[Effect of temperature and monensin on the level of intracellular sodium and pH in perfused RIF-1 cultured cells].

The direct measurement of temperature in subcutaneously (sc) implanted tumors shows that the actual tumor temperature is by 3-4 degrees C lower than the normal body temperature. Thus, the temperatures usually used for tumor hyperthermia are in fact heating the sc-tumors from 33 to 37 degrees C. The temperature increase during the perfusion of radiation-induced fibrosarcoma (RIF-1) cells from 33 to 37 degrees C caused a reversible increase in intracellular 23Na ([Na+]i) NMR signal intensity by 50-60%. This heating significantly decreased the intracellular pH (pH) in 5 min, but it returned back to the baseline level during the heating period. The 3ATP/P(i) remained generally unchanged throughout the experiment. Monensin, an antitumoral drug and Na+ ionophor, increased [Na+]i by 20% during cell superfusion without heating. When combined, monensin did not increase the heating effect on [Na+]i. However, when monensin was added to the superfusion media, the [Na+]i level did not return to baseline during post-heating recovery, but instead started to increase again. Monensin did not significantly change pH(i) and betaATP/P(i). Our data and the literature show that monensin can accelerate the processes leading to the collapse of the transmembrane Na+ gradient and thus can increase the thermo-sensitivity of tumor cells.

[Cyclic nucleotide regulation of intracellular Ca2+ release in secretory cells of salivary glands of Chironomus plumosus larvae]. / Rehuliatsiia tsyklichnymy nukleotydamy vyvil'nennia Ca2+ iz vnutrishn'oklitynnykh depo u sekretornykh klitynakh slynnykh zaloz lychynky komara-derhuna.

We investigate action of cAMP and cGMP on calcium content in tissue of salivary gland of Chironomus plumosus larvae L. using arsenaso III. In order to permeabilize cells we incubate it in medium with saponin (0.1 mg/ml during 10 min) with next incubation in necessary solution. The internal incubation solution contents (in mM): 15.3 NaCl, 129.94 KCl, 0.35 Na2HPO4, 0.44 KH2PO4, 5.55 glucose, pH 7.0. It was shown that cAMP (1 mkM) didn't cause release calcium through IP3-channels, because IP3 didn't intensify affect of cAMP, which has been established before. We observed that cAMP (100 mkM) intensified ryanodine's action to increase calcium content in saponin-treated gland tissue. We concluded that cAMP (100 mkM) activated calcium releasing though ryanodine-sensitive channels and inhibited IP3-dependent calcium releasing without any stimulation. Different concentration of cGMP didn't cause change in calcium content in saponin-treated gland tissue. But at presence of tapsigargine in incubation medium cGMP (100 mkM) inhibited Ca2+-released channels.

[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.

[Identification of Ca2+ release channels in salivary glands secretory cells of Chironomus plumosus L]. / Identyfikatsiia kanaliv vyvil'nennia Ca2+ v sekretornykh klitynakh slynnykh zaloz lychynky komara-derhuna.

The presence of two types of well-characterised Ca2+ release channels, namely IP3-receptors (Ins(1,4,5)P3Rs) and ryanodine-receptors (RyRs), was detected in the salivary glands secretory cells of Chironomus plumosus L. For this aim different blockators and activators of these Ca2+ -transport systems were used. The conditions for permeabilization of these cells by saponine were experimentally chosen for their more intensive action. It was shown that IP3 decreased calcium content in saponine-treated gland tissue by (41.14 +/- 11.75)%. The effect of IP3 was not observed under condition of heparin and eosin Y presence in the incubation medium, but heparin alone did not cause any action on calcium content in saponine-treated gland tissue. The observed effects of IP3 are supposed to be the evidences of Ins (1,4,5)P3Rs presence in the intracellular membrane of this object. It was also shown that calcium content in intact gland tissue increased by (67.12 +/- 22.60)% in presence of heparin (500 mkg/ml) in the incubation medium. This effect of heparin was also observed with presence of verapamil (100 mkM) and eosin Y (5, 20 mkM) in incubation medium. So, this effect is not connected with function of voltage-gated Ca2+ -channels and Ca2+ -pumps. Ryanodine in concentration of 5nM decreased calcium content in saponine-treated gland tissue by (35.18 +/- 3.87)% but it caused the increase of calcium content at high concentration (500 nM) by (40.72 +/- 12.52)%. It improved the presence of RyRs in intracellular membrane of secretory cells of this object. Besides, these channels, perhaps, belong to "non-sensitive" to caffeine, because caffeine did not affect calcium content in the gland tissue neither in presence nor with absence of eosin Y.

[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.

[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.

[The modification of potential-dependent calcium channels in the membrane of secretory cells in a calcium-free medium]. / Modyfikatsiia potentsialozalezhnykh kal'tsiievykh kanaliv membrany sekretornykh klityn u bezkal'tsiievomu seredovyshchi.

Effect of modification of potential-dependent calcium channels in cells of the upper lobe of the salivary gland in Chironomus larva has been ascertained in the Ca-free medium containing Ca-binding substances (EGTA or EDTA). It is shown that the inward current depends on the sodium transmembrane gradient. The amplitude of the inward sodium current decreases under the influence of verapamil as well as with an increase of external concentration of Ca2+ ions. The metabolic dependence of modified calcium channels as well as the pharmacological sensitivity are unchanged. Tau n of the sodium current through modified calcium channels increases insignificantly comparing with that of the calcium current. Maximum of the current-voltage relationship of the sodium current through modified calcium channels is shifted to a negative side by 10-15 mV as compared with the calcium current through native channels.

[Characteristics of potential-dependent calcium channel current of the secretory cell membrane]. / Kharakterystyka potentsialozalezhnoho kal'tsievoho strumu membrany sekretornykh klityn.

The parameters of inward current of potential-dependent calcium channels in cells of the upper and lower lobes of the salivary glands in Chironomus larvae have been studied. The current activation thresholds in cells of both types are near -60 mV, but in cells of the upper lobe the current reaches its maximum values at -20 mV, while in cells of the lower lobe at -10 mV. The current density in cells of the upper lobe is approximately 1.3 times higher and the inactivation time constant is 1.62 less than in cells of the lower lobe. Calcium channels in cells of the upper and lower lobes of glands also differ in metabolic dependence: introduction of cAMP, ATP and Mg2+ into cells of the lower gland lobe causes a considerable increase in the current amplitude, while the same action in cells of the upper lobe--its decrease. The current amplitude in the cells of the lower lobe decreases under the influence of nitrendipine and La, Co, Ni, Cd, Zn cations as well as a decrease of the temperature or pH in the external solution.

[The identification and properties of chloride potential-dependent channels in the membrane of secretory cells]. / Identifikatsiia i svoistva khlornykh potentsialozavisimykh kanalov membrany sekretornykh kletok.

Outward current of the salivary gland cells membrane of chironomus larva activated by the displacement of the membrane potential to the region of positive values has been registered by the voltage-clamp method under conditions of intracellular dialysis in the presence of the chloride transmembrane gradient. Activation threshold of the current is about +20 mV. Subsequent displacement of the membrane potential to the region of positive values causes an increase of the current. Time constant of the current activation is (573 +/- 34.4) ms. The current decreases with the reduction of extracellular chloride concentration, under the influence of tannin acid and temperature lowering, under conditions of alkaline medium. The current increases due to Hg2+ ions and lowering of the outward solution pH. Thus, the membrane of secretory cells contain high-threshold potential-dependent chloride channels which are characterized by the following selectivity series: Br- greater than Cl- greater than NO3- greater than SO4(2-) greater than F- greater than HCOO- greater than CH3COO-.

[The demonstration of potential-dependent potassium channels in the plasma membrane of secretory cells]. / Vyiavlenie potentsialozavisimykh kalievykh kanalov plazmaticheskoi membrany sekretornykh kletok.

Outward current of the salivary gland cells membrane of chironomus larva activated by the displacement of the membrane potential to the region of positive values has been registered by the voltage-clamp method under conditions of intracellular dialysis in the presence of only the potassium transmembrane gradient. Activation threshold of the current is about +10 mV. Subsequent displacement of the membrane potential to the region of positive values causes an increase of the current. Time constant of the current activation is (652 +/- 57) ms. The current decreases with the intracellular potassium concentration, under the influence of tetraethyl-ammonium and 4-aminopyridine. Thus, high threshold potential-dependent potassium channels are presented in the secretory cells membrane.

[Identification of depolarization-activated calcium flow in the secretory cell membrane]. / Identifikatsiia aktiviruemogo depoliarizatsiei kal'tsievogo toka membrany sekretornykh kletok.

The inward depolarization-activated current of the salivary gland cell membrane of the chironomus larva has been registered by the voltage-clamp method under conditions of the intracellular dialysis in the presence of the calcium transmembrane gradient only. An activation threshold of the current is about -50 mV, the current maximum is under -10 mV, reversion up to +10 mV leads to the current reduction. An increase in the extracellular Ca2+ concentration causes current amplification; the current decreases under the influence of verapamil, chlorpromazine, Mn2+ and Co2+. These results could indicate the existence of potential-dependent calcium channels in the secretory cell membrane.

[Effect of protein-modifying factors on sodium and potassium leakage currents of the secretory cell membranes]. / Vliianie faktorov, deistvuiushchikh na belki, na natrievyi i kalievyi toki utechki membrany sekretornykh kletok.

Potassium and sodium leakage currents decrease when pH of the surroundings is lowering. Dicyclohexylcarbodiimide, a specific reagent of the COOH-groups does not change leakage currents. Trypsin, chymotrypsin, papain and pronase increase the sodium leakage current, not altering the potassium one. Blocking agents of the SH-groups: Hg2+ and Cd2+ decrease potassium leakage current, without changing the sodium one. Results of the proteolysis testify to that the diffusion of sodium into the cells in response is accomplished through the protein components of the membrane. The action of the blocking agents of SH-groups points out that the diffusion of potassium from the cells is realized also via the protein components of the membrane.