Mechanosensitive activation of CFTR by increased cell volume and hydrostatic pressure but not shear stress.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel that is essential for electrolyte and fluid homeostasis. Preliminary evidence indicates that CFTR is a mechanosensitive channel. In lung epithelia, CFTR is exposed to different mechanical forces such as shear stress (Ss) and membrane distention. The present study questioned whether Ss and/or stretch influence CFTR activity (wild type, ∆F508, G551D). Human CFTR (hCFTR) was heterologously expressed in Xenopus oocytes and the response to the mechanical stimulus and forskolin/IBMX (FI) was measured by two-electrode voltage-clamp experiments. Ss had no influence on hCFTR activity. Injection of an intracellular analogous solution to increase cell volume alone did not affect hCFTR activity. However, hCFTR activity was augmented by injection after pre-stimulation with FI. The response to injection was similar in channels carrying the common mutations ∆F508 and G551D compared to wild type hCFTR. Stretch-induced CFTR activation was further assessed in Ussing chamber measurements using Xenopus lung preparations. Under control conditions increased hydrostatic pressure (HP) decreased the measured ion current including activation of a Cl(-) secretion that was unmasked by the CFTR inhibitor GlyH-101. These data demonstrate activation of CFTR in vitro and in a native pulmonary epithelium in response to mechanical stress. Mechanosensitive regulation of CFTR is highly relevant for pulmonary physiology that relies on ion transport processes facilitated by pulmonary epithelial cells.

Hydrostatic pressure activates ATP-sensitive K+ channels in lung epithelium by ATP release through pannexin and connexin hemichannels.

Lungs of air-breathing vertebrates are constantly exposed to mechanical forces and therefore are suitable for investigation of mechanotransduction processes in nonexcitable cells and tissues. Freshly dissected Xenopus laevis lungs were used for transepithelial short-circuit current (ISC) recordings and were exposed to increased hydrostatic pressure (HP; 5 cm fluid column, modified Ussing chamber). I(SC) values obtained under HP (I(5cm)) were normalized to values before HP (I(0cm)) application (I(5cm)/I(0cm)). Under control conditions, HP decreased I(SC) (I(5cm)/I(0cm)=0.84; n=68; P<0.0001). This effect was reversible and repeatable ≥30 times. Preincubation with ATP-sensitive K(+) channel (K(ATP)) inhibitors (HMR1098 and glibenclamide) prevented the decrease in I(SC) (I(5cm)/I(0cm): HMR1098=1.19, P<0.0001; glibenclamide=1.11, P<0.0001). Similar effects were observed with hemichannel inhibitors (I(5cm)/I(0cm): meclofenamic acid=1.09, P<0.0001; probenecid=1.0, P<0.0001). The HP effect was accompanied by release of ATP (P<0.05), determined by luciferin-luciferase luminescence in perfusion solution from the luminal side of an Ussing chamber. ATP release was abrogated by both meclofenamic acid and probenecid. RT-PCR experiments revealed the expression of pannexin and connexin hemichannels and KATP subunit transcripts in X. laevis lung. These data show an activation of KATP in pulmonary epithelial cells in response to HP that is induced by ATP release through mechanosensitive pannexin and connexin hemichannels. These findings represent a novel mechanism of mechanotransduction in nonexcitable cells.

Differential N termini in epithelial Na+ channel δ-subunit isoforms modulate channel trafficking to the membrane.

The epithelial Na(+) channel (ENaC) is a heteromultimeric ion channel that plays a key role in Na(+) reabsorption across tight epithelia. The canonical ENaC is formed by three analogous subunits, α, ß, and γ. A fourth ENaC subunit, named δ, is expressed in the nervous system of primates, where its role is unknown. The human δ-ENaC gene generates at least two splice isoforms, δ(1) and δ(2) , differing in the N-terminal sequence. Neurons in diverse areas of the human and monkey brain differentially express either δ(1) or δ(2) , with few cells coexpressing both isoforms, which suggests that they may play specific physiological roles. Here we show that heterologous expression of δ(1) in Xenopus oocytes and HEK293 cells produces higher current levels than δ(2) . Patch-clamp experiments showed no differences in single channel current magnitude and open probability between isoforms. Steady-state plasma membrane abundance accounts for the dissimilarity in macroscopic current levels. Differential trafficking between isoforms is independent of ß- and γ-subunits, PY-motif-mediated endocytosis, or the presence of additional lysine residues in δ(2)-N terminus. Analysis of δ(2)-N terminus identified two sequences that independently reduce channel abundance in the plasma membrane. The δ(1) higher abundance is consistent with an increased insertion rate into the membrane, since endocytosis rates of both isoforms are indistinguishable. Finally, we conclude that δ-ENaC undergoes dynamin-independent endocytosis as opposed to αßγ-channels.

Laminar shear stress modulates the activity of heterologously expressed P2X(4) receptors.

P2X(4) receptors are involved in mechanotransduction processes, but it is unknown whether or not P2X(4) receptors form mechanosensitive ion channels. This study questioned, whether laminar shear stress (LSS) can modulate P2X(4) receptor activity. Mouse P2X(4) receptor was cloned and heterologously expressed in Xenopus laevis oocytes. In two-electrode-voltage-clamp experiments the application of ATP (100µM) produced a transient inward current that was decreased by about 50% upon a second ATP application, corresponding to the desensitization behavior of P2X(4) receptors. In P2X(4) expressing oocytes LSS (shear forces of ~5.1dynes/cm(2)) did not produce any effect. However, LSS modulated the response of P2X(4) to ATP. With LSS (~5.1dynes/cm(2)) the desensitization of the current due to the second ATP application was diminished. Ivermectin (IVM), a compound which stabilizes the open state of P2X(4) receptors, mimicked the effect of LSS (~5.1dynes/cm(2)), since there was no additional effect of LSS after pre-incubation with IVM detected. This indicates that LSS like IVM stabilizes the open state of the receptor, although the particular mechanism remains unknown. These data demonstrate that LSS modulates the activity of P2X(4) receptors by eliminating the desensitization of the receptors in response to ATP probably by stabilizing the open state of the channel.

Thiol-reactive compounds from garlic inhibit the epithelial sodium channel (ENaC).

The epithelial sodium channel (ENaC) is a key factor in the transepithelial movement of sodium, and consequently salt and water homeostasis in various organs. Dysregulated activity of ENaC is associated with human diseases such as hypertension, the salt-wasting syndrome pseudohypoaldosteronism type 1, cystic fibrosis, pulmonary oedema or intestinal disorders. Therefore it is important to identify novel compounds that affect ENaC activity. This study investigated if garlic (Allium sativum) and its characteristic organosulfur compounds have impact on ENaCs. Human ENaCs were heterologously expressed in Xenopus oocytes and their activity was measured as transmembrane currents by the two-electrode voltage-clamp technique. The application of freshly prepared extract from 5g of fresh garlic (1% final concentration) decreased transmembrane currents of ENaC-expressing oocytes within 10 min. This effect was dose-dependent and irreversible. It was fully sensitive to the ENaC-inhibitor amiloride and was not apparent on native control oocytes. The effect of garlic was blocked by dithiothreitol and l-cysteine indicating involvement of thiol-reactive compounds. The garlic organosulsur compounds S-allylcysteine, alliin and diallyl sulfides had no effect on ENaC. By contrast, the thiol-reactive garlic compound allicin significantly inhibited ENaC to a similar extent as garlic extract. These data indicate that thiol-reactive compounds which are present in garlic inhibit ENaC.

Nitric oxide inhibits highly selective sodium channels and the Na+/K+-ATPase in H441 cells.

Nitric oxide (NO) is an important regulator of Na(+) reabsorption by pulmonary epithelial cells and therefore of alveolar fluid clearance. The mechanisms by which NO affects epithelial ion transport are poorly understood and vary from model to model. In this study, the effects of NO on sodium reabsorption by H441 cell monolayers were studied in an Ussing chamber. Two NO donors, (Z)-1-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate and diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, rapidly, reversibly, and dose-dependently reduced amiloride-sensitive, short-circuit currents across H441 cell monolayers. This effect was neutralized by the NO scavenger hemoglobin and was not observed with inactive NO donors. The effects of NO were not blocked by 8-bromoguanosine-3',5'-cyclic monophosphate or by soluble guanylate cyclase inhibitors (methylene blue and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) and were therefore independent of soluble guanylate cyclase signaling. NO targeted apical, highly selective, amiloride-sensitive Na(+) channels in basolaterally permeabilized H441 cell monolayers. NO had no effect on the activity of the human epithelial sodium channel heterologously expressed in Xenopus oocytes. NO decreased Na(+)/K(+)-ATPase activity in apically permeabilized H441 cell monolayers. The inhibition of Na(+)/K(+)-ATPase activity by NO was reversed by mercury and was mimicked by N-ethylmaleimide, which are agents that reverse and mimic, respectively, the reaction of NO with thiol groups. Consistent with these data, S-NO groups were detected on the Na(+)/K(+)-ATPase α subunit in response to NO-donor application, using a biotin-switch approach coupled to a Western blot. These data demonstrate that, in the H441 cell model, NO impairs Na(+) reabsorption by interfering with the activity of highly selective Na(+) channels and the Na(+)/K(+)-ATPase.

Expression and functional characterization of P2X receptors in mouse alveolar macrophages.

Alveolar macrophages (AM) are crucial for pulmonary host defense, and evidence emerges that ATP-gated P2X receptors are involved in inflammatory processes. This study focuses on the expression and functional characterization of P2X receptors in AM from mouse. In RT-PCR experiments, transcripts encoding the P2X1, P2X3, P2X4, P2X5, and P2X7 receptors were detected. In whole-cell patch-clamp recordings, ATP (1 mM) evoked an inward current (mouse and human AM) that was reversible upon washout, and the reversal potential was ~5 mV, indicating the activation of a non-selective conductance-a fingerprint of P2X receptors. Further characterization (mouse AM) revealed that the current was not desensitized by a second ATP application. The ATP-induced current was increased by the removal of extracellular Ca²âº (in human and mouse AM), and EC50 in mouse AM were determined with ~1 mM ATP, in the presence as well as in the absence of extracellular Ca²âº. Pharmacological characterization of mouse AM revealed that the effect was augmented by BzATP and pre-application with ivermectin, but no effect with α,ß-meATP was observed. Further, the ATP effect was reduced by PPADS (300 µM), brilliant blue G (5 µM), and about A438079 (10 µM). Although different P2X receptor transcripts were detected in mouse AM, the observed functional and pharmacological characteristics indicate primarily the participation of P2X4 and P2X7 receptors as mediators of the ATP-induced ion current in mouse AM. These suggestions were confirmed by experiments with AM from P2X7 -deficient animals, indicating a contribution of P2X4 and P2X7 receptors in pulmonary immune function.

Evidence for basolateral Cl- channels as modulators of apical Cl- secretion in pulmonary epithelia of Xenopus laevis.

Pulmonary epithelia of air-breathing vertebrates are covered by a thin, fluid layer that is essential for immune defense and gas diffusion. The composition of this layer is maintained by ion transport mechanisms, including Cl(-) transport. The present study focuses on the function of basolateral Cl(-) channels in Xenopus pulmonary epithelia, since knowledge concerning this issue is limited. Therefore, Ussing chamber measurements were performed, and transepithelial short-circuit currents (I(SC)) were monitored. Basolateral application of the Cl(-) channel inhibitor N-phenylanthranilic acid (DPC) resulted in an increase of the I(SC), indicating a DPC-sensitive Cl(-) conductance. This observation was confirmed in experiments using an apical-to-basolateral Cl(-) gradient, with and without nystatin (apical side) to permeabilize the epithelia as well as by establishing an iodide gradient. The DPC-sensitive Cl(-) conductance was influenced by procedures interfering with apical Cl(-) secretion. For example, the effect of forskolin was increased when basolateral Cl(-) channels were blocked by the simultaneous application of DPC. Activation of apical Cl(-) secretion by forskolin/IBMX and subsequent DPC application resulted in a significantly reduced DPC effect. Accordingly, DPC led to an increased apical Cl(-) secretion estimated by an increased 5-nitro-2-(3-phenylpropylamino)benzoic acid-sensitive I(SC). Furthermore, inhibition of basolateral anion exchangers responsible for Cl(-) uptake resulted in a decreased DPC-sensitive current. Taken together, we have evidence concerning the function of basolateral Cl(-) channels in Xenopus pulmonary epithelium and that these channels play a significant role in mediating apical Cl(-) secretion involving a novel Cl(-) recycling mechanism across the basolateral membrane.

The neuronal-specific SGK1.1 kinase regulates {delta}-epithelial Na+ channel independently of PY motifs and couples it to phospholipase C signaling.

The δ-subunit of the epithelial Na(+) channel (ENaC) is expressed in neurons of the human and monkey central nervous system and forms voltage-independent, amiloride-sensitive Na(+) channels when expressed in heterologous systems. It has been proposed that δ-ENaC could affect neuronal excitability and participate in the transduction of ischemic signals during hypoxia or inflammation. The regulation of δ-ENaC activity is poorly understood. ENaC channels in kidney epithelial cells are regulated by the serum- and glucocorticoid-induced kinase 1 (SGK1). Recently, a new isoform of this kinase (SGK1.1) has been described in the central nervous system. Here we show that δ-ENaC isoforms and SGK1.1 are coexpressed in pyramidal neurons of the human and monkey (Macaca fascicularis) cerebral cortex. Coexpression of δßγ-ENaC and SGK1.1 in Xenopus oocytes increases amiloride-sensitive current and channel plasma membrane abundance. The kinase also exerts its effect when δ-subunits are expressed alone, indicating that the process is not dependent on accessory subunits or the presence of PY motifs in the channel. Furthermore, SGK1.1 action depends on its enzymatic activity and binding to phosphatidylinositol(4,5)-bisphosphate. Physiological or pharmacological activation of phospholipase C abrogates SGK1.1 interaction with the plasma membrane and modulation of δ-ENaC. Our data support a physiological role for SGK1.1 in the regulation of δ-ENaC through a pathway that differs from the classical one and suggest that the kinase could serve as an integrator of different signaling pathways converging on the channel.

Basolateral Cl- uptake mechanisms in Xenopus laevis lung epithelium.

A thin liquid layer covers the lungs of air-breathing vertebrates. Active ion transport processes via the pulmonary epithelial cells regulate the maintenance of this layer. This study focuses on basolateral Cl(-) uptake mechanisms in native lungs of Xenopus laevis and the involvement of the Na(+)/K(+)/2 Cl(-) cotransporter (NKCC) and HCO(3)(-)/Cl(-) anion exchanger (AE), in particular. Western blot analysis and immunofluorescence staining revealed the expression of the NKCC protein in the Xenopus lung. Ussing chamber experiments demonstrated that the NKCC inhibitors (bumetanide and furosemide) were ineffective at blocking the cotransporter under basal conditions, as well as under pharmacologically stimulated Cl(-)-secreting conditions (forskolin and chlorzoxazone application). However, functional evidence for the NKCC was detected by generating a transepithelial Cl(-) gradient. Further, we were interested in the involvement of the HCO(3)(-)/Cl(-) anion exchanger to transepithelial ion transport processes. Basolateral application of DIDS, an inhibitor of the AE, resulted in a significantly decreased the short-circuit current (I(SC)). The effect of DIDS was diminished by acetazolamide and reduced by increased external HCO(3)(-) concentrations. Cl(-) secretion induced by forskolin was decreased by DIDS, but this effect was abolished in the presence of HCO(3)(-). These experiments indicate that the AE at least partially contributes to Cl(-) secretion. Taken together, our data show that in Xenopus lung epithelia, the AE, rather than the NKCC, is involved in basolateral Cl(-) uptake, which contrasts with the common model for Cl(-) secretion in pulmonary epithelia.

Nicotinic receptors on rat alveolar macrophages dampen ATP-induced increase in cytosolic calcium concentration.

BACKGROUND: Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine. METHODS: Rat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca2+]i) were conducted. RESULTS: Positive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, ß1, and ß2, with most stable expression being noted for subunits α9, α10, ß1, and ß2. Notably, mRNA coding for subunit α7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca2+]i revealed changes in membrane current in response to ACh and in [Ca2+]i in response to nicotine, respectively. However, nicotine (100 µM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca2+]i by 30%. This effect was blocked by α-bungarotoxin and did not depend on the presence of extracellular calcium. CONCLUSIONS: Rat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca2+-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential.

Carbon monoxide rapidly impairs alveolar fluid clearance by inhibiting epithelial sodium channels.

Carbon monoxide (CO) is currently being evaluated as a therapeutic modality in the treatment of patients with acute lung injury and acute respiratory distress syndrome. No study has assessed the effects of CO on transepithelial ion transport and alveolar fluid reabsorption, two key aspects of alveolocapillary barrier function that are perturbed in acute lung injury/acute respiratory distress syndrome. Both CO gas (250 ppm) and CO donated by the CO donor, CO-releasing molecule (CORM)-3 (100 microM in epithelial lining fluid), applied to healthy, isolated, ventilated, and perfused rabbit lungs, significantly blocked (22)Na(+) clearance from the alveolar compartment, and blocked alveolar fluid reabsorption after fluid challenge. Apical application of two CO donors, CORM-3 or CORM-A1 (100 microM), irreversibly inhibited amiloride-sensitive short-circuit currents in H441 human bronchiolar epithelial cells and primary rat alveolar type II cells by up to 40%. Using a nystatin permabilization approach, the CO effect was localized to amiloride-sensitive channels on the apical surface. This effect was abolished by hemoglobin, a scavenger of CO, and was not observed when inactive forms of CO donors were employed. The effects of CO were not blocked by 8-bromoguanosine-3',5'-cyclic guanosine monophosphate, soluble guanylate cyclase inhibitors (methylene blue and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), or inhibitors of trafficking events (phalloidin oleate, MG-132, and brefeldin A), but the amiloride affinity of H441 cells was reduced after CO exposure. These data indicate that CO rapidly inhibits sodium absorption across the airway epithelium by cyclic guanosine monophosphate- and trafficking-independent mechanisms, which may rely on critical histidine residues in amiloride-sensitive channels or associated regulatory proteins on the apical surface of lung epithelial cells.

Effect of giving birth on the cortisol level in a bonobo groups' (Pan paniscus) saliva.

This study documents the cortisol levels in the saliva of a bonobo group, especially that of a bonobo female which had given birth for the first time. During a long study in Zoological Garden Frankfurt, Germany, a bonobo baby was born on 3 August 2007. Due to the fission-fusion keeping system employed, the bonobos were divided into two groups on this day. Their behavior was observed regularly and saliva was also collected. The bonobos had been trained to chew cotton wool and to give back the samples. The cortisol response was tested twice a day before birth and three times on the day of parturition. Before birth, no observable indication behavior was seen, but an increase in the cortisol concentration of the expectant mother was found. Parturition occurred at 8 pm. The next morning, the group with the newborn was visibly more active, which correlated with the fact that their cortisol levels were increased in the morning in comparison to the second group. During the day, cortisol decreased in both groups, only it was higher throughout the day in the new mother. In the evening, the two groups showed nearly the same cortisol levels. These data indicate that there is indeed a relation between observable behavior and the cortisol level in bonobo saliva. Therefore, the cortisol level can be regarded as a suitable indicator for verifying behavioral events.

Short-term exposure to waterborne free silver has acute effects on membrane current of Xenopus oocytes.

Waterborne free silver can cause osmo- and ionoregulatory disturbances in freshwater organisms. The effects of a short-term exposure to extracellular Ag+ ions on membrane currents were investigated in voltage-clamped defolliculated Xenopus oocytes. At a holding potential of -60 mV, ionic silver (1 microM Ag+) increased inward currents (=I(Ag)) from -8+/-2 nA to -665+/-41 nA (n=74; N=27). I(Ag) activated within 2 min of silver exposure and then rose impetuously. This current was largely reversible by washout and repeatable. I(Ag) reversed around -30 mV and rectified slightly at more positive potentials. Na+-free bath conditions reduced the silver-induced current to a smaller but sustained current. The response to silver was abolished by the Cl- channel blockers DIDS and SITS, whereas niflumic acid strongly potentiated I(Ag). Intraoocyte injection of AgNO3 to about 1 mM [Ag]i strongly potentiated I(Ag). Extracellular application of either dithiothreitol (DTT), a compound known to reduce disulfide bridges, or L-cysteine abolished Ag+-activated increase of membrane current. In contrast, n-ethylmaleimide (NEM) which oxidizes SH-groups potentiated I(Ag). Hypoosmotic bath solution significantly increased I(Ag) whereas hyperosmolar conditions attenuated I(Ag). The activation of I(Ag) was largely preserved after chelation of cytosolic Ca2+ ions with BAPTA/AM. Taken together, these data suggest that Xenopus oocytes are sensitive to short-term exposure to waterborne Ag+ ions and that the elicited membrane currents result from extra- and intracellular action of Ag+ ions on peptide moieties at the oocyte membrane but may also affect conductances after internalization.

Mechano-sensitivity of epithelial sodium channels (ENaCs): laminar shear stress increases ion channel open probability.

Epithelial cells are exposed to a variety of mechanical forces, but little is known about the impact of these forces on epithelial ion channels. Here we show that mechanical activation of epithelial sodium channels (ENaCs), which are essential for electrolyte and water balance, occurs via an increased ion channel open probability. ENaC activity of heterologously expressed rat (rENaC) and Xenopus (xENaC) orthologs was measured by whole-cell as well as single-channel recordings. Laminar shear stress (LSS), producing shear forces in physiologically relevant ranges, was used to mechanically stimulate ENaCs and was able to activate ENaC currents in whole-cell recordings. Preceding pharmacological activation of rENaC with Zn2+ and xENaC with gadolinium and glibenclamide largely prevented LSS-activated currents. In contrast, proteolytic cleavage with trypsin potentiated the LSS effect on rENaC whereas the LSS effect on xENaC was reversed (inhibition of xENaC current). Further, we found that exposure of excised outside-out patches to LSS led to an increased ion channel open probability without affecting the number of active channels. We suggest that mechano-sensitivity of ENaC may represent a ubiquitous feature for the physiology of epithelia, providing a putative mechanism for coupling transepithelial Na+ reabsorption to luminal transport.

CFTR-dependent Cl- secretion in Xenopus laevis lung epithelium.

In our present study we used preparations from Xenopus laevis lungs to perform electrophysiological Ussing chamber measurements, unidirectional flux measurements, and employed molecular approaches to elucidate the presence and function of a cystic fibrosis transmembrane conductance regulator (CFTR) homolog in this tissue. Application of different CFTR blockers (NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid), niflumic acid (NFA), glibenclamide, lonidamine, CFTR(inh)-172) to the apical side of the tissues was able to significantly decrease the measured short circuit current (I(SC)) indicating a Cl(-) secretion due to luminal located CFTR channels. This was further supported by a net (36)Cl(-) secretion determined by radioactive tracer flux experiments. Further, Xenopus pulmonary epithelia responded to apical chlorzoxazone exposure - a CFTR activator - and this activated current was inhibited by CFTR(inh)-172. We performed reverse transcription-PCR (RT-PCR) and Western blot analysis and with both approaches we found characteristic signals indicating the presence of a CFTR homolog in Xenopus lung. In addition, we were able to detect CFTR in apical membranes of Xenopus lung slices with immunohistological techniques. We conclude that Xenopus lung epithelium exhibits functional CFTR channels and that this tissue represents a valuable model for the investigation of ion transport properties in pulmonary epithelia.

Annelid epithelia as models for electrogenic Na+ transport.

The electrogenic Na(+) absorption across tight epithelia from invertebrates follows the principles analog to the mechanisms found in vertebrates. Extracellular Na(+)-ions pass the apical cell membranes through highly selective Na(+) channels and follow an electrochemical gradient which is sustained by the basolateral Na(+)/K(+)-ATPases. These apical Na(+) channels are selectively blocked by amiloride and represent the rate-limiting target for the control of transcellular Na(+) uptake. Although annelids express ADH-like peptide hormones, they lack the osmoregulatory mineralocorticoid system with the vertebrate-specific key hormone aldosterone. Thus, their epithelia may represent interesting models for investigation of ion transport regulation. While the formation of urine in the nephridia of, for example, leeches had been subject to intensive studies, the investigation of ion transport across their body wall was largely neglected. We use dissected segments of integuments from the limnic leech Hirudo medicinalis and, recently, from the earthworm Lumbricus terrestris for Ussing chamber experiments. We investigate transintegumental ion transport with focus on control of electrogenic Na(+) uptake and the amiloride-sensitive part of it and identified several extracellular factors as peptide hormones, tri- and divalent cations or purinergic molecules with regulatory effects on it. Meanwhile, there exists a macroscopic view on Na(+) absorption; however, other ion transport mechanisms across annelid integuments still await scientific effort. Here we present a concise synopsis about the electrophysiology of annelid integuments to illustrate the state of science and to evaluate whether further studies in this particular field may be of interest.

Sensitivity of oocyte-expressed epithelial Na+ channel to glibenclamide.

The effect of glibenclamide on heterologously expressed amiloride-sensitive sodium channels (ENaCs) was investigated in Xenopus oocytes. The ENaC is a heteromer and consists of alpha-, beta- and gamma-subunits and the alpha- and beta-subunits have previously been shown to confer sensitivity to glibenclamide. We coexpressed either colonic rat alpha- (ralpha) or guinea-pig alpha-subunit (gpalpha) with Xenopus betagamma-subunits. The gpalphaxbetagamma was significantly stimulated by glibenclamide (100 microM) (184+/-15%), whereas the ralpha-combination was slightly down-regulated by the sulfonylurea (79+/-4%). The stimulating effect did not interfere with Na(+)-self-inhibition resulting from intracellular accumulation of Na(+)-ions. We exchanged cytosolic termini between both orthologs but the gpalpha-chimera with the termini from rat retained sensitivity to glibenclamide. The effect of glibenclamide on Xenopus ENaC (xENaC) was inhibited by ADP-beta-S but not by ATP-gamma-S, when applied intracellularly. Intracellular loading with Na(+)-ions after inhibition of Na(+)/K(+)-ATPases with ouabain prevented an up-regulation of ENaC activity by glibenclamide. Pretreatment of oocytes expressing xENaC with edelfosine (ET-18-OCH(3)) slightly reduced stimulation of I(ami) (118+/-12%; control: 132+/-9%) while phosphatidylinositol-4,5-biphosphate (PIP(2)) significantly reduced the effect of glibenclamide to 101+/-3%.

Luminal acetylcholine does not affect the activity of the CFTR in tracheal epithelia of pigs.

Fluid homeostasis mediated by the airway epithelium is required for proper lung function, and the CFTR (cystic fibrosis transmembrane conductance regulator) Cl(-) channel is crucial for these processes. Luminal acetylcholine (ACh) acts as an auto-/paracrine mediator to activate Cl(-) channels in airway epithelia and evidence exists showing that nicotinic ACh receptors activate CFTR in murine airway epithelia. The present study investigated whether or not luminal ACh regulates CFTR activity in airway epithelia of pigs, an emerging model for investigations of human airway disease and cystic fibrosis (CF) in particular. Transepithelial ion currents of freshly dissected pig tracheal preparations were measured with Ussing chambers. Application of luminal ACh (100 µM) induced an increase of the short-circuit current (I(SC)). The ACh effect was mimicked by muscarine and pilocarpine (100 µM each) and was sensitive to muscarinic receptor antagonists (atropine, 4-DAMP, pirenzepine). No changes of the I(SC) were observed by nicotine (100 µM) and ACh responses were not affected by nicotine or mecamylamine (25 µM). Luminal application of IBMX (I, 100 µM) and forskolin (F, 10 µM), increase the I(SC) and the I/F-induced current were decreased by the CFTR inhibitor GlyH-101 (GlyH, 50 µM) indicating increased CFTR activity by I/F. In contrast, GlyH did not affect the ACh-induced current, indicating that the ACh response does not involve the activation of the CFTR. Results from this study suggest that luminal ACh does not regulate the activity of the CFTR in tracheal epithelia of pigs which opposes observation from studies using mice airway epithelium.

Ion transport across Xenopus alveolar epithelium is regulated by extracellular ATP, UTP and adenosine.

Native alveolar epithelium from Xenopus lung was used for electrophysiological Ussing chamber experiments to investigate ion transport regulation. The tissue exhibits a considerable absorption of Na(+) ions and this transepithelial transport is largely up-regulated after treatment of donor animals with ACTH. Extracellular ATP, UTP and adenosine were tested for their regulating effects and all three increased I(sc), which was mainly due to a stimulation of amiloride sensitive Na(+) transport (increase of I(ami) 32% for ATP, 21% for UTP, 25% for adenosine). Solely the effect of UTP was completely abolished in the presence of amiloride. In contrast, the effects of ATP or adenosine disappeared under Cl(-)-free conditions. ATP and UTP proved to have additive effects and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), an antagonist of purinergic receptors, inhibited selectively the effect of UTP on I(sc). Further, I(sc) was increased by the P2X selective agonist beta,gamma-meATP. We were able to demonstrate, that extracellular purines and pyrimidines play a possible role as auto/paracrine messengers for alveolar ion transport regulation in Xenopus lung.