Autoradiographic characterization of [3H]inositol (1,4,5) trisphosphate and [3H]inositol (1,3,4,5) tetrakisphosphate binding sites in human brain

Autoradiographic techniques were used to investigate the characteristics of tritiated inositol(1,4,5)trisphosphate ([3H]IP3) and inositol (1,3,4,5) tetrakisphosphate ([3H]IP4) binding to human brain. In brain sections [3H]IP3 exhibited a two-site binding with KD values of 87 nM and 9.3 microM respectively for the higher and lower affinity sites. [3H]IP4 also bound to two sites with KD values of 43 nM and 1.4 microM, respectively. With the conditions fixed in this study, [3H]IP3 and [3H]IP4 autoradiography in the cortex, caudate, hippocampus and cerebellum were performed. The most prominent [3H]IP3 binding among these regions was found in the cerebellum, particularly in the molecular layer. Within the hippocampus, the subiculum and the CA1 region showed much more prominent binding than the other subfields. [3H]IP4, binding was fairly homogeneous in the regions studied, with the exception of a slightly higher binding in the molecular layer of the cerebellum.     

Molecular diversity and double regulatory mechanism of activation of phospholipase C in rat brain

Whereas evidence for a G protein-dependent stimulation of phospholipase C (PLC) is abundant, reports on the inhibition of PLC through a G protein-mediated pathway have only recently begun to appear. In the present study, cerebral cortex membranes were chosen since they have a readily measurable Gpp[NH]p and Ca2+-stimulated PLC activity. Nanomolar concentrations of Gpp[NH]p, a hydrolysis-resistant GTP analogue, inhibited basal inositol 1,4,5-trisphosphate (IP3) production, with a maximum inhibition of 25% at 10 nM. Increasing the concentrations of Gpp[NH]p to over 10 nM resulted in a reversal of the inhibitory effect and onset of stimulation of IP3 production. GDPbetaS as a G protein inhibitor and U-73122 as a putative PLC-beta inhibitor had little effect on basal IP3 production at 100 microM and 1 microM, respectively. However, GDPbetaS and U-73122 completely antagonized both the inhibition and the stimulation of IP3 production produced by lower and higher concentrations, respectively, of Gpp[NH]p. Rat cortical membranes expressed a greater amount of PLC-beta1. These data suggest that PLC-beta1 isozymes may be regulated by both inhibitory and stimulatory G protein-mediated mechanisms.     

Neurosteroid modulation of [3H]flunitrazepam binding in the medulla: an autoradiographic study

Neurosteroids bind to unique sites on the GABA(A) receptor complex and modulate receptor function. The effects of neurosteroids on GABA(A) receptors have been well characterized in forebrain regions. However, little is known about their effects on GABA(A) receptors in the medulla, especially those areas involved in autonomic reflex pathways. Stimulation of [3H]flunitrazepam binding to the GABA(A) receptor by two progesterone metabolites, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha-OH-DHP) and 3beta-hydroxy-5alpha-pregnan-20-one (3beta-OH-DHP), was studied using autoradiographic methods in the medulla and cerebellum of female rats at estrus. [3H]Flunitrazepam binding was enhanced by 3alpha-OH-DHP in every nucleus examined in the medulla and cerebellum. This effect was stereoselective since 3beta-OH-DHP had no effect on binding in any region. No differences were observed in the degree of stimulation of [3H]flunitrazepam binding by 3alpha-OH-DHP among medullary brain regions. However, in the cerebellum, the stimulation of binding was significantly greater in the granular layer than in the molecular layer. Stimulation of [3H]flunitrazepam binding by 3alpha-OH-DHP in nuclei involved in the baroreflex pathways supports previous studies which report that neurosteroids modulate autonomic regulation of blood pressure. These actions may also underlie alterations in autonomic function during pregnancy.     

Regulation of beta-adrenergic receptors by guanyl-5'-yl imidodiphosphate and other purine nucleotides

Guanyl-5'-yl imidodiphosphate (Gpp(NH)p), GTP, and other purine nucleotides selectively decrease the binding affinity of the beta-adrenergic receptors of frog erythrocyte membranes for beta-adrenergic agonists but not antagonists. Shifts in binding affinity were assessed by determining the ability of unlabeled ligands to compete with (-)-[3H]dihydroalprenolol for the membrane-bound receptors. The magnitude of the"right" shift in the binding displacement curve for any of 13 ligands tested was directly related to the intrinsic activity (maximal stimulatory capacity) of that agent for stimulation of the frog erythrocyte membrane adenylate cyclase. Thus, Gpp(NH)p-induced shifts in binding affinity were greatest for full agonists such as isoproterenol, intermediate for partial agonists such as soterenol, and no shifts were observed for antagonists such as propranolol. Shifts in binding affinity were observed only in preparations where agonist binding to the receptors leads to "coupling" of the receptors with adenylate cyclase. In solubilized preparations where the beta-adrenergic receptors and adenylate cyclase are functionally "uncoupled", Gpp(NH)p did not cause right shifts in agonist receptor binding displacement curves. In particulate preparations the Km of Gpp(NH)p for stimulation of adenylate cyclase was identical with that for its effect on beta-adrenergic agonist binding affinity, 1 to 2 muM. Moreover, the ability of several other nucleotides to cause shifts in receptor binding affinity directly paralleled their previously determined affinities for the nucleotide regulatory sites on adenylate cyclase. Gpp(NH)p also shifted agonist dose-response curves for stimulation of adenylate cyclase, but to the left. As with the effects on the receptor binding curves, the effects of Gpp(NH)p on the "apparent affinities" of agonists for enzyme stimulation were directly related to their intrinsic activities. Gpp(NH)p also markedly increased the intrinsic activity of partial agonists. These results appear to indicate that conformational alterations in adenylate cyclase caused by occupation of nucleotide regulatory sites by Gpp(NH)p are capable of inducing alterations in the beta-adrenergic receptors. These receptor alterations are induced only when the receptors are "coupled" to the enzyme by virtue of agonist binding. The nucleotide-altered conformation of the beta-adrenergic receptors is characterized by decreased binding affinity for agonist but increased functional efficacy in stimulating the enzyme.     

Thermodynamic parameters of opioid binding in the presence and absence of G-protein coupling

We have investigated the thermodynamic parameters of various opioid ligands interacting with their receptors in rat brain membranes. Affinity constants (Ka), enthalpy and entropy values were obtained from homologous displacement experiments performed at 0, 24 and 33 degrees C. It was found that all the opioid agonists tested ([3H]dihydromorphine (DHM) mu alkaloid; [3H]DAMGO mu peptide; [3H]deltorphin-B delta peptide) display endothermic binding accompanied with a large entropy increase, regardless of their chemical structure (alkaloid or peptide), or of their mu or delta receptor selectivity. In contrast, binding of the antagonist naloxone is exothermic, mainly enthalpy driven. Na+ or Mg2+ results only in quantitative changes of the thermodynamic parameters. In the presence of the GTP-analog Gpp(NH)p; or Gpp(NH)p + Na+; or Gpp(NH)p + Na- + Mg2+ the affinity of DHM binding dramatically decreases which might reflect functional uncoupling of the receptor-ligand complex and G-proteins. This altered molecular interactions are also indicated by curvilinear van't Hoff plot and entropy increase. It is concluded that the thermodynamic analysis provides means of determining the underlying driving forces of ligand binding and helps to delineate its mechanism.     

Autoradiographic visualization of group III metabotropic glutamate receptors using [3H]-L-2-amino-4-phosphonobutyrate

1. In vitro receptor autoradiography using [3H]-L-2-amino-4-phosphonobutyrate ([3H]-L-AP4) binding to sections of rat brain has been characterized and shown to most likely represent labelling of group III metabotropic glutamate receptors. 2. Specific [3H]-L-AP4 binding to rat brain sections was observed at high densities in the molecular layer of the cerebellar cortex and the outer layer of the superior colliculus. Moderate levels were observed throughout the cerebral cortex, in the molecular layer of the hippocampal dentate gyrus, in thalamus, striatum, substantia nigra and in the medial geniculate nucleus. Low levels of [3H]-L-AP4 binding were found in other regions of the hippocampal formation, in the entorhinal cortex and the granule cell layer of cerebellum. 3. Inhibitors of sodium- or calcium/chloride-dependent glutamate uptake did not displace [3H]-L-AP4 binding to rat brain sections indicating that the observed binding does not represent [3H]-L-AP4 uptake via these carriers. Furthermore, in contrast to [3H]-L-AP4 uptake into cerebellar membranes, [3H]-L-AP4 binding to brain sections was sensitive to guanosine-5'-O-(3-thio)trisphosphate-gamma-S. 4. In the molecular layer of the cerebellar cortex, [3H]-L-AP4 binding showed a maximal binding density (Bmax) of 0.52+/-0.06 pmol mg(-1) tissue and an affinity (Kd) of 346 nM. The rank order of affinity for displacement of [3H]-L-AP4 binding to rat brain sections was: L-AP4 > L-serine-O-phosphate > glutamate > (L)-2-aminomethyl-4-phosphonobutanoate > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate which is in agreement with a group III metabotropic glutamate receptor pharmacology.     

Atypical in vitro and in vivo binding of [3H]S-14506 to brain 5-HT1A receptors

The tritiated derivative of the potent 5-HT1A receptor agonist S-14506 ?1[2-(4-fluorobenzoylamino)ethyl]-4-(7-methoxynaphtyl)pipera zine? was tested for its capacity to selectively label the serotonin 5-HT1A receptors both in vitro in the rat and the mouse brain, and in vivo in the mouse. In vitro studies showed that the pharmacological profile and the distribution of [3H]S-14506 specific binding sites (Kd = 0.15 nM) in different brain regions matched perfectly those of the prototypical 5-HT1A receptor ligand [3H]8-OH-DPAT. However, in the three regions examined (hippocampus, septum, cerebral cortex), the density of [3H]S-14506 specific binding sites was significantly higher (+66-90%) than that found with [3H]8-OH-DPAT. Whereas the specific binding of [3H]8-OH-DPAT was markedly reduced by GTP and Gpp(NH)p and increased by Mn2+, that of [3H]S-14506 was essentially unaffected by these compounds. In addition, the alkylating agent N-ethylmaleimide was much less potent to inhibit the specific binding of [3H]S-14506 than that of [3H]8-OH-DPAT. Measurement of in vivo accumulation of tritium one hour after i.v. injection of [3H]S-14506 to mice revealed marked regional differences, with about 2.5 times more radioactivity in the hippocampus than in the cerebellum. Pretreatment with 5-HT1A receptor ligands prevented tritium accumulation in the hippocampus but not in the cerebellum. Autoradiograms from brain sections of injected mice confirmed the specific in vivo labeling of 5-HT1A receptors by [3H]S-14506, therefore suggesting further developments with derivatives of this molecule for positron emission tomography in vivo in man.     

The development of an instrument to measure self-consistency

These studies examined which alpha 2-adrenoceptor subtype is expressed in the hypothalamus and preoptic area and the influence of estradiol administration on alpha 2-adrenoceptors in the hypothalamus of female rats. The alpha 2-adrenoceptor antagonist [3H] RX821002 bound to a single site in hypothalamus, preoptic area, and cortex membranes, with high affinity and low nonspecific binding, as determined by Scatchard and kinetic binding analyses. Competition for [3H]RX821002 binding in the hypothalamus and preoptic area by various noradrenergic agonists and antagonists revealed a unique pharmacological specificity with a high degree of similarity to that of the alpha 2D-adrenoceptor. Norepinephrine displacement of [3H]RX821002 binding in hypothalamic membranes from ovariectomized animals was monophasic and characterized by high affinity. In contrast, norepinephrine competition for [3H]RX821002 binding sites in the hypothalamus from rats exposed to estradiol for 48 hr was biphasic, and norepinephrine bound to both a high (18%) and a low (82%) affinity site in these membranes. Thus, the formation of agonist high affinity alpha 2D-adrenoceptor complexes was inhibited by prior exposure to estrogen. In both control and estradiol-exposed hypothalamic membranes, 100 microM 5'-guanylylimidodiphosphate [Gpp(NH)p] converted the norepinephrine competition curves to ones characterized by monophasic, low affinity binding. In addition, binding of the full alpha 2-adrenoceptor agonist [3H]UK-14,304 in the hypothalamus and preoptic area of female rats was concentration-dependently diminished by Gpp(NH)p treatment. Complete loss of [3H]UK-14,304 binding was effected by 100 microM Gpp(NH)p. This suggests that [3H]UK-14,304 may be useful in labeling the agonist high affinity state of alpha 2-adrenoceptors. Decreasing the incubation temperature in saturation studies from 25 degrees to 0 degrees increased [3H]UK-14,304 binding in hypothalamic membranes of control rats but not in membranes from estradiol-treated rats. Estradiol treatment for 48 hr decreased [3H]UK-14,304 binding in hypothalamic membranes by 34% (0 degrees) to 60% (25 degrees), without changing the Kd. These results suggest that the alpha 2D-adrenoceptor is the predominant subtype in the hypothalamus and preoptic area of female rats and that estradiol treatment markedly reduces the number of alpha 2D-adrenoceptors in the agonist high affinity state.     

Age-related changes of sodium-dependent D-[3H]aspartate and [3H]FK506 binding in rat brain

We investigated age-related changes in excitatory amino acid transport sites and FK506 binding protein (FKBP) in 3-week-, and 6-, 12-, 18- and 24-month-old Fischer 344 rat brains using receptor autoradiography. Sodium-dependent D-[3H]aspartate and [3H]FK506 were used to label excitatory amino acid transport sites and immunophilin (FKBP), respectively. In immature rats (3-week-old), sodium-dependent D-[3H]aspartate binding was lower in the frontal cortex, parietal cortex, striatum, nucleus accumbens, whole hippocampus, thalamus and cerebellum as compared to adult animals (6-month-old), whereas [3H]FK506 binding was significantly lower in only the hippocampus, thalamus and cerebellum. 3[H]FK506 binding exhibited no significant change in the brain regions examined during aging. However, sodium-dependent D-[3H]aspartate binding showed a conspicuous reduction in the substantia nigra in 18-month-old rats. Thereafter, a significant reduction in sodium-dependent D-[3H]aspartate binding was found in the thalamus, substantia nigra and cerebellum in 24-month-old rats. Other regions also showed about 10-25% reduction in sodium-dependent D-[3H]aspartate binding. The results indicate that excitatory amino acid transport sites are more susceptible to aging process than immunophilin. Further, our findings demonstrate the conspicuous differences in the developmental pattern between excitatory amino acid transport sites and immunophilin in immature rat brain.     

Systemic action of human growth hormone following adenovirus-mediated gene transfer to rat submandibular glands

1. The purpose of this study was to compare the effect of NIK-247 on muscarinic receptor subtypes with that of tacrine (THA) in rats. 2. NIK-247 and tacrine dose dependently inhibited the binding of [3H]pirenzepine (M1), [3H]AF-DX 384 (M2), and [3H]4-DAMP (M3). The IC50 values for NIK-247 were 4.4 x 10(-6) M, 1.1 x 10(-5) M, and 1.5 x 10(-5) M, respectively, whereas those for tacrine were 5.8 x 10(-7) M, 2.0 x 10(-6) M, and 5.8 x 10(-6) M, respectively. 3. Gpp[NH]p, a GTP analogue, slightly shifted the curve of displacement of [3H]AF-DX. 384 binding for NIK-247 to the right. However, Gpp[NH]p did not shift the curve of displacement of [3H]pirenzepine and [3H]4-DAMP binding to the right. 4. NIK-247 moderately decreased the rate of beating in right atrial preparations, but did not decrease it below 50% of control level. 5. These findings indicate that NIK-247 is an M1 antagonist, M2 partial agonist, and M3 antagonist.     

Lobeline and nicotine evoke [3H]overflow from rat striatal slices preloaded with [3H]dopamine: differential inhibition of synaptosomal and vesicular [3H]dopamine uptake

Lobeline is currently being developed as a substitution therapy for tobacco smoking cessation. Activation of CNS dopamine (DA) systems results in the reinforcing properties of nicotine. The present study compared the effects of lobeline and nicotine on rat striatum. Both lobeline and nicotine evoked [3H]overflow from striatal slices superfused in the presence of pargyline and nomifensine in the buffer. Marked DA depletion (42-67%) and a concomitant 2-fold increase in dihydroxyphenylacetic acid (DOPAC) in slices superfused with high concentrations (30-100 microM) of lobeline were observed. The effect of nicotine (10 microM) was inhibited in a concentration-dependent manner by mecamylamine (1-100 microM). However, lobeline (0.1-100 microM)-evoked [3H]overflow was calcium-independent, and was not antagonized by mecamylamine (1-100 microM), suggesting a mechanism of action other than stimulation of nicotinic receptors. Lobeline inhibited [3H]DA uptake into synaptosomes (IC50 = 80 +/- 12 microM) and vesicles (IC50 = 0.88 +/- 0.001 microM), whereas nicotine (< or =100 microM) did not inhibit synaptosomal or vesicular [3H]DA uptake. In the absence of pargyline and nomifensine in the buffer, endogenous DA was detected in superfusate only in those slices exposed to the highest concentration (100 microM) of lobeline. However, endogenous DOPAC concentration was increased in a concentration-dependent manner, indicating that lobeline exposure resulted in increased cytosolic DA which was rapidly metabolized to DOPAC. Under these conditions, lobeline (10-100 microM) also significantly depleted (66-85%) DA content; however, no change in DOPAC content was observed. The results suggest that, unlike nicotine, lobeline increases DA release by potent inhibition of DA uptake into synaptic vesicles, and a subsequent alteration in presynaptic DA storage.     

In vivo and in vitro regulation of [3H]glyburide binding to brain sulfonylurea receptors in obesity-prone and resistant rats by glucose

Select brain neurons increase their firing rate when ambient glucose levels rise, possibly via a neuronal ATP-sensitive K+ (KATP) channel and its associated sulfonylurea receptor (SUR). We used receptor autoradiographic binding of 20 nM [3H]glyburide (in the presence or absence of Gpp(NH)p which blocks binding to low-affinity sites) to assess the in vivo and in vitro effects of altering glucose availability upon high- and low-affinity binding to brain SUR. Since the brain's ability to monitor and regulate glucose metabolism is critical to maintenance of energy balance, testing was done in chow-fed male Sprague-Dawley rats which had an underlying predisposition to develop either diet-induced obesity (DIO-prone) or to be diet-resistant (DR-prone) when subsequently fed a high-energy diet. Under control conditions, both in vivo and in vitro studies showed DIO-prone rats to have reduced levels of low-, but not high-affinity [3H]glyburide binding in most forebrain areas. As compared to equiosmolar infusions of mannitol, 60 min unilateral intracarotid glucose infusions at 4 mg/kg/min in awake rats reduced low-affinity [3H]glyburide binding in numerous hypothalamic and amygdalar areas of both DR- and DIO-prone rats with little effect on high-affinity binding. Only in the paraventricular nucleus of DR-prone rats was there a phenotype-specific downregulation of low-affinity binding. Brain sections from other rats were incubated with [3H]glyburide in the presence of 0, 5 or 10 mM glucose. The resultant in vitro effects of glucose were more variable and widespread than intracarotid infusions. Here, glucose often increased low-affinity [3H]glyburide binding, particularly in DR-prone rats at 5 mM. Again, there was little effect on high-affinity binding. Thus, glucose may affect the firing of glucose-responsive neurons by indirectly altering KATP channel function via its effects on low-affinity cell body SUR.     

Selective labelling of 5-HT7 receptor recognition sites in rat brain using [3H]5-carboxamidotryptamine

The aim of the present study was to establish a radioligand binding assay to selectively label the native 5-HT7 receptor expressed in rat brain. In rat whole brain (minus cerebellum and striatum) homogenate, (+/-)-pindolol (10 microM)-insensitive [3H]5-CT ([3H]5-carboxamidotryptamine; 0.5 nM) specific binding (defined by 5-HT, 10 microM) displayed a pharmacological profile similar to the recombinant 5-HT7 receptor, although the Hill coefficients for competition curves generated by methiothepin, ritanserin, sumatriptan, clozapine and pimozide were significantly less than unity. In homogenates of rat hypothalamus, (+/-)-pindolol (10 microM)-insensitive [3H]5-CT recognition sites also resembled, pharmacologically, the 5-HT7 receptor, although pimozide still generated Hill coefficients significantly less than unity. Subsequent studies were performed in the additional presence of WAY100635 (100 nM) to prevent [3H]5-CT binding to residual, possibly, 5-HT1A sites. Competition for this [3H]5-CT binding indicated the labelling in whole rat brain homogenate of a homogenous population of sites with the pharmacological profile of the 5-HT7 receptor. Saturation studies also indicated that (+/-)-pindolol (10 microM)/WAY 100635 (100 nM)-insensitive [3H]5-CT binding to homogenates of whole rat brain was saturable and to an apparently homogenous population of sites which were labelled with nanomolar affinity (Bmax=33.2+/-0.7 fmol mg(-1) protein, pKd=8.78+/-0.05, mean+/-S.E.M., n=3). The development of this 5-HT7 receptor binding assay will aid investigation of the rat native 5-HT7 receptor.     

Interaction between the mu-agonist dermorphin and the delta-agonist [D-Ala2, Glu4]deltorphin in supraspinal antinociception and delta-opioid receptor binding

1. In rats, the interaction between the mu-opioid agonist dermorphin and the delta-opioid agonist [D-Ala2, Glu4]deltorphin was studied in binding experiments to delta-opioid receptors and in the antinociceptive test to radiant heat. 2. When injected i.c.v., doses of [D-Ala2, Glu4]deltorphin higher than 20 nmol produced antinociception in the rat tail-flick test to radiant heat. Lower doses were inactive. None of the doses tested elicited the maximum achievable response. This partial antinociception was accomplished with an in vivo occupancy of more than 97% of brain delta-opioid receptors and of 17% of mu-opioid receptors. Naloxone (0.1 mg kg-1, s.c.), and naloxonazine (10 mg kg-1, i.v., 24 h before), but not the selective delta-opioid antagonist naltrindole, antagonized the antinociception. 3. In vitro competitive inhibition studies in rat brain membranes showed that [D-Ala2, Glu4]deltorphin displaced [3H]-naltrindole from two delta-binding sites of high and low affinity. The addition of 100 microM Gpp[NH]p produced a three fold increase in the [D-Ala2, Glu4]deltorphin Ki value for both binding sites. The addition of 10 nM dermorphin increased the Ki value of the delta-agonist for the high affinity site five times. When Gpp[NH]p was added to the incubation medium together with 10 nM dermorphin, the high affinity Ki of the delta-agonist increased 15 times. 4. Co-administration into the rat brain ventricles of subanalgesic doses of dermorphin and [D-Ala2, Glu4]deltorphin resulted in synergistic antinociceptive responses. 5. Pretreatment with naloxone or with the non-equilibrium mu-antagonists naloxonazine and beta-funaltrexamine completely abolished the antinociceptive response of the mu-delta agonist combinations. 6. Pretreatment with the delta-opioid antagonists naltrindole and DALCE reduced the antinociceptive response of the dermorphin-[D-Ala2, Glu4]deltorphin combinations to a value near that observed after the mu-agonist alone. At the dosage used, naltrindole occupied more than 98% of brain delta-opioid receptors without affecting mu-opioid-receptors. 7. These data suggest that in the rat tail-flick test to radiant heat, mu- and delta-opioid agonists co-operate positively in evoking an antinociceptive response. Although interactions between different opioid pathways cannot be excluded, in vitro binding results indicate that this co-operative antinociception is probably mediated by co-activation of the delta-opioid receptors at the cellular level by the mu- and delta-agonist.     

Perivascular localization of nitric oxide synthase in the rat adenohypophysis: potential implications for function and cell-cell interaction

Effects of activation of protein kinase C (PKC) on N-methyl-D-aspartate) NMDA receptor function were analyzed by quantitative autoradiography using [3H]MK-801 in rat brain slices. The density of [3H]MK-801 binding was highest in hippocampus and high levels were found in cortex, striatum and thalamus. Levels in brainstem and molecular layer of cerebellum were low. The receptor binding was markedly decreased in almost all areas by addition of 2. 5 mM Mg2+. After activation of PKC by 100 nM phorbol-12, 13-dibutyrate (PDBu), [3H]MK-801 binding was increased in most areas, but binding levels were not changed in brainstem and cerebellum. The elevated [3H]MK-801 binding produced by PDBu was significantly inhibited by addition of Mg2+ except in inferior colliculus and cerebellum. These results suggest that activation of PKC potentiates NMDA receptor function in a region-specific manner in the rat brain.     

Characterization of 5,7-dichlorokynurenate-insensitive D-[3H]serine binding to synaptosomal fraction isolated from rat brain tissues

To explore target sites for endogenous D-serine that are different from the glycine site of the N-methyl-D-aspartate (NMDA) type glutamate receptor, we have studied the binding of D-[3H]serine to the synaptosomal P2 fraction prepared from the rat brain and peripheral tissues in the presence of an excess concentration (100 microM) of the glycine site antagonist 5,7-dichlorokynurenate (DCK). Nonspecific binding was defined in the presence of 1 mM unlabeled D-serine. Association, dissociation, and saturation experiments indicated that D-[3H]serine bound rapidly and reversibly to a single population of recognition sites in the cerebellar P2 fraction in the presence of DCK, with a K(D) of 614 nM and a Bmax of 2.07 pmol/mg of protein. D-Serine, L-serine, and glycine produced a total inhibition of the specific DCK-insensitive D-[3H]serine binding to the cerebellum with similar Ki values. Strychnine and 7-chlorokynurenate failed to inhibit the binding at 10 microM. The profiles of displacement of the DCK-insensitive D-[3H]serine binding by various amino acids and glutamate and glycine receptor-related compounds differ from those of any other defined recognition sites. DCK-insensitive D-[3H]serine binding was at high levels in the cerebral cortex and cerebellum but very low in the kidney and liver. The present findings indicate that the DCK-insensitive D-[3H]serine binding site could be a novel candidate for a target for endogenous D-serine in mammalian brains.     

Liposomal amikacin: improved treatment of Mycobacterium avium complex infection in the beige mouse model

1. Binding of D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP 39653), a high affinity, selective antagonist at the glutamate site of the N-methyl-D-aspartate (NMDA) receptor, was investigated in rat brain by means of receptor binding and quantitative autoradiography techniques. 2. [3H]-CGP 39653 interacted with striatal and cerebellar membranes in a saturable manner and to a single binding site, with KD values of 15.5 nM and 10.0 nM and receptor binding densities (Bmax values) of 3.1 and 0.5 pmol mg-1 protein, respectively. These KD values were not significantly different from that previously reported in the cerebral cortex (10.7 nM). 3. Displacement analyses of [3H]-CGP 39653 in striatum and cerebellum, performed with L-glutamic acid, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and glycine showed a pharmacological profile similar to that reported in the cerebral cortex. L-Glutamic acid and CPP produced complete displacement of specific binding with Ki values not significantly different from the cerebral cortex. Glycine inhibited [3H]CGP 39653 binding with shallow, biphasic curves, characterized by a high and a low affinity component. Furthermore, glycine discriminated between these regions (P < 0.005, one-way ANOVA), since the apparent Ki of the high affinity component of the glycine inhibition curve (KiH) was significantly lower (Fisher's protected LSD) in the striatum than the cortex (33 nM and 104 nM, respectively). 4. Regional binding of [3H]-CGP 39653 to horizontal sections of rat brain revealed a heterogeneous distribution of binding sites, similar to that reported for other radiolabelled antagonists at the NMDA site (D-2-[3H]-amino-5-phosphonopentanoic acid ([3H]-D-AP5) and [3H]-CPP). High values of binding were detected in the hippocampal formation, cerebral cortex and thalamus, with low levels in striatum and cerebellum. 5. [3H]-CGP 39653 binding was inhibited by increasing concentrations of L-glutamic acid, CPP and glycine. L-Glutamic acid and CPP completely displaced specific binding in all regions tested, with similar IC50 values throughout. Similarly, glycine was able to inhibit the binding in all areas considered: 10 microM and 1 mM glycine reduced the binding to 80% and 65% of control (average between areas) respectively. The percentage of specific [3H]-CGP 39653 binding inhibited by 1 mM glycine varied among regions (P < 0.05, two-ways ANOVA). Multiple comparison, performed by Fisher's protected LSD method, showed that the inhibition was lower in striatum (72% of control), with respect to cortex (66% of control) and hippocampal formation (58% of control). 6. The inhibitory action of 10 microM glycine was reversed by 100 microM 7-chloro-kynurenic acid (7-CKA), a competitive antagonist of the glycine site of the NMDA receptor channel complex, in all areas tested. Moreover, reversal by 7-CKA was not the same in all regions (P < 0.05, two-ways ANOVA). In fact, in the presence of 10 microM glycine and 100 microM 7-KCA, specific [3H]-CGP 39653 binding in the striatum was 131% of control, which was significantly greater (Fisher's protected LSD) than binding in the hippocampus and the thalamus (104% and 112% of control, respectively). 7. These results demonstrate that [3H]-CGP 39653 binding can be inhibited by glycine in rat brain regions containing NMDA receptors; moreover, they suggest the existence of regionally distinct NMDA receptor subtypes with a different allosteric mechanism of [3H]-CGP 39653 binding modulation through the associated glycine site.     

Comparative autoradiographic distribution of central omega (benzodiazepine) modulatory site subtypes with high, intermediate and low affinity for zolpidem and alpidem

Pharmacological characterization of [3H]benzodiazepine binding to membrane preparations of adult rat hippocampus and neonatal rat brain have demonstrated, in addition to the omega 1 and omega 2 populations of central omega benzodiazepine binding sites associated with GABAA receptors, the existence of binding sites with microM affinity for the imidazopyridines zolpidem and alpidem. In the present study we have investigated their comparative autoradiographic distribution using [3H]flumazenil as a ligand. In the neonatal rat CNS, the imidazopyridine derivatives zolpidem and alpidem were found to discriminate two [3H]flumazenil binding site populations with an IC50 value ratio of more than 200-fold. In the different regions investigated (spinal cord, striatum, neocortex and inferior colliculus) the low affinity component had IC50 values of 20-40 microM (zolpidem) and 5-15 microM (alpidem) and accounted for ca. 50% of the total binding site population. In the adult rat, these imidazopyridine derivatives displayed a greater displacing potency in the cerebellum (IC50 = 6 and 36 nM, respectively) than in the hippocampus (IC50 = 37 and 403 nM, respectively). In the cerebellum, [3H]flumazenil binding was fully displaced by 1 microM of either compound and Hill coefficients of displacement curves were close to unity. In the hippocampus, 25% of [3H]flumazenil binding were resistant to 3 microM zolpidem or 1 microM alpidem, but were displaced by 100 microM of either compound. CL 218,872 also displayed a greater displacing potency in the cerebellum (IC50 = 83 nM) than in the hippocampus (IC50 = 711 nM) but [3H]flumazenil binding in the hippocampus was fully displaced by 10 microM of this compound. In adult rat hippocampus, zolpidem and alpidem were found to discriminate between three central omega site subtypes which display high (IC50 = 31 and 6.1 nM, for these imidazopyridine derivatives. In contrast, CL 218,872 discriminated between omega 1 and omega 2 sites but not between two omega 2 receptor subpopulations. omega 1 sites were mainly localized in layer IV of the sensorimotor cortex, cerebellum, substantia nigra, olfactory bulb and inferior colliculus. omega 2I sites were present in the cortical mantle (with higher levels in the cingulate and olfactory than in the sensorimotor cortex) and in subcortical (hippocampus, hypothalamus and nucleus accumbens) limbic structures. In the hippocampus, hypothalamus, spinal cord and nucleus accumbens, omega 2L sites accounted for more than 25% of the specific [3H]flumazenil binding; the density of these sites was minor in the cortex and in most pyramidal and extrapyramidal system structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective in vivo binding of [3H]naltriben to delta-opioid receptors in mouse brain

Naltriben (NTB) is a selective antagonist for the putative delta2-opioid receptor. We have determined the regional kinetics and pharmacological profile of [3H]naltriben in vivo in mouse brain. After i.v. administration to CD1 mice, [3H]naltriben uptake and retention were high in striatum, cortical regions and olfactory tubercles, and low in superior colliculi and cerebellum. Robust rank order correlation was found between [3H]naltriben uptake in discrete brain regions and prior delta-opioid receptor binding determinations in vitro and in vivo. [3H]Naltriben binding in vivo was saturable, and was blocked by the delta-opioid receptor antagonist naltrindole, but not by the mu-opioid receptor antagonist cyprodime or the K-opioid receptor agonist (trans)-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]ben zeneacetamide mesylate (U50,488H). (E)-7-Benzylidenenaltrexone (BNTX), a selective antagonist for the putative delta1-opioid receptor, was 9.6- to 12.9-fold less potent than naltriben as an inhibitor of [3H]naltriben binding. Thus, the sites labeled by [3H]naltriben in vivo may correspond to the delta2-opioid receptor subtype. Such assignment is not definitive, particularly considering the 4-fold higher brain uptake of naltriben as compared to (E)-7-benzylidenenaltrexone. Moreover, the regional distribution of [3H]naltriben in brains from CXB-7/BY (CXBK) mice, a strain that shows supraspinal delta1- but not delta2-opioid receptor agonist effects, was quite similar to that found for CD1 mice.