Shaun McNulty

TRPM2 channel opening in response to oxidative stress is dependent on activation of poly(ADP‐ribose) polymerase

TRPM2 (melastatin‐like transient receptor potential 2 channel) is a nonselective cation channel that is activated under conditions of oxidative stress leading to an increase in intracellular free Ca2+ concentration ([Ca2+]i) and cell death. We investigated the role of the DNA repair enzyme poly(ADP‐ribose) polymerase (PARP) on hydrogen peroxide (H2O2)‐mediated TRPM2 activation using a tetracycline‐inducible TRPM2‐expressing cell line. In whole‐cell patch‐clamp recordings, intracellular adenine 5′‐diphosphoribose (ADP‐ribose) triggered an inward current in tetracycline‐induced TRPM2‐human embryonic kidney (HEK293) cells, but not in uninduced cells. Similarly, H2O2 stimulated an increase in [Ca2+]i (pEC50 4.54±0.02) in Fluo‐4‐loaded TRPM2‐expressing HEK293 cells, but not in uninduced cells. Induction of TRPM2 expression caused an increase in susceptibility to plasma membrane damage and mitochondrial dysfunction in response to H2O2. These data demonstrate functional expression of TRPM2 following tetracycline induction in TRPM2‐HEK293 cells. PARP inhibitors SB750139‐B (patent number DE10039610‐A1 (Lubisch et al., 2001)), PJ34 (N‐(6‐oxo‐5,6‐dihydro‐phenanthridin‐2‐yl)‐N,N‐dimethylacetamide) and DPQ (3, 4‐dihydro‐5‐[4‐(1‐piperidinyl)butoxy]‐1(2H)‐isoquinolinone) inhibited H2O2‐mediated increases in [Ca2+]i (pIC50 vs 100 μM H2O2: 7.64±0.38; 6.68±0.28; 4.78±0.05, respectively), increases in mitochondrial dysfunction (pIC50 vs 300 μM H2O2: 7.32±0.23; 6.69±0.22; 5.44±0.09, respectively) and decreases in plasma membrane integrity (pIC50 vs 300 μM H2O2: 7.45±0.27; 6.35±0.18; 5.29±0.12, respectively). The order of potency of the PARP inhibitors in these assays (SB750139>PJ34>DPQ) was the same as for inhibition of isolated PARP enzyme. SB750139‐B, PJ34 and DPQ had no effect on inward currents elicited by intracellular ADP‐ribose in tetracycline‐induced TRPM2‐HEK293 cells, suggesting that PARP inhibitors are not interacting directly with the channel. SB750139‐B, PJ34 and DPQ inhibited increases in [Ca2+]i in a rat insulinoma cell line (CRI‐G1 cells) endogenously expressing TRPM2 (pIC50 vs 100 μM H2O2: 7.64±0.38; 6.68±0.28; 4.78±0.05, respectively). These data suggest that oxidative stress causes TRPM2 channel opening in both recombinant and endogenously expressing cell systems via activation of PARP enzymes.

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.

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.

Flufenamic acid is a pH-dependent antagonist of TRPM2 channels

Like a number of other TRP channels, TRPM2 is a Ca(2+)-permeable non-selective cation channel, the activity of which is regulated by intracellular and extracellular Ca(2+). A unique feature of TRPM2 is its activation by ADP-ribose and chemical species that arise during oxidative stress, for example, NAD(+) and H(2)O(2). These properties have lead to proposals that this channel may play a role in the cell death produced by pathological redox states. The lack of known antagonists of this channel have made these hypotheses difficult to test. Here, we demonstrate, using patch clamp electrophysiology, that the non-steroidal anti-inflammatory compound flufenamic acid (FFA) inhibits recombinant human TRPM2 (hTRPM2) as well as currents activated by intracellular ADP-ribose in the CRI-G1 rat insulinoma cell line. All concentrations tested in a range from 50 to 1000 microM produced complete inhibition of the TRPM2-mediated current. Following FFA removal, a small (typically 10-15%) component of current was rapidly recovered (time constant approximately 3 s), considerably longer periods in the absence of FFA produced no further current recovery. Reapplication of FFA re-antagonised the recovered current and subsequent FFA washout produced recovery of only a small percentage of the reblocked current. Decreasing extracellular pH accelerated FFA inhibition of TRPM2. Additional experiments indicated hTRPM2 activation was required for FFA antagonism to occur and that the generation of irreversible antagonism was preceded by a reversible component of block. FFA inhibition could not be induced by intracellular application of FFA. ADP-ribose activated currents in the rat insulinoma cell line CRI-G1 were also antagonised by FFA with concentration- and pH-dependent kinetics. In contrast to the observations made with hTRPM2, antagonism of ADP-ribose activated currents in CRI-G1 cells could be fully reversed following FFA removal. These experiments suggest that FFA may be a useful tool antagonist for studies of TRPM2 function.

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.

Activation of astrocyte intracellular signaling pathways by interleukin-1 in rat primary striatal cultures

The striatum has been implicated as the site of action mediating neurotoxic effects of interleukin‐1 (IL‐1) during ischemia. However, the molecular mechanisms underlying these events have yet to be fully addressed. In the present study, primary cultures of rat striatal cells were used as a model for the study of IL‐1 signaling pathways in the striatum. Immunocytochemical analyses revealed that these cultures consisted of a mixture of neurones and astrocytes and demonstrated expression of the IL‐1 type I receptor (IL‐1RI) on both cell types. Treatment with IL‐1 (3 units/ml) for 10 min increased phosphorylation of p38 MAP kinase in striatal cells. The endogenous IL‐1RI inhibitor IL‐1Ra (24 ng/ml) and the p38 MAP kinase inhibitor SB203580 (10 nM) both inhibited this response. Analysis of the effects of IL‐1 on nuclear translocation of the transcription factor NF‐kB revealed that NF‐kB became activated in a time‐dependent manner. Immunocytochemistry revealed that IL‐1 stimulated p38 phosphorylation and NF‐kB translocation in astrocytes only. TaqMan real‐time quantitative PCR analysis revealed that IL‐1 stimulated gene expression of tumor necrosis factor‐α (TNF) in striatal cultures. The p38 MAP kinase inhibitor SB203580 failed to inhibit the effects of IL‐1 on NF‐kB translocation or gene transcription. These studies have demonstrated significant aspects of the IL‐1 signaling cascade in cultured striatum. Of particular interest is the finding that IL‐1 stimulated activation of p38 MAP kinase and NF‐kB in striatal astrocytes exclusively. GLIA 37:31–42, 2002.

Characterisation of recombinant rat TRPM2 and a TRPM2-like conductance in cultured rat striatal neurones

TRPM2, a member of the TRP ion channel family, is expressed both in the brain and immune cells of the monocyte lineage. Functionally, it is unique in its activation by intracellular ADP-ribose and both oxidative and nitrosative stress. To date studies of this channel have concentrated on human recombinant channels and rodent native preparations. This provides the potential for cross-species complications in the interpretation of native tissue observations based on recombinant channel phenotype. Consequently, we have cloned and heterologously expressed rat TRPM2 (rTRPM2) in HEK293 cells. We find that, like hTRPM2, it responds to intracellular ADP-ribose in a manner dependent on extracellular Ca(2+). At the single channel level rTRPM2 is a slow gating, large conductance (84pS) channel that rapidly runs down in isolated membrane patches. Pharmacologically, rTRPM2 is rapidly and irreversibly blocked by clotrimazole (10muM), thus resembling hTRPM2 but not the TRPM2-like current of the rat-derived insulinoma CRI-G1, which exhibits reversible inhibition by this agent. We show that cultured rat striatal neurones exhibit an ADP-ribose-activated conductance at both the whole cell and single channel level. Pharmacologically this neuronal current can be irreversibly inhibited by clotrimazole. It is also sensitive to removal of extracellular Ca(2+), suggesting that it is mediated by TRPM2-containing channels. These data provide a functional characterisation of heterologously expressed rTRPM2 and demonstrate that, in addition to the previous descriptions in immune cells, microglia and insulinomas, a TRPM2-like conductance can be found in neurones derived from the rodent CNS.

Differentiation of bipolar CG-4 line oligodendrocytes is associated with regulation of CREB, MAP kinase and PKC signalling pathways.

Undifferentiated bipolar CG-4 cell line oligodendrocytes provide a model system for the O-2A progenitor cell from which oligodendrocytes are derived both in vivo and in vitro. The exchange of neuroblastoma conditioned basal media for basal media causes differentiation of undifferentiated bipolar CG-4 cells into multipolar oligodendrocyte-like cells whilst replacement with basal media containing 20% foetal bovine serum favours the formation of type-2 astrocyte-like cells. Here, we demonstrate that activation of these differentiation pathways correlates with distinct changes both in cell metabolism and in signal transduction. Exchange of neuroblastoma conditioned media for basal media correlates with stimulation of basal metabolic activity, reduced phosphorylation of p44/42 MAP kinase and reduced phosphorylation of the transcription factor CREB. In contrast, differentiation with basal medium containing 20% foetal bovine serum (FBS), into type 2 astrocyte-like cells, correlates with reduction in basal metabolic activity, increased phosphorylation of p44/42 MAP kinase and increased phosphorylation of the transcription factor CREB. Inhibition of protein kinase C blocked both the metabolic and morphological changes associated with differentiation towards mature multipolar oligodendrocyte-like cells. Inhibition of PKA and MEK did not effect metabolic activity. The rapid return of neuroblastoma conditioned basal media to cells treated with basal media, increased phosphorylation of CREB and MAP kinase. These results demonstrate that protein kinase C and p44/42 MAP kinase signalling pathways are modulated during bipolar CG-4 cell differentiation and demonstrate that the transcription factor CREB may play a pivotal role in differentiation along oligodendrocyte-or astrocyte-lineages.

Identification and characterisation of functional bombesin receptors in human astrocytes

Reverse transcription polymerase chain reaction (RT-PCR) demonstrated the presence of bombesin BB2 receptor mRNA but not bombesin BB1 receptor or bombesin BB3 receptor mRNA in cultured human astrocytes. Neuromedin C hyperpolarised human astrocytes in whole-cell current and voltage clamp recordings and increased the intracellular free Ca(2+) ion concentration ([Ca(2+)](i)) in single astrocytes. Treatment with neuromedin C caused larger and more frequent increases in [Ca(2+)](i) than those triggered by neuromedin B, with 96% and 78% of cells responding, respectively. The stimulatory effects of neuromedin C were inhibited significantly by treatment with U73122 or the bombesin BB2 receptor antagonist [D-Phe(6), des-Met(14)]bombesin-(6-14) ethylester. A Fluorometric Imaging Plate Reader (FLIPR) was used to measure [Ca(2+)](i) in cell populations. Neuromedin C was approximately 50-fold more potent than neuromedin B in elevating [Ca(2+)](i) in astrocytes and Chinese hamster ovary (CHO) cells expressing human bombesin BB2 receptors (hBB2-CHO). However, in CHO cells expressing the bombesin BB1 receptor hBB1-CHO, neuromedin B was 32-fold more potent than neuromedin C. [D-Phe(6), des-Met(14)]bombesin-(6-14) ethylester was a partial agonist in hBB1-CHO cells (E(max)=55%) but was a noncompetitive antagonist in both hBB2-CHO cells and astrocytes. These studies report the first identification of functional bombesin receptors on cultured human astrocytes and have demonstrated that the bombesin BB2 receptor contributes significantly to astrocyte physiology.