Changes in the cytosolic Ca2+ concentration and Ca(2+)-sensitivity of the contractile apparatus during angiotensin II-induced desensitization in the rabbit femoral artery

1. To investigate the underlying mechanism for the angiotensin II-induced desensitization of the contractile response during the prolonged stimulation of the vascular smooth muscle, we determined the effects of angiotensin-II on (1) cytosolic Ca2+ concentration ([Ca2+]i) and tension using fura-2-loaded medial strips of the rabbit femoral artery, (2) 45Ca2+ influx in ring preparations, and (3) Ca(2+)-sensitivity of the contractile apparatus in alpha-toxin permeabilized preparations. 2. In the presence of extracellular Ca2+, high concentrations of angiotensin-II elicited biphasic increases in [Ca2+]i and tension, which consisted of initial transient and subsequent lower and sustained phases. 3. The 45Ca2+ influx initially increased after the application of 10(-6) M angiotensin-II, and thereafter gradually decreased. At 20 min after the application, there was a discrepancy between the level of [Ca2+]i and the extent of 45Ca2+ influx. 4. The relationships between [Ca2+]i and tension suggested that the angiotensin-II-induced increase in the Ca(2+)-sensitivity of the contractile apparatus was maintained during the desensitization of smooth muscle contraction. 5. When 10(-6) M angiotensin-II was applied during the sustained phase of contraction induced by 118 mm K(+)-depolarization, at 10 min after the application, the [Ca2+]i levels were significantly lower and the tension levels were significantly higher than those prior to the application of angiotensin-II. 6. In conclusion, the decrease in [Ca2+]i, which is partially due to the inhibition of the Ca2+ influx, is mainly responsible for the desensitization evoked by high concentrations of angiotensin-II, and angiotensin-II seems to activate additional mechanisms which inhibit Ca2+ signaling during prolonged stimulation.     

Antagonism of Ca2+-induced contractions of K+-depolarized smooth muscle by local anaesthetics

Drugs known to interact with Na+ channels were compared as antagonists of Ca2+-induced contractions of K+-depolarized taenia preparations from guinea-pig caecum. Tetracaine (apparent pA2 5.3 +/- 0.2), quinidine (5.2 +/- 0.1) quinine (5.1 +/- 0.1), d-propranolol (4.7 +/- 0.1), 1-propranolol (4.7 +/- 0.1), lignocaine (4.0 +/- 0.1) and procaine (3.6 +/- 0.1) displaced cumulative concentration-response curves to Ca2+ to the right without depression the maximal response. The slopes of Arunlakshana and Schild plots were close to unity for quinidine, quinine, lignocaine and procaine. These drugs relaxed the established Ca2+-induced contractions rapidly and thus the effects of these drugs resembled the effects of low concentrations of verapamil. However, the effects of the local anaesthetics were increased in the presence of sodium salicylate (5-10 mM) which increases the negative surface charge. In contrast the effects of verapamil were decreased by salicylate. Veratridine (10-100 microM), which activates Na+ channels, had only depressant effects on Ca2+-induced contractions. Thus, drugs acting on Na+ channels can also interact with Ca2+ channels but there are qualitative as well as quantitative differences between the effects of these drugs and those of drugs such as verapamil. These findings indicate different mechanisms of action for the inhibition of Ca2+-induced contractions by local anaesthetics and verapamil.     

Effects of SC-52458, a new nonpeptide angiotensin II receptor antagonist,on increase in cytoplasmic Ca2+ concentrations and contraction induced by angiotensin II and K+-depolarization in guinea-pig taenia coli

• 1.1. The effects of angiotensin (Ang) II receptor antagonist, SC-52458, on peak and plateau components of Ang II-induced contraction were evaluated in the guinea-pig taenia coli.
• 2.2. SC-52458 suppressed both the components of and increases in cytoplasmic Ca²⁺ concentrations, [Ca²⁺]_i, coupled with the contraction by Ang II; tetrodotoxin and atropine did not affect the contractions.
• 3.3. SC-52458 inhibited a plateau component of the contraction induced by K⁺-depolarization to some extent, without affecting a peak component.
• 4.4. SC-52458 suppressed both the contraction and increase of [Ca²⁺]_i by antagonizing AT₁ receptors in the smooth muscle.

     

The effects of a novel vasodilator, LP-805, on cytosolic Ca2+ concentrations and on tension in rabbit isolated femoral arteries.

1. LP-805, 8-tert-butyl-6,7-dihydropyrrolo-[3,2-e]-5-methylpyrazolo- [1,5a]-pyrimidine-3-carbonitrile, is a newly synthesized potent vasodilator. To investigate the cellular mechanisms of vasorelaxation induced by LP-805, we simultaneously determined the effects of LP-805 on cytosolic Ca2+ concentrations ([Ca2+]i) and on tension of smooth muscle of rabbit femoral arterial strips, with or without the endothelium, using front-surface fluorometry and fura-2. 2. In the absence of the endothelium, LP-805, in a concentration-dependent manner, decreased [Ca2+]i and tension during the contraction induced by K(+)-depolarization, at relatively low concentrations ([K+]o < or = 30 mM). The decreases in [Ca2+]i and tension were fully antagonized by treatment with 2 x 10(-6) M glibenclamide. The [Ca2+]i-tension relationship in the LP-805-induced relaxation was similar to that of K(+)-depolarization-induced contractions. 3. LP-805, in a concentration-dependent manner (IC50 for inhibition of tension; 1.7 x 10(-6) M), decreased both [Ca2+]i and tension during the steady-state of contractions induced by 1 x 10(-7) M noradrenaline (NA) in the strips without the endothelium. Glibenclamide completely inhibited these reductions of [Ca2+]i and tension. At the steady-state of relaxation induced by LP-805 during NA-induced contraction, [Ca2+]i-tension relation was shifted to the left of that obtained with high K(+)-induced contraction. 4. NA induced transient increases in [Ca2+]i and tension in the absence of extracellular Ca2+. LP-805 (up to 3 x 10(-6) M) had no effect on these intracellular Ca2+ mobilisation and tension development induced by NA. 5. In strips with an intact endothelium, LP-805 decreased both [Ca2+]i and tension during contraction induced by 1 x 10(-7) M NA. The concentration-response curve for inhibition of [Ca2+]i and tension obtained in the presence of the endothelium was shifted to the left from that obtained in the absence of endothelium. IC50 for the inhibition of tension obtained in the strips with the endothelium was 4.0 x 10(-7) M. Treatment with 1 x 10(-4) M NG-nitro-L-arginine (L-NOARG) attenuated reductions of both [Ca2+]i and tension induced by LP-805 and the concentration-response curve shifted to the right and overlapped that obtained in the absence of the endothelium. Treatment with glibenclamide almost fully overcame the reduction of [Ca2+]i induced by LP-805, while the reversion of tension was 50% at most.(ABSTRACT TRUNCATED AT 400 WORDS)     

普鲁托品对兔血小板内钙的影响

AIM: To study the influence of protopine (Pro) on the cytoplasmic free Ca2+ concentration ([Ca2+]i) in rabbit platelets. METHODS: Measurement of [Ca2+]i of platelets in vitro by Fura 2-AM fluorescence technique. RESULTS: In the presence of CaCl2 1 mmol.L-1, Pro 10, 20, and 40 mumol.L-1 attenuated the rise in [Ca2+]i evoked by ADP from (420 +/- 57) to (320 +/- 26), (264 +/- 21), and (180 +/- 14) nmol.L-1, respectively, by arachidonic acid (AA) from (280 +/- 36) to (210 +/- 17), (184 +/- 21), and (143 +/- 16) nmol.L-1, respectively, and by platelet-activating factor (PAF) from (350 +/- 42) to (282 +/- 31), (223 +/- 30), and (165 +/- 15) nmol.L-1, respectively. In the presence of egtazic acid 1 mmol.L-1, Pro 10, 20, and 40 mumol.L-1 reduced the Ca2+ release induced by ADP, AA, and PAF, respectively. Pro 10, 20, and 40 mumol.L-1 also decreased ADP-, AA-, and PAF-induced Ca2+ influx. CONCLUSION: Pro inhibited not only Ca2+ release but also the influx of Ca2+.     

Changes of large conductance Ca2+-activated K+ channel current and cytosolic calcium concentrations in coronary artery smooth muscle cells of rats with diabetes

目的 探讨糖尿病大鼠冠状动脉平滑肌细胞中大电导钙离子激活钾通道(BK通道)电流及钙离子浓度的变化.方法 40只SD大鼠随机均分为正常对照组(A组)和糖尿病组(B组).采用链脲霉素腹腔内注射建立糖尿病大鼠模型,酶消化法分离冠状动脉平滑肌细胞,全细胞膜片钳实验和荧光测定方法分别检测冠状动脉平滑肌细胞BK通道电流和钙离子浓度.结果 与A组相比,当刺激电压大于60 mV时,B组冠状动脉平滑肌细胞BK通道电流密度明显下降(P＜0.05);A组冠状动脉平滑肌细胞内钙离子浓度明显低于B组[(103±23) nmol/L vs.(193±22) nmol/L](P＜0.05).结论 冠状动脉平滑肌细胞中BK通道电流下降及细胞内钙离子浓度升高可能是糖尿病冠状动脉功能损伤的重要原因.     

Zn2+ increases resting cytosolic Ca2+ levels and abolishes capacitative Ca2+ entry induced by ATP in MDCK cells

The effect of Zn2+ on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated by measuring the changes in the fluorescence of the Ca2+-sensitive dye fura-2. Zn2+ significantly increased cytoplasmic free Ca2+ levels ([Ca2+]i) at concentrations of 2-100 microM. The maximum response was obtained at concentrations of 25-100 microM. The [Ca2+]i rise induced by 100 microM Zn2+ consisted of a gradual rise and a plateau phase, and was primarily mediated by La3+-sensitive extracellular Ca2+ influx because the [Ca2+]i rise was abolished by pretreatment with 100 microM La3+ or removal of extracellular Ca2+, and that Zn2+ induced Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength which was prevented by pretreatment with 100 microM La3+. Pretreatment with 100 microM Zn2+ for 220 s did not reduce the [Ca2+]i rise induced by the endoplasmic reticulum (ER) Ca2+ pump inhibitor, thapsigargin, suggesting that Ca2+ release from the ER played a minor role in the Zn2+-induced [Ca2+]i rise. Zn2+ (100 microM) nearly abolished the capacitative Ca2+ entry induced by ATP (100 microM). We also investigated the effect of Zn2+ pretreatment on the [Ca2+]i rise induced by ATP. Zn2+ (100 microM) affected ATP-induced [Ca2+]i rise by abolishing capacitative Ca2+ entry and increasing [Ca2+]i on its own without altering Ca2+ release from intracellular sources. The effect of Zn2+ on [Ca2+]i was dissociated from changes in membrane potential.     

Contractions of rat uterine smooth muscle induced by acetylcholine and angiotensin II in Ca2+-free medium

The effects of acetylcholine (ACh, 10(-4)M) and angiotensin II (Ang II, 10(-6) M) have been studied on the mechanical and electrical activities of rat myometrial strips perfused in Ca2+-free EGTA-containing solutions. Both ACh and Ang II produced transient contractions, the amplitude of which can be taken as a measurement of the amount of Ca2+ present in a drug-sensitive Ca2+ store. The degree of filling of this store depended on the external Ca2+ concentration, and on the presence of contractile responses during the Ca2+ loading period. The existence of two pathways (either direct or transcytoplasmic) is suggested for Ca2+ uptake into the internal Ca2+ store. The rate of filling of the Ca2+ store in 2.1 mM-Ca2+-containing solution was faster (time to half-maximal response, t 1/2 = 29 +/- 2.2 s, n = 4) than the rate of depletion in Ca2+-free solution (t 1/2 = 3 +/- 0.3 min, n = 3). The gradual depletion of this store was much slower at 18 degrees C than at 35 degrees C, and in the presence of vanadate which is known to inhibit Ca2+-ATPases. Methoxyverapamil (D600, 10(-6)-10(-5) M) had no appreciable effect on the direct Ca2+ uptake or on the release of Ca2+ from the store by ACh and Ang II. Mn2+ (10(-3) M) completely inhibited the direct pathway to the internal Ca2+ store and also reduced the release of Ca2+. ACh and Ang II induced repetitive depolarizations close to zero potential which did not parallel the transient contractions as a function of the time of perfusion in Ca2+-free solution. Applications of 2 mM EGTA, 135 mM K+ or Ca2+ antagonists which suppressed or reduced the drug-induced depolarizations did not affect appreciably the drug-induced contractions. These results suggest that myometrial cells have an intracellular Ca2+ store sensitive to different stimulus substances. This store is not affected by depolarization of the plasma membrane and is certainly different from that described in voltage-clamp experiments.     

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 microM) 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.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ influx induced by the agonist U46619 is inhibited by hyperpolarization induced by the K+ channel opener cromakalim in canine coronary artery.

The fura-2 microscopic fluorimetric method was used to examine the effects of the thromboxane A2 analogue, U46619, on the force of contraction and intracellular calcium concentrations ([Ca2+]i) in canine coronary arteries. Upon cumulative application, U46619 increased [Ca2+]i and force. Depolarization by 20 mM KCl potentiated the increase in [Ca2+]i and increased the maximum force induced by U46619. In 5 mM KCl-PSS, the reduction of resting [Ca2+]i by cromakalim (3 x 10(-6) M) was greater than that by verapamil (3 x 10(-6) M). Cromakalim and verapamil inhibited the increases in [Ca2+]i and force induced by U46619 in 5 mM KCl-PSS. In 90 mM KCl-PSS in the presence of U46619, verapamil inhibited the increases in [Ca2+]i and force, whereas cromakalim did not inhibit them at all. The inhibitory effect of cromakalim was counteracted by depolarization by 20 or 25 mM KCl. Curves in the presence of U46619 which related force to [Ca2+]i were shifted to the left compared with that in the absence of U46619, suggesting that U46619 increases the Ca(2+)-sensitivity of the contractile proteins. Thus, U46619 produces Ca2+ influx through L-type Ca2+ channels, which are deactivated by hyperpolarization induced by cromakalim.     

Ca2+ release from isolated sarcoplasmic reticulum of guinea-pig psoas muscle induced by K(+)-channel blockers.

A Ca(2+)-sensitive electrode was used to measure the Ca2+ concentration of the medium containing the heavy fraction of the fragmented sarcoplasmic reticulum (SR) prepared from guinea-pig psoas muscle. Among K(+)-channel blockers tested, 4-aminopyridine (4-AP), tetraethylammonium (TEA) and charybdotoxin elicited Ca2+ release from the SR, but apamin and glibenclamide did not. These results suggest that a reduction of SR K+ conductance leads to Ca2+ release from the SR.     

Species- and temperature-dependency of the decrease in myofilament Ca2+ sensitivity induced by beta-adrenergic stimulation

Although beta-adrenergic stimulation has been shown in many studies to decrease myofilament Ca2+ sensitivity in various types of cardiac muscle such as rat and rabbit ventricles, other studies disagree with this conclusion. In the present study, we aimed to explain these contradictory findings. We examined the effect of beta-adrenoceptor stimulation on Ca2+ sensitivity using guinea pig and rat ventricles. We performed the experiment at two different temperatures and compared the results. In guinea pig ventricles, isoproterenol and forskolin did not alter the relationship between [Ca2+]i and muscle force during the relaxation phase of tetanic contraction at either 24 degrees C or 30 degrees C. In rat ventricles, in contrast, isoproterenol shifted the [Ca2+]i-force curve to the right at 24 degrees C, but not at 30 degrees C. In guinea pig ventricles permeabilized by alpha-toxin, in which the cAMP/PK-A system is intact, the addition of cAMP did not decrease Ca2+ sensitivity. These results suggest that there are species- and temperature-dependent differences in the regulation of myofilament Ca2+ sensitivity by beta-adrenergic stimulation.     

Inhibition of erythrocyte Ca2(+)-pump by Ca2+ antagonists

Inside-out vesicularized membrane fragments from human erythrocytes were prepared to study the effects of various Ca2+ channel entry blockers of plasma membrane Ca2+ transport and (Ca2+ + Mg2+)-ATPase activity concomitantly. Verapamil and diltiazem (0.01 to 5 mM) inhibited both (Ca2+ + Mg2+)-ATPase activity and initial rates of 45Ca2+ net uptake analogously. In general, the parameter affected most by these drugs, using either Ca2+ transport or (Ca2+ + Mg2+)-5'-adenosine-triphospho-hydrolase (EC 3.6.1.3) ([Ca2+ + Mg2+]-ATPase) measurements, was the stimulation by calmodulin. However, the specificity and selectivity of inhibition appeared to be highly concentration and membrane preparation dependent. Verapamil and diltiazem inhibited the calmodulin-Ca2+ transport concentration-effect relationship by changing its apparent affinity as well as the maximal velocity of the process. In a "white ghost" membrane preparation, bepridil inhibited calmodulin activation with a high degree of selectivity as opposed to its effects on calmodulin activation in the vesicular preparation. Nifedipine failed to exhibit any specificity and modestly inhibited basal and calmodulin-activated inside-out vesicular Ca2+ transport and (Ca2+ + Mg2+)-ATPase alike. Our results suggest that verapamil, diltiazem and bepridil (0.01 to 0.3 mM), but not nifedipine (1 nM to 0.01 mM), in relatively high concentrations can antagonize the calmodulin-stimulated Ca2(+)-pump, i.e. the ATPase as well as the transport process. The inhibitors differed with regard to potency, selectivity, and the type of inhibition they produced.     

A Ca2+-activated K+ channel from rabbit aorta: modulation by cromakalim

A large conductance Ca2+-activated K+ channel from rabbit aorta was incorporated into planar lipid bilayers. This channel had a conductance of 337 +/- 7 pS in symmetrical 250 mM KCl solutions and had a Na+/K+ permeability ratio of less than 0.04. In asymmetrical solutions containing 300 mM KCl cis (intracellular), 100 mM KCl trans (extracellular) or 100 mM KCl cis 500 mM KCl trans, the reversal potentials for the channel were -30 and +46 mV, respectively. This channel possessed voltage-dependent activation and cis (intracellular) Ca2+ sensitivity. Cromakalim (50 nM) added to the trans side of the bilayer significantly increased the Popen by 56% from 0.09 +/- 0.01 to 0.14 +/- 0.01 (P less than 0.01) at -40 mV without altering the single channel conductance. This effect was dose-dependent, increasing at higher cromakalim concentrations. The primary effect of cromakalim was to decrease the tau slow of the channel closed state from 266 +/- 32 to 147 +/- 17 ms which is sufficient to account for the increase in Popen of the channel in the presence of cromakalim.     

Effects of cadralazine on contractions induced by norepinephrine, serotonin, angiotensin II and K+ in rabbit aortic and renal arterial strips

Antagonistic effects of the new antihypertensive agent cadralazine (ethyl(+/-)-6-[ethyl(2-hydroxypropyl)-amino]-3-pyridazinecarbazate ) and its metabolite ISF-2405 [+/-)-6-[ethyl(2-hydroxypropyl)amino]-3-hydrazinopyridazine) on norepinephrine (NE), 5-hydroxytryptamine (serotonin), angiotensin II (angio II) and KCl induced contractions of rabbit abdominal aortic and renal arterial strips were compared with those of hydralazine. Substantially, cadralazine does not exert any effect on cumulative dose-response curves of these agonists in both vessel preparations even with the highest concentration of 10(-4) mol/l. ISF-2405 and hydralazine at concentrations of 10(-5) and 10(-6) mol/l showed non-competitive antagonism, depending not only on the dose but also on the length of the pretreatment time, on NE-induced contractions of abdominal aorta and renal artery. The two drugs attained maximal pD2 values with 60 min pretreatment without showing significant difference between the two vessel preparations, suggesting that the inhibitory effect of these drugs does not show vascular bed-related difference against NE-induced contractions. 60 min pretreatment with 10(-6) and 10(-5) mol/l of ISF-2405 and hydralazine also manifested non-competitive antagonism on contractile responses to serotonin, angio II, and K+ for both compounds. The degree of antagonistic effects of ISF-2405 and hydralazine on these agonists is similar, the order being angio II greater than serotonin greater than NE greater than K+. These results suggest that ISF-2405 and hydralazine exert direct vasodilating effects by the same mode of action at a site other than receptors against Ca2+ mobilization.     

Effect of lipoxygenase inhibitors on Ca2(+)-induced constriction of the rabbit ear artery

1. The effects of nafazatrom, nordihydroguaiaretic acid (NDGA) and quercetin on Ca2(+)-induced vasoconstriction were studied in isolated rabbit ear arteries. 2. The arteries were perfused with Ca2(+)-free and high K+ (75 mM) Krebs bicarbonate buffer. Constriction of the artery was induced by addition of Ca2+ (1.5 mM) to the perfusion fluid. 3. Indomethacin (1 microM) did not alter the response to Ca2+. 4. Nafazatrom (2 or 5 microM) produced a concentration-dependent inhibition of the constrictor response to Ca2+ ranging from 4 to 23% after 1 hr of perfusion and 26 to 62% after 3 hr. 5. Similar effects were obtained with NDGA and quercetin (0.5 and 1 microM). 6. The inhibitory effects of nafazatrom and quercetin were antagonized by Ca2+ (2.5 mM) or Bay K 8644 (1 microM), a calcium channel activator. 7. Ca2(+)-induced contractions recovered within 30 min after discontinuation of perfusion with quercetin, whereas nafazatrom and NDGA had longer durations of action. 8. These results suggest that inhibitors of lipoxygenase antagonized Ca2(+)-induced vasoconstriction and that products of lipoxygenase metabolism may facilitate Ca2+ entry into vascular smooth muscle cells.     

Effect of membrane hyperpolarization induced by a K+ channel opener on histamine-induced Ca2+ mobilization in rabbit arterial smooth muscle.

1. The role of membrane hyperpolarization on agonist-induced contraction was investigated in intact and alpha-toxin-skinned smooth muscles of rabbit mesenteric artery by use of the ATP-sensitive K+ channel opener, (-)-(3S,4R)-4-(N-acetyl-N-hydroxyamino)-6-cyano-3,4-dihydro-2,2- dimethyl-2H-1-benzopyran-3-ol (Y-26763), and either histamine (Hist) or noradrenaline (NA). 2. Hist (3 microM) and NA (10 microM) both produced a phasic, followed by a tonic increase in intracellular Ca2+ concentration ([Ca2+]i) and force. Y-26763 (10 microM) potently inhibited the NA-induced phasic and tonic increase in [Ca2+]i and force. In contrast, Y-26763 attenuated the Hist-induced phasic increase in [Ca2+]i and force but had almost no effect on the tonic response. However, ryanodine-treatment of muscles in order to inhibit the function of intracellular Ca2+ storage sites altered the action of Y-26763 which now attenuated the Hist-induced tonic increase in [Ca2+]i and force in a concentration-dependent manner (at concentrations > 1 microM). Glibenclamide (10 microM) attenuated the inhibitory action of Y-26763. 3. Hist (3 microM) depolarized the smooth muscle cells to the same extent as NA (10 microM). In the absence of either agonist, Y-26763 (over 30 nM) hyperpolarized the membrane and glibenclamide inhibited this hyperpolarization. Y-26763 (10 microM) almost abolished the NA-induced membrane depolarization, but only slightly attenuated the Hist-induced membrane depolarization in which the delta (delta) value (the difference before and after application of Hist) was not modified by any concentration of Y-26763. In ryanodine-treated smooth muscle cells, Y-26763 hyperpolarized the membrane and potently inhibited the membrane depolarization induced by Hist. 4. In ryanodine-treated muscle, Y-26763 had no measurable effect on the Hist-induced [Ca2+]i-force relationship. Y-26763 also had no apparent effect on the myofilament Ca(2+)-sensitivity in the presence of Hist in alpha-toxin-skinned smooth muscles. 5. It is concluded that the membrane hyperpolarization induced by Y-26763 may not be enough to inhibit the Hist-activated Ca2+ influx. It is also suggested that Hist prevents the membrane hyperpolarization induced by Y-26763, activating an unknown mechanism which is thought to depend on the function of intracellular Ca2+ storage sites.     

Origin of ATP for Ca2+-induced contraction in the guinea-pig femoral artery

Previously, we have described differences between the rat proximal colon and femoral artery with respect to the role of ATP newly synthesized by creatine kinase. In the present study the role of newly synthesized ATP was studied in the guinea-pig femoral artery to examine species differences. In the -toxin-permeabilized preparation of the guinea-pig femoral artery, the rapid Ca²⁺-induced contraction was suppressed when creatine kinase activity was inhibited. The contraction was restored completely by treatment with NaN₃, an inhibitor of ecto-ATPase, the enzyme that breaks down exogenous ATP. Thus, ATP newly synthesized by creatine kinase may have no role in contraction of the guinea-pig femoral artery. This is in marked contrast to the rat femoral artery, in which Ca²⁺-induced contractions are almost completely inhibited by inhibition of creatine kinase activity but only partly restored by NaN₃. To characterize the difference between the guinea-pig and rat tissue, the origin of ATP required for contraction was determined in intact preparations. Monoiodoacetic acid, an inhibitor of glycolysis, inhibited the high K⁺-induced contraction in the guinea-pig femoral artery more potently than in the rat tissue. In contrast, an inhibitor of mitochondrial respiration, carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), inhibited contraction in femoral arteries from rats, but not from guinea-pigs. These results suggest that contraction in the rat femoral artery is dependent largely on oxidative phosphorylation, while contraction in the guinea-pig tissue is dependent only on glycolysis. Because oxidative phosphorylation generates ATP and phosphocreatine, while glycolysis generates only ATP, the strong dependence of the contraction of the rat femoral artery on the oxidative phosphorylation is consistent with its dependence on ATP newly synthesized by creatine kinase from ADP and phosphocreatine, as previously shown. Thus, it is proposed that ATP, newly synthesized by creatine kinase, in addition to ATP generated by oxidative phosphorylation, is utilized for contraction in the rat femoral artery, while glycolysis produces sufficient ATP for contraction in the guinea-pig femoral artery.