Novel ligands for the investigation of imidazoline receptors and their binding proteins.

New ligands for imidazoline receptors are described so that these receptors can be more fully explored and understood. BU224, (2-(4,5-dihydroimidaz-2-yl)-quinoline, shows high affinity and is selective for the imidazoline-2 (I(2)) class of receptors. BU224 was tested in the rat Porsolt forced swim paradigm where it was found to decrease time spent immobile and increase the time spent swimming, consistent with an antidepressant profile. BU224 was tritiated and, in radioligand binding studies, was found to label a single population of saturable sites with high affinity. In vitro brain autoradiography with [(3)H]BU224 also showed a pattern of distribution similar to the known labeling of I(2) receptors. A new series of four 2BFI (2-(benzofuranyl)-2-imidazoline) derivatives were investigated as potential ligands for imaging brain I(2) receptors using positron emission tomography (PET). At least two, BU20012 and BU20013, retained high affinity and moderate selectivity and penetrated the brain when administered peripherally in the mouse. 2BFI has undergone the Mannich reaction to immobilized diaminodipropyl amine to fabricate an affinity column, which was used to isolate a protein from rabbit brain; this protein was sequenced and identified as the enzyme creatine kinase.

Identification of an I(2) binding protein from rabbit brain.

Imidazoline-2 binding proteins exist as a heterogeneous population. The aim of this study was to isolate and identify I(2) binding proteins from rabbit brain using an affinity column synthesized with a highly selective I(2) ligand, 2-(2-benzofuranyl)2-imidazoline (2BFI). The results revealed an approximately 45-kD protein to be brain creatine kinase (EC 2.7.3.2). [(3)H]-2BFI (5nM) was able to bind specifically to the purified enzyme. This study has identified brain creatine kinase as a novel I(2) binding protein.

Pharmacological characterization of recombinant human 5-hydroxytryptamine1A receptors using a novel antagonist radioligand, [3H]WAY-100635.

The ligand binding characteristics of the recombinant human 5-HT1A receptor stably expressed in a Chinese Hamster Ovary (CHO) cell line are described using a selective agonist, [3H]8-OH-DPAT, and a novel antagonist radioligand, [3H]WAY-100635. The association of [3H]WAY-100635 was a time- and temperature-dependent process. Mn2+ > Ca2+ > Mg2+ reduced the specific [3H]WAY-100635 binding in a concentration-dependent manner, whereas Na+ and K+ were ineffective. Scatchard analyses revealed a homogeneous population of [3H]WAY-100635 recognition sites (Kd = 0.32 nM; Bmax = 162 fmol/mg of protein). Addition of divalent cations to the incubation medium produced a two-fold decrease in the binding affinity of [3H]WAY-100635 with no significant change in Bmax; GTP gamma S had no effect on Kd or Bmax parameters. [3H]WAY-100635 displayed a higher affinity (2-3 fold) for the 5-HT1A site when compared with [3H] 8-OH-DPAT binding under similar incubation conditions. Furthermore, [3H] 8-OH-DPAT labelled approximately 53-61% of total 5-HT1A sites recognised by [3H]WAY-100635. The competition binding profiles of [3H]WAY-100635 and [3H]8-OH-DPAT were highly correlated and consistent with the recognition of 5-HT1A receptors. Agonist competition curves with [3H]WAY-100635 were best-resolved into high- and low-affinity binding states, whereas partial agonist and antagonist curves were best-fit to one-site binding models. A significant correlation between the respective affinities of a range of agonists and antagonists at recombinant human and rodent hippocampal 5-HT1A binding sites (previously published) was also observed using [3H]WAY-100635 (r = 0.92; P < 0.0005) and [3H]8-OH-DPAT (r = 0.96; P < 0.0005). The availability of a novel, high-affinity antagonist radioligand, [3H]WAY-100635, will provide a useful tool for the further characterisation of 5-HT1A receptor pharmacology.

Characterisation of the binding of [3H]WAY-100635, a novel 5-hydroxytryptamine1A receptor antagonist, to rat brain.

The specific binding of [3H]WAY-100635 (N-[2-[4-(2-[O-methyl-3H]methoxyphenyl)-1-piperazinyl]ethyl]-N- 2-pyridinyl) cyclohexane carboxamide trihydrochloride) to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [3H]WAY-100635 association (k+1 = 0.069 +/- 0.015 nM-1 min-1) and dissociation (k-1 = 0.023 +/- 0.001 min-1) followed monoexponential kinetics. Saturation binding isotherms of [3H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 +/- 0.051 nM and a maximal binding capacity (Bmax) of 312 +/- 12 fmol/mg of protein. The maximal number of binding sites labelled by [3H]WAY-100635 was approximately 36% higher compared with that of 8-hydroxy-2-(di-n-[3H]-propylamino) tetralin ([3H]8-OH-DPAT). The binding affinity of [3H]WAY-100635 was significantly lowered by the divalent cations CaCl2 (2.5-fold; p < 0.02) and MnCl2 (3.6-fold; p < 0.05), with no effect on Bmax. Guanyl nucleotides failed to influence the KD and Bmax parameters of [3H]WAY-100635 binding to 5-HT1A receptors. The pharmacological binding profile of [3H]WAY-100635 was closely correlated with that of [3H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine1A (5-HT1A) sites in rat hippocampus. [3H]WAY-100635 competition curves with 5-HT1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 microM guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT1A receptor. However, a residual (16 +/- 2%) high-affinity agonist binding component was still apparent in the presence of GTP gamma S, indicating the existence of GTP-insensitive sites.

The effect of toxin I, a K+ channel inhibitor, on [3H]noradrenaline release from rat cerebral cortex.

Toxin I, a K+ channel blocker found in the venom of the black mamba snake with close sequence homology to the dendrotoxins, produced a concentration-related enhancement of both spontaneous and electrically evoked [3H]noradrenaline ([3H]NA) release from slices of rat cerebral cortex. The effect of toxin I on spontaneous [3H]NA release was blocked by tetrodotoxin and reduced in the presence of either CPP ((+-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid) or CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and was abolished in the presence of both antagonists. The results suggest that the enhancement of spontaneous [3H]NA release produced by toxin I may be mediated via the release of glutamate acting on both NMDA (N-methyl-D-aspartate) and non-NMDA receptors.

Modulation of the GABAA-like autoreceptor by barbiturates but not by steroids.

In slices of cerebral cortex from rat preloaded with [3H]GABA, muscimol produced a concentration-related inhibition of K(+)-evoked release of tritium with a pIC25 value of 7.80 +/- 0.39. Dimethylbarbituric acid (10 and 100 microM) and pentobarbitone (100 microM) significantly increased this value to 8.31 +/- 0.09, 9.91 +/- 0.21 and 8.50 +/- 0.21, respectively, whereas the steroid ligands alphaxalone (1 microM) and 5 beta-pregnane-3 alpha-ol-20-one (10 nM) had no significant effect. The 5 beta-pregnane-3 alpha-ol-20-one and 5 beta-pregnane-3,20-dione produced a concentration-related increase in K(+)-evoked release of tritium alone. These data suggest that the GABAA-like autoreceptor may be modulated by barbiturates but not by steroids and thus may be different from the postsynaptic GABAA receptor.

Identification of residues important for ligand binding to the human 5-hydroxytryptamine1A serotonin receptor.

The functional significance of the conserved amino acids within transmembrane regions II and VII of the human 5-hydroxytryptamine (5-HT)1A receptor was analyzed by oligonucleotide-directed mutagenesis followed by transient expression of the mutated receptor genes in COS-1 cells. The substitution of a conserved asparagine at position 396 (transmembrane region VII) with either alanine, phenylalanine, or valine resulted in a receptor that did not bind the 5-HT1A agonist 8-hydroxy-2-(di-n-[3H]propylamino)tetralin. In contrast, replacement of Asn396 with glutamine did not affect agonist binding. In addition, serine residues at positions 391 and 393 (transmembrane domain VII) were changed to alanine. Changing the less conserved Ser391 to alanine had no effect on ligand binding. However, replacement of the conserved Ser393 with alanine reduced ligand binding by 86%. Replacement of a conserved aspartate at position 82 (transmembrane region II) with alanine also produced a receptor without detectable agonist binding. Protein immunoblotting detected receptor protein of approximately 51 kDa in both wild-type and mutant receptor-expressing cells, indicating that these mutations probably did not affect expression or processing of the protein. Importantly, the sequence of the human 5-HT1A receptor described in this paper differs from the published sequence [Nature (Lond.) 329:75-79 (1987)] in transmembrane region IV. The present sequence encodes a protein of 422 amino acids, instead of the 421-amino acid protein that has been described previously [Nature (Lond.) 329:75-79 (1987)], and has a change in the sequence in transmembrane region IV from ... RPRAL ... to ... RRAAA ..., which corresponds to the published sequence [J. Biol. Chem. 265:5825-5832 (1990)] of the rat 5-HT1A receptor. Moreover, conversion of the transmembrane region IV sequence of the present clone to that of the published sequence by site-directed mutagenesis abolished ligand binding to the receptor.

Exposure to high pressure may produce the 5-HT behavioral syndrome in rats.

In addition to the motor events associated with high pressure neurologic syndrome (HPNS), we have observed behavioral changes that resemble the 5-hydroxytryptamine (5-HT) syndrome in free-moving rats exposed to pressures up to 70 ATA. These include a flat body posture, head weaving, reciprocal forepaw treading, and hyperlocomotion. Such changes occur when brain 5-HT levels are raised or when 5-HT receptors are activated. We have therefore studied the behavior of rats at pressure treated either with saline or with one of the following drugs: p-chlorophenylalanine (pCPA) which depletes brain 5-HT by 85-90%, Wy 27587 which inhibits 5-HT reuptake, 5-hydroxytryptophan (5-HTP) and carbidopa which increase brain 5-HT synthesis, and quipazine which is a 5-HT receptor-agonist. After treatment, rats were individually exposed to pressure, and behavioral scores were made for 5 min every 10 ATA up to 70 ATA by an unbiased observer who was not aware of the treatment given. Analysis of all control rats indicated that only a flat body posture, forepaw treading, and hyperlocomotion were positively correlated with pressure, and these events were used in all subsequent analysis. Rats treated with pCPA with whole brain 5-HT levels reduced by 90% had scores significantly less than controls. Rats treated with Wy 27587 showed significantly increased scores. Rats treated with 5-HTP and quipazine failed to show a significant increase in scores. These results suggest that a modified form of the 5-HT syndrome occurs when rats are exposed to increased pressure, and the behavioral events seen are consistent with some activation of the 5-HT1A receptor subtype.

A comparison of the effects of the selective 5-HT uptake inhibitor WY 27587 on 5-HT uptake into platelets and synaptosomes in the rat.

N-[( 1-[(6-Fluoro-2-naphthalenyl)methyl]- 4-piperidinyl]amino]carbonyl]-3-pyridine carboxamide (Wy 27587), is 320 times more potent as an inhibitor of the uptake of 5-HT than of the uptake of noradrenaline into synaptosomes from the brain of the rat with a Ki against the uptake of 5-HT of 9.2 +/- 1.4 nM. It was also a potent competitive inhibitor of the uptake of 5-HT in platelets of the rat, with a Ki value of 2.9 +/- 1.5 nM. Ex vivo studies indicated that oral administration of Wy 27587 in the rat caused a prolonged inhibition of the uptake of 5-HT into platelets; inhibition reached a maximum 15 min after administration and the ID50 was 1.5 +/- 0.3 mg/kg.

Modulation of the GABA autoreceptor by benzodiazepine receptor ligands.

The effects of various benzodiazepine receptor ligands on the GABA autoreceptor have been studied in slices of cerebral cortex of the rat. The GABAA receptor agonist muscimol inhibited the K+-stimulated release of [3H]GABA with a pIC25 of 7.65 +/- 0.11. This effect was antagonised by the GABAA receptor antagonist bicuculline, which had an IC50 of 0.36 +/- 0.03 microM. Small concentrations (less than 1 microM) of the benzodiazepine full agonist clonazepam did not significantly alter K+-evoked release of [3H]GABA but shifted the concentration-effect curve for muscimol to the left. This effect was blocked by the benzodiazepine antagonist flumazenil. By contrast, the benzodiazepine full inverse agonist methyl beta-carboline-3-carboxylate shifted the muscimol concentration-effect curve to the right and this too was blocked by flumazenil. The results suggest that the GABA autoreceptor in cortical slices from the rat is modulated by a benzodiazepine receptor.

Inhibition of GABA release from slices prepared from several brain regions of rats at various times following a convulsion.

1 A method is described for the measurement of the K+-evoked release of endogenous gamma-aminobutyric acid (GABA) from slices of rat cortex, hippocampus and striatum. 2 In tissue prepared 30 min following an electroconvulsive shock, K+-evoked GABA release (above basal release) was inhibited by 45% in cortex, 50% in hippocampus and 75% in striatum. A similar inhibition of release was observed with slices prepared from rats in which a convulsion had been induced by flurothyl. There was no change in spontaneous (basal) release following either procedure. 3 An inhibition of K+-evoked endogenous GABA release was also seen in tissue prepared 4 min postictally but not 2 h after the seizure. 4 No difference was observed in the release of [3H]-GABA from preloaded cortical slices prepared from rats given a single electroconvulsive shock. 5 It is proposed that a convulsion results in an inhibition of GABA release and that this inhibition may in turn inhibit GABA synthesis as described in the preceding paper. 6 It is also proposed that changes in the endogenous releasable pool of GABA may not be detected by preloading slices with [3H]-GABA.

Inhibition of the rate of GABA synthesis in regions of rat brain following a convulsion.

1 The rate of synthesis of gamma-aminobutyric acid (GABA) in the cortex, hippocampus and striatum of rat brain was assessed by measuring the linear rate of accumulation of GABA following injection of amino-oxyacetic acid (AOAA). 2 Five min after a single electrically induced seizure there was a rise in GABA content in these brain regions and an almost total inhibition of the rate of synthesis. 3 Five min after seizure induced by the inhalant convulsant flurothyl there was no rise in GABA content in these brain regions but a similar marked degree of inhibition of GABA synthesis. 4 Two hours after the convulsion the rate of GABA synthesis had returned to control values in all three brain regions. 5 A single convulsion did not alter the glutamic acid decarboxylase activity in these brain regions either in the absence or presence of added co-factor (pyridoxal phosphate). 6 Evidence for an inhibition of GABA release following a convulsion which may be associated with the inhibition of GABA synthesis is presented in the following paper.

Serotonergic agonists stimulate inositol lipid metabolism in rabbit platelets.

The metabolism of inositol phospholipids in response to serotonergic agonists was investigated in rabbit platelets. In platelets prelabelled with [3H]-inositol, in a medium containing 10 mM LiCl which blocks the enzyme inositol-1-phosphatase, 5-hydroxytryptamine (5-HT) caused a dose-dependent accumulation of inositol phosphates (IP). This suggests a phospholipase-C-mediated breakdown of phosphoinositides. Ketanserin, a selective 5-HT2 antagonist, was a potent inhibitor of the 5-HT response, with a Ki of 28 nM, indicating that 5-HT is activating receptors of the 5-HT2 type in the platelet. Lysergic acid diethylamide (LSD) and quipazine also caused dose-related increases in inositol phosphate levels, though these were considerably less than those produced by 5-HT. These results show that relatively small changes in phosphoinositide metabolism induced by serotonergic agonists can be investigated in the rabbit platelet, and this cell may therefore be a useful model for the study of some 5-HT receptors.

The effect of repeated electroconvulsive shock on the function of THIP, a GABA agonist.

Repeated electroconvulsive shock did not alter the anticonvulsant effect of THIP in rats, although it did elevate basal rectal temperature and abolish the hypothermic response to THIP.

Investigating benzodiazepine receptor function in vivo using an intravenous infusion of DMCM.

A method for measuring seizure thresholds using an intravenous infusion of a convulsant beta-carboline benzodiazepine receptor ligand (DMCM) is reported. Seizure thresholds to DMCM are elevated by the benzodiazepine receptor agonist, flurazepam and antagonist, Ro 15-1788, and the proconvulsant beta-carboline, FG 7142. Various other anticonvulsant also antagonise DMCM seizures. Selectivity for the benzodiazepine/GABA receptor complex is demonstrated since bicuculline and pentylenetetrazol lowered thresholds whereas strychnine and N-methyl DL-aspartate did not.

Studies on [3H]diazepam and [3H]ethyl-beta-carboline carboxylate binding to rat brain in vivo. I. Regional variations in displacement.

The binding of [3H]diazepam and [3H]ethyl-beta-carboline carboxylate (beta-CCE) to rat brain membranes has been studied following injection of the ligand via a tail vein. "Ex vivo" binding was avoided by homogenising the tissue in an excess of unlabelled ligand. The dissociation rate constant for [3H]diazepam and [3H]beta-CCE was approximately 0.46 min-1 at 0 degree C. Displacement of [3H]diazepam by beta-CCE in vivo showed regional variation: the dose of beta-CCE required to inhibit 50% of [3H]diazepam binding in the cerebellum was one quarter of that required in the cortex, hippocampus, or striatum. However, when diazepam was used to displace [3H]beta-CCE in vivo the converse occurred: the dose needed for 50% inhibition in the cerebellum was more than four times that required in the other three regions. These findings support suggestions from in vitro experiments that two receptors exist with different affinities for benzodiazepines and beta-carbolines. The benzodiazepine receptor antagonist Ro 15-1788 did not differentiate between the two receptor subtypes.

Studies on [3H]diazepam and [3H]ethyl-beta-carboline carboxylate binding to rat brain in vivo. II. Effects of electroconvulsive shock.

In vivo specific binding of [3H]diazepam was not altered by a single electroconvulsive shock given 5, 30, or 60 min, or 24 h previously, nor 24 h after the last of 10 daily shocks. Similarly, in vivo [3H]ethyl-beta-carboline carboxylate binding was not changed in the brains of animals that had been given a single electroconvulsive shock 30 min previously or a series of 10 daily shocks. Brain areas examined included cerebral cortex, hippocampus, cerebellum, and striatum. However, cortical binding of [3H]diazepam was increased by 32% in animals which were present in the same room while another was being injected and killed. This may represent a response to stress and/or anxiety.