Identification of estrogen receptor mRNA and the estrogen modulation of parathyroid hormone-stimulated cyclic AMP accumulation in opossum kidney cells

Abstract

The opossum kidney (OK) cell was used as a model to test the hypothesis that estrogen directly affects proximal renal tubular epithelial cells. To demonstrate the expression of estrogen receptor in OK cells, we developed an approach using reverse transcription and the polymerase chain reaction. Analysis of the DNA amplified with nested primers revealed the predicted size fragment and restriction enzyme digestion products. To demonstrate the functional effects of estrogen, OK cells at confluence were preincubated in serum-free medium for 7-10 days with or without 17 beta-estradiol. Bovine PTH(1-34) (bPTH(1-34)) then stimulated a dose-dependent intracellular accumulation of cAMP that was maximal after 1 min and then gradually declined. Cyclic AMP in the medium slowly increased over 60 min. Preincubation with 17 beta-estradiol did not affect cell proliferation as measured by total protein content but caused an inhibition of bPTH(1-34)-stimulated intracellular cAMP accumulation that was maximal at 10(-11) M 17 beta-estradiol (71 +/- 3% control, p less than .001). bPTH(1-34) also increased cAMP release into the medium, an effect maximal using 10(-10) M 17 beta-estradiol (118 +/- 3% control, p less than .001). Preincubation with the inactive isomer 17 alpha-estradiol caused no changes in cAMP accumulation or release. Coincubation with the antiestrogen tamoxifen blocked the effects of 17 beta-estradiol. Sodium-dependent phosphate transport was: (1) inhibited by 2-h incubations with 10(-8) or 10(-10) M bPTH(1-34) and not affected by preincubation with 17 beta-estradiol, and (2) not inhibited by a 20-min incubation with 10(-8) M bPTH(1-34) unless cells were preincubated with 10(-8) M 17 beta-estradiol, suggesting that any possible effects of estrogen on phosphate transport are not directly mediated by changes in cAMP. These studies demonstrate the presence of estrogen receptor mRNA in OK cells as well as direct and specific effects of physiologic concentrations of estrogen on cAMP accumulation in these cells. This system may be a good model for further study of estrogen and PTH effects on the kidney.