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1.
We investigated reports that mitochondria isolated from hypothyroid rats have decreased ADP/O and H+/O ratios. We observed no decrease in the H+/O ratio in mitochondria from hypothyroid rats, in the presence of either 2% (w/v) fatty-acid-free bovine serum albumin or 100 nM free Ca2+. The ADP/O ratio in mitochondria isolated from hypothyroid rats in the presence of 2% fatty-acid-free bovine serum albumin was measured. Under normal experimental conditions we found no decrease in the ADP/O ratio, relative to that measured for littermate controls. At the low concentrations of mitochondrial protein used in the previously reported studies, the ADP/O ratio of mitochondria from hypothyroid rats was decreased, whereas that for control rats was only slightly decreased. The difference between the ADP/O ratios measured for mitochondria form hypothyroid rats and from control rats under these conditions was eliminated by inhibition of endogenous adenylate kinase. We suggest that the lowering of the apparent ADP/O ratio in mitochondria from hypothyroid rats at low concentrations of mitochondrial protein is an experimental artefact resulting from the breakdown of ADP to AMP.  相似文献   

2.
W E Thomas  J Mowbray 《FEBS letters》1987,223(2):279-283
Triiodothyronine in vitro at concentrations between 10(-13) and 10(-11) M very rapidly activates oxidative phosphorylation in hypothyroid rat liver mitochondria. Comparing the concentrations of hormone with estimates of the amounts of respiratory chain components present suggests that this activation may involve an amplification mechanism. Here we present evidence that while no changes in phosphorylation were detected following hormone administration, nicotinamide, an inhibitor of mono ADP-ribosylation reported to occur rapidly and reversibly in mitochondria, prevented activation by hormone. Moreover incubation with nicotinamide of euthyroid mitochondria and derived intact inner membrane vesicles revealed lowered ADP/O ratios under the same conditions as shown by hypothyroid preparations. While this lesion could be reversed simply by washing the intact mitochondria, the membrane vesicles required triiodothyronine addition.  相似文献   

3.
The mean resting concentration of cytosolic free Ca2+ [( Ca2+]i) in parenchymal liver cells, as determined with the intracellular Ca2+ indicator quin2, was lowered by about 30% in hypothyroidism (0.17 microM vs. 0.27 microM in normal cells). The [Ca2+]i level in hypothyroid cells at 10 s following stimulation by noradrenaline (1 microM) was about 64% lower than in normal cells (0.33 microM vs. 1.0 microM). The response to noradrenaline in hypothyroid cells was slower in onset (significant at 5 s vs. 3 s in euthyroid cells), and the maximum of the initial [Ca2+]i increase was reached later (14 s vs. 8 s in normal cells). In hypothyroid hepatocytes the initial increase was followed by a slow but prolonged secondary increase in [Ca2+]i. With vasopressin similar results were found. Chelation of extracellular Ca2+ with EGTA immediately prior to stimulation had no effect on the initial [Ca2+]i increase. Treatment with T3 in vivo (0.5 micrograms/100 g body weight daily during 3 days) completely restored the basal and stimulated [Ca2+]i in hypothyroid cells. The half-maximally effective dose of noradrenaline was the same in euthyroid and hypothyroid liver cells (1.8 X 10(-7) M). Hypothyroidism had no significant effect on the number of alpha 1-receptors determined by [3H]prazosin labeling in crude homogenate fractions, while the Kd for [3H]prazosin was 21% lower than in the euthyroid group. These results show that thyroid hormone has a general stimulating effect on intracellular Ca2+ mobilization by Ca2+-mobilizing hormones, probably at a site distal to the binding of the agonist to its receptor. The results also support our idea that thyroid hormone may control metabolism during rest and activation, at least partially, by altering Ca2+ homeostasis.  相似文献   

4.
The effects of the thyroid state on the cytosolic free Ca2+ concentration, [Ca2+]i, of resting and K+-depolarized cardiomyocytes were studied using the fluorescent Ca2+ indicator fura2. The mean resting [Ca2+]i in euthyroid myocytes (89 +/- 8 nM) was not significantly different from that in hyperthyroid myocytes (100 +/- 14 nM). The resting O2-consumption rate was identical for both groups when expressed per mg protein, but a 35% higher value was observed in the hyperthyroid group when expressed per cell on account of the cellular hypertrophy induced by thyroid hormone. Potassium induced depolarization (50 mM [K+]0) raised the level of [Ca2+]i by 50% in both groups. When ATP-coupled respiration was blocked with oligomycin, the 50 mM K+-induced rise in [Ca2+]i was accompanied in both groups by a 40% rise in glycolytic activity as inferred from measurement of lactate production. Ca2+-fluorescence transients were recorded from electrically stimulated myocytes of euthyroid, hyperthyroid and hypothyroid rats. The time taken to reach peak fluorescence (TPL) and that to 50% decay of peak fluorescence (RL0.5) decreased in the direction hypothyroid----hyperthyroid, indicating an increase in Ca2+ fluxes in the same direction. Isoproterenol (1 microM) enhanced the peak Ca2+ fluorescence in electrically stimulated hypothyroid and euthyroid myocytes but not in hyperthyroid myocytes. Both the TPL and RL0.5 were decreased by isoproterenol in euthyroid, but more so in hypothyroid myocytes. None of these parameters were influenced by isoproterenol in the hyperthyroid group. We conclude that (1) thyroid hormone increases neither the O2-consumption rate nor the level of [Ca2+]i of resting cardiomyocytes and (2) the effects of the beta-receptor-agonist isoproterenol on Ca2+ transients of electrically stimulated myocytes, are inversely related to the documented changes in beta-receptor density in heart tissue occurring with alterations in the thyroid state.  相似文献   

5.
Stimulation of insulin secretion in the pancreatic beta-cell by a fuel such as glucose requires the metabolism of the fuel and is accompanied by increases in oxygen consumption and intracellular free Ca2+. A very early signal for these events could be a decrease in the cytosolic ATP/ADP ratio due to fuel phosphorylation. To test this hypothesis the regulation of free Ca2+ was evaluated in permeabilized RINm5F insulinoma cells that sequester Ca2+ and maintain a low medium free Ca2+ concentration (set point), between 100 and 200 nM, in the presence of Mg2+ and ATP. ATP, creatine, creatine phosphate, and creatine phosphokinase were added to the media to achieve various constant ratios of ATP/ADP. Free Ca2 was monitored using fura-2. The results demonstrated that the steady-state free Ca2+ concentration varied inversely with the ATP/ADP ratio and orthophosphate (Pi) levels. In contrast, no correlation between free Ca2+ and the phosphorylation potential (ATP/ADP.Pi) was found. Regulation of the Ca2+ set point by the ATP/ADP ratio was observed at ratios between 5 and 50 and at Pi concentrations between 1 and 7 mM, irrespective of whether mitochondria were participating in Ca2+ sequestration or were inhibited. Increasing the ATP/ADP ratio stimulated Ca2+ uptake by the nonmitochondrial pool but did not modify Ca2+ efflux. Glucose 6-phosphate (1 mM) had no effect on the Ca2+ set point. The data suggest that variations in the cytosolic ATP/ADP ratio induced by fuel stimuli may regulate Ca2+ cycling across nonmitochondrial compartments and the plasma membrane by modulating the activity of Ca2+ -ATPases. A mechanism linking fuel metabolism and cytosolic ATP/ADP ratio to activation of the Ca2+ messenger system in pancreatic beta-cells is proposed.  相似文献   

6.
We explored the possibility that the hormone 3,3',5-tri-iodothyronine can regulate the biosynthesis of the mitochondrial calcium uniporter. To meet this objective experiments on Ca(2+) transport, and binding of the specific inhibitor Ru(360) were carried out in mitochondria isolated from euthyroid, hyperthyroid and hypothyroid rats. It was found that V(max) for Ca(2+) transport increased from 11.67+/-0.8 in euthyroid to 14.36+/-0.44 in hyperthyroid, and decreased in hypothyroid mitochondria to 8.62+/-0.63 nmol Ca(2+)/mg/s. Furthermore, the K(i) for the specific inhibitor Ru(360), depends on the thyroid status, i.e. 18, 19 and 13 nM for control, hyper- and hypothyroid mitochondria, respectively. In addition, the binding of 103Ru(360) was increased in hyperthyroid and decreased in hypothyroid mitochondria. Scatchard analysis for the binding of 103Ru(360) showed the following values: 28, 40 and 23 pmol/mg for control, hyper- and hypothyroid mitochondria, respectively. The K(d) for 103Ru(360) was found to be 30.39, 37.03 and 35.71 nM for controls, hyper- and hypothyroid groups, respectively. When hypothyroid rats were treated with thyroid hormone, mitochondrial Ca(2+) transport, as well as 103Ru(360) binding, reached similar values to those found for euthyroid mitochondria.  相似文献   

7.
Mitochondria from the livers of thyroidectomized rats have a lowered ADP/O ratio, which can be restored to normal within 15 min after intravenous injection of a near-physiological dose of tri-iodothyronine. Thyroidectomy lowered the measured delta pH, which appears to be compensated by a rise (not statistically significant) in the membrane potential, so that the protonmotive force is unaltered. A simple simulation technique is described for use in estimating H+/O ratios by the oxygen-pulse technique, which circumvents the problem that this ratio can be seriously underestimated because of re-uptake of protons from the bulk phase by the mitochondria before their expulsion is complete. By this procedure the H+/O ratio of hypothyroid mitochondria is shown to be lowered by the same factor as the ADP/O ratio, and both these ratios are very rapidly restored in parallel by hormone administration. Although these findings could be consistent with a proposal that tri-iodothyronine rapidly modulates by some mechanism the efficiency of the respiratory-chain-linked proton pumps, the kinetic properties of the proton exchange suggest that the bulk-phase protons measured may not reflect faithfully those that drive the ATP synthetase.  相似文献   

8.
The relation between Ca2+ efflux, Ca2+ mobilization from mitochondria and glycogenolysis was studied in perfused euthyroid and hypothyroid rat livers stimulated by Ca2+-mobilizing hormones. Ca2+ efflux, induced by noradrenaline (1 microM) in the absence or presence of DL-propranolol (10 microM) from livers perfused with medium containing a low concentration of Ca2+ (approx. 24 microM), was decreased by more than 50% in hypothyroidism. This correlated with an equal decrease of the fractional mobilization of mitochondrial Ca2+, which could account for 65% of the difference between the net amounts of Ca2+ expelled from the euthyroid and hypothyroid livers. With vasopressin (10 nM) similar results were found, suggesting that hypothyroidism has a general effect on mobilization of internal Ca2+. In normal Ca2+ medium (1300 microM), however, the effect of vasopressin on net Ca2+ fluxes and phosphorylase activation was not impaired in hypothyroidism, indicating that Ca2+ mobilization from the mitochondria in this case plays a minor role in phosphorylase activation. The alpha 1-adrenergic responses of Ca2+ efflux, phosphorylase activation and glucose output, glucose-6-phosphatase activity and oxygen consumption in hypothyroid rat liver were completely restored by in vivo T3 injections (0.5 micrograms per 100 g body weight, daily during 3 days). Perfusion with T3 (100 pM) during 19 min did not influence hypothyroid rat liver oxygen consumption and alpha 1-receptor-mediated Ca2+ efflux. However, this in vitro T3 treatment showed a completely recovered alpha 1-adrenergic response of phosphorylase and a partly restored glucose-6-phosphatase activity and glucose output. The results indicate that thyroid hormones may control alpha 1-adrenergic stimulation of glycogenolysis by at least two mechanisms, i.e., a long-term action on Ca2+ mobilization, and a short-term action on separate stages of the glycogenolytic process.  相似文献   

9.
We have determined the relationship between rate of respiration and protonmotive force in oligomycin-inhibited liver mitochondria isolated from euthyroid, hypothyroid and hyperthyroid rats. Respiration rate was titrated with the respiratory-chain inhibitor malonate. At any given respiration rate mitochondria isolated from hypothyroid rats had a protonmotive force greater than mitochondria isolated from euthyroid controls, and mitochondria isolated from hyperthyroid rats had a protonmotive force less than mitochondria isolated from euthyroid controls. In the absence of malonate mitochondrial respiration rate increased in the order hypothyroid less than euthyroid less than hyperthyroid, while protonmotive force increased in the order hyperthyroid less than euthyroid less than hypothyroid. These findings are consistent with a thyroid-hormone-induced increase in the proton conductance of the inner mitochondrial membrane or a decrease in the H+/O ratio of the respiratory chain at any given protonmotive force. Thus the altered proton conductance or H+/O ratio of mitochondria isolated from rats of different thyroid hormone status controls the respiration rate required to balance the backflow of protons across the inner mitochondrial membrane. We discuss the possible relevance of these findings to the control of state 3 and state 4 respiration by thyroid hormone.  相似文献   

10.
We show in the accompanying paper that the steady-state level of free Ca2+ maintained by the organelles of permeabilized RINm5F insulinoma cells varies inversely with the ATP/ADP ratio when this ratio is set by addition of creatine phosphokinase and fixed ratios of creatine to creatine phosphate. We, therefore, asked whether acute cyclic alterations in the cytosolic ATP/ADP ratio in the range known to modulate O2 consumption might be involved in regulating the physiological activity of Ca2+ -ATPases and the cytosolic free Ca2+ level. To explore this hypothesis we combined two experimental systems: 1) permeabilized RINm5F insulinoma cells that can maintain a low medium Ca2+ concentration and 2) a cell-free extract of rat skeletal muscle that spontaneously exhibits oscillatory behavior of glycolysis and linked oscillations in the ATP/ADP ratio, when provided with glucose. The free Ca2+ level maintained by the permeabilized cells oscillated in phase with the glycolytic oscillations and correlated closely with the ATP/ADP ratio but not with glucose 6-phosphate, fructose 6-phosphate, orthophosphate, or pH. When glucokinase replaced hexokinase as the glucose phosphorylating enzyme, Ca2+ oscillations were induced by increasing the glucose concentration from 2 to 8 mM. The results demonstrate a link between metabolite changes and free Ca2+ levels in a reconstituted physiological system. They support a model in which oscillations in glycolysis and the ATP/ADP ratio may cause oscillations in cytosolic free Ca2+, beta-cell electrical activity, and insulin release.  相似文献   

11.
The effects of beta-adrenergic stimulation on the relaxation rate and the Ca2+-transport rate in sarcoplasmic reticulum of hypothyroid, euthyroid and hyperthyroid rat hearts were studied. Administration of isoproterenol (0.1 microM) to perfused, electrically stimulated hearts (5 Hz) caused a decrease in the half-time of relaxation (RT 1/2) the extent of which depended on the thyroid status, i.e. hypothyroid (-24%), euthyroid (-19%) or hyperthyroid (-8%). A similar decreasing effect was found for the stimulation of Ca2+ transport in isolated SR by cyclic AMP and protein kinase, i.e. hypothyroid (75%), euthyroid (37%) and hyperthyroid (20%). These alterations were not due to differences in endogenous protein kinase activity or cyclic AMP production. Estimations of Ca2+-ATPase and phospholamban (PL) content of the sarcoplasmic reticulum were obtained by measurement of the phosphorylated forms of Ca2+-ATPase (E-P) and phospholamban (PL-P) followed by electrophoresis and autoradiography. A 3-fold decrease of PL-P, accompanied by a 2-fold increase of E-P per mg of protein was observed in sarcoplasmic reticulum preparations in the direction hypothyroid----hyperthyroid. Consequently the E-P/PL-P ratio increased from 0.32 (hypothyroid), through 0.81 (euthyroid) to 1.69 (hyperthyroid). In spite of certain limitations inherent to quantification of Ca2+-ATPase and phospholamban by their phosphorylated products, these data provide strong evidence that during thyroid-hormone mediated cardiac hypertrophy, with concomitant proliferation of the sarcoplasmic reticulum, the relative amount of phospholamban decreases with respect to Ca2+-ATPase. This could provide an explanation for the observed gradual diminishment of the beta-adrenergic effect on the relaxation rate when cardiac tissue is exposed to increasing amounts of thyroid hormone.  相似文献   

12.
Cytosolic free Ca2+ rises in pancreatic beta-cells in response to glucose stimulation and is part of the coupling to insulin secretion. This study evaluates a possible role for cytosolic long chain acyl-CoA esters in modulating Ca2+ handling by clonal beta-cells (HIT). Intact cells incubated with 20 microM free palmitic acid exhibited a 40% decrease in basal cytosolic free Ca2+. In contrast, acyl-CoA esters, up to a chain length of 16, but not the corresponding fatty acids, significantly lowered the Ca2+ set point maintained by cells permeabilized with saponin. The maximum response to the various acyl-CoA esters increased with increasing chain length, with no differences in the half-maximally effective concentration of 0.5 microM. Long chain acyl-CoA esters caused a 40-50% increase in 45Ca2+ influx into a non-mitochondrial pool in the permeabilized HIT cells, consistent with a stimulatory effect on the endoplasmic reticulum Ca(2+)-ATPase activity, but did not affect inositol 1,4,5-trisphosphate-induced Ca(2+)-efflux. Thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase activity, blocked the decrease in the Ca2+ set point caused by acyl-CoA esters. The ability of acyl-CoA esters to lower the Ca2+ set point depended on the ATP/ADP ratio (or free ADP); the Ca2+ set point was lowered by 36 +/- 3.6% at an ATP/ADP ratio of 90 and by 14 +/- 1.9% at an ATP/ADP ratio of 7. Depletion of cellular protein kinase C did not prevent the acyl-CoA-induced lowering of the Ca2+ set point. These findings suggest that the increases in long chain acyl-CoA esters may play a role in restoring cytosolic free Ca2+ through activation of Ca(2+)-ATPases.  相似文献   

13.
The effect of thyroid-hormone application on cytosolic and mitochondrial ATP/ADP ratio was investigated in rat liver in vivo and in the isolated perfused organ. In vivo the ATP/ADP ratio in livers from hypothyroid rats was 0.84 +/- 0.08 in the mitochondrial matrix and 5.6 +/- 0.9 in the cytosol, as was observed in euthyroid controls. In contrast, hyperthyroidism was followed by a significant decrease in the mitochondrial and by an increase in the cytosolic ATP/ADP ratio (to 0.34 +/- 0.06 and 11.3 +/- 2.8 respectively). In the perfused liver from hypothyroid animals, addition of L-3,3',5-tri-iodothyronine in the perfusate also provoked, within 2 h, a significant decrease in the mitochondrial ATP/ADP ratio, whereas the cytosolic ratio was unaffected. From these and previous data in the isolated perfused liver and in isolated mitochondria from hypothyroid and tri-iodothyronine-treated rats it is concluded that thyroid hormones increase mitochondrial respiration and ATP regeneration, which is associated with an acceleration of mitochondrial adenine nucleotide transport and significant alterations in the mitochondrial and cytosolic ATP/ADP ratios.  相似文献   

14.
Spermine enhances electrogenic Ca2+ uptake and inhibits Na(+)-independent Ca2+ efflux in rat brain mitochondria. As a result, Ca2+ retention by brain mitochondria increases greatly and the external free Ca2+ level at steady-state can be lowered to physiologically relevant concentrations. The stimulation of Ca2+ uptake by spermine is more pronounced at low concentrations of Ca2+, effectively lowering the apparent Km for Ca2+ uptake from 3 microM to 1.5 microM. However, the apparent Vmax is also increased. At low Ca2+ concentrations, Ca2+ uptake is diffusion-limited. Spermine strongly inhibits Ca2+ binding to anionic phospholipids and it is suggested that this increases the rate of surface diffusion which reduces the apparent Km for uptake. The same effect could inhibit the Na(+)-independent efflux if the rate of efflux is limited by Ca2+ dissociation from the efflux carrier. In brain mitochondria (but not in liver) the spermine effect depends on the presence of ADP. In a medium that contains physiological concentrations of Pi, Mg+, K+, ADP and spermine, brain mitochondria sequester Ca2+ down to 0.1 microM and below, depending on the matrix Ca2+ load. Moreover, brain mitochondria under the same conditions buffer the external medium at 0.4 microM, a concentration at which the set point becomes independent of the matrix Ca2+ content. Thus, mitochondria appear to be capable of modulating calcium oscillations in brain cells.  相似文献   

15.
The influence of hypothyroidism on the transport of phosphate and on the lipid composition in rat-liver mitochondria was examined. It was found that the rate of phosphate transport is reduced (around 40%) in mitochondria from hypothyroid rats compared to that obtained in mitochondria from normal rats. Treatment of hypothyroid rats with thyroid hormone reverses this effect completely. Kinetic analysis of the phosphate transport indicates that only the Vmax of this process is affected, while there is no change in the Km values. The lower rate of phosphate transport in mitochondria from hypothyroid rats is also demonstrated by swelling experiments. There is no significant difference either in the respiratory control ratios or in the ADP/O ratios between these two types of mitochondria. The hepatic mitochondrial lipid composition is altered significantly in hypothyroid rats. The total cholesterol increases, the phospholipids decrease and the cholesterol/phospholipid molar ratio increases (around 40%). Among the phospholipids, cardiolipin shows the greatest alteration (30% decrease in the hypothyroid rats). The phosphatidylethanolamine/phosphatidylcholine ratio also decreases. Alterations were also found in the pattern of fatty acids. These changes in lipid composition may be responsible, at least in part, for the depression of the phosphate carrier activity in mitochondria from hypothyroid rats.  相似文献   

16.
The effects of the thyroid status on the cytosolic free Ca2+ concentration ([Ca2+]i) in single cardiomyocytes were studied at rest and during contraction. The mean resting [Ca2+]i increased significantly from the hypothyroid (45 +/- 4 nM) through the euthyroid (69 +/- 12 nM) to the hyperthyroid condition (80 +/- 11 nM) at extracellular Ca2+ concentrations ([Ca2+]o) up to 2.5 mM. At [Ca2+]o above 2.5 mM the differences in [Ca2+]i between the groups became less. The amplitude of the Ca2+ transients became higher in all groups with increasing [Ca2+]o (1, 2.5 and 5 mM), and was highest at all [Ca2+]o in hyperthyroid myocytes. The beta-agonist isoprenaline elevated peak [Ca2+]i during contraction and increased the rate of the decay of the Ca2+ transients to a greater extent in hypothyroid myocytes than in hyperthyroid myocytes. Depolarization with high [K+]o induced a large but transient [Ca2+]i overshoot in hypothyroid myocytes, but not in hyperthyroid myocytes, before a new elevated steady-state [Ca2+]i was reached, which was not different between the groups. When isoprenaline was added to K+ o-depolarized myocytes after a steady state was reached, a significantly larger extra increase in [Ca2+]i was measured in the hypothyroid group (28%) compared with the hyperthyroid group (8%). It is concluded that in cardiac tissue exposed to increasing amounts of thyroid hormones (1) [Ca2+]i increases at rest and during contraction in cardiomyocytes and (2) interventions which favour Ca2+ entry into the cytosol [( Ca2+]o elevation, high [K+]o, beta-agonists) tend to have less impact on Ca2+ homoeostasis.  相似文献   

17.
The effects of the thyroid state on oxidative damage, antioxidant capacity, susceptibility to in vitro oxidative stress and Ca(2+)-induced permeabilization of mitochondria from rat tissues (liver, heart, and gastrocnemious muscle) were examined. Hypothyroidism was induced by administering methimazole in drinking water for 15 d. Hyperthyroidism was elicited by a 10 d treatment of hypothyroid rats with triiodothyronine (10 micro g/100 g body weight). Mitochondrial levels of hydroperoxides and protein-bound carbonyls significantly decreased in hypothyroid tissues and were reported above euthroid values in hypothyroid rats after T(3) treatment. Mitochondrial vitamin E levels were not affected by changes of animal thyroid state. Mitochondrial Coenzyme Q9 levels decreased in liver and heart from hypothyroid rats and increased in all hyperthyroid tissues, while Coenzyme Q10 levels decreased in hypothyroid liver and increased in all hyperthyroid tissues. The antioxidant capacity of mitochondria was not significantly different in hypothyroid and euthyroid tissues, whereas it decreased in the hyperthyroid ones. Susceptibility to in vitro oxidative challenge decreased in mitochondria from hypothyroid tissues and increased in mitochondria from hyperthyroid tissues, while susceptibility to Ca(2+)-induced swelling decreased only in hypothyroid liver mitochondria and increased in mitochondria from all hyperthyroid tissues. The tissue-dependence of the mitochondrial susceptibility to stressful conditions in altered thyroid states can be explained by different thyroid hormone-induced changes in mitochondrial ROS production and relative amounts of mitochondrial hemoproteins and antioxidants. We suggest that susceptibilities to oxidants and Ca(2+)-induced swelling may have important implications for the thyroid hormone regulation of the turnover of proteins and whole mitochondria, respectively.  相似文献   

18.
1. Examination of the distribution of L-tri-iodothyronine among rat liver tissue fractions after its intravenous injection into thyroidectomized rats focused attention on mitochondria at very short times after administration. By 15 min this fraction contained 18.5% of the tissue pool; however, the content had decreased sharply by 60 min and even further over the next 3 h. By contrast, the content in all other fractions was constant or increased over 4 h. About 60% of tissue hormone was bound to soluble protein. 2. Mitochondria isolated from thyroidectomized rats showed P/O ratios that were about 50% of those found in normal controls, with both succinate and pyruvate plus malate as substrates. There was no evidence of uncoupling; the respiratory-control ratio was about 6. 3. Mitochondria isolated 15 min after injection of tri-iodothyronine into thyroidectomized rats showed P/O ratios and respiratory-control ratios that were indistinguishable from those obtained in mitochondria from euthyroid animals. The oxidation rate was, however, not restored. 4. Incubation of homogenates of livers taken from thyroidectomized animals injected with L-tri-iodothyronine before isolation of the mitochondria restored the P/O ratio to normal; by contrast, direct addition of hormone to isolated mitochondria had no effect. The role of extramitochondrial factors in rapid tri-iodothyronine action is discussed. 5. Possible mechanisms by which tri-iodothyronine might rapidly alter phosphorylation efficiency are considered: it is concluded that control of adenine nucleotide translocase is unlikely to be involved. 6. The amounts of adenine nucleotides in liver were measured both after thyroidectomy and 15 min after intravenous tri-iodo-thyronine administration to thyroidectomized animals. The concentrations found are consistent with a decreased phosphorylation efficiency in thyroidectomized animals. Tri-iodothyronine injection resulted in very significant changes in the amounts of ATP, ADP and AMP, and in the [ATP]/[ADP] ratio, consonant with those expected from an increased efficiency of ADP phosphorylation. This suggests that the changes seen in isolated mitochondria may indeed reflect a rapid response of liver in vivo to tri-iodo-thyronine.  相似文献   

19.
The purpose of this study was to examine the effects of oxidative stress caused by hydroperoxide (H(2)O(2)) in the presence of iron ions (Fe(2+)) on mitochondria of the amoeba Acanthamoeba castellanii. We used isolated mitochondria of A. castellanii and exposed them to four levels of H(2)O(2) concentration: 0.5, 5, 15, and 25 mM. We measured basic energetics of mitochondria: oxygen consumption in phosphorylation state (state 3) and resting state (state 4), respiratory coefficient rates (RC), ADP/O ratios, membrane potential (DeltaPsi(m)), ability to accumulate Ca(2+) , and cytochrome c release. Our results show that the increasing concentrations of H(2)O(2) stimulates respiration in states 3 and 4. The highest concentration of H(2)O(2) caused a 3-fold increase in respiration in state 3 compared to the control. Respiratory coefficients and ADP/O ratios decreased with increasing stress conditions. Membrane potential significantly collapsed with increasing hydroperoxide concentration. The ability to accumulate Ca(2+) also decreased with the increasing stress treatment. The lowest stress treatment (0.5 mM H(2)O(2)) significantly decreased oxygen consumption in state 3 and 4, RC, and membrane potential. The ADP/O ratio decreased significantly under 5 mM H(2)O(2) treatment, while Ca(2+) accumulation rate decreased significantly at 15 mM H(2)O(2). We also observed cytochrome c release under increasing stress conditions. However, this release was not linear. These results indicate that as low as 0.5 mM H(2)O(2) with Fe(2+) damage the basic energetics of mitochondria of the unicellular eukaryotic organism Acanthamoeba castellanii.  相似文献   

20.
1. In uncoupled rat heart mitochondria, the kinetic parameters for oxoglutarate oxidation were very close to those found for oxoglutarate dehydrogenase activity in extracts of the mitochondria. In particular, Ca2+ greatly diminished the Km for oxoglutarate and the k0.5 value (concentration required for half-maximal effect) for this effect of Ca2+ was close to 1 microM. 2. In coupled rat heart mitochondria incubated with ADP, increases in the extramitochondrial concentration of Ca2+ greatly stimulated oxoglutarate oxidation at low concentrations of oxoglutarate, but not at saturating concentrations of oxoglutarate. The k0.5 value for the activation by extramitochondrial Ca2+ was about 20 nM. In the presence of either Mg2+ or Na+ this value was increased to about 90 nM, and in the presence of both to about 325 nM. 3. In coupled rat heart mitochondria incubated without ADP, increases in the extramitochondrial concentration of Ca2+ resulted in increases in the proportion of pyruvate dehydrogenase in its active non-phosphorylated form. The sensitivity to Ca2+ closely matched that found to affect oxoglutarate oxidation, and Mg2+ and Na+ gave similar effects. 4. Studies of others have indicated that the distribution of Ca2+ across the inner membrane of heart mitochondria is determined by a Ca2+-transporting system which is composed of a separate uptake component (inhibited by Mg2+ and Ruthenium Red) and an efflux component (stimulated by Na+). The present studies are entirely consistent with this view. They also indicate that the intramitochondrial concentration of Ca2+ within heart cells is probably about 2--3 times that in the cytoplasm, and thus the regulation of these intramitochondrial enzymes by Ca2+ is of likely physiological significance. It is suggested that the Ca2+-transporting system in heart mitochondria may be primarily concerned with the regulation of mitochondrial Ca2+ rather than cytoplasmic Ca2+; the possible role of Ca2+ as a mediator of the effects of hormones and neurotransmitters on mammalian mitochondrial oxidative metabolism is discussed.  相似文献   

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