Identification of inosine as an endogenous modulator for the benzodiazepine binding site of the GABAA receptors.

Previously we have reported the presence of endogenous ligands that are involved in the regulation of the binding of muscimol to the GABA binding site of the GABAA receptors. Here, we report the presence of multiple forms of endogenous ligands in the brain which modulate the binding of flunitrazepam (FNZP) to the benzodiazepine (BZ) binding site of the GABAA receptor. Furthermore, one of the endogenous ligands for the BZ receptors, referred to as EBZ, has been identified as inosine based on the following observations: (1) standard inosine and the EBZ have identical NMR and UV spectra; (2) the elution profile of inosine and the EBZ from a HPLC column are indistinguishable, and (3) inosine and the EBZ show identical activity in inhibiting [3H]FNZP binding.

Studies of NMDA- and non-NMDA-mediated neurotoxicity in cultured neurons.

The neurotoxic effects of various glutamate agonists were studied using whole fetal rat brain cultures. The results showed that L-glutamate (L-glu) and N-methyl-D-aspartate (NMDA) were the most potent agonists for inducing neurotoxicity, producing significant toxicity at 0.10 and 0.01 mM concentrations, respectively. Kainic acid (KA) and quisqualic acid (QA) also produced neurotoxicity, but only at a relatively high concentration (1.0 mM). No other glutamate agonist tested produced neurotoxicity in the cultures following brief incubations. The effects of each agonist were found to be Ca2+ dependent, and the selective NMDA Ca2+ channel agonist, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,1 0-imine hydrogen maleate (MK-801), blocked the toxicity produced by all the glutamate agonists. Thus, the results of this study found little or no evidence for a direct non-NMDA receptor mediated neurotoxicity. These results suggest that the neurotoxicity produced by the non-NMDA agonists may be due to one of the following mechanisms: (i) non-specific binding of non-NMDA agonists to NMDA receptor; (ii) release of L-glu via non-NMDA agonists induced depolarization of cell membrane and subsequent activation of NMDA receptor by released L-glu; (iii) inhibition of L-glu uptake by non-NMDA agonists resulting in activation of L-glu receptors including NMDA receptors.

An integral membrane protein form of brain L-glutamate decarboxylase: purification, characterization and its relationship to insulin-dependent diabetes mellitus.

A new and novel form of L-glutamate decarboxylase (GAD; EC 4.1.1.15) was purified from whole porcine brain to apparent homogeneity by a combination of column chromatographies on DE-52, ultragel AcA 34, hydroxylapatite and Sephadex G-200, and native gel electrophoresis. The purified GAD was established as an integral membrane protein based on hydrophobic interaction chromatography and membrane extraction studies. This membrane GAD (MGAD) has a native molecular weight of 120 +/- 5 kDa and is a homodimer of 60 +/- 2 kDa. Immunoprecipitation and immunoblotting tests using the sera from insulin-dependent diabetes mellitus (IDDM) patients revealed the presence of antibodies against this newly identified MGAD in IDDM. The role of MGAD in the pathogenesis of IDDM and related autoimmune disorders is also discussed.

A membrane form of brain L-glutamate decarboxylase: identification, isolation, and its relation to insulin-dependent mellitus.

A membrane form of L-glutamate decarboxylase (GAD) was identified and purified to apparent homogeneity from hog brain. The purified GAD was established as an integral membrane protein by phase-partitioning assay, charge-shift electrophoresis, and chromatography on a hydrophobic interaction column. This membrane GAD has a native molecular mass of 96 +/- 5 kDa and is a homodimer of 48 +/- 3-kDa subunits. Immunoprecipitation and immunoblotting tests revealed the presence of antibodies against this membrane GAD in sera from patients with insulin-dependent diabetes mellitus. Since this form of GAD appears to be an integral membrane protein and is presumed to have extracellular domains exposed, it seems reasonable to suggest that membrane GAD is more likely than soluble GAD to be involved in the pathogenesis of insulin-dependent diabetes and related autoimmune disorders such as stiff-man syndrome.

Isolation of endogenous modulators for the GABAA and taurine receptors.

Several endogenous brain substances which inhibit [3H]muscimol binding were isolated, and one of them has been purified to apparent homogeneity. The purification involved the extraction of brain tissue with water, followed by several steps of gel filtration column chromatography and high performance liquid chromatography (HPLC). The muscimol binding inhibitor (MBI) thus obtained appeared to be homogeneous as judged from the elution profile of an HPLC column, in which a symmetrical peak was obtained when the eluate was monitored at either 220 or 280 nm. Furthermore, the MBI activity coincided with the absorption peak. The purified MBI is not gamma-amino butyric acid (GABA), beta-alanine or taurine since these amino acids are clearly separated from the MBI in the purification procedures. The MBI has no effect on benzodiazepine (BZ) binding or glutamate binding to their respective receptors. However, the MBI is a more potent inhibitor for [3H]taurine binding than that of [3H]muscimol binding. The MBI appears to be a small molecule (< 2000 Da) that is heat and acid/base stable. The chemical nature of the MBI is currently under investigation.

Isolation and characterization of endogenous modulators for GABA system.

Pig brain extracts from both soluble and membrane fractions were found to contain potent inhibitors for GABA synthesizing enzyme, GAD, referred to as endogenous GAD inhibitors (EGIs) and for the binding of GABA agonist, muscimol, referred to as muscimol binding inhibitors (MBIs). EGIs and MBIs were first purified through gel-filtration Bio-Gel P-2 columns, in which multiple activity peaks were observed. One of them appears to be co-eluted with either L-glutamate or GABA. However, others are clearly separated from L-glutamate or GABA. EGIs were found to be low MW (less than 1,800 dalton), heat and acid-base stable, negatively charged, non hydrophobic substances. MBIs were found to be low MW (less than 1,800 dalton) neutral or positively charged substances. MBIs had no effect on [3H]flunitrazepam (FNZP) binding, indicating that they are not endogenous benzodiazepine receptor ligands and they may act specifically on GABA binding site.

Concerted enhancement of calcium influx, neurotransmitter release and protein phosphorylation by a phorbol ester in cultured brain neurons.

We have recently shown that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate enhances the depolarization induced, calcium dependent release of [3H]dopamine from cultured brain neurons in the rat. In the present study the effects of 12-O-tetradecanoyl-phorbol-13-acetate on the kinetic parameters of depolarization induced calcium influx and on Ca2+ dependent neurotransmitter release and protein phosphorylation were investigated. Depolarization induced neurotransmitter release from the neurons occurs in two phases: an initial, fast release and a subsequent slow release. At low extracellular Ca2+, 12-O-tetradecanoyl-phorbol-13-acetate enhanced the quantity of fast release and in addition, increased the rate constant of the slow release. These effects mimicked the effects of increasing the extracellular Ca2+. Various phorbol derivatives known to activate the Ca2+ activated phospholipid dependent protein kinase (protein kinase C) were also able to enhance the stimulated release of [3H]dopamine from the neurons. 12-O-tetradecanoyl-phorbol-13-acetate induced the incorporation of 32Pi into a protein with an apparent molecular weight of 45,000 daltons regardless of depolarization or of the presence of Ca2+. In addition, 12-O-tetradecanoyl-phorbol-13-acetate induced in unstimulated neurons, Ca2+ dependent increase in the amount of 32Pi incorporated into a 43,000 dalton protein and decrease in the amount incorporated into a 55,000 dalton protein. These changes mimicked the Ca2+ dependent changes in protein phosphorylation which occur upon stimulation of the neurons. Kinetic studies of depolarization induced Ca2+ uptake by the neurons indicated that 12-O-tetradecanoyl-phorbol-13-acetate enhanced the maximal influx of Ca2+ through the voltage sensitive Ca2+ channels by 40%. The results indicate that 12-O-tetradecanoyl-phorbol-13-acetate acts primarily on the regulation of stimulated Ca2+ entry into the cells. Consequently neurotransmitter release at submaximal extracellular [Ca2+] is enhanced.

Calcium permeability changes and neurotransmitter release in cultured brain neurons. II. Temporal analysis of neurotransmitter release.

The coupling between depolarization-induced calcium entry and neurotransmitter release was studied in rat brain neurons in culture. The endogenous dopamine content of the cells was determined by high performance liquid chromatography utilizing electrochemical detection. The amount of dopamine in unstimulated cells was found to be about 16 ng/mg of protein. Depolarization of the neurons by elevated K+ caused a Ca2+-dependent release of dopamine from the cells. Following 1 min of depolarization, the cellular dopamine content and the amount of [3H]dopamine in cells preloaded with the radioactive transmitter were reduced by 35%. The release of [3H]dopamine by the neurons was measured at 1.5-6-s intervals by a novel rapid dipping technique. Depolarization in the presence of Ca2+ (1.8 mM) enhanced the rate of neurotransmitter release by 90-fold (0.072 +/- 0.003 s-1) over the basal release in the presence of Ca2+. The evoked release consisted of a major rapidly terminating phase (t1/2 = 9.6 s) which comprised about 40% of the neurotransmitter content of the cells and a subsequent slower efflux (t1/2 = 575 s) which was observed during following prolonged depolarization. Predepolarization of the cells in the absence of extracellular Ca2+ did not affect the kinetics of the evoked release. The fast evoked release could be re-elicited in the cells after 20 min "rest" in reference low K+ buffer. The effects of varying the extracellular Ca2+ concentrations on the kinetic parameters of the evoked release were measured. The amount of neurotransmitter released during the fast kinetic phase was very sensitive to the external Ca2+ (from 0% in the absence of Ca2+ to 40% of the neurotransmitter content at Ca2+ 0.3 mM). The rate constant of the fast release did not depend on the extracellular Ca2+, whereas the rate constant of the slow release increased from 0.0004 +/- 0.0001 s-1 at 0.4 mM Ca2+ to 0.0012 +/- 0.0002 s-1 at 0.8 mM Ca2+. The fast evoked release was inhibited by verapamil in a concentration-dependent manner. By contrast, verapamil enhanced the basal and the slow release independent of the presence of Ca2+. Both fast and slow phases of the evoked release were blocked by Co2+. Addition of Co2+ within the first 6 s after the onset of depolarization inhibited the fast release but failed to do so when added later on.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium permeability changes and neurotransmitter release in cultured rat brain neurons. I. Effects of stimulation on calcium fluxes.

The permeability of neuronal membranes to Ca2+ is of great importance for neurotransmitter release. The temporal characteristics of Ca2+ fluxes in intact brain neurons have not been completely defined. In the present study 45Ca2+ was used to examine the kinetics of Ca2+ influx and efflux from unstimulated and depolarized rat brain neurons in culture. Under steady-state conditions three cellular exchangeable Ca2+ pools were identified in unstimulated cells: 1) a rapidly exchanging pool (t1/2 = 7 s) which represented about 10% of the total cellular Ca2+ and was unaffected by the presence of Co2+, verapamil, or tetrodotoxin; 2) a slowly exchanging pool (t1/2 = 360 s) which represented 42% of the total cellular Ca2+ and was inhibited by Co2+, but not by verapamil or tetrodotoxin; 3) a very slowly exchanging pool (t1/2 = 96 min) which represented 48% of the total cell Ca2+ was observed only in the prolonged efflux experiments. The rate of exchange of 45Ca2+ in the unstimulated cells was dependent on the extracellular Ca2+ concentration (half-saturation at 70 microM). Depolarization of the neurons with elevated K+ causes a rapid and sustained 45Ca2+ uptake. The cellular Ca2+ content increased from 56 nmol/mg protein in unstimulated cells to 81 nmol/mg protein during 5 min of depolarization. The kinetics of the net 45Ca2+ uptake by the stimulated neurons was consistent with movement of the ion with a first order rate constant of 0.0096 s-1 (t1/2 = 72 s) into a single additional compartment. The other cellular Ca2+ pools were apparently unaffected by stimulation. The stimulated 45Ca2+ uptake was inhibited by Co2+ and by the Ca2+ channel blocker verapamil but not by the Na+ channel blocker tetrodotoxin. Ca2+ uptake into this compartment was dependent on the extracellular Ca2+ concentration (half-saturation at 0.80 mM Ca2+). Predepolarization of the cells with high K+ for 10-60 s prior to the addition of the radioactive calcium did not alter the rate of 45Ca2+ incorporation into the stimulated cells. It is concluded that the rapidly exchanging, the slowly exchanging, and the depolarization-induced Ca2+ pools observed in intact brain neurons are physically as well as kinetically distinct from each other. In addition, the depolarization-induced component observed in stimulated cells represents movement of the Ca2+ ions through a single class of voltage-sensitive Ca2+ channels. These Ca2+ channels are inhibited by Co2+ ions and by verapamil and are not inactivated during depolarization of the brain neurons.