Pharmacological specificity of some psychotomimetic and antipsychotic agents for the sigma and PCP binding sites

The pharmacological specificity of representative psychotomimetic agents such as phencyclidine (PCP) analogs, opiate benzomorphans and several antipsychotic agents was assessed for the sigma and PCP binding sites. In a series of binding experiments, in rat brain membranes, sigma and PCP binding sites were labeled with [3H]-1-[1-(3-hydroxyphenyl)cyclohexyl]piperidine [( 3H]PCP-3-OH), (+) [3H]-N-allylnormetazocine [(+) [3H]SKF 10047] and (+) [3H]-3-[3-hydroxy-phenyl]-N-(1-propyl)piperidine [(+) [3H]-3-PPP]. PCP analogs inhibit potently high affinity [3H]PCP-3-OH binding and (+) [3H]SKF 10047 binding, moderately the low affinity binding component of [3H]PCP-3-OH and very weakly (+) [3H]-3-PPP binding. (+)SKF 10047 and cyclazocine are potent to moderate inhibitors of (+) [3H]SKF 10047, high affinity [3H]PCP-3-OH and (+) [3H]-3-PPP binding, but extremely weak inhibitors of low affinity [3H]PCP-3-OH binding. The antipsychotic agents display high affinity for (+) [3H]-3-PPP binding sites, moderate affinity for (+) [3H]SKF 10047 sites and have no effect on either the high or low affinity [3H]PCP-3-OH binding. The present data further support the existence of multiple sigma and PCP binding sites.     

Characterization of 5-HT transporter and receptor system in HeLaS3 cells by [(3)H]8-OH-DPAT and other serotonergic ligands

[(3)H]8-OH-DPAT is a selective ligand for labeling 5-HT(1A) receptor sites. In competition binding experiments, we found that classic biogenic amine transporter inhibitors displaced [(3)H]8-OH-DPAT binding at its high-affinity binding sites in HeLaS3 cells. [(125)I]RTI-55 and [(3)H]paroxetine are known to specifically label amine transporter sites, and this was observed in our cells. Displacement studies showed that 8-OH-DPAT displayed affinity in a dose-dependent manner for the labeled amine transporter sites. These data suggest that [(3)H]8-OH-DPAT binds to amine uptake sites in HeLaS3 cells. A variety of drugs targeting different classes of receptors did not significantly affect [(3)H]8-OH-DPAT binding. Moreover, we determined the specific binding effects of various serotonergic ligands (i.e. [(125)I]cyanopindolol, [(3)H]ketanserin/[(3)H]mesulergine, [(3)H]GR-65630, [(3)H]GR-113808 and [(3)H]LSD) that specifically labeled 5-HT(1), 5-HT(2), 5-HT(3), 5-HT(4) and 5-HT(5-7) receptors, respectively. It is suggested that HeLaS3 cells contain distinct types of the related to 5-HT receptor recognition binding sites. These observations could help elucidate the relevant characteristics of different types of 5-HT receptors and 5-HT membrane transporters in tumor cells and their role in tumorigenesis.     

Differential regulation of sigma and PCP receptors after chronic administration of haloperidol and phencyclidine in mice

The existence of multiple receptor sites for the psychotomimetic agents phencyclidine (PCP) and some opiate-benzomorphans such as (+)N-allylnormetazocine ([+]SKF 10,047) in the mammalian central nervous system is well documented. These are: 1) sigma/PCP (sigma p) site, which binds both PCP and psychotomimetic opiates but not antipsychotics such as haloperidol, 2) PCP site, which selectively binds PCP analogs, and 3) sigma/haloperidol (sigma h) site, for which certain antipsychotics and (+)SKF 10,047, but not PCP analogs, display high affinity. In this study we examined the regulation of these receptor sites after chronic treatment of mice with either PCP or haloperidol. The following radiolabeled ligands were used to assess binding to the various receptor subtypes: [3H]-1-[1-[3-hydroxyphenyl)cyclohexyl]piperidine ([3H]PCP-3-OH; sigma p and PCP sites), [3H]thienyl-phencyclidine ([3H]TCP; PCP site), (+)-[3H]SKF 10,047 (sigma p and sigma h sites), and [3H]haloperidol (sigma h and D-2 dopamine receptors). Treatment of mice for 1, 7, 14, and 21 days with PCP (10 mg.kg-1.day-1) failed to induce variations in sigma p, sigma h, and PCP receptor binding. However, similar treatment with haloperidol (4 mg.kg-1.day-1) induced: 1) complete elimination of the binding to sigma h sites, 2) up-regulation of D-2 dopamine receptors, and 3) no change in sigma p and PCP receptor binding after 14 or 21 days of treatment. However, a single day of haloperidol treatment or in vitro incubation of mouse brain membranes with haloperidol failed to alter receptor binding. This study suggests that prolonged treatment of mice with haloperidol induces a loss in sigma h receptors that are presumably associated with certain psychotomimetic effects. This phenomenon is accompanied by an up-regulation of D-2 dopamine receptors.     

Identification of three muscarinic receptor subtypes in rat lung using binding studies with selective antagonists

Heterogeneity of the muscarinic receptor population in the rat central and peripheral lung was found in competition binding experiments against [3H]quinuclidinyl benzilate [( 3H]QNB) using the selective antagonists pirenzepine, AF-DX 116 and hexahydrosiladifenidol (HHSiD). Pirenzepine displaced [3H]QNB with low affinity from preparations of central airways indicating the absence of M1 receptors in the trachea and bronchi. Muscarinic receptors in the central airways are comprised of both M2 and M3 receptors since AF-DX 116, an M2-selective antagonist, bound with high affinity to 70% of the available sites while HHSiD, an M3-selective antagonist bound with high affinity to the remaining binding sites. In the peripheral lung, pirenzepine bound with high affinity to 14% of the receptor population, AF-DX 116 bound with high affinity to 79% of the binding sites while HHSiD bound with high affinity to 18% of the binding sites. The presence of M1 receptors in the peripheral airways but not in the central airways was confirmed using [3H]telenzepine, an M1 receptor ligand. [3H]Telenzepine showed specific saturable binding to 8% of [3H]QNB labeled binding sites in homogenates of rat peripheral lung, while there was no detectable specific binding in homogenates of rat trachea or heart. The results presented here demonstrate that there are three muscarinic receptor subtypes in rat lungs, and that the distribution of the different subtypes varies within the lungs. Throughout the airways, the dominant muscarinic receptor subtype is M2. In the trachea and bronchi the remaining receptors are M3, while in the peripheral lungs, the remaining receptors are both M1 and M3.     

Substituted 1-phenyl-2-cyclopropylmethylamines with high affinity and selectivity for sigma sites

A series of 1-phenyl-2-cyclopropylmethylamines structurally related to (+)- and (-)-MPCB were synthesized and their binding affinities for sigma1, sigma2, opioid and dopamine (D2) receptors were evaluated. Substitution of the cis-N-normetazocine with different aminic moieties provided compounds with high affinity and selectivity for sigma binding sites with respect to opioid and dopamine (D2) receptors. The observed increase in sigma2 affinity as compared to the parent (+)-MPCB, supports the idea that the particular stereochemistry of (+)-cis-N-normetazocine affects sigma1 selectivity but does not affect sigma1 affinity. The (+/-)-cis isomers of methyl 2-[(1-adamantylamino)methyl]-1-phenylcyclopropane-1-carboxyl ate (18) displayed a higher affinity and selectivity for the sigma1 and sigma2 receptor subtypes compared to the (+/-)-trans 19. Interestingly, the enantiomer (-)-cis 18 displayed a preference for sigma1 receptor subtype whereas the (+)-cis 18 did for sigma2. These results prompt us to synthesize compounds with modification of nitrogen and carboxyl groups. The compounds obtained showed high affinities and selectivity for sigma sites. Moreover, modifications of carboxyl groups provided compounds with the highest affinities in the series. In particular, compound 25 with reverse-type ester showed a Ki of 0.6 and 4.05 nM for sigma1 and sigma2 binding sites, respectively.     

Synthesis and evaluation of optically pure [3H]-(+)-pentazocine, a highly potent and selective radioligand for sigma receptors

Tritium-labeled (+)-pentazocine ([3H]-1b) of specific activity 26.6 Ci/mmol was synthesized in 3 steps starting with (+)-normetazocine (2) of defined optical purity. [3H]-1b has been characterized as a highly selective ligand for labeling of sigma receptors. Competition data revealed that [3H]-1b could be displaced from guinea pig brain membrane preparations with a number of commonly used sigma receptor ligands. [3H]-1b exhibited saturable, enantioselective binding with a Kd of 5.13 +/- 0.97 nM and a Bmax of 1146 +/- 122 fmol/mg protein. Phencyclidine (PCP) displaced [3H]-1b with low affinity while MK-801 was inactive, thus indicating insignificant activity at the PCP-binding site; apomorphine failed to displace [3H]-1b indicating lack of dopamine receptor cross-reactivity. Since the affinity of [3H]-1b is about 6 times that of the two commonly employed sigma ligands ((+)-3-[3H]PPP and [3H]DTG) and since it is more selective for sigma receptors than the benzomorphan [3H]SKF-10,047, it represents the first example of a highly selective benzomorphan based sigma receptor ligand. [3H]-1b should prove useful for further study of the structure and function of sigma receptors.     

Nodulisporic acid opens insect glutamate-gated chloride channels: identification of a new high affinity modulator

Nodulisporic acid (NA) is an indole diterpene fungal product with insecticidal activity. NA activates a glutamate-gated chloride channel (GluCl) in grasshopper neurons and potentiates channel opening by glutamate. The endectocide ivermectin (IVM) induces a similar, but larger current than NA. Using Drosophila melanogaster head membranes, a high affinity binding site for NA was identified. Equilibrium binding studies show that an amide analogue, N-(2-hydroxyethyl-2,2-(3)H)nodulisporamide ([(3)H]NAmide), binds to a single population of sites in head membranes with a K(D) of 12 pM and a B(max) of 1.4 pmol/mg of protein. A similar K(D) is determined from the kinetics of ligand binding and dissociation. Four lines of evidence indicate that the binding site is a GluCl. First, NA potentiates opening of a glutamate-gated chloride current in grasshopper neurons. Second, glutamate inhibits the binding of [(3)H]NAmide by increasing the rate of dissociation 3-fold. Third, IVM potently inhibits the binding of [(3)H]NAmide and IVM binds to GluCls. Finally, the binding of [(3)H]IVM is inhibited by NA. The B(max) of [(3)H]IVM is twice that of [(3)H]NAmide, and about half of the [(3)H]IVM binding sites are inhibited by NA with high affinity (K(I) = 25 pM). In contrast, [(3)H]IVM binding to Caenorhabditis elegans membranes is not inhibited by NA at 100 nM, and there are no high affinity binding sites for NA on these membranes. Thus, half of the Drosophila IVM receptors and all of the NA receptors are associated with GluCl. NA distinguishes between nematode and insect GluCls and identifies subpopulations of IVM binding sites.     

Characterization of binding sites for [3H]-DTG, a selective sigma receptor ligand, in the sheep pineal gland

Specific binding sites for [3H]-1,3 di-ortho-tolylguanidine ([3H]-DTG), a selective radiolabeled sigma receptor ligand, were detected and characterized in sheep pineal gland membranes. The binding of [3H]-DTG to sheep pineal membranes was rapid and reversible with a rate constant for association (K+1) at 25 degrees C of 0.0052 nM-1.min-1 and rate constant for dissociation (K-1) 0.0515 min-1, giving a Kd (K-1/K+1) of 9.9 nM. Saturation studies demonstrated that [3H]-DTG binds to a single class of sites with an affinity constant (Kd) of 27 +/- 3.4 nM, and a total binding capacity (Bmax) of 1.39 +/- 0.03 pmol/mg protein. Competition experiments showed that the relative order of potency of compounds for inhibition of [3H]-DTG binding to sheep pineal membranes was as follows: trifluoperazine = DTG greater than haloperidol greater than pentazocine greater than (+)-3-PPP greater than (+/-)SKF 10,047. Some steroids (testosterone, progesterone, deoxycorticosterone) previously reported to bind to the sigma site in brain membranes were very weak inhibitors of [3H]-DTG binding in the present study. The results indicate that [3H]-DTG binding sites having the characteristics of sigma receptors are present in sheep pineal gland. The physiological importance of these sites in regulating the synthesis of the pineal hormone melatonin awaits further study.     

Histamine H(2) -like receptors in chick cerebral cortex: effects on cyclic AMP synthesis and characterization by [(3) H]tiotidine binding

In this study, histamine (HA) receptors in chick cerebral cortex were characterized using two approaches: (1) analysis of the effects of HA-ergic drugs on the cAMP-generating system, and (2) radioreceptor binding of [(3) H]tiotidine, a selective H(2) antagonist. HA was a weak activator of adenylyl cyclase in a crude membrane preparation of chick cerebrum. On the other hand, HA (0.1-1000 microm) potently and concentration dependently stimulated cAMP production in [(3) H]adenine pre-labelled slices of chick cerebral cortex, displaying an EC(50) value (concentration that produces 50% of maximum response) of 2.65 microm. The effect of HA was mimicked by agonists of HA receptors with the following rank order of potency: HA >or= 4-methylHA (H(2)) >or= N alpha,N alpha-dimethylHA (H(3) > H(2) = H(1)) > 2-methylHA (H(1)) > 2-thiazolylethylamine (H(1)) >or= R alpha-methylHA (H(3)) > amthamine, dimaprit (H(2)), immepip (H(3), H(4)). The HA-evoked increase in cAMP production in chick cerebral cortex was antagonized by selective H(2) receptor blockers (aminopotentidine >or= tiotidine > ranitidine > zolantidine), and not significantly affected by mepyramine and thioperamide, selective H(1) and H(3) /H(4) receptor blockers, respectively. A detailed analysis of the antagonistic action of aminopotentidine (vs. HA) revealed a non-competitive mode of action. The binding of [(3) H]tiotidine to chick cortical membranes was rapid, stable and reversible. Saturation analysis resulted in a linear Scatchard plot, suggesting binding to a single class of receptor binding site with high affinity [equilibrium dissociation constant (K (d)) = 4.42 nm] and high capacity [maximum number of binding sites (B (max) ) = 362 fmol/mg protein]. The relative rank order of HA-ergic drugs to inhibit [(3) H]tiotidine binding to chick cerebrum was: antagonists - tiotidine > aminopotentidine = ranitidine >or= zolantadine > thioperamide - triprolidine; agonists - HA >or= 4-methylHA > 2-methylHA >or=R alpha-methylHA - dimaprit. In conclusion, chick cerebral cortex contains H(2) -like HA receptors that are linked to the cAMP-generating system and are labelled with [(3) H]tiotidine. The pharmacological profile of these receptors is different from that described for their mammalian counterpart. It is suggested that the studied receptors represent either an avian-specific H(2) -like HA receptors or a novel subtype of HA receptors.     

Identification of Dopamine "D3'' and "D4'' Binding Sites, Labelled with [3H]2-Amino-6,7-Dihydroxy- 1,2,3,4- Tetrahydronaphthalene, as High Agonist Affinity States of the D1 and D2 Dopamine Receptors, Respectively

On the basis of affinity differences for spiperone, two binding sites for [3H](+/-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene ([3H]ADTN) in the rat brain could be distinguished: "D3" with a low and "D4" with a high affinity for spiperone. Evidence is provided that D3 and D4 sites are related to high agonist affinity states of the D1 and D2 dopamine receptors, respectively. Various well-known selective D1 and D2 agonists and antagonists showed potencies at these sites in agreement with this hypothesis. A comparison of the Bmax values for [3H]ADTN binding to D3 and D4 sites with the numbers of D1 receptors (labelled by [3H]SCH 23390) and of D2 receptors (labelled by [3H]spiperone), both in the striatum and in the mesolimbic system, indicated that under the conditions used for 3H-agonist binding experiments, both populations of D1 and D2 receptors were converted to their high agonist affinity states to a considerable, although different extent. In fact, when competition experiments with [3H]spiperone were performed under the conditions otherwise used for [3H]ADTN binding experiments (instead of the conditions usually used for antagonist binding), substantial shifts of the displacement curves of 3,4-dihydroxyphenylethylamine (dopamine) and ADTN toward higher affinities were observed. A comparison of the effects of various agonists and antagonists in the [3H]ADTN binding experiments and in functional tests revealed a significant correlation between their potencies at D4 binding sites and at D2 receptors modulating the release of [3H]acetylcholine from striatal slices. However, in the situation of the D1/D3 pair, when the measurement of adenylate cyclase activity was taken as a functional test for D1 receptors, agonists were more active in the binding than in the functional test, whereas for many antagonists the opposite was found. The results are discussed with regard to the classification and functional aspects of brain dopamine receptors.     

Comparison of [3H] phencyclidine ([3H] PCP) and [3H] N-[1-(2-thienyl) cyclohexyl] piperidine ([3H] TCP) binding properties to rat and human brain membranes

The investigation of [3H] PCP and [3H] TCP binding properties to rat cerebrum and cerebellum resulted in the demonstration of multiple binding sites for the two drugs. In the two tissue preparations PCP had a lower affinity than TCP. In membranes from the cerebrum an equal number of high affinity binding sites were present for [3H] PCP and [3H] TCP. However, low affinity binding sites were two times more numerous for [3H] PCP than for [3H] TCP. In the cerebellum, the number of high and low affinity sites labeled by the two radioligands was identical, but the number of high affinity sites was about 7 fold lower than in the cerebrum. Taken together these results may indicate that in the cerebrum [3H] PCP labels other sites than NMDA/PCP receptor(s), maybe sigma receptors and/or the dopamine uptake complex. In human cerebral cortex samples [3H] TCP also bound to two different sites. The number of high and low affinity sites were 12 and 3 times, respectively, less abundant than in the rat cerebrum. Low affinity sites were of higher affinity (5 times) than corresponding sites in the rat brain. In the human cerebellum [3H] TCP binding parameters were identical to those measured in the same region in the rat.     

Dopamine D4-like binding sites labeled by [3H]nemonapride include substantial serotonin 5-HT2A receptors in primate cerebral cortex

Dopamine D4-like binding sites are abundant in human cerebral cortex as detected by [3H]nemonapride. The extremely low density of D4 mRNA in human cerebral cortex is inconsistent with the high amount of D4-like binding sites. To investigate the nature of the D4-like receptors, [3H]nemonapride binding sites in the nonhuman primate cerebral cortex were characterized. Although [3H]nemonapride binding sites were D4-like, displaceable by clozapine but not raclopride, [3H]nemonapride binding was not displaced by selective D4 antagonists but was displaced by the selective 5-HT2A antagonist MDL100907. Using [3H]ketanserin as a 5-HT2A ligand, nemonapride showed high affinity for monkey (Ki = 10.4 nM) and cloned human (Ki = 9.4 nM) 5-HT2A receptors, while its affinity for rat receptors was lower (Ki = 140 nM). The present study demonstrates that cerebral cortical D4-like binding sites labeled by [3H]nemonapride in nonhuman primates consist of a very small portion of D4, but a substantial portion of 5-HT2A receptors. The unexpectedly high affinity of nemonapride for primate 5-HT2A receptor suggests reconsidering previous data from other studies using [3H]nemonapride, particularly those on D4-like receptors.     

(3)H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-purin-5-one ([(3)H]PSB-11), a novel high-affinity antagonist radioligand for human A(3) adenosine receptors

This study describes the preparation and binding properties of [(3)H]PSB-11, a novel, potent, and selective antagonist radioligand for human A(3) adenosine receptors (ARs). [(3)H]PSB-11 binding to membranes of Chinese hamster ovary (CHO) cells expressing the human A(3) AR was saturable and reversible. Saturation experiments showed that [(3)H]PSB-11 labeled a single class of binding sites with high affinity (K(D)=4.9 nM) and limited capacity (B(max)=3500 fmol/mg of protein). PSB-11 is highly selective versus the other adenosine receptor subtypes. The new radioligand shows an extraordinarily low degree of non-specific binding rendering it a very useful tool for studying the (patho)physiological roles of A(3 )ARs.     

Further evidence for the presence of "septide-sensitive" tachykinin binding sites in tissues possessing solely NK(1) tachykinin receptors

Binding experiments performed with [(125)I]-NKA allowed us to demonstrate the presence of "septide-sensitive" specific binding sites on membranes from rat CHO cells transfected with the NK(1) receptor cDNA (CHO-rat-NK1 cells), human astrocytoma U373 MG, or mouse cortical astrocytes, cells which express NK(1) but neither NK(2) nor NK(3) receptors. In all cases, [(125)I]-NKA was specifically bound with high affinity (2 to 5 nM) to a single population of sites. In the three preparations, pharmacological characteristics of [(125)I]-NKA binding sites were notably different from those of classical NK(1) binding sites selectively labelled with [(125)I]-BHSP. Indeed, the endogenous tachykinins NKA, NPK, and NKB and the septide-like compounds such as septide, SP(6-11), ALIE-124, [Apa(9-10)]SP, or [Lys(5)]NKA(4-10) had a much higher affinity for [(125)I]-NKA than [(125)I]-BHSP binding sites. Interestingly, differences were also found in the ratio of B(max) values for [(125)I]-NKA and [(125)I]-BHSP specific bindings from one tissue to another. These latter observations suggest that these two types of NK(1) binding sites are present on distinct NK(1) receptor isoforms (or conformers). Finally, while several tachykinins and tachykinin-related compounds stimulated cAMP formation or increased inositol phosphate accumulation in CHO-rat-NK1 cells, these compounds only increased the accumulation of inositol phosphates in the two other preparations.     

Tyrosine 62 of the gamma-aminobutyric acid type A receptor beta 2 subunit is an important determinant of high affinity agonist binding

The gamma-aminobutyric acid type A receptor (GABA(A)R) carries both high (K(D) = 10-30 nm) and low (K(D) = 0.1-1.0 microm) affinity binding sites for agonists. We have used site-directed mutagenesis to identify a specific residue in the rat beta2 subunit that is involved in high affinity agonist binding. Tyrosine residues at positions 62 and 74 were mutated to either phenylalanine or serine and the effects on ligand binding and ion channel activation were investigated after the expression of mutant subunits with wild-type alpha1 and gamma2 subunits in tsA201 cells or in Xenopus oocytes. None of the mutations affected [(3)H]Ro15-4513 binding or impaired allosteric interactions between the low affinity GABA and benzodiazepine sites. Although mutations at position 74 had little effect on [(3)H]muscimol binding, the Y62F mutation decreased the affinity of the high affinity [(3)H]muscimol binding sites by approximately 6-fold, and the Y62S mutation led to a loss of detectable high affinity binding sites. After expression in oocytes, the EC(50) values for both muscimol and GABA-induced activation of Y62F and Y62S receptors were increased by 2- and 6-fold compared with the wild-type. We conclude that Tyr-62 of the beta subunit is an important determinant for high affinity agonist binding to the GABA(A) receptor.     

Biochemical and functional characterization of high-affinity urotensin II receptors in rat cortical astrocytes

The urotensin II (UII) gene is primarily expressed in the central nervous system, but the functions of UII in the brain remain elusive. Here, we show that cultured rat astrocytes constitutively express the UII receptor (UT). Saturation and competition experiments performed with iodinated rat UII ([(125)I]rUII) revealed the presence of high- and low-affinity binding sites on astrocytes. Human UII (hUII) and the two highly active agonists hUII(4-11) and [3-iodo-Tyr9]hUII(4-11) were also very potent in displacing [(125)I]rUII from its binding sites, whereas the non-cyclic analogue [Ser5,10]hUII(4-11) and somatostatin-14 could only displace [(125)I]rUII binding at micromolar concentrations. Reciprocally, rUII failed to compete with [(125)I-Tyr0,D-Trp8]somatostatin-14 binding on astrocytes. Exposure of cultured astrocytes to rUII stimulated [(3)H]inositol incorporation and increased intracellular Ca(2+) concentration in a dose-dependent manner. The stimulatory effect of rUII on polyphosphoinositide turnover was abolished by the phospholipase C inhibitor U73122, but only reduced by 56% by pertussis toxin. The GTP analogue Gpp(NH)p caused its own biphasic displacement of [(125)I]rUII binding and provoked an affinity shift of the competition curve of rUII. Pertussis toxin shifted the competition curve towards a single lower affinity state. Taken together, these data demonstrate that rat astrocytes express high- and low-affinity UII binding sites coupled to G proteins, the high-affinity receptor exhibiting the same pharmacological and functional characteristics as UT.     

bis-Azaaromatic quaternary ammonium analogues: ligands for alpha4beta2* and alpha7* subtypes of neuronal nicotinic receptors

A series of bis-nicotinium, bis-pyridinium, bis-picolinium, bis-quinolinium and bis-isoquinolinium compounds was evaluated for their binding affinity at nicotinic acetylcholine receptors (nAChRs) using rat brain membranes. N,N'-Decane-1,12-diyl-bis-nicotinium diiodide (bNDI) exhibited the highest affinity for [(3)H]nicotine binding sites (K(i)=330 nM), but did not inhibit [(3)H]methyllycaconitine binding (K(i) >100 microM), indicative of an interaction with alpha4beta2*, but not alpha7* receptor subtypes, respectively. Also, bNDI inhibited (IC(50)=3.76 microM) nicotine-evoked (86)Rb(+) efflux from rat thalamic synaptosomes, indicating antagonist activity at alpha4beta2* nAChRs. N,N'-Dodecane-1,12-diyl-bis-quinolinium dibromide (bQDDB) exhibited highest affinity for [(3)H]methyllycaconitine binding sites (K(i)=1.61 microM), but did not inhibit [(3)H]nicotine binding (K(i)>100 microM), demonstrating an interaction with alpha7*, but not alpha4beta2* nAChRs. Thus, variation of N-n-alkyl chain length together with structural modification of the azaaromatic quaternary ammonium moiety afforded selective antagonists for the alpha4beta2* nAChR subtype, as well as ligands with selectivity at alpha7* nAChRs.     

(R)-N-[4,4-Bis(3-Methyl-2-Thienyl)but-3-en-1-yl]Nipecotic Acid Binds with High Affinity to the Brain γ-Aminobutyric Acid Uptake Carrier

(R)-N-[4,4-Bis(3-methyl-2-thienyl)but-3-en-1-yl]nipecotic acid (NO 328) has previously been shown to be a potent anticonvulsant in both mice and rats. Here, we report that NO 328 is a potent inhibitor of gamma-[3H]aminobutyric acid [( 3H]GABA) uptake in a rat forebrain synaptosomal preparation (IC50 = 67 nM) and in primary cultures of neurons and astrocytes. Inhibition of [3H]GABA uptake by NO 328 is apparently of a mixed type when NO 328 is preincubated before [3H]GABA uptake; the inhibition is apparently competitive without preincubation. NO 328 itself is not a substrate for the GABA uptake carrier, but NO 328 is a selective inhibitor of [3H]GABA uptake. Binding to benzodiazepine receptors, histamine H1 receptors, and 5-hydroxytryptamine1A receptors was inhibited by NO 328 at 5-30 microM, whereas several other receptors and uptake sites were unaffected. [3H]NO 328 showed saturable and reversible binding to rat brain membranes in the presence of NaCl. The specific binding of [3H]NO 328 was inhibited by known inhibitors of [3H]GABA uptake; GABA and the cyclic amino acid GABA uptake inhibitors were, however, less potent than expected. This indicates that the binding site is not identical to, but rather overlapping with, the GABA recognition site of the uptake carrier. The affinity constant for binding of [3H]NO 328 is 18 nM, and the Bmax is 669 pmol/g of original rat forebrain tissue. The regional distribution of NaCl-dependent [3H]NO 328 binding followed that of synaptosomal [3H]GABA uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

[3H]5-Hydroxytryptamine Binding to Brain Astroglial Cells: Differences Between Intact and Homogenized Preparations and Mature and Immature Cultures

[3H]5-hydroxytryptamine ([3H]serotonin) binds with high affinity (KD 2-12 nM) to a finite number of sites on brain astroglial cells. The number of binding sites in the C6 glioma line is decreased significantly (Bmax = 315 versus 30 fmol/mg) by homogenization. In intact primary cultures, derived from newborn rat brain, the number of binding sites is far greater in cultures of immature astrocytes than in cultures treated with dibutyryl cyclic AMP (Bmax = 1,520 versus 580 fmol/mg). A role for these receptors in development is suggested.     

Interaction of [3H](–)-SKF-10,047 with Brain σ Receptors: Characterization and Autoradiographic Visualization

The sigma opiates differ from other opiates in their stimulatory and psychotomimetic actions. The sigma opiate [3H](-)-SKF-10,047 has been used to characterize sigma receptors in rat nervous tissue. Binding of [3H](-)-SKF-10,047 to rat brain membranes was of high affinity, saturable, and reversible. Scatchard analysis revealed the apparent interaction of this drug with two distinct binding sites characterized by affinities of 0.03 and 75 nM (5 mM Tris-HCl buffer, pH 7.4, at 4 degrees C). Competition analyses involving rank order determinations for a series of opiates and other drugs indicate that the high-affinity binding site is the mu opiate receptor. The lower-affinity site (revealed after suppression of mu and delta receptor binding) has been identified as the sigma opiate/phencyclidine receptor. In vitro autoradiography has been used to visualize neuroanatomical patterns of receptors labeled using [3H](-)-SKF-10,047 in the presence of normorphine and [D-Ala2,D-Leu5]enkephalin to block mu and delta interactions, respectively. Labeling patterns differ markedly from those for mu, delta, or kappa receptors. The highest densities (determined by quantitative autoradiography) are found in the medial portion of the nucleus accumbens, amygdaloid nucleus, hippocampal formation, central gray, locus coeruleus, and the parabrachial nuclei. Receptors in these structures could account for the stimulatory, mood-altering, and analgesic properties of the sigma opiates. Although not the most selective sigma opiate ligand, [3H](-)-SKF-10,047 binds to sigma opiate receptors in brain, and this interaction can be readily distinguished from its interactions with other classes of brain opiate receptors.