Cytochemically Assayable Na+,K+-ATPase
Inhibition by Milan Hypertensive Rat Plasma
SHARON HOLLAND, JACQUELINE MILLETT, JAMSHID ALAGHBAND-ZADEH,
H U G H DE WARDENER, PATRIZIA FERRARI, AND GIUSEPPE BIANCHI
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SUMMARY The ability of plasma from 3- and 9-week-old Milan hypertensive rats and their nor motensive controls to inhibit Na + ,K+-adenosine triphosphatase (ATPase) was studied using cytochemical bioassay techniques in fresh tissue. With a validated cytochemical bioassay that measures the
capacity of biological samples to stimulate glucose-6-phosphate dehydrogenase activity in guinea pig
proximal tubules as an indication of their capacity to inhibit Na+,K+-ATPase, the mean gIucose-6phosphate dehydrogenase-stimulating ability of the plasma of the 9-week-old Milan hypertensive rats
and their normotensive controls was 586.0 ± 88 and 23.4 ± 8.3 U/ml (n = 7; p<0.001), while that of
the 3-week-old Milan hypertensive rats (before the main rise in arterial pressure) and their normotensive controls was 99.9 ± 27.4and 7.8 ± 1.8 U/ml (n = 7; p<0.001). With the use of a semiquantitative
cytochemical assay that measures Na+,K+-ATPase activity directly, plasma from the adult hypertensive rats had a much greater capacity to inhibit Na+,K+-ATPase than the plasma of the control rats.
The significantly raised levels found in the young hypertensive rats before the main rise in arterial
pressure are consistent with the hypothesis that the rise hi the ability of plasma to inhibit Na+,K+ATPase is due to an inherited renal difficulty in excreting sodium. (Hypertension 9:498-503, 1987)
KEY WORDS • plasma • glucose-6-phosphate dehydrogenase-stimulating activity
hypertensive rats • cytochemical assay • Na+,K+-ATPase-inhibiting activity
W
ITH the use of cytochemical techniques 12
we have previously demonstrated that
plasma from normal humans3 and rats4 on
a high sodium intake, from patients with essential
hypertension, 3 and from adult spontaneously hypertensive rats (SHR) 6 have a heightened ability to inhibit Na + ,K + -adenosine triphosphatase (ATPase). It
therefore appears that these two forms of hereditary
hypertension are associated with a rise in the concentration of a cytochemically bioassayable circulating
Na + ,K + -ATPase inhibitor that is controlled by salt
Milan
intake in normal humans and animals. In the following
experiments the plasma of Milan hypertensive strain
rats (MHS) was studied at 3 and 9 weeks of age (i.e.,
before and after the rise in blood pressure). Much
evidence suggests that the cause of hypertension in this
strain is a genetically determined renal abnormality7
and that, during the development of hypertension,
there is a transient period of detectable sodium retention.8 These rats may therefore be particularly suitable
for testing the hypothesis that a circulating Na + ,K + ATPase inhibitor, the concentration of which is known
to rise with a high salt intake, may be involved in the
pathogenesis of genetic hypertension.
Two cytochemical techniques9 were used. One is a
validated cytochemical bioassay that measures the capacity of biological samples to stimulate glucose-6phosphate dehydrogenase (G6PD) activity of fresh
guinea pig kidney after exposure for 2 minutes, which
is an indication, using the other cytochemical technique, of their capacity to inhibit Na + ,K + -ATPase
after exposure for 4 to 6 minutes. 2 The technique that
measures Na + ,K + -ATPase activity is direct but semiquantitative.1
From the Research Laboratories (J. Millett, H. de Wardener) and
Department of Chemical Pathology (S. Holland, J. AlaghbandZadeh), Charing Cross and Westminster Medical School, London,
England, and Centre Ricerche Farmitalia (P. Ferrari), Milan, and
Instituto di Scienze Mediche (G. Bianchi), dell'Universita di Milano, Milan, Italy.
Supported by a grant from the National Kidney Research Fund
and the Fondation Clarisse Neiman.
Address for reprints: Professor H.E. de Wardener, Research Laboratories, Charing Cross & Westminster Medical School, Fulham
Palace Road, London W6 8RF, England.
Received May 7, 1986; accepted November 3, 1986.
498
499
PLASMA SODIUM TRANSPORT INHIBITING ACTIVITY///0//W et al.
Materials and Methods
Two groups of 3-week-old rats, one comprising
MHS and the other Milan normotensive strain rats
(MNS),10' " and two groups of 9-week-old adult MHS
and MNS were studied. There were seven rats in each
group. Blood was collected in Milan in heparinized
test tubes and centrifuged, and aliquots of plasma were
then packed in solid CO 2 and transported to London,
where 6 hours later they were stored at — 20 °C. The
average sodium and potassium content of the diet for
the adult rats was 2.0 and 7.3 g/kg, respectively. The
young rats were weaned the day before the blood was
obtained.
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Cytochemical Techniques
Cytochemical procedures, including the validation
of the G6PD assay, have been described elsewhere.1' 2-9 A brief summary of the techniques is given
here.
Female guinea pigs of the Duncan-Hartley strain
(Porcellus Firgrove Farm, Cross-in-Hand, Heathfield,
Sussex, England) weighing 300 to 450 g were killed by
cervical fracture. Both kidneys were removed, cut into
segments, and placed in sealed pots in a nonproliferative culture medium under an atmosphere of 95% O 2 ,
5% CO2 at 37°C for 5 hours. The medium was then
replaced with fresh medium containing various dilutions of plasma for 2 to 6 minutes (depending on which
of the two assays was being used). The segments were
chilled by immersion in n-hexane ( —70°C), and sections, 16 ^im thick, were cut from each segment using
a cryostat microtome at — 30 °C. To demonstrate
G6PD activity, the sections were incubated at 37°C
under an atmosphere of nitrogen in a reaction medium
containing glucose-6-phosphate (5 mM), the coenzyme nicotinamide adenine dinucleotide phosphate (3
mM), neotetrazolium chloride (5 mM), potassium cyanide (10 mM), phenazine methosulfate (0.67 mM),
and poly vinyl alcohol (G18 12% wt/vol) dissolved in a
0.05 M glycylglycine sodium hydroxide buffer (pH
8.2). The reaction was stopped by immersing the sections in distilled water. The activity of the G6PD was
shown by the deposition of an intensely colored, highly localized formazan. A plasma of high potency was
used as a standard. In established cytochemical bioassays for known hormones, the quantitative effect induced by a pure hormone, when measured at a predetermined time of exposure, is only evident over a
specific range of low concentrations of the hormone.
Similarly, with increasing dilutions of the high potency plasma, stimulation of G6PD measured at the predetermined time of 2 minutes first becomes evident
and then reaches a peak of activity before returning to
baseline in a log linear relationship (Figure 1). The
amount of G6PD-stimulating activity that produces
maximum G6PD stimulation at 2 minutes is defined as
1 unit of G6PD activity. The biphasic phenomenon of
the dose response is seen in all established cytochemical assays of known hormones. 9 It is attributed to a
more rapid effect of the high concentration of hormone
on enzyme activity, so that the induced effect on en-
zyme at such concentrations, after 2 minutes of exposure in this instance, is in the recovery phase and
thus appears to be less than that seen at a lower concentration.
To demonstrate Na + ,K + -ATPase activity,1 the sections from tissue segments were preincubated at 37 °C
in a medium containing 40% (wt/vol) polypeptide
(Polypep 5115; Sigma Chemical, St. Louis, MO,
USA) and 0.1 M potassium acetate dissolved in 0.2 M
Tris at pH 7.4 for 2 minutes, to remove free phosphate.
The preincubation buffer was then replaced for 15 minutes by reaction medium containing 40% (wt/vol)
polypeptide, potassium chloride (37.5 mM), sodium
chloride (410 mM), magnesium chloride (20 mM),
ATP (10 mM), sodium acetate (2 mM), together with
lead ammonium citrate/acetate complex (32 g/L; previously dissolved by shaking in the smallest amounts
of dilute ammonia) and dissolved in 0.2 M Tris buffer,
pH 7.4. The sections were rinsed several times in 0.2
M Tris buffer, pH 7.4, and immersed in water saturated with H2S for 2 minutes. This procedure demonstrates the total ATPase activity as a brown precipitate.
The amount of Na + ,K + -ATPase (i.e., ouabain-sensitive ATPase) was determined by including ouabain
(2 X 10"* M) in the reaction medium in serial sections,
then subtracting the ouabain-insensitive ATPase activity from the total ATPase activity.
Measurement of Glucose-6-Phosphate Dehydrogenase
and Na+,K+-ATPase Activity
The colored reaction products of the enzymes in the
proximal tubule were measured using a Vickers M8S
scanning and integrating microdensitometer (Vickers
Instruments, York, England) at a wavelength of 585
nm and a magnification of x400, in 20 proximal tubules from duplicate sections from each segment.
60so-
HIGH POTENCY
STANDARD PLASMA
MEAN
4OfINTEGHATED
EXTINCTION
(xtOO)
30
2O1O-
PLASMA DILUTION tO
G6PD
OO1
(MJmuklUng units/ml)
K>'*
O1
to*
t-O
10'
FIGURE 1. Changes in glucose-6-phosphate dehydrogenase
(G6PD) activity, as shown by the amount offormazan deposit
measured by microdensitometry (expressed as mean integrated
extinction x 100) in proximal convoluted tubules in segments of
guinea pig kidney exposed to dilutions of a high potency plasma
and plasma from MHS and MNS.
500
HYPERTENSION
9, No 5,
VOL
MAY
1987
TABLE 1. Weight and Blood Pressure on Day of Experiment
9-week-old
3-week-old
Variable
MNS
48.9±7.1
117.5 ± 1.3
Weight (g)
Blood pressure (mm Hg)
MNS
273.3±4 .2
133.6±1 .0
MHS
56.4±1 .1
124.3±1 .8*
MHS
293.1 ±9.2
172±l.lt
Values arc means ± SE.
•p<0.005, tp<0.001, compared with respective value in MNS.
Statistical assessment of assay validity and potency
ratios were made with reference to the European Pharmacopoeia. '2
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Results
The weight and systolic blood pressure, measured at
the tail (W + W blood pressure recorder; Basile, Italy), are given in Table 1. To avoid disturbing the
weanling rats, on the day that blood was obtained, the
blood pressure measurements were made on another
group of rats of the same age.
Adult Rats
The mean G6PD-stimulating capacity of the hypertensive strain rat plasma was 586 ±88.8 U/ml and of
the normotensive strain plasma was 23.4 ± 8.3 U/ml
(p< 0.001; Figure 2). Figure 3 illustrates the time
course of the ability of plasma from adult MHS and
MNS to inhibit Na+,K+-ATPase directly at 2, 4, 6,
MHS
1OOO
t
and 8 minutes and demonstrates that the plasma of
MHS had a considerably greater capacity to inhibit
Na+,K+-ATPase than the plasma of the control rats.
Young Rats
The mean G6PD-stimulating activity of the hypertensive strain rat plasma was 99.9 ± 27.4 U/ml and of
the normotensive strain rat plasma was 7.8 ± 1.8 U/ml
Statistical Validation
The mean index of precision for the individual assay
results was 0.1389 (n = 28), with fiducial limits
(p<0.95) ranging from 84-119% to 13-143%
(« = 28). There was no statistical divergence from parallelism between the log-dose response of the human
plasma used as a standard and 27 of the 28 plasma
samples assayed. Values are reported as means ± SE.
Discussion
In the present study, cytochemical techniques were
used to assay the capacity of diluted whole plasma to
inhibit Na+,K+-ATPase activity and to stimulate
G6PD activity in guinea pig proximal tubule. Cytochemical bioassays of hormones are based on the prin-
PLASMA C6PO
STIMULATING
ACTIVITY
MHS
(unitl/ml)
INHIBITION
201OO
NORMOTENSIVE
1 1O3
CONTROL
MNS
O
MNS
8
o
OUABAIN
SENSITIVE
(No*— K«)
o
ACTIVITY
ATPOM
60
o
to
30
o
o
o
o
o
WEEKS
FIGURE 2. The ability of plasma obtained from 3- and 9week-oldMHS and MNS and their controls to stimulate glucose6-phosphate dehydrogenase (G6PD) activity in proximal tubules of guinea pig kidney.
.MILAN
HYPERTENSIVE
RAT
1:10 4
1OO L
TIME (mm)
FIGURE 3. Time course of the reversible effect ofplasma from
a 9-week-old MHS and a normotensive strain rat on Na+,K+ATPase activity in the proximal tubule of segments ofguinea pig
kidney.
PLASMA SODIUM TRANSPORT INHIBITING ACTIVITY/Holland et al.
PLASMA
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ciple that when a hormone binds to its receptor on a
living cell it produces a transient, rapidly reversible
effect on an enzyme that can be revealed by means of a
colored end product at the site of enzymatic activity.9
Ouabain, normal human plasma, and rat plasma induce a reversible inhibition of Na + ,K + -ATPase in
proximal tubules that is maximal at 4 to 6 minutes
(Figure 4).' In cytochemical assays of known hormones the time at which the enzymatic change is maximal is one of the features that confers specificity. With
the use of this technique the plasma of a group of saltloaded men inhibited 25 times more Na + ,K + -ATPase
than when they were on a low salt diet. 3
The cytochemical technique with which inhibition
of Na + ,K + -ATPase activity is measured, however, is
semiquantitative in that, although it is possible to obtain some quantitative information from the time
course, the small amount of baseline Na + ,K + -ATPase
activity remaining in the kidney section makes it difficult to obtain a dose versus inhibiting response of sufficient steepness for a reproducible quantitative assay.
Inhibition of Na + ,K + -ATPase in fresh tissue, however, is associated with stimulation of G6PD activity,13
and the dose-response curve, with dilutions of plasma,
is linear and of sufficient steepness to validate a quantitative assay that measures the capacity of body fluids
and extracts to stimulate G6PD as an index of their
ability to inhibit Na + ,K + -ATPase. 2 The validation criteria include a statistical evaluation of the index of
precision (A.), parallelism with the standard, and fiducial limits for each assay.12
Ouabain and normal human and rat plasma induce a
reversible rise in G6PD activity that is maximal at 2
minutes (see Figure 4). The plasma of salt-loaded humans and rats stimulates G6PD about 20 times more
than when these species are deprived of sodium.2 Tests
of selectivity have been performed2 on various sub-
8
MINUTES
i
2
K.
4
«
6
FIGURE 4. Percentage changes in glucose-6-phosphate dehydrogenase (G6PD) activity (*) and Na + ,K+ -ATPase activity
(o) in proximal tubules of segments of fresh guinea pig kidney
exposed to 1:1000 plasma and 4 X 1CH M ouabain for 2 to 8
minutes.
501
stances, many of which are known to have metabolic
effects on the proximal tubule and some of which have
been demonstrated to inhibit purified preparations of
Na + ,K + -ATPase, including fatty acids (linoleic and
oleic), 14 '" dehydroepiandrosterone sulfate,16 lysophosphatidyl choline,17 and sodium vanadate.18 None
stimulated G6PD at 2 minutes. Therefore, the cytochemical techniques that detect the presence of a substance that inhibits Na + ,K + -ATPase at 4 to 6 minutes
and stimulates G6PD at 2 minutes have a certain selectivity. The effect of ouabain is only evident at relatively high concentrations (10~3 M to 10"6 M), which
suggests that the affinity of the endogenous Na + ,K + ATPase inhibitor for the tubule cell receptor is higher
than that of ouabain itself."
The results obtained with these cytochemical techniques show that at 9 weeks, when hypertension is
fully developed, the capacity of the plasma of MHS to
inhibit Na + ,K + -ATPase activity at 4 to 6 minutes and
to stimulate G6PD activity at 2 minutes is considerably
greater than that of the plasma of MNS. This finding
supports the notion that an endogenous inhibitor of
Na + ,K + -ATPase activity is present in greater amounts
in plasma of MHS, and it is in keeping with the observation that the ouabain-sensitive Na + efflux from fresh
erythrocytes is lower in the adult MHS than in the
MNS.20
At 3 weeks the plasma of MHS has a significantly
greater capacity to stimulate G6PD than that from the
MNS, although the difference is much lower than it is
at 9 weeks. At 3 weeks the ouabain-sensitive Na +
efflux from fresh erythrocytes is similar in both
strains.20 The blood pressure of the group of 3-weekold rats on which this measurement was made was
slightly but significantly raised. But, as discussed elsewhere,7 blood pressure differences, when measured on
different occasions, between the hypertensive and the
normotensive strains at 3 weeks of age is not always
statistically significant.
The finding that the capacity of the plasma to stimulate G6PD activity is raised at 3 weeks in the MHS,
before the main rise in arterial pressure has occurred,
supports the hypothesis that the rise in the capacity of
the plasma to stimulate G6PD activity and to inhibit
Na + ,K + -ATPase activity in the adult rat is not secondary to the rise in arterial pressure per se, or to a
secondary effect of the hypertension on renal function.
The raised plasma capacity to stimulate G6PD in the
MHS probably is due to an intensified need to increase
sodium excretion because of an inherited defect in the
ability of the kidney to excrete sodium. There is some
experimental evidence that supports the existence of
such a defect in the MHS. Renal cross-transplantation
experiments between MHS and MNS have demonstrated that the arterial pressure of the recipient is determined by the kidney it receives.21 The normotensive
strain rat becomes hypertensive, while the hypertensive strain rat's blood pressure decreases or does not
rise, which demonstrates that the hypertension is due
to some genetic fault in the kidney of the MHS. Many
individual differences in renal function and sodium
HYPERTENSION
502
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balance between the MHS and the MNS are detectable
before the development of hypertension. Glomerular
filtration rate, urinary output, and renal excretion of a
sodium load are greater in the MHS, 8 ' u - v while basal
renal sodium output is equal.8 Perfused isolated kidneys from young MHS show increased tubular sodium
reabsorption and O2 consumption,24 and isolated membranes of proximal tubule cells from the kidney of
MHS have an increased sodium transport.23 Presumably these differences in sodium reabsorption give rise
to the transient cumulative sodium retention observed
in the MHS during the development of hypertension.8
Evidence for increased proximal tubule sodium reabsorption has also been obtained in patients with essential hypertension and in the SHR. 26 "
The high degree of selectivity that the cytochemical
assays confer appears to be for a substance that is
normally controlled by changes in salt intake and fluid
volume.23 The only connection that has so far
emerged with the substance in the plasma that inhibits
Na+,K+-ATPase at 4 to 6 minutes and stimulates
G6PD at 2 minutes using cytochemical techniques is
ouabain.
This work demonstrates that, using cytochemical
techniques, the ability of the plasma of MHS to stimulate G6PD and to inhibit Na+ ,K+-ATPase is concomitant with the development of hypertension, which suggests that a substance with ouabainlike bioactivity may
be involved in the rise of arterial pressure. An increase
in the plasma's cytochemically detectable Na + ,K + ATPase-inhibiting and G6PD-stimulating activity has
also been demonstrated in established essential hypertension, in adult SHR, and in normal subjects and rats
on a high sodium intake. It is possible that in these
three forms of hypertension the increase in the cytochemically detectable circulating Na+,K+-ATPase
inhibitor is due to the observed, acquired increase
in intrathoracic pressure,28'N which is due in turn
to a genetic increase in proximal tubule sodium reabsorption.26' "
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Cytochemically assayable Na+,K+-ATPase inhibition by Milan hypertensive rat plasma.
S Holland, J Millett, J Alaghband-Zadeh, H de Wardener, P Ferrari and G Bianchi
Hypertension. 1987;9:498-503
doi: 10.1161/01.HYP.9.5.498
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