Parathyroid Hormone Receptors Coupled to Cyclic Adenosine Monophosphate Formation in an Established Renal Cell Line*

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Abstract
The relationship between PTH [parathyroid hormone] binding and stimulation of cAMP formation in a cell line derived from opossum kidney (OK) was examined. In the presence of isobutylmethylxanthine (1 mM) bovine PTH(1-34) [bPTH(1-34)] (244 nM) stimulated cAMP accumulation in confluent cultures up to 40-fold over basal; this response to PTH was stable for 35 passages. The concentration of bPTH(1-34) required to raise cell cAMP levels half-maximally was 5-12 nM. Binding of [125I]bPTH(1-34) to OK cells was saturable; Scatchard analysis of competitive binding data yielded a Kd = 6 .+-. 2 nM, with 1.0 pmol binding sites/mg cell protein. Under steady-state binding conditions 89% of labeled PTH remained precipitable by 10% trichloroacetic acid, suggesting minimal metabolism of the hormone. The PTH antagonist (8Nle,18Nle,34Tyr)bPTH(3-34)amide competed for [125I]bPTH(1-34) binding sites and inhibited the action of bPTH(1-34) to raise cAMP levels. The intact PTH molecule, bPTH(1-84), and the weak agonist hPTH(1-34) were both less potent than bPTH(1-34) (6 and 30 times, respectively), with regard to binding and cAMP production. Calcitonin and arginine vasopressin did not bind to PTH receptors but raised cAMP levels in OK cell cultures 3- and 10-fold, respectively; neither glucagon nor ACTH(1-24) influenced PTH binding of cAMP in OK cells. Varying the extracellular Ca concentration in the medium bathing cells did not influence basal or PTH-stimulated cAMP generation. PTH receptors in OK cells are of high affinity, are selective for PTH, and are coupled to adenylate cyclase. This established epithelial cell line provides a model in which to study the mechanism of action of PTH in the kidney.

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