title: Protein kinase C and insulin regulation of red blood cell Na+/H+ exchange.
abstract: Insulin activation of red blood cell (RBC) Na+/H+ (NHE) and Na+/Li+ (NLiE) exchanges is mimicked by okadaic acid, thus suggesting that it may change the state of phosphorylation of serine/threonine NHE residues. To investigate the role of the serine/threonine protein kinase C (PKC) in insulin regulation, we evaluated the effect of phorbol 12-myristate 13-acetate (PMA; 1 microM) and insulin on PKC activity, membrane protein phosphorylation, and the activation kinetics of both exchangers. Our studies revealed that PMA decreased cytosolic PKC activity (4.1 +/- 0.6 to 2.3 +/- 0.5 pmol x mg protein(-1) x min(-1), n = 9, P < 0.001), increased membrane PKC activity (42.3 +/- 5 to 132 +/- 12 pmol x mg protein(-1) x min(-1), n = 11, P < 0.001), and enhanced serine phosphorylation of bands 3, 4.1, and 4.9 membrane proteins. PMA markedly reduced the Michaelis constant (Km) for intracellular H+ (415 +/- 48 to 227 +/- 38 nM, n = 11, P < 0.01) but had no effect on the maximal transport rate (Vmax) of NHE and the Km for Na+ of NLiE. NHE activation and PKC activity were affected differently by insulin (100 microU/ml) and PMA. Insulin increased the Vmax of NHE and the Km for Na+ of NLiE but had no effect on the Km for intracellular H+ and membrane PKC activity. These findings lead us to conclude that in the human RBC, NHE is modulated by PKC and insulin through different biochemical mechanisms.
authors.affiliation: Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: G
authors.initials: G
authors.lastname: Ceolotto
authors.forename: P
authors.initials: P
authors.lastname: Conlin
authors.forename: G
authors.initials: G
authors.lastname: Clari
authors.forename: A
authors.initials: A
authors.lastname: Semplicini
authors.forename: M
authors.initials: M
authors.lastname: Canessa
mesh_terms: D000328:Adult; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000097:blood*; D008094:Lithium / Q000097:blood; D008565:Membrane Proteins / Q000097:blood; D008875:Middle Aged; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011493:Protein Kinase C / Q000097:blood*; D012964:Sodium / Q000097:blood; D017923:Sodium-Hydrogen Exchangers / Q000097:blood*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D007328:Insulin; D008565:Membrane Proteins; D017923:Sodium-Hydrogen Exchangers; D010768:Phosphoserine; D008094:Lithium; D012964:Sodium; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate