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B Siegmund

Publications and source records attributed to B Siegmund.

At least 19 recordsLinked to original sources
PubMed1995

Protection of reoxygenated cardiomyocytes against hypercontracture by inhibition of Na+/H+ exchange.

Effects of Na+/H+ exchange inhibition and cytosolic acidosis on reoxygenated adult rat ventricular cardiomyocytes were investigated. Cells were incubated in anoxic media at pH 6.4 until pCa of < or = 5, intracellular pH (pHi) of 6.5, and cytosolic [Na+] of 50 mM were reached. On reoxygenation, medium pH was changed to 7.4 to activate Na+/H+ exchange. In one group, 20 microM HOE-694, an inhibitor of Na+/H+ exchange, was added. With or without HOE-694, cytosolic Ca2+ and Na+ returned to control levels within 10 min of reoxygenation. In the absence of HOE-694, the pHi renormalized (to 7.2) within 8 min, but irreversible hypercontracture and transient Ca2+ oscillations were observed. In the presence of HOE-694, pHi stayed acidotic (at 6.5), hypercontracture was prevented, and Ca2+ oscillations were attenuated. When the Na+ pump was inhibited with 0.1 mM ouabain, even partial recovery of Ca2+ control became impossible unless HOE-694 was added. Our conclusions are 1) activation of Na+/H+ exchange does not impair recovery of cytosolic Na+ and Ca2+ control unless activity of the sarcolemmal Na+ pump is critically reduced, and 2) due to prolongation of cytosolic acidosis, inhibition of Na+/H+ exchange protects against reoxygenation-induced hypercontracture and cytosolic Ca2+ oscillations.

Animals↗
PubMed1994

Importance of sodium for recovery of calcium control in reoxygenated cardiomyocytes.

The role of Na+ in the recovery from severe anoxic Ca2+ overload was investigated in isolated quiescent ventricular cardiomyocytes from adult rat. Changes of cytosolic Ca2+ and Na+ concentrations were followed by the fura 2 and Na(+)-binding benzofuran isophthalate techniques, respectively. When the fura 2 ratio (340/380 nm) reached saturation in anoxic cells, indicating a severe cytosolic Ca2+ overload, the cells were reoxygenated. This caused a rapid initial drop of cytosolic Ca2+ to a lower but still elevated level (phase I), followed by oscillatory Ca2+ transients at this level (phase II) and, within 10 min, the reestablishment of a stable cytosolic Ca2+ concentration at the normal resting level (phase III). As previously shown [B. Siegmund, R. Zude, and H. M. Piper. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1262-H1269, 1992], Ca2+ shifts in phase I and II are mainly due to uptake and release of Ca2+ by the sarcoplasmic reticulum. Phase I was unchanged, and phase II was much prolonged (> 60 min) in cells reoxygenated under Na+ pump inhibition (0.2 mM ouabain) or Na+ depletion. Phase III could only be reestablished (< 10 min) when ouabain was eluted or external Na+ replenished, respectively. The results show that full recovery of cytosolic Ca2+ control (phase III) requires an active sarcolemmal Na+ pump and the availability of external Na+. This indicates that phase III is determined by the transsarcolemmal extrusion of Ca2+ by a tandem mechanism consisting of 1) the Na+ pump, generating an extracellular-to-intracellular Na+ gradient, and 2) the sarcolemmal Na+/Ca2+ exchange, driven by that gradient to extrude Ca2+.

Animals↗
PubMed1993

Calcium and the oxygen paradox.

When myocardial cells are reoxygenated after a prolonged period of energy depletion, they rapidly hypercontract. In tissue, hypercontracture induced by reoxygenation is accompanied by cytolysis ("oxygen paradox"). Recent studies have indicated that severe cytosolic Ca2+ overload and reactivation of energy production represent the causal key factors for the deleterious hypercontracture, through the following mechanism: prolonged energy depletion leads to a progressive cytosolic Ca2+ overload in cardiomyocytes; when oxidative phosphorylation is then resumed with the resupply of oxygen, activation of the myofibrils at (still) increased cytosolic Ca2+ concentrations provokes a sustained maximal force development and consecutive mechanical cell injury. This injury can largely be prevented when the contractile machinery is inhibited during the initial phase of reoxygenation. In the model of isolated cells it has been shown that a normal cytosolic Ca2+ control can be reestablished upon reoxygenation. This seems to explain why contractile blockade is needed only temporarily for the prevention of reoxygenation induced hypercontracture and cellular deterioration. Temporary contractile blockade at the onset of reperfusion has also been shown to protect the heart in vivo against lethal reperfusion injury.

Animals↗
PubMed1992

Recovery of anoxic-reoxygenated cardiomyocytes from severe Ca2+ overload.

The ability of hypoxic-reoxygenated cardiomyocytes to recover from severe cytosolic Ca2+ overload was investigated using the fluorescent Ca2+ indicator fura-2 in ventricular cardiomyocytes from adult rats. When the fura-2 ratio (340/380 nm) reached saturation in hypoxic cardiomyocytes, indicating severe Ca2+ overload, they were reoxygenated. The cell then suddenly hypercontracted but reestablished, after a phase of Ca2+ oscillations, a normal Ca2+ control. Because these oscillations could be abolished by ryanodine (50 nM), they seem to depend on the function of the sarcoplasmic reticulum (SR). In the presence of caffeine (5 mM) and thapsigargin (100 nM), i.e., agents impairing Ca2+ sequestration in the SR, reoxygenation did not lead to Ca2+ oscillations or to a stable recovery of cytosolic Ca2+ control. The additional presence of ruthenium red (5 microM), an inhibitor of mitochondrial Ca2+ uptake, restored the ability of cells treated with caffeine or thapsigargin to reestablish a normal cytosolic Ca2+ control. The results show that cardiomyocytes are able to recover from severe hypoxic Ca2+ overload if, first, a closed sarcolemma is retained (as in isolated cardiomyocytes) and, second, the SR is available for rapid Ca2+ storage (impaired by caffeine and thapsigargin). The results also suggest that, in the case of an impairment of SR function, the inhibition of mitochondrial Ca2+ uptake (as by ruthenium red) has a protective effect.

Animals↗
PubMed1992

Prevention of the oxygen paradox in the isolated cardiomyocyte and the whole heart.

It was investigated in hypoxic-reoxygenated cardiomyocytes and isolated perfused hearts from rat whether temporary contractile blockade by 2,3-butanedionemonoxime (20 mM: BDM) during the initial phase of reoxygenation could prevent severe reoxygenation-induced cell injury. In isolated rat cardiomyocytes, reoxygenation after 120 minutes substrate-free anoxia caused sudden hypercontracture but not cytolysis. Within 15 minutes, a nearly normal free energy change of ATP hydrolysis and a normal cytosolic Ca2+ control were reestablished, in spite of irreversible hypercontracture. When BDM was present during the initial 15 minutes reoxygenation and then eluted, hypercontracture remained absent. In the isolated perfused heart, reoxygenation after 60 minutes substrate-free hypoxic perfusion provoked rapid hypercontracture and a sudden massive loss of creatine kinase ("oxygen paradox"). When BDM was present for the first 60 minutes reoxygenation and then eluted, these characteristics of the "oxygen paradox" remained virtually absent. The results demonstrate that the reoxygenation-induced hypercontracture and severe cell injury characteristic for the "oxygen paradox" can be prevented in the hypoxic-reoxygenated heart muscle cell when the contractile apparatus is temporarily paralyzed during the initial phase of reoxygenation. This time seems to be needed for the recovery of cytosolic Ca2+ control.

Animals↗
PubMed1991

Longevity of adult ventricular rat heart muscle cells in serum-free primary culture.

The study had two aims: first, to improve the longevity of isolated adult cardiomyocytes in serum-free culture, and, second, to investigate whether catecholamines which promote hypertrophy in vivo can prolong survival of isolated adult rat cardiomyocytes in serum-free culture. The basic cell culture medium consists of serum-free medium 199 with 10(-7) M insulin. In this medium 50% of the initially plated cardiomyocytes survive in elongated form for 2 days. Omission of glutamine and supplementation of the basic medium with 5 mM creatine, 2 mM carnitine and 5 mM taurine extends survival of elongated cells to 14 days. In supplemented medium, normal cell ATP content is maintained (27 nmol/mg protein after 15 days), but cells gradually atrophy and reduce their protein mass. The trophic effects of catecholamines (epinephrine, norepinephrine, phenylephrine; 10 microM, added on day 3 of culture) were investigated. After addition of catecholamines the cells spread. Spreading can be prevented by prazosin (10 microM) and phentolamine (10 microM) but not by propranolol (10 microM), indicating that spreading is stimulated via the alpha 1-adrenoreceptor. Cells also spread in the presence of the phorbol ester phorbol myristate acetate (10 microM). Catecholamines reduce the progressive cell atrophy and protein loss. With 10 microM phenylephrine, cellular ATP content remained constant at 27 nmol/mg protein until day 15. The results indicate that agents which stimulate protein kinase C (alpha 1-agonists, phorbol esters) stimulate cell spreading, protein synthesis and long-term survival of cardiomyocytes in vitro.

Animals↗
PubMed1991

Temporary contractile blockade prevents hypercontracture in anoxic-reoxygenated cardiomyocytes.

Reoxygenation after 120-min substrate-free anoxia causes sudden hypercontracture in isolated rat cardiomyocytes. Reoxygenated-hypercontracted cardiomyocytes maintain their sarcolemmal integrity as indicated by the absence of enzyme release and reestablish a nearly normal free energy change of ATP hydrolysis within 15 min [Siegmund, B., A. Koop, T. Klietz, P. Schwartz, and H. M. Piper.Am J. Physiol. 258 (Heart Circ. Physiol. 27): H285-H291, 1990]. In the same model, it was now investigated whether a temporary contractile blockade by 20 mM 2,3-butanedione monoxime (BDM) can prevent reoxygenation-induced hypercontracture. When BDM was present during 120-min anoxia and the subsequent 15-min reoxygenation, hypercontracture could be prevented. The anoxic changes of high-energy phosphate contents, the free energy change of ATP hydrolysis, and the ultrastructure of the cells remained unaffected by the presence of BDM. When BDM was applied anoxically immediately before reoxygenation, it also prevented hypercontracture. Contracture still remained absent when BDM was washed out after the first 15 min of reoxygenation. These results demonstrate that a temporary contractile blockade (15 min) at the onset of reoxygenation prevents hypercontracture in anoxic-reoxygenated cardiomyocytes. This result, the energetic recovery, and the sarcolemmal integrity of cardiomyocytes in anoxia-reoxygenation demonstrate that reoxygenation-induced hypercontracture is not based on an already irreversible cell damage.

Animals↗
PubMed1991

Prevention of the oxygen paradox in hypoxic-reoxygenated hearts.

Reoxygenation after 60 min substrate-free hypoxic perfusion (modified Tyrode solution, 37 degrees C) caused isolated Langendorff hearts (from rats) to rapidly develop hypercontracture and sarcolemmal disruptions indicated by massive and sudden loss of enzymes ("oxygen paradox"). Reoxygenation (30 min) caused an augmented loss of creatine kinase by 25.8% (lactate dehydrogenase by 40.1%) of the initial total tissue activity. It was investigated whether a temporary contractile blockade by 2,3-butanedione monoxime (BDM; 20 mM) can prevent reoxygenation-induced injury. In the presence of BDM, reoxygenation no longer caused hypercontracture or increased enzyme release. Instead, ultrastructure recovered, and contents of creatine phosphate (CrP) were partially restored (60 min hypoxia: 0.4 mumol CrP/g dry wt; after subsequent 60 min reoxygenation in presence of BDM: 7.8 mumol CrP/g dry wt). When BDM was eluted after first 20 min of reoxygenation, an attenuated but distinct increase in enzyme release was still observed. When BDM was eluted after 60 min of reoxygenation, ultrastructure did not deteriorate and increase of enzyme release remained virtually absent. During first 30 min after removal of BDM, the increased loss of creatine kinase amounted to only 5.7% (lactate dehydrogenase to 6.9%) of the initial total tissue activity. The results demonstrate that the oxygen paradox can be prevented in the hypoxic-reoxygenated heart when the contractile apparatus is temporarily paralyzed during the initial phase of reoxygenation.

Animals↗
PubMed1990

Changes in the energy metabolism of cultured lens epithelial cells in comparison with the fresh lens.

Energy metabolism of bovine cultured lens epithelial cells (CLEC) was compared to that of fresh bovine lens. CLEC contained high levels of ATP (44 nmol mg protein-1) and creatine phosphate (13 nmol mg protein-1). An ATP/ADP ratio of ten and a creatine phosphate/creatine ratio of two indicated the cells were in a well-energized state. ATP concentration in fresh epithelium was comparable to that of CLEC; however in the anterior cortex it was tenfold lower. In contrast to fresh lenses, CLEC were able to oxidize glucose, lactate and palmitic acid. Lactate was oxidized at the highest rate. In CLEC, 42% of the ATP generated by catabolizing glucose resulted from oxidative phosphorylation. Glucose (5 mM) was degraded to lactate and CO2 at a 2:1 ratio. The hexose monophosphate pathway accounted for two thirds of the CO2 produced. In the fresh whole bovine lens palmitate was not oxidized and lactate was oxidized to a lesser degree than in CLEC. Only one-tenth of the ATP generated by glucose catabolism in the fresh whole lens was derived from oxidative phosphorylation. This was also the case for a preparation of fresh epithelium, maintained in air and 100% oxygen, demonstrating that the preferential glycolytic catabolism of glucose in lens is not caused by limited oxygen diffusion. In the fresh bovine lens the epithelium accounted for one third of the glucose catabolism of the whole lens, even though it had only about 0.1% of its protein mass. In fresh human lenses, conversion of glucose into lactate was even more pronounced--the lactate/CO2 ratio was 73:1.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1990

Metabolism of exogenous substrates by coronary endothelial cells in culture.

The ability of coronary endothelial cells in 14 day confluent cultures to metabolize glucose, palmitate, lactate and various amino acids was investigated. Under aerobic conditions, 99% of glucose, (5 mM) was degraded to lactate and only 0.04% was oxidized in the Krebs cycle. One percent of the glucose catabolized was directed into the hexose monophosphate pathway, but this fraction could be increased by 81% by 0.4 mM methylene blue. Glucose oxidation in the Krebs cycle was increased at glucose concentrations lower than 1 mM, or by the uncoupler 2,4-dinitrophenol. Oxidation to CO2 of palmitate (300 microM), lactate (1 mM), and glutamine (0.5 mM) was diminished in the presence of glucose (5 mM) by 80, 66, and 48%, respectively. These results demonstrate that coronary endothelial cells utilize exogenous glucose, at physiological concentration, predominantly for glycolytic energy production. The metabolic pattern is characteristic of the Crabtree effect. In these cells, glucose not only effectively suppresses the oxidation of the substrates lactate and palmitate, i.e. of substrates preferred by the whole heart, but also of glutamine, which is a major oxidative substrate for coronary endothelial cells. Absolute rates of substrate catabolism are low as compared to those of the beating heart indicating a low energy demand of coronary endothelial cells.

Adenine Nucleotides↗
PubMed1990

Sarcolemmal integrity and metabolic competence of cardiomyocytes under anoxia-reoxygenation.

In tissue, mechanical cell-to-cell interactions may contribute to cardiomyocyte injury in anoxia-reoxygenation. In the present study, the disturbance of energy metabolism and cell injury were investigated in isolated cardiomyocytes, free of external mechanical constraints. Cardiomyocytes from adult rat, attached to culture dishes, were exposed to 120 min of anoxia and 15 min of reoxygenation in a substrate-free modified Tyrode solution. The energetic state of the cells in anoxia-reoxygenation was characterized by the free-energy change of ATP hydrolysis (delta GATP), amounting to 57 kJ/mol ATP in normoxia. After 120 min of anoxia, all cells were contracted to 65% of their length and delta GATP decreased to 41 kJ/mol. No lactate dehydrogenase was released. Reoxygenation caused a partial oxygen paradox: immediate hypercontracture of the cells, but no release of lactate dehydrogenase. delta GATP recovered to 51 kJ/mol within 15 min. The results demonstrate that anoxic cardiomyocytes can be energy depleted without losing sarcolemmal integrity. They can undergo hypercontracture, elicited by reoxygenation, and yet an almost normal delta GATP can be reestablished.

Animals↗
PubMed1989

The use of the creatine kinase reaction to determine free energy change of ATP hydrolysis in anoxic cardiomyocytes.

In isolated cardiomyocytes from adult rat heart the free energy change of ATP hydrolysis (dG) was determined under conditions of substrate-free anoxia. Changes of free cytosolic ADP concentrations, needed for the calculation of dG, were determined by two indirect methods since a direct measurement is not feasible: (i) via the mass action ratio of the creatine kinase reaction (CK) assuming near equilibrium conditions, and (ii) via quantification of the net hydrolysis of ATP to ADP by a detailed balancing of possible contribution to Pi production. Both approaches gave virtually identical results, showing that in anoxia only 6% of the ATP hydrolysed are hydrolysed to ADP and 94% completely to adenosine and further degradation products. The convergence of both methods also indicates that in this model the CK reaction is indeed catalysed near its equilibrium. Therefore estimations of free ADP and dG using its mass action ratio are valid. In anoxic cardiomyocytes dG values fell from 57 kJ/mol in normoxia to 42 kJ/mol after 120 min anoxia, corresponding to a decrease of ATP contents from 24 to 4 nmol/mg protein.

Adenosine Triphosphate↗
PubMed1989

Substrate oxidation by adult cardiomyocytes in long-term primary culture.

In medium 199 plus 20% fetal calf serum adult rat cardiomyocytes establish a long-term culture (25 days). During the first 10 days they change their gross morphology from the typical elongated in vivo shape (day 1), to a smooth spherical intermediate form (days 2 to 5), to a spread cell type beating spontaneously (days 10 to 15). During the first 10 days in culture, protein content per cell increases and the cell population decreases. By the tenth day, protein content has doubled, and about half of the cells originally plated remain. Thereafter both the protein content and the number of cells are essentially constant for the remainder of the 25-day period investigated. On days 1, 15 and 25 adenine nucleotide contents (213, 216 and 225 nmol/10(6) cells) and values of adenylate energy charge (0.91, 0.87 and 0.88) were similar. At all times in culture, palmitate (0.1 mM) is oxidized at higher rates than lactate (1 mM) and glucose (5 mM). At all times in culture glycolytic flux is sensitive to insulin with half maximal effect seen around 10(-9) M. Oxidation rates for all exogenous substrates are maximal at 15 days in culture, indicating maximal energy demand at this time. The conversion of glucose to lactate, however, progressively increases, so that at 25 days in culture, 70% of ATP derived from degradation of exogenous glucose is glycolytic. The results of this study demonstrate that oxidative metabolism of cardiomyocytes in long-term culture resembles, in its basic characteristics, that of the intact heart. In their increased glycolytic activity, however, they are clearly different.

Adenine Nucleotides↗
PubMed1989

Resistance of endothelial cells to anoxia-reoxygenation in isolated guinea pig hearts.

The release of cytosolic enzymes from myocardial and endothelial cells in the anoxic-reoxygenated guinea pig heart was investigated. Isolated hearts were perfused with Tyrode solution in the Langendorff mode. Sixty-minute anoxic perfusion with or without glucose (5 mM) was followed by 15-min normoxic perfusion with glucose. The losses of purine-nucleoside phosphorylase (PNP) from endothelial cells and of lactate dehydrogenase (LDH) and creatine kinase (CK) from the mass of myocardial cells were determined. After 30-min anoxia, the release of LDH and CK but not of PNP increased. Reoxygenation after 60-min anoxia with glucose caused a partial recovery of tissue ATP but also an increase in leakage of LDH (11% of total in 15 min) and CK (10%) and a sudden rise in coronary resistance, indicating contracture development ("oxygen paradox"). PNP release remained low (0.5%). In hearts subjected to glucose-free anoxia, ATP levels did not rise during 15-min reoxygenation, contracture development was delayed, and the release of LDH and CK was diminished (3.1 and 2.7%, respectively). Leakage of PNP was again low (0.5%). The results indicate that cardiomyocytes are more severely injured by anoxia-reoxygenation than the coronary endothelium. The rapidly developing reoxygenation-induced injury of cardiomyocytes seems to be an energy-dependent phenomenon, since it was attenuated in hearts deprived of substrate in anoxia.

Adenine Nucleotides↗
PubMed1983

Determination of meso-alanopine and D-strombine by high pressure liquid chromatography in extracts from marine invertebrates.

meso-Alanopine and D-strombine are separated by high pressure liquid chromatography using a cation exchange resin and 2.5 X 10(-5) M sulfuric acid as eluant, at a flow rate of 1.0 ml/min, 20 degrees C column temperature and a pressure of 4 500 kPa. Both opines were detected by conductivity. Separation and quantitation was possible in the range of 0.05 to 25 nmol of meso-alanopine and D-strombine. Chemically or enzymatically synthesized opines were quantitated using alanopine/strombine dehydrogenase from Crassostrea angulata. The enzyme was purified by ammonium sulfate precipitation, Sephadex G-100 filtration and fast-protein-liquid chromatography. Specific activity of the final preparation was 500 U/mg protein with glycine as substrate. The formation of meso-alanopine and D-strombine was demonstrated in neutralized perchloric acid extracts from muscle tissue of Arenicola marina L. following enhanced muscular activity and in Mytilus edulis L., Nucula nitida and Crassostrea angulata after 24 h of anoxia.

Alanine↗
PubMed1981

Pancreatic carcinoma in azaserine-treated rats: induction, classification and dietary modulation of incidence.

Pancreatic carcinomas have been induced in Wistar and W/LEW rats by administration of total azaserine doses of 150-520 mg/kg by injection or oral routes over periods of 5-52 weeks. The latent period for development of invasive carcinomas was 1-2 years, but focal abnormalities in acinar cells appear earlier. The incidence of carcinomas varied with total dose, route, and schedule of azaserine administration. The spectrum of histologic patterns of the carcinomas included well and poorly differentiated acinar cell, ductlike, and undifferentiated carcinomas. Rats fed a purified diet developed more pancreatic neoplasms than rats fed a commercial laboratory chow. Selective feeding of these diets during the administration of carcinogen and following completion of carcinogen treatment indicated that the inhibitory effect of chow on pancreatic carcinogenesis was exerted during the postinitiation phas. Supplementation of diet with 0.025% retinyl acetate during the postinitiation phase also inhibited the progression of azaserine-induced lesions in the pancreas.

Adenocarcinoma↗