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J C Hutton

Publications and source records attributed to J C Hutton.

At least 109 records · Page 6Linked to original sources
PubMed1984

Phorbol ester stimulation of insulin release and secretory-granule protein phosphorylation in a transplantable rat insulinoma.

The effects of tumour-promoting phorbol esters on protein-phosphorylation reactions and secretion in rat insulinoma tissue were investigated with the objective of assessing the possible role of Ca2+- and phospholipid-dependent protein kinases (protein kinase C) in insulin release. 4 beta-Phorbol 12-myristate 13-acetate (TPA) was a potent secretagogue at concentrations above 0.1 microM. TPA-induced release was inhibited by adrenaline or omission of Ca2+ from the extracellular medium and was augmented by theophylline. These findings suggested that TPA activated an exocytotic process. TPA enhanced the Ca2+- and phospholipid-dependent phosphorylation of histone III-S by a soluble protein fraction of the tissue. Endogenous phosphorylation reactions involving soluble and secretory-granule membrane proteins were also stimulated by TPA in tissue homogenates and reconstituted subcellular fractions. Histone phosphorylation and the granule-protein phosphorylation reactions showed similar concentration-dependencies for activation by both Ca2+ and TPA, thus indicating that the same enzyme was involved. It is concluded that the phosphorylation of cytosolic and membrane protein substrates by protein kinase C may be important in the stimulus-secretion coupling mechanism of insulin release.

Adenoma, Islet Cell↗
PubMed1984

Secretory granules.

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Adenosine Triphosphatases↗
PubMed1984

Cathepsin B-related proteases in the insulin secretory granule.

The distribution of proteases potentially reactive with peptide sequences containing pairs of basic amino acids or single basic amino acids was studied in subcellular fractions of a transplantable rat insulinoma using the affinity probes 125I-Tyr-Ala-Lys- ArgCH2Cl and 125I-Tyr-Ala-norleucine- ArgCH2Cl . Both probes labeled predominantly proteins of Mr = 39,000, 31,500, and 25,000. The Mr = 25,000 component appeared to be of lysosomal origin, while the Mr = 39,000 and 31,500 proteins were present in both the lysosomes and insulin granules. The Mr = 39,000 and 31,500 proteins were identified as precursor/product forms of the cysteine protease cathepsin B, while assays performed with fluorigenic peptide substrates suggested that the Mr = 25,000 protein was probably cathepsin L and/or H. The greater reactivity of the Mr = 39,000 form with the dibasic probe suggests that the relative proportions of the Mr = 39,000 and 31,500 forms of cathepsin B in different organelles may determine the extent to which the enzyme expresses activity as a specific (prohormone processing) endopeptidase or a more general (degradative) peptidase.

Adenoma, Islet Cell↗
PubMed1984

Involvement of protein kinase C in the phosphorylation of an insulin-granule membrane protein.

The involvement of protein kinase C in the Ca2+-dependent phosphorylation of a 29 000-Mr insulin-granule membrane protein prepared from a rat insulinoma was investigated. Protein kinase C activity towards exogenous lysine-rich histone was detected in a cytosolic fraction prepared from an insulinoma homogenate in the presence of EGTA. This activity bound reversibly to insulin granules in a Ca2+-dependent manner. Phosphatidylserine liposomes removed both protein kinase C activity and the 29 000-Mr protein-phosphorylating activity from the cytosolic fraction in a Ca2+-dependent fashion. Protein kinase C activity and the enzymic activity responsible for the phosphorylation of the 29 000-Mr granule protein behaved identically on sucrose-density-gradient centrifugation, ion-exchange chromatography, (NH4)2SO4 fractionation and gel filtration of the cytosolic fraction. These results are consistent with protein kinase C being the enzyme responsible for the phosphorylation of the 29 000-Mr insulin-granule membrane protein.

Adenoma, Islet Cell↗
PubMed1984

Pancreatic B-cells secrete a range of novel peptides besides insulin.

Insulin secretion by a transplantable rat islet B-cell tumour is accompanied by the release of two putative proinsulin cleavage intermediates, four peptides of Mr 9000-12 000 (excluding proinsulin) and peptides of Mr 21 000, 34 000 and 60 000. Granule-enriched subcellular preparations contain major peptides of identical Mr values. Of these peptides seven at least coincide in molecular weight with peptides secreted by isolated rat islets and thus may be constituents of the normal insulin secretory granule.

Adenoma, Islet Cell↗
PubMed1984

Ca2+-sensitive phosphatidylinositol 4-phosphate metabolism in a rat beta-cell tumour.

Subcellular fractions were isolated from a rat beta-cell tumour by centrifugation of homogenates on Percoll and Urografin density gradients. Fractions were incubated with [gamma-32P]ATP, and labelling of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was used to measure phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities, respectively. The distribution of enzyme markers in density gradients indicated that phosphatidylinositol kinase was located in both the plasma membrane and the secretory-granule membrane. Phosphatidylinositol 4-phosphate kinase activity was low in all fractions. Phosphatidylinositol kinase activity of secretory granules and plasma membranes was decreased to 10-20% of its initial value by raising the free [Ca2+] from 1 microM to 5 microM. The enzyme had a Km (apparent) for ATP of 110 microM (secretory granule) or 120 microM (plasma membrane) and a Ka for Mg2+ of 7 mM (secretory granule) or 6 mM (plasma membrane). Ca2+-sensitivity of phosphatidylinositol kinase in calmodulin-depleted secretory granules and plasma membranes was not affected by addition of exogenous calmodulin, although activity was stimulated by trifluoperazine in the presence of 0.1 microM or 40 microM-Ca2+. Trifluoperazine oxide had no effect on the enzyme activity of secretory granules. Plasma membranes had a phosphatidylinositol 4-phosphate phosphatase activity which was stimulated by raising the free [Ca2+] from 0.1 to 40 microM. The secretory granule showed no phosphatidylinositol 4-phosphate-degrading activity. These results suggest the presence in the tumour beta-cell of Ca2+-sensitive mechanisms responsible for the metabolism of polyphosphoinositides in the secretory granule and plasma membrane.

Adenoma, Islet Cell↗
PubMed1983

Carboxypeptidase activity in the insulin secretory granule.

Carboxypeptidase activity was studied in subcellular fractions from a transplantable rat insulinoma and found to be localised principally in the insulin secretory granule. The activity, which was specific for peptide substrates with C-terminal basic amino acids, appeared to be a single enzyme with Mr 54000. This enzyme differed with respect to size and pH optimum from other basic amino acid-specific carboxypeptidases, such as carboxypeptidases B and N, and may be a secretory granule-specific enzyme involved in propolypeptide processing.

Adenoma, Islet Cell↗
PubMed1983

5-Hydroxytryptamine transport in cells and secretory granules from a transplantable rat insulinoma.

Mechanisms of transport of 5-hydroxytryptamine in the pancreatic B-cell were investigated by using cell suspensions and secretory granules prepared from a transplantable rat insulinoma. (1) Cells incubated with 5-hydroxy[G-3H]tryptamine at concentrations ranging from 0.1 microM to 5 mM accumulated the radioisotope principally by a simple diffusion process. The incorporated radioactivity was recovered principally as the parent molecule and was recovered predominantly in soluble protein and secretory-granule fractions prepared from the tissue. (2) Isolated granules incubated in buffered iso-osmotic medium without ATP accumulated the amine to concentrations up to 38-fold that of the medium. This process was insensitive to reserpine and occurred over a wide range of 5-hydroxytryptamine concentrations (0.075 microM-25 mM). Above 5 mM, 5-hydroxytryptamine accumulation decreased in parallel with the breakdown of the delta pH across the granule membrane. Uptake was favoured by alkaline media and was reduced by the addition of (NH4)2SO4. In both cases a close correlation was observed between uptake and the transmembrane delta pH, a finding that suggested that 5-hydroxytryptamine permeated the membrane as the free base and equilibrated across the membrane with the delta pH. Binding of 5-hydroxytryptamine to granule constituents also played a part in this process. ATP caused a further doubling of granule 5-hydroxytryptamine uptake by a process that was sensitive to reserpine (0.5 microM). Inhibitor studies suggested that amine transport in this instance was linked to the activity of the granule membrane proton-translocating ATPase. (3) It was concluded that the uptake of amines driven by proton gradients across the insulin-granule membrane could account for the accumulation in vivo of amines in the B-cell.

Adenoma, Islet Cell↗
PubMed1983

Low-molecular-weight constituents of isolated insulin-secretory granules. Bivalent cations, adenine nucleotides and inorganic phosphate.

The concentrations of Zn2+, Ca2+, Mg2+, Pi and adenine nucleotides were determined in insulin-secretory granules prepared from a transplantable rat insulinoma. Differential and density-gradient centrifugation analyses revealed that Zn2+ in this tissue was principally localized in the secretory granule, a second major fraction being found in association with cytosolic proteins. Pi was principally recovered in the latter fraction, whereas Ca2+ and Mg2+ were more widely distributed. Intragranular ion-distribution experiments suggested that Zn2+ was complexed mainly to insulin and its precursor forms and remained in the granule in an insoluble state. The Zn2+/insulin ratio (0.54) was greater than that expected for insulin molecules having two centrally co-ordinated Zn2+ atoms/hexamer, but less than the maximal Zn2+-binding capacity of the molecule. Most of the granular Ca2+, Mg2+ and Pi was released in a soluble form when granules were disrupted by sonication. Simulation in vitro of the ionic composition of the granule suggested that up to 90% of its Ca2+ was complexed to Pi and adenine nucleotides. Granular macromolecules also bound Ca2+, as shown by equilibrium-dialysis studies of granule lysates. However, such binding was displaced by Mg2+. Examination of the efflux of Ca2+ from granules incubated in iso-osmotic suspensions at 37 degrees C suggested that the passive permeability of the granule membrane to Ca2+ was very low. Nevertheless, more than 50% of the granular Ca2+ was rapidly released in an ionized form on hypo-osmotic or detergent-induced disruption of the granule membrane. This may represent a potentially mobilizable pool of Ca2+ in vivo.

Adenine Nucleotides↗
PubMed1983

Ca2+-dependent binding of cytosolic components to insulin-secretory granules results in Ca2+-dependent protein phosphorylation.

1. Incubation of a rat islet cell tumour homogenate with [gamma-32P]ATP resulted in the Ca2+-dependent phosphorylation of 100000-, 57000-, 29000-, 26000- and 14000-Mr proteins. The Ca2+ concentration required was in the low-microM range. 2. Isolated insulin granules did not exhibit Ca2+-dependent protein phosphorylation, whereas a soluble protein fraction showed the Ca2+-dependent phosphorylation of a 57000-Mr protein. Combination of insulin granules with the soluble protein fraction resulted in the additional Ca2+-dependent phosphorylations of 100000-, 29000- and 10000-Mr proteins. The latter phosphorylations were not enhanced by exogenous calmodulin, but nevertheless were inhibited by trifluoperazine. Removal of endogenous calmodulin from the soluble protein fraction before incubation with insulin granules did not abolish the Ca2+-dependent phosphorylations of the 100000-, 29000- and 10000-Mr proteins but rendered the Ca2+-dependent phosphorylation of the 57000-Mr soluble protein dependent on exogenous calmodulin. 3. The components of the soluble protein fraction responsible for the interaction with insulin granules bound to intact granules in a Ca2+-dependent manner. 4. After phosphorylation, the 29000-Mr protein remained attached to granules, whereas the 100000- and 10000-Mr proteins dissociated from granules. 5. These Ca2+-dependent phenomena may be of regulatory importance in the secretory mechanism.

Adenoma, Islet Cell↗
PubMed1983

Nucleotide and bivalent cation specificity of the insulin-granule proton translocase.

1. The nucleotide and bivalent cation specificity of the proton translocase activity of insulin secretory granules was investigated by assessing the inhibitor-sensitive rates of nucleotide hydrolysis by these organelles in relation to their chemiosmotic properties. 2. The relative rates of nucleotide hydrolysis by freeze/thawed granule preparations were: Mg2+ATP (100%) greater than Mg2+GTP (55%) greater than Mg2+UTP (48%) greater than Mg2+ITP (44%) greater than Mg2+CTP (23%) greater than Mg2+TTP (20%), and by intact granules were: Mg2+ATP (100%) greater than Mg2+ITP (74%) greater than Mg2+GTP (60%) greater than Mg2+CTP (35%). Mg2+ATP, Mg2+GTP and Mg2+ITP hydrolyses were inhibited by tributyltin and stimulated, in intact granules, by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone; Mg2+CTP hydrolysis was not markedly affected by these compounds. Correspondingly, only Mg2+ATP, Mg2+GTP and Mg2+ITP produced large changes in the delta psi and delta mu H+ across the granule membrane. 3. The relative rates of maximal ATPase activity stimulated by bivalent cations in freeze/thawed granule preparations were: Mg2+ (100%) greater than Mn2+ (82%) greater than Ca2+ (40%) greater than Co2+ (36%) greater than Zn2+ (0%), and in intact granules were: Mg2+ (100%) greater than Mn2+ (85%) greater than Co2+ (61%) greater than Ca2+ (42%). Tributyltin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone affected Mg2+-, Mn2+- and Co2+-activated, but not Ca2+-activated, ATP hydrolysis. Correspondingly, only Mg2+, Mn2+ and Co2+ supported the generation of a delta psi and delta mu H+ across granule membranes in the presence of ATP. 4. The results were consistent with a single proton translocase that had its catalytic site exposed on the external face of the granule membrane. The indicated specificity (Mg2+ATP = Mn2+ATP greater than Co2+ATP greater than Mg2+GTP greater than Mg2+ITP) was similar to that of enzymes described in membrane fractions prepared from adenohypophyseal tissue, adrenal chromaffin granules and yeast vacuoles. The insulin-granule activity thus appears to be a type of proton translocase, which is characteristic of intracellular storage vesicles in eukaryotic cells.

Adenosine Triphosphatases↗
PubMed1982

Proton-translocating Mg2+-dependent ATPase activity in insulin-secretory granules.

Insulin-secretory granules isolated from a pancreatic islet-cell tumour by centrifugation on Percoll density gradients exhibited a membrane-associated Mg(2+)-dependent ATPase activity. In granule suspensions incubated in iso-osmotic media, activity was increased 2-3-fold by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K(2)SO(4) or by the addition of a detergent. Permeant anions also increased Mg(2+)-dependent ATPase activity under iso-osmotic conditions when combined with K(+) and nigericin, or NH(4) (+). It was deduced that a major component of the activity was coupled to the translocation of protons into the granule interior. The granule membrane appeared poorly permeable to H(+), K(+), NH(4) (+) and SO(4) (2-) but permeable, in increasing order, to phosphate or acetate, Cl(-), I(-) and SCN(-). Like the proton-translocating ATPase of mammalian mitochondria the granule enzyme when membrane-bound was inhibited by up to 85% by tributyltin or NN'-dicyclohexylcarbodi-imide and was solubilized in a tributyltin-insensitive form after extraction with dichloromethane. It was clearly not a mitochondrial contaminant as evidence by the distribution of marker proteins on density gradients. Unlike mitochondrial activity it was insensitive to oligomycin, efrapeptin, atractyloside, azide and oxyanions. Its properties, however, were indistinguishable from those of the proton-translocating ATPase found in the chromaffin granules of the adrenal medulla. Moreover, insulin granules and chromaffin granules exhibited similar levels of activity. This indicated that in spite of the differences in their internal composition, granules from tissues involved in polypeptide and amine hormone secretion possess catalytic components in common. Only a minor role for the ATPase in amine transport in insulin granules was apparent. Rather, its presence here may relate to the process of secretory vesicle morphogenesis or to the exocytotic mechanism.

Adenoma, Islet Cell↗
PubMed1982

The internal pH and membrane potential of the insulin-secretory granule.

The membrane potential (DeltaPsi) and the pH gradient (DeltapH) across the membrane of the insulin-secretory granule were determined in studies in vitro from the uptake of the permeant anion thio[(14)C]cyanate or the permeant base [(14)C]methylamine. Freshly prepared granules incubated in iso-osmotic medium containing sucrose and low concentrations of buffer salts exhibited an acidic internal pH and a DeltaPsi positive inside. Addition of MgATP(2-) under these conditions did not alter the DeltapH, but produced a marked increase in the DeltaPsi. Conversely, when a permeant anion was also included, ATP produced a marked increase in the DeltapH and a lesser increment in the DeltaPsi. NH(4) (+) salts reduced the DeltapH across granule membranes. In the presence of ATP this effect was accompanied by a reciprocal increase in the DeltaPsi. A similar reciprocity was evident when nigericin was added together with K(+) or on decreasing the medium pH, suggesting that these gradients were linked by a common electrogenic process. The effects of ATP were reversed by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K(+), and by the Mg(2+)-dependent ATPase inhibitor tributyltin. Uptakes of (14)C-labelled tracer molecules were also markedly reduced by cryogenic disruption of the granule membrane or hypo-osmotic incubation conditions. These results were readily interpreted within a chemiosmotic hypothesis, which proposed that the insulin granules possess an inwardly-directed electrogenic proton-translocating Mg(2+)-dependent ATPase with the additional postulate that the membrane has a low proton permeability. The intragranular pH was estimated as being between 5 and 6 in vivo. Such a value corresponds to optimal conditions for the crystallization of zinc-insulin hexamers. Several other functions related to chemiosmotic processes within insulin granules, however, may be envisaged.

Adenoma, Islet Cell↗
PubMed1982

Isolation and characterisation of insulin secretory granules from a rat islet cell tumour.

Density gradient centrifugation techniques, using iso-osmotic colloidal silica suspensions (Percoll), were developed for the isolation of insulin secretory granules from a transplantable rat islet cell tumour. These procedures were readily completed within 7 h and from each animal yielded approximately 1 mg of granule protein. The isolated granules were essentially free of other subcellular organelles as evaluated by their contents of marker proteins, electron microscopy and by electrophoretic analyses. Their susceptibilities to lysis at low osmotic strength, at pH values above 7 or in media containing sodium ions were similar to those of granules partially purified from islets. Insulin comprised 50-60% of the total granule protein when determined by immunoassay or by densitometry of electrophoretic profiles. The proinsulin content was marginally higher than that of islets, as was the ratio of insulins I to II. Electrophoretic analyses revealed that the secretory granules contained 150 or more proteins besides insulin-related peptides. The majority of these had acidic isoelectric points and were located both within the granule interior and its enveloping membrane.

Adenoma, Islet Cell↗
PubMed1981

The stimulus-secretion coupling of amino acid-induced insulin release. Metabolic interaction of L-glutamine and 2-ketoisocaproate in pancreatic islets.

1. The metabolic situation found in pancreatic islets exposed to both L-glutamine and 2-ketoisocaproate was investigated in order to assess its relevance to the synergistic effects of these nutrients upon insulin release. 2. In islet homogenates, serveral 2-keto acids could be used as partners for the transamination of L-glutamate to 2-ketoglutarate. The rate of transamination did not correlate positively with the capacity of each 2-keto acid to stimulate insulin release in the presence of L-glutamine. 3. L-Glutamine enhanced the production of L-leucine from 2-ketoisocaproate and inhibited the conversion of the 2-keto acid to acetoacetate and CO2. L-Glutamine also inhibited the oxidation of pyruvate. 4. In the presence of 2-ketoisocaproate, the rate of generation of 2-ketoglutarate from exogenous L-glutamine was increased, but the oxidative deamination of glutamate was suppressed. 5. L-Valine antagonized the effect of 2-ketoisocaproate to augment 14CO2 output from islets prelabelled with L-[U-14C]glutamine. 6. L-Glutamine did not increase the islet content of reduced pyridine nucleotides beyond the high level reached in the sole presence of 2-ketoisocaproate. 7. If allowance was made for the influence of exogenous nutrients upon the oxidation of endogenous nutrients, the insulin output evoked by L-glutamine and/or 2-keto acids tightly depended on the increment in oxidation rate attributable to these nutrients. 8. The metabolic and secretory responses to L-glutamine and 2-ketoisocaproate were best explained by a stimulation of transamination reactions between 2-ketoisocaproate and glutamate derived from exogenous glutamine.

Animals↗
PubMed1981

Isolation and characterization of calmodulin from an insulin-secreting tumour.

A major protein constituent of a rat islet cell tumour that exhibited Ca2+-dependent changes in electrophoretic mobility has been purified to homogeneity and compared in its physicochemical and biological properties with bovine brain and rat brain calmodulin (synonymous with phosphodiesterase activator protein, calcium-dependent regulator, troponin C-like protein and modulator protein). The protein, like these calmodulins, contained trimethyl-lysine, exhibited a blocked N-terminus and had an identical amino-acid composition and molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Peptide "maps' prepared after digestion of the three proteins with trypsin, papain or Staphylococcus V-8 proteinase were virtually superimposable. Ca2+ altered the electrophoretic mobilities the enhanced the native protein fluorescence in an equivalent manner with all three proteins. Equilibrium dialysis experiments demonstrated in each case the binding of 4g-atoms of calcium/mol of protein; the binding sites were equivalent and showed Kd 0.8 microM. Tumour and brain proteins were equipotent as Ca2+-dependent activators of partially purified rat brain cyclic nucleotide phosphodiesterase, and in this action were inhibited in an identical manner by trifluoperazine. The proteins also exhibited the common property of Ca2+-dependent binding to troponin I, histone H2B and myelin basic protein. The estimated tumour content of calmodulin was 450 mg/kg fresh wt., a value similar to that reported in islets of Langerhans. These results further document the validity of the islet cell tumour as an experimental model of Ca2+-mediated molecular events associated with insulin secretion. They also suggest that brain calmodulin may be substituted for endogenous calmodulin in experimental investigations into the mechanism of insulin secretion.

Adenoma, Islet Cell↗