Inhibition Of Amiloride-sensitive Na Research Articles

Inhibition of amiloride sensitive Na + transport by serotonin

The lung neuroendocrine system secretes a variety of peptides and biogenic amines in the alveolar space as well as in the surrounding tissues. Among them, serotonin has been shown to interfere with sodium transport in airways of different animal models. In this study, we investigated the effects of serotonin in human A549 and H441 adenocarcinoma cell lines. In patch-clamp whole-cell configuration, apical perfusion of serotonin (4mM and 2mM) induced an immediate and strong inhibition (70% and 45% respectively) of amiloride sensitive currents. The kinetics of inhibition suggests a direct interaction with the sodium transport protein ENaC. RTQ-PCR showed the presence of 5HT2b receptor mRNA. This receptor is linked to the phosphoinositide hydrolysis pathway. Therefore, we performed intracellular calcium measurements in Fura-2AM loaded H441 and A549 cells. These experiments showed a rapid 8–10% increase of the 340/380 epifluorescence ratio in response to 1mM apical serotonin. The A6 renal model of amiloride sensitive transport was used in parallel to investigate the putative effect basolateral serotonin. Transepithelial measurements showed an immediate and short-lived apical effect while basolateral serotonin induced a progressive inhibition of the current which was sustained for more than 90 minutes and lead to 80% inhibition of the current. Thus, serotonin inhibits independently apical and basolateral Na transport pathways through distinct mechanisms. AG is a FNRS doctoral fellow. This work was funded by grants from ULB and Fonds Defay.

Cl– Interference with the Epithelial Na+ Channel ENaC

The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A and ATP-regulated Cl- channel that also controls the activity of other membrane transport proteins, such as the epithelial Na+ channel ENaC. Previous studies demonstrated that cytosolic domains of ENaC are critical for down-regulation of ENaC by CFTR, whereas others suggested a role of cytosolic Cl- ions. We therefore examined in detail the anion dependence of ENaC and the role of its cytosolic domains for the inhibition by CFTR and the Cl- channel CLC-0. Coexpression of rat ENaC with human CFTR or the human Cl- channel CLC-0 caused inhibition of amiloride-sensitive Na+ currents after cAMP-dependent stimulation and in the presence of a 100 mM bath Cl- concentration. After activation of CFTR by 3-isobutyl-1-methylxanthine and forskolin or expression of CLC-0, the intracellular Cl- concentration was increased in Xenopus oocytes in the presence of a high bath Cl- concentration, which inhibited ENaC without changing surface expression of alpha beta gammaENaC. In contrast, a 5 mM bath Cl- concentration reduced the cytosolic Cl- concentration and enhanced ENaC activity. ENaC was also inhibited by injection of Cl- into oocytes and in inside/out macropatches by exposure to high cytosolic Cl- concentrations. The effect of Cl- was mimicked by Br-, Br-, NO3(-), and I-. Inhibition by Cl- was reduced in trimeric channels with a truncated COOH terminus of betaENaC and gammaENaC, and it was no longer detected in dimeric alpha deltaCbeta ENaC channels. Deletion of the NH2 terminus of alpha-, beta-, or gammaENaC, mutations in the NH2-terminal phosphatidylinositol bisphosphate-binding domain of betaENaC and gammaEnaC, and activation of phospholipase C, all reduced ENaC activity but allowed for Cl(-)-dependent inhibition of the remaining ENaC current. The results confirm a role of the carboxyl terminus of betaENaC for Cl(-)-dependent inhibition of the Na+ channel, which, however, may only be part of a complex regulation of ENaC by CFTR.

Control of ion transport in mammalian airways by protease activated receptors type 2 (PAR‐2)

Protease-activated receptors (PARs) are widely distributed in human airways. They couple to G- proteins and are activated after proteolytic cleavage of the N terminus of the receptor. Evidence is growing that PAR subtype 2 plays a pivotal role in inflammatory airway diseases, such as allergic asthma or bronchitis. However, nothing is known about the effects of PAR-2 on electrolyte transport in the native airways. PAR-2 is expressed in airway epithelial cells, where they are activated by mast cell tryptase, neutrophil proteinase 3, or trypsin. Recent studies produced conflicting results about the functional consequence of PAR-2 stimulation. Here we report that stimulation of PAR-2 receptors in mouse and human airways leads to a change in electrolyte transport and a shift from absorption to secretion. Although PAR-2 appears to be expressed on both sides of the epithelium, only basolateral stimulation results in inhibition of amiloride sensitive Na+ conductance and stimulation of both luminal Cl- channels and basolateral K+ channels. The present data indicate that these changes occur through activation of phospholipase C and increase in intracellular Ca2+, which activates basolateral SK4 K+ channels and luminal Ca2+-dependent Cl- channels. In addition, the present data suggest a PAR-2 mediated release of prostaglandin E2, which may contribute to the secretory response. In conclusion, these results provide further evidence for a role of PAR-2 in inflammatory airway disease: stimulation of these receptors may cause accumulation of airway surface liquid, which, however, may help to flush noxious stimuli away from the affected airways.

Purinergic P2Y6 Receptors Induce Ca2+ and CFTR Dependent Cl- Secretion in Mouse Trachea

In airways Cl^- secretion is activated and Na⁺ absorption is inhibited when P2Y₂ receptors are stimulated by ATP or UTP. Both nucleotides are subject to degradation to ADP and UDP by ecto-nucleotidases. Here we show that these metabolites change electrolyte transport by stimulation of P2Y₆ receptors in mouse trachea. Immunohistochemistry confirmed luminal and basolateral expression of P2Y ₆ receptors. In Ussing chamber experiments luminal ADP, UDP or the P2Y₆ receptor agonist INS48823 induced both transient and persistent increase in short circuit currents (I_SC). Activation of I _SC was inhibited by the P2Y₆ receptor blocker PPADS. The transient response was inhibited by DIDS, whereas the persistent I _SC was inhibited by glibenclamide and by the protein kinase A (PKA) blocker H-89. Moreover, sustained activation of I _SC by luminal UDP was inhibited by blocking basolateral K⁺ channels with 293B. Possible effects of diphosphates on P2Y₁ or adenosine receptors were excluded by the inhibitors MRS2179 and 8-SPT, respectively. Inhibition of amiloride sensitive Na⁺ absorption was only seen after blocking basolateral K⁺ channels with 293B. In contrast, Cl^- secretion activated by basolateral ADP or UDP was only transient and was blocked by the sk4 K⁺ channel blocker clotrimazole. In summary, activation of luminal P2Y₆ receptors in the airways shifts electrolyte transport towards secretion by increasing intracellular Ca⁺ and activation of PKA.

Ion Transport in an Immortalized Rat Submandibular Cell Line SMG-C6

The immortalized rat submandibular epithelial cell line, SMG-C6, cultured on porous tissue culture supports, forms polarized, tight-junction epithelia facilitating bioelectric characterization in Ussing chambers. The SMG-C6 epithelia generated transepithelial resistances of 956+/-84Omega.cm2 and potential differences (PD) of -16.9 +/- 1.5mV (apical surface negative) with a basal short-circuit current (Isc) of 23.9 +/- 1.7 microA/cm2 (n = 69). P2 nucleotide receptor agonists, ATP or UTP, applied apically or basolaterally induced a transient increase in Isc, followed by a sustained decreased below baseline value. The peak DeltaIsc increase was partly sensitive to Cl- and K+ channel inhibitors, DPC, glibenclamide, and tetraethylammonium (TEA) and was completely abolished following Ca2+ chelation with BAPTA or bilateral substitution of gluconate for Cl-. The major component of basal Isc was sensitive to apical Na+ replacement or amiloride (half-maximal inhibitory concentration 392 nM). Following pretreatment with amiloride, ATP induced a significantly greater Isc; however, the poststimulatory decline was abolished, suggesting an ATP-induced inhibition of amiloride-sensitive Na+ transport. Consistent with the ion transport properties found in Ussing chambers, SMG-C6 cells express the rat epithelial Na+ channel alpha-subunit (alpha-rENaC). Thus, cultured SMG-C6 cells produce tight polarized epithelia on permeable support with stimulated Cl- secretory conductance and an inward Isc accounted for by amiloride-sensitive Na+ absorption.

Inhibition of amiloride-sensitive Na+ channel by isothiouronium derivatives.

The effects on the amiloride-blockable Na+ channel of a family of recently synthesized isothiouronium derivatives were measured in plasma membrane vesicles from rat distal colon. Some of these derivatives act as high-affinity Na(+)-like antagonists on the Na(+)-K(+)-adenosinetriphosphatase. One of the reagents tested, 1-bromo-2,4,6-tris(isothiouronium methyl)-benzene tribromide (Br-TITU), was found to be a potent blocker of the Na+ channel. At neutral pH, Br-TITU rapidly inhibits the channel mediated 22Na+ uptake, with an inhibition constant of 94 +/- 39 nM. The inhibition observed is specific and reversible. 1,3-Dibromo-2,4,6-tris(isothiouronium methyl)benzene tribromide and Br-TITU derivatives with methyl and phenyl substitutions on the isothiouronium moiety were much less effective blockers. Incubation of cells with Br-TITU at alkaline (but not neutral) pH produces irreversible inactivation of channels, possibly due ot covalent modification of a lysine residue. This inactivation can be attenuated by amiloride but not by Na+. Thus Br-TITU may be a useful reagent in identifying essential residues of the channel protein.

Effects of a short-chain phospholipid on ion transport pathways in rabbit nasal airway epithelium.

We investigated the mechanism of interference of mucosal application of the short-chain phospholipid didecanoyl-L-alpha-phosphatidylcholine (DDPC; 0.1-0.5%) with ion transport pathways in isolated rabbit nasal airway epithelium (RNAE). Transports of Na+ and Cl- were evaluated from tracer ion fluxes, short-circuit current (Isc), and epithelial conductance (Gt) under short-circuit conditions in Ussing chambers. DDPC rapidly and reversibly abolished net Na+ absorption, reduced control Isc (approximately 110 microA/cm2) by approximately 80%, and induced a small Cl secretion. Intracellular Ca2+ concentration ([Ca2+]i) increased dose dependently and transiently (measured by fura 2 in cultured rabbit airway epithelium), but ionomycin failed to mimic the decrease in Isc. The rise in [Ca2+]i may explain a Ba(2+)-sensitive transient activation of a basolateral K+ conductance. Indomethacin-sensitive prostaglandin E2 production in RNAE increased severalfold, but cyclooxygenase and lipoxygenase inhibitors did not prevent DDPC-induced changes in Isc. DDPC initially decreased control Gt (approximately 13 mS/cm2) by approximately 25% due to inhibition of amiloride-sensitive Na+ channels, and then reversibly increased Gt to approximately 45% above control values. Passive Na+ fluxes increased more than Cl fluxes, suggesting that the increase in Gt is due to formation of a paracellular shunt conductance in parallel with unaffected, anion-selective tight junction channels. The results suggest that DDPC inhibits apical membrane Na+ channels and causes structural changes in tight junctions after incorporation in apical cell membranes.

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.

Kinetic properties of Na+/H+ exchange in cultured bovine pigmented ciliary epithelial cells.

Uptake studies with 22Na were performed in cultured bovine pigmented ciliary epithelial cells, in order to characterize mechanisms of Na+ transport. A large part of Na+ uptake was sensitive to amiloride, quinidine and harmaline. Na+ uptake was stimulated by intracellular acidification (using the NH+4 prepulse technique), and was inhibited with increasing extracellular proton concentration. Decreasing extracellular pH from 7.5 to 7.0 increased the apparent KM for Na+ from 38 to 86 mM without considerable changes in Vmax. In the presence of 5 mM Na+ half maximal inhibition of amiloride sensitive Na+ uptake by extracellular protons was observed at a hydrogen concentration of 50 nM. In the presence of 50 mM Na+ the proton concentration necessary for 50% inhibition was 139 nM. Thus, the mode of inhibition of extracellular H+ seemed to be competitive with a Ki of 20-40 nM. 10 microM amiloride increased the apparent KM for Na+ from 33 mM to 107 mM, while Vmax remained nearly unchanged. IC50 for amiloride was 6 microM at 5 mM Na+ and 36 microM in the presence of 150 mM Na+. Thus, amiloride behaves as a competitive inhibitor with a Ki of about 5 microM. The affinities of Na+ to the transport site (KM approximately 16 mM), to the inhibitory site for protons (KM approximately 21 mM), and to the inhibitory site for amiloride (KM approximately 26 mM) were in the same order of magnitude. In summary, we have presented evidence for the presence of a Na+/H+ exchanger in cultured bovine pigmented ciliary epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of serotonin on Na+-H+ exchange and intracellular calcium in isolated chicken enterocytes.

The effects of serotonin or 5-hydroxytryptamine (5-HT) on Na absorption and intracellular free Ca (Cai) in isolated chicken enterocytes was examined. The rate of initial 22Na uptake was inhibited by 50% after 90 s of stimulation with 5-HT (10(-5) M), an effect that was not additive with amiloride (10(-3) M) and was transient (less than 10 min). 5-HT similarly decreased intracellular pH (pHi) in cells loaded with the pH indicator carboxyfluorescein, an effect that was also transient, not additive with amiloride, and Na dependent. The ED50 of this effect was approximately 10(-8) M. 5-HT also stimulated a transient increase in Cai as determined by quin2 fluorescence. A maximal increase of 60 nM occurred significantly before the peak change in pHi, but the total duration of response was similar in each case. In the absence of extracellular Ca, the 5-HT effects on pHi and Cai still persisted. In cells loaded with the Ca-buffering agent MAPTAM, the 5-HT (10(-5) M) inhibitory effect of 22Na influx was partially inhibited. We conclude that 5-HT directly inhibits Na absorption by isolated enterocytes by releasing endogenous Ca, which subsequently causes an inhibition of amiloride-sensitive Na+-H+ exchange.

Ouabain inhibits intracellular pH recovery from acidosis in cultured mouse heart cells

Myocardial ischaemia induces cytosolic acidification, which promotes cardiac damage, dysfunction or arrhythmia. In this study, we investigated the effect of ouabain on the intracellular pH (pHi) in cultured mouse ventricular cells, using 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The average resting pHi in myocytes was 7.19. After myocytes were acid-loaded with NH4Cl, the pHi recovered from acidosis to the resting level within a few minutes via amiloride-sensitive Na+/H+ exchange. Ouabain inhibited this pHi recovery dose-dependently with half-maximal inhibition at 3 X 10(-5) M, but did not suppress the ionophore monensin-induced pHi elevation. The inhibition of the pHi recovery from acidosis by ouabain is possibly caused by an inhibition of amiloride-sensitive Na+/H+ exchange, which is secondary to a suppression of Na+ efflux through (Na+, K+) pump. Above results demonstrate the possibility that digitalis promotes intracellular acidosis or inhibits the pHi recovery from acidosis in ischaemic myocardium.