CALCIUM TRANSPORT AND HYPERTENSION/Wei el al.
Downloaded from http://circres.ahajournals.org/ by guest on July 31, 2017
14. Williams DO, Amsterdam EA, Mason DT: Hemodynamic effects of
nitroglyccrin in acute myocardial infarction. Circulation 51: 421-427,
1975
15. Epstein SE, Kent KM, Goldstein RE, Borer J S , Redwood DR:
Reduction of ischemic injury by nitroglycenn during acute myocardial
infarction. N Engl J Med 292: 29-36, 1975
16 Hirshfeld JW Jr, Borer J S , Goldstein RE, Barrett MJ. Epstein SE:
Reduction in seventy and extent of myocardial infarction when nitroglyccrin and mcthoxamine arc administered during coronary occlusion
Circulation 49: 291-297, 1974
17. Shell WE, Sobel BE: Protection of jeopardized ischemic myocardium by
reduction of ventricular aftcrload. N Engl J Med 291: 481-486, 1974
18. Kent KM, Smith ER, Redwood DR, Epstein SE: Beneficial electropbysiologic effects of nitroglycerin during acute myocardial infarction. Am J
Cardiol 33: 513-516, 1974
19. DeMaria AN, Vismara LA, Auditore K, Amsterdam EA, Zelis R,
Mason DT: Effects of nitroglycenn on left ventricular cavitary size and
cardiac performance determined by ultrasound in man. Am J Med 57:
754-760, 1974
20. Burggraf GW, Parker JO: Left ventricular volume changes after amyl
nitrite and nitroglycenn in man as measured by ultrasound. Circulation
49: 136-141, 1974
21. Cohn JN: Blood pressure and cardiac performance. Am J Med 55:
351-361, 1973
22. Smith ER, Redwood DR, McCarron WE, Epstein SE: Coronary artery
occlusion in the conscious dog, effects of alterations in artcnal pressure
produced by nitgoglycerin, hemorrhage, and alpha-adrenergic agonists
on the degree of myocardial ischemia Circulation 42: 51-56, 1973
23. Parker JO, West RO, Di Giorgi S The effect of nitroglycenn on
coronary blood flow and the hemodynamic response to exercise in
coronary artery disease Am J Cardiol 27: 59-66, 1971
24. Williams JF, Glick G, Braunwald E' Studies on cardiac dimensions in
intact unanestfictized man; effects of huroglycerin. Circulation 32:
767-774, 1967
25. Chnstensson B, Kanetors T, Westling H. Hemodynamic effects of
nitroglycenn in patients with coronary heart disease. Br Heart J 27:
133
511-516. 1965
26. Mason DT, Braunwald E. The effects of nitroglycerin and amyl nitrite on
artenolar and venous tone in the human forearm. Circulation 32:
755-766, 1965
27 Schlanl RC, Tsagaris TS, Robertson RJ J r Studies on acute cardiovascular effects of intravenous sodium nitroprusside. Am J Cardiol 9: 51 -59,
1962
28. MiUer RR, Amsterdam EA, Bogren HG, Massumi RA, Zelii R, Mason
DT: Electrocardiographs and cincangiographic correlations in assessment of the location, nature, and extent of abnormal left ventricular
segmental contraction in coronary artery disease Circulation 49:
447-454, 1974
29. Sandier H, Dodge HT: The use of single plane angiograms for the
calculation of left ventricular volume in man. Am Heart J 75: 325-331,
1968
30. Frank II M, Baxley WA, Dodge HT, Frimer M: Angiographic determination of the left ventricular ejection rate in man with heart disease.
Chest 66: 32-38, 1974
31. KarlinerJS, Gault JH, Eckberg D, Mulhns CB, Ross J Jr- Mean velocity
of fiber shortening; a simplified measure of left ventricular myocardial
contractility Circulation 45: 323-333, 1971
32. Mason DT, Braunwald E: Studies on digitalis X. Effects of ouabain on
forearm vascular resistance and venous tone in normal subjects and in
patients in heart failure. J Clin Invest 43: 532-543, 1964
33. Mason DT, Braunwald E: Effects of guanethtdine, reterpine, and
methyldopa on reflex venous and arterial constnetion in man. J Clin
Invest 43: 1449-1463, 1964
34. Mason DT, Zelis R, Amsterdam EA. Antianginal action of nitroglycerin
in man. In Coronary Heart Disease, edited by M Kaltcnbach, P Lichtlcn,
G Fnesinger. Stuttgart, Gcorg Thieme Verlag, 1973, pp 25-32
35 Mason DT, Zelis R, Amsterdam EA: Actions of the nitntes on the
penpheral circulation and myocardial oxygen consumption; significance
in the relief of angina pectoris. Chest 59: 296-305, 19T1
36. Graham TP Jr, Covel JW, Sonnenblick EH, Ross J Jr, Braunwald E.
Control of myocardial oxygen consumption; relative influence of contractile state and tension development. J Clin Invest 47: 375-386, 1968
Calcium Accumulation and Enzymatic Activities of
Subcellular Fractions from Aortas and Ventricles
of Genetically Hypertensive Rats
JIANN-WU WEI, P H . D . , RONALD A. JANIS, P H . D . , AND EDWIN E. DANIEL, P H . D .
SUMMARY Subcellular fractions were obtained from aortas and
ventricles of 6-roonth-otd spontaneously hypertensive and normotensive Wistar rits by the use of differential and sucrose density gradient
centrifugation. These preparations were studied to determine what
alterations in calcium accumulation and enzymatic activities might be
associated with hypertension. The total amount of calcium accumulation (in the presence of ATP and 17 n» free calcium) by the plasma
membraae-enriched fraction from hypertensive rat aortas was significantly less than that from normotensive rats ( I U ± 0.4 vs. 16.2 ±
1.6 ^moJ of calciom/g of protein, n - 8). In contrast the specific activities of the plasma membrane marker enzymes, 5-mideotrdase and
phosphodiesterase I, were 80% and 40% greater, respectively, in the
hypertensive than in the normotensive fractions. On the other hand,
various fractions from ventricles of the two types of rats were
generally similar in enzyme activities and calcium accumulation. The
decreased rate of relaxation of aortas from spontaneously hypertensive rats may be caused by the decreased rate of calcium transport
demonstrated in this study.
MANY CHANGES in the reactivity of vascular smooth
muscle in hypertensive animals and man have been reported
but little information is available concerning the possible
underlying biochemical mechanisms. 1 '' Since intracellular
calcium activity is an important determinant of smooth
muscle contractile state, an alteration in calcium regulation
is a plausible cause of supersensitivity of strips of arteries
from hypertensive animals to excitatory agents.1"' Indeed, it
has been suggested that increased sensitivity to potassium
and norepinephrine, and decreased rate of relaxation of
aortas from 6-month-old spontaneously hypertensive rats
(SHR) may be due to decreased calcium extrusion by the
cell membrane." In the present investigation we tested this
From the Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada.
Supported by the Alberta Heart Foundation and the Medical Research
Council of Canada.
Dr. Janis wai a Fellow of UK Canadian Heart Foundation; his present
address is: Department of Physiology, Northwestern University Medical
School, Chicago, Illinois 60611.
Address for reprints Dr. E. E. Daniel, Department of Neurosciences,
McMajter University Medical Center, Hamilton, Ontario, Canada.
Received Apnl 8, 1975; accepted for publication March I, 1976.
CIRCULATION RESEARCH
134
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hypothesis by isolating subcellular fractions from these
aortas and studying their calcium transporting properties.
Although the aorta is not a resistance vessel, there is some
evidence that similar changes in ionic composition and
weight occur in both large and small vessels of hypertensive
animals'- ' and alterations in contractile reactivity have been
reported for both mesenteric 7 -' and femoral artery* as well
as aorta'- 10 from SHR.
Cardiac muscle of SHR also exhibits several changes in
contractile activity and morphology associated with the
development of hypertension,"- " and sarcoplasmic reticulum from hypertrophied hearts has been reported to exhibit
decreased calcium binding." Furthermore, Shibata et al. 10
and Fujiwara et al. 12 have suggested that alterations of
contractile responses of both heart and aorta of SHR are
due to inherent differences in the musculature rather than to
secondary changes caused by hypertension. Therefore, we
also compared the calcium accumulation characteristics of
subcellular fractions from hearts of SHR and normotensive
rats.
Methods
ANIMALS AND TISSUE PREPARATIONS
Male spontaneously hypertensive Wistar rats of the
Okamoto and Aoki strain1* and Wistar normotensive rats
were 5-6 months of age and received a standard diet and tap
water. Systolic blood pressure was recorded from the tail of
prewarmed, unanesthetized rats by tail plethysmograph
using an electrosphygmomanometer and a Physiograph
Four-A (E and M Instrument Co., Houston). The rats were
killed by a blow on the head, and their beating hearts and
aortas were rapidly removed and placed in ice-cold 8%
sucrose, 40 mM histidine buffer at pH 7.0 (the pH was
adjusted with HC1; this buffer was used for all procedures
unless otherwise stated). Ventricles were freed of blood clots
and atrial tissue and sliced into 1- to 3-mm sections with
scissors; these slices were washed several times with buffer to
remove blood. All operations were carried out at 4°C unless
otherwise stated.
The methods used for the isolation of plasma membraneenriched fractions are similar to those previously developed
in this laboratory for isolating these fractions from rat heart
and myometrium."- " Slices of ventricle (3 g) were homogenized in 6 vol of buffer using a Polytron PT20 (Kinematic
G.m.b.H.) for 5 seconds (repeated once) at 15,000 rpm. The
homogenate was centrifuged at 1,000 g [average (avg)] for
10 minutes to remove whole cells, nuclei, connective tissue
and contractile elements; this step yields the 1,000 g
supernatant fraction which was the starting material for
both the mitochondrial and density gradient fractions.
Mitochondria were prepared from ventricles by centrifuging
the above supernatant for 10 minutes at 10,000 g (avg) to
yield the crude mitochondrial pellet and the 10,000 g
supernatant. The pellet was gently homogenized in sucrosehistidine buffer and centrifuged at 1,000 g (avg) for 10
minutes. The supernatant fraction was centrifuged at 10,000
g (avg) for 10 minutes and the pellet was washed and
resuspended in buffer; this centrifugation was repeated and
the final pellet was resuspended in buffer to yield a uniform
VOL. 39, No.
1, JULY
1976
suspension containing 0.4-1 mg protein/ml. Teflon-glass
homogenizers were used for suspending all pellets.
The supernatant fraction from the first 10,000 # centrifugation was spun at 113,000 g (avg) for 30 minutes to yield a
crude microsomal fraction which was suspended in 3 ml of
8% sucrose-40mM histidine buffer and carefully laid on a
sucrose gradient containing (from bottom to top) 4 ml of
45.0% sucrose and 3 ml each of 33.0% and 28.0% sucrose.
The sucrose concentrations were measured with a refractometer. After centrifugation at 112,000 g (avg) for 120
minutes, protein bands were carefully removed from the
gradient tube using Pasteur pipettes. These bands were
diluted with buffer and deionized distilled water to yield a
final sucrose concentration of 8%. The suspension was
centrifuged at 102,000 g (avg) for 30 minutes to yield the
final pellets which were used to prepare uniform suspensions
of 0.2-1 mg protein/ml. Cardiac sarcoplasmic reticulum
was prepared by homogenizing ventricular muscle in 10 mM
sodium bicarbonate and 5 mM sodium azide buffer as
described by Entman and co-workers," except that the final
pellet was suspended in sucrose-histidine buffer. The method
includes an extraction with 0.6 M KC1.
A plasma membrane-enriched fraction from aorta was
isolated by a procedure similar to that used above for
isolating this fraction from ventricle. Aortas from 12 rats
were cleaned of loosely bound fat and connective tissue and
homogenized (1.2-1.4 g of tissue) in 25 ml of sucrose-histidine buffer for 10 seconds (twice) at 15,000 rpm using a
Polytron PT 20. The remainder of the procedure (including
the sucrose gradient used) was the same as described above
for ventricle fractions except that the last 10-minute centrifugation at 10,000 g was omitted. Attempts to obtain
mitochondria by the procedure used for ventricle did not
result in a significant yield of protein.
ENZYMATIC DETERMINATIONS AND ELECTRON
MICROSCOPY
Phosphodiesterase I , " 5'-nucleotidase, cytochrome c oxidase, phosphate, and protein were determined as previously
described." Ouabain-sensitive, K + -stimulated phosphatase
was assayed" using a pH of 7.4 and I mM ouabain. ATPases
were determined under the same conditions as calcium
accumulation in the presence of 1 /JM free calcium [including
the same ethylene glycol bis(#-aminoethyl ether)-iV,/V'-tetraacetic acid (EGTA) concentration] except that calcium
was omitted in measuring Mg I + -ATPase. This resulted in a
free Ca 2+ concentration of less than 0.01 ^M. Ca 2+ -ATPase
was measured as Ca 2 + plus Mg 2+ -ATPase minus Mg 2 + ATPase.
Pellets were fixed in 2.5% glutaraldehyde buffer (pH 7.1),
postfixed in OsO, and embedded in Epon. Sections were
stained with uranyl acetate and lead citrate, and photographed on a JEM 7A electron microscope.
MEASUREMENT OF CALCIUM ACCUMULATION
Calcium accumulation* was measured by the Millipore
filtration technique" using " C a and liquid scintillation
• "Calcium accumulation" is used as an operational term for all the
processes involved in association of calcium with vesicles whether by binding
or by transport across the vesicle membrane.
CALCIUM TRANSPORT AND HYPERTENSION/Wei el al.
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counting. The reaction mixture contained: 100 mM KC1, 5
mM MgCI,; 0.1 mM CaCl,, labeled with 0.4 ^Ci/ml "Ca; 40
mM imidazole (to buffer the solution to pH 7.0); and 10-50
/ig of protein per final ml of reaction mixture (depending on
the fraction used and other additions to the reaction
mixture). Where indicated in the text, 5 mM disodium
ATP, sodium azide, or potassium oxalate were added.
Variable amounts of EGTA were added to obtain calcium
concentrations less than 17 jiM. Calculation of the amount of
EGTA to be added was according to the equations,10 and
association constants" previously published. Calcium accumulation always was studied simultaneously on freshly
prepared fractions from both types of rat. Prior to use, all
filters (diameter = 25 mm, pore size - 0.45 ^m; MathesonHiggins Co.) were washed with 10 ml of 100 mM KC1
solution followed by 10 ml of sucrose-imidazole (pH 7.0);
this procedure resulted in low background counts. The
reaction was started by adding protein to the reaction
mixture (which had been preincubated for 5 minutes at
37°C), and stopped by filtration; this was followed by a wash
with 10 ml of 8% sucrose-40 mM imidazole. The reaction
time was 10 minutes unless otherwise indicated. At this time,
calcium accumulation was either increasing or at a plateau
level. Filters were dissolved in 10 rrri of Bray's solution" and
counted to at least 2% accuracy.
MATERIALS
Solutions were prepared from distilled deionized water
and analytical grade reagents. Organic compounds were the
highest purity available from Sigma; "CaCl, was obtained
from Amersham-Searle; A23187 and X-537A, from Lilly
and Hoffman-LaRoche, respectively. These calcium ionophores were dissolved in ethanol and added to the 1-ml
reaction mixture in a volume of 10 n\.
DATA ANALYSIS
Values were compared by the Student's /-test, and
differences with P values of 0.05 or less were considered
significant. Standard errors of the means are bracketed in
the figures except where the standard error is less than the
symbol size, or where omitted for clarity.
Results
ENZYMATIC DETERMINATIONS
MICROSCOPY
AND
ELECTRON
The mean systolic blood pressure of the spontaneously
hypertensive rats used in this study was 190 ± 7.0 mm Hg
(n = 36) vs. 125 ± 5.5 mm Hg for normal Wistar rats (n 36). The enzymatic activities of the fractions from rat ventricles are shown in Table I. The fractions designated plasma
membrane were the lightest fractions from the gradient (at
the 8%/28% interface) and exhibited the highest specific
activity of plasma membrane marker enzymes (5'-nucleotidase; ouabain-sensitive, K + -stimulated phosphatase; and
phosphodiesterase I). Specific activity of cytochrome c
oxidase was lowest in these fractions. The value was only
3.6% of that in the mitochondrial-rich band obtained at the
33%/45% interface in six experiments which omitted the
135
first centrifugation at 10,000 g. Subfractionation of the band
at the 8%/28% interface using a gradient of 22% to 28%
bands of sucrose did not yield any fractions with significantly higher specific activity of plasma membrane marker
enzymes than the original fraction. Electron micrographs of
the plasma membrane fraction were essentially the same as
those previously published" for this fraction isolated by a
similar procedure, and revealed the presence of membrane
vesicles but no mitochondria or rough endoplasmic reticulum.
All denser fractions (e.g., 28%/38%) which exhibited
lower activity of 5'-nucleotidase and ouabain-sensitive,
K + -stimulated phosphatase were contaminated with broken
mitochondria. Cardiac sarcoplasmic reticulum-enriched
membrane was, therefore, prepared by differential centrifugation only.17 This fraction, designated sarcoplasmic reticulum in Table 1, showed relatively little contamination by
plasma membrane when compared to all gradient fractions
which, like this material, were low in mitochondrial contamination. The fraction designated mitochondria in Table 1
was isolated by differential centrifugation and exhibited
even lower specific activities of plasma membrane enzymes
than did the sarcoplasmic reticulum fraction (Table 1). The
only significant differences between the enzyme activities of
fractions from normotensive and hypertensive rats was the
greater ouabain-sensitive, K + -stimulated phosphatase
(Table 1) of the hypertensive plasma membrane fraction.
The lightest protein band (8%/28% interface) from the
sucrose gradient of aortas was found to exhibit the highest
specific activity of plasma membrane marker enzymes
(Table 2). This fraction was designated plasma membrane.
The protein band from the 28%/33% sucrose interface contained less plasma membrane than the 8%/28% interface
fraction (Table 2). It was designated endoplasmic reticulum
(ER) because, in relation to the lighter fraction, it was enriched in this membrane. Electron micrographs of these
fractions were essentially the same as those published for
the corresponding fractions from myometrium" except
that pieces of collagen were occasionally seen in the fractions from preparations of aorta.
The yield of protein in the 1,000 g supernatant fraction
from 12 rat aortas (1.2-1.4 g of tissue) was 10.5 ± 1.7 and
10.7 ± 1.8 ing (n - 8 each) for normotensive and
hypertensive rats, respectively. The plasma membrane and
ER fractions contained only 2.7% and 1.4% of this protein.
The protein yield of these fractions could be doubled by
increasing the homogenization time to 40 seconds, but this
resulted in a marked decrease in the specific actvity of
plasma membrane marker enzymes. Although the yield of
these fractions was the same for both hypertensive and
normotensive rats, the specific activities of their enzymes
differed greatly (Table 2). Phosphodiesterase I and 5'nucleotidase activities were increased in the plasma membrane fraction from hypertensive rats, whereas Mg 2 + ATPase and ouabain-sensitive, K+-stimulated phosphatase
activities were similar to those from normotensive rats. No
significant Ca 2+ -ATPase activity (in the presence of 1 ^ M or
17 jiM free calcium) was detected in these fractions; the very
high Mg J+ -ATPase activity would tend to mask any reasonable amount of Ca J + -ATPase.
136
CIRCULATION RESEARCH
VOL. 39, No.
1, JULY
1976
TABLE 1 Enzymatic Activities of Fractions from Rat Ventricles
Fraction (origin from gradient or otherwise)
Enzyme and type of rat
5'-Nuclcotidase
Normotensive
Hypertensive
Phosphodiesterase I
Normotensive
Hypertensive
Ouabain-sensitive, K+-stimulated phosphatase
Normotensive
Hypertensive
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Mg'+-ATPase
Normotensive
Hypertensive
Ca'+-ATPase
Normotensive
Hypertensive
Sarcoplasmtc
reticulum
(centnfugation)
Mitochondria
(centrifugation)
19.5 ± 1.41
5.4 ± 1.20
3.50 ±0.91
(9)
(4)
(5)
1.26 ±0.26
19.9 ± 1.82
4.4 ± 0.87
2.79 ±0.87
(4)
(7)
(4)
(6)
0.46 ± 0.04
(5)
0.42 ± 0.05
5.6 ± 0.3
2.4 ± 0.4
0.67 ± 0.07
(7)
(5)
(4)
4.9 ± 0.5
2.2 ±0.3
0.53 ± 0.08
(5)
(7)
(5)
(5)
0.16±0.13
2.01 ± 0.35
0.80 ±0.15
0.10 ±0.16
(8)
(8)
(7)
(5)
0.17 ±0.10
(8)
2.75 ±0.21*
1.13 ±0.10
0.15 ±0.04
(8)
(7)
(4)
36.0 ± 3.0
283 ± 7.1
75.8 ±5.2
(7)
(7)
(7)
32.2 ± 2.8
294 ± 6.4
74.5 ±4.3
(7)
(7)
(7)
3.9 ±0.3
36.4 ± 2.5
9.2 ±0.6
(7)
(7)
(7)
4.1 ±0.2
34.5 ± 2.1
8.8 ±0.5
(7)
(7)
(7)
1,000?
supernatant
fraction
Plasma
membrane
(8%/28%)
1.49 ± 0.29
(4)
Specific activities are expressed as micromoles of phosphate released per milligram of protein per hour for 5'-nucleotidase, Mg1+-ATPase and Ca' + -ATPase,
and as micromoles of p-nitrophenol released per milligram of protein per hour for phosphodiesterase I and ouabain-sensitive, K+-stimulated phosphatase. Numbers
in parentheses are the number of independent preparations tested.
• Significantly different from normotensive value.
STUDIES ON CALCIUM ACCUMULATION
Calcium accumulation was increased in all fractions of
ventricle by the addition of ATP to the reaction mixture but
there was no difference between hypertensive and normotensive membranes in this regard (Table 3). Plasma membrane
and sarcoplasmic reticulum fractions exhibited a large
increase in calcium accumulation in the presence of oxalate
but no inhibiton by azide, whereas uptake by mitochondria
was affected by azide but not oxalate. Subfractionation of
the plasma membrane preparations using a gradient of 22%
to 28% sucrose did not result in any fraction which exhibited
increased plasma membrane marker enzyme activity and
decreased oxalate stimulation, suggesting that ATP-dependent calcium transport is a property of rat ventricle plasma
membrane. However, the lack of an unequivocal marker
enzyme for cardiac sarcoplasmic reticulum makes it impossible to estimate the contribution by this membrane to the
calcium accumulation of this fraction.
A study of the dependence of calcium accumulation on the
calcium concentration (Fig. 1) also failed to reveal a
difference between the fractions from hypertensive and
normotensive hearts. Other fractions from the sucrose
gradient (at the 28% and 33% interface and within the 33%
sucrose band) which were believed to be a mixture of plasma
membrane and sarcoplasmic reticulum, also exhibited no
difference between hypertensive and control fractions (not
shown). Similarly there was no apparent difference between
these fractions in the rate of calcium accumulation as
estimated with a 30-second incubation time; for example,
the hypertensive plasma membrane fraction bound 2.5 ± 1.3
Mmol/g whereas the normotensive bound 3.6 ± 1 . 1 ^mol of
calcium/g of protein (n = 5 of each).
In contrast to heart fractions, preparations of aorta from
spontaneously hypertensive rats showed a decreased calcium
accumulation by the plasma membrane-enriched fraction in
comparison to that found for normotensive rats (Table 4,
Fig. 2). The lack of inhibition by azide indicates that this
calcium accumulation is not attributable to mitochondria
(Table 4). No significant increase in calcium uptake was
found in the presence of oxalate. However, the divalent ion
ionophores," A23187 and X-537A, markedly inhibited calcium accumulation. A study of the dependence of calcium
accumulation on free calcium concentration demonstrates
that the significant differences between hypertensive and
normotensive fractions persisted at I ^M free calcium (Fig.
3).
Discussion
Enzymological and histochemical studies of arterioles,
arteries and aortas of hypertensive animals report increased
AMPase 1 "- "
and increased alkaline phosphatase
activities." " Therefore, the increased 5'-nucleotidase activities of plasma membrane-enriched fractions from SHR
aortas observed in the present study (Table 2) were to be
expected. It is interesting to note that similar increases in
these enzymes were not seen in subcellular fractions from
CALCIUM TRANSPORT AND HYPERTENSION/Wei et al.
137
TABLE 2 Enzymatic Activities of Fractions from Rat Aortas
Fraction (origin from gradient)
Enzyme and type of rat
5'-Nucleotidase
Normotensive
Hypertensive
Phosphodiesterase I
Normotensive
Hypertensive
Ouabain-sensitive, K+-stimulatcd phosphatase
Normotensive
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Hypertensive
Mg1+-ATPase
Normotensive
Hypertensive
1,000$
supernatant
fraction
Plasma
membrane
(8%/28%
interface)
Endoplasmic
reticulum
(28%/33%
interface)
7.1 ± 1.0
(6)
11.1 ± 1.4
(6)
30.4 ± 3.9
(12)
55.6 ± 5.1'
(12)
20.8 ± 3.4
(10)
29.1 ± 2.0
(8)
0.50 ± 0.04
(5)
0.48 ±0.11
(5)
2.81 ± 0.17
(5)
3.96 ± 0.30*
(5)
1.23 ±0.51
(5)
1.01 ± 0.15
(5)
0.13 ± 0.06
(6)
0.18 ± 0.05
(6)
1.90 ± 0.27
(6)
1.76 ± 0.18
(6)
0.49 ±0.11
(6)
0.55 ±0.13
(6)
106.3 ± 10.2
(6)
92.4 ± 8.6
(6)
633 ± 14.5
(6)
595 ± 20.4
(6)
526 ± 12.7
(6)
554 ± 28.0
(6)
Values are expressed as in Table 1.
• Significantly different from normotensive.
SHR heart (Table 1), but were obtained in membrane
fractions from mesenteric arteries (Wei, Janis, and Daniel,
unpublished observation). The problem of interpreting results of studies of this type without knowing which strain of
rat is the best control has been discussed previously;"
however, the similarity between our results and those of
other workers (cited above) who used different normotensive
strains as well as different methodology suggests that the
increased specific activities of phosphodiesterase I, as well as
increased 5'-nucleotidase, represent real abnormalities associated with hypertension. We have found similar differences
between our hypertensive arteries and those of two other
normotensive strains (Wei, Janis, and Daniel, unpublished
observation). The co-purification of these plasma membrane
marker enzymes" with ouabain-sensitive, K + -phosphatase
(Table 2) suggests that the increases in AMPase activities
observed in histological studies14 " are localized, in part, at
the plasma membrane. However, the lack of a proven
marker enzyme for endoplasmic reticulum of vascular
smooth muscle makes it impossible to estimate the amount
of these intracellular membranes in the plasma membraneenriched fractions.
Previous investigations have reported that the earliest
change found in hypertrophied and failing hearts is a
decrease in the rate of calcium release, followed by a
decreased rate of accumulation and a decreased total
TABLE 3 Calcium Accumulation by Fractions from Rat Ventricles
Fraction
Addition to reaction mixture
and type of rat
None
Normotensive
Hypertensive
5 mM ATP
Normotensive
Hypertensive
5 mM ATP plus 5 mM azide
Normotensive
Hypertensive
5 mM ATP plus 5 mM oxalate
Normotensive
Hypertensive
Plasma
membrane
Sarcoplasmic
reticulum
Mitochondria
1.12 ±0.59
1.25 ±0.11
1.50 ±0.38
1.67 ±0.47
0.51 ± 0.15
0.43 ± 0.22
27.8 ± 3.9
25.9 ± 3.1
14.5 ± 2.4
15.8 ± 1.9
267 ± 43
235 ± 72
22.4 ± 4.1
25.0 ± 1.8
14.0 ± 2.7
15.2 ± 2.1
0.72 ± 0.14
0.52 ± 0.36
674 ± 85
604 ± 29
310 ± 35
295 ± 27
208 ± 5 5
190 ± 5 8
Values are expressed at micromoles of Ca I - accumulated per gram of protein during an incubation of 10 minutes at
37°C. The reaction mixture contained 17 jiM free Ca I + , 5 mM MgCl,, 100 mM KG, and 40 mM imidazole at pH 7.0.
n - 6.
CIRCULATION RESEARCH
138
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VOL. 39, N o .
1, JULY
1976
300,.
£ 20
10
03
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I
3
10
Calcium Concentration ( >iM )
30
i
3
Colcwn C«nantro1ion
FIGURE 1 Effect of calcium concentration on calcium accumulation by fractions from ventricles of normotensive (open symbols) and
spontaneously hypertensive (closed symbols) rats, a: Circles - plasma membrane-enriched; squares - sarcoplasmic reticulum. b: triangles milochondrial (n - 5-6 for each value). Calcium accumulation was measured at the end of 10 minutes after incubation in the presence of 5 mM
ATP.
amount of calcium taken u p . " Under the conditons of our
experiments, little difference was seen between the calcium
accumulation characteristics of the various fractions studied
(Table 3, Fig. 1). After we completed our study, Aoki and
co-workers" reported a small but significantly lower maximum calcium accumulation capacity of microsomes from
SHR ventricles compared with normotensive ventricles
(8.80 ± 0.33 vs. 11.95 ± 0.55 ^mol/g, n = 8-10). Their
microsomes were prepared from younger rats than those
used in our study, and calcium accumulation was determined in a reaction mixture different from ours. In sarcoplasmic reticulum fractions from SHR and normotensive
rat ventricles at 3 months of age, in which accumulation of
calcium at pH. 6.4 was determined, we found no significant
differences between the two groups (17.8 ± 1.8 vs. 18.1 ±
2.1 ^mol/g protein per 10 minutes, n = 4).
The increased specific activities of 5'-nucleotidase and
phosphodiesterase I in the plasma membrane-enriched fraction from SHR aortas (Table 2) suggests that the lower
TABLE 4
calcium accumulation by this fraction (Table 4) is not the
result of a greater contamination with nonmembranal
protein. Furthermore, inhibition of ATP-dependent calcium
accumulation by the ionophores X-537A and A23187 (Table
5) suggests that calcium is being transported across the
vesicle membrane," since these ionophores should prevent
development of a gradient of calcium across the membrane.
However, there is some evidence that these ionophores may
also act by inhibiting calcium binding." Presumably these
vesicles are composed of "inside-out" plasma membrane, as
are those of erythrocytes" which also exhibit ATP-dependent calcium transport. It should be noted that the magnitude
of this calcium accumulation is similar to that reported for
microsomal fractions from rabbit12 and bovine aorta. 31
Since completion of our study, Aoki and co-workers 30 have
reported that SHR microsomes prepared from vascular
smooth muscle pooled from aorta and several arteries also
exhibited a reduced calcium-accumulating capacity when
compared with that of preparations from normotensive rats.
Calcium Accumulation by Fractions from Rat Aortas
Fraction
Addition to reaction mixture
and type of rat
None
Normotensive
Hypertensive
5 mM ATP
Normotensive
Hypertensive
5 mM ATP plus 5 mM azide
Normotensive
Hypertensive
1000*
Plasma
membrane
Endoplasmic
reticulum
2.40 ± 0.23
2.06 ±0.12
5.40 ±0.30
5.01 ±0.67
1.89 ±0.53
2.30 ± 0.40
4.20 ± 0.45
3.92 ± 0.70
16.2 ± 1.58
11.3 ±0.39*
14.4 ± 1.60
11.76 ±0.95
3.63 ± 0.30
3.10 ±0.05
16.1 ±0.54
l l . 4 ± 1.12*
16.4 ± 3.00
12.5 ± 1.40
fraction
Values are expressed as in Table 3, and the reaction mixture is the same.
n - 4, except for addition of ATP only, where n - 8.
* Significantly different from normotensive.
CALCIUM TRANSPORT AND HYPERTENSION/Wei et al.
139
20
15
10
I 10
"3
03
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I
3
10
Time (mm)
20
FIGURE 2 Time course of calcium accumulation by plasma membrane-enriched fractions from aortas of normotensive (open circles)
and spontaneously hypertensive (closed circles) rats in the presence
of 5rnMA TP. Calcium accumulation in the absence of A TPfor this
fraction from normotensive rats is also shown (x). The ATPindependent values for hypertensive rats (not shown) were not
significantly different from those for normotensive rats (n - 4 for
each value).
A decreased ATP-dependent calcium transport of the
SHR aortic membranes is consistent with the hypothesis
that the slower rate of relaxation of the SHR aortas' is due
to a decrease in the rate of ATP-dependent calcium efflux
from the smooth muscle cell. It is not known whether the
increased sensitivity of aortas from 6-month-old SHR to
norepinephrine and potassium chloride is related to their
slower rate of relaxation after contraction induced by
potassium chloride,5 but studies with aortas from renal
hypertensive rats demonstrate that changes in relaxation
rates are seen as soon as hypertension (BP =• 140-150 mm
Hg) and vascular supersensitivity to postassium chloride
develop. 34 Additional studies will be required to determine
TABLE 5
Plasma membrane
Normotensive
Hypertensive
Endoplasmic reticulum
Normotensive
Hypertensive
30
FIGURE 3 Effect of calcium concentration on calcium accumulation by plasma membrane-enriched fractions (circles), endoplasmic
reticulum-ennched fractions (squares), and the 1,000 g supernatant
fraction (triangles) from aortas of normotensive (open symbols) and
spontaneously hypertensive (closed symbols) rats. Calcium accumulation was measured at the end of 10 minutes after incubation in the
presence of 5 mu ATP. Asterisk indicates a significant difference
from normotensive value (n = 5-8 for each value, except the plasma
membrane fraction at 17 ^M free calcium, where n - 20).
whether there are any causal relationships between the
observed alterations in enzymatic and calcium-binding activities and hypertension.
There is some evidence suggesting that increased reactivity of vascular smooth muscle to excitatory agents cannot be
explained entirely either by a decrease in the activity of
processes that lower intracellular calcium activity or by an
increase in the ratio of wall thickness to lumen size.2 For
example, the change in sensitivity to potassium chloride of
aortas from 6-month-old SHR is much greater than that to
norepinephrine,5 and the change in sensitivity of perfused
vasculature of SHR is greater to serotonin than to
norepinephrine.8 These results indicated that there are
specific changes in membrane excitability that allow for
differential changes in sensitivity to various excitatory
agents; these alterations are in addition to any changes in
calcium transport and in vascular geometry that would tend
Effect of lonophores on Calcium Accumulation
Fraction and type of rat
I
3
10
Calcium Concentration (juM )
by Fractions from Rat Aortas
95% ethanol
(1%)
X537A
(2<
A23187
Control
16.95 ±0.96
11.80 ±0.92
18.15 ± 1.25
11.83 ±0.90
4.77 ±0.34
4.58 ±0.31
4.05 ± 0.72
4.50 ± 0.29
14.23 ±0.61
11.09 ±0.64
12.93 ± 1.34
12.45 ± 1.23
3.68 ±0.27
3.58 ±0.22
3.45 ± 0.53
4.58 ± 0.47
(IO^M)
Values are expressed as in Table 3, and the reaction mixture is the same except that ethanol, or ionopborci dissolved in
ethanol, are added where indicated,
n - 4.
140
CIRCULATION RESEARCH
to produce a similar degree of hyperreactivity to all
excitatory agents.
Acknowledgments
We thank Eda Lee and James Allan for their excellent technical
assistance, and Gus Duchon for preparation of electron micrographs. Dr. R.
Hamill of Eli Lilly and Dr. J. Berger of Hoffman-LaRoche generously
•upplied the ionophores A23I87 and X-537A, respectively.
References
Downloaded from http://circres.ahajournals.org/ by guest on July 31, 2017
1. Pickering GW: High Blood Pressure, cd. 2. New York, Gnine &
Stratton, 1968
2. Folkow B- The haemodynamic consequences of adaptive structural
changes of the resistance vessels in hypertension. Clin Sci 41: 1-12, 1971
3. Somlyo AP, Somlyo AV- Vascular smooth muscle. II. Pharmacology of
normal and hypertensive vessels. Pharmacol Rev 22: 249-353, 1970
4. Hinke JAM: Effect of C a + + upon contractility of small arteries from
DCA-hypertcnsive rats Circ Res 18 (suppl I): 23-33, 1966
5. Field FP, Janis RA, Tnggle DJ: Aortic reactivity of rats with genetic and
experimental renal hypertension. Can J Physiol Pharmacol 50:
1072-1079, 1972
6. Jones AW: Altered ion transport in vascular smooth muscle from
spontaneously hypertensive rats; influence! of aldosterone, norepinephnne, and angiotensin Circ Res 33: 563-572, 1973
7 Haeusler G, Haefely W: Pre- and postjunctional supersensitivity of the
mesenteric artery preparation from normotensive and hypertensive rats.
Naunyn Schmiedebcrgs Arch Pharmacol 266: 18-33, 1970
8 Haeusler G, Finch L Vascular reactivity to 5-hydroxytryptamine and
hypertension in the rat. Naunyn Schmiedebergs Arch Pharmacol 272:
101-116, 1972
9. Holloway ET, Bohr DF Reactivity of vascular smooth muscle in
hypertensive rats. Circ Res 33: 678-685, 1973
10. Shibata S, Kurahashi K, Kuchii M: A possible etiology of contractility
impairment of vascular smooth muscle from ipontaneously hypertensive
rats. J Pharmacol Exp Ther 185: 404^tl8, 1973
11. Farmer BB, Harris RA, Jolly WW, Vail WJ: Studies on the cardiomegaly of the spontaneously hypertensive rat Circ Res 35: 102-110, 1974
12. Fujiwara M, Kuchii M, Shibata S: Differences of cardiac reactivity
between spontaneously hypertensive and normotensive rats. Eur J
Pharmacol 19: 1-11, 1972
13. Schwartz A, Sordahl LA, Entman ML, Allen JC, Reddy YS, Goldstein
MA, Luchi RJ, Wyborny LE: Abnormal biochemistry in myocardial
failure. Am J Cardiol 32: 407-422, 1973
14. Okamoto K, Aoki K Development of a strain of spontaneously
hypertensive rats. Jap Circ J 27: 282-293, 1963
15. Kidwai AM, Radcliffe MA, Duchon G, Daniel EE. Isolation of plasma
membrane from cardiac muscle. Biochem Biophys Res Commun 45:
901-910, 1971
VOL. 39, No. I, JULY 1976
16. Kidwai AM, Radcliffe MA, Daniel EE I Isolation and characterization
of plasma membrane from rat myomctnum. Biochim Biophys Acta 233:
538-549, 1971
17 Entman ML, Snow TR, Freed D, Schwartz A: Analysis of calcium
binding and release by canine cardiac relaxing system (sarcoplasmtc
reticulum). J Bio! Chem 248: 7762-7772, 1973
18. Touster O, Aronson NN, Dulaney JT, Hendnckson H: Isolation of rat
liver plasma membranes; use of nudcotide pyrophosphatase and phosphodiesterase I as marker enzymes. J Cell Biol 47: 604-618, 1970
19. Martonosi A, Feretos R: Sarcoplasmic reticulum. I. The uptake of Ca + +
by sarcoplasmic reticulum fragments. J Biol Chem 239: 648-658, 1964
20. Katz AM, Repke DI, UpshawJE, Polascik MA: Characterization of dog
cardiac microsomes Use of zonal centrifugation to fractionate fragmented sarcoplasmic reticulum, (Na + + K + )-activated ATPase and mitcchondnal fragments. Biochim Biophys Acta 205: 473-490, 1970
21 Godt RE: Calcium-activated tension of skinned muscle fibers of the frog
dependence on magnesium adenosine tnphosphatc concentration. J Gen
Physiol 63: 722-739, 1974
22 Bray GA: A simple efficient liquid scintillator for counting aqueous
solutions in a liquid scintillation counter. Anal Biochem 1: 279-285, 1960
23. Scarpa A, Baldassare J, Inesi G The effect of calcium ionophores on
fragmented sarcoplasmic reticulum. J Gen Physiol 60: 735-749, 1972
24. Oka M, Angrist A: Histoenzymatic studies of arteries in high salt
hypertension. Lab Invest 14: 1604-1615, 1965
25. Ichijima K Morphological studies on the peripheral small arteries of
spontaneously hypertensive rats. Jap Circ J 33: 785-813, 1969
26 Gardner DL, Laing CP Measurement of enzyme activity of isolated
small arteries in early rat hypertension. J Pathol Bactenol 90: 399-406,
1965
27 Ooshima A: Enzymological studies on arteries in spontaneously hypertensive rat Jap Circ J 37: 497-508, 1973
28. Lais LT, Shaffer RA, Brody MJ: Neurogenic and humoral factors
controlling vascular resistance in the spontaneously hypertensive rat.
Circ Res 35: 764-774, 1974
29. DePierre JW, Karnovsky ML' Plasma membranes of mammalian cells, a
review of methods for their characterization and isolation. J Cell Biol 56:
275-303, 1973
30 Aoki K, Ikeda N, Yamashita K, Tazumi K, Sato I, Hotta K:
Cardiovascular contraction in spontaneously hypertensive rat: calcium
interaction of myofibnls and subcellular membrane of heart and arterial
smooth muscle. Jap Circ J 38: 1115-1121, 1974
31 Weiner ML, Lee KS: Active calcium ion uptake by inside-out and right
side-out vesicles of red blood cell membranes. J Gen Physiol 59: 462-475,
1972
32. Fitzpatrick DF, Landon EJ, Dcbbas G, Hurwiu L: A calcium pump in
vascular smooth muscle. Science 176: 305-306, 1972
33. Hess ML, Ford GD: Calcium accumulation by subccllular fractions from
vascular smooth muscle. J Mol Cell Cardiol 6: 275-282, 1974
34. Field FP, Jams RA, Tnggle DJ: Relationship between aortic reactivity
and blood pressure of renal hypertensive, hyperthyroid, and hypothyroid
rats. Can J Physiol Pharmacol 51: 344-353, 1973
Calcium accumulation and enzymatic activities of subcellular fractions from aortas and
ventricles of genetically hypertensive rats.
J W Wei, R A Janis and E E Daniel
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Circ Res. 1976;39:133-140
doi: 10.1161/01.RES.39.1.133
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