SEA0400, a novel and selective inhibitor of the Na+-Ca2+ exchanger, attenuates reperfusion injury in the in vitro and in vivo cerebral ischemic models.

The effect of the newly synthesized compound 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400) on the Na+-Ca2+ exchanger (NCX) was investigated and compared against that of 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943). In addition, the effects of SEA0400 on reperfusion injury in vitro and in vivo were examined. SEA0400 was extremely more potent than KB-R7943 in inhibiting Na+-dependent Ca2+ uptake in cultured neurons, astrocytes, and microglia: IC50s of SEA0400 and KB-R7943 were 5 to 33 nM and 2 to 4 microM, respectively. SEA0400 at the concentration range that inhibited NCX exhibited negligible affinities for the Ca2+ channels, Na+ channels, K+ channels, norepinephrine transporter, and 14 receptors, and did not affect the activities of the Na+/H+ exchanger, Na+,K+-ATPase, Ca2+-ATPase, and five enzymes. SEA0400, unlike KB-R7943, did not inhibit the store-operated Ca2+ entry in cultured astrocytes. SEA0400 attenuated dose- dependently paradoxical Ca2+ challenge-induced production of reactive oxygen species, DNA ladder formation, and nuclear condensation in cultured astrocytes, whereas it did not affect thapsigargin-induced cell injury. Furthermore, administration of SEA0400 reduced infarct volumes after a transient middle cerebral artery occlusion in rat cerebral cortex and striatum. These results indicate that SEA0400 is the most potent and selective inhibitor of NCX, and suggest that the compound may exert protective effects on postischemic brain damage.     

Attenuation of ischemia/reperfusion-induced renal injury in mice deficient in Na+/Ca2+ exchanger

Using Na+/Ca2+ exchanger (NCX1)-deficient mice, the pathophysiological role of Ca2+ overload via the reverse mode of NCX1 in ischemia/reperfusion-induced renal injury was investigated. Because NCX1(-/-) homozygous mice die of heart failure before birth, we used NCX1(+/-) heterozygous mice. NCX1 protein in the kidney of heterozygous mice decreased to about half of that of wild-type mice. Expression of NCX1 protein in the tubular epithelial cells and Ca2+ influx via NCX1 in renal tubules were markedly attenuated in the heterozygous mice. Ischemia/reperfusion-induced renal dysfunction in heterozygous mice was significantly attenuated compared with cases in wild-type mice. Histological renal damage such as tubular necrosis and proteinaceous casts in tubuli in heterozygous mice were much less than that in wild-type mice. Ca2+ deposition in necrotic tubular epithelium was observed more markedly in wild-type than in heterozygous mice. Increases in renal endothelin-1 content were greater in wild-type than in heterozygous mice, and this reflected the difference in immunohistochemical endothelin-1 localization in necrotic tubular epithelium. When the preischemic treatment with KB-R7943 was performed, the renal functional parameters of both NCX1(+/+) and NCX1(+/-) acute renal failure mice were improved to the same level. These findings strongly support the view that Ca2+ overload via the reverse mode of Na+/Ca2+ exchange, followed by renal endothelin-1 overproduction, plays an important role in the pathogenesis of ischemia/reperfusion-induced renal injury.     

Inhibition of canine (NCX1.1) and Drosophila (CALX1.1) Na(+)-Ca(2+) exchangers by 7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-one (CGP-37157)

The electrophysiological effects of the benzothiazepine 7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-one (CGP-37157) (CGP) were investigated on the canine (NCX1.1) and Drosophila (CALX1.1) plasmalemmal Na+-Ca2+ exchangers. These exchangers were selected for study because they show opposite responses to cytoplasmic regulatory Ca2+, thereby allowing us to examine the role of this regulatory mechanism in the inhibitory effects of CGP. CGP blocked Na+-Ca2+ exchange current mediated by both transporters with moderate potency (IC50 values = approximately 3-17 microM) compared with other recently reported blockers of Na+-Ca2+ exchange [e.g., 2-[4-[2,5-difluorophenyl) methoxy]phenoxy]phenoxy]-5-ethoxyaniline (KB-R7943) and 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (SEA0400)]. Experiments using alpha-chymotrypsin to remove autoregulation of Na+-Ca2+ exchange showed that block by CGP was reduced, suggesting that part of the effects of this drug may require intact ionic regulatory mechanisms. For NCX1.1, the inhibition produced by CGP was greater for outward Na+-Ca2+ exchange currents compared with inward currents. When CALX1.1 was examined, the extent of inhibition was similar for both inward and outward exchange currents. Although the extent and potency of CGP-mediated inhibition of Na+-Ca2+ exchange are less than those observed with SEA0400 and KB-R7943, our data demonstrate that CGP constitutes a novel class of plasmalemmal Na+-Ca2+ exchange inhibitors. Moreover, the widespread use of CGP as a selective mitochondrial Na+-Ca2+ exchange inhibitor should be reconsidered in light of these additional inhibitory effects.     

反向模式钠钙离子交换体抑制剂降低造影剂诱导的肾小管上皮细胞凋亡

目的 探讨反向模式钠钙离子交换体抑制剂KB-R7943是否对造影剂肾损伤具有保护作用.方法 培养大鼠肾小管上皮细胞分别与不同浓度KB-R7943(10-5,10-6 mol/L)作用12 h后,加入造影剂作用1 h.采用LDH检测细胞损伤,倒置显微镜观察细胞形态变化,流式细胞仪检测细胞凋亡,共聚焦显微镜测定细胞内钙和反应氧产物水平,RT-PCR检测钠钙离子交换体mRNA表达.相同渗透压甘露醇作对照.数据以均数±标准差(x±s)表示,统计采用方差分析和q检验,简单直线相关分析两者相关性,以P<0.05为差异具有统计学意义.结果 造影剂作用1 h诱导了明显细胞损伤和细胞凋亡,细胞内钙和反应氧产物增加,KB-R7943降低了细胞内钙和反应氧产物水平,同时降低了细胞损伤和细胞凋亡并呈剂量效应;钠钙离子交换体mRNA表达无变化.结论 KB-R794对造影剂诱导的肾小管上皮细胞损伤具有保护作用.     

线粒体内膜KATP通道开放剂合用Na＋／Ca2＋交换阻滞剂对离体大鼠心肌梗死面积的影响

【目的】探讨线粒体ATP敏感性钾通道（mitoKATP通道）开放剂尼可地尔合用Na＋／Ca2＋交换阻滞剂K昏R7943对离体大鼠心肌梗死面积的影响。【方法】离体大鼠心脏应用改良的Langendorff装置灌流,行45min缺血（即将左、右冠脉灌流量减到原灌流量的5Voo）及2h再灌注。Wistar大鼠40只,随机分为4组：对照组、尼可地尔组、K＆R7943组、尼可地尔合用K昏R7943组（合用药物组）。观察各组冠脉灌流液中超氧化物歧化酶（SOD）、丙二醛（MDA）的变化,心肌梗死面积的区别。【结果】对照组心肌梗死范围为34．31％,尼可地尔组为26．35％,KBR7943组为28．74％,两者合用时心肌梗死范围为19．23％,合用组显著低于其他三组（P〈0．01）。合用药物组SOD活性显著高于其它三组,MDA显著低于其他三组（P〈0．01）。【结论】尼可地尔合用KBR7943能明显减少心肌梗死面积,能提高冠脉灌流液中SOD活性,减少MDA含量。     

Sodium hydrogen exchange 1 (NHE-1) regulates connexin 43 expression in cardiomyocytes via reverse mode sodium calcium exchange and c-Jun NH2-terminal kinase-dependent pathways

Connexin 43, the major connexin isoform in gap junctions of cardiac ventricular myocytes, undergoes changes in distribution and expression in cardiac diseases. The Na(+)-H(+) exchanger (NHE-1), a key mediator of hypertrophy and heart failure, has been shown to be localized in the cardiomyocyte gap junctional regions; however, whether NHE-1 regulates gap junction proteins in the hypertrophied cardiomyocyte is not known. To address this question, neonatal rat ventricular myocytes were treated with phenylephrine (PE) for 24 h to induce hypertrophy. Increased Cx43 expression observed with PE treatment (132.4 +/- 6.3% compared to control; P < 0.05) was further significantly augmented by the specific NHE-1 inhibitor EMD87580 [N-[2-methyl-4,5-bis(methylsulfonyl)-benzoyl]-guanidine hydrochloride] (173.2 +/- 8.7% increase compared to control; P < 0.05 versus PE), an effect that was mimicked by another NHE-1 inhibitor cariporide [4-isopropyl-3-(methylsulfonyl)benzoyl-guanidine methanesulfonate]. PE-induced hypertrophy was associated with mitogen-activated protein kinase c-Jun NH(2)-terminal kinase (JNK) 1/2 activation, whereas inhibition of JNK1/2 with either SP600125 [anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone] or small interfering RNA significantly increased PE-induced up-regulation of Cx43 protein levels. Inhibition of reverse mode Na(+)-Ca(2+) exchange (NCX) with KB-R7943 [2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate] partially reversed JNK1/2 activation (195.2 +/- 21.4 versus 143.7 +/- 14.4% with KB-R7943; P < 0.05) and augmented up-regulation of Cx43 protein (121.1 +/- 8.3 versus 215.9 +/- 25.6% with KB-R7943; P < 0.05) in the presence of PE. Our results demonstrate that NHE-1 negatively regulates Cx43 protein expression in PE-induced cardiomyocyte hypertrophy via a JNK1/2-dependent pathway, which is probably activated by reverse mode NCX activity.     

Down-regulation of Na+ pump alpha 2 isoform in isoprenaline-induced cardiac hypertrophy in rat: evidence for increased receptor binding affinity but reduced inotropic potency of digoxin

Cardiac hypertrophy in rats induces a down-regulation of Na(+),K(+)-ATPase alpha(2) isoform, although its functional consequences are poorly understood. Using a mathematical modeling approach that allows differentiation between effects elicited at the receptor and postreceptor level, we studied uptake, receptor binding kinetics, and positive inotropism of digoxin in single-pass Langendorff-perfused hearts of vehicle- and isoprenaline-pretreated rats (2.4 mg/kg per day over 4 days). Digoxin outflow concentration and left ventricular developed pressure data were measured for three consecutive doses (15, 30, and 45 microg) in the absence and presence of the reverse mode Na(+)/Ca(2+) exchange inhibitor 2-[2-[4-(4-nitrobenzyloxyl-)phenyl]ethyl isothiourea methansulfonate] (KB-R7943) (0.1 microM) in perfusate. In hypertrophied hearts, 1) the amount of alpha(2) receptors was reduced to 52% of control levels; 2) the digoxin binding affinity was increased 12-fold due to a decrease in dissociation rate constants of alpha(1) and alpha(2) receptors, and 3) inotropic responsiveness to digoxin the was attenuated on the stimulus-response level, where the coupling ratio of stimulus to response was reduced to 38% of control values. Only in the lowest dose level (15 microg) was this decrease in inotropic potency counterbalanced by the increase in receptor affinity. The Na(+),K(+)-ATPase isoform shift was not responsible for the diminished inotropic effect of digoxin. Coadministration of KB-R7943 significantly reduced cellular response generation at higher digoxin doses to the same limiting stimulus-response relationship in both the vehicle and isoprenaline group.     

Calcium channels involved in K+- and veratridine-induced increase of cytosolic calcium concentration in human cerebral cortical synaptosomes.

Human cerebral cortical synaptosomes were used to study voltage-dependent Ca(2+) channels mediating calcium influx in human axon terminals. Synaptosomes were depolarized by elevation of the extracellular K(+) concentration by 30 mM or by the addition of veratridine (10 microM). Increase in cytosolic concentration of calcium [Ca(2+)](i) induced by either stimulus was abolished in the absence of extracellular Ca(2+) ions. omega-Agatoxin IVA inhibited the K(+)-induced [Ca(2+)](i) increase concentration-dependently (IC(50): 113 nM). omega-Conotoxin GVIA (0.1 microM) inhibited K(+)-induced [Ca(2+)](i) increase by 20%. omega-Conotoxin MVIIC (0.2 microM) caused an inhibition by 85%. Nifedipine (1 microM) had no effect on K(+)-induced [Ca(2+)](i) increase. Veratridine-induced increase in [Ca(2+)](i) was inhibited by omega-conotoxin GVIA (0.1 microM) and omega-Agatoxin IVA (0.2 microM; by about 25 and 45%, respectively). Nifedipine inhibited the veratridine-evoked [Ca(2+)](i) increase concentration-dependently (IC(50): 4.9 nM); Bay K 8644 (3 microM) shifted the nifedipine concentration-response curve to the right. Mibefradil (10 microM) abolished the increase in [Ca(2+)](i) evoked by K(+) and reduced the increase evoked by veratridine by almost 90%. KB-R7943 (3 microM) an inhibitor of the Na(+)/Ca(2+) exchanger NCX1, decreased the increase in [Ca(2+)](i) evoked by veratridine by approximately 20%. It is concluded that the increase in [Ca(2+)](i) after K(+) depolarization caused by Ca(2+) influx predominantly via P/Q-type Ca(2+) channels and after veratridine depolarization via N- and P/Q-type, but also by L-type Ca(2+) channels. The toxin- and nifedipine-resistant fraction of the veratridine response may result both from influx via R-type Ca(2+) channels and by Ca(2+) inward transport via Na(+)/Ca(2+) exchanger.     

Pre- or post-ischemic treatment with a novel Na+/Ca2+ exchange inhibitor, KB-R7943, shows renal protective effects in rats with ischemic acute renal failure

We investigated the effects of pre- or post-ischemic treatment with KB-R7943, a new Na+/Ca2+ exchange inhibitor, on ischemic acute renal failure (ARF) in rats, and these were compared with the effects of verapamil. Ischemic ARF was induced by clamping the left renal pedicle for 45-min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal function markedly decreased 24 h after reperfusion. Pre-ischemic treatment with KB-R7943 or verapamil attenuated the ARF-induced renal dysfunction. The ischemia/reperfusion-induced renal dysfunction was overcome by post-ischemic treatment with KB-R7943 but not with verapamil. Histopathological examination of the kidney of ARF rats revealed severe renal damage, and suppression of the damage was seen with post-ischemic treatment with KB-R7943. KB-R7943 markedly suppressed the increment of endothelin-1 (ET-1) content in the kidney at 2, 6, and 24 h after reperfusion. No significant changes in Na+/Ca2+ exchanger protein expression in renal tissue were observed with 45-min ischemia, 6 h after reperfusion and KB-R7943 treatment. These results suggest that Ca2+ overload via the reverse mode of Na+/Ca2+ exchange, followed by ET-1 overproduction, seems to play an important role in the pathogenesis of the ischemia/reperfusion-induced ARF. KB-R7943, which is effective in both cases of pre- and post-ischemic treatments, may prove to be an effective therapeutic agent for cases of ischemic ARF.     

Mechanisms underlying ATP-induced Ca2+ mobilization in human neutrophils

The effect of ATP on Ca2+ mobilization in human neutrophils was examined by using fura-2 as a Ca2+ indicator. ATP (0.1-100 microM) caused a significant [Ca2+]i increase in a concentration-dependent manner. The [Ca2+]i signal comprised an initial rise followed by a plateau. Removal of external Ca2+ diminished the peak value of the [Ca2+]i signal. In Ca2+-free medium, pretreatment with an endoplasmic reticulum Ca2+ pump inhibitor, thapsigargin, prevented ATP from releasing Ca2+. In contrast, thapsigargin still increased [Ca2+], after pretreatment with 10 microM ATP. These results indicate that 10 microM ATP released Ca2+ mainly from thapsigargin-sensitive stores. Adding 3 mM Ca2+ induced a concentration-dependent increase in [Ca2+]i after pretreatment with ATP or thapsigargin in Ca2+-free medium, suggesting ATP induced Ca2+ influx via capacitative Ca2+ entry. ATP (10 microM)-induced Ca2+ release was abolished by inhibiting phospholipase C with 2 microM U73122, indicating that inositol-1,4,5-trisphosphate (IP3) mediates ATP-induced Ca2+ release. Conversely, ATP-induced [Ca2+]i increase was abolished by activating protein kinase C (PKC) with 10 nM phorbol myristate acetate (PMA), but was not altered by inhibiting PKC with 2 microM GF 109203X. This implies ATP-induced [Ca2+]i increase is a PMA-linked event. Together, the results suggest ATP increases [Ca2+]i in human neutrophils by releasing Ca2+ from IP3-coupled, thapsigargin-sensitive Ca2+ stores, and inducing Ca2+ influx via the process of capacitative Ca2+ entry. The ATP-induced Ca2+ signal is a PMA-linked event.     

Phospholipase C activation by Na+/Ca2+ exchange is essential for monensin-induced Ca2+ influx and arachidonic acid release in FRTL-5 thyroid cells.

BACKGROUND: Monensin, a Na+ ionophore, can increase cytosolic Ca2+ ([Ca2+]i) by reversing the Na+/Ca2+ exchange mechanism. This study provided additional insights into the mechanism of this Na+ ionophore-induced increase in [Ca2+]i, and emphasized the critical role of phospholipase C (PLC) in amplifying Na+/Ca2+ exchange-induced Ca2+ influx and subsequent arachidonic acid (AA) release in FRTL-5 thyroid cells. The possible involvement of protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and GTP-binding (G) protein in mediating monensin-induced AA release was also explored. METHODS: FRTL-5 thyroid cells were maintained in Coon's modified Ham's F-12 medium supplemented with a 6-hormone (6H) mixture. Cytosolic Ca2+ was measured by using indo-1 AM and a dual-wave-length spectrofluorometer. Release of 3H-labeled inositol trisphosphates and arachidonic acid were determined by a scintillation counter. RESULTS: In Hank's balanced salt solution with Ca2+ (HBSS+), monensin (100 mumol/L) induced a 2.3-fold sustained Ca2+ increase associated with IP3 generation and a 6-fold increase in AA release. Deletion of extracellular Ca2+, or replacement of Na+ by choline chloride in the medium, reduced the [Ca2+]i increase by 77% and completely prevented the monensin-induced rise in AA release. Similar inhibitory effects were observed in cells pretreated with a Na+ channel blocker, or Na+/Ca2+ exchange inhibitors. In HBSS without Ca2+ (HBSS-), monensin induced a 1-fold transient [Ca2+]i increase but did not increase the AA. This Ca2+ increase was not suppressed by U-73122, a PLC inhibitor. In HBSS+, U-73122 did not affect the monensin-induced initial transient peak increase of [Ca2+]i, but reduced the sustained second phase of [Ca2+]i from 400 nmol/L to 250 nmol/L, and completely blocked AA release. A phospholipase A2 (PLA2) inhibitor blocked the monensin-induced AA release without affecting the [Ca2+]i increase. Inhibition of PKC prevented 87% to 94% of the monesin-stimulated AA release. The monensin-induced AA release was also inhibited 94% by pertussis and 51% by a MAP kinase cascade inhibitor. CONCLUSIONS: The results suggest that monensin initiates an increase in [Ca2+]i via a Na+/Ca2+ exchange mechanism that triggers more pronounced and sustained [Ca2+]i increase via activation of PLC and Ca2+ influx. The PLC activation, followed by sustained Ca2+ influx and PKC activation, is a prerequisite for PLA2-mediated processes in monensin-challenged FRTL-5 thyroid cells.     

Fluvastatin and atorvastatin affect calcium homeostasis of rat skeletal muscle fibers in vivo and in vitro by impairing the sarcoplasmic reticulum/mitochondria Ca2+-release system

The mechanism by which the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) induce skeletal muscle injury is still under debate. By using fura-2 cytofluorimetry on intact extensor digitorum longus muscle fibers, here we provided the first evidence that 2 months in vivo chronic treatment of rats with fluvastatin (5 and 20 mg kg-1) and atorvastatin (5 and 10 mg kg-1) caused an alteration of calcium homeostasis. All treated animals showed a significant increase of resting cytosolic calcium [Ca2+]i, up to 60% with the higher fluvastatin dose and up to 20% with the other treatments. The [Ca2+]i rise induced by statin administration was not due to an increase of sarcolemmal permeability to calcium. Furthermore, the treatments reduced caffeine responsiveness. In vitro application of fluvastatin caused changes of [Ca2+]i, resembling the effect obtained after the in vivo administration. Indeed, fluvastatin produced a shift of mechanical threshold for contraction toward negative potentials and an increase of resting [Ca2+]i. By using ruthenium red and cyclosporine A, we determined the sequence of the statin-induced Ca2+ release mechanism. Mitochondria appeared as the cellular structure responsible for the earlier event leading to a subsequent large sarcoplasmic reticulum Ca2+ release. In conclusion, we suggest that calcium homeostasis alteration may be a crucial event for myotoxicity induced by this widely used class of hypolipidemic drugs.     

Measurement of cytoplasmic free Ca2+ concentration in rabbit aorta using the photoprotein, aequorin. Effect of atrial natriuretic peptide on agonist-induced Ca2+ signal generation.

Addition of norepinephrine, angiotensin II, or histamine leads to a transient rise in the cytoplasmic Ca2+ concentration ([Ca2+]i), as measured with aequorin, in rabbit aortic strips. Each induces a [Ca2+]i transient which peaks in 2 min and then falls either back to baseline (angiotensin II) or to a plateau (norepinephrine and histamine). The [Ca2+]i transient is due to the mobilization of Ca2+ from a caffeine-sensitive, intracellular pool. An elevation of [K+] to 35 mM leads to a monotonic sustained rise in [Ca2+]i which depends entirely on extracellular Ca2+, but an increase to 100 mM leads to a [Ca2+]i transient from the mobilization of intracellular Ca2+. Atrial natriuretic peptide does not alter basal [Ca2+]i nor inhibit the [Ca2+]i transient induced by either histamine or angiotensin II, but blocks that induced by norepinephrine, and blocks the plateau phase induced by either histamine or norepinephrine. The peptide inhibits the contractile response to all three agonists and to K+.     

脑创伤后神经元细胞内游离钙的变化及其影响因素

目的研究液压冲击伤时体外培养的单个大鼠神经细胞内游离钙 ([Ca2+ ]i)的变化,探讨尼莫地平 D-2氨基戊酸 (D-2-amino-5-phosphonovaleric acid,D-AP-5)和亚低温对创伤后细胞内 [Ca2+ ]i的影响及其机制. 方法 以 Fluo-3/AM为细胞内钙离子的荧光指示剂,用激光共聚焦显微镜测定液压冲击伤时体外培养的单个大鼠神经细胞内游离钙 ([Ca2+ ]i)的变化. 结果 液压冲击伤后脑皮质细胞内游离 Ca2+迅速升高, 24 h达高峰,随后逐渐下降, 48 h仍维持较高水平 [(411.29± 52.46),(396.53± 51.32) nmol/L],尼莫地平, D-AP-5于各时相关均降低细胞内 [Ca2+ ]i,其中 Nimodipine 10 h内应用最佳 [6 h( 98.65± 15.65) nmol/L], D-AP-5于 1～ 10 h应用较好而亚低温 30 min内显著降低细胞内 [Ca2+ ]i(t=13.74, P< 0.001),最佳时机在伤后 15 min内,伤后 1 h以上无效. 结论 创伤后神经元细胞内钙超载,尼莫地平、 D-AP-5和亚低温均降低细胞内 [Ca2+ ]i,但各自最佳时间窗不同,揭示对创伤后神经细胞 Ca2+超载应注意综合治疗,并选定各自作用最佳时间窗.     

Cytosolic Ca2+ and phosphoinositide hydrolysis linked to constitutively active alpha 1D-adrenoceptors in vascular smooth muscle

In the present study, we analyzed changes in intracellular Ca2+ levels and inositol phosphate accumulation related to a population of alpha 1d-adrenoceptors in rat aorta resembling constitutively active receptors. Following intracellular Ca2+ store depletion by noradrenaline in Ca2+-free medium and removal of the agonist, restoration of extracellular Ca2+ induced four signals: a biphasic (transient and sustained) increase in [Ca2+]i, inositol phosphate accumulation, and a contractile response in the aorta. The transient increase in Ca2+, the inositol phosphate accumulation, and the contractile response were not observed in aortae incubated with prazosin or BMY 7378 [8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione] (a selective alpha 1d-adrenoceptor ligand), relating the three signals to alpha 1d-adrenoceptor activity. In the presence of nimodipine, only the sustained increase in Ca2+ and the inositol phosphate accumulation were observed, relating both signals to calcium entry through L-channels. The four signals were abolished by Ni2+. In the rat tail artery, where alpha 1d-adrenoceptors are not functionally active, restoration of extracellular Ca2+ after store depletion induced only a sustained increase in [Ca2+]i without inositol phosphate accumulation nor contractile response. Taken together these results suggest that in the aorta, Ca2+ entry is required for the recovery of cytosolic calcium levels and the display of the membrane signals related to the constitutive activity of alpha 1d-adrenoceptors, i.e., inositol phosphate formation and Ca2+ entry through L-type channels, which maintains a contractile response once the agonist has been removed.     

Protein kinase C-dependent potentiation of intracellular calcium influx by sigma1 receptor agonists in rat hippocampal neurons

Intracellular calcium concentration ([Ca2+]i) plays a major role in neuronal excitability, especially that triggered by the N-methyl-d-aspartate (NMDA)-sensitive glutamatergic receptor. We have previously shown that sigma1 receptor agonists potentiate NMDA receptor-mediated neuronal activity in the hippocampus and recruit Ca2+-dependent second messenger cascades (e.g., protein kinase C; PKC) in brainstem motor structures. The present study therefore assessed whether the potentiating action of sigma1 agonists on the NMDA response observed in the hippocampus involves the regulation of [Ca2+]i and PKC. For this purpose, [Ca2+]i changes after NMDA receptor activation were monitored in primary cultures of embryonic rat hippocampal pyramidal neurons using microspectrofluorometry of the Ca2+-sensitive indicator Fura-2/acetoxymethyl ester in the presence of sigma1 agonists and PKC inhibitors. We show that successive activations of the sigma1 receptor by 1-min pulses of (+)-benzomorphans or (+)-N-cyclopropylmethyl-N-methyl-1,4-diphenyl-1-ethyl-but-3-en-1-ylamine hydrochloride (JO-1784) concomitantly with glutamate time dependently potentiated before inconstantly inhibiting the NMDA receptor-mediated increase of [Ca2+]i, whereas 1,3-di-o-tolyl-guanidine, a mixed sigma1/sigma2 agonist, did not significantly modify the glutamate response. Both potentiation and inhibition were prevented by the selective sigma1 antagonist N,N-dipropyl-2-[4-methoxy-3-(211phenylethoxy) phenyl]-ethylamine monohydrochloride (NE-100). Furthermore, only (+)-benzomorphans could induce [Ca2+]i influx by themselves after a brief pulse of glutamate. A pretreatment with the conventional PKC inhibitor 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo [2,3-a] pyrrolo [3,4-c] carbazole (G?-6976) prevented the potentiating effect of (+)-benzomorphans on the glutamate response. Our results provide further support for a general mechanism for the intracellular sigma1 receptor to regulate Ca2+-dependent signal transduction and protein phosphorylation.     

Endothelin receptor-coupling mechanisms in vascular smooth muscle: a role for protein kinase C

Endothelin (ET), a peptide that is released from cultured endothelial cells, is a potent vasoconstrictor that induces characteristically long-lasting contractions. We used the A10 vascular smooth muscle cell (VSMC) line to probe mechanisms underlying ET-induced contractions. Intracellular Ca2+ ([Ca2+]i) and pH were monitored in A10 monolayers using the fluorescent dyes Fura-2 and 2,7-bis-carboxyethyl-5,6-carboxyfluorescein, respectively. Synthetic porcine ET induced rapid and transient increases in [Ca2+]i (EC50 value, 0.75 nM; maximum, approximately 6-fold above basal). External Ca2+ removal did not block the ability of ET (0.5 or 50 nM) to increase initial [Ca2+]i, although [Ca2+]i returned to prestimulus levels faster as compared with that seen in the presence of external Ca2+. Total cell 45Ca2+ content decreased within 30 sec and remained below prestimulus values for at least 20 min (34 +/- 2% decrease after 5 min, n = 3) in ET-stimulated VSMC. ET stimulated a transient rise in inositol trisphosphate formation in [3H]myo-inositol labeled VSMC, peaking in 30 sec (62 +/- 20% increase, n = 3). In contrast, ET-stimulated diacylglycerol formation in [3H]arachidonic acid-labeled VSMC was sustained and biphasic, exhibiting two peaks at 15 sec (41 +/- 16% increase) and at 5 min (75 +/- 7% increase, n = 3). ET (50 nM) also induced an intracellular alkalinization of 0.17 +/- 0.02 (n = 10) pH units above basal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Halothane increases cytosolic Ca2+ and inhibits Na+/H+ exchange in L6 muscle cells

The effects of the general anesthetic halothane on the concentration of cytosolic free calcium ([Ca2+]i) and cytosolic pH (pHi), were investigated in L6 rat skeletal muscle cells. Basal [Ca2+]i was 169 +/- 8 nM, measured with the fluorescent Ca2(+)-indicator 1-[2-amino-5-(6-carboxyindol-2-yl)phenoxy]-2-(2'-amino-5- methylphenoxy)ethane-N,N,N',N'-tetra-acetate. Halothane (5.7 mM) increased [Ca2+]i to 225 +/- 15 nM in the presence of extracellular Ca2+, and from 137 +/- 6 nM to 179 +/- 9 nM in Ca2+ absence. This increase was dose-dependent. The anesthetic released about 50% of the releasable Ca2+ from intracellular stores. The resting pHi of L6 cells was 7.24 +/- 0.04, measured with the fluorescent pH indicator bis-carboxyethylcarboxyfluorescein. Halothane did not affect resting pHi, but inhibited cytoplasmic alkalinization by hypertonicity or cytoplasmic acidification: (1) The hypertonicity-induced alkalinization via activation of Na+/H+ exchange (to 7.50 +/- 0.08, initial rate 0.10 +/- 0.02 pH U/min) was inhibited with 5.7 mM halothane by 67%. (2) Acid-loaded cells (pHi 6.43 +/- 0.01 in cells) recovered towards neutrality via activation of Na+/H+ exchange (rate 0.47 pH U/min), and halothane inhibited the rate of pHi recovery by 50%. The halothane-mediated inhibition of alkalinizations after hypertonic exposure or acid-loading was also observed in bis-(o-amino-phenoxy)ethane-N,N,N',N'-tetra-acetate-loaded cells in Ca2(+)-free medium. Therefore, halothane increases [Ca2+]i and in parallel inhibits Na+/H+ exchange, compromising the ability of muscle cells to recover from imposed acidification.     

P2X receptor-stimulated calcium responses in preglomerular vascular smooth muscle cells involves 20-hydroxyeicosatetraenoic acid

The current study tested the hypothesis that endogenous 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to the increase in intracellular calcium ([Ca2+]i) elicited by P2X receptor activation in renal microvascular smooth muscle cells. Vascular smooth muscle cells obtained from rats were loaded with fura-2 and studied using standard single cell fluorescence microscopy. Basal renal myocyte [Ca2+]i averaged 96 +/- 5 nM. ATP (10 and 100 microM) increased vascular smooth muscle cell [Ca2+]i by 340 +/- 88 and 555 +/- 80 nM, respectively. The cytochrome P450 hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), or the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE), significantly attenuated the peak myocyte [Ca2+]i responses to 10 and 100 microM ATP. ATP (100 microM) increased vascular smooth muscle cell [Ca2+]i by 372 +/- 93 and 163 +/- 55 nM in the presence of DDMS or 20-HEDE, respectively. The P2X receptor agonist, alpha,beta-methylene-ATP (10 microM), increased myocyte [Ca2+]i by 78 +/- 12 nM, and this response was significantly attenuated by DDMS (40 +/- 15 nM). In contrast, the vascular smooth muscle cell [Ca2+]i evoked by the P2Y agonist, UTP (100 microM), was not altered by DDMS or 20-HEDE. The effect of 20-HETE on [Ca2+]i was also assessed, and the peak increases in [Ca2+]i averaged 62 +/- 12 and 146 +/- 70 nM at 20-HETE concentrations of 1 and 10 microM, respectively. These results demonstrate that 20-HETE plays a significant role in the renal microvascular smooth muscle cell [Ca2+]i response to P2X receptor activation.     

Activation of Ca2+-dependent K+ channels in human B lymphocytes by anti-immunoglobulin.

Many mammalian cell types exhibit Ca2+-dependent K+ channels, and activation of these channels by increasing intracellular calcium generally leads to a hyperpolarization of the plasma membrane. Their presence in B lymphocytes is as yet uncertain. Crosslinking Ig on the surface of B lymphocytes is known to increase the level of free cytoplasmic calcium ([Ca2+]i). However, rather than hyperpolarization, a depolarization has been reported to occur after treatment of B lymphocytes with anti-Ig. To determine if Ca2+-dependent K+ channels are present in B lymphocytes, and to examine the relationship between intracellular free calcium and membrane potential, we monitored [Ca2+]i by means of indo-1 and transmembrane potential using bis(1,3-diethylthiobarbituric)trimethine oxonol in human tonsillar B cells activated by anti-IgM. Treatment with anti-IgM induced a biphasic increase in [Ca2+]i and a simultaneous hyperpolarization. A similar hyperpolarization was induced by ionomycin, a Ca2+ ionophore. Delaying the development of the [Ca2+]i response by increasing the cytoplasmic Ca2+-buffering power delayed the hyperpolarization. Conversely, eliminating the sustained phase of the [Ca2+]i response by omission of external Ca2+ abolished the prolonged hyperpolarization. In fact, a sizable Na+-dependent depolarization was unmasked. This study demonstrates that in human B lymphocytes, Ca2+-dependent K+ channels can be activated by crosslinking of surface IgM. Moreover, it is likely that, by analogy with voltage-sensitive Ca2+ channels, Na+ can permeate through these ligand-gated Ca2+ "channels" in the absence of extracellular Ca2+.