The selective 5-HT1F receptor agonist lasmiditan inhibits trigeminal nociceptive processing: Implications for migraine and cluster headache.

BACKGROUND AND PURPOSE: Lasmiditan is a novel selective 5-HT1F receptor agonist, recently approved for acute treatment of migraine. 5-HT1F receptors are widely expressed in the CNS and trigeminovascular system. Here, we have explored the therapeutic effects of 5-HT1F receptor activation in preclinical models of migraine and cluster headache. EXPERIMENTAL APPROACH: Electrical stimulation of the dura mater or the superior salivatory nucleus in anaesthetised rats evoked trigeminovascular or trigeminal-autonomic reflex activation at the level of the trigeminocervical complex. Additionally, cranial autonomic manifestations in response to trigeminal-autonomic reflex activation were measured, via anterior choroidal blood flow alterations. These responses were then challenged with lasmiditan. We explored the tissue distribution of mRNA for 5-HT1F receptors in human post-mortem tissue and of several 5-HT1 receptor subtypes in specific tissue beds. KEY RESULTS: Lasmiditan dose-dependently reduced trigeminovascular activation in a preclinical model of migraine. Lasmiditan also reduced superior salivatory nucleus-evoked activation of the trigeminal-autonomic reflex, but had no effect on cranial autonomic activation. mRNA profiling in human tissue showed expression of the 5-HT1F receptor in several structures relevant for migraine and cluster headache. CONCLUSION AND IMPLICATIONS: Our data suggest that lasmiditan acts, at least in part, as an anti-migraine agent by reducing trigeminovascular activation. Furthermore, our results highlight a clear action for lasmiditan in a preclinical model of cluster headache. Given the proven translational efficacy of this model, our data support the potential utility of lasmiditan as a therapeutic option for the acute treatment of cluster headache attacks. LINKED ARTICLES: This article is part of a themed issue on Advances in Migraine and Headache Therapy (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.3/issuetoc.

Evaluation of the mGlu8 receptor as a putative therapeutic target in schizophrenia.

Aberrant glutamatergic neurotransmission may underlie the pathogenesis of schizophrenia and metabotropic glutamate receptors (mGluRs) have been implicated in the disease. We have established the localization of the group III mGluR subtype, mGluR8, in the human body and investigated the biological effects of the selective mGluR8 agonist (S)-3,4-dicarboxyphenylglycine ((S)-3,4-DCPG) in schizophrenia-related animal models. The mGlu8 receptor has a widespread CNS distribution with expression observed in key brain regions associated with schizophrenia pathogenesis including the hippocampus. (S)-3,4-DCPG inhibited synaptic transmission and increased paired-pulse facilitation in rat hippocampal slices supporting the role of mGluR8 as a presynaptic autoreceptor. Using the rat Maximal Electroshock Seizure Threshold (MEST) test, (S)-3,4-DCPG (30 mg/kg, i.p.) reduced seizure activity confirming the compound to be centrally active following systemic administration. (S)-3,4-DCPG did not reverse (locomotor) hyperactivity induced by acute administration of phenylcyclidine (PCP, 1-32 mg/kg, i.p.) or amphetamine (3-30 mg/kg, i.p.) in Sprague-Dawley rats. However, 10 nmol (i.c.v.) (S)-3.4-DCPG did reverse amphetamine-induced hyperactivity in mice although it also inhibited spontaneous locomotor activity at this dose. In addition, mGluR8 null mutant mouse behavioral phenotyping revealed an anxiety-related phenotype but no deficit in sensorimotor gating. These data provide a potential role for mGluR8 in anxiety and suggest that mGluR8 may not be a therapeutic target for schizophrenia.

Tissue distribution profiles of the human TRPM cation channel family.

Eight members of the TRP-melastatin (TRPM) subfamily have been identified, whose physiological functions and distribution are poorly characterized. Although tissue expression and distribution patterns have been reported for individual TRPM channels, comparisons between individual studies are not possible because of variations in analysis techniques and tissue selection. We report here a comparative analysis of the expression patterns of all of the human TRPM channels in selected peripheral tissues and the central nervous system (CNS) using two distinct but complimentary approaches: TaqMan and SYBR Green real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). These techniques generated comparative distribution profiles and demonstrated tissue-specific co-expression of TRPM mRNA species, indicating significant potential for the formation of heteromeric channels. TRPM channels 2, 4, 5, 6, and 7 in contrast to 1, 3, and 8 are widely distributed in the CNS and periphery. The tissues demonstrating highest expression for individual family members were brain (TRPM1), brain and bone marrow (TRPM2), brain and pituitary (TRPM3), intestine and prostate (TRPM4), intestine, pancreas, and prostate (TRPM5), intestine and brain (TRPM6), heart, pituitary, bone, and adipose tissue (TRPM7), and prostate and liver (TRPM8). The data reported here will guide the elucidation of TRPM channel physiological functions.

TRPM2 is elevated in the tMCAO stroke model, transcriptionally regulated, and functionally expressed in C13 microglia.

We report the detailed expression profile of TRPM2 mRNA within the human central nervous system (CNS) and demonstrate increased TRPM2 mRNA expression at 1 and 4 weeks following ischemic injury in the rat transient middle cerebral artery occlusion (tMCAO) stroke model. Microglial cells play a key role in pathology produced following ischemic injury in the CNS and possess TRPM2, which may contribute to stroke-related pathological responses. We show that TRPM2 mRNA is present in the human C13 microglial cell line and is reduced by antisense treatment. Activation of C13 cells by interleukin-1beta leads to a fivefold increase of TRPM2 mRNA demonstrating transcriptional regulation. To confirm mRNA distribution correlated with functional expression, we combined electrophysiology, Ca2+ imaging, and antisense approaches. C13 microglia exhibited, when stimulated with hydrogen peroxide (H2O2), increased [Ca2+]i, which was reduced by antisense treatment. Moreover, patch-clamp recordings from C13 demonstrated that increased intracellular adenosine diphosphoribose (ADPR) or extracellular H2O2 induced an inward current, consistent with activation of TRPM2. In addition we confirm the functional expression of a TRPM2-like conductance in primary microglial cultures derived from rats. Activation of TRPM2 in microglia during ischemic brain injury may mediate key aspects of microglial pathophysiological responses.

Cytochrome P450 gene induction in rats ex vivo assessed by quantitative real-time reverse transcriptase-polymerase chain reaction (TaqMan).

Drug-induced changes in expression of cytochrome P450 (P450) genes are a significant issue in the preclinical development of pharmaceuticals. For example, preclinically, P450 induction can affect safety studies by reducing the systemic exposure of a compound undergoing toxicological evaluation, thus limiting the exposure that can be safely investigated in patients. Therefore, the induction potential of candidate drugs has been studied as part of the drug development process, typically using protein and/or catalytic end points. However, measuring changes in the levels of mRNA using TaqMan technology offers the opportunity to investigate this issue with the advantages of better dynamic range and specific enzyme identification. Here, we describe the TaqMan application to study ex vivo the P450 gene induction in the rat. Initially, livers from rats dosed with the prototypic P450 inducers beta-napthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX), and clofibric acid (CLO) were analyzed for mRNA levels of CYP1A1, 1A2, 2B1, 2B2, 2E1, 3A2, 3A23, and 4A1 and compared with control animals. The maximum fold induction of mRNA varied: 2500-fold for CYP1A1 with BNF, 680-fold for CYP2B1 with PB, 59-fold for CYP3A23 with DEX, and 16-fold for CYP4A1 with CLO. This method was then applied to estimate the inductive potential of putative drug candidates undergoing rodent toxicological evaluation. We present a summary of these data that demonstrates the sensitivity and specificity of the TaqMan assay to distinguish between inducers and noninducers and that offers a highly specific alternative to the quantification of drug effects on P450 expression using immunodetection and substrate metabolism.

Quantification of apolipoprotein E receptors in human brain-derived cell lines by real-time polymerase chain reaction.

Apolipoprotein (apo) E4 is a risk factor for Alzheimer's disease (AD) and other neurodegenerative diseases, compared to wild-type apoE3. The mechanism(s) is unknown. One possibility, demonstrated in peripheral tissue cell lines, is that apoE stimulates nitric oxide synthase (NOS) via a receptor-dependent signalling pathway and that apoE4 generates inappropriate amounts of nitric oxide (NO) compared to apoE3. Prior to biochemical investigations, we have quantified the expression of several candidate receptor genes, including low-density lipoprotein-receptor (LDL-r) family members and scavenger receptor class B, types I and II (SR-BI/II), as well as the three NOS isoenzymes and protein kinase B (Akt), in 38 human cell lines, of which 12 derive from brain. Expression of apoE receptor 2 (apoER2), a known signalling receptor in brain, was readily detected in SH-SY-5Y and CCF-STTG1 cells, common models of neurons and astrocytes, respectively, and was highest in H4 neuroglioma, NT-2 precursor cells and IMR-32 neuroblastoma cells. Transcripts of the other lipoprotein receptors were widely, but variably, distributed across the different cell types. Of particular note was the predominant expression of SR-BII over SR-BI in many of the brain-derived cells. As the C-terminus of SR-BII, like apoER2, contains potential SH3 signalling motifs, we suggest that in brain SR-BII functions as a signal transducer receptor.

Atypical CTSK transcripts and ARNT transcription read-through into CTSK.

The aryl hydrocarbon receptor nuclear translocator (ARNT) and cathepsin K (CTSK) genes lie in a tandem head-to-tail arrangement on human chromosome 1. The two genes are in extremely close proximity; the usual CTSK transcription start site is less than 1.4 kb downstream of the end of the longest reported ARNT transcript. By generating an RT-PCR product that overlaps both the 3' end of ARNT and the 5' end of CTSK, we show that ARNT transcripts may extend through the ARNT-CTSK intergenic region and progress into the CTSK gene. Furthermore, by using quantitative RT-PCR from several tissues to detect the ARNT expression signature in CTSK introns, we show that ARNT transcripts can read through into CTSK as far as CTSK intron 3, extending approximately 3.7 kb downstream of the end of the longest previously described ARNT mRNA. Given that ARNT and CTSK are expressed in an overlapping range of tissues, ARNT read-through may have a negative impact on CTSK transcript levels by interfering with CTSK expression. We also present evidence for novel CTSK transcripts following sequence analysis of CTSK-derived ESTs and RT-PCR products. These transcripts show alternate 5' splicing and or 5' extension and are sometimes initiated from a cryptic alternative promoter which is upstream of the known CTSK promoter and possibly in the 3' UTR of ARNT.

C20orf9-003 (ACI-1), a gene localized on chromosome 20q13.12 encoding for a 49 kD cytoplasmic protein with a putative nucleotide binding site.

Murine NGD5 is a gene identified from NG108-15 cells which is postulated to be involved in opioid receptor function. Here we report the cloning and characterization of a cDNA C20orf9-003 (ACI-1) encoding the human orthologue of the mouse NGD5. Analysis of the genomic structure revealed that C20orf9-003 (ACI-1) contains 13 exons and 12 introns, spanning 52.5kb of genomic DNA and is a variant of C20orf9. Chromosomal localization of human C20orf9-003 (ACI-1) assigned this gene to chromosome 20q13.12. Genes at this locus have been associated with the progression and possibly the development of various cancers. In addition several linkage studies support the possibility that one or more genes affecting obesity are located in 20q13. No function can be clearly assigned to C20orf9-003 (ACI-1), however, the protein has a cytoplasmic subcellular location and the secondary structure contains a Rossman fold like feature which is found in many nucleotide binding proteins.

Characterization of the human HCN1 channel and its inhibition by capsazepine.

The human hyperpolarization-activated cyclic nucleotide-gated 1 (hHCN1) subunit was heterologously expressed in mammalian cell lines (CV-1 and CHO) and its properties investigated using whole-cell patch-clamp recordings. Activation of this recombinant channel, by membrane hyperpolarization, generated a slowly activating, noninactivating inward current. The pharmacological properties of hHCN1-mediated currents resembled those of native hyperpolarization-activated currents (I(h)), that is, blockade by Cs(+) (99% at 5 mm), ZD 7288 (98% at 100 microm) and zatebradine (92% at 10 microm). Inhibition of the hHCN1-mediated current by ZD 7288 was apparently independent of prior channel activation (i.e. non-use-dependent), whereas that induced by zatebradine was use-dependent. The VR1 receptor antagonist capsazepine inhibited hHCN1-mediated currents in a concentration-dependent (IC(50)=8 microm), reversible and apparently non-use-dependent manner. This inhibitory effect of capsazepine was voltage-independent and associated with a leftward shift in the hHCN1 activation curve as well as a dramatic slowing of the kinetics of current activation. Elevation of intracellular cAMP or extracellular K(+) significantly enhanced aspects of hHCN1 currents. However, these manipulations did not significantly affect the capsazepine-induced inhibition of hHCN1. The development of structural analogues of capsazepine may yield compounds that could selectively inhibit HCN channels and prove useful for the treatment of neurological disorders where a role for HCN channels has been described.

GPR105, a novel Gi/o-coupled UDP-glucose receptor expressed on brain glia and peripheral immune cells, is regulated by immunologic challenge: possible role in neuroimmune function.

We have recently shown that UDP-glucose, and some related UDP-sugars, are potent agonists of the novel G protein-coupled receptor GPR105 (recently re-named P2Y(14)). GPR105 is widely expressed throughout many brain regions and peripheral tissues of human and rodents, and couples to a pertussis toxin-sensitive G protein. To further characterise the role of GPR105, we demonstrate by immunohistochemistry with receptor-specific antiserum that GPR105 protein is widely distributed throughout the post mortem human brain where it is localised to glial cells, and specifically co-localises with astrocytes. Using quantitative RT-PCR we also show that GPR105 mRNA exhibits a restricted expression profile in an array of human cell lines and primary cells, with prominent expression detected in immune cells including neutrophils, lymphocytes, and megakaryocytic cells. To investigate the G protein selectivity of GPR105, we used chimeric Galpha subunits (Galpha(qi5), Galpha(qo5), and Galpha(qs5)) and an intracellular Ca(2+) mobilisation assay to demonstrate that GPR105 couples to Galpha subunits of the G(i/o) family but not to G(s) family proteins or to endogenous G(q/11) proteins in HEK-293 cells. Finally, we show that expression of GPR105 mRNA in the rat brain is up-regulated by immunologic challenge with lipopolysaccharide. Based on these observations, we propose that G(i/o)-coupled GPR105 might play an important role in peripheral and neuroimmune function in response to extracellular UDP-sugars.

Prostaglandin D2 synthase enzymes and PPARgamma are co-expressed in mouse 3T3-L1 adipocytes and human tissues.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a critical regulator of adipocyte differentiation. Whilst 15-deoxy-delta(12,14)-prostaglandin J2 (15-d-PGJ2) has been identified as a putative endogenous ligand for this transcription factor, it is unclear whether the enzymes necessary for 15-d-PGJ2 biosynthesis are co-expressed with PPARgamma. Prostaglandin D2 synthase (PGDS) enzymes represent the terminal enzymatic components responsible for 15-d-PGJ2 production. Both glutathione (GSH)-dependent and GSH-independent PGDS isoenzymes exist. We have, therefore, examined the expression of PGDS isoenzymes in mouse 3T3-L1 adipocytes, and various human tissues. The GSH-independent PGDS was found to be expressed in 3T3-L1 cells both before and after their differentiation into adipocytes. By contrast, we were unable to detect expression of the GSH-dependent PGDS at any stage during the adipose conversion of 3T3-L1 cells. Quantitative analysis of mRNA levels for PPARgamma and each PGDS isoenzyme revealed their co-expression in a number of human tissues and cell types, including adipose tissue, placenta, prostate, and macrophages. These data reveal the potential for de novo 15-d-PGJ2 synthesis in the context of PPARgamma expression, suggesting that this prostaglandin may contribute to PPARgamma signalling in vivo.

Molecular identification of high and low affinity receptors for nicotinic acid.

Nicotinic acid has been used clinically for over 40 years in the treatment of dyslipidemia producing a desirable normalization of a range of cardiovascular risk factors, including a marked elevation of high density lipoprotein and a reduction in mortality. The precise mechanism of action of nicotinic acid is unknown, although it is believed that activation of a G(i)-G protein-coupled receptor may contribute. Utilizing available information on the tissue distribution of nicotinic acid receptors, we identified candidate orphan receptors. The selected orphan receptors were screened for responses to nicotinic acid, in an assay for activation of G(i)-G proteins. Here we describe the identification of the G protein-coupled receptor HM74 as a low affinity receptor for nicotinic acid. We then describe the subsequent identification of HM74A in follow-up bioinformatics searches and demonstrate that it acts as a high affinity receptor for nicotinic acid and other compounds with related pharmacology. The discovery of HM74A as a molecular target for nicotinic acid may facilitate the discovery of superior drug molecules to treat dyslipidemia.

Resistin is expressed in human macrophages and directly regulated by PPAR gamma activators.

Resistin is a cysteine-rich protein postulated to be a molecular link between obesity and type 2 diabetes. The aim of this study was to investigate the role of PPAR gamma in the regulation of resistin expression in human primary macrophages. Fluorescent real-time PCR (Taqman) analysis of resistin expression across a range of human tissues showed that resistin is highly expressed in bone marrow compared to other tissues. Taqman analysis and Western blotting showed that rosiglitazone decreased resistin expression at both the mRNA and protein levels in human primary monocyte-derived macrophages in vitro. Resistin expression was reduced by up to 80% after exposure to 100 nM rosiglitazone for 96 h. Bioinformatics analysis of the genomic sequence upstream of the resistin coding sequence identified several putative PPAR response elements of which one was shown to bind PPAR gamma using electrophoretic mobility shift assays. Our data support a direct role for PPAR gamma in the regulation of resistin expression.

The Orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids.

GPR41 and GPR43 are related members of a homologous family of orphan G protein-coupled receptors that are tandemly encoded at a single chromosomal locus in both humans and mice. We identified the acetate anion as an agonist of human GPR43 during routine ligand bank screening in yeast. This activity was confirmed after transient transfection of GPR43 into mammalian cells using Ca(2+) mobilization and [(35)S]guanosine 5'-O-(3-thiotriphosphate) binding assays and by coexpression with GIRK G protein-regulated potassium channels in Xenopus laevis oocytes. Other short chain carboxylic acid anions such as formate, propionate, butyrate, and pentanoate also had agonist activity. GPR41 is related to GPR43 (52% similarity; 43% identity) and was activated by similar ligands but with differing specificity for carbon chain length, with pentanoate being the most potent agonist. A third family member, GPR42, is most likely a recent gene duplication of GPR41 and may be a pseudogene. GPR41 was expressed primarily in adipose tissue, whereas the highest levels of GPR43 were found in immune cells. The identity of the cognate physiological ligands for these receptors is not clear, although propionate is known to occur in vivo at high concentrations under certain pathophysiological conditions.

The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids.

GPR40 is a member of a subfamily of homologous G protein-coupled receptors that include GPR41 and GPR43 and that have no current function or ligand ascribed. Ligand fishing experiments in HEK293 cells expressing human GPR40 revealed that a range of saturated and unsaturated carboxylic acids with carbon chain lengths greater than six were able to induce an elevation of [Ca(2+)](i), measured using a fluorometric imaging plate reader. 5,8,11-Eicosatriynoic acid was the most potent fatty acid tested, with a pEC(50) of 5.7. G protein coupling of GPR40 was examined in Chinese hamster ovary cells expressing the G alpha(q/i)-responsive Gal4-Elk1 reporter system. Expression of human GPR40 led to a constitutive induction of luciferase activity, which was further increased by exposure of the cells to eicosatriynoic acid. Neither the constitutive nor ligand-mediated luciferase induction was inhibited by pertussis toxin treatment, suggesting that GPR40 was coupled to G alpha(q/11.) Expression analysis by quantitative reverse transcription-PCR showed that GPR40 was specifically expressed in brain and pancreas, with expression in rodent pancreas being localized to insulin-producing beta-cells. These data suggest that some of the physiological effects of fatty acids in pancreatic islets and brain may be mediated through a cell-surface receptor.