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...

Catecholamines have pronounced effects on the renal handling of sodium and water, dopamine-promoting sodium and water excretion, and norepinephrine-promoting sodium and water retention. In the present study, using isolated permeabilized renal tubule cells and intact rats, we have shown that these effects can be attributed to opposing actions of these transmitters on renal tubular Na+,K+-ATPase activity. The ability of each of these catecholamines to regulate Na+,K+-ATPase activity is affected by the concentration of Na+ as well as by the absence or presence of the opposing catecholamine.Hypertension represents one of the major health problems in industrialized countries (1). Sodium metabolism by the kidney represents the major determinant ofblood pressure (2, 3). It is well-established that dopamine released from intrinsic cells in the kidney is of central importance in the regulation of this metabolism (4). Moreover, dopamine is extremely widely used in intensive care units to induce sodium excretion (5). Therefore, it is of paramount importance to understand the underlying mechanism by which dopamine achieves its natriuretic action. It is generally believed that this mechanism involves inhibition of renal tubular Na+,K+-ATPase activity (6). However, previous efforts to demonstrate an inhibition of Na+,K+-ATPase activity by dopamine were unsuccessful when physiological concentrations ofNa+ were used. We now report, by using isolated permeabilized renal tubule cells, that dopamine is able to inhibit Na+,K+-ATPase activity at physiological sodium concentrations and that this inhibition requires the target cells to be subjected simultaneously to adrenergic activation. We have also found, in experiments carried out on sodium excretion in intact animals, that, for the natriuretic action of dopamine to be manifested, a background of adrenergic activity is required. MATERIALS AND METHODSThe studies were performed on male Sprague-Dawley rats (Alab, Stockholm) weighing 150-200 g. In one protocol the rats were unilaterally denervated. Under ether anesthesia, the left renal artery was dissected free from the surrounding tissue and wrapped three times for 5-min periods with cotton soaked in a solution of 10% (vol/vol) phenol in absolute alcohol. The studies were performed 8 days later. The efficacy of the denervation procedure has been assessed by examining for the presence of neuropeptide Y and calcitonin gene-related peptide by using the indirect immunofluorescence technique (7).Segments from the proximal convoluted tubule were dissected from the cortex of the collagenase-perfused rat kid-

Signaling through both angiotensin AT1 receptors (AT1R) and dopamine D1 receptors (D1R) modulates renal sodium excretion and arterial BP. AT1R and D1R form heterodimers, but whether treatment with AT1R antagonists functionally modifies D1R via allosterism is unknown. In this study, the AT1R antagonist losartan strengthened the interaction between AT1R and D1R and increased expression of D1R on the plasma membrane in vitro. In rat proximal tubule cells that express endogenous AT1R and D1R, losartan increased cAMP generation. Losartan increased cAMP in HEK 293a cells transfected with both AT1R and D1R, but it did not increase cAMP in cells transfected with either receptor alone, suggesting that losartan induces D1R activation. Furthermore, losartan did not increase cAMP in HEK 293a cells expressing AT1R and mutant S397/S398A D1R, which disrupts the physical interaction between AT1R and D1R. In vivo, administration of a D1R antagonist significantly attenuated the antihypertensive effect of losartan in rats with renal hypertension. Taken together, these data imply that losartan might exert its antihypertensive effect both by inhibiting AT1R signaling and by enhancing D1R signaling. 23: 421-428, 201223: 421-428, . doi: 10.1681 Sodium excretion and renal vascular tonus are bidirectionally regulated by dopamine, acting on dopamine D1 receptors (D1R) and angiotensin II, acting on angiotensin AT1 receptors (AT1R). [1][2][3] Several lines of evidence suggest that AT1R and D1R form a heteromeric signaling complex. [4][5][6] We recently reported that activation of either AT1R or D1R might cause internalization and/or interruption of the signaling capacity of the other receptor. 6 These observations imply that AT1R and D1R may allosterically modulate each other. Several recent studies have shown that the G-protein coupled receptor (GPCR) often forms heteromers and it has been suggested that allosteric modification within such heteromers will fine-tune receptor signal strength and offer novel opportunities for therapeutic approaches. 7-10 Because AT1R antagonists are far more commonly used therapeutically than AT1R agonists, we decided to test whether losartan, an AT1R antagonist, 11 might influence AT1R and D1R interaction and modulate D1R signaling. Losartan has been widely used to lower arterial BP and to prevent or attenuate the progression of renal disease. [12][13][14][15] Notably, the losartan binding site in AT1R does not overlap with the angiotensin binding site. 16,17 This study was performed on rat renal proximal tubule cells (RPTCs) in primary culture and on HEK 293a (HEK) cells, a cell line derived from human embryonic kidney. The endogenous expressions of D1R and AT1R are relatively high in RPTCs 1,18 and low in HEK cells. Therefore, HEK cells were used for expression of fluorescently tagged receptors and J Am Soc Nephrol 421BASIC RESEARCH mutant receptors. We performed a series of biochemical studies, which indicated that losartan strengthens the interaction between AT1R and D1R and that losartan incr...

We conclude that prolactin is a natriuretic hormone which interacts with the renal dopamine system for its effects. The natriuretic response is associated with inhibition of proximal tubular Na(+), K(+)-ATPase activity.

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.

Sodium transport correlates with varying Na+-K+-ATPase activity rates along the nephron. Whether differences in Na+-K+-ATPase regulation by protein kinase C-dependent phosphorylation are also present has not been tested. We measured the degree of Na+-K+-ATPase alpha1 subunit phosphorylation by the binding of McK-1 antibody to dephosphorylated Ser-23 and Na+-K+-ATPase activity in medullary thick ascending limb of Henle (mTAL) and proximal tubules (PCT). The degree of Na+-K+-ATPase phosphorylation at Ser-23 was lower in mTAL than in PCT (DU 13.43+/-1.99 versus 2.3+/-0.20, respectively, P<0.01) while Na+-K+-ATPase activity was higher in mTAL (3,402+/-83 vs 711+/-158 pmol/mm tubule per hour in PCT, P<0.01). PKC inhibitor RO-318220 10(-6) M decreased phosphorylation in PCT to 125+/-10% ( P<0.05). In mTAL, RO-318220 did not modify the phosphorylation degree or the activity of Na+-K+-ATPase. Both calcineurin inhibitor FK-506 10(-6) M and phorbol 12-myristate 13-acetate (PMA) 10(-6) M increased the degree of Na+-K+-ATPase phosphorylation ( P<0.05) and inhibited Na+-K+-ATPase activity to 657+/-152 and 1,448+/-347 pmol/mm tubule per hour, respectively, in mTAL ( P<0.01). Increase in [Na+]i to 30, 50 and 70 mM resulted in no changes in Na+-K+-ATPase phosphorylation degree or activity in mTAL. Conversely, in PCT increments in [Na+]i were paralleled by decreased phosphorylation (from 120+/-7 to 160+/-15% of controls, P<0.05) and increased Na+-K+-ATPase activity (from 850+/-139 to 1,874+/-203 pmol/mm tubule per hour, P<0.01). Dopamine (DA) 10(-6) M decreased both Na+-K+-ATPase dephosphorylation to 41.85+/-9.58% ( P<0.05) and Na+-K+-ATPase activity to 2,405+/-176 pmol/mm tubule per hour in mTAL ( P<0.01). RO-318220 reversed DA effects. Data suggest that regulation of the degree of Na+-K+-ATPase alpha1 subunit phosphorylation at Ser-23 and enzyme activity have different mechanisms in mTAL than in PCT, and may help us to understand the physiological heterogeneity of both segments.

Previous reports have shown a stimulatory effect of vasopressin (VP) on Na-K-ATPase and rBSC-1 expression and activity. Whether these VP-dependent mechanisms are operating in vivo in physiological conditions as well as in chronic renal failure (CRF) has been less well studied. We measured ATPase expression and activity and rBSC-1 expression in the outer medulla of controls and moderate CRF rats both before and under in vivo inhibition of VP by OPC-31260, a selective V 2-receptor antagonist. OPC-31260 decreased Na-K-ATPase activity from 11.2 Ϯ 1.5 to 3.7 Ϯ 0.8 in controls (P Ͻ 0.05) and from 19.0 Ϯ 0.8 to 2.9 Ϯ 0.5 mol P i ⅐ mg protein Ϫ1 ⅐ h Ϫ1 in moderate CRF rats (P Ͻ 0.05). CRF was associated with a significant increase in Na-K-ATPase activity (P Ͻ 0.05). Similarly, CRF was also associated with a significant increase in Na-K-ATPase expression to 164.4 Ϯ 21.5% compared with controls (P Ͻ 0.05), and OPC-31260 decreased Na-K-ATPase expression in both controls and CRF rats to 57.6 Ϯ 9.5 and 105.3 Ϯ 10.9%, respectively (P Ͻ 0.05). On the other hand, OPC-31260 decreased rBSC-I expression in both controls and CRF rats to 60.8 Ϯ 6.5 and 30.0 Ϯ 6.9%, respectively (P Ͻ 0.05), and was not influenced by CRF (95.7 Ϯ 5.2%). We conclude that 1) endogenous VP modulated Na-K-ATPase and rBSC-1 in both controls and CRF; and 2) CRF was associated with increased activity and expression of the Na-K-ATPase in the outer medulla, in contrast to the unaltered expression of the rBSC-1. The data suggest that endogenous VP could participate in the regulation of electrolyte transport at the level of the outer medulla.vasopressin; rbsc-1; sodium-potassium-adenosine 5Ј-triphosphatase; chronic renal failure THERE IS STRONG EVIDENCE that vasopressin (VP) stimulates Na-K-ATPase and Na ϩ -K ϩ -Cl Ϫ cotransporter in the kidney outer medulla and, more precisely, in the medullary thick ascending limb of Henle (mTAL) (15). However, the experimental designs so far have been mostly related to the administration of exogenous VP, to either intact animals or in vitro preparations. The question of whether the level of endogenous VP activity plays a homeostatic role at the kidney outer medulla has not yet been examined. The introduction of powerful VP inhibitors has made it possible to design experiments looking at that question. OPC-31260 is a nonpeptide V 2 -receptor inhibitor developed by Yamamura et al. (21), and its actions are limited to states where VP activity is present. This approach was recently used by Ohara et al. (18), who were able to show a VP-mediated upregulation of the aquaporin-2 water channel in pregnant rats by using OPC-31260.In addition, previous experiments from our laboratory and those of others have suggested that in chronic renal failure the remaining nephrons suffer diverse adaptive mechanisms. Thus Bertuccio et al. (1, 2) showed an increase in intracellular cAMP levels in microdissected mTAL segments in chronic renal failure (CRF) rats, which were unresponsive to VP in vitro, opposed to baseline lower levels and a dose-respon...

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