Interaction of Angiotensin II Type 1 and D ₅ Dopamine Receptors in Renal Proximal Tubule Cells

Abstract

Abstract-AngiotensinKey Words: receptors, dopamine Ⅲ receptors, angiotensin II Ⅲ rats, spontaneously hypertensive Ⅲ normotension Ⅲ kidney S everal cardiovascular diseases, including hypertension, are associated with abnormal regulation of sodium balance. This balance is regulated by natriuretic and antinatriuretic hormones and humoral agents. 1-8 Among the numerous factors involved in this process are angiotensin II and dopamine. During moderate volume expansion, renal dopamine production is increased, and dopamine, via D 1 -like (comprised of D 1 and D 5 subtypes) and D 2 -like receptors (comprised of D 2 , D 3 , and D 4 subtypes), acts to increase sodium excretion. 1,[5][6][7][8] In contrast, during salt deprivation, angiotensin II production is increased, and angiotensin II, via angiotensin II type 1 (AT 1 ) receptors, increases renal sodium transport. [2][3][4] Angiotensin II antagonizes the natriuretic response elicited by dopamine, and dopamine opposes angiotensin II-mediated sodium transport in the renal proximal tubules (RPTs). 9,10 Even a small increase in intracellular sodium concentration induces an increase in D 1 receptors and a decrease in AT 1 receptors in renal cell surface membranes. 11 Although the counter-regulation between dopamine and angiotensin II

Rat Strain Effects of AT 1 Receptor Activation on D 1 Dopamine Receptors in Immortalized Renal Proximal Tubule Cells

“…[21][22][23][24][25][26] The cells (80% confluence) were extracted in ice-cold lysis buffer (phosphate-buffered saline with 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L PMSF, 10 g/mL aprotinin, and 10 g/mL leupeptin), sonicated, kept on ice for 1 hour, and centrifuged at 16 000g for 30 minutes. The supernatants were stored at Ϫ70°C until use for immunoblotting and/or immunoprecipitation.…”

“…19,29 Rat RPT cells were treated with vehicle (dH 2 O), angiotensin II or an AT 1 receptor antagonist (losartan) at the indicated concentrations and times. Immunoblotting was performed as previously reported, 22,26 except that the transblots were probed with the D 1 (1:800) or the AT 1 receptor antibody (1:400). The amount of protein transferred onto the membranes was determined by Ponceau-S staining and immunoblotting for ␣-actin.…”

“…The immunofluorescence densities and images were acquired (Olympus AX70) at an excitation wavelength of 488 nm and 568 nm; emission was detected at 535 and 645 nm. 22,30 Immunoprecipitation RPT cells were incubated with vehicle or angiotensin II (10 Ϫ8 M) for 24 hours, as described. The cells were lysed with ice-cold lysis buffer for 1 hour and centrifuged at 16 000g for 30 minutes.…”

“…The densities of the bands were quantified by densitometry using Quantiscan (Ferguson, Mo) as previously reported. 19,22,26 Cell Surface D 1 Receptor Expression Cultured RPT cells were starved in serum-free medium for 2 hours, and then treated with angiotensin II (10 Ϫ8 M) for 15 minutes. Surface membrane proteins were biotinylated by adding sulfo-NHS-LCbiotin (final concentration 250 g/mL) into the medium 5 minutes before adding angiotensin II.…”

D3 dopamine receptor regulation of D5 receptor expression and function in renal proximal tubule cells

“…Immunoblotting was performed as previously reported, 20,23 except that the transblots were probed with the D 5 receptor (1 : 500; Research Genetics, Huntsville, AL, USA). The amino-acid sequence of the peptide for the rabbit anti-human D 5 receptor antibody corresponds to the third intracellular loop of the D 5 receptor.…”