Sumihiko Seki

Fetal and postnatal development of Ca2+ transients and Ca2+ sparks in rat cardiomyocytes

OBJECTIVE The aim of this study was to characterize the spatio-temporal dynamics of [Ca(2+)](i) in rat heart in the fetal and neonatal periods. METHODS Using confocal scanning laser microscopy and the Ca(2+) indicator fluo-3, we investigated Ca(2+) transients and Ca(2+) sparks in single ventricular myocytes freshly isolated from rat fetuses and neonates. T-tubules were labeled with a membrane-selective dye (di-8-ANEPPS). Spatial association of dihydropyridine receptors (DHPR) and ryanodine receptors (RyR) was also examined by double-labeling immunofluorescence. RESULTS Ca(2+) transients in the fetal myocytes were characterized by slower upstroke and decay of [Ca(2+)](i) compared to those in adult myocytes. The magnitude of fetal Ca(2+) transients was decreased after application of ryanodine (1 microM) or thapsigargin (1 microM). However, Ca(2+) sparks were rarely detected in the fetal myocytes. Frequent ignition of Ca(2+) sparks was established in the 6-9-day neonatal period, and was predominantly observed in the subsarcolemmal region. The developmental change in Ca(2+) sparks coincided with development of the t-tubule network. The immunofluorescence study revealed colocalization of DHPR and RyR in the postnatal period, which was, however, not observed in the fetal period. In the adult myocytes, Ca(2+) sparks disappeared after disruption of t-tubules by glycerol incubation (840 mM). CONCLUSIONS The sarcoplasmic reticulum (SR) of rat ventricular myocytes already functions early in the fetal period. However, ignition of Ca(2+) sparks depends on postnatal t-tubule formation and resultant colocalization of DHPR and RyR.

The effects of sevoflurane, isoflurane, halothane, and enflurane on hemodynamic responses during an inhaled induction of anesthesia via a mask in humans.

A rapid increase in isoflurane or desflurane concentration induces tachycardia and hypertension and increases plasma catecholamine concentration.Little information is available as to whether sevoflurane, halothane, and enflurane induce similar responses during anesthesia induction via mask. Fifty ASA physical status I patients, aged 20-40 yr, and scheduled for elective minor surgery, received one of four volatile anesthetics: sevoflurane, isoflurane, halothane, or enflurane. Anesthesia was induced with thiamylal, followed by inhalation of 0.9 minimum alveolar anesthetic concentration (MAC) of the anesthetic in 100% oxygen via mask. The inspired concentration of anesthetic was increased by 0.9 MAC every 5 min to a maximum of 2.7 MAC. Heart rate (HR) and systolic blood pressure (SBP) were measured before and every minute for 15 min during anesthetic inhalation. In the sevoflurane and isoflurane groups, venous blood samples were drawn to determine the concentrations of plasma epinephrine and norepinephrine 3 min after each increase in anesthetic concentration. Sustained increments in HR were observed after increases in inspired isoflurane concentration to 1.8 MAC and 2.7 MAC (peak changes of 15 +/- 3 and 17 +/- 3 bpm, respectively). Isoflurane also increased SBP transiently after the inspired concentration was increased to 2.7 MAC (peak change of 10 +/- 4 mm Hg). Enflurane increased HR after the inspired concentration was increased to 2.7 MAC (peak change of 9 +/- 2 bpm). In contrast, changes in sevoflurane and halothane concentrations did not induce hyperdynamic responses. Plasma norepinephrine concentration in the isoflurane group was significantly higher than that in the sevoflurane group during 2.7 MAC (P = 0.022). We propose that there is a direct relationship between airway irritation of the anesthetic and immediate cardiovascular change during an inhaled induction of anesthesia. (Anesth Analg 1996;82:821-6)

Role of Endothelium-derived Hyperpolarizing Factor in Phenylephrine-induced Oscillatory Vasomotion in Rat Small Mesenteric Artery

Background In small mesenteric arteries, endothelium-derived hyperpolarizing factor (EDHF) in addition to endothelium-derived relaxing factors (EDRFs) including NO plays an important role in acetylcholine-induced vasodilation. It has been reported that EDRFs play an important role in &agr;1-adrenoceptor agonist-induced oscillatory vasomotion and in limiting vasoconstrictor response to the agonists; however, contribution of EDHF to the &agr;1-agonist-induced oscillation is unknown. Methods Rat small mesenteric arteries were isolated and cannulated at each end with a glass micropipette. The vessels were immersed in a bath (37°C) containing physiologic saline solution. Changes in vessel diameter were measured using an optical density video detection system. Results Denudation of the endothelium and inhibition of NO synthesis caused a leftward shift in the concentration-response relation for phenylephrine in the mesenteric arteries, whereas inhibition of cyclooxygenase by indomethacin had no effect. Blockade of Ca2+-activated K+ (KCa) channels by charybdotoxin and apamin caused a further leftward shift in the concentration-response relation in the vessels pretreated with N&ohgr;-nitro-l-arginine methylester and indomethacin. Phenylephrine at concentrations higher than 10−6 m caused endothelium-dependent oscillatory vasomotion, which was reduced but not abolished after combined inhibition of the cyclooxygenase and NO synthase pathways. However, the KCa channel blockers completely abolished the remaining component of oscillation. Conclusions Endothelially-derived NO is an important modulator of sustained agonist-induced vasoconstriction. NO, as well as endothelially-derived cyclooxygenase products and EDHF, also contribute significantly to phenylephrine-induced oscillatory vasomotion.

The hemodynamic effects of landiolol, an ultra-short-acting beta1-selective blocker, on endotracheal intubation in patients with and without hypertension.

BACKGROUND:The ultra-short-acting &bgr;1-selective blocker, landiolol, is widely used in Japan. We investigated the effects of landiolol on intubation-induced adrenergic response in 88 patients. METHODS:General anesthesia was induced and maintained with target-controlled infusion of propofol at an effect–site concentration of 5 &mgr;g/mL. Muscle relaxation was obtained with 0.1 mg/kg vecuronium, and endotracheal intubation was performed 4 min after vecuronium injection. We first investigated the optimal time point for landiolol to be administered before intubation in 43 normotensive patients. Then we examined whether landiolol was as effective as fentanyl to prevent tachycardia after intubation in 45 hypertensive patients. RESULTS:Landiolol at 0.1 mg/kg was most effective against intubation-induced tachycardia when infused 4 min before intubation in normotensive patients. However, 0.2 mg/kg landiolol was necessary to prevent tachycardia after intubation in hypertensive patients. Landiolol had no significant effects on arterial blood pressure or bispectral index at any dose throughout the study period. In contrast, 2 &mgr;g/kg fentanyl frequently caused hypotension just before and 5 min after intubation. CONCLUSION:Low doses of landiolol can effectively prevent tachycardia after intubation without significant effects on arterial blood pressure.

Halothane and Enflurane Attenuate Pulmonary Vasodilation Mediated by Adenosine Triphosphate-sensitive Potassium Channels Compared to the Conscious State

Background: Adenosine triphosphate (ATP)‐sensitive potassium (K sup +ATP) channels play an important role in pulmonary vasoregulation. However, the effects of volatile anesthetics on K sup +ATP channel‐mediated pulmonary vasoregulation have not been elucidated. The purpose of the present study was to investigate the effects of halothane and enflurane anesthesia on the pulmonary vasodilator response to the selective K sup +ATP channel agonist lemakalim (BRL38227) compared with that measured in the conscious state. The authors also investigated the extent to which endogenous neurohumoral vasoconstrictor mechanisms modulate the vasodilator response to K sup +ATP channel activation. Method: Nineteen conditioned, male mongrel dogs were chronically instrumented to measure the left pulmonary vascular pressure‐flow (LPQ) relationship. LPQ plots were generated by continuously measuring the pulmonary vascular pressure gradient (pulmonary arterial pressure‐left atrial pressure) and left pulmonary blood flow during gradual (approximately 1 min) inflation of a hydraulic occluder implanted around the right main pulmonary artery. After preconstriction with the thromboxane analog, U46619 (9,11‐dideoxy‐11alpha, 9alpha ‐epoxymethano‐prostaglandin F2alpha), the pulmonary vascular dose‐response relationship for the K sup +ATP agonist lemakalim was assessed in the conscious and halothane‐anesthetized states and also in the conscious and enflurane‐anesthetized states. This protocol was repeated in conscious and halothane‐anesthetized dogs after combined neurohumoral block with antagonists of sympathetic alpha1 adrenoreceptors, arginine vasopressin V1 ‐receptors, and angiotensin II receptors. The effect of the K sup +ATP antagonist glybenclamide on the baseline LPQ relationship and on the lemakalim dose‐response relationship also was assessed in conscious dogs. Results: Compared with the conscious state, halothane, enflurane and glybenclamide had no net effect on the baseline LPQ relationship. In contrast, halothane and enflurane attenuated (P < 0.05) the pulmonary vasodilator response to lemakalim compared with the conscious state. Glybenclamide also caused a rightward shift (P < 0.05) in the lemakalim dose‐response relationship. Combined neurohumoral block did not modulate the vasodilator response to lemakalim in the conscious state. The halothane‐induced attenuation of the vasodilator response to lemakalim was apparent after combined neurohumoral block. Conclusion: These results indicate that halothane and enflurane act to reduce the magnitude of K sup +ATP channel‐mediated pulmonary vasodilation. Reflex pulmonary vasoconstriction resulting from K sup +ATP ‐mediated systematic hypotension does not alter the magnitude of the pulmonary vasodilator response to lemakalim nor is it responsible for the attenuated response to K sup +ATP channel activation during halothane anesthesia.

Contribution of KChIP2 to the developmental increase in transient outward current of rat cardiomyocytes

The Ca(2+)-independent, voltage-gated transient outward current (I(to)) displays a marked increase during development of cardiomyocytes. However, the molecular mechanism remained unclear. In rat adult ventricular myocytes, I(to) can be divided into a fast (I(to,f)) and a slow (I(to,s)) component by recovery process from inactivation. Voltage-gated K(+) channel-interacting proteins 2 (KChIP2) has recently been shown to modify membrane expressions and current densities of I(to,f). Here we examined the developmental change of I(to) and the putative molecular correlates of I(to,f) (Kv4.2 and Kv4.3) and KChIP2 in rat ventricular myocytes. Even in rat embryonic day 12 (E12) myocytes, we detected I(to). However, I(to) in E12 was solely composed of I(to,s). In postnatal day 10 (P10), we recorded much increased I(to) composed of two components (I(to,f) and I(to,s)), and I(to,f) was dominant. Thus, the developmental increase of I(to) from E12 to P10 can be explained by the dramatic appearance of I(to,f). Real-time RT-PCR revealed that Kv4.2 and Kv4.3 mRNA levels were slightly changed. By contrast, KChIP2 mRNA level increased from E12 to P10 by 731-fold. Therefore, the huge increase of KChIP2 expression was likely to be the cause of the great increase of I(to,f). In order to confirm that KChIP2 is crucial to induce I(to,f), we used adenoviral gene transfer technique. When KChIP2 was over-expressed in E12 myocytes, a great amplitude of I(to,f) appeared. Immunocytochemical experiments also demonstrated that KChIP2 enhanced the trafficking of Kv4.2 channels to cell surface. These results indicate that KChIP2 plays an important role in the generation of functional I(to,f) channels during development.

Role of intracellular Ca2+ pools in the effects of halothane and isoflurane on vascular smooth muscle contraction.

We examined the effect of halothane and isoflurane on contraction in the vascular smooth muscle of rat thoracic aorta simultaneously with the cytosolic Ca2+ levels ([Ca2+]i). Isolated spiral strips of rat thoracic aorta were suspended for isometric tension recordings in physiologic salt solution. The [Ca2+]i was measured concomitantly using fura-2-Ca2+ fluorescence. Muscle tension was elicited either by 51 mM K+ solution or 30 nM norepinephrine, and the muscle was exposed to 0%, 1%, 2%, 3% halothane or 0%, 1%, 2%, 3%, 4% isoflurane. The effects of the anesthetics were compared with the effects of verapamil, an L-type voltage-dependent Ca2+ channel blocker, also administered during K(+)-induced muscle contraction. In another series, the effects of the anesthetics on caffeine- or norepinephrine-induced muscle contraction were determined in Ca(2+)-free solution. Finally, 3% halothane or 4% isoflurane was administered during K(+)-induced contraction in muscle strips pretreated with ryanodine and caffeine. During K(+)-induced contraction, halothane evoked a transient increase followed by a decrease in both muscle tension and [Ca2+]i. The biphasic change in muscle tension was not elicited by isoflurane or by any agent under norepinephrine-induced contraction. Both halothane and isoflurane ultimately suppressed both K(+)- and norepinephrine-induced increases in muscle tension and the [Ca2+]i in a concentration-dependent manner. The slopes of the [Ca2+]i-tension regression lines under the two anesthetics were significantly steeper than that under verapamil during K(+)-induced contraction. Halothane, but not isoflurane, augmented 4 mM caffeine-induced tension and [Ca2+]i transients in the Ca(2+)-free solution in a concentration-dependent manner. However, neither anesthetic influenced norepinephrine-induced tension and [Ca2+]i transients. In the muscle strips pretreated with ryanodine and caffeine, the difference observed between the anesthetics was abolished. In conclusion, halothane, but not isoflurane, enhances Ca2+ release predominantly from the caffeine-releasable Ca2+ stores in vascular smooth muscle; this release may modify the effect of halothane. The intracellular Ca2+ pools can be affected differently by volatile anesthetic drugs, depending on the nature of the stimulus for smooth muscle contraction.

Superoxide anion scavengers restore NO-mediated pulmonary vasodilation after lung transplantation

Left lung autotransplantation (LLA) results in a chronic attenuation in endothelium-dependent, nitric oxide (NO)-mediated pulmonary vasodilation. We tested the hypothesis that this abnormality involves a decrease in the effective concentration of NO due to inactivation by superoxide anion. Size- and position-matched pulmonary arterial rings were isolated from the right (control) and left (LLA) lungs of seven dogs 1-5 mo post-LLA. The rings were suspended for isometric tension recording and contracted with phenylephrine, and cumulative dose-response curves for ACh or calcium ionophore (A-23187) were generated. Endothelium-dependent relaxation to ACh was inhibited post-LLA, with the maximum vasorelaxation response reduced from 88 +/- 5 to 63 +/- 5% (P < 0. 01) post-LLA. In contrast, after pretreatment with the superoxide anion scavengers tiron or superoxide dismutase (SOD), the dose-response relationships for ACh were similar in control and LLA rings. Oxypurinol, which inhibits superoxide anion production by endothelial xanthine oxidase, also restored the vasorelaxation response to ACh in LLA rings. The pulmonary vasorelaxant response to A-23187 was also attenuated (P < 0.01) post-LLA, and this effect was entirely reversed by pretreatment with tiron, SOD, or oxypurinol. These results indicate that the attenuated responses to these pulmonary vasorelaxants post-LLA involve inactivation of NO by superoxide anion generated by endothelial xanthine oxidase.

Propofol anesthesia enhances the pressor response to intravenous ephedrine.

The induction of anesthesia with propofol is often associated with a decrease in arterial blood pressure (BP). Although vasopressors are sometimes required to reverse the propofol-induced hypotension, little is known about the effect of propofol on these drugs. We studied the effects of propofol and sevoflurane on pressor response to IV ephedrine. Thirty adult patients were randomly assigned to one of two groups. In the Propofol group (n = 15), patients received pro-pofol 2.5 mg/kg IV for induction followed by 100 &mgr;g · kg−1 · min−1 IV for maintenance. In the Sevoflurane group (n = 15), anesthesia was induced with sevoflurane 3%–4% in oxygen and maintained with sevoflurane 2% in oxygen. All patients in both groups received ephedrine 0.1 mg/kg IV before and after the anesthetic induction. Ephedrine increased the heart rate significantly (P < 0.05) in awake patients in both study groups. In contrast, there was no increase in heart rate after the ephedrine administration under propofol or sevoflurane anesthesia. In awake patients, transient increases in mean BP were observed after IV ephedrine in both groups. In the Propofol group, 2 min after the administration of ephedrine, mean BP increased 16% ± 10% under anesthesia but increased only 4% ± 6% when the same patients were awake. The magnitudes of the pressor responses to ephedrine during propofol anesthesia were significantly greater (P < 0.05) than during the awake state. However, ephedrine 0.1 mg/kg IV showed no significant increases in BP during sevoflurane anesthesia. We conclude that propofol, not sevoflurane, anesthesia augments the pressor responses to IV ephedrine.

Halothane Attenuates Endothelium-dependent Pulmonary Vasorelaxant Response to Lemakalim, an Adenosine Triphosphate (ATP)-sensitive Potassium Channel Agonist

Background Lemakalim, an adenosine triphosphate (ATP)-sensitive potassium (K sup +ATP) channel agonist, causes profound pulmonary vasodilation in conscious dogs, which is attenuated during halothane anesthesia. The goal of the present study was to investigate the mechanism responsible for this attenuating effect of halothane. Methods Isolated canine pulmonary arterial rings were suspended for isometric tension recording in 25 ml organ baths. Rings with and without endothelium were contracted to 50% of their maximal response to phenylephrine, followed by the cumulative administration of lemakalim with or without exposure to halothane (0.5-1.5 minimum alveolar concentration [MAC] in dogs). Lemakalim dose-response curves were also generated in rings pretreated with the nitric oxide synthase inhibitor, Nw -nitro-L-arginine methyl ester (L-NAME); the cyclooxygenase inhibitor, indomethacin; or the K sup +ATP channel antagonist, glybenclamide. Results Compared with intact rings, the pulmonary vasorelaxant response to lemakalim was attenuated (P < 0.05) in endothelium-denuded rings. Halothane at 0.5 MAC had no effect on the vasorelaxant response to lemakalim. Halothane at 1 MAC attenuated (P < 0.05) the vasorelaxant response to lemakalim in intact rings, but not in endothelium-denuded rings. Halothane at 1.5 MAC attenuated (P < 0.05) the vasorelaxant response to lemakalim in both intact and endothelium-denuded rings. In endothelium-intact rings, indomethacin attenuated (P < 0.05) the vasorelaxant response to lemakalim, whereas L-NAME had no effect. Further, indomethacin, but not L-NAME, abolished the endothelium-dependent, halothane-induced attenuation of the lemakalim vasorelaxation response. Glybenclamide markedly attenuated (P < 0.05) lemakalim vasorelaxation at lemakalim doses less than 10 sup -6 M. Conclusions Lemakalim-induced pulmonary vasorelaxation involves an endothelium-dependent and vascular smooth muscle component. Further, halothane attenuates the endothelium-dependent pulmonary vasorelaxant response to lemakalim via an inhibitory effect on vasodilator metabolites of the cyclooxygenase pathway.