Ivan I. Pacheco

CaSR stimulates secretion of Wnt5a from colonic myofibroblasts to stimulate CDX2 and sucrase-isomaltase using Ror2 on intestinal epithelia

To understand whether extracellular calcium-sensing receptor (CaSR) expression on colonic myofibroblast cells (18Co) contributed to epithelial homeostasis, we activated the CaSR with 5 mM Ca(2+), screened by RT-PCR Wnt family members, and measured their secretion. Transcripts for Wnt 1, 2, 2b, 3a, 4, and 7a were either absent or unchanged whereas Wnt3 decreased and Wnt5a increased. We assessed Wnt5a secretion by Western blot. High Ca(2+) (5 mM) substantially increased Wnt5a secretion; small interfering RNA (siRNA) against the CaSR reduced this to constitutive amounts. Expression of Wnt5a plasmid but not Wnt1 or Wnt3a increased caudal homeodomain factor CDX2 transcripts and protein in HT-29 adenocarcinoma cells. Wnt5a increased activity of a sucrase-isomaltase (SI) promoter in Caco-2BBE cells. Wnt5a protein stimulation of CDX2 transcripts and protein and SI reporter were increased by overexpression of wild-type Ror2, a Wnt5a receptor, and reduced with siRNA against Ror2. CaSR activation of HT-29 cells increased Ror2 protein expression. Ror2 protein was expressed in mouse jejunum from crypt base to villus tip and in the colon on surface epithelia. Our results show that activation of a G protein-coupled receptor, the CaSR, stimulates secretion of Wnt5a from myofibroblasts. Stimulation of epithelia by the CaSR increased the expression of a receptor for Wnt5a, the tyrosine kinase Ror2, suggesting existence of a unique paracrine relationship for CDX2 homoeostasis in the intestine and revealing new contributions of CaSR-activated myofibroblasts to intestinal stem cell niche microenvironments.

Association of 72-kDa heat shock protein expression with adaptation to aspirin in rat gastric mucosa

It is well documented that gastric mucosa canincrease its resistance to mucosal damage caused byaspirin during repeated long-term administration ofaspirin. However, the underlying mechanism of thisadaptation is not well established. In the present study,we investigated the effect of long-term (chronic)administration of aspirin on expression of heat shockproteins (HSPs), which are known as endogenouscytoprotectants, in rat gastric mucosa. Rats were administeredaspirin (100 mg/kg) daily for up to 20 days. Aftervarious periods of aspirin administration, a high doseof aspirin (250 mg/kg) was administered, and the mucosal damage was assessed. Expression of heat shockproteins (HSPs) in gastric mucosa was evaluated byWestern blot. Intracellular localization of each HSP wasstudied immunohistochemically. ProstaglandinE2 (PGE2) and leukotriene B4(LTB4) levels were also investigated.Long-term aspirin administration resulted in developmentof resistance to aspirin-induced mucosal damage, and theincrease of HSP72 expression correlated with mucosal resistance to aspirin.No significant increase was observed in HSP60 and HSP90levels. Immunohistochemical study showed an increase ofHSP72 in the cytoplasm of mucosal surface cells. The PGE2 level was suppressed and nochange in the level of LTB4 was observed. Itis possible that HSP72 could play important roles ingastric mucosal adaptation when the PGE2level is suppressed by NSAIDs.

Effect of Preinduction of Heat-Shock Proteins on Acetic Acid-Induced Small Intestinal Lesions in Rats

Bowel dysfunction such as irritable bowelsyndrome caused by stress is well described. Previousreports suggest that stress is known to cause therelease of endogenous substances such as catecholamine, beta-endorphine, 5-hydroxytryptamine,corticotropin-releasing factor, andthyrotropin-releasing hormone (TRH). However, the roleplayed by these neurohormonal mediators in boweldysfunction under stress conditions is not well known. We investigatedthe influence of water-immersion stress or TRHadministration on the expression of 60-kDa, 72-kDa, and90-kDa heat-shock proteins (HSP60, HSP72, and HSP90, respectively) in rat small intestinal mucosa byWestern blot and immunohistochemical analyses. Thecytoprotective function of preinduced HSPs onexperimentally induced mucosal damage also was studied.In order to investigate the influence ofpreinduction of HSP60 on small intestinal damage, thesmall intestinal lumen was perfused with 1.5% aceticacid 1 ml/min for 15 min with or without pretreatmentwith water-immersion stress or TRH administration.Expression of HSP60 was significantly increased bywater-immersion stress or TRH administration in thesmall intestinal mucosa, whereas HSP72 and HSP90 did not increase. Interestingly, expression of thisprotein showed the biphasic peak pattern afterwater-immersion stress or TRH administration. Each peakwas observed 3-6 hr and 21-24 hr after the initiation of water-immersion stress or TRHadministration. Immunohistochemical study also showed asignificant increment of HSP60 in both the cytoplasm andnuclei of the small intestinal mucosal cells. Nohistopathologic alteration was observed in rat small intestinalmucosa after each treatment. Small intestinal damagecaused by 1.5% acetic acid perfusion was not influencedby preinduction of HSP60. We demonstrated that waterimmersion stress or TRH administrationspecifically induced HSP60, although preinduction ofthis protein did not show a cytoprotective function inthe small intestinal mucosa.

Effects of cyclosporin A on water-immersion stress-induced gastric lesion and gastric secretion in rats.

Abstract: Cyclosporin A is an immunosuppressive agent which is well known as a specific inhibitor of calcineurin (protein phosphatase 2B). In this study, we investigated the effects of cyclosporin A on water-immersion stress-induced gastric ulcer formation and gastric acid secretion in rats. We also examined the localization of calcineurin immunohistochemically. Calcineurin was specifically expressed in gastric parietal cells and chief cells of the gastric mucosa. The intraperitoneal administration of cyclosporin A dose-dependently suppressed the development of gastric mucosal lesions induced by water-immersion stress and inhibited gastric acid secretion, as assessed by pylorus ligation. These results indicated that calcineurin may play an important role in gastric acid secretion.

Corticosteroid pretreatment prevents small intestinal mucosal damage induced by acetic acid-perfusion model in rats

One of the important problems in experimentally induced small intestinal lesions is that there is no reproducible model of diffuse and stable mucosal lesion. In this paper, we studied in detail the effects of continuous perfusion of various concentrations of acetic acid on the rat small intestinal mucosa. In order to evaluate its applicability for screening of the preventive effect of drugs on gut damage, we also evaluated the efficacy of corticosteroid pretreatment in preventing acetic acid-induced mucosal lesion. Male Sprague-Dawley rats were fasted for 12 hr, and the small intestinal lumen was perfused with 1%, 1.5%, 2.5%, 3%, 3.75% (pH 2.4–2.6) acetic acid or saline (control) at 1 ml/min for 15 min. In separate experiments, the effect of preadministration of budesonide (0.5 or 0.75 mg/kg/day) and prednisone (0.75 mg/kg/day) on 1.5% acetic acid-induced mucosal damage was investigated. Macroscopic and microscopic lesions occurred diffusely in a concentration-dependent fashion. Histological findings revealed signs of transmural inflammation characterized by mucosal–submucosal edema, ulceration, and neutrophil infiltration. Mucosal–submucosal height had an inverse relation with the acetic acid concentrations perfused. Myeloperoxidase activity levels increased several-fold in the acetic acid-perfused groups. Corticosteroid pretreatment prevented microscopic damage and was associated with reduction of MPO activity levels in 1.5% acetic acid-perfused rats. We conclude that this simple and reproducible model could be applied for the screening of new drugs in the gastrointestinal tract in which large numbers of animals are taken into account.