Antony J. O'Sullivan

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.

Cyclic GMP Regulates Nicotine-Induced Secretion from Cultured Bovine Adrenal Chromaffin Cells: Effects of 8–Bromo–Cyclic GMP, Atrial Natriuretic Peptide, and Nitroprusside (Nitric Oxide)

Abstract: Methacholine, atrial natriuretic peptide (ANP), nitroprusside (nitric oxide), angiotensin II. and bradykinin raised cyclic. GMP (cGMP) levels in cultured bovine adrenal chromaffin cells. The role of cGMP in secretion from chromaffin cells was examined using 8‐bromo‐cGMP. This analogue had no effect on basal secretion or secretion due to angiotensin II, bradykinin, or a high K+ level but potentiated secretion due to low doses of nicotine. At supramaximal doses of nicotine, 8‐bromo‐cGMP inhibited secretion. These effects of 8‐bromo‐cGMP were not due to changes in the nicotine‐induced rise in cytosolic calcium concentration. A potentiation of secretion due to low doses of nicotine was also found following simultaneous addition of ANP or nitroprusside, a result suggesting that ANP and nitric oxide (endothelium‐derived relaxing factor) could be important regulators of secretion from adrenal chromaffin cells.

A comparison of bradykinin, angiotensin II and muscarinic stimulation of cultured bovine adrenal chromaffin cells.

Bradykinin, angiotensin II and a mascarnic agonist, acetyl-B-methacholine (methacholine) were all found to elict catecholamine release from cultured bovine adrenal chromaffin cells. Bradykinin was the most potent of these secretagogues and methacholine the weakest, with angiotenin II intermediate in efficacy. All three secretagogues were much less effective than nicotinic stimulation. The three secretagogues all produced a rise in cytoplasmic free calcium concentration ([Ca2+]i), measured with the fluorescent indicator fura2, which was partially independent of external calcium. In the case of bradykinin the full rise in ([Ca2+]i) may involve a component of calcium entry in addition to release of calcium from an internal store. Secretion was also found to be partially independent of external calcium. The different efficacies of the three secretagogues in elicting secretion were correlated with the rise in ([Ca2+]i) produced. The differeing efficacies of the three secretagogues may be due to the extent of release of calcium from an intracellular store which itself is less effective in eliciting secretion than a rise in [Ca2+]i following calcium entry due to nicotine. Bradykinin also stimulates calcium entry, and this may increase the efficacy of the initial rise in [Ca2+]i. Treatment with pertussis toxin resulted in an enhancement of secretion in response to all of the secretagogues.

A major role for protein kinase C in calcium-activated exocytosis in permeabilised adrenal chromaffin cells

The role of endogenously activated protein kinase C in calcium‐activated exocytosis was examined in digitonin‐permeabilised bovine adrenal chromaffin cells. Protein kinase C activity was reduced by down‐regulation following long‐term treatment with PMA or by using the inhibitor sphingosine. Both treatments resulted in a substantial reduction in catecholamine secretion elicited by micromolar calcium, indicating that endogenous activation of protein kinase C is a major requirement for calcium‐activated exocytosis in chromaffin cells.

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.

The role of cytoplasmic pH in the inhibitory action of high osmolarity on secretion from bovine adrenal chromaffin cells.

Elevated osmolarity is known to inhibit secretion from a wide range of cells including bovine adrenal chromaffin cells. The mechanism of this inhibition is unclear but the elevated osmolarity has been proposed to oppose an osmotic driving force involved in exocytotic fusion. Using the fluorescent indicators quene 1 and fura2, we monitored the effect of elevated osmolarity on cytoplasmic pH (pHi) and cytoplasmic free Ca2+ [( Ca2+]i). Elevated osmolarity increased both pHi and [Ca2+]i, but had no effect on the [Ca2+]i rise elicited by either K+ or nicotine. Elevating pHi with NH4Cl was shown to inhibit secretion from chromaffin cells. The elevation of pHi by hyperosmolar solutions is proposed as one of the mechanisms by which elevated osmolarity inhibits secretion.

Examination of the Role of ADP‐Ribosylation Factor and Phospholipase D Activation in Regulated Exocytosis in Chromaffin and PC12 Cells

Abstract: The possible role of ADP‐ribosylation factor (ARF)‐activated and constitutive phospholipase D (PLD) activity in regulated exocytosis of preformed secretory granules in adrenal chromaffin and PC12 cells was examined. With use of digitonin‐permeabilised cells, the effect of GTP analogues and exogenous ARF1 on PLD activity was determined. No evidence was seen for ARF‐stimulated PLD activity in these cell types. Exocytosis from cytosol‐depleted permeabilised chromaffin cells was not increased by adding recombinant nonmyristoylated or myristoylated ARF1, and exocytosis from both cell types was resistant to brefeldin A (BFA). Addition of bacterial PLD with demonstrably high activity in permeabilised chromaffin cells did not increase exocytosis in cytosol‐depleted chromaffin cells. Diversion of PLD activity from production of phosphatidic acid (PA) due to the presence of 4% ethanol did not inhibit exocytosis triggered by Ca2+ or poorly hydrolysable GTP analogues in permeabilised chromaffin or PC12 cells. These results indicate that exocytosis in these cell types does not appear to require a BFA‐sensitive ARF and the triggering of exocytosis does not require PLD activity and formation of PA. These findings rule out a general requirement for PLD activity during regulated exocytosis.