Okadaic acid stimulates ouabain‐sensitive ⁸⁶Rb⁺‐uptake and phosphorylation of the Na⁺/K⁺‐ATPase α‐subunit in rat hepatocytes

Abstract

Ca2+-mobilizing and cAMP-dependent hormones rapidly increase sodium, potassium-dependent adenosine triphosphatase (Na+/K +-ATPase)-mediated transport in rat hepatocytes. To explore the possible role of protein phosphatases in these responses we used a protein phosphatase inhibitor, okadaic acid. Okadaic acid stimulation of ouabain-sensitive SrRb+-uptake was maximal between two and three minutes and displayed an ECs0 of 41 + 1 nM. Inhibition of Na÷/H + exchange with an amiloride analog abolished the response to insulin, but had no effect on okadaic acid-mediated stimulation of Na+/K÷-ATPase transport. In hepatocytes metabolically-radiolabeled with 32Pi, okadaic acid stimulated the incorporation of radioactivity into several 95 kDa peptides, one of which reacted with anti-LEAVE peptide antisera, that recognizes Na+/K÷-ATPase 0t-subunits. In other experiments Na÷/K+-ATPase was immunoprecipitated from detergent-solubilized membrane fractions of metabolically-radiolabeled cells with an antisera to purified rat kidney Na÷/K+-ATPase. A 95 kDa phosphoprotein was immunoprecipitated using anti-Na+/K÷-ATPase antisera, but not by preimmune serum. Okadaic acid stimulated incorporation of radioactivity into this band by 220 _+ 28%. These findings provide support for the hypothesis that rapid stimulation of hepatic Na+/K÷-ATPase by hormones may be related to protein kinase/phosphatase-mediated changes in the phosphorylation state of the Na+/K+-ATPase ct-subunit.

Mechanisms of sodium pump regulation

“…Short-term insulin-mediated sodium pump stimulation can also be secondary to an increase in the cytoplasmic Na ϩ concentration. For example, increases in cell Na ϩ are a consequence of insulin stimulation of the Na ϩ -K ϩ -2Cl Ϫ cotransporter or Na ϩ channels in adipocytes (57,289) or of the Na ϩ /H ϩ exchanger in hepatocytes (216). Another short-term route of insulinmediated upregulation of Na ϩ -K ϩ -ATPase activity has been observed in the kidney.…”

“…Another protein phosphatase shown to modulate Na ϩ -K ϩ -ATPase activity is protein phosphatase 2A (PP2A), which increases pump plasma membrane expression in cortical collecting duct (46) and counters PKC-mediated inhibition of the Na ϩ -K ϩ -ATPase in Sf-9 infected cells (43). Paradoxically, inhibitors of PP2A stimulate the pump in hepatocytes (216). Finally, tyrosine phosphatases may also modulate Na ϩ -K ϩ -ATPase function in PCT (112) and liver (58), as evidenced by the stimulatory effect of vanadate ions acting as tyrosine phosphatase inhibitors.…”

Effects of Protein Kinase Modulators on the Sodium Pump Activities of HeLa Cells Transfected with Distinct Alpha Isoforms of Na,K‐ATPasea

“…Stimulation of protein kinases in vivo has been reported to activate as well as to inhibit pump activity in various systems, and increased phosphorylation of the Na,K‐ATPase α subunit has been demonstrated in conjunction with both of these effects, 1,2 as well as in situations in which no change in activity has been detected 3 . This study addresses the question of how the individual α isoforms of the Na,K‐ATPase are affected in vivo following activation of the endogenous protein kinases A (PKA) and C (PKC).…”

Glucagon stimulation of hepatic Na+-pump activity and α-subunit phosphorylation in rat hepatocytes

“…Hepatocytes were prepared from Sprague-Dawley rats as previously described [19,22,24,25] . Uptake reactions were initiated by adding ##NaCl (0.9i10*-1.0i10* c.p.m.\mol) to the cells along with drugs or vehicle.…”

“…The addition of protein kinase A to Na + \K + -ATPase from several sources has been reported to either stimulate [18] or inhibit [12,15] Na + \K + -ATPase enzyme activity. Lastly, okadaic acid, a protein phosphatase inhibitor, stimulates ouabain-sensitive )'Rb+ uptake and phosphorylation of the α-subunit in rat hepatocytes [19].…”