Neurotensin is localized to paracrine cells in the urinary bladder of the turtle, Chrysemys picta.

Urinary bladder from the painted turtle, Chrysemys picta, contains a substantial population of endocrine/paracrine cells scattered through the mucosal epithelium which immunostains using antisera directed toward mammalian neurotensin (NT). Radiommunoassay of 0.1 N HCl extracts of Chrysemys bladder indicated an immunoreactive NT (iNT) concentration of 161 +/- 39 pmol/g tissue (n = 9), an amount lower but comparable in magnitude to that found in mucosal scrapings of the intestine (926 +/- 125 pmol/g, n = 9). Bladder and intestinal iNT were indistinguishable during chromatography on Sephadex G-25 and HPLC on mu-Bondapak C18 where they eluted at the same position as avian NT. Similar results were obtained by immunocytochemistry and radioimmunoassay of urinary bladders from Pseudemys scripta scripta, P. scripta elegans, and P. floridana, three emydid species closely related to C. picta, but not from Sternotherus odoratus and Trionyx spinifer asper, representatives of the families Kinosternidae and Trionychidae, respectively, although positive results were obtained with intestinal preparations from these species. NT cells were not seen in urinary bladder from Rana pipiens, Bufo marinus, Necturus maculosus, or Anolis carolinensis. Thus, the presence of NT-containing cells in urinary bladder is not common among subavian vertebrates and may even be restricted to the Emydidae family of chelonians.

Insulin cells are found in the main and accessory urinary bladders of the painted turtle, Chrysemys picta.

Insulin has been localized immunocytochemically to cells in the main and accessory urinary bladders of the painted turtle, Chrysemys picta, and represents an unusual addition to the specturm of regulatory peptides associated with the urinary bladder. These stellate to fibroblastoid cells often possess neural-like processes and are similar in morphology to neurotensin cells found in Chrysemys and Pseudemys urinary bladders. Radioimmunoassay of 2M acetic acid extracts of bladder tissue indicate that the insulin concentration of accessory bladder is several-fold greater than main bladder but considerably lower than the insulin content of pancreas. Pieces of accessory bladder incubated in vitro exhibit a stable insulin release into the medium over 1 hour, but release is unaltered by known insulin secretagogues. It is tempting to postulate an endocrine or paracrine regulatory function for these cells, but at present their role in Chrysemys bladder function remains unknown.

Localization of insulin to gastroenteropancreatic cells in the turtle gastrointestinal tract.

Insulin has been localized immunocytochemically to open-type gastroenteropancreatic endocrine cells in sections of Bouin's-fixed upper, middle, and lower intestine from Chrysemys picta, Pseudemys scripta scripta, P. scripta elegans, P. floridana, Sternotherus odoratus, and Trionyx spinifer asper. Radioimmunoassay of extracts of mucosal scrapings from Chrysemys intestine indicates differential amounts of insulin-like immunoreactivity within the intestine (higher amounts in the lower intestine) and in concentrations approximately one-tenth to one-fifth that found in extracts of Chrysemys pancreas. The presence of insulin in the gastrointestinal tract of chelonians represents a major departure from the typical vertebrate condition which is characterized by an absence of insulin from the spectrum of regulatory peptides in the gut.

Development of rotorod performance in normal and congenitally hypothyroid mutant mice.

Balancing behavior on a rotating cylinder was measured in genetically hypothyroid (hyt/hyt) mutant mice and their phenotypically normal littermates (hyt/+) at 10 different ages between Day 12 and Day 55. Normal mice showed competent balancing behavior in this task by 23 days of age, but mutant hypothyroid mice were incapable of balancing throughout the period of testing. Similar results were found with animals initially tested as adults, indicating that the developmental effects are probably not attributable to different rates of learning. These results are discussed in terms of documented central nervous system abnormalities in animals deprived of thyroid hormone early in life.

The gastro-entero-pancreatic system of the turtle, Chrysemys picta.

Pancreatic endocrine cells were stained immunocytochemically for insulin, glucagon, somatostatin and pancreatic polypeptide by the PAP technique or sequentially for two hormones by the PAP followed by an indirect immunogold procedure. Pancreatic endocrine cells of Chrysemys are found scattered as single cells or small aggregates throughout the exocrine parenchyma; only the splenic region shows islets consisting of a B cell core surrounded by a loose mantle of A cells and occasional D cells. PP cells were not found in this splenic portion but were found scattered throughout the remainder of the pancreas. In contrast to the typical vertebrate islet, Chrysemys pancreatic endocrine cells are characterized by a lack of preferential association of one cell type with another and suggests that paracrine regulatory mechanisms may not be operable in this species. Insulin secretion from pieces of Chrysemys pancreas has been measured in incubation and perifusion systems employing a heterologous radioimmunoassay. Insulin release by Chrysemys B cells is enhanced by elevated levels of glucose (300 mg/dl), however, response appears to be somewhat slower compared to other vertebrate B cells. Gastrin, secretin, neurotensin, motilin, serotonin, PYY, glucagon, gastric inhibitory polypeptide, somatostatin and insulin were demonstrated immunocytochemically in open-type GEP cells of the mucosal epithelium of the Chrysemys intestine. Of these cells, gastrin, neurotensin and insulin cells appear to be the most numerous while the other types appear less frequently. Cells containing PP, bombesin, cholecystokinin and substance P could not be demonstrated. The localization of insulin to GEP cells of the turtle intestine is an unusual finding but has been confirmed by radioimmunoassay of extracts of the intestinal mucosa.

Peptide YY (PYY) immunoreactivity is co-stored with glucagon-related immunoreactants in endocrine cells of the gut and pancreas.

In this study we report the localisation of PYY immunoreactivity in intestinal mucosa endocrine (EG) cells containing glucagon-related peptides and also in foetal pancreatic A cells of rat and man. Radioimmunoassay of human foetal pancreatic extracts revealed the presence of PYY immunoreactivity, the concentration of which declined with age (from 65.42 pmol/g at week 20 to 17.0 pmol at week 40; correlation coefficient = -0.893), in contrast to the amount of glucagon which remained statistically constant throughout the same foetal period. The identity of this PYY immunoreactive material with the original 36 amino acid porcine peptide has been shown by high pressure liquid chromatography (HPLC).

Temporal changes in pancreatic islet composition in C57BL/6J-db/db (diabetes) mice.

Temporal changes in non-B cell populations were determined during the period of B cell hyperplasia in diabetes-resistant C57BL/6J mice. Pancreases from normal and db/db mice between 3 and 20 weeks of age were stained immunocytochemically for glucagon, somatostatin and pancreatic polypeptide (PP), and changes in A, D and PP cell volume densities quantified by image analysis. Further, islet volumes, D cell volumes and actual D cell numbers per islet were determined by analysis of serial sections through entire islets. The volume of db/db islets was three- and ten-fold elevated above normal by 8 and 20 weeks, respectively, due mainly to B cell hyperplasia. D cell volume density exhibited a transient increase during the initial phase of B cell hyperplasia, but then showed a gradual reduction; the average number and absolute volume of D cells per islet was comparable in db/db and normal islets from older mice. In contrast, PP cell volume density remained stable throughout, suggesting that this cell type kept pace with B cell hyperplasia. A cells showed a reduced volume density throughout and were distinguished from other islet cells which all responded positively to a degree, albeit non-coordinately, to the mitogenic stimulus exerted by db gene expression. The finding that A cells shared with certain neuroectodermally-derived cell types a differentially high concentration of sn-glycerol-3-phosphate dehydrogenase further underscored the uniqueness of the A cell from other cell types.

Immunohistochemical localization of sn-glycerol-3-phosphate dehydrogenase in Bergmann glia and oligodendroglia in the mouse cerebellum.

Immunohistochemical staining showed that the adult isozyme of sn-glycerol-3-phosphate dehydrogenase (GPDH) was localized in two populations of cells in the mouse cerebellum. Staining was not detectable until after 10 days of age, thus correlating with the onset of accumulation of the adult enzyme. The two cell populations were identified as Bergmann glia and oligodendroglia on the basis of their morphology, distribution and their nearly complete absence from cerebella of mice with mutations (Weaver, wv/wv and Jimpy, jp/Y, respectively (which are known to reduce the numbers of these cell types. Biochemical analysis of these mutants suggests that Bergmann glia contribute approximately 60% of the total cerebellar GPDH activity while the contribution of oligodendroglia in the cerebellum is negligible. In addition, immunohistochemical staining of the rat cerebellum showed a distribution of this enzyme similar to that of the mouse. However, oligodendroglia of the rat stained more intensely than did the Bergmann glia.

Ultrastructural and morphometric studies of delta cells in pancreatic islets from C57BL/Ks diabetes mice.

An ultrastructural and immunocytochemical study was undertaken to elucidate the temporal and quantitative aspects of the changes occurring in the delta cells in the pancreatic islets of C57BL/KsJ db/db (diabetes) mice. Electron microscopy revealed that prior to the major topographical redistribution of delta cells from their peripheral location to the islet interior, long delta cell filopodial extensions penetrated into the islet, greatly increasing the area of surface contact between delta cells and hypersecretory beta cells. Coincident with delta cells redistribution in islets of 8 to 10 week diabetes mice, the mean number of delta cells per islet had increased significantly. In contrast, their volume density had decreased, indicating incomplete compensation for beta cell hyperplasia which had commenced approximately 4 weeks earlier. In the 14 week mutants, numbers of delta cells per islet and islet volume reached maximum values while delta cell volume density had been restored to a control level. Delta cell volume density exhibited a 2-fold increase in the mutants at 20 weeks that coincided with massive beta cell necrosis. However, a decline in the number of delta cells per islet (173.6 +/- 20.9 at 14 weeks versus 91.2 +/- 20 weeks) suggests that islet degeneration in terminal stages of the syndrome also includes some loss of these cells.

Plasma levels of vitellogenin in Chrysemys picta during the annual gonadal cycle: measurement by specific radioimmunoassay.

A RIA for turtle (Chrysemys picta) vitellogenin is described. After dimethylformamide precipitation of vitellogenin from the plasma of estrogen-treated female turtles, antibodies were developed in rabbits. The dimethylformamide precipitate was further purified by o-triethylaminoethyl cellulose column chromatography; the vitellogenin component eluted as a single peak. This material was used for iodination by a mild chloramine method. Antibodies to turtle vitellogenin did not cross-react with plasma from male turtles or vitellogenic females of other vertebrate groups, including lizards and snakes. Limited cross-reactivity exists among the chelonians, however. Using a 1:5000 dilution of antiserum, the limit of detection was 15 ng, and the midrange was 320 +/- 45 ng. For an antiserum dilution of 1:1000, these figures were 30 and 600 +/- 37 ng, respectively. Using this assay, the seasonal pattern of plasma vitellogenin in the turtle has been described, and preliminary studies on in vitro hepatic vitellogenesis have been performed.

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.