[Characteristics of inositoltrisphosphate-sensitive Ca2+ stores in the acinar cells of rat submandibular salivary gland].

In the acinar cells of rat submandibular salivary gland activation of cholinoreceptors leads to the release of Ca2+ from endoplasmic reticulum (ER). This Ca2+ release from ER is mainly mediated by InsP3-receptors. In the present work we used Arsenazo III dye and mag-fura 2/AM to measure total cellular calcium content and Ca2+ concentration in the ER ([Ca2+]ER), respectively. We have found that application of InsP3 to the permeabilized acinar cells evoked decrease [Ca2+]ER in dose-dependent manner with EC50 1.3 +/- 0.21 mM. This InsP3-induced Ca2+ release from the ER was potentiated by Ca2+ in the physiological ranges (100-400 nM), modulated by caffeine and ATP. Low concentrations of ATP in (< 1 mM) enhanced the InsP3-induced decrease [Ca2+]ER while high concentrations of ATP markedly suppressed Ca2+ release. Caffeine (2 mM) decreased InsP3-induced Ca2+ release in the presence of Ca2+ however it has no inhibitory effect in the absence of Ca2+. This inhibitory effect of caffeine on InsP3-induced Ca2+ release is overcame by high concentration of InsP3 (20 mM) and ATP (1 mM) indicating that caffeine functionally competes with InsP3 receptor domains. We suggested that the ATP regulation of InsP3-induced Ca2+ release might also play a role in oscillations of intracellular Ca2+ and the maintenance of the cell survival during energy attenuation periods.

Changes in functioning of rat submandibular salivary gland under streptozotocin-induced diabetes are associated with alterations of Ca2+ signaling and Ca2+ transporting pumps.

Xerostomia and pathological thirst are troublesome complications of diabetes mellitus associated with impaired functioning of salivary glands; however, their cellular mechanisms are not yet determined. Isolated acinar cells were loaded with Ca2+ indicators fura-2/AM for measuring cytosolic Ca2+ concentration ([Ca2+]i) or mag-fura-2/AM-inside the endoplasmic reticulum (ER). We found a dramatic decrease in pilocarpine-stimulated saliva flow, protein content and amylase activity in rats after 6 weeks of diabetes vs. healthy animals. This was accompanied with rise in resting [Ca2+]i and increased potency of acetylcholine (ACh) and carbachol (CCh) but not norepinephrine (NE) to induce [Ca2+]i transients in acinar cells from diabetic animals. However, [Ca2+]i transients mediated by Ca2+ release from ER stores (induced by application of either ACh, CCh, NE, or ionomycin in Ca2+-free extracellular medium) were decreased under diabetes. Application of inositol-1,4,5-trisphosphate led to smaller Ca2+ release from ER under the diabetes. Both plasmalemma and ER Ca2+-ATPases activity was reduced and the latter showed the increased affinity to ATP under the diabetes. We conclude that the diabetes caused impairment of salivary cells functions that, on the cellular level, associates with Ca2+ overload, increased Ca2+-mobilizing ability of muscarinic but not adrenergic receptors, decreased Ca2+-ATPases activity and ER Ca2+ content.

[Thapsigargin-sensitive and insensitive intracellular calcium stores in acinar cells of the submandibular salivary gland in rats].

Acinar cells of rat submandibular salivary gland are characterized by heterogeneity of intracellular Ca2+ stores. In the present work we have studied this heterogeneity using Arsenazo III dye to measure a cellular total calcium content and Fura-2/AM, to determine free cytosolic calcium concentration ([Ca2+]i). We have found that the amount of Ca2+ released by inhibition of Ca2+ ATPase of the ER with thapsigargin comprises approximately 30% of total ER calcium. This result was obtained in experiments on both intact and permeabilized acinar cells. We have also shown that both Ca2+ ATPase inhibition with thapsigargin and emptying the stores with acetylcholine (ACh) led to activation of store-operated Ca2+ influx (an increase in total calcium content of approximately 14%). In permeabilized cells application of ACh after preincubation with thapsigargin led to a further decrease in total cellular calcium content (approximately 38%). At the same time in intact cells it resulted in generation of [Ca2+]i transients with gradually decreasing amplitudes. Thus, ACh is capable of producing an additional release of Ca2+ from thapsigargin-insensitive stores. This additional release is IP3-dependent since it was completely blocked by heparin. We conclude that in acinar cells of rat submandibular gland thapsigargin-sensitive and thapsigargin-insensitive Ca2+ stores could exist.

[Permeabilized salivary gland cells as a model to study Ca2+-transporting systems of endoplasmic reticulum membrane]. / Permeabilizovani klityny slynnykh zalos iak model' dlia vyvchennia kal'tsiitransportnykh system membrany endoplazmatychnoho retykuluma.

A method for chemical permeabilization of secretory cells of rat submandibular salivary gland has been elaborated. It was shown that the effects of digitonin on total calcium content in permeabilized acinar cells and protein content in their incubation medium correlated with concentration and duration of the detergent treatment. Digitonin-permeabilized acinar cells perform Ca(2+)-dependent protein secretion, which level depends on the duration of cell incubation in an intracellular buffer solution. The ability of permeabilized acinar cells to perform thapsigargin-sensitive ATP-dependent Ca2+ transport was established by using biochemical approaches and monitoring of the intrareticular calcium concentration with mag-fura 2 dye. Thapsigargin-insensitive Ca2+ store in the permeabilized acinar cells of the salivary gland was shown to be also available. Thus, these data give evidence to conclude that digitonin-permeabilized secretory cells of the submandibular salivary gland are an adequate model to study the mechanisms of Ca(2+)-dependent control of the exocytosis and membrane Ca(2+)-transporting systems of the intracellular calcium stores.