Wolf-D. Dettbarn

Effects of acetylcholine on axonal conduction of lobster nerve

Acetylcholine and related compounds have been tested for their effects on the electrical activity of lobster walking-leg nerve bundles. Changes in action and membrane potential were recorded with the “sucrose-gap” technique. ACh in concentrations of 5·10−3–10−2 M decreases the membrane potential; the simultaneously recorded action potentials initially display an increased amplitude and a marked prolongation and elevation of the descending phase which appears oscillatory. As the depolarization progresses, the spike height decreases and the effects on the descending phase decline. Finally reversible block of conduction occurs. d,l-Acetyl-β-methylcholine (10−2 M) produces similar effects on the action potential but the membrane potential is only slightly depolarized. Choline, acetate and bromide in concentrations equal to those of ACh have no effect; in 10-fold higher concentrations only choline produces minor changes in the action potential. Physostigmine (5 mM) produces effects similar to those seen with ACh while lower concentrations inhibit the action of ACh. Atropine and tetracaine reversibly block conduction and like physostigmine, also inhibit the effect of ACh. Although curare was nerve demonstrated to block conduction, it does inhibit the action of ACh. Irreversible cholinesterase inhibitors (phospholine, Paraoxon) block conduction. Paraoxon much faster than phospholine. The membrane potential is decreased by 20–30 mV, and while the effects of these inhibitors on the membrane potential are reversible, conduction remains blocked. The difficulties of interpreting some of these actions on a nerve bundle are discussed.

THE ACETYLCHOLINE SYSTEM IN PERIPHERAL NERVE

One of the characteristics of living cells is their high K t concentration in contrast to the low concentration in the outer environment. The reverse is true for the Na +. Excitable cells, such as nerve and muscle fibers, are known to utilize their ionic concentration gradients for the generation of electric currents and the propagation of impulses. It has long been realized that nerve fibers have a potential difference across their membranes, and it was suggested that the membrane was selectively permeable to K + . l Thus, the resting potential was described in terms of the Kf concentration gradient. While this may be true, in general, for the resting membrane, it was found that the conduction of a nerve impulse is associated with an action potential larger than the resting potential. The passage of an action potential involves a reversal in the polarity of the membrane. This reversal in polarity is widely attributed to a specific permeability change of the cell membrane. The rising phase of the action potential coincides with an increased permeability of Na+ which will cause the inside of the membrane to become positive. The repolarization is due to an increased efflux of K+.2 While these phenomena have been widely accepted, the molecular mechanism underlying these permeability changes is still under discussion. Some investigators accept the view that a depolarization causes an increase in Na+ and K + permeability because charged particles move to new positions in the membrane under the influence of the electric field. When the membrane repolarizes, the charged particles return to their original positions, and the only alteration that has taken place in the system is that some Na+ and K have moved down their concentration gradients. This view holds that the permeability change is a purely physical event, one for which no chemical process is needed.3 However, the theory involving chemical reactions has assumed for a long time that a shift of charged particles, i.e. conformational changes of proteins, is responsible for the permeability change^.^^^ Recent investigations of the heat production coinciding with the conduction of impulses have added strong support to the theories involving chemical reactions.s The last 30 years have witnessed a remarkable development of the role of the ACh system in nerve f ~ n c t i o n . ~ The review of these data reveals that for a long time the main emphasis was put on biochemical reactions. enzymes, their substrates and inhibitors, and proteins in general, while there were important aspects that remained unexplained. The challenge remained of applying the biochemical findings, together with electrophysiological studies, and to integrate the two lines of approach. Evidence was needed to show the direct interrelationship of these biochemical findings with the change and the control of electrical activity. The determinations of reactions and turnover rates in simple systems offer many difficulties, but they increase many-fold in the complex milieu of an intact nerve. The intricate interdependence of structure, chemistry and function has been strik-

Ester-splitting activity of the electroplax

Abstract 1. 1. The hydrolysis of acetylcholine, dl -acetyl-β-methylcholine, ethyl chloroacetate, triacetin and butyrylcholine by intact and homogenized isolated single cells and by the connective tissue between electroplax has been tested. Some connective tissue remains attached to the electroplax; a correction for this extracellular esterase was therefore applied to evaluate esterase activity of the electroplax proper. 2. 2. The electroplax enzyme displays the characteristics of acetylcholinesterase. The connective tissue enzyme has neither the characteristics of acetylcholinesterase, human-serum cholinesterase, aliesterase nor arylesterase. In some respects, however, it resembles the cholinesterase of certain piscine plasmas. 3. 3. Hydrolysis of low concentrations of acetylcholine by intact cells is mainly by acetylcholinesterase of the electroplax, whereas high concentrations of acetylcholine are mainly hydrolyzed by the connective tissue esterase. Both enzymes are inhibited by physostigmine. Ethyl chloroacetate is hydrolyzed by at least 2 enzymes in the electroplax, only one of which is inhibited by physostigmine. 4. 4. There is a strong permeability barrier to the penetration of acetylcholine to the interior enzyme of the intact electroplax. This barrier is less strong for dl -acetyl-β-methylcholine, ethyl chloroacetate, triacetin and butyrylcholine. 5. 5. Concentrations of physostigmine which block electrical activity markedly depress acetylcholine hydrolysis. Tetracaine even in much higher concentrations than required to block electrical activity only slightly inhibits acetylcholine hydrolysis. Inhibition by neostigmine is intermediate between that of tetracaine and physostigmine.

Increased cholinesterase activity of intact cells caused by snake venoms

Abstract Cottonmouth moccasin and Eastern diamondback rattlesnake venom increase the cholinesterase (ChE) activity observed in intact squid and lobster nerves and single electroplax. Cottonmouth was more effective than rattlesnake venom in lobster nerve and electroplax. Neither venom increased the activity of rabbit cerebral cortex slices or of a partially purified preparation of electroplax ChE. Both venoms are free of ChE activity. Strong permeability barriers are present in homogenized electroplax: they can be most effectively reduced by the use of the venoms. The venoms cause a reduction in the wet and dry weights of electroplax cells. They also depolarize and block electrical activity in concentrations of 50–100 μg/ml. Venoms may be useful in studies where it is necessary to measure the total ChE activity in intact cell preparations containing permeability barriers to the substrate normally employed in this enzyme assay.

EYE DROPS AND DIARRHEA. DIARRHEA AS THE FIRST SYMPTOM OF ECHOTHIOPHATE IODIDE TOXICITY.

ECHOTHIOPHATE iodide (Phospholine iodide) is administered as eye drops principally for the control of intraocular pressure in glaucoma. When introduced into the conjunctival sac, it produces an int...

HYDROLYSIS OF CHOLINE ESTERS IN INVERTEBRATE NERVE FIBERS.

The hydrolysis of acetylcholine, dl-acetyl-s-methylcholine, butyrylcholine and triacetin by various homogenized invertebrate nerve tissues has been compared. The invertebrate species used were: lobster, spider crab and squid. The enzyme of the different invertebrate nerves shows the characteristics of acetylcholinesterase (acetylcholine acetyl-hydrolase, EC 3.1.1.7) as indicated by the substrate concentration-activity relationship. The enzyme is inhibited by physostigmine. Hydrolysis of acetylcholine and acetyl-s-methylcholine by axoplasm of the giant axon of squid is only 20% and 10% of that of the envelope.

Restoration by a specific chemical reaction of “irreversibly” blocked axonal electrical activity

Abstract Experiments are reported in which the electrical activity of axons is first completely and irreversibly abolished by a well defined chemical reaction, namely the phosphorylation of a serine molecule in the active site of ACh-esterase by Paraoxon. Subsequently PAM is added which reverses the first reaction by a displacement of the phosphoryl group from the enzyme. By this second specific reaction the abolished activity is fully restored. The experiments confirm, in a striking way, the postulated inseparable association between electrical and ACh-esterase activity; they illustrate the possibility of bridging the gap between the molecular forces analyzed in vitro and the events recorded on an intact cell, since the information obtained about the reaction mechanism of an enzyme molecule has been successfully applied first to the abolition and then to the restoration of a primary cellular function, i.e., the generation and propagation of bioelectric currents.

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.

Depolarizing action of calcium-ion depletion on frog nerve and its inhibition by compounds acting on the acetylcholine system

Abstract The membrane-conductance change resulting from Ca-ion depletion has been investigated on desheathed frog tibialis nerve with the “sucrose gap” technique. Several compounds known to be acetylcholine-receptor inhibitors and thus to antagonize the depolarizing action of acetylcholine by competitive action have been tested for their effects on the depolarization produced by Ca-Ion depletion. Antagonistic effects were obtained with four different receptor inhibitors: physostigmine, procaine, dimethyl curare and atropine. The effect is rapid and reversible. The experiments suggest that the removal of Ca ions leads to depolarization of the conducting membrane by means of acetylcholine-receptor activationl; they support the view of an essential role of the acetylcholine system in ionic-conductance changes.

Electrical and esterase activity in axons

Abstract The interdependence of electrical activity and critical level of cholinesterase activity has been investigated on intact single giant axons of the squid. The Warburg manometric technique was used for measuring the enzyme activity with dimethylaminoethyl acetate as the substrate, and electrical activity was recorded with intracellular electrodes. An irreversible enzyme inhibitor (DFP) was used to block enzymic and electrical activity. The critical enzyme level at which conduction in these fibers was blocked was about 16%. The substrate-enzyme activity relatioship was tested with intact and homogenized preparations of three different types of axons (of lobster, squid and spider crab). Three different substrates were used which differed only by the removal or addition of one methyl group: acetylcholine, acetyl- DL -β-methylcholine, and dimethylaminoethyl acetate. With intact fibers, the differences between the three fibers towards the substrates were quite marked, apparently due to differences in structural permeability barriers. These results emphasize the importance of such barriers for the evaluation of pharmacological events.