title: Mechanisms of Na+-K+-ATPase phosphorylation by PKC in the medullary thick ascending limb of Henle in the rat.
abstract: Sodium transport correlates with varying Na+-K+-ATPase activity rates along the nephron. Whether differences in Na+-K+-ATPase regulation by protein kinase C-dependent phosphorylation are also present has not been tested. We measured the degree of Na+-K+-ATPase alpha1 subunit phosphorylation by the binding of McK-1 antibody to dephosphorylated Ser-23 and Na+-K+-ATPase activity in medullary thick ascending limb of Henle (mTAL) and proximal tubules (PCT). The degree of Na+-K+-ATPase phosphorylation at Ser-23 was lower in mTAL than in PCT (DU 13.43+/-1.99 versus 2.3+/-0.20, respectively, P<0.01) while Na+-K+-ATPase activity was higher in mTAL (3,402+/-83 vs 711+/-158 pmol/mm tubule per hour in PCT, P<0.01). PKC inhibitor RO-318220 10(-6) M decreased phosphorylation in PCT to 125+/-10% ( P<0.05). In mTAL, RO-318220 did not modify the phosphorylation degree or the activity of Na+-K+-ATPase. Both calcineurin inhibitor FK-506 10(-6) M and phorbol 12-myristate 13-acetate (PMA) 10(-6) M increased the degree of Na+-K+-ATPase phosphorylation ( P<0.05) and inhibited Na+-K+-ATPase activity to 657+/-152 and 1,448+/-347 pmol/mm tubule per hour, respectively, in mTAL ( P<0.01). Increase in [Na+]i to 30, 50 and 70 mM resulted in no changes in Na+-K+-ATPase phosphorylation degree or activity in mTAL. Conversely, in PCT increments in [Na+]i were paralleled by decreased phosphorylation (from 120+/-7 to 160+/-15% of controls, P<0.05) and increased Na+-K+-ATPase activity (from 850+/-139 to 1,874+/-203 pmol/mm tubule per hour, P<0.01). Dopamine (DA) 10(-6) M decreased both Na+-K+-ATPase dephosphorylation to 41.85+/-9.58% ( P<0.05) and Na+-K+-ATPase activity to 2,405+/-176 pmol/mm tubule per hour in mTAL ( P<0.01). RO-318220 reversed DA effects. Data suggest that regulation of the degree of Na+-K+-ATPase alpha1 subunit phosphorylation at Ser-23 and enzyme activity have different mechanisms in mTAL than in PCT, and may help us to understand the physiological heterogeneity of both segments.
authors.affiliation: Instituto de Investigaciones Médicas Alfredo Lanari, Universidad de Buenos Aires, 1427 Buenos Aires, Argentina.
authors.forename: Claudia A
authors.initials: CA
authors.lastname: Bertuccio
authors.forename: Sam X
authors.initials: SX
authors.lastname: Cheng
authors.forename: Elvira E
authors.initials: EE
authors.lastname: Arrizurieta
authors.forename: Rodolfo S
authors.initials: RS
authors.lastname: Martín
authors.forename: Fernando R
authors.initials: FR
authors.lastname: Ibarra
mesh_terms: D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007679:Kidney Medulla / Q000187:drug effects / Q000201:enzymology*; D008138:Loop of Henle / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase