Ruthenium Red inhibits the activation of pyruvate dehydrogenase caused by positive inotropic agents in the perfused rat heart.

The increases in the amount of active, non-phosphorylated, pyruvate dehydrogenase caused by positive inotropic agents (from a control value of about 10%, to 40% of total enzyme) in the perfused rat heart could be completely blocked by prior perfusion with 2.5 micrograms of Ruthenium Red/ml. A similar increase caused by 5 mM-pyruvate was not blocked. This concentration of Ruthenium Red caused a 25% decrease in contractile force of hearts perfused in the absence of positive inotropic agents; however, in their presence the contractile force reached the same value in the absence or presence of Ruthenium Red. Neither control nor stimulated phosphorylase a content was affected by Ruthenium Red. Verapamil (0.1 microM) also decreased control contraction (by 40%), but did not block the activation of pyruvate dehydrogenase caused by a rise in extracellular [Ca2+]. The results support the hypothesis that positive inotropic agents activate pyruvate dehydrogenase in rat heart by increasing intramitochondrial [Ca2+].     

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.     

Phosphorylation and functional modifications of sarcoplasmic reticulum and myofibrils in isolated rabbit hearts stimulated with isoprenaline.

Isoprenaline stimulation of perfused rabbit hearts was associated with simultaneous phosphorylation of proteins in the myofilaments and phospholamban in the sarcoplasmic reticulum (SR). Hearts were perfused with Krebs-Henseleit buffer containing [32P]Pi, freeze-clamped in a control condition or at the peak of the inotropic response to isoprenaline, and myofibrils and SR were prepared from the same hearts. Stimulation of 32P incorporation in troponin I (TnI) and C-protein by isoprenaline was associated with a decrease in Ca2+-sensitivity of the myofibrillar Mg2+-dependent ATPase activity. Stimulation of 32P incorporation in SR by isoprenaline was associated with an increase in the initial rates of oxalate-facilitated Ca2+ transport, assayed with SR vesicles in either microsomal fractions or homogenates from the perfused hearts. These findings provide evidence that phosphorylation of TnI, C-protein and phospholamban in the intact cell is associated with functional alterations of the myofibrils and SR which may be responsible in part for the effects of catecholamines on the mammalian myocardium.     

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.     

The alpha-adrenergic-mediated activation of the cardiac mitochondrial Ca2+ uniporter and its role in the control of intramitochondrial Ca2+ in vivo.

Administration of methoxamine (10 microM, 2 min) to perfused rat hearts increased the rate at which subsequently isolated mitochondria accumulated Ca2+. Methoxamine did not change significantly the development of delta phi with time or the basal rates of Ca2+ flux on inhibition of the uniporter with Ruthenium Red. With 200 microM-Pi, the rates of Ca2+ uptake at constant delta phi were unaffected by the small variations in endogenous [Pi] between mitochondrial preparations, and were also unaffected by changes in internal Ca2+ over the approximate range 8-43 nmol of Ca2+/mg. At low internal Ca2+ (about 8 nmol/mg of protein) the rates of Ca2+ uptake at constant delta phi were unaffected by addition of 200 microM-Pi. Under these conditions, the uniporter activity and the uniporter conductance were increased by 38-40% by methoxamine pretreatment. The endogenous Ca2+ content of mitochondria from control heart was about 1.8 nmol of Ca2+/mg of protein. Perfusion with agonist increased the Ca2+ content as follows: 10 microM-methoxamine (2 min), 48%; 1 microM-isoprenaline (2 min), 100%; 1 microM-adrenaline (2 min), 140%. The implications of the data for the adrenergic control of oxidative metabolism by intramitochondrial Ca2+ is discussed.     

Distribution of control of oxidative phosphorylation in mitochondria oxidizing NAD-linked substrates.

The flux control distribution of the net rate of state 3 respiration was determined in heart and kidney mitochondria incubated with low concentrations of pyruvate (0.5 mM) or 2-oxoglutarate (1 mM), and in conditions that led to activation of NAD-linked dehydrogenases, i.e., high substrate or Ca2+ concentrations. Control of flux was exerted by the ATP/ADP carrier (flux control coefficient, ci = 0.37) and Site 1 of the respiratory chain (ci = 0.28) when dehydrogenase activity was low. Control of the process shifted to the ATP synthase (ci = 0.32) and the Pi carrier (Ci = 0.27) when dehydrogenases were activated by high pyruvate and high Ca2+. The changes in the control exerted by the ATP/ADP carrier and the ATP synthase were not due to changes in the transmembrane potential, nor to a modification of intramitochondrial ATP/ADP ratios. Applying the summation theorem of the control analysis, it was found that at low Ca2+ and pyruvate concentrations the dehydrogenases shared the control of state 3 respiration with other steps. The NAD-linked dehydrogenases did not exert any significant control at high Ca2+ or high pyruvate concentrations.     

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.     

Phosphorylation of phospholamban in intact myocardium. Role of Ca2+-calmodulin-dependent mechanisms

Phospholamban, a putative regulator of cardiac sarcoplasmic reticulum Ca2+ transport, has been shown to be phosphorylated in vitro by cAMP-dependent protein kinase and an intrinsic Ca2+-calmodulin-dependent protein kinase activity. This study was conducted to determine if Ca2+-calmodulin-dependent phosphorylation of phospholamban occurs in response to physiologic increases in intracellular Ca2+ in intact myocardium. Isolated guinea pig and rat ventricles were perfused with 32Pi after which membrane vesicles were isolated from individual hearts by differential centrifugation. Administration of isoproterenol (10 nM) to perfused hearts stimulated 32P incorporation into phospholamban, Ca2+-ATPase activity, and Ca2+ uptake of sarcoplasmic reticulum isolated from these hearts. These biochemical changes were associated with increases in contractility and shortening of the t 1/2 of relaxation. Elevated extracellular Ca2+ produced comparable increases in contractility but failed to stimulate phospholamban phosphorylation or Ca2+ transport and did not alter the t 1/2 of relaxation. Inhibition of trans-sarcolemmal Ca2+ influx by perfusing the ventricles with reduced extracellular Ca2+ (50 microM) attenuated the increases in 32P incorporation produced by 10 nM isoproterenol. Trifluoperazine (10 microM) also attenuated isoproterenol-induced increases in 32P incorporation into phospholamban. In both cases, Ca2+ transport was reduced to a degree comparable to the reduction in phospholamban phosphorylation. These results suggest that direct physiologic increases in intracellular Ca2+ concentration do not stimulate phospholamban phosphorylation in intact functioning myocardium. Ca2+-calmodulin-dependent phosphorylation of phospholamban may occur in response to agents which stimulate cAMP-dependent mechanisms in intact myocardium.     

Relation between the O2 supply:demand ratio, MVO2, and adenosine formation in hearts stimulated with inotropic agents

Mooted controllers of adenosine formation in heart are the oxygen supply:demand ratio, myocardial oxygen consumption (MVO2), the cytosolic phosphorylation potential (log[ATP]/[ADP][Pi]). The relationship between these parameters and purine release (adenosine + inosine) into the venous effluent was examined in isovolumic rat hearts perfused at 20 and 12 mL.min-1.g-1 with a glucose containing crystalloid buffer and stimulated with inotropic agents (isoproterenol, norepinephrine, 3-isobutyl-1-methylxanthine, and ouabain). The oxygen supply:demand ratio and MVO2 were continuously determined using an oxygen electrode to monitor oxygen supply and consumption. The phosphorylation potential was calculated from phosphorus metabolite levels determined by 31P-NMR spectroscopy and HPLC analysis. Left ventricular function was assessed as the rate-pressure product. All inotropic agents increased the rate-pressure product, with increases in function being greater in the hearts perfused at 20 mL.min-1.g-1. MVO2 was linearly related to the rate-pressure product at each flow rate; however, the hearts perfused at 20 mL.min-1.g-1 exhibited approximately twofold greater MVO2 values for similar rate-pressure product values. All inotropic agents increased adenosine release into the venous effluent. While there was a significant linear relation between adenosine formation and MVO2 in hearts perfused at both flow rates and stimulated with drugs, the relations differed with adenosine release being approximately fourfold greater in hearts perfused at 12 mL.min-1.g-1 under similar conditions of MVO2. Adenosine formation correlated exponentially with the ratio of oxygen supply:demand under all conditions (r = 0.97) and the relation did not differ significantly between hearts perfused at different rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

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 steady-state free Ca2+ levels by the ATP/ADP ratio and orthophosphate in permeabilized RINm5F insulinoma cells

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

ATP-Mg/Pi carrier activity in rat liver mitochondria.

The ATP-Mg/Pi carrier in liver mitochondria is activated by micromolar Ca2+ and mediates net adenine nucleotide transport into and out of the mitochondrial matrix. The purpose of this study was to characterize certain features of ATP-Mg/Pi carrier activity that are essential for understanding how the mitochondrial adenine nucleotide content is regulated. The relative importance of ATP and ADP as transport substrates was investigated using specific trap assays to measure their separate rates of carrier-mediated efflux with Pi as the external counterion. Under energized conditions ATP efflux accounted for 88% of total ATP+ADP efflux. With oligomycin present to lower the matrix ATP/ADP ratio, ATP efflux was eliminated and ADP efflux was relatively unaffected. Mg2+ was stoichiometrically required for ATP influx and is probably transported simultaneously with ATP. Ca2+ and Mn2+ could substitute for the stoichiometric Mg2+ requirement. ADP influx and Pi-induced adenine nucleotide efflux were unaffected by external Mg2+. Experiments with Pi analogues suggested that Pi is transported as the divalent anion, HPO4(2-). The results show that ATP-Mg and divalent Pi are the major transport substrates; the most probable transport mechanism for the ATP-Mg/Pi carrier is an electroneutral exchange. The results are consistent with the hypothesis that the direction and magnitude of net adenine nucleotide movements are determined mainly by the (ATP-Mg)2- and HPO4(2-) concentration gradients across the inner mitochondrial membrane.     

Regulation of steady state level of phosphoenzyme and ATP synthesis in sarcoplasmic reticulum vesicles during reversal of the Ca2+ pump.

The role of the Ca2+ concentration gradient in ATP synthesis and membrane phosphorylation by Pi was investigated in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. The Pi concentration required to attain 50% of the maximal membrane phosphorylation varies significantly in the pH range of 5.5 to 4.5, the optimal being at pH 6.0. In the pH range of 6.0 to 7.0, this concentration of Pi was 4- to 10-fold higher in empty vesicles than in vesicles loaded with calcium phosphate, i.e. having transmembrane Ca2+ concentration gradient. ATP, ADP, and Ca2+ inhibit the membrane phosphorylation by Pi, the inhibition being greater at pH 7.0 than at pH 6.0. The pH profile for ATP synthesis shows a higher optimum than for membrane phosphorylation. The optimum pH for synthesis, but not for phosphorylation depends on whether the vesicles were previously loaded with calcium phosphate or with calcium oxalate. Addition of Ca2+ to the assay medium inhibits the extent of membrane phosphorylation and the rate of ATP synthesis to different extents. Evidence is presented that the rate of membrane phosphorylation by Pi is higher than the rate by which the phosphoprotein transfers its pohsphate to ADP for the ATP synthesis.     

ATP in equilibrium with 32Pi exchange catalyzed by plasma membrane Ca(2+)-ATPase from kidney proximal tubules.

The Ca(2+)-stimulated adenosine 5'-triphosphate-orthophosphate (ATP in equilibrium with 32Pi) exchange reaction was studied using a vesicular preparation derived from plasma membrane of kidney proximal tubules. With native inside-out vesicles, ATP in equilibrium with 32Pi was stimulated by micromolar Ca2+ concentrations. Treatment of the vesicles with the Ca2+ ionophore A23187 that abolished Ca2+ accumulation, strongly inhibited ATP in equilibrium with 32Pi. When Ca(2+)-ATPase was solubilized with the nonionic detergent octaethylene glycol mono n-dodecyl ether, maximal activation of ATP in equilibrium with 32Pi required millimolar Ca2+ concentrations. These Ca2+ concentrations inhibited ATP hydrolysis. ATP in equilibrium with 32Pi exhibited a Michaelian dependence on Pi and Mg2+, was stimulated by ATP, and depended on the ATP/ADP ratio. ATP in equilibrium with 32Pi was modified by the osmolytes urea, trimethylamine-N-oxide, and sucrose, which are representative of the methylamines and polyols that normally accumulate in renal tissue. These compounds did not modify the apparent affinity for Pi; they affected the response to ADP in the same fashion as the overall rate of ATP in equilibrium 32Pi, and their effects depended on medium pH. These data show that the Ca(2+)-ATPase from plasma membrane kidney proximal tubules can operate simultaneously in forward and backward directions. They also show that ATP in equilibrium with 32Pi is modulated by the ligands Ca2+, ATP, ADP, Pi, Mg2+, and H+, and by organic solutes found in renal tissue.     

Alpha-adrenergic stimulation of sarcolemmal protein phosphorylation and slow responses in intact myocardium

The effects of alpha- and beta-adrenergic stimulation on sarcolemmal protein phosphorylation and contractile slow responses were studied in intact myocardium. Isolated rat ventricles were perfused via the coronary arteries with 32Pi after which membrane vesicles partially enriched in sarcolemma were isolated from individual hearts. Alterations in the sarcolemmal slow inward Ca2+ current were assessed in the 32P-perfused hearts using a contractile slow response model. In this model, Na+ channels were first inactivated by partial depolarization of the hearts in 25 mM K+ after which alterations in Ca2+ channel activity produced by either alpha- or beta-adrenergic agonists could be assessed as restoration of contractions. alpha-Adrenergic stimulation (phenylephrine + propranolol) of the perfused hearts resulted in increased 32P incorporation into a 15-kDa sarcolemmal protein. This protein co-migrated with the 15-kDa sarcolemmal protein phosphorylated in hearts exposed to beta-adrenergic stimulation produced by isoproterenol. beta-Adrenergic stimulation, but not alpha-adrenergic stimulation, also resulted in phosphorylation of the sarcoplasmic reticulum protein, phospholamban. Phosphorylation of the 15-kDa protein in perfused hearts in response to either alpha- or beta-adrenergic stimulation was associated with restoration of contractions, indicative of increases in the slow inward Ca2+ current. Increases in 32P incorporation into the 15-kDa protein preceded restoration of contractions by phenylephrine. Nifedipine abolished the contractile responses to alpha-adrenergic stimulation while having no effect on increases in 15-kDa protein phosphorylation. The effects of alpha-adrenergic stimulation occurred in the absence of increases in cAMP levels. These results suggest that phosphorylation of the 15-kDa protein may be involved in increases in the slow inward current produced by stimulation of either alpha- or beta-adrenergic receptors.     

Control of mitochondrial respiration in muscle

Summary Control of mitochondrial respiration depends on ADP availability to the F₁ATPase. An electrochemical gradient of ADP and ATP across the mitochondrial inner membrane is maintained by the adenine nucleotide translocase which provides ADP to the matrix for ATP synthesis and ATP for energy-dependent processes in the cytosol. Mitochondrial respiration is responsive to the cytosolic phosphorylation potential, ATP/ADP · P_i which is in apparent equilibrium with the first two sites in the electron transport chain. Conventional measures of free adenine nucleotides is a confounding issue in determining cytosolic and mitochondrial phosphorylation potentials. The advent of phosphorus-31 nuclear magnetic resonance (P-31 NMR) allows the determination of intracellular free concentrations of ATP, creatine-P and Pi in perfused muscle in situ. In the glucose-perfused heart, there is an absence of correlation between the cytosolic phosphorylation potential as determined by P-31 NMR and cardiac oxygen consumption over a range of work loads. These data suggest that contractile work leads to increased generation of mitochondrial NADH so that ATP production keeps pace with myosin ATPase activity. The mechanism of increased ATP synthesis is referred to as stimulusre-sponse-metabolism coupling. In muscle, increased contractility is a result of interventions which increase cytosolic free Ca²⁺ concentrations. The Ca^2- signal thus generated increases glycogen breakdown and myosin ATPase in the cytosol. This signal is concomitantly transmitted to the mitochondria which respond to small increases in matrix Ca²⁺ by activation of Ca²⁺-sensitive dehydrogenases. The Ca²⁺-activated dehydrogenase activities are key rate-controlling enzymes in tricarboxylic acid cycle flux, and their activation by Ca^2- leads to increased pyridine nucleotide reduction and oxidative phosphorylation. These observations which have been consistent in preparations both in vitro and in situ do not obviate a role for ADP control of muscle respiration, but do explain, in part, the lack of dramatic fluctuations in the cytosolic phosphorylation potential over a large range of contractile activities.     

Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart

The bioenergetic basis by which the Krebs cycle substrate pyruvate increased cardiac contractile function over that observed with the Embden-Meyerhof substrate glucose was investigated in the isovolumic guinea pig heart. Alterations in the content of the high energy phosphate metabolites and the rate of high energy phosphate turnover were measured by 31P NMR. These were correlated to the changes in contractile function and rates of myocardial oxygen consumption. Maximum left ventricular developed pressure (LVDP) and high energy phosphates were observed with 16 mM glucose or 10 mM pyruvate. In hearts perfused with 16 mM glucose, the intracellular phosphocreatine (PCr) concentration was 15.2 +/- 0.6 mM with a PCr/Pi ratio of 10.3 +/- 0.9. The O2 consumption was 5.35 mumol/g wet weight/min, and these hearts exhibited a LVDP of 97 +/- 3.7 mm Hg at a constant paced rate of 200 beats/min. In contrast, when hearts were switched to 10 mM pyruvate, the PCr concentration was 18.3 +/- 0.4 mM, the PCr/Pi ratio was 30.4 +/- 2.2, the O2 consumption was 6.67 mumol/g wet weight/min, and the LDVP increased to 125 +/- 3.3 mm Hg. From NMR saturation transfer experiments, the steady-state flux of ATP synthesis from PCr was 4.9 mumol/s/g of cell water during glucose perfusion and 6.67 mumol/s/g of cell water during pyruvate perfusion. The flux of ATP synthesis from ADP was measured to be 0.99 mumol/s/g of cell water with glucose and calculated to be 1.33 mumol/s/g of cell water with pyruvate. These results suggest that pyruvate quite favorably alters myocardial metabolism in concert with the increased contractile performance. Thus, as a mechanism to augment myocardial performance, pyruvate appears to be unique.     

Oxidative phosphorylation system during steady-state hypoxia in the dog brain

The relationship between biochemical and physiological responses and tissue O2 during hypoxia was investigated in vivo in the dog brain by 31P nuclear magnetic resonance (NMR) spectroscopy. Our findings demonstrate how ATP synthesis in the brain can be maintained during hypoxia because of compensatory changes in NADH, ADP, and Pi. Eleven beagle dogs were anesthetized and mechanically ventilated, and a steady-state graded hypoxia was induced by decreasing the fraction of inspired O2 (FIO2) stepwise at 20-min intervals. Biochemical metabolites were measured using 31P-NMR and fluorescence spectroscopy. When sagittal sinus O2 partial pressure (PVO2) had decreased to 15 Torr, NADH increased by 30%, Pi increased by 50%, and phosphocreatine (PCr) decreased by 20%. In contrast, ATP remained constant. There was a 10% increase in ADP in dogs that maintained a steady temperature, but ADP decreased by as much as 30% in dogs in which body temperature decreased with the falling PVO2. PCr/Pi was logarithmically related to the phosphorylation potential during steady-state hypoxia. Compensation for the O2 lack is attributed to increases in ADP, Pi, and NADH as a result of the reciprocal relationship of the Michaelis-Menten equation. If the Michaelis-Menten constants (Km) of ADP, Pi, and O2 are the same as determined in vitro in mitochondria, the minimum brain cytosolic O2 capable of maintaining a steady-state ATP is near its Km (0.1 Torr) at a PVO2 of 7.5 Torr. At this critical O2 level, PCr/Pi is 0.9, intracellular pH is 6.75, phosphorylation potential is 38.5 mM-1, and the calculated maximum velocity of ATP formation by oxidative phosphorylation is 55% of normal.     

On the sidedness of membrane phosphorylation by Pi and ATP synthesis during reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles.

The membrane sidedness of Pi interaction in reactions which characterize reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was investigated. Vesicles previously loaded with calcium [32P]phosphate were incubated with 0.1 mM ADP and different concentrations of nonradioactive Pi. Alternatively, vesicles loaded with nonradioactive calcium phosphate were incubated in a medium containing 32Pi. The rates of Ca2+ efflux and ATP synthesis were siginficantly activated only when Pi was included in the assay medium. Although the Pi contained by the vesicles crosses the membrane at a rate proportional to the Ca2+ efflux, [gamma-32P]ATP was synthesized only when 32Pi interacted with the outer surface of the membrane. Similarly, ATP in equilibrium 32Pi or ITP in equilibrium 32Pi exchange could be measured only when the external pool of Pi was labeled. Both for ATP synthesis and for the ITP in equilibrium Pi exchange reaction, membrane phosphorylation by 32Pi was negligible unless the external pool of Pi was labeled. The ionophore X-537 A increased the rate of Ca2+ efflux but inhibited the synthesis of ATP. During reversal of the Ca2+ pump, Pi apparently interacts with the membrane only at the outer surface, and at a site different from that where Ca2+ crosses the membrane.