Somatostatin inhibits insulin and glucagon release by monolayer cell cultures of rat endocrine pancreas

Dihydrosomatostatin (0.001–1.0 ug/ml) inhibited both insulin and glucagon secretion by monolayer cell cultures of newborn rat pancreas. When cultures were incubated with somatostatin and then rinsed, the effect of somatostatin appeared to last longer on the pancreatic alpha cell than on the beta cell as indicated by a more prolonged inhibition of glucagon secretion than of insulin release. Submaximal inhibition of glucose-stimulated insulin release by somatostatin was partially reversed by increasing the concentration of glucose. We conclude that the effect of somatostatin appears to be mediated directly on the pancreatic endocrine cells.     

Removal of fibroblastoid cells from primary monolayer cultures of rat neonatal endocrine pancreas by sodium ethylmercurithiosalicylate

Anti-cytochrome b₅ immunoglobulin (AIg) from a rabbit was used to establish the role of cytochrome b₅ in the transfer of electrons from NADH or NADPH to the hepatic microsomal mono-oxidase system of the rat. AIg inhibited ethylmorphine (EM) N-demethylase when both NADH and NADPH were present, but had little effect when NADPH was the only source of electrons. Inhibition was reversed when AIg was preincubated with pure cytochrome b₅. Specificity of AIg was shown by its inhibitory effect on NADH cytochrome c reductase activity; it was without effect on NADPH-cytochrome P-450 reductase or aniline hydroxylase activities. It is concluded that the second electron required for EM N-demethylation can be donated by NADH via cytochrome b₅.     

Comparison of endocrine secretion by monolayer cultures derived by different procedures from neonatal hamster and rat pancreas.

Monolayer cultures derived from neonatal hamster or rat pancreas by two different epithelioid cell-enriching gravity sedimentation procedures varied in ability to maintain uniform levels of insulin secretion with increased culture age. Rat pancreatic cultures were superior in this respect to identically derived hamster preparations, depending on the preparative procedure employed. Quantitative differences in the temporal pattern of insulin secretion by different rat pancreatic culture preparations were ascribed to plating cell density and consequent terminal cell density as a function of preparative procedure such that reduced densities favored sustained secretory levels. These findings suggest the importance of tissue species and preparative procedure in deriving pancreatic monolayer cultures capable of sustained levels of insulin secretion with age.     

Immunohistochemical characterization of monolayer cell cultures of embryonic chicken pancreas and measurement of somatostatin release.

Monolayer cell cultures of embryonic chicken pancreas contain functionally active insulin, glucagon and somatostatin-containing cells as evidenced by immunohistochemical and radioimmunoassay techniques. Hormone release is in relation to the number of each cell type present and responds to known specific secretory stimuli. The relatively high numbers of D-cells and amounts of immunoreactive somatostatin released by this preparation makes this system a suitable model for studies of somatostatin function and secretion.     

Freeze-fracture of monolayer cultures

This paper describes a simple method for the freeze-fracturing of cells in monolayers or multi-layer tissue cultures. The method produces high quality replicas and is applicable to the study of virtually any tissue culture or organ culture system. It uses standard materials and equipment for both tissue culture and freeze-fracturing.     

Ontogeny of the endocrine pancreas

The ontogenesis of the endocrine pancreas has been a subject of controversy. The discussion essentially was about organogenesis and histogenesis of islets of Langerhans. Now, the endodermic origin seems well established by several experimental approaches. The variation of the aspects of the islets and of the number of endocrine cells are re-called as well as the functional activity during fetal life. Numerous neuropeptides have been found in endocrine pancreas, so the content of the different endocrine cell types is reviewed with respect to the classic hormones.     

Fluorimetric quantification of cell death in monolayer cultures and cell suspensions.

A fluorimetric assay using ethidium bromide (EB) was employed to quantify cell death in monolayer cell cultures (MA-104 cells) in situ and isolated cell suspensions (isolated colonic cells and Leishmania). Fluorescence of EB stained cells was measured with a photometer coupled to an inverted microscope for cell monolayers or in a spectrofluorometer for cell suspensions. Dead cells stained with trypan blue were fluorescent with EB in all preparations studied, but the latter gave an unequivocal signal. Staining with EB and fluorescein diacetate was mutually exclusive. The relationship between the number of EB fluorescent cells and the intensity of fluorescence measured in the microphotometer was linear for a large range of cell numbers (1-14000) from different types of preparations. Applicability of the method for measuring living and dead cells in two different time scales (minutes and hours) is shown using MA-104 cell monolayers infected with rotavirus and Leishmania suspensions treated with amphotericin B. The method is fast, simple, sensitive and reliable, enabling quantification of living and dead cells in monolayers and suspensions.     

Calcium-induced insulin release in monolayer culture of the endocrine pancreas. Studies with ionophore A23187.

The role of Ca2+ on insulin release has been studied by the use of ionophore A23187. The ionophore complexes divalent cations and permits Ca2+ entry into cells by acting as a carrier in the plasma membranes. Cultured cells obtained by enzymatic digestion of pancreases from newborn rats were studied on the 3rd day of culture. With Ca2+ in the incubation medium the ionophore induced sustained insulin release even in the absence of glucose. Optimal effects of the ionophore were observed at 3 and 10 mug per ml in the presence of 0.3 to 1.0 mM Ca-2+. Under these conditions the insulin release was greater than that caused by 16.7 mM glucose. A graded response was observed to changes in Ca-2+ concentration from 0.1 to 1.0 mM Ca-2+. Higher Ca-2+ concentrations caused a large amount of insulin to be released promptly, but the release was not sustained. Mg-2+ and Sr-2+ were not found to substitute for Ca-2+. Ba-2+ at 0.3 mM stimulated insulin release even in the absence of ionophore. Cyclic adenosine 3':5'-monophosphate was able to increase ionophore-induced insulin release. The alpha-adrenergic effect of epinephrine to inhibit insulin release was not observed in the presence of Ca-2+ and the ionophore, and a stimulatory effect of epinephrine was seen. This unusual stimulatory effect of epinephrine was blocked by propranolol indicating a beta-adrenergic mechanism for epinephrine. It is concluded that Ca-2+, which plays an essential role in the stimulus-secretion coupling, can alone initiate and cause sustained insulin release.     

Galanin and the endocrine pancreas

Galanin is a 29 amino acid peptide, initially isolated from the porcine small intestine. The peptide has been shown to occur in intrapancreatic nerves in close association to the islets. Its effects on islet hormone secretion and its possible mechanisms behind these effects are reviewed. Galanin has been shown to inhibit basal and stimulated insulin secretion both in vivo and in vitro under a variety of experimental conditions. The peptide has also been shown to inhibit somatostatin secretion and the secretion of pancreatic polypeptide (PP). With regard to glucagon secretion, however, results in the literature are not consistent since both stimulatory and inhibitory effects have been reported. A direct interaction with the pancreatic beta-cells has been proposed behind its inhibitory action on insulin secretion, since galanin inhibits insulin secretion from isolated beta-cells from obese, hyperglycaemic, mice. Galanin has thereby also been shown to induce repolarization and to reduce the free Ca2+ concentration, [Ca2+]i. The reduction in [Ca2+]i is probably not due to a direct interference with the voltage-activated Ca2+ channels, since there is no effect of galanin when these channels are opened by depolarization induced by high concentrations of K+. Instead, preliminary studies indicate that galanin activates the K+ channels that are regulated by ATP, in turn inducing a repolarization-induced reduction in [Ca2+]i resulting in reduced insulin secretion. However, the possibility that galanin inhibits the insulin secretory mechanism at a step distal to the regulation of cytoplasmic free Ca2+ concentration should not be overlooked.     

Substitutes for the endocrine pancreas

Since it is generally accepted that a tight metabolic control might prevent the chronic complications of diabetes mellitus, several systems have been developed in which insulin is administered in a manner that mimicks the characteristics of insulin secretion. 1) In the so-called closed-loop systems, insulin delivery is regulated by the concomitant plasma glucose level. These systems, which involve continuous measurement of plasma glucose concentration are not to date implantable and have been used so far only for short-term studies. 2) By contrast, in the "open-loop systems", no glucose sensor is needed and insulin delivery is pre-programmed to achieve a constant basal infusion with peaks of insulin delivery during the meal periods. These systems have been used in man for several months. The present achievement and limitations of both kinds of systems are discussed in this review.     

Pathology of the endocrine pancreas.

An immunocytochemical analysis of 94 pancreatic endocrine tumors revealed that 73 tumors were multicellular. Significant amounts of somatostatin and human pancreatic polypeptide were found by radioimmunoassay in extracts of 19 and 17 tumors resp., in addition to the hormone causing the clinical syndrome. Numerous tumors contained ductular structures. In the surrounding pancreatic parenchyma a proliferation of small ducts and budding-off from the ductular epithelium of endocrine cells was often observed. These features are hallmarks of nesidioblastosis of the endocrine pancreas which is a hyperplasia. In multiple endocrine neoplasia I hyperplasia of the endocrine pancreas is combined with larger nodules, currently labeled tumors. On the basis of these findings it is conceivable that pancreatic endocrine tumors are not primarily neoplastic and autonomous but that they are rather of hyperplastic origin.     

5 alpha-DHT metabolism in monolayer cultures of distinct pituitary cell populations

5 alpha-Dihydrotestosterone (DHT) metabolism into 5 alpha-androstane-3 alpha, 17 beta-diol (alpha-diol) and 5 alpha-androstane-3 beta, 17 beta-diol (beta-diol) was studied in monolayer cultures of distinct cell populations from prepubertal male rats pituitaries. Cells were characterized through immunocytochemistry with the various antihormone antisera. Centrifugal elutriation was used to prepare a gonadotrope-enriched population "G" and a gonadotrope-depleted population "L", containing most lactotropes and somatotropes. Using centrifugation on Percoll gradient, two sub-populations, P1 and P2, were prepared by further fractionation of the "L" population. Cells were incubated for 48 h with [3H]DHT (1 microM, sp. act. 0.9 Ci/mmol) and metabolites extracted from the whole cell and medium. DHT was metabolized to about the same extent (30-40%) in all cell fractions. Compared with unfractionated population, the conversion of DHT into alpha-diol increased significantly in the P1 fraction, consisting of lactotropes, somatotropes and highly depleted in gonadotropes. This increase was lower in the somatotrope-enriched P2 fraction in which the amount of lactotropes was similar to P1 but that of gonadotropes slightly higher. In contrast, the conversion of DHT into alpha-diol decreased significantly in the "G" population compared with total or "L" fractions, whereas androstanedione formation, low in every population, increased significantly. The increase in alpha-diol formation could be related either to the decrease of gonadotropes or to a role of non-immunoreactive cells. As the beta-diol formation was constant in all cell types, the beta-diol/alpha-diol amount increased significantly in gonadotropes. Then, beta-diol and DHT could be both active steroids in gonadotrope regulation inasmuch as specific binding sites were identified for these two steroids. It can be concluded that DHT action at the pituitary level is subject to complex control mechanisms involving a specific balance of its metabolites in each particular cell type.     

An improved method of eliminating fungal contamination from monolayer cell cultures

A method is described for eliminating fungal and other forms of contamination from valuable monolayer cell cultures. The method employs the following sequence of operations: several changes of medium, trypsinization and removal of cells to coverglasses in appropriate tubes with medium plus amphotericin B (Fungizone) or other antibiotic, removal of coverglasses to new tubes with medium plus antibiotic, and removal in a few days to a new culture vessel without antibiotic. If microorganisms do not show up in 3–5 days, they have been eliminated. Greater success is achieved by using the same method with coverglass fragments in small culture wells.     

Production of progenitor cells from primary human epithelial cell monolayer cultures

Primary keratinocytes derived from human epidermis are widely used in tissue engineering and regenerative medicine. An important aspect in clinical applications is the preservation of human skin keratinocyte stem cells. However, it is difficult to expand the number of human skin keratinocyte stem cells, which are undifferentiated and highly proliferative in culture by using standard cell culture methods. It is even more difficult to identify them, since universal specific markers for human skin keratinocyte stem cells have not been identified. In this paper, we show a method to produce a large number of primary progenitor human skin keratinocytes by using our novel culture techniques. Primary human skin keratinocyte monolayers are cultured using twice the volume of medium without serum and lacking essential fatty acids. Once the cells reach 70–80% confluence, they begin to float up into the overlying medium and are called “epithelial pop-up keratinocytes (ePUKs)” allowing the cells to be passaged without the use of trypsin. We analyzed the properties of ePUKs by cell size, cell viability, immunocytofluorescence biomarker staining, and cell cycle phase distribution by fluorescence-activated cell sorting (FACS). Our results showed that these ePUKs appear to be progenitor epithelial cells, which are small in size, undifferentiated, and have a high proliferative capacity. We believe that ePUKs are suitable for use in medical applications requiring a large number of primary human progenitor skin keratinocytes.     

Method of total staining of monolayer cell cultures using vanadium hematoxylin]

Original method of cytological staining for monolayer cell cultures and outgrowth zone of tissue explants with vanadium hematoxylin is described. This staining method is simple, universal, reproducible and give opportunity to stain rapidly practically all cellular elements of cultivated monolayer with high degree of cytological resolution of intracellular structures (nucleus, cytoplasmic organelles etc.).