Cellular mechanisms of acid secretion in the posterior midgut of the larval mosquito (Aedes aegypti).

The gut contents of larval mosquitoes are alkalinized by the anterior midgut and reacidified by the posterior midgut. In the present study the cellular mechanisms of reacidification were studied in isolated, perfused posterior midgut by measuring the transepithelial voltage (V(te)) and the rate of acid secretion as indicated by the color change of m-cresol purple during intervals of perfusion stop. The lumen-positive V(te) and reacidification were significantly increased by serotonin (0.2 mumol l(-1)). The V-type H(+)-ATPase inhibitor concanamycin A (10 mumol l(-1)) on the luminal side inhibited acidification and decreased V(te). On the hemolymph side the carbonic anhydrase (CA) inhibitor acetazolamide (1 mmol l(-1)) almost abolished V(te), but had no effect on acidification. Similarly, hemolymph-side DIDS (0.1 mmol l(-1)), DPC (0.5 mmol l(-1)), amiloride (1 mmol l(-1)) and ouabain (2.5 mmol l(-1)) significantly reduced V(te), whereas Ba(2+) (5 mmol l(-1)) was without effect. DPC and amiloride also reduced V(te) when applied to the luminal side of the epithelium. Unilateral substitution of gluconate for Cl(-) affected V(te) in a way consistent with a greater permeability for Cl(-) versus Na(+). Cl(-) replacement in the lumen decreased V(te), whereas replacement on the hemolymph side increased it. Bilateral replacement left the control voltage unaffected. Na(+) replacement on either side of the tissue reduced V(te) to different degrees. Omission of luminal amino acids was followed by a significant decrease in V(te). Except for concanamycin A, none of the above manipulations impaired acidification, indicating that acidification requires only the apical proton pump. However, the chemical source of secreted H(+) is still unknown and needs to be investigated.

Effect of mucosal H+ and chemical modification on transcellular K+ current in frog skin.

A transcellular K+ current (IK) was established across the skin of the frog Rana temporaria, whose apical K+ permeability had been previously stimulated by exposure to K(+)-rich media. Short-term (less than or equal to 15 s) mucosal pH-titration of IK indicated two titrated groups (A and B), with apparent pKA of 6 and pKB of 3. The height of the titration steps, A and B, varied from skin to skin. Intracellular (i) H(+)-sensitive microelectrode studies on Rana esculenta skin (which lacks apical PK) were conducted in order to assess possible changes in pHi and basolateral K+ conductance as a consequence of the rise in mucosal [H+]. Cell pH decreased only at mucosal pH lower than 5.4 which caused a drop in basolateral K+ conductance as estimated from I-V records of the serosal membranes. These effects were much too slow to account for the fast mucosal pH effects on IK (Rana temporaria). Thus, we conclude that the two-step titration curves reflect solely the interaction of external H+ with the mucosal side of apical membrane K+ channels. Exposure to the SH-reagent PCMB, and to the carboxy-modifying EEDQ markedly reduced total IK at neutral pH; however, PCMB seemed to preferentially affect titration step B while EEDQ virtually eliminated step A. When the saturating IK kinetics were studied at different mucosal pH, protons showed a 'mixed' type inhibition of K+ current in the range of titration step A; at pH values less than 5, protons blocked IK by competition with K+ ions. These results are compatible with the presence of two K+ channel populations in the apical membrane which are discernible by their different interactions with external protons and chemical modifiers.

Invertebrate epithelial Na+ channels: amiloride-induced current-noise in crab gill.

Epithelial sheets (including cuticle) from posterior gills of the freshwater-adapted euryhaline crab Eriocheir sinensis were obtained according to the method of Schwarz and Graszynski ((1989) Comp. Biochem. Physiol. 92A, 601-604; (1989) Verh. Dtsch. Zool. Ges. 82, 211 and (1989) Arch. Int. Physiol. Biochim. 97, C45). With external NaCl-saline, the outward-directed short-circuit current (Isc) could hardly be influenced by external amiloride up to 100 mumol/l but was, on the contrary, strictly dependent on apical Cl- (Onken, Graszynski and Zeiske (1991) J. Comp. Physiol. B 161, 293-301). In absence of external chloride an inward-directed, amiloride-inhibitable Isc was observed which depended on external Na+ (thus, Isc approximately INa) in a two-step, saturating mode. The Isc-block by amiloride obeyed saturation kinetics (half-maximal at less than or equal to 1 mumol/l, suggesting apical Na(+)-channels). Only for Na+ concentrations below 100 mmol/l we found an indication for a competitive interaction between Na+ and amiloride at the channel. Current fluctuation analysis revealed the presence of an amiloride-induced relaxation (Lorentzian) component in the Isc-noise (so-called 'blocker-noise'). The Lorentzian parameter-shifts with increasing amiloride concentration indicate first-order kinetics of the blocker with its apical receptor. Using a 'two-state' blocking model we calculated, for amiloride concentrations between 2 and 5 mumol/l, a mean single-channel current of 0.46 pA and a mean channel density of 250.10(6) cm-2.

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

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.

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

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.

CYCLIC AMP STIMULATION OF ELECTROGENIC UPTAKE OF Na+ AND Cl- ACROSS THE GILL EPITHELIUM OF THE CHINESE CRAB ERIOCHEIR SINENSIS

Split gill lamellae (epithelium plus cuticle) of hyperregulating Chinese crabs acclimated to fresh water were mounted in a modified Ussing chamber. Active and electrogenic absorption of sodium and chloride were measured as positive amiloride-sensitive and negative Cl--dependent short-circuit currents (INa, ICl), respectively. Both currents were characterized before and after treatment of the tissue with theophylline or dibutyryl cyclic AMP. Both drugs increased INa and ICl. A simple circuit analysis showed that INa stimulation reflected a marked increase in the transcellular Na+ conductance, whereas the respective electromotive force was unchanged. The Michaelis constant (KNa) for Na+ current saturation was decreased after INa stimulation, indicating an increased affinity of the transport mechanism for its substrate. Consequently, the affinity for the Na+ channel blocker amiloride decreased as expected for a competitive interaction between substrate and inhibitor. Analysis of the amiloride-induced current-noise revealed a marked increase in the number of apical Na+ channels after INa stimulation with theophylline, whereas there was little change in the single-channel current. Stimulation of Cl- absorption was accompanied by a substantial increase in both transcellular conductance and electromotive force, indicating an activation of the apical H+ pump that provides the driving force for active Cl- uptake via apical Cl-/HCO3- exchange and basolateral Cl- channels.

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

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.

The anterior stomach of larval mosquitoes (Aedes aegypti): effects of neuropeptides on transepithelial ion transport and muscular motility.

The present investigation studied the influence of a number of neuropeptides on semi-open preparations of the isolated and perfused anterior stomach of larval Aedes aegypti. Effects of peptides were observed on the lumen negative transepithelial voltage (Vte) that is present with serotonin in the bath; this voltage most likely reflects active HCO3- secretion involved in alkalization of the larval anterior stomach. The five different A. aegypti allatostatins (allatostatin A 1-5) all affected Vte in almost identical ways, causing a 10-15% reduction of the voltage at 10(-7) mol l(-1). A. aegypti neuropeptide F and proctolin reduced Vte at submicromolar concentrations. At 10(-6) mol l(-1), neuropeptide F reduced Vte by 30% and proctolin reduced Vte by 50%. In contrast, A. aegypti allatotropin, A. aegypti head peptides I and III and A. aegypti short neuropeptide F were without effect on Vte. During the investigation it was observed that the peristaltic contractions of the preparations caused a dynamic component of Vte. Peristaltic contractions and the correlated voltage fluctuations depended on the presence of serotonin. Peristaltic activity and Vte deflections were progressively inhibited by A. aegypti head peptides I and III by A. aegypti short neuropeptide F and by A. aegypti neuropeptide F when the peptide concentrations were increased from 10(-8) to 10(-6) mol l(-1). These observations show that physiological concentrations of some of the tested neuropeptides affect two processes that require coordination: ion transport and motility of the larval anterior stomach.

The transepithelial voltage of the isolated anterior stomach of mosquito larvae (Aedes aegypti): pharmacological characterization of the serotonin-stimulated cells.

The lumen-negative transepithelial voltage (V(te)) of the isolated and perfused anterior stomach of mosquito larvae (Aedes aegypti) was studied with a 'semi-open' preparation in which one end of the gut was ligated onto a perfusion pipette and the other end remained open to the bath. All experiments were performed with serotonin-stimulated preparations. V(te) was abolished after addition of 2.5 mmol l(-1) dinitrophenol and depended on the presence of Cl(-). Na(+) substitution experiments showed that a major part of V(te) depended on the presence of this cation in the hemolymph side of the epithelium. Addition of 10 micro mol l(-1) concanamycin (78+/-6% inhibition) or 2.5 mmol l(-1) ouabain (15+/-2% inhibition) to the bath partially inhibited V(te). DPC (0.5 mmol l(-1)) or DIDS (0.1 mmol l(-1)) reduced V(te) when applied to the hemolymph side of the epithelium (to 49+/-8% or 78+/-3% of the control, respectively). When present on both sides of the epithelium, these inhibitors caused further V(te) reductions (to 23+/-4% or 35+/-4% of the control, respectively). Hemolymph-side furosemide (0.1 mmol l(-1)) or BaCl(2) (5 mmol l(-1)) reduced V(te) by 13+/-3% or 23+/-4% of the control, respectively. When applied to the hemolymph side of the epithelium, amiloride (0.2 mmol l(-1)) significantly decreased V(te) by 35+/-6% of the control, whereas the drug caused no further effect when it was subsequently also applied to the luminal side of the epithelium. The above results are the basis for an extended model for the cellular mechanisms of NaHCO(3) secretion/HCl absorption involved in alkalization of the anterior stomach of mosquito larvae.

Active NaCl absorption across split lamellae of posterior gills of the chinese crab Eriocheir sinensis: stimulation by eyestalk extract.

Split lamellae of the posterior gills of freshwater-adapted Chinese crabs (Eriocheir sinensis) were mounted in a modified Ussing-type chamber, and active and electrogenic absorption of Na(+) and Cl(-) were measured as positive (I(Na)) or negative (I(Cl)) short-circuit currents. Haemolymph-side addition of eyestalk extract stimulated I(Cl) by increasing both the transcellular Cl(-) conductance and the electromotive force for Cl(-) absorption. The effect was dose-dependent. Boiling the eyestalk extract did not change its effectiveness. The stimulating factor passed through dialysis tubing, indicating that it has a molecular mass of less than 2 kDa. R(p)cAMPS, a blocker of protein kinase A, reduced the stimulated I(Cl). Eyestalk extract stimulated I(Na) by increasing the transcellular Na(+) conductance at constant electromotive force. Amiloride-induced current-noise analysis revealed that stimulation of I(Na) was accompanied by an increase in the apparent number of open apical Na(+) channels at a slightly reduced single-channel current. In addition to the electrophysiological experiments, whole gills were perfused in the presence and in the absence of putative transport stimulators, and the specific activities of the V-ATPase and the Na(+)/K(+)-ATPase were measured. Eyestalk extract, theophylline or dibutyryl-cyclic AMP stimulated the activity of the V-ATPase, whereas the activity of the Na(+)/K(+)-ATPase was unaffected. The simultaneous presence of R(p)cAMPS prevented the stimulation of V-ATPase by eyestalk extract or theophylline.

Electrophysiology of posterior, NaCl-absorbing gills of Chasmagnathus granulatus: rapid responses to osmotic variations.

In the present study, the influence of short-term osmotic variations on some electrophysiological properties related to NaCl absorption across posterior gills of Chasmagnathus granulatus was investigated. The transepithelial potential difference (V(te)) of isolated and perfused gills increased significantly when hyposmotic saline (699 mosmol l(-1)) was used instead of isosmotic solution (1045 mosmol l(-1)). A reduction of the concentration of Na(+) or Cl(-) at constant osmolarity did not produce any change in V(te). Transepithelial short-circuit current (I(sc)) and conductance (G(te)), measured with split gill lamellae mounted in a modified Ussing chamber, also increased after changing to hyposmotic salines (I(sc): from -89.0+/-40.8 microA cm(-2) to -179.3+/-37.0 microA cm(-2); G(te): from 40.5+/-16.9 mS cm(-2) to 47.3+/-15.8 mS cm(-2)). The observed effects of reduced osmolarity were fast, reversible and gradually dependent on the magnitude of the osmotic variation. The activity of the Na(+)/K(+)-ATPase increased significantly after perfusion with hyposmotic saline, from 18.73+/-6.35 micromol P(i) h(-1) mg(-1) to 41.84+/-14.54 micromol P(i) h(-1) mg(-1). Theophylline maintained part of the elevated V(te) induced by hyposmotic saline, suggesting that an increased cellular cyclic AMP level is involved in the response to reduced osmolarity. In summary, the results indicate that the hemolymph osmolarity regulates active transbranchial NaCl absorption by modulating the activity of the basolateral Na(+)/K(+)-ATPase and by changing a conductive pathway, probably at the apical membrane.