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.

Active electrolyte transport in mammalian buccal mucosa

1988 ◽  
Vol 255 (3) ◽  
pp. G286-G291 ◽  
Author(s):  
R. C. Orlando ◽  
N. A. Tobey ◽  
V. J. Schreiner ◽  
R. D. Readling
Keyword(s):  
Buccal Mucosa ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Electrolyte Transport ◽  
Electrical Potential Difference ◽  
Ussing Chambers ◽  
Net Fluxes

The transmural electrical potential difference (PD) was measured in vivo across the buccal mucosa of humans and experimental animals. Mean PD was -31 +/- 2 mV in humans, -34 +/- 2 mV in dogs, -39 +/- 2 mV in rabbits, and -18 +/- 1 mV in hamsters. The mechanisms responsible for this PD were explored in Ussing chambers using dog buccal mucosa. After equilibration, mean PD was -16 +/- 2 mV, short-circuit current (Isc) was 15 +/- 1 microA/cm2, and resistance was 1,090 +/- 100 omega.cm2, the latter indicating an electrically "tight" tissue. Fluxes of [14C]mannitol, a marker of paracellular permeability, varied directly with tissue conductance. The net fluxes of 22Na and 36Cl were +0.21 +/- 0.05 and -0.04 +/- 0.02 mueq/h.cm2, respectively, but only the Na+ flux differed significantly from zero. Isc was reduced by luminal amiloride, serosal ouabain, or by reducing luminal Na+ below 20 mM. This indicated that the Isc was determined primarily by active Na+ absorption and that Na+ traverses the apical membrane at least partly through amiloride-sensitive channels and exits across the basolateral membrane through Na+-K+-ATPase activity. We conclude that buccal mucosa is capable of active electrolyte transport and that this capacity contributes to generation of the buccal PD in vivo.

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

Bioelectric properties of cultured epithelial monolayers from distal lung of 18-day fetal rat

1992 ◽  
Vol 262 (5) ◽  
pp. L628-L636 ◽  
Author(s):  
P. M. Barker ◽  
A. D. Stiles ◽  
R. C. Boucher ◽  
J. T. Gatzy
Keyword(s):  
Short Circuit ◽  
In Vivo Studies ◽  
Fetal Life ◽  
Pulmonary Epithelium ◽  
Fresh Tissue ◽  
Fetal Sheep ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Fetal Rat

In vivo studies of fetal sheep suggest that the liquid present in the lumen of the lung throughout fetal life is derived from Cl- secretion by the pulmonary epithelium. Monolayer preparations of enriched epithelial cells from distal fetal rat (18-day gestation) lung, grown in serum-free media, were histologically similar to acinar (prealveolar) structures of fresh tissue. In Ussing chambers, basal transepithelial potential difference (PD), calculated equivalent short-circuit current (Ieq), and transepithelial resistance (R) were 4.4 +/- 0.3 mV (matrix positive), 35.6 +/- 1.6 microA/cm2, and 120.0 +/- 4.0 omega cm2, respectively. Ouabain (10(-3) M) eliminated 57% of basal Ieq within 30 min, amiloride (10(-4) M) induced a 13% fall in Ieq, and phlorizin (10(-4) M) had no effect on bioelectric properties. Diphenylamine-2-carboxylate (DPC, 3 x 10(-3) M) inhibited Ieq by 50%. Bumetanide had no effect on baseline bioelectric parameters. The hyperpolarization that accompanied apical or bilateral replacement of Cl- and was enhanced by terbutaline suggested an apical Cl- permselectivity. Effects of Na+ replacement on amiloride-pretreated monolayers were consistent with Na(+)-dependent Cl- secretion or amiloride-insensitive pathways. Under these growth conditions, this preparation exhibits bioelectric characteristics that are compatible with Cl- secretion and Na+ absorption. The mechanism of Cl- secretion may be similar to that of airways but is uniquely bumetanide insensitive.

NaCl transport across equine proximal colon and the effect of endogenous prostanoids

1990 ◽  
Vol 259 (1) ◽  
pp. G62-G69 ◽  
Author(s):  
L. L. Clarke ◽  
R. A. Argenzio
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Proximal Colon ◽  
Nacl Transport ◽  
Apparent Lack ◽  
Ussing Chambers ◽  
Cl Secretion

In contrast to in vivo findings, the equine proximal colon fails to demonstrate significant net absorption of Na+ and Cl- under in vitro conditions. The present study was undertaken to determine if endogenous prostanoids are responsible for this apparent lack of ion transport. Proximal colonic tissues from ponies were preincubated in either normal Ringer solution or in Ringer containing 1 microM indomethacin and studied in Ussing chambers containing these solutions. Untreated colonic mucosa demonstrated negligible Na(+)-Cl- absorption in the basal state. In contrast, indomethacin-treated colon significantly absorbed Na+ and Cl-, primarily as the result of an equivalent increase in the mucosal-to-serosal flux of these ions. Preincubation of proximal colon in 0.1 mM ibuprofen-treated Ringer yielded similar results. Treatment of indomethacin colon with 1 mM mucosal amiloride eliminated net Na(+)-Cl- absorption without affecting the short-circuit current (Isc). The Isc in control tissue was significantly greater than in indomethacin-treated tissue and was reduced by 0.1 mM serosal furosemide. Serosal addition of 0.1 microM prostaglandin E2 or 10 mM serosal plus mucosal theophylline to indomethacin-treated tissues abolished net Na(+)-Cl- absorption and increased the Isc to levels indistinguishable from control. In contrast, control tissues were essentially unaffected by these secretagogues. These findings indicated that Na(+)-Cl- absorption in equine proximal colon was electroneutral (possibly involving Na(+)-H+ exchange) and that the tissue was capable of electrogenic Cl- secretion. However, under the in vitro conditions, basal ion transport was dominated by endogenous prostanoids that abolished Na(+)-Cl- absorption and elicited near-maximal electrogenic Cl- secretion.

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.

Effect of vasoactive intestinal peptide on ion transport across dog tracheal epithelium

1983 ◽  
Vol 55 (6) ◽  
pp. 1844-1848 ◽  
Author(s):  
I. Nathanson ◽  
J. H. Widdicombe ◽  
P. J. Barnes
Keyword(s):  
Epithelial Cells ◽  
Vasoactive Intestinal Peptide ◽  
Short Circuit ◽  
Statistical Significance ◽  
Short Circuit Current ◽  
Open Circuit ◽  
Tracheal Epithelium ◽  
Cl Secretion ◽  
High Affinity ◽  
Stimulation Of

Under short-circuit conditions, vasoactive intestinal peptide (VIP) did not alter net Na+ movement but selectively stimulated net Cl- secretion across dog tracheal epithelium with a high affinity (Km congruent to 10(-8) M). The increase in Cl- secretion was not different from the rise in short-circuit current (Isc). However, stimulation of Cl- secretion was not maximal, because the addition of isoproterenol (10(-6) M) to VIP-treated tissues further increased the Isc by 54%. The effect of exogenous VIP was not blocked by a combination of atropine, phentolamine, propranolol (10(-5) or 10(-6) M), or tetrodotoxin (10(-6) M). Under open-circuit conditions, VIP caused an increase in the net secretion of Cl- and Na+, but the changes did not reach statistical significance. We conclude that VIP acts directly on receptors on the surface of epithelial cells to stimulate active Cl- secretion. The abundance of VIP nerves in the submucosa suggests that VIP may be important in regulation of fluid movement across the epithelium.

Intestinal anaphylaxis: in vivo and in vitro studies of the rat proximal colon

1988 ◽  
Vol 255 (2) ◽  
pp. G201-G205 ◽  
Author(s):  
D. Forbes ◽  
M. Patrick ◽  
M. Perdue ◽  
A. Buret ◽  
D. G. Gall
Keyword(s):  
Mast Cell ◽  
Immunoglobulin E ◽  
Short Circuit ◽  
Proximal Colon ◽  
Antigen Challenge ◽  
Food Protein ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Ige Mediated

The response of the rat proximal colon to an immunoglobulin E (IgE)-mediated hypersensitivity reaction was examined. Rats were sensitized to egg albumin (EA) by intraperitoneal injection, and serum titers of specific anti-EA IgE were measured at 14 days. Sensitized animals had titers of greater than or equal to 1:64, whereas no anti-EA IgE antibodies were detected in controls. Water and electrolyte absorption in the proximal colon, before and during antigen challenge, was measured by in vivo marker perfusion. Antigen challenge resulted in significant inhibition of water, Na+, Cl-, and K+ absorption in vivo. Proximal colonic tissue from sensitized and control animals was studied in Ussing chambers under short-circuited conditions. Antigen challenge of sensitized tissue resulted in significant increases in short-circuit current due to the induction of active Cl- secretion. No such changes were seen in control tissue. The abnormalities induced by antigen challenge in tissue from sensitized animals was blocked by doxantrazole (10(-3) M), a mast cell stabilizer. The findings indicate that IgE-mediated reactions in rat proximal colon to a food protein cause pertubations in water and electrolyte transport secondary to active Cl- secretion and these abnormalities appear to be due to mast cell degranulation.

Fasting alters basal and stimulated ion transport in piglet jejunum

1994 ◽  
Vol 267 (1) ◽  
pp. R156-R163 ◽  
Author(s):  
H. V. Carey ◽  
U. L. Hayden ◽  
K. E. Tucker
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Short Term ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Heat Stable ◽  
Active Ion Transport ◽  
Total Tissue ◽  
Net Fluxes

Three-week-old piglets were used to study the effects of short-term fasting on jejunal ion transport. A 48-h fast significantly reduced mucosal weight, villus height, and crypt depth. Fasting increased basal short-circuit current (Isc), which reflects active ion transport, and total tissue conductance (Gt) of muscle-stripped jejunal sheets mounted in Ussing chambers. Increases in Isc evoked by carbachol, serotonin, histamine, prostaglandin E2, or Escherichia coli heat-stable enterotoxin were significantly greater in the fasted piglets. Isc responses to mucosal D-glucose were also enhanced by the fast. Under basal conditions, unidirectional and net fluxes of Na+ and Cl-, as well as serosal-to-mucosal inulin fluxes, were significantly increased in fasted piglets. In fed piglets, carbachol increased net Cl- secretion by stimulating serosal-to-mucosal Cl- flux; Gt was not affected. In fasted piglets, carbachol increased net Cl- secretion by inhibiting mucosal-to-serosal fluxes with no effect on serosal-to-mucosal fluxes. In addition, carbachol significantly inhibited mucosal-to-serosal Na+ fluxes and reduced Gt in this group. Thus a 48-h fast increased unidirectional and net ion fluxes in piglet jejunum and enhanced ion transport responses to secretory agonists. The mechanism by which carbachol stimulated net Cl- secretion was also altered by the fast. These results suggest that the absence of luminal nutrition changes the ion transport characteristics of the jejunal epithelium.

p-Aminohippurate transport in canine tracheal epithelium

1985 ◽  
Vol 59 (5) ◽  
pp. 1585-1589 ◽  
Author(s):  
M. M. Cloutier ◽  
K. M. Lesniak
Keyword(s):  
Short Circuit ◽  
Organic Anion ◽  
Chloride Secretion ◽  
Open Circuit ◽  
Tracheal Epithelium ◽  
Ussing Chambers ◽  
Saturation Kinetics ◽  
Absorptive Flux ◽  
Menten Constant ◽  
Specialized System

The unidirectional fluxes of 20, 100, 500, and 2,000 microM rho-aminohippurate (PAH) were measured under open- and short-circuit conditions in canine tracheal epithelium mounted as flat sheets in Ussing chambers. In tissues pretreated with mucosal indomethacin (10(-6) M) and amiloride (10(-4) M), unidirectional PAH fluxes under short-circuit conditions increased with increasing bath concentrations but there was no significant net PAH transport. After stimulation of chloride secretion by mucosal cyclic adenosine 3′,5′ -cyclic monophosphate (cAMP 10(-3) M), there was a significant increase in the secretory flux of PAH and a significant decrease in the absorptive flux of PAH. This resulted in net PAH secretion that demonstrated saturation kinetics with an apparent Michaelis-Menten constant of 754 microM by Lineweaver-Burk analysis. Intracellular concentrations of PAH were 0.4–1.2 times bath concentrations after pretreatment with indomethacin and amiloride and increased to 2.6–3.3 times bath concentrations after cAMP. Under open-circuit conditions, secretory PAH flux decreased and absorptive flux increased resulting in net PAH absorption. We conclude from these early studies that the canine tracheal epithelium possesses a specialized system for the transport of organic anions in the airways and that this transport system may share many similarities with organic anion transport in the kidney.

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