E. Gerlach

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:

Die Inkorporation von markiertem Sauerstoff aus Wasser in die ATP-, Kreatinphosphat- und Orthophosphat-Fraktion intakter Muskeln bei Ruhe, tetanischer Reizung und Erholung

Der S~offwechsel der energiereichen Phospha t e in contrac~ilen Gewebe~ u n d die spezielle Verkni ipfung mi~ den mechanischen l~eak~ionen is t nach wie vor ein ungekl~r tes P rob l e m der Muskelphysiologie . Die For schungen der le tz ten 15 J a h r e h a b e n zwar zahlreiche EffekT~e yon A T P an isol ier ten Muskelpro~einen, Ac tomyos in f~den oder Glycerin-Wasser-ex~rahierl~en Muskelfasern gebracht~. A m lebenden Muskel war jedoch auch mi~ moder~er pap ie rch romatograph i sche r Techn ik ke in direk~er Anha lbspunk t fiir eine obliga~orische K o p p e l u n g zwischen der Spal~ung yon A T P zu A D P u n d dem mechanische~ A k t der Kon t rak~ ion zu gewinnen [F~]~cK]~s~w~ u. J ~ K ~ (1953); F~]~CK~VSTEII~, JANKE, DAVIES u. Kt~EBS (1954); F~]~O~]~ST]~I~, J ~ : w , L]~c~~ ] ~ u. B ~ v ~ (1954) ; F ~ n C K ] ~ S ~ ] ~ , JANKE, KI~USE U. LECHNEI~ (1955) ;

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.

Der Phosphat-Stoffwechsel in Tauben- und Menschen-Erythrocyten unter dem Einfluß von 2,4-Dinitrophenol, Natriumcyanid, Monojodacetat sowie von Thyroxin und Triäthylenmelamin

Vogel-Erythrocyten und die Ery~hrocyten der Si~ugetiere und des Menschen unterscheiden sich bekanntlich nicht nur in morphologiseher Hinsicht, sondern vor allem auch durch die Höhe ihres oxydativen und glykolytischen Stoffumsatzes. Während z.B. kernhaltige TaubenErythrocyten intensiv atmen, aber nur eine geringe aerobe Glykolyse aufweisen, gilt das Umgekehrte für die reißen kernlosen menschlichen Erythroeyten, die stark glykolysieren, jedoch praktisch keinen Sauerstoff verbrauchen. Nach dem Ergebnis vorausgegangener Untersuchungen über den Phosphor-Stoffwechsel ir~ diesen beiden Erythrocyten-Arten [vgl. FLECKENSTEIN U. GER:LACK (1953); GEI~LACH, FLECKENSTEIIq U. F~]~U~])T (1957) und GE~LAC~, FLECKE~STEI~ U. G~oss (1958)] finden sich beachtliche Mengen an phosphorylierten Intermedi£rprodukten des Kohlenhydrat-Abbaus nur in den glykolysierenden menschlichen Erythrocyten, nicht jedoch in den atmenden Tauben-Erythrocyten. Beiden Erythrocyten-Typen gemeinsam sind dagegen relativ große

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.

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.