Evidence for TRH-induced influx of extracellular Ca2+ in pituitary GH4C1 cells

The aim of the study was to investigate the relationship between thyrotropin-releasing hormone (TRH)-induced changes in intracellular free Ca2+ ([Ca2+]i), and influx of extracellular Ca2+ in Fura 2 loaded pituitary GH4C1 cells. Stimulating the cells with TRH in a Ca(2+)-containing buffer induced a biphasic change in [Ca2+]i. First, a transient increase in [Ca2+]i, followed by a sustained phase. In cells stimulated with TRH in a Ca(2+)-free buffer, the transient increase in [Ca(2+)]i was decreased (p less than 0.05), and the sustained phase was totally abolished. Addition of Ni2+ prior to TRH blunted the component of the TRH-induced transient increase in [Ca2+]i dependent on influx of Ca2+. In the presence of extracellular Mn2+, TRH stimulated quenching of Fura 2 fluorescence. This quenching was blocked by Ni2+. The results indicate that both the TRH-induced transient increase in [Ca2+]i as well as the sustained phase in [Ca2+]i in GH4C1 cells is dependent on influx of extracellular Ca2+.     

Rapid mobilization of cellular Ca2+ in bovine parathyroid cells evoked by extracellular divalent cations. Evidence for a cell surface calcium receptor

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in dissociated bovine parathyroid cells using the fluorescent indicator quin-2 or fura-2. Small increases in the concentration of extracellular Ca2+ produced relatively slow, monophasic increases in [Ca2+]i in quin-2-loaded cells, but rapid and transient increases followed by lower, yet sustained (steady-state), [Ca2+]i increases in fura-2-loaded cells. The different patterns of change in [Ca2+]i reported by quin-2 and fura-2 appear to result from the greater intracellular Ca2+-buffering capacity present within quin-2-loaded cells, which tends to damp rapid and transient changes in [Ca2+]i. In fura-2-loaded parathyroid cells, other divalent cations (Mg2+, Sr2+, Ba2+) also evoked transient increases in [Ca2+]i, and their competitive interactions suggest that they all affect Ca2+ transients by acting on a common site. In contrast, divalent cations failed to cause increases in steady-state levels of cytosolic Ca2+. Low concentrations of La3+ (0.5-10 microM) depressed steady-state levels of cytosolic Ca2+ elicited by extracellular Ca2+ but were without effect on transient increases in [Ca2+]i elicited by extracellular Ca2+, Mg2+ or Sr2+, suggesting that increases in the steady-state [Ca2+]i arise from the influx of extracellular Ca2+. Mg2+- and Sr2+-induced cytosolic Ca2+ transients persisted in the absence of extracellular Ca2+ but were abolished by pretreatment with ionomycin. These results show that cytosolic Ca2+ transients arise from the mobilization of cellular Ca2+ from a nonmitochondrial pool. Extracellular divalent cations thus appear to act at some site on the surface of the cell, and this site can be considered a "Ca2+ receptor" which enables the parathyroid cell to detect small changes in the concentration of extracellular Ca2+.     

Membrane potential and Na(+)-K+ pump activity modulate resting and bradykinin-stimulated changes in cytosolic free calcium in cultured endothelial cells from bovine atria

The effects of membrane potential on resting and bradykinin-stimulated changes in [Ca2+]i were measured in fura-2 loaded cultured endothelial cells from bovine atria by spectrofluorimetry. The basal and bradykinin-stimulated release of endothelium-derived relaxing factor, monitored by bioassay methods, were dependent on extracellular Ca2+. Similarly, the plateau phase of the biphasic [Ca2+]i response to bradykinin stimulation exhibited a dependence on extracellular Ca2+, whereas the initial transient [Ca2+]i peak was refractory to the removal of extracellular Ca2+. The effect of membrane depolarization on the plateau phase of the bradykinin-induced change in [Ca2+]i was determined by varying [K+]o. The resting membrane potential measured under current clamp conditions was positively correlated with the extracellular [K+] (52 mV change/10-fold change in [K+]o). The observed decrease in resting and bradykinin-stimulated changes in [Ca2+]i upon depolarization is consistent with an ion transport mechanism where the influx is linearly related to the electrochemical gradient for Ca2+ entry (Em - ECa). The inhibition of bradykinin-stimulated Ca2+ entry by isotonic K+ was not due to the absence of extracellular Na+ since Li+ substitution did not inhibit the agonist-induced Ca2+ entry. In K(+)-free solutions and in the presence of ouabain, bradykinin evoked synchronized oscillations in [Ca2+]i in confluent endothelial cell monolayers. These [Ca2+]i oscillations between the plateau and resting [Ca2+]i levels were dependent on extracellular Ca2+ and K+ concentrations. Although the mechanism(s) underlying [Ca2+]i oscillations in vascular endothelial cells is unclear, these results suggest a role of the membrane conductance.     

Receptor-stimulated phospholipase A2 activation is coupled to influx of external calcium and not to mobilization of intracellular calcium in C62B glioma cells

C62B rat glioma cells respond to muscarinic cholinergic stimulation with transient inositol phosphate formation and phospholipase A2-dependent arachidonic acid liberation. Since phospholipase A2 is a Ca2+-sensitive enzyme, we have examined the role of the agonist-stimulated Ca2+ response in production of the arachidonate signal. The fluorescent indicator fura-2 was used to monitor changes in cytoplasmic Ca2+ levels ([Ca2+]i) of C62B cells following acetylcholine treatment. In the presence of extracellular Ca2+, acetylcholine induces a biphasic [Ca2+]i response consisting of an initial transient peak that precedes arachidonate liberation and a sustained elevation that outlasts the phospholipase A2 response. The initial [Ca2+]i peak is not altered by the absence of external Ca2+ and therefore reflects intracellular Ca2+ mobilization. The sustained elevation phase is dependent on the influx of external Ca2+; it is lost in Ca2+-free medium and restored on the addition of Ca2+. Pretreating cells with phorbol dibutyrate substantially inhibits acetylcholine-stimulated inositol phosphate formation and the peak [Ca2+]i response without affecting the sustained elevation in [Ca2+]i. This suggests that the release of internal Ca2+ stores by inositol 1,4,5-trisphosphate can be blocked without interfering with Ca2+ influx. Pretreatment with phorbol also fails to affect acetylcholine-stimulated arachidonate liberation, demonstrating that phospholipase A2 activation does not require normal intracellular Ca2+ release. Stimulated arachidonate accumulation is totally inhibited in Ca2+-free medium and restored by the subsequent addition of Ca2+. Pretreatment with verapamil, a voltage-dependent Ca2+ channel inhibitor, also blocks both the sustained [Ca2+]i elevation and arachidonate liberation without altering peak intracellular Ca2+ release. We conclude that the influx of extracellular Ca2+ is tightly coupled to phospholipase A2 activation, whereas large changes in [Ca2+]i due to mobilization of internal Ca2+ stores are neither sufficient nor necessary for acetylcholine-stimulated phospholipase A2 activation.     

Na+-Ca2+ exchange and Ca2+ efflux in transfected Chinese hamster ovary cells

The objective of this study was to assess the contribution of Na+-Ca2+ exchange activity to Ca2+ efflux at various cytosolic Ca2+ concentrations ([Ca2+]i) in transfected Chinese hamster cells expressing the bovine cardiac Na+-Ca2+ exchanger. Ionomycin was added to fura-2 loaded cells and the resulting [Ca2+]i transient was monitored in Ca2+-free media with or without extracellular Na+. The presence of Na+ reduced both the amplitude and duration of the [Ca2+]i transient. Na+ had similar effects when the peak of the [Ca2+]i transient was buffered to 100 nM by cytosolic EGTA, or when Ca2+ was slowly released from internal stores with thapsigargin. Ca2+ efflux following ionomycin addition was directly measured with extracellular fura-2 and followed a biphasic time course (t(1/2) approximately = 10 s and 90s). The proportion of total efflux owing to the rapid phase was increased by Na+ and reduced by EGTA-loading. Na+ accelerated the initial rate of Ca2+ efflux by 65% in unloaded cells but only by 16% in EGTA-loaded cells. In both cases, the stimulation by Na+ was less than expected, given the pronounced effects of Na+ on the [Ca2+]i transient. We conclude that the exchanger contributes importantly to Ca2+ efflux activity at all [Ca2+]i values above 40 nM. We also suggest that Ca2+ efflux pathways may involve non-cytosolic or local routes of Ca2+ traffic.     

Sphingosine stimulates calcium mobilization in rat parotid acinar cells

In fura-2-loaded parotid acinar cells, 50-200 microM sphingosine induced an increase in cytosolic Ca2+ ([Ca2+]i). When extracellular Ca2+ was chelated by EGTA, 50 microM sphingosine failed to increase [Ca2+]i, but 100 or 200 microM sphingosine induced a slight and transient increase in [Ca2+]i. The addition of LaCl3 to the medium resulted in the same effect as chelation of extracellular Ca2+. When cells were incubated in low Ca2+ medium containing sphingosine, and extracellular Ca2+ was subsequently added, a rapid increase in [Ca2+]i depending on the concentration of sphingosine was shown. In low Ca2+ medium, a slight increase in [Ca2+]i induced by high concentrations of sphingosine was not shown after the transient increase in [Ca2+]i elicited by methacholine. Inhibitors of protein kinase C, H-7 and K252a, did not mimic the effect of sphingosine on [Ca2+]i. These results suggest that sphingosine stimulates Ca(2+)-influx and further stimulates the release of Ca2+ from agonist-sensitive intracellular pools by a mechanism that is independent of protein kinase C.     

Effect of celecoxib on Ca2+ fluxes and proliferation in MDCK renal tubular cells

The effect of celecoxib on renal tubular cells is largely unexplored. In Madin Darby canine kidney (MDCK) cells, the effect of celecoxib on intracellular CaCa2+ concentration ([Ca2+]i) and proliferation was examined by using the Ca(2 +)-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium, respectively. Celecoxib (> or =1 micro M) caused an increase of [CaCa2+]i in a concentration-dependent manner. Celecoxib-induced [CaCa2+]i increase was partly reduced by removal of extracellular CaCa2+. Celecoxib-induced CaCa2+ influx was independently suggested by MnCa2+ influx-induced fura-2 fluorescence quench. In Ca(2 +)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2 +)-ATPase, caused a monophasic [CaCa2+]i increase, after which celecoxib only induced a tiny [CaCa2+]i increase; conversely, pretreatment with celecoxib completely inhibited thapsigargin-induced [CaCa2+]i increases. U73122, an inhibitor of phospholipase C, abolished ATP (but not celecoxib)-induced [CaCa2+]i increases. Overnight incubation with 1 or 10 micro M celecoxib decreased cell viability by 80% and 100%, respectively. These data indicate that celecoxib evokes a [CaCa2+]i increase in renal tubular cells by stimulating both extracellular CaCa2+ influx and intracellular CaCa2+ release and is highly toxic to renal tubular cells in vitro.     

Two thrombin-activated Ca2+ channels in human platelets.

The regulation of extracellular Ca2+ entry into fura-2-loaded human platelets was examined following stimulation with thrombin. In the presence of external Ca2+, stimulation of platelets with thrombin resulted in a rapid increase, followed by a plateau, in intracellular Ca2+ concentration ([Ca2+]i). Pretreatment with wortmannin, a specific inhibitor of myosin light chain kinase, suppressed only the plateau phase and had no effect on the initial rapid increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thrombin induced a transient and relatively small increase in [Ca2+]i caused by Ca2+ release from internal stores. When Ca2+ was added subsequently to the Ca(2+)-free medium within 10 min after thrombin activation, a marked increase in [Ca2+]i was seen, reflecting thrombin-stimulated external Ca2+ entry. With the Ca(2+)-free medium, wortmannin did not affect either the Ca2+ mobilization from the internal stores or the rapid external Ca2+ entry at early time points (within 5 s) after thrombin stimulation, whereas it significantly inhibited Ca2+ entry when Ca2+ was added later (at 3 min). Wortmannin inhibition of this late Ca2+ entry and that of 20-kDa myosin light chain phosphorylation after thrombin stimulation were dose- and preincubation time-dependent and correlated well with each other. These results suggest that two different channels are responsible for Ca2+ entry in human platelets at the early and late phases of thrombin stimulation and that the channel responsible for the late phase of Ca2+ entry may be activated by a mechanism involving myosin light chain kinase.     

Characterization and Ca2+ Requirement of Histamine-Induced Catecholamine Secretion in Cultured Bovine Chromaffin Cells

The stimulation of cultured bovine chromaffin cells with histamine induced a continuous catecholamine secretion (EC50 = 3 x 10(-7) M) via the H1 receptor, in addition to an initial catecholamine burst due to a nonspecific stimulatory effect at higher doses (greater than or equal to 10(-4) M). The continuous secretion showed little desensitization and lasted for more than 1 h. In fura-2-loaded cells, the stimulation with histamine evoked a transient rise of intracellular free Ca2+ concentration ([Ca2+]i) which lasted only for a few minutes and was followed by a sustained [Ca2+]i rise which continued for more than 20 min. The addition of an activator for the L-type voltage-sensitive Ca2+ channel, i.e., Bay K 8644 (1 microM), facilitated the sustained [Ca2+]i rise, as well as the secretion, whereas the addition of relatively high concentrations of Ca(2+)-channel blockers (10 microM) suppressed the sustained [Ca2+]i rise and part of the secretion. Removal of extracellular Ca2+ completely abolished continuous secretion and sustained [Ca2+]i rise. When the external Ca2+ level was elevated, both sustained [Ca2+]i rise and continuous secretion were enhanced in a similar Ca(2+)-dependent manner, showing saturation with around 1-3 mM Ca2+. This Ca2+ dependence was clearly different from that observed with high K+ and nicotine, which is mediated by the L-type Ca2+ channel, in which the responses showed little or no saturation when the Ca2+ level was increased. The results indicate that stimulation with histamine induces a continuous secretion via the H1 receptor, in addition to a transient and nonspecific secretion at higher doses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histamine-Induced increases in intracellular free Ca2+ levels in hepatoma cells

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatoma cells were evaluated using fura-2 as a fluorescent Ca2+ dye. Histamine (0.2-5 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of about 1 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In Ca2+-free medium, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase of a magnitude 7-fold greater than control. Histamine (5 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 5 microM pyrilamine but was not altered by 50 microM cimetidine. Together, this study shows that histamine induced [Ca2+]i increases in human hepatoma cells by stimulating H1, but not H2, histamine receptors. The [Ca2+]i signal was caused by Ca2+ release from thapsigargin-sensitive endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, accompanied by Ca2+ entry.     

2-O-methyl PAF as a Ca2+ mobilizer in Madin Darby canine kidney cells

In Madin-Darby canine kidney (MDCK) cells, the effect of 2-O-methyl PAF, an inactive analogue of platelet activating factor (PAF), on intracellular Ca2+ concentration ([Ca2+]i) was measured by using the Ca2+-sensitive fluorescent dye fura-2. 2-O-methyl PAF (> or = 15 microM) caused a rapid rise of [Ca2+]i in a concentration-dependent manner. 2-O-methyl PAF-induced [Ca2+]i rise was partly reduced by removal of extracellular Ca2+. 2-O-methyl PAF-induced extracellular Ca2+ influx was also suggested by Mn2+ influx-induced fura-2 fluorescence quench. The 2-O-methyl PAF-induced Ca2+ influx was blocked by nifedipine, verapamil and diltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which 2-O-methyl PAF failed to increase [Ca2+]i; also, pretreatment with 2-O-methyl PAF depleted thapsigargin-sensitive Ca2+ stores. U73122, an inhibitor of phospholipase C, abolished ATP (but not 2-O-methyl PAF)-induced [Ca2+]i rise. These findings suggest that 2-O-methyl PAF evokes a rapid increase in [Ca2+]i in renal tubular cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release.     

Mechanisms of miconazole-induced rise in cytoplasmic calcium concentrations in Madin Darby canine kidney (MDCK) cells

The effect of miconazole on intracellular calcium levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was studied using fura-2 as the Ca2+ indicator. Miconazole increased [Ca2+]i dose-dependently at concentrations of 5-100 microM. The [Ca2+]i transient consisted of an initial rise, a gradual decay and an elevated plateau (220 s after addition of the drug). Removal of extracellular Ca2+ partly reduced the miconazole response. Mn2+ quench of fura-2 fluorescence confirmed that miconazole induced Ca2+ influx. The miconazole-sensitive intracellular Ca2+ store overlapped with that sensitive to thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump, because 20 microM miconazole depleted the thapsigargin (1 microM)-sensitive store, and conversely, thapsigargin abolished miconazole-induced internal Ca2+ release. Miconazole (20-50 microM) partly inhibited the capacitative Ca2+ entry induced by 1 microM thapsigargin, measured by depleting intracellular Ca2+ store in Ca(2+)-free medium followed by addition of 10 mM CaCl2. Miconazole induced capacitative Ca2+ entry on its own. Pretreatment with 0.1 mM La3+ partly inhibited 20 microM miconazole-induced Mn2+ quench of fura-2 fluorescence and [Ca2+]i rise, suggesting that miconazole induced Ca2+ influx via two pathways separable by 0.1 mM La3+. Miconazole-induced internal Ca2+ release was not altered when the cytosolic level of inositol 1,4,5-trisphosphate (IP3) was substantially inhibited by the phospholipase C inhibitor U73122.     

Fendiline increases [Ca2+]i in Madin Darby canine kidney (MDCK) cells by releasing internal Ca2+ followed by capacitative Ca2+ entry

The effect of fendiline, a documented inhibitor of L-type Ca2+ channels and calmodulin, on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated using fura-2 as a Ca2+ probe. Fendiline at 5-100 microM significantly increased [Ca2+]i concentration-dependently. The [Ca2+]i rise consisted of an initial rise and a slow decay. External Ca2+ removal partly inhibited the Ca2+ signals induced by 25-100 microM fendiline by reducing both the initial rise and the decay phase. This suggests that fendiline triggered external Ca2+ influx and internal Ca2+ release. In Ca(2+)-free medium, pretreatment with 50 microM fendiline nearly abolished the [Ca2+]i rise induced by 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor, and vice versa, pretreatment with thapsigargin prevented fendiline from releasing internal Ca2+. This indicates that the internal Ca2+ source for fendiline overlaps with that for thapsigargin. At a concentration of 50 microM, fendiline caused Mn2+ quench of fura-2 fluorescence at the 360 nm excitation wavelenghth, which was inhibited by 0.1 mM La3+ by 50%, implying that fendiline-induced Ca2+ influx has two components separable by La3+. Consistently, 0.1 mM La3+ pretreatment suppressed fendiline-induced [Ca2+]i rise, and adding La3+ during the rising phase immediately inhibited the signal. Addition of 3 mM Ca2+ increased [Ca2+]i after preincubation with 50-100 microM fendiline in Ca(2+)-free medium. However, 50-100 microM fendiline inhibited 1 microM thapsigargin-induced capacitative Ca2+ entry. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 inhibited 50 microM fendiline-induced internal Ca2+ release by 48%, but inhibition of phospholipase C with 2 microM U73122 or inhibition of phospholipase D with 0.1 mM propranolol had no effect. Collectively, we have found that fendiline increased [Ca2+]i in MDCK cells by releasing internal Ca2+ in a manner independent of inositol-1,4,5-trisphosphate (IP3), followed by external Ca2+ influx.     

Mechanisms of nordihydroguaiaretic acid-induced [Ca2+]i increases in MDCK cells

The effect of nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells has been investigated. NDGA (10-100 microM) increased [Ca2+]i concentration-dependently. The [Ca2+]i increase comprised an initial slow rise and a plateau over a time period of 5 min. Ca2+ removal partly inhibited the Ca2+ signals induced by 25-100 microM NDGA and abolished that induced by 10 microM NDGA. In Ca(2+)-free medium, pretreatment with 0.1 mM NDGA for 12 min abolished the [Ca2+]i increase induced by the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) and the endoplasmic reticulum (ER) Ca2+ pump inhibitor thapsigargin (1 microM). However, 0.1 mM NDGA still increased [Ca2+]i after Ca2+ stores had been depleted by pretreating with 2 microM CCCP, 1 microM thapsigargin and 0.1 mM cyclopiazonic acid. NDGA (50 microM) activated Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength, which was almost abolished by 50 microM La3+. This implies NDGA induced Ca2+ influx mainly via a La(3+)-sensitive pathway. Consistently, 50 microM La3+ pretreatment inhibited 0.1 mM NDGA-induced [Ca2+]i increase. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 0.1 mM NDGA in Ca(2+)-free medium, suggesting NDGA activated capacitative Ca2+ entry. Pretreatment with 0.1 mM NDGA for 200 s prior to Ca2+ did not alter 1 microM thapsigargin-induced capacitative Ca2+ entry. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 reduced 0.1 mM NDGA-induced Ca2+ release by 65%, but inhibiting phospholipase C with 2 microM U73122 had little effect. This suggests NDGA-induced Ca2+ release was independent of inositol 1,4,5-trisphosphate (IP3), but was modulated by phospholipase A2.     

Involvement of calcium and protein kinase C in the activation of the Na+/H+ exchanger in cultured bovine aortic endothelial cells stimulated by extracellular ATP

We have studied the activation of the Na+/H+ exchanger which leads to the intracellular alkalinization in cultured bovine aortic endothelial cells stimulated by extracellular ATP. The alkalinization induced by ATP was largely dependent on extracellular Ca2+ and the rate of alkalinization was decreased by about 60% in the absence of extracellular Ca2+. ATP caused a rapid and transient increase and a subsequent sustained increase of the intracellular Ca2+ concentration ([Ca2+]i) in the Ca2+ buffer, while only the rapid and transient increase of [Ca2+]i was observed in the absence of extracellular Ca2+. The Ca2+-depleted cells prepared by incubation in Ca2+-free buffer containing 0.1 mM EGTA showed only a slight increase of [Ca2+]i with no alkalinization on stimulation by ATP. The alkalinization was inhibited by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase C, but not by another isoquinoline analogue (HA 1004), which has a less inhibitory effect on the kinase. Phorbol 12-myristate 13-acetate also induced the alkalinization by the activation of the Na+/H+ exchanger. Neither dibutyryl cyclic AMP nor dibutyryl cyclic GMP affected the alkalinization induced by ATP. Treatment of the cells by pertussis and cholera toxins had no effect on the alkalinization. The results suggest that the increase in [Ca2+]i is essential for the ATP-induced activation of the Na+/H+ exchanger in cultured bovine aortic endothelial cells and a protein kinase C-dependent pathway is involved in the activation.     

Effect of diethylstilbestrol on Ca2+ handling and cell viability in human breast cancer cells

In human breast cancer cells, the effect of the widely prescribed estrogen diethylstilbestrol (DES) on intracellular Ca2+ concentrations ([Ca2+]i) and cell viability was explored by using fura-2 and trypan blue exclusion, respectively. DES caused a rise in [Ca2+]i in a concentration-dependent manner (EC50 = 15 microM). DES-induced [Ca2+]i rise was reduced by 80 % by removal of extracellular Ca2+. DES-induced Mn(2+)-associated quench of intracellular fura-2 fluorescence also suggests that DES induced extracellular Ca2+ influx. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of DES on [Ca2+]i was greatly inhibited. Conversely, pretreatment with DES to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+, whereas ionomycin added afterward still released some Ca2+. These findings suggest that in human breast cancer cells, DES increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum. Acute trypan blue exclusion studies suggest that 10-20 NM DES killed cells in a time-dependent manner.     

Secretagogue-induced mobilization of an intracellular Mg2+ pool in rat sublingual mucous acini.

The regulation of the intracellular free Mg2+ concentration ([Mg2+]i) was monitored in rat sublingual mucous acini using dual wavelength microfluorometry of the Mg(2+)-sensitive dye mag-fura-2. Acini attached to coverslips and superfused continuously with a Mg(2+)-containing medium (0.8 mM) have a steady-state [Mg2+]i of 0.35 +/- 0.01 mM. Adjusting the extracellular Mg2+ concentration to 0 and 10 mM or removing extracellular Na+ did not alter the resting [Mg2+]i. Stimulation with the Ca(2+)-mobilizing, muscarinic agonist, carbachol, induced a sustained increase in [Mg2+]i (approximately 50%; t1/2 < 20 s; Kd approximately 1.5 microM), the magnitude and the duration of which were unchanged in Mg(2+)-depleted medium indicating that the rise in [Mg2+]i was generated by Mg2+ release from an intracellular Mg2+ pool. Forskolin, which increases the intracellular cAMP content, produced a small, transient increase in the [Mg2+]i (< 10%). Muscarinic stimulation in a Ca(2+)-free medium blunted the initial increase in [Mg2+]i by approximately 50%, whereas the sustained increase in [Mg2+]i was lost. When the muscarinic-induced increase in [Ca2+]i was blocked by 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate, an inhibitor of the agonist-sensitive intracellular Ca2+ release pathway, both the initial and the sustained phases of the increase in [Mg2+]i were virtually eliminated. Thapsigargin and 2,5-di-(terbutyl)-1,4-benzohydroquinone, which increase [Ca2+]i by inhibiting microsomal Ca(2+)-ATPase, caused a dramatic increase in [Mg2+]i. Stimulation in a Na(+)-free medium or in the presence of bumetanide, an inhibitor of Na+/K+/2Cl- cotransport, blunted the agonist-induced rise in [Mg2+]i (approximately 50%), whereas ouabain, a Na+,K(+)-ATPase inhibitor, had no significant effect. FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), a mitochondrial uncoupler, mobilized an intracellular Mg2+ pool as well. The carbachol-induced increase in [Mg2+]i was markedly inhibited by FCCP (approximately 80%), suggesting that the same pool(s) of Mg2+ were primarily involved. The above results provide strong evidence that Ca(2+)-mobilizing agonists increase cytoplasmic free [Mg2+] by releasing an intracellular pool of Mg2+ that is associated with a rise in the [Na+]i.     

Effect of maprotiline on Ca(2+) movement in human neuroblastoma cells

In human neuroblastoma IMR32 cells, the effect of the anti-depressant maprotiline on baseline intracellular Ca2+ concentrations ([Ca2+]i) was explored by using the Ca2+-sensitive probe fura-2. Maprotiline at concentrations greater than 100 microM caused a rapid rise in [Ca2+]i in a concentration-dependent manner (EC50 = 200 microM). Maprotiline-induced [Ca2+]i rise was reduced by 50% by removal of extracellular Ca2+. Maprotiline-induced [Ca2+]i rises were inhibited by half by nifedipine, but was unaffected by verapamil or diiltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of maprotiline on [Ca2+]i was abolished. U73122, an inhibitor of phospholipase C, did not affect maprotiline-induced [Ca2+]i rises. These findings suggest that in human neuroblastoma cells, maprotiline increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum via a phospholiase C-independent manner.     

Characterization of histamine-induced increases in intracellular free Ca2+ concentrations in Chang liver cells.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in Chang liver cells were investigated by using fura-2 as a Ca2+ dye. Histamine (0.2-50 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 0.8 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the maximum [Ca2+]i signal and abolished the sustained phase. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase with a magnitude 7-fold greater than control. In Ca2+-free medium, after treatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. Histamine (5 microM)-induced intracellular Ca2+ release was abolished     

Depolarization of the membrane potential decreases the ATP-induced influx of extracellular Ca2+ and the refilling of intracellular Ca2+ stores in rat thyroid FRTL-5 cells.

The aim of the present study was to investigate the effect of membrane depolarization on ATP-induced changes in intracellular Ca2+ ([Ca2+]i) and the refilling of intracellular Ca2+ stores in thyroid follicular FRTL-5 cells. Depolarizing the cells with 50 mM K+, an amount sufficient to almost totally depolarize the cells as determined by bisoxonal, significantly reduced the ATP-induced uptake of 45Ca2+. This effect was not dependent on an enhanced efflux of Ca2+, as no difference in the ATP-induced efflux of 45Ca2+ was obtained between control cells and depolarized cells. The ATP-induced transient increase in [Ca2+]i in Fura-2 loaded cells was not altered by depolarization, whereas the ATP-induced plateau in [Ca2+]i was decreased compared with control cells. Furthermore, in cells stimulated with ATP in a Ca(2+)-free buffer, readdition of Ca2+ after the termination of the ATP response induced a decreased response in [Ca2+]i in depolarized cells. Refilling of intracellular Ca2+ stores was investigated by first stimulating the cells with noradrenaline (NA). The effect of NA was then terminated with prazosin, and the cells restimulated with ATP. In cells depolarized with high K+, the response to ATP was decreased compared with that seen in control cells. The results thus suggest that both the ATP-induced influx of extracellular Ca2+ and the refilling of intracellular Ca2+ stores is decreased in depolarized FRTL-5 cells.