Impaired Gsalpha and adenylyl cyclase cause beta -adrenoceptor desensitization in chronically hypoxic rat hearts

Jian-Ming Pei1, Xiao-Chun Yu1, Man-Lung Fung1,2, Jing-Jun Zhou1, Chi-Sing Cheung1, Nai-Sum Wong3, Maurrice-Ping Leung2,4, and Tak-Ming Wong1,2

Departments of 1 Physiology, 3 Biochemistry, and 4 Pediatrics; and 2 Institute of Cardiovascular Sciences and Medicine, Faculty of Medicine, University of Hong Kong, Hong Kong, China


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of beta -adrenoceptor stimulation with isoproterenol on electrically induced contraction and intracellular calcium ([Ca2+]i) transient, and cAMP in myocytes from both hypertrophied right and nonhypertrophied left ventricles of rats exposed to 10% oxygen for 4 wk, were significantly attenuated. The increased [Ca2+]i transient in response to cholera toxin was abolished, whereas increased cAMP after NaF significantly attenuated. The biologically active isoform, Gsalpha -small (45 kDa), was reduced while the biologically inactive isoform, Gsalpha -large (52 kDa), increased. The increased electrically induced [Ca2+]i transient and cAMP with 10-100 µM forskolin were significantly attenuated in chronically hypoxic rats. The content of Gialpha 2, the predominant isoform of Gi protein in the heart, was unchanged. Results indicate that impaired functions of Gs protein and adenylyl cyclase cause beta -adrenoceptor desensitization. The impaired function of the Gs protein may be due to reduced Gsalpha -small and/or increased Gsalpha -large, which does not result from changes in Gi protein. Responses to all treatments were the same for right and left ventricles, indicating that the impaired cardiac functions are not secondary to cardiac hypertrophy.

G protein; cardiac hypertrophy


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CHRONIC HYPOXIA leads to cardiac hypertrophy and reduced contractility (32, 34, 35). It is well established that the responsiveness of cardiac contractility to beta -adrenoceptor stimulation is reduced in cardiac hypertrophy (3, 44, 47) and heart failure (16). beta -adrenoceptor desensitization has been shown to be due to downregulation of the beta -adrenoceptor (9, 19). The postreceptor events, which may also be responsible for beta -adrenoceptor desensitization, are not well understood. There is evidence that the function of the Gs protein, which mediates the action of beta -adrenoceptor stimulation (15, 20, 21, 30), is impaired in the hypertrophied heart (21). On the other hand, the Gs content was unchanged in the hypoxic rat heart (18, 21). There was no study on the changes of the two Gsalpha -subunits (14, 38) derived from a single gene by alternative splicing of mRNA (23), a biologically active Gsalpha -small of an apparent molecular weight of 45 kDa (14, 31, 49) and a Gsalpha -large of an apparent molecular weight of 52 kDa. The role of adenylyl cyclase (AC), the enzyme that is activated by the Gs protein and in turn converts ATP into cAMP, is also controversial. Cardiac hypertrophy was associated with reductions in cellular cAMP (36) and chronic hypoxia with impaired AC response to forskolin, an activator of AC (30). On the other hand, it has also been reported that there was no significant impairment of activity of AC with forskolin stimulation in the right hypertrophied heart after chronic hypoxia (22, 48). The Ca2+ influx via the L-type Ca2+ channel, which is enhanced upon beta -adrenoceptor stimulation via the Gs protein/AC/cAMP cascade, is unchanged in the hypertrophied heart (10, 37, 40).

In the present study, we delineated the postreceptor signaling mechanisms in the right and left hearts of rats exposed to low oxygen for 4 wk, which has previously been shown to induce right heart hypertrophy (32, 34, 35) and reduce cardiac contractility (19, 28). With the use of electrically stimulated twitch amplitude and intracellular calcium ([Ca2+]i) transient, and cAMP in isolated ventricular myocytes as parameters, we determined the changes in ventricular myocytes of chronically hypoxic and age-matched normoxic rats subjected to manipulations that activated beta -adrenoceptor, Gs protein, or AC. The electrically induced [Ca2+]i transient represents the influx of Ca2+ via the L-type Ca2+ channel upon electrical stimulation, which then triggers release of Ca2+ from the sarcoplasmic reticulum (SR), leading to a [Ca2+]i transient and muscle contraction. A previous study in our laboratory showed that, in a similar experimental condition, the electrically induced [Ca2+]i transient is directly related to contractility (51). We measured the two Gsalpha isoforms with the low and high molecular weights in the heart of normoxic and chronically hypoxic rats. We also measured Gialpha 2, which is the predominant isoform of Gialpha in the heart (12, 23). In addition, we compared the responses of both the right hypertrophied and left nonhypertrophied ventricles to determine the relationship between hypertrophy and impaired cardiac function. Results from this study showed that the functions of both Gs protein and AC were impaired after chronic hypoxia. The impaired function of Gs protein was due to a reduction in the biologically active Gsalpha -small. The responses in the right hypertrophied and left nonhypertrophied ventricles were the same, indicating that impaired cardiac function is not secondary to hypertrophy.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Chronic hypoxia. Male Sprague-Dawley rats that weighed 100-150 g at the start of the experiment were randomly divided into two groups. One group of the rats (n = 42) was exposed to chronic hypoxia, while the control (n = 40) was maintained in room air. All the rats were kept in the same room with the same light-dark cycle. For chronic hypoxia, rats were given inspired oxygen (10% O2) in a 300-liter acrylic chamber. The hypoxic environment was established with the inflow of a mixture of room air and nitrogen that was regulated by an oxygen analyzer (model 175518A, gold edition, Vacuum Med) (1, 37a, 39). Carbon dioxide was absorbed by soda lime granules, and excess humidity was removed by a dessicator. Temperature was maintained at 19-21°C. The chamber was opened twice a week for ~1 h to clean the cages and replenish food and water. Rats were exposed to hypoxia for 4 wk, and experiments were performed immediately after removal of the rats from the chamber. The rats were weighed and decapitated, and the hearts were quickly removed. The right heart of the rats exhibited hypertrophy (Table 1), in agreement with previous findings (32, 35). The PO2 of arterial blood in the chronically hypoxic rats was reduced by ~35% compared with control.

                              
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Table 1.   Body and heart weights in normoxic and chronically hypoxic rats

Isolation of ventricular myocytes. Ventricular myocytes were isolated from the hearts of male Sprague-Dawley rats using a collagenase perfusion method described previously (11). Immediately after decapitation, the hearts were rapidly removed from the rat and perfused in a retrograde manner at a constant flow rate (10 ml/min) with an oxygenated Joklik modified Eagle's medium supplemented with 1.25 mM CaCl2 and 10 mM HEPES at pH 7.2 at 37°C for 5 min. This was followed by 5-min perfusion with the same medium free of Ca2+. Collagenase was then added to the medium to a concentration of 125 U/ml with 0.1% (wt/vol) bovine serum albumin (BSA). After 35-45 min of perfusion with the medium containing collagenase, the atria were discarded. The right ventricle (RV) and left ventricle (LV) tissues were dissociated separately by shaking in the same oxygenated collagenase-free solution for 5 min at 37°C. Ventricular tissues were cut into small pieces with a pair of scissors followed by stirring with a glass rod for 5 min. The procedure separated the ventricular myocytes from each other. The residue was filtered through 250-µm mesh screens, sedimented by centrifugation at 100 g for 1 min, and resuspended in fresh Joklik solution with 1% BSA. More than 70% of the cells were rod shaped and impermeable to trypan blue. The Ca2+ concentration of the Joklik solution was increased gradually to 1.25 mM within 40 min.

Measurement of twitch amplitude of contraction. After isolation of myocytes for 2 h, measurement of myocyte twitch amplitude was performed with the use of an optical video system, as described previously (5, 50). The myocyte was placed in a perfusion chamber at room temperature under an inverted microscope (Nikon) and perfused at a rate of 2 ml/min with a Krebs bicarbonate buffer that contained (in mM) 117 NaCl, 5 KCl, 1.2 MgSO4, 1.2 KH2PO4, 1.25 CaCl2, 25 NaHCO3, and 11 glucose, with 1% dialyzed BSA and a gas phase of 95% O2-5% CO2, pH 7.4. The myocyte was field electrically stimulated at a rate of 0.2 Hz with platinum electrodes connected to a voltage stimulator. The twitch amplitude was measured with an automatic video analyzer developed in our laboratory (51). As a prerequisite for the proper operation of this automatic analyzer, the image of cardiac myocytes projected on the video camera (SSC-M370CE; Sony) and observed on the video monitor (PVM-145E; Sony) was first rotated to align horizontally and parallel to each of the video raster lines. This was achieved by interposing a kappa -mirror (or a dove prism) between the microscope eyepiece and the video camera. The video analyzer was interposed between the video camera and the video monitor, and it generated a positionable rectangular window that was observed on the video monitor together with the image of the cell. Light-dark contrast at the edge of the myocyte provided a marker for measurement of the amplitude of motion. The amplitude of the marker was directly proportional to the dark image of contraction, and the action was in real time. The traces of twitch amplitude were recorded with the use of a two-channel amplifier recorder system. The amplitude of myocyte motion remained unchanged for at least 10 min, indicating the stability of the preparation.

Measurement of [Ca2+]i. RV and LV myocytes were incubated with 5 µM fura 2-AM in Joklik solution supplemented with 1.25 mM CaCl2 for 30 min. The unincorporated dye was removed by washing the cells twice in fresh incubation solution. The loaded cells were kept at room temperature (25°C) for 30 min before measurement of [Ca2+]i to allow the fura 2-AM in the cytosol to deesterify. A low concentration of fura 2-AM at 5 µM was loaded at a relatively low temperature of 25°C to minimize the effects of compartmentalization of the esters (42).

The ventricular myocytes loaded with fura 2-AM were transferred to the stage of an inverted microscope (Nikon) in a superfusion chamber at room temperature. The inverted microscope was coupled with a dual-wavelength excitation spectrofluorometer (Photo Technical International, South Brunswick, NJ). The myocytes were superfused with a Krebs bicarbonate buffer. The myocytes selected for the study were rod shaped and quiescent with clear striations. They exhibited a synchronous contraction (twitch) in response to suprathreshold 4-ms stimuli at 0.2 Hz delivered by a stimulator (Grass S88) through two platinum field-stimulation electrodes in the bathing fluid. There is a transient rise of [Ca2+]i associated with each contraction of a cardiac muscle cell, and this is widely termed the "[Ca2+]i transient" (2). In many mammalian species, including the rat, the [Ca2+]i transient is mainly due to release of Ca2+ from the SR into the cytoplasm triggered by the Ca2+ influx via the L-type Ca2+ channel on membrane depolarization, and this rise of [Ca2+]i activates the myofilaments to slide and produce cell shortening (4). In the present study, we measured the amplitude of electrically induced [Ca2+]i transient. Fluorescent signals obtained at 340-nm (F340) and 380-nm (F380) excitation wavelengths were stored in a computer for data processing and analysis. The fluorescence ratio (F340/F380) was used to represent [Ca2+]i changes in the myocyte.

Assay of cAMP. The measurement of cAMP was performed according to the method described previously (6). Samples that contained 3 × 106-6 × 106 freshly isolated ventricular myocytes after 1.25 mM Ca2+ loading were incubated in an atmosphere of 5% CO2-95% air for 2 h. Isoproterenol (0.1-10 µM), NaF (1-10 mM), or forskolin (1-100 µM) was added and incubated for 10 min. At the end of treatment, the cells were centrifuged for 5 s at 100 g. The medium was aspirated, the sediment was resuspended in ice-cold Krebs solution, and the cells were centrifuged again for 5 s at 100 g. The supernatant was aspirated. Ice-cold ethanol-HCl (0.5 ml) was added, mixed, and left to stand for 5 min at room temperature. The supernatant was centrifuged at 3,000 g for 5 min and collected with a pipette. The precipitate was washed with 0.5 ml of ethanol-water (2:1), mixed, and centrifuged at 3,000 g for 5 min. The supernatant was also collected and added together. Finally, it was evaporated to dryness at 55°C under a stream of nitrogen. The sediment was stored at -20°C for assay of cAMP. The pellets were neutralized in 0.1 N NaOH for protein determination by the method of Lowry et al. (25), using BSA as a standard.

For determination of cAMP, a competitive binding assay with a kit from Amersham was used. Briefly, 50 µl of 0.5 M Tris (4 mM EDTA) was added to 50 µl of each sample on ice, followed by 50 µl of [3H]cAMP and 100 µl of binding protein. The samples were vortexed for 5 s, placed in an ice bath, and allowed to incubate for 2 h. Charcoal suspension of 100 µl was added. The samples were vortexed for 10 s again and centrifuged at 12,000 g for 2 min at 4°C. Samples of 200 µl supernatant were removed for scintillation counting.

Western blotting. In the present study, we measured the levels of Gs and Gi proteins in washed particulate membrane (WPM), which was prepared according to the method of Kumar et al. (23). To prepare WPM from isolated ventricular myocytes, the freshly isolated myocytes were centrifuged at 100 g, and the pellet was washed twice with a sucrose- Tris medium (0.25 M sucrose and 25 mM Tris · HCl, pH 7.5). The washed cell pellet was suspended and homogenized in a hypotonic membrane buffer (that contained 20 mM Tris · HCl, pH 7.5, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and protease inhibitor: 0.4 mM phenylmethylsulfonyl fluoride) with an Ultra Turrax homogenizer in three bursts (5 s each) at intervals of 30 s. The homogenate was then centrifuged at 100 g for 5 min to separate the unbroken myocytes, and the supernatant was centrifuged again at 40,000 g for 25 min. The pellet was then washed once with the membrane buffer (50 mM Tris · HCl, and 150 mM NaCl, pH 7.4), recentrifuged, and finally suspended in the membrane buffer before storage at -70°C for immunoblotting of specific Gs and Gi protein isoforms. Protein was determined by the method of Bradford (7) before using these fractions.

For immunoblotting of Gs and Gi proteins, SDS gel electrophoresis of polypeptides was performed on 12% polyacrylamide gels prepared according to the method of Laemmli (24). Twenty to fifty microliters of samples, each containing 100 µg protein, was added with the same amount of the sample loading buffer (the reducing buffer), which was heated for 5 min at 95°C. The solution was added into a single lane of the gel. After gel running, electrophoresis was performed, during which polypeptides were transferred onto a nitrocellulose membrane. The membrane was then washed with a Tris buffer solution (TBS) and incubated with TBS that contained 5% nonfat dry milk (blocking buffer), a procedure that blocks the nonspecific protein binding sites on nitrocellulose. A Gs protein antibody that recognizes both the large and small isoforms of Gsalpha (26) and a Gi (Gialpha 2) antibody (31) were used for immunoblotting. The Gs and Gi protein antibodies at 1:1,000 dilution in the blocking buffer were incubated with the blot for 4 h at room temperature. After a wash with TBS that contained 0.5% Tween 20 and after several rinses in TBS, the nitrocellulose was incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG diluted at 1:5,000 in the blocking buffer for 2 h at room temperature. After several washes with TBS, the protein level on the nitrocellulose membrane was detected by a chemiluminescent substrate system. The nitrocellulose membrane was sealed with a working solution, which was prepared by mixing equal volumes of reagents A and B in the assay kit (Fuji, Hunt) in a plastic bag for further developing and fixing to the X-ray film (Kodak, Rochester, NY). Densitometric analysis was conducted on the protein bands for quantitative comparison.

Drugs and chemicals. Fura 2-AM, type I collagenase, isoproterenol, propranolol, forskolin, cholera toxin (CTX), phenylmethylsulfonyl fluoride, Tween 20, dithiothreitol, Tris, EDTA, and alkaline phosphatase-conjugated goat anti-rabbit IgG were purchased from Sigma Chemical. The [3H]cAMP assay system was purchased from Amersham International. Chemicals for SDS-PAGE, electrophoretic transfer of polypeptide, nonfat dry milk, and nitrocellulose membrane were purchased from Bio-Rad. Affinity-purified G protein antibodies for Gsalpha -subunits and Gi were purchased from Calbiochem.

All chemicals were dissolved in distilled water except fura 2-AM and forskolin, which were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was 0.1%, and at this concentration, DMSO had no effect on either [Ca2+]i or cAMP.

Statistical analysis. Values are expressed as means ± SE. In experiments concerning determination of twitch amplitude and [Ca2+]i, one to three myocytes from a single rat were used. The values obtained from more than one myocyte were averaged, and the mean was used as a single entity for statistical analysis. Paired Student's t-test was used to determine the difference between control and treatment groups. Unpaired Student's t-test was employed to determine the difference among groups. Significance level was set at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of isoproterenol on the electrically induced contraction in single ventricular myocytes of normoxic and chronically hypoxic rats. Electrical stimulation triggered contraction, and the contractile responses were significantly lower in chronically hypoxic than in normoxic rats (Fig. 1A), in keeping with the well-established fact that the contractility is reduced after chronic hypoxia (9, 41). Isoproterenol, a beta -adrenoceptor agonist, at the range of 0.01-10 µM dose dependently increased the electrically induced contraction in the isolated single ventricular myocyte (Fig. 1B). This response was blocked by 1 µM propranolol, a beta -adrenoceptor antagonist (data not shown). The response was the same in myocytes from both RV and LV (Fig. 1B).


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Fig. 1.   Effects of isoproterenol on the twitch amplitude of contraction in single ventricular myocytes of normoxic and chronically hypoxic rats. A: representative tracings showing the effect of 1 µM isoproterenol in single myocytes of right ventricle (RV) of normoxic (top) and chronically hypoxic (bottom) rats. B: group results showing the dose-related effects of isoproterenol in myocytes of both RV and left ventricle (LV) of normoxic and chronically hypoxic rats with control (Con) group as 100%. For the measurement of cell contraction, the ventricular myocyte was superfused with Krebs solution, and the myocyte was then electrically stimulated before administration of isoproterenol. The electrically induced contraction was recorded for ~10 min after administration of isoproterenol. Ten myocytes from 5 rats were used in each group. Values are means ± SE; n = 5. The baseline value of twitch amplitude of RV myocytes of chronically hypoxic rats was 8.04 ± 0.25 µm, which is significantly lower at P < 0.01 in comparison with the corresponding value in normoxic rats. No difference was found between RV and LV myocytes. *P < 0.01 vs. control; #P < 0.01 vs. corresponding control with isoproterenol in normoxic rats.

In ventricular myocytes isolated from either the right hypertrophied or left nonhypertrophied ventricle of rats subjected to chronic hypoxia, the effects of isoproterenol on the contraction of the myocyte were significantly attenuated, and the attenuation was same in myocytes from RV and LV (Fig. 1, A and B).

Effects of isoproterenol, CTX, and forskolin on the electrically induced [Ca2+]i transient in single ventricular myocytes of normoxic and chronically hypoxic rats. To correlate the contractile response to [Ca2+]i on electrical stimulation, we determined the electrically induced [Ca2+]i transient in response to beta -adrenoceptor stimulation in myocytes of normoxic and chronically hypoxic rats. The resting [Ca2+]i was not different between RV and LV myocytes isolated from hearts of both chronically hypoxic and age-matched normoxic rats (data not shown). Nor was the diastolic [Ca2+]i in the RV and LV myocytes of the two types of rats different from each other (Fig. 2, A and B). The amplitude of the [Ca2+]i transient was significantly shorter in myocytes isolated from chronically hypoxic rats than that from normoxic rats (Fig. 2, A and B). The difference was 37% in RV and 32% in LV between the two types of rats.


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Fig. 2.   Electrically induced intracellular Ca2+ ([Ca2+]i) transient in single ventricular myocytes from normoxic and chronically hypoxic rats. A: representative tracings. B: group results. Thirty myocytes from 15 rats were used in each group. Values are means ± SE; n = 15. NS, no significant difference. **P < 0.01 vs. normoxic group.

Isoproterenol, at 0.01-10 µM, increased the amplitude of the electrically induced [Ca2+]i transient in the myocytes of both normoxic and chronically hypoxic rats (Fig. 3, A and B). The increase in amplitude was significantly less in myocytes of chronically hypoxic rats, and the changes were similar between the RV and LV (Fig. 3, A and B).


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Fig. 3.   Effects of isoproterenol on electrically induced [Ca2+]i transient in single ventricular myocytes of normoxic and chronically hypoxic rats. Isoproterenol was superfused to the myocytes, and recording was performed for 900 s. A: representative tracings showing the effect of 1 µM isoproterenol in single RV myocytes of normoxic (left) and chronically hypoxic (right) rats. B: group results showing the dose-related effects of isoproterenol in both RV and LV myocytes of normoxic and chronically hypoxic rats with control group as 100%. Twelve myocytes from 8 rats were used in each group. Values are means ± SE; n = 8. *P < 0.01 vs. control; #P < 0.01 vs. corresponding value with isoproterenol in normoxic group.

In agreement with our previous findings (51), the contractile and [Ca2+]i were directly correlated to each other (Figs. 1B and 3B). The contractile response was stronger than that of the [Ca2+]i transient (Figs. 1B and 3B), which is also in agreement with the finding of a previous study (45).

To investigate the mechanism underlying the reduced beta -adrenergic response, we compared the electrically induced [Ca2+]i transient in myocytes pretreated with activators of either Gs protein or AC in chronically hypoxic and age-matched normoxic rats. Pretreatment for 6 h with CTX, which is well known to activate the Gs protein, led to an increase in the amplitude of the electrically induced [Ca2+]i transient in myocytes of the normoxic rats but not of the chronically hypoxic rats (Fig. 4, A and B). The increases in the [Ca2+]i transient were the same in LV and RV in normoxic rats (Fig. 4, A and B). Forskolin at 0.1-100 µM, known to activate AC, also increased the amplitude of the electrically induced [Ca2+]i transient in single RV and LV myocytes of both normoxic and chronically hypoxic rats (Fig. 5, A and B). The increases were significantly attenuated when the forskolin was at 10-100 µM in both RV and LV myocytes of chronically hypoxic rats (Fig. 5, A and B).


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Fig. 4.   Effects of cholera toxin (CTX) on electrically induced [Ca2+]i transient in single ventricular myocytes of normoxic and chronically hypoxic rats. Myocytes were pretreated with 20 µg/ml CTX for 6 h before experiment. A: representative tracings showing the effect of CTX in single RV myocytes of normoxic (left) and chronically hypoxic (right) rats. B: group results showing the effects of CTX in both RV and LV myocytes of normoxic and chronically hypoxic rats with control group as 100%. Twelve myocytes from 6 rats were used in each group. Values are means ± SE; n = 6 rats in each group. **P < 0.01 vs. control; ##P < 0.01 vs. corresponding value in normoxic group.



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Fig. 5.   Effects of forskolin on electrically induced [Ca2+]i transient in single ventricular myocytes of normoxic and chronically hypoxic rats. A: representative tracings showing the effect of 100 µM forskolin in single RV myocytes of normoxic (left) and chronically hypoxic (right) rats. B: group results showing the dose-related effects of forskolin in both RV and LV myocytes of normoxic and chronically hypoxic rats with control group as 100%. Twelve myocytes from 5 rats were used in each group. Values are means ± SE; n = 5 rats in each group. *P < 0.01 vs. control; #P < 0.05, ##P < 0.01 vs. corresponding value with forskolin in normoxic group.

Effects of isoproterenol, NaF, and forskolin on cAMP accumulation in ventricular myocytes of normoxic and chronically hypoxic rats. To further determine whether or not the Gs protein and AC were impaired in the heart of chronically hypoxic rats, we determined the effect of NaF, a ubiquitous stimulator of AC activity via Gsalpha (43), and forskolin on cAMP accumulation in the heart of both normoxic and chronically hypoxic rats. We also determined the effect of isoproterenol. The basal production of cAMP was not significantly changed in either RV or LV myocytes. Isoproterenol (0.1-10 µM), NaF (1-10 mM), and forskolin (1-100 µM) increased the cAMP accumulation in myocytes of both normoxic and chronically hypoxic rats (Fig. 6, A-C). Like the [Ca2+]i response, the responses to isoproterenol, NaF, and forskolin were significantly blunted in chronically hypoxic rats (Fig. 6, A-C). Again, the changes were similar in the RV and LV myocytes (Fig. 6, A-C).


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Fig. 6.   Effects of isoproterenol, NaF, and forskolin on the cAMP accumulation in ventricular myocytes of normoxic and chronically hypoxic rats. A: the dose-related effects of isoproterenol in both RV and LV myocytes of normoxic and chronically hypoxic rats. B: the dose-related effects of NaF in both RV and LV myocytes of normoxic and chronically hypoxic rats. C: the dose-related effects of forskolin in both RV and LV myocytes of normoxic and chronically hypoxic rats. Values are means ± SE; n = 6 rats in each group. *P < 0.05, **P < 0.01 vs. normoxic group.

Gsalpha protein isoforms in ventricular myocytes of normoxic and chronically hypoxic rats. Experiments on the [Ca2+]i response in the present study suggested that Gs protein might be impaired in the heart of chronically hypoxic rats. To determine whether the Gs protein was indeed impaired after chronic hypoxia, we measured the content of the Gsalpha -small (biologically active isoform) and Gsalpha -large (biologically inactive isoform) in the myocytes of chronically hypoxic rats. It was found that the density of the band of Gsalpha -small (45 kDa) in the membranes of both RV and LV myocytes was significantly lower, whereas that of the band of Gsalpha -large (52 kDa) was significantly increased in the chronically hypoxic rats (Fig. 7, A-C). Like the contractile and [Ca2+]i responses, the changes were similar in RV and LV. The results indicated that there was a change in the small and large isoforms of Gs protein distribution in the hypoxic rat heart.


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Fig. 7.   Immunoblot analysis of 2 Gsalpha isoforms in the washed plasma membrane of ventricular myocytes of normoxic and chronically hypoxic rats. A: immunoblots of Gsalpha -small (45 kDa) and Gsalpha -large (52 kDa) immunostained with a Gsalpha antibody at 1:1,000 dilution. B: group results showing changes in optical density of Gsalpha -small. C: group results showing changes in optical density of Gsalpha -large. Values are means ± SE; n = 6 rats in normoxic group, n = 8 rats in hypoxic group. **P < 0.01 vs. corresponding control.

Gialpha protein isoforms in ventricular myocytes of normoxic and chronically hypoxic rats. Interaction between Gs and Gi proteins is well known (13). In this study, we measured the content of the Gialpha 2, a predominant isoform in the heart (12), in the myocytes of normoxic and chronically hypoxic rats. It was found that the density of the band of Gialpha 2 in neither RV nor LV myocyte membranes was significantly changed in the chronically hypoxic rats (Fig. 8).


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Fig. 8.   Immunoblot analysis of Gialpha 2 in the washed plasma membrane of ventricular myocytes of normoxic and chronically hypoxic rats. A: immunoblots of Gialpha 2 immunostained with a Gialpha 2 antibody at 1:1,000 dilution. B: group results showing optical density of Gialpha 2. Values are means ± SE; n = 4 rats in each group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The most interesting observations in the present study are 1) the stimulatory actions of CTX and NaF, which stimulate the Gs protein, and of forskolin, which activates AC on the electrically induced [Ca2+]i transient and cAMP production in the ventricular myocytes, are significantly attenuated; and 2) Gsalpha -small, the predominant biologically active Gsalpha isoform in the heart, is significantly reduced in the chronically hypoxic rats. The observations indicate that, in chronically hypoxic rats, the reduced responsiveness to beta -adrenoceptor stimulation is due at least partly to an impaired function of the Gs protein, which may result from a reduction in the biologically active Gsalpha -small, and AC. So, the reduced responsiveness to beta -adrenoceptor stimulation is due not only to downregulation of the beta -adrenoceptor, as shown in previous studies (19, 27, 48), but is also due to impaired postreceptor signaling mechanisms, as shown in the present study.

In this study, we show that the biologically active isoform, Gsalpha -small, was reduced after chronic hypoxia. This is not in agreement with the previous finding that there was no change in Gsalpha -small after hypobaric hypoxia (21). It should be noted that the rats were subjected to isobaric hypoxia in this study. We also showed, for the first time, that the biologically inactive isoform, Gsalpha -large, was increased. Although the changes in contents of these two isoforms are believed to be responsible for the impaired Gs protein function, the roles of the two isoforms need further study. The mechanism underlying the alterations in the production of two isoforms in the heart after chronic hypoxia also needs further study.

As expected, forskolin at 0.1-100 µM increased dose dependently the electrically induced [Ca2+]i and cAMP in the ventricular myocyte of both normoxic and chronically hypoxic rats. It is interesting to note that only to forskolin, at high concentrations (10-100 µM), were the cardiac responses attenuated in the chronically hypoxic rats. This is in agreement with the observation in the Wistar rat subjected to 5 days of hypobaric hypoxia (29) but in disagreement with the observation in the Wistar rat subjected to 21 days of hypobaric hypoxia (22). The attenuated cardiac responses to high concentrations of forskolin in hypoxic rats suggest that only in a more active state is AC function really affected by chronic hypoxia.

It has been shown that, in cardiac hypertrophy, the Gi protein is increased (8, 17, 29). It has also been shown that the Gi protein exerts an inhibitory influence on the Gs protein in the heart (20). In this study, we showed that there was no significant change in Gialpha 2 protein, a predominant isoform in the heart. The observation is in agreement with a previous study on rats subjected to 30 days of hypoxia (21) but not in agreement with a previous study on rats subjected to 5 days of hypoxia (29).

In another study in our laboratory, we observed that, after chronic hypoxia, the cardiac responses to kappa -opioid receptor stimulation were attenuated, and the responses were similar in the right hypertrophied and the left nonhypertrophied ventricles (33). In this study, we also found that the cardiac responses to beta -adrenoceptor stimulation were similar in both right hypertrophied and left nonhypertrophied ventricles. The observations suggest that impaired cardiac responses are most likely due to hypoxia, which induces hypoxemia, but not necessarily secondary to cardiac hypertrophy.

In conclusion, the present study has provided evidence for the first time that, after chronic hypoxia, which induces right heart hypertrophy, the attenuated cardiac responses to beta -adrenoceptor stimulation are due to impaired functions of Gs protein and AC. The impaired function of the Gs protein may be due to a reduction in the biologically active Gsalpha -small and/or an increase in the Gsalpha -large. Similar cardiac responses in the right hypertrophied and the left nonhypertrophied ventricles are also unequivocal evidence, suggesting that impaired cardiac responses are due to hypoxemia, but not necessarily secondary to cardiac hypertrophy after chronic hypoxia.


    ACKNOWLEDGEMENTS

We thank Y. K. Pang for taking care of the rats subjected to chronic hypoxia and C. P. Mok for assistance.


    FOOTNOTES

This study was supported by a grant from the Institute of Cardiovascular Sciences and Medicine, Faculty of Medicine, University of Hong Kong.

This study was performed when J. M. Pei and J. J. Zhou were on leave from the Dept. of Physiology, Fourth Military Medical University, Xi'an, China.

Address for reprint requests and other correspondence: T. M. Wong, Dept. of Physiology, Faculty of Medicine, Univ. of Hong Kong, Li Shu Fan Bldg., 5 Sassoon Road, Hong Kong, China (E-mail: wongtakm{at}hkucc.hku.hk).

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 15 November 1999; accepted in final form 15 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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