James A. Hardin

Increased gastrointestinal permeability is an early lesion in the spontaneously diabetic BB rat

The BB rat spontaneously develops autoimmune diabetes. Feeding these animals a hydrolyzed casein diet significantly reduces the incidence of this disease, suggesting that a dietary antigen is involved in the pathogenesis of this disease. In other syndromes associated with luminal antigens, including celiac and Crohns disease, increased intestinal permeability has been suggested to play an etiological role. Therefore, the objective of this study was to evaluate whether increased permeability was also present in BB rats before disease development. By measuring gastrointestinal permeability, in animals on a regular or hydrolyzed casein diet, we were able to demonstrate that increased gastric and small intestinal permeability appeared before the development of both insulitis and clinical diabetes. Although hydrolysis of dietary protein significantly reduced the incidence of diabetes, it did not alter the small intestinal permeability abnormality, suggesting that this is an early event. Increased permeability appears to have an early role in the genesis of several immunological diseases and may represent a common event in these diseases.The BB rat spontaneously develops autoimmune diabetes. Feeding these animals a hydrolyzed casein diet significantly reduces the incidence of this disease, suggesting that a dietary antigen is involved in the pathogenesis of this disease. In other syndromes associated with luminal antigens, including celiac and Crohns disease, increased intestinal permeability has been suggested to play an etiological role. Therefore, the objective of this study was to evaluate whether increased permeability was also present in BB rats before disease development. By measuring gastrointestinal permeability, in animals on a regular or hydrolyzed casein diet, we were able to demonstrate that increased gastric and small intestinal permeability appeared before the development of both insulitis and clinical diabetes. Although hydrolysis of dietary protein significantly reduced the incidence of diabetes, it did not alter the small intestinal permeability abnormality, suggesting that this is an early event. Increased permeability appears to have an early role in the genesis of several immunological diseases and may represent a common event in these diseases.

Pathophysiology of small intestinal Malabsorption in gerbils infected with Giardia lamblia

Mongolian gerbils were infected with a human pathogenic Giardia lamblia strain and compared with sham-treated control animals 6 days after inoculation. Infection resulted in crypt hyperplasia associated with an increased enterocyte migration rate. Villus height was decreased in the duodenum, unchanged in the jejunum, and increased in the ileum of infected animals. Epithelial microvilli were markedly shortened, and brush border surface area decreased in the jejunum and ileum of infected animals. Thymidine kinase activity was increased in isolated duodenal villus enterocytes but did not differ in the jejunum and ileum. In vitro and in vivo experiments showed that the infection resulted in decreased jejunal glucose-stimulated electrolyte, water, and 3-O-methyl-D-glucose absorption, whereas in the ileum in vitro electrolyte and 3-O-methyl-D-glucose absorption was similar in infected and control animals. Thus, in the jejunum infection causes electrolyte, solute, and fluid malabsorption associated with decreased brush border surface area. The results indicate that the diarrhea associated with giardiasis is caused by malabsorption rather than active secretion.

Structural and functional adaptation following jejunal resection in rabbits: Effect of epidermal growth factor

BACKGROUND/AIMS Remnant small intestine undergoes adaptation following massive resection. The aim of this study was to examine the effect of epidermal growth factor (EGF) on ileal adaptation following proximal resection. METHODS New Zealand white rabbits, aged 8 weeks, underwent 2/3 proximal resection, and ileal mucosal adaptation was assessed 10 and 21 days postsurgery. In a second series of experiments, animals with resection received oral EGF (40 micrograms.kg-1.day-1) for 5 days, and the effect on adaptation was assessed 10 days postsurgery. RESULTS Transection alone stimulated mucosal hyperplasia, intestinal sucrase specific activity, and glucose transport at 10 days but not at 21 days. Resection resulted in mucosal hyperplasia at both time periods and increased disaccharidase specific activity at 10 days postresection. In contrast, 3 O-methyl-D-glucose transport was significantly decreased compared with both groups at both time periods. EGF treatment in animals with resection did not alter mucosal proliferation but did stimulate maltase specific activity and caused a 3-4-fold increase in glucose transport and phlorizin binding. CONCLUSIONS Following proximal resection, adaptation of intestinal digestive and absorptive function does not parallel mucosal hyperplasia. Administration of EGF to resected animals enhances glucose absorption and may have a therapeutic role in the management of short gut syndrome.

Intestinal permeability and postheparin plasma diamine oxidase activity in the prediction of Crohn's disease relapse

A method of detecting presymptomatic relapse of Crohns disease could allow for the selective use of maintenance or intensified medical therapy in those with an increased risk of relapse. The aim of this study was to evaluate three potential laboratory markers of relapse: intestinal and gastroduodenal permeability and plasma diamine oxidase activity. Intestinal permeability (lactulose/mannitol test), gastroduodenal permeability (urinary sucrose excretion), and postheparin plasma diamine oxidase activity were serially measured in 61 adults with Crohns disease in remission (CDAI <150) for at least 30 days. Subjects were followed periodically for clinical relapse (CDAI >150 and increased by at least 100 points or the need for steroids or surgery). Fourteen patients (23%) relapsed. A cut-off of 0.030 for the lactulose/mannitol ratio was defined. Those with ratios above the cutoff had a 7.0 times greater risk of relapse (p<0.001). Three subjects who went from a normal ratio to an abnormal ratio relapsed, whereas none of 32 subjects with a repeatedly normal ratio relapsed. Sucrose excretion and plasma diamine oxidase activity did not predict relapse. Serial testing of intestinal permeability, but not of gastroduodenal permeability or plasma diamine oxidase activity, was useful in predicting relapse in asymptomatic patients.

Intestinal anaphylaxis in the rat: Mediators responsible for the ion transport abnormalities

Antigen challenge of jejunal epithelium from rats sensitized to egg albumin induces an active Cl− secretory process secondary to release of mucosal mast cell mediators. The present study was designed to define the relative role of these mast cell mediators and the enteric nervous system in the transport abnormalities associated with intestinal anaphylaxis. Net ion transport of stripped jejural tissue from sensitized and sham-treated animals was studied in Ussing chambers. The Cl− secretory response induced by egg albumin during intestinal anaphylaxis was similar to that after addition of 5-hydroxytryptamine (5-HT), histamine, and prostaglandins D2 and E2 to jejunal tissue. Cinanserin, a 5-HT2-receptor antagonist, virtually abolished the response to 5-HT and totally abolished the response to egg albumin. Methysergide, a 5-HT1-receptor antagonist had no effect on either response. Indomethacin, an inhibitor of prostaglandin synthesis, significantly inhibited the 5-HT and egg albumin response. Diphenhydramine, an H1-receptor antagonist and cimetidine, an H2-receptor antagonist both significantly inhibited the histamine response but neither altered the response to egg albumin. Atropine, an anticholinergic, and tetrodotoxin, a nerve blocker, did not inhibit the antigen induced anaphylactic response. These results indicate that 5-HT, acting through 5-HT2 receptors is largely responsible for the transport abnormalities seen in intestinal anaphylaxis induced by egg albumin while prostaglandins appear to play a partial role. The findings do not support a role for the enteric nervous system for the egg albumin induced changes in Cl− secretion.

Role of actin in EGF-induced alterations in enterocyte SGLT1 expression

Na+-glucose cotransporter (SGLT1) expression and the role of actin in epidermal growth factor (EGF)-induced alterations in glucose transport and brush-border surface area were examined in New Zealand White rabbit jejunal loops. In separate experiments, EGF or EGF concurrent with cytochalasin D, an inhibitor of actin polymerization, was administered to the experimental loop and compared with its vehicle control. SGLT1 expression was measured by Western blot in brush-border membrane vesicles (BBMV) after 5-min and 1-h exposure. Glucose kinetics were determined by a rapid filtration technique, and brush-border surface area was examined by electron microscopy after 1-h exposure. The effect of cytochalasin D alone on BBMV glucose kinetics and brush-border surface area was also assessed. EGF resulted in a significant increase in BBMV SGLT1 expression (P < 0.05), glucose maximal uptake (Vmax; P < 0.001), and absorptive brush-border surface area (P < 0.001). These effects were abolished with concurrent cytochalasin D treatment. Cytochalasin D alone had no effect on glucose transport or brush-border surface area. The findings suggest that EGF acutely upregulates jejunal brush-border surface area and the Vmax for jejunal glucose uptake via the recruitment and insertion of SGLT1 from an internal pool into the brush border by a mechanism that is dependent on actin polymerization.Na+-glucose cotransporter (SGLT1) expression and the role of actin in epidermal growth factor (EGF)-induced alterations in glucose transport and brush-border surface area were examined in New Zealand White rabbit jejunal loops. In separate experiments, EGF or EGF concurrent with cytochalasin D, an inhibitor of actin polymerization, was administered to the experimental loop and compared with its vehicle control. SGLT1 expression was measured by Western blot in brush-border membrane vesicles (BBMV) after 5-min and 1-h exposure. Glucose kinetics were determined by a rapid filtration technique, and brush-border surface area was examined by electron microscopy after 1-h exposure. The effect of cytochalasin D alone on BBMV glucose kinetics and brush-border surface area was also assessed. EGF resulted in a significant increase in BBMV SGLT1 expression ( P < 0.05), glucose maximal uptake ( V max; P < 0.001), and absorptive brush-border surface area ( P < 0.001). These effects were abolished with concurrent cytochalasin D treatment. Cytochalasin D alone had no effect on glucose transport or brush-border surface area. The findings suggest that EGF acutely upregulates jejunal brush-border surface area and the V max for jejunal glucose uptake via the recruitment and insertion of SGLT1 from an internal pool into the brush border by a mechanism that is dependent on actin polymerization.

The role of the epidermal growth factor receptor in microbial infections of the gastrointestinal tract

The epidermal growth factor receptor (EGFr) is a transmembrane glycoprotein with an intrinsic tyrosine kinase. Ligand-binding to the EGFr activates cell signaling, phosphorylates protein kinases, and rearranges cytoskeletal proteins - responses that resemble those induced by microbial attachment to cell surfaces, a process known to be mediated by host cell receptors in a number of cases. This article critically reviews the possible role played by the EGFr in microbial colonization, and discusses how modulation of the EGF-EGFr axis may affect infection of the gastrointestinal tract.

The effect of atrial natriuretic peptide on intestinal electrolyte transport

The effect of atrial natriuretic peptide (ANP) on rat small intestinal electrolyte transport was examined. In vivo, intravenous administration of rat ANP(99-126) induced diuresis and natriuresis in conjunction with a significant decrease in intestinal water (basal, 37.1 +/- 5.7 versus ANP 28.5 +/- 6.0 microliters/cm per 20 min, P less than 0.05) and Na+ (4.0 +/- 0.7 versus 2.8 +/- 0.9 mumol/cm per 20 min, P less than 0.05) absorption (n = 9). In vitro, in Ussing chambers, in both jejunum and ileum, addition of 1.0 microM ANP to short circuited, stripped tissue produced a maximal increase in short circuit current and stimulated net Cl- secretion due to a significant increase in the unidirectional serosal to mucosal flux (JCl-sm: jejunum 17.4 +/- 1.3 versus 19.8 +/- 1.3 microEq/cm2 per h, P less than 0.01, n = 6; ileum 13.4 +/- 0.5 versus 17.2 +/- 0.6, P less than 0.01, n = 6) which was inhibited by the calcium channel antagonist verapamil (82 +/- 26%, P less than 0.05) and by the 5-HT2 receptor antagonist cinanserin (72 +/- 44%, P less than 0.05). Guanylate cyclase activity was stimulated by ANP in intact epithelium, but not in isolated crypt and villus enterocytes.

The role of nitric oxide in the regulation of macromolecular transport in rat jejunum.

1. Nitric oxide is known to affect epithelial and microvascular permeability and is a major non‐adrenergic non‐cholinergic neurotransmitter in the intestine. We have previously demonstrated neuronal regulation of macromolecular transport in the intestine. To define this regulation further the role of nitric oxide was investigated. 2. Stripped rat jejunum was mounted in Ussing chambers exposing the mucosal surface to bovine serum albumin (BSA; 2 mg ml‐1), or BSA (2 mg ml‐1) plus [125I]BSA (10 microCi). Following a 50 min equilibration, serosal fluids were sampled for four 10 min periods, and fluxes determined for intact BSA by enzyme‐linked immunosorbent assay (ELISA) and total BSA by [125I]BSA under basal conditions, and after treatment with NG‐nitro‐L‐arginine‐methyl ester (L‐NAME) alone or in conjunction with L‐arginine or decarboxylated molsidomine (SIN 1). 3. L‐NAME significantly increased intact BSA uptake. Total (intact + degraded) BSA flux was not altered. The L‐NAME effect was reversed by L‐arginine and SIN 1. Additional experiments were performed by adding the nitric oxide donors sodium nitroprusside and SIN 1 directly to control tissue. Nitric oxide donors did not further decrease intact BSA flux below levels obtained from control tissue. The L‐NAME enantiomer D‐NAME had no effect. Sodium‐free bathing solutions also had no effect on intact BSA uptake. Non‐specific permeability, as assessed by the serosal to mucosal movement of [51Cr]ethylene‐diamine‐tetraacetate ([51Cr]EDTA), was decreased with L‐NAME. 4. The findings indicate that nitric oxide downregulates intact macromolecular flux in the small intestine.

The effect of TGFα on intestinal solute transport

The effect of transforming growth factor alpha (TGF alpha) and epidermal growth factor (EGF) on 3-O-methylglucose transport was examined in vitro under short-circuited conditions in stripped rabbit jejunum. Mucosal EGF, 60 ng/ml, stimulated a significant increase in net 3-O-methylglucose transport (Jnet 0.67 +/- 0.15 vs. 0.90 +/- 0.15 microEq/cm2/h; P less than 0.03; n = 6) due to an increased mucosal to serosal flux (Jms 1.2 +/- 0.2 vs. 1.5 +/- 0.2 microEq/cm2/h; P less than 0.03). In contrast, TGF alpha, when applied to both mucosal and serosal surfaces at concentrations of either 60 (n = 6) or 150 (n = 9) ng/ml had no effect on either mucosal to serosal (Jms) or net transport (Jnet) of 3-O-methylglucose. TGF alpha did induce a significant increase in the serosal to mucosal flux (Jsm 60 ng/ml 0.44 +/- 0.02 vs. 0.51 +/- 0.03, 150 ng/ml 0.55 +/- 0.03 vs. 0.64 +/- 0.05 microEq/cm2/h; P less than 0.05). When brush border surface area was examined after exposure to either 60 ng/ml TGF alpha or saline vehicle for 2 h in in vivo isolated jejunal loops no significant difference was found (control 53 +/- 1.9; n = 35 vs. TGF alpha 52 +/- 1.9 microns 2; n = 29). Bioactivity of transforming growth factor alpha was assessed by an gastric acid secretion bioassay and found to be intact. These data provide further evidence for separate and distinct functional roles for these peptides in some biological systems.