Lateral intercellular space volume as a determinant of CFTR-mediated anion secretion across small intestinal mucosa

Studies of full-thickness, small intestinal preparations have shown that maximal anion secretion [indexed by short-circuit current (I(sc))] during intracellular cAMP (cAMP(i)) stimulation is transient and followed by a decline toward baseline. Declining I(sc) is preceded by decreases in transepithelial conductance (G(t)), which in the small intestine reflects the lateral intercellular space (LIS) volume of the paracellular pathway. We hypothesized that decreases in LIS volume limit the magnitude and duration of cAMP(i)-stimulated anion secretion. Experimental manipulations to increase the patency of the LIS (assessed by G(t) and electron microscopy) were investigated for an effect on the magnitude of cAMP(i)-stimulated anion secretion (assessed by the I(sc) and isotopic fluxes) across murine small intestine. In control studies, changes of G(t) after cAMP(i) stimulation were associated with a morphological "collapse" of the LIS, which did not occur in intestine of CFTR-null mice. Removal of the outer intestinal musculature, exposure to a serosal hypertonic solution, or increased serosal hydrostatic pressure minimized reductions in G(t) and increased the cAMP(i)-stimulated I(sc) response. Increased I(sc) primarily resulted from increased Cl(-) secretion that was largely bumetanide sensitive. However, bumetanide-insensitive I(sc) was also increased, and similar increases occurred in the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1)-null intestine, indicating that activities of non-NKCC1 anion uptake proteins are also affected by LIS volume. Thus LIS patency is an important determinant of the magnitude and duration of CFTR-mediated anion secretion in murine small intestine. Decreases in LIS volume may limit the pool of available anions to basolateral transporters involved in transepithelial secretion.     

Effect of nitric oxide on electrolyte transport across the porcine proximal colon

The effect of nitric oxide (NO) on ion transport in the porcine proximal colon was investigated in slide-stripped epithelia mounted in Ussing chambers. The serosal addition of the NO-donors sodium nitroprusside (SNP, 0.5 mM) or S-nitroso-N-acetylpenicillamine (SNAP, 0.5 mM) induced a steep increase of short-circuit current ( I(sc)). The stimulatory effect of SNP on I(sc) could not be blocked by piroxicam or tetrodotoxin. Potassium channel inhibitors (quinidine, tetraethylammonium or barium) added serosally reduced the SNP- or SNAP-induced increases of I(sc). In chloride-free solutions, the SNP-induced increase of I(sc) was smaller than in chloride-containing solutions. Cl(- )and Na(+) flux measurements demonstrated that SNP diminished Cl(-) and Na(+) net absorption. Pre-treatment with barium was able to block the inhibitory effect of SNP on NaCl net absorption totally. NO effects on paracellular pathways were assessed by measuring flux rates of [(14)C]-D-mannitol. SNP did not change unidirectional D-mannitol flux rates. In conclusion, NO inhibits NaCl net absorption in the proximal colon of pigs by acting directly on the enterocyte. The antiabsorptive (and/or prosecretory) effect of NO depends on a functional basolateral potassium conductance.     

Site-specific regulation of ion transport by prolactin in rat colon epithelium

The effect of prolactin (PRL) on ion transport across the rat colon epithelium was investigated using Ussing chamber technique. PRL (1 μg/ml) induced a sustained decrease in short-circuit current (I(sc)) in the distal colon with an EC(50) value of 100 ng/ml and increased I(sc) in the proximal colon with an EC(50) value of 49 ng/ml. In the distal colon, the PRL-induced decrease in I(sc) was not affected by Na(+) channel blocker amiloride or Cl(-) channel blockers, NPPB, DPC, or DIDS, added mucosally. However, the response was inhibited by mucosal application of K(+) channel blockers glibenclamide, quinidine, and chromanol 293B, whereas other K(+) channel blockers, Ba(2+), tetraethylammonium, clotrimazole, and apamin, failed to have effects. The PRL-induced decrease in I(sc) was also inhibited by Na(+)-K(+)-2Cl(-) transporter inhibitor bumetanide, Ba(2+), and chromanol 293B applied serosally. In the transverse and proximal colon, the PRL-induced increase in I(sc) was suppressed by DPC, glibenclamide, and bumetanide, but not by NPPB, DIDS, or amiloride. The PRL-induced changes in I(sc) in both distal and proximal colon were abolished by JAK2 inhibitor AG490, but not BAPTA-AM, the Ca(2+) chelating agent, or phosphatidylinositol 3-kinase inhibitor wortmannin. These results suggest a segment-specific effect of PRL in rat colon, by activation of K(+) secretion in the distal colon and activation of Cl(-) secretion in the transverse and proximal colon. Both PRL actions are mediated by JAK-STAT-dependent pathway, but not phosphatidylinositol 3-kinase pathway or Ca(2+) mobilization. These findings suggest a role of PRL in the regulation of electrolyte transport in mammalian colon.     

Cytokine-induced inhibition of insulin release from mouse pancreatic beta-cells deficient in inducible nitric oxide synthase

Cytokines may participate in islet destruction during the development of type 1 diabetes. Expression of inducible nitric oxide synthase (iNOS) and subsequent NO formation induced by IL-1 beta or (IL-1 beta + IFN-gamma) may impair islet function in rodent islets. Inhibition of iNOS or a deletion of the iNOS gene (iNOS -/- mice) protects against cytokine-induced beta-cell suppression, although cytokines might also induce NO-independent impairment. Presently, we exposed wild-type (wt, C57BL/6 x 129SvEv) and iNOS -/- islets to IL-1 beta (25 U/ml) and (IL-1 beta (25 U/ml) + IFN-gamma (1000 U/ml)) for 48 h. IL-1 beta and (IL-1 beta + IFN-gamma) induced a significant increase in NO formation in wt but not in iNOS -/- islets. Both IL-1 beta and (IL-1 beta + IFN-gamma) impaired glucose-stimulated insulin release and reduced the insulin content of wt islets, while (IL-1 beta + IFN-gamma) reduced glucose oxidation rates and cell viability. IL-1 beta exposure to iNOS -/- islets impaired glucose-stimulated insulin release, increased insulin accumulation and reduced the insulin content, without any increase in cell death. Exposure to (IL-1 beta + IFN-gamma) had no effect on iNOS -/- islets except reducing the insulin content. Our data suggest that IL-1 beta may inhibit glucose-stimulated insulin release by pathways that are not NO-dependent and not related to glucose metabolism or cell death.     

A schistosome-expressed immunomodulatory glycoconjugate expands peritoneal Gr1(+) macrophages that suppress naive CD4(+) T cell proliferation via an IFN-gamma and nitric oxide-dependent mechanism

Lacto-N-fucopentaose III (LNFPIII) is found in human milk and on the Th2 driving helminth parasite Schistosoma mansoni. This pentasaccharide drives Th2-type responses in vivo and in vitro when conjugated to a carrier. In an attempt to further understand early events in Th1 to Th2 switching, we examined phenotypic and functional changes in peritoneal cell populations in BALB/c and SCID mice following LNFPIII-dextran injection. We found that i.p. injection with LNFPIII-dextran resulted in rapid (<20 h) expansion of the Gr1(+) subpopulation of F4/80(+)/CD11b(+) peritoneal cells, comprising up to 75% of F4/80(+)/CD11b(+) peritoneal cells compared with 18% in uninjected or dextran-injected mice. Functionally, these cells suppressed anti-CD3- and anti-CD28-induced proliferation of naive CD4(+) T cells. LNFPIII-dextran also expanded functional Gr1(+) suppressor macrophages in SCID mice, demonstrating that expansion and function of suppressor cells did not require T cells. Suppression in both BALB/c and SCID mice was NO and IFN-gamma dependent, as addition of inhibitors of inducible NO synthase (N(G)-monomethyl-L-arginine), as well as anti-IFN-gamma Abs, restored the ability of CD4(+) T cells to proliferate in vitro. Depletion of the F4/80(+) subset of Gr1(+) cells eliminated the suppressive activity of peritoneal exudate cells showing that these cells were macrophages. Thus, LNFPIII-dextran rapidly expands the Gr1(+) suppressor macrophage population in the peritoneal cavities of otherwise naive mice. These Gr1(+) cells suppress proliferation of naive CD4(+) T cells in an NO-dependent mechanism, and may play a regulatory role in the switching of Th1- to Th2-type responses.     

Effect of luminal acidity on the apical cation channel in rabbit esophageal epithelium

Esophageal epithelial cells contain an apical cation channel that actively absorbs sodium ions (Na(+)). Since these channels are exposed in vivo to acid reflux, we sought the impact of high acidity on Na(+) channel function in Ussing-chambered rabbit epithelium. Serosal nystatin abolished short-circuit current (I(sc)) and luminal pH titrated from pH 7.0 to pH > or = 2.0 had no effect on I(sc). Circuit analysis at pH 2.0 showed small, but significant, increases in apical and shunt resistances. At pH < 2.0, I(sc) increased whereas resistance (R(T)) decreased along with an increase in fluorescein flux. The change in I(sc), but not R(T), was reversible at pH 7.4. Reducing pH from 7.0 to 1.1 with H(2)SO(4) gave a similar pattern but higher I(sc) values, suggesting shunt permselectivity. A 10:1 Na(+) gradient after nystatin increased I(sc) by approximately 4 muAmps/cm(2) and this declined at pH < or = 3.5 until it reached approximately 0.0 at pH 2.0. Impedance analysis on acid-exposed (non-nystatin treated) tissues showed compensatory changes in apical (increase) and basolateral (decrease) resistance at modest luminal acidity that were poorly reversible at pH 2.0 and associated with declines in capacitance, a reflection of lower apical membrane area. In esophageal epithelium apical cation channels transport Na(+) at gradients as low as 10:1 but do not transport H(+) at gradients of 100,000:1 (luminal pH 2.0). Luminal acid also inhibits Na(+) transport via the channels and abolishes it at pH 2.0. These effects on the channel may serve as a protective function for esophageal epithelium exposed to acid reflux.     

Na-glucose and Na-neutral amino acid cotransport are uniquely regulated by constitutive nitric oxide in rabbit small intestinal villus cells

Na-nutrient cotransport processes are not only important for the assimilation of essential nutrients but also for the absorption of Na in the mammalian small intestine. The effect of constitutive nitric oxide (cNO) on Na-glucose (SGLT-1) and Na-amino acid cotransport (NAcT) in the mammalian small intestine is unknown. Inhibition of cNO synthase with N(G)-nitro-l-arginine methyl ester (L-NAME) resulted in the inhibition of Na-stimulated (3)H-O-methyl-D-glucose uptake in villus cells. However, Na-stimulated alanine uptake was not affected in these cells. The L-NAME-induced reduction in SGLT-1 in villus cells was not secondary to an alteration in basolateral membrane Na-K-ATPase activity, which provides the favorable Na gradient for this cotransport process. In fact, SGLT-1 was inhibited in villus cell brush-border membrane (BBM) vesicles prepared from animals treated with L-NAME. Kinetic studies demonstrated that the mechanism of inhibition of SGLT-1 was secondary to a decrease in the affinity for glucose without a change in the maximal rate of uptake of glucose. Northern blot studies demonstrated no change in the mRNA levels of SGLT-1. Western blot studies demonstrated no significant change in the immunoreactive protein levels of SGLT-1 in ileal villus cell BBM from L-NAME-treated rabbits. These studies indicate that inhibition of cNO production inhibits SGLT-1 but not NAcT in the rabbit small intestine. Therefore, whereas cNO promotes Na-glucose cotransport, it does not affect NAcT in the mammalian small intestine.     

Enhancement of human monocyte function against Candida albicans by the colony-stimulating factors (CSF): IL-3, granulocyte-macrophage-CSF, and macrophage-CSF

The effect of IL-3, granulocyte-macrophage (GM)-CSF and macrophage (M)-CSF on Candida albicans growth inhibition by human peripheral blood monocytes was investigated. By using a radiolabel microassay developed in our laboratory that makes use of the incorporation of [3H]glucose into residual C. albicans, we demonstrated that rGM-CSF and rIL-3 effectively enhanced human monocyte-mediated anticandidal activity. Incubation for 24 h with either GM-CSF or IL-3 significantly enhanced monocyte antifungal responses down to 0.01 U/ml. M-CSF, at higher concentrations of 10 U/ml, could also enhance monocyte function but to a smaller degree. None of the CSF interfered directly with fungal growth, even up to 1000 U/ml. Because IFN-gamma is also a known monocyte activator, its effect on monocytes was also assessed. Monocytes were first cultured in medium for several days and then further incubated with each of the cytokines. Monocytes aged in medium were found to lose their spontaneous anticandidal activity. Such aged monocytes did not develop anticandidal activity in response to IFN-gamma but did in response to GM-CSF or IL-3. To further elucidate this difference, fresh monocytes were continuously cultured with or without cytokines for 1 to 5 days before assessing their anticandidal activity. Monocytes cultured in IFN-gamma progressively lost their activity by 2 days but monocytes in GM-CSF or IL-3 maintained their high level of anticandidal activity throughout the whole length of culture. Therefore, GM-CSF and IL-3 not only enhanced fresh monocyte anticandidal activity, but maintained monocyte function for a longer period. These results suggest that GM-CSF and IL-3 may act on monocytes via a different pathway than does IFN-gamma.     

Differences in ischemia-reperfusion-induced endothelial changes in hearts perfused at constant flow and constant pressure

Isolated hearts subjected to ischemia-reperfusion (I/R) exhibit depressed cardiac performance and alterations in subcellular function. Since hearts perfused at constant flow (CF) and constant pressure (CP) show differences in their contractile response to I/R, this study was undertaken to examine mechanisms responsible for these I/R-induced alterations in CF-perfused and CP-perfused hearts. Rat hearts, perfused at CF (10 ml/min) or CP (80 mmHg), were subjected to I/R (30 min global ischemia followed by 60 min reperfusion), and changes in cardiac function as well as sarcolemmal (SL) Na(+)-K(+)-ATPase activity, sarcoplasmic reticulum (SR) Ca(2+) uptake, and endothelial function were monitored. The I/R-induced depressions in cardiac function, SL Na(+)-K(+)-ATPase, and SR Ca(2+)-uptake activities were greater in hearts perfused at CF than in hearts perfused at CP. In hearts perfused at CF, I/R-induced increase in calpain activity and decrease in nitric oxide (NO) synthase (endothelial NO synthase) protein content in the heart as well as decrease in NO concentration of the perfusate were greater than in hearts perfused at CP. These changes in contractile activity and biochemical parameters due to I/R in hearts perfused at CF were attenuated by treatment with l-arginine, a substrate for NO synthase, while those in hearts perfused at CP were augmented by treatment with N(G)-nitro-l-arginine methyl ester, an inhibitor of NO synthase. The results indicate that the I/R-induced differences in contractile responses and alterations in subcellular organelles between hearts perfused at CF and CP may partly be attributed to greater endothelial dysfunction in CF-perfused hearts than that in CP-perfused hearts.     

DNA microarray analysis of neonatal mouse lung connects regulation of KDR with dexamethasone-induced inhibition of alveolar formation

Treating H441 cells with dexamethasone raised the abundance of mRNA encoding the epithelial Na(+) channel alpha- and beta-subunits and increased transepithelial ion transport (measured as short-circuit current, I(sc)) from <4 microA.cm(-2) to 10-20 microA.cm(-2). This dexamethasone-stimulated ion transport was blocked by amiloride analogs with a rank order of potency of benzamil >or= amiloride > EIPA and can thus be attributed to active Na(+) absorption. Studies of apically permeabilized cells showed that this increased transport activity did not reflect a rise in Na(+) pump capacity, whereas studies of basolateral permeabilized cells demonstrated that dexamethasone increased apical Na(+) conductance (G(Na)) from a negligible value to 100-200 microS.cm(-2). Experiments that explored the ionic selectivity of this dexamethasone-induced conductance showed that it was equally permeable to Na(+) and Li(+) and that the permeability to these cations was approximately fourfold greater than to K(+). There was also a small permeability to N-methyl-d-glucammonium, a nominally impermeant cation. Forskolin, an agent that increases cellular cAMP content, caused an approximately 60% increase in I(sc), and measurements made after these cells had been basolaterally permeabilized demonstrated that this response was associated with a rise in G(Na). This cAMP-dependent control over G(Na) was disrupted by brefeldin A, an inhibitor of vesicular trafficking. Dexamethasone thus stimulates Na(+) transport in H441 cells by evoking expression of an amiloride-sensitive apical conductance that displays moderate ionic selectivity and is subject to acute control via a cAMP-dependent pathway.     

Role of luminal ATP in regulating electrogenic Na(+) absorption in guinea pig distal colon

Extracellular ATP regulates a variety of functions in epithelial tissues by activating the membrane P2-receptor. The purpose of this study was to investigate the autocrine/paracrine regulation by luminal ATP of electrogenic amiloride-sensitive Na(+) absorption in the distal colon from guinea pigs treated with aldosterone by measuring the amiloride-sensitive short-circuit current (I(sc)) and (22)Na(+) flux in vitro with the Ussing chamber technique. ATP added to the luminal side inhibited the amiloride-sensitive I(sc) and (22)Na(+) absorption to a similar degree. The concentration dependence of the inhibitory effect of ATP on amiloride-sensitive I(sc) had an IC(50) value of 20-30 microM, with the maximum inhibition being approximately 50%. The effects of different nucleotides and of a nucleoside were also studied, the order of potency being ATP = UTP > ADP > adenosine. The effects of ATP were slightly, but significantly, reduced in the presence of suramin in the luminal solution. The inhibitory effect of luminal ATP was more potent in the absence of both Mg2+ and Ca2+ from the luminal solution. Pretreatment of the tissue with ionomycin or thapsigargin in the absence of serosal Ca2+ did not affect the percent inhibition of amiloride-sensitive I(sc) induced by ATP. Mechanical perturbation with a hypotonic luminal solution caused a reduction in amiloride-sensitive I(sc), this effect being prevented by the presence of hexokinase, an ATP-scavenging enzyme. These results suggest that ATP released into the luminal side by hypotonic stimulation could exert an inhibitory effect on the electrogenic Na(+) absorption. This effect was probably mediated by a P2Y(2) receptor on the apical membrane of colonic epithelial cells, and a change in the intracellular Ca2+ concentration may not be necessary for this process.     

Mechanisms mediating sodium-induced pressor responses in the PVN of Dahl rats

Intracerebroventricular infusion of Na(+)-rich artificial cerebrospinal fluid (aCSF) causes larger sympathetic and pressor responses in Dahl salt-sensitive (S) than -resistant (R) or Wistar rats. Enhanced activity of the aldosterone-"ouabain" pathway or decreased nitric oxide (NO) release may contribute to this enhanced responsiveness. Where in the brain these mechanisms interact is largely unknown. The present study evaluated whether Na(+) in the paraventricular nucleus (PVN) causes larger pressor responses in Dahl S (SS/Mcw) than R (Dahl SS.BN13) rats and whether mineralocorticoid receptors, benzamil-blockable Na(+) channels, "ouabain," angiotensin type 1 receptors, or NO mediates these enhanced responses. Na(+)-rich aCSF in the PVN caused 30-40% larger increases in blood pressure and heart rate in Dahl S than R or Wistar rats, whereas responses to ouabain, ANG II, or N(ω)-nitro-l-arginine methyl ester hydrochloride (l-NAME) in the PVN were the same. These responses to Na(+) were not affected by eplerenone, benzamil, or Fab fragments, whereas they were fully blocked by losartan, in Dahl S and R rats. l-NAME enhanced them more in Dahl R than S rats, thereby equalizing the responses in the two strains. Pressor responses to l-NAME in the PVN were attenuated by a high-salt diet in Dahl S, but not R, rats. The results indicate that acute and chronic increases in Na(+) concentration in the PVN inhibit NO release in the PVN of Dahl S, but not R, rats, thereby contributing to the enhanced pressor responses to Na(+) in Dahl S rats.     

Luminal bacterial flora determines physiological expression of intestinal epithelial cytoprotective heat shock proteins 25 and 72

Heat shock proteins (HSP) 25 and 72 are expressed normally by surface colonocytes but not by small intestinal enterocytes. We hypothesized that luminal commensal microflora maintain the observed colonocyte HSP expression. The ability of the small intestine to respond to bacteria and their products and modulate HSPs has not been determined. The effects of luminal bacterial flora in surgically created midjejunal self-filling (SFL) vs. self-emptying (SEL) small-bowel blind loops on epithelial HSP expression were studied. HSP25 and HSP72 expression were assessed by immunoblot and immunohistochemistry. SFL were chronically colonized, whereas SEL contained levels of bacteria normal for the proximal small intestine. SFL creation significantly increased HSP25 and HSP72 expression relative to corresponding sections from SEL. Metronidazole treatment, which primarily affects anaerobic bacteria as well as a diet lacking fermentable fiber, significantly decreased SFL HSP expression. Small bowel incubation with butyrate ex vivo induced a sustained and significant upregulation of HSP25 and altered HSP72 expression, confirming the role of short-chain fatty acids. To determine whether HSPs induction altered responses to an injury, effects of the oxidant, monochloramine, on epithelial resistance and short-circuit current (I(sc)) responses to carbachol and glucose were compared. Increased SFL HSP expression was associated with protection against oxidant-induced decreases in transmural resistance and I(sc) responses to glucose, but not secretory responses to carbachol. In conclusion, luminal microflora and their metabolic byproducts direct expression of HSPs in gut epithelial cells, an effect that contributes to preservation of epithelial cell viability under conditions of stress.     

Side specific effect of 5-hydroxytryptamine on NaCl transport in the apical and basolateral membrane of rat tracheal epithelia

5-Hydroxytryptamine (5-HT) can be released from mast cells and platelets through an IgE-dependent mechanism and may play a role in the pathogenesis of allergic bronchoconstriction. However, the effect of 5-HT on ion transport by the airway epithelium is still controversial. The objective of this study was to determine whether 5-hydroxytryptamine (5-HT) regulates NaCl transport by different mechanisms in the apical and basolateral membrane of tracheal epithelia. We studied the rat tracheal epithelium under short-circuit conditions in vitro. Short-circuit current (I(sc)) was measured in rat tracheal epithelial monolayers cultured on porous filters. 5-HT inhibited Na(+) absorption [measured via Na(+) short-circuit current (I(Na)(sc))] in the apical membrane and stimulated Cl(-) secretion [measured via Cl(-) short-circuit current (I(Cl)(sc))] in the basolateral membrane. Functional localization using selective 5-HT agonists and antagonists suggest that I(Cl)(sc)is stimulated by the basolateral membrane-resident 5-HT receptors, whereas I(Na)(sc) is inhibited by the apical membrane-resident 5-HT2 receptors. The basolateral addition of 5-HT increases intracellular cAMP content, but its apical addition does not. The addition of BAPTA/AM blocked the decrease of I(Na)(sc)which was induced by the apical addition of 5-HT, and 5-HT increased intracellular Ca concentrations. These results indicate that 5-HT differentially affects I(Na)(sc)and I(Cl)(sc)across rat tracheal monolayers through interactions with distinct receptors in the apical and the basolateral membrane. These effects may result in an increase of water movement towards the airway lumen.     

Contribution of villus and intervillus epithelium to intestinal transmural potential difference and response to theophylline and sugar.

A chamber design is described which permits isolation of villus or intervillus epithelium from proximal segments of Amphiuma intestine and measurement of the transpithelial potential difference (psi ms) and short-circuit current (Isc) produced by each. In media containing Cl- and 10 mequiv./l HCO3- the villus generated a basal psi ms of 0.8 mV (serosa negative) and Isc of 12 microA/cm2 while the intervillus psi ms and Isc were not different from zero. Acetazolamide altered the villus psi ms by 1.2 mV; the intervillus psi ms by only 0.3 mV. Transepithelial gradients of HCO3- appeared to generate diffusion potentials across the intervillus but not the villus epithelium. The actively transported sugar galactose elevated psi ms by 0.6 +/- 0.1 mV in the intervillus epithelium and by 1.5 +/- 0.2 mV in the villus epithelium for a response ratio (0.6/1.5) = 0.4. The response ratio for valine was 0.3. In contrast, the response ratios for theophylline (0.7) and cyclic AMP (0.7) were significantly higher. These observations indicate that the entire epithelium is responsive to theophylline and cyclic AMP while Na+-dependent solute transport and the basal electrogenic ion transport processes are primarily functions of the cells lining the intestinal villus.     

IL-15-dependent activation-induced cell death-resistant Th1 type CD8 alpha beta+NK1.1+ T cells for the development of small intestinal inflammation

To clarify the role of IL-15 at local sites, we engineered a transgenic (Tg) mouse (T3(b)-IL-15 Tg) to overexpress human IL-15 preferentially in intestinal epithelial cells by the use of T3(b)-promoter. Although IL-15 was expressed in the entire small intestine (SI) and large intestines of the Tg mice, localized inflammation developed in the proximal SI only. Histopathologic study revealed reduced villus length, marked infiltration of lymphocytes, and vacuolar degeneration of the villus epithelium, beginning at approximately 3-4 mo of age. The numbers of CD8(+) T cells, especially CD8alphabeta(+) T cells expressing NK1.1, were dramatically increased in the lamina propria of the involved SI. The severity of inflammation corresponded to increased numbers of CD8alphabeta(+)NK1.1(+) T cells and levels of production of the Th1-type cytokines IFN-gamma and TNF-alpha. Locally overexpressed IL-15 was accompanied by increased resistance of CD8alphabeta(+) NK1.1(+) T cells to activation-induced cell death. Our results suggest that chronic inflammation in the SI in this murine model is mediated by dysregulation of epithelial cell-derived IL-15. The model may contribute to understanding the role of CD8(+) T cells in human Crohn's disease involving the SI.     

Heat shock restores insulin secretion after injury by nitric oxide by maintaining glucokinase activity in rat islets

Heat shock protein (hsp), including hsp70, has been reported to restore the glucose-induced insulin release suppressed by nitric oxide (NO). However, the mechanism underlying this recovery remains unclear. In the present study, we examine the effects, in rat islets, of heat shock on insulin secretion inhibited by a small amount of NO and also on glucose metabolism, the crucial factor in insulin release. Exposure to a higher dose (15 U/ml) of interleukin-1beta (IL-1beta) abolished the insulin release by stimulation of glucose or KCl in both control and heat shocked islets. In rat islets exposed to a lower dose (1.5 U/ml) of IL-1beta, insulin secretion in response to glucose, but not to glyceraldehydes (GA), ketoisocaproate (KIC), or KCl, was selectively impaired, concomitantly with lower ATP concentrations in the presence of 16.7 mM glucose, while such suppression of insulin secretion and ATP content was not observed in heat shock-treated islets. NO production in islets exposed to 1.5 U/ml IL-1beta was significantly, but only partly, decreased by heat shock treatment. The glucose utilization rate measurement using [5-3H]-glucose and [2-3H]-glucose and the glucokinase activity in vitro were reduced in islets treated with 1.5 U/ml IL-1beta. In heat shock-treated islets, glucose utilization and glucokinase activity were not affected by 1.5 U/ml IL-1beta. These data suggest that heat shock restores glucose-induced insulin release inhibited by NO by maintaining glucokinase activity and the glucose utilization rate in islets in addition to reducing endogenous NO production.     

Early changes in glucose and phospholipid metabolism following apoptosis induction by IFN-gamma/TNF-alpha in HT-29 cells

The effects of apoptosis induction on glucose and phospholipid metabolite levels in cancer were studied using human colon adenocarcinoma cells (HT-29). Apoptosis was induced by co-incubation with 200 U/ml tumor necrosis factor (TNF)-alpha for 4, 8 or 15 h, after sensitization with 500 U/ml interferon (IFN)-gamma for 7 h. Perchloric acid extracts were analyzed by (1)H and (31)P nuclear magnetic resonance (NMR) spectroscopy. Significantly increased lactate and NTP (all nucleoside 5'-triphosphates) signals were detected 4 h after apoptosis-inducing IFN-gamma/TNF-alpha treatment, but not in cells which were TNF-alpha-treated without IFN-gamma preincubation. Simultaneous lactate and NTP changes, if confirmed in vivo, may serve as early, non-invasive markers of treatment response in some tumors.     

Gd3+-sensitive Na+ transport across the integument of Hirudo medicinalis

Leeches Hirudo medicinalis were exposed to either artificial pond water (APW; 1 mM NaCl) or to high-salinity conditions (HS; 200 mM NaCl) for several days. The aim of the study was to assess whether transepithelial ion conductances in their dorsal integuments were affected by this long-term acclimation. In voltage-clamp experiments using Ussing-type chambers, the transepithelial potential V(T) was clamped to 0 mV, and amiloride-sensitive currents (I(ami)) and total Na(+) transport (I(Na)) were determined. Apical Ca(2+)-free conditions strongly increased I(ami) to a similar magnitude in both differently acclimated integuments. Apical application of the lanthanide gadolinium <0.1 mM decreased the short-circuit current (I(sc)). In contrast, higher concentrations up to 10 mM Gd(3+) upregulated I(sc) by an additional 90% in APW integuments and by an additional 300% in HS integuments. This Gd(3+) effect was due to a doubling of I(Na) in APW and a more than sixfold increase of I(Na) in HS integuments. In summary, the macroscopic electrophysiological variables, including I(Na), were generally not affected by long-term exposure to high salinity. However, the presence of Gd(3+)-sensitive Na(+) conductances or regulating structures were greatly upregulated during HS acclimation.     

Interferon-gamma inhibits proliferation of rat vascular smooth muscle cells by nitric oxide generation.

Interferon (IFN)-gamma inhibited the proliferation of rat vascular smooth muscle cells (VSMC) and increased the cyclic GMP (cGMP) concentration in the cells. The dose dependencies of the two effects were similar (IC50 = 4 U/ml for the anti-proliferation and EC50 = 3 U/ml for cGMP formation) and the effect of IFN-gamma was enhanced by tumor necrosis factor-alpha treatment. Furthermore, NG-nitro-L-arginine, a nitric oxide (NO) synthase inhibitor, inhibited both activities induced by IFN-gamma. These findings show that the anti-proliferation and cGMP formation are closely related and that IFN-gamma inhibits the proliferation of rat VSMC by generation of NO through the induction of an NO synthase.