Catherine O. Hebb

Choline acetylase in antero- and retro-grade degeneration of a cholinergic nerve.

Feldberg (1943), and later Banister & Scrase (1950), showed that after denervation the superior cervical ganglion gradually loses its ability to synthesize acetylcholine. Neither of the methods used in these investigations permitted direct measurement of choline acetylase activity. Even though that employed by Banister & Scrase was very sensitive, the rate-determining factor in the production of ACh in their system need not have been choline acetylase; equally it could have been the activity of the enzymes which are necessary to produce acetyl coenzyme A, the substrate required by choline acetylase. The problem is resolved in the present experiments by using a system in which acetyl CoA is provided separately (Hebb, 1955). This method permits a direct measurement of ChA (choline acetylase) activity and we have been able to determine how the ChA of the cervical sympathetic nerve is affected by degenerative section.

Acetylcholine and choline acetyltransferase in the diaphragm of the rat

Although we know that skeletal muscle and many peripheral nerves contain acetylcholine (ACh) and exhibit choline acetyltransferase (ChAc) activity (acetyl-CoA:choline-O-acetyltransferase, EC 2.3.1.6), there is little evidence about how these components are distributed within a muscle. This is of some interest since it has been claimed that the spread of activity along the muscle fibres depends upon a cholinergic mechanism (Nachmansohn, 1959, 1963). If that is the case one might expect to find an even distribution of ACh and ChAc throughout the tissue without any great accumulation in the regions where motor nerve fibres and their endings are situated; particularly if neuromuscular transmission is not mediated by ACh, as is believed by the same author. To investigate this problem, we have examined the gross distribution of ACh and ChAc activity in the rat diaphragm, comparing the zone of innervation with the remainder of the muscle. In addition, we have estimated the ACh content and ChAc activity in the phrenic nerve. A preliminary account of these observations has been given at the 2nd International Pharmacological Meeting (Krnjevic, 1964).

Choline acetyltransferase activity of human brain tissue during development and at maturity.

Abstract— (1) On analysis of human brain tissue to determine its choline acetyltransferase (ChAc) content the recovery of enzyme from many regions is very poor when the tissue is acetone‐dried and then extracted in the standard manner; for this reason the method is unsuitable when quantitative recoveries are required; it is preferable to prepare sucrose homogenates and activate these with ether before incubation.

Choline acetylase in the placenta of man and other species.

It has long been known that acetylcholine (ACh) is present in the human placenta (Chang & Gaddum, 1933) and that this tissue contains choline acetylase (Comline, 1946). It has also been claimed that ACh is present in the placentas of other mammals including the cow (Heirman, 1941; Endroczi, Lissak & Nagy, 1950), the horse, the pig, the rat, the dog (Endroczi et al. 1950), the guinea-pig (Berkovich, 1950) and the rabbit (Reynolds & Foster, 1939). But the amounts which can be demonstrated with well controlled assay methods in some of these species are extremely small and of doubtful significance (see Chang & Gaddum, 1933). If it could be shown, however, that choline acetylase is present in the placenta of any of these animals it would be additional evidence in favour of the conclusion that ACh is also present. The present paper gives an account of some experiments in which the choline acetylase content ofthe placenta in twelve animal species, monkey, mongoose lemur (L. mongoz), horse, cow, sheep, goat, pig, hamster, cat, rabbit, guinea-pig and rat, has been examined. It also extends observations reported in an earlier note (Bull, Hebb & Ratkovi6, 1961) on the changes in choline acetylase which occur during development of the human placenta. METHODS

Free choline levels in the rat brain

Choline levels in nervous tissue have been investigated by a number of workers in recent years. The values have varied widely from 39.3 nmol/g (Ewetz, Sparf and Söbo, 1970) to 700 nmol/g (Smith and Saelens, 1967). Many of the values published may have been too high for one of the following reasons: (1) post mortem formation of choline, (2) hydrolysis of phospholipids (PL) by extractants and (3) inadequate assay systems. In the past we too have obtained values which we can now with confidence say were too high due to the post mortem formation of choline. In a method which employed bioassay as an end‐step after extraction of choline by acid‐ethanol the values we obtained were 138 ±27 nmol/g. Despite criticism of this method by Ewetzet al. (1970) and Schuberth, Sparf and Sundwall (1970a) we were reasonably sure that the assay system was both sensitive and specific, and that extraction with acid‐ethanol did not lead to liberation of choline from PL, especially since values for plasma choline by this method were in a number of trial extractions as low as 8 nmol/ml. In view of these results we decided to re‐investigate free choline levels in the brain using a method similar to that of Ewetzet al. (1970) in that the living animal (in this case anaesthetized) was frozen in liquid nitrogen before removing the brain, and comparing the results of three different methods of analysis applied to brain extracts prepared in this way.

Free choline in the brain of the rat.

IN RECENT years, investigators measuring the free choline concentration in brain tissue have emphasized how much lower these are when the post mortem release of choline from phospholipid, whether enzymic or chemical, is avoided (EADE et al., 1973). Values as low as 23nmol/g reported by EADE et al. (1973) have also been reported by SHEA & APRISON (1973) and DROSS & KEWITZ (1972). In the present work choline levels of the cerebrum and cerebellum have been compared using a rapid freezing extraction process.

Synthesis of acetyl‐CoA and acetyl‐dephospho‐CoA by acetyl‐CoA synthetase in the rat

THE SUBSTRATE specificity of two erzymes utilising acetylCoA for acetyltransfer reactions has been reported (BANNS et a/., 1977); choline acetyltransferase was found to be capable of using acetyl-dephospho-CoA as well as acetyl-CoA. This enzyme is cytoplasmic (see HEBB, 1972) and therefore probably uses acetyl-CoA which is synthesised either in the cytoplasm itself, or in the mitochondrion and subsequently transported into the cytoplasm. The present work examines the substrate specificity of acetyl-CoA synthetase (acetate: CoA ligase [AMP forming], EC 6.2.1.1.) which TU&K (1967) has shown t o be mainly mitochondrial. A sensitive and specific radiochemical assay has been developed for this enzyme and measures the formation of products directly without being coupled to a second enzyme or reaction.

Choline acetyltransferase in the equine spleen.

Abstract 1. 1. Choline acetyltransferase (ChAc) has been investigated in the spleens of the horse ( Equus cahallus ) and donkey ( Equus asinus ) and found to be present in amounts ranging from 0.34–6.86 μmol of ACh synthesized g −1 hr −1 . Spleens of other ungulates investigated contained much less than this. 2. 2. ACh content of the spleens studied corresponded with these findings. 3. 3. ChAc was not detected in either the splenic nerve nor in subcellular fractions which could be identified as parts of nerve endings. 4. 4. When splenic sections were stained for acetylcholinesterase very few nerve fibres could be found. On the assumption that cholinergic nerve fibres should be so stained there appear to be far too few such fibres to account for the ChAc found. 5. 5. No evidence was found for a powerful inhibitor to ACh synthesis in horse spleen as previously reported. 6. 6. The results are discussed in relation to possible roles for ACh in the post-ganglionic sympathetic release of noradrenaline.