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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...

Intracarotid infusion of 3 ,AM glycyl-L-glutamine was found to oppose the fall in the choline acetyltransferase content of the preganglionically denervated cat superior cervical ganglion; this same effect has been demonstrated previously for acetylcholinesterase content. Because choline acetyltransferase, in contrast to acetylcholinesterase, occurs exclusively in the preganglionic axons and their terminals, this finding raises the possibility that glycyl-L-glutamine opposes postsectional axonal degeneration.Studies conducted over the past half-dozen years have shown that both an endogenous small peptide of the cat central nervous system and synthetic glycyl-L-glutamine (Gly-Gln) can prevent the fall in acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7) of the cat superior cervical ganglion (SCG) that follows preganglionic denervation (for summary, see ref. 1). A similar effect of Gly-Gln was demonstrated earlier in cultured myotubes (2). This neurotrophic action was shown to take place before the aggregation of the monomeric (Gl) form of AcChoEase into higher polymers (G4, A12) (3). It was hypothesized (1) that Gly-Gln and the similar endogenous peptide enhance the transcription of the DNA coding for AcChoEase into the corresponding mRNA, in a manner analogous to that proposed for triiodothyronine in the regulation of protein synthesis (4).In extending these observations we have now measured the effect of infusions of Gly-Gln into denervated cat SCG on another enzyme of the cholinergic system, choline acetyltransferase (ChoAcTr; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). Unexpectedly, Gly-Gln was found also to maintain the ChoAcTr activity. Because this enzyme, in contrast to AcChoEase (5), is confined to the preganglionic fibers and their terminals (6-9), the present results raise the interesting possibility that Gly-Gln may oppose the degeneration of sectioned axons.No consistent effect of Gly-Gln infusion was found on the tyrosine hydroxylase [L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] content of infused SCG. METHODSSurgical and infusion procedures were identical with those reported (10). Under ketamine anesthesia (20 mg/kg, intramuscularly), 1 cm of tissue was resected from both cervical sympathetic trunks; the wound was sutured and Combiotic (penicillin/dihydrostreptomycin, 0.5 ml intramuscularly; Pfizer) was given. One day later, cats were anesthetized with sodium pentobarbital (35 mg/kg, intraperitoneally); artificial respiration was administered through a tracheal catheter attached to a Palmer pump, and a slow intravenous infusion of 5% glucose/0.45% NaCl was started. Heparin (50 units/ kg, intravenously) was administered just before bilateral ligation of the external carotid and lingual arteries. Infusion of the solution under test (Gly-Gln, incubated overnight with heat-treated cat plasma and diluted to 3 ,uM with 0.9% NaCl) was begun and continued until the time of sacrifice, -24 hr later (exactly 48 hr postdenerv...

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