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B A Stanton

Publications and source records attributed to B A Stanton.

At least 55 records · Page 3Linked to original sources
PubMed1994

Evidence for the role of actin filaments in regulating cell swelling.

Actin filaments could play a role in regulation of cell swelling by two distinct mechanisms. One is by a tensile mechanism involving the coordinated interaction of actin and actin-associated proteins with all plasma membrane domains. The actin-membrane linkage would restrain cell swelling in the event of the influx of water. In shark rectal gland cells, conditions that cause massive cell swelling (i.e., high K medium, exposure to mercurials) are associated with disruption of membrane-associated actin filaments. Under conditions that result in only moderate swelling (Na-pump inhibition, Li substitution) the actin filaments remain associated with the cell membrane. Thus, in this cell type, disruption of the actin-membrane organization is correlated with increased swelling. Another mechanism by which actin could limit cell swelling is via regulation of ion transport proteins that are activated by cell swelling. This could be accomplished by a vesicle transport and insertion mechanism that delivers ion transport units to the cell membrane or by interaction with transport proteins already present in the membrane. Cell-attached patch clamp studies of RCCT-28A cells exposed to hypotonic medium demonstrated the activation of Cl-channel activity coincident with an alteration in actin. Activation of the channel was mimicked by stretching the membrane. Exposure of inside-out patches to cytochalasins also increased Cl-channel activity. Treatment of isolated patches with phalloidin inhibited stretch-induced activation. Thus, regulation of a volume-sensitive Cl-channel appears to be directly related to the state of organization of actin filaments.

Actins↗
PubMed1994

The cytoskeleton and membrane transport.

This review summarizes recent studies demonstrating that the actin-based cytoskeleton regulates the activity of many ion channels and transport proteins. Stretch-activated ion channels; channels conductive to chloride, to sodium, and to potassium; electroneutral transport proteins, including the Na(+)-K(+)-2Cl-cotransporter, the Na+/H+ exchanger, and the organic osmolyte transporter; as well as water channels are all regulated by the actin cytoskeleton. Several potential mechanisms whereby the actin cytoskeleton and microtubules regulate transport protein activity are discussed.

Animals↗
PubMed1994

Epithelial Ca2+ channels sensitive to dihydropyridines and activated by hyperpolarizing voltages.

Parathyroid hormone (PTH) increases transcellular Ca2+ absorption in renal cortical thick ascending limbs and distal convoluted tubules (DCT). In cells isolated from these nephron segments, PTH increases Ca2+ uptake by a pathway that is sensitive to dihydropyridine-type agonists and antagonists (B. J. Bacskai and P. A. Friedman. Nature Lond. 347: 388-391, 1990). Patch-clamp techniques were used to identify Ca(2+)-permeable channels in DCT cells. Channel activity was detectable in cell-attached patches only in cells pretreated with PTH. Ca2+ channels exhibited prolonged open times (seconds), had a low single-channel conductance (2.1 pS), and open channel probability increased at hyperpolarizing voltages (-50 to -90 mV). Channel activity was sensitive to dihydropyridine-type compounds, nifedipine, and BAY K8644, as was Ca2+ uptake. However, Ca2+ entry was insensitive to verapamil or omega-conotoxin. These results demonstrate that these channels mediate PTH-stimulated apical membrane Ca2+ entry in DCT cells, which are the principal Ca(2+)-transporting cells of the kidney.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

Heterotrimeric G proteins, vesicle trafficking, and CFTR Cl- channels.

Previously (E.M. Schwiebert, N. Kizer, D. C. Gruenert, and B. A. Stanton, Proc. Natl. Acad. Sci. USA 89: 10623-10627, 1992), we showed that heterotrimeric G proteins regulate adenosine 3',5'-cyclic monophosphate (cAMP)-activated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels in human airway epithelial cells. The goal of the present study was to test the hypothesis that heterotrimeric G proteins regulate vesicle trafficking and exocytosis and that these events are critical for cAMP activation of CFTR-mediated Cl- secretion. We report that cAMP stimulates exocytosis and CFTR Cl- conductance (GCl) in normal but not in CF cells. Stimulation of the heterotrimeric G protein G alpha i-2 inhibited cAMP-activated CFTR GCl and exocytosis in normal cells. In contrast, inhibition of G alpha i-2 stimulated exocytosis and allowed cAMP to stimulate CFTR GCl in cells isolated from patients with cystic fibrosis (CF). Brefeldin A and nocodazol prevented cAMP-induced exocytosis and also blocked cAMP stimulation of CFTR GCl in normal airway epithelial cells. Our studies suggest that the heterotrimeric G protein G alpha i-2 regulates CFTR GCl in human airway epithelial cells by modulating vesicle trafficking and the delivery of CFTR Cl- channels from an intracellular vesicular pool to the plasma membrane. Inhibition of G alpha i-2 may be a useful therapeutic approach to target mutant delta F508 CFTR Cl- channels from an intracellular vesicular pool to the plasma membrane and thereby correct defective Cl- secretion in CF airway epithelial cells.

Calcium↗
PubMed1993

An immortalized cystic fibrosis tracheal epithelial cell line homozygous for the delta F508 CFTR mutation.

The development of transformed human airway epithelial cell lines has been important in advancing the understanding of the biochemical and genetic mechanisms underlying the cystic fibrosis (CF) defect. Since the most common mutation associated with CF is a phenylalanine deletion at position 508 (delta F508) in the CF transmembrane conductance regulator (CFTR) gene, a transformed airway epithelial cell line homozygous for this mutation will be important for determining the biologic significance of this mutation in the airways. We report the genotypic and phenotypic characterization of a delta F508 homozygote cell line derived from luminal epithelium in the trachea. The cells were transformed with a plasmid containing an origin of replication defective SV40 genome and have progressed through crisis. Immunocytochemical characterization of the cells shows that they express keratin, indicating epithelial cell origin, and that a calcium-dependent cell adhesion molecule, cellCAM 120/80, is present at plasma membrane junctions between cells. Electrophysiologically, the cells show no cAMP-dependent Cl transport. However, after treatment with the calcium ionophore, ionomycin, cells secrete Cl, albeit at a lower level than that observed in normal cells. Genetically, the cells express CFTR mRNA as determined by polymerase chain reaction amplification and CFTR protein as determined by Western hybridization analysis. Karyotypic analysis shows that 70% of the cells contain two copies of chromosome 7.

Adult↗
PubMed1992

GTP-binding proteins inhibit cAMP activation of chloride channels in cystic fibrosis airway epithelial cells.

Cystic fibrosis (CF) is a genetic disease characterized, in part, by defective regulation of Cl- secretion by airway epithelial cells. In CF, cAMP does not activate Cl- channels in the apical membrane of airway epithelial cells. We report here whole-cell patch-clamp studies demonstrating that pertussis toxin, which uncouples heterotrimeric GTP-binding proteins (G proteins) from their receptors, and guanosine 5'-[beta-thio]diphosphate, which prevents G proteins from interacting with their effectors, increase Cl- currents and restore cAMP-activated Cl- currents in airway epithelial cells isolated from CF patients. In contrast, the G protein activators guanosine 5'-[gamma-thio]triphosphate and AlF4- reduce Cl- currents and inhibit cAMP from activating Cl- currents in normal airway epithelial cells. In CF cells treated with pertussis toxin or guanosine 5'-[beta-thio]diphosphate and in normal cells, cAMP activates a Cl- conductance that has properties similar to CF transmembrane-conductance regulator Cl- channels. We conclude that heterotrimeric G proteins inhibit cAMP-activated Cl- currents in airway epithelial cells and that modulation of the inhibitory G protein signaling pathway may have the therapeutic potential for improving cAMP-activated Cl- secretion in CF.

Aluminum↗
PubMed1992

Conductive properties of a rabbit cortical collecting duct cell line: regulation by isoproterenol.

The present study was carried out to characterize the membrane conductive properties of RCCT-28A cells, a continuous cell line derived from rabbit cortical collecting duct (CCD). RCCT-28A cells have many phenotypic properties of acid-secreting intercalated cells (A-IC). Using the whole cell patch-clamp technique, we found that the cells are conductive to Cl-, but not to Na+ or K+. The beta-adrenergic agonists isoproterenol (2 x 10(-6) M) and adenosine 3',5'-cyclic monophosphate (cAMP, 10(-4) M) increased the whole cell Cl- conductance. Protein kinase A (150 nM) in the patch pipette (i.e., intracellular solution) also increased whole cell Cl- conductance. Because isoproterenol increases cAMP levels in these cells, we conclude that isoproterenol stimulates the Cl- conductance by increasing cell cAMP, which in turn activates protein kinase A. In contrast, vasopressin does not increase cAMP in these cells and did not increase the Cl- conductance. In conclusion, these experiments show that RCCT-28A cells, like A-IC, are conductive only to Cl-. Thus RCCT-28A cells are a good model with which to study Cl- channels in the collecting duct.

Animals↗
PubMed1992

Chloride channels in apical and basolateral membranes of CCD cells (RCCT-28A) in culture.

Previously, we found that isoproterenol activates whole cell Cl- conductance by a pathway involving adenosine 3',5'-cyclic monophosphate and protein kinase A (PKA) in a renal cell line (RCCT-28A) derived from the cortical collecting duct. The goal of the present study was to determine whether PKA activates Cl- channels in the apical and/or basolateral membrane. Using the patch clamp technique we found a 305-pS Cl- channel, described previously (22), located exclusively in the apical membrane and an outwardly rectifying Cl- channel (13/96 pS) located exclusively in the basolateral membrane. The outward rectifier was highly selective to Cl- versus cations, was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, but was not regulated by cytoplasmic pH or Ca2+. Neither isoproterenol nor PKA activated the 305-pS Cl- channel. In contrast, PKA activated a subset of outwardly rectifying channels in inside-out patches. In another subset of outwardly rectifying channels, formation of the inside-out configuration increased channel activity. These channels, however, were not sensitive to PKA. In conclusion, these experiments show that isoproterenol increases the Cl- conductance of RCCT-28A cells by activating a subset of outwardly rectifying Cl- channels located in the basolateral membrane.

Animals↗
PubMed1992

Adenosine regulates a chloride channel via protein kinase C and a G protein in a rabbit cortical collecting duct cell line.

We examined the regulation by adenosine of a 305-pS chloride (Cl-) channel in the apical membrane of a continuous cell line derived from rabbit cortical collecting duct (RCCT-28A) using the patch clamp technique. Stimulation of A1 adenosine receptors by N6-cyclohexyladenosine (CHA) activated the channel in cell-attached patches. Phorbol 12,13-didecanoate and 1-oleoyl 2-acetylglycerol, activators of protein kinase C (PKC), mimicked the effect of CHA, whereas the PKC inhibitor H7 blocked the action of CHA. Stimulation of A1 adenosine receptors also increased the production of diacylglycerol, an activator of PKC. Exogenous PKC added to the cytoplasmic face of inside-out patches also stimulated the Cl- channel. Alkaline phosphatase reversed PKC activation. These results show that stimulation of A1 adenosine receptors activates a 305-pS Cl-channel in the apical membrane by a phosphorylation-dependent pathway involving PKC. In previous studies, we showed that the protein G alpha i-3 activated the 305-pS Cl- channel (Schwiebert et al. 1990. J. Biol. Chem. 265:7725-7728). We, therefore, tested the hypothesis that PKC activates the channel by a G protein-dependent pathway. In inside-out patches, pertussis toxin blocked PKC activation of the channel. In contrast, H7 did not prevent G protein activation of the channel. We conclude that adenosine activates a 305-pS Cl- channel in the apical membrane of RCCT-28A cells by a membrane-delimited pathway involving an A1 adenosine receptor, phospholipase C, diacylglycerol, PKC, and a G protein. Because we have shown, in previous studies, that this Cl- channel participates in the regulatory volume decrease subsequent to cell swelling, adenosine release during ischemic cell swelling may activate the Cl-channel and restore cell volume.

Adenosine↗
PubMed1991

Molecular mechanisms of ANP inhibition of renal sodium transport.

ANP, a hormone secreted by the atria of mammalian hearts in response to volume expansion, increases urinary sodium excretion in part by inhibiting sodium reabsorption across the inner medullary collecting duct. A number of nephron segments may contribute to the ANP-induced natriuresis; however, this review will focus on the cellular mechanisms of ANP inhibition of electrogenic sodium reabsorption by the inner medullary collecting duct. Patch-clamp studies conducted on rat inner medullary collecting duct cells in primary culture revealed that ANP, via its second messenger cGMP, inhibits electrogenic sodium reabsorption by reducing the open probability of a cation channel located in the apical membrane. Cyclic GMP inhibits the cation channel and thereby sodium reabsorption by two mechanisms. First, cGMP inhibits the channel by a phosphorylation-independent mechanism, by binding either to an allosteric modifier site on the channel or to a regulatory subunit. Second, cGMP inhibits the channel by activating cGMP-dependent protein kinase, which by a sequential pathway involving the GTP-binding protein, Gi, inhibits the channel. These cGMP-dependent mechanisms inhibiting sodium reabsorption across the inner medullary collecting duct account for a substantial component of the natriuresis following a rise in ANP levels.

Animals↗
PubMed1990

A GTP-binding protein activates chloride channels in a renal epithelium.

Although G proteins have been shown to regulate cation channels, regulation of Cl- channels by G proteins has not been demonstrated directly. Accordingly, the objective of this study was to examine whether a G protein regulates Cl- channels in the apical membrane of rabbit kidney CCD cells grown in culture. Previous studies showed that this channel is activated by adenosine and protein kinase C and has a single channel conductance of 305 picosiemens. The PCl-:PNa+ is 9:1 and the PCl-:PHCO3- is 2:1 (Schwiebert, E.M., Light, D.B., Dietl, P., Fejes-Toth, G., Naray-Fejes-Toth, A., and Stanton, B. (1990) Kidney Int. 37,216). In the present study, Cl- channels in the apical membrane of CCD cells were studied by the patch clamp technique. GTP and guanosine 5'-O(3-thiophosphate) (GTP gamma S), a nonhydrolyzable analog of GTP, increased the single channel open probability (Po). In contrast, guanosine 5'-O-(2-thiophosphate), a nonhydrolyzable analog of GDP, and pertussis toxin (PTX) decreased the Po. GTP gamma S, but not GTP, reversed PTX inhibition of the channel. The alpha i-3-subunit of Gi increased the Po in both untreated and PTX-treated membrane patches. Because GTP gamma S activated the Cl- channel in the presence of H8, a protein kinase inhibitor, we conclude that the G protein does not activate the channel by stimulating a protein kinase. Thus, a PTX-sensitive G protein activates a Cl- channel in the apical membrane of renal CCD cells.

Adenosine↗
PubMed1990

Dual ion-channel regulation by cyclic GMP and cyclic GMP-dependent protein kinase.

Atrial natriuretic peptide, acting through its second messenger guanosine 3',5'-cyclic monophosphate (cGMP), suppresses Na+ absorption across the renal inner-medullary collecting duct and increases urinary Na+ excretion. Patch clamp studies show that cGMP reduces Na+ absorption by inhibiting an amiloride-sensitive cation channel in the apical membrane. We have now examined, using the patch clamp technique, the molecular mechanisms of cGMP inhibition. Cyclic GMP directly and specifically reduced the probability of a single channel being open (open probability, Po) by 39% (inhibition constant, Ki = 7.6 x 10(-7) M) by a phosphorylation-independent mechanism. Cyclic GMP also inhibited the channel by activating cGMP-dependent protein kinase (cGMP-kinase). Exogenous cGMP-kinase completely inhibited the channel by a phosphorylation-dependent mechanism. Activation of a pertussis toxin-sensitive G protein by GTP-gamma-S blocked cGMP-kinase inhibition of the channel. By contrast, cGMP-kinase inhibition of Po was completely reversed by GTP-gamma-S. Taken together with the results of a previous study showing that a G protein activates the cation channel, these data indicate that cGMP-kinase and a G protein sequentially regulate the cation channel. Our results show that atrial natriuretic peptide, acting through cGMP, inhibits Na+ absorption across the inner-medullary collecting duct by a dual mechanism, and that cGMP-kinase inhibits the channel by a pathway involving a G protein.

Amiloride↗
PubMed1990

The measurement of health-related quality of life: major dimensions identified by factor analysis.

The measurement of quality of life is becoming more important in the evaluation of medical technologies and pharmaceuticals. Particularly when the several available therapies have similar effects on survival, quality of life measures may help decide which should be the therapy of choice. The Recovery Study utilized a multidisciplinary array of indicators of health-related quality of life and recovery. This paper reports factor analyses of 58 outcome measures on a study group of 469 persons who had undergone coronary artery bypass or cardiac valve surgery 6-months previously. The factor analyses revealed 5 orthogonal dimensions. We have named them: low morale, symptoms of illness, neuropsychological function, interpersonal relationships, and economic-employment. The data argue that health-related quality of life is a multidimensional construct, and that these dimensions can be measured quantitatively with relatively simple interview and questionnaire approaches. The next research step is to determine whether the five dimensions of post-operative quality of life have different pre-operative predictors, and whether intervention on these predictors can improve the recovery and rehabilitation process.

Adult↗
PubMed1990

Chloride channels in the apical membrane of cortical collecting duct cells.

Ion channels in the apical membrane of cortical collecting duct (CCD) cells in culture were studied by the patch-clamp technique. CCD cells from rabbit kidney were isolated by solid-phase immunoadsorption with a monoclonal antibody. The majority of CCD cells (93%) had phenotypic characteristics similar to intercalated cells (ICC). Although Cl- channels were present in the apical membrane of the ICC cells, they were rarely active in cell-attached patches; however, channels were activated after patch excision. In inside-out patches, the channels exhibited rapid flickering, substrates, and large unitary currents. The single-channel conductance was 303 pS, the Cl(-)-to-Na+ permeability ratio was 10:1 and the Cl(-)-to-HCO3- permeability ratio was 1.5:1. The channel was inhibited by the Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, diphenylamine carboxylic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid. Although a reduction in the cytoplasmic Ca2+ concentration inhibited channel activity in both inside-out patches and cell-attached patches, alterations of Ca2+ within the physiological range did not change the channel open probability. Finally, changing the cytoplasmic pH (6.5 to 8.0) did not alter the open probability. Thus a large conductance anion channel is present in the apical membrane of CCD cells in culture. This channel may be involved in cell volume regulation or in Cl- and HCO3- secretion.

Animals↗
PubMed1990

Morphological and physiological responses to aldosterone: time course and sodium dependence.

The time course and Na+ dependence of morphological responses to a physiological elevation of aldosterone were examined in the renal cortical collecting duct. Male Sprague-Dawley rats were adrenalectomized and given a basal replacement infusion of aldosterone and dexamethasone by osmotic minipump. The animals were given additional aldosterone by either intravenous infusion or by a second implanted osmotic minipump for either 5 h or 1, 3, 7, or 14 days. Animals were fed either a Na(+)-replete or a low-Na+ diet. After these treatments we conducted a morphometric analysis on kidneys processed for electron microscopy by standard techniques. Increased aldosterone induced detectable proliferation of the basolateral membrane of principal cells after 24 h. Basolateral membrane continued to increase rapidly through 3 days of exposure and thereafter at a modest rate. Cell area and the surface density of the basolateral membrane were also increased by aldosterone treatment. The morphological response to aldosterone was markedly inhibited by maintaining animals on a low-Na+ diet. Acute exposure to elevated aldosterone levels (5 h) increased K+ secretion by principal cells without alterations in basolateral membrane area. Chronic (greater than 24 h) exposure to aldosterone induces morphological adaptations that are accompanied by further elevations in K+ secretion.

Aldosterone↗
PubMed1990

Cellular actions of thiazide diuretics in the distal tubule.

Thiazide diuretics inhibit electroneutral NaCl reabsorption across the distal tubule of the salamander, Amphiuma, and hyperpolarize the basolateral membrane voltage (Vbl) of distal tubule cells. The objective of this study was to determine whether thiazides hyperpolarize Vbl by reducing intracellular Cl- activity (Acl). To this end, distal tubules were perfused in vitro, and electrophysiological techniques were used to measure Acl and Vbl. Hydrochlorothiazide in tubular fluid reduced ACl from 17.0 to 12.6 mM and hyperpolarized Vbl by 16 mV. Reduction of Cl- in tubular fluid from 84 to 8 mM also decreased Acl from 16.1 to 9.9 mM and hyperpolarized Vbl by 12 mV. Because a previous study suggested that electroneutral NaCl reabsorption is mediated by Na+/H(+)-Cl-/HCO3- exchangers in the apical membrane, the Cl-/HCO3- exchange inhibitor, 4,4-diisothiocyanostilbene-2-2-disulphonic acid (DIDS) was added to tubule fluid. DIDS reduced Acl from 15.0 to 11.6 and hyperpolarized Vbl by 10 mV. DIDS and hydrochlorothiazide were not additive, inhibition of Na+/H+ exchange by amiloride (10(-3) M) and Na+ replacement with N-methyl-D-glucamine also reduced Acl from 17.4 to 12.9 and hyperpolarized Vbl by 16 mV. The hyperpolarization of Vbl in each experiment is referable to the fall in Acl. These data show that thiazide diuretics regulate ACl and that the hyperpolarization of Vbl is referable to the thiazide-induced reduction of Acl.

Animals↗