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The objective of this work was to evaluate the effects of testosterone (T) and 17 -estradiol (E 2 ) on coronary microvascular endothelial cells (CMECs) of male and female rats. To analyze the short-term effects of such sex steroid hormones on intracellular Ca 2+ 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 ( P 450 arom ), aminoglutethimide (4 µM), and 4-hydroxyandrostenedione (4 µM). The presence of P 450 arom was investigated by immunocytochemical and immunoblot assays using peptide-generated polyclonal antibodies raised against a 20-amino acid synthetic fragment of rat P 450 arom and by in situ hybridization to locate the aromatase mRNA in such cells. The activity of P 450 arom was demonstrated by the stereospecific loss of the tritium atom of 1 - 3 Handrostenedione. Our results indicate that both T and E 2 induced a rapid increase in Ca 2+ i . The fact that the effects of E 2 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- in these effects is suggested because U-73122 (a PLC inhibitor) blocked the effects of both T and E 2 . 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 P 450 arom , expression of the ovary-specific mRNA after in situ hybridization, and E 2 formation resulting from a significant activity of P 450 arom in CMECs. There were no gender-based differences. phospholipase C- ; cytochrome P -450 aromatase; stereospecific Address for reprint requests and other correspondence: G. Ceballos, Laboratorio Multidisciplinario, Escuela Superior de Medicina, Plan de San Luis y Diaz Mirón S/N, col. Santo Tomas, México, DF, CP 11340 (E-mail: manrey44@hotmail.com ).
AJP - Heart and Circulatory Physiology – The American Physiological Society
Published: Jul 1, 2004
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