Ca2+)i determines the effects of protein kinases A and C on activity of rat renal Na+,K+-ATPase.

摘要

1. It is well established that the activity of Na+,K+-ATPase (NKA) is regulated 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 ascribed to the intracellular concentration of Ca2+ ([Ca2+]i). 2. Rat renal NKA was stably expressed in COS cells (green monkey kidney cells). Increases in [Ca2+]i were achieved with the Ca2+ ionophore A23187 and verified by direct measurements of [Ca2+]i using fura-2 AM as an indicator. The activity of NKA was measured as ouabain-sensitive 86Rb+ uptake and 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 (120 nM) and increased it by 40.5 +/- 6.4 % at high [Ca2+]i (420 nM). The change in NKA 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 activator, on the activity of NKA was also Ca2+ dependent, being inhibitory when [Ca2+]i was low (29.3 +/- 3.6 % decrease at 120 nM Ca2+) and stimulatory when [Ca2+]i was high (36.6 +/- 10.1 % increase at 420 nM Ca2+). 5. The alpha subunit of NKA was phosphorylated under both low and high [Ca2+]i conditions upon PKA or PKC activation. PKA phosphorylates Ser943. PKC phosphorylates Ser23. 6. To see if the observed effects on NKA activity are secondary to changes in Na+ entry, we measured NKA hydrolytic activity using permeabilized membranes isolated from cells under controlled Na+ 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 PKC. 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.