In continuation of previous reports, it was found that the neurotrophic factor (NF) of the central nervous system of the cat for the maintenance of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7; AcChoEase) in the denervated cat superior cervical ganglion is a heat-stable compound of low molecular weight (<1,000) and that it is probably a peptide. Acetylcholine and nerve growth factor were eliminated as the NF; cyclic AMP produced an effect similar to that of the NF. The NF is probably not present in significant amounts in liver or skeletal muscle; it appears to be present in small intestine. It does not modify the AcChoEase content of the nondenervated cat superior cervical ganglion. Possible mechanisms of action of the NF are discussed.We have shown that arterial infusion for 24 hr of an extract of the brain, spinal cord, and sciatic nerves of cats prevents almost completely the fall in the acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7; AcChoEase) and butyrylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8; BtChoEase) contents of the cat superior cervical ganglion (SCG) that otherwise results 48 hr after preganglionic denervation (1). It was shown subsequently that ligation of the external carotid (EC) and lingual (L) arteries is necessary to obtain the full effect of the extract and to counteract partially a similar but lesser effect produced by continual anesthesia with sodium pentobarbital; although the effect was demonstrable when the infusion time was reduced to 6 hr, it was much less marked and consistent than with 24-hr infusion (2). The compound(s) responsible for the preservation of ganglionic AcChoEase and BtChoEase was designated a neurotrophic factor (NF).The present report describes studies addressed to characterization of the NF by 24-hr infusions of authentic agents and of dialyzed and enzyme-treated preparations of the central nervous system (CNS)/sciatic nerve extract. METHODSPreparation of the infusion extract (extract of the brain, spinal cord, and sciatic nerves of cats), anesthetic and surgical procedures, and methods for homogenization of ganglia, for determination of ganglionic AcChoEase, BtChoEase, and protein contents, and for calculation of statistical significance of mean differences were identical with those reported previously (1). Under sodium pentobarbital anesthesia (35 mg/kg of body weight i.p.), 1 cm was resected from both cervical sympathetic trunks; the wound was sutured, and Combiotic (penicillin and dihydrostreptomycin, Pfizer; 0.5 ml) was given i.m. The following day cats were anesthetized as before and treated with atropine (1.0 mg/kg i.p.); artificial respiration was administered through a tracheal catheter attached to a Palmer pump, and a slow i.v. infusion of 5% glucose/0.45% NaCl was started. Heparin at 50 units/kg was given i.v. just prior to bilateral ligation of the EC and L arteries and was repeated every 6 hr. Infusion of the solution under test was begun -24 hr after denervation and continued until the time of sac...

(11) showed recently that ,B-endorphin inhibits the formation of A12 AcChoEase from smaller aggregates in cultured immature rat muscle. On the other hand, the dipeptide glycyl-L-glutamine (Gly-Gln), which is formed by endopeptidase cleavage of the terminal of P-endorphin (12), was found by Haynes and Smith (13) to enhance markedly the formation of A12 and G4 AcChoEase in cultured embryonic rat and chicken skeletal muscle. Since Gly-Gln meets the criteria for the endogenous NF established for the cat SCG in vivo (8), it was examined by this procedure. Present results indicated that Gly-Gln itself, by the cat in vivo assay, is inactive as a NF, but that a metabolite is probably highly active. The three most likely metabolites were therefore tested: glycine, L-glutamine, and glycyl-L-glutamine acid (Gly-Glu). The first two were found to be inactive, the last (Gly-Glu) highly active.We have also included a comparison of the currently employed method of extraction of SCG by homogenization in distilled water for assay of AcChoEase, BtChoEase, and protein contents with a procedure widely used at present in which the enzymes are solubilized with molar NaCl/1% Triton X-100 (e.g., ref. 14). METHODSAnesthetic and surgical procedures and the methods for homogenization of ganglia and for determination of their AcChoEase, BtChoEase, and protein contents and for calculation of statistical significance of mean differences were identical with those reported (3). Under sodium pentobarbital anesthesia (35 mg/kg, intraperitoneally) 1 cm was resected from both cervical sympathetic trunks; the wound was sutured and Combiotic (penicillin/dihydrostreptomycin, Pfizer, 0.5 ml, intramuscularly) was given. The following day cats were again anesthetized as before and atropinized (1.0

Intracarotid infusion of glycyl-L-glutamine (Gly-Gln) was shown previously to oppose the fall in the acetyicholinesterase and butyrylcholinesterase contents of the cat superior cervical ganglion (SCG) that otherwise follows preganglionic denervation. However, its effect was demonstrable only on the vasculary remote left SCG but not on the directly infused right SCG. Accordingly, it was concluded that a metabolite of Gly-Gln, formed in the blood, is an active neurotrophic factor. Glycyl-L-glutamic acid and L-glutamic acid were subsequently found to have a similar but less marked effect on both SCG. In the present study an alternative explanation has been tested: that Gly-Gln must combine slowly with some component of plasma to enable it to penetrate the ganglion cells and exert its neurotrophic effect. Findings are consistent with the latter proposal.The fall in the acetylcholinesterase (AcChoEase, EC 3.1.1.7) and butyrylcholinesterase (BtChoEase, EC 3.1.1.8) contents of the cat superior cervical ganglion (SCG) that follows preganglionic denervation (1, 2) is opposed by the intracarotid infusion of an aqueous extract of cat brain, spinal cord, and sciatic nerves (3) or its dialysate (Mr cutoff, 1000) (4). Following a report that glycyl-L-glutamine (Gly-Gln) showed a similar effect on cultured rat and chicken skeletal muscle (5), this compound was tested in the cat preparation. It was found to be ineffective at the directly infused right SCG but highly active in maintaining AcChoEase and BtChoEase at the circulatory remote left SCG (6). It was therefore concluded that a metabolite of Gly-Gln, formed in the blood, is a direct neurotrophic factor for the production of this effect. Of the possible candidates tested, glycine and Lglutamine were found to be inactive, but glycyl-L-glutamic acid (Gly-Glu) was moderately active at both the right and left SCG (6). It was shown subsequently that L-glutamic acid has a similar effect, whereas pyroglutamic acid, aspartic acid, and -y-aminobutyric acid were inactive (7). However, neither Gly-Glu nor L-glutamic acid, over a wide range of concentrations, was as effective in preventing the fall in AcChoEase at the right or left SCG as was Gly-Gln at the left SCG following infusion into the right common carotid artery.Here we have tested an alternative explanation for the restriction of the neurotrophic action of Gly-Gln to the left SCG under the above conditions: that Gly-Gln combines relatively slowly with a component of plasma to permit its penetration to the cytoplasm of the neurons of the SCG. In contrast with Gly-Glu and L-glutamic acid, which are relatively lipid soluble, Gly-Gln is a highly polar compound and hence its permeation of the cell membrane should be limited. Lowry et al. (10).Heparinized blood was obtained from experimental cats by intracardiac puncture immediately following sacrifice; it was centrifuged on a Dynac centrifuge at 3500 rpm for 30 min at 5°C, and the plasma was removed and frozen until use. In the initial experimental series, freshly prepar...

In continuation of previous studies, the intraarterial fusion of L-glUtamiC acid for 24 hr was found to oppose the decrease in acetylcholinesterase and butyrylcholinesterase in the superior cervical ganglion of the cat that otherwise occurs 48 hr after preganglionic denervation. The combination of glutamic acid and raminobutyric acid, in concentrations that were inactive individually, likewise produced the same neurotrophic effect. Inactive in this respect were glycine plus L-glutamine, pyroglutamic acid, y-aminobutyric acid, and L-aspartic acid. The possible mechanisms and implications of these rmdings are discussed.Previous studies in this series have demonstrated that the 24-hr intracarotid infusion of an aqueous extract of cat brain, spinal cord, and sciatic nerves in cats opposes (1, 2) the fall in the acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7) and butyrylcholinesterase (BtChoEase; acylcholine acylhydrolase, EC 3.1.1.8) contents of the superior cervical ganglia (SCG) that otherwise occurs 48 hr after section of the cervical sympathetic trunks (1, 3, 4). The neurotrophic factor responsible for this effect was found to be a heat-stable compound of low molecular weight (Mr, <1000) and probably a peptide (5). It was postulated (6) that the neurotrophic factor might act by regulating the conversion of the G1 to the G4 and A12 molecular aggregates of the enzymes (7). Following a report (8) that glycyl-L-glutamine (Gly-Gln) exhibited such an effect in cultured embryonic rat and chicken skeletal muscle, and because this compound met the criteria indicated above, it was tested in the cat in vivo preparation. Results indicated that a metabolite of Gly-Gln is an active neurotrophic factor. Of three possible metabolites tested, glycine and L-glutamine were found to be inactive but glycyl-L-glutamic acid (GlyGlu) was significantly active (9).In the present study, we report the results of testing similarly some possible metabolites of Gly-Glu and related compounds. L-Glutamic acid was found to be an active neurotrophic factor when infused in the concentration range of 10 to 300 ,uM. The combination of 1 ,uM glutamic acid and 100 AM y-aminobutyric acid (GABA) in concentrations that were inactive individually was also effective. Combinations of glycine plus L-glutamine were inactive, as were pyroglutamic acid, GABA, and L-aspartic acid. METHODSAnesthetic and surgical procedures and the methods for homogenization of ganglia and for determination of their AcChoEase, BtChoEase, and protein contents and for calculation of statistical significance of mean differences were identical with those reported (1). Under sodium pentobarbital anesthesia (35 mg/kg intraperitoneally) 1 cm was resected from both cervical sympathetic trunks; the wound was sutured and Combiotic (penicillin/dihydrostreptomycin, 0.5 ml intramuscularly; Pfizer, New York) was given. The following day, cats were again anesthetized, ifrecovered, and atropinized (1.0 mg/kg, intraperitoneal4y); artificial respiration was admin...

Ciliary ganglia (CG) of cats were stained for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) by the bis-(thioacetoxy) aurate (I), or Au(TA)2, method for examination by electron microscopy. Acetylcholinesterase was localized along the axolemmas of the preganglionic fibers and their terminals and on the plasmalemmas of the perikarya and dendrites of the ganglion cells, as in the cat superior cervical ganglion (SCG). In contrast to the SCG, AChE was also found in significant amounts in the rough endoplasmic reticulum of the CG cells and dendrites, and in varying but high concentrations in channels of extracellular space in the complex capsular region surrounding the perikarya and dendrites. Butyrylcholinesterase was confined chiefly to the dendritic and perikaryonal plasma membranes of the ganglion cells, as in the SCG. Lysosomes and mitochondria were stained chiefly for non-cholinesterase enzymes, as indicated by the physostigmine-treated controls. The significance of these distributions is discussed.