Insulin receptors in the brain: Structural and physiological characterization

The present study was conducted to characterize insulin receptors and to determine the effects of insulin in synaptosomes prepared from adult rat brains. Binding of¹²⁵I-insulin to synaptosome insulin receptors was highly specific and time dependent: equilibrium binding was obtained within 60 minutes, and a t_1/2 of dissociation of 26 minutes. Cross-linking of¹²⁵I-insulin to its receptor followed by SDS-PAGE demonstrated that the apparent molecular weight of the alpha subunit of the receptor was 122,000 compared with 134,000 for the liver insulin receptor. In addition, insulin stimulated the dose-dependent phosphorylation of exogenous tyrosine containing substrate and a 95,000 MW plasma membrane associated protein, in a lectin-purified insulin receptor preparation. The membrane associated protein was determined to be the subunit of the insulin receptor. Incubation of synaptosomes with insulin caused a dose-dependent inhibition of specific sodium-sensitive [³H]norepinephrine uptake. Insulin inhibition of [³H]norepinephrine uptake was mediated by a decrease in active uptake sites without any effects in theK _m, and was specific for insulin since related and unrelated peptides influenced the uptake in proportion to their structural similarity with insulin. These observations indicate that synaptosomes prepared from the adult rat brain possess specific insulin receptors and insulin has inhibitory effects on norepinephrine uptake in the preparation.     

Developmental changes in high-affinity uptake of GABA by cultured neurons

High-affinity uptake of [³H]-aminobutyric acid (GABA) was studied in cultures of neonatal rat cortical neurons grown on pre-formed monolayers of non-neuronal (glial) cells. Both the maximum rate (V _max) and, to a smaller extent, theK _m of [³H]GABA uptake increased with time. In addition, in parallel with these changes, 2,4-diaminobutyric acid and cis-3-aminocyclohexane-1-carboxylic acid (ACHC), compounds which are considered typical substrate/inhibitors of GABA uptake in neurons, became progressively stronger inhibitors of [³H]GABA uptake. Consequently, the present results may mean that the studies using uptake, of [³H]GABA, [³H]ACHC, or [³H]DABA as a specific marker for GABAergic neurons differentiating during the ontogenetic development of the central nervous system may have to be interpreted with caution.     

Uptake of GABA by neuronal and nonneuronal cells in dispersed cell cultures of postnatal rat cerebellum

A study was made of the time course and kinetics of [³H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [³H]GABA, or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent K_m values for GABA neurons for high and low affinity uptake were 0.33 × 10⁻⁶ M and 41.8 × 10⁻⁴ M, respectively. For nonneuronal cells, the apparent K_m for high affinity uptake was 0.29 × 10⁻⁶ M. The apparent V_max values for GABA neurons for high and low affinity uptake were 28.7 × 10⁻⁶ mol/g DNA/min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent V_max for high affinity uptake was 0.06 × 10⁻⁶ mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 × 10⁻⁹ M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [³H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by nonneuronal cells was only slightly decreased. Most (75–85%) of the [³H]GABA (4.4 × 10⁻⁶ M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent.     

Transport of monoamine and amino acid neurotransmitters by primary astroglial cultures

The transport of [³H]l-glutamate, [³H]l-aspartate, [³H]-aminobutyric acid ([³H]GABA), [³H]dopamine, [³H]norepinephrine and [³H]5-hydroxytryptamine (³H-5-HT) was measured in primary astroglial cultures from newborn rat cerebral hemispheres. There was a high-affinity uptake with aK _m of 69.0 M for L-glutamate, 12.3 M forl-aspartate and 3.1 M for GABA. The uptake showed properties of high capacity with aV _max of 17.0 nmol·mg prot^–1·min^–1 forl-glutamate, 1.1 nmol·mg prot^–1·min^–1 forl-aspartate and 0.04 nmol·mg prot^–1·min^–1 for GABA. No high-affinity high capacity transport system was found for the monoamines studies. Autoradiographic examination demonstrated a heavy deposit of grains suggesting a prominent accumulation of [³H]l-glutamate and [³H]l-aspartate in the astroglial-like cells of the cultures, while the [³H]GABA accumulation was less intense. On the other hand, there was only a weak accumulation of grains after incubating the cultures with [³H]dopamine, [³H]norepinephrine or [³H]5-HT. Thus, astroglial cells in culture accumulate amino acid neurotransmitters and monoamines in different ways with a high-affinity high-capacity uptake of glutamate, aspartate and GABA and a diffusion-uptake of dopamine, norepinephrine and 5-HT.     

Characterization of separated cell types from developing rat cerebellum

The characteristics of [³H]GABA transport were investigated in preparations greatly enriched in different classes of cerebellar cells. In contrast to observations in situ, isolated Purkinje cells readily accumulated [³H]GABA. In comparison with astrocytes, theV _max of the high-affinity uptake process was sixfold higher (0.31 vs. 0.05 nmol/min/10⁶ cells) and the apparentK _t twofold greater (2 vs. 1 M). In contrast to these cell types, uptake was very low in granule cell-enriched preparations.cis-1,3-Aminocyclohexane carboxylic acid was a potent inhibitor of [³H]GABA uptake by the Purkinje cells and a weak blocker in astrocytes, while the converse was the case for -alanine. Diaminobutyric acid strongly inhibited uptake in both cell types. [³H]GABA transport was Na⁺ dependent in both cell classes. However, veratridine and ouabain selectively blocked [³H]GABA accumulation in the Purkinje cells, which were also more sensitive than the astrocytes to the glycolysis inhibitor, NaF. The results indicated, therefore, marked differences between Purkinje cells and astrocytes in the properties of both the [³H]GABA transport systems and the underlying metabolic processes.     

Effects of cell signaling on the development of GABA receptors in chick retina neurons

R-cognin, a cell recognition molecule, and insulin are known to play significant roles in GABAergic differentiation in the developing chick retina. In the present study, the effects of insulin and R-cognin on post-synaptic (GABAceptive) differentiation were investigated. In ovo binding of [³H]GABA and [³H]flunitrazepam ([³H]Flu) to the GABA and benzodiazepine (BZD) receptors, respectively, remained at low levels during early embryogenesis but increased sharply from mid-embryogenesis through hatching, increases which also occur in cultured neurons from early-embryonic (E7) and mid-embryonic (E11) chick retina. E7 neurons respond to insulin treatment (100 ng/ml) with increased [³H]Flu binding but no change in [³H]GABA binding. Cognin antibody (10 g/ml) treatment of E7 neurons caused no significant inhibition of the developmental increases in binding of either radioligand. Insulin in E11 cultures led to greater developmental increases in binding sites for both radioligands, but exposure to cognin antibody was without significant effect. These data, along with previous studies, indicate that GABAergic differentiation in developing chick retina is regulated, in part, by insulin and cognin-mediated cell signaling. Insulin also regulates post-synaptic (GABAceptive) differentiation whereas cognin-mediated interactions are relatively insignificant.Abbreviations BZD benzodiazepine - ChAT choline acetyltransferase - Flu flunitrazepam - GABA -aminobutyric acid - GAD glutamate decarboxylase (glutamic acid decarboxylase)     

Insulin and insulin mutants stimulate glucose uptake in rat adipocytes

A simple method to determine thein vitro biological activity of insulin by measuring glucose uptake in the rat adipocytes is presented here. In the presence of insulin, the glucose uptake is 5–6 times more than the basal control. And the uptake of D-[3-³H]-glucose is linear as the logarithm of insulin concentration from 0.2 ώg/L to 1.0 ώg/L. Glucose and 3-O-methyl-glucose inhibit D-[3-³H]-glucose uptake into adipocytes. By this method, thein vitro biological activity of [B₂-Lys]-insulin and [B₃-Lys]-insulin was measured to be 61.6% and 154% respectively, relative to that of insulin.     

Insulin and insulin mutants stimulate glucose uptake in rat adipocytes

A simple method to determine the in vitro biological activity of insulin by measuring glucose uptake in the rat adipocytes is presented here. In the presence of insulin, the glucose uptake is 5-6 times more than the basal control. And the uptake of D-[3-3H]-glucose is linear as the logarithm of insulin concentration from 0.2 μg/L to 1.0 μg/L. Glucose and 3-O-methyl-glucose inhibit D-[3-3H]-glucose uptake into adipocytes. By this method, the in vitro biological activity of [B2-Lys]-insulin and [B3-Lys]-insulin was measured to be 61.6% and 154% respectively, relative to that of insulin.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

Enzyme activities of monoamine oxidase,cathechol-o-methyltransferase and γ-aminoubutyric acid transaminase in primary astroglial cultures and adult rat brain from different brain regions

The activities of monoamine oxidase (MAO), cathechol-O-methyltransferase (COMT) and -aminobutyric acid transaminase (GABA-T) were measured in primary cultures from newborn rat cultivated from 6 different brain regions. These primary cultures contained mostly astroglial cells, evaluated by the presence of the glial fibrillary acidic protein (GFAp, -albumin) and the S-100 protein. The enzyme activities in the corresponding brain areas from adult rat were also quantified. MAO activities were on the same level in 14-day old cultures and in adult rat brain homogenates, with significantly lower values in brain stem as compared to the other brain regions examined. COMT activities were on a higher level in the cultures than in adult rat brain homogenates. Astroglial cells from hippocampus were found to have the highest and those from brain stem the lowest COMT-activities. GABA-T activities were lower in the cultures than in adult rat homogenates. No significant differences were seen in the various astroglial cultures. Accumulation of [³H]dopamine and [³H]-aminobutyric acid (GABA) visualized by autoradiography showed only a slight uptake of dopamine in comparison with the uptake of GABA. It is concluded that astroglial cells in culture have enzymatic properties similar to those of astroglial cells in different brain regions of adult rat brain. Studies are in progress to evaluate if the regional heterogeneity observed among cultivated astroglial cells is affected by in vivo differentiation until cultivation and/or time in culture.     

Glial uptake system of GABA distinct from that of taurine in the bullfrog sympathetic ganglia

The kinetics and specificity of GABA and taurine uptake were studied in the bullfrog sympathetic ganglia. GABA uptake system consisted of simple saturable component and taurine uptake system consisted of two saturable components exclusive of non-saturable influx. Taurine unaffected GABA uptake while GABA inhibited taurine uptake competitively with theK _i/K_m ratio of 38. GABA (5.14 M) uptake was inhibited by -aminovaleric acid and slightly by 2,4-diaminobutyric acid (5 mM, each) among ten structural analogs. Taurine uptake under high-affinity conditions was most strongly suppressed by hypotaurine and -alanine competitively with theK _i/K_m ratio of 1.0 and 1.9, respectively. Autoradiography showed that glial cells were heavily labeled by both [³H]GABA and [³H]taurine. These results suggest that GABA is transported by a highly specific carrier system distinct from the taurine carrier and that taurine, hypotaurine, and -alanine may share the same high-affinity carrier system in the glial cells of the bullfrog sympathetic ganglia.     

Effects of X-irradiation induced loss of cerebellar granule cells on the synaptosomal levels and the high affinity uptake of amino acids.

Abstract— Crude synaptosomal (P₂) preparations were obtained from the cerebella of rats in which the granule cell population had been selectively reduced by X-irradiation treatment and from the cerebella of control animals. In the P₂ fraction from control cerebella, the level of glutamate was greater than any other of the 5 amino acids measured and was 2-fold higher than taurine, which was present at the next highest level. The content of taurine was slightly higher than that found for aspartate and was 3-fold greater than that observed for GABA. Alanine and glycine were present in the lowest amounts. The levels of glutamate and aspartate were significantly (P < 0.05) lower by 25 and 15%, respectively, in the P₂ fraction isolated from the X-irradiated cerebella in comparison to control values. The content of taurine, GABA, glycine, and alanine were not changed by the X-irradiation treatment. The uptake of 1.0 μm -l -[³H]glutamate and l -[³H]aspartate was reduced approx 20% by X-irradiation treatment, whereas the uptake of 1.0 μm -[³H]GABA and [³H]taurine was unchanged. A more detailed kinetic analysis of l -[³H]glutamate uptake revealed there was a 20% decrease in the V_max value with X-irradiation treatment and no change in the apparent K_m value. In a second study, the uptake of l -[³H]glutamate, l -[³H]aspartate and [³H]GABA was measured using P₂ fractions obtained from the cerebella of rats in which the population of granule, stellate and basket cells had been reduced by X-irradiation treatment. The uptake of 1.0μm -l -[³H]glutamate, l -[³H]aspartate and [³H]GABA was significantly (P < 0.05) reduced to 57, 68, and 59%, respectively, of control values. A more detailed kinetic analysis of [³H]GABA uptake revealed no significant change in the apparent K_m and a 35% decrease in the V_max value. The data are discussed in terms of glutamate being the excitatory neurotransmitter released from granule cells and GABA being the inhibitory neurotransmitter released from basket cells.     

Amino acid and monoamine transport in primary astroglial cultures from defined brain regions

The uptake ofl-[³H]glutamate,l-[³H]aspartate, -[³H]aminobutric acid (GABA), [³H]dopamine,dl-[³H]norepinephrine and [³H]5-hydroxytryptamine (5-HT) was studied in astrocytes cultured from the cerebral cortex, striatum and brain stem of newborn rat and grown for 2 weeks in primary cultures. The astrocytes exhibited a high-affinityl-glutamate uptake withK _m values ranging from 11 to 110 M.V _max values were 4.5 in cerebral cortex, 39.1 in striatum, and 0.4 in brain stem, nmol per mg cell protein per min. There was a less prominent high-affinity uptake ofl-aspartate withK _m values from 88 to 187 M.V _max values were 7.4 in cerebral cortex, 37.1 in striatum, and 3.1 in brain stem, nmol per mg cell protein per min. The high-affinity GABA uptake exhibitedK _m values ranging from 5 to 17 M andV _max values were 0.01 for cerebral cortex, 0.04 for striatum, and 0.1 for brain stem, nmol per mg cell protein per min. No high-affinity, high-capacity uptake was found for the monoamines. The results demonstrate a heterogeneity among the astroglial cells cultivated from the different brain regions concerning the uptake capacity of amino acid neurotransmitters. Furthermore, amino acid transmitters and monoamines are taken up by the cells in different ways.     

The effects of insulin and tunicamycin on insulin receptors of cultured fibroblasts

The effect of down-regulation on the intracellular pool of insulin receptors and the role of glycosylation in recovery from down-regulation have been studied in fibroblastic cultures from the skin of non-diabetic mice. In control cultures, 55% of the total specific [¹²⁵I]insulin-binding activity was in the intracellular compartment. Insulin caused a time- and concentration-dependent decrease in the number of cell surface insulin receptors, with no significant change in total insulin receptors. This decrease in surface receptors was accompanied by an increase in the specific binding of [¹²⁵I]insulin in the intracellular compartment. Removal of insulin from down-regulated cells resulted in a time-dependent increase in the binding of [¹²⁵I]insulin to surface receptors, reaching 90% of that in controls by 12 h. The recovery of surface insulin receptors after removal of insulin was blocked by incubation of cultures with tunicamycin, but not by cycloheximide. These results indicate that down-regulation of surface insulin receptors by insulin is associated with translocation of receptors into the intracellular pool and suggest that protein glycosylation is important in insulin receptor recycling and externalization.     

Characterization of 5-HT transporter and receptor system in HeLaS3 cells by [H]8-OH-DPAT and other serotonergic ligands

[³H]8-OH-DPAT is a selective ligand for labeling 5-HT_1A receptor sites. In competition binding experiments, we found that classic biogenic amine transporter inhibitors displaced [³H]8-OH-DPAT binding at its high-affinity binding sites in HeLaS3 cells. [¹²⁵I]RTI-55 and [³H]paroxetine are known to specifically label amine transporter sites, and this was observed in our cells. Displacement studies showed that 8-OH-DPAT displayed affinity in a dose-dependent manner for the labeled amine transporter sites. These data suggest that [³H]8-OH-DPAT binds to amine uptake sites in HeLaS3 cells. A variety of drugs targeting different classes of receptors did not significantly affect [³H]8-OH-DPAT binding. Moreover, we determined the specific binding effects of various serotonergic ligands (i.e. [¹²⁵I]cyanopindolol, [³H]ketanserin/[³H]mesulergine, [³H]GR-65630, [³H]GR-113808 and [³H]LSD) that specifically labeled 5-HT₁, 5-HT₂, 5-HT₃, 5-HT₄ and 5-HT_5–7 receptors, respectively. It is suggested that HeLaS3 cells contain distinct types of the related to 5-HT receptor recognition binding sites. These observations could help elucidate the relevant characteristics of different types of 5-HT receptors and 5-HT membrane transporters in tumor cells and their role in tumorigenesis.     

Substrate Specificity of [3H] Muscimol Binding to a Particulate Fraction of a Neuron-enriched Culture of Embryonic Rat Brain

Abstract: The effects of some GABA analogues and some drugs on the binding of [³H]muscimol (3.08 nM) to thoroughly washed subcellular particles prepared from a neuron-enriched culture of embryonic rat brain were examined using Na+-free Tris-citrate medium and a centrifugation method. Competition for [³H]muscimol binding sites by excess(10^?5 M) unlabelled GABA provided estimates of “specific” binding. In accord with in vivo neuropharmacological studies on GABA receptors and with in vitro studies on cerebral membrane preparations, [³H]muscimol binding was potently inhibited by muscimol itself (IC₅₀, 2.5 nM), GABA (1C₅₀, 43 nM), isoguvacine (IC₅₀, 61 nM), and 3-aminopropanesulphonic acid (IC₅₀, 160 nM), and less potently inhibited by the GABA antagonist bicuculline methobromide (IC₅₀, 800 nM). δ- Aminovaleric acid (IC₅₀, 2.6 μM), the glycinelp-alanine antagonist strychnine (IC₅₀, 6.6 μM), and the predominantly glial GABA uptake inhibitors β-alanine (IC₅₀, 23 μM) and p-proline (IC₅₀, 66 μM) also inhibited [³H]muscimol binding. Other inhibitors of Na+-dependent GABA uptake, (±)-nipecotic acid, L- 2,4-diaminobutyric acid, and guvacine, as well as picrotoxinin, were relatively inactive as inhibitors of [³H]muscimol binding (IC₅₀≥ 1 mM). In addition to revealing that GABA receptors are present on neuronal membranes before the formation of most synapses, this binding of [³H]muscimol that occurs to neuronal, but not to glial, membranes might be useful as a “neuronal marker” and for the further characterization and isolation of GABA receptors.     

Effect of ferric nitrilotriacetate on predominantly cortical neuronal cell cultures

Predominately neuronal cell cultures were produced as described in previous communications. Neuronal cells were exposed to ferric nitrilotriacetate (Fe-NTA) at varying concentrations. Studies of the neuronal cells were performed at 13 and 20 days in culture. In addition to morphologic studies, biochemical assays including choline acetyltransferase (ChAT) activity, specific [³H]flunitrazepam (FLU) binding, clonazepam (CLO)-displaceable [³H]FLU binding, Ro5-4864-displaceable [³H]FLU binding, high-affinity [³H]GABA uptake, and protein determinations were performed. The data demonstrate that chelated ferric iron has an adverse effect on predominately neuronal cultures after 7 days of exposure as measured by choline acetyltransferase activity, while other measures remained unaffected; however, after 14 days of exposure all measures were significantly decreased. The effects of Fe-NTA exposure appear to be both concentration and duration-of-exposure related.     

Transgenic Murine Dopaminergic Neurons Expressing Human Cu/Zn Superoxide Dismutase Exhibit Increased Density in Culture, but No Resistance to Methylphenylpyridinium-Induced Degeneration

Abstract: Primary dopaminergic neuronal cultures with increased superoxide dismutase (SOD) activity were established for studying the role of superoxide anion (O₂^?) in 1-methyl-4-phenylpyridinium (MPP⁺)-induced degeneration of dopamine (DA) neurons. Mean SOD activity in cultures prepared from transgenic (human) Cu/Zn SOD (hSOD1) mice was 2.46–2.60 times greater than in cultures prepared from nontransgenic control mice. After 1 and 2 weeks in culture, the mean density of DA neurons [number of tyrosine hydroxylase-immunoreactive (TH-ir) cells per visual field] was significantly higher in cultures prepared from transgenic mice compared with those prepared from nontransgenic control mice (4.55–5.63 TH-ir neurons per field in hSOD1 cultures vs. 2.66–2.8 TH-ir neurons per field in control cultures). However, uptake of [³H]DA relative to uptake of [³H]GABA was only slightly greater in hSOD1 cultures than in normal cultures (14.1 nmol of DA/100 nmol of GABA vs. 12.1 nmol of DA/100 nmol of GABA). Resistance to MPP⁺ toxicity was not significantly different from that in normal cultures when based on density of surviving TH-ir cell bodies (EC₅₀ = 0.54 µM in hSOD1 and EC₅₀ = 0.37 µM in normal cultures). A more sensitive measure of DA neuron integrity and function ([³H]DA uptake) also failed to demonstrate increased resistance of hSOD1 cultures to the toxin (EC₅₀ = 73.7 nM in hSOD1 and EC₅₀ = 86.2 nM in controls). These results do not support the hypothesis that neurotoxicity of the active metabolite of MPTP, MPP⁺, is mediated by generation of O₂^? in the cytoplasm. Nevertheless, mesencephalic cultures with increased hSOD1 activity appear to survive better than normal control cultures in the oxidatively stressful environment of cell culture incubators, and such mesencephalic cells may be useful for cell grafting studies in animal models of Parkinson's disease.     

[3H]Bicuculline Methochloride Binding to Low-Affinity γ-Aminobutyric Acid Receptor Sites

Abstract: The binding of [³H]bicuculline methochloride (BMC) to mammalian brain membranes was characterized and compared with that of [³H]γ-aminobutyric acid ([³H]GABA). The radiolabeled GABA receptor antagonist showed significant displaceable binding in Tris-citrate buffer that was improved by high concentrations of chloride, iodide, or thiocyanate, reaching >50% displacement in the presence of 0.1 M SCN^?. An apparent single class of binding sites for [³H]BMC (K_D= 30 nM) was observed in 0.1 M SCN^? for fresh or frozen rat cortex or several regions of frozen and thawed bovine brain. The B_max was about 2 pmol bound/mg of crude mitochondrial plus microsomal membranes from unfrozen washed and osmotically shocked rat cortex, similar to that for [³H]GABA. Frozen membranes, however, showed decreased levels of [³H]BMC binding with no decrease or an actual increase in [³H]GABA binding sites. [³H]BMC binding was inhibited by GABA receptor specific ligands, but showed a higher affinity for antagonists and lower affinity for agonists than did [³H]GABA binding. Kinetics experiments with [³H]GABA binding revealed that low- and high-affinity sites showed a similar pharmacological specificity for a series of GABA receptor ligands, but that whereas all agonists had a higher affinity for slowly dissociating high-affinity [³H]GABA sites, bicuculline had a higher affinity for rapidly dissociating low-affinity [³H]GABA sites. This reverse potency between agonists and antagonists during assay of radioactive antagonists or agonists supports the existence of agonist- and antagonist-preferring conformational states or subpopulations of GABA receptors. The differential affinities, as well as opposite effects on agonist and antagonist binding by anions, membrane freezing, and other treatments, suggest that [³H]BMC may relatively selectively label low-affinity GABA receptor agonist sites. This study, using a new commercially available preparation of [³H]bicuculline methochloride, confirms the report of bicuculline methiodide binding by Mohler and Okada (1978), and suggests that this radioactive GABA antagonist will be a valuable probe in analyzing various aspects of GABA receptors.     

Glutamic Acid and γ-Aminobutyric Acid Modulate Each Other's Release Through Heterocarriers Sited on the Axon Terminals of Rat Brain

Abstract: The effects of γ-aminobutyric acid (GABA) on the spontaneous release of endogenous glutamic acid (Glu) or aspartic acid (Asp) and the effects of Glu on the release of endogenous GABA or [³H]GABA were studied in superfused rat cerebral cortex synaptosomes. GABA increased the outflow of Glu (EC₅₀17.2 μM) and Asp (EC₅₀ 18.4 μM). GABA was not antagonized by bicuculline or picrotoxin. Neither muscimol nor (-)-baclofen mimicked GABA. The effects of GABA were prevented by GABA uptake inhibitors and were Na⁺ dependent. Glu enhanced the release of [³H]GABA (EC₅₀ 11.5 μM) from cortical synaptosomes. Glu was not mimicked by the glutamate receptor agonists N-methyl-d -aspartic, kainic, or quisqualic acid. The Glu effect was decreased by the Glu uptake inhibitor D-threo-hydroxyaspartic acid (THA) and it was Na⁺ sensitive. Similarly to Glu, D-Asp increased [³H]GABA release (EC₅₀ 9.9 μM), an effect blocked by THA. Glu also increased the release of endogenous GABA from cortex synaptosomes. In this case the effect was in part blocked by the (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 6-cyano-7-nitroquinoxaiine-2, 3-dione, whereas the 6-cyano-7-nitroquinoxaline- 2, 3-dione-insensitive portion of the effect was prevented by THA. GABA increased the [³H]D-Asp outflow (EC₅₀ 13.7 μM) from hippocampal synaptosomes in a muscimol-, (-)- baclofen-, bicuculline-, and picrotoxin-insensitive manner. The GABA effect was abolished by blocking GABA uptake and was Na⁺ dependent. Glu increased the release of [³H]- GABA from hippocampal synaptosomes (EC₅₀ 7.1 μM) in an N-methyl-d -aspartic acid-, kainic acid-, or quisqualic acid-insensitive way. The effect of Glu was prevented by THA and was Na⁺ dependent. As in the cortex, the effect of Glu was mimicked by D-Asp in a THA-sensitive manner. It is proposed that high-affinity GABA or Glu heterocarriers are sited respectively on glutamatergic or GA- BAergic nerve terminals in rat cerebral cortex and hippocampus. The uptake of GABA may modulate Glu and Asp release, whereas the uptake of Glu may modulate the release of GABA. The existence of these heterocarriers is in keeping with the reported colocalization of GABA and Glu in some cortical and hippocampal neurons. Preliminary data suggest that these mechanisms may also be present in rat cerebellum and spinal cord.