Angiotensin II Regulation of AT ₁ and D ₃ Dopamine Receptors in Renal Proximal Tubule Cells of SHR

Abstract

Abstract-Dopamine and angiotensin II negatively interact to regulate sodium excretion and blood pressure. D 3 dopamine receptors downregulate angiotensin type 1 (AT 1 ) receptors in renal proximal tubule cells from normotensive Wistar-Kyoto rats. We determined whether AT 1 receptors regulate D 3 receptors and whether the regulation is different in cultured renal proximal tubule cells from normotensive and spontaneously hypertensive rats. Angiotensin II (10 Ϫ8M/24 hours) decreased D 3 receptors in both normotensive (control, 36Ϯ3; angiotensin II, 24Ϯ3 U) and hypertensive (control, 30Ϯ3; angiotensin II, 11Ϯ3 U; nϭ9 per group) rats; effects that were blocked by the AT 1 receptor antagonist, losartan (10 Ϫ8 M/24 hours). However, the reduction in D 3 expression was greater in hypertensive (60Ϯ10%) than in normotensive rats (32Ϯ9%). In normotensive rats, angiotensin II (10 Ϫ8 M/24hr) also decreased AT 1 receptors. In contrast, in cells from hypertensive rats, angiotensin II increased AT 1 receptors. AT 1 and D 3 receptors coimmunoprecipitated in renal proximal tubule cells from both strains. Angiotensin II decreased D 3 /AT 1 receptor co-immunoprecipitation similarly in both rat strains, but basal D 3 /AT 1 co-immunoprecipitation was 6 times higher in normotensive than in hypertensive rats. Therefore, AT 1 and D 3 receptor interaction is qualitatively and quantitatively different between normotensive and hypertensive rats; angiotensin II decreases AT 1 expression in normotensive but increases it in hypertensive rats. In addition, angiotensin II decreases D 3 expression to a greater extent in hypertensive than in normotensive rats. Aberrant interactions between D 3 and AT 1 receptors may play a role in the pathogenesis of hypertension. Key Words: receptors, dopamine Ⅲ receptors, angiotensin II Ⅲ rats, spontaneously hypertensive Ⅲ kidney T he proximal tubule is a major site of salt and water reabsorption in the mammalian nephron, with up to 60% of the filtrate reabsorbed in this segment. 1,2 Renal proximal tubule (RPT) function is under hormonal control, with angiotensin II stimulating sodium reabsorption via activation of apical Na ϩ /H ϩ exchanger and basolateral [Na ϩ ]/[K ϩ ]ATPase, and with dopamine inhibiting reabsorption by inhibiting these proteins. [3][4][5] The paracrine regulation of sodium reabsorption in the proximal tubule by the renin-angiotensin system occurs via several angiotensin receptor subtypes (AT 1 , AT 2 , and AT 4 ). [5][6][7][8] The activation of AT 1 receptors by angiotensin II increases sodium transport, whereas the activation of AT 2 and AT 4 receptors decreases sodium reabsorption in this nephron segment. [5][6][7][8] However, under physiological conditions, the major effect of angiotensin II on sodium transport is stimulatory, via AT 1 receptors. [5][6][7][8] The dopaminergic system also exerts a paracrine regulatory role on renal sodium transport in the proximal tubule via several receptor subtypes. Dopamine receptors, pharmacologically grouped into D 1 -like (D 1 and D 5 ) and D 2 -like...

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.…”

“…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.…”

“…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 densities of the bands were quantified by densitometry using Quantiscan (Ferguson, Mo) as previously reported. 19,22,26 Cell Surface D 1 …”

“…Our previous study showed that long-term exposure to angiotensin II decreases AT 1 receptor protein expression in RPT cells from WKY rats but increases it in SHRs. 26 The decreased interaction between D 1 and AT 1 receptors in renal proximal tubules in WKY rats is, therefore, not the result of decreased AT 1 receptor expression, per se, because a similar response is seen in SHRs where AT 1 expression is increased by angiotensin II. Thus, angiotensin II, via the AT 1 receptor, probably causes a decrease in the physical interaction of AT 1 and D 1 receptors in both WKY and SHR cells.…”