scholarly journals A V-ATPase drives active, electrogenic and Na+-independent Cl- absorption across the gills of Eriocheir sinensis

1995 ◽  
Vol 198 (3) ◽  
pp. 767-774 ◽  
Author(s):  
H Onken ◽  
M Putzenlechner
Keyword(s):  
Atpase Activity ◽  
Specific Activity ◽  
Short Circuit ◽  
Specific Inhibitor ◽  
Membrane Vesicles ◽  
Short Circuit Current ◽  
Eriocheir Sinensis ◽  
Similar Distribution ◽  
Fluorescence Studies ◽  
F1fo Atpase

Using biochemical and electrophysiological techniques, we have examined the proposal that an H+-ATPase is involved in Cl- uptake across the gills of the Chinese crab Eriocheir sinensis. Bafilomycin A1 (1 µmol l-1), a specific inhibitor of V-ATPases, was used to investigate the importance of this H+-translocating enzyme in Cl- transport across the gill. In homogenates of ion-transporting posterior gills, we found the activity of a bafilomycin-sensitive V-ATPase to be markedly higher than in the anterior gills, which are not involved in ion transport. A similar distribution was found for the Na+/K+- and the mitochondrial F1Fo-ATPase. After differential and density centrifugation, the specific activity of the V-ATPase was enriched by a factor of 5. Neither Na+/K+- and F1Fo-ATPase activities nor acid phosphatase activity copurified with the bafilomycin-sensitive ATPase activity, indicating that at least the major portion of V-ATPase activity is not of basolateral, mitochondrial or lysosomal origin. In fluorescence studies, using Acridine Orange or Oxonol V as dyes, membrane vesicles displayed ATP-dependent proton transport and membrane potential generation, which were markedly reduced in the presence of bafilomycin. In addition to these biochemical studies, we mounted split lamellae of posterior gills in an Ussing-type chamber and measured the negative short-circuit current (Isc), which was shown to reflect active, electrogenic, Na+-independent and ouabain-insensitive Cl- absorption. After the addition of 1 µmol l-1 bafilomycin to the external bath, this Isc was reduced to about 50­60 % of its original value. Concomitantly, the conductance of the preparation decreased by about 13 %. From these results, we conclude that an apical V-ATPase drives electrogenic Cl- uptake across the posterior gills of the Chinese crab.

Na+-K+-ATPase and Na+/Ca2+exchange activities in gills of hyperregulatingCarcinus maenas

1999 ◽  
Vol 276 (2) ◽  
pp. R490-R499 ◽  
Author(s):  
Čedomil Lucu ◽  
Gert Flik
Keyword(s):  
Atpase Activity ◽  
Specific Activity ◽  
Short Circuit ◽  
Carcinus Maenas ◽  
High Capacity ◽  
Short Circuit Current ◽  
Total Activity ◽  
Camp Content ◽  
Dependent Protein ◽  
Leaky Epithelium

Na+-K+-ATPase and Na+/Ca2+exchange activities were studied in gills of Carcinus maenas in seawater (SW) and after transfer to dilute seawater (DSW). Carcinushyperregulates its hemolymph osmolarity through active uptake of Na+, Cl−, and Ca2+. In DSW total Na+-K+-ATPase activity in posterior gills quadrupled; Na+/Ca2+exchange specific activity was unaffected, and total activity increased 1.67-fold. Short-circuit current ( Isc) in voltage-clamped posterior gill hemilamellae was −181 μA/cm2in SW and −290 μA/cm2in DSW and up to 90% ouabain sensitive; conductivity was similar in SW or DSW (42 and 46 mS/cm2, respectively) and representative of a leaky epithelium. The new steady state of hemolymph osmolarity 24 h after DSW transfer was preceded, already 3 h after transfer, by increased Na+-K+-ATPase but not Na+/Ca2+exchange activity. Western blot analysis indicated that the amount of Na+-K+-ATPase protein had increased 2.1-fold in crabs acclimated 3 wk to DSW; however, 4 h after DSW transfer no difference in the amount of Na+-K+-ATPase protein was observed. After DSW transfer branchial cAMP content decreased. A negative correlation between branchial Na+-K+-ATPase activity and cAMP content points to rapid regulation of Na+-K+-ATPase through cAMP-dependent protein kinase A activity. Ca2+transport may depend on the high-capacity Na+/Ca2+exchanger coupled to the versatile sodium pump.

Characterization of a primary cell culture model of the avian renal proximal tubule

1998 ◽  
Vol 275 (1) ◽  
pp. R220-R226 ◽  
Author(s):  
Gayle Gocek Sutterlin ◽  
Gary Laverty
Keyword(s):  
Cell Culture ◽  
Proximal Tubule ◽  
Specific Activity ◽  
Short Circuit ◽  
Gradient Centrifugation ◽  
Specific Inhibitor ◽  
Short Circuit Current ◽  
Enzymatic Digestion ◽  
Distal Marker ◽  
Mucosal Side

Methods have been developed for producing functional, transporting monolayers of avian proximal tubule (PT) cells. A highly homogenous fraction of PT fragments was prepared by enzymatic digestion (collagenase + Dispase) of chick (3- to 5-day-old) kidneys, followed by Percoll gradient centrifugation. The PT fraction was enriched in glucose-6-phosphatase, a proximal enzyme marker, and reduced in specific activity of hexokinase, a distal marker. PT fragments were grown to confluence in serum-free media on collagen-coated permeable filter supports. Electron microscopy of confluent monolayers revealed numerous microvilli and mitochondria, central cilia, and tight junctions, all characteristic of PT cells. γ-Glutamyltranspeptidase, a proximal brush-border enzyme, showed threefold higher activity on apical than on basolateral sides of the monolayer. The electrophysiological characteristics of monolayers were investigated by voltage-clamp techniques. Monolayers displayed low transepithelial resistances (40–60 Ω ⋅ cm2), lumen-negative potentials, and baseline currents of 6–12 μA/cm2(with or without 5 mM glucose). Both α-methyl-d-glucose (2 mM), a nonmetabolizable hexose, and phenylalanine (2 mM) significantly stimulated short-circuit current when added to the mucosal side of glucose-free monolayers. Phloridzin, a specific inhibitor of Na+-coupled glucose transport, significantly inhibited short-circuit current, as did 10−5M amiloride. Monolayers also expressed net secretory transport of urate. This cell culture preparation may provide a useful working model for the study of avian PT transport.

Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells

1986 ◽  
Vol 251 (2) ◽  
pp. C186-C190 ◽  
Author(s):  
J. P. Johnson ◽  
D. Jones ◽  
W. P. Wiesmann
Keyword(s):  
Enzyme Activity ◽  
Epithelial Cells ◽  
Atpase Activity ◽  
Hormonal Regulation ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Toad Urinary Bladder ◽  
A6 Cells ◽  
Cultured Epithelial Cells ◽  
Stimulation Of

Aldosterone and insulin stimulate Na+ transport through mechanisms involving protein synthesis. Na+-K+-ATPase has been implicated in the action of both hormones. We examined the effect of aldosterone and insulin on Na+-K+-ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (ISC) in TB6C cells. Aldosterone increases Na+-K+-ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in ISC, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase ISC in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na+-K+-ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na+ entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na+-K+-ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on ISC.

scholarly journals Apical adenosine regulates basolateral Ca2+-activated potassium channels in human airway Calu-3 epithelial cells

2008 ◽  
Vol 294 (6) ◽  
pp. C1443-C1453 ◽  
Author(s):  
Dong Wang ◽  
Ying Sun ◽  
Wei Zhang ◽  
Pingbo Huang
Keyword(s):  
Epithelial Cells ◽  
Adenosine Receptors ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Specific Inhibitor ◽  
Short Circuit Current ◽  
Airway Epithelial Cells ◽  
Intracellular Camp ◽  
Anion Secretion ◽  
Human Airway

In airway epithelial cells, apical adenosine regulates transepithelial anion secretion by activation of apical cystic fibrosis transmembrane conductance regulator (CFTR) via adenosine receptors and cAMP/PKA signaling. However, the potent stimulation of anion secretion by adenosine is not correlated with its modest intracellular cAMP elevation, and these uncorrelated efficacies have led to the speculation that additional signaling pathways may be involved. Here, we showed that mucosal adenosine-induced anion secretion, measured by short-circuit current ( Isc), was inhibited by the PLC-specific inhibitor U-73122 in the human airway submucosal cell line Calu-3. In addition, the Isc was suppressed by BAPTA-AM (a Ca2+ chelator) and 2-aminoethoxydiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor blocker), but not by PKC inhibitors, suggesting the involvement of PKC-independent PLC/Ca2+ signaling. Ussing chamber and patch-clamp studies indicated that the adenosine-induced PLC/Ca2+ signaling stimulated basolateral Ca2+-activated potassium (KCa) channels predominantly via A2B adenosine receptors and contributed substantially to the anion secretion. Thus, our data suggest that apical adenosine activates contralateral K+ channels via PLC/Ca2+ and thereby increases the driving force for transepithelial anion secretion, synergizing with its modulation of ipsilateral CFTR via cAMP/PKA. Furthermore, the dual activation of CFTR and KCa channels by apical adenosine resulted in a mixed secretion of chloride and bicarbonate, which may alter the anion composition in the secretion induced by secretagogues that elicit extracellular ATP/adenosine release. Our findings provide novel mechanistic insights into the regulation of anion section by adenosine, a key player in the airway surface liquid homeostasis and mucociliary clearance.

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.

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.

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.

Acid secretion by frog gastric mucosa is electroneutral

1985 ◽  
Vol 248 (2) ◽  
pp. G246-G250 ◽  
Author(s):  
S. J. Hersey ◽  
G. Sachs ◽  
D. K. Kasbekar
Keyword(s):  
Gastric Mucosa ◽  
Acid Secretion ◽  
Proton Pump ◽  
Short Circuit ◽  
Specific Inhibitor ◽  
Short Circuit Current ◽  
Potential Difference ◽  
Sulfate Solutions ◽  
High K ◽  
Gastric Proton Pump

The action of a newly developed, specific inhibitor of the gastric proton pump, omeprazole, was investigated using intact frog gastric mucosa. Omeprazole was found to inhibit acid secretion, and the inhibition could be reversed with glutathione or 2-mercaptoethanol. Inhibition was not stimulus specific, occurring with histamine, carbachol, and forskolin. With hypertonic solutions bathing the mucosal surface, omeprazole inhibition of acid secretion did not change the tissue conductance, whereas inhibition by thiocyanate or cimetidine resulted in a significant decrease in conductance. With high-K+ (greater than 80 mM) sulfate solutions bathing both surfaces, omeprazole inhibition of acid secretion resulted in no change in transtissue potential difference. In chloride solutions, omeprazole caused an increase in potential difference and short-circuit current. These results provide evidence that the proton pump in frog gastric mucosa is electroneutral.

Involvement of PI 3-kinase in IGF-I stimulation of jejunal Na+-K+-ATPase activity and nutrient absorption

2001 ◽  
Vol 280 (2) ◽  
pp. G222-G228 ◽  
Author(s):  
Andrew N. Alexander ◽  
Hannah V. Carey
Keyword(s):  
Atpase Activity ◽  
Kinase Inhibitor ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Phosphatidylinositol 3 Kinase ◽  
Cellular Mechanisms ◽  
Ussing Chambers ◽  
Igf I ◽  
Jejunal Transport ◽  
Stimulation Of

Mechanisms responsible for increased jejunal transport rates observed in tissues treated with orally administered insulin-like growth factor-I (IGF-I) were studied in 5-day-old colostrum-deprived piglets. Human recombinant IGF-I (3.5 mg · kg−1 · day−1) or control vehicle was given orogastrically for 4 days. Disaccharidase activity, fructose uptake, and Na+-glucose cotransporter SGLT-1 protein abundance were similar between groups. Oral IGF-I produced greater rates of enterocyte Na+-K+-ATPase activity with no significant differences in Na+-K+-ATPase abundance. Cellular mechanisms responsible for transport changes were studied in Ussing chambers. In control tissues, the presence of IGF-I in mucosal solutions increased basal short-circuit current ( I sc), potential difference, d-glucose-stimulated I sc, and Na+-K+-ATPase activity; these changes were abolished by preincubation of tissues with wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. The results suggest that the effect of IGF-I on jejunal ion and nutrient transport involves activation of PI 3-kinase and stimulation of Na+-K+-ATPase activity in enterocytes.

Active and electrogenic absorption of Na + and Cl- across posterior gills of Eriocheir sinensis: influence of short-term osmotic variations

1996 ◽  
Vol 199 (4) ◽  
pp. 901-910 ◽  
Author(s):  
H Onken
Keyword(s):  
Electromotive Force ◽  
Marked Decrease ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Eriocheir Sinensis ◽  
Induced Current ◽  
Na Current ◽  
Short Term ◽  
External Barrier ◽  
Short Circuit Currents

Split lamellae of posterior gills of Chinese crabs (Eriocheir sinensis) acclimated to fresh water were mounted in a modified Ussing-type chamber, and the transepithelial short-circuit current and conductance were measured. The epithelium shows independent active and electrogenic absorption mechanisms for Na+ and Cl- that can be measured as positive and negative short-circuit currents, respectively, in the absence of the counter ion. Increasing the osmolarity of the haemolymph-side saline by addition of sucrose resulted in a marked decrease in active uptake of both Na+ and Cl-. In contrast, increasing the internal osmolarity by addition of urea or moderately decreasing the haemolymph-side osmolarity resulted in a marked increase in Na+ as well as Cl- transport. Circuit analysis revealed that Na+ current changes are mostly due to alterations in the apical amiloride-sensitive Na+ conductance, while Cl- current changes are caused not only by alterations in the transcellular conductance but also by changes in the electromotive force for Cl- absorption. Osmotic perturbations in the external bath induced current changes in the same directions, but the magnitudes of the effects were smaller than those after internal osmotic variations, indicating that the external barrier has a lower water permeability than the internal barrier. Short-term osmotic perturbations did not significantly affect the leak conductance, which is not associated with active transport and which may mostly reflect the paracellular conductance.

Sign in / Sign up

Export Citation Format

Share Document