D O P A M I N E A N D T H E R E G U L A T I O N O F SODIUM
AJH
1990;3:78S-81S
Renal Dopamine and Sodium Excretion Sharath S. Hegde and Mustafa F. Lokhandwala
In the present study, we examined the renal effects of a low dose of fenoldopam in pentobarbital anes thetized rats in an attempt to unmask a direct tubu lar DA-1 receptor mediated diuresis and natriuresis. We also performed experiments to determine the possible contribution of renal dopamine (DA) and D A receptors in the natriuretic response to acute sodium loading. Fenoldopam (0.5 / / g / k g / m i n ) pro duced significant increases in urine output (UV) and urinary sodium excretion ( U V ) without altering mean blood pressure (MBP), renal blood flow (RBF) or heart rate (HR), suggesting a direct tubular site of action. This renal response to fenoldopam was completely abolished in the presence of the selec tive DA-1 receptor antagonist, SCH 23390. Isotonic NaCl loading (5% body weight for 1 h) enhanced urinary D A excretion ( U V ) which correlated sig
nificantly with the increase in U V . Pretreatment with selective DA-1 receptor antagonist SCH 23390 resulted in significant attenuation of the increase in U V whereas the increase in U V was unal tered. Pretreatment with selective DA-2 receptor an tagonist domperidone did not alter the increase in U V during isotonic NaCl loading. These results suggest that renal tubules are endowed with DA-1 receptors activation which results in diuresis and natriuresis. Furthermore, activation of these DA-1 receptors but not DA-2 receptors by endogenous DA contributes to the natriuretic response to isotonic saline loading. Am J Hypertens 1990;78S-81S
A
fects of renal DA has not been elucidated. In the present study, we attempted to identify functional responses elicited during activation of tubular DA-1 receptors by examining the effects of a low dose of fenoldopam on renal sodium and water excretion in anesthetized rats. In addition, we tested the hypothesis that acute sodium loading with isotonic sodium chloride stimulates the production of endogenous DA which in turn acts on specific DA-1 and/or DA-2 receptors to promote so dium excretion.
Na
DA
growing body of evidence from binding and biochemical studies favors the existence of tubular DA-1 receptors. Attempts to iden tify tubular DA-1 receptors in functional stud ies with selective DA-1 agonist fenoldopam have met with little success due mainly to the failure to separate the natriuretic response seen with fenoldopam from its systemic and renal hemodynamic effects. Several lines of evidence suggest that endogenous DA produced within the kidney, either from neural or nonneural sources may be playing an important physiological role in regulating sodium excretion. However, a cause and effect relation between sodium intake, sodium excretion and DA excretion has not been firmly established. In addition, the subtype of DA receptor mediating the ef1,2
3
From the Department o f Pharmacology, University of Houston, Houston, Texas. Address c o r r e s p o n d e n c e a n d reprint requests to Dr. M u s t a f a F . Lokhandwala, Department of Pharmacology, University of Houston, Houston, T X 77204-5515.
© 1990 by the American
Journal
of Hypertension,
Inc.
N a
N a
D A
N a
Dopamine, natriuresis, DA-1 receptors, DA-2 receptors, fenoldopam, isotonic. KEY WORDS:
METHODS Surgical Procedure Male Sprague-Dawley rats (Har lan, Texas) weighing between 300 to 350 g were used in the present study. They were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally). Following tracheal cannulation, the left carotid artery, right jugular vein and right femoral artery were catheterized for blood pressure measurement, drug/saline administra tion and blood sampling, respectively. In some experi-
0895-7061/'90/'$3.00
AJH-JUNE 1990-VOL 3, NO. 6, PART 2
RENAL DOPAMINE RECEPTORS AND NATRIURESIS
ments, the left jugular vein was also catheterized for measurement of central venous pressure. The left ureter was exposed and cannulated for urine collection. After the completion of surgery, an infusion of isotonic so dium chloride (NaCl) at a rate of 0.02 mL/min was begun and hemodynamic and renal parameters were allowed to stabilize over a period of 30 min before the commencement of the experiment. Experimental Protocol for Studies with Fenoldopam The protocol consisted of five consecutive 30 min urine collection periods. The initial 2 periods served as control during which saline was infused at a rate of 0.02 m L / min. During the third period, fenoldopam (0.5 / / g / k g / min) was infused for 30 min at 0.02 mL/min. Following the termination of fenoldopam infusion, saline (0.02 mL/min) was infused during the last 2 recovery periods. Arterial blood samples (0.2 mL) were collected at the midpoint of each period and the volume was replaced with an equal amount of saline. In another group of animals, the effects of fenoldopam were studied in the presence of the DA-1 receptor antagonist SCH 23390 (50 //g/kg, intravenously, 10 //g/kg/min). Experimental Protocol during Sodium Loading with Isotonic NaCl (0.9%) After the initial 2 control urine collection periods (30 min each), the rats were sodium loaded by infusing isotonic NaCl at a rate of 0.34 m L / min over a 60 min period. During this period, four con secutive 15 min urine collections were made. After 1 h of isotonic NaCl loading, the rate of infusion was reduced back to 0.02 mL/min and maintained for 60 min. Dur ing this recovery period, four consecutive 15 min urine collections were made. Blood samples (0.2 mL) were taken at the midpoint of each consecutive 30 min period following the start of the experiment. In 2 separate groups of rats, the influence of selective DA-1 blockade with SCH 23390 (50 //g/kg, intravenously, 10 / / g / k g / min) and selective DA-2 receptor blockade with domperidone (50 //g/kg, intravenously, 10 //g/kg/min) was studied. Analytical Procedures Sodium and potassium con centrations in the plasma and urine were measured using a Perkin Elmer flame photometer (Perkin Elmer, Oakbrook, Illinois). Plasma and urine creatinine con centrations were measured using a Beckman Creatinine Analyzer 2 (Beckman Instruments, Inc., Fullerton, Cali fornia). The urine samples used for DA measurements were added to a solution containing 0.1N HC1, 0.2% N a S 0 and 1 mmol/L EDTA N a and frozen at — 70 °C until ready for analysis. DA was extracted using alumina and analyzed using HPLC with electrochemi cal detection. 2
2
3
2
Statistical Analysis All data are presented as mean ± SEM. Students paired and unpaired 't' test was used for comparison within and between groups respectively. A Ρ < .05 was considered to be statistically significant.
79S
RESULTS Studies with Fenoldopam Fenoldopam (0.5 / / g / k g / min) produced a significant increase in urine output (UV) from 76.86 ± 11.86 to 97.66 ± 14.09 / / L / 3 0 min, Ρ < .05), and a significant increase in urinary sodium excretion ( U V ) (from 4.36 ± 1 . 7 6 to 6.65 ± 2.18 μΈq/30 min, Ρ < .05). This dose of fenoldopam did not produce any changes in mean blood pressure (MBP), glomerular filtration rate (GFR) or renal blood flow (RBF). In the presence of SCH 23390, fenoldopam failed to produce any significant increases in UV (from 55.87 ± 6.6 to 52.86 ± 6.6 / / L / 3 0 min) or U V (from 3.18 ± 0.57 to 3.01 ± 0.51 μΈq/30 min). Na
N a
Sodium Loading with Isotonic NaCl In the control group, acute sodium loading with isotonic NaCl re sulted in significant increases in U V (Figure 1) and this was accompanied by an increase in urinary DA excre tion ( U V ) (from 0.63 ± 0.11 to 1.58 ± 0.29 ng/min, Ρ < .05). The increase in U V was significantly corre lated with the increase in U V (r = 0.5, Ρ < .0005). In the group treated with SCH 23390, there was a signifi cant attenuation of the natriuretic response (Figure 1). However, U V still increased significantly (from 1.02 ± 0 . 1 9 to 1.74 ± 0 . 2 6 ng/min, Ρ < .05) in this group. In the group treated with domperidone, the na triuretic response did not differ significantly from that in the control group (Figure 1). Isotonic NaCl loading did not alter mean blood pressure (MBP) or heart rate (HR) significantly, however, there was an increase in central venous pressure (CVP) of 2 mm Hg, a transient increase in glomerular filtration rate (GFR) (from 0.92 ± 0.11 to 1.56 ± 0.13 mL/min, Ρ < .05) and a decrease in hema tocrit (Hct) (from 47.3 ± 1.06 to 38.12 ± 1.04, Ρ < .05). These changes in CVP, GFR and Hct were similar in the control and antagonist treated groups. N a
DA
D A
N a
D A
DISCUSSION DA-1 binding sites have been identified in the proximal tubules, collecting tubules and glomeruli devoid of arte rioles of the rat. ' Interestingly, DA and DA analogs cause inhibition of fluid and sodium reabsorption in microperfused proximal tubules and inhibition of Na+, K+-ATPase and/or Na^-H" " antiport have been sug gested as possible mechanisms. Fenoldopam is a selec tive DA-1 receptor agonist, which has been shown to produce increases in renal blood flow, water and so dium excretion in several animal species and humans. However, it is still unclear whether fenoldopam induced diuresis and natriuresis results indirectly from changes in renal hemodynamics and/or is due to direct activa tion of putative tubular DA-1 receptors. In the present study, the hemodynamic and natriuretic effects of fen oldopam (0.5 //g/kg/min) could be entirely dissociated from each other, implying a direct tubular action in the observed diuresis and natriuresis. This is consistent with 1 2
4
5
1
6
7
80S
HEGDE AND
AJH-JUNE 1990-VOL
LOKHANDWALA
15η Ο
5% BODY WEIGHT VOLUME EXPANSION I I
CONTROL SCH-23390 DOMPERIDONE
w a
12H
£
3, NO. 6, PART 2
QC
Η
FIGURE 1. Effect of DA-1 and/or DA-2 receptor blockade on the na triuretic response to sodium loading with isotonic and hypertonic sodium chloride. The top panel shows ex periments in which isotonic NaCl was infused whereas the bottom panel shows results with infusion of 15% NaCl. SCH 23390 (50 μg/kg intravenously, 10 μg/kg/min) and domperidone (50 μg/kg, intra venously, 10 μg/kgι'min) were used as DA-1 and DA-2 receptor antago nists respectively. ^Denotes statisti cally significant difference from the corresponding time period in the control group.
QC < Ζ QC 3
150 TIME(min)
/
25 η
15% HYPERTONIC SAUNE I I CONTROL w
-90 -60
SCH-23390
120 150 TIME (min)
the results of a previous study in the dog in which it was shown that fenoldopam could elicit a natriuretic re sponse even after the increase in renal blood flow was prevented by renal artery constriction. Involvement of intrarenal prostaglandins and kallikrein systems in the renal effects of fenoldopam has been previously ex cluded. The direct tubular effect appears to be me diated through stimulation of DA-1 receptors, inas much as the natriuretic and diuretic effect was antagonized by the selective DA-1 receptor antagonist SCH 23390. The possibility of functional antagonism can also be ruled out since SCH 23390 by itself produced 8
8
no changes in sodium and water excretion (data not shown). We recently performed autoradiographic stud ies with [ H]-fenoldopam in order to determine the rela tive degree of specific localization of the DA-1 agonist to vascular and tubular sites in the rat kidney. It was found that, at a concentration at which extensive bind ing of [ H]-fenoldopam to tubular structures was local ized, there was less binding in the vascular smooth muscle suggesting a far greater DA-1 receptor density in the tubules. These differences in receptor density could account for the greater efficacy of fenoldopam in elicit ing tubular than vascular responses. 3
9
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Several workers have previously demonstrated that sodium loading in animals enhances urinary DA excre tion and the accompanying natriuresis can be atten uated with DOPA decarboxylase inhibitors and nonse lective DA receptor antagonists. These observations led to the postulate that endogenous DA produced within the kidney from circulating L-dopa may be play ing a physiological role in regulating sodium excretion. In the present study, we observed that isotonic sodium loading in rats led to an increase in urinary DA excretion which was positively correlated with the accompanying natriuresis. Also, the ability of SCH 23390 to attenuate the natriuretic response without altering the accompa nying increase in U V indicates that endogenous DA may be exerting its natriuretic effect via activation of specific DA-1 receptors. In addition to DA-1 receptors, DA-2 binding sites have also been identified in the nephron using radioligand binding techniques. ' In light of these reports, we considered it possible that DA-2 receptor activation may also be mediating the renal effects of endogenously produced DA. However, the inability of domperidone to alter the natriuretic re sponse to NaCl loading rules out any possible contribu tory role of DA-2 receptors. Inadequate DA-2 receptor blockade is unlikely since the dose of domperidone em ployed is much more than that required to block the vascular effects of a DA-2 receptor agonist like quinpirole. Several previous observations lend support to the notion that tubular DA-2 receptors, if they do exist, are incapable of being activated by exogenous and endoge nously produced DA. Firstly, it has been well estab lished that the renal effects of exogenous DA can be blocked by SCH 23390 but are unaffected by domperi done. Secondly, the renal effects of y-l-glutamyl-Ldopa, a prodrug of DA which gets converted intrarenally to DA, are unaltered by domperidone. Thirdly, whereas tubular DA-1 receptor activation results in stim ulation of both adenylate cyclase and phospholipase C, tubular DA-2 receptor activation fails to alter activity of either of the enzymes. DA-2 receptors are also located prejunctionally on sympathetic nerves supplying the kidney, activation of which leads to inhibition of norepi nephrine release. Normally, such an effect would be expected to eventually result in natriuresis. However, under the conditions of NaCl loading where sympa thetic neural discharge to kidney is already considerably suppressed, endogenous DA would be unlikely to pro duce natriuresis by this mechanism. 3,10
concomitant expansion of the extracellular fluid vol ume. In conclusion, the results of our studies suggest that renal tubules are endowed with specific DA-1 receptors which can be functionally activated by DA-1 receptor agonists to produce diuresis and natriuresis. Moreover, the results also imply that acute sodium loading with isotonic NaCl stimulates the production of endogenous D A which in turn promotes sodium excretion via acti vation of DA-1 but not D A - 2 receptors. REFERENCES 1.
Felder RA, Blecher M, Eisner GM, Jose PA: Cortical tu bular and glomerular dopamine receptors in the kidney. Am J Physiol 1984;246:F557-F568.
2.
Felder CC, McKelvey AM, Gitler MS, et al: Dopamine receptor subtypes in renal brush border and basolateral membranes. Kid Int 1 9 8 9 ; 3 6 : 1 8 3 - 1 9 3 .
3.
Lee MR: Dopamine and the kidney. Clin Sci 1982; 62:439-448.
4.
Bello-Reuss E, Higashi Y, Kenda Y: Dopamine decreases fluid reabsorption in straight portions of rabbit proximal tubule. Am J Physiol 1982;242:F634-F640.
5.
Aperia A, Bertorello A, Seri I: Dopamine causes inhibi tion of Na -K+-ATPase activity in rat proximal convo luted tubule segments. Am J Physiol 1987;252:F39-F45.
D A
1 2
+
6.
Felder RA, Felder CC, Eisner GM, Jose PA: Renal Dopa mine Receptors, in Bell C, McGrath Β (eds): Peripheral Action of Dopamine. MacMillan London, Press, 1988; pp 1 2 4 - 1 4 0 .
7.
Lokhandwala MF: Preclinical and clinical studies on the cardiovascular and renal effects of fenoldopam: A DA-1 receptor agonist. Drug Dev Res 1 9 8 7 ; 1 0 : 1 2 3 - 1 2 4 .
8.
Jose PA, Eisner GM, Robillard JE: Renal hemodynamics and natriuresis induced by the dopamine-1 agonist, SK&F 82526. Am J Med Sci 1987;294(3):181-186.
9.
Hegde SS, Ricci A, Amenta F, Lokhandwala MF: Evi dence from functional and autoradiographic studies for the presence of tubular dopamine-1 receptors and their involvement in the renal effects of fenoldopam. J Phar macol Exp Ther 1 9 8 9 ; 2 5 1 : 1 2 3 7 - 1 2 4 5 .
10.
McClanahan M, Sowers JR, Beck FWJ, et al: Dopaminer gic regulation of natriuretic response to acute volume expansion in dogs. Clin Sci 1 9 8 5 ; 6 8 : 2 6 3 - 2 6 9 .
11.
Sengupta S, Lokhandwala MF: Characterization of the hypotensive action of dopamine receptor agonists fenol dopam and quinpirole in anesthetized rats. J Auton Pharmacol 1 9 8 5 ; 5 : 2 8 9 - 2 9 4 .
12.
Fredrickson ED, Bradley T, Goldberg LI: Blockade of the renal effects of dopamine in the dog by the DA-1 antago nist SCH 23390. Am J Physiol 1985;249:F236-F240.
13.
Worth DP, Harvey JN, Brown J, et al: Domperidone treatment in man inhibits the fall in plasma renin activity induced by intravenous y-l-glutamyl-L-dopa. Br J Clin Pharmacol 1 9 8 6 ; 2 1 : 4 9 7 - 5 0 2 .
14.
Lokhandwala MF, Steenberg ML: Evaluation of the ef fects of SK&F 82526 and LY 17155 on presynaptic (DA-2) and postsynaptic (DA-1) dopamine receptors in rat kidney. J Auton Pharmacol 1 9 8 4 ; 4 : 2 7 3 - 2 7 7 .
11
12
13
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14
The mechanism by which sodium loading in animals stimulates the production of kidney DA is not yet known. The natriuretic response to 1 5 % hypertonic NaCl loading can also be attenuated with SCH 23390 (Figure 1). However, some preliminary data from our laboratory (unpublished observations) suggests that the contribution by endogenous DA appears to be more significant when NaCl loading is accompanied by a
AJH
1990;3:78S-81S
Renal Dopamine and Sodium Excretion Sharath S. Hegde and Mustafa F. Lokhandwala
In the present study, we examined the renal effects of a low dose of fenoldopam in pentobarbital anes thetized rats in an attempt to unmask a direct tubu lar DA-1 receptor mediated diuresis and natriuresis. We also performed experiments to determine the possible contribution of renal dopamine (DA) and D A receptors in the natriuretic response to acute sodium loading. Fenoldopam (0.5 / / g / k g / m i n ) pro duced significant increases in urine output (UV) and urinary sodium excretion ( U V ) without altering mean blood pressure (MBP), renal blood flow (RBF) or heart rate (HR), suggesting a direct tubular site of action. This renal response to fenoldopam was completely abolished in the presence of the selec tive DA-1 receptor antagonist, SCH 23390. Isotonic NaCl loading (5% body weight for 1 h) enhanced urinary D A excretion ( U V ) which correlated sig
nificantly with the increase in U V . Pretreatment with selective DA-1 receptor antagonist SCH 23390 resulted in significant attenuation of the increase in U V whereas the increase in U V was unal tered. Pretreatment with selective DA-2 receptor an tagonist domperidone did not alter the increase in U V during isotonic NaCl loading. These results suggest that renal tubules are endowed with DA-1 receptors activation which results in diuresis and natriuresis. Furthermore, activation of these DA-1 receptors but not DA-2 receptors by endogenous DA contributes to the natriuretic response to isotonic saline loading. Am J Hypertens 1990;78S-81S
A
fects of renal DA has not been elucidated. In the present study, we attempted to identify functional responses elicited during activation of tubular DA-1 receptors by examining the effects of a low dose of fenoldopam on renal sodium and water excretion in anesthetized rats. In addition, we tested the hypothesis that acute sodium loading with isotonic sodium chloride stimulates the production of endogenous DA which in turn acts on specific DA-1 and/or DA-2 receptors to promote so dium excretion.
Na
DA
growing body of evidence from binding and biochemical studies favors the existence of tubular DA-1 receptors. Attempts to iden tify tubular DA-1 receptors in functional stud ies with selective DA-1 agonist fenoldopam have met with little success due mainly to the failure to separate the natriuretic response seen with fenoldopam from its systemic and renal hemodynamic effects. Several lines of evidence suggest that endogenous DA produced within the kidney, either from neural or nonneural sources may be playing an important physiological role in regulating sodium excretion. However, a cause and effect relation between sodium intake, sodium excretion and DA excretion has not been firmly established. In addition, the subtype of DA receptor mediating the ef1,2
3
From the Department o f Pharmacology, University of Houston, Houston, Texas. Address c o r r e s p o n d e n c e a n d reprint requests to Dr. M u s t a f a F . Lokhandwala, Department of Pharmacology, University of Houston, Houston, T X 77204-5515.
© 1990 by the American
Journal
of Hypertension,
Inc.
N a
N a
D A
N a
Dopamine, natriuresis, DA-1 receptors, DA-2 receptors, fenoldopam, isotonic. KEY WORDS:
METHODS Surgical Procedure Male Sprague-Dawley rats (Har lan, Texas) weighing between 300 to 350 g were used in the present study. They were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally). Following tracheal cannulation, the left carotid artery, right jugular vein and right femoral artery were catheterized for blood pressure measurement, drug/saline administra tion and blood sampling, respectively. In some experi-
0895-7061/'90/'$3.00
AJH-JUNE 1990-VOL 3, NO. 6, PART 2
RENAL DOPAMINE RECEPTORS AND NATRIURESIS
ments, the left jugular vein was also catheterized for measurement of central venous pressure. The left ureter was exposed and cannulated for urine collection. After the completion of surgery, an infusion of isotonic so dium chloride (NaCl) at a rate of 0.02 mL/min was begun and hemodynamic and renal parameters were allowed to stabilize over a period of 30 min before the commencement of the experiment. Experimental Protocol for Studies with Fenoldopam The protocol consisted of five consecutive 30 min urine collection periods. The initial 2 periods served as control during which saline was infused at a rate of 0.02 m L / min. During the third period, fenoldopam (0.5 / / g / k g / min) was infused for 30 min at 0.02 mL/min. Following the termination of fenoldopam infusion, saline (0.02 mL/min) was infused during the last 2 recovery periods. Arterial blood samples (0.2 mL) were collected at the midpoint of each period and the volume was replaced with an equal amount of saline. In another group of animals, the effects of fenoldopam were studied in the presence of the DA-1 receptor antagonist SCH 23390 (50 //g/kg, intravenously, 10 //g/kg/min). Experimental Protocol during Sodium Loading with Isotonic NaCl (0.9%) After the initial 2 control urine collection periods (30 min each), the rats were sodium loaded by infusing isotonic NaCl at a rate of 0.34 m L / min over a 60 min period. During this period, four con secutive 15 min urine collections were made. After 1 h of isotonic NaCl loading, the rate of infusion was reduced back to 0.02 mL/min and maintained for 60 min. Dur ing this recovery period, four consecutive 15 min urine collections were made. Blood samples (0.2 mL) were taken at the midpoint of each consecutive 30 min period following the start of the experiment. In 2 separate groups of rats, the influence of selective DA-1 blockade with SCH 23390 (50 //g/kg, intravenously, 10 / / g / k g / min) and selective DA-2 receptor blockade with domperidone (50 //g/kg, intravenously, 10 //g/kg/min) was studied. Analytical Procedures Sodium and potassium con centrations in the plasma and urine were measured using a Perkin Elmer flame photometer (Perkin Elmer, Oakbrook, Illinois). Plasma and urine creatinine con centrations were measured using a Beckman Creatinine Analyzer 2 (Beckman Instruments, Inc., Fullerton, Cali fornia). The urine samples used for DA measurements were added to a solution containing 0.1N HC1, 0.2% N a S 0 and 1 mmol/L EDTA N a and frozen at — 70 °C until ready for analysis. DA was extracted using alumina and analyzed using HPLC with electrochemi cal detection. 2
2
3
2
Statistical Analysis All data are presented as mean ± SEM. Students paired and unpaired 't' test was used for comparison within and between groups respectively. A Ρ < .05 was considered to be statistically significant.
79S
RESULTS Studies with Fenoldopam Fenoldopam (0.5 / / g / k g / min) produced a significant increase in urine output (UV) from 76.86 ± 11.86 to 97.66 ± 14.09 / / L / 3 0 min, Ρ < .05), and a significant increase in urinary sodium excretion ( U V ) (from 4.36 ± 1 . 7 6 to 6.65 ± 2.18 μΈq/30 min, Ρ < .05). This dose of fenoldopam did not produce any changes in mean blood pressure (MBP), glomerular filtration rate (GFR) or renal blood flow (RBF). In the presence of SCH 23390, fenoldopam failed to produce any significant increases in UV (from 55.87 ± 6.6 to 52.86 ± 6.6 / / L / 3 0 min) or U V (from 3.18 ± 0.57 to 3.01 ± 0.51 μΈq/30 min). Na
N a
Sodium Loading with Isotonic NaCl In the control group, acute sodium loading with isotonic NaCl re sulted in significant increases in U V (Figure 1) and this was accompanied by an increase in urinary DA excre tion ( U V ) (from 0.63 ± 0.11 to 1.58 ± 0.29 ng/min, Ρ < .05). The increase in U V was significantly corre lated with the increase in U V (r = 0.5, Ρ < .0005). In the group treated with SCH 23390, there was a signifi cant attenuation of the natriuretic response (Figure 1). However, U V still increased significantly (from 1.02 ± 0 . 1 9 to 1.74 ± 0 . 2 6 ng/min, Ρ < .05) in this group. In the group treated with domperidone, the na triuretic response did not differ significantly from that in the control group (Figure 1). Isotonic NaCl loading did not alter mean blood pressure (MBP) or heart rate (HR) significantly, however, there was an increase in central venous pressure (CVP) of 2 mm Hg, a transient increase in glomerular filtration rate (GFR) (from 0.92 ± 0.11 to 1.56 ± 0.13 mL/min, Ρ < .05) and a decrease in hema tocrit (Hct) (from 47.3 ± 1.06 to 38.12 ± 1.04, Ρ < .05). These changes in CVP, GFR and Hct were similar in the control and antagonist treated groups. N a
DA
D A
N a
D A
DISCUSSION DA-1 binding sites have been identified in the proximal tubules, collecting tubules and glomeruli devoid of arte rioles of the rat. ' Interestingly, DA and DA analogs cause inhibition of fluid and sodium reabsorption in microperfused proximal tubules and inhibition of Na+, K+-ATPase and/or Na^-H" " antiport have been sug gested as possible mechanisms. Fenoldopam is a selec tive DA-1 receptor agonist, which has been shown to produce increases in renal blood flow, water and so dium excretion in several animal species and humans. However, it is still unclear whether fenoldopam induced diuresis and natriuresis results indirectly from changes in renal hemodynamics and/or is due to direct activa tion of putative tubular DA-1 receptors. In the present study, the hemodynamic and natriuretic effects of fen oldopam (0.5 //g/kg/min) could be entirely dissociated from each other, implying a direct tubular action in the observed diuresis and natriuresis. This is consistent with 1 2
4
5
1
6
7
80S
HEGDE AND
AJH-JUNE 1990-VOL
LOKHANDWALA
15η Ο
5% BODY WEIGHT VOLUME EXPANSION I I
CONTROL SCH-23390 DOMPERIDONE
w a
12H
£
3, NO. 6, PART 2
QC
Η
FIGURE 1. Effect of DA-1 and/or DA-2 receptor blockade on the na triuretic response to sodium loading with isotonic and hypertonic sodium chloride. The top panel shows ex periments in which isotonic NaCl was infused whereas the bottom panel shows results with infusion of 15% NaCl. SCH 23390 (50 μg/kg intravenously, 10 μg/kg/min) and domperidone (50 μg/kg, intra venously, 10 μg/kgι'min) were used as DA-1 and DA-2 receptor antago nists respectively. ^Denotes statisti cally significant difference from the corresponding time period in the control group.
QC < Ζ QC 3
150 TIME(min)
/
25 η
15% HYPERTONIC SAUNE I I CONTROL w
-90 -60
SCH-23390
120 150 TIME (min)
the results of a previous study in the dog in which it was shown that fenoldopam could elicit a natriuretic re sponse even after the increase in renal blood flow was prevented by renal artery constriction. Involvement of intrarenal prostaglandins and kallikrein systems in the renal effects of fenoldopam has been previously ex cluded. The direct tubular effect appears to be me diated through stimulation of DA-1 receptors, inas much as the natriuretic and diuretic effect was antagonized by the selective DA-1 receptor antagonist SCH 23390. The possibility of functional antagonism can also be ruled out since SCH 23390 by itself produced 8
8
no changes in sodium and water excretion (data not shown). We recently performed autoradiographic stud ies with [ H]-fenoldopam in order to determine the rela tive degree of specific localization of the DA-1 agonist to vascular and tubular sites in the rat kidney. It was found that, at a concentration at which extensive bind ing of [ H]-fenoldopam to tubular structures was local ized, there was less binding in the vascular smooth muscle suggesting a far greater DA-1 receptor density in the tubules. These differences in receptor density could account for the greater efficacy of fenoldopam in elicit ing tubular than vascular responses. 3
9
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1990-VOL
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RENAL DOPAMINE RECEPTORS AND NATRIURESIS
Several workers have previously demonstrated that sodium loading in animals enhances urinary DA excre tion and the accompanying natriuresis can be atten uated with DOPA decarboxylase inhibitors and nonse lective DA receptor antagonists. These observations led to the postulate that endogenous DA produced within the kidney from circulating L-dopa may be play ing a physiological role in regulating sodium excretion. In the present study, we observed that isotonic sodium loading in rats led to an increase in urinary DA excretion which was positively correlated with the accompanying natriuresis. Also, the ability of SCH 23390 to attenuate the natriuretic response without altering the accompa nying increase in U V indicates that endogenous DA may be exerting its natriuretic effect via activation of specific DA-1 receptors. In addition to DA-1 receptors, DA-2 binding sites have also been identified in the nephron using radioligand binding techniques. ' In light of these reports, we considered it possible that DA-2 receptor activation may also be mediating the renal effects of endogenously produced DA. However, the inability of domperidone to alter the natriuretic re sponse to NaCl loading rules out any possible contribu tory role of DA-2 receptors. Inadequate DA-2 receptor blockade is unlikely since the dose of domperidone em ployed is much more than that required to block the vascular effects of a DA-2 receptor agonist like quinpirole. Several previous observations lend support to the notion that tubular DA-2 receptors, if they do exist, are incapable of being activated by exogenous and endoge nously produced DA. Firstly, it has been well estab lished that the renal effects of exogenous DA can be blocked by SCH 23390 but are unaffected by domperi done. Secondly, the renal effects of y-l-glutamyl-Ldopa, a prodrug of DA which gets converted intrarenally to DA, are unaltered by domperidone. Thirdly, whereas tubular DA-1 receptor activation results in stim ulation of both adenylate cyclase and phospholipase C, tubular DA-2 receptor activation fails to alter activity of either of the enzymes. DA-2 receptors are also located prejunctionally on sympathetic nerves supplying the kidney, activation of which leads to inhibition of norepi nephrine release. Normally, such an effect would be expected to eventually result in natriuresis. However, under the conditions of NaCl loading where sympa thetic neural discharge to kidney is already considerably suppressed, endogenous DA would be unlikely to pro duce natriuresis by this mechanism. 3,10
concomitant expansion of the extracellular fluid vol ume. In conclusion, the results of our studies suggest that renal tubules are endowed with specific DA-1 receptors which can be functionally activated by DA-1 receptor agonists to produce diuresis and natriuresis. Moreover, the results also imply that acute sodium loading with isotonic NaCl stimulates the production of endogenous D A which in turn promotes sodium excretion via acti vation of DA-1 but not D A - 2 receptors. REFERENCES 1.
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The mechanism by which sodium loading in animals stimulates the production of kidney DA is not yet known. The natriuretic response to 1 5 % hypertonic NaCl loading can also be attenuated with SCH 23390 (Figure 1). However, some preliminary data from our laboratory (unpublished observations) suggests that the contribution by endogenous DA appears to be more significant when NaCl loading is accompanied by a
