Elena Fonfria

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.

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.

Discovery and structure-activity relationships of a series of pyroglutamic acid amide antagonists of the P2X7 receptor.

A computational lead-hopping exercise identified compound 4 as a structurally distinct P2X(7) receptor antagonist. Structure-activity relationships (SAR) of a series of pyroglutamic acid amide analogues of 4 were investigated and compound 31 was identified as a potent P2X(7) antagonist with excellent in vivo activity in animal models of pain, and a profile suitable for progression to clinical studies.

Cloning and pharmacological characterization of the dog P2X7 receptor.

Background and purpose:  Human and rodent P2X7 receptors exhibit differences in their sensitivity to antagonists. In this study we have cloned and characterized the dog P2X7 receptor to determine if its antagonist sensitivity more closely resembles the human or rodent orthologues.

Agonist potency at P2X7 receptors is modulated by structurally diverse lipids.

The P2X7 receptor exhibits a high degree of plasticity with agonist potency increasing after prolonged receptor activation. In this study we investigated the ability of lipids to modulate agonist potency at P2X7 receptors.

Cloning and pharmacological characterization of the guinea pig P2X7 receptor orthologue

The human, rat, and mouse P2X7 receptors have been previously characterized, and in this study we report the cloning and pharmacological properties of the guinea pig orthologue.

Synthesis and biological activity of a series of tetrasubstituted-imidazoles as P2X7 antagonists

A series of analogues of the pyrazole lead 1 were synthesized in which the heterocyclic core was replaced with an imidazole. A number of potent antagonists were identified and structure-activity relationships (SAR) were investigated both with respect to activity at the P2X(7) receptor and in vitro metabolic stability. Compound 10 was identified as a potent P2X(7) antagonist with reduced in vitro metabolism and high solubility.

Species and response dependent differences in the effects of MAPK inhibitors on P2X7 receptor function

Recent studies have implicated the mitogen activated protein kinase (MAPK) in cellular permeability changes following P2X7 receptor activation in native tissues. In this study we have further studied the effect of MAPK inhibitors on recombinant and native P2X7 receptors.