Activation of sodium transport in rat erythrocytes by inhibition of protein phosphatases 1 and 2A

Four structurally different protein phosphatases (PPs) inhibitors – fluoride, calyculin A, okadaic acid and cantharidin – were tested for their ability to modulate unidirectional Na+ influx in rat red blood cells. Erythrocytes were incubated at 37 °C in isotonic and hypertonic media containing 1 mM ouabain and 22Na in the absence or presence of PP inhibitors. Exposure of the cells to 20 mM fluoride or 50 nM calyculin A for 1 h under isosmotic conditions caused a significant stimulation of Na+ influx, whereas addition of 200 μM cantharidin or 100 nM okadaic acid had no effect. After 2 h of treatment, however, all these PPs blockers significantly enhanced Na+ transport in rat erythrocytes. Selective inhibitors of PP-1 and PP-2A types, calyculin A, cantharidin and okadaic acid, produced similar (∼ 1.2–1.4-fold) stimulatory effects on Na+ influx in the cells. Activation of Na+ influx was unchanged with increasing calyculin A concentration from 50 to 200 nM. No additive stimulation of Na+ influx was observed when the cells were treated with combination of 20 mM fluoride and 50 nM calyculin A. Na+ influx induced by PPs blockers was inhibited by 1 mM amiloride and 200 μM bumetanide approximately in the equal extent, indicating the involvement of Na+/H+ exchange and Na–K–2Cl cotransport in sodium transport through rat erythrocytes membrane. Activation of Na+ transport in the cells induced by calyculin A and fluoride was associated with increase of intracellular Na+ content. Shrinkage of the rat erythrocytes resulted in 2-fold activation of Na+ influx. All tested PPs inhibitors additionally activated the Na+ influx by 70–100% above basal shrinkage-induced level. Amiloride and bumetanide have diminished both the shrinkage-induced and PPs-inhibitors-induced Na+ influxes. Thus, our observations clearly indicate that activities of Na+/H+ exchanger and Na–K–2Cl cotransporter in rat erythrocytes are regulated by protein phosphatases and stimulated when protein dephosphorylation is inhibited.