Depolarization potentiates endothelin-induced effects on cytosolic calcium in bovine adrenal chromaffin cells

The effects of endothelin on intracellular calcium concentrations ([Ca2+i]) in primary cultures of bovine adrenal chromaffin cells (BAM) were measured using Fura 2. Endothelin had minimal effects on [Ca2+i] over a broad dose range (1 nM to 1 microM). However, in conjunction with K+ depolarization there was a synergistic increase in [Ca2+i]. This effect was dependent on extracellular calcium as was the response to KCl alone. A partial synergistic effect was evident with endothelin and nicotinic stimulation. The effects of endothelin and angiotensin II on [Ca2+i] are only additive. Blockade of voltage sensitive calcium channels failed to alter the synergistic effects. Our results indicate that endothelin influences BAM calcium mobilization through sites regulated by membrane depolarization but differing from traditional voltage sensitive calcium channels.     

Muscarinic release of inositol trisphosphate without mobilization of calcium in bovine adrenal chromaffin cells

Chromaffin cells of bovine adrenal medulla release catecholamines in response to activation of nicotinic ACh receptors which open voltage-sensitive calcium channels. Catecholamine secretion by exocytosis requires an increase in cytosolic free calcium. The cells also possess muscarinic ACh receptors but muscarinic agents do not provoke catecholamine release. Quin-2 studies show that they do not increase cytosolic free Ca2+ concentration, but unlike the nicotinic agents, they cause phosphoinositide hydrolysis. Muscarinic stimulation leads to rapid loss of labelled phosphatidylinositol 4-phosphate and of phosphatidylinositol 4,5-bisphosphate. At the same time there is release of inositol trisphosphate, inositol bisphosphate and inositol phosphate. In a number of other cells inositol trisphosphate may act as a second messenger releasing Ca2+ from storage sites in the endoplasmic reticulum but this is not its function in bovine chromaffin cells.     

Muscarinic receptor-mediated inositol tetrakisphosphate response in bovine adrenal chromaffin cells

Inositol trisphosphate (IP3), a product of the phosphoinositide cycle, mobilizes intracellular Ca2+ in many cell types. New evidence suggests that inositol tetrakisphosphate (IP4), an IP3 derivative, may act as another second messenger to further alter calcium homeostasis. However, the function and mechanism of action of IP4 are presently unresolved. We now report evidence of muscarinic receptor-mediated accumulation of IP4 in bovine adrenal chromaffin cells, a classic neurosecretory system in which calcium movements have been well studied. Muscarine (0.4 mM) stimulated an increase in [3H]IP4 and [3H]IP3 accumulation in chromaffin cells and this effect was completely blocked by atropine (0.5 mM). [3H]IP4 accumulation was detectable within 15 sec, increased to a maximum by 30 sec and thereafter declined. 2,3-diphosphoglycerate, an inhibitor of IP3 and IP4 hydrolysis, enhanced accumulation of these inositol polyphosphates. The results provide the first evidence of a rapid inositol tetrakisphosphate response in adrenal chromaffin cells, which should facilitate the future resolution of the relationship between IP4 and calcium homeostasis.     

PTH release stimulated by high extracellular potassium is associated with a decrease in cytosolic calcium in bovine parathyroid cells

We employed the calcium (Ca⁺⁺)-sensitive, intracellular dye QUIN-2 to examine the role of cytosolic Ca⁺⁺ in the stimulation of PTH release by high extracellular potassium (K⁺) concentrations. Addition of 55 mM KCl to cells incubated with 115 mM NaCl and 5 mM KCl lowered cytosolic Ca⁺⁺ at either low (0.5 mM) extracellular Ca⁺⁺ (from 194±14 to 159±9 nM, p<.01, N=6) or high (1.5 mM) extracellular calcium (from 465±38 to 293±20 nM, p<.01, N=10). This reduction in cytosolic Ca⁺⁺ was due to high K⁺$\text{per}\text{se}$ and not to changes in tonicity since addition of 55 mM NaCl was without effect while a similar decrease in cytosolic Ca⁺⁺ occurred when cells were resuspended in 60 mM NaCl and 60 mM KCl. PTH release was significantly (p<.01) greater at 0.5 and 1.5 mM Ca⁺⁺ in QUIN-2-loaded cells incubated with 60 mM NaCl and 60 mM KCl than in those exposed to 115 mM NaCl and 5 mM KCl. In contrast to most secretory cells, therefore, stimulation of PTH release by high K⁺ is associated with a decrease rather than an increase in cytosolic Ca⁺⁺.     

Muscarinic receptors in separate populations of noradrenaline- and adrenaline-containing chromaffin cells

We have performed binding experiments of (a)[3H]quinuclidinyl benzilate to partially purified membranes from noradrenaline- and adrenaline-containing chromaffin cells and (b) [3H]N-methyl-quinuclidinyl benzilate to acutely isolated, or 48-h cultured, chromaffin cells subpopulations. Using this approach, we obtained enough evidence to conclude (1st) that muscarinic receptors are present in both noradrenaline- and adrenaline containing cells; (2nd) that noradrenaline cells contain in fact 2-3 fold higher density of those receptors; and (3rd) that those receptors undergo plastic changes upon chronic culturing of the cells.     

A delayed and sustained rise of cytosolic calcium is elicited by oxidized LDL in cultured bovine aortic endothelial cells.

Bovine aortic endothelial cells (BAEC) pulsed for 5 h with mildly oxidized low density lipoproteins (LDL), exhibited a broad, sustained and high peak of [Ca2+]i occurring several hours after the end of the pulse and reaching very high [Ca2+]i values (around 2500-3000 nmol/l) and a concomitant drop of cytosolic pH (around 0.2-0.3 pH units) without any loss of cell viability. When BAEC were continuously pulsed with oxidized LDL, the peak of [Ca2+]i was more sustained than in short pulse experiments and was associated with irreversible morphological changes usually associated with cytotoxic events (blebbing) and with a marked loss of viability. The potential involvement of these biochemical and morphological changes in atherogenesis are discussed.     

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+.     

Simultaneous measurements of cytosolic calcium and secretion in single bovine adrenal chromaffin cells by fluorescent imaging of fura-2 in cocultured cells

The cytosolic free calcium concentration ([Ca2+]i) and exocytosis of chromaffin granules were measured simultaneously from single, intact bovine adrenal chromaffin cells using a novel technique involving fluorescent imaging of cocultured cells. Chromaffin cell [Ca2+]i was monitored with fura-2. To simultaneously follow catecholamine secretion, the cells were cocultured with fura-2-loaded NIH-3T3t cells, a cell line chosen because of their irresponsiveness to chromaffin cell secretagogues but their large Ca2+ response to ATP, which is coreleased with catecholamine from the chromaffin cells. In response to the depolarizing stimulus nicotine (a potent secretagogue), chromaffin cell [Ca2+]i increased rapidly. At the peak of the response, [Ca2+]i was evenly distributed throughout the cell. This elevation in [Ca2+]i was followed by a secretory response which originated from the entire surface of the cell. In response to the inositol 1,4,5-trisphosphate (InsP3)-mobilizing agonist angiotensin II (a weak secretagogue), three different responses were observed. Approximately 30% of chromaffin cells showed no rise in [Ca2+]i and did not secrete. About 45% of the cells responded with a large (greater than 200 nM), transient elevation in [Ca2+]i and no detectable secretory response. The rise in [Ca2+]i was nonuniform, such that peak [Ca2+]i was often recorded only in one pole of the cell. And finally, approximately 25% of cells responded with a similar Ca2+-transient to that described above, but also gave a secretory response. In these cases secretion was polarized, being confined to the pole of the cell in which the rise in [Ca2+]i was greatest.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spatial localization of the stimulus-induced rise in cytosolic Ca2+ in bovine adrenal chromaffin cells. Distinct nicotinic and muscarinic patterns

The spatial distribution of the intracellular free Ca2+ (Ca2+i) rise elicited by different stimuli in bovine adrenal chromaffin cells was examined in single fura-2-loaded cells. In response to the potent secretagogues nicotine and high K+, Ca2+i was initially localized exclusively to the entire subplasmalemmal area of the cell. In response to the ineffective secretagogues, methacholine and muscarine, the rise in Ca2+i originated only in one pole of the cell and even at the peak of the response Ca2+ was still generally restricted to this same area of the cell. These results suggest that the triggering of exocytosis from these cells requires a specific spatial distribution of Ca2+i.     

Depletion and refilling of intracellular calcium pools in bovine chromaffin cells

To investigate Ca²⁺ uptake by Ca²⁺-depleted bovine chromaffin cells we depleted these cells of Ca²⁺ by incubating them in Ca²⁺-free buffer, then measured changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺ ₁)⁴⁵Ca²⁺ uptake, and Mn²⁺ uptake in response to added Ca²⁺ or MN²⁺. In depleted cells, the increase in [Ca²⁺]_i after Ca²⁺ addition, and the Mn²⁺ and⁴⁵Ca²⁺ uptakes were higher than in control cells, and were inhibited by verapamil. The size of the intracellular Ca²⁺ pools in depleted cells increased after Ca²⁺ addition. The times for [Ca²⁺]_i rise and Mn²⁺ entry to reach plateau levels were much shorter than the time for refilling of intracellular Ca²⁺ stores. In Ca²⁺-depleted cells and cells which had been loaded with BAPTA,⁴⁵Ca²⁺ uptake was much higher than in control cells. These results suggest that extracellular Ca²⁺ enters the cytoplasm first before refilling the intracellular stores. The rate of Mn²⁺ influx depended on the level of filling of the Ca²⁺ stores, suggesting that some signalling takes place between the intracellular stores and Ca²⁺ entry pathways through the plasma membrane.Abbreviations used BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid - BAPTA/AM acetoxymethyl ester of BAPTA - [Ca²⁺]_i cytosolic Ca²⁺ concentration - IP₃ inositol 1,4,5-trisphosphate - tBHQ 2,5-di-(t-butyl)-1,4-benzohydroquinone This work was included in a thesis submitted by A.-L. Sui to the Department of Biochemistry, National Yang-Ming Medical College, in partial fulfillment of the requirements for the degree of Doctor of Philosophy     

Sphingosine increases inositol trisphosphate in rat parotid acinar cells by a mechanism that is independent of protein kinase C but dependent on extracellular calcium

In rat parotid acinar cells prelabelled with [3H]-inositol, sphingosine stimulated the accumulation of [3H]-inositol polyphosphates. When the cells were exposed to sphingosine, [3H]-inositol trisphosphate (InsP3) was accumulated in a time- and dose-dependent manner. When the extracellular Ca2+ was chelated by 1 mM EGTA, the effect of sphingosine on InsP3 accumulation was completely inhibited. Ionophores, A23187 and ionomycin, had no significant effect on InsP3 accumulation. An inhibitor of protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), failed to stimulate InsP3 accumulation. In the homogenate of parotid acinar cells, InsP3 3-kinase and 5-phosphomonoesterase activities were not affected by sphingosine. These results suggest that sphingosine activates phosphoinositide turnover by a mechanism dependent upon extracellular Ca2+, but different from that of an ionophore, and independent of protein kinase C.     

GABAA receptor-mediated increase of cytosolic Ca2+ in isolated bovine adrenal chromaffin cells

We have studied the effects of GABA on cytosolic free Ca2+ concentration ([Ca2+]i) as a means of investigating the role of GABA in adrenal catecholamine (CA) secretion. It was demonstrated that GABA caused an elevation of [Ca2+]i via the GABAA receptor in a concentration-dependent manner, which was well correlated with an increase of 45Ca uptake, an increase of CA release and a depolarization of chromaffin cells assessed with bis-oxonol fluorescence. Since the GABA-induced rise of [Ca2+]i was absolutely dependent on the presence of extracellular Ca2+ and partly sensitive to nifedipine, at least one entry route for Ca2+ facilitated by GABA via a voltage-sensitive Ca2+ channel was suggested. When extracellular Cl- was lowered, GABA-induced CA release, depolarization, and rise of [Ca2+]i were all markedly enhanced. It is possible that GABA plays a modulatory role in the regulation of adrenal CA secretion as a facilitatory modulator.     

Early rise of cytosolic Ca2+ induced by NGF in PC12 and chromaffin cells

A rise of cytosolic Ca2+ is induced by NGF in rat pheochromocytoma PC12 and bovine chromaffin cells investigated (both in suspension and while attached to polyornithine-coated glass slides) by fluorescence techniques (with quin-2 and fura-2). The effect of NGF on [Ca2+]i is delayed (30-40 s of lag phase), slow (t1/2 = 40 s), relatively small (+50-75%) and persistent (over 10 min). It is due to Ca2+ influx (requires extracellular Ca2+ greater than 10 microM) through a pathway different from the voltage-gated Ca2+ channel, possibly accompanied by intracellular Ca2+ redistribution, and might play a messenger role in NGF action.     

Chromostatin receptors control calcium channel activity in adrenal chromaffin cells.

One of the functions of chromogranin A (CGA), the major soluble component of secretory granules in both adrenal medullary chromaffin cells and many other endocrine cell types appears to be that of a prohormone. CGA is the precursor of several peptides including pancreastatin, a 49-residue peptide, and a 20-residue peptide, chromostatin, which have been identified as biologically active peptides. Chromostatin produces a dose-dependent inhibition (ID50 of 5 nM) of the secretagogue-evoked catecholamine secretion from chromaffin cells. Here we report that chromostatin potently inhibits L-type calcium currents recorded with the nystatin-perforated patch technique in cultured chromaffin cells. This inhibitory effect of chromostatin on calcium currents was not observed in experiments using the classical patch-clamp whole-cell approach which induces the leakage of cytoplasmic components. Using 125I-chromostatin, we show that chromostatin exhibits a fully reversible and saturable binding to the plasma membrane of cultured chromaffin cells. Analysis of binding experiments at equilibrium indicates the existence of one class of binding sites with a Bmax of 2.7 pmol/mg of chromaffin cell proteins and an apparent Kd of 6.5 nM. This high affinity is in good correlation with the half-maximal concentration (ID50 5 nM) of chromostatin inhibiting catecholamine secretion from chromaffin cells. Specificity of the chromostatin binding was further assessed by displacement experiments with unlabeled CGA-related or -unrelated peptides. We found an excellent quantitative correlation between the affinities of the various peptides determined by binding assays and their functional potency tested on catecholamine secretion: bovine chromostatin greater than human chromostatin greater than CGA much greater than rat chromostatin, pancreastatin, CAP-14, substance P, and Leu-enkephalin. Cross-linking experiments reveal that chromostatin associates specifically with an 80-kDa plasma membrane protein. These results together with the patch-clamp experiments support the idea that chromaffin cells possess specific chromostatin receptors and that activation of such receptors leads to the inhibition of L-type voltage-sensitive calcium channels through an intracellular second messenger pathway.     

Stimulatory effect of enkephalins on calcium efflux from bovine adrenal chromaffin cells in culture

The effects of leucine- and methionine-enkephalin, opiate peptides, on Ca2+ efflux from cultured bovine adrenal chromaffin cells were examined. These enkephalins stimulated the efflux of 45Ca2+ from cells in a concentration-dependent manner (10(-8) M-10(-6) M). Leucine-enkephalin did not increase the intracellular free Ca2+ level, 45Ca2+ uptake, catecholamine secretion, cAMP level or cGMP level. The peptide-stimulated 45Ca2+ efflux was not inhibited by incubation in Ca2+-free medium, but was inhibited by incubation in Na+-free medium. These results indicate that enkephalins stimulate extracellular Na+-dependent 45Ca2+ efflux from cultured bovine adrenal chromaffin cells, probably by stimulating membrane Na+/Ca2+ exchange.     

Effects of cyclopiazonic acid on cytosolic calcium in bovine airway smooth muscle cells

In many cells, inhibition of sarcoplasmic reticulum (SR) Ca2+-ATPase activity induces a steady-state increase in cytosolic calcium concentration ([Ca2+]i) that is sustained by calcium influx. The goal was to characterize the response to inhibition of SR Ca2+-ATPase activity in bovine airway smooth muscle cells. Cells were dispersed from bovine trachealis and loaded with fura 2-AM (0.5 microM) for imaging of single cells. Cyclopiazonic acid (CPA; 5 microM) inhibited refilling of both caffeine- and carbachol-sensitive calcium stores. In the presence of extracellular calcium, CPA caused a transient increase in [Ca2+]i from 166 +/- 11 to 671 +/- 100 nM, and then [Ca2+]i decreased to a sustained level (CPA plateau; 236 +/- 19 nM) significantly above basal. The CPA plateau spontaneously declined toward basal levels after 10 min and was attenuated by discharging intracellular calcium stores. When CPA was applied during sustained stimulation with caffeine or carbachol, decreases in [Ca2+]i were observed. We concluded that the CPA plateau depended on the presence of SR calcium and that SR Ca2+-ATPase activity contributed to sustained increases in [Ca2+]i during stimulation with caffeine and, to a lesser extent, carbachol.     

Catecholamines-evoked cytosolic ca2+rise in endothelial cells from bovine adrenal medulla

The effects of catecholamines on intracellular Ca²⁺concentrations ([Ca²⁺]_i) in single acutely dissociated bovine adrenal medulla endothelial cells (BAMECs) were measured using the intracellular fluorescent probe Fluo-3 AM. 100 m epinephrine or norepinephrine induced a biphasic [Ca²⁺]_i rise with an initial peak followed by a delayed phase. 10 m phenylephrine (₁-adrenergic agonist) caused a [Ca²⁺]_i rise similar to that evoked by catecholamines. The increase in [Ca²⁺]_i induced by 10 m phenylephrine was reverted by 10 m phenoxybenzamine (-adrenergic antagonist). Neither isoproterenol (-adrenergic agonist) nor clonidine (₂-adrenergic agonist) induced [Ca²⁺]_i rise. The initial peak was insensitive to zero external Ca²⁺ and it was abolished after Ca²⁺ internal storages were emptied by 10 mM caffeine. The delayed phase was reduced to near zero by external Ca²⁺ removal. These results indicate that BAMECs possess ₁-adrenergic receptors associated to both the release of caffeine-sensitive intracellular Ca²⁺ stores and the entry of extracellular Ca²⁺ We suggest that chromaffin cell secretion may activate BAMECs in vivo through an increase in [Ca²⁺]_i which could induce the secretion of vasoactive factors allowing a rapid entry of hormones into the circulation. (Mol Cell Biochem 000: 000-000,1999)     

VIP release from enteric nerves is independent of extracellular calcium

The release of endogenous vasoactive intestinal polypeptide (VIP) from enteric nerves of isolated rat ileum and the role of extracellular calcium on the release mechanism have been investigated. Evaluation of simultaneous release of endogenous acetylcholine (ACh) and adenosine 5'-triphosphate (ATP) from enteric nerves was used to establish the reliability of the technique. Electrical field stimulation of the ileal preparation induced an increase in the release of endogenous ACh, ATP and VIP. The evoked, but not the basal, release of these substances was blocked by tetrodotoxin (TTX), indicating that the release was a result of nerve stimulation. However, while increase in release of ACh and ATP during nerve stimulation was suppressed in Ca2+-free medium and by the addition of the Ca2+ channel blocker cadmium, nerve-mediated VIP release was unaffected. Further, while K+-depolarization induced release of ACh and ATP from the ileal preparations, it did not lead to an increase in the release of VIP. These results demonstrate that, unlike ACh and ATP release, release of endogenous VIP from enteric nerves is independent of extracellular calcium. The implications of these results in terms of the mechanism of transmitter release in the gastrointestinal tract are discussed.     

L-type calcium channels are preferentially coupled to endocytosis in bovine chromaffin cells

Exocytosis and endocytosis are Ca(2+)-dependent processes. The contribution of high-voltage activated Ca(2+) channels subtypes to exocytosis has been thoroughly studied in chromaffin cells. However, similar reports concerning endocytosis are unavailable. Thus, we studied here the effects of blockers of L (nifedipine), N (omega-conotoxin GVIA) and P/Q (omega-agatoxin IVA) Ca(2+) channel on Ca(2+) currents (I(Ca)), Ca(2+) entry (Q(Ca)), as well as on the changes in membrane capacitance (C(m)) in perforated-patch voltage-clamped bovine adrenal chromaffin cells. Using 500-ms pulses to 0 or +10 mV, given from a holding potential of -80 mV and 2 mM Ca(2+) we found that omega-conotoxin GVIA affected little the exo-endocytotic responses while omega-agatoxin IVA markedly blocked those responses. However, nifedipine blocked little exocytosis but almost completely inhibited endocytosis. We conclude that L-type Ca(2+) channels seem to be selectively coupled to endocytosis.