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.

We applied recombinant forms of the Rho-related small guanosine triphosphatases (GTPases) Rac2 and Cdc42/G25K to permeabilized mast cells to test their ability to regulate exocytotic secretion. Mast cells permeabilized with streptolysin-O leak soluble (cytosol) proteins over a period of 5 min and become refractory to stimulation by Ca 2ϩ and guanosine triphosphate (GTP)␥S over about 20 -30 min. This loss of sensitivity is likely to be due to loss of key regulatory proteins that are normally tethered at intracellular locations. Exogenous proteins that retard this loss of sensitivity to stimulation may be similar, if not identical, to those secretory regulators that are lost. Recombinant Rac and Cdc42/G25K, preactivated by binding GTP␥S, retard the loss of sensitivity (rundown) and, more importantly, enable secretion to be stimulated by Ca 2ϩ alone. Investigation of the concentration dependence of each of these two GTPases applied individually to the permeabilized cells, and of Cdc42/G25K applied in the presence of an optimal concentration of Rac2, has provided evidence for a shared effector pathway and also a second effector pathway activated by Cdc42/G25K alone. Dominant negative mutant (N17) forms of Rac2 and Cdc42/G25K inhibit secretion induced by Ca 2ϩ and GTP␥S. Our data suggest that Rac2 and Cdc42 should be considered as candidates for G E , GTPases that mediate exocytosis in cells of hematopoeitic origin.

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...

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