Aberrant Regulation Of D₄Dopamine Receptor On AT₁Receptor In Renal Proximal Tubule Cells From Spontaneously Hypertensive Rats

BackgroundEssential hypertension is a clinical factor harmful to human health. It is a major riskfactor for stroke, myocardial infarction, and heart and kidney failure. It is helpful to preventand treat hypertension by clarify its pathogenesis. Hypertension occurs because of theincreased activity of prohypertensive systems and/or decreased activity of anti-hyrtensivesystems, one of which is the dopaminergic system. Therefore, a dysfunction in thedopaminergic pathway may be a mechanism in the development of essential hypertension.The dopamine system’s role in regulating blood pressure gradually has been recognized. Inthe human kidneys are the major organs responsible for the excretion of sodium. It isthrough the regulation of urinary sodium reabsorption to regulate blood pressure. In variousparts of the kidney, the renal proximal tubule is the most obvious effect, the section onurinary sodium reabsorption capacity is more than the entire kidney reabsorption of65-70%,and meanwhile, the segment is one of the important parts of synthesis of dopamine. Alldopamine receptor subtypes are directly or indirectly involved in urinary sodium excretion.It plays a role natriuretic drainage after dopamine binding to its receptor in the kidney.When the sodium overloading, the dopamine can be adjusted more than50%sodiumexcretion. Dopamine receptors are classified into D1-like （D1, D5） and D2-like （D2, D3, D₄）subtypes.Reported that systolic and diastolic blood pressures were elevated in D₄receptor-deficient （D₄-/-） mice compared with D₄wild-type （D₄+/+） littermates. Basal renaland plasma renin concentrations were similar in the2mouse strains. The protein expressionof angiotensin â…¡ type1receptor was increased in homogenates of kidney and brain of D₄-/-mice relative to D₄+/+mice. We hypothesize that D₄receptor aberrant regulation of the AT₁receptor is involved in the process of hypertension.To verify the hypothesis, renal proximal tubule cells from Wistar-Kyoto rats andspontaneously hypertensive rats were studied. We investigated the role of D₄receptor on AT₁receptor expression and function in the normal and hypertension status, and to explorethe dopamine D₄receptor in the pathogenesis of hypertension.Content：Part oneTo investigate the effect of D₄receptor on AT₁receptor in renal proximal tubule cellsfrom Wistar-Kyoto rats.1. To determine the colocalization of dopamine D₄receptor and AT₁receptor in renalproximal tubule cells from Wistar-Kyoto rats.2. To analyze AT₁receptor protein expression treated with different concentrationsand different time by PD168077, a D₄receptor agonist, in renal proximal tubule cells fromWistar-Kyoto rats.3. To detect the special effect of D₄receptor on AT₁receptor protein by L745870, a D₄receptor antagonist, in renal proximal tubule cells from Wistar-Kyoto rats.Part twoTo investigate the singnal transduction pathy of dopamine D₄receptor on AT₁receptorin renal proximal tubule cells from Wistar-Kyoto rats.Renal proximal tubule cells are treated individually with PKC inhibitor and calciumchannel blockerto investigate the signal transduction pathway.Part threeTo investigate the abnormal regulation of dopamine D₄receptor on AT₁receptorprotein expression and functional in SHR cells.1. Renal proximal tubule cells from Wistar-Kyoto rats and spontaneously hypertensiverats were treated with D₄receptor agonist PD168077and observed the abnormal regulationof dopamine D₄receptor on AT₁receptor protein expression.2. Renal proximal tubule cells from Wistar-Kyoto rats and spontaneously hypertensiverats were stimulated by D₄receptor agonist and Ang-â…¡, and Na+/K+-ATP enzyme activitywas measured.Methods：1. The renal proximal tubule cell culture.2. AT₁receptor protein expression was measured by western blot.3. The coexistence between D₄receptor and AT₁receptor was checked by immunization confocal.4. Na+/K+-ATP enzyme activity was measured as the ouabainsensitive dephosphorylationof （tris）-p-nitrophenyl phosphate by K-pnitrophenyl phosphatase.Result:1. D₄receptor-positive and AT₁receptor-positive cells coexist in renal proximal tubulecells from WKY. Activation of D₄receptor concentration-and time-dependently decreasesAT₁receptor expression in WKY cells, and the inhibitory effect can be blocked by specificD₄receptor antagonist L745870. The phenomenon suggest that the inhibition isaccomplished through the D₄receptor.2. Using PKC inhibitor and calcium channel blocker to investigate the regulatingmechanisms of D₄receptor on AT₁receptor expression, the results show that the inhibitionof D₄receptors on AT₁receptor protein is blocked by nicardipine （10-8mol/L） and PKCinhibitor. We conclude that the inhibitory effect may be through calcium channels--PKC.3. Activation of D₄receptor decreases AT₁receptor expression and inhibitsAng-â…¡-induced Na+/K+-ATP enzyme activity in WKY cells, but the effect is impaired inSHR cells.Conclusion:Stimulation of D₄receptor reduces AT₁receptor expression and inhibits AT₁receptorfunction but it is damaged in essential hypertension state. The regulation may be throughactivation of PLC--calcium channel--PKC pathway. The abnormal regulation of D₄receptoron AT₁receptor is involved in the pathogenesis of essential hypertension.