Parathyroid hormone (PTH) produces catabolic and anabolic effects on bone, but the molecular bases of its skeletal actions are poorly understood. In addition to increasing cAMP levels, PTH also raises Cai2+ in target cells, an effect that is sometimes accompanied by phospholipase C (PL-C-mediated accumulation of inositol phosphates (IPs). In UMR 106-H5 cells, PTH and thrombin elicit comparable increases in cai2+ but only the latter augments IP accumulation. The long-term goal of this proposal is to define the molecular basis and significance of the activation by PTH of these dual signalling pathways. The specific objectives are to: 1) determine whether mechanisms other than the PI turnover/IP3 pathway contribute to PTH-induced mobilization of Cai2+. Microfluorometry of UMR 106-H5 cells loaded with the calcium indicator indo-1 will be used to monitor single-cell Cai2+. Cells will be microinjected with agents expected to disrupt the PL- C/IP3/Cai2+ signalling pathway including 1,4,5-IP3 (to deplete IP3- responsive calcium pools), heparin (to block IP3 receptors), and PIP2 antibodies (to prevent PL-C mediated hydrolysis). The effects of these agents on the single-cell Cai2+ signalling. Cyclic AMP signalling will be amplified by transfecting UMR 106-H5 cells with an expression plasmid encoding wild-type G-s-alpha, the stimulatory GTP-binding component of adenylate cyclase. In a second approach, cAMP signalling will be blocked by acute transfection with an expression plasmid encoding a yeast cAMP- phosphodiesterase (PDE). The influence of PTH on the Cai2+ signalling pathway in transfected cells will be evaluated; 3) assess whether PTH- induced increments in Cai2+ modulate the adenylate cyclase/cAMP/PK-A pathway. In these studies, PTH-activation of the cAMP pathway will be compared in control UMR 106-H5 cells and in cells in which the Cai2+ response to PTH is buffered out using an intracellular calcium chelator; 4) use a novel series of PTH(1-34) analogs to obtain pharmacological evidence for or against the notion that distinct PTH receptors are coupled to the cAMP and Cai2+ signalling pathways; and 5) assess PTH action in mutant UMR 106 cell lines defective in the cAMP signalling pathway. UMR 106-H5 cells will be stably transfected with the yeast cAMP-PDE plasmid, and cell lines defective in accumulating cAMP will be evaluated for PTH-dependent biological effects. Successful completion of these studies will provide important insights into the molecular mechanisms and physiological relevance of the activation by PTH of dual cell signalling pathways.
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