Ca2+ signalling in bovine adrenal chromaffin cells.
thesisposted on 19.11.2015, 08:44 by Vassilios Konstantinos. Pappas
Cells possess two mechanisms, the inositol 1,4,5-trisphosphate (Ins(l,4,5)P3) and ryanodine receptors, by which Ca2+ in intracellular stores can be mobilised. There are also a number of pathways which can mediate Ca2+ entry across the cell plasma membrane. In this study bovine adrenal chromaffin cells were used to investigate the role of intracellular Ca2+ stores in Ca2+ signalling and the relationship between Ca2+ entry and store release. The major part of the exocytotic process in chromaffin cells is due to Ca2+ entry across the plasma membrane. Bradykinin (an Ins(l,4,5)P3 generating agonist) and nicotine (a depolarising stimulus) were found to evoke catecholamine secretion in the presence of extracellular Ca2+. Nicotinic responses were abolished in the absence of extracellular Ca2+, whereas bradykinin resulted in reduced catecholamine secretion, indicating that Ca2+ release from intracellular stores may activate secretion. Studies in permeabilised chromaffin cells showed that both Ins(l,4,5)P3 and caffeine induced Ca2+ mobilisation from intracellular Ca2+ stores. Challenge of the chromaffin cells with inositol 4,5-bisphosphorothioate resulted in depletion of the Ins(l,4,5)P3-sensitive Ca2+ stores. However, subsequent addition of caffeine stimulated Ca2+ mobilisation, indicating that the caffeine releasable stores had not been emptied. Ins(l,4,5)P3 and caffeine, when added simultaneously, resulted in a larger response than each of these agonists alone. Ryanodine pretreatment inhibited subsequent caffeine responses. Ins(l,4,5)P3 was able to stimulate Ca2+ release after prior depletion of the ryanodine-sensitive Ca2+ stores, providing a pharmacological differentiation of these stores, suggesting that the Ins(l,4,5)P3 receptor-expressing stores may be physically different from the ryanodine receptor-expressing Ca2+ stores in chromaffin cells. Experiments using epifluorescence microscopy were carried out to investigate the relationship between Ins(l,4,5)P3- and caffeine-sensitive Ca2+ stores in fura-2 loaded intact chromaffin cells. Bradykinin evoked Ca2+ responses appeared to involve activation of ryanodine receptors, probably occuring secondary to Ca2+ release via Ins(l,4,5)P3 receptors. Depletion of the Ins(l,4,5)P3- and caffeine-sensitive Ca2+ stores resulted in activation of Ca2+ entry indicating that the Ins(l,4,5)P3- and ryanodine-sensitive Ca2+ stores are both linked to the promotion of Ca2+ entry. Several aspects of Ca2+ signalling have been elucidated in this study, notably the possible expression of at least two different Ca2+ stores and the degree of physical or functional overlap between the Ins(l,4,5)P3 receptor-expressing and ryanodine-receptor expressing Ca2+ stores. Ca2+ release from the ryanodine-sensitive Ca2+ stores was found to activate Ca2+ entry across the chromaffin cells plasma membrane. These findings may have important implications for our understanding of how Ca2+ signalling occurs in adrenal chromaffin and other excitable cells in vivo.