Distribution of Na+,K+-ATPase in rat exocrine pancreas as monitored by K+-NPPase cytochemistry and [3H]-ouabain binding: a plasma membrane protein found primarily to be ductal cell associated.

We investigated the distribution of Na+,K+-ATPase in rat exocrine pancreas. By use of enzymatic dissociation techniques, pancreatic acini (containing acinar cells and centroacinar ductal cells in a ratio of about 10:1) and all major classes of pancreatic ducts were isolated and analyzed for the presence of Na+,K+-ATPase using K+-NPPase cytochemistry and [3H]-ouabain binding assays. Ultrastructural analysis demonstrated a basolateral localization of ouabain-sensitive enzyme activity in all classes of pancreatic ducts, although the degree of activity varied among the various classes. Qualitative analysis (scale of 0 to + + +) indicated the following enzyme distribution: centroacinar ductal cells (+); intralobular ducts (+ +); interlobular ducts (+ + +); main duct (+ +). In contrast, no reaction product was associated with pancreatic acinar cells even when observed adjacent to enzyme-positive centroacinar ductal cells. Parallel experiments monitoring [3H]-ouabain binding supported the cytochemical studies. When expressed as femtomoles [3H]-ouabain/microgram DNA, the following values were obtained: whole pancreas, 100.3; ducts (pooled intralobular and interlobular), 337.0; acini, 48.2. The acinar value is complicated by the fact that acini contain both acinar and centroacinar cells, but in light of the cytochemical observations we suggest that most of the [3H]-ouabain binding is due to the few ductal cells present in acini. The results suggest that Na+,K+-ATPase is primarily associated with the ductal epithelium of the exocrine pancreas and is differentially distributed among the different classes of ducts.

Distribution of sialoglycoconjugates on acinar cells of the mammalian pancreas.

Using the sialic acid-specific lectin, limulin (LPA; from Limulus polyphemus hemolymph), the distribution and nature of sialoglycoconjugates on the surface of rat pancreatic cells has been investigated. Binding of rhodaminated LPA (Rh-LPA) or horseradish peroxidase-conjugated LPA (HRP-LPA) to fixed-frozen sections of adult rat pancreas resulted in intense linear staining of the apical surface of acinar cells with fainter staining on the basal but not the lateral cell surfaces. LPA binding was specific in that it could be abolished by 1) pretreatment of tissue sections with neuraminidase or periodic acid; 2) competition with sialic acid; and 3) incubation in Ca2+ -free buffers. Pretreatment of sections with proteases abolished LPA binding to the apical surfaces of acinar cells and also enhanced LPA binding to the lateral cell surface. Lipid extraction of sections following protease treatment markedly reduced LPA binding to the acinar cell periphery. These results suggest that LPA binding sites on the acinar cell apical surface may be primarily sialoglycoproteins, while those on the basolateral surfaces may consist in part of gangliosides. Electron microscopy of collagenase-dispersed acini exposed to HRP-LPA confirmed binding of LPA to the basal plasmalemma and, in addition, revealed staining of basal lamina when present. LPA binding to the acinar cell surface was not affected by digestion of tissue sections with hyaluronidase, heparinase, collagenase, or 6 M guanidine-HCl. Control experiments indicated that rat pancreatic secretory proteins contain undetectable amounts of sialoglycoproteins and thus that the apical localization of LPA is not due to adherent secretory proteins. Islets of Langerhans were always uniformly and heavily stained with LPA conjugates; this staining was protease insensitive. Appearance of LPA binding sites was examined on embryonic pancreatic epithelia. At day 15 of gestation, Rh-LPA stained the entire periphery of the epithelial cells, including the lateral cell surface, although more intense staining was already noted on the apical surface. This pattern persisted through day 17 of gestation, but by day 19 an adult staining pattern was observed with loss of staining of the lateral cell surfaces.

Regional differences in lectin binding to colonic epithelium by fluorescent and electron microscopy.

The distribution of glycosubstances in colonic epithelium using lectins that were rhodaminated for fluorescent microscopy or coupled to colloidal gold for electron microscopy has been studied. Findings, based on lectin binding patterns, indicate that the right, transverse, and left colon of the guinea pig differ in their content of glycoconjugates within goblet cells and along the brush border. Local variations of labeling were also observed within goblet cells between the upper and lower portions of crypts. Throughout the colon, corresponding to a region of supranuclear fluorescence, Ricin II-colloidal gold labeled the Golgi complex of both enterocytes and goblet cells. Ricin II-colloidal gold also labeled small (congruent to 800 A) vesicles in the apical portion of enterocytes in all colonic segments. Microvilli were labeled by Ricin II-colloidal gold in the right and transverse colon, a finding that correlated with the observed adhesion of bacteria in those segments.

Primary monolayer cell culture of bovine parathyroids: effects of calcium, isoproterenol and growth factors.

Cell aggregates of bovine parathyroid tissue were prepared by limited collagenase digestion and placed in culture in Weymouth's MB752/1 (calcium = 3.3 mg/100 ml) containing 5% fetal bovine serum and supplemented with insulin alone, or insulin, hydrocortisone, transferrin and epidermal growth factor. Only insulin was required for the maintenance of PTH secretion over a 9-day period. The cell aggregates spread to form monolayer in 3-5 days. The majority of the cells in monolayer were polygonal with well-defined borders. Nuclei were round and the cytoplasm was free of vacuoles. Cell cultures responded to secretory stimulation by low calcium or by isoproterenol with increases in the secretion of PTH and SP-1. At low calcium, about 18% of both the cellular PTH and SP-1 was secreted per hour, and up to 50% of the cell content of these proteins was released per hour upon stimulation by isoproterenol and low calcium combined. The responses to calcium and isoproterenol decreased as a function of time in culture, and calcium responses often disappeared completely by 10 days of culture. When cells were cultured in medium containing a higher (5 mg%) than standard concentration of calcium between days 3-6 of culture, the degree of secretory inhibition attainable with high calcium was greater than that of cells cultured in the standard medium. When secreted hormonal peptides were separated by SDS-gel electrophoresis prior to RIA, it was found that the secretion of intact hormone was sensitive to calcium. For every molecule of PTH secreted into the medium, 1.5-2 mole-equivalents of carboxyl fragments were also released. Calcium control of fragment release was not as stringent as that of PTH release.

Paraphysectomy-induced stimulation of parathyroid glands in mature frogs (Rana catesbeiana): evidence for telencephalic regulation of parathyroid gland function.

The relationship of the paraphyseal-choroid plexus complex to parathyroid gland function was investigated in adult frogs. Light microscopy and morphometric analysis indicated that total parathyroid gland volume, cell volume and vascular volume doubled by 7-28 days after surgical removal of the paraphyseal-choroid plexus complex (paraphysectomy). This increase correlated with the appearance of large Golgi-associated vesicles, an increase in the apparent number of cytoplasmic dense-core granules, and PTH within the parenchymal cells as monitored by immunofluorescence. Twelve months after paraphysectomy, parathyroid glands became cystic with a central fluid-filled cavity surrounded by a stratified cuboidal cell layer. The parenchymal cells of cystic glands contained numerous cytoplasmic dense-core granules and were also positive for PTH. Radioimmunoassay of cystic parathyroid fluid indicated a PTH concentration of 2 micrograms/microliter; however, analysis by SDS-PAGE indicated a wide range of proteins in cystic fluid. The results of this study indicate that paraphysectomy induces stimulation of the parathyroid glands and suggest a role for the paraphyseal-choroid plexus complex in the regulation of amphibian parathyroid gland function.

Morphological and biochemical characterization of a human pancreatic ductal cell line (PANC-1).

Study of the products secreted by pancreatic ductal cells and analysis of the mechanisms involved in the discharge of these products have been limited by a lack of in vitro models available to experimentally approach this problem. To this aim, this investigation has been designed to determine if a human pancreatic carcinoma cell line of ductal origin (PANC-1) has maintained some of the differentiated characteristics of normal mammalian pancreatic ductal epithelium. Morphological and immunocytochemical studies indicated that, similar to isolated rat pancreatic ducts, the PANC-1 cell line contained (a) intermediate filaments of the epithelial class, (b) a basolateral plasma membrane localization of Na+, K+-ATPase, (c) complete tight junctions based on freeze-fracture analysis, (d) a cuboidal morphology when grown on Type I collagen-coated nitrocellulose filters or isolated amnion basement membrane, and (e) normal ductal epithelial ultrastructural features. Biochemical analysis indicated that, also similar to isolated rat and human pancreatic ducts, the PANC-1 cell line contained (a) gamma-glutamyltranspeptidase, (b) carbonic anhydrase, and (c) Na+, K+-ATPase based on [3H]ouabain binding assays. Comparative studies with other transformed lines indicated that PANC-1 cells have similarities to ductal lines such as MDCK cells but are markedly different from mesenchymally derived lines such as L cells. In addition, as with isolated rat and human ducts, PANC-1 cells synthesize and secrete sulfated proteins with a MW range of approximately 180K to 1 million daltons, with the predominant species being 660K daltons as indicated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. These results indicate that the PANC-1 cell line has maintained at least some of the differentiated characteristics of normal pancreatic ductal epithelial cells and may be a useful system for study of ductal secretory products as well as the mechanisms involved in the discharge of these products.

The pancreatic ductal system of the rat: cell diversity, ultrastructure, and innervation.

The morphological features and innervation of the pancreatic ductal epithelium of the rat was investigated. For the purposes of this study, pancreatic ducts were classified as either intercalated, intralobular, interlobular, or main. Epithelial cells of these different classes were classified as either principal or specialized. Principal cells make up the majority of epithelial cells in all classes of ducts. Ultrastructural analysis indicated that cytoplasmic vesicles, some containing amorphous material, were often seen in the apical portion of principal cells. Similarly, a common feature of these cells was the appearance of apical membrane projections containing various cytoplasmic organelles. These vesicles and apical membrane projections became larger as one progressed through the ductal system to the main duct. Junctional complexes contained well-developed tight junctions that, when analyzed by freeze-fracture analysis, were found to consist of three to five sealing strands in a parallel arrangement. Specialized cells could be morphologically divided into five categories: light cells, basal cells, goblet cells, endocrine cells, and brush cells. Light cells, which only differed from principal cells by their clear, lightly staining cytoplasm, were found in all classes of ducts. Basal cells, which were attached to the basement membrane of the ductal epithelium but did not extend to the lumen, were found in all classes of ducts except the intercalated ducts. Goblet cells and endocrine cells were observed in the main and interlobular ducts, while cells very similar in morphologic appearance to the brush cells of the lung were found restricted to the interlobular ducts. Localization of biogenic amines by histofluorescence indicated that adrenergic nerve fibers were associated with the main ducts and interlobular ducts. Histochemical localization of acetylcholinesterase activity indicated that cholinergic fibers were also associated with the main and interlobular ducts. These results indicate that in the rat, the pancreatic ductal system is composed of a number of cell types that are differentially distributed in the various classes of ducts, and pancreatic ducts are associated with adrenergic and cholinergic nerves.

Metal ions induce expression of metallothionein in pancreatic exocrine and endocrine cells.

Northern blot hybridization established that metallothionein (MT) mRNA levels were dramatically elevated in the rat pancreas following injection of Cd or Zn salts. To determine which pancreatic cell types express the MT gene, Northern blot hybridization analysis of RNA from preparations enriched for acini, in situ hybridization, and immunocytochemistry were used. RNA from pancreatic acini of Zn-treated rats contained high levels of MT mRNA. In control rats, in situ hybridization suggested very low levels of MT mRNA in both exocrine and endocrine cells in the pancreas, but these levels were dramatically increased in both these cell populations following metal injection. In contrast, levels of insulin-I mRNA in the endocrine cells were not affected by metal injection. A similar result with MT mRNA was obtained in mouse and chicken pancreas using Northern blot and in situ hybridization. Immunocytochemistry detected MT in the rat acinar cell cytoplasm following metal injection. Although inconsistent with in situ hybridization studies and immunocytochemical analysis of exocrine cells, immunocytochemistry for MT indicated a uniform staining pattern of islet cells that was unaffected by metal treatment. These results establish that metal ion induction of the MT genes in pancreas occurs in both endocrine and exocrine cells, which suggests that this protein has diverse physiologic functions in this organ.

High molecular weight secretory products of the human pancreatic ductal epithelium and the effect of secretin on their discharge.

Principal cells of the pancreatic ductal epithelium have been reported to secrete high molecular weight (HMW) glycoconjugates such as mucins into the ductal lumen. We used a human pancreatic carcinoma cell line of ductal origin (PANC-1) which has retained some of the morphological and biochemical characteristics of normal ductal principal cells as a source for isolation of HMW secretory products. The present study was designed to isolate these HMW secretory products, partially characterize them through biochemical and immunological approaches, and determine the effects of secretin on their synthesis and discharge from PANC-1 cells. Our results indicated that when PANC-1 cells are grown on collagen-coated beads in defined serum-free medium, at least three HMW secretory products could be isolated from the medium. These secretory products had a mass of approximately 200, 280, and 370 kDa based on sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The 200-kDa species made up proportionally less of the three in the gel-staining pattern. Polyclonal antibodies raised to the 370-kDa secretory product cross-reacted with the 280-kDa species. The 370-kDa secretory product was sulfated and wheat germ agglutinin (WGA) binding indicated that the 370-kDa species was a glycoconjugate. The 280-kDa and 200-kDa species were sulfated to a much lesser degree than the 370-kDa species and WGA binding could not be clearly demonstrated with the 280 kDa or 200 kDa species. Glycosidase and selective degradation studies, however, indicated that all three species contained glycosaminoglycan moieties. Antibodies raised to the 370-kDa secretory product localized to the epithelium of human pancreatic carcinomas but not to other cell types in this neoplastic tissue. The antibody also cross-reacted with the ductal epithelium of normal human pancreas and could be localized to centroacinar cells, the epithelium of intralobular ducts, and the epithelium of interlobular ducts. The antibody did not cross-react with goblet cells of the human small or large intestine, indicating no generalized reactivity to gastrointestinal mucins. ELISA and pulse-chase immunoprecipitation studies indicated an increase in the cellular content and synthesis of these HMW secretory products after stimulation of PANC-1 cells with 10(-8)- to 10(-11)-M secretin. We correlated secretin stimulation with the appearance of numerous membrane bound vesicles throughout the cytoplasm as monitored at the ultrastructural level. Optimal secretin concentration were in the range of 10(-10) M. The content of these vesicles was immunoreactive to the antibody raised against the 370-kDa secretory product as determined by ultrastructural immunocytochemical techniques.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative analysis of a human pancreatic undifferentiated cell line (MIA PaCa-2) to acinar and ductal cells.

Pancreatic ductal cell secretion has not been well characterized due to the difficulty in obtaining sufficient quantities of purified ductal cells. To determine if the MIA PaCa-2 cell line would provide a useful model for in vitro studies of pancreatic ductal cell secretion, the present study was designed to characterize these cells in greater detail. In this investigation, the human pancreatic undifferentiated cell line, MIA PaCa-2, was compared with PANC-1 cells (a human ductal cell line previously characterized), isolated rat and human ducts, acinar cells, and nonpancreatic cell lines. The results indicate that while the morphology of the MIA PaCa-2 cell line is nonpolarized and generally atypical of either ductal or acinar cells, the cell line has retained certain biochemical similarities to ductal cells. Additional morphological studies indicated (a) the presence of intermediate filaments characteristic of epithelial cells, (b) the absence of zymogen granules, and (c) an apparent basolateral plasma membrane localization of Na+, K+-ATPase. Similar to ductal cells, biochemical analyses indicated (a) the presence of Na+, K+-ATPase based on [3H]-ouabain binding assays, (b) high levels of carbonic anhydrase, (c) low levels of gamma-glutamyl transpeptidase, (d) nondetectable levels of amylase, and (e) protein composition and protein synthetic patterns comparable to PANC-1 cells. Finally, as with PANC-1 cells and isolated rat and human ducts, the major sulfated secretory product of MIA PaCa-2 cells was a protein with a molecular weight of approximately 660,000 to 1 million.(ABSTRACT TRUNCATED AT 250 WORDS)

Metallothionein protects against cerulein-induced acute pancreatitis: analysis using transgenic mice.

Oxidative stress has been proposed to play a role in the early events of acute pancreatitis, and metallothionein (MT) can provide protection against oxidative stress. Using transgenic mice, we characterized the effects of depletion of MT-I and -II, or overexpression of MT-I, on pancreatic responses during cerulein-induced acute pancreatitis. In MT-I/-II knockout mice, repeated injections of cerulein caused (a) higher serum amylase levels at 3 and 7 h after the initiation of acute pancreatitis; (b) earlier and stronger upregulation of oxidative stress-responsive genes, including heme oxygenase (HO)-1 and c-fos; and (c) exacerbated tissue damage (edema and polymorphonuclear neutrophil infiltration) compared with nontransgenic 129/SvCPJ mice. Total pancreatic glutathione (GSH + GSSG) content was similar between the knockout and nontransgenic 129/SvCPJ mice. Interestingly, during acute pancreatitis, CD-1 mice pretreated with L-buthionine-[S,R]-sulfoximine (BSO), which dramatically depleted pancreatic GSH, also had more severe pancreatitis, based on the same three criteria listed above, relative to untreated controls. No effects were observed with BSO treatment alone. Finally, during cerulein-induced acute pancreatitis, MT-I overexpressing transgenic mice (>20-fold increase in pancreatic MT-I content) had lower serum alpha-amylase levels between 7 and 24 h and delayed upregulation of HO-1 mRNA levels, but no difference in c-fos mRNA induction relative to the appropriate strain of nontransgenic mice. Diminished tissue damage (particularly cellular necrosis) was noted in these MT-I overexpressing transgenic mice. Total pancreatic GSH content was similar in these transgenic and nontransgenic mice during cerulein-induced acute pancreatitis. These studies suggest that pancreatic MT can function as an intracellular antioxidant as does GSH and that these intracellular antioxidants play a protective role during cerulein-induced acute pancreatitis.

Expression of bone morphogenetic proteins by osteoinductive and non-osteoinductive human osteosarcoma cells.

Freeze-dried Saos-2, human osteosarcoma cells, and extracts of Saos-2 cells contain all components necessary to induce ectopic new bone and marrow when implanted into athymic Nu/Nu nuce. On the other hand, human osteosarcoma cells of the U-2 OS strain failed to induce bone formation under the same experimental conditions. Our aim was to compare the relative expressions of known osteoinductive factors including bone morphogenetic proteins (BMPs) and transforming growth factor-beta (TGF-beta) in these two cell lines in an attempt to explain the unique bone-inducing ability of the Saos-2 cells. Saos-2 cells expressed mRNA for BMP-1, -2, -3, -4,-6, and TGF-beta 1. The non-osteoinductive U-2 OS cells expressed BMP-2, -4, -5, -6, and -7 as well as TGF-beta 1 mRNA, while levels of BMP-1 and BMP-3 mRNA were either not detectable or detectable at a very low level in U-2 OS cells. The presence of BMP-1 and -4 protein was confirmed in Saos-2 cells by immunofluorescence, and TGF beta protein was demonstrated by bioassay in both cell types. These findings suggest that Saos-2 cells are endowed qualitatively and quantitatively with sufficient amounts of many bone morphogenetic proteins-especially BMP-1, -3, and -4-to confer osteoinductivity upon these cells. However, the absence of osteoinductivity in U-2 OS cells, despite significant mRNA expression levels of several bone morphogenetic proteins, suggests that, even though expression of one or more bone morphogenetic proteins may be present, it may not necessarily be sufficient to confer osteoinductivity upon U-2 OS cells. U-2 OS cells may be non-osteoinductive because (1) they contain inhibitors to the BMPs or secrete inhibitory binding proteins, (2) they do not process BMPs correctly, or (3) the BMPs are inappropriately localized and sequestered within the U-2 OS cells. Saos 2 cells may be osteoinductive because (1) they uniquely express BMP-1, (2) they express an appropriate combination of interactive BMPs at appropriate levels, and/or (3) the Saos-2 cells elaborate as-yet-unidentified osteoinductive factor(s).

Analysis of cell surface glycoconjugates in fibroblasts from patients with cystic fibrosis.

Although the basic biochemical defect in cystic fibrosis (CF) is unknown, previous studies have indicated that errors in protein glycosylation may be involved in the pathogenesis of the disease. Utilizing human skin fibroblasts, the present study was designed to quantitatively analyze glycosylation of cell surface glycoconjugates in CF and normal cells. Cell surface glycoconjugates were analyzed using 125I-concanavilin A (Con A), 125I-WGA, and Con A-ferritin conjugates. Under our binding conditions, Con A was used as a probe for mannose residues and WGA was used as a probe for N-acetylglucosamine residues. Saturable binding of both probes was observed and appropriate sugar controls confirmed the specificity of each lectin. When compared on a DNA basis, iodinated lectin binding studies indicated that no consistent differences existed between CF and normal strains of human skin fibroblasts. Ultrastructural quantitative morphometric analysis of Con A-ferritin conjugate binding indicated that neither proteolysis of cell surface glycoconjugates or internalization of lectin probes was occurring at saturable binding concentrations. In summary, our results indicated that no consistent differences in cell surface mannose and N-acetylglucosamine residues could be detected between the normal and CF strains of human skin fibroblasts used in these studies.

Binding of wheat-germ agglutinin to glycoconjugates in the salivary glands of reserpinized rats.

Salivary glands from adult rats that had received reserpine for 1, 3 or 7 days and from saline-treated controls were treated with rhodamine-labelled wheat-germ agglutinin conjugates (WGA-TRITC) to localize and characterize the distribution of glycoconjugates. Fluorescent and morphometric analysis of the parotid, submandibular and sublingual glands indicated that each gland responded differently to reserpine treatment. Parotid gland acinar cells and ducts showed no change in pattern or intensity of WGA-TRITC staining after reserpine. Mucous acinar cells of the submandibular gland had increased WGA binding and an accumulation of WGA-positive material in duct lumina after 3-7 days of reserpine. Morphometric analysis showed that the maximal increase in submandibular acinar-cell size occurred after 1 day of reserpine treatment. In sublingual glands, there was no detectable increase in mucous acinar-cell staining, but progressive accumulation of WGA-positive material was seen in duct lumina after 7 days of reserpine. As WGA binds to N-acetyl-glucosamine and N-acetyl-neuraminic acid residues, it may be that the eventual blockage of the duct system is related to increased production and secretion of glycoconjugates that contain these carbohydrates.

BMP-1 and the astacin family of metalloproteinases: a potential link between the extracellular matrix, growth factors and pattern formation.

Members of the astacin family of metalloproteinases such as human bone morphogenetic protein 1 (BMP-1) have previously been linked to cell differentiation and pattern formation during development through a proposed role in the activation of latent growth factors of the TGF-beta superfamily. Recent findings indicate that BMP-1 is identical to pro-collagen C-proteinase, which is a metalloproteinase involved in extracellular matrix (ECM) formation. This observation suggests that a functional link may exist between astacin metalloproteinases, growth factors and cell differentiation and pattern formation during development. Taken together, current studies indicate that BMP-1 and possibly other astacin metalloproteinases are multifunctional enzymes that act directly on growth factors and the ECM. In combination, these dual actions would have profound effects on developmental processes.

Preparation and characterization of single cells from the avian salt gland.

In preparation for pulse-chase autoradiography experiments and studies of cell surface changes of relevance to plasma membrane biogenesis, we have prepared a cell suspension from the salt gland of ducklings. The method used was a modification of previous methods used for pancreas and salivary gland and included digestions with collagenase and hyaluronidase, divalent cation chelation, and dispersion by gentle pipetting. Yields were 1.13 X 10(7) cells/g gland, and cell recovery was 45% by DNA assay. Recovery of Na,K-ATPase, a marker for salt gland secretory cells was 40--47%. Cell viability was strongly indicated by trypan blue exclusion and 3H-leucine incorporation. Transmission and scanning electron microscopy revealed that most cells retained ultrastructural features characteristic of the intact gland. Smaller cells (3--8 micrometers in diameter), exhibiting few surface microvilli and relatively few cytoplasmic organelles, likely represented the undifferentiated, peripheral cells from the tips of secretory tubules. Larger cells (5--10 micrometers in diameter), exhibiting prominent surface membrane folds enclosing numerous mitochondria, likely represented the functional, secretory cells of the salt gland tubules in various stages of differentiation. The surface folds presented as microvilli and microplicae in scanning electron micrographs.

Development of an apical plasma membrane domain and tight junctions during histogenesis of the mammalian pancreas.

The role of tight junctions (zonula occludens) in the formation of apical plasma membrane (PM) domains was investigated in the embryonic rat pancreas. In the present study, lectin-rhodamine (WGA-TRITC and RCAII-TRITC) and lectin-gold (WGA-Au and RCAII-Au) conjugates were used to monitor apical PM domain formation and freeze-fracture analysis was used to monitor tight junction formation in the pancreatic epithelium of embryonic, neonatal, and adult rats. Fluorescent and TEM analysis of WGA and RCAII binding indicated that an apical PM domain is formed as early as Day 13 of gestation in the pancreatic epithelium. While apical WGA binding remained into adult life, RCAII binding was lost by 1 day after birth. In contrast, tight junctions were not observed until Day 14 of gestation. At this time, tight junctions were found to be incomplete in formation and typically consisted of linear arrays of IMPs or discontinuous arrays of sealing strands (focal adherens). Continuous tight junctions were not completely formed until Day 15 of gestation. Continued development of tight junctions during gestation was characterized by (1) an increase in the number of sealing strands and (2) a more parallel arrangement of sealing strands within each junctional complex. By 8 weeks after birth, tight junctions were more loosely organized and contained fewer sealing strands as compared to that observed in the fetus. These results suggest that lateral diffusion of apical PM glycoconjugates may be restricted even in the absence of complete tight junctional complexes during development of the rat pancreas.

Evidence for cell surface extracellular matrix binding proteins in Hydra vulgaris.

The present study was designed to identify and functionally characterize potential cell surface extracellular matrix binding proteins in Hydra vulgaris. Using [3H]-laminin as a probe, radioreceptor analysis of a dissociated mixed hydra cell preparation indicated that the average number of laminin binding sites per cell was about 10,000 with a dissociation constant of 1.49 nM. These binding sites could be displaced with unlabelled laminin in a dose-dependent manner and with high concentrations (500 nM) of unlabelled fibronectin. No displacement with type-IV collagen and type-I collagen was observed. Immunoscreening studies with a battery of antibodies raised to mammalian extracellular matrix (ECM) binding proteins indicated potential cell surface binding sites for the anti-beta 1 integrin monoclonal antibody, mAb JG22. Cell adhesion studies indicated that mAb JG22 blocked binding of hydra cells to laminin, but did not affect their binding to fibronectin, type-IV collagen, or type-I collagen. Light and electron microscopic immunocytochemical studies indicated that mAb JG22 localized to the basal plasma membrane of ectodermal and endodermal epithelial cells. Immunoprecipitation studies identified tow major bands with masses of about 196 kDa and 150 kDa under reducing conditions, and two bands with masses of > 200 kDa under non-reducing conditions. Functional studies indicated that mAb JG22 could reversibly block morphogenesis of hydra cell aggregates, and could block in vivo interstitial cell migration in hydra grafts. These observations indicate that hydra has cell surface binding sites for ECM components which are functionally important during development of this simple Cnidarian.

Cell-extracellular matrix interactions under in vivo conditions during interstitial cell migration in Hydra vulgaris.

Interstitial cell (I-cell) migration in hydra is essential for establishment of the regional cell differentiation pattern in the organism. All previous in vivo studies have indicated that cell migration in hydra is a result of cell-cell interactions and chemotaxic gradients. Recently, in vitro cell adhesion studies indicated that isolated nematocytes could bind to substrata coated with isolated hydra mesoglea, fibronectin and type IV collagen. Under these conditions, nematocytes could be observed to migrate on some of these extracellular matrix components. By modifying previously described hydra grafting techniques, two procedures were developed to test specifically the role of extracellular matrix components during in vivo I-cell migration in hydra. In one approach, the extracellular matrix structure of the apical half of the hydra graft was perturbed using beta-aminopropionitrile and beta-xyloside. In the second approach, grafts were treated with fibronectin, RGDS synthetic peptide and antibody to fibronectin after grafting was performed. In both cases, I-cell migration from the basal half to the apical half of the grafts was quantitatively analyzed. Statistical analysis indicated that beta-aminopropionitrile, fibronectin, RGDS synthetic peptide and antibody to fibronectin all were inhibitory to I-cell migration as compared to their respective controls. beta-xyloside treatment had no effect on interstitial cell migration. These results indicate the potential importance of cell-extracellular matrix interactions during in vivo I-cell migration in hydra.

Hydra and Niccolo Paganini (1782-1840)--two peas in a pod? The molecular basis of extracellular matrix structure in the invertebrate, Hydra.

The body wall of Hydra is organized as an epithelial bilayer with an intervening extracellular matrix (ECM). Molecular and biochemical analyses of Hydra ECM have established that it contains components similar to those seen in more complicated vertebrates such as human. In terms of biophysical parameters, Hydra ECM is highly flexible; a property that facilitates continuous movements along the organism's longitudinal and radial axis. A more rigid ECM, as in vertebrates, would not be compatible with this degree of movement. The flexible nature of Hydra ECM can now be explained in part by the unique structure of the organism's collagens. Interestingly, some aspects of the structural features of Hydra collagens mimic what is seen in Ehlers-Danlos syndrome, an inherited condition in humans that results in an abnormally flexible ECM that can be debilitating in extreme cases. This review will focus on structure-function relationships of the ECM of Hydra.