The stimulus-secretion coupling of glucose-induced insulin release. Does glycolysis control calcium transport in the B-cell?

1. The metabolism of glucose and the exchangeable Ca2+ pool were measured in rat pancreatic islets, in order to assess the possible significance of glycolysis in the process of glucose-induced insulin release. 2. At high glucose concentration (16.7 mM), glucose was metabolized at the following rate (pmol of glucose residue/h per islet +/- S.E.M.): 131 +/- 11 for glucose uptake, 129+/-8 for glucose utilization, as judged by the conversion of [5-3H]glucose into 3H2O,60+/-2 for lactate output and 25+/-2 for glucose oxidation. 3. The secretory pattern usually correlated with the metabolic data. For instance, the ability of different sugars (glucose, mannose, fructose, galactose, D-glyceraldehyde) to stimulate lactate output closely paralleled their relative insulinotropic capacity. A disparity between metabolic and secretory responses was, however, encountered in the presence of dibutyryl cyclic AMP and theophylline. 4. Despite this contrasting behaviour, the size of the Ca2+- exchangeable pool (net uptake of 45Ca2+) was invariably proportional to the rate of lactate output under all experimental conditions examined. It is concluded that glycolysis usually exerts a tight control on the rate constant for Ca2+ transport across the B-cell membrane.     

Evidence for a role of phosphatidylinositol turnover in stimulus–secretion coupling. Studies with rat peritoneal mast cells

Histamine secretion and phosphatidylinositol turnover were compared in antigen-sensitized rat peritoneal mast cells stimulated with a number of different ligands. A small and variable increase in the incorporation of [(32)P]P(i) and of [(3)H]inositol into phosphatidylinositol was observed when the cells were treated with immunoglobulin E-directed ligands (antigens and concanavalin A), and this was accompanied by a low amount of secretion (<10% of total cell histamine). In the presence of added phosphatidylserine, the addition of immunoglobulin E-directed ligands invariably led to an enhanced rate (approx. 4-fold) of labelling of phosphatidylinositol and, in the presence of Ca(2+), this was accompanied by the secretion of histamine. The labelling of phosphatidylinositol and histamine secretion were also stimulated by chymotrypsin and compound 48/80. Whereas the phosphatidylinositol response did not require the presence of extracellular Ca(2+), the secretion of histamine was either enhanced or dependent on extracellular Ca(2+) (depending on the ligand used). The dependence on ligand concentration for the phosphatidylinositol response and histamine secretion were similar. The increased isotopic incorporation into phosphatidylinositol continued for about 1h although histamine secretion (elicited with concanavalin A) stopped within 2min. These results support the proposition that metabolic events involving phosphatidylinositol play a necessary intermediate role in the regulation of Ca(2+) channels by ligand-activated receptors.     

Effects of trifluoperazine and pimozide on stimulus–secretion coupling in pancreatic B-cells. Suggestion for a role of calmodulin?

The possible involvement of calmodulin in insulin release was evaluated by studying the effects on intact islets of trifluoperazine and pimozide, two antipsychotic agents known to bind strongly to calmodulin in cell-free systems. Trifluoperazine (10-100mum) produced a dose- and time-dependent inhibition of the two phases of glucose-stimulated insulin release. The effect was not reversible by simple washing of the drug, but could be prevented by cytochalasin B or theophylline. Trifluoperazine also inhibited the release induced by glyceraldehyde, oxoisocaproate, tolbutamide or barium, but not that stimulated by 10mm-theophylline or 1mm-3-isobutyl-1-methylxanthine. Pimozide (0.5-10mum) also produced a dose-dependent inhibition of insulin release triggered by glucose, leucine or barium, but did not affect the release induced by methylxanthines. Glucose utilization by islet cells was not modified by trifluoperazine (25mum), which slightly increased cyclic AMP concentration in islets incubated without glucose. The drug did not prevent the increase in cyclic AMP concentration observed after 10min of glucose stimulation, but suppressed it after 60min. Basal or glucose-stimulated Ca(2+) influx (5min) was unaffected by 25mum-trifluoperazine, whereas Ca(2+)net uptake (60min) was inhibited by 20%. Glucose-stimulated Ca(2+) uptake was almost unaffected by pimozide. In a Ca(2+)-free medium, trifluoperazine decreased Ca(2+) efflux from the islets and did not prevent the further decrease by glucose; in the presence of Ca(2+), the drug again decreased Ca(2+) efflux and inhibited the stimulation normally produced by glucose. In the absence of glucose, trifluoperazine lowered the rate of Rb(+) efflux from the islets, decreased Rb(+) influx (10min), but did not affect Rb(+) net uptake (60min). It did not interfere with the ability of glucose to decrease Rb(+) efflux rate further and to increase Rb(+) net uptake. The results show thus that trifluoperazine does not alter the initial key events of the stimulus-secretion coupling. Its inhibition of insulin release suggests a role of calmodulin at late stages of the secretory process.     

Evidence for the participation of calmodulin in stimulus–secretion coupling in the pancreatic β-cell

1. The ability of a range of phenothiazines to inhibit activation of brain phosphodiesterase by purified calmodulin was studied. Trifluoperazine, prochlorperazine and 8-hydroxyprochlorperazine produced equipotent dose-dependent inhibition with half-maximum inhibition at 12mum. When tested at 10 or 50mum, 7-hydroxyprochlorperazine was a similarly potent inhibitor. However, trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were ineffective at concentrations up to 50mum, and produced only a modest inhibition at 100mum. 2. The same phenothiazines were tested for their ability to inhibit activation of brain phosphodiesterase by boiled extracts of rat islets of Langerhans. At a concentration of 20mum, 70-80% inhibition was observed with trifluoperazine, prochlorperazine, 7-hydroxyprochlorperazine or 8-hydroxyprochlorperazine, whereas trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were less effective. 3. The effect of these phenothiazines on insulin release from pancreatic islets was studied in batch-type incubations. Insulin release stimulated by glucose (20mm) was markedly inhibited by 10mum-trifluoperazine or -prochlorperazine and further inhibited at a concentration of 20mum. 8-Hydroxyprochlorperazine (20mum) was also a potent inhibitor but 7-hydroxyprochlorperazine (20mum) elicited only a modest inhibition of glucose-stimulated insulin release; no inhibition was observed with trifluoperazine-5-oxide or N-methyl-2-(trifluoromethyl)phenothiazine. 4. Trifluoperazine (20mum) markedly inhibited insulin release stimulated by leucine or 4-methyl-2-oxopentanoate in the absence of glucose, and both trifluoperazine and prochlorperazine (20mum) decreased insulin release stimulated by glibenclamide in the presence of 3.3mm-glucose. 5. None of the phenothiazines affected basal insulin release in the presence of 2mm-glucose. 6. Trifluoperazine (20mum) did not inhibit islet glucose utilization nor the incorporation of [(3)H]leucine into (pro)insulin or total islet protein. 7. Islet extracts catalysed the incorporation of (32)P from [gamma-(32)P]ATP into endogenous protein substrates. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis resolved several phosphorylated bands, but incorporation was slight. However, calmodulin in the presence of Ca(2+) greatly enhanced incorporation: the predominant phosphorylated band had an estimated mol.wt. of 55000. This enhanced incorporation was abolished by trifluoperazine, but not by cyclic AMP-dependent protein kinase inhibitor protein. 8. These results suggest that islet phosphodiesterase-stimulating activity is similar to, although not necessarily identical with, calmodulin from skeletal muscle; that islet calmodulin may play an important role in Ca(2+)-dependent stimulus-secretion coupling in the beta-cell; and that calmodulin may exert part at least of its effect on secretion via phosphorylation of endogenous islet proteins.     

The pancreatic β-cell recognition of insulin secretagogues. Effects of calcium and sodium on glucose metabolism and insulin release

The transport and oxidation of glucose, the content of fructose 1,6-diphosphate, and the release of insulin were studied in microdissected pancreatic islets of ob/ob mice incubated in Krebs-Ringer bicarbonate medium. Under control conditions glucose oxidation and insulin release showed a similar dependence on glucose concentration with the steepest slope in the range 5-12mm. The omission of Ca(2+), or the substitution of choline ions for Na(+), or the addition of diazoxide had little if any effect on glucose transport. However, Ca(2+) or Na(+) deficiency as well as diazoxide (7-chloro-3-methyl-1,2,4-benzothiadiazine 1,1-dioxide) or ouabain partially inhibited glucose oxidation. These alterations of medium composition also increased the islet content of fructose 1,6-diphosphate, as did the addition of adrenaline. Phentolamine [2-N-(3-hydroxyphenyl)-p-toluidinomethyl-2-imidazoline] counteracted the effects of adrenaline and Ca(2+) deficiency on islet fructose 1,6-diphosphate. After equilibration in Na(+)-deficient medium, the islets exhibited an increase in basal insulin release whereas the secretory response to glucose was inhibited. The inhibitory effects of Na(+) deficiency on the secretory responses to different concentrations of glucose correlated with those on (14)CO(2) production. When islets were incubated with 17mm-glucose, the sudden replacement of Na(+) by choline ions resulted in a marked but transient stimulation of insulin release that was not accompanied by a demonstrable increase of glucose oxidation. Galactose and 3-O-methylglucose had no effect on glucose oxidation or on insulin release. The results are consistent with a metabolic model of the beta-cell recognition of glucose as insulin secretagogue and with the assumption that Ca(2+) or Na(+) deficiency, or the addition of adrenaline or diazoxide, inhibit insulin release at some step distal to stimulus recognition. In addition the results suggest that these conditions create a partial metabolic block of glycolysis in the beta-cells. Hence the interrelationship between the processes of stimulus recognition and insulin discharge may involve a positive feedback of secretion on glucose metabolism.     

Establishment of variant PC12 subclones deficient in stimulation–secretion coupling

Clonal rat pheochromocytoma (PC12) cells have been widely used to study the molecular mechanism of exocytosis. We have isolated variant PC12 subclones with deficiencies in stimulation–secretion coupling, by a single cell recloning, and investigated the defects. PC12-1G2 hardly released dopamine following high-K⁺-induced depolarization, but normal release was evoked by the Ca²⁺-ionophore, ionomycin. Fura-2 fluorometry indicated that a nicardipine-sensitive component of Ca²⁺ influx was missing, suggesting that PC12-1G2 has defects in L-type Ca²⁺ channel function. PC12-2B3 was not responsive to high-K⁺-induced depolarization and ionomycin, and voltage-dependent Ca²⁺ entry was identical to that of the normal clone. Electron microscopy revealed that the number of vesicles adjacent or directly attached to the plasma membrane was decreased in PC12-2B3. The expression of presynaptic proteins was analyzed by immunoblotting using a panel of antibodies. Syntaxin 1, VAMP-2, SNAP-25, Munc18, Rab3C and Sec-6 were decreased compared to the control clone and that of synaptophysin was extremely low. PC12-D60 synthesized and released dopamine normally, but had almost lost its catecholamine-uptake activity. These results show that multiple PC12 cells variants are spontaneously generated, and that recloning can select PC12 subclones useful for the study of the molecular mechanisms of neurotransmitter release.     

The catalase–hydrogen peroxide system. A theoretical appraisal of the mechanism of catalase action

1. The mechanisms of catalase action advanced by Jones & Wynne-Jones (1962) and by Nicholls (1964) are compared in terms of their relative plausibilities and their utility for extension to accommodate more recent experimental information. 2. A revised formal mechanism is advanced that avoids the less satisfactory features of these mechanisms and attempts to account for the roles of catalase sub-units in both reversible and irreversible deactivation phenomena. 3. Theoretical studies of the redox chemistry of peroxides are used to provide the basis for a discussion of the mechanism of the redox act in catalatic action at the molecular level. It is suggested that an important feature of catalase action may be a mediation of the formation of a reactive intermediate by stereospecifically located acid-base functions in the active site. 4. A more detailed statement of this concept is attempted in terms of a hypothetical partial molecular model for the composition and stereochemistry of the active site of catalase. The utility of this model in describing the catalatic and peroxidatic actions of catalase is assessed.     

The release of oligosaccharides from glycoproteins by endo-β-N-acetylglucosaminidase of Flavobacterium sp.

Endo-β-N-acetylglucosaminidase, purified to homogenicity from the cultural filtrate of Flavobacterium sp., liberated oligosaccharides from various glycoproteins. The enzyme could liberate the carbohydrate chain from native ovalbumin. The release of oligosaccharides from ribonuclease B, yeast carboxypeptidase and a Ricinus lectin was also observed. These glycoproteins contain neutral oligosaccharides that are attached to the protein through glycosyl asparagine bonds. The treatment of glycoprotein with SDS and boiling was more effective for removal of oligosaccharides by the enzyme. The enzyme hydrolyzed all five heterogeneous ovalbumin glycopeptides, although the rate of hydrolysis decreased as the size of the sugar moiety increased. Removal of the neutral oligosaccharides did not appear to effect the enzymatic properties of the hemagglutination ability of these glycoproteins.     

Colicin E2 release: lysis,leakage or secretion? Possible role of a phospholipase.

Results presented here and by others indicate that the release of colicins from producing cells can be uncoupled from the decline in culture turbidity which usually occurs within 2-3 h after the induction of colicin synthesis. This excludes lysis as a necessary event in colicin release. Conversely, the failure to dissociate colicin release from the normally simultaneous release of a specific subset of soluble proteins argues against the idea of a specific colicin secretion system sensu-stricto. Rather, colicin release appears to be a consequence of semi-specific leakage resulting from an alteration of the permeability properties of the cell envelope. This alteration is caused by the 'lysis protein' known to be encoded by most multiple copy number Col plasmids. The finding that the expression of the lysis gene of plasmid ColE2 renders the cells exquisitely sensitive to lysozyme demonstrates that the permeability of the outer membrane must indeed be altered. Evidence is presented that this alteration could be due at least in part to the activation of the detergent-resistant phospholipase A (pldA product). Lysophosphatidylethanolamine, a product of the action of phospholipase on phosphatidylethanolamine, is a membrane perturbant which could alter the permeability properties of the envelope and allow some proteins such as colicin to leak out of the cell.     

The mode of action of adenosine 3′:5′-cyclic monophosphate in mammalian islets of Langerhans. Effects of insulin secretagogues on islet-cell protein kinase activity

1. Protein kinase activity was measured in islets of Langerhans that had been incubated in the presence of agents known to affect insulin release. 2. Glucagon, theophylline, caffeine and 3-isobutyl-1-methylxanthine, agents that raise cyclic AMP concentrations in islet cells and stimulate insulin release, increased protein kinase activity. Adrenaline and diazoxide, agents that decrease cyclic AMP concentrations and inhibit insulin secretion, decreased the activity. 3. The increase in protein kinase activity produced by different concentrations of 3-isobutyl-1-methylxanthine was apparently related to the increase in intracellular concentrations of cyclic AMP. 4. The sulphonylureas, tolbutamide and glibenclamide, agents that increase insulin release, also increased the protein kinase activity; however, leucine, arginine and xylitol, which also stimulate insulin release, were without effect on the kinase activity. 5. Increasing the glucose concentration of the incubation medium from 2 to 20mm had no effect on protein kinase activity. Further, the ability of 3-isobutyl-1-methylxanthine to increase the protein kinase activity was not affected by the glucose concentration of the incubation medium. 6. These results suggest that agents which affect insulin secretion by altering cyclic AMP concentrations may exert their effects on hormone release by altering the activity of a cyclic AMP-dependent protein kinase in islet cells.     

Immunoglobulin λ-chains. The complete amino acid sequence of a Bence-Jones protein

The total amino acid sequence of a lambda Bence-Jones protein has been established. The protein contains 211 residues, which include two methionine residues. Splitting with cyanogen bromide gave three fragments, the largest of which included the C-terminal half, which is common to other Bence-Jones proteins of the same type. The peptides obtained by tryptic, chymotryptic and peptic digestion were isolated and purified by paper-electrophoretic and chromatographic techniques. Reduction followed by carboxymethylation of the cysteine residues with radioactive iodoacetate was found to be a powerful tool in the isolation of some insoluble peptides. Unusual features of the molecule are the fact that it contains six cysteine residues and not five as observed in both kappa and lambda Bence-Jones proteins studied previously, and its size, which seems two residues smaller than the smallest Bence-Jones protein studied hitherto. The similarities and differences between this and other Bence-Jones proteins are discussed.     

The amino acid sequence of cytochrome c′ from Alcaligenes sp. N.C.I.B. 11015

The amino acid sequence of the cytochrome c' from Alcaligenes sp. N.C.I.B. 11015 (Iwasaki's ;Pseudomonas denitrificans') has been determined. This organism is the only non-photosynthetic bacterium in which the protein has been found. The protein consists of a single polypeptide chain of 127 residues, with a single haem covalently attached to two cysteines. Unlike normal cytochromes c, the haem attachment site is very close to the C-terminus. The amino acid sequence around the haem attachment site is very similar to that of Chromatium vinosum D cytochrome c'. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50022 at the British Library (Lending Division), (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.