Effect of Ca2+, ruthenium red and ageing on pregnenolone production by mitochondrial fractions from normal and luteinizing hormone treated rat testes

The effect of Ca²⁺ in vitro on pregnenolone production rates under various incubation conditions by mitochondrial fractions fractions isolated from testes of normal rats and of rats after in vivo treatment with luteinizing hormone has been investigated. Concentrations of Ca²⁺ in the range of 0.1–0.5 mM stimulated succinate supported pregnenolone production in mitochondrial fractions from both control and luteinizing hormone treated testes. When mitochondrial fractions were isolated in 0.25 M sucrose without additions, Ca²⁺ in vitro increased succinate supported pregnenolone production rates in mitochondrial fractions isolated from control testes to a greater extent than in mitochondrial fractions, from luteinizing hormone treated testes. Production rates in control mitochondrial fractions, incubated in the presence of initial Ca²⁺ concentrations of 0.7 mM and higher were almost similar to production rates in relevant luteinizing hormone treated mitochondria.Pregnenolone production from endogenous substrates in mitochondrial fractions isolated in 0.25 M sucrose from control and luteinizing hormone treated testes incubated in the absence of added succinate and Ca²⁺, was maintained during 10–20 min.After longer incubation times no further steroid synthesis took place. Addition of 0.5 mM Ca²⁺ to the incubation medium at time zero slightly stimulated initial pregnenolone production rates in control mitochondrial fractions, but had no effect during prolonged incubations. Addition of 0.5 mM Ca²⁺ to mitochondrial fractions isolated from luteinizing hormone treated glands showed no effect either on initial production rate or during prolonged incubations.Pregnenolone production rates were maintained during 90 min in the presence of 20 mM succinate in the incubation medium. Under such conditions production rates during the first 20 min in mitochondrial fractions obtained from luteinizing hormone treated glands were approx. 3 times higher than in relevant control samples. Addition of 0.5 mM Ca²⁺ to the incubation medium containing 20 mM succinate markedly stimulated initial pregnenolone production rates in control mitochondrial fractions, but gave only a small stimulation of succinate-supported production rates in luteinizing hormone treated testicular mitochondrial fractions. These results indicate that Ca²⁺ in vitro can mimic the trophic effect of luteinizing hormone in vivo on mitochondrial pregnenolone production.Ageing of mitochondrial protein for 60 min at 33°C resulted in a marked increase in pregnenolone production rates in mitochondrial fractions obtained from control testes. The same treatement hardly influenced production rates in mitochondrial fractions isolated from luteinizing hormone treated testes. Ageing may have an effect on the ultrastructure of freshly prepared mitochondria, causing a change in the amount of cholesterol readily available for the enzyme complex.The gluco- and mucoprotein specific agent Ruthenium red (50–2000 ng/ml) did not inhibit pregnenolone production in either control or hormone treated testicular mitochondrial fractions, incubated in the absence of added Ca²⁺. the presence of 200–2000 ng Ruthenium red per ml incubation mixture.The present results have been discussed in relation to the possible involvement of Ca²⁺ in the molecular mechanism of short-term action of luteinizing hormone on testicular androgen production.     

Heterogeneity of rat liver mitochondrial fractions and the effect of tri-iodothyronine on their protein turnover

1. Rat liver mitochondria were separated into heavy, light and fluffy fractions by differential centrifugation under standard conditions. 2. All mitochondrial fractions possessed soluble as well as membrane-bound enzymes typical of mitochondria. 3. The heavy fraction represented the stable mitochondrial structures and the fluffy particles appear to be loosely coupled. 4. The light mitochondrial fraction lacked the ability of coupled phosphorylation. 5. A study of mobility and isoelectric pH indicated a similarity in the basic membrane structure of all the mitochondrial fractions. 6. The turnover rates of proteins in the heavy and fluffy particles were almost identical; however, this rate was rapid for the light mitochondrial fraction. 7. On treatment with 3,3',5-tri-iodo-l-thyronine, succinoxidase activity was maximally stimulated much earlier in the light mitochondrial fraction than in the heavy fraction. The activity of the fluffy particles, however, remained almost unaffected. 8. Malate dehydrogenase activity in all the mitochondrial fractions was stimulated only at 40h after tri-iodothyronine treatment. 9. The pattern of incorporation of dl-[1-(14)C]leucine in vivo in the tri-iodothyronine-treated animals indicated a rapid initial incorporation and high synthetic ability of the light mitochondrial fraction. 10. The turnover pattern of proteins of the mitochondrial fractions from animals receiving repeated doses of tri-iodothyronine was remarkably different from the normal pattern and suggested that preformed soluble protein units may be incorporated in the light mitochondrial fraction during maturation to form the stable heavy mitochondria. 11. The amount of light-mitochondrial proteins decreased by 40% on thyroidectomy and increased by 160% on treatment with tri-iodothyronine. 12. The possible significance of these results is discussed in relation to mitochondrial genesis.     

Phosphatidate phosphohydrolase and palmitoyl-coenzyme A hydrolase in cardiac subcellular fractions of hyperthyroid rabbits and cardiomyopathic hamsters

Activities of phosphatidate phosphohydrolase and palmitoyl-CoA hydrolase were determined in cardiac subcellular fractions prepared from rabbits which has received tri-iodothyronine and from hamsters with hereditary cardiomyopathy (strain BIO 14.6). 1. Both mitochondrial and microsomal fractions of hyperthyroid rabbit hearts produced 4-5 times as much diacylglycerol 3-phosphate from glycerol 3-phosphate and palmitate as did those of euthyroid hearts. 2. Phosphatidate phosphohydrolase, measured with phosphatidate emulsion, was activated by 1mm-Mg(2+) in all but the mitochondrial fraction of euthyroid rabbit hearts. The activation was more pronounced in subcellular fractions isolated from hyperthyroid hearts, so that the measured activities were significantly increased above those of the controls. The highest activity was found in the microsomal and lysosomal fractions. 3. In the absence of Mg(2+) during incubation, the difference in phosphohydrolase activities between eu- and hyper-thyroid states was not significant. 4. The phosphohydrolase of subcellular fractions of control hamsters did not respond to addition of 0.5-8.0mm-Mg(2+). The enzyme from cardiomyopathic hearts was slightly inhibited by this bivalent cation and therefore significant increases in activity were observed only in the absence of Mg(2+) from the assay system. 5. The rate of reaction by soluble phosphatidate phosphohydrolase was similar regardless of the nature of the substrate. Both when microsomal-bound phosphatidate was used as the substrate and when phosphatidate suspension was used, the activity of soluble enzyme was lower than that of the microsomal and lysosomal enzymes measured with phosphatidate suspension; this was especially so when the assay was carried out in the absence of Mg(2+). Neither tri-iodothyronine nor cardiomyopathy influenced the soluble phosphohydrolase activity in the two species. 6. Neither tri-iodothyronine nor cardiomyopathy significantly changed palmitoyl-CoA hydrolase activities in subcellular fractions. 7. Microsomal diacylglycerol acyltransferase and myocardial triacylglycerol content were also unchanged in the hyperthyroid state.     

Effect of Ca2+, ruthenium red and ageing on pregnenolone production by mitochondrial fractions from normal and luteinizing hormone treated rat testes.

The effect of Ca2+ in vitro on pregnenolone production rates under various incubation conditions by mitochondrial fractions isolated from testes of normal rats and of rats after in vivo treatment with luteinizing hormone has been investigated. Concentrations of Ca2+ in the range of 0.1-0.5 mM stimulated succinate supported pregnenolone production in mitochondrial fractions from both control and luteinizing hormone treated testes. When mitochondrial fractions were isolated in 0.25 M sucrose without additions, Ca2+ in vitro increased succinate supported pregnenolone production rates in mitochondrial fractions isolated from control testes to a greater extent than in mitochondrial fractions, from luteinizing hormone treated testes. Production rates in control mitochondrial fraction, incubated in the presence of initial Ca2+ concentrations of 0.7 mM and higher were almost similar to production rates in relevant luteinizing hormone treated mitochondria.     

Profile of phospholipase A2 activity in subcellular fractions and lamellar bodies of developing, neonatal and adult rabbit lung. Correlation with intracellular levels of disaturated phosphatidylcholine

Phospholipase A2 activity was determined in subcellular fractions and lamellar bodies of fetal, neonatal and adult rabbit lungs. Specific activity in most fractions decreased from the 24th to the 28th day of gestation. All fractions except the mitochondrial and the nuclear fractions exhibited a sharp increase in activity in the newborn lung. Specific activity in the adult lung generally declined in comparison to neonatal values. During gestation total enzyme activity per gram of lung was concentrated in the cytosolic fraction. With the exception of the lamellar body fraction, the total content of phospholipase A2 activity increased dramatically in all fractions from the neonatal lung. The lamellar body fractions displayed both low specific activity and low total enzyme activity during gestation. Specific activity increased dramatically in the neonatal and adult lung but still accounted for only a small fraction of the activity in comparison to the other subcellular fractions. The subcellular content of disaturated phosphatidylcholine (PC) appeared to correlate well with the activity of phospholipase A2 in the neonatal mitochondrial, microsomal and cytosolic fractions. Since decreasing prenatal enzyme levels are associated with increasing disaturated PC content, the alkaline and calcium-dependent phospholipase A2 may not be directly involved in disaturated PC synthesis in the fetus. However, postnatally, the correlation between the pattern of production of disaturated PC and the activity of the phospholipase A2 indicates a role for this enzyme in surfactant-related disaturated PC synthesis.     

Hepatic enzymes, CoASH and long-chain acyl-CoA in subcellular fractions as affected by drugs inducing peroxisomes and smooth endoplasmic reticulum

1. The activities of acyl-CoA hydrolase, catalase, urate oxidase and peroxisomal palmitoyl-CoA oxidation as well as the protein content and the level of CoASH and long-chain acyl-CoA were measured in subcellular fractions of liver from rats fed diets containing phenobarbital (0.1% w/w) or clofibrate (0.3% w/w). 2. Whereas phenobarbital administration resulted in increased microsomal protein, the clofibrate-induced increase was almost entirely attributed to the mitochondrial fraction with minor contribution from the light mitochondrial fraction. 3. The specific activity of palmitoyl-CoA hydrolase in the microsomal fraction was only slightly affected while the mitochondrial enzyme was increased to a marked extent (3-4-fold) by clofibrate. 4. Phenobarbital administration mainly enhanced the microsomal palmitoyl-CoA hydrolase. 5. The increased long-chain acyl-CoA and CoASH level observed after clofibrate treatment was mainly associated with the mitochondrial, light mitochondrial and cytosolic fractions, while the slight increase in the levels of these compounds found after phenobarbital feeding was largely of microsomal origin. 6. The findings suggest that there is an intraperoxisomal CoASH and long-chain acyl-CoA pool. 7. The specific activity of palmitoyl-CoA hydrolase, catalase and peroxisomal palmitoyl-CoA oxidation was increased in the lipid-rich floating layer of the cytosol-fraction. 8. The changes distribution of the peroxisomal marker enzymes and microsomal palmitoyl-CoA hydrolase after treatment with hypolipidemic drugs may be related to the origin of peroxisomes.     

Studies on the role of calcium ions in the stimulation by adrenaline of amylase release from rat parotid

1. Mitochondrial and microsomal fractions were prepared from rat parotid glands. Both fractions were able to take up (45)Ca. The mitochondrial (45)Ca-uptake system could be driven by ATP (energy-coupled Ca(2+) uptake) or by ADP+succinate (respiration-coupled Ca(2+) uptake). Energy-coupled Ca(2+) uptake was blocked by oligomycin but not by carbonyl cyanide m-chlorophenylhydrazone; respiration-coupled Ca(2+) uptake was blocked by carbonyl cyanide m-chlorophenylhydrazone but not by oligomycin. Microsomal Ca(2+) uptake was dependent on the presence of ATP; the ATP-dependent Ca(2+) uptake was not affected by oligomycin or carbonyl cyanide m-chlorophenylhydrazone. Ca(2+) uptake by both fractions was inhibited by Ni(2+). 2. Incubation of parotid pieces with adrenaline increased the rate of release of amylase and the uptake of (45)Ca. The adrenaline-stimulated release of amylase was not dependent on the presence of extracellular Ca(2+). 3. The effect of adrenaline on the subcellular distribution of (45)Ca in parotid pieces incubated with (45)Ca was studied. In parotid tissue incubated with (45)Ca, both mitochondrial and microsomal fractions contained (45)Ca. Incubation with adrenaline increased the amount of (45)Ca incorporated into the mitochondrial fraction but not the microsomal fraction. In parotid tissue preloaded with (45)Ca subsequent incubation with adrenaline caused a decrease in the amount of (45)Ca found in both the mitochondrial and microsomal fractions. 4. From these data we conclude that the regulation of the cytosolic Ca(2+) concentration in the parotid may involve both mitochondrial and microsomal Ca(2+)-uptake systems. We suggest that the action of adrenaline on the parotid may be to increase the movement of Ca(2+) to the cytosol by increasing the flux of Ca(2+) across mitochondrial, microsomal and plasma membranes.     

Antioxidant activity of dihydrolipoate against microsomal lipid peroxidation and its dependence on alpha-tocopherol

The antioxidant effect of dihydrolipoate and lipoate was examined in microsomal fractions obtained from normal and alpha-tocopherol-deficient animals after initiation of lipid peroxidation with an NADPH/iron/ADP system. Dihydrolipoate prolonged the lag phase before the onset of low-level chemiluminescence and before the rapid accumulation of thiobarbituric acid-reactive substances in normal but not in vitamin E-deficient microsomes. Lipoate did not show such an antioxidant effect. It is concluded that the dihydrolipoate-mediated protection against lipid peroxidation by prolonging the lag phase is dependent on alpha-tocopherol. Likewise, dihydrolipoate prolonged the lag phase before the onset of the rapid loss of vitamin E during lipid peroxidation. Dihydrolipoate, like other biological thiols such as GSH, also affects the peroxidative process after the lag period. The effects included a smaller slope of the chemiluminescence increase, a lower maximal level of chemiluminescence, a slower loss of alpha-tocopherol and a slower accumulation, but unchanged maximal levels, of thiobarbituric acid-reactive substances. The biological significance may be most prominent in the mitochondrial matrix space, where lipoamide-containing ketoacid dehydrogenases are located. A potential pharmacological use of this biological dithiol in conditions associated with oxidative stress could be based on the antioxidant activity of dihydrolipoate.     

Subcellular alterations in cyclic AMP-binding and protein kinase activities during ontogeny in the rat cerebellum

Ontogenic relationships between levels of cyclic AMP-binding activity and protein kinase activity were examined in subcellular fractions of the cerebellum during the first 3 weeks of neonatal life. A progressive increase in cyclic AMP levels was paralleled by an increase in cyclic AMP bindign by the nuclear and cytosol fractions, but not by the mitochondrial or microsomal fractions. Utilization of heat-stable protein kinase inhibitor permtited distinction of the cyclic AMP-dependent from the cyclic AMP-independent form of the protein kinase population. Cyclic AMP-dependent protein kinase increased between days 4 and 20 to represent a progressively greater proportion of the protein kinase population. In all subcellular fractions alterations of cyclic AMP-dependent protein kinase during neonatal development paralleled changes in binding of cyclic AMP to protein in these fractions. In both the nuclear and cytosol fractions cyclic AMP-dependent protein kinase activity increased progressively between days 4 and 20, i.e. 64 ± 6 to 176 ± 16 and 79 ± 12 to 340 ± 12 pmol/min per mg protein, respectively. Cyclic AMP-dependent protein kinase activity in the mitochondrial fraction declined during the postnatal period studied, and in the microsomal fraction it rose to a non-sustained peak at 14 days and fell thereafter. Unlike the cyclic AMP-dependent form, cyclic AMP-independent protein kinase activity did not follow the ontogenetic pattern of cyclic AMP-binding activity. The specific activity of nuclear cyclic AMP-independent protein kinase did not change during days 4–20, and a non-sustained rise of cyclic AMP-independent protein kinase activity in both cytosol and microsomal fractions during the 7th–12th day tended to parallel more closely known patterns of postnatal proliferative growth. The findings reported herein indicate that the ontogenic pattern of cyclic AMP-dependent protein kinase varies between different subcellular fractions of the neonatal cerebellum, that these patterns parallel the changes in cyclic AMP-bidign activity, and suggest that the component parts of the cyclic AMP system may develop as a functional unit.     

Activation of NADPH oxidase and extracellular superoxide production in seizure-induced hippocampal damage

We sought to determine whether the extracellular compartment contributed to seizure-induced superoxide (O2*-) production and to determine the role of the NADPH oxidase complex as a source of this O2*- production. The translocation of NADPH oxidase subunits (p47phox, p67phox and rac1) was assessed by immunoblot analysis and NADPH-driven O2*- production was measured using 2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)-8-benzyl-3,7-dihydroimidazo [1,2-alpha] pyrazin-3-one-enhanced chemiluminescence. Kainate-induced status epilepticus resulted in a time-dependent translocation of NADPH oxidase subunits (p47phox, p67phox and rac-1) from hippocampal cytosol to membrane fractions. Hippocampal membrane fractions from kainate-injected rats showed increased NADPH-driven and diphenylene iodonium-sensitive O2*- production in comparison to vehicle-treated rats. The time-course of kainate-induced NADPH oxidase activation coincided with microglial activation in the rat hippocampus. Finally, kainate-induced neuronal damage and membrane oxygen consumption were inhibited in mice overexpressing extracellular superoxide dismutase. These results suggest that seizure activity activates the membrane NADPH oxidase complex resulting in increased formation of O2*-.     

Opposite changes with age in liver and muscle in the mitochondrial and soluble glucose-1,6-bisphosphate and 6-phosphogluconate dehydrogenase

Glucose-1,6-bisphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, was markedly decreased in liver of adult rats (2 months of age) as compared to young rats (1-2 weeks of age). This regulator was found to be present in both the mitochondrial and soluble fractions of liver. Its concentration in both these fractions was decreased with age. Concomitant to the decrease in Glc-1,6-P2, which is a potent inhibitor of 6-phosphogluconate dehydrogenase, the activity of this enzyme was markedly increased with age in both the mitochondrial and soluble fractions. However, the increase in this enzyme's activity was more pronounced in the mitochondrial fraction. The mitochondrial enzyme was more susceptible to inhibition by Glc-1,6-P2 as compared to the soluble enzyme, and this may explain the greater enhancement in its activity with age in this fraction. The tibialis anterior muscle exhibited changes with age opposite to those found in liver; Glc-1,6-P2 concentration, in both the mitochondrial and soluble fractions of muscle increased with age, and this increase was accompanied by a concomitant reduction in the activity of the mitochondrial and soluble 6-phosphogluconate dehydrogenase. Similar to liver, the mitochondrial enzyme was more affected by age, as it also exhibited a greater susceptibility to inhibition by Glc-1,6-P2.     

On the role of rat liver cytosol in suppression of low-level chemiluminescence during nonenzymatic lipid peroxidation.

1. The effect of normal rat liver cytosol on the level of Fe/ADP-ascorbate-induced lipid peroxidation in the total particulate fraction (mitochondria plus microsomes) has been studied. The intensity of lipid peroxidation was measured using chemiluminescence technique and malondialdehyde (MDA) formation. 2. Dialysed cytosol significantly decreased the level of chemiluminescence, and to a much lesser extent, the rate of MDA production. 3. Gel filtration on a Sephadex G-200 column led to appearance of at least three cytosolic fractions which suppressed the low-level chemiluminescence. 4. The discovered components differed from each other by their molecular masses, kinetics of chemiluminescence inhibition and effects on intensity of MDA formation. 5. The putative functional role of antioxidative defence factors from rat liver cytosol is discussed.     

Studies on hepatic injury and antioxidant enzyme activities in rat subcellular organelles following in vivo ischemia and reperfusion

The activities of rat hepatic subcellular antioxidant enzymes were studied during hepatic ischemia/reperfusion. Ischemia was induced for 30 min (reversible ischemia) or 60 min (irreversible ischemia). Ischemia was followed by 2 or 24 h of reperfusion. Hepatocyte peroxisomal catalase enzyme activity decreased during 60 min of ischemia and declined further during reperfusion. Peroxisomes of normal density (d = 1.225 gram/ml) were observed in control tissues. However, 60 min of ischemia also produced a second peak of catalase specific activity in subcellular fractions corresponding to newly formed low density immature peroxisomes (d = 1.12 gram/ml). The second peak was also detectable after 30 min of ischemia followed by reperfusion for 2 or 24 h. Mitochondrial and microsomal fractions responded differently. MnSOD activity in mitochondria and microsomal fractions increased significantly (p < 0.05) after 30 min of ischemia, but decreased below control values following 60 min of ischemia and remained lower during reperfusion at 2 and 24 h in both organelle fractions. Conversely, mitochondrial and microsomal glutathione peroxidase (GPx) activity increased significantly (p < 0.001) after 60 min of ischemia and was sustained during 24 h of reperfusion. In the cytosolic fraction, a significant increase in CuZnSOD activity was noted following reperfusion in animals subjected to 30 min of ischemia, but 60 min of ischemia and 24 h of reperfusion resulted in decreased CuZnSOD activity. These studies suggest that the antioxidant enzymes of various subcellular compartments respond to ischemia/reperfusion in an organelle or compartment specific manner and that the regulation of antioxidant enzyme activity in peroxisomes may differ from that in mitochondria and microsomes. The compartmentalized changes in hepatic antioxidant enzyme activity may be crucial determinant of cell survival and function during ischemia/reperfusion. Finally, a progressive decline in the level of hepatic reduced glutathione (GSH) and concomitant increase in serum glutamate pyruvate transaminase (SGPT) activity also suggest that greater tissue damage and impairment of intracellular antioxidant activity occur with longer ischemia periods, and during reperfusion.     

Elongation and desaturation of arachidonic and eicosapentaenoic acids in rat liver. Effect of clofibrate feeding.

The fatty acid elongation-desaturation ability of 5,8,11,14-eicosatetraenoic (20:4(n-6)) and 5,8,11,14,17-eicosapentaenoic (20:5(n-3)) acids was determined in both liver microsomal and light mitochondrial (rich in peroxisomes) fractions of untreated and clofibrate treated rats. The elongation and the subsequent desaturation steps were performed in the corresponding favorable media. 20:5(n-3) elongation was about 2-times more extensive than that of 20:4(n-6). Clofibrate feeding for 10 days resulted in a marked decrease in the elongation rate with the two substrates, while the delta 4 desaturation rate was increased. There were small differences in the elongation rate between the microsomal and light mitochondrial fractions, however, the relative delta 4 desaturation rate was higher in the light mitochondrial fraction than microsomes.     

The significance of promitochondrial structures in rat liver for mitochondrial biogenesis

1. The heavy, light and fluffy mitochondrial fractions obtained by differential centrifugation were further characterized with respect to their protein synthesizing ability in vitro, their nucleic acid content, buoyant density of their DNA and ultrastructure. 2. The light mitochondrial fraction synthesized proteins in vitro at a rate 4-5 times as high as heavy and fluffy mitochondria. The incorporation ability of this fraction was also maximally affected by the thyroid status of the animal. The radioactivity in leucyl-tRNA of the light mitochondrial fraction was about 3-4 times as high as that of the other two fractions. 3. The heavy, light and fluffy mitochondrial fractions contained small but consistent amounts of RNA and DNA. Although the DNA content was the same in all mitochondria fractions, the light mitochondria contained relatively more RNA. The buoyant density of DNA from all the fractions was 1.701g/cm(3). 4. Electron microscopy revealed that the heavy mitochondria have a typical mitochondrial architecture, with densely packed cristae and a well developed double membrane. Light mitochondria were also surrounded by double membranes, but were smaller in size and contained less cristae. The fluffy fraction consisted of a mixture of well formed mitochondria and those in the process of degradation. 5. The significance of these findings in relation to mammalian mitochondrial genesis is discussed.     

Studies on the sterols and sterol esters of the intracellular organelles of maize shoots

1. The composition of the esterified and unesterified sterols of the nuclear, chloroplastidic, mitochondrial and microsomal fractions of 21-day-old maize shoots was examined. 2. The microsomal and mitochondrial fractions contain the bulk of the sterols of the tissue. 3. Only 1% of the sterol isolated from all the organelles is esterified. 4. The nuclear fraction has the greatest proportion of esterified sterol and the microsomal fraction the least. 5. 4-Demethyl sterols constitute the bulk of both esterified and unesterified sterols in all organelle fractions. 6. Cholesterol is the major esterified 4-demethyl sterol of the nuclear and chloroplastidic fractions, but only the nuclear fraction has an appreciable proportion of unesterified cholesterol. 7. Sterol esters of linolenic acid are more abundant in the mitochondrial and microsomal fractions than in the other two fractions.     

The role of mitochondrial nitric oxide synthase in inflammation and septic shock

Nitric oxide and cytokines constitute the molecular markers and the intercellular messengers of inflammation and septic shock. Septic shock occurs with an exacerbated inflammatory response that damages tissue mitochondria. Skeletal muscle appears as one of the main target organs in septic shock, showing an increased nitric oxide (NO) production, an early oxidative stress, and contractile failure. Mitochondria isolated from rat and human skeletal muscle in septic shock show a markedly increased NO generation and a decreased state 3 respiration, more marked with nicotinamide adenine dinucleotide (NAD)-linked substrates than with succinate, without uncoupling or impairment of phosphorylation. One of the current hypothesis for the molecular mechanisms of septic shock is that the enhanced NO production by mitochondrial nitric oxide synthase (mtNOS) leads to excessive peroxynitrite (ONOO(-)) production and protein nitration in the mitochondrial matrix, to mitochondrial dysfunction and to contractile failure. Surface chemiluminescence is a useful assay to assess inflammation and oxidative stress in in situ liver and skeletal muscle. Liver chemiluminescence in inflammatory processes and phagocyte chemiluminescence have been found spectrally different from spontaneous liver chemiluminescence with increased 440-600 nm emission, likely due to NO and ONOO(-) participation in the reactions leading to the formation of excited species.     

Kinetics of the alanine aminotransferase reaction in the mitochondrial and cell sap fractions of rat brain.

1. A reversible transamination reaction between L-glutamate and pyruvate, or L-alanine and 2-oxoglutarate, takes place in the mitochondrial and cell sap fractions of rat brain. 2. The maximum rate of the transamination reaction in both subfractions was observed in the presence of a keto- substrate concentration of 2.5 mM only, but an amino- donor concentration of 20 mM. 3. The apparent Menten-Michaelis constants for pyruvate and 2-oxoglutarate were of a 10(-4) M and for L-glutamate and L-alanine of a 10(-3) M order and were approximately the same for both fractions. 4. The ratio of the initial rate of the L-alanine + 2-oxoglutarate to the L-glutamate + pyruvate transamination reaction in the cell sap and mitochondrial fractions amounted to up to 2. 5. The apparent equilibrium constant derived from the Haldane equation was 7.01 for cell sap alanine aminotransferase and 4 for the mitochondrial enzyme. 6. Increasing pyridoxal-5'-phosphate concentrations in the incubation medium were accompanied by only non-significant stimulation of alanine aminotransferase activity in the mitochondrial and cell sap fractions. 7. A comparison of the kinetic data obtained on mitochondrial and cell sap alanine aminotransferases in vitro with the actual substrate concentrations in the transamination reaction in nervous tissue in vivo indicates that the direction of the transamination reaction in situ seems to be determined simply by compartmentation and by dynamic changes in amino- and keto- substrates in the mitochondrial and cell sap spaces.     

Effects of oestradiol, testosterone and medroxyprogesterone on subcellular fraction marker enzyme activities from rat liver and brain

The following enzymes have been studied (subcellular fractions are shown between parentheses): NAG and beta-glucuronidase (lysosomes); SDH (mitochondrial); glucose-6-phosphatase (endoplasmic reticulum); 5'-nucleotidase and (Na+, K+)Mg2+ ATPase (plasma membranes). Alterations on their activities were observed after subcutaneous injection of sex hormones, compared with controls. NAG activity from liver was always significantly decreased in lysosomal and microsomal fractions after the hormonal treatment. In the same conditions, NAG from brain was always increased. beta-Glucuronidase behaves like NAG in brain; in liver it was not modified by testosterone and it was slightly increased in lysosomal fraction after oestradiol treatment. SDH activity was not modified in mitochondrial fractions from liver, but this activity was always significantly increased in brain. Glucose-6-phosphatase activity was always significantly decreased in microsomal fractions from liver. It was increased in brain after oestradiol and testosterone injection, but medroxyprogesterone treatment caused a decreased activity. 5'-Nucleotidase and (Na+, K+)Mg2+ ATPase from brain were significantly increased in microsomal fractions by oestradiol and testosterone. Medroxyprogesterone, however, caused an increase in ATPase, but did not affect 5'-nucleotidase. Both activities in liver were decreased by oestradiol and increased by testosterone, but medroxyprogesterone caused (Na+, K+)Mg2+ ATPase to rise and 5'-nucleotidase to fall.     

The subcellular distribution and properties of hexokinases in the guinea-pig cerebral cortex

1. Hexokinase activities were estimated in primary subcellular fractions from guinea-pig cerebral cortex and in sucrose-density-gradient subfractions of the mitochondrial and microsomal fractions. 2. Appreciable activities were observed in mitochondrial, microsomal and soluble fractions. The activity in the mitochondrial fraction was associated with the mitochondria rather than with myelin or nerve endings and that in the microsomal fraction was associated with membrane fragments. 3. Most of the mitochondrial activity was extracted in soluble form by osmotic ;shock'. The activity of the mitochondrial extract differed from the soluble activity in kinetic properties and in electrophoretic behaviour. 4. No evidence was obtained for the presence of a high-K(m) glucokinase in the brain. 5. The results are discussed in terms of relevance to considerations of glucose utilization by the brain.