Degradation of perfused adenine compounds up to uric acid in isolated rat heart

Cells are generally impermeable to nucleotides like ATP, ADP and AMP while nucleosides and bases readily cross the plasma membrane. A release of adenosine and of its catabolic derivatives by the myocardium of different animal species has been demonstrated in physiological and physiopathological conditions [2, 4, 13]. Furthermore there are many studies on the uptake of adenosine and inosine by the myocardial cells and their incorporation into tissue nucleotides [7–9, 15]. Taking into account the extracellular localization of adenosine receptors [5] and the role of this nucleoside as regulator of coronary blood flow and modulator of the positive inotropic effects of catecholamines [4] it was of interest to study a possible extracellular formation of adenosine from adenine nucleotides. Most mammalian tissues like muscle, liver and adipose tissue and cells like platelets, leukocytes and lymphocytes possess some enzyme activities associated with the cell surface membrane. Ecto-ATPase (EC 3.6.1.3/15) and ecto-5′-nucleotidase (EC 3.1.3.5) activities have been demonstrated in myocardial, smooth-muscle and endothelial cell membranes [3,4,8,12].In the present paper we report a comparative study on the ability of the isolated rat heart to breakdown exogenous adenine nucleotides, nucleosides and bases: we have determined in the coronary perfusate all the degradation products in basal conditions and after perfusion with ATP, ADP, AMP, adenosine, inosine and hypoxanthine. All the perfused compounds were degraded up to uric acid that amounted to about 10%. Nucleotides were catabolized at a higher rate than nucleosides and so adenosine may accumulate outside the cells.     

Electrophysiological and receptor binding studies to assess activation of the cardiac adenosine receptor by adenine nucleotides

Adenosine and adenine nucleotides shorten the action potential duration of atrial myocytes and activate a specific acetylcholine and adenosine receptor-operated potassium outward current referred to as IKACh,Ado. The objective of this study was to determine whether adenine nucleotides shorten the action potential duration and increase IKACh,Ado in guinea pig atrial myocytes by directly activating adenosine receptors. The potency and efficacy of AMP and adenosine in increasing IKACh,Ado and shortening atrial action potential duration were similar; the EC50 values for AMP and adenosine were 3.4 +/- 0.8 and 3.1 +/- 0.4 microM, respectively. Likewise, the maximum increases in IKACh,Ado caused by AMP and adenosine were similar (122 +/- 11% versus 123 +/- 9%). In comparison, ATP and the stable analogue of AMP, adenosine monophosphorothioate (AMPS), were significantly less potent and efficacious than adenosine and AMP, and adenosine receptor antagonist 8-(p-sulfophenyl)theophylline and abolished in the presence of adenosine deaminase and alpha, beta-methylene-ADP (APCP, an inhibitor of AMP degradation). Binding of the A1-adenosine antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX) to guinea pig atrial membranes treated with adenosine deaminase and APCP was reduced up to 60% by 100 microM concentrations of AMP, AMPS, and adenosine. Inosine inhibited binding by 43 +/- 3% at 100 microM, whereas hypoxanthine and xanthine had little (5-10% inhibition) and uric acid had no effect. Only 3% of AMP and 35% of AMPS were recovered intact after a 90-minute incubation at 21 degrees C with preparations of guinea pig atrial membranes. Percent displacement of [3H]DPCPX binding to atrial membranes by 100 microM AMP was significantly less in the presence of nucleoside phosphorylase and xanthine oxidase (to degrade inosine, hypoxanthine, and xanthine to uric acid) than in their absence (12.4 +/- 3.1% versus 49.7 +/- 1.5%). The results suggest that the observed electrophysiological actions of adenine nucleotides in cardiomyocytes are mediated by adenosine and are consistent with activation of A1-adenosine receptors.     

Functional studies in fibroblasts of adenylosuccinase-deficient children

Summary In fibroblasts of severely retarded (type I) adenylosuccinase (ASase)-deficient children, activities with the two substrates of the enzyme, succinylaminoimidazole carboxamide ribotide (succinyl-AICAR) and adenylosuccinate are decreased in parallel, to about 30% of normal. In a markedly less retarded (type II) patient, ASase activity with adenylosuccinate reaches only 3% of normal, whereas activity with succinyl-AICAR is also about 30% of normal. To assess the functional significance of a partial versus a profound deficiency of ASase, precursor incorporation studies were performed in intact fibroblasts. In cells from controls and from type I patients, incorporation of 0.2 mmol/L [¹⁴C]formate into adenine and guanine nucleotides was not accompanied by accumulation of either [¹⁴C]succinyl-AICAR or [¹⁴C]adenylosuccinate. Similarly, incorporation of 20 µmol/L [¹⁴C]hypoxanthine was not accompanied by accumulation of [¹⁴C]adenylosuccinate. In contrast, in fibroblasts of the type II patient, in accordance with the profound deficiency of ASase with adenylosuccinate, and with the inhibitory effect of Cl^– and nucleotides on the activity with succinyl-AICAR, incorporation of [¹⁴C]formate resulted in accumulation of [¹⁴C]succinyl-AICAR and [¹⁴C]adenylosuccinate, and incorporation of [¹⁴C]hypoxanthine in a marked build-up of [¹⁴C]adenylosuccinate. That both precursors were still incorporated into the adenine nucleotides of the fibroblasts of the type II patient indicates that adenylate synthesis remains possible even with 3% residual ASase activity, as also shown by their grossly normal ATP concentrations. The results suggest that the pathophysiology of ASase deficiency may be mediated at least in part by accumulation of succinyladenosine and succinyl-AICAriboside.     

Adenine and guanine nucleotide metabolism during platelet storage at 22 degrees C

Adenine and guanine nucleotide metabolism of platelet concentrates (PCs) was studied during storage for transfusion at 22 +/- 2 degrees C over a 7-day period using high-pressure liquid chromatography. There was a steady decrease in platelet adenosine triphosphate (ATP) and adenosine diphosphate (ADP), which was balanced quantitatively by an increase in plasma hypoxanthine. As expected, ammonia accumulated along with hypoxanthine but at a far greater rate. A fall in platelet guanosine triphosphate (GTP) and guanosine diphosphate (GDP) paralleled the fall in ATP + ADP. When adenine was present in the primary anticoagulant, it was carried over into the PC and metabolized. ATP, GTP, total adenine nucleotides, and total guanine nucleotides declined more slowly in the presence of adenine than in its absence. With adenine, the increase in hypoxanthine concentration was more rapid and quantitatively balanced the decrease in adenine and platelet ATP + ADP. Plasma xanthine rose during storage but at a rate that exceeded the decline in GTP + GDP. When platelet ATP + ADP was labeled with 14C-adenine at the initiation of storage, half of the radioactivity was transferred to hypoxanthine (45%) and GTP + GDP + xanthine (5%) by the time storage was completed. The isotopic data were consistent with the presence of a radioactive (metabolic) and a nonradioactive (storage) pool of ATP + ADP at the initiation of storage with each pool contributing approximately equally to the decline in ATP + ADP during storage. The results suggested a continuing synthesis of GTP + GDP from ATP + ADP, explaining the slower rate of fall of GTP + GDP relative to the rate of rise of plasma xanthine. Throughout storage, platelets were able to incorporate 14C-hypoxanthine into both adenine and guanine nucleotides but at a rate that was only one fourth the rate of hypoxanthine accumulation. All of these data should be helpful in improving the function and viability of PC as currently stored for 5 days, in devising methods for storage beyond 5 days, and in the development of synthetic media for PC storage.     

Inosine incorporation into myocardial nucleotides

Inosine and hypoxanthine, degradative products of adenine nucleotides are released and also taken up by the heart. In order to determine whether inosine can be incorporated into adenine nucleotides by direct phosphorylation or whether initial degradation to hypoxanthine is necessary, isolated guinea pig hearts were perfused with non-recirculating Krebs-Henseleit solution containing uniformly labeled inosine (0.004 μm) or 8-[¹⁴C]-hypoxanthine (0.01 μm). Enzymic hydrolysis of the adenine nucleotides from hearts perfused with inosine revealed that only the base moiety of the nucleosides was labeled, indicating that inosine was degraded to hypoxanthine prior to incorporation. Analysis of perfusates for labeled inosine and hypoxanthine indicated interconversion of these compounds by nucleoside phosphorylase which is known to be present in endothelial cells and pericytes. The reduction of the specific activity of inosine and hypoxanthine that passed through the coronary vascular bed was greater than could be accounted for by the unlabeled endogenous inosine and hypoxanthine released from the myocardium, and indicates exchange of the exogenous labeled compounds with the endogenous pool of inosine and hypoxanthine in the interstitial fluid and intracellular compartments.     

Abnormal Platelet Function in Chediak-Higashi Syndrome

Platelets in an infant with Chediak-Higashi (C-H) syndrome without bleeding manifestations and not in the accelerated phase showed abnormal function consistent with storage pool disorder as shown by abnormal aggregation, decreased storage capacity and release of [14C]5-HT, low endogenous 5-HT, reduced ATP and ADP with an increased ATP/ADP ratio, increased specific radioactivity of ADP after [14C]adenine labelling, decreased release of adenine nucleotides after stimulation, impaired secretion of acid hydrolases despite normal stores, and decreased calcium content. Incorporation of [14C]adenine into metabolic pool adenine nucleotides was normal. Nucleotide conversion to hypoxanthine in stimulated platelets was mildly impaired. Platelet cyclic-AMP (c-AMP) was initially elevated, but even when c-AMP returned to normal levels after ascorbate treatment, platelet function was not improved. Elevated intracellular c-AMP was not solely responsible for the abnormal platelet function.     

Storage of Human Platelets: Effects on Metabolically Active ATP and on the Release Reaction

During storage platelets rapidly lose their viability and their functions of aggregation, adhesion and secretion. This loss of function does not correlate with the small decrease in total ATP occurring. Platelet adenine nucleotides probably exist in two pools, one metabolically active and one inactive, so that only 40% of total ATP actually participates in the energy reactions. The metabolic pool was labelled by incubation of platelet-rich plasma (PRP) in acidified citric acid-sodium citrate-dextrose (ACD) with trace amounts of radioactive adenine. Platelets thus labelled in acidified ACD-PRP were stored in the same medium for 24 hr at 4°C, and the effect of storage on radioactive ATP and on the release reaction induced by collagen and thrombin was studied. The total amount of ATP decreased by only 10% during storage, whereas the metabolic ATP was reduced by 40–50% with a corresponding formation of hypoxanthine. The reactivity in the release reaction also dropped by 50%. When the acidified ACD-PRP was stored with 9 him inosine a 20% reduction of metabolic ATP and 25% reduction in release ability occurred. Storage with 8 mM adenosine also counteracted the loss in release ability, but 50% of the ATP radioactivity was lost. Adenosine increased the total ATP level during storage. Thus, the metabolic ATP pool might have been preserved intact under these circumstances, while exchange of radioactive ATP with nonradioactive ATP synthesized from adenosine took place. These results indicate strongly that during storage of platelets the metabolic ATP pool is lost by degradation to hypoxanthine in quantities that might well account for the loss in function.     

Extracellular ATP is a mitogen for 3T3, 3T6, and A431 cells and acts synergistically with other growth factors.

Extracellular ATP in concentrations of 5-50 microM displayed very little mitogenic activity by itself but it caused synergistic stimulation of [3H]thymidine incorporation in the presence of phorbol 12-tetradecanoate 13-acetate, epidermal growth factor, platelet-derived growth factor, insulin, adenosine, or 5'-(N-ethyl)carboxamidoadenosine. Cultures of Swiss 3T3, Swiss 3T6, A431, DDT1-MF2, and HFF cells were used. The percent of cell nuclei labeled with [3H]thymidine and cell number were also increased. ADP was equally mitogenic, while UTP and ITP were much less active. The effect of ATP was not due to hydrolysis by ectoenzymes to form adenosine, a known growth factor. Thus, the nonhydrolyzable analogue adenosine 5'-[beta, gamma-imido]triphosphate was mitogenic. In addition, it was found that ATP showed synergism in 3T6 and 3T3 cells when present for only the first hour of an incorporation assay, during which time no significant hydrolysis occurred. Furthermore, prolonged preincubation of cells with ATP reduced the mitogenic response to ATP but not to adenosine; preincubation with adenosine or N6-(R-phenylisopropyl)adenosine had the reverse effect. Finally, the effect of adenosine, but not of ATP, was inhibited by aminophylline. We conclude that extracellular ATP is a mitogen that interacts with P2 purinoceptors on the plasma membrane.     

Cloning and expression of the Rickettsia prowazekii ADP/ATP translocator in Escherichia coli.

Cosmid clone banks of Rickettsia prowazekii genomic DNA were established in Escherichia coli and screened for expression of the rickettsial carrier-mediated ADP/ATP translocator. Out of 2700 clones screened, a single clone, designated MOB286, accumulated radioactivity when incubated with [alpha-32P]ATP in 100 mM sodium phosphate buffer. This clone carried a plasmid, pMW286, containing a 9-kilobase-pair insert of rickettsial DNA, as established by DNA X DNA hybridizations. Transformation studies with purified pMW286 established that the ability of E. coli cells to accumulate radioactivity was mediated by the recombinant plasmid. Results from experiments in which [3H]ATP was substituted for [alpha-32P]ATP strongly suggested that the radiolabeled ATP was transported intact. Furthermore, [3H]ATP was incorporated into 10% (wt/vol) trichloroacetic acid-precipitable material in a time-dependent manner. Uptake of ATP was also temperature-dependent, insensitive to atractyloside, N-ethylmaleimide, and dinitrophenol, and specific for ADP and ATP. Efflux of radiolabeled nucleotide was observed in the presence of extracellular ADP or ATP but not AMP and was not observed in the absence of extracellular adenine nucleotides. The successful cloning and expression of the rickettsial ADP/ATP translocator in E. coli will permit better characterization of rickettsial bioenergetics and of the metabolic regulation of obligate intracellular parasitism.     

Pyrimidine nucleotide synthesis is preferentially supplied by exogenous cytidine in adult rat cultured cardiomyocytes.

The metabolism of pyrimidine nucleotides was studied in non-contracting myocytes isolated from adult rat hearts and compared to that observed in freshly prepared myocardium. The myocytes were cultured for up to 96 hrs in a commercial medium containing 50 microM cytidine, uridine, adenosine and adenine; 20 microM guanosine, thymidine and D-ribose; and 5 microM hypoxanthine, xanthine, guanine, thymine and uracil. Nucleotide pool sizes were measured by HPLC. Nucleotide and RNA labelling were followed by incorporation of [U-14C]-cytidine or [U-14C]-uridine added in trace amounts to the medium. The adenine nucleotide pool was 2.4-fold larger than in situ after 7 hrs of incubation and then returned to values 30% higher than that found in the myocardium after 25 hrs. Cytosine and uracil nucleotide pools after 25 hrs of culture were respectively 2 and 4-fold larger than in situ and remained at these levels thereafter. Intracellular cytidylate and uridylate equilibrated very rapidly with exogenous [U-14C]-cytidine but not with [U-14C]-uridine. We conclude that, under the experimental conditions used here, the synthesis of pyrimidine nucleotides in isolated myocytes is mainly supplied by exogenous nucleosides. Furthermore, extracellular cytidine is rapidly converted to both uracil and cytosine nucleotides while uridine serves only as the precursor for uracil nucleotide synthesis.     

Effect of varying pH [H+] on the metabolism of adenosine in isolated heart cells

We have investigated, in isolated beating cardiac muscle cells, the influence of pH variation on the uptake of radioactive adenosine and its incorporation into the adenine nucleotides. The incorporation of radioactive adenosine into the ATP, ADP and AMP fractions of the cardiac cells was significantly decreased at lower pH values (6.0 to 7.1) and was unchanged at pH values ranging from 7.4 to 8.0. The incorporation of radioactive adenosine into inosine and hypoxanthine was insignificant in comparison to the incorporation into the adenine nucleotide fraction and was unrelated to the pH changes. Theophylline, at a final concentration of 1 × 10^?4m was found to be without an effect on the pH related uptake of adenosine. Additionally, the release of adenosine due to hypoxia (95% O₂ + 5% CO₂) was not altered by the changes in the pH of the incubation medium. Our findings indicate that the increased sensitivity of adenosine at lower pH values (reported in the literature) is likely due to the inhibition in the uptake of adenosine. This would then result in increased concentrations of extracellular adenosine to which the coronary resistance vessels are exposed.We conclude that theophylline's inability to block reactive hyperemia might be related to this increased availability of adenosine due to the acidic pH which prevails during reactive hyperemia. These data show a relationship between adenosine and hydrogen ion activity and are compatible with the adenosine hypothesis for the regulation of the coronary blood flow.     

Adenine nucleotide metabolism during cardiac hypertrophy and ischemia in rats.

The effects of ischemia on the adenine nucleotides, and the production of adenosine, inosine and hypoxanthine were studied in 200 rat hearts made hypertrophic by swimming exercise, by thyroxine treatment, or by operative constriction of the abdominal dominal aorta. The results were the same irrespective of the method used for cardiac hypertrophy. In the normal heart ischemia produced a decrease in ATP and an increase in ADP, AMP, adenosine, inosine, and hypoxanthine. In the hypertrophied heart changes in ATP, AMP, adenosine, inosine and hypoxanthine were in the same direction as in the normal heart but significantly greater. ADP first increased and then fell to very low levels in the hypertrophied heart. With prolonged ischemia (5 to 20 min) adenosine, inosine, and hypoxanthine appeared in the 0.9% NaCl bathing solution of the hypertrophied hearts. When the amounts of these compounds in the medium were added to the myocardial tissue levels, the sum tended to remain constant. During the development of cardiac hypertrophy, the ATP levels, and to a lesser extent the total adenine nucleotide pool rose from the first to the third day after aortic constriction but declined from the fourth to the tenth day. These data suggest that either the “de novo” synthesis and utilization of the “salvage” pathway for the adenine nucleotides is reduced or the degradative activities dominate these synthetic pathways during cardiac hypertrophy.     

Adenine nucleotide release from isolated perfused guinea pig hearts and extracellular formation of adenosine.

The quantification of adenine nucleotides released from the heart is hampered by their rapid dephosphorylation to adenosine in the extracellular space catalyzed by highly active ectonucleotidases. To determine the total release of adenine nucleotides from isolated Langendorff-perfused guinea pig hearts, ecto 5'-nucleotidase was effectively blocked by infusion of alpha, beta-methylene-ADP (AOPCP, 50 microM). Adenine nucleotides were measured in the coronary venous effluent by the luciferin-luciferase method after enzymatic rephosphorylation to ATP. In hearts perfused at a constant flow rate (10 ml/min) with normoxic buffer (95% O2, 5% CO2) the release +/- SEM of adenine nucleotides and adenosine was 0.06 +/- 0.01 (n = 11) and 0.04 +/- 0.01 (n = 13) nmol/min. In the presence of AOPCP, the release of adenine nucleotides increased to 0.43 +/- 0.04 nmol/min (n = 9; p less than 0.05), whereas adenosine remained unchanged. Hypoxic perfusion (10% O2, 85% N2, 5% CO2) caused a threefold increase in adenine nucleotide release but a 40-fold increase in adenosine. In contrast, global ischemia (30 seconds) caused adenine nucleotide and adenosine release to rise to similar values of 1.06 +/- 0.10 and 0.80 +/- 0.14 nmol/min (n = 9). Stimulation of hearts with isoproterenol (4 nM) likewise increased the release of adenine nucleotides (0.50 +/- 0.04 nmol/min) and adenosine (0.87 +/- 0.21 nmol/min) (n = 6). To determine the cellular source of adenine nucleotides released from the heart, the coronary endothelial adenine nucleotide pool was selectively prelabeled by [3H]adenosine. Global ischemia increased the specific radioactivity of released adenine nucleotides by 57%. The findings indicate that 1) adenine nucleotides and adenosine are released at the same order of magnitude from the well-oxygenated heart; 2) beta-adrenergic stimulation and ischemia stimulate the release of adenine nucleotides and adenosine, both purines reaching vasoactive concentrations in the effluent perfusate; 3) during hypoxic perfusion only the release of adenosine is greatly enhanced; and 4) the coronary endothelium preferentially contributes to the ischemia-induced adenine nucleotide release.     

Chemical and functional correlates of postischemic renal ATP levels.

Renal energy metabolism was investigated before, during, and after ischemic insults of varying durations with in vivo 31P NMR spectroscopy. The postischemic recovery of renal ATP was found to be a biphasic process regardless of the length of the ischemia. This two-stage recovery consisted of a quick initial component immediately upon reflow followed by a slower, more gradual return toward preischemic levels. To characterize the source of each phase of the recovery, kidneys were extracted with perchloric acid at the end of the different periods of ischemia (before reflow). Concentrations of adenine nucleotides and breakdown products adenosine, inosine, and hypoxanthine were determined by 1H NMR spectroscopy. Excellent correlation was found between the residual nucleotide pool and the magnitude of the initial phase of ATP recovery. Additionally, the renal ATP content after 120 min of reflow was shown to have a strong correlation with subsequent functional recovery. These experiments show that in vivo 31P NMR can provide new and dynamic information concerning the biochemical recovery from ischemia. Furthermore, this data has the potential to predict the eventual functional recovery of the organ.     

Partial purification and characterization of the mRNA for human thymidine kinase and hypoxanthine/guanine phosphoribosyltransferase.

We used direct microinjection of poly(A)+RNA into individual hypoxanthine/guanine phosphoribosyltransferase-deficient or thymidine kinase-deficient cells and detected the specific in vivo translation products as an assay for human hypoxanthine/guanine phosphoribosyltransferase or thymidine kinase mRNAs. The incorporation of [3H]hypoxanthine or [3H]thymidine into cells in response to injected mRNA was assayed in situ by autoradiography. Methylmercuric hydroxide/agarose gel analysis showed that human hypoxanthine/guanine phosphoribosyltransferase mRNA contains approximately 1,530 nucleotides, which is twice the number required for its protein coding capacity. The mRNA for human cytoplasmic thymidine kinase is estimated to be approximately the same length; thus, the size of the cytosol thymidine kinase subunit can be predicted to be approximately 47,000 daltons, if the full coding capacity of its mRNA is utilized.     

Increased incorporation of adenosine into adenine nucleotide pools in serum-deprived mammalian cells.

The effects of serum deprivation on the incorporation of adenosine into the intracellular adenine nucleotide pools by several mamalian cell lines were studied. Cells arrested in the G1 phase of the cell cycle showed increased incorporation of exogenous adenosine into their adenine nucleotide pools as compared with growing cells. This phenomenon is unexpected because salvage pathways from all other preformed nucleosides and bases as well as the de novo synthesis of adenine nucleotides is decreased after arrest of growth by serum deprivation. The incorporation of adenosine into adenine nucleotides may serve as an intracellular signal in the regulation of growth in mammalian cells.     

The role of nonparenchymal and parenchymal liver cells in the catabolism of extracellular purines

Adenosine-degrading enzymes within the liver lobule can modulate both vascular and metabolic effects of circulating adenosine in the liver. Since it has not been fully established whether nonparenchymal cells participate in the elimination of sinusoidal purines, isolated Kupffer cells and endothelial cells were tested for their capacity to degrade extracellular purines. After perfusion and digestion of rat livers by collagenase, the resulting mixed cell population was separated by centrifugal elutriation. The isolated parenchymal and nonparenchymal cells were incubated for up to 2 hr in the presence of [8(-14)C]adenosine, [8(-14)C]guanosine and [8(-14)C]hypoxanthine (50 mumoles per liter). In the deproteinized medium, adenosine, guanosine, inosine, adenine, guanine, xanthine, hypoxanthine, uric acid and allantoin were separated by reversed-phase high-performance liquid chromatography. Radioactive peaks were collected and counted. Nonparenchymal cells catalyzed the degradation of adenosine into inosine and hypoxanthine. However, the formation of xanthine, uric acid or allantoin from adenosine could only be detected in hepatocyte suspensions. Within 15 min, adenosine was completely eliminated from the medium by Kupffer cells, whereas endothelial cells catabolized only less than half of the initial amount of the adenine nucleoside during this time period. Accordingly, incubation of nonparenchymal cells in the presence of hypoxanthine did not result in the formation of further breakdown products of the purine, whereas its catabolites slowly accumulated in the medium of hepatocytes. Guanosine conversion into guanine and xanthine was much slower in endothelial cells as compared to Kupffer cells and hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transendothelial transport and metabolism of adenosine and inosine in the intact rat aorta

This study was aimed at defining the role of vascular endothelium in the transport and metabolism of adenosine. For this purpose, endothelium-intact and endothelium-denuded isolated rat aortas, perfused at constant flow (2 ml/min), were prelabeled with 3H-adenosine or 3H-inosine for 10 minutes at concentrations of 0.012-100 microM. Sequestration of adenosine by endothelium was determined from radioactivity recovered during selective endothelial cell removal with deoxycholic acid (0.75% for 15 seconds). In the physiological concentration range of adenosine (0.012-1 microM), fractional sequestration by endothelium was 90-92% of the total adenosine incorporation by the aorta. Endothelial sequestration of inosine at 0.1 microM was 85%. At 100 microM adenosine or inosine, fractional sequestration by aortic endothelium was 33% and 39%, respectively. Analysis of the specific radioactivity of adenine nucleotides extracted from prelabeled aortas indicated that most of the adenosine was incorporated into endothelial adenine nucleotides. Incorporation of inosine into endothelial ATP was approximately 15% that of adenosine. Inhibition of aortic adenosine deaminase with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not influence sequestration of 0.1 microM adenosine, but resulted in a 49% reduction of total endothelial incorporation at 100 microM adenosine. Transfer of radioactive purines from the endothelium to underlying smooth muscle after prelabeling was equivalent to only 1%/hr of total endothelial radioactivity. Our findings suggested that 1) macrovascular endothelium of the aorta constitutes a highly effective metabolic barrier for circulating adenosine and inosine; 2) transfer of labeled adenine nucleotides from endothelium to underlying smooth muscle is rather small and most likely proceeds via dephosphorylated purine compounds; and 3) measurement of adenosine trapping in endothelial and smooth muscle compartments overestimates the transendothelial adenosine concentration gradient.     

Ribose accelerates the repletion of the ATP pool during recovery from reversible ischemia of the rat myocardium

It is a characteristic feature of the myocardium that the derangement in function [6] and the depletion of the ATP pool [1, 2, 9] that occur subsequent to oxygen deficiency persist when blood flow is restored. Of renewed interest is the inability of the heart to replenish rapidly its adenine nucleotide pool once it has been diminished during a brief period of regional ischemia [2, 9]. A hypothesis that could explain this metabolic insufficiency of the myocardium is that the biosynthesis of adenine nucleotides is very slow in the normal heart and is increased only moderately during postischemic recovery [15] so that the replenishment of adenine nucleotides is not affected appreciably. To substantiate such a hypothesis it is necessary to provide evidence that the restitution of the ATP pool can be accelerated by stimulation of this biosynthetic process. In previous studies ribose has been recognized as a substrate that enhances markedly adenine nucleotide biosynthesis in the rat heart [11, 12]. We now demonstrate that continuous i.v. infusion of ribose during recovery from a 15-min period of myocardial ischemia in rats leads to restoration of the cardiac ATP pool within 12 h, whereas 72 h are needed for ATP normalization without any intervention.     

Effect of ischemia and infarction on regional content of adenine nucleotides and derivatives in canine left ventricle.

Samples of myocardium from four areas of ischemic and infarcted canine ventricle were examined over a 20-day period for content of the three adenine nucleotides as well as inosine, hypoxanthine, adenosine, and inosine monophosphate. The adenosine triphosphate (ATP) content of central and peripheral areas within the infarct fell to 11% and 8% of control, respectively, 1 day after coronary occlusion. The total adenine nucleotide (TAN) content in these areas fell to 17% of control and showed no significant recovery during the period of study. In the functional myocardium immediately surrounding the infarct the ATP content was depressed to 58% control after 1 day, and the TAN content was also depressed. In the healthy myocardium near the apex, the ATP content was significantly depressed only at the 3-day sample period. Adenine nucleotide derivatives were detected only at 30 min in the central ischemic area. The absence of the nucleoside and nucleobase compounds formed during adenine nucleotide degradation is attributed to their high membrane diffusibility. Loss of these compounds is considered a contributing factor in the prolonged depression of adenine nucleotide content in both ischemic and non-ischemic regions.