Abnormalities of sarcoplasmic reticulum Ca2+ mobilization in aortic smooth muscle cells from streptozotocin-induced diabetic rats

1. Previously, we found that contractions in response to receptor-dependent (i.e. a(1)-adrenoceptor agonist phenylephrine) and -independent (i.e. cyclopiazonic acid) stimuli are decreased in rat aorta during late diabetes. The aim of the present study was to further investigate the changes of intracellular Ca(2+) homeostasis in diabetic aortic smooth muscle cells. Functional changes of inositol 1,4,5-trisphosphate (IP(3))- and ryanodine-sensitive Ca(2+) stores of the sarcoplasmic reticulum (SR) were evaluated using Fluo-3 acetoxymethyl ester fluorescence, western blot and organ bath techniques. 2. In aortic smooth muscle cells from diabetic rats, the Ca(2+) release and Ca(2+) influx caused by both 10 mmol/L phenylephrine (depletion of IP(3)-sensitive Ca(2+) stores) and 1 mmol/L ryanodine (depletion of ryanodine-sensitive Ca(2+) stores) were both significantly decreased compared with control. Moreover, protein expression levels of IP(3) (260 kDa) and ryanodine receptors (500 kDa) were reduced by 31.8 +/- 7.7 and 69.2 +/- 8.4%, respectively, in aortas from diabetic rats compared with those from control rats. 3. In diabetic rat aorta, phenylephrine-induced contractility was decreased to approximately two-thirds of that in controls, whereas ryanodine alone did not cause obvious contraction in aortas from either control or diabetic rats. 4. The present results suggest that the hyporeactivity of aortic smooth muscle to vasoconstrictors in diabetes results mainly from changes to the IP(3)-sensitive Ca(2+) release pathway. The SR Ca(2+) signalling pathway plays a crucial role in the development of diabetic vascular complications.     

Palytoxin-induced increase in endothelial Ca2+ concentration in the rabbit aortic valve

Palytoxin (PTX) is one of the most potent toxins isolated from marine coelenterates of the genus Palythoa. It induces depolarization in various types of cells by increasing the permeability for monovalent cations. It has been reported that PTX induces endothelium-dependent relaxation of vascular smooth muscle. In this study, we examined the effect of PTX on the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in the endothelium of rabbit aortic valves loaded with fluorescent Ca²⁺ indicators, fura-PE3 or fluo-3. PTX (10pM-300nM) irreversibly increased endothelial [Ca²⁺]_i in a concentration-dependent manner. ATP and thapsigargin also increased [Ca²⁺]_i. Imaging of [Ca²⁺]_i with a confocal microscope revealed that PTX increased [Ca²⁺]_i in all endothelial cells studied (n=13). An inorganic Ca²⁺ entry blocker, La³⁺ (30μM), had no effect on the increase in [Ca²⁺]_i induced by PTX whereas it inhibited the sustained phase of the increase in [Ca²⁺]_i induced by ATP or thapsigargin. The PTX-induced increase in [Ca²⁺]_i was partially inhibited by ouabain and was abolished by removal of external Ca²⁺ although decrease of Na⁺ concentration in the incubation medium was ineffective. Activation of protein kinase C by 1μM 12-deoxyphorbol 13-isobutyrate or inhibition of phosphatase by 10nM calyculin-A had no effect on the increase in [Ca²⁺]_i induced by PTX, whereas both agents inhibited the sustained phase of the increase in [Ca²⁺]_i induced by ATP or thapsigargin. Mn²⁺ influx, measured by the quenching of fura-PE3 fluorescence, was accelerated by ATP or thapsigargin, but not by PTX. These results suggest that PTX increases [Ca²⁺]_i in the endothelium of the rabbit aortic valve by increasing Ca²⁺ influx through a pathway which is different from that activated by ATP or thapsigargin. Received: 28 February 1997     

5-Hydroxytryptamine induces transient Ca influx through Ni-insensitive Ca channels in rat vascular smooth muscle cells

The effects of Ni²⁺, a non-selective cation channel inhibitor, on 5-hydroxytryptamine (5-HT)- and angiotensin II (Ang II)-induced intracellular Ca²⁺ dynamics in rat aortic smooth muscle cells were investigated. Ni²⁺ (1 mM) significantly inhibited the transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by Ang II (100 nM) in aortic smooth muscle cells, as measured using fura-2. However, Ni²⁺ did not suppress the transient increase in Ca²⁺ influx induced by 5-HT (10 μM), while significantly suppressed the sustained increase. Ca²⁺ influx evoked by high KCl (80 mM), thapsigargin (TG) (1 μM) or depletion of intracellular Ca²⁺ store was almost completely suppressed by Ni²⁺. Ni²⁺ had no effect on 5-HT-induced inositol triphosphate production and Ca²⁺ release from the intracellular store(s). These results suggest that 5-HT, but not Ang II, induces transient Ca²⁺ influx through Ni²⁺-insensitive Ca²⁺ channels, which are distinguishable from the voltage-dependent or store-operated Ca²⁺ channels.     

蛋白酪氨酸激酶抑制剂对脑血管平滑肌细胞Ca~(2+)池操纵性Ca~(2+)内流的影响

目的　研究蛋白酪氨酸激酶和蛋白酪氨酸磷酸酶抑制剂对牛脑血管平滑肌细胞 (CSMC)Ca2 + 池操纵性Ca2 + 内流的影响。方法　采用培养的CSMC ,在生物荧光双波长影像分析系统用Fura 2 /Am荧光探针测定单个细胞内游离Ca2 + 浓度。结果　 (1)蛋白酪氨酸激酶抑制剂 (genistein ,2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低内皮素 1(ET 1,10 -7mol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为5 6%± 2 .9%、2 5 6%± 3 9%、48 9%± 3 7% ;蛋白酪氨酸磷酸酶抑制剂 (vanadate ,2 ,4,8μmol·L-1)能浓度依赖性升高CPA刺激引起的CSMCCa2 + 内流 ,增加比率分别为8 2 %± 3 9%、18 8%± 4 9%、46 6%± 6 9% ;(2 ) genistein(2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低ATP(10 μmol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为 6 7%±2 6%、2 4 6%± 6 5 %、5 1 3 %± 6 9% ;vanadate (2 ,4,8μmol·L-1)能浓度依赖性升高ATP刺激引起的CSMCCa2 +内流 ,增加比率分别为 4 8%± 2 0 %、2 8 5 %± 4 6%、49 6%± 3 3 % ;(3 ) genistein (2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低环匹阿尼酸 (Cyclopiazonicacid ,CPA ,10 μmol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为 6 5 %± 3 0 %、2 2 5 %± 5 2 %、     

Cl~-通道阻断剂对5-HT和CPA引起的兔脑椎基底动脉平滑肌细胞Ca~(2+)内流的影响

目的　在培养的兔脑椎基底动脉平滑肌细胞上观察5 HT和CPA诱导的Ca2 + 内流的特性 ,电压依赖性Ca2 + 通道 (VDC)抑制药尼莫地平 ,非电压依赖性Ca2 + 通道抑制药SK&F963 65及Cl-通道阻断剂DIDS、NPPB对两种激动剂引起 [Ca2 + ]i 反应的影响 ,以探讨脑血管平滑肌细胞中 5 HT引起Ca2 + 内流的特性、Cl-通道与Ca2 + 内流的关系。方法　采用生物荧光双波长影像分析系统瞬即测定单细胞胞质[Ca2 + ]i 技术。结果　① 5 HT和CPA均能诱导平滑肌细胞[Ca2 + ]i 呈双相升高 ,并且 5 HT诱导的Ca2 + 释放是环匹阿尼酸 (CPA)敏感Ca2 + 池的一部分 ;②尼莫地平对 5 HT和CPA触发的Ca2 + 内流无明显影响 ,而SK&F963 65可阻止二者触发的Ca2 + 内流 ;③Cl-通道阻断剂DIDS、NPPB呈浓度依赖性抑制Ca2 + 内流 ,在SK&F963 65最大限度抑制Ca2 + 内流后 ,DIDS、NPPB可进一步抑制Ca2 + 内流 ;而Ca2 +内流被DIDS、NPPB分别最大抑制后 ,SK&F963 65也可进一步抑制Ca2 + 内流。结论　 5 HT引起的Ca2 + 内流是经SK&F963 65敏感的非VDC ,其中包含Ca2 + 释放引起的Ca2 + 内流 (CRAC)成分与非CRAC成分 ,并且这两部分Ca2 +内流均与DIDS、NPPB敏感的Cl-通道开放有关     

血管平滑肌和内皮细胞Ca2+内流机制及其与Cl-通道的关系

血管平滑肌和内皮细胞的Ｃａ２＋内流机制不同,前者是兴奋性细胞,Ｃａ２＋内流通过电压依赖性（ＶＤＣ）和非电压依赖性Ｃａ２＋通道;后者是非兴奋性细胞,Ｃａ２＋内流主要通过非ＶＤＣ途径。Ｃｌ－通道参与了这两种细胞的Ｃａ２＋调控,平滑肌细胞Ｃｌ－通道开放导致细胞膜去极化,促进ＶＤＣ开放,Ｃａ２＋内流增加;而内皮细胞Ｃｌ－通道开放导致细胞膜超极化,使Ｃａ２＋进入细胞内的电化学趋势增加,胞外Ｃａ２＋经非ＶＤＣ途径内流增加。目前对血管平滑肌和内皮细胞Ｃｌ－通道的分型、特性和功能还不清楚。     

Hypertonic saline inhibits airway smooth muscle contraction by inhibiting Ca2+ sensitization

The effects of hypertonic solution on airway smooth muscle (ASM) contraction and the underlying mechanisms are largely unknown. We found that hypertonic saline (HS) inhibited acetylcholine (ACh)‐induced contraction of ASM from the mouse trachea and human bronchi. In single mouse ASM cells (ASMCs), ACh induced an increase in intracellular Ca²⁺ that was further enhanced by 5% NaCl, indicating that the HS‐induced inhibition of ASM contraction was not mediated by a decrease in cytosolic Ca²⁺. The Rho‐associated kinase (ROCK) inhibitor Y‐27632 relaxed ACh‐induced precontraction of mouse tracheal rings. However, such inhibition was not observed after the relaxation induced by 5% NaCl. Moreover, the incubation of mouse tracheal rings with 5% NaCl decreased ACh‐induced phosphorylation of myosin light chain 20 and myosin phosphatase target subunit 1. These data indicate that HS inhibits the contraction of ASM by inhibiting Ca²⁺ sensitization, not by decreasing intracellular Ca²⁺.     

Effects of ketamine on voltage-dependent Ca2+ currents in single smooth muscle cells from rabbit portal vein.

The effects of ketamine on membrane potentials and long-lasting type (L-type) voltage-dependent Ca2+ currents were investigated in dispersed single smooth muscle cells from rabbit portal veins. The amplitude and duration of the action potentials evoked by intracellular stimulation were inhibited by ketamine and were completely blocked by 10(-3) mol/l ketamine. Ketamine, however, did not alter the resting membrane potential. Whole cell voltage clamp experiments revealed that ketamine at concentrations higher than 10(-4) mol/l reduced the peak L-type voltage-dependent Ca2+ currents. These effects of ketamine were concentration-dependent and reversible at concentrations from 10(-4) to 10(-3) mol/l. Moreover, the activation threshold of L-type Ca2+ current (approximately -30 mV) was slightly shifted to the positive potential side by ketamine. This effect can explain that the action potential was abolished by 10(-3) mol/l ketamine. It is concluded that relaxation of the vascular smooth muscle by ketamine may have to be attributed to the inhibition of L-type voltage-dependent Ca2+ current.     

A target K+ channel for the LP-805-induced hyperpolarization in smooth muscle cells of the rabbit portal vein

Summary The resting membrane potential of smooth muscle cells of the rabbit portal vein was –51.2 mV. LP-805 (8-tert-butyl-6,7-dihydropyrrolo[3,2-e] 5-methylpyrazolo [1,5-a] pyrimidine-3-carbonitrile) hyperpolarized the membrane to –62.3 mV (10 M) and inhibited the burst spike discharges as measured using the microelectrode method. In dispersed smooth muscle cells, LP-805 (10 M) generated an outward-current with a maximum amplitude of 68 pA at a holding potential of –40 mV in experiments using the voltage-clamp procedure. The reversal potential of the outward current evoked by LP-805 was –82 mV and this value was close to the equilibrium potential for K⁺ (–80 mV) in the present ionic conditions, suggesting that LP-805 activated the K⁺ channel. Generation of both the hyperpolarization and the outward c urrent by LP-805 was inhibited by glibenclamide ( 1 M). Using the cell-attached and cell-free patch-clamp (in the presence of GDP) procedures, the maxi-K⁺ channel current (150 pS) could be recorded in the absence of LP-805; application of LP-805 additionally opened a small conductance K⁺ channel current (15 pS) without change in the activity of the maxi-K⁺ channel. The maxi-K⁺ channel was sensitive to charybdotoxin (0.1 M) and to intracellular Ca²⁺ ([Ca²⁺]_i) concentration. The 15 pS channel was insensitive to [Ca²⁺]_i and charybdotoxin, but sensitive to intracellular ATP concentration. Glibenclamide (> 1 M) inhibited the 15 pS K⁺ channel activated by LP-805. These actions of LP-805 on the maxi-K⁺ and 15 pS K⁺ channels are the same as those previously observed for nicorandil and pinacidil. Thus, LP-805 is a K⁺ channel opener with a chemical structure different from those of the known openers. Correspondence to M. Kamouchi at the above address     

Role of Ca2+ stores in acetylcholine-induced all-or-none shortening of smooth muscle cells from guinea-pig taenia caecum

1. We have reported previously that isolated single smooth muscle cells from guinea-pig taenia caecum respond to acetylcholine (ACh) in an all-or-none manner. 2. To clarify the roles of intracellular Ca(2+) stores in the all-or-none response of isolated smooth muscle cells from guinea-pig taenia caecum to ACh, we examined the inositol 1,4,5-trisphosphate (IP(3))-induced contractile response in Staphylococcus aureus alpha-toxin-permeabilized smooth muscle cells and the effect of depletion of intracellular Ca(2+) stores on the all-or-none response to ACh in intact smooth muscle cells. 3. alpha-Toxin-permeabilized smooth muscle cells responded to 3-30 nmol/L or 0.3-3 nmol/L IP(3) in the presence of 0.2 micromol/L Ca(2+) with 1 mmol/L EGTA or 0.1 mmol/L EGTA, respectively, in an all-or-none manner. These results suggest that Ca(2+) release induced by IP(3) is Ca(2+) dependent and is evoked in an all-or-none manner. 4. In the presence of the Ca(2+) ionophore A23187 (0.1 micromol/L) or the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid (1 micromol/L), the shortening of intact smooth muscle cells induced by increasing concentrations of ACh showed a graded response, but not an all-or-none response. 5. In conclusion, the results suggest that Ca(2+) release from Ca(2+) stores induced by IP(3) plays an important role in the all-or-none response of intact smooth muscle cells to ACh.     

羟苯氨酮激活家兔血管平滑肌细胞钙敏感钾通道

目的研究羟苯氨酮(oxyphenamone,Oxy)扩张血管作用机理。方法用全细胞膜片钳技术,监测家兔肠系膜阻力血管平滑肌细胞钙敏感钾通道电流变化以及Oxy对其的影响。结果0.1 μmol·L^-1 Oxy明显增加钙敏感钾通道电流,冲洗后恢复至给药前水平;0.01～10 μmol·L^-1 Oxy明显增加钙敏感钾通道电流,且呈现浓度依赖性。结论 Oxy呈浓度依赖性和可逆性的增大血管平滑肌细胞钙敏感钾通道电流。     

Ca2+ channel blocking effect of iso-S-petasin in rat aortic smooth muscle cells

The purpose of the present study was to examine the mechanisms underlying the putative hypotensive actions of iso-S-petasin, a sesquiterpene extract of Petasites formosanus through both in vivo and in vitro experiments. Intravenous administration of iso-S-petasin elicited dose-dependent (0.1-1.5 mg/kg) hypotensive and bradycardiac responses in anesthetized rats. Isometric tension recording in isolated thoracic aorta revealed that iso-S-petasin (0.01-100 microM) inhibited KCl- or Bay K 8644 (1,4-dihydro-2,6-dimethyl-5-nitro-4-[2'-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid methyl ester)-induced vasoconstriction independent of endothelium. Iso-S-Petasin also attenuated Ca(2+)-induced vasoconstriction in a concentration-dependent manner in Ca(2+)-depleted/high K(+)-depolarized ring segments, indicating that iso-S-petasin inhibited Ca(2+) influx into vascular smooth muscle cells. This was confirmed by whole-cell patch-clamp recording in cultured vascular smooth muscle cells where iso-S-petasin (10-100 microM) appeared to directly inhibit the L-type voltage-dependent Ca(2+) channel (VDCC) activity. Intracellular Ca(2+) concentration ([Ca(2+)](i)) measurements using the fluorescent probe fura-2/AM (1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-amino-5'-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid pentaacetoxymethyl ester) showed suppression of the KCl-stimulated increase in [Ca(2+)](i) by iso-S-petasin (10, 100 microM). In conclusion, these results suggest that Ca(2+) antagonism of the L-type VDCC in vascular smooth muscle cells might largely account for the hypotensive action of iso-S-petasin.     

Ca~(2+)经钙激活非选择性阳离子通道进入ECV304内皮细胞

目的　研究钙离子进入ECV30 4内皮细胞株的途径和血管紧张素Ⅱ (AⅡ )对钙内流的影响。方法　用膜片钳的细胞贴附式和全细胞方式记录ECV30 4内皮细胞的通道活动。结果　 (1 )在记录单通道电流时电极液含 1 2 0mmol·L- 1 CaCl2 ,细胞浴液不含K+ 、Na+ 时 ,Ca2 + 经非选择性阳离子通道 (CAN)内流的电导为γ0 =(1 2 90± 2 1 1 ) pS(n =4)。1× 1 0 - 7mol·L- 1 AⅡ可显著增强通道电流幅度和延长通道开放时 ,其电导增大为γ1 =(2 2 1 8± 2 2 9)pS(n =4)。全细胞记录得到的结果与单通道的一致。 (2 )用全细胞方式记录到ECV30 4内皮细胞的电压依赖性钙通道电流 ,记录到该峰值电流为 (2 9 32± 3 56)pA(n =4) ,2 0 μmol·L- 1 nifedepine能抑制这个峰值电流 ,被抑制后的电流峰值为 (6 0 0± 3 94)pA(n =4)。 2 μmol·L- 1 BayK8644能显著激活通道活动。结论　Ca2 + 经CAN进入ECV30 4细胞 ,AⅡ可显著增强CAN的钙流     

Effects of [Na] and [Ca] on the responses to milrinone in rat cardiac preparations

In the present investigation we have studied the influence of changing the [Ca²⁺] and [Na⁺] on the cardiac responses to milrinone in various preparations of rat heart. Milrinone (5 × 10⁻⁵ to 8 × 10⁻⁴ M) produced a dose-dependent positive chronotropic effect on right atrium and a positive inotropic effect on left atrium and papillary muscle of the rat. A decrease in [Ca²⁺] (from 2.2 to 1.1 mM) or an increase in [Na⁺] (from 120 to 60 mM) increased the milrinone-induced inotropic effect in left atrium and papillary muscle. However, in right atrium the chronotropic effect of milrinone was significantly decreased under these conditions. Opposite changes to milrinone-induced responses were observed when [Ca²⁺] was increased (to 3.3 mM) or when the [Na⁺] was decreased to 60 mM. Nifedipine (3 × 10⁻³ M), a selective Ca²⁺ channel blocker, significantly inhibited the chronotropic response to milrinone in right atrium. However, the inotropic response to milrinone was found to be significantly greater in the presence of nifedipine. A veratridine-induced positive inotropic effect in the left atrium was also significantly increased in the presence of nifedipine. Tetrodotoxin (TTX, 1 × 10⁻⁶ M), a fast sodium channel blocker, significantly reduced the inotropic response to milrinone in left atrium and papillary muscle. A milrinone-induced dose-dependent increase in the baseline tension was observed in the right atrium which was abolished in low [Ca²⁺] and significantly increased in high [Ca²⁺]. Our data suggest the possibility that milrinone increases Ca²⁺ influx in the right atrium to cause the chronotropic effect. Milrinone also may possess an action like veratridine, involving an increased influx of Na⁺ through fast Na⁺ channels in left atrium and papillary muscle, and this action is possibly involved in the positive inotropic effect.     

A K+ single channel and whole-cell clamp study on the effects of levcromakalim in guinea pig portal vein cells

Single channel cell-attached patch and whole-cell clamp experiments on the mode of action of the K⁺ channel opener (KCO), levcromakalim, were performed in guinea pig isolated portal vein cells. At +20 mV (135/23 mM K⁺ in bath/pipette), 10 μM levcromakalim activated K⁺ channels with a chord conductance of 23.2 pS (K_KCO), which were sensitive to the blocker of ATP-dependent K⁺ channels (K_ATP), glibenclamide. Voltage steps from –80 mV to +20 mV activated 4-aminopyridine-sensitive K⁺ channels of 6.5 pS with properties of delayed rectifier K⁺ channels (K_v). In patches which upon a previous voltage step had revealed the existence of K_v, levcromakalim reduced the open-probability of K_v, but it did not concomitantly activate K_KCO. During the course of the experiments, but unrelated to the presence of levcromakalim, large conductance K⁺ channels (BK_Ca) appeared which could be inhibited by iberiotoxin, a selective blocker of BK_Ca, and by the membrane-permeant calcium buffer, BAPTA/AM, but not by glibenclamide. Whole-cell current-voltage (i-V) relations were established in response to voltage ramps from +50 mV to –100 mV; on subtraction of control i-V curves from i-V curves obtained in the presence of 10 μM levcromakalim, the KCO-induced K⁺ current remained which was proportional to voltage. This is not compatible with the upward-bent curvature predicted by the GHK current equation for purely resistive channels at high [K⁺]_i versus low [K⁺]_o. In conclusion, in the guinea pig portal vein cells, no evidence could be established for the hypotheses that KCOs may act via conversion of K_v to K_ATP (Beech and Bolton 1989; Edwards et al. 1993) or by activation of BK_Ca (Balwierczak et al. 1995). In these cells, mild inward rectification of the levcromakalim-induced current was observed which underlines their relationship to K_ATP in other tissues. Received: 1 August 1997 / Accepted: 7 June 1998     

Effects of the Na ionophore monensin on the contractile response and the movements of monovalent cations in the vascular smooth muscle of rabbit aorta

Summary The effects of monensin, a Na ionophore, on the muscle contraction and the movements of monovalent cations were investigated in rabbit aorta. Experiments were conducted in the presence of phentolamine (10^–6 M) to avoid the vasoactive effect of monensin due to the release of endogenous catecholamine. Both monensin (2×10^–5 M) and ouabain (2×10^–5 M), added separately, produced a small and slowly developing contraction, whereas simultaneous application of these agents produced more rapid and greater contraction. Verapamil (10^–6 M) decreased the contraction by 75%. Ouabain gradually increased cellular Na content. Monensin augmented the ouabain-induced Na increase. Further, the loss of cellular Na into Na deficient solution was enhanced by monensin. Low temperature (0.5°C) inhibited the monensin-induced increase in Na permeability. The relaxation of noradrenaline-contraction induced by a K-readmission was inhibited by ouabain but not by monensin. These results suggest that monensin increases Na movement down its electrochemical gradient, augments cellular Na accumulation when the Na pump is inhibited by ouabain, and induces muscle contraction, and that the contraction induced by monensin and ouabain is mainly due to an increased Ca influx through voltage sensitive Ca channels.     

Pharmacological properties of contraction caused by sodium removal in muscle strips isolated from canine coronary artery

Contractions produced by Na⁺ removal were studied in muscle strips isolated from canine coronary artery. In the presence of 20 mM K⁺ and 0.5 mM Ca⁺, rapid contractions were observed repeatedly on complete replacement of NaCl with sucrose. This contraction in the absence of Na⁺ (0-Na) was not affected by phentolamine but was strongly inhibited by verapamil. Ouabain slowly potentiated the 0-Na contraction and markedly reduced the inhibition due to verapamil. The 0-Na contraction was dependent on external Ca⁺ both with and without ouabain. Bepridil had effects very similar to those of verapamil. Amiloride and excess Mg²⁺ reduced the 0-Na contraction and the degree of their inhibition was similar after ouabain treatment. The decrease in verapamil susceptibility could suggest that the 0-Na contraction has verapamil-sensitive and -insensitive components. The former is probably due to Ca²⁺ influx through voltage-dependent channels and the latter to Ca²⁺ influx through an Na⁺-Ca²⁺ exchange process. Ouabain is considered to increase the contribution of Na⁺-Ca²⁺ exchange to the 0-Na contraction. Mg²⁺ may inhibit both verapamil-sensitive and -insensitive pathways. Amiloride probably exerts its inhibitory effect on the contractile machinery.     

Differential development of carbachol-induced desensitization in receptor-mediated Ca2+ influx and Ca2+ release pathways in smooth muscle of guinea-pig taenia caeci

1. We have found that development of carbachol (CCh)-induced desensitization to receptor agonists, but not to receptor by-passed stimulation, is transiently interrupted by a Ca2+-dependent resensitization during the early stage in the smooth muscle of guinea-pig taenia caeci. To further characterize the receptor-mediated signal transduction pathways involved in this peculiar desensitization process, we examined the desensitization processes during Ca2+ influx- and Ca2+ release-mediated contractions in response to activation of muscarinic receptors or histamine H1 receptors. 2. Desensitization treatment with 10(-4) mol/L CCh for 30 min in the presence of extracellular Ca2+ resulted in desensitization to the muscarinic agonists McN-A-343 or AHR-602, which are known to induce contraction only in the presence of extracellular Ca2+ in taenia caeci. The development of desensitization to these agonists was interrupted by a transient resensitization at 1 min. In contrast, the transient resensitization phase was lost following removal of extracellular Ca2+ during the desensitization treatment with CCh; under these conditions, the desensitization developed gradually without an apparent resensitization phase. 3. Contractions to 10(-4) mol/L CCh and 10(-4) mol/L histamine in the absence of extracellular Ca2+ were gradually desensitized without a resensitization phase following the CCh desensitization treatment, irrespective of the presence or absence of extracellular Ca2+ during CCh treatment, although the onset of the desensitization was delayed under Ca2+-free conditions. 4. These results suggest that the receptor-mediated Ca2+ influx and Ca2+ release pathways are differentially desensitized to CCh and that the transient resensitization appears to regulate the desensitization process in response to Ca2+ influx-mediated contraction. Such differential processes of desensitization in receptor-mediated bifurcated signalling pathways may determine cellular responsiveness to certain types of stimuli, depending on the different Ca2+ sources required for contraction.     

Diverse effects of monensin on capacitative Ca2+ entry and release of stored Ca2+ in vascular smooth muscle cells

The effects of monensin, an activator of Na(+)/H(+) exchanger (NHE), on capacitative Ca(2+) entry (CCE) were investigated using A7r5 cells. Capacitative Ca(2+) entry was induced by elevation of extracellular Ca(2+) concentrations of A7r5 cells in which stored Ca(2+) had been depleted by previous administration of thapsigargin. Capacitative Ca(2+) entry was abolished by pretreatment of the cells with SKF-96365 (1-[beta-(3-[4-methoxyphenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride) but was not affected by pretreatment with verapamil. Monensin significantly increased capacitative Ca(2+) entry. On the other hand, 5-hydroxytryptamine-induced inositol monophosphate accumulation and subsequent intracellular Ca(2+) release from its stores were significantly inhibited by monensin, while thapsigargin-induced Ca(2+) release was not affected by monensin. These results suggest that monensin has diverse actions on capacitative Ca(2+) entry and agonist-induced release of stored Ca(2+) in vascular smooth muscle cells.