Gender-Specific Reduction in Contraction is Associated with Increased Estrogen Receptor Expression in Single Vascular Smooth Muscle Cells of Female Rat

Gender differences in the incidence of cardiovascular disease have been related to plasma estrogen levels; however, the role of vascular estrogen receptor (ER) subtypes in these sex differences is less clear. We tested whether the gender differences in vascular smooth muscle (VSM) function reflect differential expression/activity of ER alpha, ER beta and the newly-identified GPR30. Single aortic VSM cells (VSMCs) were freshly isolated from male and female Sprague-Dawley rats, and their contraction to phenylephrine (PHE, 10(-5) M), AngII (10(-7) M) and membrane-depolarization by KCl (51 mM) was measured in the absence or presence of 10(-6) M 17 beta-estradiol (E2, stimulant of most ERs), PPT (ER alpha agonist), DPN (ER beta agonist), and ICI 182,780 (an ER alpha/ER beta antagonist with GPR30 agonistic properties). The cells were fixed and fluorescently labeled with ER alpha, ER beta or GPR30 antibody, and the subcellular distribution of ERs was examined using digital imaging microscopy. The mRNA expression and protein amount of aortic ER subtypes was examined using RT-PCR and Western blots. PHE, AngII, and KCl caused less contraction in VSMCs of females than males. Pretreatment of VSMCs with E2 reduced PHE-, AngII- and KCl-induced contraction in both males and females. PPT caused similar inhibition of PHE-, AngII- and KCl-induced contraction as E2, suggesting a role of ER alpha. DPN mainly inhibited PHE and KCl contraction, suggesting an interaction between ER beta and Ca2+ channels. ICI 182,780 did not reduce aortic VSMC contraction, suggesting little role for GPR30. RT-PCR and Western blots revealed greater expression of ER alpha and ER beta in VSMCs of females than males, but similar amounts of GPR30. The total immunofluorescence signal for ER alpha and ER beta was greater in VSMCs of females than males, and was largely localized in the nucleus. GPR30 fluorescence was similar in VSMCs of males and females, and was mainly in the cytosol. In PPT treated cells, nuclear ER alpha signal was enhanced. DPN did not affect the distribution of ER beta, and ICI 182,780 did not significantly increase GPR30 in the cell surface. Thus, ER subtypes demonstrate similar responsiveness to specific agonists in VSMCs of male and female rats. The reduced contraction in VSMCs of females could be due to gender-related increase in the expression of ER alpha and ER beta. Copyright (C) 2010 S. Karger AG, Basel