Abstract
At different concentrations, prostaglandin E2 (PGE2) can either stimulate or inhibit cAMP formation in freshly isolated rabbit cortical collecting tubule (RCCT) cells, but in cultured RCCT cells PGE2 can only stimulate cAMP synthesis (Sonnenburg, W. K., and Smith W. L. (1989) J. Biol. Chem. 263, 6155-6160). Here, we report characteristics of [3H]PGE2 binding to membrane receptor preparations from both freshly isolated and cultured RCCT cells. [3H]PGE2 binding to membranes from freshly isolated RCCT cells was saturable and partially reversible. Equilibrium binding analyses indicated that in the absence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) there is a single class of PGE2 binding sites (KD = 4.2 +/- 0.4 nM; Bmax = 583 +/- 28 fmol/mg); in the presence of 100 microM GTP gamma S, there is also only one class of binding sites but with a somewhat lower KD = 1.2 +/- 0.5 nM (Bmax = 370 +/- 40 fmol/mg). This stimulatory effect of GTP gamma S was blocked by pretreatment of the freshly isolated RCCT cells with pertussis toxin. The relative affinities of prostanoids for the [3H]PGE2-binding site were determined to be 17,18,19,20-tetranor-16-phenoxy-PGE2-methylsulfonylamide (sulprostone) approximately PGE2 approximately PGE1 approximately 16,16-dimethyl-PGE2 greater than carbacyclin approximately PGF2 alpha greater than PGD2. This is the order of potency with which prostaglandins inhibit arginine vasopressin-induced cAMP formation in fresh RCCT cells. Interestingly, [3H]PGE2 binding to membranes from cultured cells, which, unlike fresh cells, fail to show an inhibitory response to PGE2, was only 10-20% of that observed with membranes from fresh cells; moreover, binding of [3H]PGE2 to membranes from cultured cells was neither stimulated by GTP gamma S nor inhibited by sulprostone. The prostanoid binding specificities and the unusual pertussis toxin-sensitive, stimulatory effect of GTP gamma S on binding of [3H]PGE2 to membranes from freshly isolated RCCT cells are characteristics shared by a Gi-linked PGE receptor from renal medulla (Watanabe, T., Umegaki, K., and Smith, W. L. (1986) J. Biol. Chem. 261, 14340-14349). Our results suggest that the [3H]PGE2 binding site of freshly isolated RCCT cells is the PGE receptor which is coupled to a Gi to attenuate arginine vasopressin-induced cAMP synthesis in the renal collecting tubule.
Highlights
Can either stimulate or inhibit CAMP formation in freshly isolated rabbit cortical collecting tubule (RCCT) cells, but in cultured RCCT cells PGE2 can only stimulate CAMP synthesis
PH]PGE? Binding to Membranes Prepared from Freshly Isolated Rabbit Cortical Collecting Tubule Cells-Specific binding of 2 nM [“H]PGE2 to membranes prepared from freshly isolated RCCT cells was saturable and reached equilibrium in about 1 h at 30 “C (Fig. l).Nonspecific binding averaged 15 + 5% of total binding
Kn values determined for PGE2 binding to this site in the presence of GTPrS were in the range of l-5 nM, consistent with a concentration of 3 nM PGE, being able to cause half-maximal inhibition of AVPinduced CAMP formation in freshly isolated RCCT cells [1]
Summary
Materials-Reagents were obtained from the following sources:. New England Nuclear; collagenase, RPM1 1640, and fetal bovine serum from Grand Island Biological Co.; PGE,, PGE,, PGF,,,, and. MM EDTA and 40 fiM indomethacin and the membranes again collected by centrifugation at 35,000 x g for 30 min This washing procedure was again repeated and the final membrane pellet resuspended in 10 mM HEPES, pH 7.5, containing 1 mM MgCl* and 40. JLM indomethacin at a concentration of 2-4 mg of protein/ml This membrane preparation contained on the average 70% of the specific [“HIPGE, binding activity present in whole homogenates. RCCT cell or canine or rabbit outer medullary membranes (So-180 pg of protein/assay) were incubated at 30 “C in a reaction mixture containing the following components: 50 mM Tris maleate, final pH 5.5, 1 mM MgC12, and 2 nM [“H]PGE, in a final volume of 200 ~1. In duplisignificance difference test or Student-Newman-Keul’s applicable @ < 0.05) [7]
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