Inhibition of poly(ADP-ribose) synthetase (PARS) and protection against peroxynitrite-induced cytotoxicity by zinc chelation

Peroxynitrite, a potent oxidant formed by the reaction of nitric oxide and superoxide causes thymocyte necrosis, in part, via activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS). The cytotoxic PARS pathway initiated by DNA strand breaks and excessive PARS activation has been shown to deplete cellular energy pools, leading to cell necrosis. Here we have investigated the effect of tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) a heavy metal chelator on peroxynitrite-induced cytotoxicity. TPEN (10 microM) abolished cell death induced by authentic peroxynitrite (25 microM) and the peroxynitrite generating agent 3-morpholinosidnonimine (SIN-1, 250 microM). Preincubation of TPEN with equimolar Zn2+ but not Ca2+ or Mg2+ blocked the cytoprotective effect of the chelator. TPEN (10 microM) markedly reduced the peroxynitrite-induced decrease of mitochondrial transmembrane potential, secondary superoxide production and mitochondrial membrane damage, indicating that it acts proximal to mitochondrial alterations. Although TPEN (1 - 300 microM) did not scavenge peroxynitrite, it inhibited PARS activation in a dose-dependent manner. The cytoprotective effect of TPEN is only partly mediated via PARS inhibition, as the chelator also protected PARS-deficient thymocytes from peroxynitrite-induced death. While being cytoprotective against peroxynitrite-induced necrotic death, TPEN (10 microM), similar to other agents that inhibit PARS, enhanced apoptosis (at 5-6 h after exposure), as characterized by phosphatydilserine exposure, caspase activation and DNA fragmentation. In conclusion, the current data demonstrate that TPEN, most likely by zinc chelation, exerts protective effects against peroxynitrite-induced necrosis. Its effects are, in part, mediated by inhibition of PARS.     

Nimodipine and flunarizine have different effects on survival and morphology of PC12 cells during nerve growth factor deprivation

The purpose of this study was to examine the effect of antagonists of different subtypes of Ca(2+) channels (nimodipine and flunarizine) and two types of Ca(2+) chelating agents (the cell permeant Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid acetoxymethylester (BAPTA-AM) and the cell non-permeant Ca(2+) chelator EGTA) on neurite retraction and cell death of nerve growth factor (NGF)-differentiated PC12 cells after NGF deprivation. We demonstrated that flunarizine and nimodipine, but not BAPTA-AM and EGTA, provided protection against cell death due to NGF deprivation. Using time-lapse videomicroscopy and quantitative image analysis, we found that retraction of neurites was an early and fast phenomenon after removal of NGF. None of the compounds tested (flunarizine, nimodipine, BAPTA-AM, EGTA) could prevent the retraction of neurites.     

Involvement of Ca2+ in the inhibition by crocetin of angiotensin II-induced ERK1/2 activation in vascular smooth muscle cells

Crocetin, a carotenoid compound, was isolated from Gardenia jasminoids Ellis. Our recent study shows that crocetin inhibits angiotensin II-induced extracellular signal-regulated kinases 1/2 (ERK1/2) activation and subsequent proliferation in vascular smooth muscle cells (VSMCs). To further explore the mechanism involved, in the present study, we investigated the effect of Ca(2+) in the activation of ERK1/2 and whether Ca(2+) is involved in the suppression by crocetin of angiotensin II-induced ERK1/2 activation. Our findings showed that crocetin pretreatment partially attenuated both the intracellular Ca(2+) mobilization and the extracellular Ca(2+) influx induced by angiotensin II. Moreover, angiotensin II-induced ERK1/2 activation was completely abolished by acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N ',N'-tetraacetic acid (BAPTA-AM), an intracellular Ca(2+) chelator, and partially inhibited by EGTA, an extracellular Ca(2+) chelator, or verapamil, an L-type Ca(2+) channel blocker. These findings suggest that Ca(2+) may play an important role in angiotensin II-induced ERK1/2 activation in VSMCs, and Ca(2+)-dependent pathway may be involved in the inhibitory effect by crocetin of angiotensin II-induced ERK1/2 activation.     

Neuronal death signaling by beta-bungarotoxin through the activation of the N-methyl-D-aspartate (NMDA) receptor and L-type calcium channel

The aim of this study was to elucidate the mechanism of the neurotoxic effect of beta-bungarotoxin (beta-BuTX, a snake presynaptic neurotoxin isolated from the venom of Bungarus multicinctus) on cultured cerebellar granule neurons. beta-BuTX exerted a potent, time-dependent, neurotoxic effect on mature granule neurons. Mature neurons, with an abundance of neurite outgrowths, were obtained after 7-8 days in culture. By means of microspectrofluorimetry and fura-2, we measured the intracellular Ca(2+) concentration ([Ca(2+)](i)) and found it to be increased markedly. BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tertrakis(acetoxymethyl ester)], EGTA, MK801 (dizocilpine maleate), and diltiazem prevented not only the elevation of [Ca(2+)](i), but also the beta-BuTX-induced neurotoxic effect. The signaling pathway involved in the elevation of [Ca(2+)](i) in beta-BuTX-induced neurotoxicity was studied. The results obtained indicated that beta-BuTX initially increased the production of reactive oxygen species and subsequently reduced mitochondrial membrane potential and depleted ATP. All of these events in the signaling pathway were blocked by MK801, diltiazem, EGTA, and BAPTA-AM. These findings suggest that the neurotoxic effect of beta-BuTX is mediated, at least in part, by a cascade of events that include the direct or indirect activation of N-methyl-D-aspartate (NMDA) receptors and L-type calcium channels that, in turn, lead to Ca(2+) influx, oxidative stress, mitochondrial dysfunction, and ATP depletion. Therefore, we suggest that this polypeptide neurotoxin, as a result of its high potency and irreversible properties, is a useful tool to elucidate the mechanisms of neurodegenerative diseases.     

Adrenomedullin stimulates nitric oxide production from primary rat hypothalamic neurons: roles of calcium and phosphatases

Adrenomedullin (ADM) in the brain plays important roles in the maintenance of homeostasis. Although in vivo evidence has suggested that nitric oxide (NO) mediates ADM's effects in the brain, mechanisms for ADM stimulation of NO production in neurons have not been identified. In the present study, primary hypothalamic neurons were used to characterize ADM-induced NO production and to study the underlying mechanisms. Using Calcium Orange/4-amino-5-methylamino-2',7'-difluorofluorescein fluorescence live cell imaging, we found that ADM (1 or 10 nM, 5 min) significantly elevated [Ca(2+)](i) and NO production in a concentration-dependent manner. Ca(2+) and NO responses induced by 10 nM ADM were abolished by pretreatment with 50 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), an intracellular Ca(2+) chelator, or protein kinase A (PKA) inhibitors 5 microM N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and 50 microM Rp-cAMP. Furthermore, the ADM-induced NO production was significantly attenuated by a protein phosphatase 1/2A inhibitor, okadaic acid (OA; 0.1 microM), or calcineurin inhibitors, tacrolimus (FK506) (1 microM) and cyclosporin A (CsA; 0.1 microM). Using Western blotting, we found that ADM significantly decreased phosphorylation of neuronal nitric-oxide synthase (nNOS) at serine 847. This dephosphorylation was inhibited by 0.1 microM OA, 1 microM FK506, 0.1 microM CsA, or 5 microM H-89, and attenuated by 50 microM BAPTA-AM. These results suggest that, in hypothalamic neurons, ADM elevates [Ca(2+)](i) via PKA-associated mechanisms. The PKA/Ca(2+) cascade leads to protein phosphatase (PP) 1/PP2A- and calcineurin-mediated dephosphorylation of nNOS. We hypothesize that the Ca(2+) increase and nNOS dephosphorylation contribute to activation of nNOS and production of NO in hypothalamic neurons.     

Calpain activation contributes to oxidative stress-induced pancreatic acinar cell injury

Oxygen radicals have been implicated as mediators in the pathogenesis of pancreatic acinar cell necrosis. However, the sequence of events between the oxidative insult and cell damage remains unclear. In the current study, we investigated whether the Ca(2+)-regulated cytosolic cysteine protease calpain is activated by oxidative stress and contributes to oxidant-induced acinar cell damage. Isolated rat pancreatic acinar cells were exposed to hydrogen peroxide (H(2)O(2))-generated oxidative stress in the presence or absence of the Ca(2+) chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) and different calpain inhibitors including benzyloxycarbonyl-valyl-phenylalanine methyl ester. Calpain activation was studied by fluorescence spectrophotometry and immunoblotting. Cell injury was assessed by lactate dehydrogenase (LDH) release and characterization of the cellular ultrastructure including fluorescence-labeled actin filaments. Exposure of acinar cells to H(2)O(2) provoked a time- and dose-dependent increase in calpain proteolytic activity involving the ubiquitous isoforms mu- and m-calpain. The activation of calpain reflected the time course of developing cytotoxicity as demonstrated by increased LDH release. Inhibition of oxidant-induced calpain activity by BAPTA-AM and various calpain inhibitors provoked a decline in oxidant-induced cell injury. In particular, changes in the actin filament organization characterized by an increase in the basolateral actin and by a detachment of actin from the cell membrane in the region of membrane blebs were clearly reduced. In summary, our findings suggest that acinar cell damage through oxidative stress requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease. The results support the hypothesis that calpain activation may play a role in the development of acute pancreatitis.     

Na+ influx-induced decrease of (Na+ + K+)-ATPase activity in rat brain slices: role of Ca2+.

Treatment of rat brain slices with veratrine and monensin decreased (Na+ + K+)-ATPase activity in the membranes in a dose-dependent manner. The effect of monensin, like that of veratrine, was accompanied by a decrease of maximal binding sites for ouabain. The inhibitory effect of monensin on the enzyme activity was dependent on external Ca2+ at low concentrations, but not at a high concentration. The decreased enzyme activity induced by monensin was restored by subsequent incubation of the slices in a Ca(2+)-free medium containing 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM), a chelator of intracellular Ca2+. The effect of monensin at a low concentration on enzyme activity was antagonized by amiloride (1 mM), bepridil (5 microM), quinacrine (30 microM) or verapamil (30 microM), but not by nifedipine (1 microM) or omega-conotoxin (1 microM). Furthermore, the inhibitory effect of monensin at a high concentration under Ca(2+)-free conditions was blocked by BAPTA-AM (30 microM) and by bepridil (100 microM) or diazepam (500 microM), inhibitors of mitochondrial Na(+)-Ca2+ exchange. Inhibitors of calmodulin, protein kinase C, phospholipase A2 and calpain did not affect the monensin-induced decrease of enzyme activity. Dithiothreitol (10 mM) blocked the effect of monensin on enzyme activity but did not affect the ionophore-induced influx of Ca2+ in the slices.     

Critical role of calcium overloading in cadmium-induced apoptosis in mouse thymocytes

Cadmium (Cd) is a well-known environmental carcinogen and immunotoxin. Currently the direct cytotoxic effects of Cd on thymocytes are largely unexplored. The main objective of the present study was to investigate the apoptogenic property of Cd and the mechanisms involved, using primary cultured mouse thymocytes as a model. Cd-induced apoptosis in thymocytes was studied by TdT-mediated dUTP nick end-labeling assay and DNA gel electrophoresis. The results showed that Cd was able to cause apoptosis in mouse thymocytes in a time- and dose-dependent manner. Moreover, Cd exposure led to a rapid and sustained intracellular calcium (Ca2+) elevation, followed by caspase-3 activation and PARP cleavage, all of which preceded the characteristic DNA fragmentation. BAPTA-AM, a specific intracellular Ca2+ chelator, abolished Cd-induced Ca2+ overloading and subsequently inhibited caspase-3 activation, PARP cleavage, and apoptosis.It is believed that intracellular Ca2+ elevation may trigger caspase-3 activation either through mitochondria or through activation of Ca2+-dependent protease in Cd-treated thymocytes. Results from this study thus provide new information for a better understanding of the immunotoxic and immunomodulatory effects of Cd.     

Increase in intracellular Ca(2+) concentrations and the corresponding intracellular acidification are early steps for induction of apoptosis by geranylgeraniol in HL60 cells

To elucidate the mechanism of induction of apoptosis by geranylgeraniol (GGO), which is a potent inducer of apoptosis in various lines of human cancer cells, we examined the role of intracellular acidification during GGO-induced apoptosis using human leukemia HL60 cells. Flow cytometry analysis revealed that apoptosis induced in human leukemia HL60 cells by GGO was associated with intracellular acidification. Both GGO-induced intracellular acidification and apoptosis as analyzed by DNA fragmentation were inhibited by phorbol myristate acetate (TPA) and O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM), an intracellular Ca(2+) chelator, but not by ethyleneglycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). These results suggest that the early concentration change of intracellular Ca(2+) and the corresponding decrease in intracellular pH are required for the induction of apoptosis in HL60 cells by GGO.     

Ca2+-permeable AMPA receptors and intracellular Ca2+ determine motoneuron vulnerability in rat spinal cord in vivo

Excitotoxicity mediated by overactivation of glutamate receptors, particularly the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) type, has been implicated in motoneuron degeneration. AMPA receptors lacking the GluR2 subunit are permeable to Ca(2+) and the entrance of this cation might be responsible for the selective vulnerability of spinal motoneurons in amyotrophic lateral sclerosis (ALS). To evaluate this hypothesis in vivo, we have used a model of motoneuron death in which AMPA, perfused by microdialysis in the rat lumbar spinal cord, produces ipsilateral paralysis and a remarkable loss of spinal motoneurons. Perfusion of 1-naphthyl acetyl spermine, a selective blocker of the Ca(2+)-permeable AMPA receptors, and of the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), prevented the AMPA-induced paralysis and reduced by about 50% the loss of motoneurons. In addition, perfusion of pyruvate, an energy metabolic substrate, similarly prevented the paralysis and the motoneuron death. These results suggest that functional AMPA receptors lacking the GluR2 subunit are present in the rat spinal cord, and that motoneuron death is triggered by an increase of intracellular Ca(2+) via such Ca(2+)-permeable AMPA receptors. Our finding that pyruvate also protected against the excitotoxic effects of AMPA suggests that the increased intracellular Ca(2+) probably interferes with the mitochondrial energetic metabolism.     

Calcium-mediated activation of c-Jun NH2-terminal kinase (JNK) and apoptosis in response to cadmium in murine macrophages.

Cadmium is a well-known carcinogenic and immunotoxic metal commonly found in cigarette smoke and industrial effluent. An altered intracellular calcium ([Ca(2+)](i)) level has been implicated in the pathophysiology of immune dysfunction. The present study was designed to determine the possible involvement of calcium (Ca(2+)) and mitogen-activated protein kinases (MAPKs) signaling pathways on cadmium-induced cell death in J774A.1 murine macrophage cells. Cadmium caused a low-amplitude [Ca(2+)](i) elevation at 20 microM and rapid and high-amplitude [Ca(2+)](i) elevation at 500 microM. Exposure to cadmium dose-dependently induced phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and deactivated p38 MAPK. Use of the selective JNK inhibitor SP600125 suggested that activation of JNK is pro-apoptotic and pro-necrotic. Buffering of the calcium response with 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxy-methyl) ester (BAPTA-AM) and ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) completely blocked cadmium-induced apoptotic response. The pretreatment of cells with BAPTA-AM and EGTA suppressed the cadmium-induced cell injury, including growth arrest, mitochondrial activity impairment, and necrosis, and it also recovered the cadmium-altered JNK and p38 MAPK activity. Chelating [Ca(2+)](i) also reversed cadmium-induced hydrogen peroxide generation, suggesting that production of reactive oxygen species (ROS) is related to [Ca(2+)](i). The present study showed that cadmium induces a [Ca(2+)](i)-ROS-JNK-caspase-3 signaling pathway leading to apoptosis. Furthermore, cadmium-induced [Ca(2+)](i) regulates phosphorylation/dephosphorylation of JNK and p38, and it modulates signal transduction pathways to proliferation, mitochondrial activity, and necrosis.     

Inhibitory effects of loading with the calcium-chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) on amylase release and cellular ATP level in rat parotid cells.

Rat parotid cells were loaded with the Ca2(+)-chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), by incubation with the acetoxymethyl ester of BAPTA (BAPTA-AM). The BAPTA loading inhibited amylase release induced by beta-adrenergic receptor stimulation without affecting the basal release, and the IC50 value was 25 microM. Incubation of cells with BAPTA-AM also suppressed cellular ATP levels considerably. At 100 microM BAPTA-AM, the ATP level fell to about 50% of the control. A decrease in ATP levels by incubation with oligomycin, a mitochondrial inhibitor, correlated well with the inhibition of amylase release. Although these results do not exclude the possibility that cytosolic free Ca2+ is involved in the regulation of cyclic AMP-mediated amylase release, they suggest that the amylase release inhibition with BAPTA loading may be due, in part, to a decrease in cellular ATP levels. Therefore, the exact mode of the BAPTA action must be interpreted with caution.     

On the mechanism of levosimendan-induced dopamine release in the striatum of freely moving rats

The Ca(2+) sensitizer levosimendan (LEV) improves myocardial contractility by enhancing the sensitivity of the contractile apparatus to Ca(2+). In addition, LEV promotes Ca(2+) entry through L-type channels in human cardiac myocytes. In this study, which was performed using microdialysis, infusion of LEV at 0.25 microM for 160 min increased dopamine (DA) concentrations (up to fivefold baseline) in dialysates from the striatum of freely moving rats. Ca(2+) omission from the perfusion fluid abolished baseline DA release and greatly decreased LEV-induced DA release. Reintroduction of Ca(2+) in the perfusion fluid restored LEV-induced DA release. Chelation of intracellular Ca(2+) by co-infusing 1,2-bis (o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM, 0.2 mM) did not affect basal DA release and scarcely affected LEV-induced increases in dialysate DA. In addition, co-infusion of the L-type (Ca(v) 1.1-1.3) voltage-sensitive Ca(2+)-channel inhibitor nifedipine failed to inhibit LEV-induced increases in dialysate DA, which, in contrast, was inhibited by co-infusion of the N-type (Ca(v) 2.2) voltage-sensitive Ca(2+)-channel inhibitor omega-conotoxin GVIA. We conclude that LEV promotes striatal extracellular Ca(2+) entry through N-type Ca(2+) channels with a consequent increase in DA release.     

Protein tyrosine nitration and poly(ADP-ribose) polymerase activation in N-methyl-N-nitro-N-nitrosoguanidine-treated thymocytes: implication for cytotoxicity

1-Methyl-3-nitro-1-nitrosoguanidine (MNNG) is a DNA alkylating agent. DNA alkylation by MNNG is known to trigger accelerated poly(ADP-ribose) metabolism. Various nitroso compounds release nitric oxide (NO). Therefore, we set out to investigate whether MNNG functions as NO donor and whether MNNG-derived NO or secondary NO metabolites such as peroxynitrite contribute to MNNG-induced cytotoxicity. MNNG in aqueous solutions resulted in time- and concentration-dependent NO release and nitrite/nitrate formation. Moreover, various proteins in MNNG-treated thymocytes were found to be nitrated, indicating that MNNG-derived NO may combine with cellular superoxide to form peroxynitrite, a nitrating agent. MNNG also caused DNA breakage and increased poly(ADP-ribose) polymerase activity and cytotoxicity in thymocytes. MNNG-induced DNA damage (measured by the comet assay) and thymocyte death (measured by propidium iodide uptake) was prevented by the PARP inhibitor PJ-34 and by glutathione (GSH) or N-acetylcysteine (NAC). The cytoprotection provided by PJ-34 against necrotic parameters was paralleled by increased outputs in apoptotic parameters (caspase activity, DNA laddering) indicating that PARP activation diverts apoptotic death toward necrosis. As MNNG-induced cytotoxicity showed many similarities to peroxynitrite-induced cell death, we tested whether peroxynitrite was responsible for at least part of the cytotoxicity induced by MNNG. Cell-permeable enzymic antioxidants (superoxide dismutase and catalase), the NO scavenger cPTIO or the peroxynitrite decomposition catalyst FP15 failed to inhibit MNNG-induced DNA breakage and cytotoxicity. In conclusion, MNNG induces tyrosine nitration in thymocytes. Furthermore, MNNG damages DNA by a radical mechanism that does not involve NO or peroxynitrite.     

Intracellular Ca2+ regulates amphetamine-induced dopamine efflux and currents mediated by the human dopamine transporter

Although it is clear that amphetamine-induced dopamine (DA) release mediated by the dopamine transporter (DAT) is integral to the behavioral actions of this psychostimulant, the mechanism of this release is not clear. In this study, we explored the requirement for intracellular Ca(2+) in amphetamine-induced DA efflux and currents mediated by the human DAT. The patch-clamp technique in the whole-cell configuration was used on Na(+) and DA-preloaded human embryonic kidney 293 cells stably transfected with the human DAT (hDAT cells). Chelation of intracellular Ca(2+) by inclusion of 50 microM BAPTA in the whole-cell pipette reduced the voltage-dependent amphetamine-induced hDAT current, with the greatest effect seen at positive voltages. Likewise, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) reduced amphetamine-induced DA efflux as measured by amperometry. Furthermore, preincubation of the cells with 50 microM BAPTA acetoxy methyl ester (AM) or thapsigargin also blocked amphetamine-induced release of preloaded N-methyl-4-[(3)H]phenylpyridinium from superfused hDAT cells. BAPTA-AM also reduced DA release from striatal synaptosomes. Amphetamine also led to an increase in intracellular Ca(2+) that was blocked by prior treatment with 5 microM thapsigargin or 10 microM cocaine. These studies demonstrate that amphetamine-induced DAT-mediated currents and substrate efflux require internal Ca(2+) and that amphetamine can stimulate dopamine efflux by regulating cytoplasmic Ca(2+) levels through its interaction with DAT.     

Events that precede and that follow S-(1,2-dichlorovinyl)-L-cysteine-induced release of mitochondrial Ca2+ and their association with cytotoxicity to renal cells.

Previous studies showed that S-(1,2-dichlorovinyl)-L-cysteine perturbs intracellular Ca2+ homeostasis [Vamvakas et al., Mol Pharmacol 38: 455-461, 1990]. The objective of the present study was to investigate the cellular events that precede and that follow S-(1,2-dichlorovinyl)-L-cysteine-induced mitochondrial Ca2+ release. In incubations with isolated kidney mitochondria, S-(1,2-dichlorovinyl)-L-cysteine-induced Ca2+ efflux is preceded by increased oxidation of mitochondrial pyridine nucleotides and is prevented by ATP, an inhibitor of the hydrolysis of pyridine nucleotides, and by meta-iodobenzylguanidine, an acceptor of ADP-ribose moieties. In LLC-PK1 cells, elevation in the cytosolic Ca2+ concentration is followed by a several-fold increase in DNA double-strand breaks which is attributed to the activation of Ca2+- and Mg(2+)-dependent endonucleases. The formation of DNA double-strand breaks is followed by increased poly(ADP-ribosylation) of nuclear proteins. S-(1,2-Dichlorovinyl)-L-cysteine-induced cytotoxicity in LLC-PK1 cells is blocked by chelation of cytosolic Ca2+ with Quin-2, by inhibition of DNA fragmentation with aurintricarboxylic acid and by inhibition of increased poly(ADP-ribosyl)transferase activity by 3-aminobenzamide. These findings indicate that S-(1,2-dichlorovinyl)-L-cysteine bioactivation in renal cells may initiate the following cascade of events: increased oxidation and hydrolysis of mitochondrial pyridine nucleotides resulting in the modification of mitochondrial membrane proteins by pyridine nucleotide-derived ADP-ribose moieties, followed by Ca2+ release. Elevated Ca2+ concentrations may activate Ca(2+)-dependent endonucleases, which leads to DNA fragmentation followed by increased poly(ADP-ribosylation) of nuclear proteins and, finally, cytotoxicity.     

Calcium associated resistance to H(2)O(2) in Chinese hamster V79 cells

To investigate whether the difference in cellular sensitivity of Chinese hamster V79 and their H(2)O(2)-resistant variant cells (Hpr-4) to H(2)O(2) relates to the difference in intracellular Ca(2+) concentration in these cells, we measured Ca(2+) concentration by calcium ion analysis after loading these cells with Fura-2/AM. Intracellular Ca(2+) concentration increased in both Chinese hamster V79 and Hpr-4 cells as extracellular Ca(2+) concentration increased. However, the increase in intracellular Ca(2+) concentration in response to extracellular H(2)O(2) was more pronounced in Hpr-4 than V79 cells. H(2)O(2) cytotoxicity of Hpr-4 but not V79 cells was also decreased in response to the increase in extracellular Ca(2+) concentration. In parallel with the decrease in cytotoxicity in response to increasing extracellular Ca(2+) concentration, the frequency of mitochondrial DNA single strand breaks (SSB) in Hpr-4 cells also decreased without producing observable nuclear DNA SSB. Use of permeabilized V79 and Hpr-4 cells exposed to H(2)O(2) showed that mitochondrial DNA SSB decreased when extramitochondrial Ca(2+) concentration increased. These findings indicate that elevated intracellular Ca(2+) concentration may protect against H(2)O(2)-induced mitochondrial damage and cytotoxicity in these cells.     

Induction of vasorelaxation through activation of nitric oxide synthase in endothelial cells by brazilin

The vasorelaxant activity of Caesalpinia sappan L., a traditional Chinese medicine, and its major component brazilin were investigated in isolated rat aorta and human umbilical vein endothelial cells. In isolated rat aorta, C. sappan L. extract and brazilin relaxed phenylephrine-induced vasocontraction and increased cyclic guanosine 3',5'-monophosphate (cGMP) content. Induction of vasorelaxation of brazilin was endothelium-dependent and could be markedly blocked by pretreatment with nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME); N(G)-monomethyl-L-arginine acetate (L-NMMA) and guanylyl cyclase inhibitor, methylene blue; 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and nitric oxide (NO) scavenger, hemoglobin. The increasing cGMP content induced by brazilin was also blocked by pretreatment with L-NAME, methylene blue, and the removal of extracellular Ca(2+). In human umbilical vein endothelial cells, brazilin dose-dependently induced an increase in NO formation and NOS activity, which were greatly attenuated by either the removal of extracellular Ca(2+) or the chelating of intracellular Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Moreover, brazilin dose-dependently induced the influx of extracellular Ca(2+) in human umbilical vein endothelial cells. Collectively, these results suggest that brazilin induces vasorelaxation by the increasing intracellular Ca(2+) concentration in endothelial cells of blood vessels and hence activating Ca(2+)/calmodulin-dependent NO synthesis. The NO is released and then transferred into smooth muscle cells to activate guanylyl cyclase and increase cGMP content, resulting in vasorelaxation.     

Ca2+ channel blocking effect of iso-S-petasin in rat aortic smooth muscle cells

The purpose of the present study was to examine the mechanisms underlying the putative hypotensive actions of iso-S-petasin, a sesquiterpene extract of Petasites formosanus through both in vivo and in vitro experiments. Intravenous administration of iso-S-petasin elicited dose-dependent (0.1-1.5 mg/kg) hypotensive and bradycardiac responses in anesthetized rats. Isometric tension recording in isolated thoracic aorta revealed that iso-S-petasin (0.01-100 microM) inhibited KCl- or Bay K 8644 (1,4-dihydro-2,6-dimethyl-5-nitro-4-[2'-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid methyl ester)-induced vasoconstriction independent of endothelium. Iso-S-Petasin also attenuated Ca(2+)-induced vasoconstriction in a concentration-dependent manner in Ca(2+)-depleted/high K(+)-depolarized ring segments, indicating that iso-S-petasin inhibited Ca(2+) influx into vascular smooth muscle cells. This was confirmed by whole-cell patch-clamp recording in cultured vascular smooth muscle cells where iso-S-petasin (10-100 microM) appeared to directly inhibit the L-type voltage-dependent Ca(2+) channel (VDCC) activity. Intracellular Ca(2+) concentration ([Ca(2+)](i)) measurements using the fluorescent probe fura-2/AM (1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-amino-5'-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid pentaacetoxymethyl ester) showed suppression of the KCl-stimulated increase in [Ca(2+)](i) by iso-S-petasin (10, 100 microM). In conclusion, these results suggest that Ca(2+) antagonism of the L-type VDCC in vascular smooth muscle cells might largely account for the hypotensive action of iso-S-petasin.