* Department of Veterinary Medicine, National Taiwan University, Taipei, Taiwan, ROC;
Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC;
Institute of Pharmaceutical Sciences, Taipei Medical College, Taipei, Taiwan, ROC; and
§ Department of Applied Toxicology, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taichung, Taiwan, ROC
Received September 13, 1999; accepted January 25, 2000
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ABSTRACT |
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Key Words: Ca2+ release; muscular contracture; cartap.
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INTRODUCTION |
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MATERIALS AND METHODS |
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Preparation of isolated mouse phrenic nerve diaphragm.
Male ICR mice, weighing 2025 g, were purchased from the National Laboratory Animal Breeding and Research Center, Taipei. The phrenic nerve diaphragm was isolated according to the method of Bülbring (1946). The phrenic nerve diaphragm was suspended in an organ bath containing 10 ml of the Krebs solution at 37 ± 0.5° and constantly gassed with 95% O2 plus 5% CO2. The twitches were evoked by indirect stimulation of the phrenic nerve with duration of 0.05 ms at 0.2 Hz, or by direct stimulation of the muscle with a pulse of 0.5 ms at 0.2 Hz. The muscle was loaded with a resting tension of 1 g, and the changes of tension were recorded via an isometric transducer (Grass FT.03) on a Grass Model 7D polygraph (Grass Instrument Co., Quincy, USA).
Effects of nereistoxin and cartap on isolated mouse phrenic nerve diaphragm.
To compare the action of nereistoxin and cartap on the neuromuscular junction, nereistoxin at a concentration of 1 mM or cartap at a concentration of 0.1, 1, 3, 5, 10, or 20 mM was added in the organ bath with isolated mouse phrenic nerve diaphragms. Both nerve- and muscle-evoked twitches were continuously recorded for 80 min. The cartap-induced contracture force and the time interval to initiate the contracture in the isolated mouse phrenic nerve diaphragm at various concentrations were also recorded as previously stated.
Effects of AChR, Na+ channel, and Ca2+ channel blockers on cartap-induced changes in isolated mouse phrenic nerve diaphragm.
To evaluate the effects of neuromuscular blockage on cartap-induced changes, the isolated mouse phrenic nerve diaphragm was pretreated with 0.125 µM -bungarotoxin (
-BuTx), an irreversible motor endplate ACh receptor blocker; 0.32 µM tetrodotoxin (TTX), a reversible Na+ channel blocker (O'Malley et al., 1990
); 1 mM NiCl2, a nonselective Ca2+ channel blocker; 10 µM verapamil or 1 µM nifedipine, L-type Ca2+ channel blockers for 1020 min (Fleckenstein, 1983
). Thereafter, 1 mM cartap was added when the nerve- or muscle-evoked twitches had been completely inhibited.
Effects of extracellular and intracellular Ca2+ on cartap-induced changes in isolated mouse phrenic nerve diaphragm.
For the Ca2+-free experiment, the phrenic nerve diaphragm was washed three times with the Krebs solution containing 2.5 mM EGTA and then incubated in the Krebs solution without CaCl2 (Fleckenstein, 1983). For the internal Ca2+ store depletion experiment, the diaphragm was pretreated with 4 µM ryanodine, washed with the Krebs solution containing 2.5 mM EGTA, and then incubated in the Krebs solution without CaCl2 (Chu et al., 1990
). Thereafter, 1 mM cartap was added when the tension returned to baseline.
Preparation of sarcoplasmic reticulum (SR) vesicles.
The triad enriched heavy fraction of SR vesicles was prepared from rabbit hind leg and back muscles by differential centrifugation, as described by Ikemoto et al. (1984), with modifications. Briefly, the muscle was homogenized in three times its volume of ice-cold 20 mM 3-(N-morpholino)-propanesulphonic acid (MOPS), 0.1 mM EDTA, 0.1 mM EGTA, and 0.2 mM phenylmethylsulphonyl fluoride (PMSF), pH 7.0 buffer. The homogenates were centrifuged at 10,000 x g for 5 min in a JA-14 rotor (Beckman, Inst. Inc., CA, USA). The supernatant was collected and filtered through eight layers of cheesecloth and then centrifuged at 17,000 x g for 50 min. The sediment was homogenized in a solution containing 0.3 M sucrose, 150 mM KCl, 0.2 mM PMSF, and 20 mM MOPS at pH 6.8, and centrifuged at 17,000 x g for 40 min. The sediment was further homogenized in the same solution and readjusted to a final protein concentration of 2030 mg/ml by the method of Lowry et al. (1951), with bovine serum albumin as standard. The calcium content of the isolated SR was determined by EGTA titration in the medium containing 100 µM antipyrylazo (AP) III and calculated as outlined in the calcium release assay. The preparation was quickly frozen in liquid nitrogen and stored at 70° until used.
Calcium release assay.
The calcium released from SR vesicles was determined with a calcium-sensitive probe, the AP III, in a dual wavelength spectrophotometer (SLM, Aminco DW 2000) at 710790 nm with no addition of precipitating agent, as described by Palade (1987), with some modifications. The SR vesicle preparation at a protein concentration of 30 µg/ml was actively loaded with 1 mM MgATP in a reaction mixture containing 150 mM KCl, 100 µM AP III, and 20 mM MOPS at pH 6.8. Aliquots of 510 nmol CaCl2 were added sequentially until the SR vesicles were saturated. Cartap at 1, 3.3, 19.8, or 33 mM was then added. Thereafter, polylysine at 0.526 µM or A23187 at 4 µM was added. The amount of total Ca2+ released by cartap, polylysine, or A23187 was calculated according to the absorbance-concentration curve derived from the titration of buffer containing 100 µM AP III with the addition of a known concentration of Ca2+.
Ca2+-ATPase activity assay.
The Ca2+-ATPase activity was determined with a coupled-enzyme spectrophotometric ADP-release assay based on the measurement of the oxidation of NADH at 340 nm with a Beckman DU-650 spectrophotometer (CA, USA) (Warren et al., 1974). The SR vesicle preparation at a protein concentration of 20 µg/ml was incubated with cartap at 0.1, 0.3, 1, 3, or 5 mM in 1 ml of the assay mixture containing 20 mM MOPS (pH 6.8), 0.42 mM NADH, 5 mM MgCl2, 0.2 mM EGTA, 0.45 mM phospho (enol) pyruvate, 5 units/ml pyruvate kinase, 10 units/ml lactate dehydrogenase, and 4 µM A23187, for 5 min at 37°. The reaction was started by the addition of 100 µM ATP. The activity of Ca2+-ATPase was obtained as the difference between activities measured with and without the addition of 0.2 mM CaCl2.
[3H]-ryanodine binding assay.
The ryanodine binding capacity was measured according to the method reported by Pessah et al. (1987) with modifications. The SR vesicle preparation at a protein concentration of 500 µg/ml was incubated with 0.111, 0.167, 0.25, 0.333, 1, or 3 mM cartap in a buffer containing 250 mM KCl, 15 mM NaCl, 50 µM CaCl2, 10 nM [3H]-ryanodine (2.5 TBq/nM), and 20 mM Tris at pH 7.1 for 2 h at 37°. The [3H]-ryanodine binding capacity was then measured in the presence of or without 1 µM cold ryanodine. At the end of incubation, 900 µl of the reaction mixture was withdrawn and added to 5 ml with ice-scold buffer to quench the reaction, followed by rapid filtration through a glass filter (Whatman GF/B, 24 mm), and rinsed with 5 ml of the same ice-cold buffer. The [3H]-ryanodine binding capacity of SR vesicles was then detected by a ß-scintillation counter (LS 6000 IC, Beckman, Inst. Inc., CA, USA).
Statistical analysis.
Student's t-test was used to analyze the differences between control and other groups. A p value of less than 0.05 was considered significant.
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RESULTS |
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DISCUSSION |
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When the isolated mouse phrenic nerve diaphragm was placed in the Ca2+-free bathing solution, the maximal contracture force induced by cartap was reduced by approximately 47%. However, neither the nonselective Ca2+ channel blocker NiCl2 nor the L-type Ca2+ channel blockers nifedipine and verapamil (Fleckenstein, 1983) inhibited the cartap-induced contracture. The data suggest that the cartap-induced contracture is partially associated with the influx of extracellular Ca2+, but this Ca2+ influx is probably not through the Ca2+ channels on the sarcolemma. On the other hand, there was an approximately 65% inhibition in the cartap-induced contracture force of the isolated mouse phrenic nerve diaphragm when the internal Ca2+ was depleted from SR by the pretreatment of ryanodine. This result suggests that the release of internal Ca2+ also plays an important role in the cartap-induced contracture. Moreover, when the diaphragm was pretreated with the combination of ryanodine and chelation of extracellular Ca2+ with EGTA, the contracture force induced by cartap could be further reduced to nearly 93%. Thus, the cartap-induced contracture in the isolated phrenic nerve diaphragm should be a result of the combination of release of internal and influx of extracellular Ca2+.
The Ca2+ release channel, also known as ryanodine receptor, and Ca2+-ATPase on the SR membrane regulate the intracellular free Ca2+ in the skeletal muscle by controlling the release and uptake of Ca2+ into and from the myoplasm, respectively (Hosey and Lazdunski, 1988; Hymel et al., 1988
; Lai et al., 1988
). There are several chemically diverse substances that have been shown to be able to induce the release of Ca2+ from SR via the Ca2+ release channel (Fleischer and Inui, 1989
; Palade, 1987
). Ryanodine is a plant alkaloid that binds specifically to the Ca2+ release channel (Fleischer et al., 1985
). The ligand binding has been used as a probe for the channel activity (Chu et al., 1990
), based on the fact that the Ca2+ release inducers can also significantly increase the ryanodine binding (Kang et al., 1996
; Pessah et al., 1987
). The present study has demonstrated that the release of internal Ca2+ plays an important role in the cartap-induced contracture in the isolated mouse phrenic nerve diaphragm. However, instead of causing an increase in [3H]-ryanodine binding, cartap had a dose-dependent inhibitory effect on the binding of [3H]-ryanodine to SR. In addition, cartap itself could not induce Ca2+ release from the actively loaded SR vesicles. Thus, the mechanism for cartap-related internal Ca2+ release may not be through the direct activation of the ryanodine receptor channel, although cartap could indeed interact with this Ca2+ release channel. Chemicals such as xanthine derivatives (Endo, 1975
) and halothane (Ogawa and Kurebayashi, 1982
) cause muscle to contract by changing the Ca2+ sensitivity of the ryanodine receptor channel. Whether cartap has a similar potentiating action remains to be confirmed.
It is also possible that the cartap-induced contracture was secondary to the inhibition of the SR Ca2+ pump protein Ca2+-ATPase, as the inhibition of the ATPase will cause SR to unload its Ca2+ (Hosey and Lazdunski, 1988; Lai et al., 1988
). We have observed that the Ca2+-ATPase activity in the SR vesicles was inhibited by cartap at a concentration of 3 mM or higher. However, apparent muscular contracture appeared when the concentration of cartap was 1 mM and it reached the maximal level by 5 mM. Therefore, it is unlikely that the inhibitory effect of cartap on the Ca2+-ATPase activity plays a major role, but it might act in concert with other effects of cartap to induce the muscular contracture.
We have shown that cartap has an acute fatal effect in rabbits following ocular exposure (Liao et al., 1998). The isolated mouse and rat phrenic nerve diaphragms have been widely used for studying the effects of drugs or chemicals on neuromuscular transmission (Bülbring, 1946
; Kang et al., 1996
; Yan et al., 1993
). Currently, we have been trying to establish the methodology of using isolated rabbit phrenic nerve diaphragm to study the effect of cartap on its neuromuscular junction. The preliminary result has shown that cartap induced a similar contracture pattern in both isolated rabbit and mouse phrenic nerve diaphragms (unpublished data). Thus, we propose that the acute lethal effect of cartap on rabbits via ocular exposure might have resulted from respiratory failure secondary to the diaphragm contracture instead of the commonly recognized neuromuscular blockade. Cartap may exert its effect by the promotion of the extracellular Ca2+ influx and the induction of internal Ca2+ release. However, the underlying mechanism for the changes in Ca2+ flow remains unclear and needs to be further elucidated.
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NOTES |
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1 To whom correspondence should be addressed at Department of Veterinary Medicine, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei, Taiwan 106. Fax: 886-2-23661475. E-mail: pang{at}ccms.ntu.edu.tw.
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