous flow (PVF) velocity were recorded before and four minutes after 10/μg · kg−1 of phenylephrine iv. Simultaneously, arterial blood gas analysis and hemodynamic measurements were performed. The minute distance (MD) was calculated as the product of the heart rate and the sum of time-velocity integrals of PVF.ResultsPhenylephrine iv increased mean arterial blood pressure from 54 ± 8 mmHg to 73 ± 10 mmHg. This phenylephrineinduced hypertension significantly increased SaO2 and MD (92.0 ± 7.5 vs 95.0 ± 5.0% and 1318 ± 344 vs 1533 ± 425 cm · min−1, respectively). There was a significant correlation (r = 0.72) between the change in MD and the change in SaO2.ConclusionOur results suggest that the phenylephrine-induced increase in systemic blood pressure produces an increase in pulmonary blood flow in tetralogy of Fallot. Our results further suggest that this increase in pulmonary blood flow is involved in the mechanism of phenylephrine-induced improvement of arterial oxygenation in tetralogy of Fallot.RésuméObjectifOn a déjà montré qu’une augmentation de la tension artérielle améllore la saturation en oxygène du sang artériel (SaO2), chez les enfants qui présentent une tétralogie de Fallot, mais aucune étude prospective n’a démontré qu’une augmentation de la tension artérielle pouvait induire une élévation du débit sanguin pulmonaire chez ces patients. Nous voullons vérifier si une augmentation de la tension artérielle générale induite par la phényléphrine fait augmenter le débit sanguin pulmonaire et améliore l’oxygénation artérielle dans le contexte d’une tétralogie de Fallot.MéthodeChez 14 enfants porteurs d’une tétralogie de Fallot et traités consécutivement (âgés de 2–32 mois), les signaux Doppler pulsés transœsophagiens de la vitesse du flux de la veine pulmonaire gauche supérieure (DVP) ont été enregistrés avant, puis quatre minutes après l’administration iv de 10 μg · kg−1 de phényléphrine. Une analyse des gaz du sang artériel et des mesures hémodynamiques ont été réalisées simultanément. La distance minute (DM) a été calculée comme le produit de la fréquence cardiaque et de la somme des intégrales de temps-vélocité du DVR.RésultatsLa phényléphrine iv a augmenté la tension artérielle moyenne de 54 ± 8 mmHg à 73 ± 10 mmHg. L’hypertension induite par la phényiéphrine a augmenté la SaO2 et la DM de façon significative (92,0 ± 7,5 vs 95,0 ± 5,0 % et 1318 ± 344 vs 1533 ± 425 cm · min−1, respectivement). Il y avait une corrélation significative (r = 0,72) entre les modifications de la DM et celles de la SaO2.ConclusionNos résultats suggèrent que, chez les patients atteints d’une tétralogie de Fallot, l’augmentation du débit sanguin général induite par la phényiéphrine produit une élévation du débit sanguin pulmonaire. De plus, il apparaît que cette augmentation du débit sanguin pulmonaire contribue à l’amélioration de l’oxygénation artérielle induite par la phényiéphrine.

Effect of propofol on hypotonic swelling-induced membrane depolarization in human coronary artery smooth muscle cells.

BackgroundStretch (mechanical stress)–induced membrane depolarization of smooth muscle may contribute to basal vascular tone and myogenic control. Propofol induces vasodilation and inhibits myogenic control. Hypotonic swelling was used as a model of mechanical stress. The authors investigated the effects of propofol and 5-nitro-2-(3-phenylpropylamino)benzoic acid, a chloride channel and nonselective cation channel blocker, on hypotonicity-induced membrane depolarization in cultured human coronary artery smooth muscle cells. MethodsA voltage-sensitive fluorescent dye, bis-(1,3-diethylthiobarbiturate)trimethine oxonol, was used to assess relative changes in membrane potential semiquantitatively. The cells were continuously perfused with Earle’s balanced salt solution containing 200 nm bis-(1,3-diethylthiobarbiturate)trimethine oxonol and exposed sequentially to isotonic and hypotonic medium. In a second series of experiments, the cells were exposed to hypotonic media in the presence and absence of 5-nitro-2-(3-phenylpropylamino)benzoic acid or propofol. ResultsThe relative fluorescence values at 10, 20, and 30% hypotonicity were 147 ± 29, 214 ± 74, and 335 ± 102% of baseline, respectively. The changes were all significantly different from the isotonic time control group. In the presence of 200 &mgr;m 5-nitro-2-(3-phenylpropylamino)benzoic acid or 0.1, 1, 10, or 100 &mgr;g/ml propofol, the relative fluorescence values at 30% hypotonicity were 87 ± 17, 194 ± 27, 160 ± 18, 130 ± 18, and 84 ± 15%, respectively. These changes were significantly less than the 30% for the hypotonic control (246 ± 23%). ConclusionThese results suggest that volume-sensitive chloride channels and nonselective cation channels may participate in hypotonicity-induced membrane depolarization and that propofol inhibits hypotonicity-induced membrane depolarization in coronary artery smooth muscle.

Cerebral Blood Flow During Hemodilution and Hypoxia in Rats : Role of ATP-Sensitive Potassium Channels Editorial Comment: Role of ATP-Sensitive Potassium Channels

BACKGROUND AND PURPOSE Hypoxia and hemodilution both reduce arterial oxygen content (CaO(2)) and increase cerebral blood flow (CBF), but the mechanisms by which hemodilution increases CBF are largely unknown. ATP-sensitive potassium (K(ATP)) channels are activated by intravascular hypoxia, and contribute to hypoxia-mediated cerebrovasodilatation. Although CaO(2) can be reduced to equal levels by hypoxia or hemodilution, intravascular PO(2) is reduced only during hypoxia. We therefore tested the hypothesis that K(ATP) channels would be unlikely to contribute to cerebrovasodilatation during hemodilution. METHODS Glibenclamide (19.8 microg) or vehicle was injected into the cisterna magna of barbiturate-anesthetized rats. The dose of glibenclamide was chosen to yield an estimated CSF concentration of 10(-4) M. Thirty minutes later, some animals underwent either progressive isovolumic hemodilution or hypoxia (over 30 minutes) to achieve a CaO(2) of approximately 7.5 mL O(2)/dL. Other animals did not undergo hypoxia or hemodilution and served as controls. Six groups of animals were studied: control/vehicle (n=4), control/glibenclamide (n=4), hemodilution/vehicle (n=10), hemodilution/glibenclamide (n=10), hypoxia/vehicle (n=10), and hypoxia/glibenclamide (n=10). CBF was then measured with (3)H-nicotine in the forebrain, cerebellum, and brain stem. RESULTS In control/vehicle rats, CBF ranged from 72 mL. 100 g(-1). min(-1) in forebrain to 88 mL. 100 g(-1) x min(-1) in the brain stem. Glibenclamide treatment of control animals did not influence CBF in any brain area. Hemodilution increased CBF in all brain areas, with flows ranging from 128 mL. 100 g(-1) x min(-1) in forebrain to 169 mL. 100 g(-1) x min(-1) in the brain stem. Glibenclamide treatment of hemodiluted animals did not affect CBF in any brain area. Hypoxia resulted in a greater CBF than did hemodilution, ranging from 172 mL. 100 g(-1) x min(-1) in forebrain to 259 mL. 100 g(-1) x min(-1) in the brain stem. Glibenclamide treatment of hypoxic animals significantly reduced CBF in all brain areas (P<0.05). CONCLUSIONS Both hypoxia and hemodilution increased CBF. Glibenclamide treatment significantly attenuated the CBF increase during hypoxia but not after hemodilution. This finding supports our hypothesis that K(ATP) channels do not contribute to increasing CBF during hemodilution. Because intravascular PO(2) is normal during hemodilution, this finding supports the hypothesis that intravascular PO(2) is an important regulator of cerebral vascular tone and exerts its effect in part by activation of K(ATP) channels in the cerebral circulation.