LOCALIZATION, SYNTHETIC REGULATION, AND BIOLOGY OF RENAL ATRIOPEPTIN-LIKE PROHORMONE

We recently demonstrated the synthesis and secretion of an atriopeptin (AP)-like prohormone in rat neonatal and adult cortical kidney cell cultures. However, these cultures contained proximal as well as distal tubular epithelial cells; thus characterization of the peptide synthetic cell was not possible. Also, by immunohistochemical techniques, we localized this AP-like prohormone to the distal cortical nephron in adult rat kidney. In this study, we examined further details of the kidney cortical cell type that expresses and secretes this AP-like peptide in adult renal cortical cell cultures, its regulation by adenylate cyclase via adenosine 3',5'-cyclic monophosphate (cAMP) generation, and its ability to stimulate guanylate cyclase. Tubular fragments were derived from cortical tissue of adult Sprague-Dawley rats and separated into four fractions on Percoll density gradient. Cell cultures generated from fraction 3 secreted 5- to 10-fold the amount of this renal peptide compared with fractions 2 and 4. Further cell culture characterization was performed by agonist-stimulated cAMP formation, kallikrein localization, and prostaglandin E2 formation. From these analyses, it was determined that tissue band 3 was enriched for distal cortical connecting tubules. To further evaluate whether mammalian distal nephron synthesizes an AP-like protein, we determined that two immortalized mouse cell lines, derived from either the distal convoluted tubule or cortical collecting tubule, synthesized a radiolabeled AP after being pulsed with [S-35]methionine. To determine whether adenylate or guanylate cyclase activation is involved in renal AP secretion, cell cultures derived from tissue band 3 were treated with 3-isobutyl-1-methylxanthine (IBMX), dibutyryl (DB)-cGMP, DBcAMP, or forskolin. IBMX, DBcAMP, and forskolin inhibited renal AP secretion by at least 50%; DBcGMP had no effect. Finally, the addition of renal AP (10(-8) M) to primary rat papillary collecting duct cultures stimulated guanylate cyclase activity approximately 10-fold. We conclude that renal AP is synthesized and secreted from rat and mouse renal distal cortical tubules. Like cardiac AP, it stimulates renal cGMP production, and its secretion is negatively modulated by activation of adenylate cyclase.