Mechanisms of Na+ transport in human distal colonic apical membrane vesicles.

Apical membrane vesicles purified from mucosal scrapings obtained from distal segments of organ donor colons and a 22Na-uptake technique were used to characterize the mechanism(s) of Na+ transport into these vesicles. An outwardly directed H+ gradient (pH 5.5in/7.

Na+ transport in human proximal colonic apical membrane vesicles.

Background/aims: The mechanisms of Na+ movement across colonocyte plasma membranes in the human colon are not well understood. Current studies were undertaken to investigate Na+ transport pathways in apical membranes of proximal organ donor colons.

Methods: Purified apical membrane vesicles and rapid filtration 22Na-uptake techniques were used.

1,25-Dihydroxycholecalciferol rapidly activates rat colonic particulate guanylate cyclase via a protein kinase C-dependent mechanism.

The present studies were performed to determine whether the major biologically active metabolite of vitamin D3, 1,25-dihydroxycholecalciferol [1,25(OH)2D3], could influence the activities of rat colonic particulate guanylate cyclase and adenylate cyclase. To address these issues, colonocytes were harvested from Sprague-Dawley rats and suspended in Krebs-Ringer bicarbonate buffer. The cells were then treated with 1,25(OH)2D3 or other agents (see below) and crude membranes were prepared and analyzed for particulate guanylate cyclase and adenylate cyclase activities.

Inactivation of rat small intestinal brush-border membrane alkaline phosphatase by oxygen free radicals.

Background: To date, the potential effects of free radicals on the activities of intestinal brush-border membrane enzymes have received little attention. Therefore, we conducted a series of experiments to examine the effects of free radicals on various enzymatic activities of rat small intestinal brush-border membranes.

Methods: An in vitro Fe2+/ascorbate oxygen-radical generating system and rat small intestinal brush-border membranes were used for this purpose.

Supplemental dietary calcium fails to alter the acute effects of 1,2-dimethylhydrazine on O6-methylguanine, O6-alkylguanine-DNA alkyltransferase and cellular proliferation in the rat colon.

Prior studies from our laboratory have demonstrated that K-ras G to A mutations were detectable in a high percentage of carcinomas which developed in the colons of animals treated with the known colonic procarcinogen, 1,2-dimethyl-hydrazine (DMH). Moreover, in this model, the incidence of these mutations was decreased by a supplemental dietary calcium regimen which concomitantly decreased the frequency of rats with multiple tumors as well as tumor size. In an attempt to clarify the possible mechanism(s) involved in this antimutagenic effect of supplemental calcium, two groups of Sprague-Dawley rats were fed semisynthetic diets containing either 0.

Modulation of P-glycoprotein-mediated drug transport by alterations in lipid fluidity of rat liver canalicular membrane vesicles.

P-glycoprotein (P-gp) is believed to function as an ATP-dependent efflux pump for natural product anti-cancer drugs in multidrug-resistant (MDR) tumor cells and in certain normal tissues. P-gp has been localized to the apical plasma membrane of the bile canaliculus where it has been shown to transport [3H]daunomycin. In this study, we investigated whether alterations in membrane lipid fluidity of canalicular membrane vesicles (CMV) could modulate the P-gp-mediated accumulation of [3H]daunomycin and [3H]vinblastine.

Effect of vitamin D status on the rapid actions of 1,25-dihydroxycholecalciferol in rat colonic membranes.

Recent studies from our laboratory have demonstrated that the in vitro addition of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] rapidly (seconds to minutes) stimulated membrane phosphoinositide turnover, translocated protein kinase C from the cytosolic to particulate fraction, increased cytosolic calcium ([Ca2+]i), and decreased cytoplasmic pH (pHi) via inhibition of Na(+)-H+ exchange in rat colonic epithelium of dietary vitamin D-sufficient rats and in Caco-2 cells. In contrast to these prior findings, in the present experiments, 1,25(OH)2D3 failed to elicit any of these colonic biochemical responses in vitamin D-deficient animals. Bethanechol chloride also failed to alter this signal transduction pathway, [Ca2+]i, or pHi.

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).

Characterization and modulation of rat small intestinal brush-border membrane transbilayer fluidity.

In the present experiments, selective quenching by trinitrophenyl groups as well as steady-state fluorescence polarization and differential polarized phase fluorescence techniques, using three different lipid soluble fluorophores, were used to directly examine the fluidity of the exofacial and cytofacial leaflets of rat small intestinal brush-border membranes. These studies revealed that the fluidity of the exofacial hemileaflet was greater than the cytofacial hemileaflet. Differences in the distribution of phosphatidylcholine and phosphatidylethanolamine, as assessed by phospholipase A2 treatment and trinitrophenylation of aminophospholipids, were, at least partially, responsible for the asymmetrical fluidity of the hemileaflets.

Down-regulation of protein kinase C activity in 1,2-dimethylhydrazine-induced rat colonic tumors.

Recent studies by our laboratory have indicated that alterations in protein kinase C activity may be involved in the early stage(s) of malignant transformation in the 1,2-dimethylhydrazine model of colonic adenocarcinoma. In order to further evaluate the possible role of protein kinase C in this multistage process, rats were given subcutaneous weekly injections of this procarcinogen (20 mg/kg body weight) or diluent for 26 weeks. One week after receiving the last injection, animals were killed and control colonic tissue, tumor tissue and tissue at least 1 cm away from these tumors ('uninvolved mucosa') were harvested.

Differential modulation of human small intestinal brush-border membrane hemileaflet fluidity affects leucine aminopeptidase activity and transport of D-glucose and L-glutamate.

The fluidity of the exofacial (outer) and cytofacial (inner) leaflets of human proximal small intestinal brush-border membrane vesicles was studied by selective quenching by trinitrophenyl groups, steady-state fluorescence polarization, and differential polarized phase fluorometry techniques, utilizing the lipid soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene. Differences in the hemileaflet's phospholipid composition were also analyzed by trinitrophenylation of aminophospholipids and phospholipase A2 treatment of these preparations. The results of these studies demonstrated that the inner leaflet of these membranes was less fluid than its outer counterpart.

Correction of abnormal small intestinal cytosolic protein kinase C activity in streptozotocin-induced diabetes by insulin therapy.

Diabetes was induced in rats by administration of a single intraperitoneal injection of streptozotocin (50 mg/kg body wt). After 7 days, one group of diabetic animals was treated with insulin for an additional 5 days. Control, diabetic and diabetic + insulin rats were then killed, their distal small intestines were removed and the epithelial cells were examined and compared with respect to polyphosphoinositide turnover, total protein kinase C activity and cellular distribution, and 1,2-diacylglycerol mass and production.

Effect of polyamine oxidase inhibition on the colonic malignant transformation process induced by 1,2-dimethylhydrazine.

Recent studies of colon adenocarcinomas in humans and experimentally induced colonic tumors in rodents have demonstrated selective elevations in the level of N1-acetylspermidine in these malignant tissues. The exact relationship of these alterations in acetylated polyamine levels to the malignant transformation process, however, remains unclear. In order to clarify this issue, rats were given s.

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.

Intestinal D-glucose transport and membrane fluidity along crypt-villus axis of streptozocin-induced diabetic rats.

Diabetes was induced in male Lewis rats by a single injection of streptozocin (50 mg/kg body wt ip). After 10-14 days, diabetic and age- and sex-matched control animals were killed, and their proximal small intestines were removed. Villus-tip, mid-villus, and lower-villus enterocytes were harvested from each group with a method that combined divalent cation chelation with mild mechanical dissociation.

Apical plasma membrane vesicles formed from organ donor colon demonstrate Na+ and H+ conductances and Na+/H+ exchange.

Apical plasma membrane vesicles were prepared from human organ donor colon mucosal scrapings. These vesicles were enriched 10-fold in cysteine-sensitive alkaline phosphatase activity compared to starting homogenates, and showed minimal contamination of microsomal, mitochondrial or basolateral membranes. Transport studies using [22Na] uptake into proximal colonic vesicles demonstrated Na+ and H+ conductances, Na+/H+ exchange and amiloride inhibition of Na+ uptake.

Contribution of Cl(-)-OH- exchange to electroneutral NaCl absorption in rat distal colon.

Neutral NaCl absorption is the predominant Na+ absorptive process in rat distal colon. Whether this neutral NaCl absorptive process represents Na(+)-Cl- cotransport or dual ion exchanges of Na(+)-H+ and Cl(-)-OH- has been uncertain. Recent studies using rat colonic brush-border membrane vesicles (BBMVs) have described a Na(+)-H+ exchange mechanism and have proposed that net NaCl absorption occurs via a dual ion exchange process.

Protein-lipid interactions in human small intestinal brush-border membranes.

Brush-border membranes prepared from proximal and distal human small intestine were characterized with respect to lipid fluidity, lipid composition, and protein-lipid interactions. Steady-state fluorescence polarization and differential polarized phase fluorometry revealed that the "static" and "dynamic" rotational components of fluidity (assessed by r infinity values of 1,6-diphenyl-1,3,5-hexatriene and r values of 12-anthroylstearate, respectively) were greater in the distal membranes compared with their proximal counterparts. The lipid fluidity of distal brush-border membranes was also greater as measured by excimer/monomer fluorescence ratio intensities of pyrene decanoate.

Effects of dietary fish oil supplementation on membrane fluidity and enzyme activity in rat small intestine.

Rats were fed either a fat-free diet supplemented with 10% menhaden oil or a control diet for four months. Intestinal brush border membranes were isolated; phospholipid fatty acid analysis revealed that the membranes from the fish-oil fed animals had higher levels of palmitoleic (C16:1) and eicosapentaenoic (C20:5) acids and lesser levels of stearic (C18:0) linoleic (C18:2) acids compared with controls. The membranes from the fish-oil fed animals had increased levels of alkaline phosphatase activity compared with controls but disaccharidase levels were equivalent in the two groups.

Na+-H+ antiporter of rat colonic basolateral membrane vesicles.

The present experiments were conducted, using acridine orange and 22Na uptake techniques, to demonstrate the presence of an electroneutral Na+-H+ exchange process in rat colonic basolateral membrane vesicles. Results consistent with the existence of a distinct Na+-H+ antiporter in these vesicles include the following: 1) an outwardly directed Na+ gradient stimulated proton influx (Na+in, 100 mM; pHin 7.5/pHout 7.

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.

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.

Differential stimulation of S-adenosylmethionine decarboxylase by difluoromethylornithine in the rat colon and small intestine.

The effects of chronic inhibition of ornithine decarboxylase (ODC) by the specific inhibitor difluoromethylornithine (DFMO) in the rat colon and small intestine on mucosal contents of polyamines, decarboxylated S-adenosylmethionine (decarboxylated AdoMet) and S-adenosylmethionine decarboxylase (AdoMet decarboxylase) activity were studied. Administration of 1% DFMO in the drinking water for 10 or 15 weeks resulted in inhibition of ODC and decreases in intracellular putrescine and spermidine contents in both proximal and distal segments of small intestine and colon. At both time points DFMO administration resulted in a dramatic stimulation of AdoMet decarboxylase activity and a rise in decarboxylated AdoMet content in the proximal and distal small-intestinal segments compared with controls, which was not seen in either colonic segment of DFMO-treated animals.

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.

Dietary triacylglycerol modulates sodium-dependent D-glucose transport, fluidity and fatty acid composition of rat small intestinal brush-border membrane.

Rats were maintained on nutritionally complete diets enriched in unsaturated (menhaden fish oil) or saturated (butter fat) triacylglycerols. After 4 weeks, the animals were killed, proximal small intestinal brush-border membranes were prepared, and examined and compared with respect to their lipid composition, molecular species of phosphatidylcholine, lipid fluidity and sodium-dependent D-glucose transport. Membranes prepared from the two dietary groups were found to possess similar ratios of cholesterol/phospholipid (mol/mol), sphingomyelin/phosphatidylcholine (mol/mol), and protein/lipid (w/w).