Presynaptic Cholinergic Mechanisms in the Rat Cerebellum: Evidence for Nicotinic, but Not Muscarinic Autoreceptors

The present study shows that N-[3H]methylcarbamylcholine ([3H]MCC) binds to a single population of high-affinity/low-density (KD = 5.0 nM; Bmax = 8.2 fmol/mg of protein) nicotinic binding sites in the rat cerebellum. Also, there exists a single class of high-affinity binding sites (KD = 4.8 nM; Bmax = 24.2 fmol/mg of protein) in the cerebellum for the M1 specific muscarinic ligand [3H]pirenzepine. In contrast, the M2 ligand, [3H]AF-DX 116, appears to bind to two classes of binding sites, i.e., a high-affinity (KD = 3 nM)/low-capacity (Bmax = 11.7 fmol/mg of protein) class, and a second class of lower affinity (KD = 28.4 nM) and higher capacity (Bmax = 36.3 fmol/mg of protein) sites. The putative M3 selective ligand [3H]4-diphenylacetoxy-N-methylpiperidine also binds to two distinct classes of binding sites in cerebellar homogenates, one of high affinity (KD = 0.5 nM)/low capacity (Bmax = 19.5 fmol/mg of protein) and one of low affinity (KD = 57.5 nM)/high capacity (Bmax = 140.6 fmol/mg of protein). In experiments which tested the effects of cholinergic drugs on acetylcholine release from cerebellar brain slices, the nicotinic agonist MCC enhanced spontaneous acetylcholine release in a concentration-dependent manner, and the maximal increase in acetylcholine release (59.0-68.0%) occurred at 10(-7) M. The effect of MCC to increase acetylcholine release was Ca2+-dependent and tetrodotoxin-insensitive, suggesting an action on cholinergic terminals. Also, the MCC-induced increase in acetylcholine release was effectively antagonized by dihydro-beta-erythroidine, d-tubocurarine, and kappa-bungarotoxin, but was insensitive to either atropine or alpha-bungarotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic ethanol consumption reduces [3H]inositol (1,4,5) trisphosphate specific binding in mouse cerebellar membrane fragments

[3H]In(1,4,5)P3 specific binding was determined in membrane fragments from various brain regions of adult male C57/BL mice. [3H]In(1,4,5)P3 specific binding was at least 10 times higher in cerebellum than in either striatum, cerebral cortex, hippocampus, or midbrain. Ethanol added in vitro up to 500 mM to cerebellar membrane fragments of control mice had no significant effect on [3H]In(1,4,5)P3 specific binding. In contrast, the maximal number of binding sites (Bmax) for [3H]In(1,4,5)P3 was significantly decreased in cerebella from mice which had been rendered tolerant-dependent to ethanol. KD values for these mice were unchanged when compared to control values.     

Increased binding of [3H]GABA to striatal membranes following ischemia

Sodium-independent binding of [3H]gamma-aminobutyric acid ([3H]GABA) to membranes prepared from ischemic-damaged rat striatum was studied by kinetic and time-course analysis. Three days after 40 min of ischemia, [3H]GABA binding increased fourfold over control values. Scatchard analysis of the binding showed that ischemia significantly increased the affinity (KD) and the total number of binding sites (Bmax) for the high-affinity GABA receptor. These results support the conclusion that transient forebrain ischemia damages striatal GABAergic neurons.     

Bicuculline Up-Regulation of GABAA Receptors in Rat Brain

Effects of acute and subacute administration of bicuculline on [3H]muscimol, [3H]flunitrazepam, and t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to various brain regions were studied in Sprague-Dawley rats. Acute administration of bicuculline affected neither the KD nor the Bmax of the three receptor sites. In rats treated subacutely with bicuculline (2 mg/kg, i.p., daily for 10 days), [3H]muscimol binding was increased in the frontal cortex, cerebellum, striatum, and substantia nigra. Scatchard analysis revealed that subacute treatment of rats with bicuculline resulted in a significantly lower KD of high-affinity sites in the striatum and in a significantly lower KD of high- and low-affinity sites in the frontal cortex. In the cerebellum, two binding sites were apparent in controls and acutely treated animals; however, only the high-affinity site was defined in subacutely treated animals, with an increase in the Bmax value. Triton X-100 treatment of frontal cortical membranes eliminated the difference in [3H]muscimol binding between control and subacute bicuculline treatments. On the other hand, [3H]muscimol binding was significantly increased in the cerebellum from bicuculline-treated animals even after Triton X-100 treatment. The apparent Ki of bicuculline for the GABAA receptor was also decreased in the frontal cortex and the striatum following the treatment. However, subacute administration of bicuculline affected neither the KD nor the Bmax of [3H]flunitrazepam and [35S]TBPS binding in the frontal cortex and the cerebellum. These results suggest that GABAA receptors are up-regulated after subacute administration of bicuculline, with no change in benzodiazepine and picrotoxin binding sites.     

Determination of the Equilibrium Dissociation Constants and Number of Glycine Binding Sites in Several Areas of the Rat Central Nervous System, Using a Sodium-Independent System

Parameters affecting the binding of [3H]glycine to membrane fractions isolated from the cerebral cortex, midbrain, cerebellum, medulla oblongata, and spinal cord of the rat were investigated in a Na+-free medium. A [3H]glycine binding assay was established in which the binding was specific, saturable, pH-sensitive, and reversible. Conditions were chosen in an effort to minimize binding to glycine uptake sites. From data on specific [3H]glycine binding Scatchard plots were prepared and the KD and Bmax values were calculated. Two glycine binding sites (high and low affinity) were identified only in the medulla (KD: 44, 211 nM; Bmax: 361, 1076 fmol/mg protein) and spinal cord (KD: 19, 104 nM; Bmax: 105, 486 fmol/mg protein). The ranges of the KD and Bmax values for the other three areas studied were 59 to 144 nM and 882 to 3401 fmol/mg protein, respectively. When the glycine content of each area, expressed as fmol/neuron, was plotted against the respective KD (high affinity), a negative correlation was found (r = --0.90; p less than 0.05). A similar negative correlation was found between the glycine content and Bmax (r = --0.88; p less than 0.05). Hill plots indicated a slope of essentially 1.0 for all areas. GABA, taurine, strychnine, diazepam, bicuculline, and imipramine had little or no effect on [3H]glycine binding.     

Solubilisation of the γ-Aminobutyric Acid/Benzodiazepine Receptor from Rat Cerebellum: Optimal Preservation of the Modulatory Responses by Natural Brain Lipids

We have solubilised the gamma-aminobutyric acid/benzodiazepine (GABA/BDZ) receptor from rat cerebellum using 3-[(3-cholamidopropyl)dimethylammonio] 1-propane sulphonate (CHAPS) in the presence of a natural brain lipid extract and cholesteryl hemisuccinate. The soluble material shows a homogeneous [3H]flunitrazepam ([3H]FNZ) binding population with an equilibrium dissociation constant (KD) of 4.4 +/- 0.2 nM compared to a KD of 2.3 +/- 0.2 nM in cerebellar synaptosomal membranes. The receptor complex in solution retains the characteristic facilitation of [3H]flunitrazepam binding induced by GABA, the pyrazolopyridine cartazolate, and the depressant barbiturate pentobarbital to the same extent as that observed in synaptosomal membranes. Furthermore, these responses are retained both quantitatively and qualitatively when this preparation is stored for 48 h at 4 degrees C. This is contrary to the results obtained with a CHAPS-soluble preparation including asolectin in which these responses are anomalous and extremely labile on storage.     

Modulation of type 1, 2 and 3 inositol 1,4,5-trisphosphate receptors by cyclic ADP-ribose and thimerosal.

The binding of inositol 1,4,5-trisphosphate (IP3) to the IP3 receptor (IP3R) is modulated by various compounds. Until now, limited progress has been made concerning the isoform-specific effects of these modulators. In this study, we examined how [3H]IP3 binding to the three IP3R isoforms is modulated by cyclic ADP-ribose (cADPR) and by the SH-reagent thimerosal. We used rabbit cerebellum, RBL-2H3 rat mucosal mast cells and 16HBE14o- human bronchial epithelial cells as model systems for IP3R-1, -2 and -3 respectively. [3H]IP3 binding was first characterized at various pH values. We showed that [3H]IP3 binding to RBL-2H3 microsomes was more enhanced by increasing the pH from 7.4 to 8.3 than that to rabbit cerebellar microsomes. In contrast, [3H]IP3 binding to 16HBE14o- microsomes was not stimulated at alkaline pH. At pH 7.4, cADPR (50 microM) increased [3H]IP3 binding to rabbit cerebellar microsomes, RBL-2H3 and 16HBE14o- microsomes 1.5-fold, 1.3-fold and 1.8-fold respectively. The effect of cADPR on IP3 binding was abolished at pH 8.3. Scatchard analysis indicated that cADPR induced in cerebellum a decrease in IP3 affinity (KD increases from 150 nM to 252 nM) of the IP3R and a parallel increase in Bmax (from 4.8 pmol/mg to 11.1 pmol/mg). Thimerosal dose-dependently increased [3H]IP3 binding to rabbit cerebellar microsomes. The stimulatory effects of cADPR and thimerosal were not additive. Binding to cerebellar microsomes returned to control level in the presence of 500 microM thimerosal. In contrast, thimerosal (up to 500 microM) had no stimulatory effect and only a very slight, if any, inhibitory effect on [3H]IP3 binding to RBL-2H3 and 16HBE14o- microsomes respectively. These results indicate that IP3 binding to the IP3R isoforms can be differentially modulated by cADPR and thimerosal.     

Changes of Ganglioside Pattern During Cerebellar Development of Normal and Staggerer Mice

Saturable and specific binding sites for 5-[3H]hydroxytryptamine (5-HT, serotonin) characterized by a KD of 3.5-4.5 nM were detected in the chick embryo brain and were shown to develop linearly as a function of age, weight, and protein content. Saturation and displacement studies using unlabeled 5-HT as the displacing ligand suggested a single population of binding sites. However, displacement studies using 5-methoxytryptamine, lysergic acid diethylamide (LSD), 2-bromo-lysergic acid diethylamide (BOL), methysergide, and spiperone as competing ligands suggested the existence of subclasses of [3H]5-HT binding sites because the Hill coefficients were less than unity. When compared with the reported [3H]5-HT binding sites (5-HT1) in the rat forebrain, the IC50 values of the competing ligands were similar. However, the Hill coefficients for LSD and methysergide were less than unity which suggested that the [3H]5-HT binding sites in the chick embryo brain may be more similar to those found in rat spinal cord than rat forebrain. To study [3H]5-HT binding site regulation and development, various serotonergic compounds were injected into the chorioallantoic fluid of the eggs at different times during embryonic development. Multiple pretreatments with d,l-5-hydroxytryptophan, 5-HT, or BOL were found to have no significant effects on either the affinity (KD) or number (Bmax) of specific [3H]5-HT binding sites. Multiple pretreatments with p-chlorophenylalanine were found to increase the Bmax of specific [3H]5-HT binding by 23% (p less than 0.01) whereas multiple pretreatments with LSD were found to decrease the Bmax of specific binding by 45% (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

(+)-[3H]MK-801 Binding Sites in Postmortem Human Brain

The pharmacological specificity and the regional distribution of the N-methyl-D-aspartate receptor-associated 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) binding sites in human postmortem brain tissue were determined by binding studies using (+)-[3H]MK-801. Scatchard analysis revealed a high-affinity (KD = 0.9 +/- 0.2 nM, Bmax = 499 +/- 33 fmol/mg of protein) and a low-affinity (KD = 3.6 +/- 0.9 nM, Bmax = 194 +/- 44 fmol/mg of protein) binding site. The high-affinity site showed a different regional distribution of receptor density (cortex greater than hippocampus greater than striatum) compared to the low-affinity binding site (cerebellum greater than brainstem). The rank order pharmacological specificity and stereoselectivity of the high-(cortex) and low-(cerebellar) affinity binding sites were identical. However, all compounds tested showed greater potency at the high-affinity site in cortex. The results indicate that (+)-[3H]MK-801 binding in human postmortem brain tissue shows pharmacological and regional specificity.     

Diazepam Sensitivity of the Binding of an Imidazobenzodiazepine, [3H]Ro 15-4513, in Cerebellar Membranes from Two Rat Lines Developed for High and Low Alcohol Sensitivity

Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a] [1,4]benzodiazepine-3-carboxylate), a partial inverse agonist of brain benzodiazepine receptors, has been shown to antagonize some actions of ethanol. In addition to conventional benzodiazepine binding sites, Ro 15-4513 binds to a specific cerebellar protein, the binding of which has been shown to be insensitive to diazepam. The binding of [3H]Ro 15-4513 was studied in washed membranes of the cerebellum, hippocampus, and cerebral cortex of two rat lines developed for differences in their sensitivity to ethanol-induced motor impairment. Only minor differences were found in the estimated parameters (KD and Bmax) for the total specific binding between the rat lines. The main difference between the rat lines was, however, observed in the characteristics of the cerebellar binding, all of which was displaced by diazepam in most of the alcohol-sensitive [alcohol-nontolerant (ANT)] rats, in contrast to only approximately 75% displacement in most of the alcohol-insensitive [alcohol-tolerant (AT)] ones. The following cerebellar results were obtained with the major subgroups of both lines, i.e., with the AT rats chosen for the presence of the diazepam-insensitive binding and with the ANT rats chosen for its absence. The KD for the total specific [3H]Ro 15-4513 binding in the ANT animals was about half of that in the AT animals. No line difference was found in the Bmax of the binding in these rats. Photolabeling with [3H]Ro 15-4513 showed that the diazepam-insensitive binding was in a protein with a molecular weight of 55,000.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoradiographic Characterization and Localization of Quisqualate Binding Sites in Rat Brain Using the Antagonist [3H]6-Cyano-7-Nitroquinoxaline-2,3-Dione: Comparison with (R,S)-[3H]α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites

Using quantitative autoradiography, we have investigated the binding sites for the potent competitive non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitro-quinoxaline-2,3-dione ([3H]-CNQX) in rat brain sections. [3H]CNQX binding was regionally distributed, with the highest levels of binding present in hippocampus in the stratum radiatum of CA1, stratum lucidum of CA3, and molecular layer of dentate gyrus. Scatchard analysis of [3H]CNQX binding in the cerebellar molecular layer revealed an apparent single binding site with a KD = 67 +/- 9.0 nM and Bmax = 3.56 +/- 0.34 pmol/mg protein. In displacement studies, quisqualate, L-glutamate, and kainate also appeared to bind to a single class of sites. However, (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) displacement of [3H]CNQX binding revealed two binding sites in the cerebellar molecular layer. Binding of [3H]AMPA to quisqualate receptors in the presence of potassium thiocyanate produced curvilinear Scatchard plots. The curves could be resolved into two binding sites with KD1 = 9.0 +/- 3.5 nM, Bmax = 0.15 +/- 0.05 pmol/mg protein, KD2 = 278 +/- 50 nM, and Bmax = 1.54 +/- 0.20 pmol/mg protein. The heterogeneous anatomical distribution of [3H]CNQX binding sites correlated to the binding of L-[3H]glutamate to quisqualate receptors and to sites labeled with [3H]AMPA. These results suggest that the non-NMDA glutamate receptor antagonist [3H]CNQX binds with equal affinity to two states of quisqualate receptors which have different affinities for the agonist [3H]AMPA.     

Muscarinic antagonist binding site heterogeneity as evidenced by autoradiography after direct labeling with [3H]-QNB and [3H]-pirenzepine

Saturable, high affinity binding of tritiated pirenzepine [( 3H]-PZ) was obtained in slide mounted tissue sections prior to performing autoradiographic localization of these binding sites. The binding in tissue sections of rostral rat forebrain gave a KD of 18nM and a Bmax of 51 fmoles/mg tissue. These binding characteristics are similar to those previously obtained in homogenate membrane preparations and indicate the binding is taking place in a similar manner. The distribution of the binding sites labeled with [3H]-PZ represented a subpopulation of those which could be labeled with tritiated quinuclidinyl benzilate [( 3H]-QNB). Thus, [3H]-PZ and [3H]-QNB both label regions of the cerebral cortex, hippocampus, striatum and dorsal horn of the spinal cord, while sites in the cerebellum, nucleus tractus solitarius, facial nucleus and ventral horn of the spinal cord are labeled with [3H]-QNB and not by [3H]-PZ. These observations indicate separate regions of the brain where antagonists bind to subtypes of muscarinic receptors.     

Development of Benzodiazepine and Picrotoxin (t-Butylbicyclophosphorothionate) Binding Sites in Rat Cerebellar Granule Cells in Culture

The specific bindings of [3H]flunitrazepam [( 3H]FLU), [3H]CGS 8216, and t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to sites on rat cerebellar granule cells all increase from 4 to 15 days in culture, although their time courses differ. Specific [3H]FLU binding doubles, [3H]CGS 8216 binding triples, and [35S]TBPS binding increases about fourfold from 4 to 15 days in culture. Displacement studies, using the type I-selective ligand CL 218,872, indicate that at 4 days the [3H]FLU binding sites are almost entirely "type II," judging from an IC50 value near 300 nM and a pseudo-Hill number near 1. By 10 days, approximately equal numbers of type I and type II binding sites are present in the cultured cells, and this ratio remains constant thereafter (12 and 15 days). At days 10-15, both the IC50 value for CL 218,872 (near 100 nM) and the pseudo-Hill number (near 0.7) remain constant and are significantly different from the values at culture day 4. The development of specific [35S]TBPS binding parallels that of [3H]CGS 8216 binding more closely than the development of [3H]FLU binding. The [3H]CGS 8216/[3H]FLU ratio increased by a factor of 1.6 from day 4 to day 15 (p less than 0.001). Taken together, our data suggest the existence of several gamma-aminobutyric acidA (GABAA) receptor subunits, the relative proportions of which change during development. The presence of the GABA-mimetic 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridine-3-ol (THIP) in the culture medium had no apparent effect on any of the binding sites studied, although THIP was shown previously to induce low-affinity GABA binding sites.     

Development of radioimmunoassayable beta-endorphin and methionine-enkephalin binding sites in regions of rat brain

The development profiles of [3H]methionine-enkephalin ([3H]Met-enk) binding sites and radioimmunoassayable (RIA) beta-endorphin in regions of rat brain were determined. The amount [3H]Met-enk bound reached its maximum in the 1st week after birth in the cerebellum, in the brainstem at the 2nd week, and in the whole forebrain at the 3rd week, that of RIA beta-endorphin reached its highest level at day 2 postpartum (p.p.) in the cerebellum. at days 7-15 p.p. in the whole forebrain, and at days 17-21 p.p. in the brainstem. These findings suggest that both the development of RIA beta-endorphin and [3H]met-enk sites in rat brain follow a caudal to rostral sequence. Also, the close interrelationship between the elevation and decline in the amount of [3H]Met-enk bound and RIA beta-endorphin levels in each brain region suggests that these two components are important entities of the central nervous system.     

Characterization of the Binding of [3H]SR 95531, a GABAA Antagonist, to Rat Brain Membranes

A synthetic derivative of gamma-aminobutyric acid (GABA), SR 95531 [2-(3'-carboxy-2'-propyl)-3-amino-6-p-methoxyphenylpyridazinium bromide], has recently been reported, on the basis of biochemical and in vivo microiontophoretic studies, to be a potent, selective, competitive, and reversible GABAA antagonist. In the present study, the binding of [3H]SR 95531 to washed, frozen, and thawed rat brain membranes was characterized. Specific binding was linear with tissue concentrations, had a pH optimum at neutrality, and was maximal at 4 degrees C after 30 min of incubation. Pretreatment of the membranes with Triton X-100 resulted in a 50% decrease of specific binding. Addition of iodide, thiocyanate, or nitrate to the incubation mixture decreased the affinity of [3H]SR 95531 for its binding site; Na+ had no effect. Subcellular fractionation showed that 74% of the P2 binding was in synaptosomes; 31% of the total homogenate binding was in P2 and 50% in P3. The binding of [3H]SR 95531 was saturable; Scatchard analysis of the saturation isotherm revealed two apparent populations of binding sites (KD of 6.34 nM and Bmax of 0.19 pmol/mg of protein; KD of 32 nM and Bmax of 0.81 pmol/mg of protein). The binding of [3H]SR 95531 was reversible, and association and dissociation kinetics confirmed the existence of two binding sites. Only GABAA ligands were effective displacers of [3H]SR 95531. GABAA antagonists were relatively more potent in displacing [3H]SR 95531 than [3H]GABA; the inverse was true for GABAA agonists. There were marked regional differences in the distribution of binding sites: hippocampus = cerebral cortex greater than thalamus = olfactory bulb = hypothalamus = amygdala = striatum greater than pons-medulla and cerebellum. The surprisingly low density of binding sites in the cerebellum was owing to a marked reduction of Bmax values at both the high- and the low-affinity binding sites. In conclusion, the present results demonstrate specific, high-affinity, saturable, and reversible binding of [3H]SR 95531 to rat brain membranes and strongly suggest that this radioligand labels the GABAA receptor site in its antagonist conformation.     

Binding characteristics of [3H]flunitrazepam in pentobarbital-withdrawal rats

The effects of chronic pentobarbital (PB) treatment on the binding characteristics of [³H]flunitrazepam (FLU) in rat brain were examined. Saline or sodium PB (500 g/10l/hr) was infused into the lateral cerebral ventricles of rats for 6 days using osmotic pumps. Immediately before withdrawal, there were no significant differences in [³H]FLU binding constants (K_D and B_max) between saline and PB groups. However, 24 hr withdrawal caused an increase in B_max with no changes in K_D. The enhancement of [³H]FLU binding by in vitro addition of chloride ions and PB was not affected after the PB infusion. The PB enhancement of [³H]FLU binding was inhibited by the convulsant, picrotoxicin. PB withdrawal did not cause significant differences in the binding constants of [³H]Ro 15-1788, a benzodiazepine (BZ) antagonist, between the saline and PB groups. Pretreatment of membranes with 0.02 mM of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic detergent, caused decreases in both K_D and B_max in FLU binding in PB-withdrawal membrane, but not in the saline-treated membrane. The enhancement of [³H]FLU binding by chloride ions and PB was not affected by the CHAPS treatment. These results suggest that the change in BZ receptors induced by PB withdrawal is functionally linked to the GABA-BZ-barbiturate receptor complex and that PB withdrawal induces some conformational changes in BZ receptors.     

NMDA Receptors in Rat Cerebellum and Forebrain: Subtle Differences in Pharmacology and Modulation

Abstract: Binding of [³H]glutamate, [³H]glycine, and the glutamate antagonist [³H]CGS-19755 to NMDA-type glutamate receptors was examined in homogenates of rat forebrain and cerebellum. Most glutamate agonists had a higher affinity at the [³H]glutamate binding site of cerebellar NMDA receptors as compared with forebrain, whereas all the glutamate antagonists examined showed the reverse relationship. The [³H]glycine binding site of forebrain and cerebellar NMDA receptors showed a similar pharmacology in both brain regions. In the cerebellum, however, [³H]glycine bound to a second site with a 10-fold lower affinity and with a pharmacology that resembled that of the glycine/strychnine chloride channel. [³H]Glutamate binding was not affected by glycine agonists or antagonists, nor was [³H]glycine binding affected by glutamate agonists in either forebrain or cerebellum. Both CGS-19755 and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, glutamate antagonists, reduced [³H]glycine binding in cerebellum, whereas only CGS-19755 was effective in forebrain. Glycine agonists and antagonists modulated [³H]CGS-19755 binding in forebrain and cerebellum to different extents in the two brain regions. From these studies we conclude that the cerebellar NMDA receptor has a different pattern of modulation at glutamate and glycine sites and that glycine may play a more important role in the control of NMDA function in the cerebellum as compared with forebrain.     

Ontogeny of Calcium Antagonist Binding Sites in Chick Brain and Heart

The ontogeny of chick brain and heart ventricle calcium antagonist binding sites was determined, using [3H]nitrendipine ([3H]NDP), as the ligand. The binding of [3H]NDP to adult heart and brain was kinetically very similar, with the former displaying a KD of 0.28 +/- 0.02 nM and a Bmax of 138 +/- 17 fmol/mg protein, and the latter a KD of 0.30 +/- 0.02 nM and a Bmax of 160 +/- 12 fmol/mg protein. The binding site in both brain and heart was highly specific for dihydropyridine calcium antagonists, such as nifedipine, nimodipine, and nisoldipine, since these drugs were several orders of magnitude more potent as inhibitors of [3H]NDP binding than verapamil, methoxyverapamil, or diltiazem. The developmental appearance of [3H]NDP binding sites in brain was rather gradual, with adult levels being attained just prior to birth. This was in contrast to the profile in heart ventricle which showed essentially adult levels at seven days gestation. The acquisition of [3H]NDP binding sites in chick brain roughly paralleled the onset of neuronal maturation and functional activity. In both chick brain and heart, verapamil and methoxyverapamil were weak inhibitors of [3H]NDP binding. However, the inhibition of binding in both tissues was markedly biphasic, with only 50% of the binding sites being susceptible to inhibition by each agent, suggesting that multiple calcium antagonist binding sites may exist in both tissues.     

n-[3H]Butyl-β-Carboline-3-Carboxylate, a Putative Endogenous Ligand, Binds Preferentially to Subtype 1 of Central Benzodiazepine Receptors

Synthetic n-butyl beta-carboline-3-carboxylate, an endogenous central benzodiazepine receptor inhibitor found in brain, was tritium-labeled from the butenyl ester. Binding of this [3H]beta-carboline was concentrated particularly in the synaptosomal membrane fraction of the cerebral cortex; this fraction showed a single type of high-affinity site (KD = 2.7 +/- 0.1 nM) with a Bmax of 1.16 +/- 0.08 pmol/mg of protein. The number of sites labeled was about half of that obtained with [3H]flunitrazepam binding (Bmax = 2.36 +/- 0.06 pmol/mg of protein). On the other hand, in the cerebellum, both ligands bound to practically the same number of sites. When [3H]flunitrazepam binding was done in the presence of 10(-11)-10(-5) M butyl beta-carboline, the differences between the two brain regions were more apparent. In cerebellar membranes the data fitted a straight line in the Eadie-Hofstee plot; this finding and a Hill number near unity suggest a single type of binding site. In the cortical membranes the data of binding fitted a concave curve, and the Hill number was 0.6. These are characteristics of two types of binding sites with different affinities (KD1 = 0.6-1.5 nM and KD2 = 12-18 nM). The differentiation of a high- and low-affinity site in the cerebral cortex was corroborated by experiments in which [3H]butyl beta-carboline binding was displaced by the triazolopyridazine CL 218,872. These results demonstrate that in the cerebral cortex there are two subtypes of sites (1 and 2) of central benzodiazepine receptors and that CL 218,872 binds preferentially to subtype 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Late ontogenetic development of adenosine A1 receptor coupling to associated G-proteins in guinea pig cerebellum but not forebrain

The ontogenetic profile of [³H]forskolin and [³H]cyclohexyladenosine ([³H]CHA) binding sites in guinea pig forebrain and cerebellum was investigated. G-protein interactions of these binding sites were also examined by analyzing 5-guanylylimidodiphosphate (Gpp(NH)p) interactions with [³H]CHA and [³H]forskolin binding. In forebrain, similar binding characteristics of [³H]CHA and [³H]forskolin binding are observed between the developmental stages E₃₆ (the earliest time point studied) through to adult (P₂₈, the latest time point studied), although transient increased binding of both ligands is observed just prior to birth. Scatchard analysis of binding isotherms reveal that this transient rise just prior to birth is due to an increase in the number of binding sites (B_max) with little or no change in receptor affinity (K_d) In contrast, in cerebellum both [³H]CHA and [³H]forskolin binding remains at a relatively low level until just prior to birth when a dramatic increase of binding of both ligands is observed which continues to increase up to P₂₈. Scatchard analysis of binding isotherms reveal that such changes in binding of both ligands are largely due to increases in B_max and not K_d, although Scatchard analysis of [³H]CHA binding to cerebellar E₅₁ membranes reveals an absence of higher affinity [³H]CHA binding sites. Gpp(NH)p did not affect [³H]forskolin binding. Gpp (NH)_p displacement profiles of [³H]CHA binding reveal a maximum (adult) inhibition of [³H]CHA binding (approximately 80% displacement) at all time points (E₃₆ through P₂₈) in forebrain membranes, but not in cerebellar membranes. In cerebellum, displacement of [³H]CHA binding by Gpp(NH)p is much greater after birth than before birth. These results suggest that in cerebellum, but not in forebrain, postnatal coupling of adenosine A₁ receptors to associated G-proteins is much more extensive than in the pre-natal period. The extent of this inferred coupling may also coincide with the ontogenetic appearance or presence of [³H]forskolin binding sites.Abbreviations [³H]CHA N⁶-Cyclohexyl-[³H]-Adenosine - Gpp(NH)p–5 Guanylylimidodiphosphate