Liver mitochondrial pyrophosphate concentration is increased by Ca2+ and regulates the intramitochondrial volume and adenine nucleotide content.

1. The matrix pyrophosphate (PPi) content of isolated energized rat liver mitochondria incubated in the presence of ATP, Mg2+, Pi and respiratory substrate was about 100 pmol/mg of protein. 2. After incubation with sub-micromolar [Ca2+], this was increased by as much as 300%. There was a correlation between the effects of Ca2+ on PPi and on the increase in matrix volume reported previously [Halestrap, Quinlan, Whipps & Armston (1986) Biochem. J. 236, 779-787]. Half-maximal effects were seen at 0.3 microM-Ca2+. 3. Coincident with these effects, the total adenine nucleotide content increased in a carboxyatractyloside-sensitive manner. 4. Incubation with 0.2-0.5 mM-butyrate induced similar but smaller effects on mitochondrial swelling and matrix PPi and total adenine nucleotide content. Addition of butyrate after Ca2+, or vice versa, caused Ca2+-induced mitochondrial swelling to stop or reverse, while matrix PPi increased 30-fold. 5. Addition of atractyloside or the omission of ATP from incubations greatly enhanced swelling induced by Ca2+ without increasing matrix PPi. 6. Swelling of mitochondria incubated under de-energized conditions in iso-osmotic KSCN was progressively enhanced by the addition of increasing concentrations of PPi (1-20 mM) or valinomycin. 7. In iso-osmotic potassium pyrophosphate swelling was slow initially, but accelerated with time. This acceleration was inhibited by ADP, whereas carboxyatractyloside induced rapid swelling. Swelling in other iso-osmotic PPi salts showed that the rate of entry decreased in the order NH4+ greater than K+ greater than Na+ greater than Li+, whereas choline, tetramethylammonium and Tris did not enter. It is suggested that the adenine nucleotide translocase transports small univalent cations when PPi is bound and that PPi can also be transported when the transporter is in the conformation induced by carboxyatractyloside. 8. It is concluded that Ca2+ and butyrate cause swelling of energized mitochondria through this effect of PPi on K+ permeability of the mitochondrial inner membrane. 9. Freeze-clamped livers from rats treated with glucagon or phenylephrine show 30-50% increases in tissue PPi. It is proposed that Ca2+-mediated increases in mitochondrial PPi are responsible for the increase in matrix volume and total adenine nucleotide content observed after hormone treatment.     

Phosphate and calcium uptake by mitochondria and by perfused rat liver induced by the synergistic action of glucagon and vasopressin.

Co-administration of glucagon and vasopressin to rat liver perfused with buffer containing 1.3 mM-Ca2+ induces a 4-fold increase in Pi in the subsequently isolated mitochondria (from approx. 9 to approx. 40 nmol/mg of mitochondrial protein). This increase is not attributable to PPi hydrolysis, and is not observed if the perfusate Ca2+ is lowered from 1.3 mM to 50 microM. The increase in mitochondrial Pi closely parallels that of mitochondrial Ca2+; when the increase in Pi and Ca2+ accumulation is maximal, the molar ratio is close to that in Ca3(PO4)2. Measurement of changes in the perfusate Pi revealed that, whereas administration of glucagon or vasopressin alone brought about a rapid decline in perfusate Pi, the largest decrease (reflecting net retention of Pi by the liver) was observed when the hormone was co-administered in the presence of 1.3 mM-Ca2+. The synergistic action of glucagon plus vasopressin was nullified by lowering the perfusate Ca2+ to 50 microM. The data provide evidence that, whereas glucagon may be able to alter Pi fluxes directly in intact liver, any alterations induced by vasopressin are indirect and result only from its action of mobilizing Ca2+.     

Regulation of Ca2+ efflux from kidney and liver mitochondria by unsaturated fatty acids and Na+ ions.

The effects of fatty acids and monovalent cations on the Ca2+ efflux from isolated liver and kidney mitochondria were investigated by means of electrode techniques. It was shown that unsaturated fatty acids and saturated fatty acids of medium chain length (C12 and C14) induced a Ca2+ efflux from mitochondria which was not inhibited by ruthenium red, but was specifically inhibited by Na+ and Li+. The Ca2+-releasing activity of unsaturated fatty acids did not correlate with their uncoupling activity. In kidney mitochondria a spontaneous, temperature-dependent Ca2+ efflux was observed which was inhibited either by albumin or by Na+. It is suggested that the net Ca2+ accumulation by mitochondria depends on the operation of independent pump and leak pathways. The pump is driven by the membrane potential and can be inhibited by ruthenium red, the leak depends on the presence of unsaturated fatty acids and is inhibited by Na+ and Li+. It is suggested that the unsaturated fatty acids produced by mitochondrial phospholipase A2 can be essential in the regulation of the Ca2+ retention in and the Ca2+ release from the mitochondria.     

Hepatic accumulation of pyrophosphate during acetate metabolism

Accumulation of pyrophosphate induced by acetate administration was investigated in rat liver in situ and in perfused rat liver. Intraperitoneal injection of acetate into rats increased the pyrophosphate concentration in the liver to about 2 mumol/g liver, which was 200 times that in control liver. Perfusion of liver with acetate alone did not result in accumulation of pyrophosphate. However, the further addition of a Ca2+-mobilizing hormone, such as noradrenaline or angiotensin II, together with glucagon to the perfusion medium containing 1 mM acetate caused accumulation of pyrophosphate to a similar level to that observed in vivo. Acetate, glucagon and a Ca2+-mobilizing hormone were all required for accumulation of pyrophosphate in perfused liver. Omission of Ca2+ from the perfusion medium or addition of a Ca2+-antagonist reduced the accumulation significantly. The two kinds of hormones, glucagon and an alpha-agonist, either singly or in combination, did not affect the rate of acetate utilization. These results show that liver cells accumulate a large amount of pyrophosphate during acetate metabolism at high intracellular levels of Ca2+ that can be realized by the synergistic actions of the two kinds of hormones.     

Inorganic pyrophosphate is located primarily in the mitochondria of the hepatocyte and increases in parallel with the decrease in light-scattering induced by gluconeogenic hormones, butyrate and ionophore A23187.

1. The effects of a variety of hormones on the PPi content and light-scattering of isolated rat liver cells was studied. 2. The basal PPi content was about 130 pmol/mg of cell protein, and increased after hormone addition, in parallel with a decrease in light-scattering which we have observed previously [Quinlan, Thomas, Armston & Halestrap (1983) Biochem. J. 214, 395-404]. 3. The mean increases in PPi content with the agonists shown (as pmol/mg of protein) were: 0.1 microM-glucagon, 25; 20 microM-phenylephrine, 30; 25 nM-vasopressin, 127; glucagon + phenylephrine, 115; glucagon + vasopressin, 382; 100 microM-ADP, 50; 15 microM-A23187, 72; 1 mM-butyrate, 80. 4. In the absence of extracellular Ca2+, vasopressin had little effect on either the PPi content or the light-scattering of hepatocytes. 5. The magnitude of the increase in PPi content correlated with that of the decrease in light-scattering irrespective of the stimulating agent, provided that the PPi did not exceed 300 pmol/mg of protein. Above this value little additional change in light-scattering was observed. 6. Subcellular fractionation showed that over 90% of the cellular PPi was intramitochondrial in both control and stimulated cells. 7. The data support the conclusions of previous experiments using isolated liver mitochondria [Davidson & Halestrap (1987) Biochem. J. 246, 715-723] that hormones increase the mitochondrial matrix volume through a Ca2+-induced rise in matrix [PPi]. 8. It is further proposed that this increase in mitochondrial [PPi] allows entry of ADP into the mitochondria in exchange for PPi and is therefore responsible for the increase in total mitochondrial adenine nucleotides observed after hormone treatment.     

NADP-linked dismutation and concentrations of citrate, cytosolic free Ca2+ and phosphoenolpyruvate in islet B-cells stimulated with glucose

B-cells stimulated with glucose showed enhanced activities of NADP-isocitrate dehydrogenase, aconitase, ATP-citrate lyase and phosphoenolpyruvate carboxy-kinase, elevated cytosolic [NADPH]/[NADP+] ratio, and increased concentrations of glucose 6-phosphate, 6-phosphogluconate, citrate, phosphoenolpyruvate and cytosolic free Ca2+. Phosphoenolpyruvate induced release of Ca2+ accumulated in isolated mitochondria. Glucose is suggested to stimulate a cytosolic NADP-linked dismutation and synthesis of citrate, associated with increased cytosolic free Ca2+ concentration. Subsequent breakdown of cytosolic citrate may yield Pi (possibly related to the "phosphate flush") and phosphoenolpyruvate which potentiates the release of Ca2+ accumulated in the B-cell mitochondria.     

Stabilising action of carnitine on energy linked processes in rat liver mitochondria

Rat liver mitochondria exposed to stressing conditions - ageing at room temperature, incubation in the presence of t-butyl hydroperoxide or damaging concentrations of Ca2+ and phosphate- undergo a rapid fall in their membrane potential (delta psi) with a concomitant release of endogenous Mg2+ and accumulated Ca2+. Addition of L-carnitine to the incubation medium considerably delays mitochondrial deenergization. A similar, though lower, protection has also been observed in L-carnitine pretreated and subsequently washed rat liver mitochondria. Furthermore mitochondria isolated from livers of starved rats, treated with L-carnitine 30 minutes before death and exposed to the same stressing conditions show similar delay in the decrease of delta psi and concurrent energy linked processes as compared with untreated animals. Both the in vitro and in vivo results strongly indicate that the stabilising action of L-carnitine on liver mitochondria is due to the removal of membrane bound long chain acyl CoA.     

Lithium desensitizes brain mitochondria to calcium, antagonizes permeability transition, and diminishes cytochrome C release

Among the numerous effects of lithium on intracellular targets, its possible action on mitochondria remains poorly explored. In the experiments with suspension of isolated brain mitochondria, replacement of KCl by LiCl suppressed mitochondrial swelling, depolarization, and a release of cytochrome c induced by a single Ca2+ bolus. Li+ robustly protected individual brain mitochondria loaded with rhodamine 123 against Ca2+-induced depolarization. In the experiments with slow calcium infusion, replacement of KCl by LiCl in the incubation medium increased resilience of synaptic and nonsynaptic brain mitochondria as well as resilience of liver and heart mitochondria to the deleterious effect of Ca2+. In LiCl medium, mitochondria accumulated larger amounts of Ca2+ before they lost the ability to sequester Ca2+. However, lithium appeared to be ineffective if mitochondria were challenged by Sr2+ instead of Ca2+. Cyclosporin A, sanglifehrin A, and Mg2+, inhibitors of the mitochondrial permeability transition (mPT), increased mitochondrial Ca2+ capacity in KCl medium but failed to do so in LiCl medium. This suggests that the mPT might be a common target for Li+ and mPT inhibitors. In addition, lithium protected mitochondria against high Ca2+ in the presence of ATP, where cyclosporin A was reported to be ineffective. SB216763 and SB415286, inhibitors of glycogen synthase kinase-3beta, which is implicated in regulating reactive oxygen species-induced mPT in cardiac mitochondria, did not increase Ca2+ capacity of brain mitochondria. Altogether, these findings suggest that Li+ desensitizes mitochondria to elevated Ca2+ and diminishes cytochrome c release from brain mitochondria by antagonizing the Ca2+-induced mPT.     

Calcium induced release of mitochondrial cytochrome c by different mechanisms selective for brain versus liver.

Increased mitochondrial Ca2+ accumulation is a trigger for the release of cytochrome c from the mitochondrial intermembrane space into the cytosol where it can activate caspases and lead to apoptosis. This study tested the hypothesis that Ca2+-induced release of cytochrome c in vitro can occur by membrane permeability transition (MPT)-dependent and independent mechanisms, depending on the tissue from which mitochondria are isolated. Mitochondria were isolated from rat liver and brain and suspended at 37 degrees C in a K+-based medium containing oxidizable substrates, ATP, and Mg2+. Measurements of changes in mitochondrial volume (via light scattering and electron microscopy), membrane potential and the medium free [Ca2+] indicated that the addition of 0.3 - 3.2 micromol Ca2+ mg-1 protein induced the MPT in liver but not brain mitochondria. Under these conditions, a Ca2+ dose-dependent release of cytochrome c was observed with both types of mitochondria; however, the MPT inhibitor cyclosporin A was only capable of inhibiting this release from liver mitochondria. Therefore, the MPT is responsible for cytochrome c release from liver mitochondria, whereas an MPT-independent mechanism is responsible for release from brain mitochondria.     

Rapid efflux of Ca2+ from heart mitochondria in the presence of inorganic pyrophosphate

Inorganic pyrophosphate (PPi) in the intracellular concentration range causes rapid efflux of Ca2+ from rat heart mitochondria oxidizing pyruvate + malate in a low Na+ medium. Half-maximal rates of Ca2+ efflux were given by 20 microM PPi. During and after PPi-stimulated Ca2+ efflux the mitochondria retain their structural integrity and complete respiratory control. Carboxyatractyloside inhibits PPi-stimulated Ca2+ efflux, indicating PPi must enter the matrix in order to promote Ca2+ efflux. Heart mitochondria have a much higher affinity for PPi uptake and PPi-induced Ca2+ efflux than liver mitochondria.     

Role of a nonmitochondrial Ca2+ pool in the synergistic stimulation by cyclic AMP and vasopressin of Ca2+ uptake in isolated rat hepatocytes.

The subcellular distribution of 45Ca2+ accumulated by isolated rat hepatocytes exposed to dibutyryl cyclic AMP (dbcAMP) followed by vasopressin (Vp) was studied by means of a nondisruptive technique. When treated with dbcAMP followed by vasopressin, hepatocytes obtained from fed rats accumulated an amount of Ca2+ approximately fivefold higher than that attained under control conditions. Ca2+ released from the mitochondrial compartment by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) accounted for only a minor portion of the accumulated Ca2+. The largest portion was released by the Ca2+ ionophore A23187 and was attributable to a nonmitochondrial compartment. DbcAMP + Vp-treatment also caused a maximal stimulation of glucose production and a twofold increase in cellular glucose 6-phosphate levels. In hepatocytes obtained from fasted rats, dbcAMP + Vp-stimulated Ca2+ accumulation was lower, although with the same subcellular distribution, and was associated with a minimal glucose production. In the presence of gluconeogenetic substrates (lactate plus pyruvate) hepatocytes from fasted rats were comparable to cells isolated from fed animals. However, Ca2+ accumulation and glucose 6-phosphate production could be dissociated in the absence of dbcAMP, in the presence of lactate/pyruvate alone. Under this condition in fact Vp induced only a minimal accumulation of Ca2+ in hepatocytes isolated from fasted rats, although glucose production was markedly increased. Moreover, treatment of fed rat hepatocytes with 1 mM ATP caused a maximal activation of glycogenolysis, but only a moderate stimulation of cellular Ca2+ accumulation. In this case, sequestration of Ca2+ occurred mainly in the mitochondrial compartment. By contrast, the addition of ATP to dbcAMP-pretreated hepatocytes induced a large accumulation of Ca2+ in a nonmitochondrial pool. Additional experiments using the fluorescent Ca2+ indicator Fura-2 showed that dbcAMP pretreatment can enlarge and prolong the elevation of cytosolic free Ca2+ caused by Vp. A nonmitochondrial Ca2+ pool thus appears mainly responsible for the Ca2+ accumulation stimulated by dbcAMP and Vp in isolated hepatocytes, and cyclic AMP seems able to activate Ca2+ uptake in such a nonmitochondrial pool.     

Stimulation of glycine catabolism in isolated perfused rat liver by calcium mobilizing hormones and in isolated rat liver mitochondria by submicromolar concentrations of calcium.

Glucagon stimulates flux through the glycine cleavage system (GCS) in isolated rat hepatocytes (Jois, M., Hall, B., Fewer, K., and Brosnan, J. T. (1989) J. Biol. Chem. 264, 3347-3351. In the present study, flux through GCS was measured in isolated rat liver perfused with 100 nM glucagon, 1 microM epinephrine, 1 microM norepinephrine, 10 microM phenylephrine, or 100 nM vasopressin. These hormones increased flux through GCS in perfused rat liver by 100-200% above the basal rate. The possibility that the stimulation of flux by adrenergic agonists and vasopressin is mediated by increases in cytoplasmic Ca2+ which in turn could regulate mitochondrial glycine catabolism was examined by measuring flux through GCS in isolated mitochondria in the presence of 0.04-2.88 microM free Ca2+. Flux through GCS in isolated mitochondria was exquisitely sensitive to free Ca2+ in the medium; half-maximal stimulation occurred at about 0.4 microM free Ca2+ and maximal stimulation (7-fold) was reached when the free Ca2+ in the medium was 1 microM. The Vmax (nanomoles/mg protein/min) and Km (millimolar) values for the flux through GCS in intact mitochondria were 0.67 +/- 0.16 and 20.66 +/- 4.82 in the presence of 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and 3.28 +/- 0.76 and 10.98 +/- 1.91 in presence of 0.5 microM free Ca2+, respectively. The results show that the flux through GCS is sensitive to concentrations of calcium which would be achieved in the cytoplasm of hepatocytes stimulated by calcium-mobilizing hormones.     

Ca2+ and Mg2+ as modulators of mitochondrial L-glycerol-3-phosphate dehydrogenase

1. Physiological concentrations of either Ca2+ or Mg2+ stimulated L-glycerol 3-phosphate oxidation by intact mitochondria isolated from various mammalian tissues (hamster brown adipose tissue, rat brain, liver of normal and hyperthyroid rats). A higher cation concentration was required for stimulation by Mg2+ than by Ca2+. L-glycerol-3-phosphate dehydrogenase was the target of the stimulation by both cations as revealed by measurements with intact mitochondria as well as with the solubilized enzyme. With different electron acceptors Ca2+ and Mg2+ stimulation occurred at significantly different cation concentrations. 2. Substrate activation of mitochondrial L-glycerol-3-phosphate dehydrogenase was observed in intact mitochondria and with the solubilized enzyme isolated from hyperthyroid rats in the absence of Ca2+ and Mg2+. According to kinetic analysis two independent binding sites, functioning with different turnovers and with different affinities for the substrate, could account for the phenomenon. In the presence of Ca2+ or Mg2+ substrate activation could not be detected; the kinetic parameters apparently correspond to the tight substrate-binding site functioning with high turnover. 3. Thiol group(s), which in the absence of Ca2+ and Mg2+ did not participate in the functioning of the enzyme, played an essential role in the binding of these cations to the enzyme, as shown by chemical modification studies. 4. From the solubilized mitochondrial proteins L-glycerol-3-phosphate dehydrogenase was bound selectively to the hydrophobic phenyl-Sepharose 4B matrix in the presence Ca2+, and the bound enzyme could be eluted with EDTA. This suggests that Ca2+ caused an alteration in the conformation of the enzyme.     

Na+-dependent regulation of extramitochondrial Ca2+ by rat-liver mitochondria

The presence and significance of Na+-induced Ca2+ release from rat liver mitochondria was investigated by the arsenazo technique. Under the experimental conditions used, the mitochondria, as expected, avidly extracted Ca2+ from the medium. However, when the uptake pathway was blocked with ruthenium red, only a small rate of 'basal' release of Ca2+ was seen (0.3 nmol Ca2+ X min-1 X mg-1), in marked contrast to earlier reports on a rapid loss of sequestered Ca2+ from rat liver mitochondria. The addition of Na+ in 'cytosolic' levels (20 mM) led to an increase in the release rate by about 1 nmol Ca2+ X min-1 X mg-1. This effect was specific for Na+. The significance of this Na+-induced Ca2+ release, in relation to the Ca2+ uptake mechanism, was investigated (in the absence of uptake inhibitors) by following the change in the extramitochondrial Ca2+ steady-state level (set point) induced by Na+. A five-fold increase in this level, from less than 0.2 microM to more than 1 microM, was induced by less than 20 mM Na+. The presence of K+ increased the sensitivity of the Ca2+ homeostat to Na+. The effect of Na+ on the extramitochondrial level was equally well observed in an K+/organic-anion buffer as in a sucrose buffer. Liver mitochondria incubated under these circumstances actively counteracted a Ca2+ or EGTA challenge by taking up or releasing Ca2+, so that the initial level, as well as the Na+-controlled level, was regained. It was concluded that liver mitochondria should be considered Na+-sensitive, that the capacity of the Na+-induced efflux pathway was of sufficient magnitude to enable it to influence the extramitochondrial Ca2+ level biochemically and probably also physiologically, and that the mitochondria have the potential to act as active, Na+-dependent regulators of extramitochondrial ('cytosolic') Ca2+. It is suggested that changes of cytosolic Na+ could be a mediator between certain hormonal signals (notably alpha 1-adrenergic) and changes in this extramitochondrial ('cytosolic') Ca2+ steady state level.     

Effects of lysophospholipids on Ca2+ transport in rat liver mitochondria incubated at physiological Ca2+ concentrations in the presence of Mg2+, phosphate and ATP at 37 degrees C.

Lysophospholipids caused the release of 45Ca2+ from isolated rat liver mitochondria incubated at 37 degrees C in the presence of low concentrations of free Ca2+, ATP, Mg2+, and phosphate ions. The concentrations of lysophosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidic acid and lysophosphatidylinositol which gave half-maximal effects were 5, 26, 40 and 56 microM, respectively. The effects of lysophosphatidylethanolamine were not associated with a significant impairment of the integrity of the mitochondria as monitored by measurement of membrane potential and the rate of respiration. Lysophosphatidylethanolamine did not induce the release of Ca2+ from a microsomal fraction, or enhance Ca2+ inflow across the plasma membrane of intact cells, but did release Ca2+ from an homogenate prepared from isolated hepatocytes and incubated under the same conditions as isolated mitochondria. The proportion of mitochondrial 45Ca2+ released by lysophosphatidylethanolamine was not markedly affected by altering the total amount of Ca2+ in the mitochondria, the concentration of extramitochondrial Mg2+, by the addition of Ruthenium Red, or when oleoyl lysophosphatidylethanolamine was employed instead of the palmitoyl derivative. The effects of 5 microM-lysophosphatidylethanolamine were reversed by washing the mitochondria. The possibility that lysophosphatidylethanolamine acts to release Ca2+ from mitochondria in intact hepatocytes following the binding of Ca2+-dependent hormones to the plasma membrane is briefly discussed.     

Effect of bivalent metal ions on the fluctuations of ion currents in rat liver mitochondria

Ions of bivalent metals are shown to arrange in the Sr2+ greater than Ca2+ greater than Ba2+ greater than Mn2+ series as to their ability to induce ion flow vibration in the rat liver mitochondria. Application of Sr2+ results in the most stable prolonged vibrations of ion flows in mitochondria. Ca2+, Ba2+ and Mn2+ induce slightly pronounced and intensively damped vibrations. The studied Mg2+, Co2+, Ni2+, Pb2+ Fe2+ cations have effect on valinomycin-induced K+ transport in mitochondria and do not induce vibrations. It is established that the ability of bivalent cations to induce vibrations is associated with the possibility of their transfer through the mitochondrion membrane and accumulation in the matrix. Inhibitors of the electrogenic Ca2+ transport in mitochondria produce the similar effect on vibrations induced by Sr2+, Ca2+, Ba2+ and Mn2+.     

Inhibition of oxidative phosphorylation in ascites tumor mitochondria and cells by intramitochondrial Ca2+

Accumulation of Ca2+ (+ phosphate) by respiring mitochondria from Ehrlich ascites or AS30-D hepatoma tumor cells inhibits subsequent phosphorylating respiration in response to ADP. The respiratory chain is still functional since a proton-conducting uncoupler produces a normal stimulation of electron transport. The inhibition of phosphorylating respiration is caused by intramitochondrial Ca2+ (+ phosphate). ATP + Mg2+ together, but not singly, prevents the inhibitory action of Ca2+. Neither AMP, GTP, GDP, nor any other nucleoside 5'-triphosphate or 5'-diphosphate could replace ATP in this effect. Phosphorylating respiration on NAD(NADP)-linked substrates was much more susceptible to the inhibitory effect of intramitochondrial Ca2+ than succinate-linked respiration. Significant inhibition of oxidative phosphorylation is given by the endogenous Ca2+ present in freshly isolated tumor mitochondria. The phosphorylating respiration of permeabilized Ehrlich ascites tumor cells is also inhibited by Ca2+ accumulated by the mitochondria in situ. Possible causes of the Ca2+-induced inhibition of oxidative phosphorylation are considered.     

Synergistic stimulation of Ca2+ uptake by glucagon and Ca2+-mobilizing hormones in the perfused rat liver. A role for mitochondria in long-term Ca2+ homoeostasis.

A perfused liver system incorporating a Ca2+-sensitive electrode was used to study the long-term effects of glucagon and cyclic AMP on the mobilization of Ca2+ induced by phenylephrine, vasopressin and angiotensin. At 1.3 mM extracellular Ca2+ the co-administration of glucagon (10 nM) or cyclic AMP (0.2 mM) and a Ca2+-mobilizing hormone led to a synergistic potentiation of Ca2+ uptake by the liver, to a degree which was dependent on the order of hormone administration. A maximum net amount of Ca2+ influx, corresponding to approx. 3800 nmol/g of liver (the maximum rate of influx was 400 nmol/min per g of liver), was induced when cyclic AMP or glucagon was administered about 4 min before vasopressin and angiotensin. These changes are over an order of magnitude greater than those induced by Ca2+-mobilizing hormones alone [Altin & Bygrave (1985) Biochem. J. 232, 911-917]. For a maximal response the influx of Ca2+ was transient and was essentially complete after about 20 min. Removal of the hormones was followed by a gradual efflux of Ca2+ from the liver over a period of 30-50 min; thereafter, a similar response could be obtained by a second administration of hormones. Dose-response measurements indicate that the potentiation of Ca2+ influx by glucagon occurs even at low (physiological) concentrations of the hormone. By comparison with phenylephrine, the stimulation of Ca2+ influx by vasopressin and angiotensin is more sensitive to low concentrations of glucagon and cyclic AMP, and can be correlated with a 20-50-fold increase in the calcium content of mitochondria. The reversible uptake of such large quantities of Ca2+ implicates the mitochondria in long-term cellular Ca2+ regulation.     

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.     

Partial inhibition by cyclosporin A of the swelling of liver mitochondria in vivo and in vitro induced by sub-micromolar [Ca2+], but not by butyrate. Evidence for two distinct swelling mechanisms.

1. The effects of cyclosporin A on the increase in matrix PPi and consequent swelling of energized liver mitochondria incubated with 1 mM-butyrate, 30 microM-bongkrekic acid or 0.1-35 microM-Ca2+ [Halestrap (1989) Biochim. Biophys. Acta 973, 355-382] were studied. 2. Cyclosporin (1 microM) had no significant effect on the swelling induced by butyrate, bongkrekic acid or Ca2+ at concentrations of less than 0.3 microM. 3. At higher [Ca2+] (greater than 0.3 microM), swelling became progressively inhibited by cyclosporin, although the increase in matrix PPi was slightly greater in the presence than in the absence of cyclosporin. 4. Titration with cyclosporin indicated that there are 128 pmol of relevant cyclosporin-binding sites per mg of mitochondrial protein, with a Ki of about 5 nM. 5. The decrease in light-scattering by hepatocytes induced by butyrate [Davidson & Halestrap (1988) Biochem. J. 254, 379-384] was unaffected by cyclosporin, whereas that induced by vasopressin was inhibited by 20-30% without a significant change in cellular PPi content. 6. It is suggested that there are two mechanisms for the increase in mitochondrial volume induced by Ca2+: a PPi-mediated mechanism that is insensitive to cyclosporin and an additional Ca2(+)-mediated effect that is inhibited by cyclosporin. The nature of these pathways and their inter-relationship is discussed in the following paper [Halestrap & Davidson (1990) Biochem. J. 268, 153-160].