Masahisa Asano

Charybdotoxin‐sensitive K+ channels regulate the myogenic tone in the resting state of arteries from spontaneously hypertensive rats

1 To determine the possible role of Ca2+‐activated K+ (KCa) channels in the regulation of resting tone of arteries from spontaneously hypertensive rats (SHR), the effects of agents which interact with these channels on tension and 86Rb efflux were compared in endothelium‐denuded strips of carotid, femoral and mesenteric arteries from SHR and normotensive Wistar‐Kyoto rats (WKY). 2 Strips of carotid, femoral and mesenteric arteries from SHR exhibited a myogenic tone; that is, the resting tone decreased when either the Krebs solution was changed to a 0‐Ca2+ solution or 10−7 m nifedipine was added. 3 The addition of charybdotoxin (ChTX, 10−9−10−7 m), a blocker of large conductance KCa channels, to the resting strips of these arteries produced a concentration‐dependent contraction, which was significantly greater in SHR than in WKY. Relatively low concentrations of tetraethylammonium (0.05–5 mm) produced a concentration‐dependent contraction which was similar to the ChTX‐induced contraction in these strips. 4 The ChTX‐induced contractions in SHR were greatly attenuated by 10−7 m nifedipine and by 3 × 10−6 m cromakalim, a K+ channel opener. Cromakalim alone abolished the myogenic tone in SHR. 5 The addition of apamin (a blocker of small conductance KCa channels, up to 10−6 m), or of glibenclamide (a blocker of ATP‐sensitive K+ channels, up to 5 × 10−6 m), to the resting strips failed to produce a contraction. 6 In resting strips of carotid, femoral and mesenteric arteries preloaded with 86Rb, the basal 86Rb efflux rate constants were significantly greater in SHR than in WKY. The addition of 10−7 m nifedipine to the resting strips decreased the basal 86Rb efflux rate constants only in SHR. 7 The cellular Ca2+ uptake in the resting state of carotid and femoral arteries from SHR was significantly increased when compared to WKY, and this increase in SHR was significantly reduced by 10−7 m nifedipine. 8 These results suggest that the ChTX‐sensitive KCa channels were highly activated to regulate the myogenic tone in the resting state of carotid, femoral and mesenteric arteries from SHR. The increased KCa channel functions in SHR arteries appeared to be secondary to the increased Ca2+ influx via L‐type voltage‐dependent Ca2+ channels in the resting state of these arteries.

Effects of Bay K 8644 and nifedipine on femoral arteries of spontaneously hypertensive rats

1 Vasoconstrictor effects of Bay K 8644 (an agonist known to increase Ca2+ influx through the voltage‐dependent Ca2+ channels) on femoral arteries of 6 week old spontaneously hypertensive rats (SHR) were investigated, and data compared with findings in age‐matched normotensive Wistar‐Kyoto rats (WKY). 2 The addition of Bay K 8644 (1 × 10−10 − 3 × 10−7 M) elicited a dose‐dependent contraction in SHR femoral artery in the absence of any contractile agent. Maximum contraction induced by this agonist was the same as the maximum induced by either K+‐depolarization or α‐adrenoceptor stimulation. 3 Bay K 8644 was less effective in eliciting a contraction in the WKY femoral artery. 4 Increased sensitivity to K+ was also observed in the SHR femoral artery. In contrast, contractions in response to α‐adrenoceptor stimulation were the same in the SHR as those in the WKY. 5 The addition of nifedipine, a Ca2+ channel antagonist, to an unstimulated preparation produced a dose‐dependent relaxation in femoral arteries from SHR, but not from WKY. When the arteries were contracted with 60 mM K+, nifedipine produced similar relaxations in the SHR as those in the WKY, suggesting that the Ca2+ channels in the SHR femoral arteries are more activated than those in the WKY femoral arteries. 6 Contractile responses of SHR femoral arteries to Bay K 8644 were antagonized competitively by nifedipine. 7 Contractile responses to Ca2+ determined in K+‐depolarized strips were also antagonized competitively by nifedipine. However, Schild plot analysis clearly demonstrated a different pA2 value for nifedipine, suggesting that there may be a difference in the state of voltage‐dependent Ca2+ channels in SHR femoral artery between the stimulation with Bay K 8644 and K+‐depolarization.

Evidence for reduced β‐adrenoceptor coupling to adenylate cyclase in femoral arteries from spontaneously hypertensive rats

1 Arterial relaxant responses via β‐adrenoceptors have been demonstrated to be decreased in spontaneously hypertensive rats (SHR) when compared with normotensive Wistar‐Kyoto rats (WKY). To determine which process of the β‐adrenoceptor adenylate cyclase (AC) system is involved in the decreased responsiveness to β‐adrenoceptor stimulation, relaxant responses to forskolin and dibutyryl cyclic AMP (db cyclic AMP) were compared between strips of femoral and mesenteric arteries isolated from 13 week‐old SHR and age‐matched WKY. 2 The relaxant response to either forskolin, an activator of AC, or db cyclic AMP was not significantly different between the SHR and WKY, when the strips of both arteries from both strains were contracted with K+ to an equivalent magnitude (85% of the maximum). 3 Under the same conditions, however, the relaxant response to noradrenaline (NA) via β‐adrenoceptors was significantly decreased in the SHR arteries. 4 When the strips of femoral arteries were contracted with the same concentration of K+, there was a precontraction of greater magnitude in response to the K+ and a decreased relaxation in response to forskolin, db cyclic AMP or NA in the SHR. On the other hand, when the strips of mesenteric arteries were contracted with the same concentration of K+, the precontraction was smaller in magnitude and there was an increased relaxation in the SHR. 5 The relationship between the relaxant responses and the K+‐induced precontractions clearly showed that the ability of forskolin and NA to relax the K+‐contracted strips depends on the magnitude of precontraction. Therefore, a difference in magnitude of precontraction between the two groups may produce a meaningless difference. 6 The relaxant responses to forskolin and NA were significantly potentiated by the addition of isobutyl methylxanthine (IBMX), an inhibitor of cyclic AMP phosphodiesterase. Even in the presence of IBMX, relaxant responses to forskolin were the same for the two strains. The difference in the pD2 value for NA‐induced relaxation between the two strains was the same in the presence and absence of IBMX. 7 The relaxant effect of either nitroprusside or nifedipine, agents which are independent of this system, was not significantly different between the strips from SHR and WKY. These relaxations were not potentiated by IBMX. 8 From these results, it is concluded that the reduced β‐adrenoceptor coupling to AC is mainly involved in the decreased responsiveness to β‐adrenoceptor stimulation. Furthermore, for an accurate comparison to be made, it is necessary to minimize the influence of variations in the magnitude of precontraction on the relaxant responses.

Potent vasoconstrictor actions of cyclopiazonic acid and thapsigargin on femoral arteries from spontaneously hypertensive rats.

The Ca2+ buffering function of sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) was examined. Differences in the effects of cyclopiazonic acid (CPA) and thapsigargin, agents which inhibit the Ca2+‐ATPase of SR, on tension and cellular Ca2+ level were assessed in endothelium‐denuded strips of femoral arteries from 13‐week‐old SHR and normotensive Wistar‐Kyoto rats (WKY). In resting strips preloaded with fura‐PE3, the addition of CPA (10 μm) or thapsigargin (100 nm) caused an elevation of cytosolic Ca2+ level ([Ca2+]i) and a contraction. These responses were significantly greater in SHR than in WKY. The addition of verapamil (3 μm) to the resting strips caused a decrease in resting [Ca2+]i, which was significantly greater in SHR than in WKY. In SHR, but not in WKY, this decrease was accompanied by a relaxation from the resting tone, suggesting the maintenance of myogenic tone in the SHR artery. Verapamil (3 μm) abolished differences between SHR and WKY. The effects of verapamil were much greater on the contraction than on the [Ca2+]i. The resting Ca2+ influx in arteries measured after a 5 min incubation of the artery with 45Ca was not increased by CPA or thapsigargin in either SHR or WKY. The net Ca2+ entry measured after a 30 min incubation of the artery with 45Ca was decreased by CPA or thapsigargin in both SHR and WKY. The resting Ca2+ influx was significantly higher in SHR than in WKY, and was decreased by nifedipine (100 nm) in the SHR artery, but was unchanged in the WKY artery. The resting 45Ca efflux from the artery was increased during the addition of CPA (10 μm). This increase was less in SHR than in WKY. The resting 45Ca efflux was the same in SHR and WKY. These results suggest that (1) the Ca2+ influx via L‐type voltage‐dependent Ca2+ channels (VDCCs) was increased in the resting state of the SHR femoral artery, (2) the greater part of the increased Ca2+ influx was buffered by Ca2+ uptake into the SR and some Ca2+ reached the myofilaments resulting in the maintenance of the myogenic tone, and (3) therefore the functional elimination of SR by CPA or thapsigargin caused a large elevation of [Ca2+]i and a potent contraction in this artery. During this process, the contraction was mainly due to the basal Ca2+ influx via L‐type VDCCs. The present study also showed the existence of a relatively large compartment of [Ca2+]i which does not contribute to the contraction during the addition of CPA or thapsigargin.

Evidence that pinacidil may promote the opening of ATP-sensitive K+ channels yet inhibit the opening of Ca2(+)-activated K+ channels in K(+)-contracted canine mesenteric artery.

1 The effects of cromakalim and pinacidil on contraction and 86Rb efflux were investigated in strips of canine mesenteric artery. 2 Cromakalim and pinacidil relaxed arterial strips precontracted with 20.9 mm K+ with pD2 values of 6.56 and 5.88, respectively. 3 High (above 10 μm) concentrations of pinacidil, but not cromakalim, relaxed arterial strips bathed by a medium containing 65.9 mm K+, and inhibited Ca2+‐induced contractions in strips bathed by a medium containing 80 mm K+. These findings suggested that pinacidil may act as an inhibitor of Ca2+ influx. 4 In arterial strips preloaded with 86Rb, cromakalim and pinacidil increased the basal 86Rb efflux. 5 When the effects of cromakalim and pinacidil on 86Rb efflux were determined in arterial strips contracted with 65.9 mm K+, both drugs increased 86Rb efflux. The increase in 86Rb efflux induced by pinacidil was much smaller than that induced by cromakalim. Under the same conditions, nifedipine decreased 86Rb efflux. 6 After the addition of nifedipine to arterial strips contracted with 65.9 mm K+, pinacidil produced a greater increase in 86Rb efflux than in the absence of nifedipine, whereas the effects of cromakalim were the same for the two conditions. Therefore, the effects of pinacidil on 86Rb efflux may be the resultant of two opposing effects: an increased 86Rb efflux due to the opening of ATP‐sensitive K+ channels, and a decreased efflux due to the closing of Ca2+‐activated K+ channels. 7 In causing relaxation, cromakalim was competitively antagonized by glibenclamide with a pA2 value of 7.16. However, glibenclamide antagonism of pinacidil was not of the simple competitive type, suggesting that inhibition of Ca2+ influx may contribute to the relaxant action of pinacidil. 8 It may be concluded that although the ability of pinacidil to increase 86Rb efflux via ATP‐sensitive K+ channel opening was similar to that of cromakalim, the inhibition of Ca2+ influx by pinacidil may reduce the opening of Ca2+‐activated K+ channels in K+‐contracted arterial strips.

Diminished β-adrenoceptor-mediated relaxation of arteries from spontaneously hypertensive rats before and during development of hypertension

beta-Adrenoceptor agonists and other drugs were studied for their relaxant effects on femoral and mesenteric arterial strips from spontaneously hypertensive rats (SHR). The potency and efficacy of isoproterenol (ISO) in these arteries were decreased in SHR before and during the development of hypertension as compared with age-matched Wistar Kyoto rats (WKY). Reserpine and 6-hydroxydopamine inhibited the development of hypertension but did not alter the reduced ISO-induced relaxation of the arteries. These arteries from prehypertensive SHR (PHSHR) were less sensitive to salbutamol and cyclic AMP and cyclic GMP derivatives than arteries from age-matched WKY. The relaxation response to nitroprusside was less in the femoral but not in the mesenteric arteries from PHSHR than in arteries from age-matched WKY. The relaxation response to papaverine was not diminished in the PHSHR arteries. It was found that the SHR arteries had a reduced responsiveness to the beta-adrenoceptor agonists before the initiation of hypertension and that the diminished relaxation was not specific to the beta-agonists, although there was no generalized defect in vasorelaxation in PHSHR.

MECHANISM OF THE FORMATION OF MEGAMITOCHONDRIA INDUCED BY COPPER-CHELATING AGENTS

Processes of the formation of cuprizone‐induced megamitochondria in mouse liver have been studied in detail by electron microscopy. The earliest change observed was the presence of large intramitochondrial granules. The next stage was the formation of myelin flgures by which mitochondria were apparently connected. The third stage was characterized by megamitochondria connected with each other by their outer membranes. Continuity of mitochondria were further examined by serial sections, and megamitochondria were proved to be connected to each other far more frequently than expected on one plane of section. A model for the mechanism of megamitochondrial formation is proposed based on electron microscopic evidences, involving the fusion of mitochondrial membranes. Possibillty is also discussed that cuprizone‐induced megamitochondria may fuse to one single branching mitochondrion.

Possible mechanism of the potent vasoconstrictor actions of ryanodine on femoral arteries from spontaneously hypertensive rats

1 The Ca2+ buffering function of sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) was examined. Differences in the effects of ryanodine that removes the function of SR, on tension and cellular Ca2+ level were assessed in endothelium‐denuded strips of femoral arteries from 13‐week‐old SHR and normotensive Wistar‐Kyoto rats (WKY). 2 The addition of ryanodine to the resting strips caused a concentration‐dependent contraction in SHR. This contraction was extremely small in WKY. In the presence of 10−5 m ryanodine, caffeine (20 mM) failed to cause a further contraction in SHR, but it caused a small contraction in WKY. After washout of the strips with a Krebs solution, the resting tone was greatly elevated in SHR when compared with WKY. 3 The elevated resting tone in SHR strips was abolished by 10−7 m nifedipine. The ryanodine‐induced contraction was also abolished by 10−7 m nifedipine. Nifedipine itself caused a relaxation from the resting tone of SHR strips, suggesting the maintenance of myogenic tone. 4 In strips preloaded with fura‐PE3, the addition of 10−5 m ryanodine caused a large and moderate elevation of cytosolic Ca2+ level ([Ca2+]i) in SHR and WKY, respectively. After washout, the resting [Ca2+]i was greatly elevated in SHR. The ryanodine‐induced elevation of [Ca2+]i was decreased by 5 × 10−6 m verapamil in SHR. Verapamil itself caused a decrease in resting [Ca2+]i which was significantly greater in SHR than in WKY, and caused a relaxation only in SHR. 5 The resting Ca2+ influx in arteries measured by a 5 min incubation with 45Ca was significantly increased in SHR when compared with WKY. The resting Ca2+ influx was not increased by 10−5 m ryanodine in both SHR and WKY. The net cellular Ca2+ uptake in arteries measured by a 30 min incubation with 45Ca was decreased by 10−5 m ryanodine in both strains. 6 The resting Ca2+ influx was decreased by 10−7 m nifedipine in the SHR artery, but it was unchanged in the WKY artery. 7 These results suggest that (1) the Ca2+ influx via L‐type voltage‐dependent Ca2+ channels was increased in the resting state of the SHR femoral artery, (2) the greater part of the increased Ca2+ influx was buffered by Ca2+ uptake into the SR and some Ca2+ reached the myofilaments resulting in the maintenance of the myogenic tone, and (3) therefore the functional removal of SR by ryanodine caused a potent contraction in this artery.

Increased Ca2+ Influx in the Resting State Maintains the Myogenic Tone and Activates Charybdotoxin-Sensitive K+ Channels in Dog Basilar Artery

We examined whether Ca2+ channel function in the resting state alters the resting tone and Ca2+ -activated K+ (KCa) channel function in dog basilar artery: data were compared with findings in the mesenteric artery. Isolated dog basilar artery maintained a myogenic tone; that is, the resting tone decreased when either the Krebs solution was replaced with a Ca2+ -free solution or nifedipine was added. The basal 45Ca influx in the resting state of the basilar artery was significantly increased compared with that in the mesenteric artery, and this increase in the basilar artery was reduced by nifedipine. The addition of charybdotoxin (ChTX), a blocker of large-conductance KCa channels, to the resting strips caused a concentration-dependent contraction in the basilar artery but not in the mesenteric artery. The ChTX-induced contraction in the basilar artery was abolished by nifedipine. In resting strips preloaded with 86Rb, the basal 86Rb efflux rate constant was significantly greater in the basilar artery than in the mesenteric artery. The addition of nifedipine to the resting strips decreased the basal 86Rb efflux rate constant only in the basilar artery. These results suggest that the transmembrane Ca2+ influx via L-type voltage-dependent Ca2+ channels was significantly increased in the resting state of the basilar artery and that the myogenic tone was therefore maintained and the ChTX-sensitive KCa channels were highly activated.

Modification by charybdotoxin and apamin of spontaneous electrical and mechanical activity of the circular smooth muscle of the guinea-pig stomach

1 The effects of charybdotoxin and apamin, putative blockers of Ca2+‐activated K+ channels, on spontaneous electrical and mechanical activity of circular smooth muscle of the guinea‐pig stomach antrum were examined in the presence of 1 μm tetrodotoxin and 1 μm atropine. 2 Both charybdotoxin (> 3 nm) and apamin (> 3 nm) dose‐dependently increased the amplitude of spontaneous contractions without altering their frequency. The maximum effect of charybdotoxin was much greater than that of apamin. Both toxins increased the amplitude of intracellular Ca2+ oscillations measured with fura‐2. 3 When the extracellular Ca2+ concentration was lowered to 1.5 mm or less, apamin did not significantly potentiate the contractions whereas charybdotoxin still potentiated them but with less potency. 4 Charybdotoxin (30 nm) increased the amplitude of spikes and slow waves, and slightly decreased the resting membrane potential. On the other hand, apamin (100 nm) preferentially increased the slow wave amplitude with no effect on the resting membrane potential. 5 These results suggest that both toxins affect the spontaneous contraction by modifying the electrical activity and that charybdotoxin‐sensitive K+ channels and apamin‐sensitive ones are differently involved in the spontaneous electrical activity.