Природа модулюючої дії оксиду азоту на електрогенез у гладеньких м’язах основної легеневої артерії та їх скорочення-розслаблення

Показано, що внесок К_Са-каналів великої провідності у вихідний трансмембранний іонний струм поодиноких ізольованих ГМК основної легеневої артерії кроля і у формування мембранного потенціалу спокою (МПС) ГМК смужок перевищує внесок К_V-каналів.

Ключові слова: основна легенева артерія, гладеньком'язові клітини, внутрішньоклітинні кальцієві депо, кальційзалежні калієві канали великої провідності, оксид азоту, нітрогліцерин, тетраетиламоній, “сахарозний місток”, “patch-clamp”.

Аннотация

Тележкин В.С. “Природа модулирующего действия NO на электрогенез в гладких мышцах основной легочной артерии и их сокращения-расслабления”. - Рукопись.

Диссертация на соискание научной степени кандидата биологических наук по специальности 03.00.02 - биофизика. - Институт физиологии им. А.А. Богомольца НАН Украины, Киев, 2003.

Наша работа посвящена исследованию возможных механизмов действия NO на гладкомышечные клетки (ГМК) основной легочной артерии кролика. В качестве донора NO использовался нитроглицерин (НГ).

Эксперименты на многоклеточных гладкомышечных полосках производились с использованием метода “сахарозный мостик” и тензометрии, с помощью которых изучалось действие NO на вызванные электрическую и сократительную активность деэндотелизированных гладкомышечных полосок основной легочной артерии. С помощью метода фиксации потенциала (“patch-clamp”) проводились исследования фармако-биофизических характеристик выходящего трансмембранного ионного тока, входящего потенциалзависимого Са²⁺-тока и одиночных K_Ca-каналов большой проводимости мембраны изолированных одиночных ГМК основной легочной артерии кролика и влияние на них NO.

Установлено, что NO расслабляет гладкие мышцы основной легочной артерии, и этот эффект опосредован, как минимум, четырьмя известными механизмами: стимуляцией активности К_Са-каналов большой проводимости, угнетением активности высокопороговых потенциалзависимых Са²⁺-каналов L-типа, подавлением освобождения ионов Са²⁺ из инозитолтрифосфат-чувствительного компонента саркоплазматического ретикулума и влиянием на сократительный аппарат ГМК. Кроме того, результаты нашей работы указывают на то, что электрофизиологические свойства и, соответственно, реакции изолированных ГМК существенно отличаются от свойств и ответов ГМК многоклеточных препаратов.

Показано, что вклад К_Са- каналов большой проводимости в выходящий трансмембранный ионный ток одиночных изолированных ГМК основной легочной артерии кролика и в формирование мембранного потенциала покоя (МПП) ГМК полосок превосходит вклад К_V-каналов.

Ключевые слова: основная легочная артерия, гладкомышечные клетки, внутриклеточные кальциевые депо, кальцийзависимые калиевые каналы большой проводимости, оксид азота, нитроглицерин, тетраэтиламмоний, “сахарозный мостик”, “patch-clamp”.

Summary

Telezhkin V.S. “Nature of the modulatory effects of NO on the elektrogenesis in and contraction-relaxation of main pulmonary artery smooth muscles”. - Manuscript.

Thesis for A Candidate Biology degree by the speciality 03.00.02 - biophysics. - Bohomolets Institute of Physiology of The National Academy of Science of Ukraine, Kiev, 2003.

The dissertation is devoted to investigation of the mechanismes of the effects of nitric oxide (NO) on rabbit main pulmonary artery smooth muscle cells (PASMC). In our research, we used nitroglicerine (NG) as an NO donor.

The first part of the study was carried out on endothelium-denuded multicellular smooth muscle strips, by means of a sucrose-gap technique and tensometry, to measure the smooth muscle electrical and contractile activity and to investigate the modulation action of NO. All the experiments on smooth muscle strips were performed at +37 ^oC. The magnitudes of smooth muscle strip responses were normalized with respect to those (contraction and depolarization) evoked by application of the high-potassium 60 mM solution for each experiment.

NO relaxed smooth muscle strips with no hyperpolarization. NG decreased by about 20 % the muscle strip contraction induced by the high-potassium solution and caused the complete relaxation when NO acted in combination with a blocker of the voltage-gated L-type calcium channels (L-type Ca²⁺-channels), nifedipine. Such response pattern suggested that NO exerts an immediate effect on the PASMC contractile apparatus.

Our other experiments, in which we investigated the role of the intracellular calcium stores of PASMC in the NO effects, were carried out in normal and calcium-free solutions. A high-amplitude tonic contraction evoked by an б-adrenoreceptor agonist, phenilephrine, significantly depended on extracellular calcium, contrastingly to a small-amplitude transient caffeine-induced contraction. NG suppressed the phenilephrine-induced contraction approximately by 40 %, in both normal and calcium free solutions, but nearly did not reduce a contraction evoked by caffeine. These data show that NO affected calcium release from inositoltriphosphate-sensitive calcium stores but did not influence its release from the ryanodine-sensitive calcium stores in PASMC.

Investigation of the electrical and contractile responses of rabbit PASMC to applications of potassium channel blockers, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), and the NO donor NG was performed under conditions of inhibition of the adrenergic and cholinergic neuromuscular transmission with phentolamine, propranolol, and atropine. TEA and 4-AP acted in a dose-dependent manner and evoked depolarization and smooth muscle contraction. Simultaneous action of TEA and 4-AP caused a significant depolarization, generation of action potentials (AP), and increased the contraction intensity. NG applied together with TEA and 4-AP completely inhibited APs and contraction, whereas the degree of depolarization did not change. These data indicate that calcium-dependent potassium channels of large conductivity (K_Ca-channels) play more significant role in the formation of the resting membrane potential in rabbit PASMC than voltage-gated 4-AP-sensitive potassium channels (K_V-channels) do. This suggested that, in the presence of TEA and 4-AP, a supressive effect of NO on the AP generation and contraction results from inhibition of the L-type Ca²⁺-channels and from a decrease in the Ca²⁺-sensitivity of the contractile apparatus.

The second section of the study was carried out on isolated PASMC using a voltage-clamp (“patch-clamp”) technique under “whole-cell” configuration. TEA (5 mM) more than two-fold decreased the outward transmembrane current, whereas 4-AP (5 mM) at high positive potentials increased its amplitude. A simultaneous action of TEA (5 mM) and 4-AP (5 mM), however, not only significantly suppressed the transmembrane outward current, but also changed the activation threshold from -40 mV to -10 mV. NG (10 мM) increased the transmembrane current by 32.6±19.4 % (n=6) without changing the threshold activation potential and increased the probability of spontaneous outward transmembrane currents (STOCs)-like fluctuations observed at high positive potentials, that suggested the influence of K_Ca-channel activation. Application of TEA (1 mM) together with NG (10 мM) completely abolished STOCs-like fluctuations.

Experiments with recording of the activity of single channels showed the single K_Ca-channel conductance in an “outside-out” patch configuration calculated from the current-voltage plot is about 150 pS. TEA (1 mM) in an “outside-out” configuration completely inhibited unitary currents through the K_Ca-channels at a potential +40mV. In a “cell-attached” configuration experiments, NG (100 мM) enhanced the probability of channel opening to 180±5 %, compared with the controle. NG did not influence the amplitude of the single K_Ca-channel current.

The tests of the NO action on inward voltage-gated calcium currents performed in a “whole-cell” configuration showed that the current is passed through the high-threshold L-type Ca²⁺-channels. It was almost complitely inhibited by nifidipine (1 мM). NG (100 мM) decreased the current amplitude by 20 % or less.

In the path-clamp experiments, the outward transmembrane current was activated at potentials more positive than -40 mV, whereas under sucrose-gap conditions (which were quite close to the physiological ones), the potassium chanel activity was observed at the resting membrane potential (which in PASMC is about from -50 to -60 mV). So, such discrepansies suggest that the electrophisiological properties and responses of single PASMC and those of multicellular tussue preparations could significantly differ from each other.

Key words: main pulmonary artery, smooth muscle cells, intracellular calcium stores, calcium dependent potassium channels of large conductivity, nitric oxide, nitroglicerine, tetraethylammonium, sucrose-gap, patch-clamp.

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