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P Meda

Publications and source records attributed to P Meda.

At least 19 recordsLinked to original sources
PubMed1994

Heterogeneity and contact-dependent regulation of amylase release by individual acinar cells.

We have used a reverse hemolytic plaque assay to investigate the amylase release of single and aggregated pancreatic acinar cells. We have found that a minority of single acinar cells released detectable amounts of amylase under basal conditions and were modestly stimulated, in a dose-dependent manner, during a 30-min exposure to concentrations of carbamylcholine (CCh) ranging from 10(-8) to 10(-5) M. This stimulation was largely accounted for by the recruitment of additional secreting cells, rather than by a significant increase in their individual secretory output. We have also observed that aggregates comprising two to five acinar cells secreted more frequently and released more amylase than single acinar cells in the presence of each of the CCh concentrations tested. Under both basal conditions and following CCh stimulation, the proportion of secreting aggregates and their amylase output increased linearly with the aggregate size. Under basal conditions as well as in the presence of secretagogue concentrations in the 10(-8) - 10(-7) M range, individual cells contributed similarly to amylase secretion whether they were single or part of aggregates. By contrast, following stimulation by 10(-6) - 10(-5) M CCh, aggregated cells showed a much higher average secretion than single cells. Investigating the mechanism of this contact-dependent effect, we found that 10(-3) M heptanol did not significantly modify the secretion of single cells and markedly promoted the basal amylase release of acinar cell pairs. This effect was associated with a marked reduction in gap junctional communication between acinar cells, as evaluated by microinjection of Lucifer yellow, and was not observed during exposure to high concentrations of CCh, which also reduced junctional communication. These data show that pancreatic acinar cells are intrinsically heterogeneous in their ability to release amylase and that their basal as well as stimulated secretion are promoted by the establishment of direct intercellular contacts. Our experiments also suggest that junctional coupling contributes to the contact-dependent mechanism which enhances the recruitment of secreting cells and their individual output. These observations strengthen the view that direct interactions between acinar cells are essential in the control of pancreatic secretion.

Amylases↗
PubMed1994

Topography of mammalian connexins in human skin.

We have explored the expression of gap junction proteins in normal human skin by immunostaining cryostat sections (indirect immunofluorescence) or lyophilized epidermis (Western blotting) with antibodies against four mammalian connexins Cx26, Cx32, Cx40, Cx43; and by hybridizing total epidermal RNA (Northern blotting) with cRNA probes for Cx26, Cx32, and Cx43. We found that epidermal keratinocytes express Cx43 but not Cx26, Cx32, or Cx40. This expression was minimal in the basal layer, much higher in the spinous layer, reduced in the granular layer, and absent in the stratum corneum. Immunostaining for Cx43 was also observed in sebaceous glands, hairs, and eccrine sweat ducts. The two latter epidermal adnexae were also markedly labeled by antibodies against Cx26, a gap junction protein that was undetectable by immunofluorescence in interfollicular keratinocytes. Immunoblots of polyacrylamide gel electrophoresis-separated epidermal proteins and hybridization of epidermal RNA confirmed the presence of Cx43 in epidermis. These observations indicate that 1) Cx43 and Cx26 are components of human keratinocyte gap junctions; 2) these two proteins are differentially expressed in the interfollicular epidermis and the skin adnexae; 3) in interfollicular epidermis, Cx43 is a predominant gap junction protein, mostly expressed by the differentiating spinous cells; 4) Cx43 distribution is in accordance with the extensive dye coupling previously observed in this epidermal compartment.

Animals↗
PubMed1992

Basic fibroblast growth factor increases junctional communication and connexin 43 expression in microvascular endothelial cells.

We have analyzed the effect of basic fibroblast growth factor (bFGF) on junctional communication (coupling) and connexin 43 (Cx43) expression in bovine microvascular endothelial (BME) cells. In control confluent cultures, the incidence of coupling, as assessed by the intercellular transfer of microinjected Lucifer Yellow, was limited to 13% of injected cells, and decreased to 0% with time in culture. After exposure to bFGF (3ng/ml), the incidence of coupling was increased in a time-dependent manner, reaching a maximum of 38% of microinjected cells after 10-12 hours. The extent of coupling, as assessed by scrape loading, was maximally increased 2.1-fold 8-9 hours after addition of bFGF. bFGF also induced a 2-fold increase in Cx43 as assessed by Western blotting, and increased Cx43 immunolabelling at contacting interfaces of adjacent BME cells. Cx43 mRNA was likewise increased after exposure to bFGF in a time- and dose-dependent manner, with a maximal 6-7-fold increase after a 4 hour exposure to 3-10ng/ml. Finally, the increase in coupling and Cx43 mRNA expression observed after mechanically wounding a confluent monolayer of BME cells was markedly reduced by antibodies to bFGF, which have previously been shown to inhibit migration. Taken together, these results indicate that exogenous and endogenous bFGF increase intercellular communication and Cx43 expression in microvascular endothelial cells. We propose that the bFGF-mediated increase in coupling is necessary for the coordination of endothelial cells during angiogenesis and other vessel wall functions.

Animals↗
PubMed1992

Gap junctional communication of primary human keratinocytes: characterization by dual voltage clamp and dye transfer.

We have compared dye coupling in pairs of small (less than 10 microns in diameter) and large (greater than 20 microns in diameter) keratinocytes isolated from normal human epidermis, using Lucifer yellow microinjection. Under control conditions, dye coupling was found in only 1 out of the 25 small pairs tested, whereas it was evident in 75% of the large pairs (n = 52). After a 30-min incubation of the latter pairs in the presence of 10(-6) and 10(-4) M all-transretinoic acid (RA), the percentage of coupling was 53% (n = 15; NS) and 7% (n = 14; P less than 0.001), respectively. The almost complete uncoupling observed after 10(-4) M RA was not reversible even 30 min after return to control medium (n = 8). Dual whole-cell patch-clamp recordings from large keratinocyte pairs showed a macroscopic junctional conductance (gj) of 9 +/- 2 nS (n = 43), which was abolished by heptanol (3.5 mM) in a fully reversible way. Compared to heptanol, 10(-4) M RA abolished keratinocyte gj more slowly and irreversibly (n = 10). By contrast, 10(-6) M RA had no significant effect on gj (n = 8). Single-gap junctional channels were also identified between large keratinocytes. Events histograms of 152 transitions from three experiments revealed three main unitary conductances (gamma j) of 45 +/- 4, 78 +/- 4, and 106 +/- 7 pS. The dye coupling results indicate that junctional communication is markedly different in pairs of small and large cells, which showed the phenotype and keratin markers of basal and suprabasal keratinocytes, respectively. In the latter cell type, coupling is ensured by channels of three sizes and is blocked irreversibly by pharmacologic concentrations of RA.

Alcohols↗
PubMed1992

Coupling and connexin 43 expression in microvascular and large vessel endothelial cells.

Endothelial cells of the microvasculature differ both structurally and functionally from endothelial cells of larger vessels. To assess whether these cells also differ in terms of direct cell-to-cell communication, we compared gap junction-mediated intercellular coupling and connexin (Cx) expression in monolayer cultures of bovine microvascular and large vessel (aortic and pulmonary artery) endothelial cells. In confluent monolayers, junctional communication (as assessed by transfer of Lucifer Yellow) was greater between large vessel than between microvascular endothelial cells. Basal levels of connexin 43 (Cx43) and Cx43 mRNA were also greater in large vessel than in microvascular endothelial cells. When monolayers of microvascular endothelial cells were mechanically wounded, junctional communication was increased between migrating cells at the wound edge. In contrast, coupling between large vessel endothelial cells was not increased after wounding. The wound-induced increase in coupling between microvascular endothelial cells was accompanied by an increase in Cx43 and Cx43 mRNA. In contrast, Cx43 expression was unaltered after wounding monolayers of large vessel endothelial cells. These studies revealed differences in basal and wound-induced levels of coupling and Cx43 expression in microvascular and large vessel endothelial cells in vitro, raising the possibility that the role of coupling in endothelial cell function may be different in these different cell types.

Animals↗
PubMed1992

Cyclic adenosine monophosphate prevents the glucocorticoid-mediated inhibition of insulin gene expression in rodent islet cells.

Dexamethasone negatively regulates insulin gene expression in HIT-15 cells. In vivo, however, an excess of glucocorticoids results in an increase in insulin biosynthesis and peripheral hyperinsulinemia. To resolve this contradiction, we have studied the effects of dexamethasone in primary rat islet cells. We show here that dexamethasone decreases insulin mRNA levels in single islet cells, as in HIT-15 cells, but does not affect these levels in reaggregated islet cells and increases them in intact islets of Langerhans. Because cAMP is an important regulator of insulin gene expression and intracellular cAMP content may be decreased in single beta cells, we investigated whether cAMP could prevent the inhibitory effect of dexamethasone on insulin mRNA levels. In the presence of cAMP analogues, the inhibitory action of dexamethasone was not only prevented, but insulin mRNA increased to levels comparable to those observed when cAMP analogues were used alone. We conclude that the insulin gene is negatively regulated by dexamethasone in single islet cells, but that other factors such as cAMP prevent this effect when the native environment of islet cells is preserved. Our results indicate that insulin gene regulation is influenced by cell to cell contacts within the islet, and that intracellular cAMP levels might be influential in this regulation.

Animals↗
PubMed1992

Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines.

New insulin-secreting cell lines (INS-1 and INS-2) were established from cells isolated from an x-ray-induced rat transplantable insulinoma. The continuous growth of these cells was found to be dependent on the reducing agent 2-mercaptoethanol. Removal of this thiol compound caused a 15-fold drop in total cellular glutathione levels. These cells proliferated slowly (population doubling time about 100 h) and, in general, showed morphological characteristics typical of native beta-cells. Most cells stained positive for insulin and did not react with antibodies against the other islet hormones. The content of immunoreactive insulin was about 8 micrograms/10(6) cells, corresponding to 20% of the native beta-cell content. These cells synthesized both proinsulin I and II and displayed conversion rates of the two precursor hormones similar to those observed in rat islets. However, glucose failed to stimulate the rate of proinsulin biosynthesis. In static incubations, glucose stimulated insulin secretion from floating cell clusters or from attached cells. Under perifusion conditions, 10 mM but not 1 mM glucose enhanced secretion 2.2-fold. In the presence of forskolin and 3-isobutyl-1-methylxanthine, increase of glucose concentration from 2.8-20 mM caused a 4-fold enhancement of the rate of secretion. Glucose also depolarized INS-1 cells and raised the concentration of cytosolic Ca2+. This suggests that glucose is still capable of eliciting part of the ionic events at the plasma membrane, which leads to insulin secretion. The structural and functional characteristics of INS-1 cells remained unchanged over a period of 2 yr (about 80 passages). Although INS-2 cells have not been fully characterized, their insulin content was similar to that of INS-1 cells and they also remain partially sensitive to glucose as a secretagogue. INS-1 cells retain beta-cell surface antigens, as revealed by reactivity with the antigangloside monoclonal antibodies R2D6 and A2B5. These findings indicate that INS-1 cells have remained stable and retain a high degree of differentiation which should make them a suitable model for studying various aspects of beta-cell function.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Mammalian Sec23p homologue is restricted to the endoplasmic reticulum transitional cytoplasm.

The yeast Sec23 protein is required in vivo and in vitro for transport of proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. Ultrastructural localization of the Sec23p mammalian homologue (detected by antibody cross-reaction) in exocrine and endocrine pancreatic cells shows a specific distribution to the cytoplasmic zone between the transitional ER cisternae and Golgi apparatus where it appears associated with the tubular protuberances of the transitional ER cisternae, as well as with a population of vesicles, and surrounding cytoplasm. When ER-Golgi transport is interrupted with an energy poison, protuberances and transfer vesicles markedly decrease but Sec23p immunoreactive sites remain in the transitional cytoplasm not apparently tethered by membrane attachment. This unanticipated degree of organization suggests that cytosolic proteins, such as Sec23p, may be retained in specialized areas of the cytoplasm. A structure within the transitional zone may organize the flux of transport vesicles and Sec proteins so as to ensure efficient protein traffic in this limb of the secretory pathway.

Animals↗
PubMed1991

In vivo modulation of connexin 43 gene expression and junctional coupling of pancreatic B-cells.

We have explored the expression of gap junctional proteins and corresponding mRNAs by insulin-producing B-cells of native rat pancreas and of a transplantable rat insulinoma. By immunostaining cryostat sections (indirect immunofluorescence) and crude membrane preparations (Western blots) with antibodies against connexins 26, 32, and 43 and by hybridizing total islet and insulinoma RNA (Northern blot) with cRNAs for the latter two proteins, we have found that normal and tumoral B-cells express connexin 43 but do not show detectable levels of either connexin 32 or 26. By evaluating the conductance (dual patch-clamp whole-cell recording) and permeability of junctional channels (microinjection of Lucifer yellow), we have found that control B-cells show low levels of electrical and dye coupling in only a portion of the pairs studied. By studying B-cells of glibenclamide-treated rats, we have found that sustained stimulation of insulin release in vivo is associated with a two-fold increase in the level of connexin 43 gene transcripts and in the incidence of both ionic and dye coupling. These observations indicate that (1) connexin 43 is a major component of communicating channels between insulin-producing cells; (2) some but not all B-cells are electrically coupled by low conductance junctional channels; and (3) connexin 43 gene transcripts and incidence of junctional coupling are modulated in parallel during sustained stimulation of B-cell functioning in vivo.

Animals↗
PubMed1991

Distinctive gap junction channel types connect WB cells, a clonal cell line derived from rat liver.

Gap junctions, dye coupling, and junctional conductance were studied in a cell line (WB) that is derived from rat liver and displays a phenotype similar to "oval" cells. In freeze-fracture replicas, two distinctive particle sizes were detected in gap junctional plaques. Immunocytochemical studies indicated punctate staining at membrane appositions using antibodies to connexin 43 and to a brain gap junction-associated antigen (34 kDa). No staining was observed using antibodies prepared against rat liver gap junction proteins (connexins 32 and 26). Pairs of WB cells were electrically and dye coupled. Junctional conductance (gj) between cell pairs averaged approximately 10 nS; occasionally, gj was low enough that unitary junctional conductances (gamma j) could be detected. Using a CsCl-containing electrode solution, distinctive gamma j values were recorded: approximately 20-30 pS, approximately 80-90 pS, and the sum of the other sizes. The largest gamma j events were apparently due to random coincident openings or closures of the smaller channels. Several treatments reduced gj. Frequency distributions of gamma j were unaltered by 2 mM halothane or 3.5 heptanol, but the sizes of intermediate and largest events were reduced slightly by 100 nM phorbol ester, and the relative frequency of the largest events was increased by 10 microM glutaraldehyde. We conclude that the distinctive gamma j values represent openings and closures of two distinct types of gap junction channels rather than substates of a single channel type; these unitary conductances may correspond to the dual immunoreactivity and to the two particle sizes seen in freeze fracture.

Animals↗
PubMed1991

Extent and modulation of junctional communication between pancreatic acinar cells in vivo.

To assess whether junctional communication may be of physiological relevance in the control of exocrine pancreas secretion, we have studied acinar cell coupling by microinjecting Lucifer Yellow CH in the intact pancreas of anesthetized rats. Reconstructions from serial sections showed that, under control conditions, pancreatic cells are extensively coupled within each acinus but do not communicate with centroacinar cells, duct cells, and cells of neighboring acini. Intravenous infusion of acetylcholine and caerulein, or electrical stimulation of the vagus nerve, increased pancreatic secretion (P less than 0.02-0.001). Under these stimulatory conditions, the extent of acinar cell communication was decreased (P less than 0.001) by 40%. The acetylcholine-induced uncoupling was prevented by treating rats with atropine. Thus, in the intact pancreas, acinar cells intercommunicate extensively within each acinus under resting conditions and reduce their coupling during stimulation. These data support the view that modulation of cell coupling is a physiologically relevant mechanism for the regulation of exocrine pancreas secretion in vivo.

Acetylcholine↗
PubMed1991

Repeated glucose stimulation reveals distinct and lasting secretion patterns of individual rat pancreatic B cells.

To determine whether pancreatic B cells show a constant secretion pattern during repeated stimulations, we have used a sequential hemolytic plaque assay to monitor their individual insulin release during several successive 30-min incubations in the presence of 16.7 mM glucose. We have found that the total B cell secretion did not vary significantly in these successive glucose stimulations and that, under these conditions, the majority of B cells that were stimulated to release insulin during the first incubation also secreted during the second, third, and, when this was tested, during the fourth incubation. Similarly, most of the B cells that did not release detectable amounts of insulin during the first incubation did not secrete also during the two (or three) subsequent secretion tests. Together, the two groups of B cells that showed a constant secretory pattern, represented approximately 75% of the entire B cell population. The remaining 25% of B cells shifted from a secreting to a non-secreting state, or vice versa, from one incubation to another. These observations were made under three different time frames in which we tested single B cells as well as B cell clusters at rather different intervals. These findings support the existence of distinct B cell subpopulations differing lastingly in their ability to secrete insulin in response to glucose.

Animals↗
PubMed1991

Actively synthesizing beta-cells secrete preferentially after glucose stimulation.

To establish whether the heterogeneous secretion of glucose-stimulated beta-cells correlates with a different biosynthetic activity, we have studied the secretion and biosynthesis of the very same beta-cells by combining a hemolytic plaque assay with autoradiography. After a 10-min incubation in 2.8 mM glucose, 52 +/- 2% of dispersed rat beta-cells incorporated [3H] leucine into newly synthesized proteins, as revealed by autoradiographic labeling. When the incubation was performed in 16.7 mM glucose, larger (P less than 0.02) proportions (92 +/- 4%) of plaque-forming, i.e. insulin-secreting, and nonplaque-forming beta-cells (74 +/- 4%) were autoradiographically labeled. Labeled and unlabeled beta-cells were stimulated to secrete insulin during a 30-min incubation in 16.7 mM glucose, as revealed by the larger (P less than 0.001) formation of hemolytic plaques. Under these conditions, autoradiographically labeled beta-cells were recruited preferentially (P less than 0.01) and secreted more (P less than 0.04) than unlabeled beta-cells. Analogous observations were made with beta-cell pairs. Under glucose stimulation, pairs comprising two autoradiographically labeled beta-cells secreted more (P less than 0.004) than pairs comprising one or no labeled beta-cells. The data indicate that under glucose stimulation, 1) secreting and nonsecreting beta-cells increase protein biosynthesis; 2) biosynthetically active and inactive beta-cells increase insulin secretion; 3) beta-cells synthesizing new proteins release insulin preferentially; and 4) contact decreases the biosynthetic and secretory heterogeneity of beta-cells.

Animals↗
PubMed1991

Embryogenesis of the murine endocrine pancreas; early expression of pancreatic polypeptide gene.

By immunofluorescence on cytospin preparations and on semithin sections of mouse pancreatic buds, we have found glucagon and pancreatic polypeptide (PP)-containing cells at embryonal day 10.5 (E 10.5) in dorsal buds and at E 11.5 in ventral buds. Insulin-containing cells appear in dorsal buds at E 11.5, and one to two days later in ventral buds. Somatostatin-containing cells are detectable from E 13.5 in both dorsal and ventral buds. A quantitative analysis shows that up to E 15.5, PP-containing cells are relatively abundant in both buds. By PCR amplification of oligo(dT)-primed cDNAs prepared from total pancreatic RNA, we also detect PP mRNA from E 10.5 onwards, thus confirming the early expression of the PP gene in the developing mouse pancreas. Analysis of endocrine cells in situ suggests three major patterns of cell distribution in embryonic pancreas. First, individual hormone-containing cells are located within the epithelium of pancreatic ducts. In both dorsal and ventral buds, the majority of these endocrine cells contain PP, but many also contain glucagon, insulin or somatostatin. Secondly, clusters of endocrine cells are found in the pancreatic interstitium. Many of these cells contain both glucagon and PP which, by immunogold labelling of consecutive thin sections, can be shown to co-exist within individual secretory granules. Finally, starting on E 18.5, typical islets are formed with centrally located B cells and with the adult 'one cell-one hormone' phenotype. These results suggest an intriguing ontogenic relationship between A- and PP-cells, and also indicate that PP-containing cells may occupy a hitherto unexpected place in the lineage of endocrine islet cells.

Animals↗
PubMed1991

Ion channels of glucose-responsive and -unresponsive beta-cells.

To assess whether different electrophysiological characteristics could account for the heterogeneous secretion of individual beta-cells in vitro, we used patch-clamp configurations to study currents in plaque-forming (insulin-secreting) and non-plaque-forming rat pancreatic beta-cells that were distinguished in a reverse hemolytic plaque assay (RHPA) after a 30-min stimulation by 16.7 mM glucose. RHPA showed that the population of single beta-cells under study was stimulated (P less than 0.01-0.001) to secrete insulin by 16.7 mM glucose, 100 microM tolbutamide, 20 microM glyburide, or 30 mM KCl but, under these conditions, also comprised beta-cells that did not secrete detectable amounts of insulin. Under current clamp conditions, secreting and nonsecreting beta-cells showed analogous resting membrane potentials (approximately 60 mV) and were similarly depolarized by 30 mm KCl and 100 microM tolbutamide. Under voltage-clamp conditions, total membrane conductance (approximately 6 nS) was also similar in the glucose-responsive and -unresponsive beta-cells, which, when monitored in the whole-cell configuration after RHPA, showed the following currents: a voltage-dependent Na+ current, a voltage-activated Ba2+ current, a voltage-dependent K+ delayed-rectifier current, a voltage-dependent Ca(2+)-activated K+ current, and a voltage-independent and tolbutamide-sensitive K+ current. In the cell-attached configuration and the presence of 2.8 mM glucose, secreting and nonsecreting beta-cells displayed a similar single-channel activity that was abolished when glucose concentration was raised to 16.7 mM. We conclude that beta-cells studied after RHPA have an electrically normal membrane whether they release insulin in response to 16.7 mM glucose or not.

Animals↗
PubMed1990

Rapid and reversible secretion changes during uncoupling of rat insulin-producing cells.

To determine whether insulin secretion is affected by a blockage of gap junctions between B cells, we have studied the secretion of rat pancreatic islets of Langerhans, primary dispersed islet cells, and cells of the RINm5F line, during short-term exposure to heptanol. Within minutes, this alkanol blocked gap junctions between the B cells of intact islets and abolished their normal secretory response to glucose. These two changes were rapidly and fully reversible after return of the islets to control medium. We further found that heptanol had no significant effect on the glucose-stimulated secretion of single B cells but inhibited that of B cell pairs. In the clone of RINm5F cells, whose junctional coupling and D-glyceraldehyde-induced stimulation of insulin release by aggregated cells were also inhibited by heptanol, this alkanol did not perturb intracellular pH and Ca2+ and the most distal steps of the secretion pathway. In summary, a gap junction blocker affected the secretion of insulin-producing cells by a mechanism which is dependent on cell contact and is not associated with detectable pleiotropic perturbations of the cell secretory machinery. The data provide evidence for the involvement of junctional coupling in the control of insulin secretion.

1-Methyl-3-isobutylxanthine↗