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Ian C B Marshall

Publications and source records attributed to Ian C B Marshall.

5 recordsLinked to original sources
PubMed2004

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

1. 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 Ca(2+) concentration ([Ca(2+)](i)) and cell death. We investigated the role of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) on hydrogen peroxide (H(2)O(2))-mediated TRPM2 activation using a tetracycline-inducible TRPM2-expressing cell line. 2. 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, H(2)O(2) stimulated an increase in [Ca(2+)](i) (pEC(50) 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 H(2)O(2). These data demonstrate functional expression of TRPM2 following tetracycline induction in TRPM2-HEK293 cells. 3. 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 H(2)O(2)-mediated increases in [Ca(2+)](i) (pIC(50) vs 100 microm H(2)O(2): 7.64+/-0.38; 6.68+/-0.28; 4.78+/-0.05, respectively), increases in mitochondrial dysfunction (pIC(50) vs 300 microm H(2)O(2): 7.32+/-0.23; 6.69+/-0.22; 5.44+/-0.09, respectively) and decreases in plasma membrane integrity (pIC(50) vs 300 microm H(2)O(2): 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. 4. 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. 5. SB750139-B, PJ34 and DPQ inhibited increases in [Ca(2+)](i) in a rat insulinoma cell line (CRI-G1 cells) endogenously expressing TRPM2 (pIC(50) vs 100 microm H(2)O(2): 7.64+/-0.38; 6.68+/-0.28; 4.78+/-0.05, respectively). 6. These data suggest that oxidative stress causes TRPM2 channel opening in both recombinant and endogenously expressing cell systems via activation of PARP enzymes.

Adenosine Diphosphate Ribose↗
PubMed2003

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

1 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. 2 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. 3 In Ca2+ -free media, RTX caused a transient elevation in intracellular free Ca2+ concentration in hVR1-HEK293 cells (pEC(50) 6.45+/-0.05) but not in wild type cells. Capsaicin (100 microM) did not cause Ca2+ mobilization under these conditions. 4 RTX-mediated Ca2+ mobilization was inhibited by the VR1 receptor antagonist capsazepine (pIC(50) 5.84+/-0.04), the Ca2+ pump inhibitor thapsigargin (pIC(50) 7.77+/-0.04), the phospholipase C inhibitor U-73122 (pIC(50) 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 microM, 2 min). These data suggest that RTX causes Ca2+ mobilization from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in hVR1-HEK293 cells. 5 In the presence of extracellular Ca2+, both capsaicin-mediated and RTX-mediated Ca2+ rises were attenuated by U-73122 (10 microM, 30 min) and thapsigargin (1 microM, 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.

Calcium↗
PubMed2002

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.

Animals↗