Active ammonia excretion across the gills of the green shore crabCarcinus maenas: participation of Na+/K+-ATPase, V-type H+-ATPase and functional microtubules

2002 ◽  
Vol 205 (18) ◽  
pp. 2765-2775 ◽  
Author(s):  
Dirk Weihrauch ◽  
Andreas Ziegler ◽  
Dietrich Siebers ◽  
David W. Towle
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Carcinus Maenas ◽  
Specific Inhibitor ◽  
Ammonia Excretion ◽  
Short Circuit Current ◽  
Shore Crab ◽  
Dependent Manner ◽  
Microtubule Network ◽  
Experimental Conditions

SUMMARYAlthough aquatic animals are generally believed to export nitrogenous waste by diffusion of NH3 or NH4+ across external epithelia, evidence for active ammonia excretion has been found in a number of species. In the euryhaline green shore crab Carcinus maenas, active excretion of ammonia across isolated gills is reduced by inhibitors of the Na+/K+-ATPase and vacuolar-type H+-ATPase. In addition, a functional dynamic microtubule network is necessary, since application of colchicine, taxol or thiabendazole leads to almost complete blockage of active and gradient-driven ammonia excretion. Actin filaments seem not to play a role in the excretory process. The NH4+-dependent short-circuit current and the conductance of the isolated cuticle were reduced in a dose-dependent manner by amiloride,a non-specific inhibitor of the Na+/H+ exchanger and Na+ channels. Combined with an analysis of gill morphology, the strong intracellular but weak apical abundance of V-type H+-ATPase and the fact that ammonia flux rates are equal under buffered and unbuffered experimental conditions, our observations suggest a hypothetical model of transepithelial ammonia movement that features active uptake across the basolateral membrane, sequestration in acidified vesicles, vesicle transport via microtubules and exocytosis at the apical membrane.

Active absorption of Na+ and Cl- across the gill epithelium of the shore crab Carcinus maenas: voltage-clamp and ion-flux studies

1996 ◽  
Vol 199 (7) ◽  
pp. 1545-1554 ◽  
Author(s):  
S Riestenpatt ◽  
H Onken ◽  
D Siebers
Keyword(s):  
Short Circuit ◽  
Ussing Chamber ◽  
Basolateral Membrane ◽  
Carcinus Maenas ◽  
Short Circuit Current ◽  
Shore Crab ◽  
Thick Ascending Limb ◽  
Gill Epithelium ◽  
Apical Side ◽  
Shore Crabs

Mechanisms of active NaCl uptake across the posterior gills of the shore crab Carcinus maenas were examined using radiochemical and electrophysiological techniques. In order to measure short-circuit current (Isc), transepithelial conductance (Gte) and area-related unidirectional fluxes of Na+ and Cl-, single split gill lamellae (epithelium plus cuticle) of hyperregulating shore crabs were mounted in a modified Ussing chamber. The negative short-circuit current measured with haemolymph-like NaCl saline on both sides of the epithelium could be inhibited by application of basolateral ouabain (ouabain inhibitor constant KOua=56±10 µmol l-1), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; KNPPB=7.5±2.5 mmol l-1) or Cs+ (10 mmol l-1). From the apical side, Isc was nearly completely blocked by Cs+ (10 mmol l-1) or Ba2+ (15 µmol l-1), whereas apical addition of furosemide (1 mmol l-1) resulted in only a small current decrease. Cl- influxes were linearly related to negative Isc. The ratio between net influxes of Cl- and Na+ was found to be approximately 2:1. With a single membrane preparation, achieved by permeabilizing the basolateral membrane with amphotericin B, Cl- influxes which were driven by a concentration gradient were shown to depend on the presence of apical Na+ and K+. On the basis of these observations, we propose that active and electrogenic absorption of NaCl across the gill epithelium of hyperregulating shore crabs proceeds as in the thick ascending limb of Henle's loop in the mammalian nephron. Accordingly, branchial NaCl transport is mediated by apical K+ channels in cooperation with apical Na+/K+/2Cl- cotransporters and by the basolateral Na+/K+-ATPase and basolateral Cl- channels.

scholarly journals ION TRANSPORT IN THE INTESTINE OF ANGUILLA ANGUILLA: GRADIENTS AND TRANSLOCATORS

1994 ◽  
Vol 193 (1) ◽  
pp. 97-117 ◽  
Author(s):  
P Marvão ◽  
M G Emílio ◽  
K Gil Ferreira ◽  
P L Fernandes ◽  
H Gil Ferreira
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Specific Inhibitor ◽  
Anguilla Anguilla ◽  
Short Circuit Current ◽  
Inhibitory Effect ◽  
Serosal Side ◽  
Sodium Dependent ◽  
Transport Inhibitors ◽  
Experimental Strategies

The transport of Na+, K+ and Cl- across the isolated epithelium of the eel intestine was studied using a combination of four experimental strategies: short-circuiting, measurements of intracellular potentials and ion concentrations, application of a variety of transport inhibitors and measurement of unidirectional fluxes with radioactive tracers. When short-circuited, the system performs a net transport of Cl- and Na+ towards the blood side, with a stoichiometry approaching 2, and a much smaller net transport of K+ towards the lumen. The system is totally driven by the sodium pump located in the basolateral barrier and the main coupling between the fluxes of the three ions is through the operation of a furosemide-sensitive transporter in the apical barrier, probably a 2Cl-/Na+/K+ symporter. The inhibitory effect of DIDS and picrylsulphonic acid on the short-circuit current, when added to the serosal side, suggests the presence of a sodium-dependent anionic shuttle located in the basolateral membrane. The short-circuit current is inhibited by H25, a non-specific inhibitor of the K+/Cl- symport, added to the serosal side. This effect occurs after a delay of at least 5 min and may result from the diffusion of the drug to the apical barrier, where it blocks the 2Cl-/Na+/K+ symport with much higher affinity.

Amphotericin B-induced active transport of K+ and the Na+-K+ flux ratio in frog corneal epithelium

1984 ◽  
Vol 247 (5) ◽  
pp. C454-C461 ◽  
Author(s):  
O. A. Candia ◽  
P. S. Reinach ◽  
L. Alvarez
Keyword(s):  
Amphotericin B ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Flux Ratio ◽  
Short Circuit Current ◽  
Bathing Solution ◽  
Experimental Conditions ◽  
Corneal Epithelial ◽  
K Concentration ◽  
K Transport

Transepithelial unidirectional K+ fluxes across the isolated frog cornea were primarily paracellular and proportional to the K+ concentration in the bathing solution (from 2.5 to 25 mM). The net K+ flux was not different from zero. Amphotericin B (10(-5) M) elicited a large and sustained net K+ transport from the stroma- to the tear-side bathing solution of about 10 microA/cm2. Concomitantly, a net Na+ transport occurs in the opposite direction with a short-circuit current from tear to stroma of about 25 microA/cm2. The net K+ transport exhibited saturation, increasing only 35% when the K+ concentration in the bathing solution was augmented five times. Cellular K+ content measured analytically after scraping off the epithelium was reduced by amphotericin B from 0.56 to 0.10 mueq. The amphotericin B-induced K+ transport was inhibited by ouabain and low Na+ (5 mM) in the tear-side solution. Paracellular permeability determined with mannitol or estimated from the tear-to-stroma K+ flux increased four times with amphotericin B. From the net K+ transport and the short-circuit current, the Na+-K+ flux ratio was calculated and found to vary between 2.2 and possibly as high as 5.5 among corneas in the same experimental conditions. The Na+-K+ flux ratio determined in the same cornea decreased as the K+ concentration in the bathing solution increased. Such variability suggests that in corneal epithelial cells the Na+-K+ coupling ratio is sensitive to changes in the electrochemical gradient across the basolateral membrane of the cell.

scholarly journals Isolation of a neuropeptide from locust corpus cardiacum which influences ileal transport.

1992 ◽  
Vol 173 (1) ◽  
pp. 261-274 ◽  
Author(s):  
N Audsley ◽  
C McIntosh ◽  
J E Phillips
Keyword(s):  
High Performance ◽  
Schistocerca Gregaria ◽  
Short Circuit ◽  
Carcinus Maenas ◽  
Short Circuit Current ◽  
Homarus Americanus ◽  
Shore Crab ◽  
Relative Molecular Mass ◽  
Corpus Cardiacum ◽  
Orconectes Limosus

1. Schistocerca gregaria ion-transport peptide (Scg-ITP) was isolated from aqueous extracts of the corpus cardiacum by a four-step procedure, utilizing reverse-phase high-performance liquid chromatography for separation and stimulation of a Cl(-)-dependent short-circuit current (Isc) across locust ilea as the bioassay. 2. Scg-ITP has an unblocked N terminus and an apparent relative molecular mass of 7700. Thirty-one residues (of an estimated 65) were identified by sequence analysis. 3. Scg-ITP is structurally related to a crustacean family of neuropeptides which includes the crustacean hyperglycaemic hormones from the shore crab Carcinus maenas and the crayfish Orconectes limosus and moult-inhibiting hormone and vitellogenesis-inhibiting hormone from the lobster Homarus americanus. 4. Scg-ITP has no sequence homology with neuroparsins (Nps). Nps are the only other neuropeptides isolated to date that might regulate reabsorption in an insect hindgut (rectum).

Ion transport across posterior gills of hyperosmoregulating shore crabs (Carcinus maenas): amiloride blocks the cuticular Na+ conductance and induces current-noise

2002 ◽  
Vol 205 (4) ◽  
pp. 523-531 ◽  
Author(s):  
Horst Onken ◽  
Sven Riestenpatt
Keyword(s):  
Short Circuit ◽  
Noise Analysis ◽  
Carcinus Maenas ◽  
Low Frequency ◽  
Short Circuit Current ◽  
Current Noise ◽  
Corner Frequency ◽  
Shore Crab ◽  
Shore Crabs ◽  
Gill Lamellae

SUMMARYSplit gill lamellae and gill cuticles of shore crabs (Carcinus maenas) adapted to 10 ‰ salinity were mounted in a modified Ussing-type chamber. With NaCl saline on both sides, split gill lamellae generated a short-circuit current (Isc) of –301±16 μA cm–2 at a conductance (Gte) of 40±2 mS cm–2. The net influxes of Na+ and Cl– were 8.3±2.6 and 18.2±2.7 μmol cm–2 h–1, respectively. External amiloride (100 μmol l–1) reduced Gte to approximately 50 % of the original value at unchanged Isc; Cl– fluxes remained unaffected, whereas Na+ fluxes were markedly reduced by 70–80 %. The Isc in the presence of external amiloride was almost completely inhibited by internal ouabain. At a clamp voltage of 50 mV (outside-positive), a positive current was measured at unchanged Gte. Under these conditions, amiloride reduced the current and conductance at half-maximal concentrations of 3.6 and 2.0 μmol l–1, respectively. At outside-positive voltages, but not under short-circuit conditions, external amiloride induced Lorentzian components in the power density spectra. The amiloride-dependent changes in the corner frequency (linear) and of the low-frequency plateau (‘bell-shaped’) were as expected for channel blockade by amiloride with pseudo-first-order kinetics. With an outside-positive clamp voltage of 50 mV across isolated cuticles, a positive cuticular current (Icut) of 25 188±3791 μA cm–2 and a cuticular conductance (Gcut) of 547±76 mS cm–2 were measured. External amiloride reduced Icut and Gcut at half-maximal concentrations of 0.7 and 0.6 μmol l–1, respectively. Amiloride-induced current-noise analysis gave similar results to those observed with split gill lamellae. Ion-substitution experiments with isolated cuticles further support inhibition by external amiloride of the cuticular Na+ conductance of shore crab gills and not amiloride-sensitive transporters (Na+ channels or Na+/H+ antiports) in the apical membrane.

scholarly journals Enterotoxigenicity of Mature 45-Kilodalton and Processed 35-Kilodalton Forms of Hemagglutinin Protease Purified from a Cholera Toxin Gene-Negative Vibrio cholerae Non-O1, Non-O139 Strain

2006 ◽  
Vol 74 (5) ◽  
pp. 2937-2946 ◽  
Author(s):  
A. Ghosh ◽  
D. R. Saha ◽  
K. M. Hoque ◽  
M. Asakuna ◽  
S. Yamasaki ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Hela Cells ◽  
Histopathological Examination ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Toxin Gene ◽  
Dependent Manner ◽  
A Cell

ABSTRACT Cholera toxin gene-negative Vibrio cholerae non-O1, non-O139 strain PL-21 is the etiologic agent of cholera-like syndrome. Hemagglutinin protease (HAP) is one of the major secretory proteins of PL-21. The mature 45-kDa and processed 35-kDa forms of HAP were purified in the presence and absence of EDTA from culture supernatants of PL-21. Enterotoxigenicities of both forms of HAP were tested in rabbit ileal loop (RIL), Ussing chamber, and tissue culture assays. The 35-kDa HAP showed hemorrhagic fluid response in a dose-dependent manner in the RIL assay. Histopathological examination of 20 μg of purified protease-treated rabbit ileum showed the presence of erythrocytes and neutrophils in the upper part of the villous lamina propria. Treatment with 40 μg of protease resulted in gross damage of the villous epithelium with inflammation, hemorrhage, and necrosis. The 35-kDa form of HAP, when added to the lumenal surface of rat ileum loaded in an Ussing chamber, showed a decrease in the intestinal short-circuit current and a cell rounding effect on HeLa cells. The mature 45-kDa form of HAP showed an increase in intestinal short-circuit current in an Ussing chamber and a cell distending effect on HeLa cells. These results show that HAP may play a role in the pathogenesis of PL-21.

Protein kinase C potentiates cAMP-stimulated mouse duodenal mucosal bicarbonate secretion in vitro

2004 ◽  
Vol 286 (5) ◽  
pp. G814-G821 ◽  
Author(s):  
Bi-Guang Tuo ◽  
Jimmy Y. C. Chow ◽  
Kim E. Barrett ◽  
Jon I. Isenberg
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Duodenal Mucosa ◽  
Bicarbonate Secretion ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ussing Chambers ◽  
Duodenal Bicarbonate Secretion

PKC has been shown to regulate epithelial Cl- secretion in a variety of models. However, the role of PKC in duodenal mucosal bicarbonate secretion is less clear. We aimed to investigate the role of PKC in regulation of duodenal mucosal bicarbonate secretion. Bicarbonate secretion by murine duodenal mucosa was examined in vitro in Ussing chambers using a pH-stat technique. PKC isoform expression and activity were assessed by Western blotting and in vitro kinase assays, respectively. PMA (an activator of PKC) alone had no effect on duodenal bicarbonate secretion or short-circuit current ( Isc). When PMA and dibutyryl-cAMP (db-cAMP) were added simultaneously, PMA failed to alter db-cAMP-stimulated duodenal bicarbonate secretion or Isc ( P > 0.05). However, a 1-h preincubation with PMA potentiated db-cAMP-stimulated duodenal bicarbonate secretion and Isc in a concentration-dependent manner (from 10-8 to 10-5M) ( P < 0.05). PMA preincubation had no effects on carbachol- or heat-stable toxin-stimulated bicarbonate secretion. Western blot analysis revealed that PKCα, -γ, -ϵ, -θ, -μ, and -ι/λ were expressed in murine duodenal mucosa. Ro 31–8220 (an inhibitor active against PKCϵ, -α, -β, and -γ), but not Gö 6983 (an inhibitor active against PKCα, -γ, -β, and -δ), reversed the potentiating effect of PMA on db-cAMP-stimulated bicarbonate secretion. PMA also time- and concentration-dependently increased the activity of PKCϵ, an effect that was prevented by Ro 31–8220 but not Gö 6983. These results demonstrate that activation of PKC potentiates cAMP-stimulated duodenal bicarbonate secretion, whereas it does not modify basal secretion. The effect of PKC on cAMP-stimulated bicarbonate secretion is mediated by the PKCϵ isoform.

Suppression of gastric acid secretion by furosemide in isolated gastric mucosa of guinea pig

1980 ◽  
Vol 239 (6) ◽  
pp. G532-G535 ◽  
Author(s):  
A. Ayalon ◽  
A. Corcia ◽  
G. Klemperer ◽  
S. R. Caplan
Keyword(s):  
Guinea Pig ◽  
Acid Secretion ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Conductance ◽  
Short Circuit Current ◽  
Cl Transport ◽  
Consequent Reduction ◽  
Net Flux ◽  
Mucosal Side

The effect of furosemide on acid secretion and Cl- transport was studied in isolated fundic mucosa of the guinea pig. Furosemide (10(-3) M), applied to the serosal side produced an immediate effect on the short-circuit current (Isc), lowering it by 47 +/- 2%. Potential difference decreased by 29 +/- 3%, electrical conductance by 18 +/- 4%, acid secretion by 38 +/- 1%, and net flux of Cl- from serosal-to-mucosal side by 37%. Application of the drug to the mucosal side produced similar effects on acid secretion and on the electrical parameters. It is suggested that furosemide blocks the entrance of Cl-, by the Na+--Cl- cotransport mechanism, through the basolateral membrane of the secreting cell. The consequent reduction in electrogenic Cl- transport would cause Isc and acid secretion to decrease. A reduction of Cl- conductance of the apical membrane, upon mucosal application of the drug, would cause similar effects on acid secretion and Cl- transport.

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.

scholarly journals Zinc inhibits cAMP-stimulated Cl secretion via basolateral K-channel blockade in rat ileum

2005 ◽  
Vol 288 (5) ◽  
pp. G956-G963 ◽  
Author(s):  
Kazi Mirajul Hoque ◽  
Vazhaikkurichi M. Rajendran ◽  
Henry J. Binder
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
K Channel ◽  
Isolated Cells ◽  
Rat Ileum ◽  
Anion Secretion ◽  
Cl Secretion ◽  
Ion Absorption ◽  
Isotonic Buffer

Zn, an essential micronutrient and second most abundant trace element in cell and tissues, reduces stool output when administered to children with acute diarrhea. The mechanism by which Zn improves diarrhea is not known but could result from stimulating Na absorption and/or inhibiting anion secretion. The aim of this study was to investigate the direct effect of Zn on intestinal epithelial ion absorption and secretion. Rat ileum was partially stripped of serosal and muscle layers, and the mucosa was mounted in lucite chambers. Potential difference and short-circuit current were measured by conventional current-voltage clamp method.86Rb efflux and uptake were assessed for serosal K channel and Na-K-2Cl cotransport activity, respectively. Efflux experiments were performed in isolated cells preloaded with86Rb in the presence of ouabain and bumetanide, whereas uptake experiments were performed in low-Cl isotonic buffer containing Ba and ouabain. Neither mucosal nor serosal Zn affected glucose-stimulated Na absorption. In contrast, forskolin-induced Cl secretion was markedly reduced by serosal but not mucosal addition of Zn. Zn also substantially reversed the increase in Cl secretion induced by 8-bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP) with half-maximal inhibitory concentration of 0.43 mM. In contrast, serosal Zn did not alter Cl secretion stimulated by carbachol, a Ca-dependent agonist. Zn inhibited 8-BrcAMP-stimulated86Rb efflux but not carbachol-stimulated86Rb efflux. Zn had no effect on bumetanide-sensitive86Rb uptake, Na-K-ATPase, or CFTR. We conclude from these studies that Zn inhibits cAMP-induced Cl secretion by blocking basolateral membrane K channels.

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