Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.11.1.7 (peroxidase)
65,474 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The colonic epithelial cell line T84 has been shown to be a good model to investigate the regulation of Cl- secretion by the adenosine 3',5'-cyclic monophosphate (cAMP)-mediated second messenger cascade. Regulated exocytic insertion and endocytic retrieval of transport proteins, or proteins that regulate transport proteins, is one mechanism proposed to regulate plasma membrane solute permeabilities. The aims of our studies were to characterize endocytic processes in T84 cells and to investigate their regulation by known activators of Cl- secretion that are mediated by the cAMP second messenger cascade. Forskolin, an activator of adenylate cyclase, caused a marked inhibition of endocytic uptake of the fluid-phase marker horseradish peroxidase (HRP) and the adsorptive marker wheat germ agglutinin conjugated to HRP. Similar inhibition was obtained with vasoactive intestinal peptide, a secretagogue whose receptor is coupled to adenylate cyclase, and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, a membrane-permeable cAMP analogue. 1,9-Dideoxy-forskolin, a forskolin analogue that fails to activate adenylate cyclase, was without effect on endocytosis. Our data show that the net rate of endocytosis, as measured by fluid-phase uptake, is decreased by a cAMP-mediated mechanism. Because the number of Cl- channels or associated regulatory proteins in the plasma membrane reflects a balance between their exocytic insertion and endocytic retrieval, we propose that the cAMP-mediated decrease in endocytosis could contribute to the concomitant increase in plasma membrane Cl- permeability.
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PMID:Regulated endocytosis in a chloride secretory epithelial cell line. 131 84

Vasoactive intestinal peptide (VIP) and d-ala2-methionine enkephalinamide (DALA, a long-lasting enkephalin analogue) were used to investigate the peptidergic control of lacrimal gland function. To characterize the mechanism by which VIP stimulates and DALA inhibits lacrimal peroxidase secretion, the effect of these peptides on adenylate cyclase was measured. In addition, enzyme activity was measured in the presence of forskolin alone or in combination with DALA. VIP stimulated adenylate cyclase in a time- and dose-dependent manner. Negative control of adenylate cyclase was shown with the addition of DALA to membrane preparations. The enkephalin analogue inhibited basal activity approximately 65% at the maximum dose tested. The percent inhibition of VIP-stimulated activity by DALA was similar to the inhibition of basal activity. To determine if the inhibition of stimulated activity occurred at level of the VIP receptor, the effect of DALA on the response to forskolin was measured. Forskolin-stimulated adenylate cyclase activity was significantly reduced to approximately 50% in the presence of DALA. We conclude that lacrimal gland adenylate cyclase is subject to peptidergic regulation involving both stimulatory and inhibitory receptor-mediated controls.
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PMID:Peptidergic stimulation and inhibition of lacrimal gland adenylate cyclase. 217 Feb 90

Forskolin is a unique diterpene activator of adenylate cyclase which has been extensively used in the study of cAMP generating systems. This report describes the production of antibodies to forskolin and the optimization of two sensitive assay methods for such antibodies. 7-0-Hemisuccinyl 7-deacetyl forskolin, coupled to either human serum albumin or goat IgG, was injected into goats to elicit antibodies to the forskolin hapten. Two assay methods, a radioimmunoassay with [12-3H]forskolin as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) with horse radish peroxidase-labelled rabbit anti-goat IgG as an indicator, were optimized to test for the presence of forskolin antibodies in antisera and isolated IgG fractions. The titers for forskolin antisera were 4000-10000. Both assay methods can be adapted to quantify forskolin and its protein conjugates. The availability of antibodies to this diterpene will be useful in accelerating the understanding of the mechanism of adenylate cyclase activation by forskolin.
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PMID:Production and assay of antibodies to an activator of adenylate cyclase, forskolin. 369 25

Intrinsic factor (IF) is a vitamin B12 binding protein that is secreted from the gastric mucosa. We tested secretagogues which stimulate IF secretion in rat gastric perfusion and found that carbachol and cholecystokinin octapeptide (CCK-8) stimulated secretion, but histamine and tetragastrin did not. To confirm these results, we examined IF secretion from isolated rat chief cells. For this purpose, we established an enzyme immunoassay (EIA) using an avidin-biotin peroxidase complex to measure small amounts of IF. To prepare an anti-rat IF, IF was isolated from the stomach, and was injected into a rabbit for immunization. Rat gastric chief cells were isolated from the gastric mucosa with Dispase and a Percoll gradient centrifugation, and were cultured. We examined the effects of chemicals by adding them to culture dishes of chief cells in a CO2 incubator. Released IF in culture medium was determined by EIA. Carbachol, CCK-8 and secretin stimulated IF secretion from cultured chief cells, while histamine and tetragastrin did not; Forskolin and A23187 also stimulated the secretion. We concluded that carbachol and CCK-8 stimulated IF secretion via an increase of intracellular Ca2+ concentration and that secretin did so via a cAMP accumulation.
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PMID:Secretion of intrinsic factor from cultured rat gastric chief cells. 787 53

The effects of the secretagogues forskolin and carbachol on protein uptake in isolated ileum of rats were studied. The mucosal-to-serosal transport of horseradish peroxidase (HRP, mol mass 40 kDa) was measured in Ussing chambers, and afterwards tissues were processed for electron microscopy. In the absence of secretagogues, the flux of enzymatically active HRP was 5 pmol.cm-2.h-1 at a mucosal concentration of 10 microM. Electron micrographs showed vesicles filled with active HRP in enterocytes but no HRP activity in intercellular spaces. Forskolin decreased HRP activity in the cells. Carbachol increased the amount of HRP-filled vesicles in enterocytes and induced HRP filling in some intercellular spaces and tight junctions in the upper parts of the villi. The transepithelial flux of intact HRP increased more than 2.5-fold. This effect was suppressed by atropine. We conclude that cholinergic activation can increase the uptake of intact protein via endocytosis and the transepithelial passage by the induction of a diffusional paracellular pathway. We speculate that the increased transport of intact protein through the intestinal barrier may influence immunologic sensitization to food allergens.
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PMID:Carbachol, but not forskolin, increases mucosal-to-serosal transport of intact protein in rat ileum in vitro. 876 Jan 18

The transepithelial route for mucosa-to-serosa transport of the tracer macromolecule horseradish peroxidase (HRP; MW 40 kDa) and modulation of this transport by forskolin and carbachol have been studied in vi-tro in stripped goldfish intestinal epithelium mounted in Ussing-type chambers. Uptake and transport have been investigated by measuring the HRP flux from the muco-sal to serosal sides by an enzymatic method and by visualising HRP reaction products in the mucosa with electron-microscopical techniques. Both the cholinergic agonist carbachol (which is thought to increase intracellular Ca2+ and activate protein kinase C activity) and forskolin (a direct activator of adenylylcyclase) affect the amount of enzymatically active HRP in the tissue. In control tissue, HRP product is found only within the epithelial cells, the transepithelial flux reaching a constant value of about 1.5 pmoles/cm2 per h. Carbachol increases the amount of HRP product in the cells, but has no significant effect on the HRP flux compared with control values. Forskolin decreases the amount of HRP product in the cells; however, in the presence of forskolin, the lateral intercellular spaces become filled with HRP product. HRP is found in the lamina propria and the transepithelial protein flux increases more than 2.5-fold. In the presence of forskolin plus carbachol, the results are no different from the control. It is concluded that carbachol increases the endocytotic uptake of HRP, whereas forskolin inhibits the uptake but increases the paracellular permeability for HRP in goldfish intestine.
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PMID:Influence of forskolin and carbachol on intestinal absorption of horseradish peroxidase in the goldfish (Carassius auratus). 876 57

To determine whether in endothelial cells (EC) the pathways of endocytosis and transcytosis of macromolecules interconnect, the effect of Brefeldin A (BFA) on these processes was tested. To this purpose EC were grown to confluence on plastic culture dishes or on cell culture chamber inserts placed into corresponding wells, so as to obtain a dual chamber system. The cells maintained the typical characteristics of EC and had an electrical resistance in the range of 30-60 Ohm.cm2. Transendothelial transport of albumin conjugated to the fluorochrom Texas Red (Alb-TR) and of horseradish peroxidase (HRP) added to the upper compartment, in the absence or presence of BFA (0-25 micrograms/ml), was evaluated in aliquots collected from the lower compartment. At different time intervals, quantitative data were obtained by fluorimetry and spectrophotometry. In other experiments transcytosis of Alb-TR was examined in the presence of 100 microM forskolin (an inhibitor of BFA effect). The endocytosis of Alb-TR and HRP was evaluated by incubating EC with the probes, and the internalized tracers determined in the cell lysate using the methods described above. The results showed that BFA has no significant effect on transcytosis of albumin and HRP. In contradistinction, BFA (5 micrograms/ml) reduced markedly endocytosis of HRP (by 47%). Forskolin has no effect on transcytosis. The data indicate that the BFA-induced perturbance in the endocytic route does not affect the transcytotic pathway of albumin, and suggest that in EC, transcytosis of macromolecules may represent a shortcut for rapid and direct transport of some plasma molecules across the cell.
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PMID:Transcytosis of albumin in endothelial cells is brefeldin A--independent. 923 47

The effects of cocaine on endothelial cell macromolecular transport, electrical resistance, and morphology were assessed. In confluent endothelial monolayers grown on microporus filters, cocaine (0.01 to 1 mmol/L) induced a rapid concentration-dependent increase in permeability to peroxidase and low density lipoprotein. Along with increased transport, the cocaine effect was paralleled by a decrease in transendothelial electrical resistance. Alterations in membrane resistance were fully reversible following washout of the drug, providing evidence that cocaine does not cause permanent injury to the integrity of the monolayer. Cocaines major metabolites, benzoylecgonine and ecgonine methyl ester, had minimal effect on electrical resistance properties, whereas monolayer impedance was markedly depressed by the novel cocaine/alcohol metabolite, cocaine ethyl ester (cocaethylene). Morphologic studies of cocaine-treated endothelial cells revealed a marked disruption of F-actin and the formation of intercellular gaps; no evidence of cell lysis and/or detachment was noted. Forskolin, a potent activator of adenylate cyclase known to promote the endothelial cell barrier function, impaired cocaine-induced changes in electrical resistance and morphology. Cocaine, however, had no effect on resting levels of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) in confluent endothelial monolayers. In summary, the results indicate that cocaine directly induces structural defects in the endothelial cell barrier which enhance the transport of macromolecular tracers, the mechanism does not appear to involve intracellular cAMP.
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PMID:Cocaine-induced increase in the permeability function of human vascular endothelial cell monolayers. 1040 39

These studies investigated the growth characteristics and functional properties of isolated canine pancreatic ductal epithelial cells. Cells were isolated from the accessory pancreatic duct and cultured by using three conditions: on vitrogen-coated petri dishes with fibroblast conditioned medium (nonpolarized); in vitrogen-coated Transwells above a fibroblast feeder layer (polarized); or as organotypic rafts above a fibroblast-embedded collagen layer (polarized). Growth characteristics, transepithelial resistances, and carbonic anhydrase and cyclic adenosine monophosphate (AMP) responses were evaluated. Under polarized conditions, the cells grew as monolayers with columnar epithelial characteristics. The monolayers developed high transepithelial resistance and became impervious to the passage of horseradish peroxidase. Epithelial growth factor (EGF) (2 ng/ml) stimulated ductal cell growth and accelerated the formation of a high-resistance monolayer. Forskolin (10 microM) rapidly decreased transepithelial resistance. Carbonic anhydrase activity, which was lower in nonpolarized compared with polarized conditions, was stimulated by carbachol (175 microM). Secretin, however, did not stimulate carbonic anhydrase activity in these cells. Although secretin stimulated adenylyl cyclase activity in early-passage cells, this response was lost in later-passage cells. Both vasoactive intestinal polypeptide (VIP; 1 microM) and forskolin (10 microM) consistently increased adenylyl cyclase activity. Isolated canine pancreatic ductal epithelial cells proliferate in vitro, develop high-resistance epithelial monolayers, and respond to stimuli that activate adenylyl cyclase. These cells should provide a useful model for regulatory studies of ductal cell functions.
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PMID:Growth and function of isolated canine pancreatic ductal cells. 1063 Mar 86

Wortmannin is a potent inhibitor of phosphatidylinositol 3-kinase (PI3K) and membrane trafficking in many cells. To test the hypothesis that cystic fibrosis transmembrane conductance regulator (CFTR) traffics into and out of the plasma membrane during cAMP-stimulated epithelial Cl(-) secretion, we have studied the effects of wortmannin on forskolin-stimulated Cl(-) secretion by the human colonic cell line T84. At the PI3K inhibitory concentration of 100 nM, wortmannin did not affect significantly forskolin-stimulated Cl(-) secretion measured as short-circuit current (I(SC)). However, 500 nM wortmannin significantly inhibited forskolin-stimulated I(SC). cAMP activation of apical membrane CFTR Cl(-) channels in alpha-toxin-permeabilized monolayers was not reduced by 500 nM wortmannin, suggesting that inhibition of other transporters accounts for the observed reduction in T84 Cl(-) secretion. Forskolin inhibits apical endocytosis of horseradish peroxidase (HRP), but wortmannin did not alter forskolin inhibition of apical HRP endocytosis. In the absence of forskolin, wortmannin stimulated HRP endocytosis significantly. We conclude that, in T84 cells, apical fluid phase endocytosis is not dependent on PI3K activity and that CFTR does not recycle through a PI3K-dependent and wortmannin-sensitive membrane compartment.
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PMID:Inhibition of phosphatidylinositol 3-kinase does not alter forskolin-stimulated Cl(-) secretion by T84 cells. 1079 59


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