Calcineurin mediates alpha-adrenergic stimulation of Na+,K(+)-ATPase activity in renal tubule cells.

Abstract

The a-adrenergic agonist oxymetazoline increased Na+,K+-ATPase activity of single proximal convoluted tubules dissected from rat kidney. Activation of the enzyme by oxymetazoline was prevented by either the a1-adrenergic antagonist prazosin or the a2-adrenergic antagoit yohimbine and was mimicked by the calcium ionophore A23187. The effect of oxymetazoline on Na+,K+-ATPase activity was prevented by a specific peptide inhibitor of calcineurin, as wed as by FK 506, an immunosuppressant agent known to inhibit calcineurin; these results indicate that the action of oxymetazoline is mediated via activation of calcineurin (a calcium/caimodulindependent protein phosphatase). Activation of the Na+,K+-ATPase by either oxymetazoline or A23187 was aocited with a >2-fold increase in its affinity for Na+. The results provide a biochemical me s by which norepinephrine, released from renal nerve terminals, stimulates Na+ retention.Regulation of sodium excretion by renal sympathetic nerve activity plays a major role in the maintenance of Na+ homeostasis (1). However, little is known about the mechanism by which this regulation occurs. Norepinephrine promotes Na+ retention by a mechanism involving a-adrenergic, but not 3-adrenergic (2-4), receptor activation. Activation of a-adrenergic receptors is associated with increased cytosolic calcium in many cell types (5, 6). Here we present evidence that norepinephrine, acting on a-adrenergic receptors, increases Na+,K+-ATPase activity in permeabilized cells of the proximal convoluted tubule of rat kidney. Moreover, norepinephrine appears to increase this activity through a mechanism involving activation of the calcium/calmodulindependent protein phosphatase, calcineurin.MATERIALS AND METHODS Segments from 'the proximal convoluted tubule were dissected from the cortex ofcollagenase-perfused rat kidneys, in a physiological buffer containing 137 mM NaCl, 5 mM KCl, 0.8 mM MgSO4, 0.33 mM Na2HPO4, 0.44 mM KH2PO4, 0.25 mM CaCl2, 1 mM MgCl2, 10 mM Tris HCl, pH 7.4 at 40C. After dissection was completed, the tubule segments were incubated for 30 min at room temperature in the physiological buffer; in most protocols the NaCl concentration was reduced to 20 mM, and isoosmolality was maintained by the addition of 117 mM choline chloride. Oxymetazoline, prazosin, and yohimbine were obtained from Sigma, the Ca2+ ionophore A23187 from Calbiochem, and FK 506 and rapamycin were from Abbott. Test substances were added to the modified physiological buffer. To allow the peptides to enter the cell, the tubule segments were permeabilized with rapid freezing and thawed. This procedure, which allowed dextran molecules of Mr 4000 to enter the cell, was done during inspection in a stereo microscope.Measurement of Na+,K+-ATPase Activity. Segments were subjected to hypotonic shock to permeabilize the tubule cells. Na+,K+-ATPase activity was' measured as described (7) (GFSPPHRITSFEEAKG), which overlaps the first peptide, spans residues 460-475. Its amino terminus coincides with the first cleavage site of c...

G protein-coupled receptors regulate Na+,K+-ATPase activity and endocytosis by modulating the recruitment of adaptor protein 2 and clathrin

“…PCT cells incubated with DA (2.5 min at 23°C) exhibited decreased (50 Ϯ 16%, n ϭ 7; P Ͻ 0.05) NKA activity (Fig. 1A), and this effect was blocked by coincubation with 1 M Oxy (97 Ϯ 21%; n ϭ 7; not significant); Oxy alone had no significant effect on NKA activity examined under V max condition for its major substrates, as reported by others (24). Incubation of PCT cells with 1 M DA was associated with a significant increase in NKA ␣-subunit abundance in CCV and early endosomes (Fig.…”

“…(31,32). Whereas, in that study (24), the effects of DA and Oxy were determined after 30 min of incubation, we examined the effect of these agonists during 2.5 min, because within this time frame the NKA activity is significantly inhibited by DA and the ␣-subunit remains phosphorylated (after 10 min the ␣-subunit is no longer phosphorylated while being removed from the basolateral membrane) (12). Although Oxy blocked the effect of DA on NKA activity, our results show that this effect was not mediated by affecting the level of phosphorylation of the ␣-subunit, but instead, Oxy prevented the incorporation of NKA ␣-subunits into CCV and endosomes.…”

“…Because endocytosis of NKA molecules in response to DA is responsible for the decreased activity in intact PCT cells, we have now examined whether receptor agonists that counteract the effect of DA on NKA activity do so by preventing NKA subunit endocytosis. We chose to study the effect of Oxy because it had been shown to prevent the inhibitory effect of DA on NKA activity (24). PCT cells incubated with DA (2.5 min at 23°C) exhibited decreased (50 Ϯ 16%, n ϭ 7; P Ͻ 0.05) NKA activity (Fig.…”

“…It has been previously reported (24) that Oxy stimulates NKA activity. The fact that incubation with Oxy in this study did not result in stimulation of NKA could be because of different incubation times (2.5 vs. 30 min), and͞or because of measurement of NKA under different Na ϩ concentrations (20 vs. 100 mM) in the two studies.…”

“…We chose to study in renal epithelial cells the effect of Oxy, a compound postulated to counteract the effect of DA on NKA activity by activating protein phosphatases (24). Oxy action is mediated by activation ␣ 1 and ␣ 2 adrenergic receptors, and the cellular responses to it has been associated, depending on the tissue, with increased intracellular calcium (30) and with negative coupling to adenylyl cyclase.…”