Regulation of citric acid cycle by calcium

The relationship of extramitochondrial Ca2+ to intramitochondrial Ca2+ and the influence of intramitochondrial free Ca2+ concentrations on various steps of the citric acid cycle were evaluated. Ca2+ was measured using the Ca2+ sensitive fluorescent dye fura-2 trapped inside the rat heart mitochondria. The rate of utilization of specific substrates and the rate of accumulation of citric acid cycle intermediates were measured at matrix free Ca2+ ranging from 0 to 1.2 microM. A change in matrix free Ca2+ from 0 to 0.3 microM caused a 135% increase in ADP stimulated oxidation of 0.6 mM alpha-ketoglutarate (K0.5 = 0.15 microM). In the absence of ADP and the presence of 0.6 mM alpha-ketoglutarate, Ca2+ (0.3 microM) increased NAD(H) reduction from 0 to 40%. On the other hand, when pyruvate (10 microM to 5 mM) was substrate, pyruvate dehydrogenase flux was insensitive to Ca2+ and isocitrate dehydrogenase was sensitive to Ca2+ only in the presence of added ADP. In separate experiments pyruvate dehydrogenase activation (dephosphorylation) was measured. Under the conditions of the present study, pyruvate dehydrogenase was found to be almost 100% activated at all levels of Ca2+, thus explaining the Ca2+ insensitivity of the flux measurements. However, if the mitochondria were incubated in the absence of pyruvate, with excess alpha-ketoglutarate and excess ATP, the pyruvate dehydrogenase complex was only 20% active in the absence of added Ca2+ and activity increased to 100% at 2 microM Ca2+. Activation by Ca2+ required more Ca2+ (K0.5 = 1 microM) than for alpha-ketoglutarate dehydrogenase. The data suggest that in heart mitochondria alpha-ketoglutarate dehydrogenase may be a more physiologically relevant target of Ca2+ action than pyruvate dehydrogenase.     

Remodeling of the vascular tunica media is essential for development of collateral vessels in the canine heart

A method to study the export of citric acid cycle intermediates from rat liver mitochondria supplied with various individual substrates or combinations of substrates was designed to focus on the role of mitochondria in anaplerosis and cataplerosis. Under most conditions malate, citrate, and aspartate were exported in far higher amounts than isocitrate and alpha-ketoglutarate. In the presence of pyruvate alone or pyruvate in combination with most other substrates, citrate export equaled or was only slightly less than malate export. This contrasts with pancreatic islet mitochondria where citrate export is unaffected by many substrates. Malate and succinate potentiated pyruvate-induced citrate export and succinate caused massive malate export from liver mitochondria. Heart mitochondria, which possess very little or no pyruvate carboxylase, unlike liver and pancreatic islet mitochondria, did not produce malate from pyruvate. Heart mitochondria produced malate, but not citrate, from succinate. The results indicate that liver mitochondria export a larger number of metabolites from a wider range of substrates than do islet or heart mitochondria. This may reflect the multiple roles of the liver in body metabolism versus the specialized roles of the islet cell and heart.     

Pathway of carbon flow during fatty acid synthesis from lactate and pyruvate in rat adipose tissue

The metabolism of pyruvate and lactate by rat adipose tissue was studied. Pyruvate and lactate conversion to fatty acids is strongly concentration-dependent. Lactate can be used to an appreciable extent only by adipose tissue from fasted-refed rats. A number of compounds, including glucose, pyruvate, aspartate, propionate, and butyrate, stimulated lactate conversion to fatty acids. Based on studies of incorporation of lactate-2-(3)H and lactate-2-(14)C into fatty acids it was suggested that the transhydrogenation sequence of the "citrate-malate cycle"(1) was not providing all of the NADPH required for fatty acid synthesis from lactate. An alternative pathway for NADPH formation involving the conversion of isocitrate to alpha-ketoglutarate via cytosolic isocitrate dehydrogenase was proposed. Indirect support for this proposal was provided by the rapid labeling of glutamate from lactate-2-(14)C by adipose tissue incubated in vitro, as well as the demonstration that glutamate can be readily metabolized by adipose tissue via reactions localized largely in the cytosol. Furthermore, isolated adipose tissue mitochondria convert alpha-ketoglutarate to malate, or in the presence of added pyruvate, to citrate. Glutamate itself can not be metabolized by these mitochondria, a finding in keeping with the demonstration of negligible levels of NAD-glutamate dehydrogenase activity in adipose tissue mitochondria. Pyruvate stimulated alpha-ketoglutarate and malate conversion to citrate and reduced their oxidation to CO(2). It is proposed that under conditions of excess generation of NADH malate may act as a shuttle carrying reducing equivalents across the mitochondrial membrane. Malate at low concentrations increased pyruvate conversion $$Word$$ citrate and markedly decreased the formation of CO(2) by isolated adipose tissue mitochondria. Malate also stimulated citrate and isocitrate metabolism by these mitochondria, an effect that could be blocked by 2-n-butylmalonate. This potentially important role of malate in the regulation of carbon flow during lipogenesis is underlined by the observation that 2-n-butylmalonate inhibited fatty acid synthesis from pyruvate, but not from glucose and acetate, and decreased the stimulatory effect of pyruvate on acetate conversion to fatty acids.     

A comparison of the regulation of pyruvate dehydrogenase in mitochondria from rat brain and liver.

The total activity of pyruvate dehydrogenase in mitochondria isolated from rat brain and liver was 53.5 and 14.2nmol/min per mg of protein respectively. Pyruvate dehydrogenase in liver mitochondria incubated for 4 min at 37 degrees C with no additions was 30% in the active form and this activity increased with longer incubations until it was completely in the active form after 20 min. Brain mitochondrial pyruvate dehydrogenase activity was initially high and did not increase with addition of Mg2+ plus Ca2+ or partially purified pyruvate dehydrogenase phosphatase or with longer incubations. The proportion of pyruvate dehydrogenase in the active form in both brain and liver mitochondria changed inversely with changes in mitochondrial energy charge, whereas total pyruvate dehydrogenase did not change. The chelators citrate, isocitrate, EDTA, ethanedioxybis(ethylamine)tetra-acetic acid and Ruthenium Red each lowered pyruvate dehydrogenase activity in brain mitochondria, but only citrate and isocitrate did so in liver mitochondria. These chelators did not affect the energy charge of the mitochondria. Mg2+ plus Ca2+ reversed the pyruvate dehydrogenase inactivation in liver, but not brain, mitochondria. The regulation of the activation-inactivation of pyruvate dehydrogenase in mitochondria from rat brain and liver with respect to energy charge is similar and may be at least partially regulated by this parameter, and the effects of chelators differ in the two types of mitochondria.     

The use of the Ca2(+)-sensitive intramitochondrial dehydrogenases and entrapped fura-2 to study Sr2+ and Ba2+ transport across the inner membrane of mammalian mitochondria

In extracts of rat heart mitochondria, Sr2+ mimicked the activatory effects of Ca2+ on the Ca2(+)-sensitive intramitochondrial enzymes, pyruvate dehydrogenase phosphate phosphatase, isocitrate dehydrogenase (NAD+), and 2-oxoglutarate dehydrogenase, but at about tenfold higher concentrations (effective range approximately 1-100 muM) in each case. Ba2+ had no effect on extracted phosphatase, but did mimic the effect of Ca2+ on the other two enzymes with effective concentration ranges similar to those of Sr2+; as with Ca2+ and Sr2+, effective Ba2+ ranges were slightly (2-3-fold) raised by increases in ATP/ADP. In intact uncoupled rat heart mitochondria, the effects of Sr2+ and Ba2+ on the pyruvate and 2-oxoglutarate dehydrogenases were essentially similar to their effects in extracts. In fully coupled rat heart or liver mitochondria, the effective concentration ranges of extramitochondrial Sr2+, leading to activation of the matrix enzymes, were always approximately tenfold higher than those for Ca2+ under all conditions. Ba2+ did not affect pyruvate dehydrogenase in coupled mitochondria, but was shown to activate 2-oxoglutarate dehydrogenase in heart or liver mitochondria, and also isocitrate dehydrogenase (NAD+) in the latter; effective concentration ranges for extramitochondrial Ba2+ were approximately 100-fold greater than those for Ca2+, and like those for Ca2+ and Sr2+, were affected markedly by Mg2+ and spermine (which inhibit and promote mitochondrial Ca2+ uptake, respectively) but, in contrast to Ca2+ and Sr2+, they were hardly affected at all by Na+ (which promotes mitochondrial Ca2+ egress). Ba2+ effects were also blocked by ruthenium red (an inhibitor of mitochondrial Ca2+ uptake), but not so effectively as its blockage of the effects of Sr2+ and Ca2+. Ba2+ and Sr2+ both mimicked the inhibitory effects of extramitochondrial Ca2+ on the Na+/Ca2+ exchanger, but only Sr2+ could mimic Ca2+ in exchanging for internal Ca2+ by this mechanism. Both Sr2+ and Ba2+ changed the fluorescent properties of fura-2 or indo-1 in a similar manner to Ca2+, but with higher kd values. In fura-2-loaded rat heart mitochondria, increases in matrix Sr2+ and Ba2+ and the effects of the transport effectors could be readily demonstrated.     

Metabolic cycling in control of glucose-stimulated insulin secretion

Glucose-stimulated insulin secretion (GSIS) is central to normal control of metabolic fuel homeostasis, and its impairment is a key element of beta-cell failure in type 2 diabetes. Glucose exerts its effects on insulin secretion via its metabolism in beta-cells to generate stimulus/secretion coupling factors, including a rise in the ATP/ADP ratio, which serves to suppress ATP-sensitive K(+) (K(ATP)) channels and activate voltage-gated Ca(2+) channels, leading to stimulation of insulin granule exocytosis. Whereas this K(ATP) channel-dependent mechanism of GSIS has been broadly accepted for more than 30 years, it has become increasingly apparent that it does not fully describe the effects of glucose on insulin secretion. More recent studies have demonstrated an important role for cyclic pathways of pyruvate metabolism in control of insulin secretion. Three cycles occur in islet beta-cells: the pyruvate/malate, pyruvate/citrate, and pyruvate/isocitrate cycles. This review discusses recent work on the role of each of these pathways in control of insulin secretion and builds a case for the particular relevance of byproducts of the pyruvate/isocitrate cycle, NADPH and alpha-ketoglutarate, in control of GSIS.     

Hydrogen peroxide production by monoamine oxidase in isolated rat-brain mitochondria: its effect on glutathione levels and Ca2+ efflux

H2O2 production and accumulation during incubation of isolated rat-brain mitochondria with substrates of monoamine oxidase A and B were investigated. All substrates gave rise to an accumulation of H2O2 which was inhibited by malate + pyruvate or isocitrate, consistent with a need for mitochondrial NADPH to maintain glutathione in the reduced state. However, in the absence of these additions the level of reduced glutathione decreased only by about 30%, indicating that only a fraction of the mitochondrial glutathione pool was accessible to the glutathione peroxidase and glutathione reductase activities responsible for the continuous removal of H2O2 generated by monoamine oxidase. The H2O2 accumulation was also inhibited by externally added reduced glutathione or NADPH but not NADH. External NADPH was oxidized by added oxidized glutathione but not alpha-ketoglutarate + NH4+. These results suggest that the removal of H2O2 generated by monoamine oxidase proceeds by way of special fractions of glutathione peroxidase and glutathione reductase that are located in the intermembrane space of mitochondria in such a way that they can react with both intra- and extra-mitochondrial glutathione and NADPH, possibly at the contact sites between the inner and outer mitochondrial membranes. Evidence is also presented that H2O2 generated by monoamine oxidase enhances Ca2+ release from mitochondria and may thus function as a regulator of mitochondrial Ca2+ efflux.     

Regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+ ions within toluene-permeabilized rat heart mitochondria. Interactions with regulation by adenine nucleotides and NADH/NAD+ ratios.

1. Toluene-permeabilized rat heart mitochondria have been used to study the regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+, adenine and nicotinamide nucleotides, and to compare the properties of the enzymes in situ, with those in mitochondrial extracts. 2. Although K0.5 values (concn. giving half-maximal effect) for Ca2+ of 2-oxoglutarate dehydrogenase were around 1 microM under all conditions, corresponding values for NAD+-linked isocitrate dehydrogenase were in the range 5-43 microM. 3. For both enzymes, K0.5 values for Ca2+ observed in the presence of ATP were 3-10-fold higher than those in the presence of ADP, with values increasing over the ADP/ATP range 0.0-1.0. 4. 2-Oxoglutarate dehydrogenase was less sensitive to inhibition by NADH when assayed in permeabilized mitochondria than in mitochondrial extracts. Similarly, the Km of NAD+-linked isocitrate dehydrogenase for threo-Ds-isocitrate was lower in permeabilized mitochondria than in extracts under all the conditions investigated. 5. It is concluded that in the intact heart Ca2+ activation of NAD+-linked isocitrate dehydrogenase may not necessarily occur in parallel with that of the other mitochondrial Ca2+-sensitive enzymes, 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase system.     

Calcium ions and the regulation of NAD+-linked isocitrate dehydrogenase from the mitochondria of rat heart and other tissues.

The effects of Ca2+ on the activity of isocitrate dehydrogenase (NAD+) in extracts of rat heart mitochondria were explored in the presence of MgCl2 by using EGTA buffers. In the absence of ADP, Ca2+ (about 30 micrometer) resulted in a slight increase in apparent Km for threo-Ds-isocitrate; in the presence of ADP, Ca2+ (about 25 micrometer) greatly lowered the apparent Km for threo-Ds-isocitrate from 227 micrometer to 53 micrometer without changing the maximum velocity. At 100 micrometer-threo-Ds-isocitrate and 1 mM-ADP, there was an 8-fold activation by Ca2+, with a Km for Ca2+ of 1.2 micrometer. This activation was also observed with Sr2+ (Km 3.1 micrometer), but not with Mn2+ (at concentrations below 2.5 micrometer). Similar effects of Ca2+ were also observed on isocitrate dehydrogenase (NAD+) activity in extracts of mitochondria from liver, kidney, brown adipose tissue and white adipose tissue of the rat. The possible regulatory role of changes in the intramitochondrial concentration of Ca2+ is discussed.     

The export of metabolites from mitochondria and anaplerosis in insulin secretion

Combinations of insulin secretagogue-derived metabolites were added to microgram amounts of mitochondria obtained from rat and mouse pancreatic islets and the INS-1 cell line, and the export of citric acid cycle intermediates was surveyed to study anaplerosis in insulin secretion. Cellular levels of metabolites were also measured. In mitochondria from all three tissues, malate production was the most responsive to various substrates. The export of citrate and isocitrate in the presence of pyruvate and most other substrates was small and their levels in intact cells did not change with any secretagogue, except in INS-1 cells where citrate increased slightly. Changes in alpha-ketoglutarate and glutamate export from mitochondria and levels in intact cells indicate that glutamate can be consumed as a fuel secretagogue, but it is not likely produced as a messenger in insulin secretion. The citrate level may not need to increase in order to provide increased malonyl-CoA for signaling insulin secretion. Unlike some cells, insulin cells probably obtain cytosolic NADPH equivalents by exporting them from mitochondria to the cytosol via a pyruvate malate shuttle or an isocitrate shuttle. Only fuels that can enhance anaplerosis via pyruvate or alpha-ketoglutarate can be insulin secretagogues.     

A complex effect of arsenite on the formation of alpha-ketoglutarate in rat liver mitochondria

This investigation presents disturbances of the mitochondrial metabolism by arsenite, a hydrophilic dithiol reagent known as an inhibitor of mitochondrial alpha-keto acid dehydrogenases. Arsenite at concentrations of 0.1-1.0 mM was shown to induce a considerable oxidation of intramitochondrial NADPH, NADH, and glutathione without decreasing the mitochondrial membrane potential. The oxidation of NAD(P)H required the presence of phosphate and was sensitive to ruthenium red, but occurred without the addition of calcium salts. Mitochondrial reactions producing alpha-ketoglutarate from glutamate and isocitrate were modulated by arsenite through various mechanisms: (i) both glutamate transaminations, with oxaloacetate and with pyruvate, were inhibited by accumulating alpha-ketoglutarate; however, at low concentrations of alpha-ketoglutarate the aspartate aminotransferase reaction was stimulated due to the increase of NAD+ content; (ii) the oxidation of isocitrate was stimulated at its low concentration only, due to the oxidation of NADPH and NADH; this oxidation was prevented by concentrations of citrate or isocitrate greater than 1 mM; (iii) the conversion of isocitrate to citrate was suppressed, presumably as a result of the decrease of Mg2+ concentration in mitochondria. Thus the depletion of mitochondrial vicinal thiol groups in hydrophilic domains disturbs the mitochondrial metabolism not only by the inhibition of alpha-keto acid dehydrogenases but also by the oxidation of NAD(P)H and, possibly, by the change in the ion concentrations.     

Significance of the alanine aminotransferase reaction in the formation of alpha-ketoglutarate in rat liver mitochondria

The total production of alpha-ketoglutarate from glutamate and isocitrate was estimated in isolated rat liver mitochondria. Mitochondrial alanine aminotransferase converts glutamate to alpha-ketoglutarate [A.K. Groen et al. (1982) Eur. J. Biochem. 122, 87-93], thus participating in the net formation of the tricarboxylic acid cycle intermediates from glutamate. The present investigation indicates a significant contribution of the alanine aminotransferase reaction to glutamate oxidation by isolated rat liver mitochondria in the presence of bicarbonate. It amounted to 41-74 and 7-31% of the total utilization of glutamate in States 4 and 3, respectively, in various conditions in vitro, at pyruvate concentrations in the range of 0.1-10 mM. The participation of glutamate in the total production of alpha-ketoglutarate at physiological concentrations of glutamate, citrate, and isocitrate varied in the range of 72-82%. It was calculated that alpha-ketoglutarate formation by the reaction of alanine aminotransferase amounted to 30 and 5% of the total mitochondrial alpha-ketoglutarate production in States 4 and 3, respectively, at physiological concentrations of its precursors and in the presence of 0.5 mM malate and 0.1 mM pyruvate. It constituted 77-97% of the net production of the tricarboxylic acid cycle intermediates from glutamate in rat liver mitochondria. The importance of alpha-ketoglutarate production via the alanine aminotransferase reaction under various physiological conditions is discussed.     

Purification and characterization of NADP+-linked isocitrate dehydrogenase from an alkalophilic Bacillus

We have succeeded in purifying to homogeneity a very labile NADP+-linked isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) from a strain of alkalophilic Bacillus, by a simple method, with an overall yield over 76% of the original activity. The molecular weight on Sephadex G-200 was around 90,000; and that by electrophoresis on SDS-polyacrylamide gels was about 44,000. The sedimentation coefficient (s020,w) and isoelectric point of the enzyme were determined to be 3.22 S and pH 4.7, respectively. The enzyme required Mn2+ for the reaction and for stability. The optimum pH for the reaction was in the range 7.8-8.4 at 30 degrees C; the optimum temperature at pH 8.0 was 75 degrees C; the activation energy of the reaction was 6.2 kcal/mol. The Km values for threo-Ds-isocitrate, DL-isocitrate, and NADP+ were 5.4 microM, 9.9 microM, and 7.3 microM, respectively. This enzyme was inhibited by NADPH, glyceraldehyde 3-phosphate, 3-phosphoglycerate, phosphoenol pyruvate, cis-aconitate, alpha-ketoglutarate, and oxaloacetate. In addition, it was subject to a concerted inhibition by a combination of glyoxylate and oxaloacetate, and also to a cumulative inhibition by nucleoside triphosphates.     

Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive dehydrogenases within intact rat-kidney mitochondria

The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat kidney mitochondria were found to be essentially similar to those described previously for other mammalian tissues; in particular each enzyme could be activated severalfold by Ca2+ with half-maximal effects (K0.5 values) of about 1 microM and effective ranges of approx. 0.1-10 microM Ca2+. In intact mitochondria prepared from whole rat kidneys incubated in a KCl-based medium containing respiratory substrates, the amount of active, nonphosphorylated pyruvate dehydrogenase could be increased severalfold by increases in extramitochondrial [Ca2+]; these effects could be blocked by ruthenium red. Similarly, Ca2+-dependent activations of NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase could be demonstrated in intact, fully coupled, rat kidney mitochondria by either following O2 uptake (in the presence of ADP) and NAD(P)H reduction (in the absence of ADP) on presentation of non-saturating concentrations of either threo-Ds-isocitrate or 2-oxoglutarate, respectively, under appropriate conditions, or for the latter enzyme only, also by following 14CO2 production from 2-oxo[1-14C]glutarate (in the absence or presence of ADP). Effects of Na+ (as a promoter of egress) and Mg2+ (as an inhibitor of uptake) on Ca2+-transport by rat kidney mitochondria could be readily demonstrated by assaying for the Ca2+-sensitive properties of the intramitochondrial Ca2+-sensitive dehydrogenases within intact rat kidney mitochondria. In the presence of physiological concentrations of Na+ (10 mM) and Mg2+ (2 mM), activation of the enzymes was achieved by increases in extramitochondrial [Ca2+] within the expected physiological range (0.05-5 microM) and with apparent K0.5 values in the approximate range of 300-500 nM. The implications of these results on the role of the Ca2+-transport system of kidney mitochondria are discussed.     

Persistent changes in the initial rate of pyruvate transport by isolated rat liver mitochondria after preincubation with adenine nucleotides and calcium ions

1. Preincubation of isolated rat-liver mitochondria in the presence of adenine nucleotides or Ca2+ results in definite and persistent changes in the initial rate of pyruvate transport. 2. These changes in the rate of pyruvate transport are accompanied by equally persistent changes in the opposite direction of the activity of pyruvate dehydrogenase (EC 1.2.4.1). 3. Changes of the transmembrane pH gradient and of the membrane potential, brought about by the pretreatments of the mitochondria, cannot account for the observed changes in the rate of pyruvate transport. 4. It is proposed that the pretreatment of the mitochondria directly modulates the activity of the mitochondrial pyruvate carrier. The possible regulatory role of such a modulation system is discussed.     

Studies of the regulation of renal gluconeogenesis in normal and Pi depleted proximal tubule cells

Pi depletion of proximal tubule cells isolated from mouse kidney results in a decrease in the cell content of fructose-2,6-bisphosphate and an increase in the rate of gluconeogenesis from pyruvate, malate and succinate. Gluconeogenesis from glycerol is unaffected by Pi depletion. Introduction of fructose-2,6-bisphosphate into the cytosol of ATP-permeabilized cells is accompanied by a fall in gluconeogenesis. The presence of external Ca2+ stimulates gluconeogenesis. When cytosolic Ca2+ is raised to 1.8 microM by permeabilization, the resealed cells still require 2.5 mM Ca2+ in the bathing medium in order to perform gluconeogenesis at the maximum rate. Cells permeabilized in the presence of cAMP show a decreased rate of glucose production. Phorbol ester stimulates gluconeogenesis provided that the phorbol treatment is performed in the absence of Ca2+ ions. It is suggested that Pi depletion may stimulate pyruvate carboxylase activity and facilitate the entry of certain gluconeogenic substrates into mitochondria. It is also proposed that important aspects of the control of renal gluconeogenesis by parathyroid hormone are mediated by protein kinase C.     

Effects of spermine on mitochondrial Ca2+ transport and the ranges of extramitochondrial Ca2+ to which the matrix Ca2+-sensitive dehydrogenases respond.

1. Spermine has previously been reported to be an activator of mitochondrial Ca2+ uptake [Nicchitta & Williamson (1984) J. Biol. Chem. 259, 12978-12983]. This is confirmed in the present studies on rat heart, liver and kidney mitochondria by using the activities of the Ca2+-sensitive intramitochondrial dehydrogenases (pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenases) as probes for matrix Ca2+, and also, for the heart mitochondria, by using entrapped fura-2. 2. As also found previously [Damuni, Humphreys & Reed (1984) Biochem. Biophys. Res. Commun. 124, 95-99], spermine activated extracted pyruvate dehydrogenase phosphate phosphatase. However, it was found to have no effects at all on the extracted NAD+-isocitrate or 2-oxoglutarate dehydrogenases. It also had no effects on activities of the enzymes in mitochondria incubated in the absence of Ca2+, or on the Ca2+-sensitivity of the enzymes in uncoupled mitochondria. 3. Spermine clearly activated 45Ca uptake by coupled mitochondria, but had no effect on Ca2+ egress from mitochondria previously loaded with 45Ca. 4. Spermine (with effective Km values of around 0.2-0.4 mM) caused an approx. 2-3-fold decrease in the effective ranges of extramitochondrial Ca2+ in the activation of the Ca2+-sensitive matrix enzymes in coupled mitochondria from all of the tissues. The effects of spermine appeared to be largely independent of the other effectors of mitochondrial Ca2+ transport, such as Mg2+ (inhibitor of uptake) and Na+ (promoter of egrees). 5. In the most physiological circumstance, coupled mitochondria incubated with Na+ and Mg2+, the presence of saturating spermine (2 mM) resulted in an effective extramitochondrial Ca2+ range for matrix enzyme activation of from about 30-50 nM up to about 800-1200 nM, with half-maximal effects around 250-400 nM-Ca2+. The implications of these findings for the regulation of matrix and extramitochondrial Ca2+ are discussed.     

Activation of V1-receptors by vasopressin stimulates inositol phospholipid hydrolysis and arachidonate metabolism in human platelets.

The effects of Mg2+ on the activity of pyruvate dehydrogenase phosphate phosphatase within intact mitochondria prepared from control and insulin-treated rat epididymal adipose tissue was explored by incubating the mitochondria in medium containing the ionophore A23187. The apparent Ka for Mg2+ was approximately halved in the mitochondria derived from insulin-treated tissue in both the absence and the presence of Ca2+. In this system, the major effect of Ca2+ was also to decrease the apparent Ka for Mg2+, rather than to change the Vmax. of the phosphatase. Damuni, Humphreys & Reed [(1984) Biochem. Biophys. Res. Commun. 124, 95-99] have reported that spermine activates ox kidney pyruvate dehydrogenase phosphate phosphatase. Studies were carried out on phosphatase from pig heart and rat epididymal adipose tissue which confirm and extend this observation. The major effect of spermine is shown to be a decrease in the Ka for Mg2+, which is apparent in both the presence and the absence of Ca2+. Spermine did not affect the sensitivity of the phosphatase to Ca2+ at saturating concentrations of Mg2+. Other polyamines tested were not as effective as spermine. No alteration in the maximum activity or Mg2+-sensitivity of pyruvate dehydrogenase phosphate phosphatase was apparent in extracts of mitochondria from insulin-treated tissue. The close similarity of the effects of spermine and the changes in kinetic properties of pyruvate dehydrogenase phosphate phosphatase within mitochondria from insulin-treated adipose tissue suggests that insulin may activate pyruvate dehydrogenase by increasing the concentration of spermine within the mitochondria. However, it is concluded that insulin is more likely to alter the interaction of the pyruvate dehydrogenase system with some other polybasic intramitochondrial component whose action can be mimicked by spermine.     

Substrate-dependent differential regulation of mitochondrial bioenergetics in the heart and kidney cortex and outer medulla

The kinetics and efficiency of mitochondrial oxidative phosphorylation (OxPhos) can depend on the choice of respiratory substrates. Furthermore, potential differences in this substrate dependency among different tissues are not well-understood. Here, we determined the effects of different substrates on the kinetics and efficiency of OxPhos in isolated mitochondria from the heart and kidney cortex and outer medulla (OM) of Sprague-Dawley rats. The substrates were pyruvate+malate, glutamate+malate, palmitoyl-carnitine+malate, alpha-ketoglutarate+malate, and succinate±rotenone at saturating concentrations. The kinetics of OxPhos were interrogated by measuring mitochondrial bioenergetics under different ADP perturbations. Results show that the kinetics and efficiency of OxPhos are highly dependent on the substrates used, and this dependency is distinctly different between heart and kidney. Heart mitochondria showed higher respiratory rates and OxPhos efficiencies for all substrates in comparison to kidney mitochondria. Cortex mitochondria respiratory rates were higher than OM mitochondria, but OM mitochondria OxPhos efficiencies were higher than cortex mitochondria. State 3 respiration was low in heart mitochondria with succinate but increased significantly in the presence of rotenone, unlike kidney mitochondria. Similar differences were observed in mitochondrial membrane potential. Differences in H₂O₂ emission in the presence of succinate±rotenone were observed in heart mitochondria and to a lesser extent in OM mitochondria, but not in cortex mitochondria. Bioenergetics and H₂O₂ emission data with succinate±rotenone indicate that oxaloacetate accumulation and reverse electron transfer may play a more prominent regulatory role in heart mitochondria than kidney mitochondria. These studies provide novel quantitative data demonstrating that the choice of respiratory substrates affects mitochondrial responses in a tissue-specific manner.     

Effects of metabolic substrates and inhibitors on weak organic anion transport in rat renal proximal tubules

Stimulatory effects of intermediates of the tricarboxylic acid cycle on renal uptake of a weak organic anion, fluorescein, were studied with the aid of the method of contact microfluorimetry of individual convoluted proximal tubules ascending to the surface of the rat renal cortex slices. The study was undertaken for verifying the hypothesis that energization of renal excretion of anionic exenobiotics is mediated through their transport across the basolateral membrane in exchange for cytoplasmic alpha-ketoglutarate serving as a counter-anion. Effects of inhibitors of the tricarboxylic acid cycle such as fluoroacetate, malonate and 5-methoxyindole-2-carboxylate on the fluorescein uptake and renal gluconeogenesis in the presence of the metabolic substrates were investigated in order to outline metabolic pathways that could be responsible for elevation of the cytoplasmic alpha-ketoglutarate. Obtained data evidence that the stimulatory effects of the tricarboxylic acid cycle intermediates on the transport process under study depend on the metabolic state of the mitochondria and involve an activation of certain reactions but not the cycle as a whole. It has been suggested that an elevation of the cytoplasmic alpha-ketoglutarate resulting from this activation can be conditioned by export of isocitrate from the mitochondria with its subsequent transformation into alpha-ketoglutarate in the cytoplasm in the isocitrate dehydrogenase reaction.