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 17beta-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 (P450(arom)), aminoglutethimide (4 microM), and 4-hydroxyandrostenedione (4 microM). 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 [1beta-(3)H]androstenedione. 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-beta 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 P450(arom), expression of the ovary-specific mRNA after in situ hybridization, and E(2) formation resulting from a significant activity of P450(arom) in CMECs. There were no gender-based differences.
Sierra-Ramírez
Alfredo
A
Laboratorio Multidisciplinario, Sección de Posgrado, Escuela Superior de Medicina, Instituto Politécnico Nacional de México, Santo Tomas, Mexico, DF, CP 11340.
Morato
Tomás
T
Campos
Rafael
R
Rubio
Iván
I
Calzada
Claudia
C
Méndez
Enrique
E
Ceballos
Guillermo
G
D000818:Animals; D001141:Aromatase / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D003331:Coronary Vessels / Q000166:cytology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004958:Estradiol / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D004967:Estrogens / Q000096:biosynthesis*; D005260:Female; D015151:Immunoblotting; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008833:Microcirculation; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D011944:Receptors, Androgen / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism; D013739:Testosterone / Q000494:pharmacology*; D013997:Time Factors; D014316:Tritium; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D014867:Water / Q000378:metabolism
D004963:Estrenes; D047628:Estrogen Receptor alpha; D004967:Estrogens; D011760:Pyrrolidinones; D011944:Receptors, Androgen; D011960:Receptors, Estrogen; D014867:Water; D014316:Tritium; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D013739:Testosterone; D004958:Estradiol; D001141:Aromatase; D010738:Type C Phospholipases; D002118:Calcium