Effects of Cartap on Isolated Mouse Phrenic Nerve Diaphragm and Its Related Mechanism

Jiunn-Wang Liao*, Jaw-Jou Kang{dagger}, Shing-Hwa Liu{dagger}, Chian-Ren Jeng*, Yu-Wen Cheng{dagger}, Chien-Ming Hu{ddagger}, San-Fu Tsai§, Shun-Cheng Wang§ and Victor Fei Pang*,1

* Department of Veterinary Medicine, National Taiwan University, Taipei, Taiwan, ROC; {dagger} Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC; {ddagger} 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cartap, a nereistoxin analogue pesticide, is reported to have no irritation to eyes in rabbits. However, we have demonstrated recently that cartap could actually cause acute death in rabbits via ocular exposure. Our preliminary study with isolated mouse phrenic nerve diaphragms has shown that instead of neuromuscular blockade, cartap caused muscular contracture. The objective of the study was to examine the effect of cartap on the neuromuscular junction in more detail and to investigate its possible underlying mechanism with isolated mouse phrenic nerve diaphragms and sarcoplasmic reticulum (SR) vesicles. Cartap or nereistoxin at various concentrations was added in the organ bath with isolated mouse phrenic nerve diaphragm and both nerve- and muscle-evoked twitches were recorded. Instead of blocking the neuromuscular transmission as nereistoxin did, cartap caused contracture in stimulated or quiescent isolated mouse phrenic nerve diaphragm. Both the cartap-induced muscular contracture force and the time interval to initiate the contracture were dose-dependent. The contracture induced by cartap was not affected by the pretreatment of the diaphragm with the acetylcholine receptor blocker {alpha}-bungarotoxin; the Na+ channel blocker tetrodotoxin; or various Ca2+ channel blockers, NiCl2, verapamil, and nifedipine. On the contrary, the contracture was significantly inhibited when the diaphragm was pretreated with ryanodine or EGTA containing Ca2+-free Krebs solution or in combination. This suggested that both internal and extracellular Ca2+ might participate in cartap-induced skeletal muscle contracture. Moreover, cartap inhibited the [3H]-ryanodine binding to the Ca2+ release channel of SR in a dose-dependent manner. Additionally, cartap could induce a significant reduction in Ca2+-ATPase activity of SR vesicles at a relatively high dose. The results suggested that cartap might cause the influx of extracellular Ca2+ and the release of internal Ca2+, with subsequent induction of muscular contracture in the isolated mouse phrenic nerve diaphragm. Based on these findings, we propose that the acute death of rabbits following ocular exposure to cartap might have resulted from respiratory failure secondary to diaphragm contracture.

Key Words: Ca2+ release; muscular contracture; cartap.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cartap is a pesticide first introduced in Japan in 1967 and commonly used to control weevil and caterpillars (Chiba et al., 1967Go; Ray, 1991Go). Its basic chemical structure is S,S-[2-(dimethylamino)-1,3-propanediyl] dicarbamothioate and is normally used as the hydrochloride C7H15N3O2S3HCl (Ray, 1991Go; Tomlin, 1997Go). Its commercial name includes Padan®, Thiobel®, or Vegetox®. Cartap has long been recognized as an analogue of nereistoxin (Ray, 1991Go). Nereistoxin is a neurotoxic substance and was initially isolated from the marine annelid Lumbriconereis heteropoda. It inhibits the postsynaptic nicotinic acetylcholine receptor (AChR) ion channels (Eldefrawi et al., 1980Go). A recent study with clonal rat phaeochromocytoma cells has shown that cartap also acts on the nicotinic AChR channel by the single-channel patch-clamp technique (Nagata et al., 1997Go). As a result, it is generally stated that similar to nereistoxin, cartap may also produce its acute toxicity by neuromuscular blockade, leading to respiratory failure (Ray, 1991Go). Cartap is generally considered a relatively safe compound. Its oral LD50s in the rat, mouse, and monkey are 325–392, 150–225, and 100–200 mg/kg body weight (BW), respectively (Ray, 1991Go; Tomlin, 1997Go). It has also been indicated that cartap causes little or no ocular and skin irritations in the rabbit, and its dermal LD50 in the rabbit is 820 mg/kg BW (Ray, 1991Go; Tomlin, 1997Go). However, it has been reported recently in Japan that there are several human cartap poisoning cases every year (NRIPS, 1997Go). In Taiwan, cartap is one of the most extensively used pesticides for the control of rice and vegetable insects (TAIA, 1996Go). Owing to the potential of eye exposure for farmers using the commercial soluble powder (SP) of cartap product, we have reassessed its risk of eye irritation. It was found that the 50% SP as well as the 96% and 98% technical grade products could cause acute death in rabbits within 20 min following placement of 50 mg of the test compound in the lower conjunctival sac (Liao et al., 1998Go). Additionally, our preliminary study with isolated mouse phrenic nerve diaphragm showed that in either stimulated or quiescent state, cartap caused contracture instead of neuromuscular blockade as nereistoxin did (Liao et al., 1998Go). In order to further characterize the action of cartap, its effect on neuromuscular junction and the associated possible underlying mechanism were studied using isolated mouse phrenic nerve diaphragms and sarcoplasmic reticulum (SR) vesicles.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Cartap of 98% purity was obtained from Leenung Co. in Taiwan, which was authorized by Takeda Co. (Osaka, Japan). The purity of cartap was double-checked by the use of gas chromatography (GC-3600) with flame photometric detector (Varian Associates, Walnut Creek, CA, USA), as described by Nishi et al. (1971), with slight modification. Nereistoxin and ryanodine were purchased form WAKO Pure Chemical Industries Ltd. (Osaka, Japan) and Latoxan Co. (Rosans, France), respectively. Other chemicals used in the experiments were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). A 0.5 M cartap stock solution in distilled water was freshly prepared right before each experiment. It was then further diluted in a modified Krebs solution containing 130.6 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 12.5 mM NaHCO3, and 11.1 mM glucose, with 2.5 mM CaCl2 or with 2.5 mM EGTA to chelate the Ca2+ at pH 7.4 (Kang et al., 1996Go), according to the desired concentration. Cartap is stable in both distilled water and Krebs solution for more than 3 days (unpublished data) and all cartap-related assays were done within 2 h.

Preparation of isolated mouse phrenic nerve diaphragm.
Male ICR mice, weighing 20–25 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 {alpha}-bungarotoxin ({alpha}-BuTx), an irreversible motor endplate ACh receptor blocker; 0.32 µM tetrodotoxin (TTX), a reversible Na+ channel blocker (O'Malley et al., 1990Go); 1 mM NiCl2, a nonselective Ca2+ channel blocker; 10 µM verapamil or 1 µM nifedipine, L-type Ca2+ channel blockers for 10–20 min (Fleckenstein, 1983Go). 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, 1983Go). 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., 1990Go). 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 20–30 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 710–790 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 5–10 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., 1974Go). 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of Nereistoxin and Cartap on Isolated Mouse Phrenic Nerve Diaphragm
The typical traces of the effect of nereistoxin and cartap on the neuromuscular transmission of isolated mouse phrenic nerve diaphragm are shown in Figure 1Go. Nereistoxin at a concentration of 1 mM caused a complete blockage of the indirectly nerve-evoked twitches; however, it had no effects on the directly elicited muscle twitches (Fig. 1AGo). The inhibition in the indirectly elicited muscle twitches was reversible, and the twitch tension returned to the control level after nereistoxin was washed off with Krebs solution.



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FIG. 1. The effect of nereistoxin and cartap on the isolated mouse phrenic nerve diaphragm. The effect of nereistoxin (trace A) and cartap (trace B) at 1 mM each on the isolated mouse phrenic nerve diaphragm was examined as outlined in Materials and Methods. The muscle twitches evoked by stimulating the phrenic nerve with duration of 0.05 ms at 0.2 Hz are designated as the indirectly nerve-evoked twitches, and those evoked by stimulating the muscle with a pulse of 0.5 ms at 0.2 Hz are designated as the directly elicited muscle twitches. The muscle was loaded with a resting tension of 1 g, and the changes of tension were recorded via an isometric transducer on a Grass Model 7D polygraph. Both nerve- and muscle-evoked twitches were continuously recorded for 80 min. Prior to the last 10 min of recording, the diaphragm was further washed (W) with Krebs solution to evaluate the reversibility of the effect of each treatment. Results are representative of five different experiments.

 
In contrast to nereistoxin, cartap at the same concentration of 1 mM did not affect the indirectly nerve-evoked twitches but induced a marked contracture in the isolated mouse phrenic nerve diaphragm (Fig. 1BGo). The cartap-induced contracture force was in a dose-dependent manner. It was 0.9 ± 0.07 g when the concentration of cartap was 0.1 mM. The contracture force increased sharply to 3.78 ± 0.31 g as the concentration increased from 0.1 to 3 mM and reached the plateau 4.78 ± 0.52 g by 5 mM (Fig. 2AGo). On the contrary, the time interval to initiate the contracture by cartap was in reverse to the concentration. It was shortened from 17.71 ± 0.12 min at 0.1 mM to 2.88 ± 1.16 min at 20 mM (Fig. 2BGo).



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FIG. 2. The effect of cartap on the contracture of isolated mouse phrenic nerve diaphragm. The isolated mouse phrenic nerve diaphragm was prepared as described in Materials and Methods. The effect of cartap at various doses on the contracture force (A) and the time interval to initiate contracture (B) were measured. Data are presented as the mean ± SD of four different experiments; vertical bars represent SD.

 
The Role of AChR, Na+, and Extracellular and Intracellular Ca2+ Involved in Cartap-Induced Muscular Contracture of Isolated Mouse Phrenic Nerve Diaphragm
As reported by O'Malley et al. (1990), {alpha}-BuTx at 0.125 µM completely inhibited the nerve-evoked twitches (Fig. 3AGo), and TTX at 0.32 µM completely inhibited both nerve- and muscle-evoked stimulation (Fig. 3BGo) in the isolated mouse phrenic nerve diaphragm. The time period to reach the complete inhibition was longer in {alpha}BuTx than in TTX. However, the pretreatment of the phrenic nerve diaphragm with either {alpha}-BuTx (Fig. 3AGo) or TTX (Fig. 3BGo) did not affect the muscular contracture induced by 1 mM of cartap. The results indicated that the effect of cartap on the isolated mouse phrenic nerve diaphragm should be myogenic.



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FIG. 3. The effect of {alpha}-bungarotoxin ({alpha}-BuTx) and tetrodotoxin (TTX) on the muscular contracture of isolated mouse phrenic nerve diaphragm induced by cartap. The isolated mouse phrenic nerve diaphragm was pretreated with 0.125 µM {alpha}-BuTx or 0.32 µM TTX. Cartap at 1 mM was then added when the nerve-evoked (A) and nerve- and muscle-evoked twitches (B) were completely inhibited by {alpha}-BuTx and TTX, respectively. Results are representative of five different experiments.

 
The cartap-induced contracture force was significantly reduced by approximately 47% in the Ca2+-free Krebs bathing solution, consisting of Ca2+-free Krebs solution and 2.5 mM EGTA. However, no significant effects were seen in the muscular contracture following the addition of the nonselective Ca2+ blocker NiCl2, or voltage-dependent Ca2+ channel blockers verapamil and nifedipine (Table 1Go). A nearly 65% and statistically significant reduction in the cartap-induced contracture force was seen in the ryanodine-pretreated diaphragm. The reduction rate in the cartap-induced contracture force was further enhanced to about 93% with the combination of ryanodine treatment and removal of extracellular Ca2+ by EGTA (Table 1Go).


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TABLE 1 The Effect of Extracellular and Intracellular Ca2+ on the Cartap-Induced Muscular Contracture Force of Isolated Mouse Phrenic Nerve Diaphragm
 
Effects of Cartap on the Calcium Release Channel and the Activity of Ca2+-ATPase of Sarcoplasmic Reticulum
The direct effect of cartap on the Ca2+ uptake and release of SR vesicles isolated from rabbit skeletal muscle was monitored by a metallochromic Ca2+ indicator dye, antipyrilazo III, and the typical trace is shown in Figure 4Go. The Ca2+ in the reaction medium, originating from the SR vesicles, was rapidly translocated back into the SR vesicles upon the addition of Mg-ATP by the Ca2+-ATPase located on the SR membrane, as indicated by the sharp decrease in the optical absorbance. A small amount of Ca2+ was then added sequentially to ensure the saturation of the SR vesicles, and the drug-induced Ca2+ release experiments were performed after the SR was loaded with a near-maximal amount of Ca2+. The addition of cartap at 1–33 mM did not induce Ca2+ release from SR vesicles. On the contrary, polylysine at 0.526 µM and A23187 at 4 µM both induced a rapid Ca2+ release, as indicated by the sharp increase in the absorbance. The effect of cartap on the activity of Ca2+-ATPase located on the SR membrane was further evaluated. The result showed that the Ca2+-ATPase activity could be inhibited by cartap in a dose-dependent manner (Fig. 5Go). However, the reduction was not significant until the concentration of cartap reached 3 mM or higher, and the reduction rate was less than 50%, even when the concentration was increased to 5 mM.



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FIG. 4. The effect of cartap on the calcium release from sarcoplasmic reticulum (SR) vesicles. Thirty micrograms of SR vesicles was loaded with Ca2+, and the Ca2+ concentration was monitored by the absorbance difference at 710 and 790 nm according to the procedure outlined in Materials and Methods. After the sequential addition of 10 nmol of CaCl2 to saturate the SR vesicles, cartap at 1 mM was then added repeatedly to induce Ca2+ release. Calcium release induced by 0.526 µM polylysine followed by 4 µM A23187 was used as a control of the total release of calcium from the SR vesicles. 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+. Results are representative of five different experiments.

 


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FIG. 5. The effect of cartap on the Ca2+-ATPase activity of the sarcoplasmic reticulum (SR) vesicles. Cartap at the concentrations indicated was incubated with SR vesicles for 5 min in the presence of 4 µM A23187, and the ATPase activity was measured according to the procedure outlined in Materials and Methods. Data are presented as the mean ± SD of three different experiments; each experiment was done in triplicate. Vertical bars represent SD. Asterisks indicate significant differences (p < 0.05) between cartap-treated and control (0 mM) groups.

 
Effect of Cartap on the [3H]-Ryanodine Binding in Sarcoplasmic Reticulum
The direct effect of cartap on the calcium release channel of SR, namely the ryanodine receptor, was investigated. Cartap at a concentration of 0.167 mM or lower did not show a significant effect on the [3H]-ryanodine binding capacity, but a significant dose-dependent inhibition appeared at a concentration of 0.25 mM or greater (Fig. 6Go). There was an approximately 50% reduction in the binding capacity at 0.25 mM. The reduction rate gradually increased as the dose of cartap increased, and it reached almost 77% at 3 mM.



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FIG. 6. The effect of cartap on the [3H]-ryanodine binding in sarcoplasmic reticulum (SR) vesicles. Cartap at 0.111 to 3 mM was added to the assay medium and the [3H]-ryanodine binding was measured according to the procedure outlined in Materials and Methods. Data are presented as the mean ± SD of three different experiments; each experiment was done in triplicate. Vertical bars represent SD. Asterisks indicate significant differences (p < 0.05) between cartap-treated and control (0 mM) groups.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cartap and other structurally related pesticides such as bensultap, Sha chong shuang, and thiocyclam were developed as nereistoxin analogues. They have been claimed to share a common mechanism of action with nereistoxin but have a lower potency (Ray, 1991Go). The oral LD50 in mice is 118 mg/kg BW for nereistoxin (Konishi, 1972Go) and is 150–225 mg/kg BW for cartap (FAO/WHO, 1977Go). Aside from reduced weight gain or weight loss, no other effects were noted in mice, rats, and rabbits following chronic oral or dermal exposure to cartap (FAO/WHO, 1977Go). It has been reported that the nereistoxin-induced acute death in mammals is by respiratory paralysis following blockade of neuromuscular transmission (Ray, 1991Go). The mechanism of action appears to involve inhibition of the nicotinic AChR ion channel (Eldefrawi et al., 1980Go). However, our present study has shown that cartap, unlike nereistoxin, did not affect the neuromuscular transmission. In agreement with previous study (Eldefrawi et al., 1980Go), we have also demonstrated that nereistoxin could selectively inhibit the twitches evoked indirectly via the nerve in the isolated mouse phrenic nerve diaphragm in a reversible fashion. On the contrary, cartap induced a marked contracture with twitch depression in the isolated mouse phrenic nerve diaphragm. As with that reported by O'Malley et al. (1990), the nerve-evoked twitches, as well as nerve- and muscle-evoked stimulation in the isolated mouse phrenic nerve diaphragm, were completely inhibited by {alpha}-BuTx, an AChR blocker, and TTX, a Na+ channel blocker, at 0.125 and 0.32 µM, respectively. However, neither {alpha}-BuTx nor TTX affected the cartap-induced muscular contracture. These data suggest that the effect of cartap should be myogenic.

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, 1983Go) 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, 1988Go; Hymel et al., 1988Go; Lai et al., 1988Go). 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, 1989Go; Palade, 1987Go). Ryanodine is a plant alkaloid that binds specifically to the Ca2+ release channel (Fleischer et al., 1985Go). The ligand binding has been used as a probe for the channel activity (Chu et al., 1990Go), based on the fact that the Ca2+ release inducers can also significantly increase the ryanodine binding (Kang et al., 1996Go; Pessah et al., 1987Go). 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, 1975Go) and halothane (Ogawa and Kurebayashi, 1982Go) 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, 1988Go; Lai et al., 1988Go). 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., 1998Go). The isolated mouse and rat phrenic nerve diaphragms have been widely used for studying the effects of drugs or chemicals on neuromuscular transmission (Bülbring, 1946Go; Kang et al., 1996Go; Yan et al., 1993Go). 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.


    NOTES
 
This study was supported by a grant (88-AST-1.3-FAD-01) from the Council of Agriculture of the ROC.

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. Back


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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