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J Biber

Publications and source records attributed to J Biber.

At least 163 records · Page 9Linked to original sources
PubMed1993

Structural identification of brush border membrane transport systems--towards an understanding of regulatory mechanisms.

Until recently proximal tubular membrane transport systems were defined only on the basis of their function. However, in the past 4-5 years the use of molecular biology techniques has permitted structural identification of several proximal tubular transport systems. For example, sodium/D-glucose, sodium/phosphate, and sodium/sulfate cotransport systems have been identified. Also (a component of) an amino acid transport system accepting cystine and dibasic amino acids has been identified. These transport pathways may be targets for genetic alterations in proximal tubular solute reabsorption and/or be under physiological control.

Amino Acids↗
PubMed1993

Role of microtubules in the adaptive response to low phosphate of Na/Pi cotransport in opossum kidney cells.

The role of microtubules and actin microfilaments in adaptive changes of the apical Na-dependent transport of phosphate (Pi) was investigated in opossum kidney (OK) cells. Up-regulation of Na/Pi cotransport was achieved by incubating OK cells in a medium containing 0.1 mM Pi; down-regulation of Na/Pi cotransport was provoked by refeeding adapted cells with 2 mM Pi. Up-regulation of Na/Pi cotransport was found to be inhibited by approximately 50% after a pretreatment of the cells with the microtubule disrupting agents nocodozole and colchicine; indirect immunofluorescence indicated complete depolymerization of the microtubular network. No inhibition of the adaptive response was observed after treatment of the cells with cytochalasin B to depolymerize actin microfilaments. In adapted cells, depolymerization of microtubules by nocodozole led to a reversibility of Na/Pi cotransport similar to that observed after refeeding adapted cells with 2 mM Pi. No effects of the microtubule disrupting drugs were observed on Na/L-glutamic acid transport. Depolymerization of microtubules did not prevent parathyroid-hormone-mediated inhibition of Na/Pi cotransport. It is concluded that microtubules are (at least in part) involved in the correct insertion of newly synthesized apical Na/Pi cotransport systems and that microtubules are not involved in the internalization of Na/Pi cotransport systems.

Actin Cytoskeleton↗
PubMed1993

Localization of NaPi-1, a Na-Pi cotransporter, in rabbit kidney proximal tubules. I. mRNA localization by reverse transcription/polymerase chain reaction.

We have recently isolated from a rabbit cortex cDNA library a cDNA clone (NaPi-1), which, after in vitro transcription (cRNA) and injection into Xenopus laevis oocytes, expresses Na-dependent Pi uptake [Werner A, et al. (1991) Proc Natl Acad Sci USA 88:9608-9612]. The aim of the present work was to study the nephron location of the NaPi-1-related mRNA(s) by combining nephron microdissection procedures, reverse transcription (RT) and amplification of the resultant cDNA by the polymerase chain reaction (PCR). RT-PCR using NaPi-1-specific primers (different combinations) and either total kidney cortex RNA or microdissected proximal tubule segments resulted in two PCR products, both of approximately the expected length (but differing by about 30 base pairs). Restriction-enzyme analysis and nucleotide sequencing confirmed that both PCR products are related to NaPi-1 and that the "longer" PCR product has an insert of 26 base pairs containing an AluI restriction site. Nephron microdissection documents expression of NaPi-1-related mRNA(s) in superficial and deep proximal tubules (S1, S2 and S3 segments) and their absence in glomeruli, thin descending limb and thick ascending limbs of Henle's loop, distal convoluted tubules and cortical and inner medullary collecting ducts. These experiments suggest a "microheterogeneity" of NaPi-1-related mRNA(s) (which is not detected in Northern blot analysis) and proximal tubular expression of NaPi-1.

Animals↗
PubMed1993

Localization of NaPi-1, a Na/Pi cotransporter, in rabbit kidney proximal tubules. II. Localization by immunohistochemistry.

Polyclonal antibodies have been raised against a C-terminal peptide of NaPi-1, a recently cloned Na-Pi cotransport system of rabbit kidney cortex with a predicted (unglycosylated) molecular mass of 52 kDa. By Western blot analysis using brush-border membranes isolated from rabbit kidney cortex, two proteins with apparent molecular masses of 64 kDa and 35 kDa were specifically recognized (peptide protectable) by the antiserum obtained. The 64-kDa protein was found to migrate in parallel with the luminal membrane during separation by free-flow electrophoresis of brush-border and basolateral membranes. In immunofluorescence studies using cryostat sections of rabbit kidney, specific binding of antibodies was observed in proximal tubules (including S1, S2 and S3 segments) of superficial and deep nephrons. Anti-(NaPi-1)-antibody-mediated fluorescence was restricted to the brush border of proximal tubular cells. No specific immunoreaction was observed in other tubular segments. The results suggest that the native NaPi-1-related protein (Na-Pi cotransport system) has an apparent molecular mass of 64 kDa and is uniformly expressed in the apical membrane of proximal tubules of all nephron generations in the rabbit kidney. Immunohistochemical localization of the Na-Pi cotransport system NaPi-1 confirms the segmental localization within the nephron of NaPi-1-related mRNA as revealed by the reverse transcriptase/polymerase chain reaction (see preceding paper).

Amino Acid Sequence↗
PubMed1993

Effect of low-phosphate diet on sodium/phosphate cotransport mRNA and protein content and on oocyte expression of phosphate transport.

Recently, we have isolated a complementary DNA most likely related to rabbit kidney cortex brush border membrane sodium/phosphate (Na/Pi) cotransport activity [NaPi-1 (1)]. To further elucidate the cellular mechanisms involved in dietary 'adaptation' of renal Na/Pi cotransport, we have exposed young rabbits for 2 weeks to either a low phosphate (Pi) diet (LPD) or a high Pi diet (HPD). Initial linear uptake of Na/Pi cotransport in isolated brush border membrane vesicles was increased in rabbits on a LPD compared with those on a HPD. Injection of equal amounts of total mRNA isolated from kidney cortex of LPD or HPD rabbits into Xenopus laevis oocytes resulted in a higher stimulation of Na-dependent oocyte Pi uptake in LPD than HPD preparations. No difference in the content of 'specific' mRNA (NaPi-1 cDNA probe, Northern blots) and of the content of the 'specific' brush border membrane protein (NaPi-1 antipeptide antibody, Western blots) between LPD and HPD preparations was observed. We conclude that 'chronic' dietary Pi deprivation leads to a protein synthesis-dependent alteration of Na/Pi cotransport activity which does not involve a change in the total amount of a protein related to the recently cloned NaPi-1 protein.

Animals↗
PubMed1992

Poly(A)+ RNA from rabbit intestinal mucosa induces b0,+ and y+ amino acid transport activities in Xenopus laevis oocytes.

Injection of poly(A)+ RNA (mRNA) isolated from rabbit intestinal mucosa into Xenopus laevis oocytes results in an increase in sodium-independent uptake of L-[3H]leucine, L-[35S]cystine, and L-[3H]arginine. This uptake activity is related to an mRNA species corresponding to the recently isolated rabbit kidney cortex cDNA clone rBAT (related to b0,+ amino acid transporter; Bertran, J., Werner, A., Stange, G., Markovich, D., Moore, M. L., Biber, J., Testar, X., Zorzano, A., Palacin, M., and Murer, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 281, 717-723) and to a protein involved in amino acid transport via system y+. This conclusion is based on the following observations: 1) mRNA isolated from mucosa of duodenum, jejunum, and ileum, but not from colon, induces sodium-independent uptake of L-leucine, L-cystine, and L-arginine. 2) In Northern blot analysis, mRNA isolated from mucosa of duodenum, jejunum, and ileum, but not from colon, hybridizes to an rBAT cDNA probe, with signals of 2.2-2.3 kilobases and 3.7-3.9 kilobases. 3) mRNA isolated from mucosa of jejunum induces sodium-independent uptake of L-leucine and L-cysteine which shows an inhibition pattern corresponding to system b0,+; the inhibition pattern of mRNA-induced uptake of L-arginine is compatible with the contribution of system b0,+ and y+. 4) Hybrid depletion with an rBAT antisense oligonucleotide greatly prevents the mRNA-dependent induction of uptake of L-cystine (greater than 90%) and of L-leucine (approximately 75%); it reduces to about 50% the induction of L-arginine uptake. 5) After separation of mRNA on a sucrose density gradient, the fractions resulting in expression of b0,+ transport activity were also those hybridizing with rBAT cDNA; induction of transport activity from these fractions was also sensitive to hybrid depletion. 6) The mRNA-induced component of L-arginine uptake which is resistant to rBAT hybrid depletion is inhibited by L-homoserine, only in the presence of sodium; thus, it is related to a system y(+)-like activity.

Amino Acids↗
PubMed1992

Expression cloning of a cDNA from rabbit kidney cortex that induces a single transport system for cystine and dibasic and neutral amino acids.

We have isolated a cDNA clone by screening a rabbit kidney cortex cDNA library for expression of sodium-independent transport of L-arginine and L-alanine in Xenopus laevis oocytes. Expressed uptake relates to a single component of sodium-independent transport for dibasic and neutral amino acids. This transport activity resembles the functionally defined system b0,+ and carries cystine and dibasic amino acids with high affinity. The rBAT (b0,+ amino acid transporter-related) mRNA is found mainly in kidney and intestinal mucosa. It encodes a predicted 77.8-kDa protein with only one putative transmembrane domain and seven potential N-glycosylation sites. This protein could either be a constitutive element or a specific activator of system b0,+.

Amino Acid Sequence↗
PubMed1992

Stimulation of system y(+)-like amino acid transport by the heavy chain of human 4F2 surface antigen in Xenopus laevis oocytes.

A kidney cortex cDNA clone (rBAT) has recently been isolated, which upon in vitro transcription and capping complementary RNA (cRNA) and injection into Xenopus laevis oocytes induces a system b0,(+)-like amino acid transport activity. This cDNA encodes a type II membrane glycoprotein that shows significant homology to another type II membrane glycoprotein, the heavy chain of the human and mouse 4F2 surface antigen (4F2hc). Here we demonstrate that injection of human 4F2hc cRNA into oocytes results in the activation of a cation-preferring amino acid transport system that appears to be identical to the y(+)-like transport already present in the oocyte. This is based on the following results: (i) Injection of in vitro transcripts from 4F2hc cDNA (4F2hc cRNA) into oocytes stimulates up to 10-fold the sodium-independent uptake of L-arginine and up to 4.1-fold the sodium-dependent uptake of L-leucine. In contrast, 4F2hc cRNA does not increase the basal sodium-independent uptake of L-leucine. (ii) Basal and 4F2hc cRNA-stimulated sodium-independent uptake of L-arginine is completely inhibited by L-leucine in the presence of sodium. Similarly, the basal and 4F2hc cRNA-stimulated sodium-dependent uptake of L-leucine is entirely inhibited by L-arginine. (iii) The stimulation of sodium-independent uptake of L-arginine and the stimulation of sodium-dependent uptake of L-leucine induced by injection of 4F2hc cRNA are both completely inhibited by dibasic L amino acids and to a lesser extent by D-ornithine. (iv) Both basal and 4F2hc cRNA-stimulated sodium-independent uptake of L-arginine show two additional characteristics of the system y+ transport activity: inhibition of L-arginine uptake by L-homoserine only in the presence of sodium and an increase in the inhibition exerted by L-histidine as the extracellular pH decreased. Our results allow us to propose that an additional family of type II membrane glycoproteins (composed by rBAT and 4F2hc) is involved in amino acid transport, either as specific activators or as components of amino acid transport systems.

Amino Acid Sequence↗
PubMed1992

Expression of Na(+)-independent amino acid transport in Xenopus laevis oocytes by injection of rabbit kidney cortex mRNA.

Poly(A)+ mRNA was isolated from rabbit kidney cortex and injected into Xenopus laevis oocytes. Injection of mRNA resulted in a time- and dose-dependent increase in Na(+)-independent uptake of L-[3H]alanine and L-[3H]arginine. L-Alanine uptake was stimulated about 3-fold and L-arginine uptake was stimulated about 8-fold after injection of mRNA (25-50 ng, after 3-6 days) as compared with water-injected oocytes. T.I.C. of oocyte extracts suggested that the increased uptake actually represented an increase in the oocyte content of labelled L-alanine and L-arginine. The expressed L-alanine uptake, obtained by subtracting the uptake in water-injected oocytes from that in mRNA-injected oocytes, showed saturability and was inhibited completely by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) and L-arginine. The expressed L-arginine uptake in mRNA-injected oocytes also showed saturability, being completely inhibited by L-dibasic amino acids) and partially inhibited by BCH. Expression of both L-alanine and L-arginine uptake showed clear cis-inhibition by cationic (e.g. L-arginine) and neutral (e.g. L-leucine) amino acids. In all, this points to the expression of a Na(+)-independent transport system with broad specificity (i.e. b degree, (+)-like). In addition, part of the expressed uptake of L-arginine could be due to a system y(+)-like transporter. After size fractionation through a sucrose density gradient, the mRNA species encoding these increased transport activities (Na(+)-independent transport of L-alanine and of L-arginine) were found in fractions of an average mRNA chain-length of 1.8-2.4 kb. On the basis of these results, we conclude that Na(+)-independent transport system(s) for L-alanine and L-arginine from rabbit renal cortical tissues, most likely proximal tubules, are expressed in Xenopus laevis oocytes. These observations may represent the first steps towards expression and cloning of these transport pathways.

Alanine↗
PubMed1992

Effect of rabbit duodenal mRNA on phosphate transport in Xenopus laevis oocytes: dependence on 1,25-dihydroxy-vitamin-D3.

Expression of Na(+)-dependent transport of phosphate (Pi) was analysed in Xenopus laevis oocytes after injection of poly(A)-rich RNA isolated from the duodenal mucosa of rabbits with increased levels of 1,25-(OH)2-VitD3 (injection of vitamin D3 or low-Pi diet) or from control animals. In parallel, the effect of elevated levels of vitamin D3 was studied in isolated duodenal brush-border membrane vesicles. In brush-border membrane vesicles, the rate of Na(+)-Pi cotransport was found to be doubled after 1,25-(OH)2-VitD3 injections while Na(+)-D-glucose cotransport (measured as a control) was not altered. In X. laevis oocytes, Na(+)-dependent Pi uptake was increased after injection of poly(A)-rich RNA isolated from duodenal mucosa of animals with increased levels of 1,25-(OH)2-VitD3 but not after injection of poly(A)-rich RNA isolated from control animals; between the two groups of mRNA no difference in the expression of the Na(+)-D-glucose transport system was observed. Sucrose density gradient fractionation suggests that mRNA species related to the increased Na(+)-dependent Pi uptake are of average chain lengths between 2 x 10(3) and 3 x 10(3) bases (2-3 kb). It is concluded that in duodenal enterocytes 1,25-(OH)2-VitD3 increases the content of mRNA species of 2-3 kb that might be involved either directly in Na-Pi cotransport or at least in controlling its activity.

Animals↗
PubMed1992

Parathyroid hormone receptors in control of proximal tubule function.

Parathyroid hormone action on renal proximal tubule function involves phospholipase C/protein kinase C as well as adenylate cyclase/protein kinase A mediated regulatory pathways. Tissue culture experiments suggest that low concentrations of PTH affect preferentially the phospholipase C/protein kinase C pathway. In vivo, both regulatory cascades are probably involved in the regulation of proximal tubule function. It is not clear at present whether the two intracellular pathways are linked to one or two PTH receptors. A polarized distribution of PTH receptor(s) involving different second messengers appears possible in proximal tubule epithelial cells. High-affinity (Kd 10(-11)-10(-12) M) PTH receptors in the range of circulating PTH concentrations in vivo remain to be identified. Structural and functional characterization of PTH receptors as well as of the PTH-sensitive intracellular mediators and transport systems form the basis for a better understanding of PTH-dependent regulation of proximal tubule function.

Absorption↗
PubMed1991

Cloning and expression of cDNA for a Na/Pi cotransport system of kidney cortex.

A cDNA library from rabbit kidney cortex was screened for expression of Na-dependent transport of phosphate (Pi) using Xenopus laevis oocytes as an expression system. A single clone was eventually isolated (designated NaPi-1) that stimulated expression of Na/Pi cotransport approximately 700-fold compared to total mRNA. The predicted sequence of the Na/Pi cotransporter consists of 465 amino acids (relative molecular mass, 51,797); hydropathy profile predictions suggest six (possibly eight) membrane-spanning segments. In vitro translation of NaPi-1/complementary RNA in the presence of pancreatic microsomes indicated NaPi-1 to be a glycosylated protein; four potential N-glycosylation sites are present in the amino acid sequence. Northern blot analysis demonstrated the presence of NaPi-1/mRNA in kidney cortex and liver; no hybridization signal was obtained with mRNA from other tissues (including small intestine). Kinetic analysis of Na/Pi cotransport expressed by NaPi-1/complementary RNA demonstrated characteristics (sodium interaction) similar to those observed in cortical apical membranes. The alignment of 5 amino acid residues (Gly342/Ala381-Xaa-Xaa-Xaa-Xaa-Leu386-Xaa-Xaa-Xaa-P ro390- Arg391) is consistent with a motif proposed for Na-dependent transport systems. We conclude that we have cloned a cDNA for a Na/Pi cotransport system present in rabbit kidney cortex.

Amino Acid Sequence↗
PubMed1991

Functional asymmetry of phosphate transport and its regulation in opossum kidney cells: phosphate "adaptation".

The polarity (apical vs basolateral cell surface) of the up-regulatory response ("adaptation") to low medium phosphate (Pi) concentration on apical and basolateral Pi transport systems was investigated in opossum kidney (OK) cell monolayers grown on permeant supports. Incubation of cultures in low-Pi medium, given either only to the apical or simultanously to the apical and basolateral compartments, increased the rate of transport of both the apical and the basolateral Na/Pi cotransport systems. The basolateral Na-independent, 4,4-diisothiocyanatostilbene-2,2'-disulphonic-acid-sensitive Pi transport system was unaffected by Pi deprivation. Incubation with low-Pi medium from only the basolateral side failed to elicit any "adaptive" response in Pi transport. When cells were Pi-limited either apically or on both sides for short periods of time, adaptation was apparent within 2 h and close to maximal by 6 h, and the alteration in Pi transport was consistant with an increase in Jmax for both the apical and basolateral Na/Pi cotransport systems. These data suggest that apical Na-dependent Pi influx is important in signalling the adaptive response to low extracellular Pi.

Adaptation, Physiological↗
PubMed1991

Cellular mechanisms in proximal tubular reabsorption of inorganic phosphate.

Filtered inorganic phosphate (Pi) is largely reabsorbed in the proximal tubule. Na-Pi cotransport, with a stoichiometry of at least 2:1, mediates uphill transport at the apical membrane; at the basolateral membrane different types of transport systems can be involved in efflux and uptake of Pi from the interstitium. Regulation of transcellular Pi flux involves alteration of the apical Na-Pi cotransport; at least three different cellular control/sensing systems seem to participate in this regulation and are exemplified by parathyroid hormone (PTH)-dependent inhibition, Pi deprivation-dependent increase, and insulin-like growth factor I (IGF-I)-dependent increase in Na-Pi cotransport. For PTH inhibition, recent evidence suggests a role of the phospholipase C/protein kinase C-dependent regulatory cascade in inhibition of Na-Pi cotransport, at least at low PTH concentrations. In addition, an endocytic mechanism seems to be involved in this PTH action. Little is known of the cellular mechanisms in Pi deprivation-dependent and/or IGF-I-dependent increases in Na-Pi cotransport; they are dependent on de novo protein synthesis. Recent experiments involving an expression in Xenopus laevis oocytes led to the identification of an approximately 50 kDa membrane protein that is a good candidate for being involved in brush-border membrane Na-Pi cotransport activity.

Absorption↗
PubMed1990

Parathyroid hormone-induced alterations of protein content and phosphorylation in enriched apical membranes of opossum kidney cells.

Parathyroid hormone (PTH) reduces Na/Pi co-transport activity in opossum kidney (OK) cells in a process mediated by protein kinases A and C. Further, inactivation of Na/Pi transport involves irreversible inhibition, possibly via internalization, of the transport system. This study analyzed alterations of concentration and phosphorylation of membrane proteins of an apically enriched preparation induced by short (10 min) and long (3 h) term incubation with 10(-10) M PTH of monolayer cultures of the OK-cell line. To this end, an apically enriched membrane fraction was isolated from cells grown on Petri dishes and analyzed by two-dimensional gel electrophoresis. Long term exposure of the cells to PTH induced changes in apical protein concentration. Four proteins were found to be decreased and one protein was found to be increased in its concentration. Addition of 10(-10) M PTH to the cells led to transient phosphorylation of five proteins. In contrast to transient phosphorylation, phosphorylation of one protein increased over the time period of 3 h. Combined analysis of silver staining and autoradiography led to the detection of an acidic 35-kDa protein in which specific phosphorylation increased over a time period of hours. The results document for the first time alterations in apical membrane protein content and phosphorylation state mediated by PTH when added to an intact cellular system. It is concluded that the identified proteins represent possible candidates for being involved directly or indirectly in PTH alterations of membrane transport.

Animals↗
PubMed1990

Expression of rat liver Na+/L-alanine co-transport in Xenopus laevis oocytes. Effect of glucagon in vivo.

Poly(A)+ RNA (mRNA) isolated from rat liver was injected into Xenopus laevis oocytes, and expression of Na+/L-alanine transport was assayed by measuring Na(+)-dependent uptake of L-[3H]alanine. Expression of Na+/L-alanine transport was detected 3-7 days after mRNA injection, and was due to an increment of the Na(+)-dependent component. After injection of 40 ng of total mRNA, Na(+)-dependent uptake of L-alanine was 2.5-fold higher than in water-injected oocytes. In contrast with Na+/L-alanine transport by water-injected oocytes, expressed Na+/L-alanine transport was inhibited by N-methylaminoisobutyric acid, was inhibited by an extracellular pH of 6.5 and was saturated at approx. 1 mM-L-alanine. After sucrose-density-gradient fractionation, highest expression of Na+/L-alanine uptake was observed with mRNA of 1.9-2.5 kb in length. Compared with mRNA isolated from control rats, mRNA isolated from glucagon-treated rats showed a approx. 2-fold higher expression of Na+/L-alanine transport. The results demonstrate that both liver Na+/L-alanine transport systems (A and ASC) can be expressed in X. laevis oocytes. Furthermore, the data obtained with mRNA isolated from glucagon-treated rats suggest that glucagon regulates liver Na+/L-alanine transport (at least in part) via the availability of the corresponding mRNA.

Alanine↗
PubMed1990

Expression of renal transport systems for inorganic phosphate and sulfate in Xenopus laevis oocytes.

As a first step within an experimental strategy (expression cloning) leading to the structural identification of the two brush-border membrane transport systems for phosphate and sulfate, we have studied the expression of Na(+)-dependent uptake of phosphate and sulfate in Xenopus laevis oocytes injected with rabbit kidney cortex poly(A)+ RNA (mRNA). Na(+)-dependent uptake of phosphate and sulfate was stimulated in a dose- and time-dependent manner up to 20-fold as compared to water-injected controls. After fractionation of the mRNA on a sucrose gradient (or by preparative gel electrophoresis), two neighboring fractions were identified to stimulate Na(+)-dependent phosphate uptake (average size: 3.4 kilobases) and Na(+)-dependent sulfate uptake (average size: 3.7 kilobases). The two transport systems can be discriminated by their inhibition by thiosulfate, which reduced sulfate uptake, but not phosphate uptake. Kinetic characterization of the expressed Na(+)-dependent transport activities results in properties similar to those described for transport activity in renal brush-border membrane vesicles.

Animals↗