Evidence for strychnine-sensitive glycine receptors in human amygdala.

Recent studies suggested the existence of strychnine-sensitive glycine-receptors in mammalian amygdala. In the present study, we investigated the amino acid concentrations as well as immunocytochemical and pharmacological properties of glycine-receptors in fresh human amygdala tissue obtained from epilepsy surgery. High pressure liquid chromatography revealed a considerable amount of glycine and its precursors and glycine-receptors agonists L-serine and taurine in this tissue. Immunohistochemistry using the monoclonal antibody mAb4a, recognizing an epitope common to all alpha-subunit variants of glycine receptors, displayed a specific labeling at the soma and on proximal dendrites of mostly tripolar, large-sized neurons of irregular distribution and arrangement. To elucidate the pharmacological properties of the glycine-receptors found slices of human amygdala were preloaded with [(3)H]-choline and superfused. Glycine induced an overflow of [(3)H]-acetylcholine, which was inhibited by strychnine in a concentration-dependent manner. Furthermore, the glycine-induced release of [(3)H]-acetylcholine was significantly inhibited by furosemide, indicating glycine-induced actions to be attributed to chloride channels. These actions of glycine were not influenced by MK-801, D-CP-Pene or bicuculline. Thus, the effects of glycine did not seem to be mediated through NMDA or GABA receptors. These observations indicate that strychnine-sensitive, chloride-conducting glycine receptors, which elicit the release of [(3)H]-acetylcholine, are present at the soma and on proximal dendrites of neurons in human amygdala. It is hypothesized that glycine may display a regulatory role in amygdaloid functions, probably via cholinergic interneurons.

Gabapentin-lactam (8-aza-spiro[5,4]decan-9-on; GBP-L) inhibits oxygen glucose deprivation-induced [3H]glutmate release and is a neuroprotective agent in amodel of acute retinal ischemia.

The modulation of the enhanced release of [3H]glutamate following ischemia-like conditions was studied in rat hippocampal slices using a superfusion system. Ischemia was simulated by a glucose-free medium equilibrated with 95% N2 and 5% CO2. In this model the potential neuroprotective effects of several substances on [3H]glutamate release induced by ischemia-like conditions were investigated. Gabapentin-lactam (8-aza-spiro-5,4-decan-9-on; GBP-L) was synthesised and patented in our laboratory. GBP-L (100 microM) reduced the oxygen glucose deprivation-induced [3H]glutamate release by 42.5%, CI95=[33.4%, 51.5%]. The KATP channel antagonist glibenclamide (1 microM) blocked this effect completely. The high antagonist potency was reflected by an apparent pA2-value of glibenclamide of 8.3, CI95=[6.8, 9.4]. Minoxidil sulfate (10 microM), a KATP channel opener, mimicked the effect of GBP-L (inhibition by 22.8%, CI95=[13.2%, 32.5%]). Similarly to its lactam, also gabapentin (100 microM) reduced the oxygen glucose deprivation-induced [3H]glutamate release by 30.6%, CI95=[15.5%, 45.7%], whereas the "antiglutamatergic" drug riluzole was ineffective. GBP-L and gabapentin were also tested in an in vivo model of acute retinal ischemia in rats. The intraocular pressure was elevated for 1 h above the systolic blood pressure. In the control group, 17.5%, CI95=[13%, 22%], of retinal ganglion cells had survived after 2 weeks. GBP-L doubled the number of surviving ganglion cells up to 35%, CI95=[27%, 43%], while gabapentin had no effect. This difference between gabapentin and its lactam may be due to different pharmacokinetic properties: In contrast to the gamma-amino acid gabapentin, GBP-L is uncharged and therefore might diffuse more easily through biological membranes, e.g. the plasma membrane, to get access to an intracellular locus of action. Thus, the neuroprotective properties in vivo and the diminished oxygen glucose deprivation-induced [3H]glutamate efflux in vitro of the presumed KATP channel agonist GBP-L suggest that this substance might be therapeutically applied in pathological situations induced by a rise in extracellular glutamate and/or neuronal cell death.

Drug accumulation in melanin: an affinity chromatographic study.

The affinity of several drugs to melanin has been indirectly assessed using an affinity chromatographic approach based on immobilized melanin. Plots of the retention of the drugs on the affinity column versus the number of molecules applied were fitted best by nonlinear, exponential curves characteristic for each drug. These curves reflect the complexity of the binding behaviour, consisting of a variety of hydrogen bonding, hydrophobic or ionic interactions as well as cooperative or anti-cooperative interactions between the drug molecules and melanin. The nonlinear fitting procedure was based on a descriptive function and allowed to discriminate the binding behaviour according to parameter estimates which specified the investigated drugs.

Release and accumulation of neurotransmitters in the rat brain: acute effects of ethanol in vitro and effects of long-term voluntary ethanol intake.

Release from and accumulation in tissue slices of some neurotransmitters under acute ethanol in naive rats and in long-term voluntarily ethanol drinking rats were investigated. Slices of the rat caudatoputamen were prelabeled with [3H]choline and release of [3H]acetylcholine was stimulated through either N-methyl-D-aspartate (NMDA) receptors or strychnine-sensitive glycine receptors. Ethanol in vitro at 2 per thousand, 4 per thousand, and 6 per thousand (34 mM, 68 mM, and 102 mM, respectively) concentration-dependently depressed the maximum effect of the concentration-response curve of NMDA in naive rats. In contrast, voluntary ethanol consumption over months led to a significantly enhanced NMDA receptor response characterized by an increase in the maximum effect of the concentration-response curve. The glycine receptor-mediated release of [3H]acetylcholine, which is inhibited by acute ethanol in a competitive-like fashion, was not changed in animals that ingested ethanol over months. Electrically evoked release of [3H]noradrenaline ([3H]NA) and its presynaptic modulation by morphine through mu-opioid receptors in neocortical slices of the rat, preloaded with [3H]NA, was nearly identical in both ethanol-naive rats and in ethanol drinking rats. The accumulation of [3H]gamma-aminobutyric acid in rat cerebellum tissue was neither affected by acute ethanol in vitro nor after chronic ethanol consumption. In summary, long-term voluntary ethanol intake caused a significant increase in NMDA receptor function in the rat caudatoputamen, but did not result in changes in glycine-evoked [3H]acetylcholine release of electrically evoked [3H]NA release modulated by morphine or cerebellar [3H]gamma-aminobutyric acid accumulation.

Memantine is neuroprotective in a rat model of pressure-induced retinal ischemia.

PURPOSE: To quantify vitreous amino acid concentrations in pressure-induced retinal ischemia and to evaluate the neuroprotective effect of memantine, a N-methyl-D-aspartate (NMDA) antagonist, administered before and at two time intervals after ischemia. METHODS: Retinal ischemia was induced in 10 rats by elevating the intraocular pressure to 120 mm Hg. The concentrations of the amino acids of vitreous samples were measured by high-pressure liquid chromatography. In another series of 56 rats, ischemia was induced in a similar fashion. Fifteen rats received 20 mg/kg x day memantine by a subcutaneous osmotic pump starting 2 days before ischemia, 13 rats received 10 mg/kg memantine intraperitoneally (ip) 0.5 and 4.5 hours after reperfusion, 13 rats received 10 mg/kg memantine ip 3.5 and 7.5 hours after reperfusion, and 15 rats received the vehicle alone. Ischemic damage was histologically quantified 14 days after ischemia. RESULTS: Compared with the nonischemic fellow eyes, there was an elevation (P < 0.05) in the mean vitreous concentration of glutamate (223%+/-41%) and glycine (428%+/-92%). The percentage of surviving neurons in the ganglion cell layer was 33%+/-3% in the controls, 61%+/-5% (P < 0.001) when memantine was infused subcutaneously before ischemia, 52%+/-5% (P < 0.05) when memantine was injected ip 0.5 and 4.5 hours after ischemia, and 48%+/-5% (P > 0.05) when injected ip 3.5 and 7.5 hours after ischemia. CONCLUSIONS: Retinal ischemia increased vitreous concentrations of glutamate and glycine. Both amino acids were agonists at the NMDA receptor. The NMDA receptor antagonist memantine reduced ganglion cell loss when given systemically before or within 30 minutes of retinal ischemia.

Aspartate, glutamate, glutamine, glycine and gamma-aminobutyric acid in human bioptic neocortical areas: comparison to autoptic tissue.

Amino acid concentrations were determined by high performance liquid chromatography in distinct areas of human neocortex of autoptic and bioptic origin. The concentrations in autoptic tissue were similar in all cortical areas which may be explained by postmortem proteolysis, abolishing regional differences seen in bioptic tissue. Aspartate, glutamate, glycine and gamma-aminobutyric acid concentrations were lower, but glutamine levels were higher, in biopsied than in autopsied tissue. Glycine and gamma-aminobutyric acid concentrations increased with the age of biopsied patients. The differences seen suggest that only amino acid concentrations determined in bioptic tissue may yield a reliable data base for the interpretation of pathological alterations in neocortical biopsies of patients with brain diseases.

Strychnine-sensitive glycine receptors inducing [3H]-acetylcholine release in rat caudatoputamen: a new site of action of ethanol?

In the present study acute effects of ethanol on [3H]-acetylcholine ([3H]-ACh) release induced by activation of strychnine-sensitive glycine receptors in superfused slices of rat caudatoputamen were investigated. The glycine-evoked [3H]-ACh release (Ig EC50 = -4.10, CI95 = [-4.14, -4.05]) was inhibited by strychnine in a competitive manner (pA2 = 6.86, CI95 = [6.61, 7.08]). Release of [3H]-ACh could also be induced by L-serine. L-serine was less potent than glycine (Ig EC50 = -2.61, CI95 = [-2.69, -2.52]). Both glycine and L-serine showed similar maximum effects (Emax(glycine) = 1.34, CI95 = [1.24, 1.45]; Emax(L-serine) = 1.19, CI95 = [1.09, 1.32]). Ethanol at concentrations of 2%/1000 (= 34 mM) and 4%/1000 (= 68 mM) inhibited glycine-evoked [3H]-ACh release in a manner like the competitive antagonist strychnine, however with lower potency. The pA2 of ethanol was 1.19, CI95 = [0.85, 1.41], at 2%/1000 [v/v] and 1.51, CI95 = [1.19, 1.78] at 4%/1000 ethanol. Similar to its action on glycine-evoked [3H]-ACh release, ethanol at 4%/1000 [v/v] also inhibited L-serine-evoked transmitter release in a competitive-like fashion (pA2 = 0.83, CI95 = [-0.15, 1.18]). We conclude, that strychnine-sensitive glycine receptors, mediating [3H]-ACh release in the rat caudatoputamen, might represent a new site of action of ethanol.

Secondary structure and stability of the bacterial carbohydrate-specific recognition proteins K88ab, AFA-1, NFA-1, and CFA-1.

The conformation and thermodynamic stability of the four polymeric carbohydrate-specific bacterial recognition proteins K88ab, AFA-1, NFA-1, and CFA-1 and their monomeric subunits that can be obtained by variation of pH were studied by infrared spectroscopy and differential scanning microcalorimetry. For NFA-1, a pH-dependent dissociation of the polymeric form cannot be achieved due to the stronger interactions of the neighboring subunits. Generally, no alterations in secondary structure are observed between the monomeric and the polymeric proteins. All adhesins reveal a high degree of beta-sheet structure (40-55%), while the alpha-helix component is of minor importance (10-20%). The adhesins investigated in this study revealed unusually high denaturation temperatures (69-104 degrees C) and stabilizing Gibbs energies, delta G (40-125 kJ/mol), compared to common globular proteins. Statistical deconvolution of the DSC curves yields a two-state transition of K88ab, NFA-1, and the monomeric CFA-1 and the existence of intermediate states for AFA-1 and polymeric CFA-1 during the denaturation process. The irreversible denaturation of K88ab, AFA-1, and CFA-1 is explained by aggregation of the polypeptide chains forming a three-dimensional network of intermolecular beta-sheet-type structures. In contrast, denaturation of NFA-1 is completely reversible. At a physiologically relevant temperature of approximately 40 degrees C, we observe predenaturational events in the DSC curves of polymeric K88ab and NFA-1 with no concomittant changes in the secondary structure of these proteins.

Tamm-Horsfall glycoprotein: role in inhibition and promotion of renal calcium oxalate stone formation studied with Fourier-transform infrared spectroscopy.

Tamm-Horsfall glycoprotein (THP) from healthy probands inhibits the precipitation of calcium oxalate, whereas THP from individuals who repeatedly develop calcium oxalate stones has no effect or even promotes precipitation. Using Fourier-transform infrared spectroscopy, we found a structural differentiation between these functionally different THPs: a decisive difference in sialic acid content. Quantitative analysis for sialic acid showed the same results. THP from healthy probands had a high sialic acid content (51 +/- 9 g/kg), whereas THP from recurrent stone formers had a decreased sialic acid content (21 +/- 4 g/kg). This explains the dual role of THP in the precipitation of calcium oxalate and the formation of renal stones and shows the importance of glycosylation in the function of this glycoprotein.