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.

Calcium signalling: store-operated channel found at last.

The intestinal Ca(2+) transport protein CaT1 has recently been shown to have the biophysical characteristics of the Ca(2+)-release activated Ca(2+) channel that refills internal Ca(2+) stores following agonist-elicited release. This finding highlights a hitherto unrecognized link between epithelial Ca(2+) transport and Ca(2+) signalling.

[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.

[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.

[Influence of eosin y and orthovanadate on the steady-state of Ca(+)2 entry into secretory cells of exocrine glands and their spontaneous secretion]. / Vpliv eozinu Y ta ortovanadatu na bazal'nu sekretsiiu ta statsionarnii vkhid Ca(+)2 u sekretorni klitini ekzokrinnikh zaloz.

Ca(2+)-pump blocators in low concentrations (eozyn Y up to 5 mM and ortovanadate up to 40 mM) essentially increases of Ca2+ content in salivary gland of Chironomus plumosus larvae's and spontaneous protein secretion. It was shown that eozyn Y much more effectively suppresses of plasma membrane Ca(2+)-pump then ortovanadate. Eozyn Y and ortovanadate in higher concentrations essentially decrease of Ca2+ content in glands and spontaneous protein secretion. The former is evoked by suppression of endoplasm reticulum Ca(2+)-pump, decreasing of Ca2+ influx in cells following by diminishing of Ca2+ transmembrane gradient. Therefore, energydependent Ca2+ transporting systems of plasma membrane and endoplasm reticulum effectively regulate steady-state Ca2+ entry in secretory cells of Chironomus plumosus salivary glands and maintain relatively low level of spontaneous secretion.

[The demonstration of steady-state Ca2+ influx into the cells of the salivary glands in Chironomus plumosus L. larvae and its role in basal secretion]. / Dokazi statsionarnoho vkhodu Ca2+ u klityny slynnykh zaloz lychynky Chironomus plumosus L. ta ioho rol' u bazal'nii sekretsiï.

Ca2+ content in Chironomus plumosus salivary gland tissue we determined using Ca(2+)-sensitive dye arsenazo III and protein concentration in medium--by Lowry method. It was showed correlation between increasing of Ca2+ content in gland and basal secretion. So, basal secretion is Ca(2+)-dependent process. Owing to adding to medium verapamil, diltiazem and nifedipine (10(-4) M) took place decreasing of Ca2+ content and secretion. Since decreasing of Ca2+ entry in cells owing to blocators action did't exceed 53% (in the case of nifedipine) we assumed that permanent Ca2+ entry formed by potential- and receptor-operated channels. It was established dependence of continuous Ca2+ entry in cells, basal secretion and effectiveness of blockade of Ca(2+)-channels by nifedipine on the [Ca2+]e. Therefore, through the exocrine secretory cells membrane of Chironomus plumosus larvae salivary gland carry out continuous diffuse Ca2+ entry for maintain of basal secretion by cells. This Ca2+ entry provide by population of open Ca(2+)-channels, sensitive to blocators of potential dependent of Ca(2+)-conduction plasmatic membrane.

[Kinetic characteristics of Ca2+, Mg2+-ATPases in cells of the submandibular salivary gland of rats]. / Kinetychni kharakterystyky Ca2+, Mg2+-ATPases klityn pidshchelepnoï slynnoï zalozy shchuriv.

Kinetic properties of Ca2+, Mg2+-ATPases membranes from acinar cells of rat submandibular salivary glands have been investigated. It was found that kinetics of ATP hydrolysis dependent on Ca2+, Mg2+-ATPases corresponds to the first-order reaction during first 2 min. It was found that the initial velocity of the reaction (V0), maximal amount of the reaction product (Pmax) and characteristic time of the reaction (T) comprised 1.8 +/- 0.4 and 1.6 +/- 0.2 mmole Pi/min per 1 mg protein, 7.5 +/- 1.3 and 1.4 +/- 0.2 mmole Pi/mg protein and 4.1 +/- 0.7 min and 1.1 +/- 0.1 for Ca2+-ATPases from plasma and endoplasmic reticulum membranes, correspondingly. High- and low-affinity sites of ATP and Ca2+-binding in Ca2+-ATPases from plasma and endoplasmic reticulum membranes were identified. Negative cooperation in ATP binding to Ca2+-ATPase from plasma membrane and a positive cooperation for Ca2+-ATPase from endoplasmic reticulum has been found. Ca2+ binding to low-affinity sites of both Ca2+-ATPases showed no cooperation, while Ca2+ binding to high-affinity sites showed the positive cooperation. Using the Hill's coordinates we have found the values of the Mg2+ Michaelis constant (K(Mg)) which yielded 3.89 x 10(-5) and 3.80 x 10(-5) mole/l for Ca2+-ATPases from plasma and endoplasmic reticulum membranes, correspondingly. It is supposed that obtained data are important for further studies of molecular and membrane mechanisms involved in the regulation of intracellular calcium signalling and secretion by salivary acinar cells.

[Effect of p-chloromercuribenzoic acid and dithiothreitol on the Ca(2+) content of salivary glands and their protein secretion]. / Vplyv parakhlormerkuriibenzoatu ta dytiotreitolu na vmist Ca(2+) u tkanyni slynnykh zaloz ta sekretsiiu nymy zahal'noho bilka.

It has been shown, less concentrations of p-chlormercuribenzoate (1 and 2.5 mM) increased Ca2+ content in gland tissue and thereby protein secretion level that may occurred mainly by suppression Ca(2+)-pump or/and stimulation of Na(+)-Ca(2+)-exchange (both in cell plasma membrane) through modulation of SH-groups which form part of their molecules. Higher PCMB concentrations markedly decreased Ca2+ content in gland tissue as well as protein secretion. Effects of PCMB (5 and 10 mM), depending on the direction of Na(+)-Ca(2+)-exchange functioning (Ca(2+)-efflux or Ca(2+)-influx), were evoked or presumably by suppression of endoplasma reticulum Ca(2+)-pump (at conditions Na(+)-dependent Ca(2+)-efflux) or Na(+)-dependent Ca(2+)-influx into the cells that clearly confirmed when PCMB was added on the background of eosin Y (specific Ca(2+)-ATPase inhibitor). Possible role of potential dependent Ca(2+)-channnels in the mediating of PCMB effects is discussed. Introducing of dythiothreitol (DTT) increased Ca2+ content in glands and decreased secretion level obviously by protection of SH-groups of cell Ca(2+)-transporting systems and thereby diminished [Ca2+]i. Finally, we confirm important functional role of SH-groups in the regulation of Ca(2+)-homeostasis in secretory cells of exocrine glands.

[Effect of chlorpromazine on the Ca2+-transport system of secretory cell membrane in Chironomus plumosus L. larval salivary glands]. / Vplyf khlorpromazynu na ca2+-transportni systemy plazmatychnoï membrany sekretornykh klityn slynnoï lychynky Chironomus plumosus L.

Effect of chlorpromasine (specific blocking agent of calmoduline) on Na(+)-Ca(2+)-exchanger functioning, Ca(2+)-pump and potential dependent Ca(2+)-channels in plasmatic membrane of isolated salivary glands in Chironomus plumosus L. larvae was investigated. Addition of chlorpromasine in different concentrations to the incubation medium with physiological Na+ and K+ concentration increased Ca2+ content in the gland tissue and secretion of general protein by gland cells. Chlorpromasine addition to the hyposodium and hyperpotassium mediums decreased Ca2+ content in the gland tissue and protein secretion. We made a conclusion that chlorpromasine, as an inhibitor of calmoduline, blocks Na(+)-Ca(2+)-exchanger and Ca(2+)-pump of plasmatic membrane of secretory cells. Potentialdependent Ca(2+)-channels are also effectively blocked by chlorpromasine but mechanism of this process is unknown. We suppose that Ca(2+)-calmoduline complex forming leads to increase of calcium oscillations amplitude in the cells of the investigated glands and stimulation of secretion.

[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.