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N Kraus-Friedmann

Publications and source records attributed to N Kraus-Friedmann.

At least 19 recordsLinked to original sources
PubMed1992

Demonstration of ryanodine-induced metabolic effects in rat liver.

The effects of ryanodine, a plant alkaloid which alters Ca2+ sequestration in the liver, on O2 uptake and gluconeogenesis were measured. Ryanodine administration to perfused rat liver resulted in the stimulation of O2 uptake and of gluconeogenesis. Because ryanodine does not affect directly mitochondrial respiration, its stimulatory effect on O2 uptake in the whole cell is likely to be secondary to the increased cytosolic free Ca2+ levels.

Animals↗
PubMed1992

Different localization of inositol 1,4,5-trisphosphate and ryanodine binding sites in rat liver.

The distribution of inositol 1,4,5-trisphosphate and ryanodine binding sites between plasma membrane, microsomal, and mitochondrial fractions of rat liver were compared. IP3 bound mostly to the plasma membrane fraction (Kd = 6 nM; Bmax = 802 fmol/mg protein). Some IP3 binding sites were also present in the microsomal and mitochondrial fractions (Kd = 2.5 and 2.9 nM; Bmax = 35 and 23 fmol/mg protein respectively). The possibility that these binding sites are due to contamination of the fractions with plasma membrane cannot be excluded. Binding of IP3 to the plasma membrane was inhibited by heparin but not by either caffeine or tetracaine. High-affinity ryanodine binding sites were present mostly in the microsomal fraction (Kd = 13 nM; Bmax = 301 fmol/mg protein). Lower affinity binding sites were also found to be present in the mitochondrial and plasma membrane fractions. Binding of ryanodine to the microsomal fraction was inhibited by both caffeine and tetracaine but not by heparin. These data demonstrate that IP3 and ryanodine binding sites are present in different cellular compartments in the liver. These differences in the localization of the binding sites might be indicative of their functional differences.

5'-Nucleotidase↗
PubMed1991

Effects of ryanodine on calcium sequestration in the rat liver.

Ryanodine, a highly toxic alkaloid known to react specifically with the Ca2+ release channels in sarcoplasmic reticulum (SR), was employed to study Ca2+ sequestration in the liver. Ryanodine at a 200 microM concentration increased cytosolic free Ca2+ levels and phosphorylase a activity in isolated hepatocytes. These effects may involve microsomal Ca2+ sequestration, because ryanodine, in the presence of inhibitors of mitochondrial Ca2+ uptake, at concentrations of 1 nM, 1 microM, 50 microM and 100 microM decreased 45Ca2+ retention in permeabilized hepatocytes. This inhibition of Ca2+ retention by ryanodine was not due to inhibition of the microsomal Ca(2+)-ATPase. Dantrolene, a compound shown previously to inhibit ryanodine binding in the liver, also decreased 45Ca2+ retention in permeabilized hepatocytes, and activated phosphorylase a. These results show that ryanodine administration alters calcium sequestration in liver. The possibility of the existence of a ryanodine-sensitive Ca(2+)-release channel in liver is discussed.

Animals↗
PubMed1991

Characterization of high-affinity ryanodine-binding sites of rat liver endoplasmic reticulum. Differences between liver and skeletal muscle.

In this study, the binding of [3H]ryanodine to liver microsomal subfractions was investigated. The specific binding of [3H]ryanodine, as determined both by vacuum filtration and by ultracentrifugation, is to a single class of high-affinity binding sites with a Kd of 10 +/- 2.5 nM and density of 500 +/- 100 and 1200 +/- 200 fmol/mg of protein by the filtration and centrifugation methods respectively. [3H]Ryanodine binding reached equilibrium in about 1 min and 2 min at 36 degrees C and 24 degrees C respectively, and the half-time of dissociation at 37 degrees C was approx. 15 s. The binding of [3H]ryanodine is Ca(2+)-independent: it is slightly stimulated by NaCl, Mg2+, ATP and InsP3 but strongly inhibited by caffeine, diltiazem and sodium dantrolene. Thus the binding of ryanodine to endoplasmic reticulum membranes shares some of the characteristics of its binding to the sarcoplasmic reticulum but also differs from it in several important properties, such as its Ca(2+)-independence, its rapid association and dissociation, and its inhibition by caffeine. The structural similarities between the skeletal muscle and liver binding sites were further explored by employing in vitro DNA amplification techniques, using the known sequence of the skeletal muscle receptor as reference point. The data obtained with this method indicate that the liver does not process mRNA for the skeletal muscle ryanodine receptor.

Animals↗
PubMed1990

Distinct ryanodine- and inositol 1,4,5-trisphosphate-binding sites in hepatic microsomes.

A light hepatic microsomal preparation was fractionated by sucrose-density centrifugation into one rough, one intermediate and two smooth fractions. The four fractions were characterized with respect to parameters relevant to Ca2+ sequestration. Ca2(+)-ATPase activity was similar in the rough, intermediate and smooth I fractions, but lower in the smooth II fraction. Ca2+ accumulation was the highest in the smooth I and intermediate fractions. On the other hand, Ca2+ efflux from the rough fraction was several-fold faster than from the smooth I fraction. All four subfractions exhibited specific binding sites for inositol 1,4,5-trisphosphate (IP3) and ryanodine; however, the receptors were especially enriched in the smooth I fraction. The total binding sites for ryanodine in that fraction exceeded the number of binding sites for IP3 by about 10-fold. The two receptors responded differently to pharmacological agents; caffeine and dantrolene strongly inhibited ryanodine binding but not IP3 binding, whereas heparin inhibited IP3 binding only. Thus the two receptors are distinct entities. The four fractions also showed distinct gel electrophoretic patterns. The use of two different SDS/polyacrylamide-gel gradients and two protein-staining methods revealed major differences in the distribution of the bands corresponding to Mr values of (x 10(-3) 380, 320, 260, 170, 90, 29 and 21. These proteins were enriched in the smooth fraction. The results indicate that the smooth I fraction might have special importance in stimulus-evoked Ca2(+)-release processes.

Animals↗
PubMed1990

Effects of heavy metal on rat liver microsomal Ca2(+)-ATPase and Ca2+ sequestering. Relation to SH groups.

In isolated hepatic microsomal vesicles the heavy metals Cd2+, Cu2+, and Zn2+ inhibit Ca2+ uptake and evoke a prompt efflux of Ca2+ from preloaded vesicles in a dose-dependent manner. N-Ethylmaleimide also inhibits Ca2+ uptake and causes Ca2+ release, but it is less effective in these respects than the heavy metals. Measurement of mannose-6-phosphatase activity indicate that the heavy metal-induced Ca2+ efflux is not caused by a general increase in membrane permeability. Heavy metals also inhibit the Ca2(+)-ATPase activity and the formation of the phosphorylated intermediate of the enzyme. In contrast, the sulfhydryl modifying reagent, N-ethylmaleimide inhibits the Ca2(+)-ATPase activity while it has a relatively small effect on Ca2+ release. Thus, the effects of these agents on Ca2+ sequestering and Ca2(+)-ATPase activity are not strictly proportional. The sulfhydryl group reducing agent dithiothreitol protects the microsomes from the effects of heavy metals, while glutathione is less protective. Addition of vanadate to vesicles, at a concentration which completely blocked the activity of the Ca2(+)-ATPase, resulted in a small and slow release of the accumulated Ca2+. Subsequent additions of heavy metals evoked a massive Ca2+ release. Thus, the effects of heavy metals on Ca2+ efflux cannot be due entirely to their inhibition of the Ca2+ pump. The heavy metal-induced Ca2+ efflux is not inhibited either by ruthenium red or tetracaine.

Animals↗
PubMed1990

Purification of the microsomal Ca2(+)-ATPase from rat liver.

The Ca2(+)-ATPase from rat liver microsomes has been solubilized in Triton X-100 and purified to homogeneity by ficoll-sucrose treatment, column chromatography with agarose-hexane adenosine 5'-triphosphate Type 2, and high pressure liquid chromatography (HPLC). The purified enzyme obtained by this sequential procedure exhibited a 183-fold increase in specific activity. After ficoll-sucrose treatment, the activity of the Ca2(+)-ATPase was stable for at least two weeks when stored at -70 degrees C. In SDS-polyacrylamide gels, several fractions from HPLC chromatography showed a single band at a position corresponding to a molecular weight of about 107 kDa. This value is consistent with the molecular weight of the phosphoenzyme intermediate of endoplasmic reticulum (ER) Ca2(+)-ATPase. Further characterization of the ER Ca2(+)-ATPase was performed by western immunoblots. Antiserum raised against the 100-kDa sarcoplasmic reticulum (SR) Ca2(+)-ATPase cross-reacted with the purified Ca2(+)-ATPase from rat liver ER membranes.

Animals↗
PubMed1990

Mechanism of action of GTP in the induction of Ca2+ release from hepatic microsomes.

The mechanism by which GTP induces Ca2+ release from Ca2(+)-preloaded rat hepatic microsomes was studied. In the same concentration range as that for Ca2+ release, GTP inhibited the initial rate of ATP-driven Ca2+ uptake. It also inhibited the formation by ATP of the phosphorylated intermediate of Ca2(+)-ATPase, which had previously been identified by us as a 97-116 kDa protein (Fleschner, C.R., et al. (1985) Biochem. J. 226, 839). Vanadate, an inhibitor of Ca2(+)-ATPase, also caused Ca2+ release in a similar fashion, but its effect was not additive to that of GTP. Although the non-metabolizable GTP analogues, GMPPNP and GTP gamma S, did not cause Ca2+ release by themselves, GTP gamma S completely and GMPPNP partially blocked the effect of GTP. Pretreatment of vesicles with either cholera or pertussis toxin did not alter the responsiveness to GTP. These results indicate that GTP inhibits microsomal Ca2(+)-ATPase, independently of the Gs and Gi proteins. Because a decrease in Ca2+ uptake results in a net increase in Ca+ release, this effect of GTP seems to account, at least partially, for the GTP-induced Ca2+ release from microsomes.

Animals↗
PubMed1990

Hormonal stimulation of Ca2+ release from the perfused liver: effects of uncoupler.

Administration of vasopressin and glucagon evokes a transient release of Ca2+ from perfused livers. The Ca2+ is released from a pool that is depletable by the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Therefore, the mechanism of the FCCP-stimulated Ca2+ release was examined. The FCCP-stimulated Ca2+ release was associated with a decrease in ATP levels. In the presence of oligomycin, which blocked the FCCP-induced rapid ATP breakdown, FCCP did not release Ca2+ though it still stimulated respiration. The possibility that FCCP might indirectly cause a release of Ca2+ by lowering hepatic ATP was examined at two levels of organization: 1) in the whole organ, by perfusing livers with fructose, a compound that was shown previously to drastically lower ATP in the liver, and 2) in isolated microsomal vesicles by depleting ATP with glucose and hexokinase. Fructose evoked Ca2+ release from the perfused liver. Similarly, depletion of ATP by the addition of glucose and hexokinase evoked a rapid release of the accumulated Ca2+ from microsomal vesicles probably by the inhibition of the Ca2(+)-ATPase. These results demonstrate that the major mechanism by which FCCP releases Ca2+ in intact cells is by lowering ATP levels.

Animals↗
PubMed1989

Demonstration of adenylate-kinase activity in hepatic microsomes. Relevance to Ca2+ uptake.

It is demonstrated that the hepatic microsomal fraction contains significant adenylate-kinase activity. This explains a paradoxical ADP-stimulated Ca2+ uptake into microsomal vesicles which is inhibited when adenylate kinase is inhibited. The presence of adenylate kinase in the microsomal fraction helps to prevent sudden drops in ATP level, and thus has a stabilizing effect on the many ATP-dependent reactions carried out in this subcellular compartment.

Adenosine Diphosphate↗
PubMed1988

Inhibitor of anion transport, DIDS, releases Ca2+ from hepatic microsomes.

Addition of 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) to Ca2+ loaded hepatic microsomal vesicles evoked a dose-dependent release of the accumulated Ca2+. Ca2+ uptake was also inhibited. The effects of DIDS do not seem to be due to the inhibitions of either Cl- or proton fluxes. The results indicate that DIDS inhibits Ca2+ uptake and releases Ca2+ by inhibiting the Ca2+-ATPase and the formation of the phosphorylated intermediate of the enzyme, and that it might interact with a specific site on the vesicle which is involved in the translocation of Ca2+ across the microsomal and mitochondrial membranes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

Demonstration of the presence of G-proteins in hepatic microsomal fraction.

The presence of G-proteins in isolated hepatic microsomal vesicles is demonstrated. The G-proteins were identified by their capacity to be ADP-ribosylated by cholera and pertussis toxins. Cholera toxin identified 42 and 45 kDa proteins, corresponding to alpha s-1 and alpha s-2, respectively. Pertussis toxin identified a 40 kDa protein corresponding to alpha i. The microsomal G-proteins are identical to the corresponding G proteins of the plasma membrane, but are present in different proportions; the microsomes have considerably less alpha s proteins than the plasma membrane.

Adenosine Diphosphate Ribose↗
PubMed1987

Inhibition of rat liver microsomal Ca2+-ATPase by fluorescein-5'-isothiocyanate.

Rat liver microsomal fraction was incubated at pH 8.8 with fluorescein-5'-isothiocyanate in a Tris-buffered sucrose medium. This treatment completely inhibited ATP-dependent Ca2+ transport, Ca2+-ATPase activity, and Ca2+-ATPase phosphoenzyme intermediate formation. Inhibition of Ca2+ transport and phosphoenzyme intermediate formation by fluorescein-5'-isothiocyanate was partially prevented by including ATP in the treatment medium. These data taken together are consistent with the proposal that fluorescein-5'-isothiocyanate binds the Ca2+-ATPase ATP-binding site, suggesting the presence of a lysine residue in this domain. Fluorescein-5'-isothiocyanate labeling of microsomal proteins had no measurable effect on the basal, Mg2+-ATPase activity. Using fluorescein-5'-isothiocyanate-labeled microsomal fraction, we demonstrated that the Mg2+-ATPase activity was inhibited by Ca2+.

Adenosine Triphosphate↗
PubMed1987

A comparison between 45Ca2+ and atomic absorption in calcium flux determinations in perfused rat liver.

Calcium efflux from perfused rat liver following the administration of Ca2+ releasing agents was measured with two methods, 45Ca2+ labeling and atomic absorption. The values obtained with atomic absorption were usually higher than the values obtained with 45Ca2+. These indicate that the intracellular Ca2+ did not equilibrate with the perfusate ca2+ during the 90-minute labeling period. A similar conclusion was reached by measuring the liver 45Ca2+ and 40Ca2+ content. In addition, the types of albumin added to the perfusate influenced the amounts of Ca2+ released.

Animals↗
PubMed1986

Glucagon-stimulated respiration and intracellular Ca2+.

The effects of extra- and intracellular Ca2+ on glucagon-stimulated respiration were examined in perfused rat liver. Glucagon increased the uptake of O2 to a significantly greater extent in Ca2+-containing perfusate than in Ca2+-free perfusate. If, however, the livers were perfused first with Ca2+-containing perfusate for 60 min in order to load the hormone-sensitive Ca2+ pool(s) and subsequently with Ca2+-free perfusate, glucagon was able to stimulate O2 uptake to the same extent in Ca2+-free, as in Ca2+-containing perfusate. These experiments support previous observations of a connection between Ca2+ and the hormonal stimulation of respiration, but indicate a role for intracellular, rather than extracellular, Ca2+ in the process.

Animals↗
PubMed1986

The effect of Mg2+ on hepatic microsomal Ca2+ and Sr2+ transport.

The ATP-dependent uptake of Ca2+ by rat liver microsomal fraction is dependent upon Mg2+. Studies of the Mg2+ requirement of the underlying microsomal Ca2+-ATPase have been hampered by the presence of a large basal Mg2+-ATPase activity. We have examined the effect of various Mg2+ concentrations on Mg2+-ATPase activity, Ca2+ uptake, Ca2+-ATPase activity and microsomal phosphoprotein formation. Both Mg2+-ATPase activity and Ca2+ uptake were markedly stimulated by increasing Mg2+ concentration. However, the Ca2+-ATPase activity, measured concomitantly with Ca2+ uptake, was apparently unaffected by changes in the Mg2+ concentration. In order to examine the apparent paradox of Mg2+ stimulation of Ca2+ uptake but not of Ca2+-ATPase activity, we examined the formation of the Ca2+-ATPase phosphoenzyme intermediate and formation of a Mg2+-dependent phosphoprotein, which we have proposed to be an attribute of the Mg2+-ATPase activity. We found that Ca2+ apparently inhibited formation of the Mg2+-dependent phosphoprotein both in the absence and presence of exogenous Mg2+. This suggests that Ca2+ may inhibit (at least partially) the Mg2+-ATPase activity. However, inclusion of the Ca2+ inhibition of Mg2+-ATPase activity in the calculation of Ca2+-ATPase activity reveals that this effect is insufficient to totally account for the stimulation of Ca2+ uptake by Mg2+. This suggests that Mg2+, in addition to stimulation of Ca2+-ATPase activity, may have a direct stimulatory effect on Ca2+ uptake in an as yet undefined fashion. In an effort to further examine the effect of Mg2+ on the microsomal Ca2+ transport system of rat liver, the interaction of this system with Sr2+ was examined. Sr2+ was sequestered into an A23187-releasable space in an ATP-dependent manner by rat liver microsomal fraction. The uptake of Sr2+ was similar to that of Ca2+ in terms of both rate and extent. A Sr2+-dependent ATPase activity was associated with the Sr2+ uptake. Sr2+ promoted formation of a phosphoprotein which was hydroxylamine-labile and base-labile. This phosphoprotein was indistinguishable from the Ca2+-dependent ATPase phosphoenzyme intermediate. Sr2+ uptake was markedly stimulated by exogenous Mg2+, but the Sr2+-dependent ATPase activity was unaffected by increasing Mg2+ concentrations. Sr2+ uptake and Sr2+-dependent ATPase activity were concomitantly inhibited by sodium vanadate. In contrast to Ca2+, Sr2+ had no effect on Mg2+-dependent phosphoprotein formation. Taken together, these data indicate that Mg2+ stimulated Ca2+ and Sr2+ transport by increasing the Ca2+ (Sr2+)/ATP ratio.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗
PubMed1986

Effects of glucagon and vasopressin on hepatic Ca2+ release.

The effects of physiological levels of glucagon on Ca2+ efflux were examined in the perfused rat liver. Two methods were used to estimate Ca2+ efflux: prior labeling of the calcium pools with 45Ca2+ and measurement of perfusate Ca2+ with atomic absorption. According to both methods, glucagon administration at the physiological level evoked Ca2+ release. The released Ca2+ originated mostly from a carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)-depletable pool and also from an FCCP-insensitive pool from which Ca2+ could be released with A23187. Maximally effective doses of glucagon and vasopressin had no additive effect on Ca2+ release. Prior administration of vasopressin resulted in markedly reduced Ca2+ release by glucagon. These results indicate that glucagon releases Ca2+ from the same pool that vasopressin does.

Animals↗