IL-1 activates two phospholipid signaling pathways in intestinal epithelial cells.

OBJECTIVE AND DESIGN: the aim of the study was to decipher the molecular signals involved in IL-I's action on intestinal epithelial cells (IEC). MATERIALS AND METHODS: Mode-K cells, used as a model of IEC, were treated with IL-I, and PLA2 activity and PGE2, ceramide, and cyclooxygenase-2 (COX-2) levels were measured using enzyme-immuno-assay kit, EIA, thin-layer chromatography and western blotting assays respectively. RESULTS: IL-I caused a concentration- and time-dependent increase in PLA2 activity (3-fold increase), in ceramide levels (peak increase = 10.5 +/- 0.9 pmol/nmol phosphate), and in COX-2 and PGE2 levels. PGE2 increase was biphasic with an early peak at 10 min (around 5 ng/mg protein) due to increased PLA2 activity. The later peak (13.1 +/- 1.9 ng/mg protein) at 4 h was due to COX-2 induction. CONCLUSION: In conclusion, these findings demonstrate that IL-I regulates IEC function through two pathways, the PLA2 and the sphingomyelin pathways, both of which are capable of modulating the inflammatory process.

Ion transport across the cecum in normal and colitic mice.

This study was conducted to determine the ion transport mechanisms in the normal mouse cecum and compare them to an inbred mouse model of colitis. The Ussing chamber-voltage clamp technique was used to monitor the short circuit current (I(sc)). The basal I(sc) in the normal cecum was 82.6 +/- 5.8 microA/cm2. It was not affected by bumetanide, 9-anthracene carboxylate, amiloride, and phenamil or by removal of Cl- ions; but was abolished by the removal of Na+ ions. Flux measurements revealed the presence of neutral NaCl transport. In the colitic cecum, the basal current was significantly higher than the normal cecum. Basal current in the normal cecum was due primarily to Na+ absorption through a Na+ channel, while in the colitic cecum it was due to Cl- ion secretion. cAMP addition in colitic cecum did not increase Cl- secretion, further suggesting that the tissue is already secreting at a maximal rate.

Morphological and functional changes in the colonic epithelial cells in a rabbit model of colitis.

A rabbit model of TNBS-colitis was used to study the effect of intestinal inflammation on epithelial cell function. Epithelial cells were isolated using a non-enzymatic isolation method without any apparent contamination with infiltrating immune cells. The isolated cells were found to be viable using dye exclusion studies, unidirectional Na+ -fluxes, proliferation assays and morphological studies. The cells, however, showed morphological changes that suggested the presence of increased number of secretory vesicles. This increase correlated well with the increase observed in ion and water secretion as measured by the short-circuit current. Finally, in the colitic tissue the number of PGE2 receptors was greatly reduced with no changes observed in the affinity of PGE2 to its receptor. The reduced number of PGE2 receptors might be due to sensitization of the receptor. In conclusion, we have demonstrated that morphologically and functionally normal epithelial cells can be isolated from the rabbit inflamed distal colon.

The mechanisms of action of interleukin-1 on rabbit intestinal epithelial cells.

Interleukin-1 (IL-1) is an inflammatory mediator that increases Cl- secretion in intestinal epithelial cells. To identify the signal transduction pathway(s) involved in IL-1's action, cells were treated with IL-1 and the levels of cyclooxygenase (COX) enzymes, prostaglandin E2 (PGE2) and phospholipase A2-activating protein (PLAP), and the activity of phospholipase A2 (PLA2) were measured. IL-1 caused concentration- and time-dependent increases in the levels of PLA2 activity, and/or in the levels of PLAP, COX-2 and PGE2. The IL-induced increase in PGE2 levels was biphasic, with the first peak due to the increase in PLAP levels, and the second peak due to the increase in COX-2 levels. This increase in PGE2 levels may provide a mechanism for acute and chronic inflammation in the intestine.

Gap junctional communication between murine macrophages and intestinal epithelial cell lines.

In intestinal inflammation, inflammatory cells infiltrate the submucosa and are found juxtaposed to intestinal epithelial cell (IEC) basolateral membranes and may directly regulate IEC function. In this study we determined whether macrophage (M phi), P388D1 and J774A.1, are coupled by gap junctions to IEC lines, Mode-K and IEC6. Using flow cytometric analysis, we show bi-directional transfer of the fluorescent dye, calcein (700 Da) between IEC and M phi resulting in a 3.5-20-fold increase in recipient cell fluorescence. Homocellular and heterocellular dye transfer between M phi and/or IEC was detected in cocultures of P388D1, J774A.1, Mode-K, IEC6 and CMT93. However, transfer between P388D1 and Mode-K was asymmetrical in that transfer from P388D1 to Mode-K was always more efficient than transfer from Mode-K to P388D1. Dye transfer was strictly dependent on IEC-M phi adhesion which in turn was dependent on the polarity of IEC adhesion molecule expression. Both calcein dye transfer and adhesion were inhibited by the addition of heptanol to cocultures. Furthermore we demonstrate both IEC homocellular, and M phi-IEC heterocellular propagation of calcium waves in response to mechanical stimulation, typical of gap junctional communication. Finally, areas of close membrane apposition were seen in electron micrographs of IEC-M phi cocultures, suggestive of gap junction formation. These data indicate that IEC and M phi are coupled by gap junctions suggesting that gap junctional communication may provide a means by which inflammatory cells might regulate IEC function.

Regulation of electrolyte transport by nitric oxide in the mouse cecum.

The effect and role of nitric oxide (NO) in the regulation of ion transport in the mouse cecum were investigated. L-arginine, used to increase NO production, increased short-circuit current (Isc), a measure of ion transport, in a concentration-dependent manner with a maximal increase of 193.8+/-65.5 microA/cm2. This increase was not changed in Cl-- or HCO3--free buffers, but was significantly decreased in Na+-free buffer. Using immunohistochemistry, the constitutive form of nitric oxide synthase was found not to be different in the inflamed cecum. The inducible form of the enzyme, however, which was absent in the cecum of normal mice, was present in high levels in the cecum of the colitic mouse. These results suggest that NO causes an increase in Na+ absorption. The increased levels of inducible NO synthase in the inflamed cecum suggest a role for NO in the pathophysiology of inflammatory bowel disease.

Adhesion and cytosolic dye transfer between macrophages and intestinal epithelial cells.

Activated macrophages (M phi) found in the intestinal lesions of patients with inflammatory bowel disease (IBD) secrete many inflammatory mediators which can regulate intestinal epithelial cell (IEC) function. However, little is known about direct M phi-IEC interactions. Two potential mechanisms by which cells may interact are through specific receptor-ligand binding of adhesion molecules, such as integrins or cadherins, and by exchange of cytoplasmic substances through transmembraneous channels called gap junctions. We investigated whether M phi could adhere to epithelial cells in culture and form transmembrane communication channels as defined by dye transfer. Primary cultures of murine M phi and a M phi cell line, P388D1, adhered to Mode-K and IEC6, but not CMT-93 IEC. Antibody blocking studies determined that P388D1-Mode-K binding was partially dependent on beta 2 integrin (CD18) function, Mode-K constitutively expressed CD106 (VCAM-1) and cell associated fibronectin, while P388D1 expressed low levels of CD49d/CD29 (VLA4) but blocking antibodies to these surface molecules did not inhibit P388D1-Mode-K adherence. Transfer of calcein dye from M phi to IEC was quantitated by flow cytometry and was dependent on M phi-IEC adhesion. Dye transfer was concentration dependent in that the fluorescence intensity of Mode-K was proportional to the number of adherent P388D1 cells as well as the dye load of the M phi. These results indicate that M phi interact with IEC by adhesion and possibly through gap junctions and may thus regulate IEC function by direct cell-cell communication.

Regulation of ion transport by histamine in mouse cecum.

Histamine levels are elevated in inflammatory bowel disease. We investigated the mechanism by which histamine affects electrolyte transport in the mouse cecum. Using the Ussing-chamber voltage clamp technique, histamine was found to cause a transient concentration-dependent increase in short-circuit current, a measure of total ion transport across the epithelial tissue. This increase was not affected by amiloride pretreatment, but was significantly inhibited by bumetanide and completely inhibited when chloride was substituted in the bathing buffer by gluconate. A histamine-induced increase in short-circuit current was also significantly reduced by inhibitors of the cyclooxygenase pathway indicating the involvement of prostaglandin E2 in its action. Prostaglandin E2 levels were increased in histamine treated tissue and this increase was reversed by indomethacin. These data suggest that histamine causes its effect on mouse cecum largely through increasing arachidonic acid metabolism resulting in increased levels of prostaglandins which in turn increase Cl- secretion in the epithelial cells.

IL-1 beta induces synthesis of phospholipase A2-activating protein in rabbit distal colon.

In inflammatory bowel disease, the colonic mucosa is infiltrated by inflammatory cells that secrete a variety of inflammatory mediators such as interleukin-1 beta (IL-1 beta). IL-1 beta caused a delayed increase in Cl- secretion and in prostaglandin E2 (PGE2) release in rabbit distal colon. Both of these effects were abolished with cycloheximide, implying a role for protein synthesis in mediating IL-1 beta's effect. With the use of Western blot assays, the protein was identified as the phospholipase A2 (PLA2)-activating protein (PLAP). IL-1 beta caused a concentration-dependent and a time-dependent increase in PLAP levels as well as in PLA2 activity, with the maximal increase observed at an IL-1 beta concentration between 10 and 30 ng/ml reached in 2-10 min. The PLAP mRNA levels were also regulated by IL-1 beta with a similar time course. PLAP is constitutively present in the epithelial cells and in the subepithelial layer of the distal colon. These findings suggest a direct effect of IL-1 beta on intestinal epithelial cells to cause an increase in PLAP levels and phospholipase A2 activity and subsequent increase in PGE2 levels.

Characterization of PGE2 receptors in isolated rabbit colonic crypt cells.

The physiological effects of prostaglandins (PG) are mediated through their interactions with specific receptors on effector cells. In this study the properties of PGE2 receptors in the rabbit distal colon were examined. We report the presence of specific, saturable, and high-affinity binding sites of PGE2 of the EP2 subtype in isolated colonic crypts. Scatchard analysis revealed the presence of two binding sites with dissociation constants of 0.3 and 10.8 nM and corresponding maximum number of receptors of 15 and 134 fmol/10(6) cells. From competition experiments in the presence of guanosine 5'-O-(3-thiotriphosphate), PGE2 binding was decreased, suggesting that the receptor is coupled to a G protein. No PGE2 binding sites were detected in surface cells. Levels of adenosine 3',5'-cyclic monophosphate (cAMP) were measured in isolated epithelial cells after being exposed to different concentrations of PGE2. cAMP levels were significantly increased only in the crypt cells when exposed to PGE2. These data provide the first demonstration for the existence of PGE2 receptors on colonic crypt cells, which when activated lead to increased levels of cAMP.

Separation of pure populations of epithelial cells from rabbit distal colon.

A simple method using divalent chelators is described for the isolation of viable populations of surface and crypt cells from rabbit distal colon. Histological studies were performed to monitor colonocyte dissociation and determine contamination by nonepithelial cells. Cell viability was assessed by trypan blue exclusion assay and by 22Na uptake measurements. Electron microscopy was used to determine the integrity of the isolated cells. Alkaline phosphatase and [3H]thymidine uptake were measured to assess the purity of the different cell fractions. Combined fractions 4 and 5 contained the highest percentage of pure surface cells, while fractions 10, 11, and 12 were predominantly crypts. Alkaline phosphatase activity was 13 +/- 3-fold higher in the surface cells than in the crypt cells, while [3H]thymidine uptake was 8 +/- 4-fold higher in the crypt cells than in the surface cells. Amiloride-sensitive and -insensitive 22Na uptake was the same in the surface cells directly after isolation and after 3 h in culture. In this study we demonstrate a method for the preparation of highly enriched fractions of rabbit colon surface and crypt cells that remain viable and functional in short-term culture.

Regulation of electrolyte transport with IL-1beta in rabbit distal colon.

Interletrkin-1beta levels are elevated in inflammatory bowel disease. In this study the mechanism by which interleukin-1beta affects electrolyte transport in the rabbit distal colon, was investigated. Interleukin-1beta caused a delayed increase in short-circuit current (I(sc)) which was attributed to protein synthesis since the effect was inhibited by cycloheximide. The interleukin-1beta induced increase in I(sc) was not affected by amiloride treatment but was completely inhibited by bumetanide or in chloride-free buffer and by indomethacin. Prostaglandin E(2) levels increased in tissue treated with interleukin-1beta, but this increase was reversed by cycloheximide. These data suggest that interleukin-1beta causes its effect via a yet to be identified second messenger, by increasing chloride secretion through a prostaglandin E(2) mediated mechanism.

Regulation of ileal electrolyte transport by K+ channel activators.

Pinacidil (N"-cyano-N-4-pyridyl-N'-1,2,2-trimethylpropylguanidine monohydrate) and BRL 38227, a benzopyran derivative, two K+ channel activators, were found to decrease short-circuit current (ISC), a measure for ion movement across the intestinal tissue. This decrease in ISC was correlated with an increase in NaCl absorption. These results suggest the possibility of new forms of drug therapy for diarrheal diseases. The effects of pinacidil were compared to galanin which also increased NaCl absorption. Galanin increased potassium currents in whole-cell patch clamp studies. The effects of galanin and pinacidil on Isc were not additive suggesting a common pathway in their mechanism of action.

Effects of galanin on short circuit current and electrolyte transport in rabbit ileum.

Galanin decreased short circuit current (Isc) and increased active Na+ and Cl- absorption in rabbit ileum. In the absence of calcium, the galanin-induced decrease in Isc was inhibited by approximately 60%. Tetrodotoxin significantly reduced the effect of galanin on Isc, and tetrodotoxin and EGTA totally blocked the effect, indicating that the nonneuronal mediator of the effect is Ca2+ dependent. Galanin binding to basolateral membranes prepared from ileal epithelial cells was specific and of high affinity. These results suggest the involvement of this peptide in the regulation of intestinal epithelial cell function.

Electrolyte transport in piglets infected with transmissible gastroenteritis virus. Stimulation by verapamil and clonidine.

The effects of clonidine, an alpha 2-adrenergic agonist, and verapamil, a Ca2+ channel blocker, on Na+ and Cl- absorption were studied in stripped jejunal mucosa from control and transmissible-gastroenteritis-virus-infected piglets. All infected piglets developed severe diarrhea 18-24 hours after oral inoculation. Jejunum from infected animals, as compared with control jejunum, had decreased mucosal-to-serosal, serosal-to-mucosal, and net Na+ and Cl- fluxes. Clonidine and verapamil caused a decrease in short-circuit current and stimulation of Na+ and Cl- absorption in control jejunum. In infected piglets, although the jejunum exhibited severe villus atrophy, both drugs stimulated Na+ and Cl- absorption and the magnitude of Na+ and Cl- absorption was similar in control and transmissible-gastroenteritis-infected jejunum. In contrast, D-glucose stimulated Na+ absorption, and the decrease in short-circuit current caused by verapamil and clonidine, were decreased in transmissible-gastroenteritis-infected jejunum. Such pharmacological stimulation of Na+ and Cl- absorption might be useful in the management and treatment of certain viral diarrheal diseases.

Galanin inhibits a dihydropyridine-sensitive Ca2+ current in the RINm5f cell line.

Mechanisms of action of the neuropeptide galanin, a putative neuromodulator in the central and peripheral nervous systems, have been evaluated extensively in insulin-secreting cells isolated from pancreas and cell lines derived from pancreatic tumors. Galanin inhibits insulin secretion from these cells through several mechanisms, including activation of ATP-dependent K+ channels and inhibition of adenylyl cyclase leading to a decrease in cAMP. Here we report that galanin also inhibits a dihydropyridine-sensitive Ca2+ current. Both electrophysiological actions by galanin would result in less Ca2+ entry, as the action to increase K+ current would hyperpolarize the cells and the decrease in voltage-gated Ca2+ current would decrease Ca2+ influx at depolarized potentials where these channels are activated. These galanin actions would directly counter the two opposing electrophysiological responses to carbohydrate stimulation in RINm5f cells, which are to inhibit K+ current and to stimulate Ca2+ current. Given that stimulation of presynaptic nerve terminals in pancreas releases galanin, these results suggest that Ca(2+)-dependent insulin release from native pancreatic beta cells may also be regulated by similar neuropeptide effects.

Two calcium channels in basolateral membranes of rabbit ileal epithelial cells.

The actions of three different types of calcium channel blockers on short-circuit current (Isc) in rabbit ileum were studied. These included the phenylalkylamines, verapamil and (l)-desmethoxyverapamil (D888); the dihydropyridines, nifedipine and nitrendipine; and the benzothiazepine, diltiazem. All of the drugs decreased Isc, a change associated with increased Na and Cl absorption. Verapamil and D888 had the largest effects. The dihydropyridine, BAY K 8644, a calcium channel activator, increased Isc and decreased Na and Cl absorption, effects not inhibited by tetrodotoxin. The phenylalkylamines had an additional effect on Isc in the presence of a maximally inhibitory concentration of the dihydropyridines, suggesting the possibility of two distinct calcium channels, one of which is the L-type voltage-activated, dihydropyridine- and phenylalkylamine-sensitive channel, and the other is a channel only sensitive to phenylalkylamines but not to dihydropyridines. [3H]nitrendipine and [3H]D888 binding to an enriched preparation of basolateral membranes from ileal epithelial cells was characterized. Each ligand bound specifically and saturably to an apparently single population of high-affinity sites with [3H]D888 having three times as many binding sites as [3H]nitrendipine. [3H]nitrendipine binding was partially inhibited by verapamil and D888 and was increased by diltiazem; whereas [3H]D888 binding was inhibited completely by verapamil but only partially by nitrendipine and diltiazem. These transport and binding studies suggest the presence of two types of Ca2+ channels in ileal epithelial cells, one of which interacts with the dihydropyridines, the phenylalkylamines, and the benzothiazepines at three different sites and the other channel that only binds the phenylalkylamines.

Ca2+ channel blockers interact with alpha 2-adrenergic receptors in rabbit ileum.

An interaction between Ca2+ channel blockers and alpha 2-adrenergic receptors has been demonstrated in rabbit ileum by studying the effect of clonidine on active electrolyte transport, under short-circuited conditions, in the presence and absence of several Ca2+ channel blocking agents. Clonidine, verapamil, diltiazem, cadmium, and nitrendipine all decrease short-circuit current and stimulate NaCl absorption to different extents with clonidine having the largest effect. Exposure to verapamil, diltiazem, and cadmium inhibited the effects of clonidine on transport, whereas nitrendipine had no such effect. Verapamil, diltiazem, and cadmium, but not nitrendipine, also decreased the specific binding of [3H]alpha 2-adrenergic agents to a preparation of ileal basolateral membranes explaining the observed decrease in the transport effects of clonidine. The effective concentrations of the Ca2+ channel blockers that inhibited the effects of clonidine on transport were fairly similar to the concentrations needed to inhibit its specific binding. The displacement of clonidine by calcium channel blockers is ascribed to a nonspecific effect of these agents, although the possibility that their effects are exerted via their binding to the calcium channels is not excluded.

Red cell morphology in alcoholics: a new test for alcohol abuse.

Scanning electron microscopy has shown that the blood of alcoholics contains a large number of morphologically abnormal red cells. In two groups of alcoholics, the number of morphologically abnormal red cells ranged from 23.1% to 89.3% and 27.4% to 57.3% of total red cells compared to values in healthy controls of 4.5%-12.6% and 27.7%-79.5% in nonalcoholic liver disease patients. A characteristic finding was the presence of triangulocytes: these ranged from 1.2% to 18.0% of total red cells in the alcoholics as compared to 0-0.5% in healthy controls, and 0-1.3% in patients with nonalcoholic liver disease. The presence of elevated numbers of triangulocytes in blood appears to be specific to alcohol abuse. It is not, for example, elevated in nonalcoholic liver disease. No correlation was found between the number of triangulocytes or the number of morphologically abnormal red cells in blood and either the duration of alcohol abuse or the amount of alcohol consumed. Both parameters tended, however, to return to normal values during withdrawal. The mechanism by which alcohol abuse causes the morphologic abnormalities is not known. Preliminary in vitro experiments indicate that it is unlikely to arise as an effect of alcohol on circulating red cells. Based on the data presented, the measurement of the number of triangulocytes in a blood sample, although slow and laborious, may provide a highly specific test for alcohol abuse.