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

Abstract

Normal and preganglionically denervated cat superior cervical ganglia were sectioned and cultured for 24 or 48 hr, with or without preliminary inactivation of acetylcholinesterase, and in the presence or absence of 10-5 M glycyl-L-glutamine. They were then homogenized, and the molecular forms of acetylcholinesterase were analyzed by sucrose gradient sedimentation. We observed an increased proportion of the globular monomeric G, form, and to a lesser extent of the dimeric G2 and tetrameric membranous G4 forms, of acetylcholinesterase in the glycyl-L-glutamine-treated compared with the control cultures. There was only a small increase in the total acetylcholinesterase activity and no significant variation in the activity of the metabolic enzyme lactate dehydrogenase. It therefore seems likely that glycyl-L-glutamine, or the endogenous neurotrophic factor, maintains acetylcholinesterase in the preganglionically denervated ganglia in vivo by specifically increasing the biosynthesis of the monomeric G, form, but not that of other proteins; these trophic factors do not seem to promote the polymerization of G, into the more complex G2 and G4 forms.Preganglionic denervation of the cat superior cervical ganglion (SCG) results within a few days in a marked decrease in its contents of acetylcholinesterase (AcChoEase, EC 3.1.1.7) and butyrylcholinesterase (BtChoEase, EC 3.1.1.8) (1, 2). In a recent series of experiments (1, 3-6) 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.In the present study we investigated the stage at which Gly-Gln, or the unidentified endogenous neurotrophic factor, enhances the production of AcChoEase in denervated ganglia. In an effort to study this effect in vitro, we first used whole desheathed rat SCG that had been preganglionically denervated one day previously. Results were suggestive, but equivocal, probably for the reasons given in the Discussion and, consequently, are not presented.In the experiments reported here, sections of normal or preganglionically denervated cat SCG were incubated in culture medium for 24 or 48 hr, with or without inactivation of their initial AcChoEase content, and with or without the addition of Gly-Gln or nervous tissue extract. The SCGs were then homogenized in a high-salt and Triton X-100-containing extraction medium to solubilize AcChoEase. The molecular forms of the enzyme were analyzed by sedimentation in sucrose density gradients, as described previously (9). METHODSWe cultured sections of cat SCG with a number of variations: the interval between preganglionic denervation and excision, ...

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

“…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).…”

Beta-Endorphin-Induced Cardiorespiratory Depression is Inhibited by Glycyl-L-Glutamine, a Dipeptide Derived from Beta-Endorphin Processing. Appendix 1

“…Glycyl-L-glutamine also produces independent effects unrelated to inhibition of POMC peptides (Parish et al, 1983;Koelle et al, 1988;Lotwick et al, 1990;Haynes, 1991;Nyquist-Battie et al, 1993). This was first demonstrated by Parish et al (1983) who showed that glycyl-L-glutamine reduces the firing rates of brainstem neurons when applied iontophoretically.…”

Glycyl-Glutamine, an Endogenous β-Endorphin-Derived Peptide, Inhibits Morphine-Induced Conditioned Place Preference, Tolerance, Dependence, and Withdrawal

“…Glycyl-glutamine is the single most abundant ␤-endorphin-related peptide produced in some brain regions and most peripheral tissues that synthesize POMC, although relatively little is know about its physiological functions (Parish et al, 1983;Smith and Funder, 1988). Early studies showed that Gly-Gln is an inhibitory peptide that reduces the firing frequencies of brainstem neu-rons after iontophoretic application (Parish et al, 1983) and produces trophic (Koelle et al, 1988;Lotwick et al, 1990) and immune (McCain et al, 1987) effects in peripheral tissues. The simple observation that Gly-Gln is synthesized with ␤-endorphin 1-27 , an opioid receptor antagonist, prompted us to investigate whether it might also influence the pharmacological effects of opioids.…”