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

Abstract

Phorbol 12-myristate 13-acetate (PMA), a stimulator of PKC, was examined for its influence on K(+) ((86)Rb) influx in the frog erythrocyte. PMA, 0.1 microM, 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 microM staurosporine and by approximately 50% with 20 microM 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 NO(3) (-)-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 approximately 50 and approximately 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 approximately 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.

Physiological roles and molecular mechanisms of K+‐Cl− cotransport in the mammalian kidney and cardiovascular system: where are we?

“…According to the widely held view, the KCCs are activated by cell swelling through their dephosphorylation and inactivated under basal conditions through their phosphorylation. This view is supported by a large body of indirect evidence (Jennings & Schulz, 1991;Mercado et al 2000Mercado et al , 2001Mercado et al , 2016Song et al 2002;Garzon-Muvdi et al 2007;Agalakova & Gusev, 2009;Gusev & Agalakova, 2010) and the identification of conserved threonine residues that undergo dephosphorylation during cell swelling (Rinehart et al 2009;Melo et al 2013b;de Los Heros et al 2014). The localization of two such residues is shown in Fig.…”

“…; Garzon‐Muvdi et al . ; Agalakova & Gusev, ; Gusev & Agalakova, ) and the identification of conserved threonine residues that undergo dephosphorylation during cell swelling (Rinehart et al . ; Melo et al .…”

Phosphoregulation of K+‐Cl− cotransporters during cell swelling: Novel insights

“…Thus far, the implication of signaling intermediates has been mostly inferred from indirect evidence and transport studies in erythrocytes where several isoforms of the carrier are expressed (Agalakova & Gusev, ; Gusev & Agalakova, ). Even if the role of such intermediates or of potential phosphoacceptor residues was also analyzed through direct phosphorylation assays in a few studies, it is unclear that the KCC domains tested were always in their native conformational state given that they often corresponded to peptide fragments produced in foreign cell types.…”

“…A general model has emerged in which inhibition of K + -Cl − efflux is believed to occur through carrier phosphorylation by protein kinases (PKs), and activation of K + -Cl − efflux through carrier dephosphorylation by protein phosphatases (PPs). This model is supported in part by the identification of phosphoacceptor sites in both the N-and C-termini of various KCCs (Agalakova & Gusev, 2009;de Los Heros et al, 2014;Gusev & Agalakova, 2010;Melo, de los Heros, et al, 2013;Rinehart et al, 2009). Lysine-deficient protein kinases (WNKs) and SPS1-related proline/alanine-rich kinase (SPAK) are among the kinases potentially at play.…”