Effect of Carbidopa on the Excretion of Sodium, Dopamine, and Ouabain-Like Substance in the Rat
Abstract The effect of carbidopa, a competitive inhibitor of dopamine synthesis on the excretion of sodium, dopamine (DA), and endogenous sodium transport inhibitor (ESTI), was examined in rats on normal and high salt diets for 7 days. ESTI activity was measured (1) as ouabain-like substance (OLS) by radioimmunoassay using an antibody raised against ouabain, (2) by inhibition of purified Na+,K+-ATPase activity (ATPI), and (3) as digoxin-like substance (DLS) using a commercial digoxin assay. The OLS immunoassay was validated against rubidium transport studies. High salt diet caused an increase in OLS and ATPI but not DLS. Sodium excretion in rats on normal sodium intake was not affected by carbidopa treatment but was significantly lower in the high sodium diet group during the first 5 days of the study. In the low salt group, carbidopa treatment caused significant increases in OLS. We conclude that ESTI (measured as OLS) and DA are important in the natriuresis of salt loading.
Factors involved in the regulation of sodium reabsorption by the renal tubules include aldosterone, atrial natriuretic peptide, dopamine (DA), and endogenous sodium transport inhibitor (ESTI). The role of some of these factors, eg, aldosterone and atrial natriuretic peptide, in the regulation of sodium homeostasis is well established. For others, such as DA and ESTI, the roles are not clear. Most of the DA appearing in the urine is produced intrarenally,1 and high salt diet has been shown to cause an increase in the excretion of DA in animals2 and in humans.3 4
There have been several reports showing the presence of ESTI in biological material, and ESTI has been shown to increase during salt loading.2 5 6 7 The nature of this inhibitor is not fully understood. Ouabain or OLS has recently been purified from human plasma and identified as the ESTI.8 However, this view is not universally accepted.9 10 ESTI has been measured by a variety of techniques, including inhibition of purified Na+,K+-ATPase,11 inhibition of sodium transport in intact cells,12 [3H]-ouabain displacement,13 immunoassay using antibodies against digoxin,14 and immunoassay using antibodies against ouabain.15 It is not clear which of these assays is best suited for detecting changes in ESTI during salt loading.5
Epidemiological and experimental studies strongly suggest that high salt intake is an important factor in the pathogenesis of hypertension. A hypothesis to explain the mechanism of this association has been proposed.16 17 In this hypothesis it is suggested that salt-sensitive subjects have a renal abnormality in handling sodium, resulting in secretion of ESTI. The latter acting on arterioles causes an increase in blood pressure.18 The renal abnormalities in salt-sensitive subjects include defects in the DA pathway,1 and abnormalities in DA excretion have been reported in some hypertensive individuals19 20 and family members of hypertensive individuals.21
We argued that if these hypotheses are true, inhibition of DA synthesis should increase the production of ESTI. We have recently developed a sensitive and specific immunoassay for OLS.22 In this study we examined (1) the effect of salt loading on ESTI measured by three different assays and (2) the effect of carbidopa on the excretion of sodium, DA, and ESTI.
One-month-old male Sprague-Dawley rats (n=24) were studied. They were fed ad libitum throughout the study with normal rat chow containing 0.2% sodium chloride. The animals were placed in individual metabolic cages and given tap water to drink for 3 days. They were then randomly divided into four groups: group A, low salt group; group B, high salt group; group C, low salt and carbidopa; and group D, high salt and carbidopa. Carbidopa (kindly supplied by Merck, Sharp & Dohme) was dissolved in water and fed orally to groups C and D twice daily at a dose of 120 mg/kg body wt/d. Groups A and B were fed orally with a volume of water similar to that of the carbidopa solution. The low salt groups (groups A and C) were given tap water to drink and high salt groups (groups B and D) were given 0.3 mmol/L (18 g/L) of sodium chloride to drink instead of tap water. Urine was collected for 7 days.
Measurements of DA were performed on the day after the urine collection to avoid any artifact introduced by the instability of catecholamines in the urine samples. Aliquots of the urine were stored at −70°C for the measurement of urinary ESTI within 12 months. All urine aliquots were stored for the same length of time before assay.
Urinary sodium and creatinine concentrations were measured by indirect ion-selective electrode and Jaffe reaction, respectively, on a Beckman Astra-8 clinical chemistry analyzer (Beckman Instrument). The interassay coefficients of variation for both assays were less than 3%.
Urinary free DA was determined as described previously.23 Briefly, DA was extracted onto alumina, eluted with 0.1 mol/L HCl, washed with ethyl acetate, and quantitated by high-performance liquid chromatography with electrochemical detector as described by Weicker et al.24 Intra-assay and interassay coefficients of variation were 1.3% and 8.7%, respectively. The mean recovery was 91%, and the detection limit was 3 nmol/L.
Extraction of Urine ESTI
Urinary ESTI was extracted using Waters Sep-Pak C18 cartridges (Millipore). Sep-Pak cartridges were first activated by washing with methanol and water. A 300 μL aliquot of sample was applied and allowed to equilibrate with the C18 resin in the cartridge for 2 minutes before washing twice with 10 mL of water to remove salts and other interfering substances. The equilibration step was found to improve the precision of the extraction step. ESTI was eluted twice with 1 mL of methanol. The methanol eluate was evaporated to dryness and the residue reconstituted in 100 μL Tris buffer, pH 7.4. These reconstituted eluates contained no measurable DA.
ESTI in the extract was quantitated by its ability to inhibit purified dog kidney Na+,K+-ATPase activity (Sigma Chemical Co). The reconstituted extract was incubated with a 250 U/L aliquot of purified dog kidney Na+,K+-ATPase solution in the presence of 100 mmol/L of sodium and 5 mmol/L of ATP at 37°C for 2 hours. The reaction was terminated by immersing the reaction mixture in ice. The residual ATPase activity was determined by an enzyme-coupled reaction as described previously.25 Aliquots of ouabain in Tris buffer, ranging from 5 nmol/L to 50 nmol/L, were used as standards, and the results are expressed as nmol/L ouabain equivalent. Interassay coefficient of variation ranged from 18% at a concentration of 5 nmol/L to 3% at 50 nmol/L.
DLS was measured in urine extracts using a commercial kit (CEDIA digoxin kit) according to manufacturers’ instructions on a Cobas Fara analyzer (Hoffmann–La Roche and Co). The cross-reactivity of the antibody was quoted by the manufacturers as follows: ouabain 2.9%, gitalin 29.0%, and <0.1% for prednisolone, testosterone, and quinidine.
Immunoassay for OLS
Urinary concentration of OLS was measured by a radioimmunoassay as described previously.22 Briefly, ouabain was conjugated to ovalbumin and keyhole limpet haemocyanin (KLH) and antisera to ouabain was raised in goats by injecting ouabain-ovalbumin and then boosting with ouabain-KLH conjugate. The antisera showed minimal cross-reactivity against ouabagenin (4%) and other related steroids. Tritiated ouabain ([21-22-3H]-ouabain, specific activity approximately 47 μCi/mmol), was diluted to 0.3 μCi/mL with assay buffer (phosphate-buffered saline, 0.1 mol/L, pH 7.4, containing 0.1% bovine serum albumin). Aliquots of 100 μL each of tracer solution, antiserum (final dilution 1:90 000), and urine or standard ouabain were added to polystyrene tubes. After mixing, the reaction mixture was incubated for 1 hour at 37°C or overnight at 4°C. The bound and free ouabains were then separated by adding 250 μL of cold 2.5% dextran-coated charcoal. The samples were mixed, allowed to stand for 10 to 15 minutes in ice-cold water, and then centrifuged at 3000 rpm for 30 minutes at 4°C. Supernatant was transferred to scintillation vials, and after adding 4 mL of scintillant to each vial, radioactivity was counted for 5 minutes. All samples and standards were assayed in duplicate. The ouabain concentration in test samples was estimated from a standard curve covering the concentration range from 0.06 to 2.2 nmol/L.
The immunoassay for OLS was validated by testing the urine extract for its ability to inhibit the uptake of Rb, which is an analogue of potassium. For this assay, blood samples were obtained immediately before the experiment from healthy donors and the red cells washed three times with ice-cold MgCl2 (110 mmol/L, 285 mOsm/kg). The washed cells were resuspended in Ringer’s solution to give a hematocrit of 40% to 50%, and an aliquot was taken for erythrocyte counting in a Coulter counter. To 100 μL of red cell suspension, 150 μL of ouabain standard (10−2 mol/L) or urine extracts was added, and the volume was increased to 500 μL with buffer. All cell suspensions were preincubated for 3 hours at 37°C in a shaking water bath. At the end of the preincubation, 50 μL (2 to 3 Ci) of 86Rb (specific activity 1.28 mCi/mg) was added to each tube and incubated for 1 hour, after which the cells were cooled and washed three times, with 2 mL of ice-cold saline (154 mmol/L, 295 mOsm/kg). The washed cells were lysed by adding 0.5 mL of 5% trichloroacetic acid and the radioactivity determined by Cerenkov radiation detector in a liquid scintillation counter.
Urinary excretion of sodium, DA, and ESTI were expressed in relation to creatinine to correct for any collection errors and flow rate changes. All results are presented as mean±SEM. Three-way ANOVA for repeated measurements was used to examine the effect of carbidopa treatment, salt intake, and time. All analyses were performed using a computer statistical package (Winstar, Anderson Bell Corp).
Fig 1⇓ shows the relationship between immunoreactive OLS and inhibition of Rb uptake across red cell membrane by urine extract. There was a linear relationship between the concentration of OLS in the sample and percentage inhibition of Rb uptake.
Fig 2⇓ shows the changes in urinary Na/Cr ratio in the four groups. As expected, sodium excretion was higher in the high salt groups (groups B and D) compared with the low salt groups (groups A and C) (Fig 2⇓). Three-way ANOVA showed that sodium excretion was not affected by carbidopa treatment (P=.1678) or duration (P=.706) (Table 1⇓). However, inspection of Fig 2⇓ shows that urinary Na/Cr in group D (high salt carbidopa group) appears to be lower on days 1 through 6 and higher on day 7 compared with group B. Two-way ANOVA with day and treatment as independent variables for days 1 through 6 of groups B and D showed that sodium excretion was significantly lower in the carbidopa-treated group (P=.0401). A similar analysis for groups A and C showed no difference.
DA/Cr ratios in the carbidopa-treated groups (C and D) were lower than in the untreated groups (groups A and B) throughout the 7 days (Fig 3⇓). A significant effect of salt intake and carbidopa treatment was seen on DA/Cr (Table 1⇑), and there was an interaction between duration, salt, and carbidopa treatment. In the carbidopa-treated groups (groups C and D), and especially in group D (the high salt carbidopa group), there was a tendency for the DA/Cr ratio to increase toward the end of the study. When untreated groups (groups A and B) were compared, high salt diet caused a significant increase in DA/Cr (P=.0056 for treatment, P=.0002 for duration, and P=.1121 for interaction between duration and treatment). When carbidopa-treated groups (C and D) were compared, high salt diet caused a significant increase in DA/Cr ratio (P<.0001 for treatment, P<.0001 for duration and P=.0112 for interaction), especially from day 3 onward (Fig 3⇓).
OLS/Cr ratio was higher in the high salt group (P<.0001) (Fig 4⇓, Table 1⇑), whereas carbidopa caused no effect. When the low salt and high salt diets are analyzed separately to examine the effect of carbidopa treatment on OLS/Cr ratio, carbidopa had a significant effect in the low salt groups (P=.0077) but not in the high salt groups (groups B and D) (P=.2448).
Table 2⇓ shows the pattern of ATPI/Cr ratio. Within all groups ATPI/Cr changed with time. Treatment had a significant effect on ATPI/Cr ratio. In untreated groups (A and B) the peak excretions of ATPI/Cr were on day 5, while on carbidopa treatment the peaks were on day 4. High salt diet and carbidopa treatment had a significant effect on ATPI/Cr (Table 1⇑).
There is extensive evidence for the presence of a circulating ESTI,5 and until recently the nature of this substance was not known. Recent studies suggest that this inhibitor may be ouabain or OLS.8 However, others have not confirmed these findings and doubt has been expressed about the existence of endogenous OLS.9 10 We have recently developed a specific immunoassay for ouabain and showed that excretion of OLS is increased during salt loading.22 This assay was specific for ouabain and showed very little cross-reactivity to other closely related cardenolides, endogenous steroids, bile acids, and phospholipids.22 Furthermore, the concentration of OLS measured with the immunoassay was related to the inhibition of Rb uptake by the erythrocytes (Fig 1⇑). Until the development of the immunoassay more “nonspecific” methods were used. These include inhibition of purified Na+,K+-ATPase, inhibition of cellular sodium transport, displacement of radiolabeled cardiac glycosides, cytochemical assay, and immunoassay using antibodies against digoxin.5 In this study we have used two of these nonspecific assays (ATPI and DLS) and compared the results with a specific OLS immunoassay. When we examined the effects of high salt diet on the excretions of ATPI, DLS, and OLS, it was clear that DLS was not affected, while excretion of ATPI and OLS increased on high salt diet. There was a significant correlation between OLS and ATPI (r=.306, P<.001) but not between OLS and DLS (r=.101, NS). When the relationship with sodium excretion was examined, OLS gave the strongest correlation (r=.849, P<.0001; Fig 5a⇓), followed by ATPI (r=.227, P=.00343, Fig 5b⇓). DLS excretion did not correlate with sodium excretion (r=−.012, NS). These results suggest that measuring DLS does not give any indication about physiologically important sodium transport inhibitors. Measurement of ATPI is better than that of DLS but not as specific as OLS. In the ATPI assay the purified Na+,K+-ATPase enzyme is liable to be inhibited by compounds that do not inhibit sodium pump in intact cells.26
DA has been suggested to be an intrarenal hormone involved in the handling of sodium.1 Urinary DA has been shown to originate in the renal tubules, and its excretion increases during a high salt diet.1 2 A close relationship between sodium intake and DA excretion has also been shown. Although DA excretion increases on salt loading, there has been some doubt as to the physiological role of DA in the natriuresis, because the changes in DA excretion were small and followed rather than preceded the natriuresis.2 In our study the role of DA was examined, and high salt diet was shown to have a significant effect on DA excretion. Carbidopa treatment had no significant effect on sodium excretion under basal (low salt) conditions. However, on a high salt diet carbidopa treatment caused a significant reduction in sodium excretion during the first 5 days. Toward the end of the study, sodium excretion returned to the value seen in the control high salt group. These results support a role for DA in sodium excretion, at least on high salt intake.
It has been suggested that a renal defect exists in “salt sensitive” individuals that prevents sodium excretion and results in the release of an ESTI.16 17 One proposal is that this renal abnormality is a defect in intrarenal synthesis of DA.1 We postulated that if this hypothesis is true, inhibition of DA synthesis should cause an increase in ESTI. In the low salt groups, carbidopa inhibited DA synthesis, and there was an associated increase in the excretion of ESTI. In the high salt groups there was no significant difference in ESTI between the carbidopa group (group D) and the control group (group C). These results taken together suggest that on a normal diet DA is important in sodium excretion and that blocking the synthesis of DA leads to an increase in ESTI that contributes to the restoration of sodium balance. The high salt diet stimulates increases in ESTI and DA; however because ESTI may be maximally stimulated in this state, the inhibition of DA synthesis does not cause any further increase. In this situation, other mechanisms must be responsible for the restoration of sodium balance. These results support the hypothesis that increased ESTI production occurs in response to reduced sodium excretion and volume expansion.16 17 Further support for this comes from our recent observations that in non–insulin dependent diabetes mellitus patients concentration of OLS is correlated with mean blood pressure.27
We conclude that ESTI and DA are important in the natriuresis of salt loading. The relative importance of these two factors requires further study.
Selected Abbreviations and Acronyms
|ATPI||=||ATPase inhibitory activity|
|ESTI||=||endogenous sodium transport inhibitor|
Financial support from the Croucher Foundation (Hong Kong) and secretarial help of Caroline Morgan are gratefully acknowledged.
- Received September 20, 1996.
- Revision received October 29, 1996.
- Accepted April 22, 1997.
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