Wolf-Dietrich Dettbarn

Rapid and Reversible Block of Electrical Activity by Powerful Marine Biotoxins

Puffer-fish poison and clam poison reversibly inhibit conduction in single nerve fiber preparation of frog in a concentration of 3 x 10-10M. In the isolated electroplax of Electrophorus electricus higher concentrations block both transmission and conduction. Neither toxin is a potent acetylcholinesterase inhibitor. The mechanism of action of these toxins in blocking transmission and conduction has not yet been established.

Biochemical and pharmacological aspects of the synapses of the squid stellate ganglion

Abstract The giant axon and isolated single giant synapses from the squid stellate ganglion were analyzed for acetylcholine (ACh), choline acetylase, and acetylcholinesterase (ACh-esterase). ACh, choline acetylase, and ACh-esterase are all present in significant quantities in the giant axon of the squid. Isolated single giant (distal) synapses from the squid stellate ganglion were found to have much higher concentrations of ACh, choline acetylase, and ACh-esterase than the giant axon. ACh and related compounds failed to affect the synapse when it was prepared in the usual way. Effects were seen, however, in carefully cleaned preparations from which surrounding connective tissue was removed; 5 × 10 −2 M ACh plus 10 −4 M physostigmine produced a reversible block of synaptic transmission in two of eight experiments, and an irreversible block was produced in the other six; 5 × 10 −2 M choline plus 10 −4 M physostigmine had no effect on the electrical activity of the synapse. The significance of the presence of the ACh system in the giant synapse and its structural and functional interrelationships are discussed.

Sources of error in relating electrical and acetylcholinesterase activity

Abstract The desheathed vagus nerve bundle of the rabbit presents no noticeable barrier to the penetration of acetyl-β-methylcholine (MeCh), which is the specific substrate of acetylcholinesterase (AChE). An attempt was therefore made to determine the minimum level of AChE activity required for electrical activity, by exposing the intact preparation to the organophosphate inhibitor, diisopropylfluorophosphate (DFP). Conduction appeared to be completely blocked by 5 × 10 −2 M DFP but not by 5 × 10 −4 M DFP. With MeCh as substrate, the AChE activities of the intact nerve at the two DFP concentrations above were not significantly different. Intact nerves exposed to 5 × 10 −4 M and 5 × 10 −8 M DFP had higher AChE activities than nerves homogenized in these concentrations of DFP. Vagus nerves from rabbits poisoned in vivo with DFP continued to conduct and had signifiantly higher AChE activity than nerves treated with 5 × 10 −2 M DFP in vitro which were not conducting. The bundle of nerves used is composed of about 23,000 fibers, and we can only measure the average enzyme activity. The activity in single fibres after an inhibitor may vary greatly from the initial value to zero. Under these circumstances it is obviously impossible to evaluate the critical level of enzyme activity required for conduction. The importance of this difficulty in attempting to correlate electrical and enzyme activity in multifiber preparations has not been adequately appreciated in the past. It is apparent that, for an unequivocal estimate of the critical level of acetylcholinesterase activity, use of a single fiber preparation is essential.

EFFECTS OF MARINE TOXINS ON ELECTRICAL ACTIVITY AND K+ EFFLUX OF EXCITABLE MEMBRANES.

Abstract Two marine toxins clam poison and holothurin, have been tested on the Ranvier nodes of isolated myelinated nerve fiber and the monocellular electroplax preparation of Electrophorus electricus. Changes in action and resting potential were recorded with the air-gap technique or intracellular electrodes. Clam poison (10−8 M) blocks electrical activity without depolarization. Clam poison does not prevent the depolarization produced by acetylcholine or carbamylcholine. It has no effect on the increased 42K+ efflux produced by these compounds. Holothurin in a concentration of 10−5 M blocks electrical activity, and depolarizes the conducting membrane, an effect not prevented by tetracaine. Holothurin causes an initial increase in 42K+ efflux which steadily declines. The possible interactions of these toxins with the acetylcholine system are discussed.

The action of acetylcholine and cholinesterase inhibitors on single axons of the lobster

Abstract Acetylcholine (ACh), physostigmine and related substances have been tested for their effects on the electrical activity of single giant axons of the circumesophageal connectives of the lobster. Acetylcholine and physostigmine in concentrations of 5 × 10 −3 to 1 × 10 −2 M depolarize the membrane. The simultaneously recorded action potential is prolonged, and with progressive depolarization reversible block of conduction occurs. Both compounds cause initially spontaneous or repetitive firing. Neostigmine, 3-hydroxyphenyltrimethylammonium and edrophonium prevent the effect of ACh without having an effect of their own on membrane or action potential. Choline, cholinethiol and phenyltrimethylammonium do not antagonize the action of ACh, nor do they change the electrical parameters of the axon. Curare depolarizes the membrane by 15 mV, but does not cause block of conduction nor does it inhibit the ACh effect. These observations are discussed and possible mechanisms of action are suggested.

Penetration of neostigmine, physostigmine, and paraoxon into the squid giant axon

Abstract The penetration of the cholinesterase inhibitors neostigmine, physostigmine, and paraoxon into the axoplasm of the squid giant axon has been determined by measuring cholinesterase inhibition with the magnetic diver technique. Neostigmine (5 × 10 −2 M) neither penetrated significantly into control axons nor into axons pretreated with 25 μg cottonmouth moccasin venom/ml, and neostigmine did not affect conduction. Significant penetration of neostigmine (0.5–1.0 per cent) was observed after pretreatment of axons with a concentration of venom (100 mg/ml) which itself blocked conduction. The approximate levels in the axoplasm of paraoxon (10 −2 and 10 −3 M) were 5 per cent of those in the external media, whereas physostigmine (2.5 × 10 −3 and 10 −5 M) attained levels about 10 times as great. Only the higher concentrations of the two inhibitors affected conduction. The apparent discrepancy between penetrability and blocking potency is discussed.

Spontaneous reactivation of organophosphorus-inhibited electroplax cholinesterase in relation to acetylcholine-induced depolarization☆

Abstract Pretreatment of the electroplax of Electrophorus electricus with irreversible inhibitors of cholinesterase (ChE) (diisopropyl phosphorofluoridate, paraoxon or phospholine) increases the sensitivity of the membrane to acetylcholine (ACh). After washing with inhibitor-free Ringers solution, this effect disappears rapidly. Reapplication of either of the inhibitors restores the initial sensitivity to ACh. Simultaneous measurements of ChE activity show an initial inhibition which is followed by recovery of enzyme activity. The rate of reactivation of the irreversibly inhibited enzyme is highest with paraoxon and lowest with DFP. The return of enzyme activity may be responsible for the loss of ACh action on the membrane of electroplax.