Walter F. Riker

The pharmacology of Flaxedil, with observations on certain analogs.

Until as recently as 1946, the classical pharmacologic action of the natural curare alkaloids had had no counterpart in the many synthetic compounds examined in laboratories of pharmacology and physiology. True, Crum Hrown and Fraserl had described a paralytic action resulting from the yuaternization of the nitrogen atom of certain alkaloids, but this effect was soon proved to he characteristic for most quaternary amines and was recognized by physiologists as different in mechanism from the neuromuscular blockade produced by the natural curare alkaloids. In 1946, Bovet and his collaborators attempted to reproduce synthetically a simplified version of the d-tubocurarine molecule. They achieved the synthesis of a series of bis quinoline derivatives wherein the aromatic structures were connected through a methylene chain by ether linkages.‘ These compounds were actively curariform. Shortly thereafter, they examined simple mono and poly phenolic ethers of certain amino alcohols and found that these substances also possessed a striking curariform action.a In the latter series was the compound, triiodoethylate of Iris (triethylamino ethoxy) 1,2,3 benzene. This compound, named Flaxedil, exerted a potent curariform action in the rabbit. The following year! Barlow and In

Destruction of mammalian motor nerve terminals by black widow spider venom

and I’aton and Zaimis5 reported that the paralytic action of quaternary amines could be intensified by the twinning of the aniine groups through polymethylene chains of varying lengths. This represented, in principle, the distant quaternary ammonium groups found in the molecule of d-tubocurarine. Most recently, Kimura et d6 described the potent neuromuscular blocking action that results from the twinning of two atropine molecules through an amyl chain. Thus, the principle was established by which compounds with strong neuromuscular blocking action could be synthesized by establishing at least two quaternary ammonium groupings a t an optimal intramolecular,distance. Of these compounds, two have had a considerable preliminary clinical trial in the practice of anesthesiology, namely, Flaxedil and decamethonium bromide, the bis trimethylammonium decamethylene compound. The present report is concerned with the pharmacologic properties of Flaxedil, particularly as they compare with those of d-tubocurarine. The onset and development of paralysis that follows the intravenous injection of Flaxedil into the intact cat is entirely like that occurring in this animal after an equipotent dose of d-tubocurarine. The order of events is such that the respiratory musculature is usually the last to succumb. As with other compounds of this nature, resulting paralysis of the muscles of respiration is not only the direct cause of death but the

THE PRESYNAPTIC ELEMENT IN NEUROMUSCULAR TRANSMISSION

Black widow spider venom selectively poisons motor nerve endings. A progressive and irreversible failure of neuromuscular transmission occurs in the cat. Electron microscopy of the poisoned nerve-muscle junction shows a sequence of motor nerve ending damage that culminates in disruption of the prejunctional membrane and loss of all organelles, including synaptic vesicles. The postjunctional membrane was morphologically unaffected. After complete poisoning, the contractile response to exogenous acetylcholine was severely impaired, an indication that the prejunctional site is chiefly involved in the contractile response produced by exogenous acetylcholine and that the pre- and postjunctional effects of acetylcholine were separated.

THE CONSEQUENCES OF CHOLINERGIC DRUG ACTIONS ON MOTOR NERVE TERMINALS

During the past two decades the neuromuscular blocking drugs have proved to be an exceedingly popular subject for pharmacological and physiological study. Oddly, the opposite aspect, that is, the facilitation of neuromuscular transmission by drugs, has been given relatively little attention. This topic was first exposed to serious scrutiny when Brown et al. (1936) demonstrated the effect of physostigmine in enhancing the contractile response of mammalian striated muscle. Immediately thereafter Brown (1037) ascertained that the mechanism of this effect results from the conversion of each single muscle response to a repetitive one. Just two years earlier the clinical expression of neuromuscular facilitation was recorded when Walker (1934) described the dramatic alleviation of the myasthenic syndrome through the administration of either physostigmine or its newly synthesized congener neostigmine. Later research i n man (Harvey and Lilienthal, 1041) related these laboratory and clinical observations, and it. was revealed that the transmission deficit in myasthenia gravis could be temporarily repaired by neostigmine. At this time Eccles and his co-workers (1941) employed physostigmine to examine the newly discovered end-plate potential and found that the alkaloid greatly prolonged junctional negativity. This seemed a reasonable consequence of the drug’s anticholinesterase (anti-ChE) activity, and Eccles and MacFarlane thereafter (1940) showed a similar effect for other carbamates and di-isopropylfluorophosphate (DFP). On the basis of this work, the prolongation of junctional negativity is often considered synonymous with ChE inhibition. Whether or not this evidence assures a causal relationship may be questioned. In any case, the findings of Eccles et al. (1941) are important., for they disclose a mechanism by which physostigmine and similar drugs cause the junctional region to initiate repetitive discharges in response to individual nerve volleys. In 1946 W. C. Wescoe and I were engaged in an evaluation of DFP in the management of myasthenia gravis. Our efforts only impressed us the more with the remarkable therapeutic efficacy of neostigmine in this disease. Consideration of structure-activity relationship led to a search of the existing lit.erature that revealed, for neostigmine and its congeners, only a concern with the dependence of ChE inhibition on the presence of an alkyl carbamino group; strangely, the possible functional importance of the quaternary ammonium grouping was unmentioned. Simple experiment showed (Riker and Wescoe, 1946) the functional nature of the quaternary ammonium grouping. It was found that chronically denervated cat striated muscle is susceptible to direct excitation by neostigmine, just *.s it is by acetylcholine and other quaternary ammonium ions in which the N .r.tGm is substituted with 3 methyl groups. To * The work reported in this paper was supported in part by a research grant from the Nat ional Institute of Neurological Diseases and Blindness, Public Health Service, Bcthesda, Mtl. t Fellow, Lederle Faculty Fund, Pearl River, N. Y. 1 Fellow, United States Public Health Service, Bethesda, Md. Department of Phannucology, Cornell University Medical College, New Y o r k , N . Y .

Electrophysiologic and Clinical Aspects of Glucocorticoids on Certain Neural Systems

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

Neuromuscular pharmacology in rat neonates: development of responsiveness to prototypic blocking and reversal drugs.

This review considers the premise that certain neural effects of exogenously administered glucocorticoids arise from direct, non-receptor-mediated actions. It is emphasized that these effects are not regarded as alternatives to the well-documented actions mediated via cytoplasmic receptors and nuclear actions (see chapters by McEwen and Bohus); rather, these effects are considered in addition to classic mechanism. In a concluding section, data are presented to support the concept that these glucocorticoid effects result from a direct action on susceptible membranes. The evidence discussed suggests that particular actions of exogenously administrated glucocorticoids, especially with repeated high dosage, cause changes in the electrical excitability of certain susceptible neuronal structures and systems. These actions undoubtedly occur pari passu with the well-known cytoplasmic and nuclear events of these steroid hormones.

d-Tubocurarine sensitivities of a fast and a slow neuromuscular system of the rat

The neonatal pharmacology of neuromuscular drugs was studied in vivo in newborn rats and in vitro in neonatal phrenic nerve-hemidiaphragm preparations. Drugs used to probe neuromuscular development in rat neonates were physostigmine, edrophonium, neostigmine, 4-aminopyridine, d-tubocurarine (dTc), and succinylcholine. The prejunctional actions of these drugs were monitored in relation to neonatal age by the appearance of stimulus-evoked repetitive discharge initiated by motor nerve endings and the occurrence and magnitude of the resulting enhancement of twitch tension. The occurrence and incidence of drug-induced fasciculations also served to track the development of functional motor nerve endings. Each of these prejunctional actions was inoperative until the third neonatal week, indicative of incomplete motor nerve development. In contrast, 4-aminopyridine, a nonanticholinesterase, evoked these prejunctional actions in 1-wk-old rat neonates. Neostigmine and edrophonium antagonized dTc as early as the first week; presumably, postsynaptic maturation had reached a functional level. 4-Aminopyridine also antagonized dTc at week 1. Rat neonates showed resistance to dTc blockade when tested by neonatal phrenic nerve-hemidiaphragm preparations in vitro. Relationships between age and 85%-95% transmission block declined to the adult level by week 5. This result indicates that in rat neonates, pharmacodynamic rather than pharmacokinetic mechanisms predominate in the development of responsiveness to dTc.

The interactions of ouabain with post-tetanic and facilitatory drug potentiations at cat soleus neuromuscular junctions in vivo

Fast and slow neuromuscular systems in the rat were compared with respect to their sensitivity to d-tubocurarine (dTC). The fast tibialis anterior was more sensitive than the slow soleus to dTC-induced block of contractile tension when stimulated at either 0.2 or 1.0 Hz. These results in the rat contrast those made by others in the cat. Thus, relative drug sensitivities are not simply related to neuromuscular type.

Further observations on subacute denervation and mammalian motor nerve terminal function

Cat soleus motor nerve terminals, after high frequency conditioning, generate a post-tetanic repetition (PTR) which leads to a post-tetanic (PTP) of the muscle response. This property enables quantitative assessment of enhancement or depression of this nerve terminal excitability in vivo. The present study focuses on ionic mechanisms underlying the PTRs produced in this neuromuscular system either by high frequency stimulation or edrophonium. Ouabain was used as a specific probe for inhibition of Na+−K+ ATPase and its known consequences on Na+ and Ca2+ translocation. Ouabain pretreatment doubled the duration over which single stimuli, following either high frequency or edrophonium conditioning produced PTR. Ouabain in the doses used had no effectper se but as a function of dose augmented the frequency dependent responses. This pointed to Na+ loading of nerve terminals via high frequency stimulation plus ouabain inhibition of Na+−K+ ATPase. Ouabain potentiation of PTR responses evidently depends on exchange of intra-terminal sodium for external calcium. Thus, calcium entry blockers, Mn2+, and Co2+ suppressed or abolished the potentiations both before and after ouabain. Diphenylhydantoin, a Na+ and Ca2+ blocker, acted similarly. The effects of stimulation frequency, ouabain and the sequence of events leading to PTR in the soleus neuromuscular system appeared in general no different from those derived from the many in vitro microphysiologic studies of this phenomenon. Thus, EPPs were augmented and prolonged. It was concluded that intracellular Ca2+ is critical for regulating the stability of systems in which repetitive firing is both a normal and abnormal function.

Motor nerve ending disorder in myasthenia gravis

a In a previous study of neuromuscular function in the cat soleus, a slow nerve-muscle system, it was learned that 48 hours after nerve section (subacute denervation), disorders in motor nerve terminal function precede transmission loss.’ Companion studies explored the pharmacology of the 48 hour denervated junction and revealed that functional integrity of the unmyelinated terminals is essential for the a c t i o n of the neostigminelike facilitatory drugs.* This finding confirmed a number of earlier studies showing that the mammalian motor nerve terminals are the site of this facilitatory drug a ~ t i o n . ~ Because of the known functional differences between motor nerves t o tonic and phasic musc l e ~ , ~ ~ it was decided t o ascertain the physiologic and pharmacologic losses that might be exhibited by a phasic system 4 8 hours after nerve section. For this purpose, a gastrocnemius nerve-muscle preparation of the cat was chosen. The results established that the same pattern of losses occurs in this preparation as in the soleus. However, certain quantitative differences were noted.