Succinate Dehydrogenase Inhibition Research Articles

Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-α prolyl hydroxylase

Several mitochondrial proteins are tumor suppressors. These include succinate dehydrogenase ( SDH) and fumarate hydratase, both enzymes of the tricarboxylic acid (TCA) cycle. However, to date, the mechanisms by which defects in the TCA cycle contribute to tumor formation have not been elucidated. Here we describe a mitochondrion-to-cytosol signaling pathway that links mitochondrial dysfunction to oncogenic events: succinate, which accumulates as a result of SDH inhibition, inhibits HIF-α prolyl hydroxylases in the cytosol, leading to stabilization and activation of HIF-1α. These results suggest a mechanistic link between SDH mutations and HIF-1α induction, providing an explanation for the highly vascular tumors that develop in the absence of VHL mutations.

Neuroprotective effect of l-carnitine in the 3-nitropropionic acid (3-NPA)-evoked neurotoxicity in rats

A plant and fungal toxin, 3-NPA, acts as an inhibitor of mitochondrial function via irreversible inactivation of the mitochondrial inner membrane enzyme, succinate dehydrogenase (SDH). Inhibition of SDH disturbs electron transport and leads to cellular energy deficits and neuronal injury. We have shown that pretreatment with l-carnitine, while not significantly attenuating SDH inhibition, prevented hypothermia and oxidative stress-associated increased activity of free radical-scavenging enzymes. Here, a neurohistological method was applied to examine the effect of carnitine pretreatment against 3-NPA-induced neurotoxicity. Twenty adult male Sprague–Dawley rats were randomly divided into two groups ( n = 10/group). Rats in the first group were injected twice with 3-NPA at 30 mg/kg s.c., 2 days apart, and the second group of animals received l-carnitine pretreatment at 100 mg/kg 30–40 min before 3-NPA administration. Rats in both groups were perfused 7 days later and their brains harvested. Degenerating neurons were identified and localized via the fluorescent marker Fluoro-Jade B. In the three animals that survived 3-NPA dosing, one exhibited no pathology, one exhibited moderate unilateral damage to the striatum, and the third exhibited extensive bilateral neuronal degeneration in multiple forebrain regions. In the seven surviving animals that received l-carnitine prior to 3-NPA insult, six exhibited no lesions, while one exhibited a modest unilateral lesion in the striatum. It appears that l-carnitine is protective against 3-NPA-induced toxicity, as reflected by both reduced mortality and significantly reduced neuronal degeneration.

Succinate increases neuronal post-synaptic excitatory potentials in vitro and induces convulsive behavior through N-methyl- d-aspartate-mediated mechanisms

Succinate is a dicarboxylic acid that accumulates due to succinate dehydrogenase inhibition by malonate and methylmalonate exposure. These neurotoxins cause increased excitability and excitotoxic damage, which can be prevented by administering high amounts of succinate. In the present study we investigated whether succinate alters hippocampal field excitatory post-synaptic potentials. Bath application of succinate at intermediate concentrations (0.3–1 mM) increased the slope of field excitatory post-synaptic potentials in hippocampal slices, and at high concentrations (above 1 mM) did not alter or decrease field excitatory post-synaptic potentials slope. Succinate-induced enhancement of field excitatory post-synaptic potentials slope was abolished by the addition of d-2-amino-5-phosphonovaleric acid (50 μM) to the perfusate, supporting the involvement of N-methyl- d-aspartate receptors in the excitatory effect of this organic acid. Accordingly, succinate (0.8–7.5 μmol) i.c.v. administration caused dose-dependent convulsive behavior in mice. The i.c.v. co-administration of MK-801 (7 nmol) fully prevented succinate-induced convulsions, further suggesting the involvement of N-methyl- d-aspartate receptors in the convulsant action of succinate. Our data indicate that accumulation of moderate amounts of succinate may contribute to the excitotoxicity induced by succinate dehydrogenase inhibitors, through the activation of N-methyl- d-aspartate receptors.

Ammonia potentiates methylmalonic acid-induced convulsions and TBARS production

Hyperammonemia is a common finding in children with methylmalonic acidemia, an inherited metabolic disease characterized by mental retardation, convulsions, and accumulation of methylmalonic acid (MMA). Although it has been suggested that MMA induces convulsions through succinate dehydrogenase (SDH) inhibition, very little is known about the contribution of hyperammonemia to the development of convulsions in these patients. In the present study we investigated the effects of ammonium ions on the convulsant action of MMA, MMA-induced inhibition of striatal succinate dehydrogenase, and the striatal content of thiobarbituric acid-reactive substances (TBARS). Adult rats were injected with ammonium acetate (1.5 mmol/kg, sc) or sodium acetate (1.5 mmol/kg, sc), followed 5 min later by buffered MMA (3 μmol/μl) or NaCl (4.5 μmol/μl) injected into the striatum. The animals were observed in an open field for the appearance of convulsive episodes. After 30 min of behavioral evaluation, the animals were sacrificed and had their striatal TBARS content measured. Ammonium acetate pretreatment caused no behavioral effects per se, but potentiated MMA-induced convulsions and increased basal TBARS content and MMA-induced TBARS production in the striatum. Ammonium chloride had no effect on basal succinate dehydrogenase activity and did not alter MMA-induced inhibition of SDH in vitro. These results suggest that ammonia potentiates MMA-induced behavioral effects through a mechanism that does not involve further succinate dehydrogenase inhibition, but may involve facilitation of MMA-induced oxidative damage and provide evidence that ammonia and MMA may have mutually additive toxicity.

Dopamine transporter knock-out mice are hypersensitive to 3-nitropropionic acid-induced striatal damage.

Evidence suggests that dopamine is involved in the modulation of striatal excitotoxic processes. To further investigate this issue, we studied the effects of systemic 'low-dose' (total dose, 340 mg/kg in 7 days) 3-nitropropionic acid (3-NP) intoxication in dopamine transporter knock-out mice (DAT-/-) compared to wildtype (DAT+/+) mice. Systemic 'low-dose' 3-NP induced a significant impairment in a rotarod task only in DAT-/- mice. Histopathology also demonstrated a significant reduction of the striatal volume (-7%, P < 0.05), neuronal density (-12.5%, P < 0.001) and absolute number estimates of striatal neurons (-11.5%, P < 0.001) in DAT-/- compared to DAT+/+ mice, with increased glial activation, independent of the degree of succinate dehydrogenase inhibition. These findings strengthen the hypothesis for dopamine modulation of excitotoxicity within the nigrostriatal system.

Increased expression of multidrug resistance-associated proteins in bladder cancer during clinical course and drug resistance to doxorubicin.

Overexpression of the P-glycoprotein/multidrug resistance 1 (MDR1) and multidrug resistance protein 1 (MRP1) gene is closely associated with the clinical outcome of various malignancies, and it is involved in responses to some anticancer chemotherapeutic agents including doxorubicin. Six human MRP subfamily members (MRP2-7) with structural similarities to MRP1 have been identified. Recently, the relationships between MRP2 and MRP3 expression levels of some cancer cells and drug sensitivity to doxorubicin have been reported, but the relationship between the clinical samples and drug sensitivity remains unclear. We determined the expressions of the MDR1, MRP1, MRP2 and MRP3 gene in bladder cancer during the clinical course and sought to learn whether the expression was correlated with drug responses to doxorubicin. Doxorubicin, used in chemotherapeutic treatment including intravesical and systemic chemotherapy, is an important anticancer agent for the treatment of bladder cancer. We used quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for our study, and the sensitivity to doxorubicin in bladder cancer was determined using the in vitro succinate dehydrogenase inhibition test. Using 47 clinical samples of bladder cancer, we confirmed the significant correlation of MDR1, MRP1 and MRP3 mRNA levels with resistance to doxorubicin. We showed that the expression of MDR1, MRP1, MRP2 and MRP3 in recurrent tumors and residual tumors after chemotherapeutic treatment was higher than that in untreated primary tumors. In particular, the MDR1 expression in residual tumors was 5.7-fold higher than that in untreated primary tumors.

Photodynamic inactivation of isolated crayfish mechanoreceptor neuron: different death modes under different photosensitizer concentrations.

To study the mechanism of photodynamic nerve cell killing, isolated crayfish mechanoreceptor neurons were photosensitized by the sulfonated aluminum ophthalocyanine Photosens. Neuron activity was continuously recorded until irreversible abolition. Intense (10(-5) M Photosens) or weak (10(-7) M Photosens) photosensitization induced different bioelectric neuron responses: firing activation followed by irreversible depolarization block or gradual inhibition until firing abolition, respectively. These bioelectric responses were accompanied by different biochemical and morphological changes. In the case of intense photosensitization, neuron nuclei swelled and then shrank. Succinate dehydrogenase (SDH) was inhibited, and the plasma membrane was compromised just after firing cessation. Weak photosensitization did not induce these changes but caused swelling of the endoplasmic reticulum and destruction of the matrix, cristae and membranes in some of the mitochondria. Other mitochondria, however, retained the normal structure. Plasma membrane damage, SDH inhibition, nucleus shrinkage and impairment of the nuclear border occurred after 2-4 h. It is concluded that intense photosensitization induced necrotic processes during irradiation, whereas weaker impact caused delayed necrosis 2-4 h later. The observed electrophysiological neuron responses to photodynamic therapy may be considered as early hallmarks of different modes of forthcoming cell death.

Neuroprotective role of l-carnitine in the 3-nitropropionic acid induced neurotoxicity

l-carnitine (LC) plays an important regulatory role in the mitochondrial transport of long-chain free fatty acids (FFA). 3-Nitropropionic acid (3-NPA) is known to induce cellular energy deficit and oxidative stress related neurotoxicity via an irreversible inhibition of the mitochondrial enzyme succinate dehydrogenase (SDH). Protective effects of l-carnitine on the neurotoxicity induced by 3-NPA have been shown in vitro. Here, the activities of SDH as well as the activity of the antioxidant enzymes, catalase (CAT), and superoxide dismutase (SOD) were measured in order to evaluate the protective action of LC against 3-NPA-induced neurotoxicity. Male, CD Sprague–Dawley rats, 3-month old, were injected with either 50 or 100 mg/kg of LC, i.p., 30–60 min prior to 3-NPA (30 mg/kg, s.c.) or with 3-NPA alone. Enzyme activities were assayed in caudate nucleus (CN), frontal cortex (FC), and hippocampus (HIP) post sacrifice. Increased activities of CAT and SOD were observed after treatment with 3-NPA alone. Pretreatment with low or high doses of LC was associated with attenuation of these increases equivalent to, or below, the control levels. In rats treated with 3-NPA alone, SDH activity was inhibited by 62% (CN), 50% (FC), and 65% (HIP) of controls. Pretreatment with LC prior to 3-NPA attenuated decreases of SDH activity in a dose-dependent manner. However, compared with control, the activity of SDH remained significantly lower in brain regions of treated rats despite the attenuation of inhibition by LC pretreatment ( P<0.05). These data suggest protective effect of LC against 3-NPA-induced oxidative stress. It appears that the protective effect of LC against 3-NPA-induced oxidative stress is not mediated by the direct action of LC preventing the SDH inhibition but rather is achieved due to the actions of LC downstream of the SDH inhibition.

Effect of L-carnitine pretreatment on 3-nitropropionic acid-induced inhibition of rat brain succinate dehydrogenase activity.

L-Carnitine (LC) plays an important regulatory role in the mitochondrial transport of long chain free fatty acids (FFA). 3-Nitropropionic acid (3-NPA) is known to induce cellular energy deficit and oxidative stress-related neurotoxicity via an irreversible inhibition of mitochondrial succinate dehydrogenase (SDH). In the present study, activity of SDH was measured in order to evaluate neuroprotective effects of LC against the 3-NPA-induced neurotoxicity. Male, CD Sprague-Dawley rats, three months old, were injected with either 50 or 100 mg/kg of LC, i.p., 30 min prior to 3-NPA (30 mg/kg, s.c.) or with 3-NPA alone. The activity of brain SDH was quantified spectrophotometrically in caudate nucleus (CN), frontal cortex (FC), and hippocampus (HIP) 60 min after the 3-NPA injection. The SDH activity in the animals treated with 3-NPA alone was 38% (CN), 50% (FC), and 36% (HIP) that of saline controls. Pretreatment with LC prior to 3-NPA injection attenuated decreases of SDH activity by approximately 15 and 29% (LC low and high dose, respectively). Despite the attenuation of SDH inhibition, the activity of SDH in these regions remained significantly lower in treated than in control rats (p < 0.05). It appears that the protective effect of LC against 3-NPA-induced oxidative stress cannot be explained by the direct action of LC to interfere with the SDH inhibition but are rather achieved by LC actions downstream of the SDH inhibition.

SDI法による皮膚悪性腫瘍の抗癌剤感受性の検討

SDI法による皮膚悪性腫瘍の抗癌剤感受性の検討

Evaluation of usefulness of in-vitro drug sensitivity testing for adjuvant chemotherapy of stomach cancer

Background. The selection of effective drugs is the key for tumor chemotherapy. The succinic dehydrogenase inhibition (SDI) method, the simplest chemosensitivity test, is widely used at many institutions in Japan. As adequate proof of the usefulness of this test in the clinical setting has not yet been established, a prospective randomized study was undertaken. Methods. An adjuvant chemotherapy study incorporating the SDI test was performed in patients with gastrectomized stomach cancer. Fifteen test centers were set up throughout Japan, and the tests were performed on test specimens collected from neighboring institutions. In order to insure homogeneity of the test techniques, both intra- and inter-institutionally, simulation tests in tumor-bearing nude mice were carried out simultaneously. Three hundred and thirty-three eligible patients were categorized as stratum I (curative resection) or stratum II (non-curative resection) and were randomly allocated to three chemotherapy groups group A (mitomycin C), group B (doxorubicin), and group C (cisplatin), Correlations between survival rates and the inhibition index (II; a percentage value calculated from the optical density of tumor cell suspersions caltured with the anticancer drugs), which was arbitrarily divided into three categories: ≥60%; 59%–30%; and ≤29%, were analyzed in the three groups. Results. Significant differences in survival rates, in terms of the II, were shown in the stratum I group C (cisplatin), whereas no difference in terms of II was found in groups A and B. Conclusion. The SDI sensitivity test was found to be useful for cisplatin; however, the test failed to show usefulness for mitomycin C and doxorubicin.

Increased expression of an ATP-binding cassette superfamily transporter, multidrug resistance protein 2, in human colorectal carcinomas.

The expression of ATP-binding cassette superfamily transporter genes, such as P-glycoprotein/multidrug resistance (MDR) 1 and MDR protein (MRP) 1, is often up-regulated in various tumor types and is involved in responses to some anticancer chemotherapeutic agents. Five human MRP subfamily members (MRP2-6) with structural similarities to MRP1 have been identified. The relationships between MRP2-6 mRNA levels and drug resistance are not well understood. Data on 45 patients with colorectal cancer were analyzed. Of the ATP-binding cassette superfamily genes, we asked whether mRNA levels of MDR1, MRP1, MRP2, and MRP3 correlated with drug resistance to anticancer agents. For this analysis, we used quantitative reverse transcription-PCR, and the sensitivity to anticancer agents in surgically resected colon carcinomas was determined using the in vitro succinate dehydrogenase inhibition test. MDR1, MRP1, and MRP3 were highly expressed in normal colorectal mucosa, and the relative mRNA levels of MDR1, MRP1, and MRP3 in cancerous tissues compared with noncancerous tissues were decreased or unchanged. By contrast, MRP2 mRNA expression was low in normal colorectal mucosa and specifically increased in cancer regions compared with noncancerous regions. Of the anticancer agents prescribed for patients with colorectal cancers, including doxorubicin, mitomycin C, cisplatin, 5-fluorouracil, etoposide, and a camptothecin derivative, mRNA expression of MRP2 was significantly associated with resistance to cisplatin. MRP2 may be important for resistance to cisplatin treatment in colorectal cancer.

Serial 1H-NMR Spectroscopy Study of Metabolic Impairment in Primates Chronically Treated with the Succinate Dehydrogenase Inhibitor 3-Nitropropionic Acid

Previous studies in primates have shown that chronic systemic administration of the succinate dehydrogenase (SDH) inhibitor, 3-nitropropionic acid (3NP), replicates most of the motor, cognitive, and histopathological features of Huntington's disease. In the present study, serial 1H-NMR spectroscopy (1H-MRS) assessment of striatal and occipital cortex concentrations of N-acetylaspartate, phosphocreatine/creatine, choline, and lactate, were obtained every 2-weeks during the entire course of a chronic 3NP treatment in baboons. A region-selective increase in lactate was detected in the striatum of the 3NP-treated animals, either immediately before or in conjunction with a lesion in the dorsolateral putamen detected by T2-MR imaging. Absolute 1H-MRS quantitation demonstrated a progressive and region-specific decrease in striatal N-acetylaspartate, creatine, and choline, occuring as early as 3 weeks before the first detection of lactate. These results demonstrate that 1H-MRS can be used to monitor early stages of brain metabolic impairment. In addition, given that 3NP-induced SDH inhibition following systemic injection similarly affects all brain regions, the striatal selective decreases in N-acetylaspartate or creatine concentrations are not simply related to the level of mitochondrial impairment but to a preferential vulnerability of the striatum to 3NP-induced toxicity.

In vitro kinetics of the rat brain succinate dehydrogenase inhibition by hexachlorophene

In vitro kinetics of the rat brain succinate dehydrogenase inhibition by hexachlorophene

In vitro kinetics of the rat brain succinate dehydrogenase inhibition by hexachlorophene.

Brain succinate dehydrogenase (SDH) activity was inhibited by in vitro hexachlorophene (HCP) with a half inhibitory concentration (IC50) of 0.65 x 10(-3) M. The HCP exerted noncompetitive inhibition at 0.5 mM (<IC50), uncompetitive inhibition at 0.65 mM (= IC50), and mixed-type inhibition at 0.8 mM (>IC50) on SDH activity. The brain SDH demands more energy of activation (deltaE) in the presence of HCP. The ionizable groups of SDH such as the sulfhydral group of cysteine and alpha-amino groups of cysteine were not altered qualitatively in the presence of HCP.

3-Nitropropionic acid exacerbates N-methyl- d-aspartate toxicity in striatal culture by multiple mechanisms

We examined the effects of 3-nitropropionic acid-induced succinate dehydrogenase inhibition on neuronal ATP content, N-methyl- d-aspartate-induced neuronal death, resting membrane potential, and N-methyl- d-aspartate-induced changes in cytosolic calcium concentration ([Ca 2+] c) in cultured rat striatal neurons. Exposure of cultures to 3 mM 3-nitropropionic acid for 3 h did not cause overt toxicity, but reduced ATP content by 35%. Treatment with 3-nitropropionic, or removal of Mg 2+ from the medium, enhanced subsequent N-methyl- d-aspartate toxicity, reducing the LC 50 from 250 μM to 12 μM or 30 μM, respectively. Even after Mg 2+ removal, enhancement of N-methyl- d-aspartate toxicity by 3-nitropropionic acid remained pronounced, with the LC 50 further decreasing to 3 μM. The mean resting membrane potential of neurons treated with 3-nitropropionic acid was −37 mV, while that in control neurons was −61 mV. Treatment with 3-nitropropionic did not affect baseline [Ca 2+] c as determined by fura-2 microfluorimetry. N-methyl- d-aspartate (30 μM) caused a rapid rise in [Ca 2+] c, the initial magnitude of which was not affected by 3-nitropropionic acid. However, after a 1-h treatment, [Ca 2+] c was dramatically higher in 3-nitropropionic acid-treated neurons. This increased calcium load was washed out slowly and only partially, although calcium in control neurons washed out rapidly and almost completely. These results suggest that in striatal neurons, the enhancement of N-methyl- d-aspartate toxicity caused by succinate dehydrogenase inhibition may be due to synergism between partial relief of the Mg 2+ blockade of the N-methyl- d-aspartate receptor and other mechanisms, including disruption of neuronal calcium regulation. This synergism may be relevant to the neuronal death observed in neurodegenerative disorders.

Partial inhibition of brain succinate dehydrogenase by 3-nitropropionic acid is sufficient to initiate striatal degeneration in rat.

Chronic inhibition of succinate dehydrogenase (SDH) by systemic injection of the selective inhibitor 3-nitropropionic acid (3NP) has been used as an animal model for Huntington's disease (HD). However, the mechanisms by which 3NP produces lesions in the striatum are not fully characterized. A quantitative histochemical method was developed to study the level of regional SDH inhibition resulting from intraperitoneal injection of 3NP or chronic intoxication using osmotic pumps. The results showed that (a) 3NP was an irreversible SDH inhibitor in vivo, (b) the level of SDH inhibition in the striatum (the brain region most vulnerable to 3NP) was similar to that observed in other brain regions not affected by the toxin, such as the cerebral cortex, and (c) the neurotoxic threshold of SDH inhibition in the brain was 50-60% of control levels. The present study demonstrates that the selective degeneration in the striatum observed after chronic 3NP administration cannot be ascribed to a preferential inhibition of SDH in this particular brain region. This work also suggests that the partial decrease in the activity of the respiratory chain complex II-III reported in HD patients may be sufficient to induce the selective striatal degeneration observed in this disorder.

Establishment of a cisplatin-resistant gastric carcinoma cell line OCUM-2M/DDP.

A cis-diamminedichloroplatinum (CDDP)-resistant scirrhous gastric cancer cell line, OCUM-2M/DDP, was established by chronic exposure of cells of the parent scirrhous gastric cancer cell line, OCUM-2M, to CDDP at progressively increasing concentrations. The OCUM-2M/DDP cell line had an 11.3-fold higher level of resistance relative to its parent cell line as determined by a succinate dehydrogenase inhibition test. The biological and biochemical characteristics of the resistant and parent cell line were compared. There were differences in the modal chromosome number and DNA index, suggesting that some alterations of the DNA in the CDDP-resistant cells had occurred. Neither the parent nor resistant cell line expressed mdr-1 mRNA. After exposure to CDDP for 4 h, the intracellular platinum content of OCUM-2M cells was significantly higher than that of OCUM-2M/DDP cells (51.9 +/- 1.8 vs 16.4 plus 1.0 ng/mg protein, mean +/- SD, respectively). The GSH levels in OCUM-2M cells and OCUM-2M/DDP cells were 3.5 +/- 1.0 micrograms/mg protein and 16.8 +/- 1.2 micrograms/mg protein, respectively. These levels were also significantly different. These findings suggest that the possible mechanisms of acquired resistance to CDDP in OCUM-2M/DDP cells may be a decrease in intracellular CDDP accumulation and detoxication by GSH. This OCUM-2M/DDP cell line could be used in further investigations of the mechanism of CDDP resistance in gastric cancer.

In vivo vulnerability of dopamine neurons to inhibition of energy metabolism

In vitro studies indicate that mesencephalic dopamine neurons are more vulnerable than other neurons to impairment of energy metabolism. Such findings may have bearing on the loss of dopamine neurons in Parkinson's disease, in which mitochondrial deficiencies have been identified, but would only be relevant if the selective vulnerability were maintained in vivo. To examine this, rats were stereotaxically administered various concentrations of the succinate dehydrogenase inhibitor, malonate (0.25–4 μmol), either into the left substantia nigra or striatum. One week following injection, dopamine and γ-aminobutyric acid (GABA) levels in the mesencephalon and striatum were measured. Intranigral injection of malonate caused nigral dopamine and GABA to be comparably reduced at all doses tested. The 50% dose level for malonate vs. dopamine and GABA loss was 0.39 and 0.42 μmol, respectively. Tyrosine hydroxylase immunocytochemistry of the midbrains of rats which received an intranigral injection of malonate showed normal staining with 0.25 μmol malonate, but almost complete loss of tyrosine hydroxylase positive nigral pars compacta cells with 1 μmol malonate. Intrastriatal injection of malonate produced a loss of both tyrosine hydroxylase activity and dopamine. In contrast to what was seen in substantia nigra, there was a greater loss of dopamine than GABA in striatal regions nearest the injection site. In striatal regions most distal to the injection site, and which received the lowest concentration of malonate due to diffusion, dopamine levels were significantly reduced with all doses of malonate (0.5–4 μmol), whereas GABA levels were unaffected. Intrastriatal coinfusion of succinate along with malonate completely prevented the loss of dopamine and GABA indicating that succinate dehydrogenase inhibition was the cause of toxicity. These findings indicate that dopamine terminals in the striatum of adult rats are selectively more vulnerable than are the GABA neurons to a mild energy impairment.

Direct Inhibition of Plant Mitochondrial Respiration by Elevated CO2.

Doubling the concentration of atmospheric CO2 often inhibits plant respiration, but the mechanistic basis of this effect is unknown. We investigated the direct effects of increasing the concentration of CO2 by 360 [mu]L L-1 above ambient on O2 uptake in isolated mitochondria from soybean (Glycine max L. cv Ransom) cotyledons. Increasing the CO2 concentration inhibited the oxidation of succinate, external NADH, and succinate and external NADH combined. The inhibition was greater when mitochondria were preincubated for 10 min in the presence of the elevated CO2 concentration prior to the measurement of O2 uptake. Elevated CO2 concentration inhibited the salicylhydroxamic acid-resistant cytochrome pathway, but had no direct effect on the cyanide-resistant alternative pathway. We also investigated the direct effects of elevated CO2 concentration on the activities of cytochrome c oxidase and succinate dehydrogenase (SDH) and found that the activity of both enzymes was inhibited. The kinetics of inhibition of cytochrome c oxidase were time-dependent. The level of SDH inhibition depended on the concentration of succinate in the reaction mixture. Direct inhibition of respiration by elevated CO2 in plants and intact tissues may be due at least in part to the inhibition of cytochrome c oxidase and SDH.