(Hypertension. 1995;25:962-970.)
© 1995 American Heart Association, Inc.
Articles |
Treatment in Hypertensive Cardiac Hypertrophy, II
Postreceptor Events
From the Klinik III für Innere Medizin der Universität zu Köln and Bayer AG (A.K.), Wuppertal, Germany.
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Abstract We investigated the effect of pharmacological treatment with captopril, nitrendipine, and captopril plus nitrendipine on myocardial heterologous adenylyl cyclase desensitization and the underlying postreceptor defects in spontaneously hypertensive rats (SHR). In myocardial membranes from SHR, stimulation of adenylyl cyclase with guanylylimidodiphosphate (P<.001) and forskolin (P<.05) was significantly reduced, whereas no difference with forskolin was obtained in the presence of manganese chloride. Reconstitution of Gs
into
Gs-deficient S49 cyc- mouse lymphoma cells
revealed no difference between SHR and control rats. In contrast,
pertussis toxin labeling of Gi was significantly
increased in SHR. The reduction of adenylyl cyclase in SHR was
abolished after pertussis toxin treatment of membranes. Treatment with
captopril, nitrendipine, or both reduced Gi and
increased guanylylimidodiphosphate-stimulated adenylyl cyclase activity
in SHR. In summary, heterologous adenylyl cyclase desensitization due
to an increase of Gi but in the presence of an unchanged
activity of Gs or the catalyst occurs in SHR. This
alteration, which could contribute to the progression of
contractile dysfunction by producing adrenergic subsensitivity, is
sensitive to pharmacological treatment most likely because of a
reduction of sympathetic activity.
Key Words: hypertension, essential • heart hypertrophy • heart failure, congestive • rats, inbred SHR • adenylyl cyclase • pertussis toxins • angiotensin-converting enzyme inhibitors • calcium channel blockers
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Introduction |
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Stimulation of myocardial ß-adrenoceptors activates adenylyl cyclase, which leads to an increase in cAMP formation, thereby regulating intracellular Ca2+ homeostasis and force of contraction of the myocardial cell.1 After prolonged agonist stimulation, adenylyl cyclase becomes desensitized because of an uncoupling and downregulation of ß-adrenergic receptors.2 However, adenylyl cyclase activity is also modulated by other regulatory components,3 such as G proteins. G proteins, which are heterotrimers consisting of
- and ß
-subunits, are involved in
mediating the effects of neurotransmitters, hormones, drugs, light, and
membrane-bound receptors.4 The stimulatory -subunit
(Gs) dissociates from ß-subunits after activation
by agonist-occupied ß-adrenoceptors and stimulates the catalyst of
the adenylyl cyclase.3 4 Adenylyl cyclase is also under
inhibitory control by Gi proteins, a family of G protein
-subunits, which are substrates for pertussis toxin–catalyzed ADP
ribosylation.4 In recent years, it has become evident that
adenylyl cyclase desensitization can also be due to alterations on the
G protein level. In the failing human myocardium, an increase of
inhibitory G protein -subunits has been observed by pertussis
toxin–catalyzed [32P]ADP ribosylation,5 6 7
immunochemical techniques,5 and mRNA
studies.8 The content6 9 and functional
activity6 of stimulatory G protein -subunits have been
observed to be unchanged compared with nonfailing human myocardium.
Determinations of adenylyl cyclase activity with forskolin or manganese
ions also revealed an unchanged catalyst activity.5 10 11 12
Adenylyl cyclase desensitization is also known to occur in different
forms of hypertensive cardiomyopathy in rats.13 14 15
Although adenylyl cyclase desensitization is sometimes as pronounced as
in the failing human heart,16 17 18 ß-adrenoceptor
downregulation has been reported to be relatively
mild19 20 or even to be absent.17 19 As a
possible cause of heterologous adenylyl cyclase desensitization, an
increase of Gi proteins has been observed in
spontaneous16 21 22 and acquired17 18
hypertensive cardiomyopathy in rats. However, it is unknown whether the
increase of myocardial Gi and heterologous adenylyl
cyclase desensitization can be pharmacologically reversed once
established. The present study investigated the postreceptor
defects of the myocardial adenylyl cyclase system of spontaneously
hypertensive rats (SHR) at 30 weeks of age under control conditions and
after oral treatment with captopril, nitrendipine, or captopril plus
nitrendipine. Alterations of ß-adrenergic receptors and of
neuropeptide Y concentrations as measures of sympathetic activity as
well as the influence of pharmacological treatment on these parameters
have been reported previously.23 |
Methods |
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Experimental Animals
Male SHR and control Wistar-Kyoto rats (WKY) at 30 weeks of age were used. Animals were treated with captopril, nitrendipine, or captopril plus nitrendipine. Details of the treatment regimen have been reported previously.23
Adenylyl Cyclase Determinations
Adenylyl cyclase was determined according to Salomon et
al24 with some modifications as described
elsewhere.25 In brief, washed membrane fractions
(10 000g sediment) were prepared from homogenates of
rat hearts. Adenylyl cyclase activity was determined in a reaction
mixture containing 50 µmol/L [-32P]ATP
(approximately 0.3 µCi/100 µL), 50 mmol/L triethanolamine-HCl, 5
mmol/L MgCl2, 100 µmol/L EGTA, 1 mmol/L
3-isobutyl-1-methylxanthine, 5 mmol/L creatine phosphate, 0.4 mg/mL
creatine kinase, and 0.1 mmol/L cAMP at pH 7.4 in a final volume of 100
µL. The mixture was preincubated for 5 minutes at 37°C. The
incubation time was 20 minutes at the same temperature. Reactions were
stopped by the addition of 500 µL of 120 mmol/L zinc acetate. Next,
the zinc acetate was neutralized by 600 µL
Na2CO3 (144 mmol/L). After centrifugation for 5
minutes at 10 000g, 0.8 mL of the supernatant was applied
on neutral alumina columns equilibrated with 0.1 mmol/L Tris-HCl, pH
7.5. The effluent was collected and [32P]cAMP determined
by measurement of radioactivity in a liquid scintillation spectrometer
(LKB Wallac 1272 Clinigamma).
Membrane Preparation for G Protein Determinations
The method of membrane preparation has been published
elsewhere.17 18 25 In brief, myocardial tissue was chilled
in 30 mL ice-cold homogenization buffer (10 mmol/L Tris-HCl, 1 mmol/L
EDTA, 1 mmol/L dithiothreitol, pH 7.4). Connective tissue was trimmed
away and myocardial tissue minced with scissors, and membranes were
prepared with a motor-driven glass-polytetrafluoroethylene homogenizer
for 1 minute. Afterwards, the membrane preparation was homogenized by
hand for 1 minute with a glass-glass homogenizer. The homogenate was
spun at 484g (Beckman JA-20 rotor) for 10 minutes. The
supernatant was filtered through two layers of cheesecloth, diluted
with an equal volume of ice-cold 1 mol/L KCl, and stored on ice for 10
minutes. This suspension was centrifuged at 100 000g for 30
minutes. For radioligand binding experiments, the pellet was
resuspended in 50 vol incubation buffer (50 mmol/L Tris-HCl, 10 mmol/L
MgCl2, pH 7.4) and homogenized for 1 minute with a
glass-glass homogenizer. This suspension was recentrifuged at
100 000g for 45 minutes. The final pellet was resuspended
in incubation buffer (50 vol) and stored at -70°C. Storage did not
alter the results.
Pertussis Toxin–Induced [32P]ADP Ribosylation
[32P]ADP ribosylation of Gi by
pertussis toxin was performed for 12 hours at 4°C in a volume of 50
µL containing 100 mmol/L Tris-HCl (pH 8.0 at 20°C), 25 mmol/L
dithiothreitol, 2 mmol/L ATP, 1 mmol/L GTP, 50 nmol/L
[32P]NAD (800 Ci/mmol), Lubrol PX 0.5% (vol/vol), and 20
µg/mL pertussis toxin that had been activated by incubation with 50
mmol/L dithiothreitol for 1 hour at 20°C before the labeling
reaction. The experimental details have been described
earlier.16 25 26 Samples were subjected to sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (10% [wt/vol]
acrylamide, 16 cm total gel length). Gels were stained with
Coomassie blue and dried before autoradiography was performed.
Pertussis Toxin Plus NAD Treatment of Membranes
Pertussis toxin treatment was performed under the same
incubation conditions as used for [32P]ADP ribosylation,
except that [32P]NAD was replaced by 3 mmol/L NAD in the
reaction. After two washings, membranes were subjected to
[32P]ADP ribosylation or determination of adenylyl
cyclase activity. Control membranes were subjected to the same
incubation conditions except that pertussis toxin was omitted from the
medium. The same results were obtained when heat-inactivated pertussis
toxin was used. Experimental details have been described
before.25
Immunoblotting
Immunoblotting techniques were performed according to Gierschik
et al.27 The polyclonal antiserum MB1 was raised in
rabbits against the C-terminal decapeptide of retinal transducin
(KENLKDCGLF) coupled to keyhole limpet hemocyanin, as described by
Goldsmith et al.28 After electrophoretic separation,
proteins were transferred from the SDS-PAGE gel (10%, 16 cm length) to
nitrocellulose (125 mA, 12 hours, Bio-Rad Transblot apparatus) unless
otherwise indicated. Under these conditions, one immunoreactive
Gi band was detected. The sheets were immersed in 100 mL
of 3% gelatin in TBS buffer (Tris-HCl, 20 mmol/L; NaCl, 500 mmol/L; pH
7.5) and shaken for 1 hour at room temperature. Then they were
incubated in the first antibody solution (MB1) containing 100 µL of
antiserum in 50 mL of 1% gelatin in TBS (24 hours, room temperature,
shaker) to block nonspecific binding. After two washings with 100 mL
TBS containing 0.05% Tween 20 for 10 minutes, the paper was incubated
with the second antibody solution (5 µL of alkaline
phosphatase–labeled goat anti-rabbit IgG in 60 mL of 1% gelatin in
TBS) for 1 hour. After repeated washings with 0.05% Tween 20 in TBS,
the sheets were transferred to 33 mg of nitro blue tetrazolium and 15
mg of 5-bromo-4-chloro-3-indolyl phosphate in 100 mL Tris-HCl (0.1
mol/L) containing NaCl (100 mmol/L) and MgCl2 (5 mmol/L) at
pH 8.5. Color development was stopped after 10 minutes by rinsing with
water, and the nitrocellulose was dried between two sheets of filter
paper.
S49 Lymphoma cyc- Cells
S49 lymphoma cyc- cells were grown in suspension
culture in RPMI 1640 medium supplemented with 10% (vol/vol) fetal calf
serum (culture volume <100 mL) or 10% (vol/vol) horse serum (culture
volume >100 mL), NaHCO3 (44 mmol/L), glucose (5.5 mmol/L),
L-glutamine (5 mmol/L), nonessential amino acids, sodium
pyruvate (1 mmol/L), penicillin (50 U/mL), and streptomycin (50
µg/mL) in a humidified atmosphere of 90% air and 10%
CO2. The cell density was maintained at approximately
1x106 cells per milliliter. Cells ([1 to 2]x1010 cells
in 10 to 20 L medium) were harvested by centrifugation in a Beckman
type JA-10 rotor at 1000g for 20 minutes at 4°C. The
pellets were resuspended in 50 mL triethanolamine/HCl (10 mmol/L)
(pH 7.4 at 20°C). The final pellet was resuspended in 100 to
150 mL lysis buffer containing sucrose (0.25 mol/L), Tris-HCl (20
mmol/L) (pH 7.5 at 20°C), MgCl2 (1.5 mmol/L), ATP (1
mmol/L), benzamidine (3 mmol/L), leupeptin (1 µmol/L),
phenylmethylsulfonyl fluoride (1 mmol/L), and soybean trypsin inhibitor
(2 µg/mL). Cells were homogenized by nitrogen cavitation. The
cavitate was centrifuged in a JA-20 rotor at 1500g for 45
seconds at 4°C to remove unbroken cells and nuclei and filtered
through two layers of cheesecloth. A crude membrane fraction was
isolated from the resulting supernatant by centrifugation in a
JA-20 rotor at 5000g for 20 minutes at 4°C. The membranes
were washed three times with a buffer containing Tris-HCl
(20 mmol/L) (pH 7.5 at 20°C), EDTA (1 mmol/L), dithiothreitol (1
mmol/L), benzamidine (3 mmol/L), phenylmethylsulfonyl fluoride (1
mmol/L), leupeptin (10 µmol/L), and soybean trypsin inhibitor (2
µg/mL); resuspended to 10 mg protein/mL with this buffer; and stored
at -80°C. The membrane protein yield was approximately 100
mg/1010 cells.
Reconstitution of Myocardial Gs Into S49
cyc- Membranes
Reconstitution assays were performed according to Sternweis et
al.29
Miscellaneous
Protein was determined according to Lowry et al30
using bovine serum albumin as standard. SDS-PAGE was performed as
described by Lämmli.31 5'-Nucleotidase activity was
analyzed with the use of the method of Dixon and
Purdom.32
Materials
Forskolin was donated by Hoechst AG. GTP,
guanylylimidodiphosphate [Gpp(NH)p], ATP, creatine phosphate, and
creatine kinase were purchased from Boehringer Mannheim and
isobutylmethylxanthine from EGA-Chemie. [32P]ATP was from
Amersham-Buchler. Dithiothreitol was from Serva. Pertussis toxin was
from List Biological Laboratories.
Statistics
Data shown are mean±SEM. Statistical significance was estimated
with Student's t test for unpaired observations and ANOVA
according to Wallenstein et al.33 A value of
P<.05 was considered significant. Kd
values were determined graphically in each individual experiment.
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Results |
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Blood Pressure and Myocardial Hypertrophy
Oral treatment with nitrendipine, captopril, or captopril plus nitrendipine led to a reduction in blood pressure and heart weight but did not normalize these values in SHR. Treatment had no effect in WKY. The data have been detailed previously.23
Alteration of Adenylyl Cyclase Activity and Mechanisms in SHR
In Fig 1, concentration-response curves for
isoproterenol (left), Gpp(NH)p (middle), and forskolin (right)
summarize the data on adenylyl cyclase in myocardial membranes from SHR
and WKY. The effects of isoproterenol and Gpp(NH)p were strongly
reduced in SHR compared with WKY. The reduction of the Gpp(NH)p effects
pointed toward an alteration beyond ß-adrenoceptors, such as a defect
of the catalyst. Therefore, the effects of the diterpen derivative
forskolin, which directly stimulates the catalyst of the adenylyl
cyclase, were investigated. The effects of forskolin were reduced in
SHR compared with WKY (Fig 1, right), as were the effects of
isoproterenol and Gpp(NH)p. The data did not differ whether cAMP
formation was related to milligrams of membrane protein or
5'-nucleotidase activity as myocardial membrane marker (not shown).
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Studies on the Catalyst Activity
It has been reported that the effects of forskolin can depend on
the activation of G proteins by guanine nucleotides in certain
membranes.34 Manganese ions have been reported to uncouple
the effects on the catalyst from the influences of G
proteins.35 36 To investigate the effects of forskolin on
the catalyst more specifically, we studied adenylyl cyclase activity in
the presence of MnCl2 but in the absence of
MgCl2. Fig 2 summarizes the data. In the
absence of MnCl2, basal, forskolin-stimulated, and
forskolin plus Gpp(NH)p–stimulated adenylyl cyclase activities were
reduced in SHR compared with WKY. MnCl2 stimulated basal
adenylyl cyclase. Under this condition, no difference between SHR and
WKY was observed. Forskolin in the presence of MnCl2
stimulated adenylyl cyclase. Adenylyl cyclase was not further
stimulated when Gpp(NH)p was added to forskolin and
MnCl2, indicating that under these experimental
conditions the effect of forskolin on the catalyst was independent of
influences of guanine nucleotide–activated G proteins. Under this
condition, no differences between SHR and WKY were observed, indicating
that the catalyst activity was similar in both groups.
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Stimulatory G Proteins
The reduced stimulation of adenylyl cyclase activity in the
presence of unchanged catalyst activity pointed toward alterations of G
proteins. To investigate whether the stimulatory G protein -subunit
Gs is reduced or functionally impaired, we performed
functional reconstitution experiments. Gs was
solubilized from myocardial membranes and reconstituted into murine
lymphoma S49 cyc- cells, which genetically lack
Gs. Fig 3 summarizes the data. In native
S49 cyc- cell membranes, no stimulation of adenylyl
cyclase activity by isoproterenol or Gpp(NH)p was observed. When
reconstituted with Gs from myocardial membranes, basal
activity was enhanced, and isoproterenol- and Gpp(NH)p-stimulated
adenylyl cyclase activity was restored in S49 cyc- cell
membranes. The effects of reconstitution were similar when
Gs from WKY or SHR was introduced into S49
cyc- membranes. Therefore, Gs activity or
content appeared to be unchanged in SHR.
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Inhibitory G Proteins
To investigate whether an increase of inhibitory G protein
-subunit occurs in SHR, we treated myocardial membranes with
pertussis toxin plus [32P]NAD. After separation with
SDS-PAGE, Gi was identified as a 40-kD membrane protein
comigrating with Gi/Go -subunits from bovine
brain (not shown). Incorporation of radioactivity into
Gi was significantly increased in SHR compared with WKY.
The data were similar when related to 5'-nucleotidase activity as
membrane marker (Fig 4, left). However,
posttranslational modifications such as endogenous ADP ribosylation
have been reported to limit the effectiveness of [32P]ADP
ribosylation.37 Thus, one could argue that endogenous ADP
ribosylation in native rat myocardial membranes could limit the
quantification of Gi by this technique. To address this
question of endogenous ADP ribosylation, we performed Western blots in
native and pertussis toxin plus NAD–treated membranes. Fig 5
(left) shows representative autoradiography of
[32P]ADP ribosylation of native or pertussis toxin plus
NAD–treated membranes and Western blots in both conditions (Fig 5,
right). As judged from the incorporation of radioactivity into an
approximately 40-kD membrane protein, it is evident that under the
conditions used, the greatest portion of Gi is
covalently modified by pertussis toxin plus NAD treatment. Fig 5
(right) shows the immunoblots using the antiserum MB1. Pertussis toxin
plus NAD treatment reduced the electrophoretic mobility of
Gi compared with that in native membranes. In addition,
immunoreactivity of Gi toward MB1 was increased in
treated membranes. Interestingly, there was no immunoreactive material
in native membranes that comigrated with the ADP-ribosylated form of
Gi in treated membranes.
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To address the question of whether Gi is of functional
relevance for the reduced adenylyl cyclase activity in SHR, we studied
adenylyl cyclase activity in pertussis toxin plus NAD–treated
membranes compared with control membranes. In control membranes, basal
and Gpp(NH)p-stimulated adenylyl cyclase is depressed compared with WKY
membranes. After treatment with pertussis toxin plus NAD, the
differences were abolished (Fig 6).
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Taken together, these data show a heterologous adenylyl cyclase
desensitization in SHR that appears to be due to an increase of
Gi proteins (as judged from pertussis toxin–catalyzed
[32P]ADP ribosylation), whereas the functional activities
of Gs and the catalyst of the adenylyl cyclase were
unchanged. Endogenous ADP ribosylation obviously does not occur in rat
myocardial membranes. As judged from experiments on pertussis
toxin–treated membranes, the increase of Gi appears to
be causally linked to adenylyl cyclase desensitization.
Effect of Treatment on Adenylyl Cyclase Activity
Fig 7 shows concentration-response curves for
Gpp(NH)p in WKY treated orally with captopril, nitrendipine,
or captopril plus nitrendipine. None of the treatment regimens
significantly altered adenylyl cyclase activity compared with control
conditions in WKY. Adenylyl cyclase stimulation by Gpp(NH)p in
myocardial membranes of WKY was significantly stronger in the controls
(no treatment) and after treatment with captopril and nitrendipine
compared with SHR under control conditions. However, after treatment
with nitrendipine alone, Gpp(NH)p-stimulated adenylyl cyclase was not
significantly different from the effect of the guanine nucleotide in
SHR membranes (shown in Fig 7, left, for comparison). Fig 8 summarizes the effects of treatment in SHR. Treatment
with captopril, nitrendipine, or captopril plus nitrendipine
increased adenylyl cyclase activity. None of the effects were different
from those observations in untreated WKY (shown for comparison in Fig 8, left). However, only captopril and captopril plus nitrendipine
significantly increased the effects compared with untreated SHR.
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Effect of Pharmacological Treatment on Gi
Proteins
Fig 9 shows representative autoradiography
of pertussis toxin–catalyzed [32P]ADP ribosylation of
Gi proteins (approximately 40 kD) from myocardial
membranes of SHR and WKY under control conditions and after
pharmacological treatment. One pertussis toxin substrate (approximately
40 kD) was detected in myocardial membranes after treatment with
pertussis toxin plus [32P]NAD as substrate. Pertussis
toxin substrates were comigrating with purified Gi/Go -subunits
purified from bovine brain as standard. In SHR, incorporation of
[32P]ADP ribose into 40 kD was more pronounced than in
WKY membranes, which was evidence for increased myocardial
Gi levels. Captopril and nitrendipine treatment as well
as treatment with a combination of the drugs reduced incorporation of
[32P]ADP ribose into Gi in SHR. In WKY, no
changes were observed with nitrendipine or captopril, whereas with
nitrendipine plus captopril, a small increase of incorporation into
Gi was observed. Fig 10 shows the mean
values for Gi levels in WKY as judged from pertussis
toxin–induced [32P]ADP ribosylation. Neither treatment
regimen altered the Gi levels in WKY myocardial
membranes. The mean values were significantly lower than those observed
in SHR myocardial membranes. Fig 11 summarizes the data
for SHR. The Gi levels in SHR as determined by pertussis
toxin–catalyzed [32P]ADP ribosylation were significantly
reduced by treatment with captopril or nitrendipine plus captopril.
After treatment with nitrendipine alone, Gi levels were
not significantly reduced compared with those in untreated SHR.
Nevertheless, the Gi levels in SHR myocardial membranes
did not differ from those in WKY membranes after either treatment
regimen.
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Discussion |
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In this study, we examined the effects of oral treatment with captopril, nitrendipine, or captopril plus nitrendipine for 20 weeks on postreceptor events contributing to adenylyl cyclase desensitization in 30-week-old SHR. In myocardial membranes of SHR, guanine nucleotide–dependent adenylyl cyclase activity was depressed. Experiments with forskolin and MnCl2 revealed an unchanged catalyst activity. Gs
activity was unchanged in SHR as
measured by reconstitution experiments in Gs-deficient
S49 cyc- cell membranes. In contrast, Gi
was significantly increased in SHR compared with WKY, as detected by
pertussis toxin–catalyzed [32P]ADP ribosylation. No
endogenously ADP-ribosylated Gi was detected in
myocardial membranes. The difference in basal and guanine
nucleotide–stimulated adenylyl cyclase was abolished after pertussis
toxin treatment in order to inactivate Gi. Treatment
with antihypertensive drugs increased Gpp(NH)p-stimulated adenylyl
cyclase activity and reduced myocardial Gi content in
SHR but had no effect in WKY.
Data of the Framingham study have indicated that hypertension is
the most common cause of chronic heart failure.38
Myocardial hypertrophy is regarded as an adaptation to reduce wall
stress when an increased pressure load is imposed on the
myocardium.39 40 However, it has remained unresolved why
the hypertrophied heart begins to fail. Several mechanisms may
contribute to the progression from hypertensive cardiac hypertrophy to
myocardial failure, such as energetic aspects, alterations of
intracellular Ca2+ homeostasis, or adrenergic
subsensitivity due to long-term sympathetic activation.41
In heart failure, it is well established that sympathetic activation
leads to adenylyl cyclase desensitization.42 43 The
subcellular mechanism is an increase of inhibitory G protein
-subunits as well as a downregulation of
ß-adrenoceptors.1 5 6 7 Since adenylyl cyclase
desensitization has been observed in several models of hypertensive
cardiac hypertrophy, such as in spontaneous
hypertension,13 14 15 16 renal hypertension,17 and
desoxycorticosterone-induced17 or
salt-induced18 hypertension, this mechanism could be a
general feature of the hypertrophied heart in hypertension. However,
ß-adrenoceptor downregulation has been observed to be much less
pronounced in hypertensive cardiac hypertrophy than in chronic heart
failure.17 19 In several models of acquired hypertension,
ß-adrenoceptor downregulation was even absent.18 19
Thus, it is likely that postreceptor mechanisms independent of
ß-adrenoceptors might play an important role in these conditions. As
in the failing human myocardium, an increase of Gi has
been observed in SHR (this study and References 1616 , 2121 , and 2222 ), renal
hypertension,17 and salt-sensitive
hypertension.18 In SHR, these findings were reported for
pertussis toxin substrates,16 21 22 Gi
protein content,16 and Gi mRNA
levels.21 22 The increases correlated well among the
different techniques. Thus, the increased expression of myocardial
Gi is likely to represent one underlying
mechanism for adenylyl cyclase desensitization. It appears to precede
the development of heart failure and then may contribute to the
progression from cardiac hypertrophy to cardiac failure. If this
hypothesis holds true, one would expect that an inhibition of the
increased Gi levels could be useful for preventing or
delaying the development of heart failure.
In the present study, pertussis toxin–induced
[32P]ADP ribosylation was significantly increased.
However, quantification of G protein -subunits is hampered by a
number of technical and biological factors that could influence the
effectiveness of labeling in native membranes. The substrate quality of
Gi is influenced by the biophysical membrane properties.
The nonionic detergent Lubrol PX is reported to facilitate the
incorporation of [32P]ADP ribose into
Gi.44 In addition, ADP ribosylation is
enhanced by GDPßS- and ß-subunits,45 suggesting
that the GDP-liganded ß heterotrimer is the best substrate for
the ADP ribosyl transferase of pertussis toxin.45 46
Finally, preexisting covalent modification at the cysteine residue at
the fourth position from the C terminus, where Gi is ADP
ribosylated in a pertussis toxin–dependent manner47 by
endogenous ADP ribosyl transferases,48 could limit the
Gi detection in myocardial membranes. Previously, we
have systematically studied Gi proteins by pertussis
toxin labeling and radioimmunology in SHR compared with
WKY.16 [32P]ADP ribosylation was
concentration dependently enhanced by Lubrol PX with a maximum at 0.5%
(vol/vol). Under these conditions, the increase of Gi
proteins was comparable with the pertussis toxin labeling and the
radioimmunochemical method,16 whereas Western blotting
showed a far greater variability. However, endogenous ADP ribosylation
could still occur. Therefore, we have extended this investigation on
the characterization of posttranslational alterations. As in human
lung, thrombocytes, fat,49 and
myocardium,49 50 no endogenously ADP-ribosylated form of
Gi could be detected in rat myocardial membranes.
Therefore, we chose the ADP ribosylation technique to quantify
Gi in SHR compared with WKY after pharmacological
treatment.
Another important question is whether the increase of Gi
is causally related to adenylyl cyclase desensitization in SHR or
whether a functional impairment of Gs function could
have an influence. Gs was studied functionally by
reconstitution into Gs-deficient S49 cyc-
mouse lymphoma cell membranes. With this technique, no change of
Gs bioactivity was observed. It should be pointed out
that these experiments only allow conclusions to be drawn on the
functional activity of Gs and not on the actual amount
of Gs proteins. However, recent studies did not detect
any changes in Gs proteins by immunoblotting as well as
mRNA levels in SHR.51 An influence of a depressed catalyst
activity also appears unlikely because the effect of forskolin plus
MnCl2 on adenylyl cyclase was similar in SHR and WKY.
Different results were obtained by Murakami et al,52 who
observed evidence for an increased activity of the catalyst and a
slight depression of Gs activity. One potential
alteration could be the use of different SHR and control strains in the
study of Murakami et al.52 To provide direct evidence for
a role of Gi in the adenylyl cyclase desensitization, we
investigated adenylyl cyclase activity in pertussis toxin–treated
membranes. In failing human myocardium, the depressed adenylyl cyclase
activity was restored after pertussis toxin treatment.6
Similar results were obtained in cardiac membranes from SHR, in which
basal and Gpp(NH)p-stimulated adenylyl cyclase was similar compared
with SHR after treatment with pertussis toxin plus NAD (the present
study). Thus, we conclude that an increase of Gi is
causally related to adenylyl cyclase desensitization and could
represent a target for pharmacological treatment.
In the present study, the increased Gi expression in
SHR was inhibited by treatment with captopril, nitrendipine, or
captopril plus nitrendipine. Concomitantly, the depressed
Gpp(NH)p-stimulated adenylyl cyclase activity was resensitized in SHR.
Several experiments in laboratory animals or isolated cells indicate a
role of cAMP in the mechanism of increased myocardial Gi
proteins in the heart. In isolated neonatal rat cardiomyocytes, an
increase of pertussis toxin substrate has been observed after
cultivation of myocytes in norepinephrine.53 The increase
of Gi and the heterologous adenylyl cyclase
desensitization was sensitive to ß-adrenoceptor antagonists but not
to prazosin, suggesting a role of ß-adrenoceptor
stimulation.53 Treatment of rats in vivo with
isoproterenol led to an increase of Gi as judged by
pertussis toxin substrates54 and Gi2 and
Gi3 mRNA levels, whereas Gs mRNA levels
were unchanged.55 In murine S49 cyc- or
kin- cell membranes, which genetically lack
Gs- or cAMP-dependent protein kinase, Gi
was not increased after ß-adrenoceptor stimulation, but an increase
was observed in wild-type cells in which both components are
present.56 Thus, an increase of Gi
expression appears to require an intact
ß-adrenoceptor–cAMP–phosphorylation cascade. From these
observations, it appears likely that an increased myocardial
Gi protein content is due to sympathetic activation, as
occurs in hypertension57 and heart
failure.42 A reduction in sympathetic activation
would consequently attenuate the increase of Gi
proteins. Previously,23 we detected a reduction of
circulating neuropeptide Y concentrations after pharmacological
treatment as an indicator of a reduced sympathetic activity. Therefore,
the reduction of Gi (the present study) and
upregulation of ß-adrenoceptors23 with the
accompanying resensitization of isoproterenol-stimulated23
and guanine nucleotide–stimulated (the present study) adenylyl
cyclase activity in SHR argued strongly in favor of a role of
sympathetic activity in inducing adenylyl cyclase desensitization by
increased Gi protein levels and ß-adrenoceptor
downregulation. Angiotensin II induces an increase of norepinephrine
release in the myocardium by activation of presynaptic angiotensin II
receptors.58 59 Thus, the mechanism of the reduction of
the sympathetic drive and most likely the reversal of ß-adrenergic
receptor downregulation and Gi increase can be explained
by a reduction of angiotensin II formation and consequently a reduced
stimulation of presynaptic angiotensin II receptors. However, the
reduction of sympathetic activity23 and of postsynaptic
changes (the present study and Reference 2323 ) after application of
the Ca2+ antagonist nitrendipine was rather
unexpected. However, in a previous study, nisoldipine reduced
neurohumoral activation as judged by levels of circulating atrial
natriuretic peptides.60 In addition, the
Ca2+ antagonist nisoldipine has been reported to
reduce both systemic sympathetic activity61 and myocardial
norepinephrine turnover.62 These effects apparently are
not due to the hemodynamic effects of the Ca2+
antagonist because other vasodilators such as minoxidil enhanced
sympathetic activity even though they exerted similar blood
pressure–lowering effects.63
In summary, in 30-week-old SHR, an increase of Gi was
accompanied by desensitization of guanine nucleotide–stimulated
adenylyl cyclase activity. Treatment with captopril, nitrendipine, or
captopril plus nitrendipine abolished the increase of Gi
and adenylyl cyclase desensitization, most likely by a reduction of
sympathetic activity. From these data, it is tempting to speculate that
pharmacological treatment resulting in a reduction of sympathetic tone
can prevent adenylyl cyclase desensitization in hypertension and thus
could contribute to a delay in or even prevention of the progression
from cardiac hypertrophy to chronic heart failure.
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Acknowledgments |
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Experimental work was supported by the Deutsche Forschungsgemeinschaft. M.B. is a recipient of the Gerhard Hess and Heisenberg programs of the Deutsche Forschungsgemeinschaft. This work contains parts of the doctoral thesis of Claudia Gräbel (University of Munich, in preparation). We thank Evelyn Ziolkowski and Elisabeth Ronft for their excellent help.
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Footnotes |
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Reprint requests to Michael Böhm, Klinik III für Innere Medizin der Universität zu Köln, Joseph-Stelzmann Straße 9, 50924 Köln, FRG.
Received April 22, 1994; first decision July 27, 1994; accepted November 11, 1994.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Brodde O-E. ß1- and ß2-Adrenoceptors in the human heart: properties, function, and alterations in chronic heart failure. Pharmacol Rev. 1991;43:203-242. [Medline] [Order article via Infotrieve]
2. Sibley DR, Lefkowitz RJ. Molecular mechanisms of receptor desensitization using the beta-adrenergic receptor-coupled adenylate cyclase system as a model. Nature. 1985;317:124-129. [Medline] [Order article via Infotrieve]
3. Gilman AG. G proteins and dual control of adenylate cyclase. Cell. 1984;36:577-579. [Medline] [Order article via Infotrieve]
4. Gilman AG. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615-649. [Medline] [Order article via Infotrieve]
5.
Böhm M, Gierschik P, Jakobs KH, Pieske B, Schnabel P,
Ungerer M, Erdmann E. Increase of Gi in human hearts with dilated
but not ischemic cardiomyopathy. Circulation. 1990;82:1249-1265.
6. Feldman AM, Cates AE, Veazey WB, Hershberger RE, Bristow MR, Baughman KL, Baumgartner WA, Van Dop C. Increase of the 40,000-mol wt pertussis toxin substrate (G protein) in the failing human heart. J Clin Invest. 1988;82:189-197.
7. Neumann J, Schmitz W, Meyerinck L, Scholz H, Döring V, Kalmar P. Increase in myocardial Gi-proteins in heart failure. Lancet. 1988;2:936-937. [Medline] [Order article via Infotrieve]
8.
Eschenhagen T, Mende U, Nose M, Schmitz W, Scholz H, Haverich
A, Hirt S, Döring V, Kalmar P, Höppner W, Seitz HJ.
Increased messenger RNA level of the inhibitory G-protein -subunit
Gi-2 in human endstage heart failure. Circ Res. 1992;70:688-696.
9.
Schnabel P, Böhm M, Gierschik P, Jakobs KH, Erdmann E.
Improvement of cholera toxin-catalyzed ADP-ribosylation by
endogenous ADP-ribosylation factor from bovine brain provides
evidence for an unchanged amount of Gs in failing human
myocardium. J Mol Cell Cardiol. 1990;22:73-82. [Medline]
[Order article via Infotrieve]
10.
Bristow MR, Anderson FL, Port JD, Skerl L, Hershberger RE,
Larrabee P, O'Connell JB, Renlund DG, Volkamn K, Murray J, Feldman AM.
Differences in ß-adrenergic neuroeffector mechanisms in ischemic
versus idiopathic dilated cardiomyopathy.
Circulation. 1991;84:1024-1039.
11.
Hershberger RE, Feldman AM, Bristow MR.
A1-Adenosine receptor inhibition of adenylate cyclase in
failing and nonfailing human ventricular myocardium.
Circulation. 1991;83:1343-1351.
12. Böhm M, Lensche H, Erdmann E. Mechanisms of deficient cAMP generation in heart failure. In: Figulla HR, Kandolf R, McManus B, eds. Idiopathic Dilated Cardiomyopathy. Berlin, FRG: Springer-Verlag; 1993:120-131.
13. Limas C, Limas CJ. Reduced number of ß-adrenergic receptors in the myocardium of spontaneously hypertensive rats. Biochem Biophys Res Commun. 1978,83:710-714.
14. Robberecht P, Winand J, Chatelain P, Poloczek P, Camus JC, De Neef P, Christope J. Comparison of ß-adrenergic receptors and the adenylate cyclase system with muscarine receptors and guanylate cyclase activities in the heart of spontaneously hypertensive rats. Biochem Pharmacol. 1981;30:385-387. [Medline] [Order article via Infotrieve]
15. Palmer GC, Greenberg S. Alterations in myocardial adenylate cyclase in spontaneously hypertensive rats. Pharmacology. 1979;19:156-162. [Medline] [Order article via Infotrieve]
16. Böhm M, Gierschik P, Knorr A, Larisch K, Weismann K, Erdmann E. Role of altered G-protein expression in the regulation of myocardial adenylate cyclase activity and force of contraction in spontaneously hypertensive cardiomyopathy in rats. J Hypertens. 1992;10:1115-1128. [Medline] [Order article via Infotrieve]
17.
Böhm M, Gierschik P, Knorr A, Larisch K, Weismann K,
Erdmann E. Desensitization of adenylate cyclase and increase of Gi
in cardiac hypertrophy due to acquired hypertension.
Hypertension. 1992;20:103-112.
18.
Böhm M, Gierschik P, Knorr A, Schmidt U, Weismann K,
Erdmann E. Myocardial adenylyl cyclase, ß-adrenoceptors, and
G-proteins in salt-sensitive hypertension.
Hypertension. 1993;22:715-727.
19.
Michel MC, Kanczik R, Khamssi M, Knorr A, Siegl H, Beckeringh
JJ, Brodde OE. - and ß-Adrenoceptors in hypertension, I: cardiac
and renal 1-, ß1-, and
ß2-adrenoceptors in rat models of acquired hypertension.
J Cardiovasc Pharmacol. 1988;13:421-431.
20. Böhm M, Beuckelmann D, Diet F, Feiler G, Lohse MD, Erdmann E. Properties of cardiac alpha- and beta-adrenoceptors in spontaneously hypertensive rats. Naunyn Schmiedebergs Arch Pharmacol. 1988;338:383-391. [Medline] [Order article via Infotrieve]
21.
Anand-Srivastava MB, Picard S, Thibault C. Altered expression
of inhibitory guanine nucleotide regulatory proteins (Gi) in
spontaneously hypertensive rats. Am J Hypertens. 1991;4:840-843. [Medline]
[Order article via Infotrieve]
22. Thibault C, Anand-Srivastava MB. Altered expression of G-protein mRNA in spontaneously hypertensive rats. FEBS Lett. 1992;313:160-164. [Medline] [Order article via Infotrieve]
23.
Böhm M, Gräbel C, Knorr A, Erdmann E.
Treatment in hypertensive cardiac hypertrophy, I: neuropeptide Y and
ß-adrenoceptors. Hypertension. 1995;25:954-961.
24. Salomon Y, Londos C, Rodbell M. A highly sensitive adenylate cyclase assay. Ann Biochem. 1974;58:541-548.
25.
Böhm M, Schmidt U, Gierschik P, Schwinger RHG,
Böhm S, Erdmann E. Sensitization of adenylate cyclase by
halothane in human myocardium and S49 lymphoma wild-type and
cyc- cells: evidence for inactivation of the inhibitory G
protein Gi. Mol Pharmacol. 1994;45:380-389. [Abstract]
26.
Böhm M, Larisch K, Erdmann E, Camps M, Jakob KH,
Gierschik P. Failure of 32P-ADP-ribosylation by pertussis
toxin to determine Gi content in membranes from various human
tissues. Biochem J. 1991;277:223-229.
27. Gierschik P, Codina J, Simons C, Birnbaumer L, Spiegel AM. Antisera against a guanine nucleotide binding protein from retina cross react with the ß subunit of the adenylyl cyclase-associated guanine nucleotide binding proteins Ns and Ni. Proc Natl Acad Sci U S A. 1992;82:727-731.
28.
Goldsmith P, Gierschik P, Milligan G, Unson CG, Vinitsky R,
Marech HL, Spiegel AM. Antibodies directed against synthetic peptides
distinguish between GTP-binding proteins in neutrophil and brain.
J Biol Chem. 1987;262:14683-14688.
29.
Sternweis PC, Northup JK, Smigel MD, Gilman AG. The
regulatory component of adenylate cyclase. J Biol Chem. 1981;256:11517-11526.
30.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein
measurements with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.
31. Lämmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685. [Medline] [Order article via Infotrieve]
32. Dixon TF, Purdom M. Serum 5'nucleotidase. J Clin Pathol. 1954;7:341-351.
33.
Wallenstein S, Zucker CL, Fleiss JL. Some statistical methods
useful in circulation research. Circ Res. 1980;47:1-9.
34. Seamon KB, Daly JW. Forskolin: its biological and chemical properties. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1989;20:1-150.
35.
Limbird LE, Hickey AR, Lefkowitz RJ. Unique uncoupling of the
frog erythrocyte adenylate cyclase system by manganese: loss of hormone
and guanine nucleotide-sensitive enzyme activities without loss of
nucleotide-sensitive, high affinity agonist binding. J Biol
Chem. 1979;254:2677-2683.
36. Cech SY, Broaddus WC, Maguire ME. Adenylate cyclase: the role of magnesium and other divalent cations. Mol Cell Biochem. 1980;33:67-92. [Medline] [Order article via Infotrieve]
37. Hara-Yokoyama M, Furuyama S. Endogenous inhibitor of the ADP-ribosylation of (a) G-protein(s) as catalyzed by pertussis toxin is present in rat liver. FEBS Lett. 1988;234:27-30. [Medline] [Order article via Infotrieve]
38. Kannel WB, Castelli WP, McNamara PM, McKee PA, Feinleib M. Role of blood pressure in the development of congestive heart failure: the Framingham Study. N Engl J Med. 1972;287:781-787.
39. Grossman W, Jones D, McLaurin KP. Wall stress and patterns of hypertrophy in the human left ventricle. J Clin Invest. 1975;58:56-64.
40.
Spann JF Jr, Buccino RA, Sonnenblick EH, Braunwald E.
Contractile state of cardiac muscle obtained from cats with
experimentally produced ventricular hypertrophy and heart failure.
Circ Res. 1967;21:341-354.
41. Katz AM. Cardiomyopathy of overload: a major determinant of prognosis in congestive heart failure. N Engl J Med. 1990;322:100-110. [Medline] [Order article via Infotrieve]
42.
Packer M. Neurohormonal interactions and adaptations in
congestive heart failure. Circulation. 1988;77:721-730.
43. Swedberg K, Viquerat C, Rouleau J-L, Roizen M, Atherton B, Parmley WW, Chatterjee K. Comparison of myocardial catecholamine balance in chronic congestive heart failure and in angina pectoris without failure. Am J Cardiol. 1984;54:783-789. [Medline] [Order article via Infotrieve]
44. Morris SA, Horn EM, Hawley T, Manning D, Bilezikian JP. The influence of detergents on the availability of pertussis toxin substrates. Arch Biochem Biophys. 1991;290:86-92. [Medline] [Order article via Infotrieve]
45. Ribeiro-Neto F, Mattera R, Hildebrandt JD, Codina J, Field JB, Birnbaumer L, Sekura RD. ADP-ribosylation of membrane components by pertussis toxin and cholera toxin. Methods Enzymol. 1985;109:566-582. [Medline] [Order article via Infotrieve]
46.
Ribeiro-Neto F, Mattera R, Grenet D, Sekura RD, Birnbaumer L,
Field JB. Adenosine diphosphate ribosylation of G proteins by pertussis
toxin and cholera toxin in isolated membranes: different requirements
for and effects of guanine nucleotides and
Mg2+. Mol Endocrinol. 1987;1:472-481.
47.
West RE Jr, Moss J, Vaughan M, Liu T, Lui T-Y. Pertussis
toxin-catalyzed ADP-ribosylation of transducin: cysteine 347 is the
ADP-ribose acceptor site. J Biol Chem. 1985;260:14428-14430.
48.
Tanuma S, Kawashima K, Endo H. Eukaryotic mono(ADP-ribosyl)
transferase that ADP-ribosylates GTP-binding regulatory Gi
protein. J Biol Chem. 1988;263:5485-5489.
49.
Böhm M, Gräbel C, Kirchmayr R, Lensche H, Erdmann
E, Gierschik P. C-terminal modifications of pertussis toxin-sensitive
G-protein -subunits differentially affect immunoreactivity.
Biochem Pharmacol. 1993;46:2145-2154. [Medline]
[Order article via Infotrieve]
50.
Böhm M, Eschenhagen T, Gierschik P, Larisch K, Lensche
H, Mende U, Schmitz W, Schnabel P, Scholz H, Steinfath M, Erdmann E.
Radioimmunochemical quantification of Gi in right and left
ventricles from patients with ischaemic and dilated cardiomyopathy and
predominant left ventricular failure. J Mol Cell Cardiol. 1994;26:133-149. [Medline]
[Order article via Infotrieve]
51. Michel MC, Brodde OE, Insel PA. Are cardiac G-proteins altered in rat models of hypertension? J Hypertens. 1993;11:355-363. [Medline] [Order article via Infotrieve]
52. Murakami T, Katada T, Yasuda H. Reduction in the activity of the stimulatory guanine nucleotide-binding protein in the myocardium of spontaneously hypertensive rats. J Mol Cell Cardiol. 1987;19:199-208. [Medline] [Order article via Infotrieve]
53. Reithmann C, Werdan K. Noradrenaline-induced desensitization in cultured heart cells as a model for the defects of adenylate cyclase system in severe heart failure. Naunyn Schmiedebergs Arch Pharmacol. 1989;339:138-144. [Medline] [Order article via Infotrieve]
54. Mende U, Eschenhagen T, Geertz B, Schmitz W, Scholz H, Schulte am Esch J, Sempell R, Steinfath M. Isoprenaline-induced increase in the 40/41 kDa pertussis toxin substrates and functional consequences on contractile response in rat heart. Naunyn Schmiedebergs Arch Pharmacol. 1992;345:44-50. [Medline] [Order article via Infotrieve]
55.
Eschenhagen T, Mende U, Nose M, Schmitz W, Scholz H, Warnholtz
A, Wüstel J-M. Isoprenaline-induced increase in mRNA levels of
inhibitory G-protein -subunits in rat heart. Naunyn
Schmiedebergs Arch Pharmacol. 1991;343:609-615. [Medline]
[Order article via Infotrieve]
56.
Clark RB, Kunkel MW, Friedman J, Goka TJ, Johnson JA.
Activation of cAMP-dependent protein kinase is required for
heterologous desensitization of adenylyl cyclase in S49 wild-type
lymphoma cells. Proc Natl Acad Sci U S A. 1988;85:1442-1446.
57. Goldstein DS, Kopin IJ. The autonomic nervous system and catecholamines in normal blood pressure control and in hypertension. In: Laragh JH, Brenner BM, eds. Hypertension: Pathophysiology, Diagnosis and Management. New York, NY: Raven Press Publishers; 1990:711-747.
58. Zimmermann BG. Adrenergic facilitation by angiotensin: does it serve a physiological function? Clin Sci. 1981;60:343-348. [Medline] [Order article via Infotrieve]
59. Starke K. Regulation of noradrenaline release by presynaptic receptor systems. Rev Physiol Biochem Pharmacol. 1977;77:1-24. [Medline] [Order article via Infotrieve]
60. Kazda S, Stasch JP, Hirth C. Nitrendipine in experimental hypertension: effects on cardiac hypertrophy, heart failure, and atrial natriuretic peptides. J Cardiovasc Pharmacol. 1987;9:90-95.
61. Fields N, Tsoporis J, Leenen FHH. Differential effects of the calcium antagonist, nisoldipine, versus the arterial vasodilator, minoxidil, on ventricular anatomy, intravascular volume, and sympathetic activity in rats. J Cardiovasc Pharmacol. 1989;14:826-839. [Medline] [Order article via Infotrieve]
62. Tsoporis J, Fields N, Leenen FHH. Contrasting effects of calcium antagonists vs. arterial vasodilators on cardiac anatomy and sympathetic activity in spontaneously hypertensive rats. J Cardiovasc Pharmacol. 1991;17:166-168. [Medline] [Order article via Infotrieve]
63.
Tsoporis J, Leenen FHH. Effects of arterial vasodilators on
cardiac hypertrohy and sympathetic activity in rats.
Hypertension. 1988;11:376-386.
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