The Effects of Diuretics on Intracellular Ca(2+) Dynamics of Arteriole Smooth Muscles as Revealed by Laser Confocal Microscopy

The regulation of cytosolic Ca(2+) homeostasis is essential for cells, including vascular smooth muscle cells. Arterial tone, which underlies the maintenance of peripheral resistance in the circulation, is a major contributor to the control of blood pressure. Diuretics may regulate intracellular Ca(2+) concentration ([Ca(2+)](i)) and have an effect on vascular tone. In order to investigate the influence of diuretics on peripheral resistance in circulation, we investigated the alteration of [Ca(2+)](i) in testicular arterioles with respect to several categories of diuretics using real-time confocal laser scanning microscopy. In this study, hydrochlorothiazide (100 µM) and furosemide (100 µM) had no effect on the [Ca(2+)](i) dynamics. However, when spironolactone (300 µM) was applied, the [Ca(2+)](i) of smooth muscles increased. The response was considerably inhibited under either extracellular Ca(2+)-free conditions, the presence of Gd(3+), or with a treatment of diltiazem. After the thapsigargin-induced depletion of internal Ca(2+) store, the spironolactone-induced [Ca(2+)](i) dynamics was slightly inhibited. Therefore, the spironolactone-induced dynamics of [Ca(2+)](i) can be caused by either a Ca(2+) influx from extracellular fluid or Ca(2+) mobilization from internal Ca(2+) store, with the former being dominant. As tetraethylammonium, an inhibitor of the K(+) channel, slightly inhibited the spironolactone-induced [Ca(2+)](i) dynamics, the K(+) channel might play a minor role in those dynamics. Tetrodotoxin, a neurotoxic Na(+) channel blocker, had no effect, therefore the spironolactone-induced dynamics is a direct effect to smooth muscles, rather than an indirect effect via vessel nerves.