Регуляція внутрішньоклітинних СА2+-транспортувальних систем малоклітинних травних залоз

Ключові слова: слинні залози, екзокринні секреторні клітини, внутрішньоклітинні Ca²⁺-транспортувальні системи, ІФ₃-чутливі Са²⁺-канали, ріанодинчутливі Са²⁺-канали.

Бычкова С.В. Регуляция внутриклеточных Са²⁺-транспортирующих систем малоклеточных желез - Рукопись.

Диссертация на соискание научной степени кандидата биологических наук по специальности 03.00.13. - физиология человека и животных. - Львовский национальный университет имени Ивана Франко, Львов, 2004.

Диссертация посвящена исследованию функционирования внутриклеточных Са²⁺- транспортирующих системсекреторных клеток слюнных желез личинки Chironomus plumosus L. Идентифицировано ИФ₃- и рианодинчувствительные Са²⁺-каналы в мембранах внутриклеточных кальциевых депо и установлена тесная взаимосвязь между ними. Предполагается согласованное функционирование названых каналов как единая “осциллирующая единица”. Электронно-микроскопические исследования определили, что митохондрии размещены возле базолатерального полюса этих клеток и почти отсутствуют в апикальной части. Это подтверждает, что их роль в ограничении кальциевых сигналов апикальным полюсом мало возможна для этих клеток. Установлено, что цАМФ угнетает освобождение Са²⁺ ИФ₃-чувствительними каналами и усиливает освобождение Са²⁺ из рианодинчувствительного депо. Установлено, что цГМФ угнетает освобождение Са²⁺ из внутриклеточных депо только в присутствии тапсигаргина. Показано, что кальмодулин не влияет на внутриклеточные Са²⁺- транспортирующие системы при отсутствии стимуляции клетки. Однако он мощно усиливает освобождение Са²⁺ рианодинчувствительными каналами и несущественно усиливает освобождение Са²⁺ из ИФ₃-чувствительных депо. Блокатор SH-групп усиливал освобождение Са²⁺ с депо при наличии эозина Y в среде инкубации. Это указывает на важную роль SH-групп внутриклеточных каналов освобождение Са²⁺ в их функционировании. Предлагается схема взаимосвязи между внутриклеточными Са²⁺-транспортирующими системами.

Ключевые слова: слюнные железы, экзокринные секреторные клетки, внутриклеточные Са²⁺-транспортирующие системы, ИФ₃-чувствительные Са²⁺-каналы, рианодинчувствительные Са²⁺-каналы.

Bychkova S.V. Regulation of intracellular Ca²⁺-transporting systems of less-cells salivary glands. - Manuscript.

Thesis for a doctor's degree by speciality 03.00.13 - human and animal physiology. - Ivan Franko National University of Lviv, Lviv, 2004.

The dissertation is devoted to investigations of functioning of channels of Ca²⁺ release from salivary glands secretory cells' intracellular stores of Chironomus plumosus L. larvae, in particular, Ins(1,4,5)P₃Rs and RyRs. We developed experimental conditions for permeabilization of secretory cells of Chironomus plumosus L. larvae salivary gland by saponine. To check the penetration of plasma membrane after cell incubation with different concentration of saponine cells have been painted by tripan blue. In order to confirm intact of endoplasmatic membrane we testified functioning of Ca²⁺-ATPaze. After that the penetration of plasmatic membrane was testified under microscope. Using arsenazo III it was proved that thapsigargine and eozine Y statistically authentic decreased calcium content in saponine-treated glands, besides adding of ATP to incubation solution increased calcium content.

It was found that Ins(1,4,5)P₃ evokes statistically authentic decrease of Ca²⁺ content in gland tissue. This effect is completely inhibited by heparin. This improved functioning of Ins(1,4,5)P₃-indused calcium relesing mechanism in intracellular stores. It was also proved that ryanodine has concentration-dependent effect on Ca²⁺ content in saponine-treated gland tissue: in low concentration (5 nM) ryanodine causes Ca²⁺ content decrease and in higher concentration (500 nM) - its increase. So it confirmed the functioning of ryanodine-sensetive Ca²⁺-relesed channels in rest cells. We assume that Ins(1,4,5)P₃Rs and RyRs are functioning coordinately as “common oscillator unit”. The similar system was shown in pancreatic acinar cells by Cancela et al. (2000). Our data shows that some of RyRs are activated and release Ca²⁺ in the rest cells that is necessary for activation of Ins(1,4,5)P₃Rs and vice versa. At these conditions generation of Ca²⁺-signals do not take place, because released Ca²⁺ is immediately returned back into intracellular store by Ca²⁺-pumps.

We showed the functioning of mitochondrial Ca²⁺-uniporter in rest secretory cells, which was blocked by ruthenium red and was not sensitive to eozineY. We also found the correlation between ryanodine-induced Ca²⁺ release from intracellular store and Ca²⁺ accumulation by mitochondria. Using electron-microscopy it was shown that mitochondria are located mainly in basolateral pole of salivary gland secretory cells of Chironomus plumosus L. larvae. So we didn't observe mitohondrail ring in apical part of the cells as it has been postulated for pancreatic acinar cells. It allows us to suggest exists a low probability of mitochondria role in localization Ca²⁺-signals in apical pole of these cells.

The regulation of intracellular Ca²⁺-released channels by cAMP-adenilatcyclase, cGMP-gyanilatcyclase and Ca²⁺-calmoduline systems of these cells have been studied for the first. Using cAMP in different concentration and activator of adenilatcyclase - forskolin the activation of Ca²⁺ release was proved through RyRs and inhibition of Ca²⁺ release from Ins(1,4,5)P₃Rs. We also found, that cGMP inhibited Ca²⁺ release from intracellular store only at thapsigargine presence in incubation medium.

It was established, that chlorpromazine (antagonist of calmodulin) alone didn't cause any changes on Ca²⁺ content in saponine-treated glands, because calcium content in saponine-treated salivary glands didn't change neither at presence, nor at absence of heparine, tapsigargine and ryanodine (500 nM) in incubation medium. However, chlorpromazine slackened Ins(1,4,5)P₃ effect on Ca²⁺ content in salivary gland tissue and completely inhibited its decrease, evoked by ryanodine (5 nM). We conclude that calmodulin modifies Ca²⁺ release through RyRs of salivary glands secretory cells of Chironomus plumosus larvae in direct way, but Ins(1,4,5)P₃Rs might be modified by calmodulin not directly. We supposed that effect of calmodulin on Ins(1,4,5)P₃Rs is mediated by correlation between Ca²⁺-released channels or other mechanisms. It was also shown that SH-groups blockator (parahlormerkuriobenzoat) increased Ca²⁺ release at eosine Y presence in incubation medium of saponine-treated salivary glands.

A scheme of correlated functioning of intracellular Ca²⁺-transporting systems in salivary glands secretory cells of Chironomus plumosus larvae was suggested, according to which there were state of dynamic balance between the releasing of Ca²⁺ from intracellular stores and accumulation of Ca²⁺ into stores. It means that some of Ca²⁺released channels are active in rest cells, which is necessary for functioning of some of Ca²⁺-pumps of endoplasmatic reticulum.

Key words: salivary glands, secretory cells, permeabilization, RyRs, InsP₃Rs, Ca²⁺-transported systems, Ca²⁺-uniporter.

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