The Role of TRP Channels in Oxidative Stress-induced Cell Death

The transient receptor potential (TRP) protein superfamily is a diverse group of voltage-independent calcium-permeable cation channels expressed in mammalian cells. These channels have been divided into six subfamilies, and two of them, TRPC and TRPM, have members that are widely expressed and activated by oxidative stress. TRPC3 and TRPC4 are activated by oxidants, which induce Na⁺ and Ca²⁺ entry into cells through mechanisms that are dependent on phospholipase C. TRPM2 is activated by oxidative stress or TNFα, and the mechanism involves production of ADP-ribose, which binds to an ADP-ribose binding cleft in the TRPM2 C-terminus. Treatment of HEK 293T cells expressing TRPM2 with H₂O₂ resulted in Ca²⁺ influx and increased susceptibility to cell death, whereas coexpression of the dominant negative isoform TRPM2-S suppressed H₂O₂-induced Ca²⁺ influx, the increase in [Ca²⁺]_i, and onset of apoptosis. U937-ecoR monocytic cells expressing increased levels of TRPM2 also exhibited significantly increased [Ca²⁺]_i and increased apoptosis after treatment with H₂O₂ or TNFα. A dramatic increase in caspase 8, 9, 3, 7, and PARP cleavage was observed in TRPM2-expressing cells, demonstrating a downstream mechanism through which cell death is mediated. Inhibition of endogenous TRPM2 function through three approaches, depletion of TRPM2 by RNA interference, blockade of the increase in [Ca²⁺]_i through TRPM2 by calcium chelation, or expression of the dominant negative splice variant TRPM2-S protected cell viability. H₂O₂ and amyloid β-peptide also induced cell death in primary cultures of rat striatal cells, which endogenously express TRPM2. TRPM7 is activated by reactive oxygen species/nitrogen species, resulting in cation conductance and anoxic neuronal cell death, which is rescued by suppression of TRPM7 expression. TRPM2 and TRPM7 channels are physiologically important in oxidative stress-induced cell death.     

Chemical physiology of oxidative stress-activated TRPM2 and TRPC5 channels

The ability to sense and adapt to a wide variety of environmental changes is crucial for the survival of all cells. Transient receptor potential (TRP) channels play pivotal roles in these sensing and adaptation reactions. In vertebrates, there are about 30 TRP channels; these are divided into six subfamilies by homology of the protein sequences. We have previously revealed that a group of TRP channels senses oxidative stress and induces cellular signaling and gene expression. TRPM2, a member of the TRPM subfamily, is activated by reactive oxygen species (ROS) via second-messenger production. Recently, we demonstrated that Ca²⁺ influx through TRPM2 activated by ROS induces chemokine production in monocytes, which aggravates inflammatory neutrophil infiltration. Additionally, we also revealed that nitric oxide, chemical compounds containing reactive disulfide, and inflammatory mediators directly activate the TRPC, TRPV, and TRPA subfamilies via oxidative modification of cysteine residues. In this review, we describe how these TRP channels sense oxidative stress and induce adaptation reactions, and we discuss the biological importance of oxidative stress-activated TRP channels.     

TRPM2 Cation Channels,Oxidative Stress and Neurological Diseases: Where Are We Now?

The Na+ and Ca²⁺-permeable melastatin related transient receptor potential 2 (TRPM2) channels can be gated either by ADP-ribose (ADPR) in concert with Ca²⁺ or by hydrogen peroxide (H₂O₂), an experimental model for oxidative stress, binding to the channel’s enzymatic Nudix domain. Since the mechanisms that lead to TRPM2 gating in response to ADPR and H₂O₂ are not understood in neuronal cells, I summarized previous findings and important recent advances in the understanding of Ca²⁺ influx via TRPM2 channels in different neuronal cell types and disease processes. Considering that TRPM2 is activated by oxidative stress, mediated cell death and inflammation, and is highly expressed in brain, the channel has been investigated in the context of central nervous system. TRPM2 plays a role in H₂O₂ and amyloid β-peptide induced striatal cell death. Genetic variants of the TRPM2 gene confer a risk of developing Western Pacific amyotropic lateral sclerosis and parkinsonism-dementia complex and bipolar disorders. TRPM2 also contributes to traumatic brain injury processes such as oxidative stress, inflammation and neuronal death. There are a limited number of TRPM2 channel blockers and they seem to be cell specific. For example, ADPR-induced Ca²⁺ influx in rat hippocampal cells was not blocked by N-(p-amylcinnomoyl)anthralic acid (ACA), the IP₃ receptor inhibitor 2-aminoethoxydiphenyl borate or PLC inhibitor flufenamic acid (FFA). However, the Ca²⁺ entry in rat primary striatal cells was blocked by ACA and FFA. In conclusion TRPM2 channels in neuronal cells can be gated by either ADPR or H₂O₂. It seems to that the exact relationship between TRPM2 channels activation and neuronal cell death still remains to be determined.     

Sensitization of H2O2-induced TRPM2 activation and subsequent interleukin-8 (CXCL8) production by intracellular Fe2+ in human monocytic U937 cells

Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca²⁺-permeable channel. In monocytes/macrophages, H₂O₂-induced TRPM2 activation causes cell death and/or production of chemokines that aggravate inflammatory diseases. However, relatively high concentrations of H₂O₂ are required for activation of TRPM2 channels in vitro. Thus, in the present study, factors that sensitize TRPM2 channels to H₂O₂ were identified and subsequent physiological responses were examined in U937 human monocytes. Temperature increase from 30 °C to 37 °C enhanced H₂O₂-induced TRPM2-mediated increase in intracellular free Ca²⁺ ([Ca²⁺]_i) in TRPM2-expressing HEK 293 cells (TRPM2/HEK cells). The H₂O₂-induced TRPM2 activation enhanced by the higher temperature was dramatically sensitized by intracellular Fe²⁺-accumulation following pretreatment with FeSO₄. Thus intracellular Fe²⁺-accumulation sensitizes H₂O₂-induced TRPM2 activation at around body temperature. Moreover, intracellular Fe²⁺-accumulation increased poly(ADP-ribose) levels in nuclei by H₂O₂ treatment, and the sensitization of H₂O₂-induced TRPM2 activation were almost completely blocked by poly(ADP-ribose) polymerase inhibitors, suggesting that intracellular Fe²⁺-accumulation enhances H₂O₂-induced TRPM2 activation by increase of ADP-ribose production through poly(ADP-ribose) polymerase pathway. Similarly, pretreatment with FeSO₄ stimulated H₂O₂-induced TRPM2 activation at 37 °C in U937 cells and enhanced H₂O₂-induced ERK phosphorylation and interleukin-8 (CXCL8) production. Although the addition of H₂O₂ to cells under conditions of intracellular Fe²⁺-accumulation caused cell death, concentration of H₂O₂ required for CXCL8 production was lower than that resulting in cell death. These results indicate that intracellular Fe²⁺-accumulation sensitizes TRPM2 channels to H₂O₂ and subsequently produces CXCL8 at around body temperature. It is possible that sensitization of H₂O₂-induced TRPM2 channels by Fe²⁺ may implicated in hemorrhagic brain injury via aggravation of inflammation, since Fe²⁺ is released by heme degradation under intracerebral hemorrhage.     

Molecular role of catalase on oxidative stress-induced Ca2+ signaling and TRP cation channel activation in nervous system

Background: Catalase catalyzes the reduction of H₂O₂ to water and it can also remove organic hydroperoxides. Nervous system in body is especially sensitive to free radical damage due to rich content of easily oxidizible fatty acids and relatively low content of antioxidants including catalase. Recent studies indicate that reactive oxygen species actually target active channel function, in particular TRP channels. I review the effects of catalase on Ca²⁺ signaling and on TRP channel activation in neuroglial cells such as microglia and substantia nigra.

Materials: Review of the relevant literature and results from recent our basic studies, as well as critical analyses of published systematic reviews were obtained from the pubmed and the Science Citation Index.

Results: It was observed that oxidative stress-induced activations of TRPM2, TRPC3, TRPC5 and TRPV1 cation channels in neuronal cells are modulated by catalase, suggesting antioxidant-dependent activation/inhibition of the channels. I provide also, a general overview of the most important oxidative stress-associated changes in neuronal mitochondrial Ca²⁺ homeostasis due to oxidative stress-induced channel neuropathies. Catalase incubation induces protective effects on rat brain mitochondrial function and neuronal survival. A decrease in catalase activity through oxidative stress may have an important role in etiology of Parkinson’s disease and sensory pain.

Conclusion: The TRP channels can be activated by oxidative stress products, opening of nonspecific cation channels would result in Ca²⁺ influx, and then elevation of cytoplasmic free Ca²⁺ could stimulate mitochondrial Ca²⁺ uptake. Catalase modulates oxidative stress-induced Ca²⁺ influx and some TRP channels activity in neuronal cells.     

Mechanistic study of TRPM2-Ca2+-CAMK2-BECN1 signaling in oxidative stress-induced autophagy inhibition

Reactive oxygen species (ROS) have been commonly accepted as inducers of autophagy, and autophagy in turn is activated to relieve oxidative stress. Yet, whether and how oxidative stress, generated in various human pathologies, regulates autophagy remains unknown. Here, we mechanistically studied the role of TRPM2 (transient receptor potential cation channel subfamily M member 2)-mediated Ca²⁺ influx in oxidative stress-mediated autophagy regulation. On the one hand, we demonstrated that oxidative stress triggered TRPM2-dependent Ca²⁺ influx to inhibit the induction of early autophagy, which renders cells more susceptible to death. On the other hand, oxidative stress induced autophagy (and not cell death) in the absence of the TRPM2-mediated Ca²⁺ influx. Moreover, in response to oxidative stress, TRPM2-mediated Ca²⁺ influx activated CAMK2 (calcium/calmodulin dependent protein kinase II) at levels of both phosphorylation and oxidation, and the activated CAMK2 subsequently phosphorylated BECN1/Beclin 1 on Ser295. Ser295 phosphorylation of BECN1 in turn decreased the association between BECN1 and PIK3C3/VPS34, but induced binding between BECN1 and BCL2. Clinically, acetaminophen (APAP) overdose is the most common cause of acute liver failure worldwide. We demonstrated that APAP overdose also activated ROS-TRPM2-CAMK2-BECN1 signaling to suppress autophagy, thereby causing primary hepatocytes to be more vulnerable to death. Inhibiting the TRPM2-Ca²⁺-CAMK2 cascade significantly mitigated APAP-induced liver injury. In summary, our data clearly demonstrate that oxidative stress activates the TRPM2-Ca²⁺-CAMK2 cascade to phosphorylate BECN1 resulting in autophagy inhibition.     

Focus on TRP channels in cystic fibrosis

The Transient Receptor Potential (TRP) protein superfamily is a group of cation channels expressed in various cell types and involved in respiratory diseases such as cystic fibrosis (CF), the genetic disease caused by CF Transmembrane conductance Regulator (CFTR) mutations. In human airway epithelial cells, there is growing evidence for a functional link between CFTR and TRP channels. TRP channels contribute to transmitting extracellular signals into the cells and, in an indirect manner, to CFTR activity via a Ca²⁺ rise signaling. Indeed, mutated CFTR-epithelial cells are characterized by an increased Ca²⁺ influx and, on the opposite, by a decreased of magnesium influx, both being mediated by TRP channels. This increasing cellular Ca²⁺ triggers the activation of calcium-activated chloride channels (CaCC) or CFTR itself, via adenylyl cyclase, PKA and tyrosine kinases activation, but also leads to an exaltation of the inflammatory response. Another shortcoming in mutated CFTR-epithelial cells is a [Mg²⁺]_i decrease, associated with impaired TRPM7 functioning. This deregulation has to be taken into consideration in CF physiopathology, as Mg²⁺ is required for ATP hydrolysis and CFTR activity. The modulation of druggable TRP channels could supplement CF therapy either an anti-inflammatory drug or for CFTR potentiation, according to the balance between exacerbation and respite phases. The present paper focus on TRPA1, TRPC6, TRPM7, TRPV2, TRPV4, TRPV6 and ORAI 1, the proteins identified, for now, as dysfunctional channels, in CF cells.     

Vitamin E modulates oxidative stress and protein kinase C activator (PMA)-induced TRPM2 channel gate in dorsal root ganglion of rats

It is well known that Ca²⁺ influx through cation channels induces peripheral pain in dorsal root ganglion (DRG) neurons. Melastatin-like transient receptor potential 2 (TRPM2) channel is a oxidative redox sensitive Ca²⁺-permeable cation channel. There is scarce report on block of the channels. Since the mechanisms that lead to TRPM2 inhibition in response to oxidative stress and protein kinase C (PKC) activation are not understood, we investigated effects of the antioxidants on the inhibition of TRPM2 channel currents in the DRG neurons of rats. The DRG peripheral neurons were freshly isolated from rats and the neurons were incubated by phorbol 12-myristate 13-acetate (PMA) which leads to activation of PKC and cause oxidative stress. In whole-cell patch clamp experiments, TRPM2 currents in the DRG incubated with PMA were stimulated by H₂O₂. In addition, the PMA-induced activation of TRPM2 channels were blocked by nonspecific TRPM2 channels inhibitors [2-aminoethyl diphenylborinate (2-APB) and N-(p-amylcinnamoyl)anthranilic acid (ACA)]. The currents in the neurons are also totally blocked by vitamin E incubation. However, administration of catalase and vitamin C with/without the vitamin E incubation did not block the currents. In conclusion, we indicated that vitamin E modulated oxidative stress-induced TRPM2 channel activation in the DRG neurons. The results may be useful modulation of oxidative stress-induced peripheral pain in sensory neurons.     

Curcumin inhibits oxidative stress-induced TRPM2 channel activation,calcium ion entry and apoptosis values in SH-SY5Y neuroblastoma cells: Involvement of transfection procedure

Transient Receptor Potential (TRP) channels are mostly Ca²⁺ permeable cation channels. Transient Receptor Potential Melastatin-like 2 (TRPM2) is expressed in neurological tissues such as brain, dorsal root ganglia (DRG) neurons, hippocampus and also liver, heart and kidney. The SH-SY5Y cells are mostly used as a cellular model of neurodegenerative diseases, Alzheimer's and Parkinson's diseases. Curcumin, shows phenolic structure, synthesized by Curcuma longa L. (turmeric), has powerful non-enzymatically antioxidant effects compared with Vitamin E. Hence, we aimed to investigate that effects of curcumin on TRPM2 cation channel currents using the whole-cell Patch-Clamp method, Ca²⁺ signaling, apoptosis and cell viability (MTT) assays, reactive oxygen species (ROS) production, mitochondrial membrane potential levels, caspase 3 and caspase 9 activities in TRPM2 transfected SH-SY5Y neuroblastoma cells. For this aim, we designed four experimental groups named; control, curcumin, transfected and transfected?+?curcumin groups. Cytosolic free calcium concentrations were higher in transfected group compared with curcumin and transfected?+?curcumin group. Moreover, these data examined with whole-cell Patch-Clamp recordings of single cells in all groups. ROS levels were significantly higher in transfected group than in transfected?+?curcumin group. Apoptosis levels in transfected?+?curcumin group were lower than in transfected group. Procaspase 9 and procaspase 3 levels measured by western blotting and caspase 3 and caspase 9 levels by spectrophotometric methods show that TRPM2 transfected cells are more tended to apoptosis. In conclusion, curcumin strongly induces modulator effects on TRPM2-mediated Ca²⁺ influx caused by ROS and caspase 3 and 9 processes in SH-SY5Y neuroblastoma cells.     

New Molecular Mechanisms on the Activation of TRPM2 Channels by Oxidative Stress and ADP-Ribose

The Na⁺ and Ca²⁺-permeable melastatin related transient receptor potential (TRPM2) cation channels can be gated either by ADP-ribose (ADPR) in concert with Ca²⁺ or by hydrogen peroxide (H₂O₂), an experimental model for oxidative stress, and binding to the channel’s enzymatic Nudix domain. Since the mechanisms that lead to TRPM2 inhibiting in response to ADPR and H₂O₂ are not understood, I reviewed the effects of various inhibitors such as flufenamic acid and PARP inhibitors on ADPR, NAD⁺ and H₂O₂-induced TRPM2 currents. In our experimental study, TRPM2 cation channels in chinese hamster ovary transected cells were gated both by ADPR and NAD⁺. In addition, H₂O₂ seems to activate TRPM2 by changing to the hydroxyl radical in the intracellular space after passing the plasma membrane. Experimental studies with respect to patch-clamp and Ca²⁺ imaging, inhibitor roles of antioxidants are also summarized in the review.     

Plasma membrane stretch activates transient receptor potential vanilloid and ankyrin channels in Merkel cells from hamster buccal mucosa

Merkel cells (MCs) have been proposed to form a part of the MC-neurite complex with sensory neurons. Many transient receptor potential (TRP) channels have been identified in mammals; however, the activation properties of these channels in oral mucosal MCs remain to be clarified. We investigated the biophysical and pharmacological properties of TRP vanilloid (TRPV)-1, TRPV2, TRPV4, TRP ankyrin (TRPA)-1, and TRP melastatin (TRPM)-8 channels, which are sensitive to osmotic and mechanical stimuli by measurement of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) using fura-2. We also analyzed their localization patterns through immunofluorescence. MCs showed immunoreaction for TRPV1, TRPV2, TRPV4, TRPA1, and TRPM8 channels. In the presence of extracellular Ca²⁺, the hypotonic test solution evoked Ca²⁺ influx. The [Ca²⁺]_i increases were inhibited by TRPV1, TRPV2, TRPV4, or TRPA1 channel antagonists, but not by the TRPM8 channel antagonist. Application of TRPV1, TRPV2, TRPV4, TRPA1, or TRPM8 channel selective agonists elicited transient increases in [Ca²⁺]_i only in the presence of extracellular Ca²⁺. The results indicate that membrane stretching in MCs activates TRPV1, TRPV2, TRPV4, and TRPA1 channels, that it may be involved in synaptic transmission to sensory neurons, and that MCs could contribute to the mechanosensory transduction sequence.     

A critical role of the transient receptor potential melastatin 2 channel in a positive feedback mechanism for reactive oxygen species-induced delayed cell death

Transient receptor potential melastatin 2 (TRPM2) channel activation by reactive oxygen species (ROS) plays a critical role in delayed neuronal cell death, responsible for postischemia brain damage via altering intracellular Zn²⁺ homeostasis, but a mechanistic understanding is still lacking. Here, we showed that H₂O₂ induced neuroblastoma SH-SY5Y cell death with a significant delay, dependently of the TRPM2 channel and increased [Zn²⁺]_i, and therefore used this cell model to investigate the mechanisms underlying ROS-induced TRPM2-mediated delayed cell death. H₂O₂ increased concentration-dependently the [Zn²⁺]_i and caused lysosomal dysfunction and Zn²⁺ loss and, furthermore, mitochondrial Zn²⁺ accumulation, fragmentation, and ROS generation. Such effects were suppressed by preventing poly(adenosine diphosphate ribose, ADPR) polymerase-1-dependent TRPM2 channel activation with PJ34 and 3,3′,5,5′-tetra-tert-butyldiphenoquinone, inhibiting the TRPM2 channel with 2-aminoethoxydiphenyl borate (2-APB) and N-(p-amylcinnamoyl)anthranilic acid, or chelating Zn²⁺ with N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). Bafilomycin-induced lysosomal dysfunction also resulted in mitochondrial Zn²⁺ accumulation, fragmentation, and ROS generation that were inhibited by PJ34 or 2-APB, suggesting that these mitochondrial events are TRPM2 dependent and sequela of lysosomal dysfunction. Mitochondrial TRPM2 expression was detected and exposure to ADPR-induced Zn²⁺ uptake in isolated mitochondria, which was prevented by TPEN. H₂O₂-induced delayed cell death was inhibited by apocynin and diphenyleneiodonium, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase (NOX) inhibitors, GKT137831, an NOX1/4-specific inhibitor, or Gö6983, a protein kinase C (PKC) inhibitor. Moreover, inhibition of PKC/NOX prevented H₂O₂-induced ROS generation, lysosomal dysfunction and Zn²⁺ release, and mitochondrial Zn²⁺ accumulation, fragmentation and ROS generation. Collectively, these results support a critical role for the TRPM2 channel in coupling PKC/NOX-mediated ROS generation, lysosomal Zn²⁺ release, and mitochondrial Zn²⁺ accumulation, and ROS generation to form a vicious positive feedback signaling mechanism for ROS-induced delayed cell death.     

Redox Signal-mediated Enhancement of the Temperature Sensitivity of Transient Receptor Potential Melastatin 2 (TRPM2) Elevates Glucose-induced Insulin Secretion from Pancreatic Islets

Transient receptor potential melastatin 2 (TRPM2) is a thermosensitive Ca²⁺-permeable cation channel expressed by pancreatic β cells where channel function is constantly affected by body temperature. We focused on the physiological functions of redox signal-mediated TRPM2 activity at body temperature. H₂O₂, an important molecule in redox signaling, reduced the temperature threshold for TRPM2 activation in pancreatic β cells of WT mice but not in TRPM2KO cells. TRPM2-mediated [Ca²⁺]_i increases were likely caused by Ca²⁺ influx through the plasma membrane because the responses were abolished in the absence of extracellular Ca²⁺. In addition, TRPM2 activation downstream from the redox signal plus glucose stimulation enhanced glucose-induced insulin secretion. H₂O₂ application at 37 °C induced [Ca²⁺]_i increases not only in WT but also in TRPM2KO β cells. This was likely due to the effect of H₂O₂ on K_ATP channel activity. However, the N-acetylcysteine-sensitive fraction of insulin secretion by WT islets was increased by temperature elevation, and this temperature-dependent enhancement was diminished significantly in TRPM2KO islets. These data suggest that endogenous redox signals in pancreatic β cells elevate insulin secretion via TRPM2 sensitization and activity at body temperature. The results in this study could provide new therapeutic approaches for the regulation of diabetic conditions by focusing on the physiological function of TRPM2 and redox signals.     

Glutathione modulates Ca(2+) influx and oxidative toxicity through TRPM2 channel in rat dorsal root ganglion neurons

Glutathione (GSH) is the most abundant thiol antioxidant in mammalian cells and maintains thiol redox in the cells. GSH depletion has been implicated in the neurobiology of sensory neurons. Because the mechanisms that lead to melastatin-like transient receptor potential 2 (TRPM2) channel activation/inhibition in response to glutathione depletion and 2-aminoethyldiphenyl borinate (2-APB) administration are not understood, we tested the effects of 2-APB and GSH on oxidative stress and buthionine sulfoximine (BSO)-induced TRPM2 cation channel currents in dorsal root ganglion (DRG) neurons of rats. DRG neurons were freshly isolated from rats and the neurons were incubated for 24 h with BSO. In whole-cell patch clamp experiments, TRPM2 currents in the rat were consistently induced by H₂O₂ or BSO. TRPM2 channels current densities and cytosolic free Ca²⁺ content of the neurons were higher in BSO and H₂O₂ groups than in control. However, the current densities and cytosolic Ca²⁺ release were also higher in the BSO + H₂O₂ group than in the H₂O₂ alone. When intracellular GSH is introduced by pipette TRPM2 channel currents were not activated by BSO, H₂O₂ or rotenone. BSO and H₂O₂-induced Ca²⁺ gates were blocked by the 2-APB. Glutathione peroxidase activity, lipid peroxidation and GSH levels in the DRG neurons were also modulated by GSH and 2-APB inhibition. In conclusion, we observed the protective role of 2-APB and GSH on Ca²⁺ influx through a TRPM2 channel in intracellular GSH depleted DRG neurons. Since cytosolic glutathione depletion is a common feature of neuropathic pain and diseases of sensory neuron, our findings are relevant to the etiology of neuropathology in DRG neurons.     

Regulation of TRP channel TRPM2 by the tyrosine phosphatase PTPL1

TRPM2, a member of the transient receptor potential (TRP) superfamily, is a Ca²⁺-permeable channel, which mediates susceptibility to cell death following activation by oxidative stress, TNF, or -amyloid peptide. We determined that TRPM2 is rapidly tyrosine phosphorylated after stimulation with H₂O₂ or TNF. Inhibition of tyrosine phosphorylation with the tyrosine kinase inhibitors genistein or PP2 significantly reduced the increase in [Ca²⁺]_i observed after H₂O₂ or TNF treatment in TRPM2-expressing cells, suggesting that phosphorylation is important in TRPM2 activation. Utilizing a TransSignal PDZ domain array blot to identify proteins which interact with TRPM2, we identified PTPL1 as a potential binding protein. PTPL1 is a widely expressed tyrosine phosphatase, which has a role in cell survival and tumorigenesis. Immunoprecipitation and glutathione-S-transferase pull-down assays confirmed that TRPM2 and PTPL1 interact. To examine the ability of PTPL1 to modulate phosphorylation or activation of TRPM2, PTPL1 was coexpressed with TRPM2 in human embryonic kidney-293T cells. This resulted in significantly reduced TRPM2 tyrosine phosphorylation, and inhibited the rise in [Ca²⁺]_i and the loss of cell viability, which follow H₂O₂ or TNF treatment. Consistent with these findings, reduction in endogenous PTPL1 expression with small interfering RNA resulted in increased TRPM2 tyrosine phosphorylation, a significantly greater rise in [Ca²⁺]_i following H₂O₂ treatment, and enhanced susceptibility to H₂O₂-induced cell death. Endogenous TRPM2 and PTPL1 was associated in U937-ecoR cells, confirming the physiological relevance of this interaction. These data demonstrate that tyrosine phosphorylation of TRPM2 is important in its activation and function and that inhibition of TRPM2 tyrosine phosphorylation reduces Ca²⁺ influx and protects cell viability. They also suggest that modulation of TRPM2 tyrosine phosphorylation is a mechanism through which PTPL1 may mediate resistance to cell death. transient receptor potential channels; oxidative stress     

Hydrogen peroxide constricts rat arteries by activating Na+-permeable and Ca2+-permeable cation channels

Oxidative stress is associated with many cardiovascular diseases, such as hypertension and arteriosclerosis. Oxidative stress reportedly activates the L-type voltage-gated calcium channel (VDCC_L) and elevates [Ca²⁺]_i in many cells. However, how oxidative stress activates VDCC_L under clinical setting and the consequence for arteries are unclear. Here, we examined the hypothesis that hydrogen peroxide (H₂O₂) regulates membrane potential (Em) by altering Na⁺ influx through cation channels, which consequently activates VDCC_L to induce vasoconstriction in rat mesenteric arteries. To measure the tone of the endothelium-denuded arteries, a conventional isometric organ chamber was used. Membrane currents and Em were recorded by the patch-clamp technique. [Ca²⁺]_i and [Na⁺]_i were measured with microfluorometry using Fura2-AM and SBFI-AM, respectively. We found that H₂O₂ (10 and 100 µM) increased arterial contraction, and nifedipine blocked the effects of H₂O₂ on isometric contraction. H₂O₂ increased [Ca²⁺]_i as well as [Na⁺]_i, and depolarised Em. Gd³⁺ (1 µM) blocked all these H₂O₂-induced effects including Em depolarisation and increases in [Ca²⁺]_i and [Na⁺]_i. Although both nifedipine (30?nM) and low Na⁺ bath solution completely prevented the H₂O₂-induced increase in [Na⁺], they only partly inhibited the H₂O₂-induced effects on [Ca²⁺]_i and Em. Taken together, the results suggested that H₂O₂ constricts rat arteries by causing Em depolarisation and VDCC_L activation through activating Gd³⁺-and nifedipine-sensitive, Na⁺-permeable channels as well as Gd³⁺-sensitive Ca²⁺-permeable cation channels. We suggest that unidentified Na⁺-permeable cation channels as well as Ca²⁺-permeable cation channels may function as important mediators for oxidative stress-induced vascular dysfunction.     

Remodeling of calcium signaling in tumor progression

Intracellular Ca²⁺ is one of the crucial signalings that modulate various cellular functions. The dysregulation of Ca²⁺ homeostasis has been suggested as an important event in driving the expression of the malignant phenotypes, such as proliferation, migration, invasion, and metastasis. Cell migration is an early prerequisite for tumor metastasis that has a significant impact on patient prognosis. During cell migration, the exquisite spatial and temporal organization of intracellular Ca²⁺ provides a rapid and robust way for the selective activation of signaling components that play a central role in cytoskeletal reorganization, traction force generation, and focal adhesion dynamics. A number of known molecular components involved in Ca²⁺ influx pathways, including stromal interaction molecule (STIM)/Orai-mediated store-operated Ca²⁺ entry (SOCE) and the Ca²⁺-permeable transient receptor potential (TRP) channels, have been implicated in cancer cell migration and tumor metastasis. The clinical significance of these molecules, such as STIM proteins and the TRPM7 channel, in tumor progression and their diagnostic and prognostic potentials have also been demonstrated in specific cancer types. In this review, we summarize the recent advances in understanding the important roles and regulatory mechanisms of these Ca²⁺ influx pathways on malignant behaviors of tumor cells. The clinical implications in facilitating current diagnostic and therapeutic procedures are also discussed.     

Sustained CaMKII activity mediates transient oxidative stress-induced long-term facilitation of L-type Ca(2+) current in cardiomyocytes

Oxidative stress remodels Ca²⁺ signaling in cardiomyocytes, which promotes altered heart function in various heart diseases. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) was shown to be activated by oxidation, but whether and how CaMKII links oxidative stress to pathophysiological long-term changes in Ca²⁺ signaling remain unknown. Here, we present evidence demonstrating the role of CaMKII in transient oxidative stress-induced long-term facilitation (LTF) of L-type Ca²⁺ current (I_Ca,L) in rat cardiomyocytes. A 5-min exposure of 1 mM H₂O₂ induced an increase in I_Ca,L, and this increase was sustained for ~ 1 h. The CaMKII inhibitor KN-93 fully reversed H₂O₂-induced LTF of I_Ca,L, indicating that sustained CaMKII activity underlies this oxidative stress-induced memory. Simultaneous inhibition of oxidation and autophosphorylation of CaMKII prevented the maintenance of LTF, suggesting that both mechanisms contribute to sustained CaMKII activity. We further found that sarcoplasmic reticulum Ca²⁺ release and mitochondrial ROS generation have critical roles in sustaining CaMKII activity via autophosphorylation- and oxidation-dependent mechanisms. Finally, we show that long-term remodeling of the cardiac action potential is induced by H₂O₂ via CaMKII. In conclusion, CaMKII and mitochondria confer oxidative stress-induced pathological cellular memory that leads to cardiac arrhythmia.     

Identification of Direct and Indirect Effectors of the Transient Receptor Potential Melastatin 2 (TRPM2) Cation Channel

Transient receptor potential melastatin 2 (TRPM2) is a Ca²⁺-permeable cation channel involved in physiological and pathophysiological processes linked to oxidative stress. TRPM2 channels are co-activated by intracellular Ca²⁺ and ADP-ribose (ADPR) but also modulated in intact cells by several additional factors. Superfusion of TRPM2-expressing cells with H₂O₂ or intracellular dialysis of cyclic ADPR (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP) activates, whereas dialysis of AMP inhibits, TRPM2 whole-cell currents. Additionally, H₂O₂, cADPR, and NAADP enhance ADPR sensitivity of TRPM2 currents in intact cells. Because in whole-cell recordings the entire cellular machinery for nucleotide and Ca²⁺ homeostasis is intact, modulators might affect TRPM2 activity either directly, by binding to TRPM2, or indirectly, by altering the local concentrations of the primary ligands ADPR and Ca²⁺. To identify direct modulators of TRPM2, we have studied the effects of H₂O₂, AMP, cADPR, NAADP, and nicotinic acid adenine dinucleotide in inside-out patches from Xenopus oocytes expressing human TRPM2, by directly exposing the cytosolic faces of the patches to these compounds. H₂O₂ (1 mm) and enzymatically purified cADPR (10 μm) failed to activate, whereas AMP (200 μm) failed to inhibit TRPM2 currents. NAADP was a partial agonist (maximal efficacy, ∼50%), and nicotinic acid adenine dinucleotide was a full agonist, but both had very low affinities (K_0.5 = 104 and 35 μm). H₂O₂, cADPR, and NAADP did not enhance activation by ADPR. Considering intracellular concentrations of these compounds, none of them are likely to directly affect the TRPM2 channel protein in a physiological context.