Renal brush border membrane bound intrinsic factor

A highly active receptor for intrinsic factor (IF)-cobalamin (Cbl) complex has been detected and reported in mammalian kidney earlier (Seetharam, B., et al. (1988) J. Biol. Chem. 263, 4443-4449). The physiological role of this receptor in normal Cbl homeostasis is not known. In addition to binding of exogenously added IF-[57Co]Cbl, the renal apical membranes contain endogenous IF or IF-Cbl. Washing with pH 5/EDTA buffer enhanced the binding of exogenously added IF-[57Co]Cbl to renal apical but not basolateral membranes. The pH 5/EDTA extract from renal apical membranes bound [57Co]Cbl. The complex also bound to rat ileal brush border membrane and promoted ileal transport of [57Co]Cbl. On immunoblots using monospecific antiserum to IF a 62 kDa protein was identified in renal and intestinal apical membranes, serum and in tissue extracts of unperfused rat liver, kidney and heart. The 62 kDa band was eliminated from the renal apical membranes following pH 5/EDTA wash. Rat urine demonstrated unsaturated [57Co]Cbl binding (0.2 to 0.4 pmol/day) of which only 30-40% was immunoprecipitated with anti IF and could be identified on immunoblots. The identification of IF in rat renal apical membranes (160-200 ng/mg protein) and secretion of only traces of IF in urine suggest that the renal IF-Cbl receptor may play a role in sequestering IF/IF-Cbl and prevent urinary loss of Cbl.     

Synthesis and brush border expression of intrinsic factor-cobalamin receptor from rat renal cortex.

The main objective of the current study was to investigate the factors that affect brush border membrane expression of intrinsic factor-cobalamin receptor (IFCR). Because of high levels of IFCR expression (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449) in the rat kidney, we have studied the synthesis and expression of IFCR using rat cortical slices in culture. The IFCR activity in the renal apical brush border was maximum from rats between the age of 20-24 days and about 75% of the activity was lost from the isolated apical surface membranes following culture of cortical slices with nonradioactive intrinsic factor-cobalamin. However, the membrane IFCR activity recovered to 100 or 75%, respectively, when the slices were cultured with intrinsic factor-cobalamin mixed with either leupeptin or chloroquine. When these lysosomotropic agents were added during the metabolic labeling of the cortical slices with trans-35S-label neither the synthesis nor the amount of [35S]IFCR transported to the apical membrane was inhibited. However, with the addition of colchicine, the apical membrane expression of [35S]IFCR was inhibited by 75-80%. Metabolic labeling of cortical slices with trans-35S-label and immunoprecipitation of the Triton X-100 extract from the total, internal, and apical membranes revealed the presence of a 230-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With either continuous or pulse-chase labeling of the cortical slices, the amount of 230-kDa [35S]IFCR recovered in the apical membrane did not exceed 10-15% of the total labeled receptor synthesized. Based on these and our recent studies (Seetharam, S., Dahms, N., Li, N., Ramanujam, K.S., and Seetharam, B. (1991) Biochem. Biophys. Res. Commun. 177, 751-756), we propose that rat renal IFCR is synthesized as a single polypeptide chain of 220 kDa and is transported slowly to the apical membrane during which four or five N-linked oligosaccharides are processed to the complex type. Moreover, the brush border expression of IFCR is regulated by the biosynthetic and not by the endocytic pathway.     

Leupeptin and ammonium chloride inhibit intrinsic factor mediated transcytosis of [57Co]cobalamin across polarized renal epithelial cells.

The [125I] intrinsic factor (IF) mediated transcytosis of [57Co]Cyanocobalamin (Cbl) by polarized opossum kidney cells was inhibited (greater than 80%) by preincubation of the cells with lysosomotropic agents leupeptin or ammonium chloride. Inhibition of Cbl transcytosis resulted in the intracellular accumulation of both [125I]IF (48 kDa) and [57Co]Cbl. Intracellular degradation of [125I]IF occurred during normal cellular transcytosis of [57Co]Cbl and in one h following internalization the major intracellular degradation products of IF were two polypeptides of Mr 29 kDa and 19 kDa. The size of the major degradation product of IF in the basolateral media was 10 kDa. Based on these results, we suggest that IF is internalized by the renal epithelial cells and is degraded by leupeptin-sensitive acid proteases during Cbl transcytosis.     

Binding of intrinsic factor to ileal brush border membrane in the rat

The rat ileal brush border membrane binds both free [¹²⁵I]-intrinsic factor (IF) and the IF-[⁵⁷Co]cobalamin (cbl) complex. This binding is observed with IF isolated from rat stomach, but not from IF isolated from hog, canine and human stomachs. The binding of rat-IF[⁵⁷Co]cbl can be blocked with free rat IF but not with hog IF. The IF-cbl complex binds at a higher affinity (Ka=0.15 × 10⁹ M^?1) compared to that of free IF (Ka=0.9 × 10⁹ M^?1). Rat IF-cbl also binds efficiently to human and canine ileal membranes. While antibody to the canine ileal receptor blocks the binding of rat, human or hog IF-[⁵⁷Co]cbl to human and canine ileal membranes, it does not affect the binding of rat IF-[⁵⁷Co]cbl to rat ileal membranes. These findings demostrate that the rat ileal receptor is different from canine and human ileal receptors.     

Microtubules are involved in the secretion of proteins at the apical cell surface of the polarized epithelial cell, Madin-Darby canine kidney

Microtubule-disrupting drugs (nocodazole, colchicine) and cytochalasin D, which inhibits the polymerization of the actin microfilaments, were used to study the role of the cytoskeleton in protein secretion in the polarized Madin-Darby canine kidney (MDCK) epithelial cells. Two proteins were analyzed. The gp 80 glycoprotein complex, which in untreated cells is sorted into the apical pathway and lysozyme, which is released randomly at both cell surfaces in transfected MDCK cells. Our results show that cytochalasin D has no influence on the transport of the gp 80 complex and lysozyme to either cell surface. However, in the presence of nocodazole or colchicine the secretion of both proteins at the apical cell surface is reduced by 50% with a concomitant increase in the basolateral release. These data suggest that microtubules are necessary for an efficient secretion of proteins at the apical cell surface of MDCK cells. In regard to the yet unresolved discrepancy concerning the involvement of microtubules in the transport of membrane proteins to the apical surface of MDCK cells, our results are consistent with the data of Rindler et al. (Rindler, M. J., Ivanov, I. E., and Sabatini, D. D. (1987) J. Cell. Biol. 104, 231-241) who observed a nonpolarized delivery of the influenza virus hemagglutinin in the presence of nocodazole or colchicine.     

Significance of the distribution of 57Co-vitamin B12 in Spirometra mansonoides (Cestoidea) during growth and differentiation in mammalian intermediate and definitive hosts

The distribution of labeled cyanocobalamin (CN-[57Co]Cbl = [57Co]-vitamin B12) in pleurocercoids and adult tapeworms of Spirometra mansonoides was studied during development in mice 22 days days PI, respectively. Plerocercoid scolices, obtained by cutting away their bodies or by in vitro enzymatic dissolution of the bodies, were pulsed with CN- magnitude of 57Co Cbl for 1h at 37 degrees C and reimplanted subcutaneously into mice or given per os to cats. In regenerated plerocercoids, the highest concentration of magnitude of 57Co Cbl occurred in the scolex and then decreased posteriorly in the newly-formed tissues of the body. Approximately 60% of the total magnitude of 57Co Cbl present remained concentrated in the scolex following body regeneration plerocercoids and adult tapeworms of Spirometra mansonoides was studied during development in mice 22 days post-infection (PI) and in cats 16 days PI, respectively. Plerocercoid scolices, obtained by cutting away their bodies or by in vitro enzymatic dissolution of the bodies, were pulsed with CN-[57Co]Cbl for 1 h at 37 degrees C and reimplanted subcutaneously into mice or given per os to cats. In regenerated plerocercoids, the highest concentration of [57Co]Cbl occurred in the scolex and then decreased posteriorly in the newly-formed tissues of the body. Approximately 60% of the total [57Co]Cbl present remained concentrated in the scolex following body regeneration for up to 109 days PI. This high [57Co]Cbl concentration in the plerocercoid scolex was bound to protein and appears to be maintained by a complex homeostatic mechanism in association with directional transport of [57Co]Cbl to the scolex with ultimate depletion along the length of the body.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction between transcobalamin II and immobilized Cibacron Blue F3GA

Human serum transcobalamin II (TC II), a vitamin B₁₂ (Cbl) transport protein, complexes with Cibacron Blue F3GA, a reactive blue dye which can bind to proteins that require nucleotides as cofactors. Apo-TC II and holo-TC II both bind, but intrinsic factor (IF) and R-type binders of Cbl do not. Other mammalian species TC II also complex with the dye. Greater than 87% of the applied TC II-CN-[⁵⁷Co]Cbl remains bound to the dye even at pH 4.0. At pH values below this, the CN-[⁵⁷Co]Cbl dissociates off TC II which remains bound to the dye. High salt concentrations will break the TC II-dye complex. Ionic forces were considered not to be involved since complexing also occurred at pH 9.0, 2.5 pH units above the isoelectric point of TC II. Failure to dissociate the TC II-dye complex with 50% glycerol makes hydrophobic interactions unlikely. In addition to the potential uses of TC II-Cibacron Blue F3GA complexes in a total scheme for protein purification, the possibility that TC II is a nucleotide-requiring protein should be explored.     

Transepithelial transport of vinblastine by kidney-derived cell lines. Application of a new kinetic model to estimate in situ Km of the pump

We present a new transport model that may be useful for many kinds of transepithelial transport experiments. The model permits estimation of a pump Km and pump activity solely on the basis of transepithelial tracer fluxes. We apply the model to studies of a multidrug efflux pump, P-glycoprotein, which is normally located in the apical plasma membrane of certain transporting epithelia such as kidney proximal tubule cells. To determine the functional properties of this multidrug transporter in an epithelium, we studied the transepithelial transport of the chemotherapeutic drug, vinblastine, in epithelia formed by the kidney cell lines MDCK, LLC-PK1, and OK. We have previously shown that basal to apical flux of 100 nM vinblastine was about five times higher than apical to basal flux in MDCK epithelia, indicating that there is a net transepithelial transport of vinblastine across MDCK epithelia. Addition of unlabeled vinblastine reduced basal to apical flux of tracer and increased apical to basal flux of tracer in a concentration-dependent manner, a pattern expected if there is a saturable pump that extrudes vinblastine at the apical plasma membrane. The model permits estimation of a pump Km and pump activity solely on the basis of transepithelial tracer fluxes. According to the transport model the apical membrane pump has Michaelis-Menten kinetics with an apparent Km = 1.1 microM. Net basal to apical transport of vinblastine was also observed in LLC-PK1 cells and OK cells which are other kidney-derived cell lines. The order of potency of the transport is LLC-PK1 greater than MDCK greater than OK cells. The organic cation transporter is not involved in this vinblastine transport because vinblastine transport in MDCK cells was not affected by 3 mM tetramethyl- or tetraethylammonium. Inhibitors of vinblastine transport in MDCK cells was not affected by potency, were verapamil greater than vincristine greater than actinomycin D greater than daunomycin. The transport pattern we observed is that predicted to result from the function of the multidrug transporter in the apical plasma membrane.     

Cobalamin metabolism in cultured human chorionic villus cells

Cobalamin (Cbl, vitamin B₁₂) metabolism was analyzed in cultures of human chorionic villus (CV) cells obtained at 9–10 weeks of gestation. CV cells were shown to synthesize transcobalamin II (TCII) and to possess a high affinity receptor for that molecule. The cells bound and internalized radioactive cyanocobalamin (CN[⁵⁷Co]Cbl) complexed to TCII. This internalized CN[⁵⁷Co]Cbl was found to be converted to both methylCbl and adenosylCbl, the two intracellular coenzyme forms of Cbl, and bound to the two known intracellular Cbl requiring enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase. Both enzyme systems were found to be functional in the intact cell by demonstrating the incorporation of the radioactive label from both [¹⁴C]CH₃-tetrahydrofolate and [¹⁴C]propionate into acid insoluble products. MS activity was also detected in lysed cell material. CV cells were shown not to be auxotrophic for methionine since they were able to utilize homocysteine in place of methionine for cell division. Since CV cells are capable of performing many of the complex events associated with Cbl metabolism, it may be possible to use these cells to diagnose genetic defects of Cbl metabolism. © 1993 Wiley-Liss, Inc.     

Defective brush-border expression of intrinsic factor-cobalamin receptor in canine inherited intestinal cobalamin malabsorption

Ligand binding activity of intrinsic factor-cobalamin receptor (IFCR) was determined in homogenates and isolated brush-border membranes (BBM) of ileum and kidney from dogs exhibiting simple autosomal recessive inheritance of selective cobalamin malabsorption (Fyfe, J. C., Giger, U., Hall, C. A., Jezyk, P. F., Klumpp, S. A., Levine, J. S., and Patterson, D. F. (1991) Pediatr. Res. 29, 24-31). IFCR activity of affected dog ileal homogenates was 3-4-fold higher than normal whereas IFCR activity in affected dog kidney homogenates was one-tenth of normal. The recovery of IFCR activity in the BBM of ileum and renal cortex of affected dogs was 30- and 20-fold less than normal, respectively. The dissociation constant (Kd) for intrinsic factor-cobalamin was similar in BBM of both tissues and was the same in affected and normal dogs. In the affected dog ileal BBM, activities of alkaline phosphatase and sucrase-isomaltase and vesicular transport of glucose and Na(+)-taurocholate were normal. Immunoblots showed no IFCR cross-reactive material in the ileal or renal BBM of affected dogs. IFCR purified by affinity chromatography from kidney of both normal and affected dogs had an Mr = 230,000. However, amino acid analysis revealed that the affected dog IFCR had more lysine than the normal, and protease cleavage of the purified IFCRs revealed different peptide maps. Asparagine-linked oligosaccharides of both proteins were sensitive to peptide N-glycosidase F cleavage, but only the affected dog IFCR was endoglycosidase H sensitive. These results suggest that cobalamin malabsorption in this canine family is caused by inefficient BBM expression of IFCR due to a mutation of IFCR and its retention in an early biosynthetic compartment.     

Preparation of Radioactive Labelled (57Co) Sulfitocobalamin

Abstract

Farquharson and Adams (Br. J. Nutr. 36, 127-135 (1976)) have identified sulfitocobalamin (S0₃?Cbl) as one of the naturally occurring cobalamins (Cbls) in foods. We have devised a method of making radioactive labelled S0₃?Cbl for invivo and in vitro studies of this form of Cbl. ⁵⁷Co labelled cyanocobalamin (⁵⁷Co CN-Cbl) was acid photolyzed to ⁵⁷Co hydroxocobalamin (⁵⁷Co OH-Cbl) followed by ligand substitution with S0₃ ^?2 ion from aqueous sodium (meta) bisulfite in the dark. The resulting ⁵⁷Co SO₃?Cbl was purified by organic extraction and cation ex-change chromatography. The final preparation was >99% Co⁵⁷ S0₃?Cbl with an overall yield of >70%, stable for up to four weeks at 20°C in the dark, and capable of binding to the human Cbl binding proteins Transcobalamin II (TC II), Intrinsic factor (IF) and Salivary R. This method allows a simple 1 day preparation of high specific activity labelled ⁵⁷Co S0₃?Cbl for biological studies.     

Perturbation of neuronal cobalamin transport by lysosomal enzyme inhibition

Cbl (cobalamin) utilization as an enzyme cofactor is dependent on its efficient transit through lysosomes to the cytosol and mitochondria. We have previously proposed that pathophysiological perturbations in lysosomal function may inhibit intracellular Cbl transport with consequences for down-stream metabolic pathways. In the current study, we used both HT1080 fibroblasts and SH-SY5Y neurons to assess the impact that protease inhibitors, chloroquine and leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal), have on the distribution of [⁵⁷Co]Cbl in lysosomes, mitochondria and cytosol. Under standard cell culture conditions the distribution of [⁵⁷Co]Cbl in both neurons and fibroblasts was ~5% in lysosomes, 14% in mitochondria and 81% in cytosol. Treatment of cells with either 25 μM chloroquine or 40 μM leupeptin for 48 h significantly increased the lysosomal [⁵⁷Co]Cbl levels, by 4-fold in fibroblasts and 10-fold in neurons, and this was associated with reduced cytosolic and mitochondrial [⁵⁷Co]Cbl concentrations. Based on Western blotting of LAMP2 in fractions recovered from an OptiPrep density gradient, lysosomal Cbl trapping was associated with an expansion of the lysosomal compartment and an increase in a subpopulation of lysosomes with increased size and density. Moreover, the decreased mitochondrial Cbl that was associated with lysosomal Cbl trapping was correlated with decreased incorporation of [¹⁴C] propionate into cellular proteins/macromolecules, indicating an inhibition of Cbl-dependent Mm-CoA (methylmalonyl-coenzyme A) mutase activity. These results add support to the idea that lysosomal dysfunction may significantly impact upon Cbl transport and utilization.     

The metabolism of cobalamin bound to transcobalamin II and to glycoproteins that bind Cbl in HepG2 cells (human hepatoma)

The binding, internalization, processing and release of labeled cyanocobalamin (CN[57Co]Cbl) bound to human transcobalamin II (TC II) were studied in HepG2 cells, a line of hepatocytes derived from a human hepatoma. The cells bound the TC II-Cbl by specific, high affinity receptors. Within the cell, the CN-Cbl was promptly freed from TC II and the CN-Cbl converted to more active forms including adenosyl Cbl (AdoCbl) and methyl Cbl (MeCbl). Whereas free labeled Cbl was still present at 72 hours after entry, the cells also bound Cbl to an intracellular binder (ICB) presumed to represent the holo enzymes dependent on Cbl. At levels of TC II that saturated the receptors for TC II-Cbl, much of the Cbl entering the cells remained free and was converted to AdoCbl. Under these circumstances the cells released free Cbl, mostly AdoCbl. Human R type binders of Cbl, which are glycoproteins and some having a terminal galactose, were bound by the HepG2 cells. The binding was characteristic of the receptor system responsive to a terminal galactose, or asialoglycoproteins, but was inconsistent and of low affinity. Cbl bound to R binder was internalized and converted to coenzyme forms of Cbl, but the process was much less effective than when the Cbl entered via the TC II receptor system. It was concluded that the receptors for R-Cbl were unlikely to contribute to the physiologic transport of Cbl in man, but may function in some yet unknown way.     

Development of the Na(+)-dependent hexose carrier in LLC-PK1 cells is dependent on microtubules

The Na(+)-dependent hexose carrier, an endogenous apical marker, develops during differentiation of LLC-PK1, an established cell line with characteristics of the proximal tubule. This development was inhibited by the microtubule-disrupting drugs, colchicine and nocodazole, while it was insensitive to lumicolchicine. This strongly suggests that microtubules are involved in the plasma membrane expression of the Na(+)-dependent hexose carrier. We also analyzed the increase in activity of endogenous apical and basolateral membrane proteins during the polarization process. The development of three apical (Na(+)-dependent hexose carrier, gamma-glutamyltransferase and alkaline phosphatase) and one basolateral membrane protein (Na+/K(+)-ATPase) was studied during the reorganization of LLC-PK1 cells into a polarized epithelium. Colchicine inhibited the rapid, transient increase in the expression of the Na(+)-dependent hexose carrier during this polarization process. A similar result was observed for the development of the other apical proteins, while the development of Na+/K(+)-ATPase seemed to be largely insensitive to colchicine. Our results are in agreement with the model that the vesicles containing the apical membrane proteins use microtubules as tracks to reach the plasma membrane. The transport of vesicles containing basolateral membrane proteins clearly occurs by a different pathway which is independent on an intact microtubular network. Since the inhibition by the microtubule-disrupting drugs was complete, it can be concluded that after disruption of microtubules, the apical vesicles do not use the basolateral pathway by default.     

Cobalamin transport proteins and their cell-surface receptors

The primary function of cobalamin (Cbl; vitamin B12) is the formation of red blood cells and the maintenance of a healthy nervous system. Before cells can utilise dietary Cbl, the vitamin must undergo cellular transport using two distinct receptor-mediated events. First, dietary Cbl bound to gastric intrinsic factor (IF) is taken up from the apical pole of ileal epithelial cells via a 460 kDa receptor, cubilin, and is transported across the cell bound to another Cbl-binding protein, transcobalamin II (TC II). Second, plasma TC II-Cbl is taken up by cells that need Cbl via the TC II receptor (TC II-R), a 62 kDa protein that is expressed as a functional dimer in cellular plasma membranes. Human Cbl deficiency can develop as a result of acquired or inherited dysfunction in either of these two transmembrane transport events. This review focuses on the biochemical, cellular and molecular aspects of IF and TC II and their cell-surface receptors.     

Interaction of digoxin with antihypertensive drugs via MDR1

The multidrug transporter MDR1 (P-glycoprotein)-mediated interaction between digoxin and 29 antihypertensive drugs of various types was examined by using the MDR1 overexpressing LLC-GA5-COL150 cells, which were established by transfecting MDR1 cDNA into porcine kidney epithelial LLC-PK1 cells. These cells construct monolayers with tight junctions, and enable the evaluation of transcellular transport. The MDR1 was highly expressed on the apical membrane (urine side). The basal-to-apical and apical-to-basal transcellular transport of [3H]digoxin in LLC-GA5-COL150 cells was time- and temperature-dependent. The basal-to-apical transport of [3H]digoxin was markedly increased, whereas the apical-to-basal transport was decreased in LLC-GA5-COL150 cells, compared with the host LLC-PK1 cells, suggesting that [3H]digoxin was a substrate for MDR1. Most of the Ca2+ channel blockers used here markedly inhibited basal-to-apical transport and increased apical-to-basal transport. Exceptions were diltiazem, nifedipine and nitrendipine, which hardly showed inhibitory effects on transcellular transport of [3H]digoxin. Alpha-blocker doxazosin and beta-blocker carvedilol also inhibited transcellular transport of [3H]digoxin, but none of the angiotensin converting enzyme inhibitors and AT1 angiotensin II receptor antagonists used here were active. These observations will promote understanding of the digoxin-drug interactions resulting from their actions on MDR1, and which may aid in avoiding these unexpected effects of digoxin.     

Possible involvement of microtubule disruption in bipolar budding of a Sendai virus mutant, F1-R, in epithelial MDCK cells.

Envelope glycoproteins F and HN of wild-type Sendai virus are transported to the apical plasma membrane domain of polarized epithelial MDCK cells, where budding of progeny virus occurs. On the other hand, a pantropic mutant, F1-R, buds bipolarly at both the apical and basolateral domains, and the viral glycoproteins have also been shown to be transported to both of these domains (M. Tashiro, M. Yamakawa, K. Tobita, H.-D. Klenk, R. Rott, and J.T. Seto, J. Virol. 64:4672-4677, 1990). MDCK cells were infected with wild-type virus and treated with the microtubule-depolymerizing drugs colchicine and nocodazole. Budding of the virus and surface expression of the glycoproteins were found to occur in a nonpolarized fashion similar to that found in cells infected with F1-R. In uninfected cells, the drugs were shown to interfere with apical transport of a secretory cellular glycoprotein, gp80, and basolateral uptake of [35S]methionine as well as to disrupt microtubule structure, indicating that cellular polarity of MDCK cells depends on the presence of intact microtubules. Infection by the F1-R mutant partially affected the transport of gp80, uptake of [35S]methionine, and the microtubule network, whereas wild-type virus had a marginal effect. These results suggest that apical transport of the glycoproteins of wild-type Sendai virus in MDCK cells depends on intact microtubules and that bipolar budding by F1-R is possibly due, at least in part, to the disruption of microtubules. Nucleotide sequence analyses of the viral genes suggest that the mutated M protein of F1-R might be involved in the alteration of microtubules.