(Hypertension. 1997;30:1544-1548.)
© 1997 American Heart Association, Inc.
Articles |
Effect of Carbidopa on the Excretion of Sodium, Dopamine, and Ouabain-Like Substance in the Rat
From the Department of Chemical Pathology, Prince of Wales Hospital, Shatin, NT, Hong Kong (C.S.H.) and the Department of Chemical Pathology, UMDS, Guy's & St Thomas' Hospital, London, UK (A.B., Y.K.S., R.S.).
Correspondence to Prof R. Swaminathan, Department of Chemical Pathology, United Medical & Dental Schools, St Thomas' Hospital, London, SE1 7EH UK. E-mail r.swaminathan{at}umds.ac.uk
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Abstract |
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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.
Key Words: dopamine • sodium • ouabain • carbidopa • sodium transport inhibitor, endogenous
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Introduction |
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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.
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Methods |
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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.
Na+,K+-ATPI
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.
Digoxin Immunoassay
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.
Statistical Analysis
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).
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Results |
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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.
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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.
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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).
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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).
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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).
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Table 3 shows the changes in the DLS/Cr
ratio in the four groups. DLS/Cr ratio changed during the study but
high salt diet or carbidopa had no significant effect (Table 1).
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Discussion |
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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
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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.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Financial support from the Croucher Foundation (Hong Kong) and secretarial help of Caroline Morgan are gratefully acknowledged.
Received September 20, 1996; first decision October 29, 1996; accepted April 22, 1997.
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