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Preservation of erythrocytes using metabolic regulators and mutrients. V. Inosine and methylene blue.

Methylene blue and inosine have been shown to stimulate glycolytic metabolism in the erythrocytes, increasing the concentration of 2.3-diphosphoglycerate (2,3-DPG), which is necessary for hemoglobin function, by regulating oxidative metabolism and providing a five-carbon nutrient for glycolysis, respectively. However, a recent study suggested that the methylene blue effect was dependent on the presence of inosine. This study was designed to establish, if possible, the existence of a methylene blue effect and to confirm the usefulness of inosine. The optimal concentration of inosine for increasing 2,3-DPG synthesis in a CPD-adenine preservative is confirmed to be 10--15 mM. Concentrations of 2,3-DPG were maintained in the erythrocytes at normal or higher levels for 21 days of storage with 10 or 15 mM inosine, whether the methylene blue was present or not. However, when methylene blue was present, 2,3-DPG concentrations were significantly better maintained.

Adenine

Mutation of an inosine-producing strain of Bacillus subtilis to DL-methionine sulfoxide resistance for guanosine production.

An inosine-producing strain of Bacillus subtilis was mutated to resistance against the antagonist of glutamine, DL-methionine sulfoxide. Among the mutants derived, guanosine producers were observed frequently. The best strain, 14119, produced 9.6 g of guanosine per liter at a weight yield of 12% from consumed sugar. Inosine production decreased concomitantly. When resistance was increased further by exposure to higher doses of DL-methionine sulfoxide, another strain, AG169, was obtained that did not excrete inosine but produced increased amounts of xanthosine. In these strains, the specific activity of 5'-nucleotidase was lower and that of inosine 5'-monophosphate (IMP) dehydrogenase was higher than the parent strain. It is speculated that the metabolic flow from IMP to xanthosine 5'-monophosphate proceeds more smoothly than that from IMP to inosine and yields more xanthosine and guanosine.

Bacillus subtilis

Effect of inosine on ventricular regional perfusion and infarct size after coronary occlusion.

In 16 dogs inosine was infused at 0.5 mmol/min i.v. for 5 min beginning 15 min after coronary occlusion. Tracer microspheres were used to estimate flow in subepicardium and subendocardium of nonischemic, central ischemic, and borderline ischemic muscle. Estimates of flow before occlusion, 5 min after occlusion, during inosine infusion, 30 min after infusion and 60 min after infusion were obtained. Coronary occlusion reduced flow in the central ischemic regions by 75-95%. The reduction in flow was greatest in subendocardium. In the borderline regions subendocardial flow was reduced by 30% while subepicardial flow was unaffected. The major effects of inosine were seen in nonischemic and borderline ischemic regions. Flow in borderline subendocardium returned to its pre-occlusion value, and flow in nonischemic myocardium increased by approximately 60-80%. However, only in the ischemic regions was the increase in flow sustained for the entire 60 min. In 20 dogs infarct size was determined using nitro blue tetrazolium stain. In 10 controls infarct size was 20.1%, while in 10 inosine-treated dogs infarct size was 15.2% of left ventricular weight (p less than 0.01). Thus, following coronary occlusion inosine infusion was associated with an increase in perfusion of ischemic myocardium and a reduction in infarct size.

Animals

Myocardial release of lactate, inosine and hypoxanthine during atrial pacing and exercise-induced angina.

The coronary venous efflux of lactate, inosine and hypoxanthine during pacing-induced angina has been compared with myocardial extraction of the catabolites during exercise-induced angina. Inosine and hypoxanthine were analyzed by enzyme assay after separation by column chromatography. Myocardial lactate extraction at rest (15 +/- 9%, mean +/- SD) was converted to production levels (-34 +/- 26%) during pacing-induced angina (p less than 0.0005) and increased (24 +/- 13%) during exercise (p less than 0.05). The arterial values at rest (850 +/- 330 mumol/1) were unchanged during pacing and increased five-fold during exercise (4380 +/- 1860 mumol/1). The mean myocardial inosine extraction at rest (33 +/- 10%) was transformed to release values (-41 +/- 30%) during pacing (p less than 0.0005) as well as during exercise (-20 +/- 27%) (p less than 0.0005). The hypoxanthine extraction at rest (25 +/- 11%) decreased during pacing (-7.8 +/- 29%) (p less than 0.0025) and exercise (10 +/- 25%) (NS). The slight increase of arterial inosine and hypoxanthine values was not significant. Myocardially produced lactate, a sensitive marker of pacing-induced ischemia, was obscured by elevated arterial concentrations during exercise. However, inosine significantly correlated with lactate during pacing, and was useful in detecting ischemic myocardial energy deficiency during exercise-induced angina.

Angina Pectoris

Effects of fluorocarbons, chlorinated solvents, and inosine on the cardiopulmonary system.

The effects of fluorocarbons and chlorinated solvents on the cardiopulmonary system are reviewed. The new information, not hitherto reported, relates to the antagonistic action of inosine, a naturally occurring nucleoside formed in the body by deamination of adenosine. The effect of inosine on methylene chloride toxicity was investigated in open chest dogs anesthetized with pentobarbital sodium. Methylene chloride (5% in air or 50,000 ppm) elicited a decrease of ventricular contractility represented by the diminished left ventricular (dp/dt)(max) and myocardial contractile force measured directly with a Walton-Brodie strain gauge arch. Coronary blood flow decreased slightly after exposure to methylene chloride. Arterial blood pressure and heart rate did not change. The negative inotropic effect of methylene chloride was reversed or prevented to a substantial extent by intravenous infusion of inosine (5 mg/kg-min). The effect of the latter compound was also characterized by significant coronary vasodilation. It was shown by the experiments that the cardiostimulatory action of inosine was associated with improved hypoxic adaptability of the coronary blood vessels. In contrast, the effect of catecholamines (epinephrine and isoproterenol) was not accompanied by such a beneficial coronary vascular effect. On the basis of these results, the conclusion has been arrived at that inosine might be recommended as a useful antidote in methylene chloride poisoning in particular, and of poisoning by chlorinated solvents and fluorocarbons in general.

Adenosine

Further studies of renal preservation: protection of the ischemic kidney with inosine.

Preliminary data are presented on additional means of renal preservation, particularly in the case of warm ischemic insult. We hypothesize that 1 factor responsible for the failure of kidneys to recover after ischemic insult may be depletion of energy sources during the warm ischemic period. Specifically, adenosine triphosphate in the kidney is the principal source of energy for metabolic activity and membrane stability. Inosine, an adenosine triphosphate precursor, was used in a canine model and preliminary work suggested that inosine, when coupled with mannitol, may provide enhanced protection. In vivo assay of kidney slices suggests that inosine may, indeed, tend to maintain adenosine triphosphate levels in the ischemic kidney in contrast to relative adenosine triphosphate depletion in the kidneys not protected with inosine. These data are in concert with the apparently favorable survival data in inosine and mannitol-treated animals.

Adenosine Triphosphate

Tryptaminergic mechanism participating in induction of vasoconstriction by adenine nucleotides, adenosine, IMP and inosine in the isolated and blood-perfused hindlimb preparation of the rat.

The isolated right hindlimb of the recipient rat was perfused at a constant flow rate through the femoral artery with heparinized blood from the carotid artery of a donor. The preparations were under a 99.0 +/- 0.8 mmHg of mean perfusion pressure (N = 63) and 3.3 +/- 0.1 ml/min of blood flow through the right femoral artery. The actions of adenosine, adenosine tri-, die- and monophosphate, inosine monophosphate and inosine on the femoral vascular bed were investigated, respectively. These substances injected into the femoral artery, with the exception of inosine, caused a dose-dependent vasoconstriction always preceded by a temporal vasodilatation. Inosine induced only a prompt vasoconstriction. The vasoconstrictor responses to these substances were diminished or reverted to vasodilator ones after repeated administrations and such were significantly prevented by pretreatment with either reserpine or methysergide. These results indicate that all the purines tested induce a vasoconstriction in the femoral vascular bed of the rat through a common (tryptaminergic) mechanism and that such seem to be potent releasers of 5-hydroxytryptamine from peripheral tryptaminergic storage sites.

Adenine Nucleotides

Inosine nucleosidase from Azotobacter vinelandii. Purification and properties.

An enzyme catalyzing the hydrolysis of purine nucleosides was found to occur in the extract of Azotobacter vinelandii, strain O, and was highly purified by ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxylapatite chromatography and gel filtration on Sephadex G-150. A strict substrate specificity of the purified enzyme was shown with respect to the base components. The enzyme specifically attacked the nucleosides without amino groups in the purine moiety: inosine gave the maximum rate of hydrolysis and xanthosine was hydrolyzed to a lesser extent. The pH optimum of inosine hydrolysis was observed from pH 7 to 9, while xanthosine was hydrolyzed maximally at pH 7. The Km values of the enzyme for inosine were 0.65 and 0.85 mM at pH 7.1 and 9.0, respectively, and the value for xanthosine was 1.2 mM at pH 7.1. Several nucleotides inhibited the enzyme: the phosphate portions of the nucleotides were suggested to be responsible for the inhibition by nucleotides. Although the inhibition of the enzyme by nucleotides was apparently non-competitive type with respect to inosine, allosteric (cooperative) binding of the substrate was suggested in the presence of the inhibitor. The physiological significance of the enzyme was discussed in connection with the degradation and salvage pathways of purine nucleotides.

Azotobacter

An ultrastructural study on the effects of warm ischaemia on the inosine-protected kidney.

Unperfused, saline-perfused and inosine-perfused rat kidneys were subjected to various times of warm ischaemia with and without resumption of blood flow. Tissues were examined with a transmission electron microscope. Inosine has a protective effect on the epithelial cells of the proximal convoluted tubules, where microvilli are less damaged by ischaemia than those of the proximal convoluted tubular cells of the control animals. Three is also a much higher ratio of autophagic vacuoles to lysosomes present in cells of inosine-treated kidneys, reflecting their ability to sequester and digest organelles damaged during ischaemia and thus protect themselves from degradation products. A major effect of inosine seems to be the preservation of the organelles and rush border of the cells of the proximal tubule during warm ischaemia.

Animals

Utilization of exogenous purine compounds in Bacillus cereus. Translocation of the ribose moiety of inosine.

Intact cells of Bacillus cereus catalyze the breakdown of exogenous AMP to hypoxanthine and ribose 1-phosphate through the successive action of 5'-nucleotidase, adenosine deaminase, and inosine phosphorylase. Inosine hydrolase was not detectable, even in crude extracts. Inosine phosphorylase causes a "translocation" of the ribose moiety (as ribose 1-phosphate) inside the cell, while hypoxanthine remains external. Even though the equilibrium of the phosphorolytic reaction favors nucleoside synthesis, exogenous inosine (as well as adenosine and AMP) is almost quantitatively transformed into external hypoxanthine, since ribose 1-phosphate is readily metabolized inside the cell. Most likely, the translocated ribose 1-phosphate enters the sugar phosphate shunt, via its prior conversion into ribose 5-phosphate, thus supplying the energy required for the subsequent uptake of hypoxanthine in B. cereus.

Adenosine Monophosphate

Group translocation of the ribose moiety of inosine by vesicles of plasma membrane from T(3 cells transformed by Simian virus 40.

Plasma membrane vesicles are isolated from Simian virus 40-transformed Balb/c mouse 3T3 (SV-3T3) cells. These membrane vesicles contain no significant contamination by mitochondria, endoplasmic reticulum, or lysosomes as determined by marker enzyme analysis. The use of [U-14C] inosine as a transport substrate results in the accumulation of labeled ribose-1P as transport product by the plasma membrane vesicles. This suggests the action of purine nucleoside phosphorylase (the enzyme which mediates the phosphorolysis of inosine to ribose-1-P and hypoxanthine0 before, during, or after the transport step. Neither inosine nor significant amounts of hypoxanthine are found intravesicularly. The Km for inosine, the substrate in this reaction which leads to the accumulation of ribose-1-P by the plasma membrane vesicles, is 35 to 45 muM while the Vmax for ribose-1-P accumulation is 100 to 120 pmol/min/mg of plasma membrane protein...

Binding, Competitive

Interaction of metal ions with nucleic acids. Interaction of copper(II) with inosine and its derivatives.

1. The interaction of copper(II) with inosine, 2'-deoxyinosine, 1-methylinosine, 7-deazainosine, 6-methoxypurine riboside and IMP was examined. 2. Copper binds with the purine base of the nucleosides, and in IMP also with the phosphate group. Non interaction with ribose hydroxyl groups was observed. 3. In non-aqueous medium, the main site of copper binding is N-7 of inosine and 1-methylinosine, and additionally N-1 in 6-methoxypurine riboside. 4. In aqueous medium, coordination of copper with N-1 and N-7 of inosine and IMP is pH-dependent. 5. Formation of either a five-membered copper chelate with N-7 and oxygen at C-6, or a four-membered chelate of the type C(6)-O-Cu-N(1), is rather unlikely. 6. The crystalline copper-IMP complex [Cu(C10H11O8N4P).(H2O)] contains presumably two copper atoms coordinated in different manner. The phosphate group and N-7, but not the carbonyl oxygen, participate in the complex formation.

Chemical Phenomena

Adenosine phosphyorylase activity as distinct from inosine-guanosine phosphorylase activity in Sarcoma 180 cells and rat liver.

Adenosine phosphorylase (EC 2.4.2.-) activity present in Sarcoma 180 cells grown in culture and in rat liver, is shown to be distinct from inosine-guanosine phosphorylase by several criteria: (a) treatment of Sarcoma 180 cell extract with p-chloromercuribenzoate inhibited the two activities to a different extent, (b) adenine selectively protected the adenosine phosphorylase activity of Sarcoma 180 and rat liver extract against heat inactivation, while hypoxanthine selectively protected inosine-guanosine phosphorylase activity, (c) at nearly saturating substrate concentrations and using Sarcoma 180 extract, the rates of ribosylation of a mixture of adenine + hypoxanthine or adenine + guanine, but not of hypoxanthine + guanine, were found to be almost equal to the sum of their individual rates as measured separately, (d) inosine selectively inhibited the ribosylation of hypoxanthine and guanine catalysed by Sarcoma 180 and rat liver extract while 2-chloroadenosine selectively inhibited the ribosylation of adenine and N6-furfuryladenine, (e) pH vs. activity curves were similar with hypoxanthine or guanine as the substrate but they were markedly different from the curve with adenine as the substrate. The potential role of adenosine phosphorylase activity in vivo is discussed.

Adenine

Inosine: clinical results of ischaemic renal surgery.

Ten patients requiring ischaemic renal surgery for calculus or tumour were operated on with the kidney protected by perfusion with a room temperature solution of inosine. The warm ischaemic times ranged from 35 to 75 min (mean 57.1 min). Post-operative recovery was uneventful in all patients. No adverse cardiodynamic effects due to inosine were noted. There was only moderate transient depression in renal function as assessed by gamma camera renography and serum creatinine estimations. Three of the 10 patients have now been followed for over a year. The results with inosine perfusion are comparable to those obtained with hypothermic protection.

Adult

Inosine may be an endogenous ligand for benzodiazepine receptors on cultured spinal neurons.

Mouse spinal neurons grown in tissue culture were used to study the membrane effects of the benzodiazepine flurazepam and the naturally occurring purine nucleoside inosine, which competes for benzodiazepine receptor sites in the central nervous system. Application of inosine elicited two types of transmitter-like membrane effects: a rapidly desensitizing excitatory response and a nondesensitizing inhibitory response. Flurazepam produced a similar excitatory response which showed cross-desensitization with the purine excitation. Flurazepam also blocked the inhibitory inosine response. The results provide electrophysiological evidence that an endogenous purine can activate two different conductances on spinal neurons and that flurazepam can activate one of the conductances and antagonize the other.

Animals

Prevention of chronic experimental heart insufficiency by inosine.

The administration of inosine dosed 25 mg/kg daily during 10 1/2 months prevented the development of excentric cardiac hypertrophy on the background of experimental aortic stenosis in rats, reduced the lowering of the working capacity of the animals, and partially inhibited the full development of functional and morphological myocardial changes, detected by electrocardiographic, vectorcardiographic, microscopic, and electronmicroscopic examinations. Differences were found in the relative weights of the heart, thymus, liver and other organs; these findings attested to an anabolic activity of inosine. The results obtained justify the application of inosine for the prevention of excentric hypertrophy and decompensation of the heart in cardiac failures varying in etiology; any efficient preventive measure is of importance, because the degree of excentric hypertrophy is a factor influencing the survival of the patients.

Adrenal Glands

The heart production in energy-depleted human erythrocytes induced by glucose, inosine and adenine.

The heat production (HP) of glucose deprived human red blood cells was measured, using glucose, adenine and inosine as substrates. Inosine induced a significantly higher HP than glucose and adenine induced no significant HP. At low pH the HP of glucose decreased more than that of inosine, corresponding to an equally lowered lactate production. The results indicate that it should be possible to use the system developed to study the functional state both of the complete glycolytic system and the lower part of it in intact red blood cells during various clinical conditions.

Adenine

Renal function after warm ischaemia. II. Marked protective effect of intravenous inosine given prior to 60, 90 and 120 min of warm ischaemia.

Rats were given 10 or 20 mg of inosine dissolved in 1 ml of 0.9% saline, or a control solution of 1 ml of 0.9% saline, intravenously 10 or 20 min before induction of left renal warm ischemia for 60 min. Further groups of rats were treated with 20 mg inosine or with saline alone 20 min before longer periods of warm ischeamia of 90 and 120 min duration. All animals had immediate contralateral nephrectomy. In all inosine-treated groups the mean plasma creatinine was lower than in their respective control groups and these differences were statistically highly significant.

Animals