Importance of the vasoactive intestinal peptide receptor in the stimulation of cyclic adenosine 3',5'-monophosphate in gallbladder epithelial cells of man. Comparison with the guinea pig.

An EDTA procedure was used to prepare isolated epithelial cells of human gallbladder devoid of endogenous vasoactive intestinal peptide (VIP) as measured by radioimmunoassay. Specific binding sites for VIP were characterized in these cells. At 37 degrees C, the binding of (125)I-labeled VIP reached a peak within 20 min and then declined rapidly. At 15 degrees C, binding was stable between 90 and 180 min of incubation. Binding of the labeled peptide was inhibited by concentrations of native VIP of 30 pM-0.1 muM. Half-maximal inhibition was observed at 2 nM. Scatchard analysis indicated two functionally independent classes of receptor sites: 62,000 high affinity sites/cell with a dissociation constant (K(d)) of 1.3 nM, and 510,000 low affinity sites/cell with a K(d) of 16.2 nM. Secretin inhibited tracer binding but with a 1,000 times lower potency than native VIP. VIP strongly stimulated adenosine 3':5' monophosphate (cyclic AMP) production in human gallbladder epithelial cells. At 37 degrees C, 0.1 nM and 10 nM VIP raised cyclic AMP levels 44 and 100 times above the basal level, respectively. Maximal values remained constant between 60 and 90 min at 15 degrees C. The importance of the VIP-induced cyclic AMP rise was related, at least in part, to a low phosphodiesterase activity in human gallbladder epithelial cells. At equilibrium, during a 60-min incubation at 15 degrees C, cyclic AMP production was noted at concentrations of VIP as low as 3 pM. Maximal and half-maximal stimulations were observed at 10 nM and 0.2 nM VIP, respectively. Secretin also stimulated cyclic AMP production but with a 10,000 lower potency than VIP. In the guinea pig, VIP and secretin were equipotent stimulators of cyclic AMP in gallbladder epithelial cells. This particular feature was shown to be due to receptors specific for each peptide that were present in these cells.

Molecular cloning and characterization of a rat intestinal sucrase-isomaltase cDNA. Regulation of sucrase-isomaltase gene expression by sucrose feeding.

To investigate the regulation of expression of intestinal sucrase-isomaltase (SI) complex in response to sucrose feeding, we isolated a cDNA (RPSI1) encoding partially the pro-SI of rat intestinal mucosa. The clone consists of 1929 mRNA-derived nucleotides, which covered the region including the C-terminal part of the isomaltase and the N-terminal part of the sucrase in the final SI complex. Nucleotide and amino-acid sequences of RPSI1 were compared with their corresponding regions in rabbit pro-SI. A greater similarity was found in sucrase parts than in isomaltase parts of the two species. Northern blot analysis revealed that the mRNA levels of SI increased rapidly after sucrose force feeding, while those of rats fed a carbohydrate-free diet did not. These changes in mRNA levels correlated with the corresponding enzyme activities. The results demonstrate that the induction of SI activities is directly associated with an increase in SI mRNA levels. Our results also suggest that circadian modulation of SI transcription may occur in basic SI gene expression.

Characterization of vasoactive intestinal peptide receptors in human colonic epithelial cells.

Receptors, i.e. specific binding sites for vasoactive intestinal peptide (VIP), have been characterized in human colonic epithelial cells isolated by EDTA treatment using 125I-labeled porcine VIP. The binding was time and temperature dependent. Conditions of apparent equilibrium were obtained at 15 C after 45 min of incubation in the presence of 2.1-7.4 micrograms cell DNA-ml; these conditions minimized the degradation of the peptide and the binding sites. Native VIP competitively inhibited the binding of [125I]VIP in the range of 3 x 10(-11)-10(-7) M, and half-maximal inhibition was observed at 2 x 10(-9) M VIP. Scatchard analysis of these data was consistent with the existence of two classes of binding sites: 7.8 x 10(-9) high affinity sites/microgram DNA with a dissociation constant (Kd) of 1.4 x 10(-9) M, and 12.0 x 10(10) low affinity sites/microgram DNA with a Kd of 46 x 10(-9) M. Among the natural hormones structurally related to VIP, gastric inhibitory polypeptide (GIP) and glucagon had no effect on the binding of labeled porcine VIP. Porcine secretin inhibited [125I]VIP binding, but at doses 1000 times higher than those of porcine VIP. Studies of the coupling between the binding of VIP and the stimulation of cAMP formation indicated a nonlinear relationship between the two processes, with full activation of the cAMP-producing system with occupancy of only a limited number of the binding sites. The presence of binding sites with high affinity for VIP coupled with the cAMP production in human colonic epithelial cells support the concept that this peptide may contribute to the physiological regulation of the functions of the human colonic epithelium in normal and pathological conditions.

Influence of starvation on sucrase regulation by dietary sucrose in the rat.

We have studied the action of sucrose on jejunal sucrase activity. Rats (175 g) were first starved or fed a digestible carbohydrate-free diet for 60 h and then fed a high sucrose diet for varying times up to 84 h. 1) Rats starved for 60 h showed mucosal atrophy with a decrease in protein content/10 cm (18.00 +/- 1.4 versus 40.1 +/- 3 mg (controls p less than 0.001) and in villus height (357 +/- 18 versus 526 +/- 5 microns, p less than 0.001) which was fully repaired only after 60 h on the sucrose diet (528 +/- 11 microns). Rats on digestible carbohydrate-free diet showed no mucosal atrophy. 2) Starved rats had a delayed (60 h) sucrase activity response to sucrose (53 +/- 7 versus 122 +/- 4 microns/mg protein, p less than 0.001). Maximum activity was obtained after 12 h on sucrose diet in rats maintained on the carbohydrate-free diet: 38 +/- 1 versus 108 +/- 2.3 microns/mg protein, p less than 0.001. 3) Villus and crypt cell analysis after starvation and 12 h on a high sucrose diet localized the increase in sucrase activity to the villus-crypt junction. No change occurred in the upper villus. The increase was complete all along the villus by 36 h. In contrast, after the carbohydrate-free diet, sucrase activity increased maximally at all levels of the villus by 12 h on the high sucrose diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary whey proteins and their peptides or amino acids: effects on the jejunal mucosa of starved rats.

The effects of starvation (72 h) and refeeding with three liquid diets, differing only in the molecular form of the nitrogen source (whole whey proteins, WP; tryptic whey protein hydrolysate, WPH; and amino acid mixture, AAM), on the jejunal mucosal morphology and brush border enzyme activities (sucrase, S; maltase, M; and neutral aminopeptidase, NA) of male Wistar rats were studied. All three diets produced repair of the fasting-induced mucosal atrophy; the WP diet gave the most rapid growth with maximum villus height (VH) and protein content after 48 h (p less than 0.01). AAM gave the fastest and greatest stimulation of sucrase and maltase activities (p less than 0.01). There were no significant differences in NA activity. In control rats the WPH and AAM diets produced significantly greater villus height and disaccharidase activities than did the WP diet. Jejunal morphology and disaccharidase activities can be modified by the molecular form of alimentary protein and nutritional status interferes with these modifications.

Effects of dietary whey proteins, their peptides or amino-acids on the ileal mucosa of normally fed and starved rats.

The effects of three liquid diets, differing only in the molecular form of the nitrogen source (whole whey proteins, WP; trypsic whey protein hydrolysate, WPH, and amino-acid mixture, AAM) were studied on the mucosa morphology and brush border hydrolase (BBH) activities (disaccharidases, peptidases) of the ileum of normally fed male Wistar rats (controls) and during refeeding of rats starved for 72h. All three diets produced repair of the fasting induced mucosal atrophy; the AAM diet gave the most rapid response and highest villus height (p < 0.01). This was correlated with an increase in crypt mitoses (p < 0.01). Similar results were obtained in controls with AAM. The sucrase (S) and acid amino peptidase (AAP) specific activities of controls were higher (p < 0.01) on the WPH diet; neutral amino peptidase (NAP) was unaffected. Dipeptidyl peptidase IV (DDP) was lowest on AAM while glucoamylase (G) highest on WP. Fasting increased S and DDP activity, and produced no change in the other BBH. Large variations in BBH occurred during refeeding except for NAP which remained stable. Control values were restored at 96h, except for AAP. The results show that BBH and mucosa morphology of the ileum in the rat can be modified by the molecular form of the nitrogen source and that the nutritional status interferes with this adaptation. These data could have implications for the therapy of small bowel disease.

[Effect of fasting and refeeding on the adaptation of the small intestine in rats. A model for physiopathologic studies]. / Effet du jeûne et de la réalimentation sur l'adaptation de l'intestin grêle chez le rat. Un modèle d'études physio-pathologiques.

A chronological study was carried out on 50 male Wistar rats (350 g) to determine the effects of 3 days of fasting and 16 h to 9 days of refeeding on the morphology of jejunal and ileal mucosa (villus, crypt and enterocyte heights; number of mitosis), on some aspects of their functional adaptation (sucrase, maltase, protein) and on nitrogen and lipid absorptions. Three days of fasting resulted in weight loss (12 p. 100), in a jejunal mucosa atrophy (villus height: 376 +/- 18 vs. 492 +/- 4 microns in controls; enterocyte height: 31 +/- 2 vs. 41 +/- 0.3 micron in controls) and a decrease in disaccharidases activities (sucrase: 927 +/- 90 vs. 3,363 +/- 21 mU/10 cm length in controls). No change in ileal mucosa morphometry was noticed. Ad libitum refeeding caused a rapid and progressive weight gain, a jejunal morphometric regrowth identical to control values at 16 h (villus height: 521 +/- 20, enterocyte height 42 +/- 0.9 microns), and maximum at 40 h of refeeding (villus height: 601 +/- 5 microns). Disaccharidases adaptation was delayed, with a maximum at 64 h of refeeding (sucrase: 3,524 +/- 56 mU/10 cm length). Despite a 30 p. 100 increase of food consumption over the whole study (45 p. 100 during the first 16 h of refeeding), nitrogen and lipid absorption coefficients remained identical to those found in controls with an increased nitrogen balance of 70 p. 100 at 16 h and 54 p. 100 at 40 h refeeding, as compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal aminooligopeptidase in diabetic BioBreed rat: altered posttranslational processing and trafficking.

The structure of aminooligopeptidase (AOP), an intestinal brush-border digestive hydrolase, is abnormal in human diabetes and in the congenitally diabetic BioBreed Wistar (BB(d)) rat. Its assembly in the BB(d) rat was examined. After normal initial synthesis and assembly of immature AOP precursor (AOP(i)) with high-mannose N-linked chains in the endoplasmic reticulum (ER), processing of N-linked glycans in Golgi yielded a smaller than normal mature AOP precursor (AOP(m)) with persistence of some high-mannose N-linked chains. Deglycosylation analyses suggested that the mass difference could be attributed to a lower mass of N-linked with unaltered O-linked glycans in AOP(m) of the diabetic rat. Intrajejunal pulse-chase experiments revealed that the conversion of AOP(i) to AOP(m) occurred at 30 min of chase in normal rats but at 60-90 min in diabetic rats, reflecting delay in ER-to-Golgi transport or a slower processing of high-mannose chains. Once maximal transport to Golgi was achieved, the residence time in Golgi was shortened in diabetes. This altered processing of the precursor accounted for the altered structure of AOP in diabetes.

Sucrase-alpha-dextrinase in the spontaneously diabetic BioBreed Wistar rat: altered intracellular carbohydrate processing.

Sucrase-alpha-dextrinase (S-D), a glycoprotein hydrolase in the border surface of the enterocyte, is altered in congenitally diabetic BioBreed Wistar (BB(d)) rats. Its intracellular assembly was examined in the current studies. Following pulse-chase experiments, S--D was specifically immuno-isolated from ER-Golgi and brush border membranes, and examined by SDS-PAGE and autoradiography. While synthesis and co-translational glycosylation of an immature species, P(i), in the ER proceeded normally, post-translational maturation of the N-linked carbohydrate chains was altered in the BB(d) rat. The mature species, P(m), was 10 kDa larger in BB(d) than in normal rats, and approximately 25% of its N-linked chains remained immature. The difference in mass was attributed to an appreciably larger mass of the O-linked chains of P(m) in BB(d) rats. In vivo kinetics of intracellular assembly displayed a delay in the appearance of P(m) in Golgi (Wistar, 15 min; BB(d), 30--60 min). These experiments, revealing structural alterations in congenital diabetes suggest an important role for intracellular glycosylation in the orderly assembly and processing of brush border S-D in the enterocyte.

Laterobasal membranes from intestinal epithelial cells: isolation free of intracellular membrane contaminants.

A simplified method for isolating highly purified laterobasal membranes (LBM) from enterocytes is based on treatment of membranes with 8 mM CaCl2 concentration in order to aggregate intracellular membrane contaminants. The resultant LBM showed an average 15-fold enrichment and constituted 8% of the original K-stimulated phosphatase in the initial crude homogenate. It showed typical LBM migration on counter-current distribution (CCD) and was essentially free of contamination with endoplasmic reticulum and Golgi membranes. This method is highly efficient and yields sufficient purified LBM to allow comprehensive analysis of enterocyte membrane events.