Brush-border Membrane Fluidity Research Articles

Role of BBM lipid composition and fluidity in impaired renal Pi transport in aged rat.

Renal tubular phosphate (Pi) transport is impaired in the aged rat. The decrement in Pi transport is manifested as a decrease in the Vmax of the luminal brush-border membrane (BBM) sodium-dependent Pi transport. In the present study we determined the potential role of alterations in cortical BBM lipid composition and fluidity in the age-related phosphaturia observed in rats. In the aged rat there are significant increases in the BBM cholesterol (Chol, 504 vs. 422 nmol/mg protein in adult, P less than 0.01) and sphingomyelin (Sph, 41.8 vs. 37.5 mol% in adult, P less than 0.01) and a decrease in the BBM fluidity [increase in fluorescence anisotropy of diphenylhexatriene (DPH), rDPH, 0.221 vs. 0.215 in adult, P less than 0.01]. The BBM lipid compositional and fluidity alterations may also play an important role in the impaired renal adaptation to a low-Pi diet in the aged rat. In the adult rat the renal adaptation to a low-Pi diet is associated with a decrease in BBM Chol (370 to 307 nmol/mg protein, P less than 0.01) and an increase in fluidity (decrease in rDPH 0.207 to 0.201, P less than 0.05). In the aged rat the renal adaptation to a low-Pi diet is incomplete and is associated with impaired ability to lower BBM Chol (441 to 429 nmol/mg protein, P = NS) and to increase fluidity (rDPH 0.211 to 0.211, P = NS). The results of this study therefore suggest that in the aged rat the increase in BBM Chol and Sph content, and the decrease in BBM fluidity play an important role in the impaired renal tubular Pi transport and adaptation to a low-Pi diet.

Estrogen modulates ileal basolateral membrane lipid dynamics and Na+-K+-ATPase activity.

The effects of ethinyl estradiol, a synthetic estrogen with cholestatic properties and a propensity to alter hepatocyte and ileal brush-border membrane fluidity, on lipid structure and Na+-K+-ATPase activity of rabbit small intestinal basolateral membranes were determined. Utilizing the fluorophores 1,6-diphenyl-1,3,5-hexatriene and DL-12-(9-anthroyl)stearic acid, increases in fluorescence anisotropy, the reciprocal of fluidity, were found in basolateral membranes and in membrane lipid liposomes isolated from ileum. Fluidity alterations were accompanied by a marked decrease in bilayer phospholipids (0.37 vs. 0.48 mumol/mg protein; P less than 0.01) and an increase in both the cholesterol-to-phospholipid molar ratio (0.85 vs 0.61; P less than 0.02) and membrane saturated fatty acid content. Estrogen-mediated physicochemical changes were associated with a significant reduction in ileal basolateral membrane Na+-K+-ATPase specific activity (100.0 vs. 185.8 nmol Pi.min-1.mg protein-1; P less than 0.02). Control values both for fluorescence anisotropy and for Na+-K+-ATPase specific activity were restored after in vitro membrane fluidization with benzyl alcohol. The data therefore indicate that ethinyl estradiol effects on basolateral membrane lipid dynamics are confined to the ileum and are associated with inhibition of Na+-K+-ATPase activity. These structural and functional changes appear to be related, in part, to specific modifications in the availability of phospholipid after estrogen treatment.

Membrane fluidity and lipid composition of rat small intestinal brush-border membranes during postnatal maturation

Fluidity and lipid composition of rat small intestinal brush-border membranes (BBM) were studied during maturation in five age groups: newborns, sucklings (1–3 weeks), weaned (4–6 weeks), juveniles (8–10 weeks), and adults (12 weeks). Brush-border membrane fluidity was measured by steady-state fluourescence polarization. Fluorescent probes used were: 1,6-diphenyl-1,3,5-hexatriene, 1-(4-(trimethylammonium)phenyl)-6-phenyl-1,3,5-hexatriene, and a set of n-(9- antrhoyloxy) fatty acids. Fluorescence anisotropy measured with all fluorophores was increased in adult versus newborn rats ( P < 0.004). The weight ratio of saturated to cis- unsaturated fatty acids increased from birth to the suckling age ( P < 0.0004). The cholesterol to phospholipid molar ratio increased from birth to the weaned age ( P < 0.0001). Cholesterol to protein ratio and phospholipid to protein ratio decreased after the weaned age ( P < 0.004). The results not only describe maturational changes of brush-border membranes but also give a better understanding of the correlations between biophysical and biochemical data in biological membranes.

Ischemia induces surface membrane dysfunction. Mechanism of altered Na+-dependent glucose transport.

Reversible ischemia reduced renal cortical brush border membrane (BBM) Na+-dependent D-glucose uptake (336 +/- 31 vs. 138 +/- 30 pmol/mg per 2 s, P less than 0.01) but had no effect on Na+-independent glucose or Na+-dependent L-alanine uptake. The effect on D-glucose uptake was present after only 15 min of ischemia and was due to a reduction in maximum velocity (1913 +/- 251 vs. 999 +/- 130 pmol/mg per 2 s; P less than 0.01). This reduction was not due to more rapid dissipation of the Na+ gradient, altered sidedness of the vesicles, or an alteration in membrane potential. Ischemia did, however, reduce the BBM sphingomyelin-to-phosphatidylcholine (SPH/PC) and cholesterol-to-phospholipid ratios and the number of specific high-affinity Na+-dependent phlorizin binding sites (390 +/- 43 vs. 146 +/- 24 pmol/mg; P less than 0.01) without altering the binding dissociation constant (Kd). 20 mM benzyl alcohol also reduced the number of Na+-dependent phlorizin binding sites (418 +/- 65 vs. 117 +/- 46; P less than 0.01) without altering Kd. The reduction in Na+-dependent D-glucose transport correlated with ischemic-induced changes in the BBM SPH/PC and cholesterol-to-phospholipid ratios and membrane fluidity. Taken together these data indicate the cellular site responsible for ischemic-induced reduction in renal cortical transcellular glucose transport is the BBM. We propose the mechanism involves marked alterations in BBM lipids leading to large increases in BBM fluidity which reduces the binding capacity of Na+-dependent glucose carriers. These data indicate that reversible ischemia has profound effects on the surface membrane function of epithelial cells.

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 (17α-ethinyl-1,3,5-estratriene-3,17-β-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 V max for this exchange mechanism, expressed in arbitrary fluorescence units, by approx. 25% but did not influence its K m 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.

Relation between Ca 2+ uptake and fluidity of brush-border membranes isolated from rabbit small intestine and incubated with fatty acids and methyl oleate

The rate of incorporation of oleic acid into isolated brush-border membranes was found to be considerably faster than methyl oleate incorporation under similar experimental conditions. The effects of fatty acids and methyl oleate incorporation on Ca 2+ uptake and fluidity were monitored. Whereas treatment with 0.01–0.05 mM oleic acid corresponding to incorporations smaller than 90 nmol/mg protein enhanced Ca 2+ transport, exposures to higher concentrations of this fatty acid corresponding to incorporations larger than 150 nmol/mg protein, decreased uptake of this cation. On the other hand, treatment with 0.01–0.2 mM methyl oleate corresponding to incorporations of up to 220 nmol/mg protein had only a stimulatory effect on the Ca 2+ uptake. Oleic acid, linoleic acid and methyl oleate decreased the fluorescence anisotropy of membranes labelled with diphenylhexatriene in a dose-dependent manner. In contrast, palmitic acid had little or no effect on the diphenylhexatriene-reportable order of the membrane within the range of concentrations used. Monitored as a function of temperature, the anisotropy values showed a gradual melting for both the control and lipid-treated membranes. The results support the concept that saturated and cis-unsaturated fatty acids dissolve in different lipid domains and this in itself appears to be an important factor defining whether the biological function of the membrane is affected by the uptake. Incorporation of cis-unsaturated fatty acids in domains harboring the Ca 2+ uptake process increases Ca 2+ uptake in concert with increased diphenylhexatriene-monitored fluidity. However, when concentrations of such fatty acids in these domains become sufficiently great, the presence of a largely increased number of free carboxyl groups at the membrane surface causes inhibition of Ca 2+ uptake.

Modulation of lipid fluidity of small- and large-intestinal antipodal membranes by Ca2+.

A series of experiments were conducted to examine the role of Ca2+ in modulating the fluidity of rat small- and large-intestinal antipodal plasma membranes and their liposomes. This bivalent cation was found to decrease the fluidity of these preparations in a complex manner involving at least two distinct mechanisms. The first appeared to be a direct effect of Ca2+ on fluidity, was readily reversible by addition of EGTA and presumably involved binding of Ca2+ to anionic sites in the lipid bilayers of these membranes. This effect was seen with all preparations examined. In contrast, the second effect of Ca2+ on fluidity was only seen in intact small-intestinal brush-border membranes, appeared to be indirect, was time- and cation-dependent, was only minimally reversible by addition of EGTA, and appeared to involve stimulation of membrane-bound enzymes which altered this membrane's fatty acid composition. Furthermore, regional differences in this latter effect of Ca2+ on brush-border membrane fluidity were also seen in these studies, i.e. proximal greater than distal small intestine.

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.

Glucocorticoid-mediated alteration of fluidity of brush border membrane in rat small intestine.

The normal biophysical properties of the maturing mammalian enterocyte membrane are poorly understood. While the effects of glucocorticoids on maturation of intestinal enzyme function has been intensively investigated, their effects on membrane biophysical properties are not known. We used 1,6-diphenyl-1,3,5-hexatriene as a probe in fluorescence anisotropy studies to determine the fluidity of rat brush border membrane. Maturational changes and the effects of glucocorticoids administered antenatally or postnatally were determined. Fluorescence anisotropy values for 1,6-diphenyl-1,3,5-hexatriene in mature brush border membranes were higher than those values in membranes obtained from younger animals reflecting a less fluid membrane. Glucocorticoids administered to suckling rats increased the anisotropy values of 1,6-diphenyl-1,3,5-hexatriene in the membranes compared to saline-administered littermates. The anisotropy of intestinal brush border membrane was also increased in fetal rats whose mothers received dexamethasone. These alterations may relate to protein-binding properties and permeability characteristics of the enterocyte membrane.

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.

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.