Differential regulation of renal phospholipase C isoforms by catecholamines.

Dopamine and D1 agonists and NE all increase phosphatidyl inositol-specific phospholipase C (PLC) activity, but whereas dopamine produces a natriuresis, NE has an antinatriuretic effect. To determine if catecholamines differentially regulate the expression of PLC isoforms, we infused fenoldopam, a D1 agonist, or pramipexole, a D1/D2 agonist, intravenously or infused fenoldopam or NE into the renal artery of anesthetized rats. After 3-4 h of infusion, when the expected natriuresis (fenoldopam or pramipexole) or antinatriuresis (NE) occurred, the kidneys were removed for analysis of PLC isoform protein expression activity. Western blot analysis revealed that in renal cortical membranes, fenoldopam and pramipexole increased expression of PLC beta 1 and decreased expression of PLC gamma 1; PLC delta was unchanged. In the cytosol, pramipexole and fenoldopam increased expression of both PLC beta 1 and PLC gamma 1. No effects were noted in the medulla. A preferential D1 antagonist, SKF 83742, which by itself had no effect, blocked the effects of pramipexole, thus confirming the involvement of the D1 receptor. In contrast, NE also increased PLC beta 1 but did not affect PLC gamma 1 protein expression in membranes. The changes in PLC isoform expression were accompanied by similar changes in PLC isoform activity. These studies demonstrate for the first time differential regulation of PLC isoforms by catecholamines.

Disruption of the dopamine D3 receptor gene produces renin-dependent hypertension.

Since dopamine receptors are important in the regulation of renal and cardiovascular function, we studied the cardiovascular consequences of the disruption of the D3 receptor, a member of the family of D2-like receptors, expressed in renal proximal tubules and juxtaglomerular cells. Systolic and diastolic blood pressures were higher (approximately 20 mmHg) in heterozygous and homozygous than in wild-type mice. An acute saline load increased urine flow rate and sodium excretion to a similar extent in wild-type and heterozygous mice but the increase was attenuated in homozygous mice. Renal renin activity was much greater in homozygous than in wild-type mice; values for heterozygous mice were intermediate. Blockade of angiotensin II subtype-1 receptors decreased systolic blood pressure for a longer duration in mutant than in wild-type mice. Thus, disruption of the D3 receptor increases renal renin production and produces renal sodium retention and renin-dependent hypertension.

Dopamine D1 receptor and protein kinase C isoforms in spontaneously hypertensive rats.

-Dopamine, via D1-like receptors, stimulates the activity of both protein kinase A (PKA) and protein kinase C (PKC), which results in inhibition of renal sodium transport. Since D1-like receptors differentially regulate sodium transport in normotensive and hypertensive rats, they may also differentially regulate PKC expression in these rat strains. Thus, 2 different D1-like agonists (fenoldopam or SKF 38393) were infused into the renal artery of anesthetized normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) (n=5 to 6/drug/strain). Ten or 60 minutes after starting the D1-like agonist infusion, both the infused kidney and the noninfused kidney that served as control were prepared for analysis. The D1-like agonists produced a greater diuresis and natriuresis and inhibited Na+,K+-ATPase activity in proximal tubule (PT) and medullary thick ascending limb (mTAL) to a greater extent in WKY (Delta20+/-1%) than in SHR (Delta7+/-1%, P<0.001). D1-like agonists had no effect on PKC-alpha or PKC-lambda expression in either membrane or cytosol but increased PKC-theta expression in PT in both WKY and SHR at 10 minutes but not at 60 minutes. However, membranous PKC-delta expression in PT and mTAL decreased in WKY but increased in SHR with either 10 or 60 minutes of D1-like agonist infusion. D1-like agonists also decreased membranous PKC-zeta expression in PT and mTAL in WKY but increased it in PT but not in mTAL in SHR. We conclude that there is differential regulation of PKC isoform expression by D1-like agonists that inhibits membranous PKC-delta and PKC-zeta in WKY but stimulates them in SHR; this effect in SHR is similar to the stimulatory effect of norepinephrine and angiotensin II and may be a mechanism for their differential effects on sodium transport.

D1 dopamine receptor hyperphosphorylation in renal proximal tubules in hypertension.

A defect in the coupling of the D(1) receptor (D(1)R) to its G protein/effector complex in renal proximal tubules plays a role in the pathogenesis of spontaneous hypertension. As there is no mutation of the D(1)R gene in the spontaneously hypertensive rat (SHR), we tested the hypothesis that the coupling defect is associated with constitutive desensitization/phosphorylation of the D(1)R. The following experiments were performed: (1) Cell culture and membrane preparations from rat kidneys and immortalized rat renal proximal tubule cells (RPTCs); (2) immunoprecipitation and immunoblotting; (3) cyclic adenosine 3',5' monophosphate and adenylyl cyclase assays; (4) immunofluorescence and confocal microscopy; (5) biotinylation of cell surface proteins; and (6) in vitro enzyme dephosphorylation. Basal serine-phosphorylated D(1)Rs in renal proximal tubules, brush border membranes, and membranes from immortalized RPTCs were greater in SHRs (21.0+/-1.5 density units, DU) than in normotensive rats (7.4+/-2.9 DU). The increased basal serine phosphorylation of D(1)Rs in SHRs was accompanied by decreased expression of D(1)R at the cell surface, and decreased ability of a D(1)-like receptor agonist (fenoldopam) to stimulate cyclic adenosine 3',5' monophosphate (cAMP) production. Increasing protein phosphatase 2A activity with protamine enhanced the ability of fenoldopam to stimulate cAMP accumulation (17+/-4%) and alter D(1)R cell surface expression in intact cells from SHRs. Alkaline phosphatase treatment of RPTC membranes decreased D(1)R phosphorylation and enhanced fenoldopam stimulation of adenylyl cyclase activity (26+/-6%) in SHRs. Uncoupling of the D(1)R from its G protein/effector complex in renal proximal tubules in SHRs is caused, in part, by increased D(1)R serine phosphorylation.

Renal nerves and D1-dopamine receptor-mediated natriuresis.

The resistance of the spontaneously hypertensive rat (SHR) kidney to the natriuretic effect of dopamine and D1 agonists may be due to increased renal nerve activity. Therefore, we compared the effects of the intrarenal arterial infusion of the D1 agonist, SKF 38383, into the denervated (DNX) kidney of saline-loaded-anesthetized SHR and its control, the Wistar-Kyoto (WKY) rat. In both WKY and SHR, DNX of the left kidney slightly decreased urine flow (UV) and absolute (UNaV) and fractional sodium excretion (FENa) in the innervated right kidney; neither vehicle nor D1 agonist infusion exerted any effect. In the left kidney, denervation increased UV, UNaV, and FENa to a similar degree in WKY and SHR (2-fold), without affecting renal blood flow, glomerular filtration rate, or blood pressure. In WKY but not in SHR, after DNX, the D1 agonist dose-dependently increased UV, UNaV, and FENa in the denervated kidney. We conclude that the decreased natriuretic effect of D1 agonists in the SHR is not due to increased renal nerve activity. These data support our previous studies implicating a defect of the D1 receptor or its regulation in the kidney in genetic hypertension.

Role of renal dopamine D1 receptors in natriuresis induced by calcium channel blockers.

The direct tubular natriuretic effect of calcium channel blockers (CCBs) may be due to an interaction between CCBs and a renal tubular dopamine receptor. We therefore studied the effects of two chemically unrelated CCBs, diltiazem and isradipine, infused into the right renal artery of 5% saline-loaded anesthetized rats alone or in the presence of a D1 antagonist, SKF-83742. Isradipine (0.03 microgram.kg-1.min-1) or diltiazem (20 but not 10 micrograms.kg-1.min-1) alone produced an increase in urine flow and an approximate doubling of absolute and fractional sodium excretion, which was not seen in the left kidney or in the control animals (analysis of variance, Scheffé's test, P < 0.05). SKF-83742 alone given systemically or into the right renal artery did not affect these parameters but did block the actions of diltiazem or isradipine. There was no change in mean arterial pressure, renal blood flow, or glomerular filtration rate in any of the experiments. In additional studies, we found that a combined infusion of dopamine (0.1 microgram.kg-1.min-1) and diltiazem (10 micrograms.kg-1.min-1) (doses that by themselves did not alter renal function) produced a twofold or greater increase in urine flow and absolute and fractional sodium excretion; glomerular filtration rate was not significantly changed. Intrarenal arterial CCBs, without a change in renal hemodynamics, produce a natriuresis that is blocked by a D1 antagonist. Concomitant administration of diltiazem and dopamine (each in subeffective doses when used alone) produces a synergistic effect.(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.

Renal protein phosphatase 2A activity and spontaneous hypertension in rats.

The impaired renal paracrine function of dopamine in spontaneously hypertensive rats (SHR) is caused by hyperphosphorylation and desensitization of the renal D(1) dopamine receptor. Protein phosphatase 2A (PP(2A)) is critical in the regulation of G-protein-coupled receptor function. To determine whether PP(2A) expression and activity in the kidney are differentially regulated in genetic hypertension, we examined the effects of a D(1)-like agonist, fenoldopam, in renal cortical tubules and immortalized renal proximal tubule cells from normotensive Wistar-Kyoto rats (WKY) and SHR. In cortical tubules and immortalized proximal tubule cells, PP(2A) expression and activities were greater in cytosol than in membrane fractions in both WKY and SHR. Although PP(2A) expressions were similar in WKY and SHR, basal PP(2A) activity was greater in immortalized proximal tubule cells of SHR than WKY. In immortalized proximal tubule cells of WKY, fenoldopam increased membrane PP(2A) activity and expression of the regulatory subunit PP(2A)-B56alpha, effects that were blocked by the D(1)-like antagonist SCH23390. Fenoldopam had no effect on cytosolic PP(2A) activity but decreased PP(2A)-B56alpha expression. In contrast, in immortalized proximal tubule cells of SHR, fenoldopam decreased PP(2A) activity in both membranes and cytosol but predominantly in the membrane fraction, without affecting PP(2A)-B56alpha expression; this effect was blocked by the D(1)-like antagonist SCH23390. We conclude that renal PP(2A) activity and expression are differentially regulated in WKY and SHR by D(1)-like receptors. A failure of D(1)-like agonists to increase PP(2A) activity in proximal tubule membranes may be a cause of the increased phosphorylation of the D(1) receptor in the SHR.

Ontogeny of DA1 receptor-mediated natriuresis in the rat: in vivo and in vitro correlations.

The natriuretic and diuretic effects of dopamine are attenuated in the young. Because dopamine has actions on receptors (e.g., adrenergic, serotonin) other than dopamine, we studied a novel dopamine agonist, pramipexole, which has a selectivity to both DA1 and DA2-receptor subtypes. Intravenous administration of pramipexole resulted in a dose-related (1, 10, and 100 micrograms.kg-1.min-1) increase in urine flow and absolute and fractional sodium excretion and a decrease in mean arterial pressure (MAP) in three groups of rats studied. Pramipexole induced a greater decrease in MAP in 6- to 7- (n = 5) and 9- to 16- (n = 6) than in 3- to 4-wk-old (n = 8) rats; the natriuresis and diuresis were greatest in 12- to 16- and least in 3- to 4-wk-old rats. The renal effects of pramipexole were mainly due to actions at the DA1 receptor, since these effects were completely blocked by the coinfusion of a DA1 antagonist, SKF 83742. To explore further a cause of the attenuated natriuretic effect of pramipexole in the young, we studied the effect of a selective DA1-receptor agonist, fenoldopam, on amiloride-sensitive 22Na+ uptake in renal brush-border membrane vesicles. The 3-s amiloride-sensitive uptake was inhibited (45%) by fenoldopam (5 x 10(-5)M) in 9- to 16- (n = 6) but not in 3- to 4-wk-old (n = 5) rats. These studies suggest that the attenuated natriuretic effect of dopamine in the young is in part due to decreased DA1 action on the brush-border membrane Na(+)-H+ exchanger.

Effects of costimulation of dopamine D1- and D2-like receptors on renal function.

In vitro studies have suggested that dopamine D1- and D2-like receptors interact to inhibit renal sodium transport. We used Z-1046, a dopamine receptor agonist with the rank-order potency D3 >/= D4 > D2 > D5 > D1, to test the hypothesis that D1- and D2-like receptors interact to inhibit renal sodium transport in vivo in anesthetized rats. Increasing doses of Z-1046, administered via the right renal artery, increased renal blood flow (RBF), urine flow, and absolute and fractional sodium excretion without affecting glomerular filtration rate. For determination of the dopamine receptor involved in the renal functional effects of Z-1046, another group of rats received Z-1046 at 2 microgram . kg-1 . min-1 (n = 10) in the presence or absence of the D2-like receptor antagonist domperidone and/or the D1-like antagonist SCH-23390. Domperidone alone had no effect but blocked the Z-1046-mediated increase in urine flow and sodium excretion; it enhanced the increase in RBF after Z-1046. SCH-23390 by itself decreased urine flow and sodium excretion without affecting RBF and blocked the diuretic, natriuretic, and renal vasodilatory effect of Z-1046. We conclude that the renal vasodilatory effect of Z-1046 is D1-like receptor dependent, whereas the diuretic and natriuretic effects are both D1- and D2-like receptor dependent.

Differential expression of adenylyl cyclases in the rat nephron.

BACKGROUND: Adenylyl cyclases (ACs) are a family of enzymes that catalyze the formation of the second-messenger cyclic adenosine 3',5'-monophosphate (cAMP). At least nine isoforms of AC have been cloned. These isoforms differ in their tissue distribution and basal activity. AC isoforms also differ in their capacity to be stimulated or inhibited by G protein alpha(i), alpha(s) and beta/gamma subunits, protein kinase C, and intracellular calcium. The distribution of ACs in the kidney is only partially known, although it is known that ACs play important roles in kidney signal transduction. Several receptors are known to couple to AC, but their linkage to individual AC isoforms in the kidney is not known. METHODS: This study investigated the tissue distribution of AC isoforms along the nephron of Wistar-Kyoto rats using reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and immunoblotting. RESULTS: While AC VI and IX mRNA were found in all nephron segments, there was no expression of AC VIII mRNA. ACs II through V and VII mRNA were variably found in specific nephron segments. mRNA for AC isoforms II, III, VI, VII, and IX were expressed in renal proximal tubules. All of the AC isoforms studied, except VIII, were found in glomeruli. Immunoblotting and immunohistochemistry confirmed the mRNA results. AC isoforms II, III, IV, and IX were expressed in luminal rather than in basolateral membranes. However, immunohistochemical studies were not feasible for the other isoforms that could be expressed in basolateral membranes. CONCLUSION: Knowledge of the distribution of ACs may help establish the linkage between receptors and specific AC isoforms and define their functions.

The effect of docarpamine, a dopamine pro-drug, on blood pressure and catecholamine levels in spontaneously hypertensive rats.

We studied the effects of bolus intravenous injection of the dopamine prodrug, docarpamine (200 microg/kg), on mean arterial pressure (MAP) and heart rate (HR) in Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs). In WKY rats (n=18), MAP and HR increased 5 min after docarpamine and then returned to baseline levels within 15 min. In contrast, in SHRs (n=15), MAP and HR gradually decreased, reaching a nadir 20 min after injection. Five min after docarpamine, plasma dopamine and 3,4-dihydroxy phenyl acetic (DOPAC) levels increased in both WKY rats (n=5) and SHRs (n=5). The docarpamine-induced changes in MAP and HR in both rat strains (n=5/strain) were blocked by the D1-like antagonist, SCH23390. alpha-Adrenergic (n=4) and vasopressin V1 (n=3) receptor blockade also abrogated the effects of docarpamine in WKY rats. We conclude that docarpamine differentially affects MAP and HR in WKY and SHRs. In SHRs, the depressor and bradycardiac effects of docarpamine are mediated by D1-like receptors. In WKY rats, the pressor and tachycardiac responses are caused by an interaction among D1-like, alpha-adrenergic, and V1 receptors.

Adrenergic and endothelin B receptor-dependent hypertension in dopamine receptor type-2 knockout mice.

Polymorphism of the dopamine receptor type-2 (D(2)) gene is associated with essential hypertension. To assess whether D(2) receptors participate in regulation of blood pressure (BP), we studied mice in which the D(2) receptor was disrupted. In anesthetized mice, systolic and diastolic BPs (in millimeters of mercury) were higher in D(2) homozygous and heterozygous mutant mice than in D(2)+/+ littermates. BP after alpha-adrenergic blockade decreased to a greater extent in D(2)-/- mice than in D(2)+/+ mice. Epinephrine excretion was greater in D(2)-/- mice than in D(2)+/+ mice, and acute adrenalectomy decreased BP to a similar level in D(2)-/- and D(2)+/+ mice. An endothelin B (ET[B]) receptor blocker for both ET(B1) and ET(B2) receptors decreased, whereas a selective ET(B1) blocker increased, BP in D(2)-/- mice but not D(2)+/+ mice. ET(B) receptor expression was greater in D(2)-/- mice than in D(2)+/+ mice. In contrast, blockade of ET(A) and V(1) vasopressin receptors had no effect on BP in either D(2)-/- or D(2)+/+ mice. The hypotensive effect of an AT(1) antagonist was also similar in D(2)-/- and D(2)+/+ mice. Basal Na(+),K(+)-ATPase activities in renal cortex and medulla were higher in D(2)+/+ mice than in D(2)-/- mice. Urine flow and sodium excretion were higher in D(2)-/- mice than in D(2)+/+ mice before and after acute saline loading. Thus, complete loss of the D(2) receptor results in hypertension that is not due to impairment of sodium excretion. Instead, enhanced vascular reactivity in the D(2) mutant mice may be caused by increased sympathetic and ET(B) receptor activities.

Impaired renal D(1)-like and D(2)-like dopamine receptor interaction in the spontaneously hypertensive rat.

D(1)-like (D(1), D(5)) and D(2)-like (D(2), D(3), D(4)) dopamine receptors interact in the kidney to produce a natriuresis and a diuresis. Disruption of D(1) or D(3) receptors in mice results in hypertension that is caused, in part, by a decreased ability to excrete an acute saline load. We studied D(1)-like and D(2)-like receptor interaction in anesthetized spontaneously hypertensive rats (SHR) by the intrarenal infusion of Z-1046 (a novel dopamine receptor agonist with rank order potency of D(3)> or =D(4)>D(2)>D(5)>D(1)). Z-1046 increased glomerular filtration rate (GFR), urine flow, and sodium excretion in normotensive Wistar-Kyoto rats but not in SHRs. The lack of responsiveness to Z-1046 in SHRs was not an epiphenomenon, because intrarenal cholecystokinin infusion increased GFR, urine flow, and sodium excretion to a similar extent in the two rat strains. We conclude that renal D(1)-like and D(2)-like receptor interaction is impaired in SHRs. The impaired D(1)-like and D(2)-like receptor interaction in SHRs is not caused by alterations in the coding sequence of the D(3) receptor, the D(2)-like receptor expressed in rat renal tubules that has been shown to be involved in sodium transport. Because the diuretic and natriuretic effects of D(1)-like receptors are, in part, caused by an interaction with D(2)-like receptors, it is possible that the decreased Z-1046 action in SHRs is secondary to the renal D(1)-like receptor dysfunction in this rat strain.