Direct neurotrophic action of glycyl-L-glutamine in the maintenance of acetylcholinesterase and butyrylcholinesterase in the preganglionically denervated superior cervical ganglion of the cat.

Abstract

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...

Effects of glycyl-L-glutamine in vitro on the molecular forms of acetylcholinesterase in the preganglionically denervated superior cervical ganglion of the cat.

“…The same sequence occurs in vivo; at 1 day after denervation the AcChoEase content of the cat SCG is -85% of the normal value, and by 3 days it has fallen to 15% (1,2). Gly-Gln has been found to oppose the AcChoEase decrease in culture, but somewhat less effectively than after perfusion in vivo (4,6).…”

“…The inhibitory effect of high concentrations of Gly-Gln on the AcChoEase contents of denervated SCG was also found in vivo (6). It is noteworthy that in those experiments maximal enhancement was obtained with intraarterial infusion of 3 x 10-6 M Gly-Gln, and reduction of AcChoEase activity was obtained with 10-4 M-i.e., essentially the concentrations found here to produce similar effects in vitro.…”

“…In a recent series of experiments (1,(3)(4)(5)(6) it was shown that the 24-hr intracarotid infusion of a dialyzed aqueous extract of cat brain, spinal cord, and sciatic nerves, or of glycyl-L-glutamine (Gly-Gln), or of some closely related compounds, opposes the decrease of both enzymes. Gly-Gln was tested after the demonstration by Haynes and Smith (7) that this compound increases the formation of the G4 (globular tetramer) and A12 (collagen-tailed, asymmetric dodecamer) forms of AcChoEase (8) in cultured preparations of embryonic rat and chicken skeletal muscle.…”

Effect of glycyl-L-glutamine on the rate of regeneration of acetylcholinesterase in the rat gastrocnemius muscle after diisopropyl phosphorofluoridate administration.

“…At three times this concentration, the effect was minimal, and at still higher concentrations it was distinctly inhibitory. The effect was markedly reduced with the infusion of 1 ,uM Gly-Gln or with lower concentrations (8). On the assumption that the mass of the hind quarters of the rats used (400-600 g) was approximately the same as that of the head of the cat, Abbreviations: AcChoEase, acetylcholinesterase; DFP, diisopropyl phosphorofluoridate; Gly-Gln, glycyl-L-glutamine; SCG, superior cervical ganglion (ganglia).…”

Glycyl-L-glutamine opposes the fall in choline acetyltransferase in the denervated superior cervical ganglion of the cat.

“…Surgical 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.…”