Tokio Nakane

Muscarinic receptor subtypes mediating vasodilation and vasoconstriction in isolated, perfused simian coronary arteries.

Summary: Using the cannula insertion method, we investigated the vascular responses of isolated simian coronary artery to acetylcholine (ACh). When the preparation was partially precontracted by 20 m M KCl, ACh and carbachol induced vasodilation dose dependently in coronary artery with endothelium, but ACh and carbachol contracted the coronary artery after removal of the endothelium by 1 mg saponin. A selective M1 receptor agonist 4-[N-(3-chlorophenyl)carbamoyloxy]-2-butinyltrimethylammonium chloride (McN-A-343) did not affect the perfusion pressure of the precontracted coronary arteries significantly. Both these responses to ACh were inhibited by the M3 receptor antagonist 4-dipheny-lacetoxy N-methylpiperidine methobromide (4-DAMP) in a dosedependent manner, but not by a selective M2 receptor antagonist AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzo-diazepine-6-one). A selective M1 receptor antagonist pirenzepine did not affect ACh-induced vasoconstriction significantly and inhibited the vasodilation partially only at the highest dose (100 nmol). The effects of three antagonists on the vasodilative responses to carbachol were also studied and almost the same results were observed. Removal of the endothelium did not affect sodium nitroprusside (SNP)-induced vasodilation significantly. Pirenzepine, AF-DX 116, and 4-DAMP did not affect the action of isoproterenol. These data suggest that the vasodilation elicited by ACh is mediated by release of endothelium-derived relaxing factors (EDRF) consequent to the activation of M3 receptors on endothelial cells, and the constriction is mediated by stimulation of M3 receptors on smooth muscle cells in isolated simian coronary arteries

UTP induces vascular responses in the isolated and perfused canine epicardial coronary artery via UTP‐preferring P2Y receptors

1 Vasoconstrictor responses of the isolated and perfused canine epicardial coronary artery to uridine 5′‐triphosphate (UTP) were analysed pharmacologically. 2 At basal perfusion pressure, UTP induced vasoconstriction in a dose‐related manner and the vasoconstriction was sometimes followed by a slight vasodilatation at large doses (more than 10 nmol). The rank order of potency for vasoconstriction was UTP=UDP>ATP>TTPITP>> UMP. At raised perfusion pressure by 20 mm KCl, the vasoconstriction was not changed and a small vasodilatation was induced at large doses. The rank order of potency for vasodilatation was induced at large doses. The rank order of potency for vasodilatation was ATP>>ITPUDP>UTPTTP. The maximal vasodilator response to UTP was much less than that to ATP. UMP did not induce vasodilatation. 3 The P2X receptor agonist and desensitizing agent α,β‐methylene ATP (1 μm) and the P2 receptor antagonist suramin (100 μm) inhibited the vasoconstrictor responses to ATP but not those to UTP and UDP. The P2 receptor antagonist reactive blue 2 (30 μm) did not inhibit the vascular responses to UTP. 4 UTP (200 μm) desensitized the vasoconstrictor responses to UTP, but not either the vasodilator responses to UTP or the vasoconstrictor responses to ATP and UDP. UDP (200 μm) did not desensitize the vascular responses to UTP. 5 Preincubating the UDP stock solution and arterial preparation with hexokinase (10 and 1 uml−1, respectively) did not change the vasoconstrictor responses to UDP. 6 The Ca channel blocker diltiazem (1 μm) inhibited the vasoconstrictor responses to UTP but not those to ATP and UDP. Incubation in a Ca2+‐free solution containing 1 mm EGTA inhibited the vascular responses to ATP, UTP and UDP. 7 Removal of the endothelium by an intraluminal injection of saponin (1 mg) inhibited the vasodilator responses to UTP. Indomethacin, a cyclo‐oxygenase inhibitor (1 μm), inhibited the vasodilator responses to UTP, but NG‐nitro‐l‐arginine, a nitric oxide synthase inhibitor (300 μm), did not have an inhibitory effect. 8 The results suggest that (1) UTP induces vasoconstriction via UTP‐preferring P2Y receptors on the smooth muscle and vasodilatation via receptors different from those mediating the vasoconstriction induced by UTP and mediating the vasodilatation by ATP on the endothelium, through mainly the release of prostacyclin in the canine epicardial coronary artery; (2) UDP induces vasoconstriction via UDP‐preferring P2Y receptors; and (3) L‐type Ca ion channels are involved in the vasoconstriction induced by UTP, but not in that induced by UDP.

KDR-5169, a new gastrointestinal prokinetic agent, enhances gastric contractile and emptying activities in dogs and rats

KDR-5169, 4-amino-5-chloro-N-[1-(3-fluoro-4-methoxybenzyl)piperidin-4-yl]-2-(2-hydroxyethoxy)benzamide hydrochloride dihydrate, is a new prokinetic with a dual action, i.e., stimulation of the 5-HT4 receptor and antagonism of the dopamine D2 receptor. In this study, we determined in vitro activities of KDR-5169 towards both receptors and demonstrated the effect of the compound on gastrointestinal motor activity in conscious dogs and rats. In dogs, intravenous KDR-5169 stimulated upper gastrointestinal motility in the fasting state and also eliminated the depressive effect of 3,4-dihydroxyphenylalanine (L-DOPA) on this motility in the postprandial state. The effect of KDR-5169 on gastric emptying was further characterized by the use of three rat gastroparesis models (dopamine D2 receptor agonist (quinpirol)-, abdominal surgery-, or combined-situation-induced). Domperidone (a dopamine D2 receptor antagonist) was effective in the quinpirol-delay and combination-delay models, and cisapride and mosapride (5-HT4 receptor agonists) were effective in the surgery-delay model. Only KDR-5169 eliminated the delay of gastric emptying in all three models. In addition, KDR-5169 accelerated emptying to above the normal level in the combination-delay model. These results suggest that KDR-5169 would be effective in various types of gastric ileus caused by different mechanisms.

Purinergic and adrenergic transmission and their presynaptic modulation in canine isolated perfused splenic arteries

Vasoconstrictions induced by periarterial electrical stimulation were analysed pharmacologically in the canine isolated perfused splenic artery. Phentolamine enhanced the vasoconstrictions at 1 Hz but inhibited those at 10 Hz. Suramin and P2x purinoceptor desensitization with alpha,beta-methylene ATP abolished the phentolamine-enhanced and -resistant vasoconstrictions. alpha,beta-Methylene ATP inhibited the vasoconstrictions at 1 Hz and by exogenous ATP but did not change those at 10 Hz and by exogenous noradrenaline. Suramin reduced the vasoconstrictions by the electrical stimulations and alpha,beta-methylene ATP but did not affect those by exogenous ATP. Prazosin did not affect the vasoconstrictions at 1 Hz but inhibited those at 10 Hz. Rauwolscine enhanced the prazosin-resistant vasoconstrictions. These results suggest that the electrical stimulation at 1 Hz releases purinergic transmitters (ATP or a closely related compound) as a dominant candidate for the vasoconstrictions, and a co-released noradrenaline may inhibit the release of purinergic transmitters through presynaptic alpha 2-adrenoceptors in the canine splenic artery.

Differential effects of ω‐conotoxin GVIA and tetrodotoxin on vasoconstrictions evoked by electrical stimulation and nicotinic receptor stimulation in canine isolated, perfused splenic arteries

1 The effects of ω‐conotoxin GVIA (ω‐CgTX) and tetrodotoxin (TTX) on vasoconstrictions induced by acetylcholine (ACh) and nicotine were investigated and compared with those induced by periarterial electrical stimulation in the isolated and perfused canine splenic arteries. 2 ACh and nicotine at doses of 0.01 to 1 μmol constricted the splenic artery, dose‐dependently. ACh induced consistent responses, but the vasoconstrictor responses to nicotine became significantly smaller with repeated administration of nicotine. 3 Periarterial electrical stimulation produced a vasoconstriction that was abolished by either TTX (30 nmol) or ω‐CgTX (3 nmol), but the vasoconstrictor response to nicotine was not significantly affected by the same doses of TTX and ω‐CgTX. Inhibitions by TTX and ω‐CgTX of ACh‐induced vasoconstrictions were small but statistically significant, showing that the percentage inhibition was less than 15%. TTX and ω‐CgTX did not affect the vasoconstrictor responses to exogenous noradrenaline (NA). 4 ACh did not produce any vasoconstriction in the preparations treated either with α‐adrenoceptor antagonists (10 μm bunazosin and 10 μm midaglizole) or with 30 μm guanethidine. NA‐induced responses were abolished by α‐adrenoceptor antagonists, but not affected by guanethidine treatment. 5 Vascular responses to ACh were completely inhibited by 1 μmol hexamethonium. In the preparations treated with 100 nmol nicotine, ACh did not produce any vasoconstriction. However, the NA‐induced vasoconstriction was affected by neither hexamethonium nor nicotine treatment. 6 Atropine (1 μm) significantly inhibited but did not abolish the vasoconstrictor responses to ACh. The vascular responses to nicotine and NA were also significantly inhibited by atropine treatment. 7 These results indicate that (1) ACh constricts the splenic artery through the activation of presynaptic nicotinic receptors present on the sympathetic nerves; (2) differential effects of TTX and ω‐CgTX on the vascular responses to ACh and nicotine, and to electrical stimulation suggest that the receptor‐operated ion channels are mainly responsible for NA release induced by nicotinic receptor stimulation, but N‐type VOCCs are responsible for that by electrical stimulation; (3) atropine may have an inhibitory action on nicotine‐related responses, in addition to its inhibitory action on NA.

Different contributions of ATP and noradrenaline to neurotransmission in the isolated canine intermediate auricular artery.

Vasoconstrictor responses elicited by periarterial electrical nerve stimulation were analyzed pharmacologically in the canine isolated, perfused intermediate auricular artery. Phentolamine (10 microM) significantly inhibited the vasoconstrictor responses to stimulation at 5 Hz and over but not those to stimulation at frequencies below 5 Hz. Additionally administered alpha, beta-methylene ATP (1 microM) abolished the phentolamine-resistant vasoconstrictions at all frequencies used in this study. In contrast, suramin (100 microM) inhibited the vasoconstrictor responses to stimulation at 5 Hz and below but not those to stimulation at frequencies higher than 5 Hz. Phentolamine abolished the suramin-resistant vasoconstriction at all frequencies. Phentolamine and alpha, beta-methylene ATP selectively abolished the vasoconstrictor responses to exogenous noradrenaline and ATP, respectively. These results show that the co-transmission of noradrenaline and ATP exists at sympathetic nerve terminals in the canine intermediate auricular artery, and that purinergic transmission is mainly involved in the vasoconstrictor responses to low-frequency nerve stimulation.

Responses of isolated and perfused dog coronary arteries to acetylcholine, norepinephrine, KCl, and diltiazem before and after removal of the endothelial cells by saponin

SummaryThe vascular responses to acetylcholine (ACh), norepinephrine (NE), KCl, and diltiazem were examined before and after removal of endothelial cells by an intraluminal bolus injection of saponin (1 mg) in isolated and perfused dog coronary arteries. Without any precontraction, ACh induced a long-lasting vasodilation in small doses (less than 1 µg), and an initial brief vasoconstriction was occasionally accompanied in large doses. These vascular responses to ACh were not significantly affected by the pretreatment with propranolol (5×10−6 mol/l). The endothelial removal by intraluminal saponin was confirmed electron microscopically. After 20–60 min of saponin treatment, the responses to drugs were observed and compared with the control. The ACh-induced vasodilation was significantly attenuated by saponin (P<0.01), but the ACh-induced vasocon-striction was not affected by it. The vasodilation was blocked by atropine. The NE- and KCl-induced vasoconstrictions and diltiazem-induced vasodilation were not affected by saponin treatment. It is suggested that: (1) ACh produced a vasodilation in the nonpreconstricted condition of dog coronary arteries; (2) the vasodilation caused by ACh is mostly endothelium-dependent, which is considered to be mediated by muscarinic receptors; and (3) the vascular responses to NE, KCl, and diltiazem and the vasoconstriction produced by ACh are not influenced by removal of the endothelium in a relatively large epicardial coronary artery of the dog.

Postjunctional α-adrenoceptor subtypes in isolated and perfused canine epicardial coronary arteries

Summary: In isolated and perfused canine coronary arteries pretreated with propranolol (5 x I0−6 M), effects of bunazosin (E 643, a selective α1-antagonist) and DG 5128 (a selective α2-antagonist) on vasoconstrictor responses to phenylephrine, xylazine, clonidine. and norepinephrine (NE) were examined. A stainless steel cannula was inserted into the coronary artery segment (1.1 −2.4 mm OD, 1.5 cm long) and perfused with Krebs-Henseleit solution at a constant flow rate. The perfusion pressures were 60–110 mm Hg. Acetylcholine readily caused a vasodilation in used arterial preparations. Phenylephrine produced a strong vasoconstriction in a dose-dependent manner, but xylazine and clonidine induced slight vasoconstrictions and a vasodilation followed by a slight vasoconstriction in large doses. Phenylephrine-in-duced vasoconstrictions were dose-dependently attenuated by bunazosin but not by DG 5128. Neither bunazosin nor DG 5128 blocked the vascular responses to clonidine and xylazine. NE produced a slight vasoconstriction in the presence of prazosin (10−6 M), and DG 5128 did not influence that vasoconstriction. Diltiazem significantly suppressed KCI-induced vasoconstriction, but bunazosin did not. Removal of endothelium by 1 mg of saponin significantly attenuated acetylcholine (ACh)-induced vasodilation, but did not suppress clonidine-induced vasodilation. It is suggested that only α1-adrenoceptors are involved in the vasoconstrictions induced by the adrenergic agonists in canine epicardial coronary arteries and that the vascular responses to clonidine and xylazine may be direct actions

Binding of KRH-594, an antagonist of the angiotensin II type 1 receptor, to cloned human and rat angiotensin II receptors.

We studied the binding properties of KRH‐594, a new selective antagonist of angiotensin II (AII) type 1 (AT1) receptors, to rat liver membranes and to recombinant AT1 and AT2 receptors. Preincubation of rat liver membranes with KRH‐594 produced maximal inhibition of [125I]‐AII binding when the preincubation time was 1–2 h. Preincubation with KRH‐594 for 2 h decreased the Bmax value and increased the Kd value. For human AT1, human AT2, rat AT1A and rat AT1B receptors, the Ki values for KRH‐594 were 1.24, 9360, 0.67, and 1.02 nm, respectively. The rank order of Ki values for human AT1 receptors was KRH‐594 >> EXP3174 > candesartan=AII. The order of specificities for human AT1 and AT2 receptors was candesartan > EXP3174 > KRH‐594. Although a 2‐h preincubation of human AT2 receptors with KRH‐594 (30 µm) or CGP 42112 (a selective AT2 receptor antagonist; 0.3 nm) inhibited binding of [125I]‐AII, the suppression by KRH‐594 was not significant. These results indicate that KRH‐594 binds potently to AT1 receptors in an insurmountable manner, and that at a very high dose (30 µm) it may also bind to AT2 receptors, but in a surmountable manner.

Heterogeneous Distribution Of β-Adrenoceptors and Muscarinic Receptors In The Sinoatrial Node And Right Atrium Of The Dog

1. The pacemaker site is known to shift away from the sinoatrial (SA) node in response to autonomic stimuli.