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The aim of the present study was to investigate rapid effects of aldosterone and other steroids on intracellular pH of vascular smooth muscle cells and to compare these effects with those of peptide hormones. After addition of 100 nmol/L aldosterone, initial acidification is followed by significant alkalinisation occurring within two minutes, while 1 mumol/L hydrocortison does not affect intracellular pH. The initial response to 100 nmol/L angiotensin II is similar; however, subsequent alkalinization is not seen for this agonist. PDGF induces an initial acidification followed by a minor recovery so that cells remain acidified for eight minutes. Both pH recovery after angiotensin II and alkalinization after aldosterone were blocked in sodium-free medium. These results demonstrate rapid effects of aldosterone on intracellular pH in vascular smooth muscle cells, which include final alkalinization not seen after angiotensin II or PDGF.

Genomic mechanisms of steroid action have been increasingly elucidated over the past four decades. In contrast, rapid steroid actions have been widely recognized only recently, and detailed analysis of the mechanisms involved are still lacking. The present article describes rapid effects of mineralocorticoid hormones on free intracellular calcium in vascular smooth muscle cells as determined by fura 2 spectrofluorometry in single cultured cells from rat aorta. These effects are almost immediate and reach a plateau after only 3 to 5 minutes and are characterized by high specificity for mineralocorticoids versus glucocorticoids. The potent mineralocorticoids aldosterone and fludrocortisone are agonists with estimated apparent EC50 values of approximately 0.1 to 0.5 nmol/L; deoxycorticosterone acetate is an agonist with an EC50 of approximately 5 nmol/L; and progesterone, cortisol, corticosterone, and estradiol have much lower potency (EC50 values of approximately 0.5 to 5 mumol/L). The effect of aldosterone is blocked by neomycin and short-term treatment with phorbol esters but augmented by staurosporine, indicating an involvement of phospholipase C and protein kinase C. The Ca2+ effect appears to involve the release of intracellular Ca2+, as shown by the inhibitory effect of thapsigargin; intriguingly, a relatively small maximum effect (approximately 40 nmol/L increase) is consistently seen. This mechanism operates at physiological subnanomolar aldosterone concentrations and appears to be a likely candidate for rapid fine tuning of cardiovascular responsivity. It may also contribute to known clinical features of mineralocorticoid action that are difficult to explain by the traditional genomic mechanism alone.

Rapid in vitro effects of aldosterone on the intracellular concentrations of sodium, potassium and calcium, cell volume and the sodium-proton-antiport have been described in human mononuclear leukocytes and vascular smooth muscle cells (VSMC). These nongenomic effects are signalled through membrane receptors with a high affinity for aldosterone, but not for cortisol, and through the phosphoinositide pathway. In the present study, we demonstrate that free intracellular calcium is increased rapidly by aldosterone in VSMC and endothelial cells (EC) as determined by single cell imaging of Fura2-fluorescence. In VSMC, calcium elevation is localized to the perinuclear region whereas in EC, a predominant increase of subplasmalemmal calcium is seen. In VSMC, effects are half maximal at 0.1 nM aldosterone; cortisol is inactive up to 0.1 microM. These data show that intracellular signalling for aldosterone also involves calcium, but the subcellular localization of this signal varies between cell types.