Biomedical subjects

D J Garry

Publications and source records attributed to D J Garry.

25 records · Page 2Linked to original sources

PubMed1989

Effects of islet hormones on amylase secretion and localization of somatostatin binding sites.

The interaction of insulin and somatostatin on amylase secretion was examined in the isolated perfused rat pancreas. Exogenous insulin (10 mU/ml) significantly potentiated cholecystokinin- (CCK; 0.5 mU/ml) stimulated amylase secretion (12.47 +/- 2.9 micrograms/ml, n = 7). Glucose (16.7 mM) stimulated endogenous insulin secretion (523 +/- 66 microU/ml) and also significantly enhanced CCK-stimulated amylase secretion (13.41 +/- 2.8 micrograms/ml, n = 11). When somatostatin was included in the perfusion media, containing insulin and CCK, amylase secretion was reduced to 3.17 +/- 0.83 micrograms/ml (n = 7), a level comparable to that of CCK-stimulated amylase secretion alone. Similarly, addition of exogenous somatostatin to perfusion media, containing 16.7 mM glucose and CCK, reduced amylase secretion to 4.29 +/- 1.09 micrograms/ml (n = 9). The effect of somatostatin and insulin on carbamylcholine-stimulated amylase secretion was also examined. Exogenous insulin (50 mU/ml) potentiated carbamylcholine- (10(-8) M) stimulated amylase secretion, and addition of exogenous somatostatin to the media containing both insulin and carbamylcholine suppressed the insulin potentiation. Uptake of 125I-[Tyr11]somatostatin in the perfused pancreas was saturable as it decreased significantly with the addition of excess unlabeled somatostatin. Autoradiograms revealed uptake of the ligand by both the endocrine islets and the exocrine pancreas with the highest density of grains observed over the acini. These results support the hypothesis that islet peptides modulate the exocrine pancreas, that somatostatin inhibits amylase secretion by inhibiting the action of insulin, and that somatostatin may act directly on the exocrine pancreas via specific receptors on acinar cells.

Amylases↗

PubMed1988

Ultrastructural immunocytochemical localization of L-glutamate decarboxylase and GABA in rat pancreatic zymogen granules.

The ultrastructural immunohistochemical localization of gamma aminobutyric acid (GABA) and its regulating enzymes, L-glutamate decarboxylase (GAD) and gamma aminobutyrate-alpha-ketoglutarate transaminase, was determined utilizing an immunogold post-embedding protocol in pancreatic exocrine tissue. Within the acinar cell, GABA and its biosynthetic enzyme, GAD, were localized in zymogen granules. Quantitative analysis of the GABA immunoreactivity in the acinar cell revealed 1.7 +/- 0.5 gold particles/micron2 over the cytoplasm, 36.6 +/- 14.1 gold particles/micron2 over the zymogen granules, and 2.9 +/- 2.1 gold particles/micron2 over the mitochondria. Quantitative analysis of the distribution of colloidal gold particles, representing glutamate decarboxylase immunoreactivity in the acinar cells, revealed 38.4 +/- 2.5 gold particles/micron2 over the zymogen granules, 4.7 +/- 1.1 gold particles/micron2 over the mitochondria and 6.3 +/- 0.5 gold particles/micron2 over the remainder of the cytoplasm. Substitution of normal sheep serum for the sheep anti-glutamate decarboxylase serum revealed a significant (p less than 0.001) decrease of the colloidal gold particle distribution over the zymogen granules and cytoplasmic compartments of the acini. Gamma aminobutyrate-alpha-ketoglutarate transaminase, the catabolic enzyme for GABA, was not detected in the mitochondria, zymogen granules, and cytoplasm of the acinar cell, suggesting that GABA is not catabolized within the acinar cell. Preabsorption and substitution controls resulted in an absence of labeling. These results suggest that GABA may act extracellularly and/or have a role within the zymogen granule in the exocrine pancreas.

Animals↗

PubMed1988

Radioimmunoassay for rat pancreatic alpha-amylase and the effect of Phe-Met-Arg-Phe-amide on amylase secretion in the isolated perfused rat pancreas.

In this study a radioimmunoassay was developed to measure secreted amylase from the isolated perfused rat pancreas. Using Sephadex G-75 gel chromatography, rat pancreatic amylase was purified to a single migrating protein band as determined by SDS polyacrylamide gel electrophoresis. Specificity of a rat pancreatic amylase antiserum, raised in rabbits, was determined using immunodiffusion, immunoelectrophoresis, and immunoblotting techniques. Secreted amylase concentrations, obtained using the radioimmunoassay, were not significantly different than those measured with the amylase enzyme assay. The rat pancreatic amylase radioimmunoassay was used to measure the amylase secretion in the isolated perfused rat pancreas. Phe-Met-Arg-Phe-amide (FMRF-NH2) immunoreactivity has been shown to be co-localized with pancreatic polypeptide in the rat pancreatic islet, and evidence suggests that islet peptides modulate amylase secretion from the exocrine pancreas. In the present study, FMRF-NH2 significantly (p less than 0.05) suppressed cholecystokinin (CCK)-stimulated amylase secretion by 55%. The average pancreatic amylase secretion in response to CCK was 10.89 +/- 2.0 micrograms/ml/min (n = 6); with the addition of FMRF-NH2, CCK-stimulated amylase secretion was reduced to 4.79 +/- 1.6 micrograms/ml/min (n = 6). These results are consistent with the insuloacinar hypothesis in that an FMRF-NH2-like substance in the islet may act to modulate the exocrine pancreas.

Animals↗

PubMed1988

Cellular and subcellular immunolocalization of L-glutamate decarboxylase in rat pancreatic islets.

The cellular and subcellular distribution of L-glutamate decarboxylase (GAD), the biosynthetic enzyme for gamma-aminobutyric acid (GABA), was determined immunohistochemically in rat pancreatic islet using light and electron microscopic techniques. The cellular distribution of GAD was determined at the light microscopic level using an elution/re-staining protocol and a computerized digital image processing technique. At this level of resolution, immunofluorescent GAD was observed to be co-localized with immunofluorescent insulin in the islet B-cells and absent in both the A-cells, which contained glucagon, and the D-cells, which contained somatostatin. Subcellular localization of GAD was determined using an electron microscopic, colloidal gold post-embedding protocol and was compared to insulin immunoreactivity in serial sections of the same B-cell. In the same islet B-cell, GAD immunoreactivity appeared predominantly in the extragranular cytoplasm, whereas insulin immunoreactivity was associated with the secretory granules. Quantitative analysis of GAD immunoreactivity in the B-cell revealed 15.3 +/- 1.8 gold particles/micron2 in the cytoplasm, 1.7 +/- 0.2 gold particles/micron2 in the secretory granules, and 0.4 +/- 0.4 gold particles/micron2 in the mitochondria. The results of this study, localization of the biosynthetic enzyme for GABA to the B-cell cytoplasmic compartment and its absence in the secretory granules which contain insulin, are compatible with the hypothesis that GABA functions as an intracellular mediator of B-cell activity.

Animals↗

PubMed1987

Ultrastructural localization of gamma amino butyric acid immunoreactivity in B cells of the rat pancreas.

The purpose of this study was to determine the ultrastructural localization of gamma amino butyric acid (GABA) within the B cell of isolated rat islets, particularly with respect to the B cell secretion granules. GABA immunoreactivity was localized ultrastructurally, with colloidal gold, in the B cells and absent in the A cells and D cells. Quantitative analysis of the colloidal gold particle distribution in the B cell revealed 29.5 +/- 5.2 gold particles/micron 2 in the nuclei, 29.3 +/- -6.9 gold particles/micron 2 in the mitochondria and 4.9 +/- 1.0 gold particles/micron 2 in the secretion granules. Particle density in the remainder of the cytoplasm was 41.9 +/- 4.1 particles/micron 2. The paucity of gamma amino butyric acid in the B cell secretion granules differs from observations on gamma amino butyric acidergic neurons, where there is an accumulation of gamma amino butyric acid within the neurosecretory vesicles. These findings indicate that if gamma amino butyric acid is released from the islet, then it is by a nongranular mechanism. In addition, the results are compatible with the hypotheses that gamma amino butyric acid within the B cell functions in the regulation of insulin biosynthesis, and/or functions as an alternative energy source for the B cell through the gamma amino butyric acid shunt.

Animals↗

PubMed1987

Immunoreactive GABA transaminase within the pancreatic islet is localized in mitochondria of the B-cell.

Subcellular localization of gamma aminobutyrate-alpha-ketoglutarate transaminase (GABA-T) in the pancreatic islets of Langerhans was determined by use of an electron microscopic, immunogold post-embedding protocol. The objective of this study was to define the islet cell distribution and subcellular localization of GABA-T. Within the islet, GABA-T was found only in the B-cells and was localized in mitochondria; 78 mitochondria contained 336 gold particles, whereas 245 secretory granules contained only 18 gold particles. Although studies utilizing either the isolated perfused pancreas or cultured islets have shown that exogenous GABA modulates D-cell secretion, in this study immunoreactive GABA-T, the catabolic enzyme for GABA, was not detectable in A- and D-cells of the islet. Control studies substituting normal rabbit serum for the GABA-T antiserum resulted in absence of labeling. These results indicate that the high concentration of GABA present in islet B-cells is catabolized by GABA-T in the mitochondrial compartment, consistent with the possibility that GABA functions as a mediator of B-cell activity.

4-Aminobutyrate Transaminase↗

PubMed1986

Immunohistochemical colocalization of GABA and insulin in beta-cells of rat islet.

gamma-Aminobutyric acid (GABA) is found in high concentrations in the pancreatic islet. In addition, enzymes regulating the level of GABA (L-glutamate decarboxylase and GABA-alpha-ketoglutarate transaminase) have been immunohistochemically localized in the medullary cells of the islet. In this study, an immunofluorescence and elution/restaining protocol is used to determine the distribution of GABA and either insulin, glucagon, or somatostatin in a tissue section. GABA was not detected within the islet alpha- or delta-cells but was determined to be localized within the insulin-containing beta-cells.

Animals↗