Davina E. Owen

Ethanol elicits and potentiates nociceptor responses via the vanilloid receptor-1.

The vanilloid receptor-1 (VR1) is a heat-gated ion channel that is responsible for the burning sensation elicited by capsaicin. A similar sensation is reported by patients with esophagitis when they consume alcoholic beverages or are administered alcohol by injection as a medical treatment. We report here that ethanol activates primary sensory neurons, resulting in neuropeptide release or plasma extravasation in the esophagus, spinal cord or skin. Sensory neurons from trigeminal or dorsal root ganglia as well as VR1-expressing HEK293 cells responded to ethanol in a concentration-dependent and capsazepine-sensitive fashion. Ethanol potentiated the response of VR1 to capsaicin, protons and heat and lowered the threshold for heat activation of VR1 from ∼42°C to ∼34°C. This provides a likely mechanistic explanation for the ethanol-induced sensory responses that occur at body temperature and for the sensitivity of inflamed tissues to ethanol, such as might be found in esophagitis, neuralgia or wounds.

Identification and characterisation of SB-366791, a potent and selective vanilloid receptor (VR1/TRPV1) antagonist

Vanilloid receptor-1 (TRPV1) is a non-selective cation channel, predominantly expressed by peripheral sensory neurones, which is known to play a key role in the detection of noxious painful stimuli, such as capsaicin, acid and heat. To date, a number of antagonists have been used to study the physiological role of TRPV1; however, antagonists such as capsazepine are somewhat compromised by non-selective actions at other receptors and apparent modality-specific properties. SB-366791 is a novel, potent, and selective, cinnamide TRPV1 antagonist isolated via high-throughput screening of a large chemical library. In a FLIPR-based Ca(2+)-assay, SB-366791 produced a concentration-dependent inhibition of the response to capsaicin with an apparent pK(b) of 7.74 +/- 0.08. Schild analysis indicated a competitive mechanism of action with a pA2 of 7.71. In electrophysiological experiments, SB-366791 was demonstrated to be an effective antagonist of hTRPV1 when activated by different modalities, such as capsaicin, acid or noxious heat (50 degrees C). Unlike capsazepine, SB-366791 was also an effective antagonist vs. the acid-mediated activation of rTRPV1. With the aim of defining a useful tool compound, we also profiled SB-366791 in a wide range of selectivity assays. SB-366791 had a good selectivity profile exhibiting little or no effect in a panel of 47 binding assays (containing a wide range of G-protein-coupled receptors and ion channels) and a number of electrophysiological assays including hippocampal synaptic transmission and action potential firing of locus coeruleus or dorsal raphe neurones. Furthermore, unlike capsazepine, SB-366791 had no effect on either the hyperpolarisation-activated current (I(h)) or Voltage-gated Ca(2+)-channels (VGCC) in cultured rodent sensory neurones. In summary, SB-366791 is a new TRPV1 antagonist with high potency and an improved selectivity profile with respect to other commonly used TRPV1 antagonists. SB-366791 may therefore prove to be a useful tool to further study the biology of TRPV1.

Amyloid β-peptide(1–42) and hydrogen peroxide-induced toxicity are mediated by TRPM2 in rat primary striatal cultures

Amyloid β‐peptide (Aβ) is the main component of senile plaques which characterize Alzheimers disease and may induce neuronal death through mechanisms which include oxidative stress. To date, the signalling pathways linking oxidant stress, a component of several neurodegenerative diseases, to cell death in the CNS are poorly understood. Melastatin‐like transient receptor potential 2 (TRPM2) is a Ca2+‐permeant non‐selective cation channel, which responds to increases in oxidative stress levels in the cell and is activated by oxidants such as hydrogen peroxide. We demonstrate here that Aβ and hydrogen peroxide both induce death in cultured rat striatal cells which express TRPM2 endogenously. Transfection with a splice variant that acts as a dominant negative blocker of TRPM2 function (TRPM2‐S) inhibited both hydrogen peroxide‐ and Aβ‐induced increases in intracellular‐free Ca2+ and cell death. Functional inhibition of TRPM2 activation by the poly(ADP‐ribose)polymerase inhibitor SB‐750139, a modulator of intracellular pathways activating TRPM2, attenuated hydrogen peroxide‐ and Aβ‐induced cell death. Furthermore, a small interfering RNA which targets TRPM2, reduced TRPM2 mRNA levels and the toxicity induced by hydrogen peroxide and Aβ. These data demonstrate that activation of TRPM2, functionally expressed in primary cultures of rat striatum, contributes to Aβ‐ and oxidative stress‐induced striatal cell death.

Activation of vanilloid receptor 1 by resiniferatoxin mobilizes calcium from inositol 1,4,5-trisphosphate-sensitive stores

Capsaicin and resiniferatoxin (RTX) stimulate Ca2+ influx by activating vanilloid receptor 1 (VR1), a ligand‐gated Ca2+ channel on sensory neurones. We investigated whether VR1 activation could also trigger Ca2+ mobilization from intracellular Ca2+ stores. Human VR1‐transfected HEK293 cells (hVR1‐HEK293) were loaded with Fluo‐3 or a mixture of Fluo‐4 and Fura Red and imaged on a fluorometric imaging plate reader (FLIPR) and confocal microscope respectively. In Ca2+‐free media, RTX caused a transient elevation in intracellular free Ca2+ concentration in hVR1‐HEK293 cells (pEC50 6.45±0.05) but not in wild type cells. Capsaicin (100 μM) did not cause Ca2+ mobilization under these conditions. RTX‐mediated Ca2+ mobilization was inhibited by the VR1 receptor antagonist capsazepine (pIC50 5.84±0.04), the Ca2+ pump inhibitor thapsigargin (pIC50 7.77±0.04), the phospholipase C inhibitor U‐73122 (pIC50 5.35±0.05) and by depletion of inositol 1,4,5‐trisphosphate‐sensitive Ca2+ stores by pretreatment with the acetylcholine‐receptor agonist carbachol (20 μM, 2 min). These data suggest that RTX causes Ca2+ mobilization from inositol 1,4,5‐trisphosphate‐sensitive Ca2+ stores in hVR1‐HEK293 cells. In the presence of extracellular Ca2+, both capsaicin‐mediated and RTX‐mediated Ca2+ rises were a ttenuated by U‐73122 (10 μM, 30 min) and thapsigargin (1 μM, 30 min). We conclude that VR1 is able to couple to Ca2+ mobilization by a Ca2+ dependent mechanism, mediated by capsaicin and RTX, and a Ca2+ independent mechanism mediated by RTX alone.

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-1β 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.

Caspase inhibitors are functionally neuroprotective against oxygen glucose deprivation induced CA1 death in rat organotypic hippocampal slices.

We have explored the neuroprotective efficacy of the cell penetrant caspase inhibitor, Ac-YVAD-cmk, in a hippocampal slice model of neuronal cell death induced by oxygen and glucose deprivation. Organotypic hippocampal slice cultures were prepared from 8 to 10-day-old rats and maintained for 10 to 12 days in vitro. Pre-treatment with Ac-YVAD-cmk prior to 45 min oxygen and glucose deprivation was neuroprotective as measured by propidium iodide uptake, with an EC(50) between 1 and 10 micromol/l. Ac-YVAD-cmk was also able to preserve synaptic function in the organotypic hippocampal slice cultures 24 h after oxygen and glucose deprivation. Ac-YVAD-cmk prevented the increase in histone-associated DNA fragmentation induced by oxygen and glucose deprivation. Interleukin-1beta did not reverse the protective effect of Ac-YVAD-cmk, and interleukin-1 receptor antagonist alone was not protective. These results show that caspase inhibitors are neuroprotective in a hippocampal slice culture system, using structural, biochemical and electrophysiological endpoints, and that this effect is not a result of inhibition of interleukin-1beta production.

Aβ1–42 reduces synapse number and inhibits neurite outgrowth in primary cortical and hippocampal neurons: A quantitative analysis

Synaptic loss represents one of the earliest signs of neuronal damage and is observed within both Alzheimers disease patients and transgenic mouse models of the disease. We have developed a novel in vitro assay using high content screening technology to measure changes in a number of cell physiological parameters simultaneously within a neuronal population. Using Hoechst-33342 to label nuclei, betaIII-tubulin as a neuron-specific marker, and synapsin-I as an indicator of pre-synaptic sites, we have designed software to interrogate triple-labelled images, counting only those synaptic puncta associated with tubulin-positive structures. Here we demonstrate that addition of amyloid beta peptide (Abeta(1-42)), to either primary hippocampal or cortical neurons for 4 days in vitro has deleterious effects upon synapse formation, neurite outgrowth and arborisation in a concentration-dependent manner. Control reverse peptide showed no effect over the same concentration range. The effects of Abeta(1-42) were inhibited by D-KLVFFA, which contains residues 16-20 of Abeta that function as a self-recognition element during Abeta assembly and bind to the homologous region of Abeta and block its oligomerisation. These effects of Abeta(1-42) on synapse number and neurite outgrowth are similar to those described within AD patient pathology and transgenic mouse models.

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 (Ih), that is, blockade by Cs+ (99% at 5 mM), ZD 7288 (98% at 100 μM) and zatebradine (92% at 10 μM). 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 (IC50=8 μM), 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.

Activation of recombinant human TRPV1 receptors expressed in SH-SY5Y human neuroblastoma cells increases [Ca2+]i, initiates neurotransmitter release and promotes delayed cell death

The transient receptor potential (TRP) vanilloid receptor subtype 1 (TRPV1) is a ligand‐gated, Ca2+‐permeable ion channel in the TRP superfamily of channels. We report the establishment of the first neuronal model expressing recombinant human TRPV1 (SH‐SY5YhTRPV1). SH‐SY5Y human neuroblastoma cells were stably transfected with hTRPV1 using the Amaxa Biosystem (hTRPV1 in pIREShyg2 with hygromycin selection). Capsaicin, olvanil, resiniferatoxin and the endocannabinoid anandamide increased [Ca2+]i with potency (EC50) values of 2.9 nmol/L, 34.7 nmol/L, 0.9 nmol/L and 4.6 μmol/L, respectively. The putative endovanilloid N‐arachidonoyl‐dopamine increased [Ca2+]i but this response did not reach a maximum. Capsaicin, anandamide, resiniferatoxin and olvanil mediated increases in [Ca2+]i were inhibited by the TRPV1 antagonists capsazepine and iodo‐resiniferatoxin with potencies (KB) of ∼70 nmol/L and 2 nmol/L, respectively. Capsaicin stimulated the release of pre‐labelled [3H]noradrenaline from monolayers of SH‐SY5YhTRPV1 cells with an EC50 of 0.6 nmol/L indicating amplification between [Ca2+]i and release. In a perfusion system, we simultaneously measured [3H]noradrenaline release and [Ca2+]i and observed that increased [Ca2+]i preceded transmitter release. Capsaicin treatment also produced a cytotoxic response (EC50 155 nmol/L) that was antagonist‐sensitive and mirrored the [Ca2+]I response. This model displays pharmacology consistent with TRPV1 heterologously expressed in standard non‐neuronal cells and native neuronal cultures. The advantage of SH‐SY5YhTRPV1 is the ability of hTRPV1 to couple to neuronal biochemical machinery and produce large quantities of cells.