Brush-border Membrane Fluidity Research Articles

Implication of BBM lipid composition and fluidity in mitigated alkaline phosphatase activity in renal cell carcinoma

Previous study has documented reduced alkaline phosphatase (ALP) activity in brush border membrane (BBM) isolated from renal cell carcinoma (RCC). Diminished activity of ALP is associated with alteration in both increased K(m) as well as decreased V(max) of enzyme suggests that there may be a change in the conformation of enzyme as well as decreased number of ALP active molecules. The present study was conducted to find out any role of BBM lipid composition and its fluidity in diminished activity of alkaline phosphatase in renal cell carcinoma. Total phospholipids and glycolipids were significantly augmented in BBM from RCC as compared to control. Fractional analysis of total phospholipids revealed significantly increased phosphatidylethanolamine. Decreased fractions of sphingomyelin and phosphatidylinositol were observed. Cholesterol-to-total phospholipid molar ratios in tumor BBM was a significantly lower in tumor BBM. A significant reduction in polarization and microviscosity was found in BBM from RCC. Therefore, we conclude that alteration in membrane lipid composition and fluidity may play a substantial role in reduced activity of ALP in RCC.

Evaluation of the fluidity and functionality of the renal cortical brush border membrane in experimental diabetes in rats

The present study was aimed at addressing the effect of hyperglycemia on the renal cortical brush border membrane. The fluidity and the functionality of the renal cortical brush border membrane have been evaluated after 6 weeks of streptozotocin-induced diabetes in rats. Lipid peroxidation and protein oxidation were first performed to confirm a state of oxidative stress. The fluidity of the brush border membrane of diabetic rats decreased significantly by 15.76%. There was an increase in the amount of early (19.39%) and advanced (42.23%) glycation end-products suggesting the accumulation of significant amount of non-enzymic glycation products at 6 weeks of diabetes. Although, the activities of both γ-glutamyl transpeptidase and alkaline phosphatase of the brush border membrane decreased, that of the latter decreased to a significant extent with an increase in K m (81%) and no change in the V max. A study of the activities of glutathione-dependent antioxidant enzymes in the renal cortical homogenates showed that the activities of glutathione peroxidase and glyoxalase II were altered significantly. Our study seems to suggest that increased free radical generation accompanied by non-enzymic glycation may be responsible for oxidative stress and an increased rigidity of the diabetic brush border membrane. Alkaline phosphatase may thus serve as a potentially useful marker of free radical induced damage to the renal cortical brush border membrane. The results also suggest that enhanced susceptibility to oxidative stress during early stages may be an important factor in the development of secondary complications of diabetes.

Regional variations in intestinal brush border membrane fluidity and function during diabetes and the role of oxidative stress and non-enzymatic glycation.

The physical state (fluidity) of lipids modulates the activities of several membrane bound enzymes and transport proteins. Alteration of brush border membrane (BBM) fluidity is one of the several changes exhibited by the small intestine during diabetes. In the present study, an investigation of the diabetes induced regional changes in fluidity, oxidative damage, non-enzymatic glycation as well as the activities and the kinetic parameters of the enzymes alkaline phosphatase and gamma-glutamyl transpeptidase was carried out on the intestinal BBM. At the end of 6 weeks of diabetes, significant increases in the extent of both oxidative damage and non-enzymatic glycation were observed along the length of the intestine along with a simultaneous decrease in membrane fluidity. A significant correlation between the decrease in BBM fluidity and increase in non-enzymatic glycation was observed in the duodenum and jejunum. Additionally regional variations in the activities and kinetic parameters of both the enzymes were observed.

Molecular basis of renal handling of calcium in response to thyroid hormone status of rat

We investigated the effect of thyroid hormone status on renal handing of Ca 2+. Further, like kinetics of Ca 2+ transport across brush-border membrane (BBM) and basolateral membrane (BLM) of renal epithelial cells was carried out. FE Ca was decreased in hyperthyroid (Hyper-T) rats and increased in hypothyroid (Hypo-T) rats as compared to euthyroid (Eu-T) rats. Ca 2+ uptake into renal brush-border membrane vesicles (BBMV) was increased in Hyper-T rats and decreased in Hypo-T rats as compared to Eu-T rats. K m was lower in Hyper-T rats and higher in Hypo-T rats as compared to Eu-T rats whereas, V max remained unaltered. The transition temperature for calcium uptake varied inversely with the thyroid hormone status. Renal BBM of Hyper-T rats showed decreased anisotropy and polarisation of DPH as compared to EU-T rats whereas these values were increased in Hypo-T rats. Thus, the altered BBM fluidity appears to modulate Ca 2+ transport across BBM. Na +/Ca 2+ exchange activity of renal cells was increased in Hyper-T and decreased in Hypo-T rats as compared to Eu-T rats. V max for Na +/Ca 2+ exchange was increased in Hyper-T rats and deceased in Hypo-T rats as compared to Eu-T rats, whereas, [Na +] 0.5 was similar in all three groups. The c-AMP levels of renal cortex of Hyper-T rats was increased and that of Hypo-T rats decreased as compared to Eu-T rats. Thus, thyroid hormones increased Ca 2+ reabsorption in the kidney of rat. Thyroid hormone-mediated modulation of BBM fluidity appears to stimulate Ca 2+ uptake into renal BBMV. Thyroid hormones possibly activated the Na +/Ca 2+ exchanger through cAMP-dependent pathway.

Piperine modulates permeability characteristics of intestine by inducing alterations in membrane dynamics: influence on brush border membrane fluidity, ultrastructure and enzyme kinetics.

Piperine (1-Piperoyl piperidine) is a major alkaloid of Piper nigrum Linn. and Piper longum Linn. It is shown to possess bioavailability-enhancing activity with various structurally and therapeutically diverse drugs. The mechanism of enhancing the bioavailability, is, however, not understood. We hypothesize that piperine's bioavailability-enhancing property may be attributed to increased absorption, which may be due to alteration in membrane lipid dynamics and change in the conformation of enzymes in the intestine. Results of membrane fluidity studies using an apolar fluorescent probe, pyrene (which measures the fluid properties of hydrocarbon core), showed an increase in intestinal brush border membrane (BBM) fluidity. Piperine also stimulated Leucine amino peptidase and Glycyl-glycine dipeptidase activity, due to the alteration in enzyme kinetics. This suggests that piperine could modulate the membrane dynamics due to its apolar nature by interacting with surrounding lipids and hydrophobic portions in the protein vicinity, which may decrease the tendency of membrane lipids to act as stearic constrains to enzyme proteins and thus modify enzyme conformation. Ultra structural studies with piperine showed an increase in microvilli length with a prominent increase in free ribosomes and ribosomes on the endoplasmic reticulum in enterocytes, suggesting that synthesis or turnover of cytoskeletal components or membrane proteins may be involved in the observed effect. In conclusion, it is suggested that piperine may be inducing alterations in membrane dynamics and permeation characteristics, along with induction in the synthesis of proteins associated with cytoskeletal function, resulting in an increase in the small intestine absorptive surface, thus assisting efficient permeation through the epithelial barrier.

Effects of two highly monounsaturated oils on lipid composition and enzyme activities in rat jejunum.

The effects of two monounsaturated fatty acid (MUFA) oils, olive oil (OO) and high-oleic sunflower oil (HOSO), with high content in oleic acid but differing in their non-fatty acid fraction, on brush-border membrane (BBM) lipid composition and fluidity and on mucosal enzyme activities of rat jejunum were studied. Animals were given semipurified diet with linoleic acid to prevent essential fatty acid deficiency (control group) or semipurified diet containing 10% of either OO or HOSO for 12 weeks. There was a significant decrease in the content of jejunal BBM phospholipids together with an increase in the level of free cholesterol in both oil-fed rats, when compared to control group. Although the increase in the BBM free cholesterol level was not statistically significant in HOSO-fed rats, a significant decrease in the phospholipid/free cholesterol ratio was found in both OO and HOSO-fed animals compared to control group. Rat jejunal BBM had a high level of free fatty acids which was increased in BBM isolated from OO and HOSO-fed animals. There was no statistical significant difference in the phospholipid distribution between the control and the OO group. However, HOSO-fed animals showed the lowest level of phosphatidylethanolamine together with the highest phosphatidylcholine content and the phosphatidylcholine/sphingomyelin ratio. The fatty acid pattern of jejunal BBM lipids was modified according to the major fatty acids in the oils. There was a decrease in both stearic acid (18:0) and linoleic acid (18:2 n-6), together with an increase in oleic acid (18:1 n-9) in jenunal BBM isolated from both oil experimental groups. All these results were accompanied by a significant increase in the BBM fluidity (as assessed by steady-state fluorescence polarization of diphenylhexatriene) isolated from oil-fed rat, when compared to control group. OO and HOSO-fed animals had the lowest activities of sucrase and maltase, while alkaline phosphatase activity only was decreased in HOSO-fed animals. The specific activity of maltase was not modified in any experimental rats. In summary, both MUFA oils induced similar effects on jejunal BBM lipid composition, fluidity, sucrase, maltase and lactase activities. Furthermore, HOSO intake resulted in a lowest alkaline phosphatase activity which was accompanied by changes in individual phospholipid composition. All these results suggest that effects of MUFA oils on jejunal BBM lipid composition and hydrolase activities are most likely due to the presence of high content of oleic acid rather than other components contained in the non-fatty acid of olive oil.

Uptake of barbituric acid derivatives in small intestinal brush border membrane vesicles from retinyl palmitate-treated rats.

Brush border membrane was prepared from the small intestinal (jejunum) cells along the crypt-villus axis. The fluorescence spectra of 1,8-anilinonaphthalene sulfonic acid and the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene were measured in the brush border membrane vesicle suspension. The hydrophobicity of brush border membrane was found to be in the order villus tip >mid villus >lower villus. The fluidity of brush border membrane was in the order villus tip <mid villus <lower villus. The uptake of barbituric acid derivatives by brush border membrane vesicles was well correlated with their partition coefficients (isopentyl acetate/water). No significant difference was observed between the uptake of hexobarbital by brush border membrane vesicles from the villus tip and lower villus. When retinyl palmitate was administered to rats, the fluidity of brush border membrane was found to be higher in the retinyl palmitate-treated rats than in the control rats. However, no significant difference in the uptake of hexobarbital by brush border membrane vesicles was observed between the retinyl palmitate-administered rats and the control rats. Thus, the retinyl palmitate treatment seems unlikely to affect the passively transported ligands like barbituric acid derivatives in brush border membrane vesicles.

Changes in structural and functional properties of rat intestinal brush border membrane during starvation

Changes in surface area of microvilli, fluidity of brush border membrane and transport of L-amino acids through intestinal epithelial cells were studied in wellfed and starved (2,4 and 6 days) rats. The surface area of microvilli per unit area of intestinal epithelial cells increased during starvation. Studies with fluoroprobes — pyrene, 1-anilinonaphthalene-8-sulphonate and 1,6-diphenyl-1,3,5-hexatriene, showed increased fluidity of brush border membrane on progressive starvation. Transport of five amino acids representing five different transport systems was studied during starvation in everted intestinal sleeves. Transport of L-proline, glycine and L-glutamic acid which represent imino, glycine and acidic systems respectively increased significantly in Na +-dependent pathway whereas transport of L-lysine representing basic system increased significantly in Na +-independent pathway during starvation.

Developmental changes in glucose transport, lipid composition, and fluidity of jejunal BBM.

Na(+)-dependent D-glucose uptake was studied in jejunal brush-border membrane (BBM) vesicles of chickens at 2 days and 1, 2, 5-6, and 12-14 wk of age. Both initial rates and accumulation ratios of the Na(+)-dependent D-glucose transport were significantly higher during the 1st wk than at other ages. To explain the age-related changes observed in the transport of D-glucose, the phlorizin-specific binding, Na+ permeability, lipid composition, and fluidity were studied. Transporter site density was quantified using 50 mumol/l phlorizin and found to be higher during the 1st wk. During the 2nd wk it decreased and then remained constant. Permeability of Na+, studied using 22Na+, showed that fluxes were similar during the first 6 wk, and a significant decrease was observed in the oldest group. Furthermore, membrane fluidity results showed a significant age-dependent decrease that correlated well with both the increased molar ratio of cholesterol to phospholipid and the decreased ratio of lipid to protein found during development. In conclusion, changes in the density of Na(+)-dependent D-glucose transporter as well as in lipid content and fluidity might be involved in the changes observed in D-glucose uptake during the posthatching development.

Salicylic Acid Induces Changes in the Physical Properties of Model and Native Kidney Membranes

Salicylic acid (SA) can inhibit the facilitated transport of inorganic sulfate in the kidney, placenta, and erythrocytes. One mechanism of this inhibition could involve the interaction of SA with membranes, resulting in altered function of transporter protein(s) due to changes in membrane fluidity. Such membrane effects could result in altered membrane transport and consequently in changes in the pharmacokinetics and the therapeutic activity of both xenobiotics and endogenous substrates. We investigated the effect of SA on the fluidity of brush border membrane (BBM) and basolateral membrane (BLM) isolated from rat kidney and also on the physical properties (such as phase transition temperature and fluidity) of model membranes by fluorescence polarization and differential scanning calorimetry (DSC) techniques. SA decreased the lipid order parameter (S) of BBM and BLM membranes in a concentration-dependent manner, indicating that the addition of SA makes the membrane more fluid. The fluidizing effect of SA was more pronounced than that of benzyl alcohol. Studies were carried out with protein-free model membranes composed of dipalmi-toylphosphatidylcholine (DPPC) to investigate the effects of SA on the bilayer membrane lipids. SA decreased the fluorescence polarization of DPH (1,6-diphenyl 1,3,5- hexatriene) incorporated in DPPC vesicles. DSC studies demonstrated that SA broadened the phase transition temperature of DPPC vesicles and suggested that SA is located in the C1-C8 region of the acyl chain. In protein-free model membranes, SA exerted fluidizing effects through its incorporation into the cooperative hydrophobic region of the bilayer. The perturbation of membrane physical properties induced by SA and its hydrophobic localization in the membrane bilayer may be important in the SA-induced alteration of sulfate membrane transport.

Modulation of sodium-coupled uptake and membrane fluidity by cisplatin in renal proximal tubular cells in primary culture and brush-border membrane vesicles

The proximal tubule appears to be the main target for the adverse effects of cis-diamminedichloroplatinum (II) (cDDP). We evaluated the early effects of cDDP at concentrations (3 to 67 microM) lower that those which alter cell viability, on three apical transport systems and on the physical state of the brush border membrane (BBM) in rabbit proximal tubule (RPT) cells in primary culture. The maximal effect, corresponding to a 30% decrease in Na(+)-coupled uptake of phosphate (Pi) and alpha-methylglucopyranoside (MGP) and a twofold increase in Na(+)-coupled alanine uptake, was obtained at 17 microM (5 micrograms/ml) cDDP and occurred through a modification of their affinity. At this concentration, cDDP increased BBM fluidity and decreased the BBM cholesterol content by 28%, without increasing the permeability of tight junctions. To clarify the role of cDDP-induced increase in BBM fluidity on alterations of Na(+)-coupled uptake, these parameters were also investigated in BBM vesicles isolated from rabbit renal cortex directly exposed to cDDP. cDDP induced a concentration-dependent inhibition of Na(+)-coupled uptake of MGP, Pi and alanine in BBM vesicles from the renal cortex, associated with a decrease in protein sulfhydryl content, without modifying BBM fluidity. Our findings strongly suggest that the cDDP-induced increase in BBM fluidity in RPT cells results from an indirect mechanism, possibly an alteration of cholesterol metabolism, and did not play a major role in the cDDP-induced inhibition of Na+/Pi and Na+/glucose cotransport systems that may be mainly mediated through a direct chemical interaction with essential sulfhydryl groups of the transporters.

Biophysical and biochemical alterations of renal cortical membranes in diabetic rat

The objective of this study was to determine whether streptozotocin-induced diabetes mellitus in the rat causes alterations in the lipid composition and fluidity of renal brush border membranes (BBM) and basolateral membranes (BLM). Compared to membranes of non-diabetic rats, BBM and BLM of diabetic rats contained 31% and 26%, respectively, less arachidonic acid and 36% and 46%, respectively, more linoleic acid esterfied in phospholipids. These changes were accompanied by a decrease in the average number of double bonds per mole of fatty acid, a measure of fatty acid unsaturation. In diabetic rats BLM had a higher total phospholipid/protein ratio (567 ± 20 vs. 482 ± 15 nmol/mg protein, P < 0.01), less cholesterol (369 ± 30 vs. 512 ± 34 nmol/mg protein, P < 0.01), more phosphatidylcholine (+72%) and less sphingomyelin (−22%) than did BBM. These differences were identical to those observed between BLM and BBM of non-diabetic rats. In control rats BLM was more fluid than BBM as assessed by the steady state fluorescence anisotrophy of diphenylhexatriene and by glycerol permeability. In diabetic rats the fluidity of BLM was not different from that of BBM as assessed by the steady state fluorescence anisotrophy of diphenylhexatriene whereas BLM was slightly more fluid than BBM as assessed by glycerol permeability. By both measures BLM and BBM from diabetic rats were significantly less fluid than BLM and BBM from control rats. Removal of proteins and cholesterol in sequence was accompanied by an increase in membrane fluidity in both groups. However, in no instance did the removal of proteins or cholesterol abolish the difference between the fluidity of diabetic membranes and that of control membranes. From these data we conclude that the reduction in fluidity of renal BLM and BBM in the diabetic rat is due to the change in the composition of fatty acids esterified in membrane phospholipids.

Effect of starvation on glucose transport and membrane fluidity in rat intestinal epithelial cells

Studies on the surface area of microvilli (MV), fluidity of brush border membranes (BBM) and d-glucose uptake were carried out in rat intestinal epithelial cells (IEC) during progressive starvation and under re-feed conditions. The surface area of MV, fluidity of BBM and d-glucose transport through IEC membranes showed an increase during starvation when compared to well-fed controls. Re-feeding experiments restored the control values of all the three parameters within a short time. The results showed that the increase in d-glucose transport through IEC membranes during starvation is due to increased surface area of MV and increased fluidity of BBM.

Renal adaptation to dietary phosphate deprivation: role of proximal tubule brush-border membrane fluidity

With dietary phosphate (Pi) restriction, fluidity of renal proximal tubule brush-border membranes (BBM) and Na-dependent Pi transport (Na-Pi) are increased, suggesting that changes in BBM fluidity are critical for adaptation to Pi restriction. To test this hypothesis, the temporal relationship between Na-Pi transport and changes in BBM fluidity was assessed after Pi deprivation in rats. Renal cortex was obtained from rats fed either a 0.03% (-P) or a 0.6% (+P) Pi diet for 4 h or 7 days, and BBM were prepared. Na-Pi uptake by BBM was measured by use of rapid filtration, and BBM fluidity was assessed by use of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). After 4 h on the diets, Na-Pi uptake was 439 +/- 142 (SD) and 984 +/- 184 pmol.mg protein-1.5 s-1 in +P and -P, respectively (P less than 0.01, n, = 8). Na-dependent proline uptake was unchanged. DPH anisotropy and total cholesterol were similar between groups: 0.204 +/- 0.025 and 0.401 +/- 0.047 nmol/mg protein, respectively, in +P and 0.205 +/- 0.015 and 0.392 +/- 0.037 in -P (P greater than 0.05, n = 8-10). After 7 days, Na-Pi uptake was 841 +/- 291 in +P and 2,168 +/- 848 pmol.mg protein-1.5 s-1 in -P, P less than 0.01, n = 8. DPH anisotropy and BBM cholesterol were 0.175 +/- 0.019 and 443 +/- 132 nmol/mg protein, respectively, in +P and 0.162 +/- 0.020 (n = 8) and 341 +/- 128 (n = 3) in -P (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal Adaptation Symposium

The absorptive function of the intestinal tract depends on the intact villous and crypt structures which are the functional units of the small intestine. The villi increase the absorptive surface area of the intestine 7to 14-fold. The epithelial cells covering the surface of the villus are mature cells that are covered with a microvillous membrane, which increases the apical surface area 14to 4O-fold. Digestive enzymes such as disaccharides and peptidases, transport proteins such as glucose Na+ carriers, and receptors and channels for electrolyte transport are primarily associated with the microvillous membrane. The intestinal cells at the tip of the villus are renewed every 48 -72 h by migrating cells from the crypts. Like other organs the intestinal tract is subject to adaptational responses when subjected to various hormonal, neuroendocrine, nutritional, and surgical stimuli. The occurrence and importance of intestinal adaptation is well recognized for d l ages. The regulation and specific features s f the adaptation process are now only beginning to be characterized. The papers presented in this symposium are each unique in their own right and present specific aspects of small intestinal adaptation. Current evidence suggests that glucose transport across the brush border membranes occurs by a carriermediated process. This process is electrogenic and is driven by the Na+ gradient across the cell. Dr. Richard Fedorak emphasized the fact that surface messengers of transport are involved in intestinal adaptation. He used the diabetic rat model to explore regulation of the glucose transporter and Na+ -K+ ATPase. Enhanced glucose transport in the diabetic model appears to result from recruitment of glucose transporters. which in turn may be regulated by brush border membrane fluidity. It is clear that differences in chain length and double bond arrangement of fatty acids may alter membrane physical properties and affect certain membrane properties such as receptor-dependent transport and enzyme activity. The small intestinal cell, the enterocyte, has multiple membrane transpor? functions and enzyme activities that may be subject to regulation by enterocyte membrane composition. Dr. Manohar Garg presented evidence that enterocyte microsomd desaturases may regulate the enterocyte membrane composition. A9 and A6 desaturase activity, present in the microsomd fraction of enterocytes, influences the polyunsaturated fatty acid content of the enterocyte membrane. The activity of enterocytes is modified by nutrient supply and psition along the intestinal tract. Dr. Monika Keelan gave evidence for critical period programming sf intestinal transport hnction by diet. When the polyunsaturated to saturated fatty acid content of the diet was changed, jejunal microcellular membrane lipid composition was likewise accordingly modified, and this in turn influenced hexose intestinal uptake. Dr. Keelan also presented evidence that a high cholesterol diet will prevent the age-related decline in glucose transport. After resection, the remaining small intestind tract undergoes hyperplasia and hypertrophy. Dr. Vijay Grey gave evidence of the effect of a novel growth-stimulating activity derived fkom the proximal smdl intestine on the adaptive response of the small intestine following resection. The partially purified growth-stimulating protein was only present in the jejunum for 96 h post-resection and required oral feeding for its induction. Dr. Henry Koopmans gave evidence that gut signals from the lower small intestine have the potential to regulate food intake. Surgical transposition of the ileum to the upper small intestine decreased food intake. An optimally hwctioning gastrointestinal tract is a prerequisite for nutrient absorption and hence optimum health. The small intestinal tract undergoes adaptive responses to wide-ranging stimuli. This symposium presents specific aspects of intestinal adaptation that should be of interest to neonatologists, pediatric gastroenterologists, nutritionists, physiologists, and cellular biologists. We wish to thank the sponsors of this symposium, Mead Johnson Canada and Ross Laboratories.

Early selective effects of gentamicin on renal brush-border membrane Na-Pi cotransport and Na-H exchange

Gentamicin nephrotoxicity is associated with impairments in proximal tubular function. This study determined whether gentamicin administration to the rat, before a reduction in glomerular filtration rate (GFR), causes early and selective alterations in renal cortical brush-border membrane (BBM) enzyme and transport activity, lipid composition, and fluidity. Three days of gentamicin administration caused significant decreases in the Vmax of alkaline phosphatase, the Vmax of sodium gradient-dependent phosphate transport (Na-Pi cotransport), and the Vmax of pH gradient-dependent sodium transport (Na-H exchange). Gentamicin did not affect BBM-bound maltase or leucine aminopeptidase activities and sodium gradient-dependent glucose or proline transport activities. Gentamicin also caused a significant decrease in BBM sphingomyelin, significant increases in BBM phosphatidylcholine and phosphatidylinositol, a significant decrease in the phospholipid fatty acid saturation index, and a significant increase in BBM fluidity, i.e., decrease in the fluorescence anisotropy of diphenylhexatriene. These BBM functional and compositional effects of gentamicin were independent of endogenous parathyroid hormone activity. We conclude that gentamicin causes early and specific alterations in BBM enzyme and transport activity and also lipid composition, which may play an important role in the progression of renal cell injury.

Taurocholate Transport by Brush-Border Membrane Vesicles from the Developing Rabbit Ileum

To examine the ontogenesis of bile acid transport in the rabbit ileum, brush-border membrane vesicles (12- to 20-fold purified) were prepared from 14- to 49-day-old animals. Taurocholate uptake was characterized by the emergence of secondary active, Na(+)-dependent transport at the start of weaning (21 days). Transient intravesicular accumulation (overshoot) of taurocholate occurred at 5-10 s of incubation, and the overshoot maximum increased significantly from 21 days (349.2 +/- 22.4 nmol/mg protein) to 35 days (569.0 +/- 84.3 nmol/mg protein; p less than 0.001), without further increase at maturity (49 days, not equal to 607.6 +/- 136.7 nmol/mg protein). No significant taurocholate active uptake component was noted at 14 days; however, ileal vesicles from sucklings showed carrier-mediated, Na+ D-glucose cotransport. In greater than or equal to 35-day-old rabbits, osmolarity studies at 20 s of incubation showed that only approximately 12% of [14C]taurocholate uptake was secondary to bile acid-to-membrane binding. Conversely, at 20 min, greater than 95% of radiolabel incorporation represented solute bound to the external and/or internal membrane surface. Arrhenius plots establish brush-border membrane taurocholate uptake as an intrinsic, lipid-dependent process, with a slope discontinuity between 24 and 28 degrees C, similar to the membrane lipid thermotropic transition region. Steady-state fluorescence polarization studies (1,6-diphenyl-1,3,5-hexatriene) demonstrate a temporal association between the maturation of taurocholate uptake and age-related decreases in ileal brush-border membrane fluidity. These data indicate that maturation of bile acid secondary active transport in the rabbit ileum may be regulated, at least in part, by changes in brush-border membrane lipid dynamics.

Cholesterol modulates rat renal brush border membrane phosphate transport.

In dietary phosphate (Pi) deprivation and in aging there is an inverse correlation between renal proximal tubular brush border membrane (BBM) cholesterol (Chol) content, BBM fluidity, and BBM sodium gradient-dependent Pi transport activity (Na-Pi cotransport). The purpose of this study was to determine whether in vitro enrichment of renal BBM with Chol has a direct modulating effect on Na-Pi cotransport. 12 and 24 mol % increases in Chol content caused dose-dependent decreases in Na-Pi cotransport activity, 2,000 in control, vs. 1,450 in Chol (+12%), vs. 900 pmol/5 s/mg BBM protein in Chol (+24%), all P less than 0.01, which was paralleled by dose-dependent increases in the fluorescence anisotropy of diphenylhexatriene, rDPH, i.e., decrease in BBM fluidity, 0.203 in control, vs. 0.210 in Chol (+12%), vs. 0.219 in Chol (+24%), all P less than 0.01. We found that increasing ambient temperature, which increases BBM fluidity independent of changes in Chol content, increased Na-Pi cotransport. When Na-Pi cotransport was analyzed as a function of BBM fluidity, 1/rDPH, we found that at an equivalent BBM fluidity BBM Chol enrichment still resulted in a dose-dependent decrease in Na-Pi cotransport. Finally, in BBM isolated from rats fed a low Pi diet in vitro enrichment with Chol completely reversed the adaptive increases in Na-Pi cotransport and fluidity. Our study therefore, indicates that Chol is a direct modulator of renal BBM Na-Pi cotransport activity, and that in vivo alterations in BBM Chol content most likely plays an important role in the regulation of renal tubular Pi transport.

Different mechanisms of adaptive increase in Na+-Pi cotransport across renal brush-border membrane

We explored the biochemical mechanism by which thyroid hormone (T3) and low-phosphate diet (LPD) cause an adaptive increase in Na+-Pi cotransport across renal brush-border membrane (BBM). The rate of Na+-Pi cotransport was determined by 32Pi uptake by BBM vesicles (BBMV), and the number of Na+-Pi symporters was assessed by binding of [14C]phosphonoformic acid (PFA) on BBMV. In BBMV of both T3-treated rats and LPD-fed rats, the Na+ gradient-dependent 32Pi uptake increased (Vmax increased; Km Pi was not changed). The Na+-dependent [14C]PFA binding on BBMV increased (higher Vmax, no change in Km PFA) in response to T3, but it remained unchanged in rats fed LPD. Both the increase of Na+-Pi cotransport and of Na+-dependent [14C]PFA binding in response to T3 were blocked by actinomycin D or cycloheximide. Addition of benzyl alcohol to BBMV in vitro increased Na+-Pi cotransport, but [14C]PFA binding did not change; the [3H]phlorizin binding and cotransports of other solutes decreased or did not change. The exposure of BBMV to cholesterol decreased Na+-Pi cotransport without changing [14C]PFA binding. We suggest that the adaptive increase of Na+-Pi cotransport elicited by T3 is due to an increase in number of Na+-Pi cotransporters in BBM. In contrast, in response to LPD the number of Na+-Pi cotransporters is unchanged, and the increased Na+-Pi cotransport is due to faster translocation of Na+ with Pi due to enhanced fluidity of BBM.

Decrease in the fluidity of brush-border membrane vesicles induced by gentamicin a spin-labeling study

In our previous paper (Horio et al., Biochim Biophys Acta 858: 153–160, 1986), we reported that the addition of gentamicin in vitro to rabbit renal brush-border membrane vesicles decreases the apparent V max of Na +-dependent d-glucose transport without affecting the apparent K m . In the present study, we investigated the effects of gentamicin on the physical state of spin-labeled rabbit renal brush-border membranes, using electron spin resonance spectrometry. Brush-border membrane vesicles were prepared from outer cortex (mainly contains early proximal tubule) and outer medulla (containing primarily late proximal tubule), and the gentamicin toxicities in both preparations were compared. Significant decreases were observed in the membrane fluidity of 5 mM gentamici n-treated brush-border membranes. The fluidity of outer cortical brush-border membranes was affected at both 25° and 35°, whereas that of outer medullary membranes was affected only at 35°. Two different stearic acid spin labels revealed that gentamicin affected the fluidity only in the superficial region of the membranes. We also demonstrated that the gentamicin-induced decreases in Na +-dependent d-glucose transport and in the membrane fluidity were recovered by washing gentamicin-treated brush-border membranes. We suggest that gentamicin binds to the superficial region of brush-border membranes and inhibits Na + dependent d-glucose transport across brush-border membranes through the decrease in the membrane fluidity.