Pancreatic function in CCK-deficient mice: adaptation to dietary protein does not require CCK.
(17/1858)
A CCK-deficient mouse mutant generated by gene targeting in embryonic stem cells was analyzed to determine the importance of CCK for growth and function of the exocrine pancreas and for pancreatic adaptation to dietary changes. RIAs confirmed the absence of CCK in mutant mice and demonstrated that tissue concentrations of the related peptide gastrin were normal. CCK-deficient mice are viable and fertile and exhibit normal body weight. Pancreas weight and cellular morphology appeared normal, although pancreatic amylase content was elevated in CCK-deficient mice. We found that a high-protein diet increased pancreatic weight, protein, DNA, and chymotrypsinogen content similarly in CCK-deficient and wild-type mice. This result demonstrates that CCK is not required for protein-induced pancreatic hypertrophy and increased proteolytic enzyme content. This is a novel finding, since CCK has been considered the primary mediator of dietary protein-induced changes in the pancreas. Altered somatostatin concentrations in brain and duodenum of CCK-deficient mice suggest that other regulatory pathways are modified to compensate for the CCK deficiency. (+info)
Characterization of MALS/Velis-1, -2, and -3: a family of mammalian LIN-7 homologs enriched at brain synapses in association with the postsynaptic density-95/NMDA receptor postsynaptic complex.
(18/1858)
Protein assembly at the postsynaptic density (PSD) of neuronal synapses is mediated in part by protein interactions with PSD-95/discs large/zona occludens-1 (PDZ) motifs. Here, we identify MALS-1, -2, -3, a family of small synaptic proteins containing little more than a single PDZ domain. MALS-1, -2, and -3 are mammalian homologs LIN-7, a Caenorhabditis elegans protein essential for vulval development. In contrast to functions for LIN-7 in epithelial cells, MALS-1 and -2 are selectively expressed in specific neuronal populations in brain and are enriched in PSD fractions. In cultured hippocampal neurons, MALS proteins are clustered together with PSD-95 and NMDA type glutamate receptors, consistent with a postsynaptic localization for MALS proteins. Immunoprecipitation and affinity chromatography studies readily identify association of MALS with PSD-95 and an NMDA receptor subunit. The PDZ domain of MALS selectively binds to peptides terminating in E-T/S-R/X-V/I/L, which corresponds to the C terminus of NMDA type 2 receptors and numerous other ion channels at the PSD. This work suggests a role for MALS proteins in regulating recruitment of neurotransmitter receptors to the PSD. (+info)
Acute oxidative stress modulates secretion and repetitive Ca2+ spiking in rat exocrine pancreas.
(19/1858)
The effects of the oxidant tert-butylhydroperoxide (t-buOOH) on carbachol-stimulated pancreatic secretion in the vascularly perfused rat pancreas have been studied in parallel with [Ca2+]i signalling and amylase output in perifused rat pancreatic acinar cells. Perfusion of the pancreas with t-buOOH (0.1-1 mM) caused a rapid and irreversible inhibition of carbachol-stimulated (3x10-7 M) amylase and fluid secretion. Pre-perfusion of the pancreas with vitamin C and dithiothreitol or a cocktail of GSH and GSH-precursor amino acids provided only marginal protection against the deleterious effects of t-buOOH, even though GSH levels were elevated significantly. In perifused pancreatic acini, repetitive [Ca2+]i spikes evoked by carbachol (3x10-7 M) were sustained for 40 min. t-buOOH (1 mM) acutely increased the amplitude and duration of Ca2+ spikes, then attenuated Ca2+ spiking and subsequently caused a marked and sustained rise in [Ca2+]i. t-buOOH-induced alterations in carbachol-stimulated [Ca2+]i signalling and amylase release in perifused pancreatic acini were prevented by vitamin C. Although vitamin C restored impaired Ca2+ signalling and maintained amylase output in pancreatic acini, it seems likely that oxidative stress inhibits fluid secretion irreversibly in the intact pancreas, resulting in a loss of amylase output. Thus, perturbations in [Ca2+]i signalling may not fully explain the secretory block caused by oxidative stress in acute pancreatitis. (+info)
High dietary lipid levels enhance digestive tract maturation and improve dicentrarchus labrax larval development.
(20/1858)
This study was designed to determine the nutritional lipid requirement of seabass larvae and to understand the effects of dietary fat concentration on their digestive tract maturation. Seabass (Dicentrarchus labrax) larvae were fed, from d 15 to 38 of life, one of five isonitrogenous compound diets with different lipid levels, ranging from 10 to 30 g/100 g. The higher the lipid level, the greater the growth and survival of the larvae (P < 0.05). The lipolytic enzymes assayed, lipase and phospholipase A2, were stimulated by the increase in their respective dietary substrates, triglycerides and phospholipids, in 38-d-old larvae (P < 0.05). Nevertheless, a plateau in the activity of these two lipolytic enzymes was observed from 20% dietary lipids onwards. The similar mRNA levels of phospholipase A2 in the three groups fed the highest lipid levels suggested that the maximal synthesis level of lipolytic enzyme was reached at 20% dietary fat. Pancreatic secretion of trypsin and amylase were positively affected by the dietary lipid level; a possible involvement of a cholecystokinin-releasing factor is discussed. Diets containing >20% lipids led to the increase in activities of brush border membrane enzymes to the detriment of a cytosolic enzyme in enterocytes, leucine-alanine (Leu-Ala) peptidase. This enzymatic change reveals the earlier maturation of enterocytes in larva groups fed high lipid levels. (+info)
Inhibition of amylase secretion from differentiated AR4-2J pancreatic acinar cells by an actin cytoskeleton controlled protein tyrosine phosphatase activity.
(21/1858)
Disruption of the actin cytoskeleton in AR4-2J pancreatic acinar cells led to an increase in cytosolic protein tyrosine phosphatase activity, abolished bombesin-induced tyrosine phosphorylation and reduced bombesin-induced amylase secretion by about 45%. Furthermore, both tyrosine phosphorylation and amylase secretion induced by phorbol ester-induced activation of protein kinase C were abolished. An increase in the cytosolic free Ca2+ concentration by the Ca2+ ionophore A23187 had no effect on tyrosine phosphorylation but induced amylase release. Only when added together with phorbol ester, the same level of amylase secretion as with bombesin was reached. This amylase secretion was inhibited by about 40%, by actin cytoskeleton disruption similar to that induced by bombesin. We conclude that actin cytoskeleton-controlled protein tyrosine phosphatase activity downstream of protein kinase C activity regulates tyrosine phosphorylation which in part is involved in bombesin-stimulated amylase secretion. (+info)
A novel zymogen granule protein (ZG29p) and the nuclear protein MTA1p are differentially expressed by alternative transcription initiation in pancreatic acinar cells of the rat.
(22/1858)
Using a polyclonal antibody against purified zymogen granule membrane components from rat pancreas a cDNA coding for the 29 kDa protein (ZG29p) was identified by immunoscreening of a hormonally stimulated pancreas cDNA library. Western blot analysis suggests that ZG29p is a pancreas-specific protein and immunofluorescence shows that ZG29p is mainly associated with zymogen granules. Analysis of subcellular fraction applying immunoblotting revealed that ZG29p was localized mainly in the soluble fraction of zymogen granules and in a Golgi- and RER-enriched fraction, but was absent from the cytosol. In isolated zymogen granule content ZG29p was associated with protein complexes containing amylase as main constituent. The cDNA coding for ZG29p is homologous to the C-terminal region of the candidate metastasis-associated gene mta1. Northern blot analysis and RT-PCR showed that no MTA1 mRNA is present in pancreas from fasted rats and in the rat pancreas carcinoma cell line AR4-2J in its protodifferentiated state. Although no ZG29p specific mRNA was seen in the northern blot analysis, RT-PCR showed that ZG29p was expressed under both non-stimulated and stimulated conditions. The expression of MTA1 was up-regulated in the pancreas by endogenous cholecystokinin release and in AR4-2J after induction of cellular differentiation by dexamethasone. Western blotting and immunofluorescense studies indicated that MTA1p is localized in the nucleus in all tissues studied. Using genomic DNA in PCR analysis it was shown that two short introns are present flanking the sequences of the 5'end of ZG29p cDNA. One intron contains consensus elements required for pancreas specific transcription initiation, suggesting that MTA1 and ZG29 are differentially expressed by alternative transcription initiation in the pancreas. The localisation of MTA1p in the nucleus of most cell types could signify a general role in gene regulation, while the cell type specific and exclusive expression of ZG29p in pancreatic acinar cells could indicate a role in granule formation. (+info)
Expression and localization of Rab3D in rat parotid gland.
(23/1858)
Rab3 proteins (isoforms A, B, C and D) are low molecular weight GTP-binding proteins proposed to be involved in regulated exocytosis. In the present study, Rab3 protein expression and localization was examined in rat parotid gland by reverse transcription (rt) PCR, Western blotting and immunocytochemistry. An approximately 200 bp PCR product was obtained from parotid RNA by rtPCR and this fragment was cloned and sequenced. Nucleotide and deduced amino acid sequences obtained from five clones were identical to rab3D. Membrane and cytosolic fractions prepared from parotid acini were immunoblotted with antisera specific for each of the four Rab3 isoforms. A 28 kDa protein was detected with Rab3D-specific antisera in both fractions with staining being more intense in the membrane fraction. No other Rab3 isoforms were detected by immunoblotting, a result consistent with those obtained by rtPCR. Rab3D was enriched in zymogen granule membranes and Triton X-114 extraction revealed that this isoform is predominantly lipid-modified in parotid. Localization of Rab3D was done on frozen sections of parotid gland by immunofluorescence microscopy. Staining was observed primarily in the acinar cells and was adjacent to the acinar lumen. Incubation of dispersed acini with isoproterenol and substance P stimulated amylase secretion 4- and 2-fold above basal, respectively. Isoproterenol, but not substance P, induced redistribution of Rab3D from the cytosol to the membrane fraction in dispersed parotid acini. Consistent with these findings, isoproterenol injections into fasted rats also resulted in increased membrane-associated Rab3D in the parotid acini. These results indicate that Rab3D is: (1) the major Rab3 isoform expressed in rat parotid gland; (2) localized to zymogen granule membranes; and (3) involved with regulated enzyme secretion in acinar cells. (+info)
Vesicular tubular clusters between the ER and Golgi mediate concentration of soluble secretory proteins by exclusion from COPI-coated vesicles.
(24/1858)
We have determined the concentrations of the secretory proteins amylase and chymotrypsinogen and the membrane proteins KDELr and rBet1 in COPII- and COPI-coated pre-Golgi compartments of pancreatic cells by quantitative immunoelectron microscopy. COPII was confined to ER membrane buds and adjacent vesicles. COPI occurred on vesicular tubular clusters (VTCs), Golgi cisternae, the trans-Golgi network, and immature secretory granules. Both secretory proteins exhibited a first, significant concentration step in noncoated segments of VTC tubules and were excluded from COPI-coated tips. By contrast, KDELr and rBet1 showed a first, significant concentration in COPII-coated ER buds and vesicles and were prominently present in COPI-coated tips of VTC tubules. These data suggest an important role of VTCs in soluble cargo concentration by exclusion from COPI-coated domains. (+info)