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PubMed1992

ATP dependence of calcium uptake by the Na-Ca exchanger of adult heart cells.

The ATP dependence of the Na-Ca exchanger was investigated in isolated adult rat heart cells to evaluate the extent to which ATP depletion after a period of ischemia plus reperfusion in whole hearts could limit calcium uptake by Na-Ca exchange. A standard state for measurement of Na-Ca exchange activity that could be used with cells depleted of ATP to different degrees was defined. This was a state of zero sarcolemmal gradient for sodium, potassium, and pH and was achieved by incubation of the cells for 5 minutes with EDTA, EGTA, ouabain, and nigericin. Heterogeneity of cell ATP levels was minimized by using a protocol of total ATP depletion by incubation under conditions similar to ischemia, followed by reoxygenation to give partial restoration of ATP levels. No ATP was regenerated when cells were reoxygenated in the presence of rotenone, and such cells showed a very low rate of calcium uptake. Without rotenone, cells showed an almost complete restoration of Na-Ca exchange activity, in spite of a restoration of ATP levels to only one third of control values. Thus, the dependence of calcium uptake on ATP was highly nonlinear under these conditions. The calculated Km for ATP was no more than 10% of normal ATP levels. We conclude that ATP depletion after ischemia plus reperfusion is unlikely to limit the rate of calcium uptake through Na-Ca exchange in the whole heart if at least one quarter of the ATP is restored. In addition, we measured the apparent ATP dependence of calcium uptake by Na-Ca exchange in cells under conditions in which we previously had concluded that cell ATP distributions were very heterogeneous: when cells undergo contracture during incubation with oligomycin and without glucose. A linear relation between calcium uptake rate and ATP was observed at all ATP levels. This can be understood if cells in contracture that are incubated with oligomycin cannot take up calcium because of low ATP, whereas rod-shaped cells are able to retain a full uptake capability. This result further supports our conclusion that the ATP level declines catastrophically to near zero in these oligomycin-incubated cells just before contracture.

Adenosine Triphosphate↗
PubMed1993

Effect of metabolic inhibitors on Na+ transport in isolated perfused rat lungs.

Alveolar fluid absorption is a process driven by transepithelial alveolar Na+ transport. Since lungs produce significant amounts of lactate under anaerobic but also under aerobic conditions, glycolysis may conceivably contribute to producing the energy needed for transepithelial Na+ transport and fluid absorption. The effects of inhibition of oxidative phosphorylation or glycolysis on alveolar Na+ transport, fluid absorption, and preservation of alveolar epithelial barrier properties were examined using isolated, fluid-filled rat lungs. Basal lung lactate production was 65 +/- 1.0 mumol/h/g dry wt in the presence of 10 mmol/liter glucose. When oxidative phosphorylation was inhibited with rotenone, cyanide, or the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), lung lactate production increased 5- to 7-fold within 30 min (P < 0.001). No significant decrease in alveolar Na+ transport was observed over 1 h, whereas a 3-fold increase in passive epithelial permeability was observed. With rotenone and CCCP, but not cyanide, fluid absorption from airspaces was decreased but never abolished. Inhibition of aerobic glycolysis with iodoacetate did not significantly affect alveolar Na+ transport or fluid absorption. In the presence of isoproterenol or dibutyryl cyclic adenosine monophosphate (cAMP) + isobutylmethylxanthine, which have previously been shown to stimulate alveolar Na+ transport, lung lactate production increased 2-fold (P < 0.001). Inhibition of glycolysis depressed stimulated alveolar Na+ and fluid transports (P < 0.001). Inhibition of ion transport by ouabain or amiloride decreased lung lactate production (P < 0.001) under stimulated but not under unstimulated conditions. These observations suggest that glycolysis does not significantly contribute to energy provision for alveolar epithelial Na+ transport in lungs under basal, aerobic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1992

Induction of stress proteins in cultured myogenic cells. Molecular signals for the activation of heat shock transcription factor during ischemia.

Expression of major stress proteins is induced rapidly in ischemic tissues, a response that may protect cells from ischemic injury. We have shown previously that transcriptional induction of heat-shock protein 70 by hypoxia results from activation of DNA binding of a preexisting, but inactive, pool of heat shock factor (HSF). To determine the intracellular signals generated in hypoxic or ischemic cells that trigger HSF activation, we examined the effects of glucose deprivation and the metabolic inhibitor rotenone on DNA-binding activity of HSF in cultured C2 myogenic cells grown under normoxic conditions. Whole-cell extracts were examined in gel mobility shift assays using a 39-bp synthetic oligonucleotide containing a consensus heat-shock element as probe. ATP pools were determined by high-pressure liquid chromatography and intracellular pH (pHi) was measured using a fluorescent indicator. Glucose deprivation alone reduced the cellular ATP pool to 50% of control levels but failed to activate HSF. However, 2 x 10(-4) M rotenone induced DNA binding of HSF within 30 min, in association with a fall in ATP to 30% of control levels, and a fall in pHi from 7.3 to 6.9. Maneuvers (sodium propionate and amiloride) that lowered pHi to 6.7 without ATP depletion failed to activate HSF. Conversely, in studies that lowered ATP stores at normal pH (high K+/nigericin) we found induction of HSF-DNA binding activity. Our data indicate that the effects of ATP depletion alone are sufficient to induce the DNA binding of HSF when oxidative metabolism is impaired, and are consistent with a model proposed recently for transcriptional regulation of stress protein genes during ischemia.

Acidosis↗
PubMed2006

Microtubule: a common target for parkin and Parkinson's disease toxins.

Parkinson's disease (PD) is characterized by the selective loss of nigral dopaminergic (DA) neurons, which have long axons enriched with microtubules. Depolymerization of microtubules by PD toxins such as rotenone disrupts vesicular transport. The ensuing accumulation of vesicles in the cell body leads to increased cytosolic concentration of dopamine due to leakage of the vesicles. Elevated oxidative stress induced by dopamine oxidation may thus trigger the selective demise of DA neurons. Many strategies have been developed to protect DA neurons by stabilizing microtubules either directly or through intracellular signaling cascades. On the other hand, parkin, one of the most frequently mutated genes in PD, encodes for a protein-ubiquitin E3 ligase that strongly binds to microtubules. Parkin stabilizes microtubules through three domains that provide strong and independent interactions with tubulin and microtubules. These interactions anchor parkin on microtubules and may facilitate its E3 ligase activity on misfolded proteins transported along microtubules. Thus, parkin and rotenone, two prominent genetic and environmental factors linked to PD, act in an opposing manner on the same molecular target in the cell, microtubules, whose destruction underlies the selective vulnerability of dopaminergic neurons.

Animals↗
PubMed2005

Mitochondrial Ca2+ flux is a critical determinant of the Ca2+ dependence of mast cell degranulation.

An increase in intracellular Ca2+ ([Ca2+]i) is necessary for mast cell exocytosis, but there is controversy over the requirement for Ca2+ in the extracellular medium. Here, we demonstrate that mitochondrial function is a critical determinant of Ca2+ dependence. In the presence of extracellular Ca2+, mitochondrial metabolic inhibitors, including rotenone, antimycin A, and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), significantly reduced degranulation induced by immunoglobulin E (IgE) antigen or by thapsigargin, as measured by beta-hexosaminidase release. In the absence of extracellular Ca2+; however, antimycin A and FCCP, but not rotenone, enhanced, rather than reduced, degranulation to a maximum of 76% of that observed in the presence of extracellular Ca2+. This enhancement of extracellular, Ca2+-independent degranulation was concomitant with a rapid collapse of the mitochondrial transmembrane potential. Mitochondrial depolarization did not enhance degranulation induced by thapsigargin, irrespective of the presence or absence of extracellular Ca2+. IgE antigen was more effective than thapsigargin as an inducer of [Ca2+]i release, and mitochondrial depolarization augmented IgE-mediated but not thapsigargin-induced Ca2+ store release and mitochondrial Ca2+ ([Ca2+]m) release. Finally, atractyloside and bongkrekic acid [an agonist and an antagonist, respectively, of the mitochondrial permeability transition pore (mPTP)], respectively, augmented and reduced IgE-mediated Ca2+ store release, [Ca2+]m release, and/or degranulation, whereas they had no effects on thapsigargin-induced Ca2+ store release. These data suggest that the mPTP is involved in the regulation of Ca2+ signaling, thereby affecting the mode of mast cell degranulation. This finding may shed light on a new role for mitochondria in the regulation of mast cell activation.

Animals↗
PubMed1997

Leber's hereditary optic neuropathy: biochemical effect of 11778/ND4 and 3460/ND1 mutations and correlation with the mitochondrial genotype.

To clarify the bioenergetic relevance of mtDNA mutations in Leber's hereditary optic neuropathy (LHON), we investigated affected individuals and healthy carriers from six Italian LHON families harboring the 11778/ND4 and the 3460/ND1 mtDNA mutations. The enzymatic activities of mitochondrial complex I and its sensitivity to the potent inhibitors rotenone and rolliniastatin-2 were studied in mitochondrial particles from platelets, in correlation with mtDNA analysis of platelets and leukocytes. In platelets homoplasmic for mutant mtDNA, both 11778/ND4 and 3460/ND1 mutations induced resistance to rotenone and the 3460/ND1 mutation also provoked a marked decrease in the specific activity of complex I. Individuals heteroplasmic in platelets for either mutation showed normal biochemical features, indicating functional complementation of wild-type mtDNA. There was no correlation between the clinical status and mtDNA homo/heteroplasmy in platelets, but the biochemical features correlated with the mitochondrial genotype of platelets. In some cases, the degree of mtDNA heteroplasmy differed in platelets and leukocytes from the same individual with a prevalence of wild-type mtDNA in the platelets. These results imply that biochemical studies on mitochondrial diseases should always be integrated with mtDNA analysis of the same tissue investigated and also suggest that the mtDNA analysis on the leukocyte fraction, as usually performed in LHON, does not necessarily reflect the mutant genotype level of other tissues. The differential tissue heteroplasmy may be more relevant than previously thought in determining disease penetrance.

Antineoplastic Agents, Phytogenic↗
PubMedDate not supplied

Differing sensitivity of tumor cells to apoptosis induced by iron deprivation in vitro.

We studied the sensitivity of tumor cells to the induction of apoptosis by iron deprivation. Iron deprivation was achieved by the employment of a defined iron-deficient culture medium. Mouse 38C13 cells and human Raji cells die within 48 and 96 h of incubation in iron-deficient medium, respectively. On the contrary, mouse EL4 cells and human HeLa cells are completely resistant to the induction of death under the same experimental arrangement. Deoxyribonucleic acid fragmentation analysis by agarose gel electrophoresis as well as flow cytometric analysis after propidium iodide staining detected in 38C13 and Raji cells, but not in EL4 and HeLa cells, changes characteristic to apoptosis. The 38C13 cells, sensitive to iron deprivation, also displayed a similar degree of sensitivity to apoptosis induction by thiol deprivation (achieved by 2-mercaptoethanol withdrawal from the culture medium) as well as by rotenone (50 nM), hydroxyurea (50 microM), methotrexate (20 nM), and doxorubicin (100 nM). Raji cells shared with 38C13 cells a sensitivity to rotenone, methotrexate, doxorubicin, and, to a certain degree, to hydroxyurea. However, Raji cells were completely resistant to thiol deprivation. EI4 and HeLa cells, resistant to iron deprivation, also displayed a greater degree of resistance to most of the other apoptotic stimuli than did their sensitive counterparts. We conclude that some tumor cells in vitro are sensitive to apoptosis induction by iron deprivation, while other tumor cells are resistant. All the tumors found to be sensitive to iron deprivation in this study (four cell lines) are of hematopoietic origin. The mechanism of resistance to apoptosis induction by iron deprivation differs from the mechanism of resistance to thiol deprivation.

Animals↗
PubMed1985

Mitochondrial membrane potential in lymphocytes as monitored by fluorescent cation diS-C3-(5).

A lipophilic fluorescent cation diS-C3-(5) and rotenone suppress the oxygen consumption rate of thymocytes in similar concentrations. Seventy percent inhibition corresponds to an inhibitor:cytochrome a molar ratio of about 1:1. Addition of uncouplers decreases the inhibition of respiration by diS-C3-(5) (but not rotenone). FCCP in similar concentrations increases O2 consumption in the absence of diS-C3-(5) and the diS-C3-(5) fluorescence intensity in the presence of TMPD in thymocyte suspensions. In most thymocyte preparations, oligomycin (0.05-0.1 microgram/mL) increases the fluorescence of diS-C3-(5) and further addition of TMPD (50-100 microM) decreases the fluorescence. Addition of NaCN (400 microM) after oligomycin leads to a fluorescence increase that is hardly affected by subsequent addition of 0.2 microM FCCP. Nigericin (10-50 nM) decreases the diS-C3-(5) fluorescence. The data indicate that the diS-C3-(5) fluorescence associated with mitochondrial transmembrane potential (delta psi m) may be an essential part of the diS-C3-(5) fluorescence in lymphocyte suspensions. The changes of the diS-C3-(5) fluorescence intensity in the presence of TMPD after FCCP addition reflect delta psi m.

Animals↗
PubMed1985

Effect of ischemia on heart submitochondrial superoxide production.

NADH-dependent formation of superoxide anions (O2-) by rabbit cardiac submitochondrial particles (SMP) was stimulated after exposure of the isolated heart to 90 min of ischemic perfusion. This effect was more evident in the rotenone-inhibited region of the respiratory electron chain in comparison to the antimycin-inhibited region. The kinetic study of the NADH-dependent reaction showed that at the level of the rotenone-inhibited region, ischemia reduced Km value for NADH, differently from the antimycin-inhibited region where the kinetic constants remain unchanged. No significant changes of the Vmax values were observed in both SMP-producing O2- sites. The ischemic perfusions also produced a reduction of mitochondrial function, particularly evident when glutamate as substrate was studied.

Animals↗
PubMed1995

Metabolic inhibition and chloride transport in isolated toad skin.

1. When added to the Na(+)-containing solution bathing the isolated toad skin, dinitrophenol (DNP, an uncoupler of oxidative phosphorilation) caused decreases in the baseline values of short circuit current (SCC) and transepithelial conductance (G). 2. DNP also inhibited the increases in SCC and G caused by theophylline, whether added prior to the xanthine, or after the effect of the latter was fully developed. 3. In skins exposed to theophylline and bathed in Cl(-)-free (sulfate Ringer's) solution, the changes in SCC and G had a similar time course (t1/2 > 15 min). In the presence of Cl- (skins bathed in Ringer's solution), SCC decreased with a similar rate, whereas the rate of the decrease in G was greater (t1/2 < 15 min). 4. DNP also decreased the SCC induced by a Cl- concentration gradient in skins exposed to theophylline (SCCg) with a time course similar to its effect on the theophylline-increased G in the presence of Cl-. DNP was effective irrespective of the presence of ambient Na+. 5. A similar difference was observed in skins bathed in CIR and exposed to forskolin. In contrast to theophylline, however, forskolin partially overcame the inhibition of G brought about by DNP; no such recovery was observed in SCC. 6. In contrast to its influence on the responses to theophylline and forskolin, DNP failed to prevent either the increase in G or the onset of SCCg in skins exposed to dibutyryl cyclic AMP. 7. Rotenone, an inhibitor of the electron-transport chain, significantly decreased SCC and G in the unstimulated skin. It also prevented the SCC response to theophylline, and decreased it if added after the effects of the xanthine were fully developed, but failed to modify the increase in G brought about by theophylline. The time course of SCC inhibition by rotenone was similar to that caused by DNP. 8. Ouabain, an inhibitor of Na+,K(+)-ATPase, decreased SCC in the theophylline-stimulated skin, without affecting G. 9. We conclude that, whereas integrity of oxidative energy metabolism is necessary to sustain SCC in the isolated toad skin, it is not a strict requirement for the increase of Cl(-)-dependent G activated by cAMP. 10. The effect of DNP on Cl(-)-dependent G activated by cAMP is probably exerted at the cAMP generation step, by inhibition of adenyl cyclase and/or a decrease in the availability of ATP.

Animals↗
PubMed2000

Inhibition of Krebs cycle enzymes by hydrogen peroxide: A key role of [alpha]-ketoglutarate dehydrogenase in limiting NADH production under oxidative stress.

In this study we addressed the function of the Krebs cycle to determine which enzyme(s) limits the availability of reduced nicotinamide adenine dinucleotide (NADH) for the respiratory chain under H(2)O(2)-induced oxidative stress, in intact isolated nerve terminals. The enzyme that was most vulnerable to inhibition by H(2)O(2) proved to be aconitase, being completely blocked at 50 microm H(2)O(2). alpha-Ketoglutarate dehydrogenase (alpha-KGDH) was also inhibited but only at higher H(2)O(2) concentrations (>/=100 microm), and only partial inactivation was achieved. The rotenone-induced increase in reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] fluorescence reflecting the amount of NADH available for the respiratory chain was also diminished by H(2)O(2), and the effect exerted at small concentrations (</=50 microm) of the oxidant was completely prevented by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. BCNU-insensitive decline by H(2)O(2) in the rotenone-induced NAD(P)H fluorescence correlated with inhibition of alpha-ketoglutarate dehydrogenase. Decrease in the glutamate content of nerve terminals was induced by H(2)O(2) at concentrations inhibiting aconitase. It is concluded that (1) aconitase is the most sensitive enzyme in the Krebs cycle to inhibition by H(2)O(2), (2) at small H(2)O(2) concentrations (</=50 microm) when aconitase is inactivated, glutamate fuels the Krebs cycle and NADH generation is unaltered, (3) at higher H(2)O(2) concentrations (>/=100 microm) inhibition of alpha-ketoglutarate dehydrogenase limits the amount of NADH available for the respiratory chain, and (4) increased consumption of NADPH makes a contribution to the H(2)O(2)-induced decrease in the amount of reduced pyridine nucleotides. These results emphasize the importance of alpha-KGDH in impaired mitochondrial function under oxidative stress, with implications for neurodegenerative diseases and cell damage induced by ischemia/reperfusion.

Aconitate Hydratase↗
PubMed2001

Calcium-dependent inhibition of L, N, and P/Q Ca2+ channels in chromaffin cells: role of mitochondria.

The hypothesis that the buffering of Ca(2+) by mitochondria could affect the Ca(2+)-dependent inhibition of voltage-activated Ca(2+) channels, (I(Ca)), was tested in voltage-clamped bovine adrenal chromaffin cells. The protonophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), the blocker of the Ca(2+) uniporter ruthenium red (RR), and a combination of oligomycin plus rotenone were used to interfere with mitochondrial Ca(2+) buffering. In cells dialyzed with an EGTA-free solution, peak I(Ca) generated by 20 msec pulses to 0 or +10 mV, applied at 15 sec intervals, from a holding potential of -80 mV, decayed rapidly after superfusion of cells with 2 microm CCCP (tau = 16.7 +/- 3 sec; n = 8). In cells dialyzed with 14 mm EGTA, CCCP did not provoke I(Ca) loss. Cell dialysis with 4 microm ruthenium red or cell superfusion with oligomycin (3 microm) plus rotenone (4 microm) also accelerated the decay of I(Ca). After treatment with CCCP, decay of N- and P/Q-type Ca(2+) channel currents occurred faster than that of L-type Ca(2+) channel currents. These data are compatible with the idea that the elevation of the bulk cytosolic Ca(2+) concentration, [Ca(2+)](c), causes the inhibition of L- and N- as well as P/Q-type Ca(2+) channels expressed by bovine chromaffin cells. This [Ca(2+)](c) signal appears to be tightly regulated by rapid Ca(2+) uptake into mitochondria. Thus, it is plausible that mitochondria might efficiently regulate the activity of L, N, and P/Q Ca(2+) channels under physiological stimulation conditions of the cell.

Animals↗
PubMed1975

Porphyrin synthesis and mitochondrial respiration in acute intermittent porphyria: studies using cultured human fibroblasts.

The formation of variation of delta-aminolevulinic acid (ALA) and of porphyrins, as well as respiratory metabolism, have been studied in skin fibroblasts from six normal control subjects and seven patients with acute intermittent porphyria. The mean activity of ALA synthetase was the same in both groups, whereas the mean activity of uroporphyrinogen I synthetase (as measured by the conversion of porphobilinogen [PBG] to porphyrins) was significantly decreased in fibroblasts from porphyric subjects, the mean value being 52 per cent that of control subjects (p less than or equal to 0.001). The findings of decreased uroporphyrinogen synthesis without an increase in ALA synthetase in mitochondria-containing cells from subjects with acute intermittent porphyria are compatible with the concept that defective PBG ultilization is the fundamental defect in heme biosynthesis in this disease and the possibility that ALA synthetase is "irreversibly" repressed in nonhepatic tissues. Respiration of the cells was studied polarographically. The two types of cells showed similar overall rates of respiration and in general responded to substrates and inhibitors as expected. Of the inhibitors tested (rotenone, amytal, antimycin, and cyanide), only rotenone showed a differential effect: respiration of fibroblasts from porphyric patients was not as sensitive to the inhibitor as was that of the control subjects. These results are interpreted as suggesting a possible defect in mitochondrial NADH oxidation in acute intermittent porphyria.

5-Aminolevulinate Synthetase↗
PubMed1975

Respiratory properties of rat liver mitochondria immobilized on an alkylsilylated glass surface.

Rat liver mitochondria are shown to adhere to the alkylsilylated glass beads in essentially a monolayer. The amount of mitochondria bound to the beads reaches a maximum where the length of the alkyl groups covalently linked to the beads exceeds eight carbons. Mitochondria immobilized on the beads and placed in a flow system exhibit normal: (a) respiratory control, (b) phosphate to oxygen ratio, (c) uncoupling by 2,4-dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone, and (d) inhibition by cyanide, azide, rotenone, oligomycin, and antimycin. Reversibility of the effects of 2,4-dinitrophenol, cyanide, and azide was rapid and complete. Inhibition by rotenone, oligomycin was essentially irreversible. Mitochondria have been maintained in a viable state on the beads at 27 degrees for periods up to 4 hours. The use of immobilized organelles appears to offer a new technique for the study of membrane-bound particles whereby substances can be rapidly added and removed while monitoring the composition of solution flowing over the particles.

Adenosine Diphosphate↗
PubMed2002

[Disturbances in biliary secretion during tissue hypoxia and attempts at their corrections].

In acute experiments on rats in vivo the stimulating effects of hydrophilic cholic acids (CA and UDCA) on the bilification and their inhibiting by rotenone have been shown. The secretion of CA and UDCA under rotenone have been determined to be activated. The mechanisms of the influence of the cholic acids on the secretory function of the hepatocytes have being discussed. It has been concluded that NADH-dehydrogenase link in the respiratory chain is not involved in the activating effects by CA and UDCA.

Animals↗
PubMed1975

beta-Hydroxy fatty acid production during fatty acid oxidation by heart mitochondria.

The effect of various incubation conditions on the production of beta-hydroxylaurate, -myristate, and -palmitate by isolated rabbit heart mitochondria oxidizing palmityl carnitine was studied. A gas chromatographic-mass spectrometric method was developed to identify and measure submicrogram quantities of methyl esters of these beta-hydroxy fatty acids (FA). Alpha-Deuterated esters of beta-hydroxy-FA served as internal standards. The total amount of the three beta-hydroxy-FA in fresh mitochondria was 0.045 mug/mg of protein. Malate, ATP, rotenone, and 1-carnitine together effected an NADH:NAD ratio of 8 and the accumulation of 0.896 mug of beta-hydroxylpalmitate. Omission of carnitine decreased the beta-hydroxy-FA by 65 percent. Omission of palmityl carnitine resulted in no beta-hydroxy-FA. Without rotenone the NADH:NAD ratio was 0.5 and there accumulated only 0.085 mug of beta-hydroxy-FA/mg of protein/10 min. Succinate, in place of malate, effected an NADH:NAD ratio greater than 10 but only 0.465 mug of beta-hydroxy-FA/mg of protein/10 min. We conclude that elevated mitochondrial NADH:NAD ratios facilitate beta-hydroxy-FA accumulation by slowing beta-hydroxyacyl-CoA oxidation. Cardiac hypoxia may prompt beta-hydroxy-FA accumulation and/or release.

Animals↗
PubMed2002

Endoplasmic reticulum stress and the unfolded protein response in cellular models of Parkinson's disease.

6-hydroxydopamine, 1-methyl-4-phenyl-pyridinium (MPP+), and rotenone cause the death of dopaminergic neurons in vitro and in vivo and are widely used to model Parkinson's disease. To identify regulated genes in such models, we performed serial analysis of gene expression on neuronal PC12 cells exposed to 6-hydroxydopamine. This revealed a striking increase in transcripts associated with the unfolded protein response. Immunoblotting confirmed phosphorylation of the key endoplasmic reticulum stress kinases IRE1alpha and PERK (PKR-like ER kinase) and induction of their downstream targets. There was a similar response to MPP+ and rotenone, but not to other apoptotic initiators. As evidence that endoplasmic reticulum stress contributes to neuronal death, sympathetic neurons from PERK null mice in which the capacity to respond to endoplasmic reticulum stress is compromised were more sensitive to 6-hydroxydopamine. Our findings, coupled with evidence from familial forms of Parkinson's disease, raise the possibility of widespread involvement of endoplasmic reticulum stress and the unfolded protein response in the pathophysiology of this disease.

1-Methyl-4-phenylpyridinium↗
PubMed1975

Contributions of cytoplasmic factors to in vitro cellular senescence.

Mass populations of normal human lung fibroblasts were enucleated by centrifugation at greater than or equal to 25,000 g in 4 mug/ml cytochalasin B. The 1 per cent of cells that did not enucleate where rendered nonviable by treatment with mitomycin C. Whole cells were poisoned with a 99 per cent lethal dose of the sulfhydryl reagent iodoacetate. The washed cells were then mixed with the anucleate cytoplasms, fused with inactivated Sendai virus, and planted in rotenone for 20 hours. Whereas normal cells are able to survive this rotenone treatment, the 1 per cent surviving iodoacetate-treated cells cannot withstand this additional stress. However, iodoacetate treated cells that fuse to untreated cytoplasms receive sufficient amounts of active enzymes to allow them to survive. Since this selective system does not rely on using enzymatic mutants, it should permit the selection of hybrids between anucleate cytoplasms and any type of whole cell. Cytoplasmic hybrids were cultured in order to determine their proliferative capacity. The life-spans of cytoplasmic hybrids between young and old cells were compared to those of young/young and old/old controls. Cytoplasmic factors do not appear to control in vitro cellular senescence.

Cell Division↗