Postnatal development of intestinal bile salt transport. Relationship to membrane physico-chemical changes

The postnatal development of intestinal bile salt transport in the rat was examined using the villus technique. Jejunal uptake of taurocholate was linear with respect to incubation concentration at all study ages. Ileal uptake was linear with taurocholate concentration during the first 2 postnatal weeks; a curvilinear relationship indicating the presence of saturable transport appeared during the third week. With the appearance of ileal active transport at age 3 weeks, the Km (app) was constant at 0.49 mM, 0.59 mM, and 0.50 mM in 3-week, 4-week, and adult animals, respectively. The V(app) was 14.65 nmol X mg-1 (dry wt) X min-1 at 3 weeks and declined with age to 11.40 and 10.51 nmol X mg-1 (dry wt) X min-1 in 4-week and adult animals, respectively. The role of physico-chemical changes in the microvillus membrane in the development of ileal active transport was examined. With increasing postnatal age, microvillus membrane cholesterol content rose while the phospholipid content remained unchanged in both ileum and jejunum. Corresponding rises in the cholesterol/phospholipid ratio were observed in both sites. Simultaneously, the microvillus membrane fatty acid composition was changing from predominantly saturated to unsaturated species in both ileum and jejunum. The microvillus membrane fluorescence anisotropy (r) increased with postnatal age in jejunum when measured at 25 degrees C and 37 degrees C and ileum when measured at 25 degrees C; however, no change was noted in ileum when measured at 37 degrees C. Ileal active bile salt transport develops during the third postnatal week, and is associated with concurrent changes in membrane lipid composition and fluidity when measured at 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of ethinyl estradiol on intestinal membrane structure and function in the rabbit

Structural and functional properties of the small intestinal microvillus membrane were evaluated in the rabbit after administration of ethinyl estradiol, a synthetic estrogen with a demonstrated propensity to alter hepatic membrane lipid fluidity, and promote cholestasis. In the jejunum, no estrogen-induced changes in microvillus membrane total lipid, cholesterol or phospholipid content were observed. However, the ileal microvillus membrane in estradiol-treated animals demonstrates significant reductions vs. controls (per mg protein) in total lipid (0.55 milligrams vs. 0.89 milligrams) [corrected] and phospholipid (206.7 micrograms vs. 304.91 micrograms) (p less than 0.001) content, as well as modifications in specific phospholipid species. The increase in the ileal microvillus membrane cholesterol: phospholipid molar ratio (0.65 vs. 0.51, p less than 0.05) was associated with a significant decrease in membrane lipid fluidity reflected by an increase in fluorescence anisotropy measurements utilizing diphenyl hexatriene as the fluorophore (r at 25 degrees C = 0.306 vs. 0.282, p less than 0.05). Thermotropic lipid phase transitions, assessed by Arrhenius plots of both fluorescence data and ileal microvillus membrane p-nitrophenylphosphatase activity demonstrate that phase changes occur between and 24 and 28 degrees C in both treated and untreated groups. Within the temperature range studied (40-10 degrees C) no differences from control were observed in microvillus membrane alkaline phosphatase activity following estrogen treatment. These data therefore indicate that ethinyl estradiol-induced effects on microvillus membrane lipid composition and physical properties occur predominantly in the ileum and appear to be related, in part, to specific alterations in the availability of phospholipid following estrogen treatment.     

Variations in dietary triacylglycerol saturation alter the lipid composition and fluidity of rat intestinal plasma membranes

Rats were maintained on nutritionally complete diets enriched in unsaturated (corn oil) or saturated (butter fat) triacylglycerols. After 6 weeks, significant differences in the lipid composition and fluidity of a number of intestinal membranes were observed. The corn oil diet (enriched mainly in linoleic acid) increased the overall unsaturation of the acyl chains and enhanced the lipid fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, of enterocyte microvillus and basolateral membranes and of colonocyte basolateral membranes. Concomitantly, the cholesterol content and the cholesterol/phospholipid molar ratio were increased in the microvillus but not in the basolateral membranes. The increased cholesterol in ileal microvillus membranes can result from enhanced cellular biosynthesis, since ileal slices from rats fed the unsaturated diet incorporated [14C]octanoate more rapidly into digitonin-precipitable sterol. Increased fluidity of the enterocyte microvillus and basolateral membranes, respectively, enhanced the enzyme specific activities of p-nitrophenylphosphatase and (Na+ + K+)-dependent adenosine triphosphatase. The results indicate that the lipid composition, fluidity and enzyme activities of intestinal plasma membranes can be altered by dietary means. Moreover, rat enterocytes possess regulatory mechanisms which modulate the cholesterol content of the microvillus membranes so as to mitigate changes in lipid fluidity.     

L-serine and GABA uptake by synaptosomes during postnatal development of rat

Postnatal development changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Specific uptake of radiolabeled L-serine was examined and compared with that of radiolabeled GABA using synaptosomes-enriched fractions freshly prepared from cerebral cortex at different postnatal days from the birth to young adulthood. The preparations were incubated with 10 nM of [3H]L-serine and 10 nM of [3H]-GABA in either the presence or absence of NaCl, KCl or choline chloride, at 2 and 30 degrees C, for different periods up to 30 min. The uptake of [3H]l-serine was temperature dependent in synaptosomal fractions prepared from cerebral cortex of rats in postnatal days 5, 7, 13 and 21, but stronger dependence was observed in adult brain, irrespective of the presence of Na+, K+ or choline ions. At all postnatal ages studied, [3H]-GABA uptake showed a high activity in the presence of Na+ ions and at 30 degrees C. The values of Km were 90-489 microM in L-serine uptake. However, in the uptake of GABA the values of Km were 80-150 microM. The highest values of Vmax were obtained at 5 and 21 postnatal days for both transport systems. These results indicate that the uptake of l-serine and GABA are regulated differentially during postnatal development.     

Effect of external abdominal irradiation on intestinal morphology and brush border membrane enzyme and lipid composition

Previous studies have shown that external abdominal irradiation is associated with alterations in intestinal morphology and function. The activity of the jejunal brush border membrane (BBM) enzyme markers sucrase (S) and alkaline phosphate (AP) were not altered by 600 rad irradiation in the rat. In contrast, ileal BBM, AP, and AP/S were increased 3, 7/8, and 28 days postirradiation. The total lipid composition of the jejunal BBM was lower than in control animals only at 3 days postirradiation; this was due to a decrease in the total free fatty acid content. In addition to a lower total free fatty acid content, the ileal BBM contained an increased amount of total phospholipid (PL) which resulted in an increased phospholipid/cholesterol ratio at 3 days following irradiation. Variations in the BBM phospholipid composition occurred in both jejunum and ileum. In the jejunal BBM, the phospholipid composition changes did not alter the choline or amine phospholipid content; therefore, the choline/amine phospholipid ratio was unaffected by irradiation at 600 rad. In the ileal BBM, the phosphatidyl ethanolamine was increased at 3, 7/8, 14, and 28 days following irradiation. The choline/amine phospholipid ratio was not altered in the ileal BBM due to concomitant increases in lecithin content. Jejunal villus height, villus surface area, and the number of cells per villus were decreased at 3 days postirradiation, but increased by day 7/8 and 14 postirradiation to levels much higher than observed in control jejunal villi. The mucosal surface area was decreased at 3 and 7/8 days following irradiation but returned to control values by Day 14. Jejunal microvillus morphology was unaffected by irradiation. Few significant changes were observed in ileal villus morphology following irradiation at 600 rad. Ileal villus height, villus surface area, and mucosal surface area did not change, but the number of cells per villus initially decreased at 3 days and then increased beyond control values at 7/8 and 14 days postirradiation. Ileal microvillus height was significantly decreased only at 7 days postirradiation, while the number of microvilli per micron was increased only at 3 days postirradiation. This study suggests that changes in intestinal morphology and brush border composition may contribute to the altered passive permeation toward lipids which has been reported following abdominal radiation.     

Regional variation in ontogeny of class II antigens in enterocytes of mouse small intestine.

The ontogeny of major histocompatibility class II antigens in small intestine enterocytes of postnatal C3H/He mice was investigated. Cryosections of duodenal, jejunal, and ileal segments from 7-, 14-, 16-, 20-, 21-, 23-, 25-, 27-, 28-day-old and 7-week-old mice were stained for the class II antigens with MRC OX6 monoclonal antibodies by peroxidase-antiperoxidase labelling. In adults, the duodenum exhibited least expression of class II antigens that increased progressively towards the ileum. The expression in the villous epithelium was first seen in the duodenum and jejunum 21 days after birth but the ileal enterocytes did not exhibit any class II antigens. The earliest appearance (21 days postnatal) of class II antigens in the enterocytes coincides with the age of weaning which suggests that immunologic stimulation by ingested antigens after weaning may influence expression of these antigens. At day 28 after birth, the duodenum and jejunum expressed levels comparable to those in the adults. The first expression of the antigens seen in the ileum was at day 28 postpartum. Crypt epithelium of the three regions of the small intestine showed expression similar to that of corresponding regional villous enterocytes. We conclude that there is an age-dependent regional variation in the expression of class II antigens in enterocytes, and the expression increases with age. The variation in expression of the class II antigens in enterocytes of postnatal mice is attributed to the developmental status of the tissue. The nature of postnatal expression of the antigens is important since an early appearance of these antigens may have implications in autoimmunity.     

Interaction of retinol and intestinal microvillus membranes studied by fluorescence polarization

The interaction of retinol and microvillus membranes prepared from the duodenum, jejunum and ileum of the rat can be studied readily by fluorescence and fluorescence polarization. A characteristic pattern of increased retinol anisotropy in membranes from more distal segments is demonstrated. Studies with trypsin indicate that the membrane proteins influence retinol fluorescence intensity and anisotropy.     

Immunocytochemical localization of the intrinsic factor-cobalamin receptor in dog-ileum: distribution of intracellular receptor during cell maturation

Absorption of cobalamin is facilitated by the binding of the intrinsic factor-cobalamin complex (IF-cbl) to specific receptors in the ileum. The physical and biochemical characteristics of this ligand-receptor binding reaction have been extensively studied, but little is known about the cellular mechanisms or receptor synthesis, intracellular transport, and expression on the microvillus surface membrane. We attempted to delineate these mechanisms by using ultrastructural immunocytochemistry to localize the IF-cbl receptor in the crypt, mid-villus, and villus tip regions of mucosal biopsies obtained from the ileum of anesthetized dogs. Prior to initiating the ileal localization studies, the antisera to purified canine IF-cbl receptor that was employed in our studies was shown to have specificity for site (e.g., ileal enterocytes vs. other cells within the gastrointestinal tract) and immunohistochemical specificity. Receptor synthesis in endoplasmic reticulum begins in crypt enterocytes, but continues in cells throughout the villus. In the mid-villus region synthesized receptor translocates vectorially to the microvillus surface associated with membranous vesicles and then inserts into the microvillus pit. Receptor remains fixed to the microvillus pit and does not distribute uniformly over the brush border membrane. All villus tip enterocytes contained IF-cbl receptor in microvillus pits, vesicles, and endoplasmic reticulum, but in addition extensive perinuclear membrane staining was evident as well as re-internalized receptor associated with multivesicular bodies. Basolateral membranes contained no receptor at any level of the villus. These observations suggest that the IF-cbl receptor (a) translocates to the apical cell surface at the mid-villus region by transport in vesicles, (b) directly inserts into and then remains fixed in microvillus pits, (c) is elaborated on the luminal surface most extensively in villus tip cells, and (d) although reinternalized, does not move IF and/or cbl to the basolateral cell surface.     

Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Redistribution of l-[1,5,6-3H]-fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes in vivo

The biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells was studied by following the incorporation of l-[1,5,6-(3)H]fucose, given intraperitoneally with and without chase, into Golgi, lateral basal and microvillus membranes. Each membrane fraction showed distinct kinetics of incorporation of labelled fucose and was differently affected by the chase, which produced a much greater decrease in incorporation of label into Golgi and microvillus than into lateral basal membranes. The kinetic data suggest a redistribution of newly synthesized glycoproteins from the site of fucosylation, the Golgi complex, directly into both lateral basal and microvillus membranes. The observed biphasic pattern of label incorporation into the microvillus membrane fraction may be evidence for a second indirect route of incorporation. The selective effect of the chase suggests the presence of two different pools of radioactive fucose in the Golgi complex that differ in (1) their accessibility to dilution with non-radioactive fucose, and (2) their utilization for the biosynthesis of membrane glycoproteins subsequently destined for either the microvillus or the lateral basal parts of the plasmalemma. The radioactively labelled glycoproteins of the different membrane fractions were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis and identified by fluorography. The patterns of labelled glycoproteins in Golgi and lateral basal membranes were identical at all times. At least 14 bands could be identified shortly after radioactive-fucose injection. Most seemed to disappear at later times, although one of them, which was never observed in microvillus membranes, increased in relative intensity. All but two of the labelled glycoproteins present in the microvillus membrane corresponded to those observed in Golgi and lateral basal membranes shortly after fucose injection. The patterns of labelled glycoproteins in all membrane fractions were little affected by the chase. These data support a flow concept for the insertion of most surface-membrane glycoproteins of the intestinal villus cells.     

Simultaneous monoamine histofluorescence and neuropeptide immunocytochemistry: III. Ontogeny of catecholamine varicosities and neurophysin neurons in the rat supraoptic and paraventricular nuclei

The ontogeny of the rat supraoptic (SON) and paraventricular (PVN) nuclei was studied using a combined fluorescence-immunocytochemical technique for the simultaneous localization of catecholamines (CA) and neurophysin (NP). NP neurons and CA varicosities were first detected in the SON and PVN at 17 days postcoitus. The development of NP neurons which included increases in immunoreactivity in both nuclei proceeded through fetal and neonatal stages, approaching maturity by 21–28 days postnatal; the maturation of the PVN lagged behind that of the SON. CA varicosities appeared to make contact with NP neurons beginning at 21–22 days postcoitus. An apparent increase in varicosity-perikaryal contacts with age was observed in both nuclei; by 14–21 days postnatal adult-like patterns were established. The prenatal dominance of NP stain relative to CA fluorescence may suggest a possible neurotrophic role for magnocellular neurons and/or their products upon ingrowing noradrenergic axons.     

Correction of abnormal lipid fluidity and composition of rat ileal microvillus membranes in chronic streptozotocin-induced diabetes by insulin therapy

Diabetes was induced in rats by administration of streptozotocin. After 90-120 days, one group of chronic diabetic animals was treated with insulin for chronic diabetic animals was treated with insulin for 10 days. The lipid fluidity and composition of microvillus membranes prepared from ileal enterocytes of control, diabetic, and insulin-treated diabetic animals were determined. Lipid fluidity, as assessed by steady-state fluorescence polarization techniques using the probes 1,6-diphenyl-1,3,5-hexatriene, DL-2-(9-anthroyl)stearic acid and DL-12-(9-anthroyl)stearic acid, was decreased in membranes of diabetic animals compared to membranes of control and insulin-treated diabetic membranes. The differences in fluidity resulted from an increased cholesterol content and cholesterol/phospholipid molar ratio in membranes of diabetic animals. The activities of sucrase and alkaline phosphatase were also found to be higher in membranes of diabetic animals. Insulin treatment, however, failed to significantly influence the enzymatic activities of these membranes. These studies, therefore, demonstrate that alterations in the lipid fluidity, lipid composition, and certain enzymatic activities exist in microvillus membranes of enterocytes prepared from chronic streptozotocin-induced diabetic rats. Administration of insulin for 10 days to these animals restored membrane fluidity and lipid composition but not enzymatic activities to control membrane levels.     

Parental spleen cells accelerate the development of intestinal brush border structure and function in neonatal mice

The influence of parental spleen cells on the postnatal development of brush border microvillus membrane structure and the ability to transport lysine and alanine has been studied in the mouse jejunum during the second week of postnatal life. Control tissue taken from 7-11 day old mice has an unchanging crypt-villus structure and a low enterocyte migration rate of about 1 micron hr-1. Microvillus elongation in crypt enterocytes takes 6 days to complete under these conditions. Lysine and alanine transport begin 2 days after structural differentiation has ceased. Parental spleen cells injected into 1-2-day-old F1 mice cause crypt cell hyperplasia, villus shortening and a 3-6-fold increase in enterocyte migration rate after a period of 8 days. These effects are associated with large reductions in the time needed to complete microvillus membrane development and first express absorptive function. Lysine and alanine transport begin approximately 6 hr after structural differentiation has ceased under these conditions. Adaptive changes in the development of enterocyte structure and function, induced by injection of parental spleen cells, bear some resemblance to other changes found to occur normally at weaning and in adult animals subjected to controlled changes in diet and environmental temperature. The possibility that common principles govern enterocyte adaptation and that some of these still apply in an intestine undergoing an immune reaction is discussed.     

Ion requirements for taurocholate transport by ileal brush border membrane vesicles.

The ion requirements for intestinal taurocholate transport were studied using vesicles prepared from the brush borders of guinea pig small intestines. For each experimental electrolyte, parallel uptake experiments were performed with vesicles from jejunal and ileal brush border membranes to differentiate between uptake by passive fluxes and non-specific binding and uptake by the ileal bile salt active transport system. Uptake of taurocholate prior to the addition of electrolyte was the same for vesicles prepared from jejunal and ileal tissue. During the presence of a sodium gradient (extravesicular concentration greater than intravesicular), only ileal vesicles displayed the enhanced uptake which is characteristic of the overshoot phenomenon. When NaCl was replaced by KCl or LiCl, the overshoot was not observed. Replacement of NaCl with NaCNS, Na₂SO₄, or NaSO₃C₂H₄OH, however, resulted in no significant difference in the initial uptake values observed in either the jejunal or ileal vesicles. This pattern of taurocholate transport independence of relative anion permeability differs from the pattern observed by others for the Na⁺ dependent transport of D-glucose by intestinal brush border membrane vesicles. This difference may be attributed in part to the fact that, unlike the situation with glucose, the binding of a taurocholate anion and a sodium cation by the hypothetical carrier would result in an electroneutral addition.     

Lipid fluidity and composition of intestinal microvillus membranes isolated from rats of different ages

The lipid composition and fluidity of microvillus (luminal) membranes isolated from the small intestines of Fisher 344 rats aged 6, 17, and 117 weeks were compared. Lipid fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, was significantly greater in rats aged 6 weeks as compared to 17 or 117 weeks. A lipid thermotropic transition was observed at 17.5 +/- 1.3 degrees C in the membranes of the youngest group, approx. 5-6 degrees C lower than that of the older animals. The differences in lipid composition which account for the higher fluidity of the youngest preparations include a decreased cholesterol/phospholipid molar ratio in both the proximal and distal halves of the small intestine and, in the proximal half alone, increases in the lipid/protein ratio and double bond index. The foregoing reduction in cholesterol/phospholipid ratio derives mainly from a higher content of total phospholipid, and the increment in double bond index results from an increase in arachidonic acid residues. The results demonstrate an age-dependent decrease in fluidity of intestinal microvillus membranes in the early post-weaning period in the rat. This pattern was unlike that of the microvillus membrane p-nitrophenylphosphatase, whose specific activity declined progressively in the older age groups.     

Structural and biochemical differentiation of the guinea-pig colon during foetal development

Summary We have studied some aspects of the morphological and biochemical differentiation of the foetal guinea-pig colonic epithelium. At day 40 the epithelium was organised in ridges and appeared pseudo-stratified. Folding of the epithelium, followed by villus formation, occurred between days 45 and 55, and by day 50 mucus-secreting goblet cells appeared at the bases of the colonic villi. By day 55 most epithelial cells, including goblet cells, possessed numerous microvilli which, by day 65, had become organised into well developed brush-borders. Between day 55 and term (day 65–68) mucosal depth increased markedly and the colon attained its final glandular morphology.Biochemical studies showed the specific activities of the microvillar hydrolases to be much lower in the washed colon than in either foetal meconium or small intestine at all times during development. Furthermore, a membrane fraction highly enriched in microvillus hydrolase activities was prepared from foetal colonic meconium using techniques originally devised to isolate the foetal small intestinal microvillus membrane. This meconial subfraction was almost identical in polypeptide composition to the highly-purified foetal small intestinal microvillus membrane. Identification of the colonic microvillus membrane was hampered by the absence of reliable membrane markers. Nevertheless, a fraction 14-fold enriched in aminopeptidase activity was prepared from day 40 foetal colon and its polypeptide composition compared by SDS-PAGE to that of the small intestinal microvillus membrane at the same age.     

l-serine and GABA uptake by synaptosomes during postnatal development of rat

Postnatal development changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Specific uptake of radiolabeled l-serine was examined and compared with that of radiolabeled GABA using synaptosomes-enriched fractions freshly prepared from cerebral cortex at different postnatal days from the birth to young adulthood. The preparations were incubated with 10 nM of [³H]l-serine and 10 nM of [³H]-GABA in either the presence or absence of NaCl, KCl or choline chloride, at 2 and 30 °C, for different periods up to 30 min. The uptake of [³H]l-serine was temperature dependent in synaptosomal fractions prepared from cerebral cortex of rats in postnatal days 5, 7, 13 and 21, but stronger dependence was observed in adult brain, irrespective of the presence of Na⁺, K⁺ or choline ions. At all postnatal ages studied, [³H]-GABA uptake showed a high activity in the presence of Na⁺ ions and at 30 °C. The values of K_m were 90–489 μM in l-serine uptake. However, in the uptake of GABA the values of K_m were 80–150 μM. The highest values of V_max were obtained at 5 and 21 postnatal days for both transport systems. These results indicate that the uptake of l-serine and GABA are regulated differentially during postnatal development.     

Phospholipid effects on SGLT1-mediated glucose transport in rabbit ileum brush border membrane vesicles

In small intestine, sodium-glucose cotransporter SGLT1 provides the main mechanism for sugar uptake. We investigated the effect of membrane phospholipids (PL) on this transport in rabbit ileal brush border membrane vesicles (BBMV). For this, PL of different charge, length, and saturation were incorporated into BBMV. Transport was measured related to (i) membrane surface charge (membrane-bound MC540 fluorescence), (ii) membrane thickness (PL incorporation of different acyl chain length), and (iii) membrane fluidity (r_12AS, fluorescence anisotropy of 12-AS).Compared to phosphatidylcholine (PC) carrying a neutral head group, inhibition of SGLT1 increased considerably with the acidic phosphatidic acid (PA) and phosphatidylinositol (PI) that increase membrane negative surface charge. The order of PL potency was PI>PA > PE = PS > PC. Inhibition by acidic PA-oleate was 5-times more effective than with neutral PE (phosphatidylethanolamine)-oleate. Lineweaver-Burk plot indicated uncompetitive inhibition of SGLT1 by PA.When membrane thickness was increased by neutral PC of varying acyl chain length, transport was increasingly inhibited by 16:1 PC to 22:1 PC. Even more pronounced inhibition was observed with mono-unsaturated instead of saturated acyl chains which increased membrane fluidity (indicated by decreased r_12AS).In conclusion, sodium-dependent glucose transport of rabbit ileal BBMV is modulated by (i) altered membrane surface charge, (ii) length of acyl chains via membrane thickness, and (iii) saturation of PL acyl chains altering membrane fluidity. Transport was attenuated by charged PL with longer and unsaturated acyl residues. Alterations of PL may provide a principle for attenuating dietary glucose uptake.     

Protein kinase C activity in renal microvillus membranes

Protein kinase C activity was found in rabbit renal microvillus membrane vesicles. C-kinase activity was assayed by examining H1 histone phosphorylation using microvillus membrane vesicles dispersed with Triton X. Calcium-activated protein kinase activity was only demonstrable in the presence of phosphatidylserine (PS). With PS (15 micrograms/ml) the Ka for activation by calcium was 1.04 microM. This was reduced to 0.38 microM by addition of diolein (3.75 micrograms/ml). These activations were dose-dependent and their combined synergistic activation could be reproduced by the combination of PS (15 micrograms/ml) and the phorbol ester, TPA (1.17 ng/ml). During microvillus membrane purification, protein kinase C activity enriched 5-fold relative to its activity in the homogenates. The activity was not due to trapped cytosol or adventitious association with microvillus membranes during homogenization. During further purification on sucrose gradients, the C-kinase activity coenriched with brush border and not with basolateral enzyme markers. We conclude that protein kinase C is a normal component of the renal microvillus membrane.     

Modulation of intestinal brush border membrane chemical composition during postnatal development in rats: effect of gestational diabetes

There was a significant increase in fucose (52%), total hexoses (16%) and hexosamine (56%) except sialic acid, which was reduced (77%) in the microvillus membrane of infants born to rat mothers made diabetic by injecting alloxan on day 3 of gestation. Expressed on the protein basis there were a significant increase in membrane, triglyceride, total cholesterol, and phospholipids content of brush border in pups from diabetic group between 5-45 days of postnatal age. Intestinal morphology in diabetic group showed, regression of tubular glands, distorted cellular organization of mucosal cells, reduction in the mucosal cell height and number of secretory goblet cells. These findings suggest that the gestational diabetes affects the sugar and lipid composition of the intestinal brush border membrane in rats during early stages of the postnatal development, which may be associated with compromised tissue functions later in life.     

Chloride transport across rat ileal basolateral membrane vesicles.

The present study was designed to investigate Cl- transport across rat ileal basolateral membranes. Basolateral membrane vesicles were prepared by a well-validated technique. The purity of the basolateral membrane vesicles was verified by marker enzyme studies and by studies of d-glucose and calcium uptake. Cl- uptake was studied by a rapid filtration technique. Neither an outwardly directed pH gradient, nor a HCO3- gradient, or their combination could elicit any stimulation of Cl- transport when compared with no gradient. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid at 5 mM concentration did not inhibit Cl- uptake under gradient condition. Similarly, the presence of the combination of outwardly directed Na+ and HCO3- gradients did not stimulate Cl- uptake compared with the combination of K+ and HCO3- gradients or no HCO3- gradient. This is in contrast to our results in the brush border membranes, where an outwardly directed pH gradient caused an increase in Cl- uptake. Cl- uptake was stimulated in the presence of combined Na+ and K+ gradient. Bumetanide at 0.1 mM concentration inhibited the initial rate of Cl- uptake in the presence of combined Na+ and K+ gradients. Kinetic studies of bumetanide-sensitive Cl- uptake showed a Vmax of 5.6 +/- 0.7 nmol/mg protein/5 sec and a Km of 30 +/- 8.7 mM. Cl- uptake was stimulated by an inside positive membrane potential induced by the ionophore valinomycin in the setting of inwardly directed K+ gradient compared with voltage clamp condition. These studies demonstrate two processes for Cl- transport across the rat ileal basolateral membrane: one is driven by an electrogenic diffusive process and the second is a bumetanide-sensitive Na+/K+/2 Cl- process. Cl- uptake is not enhanced by pH gradient, HCO3- gradient, their combination, or outwardly directed HCO3- and Na+ gradients.