Frank G. Standaert

THE CONSEQUENCES OF CHOLINERGIC DRUG ACTIONS ON MOTOR NERVE TERMINALS

This conference occurs at the end of our first decade of investigation of motor nerve terminals. Since in large part this research has sought to understand better the action of prototypic cholinergic drugs, it seems appropriate, in this our fourth progress report to this Academy (Riker et al., 1959a; Riker & Standaert, 1966; Standaert & Roberts, 1967), to summarize our work and attempt to put it into perspective. For almost 50 years, investigative interest in the neuromuscular junction has focused on post-junctional structures, and neuromuscular function has been studied by observing muscle phenomena, that is, by recording the tension developed by the contracting muscle or by recording muscle electrical activity. These methods, however, record only the end-result of a chain of events which begins in the nerve, courses through the nerve terminal, crosses the synapse to activate the end plate and finally, triggers the muscle. Of necessity, any phenomenon recorded in the muscle is the resultant of functions of each of these structures. Many ingenious ways have been devised to resolve this resultant, but unfortunately these systems must, in some part, t e arbitrary; most commonly they depend on arbitrary assumptions of drug actions. Our concern about the adequacy of certain pharmacologic assumptions led us to undertake an examination of the primary structures in the neuromuscular junction, the motor nerve terminals. The inset in the upper right of FIGURE 1 is a sketch from Coers and Woolf (1959) of a mammalian motor nerve terminal. It shows that in proximity to the muscle, the motor nerve loses its myelin, decreases in diameter and branches repeatedly. These changes imply that the terminal may have pharmacologic properties quite different from those of the parent axon. Thus the lack of myelin suggests that ions, including quaternary ammonium compounds, will have ready access to the nerve membrane. The multiple branching suggests that the safety factor for impulse propagation is low and that blockade can occur easily. The small diameter suggests that the terminals will conduct more slowly and develop more prolonged afterpotentials than does the parent axon. The method we selected to study the nerve terminals is based on the fact that neural activity begun at any point in a nerve propagates throughout the axonantidromically as well as orthodromically-and so can be recorded at any point on the axon. We chose to record from the ventrical root portion to insure isolation of a motor axon. The recording method, described in earlier publications (Riker et al., 1957; Standaert, 1963), is shown in FIGURE 2. The politeal fossa of an anesthetized cat is dissected to expose the soleus muscle, its nerve and blood

Characterization of the train-of-four response in fast and slow muscles: effect of d-tubocurarine, pancuronium, and vecuronium.

The in vivo cat soleus and gastrocnemius muscles were used to compare isometric contraction strength and the train-of-four (T4) response (2 Hz for 2 s) of two muscle types (fast and slow) during onset of competitive neuromuscular blockade in order to determine the extent of the correlation between twitch depression and T4 fade. Prior to drug administration the muscles that were studied differed significantly in that the T4 ratio was 1.0 in the gastrocnemius and only 0.87 in the soleus. Three competetive neuromuscular-blocking agents were compared: d-Tubocurarine, pancuronium, and vecuronium. d-Tubocurarine was found to produce a close correlation between the degrees of twitch strength depression and T4 for both muscles. However, these muscles demonstrated significantly different ED50 values (105 μg/kg for gastrocnemius, 150 μg/kg for soleus). Pancuronium also produced a similar relationship between twitch strength depression and T4 decrement for each muscle. In this case, however, there was little difference in their Ed50 values for twitch depression (11.5 μg/kg for gastrocnemius, 13 μg/kg for soleus). The effects of vecuronium were quite different from the other two muscle relaxants. Although vecuronium produced a comparable correlation between twitch tension and T4 fade in fast muscle, no such relationship was found to exist in slow muscle. Even when the twitch strength was blocked to 18% of control, the soleus T4 response was depressed to only 75% of control. These results higlight major differences among competitive neuromuscular-blocking agents and suggest multiple sites of action.

A NEURAL ACTION OF PRONETHALOL

Certain effects of pronethalol, principally the antagonism of digitalisinduced arrhythmia, do not seem to be related to the drug’s action to block beta adrenergic receptors (Lucchesi, 1964, 1965; Roberts et al . , 1965; Somani et al., 1966). Although the mechanisms of these other effects remain unclear, there are several indications they may have their bases in an action on the terminal filaments of adrenergic nerves. The capacity of the drug to affect neural function is apparent in studies of the effects of pronethalol on spinal reflexes (Morales-Aguilera & Vaughan Williams, 1965; Sekiya and Vaughan Williams, 1965; Vaughan Williams, this monograph). Furthermore, it is an impressive fact that p-TM10, diphenylhydantoin and reserpine, which like pronethalol are effective against the digitalis-induced ventricular arrhythmia (Mosey & Tyler, 1954; Roberts et al . , 1963), are predominantly neurotropic agents. T o examine the possibility that pronethalol may have nerve terminal actions, we turned to a system which provides a sensitive indicator of nerve terminal activity; the soleus motor nerve terminal preparation. In addition to its simplicity and reliability this system has the advantage of being unequivocally nonadrenergic, thereby permitting the neural actions of the drug to be examined independently of its adrenergic blocking actions. Using this preparation, we demonstrated a capacity of pronethalol to depress activity in motor nerve terminals. The experimental procedure has been described previously (Standaert, 1963,1964) and is schematized in FIGURE 1. When the soleus nerve is stimulated singly it responds to the stimulus with a single action potential which, because the nerve potential propagates orthodromically and antidromically from its origin, may be recorded from any point along the axon. I t is most convenient to do so a t the ventral root, as illustrated in FIGURE 1 (left inset, trace C ) . This usual response to stimulation changes when the soleus nerve is subjected to a brief period of high-frequency stimulation. Subsequent to the high-frequency stimulation the nerve response to a single stimulus is no longer single, bu t is repetitive (left inset, trace P T ) . This post-high frequency (more commonly termed post-tetanic) repetitive activity has been thoroughly analyzed and found to originate in the most distal portion of the motor axon, the motor nerve terminal (Standaert, 1963). The repetitive activity is a highly reliable and sensitive indicator of the functional state of the soleus motor nerve terminal, but the procedure for recording it directly from the nerve is *These investigations were supported by U. S. P. H. S. grants No. NB-05706 and

The influence of sympathetic nervous activity on the antiarrhythmic efficacy of diphenylhydantoin

Abstract The efficacy of diphenylhydantoin as an antagonist for ouabain induced ventricular rhythm disorders was determined in spinal cats exhibiting signs of low and high adrenergic neural activity. In cats with high heart rates and blood pressures, diphenylhydantoin was effective in increasing the lethal dose of ouabain. On the other hand, in animals with slow heart rates and low blood pressures, diphenylhydantoin produced no increase in the lethal dose of ouabain. After diphenylhydantoin administration both groups of ouabain intoxicated animals behaved similarly with respect to heart rates, blood pressures, rate of ventricular tachycardia and lethal dose. These data indicate that diphenylhydantoin produces a reduction in adrenergic activity and that this reduction may account for the drugs antiarrhythmic activity.

The capacity of N-isopropyl-p-nitro-phenylethanolamine (INPEA) to influence the course of ouabain-induced cardiotoxicity in the cat

Abstract In previous reports we demonstrated a correlation between the capacity of drugs to depress the terminals of cat soleus nerves and their capacity to block ouabain-induced ventricular rhythm disorders. To test this correlation we examined the neurodepressant activity of INPEA, an agent which has been reported to be ineffective in antagonizing digitalis-induced arrhythmias. We found INPEA to have neurodepressant actions but that expression of these actions required more drug than had been used in previous studies of the drugs antiarrhythmic activity. When the neurodepressant doses were tested, they were found to be effective in antagonizing ouabain-induced ventricular tachycardia and death. The results are discussed with regard to the role of neural and myocardial depression in the control of digitalis-induced arrhythmias.

Motor nerve ending disorder in myasthenia gravis

Mild myasthenia gravis patients were compared with normals with respect to the capacity of their motor nerve endings (MNEs) to generate a neostigmine-induced postactivation repetition (PAR). Dose-response analyses of PAR recorded from muscle electrically and by contractile measurement disclose a loss of this pharmacologic responsiveness in myasthenia. Since mild myasthenics transmitted nerve impulse trains of 20 to 200 Hz, as did normals, it was evident that PAR is transmitted insofar as it can be generated by MNEs. The dose-response analyses support this. These data indicate an MNE disorder in the disease.