Sierra-Ramírez Alfredo, Morato Tomás, Campos Rafael, Rubio Iván, Calzada Claudia, Méndez Enrique, Ceballos Guillermo
Laboratorio Multidisciplinario, Sección de Posgrado, Escuela Superior de Medicina, Instituto Politécnico Nacional de México, Santo Tomas, Mexico, DF, CP 11340.
Am J Physiol Heart Circ Physiol. 2004 Jul;287(1):H63-71. doi: 10.1152/ajpheart.00784.2003. Epub 2004 Jan 15.
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+)) 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+). 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.
这项工作的目的是评估睾酮（T）和17β-雌二醇（E₂）对雄性和雌性大鼠冠状动脉微血管内皮细胞（CMECs）的影响。为了分析此类性类固醇激素对细胞内Ca²⁺浓度（[Ca²⁺]i）动力学的短期影响，我们使用了螯合剂fura-2乙酰氧甲酯。我们还通过使用芳香化酶细胞色素P-450芳香化酶（P450(arom)）的选择性抑制剂氨鲁米特（4 μM）和4-羟基雄烯二酮（4 μM）来探讨睾酮芳香化的可能性。使用针对大鼠P450(arom)的20个氨基酸合成片段产生的肽段多克隆抗体，通过免疫细胞化学和免疫印迹分析来研究P450(arom)的存在，并通过原位杂交来定位此类细胞中的芳香化酶mRNA。通过[1β-(³)H]雄烯二酮氚原子的立体特异性丢失来证明P450(arom)的活性。我们的结果表明，T和E₂均诱导[Ca²⁺]i迅速增加。E₂和T的作用在数毫秒内即可完成，这一事实表明它们是在膜水平发挥作用，而非通过细胞内受体。由于U-73122（一种PLC抑制剂）阻断了T和E₂的作用，提示PLC-β可能参与了这些作用。免疫细胞化学分析表明这些细胞中存在雄激素和雌激素受体。T的作用被选择性芳香化酶抑制剂阻断。我们还证明了P450(arom)与膜的结合、原位杂交后卵巢特异性mRNA的表达，以及CMECs中P450(arom)的显著活性导致的E₂形成。不存在基于性别的差异。
