Reduced ATP Levels Research Articles

ATP-dependent pHi recovery in lung macrophages: evidence for a plasma membrane H(+)-ATPase

We have previously shown that cytoplasmic pH (pHi) recovery in pulmonary macrophages, under nominally HCO3(-)-free conditions, after acute intracellular acidification is Na+ and amiloride insensitive and is blocked by nonspecific proton adenosinetriphosphatase (ATPase) inhibitors N-ethyl-maleimide and N,N'-dicyclohexylcarbodiimide [Am. J. Physiol. 257 (Cell. Physiol. 26): C65-C76, 1989]. To further delineate the mechanism of H+ extrusion across plasma membranes of pulmonary macrophages, we investigated the effects of metabolic inhibitors of oxidative phosphorylation and glycolysis on cellular ATP content and pHi recovery from an intracellular acid load under nominally HCO3(-)-free conditions. Dose-dependent reductions in ATP levels and in the rate of pHi recovery were obtained in the presence of KCN (50% inhibition, 10(-4) M). Parallel reductions in ATP content and the rate of pHi recovery were noted in the presence of antimycin A, rotenone, oligomycin, and iodoacetate. However, inhibition by iodoacetate was reduced in the presence of pyruvate. The more specific vacuolar H(+)-ATPase inhibitors, bafilomycin A1 and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, resulted in no decrement in cellular ATP levels but significantly inhibited pHi recovery. These studies demonstrate that recovery from an acid load is ATP dependent and provide support for a plasmalemmal proton ATPase, perhaps of the vacuolar type, that participates in regulation of pHi in pulmonary macrophages.

Effect of ethanol on hepatic energy metabolism and intracellular pH in chronically ethanol‐treated rats. A 31P NMR study of normoxic or hypoxic perfused liver

31P Nuclear magnetic resonance spectroscopy has been used to investigate the combined effect of 70 mM ethanol administration and hypoxia on the energetic metabolism and cytosolic pH of the perfused liver isolated from naive, fasted or chronically ethanol-treated rats. Ethanol-treatment or fasting induce a similar 25% reduction in ATP levels, and a differential enhancement of Pi levels to 130% and 225% of control values, respectively. In the naive rats, acute ethanol administration triggers profound variations in high-energy phosphorylated metabolite levels with, in particular, a 300% increase in phosphomonoesters, mostly due to sn-glycerol-3-phosphate (G3P) accumulation. A 4 min hypoxic period in the presence of ethanol induced a further accumulation of G3P. In the livers from chronically ethanol-treated rats, the G3P level remains low during acute ethanol administration, despite the presence of glycogen, indicating an attenuation of the ethanol-induced redox shift. The combination of 70 mM ethanol and hypoxia leads to an increased G3P level in the livers of chronically ethanol-treated rats which is consistent with a sustained alcohol dehydrogenase activity. The subsequent cytosolic acidosis is significantly less pronounced in the chronically ethanol-treated rats than in naive rats. These observations illustrate the adaptation of metabolic processes occurring in the livers of chronically ethanol-treated rats, which results in an increased resistance to acute ethanol exposure and hypoxic stress.

Pyruvate dehydrogenase activity in the rat cerebral cortex following cerebral ischemia.

The effect of cerebral ischemia on the activity of pyruvate dehydrogenase (PDH) enzyme complex (PDHC) was investigated in homogenates of frozen rat cerebral cortex following 15 min of bilateral common carotid occlusion ischemia and following 15 min, 60 min, and 6 h of recirculation after 15 min of ischemia. In frozen cortical tissue from the same animals, the levels of labile phosphate compounds, glucose, glycogen, lactate, and pyruvate was determined. In cortex from control animals, the rate of [1(-14)C]pyruvate decarboxylation was 9.6 +/- 0.5 nmol CO2/(min-mg protein) or 40% of the total PDHC activity. This fraction increased to 89% at the end of 15 min of ischemia. At 15 min of recirculation following 15 min of ischemia, the PDHC activity decreased to 50% of control levels and was depressed for up to 6 h post ischemia. This decrease in activity was not due to a decrease in total PDHC activity. Apart from a reduction in ATP levels, the acute changes in the levels of energy metabolites were essentially normalized at 6 h of recovery. Dichloroacetate (DCA), an inhibitor of PDH kinase, given to rats at 250 mg/kg i.p. four times over 2 h, significantly decreased blood glucose levels from 7.4 +/- 0.6 to 5.1 +/- 0.3 mmol/L and fully activated PDHC. In animals in which the plasma glucose level was maintained at control levels of 8.3 +/- 0.5 mumol/g by intravenous infusion of glucose, the active portion of PDHC increased to 95 +/- 4%. In contrast, the depressed PDHC activity at 15 min following ischemia was not affected by the DCA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Anomalous influence of reduced internal ATP levels on sodium efflux inMyxicola giant axons

Giant axons from the marine annelid, Myxicola infundibulum, were internally dialyzed with ATP-free media and with media with lower than normal ATP levels in an attempt to determine quantitatively the ATP requirement of the Na pump in these cells. This was accomplished by using 22Na ions to measure Na efflux. When [ATP]i in dialysis fluid fell to values within the range of 20-40 microM, a marked stimulation of Na efflux was observed even though an essentially normal ouabain sensitivity of Na efflux persisted; when axons were dialyzed with ATP-free solutions with ouabain present in the external medium throughout the dialysis period, the stimulation of Na efflux still occurred. The stimulation of Na efflux produced by low [ATP]i levels could be reversed by reintroducing normal ATP levels into the dialysis medium. Reversibility was complete provided axons were not depleted of ATP for periods longer than about 1 hr. Longer periods of ATP depletion led to larger and ultimately irreversible increases in Na efflux. The increases in Na efflux occasioned by ATP depletion either prevented or obscured the decrease in Na efflux expected to occur from unfueling the Na pump. Since [ATP]i levels required to significantly unfuel the Na pump lie below the levels at which the Na efflux stimulation occurred, it is problematic to quantitatively assess the influence of [ATP]i levels on Na pump rate by measurements of Na efflux in this preparation. Substitutes for ATP failed to prevent increases in Na efflux. The large increases in Na efflux observed at low [ATP]i occurred with no important changes in the resting membrane potential, and also occurred in Na-free and Ca-free external media. At least part of the increased Na efflux under these conditions may be due to a Na/Na exchange component, as a significant dependence of Na efflux on [Na]o appropriate for this kind of exchange was observed in the ATP-depleted axons. Whether the highly reproducible anomalous effect on Na efflux in Myxicola axons has some fundamental significance in its own right is a matter for future investigation. A few possible explanations of the anomalous effect of reduced ATP levels are discussed.

Phosphatidylinositol Metabolism During In Vitro Hypoxia

The effects of in vitro histotoxic hypoxia (0.5 mM KCN) on potassium-stimulated phosphatidylinositol turnover were determined. In rat cortical slices that were prelabeled with [2-3H]inositol, depolarization with 60 mM KCl increased [2-3H]inositol monophosphate and [2-3H]inositol bisphosphate accumulation in a Ca2+-dependent manner. At early times (10 s and 1 min), histotoxic hypoxia enhanced potassium-stimulated [2-3H]inositol monophosphate and inositol bisphosphate accumulation. Under basal conditions, hypoxia did not alter the accumulation of [2-3H]inositol phosphates. These results are consistent with the following hypothesis. The hypoxic-induced increase in cytosolic free calcium that we reported previously may lead to the early stimulation of inositol phosphates formation during hypoxia through activation of phospholipase C. The impairment of inositol phosphates formation during more prolonged hypoxia may be due to negative feedback regulation of the phosphatidylinositol cascade by protein kinase C or to a reduction in ATP levels.

Metabolic Interconnection between the Human Malarial Parasite Plasmodium falciparum and Its Host Erythrocyte: Regulation of ATP levels by means of an adenylate translocator and adenylate kinase

The metabolic inter-relationships between malarial parasites and their host erythrocytes are poorly understood. They have been investigated hitherto mostly by observing parasite behavior in erythrocyte variants, in metabolically altered erythrocytes, or in cell-free in vitro systems. We have studied the interconnection between the bioenergetic metabolism of host and parasite through compartment analysis of ATP in Plasmodium falciparum-infected human red blood cells, using Sendai virus-induced host cell lysis. ATP concentrations in host and parasite compartments were found to be equal. Inhibitors of mitochondrial activity reduce ATP levels to a similar extent in host and parasite compartments, although only the parasite contains functional mitochondria. It is shown that equalization of ATP levels is brought about by means of an adenylate translocator, probably localized at the parasite plasma membrane, in conjunction with adenylate kinase activity detected both in host and parasite compartments. The translocator is inhibited by compounds which are known to inhibit specifically the translocator of the inner membrane of mammalian mitochondria, with identical inhibitory constants. Addition of these inhibitors to intact infected cells causes a rapid depletion of ATP in the host compartment and a parallel increase in the parasite, suggesting that the parasite supplies ATP to its host cell rather than the reverse.

Cordycepin rapidly collapses the intermediate filament networks into juxtanuclear caps in fibroblasts and epidermal cells

The nucleoside analog 3′-deoxyadenosine (cordycepin) rapidly collapses the intermediate filaments into juxtanuclear caps in interphase fibroblasts and keratinocytes. A minimum of 80 μg/ml cordycepin or 20 μg/ml cordycepin in combination with 2 μ/ml of the deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenosine (EHNA) to inhibit its degradation is required to see these effects. This is the same concentration required for cordycepin to arrest cells at the onset of mitosis and depolymerize the microtubules to small asters. Cordycepin enters the cells rapidly and is phosphorylated to 3′-dATP with a concomitant drop in ATP levels. However, the direct reduction of ATP levels does not mimic the same rapid effects of cordycepin on either the intermediate filaments or microtubules. In addition, similar effects are not produced by a variety of other adenosine analogs with alterations in the 2′- and 3′-ribose positions. Although other pharmacological reagents result in alterations of the fibroblastic intermediate filaments, cordycepin is unusual because of the rapiditiy with which the fibroblastic intermediate filaments collapse into the juxtanuclear caps. The juxtanuclear caps have a morphology different from that of the perinuclear bundles of intermediate filaments that arise after long-term depolymerization of the microtubules. The keratin fibers in the epidermal cells retract to a perinuclear ring when treated with cordycepin

Altered calcium homeostasis and membrane integrity in myocardial cell injury.

Coronary occlusion leads to contractile arrest of ischemic myocardium and progressive metabolic derangements and ultrastructural alterations, which if of sufficient duration and severity, lead to irreversible myocardial injury. Based on a number of experimental observations, we and others have put forward the following general hypothesis regarding the pathogenesis of irreversible myocardial injury1,2. Myocardial ischemia causes oxygen deprivation which leads to depressed energy metabolism resulting in a reduced ATP level, increased lactate, and decreased pH. The metabolic changes are followed by discrete alterations in energy dependent membrane transport systems which lead to altered ionic composition of the cell, including an increase in cytosolic calcium. These membrane alterations may include altered ionic flux across the sarcolemma as well as release of calcium from mitochondria and sarcoplasmic reticulum. The elevated cytosolic calcium concentration has the potential to activate catabolic enzymes, including phospholipases. This may lead to acceleration phosphlipid degradation, increased membrane permeability, and further electrolyte derangements, including calcium accumulation. The excess cellular calcium load may also lead to ATP depletion by activation of calcium-dependent ATPases as well as by mitochondrial calcium accumulation which occurs at the expense of continued energy production. Thereafter, a vicious cycle of membrane injury and ATP depletion may ensue which terminates in irreversible cell injury. Observations pertinent to this hypothesis are reviewed in this chapter.KeywordsCoronary OcclusionTotal PhospholipidCalcium OverloadMembrane InjuryIrreversible InjuryThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Differential effects of tri- n-butylin chloride on macromolecular synthesis and ATP levels of rat thymocyte subpopulations obtained by centrifugal elutriation

Using centrifugal elutration, rat thymocytes were separated into three fractions by size. Fraction 1 (F1) consisted of a large population (>88% of all cells) of small, nonproliferating thymocytes. Fraction 2 (F2; 7% of all cells) was enriched in medium-sized thymocytes and showed an increased macromolecular synthesis. In the small fraction 3 (F3; <5% of all cells), large thymocytes, monocytes, granulocytes and cells in mitosis were concentrated. F3 demonstrated the highest proliferative activity. Fractions were characterized by size, cell morphology, DNA, RNA and protein synthesis and steady state ATP levels. The effects of the inhibitor of oxidative phosphorylaiton, tri- n-butyltin chloride (TBTC) on ATP levels and the incorporation of DNA, RNA and protein precursors were investigated for each thymocyte fraction. Although ATP levels increased with increasing thymocyte volume, TBTC reduced ATP levels in each subfraction proportionally. The incorporation of thymidine and leucine was markedly reduced in all fractions by TBTC, but not to the same extend. Dependent on the TBTC concentration, the rapidly proliferating cells of F2 and F3 were less affected compared to the noncycling cells of F1. The incorporation of uridine in the unfractionated cells and in F2 and F3 was also decreased by TBTC. However, at concentrations between 0.1 and 1 μM, TBTB stimulated uridine incorporation in the small thymocytes of F1. Therefore, fractionation of thymocyte suspensions was necessary to defect differential effects of TBTC on subpopulations. Centrifugal elutriation was found to be a useful toll with which to obtain subfractions of isolated rat thymocytes.

Methotrexate efflux in L1210 cells. Kinetic and specificity properties of the efflux system sensitive to bromosulfophthalein and its possible identity with a system which mediates the efflux of 3',5'-cyclic AMP.

Methotrexate exits L1210 mouse leukemia cells via multiple routes that include a unidirectional efflux component which is sensitive to bromosulfophthalein. This efflux component has been characterized in the present study after eliminating the contribution from the other efflux routes by treatment of the cells with an active ester of methotrexate and by reducing the assay pH to 6.2. The remaining efflux at pH 6.2 was greater than 90% sensitive to bromosulfophthalein. This route was also inhibited by probenecid, prostaglandin A1, diamide, 1-methyl-3-isobutylxanthine, various metabolic inhibitors, and by transfer of the cells to a buffer containing high concentrations of KCl. The inhibition by prostaglandin A1 was exceptionally potent and reached 50% at a concentration of 0.5 microM. An enhancement in efflux occurred upon the addition of glucose or by transfer of the cells to a non-saline buffer. When parameters relating to cellular energetics were measured, a reduction in ATP level was associated with the inhibition of efflux by probenecid, carbonylcyanide m-chlorophenylhydrazone, valinomycin, and antimycin A, whereas the increase in efflux by glucose was accompanied by an increase in intracellular ATP. Changes in ATP, however, were not associated with the inhibition by various other compounds or additions or with the enhancement in efflux by the non-anionic buffer. When the relative sensitivity of methotrexate efflux to bromosulfophthalein, 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and lactic anhydride was compared with other anion transport systems, differences in specificity indicated that methotrexate was not exiting the cells via the bicarbonate/chloride exchange carrier, the lactate/H+ co-transport system, or a system which mediates the efflux of phthalate. However, a correlation was apparent between the sensitivity of methotrexate efflux to inhibition by prostaglandin A1, probenecid, and certain metabolic inhibitors and the ability of these same compounds to inhibit the unidirectional efflux of 3',5'-cyclic AMP in other cell lines, suggesting that methotrexate may share a common efflux route with cyclic nucleotides.

Cordycepin disrupts the microtubule networks and arrests Nil 8 hamster fibroblasts at the onset of mitosis.

The nucleoside analogue 3'-deoxyadenosine (cordycepin) arrests dividing cells at the onset of mitosis in prometaphase. The microtubules in the arrested prometaphase cells depolymerize to two small asters. A minimum of 80 micrograms/ml cordycepin or 20 micrograms/ml cordycepin in combination with 2 micrograms/ml of the deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenosine (EHNA) to inhibit its degradation is required to see these effects. Analysis of cell extracts by high-pressure liquid chromatography indicates that cordycepin enters the cells rapidly and is phosphorylated to 3'-dATP. The intracellular concentration rises almost linearly from 0.7 mM after 15 min to 7 mM by 210 min. Concomitantly the ATP concentration shows a rapid drop from the 4 mM present in controls. However, the direct reduction of ATP levels does not mimic the same rapid effects of cordycepin on the microtubules. In addition, similar effects are not produced by a variety of other adenosine analogues with alterations in the 2' and 3' ribose positions. Although other pharmacological reagents arrest cells at the onset of mitosis, cordycepin is unusual because of the collapse of the microtubule networks to two small asters that radiate from the microtubule-organizing center. 3'-dATP can replace the requirement for ATP or GTP in the vitro polymerization of microtubules from microtubule protein: however, at limiting concentrations of nucleotide it requires approximately two times the concentration of 3'-dATP as ATP to support an equivalent level of microtubule polymerization. This suggests that the effects of cordycepin in vivo may be the result of the depletion of cellular ATP pools and the altered ability of 3'dATP to substitute for ATP-dependent reactions. Current experiments are testing this hypothesis.

Effects of various inhibitors of oxidative phosphorylation on energy metabolism, macromolecular synthesis and cyclic AMP production in isolated rat thymocytes. A regulating role for the cellular energy state in macromolecular synthesis and cyclic AMP production

Inhibitors of oxidative phosphorylation such as several triorganotin compounds, oligomycin, 2,4-dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone suppress energy metabolism of isolated rat thymocytes as indicated by a reduction of ATP levels, an increase in glucose consumption and by a marked accumulation of lactate. Also these compounds effectively inhibit the incorporation of DNA, RNA and protein precursors into acid-precipitable material of thymocytes. Moreover, the prostaglandin E 1-induced elevation of cAMP is markedly reduced by these inhibitors. A correlation is observed between the effects on energy metabolism, macromolecular synthesis and cAMP production, since (i) from a series of trialkyltin chlorides, tri- n-propyltin, tri- n-butyltin and tri- n-hexyltin are very effective inhibitors of these functions, while trimethyltin and tri- n-octyltin affect neither of them; (ii) other inhibitors of oxidative phosphorylation, each of them with quite different mechanisms of action, also inhibit macromolecular synthesis and cAMP production. The finding that a rise in intracellular ATP concentrations leads to a reversion of the tri- n-butyltin-induced inhibition of cAMP production and uridine incorporation, indicates a regulating role for the cellular energy state in these aspects of cellular function.

ATP Assay: Ability To Distinguish Cytostatic From Cytocidal Anticancer Drug Effects&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

A bioluminescence assay for ATP was adapted to human cancer cell lines and used to study the effect of anticancer drugs on malignant cell growth by following serial ATP measurements. Eleven drugs were tested against a colon cancer cell line (WiDR). Excellent correlation was observed between simultaneously performed soft-agar colony-forming assays and the ATP assay. In addition, cytostatic (growth inhibitory) drug effects could be distinguished from cytocidal (lethal) effects by using the ATP assay. Cytocidal drugs resulted in a reduction of ATP level below baseline levels, whereas cytostatic drugs merely yielded a reduction in the rate of increase in ATP level, i.e., slower growth. Such characterizations are not possible in colony-forming assays. Changes in ATP were correlated with the number of viable cells present. Drug concentration and duration of exposure both were important. Some drugs became cytocidal only when exposures longer than the customary 1 hour were used. The ATP assay has excellent potential as a simple, inexpensive, and rapid technique for new drug screening in cell lines, with classification of drug effects as cytostatic or cytocidal.

Inhibition of ion transport in Ehrlich cells by muzolimine

The influence of muzolimine on the transport of Na+, K+ and Cl- was studied in Ehrlich cells to test whether the diuretic inhibits the furosemide-sensitive Na+-K+-2 Cl-(-)cotransport or transport via the ouabain-sensitive Na+/K+-pump. It was shown that between 10(-5) M and 10(-3) M muzolimine pump-flux decreases with increasing drug concentration (IC50 about 0.5 mM), in contrast to the unaffected cotransport. This reduction in pump rate is only seen with respiring cells, but not during glycolytic ATP-production. Therefore, muzolimine seems to inhibit the Na+/K+-pump not directly but indirectly by interference with energy metabolism resulting in decreased ATP concentration. This reduction in ATP-level is at least partially due to activation of an ATP-consuming process of unknown nature. Whether muzolimine also inhibits respiration was not tested.

Endogenous energy supply to the plasma membrane of dark aerobic cyanobacterium Anacystis nidulans: ATPase-independent efflux of H + and Na + from respiring cells

The ejection of protons from oxygen-pulsed cells and the gradients of Na + concentration ( Na o + Na i + at 150 mM external NaCl) and proton electrochemical potential (Δ μ H + ~) across the plasma membrane of Anacystis nidulans were studied in response to dark endogenous energy supply. Saturating concentrations of the F 0F 1-ATPase inhibitors dicyclohexylcarbodiimide (F 0) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (F 1) eliminated oxidative phosphorylation and lowered the ATP level from 2.6 ± 0.15 to 0.7 ± 0.1 nmol/ mg dry wt while overall O 2 uptake and Δ μ H + ~ were much less affected. H + efflux was inhibited only 60 to 75%. Aerobic Na o + Na i + ratios (5.9 ± 0.6) under these conditions remained 50% above the anaerobic level (2.1 ± 0.2). Increasing concentrations of the electron transport inhibitors CO and KCN depressed H + efflux and O 2 uptake in parallel, with a pronounced discontinuity of the former at inhibitor concentrations, which reduced ATP levels from 2.6 to 0.8 nmol/mg dry wt, resulting in an abrupt shift of the apparent H + O ratios from 4.0 ± 0.3 to 1.9 ± 0.2. Similarly, with KCN and CO the Na o + Na i + ratios paralleled decreasing respiration rates more closely than decreasing ATP pool sizes. Ejection of protons also was observed when intact spheroplasts were pulsed with horse heart ferrocytochrome c or ferricyanide; the former reaction was inhibited, the latter was increased, by 1 m m KCN. Measurements of the proton motive force (Δ μ H + ~) across the plasma membrane showed a strong correlation with respiration rates rather than ATP levels. It is concluded that the plasma membrane of intact A. nidulans can be directly energized by proton-translocating respiratory electron transport in the membrane and that part of this energy may be used by a Na + H + antiporter for the active exclusion of Na + from the cell interior.

The effect of reducing ATP levels on reorientation of the secondary palate

The force for directing palate shelf reorientation appears to be associated with elements of the presumptive hard palate (Brinkley & Vickerman, 1979; Bulleit & Zimmerman, 1985). The palatal elements that mediate this process do not require palate cells to be metabolically active for expression of the force. This contention was demonstrated using an in vitro system that allows substantial reorientation of the hard palate to occur. ATP levels were reduced by treatment with metabolic inhibitors and the degree of reorientation was measured 1 h following pretreatment with inhibitors. Treatment of cultured embryonic heads under anoxic conditions with 2,4-dinitrophenol or KCN had no effect on the degree of reorientation occurring in vitro. These agents reduced ATP levels by 71% and 62%, respectively. Treatment of cultured heads with 2-deoxy-D-glucose under anoxia also had no effect on reorientation. This treatment reduced ATP levels in embryonic heads by 92-94%. A similar reduction was observed if ATP levels were measured in palate tissue alone. The treatment of cultured heads with 2-deoxy-D-glucose and anoxia not only reduced levels of ATP but also reduced CTP, GTP and UTP. These results indicate that palate shelf reorientation is independent of cellular metabolic activity and supports the hypothesis that reorientation is dependent on a pre-existing infrastructure within the palate shelves.

Time course of pulmonary vasoconstriction with repeated hypoxia and glucose depletion

To examine the effect of hypoxia on pulmonary vascular smooth muscle, rabbit lobar pulmonary artery was suspended in a glucose free solution and both chronologic changes in tension and ATP content were determined together at 30 min intervals after repeated hypoxic challenge (P O 2 = 11 ± 2 mm Hg). The pulmonary artery contracted and its ATP content decreased with hypoxia. This contraction was not inhibited by nifedipine, Ca + +-free EGTA, procaine, phentolamine, isopreterenol, diphenhydramine, prostaglandin E 1, atropine or nitroglycerine. Upon reoxygenation (P O 2 = 104 ± 3 mm Hg), the elevated resting tension decreased in a biphasic fashion and the ATP content of the lobar pulmonary artery increased. When hypoxic challenges were repeated, the rate of constriction on hypoxia increased, while the relaxation rate on reoxygenation, tension developed by 30 min of hypoxia and the total amount of ATP decreased. These results suggest that the ATP content in the lobar pulmonary artery is very sensitive to in vitro acute hypoxia and that the Ca ++ transport process is more easily impaired by reduction in ATP levels than is the contractile machinery.

An Investigation into the Role of Photosynthesis in Regulating ATP Levels and Rates of H+ Efflux in Isolated Meosphyll Cells

Aerated and stirred 10-ml suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used for simultaneous measurements of net H(+) efflux and steady-state ATP levels.Initial rates of medium acidification indicated values for H(+) efflux in the light and dark of 0.66 and 0.77 nanomoles H(+)/10(6) cells per minute, respectively. When the medium pH was maintained at 6.5, with a pH-stat apparatus, rates of H(+) efflux remained constant. Darkness or DCMU, however, stimulated H(+) efflux by 100% or more. Darkness increased ATP levels by 33% and a switch from dark to light reduced ATP levels by 31%. In the absence of aeration, illumination prevented the accumulation of respiratory CO(2) and the buffering capacity of the medium was about 50% less than that found in the nonilluminated nonaerated medium. As a result, rates of pH decline were similar even though the dark rate of H(+) efflux was approximately 50% greater.Proposals that photosynthesis stimulates H(+) efflux are based on changes in the rate of pH decline. The present data indicate that photosynthesis inhibits H(+) efflux and that changes in rates of pH decline should not be equated with changes in the rate of H(+) efflux.

Two separable steps in the development of platelet prothrombin-converting activity

Platelets catalyze conversion of prothrombin to thrombin in the presence of coagulation Factors Va and Xa. Bovine Factor Va was found to bind to human and bovine platelets washed by centrifugal pelleting. Factor Va was bound to sites varying in affinities but only those sites saturated at approximately 1 mM are active. Platelets in citrate or benzamidine-anticoagulated plasma or those washed by gel filtration bound substantially less Factor Va with these affinities, indicating that the binding sites increased in number or affinity or both during the centrifugal pelleting and redispersal. Thrombin production catalyzed by platelets with developed binding sites started at a low initial rate but after a lag period rapidly reached a maximum rate. Addition of 0.25 mM dinitrophenol increased the lag period and decreased the maximum rate obtained concomitantly with the reduction in ATP levels. Neither dinitrophenol nor its induced depletion of the ATP levels reduced Factor Va binding. Therefore, the activation of platelet pro thrombin-converting activity was separated into two steps; one being the development of the high-affinity binding sites and the other a step partially dependent on sufficient levels of ATP within the platelet.

Response of pulmonary energy metabolism to phosgene.

Rats were exposed to phosgene at a concentration of 1.0 ppm for 4 hours in a Rochester-type chamber. At intervals thereafter over a 4 day period, lungs were obtained for histological and biochemical assessments. Edema was estimated by histological examination and by measurement of lung wet and dry weights. In parallel studies, pulmonary mitochondrial respiratory activity was measured using Clark oxygen electrodes. The significant reduction in respiratory control index (State 3 respiration/State 4 respiration) found immediately following phosgene exposure coincided with the highest level of % lung water. There was a concomitant decrease of ATP concentration that persisted on the third day after exposure. Na-K-ATPase activity was reduced 1 day after exposure, thus a lowered ATP level preceded a reduction in Na-K-ATPase or sodium pump activity. The reduction in ATP level and Na-K-ATPase activity may play a major role in damage to lung tissue following exposure to phosgene.