Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The release of secretin was studied in secretin cell-enriched preparations isolated from canine duodenal mucosa. The crude enterocytes were isolated by treating the duodenal mucosa sequentially with collagenase and ethylenediaminetetraacetic acid. Secretin cell-enriched fraction was prepared by centrifugation of the crude enterocytes in a counterflow elutriation rotor to obtain a final preparation containing 3.2 +/- 0.3 pmol/10(6) cell of immunoreactive secretin, which was 13-fold greater than the crude cell preparation (N = 5). The cells were incubated in Hanks' balanced salt solution for 20 min at 37 degrees C under 95% O2/5% CO2 before adding various agents and further incubated for various periods of time. The amounts of secretin released into the medium and retained by the cells were then determined by a specific radioimmunoassay. The release of immunoreactive secretin was increased dose-dependently over the control by dibutyryl cyclic-3',5'-adenosine monophosphate, forskolin, 4 beta-12-O-tetradecanoylphorbol-13-acetate, the synthetic serine protease inhibitor, camostat, and the calcium ionophore, A23187. The effects of forskolin, the phorbol ester, and A23187 were time-dependent and not observed at 4 degrees C. The release of immunoreactive secretin was also stimulated by KCl in high concentration and by sodium oleate. The effect of A23187 was abolished in a Ca(2+)-free medium, while those of dibutyryl cyclic-3',5'-adenosine monophosphate and forskolin were potentiated by 3-isobutyl-1-methylxanthine, which did not have a significant effect when added alone. These results indicate that the release of secretin is regulated by both Ca(2+)- and cyclic-3',5'-adenosine monophosphate-dependent mechanisms.2+ release.
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PMID:Characterization of secretin release in secretin cell-enriched preparation isolated from canine duodenal mucosa. 842 47

Although serotonin (5-HT) release from enterochromaffin (EC) cells is considered to be regulated by multiple receptor-mediated mechanisms, little is known about the signal transduction. Here, we introduce the methods to isolate the mouse ileal crypts, which consist of various types of cells including EC cells, and to analyze the intracellular calcium dynamics. Ileal tissue was inserted with a plastic straw and the smooth muscle layers were peeled off. The mucosa were digested with collagenase and then suspended by moderate pipetting. Ileal crypts were separated by stepwise filtrations through 2 different nylon-meshes. The isolated crypt contained 0-3 EC cells as identified by immunostaining using anti-5-HT antibody followed by confocal microscopy. Isolated crypts were attached to a coverglass by an adhesive material (Cell-Tak) and loaded with fura-2/AM. Intracellular calcium dynamics in EC cells were obtained by digital video-imaging analysis of fura-2 fluorescence followed by the identification of EC cells with immunostaining of 5-HT granules. By these methods, it was suggested that norepinephrine elicited a transient elevation of intracellular calcium concentration in EC cells via alpha 2-adrenoceptors. These methods could be also useful to analyze the signal transduction system in intestinal endocrine cells that contain various intestinal hormones such as gastrin, cholecystokinin or secretin.
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PMID:[Analysis of intracellular calcium dynamics in enterochromaffin cells of small intestine]. 1067 96

The development of genetically altered murine animals has generated a need for in vitro systems in the mouse. We have now characterized a novel isolated bile duct unit (IBDU) preparation from the mouse to facilitate such studies. The mouse IBDU is isolated by portal perfusion of collagenase, blunt dissection, further enzymatic digestions, filtering through sized mesh, and culturing on Matrigel for 16-72 h. This mouse IBDU forms a central, enclosed lumen lined by polarized cytokeratin-19-positive cholangiocytes with numerous microvilli on the apical membrane. The IBDU responds to secretory stimuli, including secretin, vasoactive intestinal peptide, IBMX, and forskolin, resulting in expansion of the central lumen from secretion as quantified by videomicroscopy. The secretory response to secretin is dependent on Cl- and HCO3-in the perfusate. These findings indicate that mouse IBDUs are intact, polarized, functional bile duct secretory units that permit quantitative measurements of fluid secretion from mouse bile duct epithelium for the first time. This method should facilitate studies of cholangiocyte secretion in genetically altered murine animal models.
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PMID:Isolation of functional polarized bile duct units from mouse liver. 1120 46

The ductal system of the exocrine pancreas produces HCO(3)(-)-rich fluid in response to secretin and other stimuli. HCO(3)(-) efflux across the luminal membrane is mediated by a Cl(-)-HCO(3)(-) exchanger operating in parallel with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. Basolateral K(+) channels provide an exit pathway for K(+) and play a vital role in maintaining the membrane potential, which is a crucial component of the driving force for anion secretion. Measurements of membrane potential with intracellular microelectrodes suggested that Ba(2+)-sensitive K(+) conductance accounts for more than 60% of the total basolateral ionic conductance in resting ducts (1). To identify the Ba(2+)-sensitive K(+) channels, we isolated ducts from normal rat pancreas by collagenase digestion. We first demonstrated that the ducts did not express a vascular endothelial marker PECAM-1 (platelet/endothelial cell adhesion molecule-1), but expressed cytokeratin 20, a marker of duct cells (2), using immunofluorescent staining. In addition, monoclonal anti-CFTR antibody was detected near the luminal membrane of these cells. In cell-attached single-channel recordings, we observed three types of K(+) channels on basolateral membrane in unstimulated duct cells. The 40 pS K(+) channels are likely to mediate whole-cell inwardly rectifying K(+) (Kir) currents, which were blocked by extracellular Ba(2+) in a voltage-dependent manner. The properties of 90 pS and 170 pS K(+) channels are similar to those of Ca(2+)-activated K(+) channels. We then identified Kir2.0 and SK4/IK1 (intermediate conductance Ca(2+)-activated K(+) channel) subunits as molecular candidates of the K(+) channels using RT-PCR analysis. The present results suggest that these subunits may mediate native K(+) currents in resting duct cells. Further functional studies with specific blockers are required to evaluate which of these K(+) channels contribute to the resting membrane potential and might be involved in HCO(3)(-) secretion.
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PMID:K+ channels on resting duct cells from rat pancreas. 2022 23


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