B. Deuticke

Abbau freier Nucleotide in Herz, Skeletmuskel, Gehirn und Leber der Ratte bei Sauerstoffmangel

SummaryLack of oxygen due to ischemia causes a breakdown of adenine, guanine, and uridine nucleotides in heart, brain, liver, and skeletal muscle like in kidney [13]. In consequence of these degradative processes different nucleosides, purines, and pyrimidines, not detectable in normal tissues, accumulate in varying amounts. All these compounds were quantitated spectrophotometrically after their separation by means of special methods of paper chromatography.According to the results obtained mode and extent of nucleotide catabolism differ from organ to organ. Of special interest is the observation that the degradation of AMP leads only in heart and brain to an accumulation of vasoactive adenosine. The findings are discussed under physiological and biochemical aspects, especially with regard to the enzymatic reactions involved in nucleotide catabolism and the problems of post-anoxic recovery.ZusammenfassungSauerstoffmangel infolge Ischämie verursacht in Herz, Gehirn, Leber und Skeletmuskulatur ebenso wie in der Niere einen über die Nucleosid-Monophosphat-Stufe hinausgehenden Abbau von Adenin-, Guanin- und Uridin-Nucleotiden. Die hierbei auftretenden Nucleoside, Purine und Pyrimidine sowie die einzelnen Nucleotide wurden durch spezielle papierchromatographische Trennungsverfahren in Kombination mit spektrophotometrischen Methoden quantitativ erfaßt.Zwischen den einzelnen Organen bestehen zum Teil recht erhebliche Unterschiede hinsichtlich Art und Ausmaß der katabolen Reaktionsabläufe. Von besonderem Interesse ist die nur in Herz und Gehirn beim Abbau von AMP erfolgende Akkumulation von vasoaktivem Adenosin. Die möglichen Ursachen dieser beobachteten Organverschiedenheiten und daraus sich ergebende Konsequenzen physiologischer und biochemischer Art werden diskutiert.

Zum Verhalten von Nucleotiden und ihren dephosphorylierten Abbauprodukten in der Niere bei Ischämie und kurzzeitiger post-ischämischer Wiederdurchblutung

Der Nucleotidund Phosphat-Stoffweehscl der Warmblüterniere wird bei Sauerstoffmangel schon frühzeitig st~rkstens beeinträchtigt. Bereits wenige Sekunden nach Beginn einer Isch~mie oder Asphyxie setzt -wie wir in früheren Untersuchungen gezeigt haben s -ein rasch fortschreitender Abbau der energiereiehen Nucleosid-Triund -Diphosphate ein, der zur Anreicherung von Nucleosid-Monophosphaten und Orthophosphat führt. Der Gesamtgehalt der ~qiere an ~qucleotiden gndert sich dabei anfänglich nicht. Erst bei S~uerstoffmangel über die Dauer von 1--2 min hinaus kommt es zu einer progredienten Verminderung der Summe aller Nucleotide. Wie andernorts bereits kurz raitgeteilt wurde is, sind hierfür Dephosphorylierungsprozesse und weitere Abbauvorg~nge verantwortlich, die zur Bildung von ~queleosiden sowie von verschiedenen Purinund Pyrimidin-Basen führen. Ziel der vorliegenden Untersuchungen war zunächst das detaillierte Studium der wichtigsten Nucleotid-Abbauprozesse in der Niere nach Iseh~miezeiten von 5--60 min. Des weiteren sollte geklirrt werden, wie sich die Nueleotide und insbesondere ihre dephosphorylierten Abbauprodukte bei der Wiederdurchblutung ischgmischer Nieren verhalten. Studien dieser Art erschienen vor allem von Interesse im Hinblick auf

Phosphat-Permeabilitt und Phosphat-Stoffwechsel menschlicher Erythrocyten und Mglichkeiten ihrer experimentellen Beeinflussung

Phosphate permeability and phosphate metabolism of human erythrocytes have been studied by determining the rates of phosphate influx and efflux, and by measuring concentrations as well as 32P-labelling of intracellular phosphate compounds under different experimental conditions. The following results were obtained:Temperature coefficients of phosphate efflux and influx are of similar magnitude (4,0–4,5 between 20° and 37° C). Intracellular orthophosphate seems to be most probably the precursor pool of phosphate ions released from the red cells.The rates of phosphate influx and efflux are considerably reduced by dipyridamole, pyrazolidines, phloretin, reserpine, tannic acid et al., the efflux being in general more strongly influenced than the influx. Since under the same conditions glycolysis and concentrations of intracellular P-compounds remain unaltered, the effects observed can only result from a decrease of phosphate permeability caused by the different substances.Phosphate influx and efflux are enhanced by EDTA, EGTA, ATP, phytic acid, oxalate et al., but not by omission of Ca++ from the incubation medium. EDTA and EGTA are ineffective, however, in Ca++-free media. In most cases the enhancement of phosphate transfer is probably due to an increase of the membrane permeability only.Inhibitors of glycolysis (iodoacetate, fluoride, arsenate et al.) do not primarily affect phosphate influx; phosphate efflux, however, becomes remarkably increased due to the progressive elevation of intracellular orthophosphate levels brought about by these inhibitors. Purine nucleosides (adenosine, inosine, guanosine), which likewise have no influence on phosphate influx, considerably reduce the efflux rates. This effect results from the diminution of intracellular orthophosphate in consequence of its intensified esterification.Elevation of external orthophosphate levels causes a proportional increase of phosphate influx, thus raising the intracellular orthophosphate concentration and by this again augmenting the efflux of phosphate.From the results obtained it becomes evident that in human erythrocytes uptake and release of phosphate, which are in principle independent of cellular energy metabolism, can be experimentally affected either by alterations of membrane permeability or by changes of internal and external orthophosphate concentrations.Phosphate permeability and phosphate metabolism of human erythrocytes have been studied by determining the rates of phosphate influx and efflux, and by measuring concentrations as well as 32P-labelling of intracellular phosphate compounds under different experimental conditions. The following results were obtained:

ber die Kinetik der Phosphat-Permeation in den Menschen-Erythrocyten bei Variation von extracellulrer Phosphat-Konzentration, Anionen-Milieu und Zell-Volumen

SummaryThe kinetics of passive transfer of inorganic phosphate across the human red cell membrane have been investigated under different experimental conditions. Unidirectional steady state phosphate fluxes were calculated from the rates of P 32-uptake. The following results could be obtained:1.Elevation of extracellular phosphate levels up to 110 μmoles/ml causes an exponential increase of the rates of phosphate influx when chloride is used as concomitant anion in isotonic solutions. In contrast, when isotonicity is established by sulfate, phosphate transfer rates rise only linearily with the extracellular phosphate concentration.2.Replacement of extracellular chloride by other anions brings about characteristic changes of phosphate fluxes. The rates of penetration increase according to the following order: SCN− < NO3−< J− < Br−=Cl− < formate < propionate < pyruvate < acetate < lactate < SO4−−.3.The rates of phosphate influx are higher in hypotonic than in hypertonic sodium chloride solutions. This effect, however, is not due to the concomitant changes of cell volume, but to the fact that the extracellular chloride levels are altered parallel to the variation of osmolarity. The results are in favour of the hypothesis, that passive transmembrane flux rates of phosphate depend on the phosphate concentrations within the membrane or membrane pores. These concentrations may behave quite different from those outside, since anions are presumably distributed between membrane and medium according to a Donnan equilibrium governed by the concentration of positive charges within the membrane and the concentrations and valencies of all extracellular anions, respectively.Furthermore, additional specific anion effects on phosphate transfer are made most probable by the findings. These could be due either to varying mutual influences of anions on each other during penetration or to anion induced changes of membrane permeability.ZusammenfassungDie Kinetik der passiven Permeation von Orthophosphat durch die Membran des Menschen-Erythrocyten wurde unter verschiedenen experimentellen Bedingungen studiert. Als Maß des Phosphat-Transfer diente der mittels P32 unter Ionen-Gleichgewichtsbedingungen gemessene unidirektionale Phosphat-Influx. Es ergaben sich folgende Resultate:1.Bei Erhöhung des extracellulären Orthophosphat-Spiegels bis auf 110 μMol/ml nehmen die Phosphat-Influx-Raten in isotonischen Phosphat-Chlorid-Gemischen abszissenkonvex exponentiell, in isotonischen Phosphat-Sulfat-Gemischen linear mit der extracellulären Phosphat-Konzentration zu.2.Ersatz des extracellulären Chlorids durch andere Anionen bei konstantem Phosphat-Spiegel (1 μMol/ml) führt zu charakteristischen Veränderungen der Permeationsraten von Phosphat. Der Influx nimmt in folgender Reihenfolge zu: SCN− < NO3−< J− < Br−=Cl− < Formiat < Propionat < Pyruvat < Acetat < Lactat < SO4−−.3.Aus hypotonischen Salzlösungen nehmen Erythrocyten mehr Phosphat auf als aus hypertonischen. Dieser Effekt beruht nicht auf osmotisch bedingten Veränderungen des Zellvolumens, sondern auf den unterschiedlichen Chlorid-Konzentrationen in Medien verschiedener Tonizität. Die beobachteten Transfer-Veränderungen lassen sich zum Teil mit der Annahme erklären, daß die Permeationsrate der Phosphat-Anionen von ihrer jeweiligen Konzentration in der Erythrocyten-Membran bestimmt wird, und daß diese intramembrane Konzentration entsprechend den Gesetzen einer Donnan-Verteilung von der variablen Zahl intramembraner positiver Festladungen sowie der Konzentration und Wertigkeit aller extracellulär vorhandenen Anionen abhängt.Darüber hinaus ergeben sich aus den Befunden Hinweise auf zusätzliche spezifische Anionen-Effekte. Diese können entweder mit einer unterschiedlichen gegenseitigen Beeinflussung verschiedener Anionen bei der Penetration oder mit Anionenbedingten Änderungen der Membran-Permeabilität in Zusammenhang gebracht werden.

Paper chromatographic separation of nucleotides, nucleosides, purines, and pyrimidines

Abstract A method of paper chromatography is described for separation, identification, and quantitation of nucleotides, nucleosides, purines and pyrimidines. The method has been used for studies of nucleotide catabolism in tissues in vivo and in vitro.

Beeinflussung der Monosaccharid-Permeabilitt des Menschen-Erythrocyten durch eine Pyrimido-Pyrimidin-Verbindung

Results are reported on the influence of dipyridamole on monosaccharide penetration into human red blood cells. As could be shown by means of osmotic methods, dipyridamole (10−4 mol/l) inhibits the uptake of d-glucose, d-fructose, d-ribose, d-mannose, d-galactose, d-xylose, and l-arabinose into human erythrocytes to a different degree. The results are discussed with respect to the inhibitory effects of dipyridamole on the transfer of adenosine, orthophosphate, and monosaccharides across the human red cell membrane.Results are reported on the influence of dipyridamole on monosaccharide penetration into human red blood cells. As could be shown by means of osmotic methods, dipyridamole (10−4 mol/l) inhibits the uptake of d-glucose, d-fructose, d-ribose, d-mannose, d-galactose, d-xylose, and l-arabinose into human erythrocytes to a different degree. The results are discussed with respect to the inhibitory effects of dipyridamole on the transfer of adenosine, orthophosphate, and monosaccharides across the human red cell membrane.

Abhängigkeit der 2,3-Diphosphoglycerinsäure-Synthese in Menschen-Erythrocyten von der ADP-Konzentration

SummaryThe spontaneous decrease in 2,3-diphosphoglycerate (2,3-DPG) concentration in human erythrocytes incubated in the presence of glucose is markedly delayed or completely prevented by dipyridamole (5·10−4 M). This effect is not due to an inhibition of 2,3-DPG phosphatase, since it is abolished after interruption of 2,3-DPG synthesis. The effect of dipyridamole therefore can only be explained by an increase in 2,3-DPG formation.This increase was shown to be causally related to a diminution of ADP in the presence of dipyridamole. Since red cell glycolysis remained unchanged under these conditions, the decrease in ADP should result in an increase of 1,3-DPG concentration. The elevation of 1,3-DPG, in turn, gives rise to an enhancement of 2,3-DPG synthesis, which is responsible for the observed effect of dipyridamole on 2,3-DPG metabolism.The amount of glucose metabolized by the 2,3-DPG cycle in intact red cells under physiological conditions depends on the concentration of ADP. As ADP concentration diminishes from 0.12 to 0.07 μMol/ml erythrocytes, the proportion of glucose degraded via 2,3-DPG increases from 15 to 22%.Possible mechanisms of the action of dipyridamole on the ADP level in erythrocytes are discussed.ZusammenfassungDer spontane Abfall der 2,3-Diphosphoglycerinsäure-Konzentration (2,3-DPG) bei Inkubation menschlicher Erythrocyten in Gegenwart von Glucose wird durch Dipyridamol (5·10−4 M) verzögert bzw. aufgehoben. Dieser Effekt beruht nicht auf einer Hemmung der 2,3-DPG-Phosphatase, sondern auf einer gesteigerten Synthese von 2,3-DPG; denn nach Unterbrechung der Glykolyse beeinflußt Dipyridamol den 2,3-DPG-Abfall nicht mehr.Als Ursache der gesteigerten 2,3-DPG-Bildung ist eine Senkung der ADP-Konzentration bei unveränderter Glykolyse-Rate und eine hierdurch bedingte Erhöhung des 1,3-DPG-Spiegels anzusehen.Aus den Untersuchungen ergibt sich weiterhin, daß der Anteil des 2,3-DPG-Nebenweges am glykolytischen Gesamtdurchsatz in intakten Erythrocyten vom ADP-Spiegel abhängt und bei physiologischen ADP-Konzentrationen etwa 15 bis 22% ausmacht.

Stoffwechsel von Adenin-Nucleotiden in Nierenschnitten unter verschiedenen experimentellen Bedingungen und whrend post-anoxischer Erholung

SummaryRat kidney slices incubated aerobically in a Krebs-Ringer-solution at 30° C were shown to contain adenine nucleotides in much smaller quantities than renal tissue in vivo. The reduction of these compounds was found to be due to catabolic processes resulting in the formation of dephosphorylated degradation products. This loss of nucleotides amounted to about 45% in slices incubated in the original (primary) suspension medium and even to 65% in slices transferred to a fresh (secondary) medium prior to incubation. In both cases, however, the percentage distribution ratio of ATP, ADP and AMP proved to be very similar to that observed for normal kidneys.A close parallelism also exists between slices that are incubated anaerobically and ischemic kidneys with respect to the breakdown of ATP and ADP and the further decomposition of AMP leading to a diminution of the total amount of adenine nucleotides. During post-anoxic recovery the sum of adenine nucleotides does not increase again, though mononucleotides are intensively rephosphorylated.In further experiments, it was shown that post-anoxic recovery of kidney slices in media containing nucleosides and purines results in a pronounced increase in the sum of adenine nucleotides. Optimal effects could be obtained by mixtures of inosine, guanosine, adenine and phosphate or inosine, guanosine, aspartate and phosphate. The increase of adenine nucleotides amounted to 75% and 60%, respectively, during a 60 minute period of incubation. Various precursors, intermediates and co-factors of nucleotide synthesis de novo had no detectable effects.The restitutive actions of purine nucleosides and purines are discussed with special respect to pathways of metabolic utilisation of these compounds in renal tissue.