In situ localization with digoxigenin-labelled probes of tau-related mRNAs in the rat pancreas

Summary Two cDNA probes complementary to fetal rat brain tau cDNA were produced by the polymerase chain reaction (PCR) and labelled by digoxigenin-11-dUTP incorporation during the PCR elongation step. These probes were tested for thein situ localization of tau mRNAs in sections of rat cerebellum. The hybridization signal was consistent with the known localization of brain tau mRNAs, showing the validity of cDNA probes labelled by digoxigenin during the PCR. Using these probes, anin situ hybridization protocol was established and optimized for the localization of tau-related mRNAs in sections of pancreas. The aim was to determine whether these mRNAs were expressed in the exocrine or the endocrine part of the pancreas. A positive signal was found only in the exocrine part of the pancreas, and was distributed exclusively in the cytoplasm of acinar cells. The results described here are the first evidence for a specific expression of tau-related proteins in the exocrine pancreas.     

大鼠胚胎发育期NBC1 在胰腺的表达与定位

目的:探讨碳酸氢钠协同转运载体(NBC1)在大鼠胰腺胚胎发育期不同阶段核酸、蛋白水平的动态变化以及在腺泡和β细胞的定位表达。方法:采用高密度寡核苷酸芯片对孕12.5 d(E12.5)、E15.5、E18.5、新生和成年胰腺进行基因转录水平分析,用RT-PCR和Western blot分别验证了NBC1核酸和蛋白在E15.5、E18.5、新生和成年时期胰腺中的表达情况,用Double fluorescence immunohistochemistry分析了NBC1在E18.5、新生和成年时期胰腺腺泡和β细胞的定位表达。结果:在大鼠胰腺胚胎发育过程中,NBC1核酸、蛋白在E18.5时特异高表达,新生下降直至成年最低;在腺泡基底侧膜和β细胞膜有强烈的阳性信号,且在成年胰腺中β细胞膜阳性信号较腺泡基底侧膜强。NBC1的表达变化与其功能近似基因的表达趋势相反,而与其协同发挥作用的基因及胰腺特异基因的表达趋势一致。结论:NBC1在胰腺发育过程中不仅与结构形成而且与功能发挥相关。     

Structure and dynamics of the fusion pore in live cells.

Atomic force microscopy reveal pit-like structures typically containing three or four, approximately 150 nm in diameter depressions at the apical plasma membrane in live pancreatic acinar cells. Stimulation of secretion causes these depressions to dilate and return to their resting size following completion of the process. Exposure of acinar cells to cytochalasin B results in decreased depression size and a loss in stimulable secretion. It is hypothesized that depressions are the fusion pores, where membrane-bound secretory vesicles dock and fuse to release vesicular contents. Zymogen granules, the membrane-bound secretory vesicles in exocrine pancreas, contain the starch digesting enzyme, amylase. Using amylase-specific immunogold labeling, localization of amylase at depressions following stimulation of secretion is demonstrated. This study confirms depressions to be the fusion pores in pancreatic acinar cells. High-resolution images of the fusion pore in live pancreatic acinar cells reveal the structure in much greater detail than has previously been observed.     

Immunohistochemical localization of Zn-alpha 2-glycoprotein in normal human tissues

The Zn-alpha 2-glycoprotein (Zn-alpha 2-GP) is present at a high concentration in the seminal plasma and at significant levels in other human body fluids. Its precise localization, however, has remained unclear, as well as its physiological and pathological significance. The present study reports the immunohistochemical localization of this protein in normal adult human tissues. Localization of the reactive product to anti-human plasma Zn-alpha 2-GP antibody was demonstrated in the following cells: luminal and basal cells of the prostate gland, luminal epithelial cells of the acini and of some ducts of the mammary glands, luminal cells of the secretory portion of the eccrine and apocrine sweat glands, serous cells of the salivary, tracheal, and bronchial glands, acinar cells of the esophageal glands, exocrine acinar cells of the pancreas, hepatocytes of the liver, and epithelial cells of the proximal and distal tubules in the kidney. The present results suggest that Zn-alpha 2-GP exerts some unknown but fairly widespread exocrine function and may be produced in the various epithelial cells tested. Hepatocytes are also suggested to be a source of the protein in the blood plasma.     

Disruption of a putative SH3 domain and the proline-rich motifs in the 53-kDa substrate of the insulin receptor kinase does not alter its subcellular localization or ability to serve as a substrate

The recently identified 53-kDa substrate of the insulin receptor family was further characterized in several retroviral-generated stable cell lines overexpressing the wild type and various mutant forms of the protein. To facilitate the study of its subcellular localization in NIH3T3 cells overexpressing insulin receptor, a myc epitope-tag was added to the carboxy terminus of the 53-kDa protein. Like the endogenous protein in Chinese hamster ovary cells, the expressed myc-tagged 53-kDa protein was found partially in the particulate fraction and was tyrosine phosphorylated in insulin-stimulated cells. Immunofluorescence studies showed for the first time that a fraction of the 53-kDa protein was localized to the plasma membrane. Confocal microscopy of cells double-labeled with antibodies to the insulin receptor and the myc epitope showed the two proteins co-localize at the plasma membrane at the level of light microscopy. Further analyses of the protein sequence of the 53-kDa substrate revealed the presence of a putative SH3 domain and two proline-rich regions, putative binding sites for SH3 and WW domains. Disruption of these three motifs by the introduction of previously characterized point mutations did not affect the membrane localization of the 53-kDa protein, its ability to serve as substrate of the insulin receptor, or its colocalization with the insulin receptor, suggesting these domains are not important in the subcellular targeting of the protein and instead may function in the interaction with subsequent signaling proteins. J. Cell. Biochem. 68:139–150, 1998. © 1998 Wiley-Liss, Inc.     

In vivo localization of insulin binding to cells of the rat pancreas

The uptake of 125I-insulin by rat pancreas was studied in vivo. Following fixation and light microscope autoradiography, saturable uptake of 125I-insulin was quantitatively demonstrated on acinar and duct cells but not on blood vessels and islets of Langerhans. Electron microscopy revealed the localization of 125I-insulin to the basolateral cell membranes of acinar and duct cells.     

Cloning and immunolocalization of a rat pancreatic Na(+) bicarbonate cotransporter.

In the rat, pancreatic HCO(-)(3) secretion is believed to be mediated by duct cells with an apical Cl(-)/HCO(-)(3) exchanger acting in parallel with a cAMP-activated Cl(-) channel and protons being extruded through a basolateral Na(+)/H(+) exchanger. However, this may not be the only mechanism for HCO(-)(3) secretion by the rat pancreas. Recently, several members of electrogenic Na(+)/HCO(-)(3) cotransporters (NBC) have been cloned. Here we report the cloning of a NBC from rat pancreas (rpNBC). This rpNBC is 99% identical to the longer, more common form of NBC [pNBC; 1079 amino acids (aa); 122 kDa in human heart, pancreas, prostate, and a minor clone in kidney]. The longer NBC isoforms are identical to the rat and human kidney-specific forms (kNBC; 1035 aa; 116 kDa) at the approximately 980 C-terminal aa's and are unique (with different lengths) at the initial N-terminus. Using polyclonal antibodies to the common N- and C-termini of rat kidney NBC, a approximately 130-kDa protein band was labeled by immunoblotting of rat pancreas homogenate and was enriched in the plasma membrane fraction. Immunofluorescence and immunoperoxidase light microscopy of rat pancreatic tissue with both antibodies revealed basolateral labeling of acinar cells. Labeling of both apical and basolateral membranes was found in centroacinar cells, intra- and extralobular duct, and main duct cells. The specificity of the antibody labeling was confirmed by antibody preabsorption experiments with the fusion protein used for immunization. The data suggest that rpNBC likely plays a more important role in the transport of HCO(-)(3) by rat pancreatic acinar and duct cells than previously believed.     

Cellular Distribution and Subcellular Localization of mCLCA1/2 in Murine Gastrointestinal Epithelia

mCLCA1/2 are members of the CLCA protein family that are widely expressed in secretory epithelia, but their putative physiological role still awaits elucidation. mCLCA1/2 have 95% amino acid identity, but currently no specific antibody is available. We have generated a rabbit polyclonal antibody (pAb849) against aa 424–443 of mCLCA1/2. In HEK293 cells transfected with mCLCA1; pAb849 detected two specific protein bands at ∼125 kDa and 90 kDa, representing full-length precursor and N-terminal cleavage product, respectively. pAb849 also immunoprecipitated mCLCA1 and labeled the protein by immunostaining. But pAb849 crossreacted with mCLCA3/4/6 despite ≤80% amino acid identity of the antigenic epitope. We therefore investigated the cellular localization of mCLCA1/2 in epithelial tissues, which do not express mCLCA3/4/6 (salivary glands, pancreas, kidney) or express mCLCA3/6 with known localization (mucus cells of stomach and small intestine; villi of small intestine). mCLCA1/2 mRNA and protein expression were found in both parotid and submandibular gland, and immunohistochemistry revealed labeling in parotid acinar cells, in the luminal membrane of parotid duct cells, and in the duct cells of submandibular gland. In exocrine pancreas, mCLCA1/2 expression was restricted to acinar zymogen granule membranes, as assessed by immunoblotting, immunohistochemistry, and preembedding immunoperoxidase and immunogold electron microscopy. Moreover, mCLCA1/2 immunolabeling was present in luminal membranes of gastric parietal cells and small intestinal crypt enterocytes, whereas in the kidney, mCLCA1/2 protein was localized to proximal and distal tubules. The apical membrane localization and overall distribution pattern of mCLCA1/2 favor a transmembrane protein implicated in transepithelial ion transport and protein secretion. (J Histochem Cytochem 58:653–668, 2010)     

Histochemical and cytochemical localization of (Na+-K+)-activated adenosine triphosphatase in the acini of dog submandibular glands.

p-Nitrophenyl phosphatase (p-NPPase) activity of (Na+-K+)-activated adenosine triphosphatase ((Na+-K+)-ATPase) on the acinar cells of dog submandibular gland was demonstrated by using light microscopy. The reaction products of p-NPPase of fresh frozen sections were seen to be localized on the basal parts of acini, and disappeared when the sections were incubated in medium containing 10(-3) Mouabain or in a K-free medium. Under the electron microscope, the reaction products of ATPase were found to be localized on the basolateral plasma membrane of both serous and mucous cells. On the microvilli of the luminal plasma membrane of the acinar cell, a small quantity of the reaction products was also present. This localization of ATPase reaction products on the serous and mucous cells seemed to coincide well with that of p-NPPase activity observed on the acini under light microscopy. Possible explanations are given regarding distribution of the above mentioned enzymes in relation to the cation transport of the plasma membrane. Structural and functional asymmetrical properties of acinar cells of the dog submandibular gland are also discussed.     

Cytochemical glucose-6-phosphatase activity in the cells of mouse pancreas and submandibular gland

Ultrastructural localization of glucose-6-phosphatase activity was studied in the cells of the pancreas and submandibular gland of the mouse using a incubation medium modified from that of Wachstein & Meisel (1956). In pancreatic acinar cells, the reaction product for the enzyme activity was not found even after 90 min of incubation with three changes of the medium. However, the reaction product was localized in the endoplasmic reticulum and nuclear envelope of all other cell types composing the pancreas and submandibular gland. The reaction product appeared in moderate to abundant amounts in acinar cells and striated duct cells of the submandibular gland, and in the B cells, A and D cells of the pancreatic islet, but it was scarce in other cell types.     

Immunocytochemical localization of 67 KD Ca2+ binding protein (p67) in ventricular, skeletal, and smooth muscle cells.

By using immunocytochemical techniques, we examined the localization of a 67 kDa Ca2+ binding protein (p67) and calpactin I heavy chain (p36) in ventricular myocytes, skeletal myocytes, and intestinal smooth muscle cells. Immunofluorescence microscopy revealed that the p67 was expressed in all these muscle cells, whereas anti-p36 antibody stained cells in connective tissues but failed to stain these muscle cells. Immunogold electron microscopy was carried out to examine the subcellular localization of the p67 in muscle cells. The results showed that the p67 was exclusively confined to the plasma membrane of muscle cells and the presumptive transverse tubules of the striated myocytes. Immunoblot analysis with anti-p67 antibody showed that the p67 was indeed a constitutive protein of the sarcolemma isolated from rat hearts. These results indicate that the p67 is a sarcolemma-associated Ca2+ binding protein expressed in both striated myocytes and intestinal smooth muscle cells.