Characteristics of 5-HT3 binding sites in NG108-15, NCB-20 neuroblastoma cells and rat cerebral cortex using [3H]-quipazine and [3H]-GR65630 binding.

1. The biochemical and pharmacological properties of 5-HT3 receptors in homogenates of NG108-15 and NCB-20 neuroblastoma cells and rat cerebral cortex have been ascertained by the use of [3H]-quipazine and [3H]-GR65630 binding. 2. In NG108-15 and NCB-20 cell homogenates, [3H]-quipazine bound to a single class of high affinity (NG108-15: Kd = 6.2 +/- 1.1 nM, n = 4; NCB-20: Kd = 3.0 +/- 0.9 nM, n = 4; means +/- s.e.means) saturable (NG108-15: Bmax = 1340 +/- 220 fmol mg-1 protein; NCB-20: Bmax = 2300 +/- 200 fmol mg-1 protein) binding sites. In rat cortical homogenates, [3H]-quipazine bound to two populations of binding sites in the absence of the 5-hydroxytryptamine (5-HT) uptake inhibitor, paroxetine (Kd1 = 1.6 +/- 0.5 nM, Bmax1 = 75 +/- 14 fmol mg-1 protein; Kd2 = 500 +/- 300 nM, Bmax2 = 1840 +/- 1040 fmol mg-1 protein, n = 3), and to a single class of high affinity binding sites (Kd = 2.0 +/- 0.5 nM, n = 3; Bmax = 73 +/- 6 fmol mg-1 protein) in the presence of paroxetine. The high affinity (nanomolar) component probably represented 5-HT3 binding sites and the low affinity component represented 5-HT uptake sites. 3. [3H]-paroxetine bound with high affinity (Kd = 0.02 +/- 0.003 nM, n = 3) to a site in rat cortical homogenates in a saturable (Bmax = 323 +/- 45 fmol mg-1 protein, n = 3) and reversible manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Agonist interactions with 5-HT3 receptor recognition sites in the rat entorhinal cortex labelled by structurally diverse radioligands.

1. The pharmacological properties of 5-HT3 receptor recognition sites labelled with [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 in membranes prepared from the rat entorhinal cortex were investigated to assess the presence of cooperativity within the 5-HT3 receptor complex. 2. In rat entorhinal cortex homogenates, [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 labelled homogeneous densities of recognition sites (defined by granisetron, 10 microM) with high affinity (Bmax = 75 +/- 5, 53 +/- 5, 92 +/- 6 and 79 +/- 6 fmol mg-1 protein, respectively; pKd = 9.41 +/- 0.04, 8.69 +/- 0.14, 8.81 +/- 0.06 and 10.14 +/- 0.04 for [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330, respectively, n = 3-8). 3. Quipazine and granisetron competed for the binding of each of the radioligands in the rat entorhinal cortex preparation at low nanomolar concentrations (pIC50; quipazine 9.38-8.51, granisetron 8.62-8.03), whilst the agonists, 5-hydroxytryptamine (5-HT), phenylbiguanide (PBG) and 2-methyl-5-HT competed at sub-micromolar concentrations (pIC50; 5-HT 7.16-6.42, PBG 7.52-6.40, 2-methyl-5-HT 7.38-6.09). 4. Competition curves generated with increasing concentrations of quipazine, PBG, 5-HT and 2-methyl-5-HT displayed Hill coefficients greater than unity when the 5-HT3 receptor recognition sites in the entorhinal cortex preparation were labelled with [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330. These competing compounds displayed Hill coefficients of around unity when the sites were labelled with [3H]-(S)-zacopride. Competition for the binding of [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 by granisetron generated Hill coefficients around unity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The interaction of trichloroethanol with murine recombinant 5-HT3 receptors.

1. The effects of ethanol, chloral hydrate and trichloroethanol upon the 5-HT3 receptor have been investigated by use of electrophysiological techniques applied to recombinant 5-HT3 receptor subunits (5-HT3R-A or 5-HT3R-As) expressed in Xenopus laevis oocytes. Additionally, the influence of trichloroethanol upon the specific binding of [3H]-granisetron to membrane preparations of HEK 293 cells stably transfected with the murine 5-HT3R-As subunit and 5-HT3 receptors endogenous to NG 108-15 cell membranes was assessed. 2. Ethanol (30-300 mM), chloral hydrate (1-30 mM) and trichloroethanol (0.3-10 mM), produced a reversible, concentration-dependent, enhancement of 5-HT-mediated currents recorded from oocytes expressing either the 5-HT3R-A, or the 5-HT3R-As subunit. 3. Trichloroethanol (5 mM) produced a parallel leftward shift of the 5-HT concentration-response curve, reducing the EC50 for 5-HT from 1 +/- 0.04 microM (n = 4) to 0.5 +/- 0.01 microM (n = 4) for oocytes expressing the 5-HT3R-A. A similar shift, from 2.1 +/- 0.05 microM (n = 11) to 1.3 +/- 0.1 microM (n = 4), was observed in oocytes expressing the 5-HT3R-As subunit. Trichloroethanol (5 mM) had little or no effect upon the maximum current produced by 5-HT for either recombinant receptor. 4. Trichloroethanol (5 mM) similarly reduced the EC50 for 2-methyl-5-HT from 13 +/- 0.4 microM (n = 4) to 4.6 +/- 0.2 microM (n = 4) and from 15 +/- 2 microM (n = 4) to 5 +/- 0.4 microM (n = 4) for oocytes expressing the 5-HT3R-A and 5-HT3R-As subunit respectively. Additionally, trichloroethanol (5 mM) produced a clear enhancement of the maximal current to 2-methyl-5-HT (expressed as a percentage of the maximal current to 5-HT) from 63 +/- 0.7% (n = 4) to 101 +/- 1.6% (n = 4) and from 9 +/- 0.2% (n = 4) to 74 +/- 2% (n = 4) for oocytes expressing the 5-HT3R-A and 5-HT3R-As subunit respectively. 5. Trichloroethanol (2.5 mM) had no effect upon the Kd, or Bmax, of specific [3H]-granisetron binding to membrane homogenates of NG 108-15 cells or HEK 293 cells. Similarly, competition for [3H]-granisetron binding by the 5-HT3 receptor antagonists ondansetron and tropisetron was unaffected. However, competition for [3H]-granisetron binding by the 5-HT3 receptor agonists, 5-HT, 2-methyl-5-HT and phenylbiguanide was enhanced by trichloroethanol (2.5 mM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterisation and autoradiographic localisation of 5-HT3 receptor recognition sites identified with [3H]-(S)-zacopride in the forebrain of the rat

The pharmacological characterisation and topographical distribution of [3H]-(S)-zacopride recognition sites in the forebrain of the rat was studied using homogenate and autoradiographic radioligand binding techniques. [3H]-(S)-Zacopride labelled a single, saturable, specific binding site (defined by 10.0 microM granisetron) in homogenates prepared from the entorhinal cortex of the rat (pKD = 9.51 +/- 0.08; Bmax = 104 +/- 7 fmol mg-1 protein; mean +/- SEM, n = 8). Pharmacological characterisation of the recognition site, within the entorhinal cortex, suggested that [3H]-(S)-zacopride selectively labelled the recognition site of the 5-HT3 receptor. Specific binding of [3H]-(S)-zacopride (defined by 1.0 microM granisetron) was differentially distributed throughout the forebrain of the rat; highest densities were located within sub-nuclei of the amygdala (cortical amygdaloid nucleus, amygdalohippocampal area, posterior medial cortical amygdaloid nucleus, posterior lateral amygdaloid nucleus), cortical areas (primary olfactory cortex, entorhinal cortex) and hippocampus. Non-specific binding was distributed homogeneously, although lower in myelinated structures. It is concluded that [3H]-(S)-zacopride selectively labels 5-HT3 receptor recognition sites within the forebrain of the rat; the topographical distribution of these sites, within the limbic nuclei, is consistent with the behavioural actions in animal models of the selective 5-HT3 receptor antagonists.     

Identification and distribution of 5-HT3 recognition sites in the rat gastrointestinal tract.

1. Tritiated derivatives of the potent and selective 5-HT3 receptor antagonists GR65630 and LY278584 were used to identify 5-HT3 recognition sites in the rat gastrointestinal tract. 2. Binding studies were carried out in homogenates of the rat oesophagus, the cardia, fundus, body and antrum of the stomach, regions of the small intestine, caecum and large intestine. The specific binding of a single concentration of GR65630 (0.5 nM) defined by granisetron (10 microM) in these areas indicated that the density of 5-HT3 recognition sites varied from 2.4 +/- 1.0 to 10.1 +/- 1.0 fmol mg-1 protein. 3. Saturable binding of [3H]-GR65630 could only be demonstrated in the terminal regions of the small intestine (Bmax in the range of 13.83 +/- 4.54-21.19 +/- 0.89 fmol mg-1 protein; mean +/- s.e. mean) and of high affinity (Kd in the range of 0.42 +/- 0.18-0.79 +/- 0.24 nM). Use of [3H]-LY278584 revealed a similar binding density (Bmax 19.54 +/- 0.26 fmol mg-1 protein) and affinity (Kd 1.04 +/- 0.07 nM) in the terminal small intestine. 4. Binding of [3H]-GR65630 and [3H]-LY278584 to the terminal region of the small intestine was inhibited by 5-HT3 receptor ligands ondansetron and S-zacopride (and 5-hydroxytryptamine), but not by 5-HT1, 5-HT2, catecholamine, gamma-aminobutyric acid and opioid receptor ligands. 5. These data demonstrate that there are regional variations in the density of 5-HT3 recognition sites within the rat gastrointestinal tract. Such data are relevant to the potential use of 5-HT3 receptor ligands to modify secretory and contraction responses in the gastrointestinal system.     

Changes in [3H]-PK 11195 and [3H]-8-OH-DPAT binding following forebrain ischaemia in the gerbil.

1. A high density of [3H]-PK 11195 binding sites was present in gerbil cortical membranes (Bmax [3H]-PK 11195 1360 +/- 71 fmol mg-1 protein) in comparison to rat cortical membranes (254 +/- 21 fmol mg-1 protein). This effect was species-specific as similar findings were obtained with hippocampal membranes (Bmax 1430 +/- 111 fmol mg-1 protein in gerbil, compared to 196 +/- 31 in rat). 2. RO 5-4864, also a peripheral type benzodiazepine compound, displayed low affinity for the [3H]-PK 11195 site in the gerbil (pKi 6.57 +/- 0.02 and 6.70 +/- 0.12 in hippocampus and cortex respectively) compared to rat (pKi 8.16 +/- 0.07 and 8.48 +/- 0.02). Central benzodiazepine compounds, diazepam and flunitrazepam, also displayed this trend. 3. RO 5-4864 displaced [3H]-PK 11195 binding from gerbil and rat cortical membranes through a competitive interaction with Hill slopes close to unity. In both tissues, saturation isotherms of [3H]-PK 11195 binding indicated that the presence of RO 5-4864 caused changes in Kd without any effect on Bmax. In kinetic experiments, the presence of RO 5-4864 failed to modify the rate of dissociation of [3H]-PK 11195 from equilibrium in both rat and gerbil cortical membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]zacopride: ligand for the identification of 5-HT3 recognition sites

[3H]Zacopride displayed saturable binding to homogenates of the rat entorhinal cortex as measured by the inclusion of the 5-HT3 receptor antagonist BRL43694 in the incubation media. Scatchard analysis indicated a single high affinity binding site (KD 0.76 +/- 0.08 nM, Bmax 77.5 +/- 6.5 fmol (mg protein)-1) with a Hill slope close to unity. Other 5-HT3 receptor antagonists (zacopride, ICS 205-930, GR38032F, GR65630, metoclopramide and cocaine) also competed for the binding site displacing 60% of the total [3H]zacopride binding. 5-HT and 2-methyl-5-HT also were competitive antagonists for [3H]zacopride binding whereas 5-HT1/5-HT2 agonists and antagonists, and agents acting on other neurotransmitter receptors had Ki values greater than 10(-5) M. It is concluded that [3H]zacopride may prove a useful ligand for the study of 5-HT3 recognition sites.     

Alpha 2-adrenoceptor subtypes and imidazoline-like binding sites in the rat brain.

1. The binding of [3H]-yohimbine and [3H]-idazoxan to rat cortex and hippocampus is rapid, reversible and of high affinity. Saturation data indicate that a single population of binding sites exist for [3H]-yohimbine in the cortex (Bmax 121 +/- 10 fmol mg-1, protein; Kd 5.2 +/- 0.9 nM) and hippocampus (Bmax 72 +/- 6 fmol mg-1 protein; Kd 5.8 +/- 0.7 nM). [3H]-idazoxan labels one site in the cortex (Bmax 87 +/- 8 fmol mg-1 protein; Kd 4.1 +/- 0.9 nM) and hippocampus (Bmax 30 +/- 6 fmol mg-1 protein; Kd 3.5 +/- 0.5 nM), when 3 microM phentolamine is used to define non-specific binding. A second distinct [3H]-idazoxan binding site (Bmax 110 +/- 21 fmol mg-1 protein; Kd 3.6 +/- 0.07 nM) is identified in rat cortex if 0.3 microM cirazoline is used to define non-specific binding and 3 microM yohimbine is included to prevent binding to alpha 2-adrenoceptors. 2. Displacement studies indicate that the alpha 1-adrenoceptor antagonist prazosin and the 5-HT1 ligands 8-OH-DPAT, RU 24969 and methysergide differentiate [3H]-yohimbine binding into two components; a high and low affinity site. In contrast the displacement of [3H]-idazoxan by each ligand was monophasic. 3. The affinities of 8-OH-DPAT, RU 24969 and methysergide determined against [3H]-idazoxan binding to the cortex and hippocampus correlate significantly with the binding site displaying low affinity for prazosin and previously designated alpha 2A. In contrast, a poor correlation exists for the high affinity site for prazosin designated alpha 2B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular pharmacology of 5-HT1 and 5-HT2 recognition sites in rat and pig brain membranes: radioligand binding studies with [3H]5-HT, [3H]8-OH-DPAT, (-)[125I]iodocyanopindolol, [3H]mesulergine and [3H]ketanserin

The pharmacological characteristics of the binding of [3H]8-OH-DPAT ([3H]8-hydroxy-2(di-n-propylamino)tetralin, [125I]CYP ((-)[125I]iodocyanopindolol) (in the presence of 30 microM (-)isoprenaline) and [3H]mesulergine to 5-HT1 recognition sites were studied in rat and pig brain membranes. [3H]8-OH-DPAT bound in rat and pig cortex to the 5-HT1A recognition site characterized by high affinity for 5-CT (5-carboxamido-tryptamine), 8-OH-DPAT, 5-HT (5-hydroxytryptamine, serotonin) and low affinity for pirenperone, ketanserin and mesulergine. [125I]CYP bound in rat but not in pig cortex to the 5-HT1B site which shows high affinity for (-)21-009 (4[3-ter-butyl-amino-2-hydroxy-propoxy]indol-2-carbonic acid isopropyl ester), (+/-)ICYP (3-I-cyanopindolol), 5-HT, RU 24969 (5-methoxy-3-[1,2,3,6-tetrahydropyridon-4-yl]1H-indole) and low affinity for 8-OH-DPAT, mesulergine and pirenperone. [3H]Mesulergine bound in pig choroid plexus and in rat cortex (besides binding to 5-HT2 sites in rat cortex) to the 5-HT1C recognition site characterized by high affinity for metergoline, mesulergine, 5-HT and methergine and low affinity for (-)21-009, ICYP, 8-OH-DPAT and spiroperidol. The pharmacological profile of 5-HT1A sites in rat and pig cortex appears to be identical; 5-HT1C sites in pig choroid plexus and rat cortex show no differences. In contrast, it was not possible to label 5-HT1B sites with [125I]CYP in pig brain membranes indicating that like 5-HT2 receptors, 5-HT1 recognition sites show species differences. The pharmacological profiles of the three 5-HT1 recognition sites are clearly different from one another. Furthermore, the pharmacological profile of each individual 5-HT1 recognition site is also different from that of the 5-HT2 receptors labelled with [3H]ketanserin in rat cortex membranes although some similarities exist between 5-HT2 and 5-HT1C sites. Finally, the beta-adrenoceptor antagonist (-)21-009 which has different affinities for 5-HT1A, 5-HT1B and 5-HT1C recognition sites, yielded triphasic competition curves for [3H]5-HT binding in rat cortex membranes providing evidence that [3H]5-HT labels three distinct 5-HT1 sites in these membranes.     

Binding of [3H]dihydrotetrabenazine and [125I]azidoiodoketanserin photoaffinity labeling of the monoamine transporter of platelet 5-HT organelles

The carrier for 5-hydroxytryptamine (5-HT) of the 5-HT storage organelles of blood platelets was characterized by [3H]dihydrotetrabenazine binding and [125I]azidoiodokentanserin photoaffinity labeling. [3H]Dihydrotetrabenazine bound with high affinity to membrane preparations from different animal species. The [3H]dihydrotetrabenazine Bmax value was about 10-fold higher in rabbit (9.4 +/- 1.3 pmol/mg protein) than in human, rat and guinea-pig preparations (Bmax values = 1.1 +/- 0.2, 1.2 +/- 0.1 and 0.52 +/- 0.06 pmol/mg protein, respectively). After rabbit platelet subcellular fractionation, [3H]dihydrotetrabenazine binding was highly enriched in the fraction corresponding to pure 5-HT organelles, whereas ligand binding was much lower in the other subcellular fractions. Conversely, [3H]paroxetine binding sites were more concentrated in the lower density fractions, with no binding to the 5-HT granules. In competition experiments, [3H]dihydrotetrabenazine binding to human platelet membranes and rabbit platelet 5-HT organelles was markedly inhibited by the benzo[a]quinolizine derivatives, tetrabenazine and Ro 4-1284, and by ketanserin. In isolated rabbit platelet 5-HT organelles, reserpine showed a relatively high IC50 (930 nM), but the presence of ATP increased its potency about 10-fold. Paroxetine, methysergide and carrier substrates had little or no effect. After photoaffinity labeling of rabbit 5-HT granules with [125I]azidoiodoketanserin, the radioactivity was incorporated into several polypeptides. The presence of Ro 4-1284, reserpine and ketanserin prevented the labeling of a polypeptide of 85 kDa. The data obtained suggest that this protein represents a component of the granular carrier which binds [3H]dihydrotetrabenazine.     

Common pharmacological and physico-chemical properties of 5-HT3 binding sites in the rat cerebral cortex and NG 108-15 clonal cells

On account of the postulated existence of 5-HT3 receptor subtypes, the respective physico-chemical and pharmacological properties of specific binding sites for the potent 5-HT3 antagonist [3H]zacopride were compared using membranes from the rat posterior cortex or neuroblastoma-glioma NG 108-15 clonal cells. In both membrane preparations, [3H]zacopride bound to a single class of specific sites with a Kd close to 0.5 nM. However, the Bmax value in NG 108-15 cell membranes (970 +/- 194 fmol/mg protein) was approximately 50 times larger than that in cortical membranes (19 +/- 2 fmol/mg protein). The specific binding of [3H]zacopride was equally affected by temperature, pH and molarity of the assay medium, and equally insensitive to thiol- and disulfide-reagents (N-ethylmaleimide, p-chloromercuribenzene sulfonic acid, dithiothreitol) and GTP in cortical as well as NG 108-15 cell membranes. Determination of the molecular size of [3H]zacopride specific binding sites by radiation inactivation yielded values close to 35 kDa for both membrane preparations. Finally, a highly significant positive correlation (r = 0.979) was found between the respective pKi values of 34 different drugs for their inhibition of [3H]zacopride specific binding to cortical or NG 108-15 cell membranes. Among them, the most potent was S(-)zacopride (pKi = 9.55), followed by BRL 43964, ICS 205-930, quipazine, R(+)zacopride, GR 38032F and MDL 72222. Atypical antidepressants (mianserin, amoxapine) and neuroleptics (clotiapine, loxapine and clozapine) were active in rather low concentrations (pKi less than 6.5), suggesting that recognition of 5-HT3 sites might be relevant to part of the in vivo effects of these drugs. Such identical physico-chemical and pharmacological properties of [3H]zacopride specific binding in cortical and NG 108-15 cell membranes strongly suggest that the same 5-HT3 receptor (subtype?) exists in these two preparations.     

Allosteric modulation of 5-HT3 serotonin receptors

[(3)H]Granisetron binding to 5-HT(3) type serotonin receptors was examined in homogenates of rat forebrain and NG 108-15 cells. We have applied an allosteric model to 5-HT(3) receptor binding for the first time. Slope factors of displacement improved the modelling. Serotonin displaced [(3)H]granisetron binding with micromolar potency in forebrain and with nanomolar potency in NG 108-15 cells. Racemic and (+)verapamil, ifenprodil and GYKI-46903 were used as representative allosteric inhibitors of 5-HT(3) receptors. They displaced [(3)H]granisetron binding with great negative cooperativity (alpha>10) and exerted great negative cooperativity with serotonin binding (beta>10). Great negative cooperativity of these agents with serotonin and [(3)H]granisetron binding cannot be distinguished from dual competitive displacement. Trichloroethanol (data from literature) had no cooperativity with [(3)H]granisetron binding (alpha~1) and exhibit positive cooperativity with serotonin (beta<1) in displacement. The allosteric model can lead to a more quantitative method in vitro to develop allosteric agents for 5-HT(3) receptors.     

[3H]-thioperamide as a radioligand for the histamine H3 receptor in rat cerebral cortex.

1. The purpose of the present study was to characterize the binding of the histamine H3 receptor antagonist, [3H]-thioperamide, to rat cerebral cortical membranes. 2. The binding of [3H]-thioperamide to rat cerebral cortical membranes reached equilibrium after incubation with [3H]-thioperamide after 8-10 h at 4 degrees C. Equilibrium was maintained for up to 18 h of incubation. Addition of 1 microM (R)-alpha-methylhistamine rapidly dissociated [3H]-thioperamide from its binding sites. From these kinetic experiments a dissociation constant of 0.3 nM was obtained for [3H]-thioperamide. 3. Saturation experiments with [3H]-thioperamide using 1 microM (R)-alpha-methylhistamine to define nonspecific binding were best analysed according to a single site model. A dissociation constant (KD) of 0.80 +/- 0.06 nM (n = 3) and a maximal number of binding sites (Bmax) of 73 +/- 20 fmol mg-1 protein (n = 3) were obtained for the binding of [3H]-thioperamide to rat cerebral cortical membranes. 4. Saturation experiments with [3H]-thioperamide using 0.3 microM iodophenpropit to define nonspecific binding were best analysed according to a two site model. For the high affinity [3H]-thioperamide site a KD value of 1.1 +/- 0.3 nM (n = 3) and Bmax value of 162 +/- 108 fmol mg-1 protein (n = 3) were obtained whereas KD and Bmax values for the low affinity site were 96 +/- 19 nM and 4346 +/- 3092 fmol mg-1 protein (n = 3), respectively. 5. Using 5 nM [3H]-thioperamide, the binding was hardly displaced by H3 agonists within concentration-ranges expected to bind to the histamine H3 receptor. Under these conditions, [3H]-thioperamide binding was fully displaced by various H3-antagonists, yet most H3 antagonists showed Ki values different from those expected for the histamine H3 receptor. 6. Using 0.3 nM [3H]-thioperamide, 50-60% of the total binding was potently displaced by the H3 agonists histamine, (R)-alpha-methylhistamine, (S)-alpha-methylhistamine, imetit and immepip. Displacement of the binding of 0.3 nM [3H]-thioperamide binding exhibited clear stereoselectivity for the R and S isomers of alpha-methylhistamine. 7. Binding of 0.3 nM [3H]-thioperamide was completely displaced by several H3 antagonists (thioperamide, iodophenpropit, iodoproxyfan, and burimamide) and biphasic displacement curves were obtained; the Ki values for the high affinity site corresponded well with the expected values for the H3 receptor. Antagonists fully displaced the binding of 5 nM [3H]-thioperamide with affinities comparable to the low affinity site found with 0.3 nM [3H]-thioperamide. 8. Ondansetron and haloperidol did not displace binding of 5 nM [3H]-thioperamide at concentrations at which the former are known to bind to 5-HT3 or sigma receptors, respectively. On the other hand, nonselective cytochrome P450 inhibitors displaced the binding of 5 nM [3H]-thioperamide from both rat cerebral cortical membranes and rat liver microsomes. 9. It is concluded that the histamine H3 antagonist, [3H]-thioperamide, can be used as a radioligand to study the histamine H3 receptor in rat brain, provided that subnanomolar concentrations are used in displacement studies. Moreover, the specific binding should be defined with an H3 agonist, since most H3 antagonists share with [3H]-thioperamide a low affinity, high density, non-H3 receptor binding site(s) in rat brain. The latter is probably due to binding to cytochrome P450 isoenzymes.     

3H]rauwolscine labels alpha 2-adrenoceptors and 5-HT1A receptors in human cerebral cortex

[3H]Rauwolscine binds with high affinity to alpha 2-adrenoceptors (Kd = 4.8 +/- 1.3 nM, Bmax = 79 +/- 26 fmol/mg protein, micromolar affinity for 5-HT) as well as to 5-HT1-like receptors (Kd = 13 +/- 2.7 nM, Bmax = 147 +/- 11.4 fmol/mg protein, nanomolar affinity for 5-HT) in human brain cortex membranes. The Ki values of 11 serotonergic compounds for the latter receptors agreed closely with those previously reported for 5-HT1A sites but not with those for 5-HT1B, 5-HT1C and 5-HT1D sites.     

Characterisation of 5-HT3 recognition sites in membranes of NG 108-15 neuroblastoma-glioma cells with [3H]ICS 205-930

1. The binding characteristics of [3H]ICS 205-930, a potent and selective 5-hydroxytryptamine 5-HT3 receptor antagonist, were investigated in membranes prepared from murine neuroblastoma-glioma NG 108-15 cells. 2. [3H]ICS 205-930 bound rapidly, reversibly and stereoselectively to a homogeneous population of high affinity recognition sites: Bmax = 58 +/- 3 fmol/mg protein, pKD = 9.01 +/- 0.08 (n = 11). Non linear regression and Scatchard analysis of saturation data suggested the existence of a single class of [3H]ICS 205-930 recognition sites on NG 108-15 cells. The binding was rapid, stable and reversible. The affinity of [3H]ICS 205-930 determined in kinetic studies was in agreement with that obtained under equilibrium conditions. 3. Competition studies performed with a variety of agonists and antagonists also suggested the presence of a homogeneous population of [3H]ICS 205-930 recognition sites. All competition curves were steep and monophasic and were best fit by a 1 receptor site model. [3H]ICS 205-930 binding sites displayed the pharmacological profile of a 5-HT3 receptor. Potent 5-HT3 receptor antagonists showed nanomolar affinities for [3H]ICS 205-930 binding sites with the following rank order of potency: SDZ 206-830 greater than ICS 205-930 greater than SDZ 206-792 greater than BRL 43694 greater than quipazine greater than BRL 24924 greater than SDZ 210-204 greater than MDL 72222 greater than SDZ 210-205. Metoclopramide, mCP and mianserin showed submicromolar affinity.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]-Mesulergine labels 5-HT7 sites in rat brain and guinea-pig ileum but not rat jejunum

1. The primary aim of this investigation was to determine whether binding sites corresponding to the 5-HT7 receptor could be detected in smooth muscle of the rat jejunum. Binding studies in rat brain (whole brain minus cerebellum) and guinea-pig ileal longitudinal muscle were also undertaken in order to compare the binding characteristics of these tissues. Studies were performed using [3H]-mesulergine, as it has a high affinity for 5-HT7 receptors. 2. In the rat brain and guinea-pig ileum, pKD values for [3H]-mesulergine of 8.0 +/- 0.04 and 7.9 +/- 0.11 (n = 3) and Bmax values of 9.9 +/- 0.3 and 21.5 +/- 4.9 fmol mg(-1) protein were obtained respectively, but no binding was detected in the rat jejunum. [3H]-mesulergine binding in the rat brain and guinea-pig ileum was displaced with the agonists 5-carboxamidotryptamine (5-CT) > 5-hydroxytryptamine (5-HT) > or = 5-methoxytryptamine (5-MeOT) > sumatriptan and the antagonists risperidone > or = LSD > or = metergoline > ritanserin > > pindolol. 3. Despite the lack of [3H]-mesulergine binding in the rat jejunum, functional studies undertaken revealed a biphasic contractile response to 5-HT which was partly blocked by ondansetron (1 microM). The residual response was present in over 50% of tissues studied and was found to be inhibited by risperidone > LSD > metergoline > mesulergine = ritanserin > pindolol, but was unaffected by RS 102221 (3 microM), cinanserin (30 nM), yohimbine (0.1 microM) and GR 113808 (1 microM). In addition, the agonist order of potency was 5-CT > 5-HT > 5-MeOT > sumatriptan. 4. In conclusion, binding studies performed with [3H]-mesulergine were able to detect 5-HT7 sites in rat brain and guinea-pig ileum, but not in rat jejunum, where a functional 5-HT7-like receptor was present.     

Mu opiate receptors are selectively labelled by [3H]carfentanil in human and rat brain

[11C]Carfentanil is a potent opioid agonist currently in use as a specific PET (position emission tomography) scan radioligand for brain mu opioid receptors. In order to investigate the receptor interactions of carfentanil in detail [3H]carfentanil was used as a radioligand for labelling receptors in rat and human brain tissue homogenates. [3H]Carfentanil was found to bind saturably and with high affinity (KD = 0.08 +/- 0.01 nM) to membranes prepared from human cortical (Bmax = 42 +/- 3 fmol/mg) and thalamic (Bmax = 84 +/- 3 fmol/mg) tissues and rat cortex (Bmax = 82 +/- 4 fmol/mg) and diencephalon (Bmax = 105 +/- 5 fmol/mg). Association (1.23 +/- 0.19 X 10(10) Mol-1 X min-1 and dissociation rate (0.19 +/- 0.03 min-1) constants were determined in human cortical tissues; results from studies in rat cortical, and rat diencephalon tissue homogenates produced similar kinetic rate constants. Competition studies with a variety of drugs indicated that [3H]carfentanil interacts primarily with mu opioid receptors in the four tissues studied; the affinities of a series of non-radioactive opioid ligands were essentially identical in the four tissues (correlation coefficients = 0.88-0.93). Naloxone, morphine, DAGO [( D-Ala2-MePhe4-Gly-ol5]enkephalin), DADL [( D-Ala2-D-Leu5]enkephalin) and EKC (ehtylketazocine) potently displaced specific [3H]carfentanil binding with nM potency while the kappa agonist U-69593, the sigma agonists (+)-SKF 10047, (+)-3-PPP [3-hydroxyphenyl)-N-propylpiperidine) and haloperidol and PCP (phencyclidine) were less potent displacing agents. The higher affinities of DAGO and morphine versus DADL for the [3H]carfentanil binding sites indicates that delta opioid receptors are not being labelled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the binding of [3H]-clobenpropit to histamine H3-receptors in guinea-pig cerebral cortex membranes

1 We have investigated the binding of a novel histamine H3-receptor antagonist radioligand, [3H]- clobenpropit ([3H]-VUF9153), to guinea-pig cerebral cortex membranes. 2 Saturation isotherms for [3H]-clobenpropit appeared biphasic. Scatchard plots were curvilinear and Hill plot slopes were significantly less than unity (0.63+/-0.03; n = 12+/-s.e.mean). The radioligand appeared to label two sites in guinea-pig cerebral cortex membranes with apparent affinities (pKD') of 10.91+/-0.12 (Bmax = 5.34+/-0.85 fmol mg(-1) original wet weight) and 9.17+/-0.16 (Bmax = 23.20+/-6.70 fmol mg(-1)). 3 In the presence of metyrapone (3 mM) or sodium chloride (100 mM), [3H]-clobenpropit appeared to label a homogeneous receptor population (Bmax=3.41+/-0.46 fmol mg-1 and 3.49+/-0.44 fmol mg(-1), pKD' = 10.59+/-0.17 and 10.77+/-0.02, respectively). Scatchard plots were linear and Hill slopes were not significantly different from unity (0.91+/-0.04 and 0.99+/-0.02, respectively). Granisetron (1 microM), rilmenidine (3 microM), idazoxan (0.3 microM), pentazocine (3 microM) and 1,3-di-(2-tolyl)guanidine (0.3 microM) had no effect on the binding of [3H]-clobenpropit. 4 The specific binding of [3H]-clobenpropit appeared to reach equilibrium after 25 min at 21+/-3 degrees C and remained constant for >180 min. The estimated pKD' (10.27+/-0.27; n = 3+/-s.e.mean) was not significantly different from that estimated by saturation analysis in the presence of metyrapone. 5 A series of histamine H3-receptor ligands expressed affinity values for sites labelled with [3H]-clobenpropit which were not significantly different from those estimated when [3H]-R-alpha-MH was used to label histamine H3-receptors in guinea-pig cerebral cortex membranes.     

Characterization of a human 5-hydroxytryptamine3 receptor type A (h5-HT3R-AS) subunit stably expressed in HEK 293 cells.

1. A cloned cDNA encoding a human 5-hydroxytryptamine3 receptor type A subunit (h5-HT3R-As) was transfected into human embryonic kidney (HEK 293) cells maintained in cell culture and a stable cell line expressing a high density of the recombinant receptor was selected. 2. Membrane homogenates prepared from transfected, but not untransfected, cells exhibited a homogeneous and saturable population (Bmax = 4.49 +/- 0.46 pmol mg-1 protein) of sites that bound the radiolabelled 5-HT3 receptor antagonist, [3H]-granisetron with high affinity (pKD = 8.87 +/- 0.08). Kinetic studies (at 37 degrees C) revealed rapid association (kappa +1 4.76 +/- 0.3 x 10(8) M-1 min-1) and dissociation (kappa -1 = 0.21 +/- 0.003 min-1) of the radioligand. 3. Selective and non-selective 5-HT3 receptor ligands competed for [3H]-granisetron binding with a rank order of potency (granisetron > ondansetron > meta-chlorophenylbiguanide > 5-HT > 2-methyl-5-HT > metoclopramide > > phenylbiguanide > cocaine > (+)-tubocurarine) identical to that established for 5-HT3 receptors endogenous to the human CNS. 4. In electrophysiological recordings performed on transfected cells, voltage-clamped at a holding potential of -60 mV, locally applied 5-HT (10 microM) evoked transient inward current responses that reversed in sign at a potential of -1.0 +/- 1.1 mV. Such responses were antagonized in a reversible manner by granisetron (1 nM). 5. The construction of a stable cell line expressing a high density of recombinant human 5-HT3 receptors which display appropriate pharmacology and function will assist in the further characterization of this receptor subtype and the exploration of species differences in 5-HT3 receptor pharmacology.     

3H]rauwolscine behaves as an agonist for the 5-HT1A receptors in human frontal cortex membranes.

The alpha 2 adrenergic antagonist [3H]rauwolscine binds with comparable nanomolar affinity to alpha 2 adrenoceptors and the nonadrenergic 5-HT1A receptors sites in human frontal cortex membranes. Addition of 0.5 mM GTP into the incubation medium produces a significant decrease in the amount of [3H]rauwolscine binding sites (Bmax = 230 +/- 16 and 115 +/- 11 fmol/mg protein in the absence and presence of GTP, respectively). The affinity for [3H]rauwolscine remains unchanged (i.e. KD = 40 +/- 0.9 nM and 4.1 +/- 1 nM). This effect of GTP can be attributed to decreased binding of the radioligand to the 5-HT1A receptors. GTP decreases binding of [3H]rauwolscine to nearly the same level as the one corresponding to the alpha 2 adrenoceptors in membranes from both the human frontal cortex and hippocampus. The venom of the marine cone snail, Conus tessulatus, preferentially inhibits [3H]rauwolscine binding to 5-HT1A receptors as compared with the alpha 2 adrenoceptors. Following complete masking of the 5-HT1A receptors by this venom. GTP no longer affects the saturation binding characteristics of [3H]rauwolscine for the remaining alpha 2 adrenoceptors. Nucleotides decrease the binding of [3H]rauwolscine to the 5-HT1A receptors with an order of potencies (i.e. GTP gamma S greater than GPP(NH)P much greater than GDP greater than GTP much greater than ATP) that is typical for nucleotide-mediated receptor-G protein dissociation. This suggests that [3H]rauwolscine is a 5-HT1A receptor agonist and this conclusion is compatible with earlier functional studies, indicating that rauwolscine (as well as yohimbine) has agonistic properties at the level of 5-HT autoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)