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M A Venkatachalam

Publications and source records attributed to M A Venkatachalam.

At least 19 recordsLinked to original sources
PubMed2001

cDNA cloning and promoter analysis of rat caspase-9.

Caspase-9 is the apex caspase of the mitochondrial pathway of apoptosis, which plays a critical role in apoptotic initiation and progression. However, gene regulation of caspase-9 is largely unknown. This is in part due to the lack of information on the gene promoter. Here we have cloned the full-length cDNA of rat caspase-9 and have isolated promoter regions of this gene. The rat caspase-9 cDNA of 2058 bp predicts a protein of 454 amino acids, which contains a caspase-recruitment domain ('CARD') at the N-terminus and enzymic domains at the C-terminus. The enzyme's active site, with a characteristic motif of QACGG, was also identified. Overall, rat and human caspase-9 have 71% identity. With the cDNA sequence, we subsequently isolated the proximal 5'-flanking regions of rat caspase-9 by the procedure of genomic walking. The 2270 bp genomic segment is 'TATA-less', but contains several GC boxes. Elements binding known transcription factors such as Sp-1, Pit-1, CCAAT-enhancer-binding protein (C/EBP), glucocorticoid receptor and hypoxia-inducible factor 1 (HIF-1) were also identified. When cloned into reporter gene vectors, the genomic segment showed significant promoter activity, indicating that the 5'-flanking regions isolated by genomic walking contain the gene promoter of rat caspase-9. Of significance is that the cloned promoter segments were activated by severe hypoxia, conditions inducing caspase-9 transcription. Thus, the genomic sequences reported here contain not only the basal promoter of rat caspase-9 but also regulatory elements responsive to pathophysiological stimuli including hypoxia.

3T3 Cells↗
PubMed2001

Up-regulation of apoptosis inhibitory protein IAP-2 by hypoxia. Hif-1-independent mechanisms.

Hypoxia is a key determinant of tissue pathology during tumor development and organ ischemia. However, little is known regarding hypoxic regulation of genes that are directly involved in cell death or death resistance. Here we report the striking induction by severe hypoxia of the anti-apoptotic protein IAP-2. Hypoxic cells with IAP-2 up-regulation became resistant to apoptosis. IAP-2 was induced by hypoxia per se rather than by the secondary effects of hypoxia, including ATP depletion and cell injury. The inductive response did not relate to alterations of cellular redox status or arrest of mitochondrial respiration. On the other hand, IAP-2 induction was attenuated by actinomycin D, suggesting a role for gene transcription. In vitro nuclear run-on assays demonstrated specific increases in IAP-2 transcriptional activity after hypoxia exposure. HIF-1, the primary transcription factor that is responsible for multiple gene activation under hypoxia, does not have a role in IAP-2 expression. HIF-1 and IAP-2 were induced by different degrees of hypoxia; severe hypoxia or anoxia was required for IAP-2 induction. Moreover, cobalt chloride and desferrioxamine activated HIF-1 but not IAP-2. Finally, IAP-2 was induced by severe hypoxia in mouse embryonic stem cells that were deficient of HIF-1. Thus, this study not only provides the first demonstration of hypoxic regulation of an anti-apoptotic gene but also suggests the participation of novel hypoxia-responsive transcription mechanisms.

3T3 Cells↗
PubMed2001

Bcl-2 prevents Bax oligomerization in the mitochondrial outer membrane.

ATP depletion results in Bax translocation from cytosol to mitochondria and release of cytochrome c from mitochondria into cytosol in cultured kidney cells. Overexpression of Bcl-2 prevents cytochrome c release, without ameliorating ATP depletion or Bax translocation, with little or no association between Bcl-2 and Bax as demonstrated by immunoprecipitation (Saikumar, P., Dong, Z., Patel, Y., Hall, K., Hopfer, U., Weinberg, J. M., and Venkatachalam, M. A. (1998) Oncogene 17, 3401-3415). Now we show that translocated Bax forms homo-oligomeric structures, stabilized as chemical adducts by bifunctional cross-linkers in ATP-depleted wild type cells, but remains monomeric in Bcl-2-overexpressing cells. The protective effects of Bcl-2 did not require Bcl-2/Bax association, at least to a degree of proximity or affinity that was stable to conditions of immunoprecipitation or adduct formation by eight cross-linkers of diverse spacer lengths and chemical reactivities. On the other hand, nonionic detergents readily induced homodimers and heterodimers of Bax and Bcl-2. Moreover, associations between translocated Bax and the voltage-dependent anion channel protein or the adenine nucleotide translocator protein could not be demonstrated by immunoprecipitation of Bax, or by using bifunctional cross-linkers. Our data suggest that the in vivo actions of Bax are at least in part dependent on the formation of homo-oligomers without requiring associations with other molecules and that Bcl-2 cytoprotection involves mechanisms that prevent Bax oligomerization.

Animals↗
PubMed2001

Protection of ATP-depleted cells by impermeant strychnine derivatives: implications for glycine cytoprotection.

Glycine and structurally related amino acids with activities at chloride channel receptors in the central nervous system also have robust protective effects against cell injury by ATP depletion. The glycine receptor antagonist strychnine shares this protective activity. An essential step toward identification of the molecular targets for these compounds is to determine whether they protect cells through interactions with intracellular targets or with molecules on the outer surface of plasma membranes. Here we report cytoprotection by a cell-impermeant derivative of strychnine. A strychnine-fluorescein conjugate (SF) was synthesized, and impermeability of plasma membranes to this compound was verified by fluorescence confocal microscopy. In an injury model of Madin-Darby canine kidney cells, ATP depletion led to lactate dehydrogenase release. SF prevented lactate dehydrogenase leakage without ameliorating ATP depletion. This was accompanied by preservation of cellular ultrastructure and exclusion of vital dyes. SF protection was also shown for ATP-depleted rat hepatocytes. On the other hand, when a key structural motif in the active site of strychnine was chemically blocked, the SF lost its protective effect, establishing strychnine-related specificity for SF protection. Cytoprotective effects of the cell-impermeant strychnine derivative provide compelling evidence suggesting that molecular targets on the outer surface of plasma membranes may mediate cytoprotection by strychnine and glycine.

Adenosine Triphosphate↗
PubMed2001

Energetic determinants of tyrosine phosphorylation of focal adhesion proteins during hypoxia/reoxygenation of kidney proximal tubules.

Anaerobic mitochondrial metabolism of alpha-ketoglutarate and aspartate or alpha-ketoglutarate and malate can prevent and reverse severe mitochondrial dysfunction during reoxygenation after 60 minutes of hypoxia in kidney proximal tubules.(34) The present studies demonstrate that, during hypoxia, paxillin, focal adhesion kinase, and p130(cas) migrated faster by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their phosphotyrosine (pY) content decreased to approximately 5% of that in oxygenated tubules without changes in total protein, and the normally basal immunostaining of beta1 and alpha6 integrin subunits, pY, and paxillin was lost or markedly decreased. During reoxygenation without supplemental substrates, recovery of pY and basal localization of the focal adhesion proteins was poor. alpha-Ketoglutarate and aspartate, which maintained slightly higher levels of ATP during hypoxia, also maintained 2.5-fold higher levels of pY during this period, and promoted full recovery of pY content and basal localization of focal adhesion proteins during subsequent reoxygenation. Similarly complete recovery was made possible by provision of alpha-ketoglutarate and aspartate or alpha-ketoglutarate and malate only during reoxygenation. These data emphasize the importance of very low energy thresholds for maintaining the integrity of key structural and biochemical components required for cellular survival and reaffirm the value of approaches aimed at conserving or generating energy in cells injured by hypoxia or ischemia.

Adenosine Triphosphate↗
PubMed2000

Serine protease inhibitors suppress cytochrome c-mediatedcaspase-9 activation and apoptosis during hypoxia-reoxygenation.

We have shown that reoxygenation of hypoxic rat kidney proximaltubule cells leads to apoptosis. This is mediated by translocation ofBax from the cytosol to mitochondria, accompanied by release ofmitochondrial cytochrome c (cyt.c). The present studyhas examined the proteolytic mechanisms responsible for apoptosisduring hypoxia-reoxygenation. Caspases were activated duringhypoxia, as shown by cleavage of fluorogenic peptide substrates. By5 h caspase-3-like activity to cleave carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin was increased approx. 30-fold. Thiswas accompanied by specific processing of pro-caspase-3, -8 and -9 intoactive forms. Caspase activation during hypoxia was blocked bycarbobenzoxy-Val-Ala-Asp-fluoromethyl ketone and overexpression of Bcl-2. Of particular interest, caspase activation was also suppressed bythe chymotryptic inhibitors N-tosyl-L-phenylalaninechloromethyl ketone (TPCK) and Ala-Pro-Phe chloromethyl ketone (APF),and the general serine protease inhibitor 4-(2-aminoethyl)benzenesulphonyl fluoride. Inhibition of caspase activationby these compounds resulted in arrest of apoptosis. On the other hand,the serine protease inhibitors did not prevent release of mitochondrialcyt.c during hypoxia, suggesting that these compounds blockeda critical step in post-mitochondrial caspase activation. Furtherstudies using an in vitro reconstitution model showedthat cyt. c/dATP stimulated caspase-9 processing and downstreamcaspase activation were significantly suppressed in the presence ofTPCK and APF. Based on these results, we speculate that serineproteases may be involved in post-mitochondrial apoptotic events thatlead to activation of the initiator, caspase-9.

Adenosine Triphosphate↗
PubMed2000

Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates.

Kidney proximal tubule cells developed severe energy deficits during hypoxia/reoxygenation not attributable to cellular disruption, lack of purine precursors, the mitochondrial permeability transition, or loss of cytochrome c. Reoxygenated cells showed decreased respiration with complex I substrates, but minimal or no impairment with electron donors at complexes II and IV. This was accompanied by diminished mitochondrial membrane potential (DeltaPsi(m)). The energy deficit, respiratory inhibition, and loss of DeltaPsi(m) were strongly ameliorated by provision of alpha-ketoglutarate plus aspartate (alphaKG/ASP) supplements during either hypoxia or only during reoxygenation. Measurements of (13)C-labeled metabolites in [3-(13)C]aspartate-treated cells indicated the operation of anaerobic pathways of alphaKG/ASP metabolism to generate ATP, yielding succinate as end product. Anaerobic metabolism of alphaKG/ASP also mitigated the loss of DeltaPsi(m) that occurred during hypoxia before reoxygenation. Rotenone, but not antimycin or oligomycin, prevented this effect, indicating that electron transport in complex I, rather than F(1)F(0)-ATPase activity, had been responsible for maintenance of DeltaPsi(m) by the substrates. Thus, tubule cells subjected to hypoxia/reoxygenation can have persistent energy deficits associated with complex I dysfunction for substantial periods of time before onset of the mitochondrial permeability transition and/or loss of cytochrome c. The lesion can be prevented or reversed by citric acid cycle metabolites that anaerobically generate ATP by intramitochondrial substrate-level phosphorylation and maintain DeltaPsi(m) via electron transport in complex I. Utilization of these anaerobic pathways of mitochondrial energy metabolism known to be present in other mammalian tissues may provide strategies to limit mitochondrial dysfunction and allow cellular repair before the onset of irreversible injury by ischemia or hypoxia.

Adenosine Triphosphate↗
PubMed2000

Anaerobic and aerobic pathways for salvage of proximal tubules from hypoxia-induced mitochondrial injury.

We have further examined the mechanisms for a severe mitochondrial energetic deficit, deenergization, and impaired respiration in complex I that develop in kidney proximal tubules during hypoxia-reoxygenation, and their prevention and reversal by supplementation with alpha-ketoglutarate (alpha-KG) + aspartate. The abnormalities preceded the mitochondrial permeability transition and cytochrome c loss. Anaerobic metabolism of alpha-KG + aspartate generated ATP and maintained mitochondrial membrane potential. Other citric-acid cycle intermediates that can promote anaerobic metabolism (malate and fumarate) were also effective singly or in combination with alpha-KG. Succinate, the end product of these anaerobic pathways that can bypass complex I, was not protective when provided only during hypoxia. However, during reoxygenation, succinate also rescued the tubules, and its benefit, like that of alpha-KG + malate, persisted after the extra substrate was withdrawn. Thus proximal tubules can be salvaged from hypoxia-reoxygenation mitochondrial injury by both anaerobic metabolism of citric-acid cycle intermediates and aerobic metabolism of succinate. These results bear on the understanding of a fundamental mode of mitochondrial dysfunction during tubule injury and on strategies to prevent and reverse it.

Adenosine Triphosphate↗
PubMed1998

Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury.

We investigated mechanisms of cell death during hypoxia/reoxygenation of cultured kidney cells. During glucose-free hypoxia, cell ATP levels declined steeply resulting in the translocation of Bax from cytosol to mitochondria. Concurrently, there was cytochrome c release and caspase activation. Cells that leaked cytochrome c underwent apoptosis after reoxygenation. ATP depletion induced by a mitochondrial uncoupler resulted in similar alterations even in the presence of oxygen. Moreover, inclusion of glucose during hypoxia prevented protein translocations and reoxygenation injury by maintaining intracellular ATP. Thus, ATP depletion, rather than hypoxia per se, was the cause of protein translocations. Overexpression of Bcl-2 prevented cytochrome c release and reoxygenation injury without ameliorating ATP depletion or Bax translocation. On the other hand, caspase inhibitors did not prevent protein translocations, but inhibited apoptosis during reoxygenation. Nevertheless, they could not confer long-term viability, since mitochondria had been damaged. Omission of glucose during reoxygenation resulted in continued failure of ATP production, and cell death with necrotic morphology. In contrast, cells expressing Bcl-2 had functional mitochondria and remained viable during reoxygenation even without glucose. Therefore, Bax translocation during hypoxia is a molecular trigger for cell death during reoxygenation. If ATP is available during reoxygenation, apoptosis develops; otherwise, death occurs by necrosis. By preserving mitochondrial integrity, BCL-2 prevents both forms of cell death and ensures cell viability.

Adenosine Triphosphate↗
PubMed1998

Mechanisms of cell death in hypoxia/reoxygenation injury.

Investigation of death pathways during cell injury in vivo caused by ischemia and reperfusion is of clinical importance, but technically difficult. Heterogeneity of cell types, differences between organ systems, diversity of death paradigms and exacerbation of tissue damage caused by inflammation are only some of the variables that need to be taken into account. With respect to the identification of necrosis and apoptosis in affected organs, technical issues related to preparation artifacts, occurrence of internucleosomal DNA cleavage in necrotic as well as apoptotic cells and other overlaps in death pathways bear consideration. In that caspase independent as well as caspase dependent processes cause cell death and that caspase inhibitors can act as anti-inflammatory agents, evaluation of ischemic death mechanisms in parenchymal cells needs to be performed with caution. When the effects of inflammation are removed by appropriate in vitro studies using purified or cultured cells, several mitochondrial factors that lead to cell death can be studied. Substantial evidence exists for the participation of electron transport defects, mitochondrial permeability transitions (MPT) and release of cytochrome c from mitochondria, effected by pro-apoptotic proteins such as Bax. The anti-apoptotic protein Bcl-2 exerts an overriding protective role in this type of injury by preserving mitochondrial structure and function. In contrast, caspase inhibitors cannot offer long-term protection to ischemically injured parenchymal cells regardless of how effectively they can inhibit apoptotic events, if the cells have suffered permanent mitochondrial damage impairing respiration.

Adenosine Triphosphate↗
PubMed1998

Intracellular Ca2+ thresholds that determine survival or death of energy-deprived cells.

Increase of intracellular ionized or free Ca2+ is thought to play a central role in cell death due to ATP depletion. However, concurrently operative mechanisms of injury that do not require intracellular Ca2+ increases have made it difficult to test this hypothesis or to determine the concentrations at which intracellular Ca2+ becomes lethal. The predominant Ca2+-independent mechanism of injury during ATP depletion involves the loss of cellular glycine. This type of damage can be fully inhibited by adding the amino acid exogenously. Using glycine to suppress Ca2+-independent plasma membrane damage, we have examined the effect of intracellular Ca2+ elevations on cell viability during ATP depletion. Madin-Darby canine kidney (MDCK) cells were depleted of ATP by incubation with a mitochondrial uncoupler in glucose-free medium. Free Ca2+ concentration in the medium was varied between 26 nmol/L and 1.25 mmol/L in the presence of a Ca2+ ionophore. Measurements with the Ca2+ probes fura-2, furaptra, and fura-2FF showed that intracellular Ca2+ was clamped at extracellular levels under these conditions. Cell survival during ATP depletion was indicated by viable cells recovered 24 hours later. The results show that ATP-depleted cells can sustain high levels of intracellular Ca2+ (100 micromol/L) for prolonged periods and remain viable if plasma membrane damage is prevented by glycine. Cell death was observed only when intracellular free Ca2+ was allowed to increase beyond 100 micromol/L, and this was associated with dramatic nuclear alterations: chromatin condensation, loss of nuclear lamins, and breakdown of DNA into large 50- to 150-kb fragments. Our studies demonstrate unexpectedly high resistance of cells to calcium cytotoxicity if glycine that is lost during ATP depletion is restored. In addition, they provide insights into novel mechanisms of nuclear disintegration and DNA damage that are triggered when the high thresholds of intracellular Ca2+ required for cell death are exceeded.

Adenosine Triphosphate↗
PubMed1998

Development of porous defects in plasma membranes of adenosine triphosphate-depleted Madin-Darby canine kidney cells and its inhibition by glycine.

Studies during the past decade have led to the recognition of a fundamental, widely expressed mechanism of structural damage in energy-deprived cells, which is suppressed by physiologic levels of glycine and is independent of Ca2+ availability or alterations of cytosolic free Ca2+. To gain insight into this process, Madin-Darby canine kidney (MDCK) cells were depleted of adenosine triphosphate (ATP) by a mitochondrial uncoupler in glucose-free medium, and intracellular free Ca2+ was clamped at 100 nM to avoid calcium cytotoxicity. Although the ATP-depleted cells swelled and blebbed and their plasma membranes appeared to be under tension, they nevertheless became permeable to macromolecules. The plasma membranes of these cells retained structural continuity, as determined by morphologic observations, and confocal microscopy of a plasma membrane protein label (Biotin: Ultra Avidin-Texas Red) and a lipid label (NBD-sphingomyelin). Using fluoresceinated dextrans of graded molecular size, membrane permselectivity was examined noninvasively by confocal microscopy. Measured as inside/outside ratios of fluorescence intensity, the permeability indices showed progressively greater restriction to diffusion of increasingly larger dextran molecules across plasma membranes, with sharp break-points between 70,000 and 145,000 daltons (d). The results indicated that the membranes behaved as if they were perforated by water-filled channels or "pores," with size-exclusion limits of molecular dimensions. The membrane defects evolved from small pores permeable only to propidium iodide (668 d) and the smallest dextran (4,000 d), before enlarging with time to become permeable to larger dextrans. Inclusion of glycine during ATP depletion did not affect cell swelling or blebbing but completely prevented the development of permeability defects. Treatment of cells before ATP depletion with a membrane-impermeant homobifunctional "nearest neighbor" cross-linking agent, 3,3' dithiobis(sulfosuccinimidylpropionate), suppressed the development of permeability defects, even in the absence of glycine. These observations suggest that the cellular abnormality that is suppressed by glycine involves rearrangement of plasma membrane proteins to form water-filled pores large enough to leak macromolecules.

Adenosine Triphosphate↗
PubMed1997

Cytosolic-free calcium increases to greater than 100 micromolar in ATP-depleted proximal tubules.

Previous studies have shown that cytosolic-free Ca2+ (Caf) increases to at least low micromolar concentrations during ATP depletion of isolated kidney proximal tubules. However, peak levels could not be determined precisely with the Ca2+-sensitive fluorophore, fura-2, because of its high affinity for Ca2+. Now, we have used two low affinity Ca2+ fluorophores, mag-fura-2 (furaptra) and fura-2FF, to quantitate the full magnitude of Caf increase. Between 30 and 60 min after treatment with antimycin to deplete ATP in the presence of glycine to prevent lytic plasma membrane damage, Caf measured with mag-fura-2 exceeded 10 microM in 91% of tubules studied and 68% had increases to greater than 100 microM. Caf increases of similar magnitude that were dependent on influx of medium Ca2+ were also seen using the new low Ca2+ affinity, Mg2+-insensitive, fluorophore fura-2FF in tubules depleted of ATP by hypoxia, and these increases were reversed by reoxygenation. Total cell Ca2+ levels in antimycin-treated or hypoxic tubules did not change, suggesting that mitochondria were not buffering the increased Caf during ATP depletion. Considered in the context of the high degree of structural preservation of glycine-treated tubule cells during ATP depletion and the commonly assumed Ca2+ requirements for phospholipid hydrolysis, actin disassembly, and Ca2+-mediated structural damage, the remarkable elevations of Caf demonstrated here suggest an unexpected resistance to the deleterious effects of increased Caf during energy deprivation in the presence of glycine.

Adenosine Triphosphate↗
PubMed1997

Glycine-protected, hypoxic, proximal tubules develop severely compromised energetic function.

Glycine-treated, hypoxic, proximal tubules developed a progressive energetic defect that resulted in failure to restore ATP levels to greater than 10 to 20% of control values during reoxygenation after 60 minutes of hypoxia despite continued cytoprotection by glycine. The defect was not corrected by supplementation with exogenous purines and was not modified by lowering the pH during hypoxia or reoxygenation. In the continued presence of glycine, the failure to restore ATP was associated with impaired recovery of structural changes that developed during hypoxia and, if glycine was withdrawn, lethal membrane damage occurred. The lesion was significantly ameliorated by the presence during hypoxia of two agents known to suppress development of the mitochondrial permeability transition, cyclosporine A and butacaine, which were most effective when used in combination. The data suggest that development of the mitochondrial permeability transition in glycine-protected tubules during hypoxia contributes to continued metabolic and structural impairment and cell death that occur despite glycine replete conditions such as exist frequently during in vivo insults and may be a target for therapeutic maneuvers.

4-Aminobenzoic Acid↗
PubMed1997

Internucleosomal DNA cleavage triggered by plasma membrane damage during necrotic cell death. Involvement of serine but not cysteine proteases.

Autolytic DNA breakdown, detected as smears in electrophoretic gels, is a late event in necrosis. On the other hand, internucleosomal DNA cleavage, visualized as ladders, is thought to be a hallmark of apoptosis. We now report that this specific form of DNA fragmentation also occurs during necrosis and is an early event but appears to be triggered by proteolytic mechanisms significantly different from those documented in apoptosis. Treatment of MDCK cells with a mitochondrial uncoupler and a Ca2+ ionophore led to ATP depletion, necrotic morphology, and progressive fragmentation of DNA in an internucleosomal or ladder pattern. DNA breakdown was immediately preceded by increased permeability of the plasma membrane to macromolecules. Provision of glycine along with the noxious agents did not modify the extent of ATP depletion, but prevented plasma membrane damage. This was accompanied by complete inhibition of DNA fragmentation. Internucleosomal DNA cleavage was observed also during necrosis after rapid permeabilization of plasma membranes by detergents or streptolysin-O in hepatocytes, thymocytes, and P19, Jurkat, and MDCK cells. DNA fragmentation associated with necrosis was Ca2+/Mg2+ dependent, was suppressed by endonuclease inhibitors, and was abolished by serine protease inhibitors but not by inhibitors of interleukin-1 beta converting enzyme (ICE)-related proteases or caspases. Moreover, unlike apoptosis, it was not accompanied by caspase-mediated proteolysis. On the other hand, the cleavage-site-directed chymotryptic inhibitor N-tosyl-L-phenylalanyl-chloromethyl ketone (TPCK) suppressed DNA fragmentation not only in necrotic cells but also during Fas-mediated apoptosis, without inhibiting caspase-related proteolysis. The results suggest a novel pathway of endonuclease activation during necrosis not involving the participation of caspases. In addition, they indicate that techniques based on double-strand DNA breaks may not reliably differentiate between apoptosis and necrosis.

Adenosine Triphosphate↗