Distribution of activities of aspartate aminotransferase isoenzymes and malate dehydrogenase in guinea pig retinal layers

Distributions of activity of the cytosolic (cAAT) and mitochondrial (mAAT) isoenzymes of aspartate aminotransferase and of malate dehydrogenase (MDH) were determined in guinea pig retinal layers. The distribution of total AAT activity (tAAT = cAAT + mAAT) and of mAAT activity correlated well (r = 0.88-0.91) with the distribution of MDH activity. mAAT activity was highest in the inner segments of the photoreceptors; there was a greater than twelve-fold difference between activity in that layer and in the inner retinal layers. cAAT activity was also highest in the inner segments, but the difference between the activity in the inner segments and the other layers was not nearly as great as with mAAT. cAAT activity was also relatively high in the outer nuclear layer, outer plexiform layer, and part of the inner plexiform layer. The high activity of cAAT, mAAT, and MDH in the inner segments indicates that all of these enzymes are involved in metabolic reactions related to energy production and/or to photoreceptive processes in the outer segments and, therefore, that the enzymes are probably involved in energy-related metabolism at synapses. However, other functions, including those related to neurotransmission, are not excluded.     

Testosterone stimulation of mitochondrial aspartate aminotransferase levels and biosynthesis in rat ventral prostate

The effects of testosterone on mitochondrial aspartate aminotransferase (mAAT) synthesis in rat ventral prostate was investigated. Procedures for the isolation, purification and characterization of AAT isozymes were developed and described. Purified mAAT preparations contained no demonstrable contaminating proteins. Prostatic mAAT was characterized as a cationic protein with an estimated mol. wt of 120,000. Cytoplasmic AAT (cAAT) isozyme was identified as an anionic protein with an estimated mol. wt of 132,000. A cytosolic cationic isozyme, similar to mAAT, was also identified as pre-mAAT. Testosterone administration to castrated rats resulted in significant increases in leucine incorporation into mAAT, in the level of mAAT, and in mAAT activity. These effects of testosterone were observed within 2 h of administration. Conversely, testosterone administration had none of these effects on cAAT or on non-AAT protein pool. Testosterone treatment did appear to increase leucine incorporation into pre-mAAT. Testosterone treatment in organ cultures and in prostate epithelial cell cultures resulted in the same stimulatory effects on mAAT as observed in the in vivo studies. The hormone was effective at the physiological concentration of 2 X 10(-9) M. These results indicated that testosterone has a rapid and specific effect on the biosynthesis of mAAT. This continues to support our proposal that testosterone regulates prostate citrate production via a stimulatory effect on mAAT which results in increased mitochondrial synthesis of citrate from aspartate.     

Stability and release requirements of the complexes of GroEL with two homologous mammalian aminotransferases

The mitochondrial (mAAT) and cytosolic (cAAT) homologous isozymes of aspartate aminotransferase are two relatively large proteins that in their nonnative states interact very differently with GroEL. MgATP alone can increase the rate of GroEL-assisted reactivation of cAAT, yet the presence of GroES is mandatory for mAAT. Addition of an excess of a denatured substrate accelerates reactivation of cAAT in the presence of GroEL, but has no effect on mAAT. These competition studies suggest that the more stringent substrate mAAT forms a thermodynamically stable complex with GroEL, while rebinding affects the slow reactivation kinetics of cAAT with GroEL alone. However, the competitor appears to accelerate the release of cAAT from GroEL, most likely by displacing bound cAAT from the GroEL cavity. Moreover, cAAT, but not mAAT, shows a time-dependent increase in protease resistance while bound to GroEL at low temperature. These results suggest that folding and release of cAAT from GroEL in the absence of cofactors may occur stepwise with certain interactions being broken and reformed until the protein escapes binding. The distinct behavior of these two isozymes most likely results from differences in the structure of the nonnative states that bind to GroEL.     

Regulation of aspartate aminotransferase messenger ribonucleic acid level by testosterone

The effect of testosterone on precursor mitochondrial aspartate aminotransferase (pmAAT) mRNA was studied in rat ventral prostate and primary cell cultures of mini-pig prostate. Testosterone induced a 2-3-fold increase in pmAAT mRNA level in both rat ventral prostate and mini-pig prostate cultures. The pmAAT mRNA induction occurred 30 min after testosterone treatment and was maximal by 1.5 h. Prostatic mAAT activity was also induced by testosterone with a 1-2 h lag period. The time-course of induction of pmAAT mRNA, pmAAT activity and mAAT activity was consistent with stimulation of mRNA synthesis followed by increased synthesis and import of pmAAT into mitochondria. The effect of testosterone on pmAAT mRNA was specific because the increase in pmAAT mRNA was at least 2-fold greater than the increase in poly (A+) RNA. These results suggest that testosterone stimulated mAAT activity by induction of pmAAT mRNA. This continues to support our proposal that a major physiological effect of testosterone is increased pmAAT mRNA steady-state levels which result in increased pmAAT synthesis and increased mAAT activity. These changes ultimately result in increased citrate production by prostate epithelial cells.     

Glutamate Neurotoxicity Is Independent of Calpain I Inhibition in Primary Cultures of Cerebellar Granule Cells

Glutamate-induced neurotoxicity and calpain activity were studied in primary cultures of rat cerebellar granule neurons and glial cells. Calpain activation, as monitored by quantitative immunoblotting of spectrin, required micromolar concentrations of Ca2+ in neuronal homogenates (calpain I) and millimolar Ca2+ concentrations in glial homogenates (calpain II). Glutamate-induced toxicity and calpain activation were observed in neuronal, but not in glial, cultures. In neurons, calpain I activation by glutamate was dose-dependent and persisted after withdrawal of neurotoxic doses of glutamate. Natural (GM1) and semisynthetic (LIGA4) gangliosides or the glutamate receptor blocker MK-801 prevented calpain I activation and delayed neuronal death elicited by glutamate. GM1 and LIGA4 had no effect on calpain I activity in neuronal homogenates, however. Furthermore, two calpain I inhibitors (leupeptin and N-acetyl-Leu-Leu-norleucinal) prevented glutamate-induced spectrin degradation, but failed to affect glutamate neurotoxicity. These results thus suggest that glutamate-induced neurotoxicity is independent of calpain I activation.     

Influence of cytosolic and mitochondrial Ca2+, ATP,mitochondrial membrane potential,and calpain activity on the mechanism of neuron death induced by 3-nitropropionic acid

3-Nitropropionic acid (3NP), an irreversible inhibitor of succinate dehydrogenase, induces both rapid necrotic and slow apoptotic death in rat hippocampal neurons. Low levels of extracellular glutamate (10 microM) shift the 3NP-induced cell death mechanism to necrosis, while NMDA receptor blockade results in predominantly apoptotic death. In this study, we examined the 3NP-induced alterations in free cytosolic and mitochondrial calcium levels, ATP levels, mitochondrial membrane potential, and calpain and caspase activity, under conditions resulting in the activation of apoptotic and necrotic pathways. In the presence of 10 microM glutamate, 3NP administration resulted in a massive elevation in [Ca(2+)](c) and [Ca(2+)](m), decreased ATP, rapid mitochondrial membrane depolarization, and a rapid activation of calpain but not caspase activity. In the presence of the NMDA receptor antagonist MK-801, 3NP did not induce a significant elevation of [Ca(2+)](c) within the 24h time period examined, nor increase [Ca(2+)](m) within 1h. ATP was maintained at control levels during the first hour of treatment, but declined 64% by 16h. Calpain and caspase activity were first evident at 24h following 3NP administration. 3NP treatment alone resulted in a more rapid decline in ATP, more rapid calpain activation (within 8h), and elevated [Ca(2+)](m) as compared to the results obtained with added MK-801. Together, the results demonstrate that 3NP-induced necrotic neuron death is associated with a massive calcium influx through NMDA receptors, resulting in mitochondrial depolarization and calpain activation; while 3NP-induced apoptotic neuron death is not associated with significant elevations in [Ca(2+)](c), nor with early changes in [Ca(2+)](m), mitochondrial membrane potential, ATP levels, or calpain activity.     

Submicromolar Ca2+ regulates phosphorylating respiration by normal rat liver and AS-30D hepatoma mitochondria by different mechanisms

The stimulation of 2-oxoglutarate and NAD(+)-isocitrate dehydrogenase by Ca2+ in mitochondria from normal tissues has been proposed to mediate partially the activation of oxidative energy metabolism elicited by physiological elevations in cytosolic Ca2+. This mode of regulation may also occur in tumor cells in which several aspects of mitochondrial metabolism are known to be altered. This study provides a comparison of the stimulation by submicromolar concentrations of Ca2+ on the rates of ATP-generating (state 3) respiration under physiologically realistic conditions by mitochondria isolated from normal rat liver and from highly malignant rat AS-30D ascites hepatoma cells. The K0.5 for activation of glutamate-dependent state 3 respiration by Ca2+ in the presence of ATP at 37 degrees C was determined to be 0.70 +/- 0.05 (S.E.) microM for hepatoma mitochondria and 0.90 +/- 0.03 microM for rat liver mitochondria. This activation was also reflected by a Ca2(+)-induced shift in the oxidation-reduction state of hepatoma mitochondrial pyridine nucleotides to a more reduced level and Ca2+ stimulation of 14CO2 production from [1-14C]glutamate. Whereas the Ca2+ sensitivity of state 3 respiration by hepatoma mitochondria can be explained by the activation of 2-oxoglutarate and possibly NAD(+)-isocitrate dehydrogenases, the Ca2+ sensitivity of liver mitochondrial respiration appears to be predominantly mediated by activation of electron flow through ubiquinone and Complex III of the electron transport chain, as indicated by the specificity of the effects of Ca2+ on respiration with different oxidizable substrates. Although rat liver and hepatoma mitochondria employ different modes of Ca2(+)-activated ATP generation, these results support the hypothesis that changes in cytosolic Ca2+ play a significant role in the potentiation of energy production in tumor, as well as normal tissue.     

Study of the reorientation of coenzyme in active sites of aspartate-aminotransferase isoenzymes using linear dichroism method

Cytosolic and mitochondrial pig aspartate aminotransferases (cAAT and mAAT) and chicken cAAT were oriented in a compressed slab of polyacrylamide gel. Linear dichroism (LD) spectra of the pyridoxal and pyridoxamine forms of AATs and of complexes of the pyridoxal form with substrate analogues have been recorded. The tilt angles of the coenzyme at the intermediary steps of the transamination reaction have been calculated on the basis of reduced LD values (delta A/A), atomic coordinates of the coenzyme and directions of the transition dipole moments in the coenzyme ring. It was assumed that rotation of the coenzyme ring occurs around the C2-C5 axis in all cases except the enzyme complex with glutarate: in the latter case the direction N1-C4 was assumed to be a rotation axis. It has been found that formation of the enzyme complex with glutarate and protonation of the internal aldimine induce dissimilar reorientations of the coenzyme. As a result of protonation, the coenzyme tilts by 27 degrees in cAAT and 13 degrees in mAAT. Formation of the external aldimine with 2-methylaspartate is accompanied by tilting of the coenzyme ring by 44 degrees in cAAT and 39 degrees in mAAT. For the quinonoid complex with erythro-3-hydroxyaspartate, the tilt angles were found to be 63 degrees in cAAT and 53 degrees in mAAT. It was inferred that the basic features of the active site dynamics are similar in three AATs studied. The differences in the coenzyme tilt angles between cAAT and mAAT might be linked to catalytic peculiarities of the isoenzymes.     

Endoplasmic reticulum Ca(2+) signaling and calpains mediate renal cell death

The goal of the current study was to determine the roles of ATP content, endoplasmic reticulum (ER) Ca(2+) stores, cytosolic free Ca(2+) (Ca(2+)(f)) and calpain activity in the signaling of rabbit renal proximal tubular (RPT) cell death (oncosis). Increasing concentrations (0.3-10 microM) of the mitochondrial inhibitor antimycin A produced rapid ATP depletion that correlated to a rapid and sustained increase in Ca(2+)(f), but not phospholipase C activation. The ER Ca(2+)-ATPase inhibitors thapsigargin (5 microM) or cyclopiazonic acid (100 microM) alone produced similar but transient increases in Ca(2+)(f). Pretreatment with thapsigargin prevented antimycin A-induced increases in Ca(2+)(f) and antimycin A pretreatment prevented thapsigargin-induced increases in Ca(2+)(f). Calpain activity increased in conjunction with ER Ca(2+) release. Pretreatment, but not post-treatment, with thapsigargin or cyclopiazonic acid prevented antimycin A-induced cell death. These data demonstrate that extensive ATP depletion signals oncosis through ER Ca(2+) release, a sustained increase in Ca(2+)(f) and calpain activation. Depletion of ER Ca(2+) stores prior to toxicant exposure prevents increases in Ca(2+)(f) and oncosis.     

Calcium homeostasis in the thymocyte apoptosis II. Accumulation of calcium in mitochondria and endoplasmic reticulum

The activity of energy-dependent Ca2+-accumulation systems in rat thymocytes mitochondria and endoplasmic reticulum (ER) in control and at the early stage of X-irradiation or H2O2-induced apoptosis were determined in experiments using the model of digitonin-permeabilized cells with addition of thapsigargin and ruthenium red. The mitochondrial Ca2+-transporting system proved to be more sensitive to both apoptotic stimuli. The stationary level of Ca2+, accumulated in mitochondria and initial rate of Ca2+ accumulation in ER were reduced 15 min after H2O2 treatment. The parameters of mitochondrial Ca2+-accumulation system in irradiated cells were decreased 30 min after irradiation. Cyclosporin A almost completely inhibited DNA fragmentation in irradiated and partly--in peroxide-treated cells. The mitochondrial calcium homeostasis imbalance is suggested to be an early event in thymocytes apoptosis initiation.     

Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax

Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.     

Further studies on the effect of phosphoenolpyruvate on respiration-dependent calcium transport by rat heart mitochondria.

Phosphoenolpyruvate was found to depress extra oxygen consumption associated with Ca2+ -induced respiratory jump by rat heart mitochondria. Addition of phosphoenolpyruvate to mitochondria which have accumulated Ca2+ in the presence of glutamate and inorganic phosphate causes the release of Ca2+ from mitochondria. The phosphoenolpyruvate-stimulated Ca2+ efflux can be observed with mitochondria loaded with low initial Ca2+ concentration (0.12 mM) in the incubation medium. Measurements of mitochondrial H+ translocation produced by addition of Ca2+ to respiring mitochondria show that phosphoenolpyruvate depresses H+ ejection and enhances H+ uptake by mitochondria. The Ca2+ -releasing effect of phosphoenolpyruvate was found to be significantly stronger than that produced by rotenone when added to mitochondria loaded with Ca2+ in the presence of glutamate and inorganic phosphate. Dithiothreitol cannot overcome the effect of phosphoenolpyruvate on mitochondrial Ca2+ transport.     

Effect of Mg ions and spermine on ATP-dependent Ca2+ transport in myometrial intracellular structures. I. Comparative study of Ca2+ accumulation in mitochondria and sarcoplasmic reticulum

In experiments, which were carried out with the use of a radioactive label (45Ca2+) on the suspension of rat uterus myocytes treated by digitonin solution (0.1 mg/ml), influence of Mg ions and spermine on Mg2+, ATP-dependent Ca2+ transport in mitochondria and sarcoplasmic reticulum was investigated. Ca2+ accumulation in mitochondria (1324 +/- 174 pmol Ca2+/10(6) cells for 1 min - the control) was tested as such which was not sensitive to thapsigargin (100 nM) and was blocked by ruthenium red (10 microM). Oxalate-stimulated Ca2+ accumulation in sarcoplasmic reticulum (136 +/- 17 pmol Ca2+/10(6) cells for 1 min - the control) was tested as such which was not sensitive to ruthenium red and was blocked by thapsigargin. It has been shown, that initial speed and level of energy-dependent Ca2+ accumulation in mitochondria considerably exceeded the values of these parameters for sarcoplasmic reticulum Ca2+-accumulation system. Ca2+ accumulation kinetic in mitochondria was characterized by a steady-state phase (for 5-10 min. of incubation) while accumulation kinetic of this cation in sarcoplasmic reticulum corresponded to zero order reaction. Increase of Mg2+ concentration up to 5 mM led to activation of Ca2+-accumulation systems in mitochondria and sarcoplasmic reticulum (values of activation constants K(Mg) for Mg2+ were 2.8 and 0.6 mM, accordingly). Concentration dependence of spermine action on Ca2+ accumulation in mitochondria was described by a dome-shaped curve with a maximum at 1 mM spermine. In case of sarcoplasmic reticulum Ca2+ pump only the inhibition phase was tested at spermine concentration above 1 mM. However values of inhibition constants for both transporting systems were practically identical--5.2 +/- 0.6 and 5.7 +/- 0.7 mM, accordingly. Hence, Mg ions carry out the important role in regulation of energy-dependent Ca2+ transporting systems both in uterus smooth muscle mitochondria and sarcoplasmic reticulum. Spermine acts first of all on mitochondrial calcium uniporter.     

Mitochondrial calpain 10 is degraded by Lon protease after oxidant injury

Calpain 10 is ubiquitously expressed and is one of four mitochondrial matrix proteases. We determined that over-expression or knock-down of mitochondrial calpain 10 results in cell death, demonstrating that mitochondrial calpain 10 is required for viability. Thus, we studied calpain 10 degradation in isolated mitochondrial matrix, mitochondria and in renal proximal tubular cells (RPTC) under control and toxic conditions. Using isolated renal cortical mitochondria and mitochondrial matrix, calpain 10 underwent rapid degradation at 37°C that was blocked with Lon inhibitors but not by calpain or proteasome inhibitors. While exogenous Ca(2+) addition, Ca(2+) chelation or exogenous ATP addition had no effect on calpain 10 degradation, the oxidants tert-butyl hydroperoxide (TBHP) or H(2)O(2) increased the rate of degradation. Using RPTC, mitochondrial and cytosolic calpain 10 increased in the presence of MG132 (Lon/proteasome inhibitor) but only cytosolic calpain 10 increased in the presence of epoxomicin (proteasome inhibitor). Furthermore, TBHP and H(2)O(2) oxidized mitochondrial calpain 10, decreased mitochondrial, but not cytosolic calpain 10, and pretreatment with MG132 blocked TBHP-induced degradation of calpain 10. In summary, mitochondrial calpain 10 is selectively degraded by Lon protease under basal conditions and is enhanced under and oxidizing conditions, while cytosolic calpain 10 is degraded by the proteasome.     

MFN2 Couples Glutamate Excitotoxicity and Mitochondrial Dysfunction in Motor Neurons

Mitochondrial dysfunction plays a central role in glutamate-evoked neuronal excitotoxicity, and mitochondrial fission/fusion dynamics are essential for mitochondrial morphology and function. Here, we establish a novel mechanistic linker among glutamate excitotoxicity, mitochondrial dynamics, and mitochondrial dysfunction in spinal cord motor neurons. Ca²⁺-dependent activation of the cysteine protease calpain in response to glutamate results in the degradation of a key mitochondrial outer membrane fusion regulator, mitofusin 2 (MFN2), and leads to MFN2-mediated mitochondrial fragmentation preceding glutamate-induced neuronal death. MFN2 deficiency impairs mitochondrial function, induces motor neuronal death, and renders motor neurons vulnerable to glutamate excitotoxicity. Conversely, MFN2 overexpression blocks glutamate-induced mitochondrial fragmentation, mitochondrial dysfunction, and/or neuronal death in spinal cord motor neurons both in vitro and in mice. The inhibition of calpain activation also alleviates glutamate-induced excitotoxicity of mitochondria and neurons. Overall, these results suggest that glutamate excitotoxicity causes mitochondrial dysfunction by impairing mitochondrial dynamics via calpain-mediated MFN2 degradation in motor neurons and thus present a molecular mechanism coupling glutamate excitotoxicity and mitochondrial dysfunction.     

Calcium-induced cytochrome c release from CNS mitochondria is associated with the permeability transition and rupture of the outer membrane

The mechanisms of Ca2+-induced release of Cytochrome c (Cyt c) from rat brain mitochondria were examined quantitatively using a capture ELISA. In 75 or 125 mm KCl-based media 1.4 micromol Ca2+/mg protein caused depolarization and mitochondrial swelling. However, this resulted in partial Cyt c release only in 75 mm KCl. The release was inhibited by Ru360, an inhibitor of the Ca2+ uniporter, and by cyclosporin A plus ADP, a combination of mitochondrial permeability transition inhibitors. Transmission electron microscopy (TEM) revealed that Ca2+-induced swelling caused rupture of the outer membrane only in 75 mm KCl. Koenig's polyanion, an inhibitor of mitochondrial porin (VDAC), enhanced swelling and amplified Cyt c release. Dextran T70 that is known to enhance mitochondrial contact site formation did not prevent Cyt c release. Exposure of cultured cortical neurons to 500 microM glutamate for 5 min caused Cyt c release into the cytosol 30 min after glutamate removal. MK-801 or CsA inhibited this release. Thus, the release of Cyt c from CNS mitochondria induced by Ca2+ in vitro as well as in situ involved the mPT and appeared to require the rupture of the outer membrane.     

Tissue-specific modulation of the mitochondrial calcium uniporter by magnesium ions

This paper analyzes the kinetics of the Ca2+ uniporter of mitochondria from rat heart, kidney and liver operating in a range of Ca2+ concentrations near the steady-state value (1-4 microM). Heart mitochondria exhibit the lowest activity, and physiological Mg2+ concentrations inhibit the mitochondrial Ca2+ uniporter by approx. 50% in heart and kidney, and by 20% in liver. At physiological Ca2+ and Mg2+ concentrations the external free Ca2+ maintained by respiring mitochondria in vitro is higher in heart and kidney with respect to liver mitochondria. This behaviour could represent an adaptation of different mitochondria to their specific intracellular environment.     

Changes in mitochondrial activity evoked by cholecystokinin in isolated mouse pancreatic acinar cells

In the present study, we have employed confocal laser scanning microscopy to investigate the effect that stimulation of mouse pancreatic acinar cells with the secretagogue cholecystokinin (CCK) has on mitochondrial activity. We have monitored changes in cytosolic as well as mitochondrial Ca2+ concentrations, mitochondrial membrane potential and FAD autofluorescence by loading the cells with fluo-3, rhod-2 or JC-1, respectively. Our results show that stimulation of cells with cholecystokinin led to release of Ca2+ from intracellular stores that then accumulated into mitochondria. In the presence of the hormone a depolarization of mitochondrial membrane potential was observed, which partially recovered; in addition a transient increase in FAD autofluorescence could be observed. Similarly, treatment of cells with thapsigargin induced increases in mitochondrial Ca2+ and FAD autofluorescence, and depolarized mitochondria. Pretreament of cells with thapsigargin blocked cholecystokinin-evoked changes. Similar results were obtained when the cells were incubated in the presence of rotenone, which blocks the mitochondrial electron transport chain. Our findings are consistent with changes in mitochondrial activity in response to stimulation of pancreatic acinar cells with cholecystokinin. Following stimulation, mitochondria take up Ca2+ that could in turn activate the mitochondrial machinery that may match the energy supply necessary for the cell function during secretion, suggesting that Ca2+ can act as a regulator of mitochondrial activity.     

Kinetic model of spermine effect on the Mg2+, ATP-dependent transport of Ca ions in the smooth muscle mitochondria

In experiments, carried out with the use of a radioactive label (45Ca2+) on suspension of rat uterus myocytes treated with digitonin solution (0.1 mg/ml), influence of spermine on the Mg2+, ATP-dependent Ca2+ transport in the mitochondria was investigated. Ca2+ accumulation in the mitochondria was tested as such which was blocked by ruthenium red (10 microM) and was not sensitive to thapsigargin (100 nM). It was shown, that dependence of initial speed of Ca ions accumulation in the mitochondria on spermine concentration (0.1-10 mm) is described by a bell-shaped curve. Spermine concentration being increased in the range of 0.1-1 mM the stimulation of Ca2+ accumulation was observed, at the further increase in polyamine concentration up to 10 mM the suppression of this process took place. On the basis of the analysis of the authors' experimental results and the literature data the model of complex spermine action on Ca2+ accumulation in mitochondria was proposed and analyzed. The existence of two spermine binding sites on mitochondrial membrane--S1 and S2 occupation of which is connected to activation and inhibition of Ca(2+)-unipoter, accordingly, was taken into account. The kinetic analysis of the model which has been made in an equilibrium mode, allowed to calculate some important quantitative parameters describing spermine influence on Ca ions accumulation in mitochondria. It is supposed, that the proposed model can be useful in the further research of polyamine influence on transmembrane exchange of Ca ions in mitochondria.     

Spermine. A regulator of mitochondrial calcium cycling

Steady-state free Ca2+ concentrations have been measured with a Ca2+ electrode using suspensions of isolated rat liver mitochondria or saponin-treated hepatocytes. Mitochondria, when incubated in the presence of Mg2+ and MgATP2-, maintain a steady-state pCa2+ (-log [Ca2+]) of approximately 6.1 (0.8 microM). Addition of spermine lowered this value to a pCa2+ of 6.6 (0.25 microM). Spermine was the most effective polyamine, giving half-maximal effects at 170 microM and maximal effects at 400 microM. With saponin-permeabilized hepatocytes, spermine addition similarly showed that the mitochondria buffered the steady-state medium-free Ca2+ at a level approximating the cytosolic free Ca2+ concentration of intact hepatocytes. The initial rate of Ca2+ uptake by the mitochondrial Ca2+ uniporter was investigated using Ca2+-depleted mitochondria incubated in the presence of succinate and 0.3 mM free Mg2+. Under control conditions, Ca2+ uptake was not observed at free Ca2+ concentrations below 0.5 microM. Spermine (350 microM) increased the rate of Ca2+ uptake at all Ca2+ concentrations below 4.5 microM, but at higher Ca2+ concentrations, it was inhibitory. Spermine also affected mitochondrial Ca2+ efflux by decreasing the apparent Km from 16 to 3.8 nmol of Ca2+/mg of mitochondrial protein with no change of Vmax. Experiments with 45Ca2+ confirmed that spermine increased mitochondrial Ca2+ cycling at 0.2 microM free Ca2+. Hepatic spermine contents are reported to be about 1 mumol/g, wet weight, suggesting that this polyamine may have an important physiological role in intracellular calcium homeostasis.