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

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. Neirofiziologiya/Neurophysiology, Vol. 37, No. 4, pp. 339–346, July–August, 2005.