C-peptide stimulates Na+,K+-ATPase activity via PKC alpha in rat medullary thick ascending limb

Aims/hypothesis. C-peptide, the cleavage product of proinsulin processing exerts physiological effects including stimulation of Na+,K+-ATPase in erythrocytes and renal proximal tubules. This study was undertaken to assess the physiological effects of connecting peptide on Na+,K+-ATPase activity in the medullary thick ascending limb of Henle's loop. . Na+,K+-ATPase activity was measured as the ouabain-sensitive generation of (32)Pi from gamma[P-32]-ATP and Rb-86 uptake on isolated rat medullary thick ascending limbs. The cell-surface expression of Na+,K+-ATPase was evaluated by Western blotting of biotinylated proteins, and its phosphorylation amount was measured by autoradiography. The membrane-associated fraction of protein kinase C isoforms was evaluated by Western blotting. Results. Rat connecting peptide concentration-dependently stimulated Na+,K+-ATPase activity with a threshold at 10(-9) mol/l and a maximal effect at 10(-7) mol/l. C-peptide (10(-7) mol/l) already stimulates Na+,K+-ATPase activity after 5 min with a plateau from 15 to 60 min. C-peptide (10(-7) mol/l) stimulated Na+,K+-ATPase activity and Rb-86 uptake to the same extent, but did not alter Na+,K+-ATPase cell surface expression. The stimulation of Na+,K+-ATPase activity was associated with an increase in Na+,K+-ATPase alpha-subunit phosphorylation and both effects were abolished by a specific protein kinase C inhibitor. Furthermore, connecting peptide induced selective membrane translocation of PKC-alpha. Conclusion/interpretation. This study provides evidence that in rat medullary thick ascending limb, C-peptide stimulates Na+,K+-ATPase activity within a physiological concentration range. This effect is due to an increase in Na+,K+-ATPase turnover rate that is most likely mediated by protein kinase C-alpha phosphorylation of the Na+,K+-ATPase alpha-subunit, suggesting that C-peptide could control Na+ reabsorption during non-fasting periods.