Modulation of Na+,K+-ATPase activity is of importance for RVD.

AIM: This study was performed to examine the role of Na+,K+-ATPase activity for the adaptive response to cell swelling induced by hypoosmoticity, i.e. the regulatory volume decrease (RVD). METHODS: The studies were performed on COS-7 cells transfected with rat Na+,K+-ATPase. To study changes in cell volume, cells were loaded with the fluorescent dye calcein and the intensity of the dye, following exposure to a hypoosmotic medium, was recorded with confocal microscopy. RESULTS: Ouabain-mediated inhibition of Na+,K+-ATPase resulted in a dose dependent decrease in the rate of RVD. Total 86Rb+ uptake as well as ouabain dependent 86Rb+ uptake, used as an index of Na+,K+-ATPase dependent K+ uptake, was significantly increased during the first 2 min following exposure to hypoosmoticity. Since protein kinase C (PKC) plays an important role in the modulation of RVD, a study was carried out on COS-7 cells expressing rat Na+,K+-ATPase, where Ser23 in the catalytic alpha1 subunit of rat Na+,K+-ATPase had been mutated to Ala (S23A), abolishing a known PKC phosphorylation site. Cells expressing S23A rat Na+,K+-ATPase exhibited a significantly lower rate of RVD and showed no increase in 86Rb+ uptake during RVD. CONCLUSION: Taken together, these results suggest that a PKC-mediated transient increase in Na+,K+-ATPase activity plays an important role in RVD.

Intracellular sodium modulates the state of protein kinase C phosphorylation of rat proximal tubule Na+,K+-ATPase.

The natriuretic hormone dopamine and the antinatriuretic hormone noradrenaline, acting on alpha-adrenergic receptors, have been shown to bidirectionally modulate the activity of renal tubular Na+,K+-adenosine triphosphate (ATPase). Here we have examined whether intracellular sodium concentration influences the effects of these bidirectional forces on the state of phosphorylation of Na+,K+-ATPase. Proximal tubules dissected from rat kidney were incubated with dopamine or the alpha-adrenergic agonist, oxymetazoline, and transiently permeabilized in a medium where sodium concentration ranged between 5 and 70 mM. The variations of sodium concentration in the medium had a proportional effect on intracellular sodium. Dopamine and protein kinase C (PKC) phosphorylate the catalytic subunit of rat Na+,K+-ATPase on the Ser23 residue. The level of PKC induced Na+,K+-ATPase phosphorylation was determined using an antibody that only recognizes Na+,K+-ATPase, which is not phosphorylated on its PKC site. Under basal conditions Na+,K+-ATPase was predominantly in its phosphorylated state. When intracellular sodium was increased, Na+,K+-ATPase was predominantly in its dephosphorylated state. Phosphorylation of Na+,K+-ATPase by dopamine was most pronounced when intracellular sodium was high, and dephosphorylation by oxymetazoline was most pronounced when intracellular sodium was low. The oxymetazoline effect was mimicked by the calcium ionophore A23187. An inhibitor of the calcium-dependent protein phosphatase, calcineurin, increased the state of Na+,K+-ATPase phosphorylation. The results imply that phosphorylation of renal Na+,K+-ATPase activity is modulated by the level of intracellular sodium and that this effect involves PKC and calcium signalling pathways. The findings may have implication for the regulation of salt excretion and sodium homeostasis.

Ouabain, a steroid hormone that signals with slow calcium oscillations.

The plant-derived steroid, digoxin, a specific inhibitor of Na,K-ATPase, has been used for centuries in the treatment of heart disease. Recent studies demonstrate the presence of a digoxin analog, ouabain, in mammalian tissue, but its biological role has not been elucidated. Here, we show in renal epithelial cells that ouabain, in doses causing only partial Na,K-ATPase inhibition, acts as a biological inducer of regular, low-frequency intracellular calcium ([Ca(2+)](i)) oscillations that elicit activation of the transcription factor, NF-kappa B. Partial inhibition of Na,K-ATPase using low extracellular K(+) and depolarization of cells did not have these effects. Incubation of cells in Ca(2+)-free media, inhibition of voltage-gated calcium channels, inositol triphosphate receptor antagonism, and redistribution of actin to a thick layer adjacent to the plasma membrane abolished [Ca(2+)](i) oscillations, indicating that they were caused by a concerted action of inositol triphosphate receptors and capacitative calcium entry via plasma membrane channels. Blockade of ouabain-induced [Ca(2+)](i) oscillations prevented activation of NF-kappa B. The results demonstrate a new mechanism for steroid signaling via plasma membrane receptors and underline a novel role for the steroid hormone, ouabain, as a physiological inducer of [Ca(2+)](i) oscillations involved in transcriptional regulation in mammalian cells.

Phosphorylation of the catalytic subunit of rat renal Na+, K+-ATPase by classical PKC isoforms.

In this study we have evaluated the specificity of different PKC isozymes for the phosphorylation of the catalytic alpha1 subunit of rat renal Na+,K+-ATPase (alpha1 Na+,K+-ATPase). Using in vitro phosphotransferase assays we found that classical PKCs (cPKCs) alpha, betaI, and gamma efficiently phosphorylate alpha1 Na+,K+-ATPase. However, alpha1 Na+,K+-ATPase was a poor substrate for the novel PKCs (nPKCs) delta and epsilon. Two-dimensional phosphopeptide mapping revealed a similar pattern of phosphorylation by all cPKCs. The functional significance of this finding was evaluated by measuring Na+,K+-ATPase activity (assessed by 86Rb+ uptake) in COS-7 cells expressing the rat alpha1 Na+,K+-ATPase. 1-oleoyl-2-acetoyl-sn-glycerol (OAG), a nonselective PKC activator, inhibited Na+,K+-ATPase activity in this system. On the other hand, 12-deoxyphorbol-13-phenylacetate (DPP), which preferentially activates nPKCepsilon, did not affect 86Rb+ uptake. These results indicate a differential pattern of phosphorylation and regulation of rat renal Na+,K+-ATPase activity by PKC isoforms and suggest an important role for cPKCs in the physiological regulation of the pump.

Anatomical and physiological evidence for D1 and D2 dopamine receptor colocalization in neostriatal neurons.

Despite the importance of dopamine signaling, it remains unknown if the two major subclasses of dopamine receptors exist on the same or distinct populations of neurons. Here we used confocal microscopy to demonstrate that virtually all striatal neurons, both in vitro and in vivo, contained dopamine receptors of both classes. We also provide functional evidence for such colocalization: in essentially all neurons examined, fenoldopam, an agonist of the D1 subclass of receptors, inhibited both the Na+/K+ pump and tetrodotoxin (TTX)-sensitive sodium channels, and quinpirole, an agonist of the D2 subclass of receptors, activated TTX-sensitive sodium channels. Thus D1 and D2 classes of ligands may functionally interact in virtually all dopamine-responsive neurons within the basal ganglia.

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

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.

Beta-adrenergic stimulation of cellular K+ uptake in rat distal colon.

We recently demonstrated that the ratio between colonic K+ absorptive and K+ secretive pathways was higher in infant than in adult rats. To test the hypothesis that hormones selectively affect these pathways during ontogeny we examined the effect of adrenergic agonists on cellular K+ uptake in distal colon from infant (10-day-old) and adult (50-day-old) rats. Here we describe that adrenaline (10(-5) M) increased total and ouabain-insensitive 86Rb uptake in both age groups, but it did not affect ouabain-sensitive 86Rb uptake. This stimulation was more pronounced in adult than in infant rats. The effect of adrenaline was mediated via beta-adrenergic receptors. Incubation in vitro with beta-agonist, isoproterenol, stimulated SCH-28080-sensitive, i.e. H+, K(+)-ATPase-dependent, 86Rb uptake in adult but not in infant rats. The threshold dose of beta-agonist was at 10(-7) M, and the maximal activation was observed at 10(-5) M. In vivo inhibition of beta-adrenergic system with propranolol caused a significant decrease in H+, K(+)-ATPase-dependent 86Rb uptake in infant but not in adult colon. In conclusion, this study suggests that the higher colonic K+ absorption in infant rats may be as a result of a selective beta-adrenergic up-regulation leading to stimulation of the apical H+, K(+)-ATPase.

PKA-mediated phosphorylation and inhibition of Na(+)-K(+)-ATPase in response to beta-adrenergic hormone.

The activity of Na(+)-K(+)-ATPase can be regulated by hormones that activate adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA). Here, using a site-directed phosphorylation state-specific antibody, we show that hormonal regulation of Na(+)-K(+)-ATPase can occur via phosphorylation of Ser-943 on its alpha-subunit. cDNAs coding for wild-type rat Na(+)-K(+)-ATPase and Na(+)-K(+)-ATPase in which the PKA phosphorylation site Ser-943 was mutated to Ala were stably and transiently transfected into COS cells. In COS cells expressing wild-type Na(+)-K(+)-ATPase the beta-adrenergic agonist isoproterenol (1 microM) significantly increased the level of phosphorylation of the alpha-subunit. Phosphorylation was accompanied by a significant inhibition of the enzyme activity, as reflected by a decrease in ATP hydrolysis and 86Rb+ transport. The effect of isoproterenol was reproduced by the PKA activator forskolin used in combination with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and was abolished by the specific PKA inhibitor H-89. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, enhanced phosphorylation and inhibition of Na(+)-K(+)-ATPase induced by isoproterenol. The changes in activity of Na(+)-K(+)-ATPase linearly correlated with the extent of the alpha-subunit of Na(+)-K(+)-ATPase being phosphorylated. When Ser-943 was replaced by alanine, stimulation of the phosphorylation and inhibition of the activity of Na(+)-K(+)-ATPase induced by isoproterenol, alone or in combination with okadaic acid, were not observed. These results indicate that, in intact cells, modulation of the activity of Na(+)-K(+)-ATPase can be achieved by regulation of the state of phosphorylation of Ser-943. Moreover, they provide a biochemical mechanism by which beta-adrenergic agonists can regulate Na(+)-K(+)-ATPase activity.

20-Hydroxyeicosa-tetraenoic acid (20 HETE) activates protein kinase C. Role in regulation of rat renal Na+,K+-ATPase.

It is well documented that the activity of Na+,K+-ATPase can be inhibited by the arachidonic acid metabolite, 20-hydroxyeicosa-tetraenoic acid (20 HETE). Evidence is presented here that this effect is mediated by protein kinase C (PKC). PKC inhibitors abolished 20 HETE inhibition of rat Na+,K+-ATPase in renal tubular cells. 20 HETE caused translocation of PKC alpha from cytoplasm to membrane in COS cells. It also inhibited Na+,K+-ATPase activity in COS cells transfected with rat wild-type renal Na+,K+-ATPase alpha1 subunit, but not in cells transfected with Na+,K+-ATPase alpha1, where the PKC phosphorylation site, serine 23, had been mutated to alanine. PKC-induced phosphorylation of rat renal Na+,K+-ATPase, as well as of histone was strongly enhanced by 20 HETE at the physiologic calcium concentration of 1.3 microM, but not at the calcium concentration of 200 microM. The results indicate that phospholipase A2-arachidonic acid-20 HETE pathway can exert important biological effects via activation of PKC and that this effect may occur in the absence of a rise in intracellular calcium.