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E Pfaff

Publications and source records attributed to E Pfaff.

At least 91 records · Page 5Linked to original sources
PubMed1984

On the state of calcium ions in isolated rat liver mitochondria. I. Ion fluxes and volume changes upon Ca2+ uptake under various ionic conditions.

As to functional consequences of Ca2+ uptake in isolated rat liver mitochondria, we simultaneously measured 3H2O and [14C]sucrose spaces, monovalent cation distribution, membrane potential and delta pH across the inner membrane, and [32P]phosphate and 45Ca2+ content in parallel incubations of different ionic composition. Without added Ca2+ and phosphate, mitochondrial matrix volume, membrane potential, and delta pH depended on the concentration and permeability of monovalent cations. Despite large differences in membrane potential, maximal Ca2+ uptake was identical under all conditions. Ca2+ uptake never provoked a volume change from which an osmotic active state of mitochondrial Ca2+ could be concluded. If matrix volume shrunk this could be totally accounted for by the loss of alkali ions exchanging for calcium ions. Even phosphate taken up in conjunction with Ca2+ was osmotically silent. Volume increases here occurring if K+ was permeabilized, solely resulted from K+ uptake, though this condition may give rise to irreversible mitochondrial damage with Ca2+ and phosphate release. As mitochondrial Ca2+ is bound, an electro-chemical equilibrium across the membrane is impossible for this ion. This has to be considered in any model describing equilibria of Ca2+ with mitochondria, though present models neglect this state of mitochondrial Ca2+.

Animals↗
PubMed1984

On the state of calcium ions in isolated rat liver mitochondria. II. Effects of phosphate and pH on Ca2+-induced Ca2+ release.

At high K+ concentration, the effect of phosphate on Ca2+ uptake and release was studied in isolated rat liver mitochondria. Phosphate stimulated uptake at moderately high Ca2+ concentration, and inhibited release at high pH. At low pH, phosphate accelerated Ca2+ release. Ca2+ was released after a lag phase. The time of onset and the velocity of Ca2+ release depended on Ca2+ concentration. Ca2+ release was associated with mitochondrial swelling and destruction of the permeability barrier for sucrose and for chloride. Mg2+ inhibited Ca2+ release and the accompanying events. Ruthenium red and EGTA protected mitochondria from the destructive Ca2+ release and induced an immediate, slow release of Ca2+ and phosphate. Destructive Ca2+ release depended on the time of preincubation of respiration-inhibited mitochondria in the presence of Ca2+, prior to respiration-initiated Ca2+ uptake. The presence of phosphate and mitochondrial energization antagonized the destructive effect of calcium ions. Ca2+ release by acetoacetate also depended on pH. At pH 6.8, phosphate-stimulated Ca2+ release by acetoacetate, while it inhibited the acetoacetate effect at pH 7.6. The results suggest that an essential cause for the destruction of mitochondrial integrity is an increase in the intramitochondrial concentration of free calcium ions under the influence of phosphate.

Acetoacetates↗
PubMed1984

Bile secretion in hemoglobin-free perfused rat liver.

Hemoglobin-free perfused rat liver was demonstrated to be a suitable experimental model in studying bile secretion. Bile flow slowly decreased to more than 3 h of perfusion. Despite differences in metabolic states, the bile flow was the same in the recirculating as in the nonrecirculating mode of perfusion. Sulfobromophthalein stimulated bile flow at high rates of infusion. In bile, the ratio conjugated to unconjugated sulfobromophthalein also increased with sulfobromophthalein infusion rate. The access of [14C]insulin, [14C] sucrose, and inorganic [32P] phosphate from perfusate into bile was restricted. Bile flow, secretion of taurocholate and sulfobromophthalein, and bile pressure are compared with values from anesthetized animals and from isolated livers perfused with medium containing erythrocytes.

Animals↗
PubMed1984

On the state of calcium ions in isolated rat liver mitochondria. III. Diversity of ruthenium red action on different calcium pools.

Calcium efflux from isolated mitochondria on ruthenium red addition was shown to be biphasic. The rate of efflux from a slowly releasable pool was independent of preincubation. It could be saturated and in extrapolation revealed a maximal rate of 3.6 nmol/(min X mg protein). The efflux from a second, rapidly dischargeable pool was related to calcium added up to 300 nmol/mg protein when a final rate of 15 nmol/(min X mg protein) was reached. The magnitude of the latter pool depended on the time of preincubation in the presence of calcium and correlated with mitochondrial swelling. After ruthenium red addition, a further increase of this pool and spontaneous, destructive calcium release was prevented. Three conclusions are drawn from these results: On preincubation with calcium, part of the mitochondrial calcium develops into a rapidly dischargeable pool. This pool is responsible for mitochondrial alterations resulting in a spontaneous, destructive release of total calcium. Ruthenium red inhibits calcium release by discharging mitochondria from this destructive calcium pool. To avoid artefacts, mitochondrial parameters should be carefully controlled when ruthenium red-insensitive calcium efflux is studied.

Animals↗
PubMed1983

On the mechanisms of ATP-induced and succinate-induced redistribution of cations in isolated rat liver cells.

1. The ability of external ATP to induce calcium uptake in isolated rat liver cells was further characterized. Stimulation of calcium uptake was specific for ATP, other nucleotides or ATP metabolites had no comparable effect. ATP was dephosphorylated while stimulating calcium uptake, but there was no stoichiometry between ATP hydrolysis and calcium uptake nor did dephosphorylation depend on calcium concentration. ATP acted from outside and was dephosphorylated by an ecto-ATPase of the cells. 2. In addition to its direct action, ATP enhanced succinate-dependent calcium uptake in a cooperative fashion. This is best explained by different sites of action. ATP increases cell membrane permeability while succinate stimulates uptake into mitochondria. 3. ATP was able to lower Na+ and K+ gradients and the pH gradient between cells and incubation medium. Increasing calcium concentration counteracted this effect though calcium uptake was then stimulated. 4. Succinate alone did not affect monovalent cation gradients but raised the pH gradient. It partially counteracted the ATP effects on these gradients. 5. Since catecholamine-like actions of ATP may be mediated by an increase in cytoplasmic calcium concentration, the action of extracellular ATP can be taken as a model to study the role of calcium as a transmitter of hormone actions. From interdependence between ATP-stimulated and succinate-stimulated calcium uptake, conclusions can be drawn on the resulting cytoplasmic calcium concentration and its effect on plasma membrane permeability.

Adenosine Triphosphate↗
PubMed1983

Detection of an element of the SV40 late promoter in vectors used for expression studies in COS cells.

Plasmids containing hepatitis B virus (HBV) DNA and a 232-bp SV40 DNA fragment encoding the origin of replication were constructed. When introduced by transfection into COS cells, these plasmids directed the synthesis of hepatitis B surface antigen. S1 mapping of the mRNAs covering the S gene showed that transcriptional initiation was promoted by the interaction of HBV sequences with an SV40 promoter element: transcription started on HBV DNA but had several properties of SV40 late transcription. The detection of a promoter element in an SV40 origin fragment commonly used in the COS system is important for the interpretation of data deriving from expression studies in COS cells.

Animals↗
PubMed1983

No increase of biliary permeability in ethinylestradiol-treated rats.

Ethinylestradiol, administered to male Wistar rats for 5 days (5 mg/kg X day), decreased bile flow in anesthetized animals and in isolated perfused livers. The bile salt secretion rate was diminished. The bile-to-perfusate ratios of [14C]sucrose and [14C]inulin increased significantly, but this could be attributed to the decline of bile flow as indicated by almost identical clearance rates. Theorectical analysis according to Forker and Wheeler yielded diffusion permeability coefficients (K) for sucrose and inulin of 0.243 and 0.037 in controls and 0.273 and 0.038 in ethinylestradiol-treated rats. In contrast, 9 h of alpha-naphthylisothiocyanate treatment (250 mg/kg) caused cholestasis with heavily decreased bile salt secretion rates. Here, K-values calculated for sucrose and inulin were 0.807 and 0.175. These findings suggest that altered permeability of the paracellular pathway is the cause for alpha-naphthylisothiocyanate-induced cholestasis, but not the primary event in ethinylestradiol-induced cholestasis.

1-Naphthylisothiocyanate↗
PubMed1982

Activation by reduced glutathione of methotrexate transport into isolated rat liver cells.

The uptake of methotrexate (MTX) by isolated rat hepatocytes and its changes under the influence of exogenous GSH have been studied under various conditions: GSH concentration, pH of incubation medium, preincubation of cells prior to MTX and GSH addition, ionic composition of the incubation medium (standard saline, Na+-free, Na+ and K+-free, or ion-deficient), after prior treatment of cells by membrane -SH blockers (p-CMBS, 4-CMB and DIP2+) and ATP. It was found that GSH strongly accelerated MTX uptake. This effect depended on GSH concentration and on preincubation of cells. The GSH effect was not dependent on medium pH in spite of an observed close relationship between pH of incubate and MTX transport itself. Activation by GSH of MTX transport was connected to an increase in intracellular K+. It was also noted that while blockers of membrane -SH groups like p-CMBS and 4-CMB inhibited MTX uptake and increased the intracellular Na+/K+ ratio, both effects were partially overcome by GSH. After treatment by DIP2+, Na+/K+ ratio was unaffected, but MTX uptake inhibited. Still GSH abolished inhibition. Added ATP also inhibited MTX uptake and caused loss of cellular K+ and accumulation of Na+. Here neither effect could be reversed by GSH; consequently, high cellular amounts of K+ and MTX accumulated by previous action of GSH were depleted on subsequent ATP addition. MTX uptake was low in sucrose medium. But in this ion-deficient medium, GSH had the greatest stimulatory effect on MTX uptake. It is concluded that binding GSH can affect the redox state of the -S-S-/-SH groups of the cellular plasma membrane and that this effect of GSH might demonstrate involvement of the redox state in the control of MTX permeability.

Adenosine Triphosphate↗
PubMed1982

Antibodies against a preselected peptide recognize and neutralize foot and mouth disease virus.

A major antibody combining site on foot and mouth disease virus (FMDV) serotype O1K has been identified in a predicted surface helix of viral protein 1 (VP1) between amino acid residues 144 and 159. A hexadecapeptide covering this sequence elicits high titers of antibodies that specifically recognize and neutralize FMDV. The high quality of the immune response is attributed to a particularly stable conformation of the antigenic amino acid sequence, which is most likely an alpha-helix.

Amino Acid Sequence↗
PubMed1982

Development of intrahepatic cholestasis by alpha-naphthylisothiocyanate in rats.

Development of intrahepatic cholestasis induced by alpha-naphthylisothiocyanate was studied in rats. At various times after alpha-naphthylisothiocyanate application, livers were isolated from treated rats and perfused hemoglobin-free to assess cholestatic parameters. Unstimulated bile flow was found to only slightly decrease up to 10 h after alpha-naphthylisothiocyanate administration. In contrast, secretion into bile of sulfobromophthalein and taurocholate declined markedly between 4 and 7 h as their concentrations in the perfusate increased, and stimulation of bile flow by taurocholate decreased. The permeability of the bile-to-perfusate barrier to [14C]sucrose and [32P]orthophosphate increased in parallel with the changes in sulfobromophthalein and taurocholate distributions. This correlation of changes in the distribution of cholephilic substances with biliary accessibility for extracellular markers suggests that, in alpha-naphthylisothiocyanate-induced cholestasis, increased leakage of tight junctions may contribute to regurgitation of bile constituents into the vascular system.

1-Naphthylisothiocyanate↗
PubMed1980

Viability control and special properties of isolated rat hepatocytes.

The need for quick viability tests is stressed. Aas these should achieve more than statically categorizing dead or non-dead cells, several procedures are suggested that picture the energetic state of the cells. The almost classical criterion of this category, namely stimulation of respiration by succinate, must be questioned on the basis of the present results. It is shown, that restricted respiration by succinate is not due to limited permeability of the plasma membrane, but to competition by endogenous substrates for uptake into mitochondria. Distribution equilibria for succinate appear to be according to (delta pH)2 with regard to cytoplasm. They are attained within 5-20 s or faster. Uptake is in part regulated by the surface charge density. Permeability changes caused by effectors of surface charge, such as amphiphilic ions, are examplified for succinate, chloride, phosphate, Na+, K+, and Ca2+. Such changes repeatedly also occur after pulses of BSP. They are counterregulated by the cell within a minute in a manner dependent on BSP concentration and the state of the cells. During the preincubation phase, that is the time of readaptation after transfer of cells from 0 degree C to higher temperature, a special labile state transiently occurs, where cyclic permeability changes for Ca2+, Na+, K+ can be caused by substrate addition, especially succinate, and/or ATP. The extent of these changes and their sequence again depend on the energetic state of the cells. In a probably narrow energetic window a sequence of cation movements reminding of that after depolarization of an excitable cell, is observed. Manipulation of the Na+/K+-ratio by variation of preincubation time and by ouabain shows that this is not simply the denominator for reversible calcium uptake. As the surface charge appears to reflect the energetic state, ANS fluorescence is applied to monitor the state of the plasma membrane, though difficulties arising from a slow ANS permeation are not yet solved.

Anilino Naphthalenesulfonates↗
PubMed1979

Transient 45Ca uptake and release in isolated rat-liver cells during recovery from deenergized states.

1. Aerobic incubation of isolated rat liver cells--after dilution from the anaerobic stock suspension--transiently brings about a state, during which a reversible calcium uptake can be observed on addition of a respiratory substrate. Uptake varies greatly and can reach more than 50 nmol/mg protein, but declines to zero on prolonged preincubation, especially at higher temperature. Repeated additions of succinate or 3-hydroxybutyrate evoke new calcium transients. If ATP is simultaneously added, if greatly potentiates succinate-initiated reversible uptake. 2. If rotenone is present during the preincubation phase, calcium transients are strongly enhanced. Uptake is blocked by uncouplers and respiratory inhibitors, indicating the involvement of mitochondria. 3. Calcium uptake is not accompanied by increased oxygen consumption. The actual respiration cannot account sufficiently for the energy need of calcium uptake. Participation of cytoplasmic ATP is likely, as inhibitors of adenine nucleotide translocase affect uptake. 4. Lanthanum enhances calcium uptake in contrast to its action on mitochondria. 5. Pulse-labeling experiments indicate that the calcium taken up is removed from a rapidly exchangeable calcium pool by withdrawal into the mitochondria as a deep compartment. 6. Calcium uptake is accelerated either by increasing the phosphate level or by high temperature. It is prolonged by low temperature, high pH or high ATP concentration. Calcium release accelerates with increasing temperature, decreasing pH and a further rise in phosphate concentration. 7. The dependency on phosphate and temperature reveals a delicately poised equilibrium of uptake and release. At ambient temperature, phosphate increases uptake up to a concentration of 0.5 mM. Higher concentrations accelerate both uptake and release. At lower temperature, the accelerating effect on uptake predominates. A temperature shift during incubation results in adaptation of the calcium equilibrium to the new temperature, i.e. release of calcium at high temperature, uptake at low temperature. 8. Oxidizing metabolites inhibit succinate-stimulated calcium uptake and promote release of previously accumulated calcium. An increased sensitivity to phosphate is established. 9. With respect to isolated mitochondria, isolated liver cells appear to be a more realistic model for studying the physiological mechanism of mitochondrial calcium release, since compartmental constraints and regulations are maintained.

Aerobiosis↗