Function and regulation of a murine macrophage-specific IgG Fc receptor, Fc gamma R-alpha.

Ligand binding specificities of two cloned murine Fc gamma Rs (Fc gamma R-alpha, Fc gamma R-beta [9]) were determined by gene transfer into Fc gamma R negative cell lines. Both receptors were expressed as full-length molecules capable of IgG immune complex binding that was inhibitable by the mAb 2.4G2. The ligand binding profiles of these receptors were indistinguishable whereby both bound immune-complexed mouse IgG1, IgG2a, and IgG2b, but not IgG3. Neither receptor could bind monomeric IgG2a, indicating these receptors to be low-affinity IgG Fc receptors. Accumulation of the Fc gamma R-alpha mRNA can be induced with murine IFN-gamma at a concentration of 200 U/ml in the macrophage-like cell lines RAW 264.7 and J774a. The time course for induction indicates that the mRNA accumulation is transient but does not return to the uninduced level even after 50 h of treatment. Fc gamma R-beta mRNA was not induced by IFN-gamma, rather its expression was down modulated in mouse peritoneal macrophages. Both RAW and J774a cells lines exhibited increased receptor levels after IFN-gamma stimulation as measured by 125I-2.4G2 and ligand binding. In the absence of IFN-gamma, the RAW and J774a cell lines were minimally phagocytic, while P388D1 cells were actively phagocytic. In the presence of IFN-gamma, however, RAW 264.7 and J774a cells were induced to become actively phagocytic. Induction of Fc gamma R-alpha mRNA and protein by IFN-gamma may be part of the process by which macrophages become activated to engulf antibody-coated particles.

Cloning and expression of a glycine transporter reveal colocalization with NMDA receptors.

A complementary DNA clone encoding a transporter for glycine has been isolated from rat brain, and its functional properties have been examined in mammalian cells. The transporter displays high affinity for glycine (KM approximately 100 microM) and is dependent on external Na+ and Cl-. Northern blot analysis indicates that the distribution of the mRNA encoding the glycine transporter is restricted to the nervous system. In situ hybridization data are consistent with roles for the transporter in both glycine neurotransmission and glycine modulation of N-methyl-D-aspartate (NMDA) receptors in the hippocampus. The identification of this transporter therefore opens the study of the molecular mechanisms underlying both inhibitory glycinergic transmission and NMDA-mediated excitatory transmission.

A subfamily of 5-HT1D receptor genes.

The recent discovery and characterization of three new 5-HT1 receptor clones and the pharmacological characterization of one orphan receptor (dog RDC4) has revealed a surprising complexity within the 5-HT1D receptor subfamily. This receptor subfamily, which is believed to be the target of the anti-migraine drug sumatriptan and may regulate feeding behavior, anxiety, depression, cardiac function and movement, can now be approached on a molecular level. These cloning discoveries have also taught us an important general lesson about the molecular pharmacology of G protein-coupled receptor genes: species homologues of a gene (the equivalent gene in different species) may be highly homologous in amino acid sequence yet display very different pharmacological properties. Conversely, two different genes in the same species (intraspecies subtypes) that display only moderate degrees of transmembrane amino acid homology can display nearly indistinguishable pharmacological properties. In discussing the implications of these findings for both 5-HT receptors and G protein-linked receptors in general, Paul Hartig, Theresa Branchek and Richard Weinshank approach the question: why have so many receptor subtypes been preserved in the genome? In addition, controversy has been raging for several years over the classification of 5-HT1B receptors (found only in rat brain) and 5-HT1D receptors. Were they different subtypes or simply species homologues of the same receptor? Recent cloning studies have apparently complicated this issue, but the answer to the question is, in fact, becoming clearer.

The human serotonin 5-hydroxytryptamine1D receptor pseudogene is transcribed.

An examination of mRNA expression of the serotonin genes encoding both psi 5HT1D alpha and the related 5HT1D alpha, demonstrated that the pseudogene is transcribed, and has a tissue distribution similar to the 5HT1D alpha receptor-encoding gene. This psi 5HT1D alpha transcript is capable of being translated into a polypeptide of only 28 amino acids in length, and the psi 5HT1D alpha pseudogene most likely arose from a gene duplication or transposition event.

Two gene duplication events in the human and primate dopamine D5 receptor gene family.

The human dopamine D5 receptor (DRD5) gene family consists of the DRD5-encoding gene (DRD5) and the pseudogenes psi DRD5-1 and psi DRD5-2. Analysis of the 5' UTR of DRD5 and homologous regions in the pseudogenes revealed that the nucleotide identity (approx. 95%) extended for 1.9 kb and terminated at a monomeric Alu sequence in each of the pseudogenes. The presence of Alu sequences in the pseudogenes, at this point of divergence with DRD5, suggests that Alu sequences were involved in the evolution of the DRD5 family. This report is the first to describe a possible mechanism involved in the duplication of genes in the G-protein-coupled receptor (GPCR) family. The pseudogenes continue to share identity (approx. 98%) beyond this 5' UTR point of divergence with DRD5 for at least another 6 kb. Analysis of the 3' UTR of DRD5 and homologous regions in the pseudogenes revealed that the identity (approx. 95%) extends at least 14 kb, and the identity between the pseudogenes (approx. 98%) extends for at least 18 kb. Thus, the duplication unit that produced the first pseudogene was at least 16 kb, whereas the second pseudogene was at least 28 kb. We have also located two DRD5 pseudogenes in gorilla demonstrating that these closely related pseudogenes were present in a common ancestor of human and gorilla.

Molecular cloning of an orphan transporter. A new member of the neurotransmitter transporter family.

A complementary DNA clone predicted to encode a novel transporter was isolated from rat brain and the localization of its mRNA was examined. The cDNA, designated rB21a, predicts a protein with 12 putative transmembrane domains that exhibits significant sequence homology with neurotransmitter transporters. Expression studies have not yet identified the endogenous substrate for this transporter, but the presence of rB21a mRNA within the leptomeninges of the brain suggests the transporter may regulate CSF levels of its substrate. The cloning of rB21a provides the means to determine its physiological functions and the potential to design novel, transporter-based therapeutic agents for neurological and psychiatric disorders.

Site-directed mutagenesis of a single residue changes the binding properties of the serotonin 5-HT2 receptor from a human to a rat pharmacology.

Mesulergine displays approximately 50-fold higher affinity for the rat 5-HT2 receptor than for the human receptor. Comparison of the deduced amino acid sequences of cDNA clones encoding the human and rat 5-HT2 receptors reveals only 3 amino acid differences in their transmembrane domains. Only one of these differences (Ser----Ala at position 242 of TM5) is near to regions implicated in ligand binding by G protein-coupled receptors. We investigated the effect of mutating Ser242 of the human 5-HT2 receptor to an Ala residue as is found in the rat clone. Both [3H]mesulergine binding and mesulergine competition of [3H]ketanserin binding showed high affinity for rat membranes and the mutant human clone but low affinity for the native human clone, in agreement with previous studies of human postmortem tissue. These studies suggest that a single naturally occurring amino acid change between the human and the rat 5-HT2 receptors makes a major contribution to their pharmacological differences.

The cloned human 5-HT1E receptor couples to inhibition and activation of adenylyl cyclase via two distinct pathways in transfected BS-C-1 cells.

The pharmacological profile of coupling of the cloned human serotonin [5-hydroxytryptamine] (5-HT)1E receptors to second messengers was studied in African green monkey kidney cells (BS-C-1). At low concentrations (0.1-100 nM), 5-HT inhibited forskolin-stimulated cAMP accumulation (FSCA) by up to 90% whereas at higher concentrations it potentiated FSCA; potentiation was dependent on receptor density. Pretreatment of cells with pertussis toxin (PTx) or cholera toxin (CTx) eliminated agonist-induced inhibition and potentiation of FSCA, respectively. The potentiation of FSCA was not due to activation of phospholipase C and/or phospholipase A2 since 5-HT had no effect on inositol phosphate release, intracellular Ca2+ mobilization or arachidonic acid mobilization; neither was it affected by pretreatment with the nonselective phospholipase A2 inhibitor, quinacrine, or by the removal of extracellular Ca2+. The pharmacological profiles of the 5-HT1E receptor-mediated inhibition and potentiation of FSCA were very similar, although agonists displayed higher affinity for the former. These results indicate that the human 5-HT1E receptors can potentially couple, with similar pharmacological profiles, to multiple effector pathways. However, the potency and intrinsic activity of the compounds eliciting these responses can differ significantly, depending on the receptor density and the effector pathway studied.

Molecular cloning and pharmacological characterization of guinea pig 5-HT1B and 5-HT1D receptors.

Human 5-HT1B and 5-HT1D receptors have been implicated as molecular targets for the treatment of acute migraine based upon the pharmacological actions and clinical efficacy of sumatriptan, an agonist for human 5-HT1B/1D receptors. The guinea pig has served as an animal model to assess 5-HT1B/1D receptor function, most recently in evaluating 5-HT1B/1D receptor agonists as potential anti-migraine agents. Since two distinct, but closely-related receptors displaying "5-HT1D receptor pharmacology" have been cloned previously from most mammalian species, the genes encoding these receptors were isolated from a guinea pig liver genomic DNA library using oligonucleotide probes targeted to nonconserved regions of recombinant human 5-HT1B and 5-HT1D receptors. Sequence analysis indicates that guinea pig 5-HT1B and 5-HT1D receptors are comprised 390 and 378 amino acids, respectively. Comparison of the deduced amino acid sequences of guinea pig 5-HT1B and 5-HT1D receptor subtypes show that they display overall and transmembrane (TM) identities of 63% and 77%, respectively. Both clones contain a conserved threonine residue in TM7, a structural feature imparting "5-HT1D receptor pharmacology". Guinea pig 5-HT1B and 5-HT1D receptor genes were transiently expressed in Cos-7 cells and their binding properties were evaluated using [3H]5-HT. Both cloned receptor subtypes displayed "5-HT1D receptor pharmacology" with the following rank order of binding affinities: 5-CT > 5-HT > sumatriptan > 8-OH-DPAT > (-)-pindolol. Ketanserin displayed modest (five-fold) 5-HT1D receptor selectivity, while methiothepin exhibited a similar selectivity for the 5-HT1B subtype. In particular, ketanserin exhibits profound differences in 5-HT1D receptor affinity (and selectivity) across species. High correlations were observed between the binding affinities of serotonergic ligands for 5-HT1D binding sites measured in guinea pig cortical membranes and both cloned guinea pig 5-HT1B (r2 = 0.88) and 5-HT1D (r2 = 0.80) receptors, indicating that the development of subtype selective compounds (i.e. 5-HT1B versus 5-HT1D) using native tissues may be more difficult to achieve without the advantage of using recombinant receptor subtypes. Additionally, there is a good correspondence between binding profiles of recombinant guinea pig 5-HT1B and 5-HT1D receptor subtypes and to their respective cloned human homologs. However, species differences in binding affinities of a subset of compounds are evident. These data extend previous observations that subtype selective (i.e. 5-HT1D) compounds identified in one species may not discriminate between closely related receptors (i.e. 5-HT1B and 5-HT1D) in all animal model systems.

Cloning and characterization of the guinea pig 5-HT1F receptor subtype: a comparison of the pharmacological profile to the human species homolog.

The anti-migraine compound, sumatriptan, has been shown to have substantial affinity for the cloned human 5-HT1F receptor suggesting that, in addition to 5-HT1B/5-HT1D receptor subtypes, the 5-HT1F receptor may be a therapeutic target for the treatment of migraine. Several investigators have used the guinea pig plasma extravasation model to evaluate potential anti-migraine drugs. Since species differences in the pharmacology of serotonin receptors are well known, we compared the pharmacological profiles of the cloned human and guinea pig 5-HT1F receptors in order to validate the usefulness of the in vivo model in predicting anti-migraine activity of compounds targeted for humans. We have cloned the guinea pig 5-HT1F by homology to the human 5-HT1F receptor and evaluated its pharmacological profile using radioligand binding assays. The cloned guinea pig 5-HT1F gene exhibited 94% amino acid identity to the corresponding human homolog. High affinity (Kd approximately 10 nM) [3H]5-HT binding was detected to membranes obtained from Cos-7 cells transiently expressing the guinea pig 5-HT1F receptor. The cloned guinea pig receptor displayed typical 5-HT1F receptor pharmacology with the following rank order of binding affinities: 5-HT > sumatriptan > 1-NP = DHE > alpha-methyl 5-HT > metergoline > methiothepin > 5-CT. The pharmacological profiles of the cloned guinea pig and human 5-HT1F receptors were very similar as reflected by the high correlation (r2 = 0.72, slope = 0.76) observed between the binding affinities of compounds for these two species homologs. In situ hybridization studies in guinea pig tissue revealed 5-HT1F receptor mRNA expression in the neurons of the trigeminal ganglion, suggesting that the 5-HT1F receptor may play a role in the presynaptic inhibition of neuropeptide release at the level of the intracranial vasculature, thereby blocking the development of neurogenic inflammation. Dorsal root ganglion cells also moderately expressed the 5-HT1F transcripts. The localization of the 5-HT1F receptor to areas involved in the mediation and transfer of nociceptive information implies a role for this receptor in pain processing. These findings indicate that a selective 5-HT1F agonist may be a novel approach to treat migraine.

A single point mutation increases the affinity of serotonin 5-HT1D alpha, 5-HT1D beta, 5-HT1E and 5-HT1F receptors for beta-adrenergic antagonists.

The serotonin 5-HT1B and 5-HT1A receptors bind certain beta-adrenergic antagonists, such as propranolol and pindolol, with high affinity. Other 5-HT1 receptors that display very low affinity for beta-adrenergic antagonists, have either a threonine (T) (5-HT1D alpha, 5-HT1D beta and 5-HT1E) or an alanine (A) (5-HT1F) residue in the homologous position in the seventh transmembrane domain. In the case of the human 5-HT1D beta receptor, replacement of this T with asparagine (N), dramatically increases its ability to bind beta-adrenergic antagonists. To assess whether other 5-HT1 receptors would behave similarly, we have used site-directed mutagenesis to replace the T or A in 5-HT1D alpha, 5-HT1E and 5-HT1F receptors with N. Both the wild-type and mutant genes were expressed transiently in COS-7 cells and radioligand binding studies were performed by using [3H]5-HT and [125I]iodocyanopindolol. Using [3H]5-HT, we found that the affinities of all the mutant receptors for propranolol and pindolol were significantly increased by 100-1000 fold, 5-HT1D alpha and 5-HT1F receptors showing the highest and the 5-HT1E receptor displaying the lowest affinity. On the other hand, the affinities for 5-HT were essentially unchanged as compared to the wild-type receptors. All mutant receptors bound [125I]iodocyanopindolol with high affinity, KD values ranging between 0.04 nM (mutant 5-HT1D alpha) and 0.57 nM (mutant 5-HT1E), whereas the wild-type receptors failed to show any specific binding with this radioligand in the same concentration range used for the mutant receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Design, synthesis and evaluation of substituted triarylnipecotic acid derivatives as GABA uptake inhibitors: identification of a ligand with moderate affinity and selectivity for the cloned human GABA transporter GAT-3.

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system. Molecular biology has revealed the presence of four high-affinity GABA transporters in the brain, GAT-1, GAT-2, GAT-3, and BGT-1, the latter transporting both GABA and the osmolyte Betaine. We have shown that known GABA uptake inhibitors such as SK&F 89976-A, CI-966, and Tiagabine exhibit high affinity and selectivity for GAT-1. In the present paper we describe the design and synthesis of a novel series of triarylnipecotic acid derivatives for evaluation as GABA uptake inhibitors. The design lead for this series of compounds was the nonselective GABA uptake inhibitor EGYT-3886, [(-)-2-phenyl-2-[(dimethylamino)ethoxy]-(1R)- 1,7,7-trimethylbicyclo[2.2.1]heptane]. From this series of compounds (S)-1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-3-piperidinecarboxylic+ ++ (S)-1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-3-piperidinecarboxylic+ ++ acid, 4(S) was identified as a novel ligand with selectivity for GAT-3. 4(S) displayed an IC50 of 5 microM at GAT-3, 21 microM at GAT-2, > 200 microM at GAT-1, and 140 microM at BGT-1. This compound will be an important tool for evaluating the role of GAT-3 in neural function.

Identification of a monoclonal antibody which interacts with the parathyroid hormone receptor-adenylate cyclase system in murine bone.

We have produced monoclonal antibodies which bind specifically to mouse bone cells and then selected these monoclonal antibodies for their ability to inhibit parathyroid hormone (PTH) responses in mouse cranial bone treated with the (1-34) amino terminal peptide of bovine PTH [bPTH(1-34)]. One clone, designated 3-6, characterized as an IgM(kappa), significantly inhibited the accumulation of cAMP in response to bPTH(1-34) at concentrations of hormone between 10(-9) and 10(-7) M. This antibody was subsequently isolated by gel filtration and shown to bind to intact mouse calvariae, with saturation binding occurring at 3 micrograms/ml IgM. A maximal inhibition of approximately 70% of the cAMP accumulation produced in response to 2.5 X 10(-9) M (100 ng/ml) bPTH(1-34) was obtained with 7 micrograms/ml of the purified 3-6 IgM. At this concentration of 3-6 IgM, the half-maximal dose of PTH for activation of cAMP accumulation was increased from 5 X 10(-9) M to 2 X 10(-8) M with no reduction in maximal levels of cAMP production. The utility of this antibody as an inhibitor was further tested by its ability to block the binding of an iodinated PTH analogue, 125I-[Nle8, Nle18, Tyr34]-bPTH(1-34) to mouse cranial bone. The 3-6 IgM at a concentration of 5 X 10(-8) M inhibited 70% of the specific binding of the 125I-labeled analogue. In the absence of parathyroid hormone, 2 X 10(-8) M 3-6 IgM produced a 4-fold increase in cAMP above basal levels, as compared to 40-fold maximal increases observed with PTH, indicating a partial PTH agonist activity of this antibody. When tested for effects on other hormones, 3-6 IgM did not inhibit cAMP accumulation produced in response to salmon calcitonin, epinephrine, prostaglandin E2 or cholera toxin. We propose that the 3-6 monoclonal IgM is specific for the PTH receptor or a component of the PTH receptor-adenylate cyclase system and that this or similar antibodies will serve as useful reagents for future molecular characterization of this receptor.

Localization of messenger RNAs encoding three GABA transporters in rat brain: an in situ hybridization study.

Localization of the messenger RNAs encoding three gamma-aminobutyric acid (GABA) transporters, termed GAT-1, GAT-2, and GAT-3, has been carried out in rat brain using radiolabeled oligonucleotide probes and in situ hybridization histochemistry. Hybridization signals for GAT-1 mRNA were observed over many regions of the rat brain, including the retina, olfactory bulb, neocortex, ventral pallidum, hippocampus, and cerebellum. At the microscopic level, this signal appeared to be restricted to neuronal profiles, and the overall distribution of GAT-1 mRNA closely paralleled that seen in other studies with antibodies to GABA. Areas containing hybridization signals for GAT-3 mRNA included the retina, olfactory bulb, subfornical organ, hypothalamus, midline thalamus, and brainstem. In some regions, the hybridization signal for GAT-3 seemed to be preferentially distributed over glial cells, although hybridization signals were also observed over neurons, particularly in the retina and olfactory bulb. Notably, hybridization signal for GAT-3 mRNA was absent from the neocortex and cerebellar cortex, and was very weak in the hippocampus. In contrast to the parenchymal localization obtained for GAT-1 and GAT-3 mRNAs, hybridization signals for GAT-2 mRNA were found only over the leptomeninges (pia and arachnoid). The differential distribution of the three GABA transporters described here suggests that while each plays a role in GABA uptake, they do so via distinct cellular populations.

A structure-activity analysis of the cloned rat and human Y4 receptors for pancreatic polypeptide.

We cloned and expressed the rat Y4 receptor for pancreatic polypeptide (PP). Structure-activity profiles derived from 125I-PP binding assays and [cAMP] radioimmunoassays reveal a selective receptor interaction with rat PP vs. neuropeptide Y (NPY) or peptide YY (PYY). Rat and human Y4 receptor clones share 75% amino acid identity. Based on [cAMP] radioimmunoassay, the human Y4 receptor exhibits a less selective interaction with rat PP vs. NPY or PYY and a greater dependence on N-terminal PP residues, relative to rat Y4. Differences in sequence and structure-activity profiles suggest the rat be used with caution to model human Y4 receptor function.

Molecular biology and pharmacology of multiple NPY Y5 receptor species homologs.

NPY is a 36-amino acid peptide which exerts its physiological effects through the activation of a family of G-protein coupled receptors. In vivo and in vitro characterization of the recently cloned rat Y5 receptor suggests that it is a primary mediator of NPY-induced feeding (Gerald et al., Nature 1996;382:168-171). We now report the molecular cloning and pharmacological characterization of the human, dog and mouse homologs of the Y5 receptor. With the exception of a 21 amino acid repeat in the amino terminus of the mouse Y5 receptor, the sequence of the four species homologs appear to be highly conserved, with 88% to 97% amino acid identities between any two species. Similarly, the pharmacological profiles of the four species homologs as determined in porcine 125I-PYY binding assays show a great deal of conservation, with the following rank order of affinity: human or porcine NPY, PYY, [Leu31,Pro34]NPY, NPY(2-36), human PP > human [D-Trp32]NPY > rat PP, C2-NPY. Northern blot analysis reveals that the Y5 receptor is widely distributed in the human brain, with the strongest signals detected in the cortex, putamen and caudate nucleus. The chromosomal localization of the human Y5 receptor, previously shown to be overlapping and in the opposite orientation to the Y1 receptor, is determined to be 4q31, the same locus as previously demonstrated for the human Y1 receptor (Herzog et al., J Biol Chem 1993;268:6703-6707), suggesting that these receptors may be coregulated. These Y5 species homologs along with corresponding animal models may be useful in the search for novel therapeutics in the treatment of obesity and related feeding disorders.

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.

Tiagabine, SK&F 89976-A, CI-966, and NNC-711 are selective for the cloned GABA transporter GAT-1.

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. The synaptic action of GABA is terminated by rapid uptake into presynaptic terminals and surrounding glial cells. Molecular cloning has revealed the existence of four distinct GABA transporters termed GAT-1, GAT-2, GAT-3, and BGT-1. Pharmacological inhibition of transport provides a mechanism for increasing GABA-ergic transmission, which may be useful in the treatment of various neuropsychiatric disorders. Recently, a number of lipophilic GABA transport inhibitors have been designed and synthesized, which are capable of crossing the blood brain barrier, and which display anticonvulsive activity. We have now determined the potency of four of these compounds, SK&F 89976-A (N-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid), tiagabine ((R)-1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3- piperidencarboxylic acid), CI-966 ([1-[2-[bis 4-(trifluoromethyl)phenyl]methoxy]ethyl]-1,2,5,6-tetrahydro-3- pyridinecarboxylic acid), and NNC-711 (1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,4,6-tetrahydro-3- pyridinecarboxylic acid hydrochloride), at each of the four cloned GABA transporters, and find them to be highly selective for GAT-1. These data suggest that the anticonvulsant activity of these compounds is mediated via inhibition of uptake by GAT-1.

Differences in ligand binding profiles between cloned rabbit and human 5-HT1D alpha and 5-HT1D beta receptors: ketanserin and methiothepin distinguish rabbit 5-HT1D receptor subtypes.

The study of serotonin receptor function has been complicated by the extreme molecular diversity of serotonin receptor subtypes, the lack of selective agonists and antagonists for many of the subtypes, and divergence in the pharmacological properties of a single receptor subtype across different animal species. An example of this pharmacological diversity between species homologues is provided by the 5-HT1D receptor subfamily. To further advance the ability to characterize and pharmacologically compare functional responses mediated by native 5-HT1D receptors, we have cloned the 5-HT1D alpha and 5-HT1D beta receptor subtypes from the rabbit and evaluated their pharmacological profiles using radioligand binding assays. The deduced amino acid sequences of the rabbit 5-HT1D alpha and 5-HT1D beta receptor genes displayed 60% overall identity [75% transmembrane (TM) identity] to each other and > 90% overall identity (95% TM identity) to their corresponding human homologues. Two compounds were identified in binding assays which discriminated between the closely-related 5-HT1D receptors. Ketanserin exhibited high affinity (pKi = 7.66) and selectivity (> 20-fold) for the 5-HT1D alpha receptor while methiothepin displayed high affinity (pKi = 7.86) and selectivity (16-fold) for the 5-HT1D beta receptor subtype. The rabbit and human recombinant 5-HT1D receptors showed significant intraspecies (rabbit 5-HT1D alpha vs. 5-HT1D beta) and interspecies (i.e. rabbit vs. human 5-HT1D alpha) similarities in their ligand binding profiles. These data suggest that 5-HT1D-mediated responses in rabbit preparations may provide information relevant to the pharmacology of the 5-HT1D receptor subtypes in humans.

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.