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1 Laboratoire Réponses cellulaires et fonctionnelles à l'hypoxie, Association pour la Recherche en Physiologie de l'Environuement, Faculté de Médecine, Université Paris XIII, 93017 Bobigny, France; 2 Departamento de Ciencias Fisiológicas/Instituto de Investigaciones de la Altura (IIA),Universidad Peruana Cayetano Heredia, Lima 100, Perú; 3 Institut National de la Santé et de la Recherche Médicale Unité 400 and 4 Unité 99, 94010 Créteil, France
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Norepinephrine (NE)-induced
desensitization of the adrenergic receptor pathway may mimic the
effects of hypoxia on cardiac adrenoceptors. The mechanisms involved in
this desensitization were evaluated in male Wistar rats kept in a
hypobaric chamber (380 Torr) and in rats infused with NE (0.3 mg · kg
1 · h1) for 21 days.
Because NE treatment resulted in left ventricular (LV) hypertrophy,
whereas hypoxia resulted in right (RV) hypertrophy, the selective
hypertrophic response of hypoxia and NE was also evaluated. In hypoxia,
1-adrenergic receptors (AR) density increased by 35%,
only in the LV. In NE, 1-AR density decreased by 43% in
the RV. Both hypoxia and NE decreased 
-AR density. No difference was
found in receptor apparent affinity. Stimulated maximal activity of
adenylate cyclase decreased in both ventricles with hypoxia (LV, 41%;
RV, 36%) but only in LV with NE infusion (42%). The functional
activities of Gi and Gs proteins in cardiac
membranes were assessed by incubation with pertussis toxin (PT) and
cholera toxin (CT). PT had an important effect in abolishing the
decrease in isoproterenol-induced stimulation of adenylate cyclase in
hypoxia; however, pretreatment of the NE ventricle cells with PT failed to restore this stimulation. Although CT attenuates the basal activity
of adenylate cyclase in the RV and the isoproterenol-stimulated activity in the LV, pretreatment of NE or hypoxic cardiac membranes with CT has a less clear effect on the adenylate cyclase pathway. The
present study has demonstrated that 1) NE does not mimic the effects of hypoxia at the cellular level, i.e., hypoxia has specific effects on cardiac adrenergic signaling, and 2) changes in
- and -adrenergic pathways are chamber specific and may depend on
the type of stimulation (hypoxia or adrenergic).
adrenergic receptors; adenylate cyclase; protein kinase C; G proteins; ventricular hypertrophy
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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CHRONIC HYPOXIA
INDUCES an overall sympathetic stimulation that is reflected in
elevated plasma and urine catecholamine concentrations . The
stimulation of the adrenergic system induces a progressive blunting of
the heart chronotropic response to isoproterenol. This process produces
subsequent cardiovascular adaptations to offset a global decrease in
tissue oxygen supply (21, 23, 30, 32). These modifications
can be related, in part, to alterations in -adrenergic receptors
(-AR) signal transduction. -AR are coupled with adenylate cyclase
through guanine nucleotide binding proteins (G proteins). Activation of
-AR leads to an increased cAMP production by adenylate cyclase. In
intact animal models of chronic hypoxia, a decrease in -AR density
has been observed (12, 18, 20, 34). At the G protein
level, Gs activity was found to be decreased in both
ventricles and Gi-2, the inhibitory protein of adenylate
cyclase, increased only in the right ventricule (RV). No change was
observed in Gs mRNA levels, but an increase in
Gi-2 mRNA has been found in the RV (12).
Hypoxia-induced changes in 1-adrenergic receptors
(1-AR) have mainly been studied in vitro during acute
episodes. An enhanced inositol triphosphate response to
1-AR stimulation and a decrease in the receptor affinity
were shown to occur in cardiocytes over short periods of exposure to
hypoxia (8, 13). With a longer duration of hypoxia,
cardiac myocytes showed a differential regulation of the various
1-AR subtypes (19). Previous studies
(6, 19) have reported alterations of both - and
1-receptors in compensatory cardiac hypertrophy previous
to chronic cardiac failure. Chronic hypoxia imposes an additional load
to the right heart, which leads to a RV hypertrophy secondary to
pulmonary hypertension (28); therefore, it might be
expected that the association of both hypoxia and hypertrophy would
further alter the regulation of the receptor system.
Norepinephrine (NE), the major neurotransmitter released by sympathetic
nerves, produces positive inotropic responses. It has been proposed
that the effects of hypoxia on cardiac adrenoceptors may be mediated
indirectly, by a desensitizing effect of increased NE
(30), due, in part, to an impaired uptake-1
(22). Besides, chronic administration of NE
induces an LV hypertrophy and reduces -receptor coupling to the
contractile response without substantially compromising ventricular
function (26). Studies in rat heart muscle cells and
studies, where animals were exposed to increased catecholamine levels
for 7 to 14 days, led to a reduced sensitivity to -agonists and to a
decreased -AR density. NE-induced desensitization progresses from a
homologous to a heterologous form with increased dose and time of
exposure to NE. Events distal to the -AR in the adenylate cyclase
cascade are also affected (4, 5, 7, 29). -AR density
has also been found downregulated, and the adenylate cyclase response
has been found desensitized or unchanged in in vitro models of
catecholamine incubation in neonatal rat cardiac myocytes (7, 14,
15). Chronic infusion of isoproterenol, which also
results in myocardial hypertrophy in mice, has been shown to decrease
the adenylate cyclase mRNA levels for both isoforms in the heart (type
V and VI; Ref. 16).
It has been suggested that the effects of hypoxia on cardiac
adrenoceptors may be mediated, in part, by a desensitizing effect of
increased catecholamines (12, 30). In fact, AR regulation may result from both elevated catecholamine levels and hypoxia itself.
To investigate the possibility that agonist-induced desensitization of
the AR pathway can mimic the effects of hypoxia on cardiac adrenoceptors, we tested the effects of prolonged infusion of rats with
NE on resting heart rate (HR) and cardiac response to isoproterenol, as
well as on the characteristics of - and 1-AR and on
their effector enzymes. Furthermore, the selective hypertrophic response of hypoxia and NE enabled us to observe the characteristics of
cardiac adrenoceptor pathways from hypoxia-induced right hypertrophied ventricles and NE-induced left hypertrophied ventricles.
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MATERIAL AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals.
Male Wistar rats (200-250 g) were separated in two normoxic (NX),
two hypoxic (HX), and two NE groups. One group of each type was used
for the measurement of HR and the response to isoproterenol (
HRIso;
n = 7 in each group), as well as for AR binding (NX, HX, and NE, n = 7). The other groups (n = 7 in each group) were used for adenylate cyclase studies. HX rats
were kept on a 12:12-h light-dark cycle (room temperature, 23 ± 2°C) with free access to food and water. The chamber was brought to
normobaria for 30-40 min 3 times/wk for cleaning and food and
water replacing. They were exposed to a 5,500-m simulated altitude (380 Torr) for 21 days. After the 3-wk exposure to hypoxia, the animals were
killed by cervical dislocation and the hearts were quickly removed and dissected free of fat and large vessels. The ventricles were separated from the atria. The wet weights of the combined LV plus septum and of
the RV were determined and rapidly put into liquid nitrogen. They were
then immediately placed at 
70°C until use. All procedures were
performed in agreement with the local rules and with the regulation of
the French "Ministère de l'Agriculture" for animal care.
Surgical procedures.
Male Wistar rats were obtained from Charles River of France.
(-)NE HCl was infused at a rate of 0.3 mg · kg1 · h1 from an Alzet
minipump (model 2ML4) for a period of 21 days. The minipump was filled
with NE HCl with 0.2% ascorbic acid dissolved in isotonic saline. NE
was prepared on the day of implantation. Telemetry measurements (HR)
were obtained by a small transmitting sensor (TA 10-EA F40, Data
Sciences). A small incision was made in the intrascapular region for
implanting the minipumps and in the peritoneum for implanting the
transmitting sensors. Both incisions were made subcutaneously under
pentobarbital sodium anesthesia (6 g/100 ml ip). Fifteen rats were
operated in two different periods; the controls were not sham operated
considering that the preliminary in vivo HR measurements in
sham-operated rats had shown no differences between operated and
controls. None of the rats used in this study showed evidence of
infections at the site of the operation. Body weights were measured
before implantation of the osmotic minipumps and at days 11 and 21 after implantation.
HR and response to isoproterenol.
Resting HR and HRIso were measured in unanesthetized rats. HRIso
is defined as: HRIso = HRIso-resting HR. The signal was sent by
telemetry to a receiver placed under the cage. The average of ~150
readings recorded at the same hour each day was taken as the resting
HR. Data were collected and analyzed using DATAQUEST III data
acquisition system. For the HRIso determinations, the animals were
injected with intraperitoneal isoproterenol (0.05 mg/kg) to obtain an
HR increase of ~40%. This measurement was made only once at
day 19 of hypoxia and of NE infusion.
Preparation of membrane samples.
Preparation of membrane samples from ventricles was performed according
to the method of Baker et al. (1) with a minor modification. Briefly, the ventricles were minced and homogenized in 10 vol of ice-cold buffer (buffer A: 30 mM Tris · HCl,
100 mM NaCl, 5 mM MgCl2, 1mM EGTA, 500 µg/ml trypsin
inhibitor, and 100 µg/ml bacitracin; pH 7.5) with a polytron tissue
homogenizer (6 × 5-s bursts). The suspension was diluted with an
equal volume of ice-cold buffer A and centrifuged at 1,000 g for 10 min. Soon after, the supernatant was centrifuged
(45,000 g × 45 min × 2 times) in 4 vol of
ice-cold buffer A. The final pellet was resuspended in 3 vol
of incubation buffer (50 mM Tris · HCl, 5 mM MgCl2,
pH 7.5) and placed at 70 °C until use. The protein content was
adjusted to the convenient concentration the day of each assay
(2).
1- and 1-AR binding.
The radioligand [3H]prazosin was used to label myocardial
1-AR binding sites, and [3H]CGP-12177 was
used to label -AR. The concentration of [3H]prazosin
ranged from 0.02 to 1 nM, and the concentration of [3H]CGP-12177 ranged from 10 to 250 pM. Unlabeled
prazosin (1 µM) and propanolol (104 M) were added to
determine nonspecific binding. In displacement experiments,
[3H]prazosin concentration was 0.25 nM, corresponding to
about twice the receptor apparent affinity (Kd)
values of prazosin found in saturation binding experiments.
Displacement of [3H]prazosin binding by NE was performed
using 12 concentrations (1 nM-1 mM) of the agonist (with 0.1% ascorbic acid).
1-AR)
and at 37°C (-AR) in the incubation buffer (final vol: 250 µl).
Incubation was terminated by rapid vacuum filtration (Scatron) through
adequate filters (1-µm retention; 102 mm length, 256 mm width). The
tritiation plaques were rinsed 10 times with ice-cold incubation
buffer. The radioactivity retained on the filters was determined by
liquid scintillation spectrometry. The binding assays were carried out in triplicate; 10 points were used in each case. Nonspecific binding averaged 7% of total binding.
Adenylate cyclase assay.
Adenylate cyclase activity was determined in cardiac membranes
according to Johnson et al. (10) with minor modifications as previously described (27). The membrane preparations
were incubated (25 µg proteins) in a final volume of 60 µl of
reaction buffer (50 mM Tris · HCl, pH 7.6; 5 mM
MgCl2; 1 mM EDTA, an ATP-regenerating system consisting of
1 mg/ml creatine kinase and 1 mM phosphocreatine; and 1 mM ATP). A
concentration of 1 mM cAMP was used to quench phosphodiesterase
activities. Amounts of -[32P]ATP
[106 counts/min (cpm)/reaction, specific activity 30 Ci · min1 · mol1] were
included to give a specific radioactivity of the incubation cocktail of
40 cpm/pmol ATP. The [32P]cAMP synthetized was recovered
by chromatography on an aluminia column. Radiolabeled
[3H]cAMP (20,000 cpm/assay, specific activity 20-30
Ci · min1 · mol1) was
included to monitor the recovery of each chromatography elution.
Pertussis and cholera toxin-catalyzed
ADP-ribosylation.
Pertussis toxin (PT) catalyzes ADP-ribosylation of the -subunit of
Go and Gi proteins, which act as negative
regulators of adenylate cyclases. The modified Gi protein
uncoupled from the receptor and the cyclase. Cholera toxin (CT)
catalytic subunit by ribosylation of Gs irreversibly
activates all Gs proteins mediating the stimulation of the
adenylate cyclases. The effects of PT and CT on the basal and
isoproterenol-stimulated adenylate cyclase activities of cardiac
membranes were determined according to Sethi et al. (33).
The membrane preparations (3 mg/ml) were incubated with or without
toxin in a final volume of 100 µl for 60 min at 30°C in a
preincubation reaction mixture before the adenylate cyclase assay. The
preincubation reaction mixture consisted of 50 mM Tris · HCl
(pH, 7.6) containing 1 mM EDTA, 1 mM EGTA, 5 mM MgCl2, 5 mM
dithiothreitol (DTT), 1 mM ATP, 0.1 mM GTP, 10 mM thymidine, and 1 mM
NAD. Activated PT and CT in the preincubation mixture were 10 and 30 µg/ml, respectively.
Data analysis. Radioligand binding data were analyzed with Ligand, a weighted, nonlinear, least-square curve fitting computer program (24). For saturation experiments, equilibrium dissociation constants (Kd) and maximum numbers of binding sites were determined by nonlinear regression fitting. Displacement data were first fitted to a one- and then to a two-site model. The statistical differences between one- or two-site models were determined by comparing the residual variance between the actual and predicted data points, and F test analysis was used by the Ligand program to decide whether a model of one- or two-binding site fit was more appropriate. When the P value was >0.05, the one-site model was considered as the best fit. Even if the Hill parameter was always lower than 1, Ligand was not able to fit a two-binding fit model.
Statistical analysis. One-way ANOVA (followed by a Tukey's posttest) was used to assess the statistical significance between mean values. The effects of isoproterenol on HR were analyzed by the paired t-test because values were obtained from each animal both before and after the isoproterenol administration. The effect of PT and CT before and after membrane treatment was also analyzed by the paired t-test. P < 0.05 is considered statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Physiological data.
With NE-infusion at day 21, the animals failed to gain
weight, whereas with hypoxia, they gained weight but less than the control group (NX, 480 g ± 38 SD; HX, 451 ± 29; NE,
375 ± 9; P < 0.05). In regard to the ventricular
weight, chronic NE treatment resulted in LV hypertrophy, whereas
hypoxia resulted in RV hypertrophy as assessed by the ratio of LV and
RV wet weight to body weight (Table 1).
No significant change was found between the wet weight of the RV of the
NE group when compared with controls.
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-receptor pathway (4, 12, 20, 21). Our NE-infused and
HX-exposed animals showed as well a clearly desensitized response to
acute administration of isoproterenol (Fig.
1). In the NX rats, the HRIso increase
at min 2 of injection was 166 ± 8 bpm. In contrast,
HRIso response of NE and HX rats was significantly lower (NE,
84 ± 11 bpm; HX, 87 ± 10 bpm; P < 0.0001 vs. NX).
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Density, affinity, and distribution of cardiac adrenoceptors.
With NE infusion, no change was found on the density of
1-AR in the LV, whereas with hypoxic exposure the
density of 1-AR was increased by 35% (P < 0.05). Conversely, in the RV of the NE group, 1-AR
density was decreased by 43% (P < 0.05), but no changes were found in the HX group. No significant difference was found
in the Kd of 1A-AR among NX, HX,
or NE rats. It is worthy mentioning that the upregulation of
1-AR has been found in our particular experimental
condition (low Kd), where we have most probable
targeted the 1A-AR (6, 19). In regard to
-AR, in NE a 65% decrease in density was found in the LV
(P < 0.001), whereas a 40% decrease was found in the
RV (P < 0.01). In hypoxia, a 17% decrease in density
was found in the LV and in the RV (P < 0.05). No
significant difference was found in the affinity of [3H]CGP-12177 for -AR among the NX, HX, or NE groups.
Data are shown in Table 2. In LV, NX, and
HX, displacement curves were superimposed, while in RV there was a
rightward shift of the HX displacement curves compared with NX. Some
displacement curves of [3H]prazosin with NE displayed a
biphasic behavior for [3H]prazosin binding sites with
pseudo-Hill coefficients less than 1.0, indicating the existence of two
different affinity sites for the agonist (LV: HX, 0.86; NX, 0.78.
RV: HX, 0.55: NX, 0.77). However, as we were not able to find any
statistical difference between one- and two-site affinity models, the
data were treated as a one-site model. Hypoxia did not modify the NE
calculated dissociation constant in the LV but significantly increased
it in the RV (LV: HX, 3.4 µM ± 1.23; NX, 2.7 ± 0.55. RV:
HX, 9.6 ± 1.95: NX, 4.4 ± 1.36).
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Adenylate cyclase activity.
Figure 2 summarizes the results of
adenylate cyclase activity in membranes prepared from the hearts of NE
and HX rats. Basal activity of adenylate cyclase
(pmol · mg1 · min1) was
decreased by NE in the LV (35%) and by hypoxia in the RV (45%). There
was a significant decrease in maximal activity of adenylate cyclase
with isoproterenol stimulation only in the LV with NE infusion (42%),
whereas with hypoxia, both ventricles show a decrease in this parameter
(LV, 41%; RV, 36%). Furthermore, there was a significant impairment
of activity of adenylate cyclase with NaF and FRK stimulation again in
both ventricles with hypoxia but only in the hypertrophied ventricle
with NE infusion. It is worthy to emphasize that there was a decrease
in adenylate cyclase activity in the nonhypertrophied ventricles (in
all situations of stimulation) only in the HX group. When NE and
hypoxic hypertrophied ventricles were compared, there was a significant
decrease in maximal activity of adenylate cyclase with isoproterenol,
NaF, and FRK stimulation in both groups. However, the HX group
presented a greater decrease in adenylate cyclase activity for all the
conditions of stimulation.
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PT- and CT-catalized
ADP-ribosylation of G proteins.
The functional activities of Gi and Gs proteins
in cardiac membranes were assessed by incubation with PT and CT. Figure
3 shows the basal and
isoproterenol-stimulated adenylate activity of treated membranes. If
there is an increase of Gi, pretreatment of ventricle cells
with PT should have a considerable effect in abolishing the decrease in
isoproterenol-induced stimulation of adenylate cyclase. Incubation of
membranes of the LV with PT resulted in a greater basal adenylate
activity in NE-infused animals when compared with the respective
control group (P < 0.01). An increase was also
observed in the HX-exposed group, but this change was considerably
higher than that of NE-infused rats (P < 0.05).
Isoproterenol-stimulated adenylate activity was only increased in the
HX group (P < 0.05). As CT activates all
Gs proteins mediating the stimulation of the adenylate
cyclases, a diminution of adenylate activity in pretreated cells is an
indication of decreased levels of Gs. In LV, CT decreases isoproterenol-adenylate cyclase activity in hypoxia as expected (12). Incubation of the RV membranes with CT decreased the
basal activity in the NE and HX groups (P < 0.05) but
did not produce any change in the isoproterenol-stimulated adenylate
activity neither in the NE nor in the HX group.
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1- and -AR by NE infusion and chronic hypoxia. This
table describes as well a differential regulation of adenylate cyclase
and proteins Gi and Gs. Chronic adrenergic
stimulation (NE-RV) does not affect adenylate cyclase sensitivity or
functional activity of the G proteins, whereas hypoxic stimulation
(HX-LV) produces an attenuated sensitivity of adenylate cyclase to
hormone stimulation, an augmentation of Gi functional
activity, and a decrease of Gs functional activity. This
table also shows that changes in -AR, -AR, and adrenergic pathway
and signaling converge in someway when both ventricles are exposed to
hypertrophy (NE-LV and HX-RV).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This work answers for the first time to the question of whether
the cardiac changes observed during exposure to chronic hypoxia are
secondary to the adrenergic stimulus, or whether they are directly
related to the hypoxic stress. For this purpose, we analyzed the
changes in the transduction pathway of the 1-adenylate
cyclase cascade and in the 1-AR in animals subjected to
hypoxic and/or adrenergic stimulation. Besides, the modifications
observed in hypertrophy secondary to chronic hypoxia and chronic NE
infusion were compared. Both treatments proved to provoke the same
magnitude of decrease in HR response to isoproterenol. Our hypothesis
was that hypoxia would mimic adrenergic activation of the
1- and -pathways. The present results demonstrate
that in contrast with rats exposed to chronic hypoxia, which shows a
differential regulation of 1- and -AR in the LV, rats
exposed to NE infusion show only a -AR downregulation. In the
hypertrophied RV, hypoxic exposure decreased only the -AR density,
but NE infusion decreased both 1-AR and -AR density.
This work also describes a differential regulation of adenylate
cyclase. Rats exposed to hypoxia showed an attenuated sensitivity of
adenylate cyclase to hormone stimulation, whereas chronic adrenergic
stimulation (when not associated to hypertrophy, i.e., RV) did not
affect adenylate cyclase sensitivity. In NE-infused rats, changes in G
proteins do not parallel those observed in rats exposed to hypoxia
(Table 3). Even if the attenuated responses to acutely administered
isoproterenol confirm that both conditions cause a desensitization of
the adrenergic system, the differential results at the level of basal
heart rate (higher in the NE group) indicate that in vivo hypoxia has
also some specific effects. The regulation of adrenergic receptors in
hypoxia may result from the combination of both elevated catecholamine
levels and hypoxia itself.
One of the advantages of the chronic hypoxia model is that it
allows the comparison between LV, exposed to hypoxia and adrenergic stimulation (HX-LV), and RV, which is additionally subjected to pressure overload due to pulmonary hypertension (HX-RV; Refs. 9, 28). The model of chronic NE infusion
allows the comparison between RV (NE-RV), exposed to adrenergic
stimulation only, and LV, subjected to both adrenergic stimulation and
systemic hypertension (NE-LV). We have used a large dose of NE to avoid
the compensation by the regulatory capacity of the system. Chronic
treatment with NE caused LV (21%) but not RV hypertrophy. This level
of hypertrophy is comparable to that observed in other studies in which
similar doses of NE were used, but this response may have been
influenced by an enhanced systemic catabolic state, as demonstrated by
a decrease in weight gain (3, 17). This selective effect
suggests that the hypertrophic stimulus may be an increase in afterload produced by 1-AR-mediated vasoconstriction and/or
-AR-mediated increase in myocardial contractility. RV hypertrophy
caused by chronic hypoxia is due mainly to pulmonary hypertension
secondary to vasoconstriction and remodeling of the pulmonary arteries. LV hypertrophy is absent in chronic hypoxia, despite the elevation in
catecholamine level; thus RV hypertrophy seems not to be related to
high levels of NE. Although we have not measured catecholamine augmentation and blood pressure in our animals, there is a general agreement that plasma or urinary NE is elevated in prolonged hypoxia, as shown in humans staying for more than 1 wk at high altitude (32), and these findings are compatible with increased
sympathetic activity. At the dose of NE we presently used, an increase
in systemic pressure has been found (125-155 mmHg), which is
compatible with the LV hypertrophy found in this study (17,
36).
In HX animals, the 1-AR affinity for NE in the RV was
significantly decreased, and the magnitude of decrease in
1- and -AR density was higher with NE infusion
compared with chronic hypoxia, both in RV and LV. Thus NE may be
responsible, in part, for this reduction in intact animals, at least at
the receptor level. Our observations on 1- and -AR
differ from previous reports in cell culture models, most likely
because hypoxia-induced changes in 1- and
1-AR have been mainly studied in vitro and during acute episodes (7, 19, 31, 36). In contrast, little information is presently available regarding the regulation of these receptors during prolonged hypoxia in vivo . Thus (12, 34) caution
should be taken in extrapolating in vitro models with in vivo chronic models, taking into account that in intact animals, circulating catecholamine levels are elevated in response to hypoxia
(32).
In 21 days of hypoxia, along with the decrease in -AR density, the
catalytic unit of adenylate cyclase was desensitized, showing a
depressed response to the activators tested. However, the magnitude of
the depression of response to FSK in LV was less (21%) than that to
isoproterenol (33%), suggesting not only an uncoupling of -AR but
also a change distal to the receptor. In RV of rats exposed to NE
infusion, the response to the activators was not depressed, and there
was an insignificant change in the responses in both conditions,
suggesting that with respect to adenylate cyclase activity, hypoxia
does not mimic adrenergic activation. In contrast, in the hypertrophied
RV in hypoxia, the depression of the response to FSK was greater (13%)
than that to isoproterenol (1%), suggesting a decrease in the content
of the enzyme itself. In the hypertrophied LV of rats infused with NE,
there was a depression in the response to isoproterenol (9%); however,
no depression was found when compared with the response to FSK. In
fact, only a 42% decrease in the NE group was found in adenylate
cyclase maximal activity stimulated by forskolin when expressed per LV,
but a 73% decrease in the HX group when expressed per RV. Thus the
adenylate cyclase decrease seems to be more dependent on the degree of
hypoxic hypertrophy rather than adrenergic hypertrophy.
In chronic hypoxia, we have observed a decrease in the responsiveness
to NaF and to isoproterenol stimulation of adenylate cyclase activity.
In fact, pretreatment of hypoxic cardiac membranes with PT, which
functionally inactivates Gi proteins, not only restored but
increased the isoproterenol-induced stimulation of adenylate cyclase in
the LV. This strongly suggests an increased activation or level of
Gi proteins in desensitized membranes. Several studies
(11, 29) support the hypothesis that the regulation of the
quantity of Gi proteins may be a general regulatory
mechanism for sensitization and desensitization of adenylate cyclase at the postreceptor level. Additionally, increased activity of
Gi could result in reduced levels of cAMP, which might be
at the origin of the observed desensitization of adenylate cyclase in hypoxia (25). On the other hand, although NE infusion is
associated with a downregulation of -AR in several animal models
(4, 5, 7, 17), pretreatment of the NE ventricle cells with PT failed to restore the isoproterenol stimulation of adenylate cyclase. Our results suggest either that -AR are less coupled to
adenylate cyclase in the NE-stimulated heart or that other adenylate
cyclase regulators are more relevant in this model. Pretreatment of NE
or hypoxic cardiac membranes with CT, which functionally activates
Gs, has less clear effect on the adenylate cyclase pathway.
Although pretreatment with CT attenuated the basal activity of
adenylate cyclase in the RV and the isoproterenol-stimulated activity
in the LV, these results are in line with the less prominent role of
Gs compared with Gi in the desensitization of
-AR (11, 29). In chronically failing human
hearts, 2-AR stimulation also induces positive inotropic
and lusitropic effects and phosphorylation of regulatory proteins.
Inhibition of Gi proteins by PT causes 2-AR
to closely resemble that of 1-AR (35).
Whether the increased level of Gi in HX animals modifies
also the 2-AR/Gi coupling awaits for further study.
In conclusion, we have shown that NE does not mimic the effects of
hypoxia at the cellular level, i.e., that hypoxia has specific effects
on the transduction pathway of the 1-adenylate cyclase cascade and on 1-AR. These results also show that
changes in - and -adrenergic pathways are chamber specific. The
distinct effects of hypoxia and adrenergic stimulation seem to be due
to differential responses of the ventricles rather than various degrees of desensitization. On the contrary, the effects of hypertrophy produced by hypoxia and by adrenergic stimulation are more probably due
to different degrees of desensitization rather than differential responses of the ventricles. These distinct responses cannot be explained only on the basis of generalized changes in the hormonal profile such as increased levels of blood catecholamines in chronic hypoxia. Thus the differential changes in signal transduction in
LV and RV suggest that the regional-specific modifications in
signal transduction in the heart may be produced by differences in
local conditions such as hemodynamic or hormonal state and/or adrenergic nerve activity.
Perspectives
At high altitude and in experimental chronic hypoxia, a desensitization (blunting) of the adrenergic system and a sensitization of the cholinergic system occur. These changes are present even in animals genetically adapted to life at high altitudes, like guinea pigs, and could contribute to succesful adaptive processes in these animals. Blunting of the chronotropic response to hypoxia, mediated by-AR and M2 cholinergic receptors, may be one of the strategies to protect the myocardium. Nevertheless, these kinds of
changes have also been found in heart failure. In addition to the
interaction between these receptors, an underlying interaction between
other types of autonomic receptors might be involved in the regulation
of various cardiac functions in hypoxia. Our results show that, besides
the contribution of the adrenergic system (via 1-AR,
1-AR, A1-adenosinergic receptors, and the
M2-receptor systems) to ventricular function at high
altitude, hypoxia itself is involved in the cardiovascular changes
observed in our model. Exposure to prolonged hypoxia is a useful model
to realize how the heart adapts to a stressful environment. Hopefully,
the present study will help to understand a rather little known angle
of cardiac physiology, i.e., the interaction between different types of
autonomic receptors and signaling between both ventricles in conditions of chronic hypoxia. In addition to a better knowledge of the
physiological adaptation to hypoxia, these models may help to
understand other physiological conditions, particularly ischemia,
because in hypoxia, oxygen supply is lower than the myocardial needs.
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ACKNOWLEDGEMENTS |
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We greatly acknowledge Ana Maria Rath for implanting the transmitting sensors, Gregoire Molinatti for invaluable help, and Lucien Sambin for the technical assistance with animals.
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FOOTNOTES |
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Address for reprint requests and other correspondence: F. León-Velarde, Laboratoire de Physiologie, ARPE/UFR de Médecine, 74 rue Marcel Cachin. Université Paris XIII, 93017 Bobigny, France (E-mail: richalet{at}smbh.univ-paris13.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 January 2000; accepted in final form 13 September 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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) and
norepinephrine (NE) infusion (
), and on normoxic
animals (NX; 
) in response to isoproterenol (Iso). bpm,
Beats/min. P < 0.05 HX, NE, vs. normoxic (NX) for all
time points.
