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The formation of the crypt-villus axis during gut ontogeny requires continued reciprocal interactions between the endoderm and mesenchyme. Epimorphin/syntaxin 2 (epimorphin) is a mesenchymal protein expressed in the fetal gastrointestinal tract during villus morphogenesis. To elucidate its role in gut ontogeny, the epimorphin cDNA was transfected, in sense and antisense orientations, into a rat intestinal myofibroblast cell line, MIC 216. To determine the effects of epimorphin on the epithelium, myofibroblasts were cocultured with the Caco2 cell line. Caco2 cells spread in a simple monolayer over antisense-transfected cells lacking epimorphin. In contrast, sense-transfected myofibroblasts induced Caco2 cells to form compact, round clusters with small lumens. These morphologic differences were preserved in Transwell cocultures in which cell-cell contact was prevented, suggesting that epimorphin's effects were mediated by secreted factor(s). To determine the effects of epimorphin on crypt-villus axis formation in an in vivo model, rat gut endoderm was combined with epimorphin-transfected myofibroblasts and implanted into the chick intracoelomic cavity. The grafts in which epimorphin was overexpressed revealed multiple well-formed villi with crypt-like units, whereas those in which epimorphin expression was inhibited developed into round cystic structures without crypts or villi. Of several potential secreted morphogens, only the expression of bone morphogenetic protein 4 (Bmp4) was increased in the epimorphin-transfected cells. Incubation with noggin partially blocked the transfected myofibroblasts' effects on Caco2 colony morphology. These results indicate that mesenchymal epimorphin has profound effects on crypt-villus morphogenesis, mediated in part by secreted factor(s) including the Bmp's. [ABSTRACT FROM AUTHOR]

After intestinal injury, both the number and type of intestinal epithelial cells must be restored. Intestinal stem cells, located at the base of the intestinal crypt, repopulate the depleted crypt in a process known as compensatory proliferation. In this issue of the JCI, Brown et al. describe a new mechanism by which this process is regulated (see the related article beginning on page 258). Surprisingly, they find that a subset of stromal cells present within the intestinal tissue and expressing the proliferative factor prostaglandin-endoperoxidase synthase 2 (Ptgs2) is repositioned next to the intestinal stem cell compartment where local production of PGE(2) controls injury-induced epithelial cell proliferation.

The mechanisms by which enteropathogenic Escherichia coli (EPEC), an important cause of diarrhea among infants in developing countries, induce symptoms are not defined. EPEC have a type III secretion system required for characteristic attaching and effacing changes that modify the cytoskeleton and apical surface of host cells. Infection of polarized intestinal epithelial cell monolayers by EPEC leads to a loss of transepithelial electrical resistance, which also requires the type III secretion system. We demonstrate here that EspF, a protein that is secreted by EPEC via the type III secretion system, is not required for quantitatively and qualitatively typical attaching and effacing lesion formation in intestinal epithelial cells. However, EspF is required in a dose-dependent fashion for the loss of transepithelial electrical resistance, for increased monolayer permeability, and for redistribution of the tight junction-associated protein occludin. Furthermore, the analysis of EPEC strains expressing EspF-adenylate cyclase fusion proteins indicates that EspF is translocated via the type III secretion system to the cytoplasm of host cells, a result confirmed by immunofluorescence microscopy. These studies suggest a novel role for EspF as an effector protein that disrupts intestinal barrier function without involvement in attaching and effacing lesion formation.

The MHC class I-related Fc receptor, FcRn, mediates the intestinal absorption of maternal IgG in neonatal rodents and the transplacental transport of maternal IgG in humans by receptor-mediated transcytosis. In mice and rats, expression of FcRn in intestinal epithelial cells is limited to the suckling period. We have recently observed, however, clear expression of FcRn in the adult human intestine, suggesting a function for FcRn in intestinal IgG transport beyond neonatal life in humans. We tested this hypothesis using the polarized human intestinal T84 cell line as a model epithelium. Immunocytochemical data show that FcRn is present in T84 cells in a punctate apical pattern similar to that found in human small intestinal enterocytes. Solute flux studies show that FcRn transports IgG across T84 monolayers by receptor-mediated transcytosis. Transport is bidirectional, specific for FcRn, and dependent upon endosomal acidification. These data define a novel bidirectional mechanism of IgG transport across epithelial barriers that predicts an important effect of FcRn on IgG function in immune surveillance and host defense at mucosal surfaces.

P2X purinergic receptor (P2XR) channels bind ATP and mediate Ca(2+) influx--2 signals that stimulate secretory Cl(-) transport across epithelia. We tested the hypotheses that P2XR channels are expressed by epithelia and that P2XRs transduce extracellular ATP signals into stimulation of Cl(-) transport across epithelia. Electrophysiological data and mRNA analysis of human and mouse pulmonary epithelia and other epithelial cells indicate that multiple P2XRs are broadly expressed in these tissues and that they are active on both apical and basolateral surfaces. Because P2X-selective agonists bind multiple P2XR subtypes, and because P2X agonists stimulate Cl(-) transport across nasal mucosa of cystic fibrosis (CF) patients as well as across non-CF nasal mucosa, P2XRs may provide novel targets for extracellular nucleotide therapy of CF.

In the intestine, several growth factors stimulate migration of epithelial cells, contributing to the maintenance of tissue integrity. The Ras-like GTPase Rho regulates a signal transduction pathway linking growth factor receptors to the formation of actin stress fibers and focal adhesions, presumed to be important for motility. Using an in vitro wound-induced migration assay, we have examined the role of Rho GTPases in the migration of IEC-6 and Caco-2 cells, and provide evidence that the Rho GTPases play an essential role in the initial phase of mucosal wound healing. Treatment of the cells with Clostridium difficile toxins A and B, inhibitors of the Rho family GTPases inhibited migration in a dose-dependent fashion. Microinjection of the inhibitory exchange factor Rho-guanine nucleotide dissociation inhibitor (GDI), or Clostridium botulinum C3 ADP-ribosyl transferase (C3) toxin, a Rho-ADP-ribosylating exoenzyme, potently inhibited migration. Microinjection of RhoT19N, a dominant negative form of RhoA, or in vitro ADP-ribosylated RhoA impaired the ability of cells to migrate. Rho-GDI and C3 exoenzyme also inhibited EGF-induced migration of IEC-6 cells. These results demonstrate that Rho is required for endogenous and EGF-induced migration of small intestinal crypt cells, and that Rho proteins are essential elements of a mechanism by which growth factors induce cell migration to restitute mucosal integrity.

Previous studies in the rat have shown that antibodies to gp280, a protein > 200 kD and closely associated with the early endocytic system can induce fetal malformations. Although gp280 is thought to act as a receptor, its ligand(s) is not known. In the current study, we report that purified gp280 from rat kidney, like the intrinsic factor-Cobalamin receptor (IFCR), binds to the intrinsic factor-cobalamin (IFCbl) complex with an association constant of 0.3 x 10(9) M-1 and mediates its internalization. Furthermore, antibodies raised to purified gp280 and IFCR inhibited the binding of IF-[57Co]Cbl complex to intestinal, renal, and yolk sac apical membranes and revealed a single identically sized protein on immunoblotting of the renal membranes. Both antibodies precipitated a single radiolabeled protein > 200 kD from cellular extract from [35S]methionine-labeled yolk sac epithelial cells, and antibody to gp280 inhibited the uptake and internalization of 125IF-Cbl. Immunoelectron microscopy using the two antibodies revealed that in the kidney, both proteins were colocalized. These observations suggest that IF-Cbl complex is a ligand for gp280 and that gp280 and IFCR are identical proteins.

The acute host response to gastrointestinal infection with invasive bacteria is characterized by an accumulation of neutrophils in the lamina propria, and neutrophil transmigration to the luminal side of the crypts. Intestinal epithelial cells play an important role in the recruitment of inflammatory cells to the site of infection through the secretion of chemokines. However, little is known regarding the expression, by epithelial cells, of molecules that are involved in interactions between the epithelium and neutrophils following bacterial invasion. We report herein that expression of ICAM-1 on human colon epithelial cell lines, and on human enterocytes in an in vivo model system, is upregulated following infection with invasive bacteria. Increased ICAM-1 expression in the early period (4-9 h) after infection appeared to result mainly from a direct interaction between invaded bacteria and host epithelial cells since it co-localized to cells invaded by bacteria, and the release of soluble factors by epithelial cells played only a minor role in mediating increased ICAM-1 expression. Furthermore, ICAM-1 was expressed on the apical side of polarized intestinal epithelial cells, and increased expression was accompanied by increased neutrophil adhesion to these cells. ICAM-1 expression by intestinal epithelial cells following infection with invasive bacteria may function to maintain neutrophils that have transmigrated through the epithelium in close contact with the intestinal epithelium, thereby reducing further invasion of the mucosa by invading pathogens.

In this study we tested the hypothesis that peptone in the intestine stimulates the secretion of the CCK-releasing peptide (CCK-RP) which mediates CCK secretion, and examined the enteric neural circuitry responsible for CCK-RP secretion. We used a "donor-recipient" rat intestinal perfusion model to quantify the CCK-RP secreted in response to nutrient stimulation. Infusion of concentrated intestinal perfusate collected from donor rat perfused with 5% peptone caused a 62 +/- 10% increase in protein secretion and an elevation of plasma CCK levels to 6.9 +/- 1.8 pM in the recipient rat. The stimulatory effect of the intestinal washings was abolished when the donor rats were pretreated with atropine or hexamethonium but not with guanethidine or vagotomy. Mucosal application of lidocaine but not serosal application of benzalkonium chloride which ablates the myenteric neurons in the donor rats also abolished the stimulatory action of the intestinal washings. Furthermore, treatment of the donor rats with a 5HT3 antagonist and a substance P antagonist also prevented the secretion of CCK-RP. These observations suggest that peptone in the duodenum stimulates serotonin release which activates the sensory substance P neurons in the submucous plexus. Signals are then transmitted to cholinergic interneurons and to epithelial CCK-RP containing cells via cholinergic secretomotor neurons. This enteric neural circuitry which is responsible for the secretion of CCK-RP may in turn play an important role in the postprandial release of CCK.

The colon, unlike most organs, is normally exposed to high concentrations of ammonia, a weak base which exerts profound and diverse biological effects on mammalian cells. The impact of ammonia on intestinal cell function is largely unknown despite its concentration of 4-70 mM in the colonic lumen. The human intestinal epithelial cell line T84 was used to model electrogenic Cl- secretion, the transport event which hydrates mucosal surfaces and accounts for secretory diarrhea. Transepithelial transport and isotopic flux analysis indicated that physiologically-relevant concentrations of ammonia (as NH4Cl) markedly inhibit cyclic nucleotide-regulated Cl- secretion but not the response to the Ca2+ agonist carbachol. Inhibition by ammonia was 25-fold more potent with basolateral compared to apical exposure. Ion substitution indicated that the effect of NH4Cl was not due to altered cation composition or membrane potential. The site of action of ammonia is distal to cAMP generation and is not due simply to cytoplasmic alkalization. The results support a novel role for ammonia as an inhibitory modulator of intestinal epithelial Cl- secretion. Secretory responsiveness may be dampened in pathological conditions associated with increased mucosal permeability due to enhanced access of lumenal ammonia to the basolateral epithelial compartment.

A spatial segregation of ion transport processes between crypt and surface epithelial cells is well-accepted and integrated into physiological and pathophysiological paradigms of small and large intestinal function: Absorptive processes are believed to be located in surface (and villous) cells, whereas secretory processes are believed to be present in crypt cells. Validation of this model requires direct determination of fluid movement in intestinal crypts. This study describes the adaptation of techniques from renal tubule microperfusion to hand-dissect and perfuse single, isolated crypts from rat distal colon to measure directly fluid movement. Morphologic analyses of the isolated crypt preparation revealed no extraepithelial cellular elements derived from the lamina propria, including myofibroblasts. In the basal state, crypts exhibited net fluid absorption (mean net fluid movement = 0.34 +/- 0.01 nl.mm-1.min-1), which was Na+ and partially HCO3- dependent. Addition of 1 mM dibutyryl-cyclic AMP, 60 nM vasoactive intestinal peptide, or 0.1 mM acetylcholine to the bath (serosal) solution reversibly induced net fluid secretion (net fluid movement approximately -0.35 +/- 0.01 nl.mm-1.min-1). These observations permit speculation that absorption is a constitutive transport function in crypt cells and that secretion by crypt cells is regulated by one or more neurohumoral agonists that are released in situ from lamina propria cells. The functional, intact polarized crypt described here that both absorbs and secretes will permit future studies that dissect the mechanisms that govern fluid and electrolyte movement in the colonic crypt.

Toxin A but not toxin B, appears to mediate intestinal damage in animal models of Clostridium difficile enteritis. The purpose of this study was to investigate the electrophysiologic and morphologic effects of purified C. difficile toxins A and B on human colonic mucosa in Ussing chambers. Luminal exposure of tissues to 16-65 nM of toxin A and 0.2-29 nM of toxin B for 5 h caused dose-dependent epithelial damage. Potential difference, short-circuit current and resistance decreased by 76, 58, and 46%, respectively, with 32 nM of toxin A and by 76, 55, and 47%, respectively, with 3 nM of toxin B, when compared with baseline (P < 0.05). 3 nM of toxin A did not cause electrophysiologic changes. Permeability to [3H]mannitol increased 16-fold after exposure to 32 nM of toxin A and to 3 nM of toxin B when compared with controls (P < 0.05). Light and scanning electron microscopy after exposure to either toxin revealed patchy damage and exfoliation of superficial epithelial cells, while crypt epithelium remained intact. Fluorescent microscopy of phalloidin-stained sections showed that both toxins caused disruption and condensation of cellular F-actin. Our results demonstrate that the human colon is approximately 10 times more sensitive to the damaging effects of toxin B than toxin A, suggesting that toxin B may be more important than toxin A in the pathogenesis of C. difficile colitis in man.

In intact tissue studies, intestinal absorptogogues stimulate NaCl absorption that occurs via the dual operation of Na:H and Cl:HCO3 exchanges on the brush border membrane (BBM) of villus cells. To determine the cellular mechanism of action of an intestinal absorptogogue, the effect of clonidine was determined on Na:H and Cl:HCO3 exchange in rabbit ileal villus and crypt cells. Using 2,7-bis(carboxyethyl)-5,6-carboxy-fluorescein we have previously shown that recovery from an acid load occurs via Na:H exchange, whereas recovery from an alkaline load occurs via Cl:HCO3 exchange in both cells. In villus cells, the rate of recovery from a propionate-induced alkaline load was not altered by clonidine. However, clonidine stimulated recovery from an acid load induced by NH4Cl, Na removal, or amiloride. These data suggest that clonidine stimulates Na:H exchange in villus cells. In crypt cells, the rate of recovery from a propionate-induced alkaline load was also not altered by clonidine. However, in crypt cells, unlike the villus cells, clonidine inhibited recovery from an acid load induced by NH4Cl, Na removal, or amiloride. These data suggest that clonidine inhibits Na:H exchange in crypt cells. Stimulation of Na:H exchange on the BBM of villus cells would be expected to stimulate coupled NaCl absorption (which occurs by coupling of Na:H and Cl:HCO3 exchange). Inhibition of Na:H in crypt cells, known to be present only on the basolateral membrane, will acidify the cell and may inhibit Cl:HCO3 exchange on the BBM, resulting in the inhibition of HCO3 secretion.

Human cancers have an apparent low growth fraction, the bulk of cells presumed to being out of cycle in a G0 quiescent state due to the inability in the past to distinguish G0 from G1 cells. The allosteric M1 subunit of ribonucleotide reductase (M1-RR) is constitutively expressed by cycling cells (i.e., G1, S, G2-M). It is acquired during transition from G0 to G1, lost during exit to G0 and thus distinguishes G0 from G1 cells. To estimate the proportion of G0 and G1 cells in primary human breast (n = 5) and colorectal (n = 12) adenocarcinomas, we used both analytical DNA flow cytometry (ADFC) and immunoperoxidase staining of sections with the monoclonal antibody to M1-RR (MAb M1-RR). ADFC of fresh tumors revealed a low percentage of cells in the S phase (4.0 +/- 3.4%) but immunoperoxidase staining for M1-RR revealed an unexpectedly high proportion of positive cells (52.4 +/- 12.7%) in the G1, S, G2-M phases indicating a high G1 content of primary human tumors. Thus, human cancers are blocked in transition in G1 and are not predominantly in a G0 or quiescent differentiated state. This block was interpreted to mean that human cancers are responding to putative regulatory events at a restriction point in the G1 phase, such as relative growth factor deficiency, density inhibition, antiproliferative cytokines, or gene products. Using flow cytometry for both DNA and M1-RR content we found that human colon cancer cell lines arrest in the G1 but not G0 phase upon serum deprivation or density inhibition. Similarly, human breast cancer cell lines are arrested in G1 but not G0 phase by medroxyprogesterone acetate (MPA) or tamoxifen exposure. These findings match our in situ observations, and support the concept of a restriction point block in primary human tumors.

Proximal duodenal bicarbonate secretion is an important factor in humans and animals protecting the mucosa against acid-peptic damage. This study examined the mechanisms responsible for the central nervous system regulation of duodenal bicarbonate secretion by calcitonin gene-related peptide (CGRP) in unrestrained rats. Cerebroventricular administration of rat CGRP significantly inhibited basal duodenal bicarbonate secretion as well as the stimulatory effects of vasoactive intestinal peptide, neurotensin, a luminal PGE1 analogue, misoprostol, and hydrochloric acid. The inhibitory effects of cerebroventricular CGRP were abolished by ganglionic blockade with chlorisondamine, significantly attenuated by noradrenergic blockade with bretylium, and enhanced by vagotomy. Inhibition of duodenal bicarbonate secretion induced by CGRP coincided with significant increases in plasma norepinephrine (NE) and vasopressin concentrations. The alpha adrenergic receptor antagonist, phentolamine, and the vasopressin V1 receptor antagonist, (1-deaminopenicillamine, 2-[O-methyl]Tyr, 8-Arg)-vasopressin, given intravenously reversed the central inhibitory effect of CGRP by approximately 50% each. Pretreatment of the animals with both phentolamine and the vasopressin antagonist completely abolished the central inhibitory effect of CGRP. Peripheral vasopressin and NE significantly decreased duodenal bicarbonate secretion, and their inhibitory effects were additive and prevented by phentolamine and the vasopressin antagonist, respectively. We conclude that cerebroventricular CGRP inhibits rat duodenal bicarbonate secretion by activation of sympathetic efferents and subsequent release of NE and vasopressin that act on alpha adrenergic and vasopressin receptors, respectively.

Dimethylhydrazine (DMH) is a potent procarcinogen with selectivity for the colon. To determine whether alterations in the lipid composition and fluidity of rat colonic brush border membranes existed before the development of DMH-induced colon cancer, rats were injected s.c. with this agent (20 mg/kg body weight per wk) or diluent for 5, 10, and 15 wk. Animals were killed at these time periods and brush border membranes were prepared from proximal and distal colonocytes of each group. The "static" and "dynamic" components of fluidity of each membrane were then assessed, by steady-state fluorescence polarization techniques using limiting hindered fluorescence anisotropy and order parameter values of the fluorophore 1,6 diphenyl-1,3,5-hexatriene (DPH) and fluorescence anisotropy values of DL-2-(9-anthroyl) stearic acid and DL-12-(9-anthroyl) stearic acid, respectively. Membrane lipids were extracted and analyzed by thin-layer chromatography and gas-liquid chromatography. Phospholipid methylation activity in these membranes was also measured using S-adenosyl-L-methionine as the methyl donor. The results of these studies demonstrate that: the lipid composition and both components of fluidity of proximal DMH-treated and control membranes and their liposomes were similar at all time periods examined; at 5, 10, and 15 wk the "dynamic component of fluidity" of distal DMH-treated membranes and their liposomes was found to be higher, similar, and lower, respectively, than their control counterparts; the "static component of fluidity" of distal DMH-treated membranes and their liposomes, however, was similar to control preparations at all three time periods; and alterations in the lipid composition and phospholipid methylation activities appeared to be responsible for these differences in the "dynamic component of fluidity" at these various time periods.

Eight cases of congenital sucrase-isomaltase deficiency were studied at the subcellular and protein level with monoclonal antibodies against sucrase-isomaltase. At least three phenotypes were revealed: one in which sucrase-isomaltase protein accumulated intracellularly probably in the endoplasmic reticulum, as a membrane-associated high-mannose precursor, one in which the intracellular transport of the enzyme was apparently blocked in the Golgi apparatus, and one in which catalytically altered enzyme was transported to the cell surface. All patients expressed electrophoretically normal or near normal high-mannose sucrase-isomaltase. The results suggest that different, probably small, mutations in the sucrase-isomaltase gene lead to the synthesis of transport-incompetent or functionally altered enzyme which results in congenital sucrose intolerance.

The effects of glucose and fructose on water and sodium absorption in the human jejunum were compared to assess the relative contribution of active and passive sugar stimulation of sodium transport. The effect of fructose is assumed to be entirely passive, and the difference between the effects of fructose and glucose is assumed to be a measure of sugar-stimulated, active sodium absorption. Water and sodium movement with mannitol was the base line. Three sets of test solutions with differing sugar concentrations were studied. Fructose stimulated 66-100 per cent as much net sodium and water absorption as glucose. Fructose stimulated potassium absorption, whereas glucose stimulated potassium secretion. Urea absorption was stimulated by both sugars. Glucose and fructose stimulated sodium absorption when chloride was the major anion, but they had relatively little effect on net sodium movement when chloride was replaced by bicarbonate or sulfate. It is concluded that glucose stimulates passive and active sodium transport in the human jejunum. Stimulated active sodium absorption generates an electrical potential across the mucosa that causes sodium (and potassium) secretion and partly or completely nullifies the effect of active sodium transport on net sodium movement. Net sodium absorption sitmulated by glucose is mainly (66-100 per cent) the passive consequence of solvent flow. The accompanying anion determines the degree to which sugars stimulate sodium absorption (C1 greater than SO-4 greater than HCO3). The effects of bicarbonate and sugars on jejunal sodium absorption are not additive.

To determine the molecular species composition of lecithins of different nascent lipoproteins, high density lipoproteins (HDL), very low density lipoproteins (VLDL), and chylomicrons (CM) were isolated from the mesenteric lymph of rats. Lymph was collected at 0 degrees C with 5,5'-dithiobis-2-dinitrobenzoic acid added to inhibit lecithin-cholesterol acyl transferase. CM were separated by ultracentrifugation and HDL from VLDL by dextran SO4-MG+2 precipitation. Molecular species of lecithin were directly isolated by reverse phase high performance liquid chromatography. In fasted animals, the lecithin compositions of lymph HDL and VLDL were virtually the same and closely resembled the lecithin composition of intestinal mucosa. When bile lecithin was eliminated (by bile diversion), there was a marked change in lecithin composition of all lipoprotein and mucosal samples, which was most notable for a reduction in 16:0-species (which are predominant in bile) and a relative increase in the corresponding 18:0-species. Feeding unsaturated triglycerides (triolein, trilinolein, or a combination of triolein and trilinolein) also resulted in a change in HDL and VLDL lecithin composition. The effect was similar whether bile lecithin was present or eliminated and was notable for a reduction in 16:0-species, an increase in 18:0-species, and the emergence of large amounts of diunsaturated lecithins that corresponded to the fatty acid composition of the triglycerides fed (i.e., 18:1-18:1, 18:2-18:2, and 18:1-18:2 lecithins). When bile-diverted rats were infused via the duodenum with a mix of [14C]choline-labeled lecithins (isolated from the bile of other rats), the incorporation of infused lecithins into different lymph lipoproteins was distinctly different. Individual lecithins were incorporated to a variable extent into each lipoprotein. In fasted rats the specific activities of all major molecular species of lecithin were relatively greater in VLDL than HDL, indicating that HDL derived proportionately more of its lecithins from an endogenous pool than did VLDL. Feeding triolein changed the specific activities of more of the lecithin species of VLDL than of HDL. The specific activities of lecithins in CM were more similar to VLDL than to HDL after triolein feeding. Results thus indicate that, although the lecithins of different mesenteric lymph lipoproteins are similar and may be derived from membrane sites with the same lecithin composition, lecithins incorporated into different lipoproteins originate from different metabolic pools and/or by different mechanisms.

Expression of transforming growth factor alpha (TGF alpha), and transforming growth factor beta (TGF beta) was assessed in isolated primary rat intestinal epithelial cells as well as a rat intestinal crypt cell-derived cell line (IEC-6). A gradient in TGF beta was present, with high concentrations of a 2.5-kb transcript found in undifferentiated crypt cells and progressively lower amounts of the TGF beta transcript in increasingly differentiated villus cell populations. In contrast, the concentration of 4.5-kb TGF alpha transcript was higher in differentiated villus cells than in mitotically active, undifferentiated populations of crypt epithelial cells. The concentrations of transforming growth factors alpha and beta as determined by radioreceptor binding inhibition assay and direct assessment of transforming growth factor biological activity correlated with Northern blot analysis. Although gradients in the expression of the TGFs were present, equivalent binding was observed in the different intestinal cell populations when assessed with 125I-TGF beta and 125I-EGF (TGF alpha). No EGF transcripts were detected in any intestinal cell population, suggesting that the true ligand of the EGF receptor was TGF alpha. IEC-6 cells expressed both TGF alpha and TGF beta transcripts. In addition to the transcripts identified in the primary intestinal cells, this cell line contained an additional larger TGF alpha transcript (4.8 kb) and smaller TGF beta transcripts (2.2 and 1.8 kb). TGF alpha and TGF beta may play a significant role in the regulation of the balance between proliferative and differentiated cell compartments in the intestinal epithelium through both autocrine and paracrine mechanisms.

We used carbon monoxide (CO) as a probe to quantitatively measure intestinal unstirred water layers in vivo. CO has several features that make it uniquely well suited to measure the unstirred layer in that its tight binding to hemoglobin makes uptake diffusion limited, and its relatively high lipid solubility renders membrane resistance negligible relative to the water barriers of the unstirred layer and epithelial cell. The unique application of CO was the measurement of the absorption rate of CO both from the gas phase as well as a solute dissolved in saline. Several lines of evidence showed that a gut stripped free of saline and then filled with gas contained a negligible unstirred layer. Thus, absorption of CO from the gas phase measured resistance of just the epithelial cell. Subtraction of this value from the resistance of CO absorption from saline provided a direct measure of unstirred layer resistance. Studies in the rat showed for a 3-min absorption period that the conventionally calculated apparent unstirred layer for the jejunum was 411 micron and for the colon was 240 micron. However, this conventionally calculated unstirred layer resistance did not truly depict the situation in the rat gut, since there was a continuing depletion of CO from outer surfaces of luminal contents throughout the experiment period. This produced a continually increasing diffusion barrier with time. Calculation of expected absorption rate from unstirred cylinders with the dimensions of the rat gut indicated that there was virtually no stirring in the small intestine and minimal stirring in the colon. The technique described in this paper appears to be simpler and to require fewer assumptions for validity than other techniques previously used to measure unstirred layers in vivo.

Microbial toxins act through cyclic nucleotide dependent (cAMP or cGMP) or cyclic nucleotide independent pathways to cause intestinal ion secretion. To explore the calcium dependent, cyclic nucleotide independent pathway that is postulated to involve protein kinase C activation, we measured protein kinase C activity and phorbol ester binding in isolated intestinal epithelial cells and examined the effects of the C-kinase activators, phorbol myristate acetate, phorbol dibutyrate, and 4-beta-phorbol-12,13-didecanoate, in weaned pig jejunum in vivo. We demonstrated both protein kinase C activity and specific phorbol ester binding in porcine jejunal epithelial cells. Phorbol myristate acetate, phorbol dibutyrate, and 4-beta-phorbol-12,13-didecanoate (10(-5) M) each caused striking secretory responses at 5 h with accumulation of Na+, K+, Cl-, and HCO3- intraluminally. In contrast, 4-alpha-phorbol and 4-alpha-phorbol-12,13-didecanoate, which do not affect protein kinase C, allowed normal net absorption of all electrolytes from the intestinal lumen equivalent to controls with only Ringer's lactate. Time course studies revealed significant secretion within 30 min after exposure to the C-kinase activators. These data suggest an important role for protein kinase C activation in intestinal ion secretion.

The possibility that the autonomic nervous system may influence the function of intestinal mucosa was investigated by assessing the effect of acetyl choline on ion transport in human intestine. Isolated pieces of stripped ileal mucosa were mounted in Perspex flux-chambers and bathed in isotonic glucose Ringer's solution. Acetyl choline caused a rise in mean potential difference (8.8-12.3 mV, P less than 0.002) and short circuit current (287.7-417.2 muA-cm-2, P less than 0.01) (n = 12), observable at a concentration of 0.01 mM and maximal at 0.1 mM. This effect was enhanced by neostigmine and blocked by atropine. Isotopic flux determinations revealed a change from a small mean net Cl absorption (58) to a net Cl secretion (-4.3mueq-cm-2-h-1P less than 0.001) due predominantly to an increase in the serosal to mucosal unidirectional flux of Cl (10.63-14.35 mueq-cm-2-h-1P less than 0.05) and a smaller reduction in the mucosal to serosal flux (11.22 to 10.02 mueq-cm-2-h-1P less than 0.05). Unidirectional and net Na transport was unaffected. A similar electrical and ion transport response was observed in a single study of two pieces of jejunal mucosa. In the absence of glucose net chloride secretion was produced and again an insignificant effect on net sodium transport was noted. Acetyl choline did not provoke a sustained effect on mucosal cyclic adenine nucleotide levels although a short-lived cyclic adenine nucleotide response was seen in some tissues 20-30 s after drug addition. These studies demonstrate that acetyl choline does influence human intestinal ion transport by stimulating chloride secretion and suggest a possible mechanism by which the parasympathetic nervous system could be concerned in the control of ion transport.

THE RATE OF PASSIVE ABSORPTION INTO THE INTESTINAL MUCOSAL CELL IS DETERMINED BY AT LEAST TWO MAJOR DIFFUSION BARRIERS: an unstirred water layer and the cell membrane. This study defines the morphology and permeability characteristics of these two limiting structures. The unstirred water layer was resolved into two compartments: one behaves like a layer of water overlying the upper villi while the other probably consists of solution between villi. The superficial layer is physiologically most important during uptake of highly permeant compounds and varies in thickness from 115 to 334 mum as the rate of mixing of the bulk mucosal solution is varied. From data derived from a probe molecule whose uptake was limited by the unstirred layer, the effective surface area of this diffusion barrier also was determined to vary with stirring rate and equaled only 2.4 cm(2).100 mg(-1) in the unstirred condition but increased to 11.3 cm(2).100 mg(-1) with vigorous mixing. This latter value, however, was still only 1/170 of the anatomical area of the microvillus membrane. With these values, uptake rates for a number of passively absorbed probe molecules were corrected for unstirred layer resistance, and these data were used to calculate the incremental free energy changes associated with uptake of the -CH(2)- (-258 cal.mol(-1)), -OH (+564), and taurine (+1,463) groups. These studies, then, have defined the thickness and area of the unstirred layer in the intestine and have shown that this barrier is rate-limiting for the mucosal uptake of compounds such as fatty acids and cholesterol; in addition, the lipid membrane of the microvillus surface has been shown to be a relatively polar structure.

Pharmacologic doses of corticosteroids impair intestinal calcium absorption and contribute to negative calcium balance. However, the relationship between the impaired calcium absorption and a possible defect in the conversion of vitamin D to its physiologically active form, 1,25-dihydroxyvitamin D, is unknown. We compared fractional calcium absorption (double-isotope method, 100-mg carrier) and serum 25-hydroxyvitamin D (25-OH-D) (Haddad method) in 27 patients receiving pharmacologic doses of prednisone with 27 age-, sex-, and season-matched normal subjects. In patients receiving high daily doses of prednisone (15-100 mg/day), calcium absorption (P < 0.02) and serum 25-OH-D (P < 0.001) were decreased. However, in patients receiving low doses (8-10 mg/day) or high doses (30-100 mg) of prednisone on an alternate-day schedule, both of these parameters were normal. Calcium absorption in the patients treated with daily prednisone correlated inversely with the dose of corticosteroids (r = -0.52, P < 0.025) and, in all steroid-treated patients, correlated directly with serum 25-OH-D (r = 0.58, P < 0.01). In four patients who received high-dose corticosteroid therapy for an average of 4 wk, serum 25-OH-D decreased by 35.5% from pretreatment values. Administration of a physiologic or near-physiologic dose of synthetic 1,25-dihydroxyvitamin D(3) (0.4 mug daily for 7 days) to patients receiving high-dose corticosteroids led to an increase in calcium absorption in all patients. These results suggest that calcium malabsorption in the corticosteroid-treated patients is due to a dose-related abnormality of vitamin D metabolism and not to a direct effect of corticosteroids on depressing transmucosal intestinal absorption of calcium.

Studies were performed in rat small intestine in vivo to determine the effect of saline infusion on intestinal transport of Na(+) and H(2)O. Saline infusion decreased net Na(+) flux (J(n) (Na)) from 12.7 +/-0.8 to 6.4 +/-1.5 muEq/hr per cm in the jejunum when the intestinal perfusate contained both Na(+) and glucose. A similar fall in J(n) (Na) occurred in ileum. When mannitol was substituted for glucose in the perfusate, control absorption decreased 29% in jejunum and 18% in ileum, but saline infusion still caused a decrease in J(n) (Na) quantitatively similar to that seen when glucose was present. When choline was substituted for Na(+) in the perfusate, there was net movement of Na(+) from blood to lumen during control and this net secretion was increased further after saline infusion. These observations suggest that saline infusion has a similar effect to decrease intestinal J(n) (Na) under three widely different conditions of basal sodium transport. Permeability of intestinal mucosa to inulin was very low under basal conditions but increased fivefold after saline infusion, and the unidirectional flux of Na(+) from blood to lumen doubled. This increase in unidirectional flux of Na(+) was greater than the observed decrease in J(n) (Na).Thus, saline infusion decreased net absorption of Na(+) and H(2)O from small intestine through mechanisms which did not appear to be dependent upon the rate of Na(+) flux from lumen to blood, and in association with an increased flux of inulin and Na(+) into the intestinal lumen. The data suggest that the effect of saline infusion to decrease net absorption from the intestine could be due either to an increase in passive permeability of the epithelium which could disrupt solute gradients within the membrane or to an increase in flow of solution into the intestinal lumen.

Pressure-diameter curves of the esophagus were obtained to define its mechanical properties. The mucosal contribution to the strength of the esophagus was negligible until the outer diameter almost doubled, suggesting that small intraluminal pressures are held by the muscle layer alone. For larger deformations mucosal contribution increased and at failure the mucosa held over one-half of the failure pressure of the esophagus. The paths followed during loading and unloading are different and exhibit hysteresis. They are influenced by the rate of pressure change, being more compliant for low rates of pressure change. They are influenced by the history of loading, being different for successive loading-unloading cycles. If enough loading-unloading cycles are repeated a stable loop is reached, which does not change thereafter. Both the mucosa and the whole esophagus show increasing stiffness with increasing pressure. This behavior can be represented by a simple exponential relationship. However, at rapid rates of pressure increases, the esophageal muscles produce sigmoid loading curves, which gradually become exponential when repeating loading.

It has been confirmed that the rabbit vermiform appendix secretes spontaneously at a relatively rapid rate (1-12 ml.h(-1); 1.4+/-0.24 mul.min(-1).cm(-2)). The electrolyte composition is similar to that of ileal fluids and independent of the secretory rate. The transmural potential difference is about 12 mV, mucosa negative. Of the major electrolytes, only HCO(3) (-) is secreted grossly against its electrochemical potential difference. This finding plus the low hydraulic (or osmotic) permeability (L(p)) and high secretory pressures of the organ strongly suggest that the secretion is an active one. The passive permeability to Na(+) and Cl(-) appears to be, at most, somewhat less than for small bowel. Permeability to mannitol was estimated at 2.5 x 10(-7) cm.s(-1). On the basis of reasonable assumptions and results with luminal test solutions of differing osmolarities, it was concluded that (a) the L(p) of the appendiceal epithelium is in the lower range of values reported for small bowel and colon; (b) the L(p) is higher for osmotic absorption than for osmotic secretion; and (c) the rate of spontaneous secretion is insensitive to luminal anisotonicity over a wide range of values. But sufficiently hypotonic solutions can reverse net secretion to net absorption, more by inhibiting spontaneous secretion than increasing osmotic absorption. The rabbit vermiform appendix appears to be a useful model for the elucidation of intestinal secretory processes.

The effects of cholera enterotoxin on intestinal ion transport were examined in vitro. Addition of dialyzed filtrate of Vibrio cholerae (crude toxin) to the luminal side of isolated rabbit ileal mucosa caused a delayed and gradually progressive increase in transmural electric potential difference (PD) and shortcircuit current (SCC). A similar pattern was observed upon addition of a highly purified preparation of cholera toxin, although the changes in PD and SCC were smaller. Na and Cl fluxes across the short-circuited mucosa were determined with radioisotopes 3-4 hr after addition of crude toxin or at a comparable time in control tissues. The toxin caused a net secretory flux of Cl and reduced to zero the net absorptive flux of Na. Similar flux changes were observed when either crude or purified toxin was added in vivo and tissues were mounted in vitro 3-4 hr later. Additon of D-glucose to the luminal side of toxin-treated mucosa produced a large net absorptive flux of Na without altering the net Cl and residual ion fluxes. Adenosine 3',5'-cyclic phosphate (cyclic AMP) and theophylline had previously been shown to cause a rapid increase in SCC and ion flux changes similar to those induced by cholera toxin. Pretreatment of ileal mucosa with either crude or purified cholera toxin greatly reduced the SCC response to theophylline and dibutyryl cyclic AMP, which, together with the flux data, suggest that both cyclic AMP and cholera toxin stimulate active secretion by a common pathway. Inhibition of the SCC response to theophylline was observed after luminal but not after serosal addition of toxin. In vitro effects of cholera toxin correlated closely with in vivo effects: heating toxin destroyed both; two V. cholerae filtrates which were inactive in vivo proved also to be inactive in vitro; PD and volume flow measurements in isolated, in vivo ileal loops of rabbit revealed that the PD pattern after addition of toxin is similar to that seen in vitro and also correlates closely with changes in fluid movement. The results suggest that stimulation by cholera toxin of a cyclic AMP-dependent active secretory process of the intestinal epithelial cells is a major cause of fluid loss in cholera.

Jejunal calcium absorption was measured from test solutions containing 1.0, 2.5, 5, and 10 mM calcium (as calcium gluconate). Absorption rates increased progressively as luminal calcium concentration was increased, although there was a tendency toward saturation of the absorptive process at the higher concentrations. Calcium absorption was higher in normal young adults than in normal subjects over age 60. In both groups a 300 mg calcium diet for 4-8 wk enhanced calcium absorption relative to absorption rates after 4-8 wk on a 2,000 mg calcium diet. This adaptation was more definite and dramatic in the young than in the old subjects. Indirect estimates suggest that adaptation to a low calcium diet and the higher absorption in young than old normal subjects are mediated by an increased V(max) rather than a decreased K(m).