Role of the D1A dopamine receptor in the pathogenesis of genetic hypertension.

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 D1 receptor signal in renal proximal tubules, resulting in decreased inhibition of sodium transport (Na+/H+ exchanger [NHE] and Na+/K+ATPase activity) by dopamine. To determine if impaired D1 receptor regulation of NHE in proximal tubules is related to hypertension, studies were performed in a F2 generation from female Wistar Kyoto (WKY) and male SHR crosses. A D1 agonist, SKF 81297, inhibited (37.6 +/- 4.7%) NHE activity in brush border membranes of normotensive F2s (systolic blood pressure < 140 mm Hg, n = 7) but not in hypertensive F2s (n = 21). Furthermore, a D1 agonist, SKF 38393, when infused into the renal artery, dose dependently increased sodium excretion in normotensive F2s (n = 3) without altering renal blood flow but was inactive in hypertensive F2s (n = 21). Since the major D1 receptor gene expressed in renal proximal tubules is the D1A subtype, we determined the importance of this gene in the control of blood pressure in mice lacking functional D1A receptors. Systolic blood pressure was greater in homozygous (n = 6) and heterozygous (n = 5) mice compared to normal sex matched litter mate controls (n = 12); moreover, the mice lacking one or both D1A alleles developed diastolic hypertension. The cosegregation with hypertension of an impaired D1 receptor regulation of renal sodium transport and the development of elevated systolic and diastolic pressure in mice lacking one or both D1A alleles suggest a causal relationship of the D1A receptor gene with hypertension.

[Regulation of NHE3 by D1 dopamine receptor during development of spontaneously hypertensive rats].

OBJECTIVE: To determine if spontaneous hypertension is secondary to defective interaction among dopamine receptor, G protein, and Na(+)/H(+) exchanger 3 (NHE3). METHODS: The inhibitory effect of a D(1) dopamine agonist upon NHE3 activity and its impact upon G(s)alpha/NHE3 binding in renal brush border membrane (BBM) of spontaneously hypertensive rats (SHRs) 2 - 3 weeks before and 12 weeks after the establishment of hypertension were examined. In order to avoid the confounding influence of second messenger on D(1) receptor/NHE3 interaction, study was made in BBM devoid of cytoplasmic component. RESULTS: NHE3 activity increased with age in Wister-Kyoto (WKY) rats but not in SHRS. D1 receptor expression did change with age in both WKY rats and SHRs. The inhibitory effect of a D(1)-like agonist on NHE3 activity increased with age in WKY rats but not in SHRs. In WKY rats, another D(1)-like agonist, fedoldopam, increased G(s)alpha/NHE3 binding to the same extent in 2 week old and adult rats, but decreased the amount of G(s)(alpha)bound to NHE3 in 2 week old SHRs. CONCLUSION: The decrease of inhibitory effect of D(1)-like agonist upon NHE3 activity in SHRs precedes the development of hypertension. Spontaneous hypertension may be caused, in part, by a decreased interaction between G(s)alpha and NHE3 in BBM secondary to D(1)-like receptor function.

Renal dopamine receptors and pre- and post-cAMP-mediated Na+ transport defect in spontaneously hypertensive rats.

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)

Dopamine and diltiazem-induced natriuresis in the spontaneously hypertensive rat.

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.

Stimulation of Na(+)-K(+)-2Cl- cotransport in rat medullary thick ascending limb by dopamine.

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.

cAMP-independent, G protein-linked inhibition of Na+/H+ exchange in renal brush border by D1 dopamine agonists.

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.

Gbeta regulation of Na/H exchanger-3 activity in rat renal proximal tubules during development.

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.

Regulation of NHE3 activity by G protein subunits in renal brush-border membranes.

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.

Dopamine(1) receptor, G(salpha), and Na(+)-H(+) exchanger interactions in the kidney in hypertension.

The ability of dopamine(1) (D(1)) receptors to inhibit luminal Na(+)-H(+) 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(salpha), or effector proteins. The coupling of the D(1) receptor to G(salpha) and NHE3 was studied in renal brush border membranes (BBM), devoid of cytoplasmic second messengers. D(1) receptor, G(salpha), and NHE3 expressions were similar in SHR and their normotensive controls, Wistar-Kyoto rats (WKY). Guanosine-5'-O:-(3-thiotriphosphate) (GTPgammaS) decreased NHE activity and increased NHE3 linked with G(salpha) similarly in WKY and SHR, indicating normal G(salpha) and NHE3 regulation in SHR. However, D(1) agonists increased NHE3 linked with G(salpha) 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, GTPgammaS 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(salpha)/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(salpha)/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.

D(1) dopamine receptor regulation of NHE3 during development in spontaneously hypertensive rats.

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.