The epidermal growth factor-induced calcium signal in A431 cells

Addition of epidermal growth factor (EGF) to human A431 cells causes a 2-4-fold increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) as measured by quin-2 fluorescence. The EGF effect is rapid but transient: [Ca2+]i reaches a maximum within 30-60 s and then returns to its resting value (182 +/- 3 nM) over a 5-8-min period. The EGF-induced [Ca2+]i rise is completely dependent on extracellular Ca2+, is abolished by La3+ and Mn2+, and is not accompanied by changes in membrane potential (mean values of -64 mV). Serum also elicits a transient [Ca2+]i rise in A431 cells, but this response is not dependent on the presence of extracellular Ca2+. The tumor promoter 12-O-tetradecanoylphorbol 13-acetate completely inhibits the EGF- and serum-induced increases in [Ca2+]i without affecting basal [Ca2+]i levels. Our results, together with previous 45Ca2+ uptake data (Sawyer, S. T., and Cohen, S. (1981) Biochemistry 20, 6280-6286), suggest that while serum factors trigger the release of Ca2+ from internal stores, EGF acts by opening a voltage-independent Ca2+ channel in the plasma membrane. The data further suggest a role for protein kinase C in attenuating the Ca2+-mobilizing mechanisms of EGF and serum.     

Caffeine-Sensitive Calcium Stores in Bovine Adrenal Chromaffin Cells

Caffeine was used to study the intracellular Ca2+ pools of bovine chromaffin cells. Its effects on cytosolic Ca2+ concentration ([Ca2+]i) were examined using fura-2. Caffeine caused a transient increase in [Ca2+]i in the presence or absence of extracellular Ca2+. In the former case, the caffeine-induced [Ca2+]i increase was higher and stayed above the basal value for several minutes. In the latter case, the [Ca2+]i rise was lower and fell to the basal level within 1 min. These results suggest that caffeine increases [Ca2+]i by causing both Ca2+ influx and Ca2+ release from intracellular pools. In the absence of extracellular Ca2+, ionomycin but not caffeine caused a further increase in [Ca2+]i in cells that had been treated with caffeine. Apparently there are at least two intracellular Ca2+ pools, only one of which is sensitive to caffeine. The caffeine-induced [Ca2+]i rise became smaller when the cells were pretreated with the inositol trisphosphate-generating agonists, methacholine and bradykinin. In addition, methacholine was unable to initiate a [Ca2+]i transient after the cells had been treated with caffeine. The results indicate that the caffeine-sensitive Ca2+ pools overlap with the inositol trisphosphate-sensitive pool and that the size of the latter pool is smaller than that of the former. The caffeine-sensitive Ca2+ pools were refilled after high K+ treatment, which suggests that the caffeine-sensitive Ca2+ pools may be important in buffering the cytosolic Ca2+. The effect of caffeine on [Ca2+]i is not due to inhibition of phosphodiesterase. Our results support a Ca2+ entry model in which depletion of intracellular Ca2+ pools controls the rate of Ca2+ entry across the plasma membrane.     

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.     

The thapsigargin-sensitive intracellular Ca2+ pool is more important in plasma membrane Ca2+ entry than the IP3-sensitive intracellular Ca2+ pool in neuronal cell lines

In NG108-15 cells, bradykinin (BK) and thapsigargin (TG) caused transient increases in a cytosolic free Ca2+ concentration ([Ca2+]i), after which [Ca2+]i elevated by TG only declined to a higher, sustained level than an unstimulated level. In PC12 cells, carbachol (CCh) evoked a transient increase in [Ca2+]i followed by a sustained rise of [Ca2+]i, whereas [Ca2+]i elevated by TG almost maintained its higher level. In the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i induced by each drug we used was abolished. In addition, the rise in [Ca2+]i stimulated by TG was less affected after CCh or BK, whereas CCh or BK caused no increase in [Ca2+]i after TG. TG neither increased cellular inositol phosphates nor modified the inositol phosphates format on stimulated by CCh or BK. We conclude that TG may release Ca2+ from both IP3-sensitive and -insensitive intracellular pools and that some kinds of signalling to link the intracellular Ca2+ pools and Ca2+ entry seem to exist in neuronal cells.     

Evoked transient intracellular free Ca2+ changes and secretion in isolated bovine adrenal medullary cells

When isolated bovine adrenal medullary cells are incubated with the lipid-soluble Quin 2 acetoxymethyl ester, the ester permeates the plasma membrane and enters the cytosol, where it is hydrolysed by endogenous enzymes to yield an impermeant fluorescent indicator (Quin 2) which is sensitive to Ca2+ in the 0.1 microM range. This technique permits the average intracellular free Ca2+ level ([Ca2+]i) to be determined in a suspension of adrenal medullary cells. Unstimulated cells have a [Ca2+]i of 97 +/- 4 nM (n = 69). This level seems independent of extracellular calcium in the range 0.5-2 mM. When the extracellular calcium concentration is lowered to ca. 10(-7) M, however, [Ca2+]i decreases. A transient increase in [Ca2+]i occurs when cells are challenged with either acetylcholine or a high potassium medium. The time course of the [Ca2+]i transient rises to a maximum within seconds, and decreases to basal levels over minutes. The maximum level of [Ca2+]i associated with secretion is very variable. Hexamethonium, methyoxyverapamil, and the absence of extracellular calcium block not only the secretory response but also the [Ca2+]i transient. The action of acetylcholine leading to the Ca2+]i transient is blocked when cells are suspended in a depolarizing medium. Extracellular magnesium inhibits both the [Ca2+]i transient and the secretory response evoked by acetylcholine. Secretion is, however, more sensitive to magnesium inhibition than is calcium entry. The magnitudes of the [Ca2+]i transient and the secretory response decrease as the concentration of intracellular Quin 2 increases. Measurements of the amount of indicator titrated with calcium, as a result of an acetylcholine or potassium challenge, suggest that the increase in the apparent calcium content of the cytosol might arise from two contributing sources of calcium entry.     

Simultaneous analysis of cell Ca2+ and Ca2(+)-stimulated chloride conductance in colonic epithelial cells (HT-29).

We used perforated patch, whole-cell current recordings and video-based fluorescence ratio imaging to monitor the relation of plasma membrane ionic conductances to intracellular free Ca2+ within individual colonic epithelial cells (HT-29). The Ca2(+)-mediated agonist, neurotensin, activated K+ and Cl- conductances that showed different sensitivities to [Ca2+]i. The Cl- conductance was sensitive to increases or decreases in [Ca2+]i around the resting value of 76 +/- 32 (mean +/- SD) nM (n = 46), whereas activation of the K+ conductance required at least a 10-fold rise in [Ca2+]i. Neurotensin increased [Ca2+]i by stimulating a transient intracellular Ca2+ release, which was followed by a sustained rise in [Ca2+]i due to Ca2+ influx from the bath. The onset of the initial [Ca2+]i transient, monitored at a measurement window over the cell interior, lagged behind the rise in Cl- current during agonist stimulation. This lag was not present when the [Ca2+]i rise was due to Ca2+ entry from the bath, induced either by the agonist or by the Ca2+ ionophore ionomycin. The temporal differences in [Ca2+]i and Cl- current during the agonist-induced [Ca2+]i transient can be explained by a localized Ca2+ release from intracellular stores in the vicinity of the plasma membrane Cl- channel. Chloride currents recover toward basal values more rapidly than [Ca2+]i after the agonist-induced [Ca2+]i transient, and, during a sustained neurotensin-induced [Ca2+]i rise, Cl- currents inactivate. These findings suggest that an inhibitory pathway limits the increase in Cl- conductance that can be evoked by agonist. Because this Cl- current inhibition is not observed during a sustained [Ca2+]i rise induced by ionomycin, the inhibitory pathway may be mediated by another agonist-induced messenger, such as diacylglycerol.     

ATP-induced calcium mobilization in human neutrophils

Extracellular ATP at 10 microM increased the concentration of cytoplasmic free Ca2+ ( [Ca2+]i) 3-fold in human neutrophils. The [Ca2+]i was measured by fura-2 fluorescence. The effect was rapid but transient: [Ca2+]i reached a maximum within 10 s and then returned to its resting value after 2-3 min. The rise in [Ca2+]i elicited by ATP was unaffected by the removal of extracellular Ca2+, indicating that the primary source of Ca2+ is from intracellular stores. In contrast to ATP, neither ADP nor AMP, at concentrations as high as 100 microM, caused any detectable changes in [Ca2+]i. Among other nucleotide triphosphates tested, UTP was as effective as ATP in causing a transient rise in [Ca2+]i, and prevented a subsequent response to ATP. Similarly, ATP prevented a subsequent response to UTP but the second response could be obtained when the initially added ATP was removed by the addition of hexokinase. Phorbol myristate acetate, the activator of Ca2+, phospholipid-dependent protein kinase, completely inhibited the ATP-induced increases in [Ca2+]i without affecting the basal [Ca2+]i level. The results suggest that extracellular ATP stimulates human neutrophils by causing the release of calcium from intracellular storage pools by mechanisms which can be inhibited by phorbol myristate acetate.     

Characterization of hypoxia-induced [Ca2+]i rise in rabbit pulmonary arterial smooth muscle cells

We have investigated the effects of hypoxia on the intracellular Ca2+ concentration ([Ca2+]i) in rabbit pulmonary (PASMCs) and coronary arterial smooth muscle cells with fura-2. Perfusion of a glucose-free and hypoxic (PO2<50 mmHg) external solution increased [Ca2+]i in cultured as well as freshly isolated PASMCs. However it had no effect on [Ca2+]i in freshly isolated coronary arterial myocytes. In the absence of extracellular Ca2+, hypoxic stimulation elicited a transient [Ca2+]i increase in cultured PASMCs which was abolished by the simultaneous application of cyclopiazonic acid and ryanodine, suggesting the involvement of sarcoplasmic reticulum (SR) Ca2+ store. Pretreatment with the mitochondrial protonophore, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) enhanced the [Ca2+]i rise in response to hypoxia. A short application of caffeine gave a transient [Ca2+]i rise which was prolonged by CCCP. Decay of the caffeine-induced [Ca2+]i transients was significantly slowed by treatment of CCCP or rotenone. After full development of the hypoxia-induced [Ca2+]i rise, nifedipine did not decrease [Ca2+]i. These data suggest that the [Ca2+]i increase in response to hypoxia may be ascribed to both Ca2+ release from the SR and the subsequent activation of nifedipine-insensitive capacitative Ca2+ entry. Mitochondria appear to modulate hypoxia induced Ca2+ release from the SR.     

Regulation of cytosolic free calcium in fura-2-loaded rat parotid acinar cells

In order to analyze the factors regulating agonist-stimulated Ca2+ mobilization, cytosolic free [Ca2+] ([Ca2+]i) was measured directly in fura-2-loaded rat parotid acinar cells. Stimulation of muscarinic receptors by carbachol produced a dose-dependent rise in [Ca2+]i. In the presence of external Ca2+, the initial transient rise was followed by a maintained elevation. The maintained elevation is dependent on the presence of external Ca2+. Removal of Ca2+ by addition of EGTA caused a rapid decline in [Ca2+]i back to base line. In the absence of external Ca2+, only an initial transient peak in [Ca2+]i was seen which then declined to base line; the maintained elevation in [Ca2+]i could then be evoked by addition of Ca2+ in the continued presence of carbachol. Muscarinic receptor occupation by carbachol is required to maintain the elevated level of [Ca2+]i; addition of the muscarinic antagonist, atropine, caused [Ca2+]i to decline back to the basal level. The maintained elevation in [Ca2+]i, but not the initial transient peak, can also be blocked by Ni2+ but was unaffected by the organic Ca2+ antagonists. Total substitution of external Na+ with the impermeant cation, N-methyl-D-glucamine, had no effect on either the initial or the maintained response to carbachol; however, total substitution of Na+ with K+ attenuated the maintained response while not affecting the initial peak. Refilling of the intracellular Ca2+ store was also studied and found to take place in the absence of agonist and with no substantial elevation in [Ca2+]i. These experiments also showed that not all of the intracellular vesicular Ca2+ stores can be released by agonists. From these results, we propose a model for the regulation of [Ca2+]i.     

Acetylcholine-evoked increase in the cytoplasmic Ca2+ concentration and Ca2+ extrusion measured simultaneously in single mouse pancreatic acinar cells.

The intracellular free Ca2+ concentration ([free Ca2+]i) was measured simultaneously with the Ca2+ extrusion from single isolated mouse pancreatic acinar cells placed in a microdroplet of extracellular solution using the fluorescent probes fura-2 and fluo-3. The extracellular solution had a low total calcium concentration (15-35 microM), and acetylcholine (ACh), applied by microionophoresis, therefore only evoked a transient elevation of [free Ca2+]i lasting about 2-5 min. The initial sharp rise in [free Ca2+]i from about 100 nM toward 0.5-1 microM was followed within seconds by an increase in the total calcium concentration in the microdroplet solution ([Ca]o). The rate of this rise of [Ca]o was dependent on the [free Ca2+]i elevation, and as [free Ca2+]i gradually decreased Ca2+ extrusion declined with the same time course. Ca2+ extrusion following ACh stimulation was not influenced by removal of all Na+ in the microdroplet solution indicating that the Ca2+ extrusion is not mediated by Na(+)-Ca2+ exchange but by the Ca2+ pump. The amount of Ca2+ extruded during the ACh-evoked transient rise in [free Ca2+]i corresponded to a decrease in the total intracellular Ca concentration of about 0.7 mM which is close to previously reported values (0.5-1 mM) for the total concentration of mobilizable calcium in these cells. Our results therefore demonstrate directly the ability of the Ca2+ pump to rapidly remove the large amount of Ca2+ released from the intracellular pools during receptor activation.     

Dual-phase response of bovine pulmonary artery endothelial cells to agonists which increase free cytoplasmic calcium concentration

The regulation of free cytoplasmic calcium concentration ([Ca2+]i) was studied in bovine pulmonary artery endothelial cells (BPAEC). The cells were seeded on the inner surface of glass cuvettes, grown to confluency and loaded with INDO-1. Using a multiwavelength method for estimation of [Ca2+]i it was shown that in Ca2+ containing medium a rapid rise of [Ca2+]i occurs in response to bradykinin, ATP or thrombin followed by a much slower decrease in free cytoplasmic calcium. Binding of extracellular Ca2+ by EGTA lowered basal [Ca2+]i but had no effect on the rate of agonist-induced [Ca2+]i increase or its absolute amount. In contrast, the kinetics of [Ca2+]i decrease were entirely different. A rapid (less than 0.5 min) decrease in [Ca2+]i to the basal level was observed immediately after the maximum had been achieved. If excess Ca2+ was added to the medium after EGTA, a second [Ca2+]i rise in response to the agonists occurred. The decrease in [Ca2+]i after the second peak was several times slower than the decrease in Ca2+ free medium. It is concluded that Ca2+ entry from the external medium had no effect on the maximal increase in [Ca2+]i but provides a severalfold increase in the duration the endothelial cell responses to the agonists.     

Inhibition of glutamate-induced delayed calcium deregulation by 2-APB and La3+ in cultured cortical neurones

Exposure of neurones in culture to excitotoxic levels of glutamate results in an initial transient spike in [Ca2+]i followed by a delayed, irreversible [Ca2+]i rise governed by rapid kinetics, with Ca2+ originating from the extracellular medium. The molecular mechanism responsible for the secondary Ca2+ rise is unknown. Here, we report that the delayed Ca2+ entry in cortical neurones is diminished by 2-aminoethoxydiphenyl borate (2-APB: IC50 = 62 +/- 9 microm) and La3+ (IC50 = 7.2 +/- 3 microm), both known to inhibit transient receptor potential (TRP) and store-operated Ca2+ (SOC) channels. Application of thapsigargin, however, failed to exacerbate the delayed Ca2+ deregulation, arguing against a store depletion event as the stimulus for induction of the secondary [Ca2+]i rise. In addition, these neurones did not exhibit SOC entry. Unexpectedly, application of ryanodine or caffeine significantly inhibited glutamate-induced delayed Ca2+ deregulation. In basal Ca2+ entry experiments, La3+ and 2-APB modulated the rapid rise in [Ca2+]i caused by exposure of neurones to Ca2+ after pre-incubating in a calcium-free medium. This basal Ca2+ influx was mitigated by extracellular Mg2+ but not aggravated by thapsigargin, ryanodine or caffeine. These results indicate that 2-APB and La3+ influence non-store-operated Ca2+ influx in cortical neurones and that this route of Ca2+ entry is involved in glutamate-induced delayed Ca2+ deregulation.     

Cytoplasmic calcium transients due to single action potentials and voltage-clamp depolarizations in mouse pancreatic B-cells.

Changes in the cytoplasmic free calcium concentration ([Ca2+]i) in pancreatic B-cells play an important role in the regulation of insulin secretion. We have recorded [Ca2+]i transients evoked by single action potentials and voltage-clamp Ca2+ currents in isolated B-cells by the combination of dual wavelength emission spectrofluorimetry and the patch-clamp technique. A 500-1000 ms depolarization of the B-cell from -70 to -10 mV evoked a transient rise in [Ca2+]i from a resting value of approximately 100 nM to a peak concentration of 550 nM. Similar [Ca2+]i changes were associated with individual action potentials. The depolarization-induced [Ca2+]i transients were abolished by application of nifedipine, a blocker of L-type Ca2+ channels, indicating their dependence on influx of extracellular Ca2+. Following the voltage-clamp step, [Ca2+]i decayed with a time constant of approximately 2.5 s and summation of [Ca2+]i occurred whenever depolarizations were applied with an interval of less than 2 s. The importance of the Na(+)-Ca2+ exchange for B-cell [Ca2+]i maintenance was evidenced by the demonstration that basal [Ca2+]i rose to 200 nM and the magnitude of the depolarization-evoked [Ca2+]i transients was markedly increased after omission of extracellular Na+. However, the rate by which [Ca2+]i returned to basal was not affected, suggesting the existence of additional [Ca2+]i buffering processes.     

Internal Ca2+ mobilization and secretion in bovine adrenal chromaffin cells

Since secretion from intact bovine adrenal chromaffin cells in response to depolarization by nicotine is triggered by a rise in the concentration of intracellular Ca2+ ([Ca2+]i) to about 200-300 nM above basal, it has been assumed that the failure of the inositol 1,4,5-trisphosphate (InsP3)-mobilizing muscarinic agonists to induce secretion reflects the fact that the 50 nM rise in [Ca2+]i they elicit is insufficient to trigger the exocytotic machinery. A recent report, however, has demonstrated that some of the nicotine-induced rise in [Ca2+]i could originate from the InsP3-releasable Ca2+ store. The role of this Ca2+ store in secretion from bovine adrenal chromaffin cells is therefore unclear. In order to investigate in more detail the role of the InsP3-sensitive Ca2+ store in secretion from these cells, we have used a combination of an InsP3-mobilizing muscarinic agonist and the sesquiterpene lactone thapsigargin (TG), which releases internal Ca2+ without concomitant breakdown of inositol lipids or protein kinase C activation, to examine the events which follow depletion of the releasable Ca2+ store in these cells. Monitoring [Ca2+]i using Fura-2 demonstrated that TG released Ca2+ from the InsP3-sensitive store and, additionally, that the Ca2+ response to TG was composed of two distinct, temporally separated, components: a) a slow (1 min) increase in [Ca2+]i to approximately 50 nM above basal that was independent of extracellular Ca2+ and b) the maintenance of this level at a new steady-state that was dependent on the continual entry of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diacylglycerol increases cytosolic free Ca2+ concentration in rat pituitary cells. Relationship to thyrotropin-releasing hormone action

To elucidate possible functions of elevation of endogenous diacylglycerol induced by thyrotropin-releasing hormone in pituitary cells, we have studied the actions of two synthetic diacylglycerols, sn-1-oleoyl-2-acetylglycerol (OAG) and sn-1,2-dioctanoylglycerol (DiC8), on cytosolic free calcium concentration ([Ca2+]i) in GH4C1 cells. OAG induced an immediate increase in [Ca2+]i which gradually reached a peak that was twice the basal level after the first min; [Ca2+]i then returned to remain at basal level after 3 min. The increase in [Ca2+]i was dependent on the concentration of OAG added with two apparent potencies; half-maximal actions on [Ca2+]i were observed at 70 nM and greater than 20 microM. The increase in [Ca2+]i induced by OAG was blocked completely by chelating extracellular calcium, or by pretreatment with calcium channel blockers. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which itself induces a rise in [Ca2+]i in these cells that is similar in time course, magnitude, and drug sensitivity to that of OAG, blocked completely the actions of subsequent exposure to OAG. Analogous results were obtained using DiC8, although DiC8 induced a transient inhibition to 75% of basal levels of [Ca2+]i after the initial increase in [Ca2+]i, and DiC8 was less potent than OAG. These data indicated that diacylglycerols induce influx of extracellular calcium in these cells, possibly by activation of voltage-dependent Ca2+ channels. Furthermore, diacylglycerols and phorbol esters appear to utilize a common pathway in eliciting these actions on [Ca2+]i, possibly involving activation of a protein kinase C. These actions of diacylglycerol provide a pathway by which thyrotropin-releasing hormone may act to enhance calcium channel activity.     

Effect of the antidepressant sertraline on Ca2+fluxes in Madin-Darby canine renal tubular cells

The effect of the antidepressant sertraline on cytosolic-free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether sertraline changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Sertraline at concentrations between 1and 100 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+ implicating Ca2+ entry and release both contributed to the [Ca2+]i rise. Sertraline induced Mn2+ influx, leading to quench of fura-2 fluorescence, suggesting Ca2+ influx. This Ca2+ influx was inhibited by suppression of phospholiapase A2 but not by store-operated Ca2+ channel blockers and protein kinase C/A modulators. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitors nearly abolished sertraline-induced Ca2+ release. Conversely, pretreatment with sertraline partly reduced inhibitor-induced [Ca2+]i rise, suggesting that sertraline released Ca2+ from endoplasmic reticulum. Inhibition of phospholipase C did not much alter sertraline-induced [Ca2+]i rise. Collectively, in MDCK cells, sertraline induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive Ca2+ channels.     

Regulation of cytosolic Ca2+ in clonal human muscle cell cultures

Human muscle cells were grown in culture and clonally selected for fusion potential. The concentration of cytoplasmic ionized calcium, [Ca2+]i, was measured in monolayers of fused myotubes using the Ca2+ indicator indo-1. The contributions of independent routes of Ca2+ influx and efflux to/from the cytoplasm on [Ca2+]i were investigated. The resting [Ca2+]i was 170-190 nM in different cell clones. Acetylcholine increased [Ca2+]i by about 2-fold in the presence of absence of extracellular Ca2+. Cell depolarization by K+ elevated [Ca2+]i about 3-fold, and this increase was largely dependent on extracellular Ca2+. Replacing Na+ by N-methylglucammonium+ raised [Ca2+]i greater than 5-fold, and 50% of this increase was dependent on extracellular Ca2+. All these increases in [Ca2+]i were transient, returning to basal [Ca2+]i within 2 min. It is concluded that cells in culture [Ca2+]i can be elevated transiently by acetylcholine through Ca2+ release from intracellular stores, and by K through Ca2+ influx. The return to basal [Ca2+]i is due to Na+/Ca2+ exchange and Ca2+-ATPase activity.     

Thapsigargin increases cytoplasmic free Ca2+ without influencing steroidogenesis in chicken granulosa cells.

The effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) and progesterone production were determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye Fura-2. Thapsigargin stimulated a 4.6 +/- 0.2-fold increase in [Ca2+]i from a resting level of 55 +/- 6 nM up to 233 +/- 23 nM (n = 8) in 100% of the cells tested (n = 86). However, two different response patterns were observed. Dependent on the cell populations, a maximally effective concentration of thapsigargin (100 nM) stimulated either a rapid (within 16 +/- 2 s) transient increase in [Ca2+]i or a slowly (99 +/- 20 s) developing and sustained increase in [Ca2+]i. Both [Ca2+]i responses were concentration (0.001-1 microM)-dependent with an EC50 around 40 nM. The transient [Ca2+]i response occurred in the absence of extracellular Ca2+ and was unaffected by pretreating the cells with the Ca2+ channel blockers methoxyverapamil (50 microM) or lanthanum (1 mM). The plateau phase of the sustained [Ca2+]i response returned to resting level in the absence of extracellular Ca2+, but remained elevated in the presence of methoxyverapamil (50 microM) or lanthanum (1 mM). Despite its ability to cause transient or prolonged increases in [Ca2+]i, thapsigargin (0.001-1 microM) did not affect basal or luteinizing hormone-stimulated progesterone production by chicken granulosa cells.     

Control of the intracellular Ca(2+)-concentration and the inositol phosphate accumulation in dog thyrocyte primary culture: evidence for different kinetics of Ca(2+)-phosphatidylinositol cascade activation and for involvement in the regulation of H2O2 production

Carbachol, through a muscarinic receptor, thyrotropin-releasing hormone (TRH), prostaglandin F2 alpha (PGF2 alpha), bradykinin, and adenosine triphosphate (ATP) increased the apparent [Ca2+]i (intracellular free Ca2(+)-concentration) of dog thyrocytes in primary culture. The [Ca2+]i measured by the Quin-2 technique rose immediately after the addition of the agonists and reached a maximal value after less than 30 seconds. Afterwards, the [Ca2+]i declined to a plateau higher than the basal level when the cells were triggered with carbachol. By contrast, in most experiments with PGF2 alpha and in the case of bradykinin, TRH, and ATP, the [Ca2+]i returned to the basal value. If the extracellular Ca2+ was chelated by excess of EGTA, the addition of all agents caused a sharp reduced transient rise in the [Ca2+]i followed by a decline of the [Ca2+]i often below the basal level (especially in the case of carbachol). It is suggested that the first transient phase of these responses is due at least in part to the mobilisation of Ca2+ from intracellular stores whereas the second sustained phase of the response to carbachol mainly originates from an increased Ca2+ influx into the thyrocytes. Carbachol, bradykinin, TRH, PGF2 alpha, and ATP also increased generation of inositol phosphates in dog thyrocytes. This effect was sustained when the cells were triggered with carbachol and was more transient with bradykinin, TRH, PGF2 alpha, or ATP. All these agents and the phorbdester TPA as well as forskolin enhanced to various extent the thyrocyte H2O2 generation. This enhancement was severely reduced in the absence of extracellular Ca2+ and was mimicked by Ca2+ ionophores in the presence of extracellular Ca2+ especially in synergy with protein kinase C activators. These data suggest that the dog thyrocyte H2O2 generation, the limiting step of the thyroid hormone synthesis, is modulated by carbachol, TRH, PGF2 alpha, bradykinin, and ATP through their action on the Ca2(+)-phosphatidylinositol cascade.     

Effect of cyclosporin A on the membrane potential and Ca2+ level of human lymphoid cell lines and mouse thymocytes

The effect of the immunosuppressive cyclosporin A (CsA) on the cytosolic free Ca2+ concentration ([Ca2+]i) and membrane potential of human B and T lymphoblastoid cells and mouse thymocytes was studied in order to reveal some features of the early stage of drug-cell interaction. Cytosolic free Ca2+ concentration of the cells was measured by spectrofluorimetry using indo-1 and quin2 fluorescent calcium indicators. Membrane potential was monitored in a flow cytometer with oxonol dye. CsA applied at 2-20 micrograms/ml final concentrations caused a dose-dependent, rapid, transient rise of [Ca2+]i in all cell types. This effect could be blocked by chelating the extracellular Ca2+ with EGTA but was not sensitive to Ca2+ channel blockers verapamil and nifedipine or K+ channel blocker 4-aminopyridine. A possible explanation for the calcium mobilizing effect of CsA is an ionophore-like mode of action at the cell membrane level. Besides directly interfering with mitogenic signals, the elevation of [Ca2+]i could be responsible for an initial hyperpolarization observed in CsA-treated T lymphocytes. This hyperpolarization, however, was not detectable in B lymphoblastoid cells. A further difference between B and T cells was the diverse pattern of depolarization following CsA treatment. This variance in the behaviour of T and B lymphocytes and the diversity of membrane transport systems in its background could account for the different final outcome of the drug-cell interaction.