Dopamine‐mediated inhibition of renal Na⁺/K⁺‐ATPase in HK‐2 cells is reduced by ouabain

Abstract

1. Abnormal renal sodium handling is considered a major contributing factor in hypertension associated with chronic ouabain treatment. However, the molecular mechanisms involved in abnormal renal sodium handling have not been elucidated. Therefore, we investigated whether chronic ouabain treatment perturbs dopamine D(1) receptor function. 2. The expression and phosphorylation levels of the D(1) receptor in cells of the human proximal tubule cell line (HK-2) were determined using western blot analysis and reverse transcription polymerase chain reaction. The activity of the renal sodium/potassium pump (Na(+)/K(+)-ATPase) was measured using a colourimetric assay, and cyclic adenosine monophosphate accumulation was determined by performing a radioimmunoassay. 3. We showed that chronic ouabain treatment decreased the protein and mRNA expression levels of the D(1) receptor and increased the basal phosphorylation of the D(1) receptor in HK-2 cells. We also showed that in the presence of ouabain, HK-2 cells did not reveal the cyclic adenosine monophosphate accumulation and Na(+)/K(+)-ATPase inhibition induced by the D(1) receptor agonist fenoldopam. 4. We hypothesize that the ouabain-induced decrease in renal D(1) receptor function is responsible for the increase in renal sodium reabsorption, which eventually leads to ouabain-induced hypertension.

Human kidney-2 cells harbor functional dopamine D1 receptors that require Giαfor Gq/11αsignaling

“…4A). We found that D1R activation increases cAMP accumulation, which is in agreement with the study by Zhang and Yuan (30). In addition, D1-like receptor antagonist SCH23390 pretreatment attenuated the FD response on cAMP accumulation as well as reduced basal cAMP levels in HK2 cells.…”

“…Cell lines could mutate and may give rise to different study outcome. For example, the findings of Gildea et al (13) in HK2 cells related to the inability of FD to increase cAMP accumulation was not corroborated either by the findings of Zhang and Yuan (30) or by our present study (Fig. 4A).…”

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

“…Moreover, an increased intracellular sodium concentration induces the increased colocalization of DA receptors with NKA molecules in the region of the plasma membrane of renal epithelial cells (80). Ouabain, as a NKA inhibitor, decreased D 1 receptor-induced NKA inhibition in the human PCT cell line, which was responsible for the increase in renal sodium reabsorption and eventually led to ouabain-induced hypertension (36). This indicates that NKA inhibition is important in the regulation of DA receptor-induced sodium homeostasis and the subsequent regulation of blood pressure.…”

“…For example, DA produces a natriuresis, attributed, in part, to inhibition of NKA activity in PCT, and the impairment of this inhibition has been linked to several forms of hypertension in animals. These effects are largely mediated via D 1 or D 1 -like receptors (36) or by synergistic action of the D 1 and D 2 receptors (39) coupled to adenylyl cyclase activation and cAMP generation (40), as well as PLC/PKC signaling in rat renal PCT cells or Madin-Darby canine kidney epithelial cells (41,42). Dahl salt-sensitive rats lack the capacity to inhibit tubular NKA activity due to defective D 1 receptor adenylate cyclase coupling.…”

“…Furthermore, the DA-mediated regulation of NKA requires the PDZ-2 domain of sodium hydrogen regulatory factor-1 in OK cells (35). 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).…”