Characterization of Na-K-ATPase in dog tracheal epithelium: enzymatic and ion transport measurements

1980 ◽  
Vol 48 (6) ◽  
pp. 1008-1019 ◽  
Author(s):  
C. Westenfelder ◽  
W. R. Earnest ◽  
F. Al-Bazzaz
Keyword(s):  
Atpase Activity ◽  
Ethacrynic Acid ◽  
Specific Activity ◽  
Short Circuit ◽  
High Specific Activity ◽  
Tracheal Epithelium ◽  
Bathing Solution ◽  
Tracheal Mucosa ◽  
Ussing Chambers ◽  
Cl Secretion

The dog tracheal epithelium actively secretes Cl and absorbs Na. The possible dependency of this electrolyte transport on a Mg-dependent, Na-K-activated adenosine triphosphatase (Na-K-ATPase, EC 3.6.1.3) was examined. The characteristics of this enzyme system were investigated using homogenates of tracheal epithelium. The electrical properties and ion fluxes of this epithelium were determined in tissues mounted in Ussing chambers. Addition of Na and K produced an approximate 50% activation of basal Mg-ATPase activity. The apparent Km values for ATP, Na, K, and Mg were 0.4, 12.7, 1.9, and 1.6 mM, respectively. The total specific ATPase activity was 8.1 ± 0.4 and that of the Mg-ATPase 4.3 ± 0.1 mumol Pi. mg protein -1.h-1. Addition of ouabain (1 muM) or omission of K from the submucosal bathing solution reduced potential difference (PD) and short-circuit current (SCC) significantly. Relatively low concentrations (0.1 mM or less) of ethacrynic acid, furosemide, or 2,4-dinitrophenol (2,4-DNP) depressed SCC and PD significantly, i.e., at concentrations that were without effect on the Na-K-ATPase activity. Ethacrynic acid inhibited Cl secretion, whereas 2,4-DNP lowered both Na and Cl transport. These data demonstrate that 1) the tracheal mucosa of dogs contains a Na-K-ATPase at relatively high specific activity, 2) this enzyme is likely contained in the basal aspect of this membrane, 3) it appears to be essential for maintenance of Cl secretion, and 4) Cl secretion can be reduced (by ethacrynic acid, furosemide, and 2,4-DNP) without Na-K-ATPase inhibition.

Mechanisms of sodium and chloride transport across equine tracheal epithelium

1990 ◽  
Vol 259 (6) ◽  
pp. L459-L467 ◽  
Author(s):  
G. J. Tessier ◽  
T. R. Traynor ◽  
M. S. Kannan ◽  
S. M. O3'Grady
Keyword(s):  
Chloride Transport ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Tracheal Epithelium ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Cotransport System ◽  
Ethanesulfonic Acid

Equine tracheal epithelium, stripped of serosal muscle, mounted in Ussing chambers, and bathed in plasmalike Ringer solution generates a serosa-positive transepithelial potential of 10–22 mV and a short-circuit current (Isc) of 70–200 microA/cm2. Mucosal amiloride (10 microM) causes a 40–60% decrease in Isc and inhibits the net transepithelial Na flux by 95%. Substitution of Cl with gluconate resulted in a 30% decrease in basal Isc. Bicarbonate substitution with 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid decreased the Isc by 21%. The Cl-dependent Isc was inhibited by serosal addition of 1 mM amiloride. Bicarbonate replacement or serosal amiloride (1 mM) inhibits the net Cl flux by 72 and 69%, respectively. Bicarbonate replacement significantly reduces the effects of serosal amiloride (1 mM) on Isc, indicating its effect is HCO3 dependent. Addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 100 microM) causes a 40% increase in Isc. This effect is inhibited by subsequent addition of 10 microM serosal bumetanide. Bumetanide (10 microM) reduces net Cl secretion following stimulation with 8-BrcAMP (100 microM). Serosal addition of BaCl2 (1 mM) causes a reduction in Isc equal to that following Cl replacement in the presence or absence of 100 microM cAMP. These results suggest that 1) Na absorption depends on amiloride-inhibitable Na channels in the apical membrane, 2) Cl influx across the basolateral membrane occurs by both a Na-H/Cl-HCO3 parallel exchange mechanism under basal conditions and by a bumetanide-sensitive Na-(K?)-Cl cotransport system under cAMP-stimulated conditions, and 3) basal and cAMP-stimulated Cl secretion depends on Ba-sensitive K channels in the basolateral membrane.

Mucosal histamine inhibits Na absorption and stimulates Cl secretion across equine tracheal epithelium

1991 ◽  
Vol 261 (6) ◽  
pp. L456-L461 ◽  
Author(s):  
G. J. Tessier ◽  
T. R. Traynor ◽  
M. S. Kannan ◽  
S. M. O'Grady
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Membrane Surface ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Tracheal Epithelium ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Subsequent Addition ◽  
Over Time

When the equine tracheal epithelium is mounted in Ussing chambers and bathed in plasma-like Ringer solution, the tissue generates a lumen-negative transepithelial potential (PD) of 22 mV and a short-circuit current (Isc) of 70-200 microA/cm2. Mucosal addition of 10 microM histamine produces a transient increase in the Isc followed by a return to baseline or below. Mucosal addition of 2 microM diphenhydramine inhibits the Isc response to mucosal histamine, whereas 100 microM mucosal cimetidine produces no effect. The average initial increases in Isc over time for mucosal vs. serosal histamine addition are significantly different (17.32 +/- 2.8 and 3.76 +/- 0.69 microA/min, respectively). Pretreatment with mucosal amiloride significantly prolongs the effect of mucosal histamine on Isc over a 20-min period from 4.73 +/- 0.33 to 15.48 +/- 3.16 microA. When Cl is replaced by gluconate, mucosal histamine addition results in a gradual decrease in Isc and significantly reduces the effect of mucosal amiloride on Isc from 80.8% to 54.9%. Mucosal histamine inhibits the net transepithelial Na flux by 42% and stimulates the secretion of Cl by 106%. Subsequent addition of serosal bumetanide decreases net Cl secretion by 70% These results suggest that histamine stimulates bumetanide-sensitive Cl secretion and inhibits amiloride-sensitive Na absorption; these effects are mediated by H1 receptors at the apical membrane surface

Evidence for basolateral membrane potassium conductance in canine tracheal epithelium

1983 ◽  
Vol 244 (5) ◽  
pp. C377-C384 ◽  
Author(s):  
M. J. Welsh
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Critical Role ◽  
Electrical Potential ◽  
Electrical Conductance ◽  
Tracheal Epithelium ◽  
Bathing Solution ◽  
Electrical Potential Difference ◽  
Cl Secretion ◽  
K Concentration

The ionic dependence of the basolateral membrane conductance in canine tracheal epithelium was investigated using intracellular microelectrode techniques. Increasing the K+ concentration in the submucosal bathing solution depolarized the electrical potential difference across the basolateral membrane; neither alteration of the submucosal Na+ concentration nor the mucosal K+ concentration had a significant effect on the cellular electrical potential profile. An increase in the K+ concentration in the submucosal bathing solution also decreased the net rate of Cl-secretion. Addition of ouabain (10(-4) M) to the submucosal bathing solution decreased the short-circuit current and depolarized the intracellular voltage without altering transepithelial resistance or the cell membrane resistance ratio, suggesting that basolateral resistance was unchanged. These findings, together with the previous observation that there is no appreciable basolateral Cl- conductance, indicate that a K+ conductance accounts for the predominance of the electrical conductance at the basolateral membrane. The results also indicate that the basolateral membrane K+ conductance plays a critical role in the generation of the negative intracellular voltage that drives Cl- exit across the apical membrane and thus supports Cl- secretion.

Effects of amphotericin B on ion and fluid movement across dog tracheal epithelium

1983 ◽  
Vol 55 (4) ◽  
pp. 1257-1261 ◽  
Author(s):  
I. Nathanson ◽  
J. H. Widdicombe ◽  
J. A. Nadel
Keyword(s):  
Amphotericin B ◽  
Short Circuit ◽  
Open Circuit ◽  
Tracheal Epithelium ◽  
Fluid Movement ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Special Apparatus ◽  
Net Fluxes

Ion fluxes or fluid flow were measured across sheets of dog tracheal epithelium mounted in Ussing chambers or a special apparatus, respectively. Under short-circuit conditions, luminal amphotericin B (3 X 10(-5) M) caused an inhibition of net Cl secretion and an increase in net Na absorption across paired tissues. In paired tissues under resting open-circuit conditions, there was no significant net transepithelial flux of either Cl or Na. Amphotericin B induced significant net fluxes of both Cl and Na toward the serosal side. In separate tissues from the same animals, there was no significant transepithelial fluid movement under resting conditions. Amphotericin B caused a net absorption of fluid. The absorption of salt and fluid in amphotericin B-treated tissues was abolished by ouabain. We conclude that stimulation of active Na transport by amphotericin B leads to fluid absorption. In vivo, the movement of fluid across the dog tracheal epithelium may be dependent on a balance between active Cl secretion and active Na absorption.

Interaction between sodium and chloride transport in canine tracheal mucosa

1979 ◽  
Vol 46 (1) ◽  
pp. 111-119 ◽  
Author(s):  
F. J. Al-Bazzaz ◽  
Q. Al-Awqati
Keyword(s):  
Hormonal Regulation ◽  
Chloride Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Na Transport ◽  
Tracheal Mucosa ◽  
Electrically Conductive ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Cl Transport

Canine tracheal mucosae were dissected and mounted as flat sheets in Ussing chambers. Unidirectional isotope fluxes of 22Na and 36Cl were performed across paired mucosae from the same animal. The average spontaneous potential difference was 42 + 1.2 mV (mean +/- SE) lumen negative. The short-circuit current (SCC) 3.09 +/- 0.36 mueq/cm2.h was accounted for by a net Cl secretion of 2.46 +/- 0.26 mueq/cm2.h toward the mucosa and net Na absorption of 0.46 +/- 0.13 mueq/cm2.h toward submucosa. Removal of Cl depressed SCC but had no effect on unidirectional or net Na transport (n = 7). By contrast, removal of Na (n = 6) or the addition of ouabain (n = 7) abolished net Cl secretion and greatly reduced SCC. Theophylline (n = 6) added to the submucosal bath no significant effect on Na transport but stimulated SCC and Cl secretion, suggesting hormonal regulation of Cl transport. The results suggest that the active transport of Na and Cl in this epithelium occur by electrically conductive pathways, i.e., the transport is “electrogenic.” Further it appears that Na transport is independent of the presence of Cl but that Cl transport depends on some parameter of active Na transport.

Duramycin enhances chloride secretion in airway epithelium

1990 ◽  
Vol 259 (3) ◽  
pp. C450-C454 ◽  
Author(s):  
M. M. Cloutier ◽  
L. Guernsey ◽  
P. Mattes ◽  
B. Koeppen
Keyword(s):  
Airway Epithelium ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Bathing Solution ◽  
Maximum Increase ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Polypeptide Antibiotic ◽  
High Concentrations

The effect of duramycin, a polypeptide antibiotic, on Cl- transport in canine tracheal epithelium mounted in Ussing chambers was studied. Over a narrow concentration range, duramycin increased short-circuit current (Isc) and net Cl- secretion and had no effect on mannitol flux when added to the mucosal bathing solution. The maximum increase in Isc was observed at a duramycin concentration of 2 X 10(-6) M and was associated with an increase in both unidirectional Cl- fluxes. Higher duramycin concentrations produced a decrease in Isc. Submucosal addition of duramycin had no effect on Isc except at high concentrations. Pretreatment of tissues with mucosal amiloride (10(-4) M) to reduce basal Na+ transport had no effect on the subsequent response to duamycin. In other tissues pretreated with 10(-3) M dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), duramycin produced a further increase in Isc and net Cl- secretion similar to its effect in nonpretreated tissues. In all instances the increase in Isc was entirely accounted for by an increase in net Cl- secretion. We conclude that duramycin increases Isc and Cl- secretion in airway epithelium. Although the mechanism of activation is not known, these data demonstrate that duramycin increases Cl- secretion by a pathway other than cAMP. An understanding of the mechanism of action of duramycin may further our understanding of Cl- secretion regulation in airway epithelium.

Colonic potassium and chloride secretion: role of cAMP and calcium

1985 ◽  
Vol 248 (1) ◽  
pp. G103-G109 ◽  
Author(s):  
R. D. McCabe ◽  
P. L. Smith
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Bathing Solution ◽  
Ionophore A23187 ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Intracellular Ca

Stripped rabbit colonic mucosa was studied in vitro in Ussing chambers to further investigate the role of Ca in regulating K and Cl secretion stimulated by the divalent cation ionophore A23187, prostaglandin E1 (PGE1), or 8-bromo-cAMP (8BrcAMP). To assess the effects of these secretagogues on the paracellular shunt permeability, we measured the Na concentration dependence of the serosal-to-mucosal Na flux in the absence or presence of these stimuli. Results from these studies reveal that changes in net K and Cl secretion produced by secretory stimuli cannot be accounted for by a change in shunt permeability. The possible involvement of Ca in the secretory response of the colon to these stimuli was investigated by measuring the changes in Cl and K transport elicited by A23187, PGE1, or 8BrcAMP in the absence or presence of trifluoperazine (10(-4) M) added to the serosal bathing solution. Trifluoperazine alone did not significantly alter basal Na or Cl fluxes or short-circuit current (Isc) but did decrease transepithelial conductance (Gt) and the serosal-to-mucosal K flux. Pretreatment of the tissues with trifluoperazine significantly reduced or abolished the changes in K fluxes elicited by A23187, 8BrcAMP, or PGE1 without altering the changes in Cl transport, Isc, and Gt. These results suggest that K secretion induced by these secretagogues involves an increase in intracellular Ca concentration and may be mediated by calmodulin.

Endothelin stimulates chloride secretion across canine tracheal epithelium

1991 ◽  
Vol 261 (2) ◽  
pp. L188-L194 ◽  
Author(s):  
P. I. Plews ◽  
Z. A. Abdel-Malek ◽  
C. A. Doupnik ◽  
G. D. Leikauf
Keyword(s):  
Epithelial Cells ◽  
Short Circuit ◽  
Second Messengers ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Tracheal Epithelium ◽  
Membrane Phospholipids ◽  
Cl Secretion ◽  
Tracheal Epithelial Cells ◽  
Tracheal Epithelial

The endothelins (ET) are a group of isopeptides produced by a number of cells, including canine tracheal epithelial cells. Because these compounds are endogenous peptides that may activate eicosanoid metabolism, we investigated the effects of ET on Cl secretion in canine tracheal epithelium. Endothelin 1 (ET-1) was found to produce a dose-dependent change in short-circuit current (Isc) that increased slowly and reached a maximal value within 10-15 min. When isopeptides of ET were compared, 300 nM ET-1 and ET-2 produced comparable maximal increases in Isc, whereas ET-3 produced smaller changes in Isc (half-maximal concentrations of 2.2, 7.2, and 10.4 nM, respectively). Ionic substitution of Cl with nontransported anions, iodide and gluconate, reduced ET-1-induced changes in Isc. Furthermore, the response was inhibited by the NaCl cotransport inhibitor, furosemide. In paired tissues, ET-1 significantly increased mucosal net 36Cl flux without significant effect on 22Na flux. The increase in Isc induced by ET was diminished by pretreatment with indomethacin. The second messengers mediating the increase in Isc were investigated in cultured canine tracheal epithelial cells. ET-1 stimulated the release of [3H]arachidonate from membrane phospholipids, increased intracellular Ca2+ (occasionally producing oscillations), and increased adenosine 3',5'-cyclic monophosphate accumulation. The latter was diminished by indomethacin. Thus ET is a potent agonist of Cl secretion (with the isopeptides having the following potency: ET-1 greater than or equal to ET-2 greater than ET-3) and acts, in part, through a cyclooxygenase-dependent mechanism.

Effect of pH on chloride absorption in the flounder intestine

1988 ◽  
Vol 255 (2) ◽  
pp. G247-G252 ◽  
Author(s):  
A. N. Charney ◽  
J. I. Scheide ◽  
P. M. Ingrassia ◽  
J. A. Zadunaisky
Keyword(s):  
Small Intestine ◽  
Short Circuit ◽  
Ph Effect ◽  
Short Circuit Current ◽  
Bathing Solution ◽  
Solution Ph ◽  
Transport Pathway ◽  
Ussing Chambers ◽  
Chloride Absorption ◽  
In Series

Chloride absorption in the small intestine of the winter flounder, Pseudopleuronectes americanus, is reported to be sensitive to ambient pH. We studied this sensitivity in isolated stripped intestinal mucosa mounted in modified Ussing chambers. Unidirectional 36Cl fluxes (JClm----s, JCls----m) were measured under short-circuited conditions in bathing solutions containing various combinations of HCO3- (0-20 mM), partial pressure of CO2 (0-36 mmHg), and pH (6.77-7.85). We found that JClm----s, net 36Cl flux (JClnet), and short-circuit current (Isc) increased and JCls----m decreased predominately in response to increases in bathing solution pH. There was a linear relationship between pH and both JClnet (r = 0.92, P less than 0.01) and Isc (r = 0.96, P less than 0.005) between pH 6.77 and 7.74. The pH effect was completely reversible, did not require either CO2 or HCO3-, and was not affected by the presence of mucosal barium at 1 mM. Mucosal bumetanide (0.1 mM) completely inhibited the pH effect. These data suggest that the process by which Cl- is absorbed in the flounder intestine is sensitive to pH. The data do not indicate whether pH affects Na+-K+-2Cl- cotransport or a Cl- transport pathway in series with this process. The direction of Cl- absorption in response to pH contrasts with inverse relation of pH and Cl- absorption in mammalian small intestine.

Barium inhibition of basolateral membrane potassium conductance in tracheal epithelium

1983 ◽  
Vol 244 (6) ◽  
pp. F639-F645 ◽  
Author(s):  
M. J. Welsh
Keyword(s):  
Driving Force ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Potassium Conductance ◽  
Short Circuit Current ◽  
Electrical Circuit ◽  
Tracheal Epithelium ◽  
Bathing Solution ◽  
K Permeability

Addition of barium ion, Ba2+, to the submucosal bathing solution of canine tracheal epithelium reversibly decreased the short-circuit current and increased transepithelial resistance. The decrease in short-circuit current represented a decrease in the net rate of Cl secretion with no change in the rate of Na absorption. Intracellular microelectrode techniques and an equivalent electrical circuit analysis were used to localize the effect of Ba2+ to an inhibition of the permeability of the basolateral membrane to K. Ba2+ (2 mM) doubled basolateral membrane resistance, decreased the equivalent electromotive force at the basolateral membrane, and decreased the magnitude of the depolarization of basolateral membrane voltage produced by increasing the submucosal K concentration. The inhibition of the basolateral K permeability depolarized the negative intracellular voltage, resulting in both a decrease in the driving force for Cl exit and an estimated increase in intracellular Cl concentration. These studies indicate that there is a Ba2+-inhibitable K conductance at the basolateral membrane of tracheal epithelial cells and that the K permeability plays an important role in the generation of the negative intracellular electrical potential that provides the driving force for Cl exit from the cell.

Sign in / Sign up

Export Citation Format

Share Document