Aberrant Regulation Of D₃ Dopamine Receptor On ETB Receptor In Renal Proximal Tubule Cells From Spontaneously Hypertensive Rats

BackgroundKidney plays an important role in the long-term regulation of blood pressure and is the major organ involved in the regulation of sodium homeostasis. The proximal tubule is pre-eminent in the overall ability of the kidney to regulate sodium balance, which can reabsorpt approximately 70% of sodium, chloride and water, 100% of glucose and amino acid from ultrafiltrate. Na⁺-K⁺-ATPase, located in the basolateral membrane, can transport the sodium from urine into renal interstitium to maintain the different sodium concentration between the renal tubular fluid and interstitium. Therefore, the slight changes of Na⁺-K⁺-ATPase activity might cause significant sodium reabsorption in hypertensive states, subsequently increase blood pressure.The renal sodium excretion is regulated by many hormones or hormorals, including dopamine and endothelin systems. Endothlin, secreted in the renal proximal tubule, can regulate sodium excretion by autocrine or paracrine mechanisms, which has two receptor subtypes: ETA and ETB receptors. The renal inhibitory effect of endothelin on sodium reabsorption is via ETB receptor. The ETB receptor has been shown to decrease sodium transport in the renal medullary collecting duct and medullary thick ascending limb of Henle. However, both inhibitory and stimulatory effects of endothelin have been reported in the proximal tubule. Short-term stimulation of ETB receptors in opossum kidney cells, a renal proximal tubular cell line, activates the sodium hydrogen exchanger, NHE3. However, chronic treatment of the same opossum kidney cells by endothelin has an opposite effect on NHE3 activity. Thus, a 6-hour exposure of opossum kidney cells to endothelin-1 inhibits NHE3 expression and activity. Na⁺-K⁺-ATPase is an important sodium transporter in basolateral membranes, and our preliminary data show that ETB receptor exists in renal proximal tubules, we hypothesize that ETB receptor might have an inhibitory effect on Na⁺-K⁺-ATPase activities.Dopamine receptors are classified into D₁-like (D₁, D₅) and D₂-like (D₂, D₃, D₄) subtypes based on their structure and pharmacology. Previous studies show that activation of D₁ receptor induces diuresis, natriuresis and vasorelaxation. However, the D₂-like receptor function is not completely understood. Among D₂-like receptor, D₃ receptor is a major receptor subtype. Our previous studies show that disruption of the D₃ dopamine receptor gene in mice produces hypertension with impairment in sodium excretion, accompanied by decreased renal ETB receptor expressions, we hypothesize that there is an interaction between D₃ and ETB receptors, which may be engaged into the pathogenesis of essential hypertension. In this experiment, we will check our hypothesis in renal proximal tubule cells, and compare the difference between WKY (Wistar-Kyoto) and SHR (spontaneously hypertensive rat) cells.Objective1. To check the expression of D₃ and ETB receptors in renal proximal tubules.2. To investigate the effect of ETB receptor on Na⁺-K⁺-ATPase activity, and further check its signal pathway.3. To investigate the regulation of D₃ receptors on ETB receptor expressions, check whether there are different regulations between WKY and SHR cells, and whether the impaired regulation is involved into the pathogenesis of hypertension.Methods1. Immortalized renal proximal tubule (RPT) cells of WKY and SHR were used in this experiment. ETB and D₃ receptor localization was checked by immunohistochemistry and laser confocal microscopy.2. Effects of ETB receptor on Na⁺-K⁺-ATPaes activity were checked in RTP cells. To investigate the signal pathway of ETB receptor on Na⁺-K⁺-ATPaes activity, different blockers, such as: protein kinase A antagonist PKA 14-22, protein kinase C antagonist PKC 19-31 and calcium channel blocker, nicardipine, were pre-treated before ETB receptor agonist BQ3020.3. The regulation of D₃ receptor on ETB receptor was checked in D₃ receptor deficient mice and RPT cells. D₃ receptor protein expressions were checked by western blot, the sodium transportation by measurement of Na⁺-K⁺-ATPase activity. The difference between WKY and SHR cells was also compared in this study.Result1. D₃ and ETB receptors existed in RPT cells, and there was D₃/ETB receptor co-localization, which was distinct in WKY rats and poor in SHRs.2. Activation of ETB receptor concentration- and time-dependently decreased Na⁺-K⁺-ATPase activity. The maximal effect was about 36.1% at 15 minutes. Intracellular Ca²⁺ inflow was involved in this signal pathway, which included Gq/Ca²⁺ channel of cellular membrane-PI3K-IP₃-[Ca²⁺]i.3. Renal ETB receptor protein expression was lower in D₃ receptor deficient mice as compared with control mice. Activation of D₃ receptor increased ETB receptor protein expression in WKY cells, while decreased it in SHR cells. ETB receptor agonist, BQ3020 (10^-8M/15min), decreased Na⁺-K⁺-ATPase activity in WKY cells. However, the inhibitory effects were lost in SHR cells, and the basal levels of Na⁺-K⁺-ATPaes activity was higher in SHR cells than that in WKY cells. D₃ receptor, by itself, had no effect on Na⁺-K⁺-ATPase activity; pre-treatment with D₃ receptor agonist, PD128907 (10^-7M/24hrs), augmented the inhibitory effects of ETB receptor in WKY cells, not in SHR cells.Conclusion1. Renal ETB receptor regulates sodium reabsorption by inhibition of Na⁺-K⁺-ATPase activity, intracellular Ca²⁺ inflow was involved in this signal pathway, which included Gq/Ca²⁺ channel of cellular membrane-PI3K-IP₃-[Ca²⁺]i.2. The up-regulation of D₃ receptor on ETB receptor protein expression augments the inhibitory effect of ETB receptor on Na⁺-K⁺-ATPase activity in WKY cells; the impaired interaction between D₃ and ETB receptor is involved into the pathogenesis of hypertension.