Supplementary MaterialsSupplemental Material. are necessary for D1R and AT2R trafficking. Both 8-Br-cAMP and 8-Br-cGMP activate protein phosphatase 2A (PP2A) which is necessary for both plasma membrane recruitment of D1R and AT2R and the inhibition of NHE3-dependent Na+ transport. MK-5046 These studies provide insights into the D1R/AT2R transregulation mechanisms that play a crucial role in maintaining Na+ and ultimately blood pressure homeostasis. strong class=”kwd-title” Keywords: Dopamine 1 receptor, Angiotensin 2 receptor, Protein Phosphatase 2A, Renal sodium transport Summary This study extends previous observations about the D1R and AT2R role in regulating Na+ transport by demonstrating in human RPTCs that there is an absolute co-dependency between D1R and AT2R to inhibit Na+ transport despite their differences in second messenger linkage. INTRODUCTION The renal dopaminergic and renin-angiotensin systems are major regulators of sodium (Na+) homeostasis1C4. Both dopamine D1-like receptors (D1R) and angiotensin II type-2 receptors (AT2R) are natriuretic receptors, as stimulation by either dopamine or angiotensin III (Ang III), respectively, inhibits renal Na+ reabsorption, resulting in natriuresis4,5. Upon agonist stimulation, D1Rs are recruited by microtubules from the cytoplasm to the apical cell surface6,7. Renal tubular plasma membrane D1Rs are further stimulated by dopamine synthesized in the renal proximal tubule cell (RPTC) that is released in a paracrine manner, increasing G protein coupling and a subsequent activation of adenylyl cyclase (AC) and protein kinase A (PKA)6,8. Renal angiotensin II (Ang II) is converted to Ang III by aminopeptidase A, which stimulates AT2Rs to decrease Na+ transport through an increase in soluble guanylyl cyclase (GC) production of cGMP which stimulates protein kinase G (PKG)9. (Figure 6). Open in MK-5046 a separate window Figure 6: Suggested Model of D1R MK-5046 and AT2R Recruitment to the Apical Plasma Membrane of Human Rabbit Polyclonal to Cytochrome P450 3A7 RPTCs.Fenoldopam (FEN), adenylyl cyclase (AC), protein kinase A (PKA), angiotensin III (Ang III), guanylyl cyclase (GC), protein kinase G (PKG), and okadaic acid (OA). Previous studies have not determined whether the D1R or AT2R acts independently or collectively to inhibit renal Na+ transport. In rodents, AT2Rs are recruited to the RPTC apical plasma membrane upon D1R stimulation and the AT2R antagonist, PD-123319 (PD), blocks the D1R-mediated natriuretic response, suggesting that AT2R is necessary for D1R activity10. In human RPTCs, we and others have reported that D1R and AT2R activation increases cAMP and cGMP, respectively, leading to a decrease in Na+ transport11C14, but the interdependence of D1R and AT2R stimulation on receptor translocation to the apical plasma membrane is unknown. The goal of this study is to investigate further the relationship between the D1R and AT2R in human RPTCs. We have reported that the resensitization of the desensitized, phosphorylated D1R is dependent on its dephosphorylation by protein phosphatase 2A (PP2A)15C18. The current study tests the hypothesis that downstream activation of either D1R or AT2R converges at PP2A which is necessary for both receptors to be translocated to the apical cell surface to inhibit Na+ transport. MATERIAL AND METHODS The authors declare that the data that support the findings of this study are available from the corresponding author upon reasonable request. Pharmacological agents All pharmacological agents were purchased from Sigma-Aldrich. Fenoldopam, (FEN) is a D1R agonist; it stimulates the two D1-like MK-5046 receptors, D1R and D5R. 7-Methyl-6,7,8,9,14,15-hexahydro-5H-benz[d]indolo[2,3-g]azecine (LE30019) is a potent and selective D1R antagonist20. H-Arg-Val-Tyr-Ile-His-Pro-Phe-OH, Angiotensin III (Ang III) is the preferred endogenous AT2R agonist21. 1-[[4-(Dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditrifluoroacetate, PD-123319 (PD) is a potent and selective non-peptide antagonist to the AT2R22. 8-Bromoadenosine cyclic 3,5-monophosphate (8-Br-cAMP) is a cell-permeable cAMP analog. 8-Bromoguanosine cyclic 3,5-monophosphate (8-Br-cGMP).