Impaired [Ca2+]i and pHi responses to kappa -opioid receptor stimulation in the heart of chronically hypoxic rats

Jian-Ming Pei, Jing-Jun Zhou, Jin-Song Bian, Xiao-Chun Yu, Man-Lung Fung, and Tak-Ming Wong

Department of Physiology and Institute of Cardiovascular Sciences and Medicine, Faculty of Medicine, The University of Hong Kong, Hong Kong, China


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

kappa -Opioid receptor (kappa -OR) stimulation with U50,488H, a selective kappa -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 kappa -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 kappa -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. kappa -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 kappa -opioid receptors are present (25, 48, 50, 60, 62) and kappa -opioid peptides are produced (10, 52, 56) in the heart, suggesting that the kappa -opioid peptides may regulate the cardiac function as autocrine or paracrine hormones (28, 52). kappa -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 kappa -opioid receptor stimulation may be altered/impaired.

It has been shown that kappa -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 kappa -opioid receptor and PMA responses are also seen in other type of receptor, such as alpha -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 beta -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 kappa -opioid receptor stimulation in the heart of rats subjected to chronic hypoxia by determining the effects of U50,488H, a selective kappa -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 kappa -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 kappa -opioid receptor and PKC and measured the expression of kappa -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 kappa -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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1.   Characteristics of animal groups

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.

It should be noted that, in agreement with previous studies (26, 43), the baseline of pHi was lower in the myocyte superfused with a bicarbonate-buffered solution than with a HEPES-buffered solution, believed to be due to the Na+-independent Cl-/HCO3- exchange being an acidifying system (24).

At the end of each experiment, the calibration of BCECF signals was performed. The pHi was set to the extracellular pH with 10 µM nigericin in the calibration solution (12 mM HEPES, 140 mM KCl, 1 mM MgCl2, and 11 mM glucose). The extracellular pH was adjusted to 8, 7, 6, and 5, respectively, using KOH or HCl.

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 kappa -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 beta -actin, the internal standard, with a predicted product size of 540 bp. For kappa -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 beta -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.

The primer pair used for kappa -opioid receptor was found to be specific for its gene sequence (57). Briefly, analysis by Southern blot hybridization, using full cDNA probes for each opioid receptor type, specifically detected the PCR products generated for each receptor, with no cross-hybridization between µ-, delta -, and kappa -opioid receptor-specific PCR products. In addition, hybridization of the Northern blot to specific cDNA probes for the µ-, delta -, and kappa -opioid receptors did not reveal hybridizing bands for these receptors in any of the peripheral tissues even after 2 wk of autoradiography at -70°C. So, we only determined the PCR product semiquantitatively without Southern blot and Northern blot analyses.

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.


    RESULTS
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ABSTRACT
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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 kappa -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 kappa -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 kappa -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 kappa -opioid receptor and PKC.



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Fig. 1.   Effects of U50,488H on electrically induced intracellular Ca2+ concentration ([Ca2+]i) transient in single ventricular myocytes of the ventricles of normoxic and chronically hypoxic rats. A: dose-related effects of U50,488H in the presence and absence of 5 µM nor-binaltorphimine (nor-BNI) in normoxic rats. Top: representative tracings in right ventricular (RV) myocytes. Bottom: group results with control group as 100% in both RV and left ventricular (LV) myocytes. B: effect of 20 µM U50,488H in the presence and absence of 1 µM calphostin C in normoxic rats. Top: representative tracing. Bottom: group results with control group as 100%. C: effect of 20 µM U50,488H in normoxic and chronically hypoxic rats. Top: representative tracings in RV myocytes. Bottom: group results with control group as 100% in both RV and LV myocytes. For the measurement of electrically induced [Ca2+]i transient, the ventricular myocyte was superfused with Krebs solution; the cell was then electrically stimulated before administration of U50,488H. In experiments involving nor-BNI, the drug was administered at 5 min before and together with U50,488H. In experiments involving calphostin C, the drug was administered at 10 min before and together with U50,488H. The electrically induced [Ca2+]i transient was recorded at about 10 min after administration of U50,488H. Values are means ± SE; n = 12 in both RV and LV myocytes in A; n = 8 in B; n = 12 in both RV and LV myocytes in C. **P < 0.01 vs. control. ##P < 0.01 vs. corresponding control with U50,488H.

In the ventricular myocytes isolated from the right hypertrophied and left nonhypertrophied ventricles of rats subjected to chronic hypoxia, the effects of 20 µM U50,488H on the [Ca2+]i transient were significantly attenuated (Fig. 1C). There was no difference between the right and left ventricles.

To correlate the Ca2+ response to contraction on electrical stimulation, we also determined the electrically induced contractile response of myocytes to kappa -opioid receptor stimulation in the normoxic and chronically hypoxic rats. Electrical stimulation triggered contraction, and the contractile responses were significantly lower in chronically hypoxic than in normoxic rats (Fig. 2, A and B), in keeping with the well-established fact that the contractility is reduced following chronic hypoxia (8). U50,488H, at the range of 10-30 µM (Fig. 2, A and C), dose-dependently decreased the electrically induced contraction in the isolated single ventricular myocyte in agreement with previous studies (22, 54). The responses in electrically induced contraction and [Ca2+]i transient were directly correlated to each other (Figs. 1A and Fig. 2C), which is also in agreement with our previous study (59). The effects of 30 µM U50,488H were abolished in the presence of 5 µM nor-BNI (data not shown), which abolished the effects of U50,488H on [Ca2+]i transient (Fig. 1A). The response was similar in myocytes from both right and left ventricles (Fig. 2C).


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Fig. 2.   Effects of U50,488H on the twitch amplitude of contraction in single ventricular myocytes of the ventricles of normoxic and chronically hypoxic rats. A: representative tracings showing the effect of 30 µM U50,488H in single RV myocytes of normoxic (top) and chronically hypoxic (bottom) rats. B: group results showing the contractile responses to electrical stimulation in normoxic and chronically hypoxic rats. C: group results showing the dose-related effects of U50,488H in both RV and LV myocytes of normoxic and chronically hypoxic rats with control group as 100%. For the measurement of cell contraction, the ventricular myocyte was superfused with Krebs solution; the myocyte was then electrically stimulated before administration of U50,488H. The electrically induced contraction was recorded for about 10 min after administration of U50,488H. Values are means ± SE, for cell shortening over resting length as 100%; n = 8 in each treatment group. The resting lengths of the myocytes of the normoxic and hypoxic groups were 116.7 ± 1.3 and 128.4 ± 1.5 µm, respectively (n = 16 from 8 rats, P < 0.01). **P < 0.01 vs. control. ##P < 0.01 vs. corresponding control with U50,488H in normoxic rats. , Electrical stimulation.

In the ventricular myocyte isolated from either the right hypertrophied or left nonhypertrophied ventricle of rats subjected to chronic hypoxia, the effects of U50,488H on the contraction of the myocyte were significantly attenuated to a same extent (Fig. 2, A and C).

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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Fig. 3.   Effects of U50,488H on intracellular pH (pHi) in single ventricular myocytes of the ventricles of normoxic and chronically hypoxic rats in superfusion with HEPES-buffered solution. A: representative tracings showing the effects of 20 µM U50,488H on the pHi in the presence and absence of 5 µM nor-BNI, 1 µM calphostin C, and 10 µM ethylisopropylamiloride (EIPA) in RV myocytes of normoxic rats and the effect of U50,488H in RV myocytes of chronically hypoxic rats. B: group results showing the effects of 20 µM U50,488H on the pHi in both RV and LV myocytes of normoxic and chronically hypoxic rats. For the measurement of pHi, the ventricular myocyte was superfused with HEPES-buffered solution, and 20 µM U50,488H was then administered. In experiments involving nor-BNI, the drug was administered at 5 min before and together with U50,488H. In experiments involving calphostin C and EIPA, both drugs were administered at 10 min before and together with U50,488H, respectively. The pHi was recorded at about 10 min after administration of U50,488H. Values are means ± SE; n = 10 in each group. **P < 0.01 vs. normoxic rat.



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Fig. 4.   Effects of U50,488H on pHi in single ventricular myocytes of the ventricles of normoxic and chronically hypoxic rats in superfusion with HCO3--buffered solution. A: representative tracings showing the effects of 20 µM U50,488H on the pHi in RV myocytes of normoxic rats and chronically hypoxic rats. B: group results showing the effects of 20 µM U50,488H on the pHi in both RV and LV myocytes of normoxic and chronically hypoxic rats. Values are means ± SE; n = 8 in each group. **P < 0.01 vs. normoxic rat.

In the ventricular myocyte isolated from both the right hypertrophied ventricle and the left nonhypertrophied ventricle of rats subjected to chronic hypoxia, the effects of 20 µM U50,488H on pHi both in a HEPES-buffered solution (Fig. 3, A and B) and in a bicarbonate-buffered solution (Fig. 4, A and B) were almost completely abolished. The pHi responses to 20 µM U50,488H in myocytes of both right and left ventricles in normoxic and chronically hypoxic rats were exactly the same.

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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Fig. 5.   Effects of 4-phorbol 12-myristate 13-acetate (PMA) on electrically induced [Ca2+]i transient in single ventricular myocytes of the ventricles of normoxic and chronically hypoxic rats. A: dose-related effects of PMA in the presence and absence of 1 µM calphostin C in normoxic rats. Top: representative tracing in RV myocytes. Bottom: group results with control group as 100% in both RV and LV myocytes. B: effect of 0.1 µM PMA in normoxic and chronically hypoxic rats. Top: representative tracing in RV myocytes. Bottom: group results with control group as 100% in both RV and LV myocytes. The experimental procedure was exactly the same as described in Fig. 1, except that PMA was used instead of U50,488H. Values are means ± SE; n = 12 in each group in RV myocytes and 8 in each group in LV myocytes. **P < 0.01 vs. control. ##P < 0.01 vs. corresponding control with PMA.



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Fig. 6.   Effects of PMA on pHi in single ventricular myocytes of the ventricles of normoxic and chronically hypoxic rats in superfusion with HEPES-buffered solution. A: representative tracings showing the effects of 0.1 µM PMA on the pHi in the presence and absence of 1 µM calphostin C and 10 µM EIPA in RV myocytes of normoxic rats and the effect of 0.1 µM PMA on the pHi in RV myocytes of chronically hypoxic rats. B: group results showing the effects of 0.1 µM PMA on the pHi in both RV and LV myocytes of normoxic and chronically hypoxic rats. The experimental procedure was exactly the same as described in Fig. 3, except that PMA was used instead of U50,488H. Values are means ± SE; n = 10 in each group. **P < 0.01 vs. normoxic rat.



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Fig. 7.   Effects of PMA on pHi in single ventricular myocytes of the ventricles of normoxic and chronically hypoxic rats in superfusion with HCO3--buffered solution. A: representative tracings showing the effects of 0.1 µM PMA on the pHi in RV myocytes of normoxic rats and chronically hypoxic rats. B: group results showing the effects of 0.1 µM PMA on the pHi in both RV and LV myocytes of normoxic and chronically hypoxic rats. The experimental procedure was exactly the same as described in Fig. 3, except that PMA was used instead of U50,488H and the ventricular myocyte was superfused with HCO3--buffered solution. Values are means ± SE; n = 10 in each group in RV myocytes and 8 in each group in LV myocytes. **P < 0.01 vs. normoxic rat.

In the ventricular myocyte isolated from both right and left ventricles of rats subjected to chronic hypoxia, the effects of 0.1 µM PMA on the [Ca2+]i transient (Fig. 5B) and pHi both in a HEPES-buffered solution (Fig. 6, A and B) and in a bicarbonate-buffered solution (Fig. 7, A and B) were almost completely abolished. The [Ca2+]i and pHi responses to PMA in myocytes of both right and left ventricles in chronically hypoxic rats were exactly the same.

Expression of kappa -opioid receptor mRNA in the heart of chronically hypoxic rats. To further determine the regional difference of the kappa -opioid system between the right and left ventricles, we determined the RT-PCR product of kappa -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 kappa -opioid receptor and 540-bp product for beta -actin by using kappa -opioid receptor and beta -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 kappa -opioid receptor in the heart.


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Fig. 8.   RT-PCR analysis of kappa -opioid receptor (kappa -OR) mRNA expression in the heart tissue of normoxic and chronically hypoxic rats. A: representative tracings showing the kappa -OR and beta -actin expression in both RV and LV heart of normoxic and chronically hypoxic rats. B: group results showing the normalized opioid receptor expression. The expression levels at the number of 35-40 thermocycles were found to fall within the linear range for both beta -actin and kappa -OR cDNA amplification. Product (2 µl) from the RT was taken over the 40 thermocycles of PCR reaction. It was then analyzed by electrophoresis on a 1% agarose gel and stained with ethidium bromide. Each band was analyzed with a multi-analyst (Bio-Rad). The opioid receptor expression was normalized by dividing the density of the receptor band by the density of the beta -actin band. Values are means ± SE; n = 4 in each group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The most important observation of the present study is that the [Ca2+]i and pHi responses to kappa -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 kappa -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 kappa -opioid peptides are produced (10, 52, 56) and kappa -opioid receptor is the predominant opioid receptor (25, 46, 50, 60, 62) in the heart, it is believed that the kappa -opioid peptides may regulate the cardiac function as autocrine or paracrine hormones (28, 52). An attenuated contractile response to kappa -opioid receptor stimulation may prevent excessive reduction in contractility following chronic hypoxia. It is well known that, following chronic hypoxia, the contractile response to beta -adrenergic stimulation is attenuated (8, 27), leading to reduced contractility, which is undesirable. Since kappa -opioid receptor modulates negatively the beta -adrenoceptor (59), the modulatory action of kappa -opioid receptor on beta -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 kappa -opioid receptor stimulation activates the phospholipase C/PKC (5), the attenuated cardiac responses to kappa -opioid receptor stimulation may result, at least partly, from reduced responses to PKC activation following chronic hypoxia. It has been shown that kappa -opioid receptor stimulation activates the alpha -isoform of PKC in the heart (53). Further studies are needed to determine whether the response of the alpha -isoform in the rat heart to kappa -opioid receptor stimulation is reduced or not subjected to chronic hypoxia.

It should be noted that the [Ca2+]i response to kappa -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 kappa -opioid receptor stimulation was only partially mediated, whereas the pHi response to kappa -opioid receptor stimulation was solely mediated, by PKC and/or Na+/H+ exchange. This is in agreement with our previous observation that kappa -opioid receptor stimulation increases production of inositol trisphosphate (40, 41, 42), indicating that the [Ca2+]i response to kappa -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 kappa -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 beta -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 kappa -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 kappa -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 kappa -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 kappa -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 kappa -opioid receptor activity and identify the PKC isoform involved in the heart of rats subjected to chronic hypoxia.


    ACKNOWLEDGEMENTS

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


    FOOTNOTES

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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