sincalide and big-gastrin

This study evaluated the role played by cholecystokinin (CCK) receptors' occupation in the control of somatostatin (SS) secretion in RIN-14B cells.. The presence of the CCK receptors 1 and 2 was confirmed by immunofluorescence, and SS secretion was evaluated by enzyme-linked immunosorbent assay.. By immunofluorescence, 95% of the cell population was composed of SS cells bearing both CCK-R subtypes with 5% of beta cells (data not shown). Cerulein (Cae), a CCK-1R agonist, and pentagastrin, a CCK-2R agonist, dose-dependently increased SS release, 3-fold at 1 mumol/L Cae, 2.5-fold at 10 mumol/L pentagastrin, with occupation of both CCKRs confirmed by L-364,178 and L-365,260 inhibition of CCK receptors 1 and 2. The occupation of high-affinity CCK-1R by Cae was confirmed on SS release with JMV-180, a high-affinity CCK-1R agonist, and absence of SS release inhibition at high Cae concentration occupying the low-affinity CCK-1R. These cells release more than 60% of their SS content by constitutive secretion, confirmed by cycloheximide and brefeldin inhibiting SS synthesis and intracellular trafficking, respectively.. Both CCKR subtypes occupy RIN-14B cells and initiate SS secretion through constitutive secretion controlled at SS synthesis level. Somatostatin secretion via the CCK-1R occupation mobilizes its high-affinity sites.

Topics: Animals; Benzodiazepinones; Brefeldin A; Cell Line; Ceruletide; Cholecystokinin; Cycloheximide; Devazepide; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Gastrins; Islets of Langerhans; Pentagastrin; Phenylurea Compounds; Protein Precursors; Protein Synthesis Inhibitors; Protein Transport; Rats; Receptor, Cholecystokinin A; Receptor, Cholecystokinin B; Sincalide; Somatostatin

Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo. The aims of this study were to prepare recombinant human progastrin(6-80) and to investigate its structure and biological activities in vitro. Human progastrin(6-80) was expressed in Escherichia coli as a glutathione S-transferase fusion protein. After thrombin cleavage progastrin(6-80) was purified by reverse phase high pressure liquid chromatography and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry. Assays for metal ions by atomic emission spectroscopy revealed the presence of a single tightly bound calcium ion. Progastrin(6-80) at concentrations in the pm to nm range stimulated proliferation of the conditionally transformed mouse colon cell line YAMC. The observations that progastrin(6-80) did not bind to either the cholecystokinin (CCK)-A or the gastrin/CCK-B receptor expressed in COS cells and that antagonists selective for either receptor did not reverse the proliferative effects of progastrin(6-80) suggested that progastrin(6-80) stimulated proliferation independently of either the CCK-A or the gastrin/CCK-B receptor. We conclude that recombinant human progastrin(6-80) is biologically active and contains a single calcium ion. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin, this is the first report of selective, high affinity binding of metal ions to a prohormone.

Topics: Amino Acid Sequence; Animals; Calcium; Cell Division; COS Cells; Gastrins; Humans; Molecular Sequence Data; Peptide Fragments; Protein Precursors; Recombinant Proteins; Sincalide

A new specific method for determination of cholecystokinin, CCK-8, and CCK-33, 39 in rat plasma is described. Plasma CCK radioimmunoassay (RIA) is difficult, because of cross-reactivity with gastrin. In the rat, problems because of difficulties in separating gastrin from CCK by high performance liquid chromatography (HPLC) exist. These were solved by enzyme digestion of gastrin before HPLC separation of molecular variants of CCK from gastrin fragments. Cholecystokinin immunoreactive forms in the HPLC fractions were determined by an antibody, which recognises the carboxyl terminus of CCK and gastrin. Fasting concentrations of small (CCK-8) and large (CCK-33, 39) molecular forms of CCK averaged 1.9 (0.3) pM and were raised to 13.4 (3.8) pM in rats fed ad libitum. Cholecystokinin in lactating rats rose two-fold after suckling, compared with 2.8 fold in response to feeding. The basal ratio between CCK-8 and CCK-33, 39 was approximately 1:1, but increased in favour of CCK-8 after feeding and in response to suckling. Gastrin like immunoreactivity measured in unextracted plasma was found to rise after feeding, but was unchanged in response to suckling.

Topics: Animals; Cholecystokinin; Chromatography, High Pressure Liquid; Eating; Female; Gastric Mucosa; Gastrins; Intestinal Mucosa; Lactation; Methods; Pregnancy; Protein Precursors; Radioimmunoassay; Rats; Rats, Inbred Strains; Serine Endopeptidases; Sincalide

Extracts of rat kidney contain an enzyme (gastrinase) that is highly specific for degradation of the 34 amino acid gastrin (G34). The Michaelis constant (Km) for kidney is 0.36 +/- 0.04 microM and the Vmax is 9.5 +/- 2.4 nmol X g-1 X min-1. Extracts of liver and brain also have gastrin degrading activity but the enzymes responsible appear to be different from the kidney gastrinase. Km for the liver enzyme is 0.08 +/- 0.02 microM but its Vmax (0.10 +/- 0.02 nmol X g-1 X min-1) is only 1% of the kidney gastrinase; Km for the brain enzyme is 0.10 +/- 0.03 microM but its Vmax (0.023 +/- 0.007 nmol X g-1 X min-1) is even lower than for the liver enzyme. The liver and brain enzymes appear to be less specific than the kidney enzyme with respect to competitive inhibition by insulin and glucagon. Cholecystokinin octapeptide is less inhibitory than the other peptides even though it shares a common C-terminal pentapeptide with G34. These findings are consistent with in vivo studies which have demonstrated that the dog kidney is an important site for extraction and degradation of endogenous dog gastrin but there is little or no hepatic removal of G34.

Topics: Animals; Brain; Endopeptidases; Gastrins; Glucagon; Insulin; Kidney; Kinetics; Liver; Organ Specificity; Protein Precursors; Rats; Rats, Inbred Strains; Sincalide