Inhibition of epithelial Na currents by intracellular domains of the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis is characterized by an impaired cyclic adenosine 3,5-monophosphate (cAMP) activated Cl⁻ conductance in parallel with an enhanced amiloride sensitive Na⁺ conductance (ENaC) of the respiratory epithelium. Very recently, acute downregulation of ENaC by the cystic fibrosis transmembrane conductance regulator (CFTR) was demonstrated in several studies. The mechanism, however, by which CFTR exerts its inhibitory effect on ENaC remains obscure. We demonstrate that cytosolic domains of human CFTR are sufficient to induce inhibition of rat epithelial Na⁺ currents (rENaC) when coexpressed in Xenopus oocytes and stimulated with 3-isobutyl-1-methylxanthine (IBMX). Moreover, mutations of CFTR, which occur in cystic fibrosis, abolish CFTR-dependent downregulation of rENaC. Yeast two hybrid analysis of CFTR domains and rENaC subunits suggest direct interaction between the proteins. Enhanced Na⁺ transport as found in the airways of cystic fibrosis patients is probably due to a lack of CFTR dependent downregulation of ENaC.     

The bile acid-sensitive ion channel (BASIC), the ignored cousin of ASICs and ENaC

The DEG/ENaC gene family of ion channels is characterized by a high degree of structural similarity and an equally high degree of diversity concerning the physiological function. In humans and rodents, the DEG/ENaC family comprises 2 main subgroups: the subunits of the epithelial Na⁺ channel (ENaC) and the subunits of the acid sensing ion channels (ASICs). The bile acid-sensitive channel (BASIC), previously known as BLINaC or INaC, represents a third subgroup within the DEG/ENaC family. Although BASIC was identified more than a decade ago, very little is known about its physiological function. Recent progress in the characterization of this neglected member of the DEG/ENaC family, which is summarized in this focused review, includes the discovery of surprising species differences, its pharmacological characterization, and the identification of bile acids as putative natural activators.     

The bile acid-sensitive ion channel (BASIC), the ignored cousin of ASICs and ENaC

The DEG/ENaC gene family of ion channels is characterized by a high degree of structural similarity and an equally high degree of diversity concerning the physiological function. In humans and rodents, the DEG/ENaC family comprises 2 main subgroups: the subunits of the epithelial Na⁺ channel (ENaC) and the subunits of the acid sensing ion channels (ASICs). The bile acid-sensitive channel (BASIC), previously known as BLINaC or INaC, represents a third subgroup within the DEG/ENaC family. Although BASIC was identified more than a decade ago, very little is known about its physiological function. Recent progress in the characterization of this neglected member of the DEG/ENaC family, which is summarized in this focused review, includes the discovery of surprising species differences, its pharmacological characterization, and the identification of bile acids as putative natural activators.     

Dysfunction of mitochondria Ca uptake in cystic fibrosis airway epithelial cells

In the genetic disease cystic fibrosis (CF), the most common mutation F508del promotes the endoplasmic reticulum (ER) retention of misfolded CF proteins. Furthermore, in homozygous F508del-CFTR airway epithelial cells, the histamine Ca²⁺ mobilization is abnormally increased. Because the uptake of Ca²⁺ by mitochondria during Ca²⁺ influx or Ca²⁺ release from ER stores may be crucial for maintaining a normal Ca²⁺ homeostasis, we compared the mitochondria morphology and distribution by transmission electron microscopy technique and the mitochondria membrane potential variation (ΔΨ_mit) using a fluorescent probe (TMRE) on human CF (CF-KM4) and non-CF (MM39) tracheal serous gland cell lines. Confocal imaging of Rhod-2–AM-loaded or of the mitochondrial targeted cameleon 4mtD3cpv-transfected human CF and non-CF cells, were used to examine the ability of mitochondria to sequester intracellular Ca²⁺. The present study reveals that (i) the mitochondria network is fragmented in F508del-CFTR cells, (ii) the ΔΨ_mit of CF mitochondria is depolarized compared non-CF mitochondria, and (iii) the CF mitochondria Ca²⁺ uptake is reduced compared non-CF cells. We propose that these defects in airway epithelial F508del-CFTR cells are the consequence of mitochondrial membrane depolarization leading to a deficient mitochondrial Ca²⁺ uptake.     

Interaction of pyrethroids with the Na channel in mammalian neuronal cells in culture

The interaction of a series of pyrethroids with the Na⁺ channel of mouse neuroblastoma cells has been followed using both an electrophysiological and a ²²Na⁺ influx approach. By themselves, pyrethroids do not stimulate ²²Na⁺ entry through the Na⁺ channel (or the stimulation they give is too small to be analyzed). However, they stimulate ²²Na⁺ entry when used in conjuction with other toxins specific for the gating system of the channel. These include batrachotoxin, veratridine, dihydrograyanotoxin II or polypeptide toxins like sea anemone and scorpion toxins. This stimulatory effect is fully inhibited by tetrodotoxin with a dissociation constant of 1.6 nM for the tetrodotoxin-receptor complex. Half-maximum saturation of the pyrethroid receptor on the Na⁺ channel is observed in the micromolar range for the most active pyrethroids, Decis and RU 15525. The synergism observed between the effect of pyrethroids on ²²Na⁺ influx on the one hand, and the effects of sea anemone toxin II, Androctonus scorpion toxin II, batrachotoxin, veratridine and dihydrograyanotoxin II on the other, indicates that the binding component for pyrethroids on the Na⁺ channel is distinct from the other toxin receptors. It is also distinct from the tetrodotoxin receptor.Some of the pyrethroids used in this study bind to the Na⁺ channel but are unable to stimulate ²²Na⁺ entry. These inactive compounds behave as antagonists of the active pyrethroids.An electrophysiological approach has shown that pyrethroids by themselves are active on the Na⁺ channel of mammalian neurones, and essentially confirm the conclusions made from ²²Na⁺ flux measurements.Pyrethroids are also active on C9 cells in which Na⁺ channels are ‘silent’, that is, not activatable by electrical stimulation. Pyrethroids chemically activate the silent Na⁺ channel in a manner similar to that with veratridine, batrachotoxin, or polypeptide toxins, which are known to slow down the inactivation process of a functional Na⁺ channel.     

Quercetin and NPPB-induced diminution of aldosterone action on Na+ absorption and ENaC expression in renal epithelium

In renal epithelial A6 cells, aldosterone applied for 24 h increased the transepithelial Cl- secretion over 30-fold due to activation of the Na+/K+/2Cl- cotransporter and stimulated the transepithelial Na+ absorption, activity of epithelial Na+ channel (ENaC), and alpha-ENaC mRNA expression. The stimulatory action of aldosterone on the transepithelial Na+ absorption, ENaC activity, and alpha-ENaC mRNA expression was diminished by 24h-pretreatment with quercetin (an activator of Na+/K+/2Cl- cotransporter participating in Cl- entry into the cytosolic space) or 5-nitro 2-(3-phenylpropylamino)benzoate (NPPB) (a blocker of Cl- channel participating in Cl- release from the cytosolic space), while 24h-pretreatment with bumetanide (a blocker of Na+/K+/2Cl- cotransporter) enhanced the stimulatory action of aldosterone on transepithelial Na+ absorption. On the other hand, under the basal (aldosterone-unstimulated) condition, quercetin, NPPB or bumetanide had no effect on transepithelial Na+ absorption, activity of ENaC or alpha-ENaC mRNA expression. These observations suggest that although aldosterone shows overall its stimulatory action on ENaC (transepithelial Na+ transport), aldosterone has an inhibitory action on ENaC (transepithelial Na+ transport) via activation of the Na+/K+/2Cl- cotransporter, and that modification of activity of Cl- transporter/channel participating in the transepithelial Cl- secretion influences the aldosterone-stimulated ENaC (transepithelial Na+ transport).     

Single-file diffusion through the Ca2+-activated K+ channel of human red cells

Summary We have examined the effect of internal and external pH on Na⁺ transport across toad bladder membrane vesicles. Vesicles prepared and assayed with a recently modified procedure (Garty & Asher, 1985) exhibit large, rheogenic, amiloridesensitive fluxes. Of the total²²Na uptake measured 0.5–2.0 min after introducing tracer, 80±4% (mean±se,n=9) is blocked by the diuretic with aK ₁ of 2×10^–8 m. Thus, this amiloridesensitive flux is mediated by the apical sodium-selective channels. Varying the internal (cytosolic) pH over the physiologic range 7.0–8.0 had no effect on sodium transport; this result suggests that variation of intracellular pHin vivo has no direct apical effect on modulating sodium uptake. On the other hand,²²Na was directly and monotonically dependent on external pH. External acidification also reduced the amiloride-sensitive efflux across the walls of the vesicles. This inhibition of²²Na efflux was noted at external Na⁺ concentrations of both 0.2 m and 53mm.These results are different from those reported with whole toad bladder. A number of possible bases for these differences are considered and discussed. We suggest that the natriferic response induced by mucosal acidification of whole toad urinary bladder appears to operate indirectly through one or more factors, presumably cytosolic, present in whole cells and absent from the vesicles.     

structure of ascorbic acid and its biological function

Ascorbic acid/isoascorbic acid are present as radicals at physiological pH with the unpaired electron located in the C(4) region. Since a distinction can be made between both types of radicals, the electron spin resonance technique can be used for discrimination between the epimers of vitamin C. The radical has a cyclic side-chain structure which is formed by the hydrogen bond C(3)-O^– ... HO-C(6) ( 2.7 kJ) and which engulfs Na⁺ or K⁺ in the case of the ascorbyl or the isoascorbyl radical, respectively. The radicals Na-ASC and K-Iso-ASC are electroneutral. Red. glutathione affects both types of radicals by restoring the original electronic configuration at C(4) without changing the electroneutral bicyclic structure. In this way, the mobile carriers Na-ASC and K-Iso-ASC can transport Na⁺ and K⁺ across membranes. Its highest efficiency is around 37 C and pH 7, that is, at physiological values. The biological importance of the side chain of vitamin C is outlined and a possible transport mechanism proposed.     

Feedback inhibition of ENaC: Acute and chronic mechanisms

Intracellular [Na⁺] ([Na⁺]_i) modulates the activity of the epithelial Na channel (ENaC) to help prevent cell swelling and regulate epithelial Na⁺ transport, but the underlying mechanisms remain unclear. We show here that short-term (60–80 min) incubation of ENaC-expressing oocytes in high Na⁺ results in a 75% decrease in channel activity. When the β subunit was truncated, corresponding to a gain-of-function mutation found in Liddle's syndrome, the same maneuver reduced activity by 45% despite a larger increase in [Na⁺]_i. In both cases the inhibition occurred with little to no change in cell-surface expression of γENaC. Long-term incubation (18 hours) in high Na⁺ reduced activity by 92% and 75% in wild-type channels and Liddle's mutant, respectively, with concomitant 70% and 52% decreases in cell-surface γENaC. In the presence of Brefeldin A to inhibit forward protein trafficking, high-Na⁺ incubation decreased wt ENaC activity by 52% and 88% after 4 and 8 hour incubations, respectively. Cleaved γENaC at the cell surface had lifetimes at the surface of 6 hrs in low Na⁺ and 4 hrs in high Na⁺, suggesting that [Na⁺]_i increased the rate of retrieval of cleaved γ ENaC by 50%. This implies that enhanced retrieval of ENaC channels at the cell surface accounts for part, but not all, of the downregulation of ENaC activity shown with chronic increases in [Na⁺]_i.     

Acetylcholine-Induced Na+ influx in the mouse lacrimal gland acinar cells: Demonstration of multiple Na+ transport mechanisms by intracellular Na+ activity measurements

Summary In the isolated, superfused mouse lacrimal gland, intracellular Na⁺ activities (aNa _i ) of the acinar cells were directly measured with double-barreled Na⁺-selective microelectrodes. In the nonstimulated conditionaNa _i was 6.5±0.5 mM and membrane potential (V _m ) was –38.9±0.4 mV. Addition of 1 mM ouabain or superfusion with a K⁺-free solution slightly depolarized the membrane and caused a gradual increase inaNa _i . Stimulation with acetylcholine (ACh, 1 M) caused a membrane hyperpolarization by about 20 mV and an increase inaNa _i by about 9 mM in 5 min. The presence of amiloride (0.1 mM) reduced the ACh-induced increase inaNa _i by approximately 50%, without affectingV _m and input resistance in both nonstimulated and ACh-stimulated conditions. Acid loading the acinar cells by an addition/withdrawal of 20 mM NH₄Cl or by replacement of Tris⁺-buffer saline solution with HCO ₃ ^– /CO₂-buffered solution increasedaNa _i by a few mM. Superfusion with a Cl^–-free NO ₃ ^– solution or 1 mM furosemide or 0.5 mM bumetanide-containing solution had little effect on the restingaNa _i levels, however, it reduced the ACh-induced increase inaNa _i by about 30%. Elimination of metabolite anions (glutamate, fumarate and pyruvate) from the superfusate reduced both the restingaNa _i and the ACh-induced increase inaNa _i .The present results suggest the presence of multiple Na⁺ entry mechanisms activated by ACh, namely, Na⁺/H⁺ exchange, Na-K-Cl cotransport and organic substrate-coupled Na⁺ transport mechanisms.     

Different sensitivity to amiloride of body and tail skins of Rana catesbeiana tadpoles during metamorphosis

Regional differences in potential difference and short-circuit current between the body (dorsal) and the tail skin during metamorphosis of Rana catesbeiana tadpoles were investigated. In body skin, the potential difference and the short-circuit current across the skin develop in two successive steps. At stage XX, the potential difference and the short-circuit current across the body skins were amiloride-insensitive (1st step). At stage XXII, however, amiloride-sensitive potential difference and the short circuit current appeared (2nd step). By contrast, in tail skin the potential difference and the short-circuit current remained amiloride-insensitive (1st step) even at stage XXIII. Since the tail regresses after stage XXIII, the appearance of the second step could not be followed in vivo. To determine whether or not the second step can be induced in the tail, tail skin was cultured under conditions where the skin survives for a much longer period than it does in normally developing tadpoles. Such cultured tail skin generated the amiloride-sensitive potential difference and the short-circuit current and cultured body skin also generated them. Therefore, development of the 2nd step in the tail skin may be delayed in vivo. To characterize the differences between body and tail skin, skins were mutally grafted between body and tail at stage XIII–XV. The body skin grafted on the tail underwent both the 1st and 2nd steps by stage XXII, whereas the tail skin grafted on the body only showed the 1st step by the same stage. These results suggest that the regional specificity of the skin is already established before the prometamorphic stage.Abbreviations CMFS Ca²⁺- and Mg²⁺-free saline - CTS charcoal-treated serum - EDTA ethylene diamine tetra-acetate - I current - PD potential difference - R skin resistance - SCC short-circuit current     

No evidence for inhibition of ENaC through CFTR-mediated release of ATP

Both purinergic stimulation and activation of cystic fibrosis transmembrane conductance regulator (CFTR) increases Cl⁻ secretion and inhibit amiloride-sensitive Na⁺ transport. CFTR has been suggested to conduct adenosine 5′-triphosphate (ATP) or to control ATP release to the luminal side of epithelial tissues. Therefore, a possible mechanism on how CFTR controls the activity of epithelial Na⁺ channels (ENaC) could be by release of ATP or uridine 5′-triphosphate (UTP), which would then bind to P2Y receptors and inhibit ENaC. We examined this question in native tissues from airways and colon and in Xenopus oocytes. Inhibition of amiloride-sensitive transport by both CFTR and extracellular nucleotides was observed in colon and trachea. However, nucleotides did not inhibit ENaC in Xenopus oocytes, even after coexpression of P2Y₂ receptors. Using different tools such as hexokinase, the P2Y inhibitor suramin or the Cl⁻ channel blocker 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), we did not detect any role of a putative ATP secretion in activation of Cl⁻ transport or inhibition of amiloride sensitive short circuit currents by CFTR. In addition, N²,2′-O-dibutyrylguanosine 3′,5′-cyclic monophosphate (cGMP) and protein kinase G (PKG)-dependent phosphorylation or the nucleoside diphosphate kinase (NDPK) do not seem to play a role for the inhibition of ENaC by CFTR, which, however, requires the presence of extracellular Cl⁻.     

Effects of carbamazepine on nerve activity and transmitter release in neuroblastoma-glioma hybrid cells and the frog neuromuscular junction

Effects of the antiepileptic drug carbamazepine on nerve action potential and transmitter release in mouse neuroblastoma-glioma hybrid cells (NG108-15) and the frog neuromuscular junction were studied. Carbamazepine within a concentration range of 0.1–0.5 mmol/L reduced the peak height of the action potential of the NG108-15 cells, whereas the membrane potential and membrane resistance were unaffected. Voltage clamp revealed that the decrease in the action potential was due to the blockage of the Na⁺, delayed K⁺ and transient Ca²⁺ currents. Carbamazepine did not affect Ca²⁺-activated and A type K⁺ currents and long-lasting Ca²⁺ current. In the frog neuromuscular junction, carbamazepine decreased the mean quantal content by a parallel shift in the frequency augmentation–potentiation (FAP) relation. It is concluded that carbamazepine blocks the voltage-dependent Na⁺, delayed K⁺, and transient Ca²⁺ currents and quantal transmitter release through a decrease of nerve excitation.     

In vitro binding of inorganic mercury to the plasma membrane of rat platelet affects Na+-K+-ATPase activity and platelet aggregation

Hg²⁺ binding to ouabain-sensitive Na⁺-K⁺-ATPase of rat platelet membrane was specific with a K_a of 1.3×10⁹ moles and B_max of 3.8 nmoles/mg protein. The binding of mercury to Na⁺-K⁺-ATPase also inhibits the enzyme significantly (P<0.001), which is greater than its ouabain sensitivity. Further in the cytosol of washed platelets conjugation of reduced glutathione (GSH) to Hg²⁺ is correlated dose dependently (25, 50 and 100 pmoles) to enhanced GSH-S-transferase (GST) activity. It may be concluded from the present in vitro experiments that mercury binds specifically to thiol groups present in the platelet membrane Na⁺-K⁺-ATPase, inhibits the enzyme and induces changes in platelet function, namely, platelet aggregation by interfering with the sodium pump.     

The Na/H antiport of eukaryotic cells: Relationship between the kinetic properties of the system and its physiological function

The Na+/H+ antiport is present in the plasma membrane of virtually all vertebrate cells and it plays a central role in cell homeostasis. The pharmacological properties and the characteristics of the interaction of extracellular Na+, Li+, H+ and of intracellular H+ with the Na+/H+ antiport are reviewed herein. The kinetic properties of the system are shown to be essential for defining its four main physiological functions: transepithelial ion transport, control of the pHi, control of the intracellular Na+ concentration, and control of the cell volume. The activity of the Na+/H+ antiport can be modulated by a large number of effectors which are thought to act via protein kinases. At least three mechanisms of activation of the Na+/H+ exchanger are defined from the analysis of the kinetic properties of the system. Activation of the Na+/H+ antiport leads to very different consequences, depending upon the activity of other ion transporting systems in the membrane.