Sublethal effects of pentachlorophenol in the abalone (Haliotis rufescens) as measured by in vivo 31P NMR spectroscopy

The sublethal biochemical effects of pentachlorophenol (PCP) were investigated in live, intact red abalones (Haliotis rufescens), using a flow-through exposure system, by in vivo 31P NMR spectroscopy. Based on rangefinding tests (6-hr LC50 = 1.6 mg/L; 6-hr no-observable-effect-level (NOEL) = 0.8 mg/L), three abalones were separately exposed to a sublethal concentration (1.2 mg/L) for 5 hr, followed by a 13 hr recovery period. Effects in foot muscle included both a decrease in phosphoarginine and an increase in inorganic monophosphate concentrations ([PA] and [Pi], respectively); both foot muscle concentrations of adenosine triphosphate [ATP] and intracellular pH (pHi) also declined. Parallel in vitro experiments revealed that concentrations of glycerol 3-phosphate, lactate, citrate, succinate, malate, and alanine (Ala) all increased, while those of glyceraldehyde 3-phosphate and glutamine (Gln) remained stable. Also, these effects were not evident until 2 hr into exposure, possibly the time required for PCP to attain an effective concentration in foot muscle. During recovery, while Pi declined to pre-exposure levels, [PA] completely recovered in only one individual. Also, realkalinization of pHi was similar to recovery of [Pi], and ATP returned to near-initial levels, as did glycerol 3-phosphate, lactate, succinate, malate, and Ala; glyceraldehyde 3-phosphate, citrate, and Gln levels declined. Recovery responses corresponded to the time for PCP clearance from foot muscle. The effects of PCP were similar to those of hypoxia, fatigue, hypersalinity, and arginine kinase inhibitors, and so sublethal PCP concentrations may also inhibit electron transport and arginine kinase as well as uncouple mitochondrial oxidative phosphorylation in intact molluscs. Thus, the effects of pollutants on key biochemical processes may now be measured in intact aquatic organisms as they occur, improving our ability to accurately assess the environmental effects of pollutants in the laboratory.     

Advantages of perfluorochemical perfusion in the isolated working rabbit heart preparation using 31P-NMR

Quantitative 31P-NMR and enzymatic analysis of high-energy phosphates were used to characterize an isolated perfused working rabbit heart preparation. In this model, the left side of the heart works against a physiological after-load. Two perfusates, Krebs-Henseleit saline and the perfluorocarbon emulsion FC-43 (perfluorotributylamine), were evaluated in their ability to maintain cardiac function and high-energy phosphate metabolites over a period of 2-3 h. Adenine nucleotides ATP, ADP, phosphocreatine and inorganic phosphate (Pi) were measured by 31P-NMR while monitoring cardiac output and coronary flow. Intracellular pH was determined using the chemical shift of Pi. At the end of each experiment, hearts were freeze clamped and enzymatically assayed for adenine nucleotides, phosphocreatine and Pi. In every experiment, hearts perfused with FC-43 emulsion maintained the same rate of cardiac output as hearts perfused with Krebs-Henseleit saline, but with half the coronary flow rate: FC-43, 22 +/- 2.5 (n = 5), Krebs-Henseleit saline 42 +/- 2.7 (n = 6) ml/min, P less than 0.001. Hearts perfused with FC-43 emulsion showed higher [phosphocreatine] and [ATP] measured by 31P-NMR. For [phosphocreatine]: FC-43 3.2 +/- 0.7 (n = 5), Krebs-Henseleit saline 1.7 +/- 0.2 (n = 6) mumol/g wet wt., P less than 0.01. For [ATP]: FC-43 1.8 +/- 0.7 (n = 5), Krebs-Henseleit saline 0.9 +/- 0.2 (n = 6) mumol/g wet wt., P less than 0.02. [phosphocreatine] and [ATP] determined by 31P-NMR values were identical within experimental error to those values obtained by enzymatic analysis. Comparing [Pi] determined by both methods, 36% of Pi in FC-43-perfused hearts, and only 24% of Pi in Krebs-Henseleit saline-perfused hearts were visible by NMR, indicating that a large proportion of Pi is bound in the intact functioning heart. Similar results were obtained for [ADP]. Using the combined techniques of 31P-NMR and enzymatic assay, we have shown in this model of the isolated working rabbit heart preparation, that FC-43 emulsion maintains significantly better function and high-energy phosphate levels than Krebs-Henseleit saline.     

In vivo 31P-NMR studies on energy metabolism in and catecholamine effect on rat liver during hypovolemic shock

In vivo 31P-NMR spectroscopy (31P-MRS) was used to study the metabolism of phosphate compounds in rat liver under various conditions. The changes in hepatic concentrations of ATP and inorganic phosphate (Pi) or intracellular pH (pHi) were monitored during hypovolemic shock with or without the infusion of catecholamines. Rapid decreases in the ATP level and pHi with a concomitant increase of Pi were observed upon induction of the hypovolemic shock. Dopamine infusion markedly improved the liver ATP concentration and intracellular acidosis, but epinephrine or norepinephrine were without effects. The present results suggest that dopamine increases abdominal blood flow and improves the energy metabolism in the liver during hypovolemic shock.     

The effects of organic and inorganic pollutants on intracellular phosphate compounds in blue mussels (Mytilus edulis).

1. The effects of sublethal concentrations of organic and inorganic pollutants on intracellular energy-rich phosphates in blue mussels, Mytilus edulis, were investigated by in vivo 31P-NMR. 2. Formaldehyde (30 and 10 mg/l), phenol, pyridine, mercury and cadmium gave marked reductions in phosphoarginine and, in some cases, the ATP amounts. The reduction in high-energy phosphate was accompanied by an increase in inorganic phosphate in all groups. 3. A "phosphorus index", the product of the ratios between phosphoarginine and inorganic phosphate, and ATP and inorganic phosphate, is suggested, which might serve as an early warning ("alarm") parameter in environmental monitoring. 4. Diversity in the responses to different pollutants make phosphorus compounds in M. edulis also an interesting element in a finger print parameter system designed to distinguish between pollutants in the marine environment.     

Role of intramitochondrial pH in the energetics and regulation of mitochondrial oxidative phosphorylation.

The dependence of ATP synthesis coupled to electron transfer from 3-hydroxy-butyrate (3-OH-B) to cytochrome c on the intramitochondrial pH (pHi) was investigated. Suspensions of isolated rat liver mitochondria were incubated at constant extramitochondrial pH (pHe) with ATP, ADP, Pi, 3-OH-B, and acetoacetate (acac) (the last two were varied to maintain [3-OH-B]/[acac] constant), with or without sodium propionate to change the intramitochondrial pH. Measurements were made of the steady-state water volume of the mitochondrial matrix, transmembrane pH difference, level of cytochrome c reduction, concentration of metabolites and rate of oxygen consumption. For each experiment, conditions were used for which transmembrane pH was near maximal and minimal values and the measured extramitochondrial [ATP], [ADP], and [Pi] were used to calculate log[ATP]/[ADP][Pi]. When [3-OH-B]/[acac] and [cyt c2+]/[cyt c3+] were constant, and pHi was decreased from approx. 7.7 to 7.2, log [ATP]/[ADP][Pi] at high pHi was significantly (P less than 0.02) greater than at low pHi. The mean slope (delta log [ATP]/[ADP][Pi] divided by the change in pHi) was 1.08 +/- 0.15 (mean +/- S.E.). This agrees with the slope of 1.0 predicted if the energy available for ATP synthesis is dependent upon the pH at which 3-hydroxybutyrate dehydrogenase operates, that is, on the pH of the matrix space. The steady-state respiratory rate and reduction of cytochrome c were measured at different pHi and pHe values. Plots of respiratory rate vs.% cytochrome c reduction at different intra- and extramitochondrial pH values indicated that the respiratory rate is dependent upon pHi and not on pHe. This implies that the matrix space is the source of protons involved in the reduction of oxygen to water in coupled mitochondria.     

Studies on the mechanism of decreased NMR-measured free magnesium in stored erythrocytes

31P-NMR spectra have been recorded on erythrocytes stored at 4 degrees C in various preservation media. Storage was always associated with an upfield shift of the inorganic phosphate (Pi) resonance and a pronounced upfield shift of the ATP beta resonance, indicating decreased intracellular pH (pHi) and decreased intracellular free magnesium ([Mg2+]i). The decreased [Mg2+]i occurred in preservation media not containing citrate and even in media supplemented with Mg2+. It could not be attributed to the changes in pHi, Na+, K+, lactate, Pi or 2,3-diphosphoglycerate, that occur with storage. The decrease in [Mg2+]i was largely reversed when stored cells were incubated for 1 h at 37 degrees C in fresh plasma. Stored cells were found to contain significant amounts of inorganic pyrophosphate, up to about 200 mumol per liter cell water. Being a tight binder of Mg2+, pyrophosphate could account for some of the observed decrease in [Mg2+]i. Additional mechanisms may involve precipitation of some other Mg2+ complex during cold storage or enhancement of Mg2+ binding to membrane components.     

31P-NMR study of resting in vitro rat diaphragm exposed to hypercapnia

We have reported previously that, when exposed to hypercapnia of various intensities, the diaphragm reduces its force of twitch and tetanic contractions in the in vitro rat preparation as well as in the in vivo dog preparation. The experiments reported here with 31P nuclear magnetic resonance (31P-NMR) spectroscopy attempt to examine cellular mechanisms that might be responsible for this deterioration in mechanical performance. Specifically they describe certain characteristics of this preparation and cautions needed to study the resting in vitro rat diaphragm with such techniques. Second, they report the response of intracellular pH (pHi), phosphocreatine (PCr), ATP, and inorganic phosphate (Pi) in the resting in vitro rat diaphragm exposed to long-term normocapnia or to long-term hypercapnia. The results show that 1) to maintain a viable preparation, it was necessary to keep the diaphragm extended to an area approximating that at functional residual capacity, 2) the diaphragm seemed quite capable of maintaining a constant pHi and constant contents of ATP and Pi during normocapnia, but there was a gradual decline in PCr, and 3) during hypercapnia there was a significant decrease in pHi, but the behavior of the phosphate metabolites was exactly as during normocapnia. The results suggest that the decrease in mechanical performance of the diaphragm is probably not due to a decrease in the availability of the high-energy phosphates, although they do not completely exclude this possibility or possibilities related to regional compartmentation.     

Cocaine induces intracellular free Mg deficits, ischemia and stroke as observed by in-vivo 31P-NMR of the brain.

31P-NMR spectroscopic studies were performed in vivo on brains of rats administered cocaine. Cocaine.HCl (1-5 mg/kg) administered systemically to lightly anesthetized rats resulted in significant and progressive deficits in whole brain intracellular free Mg ([Mg2+]i). Intracellular pH (pHi) also fell in a progressive manner but only after a significant fall in brain [Mg2+]i was noted. Both [Mg2+]i and pHi returned to normal in most rats. Brains of rats that exhibited stroke-like events, however, demonstrated continued intracellular acidosis associated with progressive loss of phosphocreatine and elevation of Pi up until death. These observations are consistent with the tenet that injection of cocaine can result in severe cerebral vasospasm, ischemia and rupture of cerebral blood vessels as a consequence of depletion of brain [Mg2+]i.     

Phosphatic metabolites, intracellular pH and free [Mg2+] in single, intact porcine carotid artery segments studied by 31P-NMR

Superfused porcine carotid artery segments (approximately 7 cm lengths) were analyzed by 31P-NMR spectroscopic methods to characterize the 31P spectrum of arterial smooth muscle and to determine the influence of passive stretch (intraluminal pressurization, 95-100 mmHg) on cellular phosphatic metabolite levels, intracellular pH and free magnesium concentration ([Mg2+free]i). Equilibrated, single, intact arteries were studied under steady-state, constant flow conditions at 37 degrees C. Phosphoethanolamine, phosphocholine, inorganic phosphate (Pi), phosphocreatine (PCr) and nucleoside triphosphates (NTP), primarily ATP, were the principle metabolites detected in the 31P-NMR spectrum of intact arterial smooth muscle. The concentration of these metabolites and intracellular pH, as determined from the referenced chemical shift of Pi, were unaffected by pressurization. The PCr:Pi ratios determined for nonpressurized (flaccid) and pressurized arteries were 1.2 +/- 0.1 and 1.3 +/- 0.3, respectively. Intracellular pH averaged 7.02 +/- 0.02 (mean +/- 1 S.D.) for flaccid arteries vs. 7.03 +/- 0.05 for pressurized arteries. The upfield chemical shift of the beta-ATP peak, which has been described in other types of smooth muscle, was also observed in these experiments. Interestingly, pressurization significantly shifted the resonance position of this peak, which was interpreted to represent a change in [Mg2+free]i. The average [Mg2+free]i of flaccid artery preparations was computed to be 0.54 +/- 0.03 x 10(-3) M, as compared to 0.99 +/- 0.10 x 10(-3) M for pressurized arteries. This change in [Mg2+free]i was evident within the first hour following pressurization and persisted thereafter. These findings suggest that altering the resting length of vascular smooth muscle produces a change in [Mg2+free]i. This shift in free Mg2+ levels may act as a metabolic signal triggering a change in vascular smooth muscle metabolism, an effect which has been reported to occur in smooth muscle in response to stretch.     

The effects of organic and inorganic pollutants on intracellular phosphate compounds in blue mussels (Mythus edulis)

• 1.1. The effects of sublethal concentrations of organic and inorganic pollutants on intracellular energy-rich phosphates in blue mussels, Mytilus edulis, were investigated by in vivo ³¹P-NMR.
• 2.2. Formaldehyde (30 and 10mg/l), phenol, pyridine, mercury and cadmium gave marked reductions in phosphoarginine and, in some cases, the ATP amounts. The reduction in high-energy phosphate was accompanied by an increase in inorganic phosphate in all groups.
• 3.3. A “phosphorus index”, the product of the ratios between phosphoarginine and inorganic phosphate, and ATP and inorganic phosphate, is suggested, which might serve as an early warning (“alarm”) parameter in environmental monitoring.
• 4.4. Diversity in the responses to different pollutants make phosphorus compounds in M. edulis also an interesting element in a finger print parameter system designed to distinguish between pollutants in the marine environment.

     

In vivo 31P NMR in crustacean muscles: fatigue and recovery in the tail musculature from the prawn Palaemon elegans

31P NMR has been used to observe the in vivo phosphometabolite concentrations in the tail musculature from the prawn Palaemon elegans, at rest and after escape swimming and subsequent recovery. Muscular fatigue corresponds to a 60% breakdown of phosphoarginine, and a 45% increase of sugar phosphates. The pHi fell from 7.10 to 6.86. During recovery, the sugar phosphates and arginine phosphate are replenished after 20 minutes. The ATP concentration did not change throughout the experiment. The pHi was restored within 20 minutes.     

Muscle 31P-NMR in humans: estimate of bias and qualitative assessment of ATPase activity.

A mathematical model is developed whereby the longitudinal magnetization of phosphocreatine (PC), ATP, Pi, and total phosphate (PT) can be calculated on the basis of assumed chemical rate constants (kappa i) and spin lattice relaxation times of the muscle PC in equilibrium ATP in equilibrium Pi exchange system. By means of this model, some unexplained 31P nuclear magnetic resonance (NMR) spectroscopy results from the literature (e.g., a decrease of PT in a closed system) could be explained simply on the basis of the physiological variability of kappa i. Moreover, appropriate model simulations indicate that 1) 31P-NMR spectra obtained with short relaxation delays may be influenced to various extents by the metabolic and physicochemical status of the muscle; 2) the assessment of kappa i by standard NMR spectroscopy techniques may be extremely critical; 3) delta PC/delta Pi, as obtained from conventional 31P-NMR spectra, may represent a sensible index of kappa 2 (the pseudo first-order chemical exchange rate constant of the adenosinetriphosphatase reaction); 4) delta PC/delta Pi changes as detected from sequential (short relaxation delays) 31P-NMR spectra obtained in humans during metabolic transients (e.g., during transition from rest to work and vice versa) may represent an index of transient changes of kappa 2.     

31P-NMR studies of the metabolisms of the parasitic helminths Ascaris suum and Fasciola hepatica

31P-NMR has been applied to the study of the metabolisms of the intact parasitic helminths Ascaris suum (the intestinal roundworm) and Fasciola hepatica (the liver fluke). After calibration of the chemical shift of Pi in muscle extracts the internal pH of adult Ascaris worms and the effect of the pH of the external medium on the organism's internal pH were measured. Assignments of nearly all of the observable 31P resonances could be made. A large resonance from glycerophosphorylcholine whose function is unclear was observed but no signals from energy storage compounds such as creatine phosphate were detected. The profiles of the phosphorus-containing metabolites in both organisms were monitored as a function of time. Changes in sugar phosphate distributions but not ATP/ADP were observed. Studies of the drug closantel on Fasciola hepatica were performed. Initial effects of the drug were a decrease in glucose 6-phosphate and an increase in Pi with no substantial change in ATP levels as observed by 31P-NMR. Studies involving treatment with closantel followed by rapid freezing, extraction, and analytical determination of glycolytic intermediates confirmed NMR observations. This NMR method can serve as a simple noninvasive procedure to study parasite metabolism and drug effects on metabolism.     

Phosphorus-31 nuclear magnetic resonance of C6 glioma cells and rat astrocytes. Evidence for a modification of the longitudinal relaxation time of ATP and Pi during glucose starvation

31P-NMR spectroscopy has been used to study the energy metabolism and the NMR visibility of ATP and intracellular Pi of the C6 glioma cell line and rat astrocyte grown on microcarrier beads with the following results. 1. In vivo NMR spectra of C6 glioma cells and rat astrocytes indicate that these cells were able to maintain their level of ATP resonances during a long anoxic period (more than an hour). Both cell types were sensitive to ischemia which induced a loss of ATP resonances within 40 min. Glucose starvation induced by 40% decrease in ATP resonances correlated to a 50% increase in the intensity of the Pi signal. These changes corresponded to a new steady state which could be reversed by reperfusing the cells with a glucose-containing medium. 2. In contrast to in vivo data, 31P-NMR analyses of perchloric acid extracts of cells incubated in a glucose-free medium showed that their ATP and Pi contents were unchanged during starvation. The changes of NMR visibility of the metabolites in living C6 cells were correlated to modifications of their macroscopic longitudinal relaxation times, evolving from 0.30 +/- 0.08 s and 6.6 +/- 1.5 s in the presence of glucose to 0.68 +/- 0.26 s and 3.2 +/- 0.9 s in the absence of glucose for ATP and Pi, respectively. The changes of the NMR detectability of ATP and Pi indicate that changes in their microenvironment occur during glucose starvation, suggesting the existence of different pools of these metabolites within the cells. 3. Under various experimental conditions, i.e. anoxia, ischemia and glucose starvation, rat astrocytes in primary culture showed a very similar behavior to that of C6 cells, suggesting a similar adaptability to the nature of the energy supply for both the normal and the malignant cell.     

A 31P-NMR study of bovine adrenocortical mitochondrial metabolic activities

High-field 31P-NMR spectroscopy has been used to study the metabolic activities of coupled bovine adrenocortical mitochondria in vitro. These differentiated organelles use oxygen as a substrate to support both oxidative phosphorylation and specific steroid hydroxylation reactions. The NMR technique allowed the resolution of two inorganic phosphate signals, attributed to the matrix and external medium phosphate pools, at low and high field, respectively. These signals were used to calculate the respective Pi concentrations and to obtain the pH of the two corresponding compartments. In addition, the NMR spectra displayed resonance signals corresponding to ADP added to the medium and to ATP synthesized during oxidative phosphorylation. NMR analysis of the mitochondrial perchloric acid extracts identified the major phosphate-containing metabolites, namely NADP+, NAD+, phosphocholine, phosphoethanolamine, sn-glycero-(3)phosphocholine, AMP, ADP, ATP and Pi. Upon addition of ADP and malate to the oxygenated suspension, the kinetics of mitochondrial external Pi consumption and of ATP synthesis, along with the intra- and extraorganelle pH variations could be monitored over time periods of approximately 30 min, in the absence and presence of different steroid hydroxylation substrates. A major observation was that oxidative phosphorylation, which takes place in the absence of steroid, was markedly inhibited as soon as steroid hydroxylation was operating. These observations show the potential of 31P-NMR spectroscopy in the study of metabolic activities of isolated intact mitochondrial organelles. Such an approach appears promising for further determination of the underlying mechanisms in the balance between vital oxidative phosphorylation and differentiated steroid hydroxylation which are under hormonal control in adrenocortical mitochondria as well as in other steroidogenic cell systems.     

Acetylcholine-induced metabolic changes in the perfused rabbit mandibular salivary gland studied by 31P-NMR spectroscopy

Changes in the content of high-energy phosphates, intracellular pH (pHi) and the ratio of MgATP to total ATP ([MgATP]/[ATP]t) resulting from continuous stimulation with acetylcholine (10(-9) to 10(-4) M) were measured by 31P-NMR spectroscopy in the isolated, perfused rabbit mandibular gland at 37 degrees C. With 10(-9) to 10(-7) M acetylcholine, no significant changes in these parameters were observed. On stimulation with 10(-6) M acetylcholine, the optimal concentration for sustained secretion, the content of ATP decreased by 28 +/- 10% (mean +/- S.E.; n = 8) of its control value. pHi decreased initially by approx. 0.05 pH unit, then showed an alkalinization of 0.09 +/- 0.02 pH unit (n = 8). With 10(-5) and 10(-4) M acetylcholine, changes in ATP and pHi were similar to those induced by 10(-6) M acetylcholine: the total content of high-energy phosphates remained at approx. 70% of the control value and no decrease in [MgATP]/[ATP]t was observed. As possible causes of the reduced secretory rate observed with higher concentrations of acetylcholine (10(-5) to 10(-3) M), we can exclude depletion of high-energy phosphates, inhibition of metabolism caused by intracellular acidosis, and inhibition of ATP usage caused by a decrease in MgATP availability.     

GTP hydrolysis by tubulin occurs with water oxygen incorporation into inorganic phosphate

Tubulin assembly was conducted in [18O]H2O and the resulting mixture of GTP, GDP and Pi was examined by 31P-NMR. Two Pi signals, separated by about 0.02 ppm, were observed. By combining this mixture with a solution of Pi containing all five possible 16O and 18O isotopomers of Pi, it was shown that the two signals were due to [16O4]- and [16O3, 18O]Pi. The amount of 18O incorporated into the Pi was that expected if the hydrolysis of GTP during tubulin assembly occurs with cleavage of the gamma-phosphorus-bridge oxygen bond.     

Rates of various reactions catalyzed by ATP synthase as related to the mechanism of ATP synthesis.

The forward and reverse rates of the overall reaction catalyzed by the ATP synthase in intact rat heart mitochondria, as measured with 32P, were compared with the rates of two partial steps, as measured with 18O. Such rates have been measured previously, but their relationship to one another has not been determined, nor have the partial reactions been measured in intact mitochondria. The partial steps measured were the rate of medium Pi formation from bound ATP (in state 4 this also equals the rate of medium Pi into bound ATP) and the rate of formation of bound ATP from bound Pi within the catalytic site. The rates of both partial reactions can be measured by 31P NMR analysis of the 18O distribution in Pi and ATP released from the enzyme during incubation of intact mitochondria with highly labeled [18O]Pi. Data were obtained in state 3 and 4 conditions with variation in substrate concentrations, temperature, and mitochondrial membrane electrical potential gradient (delta psi m). Although neither binding nor release of ATP is necessary for phosphate/H2O exchange, in state 4 the rate of incorporation of at least one water oxygen atom into phosphate is approximately twice the rate of the overall reaction rate under a variety of conditions. This can be explained if the release of Pi or ATP at one catalytic site does not occur, unless ATP or Pi is bound at another catalytic site. Such coupling provides strong support for the previously proposed alternating site mechanism. In state 3 slow reversal of ATP synthesis occurs within the mitochondrial matrix and can be detected as incorporation of water oxygen atoms into medium Pi even though medium [32P]ATP does not give rise to 32Pi in state 3. These data can be explained by lack of translocation of ATP from the medium to the mitochondrial matrix. The rate of bound ATP formation from bound Pi at catalytic sites was over twice the rate of the overall reaction in both states 4 and 3. The rate of reaction at the catalytic site is considerably less sensitive to the decrease in membrane potential and the concentration of medium ADP than is the rate of medium ATP formation. This supports the view that the active catalytic site is occluded and proceeds at a rapid rate which is relatively independent of delta psi m and of media substrates.     

Stimulation of phosphate uptake in human platelets by thrombin and collagen. Changes in specific 32P labeling of metabolic ATP and polyphosphoinositides

The uptake of [32P]phosphate by human, gel-filtered blood platelets and its incorporation into cytoplasmic ATP and polyphosphoinositides was studied. In unstimulated platelets, uptake was Na+o-dependent and saturable at approximately 20 nmol/min/10(11) cells with a half-maximal rate at 0.5 mM extracellular phosphate. Upon stimulation with thrombin or collagen, net influx of [32P]Pi was accelerated 5- to 10-fold. With thrombin, [32P]Pi efflux was also increased. After the first 2 min, efflux exceeded influx, resulting in the net release of [32P]Pi from the platelets. Since the stimulus-induced burst in [32P]Pi uptake paralleled the secretory responses, it might be an integral part of stimulus-response coupling in platelets. The stimulus-induced burst in net [32P]Pi uptake led to an enhanced labeling of metabolic ATP, which was already detectable at 5 s after stimulation with thrombin. Concomitantly, the incorporation of [32P]Pi into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was accelerated. The thrombin-induced increase in specific 32P radioactivity of cytoplasmic ATP fully accounted for the simultaneous increase in specific 32P radioactivity of these phosphoinositides. In studying the extent of 32P labeling of phosphorylated compounds in response to a cellular stimulus, it is therefore essential to measure the effect of the stimulus on the specific radioactivity of cytoplasmic ATP.     

Calcineurin-induced energy wasting in a transgenic mouse model of heart failure

Overexpression of calcineurin (CLN) in the mouse heart induces severe hypertrophy that progresses to heart failure, providing an opportunity to define the relationship between energetics and contractile performance in the severely failing mouse heart. Contractile performance was studied in isolated hearts at different pacing frequencies and during dobutamine challenge. Energetics were assessed by 31P-NMR spectroscopy as ATP and phosphocreatine concentrations ([ATP] and [PCr]) and free energy of ATP hydrolysis (|Delta G( approximately ATP)|). Mitochondrial and glycolytic enzyme activities, myocardial O2 consumption, and myocyte ultrastructure were determined. In transgenic (TG) hearts at all levels of work, indexes of systolic performance were reduced and [ATP] and capacity for ATP synthesis were lower than in non-TG hearts. This is the first report showing that myocardial [ATP] is lower in a TG mouse model of heart failure. [PCr] was also lower, despite an unexpected increase in the total creatine pool. Because Pi concentration remained low, despite lower [ATP] and [PCr], |Delta G( approximately ATP)| was normal; however, chemical energy did not translate to systolic performance. This was most apparent with beta-adrenergic stimulation of TG hearts, during which, for similar changes in |Delta G( approximately ATP)|, systolic pressure decreased, rather than increased. Structural abnormalities observed for sarcomeres and mitochondria likely contribute to decreased contractile performance. On the basis of the increases in enzyme activities of proteins important for ATP supply observed after treatment with the CLN inhibitor cyclosporin A, we also conclude that CLN directed inhibition of ATP-producing pathways in non-TG and TG hearts.