Nitric oxide modulates pancreatic basal secretion and response to cerulein in the rat: Effects in acute pancreatitis

Nitric oxide synthase activity is detected in the pancreas, but the role of NO on pancreatic function has not been fully characterized. The aim of this study was to evaluate the role of NO in normal and diseased pancreatic function. Amylase and NO secretion were measured in vivo in rats and in vitro in dispersed acini, with and without NO synthesis blockade, by N^G-nitro-l-arginine methyl ester (l-NAME). Rats were subjected to cerulein-induced pancreatitis, and the effects of l-NAME or NO donors were assessed. l-NAME reduced amylase output to 60% of basal. This effect was reversed by l-arginine. The secretory response to optimal doses of cerulein induced a poor amylase secretion and a marked release of NO. High doses of cerulein in combination with l-NAME inhibited NO formation and amylase secretion. In dispersed acini, supramaximal cerulein concentrations induced NO release, but the amylase dose-response curve was not modified by NO inhibition. In acute pancreatitis, l-NAME increased amylasemia and tissue myeloperoxidase activities, whereas NO donors reduced amylasemia, lipasemia, and the histological damage score. The l-arginine/NO pathway facilitates basal and stimulated pancreatic secretion in vivo. NO donor drugs may improve the course of acute pancreatitis.     

Histamine inhibits prostaglandin E2-stimulated rabbit duodenal bicarbonate secretion via H2 receptors and enteric nerves

The gastroduodenal epithelium is protected from acid peptic damage by an adherent mucus-bicarbonate layer. Bicarbonate is secreted by the surface epithelial cells into this mucus layer. Patients with duodenal ulcer disease have impaired proximal duodenal bicarbonate secretion. Mast cells, present in large numbers in the duodenal mucosa, release a number of inflammatory mediators, including histamine. Release of such mast cell mediators has been implicated in ulcer disease. In this study, the ability of histamine to regulate bicarbonate secretion was examined. Bicarbonate secretion by rabbit proximal duodenal mucosa was examined in vitro, and the effects of histamine, its agonists, and its antagonists were studied. Histamine essentially eliminated prostaglandin E₂-stimulated duodenal mucosal bicarbonate secretion, an effect reversed both by the neurotoxin, tetrodotoxin, and the histamine H₂-receptor antagonist, cimetidine, as well as reproduced by the H₂-receptor agonist, dimaprit. In addition to the stimulatory action of histamine on gastric acid secretion, histamine expresses an additional antidefensive action by inhibiting prostaglandin E₂-stimulated duodenal epithelial bicarbonate secretion. This effect of histamine is likely mediated via H₂ receptors located on enteric nerves.     

Myenteric denervation of rat jejunum alters calcium responsiveness of intestinal smooth muscle

Long-term myenteric and extrinsic denervation of a segment of rat jejunum results in increased stress generation by the longitudinal muscle layer of the denervated segment 15 days after denervation. This study examined whether alterations in the properties of either cell membrane calcium channels and/or sarcoplasmic reticular Ca²⁺-adenosine triphosphatase (ATPase) contribute to the increased stress development. The effects of the calcium channel blocker nifedipine and the sarcoplasmic reticular Ca²⁺-ATPase inhibitor cyclopiazonic acid on the contractile activity of denervated and control smooth muscle were determined. The ability of nifedipine to inhibit KCl-induced contractions was significantly increased in denervated tissues; however, there was no difference in the potency of nifedipine when tissues were stimulated with carbachol. Calcium concentration-response curves obtained in the presence of either KCl or carbachol were determined in tissues previously depleted of calcium. Long-term denervated tissues showed an increased sensitivity to calcium and a decreased maximum contractile response after stimulation with carbachol. Cyclopiazonic acid inhibited repletion of intracellular calcium stores of control muscle but had no effect in denervated tissue. Long-term denervation of a segment of rat small intestine results in profound alterations in calcium metabolism at the cell membrane and, to a lesser extent, at the sarcoplasmic reticulum of smooth muscle cells of the longitudinal muscle layer.     

Coexpression of 5-HT2A and 5-HT4 receptors coupled to distinct signaling pathways in human intestinal muscle cells

The type and function of 5-hydroxytryptamine (5-HT) receptors on intestinal muscle cells in humans are not known. 5-HT receptors were characterized pharmacologically and by radioligand binding. Contraction, relaxation, inositol 1,4,5-triphosphate (IP₃) and adenosine 3′,5′-cyclic monophosphate (cAMP) formation, and 5-HT binding were measured in dispersed muscle cells and in cells in which only one receptor type was preserved by selective receptor protection. 5-HT binding was completely inhibited by 5-HT and partially by 5-HT_2A (ketanserin), 5-HT₄ (SDZ-205,557), and 5-HT_1p (N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide; 5-HTP-DP) receptor antagonists. 5-HT caused contraction that was inhibited by ketanserin and augmented by SDZ-205,557 and 5-HTP-DP. In the presence of ketanserin, 5-HT caused relaxation of cholecystokinin-contracted cells that was inhibited by SDZ-205,557 and 5-HTP-DP. 5-HT increased IP₃, which was inhibited by ketanserin, and cAMP, which was inhibited by SDZ-205,557 and 5-HTP-DP. In cells with only 5-HT_2A receptors, 5-HT caused contraction only, and residual binding was inhibited by ketanserin. In cells with only receptors, 5-HT caused only relaxation and residual binding was inhibited by SDZ-205,557 and 5-HTP-DP. 5-HT_2A receptors mediating contraction and 5-HT₄ receptors mediating relaxation coexist on human intestinal muscle cells. The 5-HT₄ receptors are closely similar or identical to 5-HT_1p receptors.     

Taurocholate-stimulated leukotriene C4 biosynthesis and leukotriene C4-stimulated choleresis in isolated rat liver

Cysteinyl-containing leukotrienes seem to exert a cholestatic effect. However, leukotriene inhibitors were found to reduce bile salt efflux in isolated rat hepatocytes, suggesting a role for leukotrienes in bile flow formation. In the isolated rat liver, the effects of two different concentrations of leukotriene C₄ on bile flow and bile salt excretion are analyzed, as well as the possible effect of taurocholate on the hepatic production of cysteinyl-containing leukotrienes. Leukotriene C₄ (0.25 fmol) increased bile salt excretion (+22.2%; P < 0.05), whereas a much higher dose (0.25 × 10⁶ fmol) showed the known cholestatic effect, reducing bile salt excretion (−25.9%; P < 0.01). These dose-dependent biphasic effects were specific because they could be prevented by the simultaneous administration of cysteinyl-containing leukotriene antagonists. On the other hand, taurocholate administration induced a dose-dependent increase in biliary excretion of cysteinyl-containing leukotrienes. Furthermore, taurocholate increased messenger RNA levels of 5-lipoxygenase, a key enzyme in leukotriene biosynthesis. Taurocholate increase of hepatocyte intracellular calcium was not significant, suggesting that taurocholate effects are not mediated by stimulation of calcium metabolism. These results constitute evidence for the existence of a positive feedback mechanism by which bile salts stimulate the synthesis of leukotrienes that, in turn, stimulate bile salt excretion.     

Desensitization of platelet-activating factor receptors, induced by inflammation in guinea pig ileal smooth muscle cells

Platelet-activating factor (PAF) is involved in the pathophysiology of motility changes induced by intestinal inflammation. The aim of this study was to evaluate a possible desensitization of PAF receptors in guinea pig intestinal smooth muscle cells after experimental inflammation induced by trinitrobenzenesulfonic acid (TNB). Saline or TNB (80 mg/kg) was injected in the intestinal lumen, and the animals were killed 6 days later. Smooth muscle cells from the circular layer were isolated, and cell contraction induced by PAF was measured. In cells from saline-treated animals, PAF induced a maximal cell contraction at 10 nmol/L and half-maximal contraction (EC₅₀) was 9 ± 0.2 pmol/L. After TNB injection, the maximal contraction induced by PAF was observed at 1000 nmol/L and EC₅₀ was 300 ± 70 pmol/L, indicating a 2 logmol/L right shift of the concentration-response curve of PAF. When animals were treated with the PAF antagonist, 20 mg/kg BN52021, or with 2 mg/kg indomethacin for the 6 days after TNB instillation, the right-sided shift of the concentration-response curve of PAF did not occur. Desensitization of PAF receptors occurring in intestinal smooth muscle cells after TNB instillation could be mediated in vivo by PAF itself via a prostaglandin-dependent pathway.     

Regulation of protein secretion into bile: Studies in mice with a disrupted mdr2 p-glycoprotein gene

Protein is secreted into bile via several independent pathways. The aim of this study was to investigate whether these pathways are influenced by secretion of biliary lipid. Protein secretion and biliary lipid output were studied in wildtype mice (+/+), heterozygotes (+/−), and homozygotes (−/−) for mdr2 gene disruption. Biliary lipid and protein output were varied by infusion with taurocholate (TC) and tauroursodeoxycholate (TUDC). Exocytosis and transcytosis were unaltered in (−/−) mice. Infusion with TC strongly induced secretion of alkaline phosphatase in (−/−) mice but had little effect in (+/−) and (+/+) mice. Infusion with TUDC had little effect on alkaline phosphatase output. In contrast, both TUDC and TC strongly stimulated secretion of aminopeptidase N and lysosomal enzymes in (+/+) mice but had no effect in (−/−) animals. Aminopeptidase N secretion correlated with phospholipid output, but only at high flux. At low flux, aminopeptidase N was secreted independently from both phospholipid and bile salts. The canalicular membrane enzymes alkaline phosphatase and aminopeptidase N are secreted via separate pathways. Part of alkaline phosphatase output is controlled by bile salt hydrophobicity, whereas at high lipid flux, aminopeptidase N secretion seems to be coupled to phospholipid output. Lysosomal enzymes follow the latter pathway.     

Involvement of eicosanoids and macrophage-like cells in cytokine-mediated changes in rat myenteric nerves

Proinflammatory cytokines alter function in enteric nerves, but little is known about underlying mechanisms. This study was designed to investigate the roles of prostanoids and of macrophage-like cells in cytokine-induced suppression of [³H]norepinephrine release from rat myenteric plexus. The release of ³H from jejunal longitudinal muscle-myenteric plexus preparations that had been loaded with [³H]norepinephrine was measured. Measurements of ³H release as well as concentrations of prostaglandin E₂ and leukotriene were made in preparations exposed to interleukin 1β plus interleukin 6 and in the presence or absence of piroxicam, 5-lipoxygenase inhibitor MK886, cycloheximide, or cyclosporin A. An ultrastructural analysis was also performed to investigate the presence of macrophage-like cells in the myenteric plexus. Interleukin 1β plus interleukin 6 suppressed ³H release and caused an increase in tissue prostaglandin E₂ butnot leukotriene E₄. Piroxicam and cycloheximide but not MK886 attenuated the cytokine-induced increase in prostaglandin E₂ and the suppression of [³H]norepinephrine release. Ultrastructural analysis showed macrophage-like cells in the plexus, and the cytokine effects were inhibited by cyclosporin A. Prostanoids but not leukotrienes mediate the cytokine-induced suppression of norepinephrine release, and the results of this study suggest that macrophage-like cells are also involved.     

Antioxidant activity of silybin in vivo during long-term iron overload in rats

Hepatic iron toxicity may be mediated by free radical species and lipid peroxidation of biological membranes. The antioxidant property of silybin, a main constituent of natural flavonoids, was investigated in vivo during experimental iron overload. Rats were fed a 2.5% carbonyl-iron diet and 100 mg · kg body wt⁻¹ · day⁻¹ silybin for 4 months and were assayed for accumulation of hepatic lipid peroxidation by-products by immunocytochemistry, mitochondrial energy-dependent functions, and mitochondrial malondialdehyde content. Iron overload caused a dramatic accumulation of malondialdehyde-protein adducts into iron-filled periportal hepatocytes that was decreased appreciably by silybin treatment. The same beneficial effect of silybin was found on the iron-induced accumulation of malondialdehyde in mitochondria. As to the liver functional efficiency, mitochondrial energy wasting and tissue adenosine triphosphate depletion induced by iron overload were successfully counteracted by silybin. Oral administration of silybin protects against iron-induced hepatic toxicity in vivo. This effect seems to be caused by the prominent antioxidant activity of this compound.     

Role of leukocyte-endothelial cell adhesion in radiation-induced microvascular dysfunction in rats

Recent evidence suggests an active role of endothelial cells and inflammatory cells in radiation-induced vascular dysfunction and organ damage. The aim of this study was to characterize the endothelial cell-leukocyte interactions, their molecular mechanisms, and the associated microvascular dysfunction in postcapillary venules exposed to ionizing radiation. Leukocyte rolling, adherence, and emigration and leakage of fluorescein isothiocyanate albumin in rat mesenteric venules were measured in control conditions and at 2, 4, and 6 hours after abdominal irradiation. Some animals were treated with monoclonal antibodies against leukocyte (CD18) or endothelial cell (intercellular adhesion molecule 1, P-selectin) adhesion molecules before radiation and 5 hours thereafter. In comparison with controls, irradiated animals had a marked increase in the number of rolling leukocytes at 2 hours after radiation. In animals studied 6 hours after radiation, a significant increment in the number of adherent and emigrated leukocytes was observed. This was associated with an increased permeability to fluorescein isothiocyanate albumin. Treatment with antibodies against either CD18 or intercellular adhesion molecule 1, but not P-selectin, significantly attenuated leukocyte adherence, emigration, and the increase in permeability induced by radiation. Radiation-induced leukocyte adherence and emigration involves an interaction between CD11/CD18 on leukocytes and intercellular adhesion molecule 1 on vascular endothelium. These interactions are implicated in the early increase in vascular permeability after irradiation.     

Interruption of a transforming growth factor α autocrine loop in Caco-2 cells by antisense oligodeoxynucleotides

We have previously shown that Caco-2 cell proliferation is driven by basolateral membrane epidermal growth factor receptors. The aim of this study was to investigate whether autocrine production of transforming growth factor α (TGF-α) activates these receptors and stimulates proliferation using antisense oligodeoxynucleotides. Caco-2 cells grown on microporous membranes or Jurkat cells were exposed to conventional or 5′ cholesterol-modified oligodeoxynucleotides synthesized with random, antisense, or missense base sequences. Indices of proliferation were measured, including [³H]thymidine or [³H]uridine uptake for studies of short-term stimulation and the methylthiotetrazole assay as an index of cell number increase over longer periods. Secretion of TGF-α by cells was detected using a soft agar bioassay. Incubation with antisense oligodeoxynucleotides inhibited TGF-α secretion compared with controls. Random and missense oligodeoxynucleotides had no effect on proliferation. The TGF-α antisense oligodeoxynucleotides markedly inhibited proliferation, an effect that was abolished by adding TGF-α to the medium. Oligonucleotides had no effect on Jurkat cells, a lymphocytic cell line lacking epidermal growth factor receptors. Cholesterol-modified oligodeoxynucleotides were more effective and specific than unmodified oligodeoxynucleotides. Caco-2 cell proliferation is driven by autocrine stimulation of epidermal growth factor receptors by TGF-α. This mechanism may be effectively inhibited by antisense oligodeoxynucleotides, particularly those modified by the 5′ attachment of cholesterol.     

The effects of and interactions between fermentable dietary fiber and lipid in germfree and conventional mice

Dietary fiber can stimulate intestinal epithelial cell proliferation. The aim of this study was to resolve the different roles of fermentation and intraluminal viscosity on this trophic action and to investigate reported interactions between fiber and dietary fat. Conventional and germfree mice were fed guar gum in combination with low- or high-lipid diets for 2 weeks, and crypt cell production rates were determined. Guar gum significantly stimulated proliferation in the small intestine, especially when combined with fat. Lipid itself also stimulated proliferation in the small intestine and had a direct trophic effect in the cecum and colon of the germfree mice. Fiber markedly stimulated proliferation in the cecum and colon but only in the conventional group. Interactions between lipid and bacteria and between guar gum and bacteria were also observed in the small intestine. Guar gum has a trophic effect in the small bowel, probably related to viscosity, in addition to its fermentation-related actions in the colon. Positive interaction with lipid may be associated with delayed absorption. Lipid also has its own direct actions on small bowel mucosal proliferation, which are attenuated by the presence of bacteria.     

Hansenula anomala killer toxin induces secretion and severe acute injury in the rat intestine

The yeast Hansenula anomala has been associated with gastrointestinal symptomatology and damage to the intestinal wall in humans. In vitro and in vivo, H. anomala secretes a toxin, killer toxin, which is lethal to other microorganisms. In view of the very high rate of killer phenotype expression recorded for H. anomala strains in nature, this study aimed to investigate the hypothesis that H. anomala killer toxin plays a role in the pathogenesis of H. anomala-induced enteritis. Effects of active and heat-inactivated H. anomala killer toxin on intestinal fluid homeostasis and electrolyte balance were investigated in rat small intestine using a standard intestinal perfusion technique. Sections of the perfused jejunum tracts were examined histologically. H. anomala killer toxin induced a significant secretion of water and electrolytes. No significant change was observed when either heat-inactivated H. anomala killer toxin or control growth medium were tested. Histological analysis showed ischemic degeneration of villi and sloughing of surface epithelium in 50% of active H. anomala killer toxin-perfused jejuna. This paper presents original observations compatible with the hypothesis that H. anomala killer toxin plays a role in the pathogenesis of H. anomala-induced enteritis.     

Effect of germfree state on the capacities of isolated rat colonocytes to metabolize n-Butyrate, glucose, and glutamine

Among substrates available to the colonic mucosa, n-butyrate from bacterial origin represents a major fuel. The present work investigated possible modifications of energy substrate metabolism in colonocytes isolated from germfree rats. Colonocytes isolated from germfree vs. conventional rats were incubated (30 minutes at 37°C) in the presence of ¹⁴C-labeled n-butyrate (10 mmol/L), glucose (5 mmol/L), or glutamine (5 mmol/L). ¹⁴CO₂ and metabolites generated were measured. Possible regulatory steps were also investigated. Glucose use rate was 25% lower in germfree rat colonocytes due to a reduced glycolytic capacity in these cells. Differences in 6-phosphofructo-1-kinase activity could account for this decrease. In contrast, glutamine use rate was 45% higher, and this was correlated with a higher maximum velocity of glutaminase in these cells. Nevertheless, the capacities to oxidize glucose and glutamine remained unchanged. Although the capacity to use n-butyrate was maintained in colonocytes of germfree rats, the ketogenic capacity was lower, whereas the capacity to oxidize n-butyrate was higher. The mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase protein was identified in the colonic mucosa. Moreover, the messenger RNA and amount of protein were 75% lower in the germfree state. The absence of an intestinal microflora induces specific changes in the metabolic capacities of colonocytes.     

Microsatellite instability in human colonic cancer is not a useful clinical indicator of familial colorectal cancer

Microsatellite instability is a property of most tumors occurring in the context of hereditary nonpolyposis colon cancer. Instability also occurs in 10%–15% of apparently sporadic colorectal cancers, and it has been hypothesized that this instability may indicate a genetic predisposition to colonic cancer. This study evaluated whether there is a clinically useful association between colon cancer instability and a family history of cancer. Colon cancer cases (n = 188) from a population-based study were evaluated for microsatellite instability with 10 polymerase chain reaction primer sets. Instability results were compared with family history and other clinical and biological characteristics. Microsatellite instability was found in 16.5% of tumors. It was predominantly a feature of right-sided tumors (P = 0.003) and was associated with the youngest and oldest ages at diagnosis (P = 0.01). Instability was not associated with family history of cancer, sex of the individual, or the glutathione-S-transferase mu 1 null genotype. Although some very small, and as yet undefined, proportion of colon cancer may be caused by inherited mutations leading to microsatellite instability, tumoral instability by itself is not a marker for familiality and should not be considered as evidence for an inherited syndrome.     

Shc is a substrate of the rat intestinal epidermal growth factor receptor tyrosine kinase

Epidermal growth factor (EGF) has been shown to induce intestinal proliferation and maturation; however, little information is available regarding substrates of the intestinal EGF receptor tyrosine kinase. The purpose of this study was to determine if src homologous collagen-like protein (Shc) was an in vivo substrate of the intestinal EGF receptor. Ten-day-old rats were treated with EGF or were breast-fed. In some experiments, IEC-6 cells were treated with EGF. Intestinal tissue and cell fractions were studied by immunodetection to compare the tyrosine phosphorylation state and the subcellular localization of intestinal proteins. The total tyrosine phosphorylation state of intestinal proteins was increased threefold by EGF. Tyrosine phosphorylation of the EGF receptor and Shc were rapidly increased by EGF. The association of Grb2 with Shc increased fourfold and fivefold. Plasma membrane translocation of Shc and associated phosphotyrosyl proteins was increased within 30 seconds of EGF treatment. Shc is a substrate of the intestinal EGF receptor in vivo. EGF-induced association of Shc with the adapter protein Grb2 may have implications for activation of the p21^ras signaling pathway in the intestine. The EGF-induced membrane association of Shc with two other phosphotyrosyl proteins suggests involvement of Shc in additional aspects of EGF-receptor signaling in the intestine.     

A 7-year experience of severe acetaminophen-induced hepatotoxicity (1987–1993)

Five hundred sixty patients admitted between January 1, 1987, and December 31, 1993, with severe acetaminophen-induced hepatotoxicity were studied. The aim of this study was to identify why severe acetaminophen-induced hepatotoxicity still occurs and to determine how known risk factors and advances in management have affected the pattern of illness and outcome. This was a retrospective study of the etiologic factors and the clinical course of all acetaminophen-related admissions. The number of admissions increased from 58 in 1987 to 123 in 1993. During the corresponding period, overall survival improved from just <50% to 78%. The percentage of admissions treated with N-acetylcysteine increased from 40% in 1987 to 83% in 1993. The frequency with which grade III or IV encephalopathy developed decreased from 62% in 1987 to 40% in 1993, and the percentage of these patients who developed cerebral edema decreased from 61% to 45% during the same period. There was an increase in both the number of patients transplanted and the survival of those managed medically. Severe acetaminophen-induced hepatotoxicity remains a serious condition, but the increasing use of N-acetylcysteine, advances in medical management, and the increasing availability of transplantation have resulted in a significant improvement in survival rates.     

Rat Kupffer cell-derived nitric oxide modulates induction of lymphokine-activated killer cell

Nitric oxide is now recognized to regulate immune responses and cell viability in various organs. The present study was designed to clarify whether NO released from Kupffer cells modulates the lymphokine-activated killer (LAK) activity of interleukin 2 (IL-2)-treated splenocytes. Splenocytes and Kupffer cells were isolated from male Wistar rats and cocultured for 48 hours in the presence of lipopolysaccharide (1 μg/mL). The splenocyte LAK activity and expression of IL-2 receptor were determined. Kupffer cells with lipopolysaccharide reduced the IL-2 receptor expression and LAK activity of splenocytes. The addition of either N^G-monomethyl-l-arginine, an inhibitor of NO synthesis, or aminoguanidine, an inhibitor of inducible NO synthase, to the medium reversed the suppression of IL-2 receptor expression and LAK activity by lipopolysaccharide-stimulated Kupffer cells. 8-bromoguanosine 3′,5′-cyclic monophosphate and NO donors decreased the splenocyte LAK activity and IL-2 receptor expression. Treatment with lipopolysaccharide increased the inducible NO synthase activity as well as the nitrite and nitrate levels in the culture medium of Kupffer cells but not in splenocytes. The results of this study suggest that NO produced by the inducible NO synthase of Kupffer cells in response to lipopolysaccharide modulates the IL-2 receptor expression and LAK activity of splenocytes.