guanosine-triphosphate and sulprostone

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.

Topics: Animals; Binding, Competitive; Cell Membrane; Cells, Cultured; Dinoprostone; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Kidney Cortex; Kidney Tubules; Kidney Tubules, Collecting; Kinetics; Pertussis Toxin; Prostaglandins; Rabbits; Receptors, Prostaglandin; Thionucleotides; Virulence Factors, Bordetella

We have examined the regulation by prostaglandin E2 (PGE2) of hormone-induced adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in cells isolated by immunodissection from both the medullary and cortical thick ascending limb of Henle's loop of rabbit kidney. At concentrations greater than 10(-8) M, PGE2, but not sulprostone (16-phenoxy-17,18,19,20-tetranor-PGE2 methylsulfonilamide), caused cAMP accumulation in both cortical and medullary thick limb cells. However, at concentrations of less than or equal to 10(-8) M, both PGE2 and sulprostone inhibited arginine vasopressin (AVP)-, calcitonin-, and glucagon-induced cAMP accumulation in medullary thick ascending limb (mTAL) cells. In cortical thick limb (cTAL) cells, sulprostone also inhibited AVP-, calcitonin-, and parathyroid hormone (PTH)-induced cAMP accumulation. The inhibitory effects of PGE2 and of sulprostone were blocked by pretreatment of mTAL and cTAL cells with pertussis toxin. Membranes prepared from mTAL cells exhibited a [3H]PGE2 binding activity that was stimulated on addition of the stable guanosine 5'-triphosphate (GTP) analogue, 5'-guanosine gamma-thiotriphosphate (GTP gamma S); moreover, sulprostone inhibited [3H]PGE2 binding. Our results suggest that PGE2 can function via a prostaglandin E receptor linked to a guanine nucleotide regulatory protein, Gi, to attenuate hormone-induced cAMP formation in both mTAL and cTAL cells of rabbit kidney.

Topics: Animals; Arginine Vasopressin; Cyclic AMP; Dinoprostone; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; In Vitro Techniques; Kidney Cortex; Kidney Medulla; Kidney Tubules; Kinetics; Loop of Henle; Parathyroid Hormone; Peptide Fragments; Pertussis Toxin; Rabbits; Receptors, Prostaglandin; Teriparatide; Thionucleotides; Virulence Factors, Bordetella