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[Ca 2+ ] i determines the effects of protein kinases A and C on activity of rat renal Na + ,K + ‐ATPase
Author(s) -
Cheng S. X. J.,
Aizman O.,
Nairn A. C.,
Greengard P.,
Aperia A.
Publication year - 1999
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1999.0037r.x
Subject(s) - ouabain , protein kinase c , forskolin , activator (genetics) , chemistry , phosphorylation , intracellular , protein kinase a , biophysics , medicine , microbiology and biotechnology , endocrinology , biochemistry , sodium , biology , in vitro , receptor , organic chemistry
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 Ca 2+ ([Ca 2+ ] i ). 2 Rat renal NKA was stably expressed in COS cells (green monkey kidney cells). Increases in [Ca 2+ ] i were achieved with the Ca 2+ ionophore A23187 and verified by direct measurements of [Ca 2+ ] i using fura‐2 AM as an indicator. The activity of NKA was measured as ouabain‐sensitive 86 Rb + 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 [Ca 2+ ] i (120 nM) and increased it by 40.5 ± 6.4 % at high [Ca 2+ ] i (420 nM). The change in NKA activity by forskolin correlated with the level of increase in [Ca 2+ ] i . 4 The effect of 1‐oleoyl‐2‐acetoyl‐ sn ‐glycerol (OAG), a specific PKC activator, on the activity of NKA was also Ca 2+ dependent, being inhibitory when [Ca 2+ ] i was low (29.3 ± 3.6 % decrease at 120 nM Ca 2+ ) and stimulatory when [Ca 2+ ] i was high (36.6 ± 10.1 % increase at 420 nM Ca 2+ ). 5 The α subunit of NKA was phosphorylated under both low and high [Ca 2+ ] 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 [Ca 2+ ] i and no change in activity at high [Ca 2+ ] 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 Ca 2+ nor by calmodulin, tested alone or together. 8 We conclude that effect of PKA/PKC on NKA activity is dependent on [Ca 2+ ] i . This Ca 2+ dependence may provide an explanation for the diversity of responses of NKA to activation of either PKA or PKC.