Receptor-mediated calcium influx in PC12 cells. ATP and bradykinin activate two independent pathways

In the neurosecretory cell line PC12 the cytosolic free Ca2+ concentration, [Ca2+]i, and membrane potential were affected by both external ATP and the nonapeptide bradykinin, BK. The latter caused a rapid and large release of Ca2+ from intracellular stores (Ca2+ redistribution) and, in the presence of external Ca2+, a long lasting, but moderate Ca2+ influx, which was insensitive to dihydropyridine blockers. On the contrary, ATP evoked a [Ca2+]i rise which rapidly inactivated. At least three different mechanisms accounted for the ATP-induced increase in [Ca2+]i: less than 20% of the total response was due to intracellular Ca2+ redistribution, consistent with a small increase in inositol 1,4,5-trisphosphate level; the rest (over 80%) was equally accounted for by ATP-activated cation channels and voltage-gated Ca2+ channels. ATP and BK (the latter after K+ channel blockade) caused plasma membrane depolarization. With both agonists the inward current was carried by both Na+ and Ca2+, although the BK-activated current appeared to be more selective for Ca2+. Channels triggered by ATP and BK differed not only in their cation selectivity, but also in modulation by both [Ca2+]i and drugs such as the phorbol ester phorbol 12-myristate 13-acetate and the new antagonist of ligand-activated Ca2+ influx, SK&F 96365.     

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.     

Carbachol and bradykinin elevate cyclic AMP and rapidly deplete ATP in cultured rat sympathetic neurons.

The agonists carbachol (CCh) and bradykinin (BK) and 54 mM KCl (high K+) were among the most potent stimulants of cyclic AMP (cAMP) production in cultured rat sympathetic neurons, measured with the use of a high-fidelity assay developed for small samples. The rise in cAMP evoked by CCh (through muscarinic receptors), BK, and high K+ was inhibited in Ca2(+)-depleted medium (1.3 mM Ca2+ and 2 mM BAPTA or EGTA), which also prevented the sustained rise in [Ca2+]i evoked by each of these stimuli, showing that elevation of cAMP requires extracellular Ca2+ and, possibly, Ca2+ influx. Preliminary results obtained with the novel calmodulin inhibitor CGS 9343B, which blocked the elevation of cAMP, and with the cyclogenase inhibitor indomethacin, which partially blocked the actions of the agonists but not those of high K+, suggest that calmodulin and arachidonate metabolites may be two components of the signaling pathway. In addition to their effects on cAMP metabolism, CCh, muscarine, and BK, but not nicotine, caused a 30-40% decrease in ATP levels. This effect was much greater than that evoked by high K+ and was largely inhibited by CGS 9343B but slightly enhanced in the Ca(+)-depleted medium, showing that agonists are still active in the absence of [Ca2+]o. Thus, agonists that activate phosphoinositide metabolism can also increase cAMP production and substantially deplete cells of ATP. These novel actions may have to be taken into account when the mechanisms by which such agonists regulate cell function are being considered.     

Bradykinin-evoked acetylcholine release via inositol trisphosphate-dependent elevation in free calcium in neuroblastoma x glioma hybrid NG108-15 cells

The mechanism underlying the bradykinin (BK)-induced increase of acetylcholine (ACh) release was studied in neuroblastoma x glioma hybrid NG108-15 cells and their synapses formed onto mouse muscle cells. External application of BK or iontophoretic injection of extrinsic inositol 1,4,5-trisphosphate (InsP3) into the cytoplasm of NG108-15 cells produced membrane hyperpolarization in the hybrid cells and an increase in the frequency of miniature end-plate potentials (MEPPs) in paired myotubes. Ba2+ blocked the hyperpolarization in response to BK, but facilitation of MEPPs was still observed. InsP3-dependent facilitation of MEPPs was also observed in cells where the InsP3 injections produced no detectable hyperpolarization or even depolarization. Real-time quantitative monitoring of intracellular free Ca2+ concentration [( Ca2+]i) with fura-2 in single NG108-15 cells showed that BK application or InsP3 injection induced an elevation of [Ca2+]i which coincided in time with membrane hyperpolarization recorded from the same cell. The [Ca2+]i rise produced by InsP3 injection started from the single site of injection and that produced by BK began from a deep compartment of the cytoplasm of the NG108-15 cells. The BK- and InsP3-evoked facilitation of MEPPs and the [Ca2+]i rise were relatively independent of extracellular Ca2+. These findings suggest that the BK-induced ACh release results not from membrane potential changes but from a transient InsP3-dependent elevation of [Ca2+]i.     

Differential regulation of thrombin- or ATP-induced mobilization of intracellular Ca2+ by prostacyclin receptor in mouse mastocytoma cells.

Thrombin induced an increase in [Ca2+]i in mouse mastocytoma P-815 cells. This increase was markedly reduced by prior exposure to pertussis toxin (PT) but not by removal of extracellular Ca2+, suggesting that thrombin stimulates phospholipase C via a PT-sensitive GTP-binding protein. ATP also induced an increase in [Ca2+]i. This increase was insensitive to PT but completely suppressed on removal of extracellular Ca2+, suggesting that ATP stimulates Ca2+ influx in a PT-insensitive manner. Iloprost, a stable prostacyclin analogue, increased the cellular cAMP level and dose-dependently inhibited the thrombin-induced increase in [Ca2+]i, whereas the ATP-induced increase in [Ca2+]i was markedly enhanced by iloprost. Cyclic AMP analogues, dibutyryl cAMP and 8-bromo cAMP, also inhibited the increase in [Ca2+]i induced by thrombin and promoted that by ATP, indicating that the inhibitory and stimulatory effects of iloprost are mediated by cAMP. These results suggest that the prostacyclin receptor differentially regulates two distinct Ca2+ mobilizing systems via cAMP in mastocytoma cells.     

Mitogen-induced [Ca2+]i changes in individual human fibroblasts. Image analysis reveals asynchronous responses which are characteristic for different mitogens

Temporal changes in intracellular free Ca2+ concentration ([Ca2+]i) in cultured human foreskin fibroblasts were investigated using image analysis techniques to simultaneously monitor Lys-bradykinin (BK)- or thrombin-induced elevations of [Ca2+]i in each individual cell within a microscopic field. Responses to BK are heterogeneous with respect to the shapes of the [Ca2+]i time courses. Furthermore, the onsets of these responses follow a variable lag period such that the individual cell responses occur asynchronously. The asynchrony and heterogeneity of individual cell responses are not related to cell cycle differences since noncycling cells respond in a similar manner. When cells are ranked according to order of an initial response to BK (the first cell to respond is ranked 1, the second to respond is 2, etc.), restimulation of the same cells with BK elicits a similar order of cell responses, and the shape of the [Ca2+]i time course of an individual cell is similar for both responses to BK. If cells that were stimulated with BK are washed and restimulated with thrombin (which produces [Ca2+]i changes similar to those induced by BK), the response order to thrombin does not correspond to the response order following BK stimulation. These data suggest that the asynchrony of [Ca2+]i changes induced by BK or thrombin is characteristic for each mitogen and may be determined by cell-to-cell variation in receptor number.     

Neuropeptide Y-induced intracellular Ca2+ increases in vascular smooth muscle cells

The effect of neuropeptide Y (NPY) on cytosolic free Ca2+ concentration ([Ca2+]i) was studied in cultured smooth muscle cells from porcine aorta (PASMC) and compared with the effect of bradykinin (BK) and angiotensin II (ATII) on [Ca2+]i. All peptides induced dose-dependent and transient rises in [Ca2+]i which were not blocked by extracellular EGTA, but the NPY response was different from the others' as follows. First, the [Ca2+]i rise induced by NPY was not as rapid as that induced by BK or ATII. Second, pertussis toxin abolished the [Ca2+]i rise induced by NPY, but not by BK or ATII. Third, following initial treatment with BK, PASMC were able to respond to NPY, but not to ATII. Finally, BK and ATII, but not NPY, significantly increased inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) generation. Although NPY attenuated forskolin-induced accumulation of cyclic AMP, forskolin- and 3-isobutyl-1-methyl-xanthine-induced alterations in intracellular cyclic AMP did not affect the NPY-induced [Ca2+]i rise. These results suggest that NPY increases [Ca2+]i by a pertussis toxin-sensitive GTP binding protein-involved mechanism which is not mediated by the intracellular messengers such as Ins(1,4,5)P3 and cyclic AMP.     

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.     

Manipulation of Intracellular Calcium in NCB-20 Cells

A number of lines of evidence indicate that the Ca2+ and cyclic AMP signalling systems interact in NCB-20 cells. However, to date, the regulation of [Ca2+]i homeostasis has not been studied in this cell line. The present study aimed to clarify our understanding of [Ca2+]i homeostasis in these cells and to evaluate tools that manipulate [Ca2+]i, independently of protein kinase C effects. Bradykinin, by a B2-receptor, elevated [Ca2+]i by a pertussis-toxin-insensitive mechanism. The BK-stimulated [Ca2+]i rise originated from intracellular sources, without a contribution from Ca2+ entry mechanisms. The effect of BK was precluded by pretreatment with thapsigargin and ionomycin--compounds that elevated [Ca2+]i independent of phospholipase C activation. Both compounds, however, exerted effects in addition to stimulating release of Ca2+ from BK-sensitive stores; the BK-sensitive Ca2+ pool was a subset of the thapsigargin-sensitive pool; ionomycin strongly stimulates Ca2+ entry. Activation of protein kinases A and C attenuated the duration of the BK-induced rise in [Ca2+]i, without affecting the peak [Ca2+]i, suggesting interference with the BK response at a step downstream of the activation of phospholipase C. Application of these approaches should enhance the delineation of the consequences of Ca2+ mobilization on cyclic AMP accumulation.     

Activation of the plasma membrane Ca2+ pump during agonist stimulation of pancreatic acini.

The role of internal stores and plasma membrane Ca2+ pumps in controlling [Ca2+]i during agonist stimulation and their regulation by agonists are not well understood. We report here measurements of intracellular ([Ca2+]i) and extracellular ([Ca2+]o) Ca2+ concentrations in agonist-stimulated pancreatic acini in an effort to directly address these questions. Stimulation of acini suspended in Ca(2+)-free or Ca(2+)-containing medium with Ca2+ mobilizing agonists resulted in a typical transient increase in [Ca2+]i. Thapsigargin, a specific inhibitor of internal Ca2+ pumps, inhibited the rate of [Ca2+]i reduction after agonist stimulation by approximately 40%. Under the same conditions, thapsigargin had no effect on the rate of the unidirectional Ca2+ efflux across the plasma membrane as revealed by measurements of [Ca2+]o. These findings suggest that internal Ca2+ pumps actively remove Ca2+ from the cytosol during continued agonist stimulation. The correlation between the reduction in [Ca2+]i and the increase in [Ca2+]o showed that Ca2+ efflux from cells stimulated with agonist and thapsigargin represent Ca2+ efflux across the plasma membrane. Inhibition of cells exposed to agonist and thapsigargin with a specific antagonist sharply reduced the rates of the [Ca2+]i decrease and the accompanied [Ca2+]o increase. Hence, at comparable [Ca2+]i, Ca2+ efflux from stimulated cells was about 3-fold faster than that from resting cells, indicating that agonists directly activate the plasma membrane Ca2+ pump. To study the role of [Ca2+]i increase in plasma membrane Ca2+ pump activation the acini were loaded with 1,2-bis-(2-aminophenoxyethane-N,N,N',N')-tetraacetic acid (BAPTA), and [Ca2+]o was measured during agonist stimulation. Surprisingly, although BAPTA completely prevented the increase in [Ca2+]i, Ca2+ efflux rate was reduced by only 34%. These findings provide the first evidence for Ca(2+)-independent activation of the plasma membrane Ca2+ pump by Ca2+ mobilizing agonists.     

Positive cooperativity between the thrombin and bradykinin B2 receptors enhances arachidonic acid release

Bradykinin (BK) or kallikreins activate B2 receptors (R) that couple Galpha(i) and Galpha(q) proteins to release arachidonic acid (AA) and elevate intracellular Ca2+ concentration ([Ca2+]i). Thrombin cleaves the protease-activated-receptor-1 (PAR1) that couples Galpha(i), Galpha(q), and Galpha(12/13) proteins. In Chinese hamster ovary cells stably transfected with human B2R, thrombin liberated little AA, but it significantly potentiated AA release by B2R agonists. We explored mechanisms of cooperativity between constitutively expressed PAR1 and B2R. We also examined human endothelial cells expressing both Rs constitutively. The PAR1 agonist hexapeptide (TRAP) was as effective as thrombin. Inhibitors of components of Galpha(i), Galpha(q), and Galpha(12/13) signaling pathways, and a protein kinase C (PKC)-alpha inhibitor, G?-6976, blocked potentiation, while phorbol, an activator, enhanced it. Several inhibitors, including a RhoA kinase inhibitor, a [Ca2+]i antagonist, and an inositol-(1,3,4)-trisphosphate R antagonist, reduced mobilization of [Ca2+]i by thrombin and blocked potentiation of AA release by B2R agonists. Because either a nonselective inhibitor (isotetrandrine) of phospholipase A2 (PLA2) or a Ca2+-dependent PLA2 inhibitor abolished potentiation of AA release by thrombin, while a Ca2+-independent PLA2 inhibitor did not, we concluded that the mechanism involves Ca2+-dependent PLA2 activation. Both thrombin and TRAP modified activation and phosphorylation of the B2R induced by BK. In lower concentrations they enhanced it, while higher concentrations inhibited phosphorylation and diminished B2R activation. Protection of the NH2-terminal Ser1-Phe2 bond of TRAP by an aminopeptidase inhibitor made this peptide much more active than the unprotected agonist. Thus PAR1 activation enhances AA release by B2R agonists through signal transduction pathway.     

Localization and heterogeneity of agonist-induced changes in cytosolic calcium concentration in single bovine adrenal chromaffin cells from video imaging of fura-2.

Temporal and spatial changes in the concentration of cytosolic free calcium ([Ca2+]i) in response to a variety of secretagogues have been examined in adrenal chromaffin cells using digital video imaging of fura-2-loaded cells. Depolarization of the cells with high K+ or challenge with nicotine resulted in a rapid and transient elevation of [Ca2+]i beneath the plasma membrane consistent with Ca2+ entry through channels. This was followed by a late phase in which [Ca2+]i rose within the cell interior. Agonists that act through mobilization of inositol phosphates produced an elevation in [Ca2+]i that was most marked in an internal region of the cell presumed to be the site of IP3-sensitive stores. When the same cells were challenged with nicotine or high K+, to trigger Ca2+ entry through voltage-dependent channels, the rise in [Ca2+]i was most prominent in the same localized region of the cells. These results suggest that Ca2+ entry through voltage-dependent channels results in release of Ca2+ from internal stores and that the bulk of the measured rise in [Ca2+]i is not close to the exocytotic sites on the plasma membrane. Analysis of the time courses of changes in [Ca2+]i in response to bradykinin, angiotensin II and muscarinic agonists showed that these agonists produced highly heterogeneous responses in the cell population. This heterogeneity was most marked with muscarinic agonists which in some cells elicited oscillatory changes in [Ca2+]i. Such heterogeneous changes in [Ca2+]i were relatively ineffective in eliciting catecholamine secretion from chromaffin cells. A single large Ca2+ transient, with a component of the rise in [Ca2+]i occurring beneath the plasma membrane, may be the most potent signal for secretion.     

Synchronized oscillation of Ca2+ entry and Ca2+ release in agonist-stimulated AR42J cells

Oscillation in [Ca2+]i induced by agonists has been described in many cell types and is thought to reflect Ca2+ release from and uptake into internal stores. We measured [Ca2+]i and Mn2+ entry in single cells of the pancreatic acinar cell line AR42J loaded with Fura 2 to examine the behavior of Ca2+ influx across the plasma membrane (Ca2+ entry) during agonist-evoked [Ca2+]i oscillation. Addition of extracellular Ca2+ (Ca2+out) to agonist-stimulated cells bathed in Ca2(+)-free medium resulted in a marked [Ca2+]i increase blocked by La3+. The use of Mn2+ as a congener of Ca2+ to follow unidirectional Ca2+ movement reveals an oscillatory activation of Ca2+ entry by Ca2(+)-mobilizing agonists. The frequency at which Ca2+ entry oscillated matched the frequency of Ca2+ release from intracellular stores. Ca2+ entry is activated after completion of Ca2+ release and is inactivated within the time span of each [Ca2+]i spike. These studies reveal a new aspect of [Ca2+]i oscillation in agonist-stimulated cells, that is the oscillatory activation of [Ca2+]i entry during [Ca2+]i oscillation.     

Hormone-induced increase in free cytosolic calcium and glycogen phosphorylase activation in rat hepatocytes incubated in normal and low-calcium media.

The action of alpha 1-adrenergic agonists (noradrenaline in the presence of propranolol), vasopressin and angiotensin on the intracellular free Ca2+ concentration, [Ca2+]i, was determined by using the fluorescent dye quin2 in isolated rat liver cells. In the presence of external Ca2+ (1.8 mM), 1 microM-noradrenaline induced an increase in [Ca2+]i up to about 800 nM without apparent delay, whereas 10 nM-vasopressin and 1 nM-angiotensin increased [Ca2+]i to values higher than 1500 nM with a lag period of about 6s. The successive addition of the hormones and of their specific antagonists indicated that the actions of the three Ca2+-mobilizing hormones occurred without apparent desensitization (over 6 min) and via independent receptors. The relative contributions of internal and external Ca2+ pools to the cell response were determined by studying the hormone-mediated [Ca2+]i increase and glycogen phosphorylase activation in low-Ca2+ media (22 microM). In this medium: (1) [Ca2+]i was lowered and the hormones initiated a transient instead of a sustained increase in [Ca2+]i; subsequent addition (2 min) of a second hormone promoted a lesser increase in [Ca2+]i; in contrast, the subsequent addition (2 min) of Ca2+ (1.8 mM) caused [Ca2+]i to increase to a value close to that initiated by the hormone in control conditions, the amplitude of the latter response being dependent on the concentration of Ca2+ added to the medium; (2) returning to normal Ca2+ (1.8 mM) restored the resting [Ca2+]i and allowed the hormone added 2 min later to promote a large increase in [Ca2+]i whose final amplitude was also dependent on the concentration of Ca2+ added beforehand. Similar results were found when the same protocol was applied to the glycogen phosphorylase activation. It is concluded that Ca2+ influx is required for a maximal and sustained response and to reload the hormone-sensitive stores.     

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.     

Calcium oscillation induced by bradykinin in polyoma middle T antigen-transformed NIH3T3 fibroblasts: evidence for dependence on protein kinase C

Bradykinin (BK) triggered long lasting intracellular free calcium ([Ca2+]i) oscillation in polyoma middle T-transformed cell line MT3 cells but not in the parental NIH3T3 cells. This periodic [Ca2+]i fluctuation was extracellular Ca(2+)-dependent and blocked by pretreatments with Ca2+ channel blockers, SK&F 96365 or CdCl2, suggesting a crucial role of Ca2+ entry across the plasma membrane possibly through a receptor-operated Ca2+ channel. Brief pretreatment with phorbol myristate acetate (PMA) completely abolished the BK-induced [Ca2+]i oscillation, and a protein kinase C (PKC) inhibitor, H-7, reversed the effect of PMA, indicating involvement of PKC. On the other hand, in some cells, oscillatory changes in [Ca2+]i were seen without agonist stimulation. The spontaneous oscillation was also dependent on extracellular Ca2+, but neither treatment with PMA nor H-7 had any effect under the same conditions.     

Effect of activation of muscarinic receptors on intracellular free calcium and secretion in bovine adrenal chromaffin cells

Stimulation of the nicotinic receptor of bovine chromaffin cells results in a rise in intracellular free calcium [( Ca2+]i) and subsequent release of catecholamine. This response is totally dependent on the presence of external Ca2+. Monitoring [Ca2+]i using quin-2 demonstrated a rise in [Ca2+]i in response to muscarinic agonists which was approximately 4-times less than that obtained in response to nicotinic stimulation. This atropine-sensitive [Ca2+]i rise occurred after a 10-s lag and was found to be independent of the external Ca2+, implying the existence of an intracellular Ca2+ source. Despite producing this [Ca2+]i rise low concentrations of the muscarinic agonist, methacholine (under 1 X 10(-3) M), failed to trigger secretion itself and did not effect the secretory response elicited by nicotine. Challenging the cells with higher methacholine concentrations (over 1 X 10(-3) M) resulted in the same [Ca2+]i rise, no secretion, but an inhibition of secretion due to nicotine. This latter response, however, was found to be atropine-insensitive and therefore non-muscarinic. The [Ca2+]i rise and secretion due to depolarization by 55 mM K+ were largely unaffected by prior addition 1 X 10(-2) M methacholine, inferring that high concentrations of methacholine inhibit nicotine-induced secretion by interacting with the nicotinic receptor. These results provide evidence consistent with the existence of an intracellular Ca2+ store mobilized by muscarinic receptor activation in bovine chromaffin cells and show that, despite causing a rise in [Ca2+]i, bovine chromaffin cell muscarinic stimulation does not effect secretion itself or secretion induced by either nicotine or high K+.     

Alpha 1-adrenoceptor activation elevates cytosolic calcium in rat pinealocytes by increasing net influx

The regulation of [Ca2+]i in rat pinealocytes was studied using the fluorescent indicator quin2. Pinealocyte resting [Ca2+]i was approximately 100 nM; this rapidly decreased in low Ca2+ medium (approximately 10 microM), indicating there was a high turnover of [Ca2+]i in these cells. Norepinephrine (NE, 10(-6) M) increased [Ca2+]i to approximately 350 nM within 1 min; [Ca2+]i then remained elevated for 30 min. The relative potency of adrenergic agonists was NE greater than phenylephrine much greater than isoproterenol. Phentolamine (10(-6) M) and prazosin (10(-8) M) blocked the effects of adrenergic agonists; in contrast, propranolol (10(-6) M) or yohimbine (10(-6) M) had little or no effect. These observations indicate NE acts via alpha 1-adrenoceptors to elevate [Ca2+]i. The [Ca2+]i response to NE did not occur when [Ca2+]e was reduced to approximately 10 microM by adding EGTA 5s before NE, indicating an increase in net Ca2+ influx is involved rather than mobilization of Ca2+ from intracellular stores. The effect of NE was not blocked by nifedipine (10(-6) M), which did block a K+-induced increase in [Ca2+]i, presumably involving voltage-sensitive channels. Ouabain (10(-5) M) caused a gradual increase in [Ca2+]i; this increase was not blocked by nifedipine. Together these data indicate that pinealocyte [Ca2+]i may be influenced by mechanisms regulated by alpha 1-adrenoceptors, voltage-dependent Ca2+ channels, and perhaps a Na+/Ca2+ exchange mechanism stimulated by ouabain. These studies indicate that the pinealocyte is an interesting model to use to study the adrenergic regulation of [Ca2+]i because of the rapid and prolonged changes in [Ca2+]i produced by alpha 1-adrenoceptor activation.     

The caffeine-sensitive Ca2+ store in bovine adrenal chromaffin cells; an examination of its role in triggering secretion and Ca2+ homeostasis

The effect of caffeine on catecholamine secretion and intracellular free Ca2+ concentration [( Ca2+]i) in bovine adrenal chromaffin cells was examined using single fura-2-loaded cells and cell populations. In cell populations caffeine elicited a large (approximately 200 nM) transient rise in [Ca2+]i that was independent of external Ca2+. This rise in [Ca2+]i triggered little secretion. Single cell measurements of [Ca2+]i showed that most cells responded with a large (greater than 200 nM) rise in [Ca2+]i, whereas a minority failed to respond. The latter, whose caffeine-sensitive store was empty, buffered a Ca2+ load induced by a depolarizing stimulus more effectively than those whose store was full. The caffeine-sensitive store in bovine chromaffin cells may be involved in Ca2+ homeostasis rather than in triggering exocytosis.     

P2X2 receptors are essential for [Ca2+]i increases in response to ATP in cultured rat myenteric neurons

We characterized ATP-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current in cultured rat myenteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca2+]i responses to high K+ and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [Ca2+]i that was greatly decreased by the removal of extracellular Ca2+ concentration ([Ca2+]o). The amplitude of the [Ca2+]i response to ATP was reduced by half in the presence of voltage-dependent Ca2+ channel blockers. In [Ca2+]o-free solution, ATP produced a small transient rise in [Ca2+]i similar to that induced by P2Y agonists. At -60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular Na+ concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca2+]i was ATP > or = adenosine 5'-O-3-triphosphate > or = CTP > or = 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca2+ and Mg2+ suppressed the ATP-induced current, and Zn2+, Cu2+, and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X2 receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X2 receptors, resulting in a [Ca2+]i increase dependent on [Ca2+]o in rat myenteric neurons. A small part of the ATP-induced [Ca2+]i increase may be also mediated via a P2Y receptor-related mechanism.