Naomi Kraus-Friedmann

The role of intracellular Ca2+ in the regulation of gluconeogenesis

A hypothesis for the hormonal regulation of gluconeogenesis, in which increases in cytosolic free-Ca2+ levels ([Ca2+]i) play a major role, is presented. This hypothesis is based on the observation that gluconeogenic hormones evoke a common pattern of Ca2+ redistribution, resulting in increases in [Ca2+]i. Current concepts of hormonally evoked Ca2+ fluxes are presented and discussed. It is suggested that the increase in [Ca2+]i is functionally linked to stimulation of gluconeogenesis. The stimulation of gluconeogenesis is accomplished in two ways: (1) by increasing the activities of the Krebs cycle and the electron-transfer chain, thereby supplying adenosine triphosphates (ATP) and reducing equivalents to the process; and (2) by stimulating the activities of key gluconeogenic enzymes, such as pyruvate carboxylase. The hypothesis presents a conceptual framework that ties together two interrelated manifestations of hormone action: signal transduction and metabolism.

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.

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.

Rapid stimulation of Na+,K+-ATPase by glucagon, epinephrine, vasopressin and cAMP in perfused rat liver

Hormones which promote glucose production by the liver have a pronounced effect on ion distribution in this organ. The redistribution of ions is prompt and involves several ions. Glucagon [ 1,2], epinephrine [ 1,2], norepinephrine [ 11, phenyl epinephrine [3,4] and vasopressin [ 51 affect K’ movement across the plasma membrane. A similar response is evoked by CAMP [ 1,2,6]. The effect of hormonal stimuli on K’ flux is biphasic, consisting of an initial uptake of K’, followed by release when the liver plasma membrane hyperpolarizes [7-91. The efflux of K’ and the associated hyperpolarization have been attributed to an increase in the K+ permeability of the liver cell membrane [7,8]. The uptake phase has been less studied and conflicting results on hormonal effects on Na’ fluxes render the mechanism responsible for it less clear. Gluconeogenic hormones have been reported to cause Na’ release [lo], uptake followed by release [ 11, uptake [7] or to have no effect on Na’ movements. Since K’ uptake occurs against a concentration gradient, we undertook to determine whether gluconeogenic hormones promote K+ uptake by activating the membrane-bound Na’,K’-ATPase. Here, gluconeogenie hormones are shown to stimulate the enzyme. Insulin, which antagonizes the effect of the gluconeogenie hormones [6], had no similar effect, but prevented the stimulation by glucagon.

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.

Reduction of ryanodine binding and cytosolic Ca2+ levels in liver by the immunosuppressant FK506

The mechanism of action of the immunosuppressant FK506 in the liver was studied. The hypothesis was tested that FK506 exerts its effect in the liver by interacting with the ryanodine-binding Ca2+ release channel. Two types of experiments were carried out: (1) [3H]-ryanodine binding studies with isolated microsomal fractions, and (2) cytosolic-free Ca2+ ([Ca2+]i) measurements with the intracellular Ca(2+)-indicator fura-2. The inclusion of FK506 in the incubation medium significantly decreased the binding of [3H]-ryanodine to liver microsomes. The Bmax of binding in control experiments was 405 fmol/mg protein; the presence of FK506 decreased the Bmax to 157 fmol/mg protein. Measurements of [Ca2+]i in the presence and absence of FK506 showed a decrease in [Ca2+]i in the presence of FK506. The data support the notion that FK506 interacts with the ryanodine binding Ca2+ channel in the liver and suggest a critical role for the ryanodine-binding Ca2+ channel in the hepatic responses to FK506. The interaction between FKBP-12 (FK506 binding protein) and the ryanodine-binding Ca2+ channel may be an essential link in the chain of events by which FK506 alters Ca(2+)-dependent cellular processes.

Purification of the microsomal Ca2+-ATPase from rat liver

SummaryThe Ca2+-ATPase from rat liver microsomes has been solubilized in Triton X-100 and purified to homogeneity by ficollsucrose 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°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.

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.

The hepatic microsomal Ca2+ sequestering system.

The hepatic microsomal Ca2+ sequestering system is analogous to that of sarcoplasmic reticulum and fulfills a similar role to it; namely, regulation of cytoplasmic Ca2+ concentration. It does so by taking up Ca2+ in an ATP dependent manner and releases it by either the reversal of the uptake process and/or by a different, unknown mechanism.