Malignant human gliomas express an amiloride-sensitive Na+ conductance

Human astrocytoma cells were studied using whole cellpatch-clamp recording. An inward, amiloride-sensitiveNa⁺ current was identified in fourcontinuous cell lines originally derived from human glioblastoma cells(CH235, CRT, SKMG-1, and U251-MG) and in three primary cultures ofcells obtained from glioblastoma multiforme tumors (up to 4 passages).In addition, cells freshly isolated from a resected medulloblastomatumor displayed this same characteristic inward current. In contrast,amiloride-sensitive currents were not observed in normal humanastrocytes, low-grade astrocytomas, or juvenile pilocytic astrocytomas.The only amiloride-sensitive Na⁺channels thus far molecularly identified in brain are the brain Na⁺ channels (BNaCs). RT-PCRanalyses demonstrated the presence of mRNA for either BNaC1 or BNaC2 inthese tumors and in normal astrocytes. These results indicate that thefunctional expression of amiloride-sensitive Na⁺ currents is a characteristicfeature of malignant brain tumor cells and that this pathway may be apotentially useful target for therapeutic intervention.

     

Inositol polyphosphate derivative inhibits Na+ transport and improves fluid dynamics in cystic fibrosis airway epithelia

Amiloride-sensitive, epithelial Na⁺ channel (ENaC)-mediated, active absorption of Na⁺ is elevated in the airway epithelium of cystic fibrosis (CF) patients, resulting in excess fluid removal from the airway lumen. This excess fluid/volume absorption corresponds to CF transmembrane regulator-linked defects in ENaC regulation, resulting in the reduced mucociliary clearance found in CF airways. Herein we show that INO-4995, a synthetic analog of the intracellular signaling molecule, D-myo-inositol 3,4,5,6-tetrakisphosphate, inhibits Na⁺ and fluid absorption across CF airway epithelia, thus alleviating this critical pathology. This conclusion was based on electrophysiological studies, fluid absorption, and ²²Na⁺ flux measurements in CF airway epithelia, contrasted with normal epithelia, and on electrophysiological studies in Madin-Darby canine kidney cells and 3T3 cells overexpressing ENaC. The effects of INO-4995 were long-lasting, dose-dependent, and more pronounced in epithelia from CF patients vs. controls. These findings support preclinical development of INO-4995 for CF treatment and demonstrate for the first time the therapeutic potential of inositol polyphosphate derivatives. epithelial Na⁺ channels; fluid absorption     

A role for ERK1/2 in EGF- and ATP-dependent regulation of amiloride-sensitive sodium absorption

Receptor-mediated inhibition of amiloride-sensitive sodium absorption was observed in primary and immortalized murine renal collecting duct cell (mCT12) monolayers. The addition of epidermal growth factor (EGF) to the basolateral bathing solution of polarized monolayers reduced amiloride-sensitive short-circuit current (I_sc) by 15–25%, whereas the addition of ATP to the apical bathing solution decreased I_sc by 40–60%. Direct activation of PKC with phorbol 12-myristate 13-acetate (PMA) and mobilization of intracellular calcium with 2,5-di-tert-butyl-hydroquinone (DBHQ) reduced amiloride-sensitive I_sc in mCT12 monolayers by 46 ± 4% (n = 8) and 22 ± 2% (n = 8), respectively. Exposure of mCT12 cells to EGF, ATP, PMA, and DBHQ caused an increase in phosphorylation of p42/p44 (extracellular signal-regulated kinase; ERK1/2). Pretreatment of mCT12 monolayers with an ERK kinase inhibitor (PD-98059; 30 µM) prevented phosphorylation of p42/p44 and significantly reduced EGF, ATP, and PMA-induced inhibition of amiloride-sensitive I_sc. In contrast, pretreatment of monolayers with a PKC inhibitor (bisindolylmaleimide I; GF109203x; 1 µM) almost completely blocked the PMA-induced decrease in I_sc, but did not alter the EGF- or ATP-induced inhibition of I_sc. The DBHQ-mediated decrease in I_sc was due to inhibition of basolateral Na⁺-K⁺-ATPase, but EGF-, ATP-, and PMA-induced inhibition was most likely due to reduced apical sodium entry (epithelial Na⁺ channel activity). The results of these studies demonstrate that acute inhibition of amiloride-sensitive sodium transport by extracelluar ATP and EGF involves ERK1/2 activation and suggests a role for MAP kinase signaling as a negative regulator of electrogenic sodium absorption in epithelia. mitogen-activated protein kinase; epithelial ion transport; epithelial sodium channel     

Glucocorticoids stimulate ENaC upregulation in bovine mammary epithelium

Mammary epithelia produce an isotonic, low-Na⁺ fluid that is rich in nutrients. Mechanisms that account for the low electrolyte concentration have not been elucidated, although amiloride-sensitive ion transport has been reported in some situations. We hypothesized that corticosteroid exposure modulates epithelial Na⁺ channel (ENaC) expression and/or activity in bovine mammary epithelial cells. BME-UV cells were grown to confluent monolayers on permeable supports with a standard basolateral medium and apical medium of low-electrolyte, high-lactose composition that resembles the ionic composition of milk. Ion transport was assessed in modified Ussing flux chambers. Exposure to glucocorticoids (dexamethasone, cortisol, or prednisolone), but not aldosterone, increased short-circuit current (I_sc), a sensitive measure of net ion transport, whereas apical exposure to amiloride or benzamil reduced corticosteroid-induced I_sc close to basal levels. Quantitative RT-PCR indicated a glucocorticoid-induced increase in mRNA for - and -ENaC, whereas -ENaC mRNA expression was only mildly affected. Exposure to mifepristone (a glucocorticoid receptor antagonist), but not spironolactone (a mineralocorticoid receptor antagonist), precluded both the corticosteroid-induced elevation in amiloride-sensitive I_sc and the induced changes in - and -ENaC mRNA. We conclude that Na⁺ movement across mammary epithelia is modulated by corticosteroids via a glucocorticoid receptor-mediated mechanism that regulates the expression of the - and -subunits of ENaC. ENaC expression and activity could account for the low Na⁺ concentration that is typical of milk. short-circuit current; apical cation concentration; corticosteroids; mastitis; epithelial Na⁺ channel subunits     

Development of Na+ transport in the chicken colon

To evaluate the developmental changes in colonic Na⁺ transport, Na, K-ATPase activity and the sensitivity of the short-circuit current to amiloride were investigated. The amiloride-sensitive short-circuit current which represents the electrogenic, amiloride-sensitive Na⁺ transport through Na⁺ channels, was not present in chicken embryos but rose significantly after hatching in chicks which were kept on a low-salt diet. Amiloride-sensitive short-circuit current increased gradually but the plateau was not reached during the first 15 days of life. Drinking of 0.9% NaCl totally inhibited the induction of amiloride-sensitive Na⁺ transport. Na⁺, K⁺-ATPase activity increased during development but was not influenced by changes in salt intake. Na⁺ transport in chicken colon therefore undergoes profound developmental changes. The increase of Na⁺ transport refleets not only the adaptation of colonocytes to low salt intake but also the maturation of Na⁺ absorption in colon. The possible role of aldosterone in the adaptation to low-salt intake is discussed.Abbreviations LS low-salt - HS high-salt - I _sc short-circuit current     

Steroid hormone-dependent expression of blockersensitive ENaCs in apical membranes of A6 epithelia

Weak channel blocker-induced noise analysis wasused to determine the way in which the steroids aldosterone andcorticosterone stimulated apical membraneNa⁺ entry into the cells oftissue-cultured A6 epithelia. Among groups of tissues grown on avariety of substrates, in a variety of growth media, and with cells atpassages 73-112, the steroidsstimulated both amiloride-sensitive and amiloride-insensitiveNa⁺ transport as measured byshort-circuit currents in chambers perfused with either growth mediumor a Ringer solution. From baseline rates of blocker-sensitiveshort-circuit current between 2 and 7 µA/cm², transport was stimulatedabout threefold in all groups of experiments. Single channel currentsaveraged near 0.3 pA (growth medium) and 0.5 pA (Ringer) and weredecreased 6-20% from controls by steroid due to the expecteddecreases of fractional transcellular resistance. Irrespective ofbaseline transport rates, the steroids in all groups of tissuesstimulated transport by increase of the density of blocker-sensitiveepithelial Na⁺ channels (ENaCs).Channel open probability was the same in control and stimulatedtissues, averaging ~0.3 in all groups of tissues. Accordingly,steroid-mediated increases of open channel density responsible forstimulation of Na⁺ transport aredue to increases of the apical membrane pool of functional channels andnot their open probability.

     

Self-Inhibition in Amiloride-sensitive Sodium Channels in Taste Receptor Cells

Electrophysiological recording techniques were used to study the Na⁺ dependence of currents through amiloride-sensitive sodium channels (ASSCs) in rat taste cells from the fungiform and vallate papillae. Perforated patch voltage clamp recordings were made from isolated fungiform and vallate taste receptor cells (TRCs) and Na⁺ transport was measured across lingual epithelia containing fungiform or vallate taste buds in a modified Ussing chamber. In isolated fungiform TRCs that contain Na⁺ currents sensitive to the diuretic amiloride, Na⁺ ions inhibit their own influx through ASSCs, a process known as sodium self-inhibition. Due to the interaction between self-inhibition and the driving force for Na⁺ entry, self-inhibition is most evident in whole-cell recordings at Na⁺ concentrations from 50 to 75 mM. In amiloride-sensitive cells, the Na permeability is significantly higher in extracellular solutions containing 35 mM Na⁺ than in 70 or 140 mM Na⁺. Compared with the block by amiloride, the development of self-inhibition is slow, taking up to 15 s to become maximally inhibited. Approximately one third of fungiform TRCs and all vallate TRCs lack functional ASSCs. These amiloride-insensitive TRCs show no signs of self-inhibition, tying this phenomenon to the presence of ASSCs. The sulfhydryl reagent, p-hydroxymercuribenzoate (p-HMB; 200 μM), reversibly removed self-inhibition from amiloride-sensitive Na⁺ currents, apparently by modifying cysteine residues in the ASSC. Na⁺ currents in amiloride-insensitive TRCs were unaffected by p-HMB. In sodium transport studies in fungiform taste bud–containing lingual epithelia, ∼40% of the change in short-circuit current (I_sc) after addition of 500 mM NaCl to the mucosal chamber is amiloride sensitive (0.5 mM). p-HMB significantly enhanced mucosal NaCl-induced changes in these epithelia at mucosal Na⁺ concentrations of 50 mM and above. In contrast, the vallate-containing epithelia, which are insensitive to amiloride, showed no enhancement of I_sc during p-HMB treatment. These findings suggest that sodium self-inhibition is present in ASSCs in taste receptor cells where it may play a crucial role in performance of salt-sensitive pathways in taste tissue during sodium stimulation. This phenomenon may be important in the process of TRC adaptation, in the conservation of cellular resources during chronic sodium exposure, or in the gustatory response to water.     

Modulation of Na+ transport across leech skin by pesticides and heavy metals

The aim of the present study was to investigate the effects of environmental pollutants, such as heavy metals and pesticides on ion transport across the skin of the leech (Hirudo medicinalis). We wanted to examine the suitability of this epithelium as a model system for studies concerning the mechanisms of toxic action caused by environmental pollutants. For this purpose we performed Ussing chamber experiments to test three representative heavy metals and pesticides, respectively, for their effects on current flow across leech dorsal integument. Two representatives of each substance class showed distinct effects on ion transport across this epithelium. The heavy metal ions Pb²⁺ and Hg²⁺ produced a significant inhibition of amiloride-sensitive Na⁺ transport across leech skin in concentrations below or close to their limiting values in waste water. Therefore, it seems feasible to use leech skin for future investigations of the toxic actions of these heavy metals. The fact that Pb²⁺ and Hg²⁺ exerted their effects only when applied apically points to a specific action of these divalent cations on ion channels in the apical membrane. However, this inhibition does not seem to be a general feature of divalent cations because Cd²⁺ did not influence ion transport across leech skin at all. Since current flow through amiloride-sensitive Na⁺ channels in typical vertebrate tight epithelia is stimulated by numerous divalent cations, the pronounced inhibition of amiloride-sensitive Na⁺ channels in leech skin by Pb²⁺ and Hg²⁺ might lead to a further differentiation of amiloride-sensitive Na⁺ channels. The two widespread pesticides lindane and promecarb exerted their effects only at comparativ high concentrations. This low sensitivity restricts the usefulness of leech skin as a subject for further analysis of toxicity mechanisms, at least for these two pesticides.     

Immunocytochemical localization of amiloride-sensitive sodium channels in the lower intestine of the hen

We have used polyclonal antibodies generated against purified bovine renal amiloride-sensitive Na⁺ channels to localize amiloride-sensitive Na⁺ channels within the lower intestine (colon and coprodeum) of the hen. These antibodies cross-reacted with two polypeptides exhibiting M_r's of 235 and 150 kDa on immunoblots of detergent-solubilized apical membrane fractions from both the colon and coprodeum. The apparent molecular masses of theses polypeptides are in agreement with the M_r's of 2 of the subunits of the renal high amiloride-affintiy Na⁺ channel, namely the and the (=amiloride binding) subunits. The cellular distribution of Na⁺ channels was determined by immunoperoxidase and indirect immunofluorescence cytochemical techniques. The apical (luminal) membrane and cytoplasm of villar principal cells in both colon and coprodeum exhibited immunoreactivity, whereas goblet cells were nagative. Both principal and goblet cells of the crypts were also negative. We conclude that the amiloride-sensitive Na⁺ channels are localized to the principal cells of the intestinal villi and that these cells are responsible for intestinal Na⁺ absorption.     

Na+ fluxes in Chara under salt stress

The influx and efflux of Na⁺ across the plasma membrane of Characorallina and Chara longifolia were examined under mild saltstress conditions. Na⁺ influx was found to be rapid in bothspecies with the freely exchangeable cytoplasmic Na⁺ cominginto isotopic equilibrium with external ²²Na⁺ within 1 h ofexposure to isotope. Cytoplasmlc Na⁺ concentration and Na⁺ influxwere greater in C. corallina than in C. longifolla under thesame conditions. Na⁺ influx across the tonoplast was much lowerthan the flux across the plasma membrane. Na⁺ efflux was stimulatedat pH 5 relative to pH 7 by 218% in C. coralllna and 320% inC. longifolia. In both species externally applied Li⁺ inhibitedNa⁺ efflux at pH 5 but not at pH 7. Na⁺ etflux was not significantlyinhibited by amiloride. Key words: Na⁺ influx, Na⁺ efflux, Na⁺/H⁺ antiport, Chara     

Hormonal regulation of ENaCs: insulin and aldosterone

Although a variety of hormones and other agents modulate renalNa⁺ transport acting by way of theepithelial Na⁺ channel (ENaC), themode(s), pathways, and their interrelationships in regulation of thechannel remain largely unknown. It is likely that several hormones maybe present concurrently in vivo, and it is, therefore, important tounderstand potential interactions among the various regulatory factorsas they interact with the Na⁺transport pathway to effect modulation ofNa⁺ reabsorption in distal tubulesand other native tissues. This study represents specifically adetermination of the interaction between two hormones, namely,aldosterone and insulin, which stimulate Na⁺ transport by entirelydifferent mechanisms. We have used a noninvasive pulse protocol ofblocker-induced noise analysis to determine changes in single-channelcurrent (i_Na),channel open probability (P_o), andfunctional channel density(N_T) ofamiloride-sensitive ENaCs at various time points following treatmentwith insulin for 3 h of unstimulated control and aldosterone-pretreatedA6 epithelia. Independent of threefold differences of baseline values of transport caused by aldosterone, 20 nM insulin increased by threefold and within 10-30 min the density of the pool of apical membrane ENaCs(N_T) involvedin transport. The very early (10 min) increases of channel density wereaccompanied by relatively small decreases ofi_Na(10-20%) and decreases ofP_o (28%) in the aldosterone-pretreated tissues but not the control unstimulated tissues. The early changes ofi_Na,P_o, andN_T weretransient, returning very slowly over 3 h toward their respectivecontrol values at the time of addition of insulin. We conclude thataldosterone and insulin act independently to stimulate apicalNa⁺ entry into the cells of A6epithelia by increase of channel density.

     

Reappraisal of the Role of Sodium in the Light-Dependent Active Transport of Pyruvate into Mesophyll Chloroplasts of C4 Plants

The mechanism of light-dependent active transport of pyruvatein C₄ mesophyll chloroplasts has not been clarified, particularlyin Na⁺-type C₄ species, in which the pyruvate uptake into mesophyllchloroplasts is enhanced by illumination or by making a Na⁺gradient (Na⁺-jump) across the envelope in the dark. We re-investigatedhere the effect of Na⁺ on the active transport of pyruvate inmesophyll chloroplasts of Panicum miliaceum, a Na⁺-type C₄ species,by comparing the rate of pyruvate uptake at various externalpHs under four conditions; in the light and dark together with/withoutNa⁺-jump: (1) At neutral pH, the rate of pyruvate uptake inthe dark was enhanced by Na⁺-jump but scarcely by illumination.(2) While the enhancement effect by Na⁺-jump was independentof external pH, that by illumination increased greatly at pHover 7.4, and the effects of light and Na⁺ at the alkaline pHwere synergistic. (3) The light-enhanced pyruvate uptake wasrelated to stromal alkalization induced by illumination. Infact, pyruvate uptake was induced by H⁺-jump in the medium frompH 8.0 to 6.7. (4) Stromal pH was lowered by the addition ofK⁺-pyruvate and more by Na⁺-pyruvate into the medium at pH 7.8in the light. (5) However, the pH and ATP levels in the stromawere not affected by Na⁺-jump. Thus, we discussed possibility that besides pyruvate/Na⁺ cotransportat neutral pH in the medium, pyruvate/H⁺ cotransport enhancedby the presence of Na+ operates in mesophyll chloroplasts ofNa⁺-type C4 species at alkaline medium. ¹Present address: Biological Resources Division, Japan InternationalResearch Center for Agricultural Sciences (JIRCAS), Ministryof Agriculture, Forestry and Fisheries, 2-1 Ohwashi, Tsukuba,305 Japan     

Stimulation of Transepithelial Na<Superscript>+</Superscript> Current by Extracellular Gd<Superscript>3+</Superscript> in <Emphasis Type="Italic">Xenopus laevis</Emphasis> Alveolar Epithelium

In the present study we investigated the effect of extracellular gadolinium on amiloride-sensitive Na⁺ current across Xenopus alveolar epithelium by Ussing chamber experiments and studied its direct effect on epithelial Na⁺ channels with the patch-clamp method. As observed in various epithelia, the short-circuit current (I _sc) and the amiloride-sensitive Na⁺ current (I _ami) across Xenopus alveolar epithelium was downregulated by high apical Na⁺ concentrations. Apical application of gadolinium (Gd³⁺) increased I _sc in a dose-dependent manner (EC ₅₀ = 23.5 µM). The effect of Gd³⁺ was sensitive to amiloride, which indicated the amiloride-sensitive transcellular Na⁺ transport to be upregulated. Benz-imidazolyl-guanidin (BIG) and p-hydroxy-mercuribenzonic-acid (PHMB) probably release apical Na⁺ channels from Na⁺-dependent autoregulating mechanisms. BIG did not stimulate transepithelial Na⁺ currents across Xenopus lung epithelium but, interestingly, it prevented the stimulating effect of Gd³⁺ on transepithelial Na⁺ transport. PHMB increased I _sc and this stimulation was similar to the effect of Gd³⁺. Co-application of PHMB and Gd³⁺ had no additive effects on I _sc. In cell-attached patches on Xenopus oocytes extracellular Gd³⁺ increased the open probability (NP _o) of Xenopus epithelial sodium channels (ENaC) from 0.72 to 1.79 and decreased the single-channel conductance from 5.5 to 4.6 pS. Our data indicate that Xenopus alveolar epithelium exhibits Na⁺-dependent non-hormonal control of transepithelial Na⁺ transport and that the earth metal gadolinium interferes with these mechanisms. The patch-clamp experiments indicate that Gd³⁺ directly modulates the activity of ENaCs.     

Electrophysiological Measurements on the Root of Atriplex hastata

The ion contents and membrane potentials of the cells of young,hydroponically cultured seedlings of Atriplex hastata L. var.salina, Wallr. have been measured at several different NaClconcentrations. The total tissue concentrations of Na⁺ and Cl^–increase as external NaCl increases, but there is always a markedexcess of internal Na⁺ over Cl^–; this is balanced by endogenousorganic anion formation with a concomitant extrusion of H⁺ tothe bathing solution. Membrane potentials of the root cells remain essentially invariantwith changes in external NaCl at approx. –130 mV; thereis no evidence of a radial gradient of potential across theroot. The potential seems to contain a cyanide-sensitive electrogeniccomponent, also invariant with NaCl concentration, of about–70 mV, and a diffusion component. The electrogenic componentseems likely to be a H⁺ efflux, probably through a H⁺ uniportATPase.     

Na+ channel activity in cultured renal (A6) epithelium: Regulation by solution osmolarity

Summary Solution osmolarity is known to affect Na⁺ transport rates across tight epithelia but this variable has been relatively ignored in studies of cultured renal epithelia. Using electrophysiological methods to study A6 epithelial monolayers, we observed a marked effect of solution tonicity on amiloride-sensitive Na⁺ currents (I _sc).I _sc for tissues bathed in symmetrical hyposmotic (170 mOsm), isosmotic (200 mOsm), and hyperosmotic (230 or 290 mOsm) NaCl Ringer's solutions averaged 25±2, 9±2, 3±0.4, and 0.6±0.5 A/cm², respectively. Similar results were obtained following changes in the serosal tonicity; mucosal changes did not significantly affectI _sc. The changes inI _sc were slow and reached steady-state within 30 min. Current fluctuation analysis measurements indicated that single-channel currents and Na⁺ channel blocker kinetics were similar for isosmotic and hyposmotic conditions. However, the number of conducting Na⁺ channels was approximately threefold higher for tissues bathed in hyposmotic solutions. No channel activity was detected during hyperosmotic conditions. The results suggest that Na⁺ channels in A6 epithelia are highly sensitive to relatively small changes in serosal solution tonicity. Consequently, osmotic effects may partly account for the large variability in Na⁺ transport rates for A6 epithelia reported in the literature.     

Stimulus-induced currents in isolated taste receptor cells of the larval tiger salamander

Gustatory receptor cells, isolated from the lingual epitheliumof larval tiger salamanders (Ambystoma tigrinum), possess avariety of voltage- and ion-dependent conductances, includinga transient Na⁺ -current (I_Na), a voltage-gated Ca²⁺ -current(I_A). a transient K₊ -current (I_A), a delayed rectifier K⁺ -current(I_K), and a Ca₂₊ -activated K₊ -current (I_K(Ca))- By use ofwhole-cell and excised-patch tight-seal recording techniques,we examined the effects of taste stimuli on the conductancesof taste cells from the tiger salamander. Depolarizing receptorpotentials elicited by NaCl were associated with slow, gradedinward currents which were composed of amiloride-sensitive andtetrodoxin-(TTX)-sensitive components. Potassium chloride producedmaintained inward currents, which usually showed both phasicand tonic components and were only partially blocked by tetraethylammoniumchloride (TEA). Citric and acetic acids elicited slow depolarizationsin taste cells. Under voltage-clamp, acids produced graded inwardcurrents which were composed of two components: one attributableto a transient block of voltage-dependent K₊ -channels and asmaller component which may have resulted from an increasedconductance to cations. Quinine hydrochloride elicited slowdepolarization of taste cells which was associated with a slowlydeveloping maintained inward current under voltage-clamp. Quininesuppressed both voltage-dependent inward and outward currents.In some taste cells, L-arginine elicited small outward currentswhich were attributable to an increase in K₊ conductance. Inother cells, L-arginine produced a decrease in voltage-dependentoutward currents and generated depolarizations associated withinward currents. These results indicate that several independentmechanisms, including amiloride-sensitive Na₊ -channels, andstimulus modulation of voltage-dependent K₊ -channels, are involvedin taste cell responses to chemical stimuli. More than one mechanismis typically present in a single cell. ³Present address: Department of Physiology, Tokyo Medical andDental University, 5-45 Yushima 1-chome, Bunkyo-ku, Tokyo 113,Japan     

Differential effects of phorbol ester (PMA) on blocker-sensitive ENaCs of frog skin and A6 epithelia

Activation ofprotein kinase C with phorbol 12-myristate 13-acetate (PMA) causedcomplex transient perturbations of amiloride-sensitive short-circuitNa⁺ currents(I_Na) in A6epithelia and frog skins that were tissue and concentration dependent.A noninvasive channel blocker pulse method of noise analysis (18) wasused to investigate how PMA caused time-dependent changes of apicalmembrane epithelial Na⁺ channel(ENaC) single-channel currents, channel open probabilities (P_o), andchannel densities(N_T). In A6epithelia, 5 and 50 nM PMA caused within 7 min concentration-dependentsustained decreases ofP_o (~55% belowcontrol, 50 nM) and rapid compensatory transient increases ofN_T within 7 min(~220% above control, 50 nM), resulting in either small transientincreases of I_Naat 5 nM PMA or small biphasic decreases ofI_Na at 50 nM PMA.In contrast to A6 epithelia, 50 and 500 nM PMA in frog skin causedafter a delay of at least 10 min transient increases ofN_T to~60-70% above control at 30-60 min. Unlike A6 epithelia,P_o was increased~15% above control within 7 min and remained within±10-15% of control for the duration of the 2-h experiments.Despite differences in the time courses of secondary inhibition oftransport in A6 epithelia and frog skin, the delayed downregulation oftransport was due to time-dependent decreases ofN_T from theirpreelevated levels in both tissues. WhereasP_o is decreasedwithin minutes in A6 epithelia as measured by noise analysis or bypatch clamp (8), the discrepancy in regulation ofN_T in A6epithelia as measured by noise analysis and patch clamp is most likelyexplained by the inability of on-cell patches formed before treatmentof tissues with PMA to respond to regulation of their channeldensities.

     

Epidermal growth factor increases lung liquid clearance in rat lungs

Epidermal growthfactor (EGF) has been reported to stimulate the proliferation ofepithelial cells and increase Na⁺flux andNa⁺-K⁺-ATPasefunction in alveolar epithelial cell monolayers. Increases inNa⁺-K⁺-ATPasein alveolar type II cells (AT2) have been associated with increasedactive Na⁺ transport and lungedema clearance across the rat alveolar epithelium in a model ofproliferative lung injury. Thus we tested whether administration ofaerosolized EGF to rat lungs would increase activeNa⁺ transport and lung liquidclearance. Sixteen adult Sprague-Dawley male rats were randomized tothree groups. To a group of six rats, an aerosol generated from 20 µgof EGF in saline was delivered to the lungs, to a second group of fiverats only aerosolized saline was delivered, and a third group of fiverats without treatment served as the control. Forty-eight hourspostaerosolization of rat lungs with EGF there was an ~40% increasein active Na⁺ transport and lungliquid clearance compared with control rats, in the absence of changesin²²Na⁺,[³H]mannitol, andalbumin permeabilities. TheNa⁺-K⁺-ATPaseactivity in AT2 cells harvested from these lungs was increased in ratsthat received aerosolized EGF compared with AT2 cells from both controlrats and rats receiving aerosolized saline. These results support thehypothesis that in vivo delivery of EGF aerosols upregulates alveolarepithelialNa⁺-K⁺-ATPaseand increases lung liquid clearance in rats.

     

Movement of Ions and Electrogenesis in Higher Plant Cells

During the past 10 years considerable information has accumulatedon the electrochemical relationships of higher plant cells duringtransport of mineral ions. Using the Nernst equation as a criterion,none of eight ions (K⁺, Na⁺, Ca⁺⁺, Mg⁺⁺, NO₃^–, Cl^–,H₂PO₄^–, and SO₄^–) is in a passive equilibrium. Na⁺,Ca⁺⁺, and Mg⁺⁺ are subject to an exclusion mechanism, and allof the anions appear to be pumped inwardly. K⁺ apparently approachesan electrochemical balance under certain conditions but probablyis actively accumulated. Compartmental analyses giving estimatesof amounts in the cytoplasm and vacuole and of unidirectionalfluxes permit application of the Ussing flux-ratio equation.The criterion in oat coleoptile cells suggests that at the plasmalemmaNa⁺ is pumped out while K⁺ and Cl^– are pumped in. K⁺ andCl^– appear to be coupled in active transport across thetonoplast into the vacuole. Good evidence has been found thatthe cell's electropotential arises from an electrogenic pump:CN^– (cyanide) and DNP (dinitrophenol) reversibly blockthe potential and ionic transport; cell potentials are higherthan can be accounted for by diffusion; the responses of respirationand potential to the concentration of CN^– are nearly parallel;and CN^– inhibited tissue approaches a fit to the Goldmanconstant field equation. Future objectives should be identificationof the ion, or ions, subject to the electrogenic pump and discoveryof the immediate energy source.     

How to overcome osmotic stress? Marine crabs conquer freshwater. New insights from modern electrophysiology

In the present article we review our findings on split lamella preparations of crab gills mounted in modified Ussing-chambers with respect to mechanistic and ecophysiological aspects. The leaky gill epithelium of shore crabs adapted to brackish water absorbs Na⁺ and Cl^? in a coupled mode, and shows similarities to other salt-absorbing epithelia exposed to moderately diluted media. The results so far obtained for NaCl uptake across the gills of the shore crab are compatible with a transport model where two cell types operate in parallel, one displaying cotransport-like NaCl absorption, similar to that in the thick ascending limb of Henle's loop of the mammalian mephron, and the other one with characteristics of amiloride-sensitive, channel-mediated Na⁺ uptake by frog skin. Although there is no clear evidence for the apical mechanisms in this model, it may serve as a good basis for more detailed studies in the future. The moderately tight gill epithelium of freshwater adapted Chinese crabs absorbs Na⁺ and Cl^? independently from each other, and shows similarities to other salt-absorbing epithelia exposed to freshwater. The characteristics of a positive, Na⁺-dependent short-circuit current with externally Cl^?-free saline indicate that active Na⁺ uptake proceeds in a frog-skin-like mode via apical Na⁺-channels and the basolateral Na⁺/K⁺-pump. The nature of a negative short-circuit current with external Cl^?-saline indicates that active and Na⁺-independent Cl^? uptake is driven by an apical V-type H⁺-pump and proceeds via apical Cl^?/ HCO₃ ^?-exchange and basolateral Cl^?-channels.