Involvement of calcium in the in vitro sensitivity change of the plasma membrane H+ ATPase to indole-3-acetic acid

The proton pumping activity of phase-partitioning purified plasma membrane fraction from spinach leaves was tested in vitro in the presence of exogenous indole-3-acetic acid. The sensitivity of the H+ pumping activity to the auxin was changed after flowering induction. We investigated the effect of whole spinach leaf treatments with substances affecting the phosphatidylinositol diphosphate transduction pathway on the in vitro sensitivity modification by photoperiodic induction. A role of calcium ions was supported by studies on leaves treated with a specific Ca²⁺ chelator (EGTA), a synthetic Ca²⁺ ionophore (A23187) or with calcium channel blokers (verapamil, lanthan chloride). An experiment using the transduction pathway inhibitor, lithium chloride, indicated that the intracellular concentration of Ca²⁺ was increased by inositol triphosphate.     

Differential Involvement of Intracellular Ca2+ in 1-Methyl-4-phenylpyridinium- or 6-Hydroxydopamine-Induced Cell Viability Loss in PC12 Cells

1-Methyl-4-phenylpyridinium (MPP⁺) or 6-hydroxydopamine (6-OHDA) caused a nuclear damage, the mitochondrial membrane permeability changes, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in PC12 cells. Nicardipine (a calcium channel blocker), EGTA (an extracellular calcium chelator), BAPTA-AM (a cell permeable calcium chelator) and calmodulin antagonists (W-7 and calmidazolium) attenuated the MPP⁺-induced mitochondrial damage and cell death. In contrast, the compounds did not reduce the toxicity of 6-OHDA. Treatment with MPP⁺ or 6-OHDA evoked the elevation of intracellular Ca²⁺ levels. Unlike cell injury, addition of nicardipine, BAPTA-AM and calmodulin antagonists prevented the elevation of intracellular Ca²⁺ levels due to both toxins. The results show that the MPP⁺-induced formation of the mitochondrial permeability transition seems to be mediated by elevation of intracellular Ca²⁺ levels and calmodulin action. In contrast, the 6-OHDA-induced cell death seems to be mediated by Ca²⁺-independent manner.     

Calcium antagonists and calmodulin inhibitors block cytokinin-induced bud formation in Funaria

The plant hormone cytokinin stimulates nuclear migration followed by an asymmetric cell division in target cells of the protonema of the moss Funaria hygrometrica, leading to bud formation. The role of calcium in this developmental event was investigated by examining the effects of various calcium antagonists on the cytokinin-induced division. Calcium-free medium (buffered with EGTA), the extracellular Ca²⁺ antagonist La³⁺ (lanthanum), and the Ca²⁺ channel inhibitors D 600 and verapamil all block bud formation. These inhibitions are partially reversed by washing the cells or by raising the extracellular [Ca²⁺]. The Ca²⁺ ionophore A23187 partially reversed the effects of D 600 and verapamil. Bud formation is also inhibited by the intracellular Ca²⁺ antagonist TMB-8 (8-diethylamino)ocytl 3,4,5-trimethoxybenzoate HCl), and this inhibition is partially reversed by washing or raising the extracellular [Ca²⁺]. The cross walls of both the filaments and bud initial cells formed during TMB-8 exposure exhibit a distorted morphology. High concentrations of TMB-8 block nuclear migration. The calmodulin inhibitor trifluoperazine stops cytokinin-induced budding more effectively than the related compound chlorpromazine. Low concentrations of these two compounds do not affect nuclear migration; however, the target cell does not enter mitosis. These results support the hypothesis that a rise in intracellular calcium mediates cytokinin-induced bud formation in Funaria. It is concluded that the proposed cytokinin-induced rise in intracellular calcium may be effected in part by the activation of calmodulin. The essential source of Ca²⁺ appears to be extracellular, because blocking Ca²⁺ uptake with Ca²⁺ transport inhibitors can block both nuclear migration and subsequent division.     

Calcium in the Control of Nitrogenase Activity in Vicia faba L. Root Nodules: Calcium Release from Symbiosomes and the Accompanying Decrease in Their Nitrogenase Activity Is Blocked by Verapamil

Treatment of root nodules or symbiosomes isolated from them with calcium chelator EGTA alone or together with calcium ionophore A23187 for 3 h under microaerophilic conditions considerably decreased their nitrogenase activity (NA). Under these experimental conditions, cytochemical electron-microscopic analysis revealed considerable calcium depletion of symbiosomes in the infected nodule cells treated with EGTA and A23187. Ca²⁺ channel blockers, verapamil and ruthenium red, inhibited EGTA-induced Ca²⁺ release from symbiosomes. In this case, NA insignificantly increased in the whole nodules and reached its initial level in symbiosomes. The experiments on isolated symbiosomes with arsenazo III, a Ca²⁺ indicator, demonstrated that verapamil inhibited Ca²⁺ release from them induced by valinomycin in the presence of K⁺ ions. These data suggest the presence on the peribacteroid membrane of a verapamil-sensitive transporter responsible for Ca²⁺ release from symbiosomes. A possible role of this transporter in the interaction between symbiotic partners in the infected cells of root nodules is discussed.     

Calcium transients in response to salinity and osmotic stress in the nitrogen-fixing cyanobacterium Anabaena sp. PCC7120, expressing cytosolic apoaequorin

We show here that both salinity and osmotic stress trigger transient increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in cells of the nitrogen‐fixing filamentous cyanobacterium Anabaena sp. PCC7120, which constitutively expresses apoaequorin. Isoosmolar concentrations of salt (NaCl) and osmoticum (sucrose) induced calcium transients of similar magnitude and shape, suggesting that cells sense, via Ca²⁺ signalling, mostly osmotic stress. The Ca²⁺ transients induced by NaCl and sucrose were completely blocked by the calcium chelator ethylene glycol‐bis(b‐aminoethylether)N,N,N¢,N¢‐tetraacetic acid (EGTA) and were partially inhibited by the calcium channel blocker verapamil. Increased external Ca²⁺ and the Ca²⁺ ionophore calcimycin (compound A23187) enhanced Ca²⁺ influx further, suggesting the involvement of extracellular Ca²⁺ in the observed response to salinity and osmotic stress. However, the plant hormone abscisic acid (ABA) did not provoke any effect on the Ca²⁺ transients induced by both stresses, indicating that it may not be acting upstream of Ca²⁺ in the signalling of salinity and/or osmotic stress in Anabaena sp. PCC7120.     

Inhibitors of Na+/Ca2+ exchanger prevent oxidant-induced intracellular Ca2+ increase and apoptosis in a human hepatoma cell line

Oxidative stress appears to be implicated in the pathogenesis of various diseases including hepatotoxicity. Although intracellular Ca²⁺ signals have been suggested to play a role in the oxidative damage of hepatocytes, the sources and effects of oxidant-induced intracellular Ca²⁺ increases are currently debatable. Thus, in this study we investigated the exact source and mechanism of oxidant-induced liver cell damage using HepG2 human hepatoma cells as a model liver cellular system. Treatment with 200 μM of tert-butyl hydroperoxide (tBOOH) induced a sustained increase in the level of intracellular reactive oxygen intermediates (ROI) and apoptosis, assessed by 2′,7′-dichlorofluorescein fluorescence and flow cytometry, respectively. Antioxidants, N-acetyl cysteine (NAC) or N,N′-diphenyl-p-phenylenediamine significantly inhibited both the ROI generation and apoptosis. In addition, tBOOH induced a slow and sustained increase in intracellular Ca²⁺ concentration, which was completely prevented by the antioxidants. An intracellular Ca²⁺ chelator, bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid/cetoxymethyl ester significantly suppressed the tBOOH-induced apoptosis. These results imply that activation of an intracellular Ca²⁺ signal triggered by increased ROI may mediate the tBOOH-induced apoptosis. Both intracellular Ca²⁺ increase and induction of apoptosis were significantly inhibited by an extracellular Ca²⁺ chelator or Na⁺/Ca²⁺ exchanger blockers (bepridil and benzamil), whereas neither Ca²⁺ channel antagonists (verapamil and nifedipine) nor a nonselective cation channel blocker (flufenamic acid) had an effect. These results suggest that tBOOH may increase intracellular Ca²⁺ through the activation of reverse mode of Na⁺/Ca²⁺ exchanger. However, tBOOH decreased intracellular Na⁺ concentration, which was completely prevented by NAC. These results indicate that ROI generated by tBOOH may increase intracellular Ca²⁺ concentration by direct activation of the reverse mode of Na⁺/Ca>²⁺ exchanger, rather than indirect elevation of intracellular Na⁺ levels. Taken together, these results suggest that the oxidant, tBOOH induced apoptosis in human HepG2 cells and that intracellular Ca²⁺ may mediate this action of tBOOH. These results further suggest that Na⁺/Ca²⁺ exchanger may be a target for the management of oxidative hepatotoxicity.     

K+ Channels and the Intracellular Calcium Signal in Human Melanoma Cell Proliferation

K⁺ channels, membrane voltage, and intracellular free Ca²⁺ are involved in regulating proliferation in a human melanoma cell line (SK MEL 28). Using patch-clamp techniques, we found an inwardly rectifying K⁺ channel and a calcium-activated K⁺ channel. The inwardly rectifying K⁺ channel was calcium independent, insensitive to charybdotoxin, and carried the major part of the whole-cell current. The K⁺ channel blockers quinidine, tetraethylammonium chloride and Ba²⁺ and elevated extracellular K⁺ caused a dose-dependent membrane depolarization. This depolarization was correlated to an inhibition of cell proliferation. Charybdotoxin affected neither membrane voltage nor proliferation. Basic fibroblast growth factor and fetal calf serum induced a transient peak in intracellular Ca²⁺ followed by a long-lasting Ca²⁺ influx. Depolarization by voltage clamp decreased and hyperpolarization increased intracellular Ca²⁺, illustrating a transmembrane flux of Ca²⁺ following its electrochemical gradient. We conclude that K⁺ channel blockers inhibit cell-cycle progression by membrane depolarization. This in turn reduces the driving force for the influx of Ca²⁺, a messenger in the mitogenic signal cascade of human melanoma cells. Received: 9 May 1995/Revised: 30 January 1996     

Identification of a Phosphoprotein Expressed During Somatic Embryogenesis in Wheat Leaf Base Cultures

2,4-D mediated induction of somatic embryogenesis in wheat is enhanced in the presence of Ca⁺⁺ and its removal by EGTA reduces the response significantly. Changes that occur at the polypeptide level following 2,4-D treatment were analysed. Intense cell division activity was discernable in the leaf base explants within an hour of treatment. Changes in protein profiles were prominent in the membrane fraction as compared to the soluble fraction. The protein profile of the leaf base culture with somatic embryos was distinct from the calli induced from mature embryos on a 2,4-D containing medium. The role of Ca²⁺ in the induction of somatic embryogenesis was demonstrated by the use of EGTA (a calcium chelator), verapamil, nifedipine (calcium channel blockers), W7 (calmodulin antagonist) and Li (PI inhibitor). ^{In vitro} protein phosphorylation studies showed that 2,4-D, calcium and related treatments inhibit phosphorylation of proteins. In the membrane fraction proteins, accumulation of polypeptides at the low molecular weight range was seen in samples treated with verapamil and W7, and a 30 kO polypeptide in the samples treated with calmodulin antagonist, W7. Autoradiography of membrane fraction proteins displayed the presence of a 16 kO protein phosphorylated in samples treated with verapamil, nifedipine and W7. It thus appears that 2,4-D and Ca⁺⁺ prevent the phosphorylation of this phosphoprotein. These results thus indicate the action of 2,4-D via the Ca²⁺-CaM signaling pathway in triggering the induction of somatic embryogenesis.     

Functional coupling of ion channels in cellular mechanotransduction

The major players in the processes of cellular mechanotransduction are considered to be mechanosensitive (MS) or mechano-gated ion channels. Non-selective Ca²⁺-permeable channels, whose activity is directly controlled by membrane stretch (stretch-activated channels, SACs) are ubiquitously present in mammalian cells of different origin. Ca²⁺ entry mediated by SACs presumably has a significant impact on various Ca²⁺-dependent intracellular and membrane processes. It was proposed that SACs could play a crucial role in the different cellular reactions and pathologies, including oncotransformation, increased metastatic activity and invasion of malignant cells. In the present work, coupling of ion channels in transformed fibroblasts in course of stretch activation was explored with the use of patch-clamp technique. The combination of cell-attached and inside-out single-current experiments showed that Ca²⁺ influx via SACs triggered the activity of Ca²⁺-sensitive K⁺ channels indicating functional compartmentalization of different channel types in plasma membrane. Importantly, the analysis of single channel behavior demonstrated that K⁺ currents could be activated by the rise of intracellular calcium but displayed no direct mechanosensitivity. Taken together, our data imply that local changes in Ca²⁺ concentration due to SAC activity may provide a functional link between various Ca²⁺-dependent molecules in the processes of cellular mechanotransduction.     

Constitutively active calcium channels in plasma membrane of T-lymphocytes

Compensated influx and efflux of calcium ions maintain the constancy of Ca²⁺ concentration in cytoplasm of quiescent cells under variable external conditions. In cell plasma membrane there exist several types of Ca²⁺ channels with different properties, regulation mechanisms, and pharmacology. Using fluorescent Ca²⁺-sensitive probes, we have shown here that in T-lymphocytes under resting conditions, Ca²⁺ influx occurs through special constitutively active Ca²⁺ channels, permeable to Ni²⁺ and Mn²⁺. These channels differ from the receptor-activated SOC channels, from Ca²⁺ channels activated by arachidonic acid, and from calmidazolium-activated channels. Ca²⁺ influx rate in quiescent cells increases with a rise in temperature (Q₁₀ =1.9). The strong dependence of the constitutively active channel activity on temperature coincided with the plasma membrane Ca²⁺-ATPase dependence, indicating that intracellular enzymes regulate the channel activity. To identify the constitutively active channel, we analyzed the effects of L-type Ca²⁺ channels, SOC channels, Ca²⁺-independent phospholipase A2, and calmodulin inhibitors. Of all inhibitors listed only dihydropyridine blocker of L-type voltage-dependent Ca²⁺ channels, isradipin, at a concentration of 1.5 μM completely suppressed calcium influx. However, the channels did not exhibit sensitivity to changes in membrane potential. Our observations testify to the existence of a new nonselective Ca²⁺ channel in T-lymphocyte plasma membrane and characterize the new channels pharmacologically. The results obtained are important for understanding the regulation mechanisms of Ca²⁺ channels in plasma membrane of non-excitable cells.     

钙池排空操纵的外钙内流决定甘草诱导MGC-803细胞凋亡

 用 EGTA螯合胞外 Ca2 +和异搏定抑制钙通道 ,研究胞外 Ca2 +在甘草诱导 MGC- 80 3细胞中的作用 .流式细胞仪检测凋亡峰和 DNA ladder分析均表明 ,EGTA和异博定阻断细胞凋亡 .分别以 PI或 Rh1 2 3活染后的相应荧光强度表示细胞膜通透性和线粒性膜电位 (ΔΨm) .结果表明 ,细胞膜通透性增强和线粒体 ΔΨm 下降均为细胞凋亡的早期事件 ,EGTA和异博定均可抑制细胞膜通透性增强 ,但 EGTA促进线粒体 ΔΨm 下降 ,而异博定作用相反 .进一步经 PI和 Hoechst33342荧光双染后同时观察细胞膜通透性和细胞核形态 .结果表明 ,凋亡细胞均可 PI着色 ,EGTA和异博定完全阻断染色质凝聚 ,但不能完全抑制细胞膜通透性变化 .借助 Ca2 +探针 Fluo- 3/AM研究凋亡时胞内游离钙的时相变化 ,发现 Ca2 +升高也是细胞凋亡的早期事件 . EGTA和异博定轻微促进凋亡早期 Ca2 +升高 ,但抑制随后 Ca2 +的继续升高 .所有结果提示 ,钙池排空操纵的外 Ca2 +内流在甘草诱导 MGC- 80 3细胞凋亡中发挥决定性的作用 .     

Indo-1 can simultaneously detect Ba2+ entry and Ca2+ blockade at a plasma membrane calcium channel

The fluorescent chelator Indo-1 can make simultaneous determinations of two intracellular ion concentrations, such as [Ca²⁺] and [Cd²⁺], or [Ca²⁺] and [Ba²⁺], in a normal cell suspension. The second ion can be detected even if its spectrum when bound to Indo-1 is same as for the calcium-bound or the ion-free Indo-1, as long as there is a change in height. This is because the mathematical analysis uses not only the spectral shape, but also takes into account increases in total signal intensity. For maximum accuracy, whole spectra were analyzed. When 3 mM [Ba²⁺] was added to a B cell line that had been stimulated with anti-immunoglobulin to open receptor operated calcium channels, there was a sudden drop in 400 nm Indo-1 fluorescence. Spectral analysis showed that this was due to a drop in intracellular [Ca²⁺], which was consistent with blockage of the receptor-operated calcium current by extracellular Ba²⁺. The conductance for Ba²⁺ was also observable as a slow rise in total fluorescence. There was also a slow increase in intracellular [Ca²⁺] as barium accumulated in the cell, which was tentatively attributed to blockage of the plasma membrane calcium pump by intracellular Ba²⁺.     

Differential Modulation of 7-Ketocholesterol Toxicity Against PC12 Cells by Calmodulin Antagonists and Ca<Superscript>2+</Superscript> Channel Blockers

The present study assessed the influence of intracellular Ca²⁺ and calmodulin against the neurotoxicity of oxysterol 7-ketocholesterol in relation to the mitochondria-mediated cell death process and oxidative stress in PC12 cells. Calmodulin antagonists calmidazolium and W-7 prevented the 7-ketocholesterol-induced mitochondrial damage, leading to caspase-3 activation and cell death, whereas Ca²⁺ channel blocker nicardipine, mitochondrial Ca²⁺ uptake inhibitor ruthenium red, and cell permeable Ca²⁺ chelator BAPTA-AM did not reduce it. Exposure of PC12 cells to 7-ketocholesterol caused elevation of intracellular Ca²⁺ levels. Unlike cell injury, calmodulin antagonists, nicardipine, and BAPTA-AM prevented the 7-ketocholesterol-induced elevations of intracellular Ca²⁺ levels. The results show that the cytotoxicity of 7-ketocholesterol seems to be modulated by calmodulin rather than changes in intracellular Ca²⁺ levels. Calmodulin antagonists may prevent the cytotoxicity of 7-ketocholesterol by suppressing the mitochondrial permeability transition formation, which is associated with the increased formation of reactive oxygen species and the depletion of GSH.     

Peimine inhibits the growth and motility of prostate cancer cells and induces apoptosis by disruption of intracellular calcium homeostasis through Ca2+/CaMKII/JNK pathway

Prostate cancer (PC) is one of the most common malignant tumors in man. Peimine (PM) is a bioactive substance isolated from Fritillaria. Previous studies have shown that PM could inhibit the occurrence of a variety of cancers. However, the roles of PM in PC and its related mechanism have not been elucidated. Calcium (Ca²⁺) is an important intracellular messenger involved in a variety of cell processes. In this study, we found that the appropriate doses of PM (2.5, 5, and 10 μM) significantly inhibited the growth of PC cells (DU-145, LNCap, and PC-3), but has no significant effect on normal prostate cells (RWPE-1). In addition, PM treatment inhibited the invasion and migration of PC-3 cells and blocked the epithelial-mesenchymal transition process. These effects were exhibited a dose-dependent manner. Furthermore, the current results also showed that PM treatment significantly increased the Ca²⁺ concentration, the increased Ca²⁺ promoted the phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and c-Jun N-terminal kinase (JNK), further inhibited the growth and invasion of PC-3 cells, and induced its apoptosis. Ca²⁺ chelator BAPTA-AM (1 μM) could counteract the increase of intracellular Ca²⁺ concentration. Similarly, JNK pathway inhibitor SP600125 (10 μM) also inhibited cell growth and invasion and induced apoptosis. In addition, experiments in nude mice showed that PM inhibited tumor formation through Ca²⁺/CaMKII/JNK signaling pathway. In conclusion, our results show that PM inhibits the growth and motility of prostate cancer cells and induces apoptosis by disruption of intracellular calcium homeostasis through Ca²⁺/CaMKII/JNK pathway.     

The Role of Calcium in the Volume Regulation of Rat Lacrimal Acinar Cells

Earlier studies have suggested a role for Ca²⁺ in regulatory volume decrease (RVD) in response to hypotonic stress through the activation of Ca²⁺-dependent ion channels (Kotera & Brown, 1993; Park et al., 1994). The involvement of Ca²⁺ in regulating cell volume in rat lacrimal acinar cells was therefore examined using a video-imaging technique to measure cell volume. The trivalent cation Gd³⁺ inhibited RVD, suggesting that Ca²⁺ entry is important and may be via stretch-activated cation channels. However, Fura-2 loaded cells did not show an increase in [Ca²⁺] _i during exposure to hypotonic solutions. The absence of any changes in [Ca²⁺] _i resulted from the buffering of cytosolic Ca²⁺ by Fura-2 during hypotonic shock and therefore inhibition of RVD. The intracellular Ca²⁺ chelator, BAPTA, also inhibited the RVD response to hypotonic shock. An increase in [Ca²⁺] _i induced by either acetylcholine or ionomycin, was found to decrease cell volume under isotonic conditions in lacrimal acinar cells. Cell shrinkage was inhibited by tetraethylammonium ion, an inhibitor of Ca²⁺-activated K⁺ channels. On the basis of the presented data, we suggest an involvement of intracellular Ca²⁺ in controlling cell volume in lacrimal acinar cells. Received: 20 February 1998/Revised: 1 May 1998     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

Chemico-genetic identification of drebrin as a regulator of calcium responses

Store-operated calcium channels are plasma membrane Ca²⁺ channels that are activated by depletion of intracellular Ca²⁺ stores, resulting in an increase in intracellular Ca²⁺ concentration, which is maintained for prolonged periods in some cell types. Increases in intracellular Ca²⁺ concentration serve as signals that activate a number of cellular processes, however, little is known about the regulation of these channels. We have characterized the immuno-suppressant compound BTP, which blocks store-operated channel mediated calcium influx into cells. Using an affinity purification scheme to identify potential targets of BTP, we identified the actin reorganizing protein, drebrin, and demonstrated that loss of drebrin protein expression prevents store-operated channel mediated Ca²⁺ entry, similar to BTP treatment. BTP also blocks actin rearrangements induced by drebrin. While actin cytoskeletal reorganization has been implicated in store-operated calcium channel regulation, little is known about actin-binding proteins that are involved in this process, or how actin regulates channel function. The identification of drebrin as a mediator of this process should provide new insight into the interaction between actin rearrangement and store-operated channel mediated calcium influx.     

The role of Ca2+ mobilization and heterotrimeric G protein activation in mediating tyrosine phosphorylation signaling patterns in vascular smooth muscle cells

This work investigated the role of Ca²⁺ mobilization and heterotrimeric G protein activation in mediating angiotensin II-dependent tyrosine phosphorylation signaling patterns. We demonstrate that the predominant, angiotensin II-dependent, tyrosine phosphorylation signaling patterns seen in vascular smooth muscle cells are blocked by the intracellular Ca²⁺ chelator BAPTA-AM, but not by the Ca²⁺ channel blocker verapamil. Activation of heterotrimeric G proteins with NaF resulted in a divergent signaling effect; NaF treatment was sufficient to increase tyrosine phosphorylation levels of some proteins independent of angiotensin II treatment. In the same cells, NaF alone had no effect on other cellular proteins, but greatly potentiated the ability of angiotensin II to increase the tyrosine phosphorylation levels of these proteins. Two proteins identified in these studies were paxillin and Jak2. We found that NaF treatment alone, independent of angiotensin II stimulation, was sufficient to increase the tyrosine phosphorylation levels of paxillin. Furthermore, the ability of either NaF and/or angiotensin II to increase tyrosine phosphorylation levels of paxillin is critically dependent on intracellular Ca²⁺. In contrast, angiotensin II-mediated Jak2 tyrosine phosphorylation was independent of intracellular Ca²⁺ mobilization and extracellular Ca²⁺ entry. Thus, our data suggest that angiotensin II-dependent tyrosine phosphorylation signaling cascades are mediated through a diverse set of signaling pathways that are partially dependent on Ca²⁺ mobilization and heterotrimeric G protein activation.     

Cytosolic Calmodulin Is Increased in SK-N-SH Human Neuroblastoma Cells Due to Release of Calcium from Intracellular Stores

Abstract: Muscarinic receptor stimulation elicits a redistribution of calmodulin (CaM) from the membrane fraction to cytosol in the human neuroblastoma cell line SK-N-SH. Increasing the intracellular Ca²⁺ concentration with ionomycin also elevates cytosolic CaM. The aim of this study was to investigate the roles of extracellular and intracellular Ca²⁺ pools in the muscarinic receptor-mediated increases in cytosolic CaM in SK-N-SH cells. Stimulus-mediated changes in intracellular Ca²⁺ were monitored in fura-2-loaded cells, and CaM was measured by radioimmunoassay in the 100,000-g cytosol and membrane fractions. The influx of extracellular Ca²⁺ normally seen with carbachol treatment in SK-N-SH cells was eliminated by pretreatment with the nonspecific Ca²⁺ channel blocker Ni²⁺. Blocking the influx of extracellular Ca²⁺ had no effect on carbachol-mediated increases in cytosolic CaM (168 ± 18% of control values for carbachol treatment alone vs. 163 ± 28% for Ni²⁺ and carbachol) or decreases in membrane CaM. Similarly, removal of extracellular Ca²⁺ from the medium did not affect carbachol-mediated increases in cytosolic CaM (168 ± 26% of control). On the other hand, prevention of the carbachol-mediated increase of intracellular free Ca²⁺ by pretreatment with the cell-permeant Ca²⁺ chelator BAPTA/AM did attenuate the carbachol-mediated increase in cytosolic CaM (221 ± 37% of control without BAPTA/AM vs. 136 ± 13% with BAPTA/AM). The effect of direct entry of extracellular Ca²⁺ into the cell by K⁺ depolarization was assessed. Incubation of SK-N-SH cells with 60 mM K⁺ elicited an immediate and persistent increase in intracellular free Ca²⁺ concentration, but there was no corresponding alteration in CaM localization. On the contrary, in cells where intracellular Ca²⁺ was directly elevated by thapsigargin treatment, cytosolic CaM was elevated for at least 30 min while particulate CaM was decreased. In addition, treatment with ionomycin in the absence of extracellular Ca²⁺, which releases Ca²⁺ from intracellular stores, induced an increase in cytosolic CaM (203 ± 30% of control). The mechanism for the CaM release may involve activation of the α isozyme of protein kinase C, which was translocated from cytosol to membranes much more profoundly by thapsigargin than by K⁺ depolarization. These data demonstrate that release of Ca²⁺ from the intracellular store is important for the carbachol-mediated redistribution of CaM in human neuroblastoma SK-N-SH cells.