Differential response of duodenal epithelial cells to 1,25-dihydroxyvitamin D3 according to position on the villus: a comparison of calcium uptake, calcium-binding protein, and alkaline phosphatase activity

To determine which region of the intestinal villus was primarily responsible for calcium uptake and whether cells from the different regions of the villus differed in their response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], we studied cells eluted from the duodenal villus in a sequential fashion at various times after vitamin D-deficient chicks had received 1,25-(OH)2D3. The elution scheme employed removes cells from the villus tip first and cells from the villus base last, as was documented by the distribution of alkaline phosphatase activity, sucrase activity, and cytosolic calcium-binding protein (CaBP) in the eluted fractions. Brush border membrane vesicles (BBMV) were prepared from different fractions of the villus. Calcium uptake was greatest in BBMV from cells eluted from the villus tip and least in those from the villus base. The distribution of calcium uptake and alkaline phosphatase activity in the same BBMV were parallel. After 1,25-(OH)2D3 treatment, cytosolic CaBP was observed in the cells from the villus base by 4 h and in all fractions by 8 h; at all times (from 4-24 h), cells from the villus base contained more cytosolic CaBP than did cells from the villus tip. Alkaline phosphatase activity in BBMV was stimulated in all fractions by 4 h; at all times, alkaline phosphatase activity was greatest in BBMV from cells of the villus tip. In contrast, calcium uptake by BBMV was stimulated 2 h after 1,25-(OH)2D3 administration only in cells from the villus tip and was not stimulated even by 24 h in cells from the villus base. These results indicate that the cellular response to 1,25-(OH)2D3 depends on the location of the cell on the villus and that 1,25-(OH)2D3-stimulated calcium flux across the brush border can be dissociated from 1,25-(OH)2D3-stimulated alkaline phosphatase activity and CaBP production.     

The adenosine triphosphate-dependent calcium pump in rat small intestine: effects of vitamin D deficiency and cell isolation methods

Duodenal villus cells from vitamin D-deficient (-D) and replete rats (+D) were isolated either in citrate buffers according to the method of Stern or by vibration in EDTA-containing solutions according to the method of Harrison and Webster. Basolateral plasma membrane vesicles (BLMV) were purified, and active Ca2+ transport was studied. The rates of ATP-dependent Ca2+-transport in BLMV from -D and +D animals were 2.6 +/- 1.0 and 10.6 +/- 0.6 nmol Ca2+/min X mg protein when cells had been isolated in citrate buffers. These transport rates were 9.2 +/- 0.7 and 9.6 +/- 0.5, respectively, when cells were isolated by vibration. The specific activities of other enzyme markers and vesicle parameters, such as the degree of resealing or purification factors, were not influenced by the cell isolation procedure. Addition of lipase and protease inhibitors to the citrate buffer or fasting the animals before death increase the active Ca2+ transport rates in BLM from -D rats to control levels. These results indicate that the ATP-driven Ca2+ pump in duodenal plasma membranes from vitamin D-deficient rats is more prone to inactivation during enterocyte isolation procedures. With the vibration technique, six populations of cells could be obtained that are sequentially released from villus tip to crypt base. A similar villus-crypt gradient of active Ca2+ transport was present in duodenal BLMV from -D and +D animals. In ileal BLMV from vitamin D-deficient rats a significantly lower Ca2+ transport rate was found compared to that in +D animals, even when villus cells were isolated by vibration. It is thought that the mechanisms by which 1,25-dihydroxyvitamin D3 regulate transcellular Ca2+ fluxes in duodenum and ileum must be different.     

Na+ transport in jejunal crypt cells.

As enterocytes migrate from crypts to villi they differentiate and mature. To examine the effect of epithelial differentiation on ion transport we studied 22Na+ efflux and (Na+--K+)-adenosine triphosphatase activity in suspensions of epithelial cells selectively isolated from different regions of the villus to compare crypt cells with villous tip cells. Enterocytes were isolated from rat jejunum by a dilation-vibration technique. Thymidine kinase, sucrase, and alkaline phosphatase activities were measured as markers of specific cell populations. Compared to villous cells, cells from the crypt region demonstrated lower (Na"--K+)-adenosine triphosphatase activity, lower total and passive Na+ efflux rate constants, and failure of Na+ transport to respond to an actively transported nonelectrolyte.     

Delayed enzyme expression: a defect of aging rat gut

To evaluate the effect of aging upon the small intestine, the distribution, content, and concentration of epithelial cell enzymes at different levels along the crypt-villus column were measured in aging and young adult, male, Fisher 344 rats. Specific activities of sucrase, maltase, lactase, and adenosine deaminase in mucosal homogenates were lower in the upper intestines of aging than in young animals, whereas the specific activity and content of thymidine kinase was higher. Enzyme activities were measured in cells obtained by cryostat sectioning from villus tip to crypt base. Sucrase and maltase activities were fully expressed nearest the crypt, alkaline phosphatase in cells higher on the villus, and adenosine deaminase higher still, whereas thymidine kinase activity was limited to the crypts. The ordered pattern of enzyme expression was maintained in aging rats but the initiation and duration were delayed. Because peak specific enzyme activities were similar in young and aging animals, the reduced specific activities in mucosal homogenates from aging animals were due to an increase in the proportion of relatively undifferentiated villus epithelial cells. These findings are of importance in explaining altered intestinal function during aging without a concomitant change in intestinal structure.     

Glutathione and gamma-glutamyl cycle enzymes in crypt and villus tip cells of rat jejunal mucosa.

Villus tip cells and crypt cells of rat jejunal mucosa were separated by the planning procedure of Imondi et al. and were studied with respect to their activities of the enzymes of the gamma-glutamyl cycle and glutathione content. The villus tip cells exhibit much higher gamma-glutamyl transpeptidase activities than do the crypt cells: thus, gamma-glutamyl trnaspeptidase appears to be a villus-specific enzyme. gamma-Glutamyl cyclotransferase and the enzymes required for glutathione synthesis are not specifically localized to either the crypt or villus tip cells but are present in both. The crypt cells have a high concentration of glutathione (4-5 mM) comparable to the levels found in liver and kidney; in contrast, the villus tip cells have much lower concentrations. On fasting, the glutathione concentration decreased markedly in both villus tip and crypt cells; feeding of protein, but not of sucrose, led to increased glutathione concentrations. The migration of cells from the undifferentiated crypt cell region to the villus tip is associated with structural and biochemical changes that equip the cell for its mature functional activities, which include transport. The present findings indicate that such cellular differentiation and migration is associated with a marked increase in gamma-glutamyl transpeptidase activity and in the utilization of glutathione.     

Characterization of isolated duodenal epithelial cells along a cryptvillus axis in rats fed diets with different iron content

The intestinal mucosa is characterized by cell proliferation, commitment, differentiation, digestion and absorption. These processes occur at specified locations along the crypt to villus axis. A technique is reported for the isolation of cells along this axis which allows the study of any one of these processes in an enriched population of cells. As an example, the uptake of transferrin-bound iron by enterocytes was studied. Rats were fed diets normal, high (3% carbonyl iron) or low in iron for 12 days. Cells from either the duodenum or ileum were isolated by incubating in a Ca²⁺-, Mg²⁺-free, cation chelating solution for varying periods. The incorporation of thymidine into DNA was measured in these cells as a marker of the crypt region, while alkaline phosphatase and sucrase activities marked mature enterocytes. The in vivo uptake of transferrin-bound ⁵⁹Fe was measured in cells isolated either 2 or 4 h after intravenous injection. This procedure resulted in the isolation of 10 fractions of viable cells. Earlier fractions were enriched at least 10-fold in villus cells and the last fractions in crypt cells. Cells in intermediate fractions were at various stages of development. Uptake of transferrin-bound iron into enterocytes was highest with feeding an iron-loaded diet compared with control or iron-deficient diets. However, with all diets uptake was highest in crypt cells and this fell at the crypt-villus junction to be only 25%, as high at the villus tip as the crypt. A technique for the reproducible isolation of viable enterocytes along a crypt-villus axis is described. Transferrin receptor activity changes with maturation of the enterocyte.     

Stimulation by 1,25-dihydroxyvitamin D3 of adenylate cyclase along the villus of chick duodenum

Administration of 650 pmol 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] to vitamin D-deficient chicks increased adenylate cyclase activity in the basolateral membrane of duodenal epithelial cells within 24 h. This increase in enzymatic activity was accompanied by an increase in calmodulin content of the basolateral membrane. Although neither exogenously added calmodulin (up to 10 micrograms/ml) nor calcium (from 10(-7)-10(-5) M) stimulated enzyme activity, calmodulin antagonists trifluoperazine, W7, and W13 inhibited it. When calmodulin content, adenylate cyclase activity, and alkaline phosphatase activity were measured in cells sequentially eluted from the tip to the base of the villus, cells from the midregion and base had the highest calmodulin content and adenylate cyclase activity, whereas alkaline phosphatase activity (a brush border membrane enzyme) was highest in cells eluted from the tip. Adenylate cyclase activity was increased by 1,25-(OH)2D3, particularly in cells from the midvillus. Our results indicate that the response of adenylate cyclase activity to 1,25-(OH)2D3 varies along the villus and suggest that calmodulin may be involved.     

Differentiation status of rat enterocytes after intestinal adaptation to jejunoileal bypass.

The differentiation status of epithelial cells in intestinal adaptation remains unclear. To determine whether enterocytes reach optimum maturity following adaptation after 85% shortening of the rat gut by jejunoileal bypass surgery, activities of two brush border enzymatic markers of differentiation, alkaline phosphatase and sucrase, were examined in subpopulations of epithelial cells isolated sequentially from the villus/crypt axis of normal (sham operated) and hyperplastic mucosa. In jejunal villi, adaptational hyperplasia was associated with an increase in total epithelial alkaline phosphatase, but not total sucrase, activity; alkaline phosphatase activity increased most obviously in cells at the 11-50% position (from the tip) on villi. In hyperplastic ileal villi, total alkaline phosphatase activity fell, although sucrase activity did not change significantly. Specific activity (per mg protein) of sucrase on jejunal villus epithelium was reduced by the adaptational changes to bypass; alkaline phosphatase specific activity remained unchanged. In the ileum, despite adaptational changes to bypass, there was no increase in the normally low specific activities of sucrase and alkaline phosphatase. Bypass surgery did not change the major site of expression of either enzyme on jejunal or ileal villi. In conclusion, enzymatic markers of functional differentiation are not all equally affected by adaptational hyperplasia. Hypertrophy of villi and increased cell proliferation seen in jejunum remaining exposed to luminal contents resulted in an increase in the alkaline phosphatase but not the sucrase content. This is not, therefore, the result of a simple immaturity of villus cells. Morphological adaptation in the ileum, however, is not accompanied by adaptation of brush border enzyme markers of differentiation, confirming a functional immaturity of these cells. Strategies for increasing the expression of these markers may have clinical value.     

The effects of vitamin D metabolites on the plasma and matrix vesicle membranes of growth and resting cartilage cells in vitro

This study compares the effects of vitamins 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-(OH)2D3 on populations of chondrocytes at different developmental stages. Confluent third passage chondrocytes derived from the resting zone and adjacent growth region of rat costochondral cartilage were cultured in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum and increasing concentrations of hormone. After determination of cell number, matrix vesicles and plasma membranes were isolated by differential centrifugation. The effects of hormone on alkaline phosphatase, 5'-nucleotidase, ouabain-sensitive Na+/K+-ATPase, and phospholipid composition were dependent on vitamin D metabolite and were cell specific. Growth cartilage chondrocytes responded primarily to 1,25-(OH)2D3, whereas resting zone cells responded primarily to 24,25-(OH)2D3. 1,25-(OH)2D3 inhibited growth cartilage cell number at pharmacological concentrations and had no effect on resting cartilage cell number. In contrast, 24,25-(OH)2D3 appeared to stimulate resting cartilage cell number at physiological concentrations and inhibit these cells at pharmacological doses, but had no effect on growth cartilage chondrocytes. These data were supported by [3H]thymidine incorporation studies. 1,25-(OH)2D3 stimulated alkaline phosphatase, 5'-nucleotidase activity, and Na+/K+-ATPase activity in the matrix vesicles of growth cartilage cells. 1,25-(OH)2D3 also stimulated Na+/K+-ATPase activity in the matrix vesicles and plasma membranes of resting zone cells. Incubation with 24,25-(OH)2D3 stimulated alkaline phosphatase, 5'-nucleotidase, and Na+/K+-ATPase in the matrix vesicles produced by resting zone cells. In addition, 24,25-(OH)2D3 stimulated Na+/K+-ATPase activity in the plasma membranes of resting zone cells as well as in both matrix vesicles and plasma membranes of growth cartilage cells.     

Effect of Fat Feeding on Pro-oxidant and Anti-oxidant Enzyme Systems in Rat Intestine: Possible Role in the Turnover of Enterocytes

Immature epithelial cells generated in the crypt base undergo differentiation while progressing to the villus tip, where the cells upon apoptosis are detached from the underlying muscular tissue. We previously reported that lipid peroxidation might be involved in the turnover of enterocytes across the crypt–villus axis in rat intestine (Dig Dis Sci 52:1840–1844, 2007). To examine whether long-term feeding of fat with different fatty-acid composition influences this process, in the present study we investigated the effect of feeding fish oil (n − 3) and corn oil (n − 6) polyunsaturated fatty acids on lipid per-oxidation and anti-oxidant systems in different epithelial cell fractions isolated in rat intestine. Feeding fish oil or corn oil markedly enhanced lipid per-oxidation levels of enterocytes throughout villus height compared with control, but there was no difference in the distribution profile of pro- and anti-oxidant enzyme systems and lipid per-oxidation across the crypt–villus axis under these conditions. Analysis of lipid peroxidation levels in different cell fractions revealed that the thiobarbituric acid reactive substance were 9- to 11-fold higher at the villus tip compared with at the crypt base. The activities of glutathione reductase and glutathione-S-transferase were 2- to 5-fold higher in villus tip compared to the crypt region. However, the activities of superoxide dismutase and catalase were 6- to 8-fold high at the crypt base compared with at villus tip cells. Immunocytolocalization of superoxide dismutase showed high staining in crypt base compared with that in villus, tip cells. These findings further suggest that generation of reactive oxygen species in enterocytes across the crypt–villus axis may be involved in turnover of enterocytes across the crypt–villus unit in rat intestine.     

The Profile of Antioxidant Systems and Lipid Peroxidation Across the Crypt-Villus Axis in Rat Intestine

The distribution of lipid peroxiation and profile of antioxidant-pro-oxidant enzyme systems have been studied in rat intestinal enterocyte across the length of villi. The MDA levels estimated as a measure of lipid peroxidation, under induced or uninduced in vitro conditions, indicated markedly high levels at villus tip cells compared to that in the crypt base. The activities of glutathione-S-transferase and glutathione reductase were three- to sixfold higher in villus tip cells compared to that in the crypt base. However the levels of catalase and superoxide dismutase showed a reverse pattern, being high in the crypt base and lowest in the villus tip region. Feeding coconut oil, sunflower oil, or groundnut oil did not modify the distribution pattern of these systems across crypt-villus unit in rat intestine. These findings suggest that the large amount of free radicals generated in villus tip cells may be responsible for the release of enterocytes from the villus tip as a consequence of apoptosis.     

Distribution of immunoreactive alkaline phosphatase in the adult rat ileum by immunoperoxidase staining at the light microscopic level

Our prior immunocytochemical studies using monospecific antibody to alkaline phosphatase, Bouin's fixation, and paraffin sections demonstrated a decreasing gradient of villus brush border staining from the proximal to the distal rat small intestine. In addition, we noted an unusual pattern of staining in the terminal centimeter of the adult rat ileum: the villus brush border staining was less intense than crypt brush border staining. To determine whether this pattern of staining was present throughout the entire ileum, we examined alkaline phosphatase staining in two separate jejunal sites and the entire lowest third of the intestine of adult Wistar rats. With Bouin's fixation and paraffin embedding, both conventional and germ-free rats showed the same unusual staining pattern throughout the entire ileum. This pattern suggested that bacterial proteases were not responsible for the diminished ileal brush border alkaline phosphatase. However, when acetone fixation and cryostat sections were used with the avidin-biotin-peroxidase complex system, the previously noted reversed gradient of staining between the ileal villus and crypt areas was no longer present. Rather, ileal crypt brush border staining was less than ileal villus brush border staining. With either methodology, jejunal villus brush border staining was significantly more intense than ileal brush border staining, whereas the deep crypt brush border staining was not significantly different between the two regions. The present study reinforces the need for a combination of methodologies in order to best and most accurately localize certain antigens with immunocytochemistry. It also confirms a decreasing proximal to distal gradient for villus brush border alkaline phosphatase despite similar deep crypt brush border staining throughout the small intestine.     

gamma-glutamyl transpeptidase of rat intestine: localization and possible role in amino acid transport.

gamma-Glutamyl transpeptidase (gamma-GT), an enzyme possibly involved in amino acid transport, was investigated in rat small intestine using the synthetic substrate L-gamma-glutamyl-p-nitroanilide. Enzyme localization and characteristics were correlated with features of amino acid uptake. gamma-GT activity copurified with sucrase and alkaline phosphatase. Activity was maximal at pH 8.2 and was stimulated by monovalent cations. The relative specificity of the gamma-GT reaction with diglycine and eight essential amino acids as substrates correlated well with the rate of intestinal absorption of this dipeptide and these amino acids as observed by others. gamma-GT activity was 12-fold greater in the jejunum than in the ileum, again in agreement with relative rates of amino acid absorption along the length of rat intestine. The specific activity of gamma-GT in villus tip cells was 10 times greater than in crypt cells, and amino acid uptake was 2 to 6 times greater with villus tip than with crypt cells. Bromosulfophthalein, a noncompetitive inhibitor of gamma-GT, inhibited amino acid uptake. These studies support the concept that membrane gamma-GT may be involved in amino acid and dipeptide uptake, and indicate that further investigation of such involvement may be conveniently pursued using mammalian small bowel.     

The effects of vitamin D metabolites on phospholipase A2 activity of growth zone and resting zone cartilage cells in vitro

Third passage confluent cultures of cartilage cells, initially derived from the growth zone (GC) and resting zone (RC) of rat costochondral cartilage, were incubated with either 10(-11)-10(-8) M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or 10(-9)-10(-6) M 24,25-(OH)2D3. Plasma membranes and extracellular matrix vesicles were isolated, and specific activities of phospholipase A2 and alkaline phosphatase were determined. The results demonstrate that the response to hormone is both cell and membrane specific. 1,25-(OH)2D3 produces an increase in GC matrix vesicle alkaline phosphatase and phospholipase A2 specific activities at 10(-9) and 10(-8) M, but has no effect on these enzyme activities in RC membranes. RC cultured in 24,25-(OH)2D3 exhibit increased matrix vesicle alkaline phosphatase but decreased phospholipase A2 activities at 10(-7) and 10(-6) M hormone. No effect on the RC plasma membrane enzymes or on GC plasma membrane or matrix vesicle enzymes was observed. The data suggest that changes in membrane fluidity due to phospholipase A2 activity may play a role in regulating alkaline phosphatase activity in response to vitamin D metabolites and that this regulation in GC and RC may proceed by different mechanisms.     

Regulation of intestinal epithelial cell growth by transforming growth factor type beta.

A nontransformed rat jejunal crypt cell line (IEC-6) expresses transforming growth factor type beta 1 (TGF-beta 1) mRNA, secretes latent 125I-labeled TGF-beta 1 competing activity into culture medium, and binds 125I-labeled TGF-beta 1 to specific, high-affinity (Kd = 3.7 pM) cell surface receptors. IEC-6 cell growth is markedly inhibited by TGF-beta 1 and TGF-beta 2 with half-maximal inhibition occurring between 0.1 and 1.0 ng of TGF-beta 1 per ml. TGF-beta 1-mediated growth inhibition is not associated with the appearance of biochemical markers of enterocyte differentiation such as alkaline phosphatase expression and sucrase activity. TGF-beta 1 (10 ng/ml) increases steady-state levels of its own mRNA expression within 8 hr of treatment of rapidly growing IEC-6 cells. In freshly isolated rat jejunal enterocytes that are sequentially eluted from the crypt villus axis, TGF-beta 1 mRNA expression is most abundant in terminally differentiated villus tip cells and least abundant in the less differentiated, mitotically active crypt cells. We conclude that TGF-beta 1 is an autoregulated growth inhibitor in IEC-6 cells that potentially functions in an autocrine manner. In the rat jejunal epithelium, TGF-beta 1 expression is most prominently localized to the villus tip--i.e., the region of the crypt villus unit that is characterized by the terminally differentiated phenotype. These data suggest that TGF-beta 1 may function in coordination of the rapid cell turnover typical for the intestinal epithelium.     

Chronic low vitamin intake potentiates cisplatin-induced intestinal epithelial cell apoptosis in WNIN rats

AIM:To investigate if cisplatin alters vitamin status andif VR modulates cisplatin induced intestinal apoptosis andoxidative stress in Wistar/NIN(WNIN)male rats.METHODS:Weanling,WNIN male rats(n=12 pergroup)received adlibitum for 17 wk:control diet(20%protein)or the same with 50% vitamin restriction.Theywere then sub-divided into two groups of six rats eachand administered cisplatin(2.61 mg/kg bodyweight)once a week for three wk or PBS(vehicle control).Intestinal epithelial cell(IEC)apoptosis was monitoredby morphometry,Annexin-V binding,M30 cytodeathassay and DNA fragmentation.Structural and functionalintegrity of the villus were assessed by villus height/crypt depth ratio and activities of alkaline phosphatase,lys,ala-dipeptidyl amino-peptidase,respectively.Toassess the probable mechanism(s)of altered apoptosis,oxidative stress parameters,caspase-3 activity,andexpression of Bcl-2 and Bax were determined.RESULTS:Cisplatin per se decreased plasma vitaminlevels and they were the lowest in VR animals treatedwith cisplatin.As expected VR increased only villusapoptosis,whereas cisplatin increased stem cellapoptosis in the crypt.However,cisplatin treatmentof VR rats increased apoptosis both in villus and cryptregions and was associated with higher levels of TBARS,protein carbonyls and caspase-3 activity,but lower GSHconcentrations.VR induced decrease in Bcl-2 expression was further lowered by cispiatin.Bax expression,unaffected by VR was increased on cisplatin treatment.Mucosal functional integrity was severely compromisedin cisplatin treated VR-rats.CONCLUSION:Low intake of vitamins increases thesensitivity of rats to cisplatin and promotes intestinalepithelial cell apoptosis.     

The villus gradient of brush border membrane calmodulin and the calcium-independent calmodulin-binding protein parallels that of calcium-accumulating ability

We have recently proposed that calmodulin (CaM) may mediate calcium transport across the intestinal brush border membrane. Since calcium transport across this membrane varies as a function of cellular location on the villus (the highest rates of transport occur across the brush border membrane from cells near the tip), we tested this hypothesis by determining whether CaM and its principal binding protein in the brush border membrane [a 102,000 mol wt (102K) protein] also showed this gradient of activity along the villus. Cells were sequentially eluted from the tip to the base of the villus, brush border membrane vesicles (BBMV) were prepared from the eluted cells, and CaM, CaM binding, and calcium-accumulating ability were determined for each preparation of BBMV. We observed that BBMV prepared from cells originating near the tip of the villus possessed the greatest calcium-accumulating activity, CaM content, and CaM binding by the 102K protein. All three measurements were reduced in parallel in BBMV prepared from cells originating from more basal regions of the villus. Calcium-accumulating ability correlated with CaM content (r = 0.876) and CaM binding to the 102K protein (r = 0.788); likewise, CaM correlated with CaM binding to the 102K protein (r = 0.928). When 1,25-dihydroxyvitamin D was administered to vitamin D-deficient chicks, the binding of CaM to the 102K CaM-binding protein appeared to increase more rapidly in BBMV from cells near the tip of the villus than in cells from more basal regions, comparable to our previously reported data for 1,25-dihydroxyvitamin D-stimulated calcium accumulation by similarly prepared BBMV. These data support the hypothesis that CaM and the 102K CaM-binding protein are involved in the regulation of calcium flux across the intestinal brush border membrane.     

Systemic factors are trophic in bypassed rat small intestine in the absence of luminal contents.

Mucosal histology, crypt cell proliferation and brush border enzymes were measured in rats with varying degrees of jejunoileal bypass, in order to compare the effect of systemic and luminal factors on adaptive growth and differentiation (brush border enzymes) in small intestinal epithelium. Eighty five percent jejunoileal bypass caused a functional short gut; in intestine remaining in continuity there were significant increases in segmental weight, villus area and crypt depth, compared with sham operated controls and 25% jejunoileal bypass rats. Despite villus cell hyperplasia in 85% bypass rats, mucosal sucrase and alkaline phosphatase fell in jejunum and remained low in ileum, while leucine amino peptidase rose in ileum. There was a significant fall in villus area (p less than 0.01) and crypt cell production (p less than 0.001) in self emptying loops of 25% bypass rats not exposed to luminal contents compared with control segments of sham operated rats. In contrast, self emptying loops of 85% bypass rats were not atrophied despite the much greater distance from luminal nutrients; the villus area (p less than 0.01) and crypt cell production (p less than 0.005) were higher than in 25% bypass rats, and at least as great as in sham operated rats. These results indicate that adaptive hyperplasia has a variable effect on expression of brush border enzymes which might reflect villus cell immaturity. The atrophic effect of diversion of luminal contents can be counteracted by systemic growth factors released as part of the adaptive response; thus systemic growth factors are not dependent on a permissive effect of luminal contents.     

Changes in Cell Proliferation and Differentiation of Adult Rat Small Intestine Epithelium After Adrenalectomy

The effects of 10-day bilateral adrenalectomy on morphometry, proliferation, and apoptosis were determined in the small intestine of 3-month-old Sprague–Dawley rats. The activities of sucrase, lactase, and its respective mRNA, aminopeptidase N, and intestinal alkaline phosphatase were also evaluated. Adrenalectomy lead to partial atrophy and disorganization of the epithelium, with an increased number of goblet and Paneth cells and a reduction of crypt cell proliferation paralleled by a marked increase in villus apoptosis. Biochemical assays revealed that aminopeptidase N and intestinal alkaline phosphatase activities were significantly decreased, whereas disaccharidases were increased by adrenalectomy. The corresponding induction of lactase mRNA suggests an active response of the epithelium. In conclusion, adrenalectomy modified maturation and the differentiation processes of the small intestinal mucosa, especially in the proximal part of the small intestine. This result points to an important role of adrenals and glucocorticoids in the trophic status of the adult small intestinal mucosa.     

Colchicine prevents D-galactosamine-induced hepatitis

The hepatoprotective effect of colchicine in a model of liver intoxication with galactosamine (GalN), 375 mg/kg, i.p., was studied in rats. At 0.5, 1, 3, 6, 18 and 24 h after GalN intoxication the following markers of liver damage were measured: serum activity of alanine aminotransferase, alkaline phosphatase, gamma-glutamyltranspeptidase, hepatic calcium and glycogen contents, liver lipoperoxidation, and liver plasma membrane activity of alkaline phosphatase, gamma-glutamyltranspeptidase and high-affinity Ca2+-ATPase. 24 h after GalN intoxication increases in serum levels of alanine aminotransferase, alkaline phosphatase and gamma-glutamyltranspeptidase were observed along with decreases in plasma membrane activities of alkaline phosphatase, gamma-glutamyltranspeptidase, and high-affinity Ca2+-ATPase. A sharp increase of lipoperoxidative processes measured as malondialdehyde production was also observed. Pretreatment of rats with colchicine 10 micrograms/rat/day p.o. for 7 days before GalN injection prevented partially the toxic effects of GalN. When a dose of 50 micrograms/rat/day for 7 days was given the drug prevented almost completely the damage induced by galactosamine, with the exception of glycogen and serum alkaline phosphatase that remained different from controls. Time-course experiments showed that malondialdehyde formation increased 30 min after intoxication while all other changes became apparent from 6 h after treatment, suggesting that lipoperoxidation may be a prerequisite for galactosamine-induced damage. The protection offered by colchicine was related to its capacity to inhibit lipoperoxidation. Histochemical findings paralleled the biochemical results. The possible role of lipoperoxidation in galactosamine-induced liver damage is discussed.