AUTONOMIC NEUROEFFECTOR TRANSMISSION 



221 



such as the spleen and the kidney of the sheep (129). 

 The stores of the transmitter substance can thus be 

 estimated by extracting the tissue and subjecting it to 

 chemical or biological analysis. The content of 

 adrenergic transmitter in an organ (table 2) provides 

 a measure of the relative supply of adrenergic nerves. 

 Norepinephrine was first suggested as a link in the 

 biosynthesis chain leading to epinephrine by Blaschko 

 (11). The basis for this was given by Holtz, Heise & 

 Liidtke (68) who discovered an enzyme capable of 

 decarboxylating levo-dihydroxyphenylalanine (dopa) 

 to its corresponding amine, hydroxytyramine (dopa- 

 mine). This enzyme was present in liver and kidney 

 and has also been demonstrated in the adrenals and 

 in adrenergic nerves (69). While it has been shown 

 experimentally that homogenates of the adrenal gland 

 synthesize norepinephrine from tyrosine (74), via 

 dopa (33) and dopamine (59), this sequence has not 

 been shown for adrenergic nerves although there 

 can be little doubt that this is the case. At any rate 

 it has been found that extracts of the spleen or 

 splenic nerves contain relatively large amounts of 

 dopamine (117, 132). The biosynthesis may there- 

 fore be depicted by the following .scheme: 



OH 



OH 



iOH 



CH2CHCOOH 



CH.,CHCOOH 



It appears likely that the biosynthesis is located in 

 the place of storage (see below). Analysis of extracts 

 of autonomic nerves have shown that the norepineph- 

 rine content is a function of the proportion of adrener- 

 gic fibers. These contain the transmitter along their 

 whole length and also in the jxU soma. A very 

 marked accumulation in the terminal parts must be 

 assumed for the following reasons. Splenic nerves of 

 the beef contain about 15 ng norepinephrine per gm 

 fresh tissue after removal of the sheath, while the 

 content of the whole organ is about 3 //g per gm. Since 



all of the splenic norepinephrine disappears on section 

 and degeneration of the adrenergic nerves to the 

 organ it is assumed that the norepinephrine found in 

 the organ is bound to its nerves. On the other hand 

 it is inconceivable that 20 per cent of the splenic 

 tissue consists of nerves, and it follows from this that 

 some parts of the nerves, presumably the endings, 

 contain much more of the transmitter than the main 

 nerve trunks. 



Even afte r rern oval from the body, organs retaiin 

 their adrenergic transmitter substance for a con- 

 siderable time. A beef spleen may thus be stored at 

 room temperature for 24 hours without any detectable 

 loss of norepinephrine. This indicates that it is not 

 present in a freely diffusible form and strongly sug- 

 gests that it is bound in such a way as to prevent con- 

 tact with inactivating enzymes. 



Evidence has been obtained for the storage of the 

 hormones of chromaffin cells in specific granules (12, 

 63). By increasing the acidity of the surrounding solu- 

 tion to pH5 or lower, the chromaffin cell hormones 

 are released from the granules (63). When the same 

 principle was applied to the isolated spleen by per- 

 fusi^ng it with a .solution containing acids such as as- 

 corbic, citric or lactic acid, the transmitter substance 

 was released and could be demonstrated in the per- 

 fusion fluid (40). Also other substances which have 

 been found effective in releasing the hormones from 

 isolated granules had a similar action on the perfused 

 spleen, such as detergents, digitonin and lecithinase 

 from snake venom. 



These e.xperiments add support to the hypothesis 

 (127) that the neurotransmitter is stored, and proba- 

 bly manufactured, in specific structures in the 

 adrenergic axon. Experiments by Euler & Hillarp 

 (131) have demonstrated that a microgranular frac- 

 tion rich in norepinephrine can be separated by high 

 speed centrifugation from a homogenate of beef 

 splenic nerves. The chemotransmitter is apparently 

 stored in elements surrounded by a membrane since 

 a suspension of the sediment in Ringer's solution 

 does not give off norepinephrine to the surrounding 

 fluid. If acid is added to PH4 in the suspension, the 

 norepinephrine is instantaneously released, however, 

 and can be demonstrated by ijiological and chemical 

 methods in the suspension fluid. The micrograniilar 

 stores are apparently specific for the chemotrans- 

 mitter since the histamine which is abundant in the 

 beef splenic nerves (about 100 ixg per gm nerve) is not 

 present in the same structural elements. Certain 

 cellular fractions have been found to contain more 

 than 1.5 fig norepinephrine per mg dry weight or 



