Pradeep K. Dudeja

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). In contrast to these findings, however, striking differences were noted in the total fatty acid compositions of these membranes. Plasma membranes prepared from animals fed the fish oil diet possessed higher percentages of saturated fatty acids as well as (n - 3) unsaturated fatty acids and lower percentages of monounsaturated and (n - 6) unsaturated fatty acids than those prepared from animals fed the butter fat diet. Analysis of the molecular species of phosphatidylcholine by HPLC, moreover, revealed that membranes from rats fed fish oil had higher levels of 16:0-20:5, 16:0-22:6 and 18:0-20:5 and lower levels of 18:0-18:2 and 16:0-18:1 than their butter fat counterparts. As assessed by steady-state fluorescence polarization, differential polarized phase fluorometric and excimer/monomer fluorescence intensity techniques using various fluorophores, the lipid fluidity of membranes from rats fed fish oil was also found to be significantly lower compared to membranes from rats fed butter fat. Finally, comparison of the kinetic parameters of Na+-dependent D-glucose transport revealed that fish oil-membrane vesicles had a higher maximum velocity (Vmax) than butter fat membrane vesicles but a similar Km for glucose.

Alterations in the physical state and composition of brush border membrane lipids of rat enterocytes during differentiation

The physical state of the membrane lipid of brush border membranes, prepared from rat small intestinal villus and crypt cells, was examined by steady-state fluorescence polarization using three lipid-soluble fluorophors. Membranes prepared from crypt cells were found to possess a higher lipid fluidity than those of villus cells with each probe. Analysis of the composition of these membranes revealed that those from crypt cells had lower ratios of cholesterol/phospholipid (mol/mol), protein/lipid (w/w), and saturated fatty acyl chains/unsaturated chains (w/w). Alterations in the levels of stearic (18:0) and oleic (18:1) acids were responsible for differences in the latter ratio. The results, therefore, demonstrate that alterations in the lipid composition and fluidity of brush border membranes of enterocytes occur during the process of differentiation.

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 membranes 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.

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.

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.

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 hemileaflets 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. Phosphatidylserine was located mainly in the inner hemileaflet, whereas phosphatidylethanolamine and phosphatidylcholine were more symmetrically distributed between the hemileaflets of this membrane. Moreover, in vitro addition of 2-[(2-methoxyethoxy)ethyl]-cis-8-(2-octylcyclopropyl)octanoate (final concentration, 7.5 microM) preferentially fluidized the cytofacial leaflet and concomitantly increased Na(+)-gradient-dependent D-glucose uptake, but decreased Na+, K+-dependent L-glutamic acid uptake in these membrane vesicles. In vitro addition of benzyl alcohol (final concentration, 25 mM) preferentially fluidized the exofacial leaflet and decreased leucine aminopeptidase activity in these preparations. These results, therefore, demonstrate that the hemileaflets of human small intestinal brush-border membranes have different phospholipid compositions and fluidities. Alterations of either the exofacial or cytofacial leaflet fluidity, moreover, modulate protein-mediated activities in a distinct manner.

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.

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.

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.

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.