Junichi Kurihara

Ischemic pre-conditioning affects the subcellular distribution of protein kinase C and calcium/calmodulin-dependent protein kinase II in the gerbil hippocampal CA1 neurons.

Abstract Brief ischemic episode, which in itself is not lethal, confers tolerance to subsequent ischemic insults. Since intracellular signal transduction system has been implicated in ischemic cell death, we studied the effect of pre-conditioning on the changes in the subcellular distribution of protein kinase Cγ (PKC γ) as well as CaM kinase II (CaMKII). Gerbils were pre-conditioned by a sublethal 2 min cerebral ischemia 24 h prior to lethal 5 min ischemia. The pre-conditioning generally downregulated PKCγ and CaMKII in the CA1 hippocampus. Especially at the starting point of the second lethal ischemia, the cytosolic PKCγ level was about 40% lower in the pre-conditioned group. Also, the crude synaptosomal CaMKII level at 24 h reperfusion following the second ischemia was significantly lower in the pre-conditioned group, showing enhanced recovery of CaMKII translocation. Present results suggest that ischemic pre-conditioning may downregulate calcium-mediated cell signaling system, enhancing normalization of calcium homeostasis, perturbed by the second ischemia of lethal duration. [Neurol Res 2001; 23: 751-754]

Acidosis enhances translocation of protein kinase C but not Ca2+/calmodulin-dependent protein kinase II to cell membranes during complete cerebral ischemia

Systemic hyperglycemia and hypercapnia severely aggravate ischemic brain damage when instituted prior to cerebral ischemia. An aberrant cell signaling following ischemia has been proposed to be involved in ischemic cell death, affecting protein kinase C (PKC) and the calcium calmodulin kinase II (CaMKII). Using a cardiac arrest model of global brain ischemia of 10 min duration, we investigated the effect of hyperglycemia (20 mM) and hypercapnia (pCO(2) 300 mmHg) on the subcellular redistribution of PKC (alpha, beta, gamma) and CaMKII to synaptic membranes and to the microsomes, as well as the effect on PKC activity. We confirmed the marked translocation of PKC and CaMKII to cell membranes induced by ischemia, concomitantly with a decrease in the PKC activity in both the membrane fraction and cytosol. Hyperglycemia and hypercapnia markedly enhanced the translocation of PKC-gamma to cell membranes while other PKC isoforms were less affected. There was no effect of acidosis on PKC activity, or on translocation of CaMKII to cell membranes. Our data strongly suggest that the enhanced translocation of PKC to cell membranes induced by hyperglycemia and hypercapnia may contribute to the detrimental effect of tissue acidosis on the outcome following ischemia.

Hyperglycemia and hypercapnia differently affect post-ischemic changes in protein kinases and protein phosphorylation in the rat cingulate cortex

Hyperglycemia and hypercapnia aggravate intra-ischemic acidosis and subsequent brain damage. However, hyperglycemia causes more extensive post-ischemic damage than hypercapnia, particularly in the cingulate cortex. We investigated the changes in the subcellular distribution of protein kinase Cgamma (PKCgamma) and the Ca2+/calmodulin-dependent protein kinase II (CaMKII), as well as changes in protein tyrosine phosphorylation during and following 10 min normoglycemic, hyperglycemic (plasma glucose approximately 20 mM) and hypercapnic (paCO2) approximately 300 mm Hg) global cerebral ischemia. During reperfusion period, the translocation to cell membranes of PKCgamma, but not CaMKII, was prolonged by intra-ischemic hyperglycemia, while it was only marginally affected by hypercapnia. The tyrosine-phosphorylation of proteins in the synaptosomal membranes, as well as the extracellular signal-regulated kinase (ERK) in the cytosol, markedly increased during reperfusion following hyperglycemic ischemia, but to a lesser degree following hypercapnic ischemia. Our data suggest that PKCgamma, tyrosine kinase and ERK systems are involved in the process of ischemic damage in the cingulate cortex, where hyperglycemia may affect these kinases through an additional mechanism other than exaggerated acidosis.

Effects of FK506 on the translocation of protein kinase C and CaM kinase II in the gerbil hippocampal CA1 neurons

Abstract To explore biochemical basis for cerebroprotective effect of immunosuppressant FK506, we studied changes in subcellular distribution of protein kinase Cγ (PKCγ) as well as calcium/calmodulin-dependent protein kinase II (CaMKII) after ischemia. Male Mongolian gerbils were subjected to 5 min forebrain ischemia. FK506 (1 or 3 mg kg-1) was administered at 1 min after recirculation, which was confirmed to be cerebroprotective by histological examination at seven days after ischemia. At the designated time points (before ischemia, 5 min ischemia, 1 and 24 h recovery), heads were frozen and samples were taken from CA1 subfield of hippocampus. Western blot analysis was carried out. Persistent translocations of PKCγ and CaMKII to synaptosomal P2 fraction were observed in vehicle-treated group. FK506 significantly decreased levels of PKCγ and CaMKII in P2 fraction at 24 h of recovery. The present results suggest FK506 downregulates translocated PKCγ and CaMKII, which may contribute to its survival promoting effect after cerebral ischemia.

Protective effect of beraprost sodium, a new chemically stable prostacyclin analogue, against the deterioration of baroreceptor reflex following transient global cerebral ischaemia in dogs

1 A possible cerebroprotective effect of a chemically stable prostacyclin analogue, beraprost sodium, was investigated in a canine model of cerebral ischaemia. Cerebral ischaemia was produced by the combined occlusions of the left subclavian and the brachiocephalic arteries with preceding ligations of the intercostal arteries. 2 The decrease in baroreceptor reflex sensitivity (BRS), measured by phenylephrine‐induced reflex bradycardia, following 5 min ischaemia was used to assess the cerebroprotective effect. 3 Beraprost (1 μg kg−1 min−1 i.v., infused for 15 min just before ischaemia) completely prevented the decrease in BRS. Although the lower dose of beraprost (0.1 μg kg−1 min−1 i.v.) failed to show such a protective effect, its inhibitory effect on ADP‐induced platelet aggregation was as potent as that of the higher dose. 4 The extent of decrease in BRS was inversely correlated with the extent of the residual blood flow in the medulla oblongata during ischaemia. Since beraprost did not affect the extent of the residual blood flow during ischaemia, its cerebroprotective effect could not be ascribed to the reduction of the degree of ischaemia by increasing collateral blood flow to the brain. 5 Post‐ischaemic reduction of the regional blood flow in the medulla and the cerebral cortex was completely prevented by the higher dose of beraprost. 6 The present study suggests that the cerebroprotective effect of beraprost may be independent of its anti‐aggregatory and vasodilator effects. It is possible that the protection may be due to a prostacyclin‐like cytoprotective effect through membrane stabilization.

KT3-671, an angiotensin AT1 receptor antagonist, attenuates vascular but not cardiac responses to sympathetic nerve stimulation in pithed rats.

Effects of KT3-671 on vascular and cardiac sympathetic neurotransmission were investigated in pithed rats. The pressor response to spinal stimulation (5 Hz) of the pithed rat without the adrenals was approximately 75% of that with the adrenals. Guanethidine (8 mg/kg, i.v.) decreased by about 76% the pressor response to sympathetic stimulation in the pithed rat with intact adrenals and the guanethidine-resistant response was almost completely abolished by bilateral adrenalectomy. Therefore, the following experiments were done using the pithed rat without the adrenals. KT3-671 (1–10 mg/kg, i.v.) as well as losartan (1–10 mg/kg, i.v.) inhibited dose-dependently the pressor response to sympathetic stimulation. KT3-671 was approximately four times more potent than losartan in inhibiting the pressor response. The two angiotensin II subtype 1 receptor antagonists (10 mg/kg, i.v.) did not affect the pressor response to exogenously administered norepinephrine. Neither KT3-671 nor losartan influenced the tachycardia induced by spinal stimulation and isoprenaline. Intravenous infusion of angiotensin II (100 ng/kg/min) did not affect both pressor and tachycardic responses to sympathetic stimulation. In conclusion, KT3-671 as well as losartan inhibits vascular but not cardiac sympathetic neurotransmission of the pithed rats, which may contribute to its overall antihypertensive efficacy.

テルピネン-4-オールと中鎖脂肪酸カプリン酸の併用によるCandida albicansの菌糸形発育阻止作用とマウス口腔カンジダ症への治療効果

The combined effect of terpinen-4-ol, the main component of tea tree oil, and capric acid against mycelial growth of Candida albicans and murine oral candidiasis was evaluated in vitro and in vivo. Mycelial growth of C. albicans was estimated by the Cristal violet method. Combination of these compounds revealed a potent synergistic inhibition of growth. Therapeutic efficacy of the combination was evaluated microbiologically in murine oral candidiasis, and its application of the compounds clearly demonstrated therapeutic activity. Based on these results, the combined agent of terpinen-4-ol and capric acid was discussed as a possible candidate for oral candidiasis therapy.

Protection by nicorandil against the dysfunction of the central vagal baroreflex system following transient global cerebral ischaemia in dogs

1 A possible cerebroprotective effect of nicorandil was investigated in a canine model of 5 min global cerebral ischaemia, and compared with protective effects of nitroglycerin and nicardipine. 2 Cerebral ischaemia was produced by occlusion of the left subclavian and the brachiocephalic arteries with preceding ligation of the intercostal arteries. The decrease in baroreflex sensitivity (BRS), measured by phenylephrine‐induced reflex bradycardia, was used to assess the cerebroprotective effect. 3 Nicorandil (10 or 30 μg kg−1 min−1, i.v.), nitroglycerin (3 μg kg−1 min−1, i.v.) or nicardipine (0.3 μg kg−1 min−1, i.v.) were infused for 60 min just before ischaemia. Nitroglycerin and nicardipine decreased mean arterial blood pressure to an extent similar to that induced by the lower dose of nicorandil. Blood flow in the dorsal medulla oblongata was increased by nicorandil and nicardipine, but not by nitroglycerin. 4 Nicorandil at both doses and nitroglycerin prevented the post‐ischaemic decrease in BRS. In these cases, bilateral vagotomy during the reperfusion period decreased BRS, indicating that the vagal component of BRS was protected from ischaemia. On the other hand, nicardipine failed to exert a cerebroprotective effect. 5 The present study suggests that nicorandil may possess a direct cerebroprotective effect and that its property as a nitrate might, at least in part, be important for the observed cerebral protection.

Effect of flunarizine on the attenuation of baroreflex by transient cerebral ischemia

Baroreflex sensitivity assessed by phenylephrine-induced reflex bradycardia was markedly decreased by 5- an 10-min global cerebral ischemia in anesthetized dogs. Flunarizine, 0.1 and 1 mg/kg i.v., administered 5 min prior to 5-min ischemia, completely inhibited the decrease in baroreflex sensitivity while such a protective effect of the latter dose was incomplete against 10-min ischemia. In contrast, papaverine, 0.5 mg/kg per min i.v., infused for 5 min prior to 5-min ischemia, failed to protect the decrease in baroreflex sensitivity. Flunarizine may possess a certain direct cerebroprotective effect in addition to its cerebrocirculatory effect.

Blockade of α2-adrenoceptors protects the vagal baroreflex system from transient global cerebral ischemia in dogs

Ifenprodil, phentolamine, yohimbine or idazoxan, 1 mg/kg i.v., administered 5 min prior to 5-min global cerebral ischemia, completely prevented the post-ischemic dysfunction of the vagal baroreflex in anesthetized dogs. Idazoxan, 0.1 mg/kg i.v., was cerebroprotective against mild ischemia, but ineffective against severe ischemia. The post-ischemic administration of idazoxan, 1 mg/kg i.v., failed to restore the damaged vagal baroreflex. These results suggest that blockade of alpha 2-adrenoceptors during ischemia and the early reperfusion period may protect the vagal component of baroreflex from cerebral ischemia.