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Effects of phorbol 12-myristate 13-acetate on potassium transport in the red blood cells of frog Rana temporaria

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Abstract

Phorbol 12-myristate 13-acetate (PMA), a stimulator of PKC, was examined for its influence on K+ (86Rb) influx in the frog erythrocyte. PMA, 0.1 μM, was found to accelerate ouabain-sensitive K+ influx, which was suppressed by 73% with 1 mM amiloride, indicating secondary activation of the Na+–K+-pump due to stimulation of Na+ /H+ exchange. PMA-induced stimulation of the sodium pump was completely inhibited with 1 μM staurosporine and by ~50% with 20 μM chelerythrine. In contrast to Na+–K+-pump, an activity of Cl−-dependent K+ transport (K–Cl cotransport, KCC), calculated as the difference between K+ influxes in Cl− and NO3 −-media, was substantially decreased under the influence of PMA. Staurosporine fully restored the PMA-induced inhibition of KCC, whereas chelerythrine did not exert any influence. Osmotic swelling of the frog erythrocytes was accompanied by approximately twofold stimulation of KCC. Swelling-activated KCC was inhibited by ~50 and ~83% in the presence of PMA and genistein, respectively, but not chelerythrine. Exposure of the frog erythrocytes to 5 mM fluoride (F−) also reduced the KCC activity in isotonic and hypotonic media, with maximal suppression of K+ influx in both media being observed upon simultaneous addition of PMA and F−. Furosemide and [(dihydronindenyl)oxy] alkanoic acid inhibited the K+ influx in both the media by ~50–60%. The results obtained show both the direct and indirect effects of PMA on the K+ transport in frog erythrocytes and a complicated picture of KCC regulation in frog erythrocytes with involvement of PKC, tyrosine kinase and protein phosphatase.

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Abbreviations

RBC:

Red blood cells

DMSO:

Dimethyl sulphoxide

TRIS:

Tris(hydroxymethyl)aminomethane

PMA:

Phorbol 12-myristate 13-acetate

PKC:

Protein kinase C

TK:

Tyrosine kinase

PP:

Protein phosphatase

NMDG:

N-methyl-d-glucamine

DIOA:

[(Dihydronindenyl)oxy] alkanoic acid

References

  • Adragna NC, White RE, Orlov SN, Lauf PK (2000) K–Cl cotransport in vascular smooth muscle and erythrocytes: possible implication in vasodilation. Am J Physiol 278:C381–C390

    CAS  Google Scholar 

  • Adragna NC, Di Fulvio M, Lauf PK (2004) Regulation of K–Cl cotransport: from functions to genes. J Membr Biol 201:109–137

    Article  PubMed  CAS  Google Scholar 

  • Adragna NC, Ferrell CM, Zhang J, Di Fulvio M, Temprana CF, Sharma A, Fyffe RE, Cool DR, Lauf PK (2006) Signal transduction mechanisms of K+–Cl− cotransport regulation and relationship to disease. Acta Physiol 187:125–139

    Article  CAS  Google Scholar 

  • Agalakova NI, Gusev GP (2003) Effect of protein kinase C activation on Na+–H+ exchange in erythrocytes of frog Rana temporaria. Comp Biochem Physiol A 134:11–20

    Google Scholar 

  • Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, Fukami Y (1987) Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 262:5592–5595

    PubMed  CAS  Google Scholar 

  • Bergeron MJ, Gagnon E, Caron L, Isenring P (2006) Identification of key functional domains in the C terminus of the K+–Cl− cotransporters. J Biol Chem 281:15959–15969

    Article  PubMed  CAS  Google Scholar 

  • Bize I, Dunham PB (1994) Staurosporine, a protein kinase inhibitor, activates K–Cl cotransport in LK sheep erythrocytes. Am J Physiol 266:C759–C770

    PubMed  CAS  Google Scholar 

  • Bize I, Guvenc B, Robb A, Buchbinder G, Brugnara C (1999) Serine/threonine protein phosphatases and regulation of K–Cl cotransport in human erythrocytes. Am J Physiol 277:C926–C936

    PubMed  CAS  Google Scholar 

  • Bize I, Taher S, Brugnara C (2003) Regulation of K–Cl cotransport during reticulocyte maturation and erythrocyte aging in normal and sickle erythrocytes. Am J Physiol 285:C31–C38

    Google Scholar 

  • Cohen DM (2005) SPC family kinases in cell volume regulation. Am J Physiol 288:C483–C493

    Article  CAS  Google Scholar 

  • Cossins AR, Gibson JS (1997) Volume-sensitive transport systems and volume homeostasis in vertebrate red blood cells. J Exp Biol 200:343–352

    PubMed  CAS  Google Scholar 

  • Cossins AR, Weaver YR, Lykkeboe G, Nielsen OB (1994) Role of protein phosphorylation in control of K flux pathways of trout red blood cells. Am J Physiol 267:C1641–C1650

    PubMed  CAS  Google Scholar 

  • Davies SP, Reddy H, Caivano M, Cohen P (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351:95–105

    Article  PubMed  CAS  Google Scholar 

  • De Franceschi L, Fumagalli L, Olivieri O, Corrocher R, Lowell CA, Berton G (1997) Deficiency of Src family kinases Fgr and Hck results in activation of erythrocyte K/Cl cotransport. J Clin Invest 15:220–227

    Article  Google Scholar 

  • Ferrell CM, Lauf PK, Wilson BA, Adragna NC (2000) Lithium and protein kinase C modulators regulate swelling-activated K–Cl cotransport and reveal a complete phosphatidylinositol cycle in low K sheep erythrocytes. J Membr Biol 177:81–93

    Article  PubMed  CAS  Google Scholar 

  • Flatman PW, Adragna NC, Lauf PK (1996) Role of protein kinases in regulating sheep erythrocyte K–Cl cotransport. Am J Physiol 271:C255–C263

    PubMed  CAS  Google Scholar 

  • Fujise H, Lauf PK (1988) Na+–K+ pump activities of high- and low-potassium sheep red cells with internal magnesium and calcium altered by A23187. J Physiol 405:605–614

    PubMed  CAS  Google Scholar 

  • Gamba G (2005) Molecular physiology and pathophysiology of electroneutral cation–chloride cotransporters. Physiol Rev 85:423–493

    Article  PubMed  CAS  Google Scholar 

  • Garay RP, Nazaret C, Hannaert PA, Cragoe EJ Jr (1988) Demonstration of a [K+, Cl−]-cotransport system in human red cells by its sensitivity to [(dihydroindenyl)oxy]alkanoic acids: regulation of cell swelling and distinction from the bumetanide-sensitive [Na+, K+, Cl−]-cotransport system. Mol Pharmacol 33:696–701

    PubMed  CAS  Google Scholar 

  • Godart H, Ellory JC (1996) KCl cotransport activation in human erythrocytes by high hydrostatic pressure. J Physiol 491:423–434

    PubMed  CAS  Google Scholar 

  • Gusev GP, Agalakova NI (2000) Na, K-pump activation by isoproterenol, methylxanthines, and iodoacetate in erythrocytes of the frog Rana temporaria. Zh Evol Biokhim Fiziol 36:106–111

    PubMed  CAS  Google Scholar 

  • Gusev GP, Agalakova NI, Lapin AV (1995) Potassium transport in red blood cells of frog Rana temporaria: demonstration of a K–Cl cotransport. J Comp Physiol B 165:230–237

    Article  PubMed  CAS  Google Scholar 

  • Gusev GP, Lapin AV, Agalakova NI (1997) Volume regulation in red blood cells of frog Rana temporaria after osmotic shrinkage and swelling. Membr Cell Biol 14:251–261

    CAS  Google Scholar 

  • Gusev GP, Agalakova NI, Lapin AV (1999) Kinetics of K–Cl cotransport in frog erythrocyte membrane: effect of external sodium. J Membr Biol 172:203–213

    Article  PubMed  CAS  Google Scholar 

  • Herbert JM, Augereau JM, Gleye J, Maffrand JP (1990) Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 172:993–999

    Article  PubMed  CAS  Google Scholar 

  • Holtzman EJ, Kumar S, Faaland CA, Warner F, Logue PJ, Erickson SJ, Ricken G, Waldman J, Kumar S, Dunham PB (1998) Cloning, characterization, and gene organization of K–Cl cotransporter from pig and human kidney and C. elegans. Am J Physiol 275:F550–F564

    PubMed  CAS  Google Scholar 

  • Jennings ML, Schulz RK (1991) Okadaic acid inhibition of KCl cotransport. Evidence that protein dephosphorylation is necessary for activation of transport by either cell swelling or N-ethylmaleimide. J Gen Physiol 97:799–817

    Article  PubMed  CAS  Google Scholar 

  • Kim HD, Sergeant S, Forte LR, Sohn DH, Im JH (1989) Activation of a Cl-dependent K flux by cAMP in pig red cells. Am J Physiol 256:C772–C778

    PubMed  CAS  Google Scholar 

  • Krarup T, Dunham PB (1996) Reconstitution of calyculin-inhibited K–Cl cotransport in dog erythrocyte ghosts by exogenous PP-1. Am J Physiol 270:C898–C902

    PubMed  CAS  Google Scholar 

  • Lauf PK (1984) Thiol-dependent passive K/Cl transport in sheep red cells: IV. Furosemide inhibition as a function of external Rb+, Na+, and Cl−. J Membr Biol 77:57–62

    Article  PubMed  CAS  Google Scholar 

  • Lauf PK, Adragna NC (2000) K–Cl cotransport: properties and molecular mechanism. Cell Physiol Biochem 10:341–354

    Article  PubMed  CAS  Google Scholar 

  • Lauf PK, Bauer J, Adragna NC, Fujise H, Zade-Oppen AM, Ryu KH, Delpire E (1992) Erythrocyte K–Cl cotransport: properties and regulation. Am J Physiol 263:C917–C932

    PubMed  CAS  Google Scholar 

  • Lee SK, Qing WG, Mar W, Luyengi L, Mehtas RG, Kawanishi K, Fong HHS, Beecher CWW, Kinghorn AD, Pezzuto JM (1998) Angoline and chelerythrine, benzophenanthridine alkaloids that do not inhibit protein kinase C. J Biol Chem 273:19829–19833

    Article  PubMed  CAS  Google Scholar 

  • Mercado A, Song L, Vazquez N, Mount DB, Gamba G (2000) Functional comparison of the K+–Cl− cotransporters KCC1 and KCC4. J Biol Chem 275:30326–30334

    Article  PubMed  CAS  Google Scholar 

  • O’Neill WC (1991) Swelling-activated K–Cl cotransport: metabolic dependence and inhibition by vanadate and fluoride. Am J Physiol 260:C308–C315

    PubMed  Google Scholar 

  • Sachs JR, Martin DW (1993) The role of ATP in swelling-stimulated K–Cl cotransport in human red cell ghosts. Phosphorylation–dephosphorylation events are not in the signal transduction pathway. J Gen Physiol 102:551–573

    Article  PubMed  CAS  Google Scholar 

  • Shen M-R, Chou C-Y, Ellory JC (2000) Volume-sensitive KCl cotransport associated with human cervical carcinogenesis. Eur J Physiol 440:751–760

    Article  CAS  Google Scholar 

  • Strange K, Singer TD, Morrison R, Delpire E (2000) Dependence of KCC2 K–Cl cotransporter activity on a conserved carboxy terminus tyrosine residue. Am J Physiol 279:C860–C867

    CAS  Google Scholar 

  • Su R, Shmukler BE, Chernova MN, Stuart-Tilley AK, De Franceschi L, Brugnara C, Alper SL (1999) Mouse K–Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation. Am J Physiol 277:C899–C912

    PubMed  CAS  Google Scholar 

  • Tamaoki T, Nomoto H, Takahashi I, Kato Y, Morimoto M, Tomita F (1986) Staurosporine, a potent inhibitor of phospholipid/Ca++ dependent protein kinase. Biochem Biophys Res Commun 135:397–402

    Article  PubMed  CAS  Google Scholar 

  • Weaver YR, Cossins AR (1996) Protein tyrosine phosphorylation and the regulation of KCl cotransport on trout erythrocytes. Pflugers Arch 432:727–734

    Article  PubMed  CAS  Google Scholar 

  • Yu R, Mandlekar S, Tan T-H, Kong A-NT (2000) Activation of p38 and c-Jun N-terminal kinase pathways and induction of apoptosis by chelerythrine do not require inhibition of protein kinase C. J Biol Chem 275:9612–9619

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Thorez pr. 44, 193224, St Petersburg, Russia

    Natalia Ivanovna Agalakova & G. P. Gusev

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  1. Natalia Ivanovna Agalakova
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  2. G. P. Gusev
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Correspondence to Natalia Ivanovna Agalakova.

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Communicated by G. Heldmaier.

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Agalakova, N.I., Gusev, G.P. Effects of phorbol 12-myristate 13-acetate on potassium transport in the red blood cells of frog Rana temporaria . J Comp Physiol B 179, 443–450 (2009). https://doi.org/10.1007/s00360-008-0324-2

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  • DOI: https://doi.org/10.1007/s00360-008-0324-2

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