Green tea phenolics inhibit butyrate-induced differentiation of colon cancer cells by interacting with monocarboxylate transporter 1.

Diet has a significant impact on colorectal cancer and both dietary fiber and plant-derived compounds have been independently shown to be inversely related to colon cancer risk. Butyrate (NaB), one of the principal products of dietary fiber fermentation, induces differentiation of colon cancer cell lines by inhibiting histone deacetylases (HDACs). On the other hand, (-)-epicatechin (EC) and (-)-epigallocatechin gallate (EGCG), two abundant phenolic compounds of green tea, have been shown to exhibit antitumoral properties. In this study we used colon cancer cell lines to study the cellular and molecular events that take place during co-treatment with NaB, EC and EGCG. We found that (i) polyphenols EC and EGCG fail to induce differentiation of colon adenocarcinoma cell lines; (ii) polyphenols EC and EGCG reduce NaB-induced differentiation; (iii) the effect of the polyphenols is specific for NaB, since differentiation induced by other agents, such as trichostatin A (TSA), was unaltered upon EC and EGCG treatment, and (iv) is independent of the HDAC inhibitory activity of NaB. Also, (v) polyphenols partially reduce cellular NaB; and (vi) on a molecular level, reduction of cellular NaB uptake by polyphenols is achieved by impairing the capacity of NaB to relocalize its own transporter (monocarboxylate transporter 1, MCT1) in the plasma membrane. Our findings suggest that beneficial effects of NaB on colorectal cancer may be reduced by green tea phenolic supplementation. This valuable information should be of assistance in choosing a rational design for more effective diet-driven therapeutic interventions in the prevention or treatment of colorectal cancer.

Premalignant alterations in the lipid composition and fluidity of colonic brush border membranes of rats administered 1,2 dimethylhydrazine.

Dimethylhydrazine (DMH) is a potent procarcinogen with selectivity for the colon. To determine whether alterations in the lipid composition and fluidity of rat colonic brush border membranes existed before the development of DMH-induced colon cancer, rats were injected s.c. with this agent (20 mg/kg body weight per wk) or diluent for 5, 10, and 15 wk. Animals were killed at these time periods and brush border membranes were prepared from proximal and distal colonocytes of each group. The "static" and "dynamic" components of fluidity of each membrane were then assessed, by steady-state fluorescence polarization techniques using limiting hindered fluorescence anisotropy and order parameter values of the fluorophore 1,6 diphenyl-1,3,5-hexatriene (DPH) and fluorescence anisotropy values of DL-2-(9-anthroyl) stearic acid and DL-12-(9-anthroyl) stearic acid, respectively. Membrane lipids were extracted and analyzed by thin-layer chromatography and gas-liquid chromatography. Phospholipid methylation activity in these membranes was also measured using S-adenosyl-L-methionine as the methyl donor. The results of these studies demonstrate that: the lipid composition and both components of fluidity of proximal DMH-treated and control membranes and their liposomes were similar at all time periods examined; at 5, 10, and 15 wk the "dynamic component of fluidity" of distal DMH-treated membranes and their liposomes was found to be higher, similar, and lower, respectively, than their control counterparts; the "static component of fluidity" of distal DMH-treated membranes and their liposomes, however, was similar to control preparations at all three time periods; and alterations in the lipid composition and phospholipid methylation activities appeared to be responsible for these differences in the "dynamic component of fluidity" at these various time periods.

Increased calcium absorption in prehypertensive spontaneously hypertensive rat. Role of serum 1,25-dihydroxyvitamin D3 levels and intestinal brush border membrane fluidity.

Changes in Ca absorption have been described in the spontaneously hypertensive rat (SHR) compared with Wistar-Kyoto (WKy) rats. In 3.5-wk-old SHR and age-matched WKy controls, we measured direct arterial blood pressure, Ca absorption, and serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels and small intestine brush border membrane (BBM) fluidity and lipid composition. The two objectives were (a) to define the nature of the absorptive changes before detectable hypertension and (b) to evaluate the potential mechanism(s). We found that even at this normotensive stage (106 +/- 4 vs. 107 +/- 2 torr for the female and 109 +/- 3 vs. 104 +/- 3 torr for the male), the SHR (a) absorbed more Ca (1.46 +/- 0.06 vs. 1.14 +/- 0.08 mmol/d and 1.53 +/- 0.06 vs. 1.28 +/- 0.06 mmol/d, respectively) and retained more Ca, (b) had higher serum 1,25(OH)2D3 levels (340 +/- 36 vs. 160 +/- 18 pg/ml and 230 +/- 25 vs. 150 +/- 16 pg/ml, respectively), and (c) possessed BBM with increased fluidity and with reduced fatty acyl saturation index owing to decreased stearic (32.2 +/- 2.6% vs. 38.2 +/- 0.9%) but increased linoleic acids (12.2 +/- 2.0% vs. 7.6 +/- 1.6%). These results demonstrate increased Ca absorption in prehypertensive SHR associated with increased serum 1,25(OH)2D3 levels, increased intestinal BBM fluidity, and reduced saturation index, which singly or in combination could produce the changes in intestinal Ca transport.

Correction of enhanced Na(+)-H+ exchange of rat small intestinal brush-border membranes in streptozotocin-induced diabetes by insulin or 1,25-dihydroxycholecalciferol.

Diabetes was induced in rats by administration of a single i.p. injection of streptozotocin (50 mg/kg body wt). After 7 d, diabetic rats were further treated with insulin or 1,25-dihydroxycholecalciferol [1,25(OH)2D3] for an additional 5-7 d. Control, diabetic, diabetic + insulin, and diabetic + 1,25(OH)2D3 rats were then killed, their proximal small intestines were removed, and villus-tip epithelial cells were isolated and used to prepare brush-border membrane vesicles. Preparations from each of these groups were then analyzed and compared with respect to their amiloride-sensitive, electroneutral Na(+)-H+ exchange activity, using 22Na uptake as well as acridine orange techniques. The results of these experiments demonstrated that (a) H+ gradient-dependent 22Na uptake as well as Na+ gradient-dependent transmembrane H+ fluxes were significantly increased in diabetic vesicles compared to their control counterparts, (b) kinetic studies demonstrated that this enhanced 22Na uptake in diabetes was a result of increased maximal velocity (Vmax) of this exchanger with no change in apparent affinity (Km) for Na+, (c) serum levels of 1,25(OH)2D3 were significantly lower in diabetic animals compared with their control counterparts; and (d) insulin or 1,25(OH)2D3 treatment restored the Vmax alterations to control values, without any significant changes in Km, concomitant with significantly increasing the serum levels of 1,25(OH)2D3 in diabetic animals. These results indicate that Na(+)-H+ activity is significantly increased in proximal small intestinal luminal membranes of streptozotocin-induced diabetic rats. Moreover, alterations in the serum levels of 1,25(OH)2D3 may, at least in part, explain this enhanced antiporter activity and its correction by insulin.

Premalignant alterations in the glycosphingolipid composition of colonic epithelial cells of rats treated with 1,2-dimethylhydrazine.

1,2-Dimethylhydrazine is a procarcinogen with selectivity for the colon. In weekly s.c. doses of 20 mg/kg of body weight, this agent produces colonic tumors in virtually 100% of rodents, with a latency period of approximately 6 months. To determine whether alterations in the glycosphingolipid content and composition of rat colonic epithelial cells existed before the development of dimethylhydrazine-induced colon cancer, rats were given s.c. injections of this agent (20 mg/kg body weight per week) or diluent for 5 weeks. Animals were sacrificed at this time period and colonocytes isolated from each group. Glycosphingolipids were then extracted from these cells and analyzed by thin-layer chromatography, high-performance liquid chromatography and gas-liquid chromatography. The results of these studies demonstrate that: (a) the content and relative percentages of globotriaosylceramide is increased, whereas hematoside and globotetraosylceramide are decreased in dimethylhydrazine-treated colonocytes compared to their control counterparts; and (b) differences in the enzymatic activities responsible for the biosynthesis of these glycosphingolipids appear to explain, at least partially, these compositional differences. The present data, therefore, suggest that alterations in certain glycosphingolipids may be an early event in colonic malignant transformation and, furthermore, that these alterations may prove useful in the detection of early colon cancer.

1,2-Dimethylhydrazine-induced alterations in N1-acetylspermidine levels and spermidine N1-acetyltransferase activity in rat colonic mucosa.

To determine whether alterations in the "reverse" or "conversion" pathway for putrescine formation were involved in the induction of colonic tumors by 1,2-dimethylhydrazine, male albino rats of the Sherman strain were given injections s.c. of this agent (20 mg/kg body weight/week) or diluent for 5, 10, 15, and/or 26 weeks. Animals were sacrificed at each of these time periods and polyamine levels, including N1- and N8-acetylspermidine, as well as the activities of ornithine decarboxylase, spermidine N1-acetyltransferase and polyamine oxidase were measured and compared in rat proximal and distal colonic mucosa of each group. The results of these studies demonstrated that: (a) N1- and N8-acetylspermidine levels were similar in the control and treated proximal colonic segments at all time periods examined; (b) N1- and N8-acetylspermidine levels were also similar in the control and treated distal colonic segments at 5 and 10 weeks; (c) at 15 weeks the level of N1-acetylspermidine, but not N8-acetylspermidine, however, was increased in the treated distal colonic segment secondary to increases in the activity of spermidine N1-acetyltransferase; and (d) at 26 weeks, the level of N1-acetylspermidine remained higher in treated distal "uninvolved" colonic tissue and were markedly elevated in colonic tumors in both segments. Based on these findings, it would appear that the reverse pathway for putrescine formation may be involved in the 1,2-dimethylhydrazine-induced malignant transformation process of the rat colon.

Urinary excretion of N1-acetylspermidine and other acetylated and free polyamines in the 1,2-dimethylhydrazine model of experimental rat colon cancer.

1,2-Dimethylhydrazine (DMH) is a potent procarcinogen with selectivity for the colon. Recently, it has been demonstrated that levels of N1-acetylspermidine were elevated 2-3-fold in colonic tumors induced by this agent compared to control tissues. To determine whether alterations in the urinary levels of this acetylated polyamine or other polyamines were useful biochemical markers for colon cancer in this experimental model, rats were given s.c. injections of DMH (20 mg/kg body weight/week) or diluent for 26 weeks. One week after the last injection, control and DMH-treated animals were placed in separate metabolic cages and their urine was collected for 24 h. The urinary levels (expressed as nmol/mg creatinine) of putrescine, spermidine, spermine, N1-acetylspermidine, and N8-acetylspermidine were then analyzed by high-performance liquid chromatography. Animals from each group were then sacrificed and their colons were examined for tumors. The results of these studies demonstrated that the urinary level of N1-acetylspermidine was an excellent biochemical marker for colonic tumors induced by DMH. At 18.3 nmol/mg creatinine, N1-acetylspermidine was 100% sensitive and specific for colon cancer. Moreover, urinary levels of N1-acetylspermidine were better for this purpose than the N1-acetylspermidine/N8-acetylspermidine molar ratio, a marker previously suggested to be more specific for certain cancers than free polyamines.

Identification and partial characterization of phospholipid methylation in rat small-intestinal brush-border membranes.

An earlier study (Biochim. Biophys. Acta 46 (1961) 205-216) failed to detect the enzymatic synthesis of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) via a transmethylation pathway in rat small-intestinal microsomal membranes. This pathway was therefore assumed to be absent from this organ. Recently, however, in our laboratory it has been demonstrated that this pathway for the synthesis of phosphatidylcholine is present in rat colonic brush-border and basolateral membranes. It was therefore of interest to examine whether phospholipid methylation activity was present in rat small-intestinal brush-border membranes. The results of the present experiments demonstrate for the first time that this pathway for the synthesis of phosphatidylcholine exists in these plasma membranes. Evidence to support the enzymatic nature of this reaction include: loss of activity by heat denaturation and at 0 degree C, significant inhibition by S-adenosyl-L-homocysteine and saturation kinetics. The predominant product of this brush-border membrane phospholipid methyltransferase is phosphatidyl-N-monomethylethanolamine. This enzymatic activity has an apparent Km for S-adenosyl-L-methionine of 40 microM, a Vmax of 8.4 pmol/mg protein per 5 min, and a pH optimum of 8.0.

Effect of hypothyroidism on the lipid composition and fluidity of rat colonic apical plasma membranes.

Prior studies have suggested that the lipid composition and lipid fluidity of cellular membranes of various organs are altered in the hypothyroid rat. To date, the effects of hypothyroidism on these parameters have not been examined in rat colonic apical plasma membranes. In the present experiments, male Sprague-Dawley rats were fed a pelleted diet (control group) or the same diet containing 0.1% propylthiouracil (hypothyroid group) for 3 weeks. The lipid composition and lipid fluidity of apical plasma membranes prepared from colonocytes of these two groups of animals were then examined and compared. Membranes prepared from the hypothyroid animals were found to possess a higher level of linoleic acid (18:2) and a lower level of arachidonic acid (20:4) than membranes from control animals. The molar ratio of cholesterol/phospholipid was also lower in hypothyroid membranes secondary to a decreased cholesterol content compared to their control counterparts. Moreover, the lipid fluidity of colonic apical plasma membranes, as assessed by (1) the ratio of excimer to monomer fluorescence intensities of the lipid-soluble fluorophore pyrenedecanoic acid and (2) the anisotropy values of the fluorophore DL-12-(9-anthroyloxy)stearic acid using steady-state fluorescence polarization techniques, was greater in hypothyroid animals. These data, therefore, indicate that alterations in the lipid composition and fluidity of colonic apical plasma membranes can be detected in hypothyroid rats.

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.

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.

Rat proximal small intestinal Golgi membranes: lipid composition and fluidity.

The present studies were conducted to examine and characterize the lipid composition and physical state of the membrane lipids of rat proximal small intestinal Golgi membranes. Golgi membranes were purified from isolated enterocytes; lipids were extracted from these membranes and analyzed by thin-layer and gas-liquid chromatography. The 'static' and 'dynamic' components of fluidity of Golgi membranes and their liposomes were assessed by steady-state fluorescence polarization techniques utilizing r infinity and S values of 1,6-diphenyl-1,3,5-hexatriene and r values of DL-2-(9-anthroyl)- and DL-12-(9-anthroyl)stearic acid, respectively. Additional studies were also performed on these membranes, using benzyl and methyl alcohol, to examine the relationship between alterations in lipid fluidity and glycosphingolipid glycosyltransferase activities. The results of these studies demonstrated that: (1) the principal phospholipids and neutral lipids of intestinal Golgi membranes, respectively, were phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, and unesterified cholesterol and fatty acids; (2) the major fatty acids of Golgi membranes were palmitic (16:0), stearic (18:0), linoleic (18:2), arachidonic (20:4) and oleic (18:1) acids; (3) fluorescence polarization studies using diphenylhexatriene detected a thermotropic transition at 24-26 degrees C in Golgi membranes and liposomes prepared from lipid extracts of these membranes; (4) benzyl alcohol (25 and 50 mM) but not methyl alcohol (50 mM) significantly increased the fluidity of these membranes; and (5) at these same concentrations, benzyl alcohol was also found to increase significantly the specific activity of UDP-galactosyllactosylceramide galactosyltransferase but not CMP-acetylneuraminic acid: lactosylceramide sialyltransferase. Methyl alcohol was not found to influence either enzyme's activity in these membranes.

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.

Estrogen-induced alterations of the acidic and neutral glycosphingolipids of rat kidney.

In order to determine whether female sex hormones could influence the glycosphingolipid composition of the rat kidney, male albino rats of the Sherman strain were subcutaneously administered the synthetic estrogen, ethinylestradiol (5 mg/kg body wt. per day) or vehicle for 5 days, and the ganglioside, ceramide and neutral glycosphingolipid compositions of the kidneys of these animals were analyzed and compared. The results of these experiments demonstrate that estrogen treatment: (1) increased the ceramide, acidic and neutral glycosphingolipid contents of this tissue; (2) decreased the relative percentages of glucosyl- and globotetraosylceramide and hematoside (GM3), but increased the relative percentage of globotriaosylceramide and 'other' gangliosides; (3) increased the relative percentage of N-acetyl- to N-glycolylneuraminic acid in GM3; and (4) altered the long-chain bases of GM3, glucosyl- and globotetraosylceramide in this organ. These data, therefore, demonstrate that estrogen administration induces quantitative and qualitative alterations in the gangliosides, neutral glycosphingolipids and ceramide of the rat kidney. This data as well as a discussion of the possible physiological consequences of these estrogen-induced alterations in kidney glycosphingolipids serve as the basis for this report.

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.

Regulation of Na+-H+ exchange by transmethylation reactions in rat colonic brush-border membranes.

Incubation of rat colonic brush-border membrane vesicles with 200 microM S-adenosyl-L-[Me-3H]methionine resulted in the labeling of both membrane phospholipids and proteins. This labeling was decreased approximately 50% by the methylation inhibitor S-adenosyl-L-homocysteine (2 mM). Utilizing the pH-sensitive fluorescent dye, acridine orange, as a means of determining Na+-H+ exchange, S-adenosyl-L-methionine (200 microM) significantly increased sodium-stimulated proton efflux in these vesicles at all concentrations of sodium (2.5-50 mM) tested. Examination of the kinetic parameters for sodium-stimulated proton efflux in the presence and absence of 200 microM S-adenosyl-L-methionine revealed that the methyl donor increased the Vmax for this exchange mechanism (expressed in arbitrary fluorescence units) by approx. 36% but did not influence its Km for sodium. S-Adenosyl-L-homocysteine (2 mM) inhibited S-adenosyl-L-methionine-mediated stimulation of this exchange process. The results demonstrate that methylation of membrane phospholipids and/or proteins can modulate Na+-H+ exchange in rat colonic brush-border membrane vesicles.

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.

Synthesis of phosphatidylcholine by two distinct methyltransferases in rat colonic brush-border membranes: evidence for extrinsic and intrinsic membrane activities.

The enzymatic synthesis of phosphatidylcholine from phosphatidylethanolamine via a transmethylation pathway has not been shown to occur in the small intestine and has been assumed to be absent from the entire gut. The existence of this pathway, however, has not been investigated in the large intestine. Utilizing a recently developed method for the isolation of brush-border membranes from rat colonocytes, the present studies were designed to determine whether phospholipid methylation activity was present in the large intestine. The results demonstrate that this pathway for synthesis of phosphatidylcholine exists in rat colonic plasma membranes and involves at least two distinct methyltransferases. The predominant product of the first enzyme (methyltransferase I) is phosphatidyl-N-monomethylethanolamine; phosphatidylcholine and phosphatidyl-N-monomethylethanolamine are the principal products of the second enzyme (methyltransferase II). Methyltransferase I has an apparent Km for S-adenosyl-L-methionine of 100.0 microM and a pH optimum of 8.0, while methyltransferase II has an apparent Km of 0.3 microM and a pH optimum of 6.0. Additional evidence to support the presence of two distinct enzymes includes the differential effects of ATP, Triton X-100, trypsin treatment, and temperature on their activities.

1,2-Dimethylhydrazine-induced alterations in colonic plasma membrane fluidity: restriction to the luminal region.

Recently, work in this laboratory has shown that changes in the 'dynamic' component of fluidity, lipid composition and phospholipid methylation activity of distal colonic brush-border membranes could be detected after administration of 1,2-dimethylhydrazine to rats of the Sherman strain for 5-15 weeks, i.e., before the development of colon cancer. The present experiments were therefore conducted to: determine whether similar 'premalignant' biochemical changes could be detected in basolateral membranes of Sherman rats treated with this agent; and clarify the relationship of these membrane changes to the malignant transformation process by examining the effect of 1,2-dimethylhydrazine on these biochemical parameters in colonic antipodal plasma membranes of rats of the Lobund-Wistar strain. This particular strain of rats has previously been shown to be total resistant to the induction of tumors by 1,2-dimethylhydrazine. The results of the present experiments demonstrate that similar biochemical alterations could not be detected in the colonic plasma membranes prepared from either strain of rat treated with 1,2-dimethylhydrazine. These data support the contention that the prior biochemical membrane alterations noted in brush-border membranes of 1,2-dimethylhydrazine-treated animals are, in fact, related to the malignant transformation process and, furthermore, are confined to the luminal surface of distal colonic epithelial cells.

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.