3H]Spermine binding to synaptosomal membranes from the chick retina.

The binding of [3H]spermine to synaptosomal membranes from chick retina was examined. Saturable specific binding of [3H]spermine to synaptosomal membranes from plexiform layers of retina (P1 and P2) has been characterized, and found to concentrate in the inner plexiform layer compared to the outer plexiform layer (Bmax=9.3 and 37 pmol/mg protein for P1 and P2, respectively). Kinetics of specific [3H]spermine binding yield a sigmoidal saturation curve, indicating positive cooperativity (nH: 2.4 and 3.2 for P1 and P2, respectively) with high affinity: Kapp=61 and 67 nM for P1 and P2. The time required to attain equilibrium at room temperature was less than 5 min in both fractions. Dose-response curves for spermine, spermidine, and diethylene-triamine (DET) show different potencies for inhibiting [3HDET. Our results support a role for polyamines (PA) as neurotransmitters or neuromodulators in the vertebrate retina.     

Specific [H]strychnine binding associated with glycine receptors in bovine retina

Saturable, specific [³H]strychnine binding can be demonstrated in homogenates of bovine retina. Scatchard plots revealed only one set of binding sites with a dissociation constant (K_d) of about 60 nM and a maximal number of binding sites of about 1.5 pmol/mg protein. The structural specificity of [³H]strychnine binding sites in bovine retina parallels the properties found for [³H]strychnine binding sites in the spinal cord of several vertebrates. Thus, the data do not give any evidence that specific [³H]strychnine binding in bovine retina labels taurine rather than glycine receptors and favors glycine rather than taurine as inhibitory neurotransmitter in bovine retina. The subcellular distribution of specific [³H]strychnine binding in bovine retina parallels that of sodium-dependent, high-affinity uptake of glycine and taurine. All 3 parameters are mainly found in the P₂ fractions of bovine retina homogenates, containing conventional synaptosomes, most abundant in the inner plexiform layer, but can also be found in the P₁ fractions, containing large synaptosomes from the photoreceptor cell layer.     

Characterization of adenosine receptors in brain using N cyclohexyl [H]adenosine

The presence of distinct binding sites for adenosine in both the CNS and PNS has been proposed in numerous studies. The recent availability of stable adenosine analogues such as cyclohexyladenosine, 2-chloroadenosine and diethylphenylxanthine has made the characterization of such a receptor feasible. In the present report the binding of N⁶ cyclohexyl [³H]adenosine ([³H]CHA) to rat brain synaptosomal membranes is characterized. [³H]CHA binding is saturable and exhibits a biphasic kinetic saturation profile characteristic of 2 binding sites. The high affinity site has a K_d of 0.7 nM and the low affinity site 2.4 nM. The respective B_max values are 230 and 120 fmol/mg protein in rat forebrain. The highest density of binding sites is found in the hippocampus and subcellular distribution studies indicate that the [³H]CHA site is predominantly synaptosomal. [³H]CHA binding is highly dependent on the presence of adenosine deaminase since only 30% of the binding capacity is observed in synaptosomal membranes not treated with this enzyme. Of the many cations and anions tested only copper and zinc have effects on [³H]CHA binding. Both metals are potent inhibitors of binding with copper having an IC₅₀ of 30 μM and zinc 150 μM. Sulfhydryl reducing and alkylating agents also inhibit binding indicating that the binding site is a sulfhydryl-dependent protein.     

Strychnine-insensitive [H] glycine binding to synaptosomal membranes from the chick retina

The pharmacology and kinetics of strychnine-insensitive [[3]H] glycine binding to synaptic membranes from the outer (P₁) and the inner (P₂) plexiform layers of chick retina was studied. Inhibition curves for glycine, d-serine, 1-amincyclopropanecarboxylic acid (ACPC) and strychnine were analyzed by non-linear regression. Hill slopes for glycine and d-serine were not different from unity, whereas those for ACPC were < 1 in both fractions, revealing heterogeneity of binding sites in these membranes. Non-linear regression analysis of time course and saturation experiments strengthen the idea that [[3]H] glycine binds to more than one class of sites, with similar affinities at equilibrium. Antagonists of strychnine-insensitive glycine receptors in the CNS did not inhibit [[3]H] glycine binding to these membranes, which demonstrates that NMDA receptors in the retina have different structural requirements for ligand interaction at these sites. pH affected the specific binding, in agreement with the participation of specific amino acid residues at glycine binding sites on NMDA receptors, and also with functional studies in which the modulation of affinity at this site by protons has been observed. These results support previous studies regarding CPP and MK-801 binding, and provide evidence which indicates that the pharmacophore for glycine and other NMDA-related ligands is distinct for the retina, compared to the CNS, mainly regarding the effects of glycine-site antagonists.     

Characterisation of central adenosine A1 receptors and adenosine transporters in mice lacking the adenosine A2a receptor

The present study was designed to assess whether adenosine A_2a receptor knockout mice exhibit altered purine utilisation in brain nuclei. Specifically, the properties of adenosine transporters and adenosine A₁ receptors were characterised in brain membranes and on slide-mounted sections. The B_MAX for [³H]nitrobenzylthioinosine ([³H]NBTI) binding (adenosine transporter density) was significantly reduced in brainstem membranes of homozygotes (560±52 fmol/mg protein, n=5, P<0.05, Kruskal–Wallis ANOVA) compared to wildtype (1239±213 fmol/mg protein) and heterozygous mice (1300±558 fmol/mg protein). Quantitative autoradiography data indicated that [³H]NBTI binding in the medulla oblongata of heterozygous mice was seen to decrease significantly (P<0.05) in the subpostremal nucleus tractus solitarius (NTS), medial NTS, inferior olive and area postrema (AP). On the other hand, in the homozygous mice a decrease was seen in the medial NTS and AP. In the pons, [³H]1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) (adenosine A₁ receptor density) binding increased significantly (P<0.05, Kruskal–Wallis ANOVA) in the lateral parabrachial nucleus, caudal pontine reticular nucleus and locus coeruleus of homozygotes compared to wildtype. In higher brain centres, [³H]NBTI binding was reduced in the paraventricular thalamic nucleus of both heterozygous and homozygous mice, whereas [³H]DPCPX binding was reduced in the hippocampus and lateral hypothalamus of heterozygotes. In homozygotes, [³H]DPCPX binding in the hippocampus increased compared to wildtype mice. The present study indicates that deletion of the A_2a receptor may have contributed to region-specific compensatory changes in purine utilisation in brain nuclei associated with autonomic, neuroendocrine and behavioural regulation.     

GABA binding and bicuculline in spinal cord and cortical membranes from adult rat and from mouse neurons in cell culture

Sodium-independent [³H]GABA and [³H]muscimol binding was determined in adult rat cerebral cortical and spinal cord membranes and in membranes from fetal mouse cortical and spinal cord neurons in primary dissociated cell culture. In adult rat cerebral cortical membranes, [³H]GABA bound to two sites (K_d=8nM,B_max=0.62pmol/mg protein; K_d=390nM,B_max=3.9pmol/mg protein) whereas the GABA agonist, [³H]muscimol, bound only to a high affinity site (K_d=5.6nM,B_max=1.9pmol/mg protein). In adult rat spinal cord, only a low affinity site was seen with [³H]GABA (K_d=340nM,B_max=9.8pmol/mg protein) and only a high affinity site was seen with [³H]muscimol (K_d=5.6nM,B_max=0.25pmol/mg protein). The inability to measure a high affinity [³H]GABA binding site in spinal cord probably reflects the high ratio of low to high affinity sites in spinal cord (39:1). In membranes from mouse neurons in cel; culture, [³H]GABA bound to two sites on cortical neurons (K_d=9nM,B_max=0.24pmol/mg protein; K_d=510nM,B_max=1.3pmol/mg protein) and spinal cord neurons (K_d=13nM,B_max=0.12pmol/mg protein; K_d=640nM,B_max=3.2pmol/mg protein). Again, the ratio of low to high affinity sites in cultured mouse spinal cord neurons was high (27:1).The effects of the potent GABA antagonist, (+)bicuculline, on both low and high affinity [³H]GABA binding was determined. Bicuculline appeared to inhibit binding to both sites competitively but theK_i for inhibiting the high affinity site was 5 μM and for inhibiting the low affinity site was 115 μM. Bicuculline inhibited [³H]muscimol binding in both brain and spinal cord competitively withK_is of 4μM and 10 μM respectively. Bicuculline inhibition of [³H]muscimol binding in cultured neuronal membranes was similar to that in adult rat membranes.The binding of the potent GABA agonist, muscimol, only to the high affinity site in both adult rat and cultured mouse neuronal membranes suggests that the high affinity site is the physiologically relevant postsynaptic GABA receptor. The fact that bicuculline inhibits the high affinity site (but not the low affinity site) in concentrations similar to those needed to block GABA-responses in physiological experiments²⁸ supports this hypothesis.     

D1 dopamine receptors in neurite regions of embryonic and differentiated retina are highly coupled to adenylate cyclase in the embryonic but not in the mature tissue

[³H]SCH 23390 binds stereospecifically and with high affinity to D₁ dopaminergic receptors in the developing chick retina. Autoradiographic experiments revealed that in retinas from 3-day-old chicken and embryos with 12, 14 and 16 days of development, specific labeling of [³H]SCH 23390 was mainly observed over the plexiform layers of the tissue, showing that dopaminergic D₁ receptors are localized in retina cell neurites since the initial stages of neurite formation. The total number of [³H]SCH 23390 binding sites increased 5-fold during the differentiation of the retina, while the dopamine-dependent cyclic adenosine monophosphate (AMP) accumulation was significantly decreased. Consequently, the ratio between dopamine-dependent cyclic AMP accumulation and [³H]SCH 23390 binding sites decreased 10-fold as retina differentiated, indicating that a significant portion of D₁ receptors in retinas from adult chicken are not effectively coupled to adenylate cyclase molecules.     

[H]Arginine vasopressin binding to rat brain: a homogenate and autoradiographic study

Arginine-vasopressin (AVP) has been implicated as a putative central neurotransmitter or neuromodulator in some brain functions. This study demonstrates binding of [³H]AVP to rat brain homogenates that is pH and temperature dependent, is saturable (K_d = 0.77 nM, B_max = 0.374 pmol/mg) and reversible. A number of AVP analogues competitively displaced the [³H]AVP binding, indicating that central AVP binding sites may have a resemblance to the peripheral (V₁) AVP vasopressor receptor. Homogenate binding occurred predominantly in the microsomal fraction (P₃) of the hypothalamus while in the hippocampus and septum binding was predominantly in the synaptosomal fraction (P₂). Autoradiographic methods showed displaceable [³H]AVP binding in the lateral septum, amygdala, supraoptic, paraventricular and suprachiasmatic nuclei of the hypothalamus supporting the results of homogenate binding in preparations of these regions.     

Clathrin-coated vesicles from bovine brain contain uncoupled GABAA receptors

Clathrin-coated vesicles are thought to be a vehicle for the sequestration of GABA_A receptors. For coated vesicles from bovine cerebrum, we examined the binding properties of [³H]muscimol, a GABA_A-specific agonist, [³H]flunitrazepam, a benzodiazepine agonist, and [³⁵S]t-butylbiocyclophosphorthionate (TBPS), a ligand for GABA_A receptor channels. Under standard conditions, the binding level of [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS to coated vesicles represented 12.3±1.8%, 7.9±1%, and 10.2±1.8%, respectively, of that in crude synaptic membranes. Coated vesicles showed a single [³H]flunitrazepam binding site with a K_D value (12 nM) which was 9-fold that for synaptic membranes. The allosteric coupling between binding sites was measured by the addition of GABA to [³H]flunitrazepam and [³⁵S]TBPS binding assays. For [³H]flunitrazepam binding to synaptic membranes, GABA gave an EC₅₀=2.0 μM and at saturation (100 μM) an enhancement of 122%. This stimulation was completely blocked by the GABA antagonist SR95531. In contrast, neither GABA nor SR95531 had a significant effect on [³H]flunitrazepam binding to CCVs, indicating that the allosteric interaction between GABA and benzodiazepine binding sites is abolished. Likewise, GABA displaced nearly all of the [³⁵S]TBPS binding to synaptic membranes but had no effect on binding to coated vesicles, indicating that coupling between the GABA binding sites and chloride channel is also impaired. Thus GABA_A receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The presence of major GABA_A receptor subunits on these particles was verified by quantitative immunoblotting. Relative to the levels in synaptic membranes, CCVs contained 110±14% and 29.5±3.8%, respectively, of the immunoreactivity for GABA_A receptor β2 and α1 subunits. Thus, in comparison to GABA_A receptors on synaptic membranes, those on CCVs have a reduced α1/β2-subunit ratio. It may be suggested that a selective decline in the content of α1 subunits in coated vesicles could in part account for GABA_A receptor uncoupling.     

Strychnine-insensitive [3H] glycine binding to synaptosomal membranes from the chick retina

The pharmacology and kinetics of strychnine-insensitive [[3]H] glycine binding to synaptic membranes from the outer (P₁) and the inner (P₂) plexiform layers of chick retina was studied. Inhibition curves for glycine, d-serine, 1-amincyclopropanecarboxylic acid (ACPC) and strychnine were analyzed by non-linear regression. Hill slopes for glycine and d-serine were not different from unity, whereas those for ACPC were < 1 in both fractions, revealing heterogeneity of binding sites in these membranes. Non-linear regression analysis of time course and saturation experiments strengthen the idea that [[3]H] glycine binds to more than one class of sites, with similar affinities at equilibrium. Antagonists of strychnine-insensitive glycine receptors in the CNS did not inhibit [[3]H] glycine binding to these membranes, which demonstrates that NMDA receptors in the retina have different structural requirements for ligand interaction at these sites. pH affected the specific binding, in agreement with the participation of specific amino acid residues at glycine binding sites on NMDA receptors, and also with functional studies in which the modulation of affinity at this site by protons has been observed. These results support previous studies regarding CPP and MK-801 binding, and provide evidence which indicates that the pharmacophore for glycine and other NMDA-related ligands is distinct for the retina, compared to the CNS, mainly regarding the effects of glycine-site antagonists.     

Pharmacological characterization of adenosine A1 receptors and its functional role in brown trout (Salmo trutta) brain

The adenosine receptor agonist N⁶-cyclohexyl[³H]adenosine ([³H]CHA) was used to identify and pharmacologically characterize adenosine A1 receptors in brown trout (Salmo trutta) brain. In membranes prepared from trout whole brain, the A1 receptor agonist [³H]CHA bound saturably, reversibly and with high affinity (K_d=0.69±0.04 nM; B_max=0.624±0.012 pmol/mg protein) to a single class of binding sites. In equilibrium competition experiments, the adenosine agonists and antagonists all displaced [³H]CHA from high-affinity binding sites with the rank order of potency characteristic for an adenosine A1 receptors. A1 receptor density appeared not age-related (from 3 months until 4 years), and was similar in different brain areas. The specific binding was inhibited by guanosine 5′-triphosphate (IC₅₀=0.778±0.067 μM). GTP (5 μM) induced a low affinity state of A1 receptors. In superfused trout cerebral synaptosomes, 30 mM K⁺ stimulated the release of glutamate in a calcium dependent manner. Glutamate-evoked release was dose-dependently reduced by CHA, and the inhibition was reversed by the A1 antagonist 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, 30 mM K⁺ as well as 1 mM glutamate stimulated the release of adenosine in a Ca²⁺-independent manner and tetrodotoxin insensitive. These findings show that in trout brain adenosine A1 receptors are present which are involved in the modulation of glutamate transmitter release. Moreover, the stimulation of adenosine release by K⁺ depolarisation or glutamate support the hypothesis that, as in mammalian brain, a cross-talk between adenosine and glutamate systems exists also in trout brain.     

Mercuric chloride and p-chloromercuriphenylsulfonate exert a biphasic effect on the binding of the stimulant [3H]methylphenidate to the dopamine transporter

Mercuric chloride was found to have a biphasic effect on the binding of the radiolabeled stimulant [³H]methylphenidate to membranes from a crude synaptosomal preparation of rat striatal tissue. Binding was enhanced at low concentrations of HgCl₂, reaching a maximum of 62% above control values at 2.5 μ HgCl₂. It was inhibited in a dose-dependent manner at concentrations greater than 5 μ HgC1₂, with an IC₅₀ of 7.2 μ. The increase in binding observed at the low concentrations of HgC1₂ was shown by Scatchard analysis to be due to an increase in the affinity of [³H]methylphenidate for its binding site on the dopamine transporter, while a decrease in both affinity and B_max accompanied the reduction of [³H]methylphenidate binding observed at the higher concentrations of the inorganic mercury compound. The sodium salt of the organic mercurial p-chloromercuriphenylsulfonic acid also caused an increase in [³H]methylphenidate binding (159% above controls at 2,000 μ), followed by an immediate decrease in binding at higher concentrations of the reagent. Because both of these mercury-containing compounds have a high propensity for interacting with sulfhydryl groups, these data suggest that the cysteine residues in the dopamine transporter molecule may play an important role in the regulation of stimulant binding to the uptake complex. © 1994 Wiley-Liss, Inc.     

σ Binding parameters in developing rats predict behavioral efficacy of a σ ligand

The relationship between σ binding and the behavioral efficacy of a selective σ ligand was examined in rats of varying ages (30,45,60,75,90, and 150 days old). Scatchard analyses of the binding of the σ radioligand [³H]1,3-di-o-tolyguanidine ([³H]DTG) to brain membranes revealed significant age-related differences in binding to both crude synaptosomal and microsomal fractions. The functional significance of these developmental changes in σ ligand binding was studied by determining the postural effects of rubral microinjections of DTG in age-matched littermates of rats used in the binding studies. The degree of dystonia produced by a single dose of DTG was significantly correlated with the amount of [³H]DTG bound to rat brain synaptosomal membranes at low but not at high concentrations. No significant correlation between binding to the microsomal fraction and drug efficacy was observed. These experimental results were in good agreement with predicted amounts bound as estimated from a Scatchard analysis of the data. The results suggest that σ binding sites found in brain synaptosomal membranes are functional receptors involved in the control of movement and posture.     

Effects of verapamil on the release of different neurotransmitters

The effect of verapamil on resting and depolarization-induced monoamine release was investigated in rat hippocampal synaptosomes prelabeled with [³H]-5-hydroxytryptamine (HT) or [³H]-norepinephrine (NE) and rat striatal synaptosomes prelabeled with [³H]-dopamine (DA). Verapamil (50 μM) completely abolishes high K⁺-induced [³H]-NE release, but paradoxically facilitates high K⁺-induced [³H]-5-HT and [³H]-DA release. All these high K⁺-evoked responses were Ca²⁺ dependent. Verapamil does not modify [³H]-NE baseline release, but increases dose dependently [³H]-5-HT and [³H]-DA baseline release. Verapamil (10 μM, for 5 min) increases endogenous DA release (70%) and endogenous 5-HT release (40%) independently on the presence of external Ca²⁺. The total amount of these monoamines (released plus retained by the preparation) and their metabolites (DOPAC and 5-HIAA) was similar in control and verapamil-treated synaptosomes. Verapamil displaces [³H]-spiroperidol specific binding (K_i of 2.4 × 10^?6M) and [³H]-SCH-23390 specific binding (K_i of 9 × 10^?6M) from striatal synaptosomal membranes, and [³H]-5-HT specific binding (K_i of 3 × 10^?5M) from hippocampal synaptosomal membranes. It is concluded that in addition to the Ca²⁺ antagonistic properties of verapamil on the Ca²⁺-dependent, depolarization-induced release of some neurotransmitters [gamma aminobutyric acid (GABA and NE)], another mechanism probably mediated by presynaptic receptors underlies the effects of verapamil on DA and 5-HT release from discrete brain regions. © 1995 Wiley-Liss, Inc.     

Regional differences in the enhancement by GABA of [H]zolpidem binding to ω1 sites in rat brain membranes and sections

The potential heterogeneity in the allosteric coupling between GABA and ω₁ binding sites within the native GABA_A receptor complex has been evaluated in the rat by measuring the enhancement by GABA of [³H]zolpidem binding to ω₁ site in membranes from three rat brain structures (neocortex, cerebellum and hippocampus) and brain sections. The maximal stimulatory effect of GABA was significantly higher (265 ± 47%) in cortical membranes than in cerebellar (165 ± 48%) or in hippocampal (118 ± 17%) membranes. These differences are not due either to the presence of different amounts of residual GABA in the membrane preparations or to the labeling, in presence of GABA, of binding sites other than ω₁ since: (1) the pharmacological properties of the [³H]zolpidem binding sites were similar in the three regions; (2) the degree of allosteric enhancement was unrelated to the relative proportion of ω₁ sites in each structure; and (3) GABA did not increase the B_max for [³H]zolpidem. Regional differences in the effect of 100 μM GABA on [³H]zolpidem binding were also observed by quantitative autoradiography. Regions where the strongest (3–4-fold) effects of GABA in [³H]zolpidem binding were observed included the substantia nigra, lateral geniculate body, olfactory tubercule and red nucleus. A large increase in [³H]zolpidem binding was also demonstrated in the cingulate and frontoparietal cortices with higher effects in deep (4.2-fold) rather than in superficial layers (3.3-fold). Heterogeneous subregional increases in [³H]zolpidem binding in the presence of GABA were quantified within the cerebellum, hippocampus and superior colliculus. In the cerebellum the effect of this neurotransmitter was larger in the molecular (3.8-fold) than in the granular (2.2-fold) layer. In the hippocampus the effect of GABA was also heterogeneous with larger increases in CA1 and CA2 fields (3.5-fold) than in CA3 field (2.2-fold) and dentate gyrus (2.5-fold). Finally in the deep layers of the superior colliculus GABA stimulation of [³H]zolpidem binding was greater than the superficial layer. In the other structures examined the GABA-induced increase in [³H]zolpidem binding was less than 3-fold. The smallest stimulations were quantified in the entorhinal cortex (2.1-fold), amygdala (2.4-fold) and nucleus accumbens (1.7-fold). These results suggest that [³H]zolpidem sites are associated to, at least, two GABA_A receptor subtypes that can be differentiated by their allosteric interaction between GABA and [³H]zolpidem sites.     

Quantitative light microscopic localization of [H]naltrindole binding sites in the rat brain

The binding of radiolabeled naltrindole ([³H]NTI), a selective δ-opioid antagonist, was characterized using receptor autoradiography. Receptor binding properties were established in brain paste slices which demonstrated one site receptor occupancy with an apparent K_d of 0.25 ± 0.08nM (B_max of 597.5 fmol/mg protein). Autoradiographic localization of [³H]NTI binding sites in the rat brain revealed high densities of these sites in the cortex (layers 1–3 and 6), caudate putamen, accumbens, claustrum, and internal plexiform layer of the olfactory bulb. Moderate to low levels of specific binding were observed in the hippocampus, thalamus, and substantia gelatinosa of the spinal cord.     

[H]TCP: a new tool with high affinity for the PCP receptor in rat brain

PCP binding sites have previously been demonstrated in the central nervous system with [³H]PCP. We now describe the binding properties to rat brain membranes of [³H]TCP, a PCP derivative. It is very advantageous to use [³H]TCP instead of [³H]PCP for the 3 following reasons: (i) it has a better affinity (K_d = 7.4nM) for PCP binding sites than PCP itself; (ii) it dissociates slowly from its binding sites (t1/2= 20min); (iii) the non-specific binding component obtained with [³H]TCP is much lower than that found with [³H]PCP.     

Diphenyl diselenide and diphenyl ditelluride affect the rat glutamatergic system in vitro and in vivo

The aim of this study was to investigate the possible involvement of the glutamatergic system in the toxicity of organochalcogens, since this is an important neurotransmitter system for signal transduction and neural function. The results indicated that 100 μM diphenyl diselenide (PhSe)₂ and diphenyl ditelluride (PhTe)₂ inhibit by 50 and 70% (P<0.05), respectively, [³H]glutamate binding in vitro. Acute administration of 25 μmol/kg (PhSe)₂ or 3 μmol/kg (PhTe)₂ caused a significant reduction in [³H]glutamate (30%, P<0.05) or [³H]MK-801 binding (30%, P<0.05) to rat synaptic membranes. These results suggest that (PhSe)₂ and (PhTe)₂ affect, in a rather complex way, the glutamatergic system after acute in vivo exposure in rats. In vitro, total [³H]GMP-PNP binding was inhibited about 40% at 100 μM (PhSe)₂ and (PhTe)₂. Acute exposure in vivo to (PhSe)₂ decreased the stable [³H]GMP-PNP binding to 25% and (PhTe)₂ to 68% of the control value (P<0.05, for both compounds). Simultaneously, the unstable binding of [³H]GMP-PNP was decreased about 30 and 50% (P<0.05, for both compounds) after exposure to (PhSe)₂ and (PhTe)₂, respectively. GMP-PNP stimulated adenylate cyclase (AC) activity significantly in control animals. (PhSe)₂- and (PhTe)₂-treated animals increased the basal activity of this enzyme, but GMP-PNP stimulation was totally abolished. These results suggest that the toxic effects of organochalcogens could result from action at different levels of neural signal transduction pathways, possibly involving other neurotransmitters besides the glutamatergic system.     

Characteristics of [3H]inositol(1,3,4,5)tetrakisphosphate recognition sites in human cerebellar membranes

Summary The characteristics of specific [³H]Ins(1,3,4,5)P₄ binding sites in human cerebellar membranes were determined in this study. Binding rapidly reached steady state, possessed a pH optimum of 4.5–5.1 and was greater in the absence of BSA than in its presence. Heparin inhibited both specific and pseudospecific binding of the ligand, whereas only the specific binding was inhibited by non-radioactive Ins(1,3,4,5)P₄. Calcium at a concentration of 1 mM, reduced binding by 27%. Competition studies with other inositol phosphates showed specificity for Ins(1,3,4,5)P₄ with a pI₅₀ value of 6.87 and a Hill coefficient of 0.27, indicating two sites. Ins(1,2,5,6)P₄, Ins(1,3,4,5,6)P₅, Ins(3,4,5,6)P₄ displaced binding with IC₅₀ values ranging from 0.1–1 M, Ins(1,2,5,6)P₄ and Ins(1,3,4,5,6)P₅ being the most potent. Ins(1,4)P₂ and Ins(1,5,6)P₃ had lesser effects on binding. Rosenthal analysis of [³H]Ins(1,3,4,5)P₄ saturation binding data at low ligand concentrations gave a K_D of 27 nM and a B_max of 33pmol/mg protein. It is concluded that [³H]Ins(1,3,4,5)P₄ binding sites in human cerebellar membranes have similar characteristics to these sites reported in the literature in animal cerebellar tissue, but are in greater abundance.     

Coupling between hypothalamic α2-adrenoceptors and [H]mazindol binding site in response to several hyperglycaemic stimuli in mice

The hypothalamic response to circulating glucose and insulin levels was studied in the mouse by differentially attenuating glucose-homeostasis. The administration of glucose, 2-deoxyglucose or the α₂-adrenoceptor agonist UK 14.304 was accompanied by a persistent hyperglycaemia; however, an increase in insulin levels was obtained with glucose and a decrease with the other two manipulations. Both α₂-adrenoceptors (labeled with [³H]idazoxan) and the anorectic recognition site (labeled with [³H]mazindol) were upregulated by the three treatments. A good correlation was obtained between circulating glucose levels and either hypothalamic [³H]mazindol binding (r = 0.70, P < 0.001) or [³H]idazoxan binding (r = 0.63, P < 0.001), as well as between the two binding sites (r = 0.88, P < 0.001). No correlation was obtained between circulating insulin levels and these binding sites (r = 0.18, r = 0.26, P =n.s. for [³H]mazindol and [³H]idazoxan binding, respectively). It is suggested the α₂-adrenoceptors and the anorectic binding sites are associated in their response to glucose as part of a hypothalamic center involved in the regulation of feeding mechanisms.