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A W Lowe

Publications and source records attributed to A W Lowe.

13 recordsLinked to original sources
PubMed1997

Tissue-specific alternative RNA splicing of rat vesicle-associated membrane protein-1 (VAMP-1).

The vesicle-associated membrane protein (VAMP) family is essential to vesicle-mediated protein transport. Three mammalian isoforms, VAMP-1, VAMP-2, and cellubrevin, play a role in protein transport to the plasma membrane. In this study, we describe a new rat VAMP-1 isoform produced by alternative pre-mRNA splicing. Only one VAMP-1 isoform dominates in each tissue. Analysis of the nucleotide sequence for the newly discovered isoform, VAMP-1b, reveals that its expression is determined by whether an intron is retained or removed. The predicted amino acid sequences for the VAMP-1 isoforms differ at the carboxy-terminal end of the protein. A similar process has been described for VAMPs in Drosophila melanogaster and suggests a conserved function for the carboxy-terminal domain that can be modulated.

Alternative Splicing↗
PubMed1996

Polarized GP2 secretion in MDCK cells via GPI targeting and apical membrane-restricted proteolysis.

The major zymogen granule membrane protein in the exocrine pancreas is glycoprotein 2 (GP2), a glycosyl phosphatidylinositol (GPI)-linked membrane protein. Despite its GPI anchor, GP2 is secreted into the pancreatic duct. We examined the mechanism underlying the secretion of GP2 in isolated pancreatic acini and transfected Madin-Darby canine kidney (MDCK) cells (MDCK-GP2). MDCK-GP2 cells release GP2 almost exclusively (> 95%) from the apical membrane. Using GP2 as a model, we defined a novel mechanism of polarized protein secretion in which a secretory protein is targeted via a GPI anchor to the apical plasma membrane, whereupon the mature form is released by proteolysis. Furthermore, we described two features of MDCK cells that enhance the polarized release of GP2: an apical plasma membrane-restricted distribution of the protease responsible for GP2 membrane cleavage, and a transcytotic pathway to reroute basolateral plasma membrane GP2 to the apical cell surface.

Animals↗
PubMed1995

Hierarchy of mechanisms involved in generating Na/K-ATPase polarity in MDCK epithelial cells.

We have studied mechanisms involved in generating a polarized distribution of Na/K-ATPase in the basal-lateral membrane of two clones of MDCK II cells. Both clones exhibit polarized distributions of marker proteins of the apical and basal-lateral membranes, including Na/K-ATPase, at steady state. Newly synthesized Na/K-ATPase, however, is delivered from the Golgi complex to both apical and basal-lateral membranes of one clone (II/J), and to the basal-lateral membrane of the other clone (II/G); Na/K-ATPase is selectively retained in the basal-lateral membrane resulting in the generation of complete cell surface polarity in both clones. Another basal-lateral membrane protein, E-cadherin, is sorted to the basal-lateral membrane in both MDCK clones, demonstrating that there is not a general sorting defect for basal-lateral membrane proteins in clone II/J cells. A glycosyl-phosphatidylinositol (GPI)-anchored protein (GP-2) and a glycosphingolipid (glucosylceramide, GlcCer) are preferentially transported to the apical membrane in clone II/G cells, but, in clone II/J cells, GP-2 and GlcCer are delivered equally to both apical and basal-lateral membranes, similar to Na/K-ATPase. To examine this apparent inter-relationship between sorting of GlcCer, GP-2 and Na/K-ATPase, sphingolipid synthesis was inhibited in clone II/G cells with the fungal metabolite, Fumonisin B1 (FB1). In the presence of FB1, GP-2 and Na/K-ATPase are delivered to both apical and basal-lateral membranes, similar to clone II/J cells; FB1 had no effect on sorting of E-cadherin to the basal-lateral membrane of II/G cells. Addition of exogenous ceramide, to circumvent the FB1 block, restored GP-2 and Na/K-ATPase sorting to the apical and basal-lateral membranes, respectively. These results show that the generation of complete cell surface polarity of Na/K-ATPase involves a hierarchy of sorting mechanisms in the Golgi complex and plasma membrane, and that Na/K-ATPase sorting in the Golgi complex of MDCK cells may be regulated by exclusion from an apical pathway(s). These results also provide new insights into sorting pathways for other apical and basal-lateral membrane proteins.

Animals↗
PubMed1994

GP-3, a newly characterized glycoprotein on the inner surface of the zymogen granule membrane, undergoes regulated secretion.

We have recently reported the cloning of the rat zymogen granule membrane glycoprotein GP-3 and the related pancreatic secretory lipase (Wishart, M. J., Andrews, P. C., Nichols, R., Blevins, G. T., Logsdon, C.D., and Williams, J. A. (1993) J. Biol. Chem. 268, 10303-10311). Specific antipeptide antibodies were generated against both GP-3 and secretory lipase and used for the biochemical and physiological characterization of GP-3. Western blotting confirmed that GP-3 was found exclusively in zymogen granule membranes and was absent from zymogen granule content which contains the majority of secretory lipase. Extraction of zymogen granule membranes with Triton X-114 showed GP-3 to be significantly more hydrophobic than lipase. The GP-3 amino acid sequence contains one potential N-linked glycosylation site at Asn-336. The loss of concanavalin A labeling after both chemical deglycosylation with trifluoromethanesulfonic acid and enzymatic deglycosylation with N-glycanase showed GP-3 to possess a small N-linked oligosaccharide side chain. Digestion of intact and permeabilized zymogen granules with the nonspecific protease Pronase localized GP-3 to the inner surface of zymogen granule membranes. Since GP-3 is resident on the inner surface of the zymogen granule membrane, it should appear on the outer cellular surface after exocytosis. Although membrane attachment of GP-3 was resistant to treatment with phosphatidylinositol-specific phospholipase C, we observed that GP-3 is released into the pancreatic juice and that secretion of GP-3 was greatly enhanced by cholecystokinin.

Animals↗
PubMed1994

Identification of a vesicle-associated membrane protein (VAMP)-like membrane protein in zymogen granules of the rat exocrine pancreas.

Zymogen granules of the exocrine pancreas are the secretory organelles responsible for the regulated secretion of digestive enzymes. Several proteins are associated with or are integral components of the lipid bilayer that forms the zymogen granule membrane. These proteins likely represent important components in the regulated secretion of digestive enzymes. VAMPs (vesicle-associated membrane proteins)/synaptobrevins are a family of 18-kDa integral membrane proteins originally characterized in synaptic vesicles. Polyclonal antisera raised against either a VAMP/glutathione S-transferase (GST) fusion protein or rat brain synaptic vesicles, detected an 18-kDa immunoreactive protein in zymogen granule membranes that co-migrates electrophorectically with rat brain synaptic vesicle VAMP. Rat brain synaptic vesicle VAMP was detected by both antisera. Botulinum-B toxin treatment of zymogen granule membranes did not result in cleavage of zymogen granule membrane VAMP, indicating that exocrine pancreatic VAMP is either VAMP1 or a novel VAMP-isoform. Immunofluorescent studies demonstrated that exocrine pancreatic VAMP localized with GP2, a zymogen granule membrane protein, to the apical region of pancreatic acinar cells. No significant labeling was observed in basolateral regions of pancreatic acinar cells. These results establish the presence of a VAMP protein in the zymogen granule of the rat pancreas and suggest that VAMPs have a role in exocrine secretion.

Animals↗
PubMed1994

The level of the zymogen granule protein GP2 is elevated in a rat model for acute pancreatitis.

BACKGROUND/AIMS: GP2 is the major membrane protein in pancreatic zymogen granules. It is linked to the membrane via a glycosyl-phosphatidylinositol linkage. After cleavage, a significant fraction of GP2 becomes soluble. The present study assessed whether GP2 is a useful serum marker for acute pancreatitis. METHODS: Using an anti-GP2 monoclonal antibody, an enzyme-linked immunosorbent assay was developed to measure the serum levels of GP2 in rats with cerulein-induced acute pancreatitis. RESULTS: The anti-GP2 antibody was specific because it did not cross-react with uromodulin, a structurally similar protein to GP2, or to protein extracts from nonpancreatic tissues. Eight hours after the induction of pancreatitis, the serum levels of amylase, lipase, and GP2 peaked. Peak GP2 levels were 4.2 times higher than those of controls. At 24 hours, GP2 was still 70% of the peak level, whereas amylase and lipase were 5.5% and 0.5%, respectively, of their peak levels. CONCLUSIONS: GP2 may serve as a potentially valuable marker for clinical acute pancreatitis.

Acute Disease↗
PubMed1994

Apical plasma membrane proteins are not obligatorily stored in secretory granules in exocrine cells.

Exocrine cells are epithelial cells in which secretory granules undergo fusion with the apical plasma membrane upon secretagogue stimulation. Several apical plasma membrane proteins have been found in secretory granules in cells from pancreas and salivary glands raising the possibility that incorporation into secretory granules followed by exocytosis of the granules accounts for their insertion into the apical plasma membrane. To test this hypothesis, we have expressed the influenza hemagglutinin (HA) in pancreatic AR42J cells, which make zymogen-like granules upon incubation with dexamethasone. The influenza virus HA is known to be specifically targeted to the apical plasma membrane of epithelial cells that lack a regulated pathway and is also known to be excluded from secretory granules in virally-infected pituitary AtT20 cells. Localization of the protein by immunofluorescence microscopy revealed that it accumulated at the plasma membrane of the transfected AR42J cells. HA was not observed in the amylase-rich secretory granules. By immunolabeling of ultrathin cryosections of the transfected cells, HA was also found exclusively on the cell surface, with label over secretory granules not exceeding that seen in control, untransfected cells. In addition, in cell fractionation experiments performed on radiolabeled AR42J cell transformants, HA was not detectable in the secretory granule fractions. These results indicate that HA is not efficiently stored in mature secretory granules and is likely to reach the cell surface via constitutive transport pathways.

Cell Fractionation↗
PubMed1992

Antisense oligodeoxynucleotides to the cystic fibrosis transmembrane conductance regulator inhibit cAMP-activated but not calcium-activated chloride currents.

Phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cAMP-dependent protein kinase leads to chloride flux in epithelial cells. Is CFTR also required for the calcium-dependent activation of chloride channels? We used antisense oligodeoxynucleotides to CFTR to reduce the expression of CFTR in colonic and tracheal epithelial cells. The antisense oligomers were a pair of adjacent 18-mers complementary to nucleotides 1-18 and 19-36 of CFTR mRNA. Sense and misantisense oligomers served as controls. A 48-h antisense treatment reduced the expression of CFTR protein as assayed by immunoprecipitation and autoradiography to 26% of the level in sense-treated T84 cells. Whole-cell patch clamp revealed that a 48-h antisense treatment of T84 and 56FHTE-8o- fetal tracheal epithelial cells reduced the cAMP-activated chloride current to approximately 10% of that in sense-treated cells. The half-life of functional CFTR is less than 24 h in these cells. In contrast, the calcium-activated chloride current was not affected by antisense treatment. Hence, the cAMP and calcium pathways are separate. CFTR is required for the cAMP pathway but not for the calcium pathway.

Base Sequence↗
PubMed1991

Laparoscopic cholecystectomy: initial experience.

Twenty-five consecutive patients underwent percutaneous laparoscopic cholecystectomy (PCC). The gallbladder was removed successfully in 18 patients. The mean postoperative hospital stay was 1.4 days and patients returned to normal activity at a mean 8 days after operation. Postoperative pain was minimal. Formal laparotomy was performed in 7 patients due to: bleeding (3 patients), stone spillage (3 patients) and exploration of the common bile duct (1 patient). Complications were reduced with experience and strict adherence to the described operative technique. With obvious advantages for the patient, hospitals and the community an increased demand for PCC is inevitable. However, its role in the management of cholelithiasis and overall safety have yet to be determined. There is a significant learning curve and proper training is necessary. The widespread introduction of PCC has immediate implications for surgical training.

Adolescent↗
PubMed1990

Biogenesis of synaptic vesicle-like structures in a pheochromocytoma cell line PC-12.

The presence of unique proteins in synaptic vesicles of neurons suggests selective targeting during vesicle formation. Endocrine, but not other cells, also express synaptic vesicle membrane proteins and target them selectively to small intracellular vesicles. We show that the rat pheochromocytoma cell line, PC12, has a population of small vesicles with sedimentation and density properties very similar to those of rat brain synaptic vesicles. When synaptophysin is expressed in nonneuronal cells, it is found in intracellular organelles that are not the size of synaptic vesicles. The major protein in the small vesicles isolated from PC12 cells is found to be synaptophysin, which is also the major protein in rat brain vesicles. At least two of the minor proteins in the small vesicles are also known synaptic vesicle membrane proteins. Synaptic vesicle-like structures in PC12 cells can be shown to take up an exogenous bulk phase marker, HRP. Their proteins, including synaptophysin, are labeled if the cells are surface labeled and subsequently warmed. Although the PC12 vesicles can arise by endocytosis, they seem to exclude the receptor-mediated endocytosis marker, transferrin. We conclude that PC12 cells contain synaptic vesicle-like structures that resemble authentic synaptic vesicles in physical properties, protein composition and endocytotic origin.

Animals↗
PubMed1988

Endocrine secretory granules and neuronal synaptic vesicles have three integral membrane proteins in common.

In response to an external stimulus, neuronal cells release neurotransmitters from small synaptic vesicles and endocrine cells release secretory proteins from large dense core granules. Despite these differences, endocrine cells express three proteins known to be components of synaptic vesicle membranes. To determine if all three proteins, p38, p65, and SV2, are present in endocrine dense core granule membranes, monoclonal antibodies bound to beads were used to immunoisolate organelles containing the synaptic vesicle antigens. [3H]norepinephrine was used to label both chromaffin granules purified from the bovine adrenal medulla and rat pheochromocytoma (PC12) cells. Up to 80% of the vesicular [3H]norepinephrine was immunoisolated from both labeled purified bovine chromaffin granules and PC12 postnuclear supernatants. In PC12 cells transfected with DNA encoding human growth hormone, the hormone was packaged and released with norepinephrine. 90% of the sedimentable hormone was also immunoisolated by antibodies to all three proteins. Stimulated secretion of PC12 cells via depolarization with 50 mM KCl decreased the amount of [3H]norepinephrine or human growth hormone immunoisolated. Electron microscopy of the immunoisolated fractions revealed large (greater than 100 nm diameter) dense core vesicles adherent to the beads. Thus, large dense core vesicles containing secretory proteins possess all three of the known synaptic vesicle membrane proteins.

Animals↗
PubMed1988

Targeting of secretory vesicles to cytoplasmic domains in AtT-20 and PC-12 cells.

Organelles are not uniformly distributed throughout the cytoplasm but have preferred locations that vary between tissues and during development. To investigate organelle targeting to cytoplasmic domains we have taken advantage of the mouse pituitary cell line, AtT-20, which, when induced to extend long processes, accumulates dense core secretory granules at the tips of the processes. During mitosis, these secretory granules accumulate along the plane of division. Protein synthesis is not mandatory for such redistribution of secretory granules. To explore the specificity of the redistribution we have used transfected AtT-20 cells that express the immunoglobulin kappa light chain. While the endogenous hormone ACTH is found in secretory granules, the kappa chain is a marker for organelles involved in constitutive secretion. By immunofluorescence, kappa also accumulates at the tips of growing processes, and along the midline of dividing cells, suggesting that the redistribution of vesicles is not specific for dense-core secretory granules. Since there is evidence for selective organelle transport along processes in neuronal cells, the rat pheochromocytoma cell PC-12 was transfected with DNA encoding markers for regulated and constitutive secretory vesicles. Again regulated and constitutive vesicles co-distribute, even in cells grown in the presence of nerve growth factor. We suggest that at least in the cells studied here, cytoskeletal elements normally carry exocytotic organelles to the surface; when the cytoskeletal elements coalesce in an extending process, exocytotic organelles of both the constitutive and regulated pathway are transported nonselectively to the tips of the cytoskeletal elements where they accumulate.

Adrenocorticotropic Hormone↗