Digestive Diseases and Sciences, Vol. 37, No. 3 (March 1992), pp. 417--425
Alterations in Quantitative Distribution of Na,K-ATPase Activity Along Crypt-Villus Axis in Animal Model of Malabsorption Characterized by Hyperp.roliferative Crypt Cytokinetlcs GARY E. WILD, MD, PhD, FRCP(C), and DAVID MURRAY, MB, ChB, FRCPath
The distribution of sodium- and potassium-stimulated ATPase (Na,K-ATPase) along the crypt-villus axis and crypt cytokinetics were examined in an infective model of celiac disease produced by infection of the rat with the nematode Nippostrongylus brasiliensis. In controls, levels of enzyme activity remained stable during enterocyte migration to the villous apex. In the jejunum of infected rats, the structural lesion of villous atrophy and crypt hyperplasia, observed at day 10 of infection, was associated with a threedimensional expansion of the crypts. Cell cycle time was shortened and this resulted in a markedly increased crypt cell production rate. Enterocytes emerged from the crypts at a faster rate, and this functional immaturity was paralleled by decreased Na,K-ATPase activity. Further decreases in enzyme levels were observed during enterocyte migration along the villi. This may reflect enterocyte damage or increased enzyme turnover. In the ileum of these animals, enterocyte maturation was prolonged and enzyme activity was increased at the level of the crypt villus junction with further increases noted during enterocyte transit. These changes in ileal Na,K-ATPase appear to be adaptive. KEY WORDS: nippostrongylosis; intestinal absorption; Na,K-ATPase; intestinal inflammation.
The integrity of the small intestinal epithelium is a reflection of steady-state cell kinetics wherein the loss of villous cells is balanced by crypt cell proManuscript received March 19, 1990; revised manuscript received August 2, 1991; accepted August 5, 1991. From the Department of Medicine, Division of Gastroenterology, McGill University, Montreal, Canada, and Department of Pathology, University of Toronto, Toronto, Canada. This study was presented in part at the Annual Meeting of the American Gastroenterological Association, Chicago, May 12, 1987) and published in abstract form (Gastroenterology 92:1695, 1987). These studies were supported by a grant from the Medical Research Council of Canada. Address for reprint requests: Dr. Gary Wild, Montreal General Hospital, 1650 Cedar, Montreal, Quebec, Canada H3G 1A4.
duction (1). Cell migration along the crypt-villus axis is accompanied by maturation of enzyme and transport activities (2). The small bowel mucosa in celiac disease is characterized by increased enterocyte turnover, an atrophy of the villi, and crypt hyperplasia (3-5). The hyperproliferative state is associated with a reduction in microvillar enzyme levels (6). Similar structural and functional alterations are seen in a variety of experimental animal models (7-10). We have recently examined enterocyte levels of Na,K-ATPase and sodium transport during the course of Nippostrongylus brasiliensis infection of
Digestive Diseases and Sciences, Vol. 37, No. 3 (March 1992)
WILD AND MURRAY the rat (11, 12), which is a well-characterized infective model o f celiac disease (13, 14). At the p e a k of infection (ie, day 10) the mucosal lesion of villous atrophy--crypt hyperplasia was associated with a significant reduction in jejunal enterocyte N a , K A T P a s e and sodium transport levels (12). Adaptive increases in these p a r a m e t e r s were o b s e r v e d in ileal enterocytes (12). In the present study, we have investigated the b e h a v i o r o f N a , K - A T P a s e activity during the course o f e n t e r o c y t e migration along the c r y p t villus axis in an attempt to ascertain to what extent this activity is influenced by the hyperproliferative cytokinetic state seen in this animal model of intestinal inflammation. T o this end, we focused our efforts on defining the size and kinetics of the crypt c o m p a r t m e n t in this hyperproliferative model.
M A T E R I A L S AND M E T H O D S Animals. Male Sprague-Dawley rats weighing 150 g were used in this study. Rats were injected subcutaneously with 4000 third-stage infective Nippostrongylus brasiliensis larvae as described (12). Rats were killed on the tenth day of infection. Preparation of Tissue Sections. Portions of proximal jejunum (15 cm from the py!orus) and distal ileum (3 cm from the ileocecal valve) were frozen quickly in Dry Ice and transferred to a cryostat held at - 2 0 ~ C. Serial sections (16 p~m) were cut perpendicular to the long axis of the villi. Groups of six to nine sections were transferred to small glass homogenizers. The last section in each group was taken for microscopic identification of villi and/or crypts. Biochemical determinations. The tissue sections were homogenized in double-distilled water. Aliquots (20-40 p~g protein) were assayed for Na,K-ATPase activity by measuring the release of Pi from ATP in the presence and absence of 1 mM ouabain as described previously (15). The incubation medium contained the following (final concentrations all in mM): 100 Tris HC1 (pH 7.5), 60 NaC1, 5 KCI, 2 MgC12, 2 ATP, and 0.1 EDTA. Na,KATPase activity was defined as the ouabain-sensitive fraction of the total ATPase activity and expressed as micromoles of phosphate released per hour per milligram of protein. Protein was measured according to the method of Nayyar and Glick (16). To compensate for the variation in the number of sections obtained from different animals within any particular group, activities of Na,K-ATPase were related to an idealized villus-crypt unit using the normalization procedure of Kolodovsky and Herbst (17). Morphometric Methods. Portions of jejunum and ileum adjacent to those taken for enzyme assay were processed for paraffin embedding. Villus height (V), crypt depth (C), and the V/C ratio was measured as described (15). Crypt Analyses. For the calculation of mitotic index (lm), approximately 100 completely axially sectioned
crypts were examined using criteria and counting techniques described elsewhere (18, 19). Graphs were constructed of lm as a function of crypt cell position and the normalization procedure of Wright et al (20) was applied. Crypt length was measured by counting cells from the crypt base tO the villus-crypt junction. The crypt column count (ie, number of cells in a crypt section) was measured in 50-100 circular crypt cross sections from specimens cut tangentially. The product of crypt length and column count was obtained as an estimate of the total number of crypt cells (27). The fraction of the crypt occupied by the proliferative compartment is the region over which mitoses are observed, and the upper limit of the proliferative compartment is the cell position where the value of Im is half maximal (21). Estimates of the growth fraction were derived from the ratio of the number of cells in the proliferative compartment to the total number of cells in the crypt column (20). The maturation compartment extends from the crypt cell position where Im is half maximal to the crypt-villus junction (22). The mean cell cycle time (To) and duration of mitosis (Tm) for the whole crypt were obtained using a stathmokinetic technique (23). Groups of control and experimental animals were injected intraperitoneally with 2.5 mg/kg of colchicine and killed at 30 or 150 min after injection. Groups of rats injected with saline were killed at zero time, from which the values for the native I,~ were obtained. Mitotic indices were calculated at each time interval, and regression lines were constructed by the method of least squares. The rate of entry into mitosis for the entire crypt was calculated from the slopes (R) of these lines. Correction was made for growth fraction and for spatial distribution of mitoses in crypt sections (19). The mean Tr was computed as the inverse of the rate of entry of Cells into mitosis (19). Values of Tm were derived from the ratios of native Im to R, both expressed as percentages (19). The rate of cell production per crypt and the transit time through the crypt were measured Using methods described elsewhere (5, 18). Briefly, a birth rate for each cell position was derived from the ratio of lm to Tm and summated to yield a cumulative birth rate. This in turn was multiplied by the crypt column count to obtain an estimate of the rate of cell production per crypt. The reciprocals of the cumulative birth rates at each cell position were summated over the length of the proliferative and maturation compartments to obtain an estimate of time spent in these compartments. The stem cell compartment was arbitrarily defined as those basal crypt cell positions with a transit time of greater than ten hours. Statistical Analyses. Results are expressed as the mean _+ standard error of mean (SEM). Student's t test and one-way ANOVA were used in the statistical analysis of differences. Only P values less than 0.05 were considered significant. RESULTS
Distribution of Na,K-ATPase Activity along Jejunal Crypt-Villus Axis. In controls (Figure I), analysis of variance revealed no significant differenCes Digestive Diseases and Sciences, Vol. 37, No. 3 (March 1992)
Na,K-ATPASE IN EXPERIMENTAL MALABSORPTION 6B Mainly villi
~4 0 rL b-