Roger B. Moreton

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.

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.

Multiple, coordinated Ca2+ -release events underlie the inositol trisphosphate-induced local Ca2+ spikes in mouse pancreatic acinar cells.

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 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 (∼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 (> 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.