Laureano D. Asico

G protein-coupled receptor kinase 4 gene variants in human essential hypertension

Essential hypertension has a heritability as high as 30–50%, but its genetic cause(s) has not been determined despite intensive investigation. The renal dopaminergic system exerts a pivotal role in maintaining fluid and electrolyte balance and participates in the pathogenesis of genetic hypertension. In genetic hypertension, the ability of dopamine and D1-like agonists to increase urinary sodium excretion is impaired. A defective coupling between the D1 dopamine receptor and the G protein/effector enzyme complex in the proximal tubule of the kidney is the cause of the impaired renal dopaminergic action in genetic rodent and human essential hypertension. We now report that, in human essential hypertension, single nucleotide polymorphisms of a G protein-coupled receptor kinase, GRK4γ, increase G protein-coupled receptor kinase (GRK) activity and cause the serine phosphorylation and uncoupling of the D1 receptor from its G protein/effector enzyme complex in the renal proximal tubule and in transfected Chinese hamster ovary cells. Moreover, expressing GRK4γA142V but not the wild-type gene in transgenic mice produces hypertension and impairs the diuretic and natriuretic but not the hypotensive effects of D1-like agonist stimulation. These findings provide a mechanism for the D1 receptor coupling defect in the kidney and may explain the inability of the kidney to properly excrete sodium in genetic hypertension.

Disruption of the dopamine D3 receptor gene produces renin-dependent hypertension.

Since dopamine receptors are important in the regulation of renal and cardiovascular function, we studied the cardiovascular consequences of the disruption of the D3 receptor, a member of the family of D2-like receptors, expressed in renal proximal tubules and juxtaglomerular cells. Systolic and diastolic blood pressures were higher (approximately 20 mmHg) in heterozygous and homozygous than in wild-type mice. An acute saline load increased urine flow rate and sodium excretion to a similar extent in wild-type and heterozygous mice but the increase was attenuated in homozygous mice. Renal renin activity was much greater in homozygous than in wild-type mice; values for heterozygous mice were intermediate. Blockade of angiotensin II subtype-1 receptors decreased systolic blood pressure for a longer duration in mutant than in wild-type mice. Thus, disruption of the D3 receptor increases renal renin production and produces renal sodium retention and renin-dependent hypertension.

Perturbation of D1 Dopamine and AT1 Receptor Interaction in Spontaneously Hypertensive Rats

Abstract—The dopaminergic and renin-angiotensin systems interact to regulate blood pressure. Because this interaction may be perturbed in genetic hypertension, we studied D1 dopamine and AT1 angiotensin receptors in immortalized renal proximal tubule (RPT) and A10 aortic vascular smooth muscle cells. In normotensive Wistar-Kyoto (WKY) rats, the D1-like agonist fenoldopam increased D1 receptors but decreased AT1 receptors. These effects were blocked by the D1-like antagonist SCH 23390 (10−7 mol/L per 24 hours). In spontaneously hypertensive rat (SHR) RPT cells, fenoldopam also decreased AT1 receptors but no longer stimulated D1 receptor expression. Basal levels of AT1/D1 receptor coimmunoprecipitation were greater in WKY RPT cells (29±2 density units, DU) than in SHR RPT cells (21±2 DU, n=7 per group, P <0.05). The coimmunoprecipitation of D1 and AT1 receptors was increased by fenoldopam (10−7 mol/L per 24 hours) in WKY RPT cells but decreased in SHR RPT cells. The effects of fenoldopam in RPT cells from WKY rats were similar in aortic vascular smooth muscle cells from normotensive BD IX rats, that is, fenoldopam decreased AT1 receptors and increased D1 receptors. Our studies show differential regulation of the expression of D1 and AT1 receptors in RPT cells from WKY and SHR. This regulation and D1/AT1 receptor interaction are different in RPT cells of WKY and SHR. An altered interaction of D1 and AT1 receptors may play a role in the impaired sodium excretion and enhanced vasoconstriction in hypertension.

The dopaminergic system in hypertension

Dopamine plays an important role in the pathogenesis of hypertension by regulating epithelial sodium transport, vascular smooth muscle contractility and production of reactive oxygen species and by interacting with the renin-angiotensin and sympathetic nervous systems. Dopamine receptors are classified into D(1)-like (D(1) and D(5)) and D(2)-like (D(2), D(3) and D(4)) subtypes based on their structure and pharmacology. Each of the dopamine receptor subtypes participates in the regulation of blood pressure by mechanisms specific for the subtype. Some receptors regulate blood pressure by influencing the central and/or peripheral nervous system; others influence epithelial transport and regulate the secretion and receptors of several humoral agents. This review summarizes the physiology of the different dopamine receptors in the regulation of blood pressure, and the relationship between dopamine receptor subtypes and hypertension.

Activation of D3 Dopamine Receptor Decreases Angiotensin II Type 1 Receptor Expression in Rat Renal Proximal Tubule Cells

The dopaminergic and renin angiotensin systems interact to regulate blood pressure. Disruption of the D3 dopamine receptor gene in mice produces renin-dependent hypertension. In rats, D2-like receptors reduce angiotensin II binding sites in renal proximal tubules (RPTs). Because the major D2-like receptor in RPTs is the D3 receptor, we examined whether D3 receptors regulate angiotensin II type 1 (AT1) receptors in rat RPT cells. The effect of D3 receptors on AT1 receptors was studied in vitro and in vivo. The D3 receptor agonist PD128907 decreased AT1 receptor protein and mRNA in WKY RPT cells and increased it in SHR cells. PD128907 increased D3 receptors in WKY cells but had no effect in SHR cells. D3/AT1 receptors colocalized in RPT cells; D3 receptor stimulation decreased the percent amount of D3 receptors that coimmunoprecipitated with AT1 receptors to a greater extent in WKY than in SHR cells. However, D3 receptor stimulation did not change the percent amount of AT1 receptors that coimmunoprecipitated with D3 receptors in WKY cells and markedly decreased the coimmunoprecipitation in SHR cells. The D3 receptor also regulated the AT1 receptor in vivo because AT1 receptor expression was increased in kidneys of D3 receptor–null mice compared with wild type littermates. D3 receptors may regulate AT1 receptor function by direct interaction with and regulation of AT1 receptor expression. One mechanism of hypertension may be related to increased renal expression of AT1 receptors due decreased D3 receptor regulation.

Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells

Hypertension is a multigenic disorder in which abnormal counterregulation between dopamine and Ang II plays a role. Recent studies suggest that this counterregulation results, at least in part, from regulation of the expression of both the antihypertensive dopamine 5 receptor (D5R) and the prohypertensive Ang II type 1 receptor (AT1R). In this report, we investigated the in vivo and in vitro interaction between these GPCRs. Disruption of the gene encoding D5R in mice increased both blood pressure and AT1R protein expression, and the increase in blood pressure was reversed by AT1R blockade. Activation of D5R increased the degradation of glycosylated AT1R in proteasomes in HEK cells and human renal proximal tubule cells heterologously and endogenously expressing human AT1R and D5R. Confocal microscopy, Förster/fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy revealed that activation of D5R initiated ubiquitination of the glycosylated AT1R at the plasma membrane. The regulated degradation of AT1R via a ubiquitin/proteasome pathway by activation of D5R provides what we believe to be a novel mechanism whereby blood pressure can be regulated by the interaction of 2 counterregulatory GPCRs. Our results therefore suggest that treatments for hypertension might be optimized by designing compounds that can target the AT1R and the D5R.

Angiotensin II Regulation of AT1 and D3 Dopamine Receptors in Renal Proximal Tubule Cells of SHR

Abstract—Dopamine and angiotensin II negatively interact to regulate sodium excretion and blood pressure. D3 dopamine receptors downregulate angiotensin type 1 (AT1) receptors in renal proximal tubule cells from normotensive Wistar-Kyoto rats. We determined whether AT1 receptors regulate D3 receptors and whether the regulation is different in cultured renal proximal tubule cells from normotensive and spontaneously hypertensive rats. Angiotensin II (10−8M/24 hours) decreased D3 receptors in both normotensive (control, 36±3; angiotensin II, 24±3 U) and hypertensive (control, 30±3; angiotensin II, 11±3 U; n=9 per group) rats; effects that were blocked by the AT1 receptor antagonist, losartan (10−8M/24 hours). However, the reduction in D3 expression was greater in hypertensive (60±10%) than in normotensive rats (32±9%). In normotensive rats, angiotensin II (10−8M/24hr) also decreased AT1 receptors. In contrast, in cells from hypertensive rats, angiotensin II increased AT1 receptors. AT1 and D3 receptors co-immunoprecipitated in renal proximal tubule cells from both strains. Angiotensin II decreased D3/AT1 receptor co-immunoprecipitation similarly in both rat strains, but basal D3/AT1 co-immunoprecipitation was 6 times higher in normotensive than in hypertensive rats. Therefore, AT1 and D3 receptor interaction is qualitatively and quantitatively different between normotensive and hypertensive rats; angiotensin II decreases AT1 expression in normotensive but increases it in hypertensive rats. In addition, angiotensin II decreases D3 expression to a greater extent in hypertensive than in normotensive rats. Aberrant interactions between D3 and AT1 receptors may play a role in the pathogenesis of hypertension.

Adrenergic and Endothelin B Receptor-Dependent Hypertension in Dopamine Receptor Type-2 Knockout Mice

Polymorphism of the dopamine receptor type-2 (D2) gene is associated with essential hypertension. To assess whether D2 receptors participate in regulation of blood pressure (BP), we studied mice in which the D2 receptor was disrupted. In anesthetized mice, systolic and diastolic BPs (in millimeters of mercury) were higher in D2 homozygous and heterozygous mutant mice than in D2+/+ littermates. BP after &agr;-adrenergic blockade decreased to a greater extent in D2−/− mice than in D2+/+ mice. Epinephrine excretion was greater in D2−/− mice than in D2+/+ mice, and acute adrenalectomy decreased BP to a similar level in D2−/− and D2+/+ mice. An endothelin B (ET[B]) receptor blocker for both ET(B1) and ET(B2) receptors decreased, whereas a selective ET(B1) blocker increased, BP in D2−/− mice but not D2+/+ mice. ET(B) receptor expression was greater in D2−/− mice than in D2+/+ mice. In contrast, blockade of ET(A) and V1 vasopressin receptors had no effect on BP in either D2−/− or D2+/+ mice. The hypotensive effect of an AT1 antagonist was also similar in D2−/− and D2+/+ mice. Basal Na+,K+-ATPase activities in renal cortex and medulla were higher in D2+/+ mice than in D2−/− mice. Urine flow and sodium excretion were higher in D2−/− mice than in D2+/+ mice before and after acute saline loading. Thus, complete loss of the D2 receptor results in hypertension that is not due to impairment of sodium excretion. Instead, enhanced vascular reactivity in the D2 mutant mice may be caused by increased sympathetic and ET(B) receptor activities.

Extracellular vesicle-mediated transfer of donor genomic DNA to recipient cells is a novel mechanism for genetic influence between cells

Extracellular vesicles (EVs) carry signals within or at their limiting membranes, providing a mechanism by which cells can exchange more complex information than what was previously thought. In addition to mRNAs and microRNAs, there are DNA fragments in EVs. Solexa sequencing indicated the presence of at least 16434 genomic DNA (gDNA) fragments in the EVs from human plasma. Immunofluorescence study showed direct evidence that acridine orange-stained EV DNAs could be transferred into the cells and localize to and inside the nuclear membrane. However, whether the transferred EV DNAs are functional or not is not clear. We found that EV gDNAs could be homologously or heterologously transferred from donor cells to recipient cells, and increase gDNA-coding mRNA, protein expression, and function (e.g. AT1 receptor). An endogenous promoter of the AT1 receptor, NF-κB, could be recruited to the transferred DNAs in the nucleus, and increase the transcription of AT1 receptor in the recipient cells. Moreover, the transferred EV gDNAs have pathophysiological significance. BCR/ABL hybrid gene, involved in the pathogenesis of chronic myeloid leukemia, could be transferred from K562 EVs to HEK293 cells or neutrophils. Our present study shows that the gDNAs transferred from EVs to cells have physiological significance, not only to increase the gDNA-coding mRNA and protein levels, but also to influence function in recipient cells.

Effects of costimulation of dopamine D1- and D2-like receptors on renal function.

In vitro studies have suggested that dopamine D1- and D2-like receptors interact to inhibit renal sodium transport. We used Z-1046, a dopamine receptor agonist with the rank-order potency D3 >/= D4 > D2 > D5 > D1, to test the hypothesis that D1- and D2-like receptors interact to inhibit renal sodium transport in vivo in anesthetized rats. Increasing doses of Z-1046, administered via the right renal artery, increased renal blood flow (RBF), urine flow, and absolute and fractional sodium excretion without affecting glomerular filtration rate. For determination of the dopamine receptor involved in the renal functional effects of Z-1046, another group of rats received Z-1046 at 2 microgram . kg-1 . min-1 (n = 10) in the presence or absence of the D2-like receptor antagonist domperidone and/or the D1-like antagonist SCH-23390. Domperidone alone had no effect but blocked the Z-1046-mediated increase in urine flow and sodium excretion; it enhanced the increase in RBF after Z-1046. SCH-23390 by itself decreased urine flow and sodium excretion without affecting RBF and blocked the diuretic, natriuretic, and renal vasodilatory effect of Z-1046. We conclude that the renal vasodilatory effect of Z-1046 is D1-like receptor dependent, whereas the diuretic and natriuretic effects are both D1- and D2-like receptor dependent.In vitro studies have suggested that dopamine D1- and D2-like receptors interact to inhibit renal sodium transport. We used Z-1046, a dopamine receptor agonist with the rank-order potency D3 ≥ D4 > D2 > D5 > D1, to test the hypothesis that D1- and D2-like receptors interact to inhibit renal sodium transport in vivo in anesthetized rats. Increasing doses of Z-1046, administered via the right renal artery, increased renal blood flow (RBF), urine flow, and absolute and fractional sodium excretion without affecting glomerular filtration rate. For determination of the dopamine receptor involved in the renal functional effects of Z-1046, another group of rats received Z-1046 at 2 μg ⋅ kg-1 ⋅ min-1( n = 10) in the presence or absence of the D2-like receptor antagonist domperidone and/or the D1-like antagonist SCH-23390. Domperidone alone had no effect but blocked the Z-1046-mediated increase in urine flow and sodium excretion; it enhanced the increase in RBF after Z-1046. SCH-23390 by itself decreased urine flow and sodium excretion without affecting RBF and blocked the diuretic, natriuretic, and renal vasodilatory effect of Z-1046. We conclude that the renal vasodilatory effect of Z-1046 is D1-like receptor dependent, whereas the diuretic and natriuretic effects are both D1- and D2-like receptor dependent.