AJP: Gastrointestinal and Liver Physiology recent issues

Mechanisms of gastric emptying disturbances in chronic and acute inflammation of the distal gastrointestinal tract

Keller, J., Beglinger, C., Holst, J. J., Andresen, V., Layer, P. — Wed, 21 Oct 2009 14:20:56 PDT

It is unclear why patients with inflammation of the distal bowel complain of symptoms referable to the upper gastrointestinal tract, specifically to gastric emptying (GE) disturbances. Thus we aimed to determine occurrence and putative pathomechanisms of gastric motor disorders in such patients. Thirteen healthy subjects (CON), 13 patients with Crohn's disease (CD), 10 with ulcerative colitis (UC), and 7 with diverticulitis (DIV) underwent a standardized ¹³C-octanoic acid gastric emptying breath test. Plasma glucose, CCK, peptide YY, and glucagon-like peptide-1 (GLP-1) were measured periodically and correlated with GE parameters. Results were given in means ± SD. Compared with CON, GE half time (T) was prolonged by 50% in CD (115 ± 55 vs. 182 ± 95 min, P = 0.037). Six CD, 2 DIV, and 2 UC patients had pathological T (>200 min). Postprandial plasma glucose was increased in all patients but was highest in DIV and correlated with T (r = 0.90, P = 0.006). In CD, mean postprandial CCK levels were increased threefold compared with CON (6.5 ± 6.7 vs. 2.1 ± 0.6 pmol/l, P = 0.027) and were correlated with T (r = 0.60, P = 0.041). Compared with CON, GLP-1 levels were increased in UC (25.1 ± 5.2 vs. 33.5 ± 13.0 pmol/l, P = 0.046) but markedly decreased in DIV (9.6 ± 5.2 pmol/l, P < 0.0001). We concluded that a subset of patients with CD, UC, or DIV has delayed GE. GE disturbances are most pronounced in CD and might partly be caused by excessive CCK release. In DIV there might be a pathophysiological link between decreased GLP-1 release, postprandial hyperglycemia, and delayed GE. These explorative data encourage further studies in larger patient groups.

Differential adipokine response in genetically predisposed lean and obese rats during inflammation: a role in modulating experimental colitis?

Hyland, N. P., Chambers, A. P., Keenan, C. M., Pittman, Q. J., Sharkey, K. A. — Wed, 21 Oct 2009 14:20:56 PDT

The relationship between a predisposition to obesity and the development of colitis is not well understood. Our aim was to characterize the adipokine response and the extent of colitis in diet-induced obese (DIO) rats. DIO and control, diet-resistant (DR) animals were administered either saline or trinitrobenzene sulfonic acid (TNBS) to induce colitis. Macroscopic damage scores and myeloperoxidase (MPO) activity were measured to determine the extent of inflammation. Trunk blood was collected for the analysis of plasminogen activator inhibitor-1 (PAI-1) as well as leptin, ghrelin, and adiponectin. Colonic epithelial physiology was assessed using Ussing chambers. DIO rats had a modestly increased circulating PAI-1 before TNBS treatment; however, during colitis, DR animals had more than a fourfold increase in circulating PAI-1 compared with DIO rats. Circulating leptin was higher in DIO rats compared with DR animals, in the inflamed and noninflamed states. These changes in TNBS-induced adipokine profile were accompanied by decreased macroscopic tissue damage score in DIO animals compared with DR tissues. Furthermore, TNBS-treated DR animals lost significantly more weight than DIO rats during active inflammation. Colonic epithelial physiology was comparable between groups, as was MPO activity. The factors contributing to the decreased colonic damage are almost certainly multifold, driven by both genetic and environmental factors, of which adipokines are likely to play a part given the increasing body of evidence for their role in modulating intestinal inflammation.

Mechanisms underlying modulation of monocarboxylate transporter 1 (MCT1) by somatostatin in human intestinal epithelial cells

Wed, 21 Oct 2009 14:20:56 PDT

Somatostatin (SST), an important neuropeptide of the gastrointestinal tract has been shown to stimulate sodium chloride absorption and inhibit chloride secretion in the intestine. However, the effects of SST on luminal butyrate absorption in the human intestine have not been investigated. Earlier studies from our group and others have shown that monocarboxylate transporter (MCT1) plays an important role in the transport of butyrate in the human intestine. The present studies were undertaken to examine the effects of SST on butyrate uptake utilizing postconfluent human intestinal epithelial Caco2 cells. Apical SST treatment of Caco-2 cells for 30–60 min significantly increased butyrate uptake in a dose-dependent manner with maximal increase at 50 nM (~60%, P < 0.05). SST receptor 2 agonist, seglitide, mimicked the effects of SST on butyrate uptake. SST-mediated stimulation of butyrate uptake involved the p38 MAP kinase-dependent pathway. Kinetic studies demonstrated that SST increased the maximal velocity (V_max) of the transporter by approximately twofold without any change in apparent Michaelis-Menten constant (K_m). The higher butyrate uptake in response to SST was associated with an increase in the apical membrane levels of MCT1 protein parallel to a decrease in the intracellular MCT1 pool. MCT1 has been shown to interact specifically with CD147 glycoprotein/chaperone to facilitate proper expression and function of MCT1 at the cell surface. SST significantly enhanced the membrane levels of CD147 as well as its association with MCT1. This association was completely abolished by the specific p38 MAP kinase inhibitor, SB203580. Our findings demonstrate that increased MCT1 association with CD147 at the apical membrane in response to SST is p38 MAP kinase dependent and underlies the stimulatory effects of SST on butyrate uptake.

Effects of progesterone on motility and prostaglandin levels in the distal guinea pig colon

Xiao, Z. L., Biancani, P., Behar, J. — Wed, 21 Oct 2009 14:20:56 PDT

Progesterone (P4) inhibits the gastrointestinal muscle contraction by downregulating G_q/11 proteins that mediate contraction, by upregulating G_s proteins that mediate relaxation, and by altering the pattern of cyclooxygenase (COX) enzymes and prostaglandins. We aimed to examine whether P4 treatment of guinea pigs in vivo affects basal colon motility [basal motility index (MI)] by altering the levels and actions of PGF₂ and PGE₂. Guinea pigs were treated with intramuscular (IM) P4 for 4 days. The BASAL MI, the PGF₂-induced contraction, and PGE₂-induced inhibition of contraction were examined in muscle strips and cells. The levels of PGF₂ and PGE₂ were measured by radioimmunoassay. Treatment with P4 reduced the basal MI, the levels of PGF₂, and PGF₂-induced contraction. P4 increased PGE₂ levels, and PGE₂ induced relaxation. Pretreatment with IM RU-486 (10 mg/kg per day), a P4 receptor antagonist, 1 h before P4 blocked the actions of P4. The PGF₂ antagonist Al-1180 abolished basal MI and PGF₂-induced contraction. N-ethylmaleimide, which blocks unoccupied membrane receptors, blocked Ach and VIP actions but had no effect on PGF₂ and PGE₂ effects. A COX-1 inhibitor decreased and a COX-2 inhibitor increased PGF₂ levels; GTPS increased and GDPβS decreased the levels of PGF₂. G_q/11 protein antibodies (Abs) reduced PGF₂ levels, and G_i3 Abs blocked its motor actions. Gs Abs increased PGF₂ but decreased PGE₂ levels. We concluded that P4 decreases basal MI by reducing PGF₂ levels caused by downregulation of G_q/11 and that PGF₂-induced contraction was blocked by downregulating G_i3. P4 also decreased the basal MI by increasing PGE₂ levels, and PGE₂ induced relaxation by upregulating G_s proteins.

Effect of meal volume and calorie load on postprandial gastric function and emptying: studies under physiological conditions by combined fiber-optic pressure measurement and MRI

Wed, 21 Oct 2009 14:20:56 PDT

This study assessed the effects of meal volume (MV) and calorie load (CL) on gastric function. MRI and a minimally invasive fiber-optic recording system (FORS) provided simultaneous measurement of gastric volume and pressure changes during gastric filling and emptying of a liquid nutrient meal in physiological conditions. The gastric response to 12 iso-osmolar MV-CL combinations of a multinutrient drink (MV: 200, 400, 600, 800 ml; CL: 200, 300, 400 kcal) was tested in 16 healthy subjects according to a factorial design. Total gastric volume (TGV) and gastric content volume (GCV = MV + secretion) were measured by MRI during nasogastric meal infusion and gastric emptying over 60 min. Intragastric pressure was assessed at 1 Hz by FORS. The dynamic change in postprandial gastric volumes was described by a validated three-component linear exponential model. The stomach expanded with MV, but the ratio of GCV:MV at t₀ diminished with increasing MV (P < 0.01). Postprandial changes in TGV followed those of GCV. Intragastric pressure increased with MV, and this effect was augmented further by CL (P = 0.02); however, the absolute pressure rise was <4 mmHg. A further postprandial increase of gastric volumes was observed early on before any subsequent volume decrease. This "early" increase in GCV was greater for smaller than larger MV (P < 0.01), indicating faster initial gastric emptying of larger MV. In contrast, volume change during filling and in the early postprandial period were unaffected by CL. In the later postprandial period, gastric emptying rate continued to be more rapid with high MVs (P < 0.001); however, at any given volume, gastric emptying was slowed by higher CL (P < 0.001). GCV half-emptying time decreased with CL at 18 ± 6 min for each additional 100-kcal load (P < 0.001). These findings indicate that gastric wall stress (passive strain and active tone) provides the driving force for gastric emptying, but distal resistance to gastric outflow regulates further passage of nutrients. The distinct early phase of gastric emptying with relatively rapid, uncontrolled passage of nutrients into the small bowel, modulated by meal volume but not nutrient composition, ensures that the delivery of nutrients in the later postprandial period is related to the overall calorie load of the meal.

Contribution of different triggers to the gastric accommodation reflex in humans

Vanden Berghe, P., Janssen, P., Kindt, S., Vos, R., Tack, J. — Wed, 21 Oct 2009 14:20:56 PDT

Accommodation of the stomach consists of a vagally mediated relaxation of the proximal stomach, providing the meal with a reservoir. Our aim was to study whether, similar to other vagally mediated processes, the accommodation reflex is also determined by cephalic, oropharyngeal, gastric, and intestinal phases. Eleven healthy subjects underwent in randomized order five gastric barostat studies and two satiety drinking tests. In all studies, isobaric tone measurements (at minimal distending pressure + 2 mmHg) were performed 20 min before and 20 min after a nutrient stimulus. The stimuli included only visual and olfactory exposure to a meal (cephalic stimulation), taking liquid nutrient in the mouth without swallowing (sham feeding), ingestion of a 200-ml 300-kcal nutrient meal with blocked outflow to the pylorus (gastric retention), and meal infusion through a nasointestinal tube (duodenal instillation), or normal ingestion (control). During satiety testing, subjects ingested liquid nutrient at a fixed rate of 15 ml/min until maximum satiety, with an inflated or deflated intrapyloric balloon assembly. Progressively bigger gastric relaxatory responses were seen with cephalic stimulation (18 ± 19 ml), sham feeding (54 ± 21 ml), gastric retention (95 ± 47), duodenal instillation (144 ± 33), and control (232 ± 33 ml). The amount of nutrient ingested at maximum satiety was significantly lower with an inflated intrapyloric balloon (1,223 ± 103 vs. 1,392 ± 124 ml, P < 0.05). The accommodation reflex in humans lacks a cephalic phase, but it can be activated from the oropharynx, the stomach, and the duodenum. Blocking passage to the duodenum significantly decreases the amplitude of the accommodation reflex and induces early satiety.

Chronic oxidative stress sensitizes hepatocytes to death from 4-hydroxynonenal by JNK/c-Jun overactivation

Singh, R., Wang, Y., Schattenberg, J. M., Xiang, Y., Czaja, M. J. — Wed, 21 Oct 2009 14:20:56 PDT

Sustained activation of the c-Jun NH₂-terminal kinase (JNK) signaling pathway mediates the development and progression of experimental diet-induced nonalcoholic fatty liver disease (NAFLD). Delineating the mechanism of JNK overactivation in the setting of a fatty liver is therefore essential to understanding the pathophysiology of NAFLD. Both human and experimental NAFLD are associated with oxidative stress and resultant lipid peroxidation, which have been proposed to mediate the progression of this disease from simple steatosis to steatohepatitis. The ability of oxidants and the lipid peroxidation product 4-hydroxynonenal (HNE) to activate JNK signaling suggested that these two factors may act synergistically to trigger JNK overactivation. The effect of HNE on hepatocyte injury and JNK activation was therefore examined in cells under chronic oxidant stress from overexpression of the prooxidant enzyme cytochrome P450 2E1 (CYP2E1), which occurs in NAFLD. CYP2E1-generated oxidant stress sensitized a rat hepatocyte cell line to death from normally nontoxic concentrations of HNE. CYP2E1-overexpressing cells underwent a more profound depletion of glutathione (GSH) in response to HNE secondary to decreased -glutamylcysteine synthetase activity. GSH depletion led to overactivation of JNK/c-Jun signaling at the level of mitogen-activated protein kinase kinase 4 that induced cell death. Oxidant stress and the lipid peroxidation product HNE cause synergistic overactivation of the JNK/c-Jun signaling pathway in hepatocytes, demonstrating that HNE may not be just a passive biomarker of hepatic oxidant stress but rather an active mediator of hepatocellular injury through effects on JNK signaling.

Parsing apical oxalate exchange in Caco-2BBe1 monolayers: siRNA knockdown of SLC26A6 reveals the role and properties of PAT-1

Freel, R. W., Morozumi, M., Hatch, M. — Wed, 21 Oct 2009 14:20:56 PDT

The purpose of this investigation was to quantitate the contribution of the anion exchanger PAT-1 (putative anion transporter-1), encoded by SLC26A6, to oxalate transport in a model intestinal epithelium and to discern some characteristics of this exchanger expressed in its native environment. Control (Con) Caco-2 BBe1 monolayers, 6–8 days postseeding, were compared with those transfected with a small interfering RNA targeted to SLC26A6 (A6KD). Radiotracer and Ussing chamber techniques were used to determine the transepithelial unidirectional fluxes of Ox^2–, Cl^–, and SO₄^2– whereas fluorometric/BCECF measurements of intracellular pH were used to assess HCO₃^– exchange. PAT-1 was functionally targeted to the apical membrane, and SLC26A6 knockdown reduced PAT-1 protein (>60%) and mRNA (>75%) expression in A6KD. No net flux of Ox^2–, Cl^–, or SO₄^2– was detected in Con or A6KD monolayers, yet the unidirectional fluxes in A6KD were reduced 50, 35, and 15%, respectively. Cl^–-dependent HCO₃^– efflux from A6KD was reduced 50% compared with Con. The difference between Con and A6KD properties represents that mediated solely by PAT-1, and by this approach we found that PAT-1-mediated oxalate influx and efflux are inhibited equally by mucosal DIDS (EC₅₀ ~5 µM) and that mucosal Cl^– inhibits oxalate uptake with an EC₅₀ < 20 mM. Transepithelial Cl^– gradients supported large, DIDS-sensitive net absorptive or secretory fluxes of oxalate in a direction opposite that of the imposed Cl^– gradient. The overall symmetry of PAT-1-mediated oxalate exchange suggests that vectorial oxalate transport observed in vivo is principally dependent on the magnitude and direction of counterion gradients.

Caveolin-1 mediates endotoxin inhibition of endothelin-1-induced endothelial nitric oxide synthase activity in liver sinusoidal endothelial cells

Kwok, W., Lee, S. H., Culberson, C., Korneszczuk, K., Clemens, M. G. — Wed, 21 Oct 2009 14:20:56 PDT

Endothelin-1 (ET-1) plays a key role in the regulation of endothelial nitric oxide synthase (eNOS) activation in liver sinusoidal endothelial cells (LSECs). In the presence of endotoxin, an increase in caveolin-1 (Cav-1) expression impairs ET-1/eNOS signaling; however, the molecular mechanism is unknown. The objective of this study was to investigate the molecular mechanism of Cav-1 in the regulation of LPS suppression of ET-1-mediated eNOS activation in LSECs by examining the effect of caveolae disruption using methyl-β-cyclodextrin (CD) and filipin. Treatment with 5 mM CD for 30 min increased eNOS activity (+255%, P < 0.05). A dose (0.25 µg/ml) of filipin for 30 min produced a similar effect (+111%, P < 0.05). CD induced the perinuclear localization of Cav-1 and eNOS and stimulated NO production in the same region. Readdition of 0.5 mM cholesterol to saturate CD reversed these effects. Both the combined treatment with CD and ET-1 (CD + ET-1) and with filipin and ET-1 stimulated eNOS activity; however, pretreatment with endotoxin (LPS) abrogated these effects. Following LPS pretreatment, CD + ET-1 failed to stimulate eNOS activity (+51%, P > 0.05), which contributed to the reduced levels of eNOS-Ser1177 phosphorylation and eNOS-Thr495 dephosphorylation, the LPS/CD-induced overexpression and translocation of Cav-1 in the perinuclear region, and the increased perinuclear colocalization of eNOS with Cav-1. These results supported the hypothesis that Cav-1 mediates the action of endotoxin in suppressing ET-1-mediated eNOS activation and demonstrated that the manipulation of caveolae produces significant effects on ET-1-mediated eNOS activity in LSECs.

Bifidobacterium bifidum improves intestinal integrity in a rat model of necrotizing enterocolitis

Wed, 21 Oct 2009 14:20:56 PDT

Neonatal necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. Oral administration of probiotics has been suggested as a promising strategy for prevention of NEC. However, little is known about the mechanism(s) of probiotic-mediated protection against NEC. The aim of this study was to evaluate the effects of Bifidobacterium bifidum treatment on development of NEC, cytokine regulation, and intestinal integrity in a rat model of NEC. Premature rats were divided into three groups: dam fed (DF), hand fed with formula (NEC), or hand fed with formula supplemented with 5 x 10⁶ CFU B. bifidum per day (B. bifidum). All groups were exposed to asphyxia and cold stress to develop NEC. Intestinal injury, mucin and trefoil factor 3 (Tff3) production, cytokine levels, and composition of tight junction (TJ) and adherens junction (AJ) proteins were evaluated in the terminal ileum. B. bifidum decreased the incidence of NEC from 57 to 17%. Increased levels of IL-6, mucin-3, and Tff3 in the ileum of NEC rats was normalized in B. bifidum treated rats. Reduced mucin-2 production in the NEC rats was not affected by B. bifidum. Administration of B. bifidum normalized the expression and localization of TJ and AJ proteins in the ileum compared with animals with NEC. In conclusion, administration of B. bifidum protects against NEC in the neonatal rat model. This protective effect is associated with reduction of inflammatory reaction in the ileum, regulation of main components of mucus layer, and improvement of intestinal integrity.

Impact of Roux-en-Y gastric bypass surgery on rat intestinal glucose transport

Stearns, A. T., Balakrishnan, A., Tavakkolizadeh, A. — Wed, 21 Oct 2009 14:20:56 PDT

Roux-en-Y gastric bypass (RYGB) has become the gold-standard bariatric procedure, partly because of the rapid resolution of accompanying diabetes. There is increasing evidence this is mediated by duodenal exclusion. We hypothesize that duodenal exclusion suppresses intestinal Na⁺/glucose cotransporter SGLT1-mediated glucose transport, improving glucose handling, and aimed to test this in a rodent RYGB model. Sprague-Dawley rats underwent sham procedure or duodenal exclusion by RYGB (10 cm Roux, 16 cm biliopancreatic limbs). Animals were maintained for 3 wk on a Western diet, before harvest at 10 AM, 4 PM, and 10 PM. Sections were taken from each limb for hematoxylin and eosin staining, and morphological assessment was performed. Functional glucose uptake studies, along with Western blotting and quantitative PCR, were performed on Roux limb. Histology showed morphometric changes in Roux and common limbs, with increase in villus height and crypt depth compared with BP and sham jejunum. Despite this, glucose transport was reduced by up to 68% (P < 0.001) in the Roux limb compared with sham jejunum. Normal diurnal rhythms in glucose uptake were ablated. This occurred at a posttranscriptional level, with little change in message but appearance of different weight species of Sglt1 on Western blotting. We have shown duodenal exclusion significantly influences both intestinal structure and glucose transport function, with glucose absorptive capacity reduced after RYGB. This provides a novel mechanistic explanation for some of the antidiabetic effects of RYGB.

Role of thin-filament regulatory proteins in relaxation of colonic smooth muscle contraction

Somara, S., Gilmont, R., Bitar, K. N. — Wed, 21 Oct 2009 14:20:56 PDT

Coordinated regulation of smooth muscle contraction and relaxation is required for colonic motility. Contraction is associated with phosphorylation of myosin light chain (MLC₂₀) and interaction of actin with myosin. Thin-filament regulation of actomyosin interaction is modulated by two actin-binding regulatory proteins: tropomyosin (TM) and caldesmon (CaD). TM and CaD are known to play crucial role in actomyosin interaction promoting contraction. Contraction is associated with phosphorylation of the small heat shock protein HSP27, concomitant with the phosphorylation of TM and CaD. Phosphorylation of HSP27 is attributed as being the prime modulator of thin-filament regulation of contraction. Preincubation of colonic smooth muscle cells (CSMC) with the relaxant neurotransmitter vasoactive intestinal peptide (VIP) showed inhibition in phosphorylation of HSP27 (ser78). Attenuation of HSP27 phosphorylation can result in modulation of thin-filament-mediated regulation of contraction leading to relaxation; thus the role of thin-filament regulatory proteins in a relaxation milieu was investigated. Preincubation of CSMC with VIP exhibited a decrease in phosphorylation of TM and CaD. Furthermore, CSMC preincubated with VIP showed a reduced association of TM with HSP27 and with phospho-HSP27 (ser78) whereas there was reduced dissociation of TM from CaD and from phospho-CaD. We thus propose that, in addition to alteration in phosphorylation of MLC₂₀, relaxation is associated with alterations in thin-filament-mediated regulation that results in termination of contraction.

Protease activation during in vivo pancreatitis is dependent on calcineurin activation

Wed, 21 Oct 2009 14:20:56 PDT

The premature activation of digestive proenzymes, specifically proteases, within the pancreatic acinar cell is an early and critical event during acute pancreatitis. Our previous studies demonstrate that this activation requires a distinct pathological rise in cytosolic Ca²⁺. Furthermore, we have shown that a target of aberrant Ca²⁺ in acinar cells is the Ca²⁺/calmodulin-dependent phosphatase calcineurin (PP2B). In this study, we hypothesized that PP2B mediates in vivo protease activation and pancreatitis severity. To test this, pancreatitis was induced in mice over 8 h by administering hourly intraperitoneal injections of the cholecystokinin analog caerulein (50 µg/kg). Treatment with the PP2B inhibitor FK506 at 1 and 8 h after pancreatitis induction reduced trypsin activities by greater than 50% (P < 0.005). Serum amylase and IL-6 was reduced by 86 and 84% relative to baseline (P < 0.0005) at 8 h, respectively. Histological severity of pancreatitis, graded on the basis of pancreatic edema, acinar cell vacuolization, inflammation, and apoptosis, was reduced early in the course of pancreatitis. Myeloperoxidase activity from both pancreas and lung was reduced by 93 and 83% relative to baseline, respectively (P < 0.05). These data suggest that PP2B is an important target of the aberrant acinar cell Ca²⁺ rise associated with pathological protease activation and pancreatitis.

Localization of acyl ghrelin- and des-acyl ghrelin-immunoreactive cells in the rat stomach and their responses to intragastric pH

Wed, 21 Oct 2009 14:20:56 PDT

Acyl ghrelin has a 28-amino acid sequence with O-n-octanoyl acid modification at the serine 3 position, whereas des-acyl ghrelin has no octanoyl acid modification. Although these peptides exert different physiological functions, no previous studies have shown the different localization of acyl ghrelin and des-acyl ghrelin in the stomach. Here we have developed an antibody specific for des-acyl ghrelin that does not crossreact with acyl ghrelin. Both acyl ghrelin- and des-acyl ghrelin-immunoreactive cells were distributed in the oxyntic and antral mucosa of the rat stomach, with higher density in the antral mucosa than oxyntic mucosa. Immunofluorescence double staining showed that acyl ghrelin- and des-acyl ghrelin-positive reactions overlapped in closed-type round cells, whereas des-acyl ghrelin-positive reaction was found in open-type cells in which acyl ghrelin was negative. Acyl ghrelin-/des-acyl ghrelin-positive closed-type cells contain obestatin; on the other hand, des-acyl ghrelin-positive open-type cells contain somatostatin. We measured the release of acyl ghrelin and des-acyl ghrelin in vascularly perfused rat stomach by ELISA, and the effects of different intragastric pH levels on the release of each peptide were examined. The release of des-acyl ghrelin from the perfused stomach was greater at pH 2 than at pH 4; however, the release of acyl ghrelin was not affected by intragastric pH. The present study demonstrated the differential localization of acyl ghrelin and des-acyl ghrelin in the rat stomach and their different responses to the intragastric pH.

Regulation of HSP60 and the role of MK2 in a new model of severe experimental pancreatitis

Wed, 21 Oct 2009 14:20:56 PDT

The objective of this study was to investigate the role of MAPKAP kinase 2 (MK2) and heat shock protein (HSP) HSP60 in the pathogenesis of a new model of severe acute pancreatitis (AP). MK2 plays a significant role in the regulation of cytokines. It has been shown that induction and expression of several HSPs can protect against experimental pancreatitis. Interplay between both systems seems of high interest. Mice with a homozygous deletion of the MK2 gene were used. Severe AP was induced by combined intraperitoneal injections of cerulein with lipopolysaccharide (LPS). Severity of AP was assessed by biochemical markers and histology. The serum IL-6 and lung myeloperoxidase (MPO) levels were determined for assessing the extent of systemic inflammatory response. Expression of HSP25, HSP60, HSP70, and HSP90 was analyzed by Western blotting. Repeated injections of cerulein alone or cerulein plus LPS (Cer+LPS) resulted in local inflammatory responses in the pancreas and corresponding systemic inflammatory changes with pronounced severity in the Cer+LPS group. Compared with the C57Bl wild-type mice, the MK2–/– mice presented with significant milder pancreatitis and attenuated responses of serum amylase and trypsinogen activity. Furthermore, serum IL-6 was decreased as well as lung MPO activity. Injection of LPS alone displayed neither pancreatic inflammatory responses nor alterations of pancreatic enzyme activities but evidently elevated serum IL-6 levels and increased lung MPO activity. In contrast hereto, in the MK2–/– mice, these changes were much milder. Increased expression of HSP25 and HSP60 occurred after induction of AP. Especially, HSP60 was robustly elevated after Cer+LPS treatment, in both MK2–/– and wild-type mice. Thus the homozygous deletion of the MK2 gene ameliorates the severity of acute pancreatitis and accompanying systemic inflammatory reactions in a new model of severe acute pancreatitis. Our data support the hypothesis that MK2 participates in the multifactorial regulation of early inflammatory responses in AP, independently of the regulation of stress proteins like HSP25 and HSP60 and most likely due to its effect on cytokine regulation.

Protection from diclofenac-induced small intestinal injury by the JNK inhibitor SP600125 in a mouse model of NSAID-associated enteropathy

Ramirez-Alcantara, V., LoGuidice, A., Boelsterli, U. A. — Wed, 21 Oct 2009 14:20:56 PDT

Small intestinal ulceration, bleeding, and inflammation are major adverse effects associated with the use of diclofenac (DCF) or other nonsteroidal anti-inflammatory drugs (NSAIDs). The underlying mechanisms of DCF enteropathy are poorly understood, but there is increasing evidence that topical effects are involved. The aim of this study was to explore the role of c-Jun-N-terminal kinase (JNK) in DCF-induced enterocyte death because JNK not only regulates mitochondria-mediated apoptosis but also is a key node where many of the proximal stress signals converge. Male C57BL/6J mice were injected intraperitoneally with DCF or vehicle (Solutol HS-15), and the extent of small intestinal ulceration was determined. A single dose of DCF (60 mg/kg) produced numerous ulcers in the third and fourth quartiles of the jejunum and ileum, with maximal effects after 18 h and extensive recovery after 48 h. To study the molecular pathways leading to enterocyte injury, we isolated villi-enriched mucosal fractions from DCF-treated mice. Immunoblot studies with a phosphospecific JNK antibody revealed that JNK1/2 (p46) was activated at 6 h, leading to phosphorylation of the downstream target c-Jun. The levels of the JNK-regulated proapoptotic transcription factor C/EBP homologous protein (CHOP) were also increased after DCF. The selective JNK inhibitor SP600125 (30 mg/kg ip), given both 1 h before and 1 h after DCF, blocked JNK kinase activity and afforded significant protection against DCF enteropathy. In conclusion, these data demonstrate that the JNK pathway is critically involved in the pathogenesis of DCF-induced enteropathy and suggest a potential application of JNK inhibitors in the prevention of NSAID-induced enteropathy.

The angiogenic effect of probiotic Bacillus polyfermenticus on human intestinal microvascular endothelial cells is mediated by IL-8

Im, E., Choi, Y. J., Kim, C. H., Fiocchi, C., Pothoulakis, C., Rhee, S. H. — Wed, 21 Oct 2009 14:20:56 PDT

Angiogenesis is required for wound healing and repair, but dysregulated angiogenesis is involved in gastrointestinal inflammation. Bacillus polyfermenticus (B.P.) is a probiotic bacterium clinically used for a variety of intestinal disorders in East Asia. Here we investigated the effect of B.P. on angiogenesis of human intestinal microvascular endothelial cells (HIMECs) and wound healing in intestinal mucosa. Exposure of HIMECs to the conditioned medium of B.P. cultures (B.P. CM) increased cell migration, permeability, and tube formation. Production of the proangiogenic cytokine IL-8 was increased by B.P. CM, and neutralizing antibodies against IL-8 or IL-8 receptor CXCR2 reduced tube formation as well as actin stress fiber formation. B.P. CM also increased NF-B activation, and inhibitors of NF-B suppressed B.P. CM-induced tube formation and IL-8 production. Furthermore, B.P. facilitated recovery of mice from colitis as shown by increased body weight and reduced rectal bleeding and histological severity. B.P. also increased angiogenesis and mouse IL-8 production in the mucosal layer. Collectively, these results show that B.P. increases angiogenesis of HIMECs in a NF-B/IL-8/CXCR2-dependent manner. Moreover, B.P. promotes angiogenesis in the mucosa during recovery of mice from colitis, suggesting that this probiotic may be clinically used to facilitate intestinal wound healing.

Identification and functional characterization of the intermediate-conductance Ca2+-activated K+ channel (IK-1) in biliary epithelium

Wed, 21 Oct 2009 14:20:56 PDT

In the liver, adenosine triphosphate (ATP) is an extracellular signaling molecule that is released into bile and stimulates a biliary epithelial cell secretory response via engagement of apical P2 receptors. The molecular identities of the ion channels involved in ATP-mediated secretory responses have not been fully identified. Intermediate-conductance Ca²⁺-activated K⁺ channels (IK) have been identified in biliary epithelium, but functional data are lacking. The aim of these studies therefore was to determine the location, function, and regulation of IK channels in biliary epithelial cells and to determine their potential contribution to ATP-stimulated secretion. Expression of IK-1 mRNA was found in both human Mz-Cha-1 biliary cells and polarized normal rat cholangiocyte (NRC) monolayers, and immunostaining revealed membrane localization with a predominant basolateral signal. In single Mz-Cha-1 cells, exposure to ATP activated K⁺ currents, increasing current density from 1.6 ± 0.1 to 7.6 ± 0.8 pA/pF. Currents were dependent on intracellular Ca²⁺ and sensitive to clotrimazole and TRAM-34 (specific IK channel inhibitors). Single-channel recording demonstrated that clotrimazole-sensitive K⁺ currents had a unitary conductance of 46.2 ± 1.5 pS, consistent with IK channels. In separate studies, 1-EBIO (an IK activator) stimulated K⁺ currents in single cells that were inhibited by clotrimazole. In polarized NRC monolayers, ATP significantly increased transepithelial secretion which was inhibited by clotrimazole. Lastly, ATP-stimulated K⁺ currents were inhibited by the P2Y receptor antagonist suramin and by the inositol 1,4,5-triphosphate (IP3) receptor inhibitor 2-APB. Together these studies demonstrate that IK channels are present in biliary epithelial cells and contribute to ATP-stimulated secretion through a P2Y-IP3 receptor pathway.

Angiotensin II receptors are expressed and functional in human esophageal mucosa

Wed, 21 Oct 2009 14:20:56 PDT

Only few studies have been devoted to the actions of the renin-angiotensin system (RAS) in the human gastrointestinal tract. The present study was undertaken to elucidate the expression and action of RAS in the human esophageal mucosa. Mucosal specimens with normal histological appearance were obtained from healthy subjects undergoing endoscopy and from patients undergoing esophagectomy due to neoplasm. Gene and protein expressions of angiotensin II (Ang II) receptor type 1 (AT₁) and type 2 (AT₂) and angiotensin-converting enzyme (ACE) were analyzed. In vivo functionality in healthy volunteers was reflected by assessing transmucosal potential difference (PD). Ussing chamber technique was used to analyze the different effects of Ang II on its AT₁ and AT₂ receptors. Immunoreactivity to AT₁ and AT₂ was localized to stratum superficiale and spinosum in the epithelium. ACE, AT₁, and AT₂ were found in blood vessel walls. Transmucosal PD in vivo increased following administration of the AT₁ receptor antagonist candesartan. In Ussing preparations mean basal transmural PD was –6.4 mV, epithelial current (I_ep) 34 µA/cm², and epithelial resistance (R_ep) 321 ·cm². Serosal exposure to Ang II increased PD as a result of increased I_ep, whereas R_ep was constant. Ang II given together with the selective AT₁-receptor antagonist losartan, or AT₂ agonist C21 given alone, resulted in a similar effect. Ang II given in presence of the AT₂-receptor antagonist PD123319 did not influence PD, but I_ep decreased and R_ep increased. In conclusion, Ang II receptors and ACE are expressed in the human esophageal epithelium. The results suggest that AT₂-receptor stimulation increases epithelial ion transport, whereas the AT₁ receptor inhibits ion transport and increases R_ep.

New method of manometric measurement of gastroduodenal motility in conscious mice: effects of ghrelin and Y2 depletion

Tanaka, R., Inui, A., Asakawa, A., Atsuchi, K., Ataka, K., Fujimiya, M. — Wed, 21 Oct 2009 14:20:56 PDT

Since no previous studies have reported dual measurements of stomach and duodenal motility in conscious mice, we developed a manometric method to measure the gastroduodenal motility in the physiological fed and fasted states of conscious mice. By this method we measured, for the first time, the gastroduodenal motility in Y2 knockout mice and analyzed the effects of ghrelin on the gastroduodenal motility in conscious mice. To evaluate this new method, we provide the comparison on the effects of CCK-8 examined by present and previous methods. In the fasted state of mice, phase III-like contractions with frequencies of 7.8 ± 0.5 contractions/h in the antrum and 6.6 ± 0.7 contractions/h in the duodenum were observed. This fasted pattern was disrupted and replaced by the fed pattern after feeding, with an increase of the motor index (MI) immediately after feeding. Intravenous injection of ghrelin induced the fasted pattern in the duodenum when injected in the fed state and increased %MI (114.3 ± 9.8%) compared with saline-injected controls (64.4 ± 9.6%) in the antrum. Intravenous injection of CCK-8 disrupted phase III-like contractions in both antrum and duodenum, which were replaced by fed-like motor patterns accompanied with the elevation of baseline pressure. In Y2 knockout mice, the frequency of phase III-like contractions was decreased in the antrum compared with wild-type mice and the immediate increase of MI after feeding seen in wild-type mice was disrupted in Y2 knockout mice. Our model provides a new method for studies of gastrointestinal motility in various mouse models, including transgenic and knockout ones.

Delayed restoration of Mg2+ content and transport in liver cells following ethanol withdrawal

Torres, L. M., Cefaratti, C., Berti-Mattera, L., Romani, A. — Thu, 24 Sep 2009 16:53:46 PDT

Liver cells from rats chronically fed a Lieber-De Carli diet for 3 wk presented a marked decreased in tissue Mg²⁺ content and an inability to extrude Mg²⁺ into the extracellular compartment upon stimulation with catecholamine, isoproterenol, or cell-permeant cAMP analogs. This defect in Mg²⁺ extrusion was observed in both intact cells and purified liver plasma membrane vesicles. Inhibition of adrenergic or cAMP-mediated Mg²⁺ extrusion was also observed in freshly isolated hepatocytes from control rats incubated acutely in vitro with varying doses of ethanol (EtOH) for 8 min. In this model, however, the defect in Mg²⁺ extrusion was observed in intact cells but not in plasma membrane vesicles. In the chronic model, upon removal of EtOH from the diet hepatic Mg²⁺ content and extrusion required ~10 days to return to normal level both in isolated cells and plasma membrane vesicles. In hepatocytes acutely treated with EtOH for 8 min, more than 60 min were necessary for Mg²⁺ content and extrusion to recover and return to the level observed in EtOH-untreated cells. Taken together, these data suggest that in the acute model the defect in Mg²⁺ extrusion is the result of a limited refilling of the cellular compartment(s) from which Mg²⁺ is mobilized upon adrenergic stimulation rather than a mere defect in adrenergic cellular signaling. The chronic EtOH model, instead, presents a transient but selective defect of the Mg²⁺ extrusion mechanisms in addition to the limited refilling of the cellular compartments.

Intestinal cell kinase, a MAP kinase-related kinase, regulates proliferation and G1 cell cycle progression of intestinal epithelial cells

Fu, Z., Kim, J., Vidrich, A., Sturgill, T. W., Cohn, S. M. — Thu, 24 Sep 2009 16:53:46 PDT

Intestinal cell kinase (ICK), originally cloned from the intestine and expressed in the intestinal crypt epithelium, is a highly conserved serine/threonine protein kinase that is similar to mitogen-activated protein kinases (MAPKs) in the catalytic domain and requires dual phosphorylation within a MAPK-like TDY motif for full activation. Despite these similarities to MAPKs, the biological functions of ICK remain unknown. In this study, we report that suppression of ICK expression in cultured intestinal epithelial cells by short hairpin RNA (shRNA) interference significantly impaired cellular proliferation and induced features of gene expression characteristic of colonic or enterocytic differentiation. Downregulation of ICK altered expression of cell cycle regulators (cyclin D1, c-Myc, and p21^Cip1/WAF1) of G₁-S transition, consistent with the G₁ cell cycle delay induced by ICK shRNA. ICK deficiency also led to a significant decrease in the expression and/or activity of p70 ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E), concomitant with reduced expression of their upstream regulators, the mammalian target of rapamycin (mTOR) and the regulatory associated protein of mTOR (Raptor). Furthermore, ICK interacts with the mTOR/Raptor complex in vivo and phosphorylates Raptor in vitro. These results suggest that disrupting ICK function may downregulate protein translation of specific downstream targets of eIF4E and S6K1 such as cyclin D1 and c-Myc through the mTOR/Raptor signaling pathway. Taken together, our findings demonstrate an important role for ICK in proliferation and differentiation of intestinal epithelial cells.

P2Y5 is a G{alpha}i, G{alpha}12/13 G protein-coupled receptor activated by lysophosphatidic acid that reduces intestinal cell adhesion

Lee, M., Choi, S., Hallden, G., Yo, S. J., Schichnes, D., Aponte, G. W. — Thu, 24 Sep 2009 16:53:46 PDT

P2Y5 is a G protein-coupled receptor that binds and is activated by lysophosphatidic acid (LPA). We determined that P2Y5 transcript is expressed along the intestinal mucosa and investigated the intracellular pathways induced by P2Y5 activation, which could contribute to LPA effects on intestinal cell adhesion. P2Y5 heterologously expressed in CHO and small intestinal hBRIE 380i cells was activated by LPA resulting in an increase in intracellular calcium ([Ca²⁺]_i) when the cells concurrently expressed G_6qi5myr. P2Y5 activation also increased the phosphorylation of ERK1/2 that was sensitive to pertussis toxin. Together these indicate that P2Y5 activation by LPA induces an increase in [Ca²⁺]_i and ERK1/2 phosphorylation through G_i. We discovered that P2Y5 was activated by farnesyl pyrophosphate (FPP) without a detectable change in [Ca²⁺]_i. The activation of P2Y5 by LPA or FPP induced the activity of a serum response element (SRE)-linked luciferase reporter that was inhibited by the RGS domain of p115RhoGEF, C3 exotoxin, and Y-27632, suggesting the involvement of G_12/13, Rho GTPase, and ROCK, respectively. However, only LPA-mediated induction of SRE reporter activity was sensitive to inhibitors targeting p38 MAPK, PI3K, PLC, and PKC. In addition, only LPA transactivated the epidermal growth factor receptor, leading to an induction of ERK1/2 phosphorylation. These observations correlate with our subsequent finding that P2Y5 activation by LPA, and not FPP, reduced intestinal cell adhesion. This study elucidates a mechanism whereby LPA can act as a luminal and/or serosal cue to alter mucosal integrity.

Extrinsic afferent nerve sensitivity and enteric neurotransmission in murine jejunum in vitro

Mueller, M. H., Xue, B., Glatzle, J., Hahn, J., Grundy, D., Kreis, M. E. — Thu, 24 Sep 2009 16:53:46 PDT

Enteric and extrinsic sensory neurons respond to similar stimuli. Thus they may be activated in series or in parallel. Because signal transmission via synapses or mediator release would depend on calcium, we investigated its role for extrinsic afferent sensitivity to chemical and mechanical stimulation. Extracellular multiunit afferent recordings were made in vitro from paravascular nerve bundles supplying the mouse jejunum. Intraluminal pressure and afferent nerve responses were recorded under control conditions and under four conditions designed to interfere with enteric neurotransmission. We found that phasic intestinal contractions ceased after switching perfusion to Ca²⁺-free buffer with or without a purinergic P2 receptor antagonist, pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) or cadmium (blocking all Ca²⁺-channels) but not following -conotoxin GVIA (N-type Ca²⁺-channel blocker). Luminal HCl (pH 2) and 5-HT (500 µM) evoked peak firing of 17 ± 4 impulses per second (imp/s) (n = 10) and 21 ± 4 imp/s (n = 13) under control conditions. These responses were reduced to 4 ± 2 imp/s and 5 ± 2 imp/s by cadmium (n = 7, P < 0.05), to 7 ± 2 imp/s and 6 ± 1 imp/s by Ca²⁺-free perfusion (n = 6, P < 0.05), and to 3 ± 1 imp/s and 4 ± 1 imp/s by Ca²⁺-free perfusion with PPADS (n = 6, P < 0.05). Responses were unchanged by -conotoxin GVIA. Mechanical ramp distension of the intestinal segment to 60 cmH₂O was not altered by any of the experimental conditions. We concluded that HCl and 5-HT activate extrinsic afferents via a calcium-dependent mechanism, which is unlikely to involve enteric neurons carrying N-type calcium channels. Extrinsic mechanosensitivity is independent of enteric neurotransmission. It appears that cross talk from the enteric to the extrinsic nervous system does not mediate extrinsic afferent sensitivity.

GLP-1 secretion is enhanced directly in the ileum but indirectly in the duodenum by a newly identified potent stimulator, zein hydrolysate, in rats

Hira, T., Mochida, T., Miyashita, K., Hara, H. — Thu, 24 Sep 2009 16:53:46 PDT

Glucagon-like peptide-1 (GLP-1) is released from enteroendocrine cells (L cells) in response to food ingestion. The mechanism by which dietary peptides stimulate GLP-1 secretion in the gut is unknown. In the present study, we found that a hydrolysate prepared from zein, a major corn protein [zein hydrolysate (ZeinH)], strongly stimulates GLP-1 secretion in enteroendocrine GLUTag cells. Stimulatory mechanisms of GLP-1 secretion induced by ZeinH were investigated in the rat small intestine under anesthesia. Blood was collected through a portal catheter before and after ZeinH administration into different sites of the small intestine. The duodenal, jejunal, and ileal administration of ZeinH induced dose-dependent increases in portal GLP-1 concentration. GLP-1 secretion in response to the ileal administration of ZeinH was higher than that in the duodenal or jejunal administration. Capsaicin treatment on esophageal vagal trunks abolished the GLP-1 secretion induced by duodenal ZeinH but did not affect the secretion induced by jejunal or ileal ZeinH. These results suggest that ZeinH in the jejunum or ileum directly stimulates GLP-1 secretion but duodenal ZeinH indirectly stimulates GLP-1 secretion via the vagal afferent nerve. A direct blood sampling method from the duodenal vein and ileal mesenteric vein revealed that ZeinH administered into the ligated duodenal loop enhanced GLP-1 concentration in the ileal mesenteric vein but not in the duodenal vein. This confirmed that ZeinH in the duodenum induces GLP-1 secretion from L cells located in the ileum by an indirect mechanism. These results indicate that a potent GLP-1-releasing peptide, ZeinH, induces GLP-1 secretion by direct and indirect mechanisms in the rat intestine.

Effects of electrical stimulation on isolated rodent gastric smooth muscle cells evaluated via a joint computational simulation and experimental approach

Du, P., Li, S., O'Grady, G., Cheng, L. K., Pullan, A. J., Chen, J. D. Z. — Thu, 24 Sep 2009 16:53:46 PDT

Gastric electrical stimulation (GES) involves the delivery of electrical impulses to the stomach for therapeutic purposes. New GES protocols are needed that are optimized for improved motility outcomes and energy efficiency. In this study, a biophysically based smooth muscle cell (SMC) model was modified on the basis of experimental data and employed in conjunction with experimental studies to define the effects of a large range of GES protocols on individual SMCs. For the validation studies, rat gastric SMCs were isolated and subjected to patch-clamp analysis during stimulation. Experimental results were in satisfactory agreement with simulation results. The results define the effects of a wide range of GES parameters (pulse width, amplitude, and pulse-train frequency) on isolated SMCs. The minimum pulse width required to invoke a supramechanical threshold response from SMCs (defined at –30 mV) was 65 ms (at 250-pA amplitude). The minimum amplitude required to invoke this threshold was 75 pA (at 1,000-ms pulse width). The amplitude of the invoked response beyond this threshold was proportional to the stimulation amplitude. A high-frequency train of stimuli (40 Hz; 10 ms, 150 pA) could invoke and maintain the SMC plateau phase while requiring 60% less power and accruing ~30% less intracellular Ca²⁺ concentration during the plateau phase than a comparable single-pulse protocol could in a demonstrated example. Validated computational simulations are an effective strategy for efficiently identifying effective minimum-energy GES protocols, and pulse-train protocols may also help to reduce the power consumption of future GES devices.

Hepatic uptake of {gamma}-butyrobetaine, a precursor of carnitine biosynthesis, in rats

Thu, 24 Sep 2009 16:53:46 PDT

-Butyrobetaine (GBB) is a precursor in the biosynthesis of carnitine, which plays an important role in the β-oxidation of fatty acids, and is converted to carnitine by -butyrobetaine dioxygenase (BBD) predominantly in liver. We investigated the molecular mechanism of hepatic uptake of GBB in rat hepatocytes. Cellular localization of rat Octn2 (rOctn2:Slc22A5) was studied by Western blot analysis. Uptake of deuterated GBB (d₃-GBB) was examined in HEK293 cells expressing rOctn2 (HEK293/rOctn2) and freshly isolated rat hepatocytes. d₃-GBB was quantified by use of liquid chromatography-tandem mass spectrometry. Western blot analysis demonstrated an expression of OCTN2 protein in hepatic basolateral membrane but not in bile canalicular membrane fraction. Furthermore, we found that d₃-GBB was taken up by rOctn2 in an Na⁺-dependent manner with K_m value of 13 µM. The apparent K_m value for d₃-GBB transport in freshly isolated rat hepatocytes was 9 µM. d₃-GBB uptake by the rat hepatocytes was inhibited by -aminobutyric acid (GABA) to 30% of the control, whereas it was inhibited by carnitine to 62% of the control, even at 500 µM. Furthermore, d₃-GBB uptake by rat hepatocytes was decreased by 45% with rat Gat2 (Slc6A13, a major liver GABA transporter) silenced by the microRNA method. Accordingly, the present study clearly demonstrates that GBB is taken up by hepatocytes for carnitine biosynthesis not only via Octn2 but also via the GABA transporter, possibly Gat2.

Mg2+- and MgATP-inhibited and Ca2+/calmodulin-sensitive TRPM7-like current in hepatoma and hepatocytes

Mishra, R., Rao, V., Ta, R., Shobeiri, N., Hill, C. E. — Thu, 24 Sep 2009 16:53:46 PDT

Although understood to be ubiquitously expressed, the functional identification and significance of Mg²⁺-inhibited, nonspecific cation currents has been established in only a few cell types. Here we identified an outwardly rectifying nonspecific cation current in quiescent rat hepatocytes and the proliferating and polarized rat hepatoma, WIF-B. Under whole cell recording conditions in which cells were bathed and dialyzed with Na-gluconate solutions, the latter Ca²⁺ and Mg²⁺ free, current reversed close to 0 mV, was time independent, and was greater than 10 times higher at +120 mV compared with –120 mV. Outward current at –120 mV developed slowly, from 17.7 ± 10.3 pA/pF at patch rupture to 106.6 ± 15.6 pA/pF at 12 min in WIF-B cells, and 4.9 ± 2.7 to 20.6 ± 5.6 pA/pF in rat hepatocytes. The nonspecific TRP channel inhibitor, 2-aminoethoxyphenylborate (2-APB), inhibited current (IC₅₀ = 72 ± 13 µM) and caused apoptotic cell death in WIF-B cells. Rat hepatocyte survival was more resistant to 2-APB. Dialysis of WIF-B cells with physiological concentrations of Mg²⁺ and Mg-ATP, but not ATP alone, inhibited current development, suggesting that Trpm7 rather than Trpm6 underlies this current. RT-PCR demonstrated that both Trpm6 and Trpm7 are expressed at similar levels in both cell types, suggesting that the functional differences noted are not transcript dependent. Intracellular Ca²⁺ (IC₅₀ = 125 ± 35 nM) also inhibited current development, and this could be partially relieved by the calmodulin and Ca²⁺/calmodulin-dependent kinase inhibitors W-7, staurosporine, KN-93, or calmodulin kinase II (CaMKII) inhibitory peptide. To summarize, our results show that in addition to their established Mg²⁺ sensitivity, Trpm7-like channels are inhibited by cytosolic Ca²⁺ in a CaMKII-dependent manner and may support hepatocellular survival during proliferation.

Alternative splicing of the Menkes copper Atpase (Atp7a) transcript in the rat intestinal epithelium

Collins, J. F., Hua, P., Lu, Y., Ranganathan, P. N. — Thu, 24 Sep 2009 16:53:46 PDT

The intestinal Menkes copper Atpase (Atp7a) gene is strongly induced by iron deficiency in the rat intestine. We sought to develop an in vitro model to understand the mechanism of this induction by performing molecular studies in native rat intestine and in intestinal epithelial (IEC-6) cells. IEC-6 cells express Atp7a, and induction was noted with iron deprivation. 5' Rapid amplification of cDNA ends and PCR experiments revealed three splice variants in rat intestine and IEC-6 cells; all variants were strongly induced during iron deprivation (five- to sevenfold). The splice variants presumably encode proteins that would either contain the extreme NH₂ terminus of the protein (containing copper binding domain 1) or not. We thus hypothesized that more than one version of Atp7a protein exists. Antibodies against this NH₂-terminal region of the protein were developed (named N-term) and used along with previously reported antibodies (against more COOH-terminal regions, termed 54–10) to perform immunoblotting and immunolocalization studies. Results with the 54–10 antiserum revealed an Atp7a protein variant of ~190 kDa that localized to the trans-Golgi network of IEC-6 cells and trafficked to the plasma membrane with copper loading. Using the N-term antiserum, however, we noted protein of ~97 and 64 kDa. The 97-kDa protein was cytosolic and nuclear, whereas the 64-kDa protein was nuclear specific. Immunolocalization analyses with the N-term antiserum showed strong staining of nuclei in IEC-6 and Caco-2 cells and in rat intestine. We conclude that novel Atp7a protein variants may exist in rat and human intestinal epithelial cells, with different intracellular locations and potentially distinct physiological functions.

Endocytic trafficking from the small intestinal brush border probed with FM dye

Hansen, G. H., Rasmussen, K., Niels-Christiansen, L.-L., Danielsen, E. M. — Thu, 24 Sep 2009 16:53:46 PDT

The small intestinal brush border functions as the body's main portal for uptake of dietary nutrients and simultaneously acts as the largest permeability barrier against pathogens. To enable this, the digestive enzymes of the brush border are organized in lipid raft microdomains stabilized by cross-linking galectins and intelectin, but little is known about the dynamic properties of this highly specialized membrane. Here, we probed the endocytic membrane trafficking from the brush border of organ-cultured pig intestinal mucosal explants by use of a fixable, lipophilic FM dye. The fluorescent dye readily incorporated into the brush border, and by 15 min faint but distinct punctae were detectable ~1 µm beneath the brush border, indicative of a constitutive endocytosis. The punctae represented a subpopulation of early endosomes confined to the actomyosin-rich terminal web region, and their number and intensity increased by 1 h, but trafficking further into the enterocyte was not observed except in immature epithelial cells of the crypts. A powerful ligand for receptor-mediated endocytosis, cholera toxin B subunit, increased apical endocytosis and caused membrane trafficking to proceed to compartments localized deeper into the cytoplasm of the enterocytes. Two major raft-associated brush border enzymes, alkaline phosphatase and aminopeptidase N, were excluded from endocytosis. We propose that the terminal web cytoskeleton, by inhibiting traffic from apical early endosomes further into the cell, contributes to the overall permeability barrier of the gut.

Homeostatic and therapeutic roles of VIP in smooth muscle function: myo-neuroimmune interactions

Shi, X.-Z., Sarna, S. K. — Thu, 24 Sep 2009 16:53:46 PDT

We tested the hypothesis that spontaneous release of vasoactive intestinal peptide (VIP) from enteric neurons maintains homeostasis in smooth muscle function in mild inflammatory insults and that infusion of exogenous VIP has therapeutic effects on colonic smooth muscle dysfunction in inflammation. In vitro experiments were performed on human colonic circular smooth muscle tissues and in vivo on rats. The incubation of human colonic circular smooth muscle strips with TNF- suppressed their contractile response to ACh and the expression of the pore-forming _1C subunit of Ca_v1.2 channels. VIP reversed both effects by blocking the translocation of NF-B to the nucleus and its binding to the B recognition sites on h_1C1b promoter. The translocation of NF-B was inhibited by blocking the degradation of IBβ. Induction of inflammation by a subthreshold dose of 17 mg/kg trinitrobenzene sulfonic acid (TNBS) in rats moderately decreased muscularis externa concentration of VIP, and it had little effect on the contractile response of circular smooth muscle strips to ACh. The blockade of VIP and pituitary adenylate cyclase-activating peptide receptors 1/2 during mild inflammatory insult significantly worsened the suppression of contractility and the inflammatory response. The induction of more severe inflammation by 68 mg/kg TNBS induced marked suppression of colonic circular muscle contractility and decrease in serum VIP. Exogenous infusion of VIP by an osmotic pump reversed these effects. We conclude that the spontaneous release of VIP from the enteric motor neurons maintains homeostasis in smooth muscle function in mild inflammation by blocking the activation of NF-B. The infusion of exogenous VIP mitigates colonic inflammatory response and smooth muscle dysfunction.

Effects of losartan on hepatic expression of nonphagocytic NADPH oxidase and fibrogenic genes in patients with chronic hepatitis C

Thu, 24 Sep 2009 16:53:46 PDT

Angiotensin II promotes liver fibrogenesis by stimulating nonphagocytic NADPH oxidase (NOX)-induced oxidative stress. Angiotensin II type 1 (AT1) receptor blockers attenuate experimental liver fibrosis, yet their effects in human liver fibrosis are unknown. We investigated the effects of losartan on hepatic expression of fibrogenic, inflammatory, and NOX genes in patients with chronic hepatitis C (CHC). Fourteen patients with CHC and liver fibrosis received oral losartan (50 mg/day) for 18 mo. Liver biopsies were performed at baseline and after treatment. The degree of inflammation and fibrosis was evaluated by histological analysis (METAVIR). Collagen content was measured by morphometric quantification of Sirius red staining. Overall collagen content and fibrosis stage remained stable in the whole series, yet the fibrosis stage decreased in seven patients. Inflammatory activity improved in seven patients. The effect of losartan on hepatic expression of 31 profibrogenic and inflammatory genes and components of the NOX complex was assessed by quantitative PCR. Losartan treatment was associated with a significant decrease in the expression of several profibrogenic and NOX genes including procollagen 1(I) and 1(IV), urokinase-type plasminogen activator, metalloproteinase type 2, NOX activator 1 (NOXA-1) and organizer 1 (NOXO-1), and Rac-1. Losartan was well tolerated in all patients and was effective in attenuating the activity of the systemic renin-angiotensin system. No effects on serum liver tests or viral load were observed. We conclude that prolonged administration of losartan, an oral AT1 receptor blocker, is associated with downregulation of NOX components and fibrogenic genes in patients with CHC. Controlled studies are warranted to assess the effect of AT1 receptor blockers in chronic liver injury.

Vasoactive intestinal peptide ameliorates intestinal barrier disruption associated with Citrobacter rodentium-induced colitis

Thu, 24 Sep 2009 16:53:46 PDT

Attaching and effacing bacterial pathogens attach to the apical surface of epithelial cells and disrupt epithelial barrier function, increasing permeability and allowing luminal contents access to the underlying milieu. Previous in vitro studies demonstrated that the neuropeptide vasoactive intestinal peptide (VIP) regulates epithelial paracellular permeability, and the high concentrations and close proximity of VIP-containing nerve fibers to intestinal epithelial cells would support such a function in vivo. The aim of this study was to examine whether VIP treatment modulated Citrobacter rodentium-induced disruption of intestinal barrier integrity and to identify potential mechanisms of action. Administration of VIP had no effect on bacterial attachment although histopathological scoring demonstrated a VIP-induced amelioration of colitis-induced epithelial damage compared with controls. VIP treatment prevented the infection-induced increase in mannitol flux a measure of paracellular permeability, resulting in levels similar to control mice, and immunohistochemical studies demonstrated that VIP prevented the translocation of tight junction proteins: zonula occludens-1, occludin, and claudin-3. Enteropathogenic Escherichia coli (EPEC) infection of Caco-2 monolayers confirmed a protective role for VIP on epithelial barrier function. VIP prevented EPEC-induced increase in long myosin light chain kinase (MLCK) expression and myosin light chain phosphorylation (p-MLC). Furthermore, MLCK inhibition significantly attenuated bacterial-induced epithelial damage both in vivo and in vitro. In conclusion, our results indicate that VIP protects the colonic epithelial barrier by minimizing bacterial-induced redistribution of tight junction proteins in part through actions on MLCK and MLC phosphorylation.

Mice lacking neurofibromin develop gastric hyperplasia

Lin, L., Chen, J., Richardson, J. A., Parada, L. F. — Thu, 24 Sep 2009 16:53:46 PDT

Gastrointestinal (GI) neoplasms are among many manifestations of the genetic disease neurofibromatosis type 1 (NF1). However, the physiological and pathological functions of the Nf1 gene in the GI system have not been fully studied, possibly because of a lack of mouse models. In this study, we generated conditional knockout mice with Nf1 deficiency in the GI tract. These mice develop gastric epithelial hyperplasia and inflammation together with increased cell proliferation and apoptosis. The gastric phenotypes observed in these mutant mice seem to be the consequence of loss of Nf1 in gastric fibroblasts, resulting in paracrine hyperactivation of the ERK pathway in the gastric epithelium. These mice provide a useful model to study the pathogenesis of GI lesions in a subset of patients with NF1 and to investigate the role of the Nf1 gene in the development of GI neoplasms.

Urotensin II modulates hepatic fibrosis and portal hemodynamic alterations in rats

Thu, 24 Sep 2009 16:53:46 PDT

The influence of circulating urotensin II (UII) on liver disease and portal hypertension is unknown. We aimed to evaluate whether UII executes a pathogenetic role in the development of hepatic fibrosis and portal hypertension. UII was administered by continuous infusion over 4 wk in 20 healthy rats divided into three treatment groups, controls (saline, n = 7), low dose (UII, 1 nmol·kg^–1·h^–1, n = 8), and high dose (UII, 3 nmol·kg^–1·h^–1, n = 5). Hemodynamic parameters and morphometric quantification of fibrosis were assessed, and profibrotic cytokines and fibrosis markers were assayed in hepatic tissue. UII induced a significant dose-dependent increase in portal venous pressure (5.8 ± 0.4, 6.4 ± 0.3, and 7.6 ± 0.7, respectively, P = 0.03). High-dose UII infusion was associated with an increase in hepatic transcript for transforming growth factor-β (P < 0.05) and platelet-derived growth factor-β (P = 0.06). Liver tissue hydroxyproline was elevated in the high-dose group (P < 0.05). No systemic hemodynamic alterations were noted. We concluded that UII infusion elevates portal pressure and induces hepatic fibrosis in normal rats. This response may be mediated via induction of fibrogenic cytokines. These findings have pathophysiological implications in human liver disease where increased plasma UII levels have been observed.

Upregulation of activin signaling in experimental colitis

Zhang, Y.-Q., Resta, S., Jung, B., Barrett, K. E., Sarvetnick, N. — Thu, 24 Sep 2009 16:53:46 PDT

Several lines of studies have suggested that activins are critical mediators of inflammation and tissue repair. As activins and their receptors are expressed in the gastrointestinal tract, we tested the hypothesis that activin signaling is involved in the development of colitis by using two murine models of colitis induced by dextran sodium sulfate (DSS) or in mdr1a–/– mice. By immunohistochemistry, expression of activins was found increased in both models and correlated with the severity of inflammation. Activin expression was observed in macrophages as well as in some nonmacrophage cells. Furthermore, while activin receptors are normally expressed in colonic epithelial cells, their expression was further increased in both epithelial cells and inflammatory cells in inflamed colonic mucosa. Moreover, in vitro studies showed that activin A inhibited proliferation and induced apoptosis of intestinal epithelial cells, and this growth inhibition was largely reversed by administration of the activin inhibitor, follistatin. Because we also observed an increased number of apoptotic epithelial cells in both colitis models, the upregulation of activins occurring in colitis could be involved both in the inflammatory process and in growth inhibition of the intestinal epithelium. Importantly, in vivo administration of follistatin attenuated inflammatory cell infiltration during colitis. Rectal bleeding was reduced, and the integrity of epithelium was preserved in the DSS/follistatin-treated group compared with the group treated with DSS alone. Bromodeoxyuridine incorporation studies showed an increase in proliferative epithelial cells in the DSS/follistatin-treated group, suggesting that follistatin accelerates epithelial cell proliferation/repair during colitis. Overall, our results reveal that activin signaling may play an important role in the pathogenesis and resolution of colitis. These findings suggest new therapeutic options in inflammatory bowel diseases.

Luminal L-glutamate enhances duodenal mucosal defense mechanisms via multiple glutamate receptors in rats

Akiba, Y., Watanabe, C., Mizumori, M., Kaunitz, J. D. — Thu, 24 Sep 2009 16:53:46 PDT

Presence of taste receptor families in the gastrointestinal mucosa suggests a physiological basis for local and early detection of a meal. We hypothesized that luminal l-glutamate, which is the primary nutrient conferring fundamental umami or proteinaceous taste, influences mucosal defense mechanisms in rat duodenum. We perfused the duodenal mucosa of anesthetized rats with l-glutamate (0.1–10 mM). Intracellular pH (pH_i) of the epithelial cells, blood flow, and mucus gel thickness (MGT) were simultaneously and continuously measured in vivo. Some rats were pretreated with indomethacin or capsaicin. Duodenal bicarbonate secretion (DBS) was measured with flow-through pH and CO₂ electrodes. We tested the effects of agonists or antagonists for metabotropic glutamate receptor (mGluR) 1 or 4 or calcium-sensing receptor (CaSR) on defense factors. Luminal l-glutamate dose dependently increased pH_i and MGT but had no effect on blood flow in the duodenum. l-glutamate (10 mM)-induced cellular alkalinization and mucus secretion were inhibited by pretreatment with indomethacin or capsaicin. l-glutamate effects on pH_i and MGT were mimicked by mGluR4 agonists and inhibited by an mGluR4 antagonist. CaSR agonists acidified cells with increased MGT and DBS, unlike l-glutamate. Perfusion of l-glutamate with inosinate (inosine 5'-monophosphate, 0.1 mM) enhanced DBS only in combination, suggesting synergistic activation of the l-glutamate receptor, typical of taste receptor type 1. l-leucine or l-aspartate had similar effects on DBS without any effect on pH_i and MGT. Preperfusion of l-glutamate prevented acid-induced cellular injury, suggesting that l-glutamate protects the mucosa by enhancing mucosal defenses. Luminal l-glutamate may activate multiple receptors and afferent nerves and locally enhance mucosal defenses to prevent subsequent injury attributable to acid exposure in the duodenum.

Role of endothelial nitric oxide synthase in the development of portal hypertension in the carbon tetrachloride-induced liver fibrosis model

Thu, 24 Sep 2009 16:53:46 PDT

Portal hypertension (PHT) is a complication of liver cirrhosis and directly increases mortality and morbidity by increasing the propensity of venous hemorrhage. There are two main underlying causations for PHT, increased hepatic resistance and systemic hyperdynamic circulation. Both are related to localized aberrations in endothelial nitric oxide synthase (eNOS) function and NO biosynthesis. This study investigates the importance of eNOS and systemic hyperdynamic-associated hyperemia to better understand the pathophysiology of PHT. Wild-type and eNOS^–/– mice were given the hepatotoxin CCl₄ for 4–12 wk. Hepatic fibrosis was determined histologically following collagen staining. Portal venous pressure, hepatic resistance, and hyperemia were determined by measuring splenic pulp pressure (SPP), hepatic portal-venous perfusion pressure (HPVPP), abdominal aortic flow (Qao), and portal venous flow (Qpv). Hepatic fibrosis developed equally in wild-type and eNOS^–/– CCl₄-exposed mice. SPP, Qao, and Qpv increased rapidly in wild-type CCl₄-exposed mice, but HPVPP did not. In eNOS^–/– CCl₄ mice, Qao was not increased, SPP was partially increased, and HPVPP and Qpv were increased nonsignificantly. We concluded that the systemic hyperemia component of hyperdynamic circulation is eNOS dependent and precedes increased changes in hepatic resistance. Alternative mechanisms, possibly involving cyclooxygenase, may contribute. eNOS maintains normal hepatic resistance following CCl₄-induced fibrosis. Consequently, increased portal pressure following chronic CCl₄ exposure is linked to hyperdynamic circulation in wild-type mice and increased hepatic resistance in eNOS^–/– mice.

Glucagon-like peptide-2 modulates neurally evoked mucosal chloride secretion in guinea pig small intestine in vitro

Baldassano, S., Liu, S., Qu, M.-H., Mule, F., Wood, J. D. — Thu, 24 Sep 2009 16:53:46 PDT

Glucagon-like peptide-2 (GLP-2) is an important neuroendocrine peptide in intestinal physiology. It influences digestion, absorption, epithelial growth, motility, and blood flow. We studied involvement of GLP-2 in intestinal mucosal secretory behavior. Submucosal-mucosal preparations from guinea pig ileum were mounted in Ussing chambers for measurement of short-circuit current (I_sc) as a surrogate for chloride secretion. GLP-2 action on neuronal release of acetylcholine was determined with ELISA. Enteric neuronal expression of the GLP-2 receptor (GLP-2R) was studied with immunohistochemical methods. Application of GLP-2 (0.1–100 nM) to the serosal or mucosal side of the preparations evoked no change in baseline I_sc and did not alter transepithelial ionic conductance. Transmural electrical field stimulation (EFS) evoked characteristic biphasic increases in I_sc, with an initially rapid rising phase followed by a sustained phase. Application of GLP-2 reduced the EFS-evoked biphasic responses in a concentration-dependent manner. The GLP-2R antagonist GLP-2-(3-33) significantly reversed suppression of the EFS-evoked responses by GLP-2. Tetrodotoxin, scopolamine, and hexamethonium, but not vasoactive intestinal peptide type 1 receptor (VPAC1) antagonist abolished or reduced to near zero the EFS-evoked responses. GLP-2 suppressed EFS-evoked acetylcholine release as measured by ELISA. Pretreatment with GLP-2-(3-33) offset this action of GLP-2. In the submucosal plexus, GLP-2R immunoreactivity (-IR) was expressed in choline acetyltransferase-IR neurons, somatostatin-IR neurons, neuropeptide Y-IR neurons, and vasoactive intestinal peptide-IR neurons. We conclude that submucosal neurons in the guinea pig ileum express GLP-2R. Activation of GLP-2R decreases neuronally evoked epithelial chloride secretion by suppressing acetylcholine release from secretomotor neurons.

Role of PSD95 in membrane association and catalytic activity of nNOS{alpha} in nitrergic varicosities in mice gut

Chaudhury, A., He, X.-D., Goyal, R. K. — Thu, 24 Sep 2009 16:53:46 PDT

We have recently shown that membrane association of neuronal nitric oxide synthase- (nNOS) is critical in the regulation of synthesis of NO during nitrergic neurotransmission. The purpose of this study was to examine the role of the synapse-associated proteins (SAPs) in membrane association of nNOS. Varicosities (swellings on terminal axons) were isolated from mice gastrointestinal tract and examined for nNOS, postsynaptic density protein 95 (PSD95), and membrane interactions by coimmunoprecipitation and SDS-PAGE. Our results show that PSD95 protein was present in the membrane fraction of the nerve varicosity, whereas both PSD95 and SAP97 were present in the cytosol. nNOS was associated with PSD95 but not SAP97. nNOS-PSD95 complex was bound to the membrane via palmitoylation of PSD95. Depalmitoylation of PSD95 with 2-bromopalmitate dislocates nNOS and PSD95 from the varicosity membrane and abolishes NO production. These studies show that palmitoylation of PSD95 anchors nNOS to the varicosity membrane and that it is obligatory for NO production by the enzyme. Palmitoylation of PSD95 may provide a novel target for regulation of nitrergic neurotransmission.

Interstitial cells of Cajal generate spontaneous transient depolarizations in the rat gastric fundus

Kito, Y., Sanders, K. M., Ward, S. M., Suzuki, H. — Thu, 24 Sep 2009 16:53:46 PDT

Intracellular recordings were made from isolated circular muscle bundles of rat gastric fundus. The majority of cells generated an ongoing discharge of electrical activity that were ≤10 mV in amplitude (unitary potentials). A second pattern of electrical activity was recorded in less than 1% of all impalements. This electrical activity was characterized by high frequency, large amplitude spontaneous transient depolarizations (STDs) with a maximum rate of rise (dV/dt_max) of 0.5 V/s. Injection of the fluorescent dye propidium iodide into cells and double labeling with an antibody against the Kit receptor revealed that unitary potentials were recorded from circular smooth muscle cells (CSMC), whereas STDs were generated by intramuscular interstitial cells of Cajal (ICC-IM). Sustained injection periods (>15 min) resulted in the spread of dye between CSMC, between ICC-IM, and between CSMC and ICC-IM. Two types of STDs were observed, regularly occurring continuous STDs and irregular noisy bursting STDs. The amplitude of STDs varied between the two types of STDs. Single units summed to develop STDs with a maximum amplitude of 30 mV. Sodium nitroprusside (3 µM) induced membrane hyperpolarization and abolished unitary potentials generated by CSMC. In contrast, the amplitude of STDs generated by ICC-IM was increased with membrane hyperpolarization. Hyperpolarization induced by pinacidil (10 µM) also increased the amplitude of STDs and enhanced dV/dt_max. These observations indicate that STDs generated in ICC-IM spread passively to the adjacent CSMC to evoke the discharge of unitary potentials in the gastric fundus.

Enteropathogenic Escherichia coli inhibits intestinal vitamin B1 (thiamin) uptake: studies with human-derived intestinal epithelial Caco-2 cells

Ashokkumar, B., Kumar, J. S., Hecht, G. A., Said, H. M. — Thu, 24 Sep 2009 16:53:46 PDT

Infection with the gram-negative enteropathogenic Escherichia coli (EPEC), a food-borne pathogen, represents a significant risk to human health. Whereas diarrhea is a major consequence of this infection, malnutrition also occurs especially in severe and prolonged cases, which may aggravate the health status of the infected hosts. Here we examined the effect of EPEC infection on the intestinal uptake of the water-soluble vitamin B1 (thiamin) using an established human intestinal epithelial Caco-2 cell model. The results showed that infecting Caco-2 cells with wild-type EPEC (but not with nonpathogenic E. coli, killed EPEC, or filtered supernatant) leads to a significant (P < 0.01) inhibition in thiamin uptake. Kinetic parameters of both the nanomolar (mediated by THTR-2) and the micromolar (mediated by THTR-1) saturable thiamin uptake processes were affected by EPEC infection. Cell surface expression of hTHTR-1 and -2 proteins, (determined by the biotinylation method) showed a significantly (P < 0.01) lower expression in EPEC-treated cells compared with controls. EPEC infection also affected the steady-state mRNA levels as well as promoter activity of the SLC19A2 and SLC19A3 genes. Infecting Caco-2 cells with EPEC mutants that harbor mutations in the escN gene (which encodes a putative ATPase for the EPEC type III secretion system, TTSS) or the espA, espB, or espD genes (which encode structural components of the TTSS) did not affect thiamin uptake. On the other hand, mutations in espF and espH genes (which encode effector proteins) exhibited partial inhibition in thiamin uptake. These results demonstrate for the first time that EPEC infection of human intestinal epithelial cells leads to inhibition in thiamin uptake via effects on physiological and molecular parameters of hTHTR-1 and -2. Furthermore, the inhibition appears to be dependent on a functional TTSS of EPEC.

Calcium-dependent and calcium-independent inhibition of contraction by cGMP/cGKI in intestinal smooth muscle

Frei, E., Huster, M., Smital, P., Schlossmann, J., Hofmann, F., Wegener, J. W. — Thu, 24 Sep 2009 16:53:46 PDT

cGMP-dependent protein kinase I (cGKI) induces relaxation of smooth muscle via several pathways that include inhibition of intracellular Ca²⁺ signaling and/or involve activation of myosin phosphatase. In the present study, we investigated these mechanisms comparatively in colon and jejunum longitudinal smooth muscle from mice. In simultaneous recordings from colon muscle, 8-bromo-cGMP (8-Br-cGMP) reduced both carbachol-induced tension and carbachol-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i). These effects of 8-Br-cGMP were absent in colon from mice carrying a mutated inositol-1,4,5 trisphosphate receptor I-associated G kinase substrate (IRAG) gene or lacking cGKI. However, in jejunum, 8-Br-cGMP reduced carbachol-induced tension but did not change corresponding [Ca²⁺]_i signals. This setting was also observed in jejunum from mice carrying a mutated IRAG gene, whereas no response to 8-Br-cGMP was observed in jejunum from mice lacking cGKI. After inhibition of phosphatase activity by calyculin A, 8-Br-cGMP did not relax jejunum but still relaxed colon muscle. In Western blot analysis, 8-Br-cGMP reduced the signal for phosphorylated MYPT-1 in carbachol-stimulated jejunum but not in colon. These results suggest that cGMP/cGKI signaling differentially inhibits contraction in the muscles investigated: in jejunum, inhibition is performed without changing [Ca²⁺]_i and is dependent on phosphatase activity, whereas in colon, inhibition is mediated by inhibition of [Ca²⁺]_i signals.

Extracellular activation of arginase-1 decreases enterocyte inducible nitric oxide synthase activity during systemic inflammation

Miki, K., Kumar, A., Yang, R., Killeen, M. E., Delude, R. L. — Thu, 24 Sep 2009 16:53:46 PDT

Liver dysfunction secondary to severe inflammation is associated with the release of enzymes normally sequestered within hepatocytes. The purpose of these studies was to test the hypothesis that these enzymes are released, at least in part, to modulate potentially deleterious inflammatory processes in distant tissues like the gut. Human Caco-2_BBe enterocyte-like cells were exposed to cytomix (IFN-, TNF-, and IL-1β) in the absence or presence of human liver cytosol (LC). Nitric oxide (NO^•) and inducible nitric oxide synthase (iNOS) protein production were measured by the Griess assay and Western analysis, respectively. Cytomix induced the expression of iNOS and release of NO^•. LC protein (400 µg/ml) added to the basal compartment but not apical compartment completely blocked the release of NO^• but only slightly decreased the magnitude of iNOS protein induction. Ultrafiltration and ultracentrifugation studies demonstrated that microsome-associated arginase-1 activity was the iNOS-suppressing activity in LC. Liver arginase required activation by a <10-kDa factor that was present in supernatants of cytomix-stimulated cells. The selective iNOS inhibitor l-N⁶-(1-iminoethyl)-lysine·2HCl prevented production of this factor. The biotin switch assay detected increased S-nitrosylation of arginase-1 after incubation with supernatants from immunostimulated Caco-2 cells. Serum from endotoxemic mice contained significantly greater arginase activity compared with serum from control mice. Furthermore, the ratio of mucosal monomeric to dimeric iNOS increased in endotoxemic mice compared with controls. Thus reciprocal activation of arginase-1 and modulation of mucosal iNOS activity may be protective because it would be expected to decrease NO^•-dependent intestinal barrier dysfunction on that basis.

Alterations in mechanical properties of mesenteric resistance arteries in experimental portal hypertension

Thu, 24 Sep 2009 16:53:46 PDT

Splanchnic vasodilation is the pathophysiological hallmark in the development of the hyperdynamic circulatory syndrome in liver cirrhosis and portal hypertension. This has been attributed so far mainly to a marked vascular hyporeactivity to endogenous vasoconstrictors. However, myogenic tone and vessel stiffness have not been addressed in mesenteric arteries in liver cirrhosis. CCl₄^–-induced ascitic cirrhotic (LC) and age-matched control rats, portal vein-ligated (PVL) rats, and sham-operated rats were investigated. Third-order mesenteric resistance arteries were studied under no-flow conditions using a pressure myograph measuring media thickness and lumen diameter in response to incremental increases in intramural pressure, from which wall mechanics were calculated. Electron microscopy was used for investigation of wall ultrastructure, especially the fenestrae in internal elastic lamina (IEL). In PVL animals, no significant change in passive vessel strain, stress, media-to-lumen ratio, or cross-sectional area was noted. In contrast, in LC rats, vessel strain was markedly elevated compared with healthy control rats, indicating a marked reduction in vessel stiffness. In addition, the strain-stress curve was shifted to the right, and the elastic modulus in dependency on vessel stress decreased, demonstrating predominantly structure-dependent factors to be involved. The media-to-lumen quotient was not significantly altered, but cross-sectional area was highly increased in LC rats, indicating hypertrophic outward remodeling. These findings were paralleled by enlarged fenestrae in the IEL but no change in thickness of IEL or proportion of extracellular matrix or vascular smooth muscle in LC rats. We concluded that, in long-standing severe portal hypertension such as ascitic LC but not in short-term conditions such as PVL, mesenteric resistance arteries exhibit vascular remodeling and markedly less resistant mechanical properties, leading to decreased vessel stiffness accompanied by structural changes in the IEL. This may well contribute to the maintenance and severity of splanchnic arterial vasodilation in LC.

Shedding gloomy light into the black box of the Ussing chamber

Lucas, M. L. — Thu, 24 Sep 2009 16:53:46 PDT

RETRACTION

Thu, 24 Sep 2009 16:53:46 PDT