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Valinomycin and glass electrodes were used to measure activity of potassium ions in equilibrium with adenosine triphosphate. The thermodynamic formation constant of the complex is approximately 25 times greater than indirect measurements predict.
The method for detecting the number of living cells was studied. Using an adenosine triphosphate (ATP) bioluminescence assay, the present authors reported a perfect linear relationship between lg ATP concentrations and lg luminescence counts (r = 0.9963) as well as a relationship between lg number of cells and lg ATP luminescence counts (r = 0.9922). The detectable cells ranged from 10(2) to 10(6) cells/ml, the coefficients of variation 1-3%. This method is simple, accurate and sensitive and has a high reproducibility.
The effects of temperature, adenosine triphosphate and magnesium on the calcium sensitivity of actomyosin were investigated on actomyosin obtained from six halothane-sensitive and five halothane-resistant German Landrace pigs. No difference in the contractile properties was found in actomyosin from the two types of pig. However, in both types calcium sensitivity of the actomyosin was lost at temperatures slightly greater than those occurring physiologically. Both ATP and Mg2+ protect against the loss of calcium sensitivity. These results suggest that the basic lesion in halothane-induced malignant hyperthermia does not lie in the contractile proteins. It is likely, however, that the decreases in intracellular ATP and Mg2+ concentrations which occur in pigs during malignant hyperthermia, contribute to the development of the syndrome.
Severe myocardial ischemia in dogs (perfusion 10% of normal) caused progressive deterioration in adenosine triphosphate (ATP) metabolism. Between 1/2 hour and 5 hours, myocardial ATP content fell from 55 to 6% of normal, and the sum of adenine nucleotides fell from 66 to 14% of normal. Moderate ischemia (perfusion 20 to 70%) also disturbed ATP metabolism, but to a lesser degree. Moreover, there was no significant change in the concentration of any ATP metabolite between 1/2 hour and 5 hours of moderate ischemia. ATP content was 66 and 52% of normal, and adenine nucleotide content was 73 and 59% of normal at 1/2 hour and 5 hours, respectively. Trivial ischemia (perfusion 80% or greater) barely perturbed ATP metabolism at either 1/2 hour or 5 hours. Thus, in contrast to severe or trivial ischemia, prolonged moderate ischemia produced a derangement in ATP metabolism that persisted and was relatively stable for 5 hours.
BACKGROUND/AIMS: The biliary epithelium contributes to bile formation through absorption and secretion of fluid and electrolytes. The effects of extracellular nucleotides on membrane ion transport were assessed in isolated bile duct cells from rats and Mz-ChA-1 cells from a human cholangiocarcinoma. METHODS: The rates of efflux of 125I and 86Rb were used to assess membrane Cl- and K+ permeabilities, respectively. Patch clamp recordings of whole cell currents were used to evaluate the properties of adenosine triphosphate (ATP)-activated currents. RESULTS: Purinergic receptor agonists ATP and uridine triphosphate stimulated 125I and 86Rb efflux about twofold above basal levels. The effects were reproduced by a nonhydrolyzable analogue of ATP (adenosine 5'-O-[3-thiophosphate]) and were unaffected by an adenosine receptor blocker xanthine amine congener. 125I efflux was also stimulated by adenosine and its receptor agonists 5'-N-ethylcarboxamidoadenosine, N6-(2-phenylisopropyl)adenosine; these effects were inhibited by xanthine amine congener, suggesting a separate adenosine receptor. ATP, adenosine 5'-O-(3-thiophosphate), and uridine triphosphate each stimulated release of Ca2+ from intracellular stores, whereas adenosine had no effect. In whole cell recordings of Mz-ChA-1 cells, ATP activated an early transient outward current consistent with a K+ conductance and a later, sustained inward current consistent with a Cl- conductance. CONCLUSIONS: Biliary cells possess at least two classes of nucleotide receptors that modulate membrane ion permeability through Ca(2+)-dependent and -independent pathways, and ATP may be involved in the regulation of biliary secretion.
To investigate the importance of renal tissue concentrations of adenosine triphosphate (ATP) in maintaining normal renal functions as well in the recovery from ischemic injury in vivo, fructose was administered without ischemia as well as in the setting of an ischemic insult. In nonischemic as well as kidneys recovering from 30 min of clamp ischemia, fructose caused a 50% reduction in the tissue concentration of ATP which persisted for over 1 h. In nonischemic animals, fructose had no effect on renal function as assessed by inulin clearance and sodium reabsorption and showed no evidence of histologic injury 24 h after administration. In animals subjected to 30 min of ischemia, animals given fructose showed no worsening of functional or histologic injury as compared to controls. We conclude from these data that reductions in the tissue concentration of ATP in the range observed does not adversely affect normal renal functions or recovery from a reversible ischemic insult.
Ischemia and reperfusion stimulate several adenosine triphosphate (ATP)-dependent processes involving release of substances including free radicals. This cellular response is mediated through receptors responsive to transcriptional products of gene expression; c-fos acts as a transcriptional factor involved in the regulation of genes associated with cellular proliferation and differentiation. We hypothesized that nitrone free-radical spin traps promote restoration of cytosolic ATP during reperfusion and prevent c-fos induction. Four control rats had no ischemia. Global hepatic ischemia was induced in 19 rats in four groups: saline solution, phenyl-N-tert-butyl nitrone (PBN), alpha 1-pyridyl-N-oxide N-tert-butyl nitrone (POBN), and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). ATP and intracellular pH were measured at intervals before, during, and after ischemia. At 90 minutes of reperfusion, liver c-fos mRNA was measured. A fourfold elevation of c-fos occurred in the saline-treated group (p less than 0.001). PBN and POBN groups did not differ from the saline group. DMPO resulted in significantly less induction of c-fos than did NS. ATP depletion and recovery in all treatment groups was similar to that of the saline group. We conclude that (1) nitrone spin traps do not prevent c-fos induction or alter the pattern of ATP recovery after hepatic ischemia and reperfusion and (2) c-fos induction is not necessary for restoration of ATP, but the rate of ATP restoration is inversely related to c-fos induction.
Nucleotides such as adenosine triphosphate (ATP) and uridine triphosphate (UTP) exist in the extracellular environment where they are agonists at P2 receptors. Both P2Y G-protein-coupled receptors and P2X ligand-gated ion channels are expressed by osteoblasts and osteoclasts, reflected in the diverse nucleotide-induced effects reported to occur in bone. Previous reports have implicated ATP as a proresorptive agent; however, these studies were unable to determine whether ATP mediated its actions directly on osteoclasts, or indirectly via osteoblasts. The development of techniques to generate human osteoclasts in vitro has allowed us to further investigate the intriguing role of extracellular nucleotides with regard to osteoclast activity. This study reports that nearly all P2-receptor-subtype mRNAs were expressed throughout human osteoclast development, and provides evidence for functional P2 receptor expression by these cells. In cultures of human osteoclasts alone, neither ATP nor UTP affected the quantity of resorption by these cells; however, in cocultures of osteoblast-like UMR-106 cells and human osteoclasts, ATP, but not UTP, greatly enhanced resorption, indicating a role for osteoblasts in mediating the proresorptive effects of ATP. Furthermore, ATP, but not UTP, elevated receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA and protein expression by UMR-106 cells. These data are consistent with observations that UMR-106 cells predominantly express P2Y(1) with low expression of P2Y(2), thereby explaining the response to ATP and not UTP, and further substantiating the involvement of osteoblasts in ATP-induced effects on osteoclasts. These results significantly advance our understanding of the role of P2 receptors in bone, and indicate that local-acting ATP may play a pivotal role in osteoclast activation at bone-resorbing sites by inducing elevated expression of RANKL.
BACKGROUND: Liposomally entrapped adenosine triphosphate (ATP) has been demonstrated to improve energy state and function of the cold-stored liver. The increased nitrite release associated with liposome administration led us to investigate the interactions between liposome supply and nitric oxide (NO) production through the use of L-NAME, a non-selective inhibitor of NO synthesis. METHODS: Twenty-four livers from fasted rats were stored for 18 h at +4 degrees C in University of Wisconsin solution directly (control group) or after infusion with ATP-containing liposomes (Lip-ATP), L-NAME (L-NAME) or both (Lip-ATP-L-NAME). Metabolic fluxes, cell volume and energy state were studied during reperfusion. RESULTS: After storage, nitrite release was increased by 61% in the Lip-ATP group, markedly decreased in the Lip-ATP-L-NAME group and almost abolished in the L-NAME group. The ATP content was increased by 20% in the Lip-ATP group (P < 0.05 versus control) and on reperfusion this was associated with an increase in cell volume (17%; P < 0.05) and a decrease in branched-chain amino acid release (21%; P < 0.01). The simultaneous addition of L-NAME did not affect these results, but induced a large (6-fold) increase in glucose production, possibly related to the metabolism of glycerol supplied by the liposomes. In the L-NAME group, global amino acid release was 50% lower and was associated with a dramatic decrease in urea production while the energy state deteriorated rapidly. CONCLUSIONS: The improvement in energy state and anabolic cell swelling induced by ATP-containing liposomes seems to be independent of NO synthesis. On the other hand, inhibition of NO synthesis appears to exert a detrimental effect on the liver, presumably through the decrease in hepatic energy content.
Adenosine and adenosine 5'-triphosphate (ATP) are found in every cell of the human body. These molecules are released from cells into the extracellular fluid under physiologic and pathophysiologic conditions. Outside of cells, adenosine and ATP act as physiologic regulators of cells, tissues, and organs. In the heart, extracellular adenosine and ATP exert pronounced inotropic, lusitropic, electrophysiologic, and metabolic effects, which are mediated by specific cell surface receptors. In addition, both compounds can modulate sympathetic and parasympathetic input to the heart by interacting with neural elements within and without the heart, thereby modulating autonomic neural control of cardiac functions. This article briefly reviews these indirect, neurally-mediated actions of adenosine and ATP.
Isolated dog hearts (n = 19) were preserved in cold Collins solution at 4 degrees C for 1 to 26 hours. At the end of preservation the coronary system was flushed out by cold cardioplegic solution, and the hearts were reperfused with supporting dogs. Myocardial biopsies at the end of preservation and coronary sinus effluents at the time of flush-out were analyzed for adenosine 5'-triphosphate and inosine. Both the degree of depletion in adenosine triphosphate content and the accumulation of inosine in the myocardium during preservation correlated significantly to the preservation time (correlation coefficient = -0.85 and 0.86, respectively). There was a significant correlation between the myocardial inosine level and total amount of inosine released into coronary sinus effluent (correlation coefficient = 0.91, p less than 0.001). Also, a significant correlation between the total amount of inosine in coronary sinus effluent and cellular level of adenosine triphosphate was found (correlation coefficient = -0.86, p less than 0.001). Five hearts that failed to resume beating showed significantly higher amounts of released inosine than the other hearts (3.3 +/- 0.9 versus 1.5 +/- 0.5 mumol, p less than 0.005). These results indicate that the inosine level in the coronary sinus effluent reflects the myocardial energy state, and it is suggested that the measurement of inosine level in the effluent may be applicable as a noninvasive method for assessment of myocardial viability in heart preservation.
Stimulation of human lymphocytes from 20 individuals with phytohaemagglutinin (PHA) for 60 min resulted in a fall in intracellular adenosine triphosphate (ATP) of 35% compared to unstimulated controls of 19% (p less than 0.001). There was a significant (p less than 0.01) correlation (r = 0.57) between ATP decrease at 60 min and deoxyribonucleic acid (DNA) synthesis at 72 h in PHA-stimulated cells. The technique described here is simple, rapid and has possible application in immunodiagnosis and immunologic monitoring.
Lymphocyte bioenergetics in cancer patients undergoing surgery is largely unexplored. The effect of surgery on total lymphocyte counts and adenosine triphosphate (ATP) production is reported. Lymphocyte ATP was assayed by the coupled enzymatic method using hexokinase and glucose 6-phosphate dehydrogenase and lymphocyte counts were calculated from total leukocyte counts and differential counts. Lymphocyte ATP assay and counts were carried out in 13 patients pre- and postoperatively, and lymphocyte counts were scored in six more patients similarly. In 69% of the patients lymphocyte ATP production was depressed postoperatively, and this condition appeared to be associated with cancer spread and immunosuppressive tumors. Postoperative increase in lymphocyte ATP was observed following hysterectomy, colostomy, and also after blood transfusion. The significance of the findings are discussed. Changes in postoperative lymphocyte counts correlated with lymphocyte ATP in 62% of the cases.
The antimicrobial susceptibility test for Mycobacterium tuberculosis H37Rv and 43 clinical isolates was performed using a bioluminescence assay by measuring the content of adenosine triphosphate (ATP) derived from mycobacteria. The drugs tested were isoniazid (INH), rifampicin (RFP), ethambutol (EB), streptomycin (SM), and kanamycin (KM). The ATP contents of M. tuberculosis incubated in the Middle-brook 7H9 broth medium containing antituberculous agents were measured at days of 0, 1, 3, 5, 7 and 10. A reduction of ATP content, indicating growth inhibition, was observed in susceptible strains within 5 to 10 days of incubation. Optimal concentrations to distinguish between susceptible and resistant strains were determined as being INH 0.20, RFP 0.50, EB 5.0, SM 4.0, KM 6.0 g/ml. The agreements of ATP method (evaluated at 10 days) with Vite Spectrum and MIC determinations were 81.4% and 100%, respectively. Susceptibilities to most drugs, except for EB, could be determined within 7 days. This method is simple, rapid, nonradiometric, and can be used for drug susceptibility.
The effects of hematoporphyrin derivative-induced photosensitization on the levels of adenosine triphosphate (ATP) in R3230AC mammary adenocarcinomas were studied. Enzymatically dissociated tumor cells were exposed to various doses of hematoporphyrin derivative (HPD) in vitro plus photoradiation. A drug and light dose-dependent decrease in cellular ATP levels was observed; ATP levels were reduced by 60% after treatment with 7.0 micrograms HPD per ml plus 0.72 J total energy density per cm2. Cell viability, assessed by exclusion of trypan blue, displayed an apparently coordinate behavior to ATP levels. The effects of hematoporphyrin derivative plus photoradiation were examined in the presence of oligomycin, an inhibitor of mitochondrial oxidative phosphorylation, or iodoacetate, an inhibitor of glycolysis, experiments designed to elucidate the site of action leading to reduced ATP levels. The results indicated that HPD-induced photosensitization had little additive effects to oligomycin-sensitive ATP production, whereas significant further reduction in ATP levels was obtained by HPD-induced photosensitization in the presence of iodoacetate. Taken together, along with earlier studies of selected mitochondrial enzymes, we conclude that HPD plus photoradiation inhibits mitochondrial function leading to reduction in cellular ATP levels and loss of viability.
Oxidative phosphorylation within the inner mitochondrial membrane generates the majority of cellular adenosine triphosphate (ATP) required for normal physiological functions (including regulation of cell volume and solute concentration, maintenance of cellular architecture, and synthesis of essential macromolecules). Its efficient functioning depends on the maintenance of an electrochemical gradient and is tightly coupled to the energetic demands of the cell and/or tissue. Commitment to and completion of the cell division cycle are sensitive to changes in the availability of mitochondrially derived ATP, although the relationship between cell cycle and mitochondrial physiology is poorly understood. Using vital, mitochondrial-specific fluorochromes to differentiate between mitochondrial mass (10-N-nonyl acridine orange) and mitochondrial membrane potential (Rhodamine 123), together with a quantification of total cellular ATP levels, it was possible to generate profiles of these mitochondrial characteristics in HL-60 cells at different stages of their cell cycle. The data suggest that the availability of ATP changes in a cell cycle-specific manner and cannot be predicted by changes in mitochondrial mass or membrane potential. Furthermore, transition points in the cell cycle where ATP availability is low with respect to the amount of functional inner mitochondrial membrane have been observed. We suggest that these cell cycle phase transitions are sensitive to inhibition of mitochondrial activity because the basal levels of available ATP at these points are nearer to a theoretical "minimal threshold" below which cell cycle progression is inhibited.
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The neuromuscular blocking effect of atracurium under the influence of controlled hypotension by adenosine triphosphate (ATP) or nitroglycerin (NTG) was studied in mongrel dogs under halothane anesthesia. Under hypotensive state (60 +/- 5 mmHg) elicited by ATP (0.5 mg/kg/min) or NTG (1 microgram/kg/min), the neuromuscular blockade produced by atracurium (30 micrograms/kg, i.v.) was significantly potentiated and prolonged. The maximal depression of twitch contraction of the gastrocnemius-soleus muscle increased from 10 +/- 3% to 36 +/- 10% (ATP group) and 56.0 +/- 2.4% (NTG group), while the duration of neuromuscular blockade was prolonged from 663 +/- 96 s to 1060 +/- 277 s (ATP group), and 1375 +/- 441 s (NTG group). The potentiation and prolongation of neuromuscular blockade by atracurium was still apparent upon reversal of the hypotensive effect of ATP, but not of NTG, by dopamine infusion. We suggest that ATP may prolong and augment the effect of atracurium by reducing the presynaptic release of acetylcholine at the neuromuscular junction.