Negative allosteric modulation of metabotropic glutamate receptor 5 results in broad spectrum activity relevant to treatment resistant depression.

Evidence suggests that 30-50% of patients suffering from major depressive disorder (MDD) are classified as suffering from treatment resistant depression (TRD) as they have an inadequate response to standard antidepressants. A key feature of this patient population is the increased incidence of co-morbid symptoms like anxiety and pain. Recognizing that current standards of care are largely focused on monoaminergic mechanisms of action (MOAs), innovative approaches to drug discovery for TRD are targeting glutamate hyperfunction. Here we describe the in vitro and in vivo profile of GRN-529, a novel negative allosteric modulator (NAM) of metabotropic glutamate receptor 5 (mGluR5). In cell based pharmacology assays, GRN-529 is a high affinity (Ki 5.4 nM), potent (IC50 3.1 nM) and selective (>1000-fold selective vs mGluR1) mGluR5 NAM. Acute administration of GRN-529 (0.1-30 mg/kg p.o.) had dose-dependent efficacy across a therapeutically relevant battery of animal models, comprising depression (decreased immobility time in tail suspension and forced swim tests) and 2 of the co-morbid symptoms overrepresented in TRD, namely anxiety (attenuation of stress-induced hyperthermia, and increased punished crossings in the four plate test) and pain (reversal of hyperalgesia due to sciatic nerve ligation or inflammation). The potential side effect liability of GRN-529 was also assessed using preclinical models: GRN-529 had no effect on rat sexual behavior or motor co-ordination (rotarod), however it impaired cognition in mice (social odor recognition). Efficacy and side effects of GRN-529 were compared to standard of care agents (antidepressant, anxiolytic or analgesics) and the tool mGluR5 NAM, MTEP. To assess the relationship between target occupancy and efficacy, ex vivo receptor occupancy was measured in parallel with efficacy testing. This revealed a strong correlation between target engagement, exposure and efficacy across behavioral endpoints, which supports the potential translational value of PET imaging to dose selection in patients. Collectively this broad spectrum profile of efficacy of GRN-529 supports our hypothesis that negative allosteric modulation of mGluR5 could represent an innovative therapeutic approach to the treatment of TRD. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Concomitant blockade of 5-HT(1A) receptor and 5-HT transporter: use of the Hunter Serotonin toxicity criteria in a clinical pharmacology study.

There is a potential risk that 5-HT(1A) receptor blockade combined with blockade of the 5-HT transporter by an SSRI may cause a toxic increase in 5-HT within the synapse, sparking concern for 'serotonin syndrome', a rare but potentially life threatening condition. We evaluated the safety and pharmacodynamics of the combination of the 5-HT(1A) antagonist lecozotan and the SSRI citalopram in a well-controlled Clinical Pharmacology Unit setting using the Hunter Serotonin Toxicity Criteria (HSTC), a set of validated decision rules featuring neurological and body temperature measurements, to detect any clinically relevant serotonin toxicity. Forty-three young healthy male subjects were randomized, to 2 parallel double-blind treatment groups following a 10-day citalopram 40 mg run-in period: citalopram 40 mg/lecozotan 10mg or citalopram 40 mg/placebo for 9 days. Overall, the combined administration of active drugs was well tolerated, however, one subject experienced moderate hyperreflexia, tremor of the hands, and sweating of hands and feet after 3 days of combined treatment. The event prompted treatment withdrawal and was regarded as mild serotonin toxicity, as per the HSTC. The onset of the event was around the time of peak plasma concentrations (t(max)) of both lecozotan and citalopram, and its time course corresponds to the well-defined PK profile of lecozotan. No evidence of a PK interaction was detected trough lecozotan and citalopram plasma concentrations analysis. The utility of the HSTC in detecting the non-discrete group of symptoms commonly referred to as "serotonin toxicity" was demonstrated in this clinical pharmacology study combining two 5-HT agents in a clinically controlled setting.

Preclinical characterization of WAY-211612: a dual 5-HT uptake inhibitor and 5-HT (1A) receptor antagonist and potential novel antidepressant.

BACKGROUND AND PURPOSE: As a combination of 5-HT selective reuptake inhibitor (SSRI) with 5-HT(1A) receptor antagonism may yield a rapidly acting antidepressant, WAY-211612, a compound with both SSRI and 5-HT(1A) receptor antagonist activities, was evaluated in preclinical models. EXPERIMENTAL APPROACH: Occupancy studies confirmed the mechanism of action of WAY-211612, while its in vivo profile was characterized in microdialysis and behavioural models. KEY RESULTS: WAY-211612 inhibited 5-HT reuptake (K(i) = 1.5 nmol.L(-1); K(B) = 17.7 nmol.L(-1)) and exhibited full 5-HT(1A) receptor antagonist activity (K(i) = 1.2 nmol.L(-1); K(B) = 6.3 nmol.L(-1); I(max) 100% in adenyl cyclase assays; K(B) = 19.8 nmol.L(-1); I(max) 100% in GTPgammaS). WAY-211612 (3 and 30 mg.kg(-1), po) occupied 5-HT reuptake sites in rat prefrontal cortex (56.6% and 73.6% respectively) and hippocampus (52.2% and 78.5%), and 5-HT(1A) receptors in the prefrontal cortex (6.7% and 44.7%), hippocampus (8.3% and 48.6%) and dorsal raphe (15% and 83%). Acute or chronic treatment with WAY-211612 (3-30 mg.kg(-1), po) raised levels of cortical 5-HT approximately twofold, as also observed with a combination of an SSRI (fluoxetine; 30 mg.kg(-1), s.c.) and a 5-HT(1A) antagonist (WAY-100635; 0.3 mg.kg(-1), s.c). WAY-211612 (3.3-30 mg.kg(-1), s.c.) decreased aggressive behaviour in the resident-intruder model, while increasing the number of punished crossings (3-30 mg.kg(-1), i.p. and 10-56 mg.kg(-1), po) in the mouse four-plate model and decreased adjunctive drinking behaviour (56 mg.kg(-1), i.p.) in the rat scheduled-induced polydipsia model. CONCLUSIONS AND IMPLICATIONS: These findings suggest that WAY-211612 may represent a novel antidepressant.

Lecozotan (SRA-333): a selective serotonin 1A receptor antagonist that enhances the stimulated release of glutamate and acetylcholine in the hippocampus and possesses cognitive-enhancing properties.

Recent data has suggested that the 5-hydroxytryptamine (5-HT)(1A) receptor is involved in cognitive processing. A novel 5-HT(1A) receptor antagonist, 4-cyano-N-{2R-[4-(2,3-dihydrobenzo[1,4]-dioxin-5-yl)-piperazin-1-yl]-propyl}-N-pyridin-2-yl-benzamide HCl (lecozotan), which has been characterized in multiple in vitro and in vivo pharmacological assays as a drug to treat cognitive dysfunction, is reported. In vitro binding and intrinsic activity determinations demonstrated that lecozotan is a potent and selective 5-HT(1A) receptor antagonist. Using in vivo microdialysis, lecozotan (0.3 mg/kg s.c.) antagonized the decrease in hippocampal extracellular 5-HT induced by a challenge dose (0.3 mg/kg s.c.) of 8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT) and had no effects alone at doses 10-fold higher. Lecozotan significantly potentiated the potassium chloride-stimulated release of glutamate and acetylcholine in the dentate gyrus of the hippocampus. Chronic administration of lecozotan did not induce 5-HT(1A) receptor tolerance or desensitization in a behavioral model indicative of 5-HT(1A) receptor function. In drug discrimination studies, lecozotan (0.01-1 mg/kg i.m.) did not substitute for 8-OH-DPAT and produced a dose-related blockade of the 5-HT(1A) agonist discriminative stimulus cue. In aged rhesus monkeys, lecozotan produced a significant improvement in task performance efficiency at an optimal dose (1 mg/kg p.o.). Learning deficits induced by the glutamatergic antagonist MK-801 [(-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate] (assessed by perceptually complex and visual spatial discrimination) and by specific cholinergic lesions of the hippocampus (assessed by visual spatial discrimination) were reversed by lecozotan (2 mg/kg i.m.) in marmosets. The heterosynaptic nature of the effects of lecozotan imbues this compound with a novel mechanism of action directed at the biochemical pathologies underlying cognitive loss in Alzheimer's disease.

Effect of chronic fluoxetine and WAY-100635 treatment on serotonergic neurotransmission in the frontal cortex.

Clinical augmentation strategies have shown that some improvement in antidepressant efficacy can be achieved by combining the beta-adrenergic/serotonin (5-HT)1A/1B receptor antagonist (+/-)pindolol with a selective serotonin reuptake inhibitor (SSRI). This has lead to the hypothesis that a combination of a 5-HT1A receptor antagonist with an SSRI will lead to a faster onset of antidepressant action. Although there is a significant accumulation of acute preclinical data supporting this rationale, until recently, there have been no investigations examining the chronic effects of combining an SSRI with a 5-HT1A receptor antagonist. Here, we determined the chronic effects of fluoxetine (10 mg/kg o.d.), administered in combination with the selective 5-HT1A receptor antagonist WAY-100635 (1 mg/kg b.i.d.), on serotonergic neurotransmission in the frontal cortex using in-vivo microdialysis. Following chronic administration of fluoxetine +/- WAY-100635, functional changes in serotonergic neurotransmission, as well as 5-HT1A autoreceptors, were assessed by administering fluoxetine or (+/-) 8-hydroxy-2-(di-n-propylamino)tetralin [(+/-) 8-OH-DPAT] 24 h after the last chronic dose. Chronic administration of WAY-100635 alone produced no detectable change in the functional status of the 5-HT1A receptor. However, fluoxetine alone produced a time-dependent adaptation in serotonergic transmission such that fluoxetine (acutely administered on day 15) was able to produce a two-fold increase in extracellular 5-HT levels but the decrease in response to 8-OH-DPAT was completely attenuated. These data indicate that the fluoxetine-induced adaptation was mediated by desensitization of the 5-HT1A receptor. WAY-100635 given chronically in combination with fluoxetine blocked the SSRI-induced desensitization of the 5-HT1A receptor. Furthermore, chronic treatment with this combination produced no tolerance in terms of its ability to acutely increase forebrain 5-HT levels. These data suggest that augmentation of an SSRI by combined pharmacotherapy with a 5-HT1A antagonist would be effective upon prolonged exposure.

The potential utility of 5-HT1A receptor antagonists in the treatment of cognitive dysfunction associated with Alzheimer s disease.

The 5-HT1A receptor has been extensively studied over the last two decades. There is a plethora of information describing its anatomical, physiological and biochemical roles in the brain. In addition, the development of selective pharmacological tools coupled with our understanding of psychiatric pathology has lead to multiple hypotheses for the therapeutic utility of 5-HT1A agents and in particular 5-HT1A receptor antagonists. Over the last decade it has been suggested that 5-HT1A receptor antagonists may have therapeutic utility in such diseases as depression, anxiety, drug and nicotine withdrawal as well as schizophrenia. However, a very compelling rationale has been developed for the therapeutic potential of 5-HT1A receptor antagonists in Alzheimer s disease and potentially other diseases with associated cognitive dysfunction. Receptor blockade by a 5-HT1A receptor antagonist appears to enhance activation and signaling through heterosynaptic neuronal circuits known to be involved in cognitive processes and, as such, represents a novel therapeutic approach to the treatment of cognitive deficits associated with Alzheimer s disease and potentially other disorders with underlying cognitive dysfunction.

Studies towards the next generation of antidepressants. Part 1: Indolylcyclohexylamines as potent serotonin reuptake inhibitors.

A series of indolylcyclohexylamines possessing potent and selective serotonin reuptake inhibition is reported. The most interesting compounds proved to have subnanomolar 5-HT transporter activity, and exhibited moderate 5-HT(1A) affinity.

1,2,5-Thiadiazole derivatives are potent and selective ligands at human 5-HT1A receptors.

Amino acid derivatives of 1,2,5-thiadiazol-3-yl-piperazine related to (+)-WAY-100135 and WAY-100635 are potent 5-HT1A receptor agonists and antagonists, which have selective affinity for 5-HT1A receptors versus alpha1 and dopamine (D2, D3, and D4) receptors.

Effects of chronic fluoxetine treatment in the presence and absence of (+/-)pindolol: a microdialysis study.

Using in vivo microdialysis in the frontal cortex of the freely moving rat we evaluated the effects of chronic treatment with the serotonin specific reuptake inhibitor (SSRI) fluoxetine in the presence and absence of the 5-HT(1A)/beta-adrenergic antagonist (+/-)pindolol. Chronic vehicle treated animals produced no significant response to a challenge with fluoxetine (10 mg kg(-1)) on day 8 and 15. Alternatively, a significant (P<0.05) decrease in extracellular 5-HT was observed in control animals upon challenge with the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.03 and 0.1 mg kg(-1)). Conversely, animals treated with fluoxetine (10 mg kg(-1) o.d.) for 7 and 14 days produced a significant (P<0.05) 2 fold increase in extracellular 5-HT when challenged with fluoxetine (10 mg kg(-1)) on day 8 and 15. Moreover, no significant decrease in extracellular 5-HT was observed upon challenge with either dose of 8-OH-DPAT. Animals chronically treated with (+/-)pindolol (10 or 20 mg kg(-1) b.i.d.) produced a significant dose-related increase in extracellular 5-HT upon challenge with fluoxetine on day 15 only. Furthermore, both doses produced a significantly blunted response to the low dose challenge of 8-OH-DPAT (0.03 mg kg(-1)). In addition, 20 mg kg(-1) (+/-)pindolol treated animals also had no response to the higher 0.1 mg kg(-1) dose of 8-OH-DPAT. Animals treated for 14 days with a combination of (+/-)pindolol (10 or 20 mg kg(-1)) and fluoxetine were not significantly different from vehicle treated animals when challenged with fluoxetine or 8-OH-DPAT. Taken together it would therefore appear that although (+/-)pindolol alone has sufficient intrinsic activity to produce a desensitization of the 5-HT(1A) receptor, when given in combination with fluoxetine it is able to prevent the desensitization induced by not only fluoxetine but also itself. This may suggest that the clinical augmentation of antidepressant action by pindolol, when co-administered with a SSRI, is via antagonism of the 5-HT(1A) receptor.

Cloning, mRNA localization and evolutionary conservation of a human 5-HT7 receptor pseudogene.

Initial experiments designed to clone novel serotonin receptor subtypes in the substantia nigra have led to the discovery of a transcribed human 5-HT7 receptor pseudogene that is expressed in a wide range of tissues. The original clone (S771) possessed greater than 90% homology to the 5-HT7 receptor sequence and was identified by a degenerate PCR approach. Expression of the pseudogene transcript was detected throughout the brain and peripheral tissues in general agreement with 5-HT7 mRNA localization. Interestingly, the transcript was detected in tissues not known to express the 5-HT7 receptor (i.e. liver and kidney). Analysis of genomic DNA explained the genesis of the human pseudogene via a processed parental transcript (retrotransposition) and led to the discovery of a species homologue in the rhesus monkey.

Identification of a human 5-HT6 receptor variant produced by alternative splicing.

The complexity of the 5-hydroxytryptamine (5-HT) (serotonin) receptor family has been increased by the findings that isoforms or splice variants exist for subtypes such as the 5-HT2B, 5-HT2C, 5-HT4 and 5-HT7 subtypes. Further molecular biological studies in our laboratory have demonstrated that a splice variant of the 5-HT6 receptor exists in the human brain. Experiments performed using a degenerate PCR approach from human caudate cDNA revealed a 5-HT6 receptor clone with a 289 bp deletion of the region coding for transmembrane IV through the third intracellular loop. This deletion produces a frameshift creating a downstream stop codon which results in a truncated protein containing 10 unique amino acids at its carboxyl end. The variant transcript occurs as a result of alternative splicing using an upstream donor site and the acceptor site from the first intron in the 5-HT6 receptor gene. The splicing pattern seen for this transcript was not detected in rat or mouse whole brain cDNA by PCR due to the lack of a consensus 5' donor site. Coexpression of the variant 5-HT6 transcript and the full length 5-HT6 transcript was observed in caudate and substantia nigra but not in hippocampus, cortex, cerebellum and thalamus. Transient transfection of a 5-HT6 variant construct into Cos-7 cells demonstrated that a truncated receptor was translocated to the membrane but appeared nonfunctional.

Characterization of 5-HT1A receptor functional coupling in cells expressing the human 5-HT1A receptor as assessed with the cytosensor microphysiometer.

The functional activity of a series of 5-HT1A receptor ligands has been evaluated in a cell line expressing the human 5-HT1A receptor (h5-HT1A x CHO) using the agonist-stimulated increase in extracellular acidification rate, measured with the microphysiometer, as a functional assay. Both 5-CT and 8-OH-DPAT were potent agonists in stimulating an increase in extracellular acidification rate in h5-HT1A x CHO cells with estimated EC50 values of 1.2 and 7.8 nM, respectively. Additionally, these two 5-HT1A receptor agonists elicited a similar maximum response. Concentration-dependent agonist activity was also observed in the presence of buspirone, ipsapirone, BMY7378, NAN-190 and WAY100135, and each of these compounds behaved as partial 5-HT1A receptor agonists. The selective 5-HT1A receptor antagonist WAY100635 produced a potent (IC50, 2.3 nM) and complete block of the 8-OH-DPAT-stimulated response. An evaluation of the inhibitory activity of a series of 5-HT1A receptor antagonists produced the following rank order of potency; WAY100635 > LY206130 (IC50, 7.1 nM) > WAY100135 (30.8 nM) > pindolol (76.2 nM) > (-)UH-301 (92.8 nM). Parallel studies on the inhibition of forskolin-stimulated adenylyl cyclase activity in hS-HT1A x CHO cells revealed that agonist potencies were generally similar between the two functional assays and were in good agreement with the estimated 5-HT1A receptor binding affinities. However, the relative efficacies determined for the partial agonists in the cAMP assay were substantially greater than those observed with the microphysiometer. Finally, antagonists were considerably weaker in the cAMP assay compared with the microphysiometer. The evaluation of 5-HT1A ligands using the microphysiometer, which represents a very distinct indice of 5-HT1A receptor function compared with the cAMP assay, results in a different profile of functional activity.

The potassium channel blockers 4-aminopyridine and tetraethylammonium increase the spontaneous basal release of [3H]5-hydroxytryptamine in rat hippocampal slices.

Previous investigations have demonstrated that compounds capable of blocking presynaptic potassium channels can stimulate neurotransmitter release at both peripheral and central synapses. This study examined the in vitro effects of the "classical" potassium channel blockers 4-aminopyridine (4-AP) and tetraethylammonium (TEA) on the spontaneous basal release of [3H]5-hydroxytryptamine ([3H]5-HT) from rat hippocampal slices using an automated superfusion apparatus. 4-AP and structural analogs increased the spontaneous basal release of [3H]5-HT in a concentration-related manner. The rank order of potencies from the estimated EC50 values indicated that 3,4-diaminopyridine (0.88 mM) approximately 4-AP (1.2 mM) > 2-AP (89 mM) > 3-AP (100 mM) > pyridine (256 mM). TEA stimulated [3H]5-HT release with an estimated EC50 value of 63 mM and was less efficacious than the pyridine congeners. The enhancement of release induced by 1 mM 4-AP was additive with 100 mM TEA and 5 microM veratridine but not with 3,4-diaminopyridine or KCl (25 and 50 mM). The release induced by 4-AP (0.3, 1 and 10 mM) and TEA (30, 100 and 300 mM) was significantly attenuated in a calcium-free buffer containing 1 mM ethylene glycol-bis(b-aminoethyl ether N,N,N',N'-tetraacetic acid. Tetrodotoxin (1 microM), a sodium channel blocker, was unable to block the response to 4-AP (1 mM) and TEA (100 mM). Notably, this concentration of tetrodotoxin reduced the stimulation of [3H]5-HT release produced by the sodium channel opener veratridine (5 microM). Taken together, the results demonstrate that potassium channel blockade can enhance the spontaneous basal release of [3H]5-HT in rat hippocampal slices. These effects are at least partly dependent on extracellular calcium and do not appear to be mediated by modulating sodium channel function.

Pharmacological characterizations of recombinant human 5-HT1D alpha and 5-HT1D beta receptor subtypes coupled to adenylate cyclase inhibition in clonal cell lines: apparent differences in drug intrinsic efficacies between human 5-HT1D subtypes.

Recombinant human 5-HT1D alpha and 5-HT1D beta receptor subtypes were stably expressed in NIH-3T3 fibroblasts (1D alpha cell line) and Y-1 adrenocortical tumor cells (1D beta cell line), respectively, for pharmacological evaluations of serotonergic compounds to inhibit forskolin-stimulated cAMP accumulation (FSCA). [3H]LSD saturation studies indicated that 5-HT1D receptor expression levels were slightly higher in the 1D beta cell line (Bmax = 1334 +/- 134 fmol/mg protein) than in the 1D alpha cell line (Bmax = 900 +/- 218 fmol/mg protein). 5-HT inhibited FSCA with similar potencies (EC50 approximately 2 nM) in both assay systems. The rank order of agonist potencies in both clonal cell lines matched their pharmacological profiles previously determined in binding studies: dihydroergotamine > or = 5-carboxamidotryptamine (5-CT) > LSD > or = 5-HT > sumatriptan > 1-naphthylpiperazine (1-NP) > yohimbine > 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH DPAT) > 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), with Ki/EC50 ratios greater than unity. Methiothepin acted as a silent antagonist at both human 5-HT1D alpha and 5-HT1D beta receptors with apparent dissociation constants (Kb values) of 12 +/- 1 nM and 3 +/- 1 nM, respectively. Whereas GR 127,935, metergoline, DOI, and quipazine acted as full agonists in the 1D alpha cell line, these compounds behaved as partial agonists in the 1D beta cell line. To determine whether high levels of receptor reserve might mask partial agonist activity in the two second messenger assay systems, studies were performed using the irreversible receptor alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The relationships between receptor occupancy and inhibition of FSCA were determined for 5-HT, sumatriptan, and 1-NP in both clonal cell lines after partial receptor inactivation using Furchgott analysis. Hyperbolic relationships between receptor occupancy and second messenger response were determined for 5-HT in both transfected cell lines. Steep hyperbolic relationships were also found for sumatriptan and 1-NP in the 1D beta cell line whereas nearly linear relationships were observed for these two compounds in the 1D alpha cell line. Moreover, KA/EC50 ratios of these compounds were significantly larger in the 1D beta (10-32) as compared to the 1D alpha (0.9-2.5) cell line. These data are consistent with the hypothesis that the two heterologous expression systems contain a differential amount of receptor reserve. Despite the presence of an apparently larger-receptor reserve in the 1D beta cell line, GR 127,935, metergoline, DOI, and quipazine behaved as partial agonists. Although the potencies (EC50 values) of compounds matched their respective affinity constants (Ki values) for the closely-related 5-HT1D subtypes, differences in intrinsic activities were observed for a few compounds between the two 5-HT1D receptor expression systems. Since receptor reserve is dependent on the properties of both the assay system and drug, the observed variations in intrinsic activity, although influenced by the variable amounts of receptor reserve in the two transfected cell lines, reflect primarily system-independent differences in the intrinsic efficacy of the tested compounds at the two human 5-HT1D receptors. Higher intrinsic efficacies of compounds at the human 5-HT1D alpha receptor relative to the human 5-HT1D beta subtype may be responsible for the higher intrinsic activities observed in the 1D alpha cell line, even though receptor reserve is apparently lower in this system.

Voltage-gated K+ channel beta subunits: expression and distribution of Kv beta 1 and Kv beta 2 in adult rat brain.

Recent cloning of K+ channel beta subunits revealed that these cytoplasmic polypeptides can dramatically alter the kinetics of current inactivation and promote efficient glycosylation and surface expression of the channel-forming alpha subunits. Here, we examined the expression, distribution, and association of two of these beta subunits, Kv beta 1 and Kv beta 2, in adult rat brain. In situ hybridization using cRNA probes revealed that these beta-subunit genes are heterogeneously expressed, with high densities of Kv beta 1 mRNA in the striatum, CA1 subfield of the hippocampus, and cerebellar Purkinje cells, and high densities of Kv beta 2 mRNA in the cerebral cortex, cerebellum, and brainstem. Immunohistochemical staining using subunit-specific monoclonal and affinity-purified polyclonal antibodies revealed that the Kv beta 1 and Kv beta 2 polypeptides frequently co-localize and are concentrated in neuronal perikarya, dendrites, and terminal fields, and in the juxtaparanodal region of myelinated axons. Immunoblot and reciprocal co-immunoprecipitation analyses indicated that Kv beta 2 is the major beta subunit present in rat brain membranes, and that most K+ channel complexes containing Kv beta 1 also contain Kv beta 2. Taken together, these data suggest that Kv beta 2 is a component of almost all K+ channel complexes containing Kv 1 alpha subunits, and that individual channels may contain two or more biochemically and functionally distinct beta-subunit polypeptides.

Beta subunits promote K+ channel surface expression through effects early in biosynthesis.

Voltage-gated K+ channels are protein complexes composed of ion-conducting integral membrane alpha subunits and cytoplasmic beta subunits. Here, we show that, in transfected mammalian cells, the predominant beta subunit isoform in brain, Kv beta 2, associates with the Kv1.2 alpha subunit early in channel biosynthesis and that Kv beta 2 exerts multiple chaperone-like effects on associated Kv1.2 including promotion of cotranslational N-linked glycosylation of the nascent Kv1.2 polypeptide, increased stability of Kv beta 2/Kv1.2 complexes, and increased efficiency of cell surface expression of Kv1.2. Taken together, these results indicate that while some cytoplasmic K+ channel beta subunits affect the inactivation kinetics of alpha subunits, a more general, and perhaps more fundamental, role is to mediate the biosynthetic maturation and surface expression of voltage-gated K+ channel complexes. These findings provide a molecular basis for recent genetic studies indicating that beta subunits are key determinants of neuronal excitability.

Ketanserin and ritanserin discriminate between recombinant human 5-HT1D alpha and 5-HT1D beta receptor subtypes.

Compounds able to discriminate functionally between the closely related cloned human 5-HT1D alpha and 5-HT1D beta receptor subtypes have not been reported previously. In [3H]5-HT competition assays, the classical 5-HT2A receptor antagonists, ritanserin and ketanserin, displayed moderate affinity (pKi = 7.30 and 7.17, respectively) and marked selectivity (22- and 71-fold, respectively) for the recombinant human 5-HT1D alpha subtype relative to the 5-HT1D beta receptor. In contrast, the nonselective 5-HT1/2 receptor antagonist, methiothepin, exhibited similar binding affinities (pKi = 7.64-8.01) for both recombinant 5-HT1D subtypes. The antagonistic properties of these compounds were evaluated for their ability to block 5-HT-induced inhibition of forskolin-stimulated cAMP accumulation in intact cells stably expressing either 5-HT1D alpha or 5-HT1D beta receptors. All three compounds behaved as antagonists devoid of intrinsic activity in the functional assays. The apparent pKb values determined in functional assays closely matched their pKi values obtained in binding assays. Since ketanserin exhibits significant selectivity for the human 5-HT1D alpha receptor, this antagonist can be used as a pharmacological tool to discriminate between 5-HT1D alpha and 5-HT1D beta receptor-mediated responses in human tissues.

The 5-HT4 receptor: molecular cloning and pharmacological characterization of two splice variants.

Molecular cloning efforts have provided primary amino acid sequence and signal transduction data for a large collection of serotonin receptor subtypes. These include five 5-HT1-like receptors, three 5-HT2 receptors, one 5-HT3 receptor, two 5-HT5 receptors, one 5-HT6 receptor and one 5-HT7 receptor. Molecular biological information on the 5-HT4 receptor is notably absent from this list. We now report the cloning of the pharmacologically defined 5-HT4 receptor. Using degenerate oligonucleotide primers, we identified a rat brain PCR fragment which encoded a '5-HT receptor-like' amino acid sequence. The corresponding full length cDNA was isolated from a rat brain cDNA library. Transiently expressed in COS-7 cells, this receptor stimulates adenylyl cyclase activity and is sensitive to the benzamide derivative cisapride. The response is also blocked by ICS-205930. Interestingly, we isolated two splice variants of the receptor, 5-HT4L and 5-HT4S, differing in the length and sequence of their C-termini. In rat brain, the 5-HT4S transcripts are restricted to the striatum, but the 5-HT4L transcripts are expressed throughout the brain, except in the cerebellum where it was barely detectable. In peripheral tissues, differential expression was also observed in the atrium of the heart where only the 5-HT4S isoform was detectable.

Differential effects of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) on various 5-HT receptor binding sites in the rat brain.

The effects of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), an alkylating agent producing irreversible blockade of various membrane bound receptors in brain, were investigated on four different types of serotonin receptors, 5-HT1A, 5-HT1B, 5-HT2A and 5-HT3, in various brain regions in the rat. In addition, the fate of central benzodiazepine- and "R"-zacopride-specific binding sites was also examined in rats treated with EEDQ. Membrane binding assays and/or quantitative autoradiography with appropriate radioligands indicated that EEDQ inactivated 5-HT1A, 5-HT1B and 5-HT2A sites, but was poorly active on 5-HT3, benzodiazepine and "R" sites. Among the receptors affected by EEDQ, hippocampal 5-HT1A sites were the most sensitive to the alkylating agent (ID50 approximately 1 mg/kg i.p.), followed by the cortical 5-HT2A (ID50 approximately 3 mg/kg i.p.) and the striatal 5-HT1B (ID50 approximately 6 mg/kg i.p.) sites. Pretreatment by selective ligands partially protected hippocampal 5-HT1A sites from irreversible inactivation by EEDQ (10 mg/kg i.p.) with the following order of efficacy: WAY 100635 > spiperone > BMY 7378 > ipsapirone. Similarly, pretreatment by spiperone (5 mg/kg i.p.) also reduced the ability of EEDQ to inactivated cortical 5-HT2A receptors. Analyses of the time-course recovery of respective binding sites after EEDQ administration showed that the turnover rate of 5-HT1A sites did not significantly differ in the dorsal raphe nucleus and in various forebrain areas (hippocampus, septum, cerebral cortex; half-life: approximately 4 days), but was lower than that of cortical 5-HT2A sites (half-life: 2.9 days).

Cell-specific coupling of the cloned human 5-HT1F receptor to multiple signal transduction pathways.

We recently described the cloning of a fifth member of the 5-hydroxytryptamine (5-HT)1 (serotonin1) receptor class that inhibits adenylyl cyclase, namely the human 5-HT1F receptor (Adham et al. 1993a). In the present study we have examined in greater detail the functional coupling of the 5-HT1F receptor in two different cell lines, NIH-3T3 and LM(tk-) fibroblasts (receptor densities of 1.7 and 4.4 pmol/mg protein, respectively). The maximal inhibitory response elicited by 5-HT was significantly greater in NIH-3T3 as compared to LM(tk-) cells, whereas the EC50 values were comparable. To investigate the relationship between receptor occupancy and inhibition of cAMP accumulation mediated by 5-HT1F receptors in NIH-3T3 cells (and hence the degree of receptor reserve), we used the irreversible receptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The half-maximal response required only about 10% receptor occupancy, consistent with a receptor reserve of 90% (88 +/- 2.1%, n = 4) for 5-HT-induced inhibition of FSCA. Despite the presence of such a high degree of receptor reserve, a range of intrinsic activities was displayed by structurally diverse classes of compounds. For example, sumatriptan and lysergol were as efficacious as 5-HT itself and thus acted as full agonists, whereas metergoline and 1-NP behaved as partial agonists and as shown previously (Adham et al. 1993a), methiothepin was a silent antagonist (Kb = 438 nM). We have also investigated activation of additional signal transduction pathways by the 5-HT1F receptor and found that the responses differ in the two cell lines with respect to stimulation of phospholipase C. For example, in NIH-3T3 cells no elevation of inositol phosphates (IP) of [Ca2+]i was observed even at very high agonist concentrations (100 microM). In contrast, in LM(tk-) cells concentrations of 5-HT as low as 10 nM induced stimulation of IP and a rapid increase of [Ca2+]i. The 5-HT1F receptor failed to alter arachidonic acid release in either cell line. The maximal increase in IP accumulation in LM(tk-) cells was modest, averaging about 100% above basal. The increases of IP and [Ca2+]i required 5-HT concentrations less than one order of magnitude greater than those inhibiting FSCA (EC50 = 17, 55 and 8 nM, respectively), and both responses were blocked by 100 microM methiothepin. All three responses (cAMP, IP, and [Ca2+]i) were sensitive to pertussis toxin pre-treatment, suggesting the involvement of Gi/Go protein(s) in these signal transduction pathways.(ABSTRACT TRUNCATED AT 400 WORDS)