| BackgroundReactive oxygen species(ROS) are intermediates in reduction-oxidation(Redox)reactions involved in multi-function like intracellular signaling transduction and cell defense. A balance between the production and breakdown of ROS is important in keeping healthy status. Excessive ROS production is associated with various diseases such as hypertension, atherosclerosis, heart failure, diabetes and chronic kidney diseases.Peripheral dopamine is known as an important regulator of blood pressure, exerts its actions via membrane dopamine receptors, which belong to G protein-coupled receptor superfamily. Dopamine receptors are divided into D1-like(D1R and D5R) and D2-like(D2R,D3 R and D4R) receptors, according to their structure and pharmacology. D1-like receptors couple to Gαs and stimulate adenylyl cyclase activity whereas D2-like receptors couple to Gαi/Gαo and inhibit adenylyl cyclase activity. Abnormalities of dopamine production and receptor function are responsible for hypertension. Dopamine receptors contribute to blood pressure control via influencing sodium transport, vascular smooth muscle contractility, and production of reactive oxygen species. Dopamine D1-like receptors are important in the regulation of urinary sodium excretion, and also responsible for oxidative stress. D1 R can decrease ROS production via inhibiting NADPH oxidase activity, D5 R is involved in negative regulation of NADPH oxidase activity and expression, or positive regulation of antioxidants like superoxide dismutase(SOD), heme oxygenase(HO), which are known to decrease ROS production and lower blood pressure.Paraoxonases(PONs), classified as lactonases, contain three members: PON1, PON2,and PON3, sharing approximately 65% similarity at the amino acid level but with different physiological function. PON1 and PON3 are predominantly expressed in the liver and are associated with circulating high density lipoproteins(HDL). PON1 and PON3 can prevent low density lipoproteins(LDL) oxidation and decrease oxidative stress. By contrast, PON2 is not found in serum, but located in various tissues. PON2 has the greatest lactonase activity, protects against atherosclerosis by reducing LDL oxidation or decreasing hydroperoxides production. Also, PON2 inhibits the expression and activity of NADPH oxidase, as well as decreases ROS production in mouse kidney and human renal proximal tubule(h RPT) cells.Lipid rafts(LRs) are membrane microdomains composed of sphingolipids, cholesterol,glycolipids, and specific proteins that serve as a spatial and temporal platforms to recruit signaling components, form new mixtures of signaling molecules and enzymes, and increase the efficiency of signal transduction. Many signaling molecules, including dopamine receptors, are associated with LRs. Membrane LRs participate in redox signaling in several cells. In bovine coronary endothelial cell, activation of death receptor induces LRs cluster in the cell membrane, resulting in the aggregation of the NADPH oxidase subunits, activating NADPH oxidase, increasing ROS production and impairing endothelial function. However, in h RPT cells, activation of D1-like receptors dispersing NADPH oxidase subunits in LRs and non-lipid rafts(non-LRs), resulting in decreased NADPH oxidase activity. In h RPT cells, PON2 was also found associated with membrane microdomains and may interact with D2 R in LRs and non-LRs.Based on the above, we presume that D1 R and D5 R inhibit ROS production, part by upregulating PON2 expression in LRs. Our study consists of the short-term and long-term effect of activation of D1-like receptors on oxidative stress, involving the regulation of PON2. The role of D1 R or D5 R in the regulation of oxidative stress need to be checked,respectively. This will help to indentify a mechanism by which dopamine D1-like receptors regulate oxidative stress, providing theoretical support to antioxidant therapeutic approach.ObjectiveTo uncover the role of PON2 in the antioxidant effect of D1 R and D5 R in kidney cells.Methods1. HEK-293 cells stably transfected with h D1R(HEK-h D1R) or h D5R(HEK-h D5R)treated with or without D1-like receptor agonist fenoldopam(1 μM, 15 min), and the sections of mice kidneys were used in this experiment. The colocalization of D1 R and PON2,D5 R and PON2 were checked by laser confocal microscopy, using immunofluorescence staining.2. HEK-h D1 R and HEK-h D5 R cells treated with or without fenoldopam(1 μM, 15 min)were used to check the physical linkage of D1 R and PON2, D5 R and PON2 by coimmunoprecipitation.3. HEK-h D1 R and HEK-h D5 R cells transfected with PON2-si RNA or scrambled si RNA were subject to PON2 expression analysis, detection of ROS production and assay of NADPH oxidase activity, using immunoblotting, 2’,7’-dichlorofluoresceindiacetate(H2DCFDA) fluorescence and lucigenin luminescence, respectively.4. HEK-h D1 R and HEK-h D5 R and h RPT cells treated with or without fenoldopam(1μM, 15min) or methyl-β-cyclodextrin(βCD)( 2%, 60 min) were used to check PON2 expression in LRs and non-LRs by sucrose gradient centrifugation.5. HEK-h D1 R and HEK-h D5 R cells were used in this experiment. Cells were treated by fenoldopam(1 μM) at varying durations(2-24 h), or fenoldopam in combination with a D1-like receptor antagonist, SCH 23390(1 μM,24 h). The protein expression and m RNA of PON2 were checked using immunoblotting and quantitative PCR(q PCR), respectively.6. HEK-h D5 R cells transfected with PON2-si RNA or scrambled si RNA were used to check the NOX2 protein expression and the difference in NADPH oxidase activity, with or without fenoldopam(1 μM, 24 h) treatment.7. Renal cortical homogenates of D5R-/- and D5R+/+ mice were used to check the PON2 protein expression by immunoblotting. Human RPT cells transfected with D5R-si RNA or scrambled si RNA were used to check the protein expression of D5 R, NOX2 and ROS production.8. Human RPT cells transfected with PON2-si RNA and scrambled si RNA were used to check the difference in ROS production or NADPH oxidase activity, with or without fenoldopam(1 μM, 24 h) treatment, using H2 DCFDA fluorescence and lucigenin luminescence, respectively.Results1. To observe whether the interaction between D1-like receptors and PON2 are associated with physical interaction, firstly, the colocalization of D1 R and PON2, D5 R and PON2 were checked. The result showed that D1 R or D5 R colocalized with PON2 in the brush borders of RPTs in the mouse kidney. In h RPT cells, in the basal state, D1 R or D5 R was expressed at the plasma membrane and cytoplasm, while PON2 was found mainly inthe cytoplasm, there was some colocalization of D1 R and PON2, D5 R and PON2 in the cytoplasm. Fenoldopam(1 μM, 15 min) promoted the internalization of some D1 Rs or D5 Rs and result in increased colocalization between D1 R and PON2, D5 R and PON2 in the cytoplasm. In HEK-h D1 R and HEK-h D5 R cells, fenoldopam(1 μM, 15 min) also increased the colocalization in the cytosol of D1 R with PON2 in HEK-h D1 R cells, as well as D5 R with PON2 in HEK-h D5 R cells.2. D1 R and PON2 as well as D5 R and PON2 coimmunoprecipitated in homogenates of HEK-h D1 R, and HEK-h D5 R, respectively. Fenoldopam(1 μM, 15 min) increased PON2 coimmunoprecipitation with D1 R in HEK-h D1 R cells and with D5 R in HEK-h D5 R cells. It demonstrates that D1-like receptors physically interact with PON2.3. PON2-si RNA decreased PON2 expression to a similar extent in HEK-h D1 R and HEK-h D5 R cells, PON2-si RNA increased basal ROS production to a similar extent in HEK-h D1 R and HEK-h D5 R cells. Apocynin completely reversed the increased ROS production in PON2-si RNA-transfected cells. PON2-si RNA impaired the ability of fenoldopam(1 μM, 15 min) to lower NADPH activity. It demonstrates that PON2 is involved in the inhibitory effect of D1-like receptor on NADPH oxidase activity.4. We presumed that the regulation of D1-like receptor on PON2 may be associated with membrane LRs. Fenoldopam treatment(1 μM, 15 min) of HEK-h D1 R cells increased PON2 protein in both LRs and non-LRs. in HEK-h D5 R cells, fenoldopam upregulated PON2 only in non-LRs, expression in LRs was not affected. In h RPT cells, fenoldopam increased PON2 protein only in non-LRs. βCD treatment shifted PON2 protein expression from LRs to non-LRs in HEK-h D1 R, HEK-h D5 R, and h RPT cells. It shows the differential role of D1 R and D5 R on the regulation of PON2, which may be associated with different antioxidant mechanism of D1 R and D5 R.5. To observe the long-term effect of D1-like receptors on PON2, fistly, the PON2 protein expression were checked. Fenoldopam upregulated PON2 protein expression in a time-dependent manner in HEK-h D5 R cells but had no effect in HEK-h D1 R cells. The fenoldopam-induced increase in PON2 protein in HEK-h D5 R cells was completely prevented by SCH23390. Fenoldopam also increased PON2 m RNA at 24 h in HEK-h D5 R cells but not in HEK-h D1 R cells. This also shows the differential regulation of D1 R and D5 R on PON2 expression.6. PON2-si RNA decreased PON2 protein but result in an increase in NOX2 protein in HEK-h D5 R cells. PON2-si RNA impaired the ability of fenoldopam(1 μM, 24 h) to inhibit NADPH oxidase activity. It demonstrates that PON2 are involved in the inhibitory effect of D5 R on NADPH oxidase activity.7. PON2 protein was decreased in D5-/- mice relative to D5+/+ mice. D5R-si RNA decreased D5 R protein and result in a decrease in PON2 protein, an increase in NOX2 expression and ROS production in h RPT cells. Apocynin completely reversed the increase in ROS production in D5R-si RNA-transfected cells.8. In h RPT cells, PON2-si RNA impaired the ability of fenoldopam(1 μM, 24 h) to inhibit ROS production and NADPH oxidase activity.ConclusionsD1R and D5 R differentially regulate PON2, are associated with oxidative stress by short term or long term stimulation, respectively. By the short-term stimulation D1 R increase PON2 protein in membrane LRs but D5 R only increase the PON2 in non-LRs,other mechanisms besides dispersal in membranes are may be involved. By the long-term stimulation, D5 R increase the PON2 expression, involving the antioxidant effect but D1 R not. Our study help to explain the new mechanism among which D1-like receptor regulate oxidative stress, with D1R- and D5R-specific effect.BackgroundEssential hypertension(EH) is one of the most common chronic diseases, affecting more than one billion people. EH raises mortality and morbidity of serious diseases such as stroke, myocardial infarction, heart failure and kidney failure. The genetic and environmental factors contribute to the pathogenesis of EH. Kidney plays a key role in the long-term regulation of blood pressure and sodium excretion. The proximal tubule(PT) is the major site of salt and water reabsorption in the mammalian nephron and responsible for reabsorbing more than 65% of filtered sodium and water. Na+-K+-ATPase and Na+-H+-exchanger-3(NHE3) are major sodium transporters of PT, are associated with sodium absorption. Renal PT(RPT) function is under hormonal control, such as angiotensin II, dopamine and insulin, involving NHE3 and Na+-K+-ATPase.Dopamine is now recognized as an important regulator of water and sodium excretion,acting via binding with dopamine receptors, which belong to the G protein-coupled receptor(GPCR) superfamily. Dopamine receptors are divided into two groups: D1-like receptors(D1R and D5R) and D2-like receptors(D2R, D3 R, and D4R). All dopamine receptors are located along the nephron and with highest expression on PT. Activation of dopamine receptors, especially the D1 R and D3 R, induces natriuresis and diuresis. In renal proximal tubule cells from Wistar-Kyoto(WKY) rats(WKY RPT cells), activation of D3 R can inhibit Na+-K+-ATPase activity.The renin-angiotensin system(RAS) is a major contributor to renal sodium transport and blood pressure. Angiotensin II is the primary peptide that mediates the effects of the RAS by binding to two receptors, angiotensin II type 1 receptor(AT1R) and angiotensin II type 2 receptor(AT2R), which have opposing effects. The renal expression of AT1 R is greater than AT2 R, which accounts for approximately 5% of total angiotensin II receptorbinding in the RPT. Under normal circumstances, the AT1 R masks the renal effects of the AT2 R. Activation of AT2 R can decrease renal sodium transport by inhibiting Na+-K+-ATPase and NHE3 activities in RPTs.Dopaminergic system interacts with RAS and endothelin system to regulate sodium excretion and blood pressure. Inhibition of renal proximal tubular angiotensin II production or blockade of AT1 R increases the natriuretic effect of the D1-like agonist, fenoldopam.D1-like and D2-like receptor agonists also antagonize the stimulatory effect of angiotensin II,acting via AT1 R, on renal proximal tubular luminal sodium transport. Both D1-like receptors,D1 R and D5 R are involved in this interaction with the AT1 R. All the D2-like receptors, i.e.,D2 R, D3 R, and D4 R have also been reported to negatively regulate AT1 R expression. AT2 R may mediate the natriuresis induced by D1-like receptors. However, an interaction between D2-like receptors and the AT2 receptor has not been reported. In RPT cells from WKY rats,D3 R negatively regulate AT1 receptors protein expression whereas in RPT cells from spontaneously hypertensive rat(SHR), the interaction is impaired. D3 R, as with AT2 receptors, are located mainly in RPTs also decrease sodium transport via inhibition sodium transporters. Taken together, we tested the hypothesis that D3 R and AT2 R interact to regulate renal sodium transport in vivo and in vitro, involving natriuresis and diuresis in vivo and inhibit Na+-K+-ATPase activity in vitro. This will help to enrich the mechanism involving the interaction between renal dopaminergic system and RAS on regulation of sodium excretion and blood pressure.ObjectiveTo uncover the interaction of renal dopamine D3 receptor with angiotensin II type 2receptor on natriuresis and diuresis in Wistar rats and Na+-K+-ATPase activity in WKY RPT cells.Methods1. Wistar rats were used to perform intrarenal arterial infusion with D3 R agonist PD128907, D3 R antagonist U99194 A, AT2 R agonist CGP42112 A, AT2 R antagonist PD123319, alone or in combination, urines were collected and measured using an electrolyte analyzer.2. WKY RPT cells were treated with PD128907, U99194 A, CGP42112 A and PD123319, alone or in combination. Ouabain was used to assay Na+-K+-ATPase activity.3. WKY RPT cells treated with PD128907, CGP42112 A alone or together, and thesections of Wistar rat kidneys were used to check the colocalization of D3 R and AT2 R by laser confocal microscopy, using immunofluorescence staining.4. WKY RPT cells treated with PD128907, CGP42112 A alone or in combination, and renal cortex homogenates of Wistar rats were used to check the physical linkage of D3 R and AT2 R by coimmunoprecipitation.5. WKY RPT cells were used in this experiment to explore the mechanism among which D3 R interact with AT2 R.Results1. To observe whether the interaction between D3 R and AT2 R promote water and sodium excretion in vivo, Wistar rats were used to intrarenal arterially infused with PD128907 and CGP42112 A alone or in combination. PD128907 increased urine flow(V)and absolute sodium excretion(UNa V) and the effects were completely blocked by U99194 A. CGP42112 A also induced a diuresis and natriuresis in a dose-dependent manner and the effects were completely blocked by PD123319. The simultaneous infusion of both PD128907 and CGP42112A(0.5 μg/kg/min) produced a greater than an additive increase in V and UNa V. The enhanced increase in V and UNa V were blocked by either D3 R or AT2 R antagonist. This demonstrates that co-stimulation of D3 R or AT2 R induces synergistic effect on natriuresis and dieresis, which is D3 R or AT2 R speicifc, respectively.2. To observe whether the interaction between D3 R and AT2 R affect Na+-K+-ATPase activity in vitro, WKY RPT cells treated with PD128907 and CGP42112 A, alone or in combination were used to check the Na+-K+-ATPase activity using oubain method.PD128907 inhibited Na+-K+-ATPase activity in a concentration-dependent manner, the effect was blocked by U99194 A. CGP42112 A also inhibited Na+-K+-ATPase activity in a concentration-dependent manner, PD123319 completely blocked the effect. The co-stimulation of PD128907(10-8M) and CGP42112A(10-10M) inhibited Na+-K+-ATPase activity to a greater extent more that an additive effect. This shows that co-stimulation of D3 R or AT2 R results in an amplified inhibitory effect on Na+-K+-ATPase activity, which is D3 R or AT2 R speicifc, respectively.3. The physical interaction between D3 R and AT2 R were checked by laser confocal microscopy, using fluorescence staining. D3 R colocalized with AT2 R in RPTs of Wistar rats.In WKY RPT cells, in basal conditions, D3 R and AT2 R are slight intracellular co-localization. Either PD128907 or CGP42112 A resulted in granular staining at the membrane and cytoplasm. Simultaneous stimulation by PD128907 and CGP42112 A led to a strong granular staining of both receptors at the membrane and cytoplasm, and enhanced their co-localization.4. The physical interaction between D3 R and AT2 R were also checked by coimmunoprecipitation. D3 R an AT2 R were found co-immunoprecipitated in renal cortex homogenates of Wistar rats and WKY RPT cells. Simultaneous stimulation by PD128907 and CGP42112A promoted their co-immunopreciptation to a greater extent than an additive increase. It shows there is a physical interaction between D3 R and AT2 R, enhanced by co-stimulation of D3 R an AT2 R.5. Mitogen-activated protein kinases(MAPK) and extracellular signal-regulated kinase(ERK) are involved in the signaling pathway of both D3 R an AT2 R, to explore whether MAPK-ERK signaling are involved in the enhanced effect observed above, we checked the expression of phosphor-ERK in WKY RPT cells treated with PD128907 and CGP42112 A,alone or in combination. It shows that PD128907 in combination with CGP42112 A enhanced the protein expression of phosphor-ERK. Furthermore, the MAPK inhibitor PD98059 was used in combination with PD128907 and CGP42112 A. PD98059 reduced the enhanced effect of PD128907 and CGP42112 A on Na+-K+-ATPase activity, as well as the co-localization and co-immunoprecipitation of D3 R and AT2 R in WKY RPT cells. It suggests that MAPK-ERK signal pathway is associated with the synergistic effect of D3 R and AT2 R.ConclusionsOur current study shows that there is interaction between D3 R and AT2 R, enhanced by co-stimulation of D3 R and AT2 R, leading to amplified inhibitory effect on Na+-K+-ATPase activity and enhanced diuresis and natriuresis, which may be associated with MAPK-ERK signal pathway. We demonstrate that dopamine receptor interacts with angiotensin Ⅱreceptor to play a synergistic effect in spite of the antagonized effect reported previously.The synergistic effect is associated with enhanced interaction between receptors, as well as the amplified common signaling pathway. This suggests the synergy between peripheral dopamine system and RAS in addition to the antagonism in regulation of water and sodiumexcretion. This study raises the new mechanism between dopamine system and RAS,providing new evidence to the prevention and treatment of hypertension.
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