Renal sodium metabolism, a major determinant of blood pressure, is regulated with great precision by a variety of endocrine, autocrine, and neuronal factors. Although these factors are known to regulate sodium metabolism by affecting the rate of tubular sodium reabsorption, the molecular mechanisms by which they act are poorly understood. Na+,K(+)-ATPase plays a pivotal role for sodium reabsorption in all tubular segments. The activity of this enzyme can be dynamically regulated by phosphorylation and dephosphorylation. Here we summarize both old and new evidence that several major substances believed to be involved in the regulation of sodium metabolism and blood pressure, i.e., the antidiuretic agents angiotensin II and norepinephrine, and the diuretic agents dopamine and atrial natriuretic peptide (ANP), may achieve their effects through a common pathway that involves reversible activation/deactivation of renal tubular Na+,K(+)-ATPase. Regulation of Na+,K(+)-ATPase activity was studied using a preparation of single proximal tubule (PT) segments, dissected from rat kidneys. Na+,K(+)-ATPase activity was stimulated by angiotensin II and the alpha-adrenergic agonist, oxymetazoline, at physiological, nonsaturating Na+ concentrations. These stimulatory effects were blocked by dopamine and ANP as well as by their respective second messengers, cAMP and cGMP. They were also blocked by the specific protein phosphatase 2B inhibitor FK506. These results indicate that regulation of sodium excretion by norepinephrine, angiotensin II, dopamine, and ANP can be accounted for by a bidirectionally regulated intracellular protein phosphorylation cascade that modulates the activity of renal tubular Na+,K(+)-ATPase.

Sodium excretion is bidirectionally regulated by dopamine, acting on D1-like receptors (D1R) and angiotensin II, acting on AT1 receptors (AT1R). Since sodium excretion has to be regulated with great precision within a short frame of time, we tested the short-term effects of agonist binding on the function of the reciprocal receptor within the D1R-AT1R complex in renal proximal tubule cells. Exposure of rat renal proximal tubule cells to a D1 agonist was found to result in a rapid partial internalization of AT1R and complete abolishment of AT1R signaling. Similarly, exposure of rat proximal tubule cells and renal tissue to angiotensin II resulted in a rapid partial internalization of D1R and abolishment of D1R signaling. D1R and AT1R were, by use of coimmunoprecipitation studies and glutathione-S-transferase pull-down assays, shown to be partners in a multiprotein complex. Na ϩ -K ϩ -ATPase, the target for both receptors, was included in this complex, and a region in the COOH-terminal tail of D1R (residues 397-416) was found to interact with both AT1R and Na ϩ -K ϩ -ATPase. Results indicate that AT1R and D1R function as a unit of opposites, which should provide a highly versatile and sensitive system for short-term regulation of sodium excretion.AT 1 receptors; Na ϩ -K ϩ -ATPase; calcium signaling RENAL SODIUM EXCRETION IS bidirectionally regulated by angiotensin II (ANG II) and dopamine (13). Long-term dopamine exposure is known to decrease AT 1 receptors (AT1R) in renal proximal tubular cells (7). Furthermore, studies by Zeng et al. (21) have shown that long-term stimulation of AT1R results in an upregulation of D1-like receptors (D1R). This effect was not observed in spontaneously hypertensive rats, indicating that the interaction between AT1R and D1R has an impact on blood pressure regulation. Since sodium excretion must be regulated with great precision over a short period of time, it is important that control mechanisms are able to exert their effects within a short time frame. The aim of the current study has been to explore the short-term effects of ANG II exposure on D1R and the short-term effects of a D1-agonist on AT1R. Our approach has been to test the hypothesis that AT1R and D1R form a heteromeric signaling complex, where activation of either receptor may cause internalization and/or interruption of the signaling capacity of the other.The studies were performed using rat proximal tubule cells, since these cells express both AT1R and D1R in both the apical and the basolateral membrane (12,18). Previous studies from our laboratory have shown that in these cells Na ϩ -K ϩ -ATPase, the enzyme responsible for active sodium transport, is bidirectionally regulated by ANG II and dopamine (2). MATERIALS AND METHODS Cells and tissue.All studies were performed using outer cortical tissue from young (3-5 wk) male Sprague-Dawley rats. Immediately after the animals were killed, 250-m slices were taken from the outer renal cortex using a microtome. The outer 250-m region of the rat renal cortex contains Ͼ90% proxi...

There is a great deal of evidence for synergistic interactions between G protein-coupled signal transduction pathways in various tissues. As two specific examples, the potent effects of the biogenic amines norepinephrine and dopamine on sodium transporters and natriuresis can be modulated by neuropeptide Y and atrial natriuretic peptide, respectively. Here, we report, using a renal epithelial cell line, that both types of modulation involve recruitment of receptors from the interior of the cell to the plasma membrane. The results indicate that recruitment of G protein-coupled receptors may be a ubiquitous mechanism for receptor sensitization and may play a role in the modulation of signal transduction comparable to that of the well established phenomenon of receptor endocytosis and desensitization.There are many examples documenting that the cellular response to catecholamines and other small molecules can be enhanced by peptide hormones. In the nervous system, the efficacy of synaptic transmission is modulated both by peptide neurotransmitters and by biogenic amines (1, 2). In the kidney, the potent effects of the biogenic amines norepinephrine and dopamine (DA) on sodium transporters and natriuresis can be modulated by neuropeptide Y (NPY; refs. 3 and 4) and atrial natriuretic peptide (ANP; refs. 5-8), respectively. Little is known about the molecular basis for such heterologous sensitization. Here, we report that one cellular mechanism by which peptide hormones induce heterologous sensitization involves recruitment of catecholamine receptors from the interior of the cell to the plasma membrane. We show by the use of confocal microscopy and subcellular fractionation that the well established ability of ANP to potentiate the effects of DA and the ability of NPY to potentiate the effects of norepinephrine are attributable to recruitment of these two classes of receptors to the plasma membrane. MATERIALS AND METHODS Na؉ ,K ؉ -ATPase Activity in Single Proximal Tubular Segments. Renal proximal tubular segments were microdissected from a collagenase-treated rat kidney. Individual segments were incubated with the indicated drugs for 30 min at room temperature. Na ϩ ,K ϩ -ATPase activity was measured in single segments by ouabain-sensitive ATP hydrolysis (9). Assays were performed in the presence of 70 mM Na ϩ (V max conditions). This method permits direct measurements of Na ϩ ,K ϩ -ATPase activity in intact permeable cells, wherein effects of changes in Na ϩ influx are eliminated. Results were calculated relative to the length of the tubular segments.Antibodies. Na ϩ ,K ϩ -ATPase, a plasma membrane marker enzyme, was probed with a monoclonal antibody (a kind gift from M. Caplan, Yale University, New Haven, CT). D1 DA receptors were probed with affinity-purified rabbit anti-D1 rat DA receptor antibodies, which recognize the third extracellular loop of the receptor (a kind gift from R. M. Carey, University of Virginia, Charlottesville, VA; ref. 10). ␣ 1A -Adrenergic receptors were probed with affinity-purified...

The enzyme catechol-O-methyltransferase (COMT), which plays an important role for dopamine metabolism, is abundantly expressed in the kidney. To test whether the natriuretic effects of dopamine may be related to the rate of dopamine metabolism, rats were treated with nitecapone, a peripheral inhibitor of COMT. Nitecapone, given by gavage, induced a highly significant (5.6-fold) increase in sodium excretion, which was associated with an inhibition of the Na+,K+-ATPase activity in both the proximal convoluted and proximal straight tubules (PCT and PST, respectively). These effects were completely abolished if the rats were also treated with a specific dopamine 1 antagonist, SCH 23390. Furthermore, the natriuretic effect of nitecapone was also observed in rats on a high salt diet. The kidney-specific pro-drug to dopamine, glu-dopa, induced a significant, but less pronounced increase in urinary sodium excretion, associated with a dopamine-dependent inhibition of the Na+,K+-ATPase activity in the PCT but not in the PST. Nitecapone and glu-dopa had an additive natriuretic effect. It is concluded that COMT plays an important role in determining the natriuretic effects of the renal dopamine system.

There is a great need for treatment that arrests progression of chronic kidney disease. Increased albumin in primary urine leads to apoptosis and fibrosis of podocytes and tubular cells and is a major cause of functional deterioration. There have been many attempts to target fibrosis but few to target apoptosis, because of lack of appropriate agents. Our group has described an ouabain activated Na,K-ATPase/IP3R signalosome, which protects from apoptosis. Here we show that albumin uptake in primary rat renal epithelial cells is accompanied by a time and dose dependent mitochondrial accumulation of the apoptotic factor Bax, down-regulation of the anti-apoptotic factor Bcl-xL and mitochondrial membrane depolarization. Ouabain opposes these effects and protects from apoptoss in albumin-exposed proximal tubule cells and podocytes. The efficacy of ouabain as an anti-apoptotic and kidney-protective therapeutic tool is tested in rats with passive Heymann nephritis, a model of proteinuric chronic kidney disease. Chronic ouabain treatment preserves renal function, protects from renal cortical apoptosis, up-regulation of Bax, down-regulation of Bcl-xL and rescues from glomerular tubular disconnection and podocyte loss. Thus we have identified a novel clinically feasible therapeutic tool, which has the potential to protect from apoptosis and rescue from loss of functional tissue in chronic proteinuric kidney disease.

The molecular mechanisms underlying the regulation of sodium excretion are incompletely known. Here we propose a general model for a bi-directional control of tubular sodium transporters by natriuretic and antinatriuretic factors. The model is based on experimental data from studies on the regulation of the activity of Na+,K+-ATPase, the enzyme that provides the electrochemical gradient necessary for tubular reabsorption of electrolytes and solutes in all tubular segments. Regulation is carried out to a large extent by autocrine and paracrine factors. Of particular interest are the two catecholamines, dopamine and norepinephrine. Dopamine is produced in proximal tubular cells and inhibits Na+,K+-ATPase activity in several tubule segments. Renal dopamine availability is regulated by the degrading enzyme, catechol-O-methyl transferase. Renal sympathetic nerve endings contain norepinephrine and neuropeptide Y (NPY). Activation of alpha-adrenergic receptors increase and activation of beta-adrenergic receptors decrease Na+,K+-ATPase activity. alpha-Adrenergic stimulation increases the Na+ affinity of the enzyme and thereby the driving force for transcellular Na+ transport. NPY acts as a master hormone by synergizing the alpha- and antagonizing the beta-adrenergic effects. Dopamine and norepinephrine control Na+,K+-ATPase activity by exerting opposing forces on a common intracellular signaling system of second messengers, protein kinases and protein phosphatases, ultimately determining the phosphorylation state of Na+,K+-ATPase and thereby its activity. Important crossroads in this network are localized and functionally defined. Phosphorylation sites for protein kinase A and C have been identified and their functional significance has been verified.

No abstract

Renal dopamine1 receptor (D1R) can be recruited from intracellular compartments to the plasma membrane by D1R agonists and endogenous dopamine. This study examines the role of the cytoskeleton for renal D1R recruitment. The studies were performed in LLCPK-1 cells that have the capacity to form dopamine from L-dopa. In approximately 50% of the cells treated with L-dopa the D1R was found to be translocated from intracellular compartments towards the plasma membrane. Disruption of the microtubulin network by nocodazole significantly prevented translocation. In contrast, depolymerization of actin had no effect. In control cells D1R colocalized with NBD-C(6)-ceramide, a trans-Golgi fluorescent marker. This colocalization was disrupted in L-dopa-treated cells. Tetanus toxin, an inhibitor of exocytosis, prevented L-dopa-induced receptor recruitment. L-Dopa treatment resulted in activation of protein kinase C (PKC). To test the functional effect of D1R recruitment, the capacity of D1R agonists to activate PKC was studied. Activation of D1R significantly translocated PKC-alpha from intracellular compartments to the plasma membrane. Disruption of microtubules abolished D1R-mediated - but not phorbol-ester-mediated - translocation of PKC. We conclude that renal D1R recruitment requires an intact microtubulin network and occurs via Golgi-derived vesicles. These newly recruited receptors couple to the PKC signaling pathway.