Satoru Sunano

Endothelium-derived relaxing, contracting and hyperpolarizing factors of mesenteric arteries of hypertensive and normotensive rats

Differences in the acetylcholine (ACh)‐induced endothelium‐dependent relaxation and hyperpolarization of the mesenteric arteries of Wistar Kyoto rats (WKY) and stroke‐prone spontaneously hypertensive rats (SHRSP) were studied. Relaxation was impaired in preparations from SHRSP and tendency to reverse the relaxation was observed at high concentrations of ACh in these preparations. Relaxation was partly blocked by NG‐nitro‐L‐arginine (L‐NOARG, 100 μM) and, in the presence of L‐NOARG, tendency to reverse the relaxation was observed in response to higher concentrations of ACh, even in preparations from WKY. The relaxation remaining in the presence of L‐NOARG was also smaller in preparations from SHRSP. The tendency to reverse the relaxation observed at higher concentrations of ACh in preparations from SHRSP or WKY in the presence of L‐NOARG were abolished by indomethacin (10 μM). Elevating the K+ concentration of the incubation medium decreased relaxation in the presence of both indomethacin and L‐NOARG. Relaxation in the presence of L‐NOARG and indomethacin was reduced by the application of both apamin (5 μM) and charybdotoxin (0.1 μM). This suggests that the relaxation induced by ACh is brought about by both endothelium‐derived relaxing factor (EDRF, nitric oxide (NO)) and hyperpolarizing factor (EDHF), which activates Ca2+‐sensitive K+ channels. Electrophysiological measurement revealed that ACh induced endothelium‐dependent hyperpolarization of the smooth muscle of both preparations in the presence of L‐NOARG and indomethacin; the hyperpolarization being smaller in the preparation from SHRSP than that from WKY. These results suggest that the release of both NO and EDHF is reduced in preparations from SHRSP. In addition, indomethacin‐sensitive endothelium‐derived contracting factor (EDCF) is released from both preparations; the release being increased in preparations from SHRSP.

Involvement of α-adrenoceptors in the endothelium-dependent depression of noradrenaline-induced contraction in rat aorta

The involvement of endothelium-derived nitric oxide (NO) in the depressant action of the endothelium on noradrenaline-induced contractions and characterization of the receptor involved in the release of NO were studied using rat aorta. The noradrenaline-induced contraction was significantly potentiated by endothelium removal and in the presence of NG-nitro-L-arginine (L-NNA) or NG-monomethyl-L-arginine (L-NMMA). The contraction induced by phenylephrine was also potentiated in the presence of L-NNA. Clonidine could induce contraction only in endothelium-denuded preparations or in the presence of L-NNA. The potentiating action of L-NNA on noradrenaline-induced contractions could also be observed in the presence of yohimbine or rauwolscine, although dose-response curves were shifted to the right. The depression of noradrenaline-induced contractions observed in the presence of the endothelium was increased by repeated stimulation. The depression was prevented by L-NNA and this effect was reversed by L-arginine. These results indicate the possibility that NO can be released through stimulation of alpha 1- and alpha 2-adrenoceptors on the endothelium and depresses noradrenaline-induced contractions of smooth muscle, although the contribution of the respective adrenoceptors remains to be investigated. The release of NO was increased when the stimulation was applied repeatedly.

Blood pressure and impairment of endothelium-dependent relaxation in spontaneously hypertensive rats

Correlation between hypertension and impairment of endothelium-dependent relaxation was demonstrated using aortae from certain strains of rats with various levels of spontaneous hypertension. It was also observed that the impairment of endothelium-dependent relaxation is the secondary change due to hypertension, and the level and duration of hypertension is the determinant factor of the impairment.

Effects of cyclopiazonic acid and thapsigargin on electromechanical activities and intracellular Ca2+ in smooth muscle of carotid artery of hypertensive rats

1 The effects of cyclopiazonic acid (CPA) and thapsigargin (TG), both of which are known to inhibit sarcoplasmic reticular Ca2+‐ATPase, on the mechanical activities, intracellular Ca2+ level and electrical activities of smooth muscle of the carotid artery of stroke‐prone spontaneously hypertensive rats (SHRSP) and Wistar Kyoto rats (WKY) were compared. 2 Both CPA and TG induced elevation of tension of the smooth muscle, which was composed of a phasic and a tonic component. The level of tension attained, especially the tonic component, was greater in the preparation from SHRSP. 3 The elevation of tension was associated with an increased intracellular Ca2+ level. Both the elevation of tension and the increase in intracellular Ca2+ were diminished by the removal of extracellular Ca2+ or by the application of verapamil. 4 The resting membrane potential of the preparations from SHRSP were depolarized to a greater extent than those from WKY. CPA depolarized the smooth muscle from both SHRSP and WKY, and the final level was also more depolarized in the preparation from SHRSP. 5 These results indicate that the elevation of tension induced by these drugs is mainly due to increased Ca2+ influx through voltage‐dependent Ca2+ channels, and the difference in the action between the preparation from SHRSP and that from WKY can be explained mainly by the changes in the channels. 6 Thus, differences in the action of these drugs on the tension of smooth muscle between preparations from WKY and SHRSP can mainly be explained by the difference in the membrane potential which is related to the difference in voltage‐dependent Ca2+ influx.

TENSION OSCILLATION IN ARTERIES AND ITS ABNORMALITY IN HYPERTENSIVE ANIMALS

1. The mechanisms of oscillatory contraction of arterial smooth muscle in vitro are discussed.

Endothelium-dependent relaxation by α2-adrenoceptor agonists in spontaneously hypertensive rat aorta

Differences in alpha(2)-adrenoceptor-induced relaxation of the aorta between stroke-prone spontaneously hypertensive rats (SHRSP) and control normotensive Wistar Kyoto rats (WKY) were studied. Changes in the tension of ring preparations of the aortas were measured isometrically. Relaxation was observed in the preparations precontracted in the presence of ONO-11113, a thromboxane A(2) analogue. The alpha(2)-agonist clonidine and UK-14304 induced dose-dependent relaxation in both the WKY and SHRSP preparations. The relaxation was impaired in the SHRSP preparation. A modified sandwich experiment showed that the relaxing substance from the SHRSP endothelium was decreased. Acetylcholine (ACh) also induced dose-dependent relaxation, and the relaxation was impaired in the SHRSP preparations. alpha(2)-Agonists induced a greater degree of impairment in the relaxation than did ACh. The relaxation induced by alpha(2)-agonists and by ACh was blocked by N G-nitro-L-arginine (L-NNA). Indomethacin improved the relaxation induced by ACh but not that induced by alpha(2)-agonists in the SHRSP aortas. These results suggest that the impairment of relaxation by alpha(2)-agonists in SHRSP is not caused by the increase in the release of endothelium-derived contracting factor (EDCF) but by the reduction in the release of nitric oxide (NO). Alteration of the alpha(2)-adrenoceptors and/or the intracellular mechanism through which NO is synthesized by stimulation of the alpha(2)-adrenoceptors may be the cause of the reduction in relaxation.

Effects of chronic treatment with SQ29852 on spontaneous smooth muscle tone and endothelium-dependent relaxation in aorta of stroke-prone spontaneously hypertensive rats.

The effects of chronic treatment with SQ29852, an angiotensin-converting enzyme inhibitor, on spontaneous smooth muscle tone and endothelium-dependent relaxation of aorta from stroke-prone spontaneously hypertensive rats (SHRSP) were studied and compared with those of captopril. Endothelium-removed aorta from 16-week-old SHRSP exhibited a high amplitude of spontaneously developed active tension (active tone), whereas no active tone was observed in the preparation from control normotensive Wistar-Kyoto (WKY) rats. Treatment with SQ29852 or captopril at age 5–16 weeks prevented the development of hypertension. No active tone could be detected in the preparation from SQ29852-treated SHRSP. Endothelium-dependent relaxation was markedly reduced in the preparation from non-treated SHRSP compared with WKY rats. Treatment with SQ29852 prevented the impairment of endothelium-dependent relaxation. It was also shown that norepinephrine-induced contraction was markedly depressed in en-dothelium-intact aorta from SQ29852-treated rats. The effects of SQ29852 were more prominent than those of hydralazine when blood pressure was maintained at similar levels. It was suggested that SQ29852 exerts an action on both vascular smooth muscle and endothelium that is mediated by the inhibition of angiotension-converting enzyme in addition to indirect actions of SQ29852 that are brought about by blood pressure lowering.

Effects of NG-nitro-L-arginine on electrical and mechanical responses to stimulation of non-adrenergic, non-cholinergic inhibitory nerves in circular muscle of the rat gastric fundus

The effects of L-nitroarginine (L-NNA), an inhibitor of nitric oxide (NO) synthesis, on non-adrenergic, non-cholinergic inhibitory transmission in the circular smooth muscle of the rat gastric fundus were studied. The relaxation in response to nerve stimulation in the presence of guanethidine and atropine was depressed by the application of L-NNA (10(-4) M), and the depression was reversed by the addition of L-arginine (10(-4) M). The time course of the relaxation was altered from a monophasic to biphasic one, both of which were depressed. Stimulation of non-adrenergic, non-cholinergic nerves initiated inhibitory junctional potentials (i.j.p.). In addition, slow after-hyperpolarization was observed when stimuli were applied repeatedly at frequencies higher than 0.5 Hz. L-NNA reduced the i.j.p. partially and abolished the after-hyperpolarization, indicating the partial involvement of NO in non-adrenergic, non-cholinergic inhibitory transmission. The effects of L-NNA on the nerve-mediated relaxation are thought to be due to changes in the i.j.p. and slow after-hyperpolarization.

Attenuation of intrinsic active tone by endothelium-derived nitric oxide in aortae of spontaneously hypertensive rats with different levels of blood pressure

The influences of endothelium on the basal tone of aortae from various strains of spontaneously hypertensive rats with different blood pressure (SHR, SHRSP, M-SHRSP) were studied. Endothelium-intact preparations of aortae from spontaneously hypertensive rats exhibited spontaneous active tone, which was greater in the order of SHR < SHRSP < M-SHRSP. The active tone of the M-SHRSP preparations was about 40% of high-K(+)-induced contraction, while that of normotensive WKY was less than 5%. The active tone was enhanced by the removal of endothelium. The active tone was sensitive to extracellular Ca2+ and abolished by verapamil. The application of N(G)-monomethyl-L-arginine caused the increase in the active tone which was counteracted by L-arginine. These results indicate that the active tone of smooth muscle increases as the blood pressure of the rat increases, and that endothelium attenuates the active tone by releasing nitric oxide (NO) spontaneously. It was also demonstrated that the attenuating action of endothelium was impaired depending on the blood pressure level.

Caffeine-induced contraction in arteries from stroke-prone spontaneously hypertensive rats

Differences in caffeine-induced contraction in smooth muscle of resistance vessels from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar Kyoto rats (WKY) were investigated by using mesenteric artery preparations. The contraction induced by caffeine (10 mM) was greater in SHRSP preparations, both in the presence and absence of Ca (10 min after Ca removal). Caffeine-induced contraction was gradually decreased by the removal of extracellular Ca. No significant difference was observed in the time course of the decay of the contraction between SHRSP and WKY preparations, and the contraction disappeared when the time in Ca-free solution exceeded 80 min. The contraction induced by high-K-Tyrodes solution was completely abolished within 10 min after Ca removal, both in SHRSP and WKY preparations. Caffeine-induced contraction could be blocked by procaine or ryanodine. The results suggest that caffeine induces contraction by releasing Ca from sarcoplasmic reticulum, and that the release of Ca is greater in SHRSP vascular smooth muscle. It is also suggested that sarcoplasmic reticulum is leaky for stored Ca when extracellular Ca is removed, and that the rate of leakage does not differ between smooth muscle cells of SHRSP and WKY mesenteric arteries.