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


METHODS
Na(+),K(+)-ATPase activity was measured as the ouabain-sensitive generation of (32)Pi from gamma[(32)P]-ATP and (86)Rb 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 (86)Rb 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.


CONCLUSIONS
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.
Tsimaratos
M
M
Paediatric Nephrology Unit, Children's Hospital la Timone, Marseille, France. michel.tsimaratos@ap-hm.fr
Roger
F
F
Chabardès
D
D
Mordasini
D
D
Hasler
U
U
Doucet
A
A
Martin
P-Y
PY
Féraille
E
E
D000818:Animals; D002096:C-Peptide / Q000008:administration & dosage / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D007679:Kidney Medulla; D008138:Loop of Henle / Q000378:metabolism*; D008297:Male; D009994:Osmolar Concentration; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051381:Rats; D017208:Rats, Wistar; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013997:Time Factors
D002096:C-Peptide; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D000254:Sodium-Potassium-Exchanging ATPase