418 ANNALS NEW YORK ACADEMY OF SCIENCES 



activity disappears, the measurement of the CO2 liberation falls into 

 a range where precise evaluation becomes difficult. Moreover, in such 

 a preparation, the retention of CO2 by the protein becomes an impor- 

 tant factor. Finally, even in the thin lipoid membrane of the lobster 

 nerve after prolonged washing, sufficient excess of DFP is retained 

 to inhibit 20-40 per cent of the remaining esterase activity. At 

 least this amount, if not more, may be retained in the relatively greater 

 amount of myelin and fat in the bullfrog sciatic nerve. When this 

 nerve is then ground, the retained DFP may come in contact with the 

 cholinesterase and destroy a considerable fraction of the enzyme still 

 present in the intact nerve. 



DISCUSSION 



It may be of interest to discuss the neuro-humoral theory in the light 

 of recent developments, and to analyze the two basic experiments which 

 form the main support for the hypothesis that the ester is actually 

 liberated at the nerve ending and, having crossed the synapse or motor 

 end-plates, acts directly on the second neuron or on the muscle fiber. 

 The two observations are: (1) The stimulating action of ACh when 

 applied to synaptic regions; (2) the appearance of the ester in the 

 perfusion fluid, following nerve stimulation. It has just been explained 

 why the effect of ACh applied externally is limited to the nerve ending. 

 In any case, a stimulating effect is not necessarily a physiological effect, 

 but may well be a pharmacological one. The same action may, indeed, 

 be produced by other compounds. The observation of Otto Loewi that 

 a compound appears in the perfusion fluid, following nerve stimulation, 

 was important because it suggested that the compound may be con- 

 nected with nerve activity. The importance of this observation need 

 not be minimized because a quarter of a century later the original in- 

 terpretation has to be changed. In fact, by the new development, the 

 role of ACh became more general and more important than could orig- 

 inally have been anticipated. The appearance of a compound in the per- 

 fusion fluid, however, is not sufficient evidence for concluding that the 

 compound acts outside the cell. Many compounds of intermediate 

 cell metabolism may appear outside the cell. This is due to the fact 

 that all enzymatic reactions follow a logarithmic curve. Therefore, 

 if even the greatest part of a compound is rapidly metabolized by the 

 intracellular enzymes, a small fraction may persist long enough to 

 escape enzymatic action and leak out from the cell. This, apparently, 

 may happen also to ACh, in spite of the high concentration of cholin- 



