Linked oscillations of free Ca2+ and the ATP/ADP ratio in permeabilized RINm5F insulinoma cells supplemented with a glycolyzing cell-free muscle extract

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

Phosphate free perfusion prevents washout of tissue creatine in Langendorff perfused rabbit heart.

Rabbit hearts were perfused with Krebs-Henseleit bicarbonate buffer supplemented with 15 mM glucose and 10 mU/ml of insulin +/- Pi. At the end of 60 min the hearts were freeze-clamped and the content of ATP, creatine phosphate, creatine, lactate, pyruvate, DHAP and 3-P glycerate were determined enzymatically in neutralized perchloric acid tissue extracts. The free cytosolic ADP and Pi and the cytosolic NAD+ redox and phosphorylation potentials were calculated from the measured metabolite concentrations. Pi free perfusion resulted in increased creatine, free cytosolic ADP and cytosolic phosphorylation potential, decreased calculated free Pi and no change in cardiac ATP and creatine phosphate content. The increase in the cytosolic phosphorylation potential was due to the lowering of cytosolic free Pi. The increase in ADP was due to the increase in creatine. The increase in creatine appeared to be due to an inhibition of creatine efflux from the heart during Pi free perfusion which was mediated by an enhanced Na+ electrochemical gradient.     

Acyl-CoA esters modulate intracellular Ca2+ handling by permeabilized clonal pancreatic beta-cells.

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

Pyruvate-enhanced phosphorylation potential and inotropism in normoxic and postischemic isolated working heart. Near-complete prevention of reperfusion contractile failure

Bioenergetic and hemodynamic consequences of cellular redox manipulations by 0.2-20 mM pyruvate were compared with those due to adrenergic stress (0.7-1.1 microM norepinephrine) using isolated working guinea-pig hearts under the conditions of normoxia, low-flow ischemia, and reperfusion. 5 mM glucose (+ 5 U/l insulin) + 5 mM lactate were the basal energy-yielding substrates. To stabilize left ventricular enddiastolic pressure, ventricular filling pressure was held at 12 cmH2O under all conditions; this preload control minimized Frank-Starling effects on ventricular inotropism. Global low-flow ischemia was induced by reducing aortic pressure to levels (20-10 cmH2O) below the coronary autoregulatory reserve. Reactants of the creatine kinase, including H+ and other key metabolites, were measured by enzymatic, HPLC, and polarographic techniques. In normoxic hearts, norepinephrine stimulations of inotropism, heart rate x pressure product, and oxygen consumption (MVO2) were associated with a fall in the cytosolic phosphorylation potential [( ATP]/[( ADP].[Pi]] as judged by the creatine kinase equilibrium. In contrast, infusion of excess pyruvate (5 mM) markedly increased [ATP]/[( ADP].[Pi]) and ventricular work output, while intracellular phosphate decreased; MVO2 remained constant under the same conditions. During reperfusion following ischemia, pyruvate effected striking and concentration-dependent increases in MVO2, phosphorylation potential, and inotropism. Pyruvate dehydrogenase flux was augmented during reperfusion hyperemia followed by near-complete recoveries of [ATP]/([ADP].[Pi]), contractile force, heart rate x pressure product, and MVO2 in the presence of 5-10 mM pyruvate. Pyruvate also attenuated ischemic adenylate degradation. Omission of glucose from the perfusion medium rendered pyruvate ineffective in postischemic hearts. Similarly, excess lactate (5-15 mM) or acetate (5 mM) failed to reenergize reperfused hearts and severe depressions of MVO2 and inotropism developed despite the presence of glucose. Apparently, subcellular redox manipulations by pyruvate dissociated stimulated mitochondrial respiration and increased inotropism from low cytosolic phosphorylation potentials. This was evidence against the extramitochondrial [ADP].[Pi]/[ATP] ratio being the primary factor in the control of mitochondrial respiration. The mechanism of pyruvate enhancement of inotropism during normoxia and reperfusion is probably multifactorial. Thermodynamic effects on subcellular [NADH]/[NAD+] ratios are coupled with a rise in the cytosolic [ATP]/[( ADP].[Pi]) ratio at constant (normoxia) or increased (reperfusion) MVO2.(ABSTRACT TRUNCATED AT 400 WORDS)     

The interactions of ATP, ADP, and inorganic phosphate with the sheep cardiac ryanodine receptor.

The effects of ATP, ADP, and inorganic phosphate (Pi) on the gating of native sheep cardiac ryanodine receptor channels incorporated into planar phospholipid bilayers were investigated. We demonstrate that ATP and ADP can activate the channel by Ca2+-dependent and Ca2+-independent mechanisms. ATP and ADP appear to compete for the same site/s on the cardiac ryanodine receptor, and in the presence of cytosolic Ca2+ both agents tend to inactivate the channel at supramaximal concentrations. Our results reveal that ATP not only has a greater affinity for the adenine nucleotide site/s than ADP, but also has a greater efficacy. The EC50 value for channel activation is approximately 0.2 mM for ATP compared to 1.2 mM for ADP. Most interesting is the fact that, even in the presence of cytosolic Ca2+, ADP cannot activate the channel much above an open probability (Po) of 0.5, and therefore acts as a partial agonist at the adenine nucleotide binding site on the channel. We demonstrate that Pi also increases Po in a concentration and Ca2+-dependent manner, but unlike ATP and ADP, has no effect in the absence of activating cytosolic [Ca2+]. We demonstrate that Pi does not interact with the adenine nucleotide site/s but binds to a distinct domain on the channel to produce an increase in Po.     

Combined glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase in catecholamine-stimulated guinea-pig cardiac muscle. Comparison with mass-action ratio of creatine kinase.

The steady-state reactant levels of triose-phosphate isomerase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system were examined in guinea-pig cardiac muscle. Key glycolytic intermediates, including glyceraldehyde 3-phosphate were directly measured and compared with those of creatine kinase. Non-working Langendorff hearts as well as isolated working hearts were perfused with 5 mM glucose (plus insulin) under normoxia conditions to maintain lactate dehydrogenase near-equilibrium. The cytosolic phosphorylation potential ([ATP]/([ADP].[Pi])) was derived from creatine kinase and the free [NAD+]/([NADH].[H+]) ratio from lactate dehydrogenase. In Langendorff hearts glycolysis was varied from near-zero flux (hyperkalemic cardiac arrest) to higher than normal flux (normal and maximum catecholamine stimulation). The triose-phosphate isomerase was near-equilibrium only in control or potassium-arrested Langendorff hearts as well as in postischemic 'stunned' hearts. However, when glycolytic flux increased due to norepinephrine or due to physiological pressure-volume work the enzyme was displaced from equilibrium. The alternative phosphorylation ratio [ATP]'/([ADP]).[Pi]) was derived from the magnesium-dependent glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system assigning free magnesium different values in the physiological range (0.1-2.0 mM). As predicted, [ATP]/([ADP].[Pi]) and [ATP]'/([ADP]'.[Pi]') were in excellent agreement when glycolysis was virtually halted by hyperkalemic arrest (flux approximately 0.2 mumol C3.min-1.g dry mass-1). However, the equality between the two phosphorylation ratios was not abolished upon resumption of spontaneous beating and also not during adrenergic stimulation (flux approximately 5-14 mumol C3.min-1.g dry mass-1). In contrast, when flux increased due to transition from no-work to physiological pressure-volume work (rate increase from approximately 3 to 11 mumol C3.min-1.g dry mass-1), the two ratios were markedly different indicating disequilibrium of the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. Only during adrenergic stimulation or postischemic myocardial 'stunning', not due to hydraulic work load per se, glyceraldehyde-3-phosphate levels increased from about 4 microM to greater than or equal to 16 microM. Thus the guinea-pig cardiac glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system can realize the potential for near-equilibrium catalysis at significant flux provided glyceraldehyde-3-phosphate levels rise, e.g., due to 'stunning' or adrenergic hormones.     

Contribution of the translocator of adenine nucleotides and the ATP synthase to the control of oxidative phosphorylation and arsenylation in liver mitochondria

The regulation of the rate of mitochondrial oxidative phosphorylation and arsenylation was studied at two external free Ca2+ concentrations. The rate of arsenate-stimulated respiration in absence of added ADP was not affected by external 10(-9) and 10(-6) M Ca2+ levels or carboxyatractyloside, while state 3 respiration was profoundly modified. In addition, the kinetic analysis showed that the rate of arsenylation in the presence of ADP was more efficient (Vm/Km ratio 3.5 times higher) in the catalytic process than phosphorylation. Therefore, this suggests that the activity of the ATP/ADP carrier is importantly controlled by Ca2+. The evaluation of the control in phosphorylation showed that the flux-control coefficients (Ci) exerted by the ATP/ADP carrier (ranged between 0.23 and 0.48) and the ATP synthase (0.05-0.57) were modified in a reciprocal way by Ca2+ and Pi concentrations. This suggests that these two enzymes are coupling sequentially through a common intermediate, the intramitochondrial ATP/ADP ratio. Other important steps controlling phosphorylation were the b-c1 complex (Ci = 0.30) and the cytochrome oxidase (Ci = 0.23) but they were not modified by Ca2+. It was also found that the main step controlling arsenylation was the ATP synthase (Ci = 0.74). The increment in the inorganic arsenate concentration induced a diminution in the control exerted by the ATP synthase (from 0.73 to 0.56). The results suggest that Ca2+ and Pi (or inorganic arsenate) could be regulated by ATP synthesis through an activating effect on ATP/ADP carrier and/or ATP synthase.     

Ca2+ translocation and catalytic activity of the sarcoplasmic reticulum ATPase. Modulation by ATP, Ca2+, and Pi

The ratio between Ca2+ uptake and Ca(2+)-dependent ATP hydrolysis measured in sarcoplasmic reticulum vesicles of rabbit skeletal muscle was found to vary greatly depending on the concentrations of oxalate or Pi used. In the presence of 5 mM oxalate, 20 mM Pi, and 1 mM Pi, the ratios found were in the range of 1.4-2.3, 0.6-0.8, and 0.01-0.10, respectively. The rates of Ca2+ exchange and ATP synthesis were measured at the steady state by adding trace amounts of 45Ca and 32Pi, after the vesicles had been loaded with Ca2+. In the presence of 1 mM Pi, 10 mM MgCl2, and 0.2 mM CaCl2, the ratio between Ca2+ exchange and ATP synthesis varied from 9 to 14. This ratio approached two when Ca2+ in the medium was reduced to a very low level, or when in the presence of Ca2+, dimethyl sulfoxide was added to the assay medium, or when the Pi concentration was raised from 1 to 20 mM. A ratio of two was also measured when the steady state was attained using ITP instead of ATP. In all the conditions that led to a ratio close to two, there was an increase in the fraction of enzyme phosphorylated by Pi. It is proposed that the coupling between Ca2+ translocation and ATP hydrolysis or synthesis is modulated by the phosphorylation of the ATPase by Pi.     

Cytosolic adenylates and adenosine release in perfused working heart. Comparison of whole tissue with cytosolic non-aqueous fractionation analyses

Free cytosolic adenylates were examined in relation to adenosine plus inosine released from perfused working guinea-pig hearts. Whole-tissue adenylate data from freeze-clamped hearts were quantitatively compared with corresponding values obtained by subcellular fractionation of homogenized myocardium in non-aqueous media. Adenosine and inosine in venous cardiac effluents were measured by high-performance liquid chromatography. Hearts, perfused at their natural flows, were subjected to various workloads, substrates and catecholamines to alter myocardial energy metabolism and respiration over a wide physiological range. Non-aqueous cytosolic ATP and creatine phosphate (CrP) accounted for more than 80% of the respective total myocardium content. The cytosolic CrP/Pi ratio was in near-quantitative agreement with the overall tissue CrP/Pi ratio when the latter parameter was corrected for extracellular Pi. This was conclusive evidence that ATP, CrP and Pi were predominantly located in the cytosol of the well-oxygenated cardiomyocyte. Measured myocardial oxygen uptake (MVO2) was reciprocally related to the phosphorylation state of CrP [( CrP]/[Cr] X [Pi]) and hence that of ATP [( ATP]/[ADP] X [Pi]) assuming the creatine kinase at near-equilibrium at a near-constant pH of 7.2. On the other hand, calculated mean free cytosolic ADP concentrations increased essentially linearly up to threefold with increasing MVO2 in the presence of virtually unchanged or only slightly decreased ATP levels; this was found both according to the whole tissue and the special subcellular fractionation data. Employing the myokinase mass-action ratio and substituting total cardiac ADP by the mean free cytosolic ADP concentrations, the mean free cytosolic AMP concentrations proved to be in the nanomolar range, i.e. up to three orders of magnitude lower than the overall tissue AMP content. We propose, therefore, that in the normoxic heart, AMP is located predominantly in the mitochondrial compartment. Nevertheless, both free cytosolic AMP concentration and release of adenosine plus inosine were apparently square or even higher-power functions of the rate of cardiac respiration. On the other hand, the mean purine nucleoside release seemed linearly correlated (r = 0.920) with the calculated free cytosolic AMP concentration. Our observations seem to suggest that the concentrations of free ADP and AMP in the cytosol are major determinants of the production of inosine and coronary vasodilator adenosine.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of ATP and free ADP in metabolic coupling during fuel-stimulated insulin release from islet beta-cells in the isolated perfused rat pancreas.

The isolated perfused rat pancreas was used to test the hypothesis that total cellular ATP or the ratio of ATP/free ADP plays the primary role in coupling intermediary metabolism to the biophysical events that are the basis of glucose-stimulated insulin release. The pancreas was preperfused for 20 min with 4.0 mM of a physiological mixture of 20 amino acids plus 4.2 mM glucose, and insulin release was then stimulated for 150 s by suddenly increasing the glucose to 8.3 mM. The pancreas was sampled at 24, 48, 72, and 150 s after the switch. The content of total ATP, ADP, AMP, Pi, phosphocreatine, and creatine were measured in beta-cell enriched cores of pancreatic islets microdissected from freeze-dried pancreas cryostat sections. Metabolites were measured by quantitative histochemical enzymatic cycling techniques. Modeling studies were carried out to assess the impact of biochemical analytical results on the membrane potential of the beta-cells. The level of free ADP was calculated using the creatine kinase equilibrium reaction and an intracellular pH of 7.2. First phase insulin release was stimulated at least 10-fold with the maximum reached 45 s after adding high glucose. The biochemical analytical data demonstrate that the total cellular level of the putative coupling factor ATP and of the ratios ATP/free ADP and ATP/free ADP x Pi are not significantly influenced by a glucose level change that causes a more than 10-fold surge of insulin release. The strength and limitations of the present experimental strategy and the implications of the results for our understanding of metabolic coupling in glucose-stimulated insulin release are discussed.     

Cardiac contractile function, oxygen consumption rate and cytosolic phosphates during inhibition of electron flux by amytal--a 31P-NMR study

In order to investigate the potential role of cytosolic phosphates ([ATP], [ADP] and [Pi]) in the integration of mitochondrial respiration and mechanical function in the perfused heart, inhibition of the substrate end of the respiratory chain by amytal has been employed. A stepwise increase in amytal concentration (from 0.2 to 1.2 mM) resulted in the progressive abolition of the cardiac oxygen consumption, rate (VO2) in hearts oxidizing pyruvate (5 mM). The inhibition curve for VO2 was S-shaped, with K0.5 = 1.1 mM, and independent of the initial VO2 values varied by coronary flow and isoproterenol (Iso) addition. ADP-stimulated respiration of isolated mitochondria (malate + pyruvate) was twice as sensitive to amytal inhibition, whereas state 2 respiration (before ADP addition) had the same sensitivity as cardiac VO2. Decrease in VO2 was followed by a decline in phosphocreatine (PCr) content and augmentation of Pi at nearly constant ATP level and intracellular pH as assessed by the 31P-NMR method. These changes were associated with an elevation of cytosolic free [ADP] and a reduction of the [ATP]/[ADP] ratio and ATP affinity calculated from creatine kinase equilibrium. Concomitantly, pressure-rate product (PRP), maximal rates of contraction and relaxation fell down and the end diastolic pressure (EDP) rose at all initial loads. Amytal-inhibited hearts retained the capability to respond to Iso stimulation (0.1 microM, about 50% enhancement of PRP) even at 1 mM amytal, but their response to elevation of coronary flow was greatly diminished. Alterations in the PRP value induced by the inhibitor at a fixed coronary flow correlated negatively with cytosolic [ADP] and [Pi], and positively with [ATP]/[ADP] and A(ATP). In contrast, EDP correlated with all these parameters in the opposite manner. However, when PRP was varied by coronary flow in the absence of the inhibitor or at its fixed concentrations, such correlations were absent. These data imply that cytosolic phosphates can serve as a feedback between energy production and utilization when the control point(s) is (are) at the mitochondria. In contrast, other regulatory mechanisms should be involved when control is distributed among different steps located both in energy producing and utilizing systems.     

Regulation of Ca2+ transport in brain mitochondria. II. The mechanism of the adenine nucleotides enhancement of Ca2+ uptake and retention

ADP greatly enhances the rate of Ca2+ uptake and retention in Ca2+ loaded mitochondria. Atractyloside, a specific inhibitor of the ADP/ATP translocator, completely inhibits the ADP effect, while bongkrekate, another specific inhibitor of the translocator enhances the effect of ADP. These results indicate that locking the ADP/ATP translocator in the M-state is sufficient to produce the ADP effect. Cyclosporin A, a specific inhibitor of the Ca2(+)-induced membrane permeabilization does not substitute for ADP, indicating that ADP directly affect the rate of electrogenic Ca2+ uptake. The effect of the translocator conformation on the rate of electrogenic Ca2+ uptake is independent of the concentration of Pi and is not caused by changes in membrane potential. However, locking the carrier in the M-state appears to increase the negative surface charge on the matrix face of the inner membrane. This may lead to an enhanced rate of Ca2+ dissociation from the electrogenic carrier at the matrix surface. The rate of Na(+)-independent Ca2+ efflux is only slightly inhibited by locking the carrier in the M-state, presumably due to the same mechanism. In the presence of ADP, Pi inhibits the Na(+)-independent efflux. In the presence of physiological concentrations of spermine, Pi and Mg2+, the rate of Ca2+ uptake, Ca2+ retention and Ca2+ set points depend sharply on ADP concentration at the physiological range of ADP. Thus, changes of cytosolic ADP concentration may lead to change in the rate of Ca2+ uptake by mitochondria and thus modulate the excitation-relaxation cycles of cytoplasmic free calcium.     

Glucose requirement for postischemic recovery of perfused working heart

The quantitative importance of glycolysis in cardiomyocyte reenergization and contractile recovery was examined in postischemic, preload-controlled, isolated working guinea pig hearts. A 25-min global but low-flow ischemia with concurrent norepinephrine infusion to exhaust cellular glycogen stores was followed by a 15-min reperfusion. With 5 mM pyruvate as sole reperfusion substrate, severe contractile failure developed despite normal sarcolemmal pyruvate transport rate and high intracellular pyruvate concentrations near 2 mM. Reperfusion dysfunction was characterized by a low cytosolic phosphorylation potential [( ATP]/[( ADP][Pi]) due to accumulations of inorganic phosphate (Pi) and lactate. In contrast, with 5 mM glucose plus pyruvate as substrates, but not with glucose as sole substrate, reperfusion phosphorylation potential and function recovered to near normal. During the critical ischemia-reperfusion transition at 30 s reperfusion the cytosolic creatine kinase appeared displaced from equilibrium, regardless of the substrate supply. When under these conditions glucose and pyruvate were coinfused, glycolytic flux was near maximum, the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction was enhanced, accumulation of Pi was attenuated, ATP content was slightly increased, and adenosine release was low. Thus, glucose prevented deterioration of the phosphorylation potential to levels incompatible with reperfusion recovery. Immediate energetic support due to maximum glycolytic ATP production and enhancement of the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction appeared to act in concert to prevent detrimental collapse of [ATP]/[( ADP][Pi]) during creatine kinase dysfunction in the ischemia-reperfusion transition. Dichloroacetate (2 mM) plus glucose stimulated glycolysis but failed fully to reenergize the reperfused heart; conversely, 10 mM 2-deoxyglucose plus pyruvate inhibited glycolysis and produced virtually instantaneous de-energization during reperfusion. The following conclusions were reached. (1) A functional glycolysis is required to prevent energetic and contractile collapse of the low-flow ischemic or reperfused heart (2). Glucose stabilization of energetics in pyruvate-perfused hearts is due in part to intensification of glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase activity. (3) 2-Deoxyglucose depletes the glyceraldehyde-3-phosphate pool and effects intracellular phosphate fixation in the form of 2-deoxyglucose 6-phosphate, but the cytosolic phosphorylation potential is not increased and reperfusion failure occurs instantly. (4) Consistent correlations exist between cytosolic ATP phosphorylation potential and reperfusion contractile function. The findings depict glycolysis as a highly adaptive emergency mechanism which can prevent deleterious myocyte deenergization during forced ischemia-reperfusion transitions in presence of excess oxidative substrate.     

Calcium stimulates ATP-Mg/Pi carrier activity in rat liver mitochondria

Adenine nucleotide transport over the carboxyatractyloside-insensitive ATP-Mg/Pi carrier was assayed in isolated rat liver mitochondria with the aim of investigating a possible regulatory role for Ca2+ on carrier activity. Net changes in the matrix adenine nucleotide content (ATP + ADP + AMP) occur when ATP-Mg exchanges for Pi over this carrier. The rates of net accumulation and net loss of adenine nucleotides were inhibited when free Ca2+ was chelated with EGTA and stimulated when buffered [Ca2+]free was increased from 1.0 to 4.0 microM. The unidirectional components of net change were similarly dependent on Ca2+; ATP influx and efflux were inhibited by EGTA in a concentration-dependent manner and stimulated by buffered free Ca2+ in the range 0.6-2.0 microM. For ATP influx, increasing the medium [Ca2+]free from 1.0 to 2.0 microM lowered the apparent Km for ATP from 4.44 to 2.44 mM with no effect on the apparent Vmax (3.55 and 3.76 nmol/min/mg with 1.0 and 2.0 microM [Ca2+]free, respectively). Stimulation of influx and efflux by [Ca2+]free was unaffected by either ruthenium red or the Ca2+ ionophore A23187. Calmodulin antagonists inhibited transport activity. In isolated hepatocytes, glucagon or vasopressin promoted an increased mitochondrial adenine nucleotide content. The effect of both hormones was blocked by EGTA, and for vasopressin, the effect was blocked also by neomycin. The results suggest that the increase in mitochondrial adenine nucleotide content that follows hormonal stimulation of hepatocytes is mediated by an increase in cytosolic [Ca2+]free that activates the ATP-Mg/Pi carrier.     

Oscillations in cytosolic free Ca2+, oxygen consumption, and insulin secretion in glucose-stimulated rat pancreatic islets

Insulin secretion in the intact organism, and by the perfused pancreas and groups of isolated perifused islets, is pulsatile. We have proposed a metabolic model of glucose-induced insulin secretion in which oscillations in the ATP/ADP ratio drive alterations in metabolic and electrical events that lead to insulin release. A key prediction of our model is that metabolically driven Ca2+ oscillations will also occur. Using the fluorescent Ca2+ probe, fura 2, digital image analysis, and sensitive O2 electrodes, we investigated cytosolic free Ca2+ responses and O2 consumption in perifused rat islets that had been maintained in culture for 1-4 days. We found that elevated ambient glucose increased the average cytosolic free Ca2+ level, the ATP/ADP ratio, and oxygen consumption, as previously found in freshly isolated islets. Oscillatory patterns were obtained for Ca2+, O2 consumption, and insulin secretion in the presence of 10 and 20 mM glucose. Very low amplitude oscillations in cytosolic free Ca2+ were observed at 3 mM nonstimulatory glucose levels. Evaluation of the Ca2+ responses of a large series of individual islets, monitored by digital image analysis and perifused at both 3 and 10 mM glucose, indicated that the rise in glucose concentration caused more than a doubling of the average cytosolic free Ca2+ value and a 4-fold increase in the amplitude of the oscillations with little change in period. The pattern of Ca2+ change within the islets was consistent with recruitment of responding cells. The coexistence of oscillations with similar periods in insulin secretion, oxygen consumption, and cytosolic free Ca2+ is consistent with the model of metabolically driven pulsatile insulin secretion.     

Adenine nucleotide compartmentation in foetal rat hepatocytes. Effects of atractyloside, oligomycin, calcium ionophore and adrenergic agonists

In the presence of lactate plus pyruvate, or glucose or alanine as substrates, ATP/ADP ratios in the cytosol were higher than in mitochondria in isolated rat foetal hepatocytes. The cytosolic ATP/ADP ratios were dependent on substrate (lactate + pyruvate greater than glucose greater than alanine). Oleate increased the cytosolic ATP/ADP ratios in the presence of the other substrates studied. Atractyloside decreased the cytosolic ATP/ADP ratios, oligomycin decreasing these values in both compartments. Isoproterenol, phenylephrine and Ca2+ ionophore decreased the cytosolic ATP/ADP ratio, without altering this value in mitochondria.     

Ca2+, NAD(P)H and membrane potential changes in pancreatic beta-cells by methyl succinate: comparison with glucose

The present study was undertaken to determine the main metabolic secretory signals generated by the mitochondrial substrate MeS (methyl succinate) compared with glucose in mouse and rat islets and to understand the differences. Glycolysis and mitochondrial metabolism both have key roles in the stimulation of insulin secretion by glucose. Both fuels elicited comparable oscillatory patterns of Ca2+ and changes in plasma and mitochondrial membrane potential in rat islet cells and clonal pancreatic beta-cells (INS-1). Saturation of the Ca2+ signal occurred between 5 and 6 mM MeS, while secretion reached its maximum at 15 mM, suggesting operation of a K(ATP)-channel-independent pathway. Additional responses to MeS and glucose included elevated NAD(P)H autofluorescence in INS-1 cells and islets and increases in assayed NADH and NADPH and the ATP/ADP ratio. Increased NADPH and ATP/ADP ratios occurred more rapidly with MeS, although similar levels were reached after 5 min of exposure to each fuel, whereas NADH increased more with MeS than with glucose. Reversal of MeS-induced cell depolarization by Methylene Blue completely inhibited MeS-stimulated secretion, whereas basal secretion and KCl-induced changes in these parameters were not affected. MeS had no effect on secretion or signals in the mouse islets, in contrast with glucose, possibly due to a lack of malic enzyme. The data are consistent with the common intermediates being pyruvate, cytosolic NADPH or both, and suggest that cytosolic NADPH production could account for the more rapid onset of MeS-induced secretion compared with glucose stimulation.     

A P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) ³¹P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5–12-times the steady-state rates of ATP utilization (estimated from rates of O₂-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a K_m for cytosolic ADP of 35 μM and an apparent V_max of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr /ag ATP or ATP /ag ADP + P_i interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.     

Effect of ADP on the rate of acetyl phosphate hydrolysis by the Ca2+-ATPase of sarcoplasmic reticulum

Hydrolysis of acetyl phosphate is inhibited by high concentrations of Pi and MgCl2, probably due to an increase in the steady-state level of phosphoenzyme formed from Pi in the medium. A dual effect of ADP during steady-state hydrolysis of acetyl phosphate was observed. ADP inhibited hydrolysis in the presence of 5 mM MgCl2 and no added Pi, whereas it stimulated hydrolysis when phosphoenzyme formation by Pi was favored by including 6 mM Pi and 20 mM MgCl2 in the assay medium. ATP inhibited acetyl phosphate hydrolysis in both of these assay media. When phosphoenzyme formation by Pi in the presence of acetyl phosphate was stimulated at Ca2+ concentrations sufficient to saturate the low-affinity Ca2+-binding sites, ADP stimulated acetyl phosphate hydrolysis and also promoted ATP synthesis by reversal of the catalytic cycle. The rate of ATP synthesis was dependent on ADP, Pi and Ca2+. Phosphoenzyme formation by Pi and MgCl2, whether in the absence of Ca2+ and acetyl phosphate, or during acetyl phosphate hydrolysis, was inhibited by ADP and ATP. These results suggest that ADP interacts with different intermediates of the catalytic cycle and that expression of inhibition or activation of acetyl phosphate hydrolysis depends on the steady-state level of phosphoenzyme formed by Pi.     

The role of ADP in the modulation of the calcium-efflux pathway in rat brain mitochondria.

The role of ADP in the regulation of Ca2+ efflux in rat brain mitochondria was investigated. ADP was shown to inhibit Ruthenium-Red-insensitive H+- and Na+-dependent Ca2+-efflux rates if Pi was present, but had no effect in the absence of Pi. The primary effect of ADP is an inhibition of Pi efflux, and therefore it allows the formation of a matrix Ca2+-Pi complex at concentrations above 0.2 mM-Pi and 25 nmol of Ca2+/mg of protein, which maintains a constant free matrix Ca2+ concentration. ADP inhibition of Pi and Ca2+ efflux is nucleotide-specific, since in the presence of oligomycin and an inhibitor of adenylate kinase ATP does not substitute for ADP, is dependent on the amount of ADP present, and requires ADP concentrations in excess of the concentrations of translocase binding sites. Brain mitochondria incubated with 0.2 mM-Pi and ADP showed Ca2+-efflux rates dependent on Ca2+ loads at Ca2+ concentrations below those required for the formation of a Pi-Ca2+ complex, and behaved as perfect cytosolic buffers exclusively at high Ca2+ loads. The possible role of brain mitochondrial Ca2+ in the regulation of the tricarboxylic acid-cycle enzymes and in buffering cytosolic Ca2+ is discussed.