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Biomedical subjects

P Meda

Publications and source records attributed to P Meda.

At least 109 records · Page 6Linked to original sources
PubMed1986

Heterogeneity and contact-dependent regulation of hormone secretion by individual B cells.

A reverse hemolytic plaque assay was developed to visualize insulin release from individual adult pancreatic B cells. Cells obtained by mechanical dispersion of isolated rat islets of Langerhans were mixed with protein A-coated sheep red blood cells and incubated in the presence of an anti-insulin serum, under conditions known to affect insulin release. The cell mixture was further incubated with complement and finally fixed. Insulin release was revealed by the presence of hemolytic plaques which resulted from the complement-mediated lysis of red blood cells bearing insulin-anti-insulin complexes bound to protein A. Quantitation of hemolytic plaques around trypan blue-unstained and immunohistochemically identified B cells showed that stimulation of insulin release results in the recruitment of increasing numbers of secreting B cells as well as in the enhanced response of individual B cells. Reverse changes occur upon inhibition of insulin release. Comparison of freshly dispersed and one-day-cultured preparations did not reveal significant differences in the secretory response of undamaged B cells. In both preparations, single B cells responded to secretagogues in smaller proportions and to a lesser extent than clusters in which B cells had either maintained or restored contacts and junctional communication with their neighbours. However, the overall preponderant response of clusters was less than expected from the number of individually secreting B cells they contained. The data show that B cells are heterogeneous in terms of their ability to release insulin and provide evidence that cell-to-cell adhesion and/or junctional communication regulate hormone secretion from individual B cells.

Animals↗
PubMed1986

Blockage of cell-to-cell communication within pancreatic acini is associated with increased basal release of amylase.

To assess whether junctional coupling is involved in the secretory activity of pancreatic acinar cells, dispersed rat acini were incubated for 30 min in the presence of either heptanol (3.5 mM) or octanol (1.0 mM). Exposure to either alkanol caused a marked uncoupling of the acinar cells which, in control acini, were extensively coupled. Uncoupling was associated with an increased basal release of amylase that was at least twice that of controls. By contrast, carbamylcholine (10(-5) M)-induced maximal amylase secretion, cytosolic pH, and free Ca2+, as well as the structure of gap junctions joining the acinar cells, were unaffected. Both uncoupling and the alteration of basal secretion were already observed after only 5 min of exposure to heptanol, they both persisted throughout the 30-min exposure to the alkanols, and were reversible after removal of either heptanol or octanol. Since neither of the two uncouplers appeared to alter unspecifically the secretory machinery and the nonjunctional membrane of acinar cells, the data are consistent with the view that junctional coupling participates in the control of the basal secretion of acinar cells.

Alcohols↗
PubMed1986

Direct identification of electrophysiologically monitored cells within intact mouse islets of Langerhans.

Cells found to be electrically active within microdissected mouse islets of Langerhans perifused with high (greater than or equal to 11.1 mM) glucose concentrations were labeled by injecting Lucifer yellow through the recording electrode. After fixation, these cells were located by fluorescence microscopy on sections serially cut throughout the islets and were subsequently identified by immunofluororescence staining with specific anti-islet hormone sera. Electrophysiologic control confirmed that the electrode tip had remained within the same cell throughout the experiment and showed that Lucifer yellow labeling did not affect the electrical activity of the impaled cell. Upon individual impalements, Lucifer yellow labeled either the impaled cell alone or this cell and some of its neighbors to which it was dye coupled. Immunofluorescence staining of the Lucifer yellow-labeled cells revealed that glucose-induced electrical activity was recorded from individual B-cells or groups of dye-coupled B-cells as well as from A-cells coupled to B-cells.

Action Potentials↗
PubMed1984

Dispersed adult rat pancreatic islet cells in culture: A, B, and D cell function.

The availability of suitably characterized dispersed islet cell preparations may assist in studies of islet function. Since freshly dispersed adult rat islet cells failed to respond appropriately to secretagogues (no alteration in insulin, glucagon, or somatostatin release after glucose change; modest response to IBMX), these cells were established in primary monolayer culture. We then tested the hypothesis that islet function is at least partially determined by islet structure. B cells which had attached to Petri dishes during a culture period of four days were well preserved at the ultrastructural level, with mitochondria clustered at the cell face attached to the Petri dish and secretory granules concentrated towards the portion of the cell facing the medium. Since it was not possible to estimate cellular hormone content or hormone release as a function of the number of specific types of cells, fractional rates of release and hormone content ratios were compared with those for intact islets maintained in culture in parallel. Whereas the ratio of somatostatin:insulin content was similar for islets and cells (approximately 0.7:100), the dispersed cell population appeared depleted in glucagon (glucagon:insulin ratios being 17:100 for islets and 4:100 for cells) reflecting either degranulation or relative loss of A cells. In contrast to the lack of responsiveness seen with freshly dispersed islet cells, the cultured cells released insulin in response to glucose and glucose plus IBMX in a fashion comparable to that seen with cultured islets. Proinsulin biosynthesis (incorporation of [3H] leucine) was higher in cultured cells than islets. Somatostatin release was lower from dispersed cells than from islets while the opposite was true for glucagon.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1984

The topography of electrical synchrony among beta-cells in the mouse islet of Langerhans.

beta-Cells in microdissected islets of Langerhans produce rhythmical bursts of electrical activity. This was monitored with two micro-electrodes simultaneously and the frequency and phase (collectively referred to as synchrony) of the two signals was investigated. At any instant two impaled cells produced bursts of the same frequency even when separated by up to 400 micron. When the electrode tips were separated by less than about 20 micron and current injection showed the cells to be ionically coupled the two signals were in phase and had almost identical shape. The phase relations between cells further apart were variable, the leading cell usually being located deeper within the islet than the other impaled cell. Increasing the glucose concentration increased electrical activity, reduced any phase lags and made the shape of the bursts more similar. There was less lag between the responses from two cells when the glucose concentration was suddenly reduced, than when it was suddenly increased. Qualitatively similar observations were made in glibenclamide-treated mice, a treatment previously shown to increase dye coupling between islet cells. However, the response to increasing glucose concentrations showed less phase lag; likewise the phase lag between bursts was reduced. Furthermore the response to current injected into one cell could be detected at much larger distances (up to 80 micron) than in control islets. This suggests that electrical coupling of beta-cells was improved in sulphonylurea-treated mice. Electron microscopy of both control and glibenclamide-treated mouse islets fixed at the end of each electrophysiological experiment showed the region impaled by the electrodes to be well preserved and, whenever the electrodes penetrated at least 20 micron into the islet, to contain a large proportion of beta-cells. The data support the view that, within an islet, most but not necessarily all cells are electrically synchronized, and that the coupling can be modulated by natural and pharmacological secretagogues.

Action Potentials↗
PubMed1983

Short and reversible uncoupling evokes little change in the gap junctions of pancreatic acinar cells.

Three different preparations of mouse pancreatic fragments where all the cells tested electrophysiologically showed (a) complete electrical coupling (control), (b) complete uncoupling (after 1-to 2-min exposure to 100% CO2), or (c) complete recoupling (1-2 min after removal of 100% CO2) were fixed, with the electrodes in situ, with 0.2% glutaraldehyde and freeze-fractured for quantitative analysis of acinar cell gap junctions. No obvious difference was observed between gap junctions of coupled and uncoupled acinar cells. However, quantitation revealed a small (2.3-5.6%) increase in particle diameter and spacing within junctions of uncoupled cells. Such increase was rapidly reversed upon cell recoupling. In all preparations, most of the gap junctions were made up of disordered arrays of particles but a few of them showed a more tight packing of their particles of which most had lost the usual globular appearance. These "amorphous" gap junctions had larger particle diameter but smaller particle spacing than the other gap junctions and these parameters were not modified during cell uncoupling. However, "amorphous" gap junctions were more frequent in the latter condition.

Animals↗
PubMed1983

Cellular mechanisms of insulin release. Effects of retinoids on rat islet cell-to-cell adhesion, reaggregation, and insulin release.

We studied the effects of retinoids on islet cell-to-cell adhesiveness and glucose-induced insulin release from rat islets. For adhesion studies, islets were dispersed using low concentrations of trypsin. Thirteen cis-retinoic acid (13 cis-RA) was added to a suspension of 15 X 10(5) islet cells and adhesion of cells was quantitated using a hemocytometer. For functional studies, we measured biphasic insulin release from collagenase-isolated perifused islets, dispersed cells, and single large aggregates (clumps) of islet cells. Thirteen cis-RA (10(-4) M) stimulated insulin secretion at 9.7, 12.5, 16.7, and 27.7 mM glucose. Maximal effects of 13 cis-RA (174% of control) were evident during second phase release at 9.7 mM glucose. Thirteen cis-RA (10(-7) and 10(-6) M) caused cells to adhere to each other, and at higher concentrations, 13 cis-RA caused dispersed cells to reaggregate into a single clump. These retinoid-induced clumps were perifused in a Bio-Gel P-2 gel column. Secretion from the clump was twofold greater than from an equal number of perifused dispersed cells. Electron microscopic and freeze-fracture examination of the clump showed reaggregated cells to be intact and the presence of gap junctions between cells. In conclusion, 13 cis-RA has marked effects on islet cell-to-cell adhesiveness. Trypsin-dispersed cells reaggregated by 13 cis-RA have anatomical contacts and secrete more insulin as an aggregate than as dispersed cells. Thirteen cis-RA increases insulin release possibly by increasing adhesion or interactions between beta-cells.

Animals↗
PubMed1983

In vivo modulation of gap junctions and dye coupling between B-cells of the intact pancreatic islet.

The extent of gap junctions and dye coupling between insulin-producing B-cells was analyzed on islets of Langerhans isolated from adult rats treated for one day with glibenclamide, an insulin secretagogue, or diazoxide, a blocker of insulin release, or a combination of the two drugs. Glibenclamide treatment was associated with a marked depletion of the islet insulin content, an effect which was blocked by pretreatment of the rats with diazoxide. Diazoxide alone caused a marked increase in the plasma glucose level, and a decrease in the level of circulating insulin and in the hormone content of the B-cells. Quantitative analysis showed that (1) under control conditions, B-cells are connected by minute gap junctions (as evaluated on freeze-fracture replicas) and show a nonuniform and apparently restricted dye coupling (as determined by microinjection of the low-molecular-weight fluorescent probe Lucifer Yellow CH); (2) each of the three treatments tested significantly increased the relative and absolute gap junction area of the B-cells and the number of detectable, dye coupled B-cells per microinjection. After treatment with glibenclamide alone or with diazoxide plus glibenclamide, a 1.5-1.8-fold increase in gap junction area and a 2.7-3.7-fold increase in the number of dye-coupled B-cells were observed. In contrast, following treatment with diazoxide alone, gap junctions and dye coupling were found increased 1.8 and 8.7 times, respectively, as compared with control values.

Animals↗
PubMed1982

Direct communication of homologous and heterologous endocrine islet cells in culture.

The transfer of 6-carboxyfluorescein between islet cells in monolayer culture was observed by fluorescence microscopy, and the endocrine cells involved in this transfer were identified by immunohistochemistry and electron microscopy. The results show that carboxyfluorescein was directly exchanged between homologous B-cells and also between B- and A- or D-cells. Successive microinjections of the probe into different cells of the same cluster showed the existence of separate territories, each formed by 2-8 communicating cells. Intercellular communication was not observed after every dye microinjection, and communicating and noncommunicating islet cells were found to coexist within the same cluster. The data indicate that the exchange of exogenous cytoplasmic molecules occurs between different types of endocrine islet cells. However, within a single cluster, all islet cells are not metabolically coupled to one another, at a given time.

Animals↗
PubMed1982

The possible importance of contact between pancreatic islet cells for the control of insulin release.

Insulin secretion was studied using adult rat intact islets, dissociated isolated islet cells, and isolated islet cells that had been allowed to reaggregate. The three preparations were maintained for 2 h in tisue culture medium at 37 C in order to allow for equilibration after the isolation procedures and to permit restoration of contacts between the reaggregated cells. The isolated cells appeared well preserved at the ultrastructural level. Evidence for intimate contacts between the reaggregated cells was demonstrated by the reappearance of gap junctions between adjacent cells. Basal insulin release (2.8 mM glucose) during 30 min was elevated in the isolated cell suspension but was restored to the level found in intact islets when isolated cells from the same population were reaggregated. The elevated basal release did not appear to be due to cell damage since there was a commensurate increase in the rate of insulin biosynthesis relative to intact islets. Although there was a marked stimulation of insulin release from the intact islets at 16.7 mM glucose, the response was smaller in the reaggregated cells and in the isolated cell suspension. All three preparations responded to elevated cAMP levels evoked by glucagon in the presence of 16.7 mM glucose. Similar results were obtained when insulin release was studied in various preparations derived from newborn rat pancreatic endocrine cells. Thus, basal insulin release was elevated in isolated cells, whereas cultures with cell contacts displayed lower basal insulin release. Taken together, the restoration of lower basal insulin release and the parallel appearance of contacts between the reaggregated cells suggest that these two phenomena are interrelated. Thus, cell contacts may be important in regulating basal insulin release. Whether such regulation is a consequence merely of cell association or of direct communication between cells remains to be established.

Aging↗
PubMed1981

[Heterocellular coupling in cultures of endocrine pancreatic cells].

The intracellular injection of 6-carboxyfluorescein in endocrine cells of cultured Rat pancreas followed by the characterization, by electron microscopy, of the injected cells shows that the dye is transferred from the injected cell into adjacent cells, presumably by gap junctions (nexus) = intercellular coupling. The transfer occurs not only between insulin-containing cells (B-cells), but also between B-cells and glucagon containing cells (A-cells) or somatostatin-containing cells (D-cells). The demonstration of homo- and heterocellular coupling in the endocrine pancreas supports the hypothesis that a direct exchange of molecules between islet cells may participate in the regulation of the secretory behavior of the islet of Langerhans.

Animals↗
PubMed1981

Co-culture of nerve cells and pancreatic islets.

Nerve cell dissociated from the cerebral cortex of mice were co-cultured with isolated pancreatic islets of Langerhans. After two weeks of co-culture, numerous axons had invaded the islets and differentiated nerve endings were closely juxtaposed to the endocrine islet cells. In contrast, no remnants of nerve cells were observed in isolated islets cultured alone for the same duration. The attachment and/or survival and differentiation of nerve cells were markedly promoted in the presence of islets. The latter effects were probably due to insulin secreted by the islets in the culture medium.

Animals↗
PubMed1981

Heterogeneous distribution of filipin--cholesterol complexes across the cisternae of the Golgi apparatus.

The Golgi apparatus is a key element in the ordered movement of secretory polypeptides from the rough endoplasmic reticulum to the plasma membrane during secretion. It has been shown that cisternae that receive membranes from the reticulum are morphologically similar to the latter and that cisternae liberating secretory granules resemble that plasma membrane. By using an ultrastructural probe for membrane cholesterol, filipin, on freeze-fractured and thin-sectioned exocrine and endocrine pancreatic cells, we have shown that an enrichment in filipin-cholesterol complexes takes places across the stacked cisternae of the Golgi apparatus; the reticulum-related (forming) cisternae are poor in such complexes, but the secretory granule-related (maturing) cisternae contain numerous complexes. Secretory granule membrane is also richly labeled with filipin-cholesterol complexes. The heterogeneous cholesterol distribution in the membranes of the Golgi apparatus, as shown by filipin, emphasizes the polarity of this organelle, in agreement with its role in organizing the traffic of the secretory polypeptides from the rough endoplasmic reticulum to the plasma membrane.

Animals↗
PubMed1981

Tetraethylammonium modifies gap junctions between pancreatic beta-cells.

Gap junctions between pancreatic beta-cells were quantitatively assessed in freeze-fracture replicas of isolated rat islets of Langerhans incubated for 90 min with or without the potassium conductance blocker tetraethylammonium (TEA). The results show that TEA increases the median number of particles per beta-cell gap junction but not the frequency of gap junctions at both nonstimulating and threshold-stimulating concentrations of glucose. TEA increased the relative gap junctional area at both concentrations of glucose. TEA had no effect on insulin release at a basal concentration of glucose but potentiated that release at the threshold glucose level. Thus TEA modifies beta-cell gap junctions independently of its effect on insulin release. However, the junctional changes observed were greater when insulin release was also elevated.

Animals↗
PubMed1980

Gap junction development is correlated with insulin content in the pancreatic B cell.

The development of gap junctions between insulin-containing B cells was quantitatively analyzed in islets of Langerhans isolated from rats treated with the sulfonylurea glibenclamid for 1, 2, or 7 days. Glibenclamid treatment was associated with a marked depletion of the insulin content of B cells and with an increase in the number and size of gap junctions between these cells. A significance correlation was found between these two events.

Animals↗
PubMed1980

Multichannel analysis of intracellular control and intercellular transfer of molecules.

The metabolic regulation and exchanges within intracellular organelles or a cell cluster are studied by multichannel microfluorometry and microinjection of metabolites or tracers. The determination of structure-function relationships relies on the retrieval of cells after microfluorometry, for subsequent morphological evaluation. Rate constants of coenzyme reduction-reoxidation were deduced from a mathematical model of NAD(P) in equilibrium with NAD(P)H transients due to microinjection of metabolites into cultured cells belonging to a variety of normal or malignant lines. Nuclear and cytoplasmic sites operate synchronously or not, depending upon metabolic demand or pathological alterations. Intercellular transit times are determined for tracers and metabolites. Within cell clusters 'communicating territories' are described, which can show metabolically a multicellular integrated state. Microfluorometry in conjunction with ultrastructural and other studies can be used to develop a cybernetic model of the living cell, also yielding dynamic models of cooperative and regulatory interactions between different kinds of specialised cells within a cell cluster.

Animals↗
PubMed1980

Nonrandom distribution of gap junctions between pancreatic beta-cells.

The numerical and spatial distribution of gap junctions between insulin-containing cells (beta-cells) under resting and stimulated conditions of insulin secretion were quantitatively analyzed in freeze-fracture replicas of isolated rat islets of Langerhans. The results show that the beta-cells located at the periphery of the islet have twice as many gap junctions per unit membrane area as the beta-cells situated in the islet center. In both locations, gap junctions assumed a nonrandom clustering on the beta-cell membranes. During stimulation of insulin secretion, the gap junctions were found increased between the central and between the peripheral beta-cells. The degree of their clustering was also modified. The latter change depended both on the location of the gap junctions in the islet and on the type of stimulation used (high glucose or glibenclamide).

Animals↗