Camp. &o&-m. Physiol. Vol. 96C, No. I, pp. 193-197, 1990 Printed in Great Britain

0306~4492/90 $3.00 + 0.00 0 1990 Pergamon Press plc

THE EFFECTS OF PENTACHLOROPHENOL (PCP) AT THE TOAD NEUROMUSCULAR JUNCTION

GONZALO A. MONTOYA* and LADISLAO QUEVEDO

Department of Physiological Sciences, Faculty of Biological Sciences and Natural Resources, University of Concepcibn, P.O. Box 2407, ConcepcGn, Chile

(Received 24 January 1990)

Abstract-l. Effects of PCP at the frog neuromuscular junction were studied in vitro in sciatic nerve sartorius muscle of the toad Pleurodema-thaul.

2. Within the concentration 0.003-0.1 mM. PCP caused a dose-time-deDendent block of evoked transmitter release acompanied by an increase’in the rate of spontaneous quanta1 release.

3 PCP induced an increase in miniature endplate potential (MEPP) frequency and it was not antagonized in a Ca2+-free medium, indicating that it does not depend upon Ca2+ influx from the external medium, but may act by releasing Ca2+ from intraterminal stores.

4. The preseni data, -together with previous results concerning PCP at eighth sympathetic ganglia indicate that 3,4_diaminopyridine (3,4-DAP) counteracts the effects of PCP on synaptic transmission. This result suggests that PCP interfering Ca 2+ influx occurs during depolarization of motor nerve terminals.

INTRODUCTION

The major application of pentachlorophenol (PCP) is in wood industries as a wood preservative. It is also employed as a biocide in agriculture and various industries. No precise estimates can be made of the total world production of PCP. According to the data profile of IRPTC (1983), 90,000 tonnes of PCP per year are produced. During the last few years pro- duction has decreased, not only because highly toxic chlorinated dioxins, which are formed in the pro- duction process, have been found in PCP prep- arations, but also because environmental pollution with PCP is considered a serious problem. Its recalci- trance in soil water and sewage led to the assumption that PCP is an example of a non-degradable ‘man- made’ substance (Alexander, 1965). In recent years, however, biodegradation of PCP in the environment and in the laboratory has been described by various groups (Mikesell and Boyd, 1986; Mileski et nl., 1988; Rott et al., 1979). PCP was also found, along with a metabolite, in the urine of exposed workers (Ahlborg et al., 1974; Edgerton and Moseman, 1979).

According to several studies, PCP alters the micro- somal electron transport system (Arrhenius et al., 1977), the transport of protons across the phospho- lipid bilayer membranes (Smejtek et al., 1987), un- couples oxidative phosphorylation in mitochondria (Weinbach and Garbus, 1965), inhibits amino acid transport across the cell membrane (Brummett and Ordal, 1977; Hissin and Hilf, 1978), and induces electrical conductivity in model membranes (Smejtek et al., 1976).

In a previous study axonal conduction and gangli- onic transmission were blocked by PCP (Montoya et al., 1988). In the present study we have investigated

*Permanent address: Departamento de Ciencias Fisio- lbgicas, Facultad de Ciencias Biolbgicas y de Rucursos Naturales, Universidad de ConcepcGn, Casilla 2407, ConcepcGn, Chile.

the action of PCP on the toad neuromuscular junc- tion to establish its effects on spontaneous and evoked transmitter release.

MATERIALS AND METHODS

Experiments were performed on isolated sciatic nerve- sartorius muscle preparations of the toad Pleurodema-thaul at room temperature (2&22”C). Intracellular recordings were made for junctional and extra-junctional regions of muscle fibers. Endplate localization was determined by placing the microelectrode along the muscle fiber in order to find the position of maximum amplitude and minimum rise time of synaptic potentials. Intracellular recording of miniature endplate potential (MEPP), muscle action poten- tial and resting membrane potentials were obtained using 3M KCl-filled microelectrodes having tip resistances of 610 Ma. Amplification and recordings were made using coventional techniques (Molg6 et al., 1977).

The mean + SEM MEPP frequency was calculated from the number of MEPPS recorded during a l-2 min period. The motor nerve was stimulated through a suction electrode by supramaximal current pulses of 0.08 msec duration at 0.2-0.5 Hz.

Indirectly elicited muscle action potentials were recorded from junctional and extra-junctional regions of muscle fibers in which excitation-contraction was uncoupled using the formamide method (Del Castillo and Escalona de Motta, 1978); the isolated preparation was soaked in a Ringer solution containing 2M formamide for 12-l 5 min and then brought back to normal Ringer solution. In these con- ditions, nerve stimulation evoked action potentials without contraction in the muscle fibers, and without a fall in resting potential.

The standard Ringer’s solution used in the present exper- iments had the following composition (mM): NaCl 115; KC1 2.0; CaCl, 1.8; Tris-maleate buffer 2.0. This solution was modified by adding Mg*+ and/or by reducing the Ca2+ concentration as indicated in the Results section. All prep- arations were kept for 1 hr in solutions with altered Ca2+ or Mg2+ concentrations to allow for equilibration. The osmo- larity of the modified Ringer’s solution was maintained by varying the Na+ concentration. All solutions were adjusted to pH 7.2 before use. The solutions flowed continuously

194 GONZALO A. MONTOYA and LADISLAO QUEVEW

CONTROL 1 A 5 C D

- -

3uM

PCNTACHLOROPHENOL

1OuM 30uM 1OOuM

RECOVERY

- 30’

Fig. 1. Concentration effect characteristics of PCP on neuromuscular transmission block. Actions of PCP on EPP. Control EPPs are in PCP-free Ringer’s solution. Each of the four columns below the Control show EPPs recorded at 0.003, 0.01, 0.03 and 0.1 (mM), 30min after perfusion of the junction in PCP concentration indicated at the top of the second column. Each dose-response was made on a different sciatic sartorious muscle preparation depressed by 0.005 mM of (+)tubocurarine, present throughout the experiment. The resting membrane potential during measurements was -85.0 mV. Calibrations were

I mV and 2 msec.

over preparations placed in a 5 ml organ bath at a constant flow rate of 8 ml min-‘.

Drugs used were: pentachlorophenol (Sigma Chemical, St Louis, MO, U.S.A.): 3,4 diaminopyridine (3,4-DAP) (Aldrich Chem. Co., Belgium) and (+)Tubocurarine chlorhidrate (Fluxa A.G. Buchs S.G., Switzerland).

RESULTS

In junctions equilibrated for 1 hr in standard Ringer Solution containing 0.005 mM (+)tubocur- arine, the addition to the medium of (0.01-0.1 mM) PCP decreased the average EPP amplitude evoked by nerve stimulation at 0.2 Hz (Fig. 1). This effect occurred after a delay of 5-10min and the steady state effect was reached after a 15-30 min exposure

CONTROL

PCP 10 uM

PCP 10 UM +

3,4-DAP 5 uM

RECOVERY

I+= L 15’

20’

J 22’

A 30’

Fig. 2. Records show EPPs in PCP (0.01 mM) during 15 min on sciatic sartorius preparation depressed by 0.005 mM (+)tubocurarine, preieni throughouithe experiment. When 3,4-DAP was added to the solution a marked increase occurred in the amplitude of successive EPPs as shown after

15, 20 or 22 min.

time. At the lower concentrations (0.003 mM) the preparation was recovered, but at higher concen- trations (0.01-0.1 mM) the reduction in endplate potential (EPP) amplitude was concentration-depen- dent, and the block of neuromuscular transmission was irreversible in both normal or formamide-treated junctions, since no recovery of the EPPS or indirectly elicited action potential was observed, even 180 min after wash-out of the drug from the medium (Figs 1 and 3).

When Ca*+ influx into the axon terminal was facilitated with 3,4-DAP (Molg6 et al., 1985) which prolongs the plateau phase of the action potential and prevents K+ efflux, quanta1 release was increased, restoring the EPP amplitude in preparations contain- ing PCP at 0.01 mM (Fig. 2). At 15 min after the addition of PCP, the EPPs amplitudes were 54 f 1% control. 3,4-DAP caused a marked increase in EPP amplitude after 2&25 min, which persisted even 30min after washing (Fig. 2).

Nerve stimulation of sartorius muscle treated with formamide, which uncouples excitation and con- traction (see Materials and Methods), elicited action potentials that were conducted from junctional to extra-junctional areas of the muscle fiber without contraction. Under those conditions, addition of PCP at 0.03 mM to the normal Ringer solution bathing the neuromuscular preparation decreased the amplitude of action potentials at 0.1 Hz nerve stimulation after about 20-30min. The rate of inhibition of neuromuscular transmission by 0.03mM PCP was enhanced by increasing the frequency of nerve stimu- lation from 0.1 to 2 Hz. The effects described in the presence of PCP cannot be due to the amount of formamide treatment. In control experiments performed in formamide-treated junctions bathed in normal Ringer, we observed that indirect elicited action potential could be evoked at 0.1 Hz for more than 60min without any impairment of synaptic transmission.

PCP, when added to standard solution and applied for 15-30 min to isolated neuromuscular prep- arations, had no consistent effect on spontaneous

A

PCP on neuromuscular junction

CONTROL

195

70.5

CONTROL

PCP 3uw

2Wr

“i 5’ J

A-

: A

15‘ A

B CONTROL

0.1 HZ

C

i d ’

PCP 30 UM

2Hr

RECOVERY

10’ __A-

Fig. 3. Indirectly elicited muscle action potentials recorded in junctional (A) and extra-junctional regions (B) of neuro- muscular preparations pre-treated with formamide to uncouple excitation-contraction. Calibration in mV and msec. (A) Junctional action potentials recorded before (control), and after 5, 7, 10 and 15 min of PCP (0.003 mM), to different frequencies (0.1 and 2.0 Hz). Records A and B are different preparations; B and C were recorded in the same preparations. Note that in column C the action potential was decreased before B because it was partially recovered. (B) Extra-junctional action potentials recorded before (control), and after 5, 6, 7 and 20min of PCP (0.03 mM), to different frequencies (0.1 and 2.0 Hz). The recordings were obtained in two different

preparations.

A

PCP 10uM

45’ 55

, \-- I \ u,

Fig. 4. MEPP frequency at a toad endplate followed for 60 min after PCP (0.01 mM) application at zero time. MEPP frequency increased after 40 min in one preparation bathed with a medium in which Ca*+ was 1 mM and M$+ 10 mM.

MEPP frequency in concentrations that decreased EPP amplitude. In junctions bathed during pro- longed periods (45-60 min) in the presence of PCP (0.01 mM), a gradual significant increase in spon- taneous MEPP frequency that reached about 10 times the basal level was observed [i.e. control MEPP 0.9 + 0.2 Hz, while in the presence of PCP it was 9.3 f 1.2 Hz, n = 4 (Fig. 4)].

In order to study the contribution which external Ca2+ may have on the increase of spontaneous MEPP frequency induced by PCP, the neuromuscular prep- arations were exposed for 1 hr to a Ca2+-free medium containing 2 mM Mg2+ and 1 mM EGTA to reduce external Ca2+ levels to less than 10nM (Portzehl et al., 1964). Under those conditions, MEPP frequency in most junctions that were examined was lower than 0.2/set. Addition of 0.01 mM PCP to the Ca2+-free medium caused a similar increase in MEPP frequency as that observed in a normal Ringer solution, but with a longer latency.

DISCUSSION

PCP was found to block irreversibly transmitter release evoked by nerve impulse. Although we cannot exclude the fact that PCP may block action potential invasion in motor terminals, the drug seems to have a more generalized effect on the nerve terminal depolarization-transmitter release coupling process. Our results demonstrate that when evoked trans- mitter release by nerve impulse is decreased by PCP, it is possible to induce recovery in the presence of 3,4-DAP (Fig. 2), which might enhance Ca2+ influx by blocking voltage-sensitive K channels in the nerve terminal (Mallart, 1984; Molgo et al., 1985). On this basis it may be suggested that a reduction of Ca2+ entry is involved in the PCP action. This is consistent

196 GONZALO A. MONTOYA and LADISLAO QUEVED~

with Fig. 3A and B, for muscle-formamide blockade is dependent upon the frequency of nerve stimulation (0.1-2 Hz) in the presence of PCP. Similarly, 3,4- DAP antagonizes the effect of the PCP on synaptic transmission in the eighth sympathetic ganglion (Montoya et al., 1988).

The present study has shown that PCP is able to stimulate spontaneous transmitter release recorded as MEPP at the frog neuromuscular junction. Our results with calcium-depleted nerve terminals indicate that PCP by itself can activate the transmitter release process. This action of PCP to increase MEPP fre- quency is similar to that previously reported with other uncoupling oxidative phosphorylation, for example carbonyl cyanide m-chlorophenylhydrazone (CCCP) and carbonyl cyanide p-trifluoro metoxy- phenylhydrazone (FCCP) (Molgo and Pecot- Dechavassine, 1988). The present results, showing that spontaneous transmitter release is increased by PCP, support the view that the drug also increases cytosolic calcium levels in motor endings at the time when evoked transmitter release is blocked. Whether high intraterminal Ca2+ could inhibit the evoked release by reducing the driving force for Ca2+, by inactivating nerve terminal voltage-sensitive Ca2+ channels, or by inhibiting some step in the depolar- ization-transmitter release coupling mechanism, remains to be determined.

It is well known that large quantities of Ca2+ are stored within nerve terminals in a sequestered form (Blaustein et al., 1980). A number of metabolic inihibitors which increase spontaneous quanta1 acetylcholine release are thought to free this intern- ally bound Ca2+ (Alnaes and Rahamimoff, 1975; Glagoleva et al., 1970; Nachshen, 1975; Molgo and Pecot-Dechavassine, 1988). Since PCP enhanced spontaneous transmitter release even in the absence of extracellular Ca2+, the drug may act by releasing Ca2+ from mitochondria and other intraterminal structures. This is not surprising since there is direct experimental evidence that PCP uncouples oxidative phosphorylation in liver mitochondria (Masini et al.,

1985). PCP may reduce intraterminal adenosine 5’-

triphosphate (ATP) levels, and thereby block Ca2+ regulatory mechanisms that are ATP-dependent (Komulainen and Bondy, 1988). If the PCP site of action is indeed intracellular, a delayed onset of the action of spontaneous quanta1 release could reflect the time required to affect these cellular processes in motor endings. In addition, our previous results when PCP ionization was increased show that lower per cent reached the site of action, reducing its capacity to block the axonal conduction and ganglionic trans- mission (Montoya et al., 1988).

Acknowledgement-These studies were supported by grants FONDECYT No. 89625.

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