Hormone effects on muscarinic cholinergic binding in bovine and rat sympathetic superior cervical ganglia

Muscarinic receptors were assessed by [3H]-quinuclidinyl benzilate (QNB) binding in 900 xg supernatants of bovine superior cervical ganglia (SCG). At 30 degrees C half maximal binding was reached within 3 min and equilibrium within 30 min. Scatchard analysis revealed a single population of binding sites with dissociation constant (Kd) = 0.15 +/- 0.01 nM and site concentration (Bmax) = 101 +/- 4 fmoles/mg prot. Binding was specific for muscarinic drugs. Incubation of bovine SCG with different hormones (10(-7)M) indicated that LH, TRH and testosterone depressed significantly Bmax, and that prolactin decreased both Kd and Bmax of [3H] -QNB binding. Several other hormones tested (TSH, GH, FSH, LHRH, angiotensin II, bradykinin, melatonin, estradiol, thyroxine and triiodothyronine) did not affect QNB binding. Hormone effects were not due to a direct interference with radioligand binding to membrane. The injection of LH to orchidectomized rats depressed Bmax of SCG QNB binding without changing the Kd. These results suggest that muscarinic cholinergic neurotransmission in SCG may be affected by hormones.     

Stimulation of δ-Opioid Receptors Reduces the In Vivo Binding of the Cholecystokinin (CCK)-B-Selective Agonist [3H]pBC 264: Evidence for a Physiological Regulation of CCKergic Systems by Endogenous Enkephalins

Cholecystokinin (CCK) and enkephalins appear to be colocalized in several brain structures, and a physiological interaction between these peptides has been suggested by a large number of pharmacological studies. In this work we have shown, by in vivo binding experiments, that the endogenous enkephalins, protected from degrading enzymes by mixed inhibitors such as kelatorphan and N-[(R,S)-2-benzyl-3-[(S)-2-amino-4-methylthiobutyldithio]-1-oxo pro pyl]- L-phenylalanine benzyl ester (RB 101), a systemically active prodrug, modulate CCK release in mouse brain, leading to an overall increase in the extracellular levels of CCK. This was quantified by measuring the effects of both inhibitors on the in vivo binding of [3H]propionyl-Tyr(SO3H)-gNle-mGly-Trp-(N-Me)Nle-Asp-Phe-NH2 ([3H]pBC 264), a selective and highly potent CCK-B agonist. Thus, intracerebroventricular injection of kelatorphan produced a dose-dependent inhibition of the in vivo binding of [3H]pBC 264 with a maximal effect (40%) at 50 nmol. A similar response was observed after intravenous injection of RB 101 (40 mg/kg). The specific binding of [3H]pBC 264 was also inhibited (25%) by intravenous injection of the selective delta-opioid agonist H-Tyr-D-Cys(StBu)-Gly-Phe-Leu-Thr(OtBu)-OH (BUBUC; 2 mg/kg) but not by the mu-agonist H-Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol (5 mg/kg), suggesting a preferential involvement of delta-opioid receptors in the modulation of CCK release. This was confirmed by using the selective delta-opioid antagonist naltrindole, which prevented the inhibitory effects of BUBUC and of enkephalin-degrading enzyme inhibitors on [3H]pBC 264 binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

In Vivo Labeling of Serotonin Uptake Sites with [3H]Paroxetine

Previous work has shown that [3H]paroxetine is a potent and selective in vitro label for serotonin uptake sites in the mammalian brain. In the present study, [3H]paroxetine was tested in mice as an in vivo label for serotonin uptake sites. Maximum tritium concentration in the whole brain (1.4% of the intravenous dose) was reached 1 h after injection into a tail vein. Distribution of the tracer at 3 h after injection followed the distribution of serotonin uptake sites known from previous in vitro binding studies (r = 0.85). The areas of highest [3H]paroxetine concentration, in decreasing order, were: hypothalamus greater than frontal cortex greater than olfactory tubercles greater than thalamus greater than upper colliculi greater than brainstem greater than hippocampus greater than striatum greater than cerebellum. Preinjection of carrier paroxetine (1 mg/kg) significantly decreased [3H]paroxetine concentration in all areas except in the cerebellum, which is known to contain a relatively low number of specific binding sites. Kinetic studies showed highest specific [3H]paroxetine binding (tissue minus cerebellum) at 2 h after injection and slow clearance of activity thereafter (half-time of dissociation from the hypothalamus, 215 min). The specificity of in vivo [3H]paroxetine binding was studied by preinjecting monoamine uptake blockers or receptor antagonists 5 min before administration of [3H]paroxetine. Serotonergic or muscarinic cholinergic receptor antagonists and dopamine or norepinephrine uptake blockers did not reduce the in vivo binding of [3H]paroxetine. In contrast, there was an excellent correlation (r = 0.99) between the in vivo inhibitory potencies of serotonin uptake blockers in this study and previously published in vitro data on inhibition of [3H] serotonin uptake in brain synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative Analysis of Nicotine-Like Receptor-Ligand Interactions in Rodent Brain Homogenate

The effects of different variables such as incubation time, temperature, tissue protein content, and pH on the interactions of various labelled nicotinic ligands with nicotine-like binding sites in vitro were studied in rodent brain preparations. The ligands tested were alpha-[3H]bungarotoxin (alpha-[3H]BTX), [3H]tubocurarine ([3H]TC), and [3H]nicotine ([3H]NIC). The regional distribution of the labelled nicotinic ligand binding was also studied and affinity constants and maximal binding (Bmax) values for the equilibrium [3H]NIC binding are given. Association kinetics for [3H]NIC and [3H]TC binding to brain homogenate were similar, with maximal binding within 5-10 min of incubation, followed by a continuous decrease. In contrast, the binding of alpha-[3H]BTX to brain homogenate was much slower, reaching equilibrium after 30-60 min of incubation. Scatchard analysis of equilibrium binding data for [3H]NIC in the hippocampus indicated two binding sites: a high-affinity site (Bmax, 60 pmol/g protein; KD, 6 nM) and a low-affinity site (Bmax, 230 pmol/g protein; KD, 125 nM). The data for the high-affinity [3H]NIC binding site are very similar to previously found data for the high-affinity binding site of [3H]TC and the binding site of alpha-[3H]BTX. Each ligand showed regional differences in binding, and the binding pattern also differed between the ligands.     

Selective inhibition of the binding of 3H-(3-MeHis2) thyrotropin releasing hormone to rat amygdala membranes by some naturally occurring cannabinoids

The effect of naturally occurring cannabinoids, delta 9-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD), on the brain receptors for thyrotropin releasing hormone (TRH) was investigated. TRH receptors were labeled with 3H-(3-MeHis2)TRH (3H-MeTRH). 3H-MeTRH bound specifically to rat brain membranes at a single high affinity site with a Bmax value of 49.2 +/- 0.96 fmol per mg protein and a Kd value of 3.83 +/- 0.12 nM. The binding of 3H-MeTRH to whole brain membranes was inhibited when rats were injected intraperitoneally with 3 to 30 mg/kg of THC. The extent of inhibition in the binding at 10 and 30 mg/kg was similar. THC (10 mg/kg) significantly inhibited the binding of 3H-MeTRH to amygdala membranes but did not affect the binding to membranes prepared from hippocampus, septum, cortex, striatum and the rest of the brain. THC, CBN and CBD in doses of 3 to 30 mg/kg did not affect the binding of 3H-MeTRH to hypothalamic membranes. All the three cannabinoids at 30 mg/kg inhibited the binding of 3H-MeTRH to amygdala membranes. The inhibition in the binding of 3H-MeTRH by the cannabinoids was due to changes in the Kd values but the Bmax values remained unchanged. It is concluded that both psychotomimetic and nonpsychotomimetic cannabinoids inhibit the binding of 3H-MeTR to amygdala membranes selectively, which is accomplished by decreases in the affinity of the ligand to receptors, and the amygdala may be an important brain area in some of the actions of cannabinoids.     

Solubilized Rat Brain Adenosine Receptors Have Two High-Affinity Binding Sites for l, 3-Dipropyl-8-Cyclopentylxanthine

The specific binding of L-N6-[3H]phenylisopropyladenosine (L-[3H]PIA) to solubilized receptors from rat brain membranes was studied. The interaction of these receptors with relatively low concentrations of L-[3H]PIA (0.5-12.0 nM) in the presence of Mg2+ showed the existence of two binding sites for this agonist, with respective dissociation constant (KD) values of 0.24 and 3.56 nM and respective receptor number (Bmax) values of 0.28 +/- 0.03 and 0.66 +/- 0.05 pmol/mg of protein. In the presence of GTP, the binding of L-[3H]PIA also showed two sites with KD values of 24.7 and 811.5 nM and Bmax values of 0.27 +/- 0.09 and 0.93 +/- 0.28 pmol/mg of protein for the first and the second binding site, respectively. Inhibition of specific L-[3H]PIA binding by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (0.1-300 nM) performed with the same preparations revealed two DPCPX binding sites with Ki values of 0.29 and 13.5 nM, respectively. [3H]DPCPX saturation binding experiments also showed two binding sites with respective KD values of 0.81 and 10.7 nM and respective Bmax values of 0.19 +/- 0.02 and 0.74 +/- 0.06 pmol/mg of protein. The results suggest that solubilized membranes from rat brain possess two adenosine receptor subtypes: one of high affinity with characteristics of the A1 subtype and another with lower affinity with characteristics of the A3 subtype of adenosine receptor.     

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.     

Thyrotropin releasing hormone (TRH) binding sites in the adult human brain: localization and characterization

In the current study, we found evidence for the existence of binding sites for TRH in synaptic membrane preparations of several regions of the postmortem adult human brain. High levels of specific binding (fmol [3H]Me-TRH/mg protein/2 hr) were found in limbic structures: amygdala (7.1 +/- 0.6, Mean +/- SE), hippocampus (2.8 +/- 0.3), and temporal cortex (2.4 +/- 0.8). Intermediate levels of binding were found in the hypothalamus and nucleus accumbens whereas binding was low to undetectable in frontal and occipital cortex, cerebellum, pons, medulla and corpus striatum. Binding of the radioligand was linear over protein concentrations of 0.05-1.5 mg, and greater than 6 hr of incubation was required to achieve maximal binding. In the amygdala, binding was inhibited in the presence of TRH and Me-TRH but not in the presence of up to 1 microM concentrations of cyclo (His-Pro), TRH-OH, pGlu-His or peptides unrelated to TRH. Pretreatment of amygdala synaptic membranes with detergents, proteases or phospholipases disrupted [3H]Me-TRH binding; pretreatment with DNase or collagenase had no effect on binding. Saturation and association/dissociation analyses of the binding of [3H]Me-TRH to purified amygdala synaptic membranes revealed the presence of a high affinity (KD = 2.0 nM), low capacity (Bmax = 180 +/- 16 fmoles/mg protein) binding site. These results demonstrate that a highly specific membrane associated receptor for TRH is present in the adult human brain. The specific role that this receptor plays in brain function remains to be elucidated.     

Equilibrium binding characteristics of [3H]thiomuscimol.

The equilibrium binding characteristics of the tritiated GABAA agonist, 5-aminomethyl-3-isothiazolol (thiomuscimol) are described. Using the filtration technique to separate bound- from free-ligand, [3H]thiomuscimol was shown to bind to the GABA(A) receptor site(s) in a saturable manner with a Kd value of 28+/-6.0 nM and a Bmax value of 50+/-4.0 fmol/mg original tissue. In parallel binding experiments, the Kd and Bmax values for [3H]muscimol were determined to be 5.4+/-2.8 nM and 82+/-11 fmol/mg original tissue, respectively. In binding assays using the centrifugation technique, Kd and Bmax values for [3H]thiomuscimol were found to be 116+/-22 nM and 154 13 fmol/mg original tissue, respectively, whereas a Kd value of 16+/-1.8 nM and a Bmax value of 155+/-8.0 fmol/mg original tissue were determined for [3H]muscimol. In comparative inhibition studies using the GABA(A) antagonist SR 95531 and a series of specific GABAA agonists, the binding sites for [3H]thiomuscimol and [3H]muscimol were shown to exhibit similar pharmacological profiles. Autoradiographic studies disclosed similar regional distribution of [3H]thiomuscimol and [3H]muscimol binding sites in rat brain. Highest densities of binding sites were detected in cortex, hippocampus, and cerebellum, whereas low densities were measured in the midbrain structures of rat cortex. In conclusion, the equilibrium GABA(A) receptor binding characteristics of [3H]thiomuscimol are very similar to those of [3H]muscimol.     

Relationship between levels and uptake of serotonin and high affinity [3H]imipramine recognition sites in the rat brain

High affinity [3H]imipramine binding, endogenous levels of serotonin and noradrenaline, and serotonin uptake were determined in brain regions of rats with selective destruction of serotonergic neurons by 5,7-dihydroxytryptamine (5,7-DHT), of adrenergic neurons by 6-hydroxydopamine (6-OHDA), and of rats treated with reserpine. Neonatal treatment with 5,7-DHT resulted in a significant decrease of both serotonin levels and density (Bmax) of high affinity [3H]imipramine binding sites in the hippocampus. In contrast, an elevation of serotonin levels and an increase in Bmax of [3H]imipramine binding were noted in the pons--medulla region. No changes were observed in the noradrenaline content in either of these regions. Intracerebral 6-OHDA lesion produced a drastic suppression of noradrenaline levels in cerebral cortex but failed to alter the binding affinity (KD) or density (Bmax) of [3H]imipramine recognition sites. A single injection of reserpine (2.5 mg/kg) resulted in marked depletion of both serotonin (by 57%) and noradrenaline (by 86%) content and serotonin uptake (by 87%) in the cerebral cortex but had no significant influence of the parameters of high affinity [3H]imipramine binding in this brain region. The results suggest that high affinity [3H]imipramine binding in the brain is directly related to the integrity of serotonergic neurons but not to the magnitude of the uptake or the endogenous levels of the transmitter, and is not affected by damage to noradrenergic neurons or by low levels of noradrenaline.     

Solubilization of receptors for thyrotropin-releasing hormone from GH4C1 rat pituitary cells: demonstration of guanyl nucleotide sensitivity

TRH receptors have been solubilized from GH4C1 cells using the plant glycoside digitonin. Solubilized receptors retain the principal binding characteristics exhibited by the TRH receptor in intact pituitary cells and their membranes. The binding of the methylhistidyl derivative of TRH [( 3H]MeTRH) attained equilibrium within 2-3 h at 4 C, and it was reversible, dissociating with a t1/2 of 7 h. Analysis of [3H]MeTRH binding to soluble receptors at 4 C yielded a dissociation constant (Kd) of 3.8 nM and a total binding capacity (Bmax) of 3.9 pmol/mg protein. Peptides known to interact with non-TRH receptors on GH cells failed to interfere with the binding of [3H]MeTRH, indicating that the TRH binding was specific. Chlordiazepoxide, a competitive antagonist for TRH action in GH cells, inhibited TRH binding to soluble receptors with an IC50 of 11 microM. When [3H]MeTRH was bound to membranes and the membrane proteins were then solubilized, we found enhanced dissociation of the prebound [3H]MeTRH from its solubilized receptor by guanyl nucleotides. Maximal enhancement of [3H]MeTRH dissociation by 10 microM GTP gamma S occurred within about 45 min at 22 C. GTP gamma S, GTP, GDP beta S, and GDP were all effectors of [3H]MeTRH dissociation, exhibiting EC50s in the range of 14-450 nM. The rank order of potency of the tested nucleotides was GTP gamma S greater than GTP congruent to GDP beta S greater than GDP much greater than ATP gamma S greater than GMP. We conclude that TRH receptors have been solubilized from GH cells with digitonin and retain the binding characteristics of TRH receptors in intact pituitary cells. Furthermore, prebinding [3H]MeTRH to GH4C1 cell membranes results in the solubilization of a complex in which the TRH receptor is linked functionally to a GTP binding protein.     

Solubilization of an Adenosine Uptake Site in Brain

Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine [( 3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 +/- 0.02 nM and 133 +/- 18 fmol/mg protein, respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep greater than hexobendine greater than dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine [( 3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high-affinity site with a KD of 0.32 +/- 0.06 nM and a Bmax of 105 +/- 30 fmol/mg protein and a lower-affinity site with a KD of 5.50 +/- 0.52 nM and Bmax of 300 +/- 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake [( 3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.     

A Comparison of [3H]MK-801 and N-[1-(2-Thienyl)cyclohexyl]-3,4-[3H]Piperidine Binding to the N-Methyl-D-Aspartate Receptor Complex in Human Brain

The binding of (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate ([3H]MK-801) and N-[1-(2-thienyl)cyclohexyl]-3,4-[3H]piperidine ([3H]TCP) to the N-methyl-D-aspartate (NMDA) receptor complex of human brain has been investigated. Significant differences were noted between the binding of the two ligands in the same tissue samples. Binding of both ligands was stimulated by addition of glutamic acid or glycine. However, addition of both compounds resulted in an additional effect with [3H]MK-801 but not [3H]TCP binding. Saturation analysis revealed approximately twice as many high-affinity sites for [3H]MK-801 (Bmax, 1,500 +/- 300 fmol/mg of protein) than for [3H]TCP (Bmax, 660 +/- 170 fmol/mg of protein). In addition, a low-affinity site was detected for [3H]MK-801 binding but not [3H]TCP binding. The pharmacology of the high-affinity [3H]MK-801 and [3H]TCP binding sites was similar with rank order of potency of inhibitors being MK801 greater than TCP greater than phencyclidine greater than N-allylnormetazocine (SKF 10047). 2-Amino-5-phosphonopentanoate inhibited binding of both ligands with comparable potency whereas both 7-chlorokynurenic acid and ZnCl2 were more potent inhibitors of [3H]MK-801 than of [3H]TCP binding. All compounds examined exhibited Hill coefficients of significantly less than unity. Saturation analysis performed in the striatum revealed that the number of binding sites was the same for both [3H]MK-801 (Bmax, 1,403 +/- 394 fmol/mg) and [3H]TCP (Bmax, 1,292 +/- 305 fmol/mg). Addition of glutamate or glycine stimulated striatal binding but there was no further increase on addition of both together.(ABSTRACT TRUNCATED AT 250 WORDS)     

MIF-1 attenuates spiroperidol alteration of striatal dopamine D2 receptor ontogeny

Long-term postnatal treatment of rats with the dopamine D2 receptor antagonist, spiroperidol, results in the impaired development of striatal D2 receptors. Because the tripeptide prolyl-leucyl-glycinamide (MIF-1) attenuates haloperidol-induced up-regulation of striatal dopamine D2 receptors in adult rats, we studied the effect of MIF-1 on the spiroperidol-induced alteration of striatal D2 ontogeny. Postnatal treatment of rats with spiroperidol (1.0 mg/kg/day, IP, x32 days from birth) resulted in a 74% decrease in the Bmax for [3H]spiroperidol binding with no change in the Kd at 5 weeks. When rats were studied at 8 weeks, in the absence of additional treatment, total specific [3H]spiroperidol binding was reduced by 59%. While MIF-1 alone (1.0 mg/kg/day, IP, x32 days from birth) had no effect on [3H]spiroperidol binding, MIF-1 completely attenuated the ontogenic impairment of striatal D2 receptors that was produced by spiroperidol treatment. At 5 weeks the Bmax for [3H]spiroperidol binding was at the saline control level in the group of rats cotreated with spiroperidol and MIF-1. At 8 weeks, with no additional treatments, the specific binding of [3H]spiroperidol to striatum was also at control levels in the group cotreated with spiroperidol and MIF-1. These findings demonstrate that MIF-1 attenuates spiroperidol-induced impairment of development of striatal dopamine D2 receptors in rats.     

Pharmacological characterization and autoradiographic localization of substance P receptors in guinea pig brain

[3H]Substance P ([3H]SP) was used to characterize substance P (SP) receptor binding sites in guinea pig brain using membrane preparations and in vitro receptor autoradiography. Curvilinear Scatchard analysis shows that [3H]SP binds to a high affinity site (Kd = 0.5 nM) with a Bmax of 16.4 fmol/mg protein and a low affinity site (Kd = 29.6 nM) with a Bmax of 189.1 fmol/mg protein. Monovalent cations generally inhibit [3H]SP binding while divalent cations substantially increased it. The ligand selectivity pattern is generally similar to the one observed in rat brain membrane preparation with SP being more potent than SP fragments and other tachykinins. However, the potency of various nucleotides is different with GMP-PNP greater than GDP greater than GTP. The autoradiographic distribution of [3H]SP binding sites shows that high amounts of sites are present in the hippocampus, striatum, olfactory bulb, central nucleus of the amygdala, certain thalamic nuclei and superior colliculus. The cortex is moderately enriched in [3H]SP binding sites while the substantia nigra contains only very low amounts of sites. Thus, the autoradiographic distribution of SP binding sites is fairly similar in both rat and guinea pig brain.     

Administration of chlordiazepoxide affects [3H][3-MeHis2]thyrotropin-releasing hormone binding in rat brain.

The effects of acute and subchronic administration of chlordiazepoxide (CDZ) on [3H][3-methyl-histidyl2]thyrotropin-releasing hormone binding to thyrotropin-releasing hormone (TRH) receptors in membrane preparations from various regions of rat brain were examined. Acute administration of CDZ (50 mg/kg x 3 within 24 h) did not alter either the equilibrium dissociation constant (Kd) or the maximum number of binding sites (Bmax) in cerebellum (CB), olfactory bulbs (OB), frontal cortex (Cx), hypothalamus (HT) or corpus striatum (ST). However, the Kds of the pyriform cortex/amygdala (PC/A) (Kd = 3.6 +/- 0.1 nM compared to 1.9 +/- 0.1 nM in the control group; p less than 0.01) and the hippocampus (HP) (Kd = 7.8 +/- 0.7 nM compared to 2.1 +/- 0.1 nM in the control group; p less than 0.01) were increased. There were no changes in Bmax. Subchronic administration of CDZ (50 mg/kg/day for 7 days) increased the Kd of the PC/A complex (p less than 0.05), the OB (p less than 0.05) and the HP (p less than 0.01) without altering in Bmax. These results, showing regional differences in the response of TRH receptors to acute and subchronic CDZ administration, suggest that reduced affinity of TRH receptors in the PC/A complex, OB and HP may be related to some of the neurobiological actions of CDZ and/or its metabolites.     

Quantitative autoradiography of [3H]CTOP binding to mu opioid receptors in rat brain

[3H]H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 ([3H]CTOP), a potent and highly selective mu opioid antagonist, was used to localize the mu receptors in rat brain by light microscopic autoradiography. Radioligand binding studies with [3H]CTOP using slide-mounted tissue sections of rat brain produced a Kd value of 1.1 nM with a Bmax value of 79.1 fmol/mg protein. Mu opioid agonists and antagonists inhibited [3H]CTOP binding with high affinity (IC50 values of 0.2-2.4 nM), while the delta agonist DPDPE, delta antagonist ICI 174,864, and kappa agonist U 69, 593 were very weak inhibitors of [3H]CTOP binding (IC50 values of 234-3631 nM). Light microscopic autoradiography of [3H]CTOP binding sites revealed regions of high density (nucleus of the solitary tract, clusters in the caudate-putamen, interpeduncular nucleus, superior and inferior colliculus, subiculum, substantia nigra zona reticulata, medial geniculate, locus coeruleus and dorsal motor nucleus of the vagus) and regions of moderate labeling (areas outside of clusters in the caudate-putamen, cingulate cortex, claustrum and nucleus accumbens). The cerebral cortex (parietal) showed a low density of [3H]CTOP binding.     

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.     

Effects of chronic antidepressant treatment on dopamine-related [3H]SCH 23390 and [3H]Spiperone binding in the rat striatum

1. The effects of chronic administration of antidepressants on dopamine-related [3H]SCH 23390 and [3H]spiperone binding to rat striatal membranes were assessed. 2. The monoamine oxidase inhibitors phenelzine (5 or 10 mg kg-1/day) and tranylcypromine (1 mg kg-1/day) and the tricyclic desipramine (10 mg kg-1/day) were administered for 28 days by constant subcutaneous infusion using Alzet (2ML4) osmotic minipumps. 3. These treatments did not alter Kd estimates for either [3H]SCH 23390 or [3H]spiperone binding sites. The monoamine oxidase inhibitors induced a decrease in the Bmax values for both [3H]SCH 23990 and [3H]spiperone binding sites. Desipramine induced a decrease in the Bmax value for [3H]SCH 23390 binding but had no effect on the Bmax value for [3H]spiperone binding.     

Thyrotropin-releasing hormone (TRH) and its analog (DN-1417): interaction with pentobarbital in choline uptake and acetylcholine synthesis of rat brain slices

TRH or its analog DN-1417 (gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-proliamide) given 15 min after intravenous (i.v.) administration of pentobarbital (30 mg/kg) markedly shortened the pentobarbital-induced sleeping time in rats. This effect was almost completely abolished by intracerebroventricular pretreatment with atropine methylbromide (20 micrograms/rat), thereby suggesting the involvement of cholinergic mechanism. The action mechanism was investigated using rat brain slices. TRH (10(-6)-10(-4)M) or DN-1417 (10(-7)-10(-5)M) caused significant increases in the uptake of [3H]-choline into striatal slices. TRH(10(-4)M) or DN-1417(10(-5)M) also stimulated the conversion of [3H]-choline to [3H]-acetylcholine in striatal slices. A 30% reduction of acetylcholine synthesis from [3H]-choline in hippocampal slices and a 40% reduction of [3H]-choline uptake in slices of cerebral cortex, hippocampus and hypothalamus were observed in rats pretreated with pentobarbital (60 mg/kg, i.v.). TRH or DN-1417 (20 mg/kg, i.v.) given 15 min after the administration of pentobarbital markedly reversed both of the pentobarbital effects. Direct application of pentobarbital (5 X 10(-4)M) to slices in vitro also caused a 20-40% reduction of [3H]-choline uptake of cerebral cortex, hippocampus and diencephalon. A concomitant application of TRH(10(-4)M) or DN-1417(10(-5)M) and pentobarbital abolished the pentobarbital effect. These results provide neurochemical evidence that the antagonistic effects of TRH and DN-1417 on pentobarbital-induced narcosis are closely related to alterations in the rat brain choline uptake and acetylcholine synthesis, which are considered to be measures of the activity of cholinergic neurons.