(Hypertension. 1995;25:954-961.)
© 1995 American Heart Association, Inc.
Articles |
Treatment in Hypertensive Cardiac Hypertrophy, I
Neuropeptide Y and ß-Adrenoceptors
From the Klinik III für Innere Medizin der Universität zu Köln and Bayer AG (A.K.), Wuppertal, Germany.
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Abstract In the present study, we investigated serum and myocardial neuropeptide Y concentrations as measures of sympathetic activity as well as myocardial ß-adrenoceptors and ß-adrenoceptor–stimulated adenylyl cyclase activity in spontaneously hypertensive rats (SHR). SHR and control rats at 10 weeks of age were kept on oral treatment with captopril, nitrendipine, or both for 20 weeks. Treatment only slightly reduced but did not normalize blood pressure and cardiac hypertrophy in SHR. The elevated serum concentration of neuropeptide Y, the reduced number of ß-adrenoceptors, and the depressed ß-adrenoceptor–stimulated adenylyl cyclase activity were partly normalized compared with the values observed in control rats. We conclude that antihypertensive treatment, at doses that failed to normalize systolic pressure and to reverse cardiac hypertrophy completely, is able to reduce sympathetic activity in SHR, thereby resensitizing the depressed ß-adrenoceptor–adenylyl cyclase system.
Key Words: hypertension, essential • cardiac hypertrophy • heart failure, congestive • rats, inbred SHR • adenylyl cyclase • receptors, adrenergic • sympathetic nervous system • angiotensin-converting enzyme inhibitors • calcium channel blockers
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Introduction |
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An increase in myocardial force of contraction is mediated by the stimulation of myocardial ß-adrenoceptors,1 which leads to enhanced formation of cAMP from ATP.2 After prolonged stimulation of myocardial ß-adrenoceptors, the ß-adrenoceptor–adenylyl cyclase system becomes desensitized.3 4 ß-Adrenoceptor–adenylyl cyclase desensitization can be brought about by several mechanisms. In the failing human myocardium, a downregulation of ß1-adrenoceptors,5 6 7 an uncoupling of ß2-adrenoceptors,8 and an increase of inhibitory G proteins9 10 11 have been observed. The stimulatory G protein (Gs
) and the catalyst of
the adenylyl cyclase are apparently unchanged in heart
failure.9 11 12 The mechanism of adenylyl cyclase
desensitization in heart failure is presumably due to increased
sympathetic nervous system activity, resulting in increased
norepinephrine release from the heart and depleted myocardial stores of
norepinephrine and neuropeptide Y, which is released with
norepinephrine from sympathetic nerve terminals.7 Arterial
hypertension is reported to represent the most common cause of
chronic heart failure.13 In this respect, it is intriguing
that a desensitization of myocardial adenylyl cyclase activity occurs
in several models of genetic14 15 16 17 and
acquired18 19 20 21 hypertension with cardiac hypertrophy in the
absence of heart failure. Thus, one might speculate that the
desensitization of cardiac adenylyl cyclase occurs already in
hypertensive cardiac hypertrophy and could represent an
important pathogenetic mechanism contributing to the progression from
hypertrophy to failure. If this hypothesis is pathophysiologically
relevant, one would expect that pharmacological treatment in early
stages would be useful in delaying the development of contractile
failure. However, limited data are available on the effects of
antihypertensive drug treatment on alterations of neuroeffector
mechanisms in hypertensive cardiac hypertrophy. In this part of our
studies, we investigated the effects of captopril, nitrendipine, or a
combination of the two on myocardial neuropeptide Y levels, myocardial
ß-adrenoceptors, and ß-adrenoceptor–mediated stimulation of
adenylyl cyclase activity. Low doses of antihypertensive drugs were
used that did not completely normalize blood pressure (BP) or
completely reverse cardiac hypertrophy. This approach was chosen to
differentiate between the direct effects of the drugs on sympathetic
mechanisms and the secondary effects of BP reduction and regression of
cardiac hypertrophy. |
Methods |
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Experimental Animals
Male Wistar spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were used. Rats were given a normal laboratory animal diet (sniff, sniff Versuchstierdiäten GmbH) and treated with nitrendipine, captopril, or nitrendipine plus captopril orally. The doses were 1000 parts per mouth (ppm, ie, 1000 mg per 1 kg of food) nitrendipine, 300 ppm captopril (ie, 300 mg per 1 kg of food), and 1000 ppm nitrendipine plus 300 ppm captopril. Daily food intake was approximately 25 g, equivalent to a daily oral dose of 7.5 mg captopril or 25 mg nitrendipine in each animal. For biochemical experiments, SHR and WKY at 30 weeks of age were used. Treatment of rats started at 10 weeks, when hypertension was completely established in SHR. Thus, pharmacological treatment was continued for 20 weeks. During the treatment period, systolic BP was measured by the tail-cuff method according to Pfeffer et al22 every 2 weeks. At the end of the experiments, rats were weighed and killed by decapitation at 9 to 10 AM. After thoracotomy, the hearts were quickly removed. Blood samples from each rat were collected in prechilled tubes after decapitation. The animal experiments were approved by the institutional committee and were in accordance with guidelines for experimental research (Nordrhein-Westfalen, Germany).
Neuropeptide Y Determinations
For neuropeptide Y measurements, tissue samples were homogenized
with a polytron device in 0.1 mol/L Tris-HCl at pH 7.4. After
centrifugation (10 000g, 30 minutes), neuropeptide Y was
determined with a commercially available radioimmunoassay (Amersham
Buchler). In brief, the assay was performed in a final volume of 500
µL in 3.5-mL polypropylene tubes containing 100 µL test sample, 100
µL 125I–neuropeptide Y, and 100 µL antiserum. Samples
were diluted to 500 µL with assay buffer consisting of 50 mmol/L
sodium phosphate buffer with 0.3% bovine serum albumin and 10 mmol/L
EDTA at pH 7.4. The assay was performed at +4°C for 24 hours. For
separation of free and bound neuropeptide, samples were supplemented
with 0.25 mL activated charcoal and dextran (molecular weight, 60 000
to 90 000 D) in separation buffer (50 mmol/L sodium phosphate buffer
with 0.2% gelatin and 10 mmol/L EDTA at pH 7.4). Samples were vortexed
and centrifuged for 10 minutes with 20 000g at 4°C.
Radioactivity of the supernatant and pellet was determined with a gamma
scintillation counter. Neuropeptide Y was quantified using a standard
curve (10 to 1000 fmol per tube). This technique is similar to the
radioimmunoassay described elsewhere.23
Membrane Preparation
Myocardial tissue was chilled in 30 mL ice-cold homogenization
buffer (10 mmol/L Tris-HCl, 1 mmol/L Na2EDTA, 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. Afterward, the membrane preparation was homogenized by hand for
1 minute with a glass-glass homogenizer. The homogenate was spun at
484g (JA 20 rotor, Beckman) 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. As determined in
independent experiments on rat and human myocardial membranes, storage
of the preparations up to 1.5 years did not alter the recovery of
ß-adrenoceptors.
Radioligand Binding Studies
Assays were performed in a total volume of 250 µL incubation
buffer. The incubation was carried out at 37°C for 60 minutes. These
conditions allowed complete equilibration of the receptors with the
radioligand. The reaction was terminated by rapid vacuum filtration
through Whatman GF/C filters, and filters were immediately washed three
times with 6 mL ice-cold incubation buffer. All experiments were
performed in triplicate. Radioactivity was determined in a gamma
counter (LKB Wallac). Myocardial ß-adrenoceptors were studied using
125I-cyanopindolol (125I-Cyp) as radiolabeled
ligand as described previously.11 Specific activity was
2000 Ci/mmol. (-)-Propranolol (1 µmol/L) was used for determination
of nonspecific binding. Experiments on WKY and SHR were conducted on
the same day in parallel.
Adenylyl Cyclase Determinations
Adenylyl cyclase was determined according to Salomon et
al24 with slight modifications as published
elsewhere.25 Particulate 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 per 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. 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).
Miscellaneous
Protein was determined according to Lowry et al26
using bovine serum albumin as standard. 5'-Nucleotidase activity was
measured according to Dixon and Purdom.27
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.28 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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BP and Myocardial Hypertrophy
As shown in Fig 1, systolic BP was significantly increased in SHR compared with WKY. Treatment of SHR with captopril, nitrendipine, or both led to only a slight, albeit significant, reduction of systolic BP. However, systolic BP values were still significantly higher in treated SHR than in WKY. Pharmacological treatment in WKY had no significant effect on BP. Consistent with the small effects on BP, treatment did not normalize heart weight in SHR (Fig 2). After treatment with nitrendipine or nitrendipine plus captopril, a significant reduction of relative heart weights was observed in SHR. During the time course of treatment, there were no significant effects of the different treatment regimens on body weight development in the different groups (not shown). Body weights were comparable in the different groups (Table). Treatment of SHR with low-dose nitrendipine plus captopril but not with captopril alone slightly but significantly reduced cardiac hypertrophy and BP. Thus, alterations of sympathetic neuroeffector mechanisms with treatment appear to represent largely direct effects of the drugs rather than indirect effects induced by a reduction of hypertrophy or myocardial pressure load.
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Serum and Myocardial Neuropeptide Y Concentrations
We studied serum and myocardial concentrations of neuropeptide Y
as parameters of sympathetic nervous system activity. Fig 3 summarizes the data. In SHR, serum neuropeptide Y
concentration increased significantly compared with WKY (Fig 3, left).
The data were similar when related to milliliters of serum (SHR:
control, 247.2±67.8 fmol/mL; captopril, 91.1±17.8; nitrendipine,
86.6±14.9; nitrendipine plus captopril, 108.1±19.7; WKY: control,
48.1±7.2; captopril, 46.5±7.9; nitrendipine plus captopril,
42.6±5.2; n=8 to 10). Serum protein concentration did not differ among
the groups (not shown). Correspondingly, neuropeptide Y concentrations
in the myocardium were significantly less in SHR than WKY (Fig 3,
right). Fig 4, top, summarizes the effect of
pharmacological treatment on neuropeptide Y concentration in WKY serum.
Captopril, nitrendipine, and captopril plus nitrendipine did not
significantly reduce neuropeptide Y concentration in WKY serum,
although there was a tendency toward a reduction in the WKY group on
captopril plus nitrendipine (P=NS). The data in the
treatment groups were still significantly lower than in SHR (Fig 4, top
left, shown for comparison). Fig 4, bottom, shows the data for SHR.
Treatment with captopril, nitrendipine, and nitrendipine plus captopril
significantly reduced neuropeptide Y serum concentration in SHR. In the
group of SHR on nitrendipine, the data were similar to those in WKY
(Fig 4, bottom left, shown for comparison). Data for treated or
untreated WKY and SHR were similar when related to milligrams of
protein or milliliters of volume (not shown). To investigate whether
the alterations in serum neuropeptide Y concentration corresponded to
those in the myocardium, we also measured myocardial neuropeptide Y
content. Fig 5, top, demonstrates that treatment with
antihypertensive drugs significantly reduced neuropeptide Y stores in
WKY; however, they were still elevated compared with those in control
SHR (Fig 5, top left, shown for comparison). The results for SHR are
summarized in Fig 5, bottom. Treatment of SHR with captopril,
nitrendipine, or captopril plus nitrendipine did not significantly
alter myocardial neuropeptide Y concentration. In all treated groups,
neuropeptide Y levels remained reduced compared with WKY (Fig 5, bottom
left, shown for comparison). Data for treated or untreated WKY and SHR
were similar when related to milligrams of protein or milligrams wet
weight (not shown).
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Myocardial ß-Adrenoceptors
To investigate whether the alterations of sympathetic nervous
system activity after pharmacological treatment have an effect on
postsynaptic sympathetic neuroeffector mechanisms, we investigated the
density of myocardial ß-adrenergic receptors using radioligand
binding experiments. Fig 6 shows a typical saturation
experiment of 125I-Cyp binding to myocardial membranes of
SHR and WKY. Specific binding was monophasic and saturable in both
groups. Transformation of binding data revealed one class of binding
sites. As depicted in Fig 6, the ß-adrenergic receptor density was
reduced in SHR, whereas the antagonist affinity as judged from the
slope of the transformed data (Fig 6 inset) was similar. Fig 7, top, summarizes the binding data for WKY. In SHR,
there was a significant reduction in the ß-adrenoceptor density
compared with WKY. Treatment with captopril, nitrendipine, or captopril
plus nitrendipine had no significant effect on ß-adrenoceptor density
in WKY. Fig 7, bottom, summarizes the data for SHR. After treatment of
SHR with antihypertensive drugs, the number of ß-adrenoceptors was
significantly increased. Captopril plus nitrendipine treatment produced
an increase of receptor numbers, resulting in similar densities
compared with WKY (Fig 7, bottom left, shown for comparison). The
antagonist affinities as judged from the Kd
values did not differ among the groups.
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Adenylyl Cyclase Activity
To investigate whether the decline in the number of ß-adrenergic
receptors in SHR and their upregulation after pharmacological treatment
have a consequence for myocardial cAMP production, we determined
adenylyl cyclase activity in myocardial membrane preparations from all
groups. Concentration-response curves summarize the effects of
isoproterenol on adenylyl cyclase activity in myocardial membranes of
SHR and WKY (Fig 8). The effects of isoproterenol on
adenylyl cyclase were significantly reduced in SHR compared with WKY
(Fig 8, top left). After pharmacological treatment in WKY, the response
to isoproterenol did not differ between treated and untreated rats.
Although the effect of isoproterenol was similar in all WKY groups, the
effects of isoproterenol in WKY after nitrendipine and nitrendipine
plus captopril were not statistically different from the effects in
SHR. This finding could reflect a weak desensitization of
ß-adrenoceptor–stimulated adenylyl cyclase in WKY on nitrendipine or
nitrendipine plus captopril. Fig 8, bottom, shows the results for SHR.
Treatment of SHR with captopril and nitrendipine significantly
increased the effects of isoproterenol compared with untreated rats.
Treatment of SHR with captopril or nitrendipine resulted in
isoproterenol-stimulated adenylyl cyclase activity that was similar to
that observed in WKY. No significant effect was observed after
treatment with nitrendipine plus captopril.
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Discussion |
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We studied the effect of antihypertensive drugs on sympathetic neuroeffector mechanisms in SHR and control WKY. Although no changes in systolic BP or heart weight were observed in WKY, treatment with captopril, nitrendipine, or captopril plus nitrendipine led to small reductions in systolic BP and myocardial hypertrophy (except captopril). In SHR, serum neuropeptide Y concentrations were significantly increased, and myocardial neuropeptide Y concentrations were reduced by 85% compared with WKY. Treatment reduced serum concentrations but had no effect on myocardial neuropeptide Y concentrations in SHR. Consistent with the apparent sympathetic activation, the number of ß-adrenoceptors and amount of isoproterenol-stimulated adenylyl cyclase activity were reduced in SHR. After pharmacological treatment, the observed alterations were completely or partially reversed in SHR.
Alterations in the sympathetic nervous system have been suggested to play an important role in the pathogenesis of hypertension in animal models and humans.29 30 Sympathetic activation produces a marked desensitization of the ß-adrenoceptor–adenylyl cyclase system and is regarded as one important mechanism involved in the dysregulation of myocardial force of contraction in hypertensive cardiac hypertrophy.14 15 16 17 18 19 30 31 Thus, it appears most important to study whether and how alterations of sympathetic neuroeffector mechanisms can be modulated by pharmacological treatment. SHR have often been used as a model for hypertensive cardiac hypertrophy.30 Adenylyl cyclase desensitization,15 16 partially caused by a reduction of cardiac ß-adrenoceptors,14 31 32 has been suggested to be involved in the reduced ß-adrenergic effects on myocardial force of contraction observed in this model.31 33 A reduced gene expression of ß-adrenergic receptors has been suggested to play a role in the reduced receptor number in SHR.34 In the present study, we used SHR to characterize the effect of pharmacological treatment with antihypertensive drugs on sympathetic activation, myocardial ß-adrenoceptors, and adenylyl cyclase activity.
In the failing myocardium, ß-adrenergic desensitization occurs,7 probably because of increased sympathetic nervous system activity.35 In this condition, the catecholamine drive originates from the sympathetic neurons, because norepinephrine is released in considerable amounts from the myocardium.36 Thus, the heart becomes an endocrine organ contributing to the elevated peripheral norepinephrine concentrations in this condition.36 37 Paradoxically, myocardial norepinephrine stores become depleted,23 38 which might be related to the fact that catecholamine uptake becomes the major determinant of myocardial norepinephrine stores, when norepinephrine release from the heart to the periphery is activated.39 It has been observed that norepinephrine and neuropeptide Y coexist in sympathetic neurons, where they are stored in vesicles of different sizes.40 41 In the heart, neuropeptide Y–containing vesicles in sympathetic nerves have been observed in the myocardial interstitium and coronary arteries.42 The fact that circulating neuropeptide Y correlates better with circulating norepinephrine than epinephrine suggests that its origin is indeed the sympathetic nerve terminal rather than the adrenal medulla.43 Since neuropeptide Y is stored and released with norepinephrine,41 one might suggest that the peptide represents a valuable marker of myocardial sympathetic activity. The approach of measuring neuropeptide Y concentrations as a marker of sympathetic activity might be superior to that of studying norepinephrine concentrations. This is because during short-term stress, neuropeptide Y concentrations are not altered, and thus, this parameter might represent a valuable measure of long-term changes of sympathetic nervous system activity. This was the reason we determined neuropeptide Y rather than norepinephrine levels. The amount of tissue did not allow the measurement of both parameters.43 44 Therefore, it seems unlikely that the observations described here have been influenced by exogenous factors such as stress during death of the rats.45 Although sympathetic activation has been suggested to play a pathophysiological role in the syndrome in SHR, measurements of peripheral or myocardial neuropeptide Y concentrations have so far not been available. In the present article, we report that serum neuropeptide Y concentrations are increased by a factor of approximately 4.5, whereas the myocardial stores of neuropeptide Y were diminished by approximately 75% to 80% in SHR. This finding is similar to data obtained in failing human hearts of patients with terminal heart failure.23 Thus, strong evidence is provided that not only in the failing human heart35 but also in spontaneous hypertensive cardiomyopathy of SHR a strong activation of the sympathetic nervous system occurs29 that involves the activation of myocardial sympathetic nerves as shown in the hearts from SHR. However, one has to keep in mind that this technique provides only an indirect measurement of sympathetic activity. Alterations of neuropeptide Y clearance in the different rat groups studied herein could potentially influence these results and represent a limitation of this technique.
Since sympathetic activation is regarded as the cause of adenylyl cyclase desensitization in SHR, it appeared worthwhile to investigate the influence of hypertensive drugs on neurosympathetic mechanisms. In SHR, treatment with the Ca2+ antagonist nitrendipine, the angiotensin-converting enzyme inhibitor captopril, and a combination of both reduced serum concentrations of neuropeptide Y. This finding is compatible with a reduction of sympathetic nervous system activity. Since myocardial neuropeptide Y concentrations did not recover after treatment, although serum concentrations were reduced, it is likely that the antihypertensive drugs act primarily on peripheral sites rather than on myocardial sympathetic nerves. In WKY after treatment, a small but significant reduction of neuropeptide Y concentrations in the heart occurred. This could be an indicator of myocardial sympathetic activation in normotensive animals. Alternatively, direct stimulatory effects of myocardial nerves in SHR could have masked an increasing effect of myocardial neuropeptide Y caused by systemic decline of sympathetic activity. However, in normotensive WKY, no alterations of serum neuropeptide Y concentrations were observed. These data show that in rats the neurotransmitter release from the heart is likely to be too small to produce detectable changes in the general circulation.
From the observed evidence for an increase of general and myocardial sympathetic activity, one would expect that sympathetic activation could represent a mechanism to produce alterations of the postsynaptic ß-adrenoceptor–adenylyl cyclase system. As shown in radioligand binding experiments, the number of ß-adrenoceptors was significantly reduced in SHR. Similar results were obtained when binding data were related to 5'-nucleotidase activity as membrane marker. Similar findings have been reported previously.14 15 16 17 31 32 34 However, it is noteworthy that the decline in the number of ß-adrenoceptors is rather small and much less pronounced than in the failing human heart,7 in which, as judged from neuropeptide Y studies, a similar sympathetic activation occurs. In this respect, it is noteworthy that in salt-sensitive hypertension21 as well as in renal46 47 and deoxycorticosteroid-induced20 47 hypertension, no ß-adrenoceptor downregulation occurs although adenylyl cyclase was strongly desensitized. Thus, one might speculate that downregulation of ß-adrenoceptors in rats or in hypertension is less pronounced than in other species, such as humans, or in other conditions, such as heart failure, or both. After pharmacological treatment, the number of ß-adrenoceptors was significantly increased in SHR and was not statistically different from the number in WKY controls. This finding corresponds to the reduction of serum neuropeptide Y concentrations after pharmacological treatment. The lack of ß-adrenoceptor changes in WKY is in agreement with the unchanged serum neuropeptide Y concentrations. Since myocardial neuropeptide Y concentrations did not increase although ß-adrenoceptors increased in SHR and since neuropeptide Y levels decreased although ß-adrenoceptors were unchanged in WKY, strong evidence is provided that serum neuropeptide Y levels better correspond to ß-adrenoceptor changes than myocardial neuropeptide Y levels. However, not only BP but serum levels of neuropeptide Y were not normalized by the pharmacological treatment regimens. The BP reduction was only 6% to 9%, whereas serum levels were reduced by 40% to 60% and myocardial levels increased by 300% to 500% in SHR after treatment with antihypertensive drugs compared with control SHR. Although these treatments did not restore the values completely toward those in WKY, we think that the sympathetic activity as judged from cardiac and peripheral neuropeptide Y levels is more sensitive to pharmacological treatment than the BP-lowering effects of these agents. However, it cannot completely be ruled out that small BP alterations have a pronounced effect on sympathetic activity, thereby influencing postsynaptic mechanisms indirectly. In addition, the small reduction of cardiac mass (significant only for nitrendipine or nitrendipine plus captopril) could have altered local sympathetic activity in the heart. This is also a hypothetical mechanism. However, similar ß-adrenoceptor changes were observed with captopril, which did not produce a reduction in cardiac mass. From these observations it is likely that a reduction of sympathetic activity by pharmacological treatment with antihypertensive drugs is able to upregulate ß-adrenoceptors, even when BP is not normalized and cardiac hypertrophy is not completely reversed.
To investigate whether the reduction in the number of ß-adrenoceptors is functionally relevant, we determined adenylyl cyclase activity. Isoproterenol-stimulated adenylyl cyclase activity was markedly reduced in SHR compared with WKY. An adenylyl cyclase desensitization in myocardial membranes from SHR has been observed previously by others14 15 16 and in our own laboratory.17 The ß-adrenergic adenylyl cyclase desensitization was accompanied by reduced positive inotropic effects of ß-adrenoceptor agonists.17 33 However, it is noteworthy that the decline in the isoproterenol-stimulated adenylyl cyclase activity was more pronounced than the reduction of ß-adrenoceptors. This finding indicates changes of postreceptor events (see Böhm et al48 ). Since the numbers of ß-adrenoceptors were upregulated after pharmacological treatment, one might suggest that ß-adrenoceptor–stimulated adenylyl cyclase would also recover. Indeed, adenylyl cyclase activity was increased in SHR after drug treatment, the effect being more pronounced with captopril or nitrendipine. In WKY after treatment with nitrendipine plus captopril, isoproterenol-stimulated adenylyl cyclase was not significantly altered. Thus, the findings in studies on adenylyl cyclase closely correspond to those obtained in ß-adrenoceptor studies.
Neuropeptide Y levels were higher in WKY than in SHR but were observed to be reduced in WKY after drug treatment. This observation could reflect an increase in myocardial sympathetic activity. However, the number of ß-adrenoceptors or amount of isoproterenol-stimulated adenylyl cyclase activity did not change in treated WKY compared with nontreated rats. This finding could indicate that the postsynaptic ß-adrenoceptor–adenylyl cyclase system is relatively insensitive to slight or moderate stimulations of sympathetic nerve terminals in WKY. Consistently, a reduction but not normalization of neuropeptide Y in SHR led to a complete recovery of myocardial ß-adrenergic receptors. Therefore, these findings provide evidence that in rat myocardium ß-adrenoceptors and ß-adrenoceptor–mediated responses are reduced only when a marked sympathetic stimulation occurs. In this respect, it is interesting to note that in SHR the downregulation of ß-adrenoceptors is much less pronounced14 15 17 31 32 34 47 than in the failing human heart.5 6 7 In addition, in several other rat models of hypertension, no ß-adrenoceptor reduction occurs although adenylyl cyclase is markedly desensitized.20 21 30 46 47 These observations favor the notion that ß-adrenoceptors in rat models of hypertensive cardiac hypertrophy are relatively resistant to sympathetic stimulation.
The angiotensin-converting enzyme inhibitor captopril was used to inhibit sympathetic activation, which facilitates norepinephrine release from sympathetic terminals,49 by reducing angiotensin II effects on presynaptic receptors. With this agent, the effects on neuropeptide Y levels and postsynaptic changes can be well explained. However, nitrendipine was used as an agent with selective effects on vascular smooth muscle. We expected no effects on sympathetic activity at this low dose. However, as with the other agents, there was evidence that nitrendipine also reduces sympathetic activity and thus could allow postsynaptic events to recover. At present, the mechanism of this effect remains unclear. However, one previous report observed evidence for a reduction of neurohumoral activation as judged by the decline of atrial natriuretic factor levels in SHR on nitrendipine treatment.50 Thus, effects of antihypertensive agents on sympathetic neuroeffector mechanisms must be investigated for each drug.
In summary, neuropeptide Y levels are markedly increased in the serum and strongly reduced in the myocardium of SHR. Thus, an activation of the sympathetic nervous system and in particular of myocardial sympathetic nerves occurs in SHR. Correspondingly, the number of ß-adrenoceptors and amount of isoproterenol-stimulated adenylyl cyclase activity was reduced in SHR. Treatment of SHR reduced peripheral neuropeptide Y levels, increased ß-adrenoceptors, and restored isoproterenol-stimulated adenylyl cyclase activity even at doses that did not normalize BP or reverse myocardial hypertrophy. Since ß-adrenoceptor downregulation and adenylyl cyclase desensitization are alterations that occur already in myocardial hypertrophy before the development of heart failure, they could contribute to the development of myocardial failure in later stages. These alterations can be favorably influenced by treatment with antihypertensive agents.
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Acknowledgments |
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This 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 18, 1994; accepted December 14, 1994.
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