Renal cells exposed to cadmium in vitro and in vivo: normalizing gene expression data.

Cadmium (Cd) is a toxic metal with a long half-life in biological systems. This half-life is partly as a result of metallothioneins (MTs), metal-binding proteins with a high affinity for Cd. The high retention properties of the kidneys reside in proximal tubular cells that possess transport mechanisms for Cd-MT uptake, ultimately leading to more Cd accumulation. Researchers have studied MT-metal interactions using various techniques including quantitative real-time PCR (qPCR), an efficient tool for quantifying gene expression. Often a poor choice of reference genes, which is represented by their instability and condition dependency, leads to inefficient normalization of gene expression data and misinterpretations. This study demonstrates the importance of an efficient normalization strategy in toxicological research. A selection of stable reference genes was proposed in order to acquire reliable and reproducible gene quantification under metal stress using MT expression as an example. Moreover, in vitro and in vivo setups were compared to identify the influence of toxicological compounds in function of the experimental design. This study shows that glyceraldehyde-3-phosphate dehydrogenase (Gapdh), tyrosine monooxygenase/tryptophan5-monooxygenase activation-protein, zeta polypeptide (Ywhaz) and beta-actin (Actb) are the most stable reference genes in a kidney proximal tubular cell line exposed to moderate and high Cd concentrations, applied as CdCl2 . A slightly different sequence in reference gene stability was found in renal cells isolated from rats in vivo exposed to Cd. It was further shown that three reference genes are required for efficient normalization in this experimental setup. This study demonstrates the importance of an efficient normalization strategy in toxicological research.

Store-operated Ca2+-channels are sensitive to changes in extracellular pH.

The sensitivity of store-operated Ca(2+)-entry to changes in the extra- and intracellular pH (pH(o) and pH(i), respectively) was investigated in SH-SY5Y human neuroblastoma cells. The intracellular Ca(2+)-stores were depleted either with 1 mM carbachol (CCH) or with 2 microM thapsigargin (TG). Extracellular acidification suppressed both the CCH- and TG-mediated Ca(2+)-entry while external alkalinization augmented both the CCH- and the TG-induced Ca(2+)-influx. Mn(2+)-quenching experiments revealed that the rates of Ca(2+)-entry at the thapsigargin- or carbachol-induced plateau were both accelerated at pH(o) 8.2 and slowed down at pH(o) 6.8 with respect to the control at pH(o) 7.4. Alteration of pH(o) between 6.8 and 8.2 did not have any significant prompt effect on pH(i) and changes in pH(i) left the CCH-induced Ca(2+)-entry unaffected. These findings demonstrate that physiologically relevant changes in pH(o) affect the store-operated Ca(2+)-entry in SH-SY5Y cells and suggest that endogenous pH(o) shifts may regulate cell activity in situ via modulating the store-operated Ca(2+)-entry.

The effects of endogenous diuretic and antidiuretic peptides and their second messengers in the Malpighian tubules of Tenebrio molitor: an electrophysiological study.

The Malpighian tubules of Tenebrio molitor provide a model system for interpreting the actions of endogenous diuretic and antidiuretic peptides. The effects of diuretic (Tenmo-DH(37)) and antidiuretic (Tenmo-ADFa) peptides and their respective second messengers (cyclic AMP and cyclic GMP) on basolateral (V(bl)) and transepithelial (V(te)) potentials of Tenebrio Malpighian tubules were determined using conventional microelectrodes. In the presence of 6 mmol l(-1) Ba(2+), Tenmo-DH(37) (100 nmol l(-1)) reversibly hyperpolarized V(bl) and depolarized V(te). A similar response was seen with the addition of 1 mmol l(-1) cyclic AMP; however, the apical membrane potential (V(ap)) then showed a hyperpolarization, whereas a depolarization of V(ap) was observed with Tenmo-DH(37). Bafilomycin A(1) (5 micromol l(-1)) inhibited fluid secretion of stimulated tubules and reversed the hyperpolarization of V(bl) in response to Tenmo-DH(37). In response to 100 nmol l(-1) Tenmo-ADFa or 1 mmol l(-1) cyclic GMP, V(bl) and V(te) depolarized, although cyclic GMP affected membrane potentials somewhat differently by causing an initial hyperpolarization of V(bl) and V(te). In high [K(+)]-low [Na(+)] Ringer, 1 mmol l(-1) amiloride decreased fluid secretion rates, and depolarized both V(bl) and V(te). Amiloride significantly decreased luminal pH in paired experiments, indicating the presence of a K(+)/nH(+) exchanger in tubule cells of Tenebrio. The results suggest that the endogenous factors and their second messengers stimulate/inhibit fluid secretion by acting on the apical V-ATPase, basolateral K(+) transport, and possibly Cl(-) transport.

Characterization of Na(+) currents in isolated dorsal unpaired median neurons of Locusta migratoria and effect of the alpha-like scorpion toxin BmK M1.

A primary cell culture was developed for efferent dorsal unpaired median (DUM) neurons of the locust. The isolated somata were able to generate Tetrodotoxin (TTX)-sensitive action potentials in vitro. The alpha-like scorpion toxin BmK M1, from the Asian scorpion Buthus martensi Karsch, prolonged the duration of the action potential up to 50 times. To investigate the mechanism of action of BmK M1, the TTX-sensitive voltage gated Na(+) currents were studied in detail using the whole cell patch clamp technique. BmK M1 slowed down and partially inhibited the inactivation of the TTX-sensitive Na(+) current in a dose dependent manner (EC50=326.8+/-34.5 nM). Voltage and time dependence of the Na(+) current were described in terms of the Hodgkin-Huxley model and compared in control conditions and in the presence of 500 nM BmK M1. The BmK M1 shifted steady state inactivation by 10.8 mV to less negative potentials. The steady state activation was shifted by 5.5 mV to more negative potentials, making the activation window larger. Moreover, BmK M1 increased the fast time constant of inactivation, leaving the activation time constant unchanged. In summary, BmK M1 primarily affected the inactivation parameters of the voltage gated Na(+) current in isolated locust DUM neurons.

K(+) transport in Malpighian tubules of Tenebrio molitor L: a study of electrochemical gradients and basal K(+) uptake mechanisms.

Malpighian tubules of the mealworm Tenebrio molitor were isolated for intracellular measurement of basolateral (V(bl)) and, indirectly, apical (V(ap)) membrane potentials. In control Ringer (50 mmol l(-1) K(+), 140 mmol l(-1) Na(+)), V(bl) was 24 mV, cell negative, and V(ap) was 48 mV, cell negative with reference to the lumen. Ion substitution experiments involving K(+) and Na(+) indicated that both V(bl) and V(ap) were sensitive to the bathing K(+) concentration, with the change in V(ap) being 60-77% that of V(bl). A 10-fold drop in bath [K(+)] irreversibly decreased fluid secretion rates from 6.38+/-0.95 nl x min(-1) (mean +/- S.E.M.) to 1.48+/-0.52 nl x min(-1) (N=8). In the presence of 6 mmol l(-1) Ba(2+), a blocker of basal K(+) channels, fluid secretion rates reversibly decreased and the hyperpolarization of both V(bl) and V(ap) seen in 50 mmol l(-1) and 140 mmol l(-1) K(+) indicated a favourable electrochemical gradient for basal K(+) entry. In 5 mmol l(-1) K(+), Ba(2+) induced two different responses: V(bl) either hyperpolarized by approximately 10 mV or depolarised by approximately 14 mV, according to the electrochemical gradient for K(+), which was either inward or outward in low bath [K(+)]. Rubidium, a 'permeant' potassium substitute, caused a hyperpolarization of V(bl), indicating the specificity of K(+) channels found in Tenebrio tubule cells. Other possible K(+) uptake mechanisms located in the basolateral membrane were investigated. Blocking of the putative electroneutral Na(+)/K(+)/2Cl(-) cotransporter by 10 micromol l(-1) bumetanide reversibly decreased fluid secretion rates, with no detectable change in membrane potentials. Ouabain (1 mmol l(-1)), an Na(+)/K(+)-ATPase inhibitor, irreversibly decreased fluid secretion rates but had no effect on electrical potential differences either in the absence or presence of Ba(2+). The results implicate K(+) channels, the Na(+)/K(+)/2Cl(-) contransporter and the Na(+)/K(+)-ATPase in basal K(+) and fluid transport of Tenebrio tubule cells.

K(+) transport in Malpighian tubules of Tenebrio molitor L: is a K(ATP) channel involved?

The presence of ATP-regulated K(+) (K(ATP)) channels in Tenebrio molitor Malpighian tubules was investigated by examining the effect of glibenclamide on both fluid secretion and basolateral membrane potentials (V(bl)). Glibenclamide, a K(ATP) channel blocker, slowed fluid secretion of Tenebrio tubules. In low bath K(+) concentration (5 mmol l(-1)), glibenclamide either hyperpolarized or depolarized V(bl), resembling the effect seen with Ba(2+). Subsequent addition of 6 mmol l(-1) Ba(2+) caused a further hyper- or depolarization of V(bl). In control Ringer (50 mmol l(-1) KCl, 90 mmol l(-1) NaCl), glibenclamide had no visible effect on V(bl). The effect of ouabain was investigated in low bath [K(+)] in the presence of Ba(2+). V(bl) responded by a small but significant hyperpolarization from -51+/-4 mV to -56+/-4 mV (n=16, P<0.001) in response to 1 mmol l(-1) ouabain. Repeating the experiments in the presence of both glibenclamide and Ba(2+) resulted in a depolarization of V(bl) when ouabain was added. In low bath [K(+)] (high Na(+)), the Na(+)/K(+)-ATPase is expected to function at a high rate. In the presence of Ba(2+), replacing Na(+) by K(+) rapidly depolarized V(bl), but this was followed by a repolarization. Repeating the experiments in the presence of glibenclamide markedly reduced the depolarizing effect and abolished the repolarization, with a gradual decrease in the sensitivity of V(bl) to the surrounding [K(+)]. These results suggest the presence of K(ATP) channels in the basolateral membrane. Glibenclamide had no visible effect on V(bl) in high K(+) or in the absence of Ba(2+), indicating that other highly conductive K(+) channels may mask the effect on K(ATP) channels. This is the first demonstration of the presence of K(ATP) channels in an insect epithelium.

Effect of Bacillus thuringiensis Cry1 toxins in insect hemolymph and their neurotoxicity in brain cells of Lymantria dispar.

Little information is available on the systemic effects of Bacillus thuringiensis toxins in the hemocoel of insects. In order to test whether B. thuringiensis-activated toxins elicit a toxic response in the hemocoel, we measured the effect of intrahemocoelic injections of several Cry1 toxins on the food intake, growth, and survival of Lymantria dispar (Lepidoptera) and Neobellieria bullata (Diptera) larvae. Injection of Cry1C was highly toxic to the Lymantria larvae and resulted in the complete inhibition of food intake, growth arrest, and death in a dose-dependent manner. Cry1Aa and Cry1Ab (5 microg/0.2 g [fresh weight] [g fresh wt]) also affected growth and food intake but were less toxic than Cry1C (0.5 microg/0.2 g fresh wt). Cry1E and Cry1Ac (5 microg/0.2 g fresh wt) had no toxic effect upon injection. Cry1C was also highly toxic to N. bullata larvae upon injection. Injection of 5 microg/0.2 g fresh wt resulted in rapid paralysis, followed by hemocytic melanization and death. Lower concentrations delayed pupariation or gave rise to malformation of the puparium. Finally, Cry1C was toxic to brain cells of Lymantria in vitro. The addition of Cry1C (20 microg/ml) to primary cultures of Lymantria brain cells resulted in rapid lysis of the cultured neurons.

An antidiuretic factor in the forest ant: purification and physiological effects on the Malpighian tubules.

Formica polyctena antidiuretic factor (FopADF) was purified from a 15% trifluoroacetic acid (TFA) extract of the abdomens of 150,000 worker ants. After solid phase extraction of the crude extract and reversed-phase HPLC on two C(18) columns, an antidiuretic factor was isolated. Tested at a concentration of 1.0 ant-equivalents/µl (ant-eq/µl), the factor reversibly inhibited fluid secretion of isolated Malpighian tubules to 29+/-5% (mean+/-SE, n=24) of the control value. The same concentration of FopADF reversibly depolarized both the basolateral membrane potential (V(bl)), from -21+/-2 mV to -3+/-1 mV (n=5), and the apical membrane potential (V(ap)), from -65+/-5 mV to -20+/-5 mV (n=5). Similar effects on fluid secretion and V(ap) were caused by a TFA extract of the haemolymph of ants with non-secreting tubules. Unfortunately, further purification of FopADF on a C(4) column led to a loss of activity in the fluid secretion assay. This is the first time an endogenous antidiuretic factor acting directly on Malpighian tubules has been partially purified and shown to depolarize the tubule cell membranes.

Effect of Bacillus thuringiensis toxins on the midgut of the nun moth Lymantria monacha.

Three steps of the proposed mode of action of Bacillus thuringiensis toxins have been studied in Lymantria monacha. We demonstrated that only the toxins that caused typical pathological changes in midgut epithelial cells and bound to the midgut brush border membrane were able to drastically reduce the midgut transepithelial voltage of the nun moth.

Intracellular and luminal pH measurements of Malpighian tubules of the mosquito Aedes aegypti: the effects of cAMP.

In order to understand the critical role that hydrogen ions play in fluid secretion in Malpighian tubules, intracellular and luminal pH and K+ measurements were performed in isolated Malpighian tubules of the yellow fever mosquito (Aedes aegypti). The intracellular pH was 7.03+/-0.05 (n=15 Malpighian tubules (MT)) and the luminal pH was 7.19+/-0.09 (n=99 MT) when bathed in saline at a pH of 7.0. The lumen potential is positive, thus net proton secretion into the lumen is active. The intracellular and the luminal K+ concentrations were 75+/- 9 mM (n=15) and 102+/-13 mM (n=9 MT) respectively. Cyclic AMP analogues accelerated fluid secretion and at the same time acidified the cell without affecting the luminal pH. Both effects were abolished by an isomer of adenosine-3',5' cyclic monophosphothioate (cAMPS), the Rp-cAMPS, known to inhibit protein kinase A. The results suggest that in the presence of cAMP the properties of the cation/H+ exchanger are affected and that this may be a result of phosphorylation of a Na+/2H+ antiporter located on the apical membrane.

Partial identification of a peptide that stimulates the primary urine production of single isolated Malpighian tubules of the forest ant, Formica polyctena.

A peptide was purified from a 10% trifluoroacetic acid (TFA) head/thorax extract of 300,000 ants with high performance liquid chromatography (HPLC). Fluid secretion assay of single isolated Malpighian tubules was used as a bioassay. The purity of F. polyctena diuretic peptide (FopDP) after a two step HPLC protocol was confirmed by means of mass spectrometry and revealed a molecular mass of 7514 daltons. Due to lack of material, no enzymatic digestion could be performed and the sequence of only the first 25 amino acids could be determined: VPKYENCVSEVLPAGDRQRCVKVTC. A computer search of sequence data banks did not reveal any significant similarity between FopDP and other known insect diuretic peptides.FopDP had no effect on the basolateral membrane potential and depolarised the apical membrane potential of the Malpighian tubule cells. This effect as well as the stimulatory effect on the primary urine formation in the Malpighian tubule of the ant, could be mimicked with A23187, a calcium ionophore, and by thapsigargin, an inhibitor of the endoplasmic reticulum calcium ATPase. FopDP did not stimulate the cAMP content. The results suggest that FopDP uses an increase of intracellular calcium as cellular transduction mechanism.

Changes in Permeability of Brush Border Membrane Vesicles from Spodoptera littoralis Midgut Induced by Insecticidal Crystal Proteins from Bacillus thuringiensis.

Bacillus thuringiensis insecticidal crystal proteins (ICPs) are thought to induce pore formation in midgut cell membranes of susceptible insects. Cry1Ca, which is significantly active in Spodoptera littoralis, made brush border membrane vesicles permeable to KCl (osmotic swelling was monitored by the light scattering technique); the marginally active ICPs Cry1Aa, Cry1Ab, and Cry1Ac did not.

Rapid activation of KATP channels by aldosterone in principal cells of frog skin.

1. In epithelial cells of frog skin, potassium ions are recycled across the basolateral membrane via an inward-rectifier, ATP-sensitive K+ channel (KATP channel). In this study, we show that aldosterone has a stimulatory effect on KATP channel activity and we have investigated the involvement of Na+-H+ exchange and intracellular pH (pHi) in this phenomenon. 2. Aldosterone (10 nM) produced an increase in the open probability of the KATP channel within 15 min from 0.21 +/- 0.05 to 0.93 +/- 0.10 (n = 8), measured in cell-attached patches. Aldosterone also increased the tolbutamide-sensitive K+ current across the basolateral membrane within 30 min from 17.2 +/- 1.9 to 30.3 +/- 1.6 microA cm-2 (n = 8) in nystatin-permeabilized whole skins. 3. The KATP channel is very sensitive to variations in cytosolic pH within the physiological range 7.0-7.4. 4. The intracellular pH of principal cells is regulated by Na+-H+ exchange, and the stimulatory effect of aldosterone on KATP channel activity was abolished by amiloride (100 microM) added on the basolateral side of the epithelium either before or after aldosterone treatment. 5. We propose that aldosterone activates the KATP channels via stimulation of Na+-H+ exchange. The rapidity of aldosterone activation of KATP channels is presented as evidence for a novel non-genomic steroid hormone effect on epithelial ion transport.

Cross-talk between ATP-regulated K+ channels and Na+ transport via cellular metabolism in frog skin principal cells.

Isolated frog skin epithelium, mounted in an Ussing chamber and bathed in standard NaCl Ringer solution, recycles K+ across the basolateral membrane of principal cells through an inward-rectifier K+ channel (Kir) operating in parallel with a Na+-K+-ATPase pump. Here we report on the metabolic control of the Kir channel using patch clamping, short-circuit current measurement and enzymatic determination of cellular (ATP (ATPi). 2. The constitutively active Kir channel in the basolateral membrane has the characteristics of an ATP-regulated K+ channel and is now classed as a KATP channel. In excised inside-out patches the open probability (Po) of KATP channels was reduced by ATPi with half-maximum inhibition at an ATPi concentration of 50 microM. 3. ATPi measured (under normal Na+ transport conditions) with luciferin-luciferase was 1.50 +/- 0.23 mM (mean +/- S.E.M.; range, 0.4-3.3 mM n = 11). Thus the KATP channel would be expected to be inactive in intact cells if ATPi was the sole regulator of channel activity. KATP channels which were inactivated by 1 mM ATPi in excised patches could be reactivated by addition of 100 microM ADP on the cytosolic side. When added alone, ADP blocks this channel with half-maximal inhibition at [ADPi] > 5 mM. 4. Sulphonylureas inhibit single KATP channels in cell-attached patches as well as the total basolateral K+ current measured in frog skin epithelia perforated with nystatin on the apical side. 5. Na+-K+-ATPase activity is a major determinant of cytosolic ATP. Blocking the pump activity with ouabain produced a time-dependent increase in ATPi and reduced the open probability of KATP channels in cell-attached membranes. 6. We conclude that the ratio of ATP/ADP is an important metabolic coupling factor between the rate of Na+-K+ pumping and K+ recycling.

Maxi K+ channels in the basolateral membrane of the exocrine frog skin gland regulated by intracellular calcium and pH.

With the single-channel patch-clamp technique we have identified Ca2+-sensitive, high-conductance (maxi) K+ channels in the basolateral membrane (BLM) of exocrine gland cells in frog skin. Under resting conditions, maxi K+ channels were normally quiescent, but they were activated by muscarinic agonists or by high serosal K+. In excised inside-out patches and with symmetrical 140mmol/l K+, single-channel conductance was 200pS and the channel exhibited a high selectivity for K+ over Na+. Depolarization of the BLM increased maxi K+ channel activity. Increasing cytosolic free Ca2+ (by addition of 100nmol/l thapsigargin to the bathing solution of cell-attached patches also increased channel activity, whereas thapsigargin had no effect when added to excised inside-out patches. An increase in cytosolic free Ca2+ directly activated channel activity in a voltage-dependent manner. Maxi K+ channel activity was sensitive to changes in intracellular pH, with maximal activity at pH 7.4 and decreasing activities following acidification and alkalinization. Maxi K+ channel outward current was reversibly blocked by micromolar concentrations of Ba2+ from the cytosolic and extracellular site, and was irreversibly blocked by micromolar concentrations of charybdotoxin and kaliotoxin from the extracellular site in outside-out patches.

Mechanisms of K+ uptake across the basal membrane of malpighian tubules of Formica polyctena: the effect of ions and inhibitors.

In the presence of 6 mmol l-1 Ba2+, known to block the K+ channels in the basal membrane, a rise in bath [K+] ([K+]bl) induced an increase in intracellular K+ concentration ([K+]i) similar in amount and in time course to that obtained in the absence of Ba2+. The presence of active and passive (other than through K+ channels) K+ uptake mechanisms across the basal membrane was investigated in different bath K+ concentrations. Dihydro-ouabain (10(-3) mol l-1), a blocker of the Na+/K(+)-ATPase, tested in low bath [K+], and Sch28080 (10(-4) mol l-1), a K+/H(+)-ATPase inhibitor, were without effect on fluid secretion. Dihydro-ouabain was also without effect on electrical potential differences either in the absence or in the presence of Ba2+. Vanadate (10(-3) mol l-1), in contrast, strongly reduced fluid secretion not only in control solution but also in high-K+, Na(+)-free medium and reduced the transepithelial and the apical membrane potential differences but not the basal membrane potential difference of [K+]i. Omitting Na+ from the bathing medium, replacing Cl- by Br- or applying bumetanide (10(-5) mol l-1) inhibited fluid secretion only in a low-K+ (10 mmol l-1) medium. In 51 mmol l-1 [K+]bl, omitting Na+ was without effect and 10(-4) mol l-1 bumetanide was needed to inhibit secretion. Replacing Cl- by Br- stimulated fluid secretion at this K+ concentration. Bumetanide (10(-4) mol l-1) had no effect in 113 mmol l-1 [K+]bl. Bumetanide (10(-4) mol l-1) in 51 mmol l-1 [K+]bl did not affect membrane potentials, did not lower [K+]i and did not affect the rise in [K+]i observed on an increase in [K+]bl. The results were summarized in a model proposing that K+ channels play a dominant role in high-K+ (113 mmol l-1) bathing medium. A K+/Cl- cotransporter may become more important in 51 mmol l-1 [K+]bl and a K+/Na+/2Cl- cotransporter may gain in importance in 10 mmol l-1 [K+]bl. Active mechanisms for K+ uptake across the basal membrane seem to play no detectable role in sustaining fluid secretion. The response to vanadate might be due to an effect on the apical electrogenic H+ pump.

Effects of dinitrophenol on active-transport processes and cell membranes in the Malpighian tubule of Formica.

In Formica Malpighian tubules KCl secretion is driven by a V-type H+ ATPase in the luminal membrane in parallel with a H+/K+ antiporter. The effect of the protonophore dinitrophenol (DNP) was investigated on the isolated, symmetrically perfused tubule. DNP was applied in two different concentrations: 0.2 mmol/l and 1 mmol/l. The effects were fast and rapidly reversible. The equivalent short-circuit current (Isc) was reduced significantly to respectively 25 +/- 3% Cn = 4) and -3 +/- 7% (n = 11) of the control value when 0.2 mmol/l or 1 mmol/l was added to the bath. When 1 mmol/l DNP was applied the transepithelial resistance (Rte) decreased significantly to 74 +/- 11% of the control value (n = 11), and the luminal over basolateral voltage divider ratio (VDR), providing an estimate of luminal over basolateral membrane resistance, decreased to 37 +/- 12% of the control (n = 6). A concentration of 1 mmol/l DNP was also applied from the lumen. The decrease in Isc was significant, but much less pronounced (74 +/- 5% of control; n = 6) and no significant changes in Rte and VDR were observed. It is argued that, when the concentration in the bath is high enough, DNP may cross the cell and have a protonophoric effect not only on the mitochondria but also across the luminal cell membrane explaining the drop in transepithelial and in relative luminal membrane resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium.

UNLABELLED: Inward-rectifier K channel: using macroscopic voltage clamp and single-channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current-voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell-attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. CONCLUSIONS: under physiological ionic gradients, a 15-pS inward-rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.

Electrophysiological evidence for the presence of an apical H(+)-ATPase in Malpighian tubules of Formica polyctena: intracellular and luminal pH measurements.

Cellular and luminal pH of isolated ant Malpighian tubules were measured in different bath K+ concentrations using double-barrelled pH microelectrodes. The electrochemical gradient for H+ across the basolateral and the apical cell membranes was estimated. In control Ringer (51 mmol/l K+) cell and luminal pH were alkaline with respect to the basolateral solution: 7.77 and 7.36, respectively, versus 7.25. On lowering basolateral K+ concentration to 5 mmol/l or increasing it to 113 mmol/l, luminal pH and to a lesser extent cell pH followed: luminal pH changed to 7.14 and 7.43 and cell pH to 7.69 and 7.82, respectively. In all conditions a cell inward electrochemical gradient for protons across both membranes was observed. Increasing basolateral K+ concentration, which was positively correlated with secretion rate, decreased the cell inwardly directed apical proton gradient; moreover, the apical membrane potential difference decreased as well, from -93 mV in 5 mmol/l K+ to -65 mV in 113 mmol/l K+. Therefore the turnover rate of the electrogenic active proton pump at the apical membrane is facilitated in a high basolateral K+ concentration. The calculated electromotive force of this pump is -159 mV. Comparing the proton with the K+ electrochemical gradient, taken from another study in the same experimental conditions, we find that the apical proton electrochemical gradient can drive K+ extrusion into the lumen for each value of secretion rate.

Characteristics of the luminal proton pump in malpighian tubules of the ant.

The active pump mechanisms involved in K+ secretion of the malpighian tubules of the ant and present in the luminal membrane were investigated on isolated, luminally perfused tubules of Formica. The specific blocker for vacuolar type ATPases, bafilomycin A1, was found to half-maximally inhibit secretion at a concentration of 10(-5) mol/l when added to the lumen. N-Ethylmaleimide reduced the calculated short circuit current (Isc) to 78 and 21% of control value when added at 5 x 10(-4) mol/l, respectively, to the lumen and the bath. Reducing luminal pH inhibited Isc with a half-maximal inhibition at a luminal pH of 4.5. Acidified omeprazole, Schering compound 28080 and vanadate (both 10(-3) and 10(-4) mol/l) inhibited Isc only partially. The present data suggest that the luminal membrane of ant malpighian tubules contains a H+ pump. This pump is only poorly bafilomycin-sensitive. Furthermore, additional active transport systems responsible for secretion may be present. Part of these results have been published as abstracts.