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Mechanisms Underlying Leakage of Calcium from the Endoplasmic Reticulum of Acinar Cells of the Submandibular Salivary Gland

Research paper by O. V. Kopach, I. A. Kruglikov, P. G. Kostyuk, N. V. Voitenko, N. V. Fedirko

Indexed on: 01 Jul '05Published on: 01 Jul '05Published in: Neurophysiology



Abstract

In the resting state, the Ca2+ concentration in agonist-sensitive intracellular stores reflects the balance between active uptake of Ca2+, which is mediated by Ca2+-ATPase (SERCA), and passive leakage of Ca2+. The mechanisms underlying such a leakage in cells of the submaxillary salivary gland were not studied. In our experiments, we examined possible pathways of passive leakage of Ca2+ from the endoplasmic reticulum (ER) of acinar cells obtained from the rat submaxillary salivary gland; direct measurements of the concentration of Ca2+ in the ER ([Ca2+]ER) using a low-affinity calcium-sensitive dye, mag-fura 2/AM, were performed. The cellular membrane was permeabilized with the help of β-escin (40 μg/ml); the Ca2+ concentration in the cytoplasm ([Ca2+]i) was clamped at its level typical of the resting state (∼100 nM) using an EGTA/Ca2+ buffer. Incubation of permeabilized acinar cells in a calcium-free intracellular milieu, as well as application of thapsigargin, resulted in complete inhibition of the uptake of Ca2+ with the involvement of SERCA. This effect was observed 1 min after the beginning of superfusion of the cells with the corresponding solutions and was accompanied by the leakage of Ca2+ from the ER; this is confirmed by a gradual drop in the [Ca2+]ER. Such a leakage of Ca2+ remained unchanged in the presence of thapsigargin, heparin, and ruthenium red; therefore, it is not mediated by SERCA, inositol 1,4,5-trisphosphate-sensitive receptors (InsP3R), or ryanodine receptors (RyRs). At the same time, an antibiotic, puromycin (0.1 to 1.0 mM), which disconnects polypeptides from the ER-ribosome translocon complex, caused intensification of passive leakage of Ca2+ from the ER. This effect did not depend on the functioning of SERCA, InsP3R, or RyR. Therefore, passive leakage of Ca2+ from the ER in acinar cells of the submaxillary salivary gland is realized through pores of the translocon complex of the ER membrane.