J. Del Castillo

Acetylcholine sensitivity of the spine-test articular capsule of the sea urchin Evcidaris tribuloides

1. The changes in the consistence of the spine-test articular capsule, or ligament, of the primary spines of Eucidaris tribuloides induced by acetylcholine (ACh) have been studied. Two complementary techniques were used: (a) forced-vibration, which detects variations in the stiffness of the ligament along a single diametral plane; and (b) forced-rotation which records the spatial distribution of those changes. 2. ACh (1 microM to 1 mM) caused a rapid increase in the resistive force opposed by the ligament to passive stretching. Similar effects were elicited by several monoquaternary, N-substituted derivatives of trimethylammonium. 3. The opposite effect, i.e. softening, was induced by decamethonium, dimethylphenylpiperazine, and 2-ketoamyltrimethylammonium. 4. The involvement in these effects of ACh-binding groups with pharmacological properties similar to those of the anionic sites of nicotinic ACh receptors is suggested.

Correlation Between Amplitudes and Rise Times of the Miniature Endplate Potentials in Frog Muscle

An investigation of the relationship between the amplitudes and rise times of the miniature end-plate potentials recorded from the neuromuscular junctions of the frog sartorius has shown the existence of a positive correlation between these two parameters. This finding is consistent with the occurrence of a process of “spreading activation”, i.e. the progressive recruitment of end-plate receptors by a wave of acetylcholine molecules diffusing tangentially within the synaptic space. It appears that the initial concentration of transmitter molecules within the synaptic cleft must be large enough to saturate the receptors of the end-plate membrane, immediately subjacent to the site of release. This idea is supported by the result of calculations using a highly simplified model of the myoneural junction.

Pharmacological sensitivity of the articular capsule of the primary spines of Eucidaris tribuloides

1. This paper describes the effects of several cholinergic agonists and antagonists, and of beta-phenylethylamine (PEA) and some of its derivatives, on the articular capsule, or ligament, of the primary spines of Eucidaris tribuloides. 2. Carbamylcholine (CCh), methacholine (MeACh), nicotine, and muscarine exert a stiffening effect similar to that of acetylcholine (ACh), although the time course of their actions varies widely. 3. Atropine induced stiffening and blocked and responses to muscarine and MeACh. The responses to MeACh were blocked also by 4-diphenylacetoxy-N-methylpiperidine, suggesting the presence in the ligament of type M3 muscarinic receptors, in addition to nicotinic ones. d-Tubocurarine induced stiffness of the ligament and failed to block the responses to ACh and nicotine. 4. While ACh induced only a slight desensitization, CCh caused a long-lasting blockade of the stiffening effects of the cholinergic agonists. This shows that the receptors for ACh have a site or sites that recognize the ester moieties of these molecules. 5. Eserine and neostigmine potentiate the responses to acetylcholine, indicating the presence of acetylcholinesterase in the ligament. 6. beta-Phenylethylamine, epinephrine, norepinephrine, and dopamine induce diphasic responses; usually a brief softening followed by a slow and irreversible stiffening of the ligament. 7. In contrast to the above, tyramine and octopamine elicit a simple softening of ligaments which are stiff as a result of handling or by exposure to cholinergic agonists. However, tyramine and octopamine do not soften ligaments which become stiff as a result of exposure to adrenergic agonists.

On the modification of sulfhydryl groups by 4-cyclopentene-1,3-dione

4-Cyclopentene-1,3-dione reacts quantitatively with sulfhydryl groups of both cysteine and the sulfhydryl containing protein pinguinain. A 10fold excess of the reagent totally modifies cysteine after 15 min at room temperature and the single sulfhydryl group in native pinguinain is totally modified by a 48–86fold excess of reagent.

Quantal nature of the end-plate potential.

THE liberation of acetylcholine from the motor nerve endings in skeletal muscle is believed to take place in a quantal manner. The arrival of the action potential at the nerve terminals elicits the release of a number of packets of acetylcholine molecules of fairly uniform size, each of them producing a unit of depolarization in the post-synaptic membrane. This view is derived from the statistical analysis of the random step-wise fluctuations in the amplitude of successive end-plate potentials recorded from single neuromuscular junctions blocked by excess magnesium ions or lack of calcium ions1. The average magnitude of the steps was found to be identical with the mean size of the miniature end-plate potentials recorded from the same junction and due to the spontaneous extrusion of single acetylcholine quanta from the resting nerve endings.

Induction of electrical excitability in crustacean muscle by 4-cyclopentene-1,3-dione

4-Cyclopentene-1,3-dione induces electrical activity in inexcitable crustacean muscle. This effect is blocked by previous treatment with p-chloromercuribenzoic acid. These results suggest that crustacean muscle becomes excitable when certain-CH2-SH side chains are converted to thioethers having carbonyl groups.

Carbonyl Binding Sites in the Cholinergic Receptors of the Motor-End-Plate

The microelectro-osmotic application of either methylacetate (MA) or ethylacetate (EA) to the chemosensitive areas of the end-plate membrane in frog muscle is followed by depolarizations similar to those elicited by the iontophoretic application of acetylcholine from the same multi-barrelled micropipettes. The chemical function responsible for the cholinergic activity of MA and EA, well-known substrates of acetycholinesterase, appears to be their carbonyl group. If the carbonyl oxygen of EA is replaced by two hydrogens, the resulting compound is ethyl ether, which not only lacks depolarizing action but is a cholinergic blocking agent. On the contrary, if the ether oxygen of MA is removed, the resulting compound, acetone, retains the depolarizing action. These results demonstrate the presence of a carbonyl binding site in the cholinergic end-plate receptors and also that drug-receptor interactions restricted to this site are sufficient to induce receptor activation.