Cyclic AMP production in isolated colonic epithelial crypts: a highly sensitive model for the evaluation of vasoactive intestinal peptide action in human intestine.
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Biomedical subjects
Publications and source records attributed to C Dupont.
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This study was undertaken to assess the role of vasoactive intestinal peptide (VIP) in the control of cyclic adenosine 3':5'-monophosphate production in colonic tumor cells. Seven human colorectal adenocarcinoma cell lines in culture were investigated (HT-29, HRT-18, SW-480, Caco-2, CO-115, CO-125, and HCT-8R). These cell-lines had a cyclic adenosine 3':5'-monophosphate production system which was very sensitive to VIP but less so to prostaglandin E1 and/or isoproterenol. Nonintestinal human malignant epithelial cells, such as HeLa (cervix) and Caki-1 and Caki-2 (kidney), by contrast, did not respond to VIP. The dose-response relationships of malignant colorectal cells were compared to those obtained with epithelial cells of normal human colon and showed that: (a) maximal responses were observed with 0.1 micro M VIP in both malignant and normal cells; (b) half-maximal responses were elicited by VIP concentrations in the 0.3 to 2 nM range in malignant cells (1.2 nM in normal cells), thus indicating the high apparent affinity of the cells to VIP; and (c) the magnitudes of the responses (stimulated:basal ratios) were highly variable in malignant cells, ranging from 225 in HT-29 cells to 3.5 in Caco-2 cells, but were more constant, in the order of 25, in normal cells. Secretin, a VIP agonist in intestinal tissue, stimulated cyclic adenosine 3':5'-monophosphate accumulation in all colorectal cells, but with a 1000- to 5000-fold lower potency than did VIP. These results show that the VIP-sensitive adenylate cyclase system operates in malignant as well as in normal colon epithelial cells.
Three cases of a primary disorder of intestinal motility which was responsible for neonatal functional intestinal obstruction are reported. This serious condition affected the small intestine and the colon and was difficult to treat. Early operation and prolonged intravenous alimentation was necessary. It is distinct from Hirschsprung's disease because of the presence of ganglion cells and because of the abnormalities of the myenteric plexus on silver staining. However the clinical symptoms and the decreased intraparietal cell V.I.P. concentration in pathological areas are similar in both conditions.
Porcine vasoactive intestinal peptide stimulated adenosine 3':5'-monophosphate (cyclic AMP) production in rat intestinal epithelial cells. The stimulation was dependent on time and temperature and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Under optimal conditions (at 15 degrees C, with 0.2 mM 3-isobutyl-1-methylaxanthine, at a cell concentration up to 18 microgram DNA/ml), the cyclic AMP production produced by vasoactive intestinal peptide was constant for 10 min and stopped after 15 min incubation, at either low (1 nM) or high (30 nM) concentration of the peptide. This plateau effect was demonstrated not to be due to an inactivation of vasoactive intestinal peptide in the medium nor to an alteration of receptors for the peptide. Cyclic AMP production was sensitive to a concentration as low as 0.1 nM vasoactive intestinal peptide. Maximal stimulation of cyclic AMP levels by vasoactive intestinal peptide was observed with 30 nM vasoactive intestinal peptide and represented an 11-fold increased above basal. The dorse-response curve was monophasic with a Km of 2.3 x 10(-9) M. No cooperative effects were detected by Hill analysis. The positive non-linear relationship observed between stimulation of cyclic AMP production and occupancy of binding site was not time-dependent as indicated by experiments performed after 15, 45 and 120 min incubation. Maximal and half-maximal responses were obtained at about 70% and 7% occupation of binding sites, respectively. Chicken vasoactive intestinal peptide and porcine secretin were agonists of porcine vasoactive intestinal peptide with a 6-times and a 120-times lower potency, respectively. Among secretin analogs that were found to have low affinity for vasoactive intestinal peptide binding sites, [4-alanine, 5-valine]secretin, that resembles vasoactive intestinal peptide at the first seven amino acids at the N-terminal end, was a partial agonist of vasoactive peptide at the first seven amino acids at the N-terminal end, was a partial agonist of vasoactive intestinal peptide and others failed to stimulate cyclic AMP production. Glucagon (10microM), gastric inhibitory peptide (0.1 microM), substance, P, neurotensin, octapeptide of cholecystokinin, bovine pancreatic polypeptide, human gastrin I with leucine at residue 15, Leu-enkephalinand somatostatin (1 microM) did not alter cyclicAMP levels. Non-peptide mediators such as dopamine, serotonin, acetylcholine and histamine, tested at 10 microM, were also ineffective. Prostaglandins E2, E1 and isoproterenol, tested at 10 microM, induced an increase of cyclic AMP levels above basal but were 9.5, 13.7 and 17.5 times less efficient than vasoactive intestinal peptide, respectively. Thus vasoactive intestinal peptide is a unique stimulus of cyclic AMP production in rat intestinal epithelial cells.
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A vasoactive intestinal peptide-sensitive adenylate cyclase in intestinal epithelial cell membranes was characterized. Stimulation of adenylate cyclase activity was a function of vasoactive intestinal peptide concentration over a range of 1 . 10(-10)-1 . 10(-7) M and was increased six-times by a maximally stimulating concentration of vasoactive intestinal peptide. Half-maximal stimulation was observed with 4.1 +/- 0.7 nM vasoactive intestinal peptide. Fluoride ion stimulated adenylate cyclase activity to a higher extent than did vasoactive intestinal peptide. Under standard assay conditions, basal, vasoactive intestinal peptide- and fluoride-stimulated adenylate cyclase activities were proportional to time of incubation up to 15 min and to membrane concentration up to 60 microgram protein per assay. The vasoactive intestinal peptide-sensitive enzyme required 5-10 mM Mg2+ and was inhibited by 1 . 10(-5) M Ca2+. At sufficiently high concentrations, both ATP (3 mM) and Mg2+ (40 mM) inhibited the enzyme. Secretin also stimulated the adenylate cyclase activity from intestinal epithelial cell membranes but its effectiveness was 1/1000 that of vasoactive intestinal peptide. Prostaglandins E1 and E2 at 1 . 10(-5) M induced a two-fold increase of cyclic AMP production. Vasoactive intestinal peptide was the most potent stimulator of adenylate cyclase activity, suggesting an important physiological role of this peptide in the cyclic AMP-dependent regulation of the intestinal epithelial cell function.
Emergence of gram-negative bacteria resistnant to a number of antibiotics in intensive care nurseries for neonates emphasizes the need for alternatives in antibiotic combinations. One commonly used combination, gentamicin-ampicillin, and two newer combinations, tobramycin-cephalothin and amikacinampicillin, were evaluated prospectively in 60 newborns in such a nursery. Subjects were randomly assigned to one of the above therapy groups. Dosages in mg/kg.d were 100 for ampicillin and cephalothin, 6 for gentamicin and tobramycin and 15 for amikacin. Aminoglycoside serum concentrations, clinical tolerance and toxicity were monitored. Aminoglycoside concentrations after intravenous administration of the drugs were within the expected range (gentamicin and tobramycin 4 to 6 microgram/mL and amikacin 15 to 20 microgram/mL). There was no hematologic, renal or hepatic toxicity attributable to antibiotic therapy and the combinations were tolerated equally; no bilirubin displacement was detected in vitro or in vivo.
Human colonic adenocarcinoma plasma membranes exhibit specific receptors for VIP. Adenylate cyclase activity is stimulated by a VIP concentration as low as 10(-10) mol/1.
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Using a one-compartment model, the pharmacokinetic disposition of furosemide was studied in eight premature and term neonates with fluid overload. Following a single intravenous injection of furosemide (1 to 1.5 mg/kg), multiple blood samples were obtained from a heelstick or an arterial catheter and analyzed for furosemide by gas liquid chromatography. The mean (+/- SE) apparent volume of distribution was 829.2 +/- 118.9 ml/kg; t1/2 was 7.7 +/- 1.0 hour; elimination rate constant was 0.102 +/- 0.013/hour and plasma clearance was 81.61 +/- 14.98 ml/kg/hour. Compared to the disposition of furosemide in normal adults. AVd is almost fourfold greater in the neonate with an eightfold prolongation in plasma t1/2, an eightfold decrease in Ke1, and a twofold decrease in plasma clearance. Neither gestational and postnatal age nor birth weight correlated with the pharmacokinetic variables. No significant change in reserve bilirubin-binding capacity after an intravenous dose of furosemide was noted. Slow elimination of furosemide may partly explain the prolonged diuretic and saluretic effect of furosemide in the neonate.
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Compared study was performed in rats under chloral or urethanne anaesthesia after venous infusion of isoproterenol. Cardiovascular haemodynamics and capillary regional blood flows of digestive tract showed changes; cardiac output was increased only under chloral anaesthesia; nutritional flows were different according to anaesthesia.
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The Sephadex gel elution method for assay of the reserve bilirubin binding capacity (RBBC) of the serum of jaundiced newborns has been modified to permit its application on a routine basis in the clinical chemistry laboratory. The volume of sample required has been reduced to 0.2 ml. of serum or less, the time required to perform the assay decreased to 30 to 45 minutes, and the reliability of the results enhanced by unidormly higher absorbance readings. The RBBC as determined by this modified micromethod agrees within 1 mg./dl. with that obtained from the original method. The results are not affected by flow rates through the columns, which vary by as much as a factor of 5, but are interfered with by sample hemolysis, lipemia, and a high proportion of direct-reacting bilirubin. The RBBC assay is a useful tool in the management of neonatal hyperbilirubinemia, and the simplification of the method should permit its greater use on a routine basis.
The stimulatory effects of isoproterenol and secretin on external pancreatic secretion were compared in the rat. 1. In acute fistula, pylorus ligation, atropine, glucagon did not change either of the stimulated secretions. Propranolol inhibited isoproterenol-induced secretion and did not change secretin induced stimulation. Theophylline alone displayed a large hydrelatic stimulatory effect, without increasing protein excretion; the effect of theophylline was additive with the effects of isoproterenol or secretin but no evidence was found of potentiation or prolongation of action. Isoproterenol did not increase pancreatic blood flow and induced no variations in the plasma levels of immunoreactive secretin. Combining various doses of isoproterenol and secretin did not allow to reach secretory levels greater than the maximal response to secretin. 2. In conscious rats chronic fistulae, isoproterenol and secretin had a distinct effect.