ABSTRACT
ATP-dependent potassium (K ATP) channels occupy a key position in the control of insulin release from the pancreatic beta cell since they couple cell polarity to metabolism. These channels close when more ATP is produced via glucose metabolism. They are also controlled by sulfonylureas, a class of drugs used in type 2 diabetic patients for triggering insulin secretion from beta cells that have lost part of their sensitivity to glucose. We have demonstrated the existence of endogenous counterparts to sulfonylureas which we have called 'endosulfines.' In this review, we describe the discovery, isolation, cloning, and biological features of the high-molecular-mass form, alpha-endosulfine, and discuss its possible role in the physiology of the beta cell as well as in pathology.
Subject(s)
Drosophila Proteins , Insulin/metabolism , Peptides/physiology , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , Glucose/metabolism , Humans , Insulin Secretion , Intercellular Signaling Peptides and Proteins , Models, Biological , Molecular Sequence Data , Peptides/chemistry , Peptides/genetics , Potassium Channels/physiology , Sequence Homology, Amino AcidABSTRACT
Glycogen synthase (EC 2.4.1.11) activity was studied in cell extracts from wild-type Chinese hamster ovary (CHO) cells and three mutants resistant to cyclic AMP effects on cell shape and cell growth. Based on the capacity of crude extracts to phosphorylate exogenous histone, two of the mutants appeared to have altered cyclic AMP-dependent protein kinase (EC 2.7.1.37) and one of them had apparently normal amounts of kinase activity. Glycogen synthase activity was present in comparable amounts in wild-type and all three mutant strains in a presumably inactive phosphorylated form since activity was virtually completely dependent upon the presence of glucose 6-phosphate. The enzyme could be partially dephosphorylated by endogenous phosphatases and rephosphorylated by exogenous cyclic AMP-dependent protein kinase. Attempts to find culture conditions (e.g. glucose starvation) or cell treatment (e.g. insulin) which might activate glycogen synthase in intact cells were unsuccessful. since glycogen synthase activity present in CHO cells was independent of the level of cyclic AMP-dependent kinase, we conclude that cyclic AMP-dependent protein kinase does not play a critical role in regulating the state of phosphorylation of the synthase.
Subject(s)
Cyclic AMP/pharmacology , Glycogen Synthase/metabolism , Protein Kinases/metabolism , Animals , Cell Line , Cricetinae , Cricetulus , Female , Glucosephosphate Dehydrogenase/pharmacology , Histones/metabolism , Mutation , Ovary/enzymology , Phenotype , PhosphorylationABSTRACT
The effect of 8-bromo cyclic adenosine 3':5'-monophosphate (8-Br-cAMP) on sugar and amino acid transport was investigated in wild-type Chinese hamster ovary (CHO) cells and in mutants selected for resistance to cAMP inhibition of cell growth. In wild type cells, both 3-O-methyl-D-glucose and alpha-aminoisobutyric acid transport were decreased in cells treated for 24 h with 8-Br-cAMP; kinetic analysis indicated that a decrease in Vmax, without a significant change in Km, accounted for the lower transport capacity of 8-Br-cAMP treated cells. Among the different transport systems contributing to amino acid entry, "alanine" preferring transport system (system A) appeared to be specifically affected. The sensitivity of transport processes to 8-Br-cAMP was tested in three cAMP-resistant cell lines. When tested for their capacity to phosphorylate histones in crude extracts, one strain had apparently normal amounts of protein kinase activity, one strain had a decreased enzyme sensitivity to cAMP, and one strain had little or no enzyme activity. In all three mutants, no effect of 8-Br-cAMP on 3-O-methyl glucose and alpha-aminoisobutyric acid transport could be observed, regardless of the level of cAMP-dependent protein kinase activity. These data do not indicate whether the effect of cAMP on nutrient transport in CHO cells is the cause or consequence of growth inhibition. However, they support the conclusion that, in CHO cells, the presence of a normally functioning cAMP-dependent protein kinase appears to be necessary but may not be sufficient to observe the effects of cAMP on nutrient transport as well as cell shape and cell growth.
Subject(s)
Amino Acids/metabolism , Cyclic AMP/analogs & derivatives , Cyclic AMP/pharmacology , Deoxy Sugars/metabolism , Deoxyglucose/metabolism , Methylglucosides/metabolism , Methylglycosides/metabolism , 8-Bromo Cyclic Adenosine Monophosphate , Aminoisobutyric Acids/metabolism , Animals , Biological Transport/drug effects , Cell Line , Cricetinae , Cricetulus , Drug Resistance , Female , Kinetics , Ovary , Protein Kinases/metabolismABSTRACT
From mutagenized Chinese hamster ovary (CHO) cells we have isolated, in a single step, 11 independent mutants resistant to the growth-inhibitory effects of 8-Br-cyclic AMP, cholera toxin, and methylisobutylxanthine. Two major classes and several subclasses of mutants were obtained. Mutants from all classes have a normal doubling time. None of the mutants respond to cyclic AMP treatment with increased flattening and elongation as do the parental cells. Members of the first class have an altered protein kinase activity which has either an increased Ka for cyclic AMP or an absent response to cyclic AMP. Most of those mutations which result in a protein kinase with increased Ka for cyclic AMP (6/11) are dominant in somatic cell hybrids. Those mutations which result in a protein kinase with little or no response to cyclic AMP (3/11) are recessive. Members of the second major class (2/11) have normal levels of basal and cyclic AMP-dependent protein kinase activity. One is recessive and one is dominant by genetic tests. The basis for the defect in this second class of mutants has not been determined.
Subject(s)
Cell Line , Cyclic AMP/analogs & derivatives , Genes, Dominant , Genes, Recessive , 8-Bromo Cyclic Adenosine Monophosphate , Animals , Cell Division/drug effects , Cell Membrane/drug effects , Cricetinae , Cyclic AMP/pharmacology , Drug Resistance , Female , Hybrid Cells , Mutation , Ovary , Protein Kinases/metabolismABSTRACT
By quantitative electron microscopic autoradiographic technique, we have previously shown that 125I-insulin initially localizes to the plasma membrane of isolated rat hepatocytes and is subsequently internalized in a limited region of the peripheral cytoplasm. In the present study, we have shown that when cells are incubated at 20 degrees C, steady state binding is reached by 60 minutes and maintained up until 120 minutes of incubation while at 37 degrees C steady state binding is reached by 10 minutes and maintained for 30 minutes. Under both of these conditions, internalization of the labelled material occurs as a constant function of the binding. These data suggest that under normal conditions the binding of the ligand is an important rate limiting determinant of the internalization process.
Subject(s)
Insulin/metabolism , Liver/metabolism , Receptor, Insulin/metabolism , Animals , Biological Transport , Cell Membrane/metabolism , Cell Membrane/ultrastructure , In Vitro Techniques , Kinetics , Liver/ultrastructure , RatsSubject(s)
Aminoisobutyric Acids/metabolism , Insulin/pharmacology , Liver/metabolism , Animals , Biological Transport/drug effects , Cyclic AMP/pharmacology , Glucagon/pharmacology , In Vitro Techniques , Insulin/metabolism , Kinetics , Liver/drug effects , Male , Methylamines/pharmacology , Rats , Receptor, Insulin/metabolismABSTRACT
Insulin labeled with iodine-125 binds to receptors on isolated rat hepatocytes. At low temperatures initial binding is restricted to the plasma membrane as detected by direct quantitative autoradiographic analysis with the electron microscope. With increasing time and temperature of incubation there is a systematic and progressive translocation of autoradiographic grains to a highly limited area of the cell periphery representing no more than 15% of the radius of the cell.
