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.

Evidence for Involvement of 17β-Estradiol in Intestinal Calcium Absorption Independent of 1,25-Dihydroxyvitamin D3 Level in the Rat

“…(54) In laying hens, 17␤-E 2 reduces PTH receptor binding affinity in calvaria and kidney. (55) However, estrogen substitution in postmenopausal women may result in either unchanged (19,49,54) or increased renal 1␣-hydroxylase activity, either due to increased serum PTH levels (30) or an increased renal responsiveness to PTH. (56) Furthermore, Ash et al have shown in a rat model that estrogens promote PTH-induced synthesis of calcitriol.…”

“…(8)(9)(10)(11) These effects can be direct and/ or indirect via regulation of and/or interaction with other factors influencing bone metabolism, e.g., parathyroid hormone (PTH) and insulin-like growth factor I. (12)(13)(14)(15)(16)(17) Menopause is associated with decreased calcium absorption, (18) which can be normalized by 17␤-E 2 supplementation. (19,20) A positive regulation of intestinal calcium absorption by estradiol has been suggested by Arjmandi et al (21) Therefore, modulation of intestinal calcium absorption could be a target for estradiol and selective estrogen receptor modulators in postmenopausal osteoporosis.…”

Effect of estrogen on calcium and sodium transport by the nephron luminal membranes

“…In the kidney, however, the action of estrogen on adenylate cyclase is less well defined; Imanaka et al (1998) reported that estrogen administration to elderly women enhances cAMP excretion induced by PTH loading. In contrast, 17 -estradiol has been shown to inhibit the PTH-dependent cAMP release in opossum kidney cells (Stock et al 1992). In the renal distal tubule, we previously showed that either cAMP alone, or the combined cAMP and protein kinase C activation, increases rather than decreases Ca 2+ transport by the luminal membrane (Hilal et al 1997).…”

“…Moreover, in vivo regulation of calciuria is complex. In addition to its effect on bone, estrogen has been shown to interfere with the PTH-dependent cAMP synthesis in opossum kidney cells (Stock et al 1992) and to stimulate both the vitamin D active metabolite 1,25(OH 2 )D 3 (Tanaka et al 1978, Cheema et al 1989, Castillo et al 1977 and calbindin 28K (Gill & Christakos 1995) expression. Not only does 1,25(OH 2 )D 3 increase Ca 2+ intestinal absorption but it also directly regulates PTH secretion by the gland (Chertow et al 1980, Russel et al 1986, Silver et al 1986, which is a site of receptors for this hormone (Brumbaugh et al 1975).…”

Influence of Estrogen and Xenoestrogens on Basolateral Uptake of Tetraethylammonium by Opossum Kidney Cells in Culture

“…The localization of estrogen receptors in OK cells (Stock et al, 1992), along with the expression of OC transport function (McKinney et al, 1990;Yuan et al, 1991;Endo et al, 2000), makes this renal culture an appropriate model system for investigating estrogen regulation of OC transport. The data reported here demonstrate that the endogenous estrogen 17␤-E 2 and the synthetic pharmaceutical estrogen DES reduce TEA transport across the basolateral membrane of OK cells.…”