Caveolin-1 and Dopamine-Mediated Internalization of NaKATPase in Human Renal Proximal Tubule Cells

Abstract

Abstract-In moderate sodium-replete states, dopamine 1-like receptors (D 1 R/D 5 R) are responsible for regulating Ͼ50% of renal sodium excretion. This is partly mediated by internalization and inactivation of NaKATPase, when associated with adapter protein 2. We used dopaminergic stimulation via fenoldopam (D 1 -like receptor agonist) to study the interaction among D 1 -like receptors, caveolin-1 (CAV1), and the G protein-coupled receptor kinase type 4 in cultured human renal proximal tubule cells (RPTCs). We compared 2 groups of RPTCs, 1 of cell lines that were isolated from normal subjects (nRPTCs) and a second group of cell lines that have D 1 -like receptors that are uncoupled (uncoupled RPTCs) from adenylyl cyclase second messengers. In nRPTCs, fenoldopam increased the plasma membrane expression of D 1 R (10.0-fold) and CAV1 (1.3-fold) and markedly decreased G protein-coupled receptor kinase type 4 by 94Ϯ8%; no effects were seen in uncoupled RPTCs. Fenoldopam also increased the association of adapter protein 2 and NaKATPase by 53Ϯ9% in nRPTCs but not in uncoupled RPTCs. When CAV1 expression was reduced by 86.0Ϯ8.5% using small interfering RNA, restimulation of the D 1 -like receptors with fenoldopam in nRPTCs resulted in only a 7Ϯ9% increase in association between adapter protein 2 and NaKATPase. Basal CAV1 expression and association with G protein-coupled receptor kinase type 4 was decreased in uncoupled RPTCs (58Ϯ5% decrease in association) relative to nRPTCs. We conclude that the scaffolding protein CAV1 is necessary for the association of D 1 -like receptors with G protein-coupled receptor kinase type 4 and the adapter protein 2-associated reduction in plasma membrane NaKATPase.

Inhibition of renal caveolin-1 reduces natriuresis and produces hypertension in sodium-loaded rats

“…Following the addition of both Na-K-ATPase and AP2 mouse monoclonal antibodies, goat anti-mouse IR Dye 800 secondary antibody was added, and the cells were imaged using an Odyssey infrared imaging system. The specificity of this immuno dot-blot method was previously verified by Western blot analysis (16).…”

“…Measurement of Na-K-ATPase internalization by association with AP2 was performed as previously reported (16). Immediately following the acute experimental conditions in protocols 1 and 2, the kidneys were excised, decapsulated, and snap-frozen in liquid nitrogen.…”

“…In numerous cell lines that were derived from kidney tissue from hypertensive subjects, the D 1 -like receptors have diminished agonist-induced recruitment to the plasma membrane and coupling to adenylyl cyclase. We compared these uncoupled cell lines to their normally coupled counterparts and recently reported that CAV1 and lipid rafts are necessary for the D 1 -like receptor-dependent reduction in Na ϩ transport in normally coupled RPTCs (16). Blocking CAV1 expression in D 1 -like receptor-uncoupled human RPTCs had no effect on Na-K-ATPase or Na ϩ transport.…”

Crosstalk between dopamine receptors and the Na+/K+-ATPase (Review)

“…Ouabain, as an NKA inhibitor, was previously shown to decrease D 1 receptor-induced NKA inhibition in a human PCT line, resulting in increased renal sodium reabsorption and eventual ouabain-induced hypertension (36). In human renal PCT cells, the scaffolding protein, caveolin-1, was revealed to be necessary for the association of D 1 -like receptors with G protein-coupled receptor kinase type 4 (GRK4) and the AP-2-associated reduction in plasma membrane NKA (37), among which the presence of gene variants at 3 exons in GRK4 is consistent with the known effects of GRK4 variants on uncoupling D 1 receptors from adenylate cyclase, demonstrating that GRK4 is a potential therapeutic target (38).…”

D1-like dopamine receptors downregulate Na+-K+-ATPase activity and increase cAMP production in the posterior gills of the blue crabCallinectes sapidus

“…In human lung epithelia, dopamine via D 1 -like dopamine receptors increases sodium transport by stimulating the rapid recruitment of Na ϩ -K ϩ -ATPase from cellular endosomes to the basolateral membrane (5). In the proximal and distal tubules of the mammalian kidney, however, dopamine decreases ion transport by acting on D 1 -like dopamine receptors to increase cAMP, which leads to the phosphorylation of Na ϩ -K ϩ -ATPase, resulting in its internalization and inactivation (2,4,9,11,22,26). Altered arachidonic metabolism may result in the failure of dopamine to inhibit Na ϩ -K ϩ -ATPase (28).…”