Inhibition of nucleoside diphosphate kinase in rat liver mitochondria by added 3'-azido-3'-deoxythymidine. (1/690)

The effect of 3'-azido-3'-deoxythymidine on nucleoside diphosphate kinase of isolated rat liver mitochondria has been studied. This is done by monitoring the increase in the rate of oxygen uptake by nucleoside diphosphate (TDP, UDP, CDP or GDP) addition to mitochondria in state 4. It is shown that 3'-azido-3'-deoxythymidine inhibits the mitochondrial nucleoside diphosphate kinase in a competitive manner, with a Ki value of about 10 microM as measured for each tested nucleoside diphosphate. It is also shown that high concentrations of GDP prevent 3'-azido-3'-deoxythymidine inhibition of the nucleoside diphosphate kinase.  (+info)

Dual actions of the metabolic inhibitor, sodium azide on K(ATP) channel currents in the rat CRI-G1 insulinoma cell line. (2/690)

1. The effects of various inhibitors of the mitochondrial electron transport chain on the activity of ATP-sensitive K+ channels were examined in the Cambridge rat insulinoma G1 (CRI-G1) cell line using a combination of whole cell and single channel recording techniques. 2. Whole cell current clamp recordings, with 5 mM ATP in the pipette, demonstrate that the mitochondrial uncoupler sodium azide (3 mM) rapidly hyperpolarizes CRI-G1 cells with a concomitant increase in K+ conductance. This is due to activation of K(ATP) channels as the sulphonylurea tolbutamide (100 microM) completely reversed the actions of azide. Other inhibitors of the mitochondrial electron transport chain, rotenone (10 microM) or oligomycin (2 microM) did not hyperpolarize CRI-G1 cells or increase K+ conductance. 3. In cell-attached recordings, bath application of 3 mM sodium azide (in the absence of glucose) resulted in a rapid increase in K(ATP) channel activity, an action readily reversible by tolbutamide (100 microM). Application of sodium azide (3 mM), in the presence of Mg-ATP, to the intracellular surface of excised inside-out patches also increased K(ATP) channel activity, in a reversible manner. 4. In contrast, rotenone (10 microM) or oligomycin (2 microM) did not increase K(ATP) channel activity in either cell-attached, in the absence of glucose, or inside-out membrane patch recordings. 5. Addition of sodium azide (3 mM) to the intracellular surface of inside-out membrane patches in the presence of Mg-free ATP or the non-hydrolysable analogue 5'-adenylylimidodiphosphate (AMP-PNP) inhibited, rather than increased, K(ATP) channel activity. 6. In conclusion, sodium azide, but not rotenone or oligomycin, directly activates K(ATP) channels in CRI-G1 insulin secreting cells. This action of azide is similar to that reported previously for diazoxide.  (+info)

Mitochondrial regulation of the cytosolic Ca2+ concentration and the InsP3-sensitive Ca2+ store in guinea-pig colonic smooth muscle. (3/690)

1. Mitochondrial regulation of the cytosolic Ca2+ concentration ([Ca2+]c) in guinea-pig single colonic myocytes has been examined, using whole-cell recording, flash photolysis of caged InsP3 and microfluorimetry. 2. Depolarization increased [Ca2+]c and triggered contraction. Resting [Ca2+]c was virtually restored some 4 s after the end of depolarization, a time when the muscle had shortened to 50 % of its fully relaxed length. The muscle then slowly relaxed (t = 17 s). 3. The decline in the Ca2+ transient was monophasic but often undershot or overshot resting levels, depending on resting [Ca2+]c. The extent of the overshoot or undershoot increased with increasing peak [Ca2+]c. 4. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 5 microM), which dissipates the mitochondrial proton electrochemical gradient and therefore prevents mitochondrial Ca2+ accumulation, slowed Ca2+ removal at high ( > 300 nM) but not at lower [Ca2+]c and abolished [Ca2+]c overshoots. Oligomycin B (5 microM), which prevents mitchondrial ATP production, affected neither the rate of decline nor the magnitude of the overshoot. 5. During depolarization, the global rhod-2 signal (which represents the mitochondrial matrix Ca2+ concentration, [Ca2+]m) rose slowly in a CCCP-sensitive manner during and for about 3 s after depolarization had ended. [Ca2+]m then slowly decreased over tens of seconds. 6. Inhibition of sarcoplasmic reticulum Ca2+ uptake with thapsigargin (100 nM) reduced the undershoot and increased the overshoot. 7. Flash photolysis of caged InsP3 (20 microM) evoked reproducible increases in [Ca2+]c. CCCP (5 microM) reduced the magnitude of the [Ca2+]c transients evoked by flash photolysis of caged InsP3. Oligomycin B (5 microM) did not reduce the inhibition of the InsP3-induced Ca2+ transient by CCCP thus minimizing the possibility that CCCP lowered ATP levels by reversing the mitochondrial ATP synthase and so reducing SR Ca2+ refilling. 8. While CCCP reduced the magnitude of the InsP3-evoked Ca2+ signal, the internal Ca2+ store content, as assessed by the magnitude of ionomycin-evoked Ca2+ release, did not decrease significantly. 9. [Ca2+]c decline in smooth muscle, following depolarization, may involve mitochondrial Ca2+ uptake. Following InsP3-evoked Ca2+ release, mitochondrial uptake of Ca2+ may regulate the local [Ca2+]c near the InsP3 receptor so maintaining the sensitivity of the InsP3 receptor to release Ca2+ from the SR.  (+info)

Oligomycin induces a decrease in the cellular content of a pathogenic mutation in the human mitochondrial ATPase 6 gene. (4/690)

A T --> G mutation at position 8993 in human mitochondrial DNA is associated with the syndrome neuropathy, ataxia, and retinitis pigmentosa and with a maternally inherited form of Leigh's syndrome. The mutation substitutes an arginine for a leucine at amino acid position 156 in ATPase 6, a component of the F0 portion of the mitochondrial ATP synthase complex. Fibroblasts harboring high levels of the T8993G mutation have decreased ATP synthesis activity, but do not display any growth defect under standard culture conditions. Combining the notions that cells with respiratory chain defects grow poorly in medium containing galactose as the major carbon source, and that resistance to oligomycin, a mitochondrial inhibitor, is associated with mutations in the ATPase 6 gene in the same transmembrane domain where the T8993G amino acid substitution is located, we created selective culture conditions using galactose and oligomycin that elicited a pathological phenotype in T8993G cells and that allowed for the rapid selection of wild-type over T8993G mutant cells. We then generated cytoplasmic hybrid clones containing heteroplasmic levels of the T8993G mutation, and showed that selection in galactose-oligomycin caused a significant increase in the fraction of wild-type molecules (from 16 to 28%) in these cells.  (+info)

Bioenergetic and structural consequences of allotopic expression of subunit 8 of yeast mitochondrial ATP synthase. The hydrophobic character of residues 23 and 24 is essential for maximal activity and structural stability of the enzyme complex. (5/690)

Subunit 8 (Y8), a mitochondrially encoded subunit of the F0 sector of the F1F0-ATP synthase is essential for oxidative phosphorylation. We have previously introduced the technique of allotopic expression to study the structure/function of Y8, whereby an artificial Y8 gene is expressed in the nucleus of cells lacking a functional mitochondrial Y8, thus generating assembly of a functional F1F0-ATPase complex. In this paper we show that when a gene encoding an essentially unmodified version of Y8 is allotopically expressed, ATP synthesis and hydrolysis rates, as well as efficiency of oxidative phosphorylation, were similar to those of the parental wild-type strain in which Y8 is naturally expressed in mitochondria. We then tested the requirement for the hydrophobicity of the central domain (residues 14-32), which possibly represents a transmembrane stem, by introducing adjacent negative charges at different positions of Y8. One of the variants thus generated, which carries the double substitution Leu23-->Asp, Leu24-->Asp, when expressed in a strain lacking endogenous Y8, gave rise to cells which grew very slowly by oxidative phosphorylation. Measurement of bioenergetic parameters showed two major defects in these cells relative to control cells allotopically expressing unmodified Y8. First, the activity of the F1F0-ATP synthase was significantly decreased. ATP synthesis and state 3 of respiration were reduced by approximately 30-40%. ATP hydrolysis was reduced by approximately 30% and was almost insensitive to the F0 inhibitor oligomycin. Second, the physical coupling between the two sectors of the enzyme, as well as the stability of the F1 sector itself, were affected as shown by decreased recovery of F0 sector [8, 9, b, oligomycin sensitivity-conferring protein (OSCP), d, h and f] and F1 sector (alpha, gamma, delta) subunits in immunoprecipitates of ATP synthase. This study indicates that Y8 not only performs an important role in the structure of the mitochondrial complex but also in its activity. We conclude that the hydrophobic character of amino acids 23 and 24 in the middle of the putative transmembrane stem of Y8 is essential for coupling proton transport through F0 to ATP synthesis on F1.  (+info)

ATP-dependent steps in apoptotic signal transduction. (6/690)

Apoptotic changes of the nucleus induced by Fas (Apo1/CD95) stimulation are completely blocked by reducing intracellular ATP level. In this study, we examined the ATP-dependent step(s) of Fas-mediated apoptotic signal transduction using two cell lines. In SKW6.4 (type I) cells characterized by rapid formation of the death-inducing signaling complex on Fas treatment, the activation of caspases 8, 9, and 3, cleavage of DFF45 (ICAD), and release of cytochrome c from the mitochondria to the cytoplasm were not affected by reduction of intracellular ATP, although chromatin condensation and nuclear fragmentation were inhibited. On the other hand, in the Fas-mediated apoptosis of Jurkat (type II) cells, which is characterized by involvement of mitochondria and, thus, shares signal transduction mechanisms with apoptosis induced by other stimuli such as genotoxins, activation of the three caspases, cleavage of DFF45 (ICAD), and nuclear changes were blocked by reduction of intracellular ATP, whereas release of cytochrome c was not affected. These results suggested that the ATP-dependent step(s) of Fas-mediated apoptotic signal transduction in type I cells are only located downstream of caspase 3 activation, whereas the activation of caspase 9 by released cytochrome c is the most upstream ATP-dependent step in type II cells. These observations also confirm the existence of two pathways for Fas-mediated apoptotic signal transduction and suggest that the Apaf-1 (Ced-4 homologue) system for caspase 9 activation operates in an ATP-dependent manner in vivo.  (+info)

Oxidative stress inhibits apoptosis in human lymphoma cells. (7/690)

Apoptosis and necrosis are two forms of cell death that are induced under different conditions and that differ in morphological and biochemical features. In this report, we show that, in the presence of oxidative stress, human B lymphoma cells are unable to undergo apoptosis and die instead by a form of necrosis. This was established using the chemotherapy drug VP-16 or the calcium ionophore A23187 to induce apoptosis in Burkitt's lymphoma cell lines and by measuring classical markers of apoptotic death, including cell morphology, annexin V binding, DNA ladder formation, and caspase activation. In the presence of relatively low levels of H2O2 (75-100 microM), VP-16 and A23187 were unable to induce apoptosis in these cells. Instead, the cells underwent non-apoptotic cell death with mild cytoplasmic swelling and nuclear shrinkage, similar to the death observed when they were treated with H2O2 alone. We found that H2O2 inhibits apoptosis by depleting the cells of ATP. The effects of H2O2 can be overcome by inhibitors of poly(ADP)-ribosylation, which also preserve cellular ATP levels, and can be mimicked by agents such as oligomycin, which inhibit ATP synthesis. The results show that oxidants can manipulate cell death pathways, diverting the cell away from apoptosis. The potential physiological ramifications of this finding will be discussed.  (+info)

The nop-1 gene of Neurospora crassa encodes a seven transmembrane helix retinal-binding protein homologous to archaeal rhodopsins. (8/690)

Opsins are a class of retinal-binding, seven transmembrane helix proteins that function as light-responsive ion pumps or sensory receptors. Previously, genes encoding opsins had been identified in animals and the Archaea but not in fungi or other eukaryotic microorganisms. Here, we report the identification and mutational analysis of an opsin gene, nop-1, from the eukaryotic filamentous fungus Neurospora crassa. The nop-1 amino acid sequence predicts a protein that shares up to 81.8% amino acid identity with archaeal opsins in the 22 retinal binding pocket residues, including the conserved lysine residue that forms a Schiff base linkage with retinal. Evolutionary analysis revealed relatedness not only between NOP-1 and archaeal opsins but also between NOP-1 and several fungal opsin-related proteins that lack the Schiff base lysine residue. The results provide evidence for a eukaryotic opsin family homologous to the archaeal opsins, providing a plausible link between archaeal and visual opsins. Extensive analysis of Deltanop-1 strains did not reveal obvious defects in light-regulated processes under normal laboratory conditions. However, results from Northern analysis support light and conidiation-based regulation of nop-1 gene expression, and NOP-1 protein heterologously expressed in Pichia pastoris is labeled by using all-trans [3H]retinal, suggesting that NOP-1 functions as a rhodopsin in N. crassa photobiology.  (+info)