Functional expression of SLC15 peptide transporters in rat thyroid follicular cells.

Peptide transport and expression of SoLute Carrier 15 (SLC15) peptide transporters was assessed in rat thyroid tissue and a rat thyroid cell line (PC Cl3 cells). Peptide transport was studied by monitoring the uptake of the fluorophore-conjugated dipeptide beta-Ala-Lys-N(epsilon)-7-amino-4-methyl-coumarin-3-acetic acid (Ala-Lys-AMCA). Expression of SLC15-specific mRNA transcripts was analyzed by RT-PCR. Of the two SLC15 transporters expressed in thyroid follicular cells, namely PEPT2 (SLC15A2) and PHT1 (SLC15A4), only PEPT2 was involved in peptide transport at the plasma membrane, with PHT1 most likely being intracellular. Interestingly, at the mRNA level PEPT2 was up-regulated under TSH stimulation. These findings represent the first evidence that peptide transport occurs in thyroid follicular cells. SLC15 transporters could participate to recycling of peptides derived from extracellular and lysosomal thyroglobulin proteolysis, both essential steps for thyroid hormone synthesis.

PKC-epsilon-dependent cytosol-to-membrane translocation of pendrin in rat thyroid PC Cl3 cells.

We studied the expression and the hormonal regulation of the PDS gene product, pendrin, which is, in thyrocytes, responsible for the iodide transport out of the cell. We show that PC Cl3 cells, a fully differentiated thyroid cell line, grown without TSH and insulin, express very low level of PDS mRNA; such expression is greatly increased after stimulation with insulin or TSH. (125)I pre-loaded cells showed an (125)I efflux accelerated in chloride-containing buffer with respect to chloride-free buffer, suggesting that this efflux is chloride dependent. By immunoblotting, pendrin was found in agonists-stimulated cells, whereas it was barely detectable in un-stimulated cells. An increase in both PDS mRNA and protein was also obtained using phorbol ester PMA, or using 8-Br-cAMP and forskolin. Stimulation with insulin (1 microg/ml; 0-40 min) provoked the cytosol-to-membrane translocation of pendrin and a decrease of intracellular I(-) content in (125)I pre-loaded cells. Insulin- or PMA-treated cells also showed a cytosol-to-membrane translocation of PKC-delta and -epsilon. Inhibition of both PKC-delta and -epsilon activities by GF109203X blocked pendrin translocation, whilst the inhibition of PKA did not. The selective inhibition of PKC-delta by rottlerin did not affect the insulin-provoked translocation of pendrin whilst it was inhibited by a PKC-epsilon translocation inhibitor peptide and also by PKC-epsilon downregulation using the small interfering RNA, thus indicating that such translocation was due to PKC-epsilon activity. In conclusion, our study demonstrates that, in PC Cl3 cells, pendrin expression and localisation are regulated by insulin and influenced by a PKC-epsilon-dependent intracellular pathway.

Differential expression of Na+/D-glucose cotransport in isolated cells of Marsupenaeus japonicus hepatopancreas.

D-Glucose absorptive processes at the gastrointestinal tract of decapod crustaceans are largely under-investigated. We have studied Na(+)-dependent D-glucose transport (Na(+)/D-glucose cotransport) in the hepatopancreas of the Kuruma prawn, Marsupenaeus japonicus, using both brush-border membrane vesicles and purified R and B hepatopancreatic cell suspensions. As assessed by brush-border membrane vesicle studies, Na(+)/D-glucose cotransport was inhibited by phloridzin and responsive to the (inside negative) membrane potential. Furthermore, it was strongly activated by protons (although only in the presence of an inside-negative membrane potential), which correlates with the fact that the lumen of crustacean hepatopancreatic tubules is acidic. When assayed in purified R and B cell suspensions, Na(+)/D-glucose cotransport activity was restricted to B cells only. Mab 13, a monoclonal antibody recognizing an 80- to 85-KDa protein at the brush-border membrane location, inhibited Na(+)/D-glucose cotransport in brush-border membrane vesicles as well as in enriched B cell suspensions. Primers designed after comparison of highly homologous regions of various mammalian sodium-glucose transporter) nucleotide sequences failed to produce RT-PCR amplification products from Kuruma prawn hepatopancreatic RNA. The molecular nature of this Na(+)/D-glucose cotransport system is still to be established.

Changes in cell type composition and enzymatic activities in the hepatopancreas of Marsupenaeus japonicus during the moulting cycle.

The goal of the present study was to evaluate the changes in the cell type composition and ATPase activities (total ATPase, ouabain-sensitive Na+/K(+)-ATPase, furosemide-sensitive Na(+)-ATPase) that occur during the different stages of the moulting cycle in the hepatopancreas of the Marsupenaeus japonicus. The results clearly suggest that the number of resorptive and fibrillar cell types changes significantly during the different stages. An inverse correlation between resorptive and fibrillar cells is observed during moulting (both in normally fed and fasted animals). Fasting, but not the moulting cycle, affects the number of blister-like cells. In the resorptive cells the enzymatic activities (total ATPases and ouabain-sensitive Na+/K(+)-ATPase) also change during the moulting in a cyclical manner. All these results are in agreement with and confirm the different functions carried out by the two cell types within the hepatopancreas.

Characterisation of intestinal peptide transporter of the Antarctic haemoglobinless teleost Chionodraco hamatus.

H(+)/peptide cotransport was studied in brush-border membrane vesicles (BBMV) from the intestine of the haemoglobinless Antarctic teleost Chionodraco hamatus by monitoring peptide-dependent intravesicular acidification with the pH-sensitive dye Acridine Orange. Diethylpyrocarbonate-inhibited intravesicular acidification was specifically achieved in the presence of extravesicular glycyl-L-proline (Gly-L-Pro) as well as of glycyl-L-alanine (Gly-L-Ala) and D-phenylalanyl-L-alanine (D-Phe-L-Ala). H(+)/Gly-L-Pro cotransport displayed saturable kinetics, involving a single carrier system with an apparent substrate affinity (K(m,app)) of 0.806+/-0.161 mmol l(-1). Using degenerated primers from eel and human (PepT1) transporter sequence, a reverse transcription-polymerase chain reaction (RT-PCR) signal was detected in C. hamatus intestine. RT-PCR paralleled kinetic analysis, confirming the hypothesis of the existence of a PepT1-type transport system in the brush-border membranes of icefish intestine. Functional expression of H(+)/peptide cotransport was successfully performed in Xenopus laevis oocytes after injection of poly(A)(+) RNA (mRNA) isolated from icefish intestinal mucosa. Injection of mRNA stimulated D-Phe-L-Ala uptake in a dose-dependent manner and an excess of glycyl-L-glutamine inhibited this transport. H(+)/peptide cotransport in the Antarctic teleost BBMV exhibited a marked difference in temperature optimum with respect to the temperate teleost Anguilla anguilla, the maximal activity rate occurring at approximately 0 degrees C for the former and 25 degrees C for the latter. Temperature dependence of icefish and eel intestinal mRNA-stimulated uptake in the heterologous system (oocytes) was comparable.

Cell shape and plasma membrane alterations after static magnetic fields exposure.

The biological effects of static magnetic fields (MFs) with intensity of 6 mT were investigated in lymphocytes and U937 cells in the presence or absence of apoptosis-inducing drugs by transmission (TEM) and scanning (SEM) electron microscopy. Lectin cytochemistry of ConA-FITC conjugates was used to analyze plasma membrane structural modifications. Static MFs modified cell shape, plasma membrane and increased the level of intracellular [Ca++] which plays an antiapoptotic role in both cell types. Modifications induced by the exposure to static MFs were irrespective of the presence or absence of apoptotic drugs or the cell type. Abundant lamellar-shaped microvilli were observed upon 24 hrs of continuous exposure to static MFs in contrast to the normally rough surface of U937 cells having numerous short microvilli. Conversely, lymphocytes lost their round shape and became irregularly elongated; lamellar shaped microvilli were found when cells were simultaneously exposed to static MFs and apoptosis-inducing drugs. In our experiments, static MFs reduced the smoothness of the cell surface and partially impeded changes in distribution of cell surface glycans, both features being typical of apoptotic cells. Cell shape and plasma membrane structure modifications upon static MFs exposure were time-dependent. Lamellar microvilli were clearly observed before the distortion of cell shape, which was found at long times of exposure. MFs exposure promoted the rearrangement of F-actin filaments which, in turn, could be responsible for the cell surface modifications. Here we report data that support biological effects of static MFs on U937 cells and human lymphocytes. However, the involvement of these modifications in the onset of diseases needs to be further elucidated.

D-glucose transport in decapod crustacean hepatopancreas.

Physiological mechanisms of gastrointestinal absorption of organic solutes among crustaceans remain severely underinvestigated, in spite of the considerable relevance of characterizing the routes of nutrient absorption for both nutritional purposes and formulation of balanced diets in aquaculture. Several lines of evidence attribute a primary absorptive role to the digestive gland (hepatopancreas) and a secondary role to the midgut (intestine). Among absorbed organic solutes, the importance of D-glucose in crustacean metabolism is paramount. Its plasma levels are finely tuned by hormones (crustacean hyperglycemic hormone, insulin-like peptides and insulin-like growth factors) and the function of certain organs (i.e. brain and muscle) largely depends on a balanced D-glucose supply. In the last few decades, D-glucose absorptive processes of the gastrointestinal tract of crustaceans have been described and transport mechanisms investigated, but not fully disclosed. We briefly review our present knowledge of D-glucose transport processes in the crustacean hepatopancreas. A discussion of previous results from experiments with hepatopancreatic epithelial brush-border membrane vesicles is presented. In addition, recent advances in our understandings of hepatopancreatic D-glucose transport are shown, as obtained (1) after isolation of purified R-, F-, B- and E-cell suspensions from the whole organ by centrifugal elutriation, and (2) by protein expression in hepatopancreatic mRNA-injected Xenopus laevis oocytes. In a perspective, the applicability of these novel methods to the study of hepatopancreatic absorptive function will certainly improve our knowledge of this structurally complex organ.

Characterisation of the H(+)/peptide cotransporter of eel intestinal brush-border membranes.

H(+)/peptide cotransport in brush-border membrane vesicles (BBMVs) from eel (Anguilla anguilla) intestine was studied by measuring d-[(3)H]-phenylalanyl-l-alanine uptake and by monitoring peptide-dependent intravesicular acidification using the pH-sensitive dye Acridine Orange. d-[(3)H]-phenylalanyl-l-alanine influx was greatly stimulated by an inside-negative membrane potential and enhanced by an inwardly directed H(+) gradient. In parallel, vesicular H(+) influx was significantly increased in the presence of extravesicular d-phenylalanyl-l-alanine or a series of glycyl and l-prolyl peptides. H(+)/peptide cotransport displayed saturable kinetics involving a single carrier system with apparent substrate affinities of 0.9-2.6 mmol l(-1) depending on the particular peptide. All substrates tested competed with this system. Pre-incubation of BBMVs with dipeptides prevented diethylpyrocarbonate inhibition of transport activity, suggesting that the substrates mask histidine residues involved in the catalytic function of the transporter. Using human PepT1-specific primers, a reverse transcription-polymerase chain reaction (RT-PCR) signal was detected in eel intestine. Our results suggest that, in eel intestine, a brush-border membrane 'low-affinity'-type H(+)/peptide cotransport system is present that shares kinetic features with the mammalian intestinal PepT1-type transporters.

Electroneutral Na+/H+ exchange in brush-border membrane vesicles from Penaeus japonicus hepatopancreas.

An electroneutral Na+/H+ exchange mechanism (dimethylamiloride inhibitable, Li+ sensitive, and Ca2+ insensitive) was identified in brush-border membrane vesicles (BBMV) from Kuruma prawn hepatopancreas by monitoring Na(+)-dependent H+ fluxes with the pH-sensitive dye acridine orange and measuring 22Na+ uptake. Kinetic parameters measured under short-circuited conditions were the Na+ concentration that yielded one-half of the maximal dissipation rate (Fmax) of the preset transmembrane delta pH (KNa) = 15 +/- 2 mM and Fmax = 3,626 +/- 197 delta F.min-1.mg protein-1, with a Hill coefficient for Na+ of approximately 1. In addition, the inhibitory constant for dimethylamiloride was found to be approximately 1 microM. The electroneutral nature of the antiporter was assessed in that an inside-negative transmembrane electrical potential neither affected kinetic parameters nor stimulated pH-dependent (intracellular pH > extracellular pH) 22Na+ uptake. In contrast, electrogenic pH-dependent 22Na+ uptake was observed in lobster hepatopancreatic BBMV. Substitution of chloride with gluconate resulted in increasing KNa and decreasing delta Fmax, which suggests a possible role of chloride in the operational mechanism of the antiporter. These results indicate that a Na+/H+ exchanger, resembling the electroneutral Na+/H+ antiporter model, is present in hepatopancreatic BBMV from the Kuruma prawn Penaeus japonicus.

The bacteriophage T7 binary system activates transient transgene expression in zebrafish (Danio rerio) embryos.

The bacteriophage T7 binary expression system is widely used in vitro for high level selective expression of cloned genes but its application to in vivo models has not yet been investigated. In the present work, we show that coinjection into fertilized zebrafish eggs of pE1T7R, an expression plasmid bearing the T7 RNA polymerase gene driven by the cytomegalovirus (CMV) promoter, together with reporter vectors containing the Escherichia coli lacZ gene driven by the T7 promoter, resulted in the efficient expression of the reporter gene in 24-h mosaic transgenic embryos. Conversely, embryos receiving an unrelated CMV-expression plasmid, instead of pE1T7R, lacked significant reporter gene activity, indicating the strict requirement of T7 polymerase to activate the T7 promoter in these embryos. The present study demonstrates the possibility of applying efficiently the bacteriophage T7 binary system in vivo to a vertebrate model.

H(+)-glycyl-L-proline cotransport in brush-border membrane vesicles of eel (Anguilla anguilla) intestine.

A plasma membrane H(+)-glycyl-L-proline (Gly-L-Pro) cotransport mechanism has been identified in isolated eel intestinal brush-border membrane vesicles (BBMV) by both measuring radiolabeled Gly-L-Pro uptake and monitoring Gly-L-Pro-dependent H+ influx with the pH-sensitive dye acridine orange. The application of an inside negative membrane potential resulted in increasing Gly-L-Pro uptake, as well as the application of inwardly directed H+ gradient (although only when an inside negative membrane potential was present). Furthermore, vesicular H+ influx was found specifically associated with the presence of Gly-L-Pro in the extravesicular medium. The carrier-mediated nature of H(+)-Gly-L-Pro cotransport was assessed, and its concentration that yielded one-half maximal Gly-L-Pro influx was approximately 1.30 mM when measured by either radioactive or fluorescent tracers. Different dipeptides strongly inhibited Gly-L-Pro uptake by eel intestinal BBMV, as well as the cephalosporin antibiotic cephalexin, suggesting that dipeptide molecules and cephalosporin antibiotics may share a common transport system in eel intestinal BBMV.

Thyroid hormone stimulation of Na/Pi-cotransport in opossum kidney cells.

Thyroid hormone (T3), a known stimulator of renal proximal tubular brush border membrane Na-dependent phosphate (Pi) uptake (Na/Pi-cotransport), stimulated Na-dependent Pi transport in opossum kidney (OK) cells. Na/Pi-cotransport was stimulated in a time- and dose-dependent manner with maximal effects (57%) at 24 h and 10(-10) M T3. This stimulation was related to an increase in the apparent capacity (Vmax) of Na/Pi-cotransport. Treatment with T3 had no effect on Na-independent transport of Pi or of L-arginine. The stimulation of Na/Pi-cotransport was paralleled by an increase in the messenger ribonucleic acid (mRNA) encoding the OK cell apical Na/Pi-cotransporter (termed NaPi-4); the mRNA levels related to the activity of Na-independent L-arginine transport (rBAT) were unaffected by T3. Actinomycin D (10(-7) M) completely prevented the stimulatory effect of T3 on OK cell Na/Pi-cotransport and on NaPi-4 mRNA content. In conclusion, T3 stimulates apical Na/Pi-cotransport in OK cells most likely by enhancing its transcription.

Regulation of opossum kidney (OK) cell Na/Pi cotransport by Pi deprivation involves mRNA stability.

Renal proximal tubular Na-dependent phosphate transport (Na/Pi cotransport) has been studied extensively in the opossum kidney (OK) cell line. Recently, we cloned a complementary deoxyribonucleic acid (cDNA) (NaPi-4) from OK cells encoding an apical NaPi cotransport system. OK cells exposed to a low-Pi medium, as compared to high-Pi media, responded with an increase in Na/Pi cotransport, which was followed by an increase in NaPi-4 messenger ribonucleic acid (mRNA) abundance; maximal stimulation of Na/Pi cotransport was reached in 2 h, with no further increase for up to 16 h. NAPi-4 mRNA abundance was unaltered for 2 h, then increased to a maximum after 6-16 h in cells treated with low Pi medium. NaPi-4 mRNA decay rate was lowered by low-Pi media when compared to high-Pi media, with no increase in the NaPi-4 mRNA transcription rate. These data suggest that the upregulation of Na/Pi cotransport in OK cells by low-Pi media involves two regulatory mechanisms: an immediate (early) increase (after 2 h) in the expression of Na/Pi cotransport, independent of mRNA synthesis or stability, and a delayed (late) effect (after 4-6 h), resulting in an increase in NaPi-4 mRNA abundance, due to an increased stability.

Cloning of a rabbit renal Na-Pi cotransporter, which is regulated by dietary phosphate.

Previously, we isolated a cDNA (NaPi-1) related to a rabbit renal proximal tubular Na-Pi cotransporter (A. Werner, M.L. Moore, N. Mantei, J. Biber, G. Semenza, and H. Murer. Proc. Natl. Acad. Sci. USA 88:9608-9612, 1991.). In this study, we isolated an additional (rabbit renal) cDNA (NaPi-6), which induces Na-dependent Pi uptake in Xenopus laevis oocytes. Substrate specificity and kinetic properties corresponded to those known for rabbit renal brush-border membrane (BBM) Na-Pi cotransport. NaPi-6 was cloned by homology using NaPi-2 cDNA, a rat renal BBM Na-Pi cotransporter (S. Magagnin, A. Werner, D. Markovich, V. Sorribas, G. Stange, J. Biber, and H. Murer. Proc. Natl. Acad. Sci. USA 90: 5979-5983, 1993). NaPi-6 encodes a protein of 642 amino acids, exhibiting at least eight transmembrane domains. NaPi-6 mRNA and protein in kidneys of rabbits fed a low-Pi diet (LPD; 0.11% Pi) for 1 wk were increased by 1.5- and 4-fold, respectively, compared with those of rabbits fed a high-Pi diet (HPD; 1.20% Pi). This effect was correlated with an increase in Na-Pi cotransport of BBM vesicles isolated from animals adapted to LPD (2.5-fold with respect to HPD). In contrast, NaPi-1 mRNA and protein were not altered in response to LPD. Thus rabbit proximal tubular BBMs contain two different Na-Pi cotransport systems: NaPi-1 (type I) and NaPi-6 (type II). Only the type II transport system seems to be under regulatory control in response to low-Pi dietary intake.

Characterization of plasma membrane Na+/H+ exchange in eel (Anguilla anguilla) intestinal epithelial cells.

The ability of eel intestinal epithelial cells to recover from an acute acid load was analysed using the fluorescent dye 2',7'-bis-carboxy-ethyl-5,6-carboxyfluorescein (BCECF) and cell suspensions. Under these experimental conditions (bicarbonate-free solutions) the resting pHi in cells prepared from sea-water (7.52 +/- 0.031) and fresh-water (7.50 +/- 0.094) adapted animals proved to be similar. The recovery rate (following an acid load) increases by increasing the Na ion concentration in the extracellular medium. This pHi recovery is competitively inhibited by the specific inhibitor dimethylamiloride (DMA) with a low Ki in sea- (1.2 microM) as well as in fresh-water (1.3 microM) adapted animals, indicating the presence of a specific Na/H exchange activity in these cells. Using basolateral membrane vesicles it could be demonstrated that this activity is located on the basolateral side of the enterocyte membrane. The kinetic parameters (Kapp and Jmax) of this exchanger are similar in fresh-water and sea-water adapted animals suggesting that no salinity adaptation occurs, thus excluding the involvement of the antiporter in the osmoregulatory processes. These results are in agreement with the presence in the plasma membrane of the eel enterocytes of a Na/H-1 (housekeeper) form of the antiporter.

cDNA cloning of a rat small-intestinal Na+/SO4(2-) cotransporter.

We have isolated a cDNA (ileal NaSi-1) from rat small intestine by homology screening with a cDNA (renal NaSi-1) encoding rat kidney cortex Na(+)-SO4(2-) cotransport. Ileal NaSi-1 cRNA specifically stimulates Na(+)-dependent SO4(2-) uptake in a time- and dose-dependent manner in Xenopus laevis oocytes, with kinetic parameters almost identical to those of the renal NaSi-1. Ileal NaSi-1 cDNA contains 2722 base pairs (bp), almost 500 bp more than the renal NaSi-1 cDNA; however, it encodes a protein of 595 amino acids identical to the renal NaSi-1 protein. Northern blot analysis shows strong signals in rat lower small intestine and kidney cortex (2.9 x 10(3) and 2.3 x 10(3) bases), with the ileal NaSi-1 corresponding to the longer transcript. We conclude that we have identified a rat ileal cDNA that encodes a membrane protein most likely involved in brush-border Na(+)-SO4(2-) cotransport. It differs to the renal NaSi-1 only in the length of the 3' untranslated region, suggesting that the major difference lies in the differential use of polyadenylation signals.

Expression of rat ileal Na(+)-sulphate cotransport in Xenopus laevis oocytes: functional characterization.

Small-intestinal sulphate absorption is a Na(+)-dependent process having its highest rate in the ileum; it involves brush-border membrane Na(+)-sulphate cotransport. Injection of rat ileal mRNA into Xenopus laevis oocytes induced Na(+)-dependent sulphate uptake in a dose-dependent manner, with no apparent effect on Na(+)-independent sulphate uptake. For mRNA-induced transport, the apparent Km value for sulphate interaction was 0.6 +/- 0.2 mM and that for sodium interaction was 25 +/- 2 mM (Hill coefficient: 2.3 +/- 0.3). mRNA-induced transport, was inhibited by thiosulphate, but not by phosphate or 4,4,'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). Using a rat renal Na(+)-sulphate cotransporter cDNA as a probe [NaSi-1; Markovich et al. (1993) Proc Natl Acad Sci USA 90:8073-8077], the highest hybridization signals (2.3 kb and 2.9 kb) were obtained in size fractions showing the highest expression of Na(+)-dependent sulphate transport in oocytes. Hybrid depletion experiments using antisense oligonucleotides (from the NaSi-1 cDNA sequence), provided further evidence that rat small-intestinal (ileal) Na(+)-sulphate cotransport is closely related to rat proximal-tubular brush-border membrane Na(+)-sulphate cotransport.

Angiotensin II receptor subtypes in eel (Anguilla anguilla).

Previous studies have shown the effects of angiotensin II (Ang II) in teleosts, and Ang II-binding sites have also been localized in tissues from rainbow trout. The purpose of this study was to extend these findings and to provide an analysis of Ang II receptor (Ang II-R) isoforms in three tissues obtained from European eel (Anguilla anguilla). Ang II-Rs were identified in eel liver, kidney and intestine membranes by the binding of either 0.5 nmol human 125I-labelled Tyr4-Ile5-Ang II/l or increasing concentrations (1-120 nmol/l) of [3,5-3H]Tyr4-Ile5-Ang II. Using an isoelectric focusing technique, two Ang II-binding sites were identified in liver membranes. These migrated to isoelectric points (pI values) 6.5 and 6.7. Seventy per cent of binding to both sites was displaced by a 10,000-fold excess of unlabelled human Ang II. In both whole plasma membranes and brush border membranes from intestine, only one form of the Ang II-R was found, with pI 6.5 and high affinity (Kd = 3.4 nmol/l) for the [3,5-3H]Tyr4-Ile5-Ang II. Similarly, only the isoform focusing at pI 6.5 was observed in renal tubular epithelial brush border membranes. Reduction of disulphide bridges with dithiothreitol significantly enhanced Ang II binding to the isoform at pI 6.5 in liver (P < 0.05) and kidney (P < 0.01), while in liver the binding to the isoform of pI 6.7 was significantly reduced (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

H+/glycyl-glycine cotransport in eel intestinal brush-border membrane vesicles: studies with the pH-sensitive dye Acridine orange.

Monitoring the fluorescence quenching of the pH-sensitive dye Acridine orange, proton accumulation in the presence of an inside-negative transmembrane potential was measured in eel (Anguilla anguilla) intestinal brush-border membrane vesicles. It was demonstrated that the proton accumulation was specifically increased by the presence of the dipeptide glycyl-glycine in the extravesicular space, showing saturation kinetics at increasing dipeptide concentrations and was specifically inhibited by diethylpyrocarbonate. Data reported suggest the presence of an electrical-potential-dependent H+/glycyl-glycine cotransport system in the eel intestinal brush-border membrane vesicles.