Biomedical subjects

C M Fuller

Publications and source records attributed to C M Fuller.

At least 55 records · Page 3Linked to original sources

PubMed1996

Regulation of epithelial sodium channels by the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis airway epithelia exhibit enhanced Na+ reabsorption in parallel with diminished Cl- secretion. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) directly affects epithelial Na+ channel activity by co-incorporating into planar lipid bilayers immunopurified bovine tracheal CFTR and either heterologously expressed rat epithelial Na+ channel ( alpha,b eta,gamma-rENaC) or an immunopurified bovine renal Na+ channel protein complex. The single channel open probability (Po) of rENaC was decreased by 24% in the presence of CFTR. Protein kinase A (PKA) plus ATP activated CFTR, but did not have any effect on rENaC. CFTR also decreased the extent of elevation of the renal Na+ channel Po following PKA-mediated phosphorylation. Moreover, the presence of CFTR prohibited the inward rectification of the gating of this renal Na+ channel normally induced by PKA-mediated phosphorylation, thus down-regulating inward Na+ current. This interaction between CFTR and Na+ channels occurs independently of whether or not wild-type CFTR is conducting anions. However, the nonconductive CFTR mutant, G551D CFTR, cannot substitute for the wild-type molecule. Our results indicate that CFTR can directly down-regulate single Na+ channel activity, thus accounting, at least in part, for the observed differences in Na+ transport between normal and cystic fibrosis-affected airway epithelia.

Adenosine Triphosphate↗

PubMed1996

G-protein regulation of outwardly rectified epithelial chloride channels incorporated into planar bilayer membranes.

Experiments were designed to test if immunopurified outwardly rectified chloride channels (ORCCs) and the cystic fibrosis transmembrane conductance regulator (CFTR) incorporated into planar lipid bilayers are regulated by G-proteins. pertussis toxin (PTX) (100 ng/ml) + NAD (1 mM) + ATP (1 mM) treatment of ORCC and CFTR in bilayers resulted in a 2-fold increase in single channel open probability (Po) of ORCC but not of CFTR. Neither PTX, NAD, nor ATP alone affected the biophysical properties of either channel. Further, PTX conferred a linearity to the ORCC current-voltage curve, with a slope conductance of 80 +/- 3 picosiemens (pS) in the +/- 100 mV range of holding potentials. PKA-mediated phosphorylation of these PTX + NAD-treated channels further increased the Po of the linear 80-pS channels from 0.66 +/- 0.05 to >0.9, and revealed the presence of a small (16 +/- 2 pS) linear channel in the membrane. PTX treatment of a CFTR-immunodepleted protein preparation incorporated into bilayer membranes resulted in a similar increase in the Po of the larger conductance channel and restored PKA-sensitivity that was lost after CFTR immunodepletion. The addition of guanosine 5'-3-O-(thio)triphosphate (100 mum) to the cytoplasmic bathing solutions decreased the activity of the ORCC and increased its rectification at both negative and positive voltages. ADP-ribosylation of immunopurified material revealed the presence of a 41-kDa protein. These results demonstrate copurification of a channel-associated G-protein that is involved in the regulation of ORCC function.

Adenosine Diphosphate Ribose↗

PubMed1996

Triple-barrel organization of ENaC, a cloned epithelial Na+ channel.

A cloned rat epithelial Na+ channel (rENaC) was studied in planar lipid bilayers. Two forms of the channel were examined: channels produced by the alpha subunit alone and those formed by alpha, beta, and gamma subunits. The protein was derived from two sources: either from in vitro translation reaction followed by Sephadex column purification or from heterologous expression in Xenopus oocytes and isolation of plasma membranes. We found that either alpha-rENaC alone or alpha- in combination with beta- and gamma-rENaC, produced highly Na(+)-selective (PNa/PK = 10), amiloride-sensitive (Kamili = 170 nM), and mechanosensitive cation channels in planar bilayers. alpha-rENaC displayed a complicated gating mechanism: there was a nearly constitutively open 13-picosiemens (pS) state and a second 40-pS level that was achieved from the 13-pS level by a 26-pS transition. alpha-, beta-, gamma-rENaC showed primarily the 13-pS level. alpha-rENaC and alpha,beta,gamma-rENaC channels studied by patch clamp displayed the same gating pattern, albeit with > 2-fold lowered conductance levels, i.e. 6 and 18 pS, respectively. Upon treatment of either channel with the sulfhydryl reducing agent dithiothreitol, both channels fluctuated among three independent 13-pS sublevels. Bathing each channel with a high salt solution (1.5 M NaCl) produced stochastic openings of 19 and 38 pS in magnitude between all three conductance levels. Different combinations of alpha-, beta-, and gamma-rENaC in the reconstitution mixture did not produce channels of intermediate conductance levels. These findings suggest that functional ENaC is composed of three identical conducting elements and that their gating is concerted.

Animals↗

PubMed1996

Associated proteins and renal epithelial Na+ channel function.

The hypothesis that amiloride-sensitive Na+ channel complexes immunopurified from bovine renal papillary collecting tubules contain, as their core conduction component, an ENaC subunit, was tested by functional and immunological criteria. Disulfide bond reduction with dithiothreitol (DTT) of renal Na+ channels incorporated into planar lipid bilayers caused a reduction of single channel conductance from 40 pS to 13 pS, and uncoupled PKA regulation of this channel. The cation permeability sequence, as assessed from bi-ionic reversal potential measurements, and apparent amiloride equilibrium dissociation constant (K(amil)i) of the Na+ channels were unaltered by DTT treatment. Like ENaC, the DTT treated renal channel became mechanosensitive, and displayed a substantial decrease in K(amil)i following stretch (0.44 +/- 0.12 microM versus 6.9 +/- 1.0 microM). Moreover, stretch activation induced a loss in the channel's ability to discriminate between monovalent cations, and even allowed Ca2+ to permeate. Polyclonal antibodies generated against a fusion protein of alpha bENaC recognized a 70 kDa polypeptide component of the renal Na+ channel complex. These data suggest that ENaC is present in the immunopurified renal Na+ channel protein complex, and that PKA sensitivity is conferred by other associated proteins.

Amiloride↗

PubMed1996

Diversity and regulation of amiloride-sensitive Na+ channels.

Amiloride-sensitive Na+ channels play a vital role in many important physiological processes such as delineation of the final urine composition, sensory transduction, and whole-body Na+ homeostasis. These channels display a wide range of biophysical properties, and are regulated by cAMP-mediated second messenger systems. The first of these channels has recently been cloned. This cloned amiloride-sensitive Na+ channel is termined ENaC (Epithelial Na+ Channel) and, in heterologous cellular expression systems, displays a single channel conductance of 4 to 7 pS, a high PNa/PK (> 10), a high amiloride sensitivity (Ki(amil) = 150 nM), and relatively long open and closed times. ENaC may form the core conduction element of many of these functionally diverse forms of Na+ channel. The kinetic and regulatory differences between these channels may be due, in large measure, to unique polypeptides that associate with the core element, forming a functional channel unit.

Amiloride↗

PubMed1996

Protein kinase regulation of a cloned epithelial Na+ channel.

We examined the regulation of a cloned epithelial Na+ channel (alpha beta gamma-rENaC) by protein kinase A (PKA) and protein kinase C (PKC). Experiments were performed in Xenopus oocytes and in planar lipid bilayers. At a holding potential of -100 mV, amiloride-sensitive current averaged -1,279 +/- 111 nA (n = 7) in alpha beta gamma-rENaC-expressing oocytes. Currents in water-injected oocytes were essentially unresponsive to 10 microM amiloride. A 1-h stimulation of PKC with 100 nM of PMA inhibited whole-cell currents in Xenopus oocytes to 17.1 +/- 1.8, and 22.1 +/- 2.6% of control (n = 7), at holding potentials of -100 and +40 mV, respectively. Direct injection of purified PKC resulted in similar inhibition to that observed with PMA. Additionally, the inactive phorbol ester, phorbol-12-myristate-13-acetate, 4-O-methyl, was without effect on alpha beta gamma-rENaC currents. Pretreatment with the microtubule inhibitor colchicine (100 microM) did not modify the inhibitory effect of PMA; however, pretreatment with 20 microM cytochalasin B decreased the inhibitory action of PMA to < 20% of that previously observed. In vitro-synthesized alpha beta gamma-rENaC formed an amiloride-sensitive Na(+)-selective channel when incorporated into planar lipid bilayers. Addition of PKC, diacyl-glycerol, and Mg-ATP to the side opposite that which amiloride blocked, decreased the channel's open probability (Po) from 0.44 +/- 0.06 to 0.13 +/- 0.03 (n = 9). To study the effects of PKA on alpha beta gamma-rENaC expressed in Xenopus oocytes, cAMP levels were elevated with 10 microM forskolin and 1 mM isobutyl-methyl-xanthine. This cAMP-elevating cocktail did not cause any stimulation of alpha beta gamma-rENaC currents in either the inward or outward directions. This lack of activation was also observed in oocytes preinhibited with PMA and in oocytes pretreated with cytochalasin B and PMA. Neither alpha-rENaC nor alpha beta gamma-rENaC incorporated into planar lipid bilayers could be activated with PKA and Mg-ATP added to either side of the membrane, as Po remained at 0.63 +/- 0.06 (n = 7) and 0.45 +/- 0.05 (n = 9), respectively. We conclude that: alpha beta gamma-rENaC is inhibited by PKC, and that alpha beta gamma-rENaC is not activated by PKA.

Animals↗

PubMed1996

Liddle's disease: abnormal regulation of amiloride-sensitive Na+ channels by beta-subunit mutation.

Liddle's disease is an autosomal dominant genetic disorder characterized by severe low renin hypertension ("pseudoaldosteronism") that has been genetically linked to a locus on chromosome 16 encoding the beta-subunit of an amiloride-sensitive Na+ channel (ASSC) (15). Peripheral blood lymphocytes (PBL) express ASSC that are functionally indistinguishable from those expressed by Na(+)-reabsorbing renal epithelial cells (3, 5). The amiloride-sensitive Na+ conductance in PBL from affected and unaffected individuals from the original Liddle's pedigree was examined using whole cell patch clamp. Typically, the basal Na+ currents in cells from affected individuals were maximally activated. Basal Na+ currents in cells from unaffected individuals were minimal and could be maximally activated by superfusion with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Affected cells could not be further stimulated with CPT-cAMP. Superfusion with a supermaximal concentration of amiloride (2 microM) inhibited both the cAMP-activated Na+ conductance in unaffected cells and the constitutively activated inward conductance in affected cells. Cytosolic addition of a peptide identical to the terminal 10 amino acids of the truncated beta-subunit normalized the cAMP-mediated but not the pertussis toxin-induced regulation of the mutant ASSC. The findings show that lymphocyte ASSC are constitutively activated in affected individuals, that a mutation of the beta-subunit alters ASSC responsiveness to specific regulatory effectors, and that the cellular mechanism responsible for the pathophysiology of Liddle's disease is abnormal regulation of Na+ channel activity. These findings have important diagnostic and therapeutic implications and provide a cellular phenotype for the diagnosis of pseudoaldosteronism.

Amiloride↗

PubMed1996

Peptide block of constitutively activated Na+ channels in Liddle's disease.

Hypertension is a multifactorial disorder that results in an increased risk of cardiovascular and end-stage renal disease. Liddle's disease represents a specific hypertensive disease and expresses itself in the human population as an autosomal dominant trait. Recent experimental evidence indicates that patients with Liddle's disease have constitutively active amiloride-sensitive Na+ channels and that these channels are phenotypically expressed in lymphocytes obtained from normal and affected members of the original Liddle's kindred. Linkage analysis indicates that this disease results from a deletion of the carboxy-terminal region of the beta-subunit of a recently cloned epithelial Na+ channel (ENaC). We report the successful immunopurification and reconstitution of both normal and constitutively active lymphocyte Na+ channels into planar lipid bilayers. These channels display all of the characteristics typical of renal Na+ channels, including sensitivity to protein kinase A phosphorylation. We demonstrate that gating of normal Na+ channels is removed by cytoplasmic trypsin digestion and that the constitutively active Liddle's Na+ channels are blocked by a beta- or gamma-ENaC carboxy-terminal peptide in a GTP-dependent fashion.

Amiloride↗

PubMed1996

Apical recruitment of CFTR in T-84 cells is dependent on cAMP and microtubules but not Ca2+ or microfilaments.

Previous studies from this laboratory have demonstrated that chloride transport induced by forskolin, but not ionomycin, in T84 cells is highly dependent on an intact microtubular network. Using an antibody raised against a region of the R domain of CFTR, we now show by indirect immunofluorescence that forskolin causes relocation of CFTR to the apical domain of T84 cells. T84 cells grown on transparent filters were incubated with agonists and/or cytoskeletal inhibitors prior to fixation, permeabilization, and staining with the antibody. A 30 second stimulation with forskolin (10 microM) caused a twofold increase in relative fluorescence intensity at the apical surface. In contrast, a 30 second exposure to ionomycin (2 microM), had no effect on the distribution of CFTR-related fluorescence. Incubation of the cells with nocodazole (33 microM), a microtubule disrupting agent, prevented the forskolin-induced rise in CFTR fluorescence at the apical surface. However, incubation of the cells with cytochalasin D, an actin inhibitor, was without effect on forskolin-related re-distribution of CFTR-associated fluorescence. In double label experiments using antibodies against both beta-tubulin and actin, CFTR-related fluorescence was found to co-localize with the microtubule network, but not with actin filaments. These observations are consistent with the microtubule-dependent acute recruitment of CFTR to the apical plasma membrane of T84 cells in response to elevations in intracellular cAMP.

Actin Cytoskeleton↗

PubMed1995

Cloning of an epithelial chloride channel from bovine trachea.

We have isolated and cloned a novel epithelial Cl- channel protein from a bovine tracheal cDNA expression library using an antibody probe. The antibody (alpha p38) was raised against a 38-kDa component of a homopolymeric protein that behaves as a Ca2+/calmodulin kinase II-, DIDS-, and dithiothreitol (DTT)-sensitive, anion-selective channel when incorporated into planar lipid bilayers. The full-length cDNA is 3001 base pairs long and codes for a 903-amino acid protein. The clone does not show any significant homology to any other previously reported Cl- channel sequence. Northern analysis of bovine tracheal mRNA with a cDNA probe corresponding to the cloned sequence revealed a band at 3.1 kilobases, suggesting that close to the full-length sequence has been cloned. The full-length open reading frame (2712 base pairs) has been expressed in Xenopus oocytes and in mammalian COS-7 cells. In oocytes, expression of the clone was associated with the appearance of a novel DIDS-, and DTT-sensitive, anion-selective conductance that was outwardly rectified and exhibited a reversal potential close to 0 mV. Whole-cell patch clamp studies in COS-7 cells transfected with the clone identified an ionomycin-, and DTT-sensitive chloride conductance that was not apparent in mock-transfected or control cells. In vitro translation studies have shown that the primary transcript codes for a protein migrating at 140 kDa under reduced conditions, significantly larger than the polypeptide recognized by alpha p38. We therefore suggest that either the 140-kDa translated product is a prepro form of the 38-kDa subunit of the previously identified bovine tracheal anion channel and that the primary transcript is post-translationally cleaved to yield the final product, or that the cloned channel and the previously identified bovine tracheal anion channel protein share an epitope that is recognized by the alpha p38 antibody.

Amino Acid Sequence↗

PubMed1995

Interaction between cystic fibrosis transmembrane conductance regulator and outwardly rectified chloride channels.

We have previously described a protocol for the simultaneous isolation and reconstitution of a protein kinase A (PKA)-sensitive outwardly rectified chloride channel (ORCC) and the cystic fibrosis transmembrane conductance regulator (CFTR) from bovine tracheal epithelium. Immunoprecipitation of CFTR from this preparation prevented PKA activation of the ORCC, suggesting that CFTR regulated the ORCC and that this regulatory relationship was preserved throughout the purification procedure. We now report the purification of CFTR from bovine tracheal epithelia and the purification of a CFTR conduction mutant (G551D CFTR) from retrovirally transduced mouse L cells using a combination of alkali stripping, Triton-X extraction, and immunoaffinity chromatography. Immunopurified CFTR proteins were reconstituted in the absence and presence of ORCC. To test the hypothesis that only functional CFTR can support activation of ORCC by PKA and ATP, we used an inhibitory anti-CFTR505-511 peptide antibody or G551D CFTR. When anti-CFTR505-511 peptide antibodies were present prior to the addition of PKA and ATP, activation of both the ORCC and CFTR was prevented. If the antibody was added after activation of the ORCC and CFTR Cl- channels by PKA and ATP, only the CFTR Cl- channel was inhibited. When ORCC and G551D CFTR were co-incorporated into planar bilayers, only the ORCC was recorded and this channel could not be further activated by the addition of PKA and ATP. Thus, functional CFTR is required for activation of the ORCC by PKA and ATP. We also tested the hypothesis that PKA activation of ORCC was dependent on the extracellular presence of ATP. We added ATP on the presumed extracellular side of the lipid bilayer under conditions where it was not possible to activate the ORCC, i.e. in the presence of inhibitory anti-CFTR505-511 antibody or G551D CFTR. In both cases the ORCC regained PKA sensitivity. Moreover, the addition of hexokinase + glucose to the extracellular side prevented activation of the ORCCs by PKA and ATP in the presence of CFTR. These experiments confirm that both the presence of CFTR as well as the presence of ATP on the extracellular side is required for activation of the ORCC by PKA and ATP.

Adenosine Triphosphate↗

PubMed1995

Cloning of a bovine renal epithelial Na+ channel subunit.

A bovine homologue of the rat and human epithelial Na+ channel subunits, alpha-rENaC and alpha-hENaC, was cloned. The cDNA clone, termed alpha-bENaC, was isolated from a bovine renal papillary collecting duct cDNA expression library. The bovine cDNA is 3,584 base pairs (bp) long, has an open reading frame of 2,094 bp encoding a 697-amino acid protein, and is 75-85% homologous to its rat and human counterparts. In vitro translation of the transcribed cRNA yields an 80-kDa polypeptide and one at 92 kDa in the presence of pancreatic microsomes. The clone exhibits consensus sequences for N-linked glycosylation and for phosphorylation by protein kinase C, but not for protein kinase A. After expression in Xenopus laevis oocytes, a small amiloride-sensitive Na+ conductance that exhibited inward rectification and a reversal potential greater than +30 mV, consistent with the predicted equilibrium potential for Na+, was identified. The expressed alpha-bENaC-associated Na+ current was not responsive to elevations in adenosine 3',5'-cyclic monophosphate but could be stimulated by phorbol 12-myristate 13-acetate, an activator of protein kinase C. alpha-bENaC also formed amiloride-sensitive chimeric channels when coexpressed with the rat beta- and gamma-ENaC subunits in Xenopus oocytes. alpha-bENaC therefore represents a novel isoform of a growing family of epithelial Na+ channels.

Amino Acid Sequence↗

PubMed1994

Phosphorylation and activation of a bovine tracheal anion channel by Ca2+/calmodulin-dependent protein kinase II.

Secretion of Cl- by epithelial cells is fundamental to the processes of fluid and electrolyte transport by epithelia such as those of the airways, sweat-ducts, and gastrointestinal tract. In the present study, we show that a novel Cl- channel protein, immunoaffinity purified from bovine tracheal apical membrane vesicles, is sensitive to phosphorylation by Ca2+/calmodulin protein kinase II (CaMK II). The channel protein, which migrates with an M(r) of 140,000 under nonreducing conditions, is phosphorylated in vitro by CaMK II in a Ca(2+)- and calmodulin-dependent manner. When reconstituted into planar lipid bilayers, the protein behaves as an anion-selective, 4,4'-diisothiocyanostilbene- and dithiothreitol-sensitive channel. The open probability of this channel is significantly increased by Ca2+ alone but only at levels of Ca2+ (5-10 microM) that lie outside the physiological range. Addition of CaMK II to the presumptive cytoplasmic side of the bilayer in the presence of ATP and calmodulin dramatically increased the sensitivity of the channel to free Ca2+, shifting the dose-response curve for Ca(2+)-dependent channel activation to lower [Ca2+]i, the maximum increase in channel Po occurring between 0.6 and 1 microM. The addition of kinase in the absence of ATP or calmodulin or the addition of ATP or calmodulin in the absence of kinase was without effect on channel Po. Increasing [Ca2+] above 1 microM decreased channel mean current, causing a flickery block that was maximal at 2 microM. Increasing [Ca2+] as high as 10 microM in the presence of kinase did not further alter channel behavior. In contrast to CaMK II, the addition of the catalytic subunit of protein kinase A either alone or together with ATP had no effect on channel Po. These observations suggest that a novel Ca(2+)-sensitive anion channel isolated from bovine airway epithelium is regulated by CaMK II phosphorylation.

Adenosine Triphosphate↗

PubMed1994

Forskolin- but not ionomycin-evoked Cl- secretion in colonic epithelia depends on intact microtubules.

Several transport proteins are known to be trafficked to the cell membrane in response to appropriate secretagogues. In several cases, the response has been shown to be dependent on the cytoskeleton. We tested the hypothesis that the forskolin- and/or ionomycin-sensitive Cl- secretory response in colonic epithelia is dependent on an intact cytoskeleton. Using 125I- efflux as an assay for Cl- transport in the colonic epithelial cell line T84, we found that preincubation of the tissue for 3 h with either of two inhibitors of microtubule polymerization, nocodazole or colchicine, disrupted the cellular tubulin architecture and also reduced the forskolin- but not the ionomycin-evoked I- efflux. In contrast, brief exposure (4 min) to nocodazole was without effect on the forskolin-sensitive efflux, suggesting that the drug is not acting to block the stimulus-response pathway. An inactive structural analogue of colchicine, beta-lumicolchicine, had no inhibitory effect on either the forskolin-sensitive efflux or on microtubular structure. In a second model of Cl- secretion, the stripped rat colon, both colchicine and nocodazole reduced the forskolin-dependent short-circuit current by an average of 30-40%, suggesting a similar mechanism for insertion of Cl- channels into the plasma membrane. These findings suggest that the Cl- secretory response is dependent on microtubules and has a physiological role in the adenosine 3',5'-cyclic monophosphate-dependent, but not the Ca(2+)-dependent, Cl- secretion in colonic epithelia.

Animals↗

PubMed1992

Functional reconstitution of a chloride channel protein from bovine trachea.

We characterized the electrophysiological properties of a chloride channel protein isolated from bovine trachea after incorporation into planar lipid bilayers, and studied the effects of thiol-modulating agents on channel regulation both in bilayers and vesicular iodide uptake studies. Our experiments showed that this protein formed perfectly anion-selective channels in the bilayer, with an anion permeability sequence of I- (2.1) > NO3- (1.7) > Br- (1.2) > Cl- (1.0). The conductance of this channel was 25-30 picosiemens in 150 mM Cl-, and saturated with increasing chloride concentration. This channel could be completely inhibited by 4,4'-bis(isothiocyano)-2,2'-stilbenedisulfonate. Immunoblot analysis, using polyclonal antibodies (anti-p38), revealed one major band at 140 kDa. Upon reduction with dithiothreitol, 64- and 38-kDa polypeptides were observed. Functional experiments showed that reduction was accompanied by loss of 125I- uptake and single-channel activity. In the presence of dithiothreitol, only the low molecular mass protein forms (64 and 38 kDa) were detected by anti-p38 antibodies on Western blots. Cross-linking of S-S bonds with Cu(2+)-o-phenanthroline led to activation of chloride channels in vesicles and bilayers. Over-aggregation of chloride channels by this S-S cross-linking reagent caused inhibition of 125I- uptake by 80-100% and the abolishment of single-channel activity. We propose that the native chloride channel from bovine trachea can exist in vivo in different structural and functional forms depending upon its thiol-disulfide oxidation reduction status. The oxidized form has a molecular mass of 140 kDa and represents a fully active chloride channel. Inactivation of this channel might occur by over-aggregation of protein subunits, or by dissociation of the 140-kDa subunit by disulfide bond reduction.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1992

Peroxynitrite inhibits sodium uptake in rat colonic membrane vesicles.

Peroxynitrite (ONOO-) is a potent oxidizing agent that initiates lipid peroxidation and sulfhydryl oxidation and may be responsible for a portion of the cytotoxicity attributed to superoxide anion (.O2-). We quantified the extent to which ONOO-, xanthine plus xanthine oxidase (XO) and hydrogen peroxide (H2O2), decreased sodium (Na+) uptake into membrane vesicles derived from colonic cells of dexamethasone-treated rats. Carrier-free 22Na+ uptake into vesicles was measured in the presence of an inside-negative membrane potential, produced by the addition of the potassium ionophore valinomycin (10 microM) after removal of all external potassium by cation exchange chromatography. Preincubation of vesicles with either 100 microM or 1 mM ONOO- for 30 s decreased the amiloride-blockable fraction of Na+ uptake by 27 +/- 7% and 65 +/- 2%, respectively (means +/- S.E.; n greater than or equal to 5; P less than 0.05 from control). However, the amiloride-insensitive part of Na+ uptake was not affected, indicating that there was no overt destruction of these vesicles by these ONOO- concentrations. Decomposed ONOO-, hydrogen peroxide (1 microM-10 mM), or xanthine (500 microM) plus XO (10-30 mU/ml), either in the absence or in the presence of 100 microM FeEDTA, did not decrease Na+ uptake. These data suggest that ONOO- is a potent injurious agent that can compromise Na+ uptake across epithelial cells, possibly by damaging Na+ channels.

Amiloride↗

PubMed1992

Identification of a membrane protein from T84 cells using antibodies made against a DIDS-binding peptide.

The outwardly rectified chloride channel of secretory epithelial cells is inhibited by disulfonic stilbene (DS) compounds such as 4,4'-diisothiostilbene-2,2'-disulfonic acid (DIDS) [R. J. Bridges, R. T. Worrell, R. A. Frizzell, and D. J. Benos, Am. J. Physiol. 256 (Cell Physiol. 25): C902-C912, 1989]. A 13-amino acid peptide (P49) corresponding to the putative DS binding site region of the murine anion exchange protein was synthesized, and polyclonal antibodies were generated against it and then purified over a P49 affinity column. The resulting monospecific antibodies reacted on Western blots with a 95- to 100-kDa protein from human erythrocytes and a 55- to 60-kDa protein from the human colonic tumor cell line, T84. The reaction with T84 protein did not appear to represent recognition of an anion exchanger because anion efflux from T84 cells was independent of external Cl-. In addition, monoclonal antibodies raised against human band 3 recognized the band 3 protein in human red cell ghost preparations but recognized nothing in T84 cell membrane preparations. In T84 cells, DIDS protected the 60-kDa protein from antibody binding. The anti-P49 antibody blocked outwardly rectified Cl- channels incorporated into planar lipid bilayer membranes from rat colon. Immunocytochemical data reveal specific binding of the anti-P49 antibody to perinuclear cytoplasmic vesicles. Forskolin caused these antibody-labeled vesicles to migrate from the perinuclear region to the plasma membrane under conditions and with a time course identical to that seen for stimulation of Cl- transport in these cells. Our results suggest that the protein may be a part of a chloride channel complex of secretory epithelial cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1992

Antibodies against the cystic fibrosis transmembrane regulator.

Rabbit polyclonal antibodies have been raised against high-performance liquid chromatography purified synthetic peptides corresponding to two discrete regions of the cystic fibrosis transmembrane regulator (CFTR) protein: the R-domain (residues 785-796) and the extreme COOH-terminus (residues 1467-1480). Antibodies (Ab) to each of these peptides were affinity purified either by passage over a peptide-derivatized agarose matrix (Ab 785) to produce monospecific polyclonal antibodies or by protein A affinity chromatography to purify the immunoglobulin G1 fraction free of other serum proteins (Ab 1467). These antibodies recognize a candidate CFTR protein in the colonic cell line T84, as determined by Western blot analysis and by immunoprecipitation and labeling of the precipitate with [gamma-32P]ATP in the presence of protein kinase A. Both antibodies precipitated CFTR-related polypeptides from four mammalian cell types (HeLa, Bsc-40, HEp-2, and Chinese hamster ovary cells) transfected with the full-length CFTR cDNA clone using a vaccinia T7 protein expression system. Similar results were observed using a yeast CFTR expression system. In each case the Mr values of the bands observed were consistent with that expected for the CFTR protein. These antibodies should be useful probes for the immunocytochemical localization, immunoaffinity purification, and ultimately the functional characterization of the CFTR protein.

Animals↗