Henry B. Higman

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.

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.

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.

An improved isolated single electroplax preparation. I. Effect of compounds acting primarily at the synapses.

Abstract Acetylcholine and related structures have been tested as to their effects on the electrical activity of an improved isolated single electroplax preparation. As a measure for comparing the relative strength of the compounds, the concentration which produced 50% decrease in spike height within 20 min or less (usually from 5–15 min) was used. This concentration is referred to as the minimum active concentration. (a) Acetylcholine, decamethonium and d-tubocurarine were about 5–10 times as active as in previous preparations; no marked increase in sensitivity was observed with neostigmine and dimethylaminoethyl acetate. (b) Among the newly tested compounds succinylcholine proved to be twice as strong as acetylcholine, propionylcholine about 100 times weaker, and benzoylcholine several hundred fold weaker; the S-analogue of acetylcholine was about 3–5 times weaker. Dimethyl curare is only about half as strong as curare. (c) The decrease in spike height caused by the various quaternary compounds tested showed much better reversibility than in the previous preparation. (d) Curare in concentrations about 1000 times as high as those required to block the indirect response, abolished the propagated spike evoked by direct stimulation. In previous preparations no effect was obtained with curare outside the synaptic region even in higher concentrations than those applied in the present studies. (e) Following block of the indirect spike by curare, acetylcholine when applied in higher concentrations was able to overcome the block thys adding new evidence for the competitive action between acetylcholine and curare for the receptor. Dimethylaminoethyl acetate and the S-analogue of acetylcholine did not affect the direct spike in presence of curare in the concentrations tested. (f) Dimethylaminoethyl acetate acts much more strongly at pH 6 to 8, where it is present mostly in the cationic (protonated) form, than at pH 9, where it is present mostly in its uncharged form. The role of the Coulombic forces in the receptor protein thus appears similar to that previously observed with acetylcholinesterase. (g) The various changes of the response to acetylcholine and related structures after the removal of the adjacent membrane are a new demonstration that the effect of externally applied compounds on biological membranes depends to a great extent on the permeability barriers protecting the active membrane.

An improved isolated single electroplax preparation: II. Compounds acting on the conducting membrane

Abstract Effects on the electrical activity of an isolated single electroplax preparation were studied with several compounds which act at the conducting membrane as well as at the synapses. Two types of compounds were tested: 1. 1. Tertiary amines. Some of them are potent inhibitors of acetylcholinesterase and of the acetylcholine receptor (physostigmine and the tertiary analogue of Prostigmine). Others are mainly receptor inhibitors: procaine, tetracaine, and dibucaine (“local anesthetics”) and atropine. All these compounds block electrical activity without depolarization. Their reaction with the receptor protein has been recently demonstrated in solution. 2. 2. Lipid soluble quaternary ammonium ions: pyridine aldoxime dodeciodide, noracetylcholine, and norcholine. These compounds act on the acetylcholine system, but the affinities to the various protein members seem to be influenced by concentration and structure; dependeng on a variety of factors block of response may be observed with or without depolarization. Atropine, norACh and norCh affected the indirect response in about 0.1 the concentration required to affect the directly evoked spike. The other compounds affected the response to both types of stimulation about equally. All compounds tested affected the directly evoked spike in approximately the same concentration in the presence as in the absence of curare. Physostigmine and T.P. are twice as potent blockers of electrical activity at pH 6 as at pH 9. At pH 6 the molecules, in solution, are present in their cationic, at pH 9 in their uncharged form.

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.

Choline acetylase and cholinesterase activity in denervated electroplax

Abstract 1. 1. Following denervation of the Sachs electric organ of Electrophorus, choline acetylase (acetyl-CoA: choline O -acetyl transferase, EC 2.3.1.6) activity decreased about 50% in one week and 90% in four weeks, no activity was detectable after 74–97 days. In the Main electric organ 97 days after denervation choline acetylase activity is less than 1% that of control tissue. The medium for testing choline acetylase activity was improved by varying some of the components. 2. 2. Tested 78 days after denervation cholinesterase activity of the Sachs organ was not significantly decreased. 3. 3. Structural changes were microscopically detectable in the terminal neural innervation of the electroplax as early as 2 days following denervation, while after 20 days there was a virtual absence of nerve fibers. 4. 4. The changes in electrical activity of the denervated cells are discussed in reference to the changes in structure and enzyme activities.

The active structure of local anesthetics effects on electrical and cholinesterase activity

Abstract The potency of several local anesthetics as a function of pH has been tested both as to their inhibitory effect on purified acetylcholinesterase (EC 3.1.1.7) prepared from electric tissue, and their blocking action on the electrical activity of the isolated single electroplax. The inhibition of acetylcholinesterase in solution by the tertiary nitrogen derivatives procaine, dibucaine and tetracaine and the quaternary analogue of tetracaine decreases with an increase in pH from 6 to 9. When, however, the pH is raised from 7.5 to 9 the inhibiting effect of the tertiary compounds procaine and tetracaine decreases more strongly than that of the quaternary tetracaine. Carbamylcholine and quaternary tetracaine block electrical activity of the electroplax about equally well at pH 6 or 9. In contrast the tertiary tetracaine is more potent at the acid pH. The results provide further evidence that the cationic form is the more active structure of tertiary nitrogen-containing local anesthetics and support the view of the presence of an anionic group in the active site of the receptor comparable to that existing in the esterase.