Since dopamine produced by the kidney is an intrarenal regulator of sodium transport, an abnormality of the dopaminergic system may be important in the pathogenesis of hypertension. In the spontaneously hypertensive rat (SHR), in spite of normal renal production of dopamine and receptor density, there is defective transduction of the D 1 receptor signal in renal proximal tubules, resulting in decreased inhibition of sodium transport (Na

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.

Abstract-The ability of dopamine 1 (D 1 ) receptors to inhibit luminal Na ϩ -H ϩ exchanger (NHE) activity in renal proximal tubules and induce a natriuresis is impaired in spontaneously hypertensive rats (SHR). However, it is not clear whether the defect is at the level of the D 1 receptor, G s␣ , or effector proteins. The coupling of the D 1 receptor to G s␣ and NHE3 was studied in renal brush border membranes (BBM), devoid of cytoplasmic second messengers. D 1 receptor, G s␣ , and NHE3 expressions were similar in SHR and their normotensive controls, Wistar-Kyoto rats (WKY). Guanosine-5Ј-O-(3-thiotriphosphate) (GTP␥S) decreased NHE activity and increased NHE3 linked with G s␣ similarly in WKY and SHR, indicating normal G s␣ and NHE3 regulation in SHR. However, D 1 agonists increased NHE3 linked with G s␣ in WKY but not in SHR, and the inhibitory effects of D 1 agonists on NHE activity were less in SHR than in WKY. Moreover, GTP␥S enhanced the inhibitory effect of D 1 agonist on NHE activity in WKY but not in SHR, suggesting an uncoupling of the D 1 receptor from G s␣ /NHE3 in SHR. Similar results were obtained with the use of immortalized renal proximal tubule cells from WKY and SHR. We conclude that the defective D 1 receptor function in renal proximal tubules in SHR is proximal to G s␣ /effectors and presumably at the receptor level. The mechanism(s) responsible for the uncoupling of the D 1 receptor from G proteins remains to be determined. Because the primary structure of the D 1 receptor is not different between normotensive and hypertensive rats, differences in D 1 receptor posttranslational modification are possible. Key Words: dopamine Ⅲ receptors, dopamine Ⅲ G protein Ⅲ rats, inbred SHR R enal dopamine production, dopamine receptors, and dopamine receptor regulation are important in the pathogenesis of hypertension. 1 Dopamine, produced by renal proximal tubules, is an important paracrine/autocrine inhibitor of renal sodium transport under conditions of sodium loading. 1 The inhibition of sodium transport in renal proximal tubules by dopamine is exerted via the Na ϩ -H ϩ exchanger (NHE) at the luminal or brush border membrane (BBM) and via Na ϩ ,K ϩ -ATPase at the basolateral membrane. 1 The major transport of sodium across the luminal membrane of renal proximal tubules is caused by NHE activity. 2 Five of the 6 isoforms of NHE are expressed in the kidney. 3,4 However, the NHE3 isoform predominates in the BBM of rat renal proximal tubules. 2,5 There is an impaired ability of dopamine 1 (D 1) -like receptors to inhibit NHE activity in BBM of the spontaneously hypertensive rat (SHR). 1 The decreased inhibitory effect of D 1 -like receptors on NHE activity in renal proximal tubules and failure to induce a natriuresis cosegregate with hypertension in SHR and normotensive WistarKyoto rat (WKY) crossbreeds. 6 However, it is not clear whether the impaired inhibitory action of D 1 -like receptors on NHE activity is at the receptors, G proteins, signal transducers, or effectors. G proteins can regulate NHE acti...

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.

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)