bafilomycin-a1 and phenylamil

The anal papillae of freshwater mosquito larvae are important sites of NaCl uptake, thereby acting to offset the dilution of the hemolymph by the dilute habitat. The ion-transport mechanisms in the anal papillae are not well understood. In this study, the scanning ion-selective electrode technique (SIET) was utilized to measure ion fluxes at the anal papillae, and pharmacological inhibitors of ion transport were utilized to identify ion-transport mechanisms. Na(+) uptake by the anal papillae was inhibited by bafilomycin and phenamil but not by HMA. Cl(-) uptake was inhibited by methazolamide, SITS and DIDS but not by bafilomycin. H(+) secretion was inhibited by bafilomycin and methazolamide. Ouabain and bumetanide had no effect on NaCl uptake or H(+) secretion. Together, the results suggest that Na(+) uptake at the apical membrane occurs through a Na(+) channel that is driven by a V-type H(+)-ATPase and that Cl(-) uptake occurs through a Cl(-)/HCO(3)(-) exchanger, with carbonic anhydrase providing H(+) and HCO(3)(-) to the V-type H(+)-ATPase and exchanger, respectively.

Topics: Aedes; Amiloride; Anal Canal; Animals; Bumetanide; Carbonic Anhydrases; Chlorides; Ion Transport; Ion-Selective Electrodes; Larva; Macrolides; Methazolamide; Microelectrodes; Models, Biological; Ouabain; Sodium; Sodium-Potassium-Chloride Symporters; Sodium-Potassium-Exchanging ATPase; Vacuolar Proton-Translocating ATPases

The mechanism of Pb-induced disruption of Na(+) and Cl(-) balance was investigated in the freshwater rainbow trout (Oncorhynchus mykiss). Na(+) and Cl(-) influx rates were reduced immediately in the presence of 2.40 +/- 0.24 and 1.25 +/- 0.14 muM Pb, with a small increase in efflux rates occurring after 24-h exposure. Waterborne Pb caused a significant decrease in the maximal rate of Na(+) influx without a change in transporter affinity, suggesting a noncompetitive disruption of Na(+) uptake by Pb. Phenamil and bafilomycin markedly reduced Na(+) influx rate but did not affect Pb accumulation at the gill. Time-course analysis in rainbow trout exposed to 0, 0.48, 2.4, and 4.8 microM Pb revealed time- and concentration-dependent branchial Pb accumulation. Na(+)-K(+)-ATPase activity was significantly reduced, with 4.8 microM exposure resulting in immediate enzyme inhibition and 0.48 and 2.4 microM exposures inhibiting activity by 24 h. Reduced activity was weakly correlated with gill Pb accumulation after 3- and 8-h exposures; this relationship strengthened by 24 h. Reduced Na(+) uptake was correlated with gill Pb burden after exposures of 3, 8, and 24 h. Immediate inhibition of branchial carbonic anhydrase activity occurred after 3-h exposure to 0.82 +/- 0.05 or 4.30 +/- 0.05 microM Pb and continued for up to 24 h. We conclude that Pb-induced disruption of Na(+) and Cl(-) homeostasis is in part a result of rapid inhibition of carbonic anhydrase activity and of binding of Pb with Na(+)-K(+)-ATPase, causing noncompetitive inhibition of Na(+) and Cl(-) influx.

Topics: Amiloride; Animals; Carbonic Anhydrases; Chlorides; Enzyme Inhibitors; Gills; Lead; Macrolides; Oncorhynchus mykiss; Proton-Translocating ATPases; Sodium; Sodium Channel Blockers; Sodium-Potassium-Exchanging ATPase; Time Factors; Water-Electrolyte Balance

Percoll density-gradient separation, combined with peanut lectin agglutinin (PNA) binding and magnetic bead separation, was used to separate dispersed fish gill cells into sub-populations. Functional characterization of each of the sub-populations was performed to determine which displayed acid-activated phenamil- and bafilomycin-sensitive Na(+) uptake. Analysis of the mechanism(s) of (22)Na(+) influx was performed in control and acid-activated (addition of 10 mmoll(-1) proprionic acid) cells using a variety of Na(+) transport inhibitors (ouabain, phenamil, HOE-694 and bumetanide) and a V-type ATPase inhibitor (bafilomycin). We found that cells migrating to a 1.03-1.05 g ml(-1) Percoll interface [pavement cells (PVCs)] possessed the lowest rates of Na(+) uptake and that influx was unchanged during either bafilomycin (10 nmoll(-1)) treatment or internal acidification with addition of proprionic acid (10 mmoll(-1)). Mitochondria-rich (MR) cells that migrated to the 1.05-1.09 g ml(-1) interface of the Percoll gradient demonstrated acidification-activated bafilomycin and phenamil-sensitive Na(+) influx. Further separation of the MR fraction into PNA(+) and PNA(-) fractions using magnetic separation demonstrated that only the PNA(-) cells (alpha-MR cells) demonstrated phenamil-and bafilomycin-sensitive acid-activated (22)Na(+) uptake. We confirm the coupling of a V-type H(+)-ATPase with phenamil-sensitive Na(+) uptake activity and conclude that high-density alpha-MR cells function in branchial Na(+) uptake in freshwater fish.

Topics: Amiloride; Animals; Anti-Bacterial Agents; Biological Transport; Bumetanide; Epithelial Cells; Female; Gills; Guanidines; Hydrogen-Ion Concentration; Macrolides; Male; Mitochondria; Models, Biological; Oncorhynchus mykiss; Ouabain; Peanut Agglutinin; Sodium; Sodium Radioisotopes; Sulfones

Several components of branchial copper uptake were identified in juvenile freshwater rainbow trout (Oncorhynchus mykiss) using (64)Cu. On the basis of competitive interactions between sodium and copper uptake, inhibition of copper uptake by a proton pump inhibitor (bafilomycin A1, 2 micromol l(-1)) and a Na(+) channel blocker (phenamil, 100 micromol l(-1)), it appears that a proportion of the branchial copper uptake occurs via an apical Na(+) channel. This sodium-sensitive copper uptake demonstrates saturation kinetics, with a K(m) of 7.1 nmol l(-1) and a J(max) of 21.2 pmol g(-1) h(-1), and is characterized by an IC(50) of 104 micromol l(-1) sodium. On the basis of residual copper uptake in the presence of high sodium concentrations (20 mmol l(-1)) and differential inhibition of sodium and copper uptake by phenamil (100 micromol l(-1)), a sodium-insensitive component of copper uptake is also present in trout gills. It demonstrates saturation kinetics with a comparably low K(m) (9.6 nmol l(-1)) but a lower maximum transport capacity (J(max)=3.5 pmol g(-1) h(-1)) than the sodium-insensitive system. Sodium uptake exhibits saturation kinetics with a K(m) of 69 micromol l(-1). Copper reduced branchial sodium transport affinity but increased the maximal sodium transport capacity.

Topics: Amiloride; Animals; Anti-Bacterial Agents; Biological Transport; Calcium; Copper; Copper Radioisotopes; Gills; Kinetics; Macrolides; Oncorhynchus mykiss; Potassium; Proton Pump Inhibitors; Sodium; Sodium Channel Blockers

The branchial uptake mechanism of the nonessential heavy metal silver from very dilute media by the gills of freshwater rainbow trout was investigated. At concentrations >36 nM AgNO(3), silver rapidly entered the gills, reaching a peak at 1 h, after which time there was a steady decline in gill silver concentration and a resulting increase in body silver accumulation. Below 36 nM AgNO(3), there was only a very gradual increase in gill and body silver concentration over the 48-h exposure period. Increasing water sodium concentration ([Na(+)]; 0.05 to 21 mM) significantly reduced silver uptake, although, in contrast, increasing ambient [Ca(2+)] or [K(+)] up to 10 mM did not reduce silver uptake. Kinetic analysis of silver uptake at varying [Na(+)] showed a significant decrease in maximal silver transport capacity (173 +/- 34 pmol. g(-1). h(-1) at 0.1 mM [Na(+)] compared with 35 +/- 9 at 13 mM [Na(+)]) and only a slight decrease in the affinity for silver transport (K(m); 55 +/- 27 nM at 0.1 mM [Na(+)] compared with 91 +/- 47 nM at 13 mM [Na(+)]). Phenamil (a specific blocker of Na(+) channels), at a concentration of 100 microM, blocked Na(+) uptake by 78% of control values (58% after washout), and bafilomycin A(1) (a specific blocker of V-type ATPase), at a concentration of 2 microM, inhibited Na(+) uptake by 57% of control values, demonstrating the presence of a proton-coupled Na(+) channel in the apical membrane of the gills. Phenamil (after washout) and bafilomycin A(1) also blocked silver uptake by 62 and 79% of control values, respectively, indicating that Ag(+) is able to enter the apical membrane via the proton-coupled Na(+) channel.

Topics: Amiloride; Animals; Anti-Bacterial Agents; Binding, Competitive; Biological Transport; Cations; Cell Membrane; Enzyme Inhibitors; Gills; Kinetics; Macrolides; Oncorhynchus mykiss; Protons; Silver Nitrate; Sodium Channel Blockers; Sodium Channels; Time Factors