Camp. Biochem. Physiol. Vol. 92C. No. 2. pp. 309-313. 1989 Printed in Great Britain

0306-492/89 S3.00 + 0.00 G 1989 Pergamon Ptwa pk

BLOCKING OF REPETITIVE RESPONSE FROM A NEUROMUSCULAR PREPARATION OF

ZWV,!33RIO MOLITOR BY PHOSPHOTRIESTERS

TAKASHI ABE,* NOBIJFIJMI KAWAI~ and AKIKO MtwAt

*Institute of Physical and Chemical Research. Hirosawa 2-l. Wako-shi, Saitama 351, Japan, and tTokyo Metropolitan Institute for Neurosciences, 2-6, Musashidai. Fuchu-shi, Tokyo 183, Japan

(Receiced 6 April 1988)

Abstract-l. Diethylphosphate esters of m-cresol, o-, m- and p-nitrophenols. 2-nitro-p-, 4-nitro-m- and 5-nitro-o-cresols, thymol, 3,5-xylenol, a-naphthol, p-hydroxybiphenol, p-cyanophenol, pchlorophenol and 3.4,s~trichlorophenol were synthesized and tested for their inhibitory activity toward the neuro- muscular preparation of meal worm. T. moliror.

2. Remarkable depression of excitatory postsynaptic potentials (EPSPs). possibly due to acetyl- cholinesterase inhibition, was observed to occur by p-nitrophenol. 2-nitro-pcresol, p-chlorophenol and 3,4,5-trichlorophenol derivatives of diethylphosphate ester, each at a concentration from IO-J-IO-6 M.

3. The spontaneous repetitive miniature end-plate potentials (MEPPs) on a post synaptic membrane was predominantly induced by p-cyanophenol, m-cresol, Cnitro-m- and 5-nitro-ocresols esters of the diethylphosphate. Following inducement of the spontaneous excitation, neural transmission was blocked completely.

The effect of many organophosphates is acctyl- cholinesterase inhibition, the major functional target being neural tissue. They actually block the impulses of the cholinergic nerve (Werner, 1963). This property has found extensive application in the prep- aration of insecticides. Thus, in spite of their major effect which is certainly acetylcholinesterase in- hibition, some organophosphates show side effects other than acetylcholinesterase inhibition, such as membrane depolarization (Abe, 1983). respiratory inhibition (Parker, 1958) and induction of repetitive response in choline@ synapse (Clark, 1980) or in amine@ neuromuscular preparation (Abe, I98 I). However, part of its effect still indistinct, particularly in insects. This may be due to the complexity of the neural mechanism in insects.

Many organophospho-insecticides show a knock- down effect in insects, but in some cases this effect is distinct from the neural depressive effect of acetyl- cholinesterase inhibition. This is evident from the finding that the knockdown effect exerts very quickly while depression of EPSPs occurs slowly. In this study, we found immediate blocking effect accom- panying spontaneous repetitive response induced by organophosphates which applied to a glutaminergic neuromuscular preparation of meal worm.

.UATERIALS AND METHODS

Marerials

m-cresol, m-xylenol, p-cyanophenol, p-chlorophenol and 3,4,5-trichlorophenol were purchased from Tokyo Kasei Chemical Co. Thymol and triethylamine were provided through the courtesy of Wako Chemical Co. Diethylchloro- phosphate was from Aldrich Chemical Co.

Melhodq

Synlhesis of dieihylphosphate phenylesters. In our previous paper, we developed a new method for the synthesis of phosphotriesters using dicyclohexylcarbodiimide and char- acterized their physico-chemical properties (Ahe. 1984). Other phosphotriesters used in the present study were synthesized from diethylchlorophosphate and phenol deriv- atives using triethylamine (Table I).

Physical analysis of the synthetic compounrls. Infrared and NMR spectra were obtained and elemental analysis conduc- ted by the same procedures as in our previous paper (Abe, 1984). The elemental analysis, IR and NMR speetrometric data of the compounds are listed in Table I.

Recording ofpostsynapric membrane potentials. The neuro- muscular preparation of a meal worm was prepared according to the method of Yamamoto er al. (Yamamoto, 1979). The membrane potential of the ventral longitudinal muscle was measured with a glass microelectrode filled with 3 M KCI. The potential was recorded both by CR0 and an ink writer. The excitatory nerve was stimulated with a pair of silver wires.

For application of synthetic chemicals, the compounds were dissolved in a small amount of acetone and diluted to a concentration of IO-’ M with a bathing solution. One ~1 of the sample was applied to the preparation previously dipped in IOml of the bathing medium. The effective concentration of each chemical was finally around IO-’ M or 10m6M bv dilutina with the bathina solution. This solution contained 70 mMol NaCI, 30 mM;l KCI, 0.5 mMol CaCI,, 14.5 mMol MgCI,, 475 mMol sucrose and 5 mMol Tris-HCI, pH 7.2.

RESULTS

Inhibition of EPSPs on the meal worm neuromuscular preparation by paraoxon

When IO-’ M paraoxon (diethylphosphate p- nitrophenyl ester) was applied to ventral longitudinal muscular fibers of the meal worm, the muscular

309

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Phosphotriesters on insect 311

A 10

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Fig. 1. Inhibitory effects of paraoxon and diethylphosphate p-cyanophenylester on meal worm neuro- muscular preparation. A. Depression of EPSPs by paraoxon. B. Inducement of repetitive MEPPs and

blocking by diethylphosphate p-cyanophenol. The chemicals were administered at 0 sec.

membrane became depolarized, as was also observed for a crustacean muscular membrane (Abe, 1983). A few minutes later, the EPSPs in the muscular fiber slowly increased to about twice that of the first minute as shown in Fig. 1A. This was followed by rapid decrease and complete blocking of EPSPs. The slow depression of EPSPs by paraoxon is probably a typical synaptic block following acetylchohnesterase inhibition, since earlier increase in and continuous inhibition of EPSPs are influenced by excessive accu- mulation of acetylcholine on cholinergic synapses in which enzymic inhibition of acetylcholine decom- position occurs.

Blocking of neuromuscular transmission following inducement of repetitive response on meal worm neuromuscular junction by diethylphosphate cyanophenylester

To the neuromuscular preparation of T. molitor, 10m3 M diethylphosphate cyanophenylester was applied according to the procedure in Materials and Methods. Repetitive response was observed in a few seconds following administration of this compound as shown in Fig. 1B. The rapid responses of repetitive giant MEPPs and MEPPs were found and unexpectedly blocked completely by continuous excitations. By single axonal excitation, the repetitive MEPPs induced subsequently were observed and then neuromuscular transmission was quickly blocked. The blocking was completely different from the slow depression of EPSPs on cholinergic nerve by paraoxon (see in Fig. IA). This is very similar to spontaneous excitation of crayfish end-plate potentials by ED0 (Chau, 1975). Thus, the blocking is probably induced by the organophosphate on the aminergic neuromuscular junction of meal worm, and would correspond to the knockdown effect of organophospho-insecticides.

Inhibitory effects of synthetic diethylphosphatephenvl- esters on the neuromuscular preparation of a meaI worm

Fourteen derivatives of diethylphosphate phenyl- esters were examined for their effects on the neuro- muscular preparation of T. molitor (see Table 2).

The slow depression of EPSPs was predominantly observed for derivatives of o- and m-nitrophenols, 2-nitro-p- and 4-nitro-m-cresols, thymol, a-naphthol, p-chlorophenol and 3,4,5-trichlorophenol. Among these compounds, 2-nitro-p-cresol, p-chlorophenol and 3,4,5-trichlorophenol inhibited greatly neuro- muscular transmission. While slow depression could not be confirmed for derivatives of m-cresol, 4-nitro-m- and 5-nitro-o -cresols, they predominantly induced spontaneous repetitive excitations similar to that of p-cyanophenol derivative. The repetitive MEPPs increased in the muscular cells were quickly blocked.

The slow depression of EPSPs was generally invalidated by means of the fast blocking which followed the repetitive response. Those two types of neuromuscular blocking were not observed in the derivatives of 3,5-xylenol and p-hydroxybiphenol.

DISCUSSION

The major target of organophosphates, well known to be acetylcholinesterase inhibitors, is the cholinergic

Table 2. Effect of diethylphosphate phenylesters on “euro- muscular preparation of T. molilor

Dominant effect Derivatives of on neuromuscular diethylphosphates membrane of meal worm

m-cresol Repe. Block o-nitrophenol Depress. (+) WI-nitrophenol Depress. (+ +) p-nitrophenol Depress. (t + + ) 2-nitro-p-cresol Depress. ( + + +) 4-nitro-m-cresol Repe. Block 5-nitro-o-cresol Repe. Block Thymol Depress. (+) 3,5-xylenol No Detec. a-naphthol Depress. (f +) p-hydroxybiphenol No Detec. p-cyanophenol Repe. Block p-chlorophenol Depress. (+ + +) 3,4,5-trichlorophenol Depress. (+ + +)

Depress. and parentheses show depression of EPSPs and its intensity, respectively. Repe. Block is abbreviation of blocking accompanied by repetitive response. No Detec. is not detectable. See details in Materials and Methods.

312 TAKASHI ABE et al.

nerve. In many studies, EPSPs have been found to be blocked by the organophosphates (Werner, 1963) and similar blocking of slow inhibition was observed in the present experiment of a meal worm neuro- muscular preparation. It is because L-glutamic acid, not acetylcholine, is transmitted in neuromuscular junction of an insect, in other words an aminergic transmission (Usherwood, 1968), that the slow inhibition by organophosphates may possibly have resulted from the inhibition of the cholinergic synapse.

However, some organophosphates, particularly highly toxic compounds, exert various side effects other than acetylcholinesterase inhibition, such as depolarization of muscular membranes (Abe, 1983), respiratory inhibition (Parker, 1958) and repetitive response of cholinergic neuromuscular junction (Clark, 1984). Such effects might reflect high toxicity. In this study, a side effect such as the repetitive excitation in the meal worm neuromuscular membrane was observed following administration of various organophosphates. Our neuromuscular preparation of T. molitor was a neuromuscular com- plex of axons, axonal synapses, and neuromuscular junctions in the bathing solution, but was not con- tained central nervous system. It is apparent that organophosphates administrated to the preparation affect not only neuromuscular junctions but axons and axonal synapses as well. Therefore, two active mechanisms would possibly be presumed. One is that, if the repetitive response is induced on meal worm axons by organophosphates, as in the case of the periferal nerve of the cockroach (Becht, 1958) and Xenopus by DDT (Akkermans, 1975) it remarkably accelerates excretion of an autoneurotoxin from the nerves (Sternberg, 1963) which subsequently paralyzes axonal transmission. On the other hand, possibly on excess amount of acetylcholine accumu- lated in the axonal synapse by severe repetitive responses may inhibit transmission. The temporary increase in MEPPs at the repetitive response (Fig. 1B) is consistent with this possibility. Assuming that the organophosphates do not block aminergic trans- mission, paralysis of the transmission subsequent to the repetitive response should ocur in the cholinergic synapse of meal worm, not the neuromuscular junc- tion. The other possible mechanism is blocking of the neuromuscular junction. The blocking of this junc- tion by EDO, a derivative of DDT, has been observed in crayfish, where a single or continuous excitation of the axon occurred the repetitive response of the presynaptic membrane and evoked the repetitive MEPPs. After repetitive excitation, transmission is completely blocked (Chau, 1975). This process of blocking is very similar to that of meal worm. If anthropods are in general possessed of aminergic neuromuscular transmission, meaning that the meal worm neuromuscular junction would have a mechanism of chemical transmission similar to that of the crayfish, the organophosphates should likely affect ED0 and DCC (Chau, 1975). The MEPP is commonly observed not only in aminergic synapse, but also in cholinergic one. In the experiment of frog neuromuscular junction, its frequency and acetyl- choline release are increased by low concentration of Ca*+ or Mn*+ (divalent cations) and by increasing

the tonicity (Narita, 1983). While, the giant MEPP is induced by high concentration of K+, and the frequency and acetylcholine release are increased at high concentration of Cl- (Molenaar, 1987). These repetitive excitations are reversibly caused by imbal- ance of ions or tonicity. In intoxication of paraoxon, the repetitive response is induced without blocking the transmission by a single nerve stimulation in muscular preparation of rat diaphragm and mouse omohyoideus (Clark, 1964). For generation of the repetitive response, a long term exposure of paraoxon is necessary, and it is prevented by pretreatment of dithiothreitol. Thus, the cholinergic repetitive response induced by paraoxon or ionic imbalance in vertebrates markedly differs from the aminergic junctional block following MEPPs in insects. This fact is probably caused by a fundamental difference of the synaptic membrane transmission, for example difference of the transmitter decomposition and func- tional complexity of aminergic receptor (Abe, 1983). Therefore, this blocking may also be induced by those organophosphates on glutaminergic hippocampus or olfactory cortex of central nervous system. Moreover, all the data of the present study indicates the mechanism for spontaneous excitation by organo- phosphates to differ completely from that of acetyl- cholinesterase inhibition on cholinergic junction; that is, the phenomenon will occurs on aminergic nerve as a side effect regardless of acetylcholinesterase inhibition. The MEPP is commonly observed in neuronal phenomena accompanying tetanus, tremor and twitch, so the blocking followed by MEPPs will correspond to the knockdown in insects.

The molecular similarity between ED0 and organophosphates is not with respect to skeletal structure. In consideration of the structural specificity of DDT derivatives as a factor for bringing about blocking and repetitive response, the active structure of these compounds and organophosphates may be determined by a conformational balance between hydrophobic and ionic regions in the molecule.

Acknowledgement-We would like to thank Dr Haruo Honma for elemental analysis.

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in low calcium and in manganese saline solutions. Bruin Res. 289, 7!%85.