Hidekazu Hashimoto

Prostaglandin H2 may be the endothelium-derived contracting factor released by acetylcholine in the aorta of the rat.

The present experiment was performed to identify endothelium-derived contracting factor produced by acetylcholine stimulation in the aorta of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. The rings of the thoracic aorta were obtained from age-matched SHR and WKY rats, and changes in isometric tension were recorded. The relaxant responses to acetylcholine in the aortic rings from SHR were significantly weaker than those from WKY rats. The relaxant responses to acetylcholine were significantly enhanced by pretreatment with a cyclooxygenase inhibitor (indomethacin) or thromboxane A2/prostaglandin H2 receptor antagonist (ONO-3708) in aortic rings from both SHR and WKY rats. A thromboxane A2 synthetase inhibitor (OKY-046) did not affect the acetylcholine-induced relaxation in the aortic rings from SHR or WKY rats. In the organ bath solution, after acetylcholine stimulation, prostaglandin E2 and 6-keto-prostaglandin F1 alpha concentrations increased but not prostaglandin F2 alpha and thromboxane B2 concentrations. Exogenous prostaglandin H2, a stable analogue of thromboxane A2, and prostaglandin F2 alpha induced contractions of the SHR rings at a lower concentration than prostaglandin E2, prostaglandin D2, and prostaglandin I2. These contractile responses to various prostaglandins were markedly inhibited by pretreatment with ONO-3708. A prostacyclin synthetase inhibitor did not affect the relaxant responses to acetylcholine in the SHR rings. These results show that endothelium-derived contracting factor is produced and released by acetylcholine stimulation not only in the aorta of SHR but also in those of WKY rats and suggest that prostaglandin H2, a precursor of the released prostaglandins, is a strong candidate for endothelium-derived contracting factor produced by acetylcholine stimulation.

Long-term inhibition of NO synthesis promotes atherosclerosis in the hypercholesterolemic rabbit thoracic aorta. PGH2 does not contribute to impaired endothelium-dependent relaxation.

We examined whether prostaglandin (PG) H2, as an endothelium-dependent contracting factor, or the disturbed production of endothelium-derived relaxing factor, impairs endothelium-dependent relaxation and whether long-term inhibition of nitric oxide (NO) synthesis aggravates atherosclerosis in hypercholesterolemic rabbits. Male New Zealand White rabbits were fed one of the following diets: (1) standard chow; (2) 2% cholesterol-supplemented chow; (3) standard chow with 80 micrograms/mL N omega-nitro-L-arginine methylester (L-NAME), an NO synthetase inhibitor, in their drinking water; or (4) 2% cholesterol-supplemented chow with 80 or 160 micrograms/mL L-NAME in their drinking water. The rabbits were fed these diets for 8 or 12 weeks. Then aortic rings were obtained, and changes in isometric tension were recorded. Intimal atherosclerotic areas of the thoracic aortas were subsequently measured by planimetry. The cholesterol-supplemented diet significantly impaired endothelium-dependent aortic relaxation to acetylcholine. Pretreatment with the thromboxane A2/PGH2 receptor antagonist ONO-3708 did not reverse this impaired response. Vessels from both normocholesterolemic and hypercholesterolemic rabbits given L-NAME showed more impaired endothelium-dependent relaxation than those from their dietary counterparts not given L-NAME. Morphometric analysis revealed marked enlargement of intimal atherosclerotic areas in aortas from L-NAME-treated hypercholesterolemic rabbits compared with those from untreated hypercholesterolemic rabbits. These findings suggest that PGH2 does not contribute to impaired endothelium-dependent relaxation and that long-term administration of L-NAME promotes atherosclerosis by inhibition of NO synthesis in the hypercholesterolemic rabbit thoracic aorta.

Prostaglandin H2 as an endothelium-derived contracting factor and its interaction with endothelium-derived nitric oxide.

The possibility that prostaglandin H2 is an endothelium-derived contracting factor (EDCF) was evaluated in rings of thoracic aorta of spontaneously hypertensive rats (SHR). When the aortic rings were contracted with norepinephrine (10(-7) mol/l) and treated with acetylcholine (10(-5) mol/l), a relaxant response with a peak after approximately 1 min and a contractile response with a peak after approximately 7 min were observed. When these rings were pretreated with a thromboxane A2/prostaglandin H2 receptor antagonist (ONO-3708), the later contractile response was clearly inhibited and only a sustained relaxant response was observed. This relaxant response was completely inhibited by pretreatment with an inhibitor of nitric oxide production (N-nitroarginine methylester; NNM). When aortic rings in the basal condition were treated with NNM and then with acetylcholine, a contractile response with a peak after 7 min was observed, but this reaction was completely inhibited by pretreatment with ONO-3708. The rate of 6-keto-prostaglandin F1 alpha production showed a peak of 1.4 x 10(-6) mol/l per min per tissue, 2-4 min after administration of acetylcholine. With exogenous prostaglandin H2 (5 x 10(-7) mol/l), a peak contraction was observed after approximately 4 min, the degree and pattern of which were similar to that induced by acetylcholine. Endogenous prostaglandin H2 is considered to be produced by the aortic rings in an amount sufficient to induce vascular contraction within 30 s, and the pattern of this contraction induced by acetylcholine resembles that induced by exogenous prostaglandin H2. These findings most strongly suggest that prostaglandin H2 is an EDCF.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of Prostaglandin H2 as an Endothelium-Derived Contracting Factor in Diabetic State

This study investigated the possible involvement of prostaglandin H2, an acetylcholine-induced endothelium-derived contracting factor in rat aorta, in the development of abnormality of the vasculature in diabetes. Rings of thoracic aorta were prepared from control Wistar-Kyoto and STZ-induced diabetic rats to examine the changes in isometric tension. In 10−7 M norepinephrine-precontracted rings, acetylcholine induced relaxations, which were significantly impaired in diabetic rats. Inhibition of thromboxane A2-prostaglandin H2 receptors with ONO-3708 (10−6) M) prevented the development of the impairment of relaxation in diabetic rats. Thromboxane A2 synthesis inhibition with OKY-046 (10−5 M) did not affect the acetylcholine-induced relaxation in both control and diabetic rats. In aortic rings under resting tension, acetylcholine induced a contraction that was greater in diabetic than control rats, when the nitric oxide production was inhibited by NGxs-nitro-L-arginine methylester (10−4 M). This acetylcholine-induced contraction was observed only in the rings with intact endothelium and was completely abolished by ONO-3708 (10−6 M). The concentration of 6-keto-prostaglandin F1α in the solution bathing diabetic rat aortic rings increased significantly after acetylcholine (10−5 M) administration. Prostacyclin (10−9 · 10−6 M) did not induce contractions at all. Prostacyclin is unlikely to mediate contractions because of its low contractile potency. These findings suggest that the impairment of acetylcholine-induced relaxation in the diabetic state is not caused by the diminished production of an endothelium-derived relaxing factor or nitric oxide but rather by the increased endothelium-derived contracting factor or prostaglandin H2, which may be responsible for abnormalities of the vasculature in diabetes.

Altered Acetylcholine and Norepinephrine Concentrations in Diabetic Rat Hearts: Role of Parasympathetic Nervous System in Diabetic Cardiomyopathy

The concentrations of acetylcholine (ACh) as a parasympathetic marker and norepinephrine (NE) as a sympathetic marker were investigated in the hearts of rats 2, 4, and 8 wk after the inductionof diabetes by an injection of streptozocin (STZ; 65 mg/kg i.v.). ACh and NE were measured by high-performance liquid chromatography with electrochemical detection. Diabetic rats showed low body weight and heart weight at 2, 4, and 8 wk and higher heart-to-body weight ratio and bradycardiaat 8 wk, almost all of which were normalized after insulin treatment. Myocardial ACh and NE concentrations in the diabetic rats at 2 and 4 wk were not significantly different from those in age-matched control rats. However, ACh and NE concentrations in the diabetic rats at 8 wk significantly increased compared with the control rats. Diabetic rats at 8 wk also had increased myocardial choline concentration and choline acetyltransferase activity and decreased acetylcholinesterase activity. Insulin treatment normalized all of these changes in the diabetic rats. Thus, in STZ-induced diabetes (STZ-D), the concentrations of both cholinergic and noradrenergic neurotransmitters in the myocardium increased. The results of this study confirm a previously reported increase in sympathetic activity to the heart and also indicate that there is an increase in the synthesis and a decrease in the metabolism of ACh in STZ-D and that adequate insulin treatment normalizes these changes.

Correlation with blood pressure of the acetylcholine-induced endothelium-derived contracting factor in the rat aorta.

To examine a relation between the production of acetylcholine-induced endothelium-derived contracting factor and an increase in blood pressure, endothelium-dependent contraction and relaxation were evaluated by measuring the isometric tension of aortic rings from spontaneously hypertensive rats and Wistar-Kyoto rats at 5, 10, 20, and 30 weeks of age. In norepinephrine-precontracted rings, acetylcholine (10(-8) to 10(-5) M)-induced relaxations diminished at the doses of 10(-6) to 10(-5) M in both strains except at 5 weeks of age. Treatment with a thromboxane A2/prostaglandin H2 antagonist (ONO-3708) prevented this reduction in acetylcholine-induced relaxations in both strains and induced dose-dependent relaxations, which were completely inhibited by treatment with a nitric oxide inhibitor, NG-nitro-L-arginine methyl ester. In aorta treated with NG-nitro-L-arginine methyl ester without precontraction, acetylcholine induced dose-dependent contractions, which were greater in spontaneously hypertensive rats than in Wistar-Kyoto rats. These acetylcholine-induced contractions, which were observed only in rings with endothelium, were completely inhibited by treatment with ONO-3708 but not with a thromboxane A2 synthetase inhibitor (OKY-046). There was a statistically significant correlation between the acetylcholine-induced contractions and blood pressure. Release of 6-ketoprostaglandin F1 alpha by acetylcholine from the aorta was greater in spontaneously hypertensive rats. In vivo administration of another thromboxane A2/prostaglandin H2 antagonist (ONO-8809) (10 or 30 micrograms per body per day) for 3 weeks (5-8 weeks of age) did not affect blood pressure in either rat strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of 1,2-Diacylglycerol Content in Myocardium From Diabetic Rats

1,2-Diacylglycerol has been proposed to be a secondary messenger; therefore, in this study we evaluated the amount of 1,2-diacylglycerol in heart tissue from streptozocin-induced diabetic rats and examined the effect of insulin treatment on 1,2-diacylglycerol content. Diabetic rats had lower body and ventricular weights and higher ratios of ventricular to body weight, all of which shifted toward normal values after 4 wk of untreated diabetes followed by 4 wk of insulin treatment. The contents of major phospholipids were significantly depressed in the diabetic rat hearts. In contrast, the triglyceride and cholesterol contents in the myocardium were increased by streptozocin injection and completely normalized by insulin treatment, and glucose levels returned to normal. The 1,2-diacylglycerol content in the myocardium was also significantly elevated in the diabetic rats compared with age-matched controls. Moreover, the 1,2-diacylglycerol content was significantly higher in rats with 4 wk of diabetes than in those with 8 wk of diabetes. Insulin treatment in the diabetic rats, however, did not produce any decrease in 1,2-diacylglycerol content. The results of this study suggest that the development of cardiomyopathy induced by streptozocin injection is associated with a high 1,2-diacylglycerol level, which may result in the activation of protein kinase C. Insulin is one of the agonists that generates 1,2-diacylglycerol in myocytes; however, the relationship between the sustained 1,2-diacylglycerol level and the normalization of diabetes by insulin administration is unclear.

Alteration of 1,2-diacylglycerol content in ischemic and reperfused heart

The myocardial 1,2-diacylglycerol (DG) and phospholipid levels during ischemia and reperfusion were studied in open-chest dogs by means of sequential epicardial minibiopsies, followed by quantification based on mass measurement technique. 1,2-DG level increased as early as 5 min after coronary ligation but decreased at 30 min. Also as early as 2 min after postischemic (35 min) reperfusion, 1,2-DG level increased transiently compared to pre-reperfusion level. Prazosin inhibited these changes significantly. A significant change in the incidence of reperfusion-induced ventricular tachycardia (VT) was not obtained in the prazosin-treated group. However, the 1,2-DG level 2 min after reperfusion was significantly higher in the ischemic myocardium developed reperfusion-induced VT than in the undeveloped one. Phospholipid levels remained unchanged during ischemia and reperfusion. These results suggest that α1-adrenergic stimulation occurs early in ischemia and reperfusion and leads to 1,2-DG accumulation, which may be involved in the pathogenesis of ischemic and reperfusion injury.

Prostaglandin H2 as an endothelium-derived contracting factor modulates endothelin-1-induced contraction

Objective To investigate the possible involvement of prostaglandin H2, an endothelium-derived contracting factor in the rat aorta, in the development of the contraction induced by endothelin-1 Methods The aortic rings from spontaneously hypertensive rats (SHR) were prepared, and the changes of isometric tension of these rings developed by endothelin-1 were recorded with or without the treatment of several inhibitors or an antagonist. The concentrations of prostaglandins and thromboxane B2 in the bath solution with the rings contracted by endothelin-1 were measured by radioimmunoassay. The effects of a thromboxane A2/prostaglandin H2 receptor antagonist (ONO-3708) on endothelin-1-induced contraction were compared in SHR and Wistar-Kyoto (WKY) rats Results Indomethacin (10-5mol/l) and ONO-3708 (10-6mol/l) significantly diminished endothelin-1 (3×10-8mol/l)-induced contractions in the aortic rings from SHR with but not without endothelium. The thromboxane A2 synthetase inhibitors OKY-046 (10-5 mol/l) and RS-5186 (10-5mol/l) did not attenuate the contractions either with or without endothelium. Endothelin-1 significantly increased the release of 6-keto-prostaglandin F1α, which is the metabolite of prostaglandin I2 and its precursor prostaglandin H2, from rings with endothelium of SHR, but the concentration of thromboxane B2 from aortic rings was unchanged. In the rings without endothelium the endothelin-1-induced release of 6-keto-prostaglandin F1α was also observed. The half-maximal effective concentration of endothelin-1 for rings from SHR was shifted to the right by ONO-3708, but that of WKY rats was not changed, and significantly greater amounts of 6-keto-prostaglandin F1α were released in the rings from SHR than in those from WKY rats by endothelin-1 Conclusions Endothelin-1 induced the release of prostaglandin H2 from endothelial cells in the rat aorta, the effect being greater in the hypertensive state. The released prostaglandin H2, an endothelium-derived contracting factor, modulated the vasoconstriction that is induced by endothelin-1, another endothelium-derived contracting factor, in addition to the direct vasoconstrictive action of endothelin-1 on vascular smooth muscle

The effects of lipoxygenase inhibitor and peptidoleukotriene antagonist on myocardial injury in a canine coronary occlusion-reperfusion model

UNLABELLED We studied effects of lipoxygenase inhibitor (AA-861) and peptidoleukotriene antagonist (ONO-1078) on infarct size, polymorphonuclear leukocyte (PMNs) infiltration, gross myocardial hemorrhage and ventricular arrhythmias in canine coronary occlusion (2 hr)-reperfusion (5 hr) model. Infarct size (IS) and risk area (RA) were determined by dual staining technique. Thirty minutes before coronary occlusion dogs were randomly assigned to one of the following three groups: lipoxygenase inhibitor group (n = 11) receiving AA-861 3 mg/kg i.v., peptidoleukotriene antagonist group (n = 11) receiving continuous intravenous infusion of ONO-1078 1 micrograms/kg/min and vehicle control group (n = 15). Both AA-861 and ONO-1078 reduced infarct size [AA-861: 21.8 +/- 1.3% of RA (mean +/- SEM), ONO-1078: 22.5 +/- 4.4% vs CONTROL 54.0 +/- 6.4%, p less than 0.01 and p less than 0.01, respectively] and area of gross myocardial hemorrhage (AA-861: 5.1 +/- 2.4% of IS, ONO-1078: 5.2 +/- 2.5% vs CONTROL 22.3 +/- 3.9%, p less than 0.01 and p less than 0.01, respectively). Both drugs also decreased frequency of ventricular premature contractions both during occlusion and during reperfusion, and that of ventricular tachycardia during reperfusion. AA-861 inhibited PMNs recruitment into infarcted area. However, ONO-1078 had no significant influence on degree of PMNs infiltration. These results suggest that lipoxygenase products, especially peptidoleukotrienes (LTC4, D4 and E4) may play important roles in the pathogenesis of myocardial ischemic and reperfusion injuries.