Reverse Hemolytic Plaque Assay Research Articles

Intercellular contacts affect secretion and biosynthesis of pancreatic islet cells.

Cell protein biosynthesis is regulated by different factors, but implication of intercellular contacts on alpha and beta cell protein biosyntheses activity has not been yet investigated. Islet cell biosynthetic activity is essential in regulating not only the hormonal reserve within cells but also in renewing all the proteins involved in the control of secretion. Here we aimed to assess whether intercellular interactions affected similarly secretion and protein biosynthesis of rat alpha and beta cells. Insulin and glucagon secretion were analyzed by ELISA or reverse hemolytic plaque assay, and protein biosynthesis evaluated at single cell level using bioorthogonal noncanonical amino acid tagging. Regarding beta cells, we showed a positive correlation between insulin secretion and protein biosynthesis. We also observed that homologous contacts increased both activities at low or moderate glucose concentrations. By contrast, at high glucose concentration, homologous contacts increased insulin secretion and not protein biosynthesis. In addition, heterogeneous contacts between beta and alpha cells had no impact on insulin secretion and protein biosynthesis. Regarding alpha cells, we showed that when they were in contact with beta cells, they increased their glucagon secretion in response to a drop of glucose concentration, but, on the other hand, they decreased their protein biosynthesis under any glucose concentrations. Altogether, these results emphasize the role of intercellular contacts on the function of islet cells, showing that intercellular contacts increased protein biosynthesis in beta cells, except at high glucose, and decreased protein biosynthesis in alpha cells even when glucagon secretion is stimulated.

Rat Pancreatic Beta-Cell Culture.

In this chapter, we describe the methods used to culture mainly rat pancreatic beta cells. We consider necessary to use this approach to get more information about physiological, biophysical, and molecular biology characteristics of primary beta cells. Most of the literature published has been developed in murine and human beta-cell lines. However, there are many differences between tumoral cell lines and native cells because, in contrast to cell lines, primary cells do not divide. Moreover, cell lines can be in various stages of the cell cycle and thus have a different sensitivity to glucose, compared to primary cells. Finally, for these reasons, cell lines can be heterogeneous, as the primary cells are. The main problem in using primary beta cells is that despite that they are a majority within a culture they appear mixed with other kinds of pancreatic islet cells. If one needs to identify single cells or has an only beta-cell composition, it is necessary to process the sample further. For example, one may obtain an enriched population of beta cells using fluorescence-activated cell sorting or identify single cells with the reverse hemolytic plaque assay. The other problem is that cells change with time in culture, becoming old and losing some characteristics, and so must be used preferentially during the first week. The development of human beta-cell cultures is of importance in medicine because we hope one day to be able to transplant viable beta cells to patients with diabetes mellitus type 1.

Dual Effect of Palmitate on Voltage-Gated Calcium Channels and Insulin Secretion in Pancreatic Beta Cells of Rats

A variety of signaling molecules modify voltage-gated calcium channels activity in pancreatic beta cells, among them are, free fatty acids (FFA). Acute exposure to FFA increases glucose-stimulated insulin secretion, while chronic exposure increases basal insulin secretion, but decreases glucose-stimulated (Olofsson et al., 2004; Zhou and Grill, 1994). Preliminary studies in our laboratory showed that pre-incubation with 1 mM palmitate during 48-72 hours reduced barium currents in beta cells. In this work, we analyze the effect of different palmitate concentrations on calcium currents and insulin secretion in beta cells of adult male Wistar rats. Electrophysiological recordings were performed using whole-cell voltage clamp technique. Reverse hemolytic plaque assay measured insulin secretion by isolated beta cells. Pre-incubation with 0.25 and 0.5 mM palmitate during 48-72 h increased the high voltage-activated (HVA) calcium current without affecting the LVA current. An acute 5 min pre-incubation with 0.5 mM palmitate also increased HVA current. In contrast, 24 h pre-incubation with 1 mM decreased both, LVA and HVA currents. A similar effect was observed during an acute incubation. Chronic pre-incubation with 0.25, 0.5 and 1 mM palmitate decreased the percentage of insulin secretory cells, immunoplaque area and insulin secretion index in a glucose-stimulated condition (15.6 mM). Chronic pre-incubation with 0.5 and 1 mM palmitate, also decreased the secretion index at basal glucose (5.6 mM). However, acute pre-incubation with 1 mM palmitate increased the immunoplaque area in 5.6 mM glucose. Together these results show that palmitate has a dual effect, depending on time and concentration on the calcium currents and insulin secretion in beta cells. Partially supported by DGAPA PAPIIT-UNAM Grants: IN213114, M Hiriart; IV100116 to A. Frank and M Hiriart, and IN211416 to M Velasco-Torres.

Transcriptome landmarks of the functional maturity of rat beta-cells, from lactation to adulthood.

Research on the postnatal development of pancreatic beta-cells has become an important subject in recent years. Understanding the mechanisms that govern beta-cell postnatal maturation could bring new opportunities to therapeutic approaches for diabetes. The weaning period consists of a critical postnatal window for structural and physiologic maturation of rat beta-cells. To investigate transcriptome changes involved in the maturation of beta-cells neighboring this period, we performed microarray analysis in fluorescence-activated cell-sorted (FACS) beta-cell-enriched populations. Our results showed a variety of gene sets including those involved in the integration of metabolism, modulation of electrical activity, and regulation of the cell cycle that play important roles in the maturation process. These observations were validated using reverse hemolytic plaque assay, electrophysiological recordings, and flow cytometry analysis. Moreover, we suggest some unexplored pathways such as sphingolipid metabolism, insulin-vesicle trafficking, regulation of transcription/transduction by miRNA-30, trafficking proteins, and cell cycle proteins that could play important roles in the process mentioned above for further investigation.

Cadherin engagement improves insulin secretion of single human β-cells.

The aim of this study was to assess whether cadherin-mediated adhesion of human islet cells was affected by insulin secretagogues and explore the role of cadherins in the secretory activity of β-cells. Experiments were carried out with single islet cells adherent to chimeric proteins made of functional E-, N-, or P-cadherin ectodomains fused to the Fc fragment of immunoglobulin (E-cad/Fc, N-cad/Fc, and P-cad/Fc) and immobilized on an inert substrate. We observed that cadherin expression in islet cells was not affected by insulin secretagogues. Adhesion tests showed that islet cells attached to N-cad/Fc and E-cad/Fc acquired, in a time- and secretagogue-dependent manner, a spreading form that was inhibited by blocking cadherin antibodies. By reverse hemolytic plaque assay, we showed that glucose-stimulated insulin secretion of single β-cells was increased by N-cad/Fc and E-cad/Fc adhesion compared with control. In the presence of E-cad/Fc and after glucose stimulation, we showed that total insulin secretion was six times higher in spreading β-cells compared with round β-cells. Furthermore, cadherin-mediated adhesion induced an asymmetric distribution of cortical actin in β-cells. Our results demonstrate that adhesion of β-cells to E- and N-cadherins is regulated by insulin secretagogues and that E- and N-cadherin engagement promotes stimulated insulin secretion.

Hyperinsulinemia is Associated with Increased Soluble Insulin Receptors Release from Hepatocytes.

It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR) has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration, and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l−1 insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia, the amount of this soluble receptor increases and this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance.

Subpopulations of GFP-Marked Mouse Pancreatic β-Cells Differ in Size, Granularity, and Insulin Secretion

There is growing information about the heterogeneity of pancreatic β-cells and how it relates to insulin secretion. This study used the approach of flow cytometry to sort and analyze β-cells from transgenic mice expressing green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter. Three populations of β-cells with differing GFP brightness could be identified, which were classified as GFP-low, GFP-medium, and GFP-bright. The GFP-medium population comprised about 70% of the total. The GFP-low population had less insulin secretion as determined by the reverse hemolytic plaque assay and reduced insulin gene expression. Additionally, all three subpopulations of β-cells were found in mice of varying ages (embryonic d 15.5 and postnatal wk 1-9). The three populations from the youngest had larger cells (forward scatter) and less granularity (side scatter) than those from the adults. This approach opens up new ways to advance knowledge about β-cell heterogeneity.

Mafa expression enhances glucose-responsive insulin secretion in neonatal rat beta cells.

Neonatal beta cells lack glucose-stimulated insulin secretion and are thus functionally immature. We hypothesised that this lack of glucose responsiveness results from a generalised low expression of genes characteristic of mature functional beta cells. Important glucose-responsive transcription factors, Mafa and Pdx1, regulate genes involved in insulin synthesis and secretion, and have been implicated in late beta cell development. The aim of this study was to assess whether Mafa and/or Pdx1 regulates the postnatal functional maturation of beta cells. By quantitative PCR we evaluated expression of these and other beta cell genes over the first month compared with adult. After infection with adenovirus expressing MAFA, Pdx1 or green fluorescent protein (Gfp), P2 rat islets were evaluated by RT-PCR and insulin secretion with static incubation and reverse haemolytic plaque assay (RHPA). At P2 most beta cell genes were expressed at about 10% of adult, but by P7 Pdx1 and Neurod1 no longer differ from adult; by contrast, Mafa expression remained significantly lower than adult through P21. Overexpression of Pdx1 increased Mafa, Neurod1, glucokinase (Gck) mRNA and insulin content but failed to enhance glucose responsiveness. Similar overexpression of MAFA resulted in increased Neurod1, Nkx6-1, Gck and Glp1r mRNAs and no change in insulin content but, importantly, acquisition of glucose-responsive insulin secretion. Both the percentage of secreting beta cells and the amount of insulin secreted per beta cell increased, approaching that of adult beta cells. In the process of functional maturation acquiring glucose-responsive insulin secretion, neonatal beta cells undergo a coordinated gene expression programme in which Mafa plays a crucial role.

Role of CB1 cannabinoid receptor in the modulation of pituitary hormone secretion

Event Abstract Back to Event Role of CB1 cannabinoid receptor in the modulation of pituitary hormone secretion Ibolya Bodnár1, Márk Oláh1*, Dániel Hechtl1, Tibor Wenger2, Miklós Góth3, Valéria Rettoni4 and György M. Nagy1 1 Neuromorphological and Neuroendocrine Research Laboratory, Hungarian Academy of Sciences–Semmelweis University, Department of Human Morphology and Developmental Biology, Hungary 2 Semmelweis University, Department of Human Morphology and Developmental Biology, Hungary 3 State Health Centre, Division of Internal Medicine, Hungary 4 CONICET, Universidad de Buenos Aires, Facultad de Medicina, Centro de Estudios Farmacológicos y Botánicos, Argentina Background and Aims: It is known that cannabinoids can modulate anterior pituitary hormone secretion through a supposed crosstalk between CB1 receptor (CB1R) and different G-protein coupled receptors (GPCRs). We have investigated the significance of CB1 receptor in dopamine- (DA) and angiotensin II (AII) induced prolactin (PRL) and adrenocorticotroph hormone (ACTH) release as well as ghrelin- and growth hormone-releasing hormone (GHRH) induced growth hormone (GH) release using pituitary cell cultures derived from wild type (WT) and CB1 knock-out (CB1KO) mice. Dispersed anterior lobe cells were used immediately after dispersion in the PRL reverse hemolytic plaque assay (RHPA) and/or following 24 hours culture, then the concentration of PRL, ACTH and GH of the culture media was measured by specific RIA. Cells were treated with DA or TRH and with angiotensin II (AII), AM251 (CB1 antagonist), ghrelin or GHRH in the RHPA and in primary cultures, respectively. Results: DA exhibited similar inhibition on PRL release in both WT and CB1KO animals however the number of PRL cells were almost doubled in CB1KO mice detected in the RHPA. AII could strongly stimulate PRL and ACTH secretion in WT animals, while in the CB1KO group PRL release was absent and ACTH release was enhanced. Pretreatment of cultured cells with AM251 prevented AII induced PRL but not ACTH response in WT mice. Basal release of GH was significantly higher in CB1KO mice but the stimulating effects of ghrelin and GHRH were the same in both groups. Conclusion: Our results clearly show that the lack of CB1R profoundly affects secretagogues induced pituitary hormone release in vitro, therefore, indicates an intra-pituitary role of the endocannabinoid system in hormone release. This work was supported by the OTKA-68170 and 68660, ETT-458/09 and Hungarian–Argentinean Intergovermental S&T Cooperation Programme ARG 20/2006. Conference: IBRO International Workshop 2010, Pécs, Hungary, 21 Jan - 23 Jan, 2010. Presentation Type: Poster Presentation Topic: Homeostatic and neuroendocrine systems Citation: Bodnár I, Oláh M, Hechtl D, Wenger T, Góth M, Rettoni V and Nagy GM (2010). Role of CB1 cannabinoid receptor in the modulation of pituitary hormone secretion. Front. Neurosci. Conference Abstract: IBRO International Workshop 2010. doi: 10.3389/conf.fnins.2010.10.00110 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 23 Apr 2010; Published Online: 23 Apr 2010. * Correspondence: Márk Oláh, Neuromorphological and Neuroendocrine Research Laboratory, Hungarian Academy of Sciences–Semmelweis University, Department of Human Morphology and Developmental Biology, Budapest, Hungary, olahmark@ana2.sote.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Ibolya Bodnár Márk Oláh Dániel Hechtl Tibor Wenger Miklós Góth Valéria Rettoni György M Nagy Google Ibolya Bodnár Márk Oláh Dániel Hechtl Tibor Wenger Miklós Góth Valéria Rettoni György M Nagy Google Scholar Ibolya Bodnár Márk Oláh Dániel Hechtl Tibor Wenger Miklós Góth Valéria Rettoni György M Nagy PubMed Ibolya Bodnár Márk Oláh Dániel Hechtl Tibor Wenger Miklós Góth Valéria Rettoni György M Nagy Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Physalia physalis Poison Depolarizes Beta Cell Membrane and Increases Insulin Secretion

Peptide toxins isolated from marine cnidarians are considered as useful tools for studying ionic channels and promising drugs for therapeutics. Hemolytic and cardiotoxic activities have been described in Physalia physalis venom, and two toxins with anticholinergic and antiglutamatergic effects have been isolated in its high molecular weight fraction.In this work, we explored some crude extract of P. physalis and its low molecular weight fractions on ionic currents and insulin secretion of pancreatic rat beta-cells. P. physalis specimens were collected at the north littoral of La Habana, Cuba. Crude extract were purified by a gel filtration (Superdex 200). Mass spectrometry MALDI-TOF and RP-HPLC (C18 column) were performed on each fraction collected from gel filtration. The biological activity on insulin secretion was tested in a reverse hemolytic plaque assay on primary cultures of pancreatic beta cells from male Wistar rats.In basal glucose (5.6 mM), the crude extract (25 protein ug/mL) increased by 77% the average immunoplaque area, which is directly proportional to insulin secreted by isolated cells, without disrupting of beta cells or erythrocytes integrity. Low molecular fractions from gel filtration did not exceed of 20 kDa, and most of them eluted before the 20 % of acetonitrile on RP-HPLC. These fractions increased both, the percentage of insulin-secreting cells and the average immunoplaque area at a basal glucose level, suggesting a direct effect on TRP-type channels that are responsible for beta cell depolarization.Physalia physalis poison contains polar bioactive compounds capable of enhance the secretory behavior of pancreatic beta cells from male Wistar rats by depolarizing the membrane, in non stimulant glucose conditions.Supported by Instituto de Ciencia y Tecnologia del DF, ICYT (PICSD 08-72).

Paracrine cell to cell interactions determine the effects of pituitary adenylate cyclase activating polypeptide (PACAP) on in vitro prolactin release from rat pituitary cells

In static cultures of dispersed rat pituitary cells and in the reverse hemolytic plaque assay PACAP inhibits prolactin (Prl) secretion, while in vivo application of PACAP stimulates Prl release in rats. To elucidate the mechanism of this contradictory action, we compared the in vitro effects of PACAP on Prl secretion in cultures of dispersed or reaggregated cells and in pituitary fragments. While in monolayer cultures Prl release was inhibited by PACAP, in cultures of aggregated cells and in pituitary fragments Prl release was stimulated. Dopamine (DA) inhibited Prl release in either type of culture. PACAP also stimulated interleukin 6 (IL6) release under each of the experimental conditions. We conclude that PACAP has a direct inhibitory action on lactotropes. In addition, PACAP may induce the release of a paracrine acting factor within the pituitary which stimulates Prl release and which may be IL6. In the intact pituitary tissue and in reaggregated cells this paracrine factor stimulates Prl release more potently than PACAP directly inhibits Prl secretion resulting in a net effect of enhanced hormone release. In monolayer cultures, however, the direct inhibition is dominant, because the stimulatory paracrine factor is diluted in the culture medium. Therefore we suggest that paracrine cell to cell communication is crucial for the action of PACAP on Prl release.

Insulin secretion from human beta cells is heterogeneous and dependent on cell-to-cell contacts

We assessed the heterogeneity of insulin secretion from human isolated beta cells and its regulation by cell-to-cell contacts. Insulin secretion from single and paired cells was assessed by a reverse haemolytic plaque assay. The percentage of plaque-forming cells, the mean plaque area and the total plaque development were evaluated after 1 h of stimulation with different secretagogues. Not all beta cells were surrounded by a haemolytic plaque under all conditions tested. A small fraction of the beta cell population (20%) secreted more than 90% and 70% of total insulin at 2.2 and 22.2 mmol/l glucose, respectively. Plaque-forming cells, mean plaque area and total plaque development were increased at 12.2 and 22.2 compared with 2.2 mmol/l glucose. Insulin secretion of single beta cells was similar at 12.2 and 22.2 mmol/l glucose. Insulin secretion of beta cell pairs was increased compared with that of single beta cells and was higher at 22.2 than at 12.2 mmol/l glucose. Insulin secretion of beta cells in contact with alpha cells was also increased compared with single beta cells, but was similar at 22.2 compared with 12.2 mmol/l glucose. Delta and other non-beta cells did not increase insulin secretion of contacting beta cells compared with that of single beta cells. Differences in insulin secretion between 22.2 and 12.2 mmol/l glucose were observed in murine but not in human islets. Human beta cells are highly heterogeneous in terms of insulin secretion so that a small fraction of beta cells contributes to the majority of insulin secreted. Homologous and heterologous intercellular contacts have a significant impact on insulin secretion and this could be related to the particular architecture of human islets.

Defective IL-6 secretion in HIV-infected haemophilia patients.

To study the role of IL-6 in HIV-induced B cell defects, in vitro B cell responses and IL-6 secretion were determined simultaneously in 67 haemophilia patients. Twenty-three patients were HIV- (Group 1), 27 HIV+ stage CDC II, III (Group 2), and 17 were HIV+ stage CDC IV (Group 3). Pokeweed mitogen (PWM) was used for T cell-dependent and Staphylococcus aureus Cowan I (SAC I) for T cell-independent B cell stimulation. B cell differentiation was assessed in a reverse haemolytic plaque assay and by ELISA determination of IgG and IgM in culture supernatants. An ELISA was used to measure IL-6 in plasma and culture supernatants. HIV- patients showed impaired immunoglobulin-secreting cell (ISC) responses after T cell-independent and T cell-dependent stimulation (P < 0.0001 and P < 0.01, respectively), whereas IL-6 secretion, IgM and IgG responses were comparable to those in healthy controls. HIV+ patients at stage CDC II, III or IV demonstrated significantly reduced mitogen-stimulated IL-6 secretion (P < 0.05, PWM; P < or = 0.001, SAC I) as well as impaired ISC and IgG responses (P < 0.01, PWM; P < or = 0.0001, SAC I). CDC IV patients showed reduced IgM responses in addition (P < 0.02, PWM; P < 0.0005, SAC I). Plasma IL-6 levels were elevated both in HIV+ patients (CDC II, III patients: 165 +/- 73 pg/ml, P < 0.005; CDC IV patients: 58 +/- 18 pg/ml, P < 0.0001) and in HIV- patients (283 +/- 65 pg/ml, P < 0.0001) which appeared to be a T cell effect induced by treatment with haemophilia factor concentrates. Our data provide evidence for different types of B cell deficiencies in HIV- patients (impaired ISC response only) and HIV+ patients (impaired ISC as well as IL-6 and IgM/IgG responses). The defective IL-6 secretion in HIV+ patients is likely to affect terminal B cell differentiation and this may explain the reduced immunoglobulin secretion in these patients in response to antigenic challenge.

Differential expression of E-cadherin at the surface of rat β-cells as a marker of functional heterogeneity

The aim of this study was to assess whether the expression of E-cadherin at the surface of rat beta-cells is regulated by insulin secretagogues and correlates with insulin secretion. When cultured under standard conditions, virtually all beta-cells expressed E-cadherin observed by immunofluorescence, but heterogeneous staining was observed. Using fluorescence-activated cell sorting (FACS), two beta-cell sub-populations were sorted: one that was poorly labeled ('ECad-low') and another that was highly labeled ('ECad-high'). After 1-h stimulation with 16.7 mM glucose, insulin secretion (reverse hemolytic plaque assay) from individual ECad-high beta-cells was higher than that from ECad-low beta-cells. Ca2+-dependent beta-cell aggregation was increased at 16.7 mM glucose when compared with 2.8 mM glucose. E-cadherin at the surface of beta-cells was increased after 18 h at 11.1 and 22.2 mM glucose when compared with 2.8 mM glucose, with the greatest increase at 22.2 mM glucose + 0.5 mM isobutylmethylxanthine (IBMX). While no labeling was detected on freshly trypsinized cells, the proportion of stained cells increased in a time-dependent manner during culture for 1, 3, and 24 h. This recovery was faster when cells were incubated at 16.7 vs 2.8 mM glucose. Cycloheximide inhibited expression of E-cadherin at 2.8 mM glucose, but not at 16.7 mM, while depolymerization of actin by either cytochalasin B or latrunculin B increased surface E-cadherin at low glucose. In conclusion, these results show that expression of E-cadherin at the surface of islet beta-cells is controlled by secretagogues including glucose, correlates with insulin secretion, and can serve as a surface marker of beta-cell function.

The increase in prolactin-secreting cells in incubating chicken hens can be mimicked by extended treatment of pituitary cells in vitro with vasoactive intestinal polypeptide (VIP)

Incubation of eggs by birds and lactation in mammals are regulated by pituitary prolactin (PRL) and associated with an increase in pituitary PRL-producing cells or lactotrophs. However, the mechanisms controlling this increase in lactotroph numbers are not known. PRL secretion in birds is regulated by vasoactive intestinal polypeptide (VIP). This study was designed to determine whether VIP treatment could modulate lactotroph abundance in culture. Anterior pituitary cells were isolated from laying Japanese White Silkie hens and cultured for 2 or 6 days in the absence or presence of VIP. PRL-secreting cells were identified by reverse hemolytic plaque assay. Treatment with VIP for 6 days substantially increased the abundance of PRL-secreting cells from 47.5% under basal conditions to 70.6% of all pituitary cells following VIP stimulation. However, 2-day VIP treatment had no effect. Furthermore, the extent to which the hens were allowed to accumulate eggs in a clutch prior to isolation of the pituitaries did not affect the lactotroph response to VIP in vitro . These findings indicate that chronic VIP stimulation may be responsible for the increased abundance of lactotrophs found in the pituitary glands of incubating hens.

Expression and secretion of alpha1-proteinase inhibitor are regulated by proinflammatory cytokines in human pancreatic islet cells

Alpha1-proteinase inhibitor (alpha1-PI) has been considered a key player in inflammatory processes. In humans, the main production site of alpha1-PI is the liver, but other tissues, including pancreatic islets, also synthesise this molecule. The aims of this study were to assess the islet cell types that produce alpha1-PI, to determine whether alpha1-PI is actually secreted by islet cells, and to assess how its production and/or secretion are regulated. Expression of alpha1-PI in human islet cells was assessed by immunofluorescence, electron microscopy and western blotting. Release of alpha1-PI was analysed by reverse haemolytic plaque assay and ELISA. The effects of cytokines on alpha1-PI synthesis and secretion were tested. Immunofluorescence showed that alpha and delta cells do express alpha1-PI, whereas beta cells do not. By electron microscopy, we demonstrated a colocalisation of alpha1-PI with glucagon and somatostatin within secretory granules. Immunolabelling also revealed localisation of alpha1-PI within the Golgi apparatus, related vesicles and lysosomal structures. The expression of alpha1-PI in islet cells was also demonstrated by western blotting and ELISA of protein extracts. ELISA and reverse haemolytic plaque assay showed that alpha1-PI is secreted into the culture medium. Treatment of islet cells with IL-1beta and oncostatin M for 4 days increased the production and release of alpha1-PI. Our results demonstrate that alpha1-PI is expressed by the alpha and delta cells of human islets, and that proinflammatory cytokines enhance the production and release of this inhibitor.

Thapsigargin-sensitive cationic current leads to membrane depolarization, calcium entry, and insulin secretion in rat pancreatic β-cells

Glucose-induced insulin secretion by pancreatic beta-cells depends on membrane depolarization and [Ca2+]i increase. We correlated voltage- and current-clamp recordings, [Ca2+]i measurements, and insulin reverse hemolytic plaque assay to analyze the activity of a thapsigargin-sensitive cationic channel that can be important for membrane depolarization in single rat pancreatic beta-cells. We demonstrate the presence of a thapsigargin-sensitive cationic current, which is mainly carried by Na+. Moreover, in basal glucose concentration (5.6 mM), thapsigargin depolarizes the plasma membrane, producing electrical activity and increasing [Ca2+]i. The latter is prevented by nifedipine, indicating that Ca2+ enters the cell through L-type Ca2+ channels, which are activated by membrane depolarization. Thapsigargin also increased insulin secretion by increasing the percentage of cells secreting insulin and amplifying hormone secretion by individual beta-cells. Nifedipine blocked the increase completely in 5.6 mM glucose and partially in 15.6 mM glucose. We conclude that thapsigargin potentiates a cationic current that depolarizes the cell membrane. This, in turn, increases Ca2+ entry through L-type Ca2+ channels promoting insulin secretion.

Reverse hemolytic plaque assays: versatility in the study of secretion

The reverse hemolytic plaque assay has been used for several years to study hormone release from various endocrine cell types. The basic method utilizes a monolayer (consisting of indicator erythrocytes and the cells under study) that is fixed to the floor of an incubation chamber. Antibody directed against a peptide or protein is added to the chamber. Peptides released from the cells under study complex with the antibody and bind to protein-A on the surface of the indicator erythrocytes. The addition of complement causes the indicator cells to lyse, forming a “plaque” or zone of hemolysis surrounding the secreting cells. The size or rate of formation of these plaques can be used as indices to monitor peptide or protein release. In addition to this standard procedure, the plaque assay can be modified by using loose or unattached indicator cells and is termed the loose plaque assay (LPA). The LPA for a particular peptide can be used alone, sequentially with an assay directed toward another peptide, or repeatedly on the same cells to monitor release over time. In light of the fact that plaque assays do not compromise the function of living cells, it is possible to combine these plaque assays with other procedures such as immunocytochemistry, in situ hybridization, fluorescent microscopy, electrophysiology, and electron microscopy to explore other facets of the secretory process in conjunction with release. When taken together, the plaque assay has been quite useful in the study of endocrine cell secretion. Moreover, with the many adaptations possible, it should be particularly valuable in the future for the study of peptide release in other cell types such as neurons.

Ghrelin-induced GH secretion in domestic fowl in vivo and in vitro

Although avian and mammalian species differ significantly in their regulation of GH secretion, preliminary studies have demonstrated in vivo GH responses to ghrelin in chickens, as in mammals. However, the relative potency of ghrelin as a GH-releasing hormone (GHRH) in birds is uncertain, as is its site of action. The intravenous administration of human ghrelin to immature chickens promptly increased the circulating GH concentration (within 10 min), although this was transitory and was only maintained for 20 min. This GH response was dose-related with an EC50 of approximately 3.0 microg/kg, comparable with the reported potency of human GHRH in chickens. When incubated with dispersed pituitary cells, human ghrelin induced dose-dependent GH release over a range of 10(-6) to 10(-9) M, with an EC50 of 7.0 x 10(-8) M, comparable with that induced by human GHRH (EC50 6.0 x 10(-8) M), although it was less effective at doses of 10(-6) to 10(-8) M. This was due to direct effects on pituitary somatotrophs, since human ghrelin increased GH release (determined by the reverse hemolytic plaque assay) from individual pituitary cells. The incubation of these cells with human ghrelin induced a dose-dependent increase in the numbers of somatotrophs secreting GH and in the amount of GH released by each cell. In summary, these results demonstrated that ghrelin is a dose-related GH-releasing factor in chickens with a potency comparable with that induced by human GHRH. The GH-releasing action of ghrelin is due, at least in part, to stimulatory actions on the numbers of somatotrophs induced to release GH and upon the amount of GH released from individual somatotrophs.

Thyroid hormones interact with glucocorticoids to affect somatotroph abundance in chicken embryonic pituitary cells in vitro.

Our laboratory has reported that somatotroph differentiation occurs between d 14 and d 16 of chicken embryonic development and that corticosterone (CORT) can induce somatotroph differentiation at an earlier age in vitro and in vivo. The objective of the present study was to test for thyroid hormone-CORT interactions on somatotroph differentiation in vitro. Pituitary cells from d 11 chicken embryos were treated with CORT and thyroid hormones, and GH-producing somatotrophs were detected by reverse hemolytic plaque assays and immunocytochemistry. We found that thyroid hormones can act synergistically with CORT to further augment the abundance of somatotrophs in vitro but have little to no effect on their own. Both T(4) and T(3) could act synergistically with CORT to increase somatotroph abundance, but the effects of T(3) were biphasic, inhibiting CORT actions at higher concentrations. The monodeiodination inhibitor iopanoic acid inhibited the synergistic effect of T(4) on CORT induction of GH cells in vitro but not the synergistic effect of CORT and T(3) or the effect of CORT alone. Furthermore, T(3) treatment overcame the iopanoic acid-induced reduction in the T(4)-CORT effect. Our findings indicate that thyroid hormones act synergistically with CORT to further augment the abundance of somatotrophs in vitro and that conversion of T(4) to T(3) within the pituitary is involved in T(4) modulation of somatotroph abundance. Somatotroph differentiation during normal development may be regulated by complex interactions of hormones produced by the embryonic thyroid and adrenal glands.