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D C Dawson

Publications and source records attributed to D C Dawson.

At least 37 records · Page 2Linked to original sources
PubMed1993

Mercury blockade of thiazide-sensitive NaCl cotransport in flounder urinary bladder.

The effects of HgCl2 on ion transport were investigated using isolated sheets of flounder urinary bladder, a model epithelium that is capable of electrically silent NaCl absorption and electrogenic K secretion. Exposure of the mucosal surface of the bladder to submicromolar doses of HgCl2 reduced K secretion, but the effect was not due to blockade of apical K channels. Rather, the effects of HgCl2 were virtually identical to those seen with experimental maneuvers that blocked the thiazide-sensitive NaCl cotransporter in the apical membrane, e.g., hydrochlorothiazide, Cl-free solutions, and Na-free solutions. Mucosal HgCl2 also blocked 22Na absorption, suggesting that the effect of the metal was mediated by blockade of NaCl entry. The effects of HgCl2 had a rapid onset and were readily reversed by washing, suggesting a noncovalent binding reaction. The abundance of polyanionic Hg complexes in salt solutions prompts the speculation that one of these may bind to the Cl-binding site on the cotransporter, thereby blocking it. The results provide the first evidence that the thiazide-sensitive NaCl cotransporter is a specific site of action for inorganic mercury.

Animals↗
PubMed1993

Expression of an abundant alternatively spliced form of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is not associated with a cAMP-activated chloride conductance.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cAMP-activated chloride (Cl-) channel, and expression of the full length gene in vitro is sufficient to correct the Cl- conductance defect that is characteristic of cystic fibrosis (CF) epithelial cells. Alternatively spliced forms of CFTR mRNA have been identified in several tissues from normal individuals. One of the alternative transcripts, often present at high levels, results in the in-frame deletion of exon 9. Translation of this transcript would result in a CFTR protein missing the amino terminal portion of the first nucleotide binding fold (NBF). To evaluate the possible function of this form of CFTR, a cDNA representing this transcript (CFTR delta 9) was transduced into CFPAC cells, which are derived from a CF patient. CFTR delta 9 RNA was expressed in the transduced cell lines, but only immature, incompletely glycosylated protein was detectable by Western blot analysis. No increase in cAMP-activated anion permeability was detectable by 125I efflux assay or by means of the halide sensitive dye 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ). In a second assay system, in vitro synthesized mRNA representing CFTR delta D9 was injected into Xenopus oocytes, but expression of this alternatively spliced form of CFTR was not associated with the appearance of Cl- conductance. These results suggest that the protein produced by the CFTR delta 9 transcript is not properly processed and is not capable of generating Cl- conductance in response to cAMP. Whether this alternative transcript has some other function or represents 'noise' in the mRNA splicing mechanism remains unresolved.

Alternative Splicing↗
PubMed1993

Tetraethylammonium-sensitive apical K+ channels mediating K+ secretion by turtle colon.

1. Apical membrane K+ channels in turtle colon were identified and characterized using current fluctuation analysis. 2. Under short-circuit conditions in NaCl-Ringer solution, the power density spectrum (PDS) of the short-circuit current (Isc) sometimes exhibited a clearly discernible Lorentzian component, indicating spontaneous fluctuations produced by a population of apical ion channels. The Lorentzian component had a characteristic corner frequency (fc) which averaged 10.2 +/- 0.9 Hz (mean +/- S.E.M., n = 20). 3. The power of the spontaneous fluctuations was enhanced (So increased) by manoeuvres that depolarize the apical membrane electrical potential (Va). Discernible fluctuations were enhanced or induced by raising the serosal K+ concentration ([K+]s = 50-115 mM, Na+ replacement), by clamping the transepithelial potential (Vt) to serosa-positive values, or by blocking basolateral K+ channels with Ba2+. 4. Mucosal amiloride (100 microM) attenuated the spontaneous fluctuations observed in NaCl-Ringer solution but had no effect in the presence of serosal high K+, indicating that amiloride did not block the K(+)-permeable channels but these channels resided in the same cells as the amiloride-sensitive Na+ channels. 5. Raising the mucosal K+ concentration attenuated spontaneous fluctuations. 6. In the presence of serosal high K+ and mucosal amiloride, the spontaneous fluctuations were often accompanied by a reversed Isc consistent with K+ secretion. These conditions were used to test the effects of putative channel blockers. 7. Mucosal Ba2+ and tetraethylammonium (TEA+) were effective inhibitors of the spontaneous fluctuations and the reversed Isc. At a concentration of 10 mM, TEA+ was maximally effective but the TEA+ analogues tetramethylammonium (TMA+) and tetrapropylammonium (TPrA+) were much less effective. Mucosal Rb+ or Cs+ did not inhibit at a concentration of 10 mM. 8. Mucosal lidocaine (200 microM), quinidine (200 microM), or diphenylamine-2-carboxylate (DPC, 1 mM) had little or no effect on the spontaneous fluctuations and reversed Isc. Quinine (100 microM), 4-aminopyridine (1 mM), and apamin (100 nM) were also without effect. 9. Mucosal TEA+ (10 mM) abolished the active secretory K+ flux measured in the presence of serosa-positive transepithelial potentials. 10. These experiments identified a population of TEA(+)-sensitive, apical K+ channels which mediate active K+ secretion in turtle colon. Sensitivity to external TEA+ distinguishes these channels from basolateral K+ channels in turtle colon and demonstrates similarity to apical K+ channels in mammalian colon.

Amiloride↗
PubMed1993

Selective block of specific K(+)-conducting channels by diphenylamine-2-carboxylate in turtle colon epithelial cells.

1. The conduction and gating properties of K(+)-conducting channels were studied in isolated turtle colon cells in an attempt to identify the single channels responsible for specific components of the macroscopic conductance of the basolateral membrane. Three types of Ca(2+)-activated channel were identified, two of which were selective for K+ over Na+ and a third which was selective for monovalent cations over anions, but did not discriminate between K+ and Na+. 2. One of the K(+)-selective channels was a large-conductance 'maxi' K+ channel. A second was characterized by a lower conductance and pronounced inward rectification. 3. The inward-rectifying K+ channel was selectively blocked by diphenylamine-2-carboxylate (DPC). Neither the maxi K+ channel nor a previously identified K(+)-selective channel thought to be activated by cell swelling was affected by this compound. DPC also blocked the non-selective cation channel. 4. An inward-rectifying, DPC-sensitive current was prominent in whole cell-recordings, and DPC blocked basolateral K+ currents in colonic cell layers apically permeabilized with amphotericin-B. In addition, the compound blocked active Na+ absorption. 5. The selective block of a class of epithelial K+ channels by DPC may be a useful tool for determining the contribution of this specific subpopulation to macroscopic conductance and transepithelial salt transport.

Animals↗
PubMed1992

Basolateral Na-H antiporter: uncoupled Na transport produces an amiloride-sensitive conductance.

An amiloride-inhibitable, Na(+)-H+ antiporter was identified in the basolateral membrane of turtle colon by measuring 22Na+ fluxes across isolated tissues apically permeabilized with the pore-forming antibiotic amphotericin B. In cells shrunken by exposure to Cl(-)-free (gluconate) solutions and treated with ouabain to block the Na-K-ATPase, Na+ movement across the basolateral membrane was due entirely to the antiporter. Elevation of cytosolic Na+ was associated with an amiloride-inhibitable outward current across the basolateral membrane. The sensitivity of the current to various amiloride analogues paralleled that of Na+ exchange rather than that of the apical Na+ channel. Furthermore, cell volume changes altered basolateral Na+ exchange and basolateral Na+ conductance in a parallel fashion. We propose that this amiloride-sensitive basolateral Na+ conductance represents an altered operating mode of a basolateral Na(+)-H+ exchanger.

Amiloride↗
PubMed1991

Chloride conductance expressed by delta F508 and other mutant CFTRs in Xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR) is associated with expression of a chloride conductance that is defective in cystic fibrosis (CF). Xenopus oocytes injected with RNA coding for CFTR that contained mutations in the first nucleotide binding fold (NBF1) expressed chloride currents in response to raising adenosine 3',5'-monophosphate (cAMP) with forskolin and 3-isobutyl-1-methylxanthine (IBMX). The mutant CFTRs were less sensitive than wild-type CFTR to this activating stimulus, and the reduction in sensitivity correlated with the severity of cystic fibrosis in patients carrying the corresponding mutations. This demonstration provides the basis for detailed analyses of NBF1 function and suggests potential pharmacologic treatments for cystic fibrosis.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Intracellular pH regulates basolateral K+ and Cl- conductances in colonic epithelial cells by modulating Ca2+ activation.

The role of intracellular pH as a modulator of basolateral K+ and Cl- conductances in epithelial cells was studied using digitonin-permeabilized colonic cell layers so that cytosolic pH could be clamped at specific values, while basolateral K+ and Cl- conductances were activated by stepwise increases in intracellular free Ca2+. Increasing the intracellular pH from 6.6 to 8.0 enhanced the sensitivity of both ionic conductances to intracellular Ca2+, but changing extracellular pH had no effect. Maximal K+ and Cl- currents activated by Ca2+ were not affected by changes in intracellular pH, suggesting that protons do not alter the conduction properties of the channels. Hill analysis of the Ca2+ activation process revealed that raising the cytosolic pH from 6.6 to 8.0 reduced the K1/2 for Ca2+ activation. In the absence of Ca2+, changes in intracellular pH did not have a significant effect on the basolateral K+ and Cl- conductances. These results are consistent with the notion that changes in cytosolic pH can modulate basolateral conductances by modifying the action of calcium, perhaps by acting at or near the activation site to provide a mechanism of variable "gain control."

Animals↗
PubMed1990

Basolateral K conductance: role in regulation of NaCl absorption and secretion.

In this review we explore the possible role of basolateral K conductance (gK) in the regulation of salt absorption and secretion. This inquiry is prompted by a growing body of evidence which, taken together, suggests that basolateral gK is very labile and that alterations in basolateral gK may be a key feature in both stimulatory and inhibitory regulatory mechanisms. We first consider the role of basolateral gK in relation to models for salt absorption and secretion, particularly in relation to the maintenance of cellular charge balance and the obligatory coupling between the apical and basolateral membranes that is produced by transcellular current flow. Next, we review some of the experimental evidence that suggests that changes in basolateral gK are associated with transport regulation. The cellular mechanisms that are known to impact on K channel regulation are considered in a general way, and finally, we consider the use of integrated models for understanding possible coordinate regulation of apical and basolateral cell membranes.

Animals↗
PubMed1990

Cholinergic modulation of apical Na+ channels in turtle colon: analysis of CDPC-induced fluctuations.

Current fluctuation analysis was used to investigate the properties of apical Na+ channels during muscarinic inhibition of active Na+ absorption. A reversible Na+ channel blocker, 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC), was used to induce fluctuations in the short-circuit current (I(sc)). Power density spectra of the CDPC-induced fluctuations exhibited a clearly discernible Lorentzian component, characterized by a corner frequency that was linearly related to CDPC concentration between 20 and 100 microM. The on (k'on) and off (k(off)) rate coefficients for the CDPC blocking reaction were k'on = 11.1 +/- 0.8 rad.s-1.microM-1 and k(off) = 744 +/- 53 rad/s, and the microscopic inhibition constant was 67 microM (n = 11). CDPC blocking kinetics were not significantly different after inhibition of Isc by 5 microM serosal carbachol. Single-channel Na+ current (iNa) and the density of open and blocked Na+ channels (N(ob)) were estimated from the fluctuations induced by 40 microM CDPC. Under control conditions, iNa was 0.43 +/- 0.05 pA and N(ob) was 251 +/- 42 X 10(6)/cm2 (n = 10). After exposure to serosal carbachol (2-10 microM) for 60 min, Na+ current and N(ob) were reduced by approximately 50%, but iNa was not changed significantly. These results indicate that muscarinic inhibition of electrogenic Na+ absorption was associated with a reduction in the number of open Na+ channels in the apical membrane. They also suggest that this downregulation of transport involved a coordinated decrease in both apical and basolateral membrane conductances.

Amiloride↗
PubMed1989

Mechanism of active K+ secretion by flounder urinary bladder.

We investigated the mechanism of active K+ transport by the urinary bladder of the winter flounder by measuring transepithelial properties in Ussing Chambers and by determining the cellular electrical potential profile using conventional microelectrodes. In the absence of transmural electrochemical potential gradients isolated bladders can exhibit a serosa-to-mucosa short circuit which is due entirely to net K+ secretion. The properties of transcellular K+ movement can be adequately described by a model which provides for active K+ uptake across the basolateral membrane via an electrogenic Na/K ATPase and K+ exit from the cell across the apical membrane down an electrochemical potential gradient via K+ channels which are blocked by mucosal barium. The conductance of the apical membranes of the transporting cells appears to be due almost solely to K+ while that of the basolateral membrane may be due largely to C1-.

Absorption↗
PubMed1989

Two K+ channel types, muscarinic agonist-activated and inwardly rectifying, in a Cl- secretory epithelium: the avian salt gland.

Patches of membrane on cells isolated from the nasal salt gland of the domestic duck typically contained two types of K+ channel. One was a large-conductance ("maxi") K+ channel which was activated by intracellular calcium and/or depolarizing membrane voltages, and the other was a smaller-conductance K+ channel which exhibited at least two conductance levels and displayed pronounced inward rectification. Barium blocked both channels, but tetraethylammonium chloride and quinidine selectively blocked the larger K+ channel. The large K+ channel did not appear to open under resting conditions but could be activated by application of the muscarinic agonist, carbachol. The smaller channels were open under resting conditions but the gating was not affected by carbachol. Both of these channels reside in the basolateral membranes of the Cl- secretory cells but they appear to play different roles in the life of the cell.

Animals↗
PubMed1989

Mechanism of ion transport by avian salt gland primary cell cultures.

Confluent sheets formed from primary culture of avian salt gland secretory cells exhibit a short-circuit current (Isc) in response to cholinergic and beta-adrenergic stimulation [Lowy, R. J., D. C. Dawson, and S. A. Ernst. Am J. Physiol. 249 (Cell Physiol. 18): C41-C47, 1985]. To establish the ionic basis for the Isc, transmural fluxes of 22Na and 36Cl were measured. Under short-circuit conditions there was little net flux of either ion in the absence of agonists. Addition of carbachol elevated net serosal-to-mucosal Cl flux to 1.71 mu eq.h-1.cm-2, whereas a smaller increase to 0.85 mu eq.h-1.cm-2 occurred with isoproterenol. Neither agonist altered net Na flux. The stimulated Isc accounted for 70% of the net Cl flux induced by carbachol and nearly 100% of that induced by isoproterenol. Replacement of Cl by gluconate or Na by choline abolished (carbachol) or greatly reduced (isoproterenol) the Isc, which could be restored in a dose-dependent fashion by ion restitution. Active ion transport was preferentially inhibited by basal (vs. apical) addition of ouabain, furosemide, or barium. The results provide evidence that cholinergic and beta-adrenergic agonists elicit active transmural Cl secretion. They further suggest that transport is dependent on the Na+-K+-adenosine-triphosphatase, a Na-Cl cotransport process, and a basal K conductance, all features of a secondary active Cl secretory mechanism.

Animals↗
PubMed1988

Calcium: a program in BASIC for calculating the composition of solutions with specified free concentrations of calcium, magnesium and other divalent cations.

A BASIC program is presented which facilitates the formulation of biologically relevant chemical solutions containing specified free concentrations of as many as three divalent metal cations (Ca2+, Mg2+ and the choice of a third divalent cation) at any pH in the presence of as many as three ligands (EGTA, ATP and GTP). The program uses the law of mass action and the absolute stability (association) constants found in the literature to calculate the total concentration of divalent metal cation needed to achieve a desired free concentration. The user enters the pH, the concentrations of the ligands used and the desired free concentrations of the divalent cations. This program was developed for use in a wide range of biological applications, particularly the rapid design of solutions which mimic certain aspects of intracellular fluid.

Adenosine Triphosphate↗
PubMed1988

Digitonin-permeabilized colonic cell layers. Demonstration of calcium-activated basolateral K+ and Cl- conductances.

Sheets of isolated turtle colon were exposed to digitonin on the mucosal side to chemically remove the apical membrane as a permeability barrier. Increases in the mucosal uptake of 86Rb, [3H]mannitol, and 45Ca-EGTA, and the appearance of the cytosolic marker enzyme lactate dehydrogenase in the mucosal bath confirmed the permeabilizing effect of the detergent. Basolateral K+ and Cl- currents were generated by imposing transmural ion gradients, and cytosolic free Ca2+ was manipulated by means of a Ca2+-EGTA buffer system in the mucosal bathing solution. Raising the cytosolic free Ca2+ concentration from the nanomolar to the micromolar range activated basolateral conductances for K+ and Cl-. Differences in ion selectivity, blocker specificity, calcium activation kinetics, and divalent cation activation selectivity indicated that the Ca2+-induced increases in the K+ and Cl- conductances were due to separate populations of channels. The results are consistent with the notion that the apical membranes of turtle colon epithelial cells can be functionally removed under conditions that preserve some of the conductive properties of the basolateral membrane, specifically Ca2+-activated conductive pathways for K+ and Cl-. This permeabilized preparation should offer a means for the identification of macroscopic currents that are due to presumed Ca2+-activated channels, and may also provide a model system for the functional reconstitution of channel regulatory mechanisms.

Animals↗
PubMed1988

Osmotically induced basolateral K+ conductance in turtle colon: lidocaine-induced K+ channel noise.

The basolateral membrane of amphotericin-treated turtle colon can exhibit two distinct types of K+ conductance, one of which is associated with cell swelling and is blocked by quinidine or lidocaine. Fluctuations in basolateral K+ currents were analyzed under swelling (mucosal KCl) and nonswelling (mucosal K gluconate) conditions. Under nonswelling conditions, it was not possible to detect a spontaneous Lorentzian component in the power density spectrum (PDS) and the addition of lidocaine neither inhibited the macroscopic current nor induced a Lorentzian component in the PDS. Under swelling conditions, however, lidocaine induced a Lorentzian component in the PDS and the corner frequency increased linearly with blocker concentration as expected for reversible blockade of the channel. The gating and conductance properties of osmotically induced channels estimated from a two-state model were similar to those determined recently in single-channel recordings from isolated colonic cells.

Animals↗