Parathyroid hormone (PTH) regulates renal phosphate reabsorption utilizing adenylyl cyclase- and phospholipase C-generated second messengers. Understanding how signal transduction pathways interact is particularly important in disease states where renal PTH resistance is manifested by changes in receptor coupling to adenylyl cyclase and/or phospholipase C. In pseudohypoparathyroidism type I (PHP-I), PTH resistance is characterized by impaired cAMP production, hyperphosphatemia and a genetically acquired reduction in Gsalpha. PHP-II is distinguished by normal cAMP responses but a reduced phosphaturic response to PTH. PTH resistance in PHP-II may reflect changes in protein kinase A (PKA) or in the phospholipase C/protein kinase C (PKC) pathway. It is postulated that PTH regulation of renal Na+- dependent phosphate reabsorption requires activation of both adenylyl cyclase and phospholipase C. This postulate will be tested using 2 clonal populations of the renal OK cells isolated in this laboratory. OK/E cells retain PTH stimulated adenylyl cyclase, and phosphate transport is highly response to PTH inhibition. OK/H cells respond to PTH with increases in intracellular cAMP, but PKA is partially resistant to cAMP activation, there is no PTH-induced change intracellular Ca2+, and PTH does not inhibit phosphate transport. Site(s) for PTH resistance in OK/H cells will be assessed by comparing PKA and PKC activation in both OK clones. PKA isozymes will be identified by DEAE-Sephacel column chromatography and PKA type-directed site-selective cAMP analogs. To determine if the receptor-G- adenylyl cyclase is affected in OK/H cells, PTH-, GTPgammaS- and forskolin stimulation of adenylyl cyclase will be measured in both clones. Agonist- induced activation of the phospholipase C will be measured by [3H]IP production. Phospholipase C will be activated in the presence of PTH- stimulated cAMP production to determine if phosphate transport regulation can be restored to OK/H cells. Bacterial toxin-mediated ADP-ribosylation, Western analysis and [alpha32P]GTP-binding will be used to identify G proteins in both clones since loss of a G protein may contribute to PTH resistance in OK/H cells. To assess how PKC influences the PTH receptor- Gs-adenylyl cyclase complex and phosphate transport, PTH stimulation of adenylyl cyclase and competitive inhibition binding of [125I]hPTH-related peptide binding will be evaluated in OK/E cells treated with various PKC activators. These studied should provide new information concerning the importance of each signal transduction pathway in PTH regulation of renal Na+-dependent phosphate transport.
