Measurement of cytosolic free Ca2+ in individual pancreatic acini

The kinetics of changes in cytosolic free Ca2+ ([Ca2+](i)) were determined in individual rat pancreatic acini by microfluorimetry. Three major findings are reported. First, at maximal stimulatory concentrations for amylase release, both caerulein and bombesin induced an initial rise in [Ca2+](i) followed by prolonged secondary oscillations of smaller amplitude. The latter effect was not observed with supramaximal doses of caerulein. Second, these cyclic changes were dependent, at least in part, on extracellular Ca2+. Finally, comparison of the threshold doses for [Ca2+](i) mobilization and enzyme discharge demonstrated that pathways independent of an elevation of [Ca2+](i) control the secretory activity of pancreatic acini at low, picomolar agonist concentrations.link_to_subscribed_fulltex

Glucose-6-phosphatase in the insulin secreting cell line INS-1.

The glucose-6-phosphatase system of the glucose sensitive insulin secreting rat insulinoma cells (INS-1) was investigated. INS-1 cells contain easily detectable levels of glucose-6-phosphatase enzyme protein (assessed by Western blotting) and have a very significant enzymatic activity. The features of the enzyme (Km and Vmax values, sensitivity to acidic pH, partial latency, and double immunoreactive band) are similar to those of the hepatic form. On the other hand, hardly detectable levels of glucose-6-phosphatase activity and protein were present in the parent glucose insensitive RINm5F cell line. The mRNA of the glucose-6-phosphate transporter was also more abundant in the INS-1 cells. The results support the view that the glucose-6-phosphatase system of the beta-cell is associated with the regulation of insulin secretion

Mitochondrial cAMP and Ca(2+) metabolism in adrenocortical cells.

The biological effects of physiological stimuli of adrenocortical glomerulosa cells are predominantly mediated by the Ca(2+) and the cAMP signal transduction pathways. The complex interplay between these signalling systems fine-tunes aldosterone secretion. In addition to the well-known cytosolic interactions, a novel intramitochondrial Ca(2+)-cAMP interplay has been recently recognised. The cytosolic Ca(2+) signal is rapidly transferred into the mitochondrial matrix where it activates Ca(2+)-sensitive dehydrogenases, thus enhancing the formation of NADPH, a cofactor of steroid synthesis. Quite a few cell types, including H295R adrenocortical cells, express the soluble adenylyl cyclase within the mitochondria and the elevation of mitochondrial [Ca(2+)] activates the enzyme, thus resulting in the Ca(2+)-dependent formation of cAMP within the mitochondrial matrix. On the other hand, mitochondrial cAMP (mt-cAMP) potentiates the transfer of cytosolic Ca(2+) into the mitochondrial matrix. This cAMP-mediated positive feedback control of mitochondrial Ca(2+) uptake may facilitate the rapid hormonal response to emergency situations since knockdown of soluble adenylyl cyclase attenuates aldosterone production whereas overexpression of the enzyme facilitates steroidogenesis in vitro. Moreover, the mitochondrial Ca(2+)-mt-cAMP-Ca(2+) uptake feedback loop is not a unique feature of adrenocortical cells; a similar signalling system has been described in HeLa cells as well