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Am J Physiol Regul Integr Comp Physiol 280: R274-R281, 2001;
0363-6119/01 $5.00
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Vol. 280, Issue 1, R274-R281, January 2001

Differential alterations in cardiac adrenergic signaling in chronic hypoxia or norepinephrine infusion

F. León-Velarde1,2,3, M.-C. Bourin4, R. Germack1, K. Mohammadi3, B. Crozatier3, and J.-P. Richalet1,3

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 · h-1) 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, alpha 1-adrenergic receptors (AR) density increased by 35%, only in the LV. In NE, alpha 1-AR density decreased by 43% in the RV. Both hypoxia and NE decreased beta -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 alpha - and beta -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
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DISCUSSION
REFERENCES

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 beta -adrenergic receptors (beta -AR) signal transduction. beta -AR are coupled with adenylate cyclase through guanine nucleotide binding proteins (G proteins). Activation of beta -AR leads to an increased cAMP production by adenylate cyclase. In intact animal models of chronic hypoxia, a decrease in beta -AR density has been observed (12, 18, 20, 34). At the G protein level, Galpha s activity was found to be decreased in both ventricles and Galpha i-2, the inhibitory protein of adenylate cyclase, increased only in the right ventricule (RV). No change was observed in Galpha s mRNA levels, but an increase in Galpha i-2 mRNA has been found in the RV (12).

Hypoxia-induced changes in alpha 1-adrenergic receptors (alpha 1-AR) have mainly been studied in vitro during acute episodes. An enhanced inositol triphosphate response to alpha 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 alpha 1-AR subtypes (19). Previous studies (6, 19) have reported alterations of both beta - and alpha 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 beta -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 beta -agonists and to a decreased beta -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 beta -AR in the adenylate cyclase cascade are also affected (4, 5, 7, 29). beta -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 beta - and alpha 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.


    MATERIAL AND METHODS
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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 (Delta 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 · kg-1 · h-1 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 Delta HRIso were measured in unanesthetized rats. Delta HRIso is defined as: Delta 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 Delta 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).

alpha 1- and beta 1-AR binding. The radioligand [3H]prazosin was used to label myocardial alpha 1-AR binding sites, and [3H]CGP-12177 was used to label beta -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 (10-4 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).

Triplicate of samples (100 µl; 60-75 µg) of the membrane preparations was incubated for 45 min at 25°C (alpha 1-AR) and at 37°C (beta -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 alpha -[32P]ATP [106 counts/min (cpm)/reaction, specific activity 30 Ci · min-1 · mol-1] 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 · min-1 · mol-1) was included to monitor the recovery of each chromatography elution.

After a 10-min incubation period in a shaking water bath at 37°C, the reaction was terminated by adding 200 µl of 0.5 N HCl followed by inmediate boiling for 6 min. The pH of the assay mixture was adjusted to 7.6 with 250 µl of 1.5 M imidazole-HCl. Samples were then eluted with 2 ml of 10 mM imidazole-HCl through an alumina column that retains [32P]ATP. The 3H and 32P activities of the eluate were then counted after addition of a scintillation cocktail. After incubation with [32P]ATP, the level of [32P]cAMP was measured. Basal adenylate cyclase activity as well as the activities after stimulation with 10 mM sodium fluoride (NaF) and 30 µM (-)isoproterenol in the presence of 10 µM GTP and 50 µM forskolin (FRK) were measured. All determinations were performed in triplicate and expressed as picomoles of cAMP synthesized per minute and milligram of protein.

Pertussis and cholera toxin-catalyzed ADP-ribosylation. Pertussis toxin (PT) catalyzes ADP-ribosylation of the alpha -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 Galpha s 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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ABSTRACT
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RESULTS
DISCUSSION
REFERENCES

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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  		Table 1.   BWs and LV and RV fresh weights and LV/BW and RV/BW in NX, HX, and NE-infused rats

NE infusion had no effect on HR during the first 4 days of exposure [360 ± 43 beats/min (bpm)], whereas the HX group increased its mean HR at the same period to 465 ± 25 bpm (P < 0.01). After a 7-day treatment NE rats started to raise their HRs, but HX animals greatly decreased their initial increased HR progressing toward the NX values at the end of exposure. The NE-infused animals presented the highest average mean HR for days 18-19 (417 ± 32 bpm; P < 0.05) when compared with HX (346 ± 13 bpm) and NX (342 ± 12 bpm) rats. NE and hypoxia produce desensitized responses to stimulation at various levels of the beta -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 Delta HRIso increase at min 2 of injection was 166 ± 8 bpm. In contrast, Delta 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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  		Fig. 1.   Change in heart rate (HR) from baseline (b) values on day 19 of chronic hypoxic (HX; ) and norepinephrine (NE) infusion (), and on normoxic animals (NX; open circle ) in response to isoproterenol (Iso). bpm, Beats/min. P < 0.05 HX, NE, vs. normoxic (NX) for all time points.

Density, affinity, and distribution of cardiac adrenoceptors. With NE infusion, no change was found on the density of alpha 1-AR in the LV, whereas with hypoxic exposure the density of alpha 1-AR was increased by 35% (P < 0.05). Conversely, in the RV of the NE group, alpha 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 alpha 1A-AR among NX, HX, or NE rats. It is worthy mentioning that the upregulation of alpha 1-AR has been found in our particular experimental condition (low Kd), where we have most probable targeted the alpha 1A-AR (6, 19). In regard to beta -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 beta -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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  		Table 2.   Effects of 21 days of hypoxia and NE infusion on alpha 1- and beta -AR density and affinity

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 · mg-1 · min-1) 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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  		Fig. 2.   Effect of chronic HX and NE infusion on adenylate cyclase (AC) activity stimulated by sodium fluoride (NaF, 10 mM), Iso 30 µM + 10 µM GTP, and forskolin (FRK, 50 µM) in left (LV; A) and right (RV; B) ventricles. AC activity was assayed in heart membranes of NX (open bars), HX (dotted bars), and NE-infused (lined bars) rats. Data are means ± SE for 5 separate experiments. *P < 0.05; **P < 0.001 for HX and NE vs. NX.

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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  		Fig. 3.   Basal (Base AC) and Iso-stimulated adenylate activity of pertussis toxin (PT)- and cholera toxin (CT)-treated membranes in LV (A) and RV (B). PT and CT were assayed in heart membranes of NX (open bars), HX (dotted bars), and NE-infused (lined bars) rats. C, control. Data are means ± SE for 5 separate experiments. *P < 0.05; **P < 0.01.

Table 3 summarizes the results of the study. It shows that there is a differential regulation of alpha 1- and beta -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 alpha -AR, beta -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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  		Table 3.   Differential regulation of alpha 1- and beta -adrenergic receptors and signaling by chronic HX and NE infusion


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta 1-adenylate cyclase cascade and in the alpha 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 alpha 1- and beta -pathways. The present results demonstrate that in contrast with rats exposed to chronic hypoxia, which shows a differential regulation of alpha 1- and beta -AR in the LV, rats exposed to NE infusion show only a beta -AR downregulation. In the hypertrophied RV, hypoxic exposure decreased only the beta -AR density, but NE infusion decreased both alpha 1-AR and beta -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 alpha 1-AR-mediated vasoconstriction and/or beta -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 alpha 1-AR affinity for NE in the RV was significantly decreased, and the magnitude of decrease in alpha 1- and beta -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 alpha 1- and beta -AR differ from previous reports in cell culture models, most likely because hypoxia-induced changes in alpha 1- and beta 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 beta -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 beta -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 Galpha i 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 beta -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 beta -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 beta -AR (11, 29). In chronically failing human hearts, beta 2-AR stimulation also induces positive inotropic and lusitropic effects and phosphorylation of regulatory proteins. Inhibition of Gi proteins by PT causes beta 2-AR to closely resemble that of beta 1-AR (35). Whether the increased level of Gi in HX animals modifies also the beta 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 beta 1-adenylate cyclase cascade and on alpha 1-AR. These results also show that changes in alpha - and beta -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 beta -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 beta 1-AR, alpha 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Regul Integr Comp Physiol 280(1):R274-R281
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