Department of Physiology and Institute of Cardiovascular Sciences and Medicine, Faculty of Medicine, The University of Hong Kong, Hong Kong, China
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ABSTRACT |
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-Opioid receptor (
-OR)
stimulation with U50,488H, a selective
-OR agonist, or activation of
protein kinase C (PKC) with 4-phorbol 12-myristate 13-acetate (PMA), an
activator of PKC, decreased the electrically induced intracellular
Ca2+ ([Ca2+]i) transient and
increased the intracellular pH (pHi) in single ventricular
myocytes of rats subjected to 10% oxygen for 4 wk. The effects of
U50,488H were abolished by nor-binaltorphimine, a selective
-OR
antagonist, and calphostin C, a specific inhibitor of PKC, while the
effects of PMA were abolished by calphostin C and
ethylisopropylamiloride (EIPA), a potent Na+/H+
exchange blocker. In both right hypertrophied and left
nonhypertrophied ventricles of chronically hypoxic rats, the effects of
U50,488H or PMA on [Ca2+]i transient and
pHi were significantly attenuated and completely abolished,
respectively. Results are first evidence that the
[Ca2+]i and pHi responses to
-OR stimulation are attenuated in the chronically hypoxic rat heart,
which may be due to reduced responses to PKC activation. Responses to
all treatments were the same for right and left ventricles, indicating
that the functional impairment is independent of hypertrophy.
-OR
mRNA expression was the same in right and left ventricles of both
normoxic and hypoxic rats, indicating no regional specificity.
hypoxia; hypertrophy; protein kinase C; intracellular calcium; intracellular pH; heart
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INTRODUCTION |
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THERE IS SUBSTANTIAL
EVIDENCE that following chronic hypoxia, which causes cardiac
hypertrophy and failure, the cardiac contraction and
intracellular Ca2+ ([Ca2+]i)
homeostasis is altered (1, 2, 4, 16, 17, 18, 29, 31, 45),
suggesting that the altered Ca2+ homeostasis may be
responsible for hypertrophy and impaired function in the heart
(7). There is evidence that an altered intracellular pH
(pHi) may also contribute to cardiac hypertrophy (38,
47). The -opioid receptors are present (25, 48, 50, 60,
62) and
-opioid peptides are produced (10, 52,
56) in the heart, suggesting that the
-opioid peptides may
regulate the cardiac function as autocrine or paracrine hormones
(28, 52).
-Opioid receptor stimulation decreases the
cardiac contractility and [Ca2+]i transient
as a result of depletion of Ca2+ from the sarcoplasmic
reticulum (SR) (5, 33, 41) and increases pHi
due to an increased Na+/H+ exchange activity
(15, 51). It is likely that, following chronic hypoxia,
the [Ca2+]i and pHi responses to
-opioid receptor stimulation may be altered/impaired.
It has been shown that -opioid receptor stimulation activates the
protein kinase C (PKC) (5, 51), which increases the [Ca2+]i by inhibiting the Ca2+
reuptake by SR (36) and increases pHi by
activating the Na+/H+ exchange
(21). Activation of PKC by 4-phorbol 12-myristate 13-acetate (PMA), a phorbol ester, also exerts a negative inotropic effect (11), which is due to a decrease in the amplitude
of the [Ca2+]i transient (11).
These similarities of the
-opioid receptor and PMA responses
are also seen in other type of receptor, such as
-adrenergic
receptor in rat ventricular myocytes (12, 20). It has been
shown that most isoforms of PKC were reduced following cardiac
hypertrophy and failure (37). In addition, a recent study
showed that the contractile, [Ca2+]i and
pHi responses to PMA, an activator of PKC, were
abolished in the hypertrophied and failing heart (19). The
observations suggest that PKC pathway may be impaired in cardiac
hypertrophy and failure.
It is widely believed that, following chronic hypoxia, cardiac
hypertrophy occurs, which eventually leads to cardiac failure. It was
recently reported that the poor contraction-relaxation exhibited by the
myocytes in the human hypertrophied left ventricle was not correlated
with the size of the myocytes (13), suggesting that the
impaired cardiac function may not necessarily be secondary to cardiac
hypertrophy. In support of this, we recently found that cardiac
responses to -adrenoceptor stimulation and Gs protein stimulation were the same for left nonhypertrophied and right hypertrophied ventricles of the chronically hypoxic rat
(34).
The purpose of the present study is therefore mainly twofold. First, we
attempted to determine the [Ca2+]i and
pHi responses to -opioid receptor stimulation in the
heart of rats subjected to chronic hypoxia by determining the effects of U50,488H, a selective
-opioid receptor agonist, on electrically induced [Ca2+]i transient and pHi
in isolated myocytes of the heart of rats chronically exposed to
hypoxia for 4 wk. Electrically induced [Ca2+]i transient results from an influx of
Ca2+ upon membrane depolarization, which triggers a sudden
release of Ca2+ from the SR via a Ca2+-induced
Ca2+ release mechanism. A previous study in our laboratory
has shown that electrically induced [Ca2+]i
transient is directly related to the contraction in single ventricular
myocytes (59). In this study we also correlated the
contractile responses to [Ca2+]i responses by
measuring the electrically induced cell shortening in the myocyte.
Secondly, we also determined cardiac responses to activation of PKC,
which mediates the action of
-opioid receptor stimulation (5,
15, 51), by determining the [Ca2+]i
and pHi changes in response to administration of PMA. In
addition, we compared the [Ca2+]i and
pHi responses to activation of
-opioid receptor and PKC and measured the expression of
-opioid receptor gene by RT-PCR in
left and right ventricles in normoxic and chronically hypoxic rats. We
employed a procedure in which rats were exposed to 10% oxygen for 4 wk
in isobaric condition, shown to cause systemic hypoxemia, pulmonary
hypertension, and right heart hypertrophy (32, 35). The
results showed for the first time that, following chronic hypoxia,
[Ca2+]i and pHi responses to
-opioid receptor stimulation were impaired, which may be due mainly
to reduced responses to PKC activation. Results from this study also
indicate that cardiac hypertrophy is not necessarily a prerequisite of
cardiac failure.
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MATERIALS AND METHODS |
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Chronic hypoxia and assessment of right ventricular hypertrophy. The experimental protocol for preparing chronically hypoxic rats was approved by the Committee on the Use of Life Animal in Teaching and Research of the University of Hong Kong. For exposure of rats to hypoxia, Sprague-Dawley rats of 100-150 g were placed inside an acrylic chamber supplied with 10% oxygen for 4 wk. The oxygen level was continuously monitored by an oxygen analyzer (model 175518A; Gold Edition, Vacuum Med) and was maintained by a servo-feedback of solenoid valves that regulate the inflow of pure nitrogen. Nitrogen was mixed with room air, which was continuously vented to the chamber by a pump at 2 l/min. The humidity and carbon dioxide level were maintained by a desiccator and soda lime placed inside the chamber. Every 2-3 days, the chamber was opened for 30 min for regular maintenance. For normoxic controls, rat pups were kept in the same housing condition with breathing in room air. All animals were freely accessible to water and chow. The PO2 of arterial blood in the chronically hypoxic rats was reduced by ~35% compared with control.
The rats were weighed, then decapitated, and the hearts were quickly removed. The heart weight was determined. The right ventricular (RV) and left ventricular (LV) + septum weights were also determined after a collagenase perfusion. The RV hypertrophy was indicated by calculating the ratio of whole heart weight to body weight and the ratio of RV weight to body weight (Table 1) in agreement with previous observations (32, 35).
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Isolation of ventricular myocytes. RV and LV myocytes were isolated from the heart of male Sprague-Dawley rats, using a collagenase perfusion method described previously (14). Immediately after decapitation, the heart was rapidly removed from the rat and perfused in a retrograde manner at a constant flow rate (10 ml/min) with oxygenated Joklik modified Eagle's medium supplemented with 1.25 mM CaCl2 and 10 mM HEPES, pH 7.2, at 37°C for 5 min followed by 5 min 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) BSA. After 35-45 min of perfusion with medium containing collagenase, the atria were discarded and the RV and LV tissues were dissociated by shaking in the same oxygenated collagenase-free solution for 5 min at 37°C, respectively. The ventricular tissue was cut into small pieces with a pair of scissors, followed by stirring with a glass rod, which 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. Ca2+ concentration of the Joklik solution was increased gradually to 1.25 mM in 40 min.
Measurement of twitch amplitude of contraction. After isolation of myocytes for 2 h, measurement of myocyte twitch amplitude was performed using an optical video system as described previously (3, 58). 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 (KB buffer) containing (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 automatic video analyzer developed in our laboratory (59). As a prerequisite for the proper operation of this automatic analyzer, the image of cardiac myocytes projected on the video camera (Sony model SSC-M370CE) and observed on the video monitor (Sony model PVM-145E) was first rotated to align horizontally and parallel to each of the video raster lines. This was achieved by interposing a k-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 using 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 fura 2-AM (5 µM) 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 measuring [Ca2+]i to allow the fura 2-AM in the cytosol to deesterify. We loaded with a low concentration of fura 2-AM and at a relatively low temperature of 25°C, to minimize the effects of compartmentalization of the esters (44).
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). The myocytes were perfused with KB buffer containing 1% dialyzed BSA and a gas phase of 95% O2-5% CO2, pH 7.4. The myocytes selected for the study were rod shaped and quiescent with clear striations. They exhibited synchronous contractions (twitches) 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. Fluorescent signals obtained at 340 nm (F340) and at 380 nm (F380) excitation wavelengths were stored in a computer for data processing and analysis. The fluorescence (F340/F380) ratio was used to represent [Ca2+]i changes in the myocytes. The baseline of [Ca2+]i fluorescence ratios is presented in Table 1.Measurement of pHi.
The pHi was measured in the single myocyte as described
previously (9). The apparatus and optical arrangement used
for the measurement of fluorescent light emission and preparation procedure were similar to those described in Measurement of
[Ca2+]i except that the cells were
loaded with the membrane-permeant acetoxymethyl ester of
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) as the fluorescence indicator at 5 µM for 30 min. The loaded cells were transferred to the stage of an inverted microscope in
the superfusion chamber at room temperature. Myocytes were continuously
superfused with oxygenated bicarbonate-buffered KB solution or
HEPES-buffered normal Tyrode solution of the following composition (in
mM): 137 NaCl, 3.7 KCl, 0.5 MgCl2, 4.0 HEPES (free acid),
1.25 CaCl2, and 11 glucose with a final pH of 7.4. Superfusion with a HEPES-buffered solution was to eliminate effects of
Cl/HCO3
exchange and
Na+/HCO3
exchange on pHi
(6). The pH-dependent signal of BCECF was obtained by
illuminating at 490 and 435 nm, and the fluorescent emission wavelength
was measured at 520 nm. The ratio of F490 over
F435 was used to represent the pHi. The
baseline of pHi fluorescence ratios is presented in Table
1.
Total RNA isolation and RT-PCR.
Total RNA was isolated from right and left heart tissues of normoxic
and chronically hypoxic rats by using TRIzol reagent [GIBCO BRL; Life
Technologies (Pacific), Hong Kong], and RNA integrity was confirmed by
agarose gel electrophoresis. RT-PCR was performed with a ThermoScript
RT-PCR reagent system (GIBCO BRL). Total RNA (4 µg) was added to
oligo(dT) primers, denatured at 90°C for 5 min, and quenched on ice.
RT was carried out by addition of 15 units of an avian RNase
H reverse transcriptase, 10 mM dNTPs, and 100 mM DTT at
50°C for 60 min in a total reaction volume of 20 µl. The reaction
was terminated by a 5-min incubation at 90°C. Primers for PCR were
designated according to the published sequences (57). The
primers of sense, 5'-CCG CTG TCT ACT CTG TGG TGT-3', and antisense,
5'-ATG TTG ATG ATC TTT GCT TTC-3', were used for
-opioid receptor
with a predicted product size of 352 bp, whereas the primers of the
sense, 5'-GTG GGG CGC CCC AGG CAC CA-3', and antisense, 5'-CTC CTT AAT
GTC ACG CAC GAT TTC-3', were used for
-actin, the internal
standard, with a predicted product size of 540 bp. For
-opioid
receptor, the PCR amplification was carried out as follows: 1 min at
95°C for denaturing, 1 min at 60°C for annealing, and 1 min at
72°C for extension for 40 cycles followed by a 7-min extension. For
-actin, following the denaturation at 95°C for 5 min, PCR was proceeding under the following conditions: 30 s at 95°C for
denaturing, 45 s at 60°C for annealing, and 1 min at 72°C for
extension for 40 cycles followed by a 7-min extension.
Drugs and chemicals. U50,488H (trans-3,4-dichloro-N-methyl-[2-(1-pyrrolidinyl)cyclohexy]benzeacetamidel), PMA, fura 2-AM, type I collagenase, nigericin and calphostin C were purchased from Sigma Chemicals. Nor-binaltorphimine (nor-BNI) was purchased from Tocris Cookson. BCECF-AM and ethylisopropylamiloride (EIPA) were purchased from RBI.
Fura 2-AM, BCECF-AM, PMA, calphostin C, and EIPA were dissolved in dimethyl sulfoxide (DMSO), nigericin was dissolved in the ethanol, and the rest were dissolved in distilled water. U50,488H at the dose range of 10-30 µM was administered for 10 min, because preliminary studies showed that the effects of the opioid were obvious at 2-3 min and reached maximum before 10 min. The dose range used in the present study has been shown to decrease electrically induced [Ca2+]i transient, effects antagonized by 1-5 µM nor-BNI (59, 61, 62), which itself had no effect on any of the preparations studied. The concentrations of the PKC agonist and antagonist used were based on previous studies (11, 39, 49). In a preliminary experiment, 10 µM EIPA did not alter the autofluorescence of the cell at the BCECF excitation wavelength as previously reported (30). The final concentration of DMSO was 0.1%, and at this concentration DMSO had no effect on either [Ca2+]i or pHi (30).Statistical analysis. Values are means ± SE. Paired Student's t-test was used to determine the difference between control and drug treatment groups. Unpaired Student's t-test was employed to determine the difference among groups. Significance level was set at P < 0.05.
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RESULTS |
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Effects of U50,488H on electrically induced
[Ca2+]i transient and contraction in single
ventricular myocytes.
In this series of experiments, we first determined the
[Ca2+]i response to -opioid receptor
stimulation in the ventricular myocyte of chronically hypoxic rats. In
agreement with previous studies in our laboratory (33,
61), U50,488H, a selective
-opioid receptor agonist
(22), at the range of 10-30 µM (Fig. 1A)
dose-dependently decreased the electrically induced
[Ca2+]i transient in the isolated single
ventricular myocyte. The effect of U50,488H at 30 µM on the
[Ca2+]i transient (Fig. 1A) was
completely abolished in the presence of 5 µM nor-BNI, a selective
-opioid receptor antagonist (46). The effects of
U50,488H at 20 µM, a dose approximately equal to the
EC50, on the [Ca2+]i transient
(Fig. 1B) were significantly attenuated in the presence of
1 µM calphostin C, a specific PKC inhibitor (23),
indicating that effects were mediated via the
-opioid receptor and
PKC.
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Effects of U50,488H on pHi in single ventricular
myocytes.
In agreement with previous studies in our laboratory (15),
20 µM U50,488H increased the pHi of the RV myocyte both
in a HEPES-buffered solution (Fig. 3,
A and B) and in a bicarbonate-buffered solution
(Fig. 4, A and B).
The effects of 20 µM U50,488H on pHi (Fig. 3A)
were completely abolished in the presence of 5 µM nor-BNI. The
effects of 20 µM U50,488H on the pHi (Fig.
3A) were completely abolished in the presence of 1 µM
calphostin C and 10 µM EIPA (Fig. 3A), a potent
Na+/H+ exchange blocker (30),
indicating that the effect was also mediated via PKC and
Na+/H+ exchanger.
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Effects of PMA on electrically induced
[Ca2+]i transient and pHi in
single ventricular myocytes.
In this series of experiments, we determined whether PKC was impaired
in the heart following chronic hypoxia. We determined the effects of
activation of PKC on the electrically induced
[Ca2+]i transient and pHi in the
isolated RV myocyte. In agreement with our previous observation
(5), PMA, an activator of PKC, at 0.01-1 µM
dose-dependently decreased the electrically induced [Ca2+]i transient in the isolated single
ventricular myocyte (Fig. 5A). PMA
(0.1 µM) also increased the pHi both in a
HEPES-buffered solution (Fig. 6, A and B) and in
a bicarbonate-buffered solution (Fig. 7, A and
B). The effects of 0.1 µM PMA on both the
[Ca2+]i transient (Fig. 5A) and
pHi (Fig. 6A) were
completely abolished by 1 µM calphostin C. The effect of the PKC
activator on pHi was also abolished by 10 µM EIPA (Fig.
6A), indicating that the effect was mediated by the
Na+/H+ exchange. There was no difference in
[Ca2+]i and pHi responses to PMA
between the right and left ventricles of normoxic rats.
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Expression of -opioid receptor mRNA in the heart of chronically
hypoxic rats.
To further determine the regional difference of the
-opioid system
between the right and left ventricles, we determined the RT-PCR product
of
-opioid receptor mRNA in the heart of normoxic and chronically
hypoxic rats. As shown in Fig.
8A, RT-PCR yielded a 352-bp
product for
-opioid receptor and 540-bp product for
-actin by
using
-opioid receptor and
-actin-specific primers, respectively.
No significant difference was found between the right hypertrophied and
the left nonhypertrophied ventricles nor between normoxic and
chronically hypoxic rats (Fig. 8B). The result indicates a
lack of regional specificity in
-opioid receptor in the heart.
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DISCUSSION |
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The most important observation of the present study is that the
[Ca2+]i and pHi responses to
-opioid receptor stimulation were significantly attenuated and
almost completely abolished, respectively, in the heart subjected to
chronic hypoxia. The [Ca2+]i
response was directly correlated with the contractile response. The
novel finding is that cardiac responses to
-opioid receptor stimulation were impaired in the heart following chronic hypoxia. In
the present study the contractile response to electrical stimulation is
attenuated in the heart of chronically hypoxic rat, indicating a
reduction in cardiac contractility in agreement with well-established observations (8). Since
-opioid peptides are produced
(10, 52, 56) and
-opioid receptor is the predominant
opioid receptor (25, 46, 50, 60, 62) in the heart, it is
believed that the
-opioid peptides may regulate the cardiac function
as autocrine or paracrine hormones (28, 52). An attenuated
contractile response to
-opioid receptor stimulation may prevent
excessive reduction in contractility following chronic hypoxia. It is
well known that, following chronic hypoxia, the contractile response to
-adrenergic stimulation is attenuated (8, 27), leading to reduced contractility, which is undesirable. Since
-opioid receptor modulates negatively the
-adrenoceptor (59),
the modulatory action of
-opioid receptor on
-adrenoceptor may
also be attenuated, which also prevents excessive reduction in
contractility following chronic hypoxia.
Another important observation in the present study is that the
[Ca2+]i and pHi responses to
activation of PKC by PMA were almost completely abolished in the heart
of chronically hypoxic rats in agreement with the previous observation
that the contractile, [Ca2+]i, and
pHi responses to phorbol ester, an activator of
PKC, are abolished in hypertrophied heart induced by aortic banding
(19). Because -opioid receptor stimulation activates
the phospholipase C/PKC (5), the attenuated cardiac
responses to
-opioid receptor stimulation may result, at least
partly, from reduced responses to PKC activation following chronic
hypoxia. It has been shown that
-opioid receptor stimulation
activates the
-isoform of PKC in the heart (53).
Further studies are needed to determine whether the response of the
-isoform in the rat heart to
-opioid receptor stimulation is
reduced or not subjected to chronic hypoxia.
It should be noted that the [Ca2+]i response
to -opioid receptor stimulation was attenuated, whereas the
pHi response completely abolished by calphostin C, the PKC
inhibitor. On the other hand, both the
[Ca2+]i and pHi responses to
activation of PKC were completely abolished by calphostin C. The
observations indicate that the [Ca2+]i
response to
-opioid receptor stimulation was only partially mediated, whereas the pHi response to
-opioid receptor
stimulation was solely mediated, by PKC and/or
Na+/H+ exchange. This is in agreement
with our previous observation that
-opioid receptor stimulation
increases production of inositol trisphosphate (40, 41,
42), indicating that the [Ca2+]i
response to
-opioid receptor stimulation may be mediated via inositol trisphosphate as well.
It is important to note that there was no difference in
[Ca2+]i and pHi responses to
-opioid receptor stimulation and PKC activation in myocytes from the
right hypertrophied and the left nonhypertrophied ventricles following
chronic hypoxia. The observation is in agreement with the previous
observation that, in ventricular myocytes of human left hypertrophied
heart, the poor contraction-relaxation is not correlated to the size of
the myocyte (13) and with our recent observation that the
cardiac responses to stimulation of
-adrenoceptor and Gs
protein are the same for the left nonhypertrophied and right
hypertrophied ventricles of chronically hypoxic rats. The observations
are unequivocal evidence that the impaired cardiac functions are not
secondary to cardiac hypertrophy. It is therefore likely that hypoxemia
is the cause of impaired function of both right and left ventricles.
In this study we found that the mRNA expression of -opioid receptor
was not altered in the heart of rats subjected to chronic hypoxia. The
observation suggests that there may not be any downregulation of
cardiac
-opioid receptor following chronic hypoxia. However, further
study on receptor activity is needed to verify the suggestion. There is
also no difference between the right and left ventricles, indicating no
regional specificity.
In the present study the rats were subjected to isobaric hypoxia with 10% oxygen for 4 wk. The treatment would give an arterial partial pressure oxygen at about 40-50 Torr and an oxygen saturation of blood at about 75-85% (32). These values are close to those of patients with congenital heart diseases and chronic pulmonary diseases and are equivalent to conditions at high altitude of about 5,000 meters in human habitation. Previous studies have shown that these chronically hypoxic rats showed significant increases in hematocrit, pulmonary arterial pressure, and right heart size (32, 35). Therefore, findings from studies using rats subjected to 4 wk with 10% oxygen will provide clinically relevant information. It should be cautioned, however, that different species might employ different compensatory mechanisms in response to hypoxia. Studies on human subjects are necessary.
In conclusion, the present study has provided evidence for the first
time that the contractile, [Ca2+]i, and
pHi responses to -opioid receptor stimulation
are impaired in the heart of rats subjected chronic hypoxia. The
impaired responses may be due, at least partly, to reduced responses to
PKC activation. The responses to both
-opioid receptor stimulation
and PKC activator in the right hypertrophied and left nonhypertrophied
ventricles were exactly the same, indicating that hypoxemia rather than
hypertrophy may be responsible for the impaired cardiac function.
Further studies are needed to investigate the
-opioid receptor
activity and identify the PKC isoform involved in the heart of rats
subjected to chronic hypoxia.
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ACKNOWLEDGEMENTS |
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We thank Y. K. Pang for taking care of the rats and C. P. Mok for assistance.
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FOOTNOTES |
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The study was supported by Research Grants Council, Hong Kong, and the Institute of Cardiovascular Sciences and Medicine, Faculty of Medicine, The University of Hong Kong. The study was performed when J.-M. Pei and J.-J. Zhou were on leave from the Department 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, The Univ. of Hong Kong, Li Shu Fan Bldg., 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 22 October 1999; accepted in final form 12 June 2000.
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