Effects of indapamide on Ca2+ entry into vascular smooth muscle cells.

Part of the antihypertensive action of indapamide has been attributed to a direct inhibitory action on Ca2+ entry into vascular smooth muscle cells. The present study has been performed to identify the possible mechanisms involved. To this end the effect of indapamide on intracellular Ca2+ activity - [Ca2+]i - has been tested under control conditions and under conditions known to increase [Ca2+]i such as osmotic cell swelling (mimicking mechanical stress), depolarization (increase of extracellular K+ concentration) and oxidative stress (H2O2). Indapamide (10 micromol/l) was without effect on control [Ca2+]i, but significantly blunted the increase of [Ca2+]i following potassium-induced depolarization or following osmotic cell swelling. It did not significantly modify the increase of [Ca2+]i induced by H2O2. The effects on cell membrane potential induced by increased [K+], osmotic cell swelling, or H2O2 were not significantly modified by indapamide (10 micromol/l). Voltage-gated Ca2+ currents were not significantly modified by 10 micromol/l indapamide, but were significantly reduced by 100 micromol/l and blunted by 1 mmol/l. In conclusion, indapamide at high concentrations (100 micromol/l) inhibits voltage-gated Ca2+ channels, an effect which blunts the increase of [Ca2+]i during depolarization of the cell membrane at increased extracellular [K+] or osmotic stress. Whether these effects at high concentrations of indapamide are relevant to the antihypertensive action, however, cannot be established from these in vitro studies.

Ca2+ entry and vasoconstriction during osmotic swelling of vascular smooth muscle cells.

Exposure of aortic strips from guinea-pigs to hypotonic extracellular fluid is followed by marked vasoconstriction, which is inhibited by D-600 (3 microM), a blocker of voltage-sensitive Ca2+ channels. Conventional electrophysiology, patch-clamp studies, pH determination with 2',7' bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and Ca2+ measurements with Fura-2 have been performed on smooth muscle cells cultured either from rat or human aorta to further elucidate the underlying mechanisms. Exposure of the cells to a 25% hypotonic extracellular fluid leads to a rapid and fully reversible depolarization, paralleled by an increase of the selectivity and conductance of the cell membrane to Cl-, an acidification of the cytoplasm and an increase of intracellular Ca2+ concentration ([Ca2+]i). The latter is inhibited by the Ca2+ channel blocker D-600 (1-3 microM). It is concluded that osmotic cell swelling leads to the activation of an anion channel. The subsequent depolarization of the cell membrane activates voltage-sensitive Ca2+ channels which increases [Ca2+]i, thus stimulating the contraction of vascular smooth muscle cells.