Kaoru Masuzawa

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.

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.

MECHANISM OF THE ENHANCED VASOCONSTRICTOR RESPONSES TO ENDOTHELIN-1 IN CANINE CEREBRAL ARTERIES

Vasoconstrictor effects of endothelin-1 (ET) were investigated in endothelium-denuded strips of cerebral (basilar and posterior cerebral) and mesenteric arteries of the dog. ET produced a concentration-dependent contraction in these arteries. Contractile responses to lower concentrations (below 3 × 10−10 M) of ET were significantly greater in the cerebral arteries than in the mesenteric artery. Inhibition by nifedipine of the contractile responses to ET was greater in the basilar artery than in the mesenteric artery. After the inhibition by 10−7 M nifedipine, the remaining responses to ET were similar in the two arteries. Cerebral arteries, but not the mesenteric artery, relaxed significantly from the resting level when placed in a Ca2+ -free solution containing 0.1 mM EGTA (0-Ca solution). Readdition of Ca2+ to the cerebral arteries placed in the 0-Ca solution caused a biphasic contraction that was sensitive to nifedipine. When 10−9 M ET was introduced before the Ca2+-induced contraction, this peptide produced only a very small contraction, but enhanced the Ca2+-induced contraction. The extent of the enhancement induced by ET was much greater in the cerebral arteries than in the mesenteric artery. These results indicate that the enhanced responses to ET in the cerebral arteries were dependent to a large extent on Ca2+ influx through voltage-dependent Ca2+ channels (VDCs). It is likely that the VDCs in these arteries are more activated in the resting state than those in the mesenteric artery.

Decreased arterial responsiveness to multiple cyclic AMP‐generating receptor agonists in spontaneously hypertensive rats

1 Arterial relaxant responses via β‐adrenoceptors are decreased in spontaneously hypertensive rats (SHR) when compared with normotensive Wistar‐Kyoto rats (WKY). Recent studies from this laboratory proposed that a reduced function of stimulatory guanosine 5′‐triphosphate (GTP)‐binding protein (Gs) is responsible for the decreased β‐adrenoceptor responsiveness in the SHR femoral artery. Since the Gs is common to all tissues, as opposed to receptors, which are tissue specific, the reduced function of Gs should lead to resistance to multiple receptors that act by activating adenylate cyclase (AC). To test this hypothesis, relaxant responses via β‐adrenoceptors, A2‐adenosine, H2‐histamine and D1‐dopamine receptors were compared between arterial strips from 13 week‐old WKY and age‐matched SHR. 2 The relaxant responses to noradrenaline (NA) via β‐adrenoceptors in femoral, mesenteric, renal and carotid arteries were significantly decreased in the SHR, when compared with the respective arteries from WKY. 3 However, under the same conditions arterial relaxant responses to forskolin, an activator of AC, were not significantly different between the WKY and SHR. 4 The relaxant responses due to activation of A2‐adenosine. H2‐histamine and D1‐dopamine receptors were significantly decreased in the SHR arteries. 5 Nitroprusside and nifedipine, agents which are independent of the Gs·AC system, produced similar arterial relaxations in the WKY and SHR. 6 These results support the hypothesis that a reduced function of Gs in the SHR is responsible for the decreased arterial responsiveness to a variety of receptor agonists whose mechanism of action involves AC activation.

Decreased responsiveness to beta-adrenoceptor agonists in arterial strips from spontaneously hypertensive rats is not associated with alterations in beta-adrenoceptors.

Beta-adrenoceptors in femoral and mesenteric arteries from 13-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were studied using radioligand binding assays and were compared with in vitro measurements of beta-adrenoceptor-mediated relaxation. The relaxant responses to noradrenaline via beta-adrenoceptors were significantly decreased in the SHR femoral artery when compared with the WKY femoral artery. However, under the same conditions, arterial relaxant responses to forskolin, an activator of adenylate cyclase, were not significantly different between SHR and WKY rats. Specific binding of 125I-iodocyanopindolol to membranes prepared from femoral arteries of SHR and WKY rats was saturable and of high affinity. Neither the equilibrium dissociation constant of 125I-iodocyanopindolol, nor the maximum number of binding sites were significantly different between SHR and WKY rats. Similar results were obtained in the case of mesenteric arteries from SHR and WKY rats. These results indicate that the decreased responsiveness to beta-adrenoceptor stimulation in SHR arteries is not associated with alterations in beta-adrenoceptors and further support the hypothesis that a reduced function of the stimulatory guanosine triphosphate-binding protein is responsible for the decreased responsiveness to a variety of receptor agonists whose mechanism of action involves adenylate cyclase activation.

Role of stimulatory GTP-binding protein (Gs) in reduced β-adrenoceptor coupling in the femoral artery of spontaneously hypertensive rats

1 Arterial relaxant responses to β‐adrenoceptor agonists are decreased in spontaneously hypertensive rats (SHR) when compared with normotensive Wistar‐Kyoto rats (WKY). To establish which component of the β‐adrenoceptor · adenylate cyclase (AC) system is impaired in the SHR arteries, effects of two activators of AC ‐ cholera toxin (CTX) and forskolin ‐ and of dibutyryl cyclic AMP (db cyclic AMP) were compared between strips of femoral arteries isolated from 13 week‐old SHR and age‐matched WKY. 2 In the absence of timolol, a β‐adrenoceptor antagonist, contractile responses of the strips to noradrenaline (NA) were significantly greater in the SHR than in the WKY. Timolol augmented the contractile responses to NA to a smaller extent in the SHR than in the WKY. 3 After blockade by timolol of β‐adrenoceptors, contractile responses of the strips to NA through the activation of α‐adrenoceptors were not significantly different between the two strains. 4 Pre‐treatment of the strips with CTX, an activator of the stimulatory GTP‐binding protein (Gs), produced a slow‐onset and long‐lived antagonism of the α‐adrenoceptor‐mediated contractions. The antagonism was much smaller in the SHR than in the WKY. 5 The dose‐response curves of the strips from both strains for α‐adrenoceptor stimulation with NA determined after pretreatment with CTX were comparable to those determined in the absence of timolol. 6 Forskolin, an activator of the catalytic subunit of AC, and DB cyclic AMP also antagonized the α‐adrenoceptor‐mediated contractions. However, these antagonisms were not significantly different between the two strains. 7 Isobutyl methylxanthine (IBMX), an inhibitor of cyclic AMP phosphodiesterase, produced a similar antagonism of the α‐adrenoceptor‐mediated contractions between the two strains. 8 These results suggest that a reduced function of Gs is the main factor responsible for the decreased responsiveness to β‐adrenoceptor stimulation in the SHR femoral artery.

The diverse effects of cromakalim on tension and 86Rb efflux in canine arterial smooth muscle.

1 To characterize further the K+ channels opened by cromakalim in arterial smooth muscle, the effects of cromakalim on tension and 86Rb efflux were compared in endothelium‐denuded strips of coronary, mesenteric and middle cerebral (MC) arteries of the dog. 2 Cromakalim relaxed strips precontracted with 20.9 mm K+. The maximum relaxation induced by cromakalim varied in the arteries used; 94% in the coronary artery, 60% in the mesenteric artery and only 38% in the MC artery. Cromakalim failed to relax arterial strips precontracted with 65.9 mm K+. 3 When the effects of cromakalim on 86Rb efflux were determined in 20.9 mm K+‐contracted strips, cromakalim‐induced relaxations were accompanied by a large increase in 86Rb efflux in the coronary artery, by a small increase in the mesenteric artery but by an apparent decrease in the MC artery. 4 When 10−7m nifedipine was added to 20.9 mm K+‐contracted strips, to inactivate Ca2+‐activated K+ (KCa) channels, cromakalim produced a greater increase (measured from the point at which cromakalim was administered) in 86Rb efflux than in the absence of nifedipine, suggesting that the effects of cromakalim on 86Rb efflux from the 20.9 mm K+‐contracted strips may be the resultant of two opposing effects: an increased 86Rb efflux perhaps due to the opening of ATP‐sensitive K+ (KATP) channels, and a decreased efflux due to the closing of KCa channels. 5 After the inactivation of KCa channels in 20.9 mm K+‐contracted strips, the cromakalim‐induced increase in 86Rb efflux measured as area under the curve was eight times greater in the coronary artery than in the MC artery. The increase in 86Rb efflux in the mesenteric artery was intermediate between these extremes. 6 Cromakalim also increased the 86Rb efflux from 65.9 mm K+‐contracted strips. This increase was not augmented by the addition of nifedipine. Under these conditions, a similar variation in efflux response (as area under the curve) for cromakalim was noted in the arteries used. 7 The relaxant responses of each artery to cromakalim were competitively antagonized by glibenclamide, a blocker of KATP channels. The cromakalim‐induced increase in 86Rb efflux was also inhibited by glibenclamide. 8 These results suggest that cromakalim‐opened K+ channels in the three arteries may differ in terms of their frequency of occurrence in the plasmalemma, their permeability to 86Rb and their ability to modulate tension development. On the other hand, the activities of KCa and voltage‐sensitive K+ channels, estimated from the effects of nifedipine, were similar in the three arteries.