褪黑素对谷氨酸所致大鼠神经细胞Ca~(2+)内流的影响

目的 :研究褪黑素对谷氨酸引起的神经细胞Ca2 +内流的抑制作用。方法 :用Ca2 +敏感荧光指示剂Fura -2/AM负载大鼠脑细胞 ,测定神经细胞内游离Ca2 +浓度。结果 :谷氨酸500μmol/L可促进Ca2 +内流 ,显著增加细胞内游离Ca2 +浓度 (P<0. 01) ;应用褪黑素10 -5mol/L后可显著抑制Ca2 +内流 ,降低细胞内游离Ca2 +浓度 (P<0. 01)。结论 :褪黑素可通过抑制谷氨酸引起的Ca2 +内流保护神经细胞。     

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 %、     

酪氨酸激酶抑制剂对hTrp3蛋白介导的α_(1B)-AR引起的Ca~(2+)内流的影响

目的　探讨Trp3(transientreceptorpotential 3)蛋白是否参与α1B AR引起的Ca2 + 内流以及酪氨酸激酶对其调控作用。方法　采用脂质体转染 ,将hTrp3cDNA分别转染到HEK2 93细胞和已有α1B受体稳定表达的HEK2 93细胞 ;Westernblot方法检测Trp3蛋白表达情况 ;Fura 2 /AM荧光分光光度法 ,测定胞浆游离Ca2 + 浓度。结果　HEK2 93细胞上可检测到hTrp3的内源性表达 ,转染后其表达增加。α1B HEK2 93细胞转染hTrp3cDNA后 ,α1B AR引起的Ca2 +内流显著增加 (P <0 0 1) ;转染hTrp3cDNA对thapsigargin诱导的Ca2 + 内流无作用。 5～ 30 μmol·L-1genistein对转染细胞α1B AR诱发的Ca2 + 内流有抑制作用 ,最大抑制率达(75 2± 12 6 ) %。结论　Trp3cDNA转染可能主要通过非CRAC(calciumreleaseactivatedcalcium )途径增加α1B AR引起的Ca2 + 内流 ,这一过程很大程度上依赖酪氨酸激酶的调控     

库容性Ca~(2+)内流介导大鼠远端结肠平滑肌收缩

目的探讨库容性Ca2+内流(capac itative Ca2+entry,CCE)是否参与大鼠远端结肠平滑肌兴奋-收缩偶联过程。方法利用器官离体装置、张力换能器、Powerlab 4/25T数据采集分析系统测定远端结肠平滑肌的张力。结果毒胡萝卜素(thapsigargin,TG,10 nmol.L-1~1μmol.L-1)诱导结肠平滑肌条产生持续的张力性收缩,不同浓度TG所致的同步收缩反应张力不同。在无钙Krebs液(包含1 mmol.L-1EDTA)中使用TG将肌条培养35 m in后,再加入Ca2+2.5mmol.L-1,比未使用TG处理的肌条产生的收缩张力明显提高(99%±28%vs70%±8%)。且TG耗竭胞内钙库后再复钙所致的收缩效应,不受L型钙通道阻断剂verapam il影响,但可被SOC通道阻断剂La3+减弱。结论TG耗竭胞内钙库后再复钙诱导的大鼠远端结肠平滑肌收缩反应由CCE介导,提示CCE是提供大鼠远端结肠平滑肌收缩的激活信号Ca2+的来源之一,参与完成结肠平滑肌兴奋-收缩偶联过程。     

大明胶囊对糖尿病大鼠心肌L型Ca~(2+)通道蛋白mRNA表达的影响

目的探讨大明胶囊对2型糖尿病大鼠心肌L型Ca2+通道蛋白mRNA表达的影响。方法建立2型糖尿病大鼠模型,筛选空腹血糖值大于16.7mmol·L-1的大鼠随机分组:大明胶囊大(200mg·kg-1·d-1)、中(100mg·kg-1·d-1)、小(50mg·kg-1·d-1)剂量组、糖尿病模型组、苯乙双胍(75mg·kg-1·d-1)组,连续用药14d,用快速血糖仪测空腹血糖值。提取心肌总RNA,采用逆转录聚合酶链式反应(RTPCR)的方法,观察大明胶囊治疗后2型糖尿病大鼠心肌L型Ca2+通道蛋白mRNA水平的变化。结果2型糖尿病组大鼠心肌L型Ca2+通道蛋白mRNA表达高于正常组大鼠(P<0.01),经大明胶囊治疗后的心肌L型Ca2+通道蛋白mRNA表达降低(P<0.05),空腹血糖也明显下降。结论大明胶囊对2型糖尿病大鼠有明显的降糖作用,并且可以降低2型糖尿病大鼠心肌L型Ca2+通道蛋白mRNA的表达。     

牛磺酸对大鼠心肌Ca~(2+)调节作用的研究

研究了牛磺酸对大鼠离体左心室肌~(45)Ca内流的影响。对照组~(45)Ca内流与KH液中Ca_-~(2+)浓度有关。当Ca~(2+)浓度分别为0.62(低Ca~(2+)),1.25(正常Ca_-~(2+))和1.87(高Ca_-~(2+))mmol/L时,~(45)Ca内流相应为1.02±0.25,1.37±0.14,和1.45±0.14μmol/g。加入牛磺酸后,上述情况显著改变。低Ca_-~(2+),Tau 10、20、40mmol/L能分别使~(45)Ca内流增加为1.11±0.11,1.45±0.12和1.48±0.09μmol/g:正常Ca~(2+)时,Tau 10、20、40mmol/L能分别使~(45)Ca内流减少为1.19±0.07,1.14±0.23和0.97±0.24μmol/g:高Ca~(2+)时、Tau 10、20、40mmol/L能使~(45)Ca内流显著降低为1.12±0.05,0.58±0.18和0.53±0.10μmol/g。结果表明不同Ca~(2+)浓度能影响心肌~(45)Ca内流,牛磺酸对心肌~(45)Ca内流有双向调节作用,这可能在其抗心律失常机制中起重要作用。     

舒郁胶囊对大鼠海马原代培养神经元Ca~(2+)-CaM通路的作用

目的观察海马神经元内Ca2+浓度和CaM蛋白水平表达变化,探索复方舒郁胶囊治疗抑郁情绪的微观作用机制。方法采用孤养结合慢性温和刺激复制抑郁情绪大鼠模型,以调肝方药舒郁胶囊及其君药柴胡提取物进行干预,氟西汀作为阳性对照药,制备各组大鼠血清;运用原代无血清培养技术培养大鼠海马神经元,以制备的各组大鼠含药血清孵育神经元;采用荧光探针Fluo-3/AM染色和细胞免疫荧光技术,分别测定海马神经元胞内[Ca2+]i和CaM蛋白表达水平。结果与正常组相比,模型血清组细胞内[Ca2+]i和CaM蛋白表达水平明显降低(P<0.05);各含药血清组神经元胞内[Ca2+]i和CaM蛋白水平较模型血清组明显升高(P<0.05),与正常血清组差异无显著性。结论舒郁胶囊调控海马神经元胞内Ca2+-CaM体系的变化可能是其治疗抑郁情绪的作用机制之一。     

牛磺酸对脑挫伤大鼠氧应激与Ca~(2+)超载损伤的影响

目的 :探讨牛磺酸对大鼠脑挫伤导致的氧应激和细胞Ca2 + 超载损伤的影响。方法 :将SD大鼠随机分为正常对照组、脑挫伤模型组、牛磺酸 (高、中、低剂量 )治疗组与尼莫地平组。在给药7d后进行脑挫伤造模 ,造模后2h切取活体脑片以荧光标记法测定胞内Ca2 +水平 ;造模后24h分离大鼠患侧大脑皮层 ,以比色法测定脑匀浆中超氧化物歧化酶 (SOD )活性与丙二醛 (MDA )含量。结果 :与模型组比较 ,牛磺酸各剂量组皮层胞内Ca2 + 、MDA水平显著降低 ,高剂量牛磺酸组皮层SOD水平显著升高。结论 :牛磺酸对大鼠脑挫伤导致的氧应激和细胞Ca2 +超载损伤有较好的保护作用。     

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     

人参皂苷Rg_2对培养心肌细胞内游离Ca~(2+)含量的影响

众所周知 ,Ca2 + 在心肌的舒缩过程中具有调控作用 ,文献[1] 曾研究了人参皂苷Rg2 对休克动物心功能的改善作用 ,该作用是否与Ca2 + 有关 ,故观察了Rg2 对体外培养心肌细胞内游离Ca2 + 含量的影响。1　材料与方法　　药物 :人参皂苷Rg2 (Rg2 )由吉林省中医中药研究院制备 ,纯度为 98 5 % ,Fura 2 /AM和DME/F12培养基购自Sigma公司。动物 :新生 4d的Wistar大鼠乳鼠 ,由中山医科大学实验动物中心提供。心肌细胞培养参照文献[2 ] 进行 ,取乳鼠心肌 ,用胰蛋白酶反复消化 ,制成单细胞悬液。用DME/F12培养液调细胞浓度 ,接种培养皿 ,形成…     

Activation of a Ca2+-dependent K+ current by the oncogenic receptor protein tyrosine kinase v-Fms in mouse fibroblasts

We investigated the effects of the receptor-coupled protein tyrosine kinase (RTK) v-Fms on the membrane current properties of NIH3T3 mouse fibroblasts. We found that v-Fms, the oncogenic variant of the macrophage colony-stimulating factor receptor c-Fms, activates a K⁺ current that is absent in control cells. The activation of the K⁺ current was Ca²⁺-dependent, voltage-independent, and was completely blocked by the K⁺ channel blockers charybdotoxin, margatoxin and iberiotoxin with IC₅₀ values of 3nM, 18 nM and 76nM, respectively. To identify signalling components that mediate the activation of this K⁺ current, NIH3T3 cells that express different mutants of the wildtype v-Fms receptor were examined. Mutation of the binding site for the Ras-GTPase-activating protein led to a complete abolishment of the K⁺ current. A reduction of 76% and 63%, respectively, was observed upon mutation of either of the two binding sites for the growth factor receptor binding protein 2. Mutation of the ATP binding lobe, which disrupts the protein tyrosine kinase activity of v-Fms, led to a 55% reduction of the K⁺ current. Treatment of wild-type v-Fms cells with Clostridium sordellii lethal toxin or a farnesyl protein transferase inhibitor, both known to inhibit the biological function of Ras, reduced the K⁺ current amplitude to 17% and 6% of the control value, respectively. This is the first report showing that an oncogenic RTK can modulate K⁺ channel activity. Our results indicate that this effect is dependent on the binding of certain Ras-regulating proteins to the v-Fms receptor and is not abolished by disruption of its intrinsic protein tyrosine kinase activity. Furthermore, our data suggest that Ras plays a key role for K⁺ channel activation by the oncogenic RTK v-Fms. Received: 19 November 1997 / Accepted: 21 January 1998     

Evaluation of N-type Ca2+ channel currents in cultured rat superior cervical ganglion neurons

Electrophysiological studies of the effects of test compounds on ion channel currents have been useful in the identification of novel therapeutic agents. Here, we examined the use of cultured superior cervical ganglion (SCG) neurons as a model system for the electrophysiological evaluation of N-type Ca²⁺ channels in vitro. As previously reported, Ca²⁺ channel currents in acutely dissociated preparations of SCG neurons were mainly N-type, as defined by inhibition of Ca²⁺ channel current with the specific N-type Ca²⁺ channel blocker, ω-conopeptides GVIA or MVIIA. However, a cultured preparation that could be used over an extended period of time would be more useful for drug discovery since acutely dissociated preparations require daily dissections. We found that with extended time in culture the amplitudes of Ca²⁺ channel currents increased with time. While there was a reduction in the percentage of N-type Ca²⁺ channel component, the majority (70%) of Ca²⁺ channel currents in long-term cultured SCG neurons remained N-type. A portion of the MVIIA-resistant Ca²⁺ channel current component was blocked by the ω-conopeptide MVIIC and ω-agatoxin IIIA, but not by ω-agatoxin IVA, a pharmacological profile similar to Q-type Ca²⁺ channel current. These studies suggest that cultured SCG neurons would be useful for the study of N-type Ca²⁺ currents even after prolonged time in culture, and may also be used to indicate the selectivity of test compounds for other Ca²⁺ channel subtypes. Drug Dev. Res. 41:85–90, 1997. © 1997 Wiley-Liss, Inc.     

Suppression of agonist induced Ca2+ oscillations in cultured hepatocytes by nafenopin: possible involvement of protein kinase C

The alpha₁-agonist phenylephrine (5 μM) induces an increase in the free cytosolic Ca²⁺ concentration, followed by repetitive transients of the cytoplasmic Ca²⁺ concentration, in single Fura-2 loaded hepatocytes. The tumor promoting, hypolipidemic drug nafenopin suppressed the cellular Ca²⁺ response to phenylephrine. The effect of nafenopin on the Ca²⁺ increase and Ca²⁺ oscillations was largely prevented by the specific protein kinase C inhibitor Gö 6976. This finding suggests involvement of protein kinase C in the action of nafenopin on phenylephrine induced Ca²⁺ mobilization.     

KN-62拮抗谷氨酸对神经细胞的兴奋毒作用(英文)

目的:研究钙/钙调素依赖性蛋白激酶Ⅱ抑制剂KN-62对谷氨酸介导皮质神经元损伤及CCDPK Ⅱ活性下降的影响。方法:LDH释放代表神经元损伤,~(32)P掺入法测CCDPK Ⅱ活性,放射自显影显示后磷酸化水平变化。结果:只有在谷氨酸(100μmol·L~(-1),10min)作用前加KN-62可部分保护神经元损伤,明显拮抗CCDPK Ⅱ活性下降,活性由48.0％恢复到90.6％,显著抑制内源蛋白后磷酸化水平下降。结论:KN-62对CCDPK Ⅱ活性的保护作用是通过抑制CCDPK Ⅱ的自身磷酸化而实现的。     

蛋白酪氨酸激酶抑制剂的研究进展

目的综述蛋白酪氨酸激酶(protein tyrosine kinase,PTK)抑制剂类抗肿瘤药物的研究进展。方法根据已报道的PTK抑制剂的相关文献,将其分为国外已经上市、国外处于临床研究和我国自主研发的PTK抑制剂进行具体介绍。结果 PTK在细胞内的信号转导中起着重要的作用,与肿瘤细胞的生长、增殖、分化和凋亡密切相关,目前已经有多种结构的PTK抑制剂类抗肿瘤药物上市或进入临床研究。结论随着PTK的作用机制及构效关系研究的不断深入,该类药物终将成为治疗肿瘤的有效药物。     

Capacitative Ca2+ entry associated with α1-adrenoceptors in rat aorta

In rat aorta, depletion of internal Ca²⁺ stores by addition of noradrenaline (1μM) induces a biphasic response (an initial phasic response and a tonic one) mediated by two different intracellular Ca²⁺ pools. This response cannot be repeated, suggesting a depletion of internal Ca²⁺ stores sensitive to noradrenaline. In absence of the agonist, this depletion is the signal for the entry of extracellular Ca²⁺, not only to refill the stores but also, under our experimental conditions, to activate the contractile proteins thus inducing an increase in the resting tone (IRT) that constitutes functional evidence of this Ca²⁺ entry. The ionic channels involved in the mechanism of the IRT have been studied in the present work. The fact that the addition of nimodipine (10^–15– 10^–11M) selectively inhibits the IRT suggests that this mechanical response is mediated by Ca²⁺ influx through dihydropyridine-sensitive Ca²⁺ channels. Moreover, the inhibitory action of nimodipine is attenuated by glibenclamide (10μM). Cromakalim (10^–10–10^–6M) also inhibits the IRT concentration dependently, and this inhibition is antagonized by glibenclamide (10μM). These results relate the ATP-dependent K⁺ channels to the mechanism of the IRT. The refilling of the two internal Ca²⁺ compartments sensitive to noradrenaline was, like the IRT, altered in presence of the compounds tested, since the subsequent contractile response to noradrenaline was decreased. The present results suggest that nimodipine treatment inhibits the refilling of the Ca²⁺ compartment responsible for the tonic contraction induced by noradrenaline in Ca²⁺-free medium, whereas the refilling of the Ca²⁺ pool responsible for the phasic response to noradrenaline remained unaltered. Both the phasic and tonic responses to noradrenaline in Ca²⁺-free medium decreased after treatment with cromakalim. We can therefore assume that the refilling of both Ca²⁺ compartments sensitive to noradrenaline was inhibited. In conclusion, these results are consistent with the contraction of the rat aorta in response to noradrenaline in Ca²⁺-free medium consisting of an initial phasic response and a tonic one. The former is due to the release of internal Ca²⁺ from a compartment refilled through a special channel that is cromakalim but not dihydropyridine sensitive. The tonic response is due to Ca²⁺ release from another compartment refilled through a cromakalim- and dihydropyridine-sensitive Ca²⁺ channel. The Ca²⁺ entry through this latter channel intervenes in the IRT observed during the refilling of these stores previously depleted by noradrenaline, and the opening state of this channel is also modulated by ATP-dependent K²⁺ channels. Received: 7 May 1996 / Accepted: 30 January 1997     

Ca2+ channel activating action of maitotoxin in cultured brainstem neurons

The actions of maitotoxin were studied using cultured brainstem cells and adrenal chromaffin cells. Maitotoxin induced a profound increase in the Ca2+ influx into cultured brainstem cells after a brief lag period. The maitotoxin-induced Ca2+ influx was suppressed by various voltage-dependent Ca2+ channel blockers such as Co2+, Mn2+, verapamil and diltiazem. Maitotoxin-catecholamine release in brainstem cells initiated to increase after a lag period of about 1 min and the increase continued even at 4 min after treatment, while in the adrenal chromaffin cells the release started after an about 1-min lag period to attain a maximum within first 2-min and gradually decrease thereafter. These results suggest that maitotoxin acts on Ca2+ channels to increase the Ca2+ influx, accompanied by enhancement of catecholamine release in the brainstem cells with a different temporal profile from that in the adrenal chromaffin cells.     

Competitive binding of postsynaptic density 95 and Ca^2＋-calmodulin dependent protein kinase Ⅱ to N-methyl-D-aspartate receptor subunit 2B in rat brain

AIM: To investigate the interactions among postsynaptic density 95 (PSD-95), Ca^2 -calmodulin dependent protein kinase Ⅱα (CaMKⅡα), and N-methyl-D-aspartate receptor subunit 2B (NR2B) during ischemia and reperfusion in hippocampus of rats. METHODS: Brain ischemia was induced by four-vessel occlusion procedure in rats. Immunoprecipitation and immunoblotting were performed to study the interactions and phosphorylation of proteins. The association-dissociation of PSD-95 and CaMKⅡα to and from N-methyl-D-aspartate (NMDA) receptor induced by ischemia and reperfusion and the effects of 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-piperazine (KN-62, a selective inhibitor of CaMKⅡ) on these protein interactions were investigated. Coimmunoprecipitation and immunoblotting were performed for the studies of interactions among proteins. RESULTS: The alternations of the binding level of PSD-95 and CaMKⅡα to NR2B during ischemia and reperfusion demonstrated the negative correlation to each other. Pre-administration of KN62 through both cerebral ventricles inhibited the 10min ischemia-induced increase of the binding of PSD-95 to NR2B and, on the contrary, promoted the binding of CaMKⅡα to NR2B. CONCLUSION: PSD-95 competes with CaMKⅡ to bind to NR2B during ischemia and reperfusion in rat hippocampus.     

Involvement of different Ca2+ pools during the canine bronchial sustained contraction in Ca2+-free medium: lack of effect of PKC inhibition

We evaluated the role of protein kinase C (PKC) in the sustained bronchial contraction (SBC) induced by carbachol (Cch) or histamine in a Ca²⁺-free medium and the possibility that each agonist uses a different Ca²⁺ store for this response. We studied third-order bronchi and airway smooth muscle (ASM) from first-order bronchi dissected free of cartilage and epithelium. Bronchial and ASM responsiveness to Cch or histamine were evaluated in Krebs solution (2.5 mM Ca²⁺) and in Ca²⁺-free medium. Cch and histamine induced an SBC in bronchial tissues in Ca²⁺-free medium. In ASM each agonist produced a transient contraction, but the response to histamine was much smaller. Cch induced a concentration-dependent accumulation of inositol phosphates (IPs) in both bronchi and ASM; however, histamine did not induce significant accumulation of IPs. Repeated exposure to histamine in bronchial rings abolished contractile responses in Ca²⁺-free media, but Cch added afterwards still produced a sustained contraction. This response was blocked when bronchial tissues were preincubated with 10 μM cyclopiazonic acid (CPA). Brief incubation of these preparations with a high EGTA concentration (1 mM) abolished the histamine-induced SBC. The SBC induced by Cch or histamine in Ca²⁺-free medium was not affected by the preincubation of the tissues with calphostin C, chelerythrine or staurosporine. We concluded that Cch mobilizes Ca²⁺ from two different sources during the SBC in Ca²⁺-free medium: from a CPA-sensitive one from sarcoplasmic reticulum (SR) and from a putative extracellular membrane Ca²⁺ pool sensitive to 1 mM EGTA, and neither process involved PKC activation. Histamine appeared to utilize the extracellular membrane pool only. Received: 12 March 1998 / Accepted: 2 September 1998