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PubMed1977

Reduction of reactivity of Escherichia coli enterotoxins by intestinal mucosal components.

Incubation studies involving rabbit and piglet small intestinal mucosal components and Escherichia coli and Vibrio cholerae enterotoxins were conducted at 37 and 4 degrees C. Mucosal homogenate cytosol from rabbits did not significantly alter the reactivities of either cholera enterotoxin (CT) or E. coli labile enterotoxin (LT). However, mucosal homogenate cytosol from piglets was capable of neutralizing LT, though it had no effect on E. coli stable enterotoxin. LT became bound to piglet and rabbit microvillous membranes at 4 degrees C in the presence of a protective protein. In rabbits, the binding of LT was not dependent upon an intact glycocalyx or free unbound CT-receptors, although some binding was apparently associated with the glycocalyx and CT-receptors. These results indicated the presence of two different LT-receptors in microvillous membranes one being associated with the membrane proper and the other with the glycocalyx. Stable enterotoxin did not bind to in vitro preparations of piglet mucosal components, which is evidence for a different mechanism for inducing intestinal secretion.

Animals↗
PubMed2002

Morphological and immune response alterations in the intestinal mucosa of the mouse after short periods on a low-magnesium diet.

The importance of Mg for the immune function is well recognized; however, there is no information available about the effect of Mg intake on the modulation of local immune response in the intestine. Thus, in the present study the hypothesis that short periods of Mg deprivation can affect intestinal mucosa and local immune response was tested. For this purpose, OF1 female mice were fed a semipurified diet (1000 mg Mg/kg diet). For 3 d before immunization and 1 d after, half of the animals were fed a Mg-deficient diet (30 mg Mg/kg diet), three immunizations per os were performed every 3 weeks with Escherichia coli producing the CS31A capsule-like protein (1010 or bacteria per animal). Mice were killed 10 d after the last immunization. The level of specific anti CS31A immunoglobulin (Ig) G and IgA in the serum and secretory IgA in the intestinal secretions and faeces were measured by ELISA. The results indicated that administration of a high dose of immunogen with a low-Mg diet led to lower specific IgA levels in the intestinal mucus and serum. Administration of a low dose of immunogen with a low-Mg diet led to lower IgA and IgG levels in the serum and secretory IgA coproantibodies. To assess alterations of intestinal mucosa caused by a low-Mg diet for a short period, histological and scanning electron microscopy analyses were performed on samples from mice (not submitted to the vaccination protocol) after 3 d on the Mg-deficient diet. These analyses showed several alterations, suggesting perturbations in the growth of the intestinal mucosa. These changes were accompanied by modifications in the expression of several genes involved in cell growth and stress response. From this present work, it may be concluded that short periods of Mg deprivation can affect the intestinal mucosa and local immune response of the intestine.

Animals↗
PubMed2001

[Vitamin E deficiency. Etiopathogenesis, clinical, histopathologic, and electrical features, and main etiologies].

Vitamin E deficiency is frequently observed in human pathology. In most cases, deficiency is moderate and asymptomatic. Severe deficiency is rare and presents as a progressive neurological syndrome including cerebellar ataxia and posterior cord injury. Neuropathological and electrophysiological features confirm spinocerebellar degeneration. The pathophysiology of vitamin E deficiency nervous dysfunction is still unknown. Oxidative alterations due to the lack of the main lipid-soluble antioxidant could be involved. A few causes of vitamin E deficiency are recognized (malnutrition, impaired lipid absorption, specific anomaly of hepatic or intestinal secretion of vitamin E, excessive endogenous consumption), but diseases associated with vitamin E deficiency are numerous and divers. Symptomatic severe deficiency is constantly observed in ataxia with vitamin E deficiency and abetalipoproteinemia. Intestinal, hepatobiliary, and pancreatic diseases are often associated with more or less marked deficiency. Other pathological circumstances such as malnutrition, alcoholism, hemolytic anemia, renal failure and hemodialysis could be associated with moderated and asymptomatic vitamin E deficiency. Oral or regular administration of high-dose vitamin E is required for patients with symptomatic severe deficiency. An adapted supplementation is recommended for patients with a marginal vitamin E status.

Electrophysiology↗
PubMed1996

5-hydroxytryptamine release into human jejunum by cholera toxin.

BACKGROUND: Cholera toxin produces intestinal secretion by activation of the adenylate cyclase complex. However animal studies have shown 5-hydroxytryptamine may be released after exposure to cholera toxin, and thereby contribute to the secretory state. AIM: To determine whether cholera toxin releases 5-hydroxytryptamine in human jejunum. SUBJECTS: Seven male subjects were given a subclinical dose of cholera toxin in a paired, controlled, randomised, double blind study. METHODS: A closed 10 cm segment of upper jejunum was exposed to 15 micrograms of cholera toxin for two hours prior to closed segment perfusion with plasma electrolyte solution containing a non-absorbable volume marker, [14C]-polyethylene glycol. 5-Hydroxytryptamine in jejunal effluent and 5-hydroxyindoleacetic acid in urine (up to seven hours after cholera toxin) were measured by high performance liquid chromatography with fluorimetric detection. RESULTS: In contrast with controls, all subjects secreted fluid in response to cholera toxin, median-2.1 ml/cm/h (interquartile range-4.1 to -0.1). During seven hours following cholera toxin, 5-hydroxytryptamine was secreted into the lumen (range 31 to 395 nmol/l) but not in control experiments. After exposure to cholera toxin median urinary 5-hydroxyindoleacetic acid was 5.7 (4.1 to 6.3), which was similar to controls 4.9 (4.1 to 6.3), which was similar to controls 4.9 (4.1 to 6.2). CONCLUSION: Thus, cholera toxin induced a secretory state and promoted the release of 5-hydroxytryptamine into the intestinal lumen, but quantitative changes in urinary 5-hydroxyindoleacetic acid were not detectable. As an intestinal secretagogue, these findings suggest that 5-hydroxytryptamine may play a part in mediating cholera toxin induced secretion in humans.

Adult↗
PubMed1987

The influence of digoxin-specific antibody fragments on digoxin disposition in the rat.

Pentobarbitone-anaesthetized bile duct-cannulated female rats were injected intravenously with an equimolar dose of digoxin-specific sheep antibody fragments (DS-Fab) at 2 or 60 min after a dose of [3H]digoxin. The plasma drug levels were promptly elevated by 7-fold or 12-30-fold when the DS-Fab were given at 2 or 60 min respectively. When tissue drug concentrations were measured 2 min after a dose of DS-Fab (given 60 min after digoxin) which caused a 30-fold increase in plasma concentration, reductions could be detected if corrections were made for the presence in the tissues of high plasma concentrations of DS-Fab-bound drug. For instance, reductions in the heart, liver and small intestine were 63, 58 and 48% respectively. However, by 120 min after digoxin injection the only detectable effects on tissue drug concentration were in the kidney, where concentrations had increased 14-fold or 7-fold when the DS-Fab were given at 2 or 60 min respectively. Over the 120 min period the urinary excretion of digoxin-derived radioactivity was enhanced, and in the case where DS-Fab were given at 2 min, a 3-fold increase in urinary excretion was seen, which resulted in a net increase in the overall drug elimination. This greater urinary elimination was accompanied by a marked increase in the amount of bound drug in the urine (control and experimental values were 4 and 36% respectively). The cumulative biliary excretion of radioactivity seemed to be slightly reduced by DS-Fab administration at 2 or 60 min, although this was not statistically significant. A lack of significant drug-specific binding in the bile suggested that the liver is not involved in the elimination of hapten-DS-Fab complexes. There was little effect on the intestinal secretion of the drug.

Animals↗
PubMed1998

Effect of different beta-adrenergic agonists on the intestinal absorption of galactose and phenylalanine.

Nutrient transport across the mammalian small intestine is regulated by several factors, including intrinsic and extrinsic neural pathways, paracrine modulators, circulating hormones and luminal agents. Because beta-adrenoceptors seem to regulate gastrointestinal functions such as bicarbonate and acid secretion, intestinal motility and gastrointestinal mucosal blood flow, we have investigated the effects of different beta-adrenergic agonists on nutrient absorption by the rat jejunum in-vitro. When intestinal everted sacs were used the beta2-agonist salbutamol had no effect either on galactose uptake by the tissue or mucosal-to-serosal flux whereas mixed beta1- and beta2-agonists (isoproterenol and orciprenaline) and beta3-agonists (BRL 35135, Trecadrine, ICI 198157 and ZD 7114) inhibited galactose uptake and transfer of D-galactose from the mucosal-to-serosal media across the intestinal wall (although the inhibiting effects of isoproterenol and Trecadrine were not statistically significant). In intestinal everted rings both Trecadrine and BRL 35135 clearly reduced galactose uptake, the effect being a result of inhibition of the phlorizin-sensitive component. Total uptake of phenylalanine by the intestinal rings was also reduced by those beta3-adrenergic agonists. These results suggest that beta1- and beta3-adrenergic receptors could be involved in the regulation of intestinal active transport of sugars and amino acids.

Adrenergic beta-Agonists↗
PubMed1989

Failure of pertussis toxin to inhibit activation of guanylate cyclase by the heat-stable enterotoxin of Escherichia coli (STa) in the T84 cell line.

The heat-stable enterotoxin (STa) of Escherichia coli causes intestinal secretion by stimulating guanylate cyclase, an enzyme believed to be distinct from the STa receptor. Pertussis toxin (PT) has been reported to block the ability of STa to stimulate guanylate cyclase in rat intestinal mucosa (S. A. Epstein, R. A. Giannella, and H. J. Brandwein, FEBS Lett. 203:44-48, 1986). This suggested that a guanine nucleotide regulatory protein (G protein) coupled the STa receptor to guanylate cyclase, a function not previously recognized for G proteins. We sought to explore this phenomenon and, if possible, to identify this G protein. Initial experiments with the human colon carcinoma cell line T84 revealed that higher-than-expected concentrations (1 micrograms/ml) of PT were needed to intoxicate cells, as assessed by ADP-ribosylation of endogenous PT substrate, but that 99 to 100% intoxication could be achieved. Homogenates made from fully intoxicated cells did not differ from controls in basal or STa-stimulated guanylate cyclase activity, and cyclic GMP accumulation in intact T84 cells was not changed by PT treatment. We conclude that a PT-sensitive G protein is not involved in the stimulation of cyclic GMP production by the enterotoxin STa.

Adenosine Diphosphate Ribose↗
PubMed1973

Effects of cycloheximide on the response of intestinal mucosa to cholera enterotoxin.

Prior studies have indicated that effects of cholera enterotoxin (CT) on the small intestine are delayed in onset and involve an interaction with adenyl cyclase in the mucosa. It has also been shown that the administration of cycloheximide to rabbits in doses which inhibit crypt cell mitoses (20 mg/kg), diminishes CT-induced fluid production in jejunal loops. These latter studies have been interpreted as indications that CT-related intestinal secretion is a crypt cell function and that it is mediated by a CT-induced protein. The present study was undertaken to delineate more precisely the nature of the interaction in the intestine between cycloheximide and cholera toxin. Pretreatment of rabbits with cycloheximide reduced by 60% the secretory response to CT in isolated ileal loops with intact blood supply. Sodium and chloride flux measurements on mucosa isolated from these and control loops indicated that this antisecretory effect of cycloheximide persists in vitro. Measurements of radioactive leucine incorporation into mucosal protein indicated that the dose of cycloheximide employed inhibited protein synthesis by 90%. This inhibitory effect was shown to be independent of any effect of cycloheximide on amino acid uptake across the brush border. Measurements of adenyl cyclase activity and cyclic AMP levels in ileal mucosa of cycloheximide pretreated and control animals indicated that cycloheximide did not diminish the CT-induced increases in these parameters. These observations demonstrate that cycloheximide reduces CT-induced intestinal fluid production without interfering with the CT-induced augmentation of adenyl cyclase activity or the consequent rise in cyclic. AMP concentration. Since the antisecretory effect of cycloheximide persists in vitro, it probably involves a direct interaction of the antibiotic with mucosal cell ion transport mechanisms rather than an indirect effect mediated by other humoral or neurogenic factors. The present observations also suggest that the secretory response of the intestine to CT involves neither the synthesis of new adenyl cyclase nor that of a protein modifying its activity.

Adenylyl Cyclases↗
PubMed1982

Blood flow an intestinal absorption.

Intestinal blood flow interacts with intestinal absorption at several levels and affects absorption through several mechanisms. The precise relationships are difficult to define because, at present, there is not an adequate technique available for measuring blood flow to the absorptive site. The absorption of certain nutrients requires O2 delivered through the blood for maintenance of carrier-mediated transcellular transport. Both altered capillary pressure, which may or may not accompany altered blood flow because of myogenic autoregulation, and changes in absorption can influence tissue hydrostatic and colloid osmotic pressure and also epithelial and interstitial space conductance. These latter, in turn, can change the rate of passive ultrafiltration for a constant driving force. Changes in blood flow can also influence the rate of washout of absorbed substances. Countercurrent exchange in the villous vasculature can buffer the rate of absorption of some substances and may also facilitate the absorption of water. Regulatory agents such as hormones or neurotransmitters can affect blood flow and absorption both independently and interactively. Agents that cause active intestinal secretion may have their effects modified by consequent changes in blood flow.

Animals↗
PubMed1991

Biologically active peptides in the gastrointestinal lumen.

The release of a variety of biologically active peptides into the gastrointestinal lumen via gastric, duodenal and intestinal secretions, as well as in the saliva, pancreatic juice and bile, has been explored. The key features of luminal secretion of peptides such as secretion at high concentrations, neurohormonal regulation, luminal orientation of stimulated secretion, stability of peptides in the gastrointestinal lumen, altered secretion under pathophysiological conditions, and biological activity of luminally administered peptides are discussed. This review develops a detailed picture of the current understanding of luminal secretion of peptides and their possible biological functions under normal and pathophysiological conditions.

Animals↗
PubMed1977

Digestibilities of nitrogen compounds in rumen bacteria and in other components of digesta in the small intestine of the young steer.

1. Mixed bacteria were obtained from the rumen contents of steers and were taken at different times after the animals had been given different feeds. The feeds contained [15N]urea and in some experiments Na2 35SO4 so that the bacteria were labelled with these isotopes. 2. Samples of labelled bacteria were subjected to a simulated abomasal digestion with pepsin and the digests were infused with a non-absorbed marker, polyethylene glycol, into the duodenums of test steers equipped with re-entrant ileal cannulas. Except for samples taken after a 24 h fast, which sometimes gave somewhat lower values, mean values for digestibilities of 15N and 35S in the small intestine were 0-79 and 0-85 respectively. 3. The corresponding value for the digestibility of 15N in similarly-treated wheat-leaf protein concentrate was 0-86. 4. Net digestibilities of total N in the small intestine of the test steers when they were given either a mainly protein-free diet of straw, tapioca and urea or a diet of flaked maize and hay were approximately 0-62. 5. From these and other values it was calculated that approximately 5-2 g intestinally-secreted endogenous N/d flowed through the ileum of a 100 kg steer.

Animals↗
PubMed1993

Peptide YY is a physiological regulator of water and electrolyte absorption in the canine small bowel in vivo.

BACKGROUND: Peptide YY (PYY), a hormone released following a meal, is one potential mediator of intestinal absorption. Although PYY inhibits 5'-cyclic adenosine monophosphate (cAMP)-stimulated small intestinal secretion in vitro, its effects on fluid and electrolyte transport in vivo are unknown. METHODS: This study examines the effects of physiological doses of PYY in dogs (n = 6) with jejunal and ileal exteriorized, neurovascularly intact intestinal loops (Thiry-Vella fistulas). RESULTS: Plasma PYY levels increased after a meal from 155 +/- 15 to 324 +/- 26 pmol/L at 30 minutes and remained elevated for 2 hours. PYY infused intravenously in unfed animals at 25, 50, 100, and 200 pmol.kg-1.h-1, produced a dose-dependent increase in plasma PYY levels. At 100 pmol.kg-1.h-1, PYY plasma concentrations were similar to those of fed animals (317 +/- 39 pmol/L). PYY infusion resulted in a dose-dependent increase in water and electrolyte absorption at all doses in both the jejunum and ileum. Although the relative increase in absorption was similar, the magnitude was greater in the ileum. CONCLUSIONS: Physiological concentrations of PYY produced an increase in small bowel absorption of water and electrolytes in vivo. The postprandial release of PYY may mediate the increase in absorption following a meal. Such a proabsorptive agent may have considerable potential for clinical use in malabsorptive states.

Animals↗
PubMed1996

Immune response to Clostridium difficile infection.

Clostridium difficile produces two toxins (A and B) which cause antibiotic-associated diarrhoea and pseudomembranous colitis. One of the most puzzling aspects of C. difficile infection is the wide spectrum of clinical presentation which ranges from asymptomatic carriage to fulminant, life-threatening colitis. This review examines the hypothesis that immune responses to C. difficile underlie these dramatic variations in disease presentation and course. Animals can be protected from C. difficile colitis by passive or active immunization against toxins A and B. Human antibody responses to these toxins are evident in approximately 60% of the general population. A number of clinical studies indicate that antitoxin responses in both serum and intestinal secretions may be protective whereas an inadequate immune response predisposes to severe or recurrent C. difficile diarrhoea. Thus there is now considerable interest in developing methods for passive and active immunization to combat this prevalent nosocomial intestinal pathogen.

Animals↗
PubMed1984

Absorption, secretion and excretion of dimethylamine in rats.

The dimethylamine (DMA) concentration in the gastrointestinal tract of Wistar male rats fed a commercial diet containing 23.6 mg/kg DMA was highest (11.2 +/- 2.1 mg/kg) in the stomach and decreased from the upper region to the lower region. In contrast, the highest DMA concentration (6.6 +/- 2.5 mg/kg) in the upper small intestine was observed in rats fed a low-DMA diet containing 1.0 mg/kg DMA. DMA absorption was observed in the intestines and the absorbtion curves were monoexponential. The biological half-lives (t1/2) of DMA in the ligated stomach, upper and lower small intestine, caecum and large intestine were 198, 8.3, 11.6, 31.5 and 11.0 min, respectively. The DMA concentration in blood increased from 0.3 +/- 0.1 mg/kg to 3.0 +/- 1.0 mg/kg 5 min after injection of 250 micrograms DMA into the ligated upper small intestine. The disappearance curve of DMA in blood was monoexponential and the half-life for the initial 15 min was 12.5 min when 250 micrograms DMA were injected through a femoral vein. Intestinal secretion of DMA (15.6 +/- 12.6 mg/kg) was observed 15 min after the injection. Urinary DMA increased from 17.3 +/- 9.4 to 139 +/- 23 mg/kg within 30 min of intravenous injection of DMA. These results show that the behaviour of DMA in rats is as follows: (Formula: see text)

Animals↗
PubMed1992

Jejunal toxin inactivation regulates susceptibility of the immature rat to STa.

The immature rat jejunum demonstrates an increased response, sensitivity, and susceptibility to heat-stable enterotoxin. In the present study, the hypothesis that this increased secretory response results from diminished jejunal toxin inactivation and persistence of biologically active toxin was tested. A modification of toxin injected into ligated jejunal loops was observed in both adult and immature rats. However, in the immature jejunum there was also persistence of biologically active toxin and continued toxin-induced secretion. In contrast, in the adult jejunum there was inactivation of toxin and cessation of secretion. Incubation of toxin with luminal fluid, pancreatic fluid, or jejunal brush border membranes resulted in toxin alteration. However, a completely inactive toxin species was generated only after incubation with jejunal organ culture slices. It is concluded that (a) incomplete toxin inactivation in the immature rat jejunum contributes to prolonged intestinal secretion and that (b) the inactivation process is multifactorial, but it is likely that final toxin inactivation requires the participation of the enterocyte. These findings may help explain the increased responsiveness of the immature intestine to heat-stable enterotoxin.

Animals↗
PubMed1991

Jejunal permeability to water and electrolytes in patients with chronic intrahepatic hypertension: evidence for a role of aldosterone.

Acute prehepatic portal hypertension induces intestinal secretion in animal models. In the course of chronic liver disease, however, these changes are not observed, despite higher portal pressures than those found in experimental studies. Eight patients without diarrhoea and with chronic alcoholic liver disease were examined for evidence of increased jejunal secretion; their suprahepatic wedge pressure was raised from 21 to 45 mmHg (mean 34.6 mmHg). Jejunal perfusion with a triple lumen catheter and a proximal occluding balloon was used to study net flows of water and chloride as well as net and unidirectional flows of sodium and potassium. No statistical difference in intestinal flows of water and electrolytes was noted between cirrhotic patients and control subjects after infusion with a 30 mmol/l glucose solution. Infusion with a 30 mmol/l mannitol solution resulted in a lower absorption of water, Na, K, and Cl than with the glucose solution. A higher rate of Na secretion was observed in cirrhotic patients than control subjects after infusion with 30 mmol/l mannitol (p less than 0.01). In addition, the rate of Na secretion was higher in cirrhotic patients than in control subjects (p less than 0.05). There was no correlation between the net flow of Na and the suprahepatic wedge pressure. A second perfusion with a 30 mmol/l glucose solution was given 75 minutes after a bolus injection of spironolactone (400 mg). Net flows of Na and Cl were lower in cirrhotic patients than in control subjects (p less than 0.05) because of a lower absorption of Na. Patients with gradually developing portal hypertension have moderate jejunal secretions of H2O and electrolytes which we assume are partly masked by increased absorption resulting from hyperaldosteronism. In contrast to animal models, this mechanism may be part of the jejunal adaptation to permeability in acute portal hypertension.

Adult↗
PubMed2002

NaCl and fluid secretion by the intestine of the teleost Fundulus heteroclitus: involvement of CFTR.

Sections of posterior intestine of the euryhaline killifish Fundulus heteroclitus adapted to sea water were stimulated by the calcium ionophore ionomycin (1 micromol l(-1)) in combination with agents to elevate intracellular cyclic AMP levels, 0.5 mmol l(-1) dibutyryl-cyclic AMP (db-cAMP) with 0.1 mmol l(-1) 3-isobutyl-1-methylxanthine (IBMX). Intestinal bag preparations from recently fed animals (but not from overnight unfed animals) changed from fluid absorption (+18.9+/-8.30 microl cm(-2) h(-1), N=8) in the untreated control period to net fluid secretion after stimulation (-7.43+/-1.30 microl cm(-2) h(-1), N=8, P<0.01; means +/- S.E.M.), indicative of the capacity of teleost intestine to undergo secretion. Posterior intestinal pieces mounted in vitro in Ussing-style membrane chambers showed net Cl(-) uptake (+2.245+/-0.633 microequiv cm(-2) h(-1), N=7) that turned to net secretion following stimulation by ionomycin + db-cAMP + IBMX (-3.809+/-1.22 microequiv cm(-2) h(-1), N=7, P<0.01). Mucosal application of the anion channel blocker 1 mmol l(-1) diphenylamine-2-carboxylate (DPC) after ionomycin + db-cAMP + IBMX treatment significantly reduced serosal-to-mucosal unidirectional Cl(-) flux (P<0.001), net Cl(-) flux (P<0.05), short-circuit current (I(sc), P<0.001) and tissue conductance (G(t), P<0.001), while 0.1 mmol l(-1) 4,4'-diisothiocyano-2,2'-stilbene-disulphonic acid (DIDS, a blocker of anion exchange) was without effect. Stimulation by db-cAMP + IBMX (no ionomycin) significantly increased unidirectional fluxes, I(sc) and G(t) but did not produce net Cl(-) secretion. Ionomycin alone produced a transient increase in I(sc) but had no effect on G(t) and caused no significant changes in unidirectional or net Cl(-) fluxes. Addition of db-cAMP + IBMX after ionomycin treatment produced net secretion of Cl(-) and large increases in unidirectional fluxes and G(t). Cystic fibrosis transmembrane conductance regulator (CFTR) was immunocytochemically localized with a monoclonal mouse antibody to the carboxy terminus and found to be present in the cytoplasm and basolateral membranes of all enterocytes and in the brush-border membrane of some cells, whereas NKCC immunofluorescence, demonstrating the presence of the Na(+)/K(+)/2Cl(-) cotransporter, was present in the cytoplasm and brush-border membrane. We conclude that the teleost intestine is capable of salt and fluid secretion only if intracellular Ca(2+) and cyclic AMP pathways are stimulated together and that this secretion appears to involve activation of CFTR ion channels in the apical membrane of a subpopulation of enterocytes.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Role of ascorbic acid in procollagen expression and secretion by human intestinal smooth muscle cells.

The role of ascorbate in the production and secretion of procollagen by human intestinal smooth muscle cells and the conditions in culture for optimal ascorbate bioefficacy were studied. Procollagen synthesis and secretion were determined by the incubation of cells with L-[5-3H]proline, and the quantitation of radiolabelled procollagen bands in the cell layer and the culture medium by polyacrylamide slab gel electrophoresis and densitometry. When cells were cultured without ascorbate in the culture medium, procollagen secretion into the medium was 75% less than in cells receiving fresh ascorbate daily. In the cell layer, in contrast, procollagen accumulation was fourfold greater in the scorbutic cells than in the ascorbate-replete cells. These findings contrasted with those in a control line of scorbutic human dermal fibroblasts in which a 95% decrease in procollagen secretion was not associated with any procollagen accumulation in the cells. In the intestinal smooth muscle cells, the absence of ascorbate resulted in a 25 and 50% decrease in steady-state levels of procollagen I and III mRNA, respectively, compared to a 40 and 75% decrease in fibroblasts. Heat inactivation of the serum in the culture medium augmented the promotion of procollagen secretion by ascorbate two- to fourfold. L-ascorbate phosphate did not increase the activity of L-ascorbate when replaced in medium either daily or every 4 days, and its efficacy was not augmented by serum heat inactivation. The changing of culture medium induced collagen secretion in the absence of ascorbate, but this process was markedly enhanced by ascorbate and induced a transient decrease in the steady-state levels of both procollagen and nonprocollagen mRNAs. The predominant action of L-ascorbate on HISM cells in vitro is to promote procollagen secretion and not procollagen synthesis. L-ascorbate-phosphate is not an adequate substitute for L-ascorbate in this cell line.

Ascorbic Acid↗