The role of sphingomyelin synthetase and sphingomyelinase in 1,2-dimethylhydrazine-induced lipid alterations of rat colonic plasma membranes

Recently, our laboratory, utilizing the 1,2-dimethylhydrazine model of colonic adenocarcinoma, demonstrated alterations in the 'dynamic component' of fluidity in brush-border membranes prepared from distal colonocytes of rats administered this agent for 5, 10 and 15 weeks, i.e., before the development of colon cancer. Furthermore, changes in the sphingomyelin content and sphingomyelin/phosphatidylcholine molar ratio of these membranes appeared, at least partially, to be responsible for these fluidity alterations. In an attempt to elucidate the mechanism(s) involved in these dimethylhydrazine-induced lipid changes, in the present studies the activities of sphingomyelin synthetase and magnesium-dependent neutral sphingomyelinase, enzymes involved in the synthesis and degradation of this phospholipid, respectively, were examined and compared in distal colonic brush-border membranes prepared from rats after 5, 10 or 15 weeks administration of dimethylhydrazine or diluent. The results of these studies demonstrate that alterations in both these enzymatic activities can be detected after administration of dimethylhydrazine and appear to, at least in part, be responsible for the changes in membrane sphingomyelin composition noted previously. These results as well as a discussion of their possible serve as the basis for the present report.     

1,2-Dimethylhydrazine-induced premalignant alterations in the S-adenosylmethionine/S-adenosylhomocysteine ratio and membrane lipid lateral diffusion of the rat distal colon

Prior studies by our laboratory, utilizing the 1,2-dimethylhydrazine experimental model of colonic cancer, had shown that administration of this procarcinogen for 5 weeks was found to increase phospholipid methyltransferase activity and the fluidity of rat distal colonic brush-border membranes. The present studies were conducted to further explore these 'premalignant' colonic phenomena. Male albino rats of the Sherman strain were subcutaneously injected with dimethylhydrazine (20 mg/kg body weight per week) or diluent for 5 weeks. Animals from each group were killed, distal colonic tissue harvested and the levels of S-adenosylmethionine, S-adenosylhomocysteine and decarboxylated S-adenosylmethionine measured by high performance liquid chromatography. The activity of methionine adenosyltransferase was also examined in these tissues. Additionally, brush-border membranes were isolated from the distal colonocytes of control and treated-animals and examined and compared with respect to their phospholipid methylation activities as well as their lipid fluidity as assessed by the rotational mobilities of the probes 1,6-diphenyl-1,3,5-hexatriene and DL-12-(9-anthroyl)stearic acid and translational mobility of the fluorophore pyrenedecanoic acid. The results of these studies demonstrated: (1) phospholipid methyltransferase activity in rat colonic plasma membranes was increased concomitantly with increases in the cellular levels of S-adenosylmethionine and the S-adenosylmethionine/S-adenosylhomocysteine ratio in the distal colonic segment of treated-animals; and (2) the lateral diffusion of rat distal colonic brush-border membrane lipids, as assessed by the ratio of excimer/monomer fluorescence intensities of the fluorophore pyrenedecanoate, was also increased after dimethylhydrazine administration to these animals for 5 weeks.     

1,2-Dimethylhydrazine-induced alterations in Na+-H+ exchange in rat colonic brush-border membrane vesicles

1,2-Dimethylhydrazine, in weekly subcutaneous (s.c.) doses of 20 mg/kg body weight, produces colonic tumors in virtually 100% of rodents, with a latency period of approximately 6 months. To determine whether alterations in Na+-H+ exchange existed before the development of dimethylhydrazine-induced colon cancer, rats were given s.c. injections of this agent (20 mg/kg body wt. per per week) or diluent for 5 weeks. Animals were then killed, rat colonic brush-border membrane vesicles prepared and amiloride-sensitive sodium-stimulated proton efflux was measured and compared in control and treated-preparations. The results of these studies demonstrated that dimethylhydrazine treatment: (1) significantly increased the Vmax of this exchange without altering the Km for sodium of this exchange process, utilizing the fluorescent pH-sensitive dye, acridine orange; 22Na flux experiments also demonstrated an increase in amiloride-sensitive proton-stimulated sodium influx across treated-membrane vesicles; (2) did not appear to significantly influence Na+ permeability or proton conductance in treated-preparations compared to their control counterparts; and (3) did not significantly affect the kinetic parameters of amiloride-sensitive sodium-stimulated proton efflux in renal cortex brush-border membrane vesicles using acridine orange. This data, therefore, suggests that alterations in Na+-H+ exchange in rat colonic brush-border membranes may be involved in the malignant transformation process induced by this procarcinogen in the large intestine.     

1,2-Dimethylhydrazine-induced alterations in lipid peroxidation in preneoplastic and neoplastic colonic tissues

To determine whether alterations in lipid peroxidation existed in the preneoplastic and neoplastic colonic tissues of animals treated with the procarcinogen 1,2-dimethylhydrazine, rats were injected subcutaneously with this agent (20 mg/kg body weight per week) or diluent for 5, 10, 15 and 26 weeks. At each of these time periods, animals from both groups were sacrificed, their distal colonic mucosa and/or tumors harvested, and examined and compared with respect to malondialdehyde and lipofuscin-like pigments levels. Additionally, at 26 weeks, the fatty acid composition of microsomes prepared from control, 'uninvolved' and tumor colonic tissues were analyzed and compared. The results of these experiments demonstrated that: (1) the levels of these products of lipid peroxidation were similar in the distal colons of all animals at 5 and 10 weeks; (2) at 15 weeks, however, lipid peroxidation was decreased in the distal colons of animals treated with dimethylhydrazine; (3) at 26 weeks, the levels of these products of lipid peroxidation remained lower in dimethylhydrazine-treated distal 'uninvolved' colonic mucosa and was, moreover, markedly decreased in colonic tumors; and (4) at this latter time period, differences in the fatty acid composition between tumor, 'uninvolved' and control tissues were found. These differences, however, did not appear to underlie the changes noted in the lipid peroxidation products seen in these tissues. Taken together, these findings suggest that alterations in lipid peroxidation may be involved in the colonic malignant transformation process in this experimental model.     

N-acetylneuraminic acid and N-glycolylneuraminic acid in glycopeptides of colonic tumor and mucosa in rats treated with carrageenan and 1,2-dimethylhydrazine

N-linked glycopeptides were prepared from colonic tumor (adenocarcinoma) and mucosa in rats treated with carrageenan, an indigestible polysaccharide, and 1,2-dimethylhydrazine. Sialic acids, N-acetylneuraminic acid and N-glycolylneuraminic acid, obtained by acid hydrolysis of the glycopeptides were determined by HPLC. The N-acetylneuraminic acid/N-glycolylneuraminic acid ratio in colonic tumor was 25.2, while each treated mucosa had the values between 0.29 and 0.55. Thus, necessity which observes the qualitative change of sialic acid in malignant transformation was suggested.     

1,2-Dimethylhydrazine-induced alterations in N1-acetylspermidine levels in rat distal colonic mucosa: effects of 2-difluoromethylornithine

Recently, our laboratory has demonstrated that N1-acetylspermidine levels were increased in the distal colonic mucosa of rats administered 1,2-dimethylhydrazine for 15 and 26 weeks. In order to further explore the possible role of this acetylated polyamine in the malignant transformation process induced by this carcinogen, groups of rats were subcutaneously injected weekly with dimethylhydrazine (20 mg/kg body wt.) or diluent for 5, 10, 15 and 26 weeks +/- 1% 2-difluoromethylornithine in the drinking water. The latter agent, an irreversible inhibitor of ornithine decarboxylase, has previously been shown to inhibit colonic tumor formation in this experimental model. At each of these time periods, rats from each group were killed, their proximal and distal colonic mucosa harvested and examined, and compared with respect to polyamine levels, including N1-acetylspermidine, as well as the activities of ornithine decarboxylase, S-adenosylmethionine decarboxylase, spermidine N1-acetyltransferase and polyamine oxidase. The results of these experiments demonstrated that: (1) N1-acetylspermidine levels in the proximal colonic segment of all animals were similar at each time point; (2) N1-acetylspermidine levels were also similar in the distal colons of all animals at 5 and 10 weeks. At 15 weeks, however, the level of N1-acetylspermidine was increased in the dimethylhydrazine-treated distal colonic segment secondary to increases in the activity of spermidine N1-acetyltransferase; and (3) at 26 weeks, the level of this acetylated polyamine remained higher in dimethylhydrazine-treated distal 'uninvolved' colonic mucosa and was markedly elevated in colonic tumors; (4) co-administration of difluoromethylornithine decreased the elevated levels of N1-acetylspermidine to control values in the distal colons of animals treated with carcinogen for 15 and 26 weeks; and (5) difluoromethylornithine markedly reduced the number of tumors induced by dimethylhydrazine in the distal but not proximal colonic mucosa at 26 weeks.     

Modulation of rat distal colonic brush-border membrane Na+-H+ exchange by dexamethasone: role of lipid fluidity

Earlier studies by our laboratory have suggested a relationship between an amiloride-sensitive Na+-H+ exchange process and the physical state of the lipids of rat colonic brush-border membrane vesicles. To further assess this possible relationship, a series of experiments were performed to examine the effect of dexamethasone administration (100 micrograms/100 g body wt. per day) subcutaneously for 4 days on Na+-H+ exchange, lipid composition and lipid fluidity of rat distal colonic brush-border membrane vesicles. The results of these studies demonstrate that dexamethasone treatment significantly: (1) increased the Vmax of the Na+-H+ exchange without altering the Km for sodium of this exchange process, utilizing the fluorescent pH-sensitive dye, acridine orange. 22Na flux experiments also demonstrated an increase in amiloride-sensitive proton-stimulated sodium influx across dexamethasone-treated brush-border membrane vesicles; (2) increased the lipid fluidity of treated-membrane vesicles compared to their control counterparts, as assessed by steady-state fluorescence polarization techniques using three different lipid-soluble fluorophores; and (3) increased the phospholipid content of treated-membrane vesicles thereby, decreasing the cholesterol/phospholipid molar ratio of treated compared to control preparations. This data, therefore, demonstrates that dexamethasone administration can modulate amiloride-sensitive Na+-H+ exchange in rat colonic distal brush-border membrane vesicles. Moreover, it adds support to the contention that a direct relationship exists between Na+-H+ exchange activity and the physical state of the lipids of rat colonic apical plasma membranes.     

Modulation of Na+-H+ exchange by ethinyl estradiol in rat colonic brush-border membrane vesicles

Prior studies by our laboratory have suggested that a relationship may exist between rat colonic brush-border membrane vesicular fluidity and Na+-H+ exchange. To further explore this possible relationship, in the present studies the effects of ethinyl estradiol (17 alpha-ethinyl-1,3,5-estratriene-3,17-beta-diol) administration subcutaneously (5 mg/kg body wt. per day) for 5 days, on rat colonic brush-border membrane fluidity and Na+-H+ exchange were examined. This treatment regimen has previously been shown to decrease the lipid fluidity of rat hepatic and rabbit small intestinal plasma membranes. In agreement with these prior studies, the present results demonstrate that this agent decreases the lipid fluidity of treated-rat colonic brush-border membranes compared to control membranes, as assessed by steady-state fluorescence polarization techniques using three different fluorophores. An increase in the cholesterol content and cholesterol/phospholipid molar ratio of treated-membranes appear to, at least partially, be responsible for the fluidity differences. Furthermore, examination of the kinetic parameters for amiloride-sensitive sodium-stimulated proton efflux in treated and control membrane vesicles, utilizing the pH-sensitive fluorescent dye, Acridine orange, revealed that ethinyl estradiol administration decreased the Vmax for this exchange mechanism, expressed in arbitrary fluorescence units, by approx. 25% but did not influence its Km for sodium. These data, therefore, lend further support to the contention that alterations in fluidity may modulate Na+-H+ exchange in rat colonic brush-border membrane vesicles.     

Regional differences in the lipid composition and fluidity of rat colonic brush-border membranes

The lipid composition and fluidity of brush-border membranes prepared from rat proximal and distal colonocytes were determined. Fluidity, as assessed by steady-state fluorescence polarization techniques using the fluorophores 1,6-diphenyl-1,3,5-hexatriene, DL-2(9-anthroyl)stearic acid and DL-12(9-anthroyl)stearic acid, was decreased in distal compared to proximal plasma membranes. This pattern was similar to that previously described for both antipodal plasma membranes in rat enterocytes of the small intestine. The decrease in fluidity of the distal as compared to the proximal membranes resulted from an increase in cholesterol content, cholesterol/phospholipid molar ratio and degree of saturation of the fatty acid residues in the distal membranes. The specific activities of total alkaline phosphatase and cysteine-sensitive alkaline phosphatase, enzymes previously shown to be functionally dependent on the physical state of the colonic brush-border membrane's lipid, were also significantly lower in distal as compared to proximal clonic plasma membranes. These studies, therefore, demonstrate that differences in the lipid fluidity, lipid composition and certain enzymatic activities exist in brush-border membranes prepared from rat proximal and distal colonocytes. The regional variation in rat colonic luminal membrane lipid fluidity and composition may, at least partially, be responsible for differences in these enzymatic activities as well as in sodium and water absorption along the length of this organ.     

Premalignant alterations in rat colonic N1-acetylspermidine levels induced by 1,2-dimethylhydrazine: effects of a high corn oil dietary regimen

Recently, our laboratory has demonstrated that elevations in the levels of N1-acetylspermidine could be detected in the colonic mucosa of rats after administration of 1,2-dimethylhydrazine for 15 weeks, i.e., before the development of colon tumors. Since prior studies have indicated that diets high in fat, particularly unsaturated fat, promote the development of dimethylhydrazine-induced tumors, it was of interest to examine the effect of a corn oil dietary regimen (20% by weight) on colonic N1-acetylspermidine levels in this model of colonic adenocarcinoma. Four groups of rats were used in these studies: chow, chow + carcinogen, corn oil and corn oil + carcinogen. The carcinogen groups received weekly s.c. injections of 1,2-dimethylhydrazine (20 mg/kg body wt) for 15 weeks, while the control groups received diluent. 1 week after the last injection, animals from each group were killed, and their proximal and distal colons were resected, examined and compared with respect to polyamine levels, including N1-acetylspermidine, as well as the activities of ornithine decarboxylase, spermidine N1-acetyltransferase, and polyamine oxidase. In view of previous studies which suggested that N1-acetylspermidine levels may be elevated in the urine of patients with various malignancies, it was also of interest to examine and compare the urinary levels of this acetylated polyamine in animals from each group. The results of these experiments demonstrated that: (1) the levels of N1-acetylspermidine in the distal colonic segment were found to be increased approx. 25 and 80% in the chow + carcinogen and corn oil + carcinogen groups, respectively, compared to their control counterparts; (2) the activities of spermidine N1-acetyltransferase in the distal colonic segments of chow + carcinogen and corn oil + carcinogen animals were increased 1.5- and 2-fold, respectively, compared to control values; (3) dimethylhydrazine administration did not affect the levels of this acetylated polyamine or spermidine N1-acetyltransferase activities in the proximal colon, but, in general, did increase the levels of putrescine and spermidine as well as ornithine decarboxylase activities in both colonic segments of animals fed chow or corn oil diets; and (4) elevated urinary levels of N1-acetylspermidine did not appear to be a reliable 'premalignant' marker in this experimental model of colonic adenocarcinoma.     

Isolation of brush-border membranes from rat and rabbit colonocytes: Is alkaline phosphatase a marker enzyme?

Summary A method for the isolation of brush-border membranes of large intestinal epithelial cells was developed, which is based on the purification of intact brush-border caps by Percoll^® density-gradient centrifugation followed by separation of the vesiculated brush-border membranes on sucrose gradients. The procedure has two major advantages in comparison to known methods: 1) its first step does not depend on the determination of marker enzymes and 2) the method is applicable to rats as well as rabbits without major modifications. Due to the lack of an accepted marker for the colonic brush-border membrane the validity of the isolation procedure was tested by its application to the small intestine. Rat small intestinal brush-border membranes were enriched 21-fold when compared to the homogenate. The method was used to evaluate alkaline phosphatase as a marker enzyme for the colonic brush-border membrane. The results suggest that alkaline phosphatase is not exclusively localized in the brush-border membrane since this enzyme was also associated with membranes having different physical properties.     

Lectin histochemistry of 1,2-dimethylhydrazine-induced rat colon neoplasia

Lectins linked to fluorescein were used as carbohydrate probes to examine the goblet cell mucin and epithelial cell surface glycoconjugate alterations in an experimental rodent model of colonic neoplasia induced with parenteral 1,2-dimethylhydrazine dihydrochloride. Lectins derived from Triticum vulgare (WGA), Ricinus communis (RCA1), and Limulus polyphemus (LPA) showed reduced labeling of goblet cell mucin in these tumors, while binding with peanut lectin from Arachis hypogaea (PNA), a lectin ordinarily failing to bind to mucin in normal colon, was positive. In addition, RCA1 and LPA showed increased cell surface labeling of neoplastic epithelial cells. Finally, alterations were observed in lectin binding to "transitional" colonic mucosa adjacent to colonic tumors from carcinogen-treated rats. These findings indicate that significant alterations in both membrane and mucin glycoconjugates occur in colonic tumors and mucosa adjacent to tumors in a chemically induced experimental animal model of human colon cancer.     

Inhibition of the Na+,K(+)-ATPase pump during induction of experimental colon cancer.

During the development of large bowel cancer alterations in colonic epithelial ion transport have been observed some of which result in altered intracellular ionic composition. In many tumors intracellular sodium and potassium become elevated and depressed, respectively. This observation suggests that mechanisms governing intracellular homeostasis for sodium and potassium are no longer tightly regulated. Changes in cell membrane permeability, sodium, potassium-ATPase K(+)-ATPase) pump activity, or both may be responsible for these alterations. It is not known when during initiation and development of cancer such changes may occur. To assess whether there are changes in the Na+, K(+)-ATPase pump early during the induction of large bowel cancer and prior to any notable histological changes, we measured the kinetics of the Na+, K(+)-pump in distal colonic mucosa of CF1 mice one week following only four weekly injections of the carcinogen 1,2-dimethyhydrazine (DMH). The kinetics of the pump were found to be best described by a model of highly cooperative binding. The VMAX of the pump in premalignant mucosa was lower for both sodium and potassium substrate activation (55-65% of control) with little change in other kinetic parameters. Depression of VMAX could not be attributed to an increased barium blockable potassium conductance of the basolateral membrane. Na+,K(+)-ATPase activity was also decreased by 50% in the distal colon of DMH treated mice, but was not affected in the less cancer susceptible proximal colon. These data demonstrate that alterations occur in the Na+,K(+)-pump in premalignant mucosa months before gross tumors develop, and these changes may partially explain the altered levels of Na+ and K+ in the cytoplasm of pre-malignant and malignant colonocytes.     

Individual variability of pathological parameters in chemically induced rat colon tumors.

The development of tumorigenic conditions in the carcinogen-exposed rat colon was studied using selected morphological, histochemical, immunohistochemical and biochemical methods of analysis. Rats were treated with two carcinogens: 1,2-dimethylhydrazine and N-methyl-N'-nitro-N-nitrosoguanidine alone or with deoxycholic acid as a tumor promoter. It was found that 3 months after treatment of animals with the carcinogens the following changes were developed in colonic tissue: infiltration of lymphocytes in the mucous membrane, high increase in mitotic index among epithelial cells, negative reactions of colonic cells for neutral mucopolysaccharides and sulfomucins and positive reactions to carboxyl groups, nonsulfated acid mucosubstances and tissue polypeptide antigens. An increase in the activity of ornithine decarboxylase in colonic tissue was developed within the same time period and has been seen only in those tissues which were characterized by the development of precancerous conditions. Individual variations were observed in the manifestation of the studied parameters in rat neoplastic colonic tissues. It is suggested that these differences reflect an individual sensitivity of animals to carcinogens and the magnitude of the dysplastic processes induced in the colon.     

Alterations in surface ultrastructure and anionic sites of rat dimethylhydrazine-induced intestinal tumors

The relative roles of cell surface shedding and electronegative charge as determinants of metastatic capacity were studied in experimentally produced intestinal tumors. The ultrastructural organization and distribution of anionic sites on the luminal plasma membrane surface components were examined in small intestinal and colonic tumors induced in male Sprague-Dawley rats with 1,2-dimethylhydrazine. The overall distribution of negatively charged groups was demonstrated with ruthenium red staining. Compared to normal epithelial cells, neoplastic cells revealed evidence of decreased cell surface shedding as manifested by decreased numbers of membrane-bound bodies, and an increased quantity of glycocalyx. Malignant cell surfaces were directly exposed to the intestinal lumen as a result of losing the enteric surface coat covering. The exposed microvilli appeared damaged with shortening and blunting. The glycocalyx and surface coat both reacted strongly with ruthenium red indicating the presence of anionic sites. As a result of surface coat loss, the malignant cell surface components revealed an overall decrease in net negative charge. These alterations in cell surface component ultrastructure and electronegative charge appear to be consistent with the low capacity for chemically induced rat intestinal tumors to metastasize.     

Studies on the kinetics of glycosidases from chemically-induced rat colonic tumours and normal rat colon.

K-m values of beta-N-acetylglucosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase EC 3.2.1.30), beta-N-acetylgalactosaminidase (EC 3.2.1.53), beta-galactosidase (beta-D-galactoside galactohydrolase EC 3.2.1.23) and alpha-L-fucosidase (alpha-L-fucoside fucohydrolase EC 3.2.1.51) of distal colonic tumours, induced in rats by 1,2-dimethylhydrazine, were found to be significantly different compared with the values for the enzymes of the colonic mucosa of the control and tumour-bearing animals and of the proximal colonic tumours. The inhibition kinetics data also showed a significant difference between the enzymes of the distal colon tumours and of other experimental tissues. The data on the effect of pH on enzyme kinetics (pK values) showed no significant difference in the catalytic groups of the active centres of enzymes from tumours and from the control colonic mucosa. Tumour beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase compared with the enzymes from other experimental tissues were found to be different in their thermal inactivation kinetics. K-m values of 14 days old foetal intestinal beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase were significantly different from the values obtained for the adult mucosal enzymes but were similar to those of the distal colonic tumour enzymes.     

Dexamethasone-induced alterations in lipid composition and fluidity of rat proximal-small-intestinal brush-border membranes.

A series of experiments were conducted to examine the possible effects of subcutaneous administration of the synthetic glucocorticoid dexamethasone (100 micrograms/day per 100 g body wt.) on the lipid fluidity and lipid composition of rat proximal-small-intestinal brush-border membranes. After 4 days of treatment, membranes and their liposomes prepared from treated animals possessed a greater fluidity than did their control (diluent, 0.9% NaCl) counterparts, as assessed by steady-state fluorescence-polarization techniques using several different fluorophores. Examination of the effects of temperature on the anisotropy values of 1,6-diphenylhexa-1,3,5-triene, using Arrhenius plots, moreover, revealed that the mean break-point temperatures of the treated preparations were approx. 3-4 degrees C lower than those of their control-preparation counterparts. Changes in the sphingomyelin/phosphatidylcholine (PC) molar ratio as well as in certain of the fatty acids of the PC fraction of treated membranes, secondary to alterations in membrane PC levels and in lysophosphatidylcholine acyltransferase activities respectively, were also noted after dexamethasone administration. These compositional alterations appeared to be responsible, at least in part, for the differences in fluidity noted between treated and control plasma membranes. These results therefore demonstrate that dexamethasone administration can modulate the lipid fluidity and lipid composition of rat proximal-small-intestinal brush-border membranes.     

Kinetic studies of D-glucose transport in renal brush-border membrane vesicles of streptozotocin-induced diabetic rats

Renal brush-border membrane vesicles prepared from streptozotocin-induced 4-day-diabetic rats possessed a Na+-dependent D-glucose transport system that exhibited apparent Kt and Vmax values about 2-fold greater than normal. Apparently, hyperglycemia and probably other stimuli cause the induction and membrane incorporation of a low-affinity transporter in these membranes; this increased sugar-transport capacity is retained for at least 4 weeks so long as the animals maintained or increased their body weight. Membranes prepared from 28-day-diabetic, severely ill ketoacidotic animals lose this enhanced transport ability and the decrease in Vmax was found to correlate directly with the weight loss. Furthermore, the transporter in brush-border membranes prepared from these cachectic animals had an even lower affinity for glucose than those from the acute hyperglycemic animals. That these changes in the diabetic animals represent major alterations in renal brush-border membrane construction is further supported by our observation that the specific activity of the marker enzymes, alkaline phosphatase and neutral alpha-glucosidase, are profoundly increased and decreased, respectively, in this condition.     

A novel marker glycoprotein for the microvillus membrane of surface colonocytes of rat large intestine and its presence in small-intestinal crypt cells

Murine mAbs were produced against purified microvillus membranes of rat colonocytes in order to establish a marker protein for this membrane. The majority of antibodies binding to the colonic microvillus membrane recognized a single protein with a mean apparent Mr of 120 kD in both proximal and distal colon samples. The antigen is membrane bound as probed by phase-partitioning studies using Triton X-114 and by the sodium carbonate extraction procedure and is extensively glycosylated as assessed by endoglycosidase F digestion. Localization studies in adult rats by light and electron microscopy revealed the microvillus membrane of surface colonocytes as the principal site of the immunoreaction. The antigen was not detectable in kidney or liver by immunoprecipitation but was present in the small intestine, where it was predominantly confined to the apical membrane of crypt cells and much less to the microvillus membrane of differentiated enterocytes. During fetal development, the antigen appears first in the colon at day 15 and 1-2 d later in the small intestine. In both segments, it initially covers the whole luminal surface but an adult-like localization pattern develops soon after birth. The antibodies were also used to develop a radiometric assay for the quantification of the antigen in subcellular fractions of colonocytes in order to assess the validity of a previously developed method for the purification of colonic brush-border membranes (Stieger, B., A. Marxer, and H.P. Hauri. 1986. J. Membr. Biol. 91:19-31.). The results suggest that we have identified a valuable marker glycoprotein for the colonic microvillus membrane, which in adult rats may also serve as a marker for early differentiation of enterocyte progenitor cells in small-intestinal crypt cells.     

Modulation of rat distal colonic brush-border membrane Na−H exchange by dexamethasone: role of lipid fluidity

Earlier studies by our laboratory have suggested a relationship between an amiloride-sensitive Na⁺−H⁺ exchange process and the physical state of the lipids of rat colonic brush-border membrane vesicles. To further assess this possible relationship, a series of experiments were performed to examine the effect of dexamethasone administration (100 μg/100 g body wt. per day) subcutaneously for 4 days on Na⁺−H⁺ exchange, lipid composition and lipid fluidity of rat distal colonic brush-border membrane vesicles. The results of these studies demonstrate that dexamethasone treatment significantly: (1) increased the V_max of the Na⁺−H⁺ exchange without altering the K_m for sodium of this exchange process, utilizing the fluorescent pH-sensitive dye, acridine orange. ²²Na flux experiments also demonstrated an increase in amiloride-sensitive proton-stimulated sodium influx across dexamethasone-treated brush-border membrane vesicles; (2) increased the lipid fluidity of treated-membrane vesicles compared to their control counterparts, as assessed by steady-state fluorescence polarization techniques using three different lipid-soluble fluorophores; and (3) increased the phospholipid content of treated-membrane vesicles thereby, decreasing the cholesterol/phospholipid molar ratio of treated compared to control preparations. This data, therefore, demonstrates that dexamethasone administration can modulate amiloride-sensitive Na⁺−H⁺ exchange in rat colonic distal brush-border membrane vesicles. Moreover, it adds support to the contention that a direct relationship exists between Na⁺−H⁺ exchange activity and the physical state of the lipids of rat colonic apical plasma membranes.