Biomedical subjects

T R Kleyman

Publications and source records attributed to T R Kleyman.

At least 37 records · Page 2Linked to original sources

PubMed1997

Expression of the cystic fibrosis phenotype in a renal amphibian epithelial cell line.

Mutations in a Cl- channel (cystic fibrosis transmembrane conductance regulator or CFTR) are responsible for the cystic fibrosis (CF) phenotype. Increased Na+ transport rates are observed in CF airway epithelium, and recent studies suggest that this is due to an increase in Na+ channel open probability (Po). The Xenopus renal epithelial cell line, A6, expresses both cAMP-activated 8-picosiemen (pS) Cl- channels and amiloride-sensitive 4-pS Na+ channels, and provides a model system for examining the interactions of CFTR and epithelial Na+ channels. A6 cells express CFTR mRNA, as demonstrated by reverse transcriptase-polymerase chain reaction and partial sequence analysis. A phosphorothioate antisense oligonucleotide, complementary to the 5' end of the open reading frame of Xenopus CFTR, was used to inhibit functional expression of CFTR in A6 cells. Parallel studies utilized the corresponding sense oligonucleotide as a control. CFTR protein expression was markedly reduced in cells incubated with the antisense oligonucleotide. Incubation of A6 cells with the antisense oligonucleotide led to inhibition of forskolin-activated amiloride-insensitive short circuit current (Isc). After a 30-min exposure to 10 microM forskolin, 8-pS Cl- channel activity was detected in only 1 of 31 (3%) cell-attached patches on cells treated with antisense oligonucleotide, compared to 5 of 19 (26%) patches from control cells. A shift in the single-channel current-voltage relationship derived from antisense-treated cells was also consistent with a reduction in Cl- reabsorption. Both amiloride-sensitive Isc and Na+ channel Po were significantly increased in antisense-treated, forskolin-stimulated A6 cells, when compared with forskolin-stimulated controls. These data suggest that the regulation of Na+ channels by CFTR is not limited to respiratory epithelia and to epithelial cells in culture overexpressing CFTR and epithelial Na+ channels.

Amiloride↗

PubMed1997

An aldosterone regulated chicken intestine protein with high affinity to amiloride.

The pattern of chicken intestine amiloride-binding proteins was determined using the photoreactive amiloride analogue 2'-methoxy-5'-nitrobenzamil (NMBA) and a polyclonal anti-amiloride antibody. At 10(-7)M, NMBA inhibits approximately 62% of the Na+ channel activity. At this concentration the amiloride analogue labels a number of membrane proteins, and in particular a 40-45 kDa polypeptide denoted ABP40. Incorporation of NMBA into ABP40 could be prevented by a 100-fold excess of benzamil, but not by a 1000-fold excess of 5-(N-ethyl-N-isopropyl)-amiloride. Labeling of ABP40 was intense in membranes derived from salt-deprived chickens and approximately 5-fold weaker in membranes from salt-repleted animals. Because of its small size, ABP40 is not likely to be an avian Na+ channel subunit, yet this amiloride-binding protein could be involved in the response to aldosterone.

Aldosterone↗

PubMed1997

Delivery of newly synthesized Na(+)-K(+)-ATPase to the plasma membrane of A6 epithelia.

Na(+)-K(+)-ATPase is localized to the basolateral cell surface of most epithelial cells. Conflicting results regarding the intracellular trafficking of Na(+)-K(+)-ATPase in Madin-Darby canine kidney cells have been reported, with delivery to both apical and basolateral membranes or exclusively to the basolateral cell surface. We examined the delivery and steady-state distribution of Na(+)-K(+)-ATPase in the amphibian epithelial cell line A6 using an antibody raised against Na(+)-K(+)-ATPase alpha-subunit and sulfo-N-hydroxysuccinimidobiotin to tag cell surface proteins. The steady-state distribution of the Na(+)-K(+)-ATPase was basolateral, as confirmed by immunocytochemistry. Delivery of newly synthesized Na(+)-K(+)-ATPase to the cell surface was examined using [35S]methionine and [35S]cysteine in a pulse-chase protocol. After a 20-min pulse, the alpha-subunit and core glycosylated beta-subunit were present at both apical and basolateral cell surfaces. The alpha-subunit and core glycosylated beta-subunit delivered to the apical cell surface were degraded within 2 h. Mature alpha/beta-heterodimer was found almost exclusively at the basolateral surface after a 1- to 24-h chase. These data suggest that immature Na(+)-K(+)-ATPase alpha-subunit and core glycosylated beta-subunits are not retained in the endoplasmic reticulum of A6 cells and apparently lack sorting signals. Mature Na(+)-K(+)-ATPase is targeted to the basolateral surface, suggesting that basolateral targeting of the protein is conformation dependent.

Animals↗

PubMed1996

Renal epithelial protein (Apx) is an actin cytoskeleton-regulated Na+ channel.

Apx, the amphibian protein associated with renal amiloride-sensitive Na+ channel activity and with properties consistent with the pore-forming 150-kDa subunit of an epithelial Na+ channel complex initially purified by Benos et al. (Benos, D. J., Saccomani, G., and Sariban-Sohraby, S.(1987) J. Biol. Chem. 262, 10613-10618), has previously failed to generate amiloride-sensitive Na+ currents (Staub, O., Verrey, F., Kleyman, T. R., Benos, D. J., Rossier, B. C., and Kraehenbuhl, J.-P.(1992) J. Cell Biol. 119, 1497-1506). Renal epithelial Na+ channel activity is tonically inhibited by endogenous actin filaments (Cantiello, H. F., Stow, J., Prat, A. G., and Ausiello, D. A.(1991) Am. J. Physiol. 261, C882-C888). Thus, Apx was expressed and its function examined in human melanoma cells with a defective actin-based cytoskeleton. Apx-transfection was associated with a 60-900% increase in amiloride-sensitive (Ki = 3 microM) Na+ currents. Single channel Na+ currents had a similar functional fingerprint to the vasopressin-sensitive, and actin-regulated epithelial Na+ channel of A6 cells, including a 6-7 pS single channel conductance and a perm-selectivity of Na+:K+ of 4:1. Na+ channel activity was either spontaneous, or induced by addition of actin or protein kinase A plus ATP to the bathing solution of excised inside-out patches. Therefore, Apx may be responsible for the ionic conductance involved in the vasopressin-activated Na+ reabsorption in the amphibian kidney.

Actins↗

PubMed1996

Photolabile amiloride derivatives as cation site probes of the Na,K-ATPase.

Treatment of purified canine renal Na,K-ATPase with a range of photoactivatable amiloride derivatives results in inhibition of ATPase activity prior to illumination. Inhibition by amiloride derivatives substituted on a guanidium N could not be prevented by the presence of either K or Na; however, these cations could protect the enzyme against inhibition by derivatives substituted on the 5-position of the pyrazine ring. In the case of 5-(N-ethyl-[2'-methoxy-4'-nitrobenzyl])amiloride (NENMBA), the presence of monovalent cations (Na, K, and Rb) protected the enzyme effectively against inhibition, with concentrations in the millimolar range. ATP did not prevent inhibition; furthermore, native and NENMBA-treated enzyme exhibited normal levels of high affinity [3H]ADP (and hence ATP) binding. The rate of inhibition increased with increasing concentrations of NENMBA. Extensive washing of NENMBA-inhibited enzyme did not restore ATPase activity, showing that NENMBA has an extremely slow off-rate for dissociation from its inhibitory site. Partially inhibited enzyme could be rapidly pelleted and resuspended in NENMBA-free buffer and inhibition was observed to continue, albeit at a somewhat diminished rate, suggesting that NENMBA gains access to its inhibitory site after partitioning into the lipid phase rather than directly from the aqueous solution. Photolysis of NENMBA-inhibited enzyme resulted in covalent incorporation of the reagent into the alpha-subunit of the Na,K-ATPase, as observed by separation of labeled protein on a Laemmli gel and Western analysis using a polyclonal amiloride antibody. Almost all of the covalent labeling could be prevented by the presence of Rb in the incubation and labeling medium. These results suggest that NENMBA inhibits the Na, K-ATPase by disruption of the cation transport domain rather than the catalytic domain of the enzyme and that it promises to be a useful tool for cation site localization.

Affinity Labels↗

PubMed1996

The chemokine C10: immunological and functional analysis of the sequence encoded by the novel second exon.

The gene for C10, a member of the beta-chemokine family of cytokines, contains a novel second exon encoding a 16 amino acid sequence which is inserted into the amino-terminal region of the protein. We propose a model of C10 structure in which the sequence encoded by the second exon is located on the surface of the protein where it interacts with receptors and defines the predominant epitope of the C10 protein. To test this model we produced C10 protein without the sequence encoded by the second exon, termed C10(-). Antisera generated against purified C10 recognized C10 protein but reacted only weakly with C10(-) protein. Recognition of C10 protein by C10 antisera was not blocked by pre-incubation with a peptide encoding the C10 second exon, indicating that the predominant epitope was not simply the amino acid sequence encoded by the second exon. Antibodies generated against a peptide encoding the C10 second exon sequence recognized C10 on Western blots; this reaction was competed both by C10 second exon peptide and by C10 protein. Functional studies demonstrated that both C10/FLAG and C10(-)/FLAG elicit chemotaxis of mouse PECs and human PBMCs, but that C10(-)/FLAG appears to be more potent than C10/FLAG. Thus these data are consistent with our model of C10 in which the sequence encoded by the second exon is located on the surface, participates in defining the predominant epitope, and plays a key role in recognizing or activating chemokine receptors.

Animals↗

PubMed1996

Antikaliuretic action of trimethoprim is minimized by raising urine pH.

This study was designed to test the hypothesis that the antikaliuresis caused by trimethoprim could be diminished by alkalinizing the luminal fluid in the CCD, thereby converting trimethoprim from its cationic, active form to an electroneutral, inactive, form. Trimethoprim-induced inhibition of transepithelial Na+ transport was examined in A6 distal nephron cells by analysis of short circuit current. The voltage-dependence of the trimethoprim-induced block of Na+ channels was examined with patch clamp recordings of A6 cells. The antikaliuretic effect of trimethoprim was examined in vivo in rats pretreated with deoxycorticosterone and with NH4Cl to lower urine pH, and in rats also receiving acetazolamide to raise urine pH. We found that the concentration of trimethoprim required to inhibit the amiloride sensitive component of short circuit current by 50% (IC50) was 340 microM (at pH 8.2) and 50 microM (at pH 6.3). The IC50S of protonated trimethoprim were similar (34 microM at pH 8.2 and 45 microM at pH 6.3). The mean time open for the high selectivity, Na+ channel was reduced from 1679 +/- 387 msec to 502 +/- 98 msec with addition of 10-5 M trimethoprim to patch pipette solution at the resting membrane potential (-Vpipette = 0 mV). further decreases in mean time open were observed as -Vpipette was reduced (that is, apical membrane hyperpolarization) to -40 mV (mean time open = 217 +/- 85 msec) and to -80 mV (mean time open = 69 +/- 13 msec). In vivo, trimethoprim caused a > 50% reduction in potassium (K+) excretion due primarily to a fall in the [K+] in the lumen of the terminal CCD. This effect of trimethoprim was markedly attenuated in an alkaline urine induced by acetazolamide. We conclude that it is the charged, protonated species of trimethoprim which blocks epithelial Na+ channels. Increasing urinary pH decreases the concentration of the charged species of trimethoprim and minimizes its antikaliuretic effect.

Animals↗

PubMed1996

Lupus autoantibodies interact directly with distinct glomerular and vascular cell surface antigens.

We have identified monoclonal anti-DNA antibodies derived from lupus prone MRL-lpr/lpr mice that produce glomerular immune deposits and nephritis after passive transfer to normal mice. Particularly noteworthy is that the location of immune deposition varied among nephritogenic Ig, and this was associated with distinctive histologies and clinical disease profiles. Although their autoantigen binding properties differed, they were highly cross-reactive, in a manner similar to Ig deposited in glomeruli of lupus mice. This antigen binding profile was also typical of other previously described nephritogenic autoantibodies that bound directly to glomerular antigens to initiate immune deposit formation. In this study, we questioned whether ligation of different glomerular antigens by individual autoantibodies could contribute to the observed differences in the location of immune deposits. To examine this possibility, monoclonal anti-DNA antibodies (IgG2a) that produced glomerular immune deposits in different locations were evaluated. H221 produced mesangial, intracapillary (that is, intraluminal or within the capillary lumen) and subendothelial deposits associated with heavy proteinuria, whereas H147 produced mesangial, subendothelial and linear basement membrane deposits associated with proliferative glomerulonephritis. Initially, the capacity of H221 and H147 to bind directly to glomerular and vascular cell surfaces was evaluated. As demonstrated by FACS, H221 bound preferentially to mesangial cells whereas H147 bound preferentially to endothelial cells. To identify possible target cell surface antigens, Western blots, immunoprecipitation of surface labeled cells, and 2D gel electrophoresis were employed. H221 reacted with a 108 kDa protein on mesangial cells not identified by H147, whereas H147 reacted with a 45 kDa protein on endothelial cells not identified by H221. These results support the hypothesis that some nephritogenic lupus autoantibodies initiate immune deposit formation through direct interaction with glomerular antigens. Furthermore, they suggest that the site of immune deposition is determined by both antigen binding properties of the relevant antibody and the location of its target ligand within the glomerulus. In a given individual, therefore, the predominant autoantibody-glomerular antigen interaction may influence the morphologic and clinical phenotype expressed. Variation in the predominant interaction may also contribute to variations in disease expression among individuals with lupus nephritis.

Animals↗

PubMed1996

Biosynthesis and cell surface delivery of the NHE1 isoform of Na+/H+ exchanger in A6 cells.

The Na+/H+ exchanger isoform NHE1 is localized to the basolateral membrane of renal and intestinal epithelia. We examined the plasma membrane distribution, biosynthesis, and cell surface delivery of NHE1 in A6 epithelia. NHE1 was localized to the basolateral membrane. Studies of NHE1 biosynthesis with a pulse-chase protocol demonstrated that a core glycosylated, endoglycosidase H-sensitive, 90-kDa NHE1 was present 0-5 h into the chase period and that mature 110-kDa NHE1 was present 1-24 h into the chase period. Studies of plasma membrane delivery of newly synthesized NHE1 demonstrated that the 90-kDa NHE1 was detected at both apical and basolateral membranes 2-5 h into the chase period. The 110-kDa NHE1 was observed at the basolateral membrane 5-24 h into the chase period. These results suggest that NHE1 is expressed primarily at the basolateral membrane of A6 cells, that core glycosylated NHE1 is delivered to the plasma membrane in a nonpolarized manner, and that nature 110-kDa NHE1 is delivered to the basolateral membrane.

Animals↗

PubMed1995

Epithelial sodium channel genes Scnn1b and Scnn1g are closely linked on distal mouse chromosome 7.

The chromosomal localizations of Scnn1b and Scnn1g, genes corresponding to the beta- and gamma-subunits, respectively, of an epithelial non-voltage-gated amiloride-sensitive sodium channel, were determined by analyses of two sets of multilocus crosses using probes generated by polymerase chain reaction and a mouse kidney cortical collecting tubule cell line (M1). Scnn1b and Scnn1g were determined to be closely linked on distal mouse chromosome 7, showing no recombination with Zp2, whereas the gene for the alpha-subunit, Scnn1a, was confirmed to map to distal mouse chromosome 6.

Amino Acid Sequence↗

PubMed1995

A mechanism for pentamidine-induced hyperkalemia: inhibition of distal nephron sodium transport.

OBJECTIVES: To determine whether pentamidine directly affects the transport of renal ions and thus provides a mechanism for hyperkalemia, which develops in as many as 100% of patients with the acquired immunodeficiency syndrome (AIDS) who receive pentamidine for more than 6 days. DESIGN: Transepithelial and single-channel electrical measurements were made on two models of distal-nephron ion transport: an amphibian distal-nephron cell line (A6) and primary cultures of rabbit cortical collecting tubules. RESULTS: Luminal bath application of pentamidine to A6 monolayers inhibited the amiloride-sensitive, short-circuit current with a 50% inhibitory concentration of 700 microM (five experiments). In the principal cell apical membranes of cortical collecting tubule primary cultures, amiloride-sensitive, 4-picosiemen Na+ channels in cell-attached patches were also identified. When the luminal membrane was directly exposed to 1.0 microM of pentamidine in the patch pipette solution, channel activity decreased by 40% (11 experiments). Channel inhibition rapidly reversed with washout of intrapipette pentamidine (four experiments). In contrast, replacement of either the luminal bath outside the patch pipette (four experiments) or the serosal bath (five experiments) with pentamidine did not significantly affect Na+ channel activity in the patches. CONCLUSIONS: Luminal or "urinary" pentamidine inhibits distal nephron reabsorption of Na+ by blocking apical Na+ channels in a manner similar to "potassium-sparing" diuretics (for example, amiloride and triamterene). This results in a decrease in the electrochemical gradients that drive secretion of distal nephron K+. Because pentamidine is eliminated through urinary excretion, this renal tubular effect provides a mechanism for pentamidine-induced hyperkalemia.

Animals↗

PubMed1995

Defining topological similarities among ion transport proteins with anti-amiloride antibodies.

The structural features of amiloride binding sites on amiloride-sensitive transport proteins have received limited characterization. An antibody that recognizes limited regions of amiloride and can mimic, in binding specificity, certain amiloride-sensitive transport proteins was used as a model to elucidate potential amino acid residue relationships that might define putative amiloride contact sites. Analysis of the structure of this antibody has allowed us to identify sequence relationships among several Na+ selective transport proteins. A structure-based relational database was employed to re-examine sequence homologies among these ion transport proteins. A search of the protein sequence databank identified representative amino acid tracts among amiloride sensitive proteins involving planar residues that might be involved in interacting with amiloride. Computer models of sites within transmembrane domains of NHE1 and NHE2 isoforms of the Na+/H+ exchanger reflective of these planar tracts indicate that amiloride probably spans two helices for interaction with the Na+/H+ exchanger. Structural analysis of this monoclonal anti-amiloride antibody appears to mimic some of the salient features of amiloride binding sites on these proteins.

Amiloride↗

PubMed1994

Utilization of amiloride analogs for characterization and labeling of the plasma membrane Na+/H+ antiporter from Dunaliella salina.

The interactions of amiloride analogs with the Na+/H+ antiporter from plasma membrane of the halotolerant alga Dunaliella salina [Katz et al. (1989) Biochem. Biophys. Acta 983, 9-14] have been investigated. Analogs bearing hydrophobic substitutions at the guanidino moiety of amiloride, such as benzamil, are the most effective inhibitors of Na+ uptake in plasma membrane vesicles, whereas substituents of the 5-amino group are less effective inhibitors than amiloride. This order of specificity is opposite to that found for most Na+/H+ antiporters. The photoaffinity amiloride analog 2'-methoxy-5'-nitrobenzamil (NMBA), a competitive inhibitor with respect to Na+ with Ki = 10 microM, photolabels upon illumination two polypeptides of apparent MW 30 and 50 kDa in purified plasma membrane vesicles. Similar labeling is obtained by immunodetection with antiamiloride antibodies and by incorporation of [125I]NMBA. The specificity of the labeling was ascertained by competition with benzamil. Plasma membrane preparations from high-salt or ammonia-adapted cells, which have higher Na+/H+ antiporter activity [Katz et al. (1992) Plant Physiol. 100, 1224-1229], also show increased incorporation of NMBA into the 30- and 50-kDa polypeptides. It is suggested that: (1) the structure of the Na+ binding site of the D. salina Na+/H+ antiporter differs from that of most Na+/H+ antiporters and (2) the 50- and/or 30-kDa polypeptides are subunits of the plasma membrane antiporter of this alga.

Amiloride↗

PubMed1994

Topology of an amiloride-binding protein.

Amiloride and structurally related compounds inhibit many transport proteins, enzymes, and drug or hormone receptors, although the topology of amiloride binding sites on these proteins has not been defined. We have previously raised and characterized a monoclonal antiamiloride antibody (mAb BA7.1) which is similar to epithelial Na+ channels in its specificity of binding of amiloride and amiloride analogs, suggesting that their amiloride binding sites may be similar in topology, mAb BA7.1 was used as a model system to analyze the three-dimensional conformation of an amiloride binding site. The photoactive amiloride analog 2'-methoxy-5'-nitrobenzamil specifically labeled the heavy chain of mAb BA7.1, suggesting that the heavy chain participates in amiloride binding. The nucleotide sequences of the variable regions of the heavy and light chains of mAb BA7.1 were determined and amino acid sequences deduced to analyze the structure of the amiloride binding site. A comparative modeling approach was used to construct a model of the amiloride binding domain of mAb BA7.1, and a docking procedure was used to place amiloride within this domain. The model indicated that planar aromatic amino acid resides form a pocket into which amiloride, a planar molecule, inserts. Constraints on amiloride binding predicted by this model correlated with the measured specificity of binding of amiloride analogs with mAb BA7.1. These results provide a potential guide for the identification of motifs or amino acid contact residues present within other amiloride-sensitive proteins.

Amiloride↗

PubMed1994

K(+)-sparing diuretic actions of trimethoprim: inhibition of Na+ channels in A6 distal nephron cells.

Hyperkalemia complicates trimethoprim-sulfamethoxazole (TMP-SMX) therapy in over 20% of HIV-infected patients. TMP is a heterocyclic weak base, similar to amiloride, a "K(+)-sparing" diuretic and Na+ channel blocker. Apical TMP is known to inhibit amiloride-sensitive short circuit current in A6 cells, a tissue culture model for mammalian cortical collecting tubule principal cells [1]. We used cell-attached patch clamp techniques to investigate the effect of TMP on the 4 pS, highly selective Na+ channel in the apical membrane of A6 cells grown on permeable supports in the presence of 1.5 microM aldosterone. Baseline channel activity at resting membrane potential, measured as NPo (N of channels x open probability), was 1.09 +/- 0.50 (N = 18). NPo (0.92 +/- 0.38; N = 9) was unchanged when 10(-3) M TMP was added to the basolateral bath for 30 minutes. However, apical exposure with pipettes containing 10(-3) or 10(-5) M TMP reduced NPo approximately tenfold (0.12 +/- 0.08; N = 7 and 0.18 +/- 0.14; N = 12, respectively). Kinetic analysis revealed the appearance of a new closed state after apical TMP treatment. Another group of A6 cells were pretreated with 10(-3) M apical TMP for 30 minutes prior to patching with pipettes filled with TMP-free saline. NPo progressively rose from 0.07 +/- 0.09 to 0.87 +/- 0.23 (N = 5) as the residual TMP was diluted within the pipette. Apical or basolateral pretreatment (30 min) with 10(-3) M SMX did not change Na+ channel activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Characterization and localization of epithelial Na+ channels in toad urinary bladder.

The toad urinary bladder and epithelial cell lines derived from the urinary bladder, including TBM, serve as model systems for the study of transepithelial Na+ transport. We examined biochemical characteristics of epithelial Na+ channels in toad urinary bladder and TBM cells and their cellular localization in the urinary bladder. The radiolabeled amiloride analogue [3H]benzamil bound to a single class of high-affinity binding sites in membrane vesicles from toad urinary bladder with a dissociation constant (Kd) of 10 nM. Photoactive benzamil analogues specifically labeled a 135,000-Da polypeptide in toad urinary bladder and TBM cells. A monoclonal anti-Na+ channel antibody directed against the amiloride-binding component of the channel specifically recognized a 135,000-Da polypeptide in TBM cells. Polyclonal anti-Na+ channel antibodies generated against purified bovine epithelial Na+ channel specifically recognized a 235,000-Da polypeptide in toad urinary bladder and localized Na+ channels to the apical plasma membrane of urinary bladder epithelial cells. The biochemical characteristics and the cellular localization of epithelial Na+ channels in toad urinary bladder are similar to those previously described in mammalian kidney and in the A6 cell line.

Amiloride↗

PubMed1994

Anti-idiotypic antibodies to delineate epitope specificity of anti-amiloride antibodies.

Amiloride and related compounds have found widespread use as cation transport inhibitors. We have previously raised a series of polyclonal anti-amiloride antibodies using different amiloride-protein conjugates as immunogens, where amiloride was coupled to protein either through its guanidino moiety or through its 5-aminopyrazinyl moiety. The anti-amiloride antibodies recognized distinct sites on amiloride, and the site of attachment of amiloride to carrier protein was a critical factor in determining which part of the amiloride molecule was recognized by the anti-amiloride antibody. The specificity of binding of amiloride analogues to these polyclonal anti-amiloride antibodies mimicked the specificity of binding of amiloride analogues to selected isoforms of the epithelial Na+ channel or the Na+/H+ exchanger, suggesting that antigen binding site of these antibodies might be similar in structure to amiloride binding sites on selected Na+ transport proteins. We previously generated monoclonal anti-idiotypic antibodies RA2.4 and RA6.3 by an auto-anti-idiotypic approach, using amiloride coupled to albumin through the guanidinium moiety (amiloride-A1). We have now raised a series of monoclonal anti-idiotypic antibodies, T6, T26, T40, and T181, using amiloride coupled to keyhole limpet hemocyanin through the 5-aminopyrazinyl moiety (amiloride-A5) as an immunogen with the same auto-anti-idiotypic approach. These monoclonal anti-idiotypic antibodies recognized both polyclonal anti-amiloride-A1 and anti-amiloride-A5 antibodies, suggesting that idiotype-anti-idiotype interaction was not epitope restricted.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗

PubMed1994

Arginine vasopressin and forskolin regulate apical cell surface expression of epithelial Na+ channels in A6 cells.

Both arginine vasopressin (AVP) and forskolin regulate vectorial Na+ transport across high-resistance epithelia by increasing the Na+ conductance of the apical membrane mediated by amiloride-sensitive Na+ channels. Pretreatment of A6 cells with brefeldin A partially inhibited the increase in Na+ transport in response to forskolin, suggesting recruitment of Na+ channels from an intracellular pool. The activation of Cl- secretion was not affected. Apical cell surface expression of Na+ channels was examined following activation of transepithelial Na+ transport across the epithelial cell line A6 by AVP or forskolin. Apical cell surface radioiodinated Na+ channels were immunoprecipitated to quantify the biochemical pool of Na+ channels at the apical plasma membrane and to determine whether an increment in the biochemical pool of Na+ channels expressed at the apical cell surface is a potential mechanism by which AVP and forskolin increase apical membrane Na+ conductance. The activation of Na+ transport across A6 cells by AVP was accompanied by a significant increase in the biochemical pool of Na+ channels at the apical plasma membrane within 5 min after addition of hormone, which was sustained for at least 30 min. The increase in apical cell surface expression of Na+ channels was also observed 30 min after application of forskolin. No changes in the oligomeric subunit composition of the channel were noted. Brefeldin A inhibited the forskolin-stimulated increase in apical cell surface expression of Na+ channels. These results suggest that AVP and forskolin regulate Na+ transport, in part, via rapid recruitment of Na+ channels to the cell surface, perhaps from a pool of channels in the subapical cytoplasm.

Amiloride↗