| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Hypertension. 1999;34:666-672.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
Aging, High Salt Intake, and Renal Dopaminergic Activity in Fischer 344 Rats
From the Institute for Cardiovascular Studies (T.H., V.K., M.F.L.), College of Pharmacy, University of Houston, Houston, Tex; and the Institute of Pharmacology and Therapeutics (M.A.V., M.P.S.,J.T.G., M.P., P.S.), Faculty of Medicine, University of Porto, Portugal.
Correspondence to M.F. Lokhandwala, Institute for Cardiovascular Studies, College of Pharmacy, University of Houston, TX 77204-5511. E-mail MLokhandwala{at}uh.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—The present study examined renal dopaminergic activity and its response to high salt (HS) intake in adult (6-month-old) and old (24-month-old) Fischer 344 rats. Daily urinary excretion of L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine, and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid was similar in adult and old rats; by contrast, daily urinary excretion of norepinephrine in old rats was almost twice that in adult animals. HS intake (1% NaCl) over a period of 24 hours resulted in a 2-fold increase in the urinary excretion of dopamine, DOPAC, and norepinephrine in adult animals but not in old animals. Norepinephrine and L-DOPA plasma levels did not change during HS intake and were similar in both groups of rats. The natriuretic response to an HS intake in old rats (from 4.7±0.4 to 10.7±2.0 nmol · kg-1 · d-1;
=6.0±0.9 nmol · kg-1 ·
d-1) was less than in adult rats (from 5.2±0.4 to
13.5±2.5 nmol · kg-1 · d-1;
=8.3±0.8 nmol · kg-1 ·
d-1). A diuretic response to HS intake was
observed in adult rats (from 20.9±2.3 to 37.6±2.8 mL ·
kg-1 · d-1) but not in old rats (from
37.7±5.7 to 42.3±6.0 mL · kg-1 ·
d-1). Dopamine levels and dopamine/L-DOPA ratios in the
renal cortex of old rats were greater than in adult rats. HS intake
increased both dopamine levels and dopamine/L-DOPA ratios in the renal
cortex of adult rats but not in old rats. Aromatic L-amino
acid decarboxylase activity was higher in old rats than in adult rats;
HS intake increased L-amino acid decarboxylase activity
(nmol · mg protein-1 · l5
min-1) in adult rats (from 67±1 to 93±1) but not in old
rats (from 86±2 to 87±2). Dopamine inhibited
Na+,K+-ATPase activity in proximal tubules
obtained from adult rats, but it failed to exert such an
inhibitory effect in old rats. It is concluded that renal
dopaminergic tonus in old rats is higher than in adult rats but fails
to respond to HS intake as observed in adult rats. This may be due in
part to the inability of dopamine to inhibit
Na+,K+-ATPase activity in old rats.
Key Words: aging • kidney • dopamine • sodium • Na+,K+-ATPase
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Dopamine exerts natriuretic and diuretic effects by activating D1-like receptors located at various regions in the nephron.1 2 At the level of the proximal tubule, the overall increase in sodium excretion produced by dopamine and D1 receptor agonists results from inhibition of Na+,K+-adenosine triphosphatase (Na+,K+-ATPase)3 and the Na+/H+ exchanger.4 Inhibition of Na+,K+-ATPase by dopamine occurs via a phospholipase C–coupled protein kinase C–linked pathway.5 6 Stimulation of D1 receptors leads to inhibition of the Na+/H+ exchanger as a result of increased adenosine 3',5'cyclic monophosphate formation and subsequently via protein kinase A–mediated phosphorylation.4
The physiological importance of the renal actions of dopamine depends on the sources of the amine in the kidney and on the availability of this dopamine to activate the amine-specific receptors. The proximal tubules are endowed with high aromatic L-amino acid decarboxylase (AADC) activity, and epithelial cells of the proximal tubules synthesize dopamine from circulating or filtered L-3,4-dihydroxyphenylalanine (L-DOPA).7 This nonneuronal renal dopaminergic system appears to be highly dynamic, and the basic mechanisms for the regulation of this system are thought to depend mainly on the availability of L-DOPA, its rapid decarboxylation into dopamine, and the precise and accurate cell-outward amine-transfer mechanisms.8 High levels of metabolic enzymes such as types A and B monoamine oxidases (MAO-A and MAO-B) and catechol-O-methyltransferase (COMT) have also been considered as important determinants in the overall availability of renal dopamine.9 10
The aging kidney undergoes structural changes that result in
quantitative alterations in some renal functions such as decline in
renal blood flow and glomerular filtration
rate.11 High blood pressure or edema formation may be
linked to abnormalities in the function of the renal dopaminergic
system, such as decreased ability to synthesize
dopamine12 13 and deficient coupling of dopamine receptors
to effector mechanisms.6 14 15 16 Despite their normal blood
pressure, an increasing number of studies have revealed that old rats
may present particular deficiencies in the renal handling of
L-DOPA, its subsequent conversion to dopamine,17 18 and at
the level of receptor number or coupling to G
proteins.19 20 21 Recently, 24-month-old Fischer 344 rats
were reported to have an impairment in dopamine receptor–mediated
inhibition of renal
Na+,K+-ATPase.21
This deficient response to dopamine is accompanied by a decreased
number of dopamine receptors and a marked attenuation of
D1-receptor–mediated activation of G proteins
but a significant increase in the levels of Gs
and Gq/11 in the basolateral membranes.21
Because tubular epithelial cells responsible for the production of dopamine are endowed with specific receptors for the amine, it was thought appropriate to evaluate the status of tubular renal dopaminergic system activity in 24-month-old Fischer 344 rats and compare it with that in adult animals (6 months old). Because one of the important factors regulating the synthesis of dopamine is the amount of sodium delivered to the kidney, it was decided to assess the activity of the renal dopaminergic system in adult and old Fisher 344 rats during normal salt (NS) and high salt (HS) intake. We report here the tissue levels and the urinary excretion of water and electrolytes, dopamine, its immediate precursor L-DOPA, and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA; 4-hydroxy-3-methoxyphenylacetic acid). The same animals were used for the study of the activities of enzymes involved in the synthesis and degradation of dopamine, namely, aromatic AADC, MAO, and COMT. In addition, we also examined the effect of dopamine on proximal tubular Na+,K+-ATPase activity in these different groups of rats.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Experimental Protocol
Normotensive, male Fischer 344 rats (Harlan Sprague Dawley, Indianapolis, Ind) aged 6 (adult) and 24 (old) months old and weighing 350 and 450 g, respectively, were used for all experiments. Animals were kept under controlled conditions (12-hour light/dark cycle and room temperature at 22±2°C); fluid intake and food consumption were monitored daily. All animals were fed ad libitum with ordinary rat chow containing 0.4% sodium (Purina Mills, St. Louis, Mo). Old (n=12) and adult (n=12) animals were subdivided in 2 groups (6 in each group) according to their daily sodium intake, ie, normal salt (NS) and high salt (HS). Rats on NS intake received tap water, and their daily sodium intake averaged 0.5 mmol/100 g of body weight. Rats on HS intake had 1.0% NaCl in their drinking water, and their daily sodium intake averaged 5.0 mmol/100 g of body weight. All groups of rats were maintained in metabolic cages for the duration of the study (24 hours). The vials collecting 24-hour urine contained 1 mL of 6 mol/L HCl to prevent decomposition of monoamines and amine metabolites. The animals were housed in an AAALAC-accredited facility and all of the protocols were approved by the Institutional Animal Care and Use Committee.
After completion of this protocol, rats were anesthetized with
sodium pentobarbital (50 mg/kg IP). Blood from the vena cava was
collected in tubes containing heparin. The kidneys were rapidly removed
through an abdominal midline incision, rinsed free of blood with normal
saline, cut in half, and placed in ice-cold saline. Thereafter, the
outer cortex was cut off, and fragments weighing 
100 mg were placed
in vials containing 500 µL of 0.2 mol/L HClO4.
The samples were stored at -80°C until quantification of
catecholamines and metabolites.
In Vitro Studies
In vitro studies included the assay of enzymes involved in the
synthesis (AADC) and metabolism (MAO and COMT) of renal
dopamine. For these studies, renal tissues were obtained from the same
4 experimental groups of rats mentioned above.
AADC Activity
AADC activity was determined in renal tissues as previously
described.22 In brief, fragments of renal cortex were
homogenized, and aliquots of 500 µL of cell
homogenate plus 400 µL of incubation medium were
preincubated for 20 minutes. Thereafter, L-DOPA (50 to 5000
µmol/L) was added to the medium for an additional 15 minutes; the
final reaction volume was 1 mL. The incubation medium contained the
following (in mmol/L):
NaH2PO4 0.35,
Na2HPO4 0.15,
Na2B4O7
0.11, and pyridoxal phosphate 0.2, pH 7.2; tolcapone (1 µmol/L)
and pargyline (100 µmol/L) were also added to the medium to
inhibit the enzymes COMT and MAO, respectively. The assay of dopamine
was performed by high-performance liquid
chromatography (HPLC) with electrochemical
detection.
MAO Activity
MAO activity was determined in renal tissue
homogenates as previously described23 with
[3H]5-hydroxytryptamine
([3H]5-HT) as a preferential substrate for
MAO-A and [14C]ß-phenylethylamine
([14C]ß-PEA) as a preferential substrate for
MAO-B. The reaction mixture contained 50 µL of tissue
homogenate, 50 µL of 67 mmol/L phosphate buffer, and
increasing concentrations of [3H]5-HT (75 to
3000 µmol/L) and [14C]ß-PEA (10 to
500 µmol/L). After 20 minutes of incubation at 37°C, the
reaction was stopped by the addition of 10 µL of 3 mol/L HCl. The
deaminated products were extracted with ethyl acetate (500 µL)
and measured by liquid scintillation counting. MAO activity was
expressed in nanomoles of substrate metabolized per milligram of
protein per hour.
COMT Activity
COMT activity was evaluated by the ability of tissue
homogenates to methylate epinephrine to
metanephrine.10 Aliquots of 100 µL of the
homogenate were preincubated for 20 minutes with 100 µL
of phosphate buffer (0.5 mmol/L). The reaction mixture was
incubated for 5 minutes with increasing concentrations of
epinephrine (1 to 2000 µmol/L; 50 µL) in the presence
of a saturating concentration of the methyl donor
S-adenosyl-L-methionine (250
µmol/L); the incubation medium also contained pargyline (100
µmol/L), MgCI2 (100 µmol/L), and EGTA
(1 mmol/L). The assay of metanephrine was performed by HPLC with
electrochemical detection.
Na+,K+-ATPase Activity
The isolated rat renal proximal tubules were incubated with
dopamine for 20 minutes at 37°C. The tubules were
permeabilized by rapid freezing in dry ice–acetone and
subsequent thawing. The
Na+,K+-ATPase activity was
measured as described earlier14 and expressed as
nanomoles of inorganic phosphate per milligram protein per minute
and determined as the difference between total and ouabain-insensitive
ATPase.
Alkaline Phosphatase and 
-Glutamyl Transferase
Activities
Alkaline phosphatase (ALKP) and -glutamyl transferase
(-GT) activities in homogenates of renal cortex were
measured by using standard enzymic techniques (p-nitrophenyl
phosphate and
L--glutamyl-p-nitroanilide as
substrates for ALKP and -GT, respectively) on a Kodak Ektachem 250
analyzer (Eastman Kodak Co, Clinical Diagnostics
Division, Rochester, NY).
Assay of Catecholamines
The assay of catecholamines and their metabolites in
urine, plasma samples, and renal tissues was performed by HPLC with
electrochemical detection.13 In brief, aliquots of 0.5 mL
of acidified urine or plasma and 1.5 mL of HClO4,
in which tissues had been kept or renal homogenates
acidified, were placed in 5-mL conical-base glass vials with 50
mg alumina, and the pH of the samples was adjusted to pH 8.6 by
addition of Tris buffer. The adsorbed catecholamines were
then eluted from the alumina with 200 µL of 0.2 mol/L
HClO4 on Costar Spin-X microfilters; 50 µL of
the eluate was injected into an HPLC (Gilson Medical Electronics,
Villiers le Bel, France). The lower limit of detection of L-DOPA,
dopamine, norepinephrine, epinephrine,
metanephrine, 3,4-dihydroxymandelic acid, DOPAC, HVA, 5-HT, and
5-hydroxyindoleacetic acid ranged from 350 to 1000 fmol.
Plasma and Urine Ionogram
Urinary sodium and potassium were measured by flame photometry
(model FML3) connected to a diluter (model A 6241, Radiometer,
Copenhagen, Denmark), and urine and plasma osmolality values were
measured by means of an osmometer (model 3 MO, Advanced Instruments,
Inc).
Statistics
Results are expressed as mean±SEM of values for the indicated
number of determinations. Vmax and
Km values for the decarboxylation of
L-DOPA, the O-methylation of epinephrine, or the
deamination of [3H]5-HT and
[14C]ß-PEA were calculated from nonlinear
regression analysis by using the GraphPad Prism statistics
software package.24 Geometric means are given with
95% confidence limits, and arithmetic means are given with SEM.
Statistical analysis was performed by 1-way ANOVA followed by
the Newman-Keuls post hoc test. A value of P<0.05 was
assumed to denote a significant difference.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Effect of HS Intake on Urinary Sodium and Dopamine Excretion
Old rats were
100 g heavier than adult rats, but HS intake for
24 hours did not affect body weight in both groups of rats. Liquid
intake in both old and adult rats did not differ, either during NS
intake or HS intake. The urine volume in old rats on NS intake was
almost twice that in adult rats; however, during HS intake, adult rats
responded with an increase in urine volume, but similar
diuresis was not seen in old rats (Figure 1). Fractional excretion of sodium during
HS intake increased significantly, and this increase was similar in
adult and old rats (Figure 1). Urinary excretion of potassium
(mmol · kg-1 · 24
h-1) and the fractional excretion of potassium
did not change during HS intake, and these values were also similar
between adult and old rats (Urinary K+ from NS
adult rats 7.1±0.7, HS adult rats 10.7±1.9, NS old rats 7.0±0.7, and
HS old rats 7.0±0.7; fractional excretion of K+
from NS adult rats 34.7±5.1, HS adult rats 21.9±3.8, NS old rats
35.5±5.7, and HS old rats 32.2±8.0). Urine osmolality in old rats on
an NS intake was considerably lower than that in adult rats. During HS
intake, adult rats responded with a slight decrease in urine osmolality
and an increase in the osmolar clearance, whereas old rats showed no
change when compared with values observed on an NS intake (Figure 1). There were no significant changes in plasma sodium,
potassium, and chloride levels and plasma osmolality between adult and
old rats on both NS and HS intakes.
|
Data on the urinary excretion of catecholamines and metabolites is given in Figure 2. The urinary excretion of dopamine and DOPAC in adult rats on an NS intake did not differ from that in old rats. However, when adult rats were placed on an HS intake, the urinary excretion of both dopamine and DOPAC was twice that observed during NS intake, and such an increase was not observed in old rats (Figure 2). Old rats failed to respond to an HS intake by an increase in the urinary excretion of dopamine and of its deaminated metabolite, despite the observation that their basal excretion during NS intake did not differ from that in adult rats (Figure 2). In both adult and old rats, the urinary excretion of L-DOPA and HVA were similar during NS intake and not affected by HS intake. It is interesting to note that the urinary excretion of norepinephrine, but not that of epinephrine, in adult rats on an NS intake was half that in old rats, and that HS intake produced a significant increase in the urinary excretion of this amine in adult but not in old animals (Figure 2). The urinary excretion of 5-hydroxyindoleacetic acid (in nmol · kg-1 · 24 h-1) in both adult (281±43) and old (354±36) rats on an NS intake did not significantly differ from that observed during HS intake (adult rats 508±127, old rats 458±28). There were no significant changes in plasma L-DOPA, norepinephrine, and DOPAC levels between adult and old rats on both the NS and HS intake.
|
Data on tissue levels of catecholamines and metabolites are given in the Table. It was found that tissue levels of dopamine and of its deaminated metabolite DOPAC in fragments from the renal cortex of adult rats on an NS intake were significantly lower than those in old rats under the same experimental conditions. Placing adult but not old rats on an HS intake resulted in an increase in dopamine tissue levels; DOPAC tissues levels, however, were not affected by different regimens in sodium intake. Also, no significant changes were observed between adult and old rats on both NS and HS intakes in terms of norepinephrine tissue levels. By contrast, tissue levels of epinephrine in adult rats on an NS intake were higher than in old rats, and HS intake was found to significantly reduce epinephrine levels in both adult and old rats.
|
In Vitro Studies
AADC Activity
Incubation of homogenates of renal cortex with L-DOPA
(50 to 5000 µmol/L) resulted in a concentration-dependent
formation of dopamine (Figure 3). The
Vmax values for AADC with the use of L-DOPA as
the substrate in adult rats on an NS intake were found to be
significantly (P<0.01) lower than those observed in old
rats under the same salt regimen. The effect of HS intake was a marked
increase in Vmax values in adult but not in old
rats (Figure 3). In all experimental conditions, the
decarboxylation reaction was a saturable process, with
Km values of the same magnitude.
|
MAO Activity
Homogenates of renal cortex from both adult and old
rats were found to deaminate quite actively both
[3H]5-HT and
[14C]ß-PEA. Deamination of
[14C]ß-PEA was a high-affinity process when
compared with that for [3H]5-HT, as expected
for specific substrates of MAO-B and MAO-A, respectively. It is
interesting to note that Vmax and
Km values were similar in both age groups
and were not affected by HS intake (data not shown).
COMT Activity
Incubation of homogenates of rat renal cortex in the
presence of increasing concentrations of epinephrine resulted
in a concentration-dependent formation of metanephrine (data not
shown). The kinetics, Vmax, and
Km values for COMT were similar in adult
and old rats. In all experimental conditions, the
O-methylation reaction was a saturable process, with
Km values of the same magnitude (data not
shown).
Na+,K+-ATPase Activity
Basal Na+,K+-ATPase
activity (in nmol · mg protein-1 ·
min-1) in the proximal tubules of adult rats on
an NS intake was similar to that observed in old rats under the same
sodium regimen (adults 127±4, old rats 105±17) and was found not to
be significantly changed with the increase in sodium intake (adult
120±5, old 120±20). As shown in Figure 4, dopamine caused a
concentration-dependent reduction of
Na+,K+-ATPase activity in
the proximal tubules of adult rats on either the NS or HS intake
(Figure 4). However, it failed to inhibit enzyme activity in
either group of old rats (NS or HS intake).
|
ALKP and -GT Activities
There were no significant changes in ALKP between adult and old
rats on both NS and HS intakes. However -GT activity in adult rats
(2.01±0.17 U/mg protein) on the NS intake was significantly greater
than that observed in old rats (1.64±0.11 U/mg protein) under the same
experimental regimen. However, HS intake failed to alter -GT
activity in either group of rats.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The findings of the present study show that old Fischer 344 rats have an increased renal dopaminergic tonus; however, the animals failed to respond to an HS intake with a subsequent increase in dopamine synthesis, which is accompanied by changes in water and electrolyte metabolism. This increased renal dopaminergic tonus, when viewed collectively with the failure of dopamine to inhibit Na+,K+-ATPase activity as shown in the present and previous studies,21 and a marked decrease in the number of dopamine receptors may be related to an attempt of the renal epithelial cells from old Fischer 344 rats to induce a compensatory response and overcome the reduced expression of dopamine receptors and the functional consequences of this phenomenon.
The increased renal dopaminergic tonus in old Fischer 344 rats was evidenced by high AADC activity, high dopamine and DOPAC levels, and high tissue dopamine/L-DOPA ratios. It is interesting to note that the renal dopaminergic system in old rats failed to respond to an HS intake with an increase in AADC or urinary excretion of dopamine and its deaminated metabolite DOPAC, as it did in the adult rats. On the other hand, the presence of an increased dopaminergic activity in old rats was accompanied by an increase in the urinary excretion of neither dopamine nor of its metabolites. Our present findings of a similar urinary excretion of dopamine and DOPAC in adult and old rats are at variance with a previous report showing that urinary dopamine and DOPAC excretion values were lower in old than adult rats.17 This discrepancy may be due to differences in the strain of rats; ie, we used Fischer 344 animals, whereas Armando et al17 used Wistar rats. Also, the age of the animals in the 2 studies was different, in that although we used 6- and 24-month-old rats, the previous study used 3- and 12-month-old rats.17 Unlike the findings of an increase in urinary dopamine excretion during HS intake in Fischer 344 adult rats, in our previous study in Wistar rats placed on an HS intake, we did not see a significant increase in urinary excretion of dopamine.25 We believe this discrepancy is related to the strain of animal used, as we have repeatedly failed to observe this type of an effect of an increase in urinary dopamine when Wistar rats are placed on an HS intake (M.A.V.-C., unpublished observation, 1999). In a previous study,18 we have shown that old rats decarboxylate less L-DOPA in kidney slices, but this was not true of kidney homogenates. Also, old Wistar rats had a reduced ability to take up L-DOPA into tubular cells, similar to findings of another group of investigators.17 Again, these discrepancies are most likely due to the different strains of animal used in the different studies. It is also interesting to note that although we were able to detect basal levels of DOPAC in the present study in Fischer 344 rats, we were unable to detect basal DOPAC levels in Wistar rats,18 which may be due to the fact that basal DOPAC levels in Wistar rats are beyond the limit of detection of our assay procedure. The lack of correlation between renal AADC activity and urinary levels of dopamine contrasts with the positive correlation between renal AADC activity and the tissue levels of dopamine and DOPAC and the dopamine/L-DOPA tissue ratios. One possible explanation could be deficient renal delivery of L-DOPA in old rats, particularly during HS intake, as a result of a reduced ability to mobilize L-DOPA from internal stores. Alternatively, it may be related to a reduced ability to take up L-DOPA at the level of the renal tubular epithelial cells. The finding that urinary and plasma levels of L-DOPA were quite similar between old and adult rats on NS and HS intakes suggests that deficient delivery of L-DOPA to the kidney could not account for this phenomena. On the other hand, a deficient uptake of L-DOPA by renal tubular epithelial cells is supported by the finding of reduced dopamine/L-DOPA ratios in both the urine and renal tissues. This suggestion is substantiated by the observation that renal tubular cells from old rats take up less exogenous L-DOPA.2 18 The inability of old rats on an HS intake to increase urinary dopamine excretion may be related to the reduced uptake of L-DOPA by tubular cells. In fact, several groups have shown an association between renal delivery of sodium and the ability to form dopamine,26 27 and in vitro studies have shown that L-DOPA uptake by renal epithelial cells is a sodium-dependent process.17 28 29 If the uptake of L-DOPA by tubular epithelial cells in old rats became defective, then it would not be able to respond to an increase in sodium by synthesizing more dopamine.
As mentioned above (see the introductory section), there is evidence to suggest that the renal availability of dopamine may depend on the activity of metabolic processes involved in the degradation of the amine, and these include deamination to DOPAC by MAO and methylation to HVA by COMT. The urinary excretion of DOPAC followed quite closely the urinary excretion of the parent amine in all of the 4 experimental conditions. This suggests that deamination of dopamine was not compromised and that an HS intake did not interfere with this process. This finding further supports our previous suggestion that urinary DOPAC is a good marker of renal production of dopamine and simultaneously a good index of cell integrity and viability,9 30 since MAO is a mitochondrial enzyme and is quite sensitive to changes in tissue oxygen tension.29 However, DOPAC/dopamine tissue ratios, another marker of the deamination process, in old rats were found to be greater than those in adult rats. This was observed only during NS intake and not during HS intake and, therefore, may not represent a difference in the activity of renal MAO between old and adult rats. Similarly, no changes in COMT activity were observed between these 4 groups of rats, except for the observation of a lower Vmax value in old rats on HS intake compared with those on NS intake. This change may not have dramatic consequences on the O-methylation of dopamine, since this was not accompanied by changes in urinary levels of HVA and DOPAC.
These findings reported here for old Fischer rats are different from the observations made in old Wistar rats17 in several aspects, but both studies show that aging is accompanied by a 2.3-fold (Wistar rats) and a 1.3-fold (Fischer 344 rats) increase in AADC. Obviously, the increase in AADC activity was greater in Wistar than in Fischer 344 rats, but this difference may be due to the fact that AADC activity in the study of Armando et al17 was performed with a single concentration of substrate (100 µmol/L L-DOPA), which is clearly a nonsaturating concentration. The same criticism applies to determination of MAO activity, wherein a single, nonsaturating concentration of tyramine was used.17 By contrast, COMT activity in the study of Armando et al17 was performed with a saturating concentration of substrate, and their result is quite similar to that reported here, ie, no change in COMT activity between old and adult rats.
Another interesting observation of the present study was that urinary norepinephrine excretion was almost twice as high in old rats compared with adult rats. One possible explanation could be a selective increase in renal sympathetic tone in old rats; the mechanism of and reasons for such an increase remain to be determined. In a previous study,17 no differences were found in urinary norepinephrine between adult and old rats, which may be due to differences in the strain and age of the animals. The finding that plasma and tissue levels of norepinephrine in adult and old rats were similar can be explained on the basis that circulating levels of norepinephrine mainly reflect release of the amine during a limited period of time, whereas urinary levels give a better indication over a longer period of time. Similarly, tissue levels do not always reflect the rate of norepinephrine synthesis or release. The reason for the increase in urinary excretion of norepinephrine in adult rats placed on an HS intake remains to be determined.
In terms of urinary excretion of electrolytes, we found that old rats had larger urine volumes over the 24-hour period than did adult rats; however, the urine from the former group had a lower osmolality compared with that from adult rats. During an increase in sodium intake, old rats appear to slightly retain sodium, as evidenced by a less-pronounced increase in urinary excretion of sodium compared with adult rats. This may be due to the inability of dopamine in old rats, on either an NS or HS intake, to inhibit Na+,K+-ATPase (Figure 4). The basal activity of Na+,K+-ATPase was not different between rats on either NS or HS intake, and although this finding agrees with 1 report,31 it differs from other published reports in which the authors found a decrease in basal Na+,K+-ATPase activity in rats placed on an HS intake.32 However, it should be noted that Bertorello et al32 observed a decrease in basal Na+,K+-ATPase activity in rats after 10 days of HS intake, whereas in the present study, we conducted our measurements after 24 hour of HS intake. It is likely that the exposure to HS intake needs to be of longer duration before changes in basal Na+,K+-ATPase activity in rats can be observed. Although in the present study dopamine was able to inhibit Na+,K+-ATPase in rats placed on both NS and HS intakes, Bertorello et al32 found that the inhibitory effect of dopamine on Na+,K+-ATPase was diminished in rats placed on an HS intake. Again, this discrepancy may be due to differences in the amount of time that the rats were exposed to HS intake.
In conclusion, it is suggested that old Fischer 344 rats have an increased renal dopaminergic tonus, which does not respond to HS intake by a subsequent increase in dopamine synthesis, and is accompanied by changes in water and electrolyte metabolism. The failure of dopamine to inhibit Na+,K+-ATPase may be one explanation for this derangement and for the water and electrolyte excretion observed in old rats.
|
Acknowledgments |
|---|
This study was supported in part by National Institutes of Health grant AG-15031 from the National Institute on Aging, Bethesda, Md (to M.F.L.), and by grant PECS/C/SAU/24/95 from the Fundacao para a Ciencia e a Tecnologia (to P.S.S.). M.A. Vieira-Coelho is a recipient of a Praxis XXI fellowship (BD/5973/95) from the Fundacao para a Ciencia e a Tecnologia.
Received March 16, 1999; first decision April 7, 1999; accepted June 3, 1999.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Jose PA, Raymond JR, Bates MD, Aperia A, Felder RA, Carey RM. The renal dopamine receptors. J Am Soc Nephrol. 1992;2:1265–1278.[Abstract]
- Lokhandwala MF, Amenta F. Anatomical distribution and function of dopamine receptors in the kidney. FASEB J. 1991;5:3023–3030.[Abstract]
- Seri I, Kone BC, Gullaris SR, Aperia A, Brenner BM, Ballermann BJ. Influence of Na+ intake on dopamine-induced inhibition of renal cortical Na+,K+-ATPase. Am J Physiol. 1990;58:F52–F60.
-
Felder CC, Campbell T, Albrecht F, Jose PA. Dopamine
inhibits Na+,H+-exchanger
activity in renal BBMV by stimulation of adenylate cyclase.
Am J Physiol. 1990;259:F297–F303.
[Abstract/Free Full Text] -
Felder CC, Jose PA, Axelrod J. The dopamine-1 agonist,
SKF 82526, stimulates phospholipase C activity independent of
adenylate cyclase. J Pharmacol Exp Ther. 1989;248:171–175.
[Abstract/Free Full Text] - Kansra V, Chen CJ, Lokhandwala MF. Dopamine fails to stimulate protein kinase C activity in renal proximal tubules of spontaneously hypertensive rats. Clin Exp Hypertens. 1995;17:837–845.
- Soares-da-Silva P, Fernandes MF. Regulation of dopamine synthesis in the rat kidney. J Auton Pharmacol. 1990;10:s25–s30.
-
Soares-da-Silva P. Source and handling of renal
dopamine: its physiological importance. News
Physiol Sci. 1994;9:128–134.
[Abstract/Free Full Text] -
Fernandes MH, Soares-da-Silva P. Effects of MAO-A and
MAO-B selective inhibitors Ro 41-1049 and Ro 19-6327 on the
deamination of newly formed dopamine in the rat kidney. J
Pharmacol Exp Ther. 1990;255:1309–1313.
[Abstract/Free Full Text] - Vieira-Coelho MA, Soares-da-Silva P. Ontogenic aspects of liver and kidney catechol-O-methyltransferase sensitivity to tolcapone. Br J Pharmacol. 1996;117:516–520.[Medline] [Order article via Infotrieve]
- Lindeman RD. Overview: renal physiology and pathophysiology of aging. Am J Kidney Dis. 1990;16:275–282.[Medline] [Order article via Infotrieve]
-
Kuchel O, Shigetomi S. Defective dopamine generation
from dihydroxyphenylalanine in stable essential hypertensive patients.
Hypertension. 1992;19:634–638.
[Abstract/Free Full Text] - Soares-da-Silva P, Pestana M, Vieira-Coelho MA, Fernandes MH, Albino-Teixeira A. Assessment of renal dopaminergic system activity in the nitric oxide-deprived hypertensive rat model. Br J Pharmacol. 1995;114:1403–1413.[Medline] [Order article via Infotrieve]
-
Chen C, Beach RE, Lokhandwala MF. Dopamine fails to
inhibit renal tubular sodium pump in hypertensive rats.
Hypertension. 1993;21:364–372.
[Abstract/Free Full Text] -
Hussain T, Lokhandwala MF. Renal dopamine
DA1 receptor coupling with
Gs and Gq/11 proteins in spontaneously
hypertensive rats. Am J Physiol. 1997;41:F339–F346.
- Kinoshita S, Sidhu A, Felder RA. Defective dopamine-1 receptor adenylate cyclase coupling in the proximal convoluted tubule from the spontaneously hypertensive rat. J Clin Invest. 1989;84:1849–1856.
-
Armando I, Nowicki S, Aguirre J, Barontini M. A
decreased tubular uptake of dopa results in defective renal dopamine
production in aged rats. Am J Physiol. 1995;268:F1087–F1092.
[Abstract/Free Full Text] - Soares-da-Silva P, Fernandes MH. A study on the renal synthesis of dopamine in aged rats. Acta Physiol Scand. 1991;143:287–293.[Medline] [Order article via Infotrieve]
- Galbusera M, Garattini S, Remuzzi G, Mennini T. Catecholamine receptor binding in rat kidney: effect of aging. Kidney Int. 1988;33:1073–1077.[Medline] [Order article via Infotrieve]
- Ricci A. The renal dopaminergic system in ageing. J Auton Pharmacol. 1990;10:s19–s24.
- Kansra V, Hussain T, Lokhandwala MF. Alterations in dopamine DA1 receptor and G proteins in renal proximal tubules of old rats. Am J Physiol. 1997;42:F53–F59.
- Soares-da-Silva P, Serrao MP, Vieira-Coelho MA. Apical and basolateral uptake and intracellular fate of dopamine precursor L-dopa in LLC-PK1 cells. Am J Physiol. 1998;274:F243–F251.
- Fernandes MH, Soares-da-Silva P. Type A and B monoamine oxidase activities in the human and rat kidney. Acta Physiol Scand. 1992;145:363–367.[Medline] [Order article via Infotrieve]
- Motulsky HJ, Spannard P, Neubig R. GraphPad Prism (Version JO). San Diego, Calif: GraphPad Prism Software Inc; 1994.
- Vieira-Coelho MA, Pestana M, Soares-da-Silva P. High sodium increases the urinary excretion of L-3,4-dihydroxyphenylalanine but fails to increase the urinary excretion of dopamine and amine metabolites in Wistar rats. Gen Pharmacol. 1996;27:1421–1427.[Medline] [Order article via Infotrieve]
- Goldstein DS, Stull R, Eisenhofer G, Gill J Jr. Urinary excretion of dihydroxyphenylalanine and dopamine during alterations of dietary salt intake in humans. Clin Sci. 1989;76:517–522.[Medline] [Order article via Infotrieve]
- Grossman E, Hoffman A, Tamrat M, Armando I, Keiser HR, Goldstein DS. Endogenous dopa and dopamine responses to dietary salt loading in salt-sensitive rats. J Hypertens. 1991;9:259–263.[Medline] [Order article via Infotrieve]
-
Hayashi M, Yamaji Y, Kitajima W, Saruta T. Effects of
high salt intake on dopamine production in rat kidney.
Am J Physiol. 1991;260:E675–E679.
[Abstract/Free Full Text] - Soares-da-Silva P, Fernandes MH. Sodium-dependence and ouabain sensitivity of the synthesis of dopamine in renal tissues of the rat. Br J Pharmacol. 1992;105:811–816.[Medline] [Order article via Infotrieve]
-
Pestana M, Faria MS, Oliveira JG, Baldaja J, Santos A,
Guerra LE, Soares-da-Silva P. Assessment of renal dopaminergic system
activity during the recovery of renal function in human kidney
transplant recipients. Nephrol Dial Transplant. 1997;12:2667–2672.
[Abstract/Free Full Text] - Wald H, Epstein FH, Popovtzer MM. Effect of chronic salt loading on renal Na+,K+-ATPase activity in the rat. Proc Soc Exp Biol Med. 1983;172:291–296.[Abstract]
-
Bertorello A, Hokfelt T, Goldstein M, Aperia A.
Proximal tubule
Na+,K+-ATPase activity is
inhibited during high-salt diet: evidence for DA-mediated effect.
Am J Physiol. 1988;254:F795–F801.
[Abstract/Free Full Text]
This article has been cited by other articles:
 |
|
 |
|
  M. J. Pinho, M. P. Serrao, and P. Soares-da-Silva High-salt intake and the renal expression of amino acid transporters in spontaneously hypertensive rats Am J Physiol Renal Physiol, May 1, 2007; 292(5): F1452 - F1463. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. SOARES-DA-SILVA, M. P. SERRAO, M. J. PINHO, and M. J. BONIFACIO Cloning and gene silencing of LAT2, the L-3,4-dihydroxyphenylalanine (L-DOPA) transporter, in pig renal LLC-PK1 epithelial cells FASEB J, October 1, 2004; 18(13): 1489 - 1498. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Soares-da-Silva and M. P. Serrao High- and low-affinity transport of L-leucine and L-DOPA by the hetero amino acid exchangers LAT1 and LAT2 in LLC-PK1 renal cells Am J Physiol Renal Physiol, August 1, 2004; 287(2): F252 - F261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Pinho, P. Gomes, M. P. Serrao, M. J. Bonifacio, and P. Soares-da-Silva Organ-Specific Overexpression of Renal LAT2 and Enhanced Tubular L-DOPA Uptake Precede the Onset of Hypertension Hypertension, October 1, 2003; 42(4): 613 - 618. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Vieira-Coelho, P. Serrao, J. T. Guimaraes, M. Pestana, and P. Soares-Da-Silva Concerted action of dopamine on renal and intestinal Na+-K+-ATPase in the rat remnant kidney Am J Physiol Renal Physiol, December 1, 2000; 279(6): F1033 - F1044. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||