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Nuclear factor erythyroid factor 2 (Nrf2) transcribes genes in cultured endothelial cells that reduce reactive oxygen species (ROS) and generate nitric oxide (NO) or metabolize asymmetric dimethylarginine (ADMA), which inhibits NO synthase (NOS). Therefore, we undertook a functional study to test the hypothesis that activation of Nrf2 by tert-butylhydroquinone (tBHQ) preserves microvascular endothelial function during oxidative stress. Wild-type CB57BL/6 (wt), Nrf2 wt (+/+), or knockout (-/-) mice received vehicle (Veh) or tBHQ (0.1%; activator of Nrf2) during 14-day infusions of ANG II (to induce oxidative stress) or sham. MAP was recorded by telemetry. Mesenteric resistance arterioles were studied on isometric myographs and vascular NO and ROS by fluorescence microscopy. ANG II increased the mean arterial pressure (112 ± 5 vs. 145 ± 5 mmHg; P < 0.01) and excretion of 8-isoprostane F 2α (2.8 ± 0.3 vs. 3.8 ± 0.3 ng/mg creatinine; P < 0.05) at 12-14 days. However, 12 days of ANG II reduced endothelium-derived relaxation (27 ± 5 vs. 17 ± 3%; P < 0.01) and NO (0.38 ± 0.07 vs. 0.18 ± 0.03 units; P < 0.01) but increased microvascular remodeling, endothelium-derived contractions (7.5 ± 0.5 vs. 13.0 ± 1.7%; P < 0.01), superoxide (0.09 ± 0.03 vs. 0.29 ± 0.08 units; P < 0.05), and contractions to U-46,619 (87 ± 6 vs. 118 ± 3%; P < 0.05), and endothelin-1(89 ± 4 vs. 123 ± 12%; P < 0.05). tBHQ prevented all of these effects of ANG II at 12-14 days in Nrf2 +/+ mice but not in Nrf2 -/- mice. In conclusion, tBHQ activates Nrf2 to prevent microvascular endothelial dysfunction, remodeling, and contractility, and moderate ADMA and hypertension at 12-14 days of ANG II infusion, thereby preserving endothelial function and preventing hypertension.

Dopamine decreases Na-K-ATPase (NKA) activity by PKC-dependent phosphorylation and endocytosis of the NKA α1. Dopamine-mediated regulation of NKA is impaired in aging and some forms of hypertension. Using opossum (OK) proximal tubule cells (PTCs), we demonstrated that sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) associates with NKA α1 and dopamine-1 receptor (D1R). This association is required for the dopamine-mediated regulation of NKA. In OK cells, dopamine decreases NHERF-1 association with NKA α1 but increases its association with D1R. However, it is not known whether NHERF-1 plays a role in dopamine-mediated NKA regulation in animal models of hypertension. We hypothesized that defective dopamine-mediated regulation of NKA results from the decrease in NHERF-1 expression in rat renal PTCs isolated from animal models of hypertension [spontaneously hypertensive rats (SHRs) and aged F344 rats]. To test this hypothesis, we isolated and cultured renal PTCs from 22-mo-old F344 rats and their controls, normotensive 4-mo-old F344 rats, and SHRs and their controls, normotensive Wistar-Kyoto (WKY) rats. The results demonstrate that in both hypertensive models (SHR and aged F344), NHERF-1 expression, dopamine-mediated phosphorylation of NKA, and ouabain-inhibitable K + transport are reduced. Transfection of NHERF-1 into PTCs from aged F344 and SHRs restored dopamine-mediated inhibition of NKA. These results suggest that decreased renal NHERF-1 expression contributes to the impaired dopamine-mediated inhibition of NKA in PTCs from animal models of hypertension.

Gastrin is a peptide hormone that is involved in the regulation of sodium balance and blood pressure. Dopamine, which is also involved in the regulation of sodium balance and blood pressure, directly or indirectly interacts with other blood pressure-regulating hormones, including gastrin. This study aimed to determine the mechanisms of the interaction between gastrin and dopamine and tested the hypothesis that gastrin produced in the kidney increases renal dopamine production to keep blood pressure within the normal range. We show that in human and mouse renal proximal tubule cells (hRPTCs and mRPTCs, respectively), gastrin stimulates renal dopamine production by increasing the cellular uptake of l-DOPA via the l-type amino acid transporter (LAT) at the plasma membrane. The uptake of l-DOPA in RPTCs from C57Bl/6J mice is lower than in RPTCs from normotensive humans. l-DOPA uptake in renal cortical slices is also lower in salt-sensitive C57Bl/6J than in salt-resistant BALB/c mice. The deficient renal cortical uptake of l-DOPA in C57Bl/6J mice may be due to decreased LAT-1 activity that is related to its decreased expression at the plasma membrane, relative to BALB/c mice. We also show that renal-selective silencing of Gast by the renal subcapsular injection of Gast siRNA in BALB/c mice decreases renal dopamine production and increases blood pressure. These results highlight the importance of renal gastrin in stimulating renal dopamine production, which may give a new perspective in the prevention and treatment of hypertension., (Copyright © 2017 the American Physiological Society.)

The natriuretic effect of gastrin suggests a role in the coordinated regulation of sodium balance by the gastrointestinal tract and the kidney. The renal molecular targets and signal transduction pathways for such an effect of gastrin are largely unknown. Recently, we reported that gastrin induces NHE3 phosphorylation and internalization via phosphatidylinositol (PI) 3-kinase and PKCα. In this study, we show that gastrin induced the phosphorylation of human Na(+),K(+)-ATPase at serine 16, resulting in its endocytosis via Rab5 and Rab7 endosomes. The gastrin-stimulated phosphorylation of Na(+),K(+)-ATPase was dependent on PI 3-kinase because the phosphorylation was blocked by the PI 3-kinase inhibitor wortmannin. The phosphorylation of Na(+),K(+)-ATPase was also blocked by chelerythrine, a pan-PKC inhibitor, Gö-6976, a conventional PKC (cPKC) inhibitor, and BAPTA-AM, an intracellular calcium chelator, suggesting the importance of cPKC and intracellular calcium in the gastrin signaling pathway. The gastrin-mediated phosphorylation of Na(+),K(+)-ATPase was also inhibited by U-73122, a phospholipase C (PLC) inhibitor. These results suggest that gastrin regulates sodium hydrogen exchanger and pump in renal proximal tubule cells at the apical and basolateral membranes., (Copyright © 2016 the American Physiological Society.)

Lack or downregulation of the dopamine D2 receptor (D2R) results in increased renal expression of injury markers and proinflammatory factors that is independent of a blood pressure increase. This study aimed to determine the mechanisms involved in the regulation of renal inflammation by D2Rs. Silencing D2Rs in mouse renal proximal tubule cells increased the expression of the proinflammatory TNF-α, monocyte chemoattractant protein-1 (MCP-1), and IL-6. D2R downregulation also increased Akt phosphorylation and activity, and glycogen synthase kinase-3β (GSK3β) phosphorylation and cyclin D1 expression, downstream targets of Akt; however. phosphatidylinositol 3-kinase (PI3K) activity was not affected. Conversely, D2R stimulation decreased Akt and GSK3β phosphorylation and cyclin D1 expression. Increased phospho-Akt, in the absence of increased PI3K activity, may result from decreased Akt dephosphorylation. Inhibition of protein phosphatase 2A (PP2A) with okadaic acid reproduced the effects of D2R downregulation on Akt, GSK3β, and cyclin D1. The PP2A catalytic subunit and regulatory subunit PPP2R2C coimmunoprecipitated with the D2R. Basal phosphatase activity and the expression of PPP2R2C were decreased by D2R silencing that also blunted the increase in phosphatase activity induced by D2R stimulation. Similarly, silencing PPP2R2C also increased the phosphorylation of Akt and GSK3β. Moreover, downregulation of PPP2R2C resulted in increased expression of TNF-α, MCP-1, and IL-6, indicating that decreased phosphatase activity may be responsible for the D2R effect on inflammatory factors. Indeed, the increase in NF-κB reporter activity induced by D2R silencing was blunted by increasing PP2A activity with protamine. Our results show that D2R controls renal inflammation, at least in part, by modulation of the Akt pathway through effects on PP2A activity/expression., (Copyright © 2016 the American Physiological Society.)

The electrogenic sodium bicarbonate cotransporter (NBCe2) is encoded by SLC4A5, variants of which have been associated with salt sensitivity of blood pressure, which affects 25% of the adult population. NBCe2 is thought to mediate sodium bicarbonate cotransport primarily in the renal collecting duct, but NBCe2 mRNA is also found in the rodent renal proximal tubule (RPT). The protein expression or function of NBCe2 has not been demonstrated in the human RPT. We validated an NBCe2 antibody by shRNA and Western blot analysis, as well as overexpression of an epitope-tagged NBCe2 construct in both RPT cells (RPTCs) and human embryonic kidney 293 (HEK293) cells. Using this validated NBCe2 antibody, we found NBCe2 protein expression in the RPT of fresh and frozen human kidney slices, RPTCs isolated from human urine, and isolated RPTC apical membrane. Under basal conditions, NBCe2 was primarily found in the Golgi, while NBCe1 was primarily found at the basolateral membrane. Following an acute short-term increase in intracellular sodium, NBCe2 expression was increased at the apical membrane in cultured slices of human kidney and polarized, immortalized RPTCs. Sodium bicarbonate transport was increased by monensin and overexpression of NBCe2, decreased by NBCe2 shRNA, but not by NBCe1 shRNA, and blocked by 2,2'-(1,2-ethenediyl)bis[5-isothiocyanato-benzenesulfonic acid]. NBCe2 could be important in apical sodium and bicarbonate cotransport under high-salt conditions; the implication of the ex vivo studies to the in vivo situation when salt intake is increased remains unclear. Therefore, future studies will examine the role of NBCe2 in mediating increased renal sodium transport in humans whose blood pressures are elevated by an increase in sodium intake., (Copyright © 2015 the American Physiological Society.)

The homeostatic control of blood pressure hinges upon the delicate balance between prohypertensinogenic and antihypertensinogenic systems. D₁-like dopamine receptors [dopamine D₁ and D₅ receptors (D₁Rs and D₅Rs, respectively)] and the α₁A-adrenergic receptor (α₁A-AR) are expressed in the renal proximal tubule and engender opposing effects on Na(+) transport, i.e., natriuresis (via D₁Rs and D5Rs) or antinatriuresis (via α₁A-ARs). We tested the hypothesis that the D₁R/D₅R regulates the α₁A-AR. D₁-like dopamine receptors coimmunoprecipitated, colocalized, and cofractionated with α₁A-ARs in lipid rafts in immortalized human renal proximal tubule cells. Long-term treatment with the D₁R/D₅R agonist fenoldopam resulted in decreased D₁R and D₅R expression but increased α₁A-AR abundance in the plasma membrane. Short-term fenoldopam treatment stimulated the translocation of Na(+)-K(+)-ATPase from the plasma membrane to the cytosol that was partially reversed by an α₁A-AR agonist, which by itself induced Na(+)-K(+)-ATPase translocation from the cytosol to the plasma membrane. The α₁A-AR-specific agonist A610603 also minimized the ability of fenoldopam to inhibit Na(+)-K(+)-ATPase activity. To determine the interaction among D₁Rs, D₅Rs, and α₁A-ARs in vivo, we used phenylephrine and A610603 to decrease Na(+) excretion in several D1-like dopamine receptor knockout mouse strains. Phenylephrine and A61603 treatment resulted in a partial reduction of urinary Na(+) excretion in wild-type mice and its abolition in D1R knockout, D₅R knockout, and D₁R-D₅R double-knockout mice. Our results demonstrate the ability of the D₁-like dopamine receptors to regulate the expression and activity of α₁A-AR. Elucidating the intricacies of the interaction among these receptors is crucial for a better understanding of the crosstalk between anti- and pro-hypertensive systems.

Dopamine-mediated regulation of Na(+)-K(+)-ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-like receptor is expressed in the blue crab Callinectes sapidus and regulates Na(+)-K(+)-ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1βR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1βR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na(+)-K(+)-ATPase and D1βR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na(+)-K(+)-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na(+)-K(+)-ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na(+)-K(+)-ATPase activity by itself. Fenoldopam minimally decreased D1βR protein expression (10%) but did not affect Na(+)-K(+)-ATPase α-subunit protein expression. This study shows the presence of functional D1βR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis., (Copyright © 2014 the American Physiological Society.)

The dopaminergic and sympathetic systems interact to regulate blood pressure. Our previous studies showed regulation of α1-adrenergic receptor function by D1-like dopamine receptors in vascular smooth muscle cells. Because renalase could regulate circulating epinephrine levels and dopamine production in renal proximal tubules (RPTs), we tested the hypothesis that D1-like receptors regulate renalase expression in kidney. The effect of D1-like receptor stimulation on renalase expression and function was measured in immortalized RPT cells from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs). We found that the D1-like receptor agonist fenoldopam (10(-7)-10(-5) mol/l) increased renalase protein expression and function in WKY RPT cells but decreased them in SHR cells. Fenoldopam also increased renalase mRNA levels in WKY but not in SHR cells. In contrast, fenoldopam increased the degradation of renalase protein in SHR cells but not in WKY cells. The regulation of renalase by the D1-like receptor was mainly via the D5 receptor because silencing of the D5 but not D1 receptor by antisense oligonucleotides blocked the stimulatory effect of the D1-like receptor on renalase expression in WKY cells. Moreover, inhibition of PKC, by the PKC inhibitor 19-31, blocked the stimulatory effect of fenoldopam on renalase expression while stimulation of PKC, by a PKC agonist (PMA), increased renalase expression, indicating that PKC is involved in the process. Our studies suggest that the D5 receptor positively regulates renalase expression in WKY but not SHR RPT cells; aberrant regulation of renalase by the D5 receptor may be involved in the pathogenesis of hypertension.

Intraperitoneal injection of all-trans-retinoic acid (ATRA) results in a reduction of blood pressure in spontaneously hypertensive rats. However, the mechanisms involved in this effect are not clear. We hypothesized that ATRA may relax resistance arteries. In this study, we found that ATRA relaxed phenylephrine-preconstricted mesenteric arterial rings, which were abrogated by the removal of the endothelium. Pretreatment of endothelium-intact arterial rings with an inhibitor of endothelial nitric oxide (NO) synthase, N(G)-nitro-l-arginine methyl ester (l-NAME), or soluble guanylyl cyclase, 1H-[1,2,4]-oxadiazole-[4,3-α]-quinoxaline-1-one, reduced the vasorelaxant effect of ATRA. Incubation of mesenteric arterial rings with ATRA increased the production of NO and cGMP, which were blocked by N(G)-nitro-l-arginine methyl ester. The vasorelaxant effect of ATRA was markedly attenuated in the presence of an inhibitor of big conductance calcium-activated potassium channels (charybdotoxin), but not with an inhibitor of voltage-dependent potassium channel (4-aminopyridine) or ATP-sensitive potassium channel (glibenclamide). Activation of retinoic acid receptors (RARs) with CH55 or retinoic X receptors (RXRs) with LGD1069 induced the vasorelaxation of phenylephrine-preconstricted mesenteric arterial rings. The RAR (BMS493) and RXR (UVI3003) antagonists blocked the ATRA-induced vasorelaxation. The vasorelaxant effect ATRA is physiologically relevant because the intravenous infusion of ATRA decreased blood pressure in normotensive rats. We conclude that ATRA relaxes resistance vessels via both RARs and RXRs receptors that are mediated by the endothelium-dependent NO-cGMP pathway, which may participate in the control of blood pressure.

We tested the hypothesis that reactive oxygen species (ROS) contributed to renal hypoxia in C57BL/6 mice with &frac56; surgical reduction of renal mass (RRM). ROS can activate the mitochondrial uncoupling protein 2 (UCP-2) and increase O(2) usage. However, UCP-2 can be inactivated by glutathionylation. Mice were fed normal (NS)- or high-salt (HS) diets, and HS mice received the antioxidant drug tempol or vehicle for 3 mo. Since salt intake did not affect the tubular Na(+) transport per O(2) consumed (T(Na/)Q(O2)), further studies were confined to HS mice. RRM mice had increased excretion of 8-isoprostane F(2α) and H(2)O(2), renal expression of UCP-2 and renal O(2) extraction, and reduced T(Na/)Q(O2) (sham: 20 ± 2 vs. RRM: 10 ± 1 μmol/μmol; P < 0.05) and cortical Po(2) (sham: 43 ± 2, RRM: 29 ± 2 mmHg; P < 0.02). Tempol normalized all these parameters while further increasing compensatory renal growth and glomerular volume. RRM mice had preserved blood pressure, glomeruli, and patchy tubulointerstitial fibrosis. The patterns of protein expression in the renal cortex suggested that RRM kidneys had increased ROS from upregulated p22(phox), NOX-2, and -4 and that ROS-dependent increases in UCP-2 led to hypoxia that activated transforming growth factor-β whereas erythroid-related factor 2 (Nrf-2), glutathione peroxidase-1, and glutathione-S-transferase mu-1 were upregulated independently of ROS. We conclude that RRM activated distinct processes: a ROS-dependent activation of UCP-2 leading to inefficient renal O(2) usage and cortical hypoxia that was offset by Nrf-2-dependent glutathionylation. Thus hypoxia in RRM may be the outcome of NADPH oxidase-initiated ROS generation, leading to mitochondrial uncoupling counteracted by defense pathways coordinated by Nrf-2.

Response gene to complement 32 (RGC-32) is activated by transforming growth factor- β (TGF-β) and plays an important role in smooth muscle cell (SMC) differentiation from neural crest Monc-1 cells. The molecular mechanism governing TGF-β activation of RGC-32, however, remains to be determined. The present studies indicate that TGF-β regulates RGC-32 gene transcription. Sequence analysis revealed a Smad binding element (SBE) located in the region from -1344 to -1337 bp upstream of the transcription start site of RGC-32 gene. A polyomavirus enhancer activator (PEA3) binding site is adjacent to the SBE. Mutation at either SBE or PEA3 site significantly inhibited RGC-32 promoter activity. Mutations at both sites completely abolished TGF-β-induced promoter activity. Biochemically, TGF-β stimulated recruitment of Smad2, Smad4, and PEA3 to the RGC-32 promoter, as revealed by gel shift and chromatin immunoprecipitation analyses. Functionally, Smad2, but not Smad3, activated RGC-32 promoter. PEA3 appeared to enhance Smad2 activity. In agreement with their function, Smad2, but not Smad3, physically interacted with PEA3. In TGF-β-induced SMC differentiation of Monc-1 cells, knockdown of Smad2 by short hairpin RNA resulted in downregulation of RGC-32 and SMC marker genes. The downregulation of SMC markers, however, was rescued by exogenously introduced RGC-32. These results demonstrate that Smad2 regulation of RGC-32 transcription is essential for SMC differentiation from neural crest cells.

Previous studies demonstrate that response gene to complement 32 (RGC-32) mediates transforming growth factor-β(1)-induced epithelial-mesenchymal transition (EMT) of human renal proximal tubular cells. However, the mechanisms underlying RGC-32 function remain largely unknown. In the present study, we found that RGC-32 function in EMT is associated with Smad3. Coexpression of RGC-32 and Smad3, but not Smad2, induces a higher mesenchymal marker α-smooth muscle actin (α-SMA) protein expression as compared with RGC-32 or Smad3 alone, while knockdown of Smad3 using short hairpin interfering RNA blocks RGC-32-induced α-SMA expression. These data suggest that RGC-32 interacts with Smad3, but not Smad2, in the regulation of EMT. In addition to α-SMA, RGC-32 and Smad3 also synergistically activate the expression of extracellular matrix protein fibronectin and downregulate the epithelial marker E-cadherin. RGC-32 colocalizes with Smad3 in the nuclei of renal proximal tubular cells. Coimmunoprecipitation assays showed that Smad3, but not Smad2, physically interacts with RGC-32 in renal proximal tubular cells. Mechanistically, RGC-32 and Smad3 coordinate the induction of EMT by regulating the EMT regulators Slug and Snail. Taken together, our data demonstrate for the first time that RGC-32 interacts with Smad3 to mediate the EMT of human renal proximal tubular cells.

Dopamine produced by renal proximal tubules increases sodium excretion via a decrease in renal sodium reabsorption. Dopamine natriuresis is impaired in obese Zucker rats; however, the mechanism is not fully understood. To test the hypothesis that renal expression of one or more of the subtypes are altered in these rats, we measured whole kidney protein levels by immunoblotting of D1-like (D1R and D5R) and D2-like (D2R, D3R, and D4R) dopamine receptors in both male and female obese and lean Zucker rats. In obese males on 1% NaCl diet, D1R, D2R, D4R, and D5R were decreased, while D3R was increased, relative to lean rats. Under a 4% NaCl diet, D2R and D3R levels in obese rats were restored to lean levels. 4% NaCl diet reduced D5R in both body types, relative to 1% NaCl diet. Female rats had higher expression of D1R and D3R than did male; however, the sex difference for D1R was markedly blunted in obese rats. In obese rats, dietary candesartan (angiotensin II type 1 receptor blocker) normalized downregulated D1R and D2R, but either decreased (D3R), did not affect (D4R), or further downregulated (D5R) the other subtypes. Candesartan also decreased D4R in lean rats. In summary, reduced renal protein levels of D1R, D2R, D4R, and D5R in obese Zucker rats could induce salt sensitivity and elevate blood pressure. Increased angiotensin II type 1 receptor activity may be mechanistically involved in the decreased expression of D1R and D2R in obese rats. Finally, reduced D1R and D3R in male rats may contribute to sex differences in blood pressure.

The C57Bl/6J mouse strain, the genetic background of many transgenic and gene knockout models, is salt sensitive and resistant to renal injury. We tested the hypothesis that renal dopaminergic function is defective in C57Bl/6J mice. On normal NaCl (0.8%, 1 wk) diet, anesthetized and conscious (telemetry) blood pressures were similar in C57Bl/6J and SJL/J mice. High NaCl (6%, 1 wk) increased blood pressure (approximately 30%) in C57Bl/6J but not in SJL/J mice and urinary dopamine to greater extent in SJL/J than in C57Bl/6J mice. Absolute and fractional sodium excretions were lower in SJL/J than in C57Bl/6J mice. The blood pressure-natriuresis plot was shifted to the right in C57Bl/6J mice. Renal expressions of D(1)-like (D(1)R and D(5)R) and angiotensin II AT(1) receptors were similar on normal salt, but high salt increased D(5)R only in C57Bl/6J. GRK4 expression was lower on normal but higher on high salt in C57Bl/6J than in SJL/J mice. Salt increased the excretion of microalbumin and 8-isoprostane (oxidative stress marker) and the degree of renal injury to a greater extent in SJL/J than in C57Bl/6J mice. A D(1)-like receptor agonist increased sodium excretion whereas a D(1)-like receptor antagonist decreased sodium excretion in SJL/J but not in C57Bl/6J mice. In contrast, parathyroid hormone had a similar natriuretic effect in both strains. These results show that defective D(1)-like receptor function is a major cause of salt sensitivity in C57Bl/6J mice, decreased renal dopamine production might also contribute. The relative resistance to renal injury of C57Bl/6J may be a consequence of decreased production of reactive oxygen species.

It has been reported that mechanical strain activates extracellular signal-regulated protein kinases (ERK) without the involvement of angiotensin II (Ang II) in cardiomyocytes. We examined the effects of mechanical strain on ERK phosphorylation levels in the absence of Ang II using rat mesangial cells. The ratio of phosphorylated ERK (p-ERK) to total ERK expression was increased by cyclic mechanical strain in a time- and elongation strength-dependent manner. With olmesartan [Ang II type 1 receptor (AT1R) antagonist] pretreatment, p-ERK plateau levels decreased in a dose-dependent manner (EC(50) = 1.3 x 10(-8) M, maximal inhibition 50.6 +/- 11.0% at 10(-5) M); a similar effect was observed with RNA interference against Ang II type 1A receptor (AT(1A)R) and Tempol, a superoxide dismutase mimetic. In addition to the inhibition of p-ERK levels, olmesartan blocked the increase in cell surface and phosphorylated p47(phox) induced by mechanical strain and also lowered the mRNA expression levels of NADPH oxidase subunits. These results demonstrate that mechanical strain stimulates AT1R to phosphorylate ERK in mesangial cells in the absence of Ang II. This mechanotransduction mechanism is involved in the oxidative stress caused by NADPH oxidase and is blocked by olmesartan. The inverse agonistic activity of this AT1R blocker may be useful for the prevention of mesangial proliferation and renal damage caused by mechanical strain/oxidative stress regardless of circulating or tissue Ang II levels.

The sympathetic nervous system plays an important role in the regulation of blood pressure. There is increasing evidence for positive and negative interactions between dopamine and adrenergic receptors; the activation of the alpha-adrenergic receptor induces vasoconstriction, whereas the activation of dopamine receptor induces vasorelaxation. We hypothesize that the D1-like receptor and/or D3 receptor also inhibit alpha1-adrenergic receptor-mediated proliferation in vascular smooth muscle cells (VSMCs). In this study, VSMC proliferation was determined by measuring [3H]thymidine incorporation, cell number, and uptake of 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT). Norepinephrine increased VSMC number and MTT uptake, as well as [3H]thymidine incorporation via the alpha1-adrenergic receptor in aortic VSMCs from Sprague-Dawley rats. The proliferative effects of norepinephrine were attenuated by the activation of D1-like receptors or D3 receptors, although a D1-like receptor agonist, fenoldopam, and a D3 receptor agonist, PD-128907, by themselves, at low concentrations, had no effect on VSMC proliferation. Simultaneous stimulation of both D1-like and D3 receptors had an additive inhibitory effect. The inhibitory effect of D3 receptor was via protein kinase A, whereas the D1-like receptor effect was via protein kinase C-zeta. The interaction between alpha1-adrenergic and dopamine receptors, especially D1-like and D3 receptors in VSMCs, could be involved in the pathogenesis of hypertension.

Dopamine plays an important role in the pathogenesis of hypertension by regulating epithelial sodium transport and by interacting with vasoactive hormones/humoral factors, such as aldosterone, angiotensin, catecholamines, endothelin, oxytocin, prolactin pro-opiomelancortin, reactive oxygen species, renin, and vasopressin. 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. In recent years, mice deficient in one or more of the five dopamine receptor subtypes have been generated, leading to a better understanding of the physiological role of each of the dopamine receptor subtypes. This review summarizes the results from studies of various dopamine receptor mutant mice on the role of individual dopamine receptor subtypes and their interactions with other G protein-coupled receptors in the regulation of blood pressure.

This study examined the inward transport of l-[(14)C]alanine, an ASCT2 preferential substrate, in monolayers of immortalized renal proximal tubular epithelial (PTE) cells from Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. The expression of ASCT2 in WKY and SHR PTE cells and kidney cortices from WKY and SHR was also evaluated. l-[(14)C]alanine uptake was highly dependent on extracellular Na(+). Replacement of NaCl by LiCl or choline chloride abolished transport activity in SHR and WKY PTE cells. In the presence of the system L inhibitor BCH, Na(+)-dependent l-alanine uptake in WKY and SHR PTE cells was inhibited by alanine, serine, and cysteine, which is consistent with amino acid transport through ASCT2. The saturable component of Na(+)-dependent l-alanine transport under V(max) conditions in SHR PTE cells was one-half of that in WKY PTE cells, with similar K(m) values. Differences in magnitude of Na(+)-dependent l-alanine uptake through ASCT2 between WKY and SHR PTE cells correlated positively with differences in ASCT2 protein expression, this being more abundant in WKY PTE cells. Abundance of ASCT2 transcript and protein in kidney cortices of SHR rats was also lower than that in normotensive WKY rats. In conclusion, immortalized SHR and WKY PTE cells take up l-alanine mainly through a high-affinity Na(+)-dependent amino acid transporter, with functional features of ASCT2 transport. The activity and expression of the ASCT2 transporter were considerably lower in the SHR cells.

G protein-coupled receptor (GPCR) kinases (GRKs) regulate the sensitivity of GPCRs, including dopamine receptors. The GRK4 locus is linked to, and some of its polymorphisms are associated with, human essential hypertension. Transgenic mice overexpressing human (h) GRK4gamma A142V on a mixed genetic background (C57BL/6J and SJL/J) have impaired renal D(1)-dopamine receptor (D(1)R) function and increased blood pressure. We now report that hGRK4gamma A142V transgenic mice, in C57BL/6J background, are hypertensive and have higher blood pressures than hGRK4gamma wild-type transgenic and nontransgenic mice. The hypertensive phenotype is stable because blood pressures in transgenic founders and F6 offspring are similarly increased. To determine whether the hypertension is associated with increased production of reactive oxygen species (ROS), we measured renal NADPH oxidase (Nox2 and Nox4) and heme oxygenase (HO-1 and HO-2) protein expressions and urinary excretion of 8-isoprostane and compared the effect of Tempol on blood pressure in hGRK4gamma A142V transgenic mice and D(5)R knockout (D(5)(-/-)) mice in which hypertension is mediated by increased ROS. The expressions of Nox isoforms and HO-2 and the urinary excretion of 8-isoprostane were similar in hGRK4gamma A142V transgenic mice and their controls. HO-1 expression was increased in hGRK4gamma A142V relative to hGRK4gamma wild-type transgenic mice. In contrast with the hypotensive effect of Tempol in D(5)(-/-) mice, it had no effect in hGRK4gamma A142V transgenic mice. We conclude that the elevated blood pressure of hGRK4gamma A142V transgenic mice is due mainly to the effect of hGRK4gamma A142V transgene acting via D(1)R and increased ROS production is not a contributor.

Activation of D1-like receptors (D1 and/or D5) induces antioxidant responses; however, the mechanism(s) involved in their antioxidant actions are not known. We hypothesized that stimulation of the D5 receptor inhibits NADPH oxidase activity, and thus the production of reactive oxygen species (ROS). We investigated this issue in D5 receptor-deficient (D5-/-) and wild-type (D5+/+) mice. NADPH oxidase protein expression (gp91(phox), p47(phox), and Nox 4) and activity in kidney and brain, as well as plasma thiobarbituric acid-reactive substances (TBARS) were higher in D5-/- than in D5+/+ mice. Furthermore, apocynin, an NADPH oxidase inhibitor, normalized blood pressure, renal NADPH oxidase activity, and plasma TBARS in D5-/- mice. In HEK-293 cells that heterologously expressed human D5 receptor, its agonist fenoldopam decreased NADPH oxidase activity, expression of one of its subunits (gp91(phox)), and ROS production. The inhibitory effect of the D5 receptor activation on NADPH oxidase activity was independent of cAMP/PKA but was partially dependent on phospholipase D2. The ability of D5 receptor stimulation to decrease ROS production may explain, in part, the antihypertensive action of D5 receptor activation.

D(1)-like receptors have been reported to decrease oxidative stress in vascular smooth muscle cells by decreasing phospholipase D (PLD) activity. However, the PLD isoform regulated by D(1)-like receptors (D(1) or D(5)) and whether abnormal regulation of PLD by D(1)-like receptors plays a role in the pathogenesis of hypertension are unknown. The hypothesis that the D(5) receptor is the D(1)-like receptor that inhibits PLD activity and serves to regulate blood pressure was tested using D(5) receptor mutant mice (D(5)(-/-)). We found that in the mouse kidney, PLD2, like the D(5) receptor, is mainly expressed in renal brush-border membranes, whereas PLD1 is mainly expressed in renal vessels with faint staining in brush-border membranes and collecting ducts. Total renal PLD activity is increased in D(5)(-/-) mice relative to congenic D(5) wild-type (D(5)(+/+)) mice. PLD2, but not PLD1, expression is greater in D(5)(-/-) than in D(5)(+/+) mice. The D(5) receptor agonist fenoldopam decreases PLD2, but not PLD1, expression and activity in human embryonic kidney-293 cells heterologously expressing the human D(5) receptor, effects that are blocked by the D(5) receptor antagonist SCH-23390. These studies show that the D(5) receptor regulates PLD2 activity and expression. The hypertension in the D(5)(-/-) mice is associated with increased PLD expression and activity. Impaired D(5) receptor regulation of PLD2 may play a role in the pathogenesis of hypertension.

Abnormalities in dopamine production and receptor function have been described in human essential hypertension and rodent models of genetic hypertension. Under normal conditions, D(1)-like receptors (D(1) and D(5)) inhibit sodium transport in the kidney and intestine. However, in the Dahl salt-sensitive and spontaneously hypertensive rats (SHRs) and in humans with essential hypertension, the D(1)-like receptor-mediated inhibition of epithelial sodium transport is impaired because of an uncoupling of the D(1)-like receptor from its G protein/effector complex. The uncoupling is receptor specific, organ selective, nephron-segment specific, precedes the onset of hypertension, and cosegregates with the hypertensive phenotype. The defective transduction of the renal dopaminergic signal is caused by activating variants of G protein-coupled receptor kinase type 4 (GRK4: R65L, A142V, A486V). The GRK4 locus is linked to and GRK4 gene variants are associated with human essential hypertension, especially in salt-sensitive hypertensive subjects. Indeed, the presence of three or more GRK4 variants impairs the natriuretic response to dopaminergic stimulation in humans. In genetically hypertensive rats, renal inhibition of GRK4 expression ameliorates the hypertension. In mice, overexpression of GRK4 variants causes hypertension either with or without salt sensitivity according to the variant. GRK4 gene variants, by preventing the natriuretic function of the dopaminergic system and by allowing the antinatriuretic factors (e.g., angiotensin II type 1 receptor) to predominate, may be responsible for salt sensitivity. Subclasses of hypertension may occur because of additional perturbations caused by variants of other genes, the quantitative interaction of which may vary depending upon the genetic background.

This study evaluated the transduction pathway associated with type 3 Na(+)/H(+) exchanger (NHE3) activity-induced inhibition during dopamine D(3) receptor activation in immortalized renal proximal tubular epithelial cells from the spontaneously hypertensive rat. The dopamine D(3) receptor agonist 7-OH-DPAT decreased NHE3 activity, which was prevented by the D(2)-like receptor antagonist S-sulpiride, pertussis toxin (PTX; overnight treatment), and the PKC inhibitor chelerythrine, but not by cholera toxin (overnight treatment), the MAPK inhibitor PD-098059, or the p38 inhibitor SB-203580. The PKA inhibitor H-89 abolished the inhibitory effects of forskolin on NHE3 activity, but not that of 7-OH-DPAT. The phospholipase C (PLC) inhibitor U-73122 prevented the inhibitory effects of 7-OH-DPAT, whereas PDBu and 7-OH-DPAT increased PLC activity and reduced NHE3 activity; downregulation of PKC abolished the inhibitory effects of both PDBu and 7-OH-DPAT on NHE activity. The inhibition of NHE3 activity by GTPgammaS and the prevention of the effect of 7-OH-DPAT by PTX suggest an involvement of a G(i/o) protein coupled to the dopamine D(3) receptor. Indeed, the 7-OH-DPAT-induced decrease in NHE3 activity was abolished in cells treated overnight with the anti-G(i)alpha3 antibody, but not in cells treated with antibodies against G(q/11), G(s)alpha, G(beta), and G(i)alpha1,2 proteins. The calcium ionophore A-23187 and the Ca(2+)-ATPase inhibitor thapsigargin increased intracellular Ca(2+) but did not affect NHE3 activity. However, the inhibitory effects of PDBu and 7-OH-DPAT on NHE3 activity were completely abolished by A-23287 and thapsigargin. It is concluded that inhibition of NHE3 activity by dopamine D(3) receptors coupled to G(i)alpha3 proteins is a PLC-PKC-mediated event, modulated by intracellular Ca(2+).

Dopamine-induced inhibition of Na(+)-K(+)-ATPase has been suggested to play a role in the regulation of Na(+) absorption at the intestinal level, and these effects were mediated by dopamine D(1)-like receptors. The aim of this work was to evaluate the effect of the activation of the D(1)-like receptors on the activity of the Na(+)/H(+) exchanger (NHE) in the rat intestinal epithelial cell line IEC-6. The presence of D(1) receptors was confirmed by immunoblotting. The dopamine D(1)-like receptor agonist SKF-38393 produced a concentration-dependent inhibition of NHE activity and stimulation of adenylyl cyclase (AC), this being antagonized by the D(1) selective antagonist SKF-83566. Effects of SKF-38393 on NHE and AC activities were maximal at 5 min of exposure to the agonist and rapidly diminished with no effect at 25 min. Exposure of cells for 25 min to dibutyryl-cAMP (0.5 mM) or to the AC activator forskolin (3 microM) effectively inhibited NHE activity. Pretreatment of cells with heparin (1 microM), a nonselective G protein-coupled receptor kinase (GRK) inhibitor, prevented the loss of effects on NHE activity after 25 min exposure to SKF-38393. The presence of GRK4, GRK6A, and GRK6B was confirmed by immunoblotting. Overnight treatment with the anti-GRK4-6 antibody complexed with Lipofectin was also effective in preventing loss of the effects of SKF-38393 on NHE and AC activities. It is concluded that dopamine D(1) receptors in IEC-6 rapidly desensitize to D(1)-like agonist stimulation and GRK4 and 6 appear to be involved in agonist-mediated responsiveness and desensitization.

The sensitivity of the Cl(-)/HCO(3)(-) exchanger to dopamine D(1)- and D(2)-like receptor stimulation in immortalized renal proximal tubular epithelial cells from the spontaneous hypertensive rat (SHR) and Wistar-Kyoto rat (WKY) was examined. The activity of the Cl(-)/HCO(3)(-) exchanger (in pH U/s) in SHR cells (0.00191) was greater than in WKY cells (0.00126). The activity of Cl(-)/HCO(3)(-) exchanger was exclusively observed at the apical cell side and probably occurs through the SLC26A6 anion transporter that is expressed in both WKY and SHR cells. Stimulation of D(1)-like receptors with SKF-38393 markedly attenuated the HCO(3)(-)-dependent intracellular pH recovery in WKY cells but not in SHR cells. Stimulation of D(2)-like receptors with quinerolane did not alter Cl(-)/HCO(3)(-) exchanger activity in both WKY and SHR cells. The selective D(1)-like receptor antagonist SKF-83566 prevented the effect of SKF-38393. Both WKY and SHR cells responded to dibutyryl-cAMP (DBcAMP) with inhibition of the Cl(-)/HCO(3)(-) exchanger, and downregulation of PKA (overnight exposure to DBcAMP) abolished the inhibitory effect of both DBcAMP and SKF-38393 in WKY cells. Both SHR and WKY cells responded to forskolin with increases in the formation of cAMP. However, only WKY responded to SKF-38393 with increases in the formation of cAMP that was prevented by SKF-83566. It is concluded that WKY cells respond to D(1)-like dopamine receptor stimulation with inhibition of the apical Cl(-)/HCO(3)(-) (SLC26A6) exchanger and SHR cells have a defective D(1)-like dopamine response.

The present study describes characteristic features of two clonal subpopulations of opossum kidney (OK) cells (OK(LC) and OK(HC)) that are functionally different but morphologically identical. The most impressive differences between OK(HC) and OK(LC) cells are the overexpression of Na+-K+-ATPase and type 3 Na+/H+ exchanger by the former, accompanied by an increased Na+-K+-ATPase activity (57.6 +/- 5.6 vs. 30.0 +/- 0.1 nmol P(i). mg protein(-1). min(-1)); the increased ability to translocate Na+ from the apical to the basolateral surface; and the increased Na+-dependent pH(i) recovery (0.254 +/- 0.016 vs. 0.094 +/- 0.011 pH units/s). Vmax values (in pH units/s) for Na+-dependent pHi recovery in OK(HC) cells (0.00521 +/- 0.0004) were twice (P < 0.05) those in OK(LC) (0.00202 +/- 0.0001), with similar Km values (in mM) for Na+ (OK(LC), 21.0 +/- 5.5; OK(HC), 14.0 +/- 5.6). In addition, we measured the activities of transporters (organic ions, alpha-methyl-D-glucoside, L-type amino acids, and Na+ and enzymes (adenylyl cyclase, aromatic L-amino acid decarboxylase, and catechol-O-methyltransferase). The cells were also characterized morphologically by optical and scanning electron microscopy and karyotyped. It is suggested that OK(LC) and OK(HC) cells constitute an interesting cell model for the study of renal epithelial physiology and pathophysiology, namely, hypertension.

D(1)-like (D(1), D(5)) and D(2)-like (D(2), D(3), D(4)) dopamine receptors interact in the kidney to produce a natriuresis and a diuresis. Disruption of D(1) or D(3) receptors in mice results in hypertension that is caused, in part, by a decreased ability to excrete an acute saline load. We studied D(1)-like and D(2)-like receptor interaction in anesthetized spontaneously hypertensive rats (SHR) by the intrarenal infusion of Z-1046 (a novel dopamine receptor agonist with rank order potency of D(3)> or =D(4)>D(2)>D(5)>D(1)). Z-1046 increased glomerular filtration rate (GFR), urine flow, and sodium excretion in normotensive Wistar-Kyoto rats but not in SHRs. The lack of responsiveness to Z-1046 in SHRs was not an epiphenomenon, because intrarenal cholecystokinin infusion increased GFR, urine flow, and sodium excretion to a similar extent in the two rat strains. We conclude that renal D(1)-like and D(2)-like receptor interaction is impaired in SHRs. The impaired D(1)-like and D(2)-like receptor interaction in SHRs is not caused by alterations in the coding sequence of the D(3) receptor, the D(2)-like receptor expressed in rat renal tubules that has been shown to be involved in sodium transport. Because the diuretic and natriuretic effects of D(1)-like receptors are, in part, caused by an interaction with D(2)-like receptors, it is possible that the decreased Z-1046 action in SHRs is secondary to the renal D(1)-like receptor dysfunction in this rat strain.

To determine if the defective interactions among D(1)-like receptors, G proteins, and Na(+)/H(+) exchanger 3 (NHE3) are consequences of hypertension, we studied these interactions in rats, before (2--3 wk) and after (12 wk) the establishment of hypertension. To eliminate the confounding influence of second messenger action on D(1) receptor-NHE3 interaction, studies were performed in renal brush-border membranes (BBM) devoid of cytoplasmic second messengers. NHE3 activity increased with age in Wistar-Kyoto (WKY) rats (3 wk = 1.48 +/- 0.39, n = 13; 12 wk = 2.83 +/- 0.15, n = 16, P < 0.05) but not in spontaneously hypertensive rats (SHRs; 3 wk = 2.52 +/- 0.37, n = 11; 12 wk = 2.81 +/- 0.20, n = 16). D(1) receptor protein tended to decrease, whereas NHE3 protein tended to increase with age in both WKY and SHRs. However, the inhibitory effect of a D(1)-like agonist, SKF-81297, on NHE3 activity increased with age in WKY rats (3 wk = -40.7 +/- 5.3%, n = 10, 12 wk = -58.7 +/- 4.6%, n = 12, P < 0.05) but not in SHRs (3 wk = -27.6 +/- 5.9%, n = 11, 12 wk = -25.1 +/- 3.2%, n = 11). The decreased inhibitory effect of another D(1)-like agonist, fenoldopam, on NHE3 activity in SHRs was not caused by increased activity and binding of G beta gamma to NHE3 as has been reported in young WKY rats. G(s)alpha mediates, in part, the inhibitory effect of D(1)-like agonists on NHE3 activity. In WKY rats, fenoldopam increased G(s)alpha/NHE3 binding to the same extent in 2-wk-old (1.5-fold, n = 4) and adult (1.5-fold, n = 4) rats. In contrast, in SHRs, fenoldopam decreased the amount of G(s)alpha bound to NHE3 in 2-wk-old SHRs and had no effect in 4-wk-old and adult SHRs. These studies indicate that the decreased inhibitory effect of D(1)-like agonists on NHE3 activity in SHRs (compared with WKY rats) precedes the development of hypertension. This may be caused, in part, by a decreased interaction between G(s)alpha and NHE3 in BBM secondary to impaired D(1)-like receptor function.

The decreased natriuretic action of dopamine in the young has been attributed to decreased generation of cAMP by the activated renal D(1)-like receptor. However, sodium/hydrogen exchanger (NHE) 3 activity in renal brush-border membrane vesicles (BBMV) can be modulated independent of cytoplasmic second messengers. We therefore studied D(1)-like receptor regulation of NHE activity in BBMVs in 2-, 4-, and 12-wk-old (adult) rats. Basal NHE activity was least in 2-wk-old compared with 4- and 12-wk-old rats. D(1)-like agonist (SKF-81297) inhibition of NHE activity was also least in 2-wk-old (-1 +/- 9%, n = 3) compared with 4 (-15 +/- 5%, n = 6)- and 12 (-65 +/- 4%, n = 6)-wk-old rats. The decreased response to the D(1)-like agonist in BBMV was not caused by decreased D(1) receptors or NHE3 expression in the young. G(s)alpha, which inhibits NHE3 activity by itself, coimmunoprecipitated with NHE3 to the same extent in 2-wk-old and adult rats. G(s)alpha function was also not impaired in the young because guanosine 5'-O-(3-thiotriphosphate) decreased NHE activity to a similar extent in 4-wk-old and adult rats. Galpha(i-3) protein expression in BBMV also did not change with age. In contrast, Gbeta expression and the amount of Gbeta that coimmunoprecipitated with NHE3 in BBMV was greatest in 2-wk-old rats and decreased with age. Gbeta common antibodies did not affect D(1)-like agonist inhibition of NHE activity in adult rats (8%) but markedly increased it (48%)in 4-wk-old rats. We conclude that the decreased inhibitory effect of D(1)-like receptors on NHE activity in BBMV in young rats is caused, in part, by the increased expression and activity of the G protein subunit Gbeta/gamma. The direct regulation of NHE activity by G protein subunits may be an important step in the maturation of renal tubular ion transport.

NHE3 activity is regulated by phosphorylation/dephosphorylation processes and membrane recycling in intact cells. However, the Na(+)/H(+) exchanger (NHE) can also be regulated by G proteins independent of cytoplasmic second messengers, but the G protein subunits involved in this regulation are not known. Therefore, we studied G protein subunit regulation of NHE3 activity in renal brush-border membrane vesicles (BBMV) in a system devoid of cytoplasmic components and second messengers. Basal NHE3 activity was not regulated by G(s)alpha or G(i)alpha, because antibodies to these G proteins by themselves were without effect. The inhibitory effect of D(1)-like agonists on NHE3 activity was mediated, in part, by G(s)alpha, because it was partially reversed by anti-G(s)alpha antibodies. Moreover, the amount of G(s)alpha that coimmunoprecipitated with NHE3 was increased by fenoldopam in both brush-border membranes and renal proximal tubule cells. Furthermore, guanosine 5'-O-(3-thiotriphosphate) but not guanosine 5'-O-(2-thiodiphosphate), the inactive analog of GDP, increased the amount of G(s)alpha that coimmunoprecipitated with NHE3. The alpha(2)-adrenergic agonist, UK-14304 or pertussis toxin (PTX) alone had no effect on NHE3 activity, but UK-14304 and PTX treatment attenuated the D(1)-like receptor-mediated NHE3 inhibition. The ability of UK-14304 to attenuate the D(1)-like agonist effect was not due to G(i)alpha, because the attenuation was not blocked by anti-G(i)alpha antibodies or by PTX. Anti-Gbeta(common) antibodies, by themselves, slightly inhibited NHE3 activity but had little effect on D(1)-like receptor-mediated NHE3 inhibition. However, anti-Gbeta(common) antibodies reversed the effects of UK-14304 and PTX on D(1)-like agonist-mediated NHE3 inhibition. These studies provide concrete evidence of a direct regulatory role for G(s)alpha, independent of second messengers, in the D(1)-like-mediated inhibition of NHE3 activity in rat renal BBMV. In addition, beta/gamma dimers of heterotrimeric G proteins appear to have a stimulatory effect on NHE3 activity in BBMV.

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.

An attenuated natriuretic response to dopamine and D1 agonists in genetic hypertension has been attributed to an uncoupling of the renal D1 dopamine receptor from its G protein-effector protein complex. We have reported that in normotensive Wistar-Kyoto (WKY) rats the natriuresis induced by calcium channel blockers is caused in part by activation of renal D1 dopamine receptors. We tested the interaction between the renal D1 receptor and a calcium channel blocker, diltiazem, infused into a renal artery of anesthetized spontaneously hypertensive rats (SHR) acutely loaded with 5% saline. Diltiazem produced a 50% increase in renal blood flow and nearly tripled absolute and fractional sodium excretion; urine flow rate more than doubled, but glomerular filtration rate did not change. However, the D1 receptor antagonist SKF-83742, which had no effect by itself, did not diminish the response to diltiazem. In a separate group of concurrent experiments, we found that the diltiazem-induced natriuresis was associated with a decrease in Na(+)-K(+)-adenosinetriphosphatase activity in the renal medulla of SHR. In contrast, in WKY rats, no changes were noted in the renal medulla but a decrease in Na(+)-K(+)-adenosinetriphosphatase activity was noted in the renal cortex. Diltiazem had no effect on urinary dopamine excretion in either rat strain. We conclude that diltiazem induces natriuresis differently in SHR and WKY rats; it is independent of D1 receptors in SHR and is in great part mediated by renal hemodynamic, rather than by cortical tubular, effects. These studies support previous findings of a defective renal cortical tubular D1 mechanism in SHR.

Dopamine receptors are present in the medullary thick ascending limb (mTAL) of Henle, but their effect on ion transport in this nephron segment has not been tested. Therefore, we studied the short-term effects of dopamine on Na(+)-K(+)-2Cl- cotransport (assessed by 100 microM bumetanide-sensitive 86Rb uptake) in rat mTAL tubular suspensions. Dopamine (1 microM) stimulated bumetanide-sensitive 86Rb uptake (72.1 +/- 10.6% vs. control, n = 5) by increasing total 86Rb uptake and by decreasing bumetanide-insensitive 86Rb uptake; this effect was concentration dependent. The dopamine-induced stimulation of Na(+)-K(+)-2Cl- cotransport activity was mimicked by calyculin A, a protein phosphatase (PP) inhibitor, and Sp isomer of adenosine 3',5'-cyclic monophosphothioate (Sp-cAMP[S]), a protein kinase A (PKA) agonist, and blocked by Rp isomer of 8-(4-chlorophenylthio)-cAMP[S] (Rp-8-CPT-cAMP[S]), a PKA inhibitor (n = 5). Dopamine did not increase the stimulatory effect of the PP inhibitor. However, the stimulatory effect of the PP inhibitor and PKA agonist was additive and approached the stimulatory effect of dopamine. The stimulatory effects of dopamine, PP inhibitor, and PKA agonist persisted even when intracellular sodium was clamped by 5 microM monensin. When K+ channels were blocked by 1 mM BaCl2, the effects of dopamine and calyculin A on the cotransport were no longer apparent, although the stimulatory effect of the PKA agonist was attenuated. We conclude that dopamine stimulates Na(+)-K(+)-2Cl- cotransport activity. This action is mediated mainly by PKA-dependent phosphorylation/dephosphorylation processes and modulated by dopamine actions on K+ channels.

We have studied the role of glucocorticoids in inducing the maturation in activity of the proximal tubule Na+/H+ exchanger that follows birth. Renal cortical microvillus membrane vesicles were prepared from 132-day gestation sheep fetuses (n = 8) that had received intraperitoneal cortisol (13 micrograms.kg-1.h-1) for the previous 48 h. Membrane vesicles were also obtained from sham-operated twin controls (n = 8). Amiloride-sensitive uptake of 22Na+ by these vesicles was measured, and Woolf-Augustinsson-Hofstee plots were used to determine the Michaelis constant (Km) and maximal velocity (Vmax). There was no significant difference in Km; however, the Vmax was 61% higher in cortisol-treated fetuses. Posttreatment circulating cortisol levels were significantly higher in the treated fetuses. Total RNA was collected from renal cortex of the eight pairs of twins when killed. Renal cortex Na+/H+ exchanger 3 (NHE3) mRNA levels were approximately fourfold higher in cortisol-treated than in control fetuses. Although proximal tubule Na+/H+ exchanger activity and renal cortex NHE3 mRNA levels increased significantly in cortisol-treated fetuses, cortisol infusion did not stimulate renal sodium reabsorption in the fetus but rather produced a natriuresis. These results demonstrate that glucocorticoids can induce an increase in both Na+/H+ exchanger activity and NHE3 mRNA levels during the last trimester of gestation in sheep. However, these changes are not associated with an increased ability of the fetal kidney to reabsorb sodium.

In the brain, dopamine, via protein kinase A (PKA) activation of dopamine- and cAMP-regulated phosphoprotein (DARPP-32), inhibits protein phosphatase 1 (PP1) activity and keeps Na(+)-K(+)-adenosinetriphosphatase (ATPase) in its phosphorylated inactive state. In the present study, we examined the relationship among dopamine, PP1, and Na(+)-K(+)-ATPase activities in renal proximal tubules. PP1 activity in proximal tubules was not decreased by dopamine (5 x 10(-9)-10(-4) M), fenoldopam (5 x 10(-6) M), or norepinephrine (5 x 10(-7) M). In contrast, in the medullary thick ascending limb of Henle and in the brain striatum, PP1 activity was decreased by fenoldopam (5 x 10(-6) M). We also showed that the ability of dopamine (10(-6) M) to inhibit Na(+)-K(+)-ATPase activity in proximal tubules (assessed by ouabain-sensitive 86Rb uptake) occurred in the absence or presence of a sodium clamp with 5 microM monensin. Thus the inhibitory effect of dopamine on Na(+)-K(+)-ATPase activity in proximal tubules is not regulated by PP1 activity. Tautomycin and okadaic acid by themselves, at concentrations that inhibited PP1 activity, had no effect on Na(+)-K(+)-ATPase activity in proximal tubules. The ability of a dopamine D1 agonist, fenoldopam, to inhibit PP1 activity in brain striatum and in medullary thick ascending limb, but not in proximal tubules, suggests differential organ and nephron segment regulation of PP activity.

The direct tubular natriuretic effect of calcium channel blockers (CCBs) may be due to an interaction between CCBs and a renal tubular dopamine receptor. We therefore studied the effects of two chemically unrelated CCBs, diltiazem and isradipine, infused into the right renal artery of 5% saline-loaded anesthetized rats alone or in the presence of a D1 antagonist, SKF-83742. Isradipine (0.03 microgram.kg-1.min-1) or diltiazem (20 but not 10 micrograms.kg-1.min-1) alone produced an increase in urine flow and an approximate doubling of absolute and fractional sodium excretion, which was not seen in the left kidney or in the control animals (analysis of variance, Scheffé's test, P < 0.05). SKF-83742 alone given systemically or into the right renal artery did not affect these parameters but did block the actions of diltiazem or isradipine. There was no change in mean arterial pressure, renal blood flow, or glomerular filtration rate in any of the experiments. In additional studies, we found that a combined infusion of dopamine (0.1 microgram.kg-1.min-1) and diltiazem (10 micrograms.kg-1.min-1) (doses that by themselves did not alter renal function) produced a twofold or greater increase in urine flow and absolute and fractional sodium excretion; glomerular filtration rate was not significantly changed. Intrarenal arterial CCBs, without a change in renal hemodynamics, produce a natriuresis that is blocked by a D1 antagonist. Concomitant administration of diltiazem and dopamine (each in subeffective doses when used alone) produces a synergistic effect.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study was designed to examine the effect of direct intrarenal infusion of the alpha 1-adrenoceptor agonist, phenylephrine, on urinary flow rate (UFR) and on renal Na and Cl excretion in conscious and chronically instrumented fetal (128-133 days gestation, term 145 days), newborn (6-12 days), and adult sheep. Five different renal concentrations of phenylephrine, varying from 5 +/- 1 to 72 +/- 2 ng/ml, were studied. Low renal phenylephrine concentration (< or = 12 +/- 1 ng/ml) induced a significant renal vasoconstrictor response in fetuses but not in newborn and adult sheep. The effects of intrarenal phenylephrine infusion on UFR and fractional excretion of Na (FENa) was greater (P < 0.05) in newborn lambs than in fetal and adult sheep. At a renal concentration of phenylephrine between 9 +/- 1 and 12 +/- 1 ng/ml, the percent decrease in UFR was greater (P < 0.05) in newborn lambs (-19.1 +/- 4.7%) than in fetal (9.8 +/- 8.9%) and adult sheep (-3.3 +/- 3.9). The percent decrease in FENa at renal concentration of phenylephrine between 18 +/- 1 and 24 +/- 1 ng/ml was also significantly (P < 0.05) larger in newborn lambs (-20.2 +/- 2.8%) than in fetal (-8.0 +/- 3.1%) and adult sheep (-11.2 +/- 2.6%). In summary, the present results indicate that the fetal kidney has a limited ability to increase sodium reabsorption in response to stimulation of alpha-adrenoceptors and that the effect of renal alpha-adrenoceptor stimulation on urinary volume and urinary sodium excretion increases during the newborn period.

The D2, D3, and D4 dopamine receptors cloned from brain correspond to the classically described "D2" receptors. Although radioligand binding and biochemical and functional studies have demonstrated the presence of D2-like receptors in the kidney, the expression of D2, D3, or D4 receptor genes has not been conclusively demonstrated in the kidney. Since Northern blot analysis may have precluded demonstration of dopamine receptor mRNAs because of their relative low abundance, the expression of the D2long and D3 receptor genes was studied by reverse transcription-polymerase chain reaction (RT-PCR). We were able to amplify PCR products of the predicted size using mRNA from glomeruli, proximal tubules, outer medulla, inner medulla, and renal microvessels from normotensive Wistar-Kyoto rats (WKY). Specificities of the amplified products were confirmed by restriction analysis and by sequencing the D2long product and Southern blotting the D3 product. Because some studies have suggested that D2-like receptor actions may be different between WKY and spontaneously hypertensive rats (SHR), similar studies were performed in this rat strain. In the SHR, as in WKY, PCR products of the predicted size were amplified, and restriction enzyme digestion patterns were as predicted from the cDNA sequence. The PCR-generated cDNA from the glomeruli of SHR was subcloned and sequenced and was revealed to be identical to the D2long receptor cDNA from WKY. We conclude that the D2long and D3 receptor genes are expressed in specific regions of the kidney including the glomeruli. No differences in the sequence of the D2long receptor cDNA in part of the 3rd cytoplasmic loop were noted between WKY and SHR. These studies do not rule out the possibility that mutations in other segments of the receptor exist in the SHR.

When D1 dopamine agonists are incubated with renal cortical tissue, Na+/H+ exchange activity is inhibited, presumably due to D1 receptor-mediated stimulation of adenylyl cyclase and subsequent increase in protein kinase A activity. Although the role of adenosine 3',5'-cyclic monophosphate (cAMP) and cAMP-dependent protein kinase in the regulation of Na+/H+ exchange activity is well established, receptors functionally coupled to adenylyl cyclase can regulate Na+/H+ exchange activity independently of changes of cAMP accumulation. The current studies were designed to determine whether D1 agonists can inhibit Na+/H+ exchange activity independently of changes of cAMP accumulation and also to determine the role of G proteins in this process. The D1 agonist, fenoldopam, inhibited Na+/H+ exchange activity in a time-related and concentration-dependent manner. The 50% inhibitory concentration was 5-34 microM. Occupation of the renal D1 receptor mediates this action, since the D1 antagonist, SKF 83742, partially blocks the effect. This action, however, was independent of adenylyl cyclase, protein kinase A, and protein kinase C activity. Inhibition of adenylyl cyclase with dideoxyadenosine or inhibition of protein kinase A and C with the isoquinolines N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (H-4) and 1-(5-isoquinolinesfulfonyl)-2-methylpiperazine (H-7) did not block the effect of fenoldopam on the exchanger. The action of fenoldopam is not due to an amiloride-like action on the exchanger, because kinetic analysis of the inhibitory action was noncompetitive and the effect of fenoldopam was time dependent. The process involved G proteins, since guanosine 5'-O-(2-thiodiphosphate) prevented while guanosine 5'-O-(3-thiotriphosphate) increased the inhibitory effect of fenoldopam.

The coupling between the dopamine1 (DA1) receptor and the G protein/adenylyl cyclase (AC) enzyme complex is defective in the proximal convoluted tubule (PCT) of 20-wk-old spontaneously hypertensive rats (SHRs). Because this coupling defect could have been due to desensitization secondary to elevated renal dopamine levels in the adult animal, we studied the interaction between DA1 receptors and AC in PCT of rats as early as 3 wk of age, a time when renal dopamine levels are similar in SHRs and their normotensive controls (Wistar-Kyoto rats, WKYs). Maximum receptor density did not change with age and was similar in WKYs and SHRs in all the age groups studied (3, 8, and 20 wk). Basal-, forskolin-, and guanyl nucleotide-stimulated AC activities were also similar in WKYs and SHRs and did not change with age. However, the DA1 agonist-stimulated AC activity was greater in WKYs than in SHRs and increased with age in WKYs but not in SHRs. Moreover, the ability of a nonhydrolyzable analogue of GTP, Gpp(NH)p, to enhance DA1 agonist (SND-919-C12, 1 microM)-stimulated AC activity increased with age in WKY but not in SHRs. To determine if the defect noted in the PCT of SHRs is due to a defective D1A receptor gene, parallel studies were performed in the striatum, since this receptor is expressed predominantly in the latter tissue. In contrast to the results in PCT, radioligand binding and AC studies in striatum revealed no differences between WKYs and SHRs.(ABSTRACT TRUNCATED AT 250 WORDS)

The existence of dopamine receptor subtypes outside the central nervous system has been suggested by pharmacological and biochemical techniques. Whether the renal dopamine receptors are distinct from those cloned from the brain remains to be determined. Indeed, the expression of any of these receptor genes in the kidney has not been demonstrated definitively. In this study, we amplified D1A receptor cDNA from microdissected proximal convoluted tubules of the rat kidney by reverse transcription-polymerase chain reaction; primers were based on the sequence of rat D1A cDNA cloned from the brain and corresponded to the third cytoplasmic loop of the receptor. Specificity of the amplified products was verified by restriction analysis, Southern blots, and sequencing. Furthermore, solution hybridization indicated the presence of a single ribonuclease-protected RNA species corresponding to the D1A receptor mRNA in proximal tubules. These studies report for the first time the presence of D1A receptor message in the kidney.

We have reported defective coupling of the renal tubular DA1 dopamine receptor to adenylyl cyclase in both the spontaneously hypertensive rat (SHR) and the Dahl salt-sensitive rat. Since Na+, 5'-guanyl imidodiphosphate [Gpp(NH)p], and N-ethylmaleimide (NEM) reduce agonist affinity for brain D1 dopamine receptors, we compared the effects of these agents on agonist affinity in proximal tubules from SHR and its normotensive control, the Wistar-Kyoto rat (WKY), to delineate further the site of the DA1-adenylyl cyclase coupling defect. In WKY, the D1/DA1 agonist, fenoldopam, competed for 125I-Sch 23982 at a high-affinity site (KiH = 1.8 +/- 0.8 x 10(-8) M) and a low-affinity site (KiL = 7.6 +/- 1.1 x 10(-5) M, n = 6). Na+ (150 mM) or Gpp(NH)p (10(-4) M) converted KiH to KiL. NEM, which alkylates sulfhydryl groups, also converted all the binding to KiL; this effect could be prevented by prior treatment with 10(-4) M fenoldopam. In contrast, in SHR, fenoldopam detected only a KiL (7.8 +/- 1.4 x 10(-5) M, n = 6). Neither Na+, Gpp(NH)p, nor NEM had any effect on KiL. To study a functional expression of these binding sites, the effect of 5 x 10(-5) M fenoldopam or 8-(chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (8-CPT-cAMP) on Na+/H+ exchange activity in proximal tubular brush-border membrane vesicles was tested. In WKY, the inhibitory effects of these agents on the exchanger increased with the age of the rat.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study was designed to characterize the developmental changes in the renal responses to dopamine DA1-receptor activation in chronically instrumented preterm (109-115 days) and near-term (130-140 days, full term 145 days) fetal sheep. Cumulative doses of the selective DA1-agonist fenoldopam increased mean arterial blood pressure (MABP) in both preterm (+16 +/- 3%) and near-term fetuses (+16 +/- 3%) but had no significant effect on renal blood flow velocity. Infusion of the DA1-antagonist SCH-23390 did not affect the increase in MABP, suggesting that the effect of fenoldopam on MABP was not directly related to activation of DA1-receptors. Fenoldopam infusion had no significant effects on renal function parameters in preterm fetuses. In near-term fetuses, however, fenoldopam increased urinary flow rate (82.6 +/- 20.9%, P < 0.003), glomerular filtration rate (GFR; 16.6 +/- 4.9%, P < 0.01), urinary sodium excretion (40.1 +/- 14.9%, P < 0.02), and fractional excretion of sodium (26.8 +/- 11.2%, P < 0.03). Infusion of the DA1-antagonist SCH-23390 blocked the fenoldopam-induced diuresis and natriuresis but had no significant effect on the rise in GFR. Fenoldopam infusion had no significant effects on plasma renin activity and plasma aldosterone concentration and on urinary prostaglandin (PG) excretion (PGE2, PGF2 alpha, and 6-keto-PGF1 alpha). Taken together, these results suggest that the renal effect of DA1-receptor activation is age dependent and that stimulation of DA1-receptor in near-term fetuses is associated with a diuresis and natriuresis that seem to be independent of renal hemodynamics and adrenal effects.

The natriuretic and diuretic effects of dopamine are attenuated in the young. Because dopamine has actions on receptors (e.g., adrenergic, serotonin) other than dopamine, we studied a novel dopamine agonist, pramipexole, which has a selectivity to both DA1 and DA2-receptor subtypes. Intravenous administration of pramipexole resulted in a dose-related (1, 10, and 100 micrograms.kg-1.min-1) increase in urine flow and absolute and fractional sodium excretion and a decrease in mean arterial pressure (MAP) in three groups of rats studied. Pramipexole induced a greater decrease in MAP in 6- to 7- (n = 5) and 9- to 16- (n = 6) than in 3- to 4-wk-old (n = 8) rats; the natriuresis and diuresis were greatest in 12- to 16- and least in 3- to 4-wk-old rats. The renal effects of pramipexole were mainly due to actions at the DA1 receptor, since these effects were completely blocked by the coinfusion of a DA1 antagonist, SKF 83742. To explore further a cause of the attenuated natriuretic effect of pramipexole in the young, we studied the effect of a selective DA1-receptor agonist, fenoldopam, on amiloride-sensitive 22Na+ uptake in renal brush-border membrane vesicles. The 3-s amiloride-sensitive uptake was inhibited (45%) by fenoldopam (5 x 10(-5)M) in 9- to 16- (n = 6) but not in 3- to 4-wk-old (n = 5) rats. These studies suggest that the attenuated natriuretic effect of dopamine in the young is in part due to decreased DA1 action on the brush-border membrane Na(+)-H+ exchanger.

Intrarenal arterial infusion of alpha 1-adrenergic agonists decreases renal blood flow, glomerular filtration rate, and water and sodium excretion to a greater extent in fetus and newborn than in adult sheep. In vitro renal vascular effects of alpha 1- and alpha 2-adrenergic agonists are also greater in fetus than newborn or adult. The present studies were designed to examine the ontogeny of renal alpha-adrenoceptor subtypes in sheep for whom the renal effects of alpha-adrenergic agonists have been described at similar postconceptional ages. With the use of radioligand-binding techniques, specific binding of [3H]prazosin (alpha 1-adrenergic antagonist), [3H]idazoxan, and [3H]rauwolscine (alpha 2-adrenergic antagonists) was studied in fetus, lamb, and adult sheep kidneys. The specific binding of the three radioligands was greatest in fetal and least in adult kidneys. Analysis of Scatchard plots revealed a greater renal alpha 1-adrenoceptor density in fetuses than in lamb or adults. Renal alpha 2-adrenoceptor density was also greater in fetuses than in lambs. These studies suggest that the increased renal alpha 1- and alpha 2-adrenergic effects in fetal sheep are related to increased alpha-adrenoceptor density. Competition experiments and rank adrenergic antagonist potency suggested the presence of only the alpha 1b-adrenoceptor in fetal and adult sheep kidneys. The alpha 2-adrenoceptor that was found only in the fetal sheep had a low affinity to rauwolscine, which is unlike that described in most species for alpha 2-adrenoceptors.

To determine a renal tubular mechanism for the natriuretic effect of dopamine (DA) and DA-1 agonists, we measured Na(+)-H+ exchange activity (amiloride sensitive) in rat renal cortical brush-border membrane vesicles (BBMV). Renal cortical tissues were preincubated with ligands before BBMV preparation to study Na(+)-H+ exchange activity in the absence of the added ligands that may compete for ion binding sites of the exchanger. DA and DA-1 agonist-inhibited Na(+)-H+ exchange activity was concentration and time dependent. The inhibitory effect was not due to increased permeability, collapse of the proton gradient, or change in vesicle size and did not extend to Na(+)-glucose symport. DA-2 agonists had no effect, whereas alpha-adrenergic agonists increased Na(+)-H+ exchange activity. Kinetic analysis revealed that the DA-1 agonist inhibited Na(+)-H+ exchange activity by a noncompetitive process. 2',5'-Dideoxyadenosine inhibited adenylate cyclase activity and reversed the inhibitory effect of DA-1 agonist on the exchanger. H4, an isoquinoline sulfonamide, which inhibits protein kinase A, also reversed the inhibitory effect of DA-1 agonist on the exchanger. Thus the DA-1 agonist-mediated inhibition of Na(+)-H+ exchange activity in BBMV is a receptor-mediated adenylate cyclase-linked process.