pH-dependent fluoride transport in intestinal brush border membrane vesicles

Ciliary neurotrophic factor (CNTF) exerts a multiplicity of effects on a broad spectrum of target cells, including retinal neurons. To investigate how this functional complexity relates to the regulation of CNTF receptor alpha (CNTFR alpha) expression, we have studied the developmental expression of the receptor protein in chick retina by using immunocytochemistry. During the course of development, the receptor is expressed in all retinal layers, but three levels of specificity can be observed. First, the expression is regulated temporally with immunoreactivity observed in ganglion cells (embryonic day 8 [E8] to adult), photoreceptor precursors (E8-E12), amacrine cells (E10 to adult), bipolar cells (E12-E18), differentiated rods (E18 to adult), and horizontal cells (adult). Second, expression is restricted to distinct subpopulations of principal retinal neurons: preferentially, large ganglion cells; subpopulations of amacrine cells, including a particular type of cholinergic neuron; a distinctly located type of bipolar cell; and rod photoreceptors. Third, expression exhibits subcellular restriction: it is confined largely to dendrites in mature amacrine cells and is restricted entirely to outer segments in mature rods. These data correlate with CNTF effects on the survival of ganglion cells and mature photoreceptors, the in vitro differentiation of photoreceptor precursors and cholinergic amacrine cells, and the number of bipolar cells in culture described here or in previous studies. Thus, our results demonstrate an exceptional degree of complexity with respect to the regulation of neuronal CNTFR alpha expression in a defined model system. This suggests that the same signaling pathway is used to mediate a variety of regulatory influences, depending on the developmental stage and cell type.     

Amplitude-dependent modulation of brush border enzymes and proliferation by cyclic strain in human intestinal Caco-2 monolayers

Little is known about the effects of repetitive deformation during peristaltic distension and contraction or repetitive villus shortening on the proliferation and differentiation of the intestinal epithelium. We sought to characterize the effects of repetitive deformation of a physiologically relevant magnitude and frequency on the proliferation and differentiation of human intestinal epithelial Caco-2 cells, a common cell culture model for intestinal epithelial biology. Human intestinal epithelial Caco-2 cells were cultured on collagen-coated membranes deformed by -20 kPa vacuum at 10 cycles/minute, producing an average 10% strain on the adherent cells. Proliferation was assessed by cell counting and 3H-thymidine incorporation. Alkaline phosphatase and dipeptidyl dipeptidase specific activity were measured in cell lysates. Since cells at the membrane periphery experience higher strain than cells in the center, the topography of brush border enzyme histochemical and immunohistochemical staining was analyzed for strain-dependence. Cyclic strain stimulated proliferation compared to static cells. Proliferation was highest in the membrane periphery where strain was maximal. Strain also modulated differentiation independently of its mitogenic effects, selectively stimulating dipeptidyl dipeptidase while inhibiting alkaline phosphatase. Strain-associated enzyme changes were also maximal in areas of greatest strain. The PKC inhibitors staurosporine and calphostin C ablated strain mitogenic effects while intracellular PKC activity was increased by strain. The strain-associated brush border enzyme changes were attenuated but not blocked by PKC inhibition. Thus, strain of a physiologically relevant frequency and magnitude promotes proliferation and modulates the differentiation of a well-differentiated human intestinal epithelial cell line in an amplitude-dependent fashion. PKC may be involved in coupling strain to increased proliferation.     

Inhibitory effects of angiotensin-converting enzyme inhibitor on cefroxadine uptake by rabbit small intestinal brush border membrane vesicles

Effects of angiotensin-converting enzyme (ACE) inhibitors, captopril, enalapril maleate and quinapril, on the uptake of aminocephalosporin antibiotic, cefroxadine, by rabbit small intestinal brush border membrane vesicles were examined. These ACE inhibitors significantly inhibited the uptake of cefroxadine, which is transported by H+/dipeptide transporter in the membrane, in the order of captopril < enalapril < quinapril in the presence of an inward H+ gradient. Inhibitory effect of quinapril was more marked than that of aminocephalosporin cephradine, while in the absence of an inward H+ gradient inhibition by these ACE inhibitors was much less. Dixon plot analysis showed that the inhibition by enalapril and quinapril in the presence of an inward H+ gradient occurred in a competitive manner and estimated inhibition constants of these two drugs to be 5.3 mM and 0.46 mM, respectively. These results suggested the strong affinity of these ACE inhibitors, especially quinapril, on the H+/dipeptide transporter.     

Refeeding varying fatty acid and cholesterol diets alters phospholipids in rat intestinal brush border membrane

The epidermis is an attractive site for therapeutic gene delivery because it is accessible and capable of delivering polypeptides to the systemic circulation. A number of difficulties, however, have emerged in attempts at cutaneous gene delivery, and central among these is an inability to sustain therapeutic gene production. We have examined two major potential contributing factors, viral vector stamina and involvement of long-lived epidermal progenitor cells. Human keratinocytes were either untreated or transduced with a retroviral vector for beta-galactosidase (beta-Gal) at > 99% efficiency and then grafted onto immunodeficient mice to regenerate human epidermis. Human epidermis was monitored in vivo after grafting for clinical and histologic appearance as well as for gene expression. Although integrated vector sequences persisted unchanged in engineered epidermis at 10 weeks post-grafting, retroviral long terminal repeat (LTR)-driven beta-Gal expression ceased in vivo after approximately 4 weeks. Endogenous cellular promoters, however, maintained consistently normal gene expression levels without evidence of time-dependent decline, as determined by immunostaining with species-specific antibodies for human involucrin, filaggrin, keratinocyte transglutaminase, keratin 10, type VII collagen, and Laminin 5 proteins out to week 14 post-grafting. Transduced human keratinocytes generated multilayer epidermis sustained through multiple epidermal turnover cycles; this epidermis demonstrated retention of a spatially appropriate pattern of basal and suprabasal epidermal marker gene expression. These results confirm previous findings suggesting that viral promoter-driven gene expression is not durable and demonstrate that keratinocytes passaged in vitro can regenerate and sustain normal epidermis for prolonged periods.     

Protein turnover in intestinal mucosal villus and crypt brush border membranes

Following a concomitant oral dose of salicylamide and acetaminophen (5 mg/kg of each) the urinary excretion of glucuronide and sulfate conjugates of the drugs were followed in children (ages seven to ten years) and adults. No significant difference were observed between the two age groups in the half-lives for appearance of salicylamide conjugates in urine. Age-related changes in the metabolic pathways, however, were observed. The mean percentage of dose excreted as salicylamide sulfate was significantly higher in children (78%) than in adults (36%). In contrast, salicylamide glucuronide was the major excretory product in adults. Similar age-related differences were observed for acetaminophen conjugation. Pharmacokinetic analysis indicated that the deficiency in glucuronide conjugation of these drugs in children is accompanied by a higher rate of sulfate formation.     

Changes in membrane-bound hydrolases by metronidazole in rat renal brush border

The antiprotozoal drug metronidazole, when administered orally at a dose level of 100 mg/kg body wt. daily for 7 days to rats, brought about significant elevation of renal brush-border-membrane-bound hydrolytic enzymes, such as alkaline phosphatase, maltase, sucrase, and leucine aminopeptidase (LAP). Kinetic analysis of the enzymes (substrate saturation) indicated that the drug produced an increase in the maximum of apparent initial enzyme velocity (Vmax), while the substrate affinity constant (Km) remained unaltered. These changes were not recovered to the normal level even after the drug regimen was stopped and the animals were allowed to recover for a period of 7 days. Lipid analysis of brush border membrane (BBM) revealed a significant elevation in the cholesterol, phospholipid, and ganglioside levels, while no marked change was recorded in triglyceride, free fatty acid and plasmalogen. Study of the temperature-dependent parameters of the enzymes showed that metronidazole induced a shift in the transition temperature (To) in LAP with nearly total reversibility in the recovery group. No such change was seen in the other enzymes. However, there also was a lowering in the energy of activation (Ea) below To, which returned to normal after the treatment was withdrawn.     

Evidence that L-sucrose is resistant to hydrolysis catalyzed by jejunal brush border enzymes

The sucrase-isomaltase complex of the intestinal brush border membrane (BBM) catalyzes the hydrolysis of sucrose. The stereospecificity of this enzyme, however, is not known. To investigate this, BBM of hamster jejunum was incubated with D-sucrose or L-sucrose, and the reaction mixture was analyzed using a gas-liquid chromatograph. It was found that D-sucrose was hydrolyzed to its monomers, but L-sucrose remained unhydrolyzed. It is concluded that the sucrase-isomaltase of intestinal BBM of hamster jejunum does not hydrolyze L-sucrose and therefore this enzyme is stereospecific.     

Studies of brush border enzymes, basement membrane components, and electrophysiology of tissue-engineered neointestine

BACKGROUND/PURPOSE: Previous studies have shown that intestinal crypt cell transplantation using biodegradable scaffolds can generate stratified epithelium reminiscent of embryonic gut. The authors propose to tissue engineer small intestine on biodegradable scaffolds by transplanting intestinal epithelial organoid units, which maintain the epithelial mesenchymal cell-cell interaction necessary for epithelial survival, proliferation, and differentiation. METHODS: Intestinal epithelial organoid units were isolated from neonatal Lewis rats by enzyme digestion and differential sedimentation. Organoid units were seeded on to tubular scaffolds made of nonwoven polyglycolic acid (PGA) sprayed with 5% polylactic acid (PLA). Polymers either were coated (28 constructs) or noncoated (33 constructs) with collagen type I. A total of 61 organoid unit polymer constructs were implanted into 61 animals. Animals were killed and constructs harvested at 2, 6, 7, 8, 9, 10, 12, and 14 weeks. RESULTS: Histological analysis showed formation of neomucosa characterized by columnar epithelium with goblet, and paneth cells were evident in 47 of the 61 constructs. The outer walls were composed of fibrovascular tissue, degradable polymer, extracellular matrix, and smooth muscle-like cells. Immunofluorescent microscopy showed apical staining of brush border enzymes, sucrase and lactase, and basolateral staining for laminin, indicating the establishment of cell polarity. Electrophysiology of Ussing-chambered neomucosa and adult ileal mucosa exhibited similar transepithelial resistance. CONCLUSION: These results suggest that intestinal crypt cells heterotopically transplanted as epithelial organoid units on PGA-PLA tubular scaffolds can survive, reorganize, and regenerate complex composite tissue resembling small intestine demonstrating organ morphogenesis, cytodifferentiation, and phenotypic maturation.     

The lipid composition of the membrane of the brush border in the rat small intestine in the late period after gamma irradiation

The lipid composition of membrane of the small intestine brush border was studied 1, 2, 3 and 6 months after the whole-body fractionated gamma irradiation of the one-month-old rats of 80 g weight with a dose of 6 Gy (2 Gy x 3 at a week intervals). Three months after exposure the amount of cholesterol, total phospholipids, phosphatidylcholine, phosphatidyl-ethanolamine in brush border membrane was the same as in control. The role of phospholipids and cholesterol catabolism suppression in membrane regulatory function disturbances after irradiation is discussed.     

Effect of vitamin D metabolites and analogs on renal and intestinal calbindin-D in the rat

The effects of endotoxin (20 mg kg-1 i.p.) on the mesenteric vascular responses to acetylcholine, bradykinin, sodium nitroprusside, and to transient occlusion of the superior mesenteric artery were examined in rats anesthetized with pentobarbitone. Mesenteric vasodilator responses to close arterial injections of acetylcholine and bradykinin were reduced at 1.5 h after endotoxin and almost abolished by 4 h; responses to sodium nitroprusside were unaffected. Occlusion of the superior mesenteric artery for 30, 60, or 120 s produced, on release of the occlusion, a time-dependent vasodilator response in the mesenteric circulation (post-occlusion hyperemia). This hyperemia was markedly reduced by nitro-L-arginine methyl ester (L-NAME); L-NAME did not modify acetylcholine-induced vasodilation. Endotoxin-pretreatment did not modify mesenteric post-occlusion hyperemia 1.5 h after administration but markedly reduced the response by 2.5 h. The administration of L-NAME to endotoxin-treated rats did not further attenuate the hyperemic responses. Mesenteric vasoconstrictor responses to phenylephrine were not modified by endotoxin, although systemic pressor responses to this agent were impaired. We concluded that endotoxin impairs endothelium and nitric oxide-dependent vasodilator responses in the mesenteric circulation.     

Lipids in total parenteral nutrition solutions differentially modify lipids in piglet intestinal brush border and microsomal membranes

BACKGROUND: Fats in the diet modify the lipid composition and function of the intestinal brush border membrane (BBM) as well as the enterocyte microsomal membrane (EMM). METHODS: This study was undertaken in pigs to establish the effect of 3 weeks of total parenteral nutrition (TPN) on the fatty acids in the major phospholipids, (phosphatidylcholine [PC] and phosphatidylethenolamine [PE] in the jejunal and ileal BBM and EMM. RESULTS: In a comparison of 21-day-old milk-fed piglets and newborn animals, there were differences in the major fatty acids (palmitic, 16:0; stearic, 18:0; oleic, 18:1 omega 9, and linoleic acid, 18:2 omega 6) in PC and PE in BBM and EMM. Age-matched (3-week-old) animals fed a lipid-free glucose-containing TPN solution had different membrane fatty acids than did milk-fed piglets, or animals given a soybean oil-containing TPN solution for 21 days. Substituting fish oil or fish oil plus soybean oil altered BBM and EMM fatty acids, compared with the soybean oil-based TPN solutions. These changes varied between the class of phospholipids (PC vs PE), between intestinal site (jejunum vs ileum), and between the type of membrane (BBM vs EMM). CONCLUSIONS: The jejunum and ileum have distinctive control mechanisms for varying their membrane lipids in response to TPN. There is some postmicrosomal modification of lipids between the EMM and BBM. It remains to be established whether the lipid content of the membranes of other organs, and therefore their function, is modified by the lipid composition of parenterally infused lipids.     

Lipid and fatty acid composition of brush border membrane of rat intestine during starvation

Alterations in the lipid and fatty acid composition of brush border membrane (BBM) of small intestine were studied in well-fed, starved, and refed rats. The ratios of cholesterol/phospholipid (mol/mol), sphingomyelin/phosphatidylcholine (mol/mol), protein/lipid (w/w), and free fatty acids (w/w) decreased whereas the total phospholipid (w/w) ratio and the double-bond index increased in BBM of the intestine of the starved rat compared to that of the well-fed rat. Analyses of fatty acids showed higher percentage of stearic and arachidonic acids whereas oleic and linoleic acids decreased under starvation. The acyl chain of starved rat BBM was less ordered compared with that of well-fed rat BBM. On refeeding, these changes were restored to well-fed levels. The change in membrane state under starvation is associated with alterations in the lipid and fatty acid composition of BBM and may be responsible for functional changes that occur under nutritional stress.     

Effect of aging on the intestinal transport of hydrophilic drugs in the rat small intestine

The effect of aging on the intestinal transport of hydrophilic drugs (and probe compounds) was investigated in the rat small intestine. Passive transport was suggested to be unchanged with aging from 8 (young) to 54 (old) and further to 101 (very old) weeks old, as shown for D-xylose and urea in single-pass intestinal perfusion (under urethane anesthesia), where steady-state transport across the intestinal membrane into the blood stream was evaluated. The passive transports of cephradine, 5-fluorouracil (5-FU) and L-glucose were also unchanged, though they were compared only between the young and the old. Consistently, the passive uptake in the intestinal everted sacs, where the entry process into the membrane was evaluated for 5-FU, D-xylose, urea and polyethylene glycol (PEG) 900, was unchanged with aging from the young to the very old. The carrier-mediated transport of cephradine was also unchanged with aging from the young to the old in perfusion under anesthesia, though that of D-glucose was declined by about 50% with aging from the young to the old and thereafter remained constant in the very old. In perfusion in unanesthetized rats, age independency in passive transport (examined for cephradine, L-glucose and D-xylose) and an age-dependent decline in D-glucose transport were also observed, suggesting that the findings under anesthesia are not qualitatively distorted. These results suggest that, although carrier-mediated transport may moderately decline with aging, the barrier function of the intestinal membrane to passive permeation of hydrophilic drugs (with molecular weight below 1000) may be unaffected by aging, supporting the suggestion from our previous in vivo studies that age-dependent increases in the orally absorbed fraction may be predicted for incompletely absorbed drugs because of delayed intestinal transit rather than increased intestinal transport (membrane permeability).     

Establishment of regional differences in brush border enzymatic activities during the development of fetal mouse small intestine

In order to study the establishment of regional differences in brush border enzymic activities during the development of fetal mouse small intestine we have followed (1) the differentiation of microvilli by morphometry, and (2) the developmental pattern of three brush border enzymes (lactase, glucoamylase and alkaline phosphatase). From day 16 to day 19 of gestation, the height of duodenal microvilli increases 2.4 times on the absorptive cells located near the tip of the villi. During the same period in the upper half of the duodenal villi, the number of microvilli per square micrometer rises by a factor of 2.4 and the microvillous surface area increases by a factor of 5.2. The differentiation of ileal microvilli follows a similar pattern but they are always shorter and less numerous than those of the duodenum. Lactase activity appears at 18 days of gestation; the other two brush border enzymes are first detected at 16 days of gestation. Afterwards all three enzyme activities increase rapidly and a decreasing gradient of activity is established from the proximal to the distal segment of the small intestine. Hence, the structural development of the microvilli and the appearance of brush border enzyme activities occur simultaneously and a proximo-distal gradient is already established at 16 days of gestation.     

Six putative IQ motifs of the recombinant chicken intestinal brush border myosin I are involved in calmodulin binding

A case of Ivemark's renal-hepatic dysplasia syndrome is presented. The findings are suggestive of a ductal obstructive process anatomically located at the junction of ducts with the parenchyma. The differential diagnosis includes Meckel syndrome, short rib polydactyly syndromes, and glutaric aciduria type 2. The molecular basis for this syndrome remains unknown.     

Zipper protein, a B-G protein with the ability to regulate actin/myosin 1 interactions in the intestinal brush border

We recently identified a 28-kDa protein in the intestinal brush border that resembled tropomyosin in terms of size, homology, and alpha helical content. This protein contained 27 heptad repeats, nearly all of which began with leucine, leading to its name zipper protein. Subsequent analysis, however, indicated that both a 49-kDa and a 28-kDa immunoreactive protein existed in intestinal brush-border extracts. Using 5'-rapid amplification of cDNA ends analysis, we extended the N-terminal sequence of zipper protein to the apparent translation start site. This additional sequence contained a putative transmembrane domain and two potential tryptic cleavage sites C-terminal to the transmembrane domain which would release a 28-kDa cytoplasmic protein if utilized. The additional sequence was highly homologous to members of the B-G protein family, a family with no known function. Immunoelectron microscopy showed that zipper protein was confined to the membrane of the microvillus where it was in close association with brush-border myosin 1 (BBM1). Recombinant zipper protein (28-kDa cytoplasmic portion) blocked the binding of actin to BBM1 and inhibited actin-stimulated BBM1 ATPase activity. In contrast, zipper protein had no effect on endogenous or K/EDTA-stimulated BBM1 ATPase activity. Furthermore, zipper protein displaced tropomyosin from binding to actin, suggesting that these homologous proteins bind to the same sites on the actin molecule. We conclude that zipper protein is a transmembrane protein of the B-G family localized to the intestinal epithelial cell microvillus. The extended cytoplasmic tail either in the intact molecule or after tryptic cleavage may participate in regulating the binding and, thus, activation of BBM1 by actin in a manner similar to tropomyosin.     

Sucrase-isomaltase and other brush border glycosidases in colorectal tumors

CD28 ligation delivers a costimulatory signal important in T cell activation. This study demonstrates that the disruption of the CD28/B7 pathway early in the nonobese diabetic mouse strain, using CD28-/- and CTLA41g transgenic mice, promoted the development and progression of spontaneous autoimmune diabetes. Functional analyses of T cells isolated from CD28-deficient mice demonstrated that the GAD-specific T cells produced enhanced Th1-type cytokines (IL-2 and IFN gamma) and diminished Th2-type cytokine, IL-4. Moreover, there was a significant decrease in serum levels of anti-GAD antibodies of the IgG1 isotype consistent with a profound suppression of Th2-type responses in these animals. Thus, the early differentiation of naive diabetogenic T cells into the Th2 subset is dependent upon CD28 signaling and extends our understanding of the importance of Th1/Th2 balance in the regulation of this spontaneous autoimmune disease.     

Characteristics of pantothenic acid transport in membrane vesicles of the brush border of small intestine epithelial cells in rats

Ferric nitrilotriacetate (Fe-NTA) induces renal proximal tubular damage that ultimately leads to a high incidence of renal cell carcinoma (RCC) in rats. The RCCs are characterized by 1) high incidence of pulmonary metastasis and peritoneal invasion, 2) high incidence of tumor-associated mortality and 3) possible involvement of reactive oxygen species in carcinogenesis. The present study investigated the possible role of Tsc2 and VHL tumor suppressor genes in this model. Thirty-four Fe-NTA-induced primary RCCs and 20 other primary or metastatic tumors of rats were searched for genetic alteration in all the coding exons of both genes by polymerase chain reaction-single-strand-conformation polymorphism analysis and sequencing in conjunction with morphological evaluation. In the Fe-NTA-induced RCCs, frequency of metastasis or invasion was proportionally associated with the nuclear grade of the tumor (grades 1-3). Only one Fe-NTA-induced RCC of grade 1 revealed missense mutations with loss of heterozygosity in exon 10 of the Tsc2 gene (codons 334, GTG (Val) to GCG (Ala), and 336, TAT (Tyr) to CAT (His). No mutation was found in the VHL gene. The results suggest that 1) high-grade RCCs can develop in the absence of mutations in the Tsc2 and VHL genes in rats, and that 2) Tsc2 gene somatic mutation can nonetheless be one of the causes of non-Eker rat RCCs.     

Ketone body transport in renal brush border membrane vesicles

Ketone body uptake by renal brush border vesicles has been investigated. Ketone bodies enter into the brush border vesicles by a carrier-mediated process. The uptake is dependent on an Na+ gradient ([Na+]outside>[Na+]inside) and is electroneutral. The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. A pH gradient (alkaline inside) also stimulates the ketone body uptake. Acetoacetate and 3-hydroxybutyrate share the same carrier as demonstrated by the accelerated exchange diffusion and mutual inhibitory effects.