Effect of Exogenous Gangliosides on Amino Acid Uptake and Na+,K+-ATPase Activity in Superior Cervical and Nodose Ganglia of Rats

The effects of some gangliosides on active uptake of nonmetabolizable alpha-aminoisobutyric acid (AIB) and Na+, K+-ATPase and Ca2+, Mg2+-ATPase activities in superior cervical ganglia (SCG) and nodose ganglia (NG) excised from adult rats were examined during aerobic incubation at 37 degrees C for 2 h. In NG, amino acid uptake was greatly accelerated with the addition of galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylgluc osyl ceramide (GM1) (85%) and also with N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide (GM2) or [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetyl- neuraminyl]-galactosylglucosyl ceramide (GD1a) (43% each) compared with a nonaddition control at a 5 nM concentration. Under identical conditions, Na+, K+-ATPase activity was strongly stimulated with GM1 (180%) and GD1a (93%), whereas Ca2+, Mg2+-ATPase activity showed no change. In SCG, on the other hand, AIB uptake was apparently inhibited (-27%) by addition of GM1, with a slight decrease in Na+, K+-ATPase but no change in Ca2+, Mg2+-ATPase activity in the tissue. Both asialo-GM1, in which N-acetylneuraminic acid is deficient, and Forssman glycolipid, which is not present in nervous tissue, failed to produce any significant increase in both SCG and NG not only in amino acid uptake, but also in Na+, K+-ATPase activity. A kinetic study of active AIB uptake showed that GM1 ganglioside produced an increase in Km with no change in Vmax in SCG, whereas it caused a decrease in Km with a slight increase in Vmax in NG. Treatment of NG and SCG with neuraminidase from Vibrio cholerae, an enzyme that split off sialic acid from polysialoganglioside, leaving GM1 intact, caused little inhibition of the amino acid uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Appearance of retrogradely labeled neurons in the rat superior cervical ganglion after injection of wheat-germ agglutinin-horseradish peroxidase conjugate into the contralateral ganglion

Summary Injection of wheat-germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) into the superior cervical ganglion (SCG) of the rat results in accumulation of WGA-HRP in sympathetic postganglionic neurons in the contralateral SCG. The sympathetic pathways involved and the mechanism underlying the labeling were investigated. The labeling in neurons in the contralateral SCG was apparent 6 h after injection and increased in intensity with longer survival times. The number of labeled neurons reached 1300 at 72 h after the injection. Transection of the external (ECN) or internal carotid nerves (ICN) resulted in considerable reduction in the number of labeled neurons. Combined transection of both ECN and ICN virtually eliminated labeling in the contralateral SCG. This provides strong evidence that these two nerves are the major pathways for WGA-HRP transport out of the SCG. No labeling was observed in the contralateral SCG following injection of horseradish peroxidase (HRP). Therefore, it seems unlikely that a direct nerve connection exists between the bilateral ganglia. Instead, the labeling of contralateral SCG neurons appears to depend on the transneuronal transport capacity of WGA-HRP, which conveys the marker in an anterograde direction along the postganglionic fibers to terminals in sympathetic target organs, and then delivers it transneuronally to contralateral SCG neurons. We suggest that the sympathetic nerve fibers originating in the bilateral SCGs run intermingled and are in close contact in their peripheral target organs.     

Calcium-Dependent Evoked Release of N[3H]Acetylaspartylglutamate from the Optic Pathway

N-Acetylaspartylglutamate (NAAG) is a neuropeptide localized to several putative glutamatergic neuronal systems, including the rodent optic pathway. To determine whether the peptide is released by depolarization, the superior colliculus of the rat was perfused with 2 microCi of [3H]NAAG, then with Krebs-bicarbonate buffer for 1 h, using a microdialysis system. Subsequently, 10-min fractions were collected and analyzed by HPLC for [3H]NAAG. Addition of 100 microM veratridine resulted in a several-fold increase in the evoked release of [3H]NAAG that was virtually abolished by coperfusion with Ca2+-free Krebs buffer containing 1 mM EGTA. When [3H]glutamate was used as the precursor, veratridine depolarization resulted in only an 80% increase in the release of [3H]NAAG. Prior enucleation of the right eye reduced the spontaneous release of [3H]NAAG by 50%, and the veratridine-evoked release by greater than 85%, from the left superior colliculus. These results suggest that NAAG is released upon depolarization and may serve as a neurotransmitter/neuromodulator in the optic tract.     

Stimulation of Amino Acid Uptake and Na+, K+-ATPase Activity by Norepinephrine in Superior Cervical Sympathetic Ganglia Excised from Adult Rats

Active uptake of a labelled nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB), into isolated superior cervical sympathetic ganglia (SCG) excised from adult rats was considerably stimulated by the addition of either norepinephrine (NE, 50 microM) or 3,4-dihydroxyphenylethylamine (dopamine, DA, 100 microM) to the medium during aerobic incubation for 2 h at 37 degrees C. The NE-induced increase in AIB uptake was significantly antagonized by the addition of an alpha 1-adrenoceptor antagonist (prazosin, 10 microM) in SCG axotomized 1 week prior to the examination, in which most of the ganglionic neurons had degenerated and reactive proliferation of the satellite glial components was in progress. The addition of neither acetylcholine (ACh, 1 mM) plus eserine (0.1 mM) nor cyclic nucleotides (1 mM) changed the AIB uptake by the SCG. In the axotomized SCG, the NE-evoked increase in AIB uptake was much more pronounced than that of intact or denervated SCG. A kinetic study of the active AIB uptake in the SCG showed that NE produced a decrease of the Km value and an increase in the Vmax, especially in the axotomized SCG. Ganglionic Na+, K+-ATPase activity was greatly stimulated in the presence of NE, but not by ACh. These results strongly suggest that the NE-induced enhancement of active AIB uptake in the isolated SCG is occurring in glial cells rather than in neuronal cells, with a possible alteration of membrane properties for amino acid uptake and with an apparent regulation by the stimulated transport enzyme Na+, K+-ATPase.     

Phorbol 12,13-Dibutyrate Increases Tyrosine Hydroxylase Activity in the Superior Cervical Ganglion of the Rat

Phorbol 12,13-dibutyrate (PDBu) increased the production of 3,4-dihydroxyphenylalanine (DOPA) in the superior cervical ganglion of the rat. This effect occurred without a detectable lag and persisted for at least 90 min of incubation. The action of PDBu was half-maximal at a concentration of approximately 0.1 microM; at high concentrations, PDBu produced about a twofold increase in DOPA accumulation. PDBu increased DOPA production in decentralized ganglia and in ganglia incubated in a Ca2+-free medium. The action of PDBu was additive with the actions of dimethylphenylpiperazinium, muscarine, and 8-Br-cyclic AMP, all of which also increase DOPA accumulation, and was not inhibited by the cholinergic antagonists hexamethonium (3 mM) and atropine (6 microM). Finally, PDBu did not increase the content of cyclic AMP in the ganglion. Thus, the action of PDBu does not appear to be mediated by the release of neurotransmitters from preganglionic nerve terminals, by the stimulation of cholinergic receptors in the ganglion, or by an increase in ganglionic cyclic AMP. PDBu also increased the incorporation of 32Pi into tyrosine hydroxylase. PDBu activates protein kinase C, which in turn may phosphorylate tyrosine hydroxylase and increase the rate of DOPA synthesis in the ganglion.     

A Bioluminescence Method for the Measurement of l-Glutamate: Applications to the Study of Changes in the Release of l-Glutamate from Lateral Geniculate Nucleus and Superior Colliculus After Visual Cortex Ablation in Rats

We have developed a rapid, simple, specific, and very sensitive bioluminescence method for the measurement of L-glutamate (L-Glu). Oxidation of L-Glu by glutamate dehydrogenase has been coupled with bacterial FMN reductase and luciferase. Light production (i.e., peak height or integral) was linear from less than 0.5 to 500 pmol of L-Glu. Potential interfering substances that may be encountered in brain tissue have been identified. The most potent inhibitors were ascorbate and the biogenic amines. Procedures that conferred long-term stability of the reagent mixture (greater than 8 h) were established. Bioluminescence analysis of L-Glu content in brain tissue extracts, fractions from release experiments, and human CSF corroborated respective results obtained by HPLC analysis. In this study, we have applied the method to monitor changes in the KCl-evoked release of endogenous L-Glu from milligram amounts of brain tissue, i.e., from lateral geniculate nucleus and superior colliculus after visual cortex ablation.     

Effects of Neuronal Activity on Inositol Phospholipid Metabolism in the Rat Autonomic Nervous System

The effect of nerve stimulation on inositol phospholipid hydrolysis in autonomic tissue was assessed by direct measurement of [3H]inositol phosphate production in ganglia that had been preincubated with [3H]inositol. Within minutes, stimulation of the preganglionic nerve increased the [3H]inositol phosphate content of the superior cervical sympathetic ganglion indicating increased hydrolysis of inositol phospholipids. This effect was blocked in a low Ca2+, high Mg2+ medium. It was also greatly reduced when nicotinic and muscarinic antagonists were present together in normal medium. However, neither the nicotinic antagonist nor the muscarinic antagonist alone appeared to be as effective as both in combination. In other experiments, stimulation of the vagus nerve caused dramatic increases in [3H]inositol phosphate in the nodose ganglion but did not increase [3H]inositol phosphate in the nerve itself. This effect was insensitive to the cholinergic antagonists. Thus, neuronal activity increased inositol phospholipid hydrolysis in a sympathetic ganglion rich in synapses, as well as in a sensory ganglion that contains few synapses. In the sympathetic ganglion, synaptic stimulation activated inositol phospholipid hydrolysis and this was primarily due to cholinergic transmission; both nicotinic and muscarinic pathways appeared to be involved.     

Stimulation of DOPA Synthesis in the Superior Cervical Ganglion by Veratridine

We have investigated the effect of veratridine on DOPA (3,4-dihydroxyphenylalanine) accumulation by the superior cervical ganglion of the rat. Incubation of the ganglion with veratridine (50 microM) causes a 10-fold increase in the rate of DOPA accumulation. Veratridine-stimulated DOPA accumulation is blocked by tetrodotoxin, but not by cholinergic or adrenergic antagonists or by decentralization of the ganglion. The cyclic nucleotide 8-bromo cyclic GMP does not increase DOPA accumulation, and 8-bromo cyclic AMP causes only a 2-fold increase in DOPA accumulation, which is additive with the effect of veratridine. Thus, the action of veratridine appears to be independent of these cyclic nucleotides. The effect of veratridine on DOPA accumulation is probably due to a stable modification of tyrosine hydroxylase, since an increase in tyrosine hydroxylase activity can be measured in cell-free extracts of veratridine-treated ganglia. Both the increase in DOPA accumulation and the stable activation of tyrosine hydroxylase are dependent upon extracellular Ca2+. The activation of tyrosine hydroxylase by veratridine may be mediated by the depolarization of, and the subsequent entry of Ca2+ into, ganglionic neurons.     

Activation of Tyrosine Hydroxylase in the Superior Cervical Ganglion by Nicotinic and Muscarinic Agonists

Both dimethylphenylpiperazinium (DMPP), a nicotinic agonist, and bethanechol, a muscarinic agonist, increase 3,4-dihydroxyphenylalanine (DOPA) synthesis in the superior cervical ganglion of the rat. DMPP causes approximately a fivefold increase in DOPA accumulation in intact ganglia whereas bethanechol causes about a two-fold increase in DOPA accumulation. These effects are additive with each other and with the increase in DOPA accumulation produced by 8-bromo cyclic AMP. The action of DMPP is dependent on extracellular Ca2+ while the actions of bethanechol and 8-bromo cyclic AMP are not dependent on extracellular Ca2+. Cholinergic agonists and cyclic nucleotides produce a stable activation of tyrosine hydroxylase (TH) in the ganglion. The activation of TH by nicotinic and muscarinic agonists can be detected after 5 min of incubation of the ganglia with these agents. The nicotinic response disappears after 30 min of incubation, whereas the muscarinic response persists for at least 30 min. The Ca2+ dependence of the TH activation produced by these agents is similar to the Ca2+ dependence of their effects on DOPA accumulation in intact ganglia. These data are consistent with the hypothesis that nicotinic agonists, muscarinic agonists, and cyclic AMP analogues increase TH activity by three distinct mechanisms. The activation of TH presumably underlies the increase in DOPA synthesis produced by these agents.     

Epinephrine: A Potential Neurotransmitter in Retina

Abstract: Dopamine (DA), norepinephrine (NE), and epinephrine (EPI) are present in rat retina. DA is the major catecholamine, whereas NE and EPI represent ∼5% of the DA content. DA is contained in a subpopulation of amacrine cells and has been the subject of numerous studies. We investigated the origin and properties of NE and EPI in retina. Following superior cervical ganglionectomy, there was a decrease in NE content, but no decrease in EPI or phenylethanolamine- N -methyltransferase (PNMT) activity. PNMT in retina has many of the substrate-specificity and inhibitor-sensitivity characteristics of other tissues. Enzyme activity is enhanced in newborn rats by treatment with dexamethasone. Exposure to a lighted environment increases retinal EPI in normal and superior cervical ganglionectomized rats. EPI content increased for more than 2 h in a lighted environment. We conclude that most of the NE is contained within the sympathetic neurons that innervate the eye from the superior cervical ganglion, whereas EPI is contained in retinal elements that are responsive to photic stimulation.