Yuji Okada

Characterization of prostanoid receptors mediating contraction of the gastric fundus and ileum: studies using mice deficient in prostanoid receptors

Receptors mediating prostanoid‐induced contractions of longitudinal sections of gastric fundus and ileum were characterized by using tissues obtained from mice deficient in each type and subtype of prostanoid receptors. The fundus and ileum from mice deficient in either EP3 (EP3−/− mice), EP1 (EP1−/− mice) and FP (FP−/− mice) all showed decreased contraction to PGE2 compared to the tissues from wild‐type mice, whereas contraction of the fundus slightly increased in EP4−/− mice. 17‐phenyl‐PGE2 also showed decreased contraction of the fundus from EP3−/−, EP1−/− and FP−/− mice. Sulprostone showed decreased contraction of the fundus from EP3−/− and FP−/− mice, and decreased contraction of the ileum to this compound was seen in tissues from EP3−/−, EP1−/− and FP−/− mice. In DP−/− mice, sulprostone showed increased contraction. DI‐004 and AE‐248 caused the small but concentration‐dependent contraction of both tissues, and these contractions were abolished in tissues obtained from EP1−/− and EP3−/− mice, respectively, but not affected in other mice. Contractions of both fundus and ileum to PGF2α was absent at lower concentrations (10−9 to 10−7 M), and suppressed at higher concentrations (10−6 to 10−5 M) of the agonist in the FP−/− mice. Suppression of the contractions at the higher PGF2α concentrations was also seen in the fundus from EP3−/−, EP1−/− and TP−/− mice and in the ileum from EP3−/− and TP−/− mice. Contraction of the fundus to PGD2 was significantly enhanced in DP−/− mice, and contractions of the fundus and ileum to this PG decreased in FP−/− and EP3−/− mice. Contractions of both tissues to I‐BOP was absent at 10−9 to 10−7 M and much suppressed at higher concentrations in TP−/− mice. Slight suppression to this agonist was also observed in the tissues from EP3−/− mice. PGI2 induced small relaxation of both tissues from wild‐type mice. These relaxation reactions were much potentiated in EP3−/− mice. On the other hand, significant contraction to PGI2 was observed in both tissues obtained from IP−/− mice. These results show that contractions of the fundus and ileum induced by each prostanoid agonist are mediated by actions of this agonist on multiple types of prostanoid receptors and in some cases modified by its action on relaxant receptors.

Effects of the prostanoids on the proliferation or hypertrophy of cultured murine aortic smooth muscle cells

Effects of the prostanoids on the growth of cultured aortic vascular smooth muscle cells (VSMCs) were examined using mice lacking prostanoid receptors. Proliferation of VSMCs was assessed by measuring [3H]‐thymidine incorporation and the cell number, and their hypertrophy by [14C]‐leucine incorporation and protein content. In VSMCs from wild‐type mice, expressions of mRNAs for the EP4 and TP were most abundant, followed by those for the IP, EP3 and FP, when examined by competitive reverse transcriptase‐PCR. Those for the EP1, EP2 and DP, however, could not be detected. AE1‐329, an EP4 agonist, and cicaprost, an IP agonist, inhibited platelet derived growth factor (PDGF)‐induced proliferation of VSMCs from wild‐type mice; these inhibitory effects disappeared completely in VSMCs from EP4−/− and IP−/− mice, respectively. In accordance with these effects, AE1‐329 and cicaprost stimulated cAMP production in VSMCs from wild‐type mice, which were absent in VSMCs from EP4−/− and IP−/− mice, respectively. Effects of PGE2 on cell proliferation and adenylate cyclase were almost similar with those of AE1‐329 in VSMCs from wild‐type mice, which disappeared in VSMCs from EP4−/− mice. PGD2 inhibited PDGF‐induced proliferation of VSMCs from both wild‐type and DP−/− mice to a similar extent. This action of PGD2 was also observed in VSMCs from EP4−/− and IP−/− mice. In VSMCs from wild‐type mice, I‐BOP, a TP agonist, showed potentiation of PDGF‐induced hypertrophy. I‐BOP failed to show this action in VSMCs from TP−/− mice. The specific agonists for the EP1, EP2 or EP3, and PGF2α showed little effect on the growth of VSMCs. These results show that PGE2, PGI2 and TXA2 modulate PDGF‐induced proliferation or hypertrophy of VSMCs via the EP4, IP and TP, respectively, and that the inhibitory effect of PGD2 on PDGF‐induced proliferation is not mediated by the DP, EP4 or IP.

Effects of KRN4884, a novel K channel opener, on the cardiovascular system in anesthetized dogs : a comparison with levcromakalim, nilvadipine, and nifedipine

Pharmacological profiles of KRN4884, 5-amino-N-[2-(2-chlorophenyl)ethyl]-N‘-cyano-3-pyridinecarboxamidine, were evaluated in in vitro and in vivo experiments. In rat isolated aorta, KRN4884 (10−10-10−5 M) produced a concentration-dependent relaxation. Pretreatment with glibenclamide (10−7-10−6 M) produced a rightward shift of the concentration-response curve for KRN4884. In anesthetized dogs, KRN4884 (3 and 10 μg/kg intravenously, i.v.), levcromakalim (3 and 10 μg/kg i.v.), nilvadipine (1–10 μg/kg i.v.), and nifedipine (1–10 μg/kg i.v.) produced decreases in mean blood pressure (MBP), total peripheral vascular resistance (TPR), and coronary vascular resistance (CVR), and increases in aortic blood flow (AoF) and coronary blood flow (CBF). The percentage decrease in CVR was greater than that in TPR with KRN4884 and levcromakalim, but nilvadipine and nifedipine showed no significant differences between CVR and TPR in percentage decreases. Heart rate (HR) was slightly increased by KRN4884 but was not affected by levcromakalim, nilvadipine, or nifedipine. Left ventricular dP/dt (LVdP/dt) was reduced only by nifedipine in a dose-dependent manner. The duration of the hypotensive action of KRN4884 was longer than those of levcromakalim and nifedipine and was similar to that of nilvadipine. The duration of the decreases in TPR and CVR induced by KRN4884 was longer than those induced by levcromakalim and nifedipine and shorter than that induced by nilvadipine. These results suggest that the cardiovascular effects of KRN4884 are very similar to those of the K channel opener levcromakalim and Ca channel blockers such as nilvadipine and nifedipine. However, the hypotensive effects of KRN4884 are long-acting in comparison with those of levcromakalim and the selective effect of KRN4884 on coronary vasculature is greater than those of nilvadipine and nifedipine.

Vasorelaxant mechanism of KRN2391 and nicorandil in porcine coronary arteries of different sizes

1 The relaxant mechanisms of action of KRN2391, a novel vasodilator, and nicorandil on epi‐myocardial coronary artery (2.5–3.0 mm outer diameter) and mid‐myocardial coronary artery (0.8–1.0 mm outer diameter) were investigated in porcine isolated coronary arteries. In addition, the vasorelaxant responses of KRN2391 and nicorandil were compared with those of nitroglycerin and cromakalim, a K+ channel opener, in epi‐ and mid‐myocardial coronary arteries. 2 Nitroglycerin showed a more potent relaxant effect on epi‐myocardial coronary arteries than on mid‐myocardial coronary arteries, whereas cromakalim produced greater relaxation responses in mid‐myocardial coronary arteries. There was no difference between epi‐ and mid‐myocardial coronary arteries in terms of the relaxant effect of KRN2391 and nicorandil. 3 Relaxation induced by KRN2391 in epi‐ and mid‐myocardial coronary arteries was inhibited by oxyhaemoglobin, a pharmacological antagonist of nitrovasodilators, and glibenclamide, a pharmacological antagonist of K+ channel opening drugs. However, the inhibitory effect of glibenclamide on KRN2391‐induced relaxation was greater in mid‐myocardial coronary artery than in epi‐myocardial coronary artery. 4 Relaxation induced by nicorandil was inhibited by oxyhaemoglobin alone in epi‐myocardial coronary arteries and by both oxyhaemoglobin and glibenclamide in mid‐myocardial coronary arteries. 5 In epi‐ and mid‐myocardial coronary arteries, relaxation induced by cromakalim was inhibited by glibenclamide but not by oxyhaemoglobin, whereas relaxation induced by nitroglycerin was inhibited by oxyhaemoglobin but not by glibenclamide. 6 These results suggest that KRN2391 and nicorandil exhibit a dual mechanism of action acting partly as a nitrate and partly as a K+ channel opener. The mechanism of action of these drugs depend on the segment of coronary artery studied. Furthermore, the dual mechanism of action of KRN2391 and nicorandil seems to contribute to the equipotent relaxant effect between epi‐ and mid‐myocardial coronary arteries.

Effects of the potassium channel openers KRN4884 and levcromakalim on the contraction of rat aorta induced by A23187, compared with nifedipine.

We examined the different vasodilatory effects of the K+ channel openers levcromakalim and 5-amino-N2-[2-(2-chlorophenyl)ethyl]-N′-cyano-3-pyridinecarboxamidine (KRN4884), and the Ca2+ channel blocker nifedipine in the rat aorta. KRN4884 (10−10-10−5 M) and nifedipine (10−10−10−5 M) produced concentration-dependent relaxation in the rat aorta precontracted by 25 mM KCl. The K+ channel blocker glibenclamide (1 μM) inhibited the relaxation induced by KRN4884 but did not influence nifedipine-induced relaxation. KRN 4884 had almost no effect on contraction induced by 80 mM KCl, whereas nifedipine completely relaxed the muscle precontracted by 80 mM KCl, whereas nifedipine completely relaxed the muscle precontracted by 80 mM KCl. These results indicate that KRN4884 is a K+ channel opener. We investigated the relaxant effects of KRN4884 (10−10-10−5 M), levcromakalim (10−9-10−5 M) and nifedipine (10−9-10−5 M) on A23187 (1 μM)-induced contraction. KRN4884 and levcromakalim had a potent relaxant effect but nifedipine only a weak effect on the smooth muscle contracted by A23187. Glibenclamide (1 μM) inhibited the relaxation induced by KRN4884 and levcromakalim, but did not influence the nifedipine-induced relaxation. KRN 4884 (1 μM) produced a larger relaxation of A23187-induced contraction but had little effect on the increase in intracellular [Ca2+] induced by A23187. These results suggest that KRN4884 is a specific K+ channel opener and its vasodilating mechanisms involve not only deactivation of Ca2+ channels but also a decrease in the Ca2+ sensitivity of contractile elements.

Effect of KRN4884 on lipid metabolism in hyperlipidemic rats

1. KRN4884 is a novel pyridinecarboxamidine type potassium channel opener. 2. To determine whether KRN4884 affects lipid metabolism, we investigated its effects on serum lipid levels by using two types of hyperlipidemic rats: genetically hyperlipidemic obese Zucker rats and diet-induced hyperlipidemic rats fed a high fat diet. KRN4884 dose dependently decreased systolic blood pressure in Zucker rats. 3. Oral administration of KRN4884 (1-10 mg/(kg day) for 14 days dose dependently reduced serum triglyceride levels in Zucker rats. The reductions in serum triglyceride were associated with reductions in triglyceride in chylomicron and very low density lipoprotein. 4. KRN4884 produced no change in serum insulin and glucose levels in Zucker rats. 5. KRN4884 exhibited a similar triglyceride lowering effect in diet-induced hyperlipidemic rats. 6. These results suggest that KRN4884 has a beneficial effect on serum triglyceride levels as well as a hypotensive effect.

Effects of KRN4884, a novel pyridinecarboxamidine type KATP channel opener, on serum triglyceride levels in rats

The effects of KRN4884, a novel pyridinecarboxamidine type KATP channel opener, on serum triglyceride levels were investigated in Sprague‐Dawley rats. Oral administration of KRN4884 (3 mg kg−1) for 10 days caused a significant reduction in serum triglyceride levels, which was comparable to that of clofibrate (160 mg kg−1). Reduction in serum triglyceride levels by KRN4884 and clofibrate were accompanied by a reduction in triglyceride levels both in chylomicron and in very low density lipoprotein. KRN4884 treatment did not affect serum concentrations of total cholesterol and phospholipid, but did increase free fatty acid levels. Clofibrate reduced total cholesterol, phospholipid and free fatty acid levels. Administration of clofibrate significantly decreased triglyceride secretion rate as measured by the Triton WR‐1339 injection procedure, while KRN4884 did not. Rats receiving KRN4884 exhibited an increase in lipoprotein lipase (LPL) activity both in adipose tissue and in skeletal muscle. There was an inverse correlation between serum triglyceride levels and tissue LPL activities. KRN4884 did not change hepatic triglyceride lipase (HTGL) activity. Clofibrate affected neither LPL nor HTGL activities. It is concluded that administration of KRN4884 results in reduced serum triglyceride levels which may be due to the enhancement of LPL activity in peripheral tissues.

Protective effect of the K+ channel opener KRN4884 on peripheral occlusive arterial disease in rats.

1. The effect of the potassium channel opener KRN4884 on the peripheral arterial occlusion model induced by laurate was examined and compared with that of beraprost sodium and nilvadipine. 2. KRN4884 or beraprost sodium prevented macroscopic changes in the paw after the injection of laurate. In contrast, nilvadipine did not improve the lesions. 3. KRN4884 produced a dose-dependent increase in gastrocnemius blood flow in the chronic femoral artery-ligated rats. The effect of KRN4884 on the blood flow was stronger in the hypoxic muscle than in the normal muscle. 4. KRN4884 did not have a direct antiplatelet aggregation activity. 5. These findings suggest that KRN4884 is useful for the therapy of peripheral arterial occlusive disease and that the effect of KRN4884 is associated with an increase in blood flow in ischemic skeletal muscle.

Effect of Kil769, a Novel K+-channel Opener, on Sensitivity to Ca2+ of Contractile Elements and Inositol Phosphate Formation in Porcine Coronary Artery

To determine whether Kil769, a novel K+‐channel opener, acts intracellularly in vasorelaxation, we compared the effects of Kil 769 on force of contraction, intracellular Ca2+ concentration ([Ca2+]i) and inositol phosphate (IP1) formation with those of Ca2+‐channel blockers in isolated porcine coronary artery.

Comparative analysis of vasodilating Mechanisms of Kil769, Ki3315 and KRN2391, pyridinecarboximidamide derivatives, in porcine isolated coronary artery

1. The vasodilating mechanisms of pyridinecarboximidamide derivatives which have a nitroxyl group (KRN2391), a phenyl group (Ki1769) or a hydroxyl group (Ki3315) were studied in porcine isolated coronary artery. 2. KRN2391 (10(-6) M) increased cyclic GMP formation but did not affect intracellular cyclic AMP level. Ki1769 (10(-5) M) and Ki3315 (10(-3) M) had no effect on intracellular cyclic GMP and cyclic AMP levels. 3. Despite producing submaximal relaxation at KRN2391 (10(-6) M) and nitroglycerin (10(-6) M), the increase in cyclic GMP caused by KRN2391 was lower than that caused by nitroglycerin. 4. Methylene blue (10(-5) M) inhibited KRN2391- and nitroglycerin-induced relaxations but did not affect Ki1769- and Ki3315-induced relaxation. 5. Glibenclamide (10(-6) M) inhibited KRN2391-, Ki1769- and Ki3315-induced relaxation but did not affect nitroglycerin-induced relaxation. 6. These results suggest that the nitroxyl group of KRN239 contributes to its nitrate action and the pyridinecarboximidamide moiety plays an important role of developing a K channel opening action.