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1.
J Neuroimmunol ; 246(1-2): 69-77, 2012 May 15.
Article in English | MEDLINE | ID: mdl-22498097

ABSTRACT

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor that has been shown to have anti-inflammatory and matrix metalloproteinase (MMP) inhibitor properties. PPARγ agonists have been shown to have neuroprotective effects in various neurodegeneration models where inflammation is implicated, including models of Parkinson's disease. However, no studies have looked at the effects of partial PPARγ agonists. EXPERIMENTAL APPROACH: The neuroprotective effects of the PPARγ full agonist, pioglitazone (20 mg/kg), partial PPARγ agonist GW855266X (15 mg/kg) and PPAR-δ full agonist GW610742X (10 mg/kg) were investigated in the 6-hydroxydopamine (6-OHDA) model of Parkinson's disease when administered prior to or post 6-OHDA lesioning. The integrity of the nigrostriatal system was assessed by assessing the numbers dopaminergic neurons in the substantia nigra (SN) and by assessing striatal dopamine content. The degree of microglia activation in the SN was also immunohistochemistry assessed utilizing the marker OX-6 for activated microglia and CD-68 a marker for phagocytic microglia. Additionally we performed immunocytochemistry for MMP3 in the SN. Finally, we investigated whether a period of drug withdrawal for a further 7 days affected the neuroprotection produced by the PPARγ agonists. KEY RESULTS: Both pioglitazone and GW855266X protected against 6-OHDA induced loss of dopaminergic neurons in the substantia nigra and depletion of striatal dopamine when administered orally twice daily for either 1) 7 day prior to and 7 days post lesioning or 2) for 7 days starting 2 days post lesioning when neurons will be severely traumatized. 6-OHDA lesioning was associated with an increase in microglia activation and in numbers of MMP-3 immunoreactive cells which was attenuated by pioglitazone and GW855266X. Neuroprotective effects were not replicated using the PPARδ agonist GW610742X. Subsequent withdrawal of both pioglitazone and GW855266X, for a further 7 days negated any neuroprotective effect suggesting that long-term administration may be required to attenuate the inflammatory response. CONCLUSIONS AND IMPLICATIONS: For the first time a partial PPAR-γ agonist has been shown to be neuroprotectory when administered post lesioning in a parkinsonian model. Effects may be via the inhibition of microglial and MMP activation and support further research.


Subject(s)
Dopaminergic Neurons/immunology , Growth Inhibitors/pharmacology , Matrix Metalloproteinase Inhibitors , Microglia/immunology , PPAR gamma/agonists , Parkinsonian Disorders/immunology , Protease Inhibitors/pharmacology , Animals , Disease Models, Animal , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/enzymology , Male , Matrix Metalloproteinase 3/biosynthesis , Microglia/drug effects , Microglia/enzymology , Neuroprotective Agents/pharmacology , Oxidopamine/toxicity , PPAR delta/agonists , PPAR delta/pharmacology , PPAR gamma/pharmacology , Parkinsonian Disorders/chemically induced , Parkinsonian Disorders/enzymology , Rats , Rats, Sprague-Dawley
2.
Br J Pharmacol ; 157(1): 104-17, 2009 May.
Article in English | MEDLINE | ID: mdl-19413575

ABSTRACT

BACKGROUND AND PURPOSE: Histamine H3 receptor antagonists are currently being evaluated in clinical trials for a number of central nervous system disorders including narcolepsy. These agents can increase wakefulness (W) in cats and rodents following acute administration, but their effects after repeat dosing have not been reported previously. EXPERIMENTAL APPROACH: EEG and EMG recordings were used to investigate the effects of acute and repeat administration of the novel H3 antagonist GSK189254 on the sleep-wake cycle in wild-type (Ox+/+) and orexin knockout (Ox-/-) mice, the latter being genetically susceptible to narcoleptic episodes. In addition, we investigated H3 and H1 receptor expression in this model using radioligand binding and autoradiography. KEY RESULTS: In Ox+/+ and Ox-/- mice, acute administration of GSK189254 (3 and 10 mg x kg(-1) p.o.) increased W and decreased slow wave and paradoxical sleep to a similar degree to modafinil (64 mg x kg(-1)), while it reduced narcoleptic episodes in Ox-/- mice. After twice daily dosing for 8 days, the effect of GSK189254 (10 mg x kg(-1)) on W in both Ox+/+ and Ox-/- mice was significantly reduced, while the effect on narcoleptic episodes in Ox-/- mice was significantly increased. Binding studies revealed no significant differences in H3 or H1 receptor expression between Ox+/+ and Ox-/- mice. CONCLUSIONS AND IMPLICATIONS: These studies provide further evidence to support the potential use of H3 antagonists in the treatment of narcolepsy and excessive daytime sleepiness. Moreover, the differential effects observed on W and narcoleptic episodes following repeat dosing could have important implications in clinical studies.


Subject(s)
Benzazepines/pharmacology , Histamine H3 Antagonists/pharmacology , Intracellular Signaling Peptides and Proteins/genetics , Narcolepsy/drug therapy , Neuropeptides/genetics , Niacinamide/analogs & derivatives , Sleep/drug effects , Wakefulness/drug effects , Animals , Autoradiography , Benzazepines/administration & dosage , Benzazepines/therapeutic use , Benzhydryl Compounds/pharmacology , Brain/metabolism , Dose-Response Relationship, Drug , Electroencephalography , Electromyography , Histamine H3 Antagonists/administration & dosage , Histamine H3 Antagonists/therapeutic use , Mice , Mice, Knockout , Modafinil , Narcolepsy/genetics , Niacinamide/administration & dosage , Niacinamide/pharmacology , Niacinamide/therapeutic use , Orexins , Radioligand Assay , Receptors, Histamine H3/metabolism
3.
Br J Pharmacol ; 157(1): 130-8, 2009 May.
Article in English | MEDLINE | ID: mdl-19222483

ABSTRACT

BACKGROUND AND PURPOSE: Histamine H3 receptor antagonists are currently being evaluated for their potential use in a number of central nervous system disorders including Alzheimer's Disease (AD). To date, little is known about the state of H3 receptors in AD. EXPERIMENTAL APPROACH: In the present study we used the radiolabelled H3 receptor antagonist [3H]GSK189254 to investigate H3 receptor binding in the amyloid over-expressing double mutant APPswe x PSI.MI46V (TASTPM) transgenic mouse model of AD and in post-mortem human AD brain samples. KEY RESULTS: No significant differences in specific H3 receptor binding were observed between wild type and TASTPM mice in the cortex, hippocampus or hypothalamus. Specific [3H]GSK189254 binding was detected in sections of human medial frontal cortex from AD brains of varying disease severity (Braak stages I-VI). With more quantitative analysis in a larger cohort, we observed that H3 receptor densities were not significantly different between AD and age-matched control brains in both frontal and temporal cortical regions. However, within the AD group, [3H]GSK189254 binding density in frontal cortex was higher in individuals with more severe dementia prior to death. CONCLUSIONS AND IMPLICATIONS: The maintenance of H3 receptor integrity observed in the various stages of AD in this study is important, given the potential use of H3 antagonists as a novel therapeutic approach for the symptomatic treatment of AD.


Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Protein Precursor/biosynthesis , Brain/metabolism , Receptors, Histamine H3/metabolism , Aged , Alzheimer Disease/pathology , Amyloid beta-Protein Precursor/genetics , Animals , Autoradiography , Benzazepines/pharmacology , Brain/pathology , Female , Histamine H3 Antagonists/pharmacology , Humans , Male , Mice , Mice, Transgenic , Mutation , Neocortex/metabolism , Niacinamide/analogs & derivatives , Niacinamide/pharmacology , Radioligand Assay , Severity of Illness Index
4.
Br J Pharmacol ; 154(1): 226-33, 2008 May.
Article in English | MEDLINE | ID: mdl-18332857

ABSTRACT

BACKGROUND AND PURPOSE: The peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist pioglitazone has previously been shown to attenuate dopaminergic cell loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease, an effect attributed to its anti-inflammatory properties. In the present investigation, we provide evidence that pioglitazone is effective in the MPTP mouse model, not via an anti-inflammatory action, but through inhibition of MAO-B, the enzyme required to biotransform MPTP to its active neurotoxic metabolite 1-methyl-4-phenylpyridinium (MPP+). EXPERIMENTAL APPROACH: Mice were treated with pioglitazone (20 mg kg(-1) b.i.d. (twice a day), p.o., for 7 days), prior and post or post-MPTP (30 mg kg(-1) s.c.) treatment. Mice were then assessed for motor impairments on a beam-walking apparatus and for reductions in TH immunoreactivity in the substantia nigra and depletions in striatal dopamine. The effects of pioglitazone on striatal MPP+ levels and MAO-B activity were also assessed. KEY RESULTS: Mice treated with MPTP showed deficits in motor performance, marked depletions in striatal dopamine levels and a concomitant reduction in TH immunoreactivity in the substantia nigra. Pretreatment with pioglitazone completely prevented these effects of MPTP. However, pretreatment with pioglitazone also significantly inhibited the MPTP-induced production of striatal MPP+ and the activity of MAO-B in the striatum. CONCLUSIONS AND IMPLICATIONS: The neuroprotection observed with pioglitazone pretreatment in the MPTP mouse model was due to the blockade of the conversion of MPTP to its active toxic metabolite MPP+, via inhibition of MAO-B.


Subject(s)
Hypoglycemic Agents/therapeutic use , MPTP Poisoning/drug therapy , Monoamine Oxidase Inhibitors/pharmacology , Monoamine Oxidase/metabolism , PPAR gamma/agonists , Thiazolidinediones/therapeutic use , Animals , Cell Count , Chromatography, High Pressure Liquid , Dopamine/metabolism , Dopamine/physiology , Electrochemistry , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Nerve Degeneration/pathology , Neuroglia/drug effects , Pioglitazone , Postural Balance/drug effects , Psychomotor Performance/drug effects , Selegiline/pharmacology , Serotonin/metabolism , Substantia Nigra/drug effects
5.
Eur J Neurosci ; 23(7): 1766-74, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16623833

ABSTRACT

Dysfunction of the ubiquitin-proteasome system occurs in the substantia nigra (SN) in Parkinson's disease (PD). However, it is unknown whether this is a primary cause or a secondary consequence of other components of the pathogenic process. We have investigated in nonhuman primates whether initiating cell death through mitochondrial complex I inhibition using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) altered proteasomal activity or the proteasomal components in the SN. Chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolase (PGPH) activating of 20S proteasome were decreased in SN homogenates of MPTP-treated marmosets compared to naïve animals. Western blotting revealed a marked decrease in the expression of 20S-alpha subunits, but no change in 20S-beta subunits in the SN of MPTP-treated marmoset compared to naïve animals. There was a marked decrease in the expression of the proteasome activator 700 (PA700) and proteasome activator 28 (PA28) regulatory complexes. The 20S-alpha4 subunit immunoreactivity was decreased in the nucleus of colocalized tyrosine hydroxylase (TH)-positive cells of MPTP-treated animals compared to naïve animals but no difference in the intensity of 20S-beta1i subunit staining. Immunoreactivity for PA700-Rpt5 and PA28-alpha subunits within surviving TH-positive cells of MPTP-treated marmoset was reduced compared to naïve controls. Overall, the changes in proteasomal function and structure occurring follow MPTP-induced destruction of the SN in common marmosets were very similar to those found in PD. This suggests that altered proteasomal function in PD could be a consequence of other pathogenic processes occurring in SN as opposed to initiating cell death as previously suggested.


Subject(s)
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Proteasome Endopeptidase Complex/metabolism , Animals , Blotting, Western , Brain/drug effects , Brain/enzymology , Callithrix , Immunohistochemistry , Proteasome Endopeptidase Complex/biosynthesis , Protein Subunits/biosynthesis , Protein Subunits/metabolism , Tyrosine 3-Monooxygenase/metabolism
6.
Mech Ageing Dev ; 126(6-7): 760-6, 2005.
Article in English | MEDLINE | ID: mdl-15888331

ABSTRACT

Age-related increase in protein oxidation in brain coupled to an impairment of proteasomal activity may underline neuronal loss but differences in susceptibility between species and brain regions remain unexplained. We now investigate differences in proteasomal activity, measured as chymotrypsin-, trypsin- and peptidylglutamyl-like hydrolysing activities between brain regions in rats, mice and common marmosets. In aged rats and mice, proteasomal activity was decreased in the cortex, striatum, cerebellum, globus pallidus and substantia nigra overall when compared to young animals. However, in the aged brain only chymotrypsin-like activity was decreased in the cortex and the globus pallidus while only trypsin-like activity was reduced in the cerebellum. In contrast, in the striatum, both chymotrypsin-like and trypsin-like activities were reduced and in the substantia nigra, all the three catalytic activities of proteasome were significantly impaired. Chymotrypsin-like and trypsin-like activities were significantly higher in all the brain regions of marmosets compared to those of mice and rats. Peptidylglutamyl-like activity was only significantly higher in the cerebellum and striatum of marmoset compared to rodents. The data suggest that there is higher proteasome activity in common marmoset brain compared to rat and mouse and that the basal ganglia are more prone to an age-related decrease in proteasomal activity. This may explain the involvement of altered ubiquitin-proteasome system activity in Parkinson's disease and the relationship to ageing.


Subject(s)
Aging/physiology , Brain/enzymology , Callithrix/physiology , Proteasome Endopeptidase Complex/metabolism , Animals , Female , Mice , Parkinson Disease/metabolism , Rats , Rats, Sprague-Dawley , Species Specificity
7.
Neuroscience ; 129(1): 49-54, 2004.
Article in English | MEDLINE | ID: mdl-15489027

ABSTRACT

5-HT(4) receptors are widely distributed in both peripheral and central nervous systems where they couple, via a G-protein, to the activation of adenylate cyclase. In the brain, the highest 5-HT(4) receptor densities are found in the limbic system, including the hippocampus and frontal cortex. It has been suggested that activation of these receptors may be of therapeutic benefit in diseases that produce cognitive deficits such as Alzheimer's disease (AD). Previous electrophysiological studies have shown that the 5-HT(4) agonist, Zacopride, can increase population spike amplitude recorded in region CA1 of rat hippocampal slices in a cyclic AMP (cAMP)/cAMP-dependent protein kinase A-dependent manner. We report here that the 5-HT(4) agonist, Prucalopride, and the 5-HT(4) partial agonist, SL65.0155, produce a similar effect in rat hippocampal slices and that the specific 5-HT(4) antagonist, GR113808, blocks these effects. To investigate the potential use of 5-HT(4) agonists in the treatment of AD, Prucalopride was applied to hippocampal slices from a transgenic mouse line that overexpresses the Abeta peptide. Despite the deficit in synaptic transmission present in these mice, the percentage increase of the CA1 population spike induced by Prucalopride was the same as that observed in wild-type mice. These data support 5-HT(4) receptors as a target for cognitive enhancement and suggest that a partial agonist would be sufficient to produce benefits, while reducing potential peripheral side effects. In addition, we show that 5-HT(4) receptors remain functional in the presence of excess Abeta peptide and may therefore be a useful target in AD.


Subject(s)
Alzheimer Disease/physiopathology , Amyloid beta-Peptides/genetics , Hippocampus/metabolism , Neurons/metabolism , Serotonin Receptor Agonists/pharmacology , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Animals , Animals, Genetically Modified , Benzofurans/pharmacology , Dioxanes/pharmacology , Disease Models, Animal , Excitatory Postsynaptic Potentials/physiology , Hippocampus/drug effects , Humans , Indoles/pharmacology , Male , Neurons/drug effects , Organ Culture Techniques , Oxadiazoles/pharmacology , Patch-Clamp Techniques , Rats , Receptors, Serotonin, 5-HT4/metabolism , Serotonin Antagonists/pharmacology , Sulfonamides/pharmacology
8.
Brain Res Mol Brain Res ; 110(2): 305-17, 2003 Feb 20.
Article in English | MEDLINE | ID: mdl-12591167

ABSTRACT

Using a homology-based bioinformatics approach we have analysed human genomic sequence and identified the human and rodent orthologues of a novel putative seven transmembrane G protein coupled receptor, termed GABA(BL). The amino acid sequence homology of these cDNAs compared to GABA(B1) and GABA(B2) led us to postulate that GABA(BL) was a putative novel GABA(B) receptor subunit. The C-terminal sequence of GABA(BL) contained a putative coiled-coil domain, di-leucine and several RXR(R) ER retention motifs, all of which have been shown to be critical in GABA(B) receptor subunit function. In addition, the distribution of GABA(BL) in the central nervous system was reminiscent of that of the other known GABA(B) subunits. However, we were unable to detect receptor function in response to any GABA(B) ligands when GABA(BL) was expressed in isolation or in the presence of either GABA(B1) or GABA(B2). Therefore, if GABA(BL) is indeed a GABA(B) receptor subunit, its partner is a potentially novel receptor subunit or chaperone protein which has yet to be identified.


Subject(s)
Brain/metabolism , GTP-Binding Proteins/isolation & purification , Protein Subunits/isolation & purification , Receptors, GABA-B/isolation & purification , Amino Acid Sequence/genetics , Animals , Base Sequence/genetics , Cells, Cultured , Chromosome Mapping , Chromosomes, Human, Pair 3/genetics , Cloning, Molecular , DNA, Complementary/analysis , DNA, Complementary/genetics , GTP-Binding Proteins/genetics , Humans , Immunohistochemistry , Male , Mice , Molecular Sequence Data , Molecular Structure , Phylogeny , Protein Structure, Tertiary/genetics , Protein Subunits/genetics , Rats , Receptors, GABA-B/genetics
9.
Neuroscience ; 105(2): 353-64, 2001.
Article in English | MEDLINE | ID: mdl-11672603

ABSTRACT

Destruction of the nigro-striatal pathway in Parkinson's disease and treatment with L-DOPA lead to persistent alterations in basal ganglia output pathways that are poorly characterised. Differential display mRNA analysis was used to study the effects of 6-hydroxydopamine-induced lesions of the medial forebrain bundle on gene expression in the rat striatum. One up-regulated cDNA identified in two independent groups of 6-hydroxydopamine-lesioned animals was cloned and sequence analysis showed 97% homology to secretogranin II. Differential up-regulation of secretogranin II following 6-hydroxydopamine lesioning was confirmed in a further group of 6-hydroxydopamine-lesioned rats using TaqMan real time quantitative reverse transcription-polymerase chain reaction. Following chronic L-DOPA treatment of 6-hydroxydopamine-lesioned rats, secretogranin II mRNA was further up-regulated to a similar degree to that observed for preproenkephalin A mRNA expression. Immunohistochemical analysis confirmed the increase in secretogranin II peptide levels in striatal neurones in 6-hydroxydopamine-lesioned rats following chronic L-DOPA treatment. The increase in secretogranin II mRNA occurring following destruction of the nigro-striatal pathway and chronic L-DOPA treatment may result in an increase in secretoneurin levels, which could be important for the regulation of striatal output pathways.


Subject(s)
Levodopa/pharmacology , Neostriatum/metabolism , Neuropeptides/metabolism , Parkinsonian Disorders/metabolism , Proteins/genetics , RNA, Messenger/metabolism , Up-Regulation/physiology , Animals , Apomorphine/pharmacology , Chromogranins , DNA, Complementary/analysis , Dopamine Agonists/pharmacology , Drug Administration Schedule , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Immunohistochemistry , In Situ Hybridization , Male , Neostriatum/drug effects , Neostriatum/physiopathology , Neurons/drug effects , Neurons/metabolism , Neuropeptides/drug effects , Oxidopamine/pharmacology , Parkinsonian Disorders/chemically induced , Parkinsonian Disorders/drug therapy , RNA, Messenger/drug effects , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Secretogranin II , Sympatholytics/pharmacology , Up-Regulation/drug effects
10.
J Physiol ; 533(Pt 2): 467-78, 2001 Jun 01.
Article in English | MEDLINE | ID: mdl-11389205

ABSTRACT

1. Voltage-sensitive Ca(2+) channels (VSCCs) are often heteromultimeric complexes. The VSCC subtype specifically expressed by skeletal muscle has long been known to contain a gamma subunit, gamma(1), that is only expressed in this tissue. Recent work, initiated by the identification of the mutation present in the stargazer mouse, has led to the identification of a series of novel potential Ca(2+) channel gamma subunits expressed in the CNS. 2. Based on bioinformatic techniques we identified and cloned the human gamma(2), gamma(3) and gamma(4) subunits. 3. TaqMan analysis was used to quantitatively characterise the mRNA expression patterns of all the gamma subunits. All three subunits were extensively expressed in adult brain with overlapping but subunit-specific distributions. gamma(2) and gamma(3) were almost entirely restricted to the brain, but gamma(4) expression was seen in a broad range of peripheral tissues. 4. Using a myc epitope the gamma(2) subunit was tagged both intracellularly at the C-terminus and on a predicted extracellular site between the first and second transmembrane domains. The cellular distribution was then examined immunocytochemically, which indicated that a substantial proportion of the cellular pool of the gamma(2) subunit was present on the plasma membrane and provided initial evidence for the predicted transmembrane topology of the gamma subunits. 5. Using co-transfection techniques we investigated the functional effects of each of the gamma subunits on the biophysics of the T-type VSCC encoded by the alpha(1I) subunit. This revealed a substantially slowed rate of deactivation in the presence of gamma(2). In contrast, there was no significant corresponding effect of either gamma(3) or gamma(4) on alpha(1I) subunit-mediated currents.


Subject(s)
Calcium Channels, T-Type/genetics , Calcium Channels, T-Type/metabolism , Calcium Channels/genetics , Calcium Channels/metabolism , Neurons/chemistry , Animals , Calcium Channels/chemistry , Calcium Channels, T-Type/chemistry , Cell Line , Cloning, Molecular , DNA, Complementary , Electrophysiology , Gene Expression/physiology , Humans , Kidney/cytology , Kinetics , Membrane Potentials/physiology , Mice , Mice, Neurologic Mutants , Molecular Sequence Data , RNA, Messenger/analysis , Sequence Homology, Amino Acid , Taq Polymerase , Transfection
11.
Brain Res Mol Brain Res ; 90(2): 125-34, 2001 Jun 20.
Article in English | MEDLINE | ID: mdl-11406291

ABSTRACT

5-HT4 receptors mediate several physiological effects of 5-HT, particularly in the central nervous system (CNS), heart and gut. Recently, several C-terminal splice variants of the human 5-HT4 (h5-HT4) receptor have been described, namely h5-HT4(a), h5-HT4(b), h5-HT4(c), h5-HT4(d) and h5-HT4(g). Previous tissue distribution data suggest some degree of specificity in the mRNA expression patterns of the different h5-HT4 receptor splice variants. However, comparison of the mRNA expression profiles of these splice variants is difficult due to the non-quantitative methods used, and in addition, there is very limited data on the expression of each splice variant in human CNS subregions. In the present study we used a single technique, TaqMan real time quantitative RT-PCR, to investigate the mRNA distribution of 5-HT4 receptor C-terminal splice variants in multiple human CNS and peripheral tissues. Using a primer/probe set that amplified all 5-HT4 splice variants (5-HT4pan), the highest CNS expression of 5-HT4 receptor mRNA was observed in basal ganglia, amygdala and hippocampus, consistent with previous studies. h5-HT4(a), h5-HT4(b), h5-HT4(c) and h5-HT4(g) were predominantly expressed in various CNS tissues, compared to most peripheral tissues, but there were differences in expression levels and distribution patterns of each variant. The distribution profile and expression levels observed for the 5-HT4(b) splice variant were virtually identical to that obtained with the 5-HT4pan primer/probe set, whilst the other splice variants were expressed at much lower levels and with different expression patterns obtained with both 5-HT4(b) and 5-HT4pan primer/probe sets. Highest levels of 5-HT4(g) were observed in the hypothalamus and cortex, whilst the 5-HT4(a) variant was highest in the amygdala. 5-HT4(c) expression was highest in the pituitary gland whilst 5-HT4(d) mRNA was only detected in the small intestine at very low levels and not in the CNS. In conclusion, we have shown quantitative differences in the mRNA distribution profiles of the 5-HT4 receptor C-terminal splice variants in human CNS subregions as well as peripheral tissues. In addition, our data suggests that the h5-HT4(b) variant is the most predominant form of the 5-HT4 receptor in humans.


Subject(s)
Alternative Splicing/genetics , Central Nervous System/physiology , Receptors, Serotonin/genetics , Reverse Transcriptase Polymerase Chain Reaction , Antisense Elements (Genetics) , Gene Expression , Humans , RNA, Messenger/analysis , Receptors, Serotonin, 5-HT4 , Taq Polymerase
12.
Brain Res Mol Brain Res ; 86(1-2): 101-14, 2001 Jan 31.
Article in English | MEDLINE | ID: mdl-11165377

ABSTRACT

Potassium channels are amongst the most heterogeneous class of ion channels known and are responsible for mediating a diverse range of biological functions. The most recently described family of K+ channels, the 'two pore-domain family', contain four membrane spanning domains and two pore-forming domains, suggesting that two channel subunits associate to form a functional K+ pore. Several sub-families of the two pore domain potassium channel family have been described, including the weakly inward rectifying K+ channel (TWIK), the acid-sensitive K+ channel (TASK), the TWIK-related K+ channel (TREK) and the TWIK-related arachidonic acid stimulated K+ channel (TRAAK). However, comparison of the mRNA expression of these channels has been difficult due to the differences in methods used and the species studied. In the present study, we used a single technique, TaqMan semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), to investigate the mRNA distribution of all currently known two pore potassium channels in human central nervous system (CNS) and peripheral tissues. TWIK-1 and the TWIK-1-like channel KCNK7 were predominantly expressed in the CNS, in contrast to TWIK-2 which was preferentially expressed in peripheral tissues such as pancreas, stomach, spleen and uterus. TASK-1 was expressed in the CNS and some peripheral tissues, whereas TASK-2 was exclusively expressed in the periphery except for mRNA expression observed in dorsal root ganglion and spinal cord. In addition, mRNA expression of the recently identified TASK-3, was almost completely exclusive to cerebellum with little or no mRNA detected in any other tissues. TREK-1 and TRAAK mRNA expression was predominantly CNS specific in contrast to the closely related TREK-2, which was expressed in both CNS and peripheral tissues. Studying the mRNA expression profiles of known two pore domain K+ channels will aid in the understanding of the biological roles of these channels. Furthermore, identification of common areas of expression may help identify which channels, if any, associate to form heteromeric K+ channel complexes.


Subject(s)
Central Nervous System/physiology , Ganglia, Spinal/physiology , Nerve Tissue Proteins , Potassium Channels, Tandem Pore Domain , Potassium Channels/chemistry , Potassium Channels/genetics , Central Nervous System/chemistry , Ganglia, Spinal/chemistry , Gene Expression/physiology , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Protein Structure, Tertiary , RNA, Messenger/analysis , Sequence Homology, Amino Acid
13.
Genomics ; 70(2): 241-52, 2000 Dec 01.
Article in English | MEDLINE | ID: mdl-11112352

ABSTRACT

Orphan transporters form a growing subfamily of genes related by sequence similarity to the Na+/Cl- -dependent neurotransmitter superfamily. Using a combination of database similarity searching and cloning methods, we have identified and characterized two novel human orphan transporter genes, v7-3 and NTT5. Similar to other known orphan transporters, v7-3 and NTT5 contain 12 predicted transmembrane domains, intracellular N- and C-terminal domains, and large extracellular loops between transmembrane (TM) domains 3 and 4 and between TM domains 7 and 8. Residues within the extracellular loops are also predicted to contain sites for N-linked glycosylation. Human v7-3, the species orthologue of rat v7-3, contains an open reading frame (ORF) of 730 amino acids. Human NTT5 is a new member of the orphan transporter family and has an ORF of 736 amino acids. The amino acid sequences of human v7-3 and NTT5 are greater than 50% similar to other known orphan neurotransmitter transporters and also show sequence similarity to the human serotonin and dopamine transporters. Radiation hybrid mapping located the human v7-3 and NTT5 genes on chromosomes 12q21.3-q21.4 and 19q13.1-q13.4, respectively. Human mRNA distribution analysis by TaqMan reverse transcription-polymerase chain reaction showed that v7-3 mRNA is predominantly expressed in neuronal tissues, particularly amygdala, putamen, and corpus callosum, with low-level expression in peripheral tissues. In contrast, NTT5 mRNA was highly expressed in peripheral tissues, particularly in testis, pancreas, and prostate. Transient transfection with epitope-tagged transporter constructs demonstrated v7-3 to be expressed at the cell surface, whereas NTT5 was predominantly intracellular, suggestive of a vesicular location. Although the substrates transported by these transporters remain unknown, their specific but widespread distribution suggests that they may mediate distinct and important functions within the brain and the periphery.


Subject(s)
Membrane Transport Proteins/metabolism , Multienzyme Complexes/genetics , Multigene Family , Neurotransmitter Agents/metabolism , Protein Serine-Threonine Kinases/genetics , Sodium Chloride/metabolism , AMP-Activated Protein Kinases , Amino Acid Sequence , Base Sequence , Cells, Cultured , Chromosome Mapping , Cloning, Molecular , DNA, Complementary , Humans , Membrane Transport Proteins/genetics , Molecular Sequence Data , Plasma Membrane Neurotransmitter Transport Proteins , RNA, Messenger/genetics , Sequence Homology, Amino Acid , Subcellular Fractions/metabolism
14.
Neuroscience ; 100(1): 155-70, 2000.
Article in English | MEDLINE | ID: mdl-10996466

ABSTRACT

GABA(B) receptors are G-protein-coupled receptors that mediate the slow and prolonged synaptic actions of GABA in the CNS via the modulation of ion channels. Unusually, GABA(B) receptors form functional heterodimers composed of GABA(B1) and GABA(B2) subunits. The GABA(B1) subunit is essential for ligand binding, whereas the GABA(B2) subunit is essential for functional expression of the receptor dimer at the cell surface. We have used real-time reverse transcriptase-polymerase chain reaction to analyse expression levels of these subunits, and their associated splice variants, in the CNS and peripheral tissues of human and rat. GABA(B1) subunit splice variants were expressed throughout the CNS and peripheral tissues, whereas surprisingly GABA(B2) subunit splice variants were neural specific. Using novel antisera specific to individual GABA(B) receptor subunits, we have confirmed these findings at the protein level. Analysis by immunoblotting demonstrated the presence of the GABA(B1) subunit, but not the GABA(B2) subunit, in uterus and spleen. Furthermore, we have shown the first immunocytochemical analysis of the GABA(B2) subunit in the brain and spinal cord using a GABA(B2)-specific antibody. We have, therefore, identified areas of non-overlap between GABA(B1) and GABA(B2) subunit expression in tissues known to contain functional GABA(B) receptors. Such areas are of interest as they may well contain novel GABA(B) receptor subunit isoforms, expression of which would enable the GABA(B1) subunit to reach the cell surface and form functional GABA(B) receptors.


Subject(s)
Central Nervous System/metabolism , Receptors, GABA-B/metabolism , Animals , Brain/metabolism , Female , Humans , Male , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, GABA-B/genetics , Spinal Cord/metabolism , Spleen/metabolism , Tissue Distribution , Uterus/metabolism
15.
J Pharmacol Exp Ther ; 295(1): 373-81, 2000 Oct.
Article in English | MEDLINE | ID: mdl-10992004

ABSTRACT

The pharmacological and pharmacokinetic profile of SB-222200 [(S)-(-)-N-(alpha-ethylbenzyl)-3-methyl-2-phenylquinoline-4-car boxami de], a human NK-3 receptor (hNK-3R) antagonist, was determined. SB-222200 inhibited (125)I-[MePhe(7)]neurokinin B (NKB) binding to Chinese hamster ovary (CHO) cell membranes stably expressing the hNK-3 receptor (CHO-hNK-3R) with a K(i) = 4.4 nM and antagonized NKB-induced Ca(2+) mobilization in HEK 293 cells stably expressing the hNK-3 receptor (HEK 293-hNK-3R) with an IC(50) = 18.4 nM. SB-222200 was selective for hNK-3 receptors compared with hNK-1 (K(i) > 100,000 nM) and hNK-2 receptors (K(i) = 250 nM). In HEK 293 cells transiently expressing murine NK-3 receptors (HEK 293-mNK-3R), SB-222200 inhibited binding of (125)I-[MePhe(7)]NKB (K(i) = 174 nM) and antagonized NKB (1 nM)-induced calcium mobilization (IC(50) = 265 nM). In mice oral administration of SB-222200 produced dose-dependent inhibition of behavioral responses induced by i.p. or intracerebral ventricular administration of the NK-3 receptor-selective agonist, senktide, with ED(50) values of approximately 5 mg/kg. SB-222200 effectively crossed the blood-brain barrier in the mouse and rat. The inhibitory effect of SB-222200 against senktide-induced behavioral responses in the mouse correlated significantly with brain, but not plasma, concentrations of the compound. Pharmacokinetic evaluation of SB-222200 in rat after oral administration (8 mg/kg) indicated sustained plasma concentrations (C(max) = about 400 ng/ml) and bioavailability of 46%. The preclinical profile of SB-222200, demonstrating high affinity, selectivity, reversibility, oral activity, and central nervous system penetration, suggests that it will be a useful tool compound to define the physiological and pathophysiological roles of NK-3 receptors, in particular in the central nervous system.


Subject(s)
Brain/drug effects , Quinolines/pharmacology , Receptors, Neurokinin-3/antagonists & inhibitors , Animals , Brain/metabolism , CHO Cells , Calcium/metabolism , Cricetinae , Dose-Response Relationship, Drug , Humans , In Vitro Techniques , Iris/drug effects , Iris/physiology , Male , Mice , Mice, Inbred BALB C , Peptide Fragments/pharmacology , Quinolines/pharmacokinetics , Rabbits , Rats , Rats, Sprague-Dawley , Substance P/analogs & derivatives , Substance P/pharmacology
16.
Genomics ; 67(1): 8-18, 2000 Jul 01.
Article in English | MEDLINE | ID: mdl-10945465

ABSTRACT

Using homology searching of public databases with a metabotropic glutamate receptor sequence from Caenorhabditis elegans, two novel protein sequences (named RAIG-2 (HGMW-approved symbol GPRC5B) and RAIG-3 (HGMW-approved symbol GPRC5C) were identified containing seven putative transmembrane domains characteristic of G-protein-coupled receptors (GPCRs). RAIG-2 and RAIG-3 encode open reading frames of 403 and 442 amino acid polypeptides, respectively, and show 58% similarity to the recently identified retinoic acid-inducible gene-1 (RAIG-1, HGMW-approved symbol RAI3). Analysis of the three protein sequences places them within the type 3 GPCR family, which includes metabotropic glutamate receptors, GABA(B) receptors, calcium-sensing receptors, and pheromone receptors. However, in contrast to other type 3 GPCRs, RAIG-1, RAIG-2, and RAIG-3 have only short N-terminal domains. RAIG-2 and RAIG-3 cDNA sequences were cloned into the mammalian expression vector pcDNA3 with c-myc or HA epitope tags inserted at their N-termini, respectively. Transient transfection experiments in HEK239T cells using these constructs demonstrated RAIG-2 and RAIG-3 expression at the cell surface. Distribution profiles of mRNA expression obtained by semiquantitative Taq-Man PCR analysis showed RAIG-2 to be predominantly expressed in human brain areas and RAIG-3 to be predominantly expressed in peripheral tissues. In addition, expression of RAIG-2 and RAIG-3 mRNA was increased following treatment with all-trans-retinoic acid in a manner similar to that previously described for RAIG-1. Finally, RAIG-2 was mapped to chromosome 16p12 (D16S405-D16S3045) and RAIG-3 to chromosome 17q25 (D17S1352-D17S785). These results suggest that RAIG-1, RAIG-2, and RAIG-3 represent a novel family of retinoic acid-inducible receptors, most closely related to the type 3 GPCR subfamily, and provide further evidence for a linkage between retinoic acid and G-protein-coupled receptor signal transduction pathways.


Subject(s)
GTP-Binding Proteins/metabolism , Receptors, Cell Surface/genetics , Receptors, G-Protein-Coupled , Amino Acid Sequence , Base Sequence , Brain/physiology , Chromosomes, Human, Pair 16 , Chromosomes, Human, Pair 17 , Cloning, Molecular , DNA Primers/chemistry , GTP-Binding Proteins/genetics , Gene Expression Regulation , Humans , Molecular Sequence Data , Multigene Family , Phylogeny , RNA, Messenger/analysis , Receptors, Cell Surface/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Signal Transduction , Tissue Distribution , Transfection , Tretinoin/pharmacology , Tumor Cells, Cultured/cytology , Tumor Cells, Cultured/drug effects
17.
J Pharmacol Exp Ther ; 294(3): 1154-65, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10945872

ABSTRACT

SB-277011-A (trans-N-[4-[2-(6-cyano-1,2,3, 4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolininecarboxamide), is a brain-penetrant, high-affinity, and selective dopamine D(3) receptor antagonist. Radioligand-binding experiments in Chinese hamster ovary (CHO) cells transfected with human dopamine D(3) or D(2 long) (hD(3), hD(2)) receptors showed SB-277011-A to have high affinity for the hD(3) receptor (pK(i) = 7.95) with 100-fold selectivity over the hD(2) receptor and over 66 other receptors, enzymes, and ion channels. Similar radioligand-binding data for SB-277011-A were obtained from CHO cells transfected with rat dopamine D(3) or D(2). In the microphysiometer functional assay, SB-277011-A antagonized quinpirole-induced increases in acidification in CHO cells overexpressing the hD(3) receptor (pK(b) = 8.3) and was 80-fold selective over hD(2) receptors. Central nervous system penetration studies showed that SB-277011-A readily entered the brain. In in vivo microdialysis studies, SB-277011-A (2. 8 mg/kg p.o.) reversed the quinelorane-induced reduction of dopamine efflux in the nucleus accumbens but not striatum, a regional selectivity consistent with the distribution of the dopamine D(3) receptor in rat brain. SB-277011-A (2-42.3 mg/kg p.o.) did not affect spontaneous locomotion, or stimulant-induced hyperlocomotion. SB-277011-A (4.1-42.2 mg/kg p.o.) did not reverse prepulse inhibition deficits in apomorphine- or quinpirole-treated rats, but did significantly reverse the prepulse inhibition deficit in isolation-reared rats at a dose of 3 mg/kg p.o. SB-277011-A (2.5-78. 8 mg/kg p.o.) was noncataleptogenic and did not raise plasma prolactin levels. Thus, dopamine D(3) receptor blockade produces few of the behavioral effects characteristic of nonselective dopamine receptor antagonists. The effect of SB-277011-A on isolation-induced prepulse inhibition deficit suggests that blockade of dopamine D(3) receptors may benefit the treatment of schizophrenia.


Subject(s)
Dopamine Antagonists/pharmacology , Nitriles/pharmacology , Quinolines/pharmacology , Receptors, Dopamine D2/drug effects , Tetrahydroisoquinolines , Animals , Brain/metabolism , CHO Cells , Catalepsy/chemically induced , Cricetinae , Dopamine Antagonists/metabolism , Dopamine Antagonists/toxicity , Humans , Male , Microdialysis , Motor Activity/drug effects , Nitriles/metabolism , Nitriles/toxicity , Prolactin/blood , Quinolines/metabolism , Quinolines/toxicity , Radioligand Assay , Rats , Rats, Inbred Strains , Rats, Sprague-Dawley , Receptors, Dopamine D2/metabolism , Receptors, Dopamine D3 , Reflex, Startle/drug effects , Reverse Transcriptase Polymerase Chain Reaction , Transfection
18.
J Neurosci Methods ; 98(1): 9-20, 2000 May 15.
Article in English | MEDLINE | ID: mdl-10837866

ABSTRACT

TaqMan reverse transcription polymerase chain reaction (RT-PCR) is a recently developed technique which allows the measurement of an accumulating PCR product in real time. In the present study we have validated the use of TaqMan RT-PCR for mRNA localisation studies in human and rat tissues, and for the investigation of gene expression changes in CNS animal models. In human brain, D(2) receptor mRNA was enriched in caudate nucleus and putamen, whilst in rat brain, highest levels of D(2) receptor mRNA expression were observed in striatum and nucleus accumbens, consistent with the known distribution of this receptor in basal ganglia. In a rat model of permanent middle cerebral artery occlusion (pMCAO), endogenous interleukin-1 receptor antagonist (IL-1ra) mRNA was upregulated over 30-fold at 24 h post-lesion in both striatum and cortex ipsilateral to artery occlusion. Brain-derived neurotrophic factor (BDNF) mRNA was transiently upregulated 3.7-fold at 3 h, but not at 24 h or 3 days after induction of cortical spreading depression (CSD) in rats. Our observations in these two animal models using TaqMan RT-PCR were consistent with previous reports using other techniques. In conclusion, TaqMan RT-PCR assays provide a rapid and reliable method for semi-quantitative analysis of gene expression in the nervous system.


Subject(s)
Brain Chemistry/genetics , Gene Expression/physiology , Infarction, Middle Cerebral Artery/genetics , Actins/genetics , Animals , Cortical Spreading Depression , DNA Primers , Disease Models, Animal , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Humans , Infarction, Middle Cerebral Artery/physiopathology , Peptidylprolyl Isomerase/genetics , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Receptors, Dopamine D2/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Taq Polymerase
19.
Br J Pharmacol ; 130(3): 539-48, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10821781

ABSTRACT

The novel 5-HT(7) receptor antagonist, SB-269970-A, potently displaced [(3)H]-5-CT from human 5-HT(7(a)) (pK(i) 8.9+/-0.1) and 5-HT(7) receptors in guinea-pig cortex (pK(i) 8.3+/-0.2). 5-CT stimulated adenylyl cyclase activity in 5-HT(7(a))/HEK293 membranes (pEC(50) 7.5+/-0.1) and SB-269970-A (0.03 - 1 microM) inhibited the 5-CT concentration-response with no significant alteration in the maximal response. The pA(2) (8.5+/-0.2) for SB-269970-A agreed well with the pK(i) determined from [(3)H]-5-CT binding studies. 5-CT-stimulated adenylyl cyclase activity in guinea-pig hippocampal membranes (pEC(50) of 8.4+/-0.2) was inhibited by SB-269970-A (0.3 microM) with a pK(B) (8.3+/-0.1) in good agreement with its antagonist potency at the human cloned 5-HT(7(a)) receptor and its binding affinity at guinea-pig cortical membranes. 5-HT(7) receptor mRNA was highly expressed in human hypothalamus, amygdala, thalamus, hippocampus and testis. SB-269970-A was CNS penetrant (steady-state brain : blood ratio of ca. 0.83 : 1 in rats) but was rapidly cleared from the blood (CLb=ca. 140 ml min(-1) kg(-1)). Following a single dose (3 mg kg(-1)) SB-269970 was detectable in rat brain at 30 (87 nM) and 60 min (58 nM). In guinea-pigs, brain levels averaged 31 and 51 nM respectively at 30 and 60 min after dosing, although the compound was undetectable in one of the three animals tested. 5-CT (0.3 mg kg(-1) i.p.) induced hypothermia in guinea-pigs was blocked by SB-269970-A (ED(50) 2.96 mg kg(-1) i.p.) and the non-selective 5-HT(7) receptor antagonist metergoline (0.3 - 3 mg kg(-1) s.c.), suggesting a role for 5-HT(7) receptor stimulation in 5-CT induced hypothermia in guinea-pigs. SB-269970-A (30 mg kg(-1)) administered at the start of the sleep period, significantly reduced time spent in Paradoxical Sleep (PS) during the first 3 h of EEG recording in conscious rats.


Subject(s)
Phenols/pharmacology , Receptors, Serotonin/drug effects , Serotonin Antagonists/pharmacology , Sulfonamides/pharmacology , Adenylyl Cyclases/metabolism , Animals , Body Temperature/drug effects , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Guinea Pigs , Hippocampus/metabolism , Humans , Male , Membranes/metabolism , Phenols/pharmacokinetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Serotonin Antagonists/pharmacokinetics , Sleep/drug effects , Sleep Stages/drug effects , Sulfonamides/pharmacokinetics
20.
Pflugers Arch ; 439(6): 714-22, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10784345

ABSTRACT

We have cloned human TREK-1, one of the newly emerging mammalian family of 2-P domain potassium channels. The channel has 411 amino acids with a 41-amino-acid extension at the C-terminus when compared with the cloned mouse TREK-1 channel. Expression of hTREK-1 produced a substantial hyperpolarising shift in resting membrane potential accompanied by the induction of large, outwardly rectifying, non-inactivating currents which were potassium selective. Pharmacologically, hTREK-1-mediated currents were only blocked to a limited extent by classic potassium channel blockers or open channel pore blockers known to potently inhibit other channels. The channel was reversibly potentiated by arachidonic acid. CNS distribution of hTREK-1 is widespread with higher levels being observed in caudate, putamen, amygdala, thalamus and spinal cord. Only low levels of expression were seen in the majority of peripheral regions. Thus, hTREK-1, although functionally and pharmacologically similar to mouse TREK-1, appears to have a more CNS-specific distribution.


Subject(s)
Cloning, Molecular , Potassium Channels, Tandem Pore Domain , Potassium Channels/genetics , Potassium Channels/metabolism , Amino Acid Sequence/genetics , Animals , Base Sequence/genetics , Cell Line , Humans , Mice , Molecular Sequence Data , Oocytes/metabolism , Peptide Fragments/metabolism , Peptide Fragments/physiology , Tissue Distribution , Xenopus laevis/metabolism
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