Effects of detergents on binding of 5-hydroxytryptamine3 receptor antagonist [H]GR65630 to rat cortical membranes

In the absence of detergent, specific binding of [³H]GR65630, a 5-hydroxytryptamine₃ (5-HT₃) antagonist, determined in the presence of 5-HT₃ receptor antagonist ICS205-930, was at most 30% of the total binding. To decrease the level of nonspecific binding, the effects of detergents on [³H]GR65630 binding to rat cortical membranes were investigated. The use of a detergent (0.1% Lubrol PX or Triton X-100) decreased nonspecific binding, increasing the proportion of specific binding to 70% of total binding. In the presence of 0.1% Triton X-100, binding of [³H]GR65630 was rapid, reversible and saturable at 25°C. The rank order of 5-HT₃ receptor active drugs in inhibiting [³H]GR65630 binding was quipazine > ICS205-930 > 2-methyl-5-HT = 5-HT > metoclopramide, which confirmed that [³H]GR65630 efficiently labeled 5-HT₃ receptors in the presence of Triton X-100. Triton X-100 improved 5-HT₃ receptor binding with rat brain membranes.     

[H]lysergic acid diethylamide (LSD): differential agonist and antagonist binding properties at 5-HT receptor subtypes in rat brain

[³H]Lysergic acid diethylamide (LSD) in the presence of 40 nM ketanserin labeled the 5-HT_1A receptor subtype in rat hippocampal membranes. In the presence of guanosine triphosphate (GTP), the B_max and affinity of [³H]LSD binding to the 5-HT_1A binding site were significantly decreased. [³H]LSD in the presence of 40 nM WB4101 labeled the 5-HT₂ receptor subtype in homogenates of rat frontal cortex. In contrast to the effect on [³H]LSD binding to the 5-HT_1A binding site, GTP produced no significant effect on either the B_max or the K_D of [³H]LSD binding to the 5-HT₂ binding site. Competition of 5-HT for [³H]LSD binding to the 5-HT₂ binding site was best described by a computer-derived model assuming two binding sites. In the presence of GTP, the 5-HT competition curve was shifted significantly to the right with an approx. 3-fold increase in the IC₅₀. These binding characteristics are consistent with [³H]LSD acting as an antagonist at the 5-HT₂ receptor which has multiple affinity states for agonists and is coupled to a guanine nucleotide regulatory subunit. Thus, [³H]LSD has binding characteristics consistent with it acting as an agonist at the 5-HT_1A receptor subtype but as an antagonist at the 5-HT₂ receptor subtype in rat brain.     

Identification of a novel 5-HT1 binding site in rat spinal cord

High affinity, specific [³H]5-hydroxytryptamine (5-HT) binding to spinal cord synaptosomes was examined to identify the 5-HT receptor subtypes present. Computer nonlinear regression analysis of competition studies employing 8-OH-DPAT indicated that this 5-HT_1A selective agonist demonstrated high affinity competition (K_{i = 1.3 nM}) for 24.6 ± 0.7% of the total [³H]5-HT binding sites. Competition studies employing the 5-HT_1B selective agonist RU24969, in the presence of 100 nM 8-OH-DPAT, indicated that RU24969 demonstrated high affinity (K_{i = 1.1 nM}) competitive inhibition for 26.2 ± 1.4% of all [³H]5-HT binding sites. Neither 5-HT_1C, 5-HT_1D, 5-HT₂ nor 5-HT₃ selective compounds demonstrated any high affinity competition for the residual 49% of specific [³H]5-HT binding. Therefore, three major classes of [³H]5-HT binding sites could be demonstrated in spinal cord synaptosomes: 5-HT_1A, 5-HT_1B and a novel [³H]5-HT binding site which respectively represented 25, 26 and 49% of spinal cord synaptosomal [³H]5-HT binding. Further studies focusing on the function of the latter binding site are needed to determine if the presently identified novel binding site is the major 5-HT₁ receptor subtype present in spinal cord.     

Cortical 5-HT(1A) receptor downregulation by antidepressants in rat brain

Total 5-HT binding sites and 5-HT_1A receptor density was measured in brain regions of rats treated with imipramine (5 mg/kg body wt), desipramine (10 mg/kg body wt) and clomipramine (10 mg/kg body wt), for 40 days, using [³H]5-HT and [³H]8-OH-DPAT, respectively. It was observed that chronic exposure to tricyclic antidepressants (TCAs) results in significant downregulation of total [³H]5-HT binding sites in cortex (42–76%) and hippocampus (35–67%). The 5-HT_1A receptor density was, however, decreased significantly (32–60%) only in cortex with all the three drugs. Interestingly, in hippocampus imipramine treatment increased the 5-HT_1A receptor density (14%). The affinity of [³H]8-OH-DPAT was increased only with imipramine treatment both in cortex and hippocampus. The affinity of [³H]5-HT to 5-HT binding sites in cortex was increased with imipramine treatment and decreased with desipramine and clomipramine treatment. 5-HT sensitive adenylyl cyclase (AC) activity was significantly increased in cortex with imipramine (72%) and clomipramine (17%) treatment, whereas in hippocampus only imipramine treatment significantly increased AC activity (50%). In conclusion, chronic treatment with TCAs results in downregulation of cortical 5-HT_1A receptors along with concomitant increase in 5-HT stimulated AC activity suggesting the involvement of cortical 5-HT_1A receptors in the mechanism of action of TCAs.     

Short-term effects of 3,4-methylen-dioxy-metamphetamine (MDMA) on 5-HT(1A) receptors in the rat hippocampus

The first effects of 3,4-methylen-dioxy-metamphetamine (MDMA, “ecstasy”), on serotonin 1A (5-HT_1A) receptors in rat hippocampus were determined by means of [³H]-8-hydroxy-dipropylamino-tetralin ([³H]-8-OH-DPAT) and 5′guanosine-(γ-[³⁵S]-thio)triphosphate ([³⁵S]-GTPγS) binding as well as inhibition of forskolin (FK)-stimulated adenylyl cyclase (AC) activity. The study was completed by [³⁵S]-GTPγS functional autoradiography experiments carried out in frontal sections of rat brain, including the hippocampal region. Results showed that MDMA was either able to displace [³H]-8-OH-DPAT binding (K_i  500 nM) or to reduce the number of specific sites (B_max) without affecting K_d. The drug also failed to change the [³⁵S]-GTPγS binding or to inhibit AC velocity, underlying its behavior as a non-competitive 5-HT_1A receptor antagonist. Further, MDMA (1 or 100 μM), partially antagonized either [³⁵S]-GTPγS binding stimulation of the agonists 5CT and 8-OH-DPAT or the AC inhibition induced by 5CT and DP-5CT. However, in contrast to binding studies, in AC assays the amphetamine displayed an effect also on EC₅₀, always being less potent than the reference antagonist WAY100,635. In functional autoradiography, MDMA behaved either as a partial 5-HT_1A antagonist in limbic areas or, added alone, as an agonist, increasing the coupling signal presumably through 5-HT release from synapses. Interestingly, the selective 5-HT re-uptake inhibitor (SSRI) fluoxetine had no effect on MDMA [³⁵S]-GTPγS binding activation. This latter finding indicates that the amphetamine can release 5-HT via alternative mechanisms to 5-HT transporter binding, probably via membrane synaptic receptors or vesicular transporters. The release of other transmitters is not excluded. Therefore, our results encourage at extending the study of MDMA biochemical profiles, in the attempt to elucidate those amphetamine-induced pathways with a potential for neurotoxicity or psycho-stimulant activity.     

Influence of lipid peroxidation on [H]ketanserin binding to 5-HT2 prefrontal cortex receptors

The influence of lipid peroxidation on 5-HT₂ receptor binding was examined in prefrontal cortex membranes from sheep brain. Lipid peroxidation was induced with ascorbic acid and ferrous sulphate and measured by the thiobarbituric acid method. In lipid-peroxidized membranes, [³H]ketanserin specific binding was inhibited. The B_max values decreased by 80%, from 50.1±3.5 fmol/mg protein in control membranes to 10.1±2.0 fmol/mg protein in peroxidized membranes, indicating a decrease in the number of 5-HT₂ binding sites. However, the K_D values for the [³H]ketanserin specific binding did not significantly change. In order to further characterize [³H]ketanserin binding, the inhibition potency (IC₅₀ values) of antagonists or agonists of serotonin and dopamine receptors for [³H]ketanserin specific binding was determined. In control membranes, the order of the inhibition potency of the drugs tested was the following: ketanserin (−log [IC₅₀] = 8.56±0.70) ritanserin (−log [IC₅₀] = 8.13±0.30) methysergide (−log [IC₅₀] = 7.42±0.50) spiperone (−log [IC₅₀] = 7.23±0.18) serotonin (−log [IC₅₀] = 6.99±0.65) haloperidol (−log [IC₅₀] = 6.95±0.65) dopamine (−log [IC₅₀] = 5.82±0.76). After membrane lipid peroxidation, the IC₅₀ value for ritanserin was significantly increased, suggesting a decreased capacity for displacing [³H]ketanserin specific binding. Other antagonists of 5-HT₂ receptors showed apparent increases in IC₅₀ values upon peroxidation, whereas spiperone was shown to be the most potent drug (−log [IC₅₀] = 7.19±1.06) in inhibiting [³H]ketanserin specific binding. A decrease in polyunsaturated fatty acids, namely docosahexaenoic acid (22:6) was also observed in peroxidized membranes. These results indicate a modulating role of the surrounding lipids and of the physical properties of the membranes on the binding activity of 5-HT₂ receptors upon the lipid peroxidation process, which can be involved in the tissue impairment that occurs during the aging process and in post-ischemic situations.     

Visualization and quantification of central 5-HT1A receptors with specific antibodies

Polyclonal antibodies were raised by the repeated injection of rabbits with synthetic peptides corresponding to selective portions (peptide 1: aminoacid residues 12–23, and peptide 2: aminoacid residues 243–268) of the aminoacid sequence of the rat 5-HT_1A receptor. Both antisera allowed the immunoprecipitation of 5-HT_1A receptors but not of other 5-HT receptor types and adrenergic receptors solubilized from rat hippocampal membranes. Immunoblots demonstrated that a single protein of 63 kDa, corresponding to the molecular weight of the rat 5-HT_1A receptor binding subunit, was recognized by each antiserum. Immunoautoradiographic labelling of rat brain sections with the anti-peptide 2-antiserum exhibited the same regional distribution as 5-HT_1A sites labelled by selective radioligands such as [³H]8-OH-DPAT and [¹²⁵I]BH-8-MeO-N-PAT. However regional differences apparently existed between the respective intensity of labelling by the agonist radioligands and the antiserum, which might be explained by variations in the degree of coupling of 5-HT_1A receptor binding subunits with G proteins from one brain area to another.     

Autoradiographic comparison of [I]LSD-labeled 5-HT2A receptor distribution in rat and guinea pig brain

Although the density and distribution of 5-HT_2A(5-hydroxytryptamine-2A) receptors is well established for rat brain, the 5-HT_2A receptor distribution and density in guinea pig brain has not been extensively studied. In the present in vitro study, we have utilized ¹²⁵I-lysergic acid diethylamide ([¹²⁵I]LSD) to quantify and compare 5-HT_2A receptor density in coronal sections of rat and guinea pig brain. Spiperone (1 μM) and sulpiride (1 μM) were used to displace [¹²⁵I]LSD binding from 5-HT_2A and D₂ binding sites, respectively. Ligand binding was quantified by computer-aided image analysis densitometry (MCID). Similar to the rat, areas of highest specific 5-HT_2A receptor binding (fmol/mg protein) in guinea pig brain included the claustrum and Layer 4 of the cerebral cortex. Significant binding was also found in remaining neocortical layers, islands of Calleja, caudate putamen, olfactory bulb, nucleus accumbens, and choroid plexus. While the rat brain exhibited a high level of specific binding in the tenia tecta and mammillary nuclei, little binding was observed in these regions in the guinea pig. In both rat and guinea pig, low specific binding was found in amygdaloid, thalamic, or cerebellar areas. These studies indicate a general similarity between 5-HT_2A binding site distribution and relative density in guinea pig and rat brain but point to a few brain regions where significant differences exist.     

Phenylpiperazine derivatives with strong affinity for 5HT1A, D2A and D3 receptors

Four 7-[3-(4-phenyl-1-piperazinyl)propoxy]coumarins were synthesized. The affinities of these compounds for DA (D_2A, D₃) and 5HT_1A receptors were evaluated for their ability to displace [³H]-raclopride and [³H]-8-OH-DPAT respectively from their specific binding sites. The affinities of the target compounds were all in the nanomolar range and followed the order 5-HT_1A > D₂ > D₃.     

Binding activities by propiverine and its N-oxide metabolites of L-type calcium channel antagonist receptors in the rat bladder and brain

The present study was undertaken to characterize the binding activities of propiverine and its N-oxide metabolites (1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide: P-4(N → O), 1-methyl-4-piperidyl benzilate N-oxide: DPr-P-4(N → O)) toward L-type calcium channel antagonist receptors in the rat bladder and brain. Propiverine and P-4(N → O) inhibited specific (+)-[³H]PN 200–110 binding in the rat bladder in a concentration-dependent manner. Compared with that for propiverine, the K_i value for P-4(N → O) in the bladder was significantly greater. Scatchard analysis has revealed that propiverine increased significantly K_d values for bladder (+)-[³H]PN 200–110 binding. DPr-P-4(N → O) had little inhibitory effects on the bladder (+)-[³H]PN 200–110 binding. Oxybutynin and N-desethyl-oxybutynin (DEOB) also inhibited specific (+)-[³H]PN 200–110 binding in the rat bladder. Propiverine, oxybutynin and their metabolites inhibited specific [N-methyl-³H]scopolamine methyl chloride ([³H]NMS) binding in the rat bladder. The ratios of K_i values for (+)-[³H]PN 200–110 to [³H]NMS were markedly smaller for propiverine and P-4(N → O) than oxybutynin and DEOB. Propiverine and P-4(N → O) inhibited specific binding of (+)-[³H]PN 200–110, [³H]diltiazem and [³H]verapamil in the rat cerebral cortex in a concentration-dependent manner. The K_i values of propiverine and P-4(N → O) for [³H]diltiazem were significantly smaller than those for (+)-[³H]PN 200–110 and [³H]verapamil. Further, their K_i values for [³H]verapamil were significantly smaller than those for (+)-[³H]PN 200–110. The K_i values of propiverine for each radioligand in the cerebral cortex were significantly (P < 0.05) smaller than those of P-4(N → O). In conclusion, the present study has shown that propiverine and P-4(N → O) exert a significant binding activity of L-type calcium channel antagonist receptors in the bladder and these effects may be pharmacologically relevant in the treatment of overactive bladder after oral administration of propiverine.     

No Orcadian Rhythms of Serotoninergic, Alpha-, Beta-Adrenergic and Imipramine Binding Sites in Rat Brain Regions

B_max values of the specific binding of [³H]-WB 4101, [³H]-dihydroalprenolol, [³H]-spiperone and [³H]-imipramine to various rat brain regions were determined at 4 hr intervals over 24 hr under circadian conditions. No significant circadian rhythm of binding sites number was found for any receptor investigated in cerebral cortex, hypothalamus or brain stem. Some methodological issues are discussed.     

Central serotonin1A receptors: Respective distributions of encoding mRNA, receptor protein and binding sites by in situ hybridization histochemistry, radioimmunohistochemistry and autoradiographic mapping in the rat brain

The regional distribution of the mRNA encoding the 5-HT_1A serotonin receptor (whose selective agonists are potential anxiolytic and antidepressant drugs) was investigated in rat brain sections by in situ hybridization histochemistry using two sets of [³²P]labelled nucleoprobes, a riboprobe of 156 bases and oligoprobes of 30 bases corresponding to highly selective portions within the third intracellular loop and the N terminus domain of the amino acid sequence. These probes allowed the visualization of the 5-HT_1A mRNA mainly in the limbic regions: dentate gyrus and area CA₁ of the hippocampus, amygdala, entorhinal cortex, lateral septum and the dorsal raphe nucleus. These structures were also those which could be labelled by the specific 5-HT_1A radioligand [¹²⁵I]BH-8-MeO-N-PAT and antibodies raised against a synthetic 26 amino acid peptide whose sequence was taken from the most selective portion of the rat 5-HT_1A receptor protein. These data suggest that the 5-HT_1A receptors are not transported to a long distance from their site of synthesis, as it has been already reported for the somato-dendritic 5-HT_1A autoreceptors in the dorsal raphe nucleus. Combined autoradiographic quantification of the 5-HT_1A binding sites (labelled by a selective radioligand such as [¹²⁵I]BH-8-MeO-N-PAT, the 5-HT_1A receptor binding subunit (by radioimmunohistochemistry) and the 5-HT_1A mRNA on adjacent brain sections should be a relevant approach for assessing the molecular mechanisms responsible for the functional alterations of these receptors under various pathological and pharmacological conditions.     

[H]WAY–100635 for 5–HT1A receptor autoradiography in human brain: a comparison with [H]8–OH–DPAT and demonstration of increased binding in the frontal cortex in schizophrenia

WAY–100635 is the first selective, silent 5–HT_1A (5-hydroxytryptamine_1A, serotonin-1A) receptor antagonist. We have investigated the use of [³H]WAY–100635 as a quantitative autoradiographic ligand in post-mortem human hippocampus, raphe and four cortical regions, and compared it with the 5–HT_1A receptor agonist, [³H]8–OH–DPAT. Saturation studies showed an average Kd for [³H]WAY–100635 binding in hippocampus of 1.1 nM. The regional and laminar distributions of [³H]WAY–100635 binding and [³H]8–OH–DPAT binding were similar. The density of [³H]WAY–100635 binding sites was 60–70% more than that of [³H]8–OH–DPAT in all areas examined except the cingulate gyrus where it was 165% higher. [³H]WAY–100635 binding was robust and was not affected by the post-mortem interval, freezer storage time or brain pH (agonal state). Using [³H]WAY–100635, we confirmed an increase of 5–HT_1A receptor binding sites in the frontal cortex in schizophrenia, previously demonstrated with [³H]8–OH–DPAT. Compared to [³H]8–OH–DPAT, [³H]WAY–100635 has two advantages: it has a higher selectivity and affinity for the 5–HT_1A receptor, and it recognizes 5–HT_1A receptors whether or not they are coupled to a G-protein, whereas [³H]8–OH–DPAT primarily detects coupled receptors. Given these considerations, the [³H]WAY–100635 binding data in schizophrenia clarify two points. First, they indicate that the elevated [³H]8–OH–DPAT binding seen in the same cases is attributable to an increase of 5–HT_1A receptors rather than any other binding site. Second, the enhanced [³H]8–OH–DPAT binding in schizophrenia reflects an increased density of 5–HT_1A receptors, not an increased percentage of 5–HT_1A receptors which are G-protein-coupled. We conclude that [³H]WAY–100635 is a valuable autoradiographic ligand for the qualitative and quantitative study of 5–HT_1A receptors in the human brain.     

A differential interaction of putative μ-selective agonists with opiate binding sites in rat brain

The availability of tritium-labelled sufentanil ([³H]SUF) allowed for a further radioligand analysis of opiate binding sites in rat brain. A comparison of the binding characteristics of [³H]SUF and [³H]dihydromorphine ([³H]DHM) revealed a very similar potency in their mutual displacement by unlabelled analogues. Furthermore, a series of putative μ-opiate agonists displayed equal potencies in displacing either [³H]SUF and [³H]DHM, the only striking exception being the highly μ-selective opioid peptide morphiceptin which was 33 times less potent in inhibiting [³H]SUF as compared to [³H]DHM binding. Additional experiments revealed further pronounced differences in [³H]SUF and [³H]DHM binding characteristics: the total amount of binding sites for [³H]SUF was 4 times higher than that for [³H]DHM and the regional distribution within particular brain areas displayed considerable differences. Furthermore, the binding of [³H]SUF was differentially modulated by sodium and GTP as compared to [³H]DHM binding. These data suggest that in rat brain, [³H]SUF interacts both with μ-opiate sites recognizing [³H]DHM and another type of opiate site, which cannot be equated with any of the, as yet, described δ- or κ-binding sites, and rather, represents a subclass of μ-opiate receptor sites. These experiments, thus, support the notion of subclasses (isoreceptors) for different types of opiate receptors.     

Selective changes of neurotransmitter receptors in middle-aged gerbil brain

Age-related alterations in major neurotransmitter receptors and voltage dependent calcium channels were analyzed by receptor autoradiography in the gerbil brain. [³H]Quinuclidinyl benzilate (QNB). [³H]cyclohexyladenosine (CHA), [³H]muscimol, [³H]MK-801, [³H]SCH 23390, [³H]naloxone, and [³H]PN200-110 were used to label muscarinic acetylcholine receptors, adenosine A₁ receptors, γ-aminobutyric acid_A (GABA_A) receptors, (NMDA) receptors, dopamine D₁ receptors, opioid receptors, and voltage dependent calcium channels, respectively. In middle-aged gerbils (16 months old), the hippocampus exhibited a significant elevation in [³H]QNB, [³H]MK-801, [³H]SCH 23390, [³H]naloxone, and [³H]PN200-110 binding, whereas [³H]CHA and [³H]muscimol binding showed a significant reduction in this area, compared with that of young animals (1 month). On the other hand, the cerebellum showed a significant alteration in [³H]QNB, [³H]CHA, and [³H]naloxone binding and the striatum also exhibited a significant alteration in [³H]SCH 23390 and [³H]CHA binding in middle-aged gerbils. The neocortex showed a significant elevation only in [³H]CHA binding in middle-aged animals. The nucleus accumbens and thalamus also showed a significant alteration only in [³H]muscimol binding. However, the hypothalamus and substantia nigra exhibited no significant alteration in these bindings in middle-aged gerbils. These results demonstrate the age-related alterations of various neurotransmitter receptors and voltage dependent calcium channels in most brain regions. Furthermore, they suggest that the hippocampus is most susceptible to aging processes and is altered at an early stage of senescence.     

Tris-azaaromatic quaternary ammonium salts: Novel templates as antagonists at nicotinic receptors mediating nicotine-evoked dopamine release

A series of tris-azaaromatic quaternary ammonium salts has been synthesized and evaluated for their ability to inhibit neuronal nicotinic acetylcholine receptors (nAChRs) mediating nicotine-evoked [³H]dopamine release from superfused rat striatal slices and for inhibition of [³H]nicotine and [³H]methyllycaconitine binding to whole rat brain membranes. The 3-picolinium compound 1,3,5-tri-{5-[1-(3-picolinium)]-pent-1-ynyl}benzene tribromide (tPy3PiB), 3b, exhibited high potency and selectivity for nAChR subtypes mediating nicotine-evoked [³H]dopamine release with an IC₅₀ of 0.2 nM and I_max of 67%.     

Muscarinic cholinergic components in the carp brain

The activity of the muscarinic cholinergic system (acetylcholine, ACh; acetylcholinesterase, AChE; choline acetyltransferase, ChAT; muscarinic acetylcholine receptors) was studied in the carp brain. The ACh content (13.9 ± 1.1 nmol/g wet tissue) was estimated by gas chromatography after microwave irradiation focused to the head. The AChE and ChAT activities were 153 ± 13 nmol/min/mg protein and 817 ± 50 pmol/min/mg protein, respectively. The characteristics of [³H](−)quinuclidinyl benzilate ([³H](−)QNB) and [³H]pirenzepine ([³H]PZ) binding were also studied in brain membranes. Their specific binding was linearly dependent on the protein content and they appeared to bind with high affinity to a single, saturable binding site. A dissociation constant (K_d) of 47 ± 6.3 pM and a maximum number of binding sites (B_max) of 627 ± 65 fmol/mg protein were obtained for [³H](−)QNB, with a K_d value of 3.85 ± 0.67 nM and a B_max value of 95.3 ± 6.25 fmol/mg protein for [³H]PZ binding. The [³H]PZ binding amounted to only 15% of the [³H](−)QNB-labeled sites, as estimated from the ratio of the B_max values of [³H](−)QNB and [³H]PZ, suggesting a low density of M₁ subtype. Atropine sulfate, atropine methylnitrate and PZ inhibited the binding of both radioligands with Hill slopes (n_H) close to unity. The n_H value of AF-DX 116 was close to 1 against [³H](−)QNB binding, while it was 0.75 against [³H]PZ binding. The displacement curves of oxotremorine and carbachol were shallow for the binding of both radioligands. The rank order of potency of muscarinic ligands against [³H](−)QNB binding (K_i nM) was atropine sulfate (0.55) > atropine methylnitrate (1.61) > PZ (61.19) > oxotremorine (156.3) > AF-DX 116 (307) > carbachol (1301), while in the case of [³H]PZ binding it was atropine sulfate (0.24) > atropine methylnitrate (0.34) > PZ (10.38) > AF-DX 116 (55.87) > oxotremorine (62.79) > carbachol (1696). The results indicate the presence of a well-developed muscarinic cholinergic system with predominantly M₂ receptors in the carp brain.     

Demonstration of steroid hormone receptors and steroid action in primary cultures of rat glial cells

Primary cultures of rat glial cells were established from newborn rat forebrains. A mixed population of oligodendrocytes and astrocytes was obtained, as confirmed by indirect immunofluorescence staining with specific markers for each cell type. Receptors were measured 3 weeks after primary culture in glial cells cultured in the presence or not of 50 nM estradiol and we have identified progesterone, glucocorticoid, estrogen, and androgen receptors (PR, GR, ER and AR), but only PR was inducible by the estrogen treatment. This estrogen-induction of PR was more dramatic in glial cells derived from female offsprings than from males, as measured by binding studies and by immunohistochemical techniques with the KC 146 anti-PR monoclonal antibody. The antiestrogen tamoxifen inhibited the estrogen induction, but had no effect by itself on PR concentration. Specific binding sites for PR, GR, ER and AR were measured by whole cell assays after labeling cells with, respectively, [³H]R5020, [³H]dexamethasone, [³H]OH-tamoxifen or [³H]R1881. PR and GR were also analyzed by ultracentrifugation and after exposure of cells to agonists, both receptors were recovered from cytosol as a 9S form, and from the nuclear high-salt, tungstate ions-containing fraction as a 4–6S form. In contrast, when the antiprogestin- and antiglucocorticosteroid RU486 was used as a ligand, a non-activated 8.5S receptor complex was found for both receptors in this nuclear fraction. The 8.5S complex of the GR was further analyzed in the presense of specific antibodies and, in addition to GR, the presence of the heat shock protein hsp90 and of a 59 kDa protein was found.

During primary culture, the effects of progesterone (P) and estradiol (E₂) were tested on glial cell multiplication, morphology and differentiation. Cell growth was inhibited by P and stimulated by E₂. Both hormones induced dramatic morphologic changes in oligodendrocytes and astrocytes and increased synthesis of the myelin basic protein in oligodendrocytes and of the glial fibrillary acidic protein in astrocytes.     

Purine enzyme inhibition fails to alter benzodiazepine receptor binding in brain

Binding of [³H]flunitrazepam to benzodiazepine receptors in brain from several species, including human, was measured in vitro in the presence and absence of purine-metabolizing enzyme inhibitors. Incubation with potent inhibitors of either adenosine deaminase (2′-deoxycoformycin and erythro-9-(2-hydroxy-3-nonyl)-adenine) or guanine deaminase (5-amino-4-imidazole carboxamide) failed to alter [³H]flunitrazepam binding in homogenates of several different regions of human, rabbit, rat or guinea pig brain. These findings are in contrast to those of Norstrand et al. [Enzyme 29, 61–65 (1983)] who reported substantial alterations in [³H]flunitrazepam binding to human brain membranes in the presence of erythro-9-(2-hydroxy-3-nonyl)-adenine (increase) and 5-amino-4-imidazole carboxamide (decrease). In our studies, [³H]flunitrazepam binding was also unaltered in more anatomically intact brain sections following treatment with purine enzyme inhibitors. Furthermore, in vivo administration of erythro-9-(2-hydroxy-3-nonyl)-adenine to mice at a dose (200 mg/kg, i.p.) known to almost totally inhibit central adenosine deaminase activity also failed to alter brain [³H]flunitrazepam binding measured ex vivo, 30–120 min post injection.

While previous studies have shown that purines such as inosine interact with benzodiazepine receptors, our results raise some questions about the role of endogenous purines in regulating benzodiazepine receptors, at least in vitro and also acutely _vivo following purine enzyme inhibitor administration.     

Autoradiography of CCK receptors in the rat brain using [H]Boc[Nle]CCK27–33 and [I]bolton-hunter CCK8. Functional significance of subregional distributions

[³H]Boc[Nle^28,31]CCK₂₇–₃₃ ([³H]BDNL-CCK₇) is a new ligand for cholecystokinin (CCK) receptors, endowed with a high specific activity (100 Ci/mmol). Binding sites for this ligand were visualized in the rat brain by autoradiography [³H]BDNL-CCK₇ binds specifically to an apparent single class of CCK receptors on rat striatum sections with a K_d of 1.76 nM and a B_max of 57 fmol/mg protein. Unsulfated CCK₈ was two times less potent than sulfated CCK₈ to displace binding of [³H]BDNL-CCK₇. Binding sites for [³H]BDNL-CCK₇ were present in many brain regions, the highest concentrations occurring in cortex, olfactory bulbs, nucleus accumbens, and medium to high concentrations in striatum, hippocampus, and several nuclei of thalamus, hypothalamus and amygdala. In the same experimental conditions, the binding sites for [¹²⁵I]BH-CCK₈ showed similar specificity and localization. We thus used both ligands to investigate the subregional distributions of CCK receptors in nucleus accumbens and hippocampus, where a highly organized topography of action of CCK has been reported. In nucleus accumbens, the CCK binding sites were concentrated in the anterior portion of the nucleus, whereas very low densities were observed within medial posterior nucleus accumbens, where injection of CCK has been shown to potentiate dopamine-induced hyperlocomotion. p]In hippocampus, CCK receptors were concentrated in the polymorphic zone of the hilus of the dentate gyrus and in stratum lacunosum moleculare of Ammon's horn. Very few receptors were observed in other regions of hippocampus, including stratum pyramidale and stratum moleculare. This is in contrast with the presence of numerous CCK terminals and the potent effect of CCK in these areas. The distributions of CCK receptors reported here in both nucleus accumbens and hippocampus were discussed in correlation with the distribution of CCK neurons and terminals, the related anatomical pathways, and the pharmacological profiles of the effects of CCK in these regions.