Itsuo Katoh

Equilibrium in the hydrolysis and synthesis of cannabimimetic anandamide demonstrated by a purified enzyme.

Anandamide, an endogenous ligand for cannabinoid receptors, loses its biological activities when it is hydrolyzed to arachidonic acid and ethanolamine by anandamide amidohydrolase. We overexpressed a recombinant rat enzyme with a hexahistidine tag in a baculovirus-insect cell expression system, and purified the enzyme with the aid of a Ni-charged resin to a specific activity as high as 5.7 micromol/min/mg protein. The purified recombinant enzyme catalyzed not only the hydrolysis of anandamide and palmitoylethanolamide, but also their reverse synthetic reactions. In order to attain an equilibrium of the anandamide hydrolysis and its reverse reaction within 10 min, we utilized a large amount of the purified enzyme. The equilibrium constant ([arachidonic acid][ethanolamine])/([anandamide][water]) was calculated as 4x10(-3) (37 degrees C, pH 9.0). These experimental results with a purified enzyme preparation quantitatively confirmed the reversibility of the enzyme reaction previously observed with crude enzyme preparations.

Extracellulary administered lysophosphatidylcholine causes Ca2+ efflux from freshly isolated adult rat cardiomyocytes

Abstract It has previously been reported that ischemia and reperfusion of the heart cause accumulation of lyophosphatidylcholine (LPC) within the myocardium. While it is known that LPC causes the transient increase of intracellular free Ca2+ concentration ([Ca2+]i) during contraction of cardiac cells, little is known about the mechanism for decreasing [Ca2+]i in cardiomyocytes during LPC accumulation. Since cumulative elevation in [Ca2+]i leads to irreversible injury to cardiomyocytes, elevated [Ca2+]i must be restored to an unstimulated level to maintain cell functions. In the present study, we therefore examined the effect of LPC on Ca2+ efflux from freshly isolated adult rat cardiomyocytes. LPC stimulated the efflux of 45Ca2+ from the cells in a concentration‐dependent manner (10–7 M – 10–5 M). Other lysophospholipids, which are generated from phopholipids of the cell membrane, failed to induce 45Ca2+ efflux from the cells. Dilazep and K‐7259, which are known to inhibit the increase in [Ca2+]i caused by LPC, likewise reduced 45Ca2+ efflux caused by LPC addition. Furthermore, the LPC‐stimulated 45Ca2+ efflux was not affected by removal of extracellular Ca2+, but was dependent on the presence of extracellular Na+. On the other hand, inhibitors of Na+/Ca2+ exchange, amiloride and 5‐(N,N‐dimethyl)‐amiloride, inhibited LPC induced 45Ca2+ efflux. These results suggest that LPC stimulates extracellular Na+‐dependent 45Ca2+ efflux from freshly isolated adult rat cardiomyocytes, probably through Na+/Ca2+ exchange on the plasma membrane of cells.

Changes in plasma free and sulfoconjugated catecholamines before and after acute physical exercise: Experimental and clinical studies

To elucidate whether sulfoconjugated catecholamines in plasma, especially dopamine, serve as a source of free catecholamines, we examined the change in afterload on the deconjugating activity of catecholamines in isolated Langendorff perfused rat hearts. Dopamine-sulfate was administered under ordinary or high-work-load conditions. Free dopamine in the effluent was increased by the high-work-load of the hearts, whereas conjugated dopamine showed an apparent decrease. These results indicate the possibility that deconjugation of sulfoconjugated catecholamines is accelerated by a high-work-load. To obtain further evidence in humans, we also examined the changes in the plasma levels of free and sulfoconjugated catecholamines in healthy volunteers before and after marathon running. Free dopamine increased 1.99-fold from the baseline value after exercise, whereas conjugated dopamine decreased by 12%. Similarly, the plasma levels of free noradrenaline and adrenaline increased after exercise to 2.45- and 1.51-fold their respective baseline values, while conjugated noradrenaline and adrenaline both decreased. These clinical results, as well as those of the experimental studies, suggest that the increase in plasma free catecholamines after exercise is due not only to increased release from the sympathoadrenal system but also to accelerated conversion from sulfoconjugated catecholamines in the plasma.

Achieving optimal pulmonary blood flow in the first-stage of palliation in early infancy for complex cardiac defects with hypoplastic left ventricles

The aim of the study was to determine the optimal size and technique for construction of the systemic-to-pulmonary arterial shunt which will provide suitable pulmonary blood flow in first-stage Norwood palliation for hypoplastic left heart syndrome in neonates. Our clinical experience suggested that an arterial oxygen tension of about 30 mm Hg immediately after cardiopulmonary bypass, with the patients being ventilated at the lowest possible mean airway pressure with an FiO 2 of 1.0, provided a suitable pulmonary-to-systemic flow ratio. We also aimed to clarify the characteristics of pulmonary blood flow in accordance with the size of the shunt and the change in the pulmonary vascular resistance in a simplified rigid model of the Norwood procedure. A hole of2.0 mm diameter proved adequate to provide a suitable pulmonary blood flow of 200−300 mlx002F;min in the presence of a pressure gradient of 20−40 mm Hg between the systemic and pulmonary circulations in neonates weighing 3 kg. A short central shunt with a prosthesis of4 mm in diameter produced an excessive flow of pulmonary blood. Our data suggest that using a smaller shunt than that commonly used is necessary to decrease the early and intermediate postoperative mortality. A prosthesis of 3.0 or 3.5 mm in diameter arising from the brachiocephalic artery would be acceptable and can be recommended for first-stage Norwood palliation in small infants, especially in view of the operative difficulties encountered in taking down the shunt at the time of subsequent operations.

Viability of cryopreserved semilunar valves: an evaluation of cytosolic and mitochondrial activities

BACKGROUND Despite long-standing, widespread use of cryopreserved allografts, the basic cellular biology of these tissues is still yet unknown. The present investigation was undertaken to study cryopreserved heart valves from the standpoint of cytosolic esterase and mitochondrial dehydrogenase activities. METHODS Cryopreserved porcine aortic cusps were observed in an unfixed fresh condition with a confocal laser scanning microscope using fluorescent dye. Porcine cusps and cultured human umbilical vein endothelial cells were divided into three groups, including fresh, cold-preserved, and cryopreserved specimens, and cytosolic esterase activity and mitochondrial dehydrogenase activity were analyzed in each. RESULTS Confocal laser scanning microscope findings disclosed a widely distributed fluorescence in the cusp. Cytosolic esterase activity within human umbilical vein endothelial cells (28%+/-9.0%) after cryopreservation was significantly less than that it was in the cusps (72%+/-21%). Mitochondrial dehydrogenase activity of cryopreserved human umbilical vein endothelial cells and that of cusps fell to 44%+/-6.1% and 64%+/-17% respectively; the difference between the two values was not significant. CONCLUSIONS Cryopreservation appeared to produce serious damage to cytosolic and mitochondrial functions of endothelial cells. The cytosolic function of cusps, mainly consisting of fibroblasts, was comparatively preserved after cryopreservation, but mitochondrial function of the cusps was more diminished.

Characterization of palytoxin-induced catecholamine secretion from cultured bovine adrenal chromaffin cells: Effects of Na+- and Ca2+-channel blockers

The effect of palytoxin (PTX), a potent marine toxin, on catecholamine (CA) secretion from cultured bovine adrenal chromaffin cells was examined. PTX at concentrations of over 10(-10) M induced CA secretion concentration-dependently. About 40-50% of the total cellular CA was secreted during 20-min incubation with 3 x 10(-8) M PTX. PTX-induced CA secretion was dependent on both extracellular Na+ and Ca2+. PTX caused increases in [22Na](+)- and [45Ca](2+)-influxes into the cells. Increase in [22Na](+)-influx was observed at concentrations of over 10(-11) M PTX and was maximal at 10(-10) M PTX and then gradually decreased at higher concentrations that induced [45Ca](2+)-influx and CA secretion. On the other hand, increase in [45Ca](2+)-influx was observed at concentrations of over 10(-10) M PTX and increased with increase in concentration of PTX. This concentration-response curve for PTX-induced [45Ca](2+)-influx was similar to that for PTX-induced CA secretion. The CA secretion and [22Na](+)- and [45Ca](2+)-influxes induced by PTX were not affected by tetrodotoxin (TTX), but were significantly inhibited by quinidine and aprindine(mexiletine), antiarrythmic drugs known to block Na(+)-channels. Ca(2+)-channel blockers such as nifedipine, verapamil, Co2+, Cd2+, inhibited both CA secretion and [45Ca](2+)-influx induced by PTX. These results indicate that PTX-induced CA secretion is mediated by activation of Na(+)-dependent, TTX-insensitive voltage-dependent Ca(2+)-channels, and is inhibited by quinidine and aprindine through their inhibitory effects on the Na(+)- and Ca(2+)-influxes into the cells induced by PTX.

Physiological significance of plasma sulfoconjugated dopamine in patients with hypertension--clinical and experimental studies.

Sulfoconjugated catecholamines, especially dopamine sulfate, have recently attracted much attention because of the possibility of their conversion to active free dopamine by tissue arylsulfatase. In the present study, we have measured the plasma levels of free and sulfoconjugated dopamine in patients with hypertension and have investigated the physiological significance of sulfoconjugation. Results showed that the plasma level of dopamine sulfate in patients with essential hypertension was higher than the level in control subjects, and was highest in patients with renal hypertension. However, the plasma level of free dopamine showed no significant difference between patients with hypertension and normal subjects. Moreover, after normalization of blood pressure in hypertensive patients with medication, the plasma levels of conjugated dopamine decreased to almost the control value. In the experimental study, dopamine sulfate inhibited angiotensin II-induced aldosterone release from bovine adrenal cortical cells to a similar extent as produced by free dopamine. From these results, we have concluded that plasma sulfoconjugated dopamine may regulate free dopamine in the plasma of patients with hypertension, and it may have some physiological effects on blood pressure regulation.

Technique for Constructing the Pulmonary Trunk for Tetralogy of Fallot With Pulmonary Atresia

In expectation of the growth of a new pulmonary arterial trunk in total correction of tetralogy of Fallot with pulmonary atresia, we used pedicled autologous pericardium combined with left atrial appendage as the posterior wall of a new pulmonary arterial trunk. In cases of long discontinuity between the right ventricular infundibulum and left pulmonary artery, our technique could be recommended for early repair of tetralogy of Fallot with pulmonary atresia.

New operative method for distal aortopulmonary septal defect

A new technique is described for repairing distal aortopulmonary septal defect with aortic origin of the right pulmonary artery in a 26-day-old neonate. To prevent the narrowing of the proximal right pulmonary artery in the distal aortopulmonary septal defect caused by conventional intraluminal prosthetic patch reconstruction, rerouting of the right pulmonary artery using native aortic wall tissue without artificial material was performed. This method seems to be superior to the conventional method, especially during the neonatal period.

Effect of angiotensin II on Ca2+ efflux from freshly isolated adult rat cardiomyocytes: possible involvement of Na+/Ca2+ exchanger.

In the present study, we examined the effect of angiotensin II on Ca2+ efflux from freshly isolated adult rat cardiomyocytes. Angiotensin II stimulated the efflux of 45Ca2+ from the cells in a concentration-dependent manner, at least in pharmacological doses of 10(-8) M to 10(-5) M. The 45Ca2+ efflux was inhibited by the type 1 angiotensin II receptor antagonist losartan, but not by the type 2 antagonist PD 123319. Angiotensin II also induced an increase in cytosolic free calcium ([Ca2+]i) and inositol trisphosphate formation within the cardiomyocytes. Angiotensin II-induced 45Ca2+ efflux and the increase in [Ca2+]i were both inhibited by thapsigargin, a specific inhibitor of the sarcoplasmic reticulum Ca2+ pump. The 45Ca2+ efflux was not affected by removal of the extracellular Ca2+ but was dependent on the presence of extracellular Na+. In addition, angiotensin II caused 22Na+ influx into the cells. These results indicate that angiotensin II stimulates Na+-dependent 45Ca2+ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on the plasma membrane type 1 angiotensin II receptors. Angiotensin II-induced increase in [Ca2+]i may cause an activation of Na+/Ca2+ exchange which finally results in the stimulation of 45Ca2+ efflux from the cells. Since it is reported that Na+/Ca2+ exchange is important in calcium homeostasis within the cells, angiotensin II may play some role in the reduction of intracellular Ca2+ from isolated adult rat cardiomyocytes.