Rapid Nongenomic Effects of 3,5,3'-Triiodo L-Thyronine on the Intracellular pH of L-6 Myoblasts are Mediated by Intracellular Calcium Mobilization and Kinase Pathways

Low physiological concentrations of 17beta-estradiol increased the intracellular pH of rat aortic smooth muscle cells by a rapid nongenomic mechanism. This effect was due to stimulation of the Na+/H+ exchanger activity, measured using the intracellular pH-sensitive fluorescent probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. The 17beta-estradiol gave rise to a bell-shaped dose response, with a maximum at 10(-12)M and no significant effect at 10(-9)M. The specificity of the effect was verified by the use of the Na+/H+ exchanger inhibitor 5-(ethyl-N-isopropyl) amiloride and the lack of effect of the isomer 17alpha-estradiol. Inhibitors of the nuclear estrogen receptors, tamoxifen and ICI 182,780, completely prevented activation of the exchanger by 17beta-estradiol. The effect of low estrogen concentrations on the intracellular pH was mimicked by both norepinephrine and phenylephrine, suggesting a connection between the increase of intracellular pH and the muscle contraction process. The transduction mechanism for this nongenomic effect of estrogens did not involve modulation of the cAMP content, whereas inositol 1,4,5-trisphosphate, protein kinase C and MAPK pathways appear to play a role, as indicated by both pharmacological approaches and immunoblot experiments on protein kinase C translocation and ERK phosphorylation. These results for the first time provide evidence for a nongenomic effect of low physiological concentrations of 17beta-estradiol on intracellular pH that, together with other factors, may contribute to the development of hypertension and atherosclerosis in men and postmenopausal women and increase the risk of cardiovascular disease. Paradoxically, the lack of stimulation at high physiological estradiol levels could explain the protective effects found in premenopausal women.