Dmt-Tic-NH-CH2-Bid (UFP-502), a potent DOP receptor agonist: in vitro and in vivo studies

Knockout and pharmacological studies demonstrated that the activation of delta opioid peptide (DOP) receptors produces antidepressant-like effects in rodents. Here we report the results obtained with the novel DOP ligand H-Dmt-Tic-NH-CH₂-Bid (UFP-502). UFP-502 bound with high affinity (pK_i 9.43) to recombinant DOP receptors displaying moderate selectivity over MOP and KOP. In CHO_hDOP [³⁵S]GTPγS binding and mouse vas deferens experiments, UFP-502 behaved as a potent (pEC₅₀ 10.09 and 10.70, respectively) full agonist. In these preparations, naloxone, naltrindole and N,N(CH₃)₂Dmt-Tic-OH showed similar pA₂ values against UFP-502 and DPDPE and the same rank order of potency. In vivo in mice, UFP-502 mimicked DPDPE actions, producing a significant reduction of immobility time after intracerebroventricular administration in the forced swimming test and a clear antinociceptive effect after intrathecal injection in the tail withdrawal assay. However, while the effects of DPDPE were fully prevented by naltrindole those evoked by UFP-502 were unaffected (tail withdrawal assay) or only partially reversed (forced swimming test). In conclusion, UFP-502 represents a novel and useful chemical template for the design of selective agonists for the DOP receptor.     

Anxiolytic- and antidepressant-like activities of H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512), a novel selective delta opioid receptor agonist

Knockout and pharmacological studies have shown that delta opioid peptide (DOP) receptor signalling regulates emotional responses. In the present study, the in vitro and in vivo pharmacological profile of the DOP ligand, H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512) was investigated. In receptor binding experiments performed on membranes of CHO cells expressing the human recombinant opioid receptors, UFP-512 displayed very high affinity (pKi 10.20) and selectivity (>150-fold) for DOP sites. In functional studies ([35S]GTP gamma S binding in CHOhDOP membranes and electrically stimulated mouse vas deferens) UFP-512 behaved as a DOP selective full agonist showing potency values more than 100-fold higher than DPDPE. In vivo, in the mouse forced swimming test, UFP-512 reduced immobility time both after intracerebroventricular (i.c.v.) and intraperitoneal (i.p.) administration. Similar effects were recorded in rats. Moreover, UFP-512 evoked anxiolytic-like effects in the mouse elevated plus maze and light-dark aversion assays. All these in vivo actions of UFP-512 were fully prevented by the selective DOP antagonist naltrindole (3 mg/kg, s.c.). In conclusion, the present findings demonstrate that UFP-512 behaves as a highly potent and selective agonist at DOP receptors and corroborate the proposal that the selective activation of DOP receptors elicits robust anxiolytic- and antidepressant-like effects in rodents.     

Role of the delta-opioid receptor in (1DMe)NPYF mediated antinociception

A selective delta-opioid antagonist, naltrindole, was used to study the role of the delta-opioid receptor in the antinociceptive actions of a synthetic NPFF analog, (1DMe)NPYF. I.t. (1DMe)NPYF (5 nmol) produced antinociception in the tail flick test and (1DMe)NPYF (0.5 nmol) potentiated the antinociceptive effect of i.t. morphine 7.8 nmol. (1DMe)NPYF (5 nmol) had an antihyperalgesic effect in carrageenan inflammation and it significantly reduced mechanical allodynia in the spinal nerve ligation model. All these effects were prevented or significantly reduced by pretreatment with naltrindole (28 nmol) (P < 0.01-0.001). These data suggest that activation of spinal delta-opioid receptors plays an important role in mediating the spinal antinociceptive effects of (1DMe)NPYF.     

Role of serotonin receptors in the delta-opioid immunosuppression

The present study indicates involvement of serotoninergic (5-HTergic) mechanisms in immunosuppression by DSLET (100 mkg/kg), a selective agonist of the delta2-opioid receptors, in CBA mice. 8-OH-DPAT (0.1 mg/kg), a selective agonist of the 5-HT(1A)-autoreceptors, WAY-100635 (1, 3 mg/kg) and ketanserin (1, 3 mg/kg), a selective antagonists of the postsynaptic 5-HT(1A)- and 5-HT(2A)-receptors, respectively, prevented immunosuppressive effect of DSLET. A possible differential role for 5-HT-receptors in delta-opioid immunosuppression is suggested.     

Role of delta-opioid receptor function in neurogenesis and neuroprotection

The present study was undertaken to evaluate the implication of delta-opioid receptor function in neurogenesis and neuroprotection. We found that the stimulation of delta-opioid receptors by the selective delta-opioid receptor agonist SNC80 [(+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide] (10 nm) promoted neural differentiation from multipotent neural stem cells obtained from embryonic C3H mouse forebrains. In contrast, either a selective micro-opioid receptor agonist, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), or a specific kappa-opioid receptor agonist, (-)-trans-(1S,2S)-U-50488 hydrochloride (U50,488H), had no such effect. In addition to neural differentiation, the increase in cleaved caspase 3-like immunoreactivity induced by H2O2 (3 microm) was suppressed by treatment with SNC80 in cortical neuron/glia co-cultures. These effects of SNC80 were abolished by a Trk-dependent tyrosine kinase inhibitor: (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cycloocta(cde)trinden-1-one (K-252a). The SNC80-induced neural differentiation was also inhibited by treatment with the protein kinase C (PKC) inhibitor, phosphatidylinositol 3-kinase (PI3K) inhibitor, mitogen-activated protein kinase kinase (MEK) inhibitor or Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These findings raise the possibility that delta-opioid receptors play a crucial role in neurogenesis and neuroprotection, mainly through the activation of Trk-dependent tyrosine kinase, which could be linked to PI3K, PKC, CaMKII and MEK.     

Molecular determinants of receptor affinity and selectivity of the natural delta-opioid agonist, dermenkephalin

Processing of the polyprotein precursor pro-dermorphin generates two distantly related D-amino acid-containing peptides, dermorphin and dermenkephalin, which are among the most selective high affinity agonists described, respectively, for the mu- and delta-opioid receptors. Dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, is a linear, potentially flexible peptide devoid of structural homology with either enkephalins, endorphins, or dynorphins and, as such, represents a useful tool for identifying determinants of high affinity and selective binding of opioids to the delta-receptor. A series of selected dermenkephalin analogs and homologs was investigated for affinity at the mu- and delta-sites in the brain. Whereas dermenkephalin has high affinity and specificity for the delta-opioid receptors, its tetrapeptide amino end, dermenkephalin-[1-4]-NH2 binds almost exclusively at the mu-receptors. Dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, is only marginally more selective for the u-sites than is dermenkephalin-[1-4]-NH2. Using dermorphin-dermenkephalin peptide hybrids and C-terminal deletion analogs of dermenkephalin, we showed the critical role that the C-terminal residues Met6 and Asp7 play in specifying correct addressing of dermenkephalin toward delta-receptors. The potent mu-deteminant located within the amino end of dermenkephalin is over-whelmed by the powerful delta-directing ability of the carboxy end. The negatively charged side chain of Asp7 makes a significant contribution to the delta-addressing ability of the C-terminal region, a finding consistent with Schwyzer's membrane selection model (Schwyzer, R. (1986) Biochemistry 25, 6335-6342). The Leu residue in position 5 and D-configuration about the alpha-carbon of Met2 were found to be of crucial importance for high affinity binding to delta-receptors. Whereas the Met residue in position 6 in dermenkephalin could safely be oxidized or replaced with D-Met, oxidation of Met2 led to deleterious effects, this analog being 1/100 as potent as dermenkephalin at delta-sites. Overall, the data collected demonstrate that highest levels of selectivity and affinity for the delta-opioid receptors can be achieved with small-sized, potentially flexible, linear peptides and further support the model according to which, in addition to optimum accommodation at the receptor, selection for delta-receptors is reduced by the effective positive charge of the molecule. Dermenkephalin may provide a starting point for the design of agonists and antagonists with nearly total specificity for the delta-sites. Such pharmacological agents could be used to explore the ill-defined physiological role and behavioral actions conveyed by delta-opioid receptors.     

Coordination chemistry of Re complexes with 2(2′-pyridyl)benzimidazole

Two new Re(III) and Re(IV) complexes with 2(2′-pyridyl)benzimidazole (pbimz) were prepared and their crystal and molecular structures established by single-crystal X-ray diffraction. Reaction of [ReOCl₂(OEt)(PPh₃)₂] with the ligand gave red cis(Cl),trans(P)-[ReCl₂(PPh₃)₂(pbimz)]Cl (1), while red [ReCl₄(pbimz)] · OPPh₃ (2) was obtained from [ReCl₃(PhC(O)C(O)Ph)(PPh₃)] and pbimz in the presence of perchlorate. The compounds were characterized by elemental analysis, FAB-MS, UV-Vis, IR, NMR spectroscopy and magnetic susceptibility measurements.     

Role of delta-opioid receptor agonists on infarct size reduction in swine

Opioids are involved in cardiac ischemic preconditioning. Important species differences in cellular signaling mechanisms, antiarrhythmic, and antistunning effects have been described. The role of the delta-opioid receptor activation in swine remains unknown. Forty minutes before a 45-min occlusion and 180-min reperfusion of the left anterior descending coronary artery, open-chest, pentobarbital-anesthetized swine received either 1) saline (controls); 2) [D-Ala(2),D-Leu(5)]enkephalin (DADLE); 3) [D-Pen(2,5)]enkephalin (DPDPE); 4) deltorphin-D, a novel delta(2)-opioid agonist; or 5) ischemic preconditioning (IP). Assessed were 1) infarct size to area at risk (IS, triphenyltetrazolium staining), 2) regional and global myocardial function (sonomicrometry, ventricular pressure catheters), and 3) arrhythmias (electrocardiogram analyses). It was found that DPDPE and deltorphin-D pretreatment reduced IS from 64.7 +/- 5 to 36.5 +/- 6% and 27.4 +/- 11% (P < 0.01), respectively, whereas DADLE had no effect (66.8 +/- 3%). Both IP and DADLE had a proarrhythmic effect (P < 0.01). However, no differences in global or regional myocardial function or arrhythmia scores were observed between groups. This suggests that delta-receptor-specific opioids provide cardioprotection in swine.     

The gastric effects of UFP-112, a new nociceptin/orphanin receptor agonist, in physiological and pathological conditions

Nociceptin/orphanin FQ (N/OFQ), the endogenous NOP receptor ligand, centrally modulates gastric motor and secretory functions and prevents ethanol-induced gastric lesions in rats. A recently synthesized N/OFQ analog, [(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-112), acts as a highly potent and selective peptide agonist for NOP receptors and produces longer-lasting in vitro and in vivo effects in mice than the natural ligand N/OFQ. In this study, we evaluated the effects of centrally (intracerebroventricularly/icv) and peripherally (intraperitoneally/ip) injected UFP-112 on gastric emptying and gastric acid secretion, and on the development of gastric mucosal lesions induced by 50% ethanol in the rat. When injected icv, it dose-dependently delayed gastric emptying of a phenol red meal (by up to 70%), decreased gastric secretion in water-loaded rats after 90 pylorus ligature, and reduced ethanol-induced gastric lesions (by up to 87%). In all three assays, UFP-112 was more effective than N/OFQ. The highly selective NOP receptor antagonist, UFP-101, decreased the efficacy of UFP-112, thus confirming that central NOP receptors mediate inhibitory control on these functional and pathological conditions in rats. Ip injected N/OFQ and UFP-112 induced non-dose-related gastric hypersecretory and antiulcer effects, which UFP-101 partially abolished. Ip N/OFQ appeared equiactive but about 30-100 times less potent than ip UFP-112 in stimulating gastric acid secretion and preventing lesion formation. When ip injected, both UFP-112 and N/OFQ left gastric emptying in rats unchanged, suggesting that peripheral NOP receptors have a role in mediating gastric hypersecretory and antiulcer effects but are not involved in regulating gastric motility. In addition, the inhibitory effects induced by this novel NOP receptor agonist lasted longer than those induced by N/OFQ. In conclusion, UFP-112 is a promising new pharmacological tool for studying the functional roles of the central and peripheral N/OFQ receptor system.     

Anti-inflammatory effect of the ghrelin agonist growth hormone-releasing peptide-2 (GHRP-2) in arthritic rats

Chronic arthritis induces hypermetabolism and cachexia. Ghrelin is a gastrointestinal hormone that has been proposed as a treatment to prevent cachexia. The aim of this work was to examine the effect of administration of the ghrelin agonist growth hormone-releasing peptide-2 (GHRP-2) to arthritic rats. Male Wistar rats were injected with Freund's adjuvant, and 15 days later arthritic and control rats were daily injected with GHRP-2 (100 microg/kg) or with saline for 8 days. Arthritis induced an increase in serum ghrelin (P < 0.01) and a decrease in serum concentrations of leptin (P < 0.01), whereas GHRP-2 administration increased serum concentrations of leptin. GHRP-2 increased food intake in control rats but not in arthritic rats. However, in arthritic rats GHRP-2 administration ameliorated the external symptoms of arthritis, as it decreased the arthritis score (10.4 +/- 0.8 vs. 13.42 +/- 0.47, P < 0.01) and the paw volume. In addition, circulating IL-6 and nitrites/nitrates were increased by arthritis, and GHRP-2 treatment decreased the serum IL-6 levels (P < 0.01). To elucidate whether GHRP-2 is able to modulate IL-6 release directly on immune cells, peritoneal macrophage cultures were incubated with GHRP-2 or ghrelin, the endogenous ligand of the growth hormone (GH) secretagogue receptor. Both GHRP-2 (10(-7) M) and ghrelin (10(-7) M) prevented endotoxin-induced IL-6 and decreased nitrite/nitrate release from peritoneal macrophages in vitro. These data suggest that GHRP-2 administration has an anti-inflammatory effect in arthritic rats that seems to be mediated by ghrelin receptors directly on immune cells.     

Membrane partitioning of various delta-opioid receptor forms before and after agonist activations: the effect of cholesterol

Lipid rafts depicted as densely packed and thicker membrane microdomains, based on the dynamic clustering of cholesterol and sphingolipids, may help as platforms involved in a wide variety of cellular processes. The reasons why proteins segregate into rafts are yet to be clarified. The human delta opioid receptor (hDOR) reconstituted in a model system has been characterised after ligand binding by an elongation of its transmembrane part, inducing rearrangement of its lipid microenvironment [Alves, Salamon, Hruby, and Tollin (2005) Biochemistry 44, 9168-9178]. We used hDOR to understand better the correlation between its function and its membrane microdomain localisation. A fusion protein of hDOR with the Green Fluorescent Protein (DOR*) allows precise receptor membrane quantification. Here we report that (i) a fraction of the total receptor pool requires cholesterol for binding activity, (ii) G-proteins stabilize a high affinity state conformation which does not seem modulated by cholesterol. In relation to its distribution, and (iii) a fraction of DOR* is constitutively associated with detergent-resistant membranes (DRM) characterised by an enrichment in lipids and proteins raft markers. (iv) An increase in the quantity of DOR* was observed upon agonist addition. (v) This DRM relocation is prevented by uncoupling the receptor-G-protein interaction.     

Spinal antinociceptive effects of [D-Ala2]deltorphin II, a novel and highly selective delta-opioid receptor agonist.

Pharmacological assays in isolated tissues and binding tests have recently shown that two peptides, with the sequence Tyr-D-Ala-Phe-Asp-(or Glu)- Val-Val-Gly-NH2, isolated from skin extracts of Phyllomedusa bicolor and named [D-Ala2]deltorphin I and II, respectively, possess a higher affinity and selectivity for delta-opioid receptors than any other known natural compound. Since much evidence supports the role of spinal delta-opioid sites in producing antinociceptive effects, we investigated whether analgesia might be detected by direct spinal cord administration of [D-Ala2]deltorphin II (DADELT II) in the rat. The thermal antinociceptive effects of intrathecal DADELT II and dermorphin, a potent mu-selective agonist, were compared at different postinjection times by means of the tail-flick test. The DADELT II produced a dose-related inhibition of the tail-flick response, which lasted 10-60 min depending on the dose and appeared to be of shorter duration than the analgesia produced in rats after intrathecal injection of dermorphin (20-120 min). The analgesic effect of infused or injected DADELT II was completely abolished by naltrindole, the highly selective delta antagonist. These results confirm the involvement of delta receptors in spinal analgesic activity in the rat.     

A pseudopeptide that is a potent cholecystokinin agonist in the peripheral system is able to inhibit the dopamine-like effects of cholecystokinin in the striatum

There are no known specific effective cholecystokinin (CCK) receptor antagonists of both peripheral and central nervous systems. Here, we describe experiments which demonstrate that a synthetic pseudopeptide analogue of CCK-7 is a potent agonist in the peripheral system and behaves as a selective and highly potent inhibitor of the dopamine-like effects of CCK in the striatum. This compound, t-butyloxycarbonyl-Tyr (SO3H)-Nle psi (COCH2)Gly-Trp-Nle-Asp-Phe-NH2, is able to stimulate enzyme secretion from rat pancreatic acini, with high efficacy and potency. It is also very potent in inhibiting the binding of labeled CCK-8 to rat pancreatic acini (IC50 = 5 nM) and to guinea pig and mouse brain membranes (IC50 = 0.7 nM). However, this compound is able to antagonize the effects of intrastriatally injected t-butyloxycarbonyl-[Nle28,31] CCK-8 in mice, with high potency.     

Unusual hexamers and infinite water chains trapped in the complexes of 2-(4-pyridyl)benzimidazole

Unusual hexamers and water chains have been observed in the complexes of (HPyBIm)⁺(Hterephate)⁻(PyBIm) · 4H₂O (1) and [Ag₂(PyBIm)₂(μ₂-SO₄)] · 4H₂O (2), respectively (PyBIm = 2-(4-pyridyl)benzimidazole). In 1, a chair-shaped hexamer (not water hexamer) formed by the water molecules and carboxylate groups as well as one-dimensional water chain are being observed. While in 2, a water hexamer-shaped as parallelogram is obtained; more interestingly, the parallelogram-shaped water hexamers are further aggregated into tape like infinite water chain via hydrogen-bonding interactions.     

Constitutive precoupling to G(i) and increased agonist potency in the alpha(2B)-adrenoceptor

The human alpha(2B)-adrenoceptor (alpha(2B)-AR) was mutated by substituting the D(3.49) aspartate in position 109 with an alanine (alpha(2B)-D109A) in the conserved DRY sequence at the cytoplasmic face of TM3. We studied the effects of the mutation on agonist binding and on receptor activation in CHO cells, including possible inverse agonism monitored by measuring intracellular Ca(2+) concentrations ([Ca(2+)](i)). The mutated receptor had increased binding affinity for agonists, especially dexmedetomidine (3.8-fold). The increased affinity was abolished by pretreatment of the cells with pertussis toxin. The mutation produced constitutive receptor activity evidenced as increased basal [Ca(2+)](i) and increased potency and efficacy of agonists to elicit Ca(2+) responses. The imidazoline derivative RX821002 functioned as an inverse agonist only through the alpha(2B)-D109A, reducing [Ca(2+)](i). The results thus indicate that this mutation causes constitutive receptor-G(i)-protein precoupling, and that the D(3.49) aspartate residue of the DRY motif is involved in controlling coupled and uncoupled conformations of alpha(2B)-AR.     

Stimulation of melanogenesis in murine melanoma cells by 2-mercapto-1-(beta-4-pyridethyl) benzimidazole (MPB)

The effects of 2-mercapto-1-(beta-4-pyridethyl) benzimidazole (MPB), one of the benzimidazole derivatives designed for a nucleic acid analogue, on melanogenesis of murine B16-F10 melanoma cell lines were investigated. MPB (40 microM) induced a striking dendricity in B16 melanoma cells within 12 h and maximal dendricity between 48 and 72 h. The stimulation of melanin synthesis was observed after only 2 days of treatment together with a dose-dependent growth inhibition. Moreover, MPB increased the activity of tyrosinase through the expression of tyrosinase mRNA without increasing the intracellular cyclic AMP content. MPB-induced melanogenesis was inhibited by novel protein kinase A inhibitors, KT-5720 and H-85. These findings indicate that MPB stimulated B16 cells to terminally differentiate and may be a useful drug in studying the regulation of melanogenesis.     

Synthesis, X-ray crystal structures and biomimetic and anticancer activities of novel copper(II)benzoate complexes incorporating 2-(4'-thiazolyl)benzimidazole (thiabendazole), 2-(2-pyridyl)benzimidazole and 1,10-phenanthroline as chelating nitrogen donor ligands

Cu(BZA)(2)(EtOH)(0.5) (1) was generated by the reaction of copper(II) hydroxide with benzoic acid (BZAH). [Cu(TBZH)(2)(BZA)](BZA).0.5TBZH.H(2)O (2) and [Cu(2-PyBZIMH)(2-PyBZIM)(BZA)].1.66EtOH (3) were obtained when 1 reacted with Thiabendazole (TBZH) and 2-(2-pyridyl)benzimidazole (2-PyBZIMH), respectively. [Cu(BZA)(2)(phen)(H(2)O)] (4) was isolated from the reaction of benzoic acid and 1,10-phenanthroline (phen) with copper(II)acetate dihydrate. Molecular structures of 2, 3 and 4 were determined crystallographically. 2 and 3 are hydrogen bonded dimers and trimers, respectively. The copper centres in complexes 2 and 3 are bis-chelate derivatives that have N(4)O ligation and their geometry is very similar being approximately square-pyramidal. However whereas in complex 2 both TBZH ligands are neutral in 3 one of the 2-PyBZIMH chelators is deprotonated on each copper. The structural results for 4 represent a re-examination of this crystallographically known compound for which no hydrogen atom coordinates have been previously reported. It crystallises as a hydrogen bonded dimmer and is a mono-chelate of phen with each copper centre possessing N(2)O(3) ligation and square pyramidal geometry. The catalase and superoxide dismutase (SOD) activities of the four complexes along with those of the known phenanthroline complexes [Cu(mal)(phen)(2)] and [Cu(phendione)(3)](ClO(4))(2) (malH(2)=malonic acid and phendione=1,10-phenanthroline-5,6-dione) were investigated. Complexes 1-4, the metal free ligands and a simple copper(II) salt were assessed for their cancer chemotherapeutic potential against the hepatocellular carcinoma (Hep-G(2)) and kidney adenocarcinoma (A-498) cell lines. TBZH, 2-PyBZIMH and benzoic acid when uncoordinated to a metal centre offer poor chemotherapeutic potential. copper(II) benzoate is significantly more active than the free acid. The bis-chelate derivatives [Cu(TBZH)(2)(BZA)](BZA).0.5TBZH.H(2)O (2) and [Cu(2-PyBZIMH)(2-PyBZIM)(BZA)].1.66EtOH (3) elicit a significant cytotoxic response to the cancer cell lines tested. Replacing TBZH and 2-PyBZIMH with phen to give [Cu(BZA)(2)(phen)(H(2)O)] (4) does not significantly increase the anti-cancer activity.     

Role of TRPM2 in H(2)O(2)-Induced Cell Apoptosis in Endothelial Cells

Melastatin-like transient receptor potential channel 2 (TRPM2) is an oxidant-sensitive and cationic non-selective channel that is expressed in mammalian vascular endothelium. Here we investigated the functional role of TRPM2 channels in hydrogen peroxide (H(2)O(2))-induced cytosolic Ca(2+) ([Ca(2+)](i)) elavation, whole-cell current increase, and apoptotic cell death in murine heart microvessel endothelial cell line H5V. A TRPM2 blocking antibody (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the [Ca(2+)](i) rise and whole-cell current change in response to H(2)O(2). Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect. H(2)O(2)-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect against H(2)O(2)-induced apoptotic cell death. TM2E3 and TRPM2-specific shRNA also protect the cells from tumor necrosis factor (TNF)-α-induced cell death in MTT assay. In contrast, overexpression of TRPM2 in H5V cells resulted in an increased response in [Ca(2+)](i) and whole-cell currents to H(2)O(2). TRPM2 overexpression also aggravated the H(2)O(2)-induced apoptotic cell death. Downstream pathways following TRPM2 activation was examined. Results showed that TRPM2 activity stimulated caspase-8, caspase-9 and caspase-3. These findings strongly suggest that TRPM2 channel mediates cellular Ca(2+) overload in response to H(2)O(2) and contribute to oxidant-induced apoptotic cell death in vascular endothelial cells. Down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death.