External Ni2 + and ENaC in A6 cells: Na+ current stimulation by competition at a binding site for amiloride and Na+.

In cultured A6 monolayers from distal Xenopus kidney, external Ni2+ stimulated active Na+ uptake via the epithelial Na+ channel, ENaC. Transepithelial capacitance measurements ruled out exocytosis of ENaC-containing vesicles underlying the Ni2+ effect. Na+ current noise analysis was performed using the neutral Na(+) -channel blocker 6-chloro-3,5-diamino-pyrazine-2-carboxamide (CDPC) and amiloride. The analysis of CDPC-induced noise in terms of a three-state channel model revealed that Ni2+ elicits an increase in the number of open channels as well as in the spontaneous open probability. While Ni2+ had no influence on CDPC-blocker kinetics, the macroscopic and microscopic blocking kinetics of amiloride were affected. Ni2+ turned out to compete with amiloride for a putative binding site but not with CDPC. Moreover, external Na(+)--known to compete with amiloride and so producing the "self-inhibition" phenomenon--and Ni2+ exerted mutually exclusive analogous effects on amiloride kinetics. Na+ current kinetics revealed that Ni2+ prevents ENaC to be downregulated by self-inhibition. Co2+ behaved similarly to Ni2+, whereas Zn2+ did not. Attempts to disclose the chemical nature of the site reacting with Ni2+ suggested cysteine but not histidine as reaction partner.

Effects of extracellular Mg2+ on transepithelial capacitance and Na+ transport in A6 cells under different osmotic conditions.

The electrophysiological characteristics of monolayers of cultured renal epithelial A6 cells were studied under short-circuit conditions. Replacing basolateral isosmotic (260 mOsm/kg H2O) media by hyposmotic (140 mOsm/kg H2O) solutions transiently increased the transepithelial capacitance (C(T)) by 57.3+/-2.3% after 16 min. The transepithelial Na+ current (I(Na)) increased concomitantly from 4.2+/-0.7 to 26.1+/-2.6 microA/cm2 with a time course that was noticeably slower, reaching its maximum after 60 min of hypotonicity. The transepithelial conductance (G(T)) increased synchronously with I(Na). Analysis of blocker-induced noise in I(Na), using the amiloride analogue 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC), showed that the hypotonic shock increased Na+ channel density (N(T)) at the apical border. The presence of 10 mM Mg2+ on both sides of the epithelium suppressed the hypotonicity-induced C(T) increase to 14.3+/-0.5%, whereas the I(Na) increase was even larger than without Mg2+. Both effects of Mg2+ were located at an extracellular, basolateral site, because apical administration was without effect, whereas the acute basolateral addition of Mg2+ at the moment of the hypotonic shock was sufficient. Interaction between Mg2+ and Ca2+ influenced the behaviour of C(T). At constant osmolality (200 mOsm/kg H2O) 10 mM Mg2+ increased I(Na), leaving C(T) unaffected, whereas 10 mM Ca2+ stimulated both I(Na) and CT. In the presence of 1 mM Mg2+, however, the Ca(2+)-induced CT increase was abolished. The failure of CT to increase during stimulation of I(Na) by Mg2+ suggests that the divalent cation activates pre-existing channels in the apical membrane. Noise analysis showed that the natriferic effects of Mg2+ were also mediated by an increase in NT. The moderate initial increase in CT in the presence of Mg2+ under hypotonic conditions, occurring in parallel with increases in GT and I(Na), reflects most likely Na+ channel insertion induced by the hypotonic treatment. However, the large, transient, Mg(2+)-sensitive increase in CT, not correlated with increases in GT and I(Na), seems to be unrelated to Na+ channel recruitment.

Structure and regulation of insect plasma membrane H(+)V-ATPase.

H(+) V-ATPases (V-ATPases) are found in two principal locations, in endomembranes and in plasma membranes. The plasma membrane V-ATPase from the midgut of larval Manduca sexta is the sole energizer of all transepithelial secondary transport processes. At least two properties make the lepidopteran midgut a model tissue for studies of general aspects of V-ATPases. First, it is a rich source for purification of the enzyme and therefore for structural studies: 20 larvae provide up to 0.5 mg of holoenzyme, and soluble, cytosolic V(1) complexes can be obtained in even greater amounts of up to 2 mg. Second, midgut ion-tranport processes are strictly controlled by the regulation of the V-ATPase, which is the sole energizer of all ion transport in this epithelium. Recent advances in our understanding the structure of the V(1) and V(o) complexes and of the regulation of the enzyme's biosynthesis and ion-transport activity will be discussed.

Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport.

We report, for the epithelial Na+ channel (ENaC) in A6 cells, the modulation by cell pH (pHc) of the transepithelial Na+ current (INa), the current through the individual Na+ channel (i), the open Na+ channel density (No), and the kinetic parameters of the relationship between I(Na) and the apical Na+ concentration. The i and N) were evaluated from the Lorentzian INa noise induced by the apical Na+ channel blocker 6-chloro-3, 5-diaminopyrazine-2-carboxamide. pHc shifts were induced, under strict and volume-controlled experimental conditions, by apical/basolateral NH4Cl pulses or basolateral arrest of the Na+/H+ exchanger (Na+ removal; block by ethylisopropylamiloride) and were measured with the pH-sensitive probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. The changes in pHc were positively correlated to changes in INa and the apically dominated transepithelial conductance. The sole pHc-sensitive parameter underlying INa was No. Only the saturation value of the INa kinetics was subject to changes in pHc. pHc-dependent changes in No may be caused by influencing Po, the ENaC open probability, or/and the total channel number, NT = No/Po.

Secretory apical Cl- channels in A6 cells: possible control by cell Ca2+ and cAMP.

Distal kidney cells (A6) from Xenopus laevis were cultured to confluency on porous supports. Tissues were mounted in Ussing-type chambers to measure short-circuit current (Isc), transepithelial conductance and capacitance, and to analyse the fluctuation in Isc. In the absence of apical NaCl, but with normal basolateral NaCl Ringer's solution, extracellular addition of ATP, oxytocin, a membrane-permeant cAMP derivative, and forskolin produced a transient increase of the electrical parameters. Noise analysis revealed a spontaneous Lorentzian component. All responses depend strictly on the presence of basolateral Cl- and are caused by the activation of an apical (CFTR type) Cl- permeability. Repetitive treatment with ATP (or oxytocin) resulted in refractoriness. ATP and oxytocin acted antagonistically, whereas cAMP and ATP had additive effects. Incubation with the vesicular Ca2+ pump inhibitor thapsigargin or application of the Ca2+ channel blocker nifedipine elicited finite but variable Cl- channel activity. After treatment with nifedipine or thapsigargin, the response to oxytocin was severely impaired. We speculate that not only cAMP but also cell Ca2+ plays a crucial role in the activation of CFTR in A6. Ca2+ may be multifunctional but the rise in capacitance (apical area) observed with all stimulants strongly suggests its involvement in, and contribution to, exocytosis in the process of the CFTR-mediated transcellular Cl- movements.

Apical Cl- channels in A6 cells.

Short-circuit current (Isc), transepithelial conductance (Gt), electrical capacitance (CT) and the fluctuation in Isc were analyzed in polarized epithelial cells from the distal nephron of Xenopus laevis (A6 cell line). Tissues were incubated with Na+- and Cl--free solutions on the apical surface. Basolateral perfusate was NaCl-Ringer. Agents that increase cellular cAMP evoked increases in Gt, CT, Isc and generated a Lorentzian Isc-noise. The responses could be related to active, electrogenic secretion of Cl-. Arginine-vasotocin and oxytocin caused a typical peak-plateau response pattern. Stimulation with a membrane-permeant nonhydrolyzable cAMP analogue or forskolin showed stable increases in Gt with only moderate peaking of Isc. Phosphodiesterase inhibitors also stimulated Cl- secretion with peaking responses in Gt and Isc. All stimulants elicited a spontaneous Lorentzian noise, originating from the activated apical Cl- channel, with almost identical corner frequency (40-50 Hz). Repetitive challenge with the hormones led to a refractory behavior of all parameters. Activation of the cAMP route could overcome this refractoriness. All agents caused CT, a measure of apical membrane area, to increase in a manner roughly synchronous with Gt. These results suggest that activation of the cAMP-messenger route may, at least partly, involve exocytosis of a vesicular Cl- channel pool. Apical flufenamate depressed Cl- current and conductance and apparently generated blocker-noise. However, blocking kinetics extracted from noise experiments could not be reconciled with those obtained from current inhibition, suggesting the drug does not act as simple open-channel inhibitor.

Na+ dependence of single-channel current and channel density generate saturation of Na+ uptake in A6 cells.

In high-resistance, salt-absorbing epithelia the apical amiloride-sensitive Na+ channel is the key site for regulation of salt and water balance. The saturation of macroscopic Na+ transport through these channels was investigated using A6 epithelial monolayers. The relation between transepithelial Na+ transport (INa) and apical Na+ concentration ([Na+]ap) under short-circuit conditions was studied. Michaelis-Menten analysis of the saturable short-circuit current (Isc) yielded an apparent Michaelis-Menten constant (KmI) of 5 mmol/l and a maximal current (Imax) of 8 microA/cm2. The microscopic parameters underlying INa, namely the single-channel current (i) and the open channel density (No), were investigated by the analysis of current fluctuations induced by the electroneutral amiloride analogue CDPC (6-chloro-3, 5-diaminopyrazine-2-carboxamide). A two-state model analysis yielded the absolute values of i (0.18 +/- 0.01 pA) and No (65.38 +/- 9.57 million channels/cm2 of epithelium) at [Na+]ap = 110 mmol/l containing 50 mumol/l CDPC. Our data indicate that in A6 cells both i and No depend on [Na+]ap. Between 3 and approximately 20 mmol/l the density of conducting pores, No, decreases sharply and behaves again as an almost [Na+]ap-independent parameter at higher [Na+]ap. The single-channel current clearly saturates with an apparent Michaelis-Menten constant, Kmi, of approximately 17 mmol/l. Thus, the [Na+]ap dependence of No as well as the limited transport capacity of the amiloride-sensitive Na+ channel are both responsible for the saturation of INa.

The H+ pump in frog skin (Rana esculenta): identification and localization of a V-ATPase.

We here report on studies on the frog skin epithelium to identify the nature of its excretory H+ pump by comparing transport studies, using inhibitors highly specific for V-ATPases, with results from immunocytochemistry using V-ATPase-directed antibodies. Bafilomycin A1 (10 microM) blocked H+ excretion (69 +/- 8% inhibition) and therefore Na+ absorption (61 +/- 17% inhibition after 60 min application, n = 6) in open-circuited skins bathed on their apical side with a 1 mm Na2SO4 solution, "low-Na+ conditions" under which H+ and Na+ fluxes are coupled 1:1. The electrogenic outward H+ current measured in absence of Na+ transport (in the presence of 50 microM amiloride) was also blocked by 10 microM bafilomycin A1 or 5 microM concanamycin A. In contrast, no effects were found on the large and dominant Na+ transport (short-circuit current), which develops with apical solutions containing 115 mm Na+ ("high-Na+ conditions"), demonstrating a specific action on H+ transport. In immunocytochemistry, V-ATPase-like immunoreactivity to the monoclonal antibody E11 directed to the 31-kDa subunit E of the bovine renal V-ATPase was localized only in mitochondria-rich cells (i) in their apical region which corresponds to apical plasma membrane infoldings, and (ii) intracellularly in their neck region and apically around the nucleus. In membrane extracts of the isolated frog skin epithelium, the selectivity of the antibody binding was tested with immunoblots. The antibody labeled exclusively a band of about 31 kDa, very likely the corresponding subunit E of the frog V-ATPase. Our investigations now deliver conclusive evidence that H+ excretion is mediated by a V-ATPase being the electrogenic H+ pump in frog skin.

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.

K+ CHANNEL PERMEATION AND BLOCK IN THE MIDGUT EPITHELIUM OF THE TOBACCO HORNWORM MANDUCA SEXTA

The K+-secreting larval midgut of Manduca sexta in vitro was voltage- or current-clamped. In contrast to Tl+, NH4+ and Na+, both Rb+ and K+ generated a short-circuit current, although with different saturation kinetics. The dependence of the short-circuit current on Rb+/K+ mole fraction gave no evidence for multi-ion occupation of the basolateral K+ channels. After 'functionally' eliminating the apical membranes using the ionophore amphotericin B and the 'apical K+ pump' blockers trimethyltin chloride or Tl+, the K+ channels could be more closely investigated. By measuring zero-current potentials, permeability ratios PX/PK were estimated using an adapted version of the Goldman­Hodgkin­Katz voltage equation. Their sequence was K+ (1) = Tl+ > Rb+ (0.38) > NH4+ (~0.3) > Cs+ (0.03) > Na+ (~0). The K+ channels could not be blocked by basally applied Cs+, Na+ or tetraethylammonium. Blockade of K+ current by Ba2+ was typically voltage-dependent, but only at moderate transbasal voltages. The relative electrical distance delta of the Ba2+ binding site from the basal channel opening was determined to be 0.2. At zero transbasal voltage, the apparent inhibition constant for barium KBa* was 1.7 mmol l-1.

AN INVESTIGATION OF THE MIDGUT K+ PUMP OF THE TOBACCO HORNWORM (MANDUCA SEXTA) USING SPECIFIC INHIBITORS AND AMPHOTERICIN B

Active K+ secretion in isolated posterior midguts of Manduca sexta was studied by measuring the short-circuit current. One aim of this study was to verify the postulate from biochemical reports that the cooperative apical arrangement of a vacuolar-type H+-ATPase (V-ATPase) and a K+/H+ antiporter drive the short-circuit current. Hence, we tested several specific inhibitors of the V-ATPase on the in vitro midgut preparation. Nitrate, bafilomycin A1, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) and amiloride all reduced the short-circuit current. This suggests that the H+-ATPase is involved in transepithelial K+ secretion. However, even at relatively high doses of these inhibitors, the block of the short-circuit current was not complete. Two other agents, thallium ions (Tl+, at millimolar concentrations) and trimethyltin chloride (TMT, 50 µmol l-1), did abolish the short-circuit current. Apical, but not basal, use of the ionophore amphotericin B largely eliminated the short-circuit current. This supports the view that the current-generating source resides in the apical membranes. An apical (and probably intracellular) site of action for NO3-, Tl+ and TMT is suggested by the observation that basal amphotericin B is needed for blockage by NO3- but does not, however, influence the effect of Tl+ and TMT. Likely sites of action are the V-ATPase (for nitrate and TMT) and the K+/H+ antiporter (for Tl+).

INSECT ION HOMEOSTASIS.

The constant composition of body fluids in insects is maintained by the cooperative interaction of gastrointestinal and urinary tissues. Water follows ionic movements, which are driven by the basolateral Na+/K+-ATPase and/or the apical 'K+(or Na+) pump'. The latter now is thought to be the functional expression of a parallel arrangement of a proton-motive V-ATPase and a K+(or Na+)/nH+ antiport. This review focuses on the pathways for the movement of monovalent inorganic ions through epithelia involved in ion homeostasis. A graphical summary compares the principal findings with respect to cation secretion in lepidopteran caterpillar midgut goblet cells (K+) and in brush-border cells of Malpighian tubules (K+, Na+).

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.

K+ current stimulation by Cl- in the midgut epithelium of tobacco hornworm (Manduca sexta). I. Kinetics and effect of Cl(-)-site-specific agents.

Goblet cells in the midgut epithelium of the tobacco hornworm (Manduca sexta larva, 5th instar) actively secrete K+. This can be measured as short-circuit current (Isc) when the tissue is mounted in an Ussing chamber and bathed in K(+)-rich standard saline containing 32 mmol K+.l-1. Isc depends strictly on basolateral (i.e. haemolymph side) K+ and is therefore termed K+ current, IK. Basolateral, but not apical, chloride, bromide and iodide stimulate IK when compared to the baseline current recorded with gluconate-, nitrate- or thiocyanate-containing salines. So-called "Cl(-)-specific" transport inhibitors (frusemide, 9-anthracene carboxylic acid, diphenylamine carboxylic acid and 4,4'-diisothiocyana-to-stilbene-2,2'-disulphonic acid) reduce IK when added to the basolateral bath, whether Cl- or gluconate is the principal ambient anion. Cl- stimulates IK according to saturation kinetics. The Michaelis-Menten-type, K+ concentration-dependent, saturation of IK is altered in a highly specific manner when gluconate is replaced by Cl-: maximal K+ current, as well as the apparent Michaelis constant, are increased by a factor of 4. Since IK develops in these conditions exclusively via basolateral, Ba(2+)-blockable K+ channels, these results can be understood if it is assumed that haemolymph Cl- interferes with the K+ channel by simultaneously lowering the binding affinity for K+ ions and increasing their subsequent transfer rate across the basolateral goblet cell membrane.

K+ current stimulation by Cl- in the midgut epithelium of tobacco hornworm (Manduca sexta). II. Analysis of Ba(2+)-induced K+ channel conduction noise.

Chloride-stimulated K+ secretion by Manduca sexta midgut (5th-instar larvae) was measured as K(+)-carried short-circuit current of the tissue mounted in an Ussing chamber. "Microscopic" parameters, such as single-channel current and channel density for the rate-determining passive transport step across the basolateral goblet cell membrane (i.e. K+ channels), were estimated by means of current-fluctuation analysis of the K+ channel blockade by haemolymph-side Ba2+ ions. Ba2+ was equally effective with Cl- or gluconate (Glu-) as the principal ambient anion. The Ba(2+)-induced K+ channel conduction noise is reflected by a Lorentzian, or relaxation, noise component in the power spectrum of the K+ current fluctuations. A reduced Lorentzian plateau value, but an unchanged corner frequency, were observed when Cl- was replaced by Glu-. The results from the analysis of a "two-state" model of K+ channel block by Ba2+, with respect to the anion-replacement effects, suggest that the observed changes in K+ current and Lorentzian plateau value mirror a complex change of the underlying parameters: Cl- omission reduces single channel current but increases channel density so that the product of single channel current and channel density is smaller in Glu- than in Cl-. It seems likely that basolateral K+ channels (1) are subject to anionic gating ligands, and (2) depend on anions with respect to the rate of K+ transfer through an open K+ channel.

A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane.

In this paper we demonstrate that a vacuolar-type H(+)-ATPase energizes secondary active transport in an insect plasma membrane and thus we provide an alternative to the classical concept of plasma membrane energization in animal cells by the Na+/K(+)-ATPase. We investigated ATP-dependent and -independent vesicle acidification, monitored with fluorescent acridine orange, in a highly purified K(+)-transporting goblet cell apical membrane preparation of tobacco hornworm (Manduca sexta) midgut. ATP-dependent proton transport was shown to be catalyzed by a vacuolar-type ATPase as deduced from its sensitivity to submicromolar concentrations of bafilomycin A1. ATP-independent amiloride-sensitive proton transport into the vesicle interior was dependent on an outward-directed K+ gradient across the vesicle membrane. This K(+)-dependent proton transport may be interpreted as K+/H+ antiport because it exhibited the same sensitivity to amiloride and the same cation specificity as the K(+)-dependent dissipation of a pH gradient generated by the vacuolar-type proton pump. The vacuolar-type ATPase is exclusively a proton pump because it could acidify vesicles independent of the extravesicular K+ concentration, provided that the antiport was inhibited by amiloride. Polyclonal antibodies against the purified vacuolar-type ATPase inhibited ATPase activity and ATP-dependent proton transport, but not K+/H+ antiport, suggesting that the antiporter and the ATPase are two different molecular entities. Experiments in which fluorescent oxonol V was used as an indicator of a vesicle-interior positive membrane potential provided evidence for the electrogenicity of K+/H+ antiport and suggested that more than one H+ is exchanged for one K+ during a reaction cycle. Both the generation of the K+ gradient-dependent membrane potential and the vesicle acidification were sensitive to harmaline, a typical inhibitor of Na(+)-dependent transport processes including Na+/H+ antiport. Our results led to the hypothesis that active and electrogenic K+ secretion in the tobacco hornworm midgut results from electrogenic K+/nH+ antiport which is energized by the electrical component of the proton-motive force generated by the electrogenic vacuolar-type proton pump.

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.

The chloride-stimulated K(+)-secretion by insect midgut and its modification in the presence of osmotic gradients: a short-circuit current and noise-analysis study.

K(+)-secretion in the midgut of the larval moth, Manduca sexta, was studied by measuring the kinetics of the lumen-directed short-circuit current (Isc) and the conduction noise from basolateral K+ channel block by Ba2+. Hemolymph chloride as well as hypotonicity both stimulate this K+ current (IK). The kinetic nature of the stimulation is, however, different in each case. Analysis of blocker noise supports, to a large degree, the interpretation obtained from kinetics, namely: chloride ions do not act via changes in cell volume but influence ion turnover and channel number.

A novel synergistic stimulation of Na+-transport across frog skin (Xenopus laevis) by external Cd2+- and Ca2+-ions.

Isolated skin of the clawed frog Xenopus laevis was mounted in an Ussing-chamber. The transcellular sodium-current (INa) was identified either as amiloride-blockable (10(-3) mol/l) short-circuit current (ISC), or by correcting ISC for the shunt-current obtained with mucosal Tris. A dose of 10 mmol/l Cd2+ applied to the mucosal side increased the current by about 70%. The half-maximal effect was reached at a Cd2+-concentration of 2.6 mmol/l (in NaCl-Ringer). The quick and fully reversible effect of Cd2+ could not be seen when 10(-3) mol/l amiloride was placed in the outer, Na+-containing solution, nor when Na+ was replaced by Tris. This suggests that Cd2+ stimulates INa. Cd2+ interfered with the Na+-current self-inhibition, and therefore with the saturation of INa by increasing the apparent Michaelis constant (KNa) of this process. The "INa recline" after stepping up mucosal [Na+] was much reduced in presence of Cd2+. Ca2+-ions on the mucosal side had an identical effect to Cd2+, and 10 mmol/l Ca2+ increase INa by about 100%. The half-maximal effect was obtained with 4.4 mmol/l Ca2+. The mechanism of INa-stimulation by Ca2+ did not seem to differ from that of Cd2+. Thus, although of low Na+-transport capacity, Xenopus skin appears to be as good a model for Na+-transporting epithelia as Ranidae skin, with the exception of the calcium effect which, so far, has not been reported for Ranidae.

Current-noise analysis of the basolateral route for K+ ions across a K+-secreting insect midgut epithelium (Manduca sexta).

The isolated midgut of a lepidopteran larva (Manduca sexta), 5th instar was investigated with voltage-clamp and fluctuation analysis techniques. With high K+ insect saline on both sides the outward-directed short-circuit current (Isc) was carried by K+ (IK) from serosal to mucosal compartment. IK could be blocked, in a dose-dependent manner by serosal Ba2+ ions. There was no current with serosal Na+. Noise analysis of IK revealed a Lorentzian component in the power spectrum when Ba2+ was present in the serosal solution. The Ba2+/receptor kinetics show pseudo-first order characteristics only at low [Ba2+]s. For [Ba2+]s greater than KBa, the apparent Ba2+ association rate decreases with a hyperbolic course as a function of serosal [Ba2+] which could indicate some "substrate-inhibition"-like interaction of Ba2+ at its receptor site. It is concluded that the serosal membranes of the K+-secreting intestinal cells contain the common type of Ba2+-blockable K+ channel which provides the serosal pathway for K+ during secretion which is ultimately driven by the mucosally-located electrogenic K+-ATPase.