Andrew D. Medhurst

Cloning and functional expression of a human orthologue of rat vanilloid receptor-1

&NA; Capsaicin, resiniferatoxin, protons or heat have been shown to activate an ion channel, termed the rat vanilloid receptor‐1 (rVR1), originally isolated by expression cloning for a capsaicin sensitive phenotype. Here we describe the cloning of a human vanilloid receptor‐1 (hVR1) cDNA containing a 2517 bp open reading frame that encodes a protein with 92% homology to the rat vanilloid receptor‐1. Oocytes or mammalian cells expressing this cDNA respond to capsaicin, pH and temperature by generating inward membrane currents. Mammalian cells transfected with human VR1 respond to capsaicin with an increase in intracellular calcium. The human VR1 has a chromosomal location of 17p13 and is expressed in human dorsal root ganglia and also at low levels throughout a wide range of CNS and peripheral tissues. Together the sequence homology, similar expression profile and functional properties confirm that the cloned cDNA represents the human orthologue of rat VR1.

Pharmacological and immunohistochemical characterization of the APJ receptor and its endogenous ligand apelin

Apelin peptides have recently been identified to be the endogenous ligands for the G protein‐coupled receptor APJ. However, little is known about the physiological roles of this ligand‐receptor pairing. In the present study we investigated the pharmacology of several apelin analogues at the human recombinant APJ receptor using radioligand binding and functional assays. This has led to the identification of key residues in the apelin peptide required for functional potency and binding affinity through structure–activity studies. In particular, we have identified that replacement of leucine in position 5, or arginine in position 2 and 4 of the C‐terminal apelin peptide, apelin‐13, resulted in significant changes in pharmacology. We also investigated the detailed localization of pre‐proapelin and APJ receptor mRNA in a wide range of human, rat and mouse tissues using quantitative RT–PCR, and carried out a detailed immunohistochemical study of the distribution of the APJ receptor in rat brain and spinal cord. Interestingly, the APJ receptor was not only co‐localized in white matter with GFAP in the spinal cord, but was also clearly localized on neurones in the brain, suggesting that this receptor and its peptide may be involved in a wide range of biological process yet to be determined.

The use of TaqMan RT-PCR assays for semiquantitative analysis of gene expression in CNS tissues and disease models

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.

Characterization of SB-269970-A, a selective 5-HT7 receptor antagonist

The novel 5‐HT7 receptor antagonist, SB‐269970‐A, potently displaced [3H]‐5‐CT from human 5‐HT7(a) (pKi 8.9±0.1) and 5‐HT7 receptors in guinea‐pig cortex (pKi 8.3±0.2). 5‐CT stimulated adenylyl cyclase activity in 5‐HT7(a)/HEK293 membranes (pEC50 7.5±0.1) and SB‐269970‐A (0.03–1 μM) inhibited the 5‐CT concentration‐response with no significant alteration in the maximal response. The pA2 (8.5±0.2) for SB‐269970‐A agreed well with the pKi determined from [3H]‐5‐CT binding studies. 5‐CT‐stimulated adenylyl cyclase activity in guinea‐pig hippocampal membranes (pEC50 of 8.4±0.2) was inhibited by SB‐269970‐A (0.3 μM) with a pKB (8.3±0.1) in good agreement with its antagonist potency at the human cloned 5‐HT7(a) receptor and its binding affinity at guinea‐pig cortical membranes. 5‐HT7 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 (ED50 2.96 mg kg−1 i.p.) and the non‐selective 5‐HT7 receptor antagonist metergoline (0.3–3 mg kg−1 s.c.), suggesting a role for 5‐HT7 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.

GSK189254, a Novel H3 Receptor Antagonist That Binds to Histamine H3 Receptors in Alzheimer's Disease Brain and Improves Cognitive Performance in Preclinical Models

6-[(3-Cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254) is a novel histamine H3 receptor antagonist with high affinity for human (pKi = 9.59 –9.90) and rat (pKi = 8.51–9.17) H3 receptors. GSK189254 is >10,000-fold selective for human H3 receptors versus other targets tested, and it exhibited potent functional antagonism (pA2 = 9.06 versus agonist-induced changes in cAMP) and inverse agonism [pIC50 = 8.20 versus basal guanosine 5′-O-(3-[35S]thio)triphosphate binding] at the human recombinant H3 receptor. In vitro autoradiography demonstrated specific [3H]GSK189254 binding in rat and human brain areas, including cortex and hippocampus. In addition, dense H3 binding was detected in medial temporal cortex samples from severe cases of Alzheimers disease, suggesting for the first time that H3 receptors are preserved in late-stage disease. After oral administration, GSK189254 inhibited cortical ex vivo R-(–)-α-methyl[imidazole-2,5(n)-3H]histamine dihydrochloride ([3H]R-α-methylhistamine) binding (ED50 = 0.17 mg/kg) and increased c-Fos immunoreactivity in prefrontal and somatosensory cortex (3 mg/kg). Microdialysis studies demonstrated that GSK189254 (0.3–3 mg/kg p.o.) increased the release of acetylcholine, noradrenaline, and dopamine in the anterior cingulate cortex and acetylcholine in the dorsal hippocampus. Functional antagonism of central H3 receptors was demonstrated by blockade of R-α-methylhistamine-induced dipsogenia in rats (ID50 = 0.03 mg/kg p.o.). GSK189254 significantly improved performance of rats in diverse cognition paradigms, including passive avoidance (1 and 3 mg/kg p.o.), water maze (1 and 3 mg/kg p.o.), object recognition (0.3 and 1 mg/kg p.o.), and attentional set shift (1 mg/kg p.o.). These data suggest that GSK189254 may have therapeutic potential for the symptomatic treatment of dementia in Alzheimers disease and other cognitive disorders.

The expression of GABAB1 and GABAB2 receptor subunits in the CNS differs from that in peripheral tissues

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.

Relationship between microglial activation and dopaminergic neuronal loss in the substantia nigra: a time course study in a 6‐hydroxydopamine model of Parkinson’s disease

Cellular interactions between activated microglia and degenerating neurons in in vivo models of Parkinson’s disease are not well defined. This time course study assesses the dynamics of morphological and immunophenotypic properties of activated microglia in a 6‐hydroxydopamine (6‐OHDA) model of Parkinson’s disease. Neurodegeneration in the substantia nigra pars compacta (SNc) was induced by unilateral injection of 6‐OHDA into the medial forebrain bundle. Activated microglia, identified using monoclonal antibodies: clone of antibody that detects major histocompatibility complex (MHC) class II antigens (OX6) for MHC class II, clone of antibody that detects cell surface antigen‐cluster of differentiation 11b – anti‐complement receptor 3, a marker for complement receptor 3 and CD 68 for phagocytic activity. Activation of microglia in the lesioned SNc was rapid with cells possessing amoeboid or ramified morphology appeared on day 1, whilst antibody clone that detects macrophage‐myeloid associated antigen immunoreactivity was observed at day 3 post‐lesion when there was no apparent loss of tyrosine hydroxylase (TH)+ve dopaminergic (DA) SNc neurons. Thereafter, OX6 and antibody clone that detects macrophage‐myeloid associated antigen activated microglia selectively adhered to degenerating axons, dendrites and apoptotic (caspase 3+ve) DA neurons in the SNc were observed at day 7. This was followed by progressive loss of TH+ve SNc neurons, with the peak of TH+ve cell loss (51%) being observed at day 9. This study suggests that activation of microglia precedes DA neuronal cell loss and neurons undergoing degeneration may be phagocytosed prematurely by phagocytic microglia.

The use of quantitative RT-PCR to measure mRNA expression in a rat model of focal ischemia--caspase-3 as a case study.

Quantitative reverse transcription and polymerisation chain reaction (RT-PCR) using Taqman¿trade mark omitted¿ fluorogenic probes has been used to measure changes in gene expression in the cerebral cortex of rats in the permanent middle cerebral artery occlusion (pMCAO) model of focal ischemia. The mRNA levels of three housekeeping genes have been analysed in this model to determine which gene showed least change following experimental insult. In the lesioned cortex, beta-actin mRNA increased at 24 h, while the levels of cyclophilin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) did not change. We have also used this methodology to examine modulations in the level of caspase-3 mRNA during focal ischemia in the rat. Caspase-3 mRNA showed a 41% increase at 6 h post-MCAO, which was specific to the lesioned cortex. This change became more pronounced with time, showing an increase of 220% at 24 h. This methodology enables changes in mRNA expression to be analysed more sensitively and quantitatively than other available techniques and highlights the need for careful choice of control or housekeeping genes used for RNA comparisons.

Cloning and Functional Expression of Human Short TRP7, a Candidate Protein for Store-operated Ca2+ Influx

The regulation and control of plasma membrane Ca2+ fluxes is critical for the initiation and maintenance of a variety of signal transduction cascades. Recently, the study of transient receptor potential channels (TRPs) has suggested that these proteins have an important role to play in mediating capacitative calcium entry. In this study, we have isolated a cDNA from human brain that encodes a novel transient receptor potential channel termed human TRP7 (hTRP7). hTRP7 is a member of the short TRP channel family and is 98% homologous to mouse TRP7 (mTRP7). At the mRNA level hTRP7 was widely expressed in tissues of the central nervous system, as well as some peripheral tissues such as pituitary gland and kidney. However, in contrast to mTRP7, which is highly expressed in heart and lung, hTRP7 was undetectable in these tissues. For functional analysis, we heterologously expressed hTRP7 cDNA in an human embryonic kidney cell line. In comparison with untransfected cells depletion of intracellular calcium stores in hTRP7-expressing cells, using either carbachol or thapsigargin, produced a marked increase in the subsequent level of Ca2+ influx. This increased Ca2+ entry was blocked by inhibitors of capacitative calcium entry such as La3+ and Gd3+. Furthermore, transient transfection of an hTRP7 antisense expression construct into cells expressing hTRP7 eliminated the augmented store-operated Ca2+ entry. Our findings suggest that hTRP7 is a store-operated calcium channel, a finding in stark contrast to the mouse orthologue, mTRP7, which is reported to enhance Ca2+ influx independently of store depletion, and suggests that human and mouse TRP7 channels may fulfil different physiological roles.

The PPARγ agonist pioglitazone is effective in the MPTP mouse model of Parkinson's disease through inhibition of monoamine oxidase B

Background and purpose: The peroxisome proliferator‐activated receptor‐γ (PPARγ) 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 Parkinsons 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+).