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This study describes the sequential morphological changes in pancreatic islets from 1-, 6-, and 18-month-old male eSS rats, as compared to aged-matched control animals. Spontaneous diabetes mellitus was confirmed in 6- and 18-month-old eSS rats after an oral glucose tolerance test. Light microscopic immunocytochemical and morphometric techniques were used to study islet-cell populations. The pancreas was normal, and the morphometric methods did not reveal significant changes in islets from 1-month-old rats. However, 6-month-old eSS animals showed disruption of islet architecture and fibrosis in the stroma. The volume density (Vvi) of endocrine tissue and the Vvi and percentage of B cells were increased, whereas the Vvi of exocrine tissue and the Vvi and percentage of A cells were diminished. Eighteen-month-old eSS rats also exhibited conspicuous islet lesions. Nevertheless, the Vvi of endocrine tissue and the Vvi and percentage of B cells were diminished, while the Vvi of exocrine tissue and the Vvi and percentage of D cells were increased. Our results provide further quantitative evidence for the sequential morphological events occurring in the pancreatic islets of a useful animal model of diabetes mellitus.

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The perimeter, cell area and volume density (Vvi) of B cells and exocytotic images present in these cells were measured in rat pancreas perfused with 3.3 or 16.6 mM glucose. Four minutes after the beginning of 16.6-mM glucose perfusion and coincident with the appearance at the apex of the first phase of insulin secretion, all these parameters underwent a significant increase. The changes observed in the perimeter, the cell area and the Vvi of B cells suggest an increase in their surface area. An imbalance in the rate of endocytosis:exocytosis processes with a relative predominance of the latter would increase the length of the plasma membrane and could be responsible, at least partly, for the changes in the B cell size.

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The present study describes the cytopathology of the pancreatic islets in 18-old male eSS rats with spontaneous diabetes mellitus as compared to aged-matched normal animals. Light-microscopic immunocytochemical and morphometric techniques were used to study islet-cell populations, while quantitative methods were employed specifically for the analysis of B-cell ultrastructure. The diabetic rats showed disruption of the islet structure and fibrosis in the stroma. The volume density (Vvi) of endocrine tissue and the Vvi and percentage of B cells were diminished, whereas the Vvi of exocrine tissue and the Vvi and percentage of D cells were increased. The number of medium and large islets as well as their mean volume (micron3) decreased in these animals. Pancreatic B cells from eSS rats showed an increase in the Vvi of endoplasmic reticulum, immature secretory granules and lysosomes. Conversely, the Vvi of total secretory granules and microtubules appeared diminished. The current observations contribute to our understanding of this useful animal model of diabetes mellitus, in the attempt to clarify the pathogenesis of the disease.

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Insulin secretion and B-cell calcium distribution, assessed with the pyroantimonate precipitation technique, were studied in rat pancreases perfused with glucose (3.3 or 16.6 mM) alone or together with verapamil or trifluoperazine (TFP). Total calcium pyroantimonate precipitates (CPP), and those bound to every B cell structure, at every sampling period, were larger at 16.6 mM glucose concentration. The largest percentage of CPP was located, at early stages of the glucose stimulatory period, mainly within the clear halo of the B granules, while later on they shifted to the plasma membrane. Verapamil and TFP diminished the second phase of glucose-induced insulin release and greatly affected the above mentioned pattern of B cell CPP distribution. The main changes consist in a diminution in the total number of CPP all throughout the perfusion-time as well as an alteration in the percentage distribution of the CPP within the different B cell organelles, i.e., an early diminution in the CPP present in the B granules and of those attached to the plasma membrane and to the mitochondria at the end of the perfusion, were the most striking changes observed. The results suggest that during the glucose stimulus, different B cell structures take over the control of available calcium within the cell, following a chronological sequence. Such sequence might be determined by the different Ca2(+)-set point of those structures. Intracellular provision of calcium might be sufficient to maintain the early phase of insulin secretion when the cation entrance is blocked. Conversely, this substitution might not be enough to sustain the second phase of insulin release. The different amounts of CPP in every B cell organelle, besides their buffering capacity to control free Ca2+ availability, might also be coupled to a regulatory role of the cation upon their respective metabolic functions. In some cases, this latter effect may be the main role for the cation distribution. Our results support the concept that the level of free calcium, acting as the coupler for the stimulus:secretion process, might be regulated by different calcium pools located in the B cell.

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Insulin secretion and the pattern of calcium distribution in B cells, assessed with the pyroantimonate precipitation technique, were simultaneously studied in rat pancreases perfused with 3.3 and 16.6 mM glucose solutions of pH 7.4 and 7.8. We have previously demonstrated the blocking effect of the latter pH upon glucose-induced insulin secretion. Glucose (16.6 mM) caused an increase in the total number of calcium pyroantimonate precipitates (CPP), as well as their number bound to different B cell structures, at every sampling period studied, with respect to the 3.3 mM glucose experiments. Extracellular alkalosis strongly inhibited both phases of the B cell response to the glucose stimulus, and greatly affected the distribution of CPP in the cells with respect to the pH 7.4 ones. During the first phase of glucose induced-insulin secretion, most of the CPP appeared within B granules at pH 7.4, while on the development of the second phase of secretion, they appeared mainly attached to the cell plasma membranes. Conversely, in pH 7.8 experiments, at the first minutes of the glucose challenge, CPP appeared principally located in the cytoplasm, being almost absent from the plasma membrane during the second phase of insulin secretion. These observations suggest that during the glucose stimulus, the cell calcium distribution within the B cells followed a clear chronological sequence. Such sequence might be determined, at least in part, according to the different Ca2(+)-set points of the different B cell structures. In our case, the extracellular alkalosis might interfere with the normal intracellular calcium fluxes, which in consequence might impair release of insulin by affecting several B cell functions.

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RESUMEN

Insulin secretion and B-cell calcium distribution, assessed with the pyroantimonate precipitation technique, were studied in rat pancreases perfused with glucose (3.3 or 16.6 mM) alone or together with verapamil or trifluoperazine (TFP). Total calcium pyroantimonate precipitates (CPP), and those bound to every B cell structure, at every sampling period, were larger at 16.6 mM glucose concentration. The largest percentage of CPP was located, at early stages of the glucose stimulatory period, mainly within the clear halo of the B granules, while later on they shifted to the plasma membrane. Verapamil and TFP diminished the second phase of glucose-induced insulin release and greatly affected the above mentioned pattern of B cell CPP distribution. The main changes consist in a diminution in the total number of CPP all throughout the perfusion-time as well as an alteration in the percentage distribution of the CPP within the different B cell organelles, i.e., an early diminution in the CPP present in the B granules and of those attached to the plasma membrane and to the mitochondria at the end of the perfusion, were the most striking changes observed. The results suggest that during the glucose stimulus, different B cell structures take over the control of available calcium within the cell, following a chronological sequence. Such sequence might be determined by the different Ca2(+)-set point of those structures. Intracellular provision of calcium might be sufficient to maintain the early phase of insulin secretion when the cation entrance is blocked. Conversely, this substitution might not be enough to sustain the second phase of insulin release. The different amounts of CPP in every B cell organelle, besides their buffering capacity to control free Ca2+ availability, might also be coupled to a regulatory role of the cation upon their respective metabolic functions. In some cases, this latter effect may be the main role for the cation distribution. Our results support the concept that the level of free calcium, acting as the coupler for the stimulus:secretion process, might be regulated by different calcium pools located in the B cell.

RESUMEN

Insulin secretion and the pattern of calcium distribution in B cells, assessed with the pyroantimonate precipitation technique, were simultaneously studied in rat pancreases perfused with 3.3 and 16.6 mM glucose solutions of pH 7.4 and 7.8. We have previously demonstrated the blocking effect of the latter pH upon glucose-induced insulin secretion. Glucose (16.6 mM) caused an increase in the total number of calcium pyroantimonate precipitates (CPP), as well as their number bound to different B cell structures, at every sampling period studied, with respect to the 3.3 mM glucose experiments. Extracellular alkalosis strongly inhibited both phases of the B cell response to the glucose stimulus, and greatly affected the distribution of CPP in the cells with respect to the pH 7.4 ones. During the first phase of glucose induced-insulin secretion, most of the CPP appeared within B granules at pH 7.4, while on the development of the second phase of secretion, they appeared mainly attached to the cell plasma membranes. Conversely, in pH 7.8 experiments, at the first minutes of the glucose challenge, CPP appeared principally located in the cytoplasm, being almost absent from the plasma membrane during the second phase of insulin secretion. These observations suggest that during the glucose stimulus, the cell calcium distribution within the B cells followed a clear chronological sequence. Such sequence might be determined, at least in part, according to the different Ca2(+)-set points of the different B cell structures. In our case, the extracellular alkalosis might interfere with the normal intracellular calcium fluxes, which in consequence might impair release of insulin by affecting several B cell functions.

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Using the pyroantimonate technique, the ultracytochemical distribution of calcium within B cells was studied in isolated rat pancreatic islets incubated during 5, 15 and 30 min with 8.3 mM glucose alone or together with 76 microgram glicazide. Glucose alone produced a continuous increment in the total number of calcium pyroantimonate precipitates (CPP) throughout the incubation period studied. The CPP were mainly associated to the cytoplasmic matrix and the secretory granules at 5 and 15 min and almost evenly distributed between these structures and the plasma membrane at 30 min. Gliclazide plus glucose produced a significant increment, above the glucose values, of the total CPP at 5 min and a later decrease of such values at 15 and 30 min. At 5 min, the incremented total CPP was mainly associated to the secretory granules and the cytoplasmic matrix. The increment in CPP preceded the largest effect of gliclazide on insulin secretion. The latter diminution of CPP induced by gliclazide could contribute to the failure of this drug, as well as other oral hypoglycemic agents, to elicit a second phase of insulin secretion. Changes induced by gliclazide upon B-cell CPP content and distribution might suggest that beyond the effective role of cytosolic calcium in the control of insulin secretion, the cation might reach a threshold concentration in some cell structures to assure the normal development of the secretory process.

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Cell-tight junctions were counted in rat isolated islets incubated for different periods of time in the presence of different extracellular glucose concentrations. The number of tight junctions increased as a function of the length of the incubation period and the concentration of glucose. These results would suggest the involvement of tight junctions in the regulatory process of glucose-induced insulin secretion. Key words: Tight junctions, islet cell ultrastructure, isolated islets, islet incubation, glucose-induced insulin secretion, islet cell secretory function.

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Cell-tight junctions were counted in rat isolated islets incubated for different periods of time in the presence of different extracellular glucose concentrations. The number of tight junctions increased as a function of the length of the incubation period and the concentration of glucose. These results would suggest the involvement of tight junctions in the regulatory process of glucose-induced insulin secretion. Key words: Tight junctions, islet cell ultrastructure, isolated islets, islet incubation, glucose-induced insulin secretion, islet cell secretory function.

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Dentro de la patologia urológica, la estenosis uretral masculina es uno de los capítulos que mayores controversias sucita. Básicamente han sido tres las fórmulas propuestas en su terapéutica: dilataciones periódicas, uretrotomia ciega y numerosisimas técnicas de uretroplastia cruenta. Durante la última década y merced sobre todo a los trabajos desarrollados por Sachse, la uretrotomia interna, bajo visión directa y con corte frio, ha surgido con fuerza mostrándose, cada vez más, como alternativa validad frente a otros procederes más agresivos. . Presentamos en esta revisión, los resultados obtenidos en 45 pacientes tratados con esta técnica

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RESUMEN

Dentro de la patologia urológica, la estenosis uretral masculina es uno de los capítulos que mayores controversias sucita. Básicamente han sido tres las fórmulas propuestas en su terapéutica: dilataciones periódicas, uretrotomia ciega y numerosisimas técnicas de uretroplastia cruenta. Durante la última década y merced sobre todo a los trabajos desarrollados por Sachse, la uretrotomia interna, bajo visión directa y con corte frio, ha surgido con fuerza mostrándose, cada vez más, como alternativa validad frente a otros procederes más agresivos. . Presentamos en esta revisión, los resultados obtenidos en 45 pacientes tratados con esta técnica (AU)

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Changes induced in the endocrine pancreas were studied in rats 14 days after ovariectomy. The study was performed in age matched control (C) and ovariectomized (O) rats. The pancreases were stained using the p-aldehyde-fuchsin and the peroxidase (using antiinsulin serum) methods. Morphometric analysis was done using the Weibel technique. The results obtained show a diminution in the total volume of the endocrine pancreas accompanied by an increment in the individual islet volume following the ovariectomy. The last change would be the consequence of an increment in the number of islet cells (mainly the B cell population), without change in cellular volume, i.e., hyperplasia without hypertrophy. These changes might explain the apparent discrepancy regarding the effect of ovariectomy upon insulin secretion when this process is studied using either the total pancreas preparation or the isolated islet model.

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The acute ultrastructural changes induced by glucose upon the B cells were studied in normal rat pancreas perfused with 3.3 and 16.6 mmol/l glucose. A significant increment in the volume of the RER, microtubule, mitochondria, lysosomes and B granules was induced by 16.6 mmol/l glucose, while no significant changes were detected in the total B cell volume or in the size of the nucleus, cytoplasm and Golgi complex. The number of secretory granules was greatly reduced in B cells obtained from pancreas perfused with 16.6 mmol/l glucose, while its diameter was significantly enhanced. In these cells both, the number of pale granules as well as those attached to the cell membrane, were increased. All these data suggest that the increase in the extracellular glucose concentration produces not only the classical biphasic secretion on insulin, but also induces significant and measurable changes in the volume of several B cell organelles. Such ultrastructural changes correlate well with the well-known effect of glucose upon the metabolism of these cells.

Asunto(s)

RESUMEN

Changes induced in the endocrine pancreas were studied in rats 14 days after ovariectomy. The study was performed in age matched control (C) and ovariectomized (O) rats. The pancreases were stained using the p-aldehyde-fuchsin and the peroxidase (using antiinsulin serum) methods. Morphometric analysis was done using the Weibel technique. The results obtained show a diminution in the total volume of the endocrine pancreas accompanied by an increment in the individual islet volume following the ovariectomy. The last change would be the consequence of an increment in the number of islet cells (mainly the B cell population), without change in cellular volume, i.e., hyperplasia without hypertrophy. These changes might explain the apparent discrepancy regarding the effect of ovariectomy upon insulin secretion when this process is studied using either the total pancreas preparation or the isolated islet model.

RESUMEN

The acute ultrastructural changes induced by glucose upon the B cells were studied in normal rat pancreas perfused with 3.3 and 16.6 mmol/l glucose. A significant increment in the volume of the RER, microtubule, mitochondria, lysosomes and B granules was induced by 16.6 mmol/l glucose, while no significant changes were detected in the total B cell volume or in the size of the nucleus, cytoplasm and Golgi complex. The number of secretory granules was greatly reduced in B cells obtained from pancreas perfused with 16.6 mmol/l glucose, while its diameter was significantly enhanced. In these cells both, the number of pale granules as well as those attached to the cell membrane, were increased. All these data suggest that the increase in the extracellular glucose concentration produces not only the classical biphasic secretion on insulin, but also induces significant and measurable changes in the volume of several B cell organelles. Such ultrastructural changes correlate well with the well-known effect of glucose upon the metabolism of these cells.