Eva Bartels

Effects of blocking sulfhydryl groups and of reducing disulfide bonds on the acetylcholine-activated permeability system of the electroplax

Abstract The depolarization of the single-cell preparation of the electroplax of Elecrophorus electricus by acetylcholine, carbamylcholine, and trimethylbutylammonium ion is inhibited by prior treatment of the electroplax with p- chloromercuribenzoate (PCMB) or with 1,4-dithiothreitol (DTT). The inhibition by either agent is characterized by an increase in the concentration of carbamylcholine eliciting a half-maximal response with no great change in the maximum response. The inhibition due to PCMB is reversed by subsequent treatment with thiol compounds, and that due to DTT is reversed by oxidizing agents and by cysteine (but not by other thiol compounds). Washing alone has no effect on the inhibition due to either agent. Treatment of the electroplax with N- ethylmaleimide (NEM) after DTT blocks the reversal of the inhibition by oxidizing agents and by cysteine, added subsequently. NEM alone under the same conditions has no direct effect on the depolarization. It is concluded that PCMB is reacting with sulfhydryl groups and DTT with disulfide bonds, present in one or more component of the acetylcholine-activated permeability system at the junctional regions of the innervated membrane of the electroplax. The evidence is taken as support for the view that some of the components of this system are proteins.

Apparent dissociation constants between carbamylcholine, d-tubocurarine and the receptor

Abstract The depolarizing action of carbamylcholine on the monocellular electroplax preparation has been analyzed as a function of concentration. The apparent dissociation constant between carbamylcholine and the acetylcholine receptor was found to be 4.4·10 −5 M. The depolarization due to carbamylcholine is inhibited competitively by d -tubocurarine. The apparent dissociation constant between d -tubocurarine and the acetylcholine receptor was found to be 2.4·10 −7 M.

Organophosphate inhibitors of acetylcholine-receptor and -esterase tested on the electroplax

Abstract The organophosphates—DFP, phospholine, and Paraoxon, long known to be potent inhibitors of acetylcholine (AcCh)-esterase—have been applied to the monocellular electroplax preparation. The experiments show that the compounds act at high concentrations as inhibitors of the AcCh-receptor. The effects are rapidly reversible. At low concentrations the organophosphates potentiate the action of AcCh. Occasionally they may cause spontaneous depolarizations of the electroplax membrane. These effects are due to the inhibition of AcCh-esterase, since they are rapidly reversed by pyridine-2-aldoxime methiodide, a potent and specific reactivator of phosphorylated enzyme. A reversal takes place also in Ringers solution, but much more slowly. Ambenonium, a bisquaternary nitrogen derivative and a potent inhibitor of AcCh-esterase, also causes spontaneous depolarizations. It potentiates the action of AcCh, but to a lesser extent than the organophosphates.

Sulfur and selenium isologs related to acetylcholine and choline IV. Activity in the electroplax preparation

Abstract Studies using the isolated single cell electroplax preparation indicated that the hydrolysis products of acetylthiocholine and acetylselenocholine, cholinethiol and cholineselenol, are powerful depolarizing agents. The activity of cholinethiol was greater at pH 6 than at pH 9. Methylation to form methylthiocholine and methylselenocholine increased activity further. Choline disulfide and choline diseleaide were antidepolarizing blocking agents. Differences between the active site of the depolarizing receptor and that of acetylcholinesterase are discussed.

New method for recording electrical characteristics of the monocellular electroplax

Abstract 1. A new and improved method has been developed for recording the electrical characteristics of a monocellular electroplax preparation. By means of a switching device, transmembrane potentials across the two faces of the electroplax may be recorded separately; also the transcellular potential may be determined. 2. The depolarizing action of acetylcholine and related compounds is much stronger and more rapid in onset than appeared from previous work. 3. The block of the propagated spike by acetylcholine and related compounds is associated with the simultaneous development of a transcellular potential and a depolarization of the membrane. 4. Earlier results showing competition between tetracaine and carbamylcholine for the acetylcholine receptor have been confirmed.

Relationship between acetylcholine and local anesthetics

Abstract The effects of acetylcholine, local anesthetics and intermediary forms on the excitable membrane of the monocellular electroplax preparation have been investigated. It has been shown that small modifications of structure may markedly modify the effects on the electrical parameters and transform the activator into an inhibitor. 1. 1. Acetylcholine (2.5 μM) has a strong depolarizing action. This action is, however, limited to the synaptic membrane. The depolarization of the conducting membrane is known to be a spread of depolarization from the synapse; the compound does not reach the conducting membrane even in high concentrations. 2. 2. If the methyl on the carbon of the carbonyl group is replaced by cyclohexyl (hexahydrobenzoylcholine), the depolarizing action is markedly decreased, by a factor of 200, but the fundamental properties are still similar to acetylcholine. Combined with carbamylcholine in low concentration it has an additive effect. 3. 3. If the methyl is replaced by a phenyl radical (benzoylcholine) a marked change of the properties of the molecule in regard to its action on the excitable membrane is observed. The depolarizing action is similar to that of hexahydrobenzoylcholine, but it also exhibits some inhibitory effects. It counteracts the depolarizing action of carbamylcholine. Moreover, in contrast to acetylcholine and hexahydrobenzoylcholine, benzoylcholine blocks the directly evoked action potential in the presence of curare. The presence of the phenyl radical but not its saturated analog enables the compound to penetrate the barriers surrounding the conducting membrane. The compound may be considered as the transitory form between acetylcholine and the local anesthetics. 4. 4. The tertiary analogue of benzoylcholine has no depolarizing effect but only a weak inhibitory action on the synaptic membrane. 5. 5. Replacing the phenyl by a p-aminophenyl radical (procaine, tetracaine, etc.) deprives the compound of its depolarizing action. These compounds are receptor inhibitors. Their ability to penetrate through the barriers protecting the conducting membrane is greatly increased. These compounds block all excitable membranes, but without depolarization, hence their usefulness as local anesthetics. These studies offer a further elucidation of the mode of action of local anesthetics and their close relationship to acetylcholine.

CORRELATION BETWEEN ELECTRICAL ACTIVITY AND SPLITTING OF PHOSPHOLIPIDS BY SNAKE VENOM IN THE SINGLE ELECTROPLAX

Abstract— Cottonmouth moccasin snake venom (SV) was applied to the innervated membrane of the isolated single cell of the Sachs electric organ (electroplax) of the electric eel, Electrophorus electricus. Concentrations as low as 0.05 μg/ml irreversibly antagonized depolarization by carbamylcholine, whereas concentrations of 0.1 mg/ml or higher were required to directly and irreversibly depolarize and block electrical excitation. The active component of the venom was stable to boiling at acid pH, destroyed by boiling at alkaline pH and nondialyzable and corresponded to those fractions containing maximum phospholipase A activity demonstrable when isolated by paper electrophoresis and Sephadex filtration. Phospholipase C and lysolecithin in concentrations of 1 mg/ml and 0.2 mg/ml, respectively, depolarized and blocked electrical excitation, whereas lower concentrations did not antagonize depolarization by carbamylcholine. Triton X‐100 (0.01 mg/ml) antagonized carbamylcholine, whereas 10‐fold higher concentrations directly blocked electrical excitation. Hyaluronidase had no effect on resting or action potential but decreased the depolarizing response to carbamylcholine.

DIFFERENCE BETWEEN TETRACAINE AND D-TUBOCURARINE IN THE COMPETITION WITH CARBAMYLCHOLINE.

The antagonistic action of tetracaine and tetracaine methiodide on depolarizations produced by carbamylcholine using monocellular electroplax preparation has been studied. A two-fold action for these two compounds has been demonstrated. One action occurs at the synapse and results in competition with d-tubocurarine and carbamylcholine. The second action is localized at the conducting membrane and is responsible for preventing the depolarizing currents produced at the synapses by carbamylcholine from spreading along the conducting membrane. The effect of tetracaine on the conducting membrane occurs at a concentration of 3.3·10−5 M, while 5·10−5 M is required to demonstrate competition at the synapse. Tetracaine methiodide 1·10−5 M produces an effect on the conducting membrane and 2·10−5 M on the synapse. It is suggested that the effect of these two compounds on the conducting membrane is similar to the competition demonstrated at the synapse, but the competition occurs between acetylcholine, which is released within the membrane by the action of carbamylcholine at the synapse, and tetracaine. It was also found that, while quaternary tetracaine methiodide apparently has a higher affinity for the receptor compared to tertiary tetracaine, it is a weaker blocking agent. The effects of the two compounds on the conducting membrane, however, are additive.

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.

Correlation of membrane potential and potassium flux in the electroplax of electrophorus

Abstract 1. 1. Exposure of the innervated membrane to high external potassium concentrations or to carbamylcholine results in an initial rapid depolarization with a further gradual decline to a steady level which varies with the concentration of the depolarizing agent. 2. 2. The time course for repolarization following withdrawal of the depolarizing agent is slower than for depolarization. 3. 3. Depolarization of the innervated membrane by high external potassium concentrations is followed by a transitory increase in potassium efflux but a sustained influx. The influx and efflux increase to the same level when both membranes are simultaneously depolarized by high external potassium concentrations. 4. 4. Depolarization of the innervated membrane by the action of carbomylcholine at the synapses is followed by a transitory increase in potassium efflux and little change in the influx. 5. 5. Repolarization is followed by a less marked but more prolonged transitory decrease in the efflux.