Acidosis antagonizes intracellular calcium response to kappa -opioid receptor stimulation in the rat heart

Jian-Ming Pei, Xiao-Chun Yu, Jin-Song Bian, 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

To study the effects of kappa -opioid receptor stimulation on intracellular Ca2+ concentration ([Ca2+]i) homeostasis during extracellular acidosis, we determined the effects of kappa -opioid receptor stimulation on [Ca2+]i responses during extracellular acidosis in isolated single rat ventricular myocytes, by a spectrofluorometric method. U-50488H (10-30 µM), a selective kappa -opioid receptor agonist, dose dependently decreased the electrically induced [Ca2+]i transient, which results from the influx of Ca2+ and the subsequent mobilization of Ca2+ from the sarcoplasmic reticulum (SR). U-50488H (30 µM) also increased the resting [Ca2+]i and inhibited the [Ca2+]i transient induced by caffeine, which mobilizes Ca2+ from the SR, indicating that the effects of the kappa -opioid receptor agonist involved mobilization of Ca2+ from its intracellular pool into the cytoplasm. The Ca2+ responses to 30 µM U-50488H were abolished by 5 µM nor-binaltorphimine, a selective kappa -opioid receptor antagonist, indicating that the event was mediated by the kappa -opioid receptor. The effects of the agonist on [Ca2+]i and the electrically induced [Ca2+]i transient were significantly attenuated when the extracellular pH (pHe) was lowered to 6.8, which itself reduced intracellular pH (pHi) and increased [Ca2+]i. The inhibitory effects of U-50488H were restored during extracellular acidosis in the presence of 10 µM ethylisopropyl amiloride, a potent Na+/H+ exchange blocker, or 0.2 mM Ni2+, a putative Na+/Ca2+ exchange blocker. The observations indicate that acidosis may antagonize the effects of kappa -opioid receptor stimulation via Na+/H+ and Na+/Ca2+ exchanges. When glucose at 50 mM, known to activate the Na+/H+ exchange, was added, both the resting [Ca2+]i and pHi increased. Interestingly, the effects of U-50488H on [Ca2+]i and the electrically induced [Ca2+]i transient during superfusion with glucose were significantly attenuated; this mimicked the responses during extracellular acidosis. When a high-Ca2+ (3 mM) solution was superfused, the resting [Ca2+]i increased; the increase was abolished by 0.2 mM Ni2+, but the pHi remained unchanged. Like the responses to superfusion with high-concentration glucose and extracellular acidosis, the responses of the [Ca2+]i and electrically induced [Ca2+]i transients to 30 µM U-50488H were also significantly attenuated. Results from the present study demonstrated for the first time that extracellular acidosis antagonizes the effects of kappa -opioid receptor stimulation on the mobilization of Ca2+ from SR. Activation of both Na+/H+ and Na+/Ca2+ exchanges, leading to an elevation of [Ca2+]i, may be responsible for the antagonistic action of extracellular acidosis against kappa -opioid receptor stimulation.

sodium/hydrogen exchange; sodium/calcium exchange; sarcoplasmic reticulum


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACIDOSIS IS WELL KNOWN TO affect the cardiac functions (9, 12, 24). During myocardial ischemia, extracellular acidosis occurs with a lowered intracellular pH (pHi), which decreases contractility (3, 33). It has been shown that intracellular acidosis activates Na+/H+ exchange, leading to an increased extrusion of H+ in exchange for an influx of Na+ (9, 25, 29). The increased activity of Na+/H+ exchange in turn activates the reverse mode of Na+/Ca2+ exchange, leading to an increased influx of Ca2+ and an elevation of intracellular Ca2+ concentration ([Ca2+]i) (5, 30). The altered [Ca2+]i homeostasis may account, at least in part, for the altered contractility during extracellular acidosis (31, 42).

Both kappa -opioid receptors (27, 44, 45, 53, 54) and kappa -opioid peptides (48) are present in the heart. Previous studies have shown that during myocardial ischemia the kappa -opioid receptor is activated presumably because of an increased release of kappa -opioid peptides from the heart (50, 51). It has been shown previously that kappa -opioid receptor stimulation with a selective kappa -opioid receptor agonist increases [Ca2+]i by mobilizing Ca2+ from the sarcoplasmic reticulum (SR) via the phospholipase C/Ca2+ pathway (7, 36-38, 43, 46, 47). It has been shown that kappa -opioid receptor stimulation also negatively modulates the stimulatory effects of beta -adrenoreceptor stimulation on cardiac contractility (52). Depletion of Ca2+ from SR and negative modulation of the beta -adrenoreceptor are responsible, at least in part, for the inhibitory action of kappa -opioid receptor stimulation on cardiac contractility.

Because both acidosis and kappa -opioid receptor activation occur during myocardial ischemia, it would be important to study the interaction between acidosis and kappa -opioid receptor stimulation and the underlying mechanisms. The effect of acidosis on kappa -opioid receptor stimulation is, however, not known. In the present study we determined the Ca2+ responses to kappa -opioid receptor stimulation in the heart during extracellular acidosis. We measured the [Ca2+]i transients induced electrically and with caffeine, in addition to the resting [Ca2+]i, in a single isolated ventricular myocyte preparation. The electrically induced [Ca2+]i transient results from the influx of Ca2+ on membrane depolarization, which triggers Ca2+ release from the SR via a Ca2+-induced Ca2+-release mechanism, and has been shown to correlate directly to the contraction of the myocyte (52). The caffeine-induced [Ca2+]i transient is an index of the Ca2+ content in the SR because caffeine depletes SR of Ca2+ (4, 40). We also determined the Ca2+ responses to kappa -opioid receptor stimulation after manipulations that stimulate or mimic Na+/H+ exchange and Na+/Ca2+ exchange in normal extracellular pH (pHe). In addition, we determined the Ca2+ responses during extracellular acidosis when these two exchanges were inhibited. Results from the present study showed that extracellular acidosis antagonizes the effects of kappa -opioid receptor stimulation on Ca2+ responses in the heart. This action may result from activation of Na+/H+ and Na+/Ca2+ exchanges, which elevates the [Ca2+]i.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Isolation of ventricular myocytes. Ventricular myocytes were isolated from the hearts of male Sprague-Dawley rats (190-210 g), by a collagenase perfusion method described previously (14). Immediately after decapitation, the hearts were rapidly removed from the rats and perfused in a retrograde manner at a constant flow rate (10 ml/min) with oxygenated Joklik MEM supplemented with 1.25 mM CaCl2 and 10 mM HEPES, pH 7.2, at 37°C for 5 min; this was 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 a medium containing collagenase, the atria were discarded. The ventricular tissue was transferred to an oxygenated solution that was the same as that described above but without collagenase; the tissue was cut into small pieces with a pair of scissors, and then the solution was stirred with a glass rod for 5 min to separate 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 2% 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 in 40 min.

Measurement of [Ca2+]i. Ventricular 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 (24-26°C) for 30 min before measurements of [Ca2+]i to allow the fura 2-AM in the cytosol to deesterify. Loading with a low concentration of fura 2-AM and at a relatively low temperature of 24-26°C was done to minimize the effects of the compartmentalization of the esters (39).

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 a Krebs bicarbonate buffer containing (in mM) 118 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. To produce extracellular acidosis, HCl was used to adjust to pH 6.8, which was chosen on the basis of previous studies (1, 11, 28). The myocytes selected for the study were rod shaped with clear striations. They were quiescent but 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. Fluorescence signals obtained at 340-nm (F340) and at 380-nm (F380) excitation wavelengths were stored in a computer for data processing and analysis. The F340/F380 ratio was used to represent [Ca2+]i changes in the myocytes. In some experiments, the resting [Ca2+]i was observed while the electrical stimulation was off.

Measurement of pHi. The pHi was measured in a single myocyte as described previously (10). The apparatus and optical arrangement used for the measurement of fluorescent light emission and the preparation procedure were similar to those described in the previous section except that the cells were loaded with the membrane-permeable 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 a Krebs solution (normal solution; pHe = 7.4) or an acidic solution (pHe = 6.8), as described above, according to the requirement of the experiment. The pH-dependent signal of BCECF was obtained by illuminating at 490 and 435 nm, and the fluorescence emission wavelength was measured at 520 nM. The ratio of fluorescence at 490 nm (F490) to F435 was used to represent pHi.

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

Drugs and chemicals. U-50488H {trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide}, fura 2-AM, type I collagenase, and nigericin were purchased from Sigma. The nor-binaltorphimine (nor-BNI) was purchased from Tocris Cookson. BCECF-AM and ethylisopropyl amiloride (EIPA) were purchased from Research Biochemicals Incorporated.

Fura 2-AM, BCECF-AM, and EIPA were dissolved in DMSO, nigericin was dissolved in ethanol, and the rest were dissolved in distilled water.

U-50488H 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. In experiments conducted during extracellular acidosis the kappa -opioid agonist was administered for 10 min after the perfusion of a solution at pHe 6.8 for 10 min because we also found that the effect of acidosis reached the maximum at ~10 min. The dose range used in the present study has been shown to increase the [Ca2+]i and inositol 1,4,5-trisphosphate level, effects antagonized by 1-5 mM nor-BNI (51, 54, 55), which itself had no effect on any of the preparations studied. In a preliminary experiment, 10 µM EIPA did not alter the autofluorescence of the cell at the BCECF excitation wavelength as previously reported (29). The final concentration of DMSO was 0.1%, and at this concentration DMSO had no effect on either [Ca2+]i or pHi (29).

Statistical analysis. Values are expressed as means ± SE. The paired Student's t-test was used to determine the difference between control and drug treatment groups. The unpaired Student's t-test was employed to determine the difference among groups. The significance level was set at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of extracellular acidosis on pHi and [Ca2+]i. When the ventricular myocyte was superfused with a Krebs solution at pHe 6.8, the pHi dropped slowly to a steady state (from 7.03 ± 0.03 to 6.65 ± 0.04; P < 0.01) with a half time of 2.2 ± 0.42 min (n = 5). After this short period, during which the pHi remained at the low level, the pHi rose gradually by 0.14 ± 0.04 (n = 8; P < 0.01) within 10 min during acidosis (Fig. 1). The recovery in pHi was abolished by 10 µM EIPA, a blocker of Na+/H+ exchange (29) (Fig. 1).


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Fig. 1.   Typical tracings from 4-8 experiments showing time course changes in intracellular pH (pHi) and resting fura 2 fluorescence ratio during extracellular acidosis in presence and absence of ethylisopropyl amiloride (EIPA) and Ni2+ in single ventricular myocyte. pHe, extracellular pH.

As shown in Fig. 1, extracellular acidosis also gradually increased the fura 2 fluorescence ratio from 0.95 ± 0.07 to 1.1 ± 0.08 (n = 4; P < 0.01) within 10 min, indicating a gradual increase in the resting [Ca2+]i. The elevation was blocked by 10 µM EIPA and 0.2 mM Ni2+, a putative blocker of Na+/Ca2+ exchange (6).

[Ca2+]i responses to U-50488H during extracellular acidosis. In this series of experiments we determined the resting [Ca2+]i and the [Ca2+]i transients induced electrically and with caffeine in a single ventricular myocyte. In view of the fact that the changes in the electrically induced [Ca2+]i transient were large and easily quantified, the dose-related response was determined by observing the responses of the electrically induced [Ca2+]i transient. In agreement with previous observations (23, 38), 10-30 µM U-50488H, a selective kappa -opioid receptor agonist, suppressed the electrically induced [Ca2+]i transient in a concentration-dependent manner (Fig. 2). The effect of the kappa -agonist at 30 µM was abolished by 5 µM nor-BNI, a selective kappa -opioid receptor antagonist (Fig. 2).



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Fig. 2.   Effects of kappa -opioid receptor stimulation with U-50488H on electrically induced intracellular Ca2+ concentration ([Ca2+]i) transient during extracellular acidosis in presence and absence of nor-BNI (5 µM) in a single ventricular myocyte. The ventricular myocyte was superfused with a solution at pHe 6.8 for 10 min when effects of low pH reached a plateau as shown in Fig. 1. U-50488H was then administered. For measurement of electrically induced [Ca2+]i transient, cell was electrically stimulated before administration of U-50488H. In experiments involving nor-BNI, drug was administered 5 min before and together with U-50488H. Both resting [Ca2+]i and electrically induced [Ca2+]i transients were recorded at ~10 min after administration of U-50488H. Same procedure was employed for measurement of caffeine-induced [Ca2+]i transient except that myocyte was given a bolus dose of 10 mM caffeine after electrical stimulation. Shown are representative tracings of effects of 30 µM U-50488H at pHe 7.4 (A) and 6.8 (B). C: dose-related effects of U-50488H. Values are means ± SE; n = 4. * P < 0.05, ** P < 0.01 vs. corresponding control without U-50488H; ++ P < 0.01 vs. corresponding control group in normal pH.

U-50488H at 30 µM also increased the resting [Ca2+]i (Fig. 3) and inhibited the caffeine-induced [Ca2+]i transient (Fig. 4). The effect of 30 µM U-50488H on the resting [Ca2+]i was also abolished by 5 µM nor-BNI (Fig. 3). The observations are in agreement with previous findings (37, 47).


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Fig. 3.   Effects of kappa -opioid receptor stimulation with U-50488H (U50) on resting [Ca2+]i in presence of 5 µM nor-BNI and in absence of nor-BNI. A: representative tracings showing effect of 30 µM U-50488H. B: group results. Values are means ± SE; n = 6. ** P < 0.01 vs. corresponding control.




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Fig. 4.   Effects of kappa -opioid receptor stimulation with U-50488H on caffeine-induced [Ca2+]i transient. A-C: representative tracings showing effect of 30 µM U-50488H at pH 7.4 (A and B) and at pH 6.8 (C). D: group results showing effects of 30 µM U-50488H on [Ca2+]i transients induced both electrically and with caffeine. ES, electrical stimulation; con, control. Values are means ± SE; n = 4. * P < 0.05, ** P < 0.01 vs. corresponding control without U-50488H; ++ P < 0.01 vs. corresponding control group in normal pH.

After the ventricular myocytes had been superfused with a solution at pHe 6.8 for 10 min, during which time the cells stabilized, the effects of 30 µM U-50488H on the resting [Ca2+]i (Fig. 3) were abolished, and those on the [Ca2+]i transients induced electrically (Fig. 2) and with caffeine (Fig. 4) were significantly attenuated.

The attenuating effect of the kappa -opioid receptor agonist on the electrically induced [Ca2+]i transient during extracellular acidosis was restored by 10 µM EIPA and 0.2 mM Ni2+(Fig. 5).



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Fig. 5.   Effects of kappa -opioid receptor stimulation with U-50488H on electrically induced [Ca2+]i transient upon blockade of Na+/H+ or Na+/Ca2+ exchange during extracellular acidosis in a single ventricular myocyte. A-D: representative tracings at pH 7.4 (A) and at pH 6.8 either without EIPA or Ni2+ (B), with EIPA (C), or with Ni2+ (D). E: group results showing effects of 30 µM U-50488H at pH 7.4 and at pH 6.8 in presence of either 10 µM EIPA, a Na+/H+ exchange blocker, or 0.2 mM Ni2+, a Na+/Ca2+ exchange blocker. Experimental procedure was exactly the same as that described in legend for Fig. 2. EIPA and Ni2+ were administered together with the acidic solution. Values are means ± SE; n = 8. * P < 0.05, ** P < 0.01 vs. corresponding control without U-50488H. ++ P <0.01 vs. group at pHe 7.4.

Effects of U-50488H on [Ca2+]i and electrically induced [Ca2+]i transients during superfusion with a high-glucose solution. To further delineate the role of Na+/H+ exchange in mediating the action of extracellular acidosis on the Ca2+ responses to kappa -opioid receptor stimulation, a solution containing a high glucose concentration, known to activate Na+/H+ exchange as a result of acute osmotic stimulation (49), was superfused. Both the [Ca2+]i and electrically induced [Ca2+]i transient in response to U-50488H were determined. In agreement with previous observations (34, 41), both the pHi (Fig. 6A) and the fura 2 fluorescence ratio (Fig. 6B) increased gradually on superfusion with a 50 mM glucose solution. The pHi and the resting fura 2 fluorescence ratio increased by 0.14 ± 0.04 (n = 5; P < 0.01) and 0.12 ± 0.05 (n = 5; P < 0.01), respectively, within 10 min. More importantly, the high glucose concentration abolished the effects of 30 µM U-50488H on [Ca2+]i (Fig. 6B), and significantly attenuated its effects on the electrically induced [Ca2+]i transient (Fig. 6, C and D), in the ventricular myocyte. The effects were similar to those of extracellular acidosis.


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Fig. 6.   Effects of kappa -opioid receptor stimulation with U-50488H on resting [Ca2+]i and electrically induced [Ca2+]i transient during extracellular high-glucose (50 mM) perfusion in single ventricular myocyte. Experimental procedure was same as that in legend for Fig. 2 except that high-glucose solution was superfused for 10 min before electrical stimulation and/or administration of U-50488H. A: typical tracing of 5 experiments on changes in pHi. B: typical tracing of 6 experiments on changes in resting fura 2 fluorescence ratio. C: typical tracings showing effects of 30 µM U-50488H on electrically induced [Ca2+]i transient. D: group results showing effects of 30 µM U-50488H on electrically induced [Ca2+]i transient. Values are means ± SE; n = 6 in all groups. Hglucose, high glucose. ** P < 0.01 vs. corresponding control without U-50488H. ++ P < 0.01 vs. corresponding group without high glucose.

Effects of U-50488H on [Ca2+]i and electrically induced [Ca2+]i transients in extracellular high Ca2+. This series of experiments was performed to further determine the roles of [Ca2+]i and Na+/Ca2+ exchange in mediating the action of acidosis. Superfusion with a high (3 mM) extracellular Ca2+ solution gradually increased the resting fura 2 fluorescence ratio by 0.21 ± 0.06 within 10 min (n = 5; P < 0.01; Fig. 7B), whereas the pHi remained the same (Fig. 7A). The rise in [Ca2+]i was abolished by 0.2 mM Ni2+ (Fig. 7B).



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Fig. 7.   Effects of kappa -opioid receptor stimulation with U-50488H on resting [Ca2+]i and electrically induced [Ca2+]i transient during high extracellular Ca2+ concentration ([Ca2+]o; 3 mM) superfusion in single ventricular myocyte. Experimental procedure was same as that described in legend for Fig. 2 except that high-Ca2+ solution was superfused for 10 min before electrical stimulation and/or administration of U-50488H. A: typical tracing of 4 experiments on changes in pHi. B: typical tracing of 6 experiments on changes in resting fura 2 fluorescence ratio in presence of 0.2 mM Ni2+ or in absence of Ni2+. C: typical tracings showing effects of 30 µM U-50488H on electrically induced [Ca2+]i transient. D: group results showing effects of 30 µM U-50488H on electrically induced [Ca2+]i transient. Values are means ± SE; n = 6 in all groups. Hcalcium, high calcium. * P < 0.05, ** P < 0.01 vs. corresponding control without U-50488H. ++ P < 0.01 vs. corresponding group without high [Ca2+]o.

In the 3 mM Ca2+ solution, the effects of 30 µM U-50488H on [Ca2+]i in the ventricular myocyte were abolished (Fig. 7B) and those on the electrically induced [Ca2+]i transient significantly attenuated (Fig. 7, C and D).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The most important finding of the present study was that the effects of kappa -opioid receptor stimulation with the selective kappa -opioid receptor agonist U-50488H on resting [Ca2+]i and on [Ca2+]i transients induced electrically and with caffeine were significantly attenuated during extracellular acidosis, which decreased pHi (13, 20) but increased the resting [Ca2+]i (10, 18, 21) via activation of the Na+/H+ and Na+/Ca2+ exchanges (2, 15, 22, 32, 35). The observations demonstrate for the first time that extracellular acidosis antagonizes the Ca2+ responses to kappa -opioid receptor stimulation in the heart. This is also the first demonstration that extracellular acidosis antagonizes the Ca2+ response to stimulation of a receptor.

Previous studies have shown that kappa -opioid receptor stimulation increases the resting [Ca2+]i and decreases the electrically induced [Ca2+]i transient, which is due to an initial influx of Ca2+ upon membrane depolarization and a subsequent release of Ca2+ from the SR, and the caffeine-induced [Ca2+]i transient, which is an index of Ca2+ content in the SR (37, 38, 47). The observations indicate that kappa -opioid receptor stimulation mobilizes Ca2+ from its intracellular store, leading to an increase in the cytosolic Ca2+. In the present study we found that extracellular acidosis antagonizes all these responses to kappa -opioid receptor stimulation, suggesting that extracellular acidosis may inhibit mobilization of Ca2+ from the intracellular store upon kappa -opioid receptor stimulation.

In the present study we made two interesting observations. First, blockade of these two exchanges by their respective blockers, EIPA and Ni2+, restored the effects of U-50488H during extracellular acidosis. Second, activation of Na+/H+ exchange with an osmotic stimulation by a high extracellular glucose concentration, which increased pHi and resting [Ca2+]i (34, 41, 49), or an increase in extracellular Ca2+ concentration ([Ca2+]o), known to activate the reverse mode of Na+/Ca2+ exchange and to increase the resting [Ca2+]i (26), mimicked the influence of extracellular acidosis on the Ca2+ responses to U-50488H. The observations suggest that extracellular acidosis may activate Na+/H+ exchange, which in turn may activate Na+/Ca2+exchange. In summary, extracellular acidosis may antagonize the effects of kappa -opioid receptor stimulation on Ca2+ mobilization from its intracellular store via Na+/H+ and Na+/Ca2+ exchanges, thus reducing the inhibitory effects of kappa -opioid receptor stimulation on muscle contraction. On the other hand, acidosis is well established to inhibit the binding of Ca2+ to troponin, thus decreasing contractility (8, 19, 31). However, the functional implication of the antagonism of extracellular acidosis against kappa -opioid receptor stimulation and its effect on the contractility of the heart, especially during myocardial ischemia, need further study.

Another interesting observation in the present study is that extracellular acidosis, which reduced pHi and elevated [Ca2+]i, high extracellular glucose, which increased both pHi and [Ca2+]i, and high [Ca2+]o, which only elevated resting [Ca2+]i without affecting pHi, all attenuated the Ca2+ response to U-50488H. The observations suggest that the elevation of resting [Ca2+]i, not pHi, may mediate the inhibitory action of extracellular acidosis on the Ca2+ response to kappa -opioid receptor stimulation. Because it was found that cytosolic Ca2+ inhibits the release of Ca2+ from the SR (16, 17), further studies are needed to determine whether or not the elevation in the resting [Ca2+]i during extracellular acidosis inhibits directly the mobilization of Ca2+ from the SR on kappa -opioid receptor stimulation.

One possible shortcoming of the present study was the use of a high glucose concentration to activate Na+/H+ exchange because a high glucose concentration has metabolic effects. In the present study we found that a high glucose concentration, which increased [Ca2+]i, attenuated the action of kappa -opioid receptor stimulation, an effect similar to that of extracellular acidosis and a high [Ca2+]o. In view of the fact that there is no specific activator of Na+/H+ exchange, a high glucose concentration is the only choice available.

In conclusion, the present study has provided evidence for the first time that acidosis antagonizes the effects of kappa -opioid receptor stimulation on mobilization of Ca2+ from SR in the heart. Both Na+/H+ exchange and Na+/Ca2+ exchange are involved in the process, leading to an elevation of [Ca2+]i, which may inhibit Ca2+ release from the SR induced by kappa -opioid receptor stimulation. Further study is needed to determine the physiological implication of the antagonism of extracellular acidosis on the effects of kappa -opioid receptor stimulation and to verify the role of [Ca2+]i in the mobilization of Ca2+ from its intracellular store and delineate the underlying mechanisms.


    ACKNOWLEDGEMENTS

We thank Dr. Fanny Mo for advice on the measurement of intracellular pH, Dr. I. Bruce for advice on English, and C. P. Mok for assistance.


    FOOTNOTES

This study was supported by the Research Grant Council, Hong Kong, China.

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

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. §1734 solely to indicate this fact.

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).

Received 25 January 1999; accepted in final form 27 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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