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.

Many cellular functions are regulated by activation of cell-surface receptors that mobilize calcium from internal stores sensitive to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). The nature of these internal calcium stores and their localization in cells is not clear and has been a subject of debate. It was originally suggested that the Ins(1,4,5)P3-sensitive store is the endoplasmic reticulum, but a new organelle, the calciosome, identified by its possession of the calcium-binding protein, calsequestrin, and a Ca2+-ATPase-like protein of relative molecular mass 100,000 (100K), has been described as a potential Ins(1,4,5)P3-sensitive calcium store. Direct evidence on whether the calciosome is the Ins(1,4,5)P3-sensitive store is lacking. Using monoclonal antibodies raised against the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum, we show that bovine adrenal chromaffin cells contain two Ca2+-ATPase-like proteins with distinct subcellular distributions. A 100K Ca2+-ATPase-like protein is diffusely distributed, whereas a 140K Ca2+-ATPase-like protein is restricted to a region in close proximity to the nucleus. In addition, Ins(1,4,5)P3-generating agonists result in a highly localized rise in cytosolic calcium concentration ([Ca2+]i) initiated in a region close to the nucleus, whereas caffeine results in a rise in [Ca2+]i throughout the cytoplasm. Our results indicate that chromaffin cells possess two calcium stores with distinct Ca2+-ATPases and that the organelle with the 100K Ca2+-ATPase is not the Ins(1,4,5)P3-sensitive store.

Abstract. 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. Chromaflin cell [Ca2+]i was monitored with fura-2. To simultaneously follow catecholamine secretion, the cells were cocultured with fura-2-1oaded NIH-3T3' cells, a cell line chosen because of their irresponsiveness to chromaffin cell secretagogues but their large Ca 2+ 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 chromaftin cells showed no rise in [Ca2+]i and did not secrete. About 45% of the cells responded with a large (>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, ~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. Exocytosis in response to nicotine occurred over the entire surface of the cell, whereas exocytosis due to angiotensin II was polarized, as was confirmed by immunofluorescent localization of dopamine-B-hydroxylase, a chromaflin granule protein that becomes incorporated into the plasma membrane during fusion.These results directly demonstrate, for the first time, that intact chromaffin cells can undergo a large, agonist-induced transient rise in [Ca2+]i without this stimulating secretion and, furthermore, show that the location of exocytosis around the cell can vary depending on the nature of the stimulus. XOCYTOSlS, the process by which intracellular vesicles fuse with the inner surface of the plasma membrane and release their contents into the surrounding medium, is the mechanism underlying the secretion of many physiologically important mediators such as hormones, enzymes, and neurotransmitters. The process is often regulated by an external signal which stimulates release by altering the level of an intracellular second messenger.The pivotal role that Ca 2÷ plays in triggering exocytosis was first noted nearly 30 yr ago with the demonstration that depolarized chromaffin cells would not secrete catecholamines in the absence of extraceUular Ca 2÷ (13). The involvement of Ca 2÷ in exocytosis was advanced by subsequent information obtained from studies with 45Ca 2÷ (12) All reprint requests should be...

Stimulation of single HeLa cells with histamine evoked repetitive increases of the intracellular calcium ion concentration (Ca2+ spikes). The frequency of Ca2+ spiking increased as the extracellular hormone concentration was elevated. In addition, the frequency of Ca2+ spiking could be accelerated by increasing the extracellular Ca2+ concentration ([Ca2+]0) in the presence of a constant hormone concentration. The range of [Ca2+]0 over which the spiking frequency could be titrated was nominally-zero to 10mM, being half-maximally effective at approx. 1 and 2.5mM for 37 and 22 degrees C respectively. The effect of [Ca2+]0 on inositol phosphates production was also examined. Changes of [Ca2+]0 over a range which had been found to affect the frequency of Ca2+ spiking did not have any effect on the rate of myo-inositol 1,4,5-trisphosphate (InsP3) production, although an increase in inositol phosphates production was observed as [Ca2+]0 was increased from zero to values giving less than half-maximal Ca2+ spike frequency. These data suggest that at low Ca2+ spike frequency, Ca2+-stimulated activation of phospholipase C may contribute to Ca2+ spiking in HeLa cells, but under some conditions the availability of Ca2+ to the intracellular stores, rather than changes in the rate of InsP3 production, determines the Ca2+ spike frequency.

Ca2+ wave initiation and non‐propagating Ca2+ spikes occur as a result of localized Ca2+ release from the more sensitive intracellular Ca2+ stores. Using high spatial and temporal Ca2+ ‐imaging techniques we have investigated inositol 1,4,5 triphosphate (InsP3)‐induced local Ca2+ spiking, which occurs at the site of Ca2+ wave initiation in pancreatic acinar cells. The spatial and temporal organization of a single spike suggested discrete hot spots of Ca2+ release. Further analysis of long trains of Ca2+ spikes demonstrated that these hot spots showed regenerative Ca2+ ‐release events which were consistently active from spike to spike. Regions adjacent to these hot spots also showed regenerative Ca2+ ‐release events of similar amplitude but with a much lower frequency of occurrence. We conclude that the InsP3‐induced non‐propagating Ca2+ spikes can be devolved into smaller components of release. Our results are consistent with a model of coordinated activity of pacemaker hot spots of Ca2+ release that recruit and entrain active Ca2+ ‐release events from surrounding regions.

The spatial distribution of the intracellular free Caz+ (Ca?') 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+, Ca? was initially localized exclusively to the entire subplasmalemmal area of the cell. In response to the ineffective secretagogues, methacholine and muscarine, the rise in CaS_ originated only in one pole of the cell and even at the peak of the response Ca*+ 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:.

Low caffeine concentrations were unable to completely release the caffeine- and ryanodine-sensitive intracellular Ca2+ pool in intact adrenal chromaffin cells. This 'quantal' Ca2+ release is the same as that previously observed with inositol Ins(1,4,5)P3-induced Ca2+ release. The molecular mechanism underlying quantal Ca2+ release from the ryanodine receptor was investigated using fura-2 imaging of single chromaffin cells. Our data indicate that the intracellular caffeine-sensitive Ca2+ pool is composed of functionally discrete stores, that possess heterogeneous sensitivities to caffeine. These stores are mobilized by caffeine in a concentration-dependent fashion, and, when stimulated, individual stores release their Ca2+ in an 'all-or-none' manner. Such quantal Ca2+ release may be responsible for graded Ca2+ responses in single cells.

The effect of caffeine on catecholamine secretion and intracellular free Ca 2+ concentration ([Ca2+]i) in bovine adrenal chromaffin cells was examined using single fura-2-1oaded cells and cell populations. In cell populations caffeine elicited a large (~ 200 nM) transient rise in [Ca2+]i that was independent of external Ca 2+ . This rise in [CaZ+]i triggered little secretion. Single cell measurements of [Ca2+]i showed that most cells responded with a large (> 200 nM) rise in [Ca2+]~, whereas a minority failed to respond. The latter, whose caffeine-sensitive store was empty, buffered a Ca z+ 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 Ca 2+ homeostasis rather than in triggering exocytosis.