[Ca2+](i) determines the effects of protein kinases A and C on activity ofrat renal Na+, K+-ATPase

1. It is well established that the activity of Na+, K+-ATPase (NKA) is regu lated by protein kinases A (PKA) and C (PKC), but results on their effects have been conflicting. The aim of this study was to examine if this is ascr ibed to the intracellular concentration of Ca2+ ([Ca2+](i)). 2. Rat renal NKA was stably expressed in COS cells (green monkey kidney cel ls). Increases in [Ca2+](i) were achieved with the Ca2+ ionophore A23187 an d verified by direct measurements of [Ca2+](i) using fura-2, AM as an indic ator. The acting of NKA was measured as ouabain-sensitive Rb-86(+) uptake a nd the state of phosphorylation of NKA was monitored with two site-directed phosphorylation state-specific antibodies 3. Activation of PKA with forskolin decreased NKA activity by 45.5 +/- 8.9% at low [Ca2+](i) [Ca2+](i) (420 nM). The change in NKA (120 nM) and includ ed it by 40.5 +/- 6.4% at high [Ca2+](i) a activity by forskolin correlated with the level of increase in [Ca2+](i). 4. The effect of 1-oleoyl-2-acetoyl-sn-glycerol (OAG), a specific PKC activ ator, on the activity of NKA was also Ca2+ dependent, being inhibitory when [Ca2+], was low (29.3 +/- 3.6 % decrease at 120 nM Ca2+) and stimulatory w hen [Ca2+](i) was high (36.8 +/- 10.1% increase at 420 nar Ca2+). 5. The alpha subunit of NKA was phosphorylated under both low and high [Ca2 +](i) PKA or PKC activation. PKA phosphorylated Ser943 . PKC phosphorylates Ser23. 6. To see if the observed effects on NEA activity are secondary to changes in Na+ entry, we measured NKA hydrolytic activity using permeabilized membr anes isolated from cells under controlled conditions. A decreased activity at low [Ca2+](i) and no change in activity at high [Ca2+](i) were observed following forskolin or OAG treatment. 7. Purified NKA from rat renal cortex was phosphorylated and inhibited by P KC. This phosphorylation-associated inhibition of NKA was neither affected by Ca2+ nor by calmodulin, tested alone or together. 8. We conclude that effect of PKA/PKC on NKA activity is dependent on [Ca2](i). This Ca2+ dependence may provide an explanation for the diversity of responses of NKA to activation of either PKA or PKC.