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

Abstract

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.

Role of aromatase in sex-specific cerebrovascular endothelial function in mice

“…Surprisingly, despite the observed sex difference in aromatase protein expression in cerebral vessels, we did not detect any sex differences in aromatase mRNA expression in microvascular endothelial cells. A lack of sex difference in aromatase expression has also been reported in rat coronary artery endothelial cells (50). Our data suggests that the sex difference in aromatase in cerebral vessels may be due to either increased expression in vascular smooth muscle cells from female mice or due to sex differences in post-transcriptional regulation of aromatase.…”

Regulation of adrenomedullin release from human endothelial cells by sex steroids and angiotensin-II

“…The latter possibility is supported by investigations of other vascular systems, including prostacyclin production in endothelial cells (Mikkola et al 1995) and AT1 receptor gene expression in smooth muscle cells (Nickenig et al 1998), which have revealed that oestradiol may have a maximum effect at 12 h or greater. Endothelial cells can convert testosterone to oestradiol (Mukherjee et al 2002) and it has been suggested that effects attributed to testosterone may be in part a result of its conversion to oestradiol (Sierra-Ramirez et al 2004). However, our observation that testosterone increased the number of adrenomedullin-secreting endothelial cells, and that oestradiol had no significant effect, indicates that the actions of testosterone exhibited here were unlikely to be dependent on its conversion to oestradiol.…”

“…For example, both vasodilatory (Webb et al 1999) and anti-vasodilatory effects have been observed (Hutchison et al 1997, Ceballos et al 1999 and testosterone may be involved in the development of some forms of experimental hypertension (Song et al 2004, Reckelhoff 2005. Endothelial cells have receptors for both oestradiol (Losordo et al 1994) and testosterone (Sierra-Ramirez et al 2004), by which these steroids can directly influence blood vessels and hence the development of vascular disease and hypertension.…”

Effect of estradiol and dihydrotestosterone on hypergravity-induced MAPK signaling and occludin expression in human umbilical vein endothelial cells

“…This may be because of the availability of more membrane AR than ER in HUVECs. Although the existence of membrane ER and AR has been reported in various cell types, including ECs, in several studies (Armen and Gay 2000;Rubio-Gayosso et al 2002;Sierra-Ramirez et al 2004;Kampa et al 2005), none of them have analyzed ER and AR. However, in agreement in principle with our results, AR is expressed at higher levels than ER and the progesterone receptor in the ECs of the oral mucosa (Ojanotko-Harri et al 1992).…”