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 ([Ca ²⁺]_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 (P450_arom), aminoglutethimide (4 μM), and 4-hydroxyandrostenedione (4 μM). The presence of P450_arom was investigated by immunocytochemical and immunoblot assays using peptide-generated polyclonal antibodies raised against a 20-amino acid synthetic fragment of rat P450_arom and by in situ hybridization to locate the aromatase mRNA in such cells. The activity of P450_arom 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 in [Ca²⁺]_i. The fact that the effects of E₂ and T were carried out within milliseconds suggests that they were exerted at the membrane level and not through intracellular receptors. The possibility of involvement of PLC-β in these effects is suggested because U-73122 (a PLC inhibitor) blocked the effects of both T and E₂. Immunocytochemical assays indicated the expression of androgenic and estrogenic receptors in these cells. The effects of T were blocked by the selective aromatase inhibitors. We also demonstrated membrane association of P450 _arom, expression of the ovary-specific mRNA after in situ hybridization, and E₂ formation resulting from a significant activity of P450_arom in CMECs. There were no gender-based differences.