Non-lysosomal cycling pathway for atrial natriuretic peptide activated by protein kinase C in human NPE cells

bound receptors which mediate this effect have not been well studied in the eye. Endocytosis of [I-125]ANP bound to natriuretic peptide C receptors wa s characterized in fetal human nonpigmented ciliary epithelial (NPE) cells, [I-125]ANP which bound to cells at 4 degrees C was detected in the cell in terior after a temperature shift to 37 degrees C. Appearance of ligand with in the cell peaked at 5 min, and then declined towards zero over 20 min. Th e endocytosis inhibitor phenylarsine oxide blocked the appearance of intern alized ligand, whereas the lysosomotropic drug chloroquine had no effect on internalization but blocked subsequent loss of internalized ligand. Chloro quine also blocked the accumulation of degraded ligand in the extracellular medium. Treatment with phorbol 12-myristate, 13-acetate accelerated the loss of int ernalized ligand from cells and increased the accumulation of ligand in the extracellular medium. Ligand in the medium was also increased by dioctanoy lglycerol but not by 4 alpha, phorbol didecanoate, an isomer which does not activate protein kinase C. The protein kinase inhibitors staurosporine and bisindolylymaleimide blocked the increase in ligand. Phorbol eater-stimulated loss of internalized ligand occurred in the presen ce of chloroquine. TCA precipitation of ligand in the extracellular medium showed that both degraded and undegraded [I-125]ANP were present. However, in the presence of chloroquine only, undegraded ANP was detected in the med ium, and phorbol esters stimulated its rate of appearance by similar to 2 f old. A similar stimulation occurred when cells containing internalized liga nd, but stripped of membrane-bound ligand, were exposed to phorbol esters. The data suggest that ANP bound to natriuretic peptide C receptors on NPE c ells is endocytosed, and that protein kinase C activates a non-lysosomal pa thway for ANP retroendocytosis in these cells. (C) 1998 Academic Press.