Salivary epidermal growth factor and intestinal adaptation in male and female mice

Salivary epidermal growth factor (sEGF) levels are increased in male mice after small bowel resection (SBR) and may be important during intestinal adaptation. Since males have greater sEGF than females, the influence of sex on postresection adaptation was tested. Females had lower sEGF; however, sEGF substantially increased in both sexes after a massive (50%) SBR. Adaptive increases in DNA and protein content, villus height, and crypt depth, as well as crypt cell proliferation rates in the remnant ileum, were not different between males and females. Although significant postresection increases in sEGF were identified, EGF mRNA and protein did not change within the submandibular gland. Glandular kallikrein-13 and ileal EGF receptor expression were greater after SBR in female mice. Intestinal adaptation is equivalent in female and male mice after SBR. Despite lower sEGF, females demonstrated increased expression of a kallikrein responsible for sEGF precursor cleavage as well as amplified ileal EGF receptor expression. These results endorse an important differential response between sexes regarding sEGF mobilization and intestinal receptor availability during adaptation.     

Increased apoptosis and accelerated epithelial migration following inhibition of hedgehog signaling in adaptive small bowel postresection

The intestinal epithelium undergoes a marked adaptive response following loss of functional small bowel surface area characterized by increased crypt cell proliferation and increased enterocyte migration from crypt to villus tip, resulting in villus hyperplasia and enhanced nutrient absorption. Hedgehog (Hh) signaling plays a critical role in regulating epithelial-mesenchymal interactions during morphogenesis of the embryonic intestine. Our previous studies showed that blocking Hh signaling in neonatal mice results in increased small intestinal epithelial crypt cell proliferation and altered enterocyte fat absorption and morphology. Hh family members are also expressed in the adult intestine, but their role in the mature small bowel is unclear. With the use of a model of intestinal adaptation following partial small bowel resection, the role of Hh signaling in the adult gut was examined by determining the effects of blocking Hh signaling on the regenerative response following loss of functional surface area. Hh-inactivating monoclonal antibodies or control antibodies were administered to mice that sustained a 50% intestinal resection. mRNA analyses of the preoperative ileum by quantitative real-time PCR revealed that Indian hedgehog was the most abundant Hh family member. The Hh receptor Patched was more abundant than Patched 2. Analyses of downstream targets of Hh signaling demonstrated that Gli3 was twofold more abundant than Gli1 and Gli2 and that bone morphogenetic protein (BMP)2 was most highly expressed compared with BMP1, -4, and -7. Following intestinal resection, the expression of Hh, Patched, Gli, and most BMP genes was markedly downregulated in the remnant ileum, and, in anti-Hh antibody-treated mice, expression of Patched 2 and Gli 1 was further suppressed. In Hh antibody-treated mice following resection, the enterocyte migration rate from crypt to villus tip was increased, and by 2 wk postoperation, apoptosis was increased in the adaptive gut. However, crypt cell proliferation, villus height, and crypt depth were not augmented. These data indicate that Hh signaling plays a role in adult gut epithelial homeostasis by regulating epithelial cell migration from crypt to villus tip and by enhancing apoptosis.     

Alanyl-glutamine promotes intestinal epithelial cell homeostasis in vitro and in a murine model of weanling undernutrition

Alanyl-glutamine (Ala-Gln) has recently been shown to enhance catch-up growth and gut integrity in undernourished children from Northeast Brazil. We hypothesized that the intestinal epithelial effects of Ala-Gln in malnourished weanling mice and mouse small intestinal epithelial (MSIE) cells would include modulation of barrier function, proliferation, and apoptosis. Dams of 10-day-old suckling C57BL/6 pups were randomized to a standard diet or an isocaloric Northeast Brazil "regional basic diet," moderately deficient in protein, fat, and minerals. Upon weaning to their dam's diet on day of life 21, pups were randomized to Ala-Gln solution or water. At 6 wk of age, mice were killed, and jejunal tissue was collected for morphology, immunohistochemistry, and Ussing chamber analysis of transmucosal resistance and permeability. Proliferation of MSIE cells in the presence or absence of Ala-Gln was measured by MTS and bromodeoxyuridine assays. MSIE apoptosis was assessed by annexin and 7-amino-actinomycin D staining. Pups of regional basic diet-fed dams exhibited failure to thrive. Jejunal specimens from undernourished weanlings showed decreased villous height and crypt depth, decreased transmucosal resistance, increased permeability to FITC-dextran, increased claudin-3 expression, and decreased epithelial proliferation and increased epithelial apoptosis (as measured by bromodeoxyuridine and cleaved caspase-3 staining, respectively). Undernourished weanlings supplemented with Ala-Gln showed improvements in weight velocity, villous height, crypt depth, transmucosal resistance, and epithelial proliferation/apoptosis compared with unsupplemented controls. Similarly, Ala-Gln increased proliferation and reduced apoptosis in MSIE cells. In summary, Ala-Gln promotes intestinal epithelial homeostasis in a mouse model of malnutrition-associated enteropathy, mimicking key features of the human disease.     

Ret heterozygous mice have enhanced intestinal adaptation after massive small bowel resection

Intestinal adaptation is an important compensatory response to massive small bowel resection (SBR) and occurs because of a proliferative stimulus to crypt enterocytes by poorly understood mechanisms. Recent studies suggest the enteric nervous system (ENS) influences enterocyte proliferation. We, therefore, sought to determine whether ENS dysfunction alters resection-induced adaptation responses. Ret+/- mice with abnormal ENS function and wild-type (WT) littermates underwent sham surgery or 50% SBR. After 7 days, ileal morphology, enterocyte proliferation, apoptosis, and selected signaling proteins were characterized. Crypt depth and villus height were equivalent at baseline in WT and Ret+/- mice. In contrast after SBR, Ret+/- mice had longer villi (Ret+/- 426.7 ± 46.0 μm vs. WT 306.5 ± 7.7 μm, P < 0.001) and deeper crypts (Ret+/- 119 ± 3.4 μm vs. WT 82.4 ± 3.1 μm, P < 0.001) than WT. Crypt enterocyte proliferation was higher in Ret+/- (48.8 ± 1.3%) than WT (39.9 ± 2.1%; P < 0.001) after resection, but apoptosis rates were similar. Remnant bowel of Ret+/- mice also had higher levels of glucagon-like peptide 2 (6.2-fold, P = 0.005) and amphiregulin (4.6-fold, P < 0.001) mRNA after SBR, but serum glucagon-like peptide 2 protein levels were equal in WT and Ret+/- mice, and there was no evidence of increased c-Fos nuclear localization in submucosal neurons. Western blot confirmed higher crypt epidermal growth factor receptor (EGFR) protein levels (1.44-fold; P < 0.001) and more phosphorylated EGFR (2-fold; P = 0.003) in Ret+/- than WT mice after SBR. These data suggest that Ret heterozygosity enhances intestinal adaptation after massive SBR, likely via enhanced EGFR signaling. Reducing Ret activity or altering ENS function may provide a novel strategy to enhance adaptation attenuating morbidity in patients with short bowel syndrome.     

Hyaluronic acid regulates normal intestinal and colonic growth in mice

Hyaluronic acid (HA), a component of the extracellular matrix, affects gastrointestinal epithelial proliferation in injury models, but its role in normal growth is unknown. We sought to determine the effects of exogenous HA on intestinal and colonic growth by intraperitoneal injection of HA twice a week into C57BL/6 mice from 3 to 8 wk of age. Similarly, to determine the effects of endogenous HA on intestinal and colonic growth, we administered PEP-1, a peptide that blocks the binding of HA to its receptors, on the same schedule. In mice treated with exogenous HA, villus height and crypt depth in the intestine, crypt depth in the colon, and epithelial proliferation in the intestine and colon were increased. In mice treated with PEP-1, intestinal and colonic length were markedly decreased and crypt depth and villus height in the intestine, crypt depth in the colon, and epithelial proliferation in the intestine and colon were decreased. Administration of HA was associated with increased levels of EGF (intestine) and IGF-I (colon), whereas administration of PEP-1 was associated with decreased levels of IGF-I (intestine) and epiregulin (colon). Exogenous HA increases intestinal and colonic epithelial proliferation, resulting in hyperplasia. Blocking the binding of endogenous HA to its receptors results in decreased intestinal and colonic length and a mucosal picture of hypoplasia, suggesting that endogenous HA contributes to the regulation of normal intestinal and colonic growth.     

胰高血糖素样肽-2对小鼠小肠缺血/再灌注损伤的保护作用

目的:观察胰高血糖素样肽-2(GLP-2)对缺血/再灌注损伤小鼠小肠的保护效应.方法:采用肠缺血/再灌注(I/R)模型,将32只小鼠随机分为4组(n=8)假手术(Sham)组、I/R组、I/R GLP-2保护组和I/R 谷氨酰胺(GLN)阳性对照组.光镜观察小肠黏膜形态学改变.检测小肠绒毛高度和隐窝深度;小肠组织二胺氧化酶(DAO)活性;肠系膜淋巴结(MLN)细菌易位率.结果:与假手术组相比,I/R组部分小肠绒毛坏死脱落,绒毛高度下降,隐窝变浅(P＜0 01);小肠组织DAO活性降低(P＜0.01);MLN细菌易位率增加(P＜0.05).与I/R组比,GLP-2组肠绒毛损害明显减轻,DAO活性回升(P＜0.01),细菌易位率回降(P＜0.05).结论:GLP-2对缺血/再灌注损伤小鼠小肠的形态结构及肠屏障功能具有保护作用.     

p38 MAPK regulates Bax activity and apoptosis in enterocytes at baseline and after intestinal resection

Increased apoptosis in crypt enterocytes is a key feature of intestinal adaptation following massive small bowel resection (SBR). Expression of the proapoptotic factor Bax has been shown to be required for resection-induced apoptosis. It has also been demonstrated that p38-α MAPK (p38) is necessary for Bax activation and apoptosis in vitro. The present studies were designed to test the hypothesis that p38 is a key regulator of Bax activation during adaptation after SBR in vivo. Enterocyte expression of p38 was deleted by tamoxifen administration to activate villin-Cre in adult mice with a floxed Mapk14 (p38-α) gene. Proximal 50% SBR or sham operations were performed on wild-type (WT) and p38 intestinal knockout (p38-IKO) mice under isoflurane anesthesia. Mice were killed 3 or 7 days after operation, and adaptation was analyzed by measuring intestinal morphology, proliferation, and apoptosis. Bax activity was quantified by immunoprecipitation, followed by Western blotting. After SBR, p38-IKO mice had deeper crypts, longer villi, and accelerated proliferation compared with WT controls. Rates of crypt apoptosis were significantly lower in p38-IKO mice, both at baseline and after SBR. Levels of activated Bax were twofold higher in WT mice after SBR relative to sham. In contrast, activated Bax levels were reduced by 67% in mice after p38 MAPK deletion. Deleted p38 expression within the intestinal epithelium leads to enhanced adaptation and reduced levels of enterocyte apoptosis after massive intestinal resection. p38-regulated Bax activation appears to be an important mechanism underlying resection-induced apoptosis.     

Rapid expansion of intestinal secretory lineages following a massive small bowel resection in mice

Following massive small bowel resection (SBR) in mice, there are sustained increases in crypt depth and villus height, resulting in enhanced mucosal surface area. The early mechanisms responsible for resetting and sustaining this increase are presently not understood. We hypothesized that expansion of secretory lineages is an early and sustained component of the adaptive response. This was assessed in the ileum by quantitative morphometry at 12 h, 36 h, 7 days, and 28 days and by quantitative RT-PCR of marker mRNAs for proliferation and differentiated goblet, Paneth cell, and enterocyte genes at 12 h after 50% SBR or sham operation. As predicted, SBR elicited increases of both crypt and villus epithelial cells, which were sustained though the 28 days of the experiment. Significant increases in the overall number and percentage of both Paneth and goblet cells within intestinal epithelium occurred by 12 h and were sustained up to 28 days after SBR. The increases of goblet cells after SBR were initially observed within villi at 12 h, with marked increases occurring in crypts at 36 h and 7 days. Consistent with this finding, qRT-PCR demonstrated significant increases in the expression of mRNAs associated with proliferation (c-myc) and differentiated goblet cells (Tff3, Muc2) and Paneth cells (lysozyme), whereas mRNA associated with differentiated enterocytes (sucrase-isomaltase) remained unchanged. From these data, we speculate that early expansion of intestinal secretory lineages within the epithelium of the ileum occurs following SBR, possibly serving to amplify the signal responsible for initiating and sustaining intestinal adaptation.     

Bringing target-matched PI3King from the bench to the clinic

Caveolin-1 (Cav-1) is a protein marker for caveolae organelles, and acts as a scaffolding protein to negatively regulate the activity of signaling molecules by binding to and releasing them in a timely fashion. We have previously shown that loss of Cav-1 promotes the proliferation of mouse embryo fibroblasts (MEFs) in vitro. Here, to investigate the in vivo relevance of these findings, we evaluated the turnover rates of small intestine crypt stem cells from WT and Cav-1 deficient mice. Interestingly, we show that Cav-1 null crypt stem cells display higher proliferation rates, as judged by BrdU and PCNA staining. In addition, we show that Wnt/?-catenin signaling, which normally controls intestinal stem cell self-renewal, is up-regulated in Cav-1 deficient crypt stem cells. Because the small intestine constitutes one of the main targets of radiation, we next evaluated the role of Cav-1 in radiation-induced damage. Interestingly, after exposure to 15 Gy of ?-radiation, Cav-1 deficient mice displayed a decreased survival rate, as compared to WT mice. Our results show that after radiation treatment, Cav-1 null crypt stem cells of the small intestine exhibit far more apoptosis and accelerated proliferation, leading to a faster depletion of crypts and villi. As a consequence, six days after radiation treatment, Cav-1 -/- mice lost all their crypt and villus structures, while WT mice still showed some crypts and intact villi. In summary, we show that ablation of Cav-1 gene expression induces an abnormal amplification of crypt stem cells, resulting in increased susceptibility to ?-radiation. Thus, our studies provide the first evidence that Cav-1 normally regulates the proliferation of intestinal stem cells in vivo.     

Keratinocyte Growth Factor Improves Epithelial Structure and Function in a Mouse Model of Intestinal Ischemia/Reperfusion

Background

Intestinal ischemia/reperfusion (I/R) induces the desquamation of the intestinal epithelium, increases the intestinal permeability, and in patients often causes fatal conditions including sepsis and multiple organ failure. Keratinocyte growth factor (KGF) increases intestinal growth, although little is known about KGF activity on intestinal function after intestinal I/R. We hypothesized that KGF administration would improve the intestinal function in a mouse model of intestinal I/R.

Methods

Adult C57BL/6J mice were randomized to three groups: Sham, I/R group and I/R+KGF group. Mice were killed on day 5, and the small bowel was harvested for histology, wet weight, RNA and protein content analysis. Epithelial cell (EC) proliferation was detected by immunohistochemistry for PCNA, and apoptosis was determined by TUNEL staining. The expressions of Claudin-1 and ZO-1 were detected by immunohistochemistry. Epithelial barrier function was assessed with transepithelial resistance (TER).

Results

KGF significantly increased the intestinal wet weight, contents of intestinal protein and RNA, villus height, crypt depth and crypt cell proliferation, while KGF resulted in the decrease of epithelial apoptosis. KGF also stimulated the recovery of mucosal structures and attenuated the disrupted distribution of TJ proteins. Moreover, KGF attenuated the intestinal I/R-induced decrease in TER and maintained the intestinal barrier function.

Conclusion

KGF administration improves the epithelial structure and barrier function in a mouse model of intestinal I/R. This suggests that KGF may have clinical applicability.     

Prosurvival and antiapoptotic effects of PGE2 in radiation injury are mediated by EP2 receptor in intestine

The biological activities of PGE(2) are mediated through EP receptors (EP(1)-EP(4)), plasma membrane G protein-coupled receptors that differ in ligand binding and signal-transduction pathways. We investigated gastrointestinal EP(2) receptor expression in adult mice before and after radiation injury and evaluated intestinal stem cell survival and crypt epithelial apoptosis after radiation injury in EP(2) null mice. EP(2) was expressed throughout the gut. Intestinal EP(2) mRNA increased fivefold after gamma-irradiation. Crypt survival was diminished in EP(2)-/- mice (4.06 crypts/cross section) compared with wild-type littermates (8.15 crypts/cross section). Radiation-induced apoptosis was significantly increased in EP(2)-/- mice compared with wild-type littermates. Apoptosis was 1.6-fold higher in EP(2) (-/-) mice (5.9 apoptotic cells/crypt) than in wild-type mice (3.5 apoptotic cells/crypt). The EP(2) receptor is expressed in mouse gastrointestinal epithelial cells and is upregulated following radiation injury. The effects of PGE(2) on both crypt epithelial apoptosis and intestinal crypt stem cell survival are mediated through the EP(2) receptor.     

Role of VEGF in small bowel adaptation after resection: the adaptive response is angiogenesis dependent

Previous work in our group has demonstrated that mouse salivary gland has the highest concentration of salivary-derived VEGF protein compared with other organs and is essential for normal palatal mucosal wound healing. We hypothesize that salivary VEGF plays an important role in maintaining the integrity of the gastrointestinal mucosa following small bowel resection (SBR). Thirty-five 8- to 10-wk-old C57BL/6 female mice were divided into seven treatment groups: 1) sham (transaction and anastomosis, n = 5); 2) SBR (n = 8); 3) sialoadenectomy and small bowel resection (SAL+SBR, n = 8); 4) sialoadenectomy and small bowel resection with EGF supplementation (SAL+SBR+EGF, n = 9); 5) sialoadenectomy and small bowel resection with VEGF supplementation (SAL+SBR+VEGF, n = 9); 6) sialoadenectomy and small bowel resection supplemented with EGF and VEGF (SAL+ SBR+VEGF+EGF, n = 6); 7) selective inhibition of VEGF in the submandibular gland by Ad-VEGF-Trap following small bowel resection (Ad-VEGF-Trap+SBR, n = 7). Adaptation was after 3 days by ileal villus height and crypt depth. The microvascular response was evaluated by CD31 immunostaining and for villus-vessel area ratio by FITC-labeled von Willebrand factor immunostaining. The adaptive response after SBR was significantly attenuated in the SAL group in terms of villus height (250.4 +/- 8.816 vs. 310 +/- 19.35, P = 0.01) and crypt depth (100.021 +/- 4.025 vs. 120.541 +/- 2.82, P = 0.01). This response was partially corrected by orogastric VEGF or EGF alone. The adaptive response was completely restored when both were administered together, suggesting that salivary VEGF and EGF both contribute to intestinal adaptation. VEGF increases the vascular density (6.4 +/- 0.29 vs. 6.1 +/- 0.29 vs. 5.96 +/- 0.20) and villus-vessel area ratio (0.713 +/- 0.01 vs. 0.73 +/- 0.01) in the adapting bowel. Supplementation of both EGF and VEGF fully rescues adaptation, suggesting that the adaptive response may be dependent on VEGF-driven angiogenesis. These results support a previously unrecognized role for VEGF in the small bowel adaptive response.     

Proliferation, not apoptosis, alters epithelial cell migration in small intestine of CFTR null mice

Expression of a mutated cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to enhance proliferation within CF airways, and cells expressing a mutated CFTR have been shown to be less susceptible to apoptosis. Because the CFTR is expressed in the epithelial cells lining the gastrointestinal tract and all CF mouse models are characterized by gastrointestinal obstruction, we hypothesized that CFTR null mice would have increased epithelial cell proliferation and reduced apoptosis within the small intestine. The rate of intestinal epithelial cell migration from crypt to villus was increased in CFTR null mice relative to mice expressing the wild-type CFTR. This difference in migration could be explained by an increase in epithelial cell proliferation but not by a difference in apoptosis within the crypts of Lieberkühn. In addition, using two independent sets of CF cell lines, we found that epithelial cell susceptibility to apoptosis was unrelated to the presence of a functional CFTR. Thus increased proliferation but not alterations in apoptosis within epithelial cells might contribute to the pathophysiology of CF.     

Intestinal intraepithelial lymphocyte derived angiotensin converting enzyme modulates epithelial cell apoptosis

Background & Aims: Intestinal adaptation in short bowel syndrome (SBS) consists of increased epithelial cell (EC) proliferation as well as apoptosis. Previous microarray analyses of intraepithelial lymphocytes (IEL) gene expression after SBS showed an increased expression of angiotensin converting enzyme (ACE). Because ACE has been shown to promote alveolar EC apoptosis, we examined if IEL-derived ACE plays a role in intestinal EC apoptosis. Methods: Mice underwent either a 70% mid-intestinal resection (SBS group) or a transection (Sham group) and were studied at 7 days. ACE expression was measured, and ACE inhibition (ACE-I, enalaprilat) was used to assess ACE function. Results: IEL-derived ACE was significantly elevated in SBS mice. The addition of an ACE-I to SBS mice resulted in a significant decline in EC apoptosis. To address a possible mechanism, tumor necrosis factor alpha (TNF-α) mRNA expression was measured. TNF-α was significantly increased in SBS mice, and decreased with ACE-I. Interestingly, ACE-I was not able to decrease EC apoptosis in TNF-α knockout mice. Conclusions: This study shows a previously undescribed expression of ACE by IEL. SBS was associated with an increase in IEL-derived ACE. ACE appears to be associated with an up-regulation of intestinal EC apoptosis. ACE-I significantly decreased EC apoptosis.     

Selective sparing of goblet cells and paneth cells in the intestine of methotrexate-treated rats

Proliferation, differentiation, and cell death were studied in small intestinal and colonic epithelia of rats after treatment with methotrexate. Days 1-2 after treatment were characterized by decreased proliferation, increased apoptosis, and decreased numbers and depths of small intestinal crypts in a proximal-to-distal decreasing gradient along the small intestine. The remaining crypt epithelium appeared flattened, except for Paneth cells, in which lysozyme protein and mRNA expression was increased. Regeneration through increased proliferation during days 3-4 coincided with villus atrophy, showing decreased numbers of villus enterocytes and decreased expression of the enterocyte-specific genes sucrase-isomaltase and carbamoyl phosphate synthase I. Remarkably, goblet cells were spared at villus tips and remained functional, displaying Muc2 and trefoil factor 3 expression. On days 8-10, all parameters had returned to normal in the whole small intestine. No methotrexate-induced changes were seen in epithelial morphology, proliferation, apoptosis, Muc2, and TFF3 immunostaining in the colon. The observed small intestinal sparing of Paneth cells and goblet cells following exposure to methotrexate is likely to contribute to epithelial defense during increased vulnerability of the intestinal epithelium.     

Radioprotection to small intestine of the mice against ionizing radiation by semiquinone glucoside derivative (SQGD) isolated from Bacillus sp. INM-1

Ionizing irradiation induces severe damage to the intestinal crypt cells which are responsible for renovation and maintenance of the intestinal cellular architecture. Therefore, protection of intestinal cells and tissue against lethal irradiation using a semiquinone glucoside derivative (SQGD) isolated from radioresistant bacterium Bacillus sp. INM-1 is the prime focus of the present investigation. BALB/c mice were administered by SQGD (50?mg/kg.b.wt. i.p.) 2?h before whole body irradiation (10?Gy), and histological analysis of the jejunum section was carried out and compared to the irradiated mice. Significant (p?<?0.0001) increase in villus length, number of cells per villus, crypts numbers per villus section, total cells counts and mitotic cell counts per crypt and low goblet cells per villus section, and low apoptotic index per crypt section were observed in the irradiated mice pre-treated by SQGD at 48–168?h. Significant induction in NF-kβ at 24?h and Bcl-2/Bax ratio was observed in irradiated mice pre-treated by SQGD compared to only irradiated animals. SQGD pre-treatment before irradiation was found instrumental to reverse the radiation-induced degenerative changes by replenishment of the damaged cells by enhancing mitotic, proliferating, pro-survival, and apoptosis inhibitory activities probably through modulation of cell cycle arrest in G₁/S phase in the intestinal cellular milieu.     

Roles for p21waf1/cip1 and p27kip1 during the adaptation response to massive intestinal resection

The magnitude of gut adaptation is a decisive factor in determining whether patients are able to live independent of parenteral nutrition after massive small bowel loss. We previously established that the cyclin-dependent kinase inhibitor (CDKI) p21(waf1/cip1) is necessary for enterocyte proliferation and a normal adaptation response. In the present study, we have further elucidated the role of this CDKI in the context of p27(kip1), another member of the Cip/Kip CDKI family. Small bowel resections (SBRs) or sham operations were performed in control (C57/BL6), p21(waf1/cip1)-null, p27(kip1)-null, and p21(waf1/cip1)/p27(kip1) double-null mice. Morphological (villus height/crypt depth) alterations in the mucosa, the kinetics of enterocyte turnover (rates of enterocyte proliferation and apoptosis), and the protein expression of various cell cycle-regulatory proteins were recorded at various postoperative times. Enterocyte compartment-specific mRNA expression was investigated using laser capture microdissection. Resection-induced adaptation in control mice coincided with increased protein expression of p21(waf1/cip1) and decreased p27(kip1) within 3 days postoperatively. Identical changes in mRNA expression were detected in crypt but not in villus enterocytes. Adaptation occurred normally in control and p27(kip1)-null mice; however, mice deficient in both p21(waf1/cip1) and p27(kip1) failed to increase baseline rates of enterocyte proliferation and adaptation. The expression of p21(waf1/cip1) protein and mRNA in the proliferative crypt compartment is necessary for resection-induced enterocyte proliferation and adaptation. The finding that deficient expression of p27(kip1) does not affect adaptation suggests that these similar CDKI family members display distinctive cellular functions during the complex process of intestinal adaptation.     

(Na---K)-stimulated adenosinetriphosphatase in isolated intestinal villus tip and crypt cells

The migration of intestinal epithelial cells from the crypt area to the villus tip is associated with progressive differentiation of these cells. The distribution of (Na⁺---K⁺) stimulated adenosinetriphosphatase ((Na⁺---K⁺)-ATPase; EC 3.6.1.3) along the intestinal villus may have functional as well as developmental implications. To define this distribution, rat jejunal and ileal segments were incubated in vitro with a citrate solution that dissociates epithelial cells sequentially from villus tip to crypt area. ATPase activity in cell collections from villus tips and crypt areas were compared. The specific activity of (Na⁺---K⁺)-ATPase was higher in the villus tip than in the crypt cells of both jejunum and ileum. Crypt cell (Na⁺---K⁺)-ATPase activity in the jejunum and ileum were similar. Thus, (Na⁺---K⁺)-ATPase activity of villus tip cells in the jejunum was greater than in the ileum. There was no difference in villus tip and crypt cell Mg²⁺-ATPase activity in either jejunum or ileum. The steep gradient for (Na⁺---K⁺)-ATPase along the intestinal villus may signify an improtant difference in Na⁺ transport between the villus tip and crypt area. The higher level of (Na⁺---K⁺)-ATPase activity in the jejunal villi is consistent with the more important role of the jejunum in Na⁺ and substrate-linked Na⁺ transport.     

Apoptotic process in the monkey small intestinal epithelium: 2. Possible role of oxidative stress

Recent findings suggest that intracellular oxidants are involved in the induction of apoptosis and this type of cell death can be inhibited by various antioxidants. In our accompanying paper, we have shown apoptosis in the villus tip cells of the monkey small intestinal epithelium. The aim of the present study was to evaluate the possible relationship between oxidative stress, antioxidant levels and the apoptotic process in the monkey small intestinal epithelium. Monkey small intestinal epithelial cells were isolated into different fractions consisting of villus, middle and crypt cells. Mitochondrial function was assessed by the reduction of the tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), with and without succinate. The extent of lipid peroxidation was assessed by measuring the formation of conjugated diene, depletion of polyunsaturated fatty acids and α-tocopherol. Level of antioxidant enzymes like, superoxide dismutase (SOD), catalase, glutathione S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase were also quantitated in various cell fractions. MTT reduction was significantly decreased in villus cells as compared to the cells from other fractions and this was evident even in presence of the respiratory substrate, succinate. Increased formation of conjugated diene and depletion of polyunsaturated fatty acids were seen in villus and crypt cells as compared to middle fraction cells. The α-tocopherol level was decreased in both villus and crypt cells as compared to cells from middle region. Significant decrease of SOD activity was seen in the villus tip cells and a slight decrease was seen in the crypt fractions. Glutathione dependent enzymes like GST, GPx and GSH reductase showed higher activity in the villus fractions. A similar observation was also seen in the catalase activity. This study has shown that although oxidative stress is seen in both villus and crypt cells, decreased mitochondrial function was seen in villus tip cells which may be responsible for apoptotic process in the intestinal epithelium.     

Stimulation of colonic mucosal growth associated with oxidized redox status in rats

Limited data in animal models suggest that colonic mucosa undergoes adaptive growth following massive small bowel resection (SBR). In vitro data suggest that intestinal cell growth is regulated by reactive oxygen species and redox couples [e.g., glutathione (GSH)/glutathione disulfide (GSSG) and cysteine (Cys)/cystine (CySS) redox]. We investigated the effects of SBR and alterations in redox on colonic growth indexes in rats after either small bowel transection (TX) or 80% midjejunoileal resection (RX). Rats were pair fed +/- blockade of endogenous GSH synthesis with buthionine sulfoximine (BSO). Indexes of colonic growth, proliferation, and apoptosis and GSH/GSSG and Cys/CySS redox potentials (E(h)) were determined. RX significantly increased colonic crypt depth, number of cells per crypt, and epithelial cell proliferation [crypt cell bromodeoxyuridine (BrdU) incorporation]. Administration of BSO markedly decreased colonic mucosal GSH, GSSG, and Cys concentrations in both TX and RX groups, with a resultant oxidation of GSH/GSSG and Cys/CySS E(h). BSO did not alter colonic crypt cell apoptosis but significantly increased all colonic mucosal growth indexes (crypt depth, cells/crypt, and BrdU incorporation) in both TX and RX groups in a time- and dose-dependent manner. BSO significantly decreased plasma GSH and GSSG, oxidized GSH/GSSG E(h), and increased plasma Cys and CySS concentrations. Collectively, these data provide in vivo evidence indicating that oxidized colonic mucosal redox status stimulates colonic mucosal growth in rats. The data also suggest that GSH is required to maintain normal colonic and plasma Cys/CySS homeostasis in these animal models.