Parathyroid hormone and parathyroid hormone-related peptide inhibit the apical Na+/H+ exchanger NHE-3 isoform in renal cells (OK) via a dual signaling cascade involving protein kinase A and C.
Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP) interact with a common G protein-coupled receptor and stimulate production of diverse second messengers (i.e. cAMP, diacylglycerol, and inositol 1,4,5-trisphosphate) that varies depending on the target cell. In renal proximal tubule OK cells, PTH inhibits the activity of the apical membrane Na+/H+ exchanger, although it is unclear whether the signal is transmitted through protein kinase A (PKA) and/or protein kinase C (PKC). To delineate the signaling circuitry, a series of synthetic PTH and PTHRP fragments were used that stimulate the adenylate cyclase-cAMP-PKA and/or phospholipase C-diacylglycerol-PKC pathways. Human PTH-(1-34) and PTHRP-(1-34) stimulated adenylate cyclase and PKC activity, whereas the PTH analogues, PTH-(3-34), PTH-(28-42), and PTH-(28-48), selectively enhanced only PKC activity. However, each peptide fragment inhibited Na+/H+ exchanger activity by 40-50%, suggesting that PKC and possibly PKA were capable of transducing the PTH/PTHRP signal to the transporter. This was corroborated when forskolin and phorbol 12-myristate 13-acetate (PMA), direct agonists of adenylate cyclase and PKC, respectively, both inhibited the Na+/H+ exchanger. The specific PKA antagonist, H-89, abolished the forskolin-mediated suppression of Na+/H+ exchanger activity, but did not prevent the inhibitory effects of PTH-(1-34) or PMA. In comparison, the potent PKC inhibitor, chelerythrine chloride, prevented the inhibition of Na+/H+ exchanger activity mediated by PTH-(28-48) and PMA but did not avert the negative regulation caused by PTH-(1-34) or forskolin. However, inhibition of both PKA and PKC prevented PTH-(1-34)-mediated suppression of Na+/H+ exchanger activity, indicating that PTH-(1-34) acted through both signaling pathways. In addition, Northern blot analysis revealed the presence of only the NHE-3 isoform of the Na+/H+ exchanger in OK cells. In summary, these results demonstrated that NHE-3 is expressed in OK cells and that activation of the PTH receptor can stimulate both the PKA and PKC pathways, each of which can independently lead to inhibition of NHE-3 activity.
Azarani
A
A
Department of Physiology, McGill University, Montreal, Quebec, Canada.
Goltzman
D
D
Orlowski
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010281:Parathyroid Hormone / Q000494:pharmacology*; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000494:pharmacology*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors*; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D009842:Oligopeptides; C470121:PTHLH protein, human; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D011506:Proteins; C000613385:SLC9A3 protein, human; C000613387:Slc9a3 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D005576:Colforsin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate