Acute effects of testosterone on intracellular Ca2+ kinetics in rat coronary endothelial cells are exerted via aromatization to estrogens

The objective of this work was to evaluate the effects of testosterone (T) and 17β-estradiol (E2) on coronary microvascular endothelial cells (CMECs) of male and female rats. To analyze the short-term effects of such sex steroid hormones on intracellular Ca2+ concentration ([Ca2+]i) kinetics, we used the chelating agent fura-2 acetoxymethyl ester. We also explored the possibility of testosterone aromatization by using selective inhibitors of the aromatase enzyme cytochrome P-450 aromatase (P450arom), aminoglutethimide (4 μM), and 4-hydroxyandrostenedione (4 μM). The presence of P450arom was investigated by immunocytochemical and immunoblot assays using peptide-generated polyclonal antibodies raised against a 20-amino acid synthetic fragment of rat P450arom and by in situ hybridization to locate the aromatase mRNA in such cells. The activity of P450arom was demonstrated by the stereospecific loss of the tritium atom of [1β-3H]androstenedione. Our results indicate that both T and E2 induced a rapid increase...