Contrasting effects of cPLA2 on epithelial Na+ transport

Activity of the epithelial Na+ channel (ENaC) is the limiting step for discretionary Na+ reabsorption in the cortical collecting duct. Xenopus laevis kidney A6 cells were used to investigate the effects of cytosolic phospholipase A2 (cPLA2) activity on Na+ transport. Application of aristolochic acid, a cPLA2 inhibitor, to the apical membrane of monolayers produced a decrease in apical [3H]arachidonic acid (AA) release and led to an approximate twofold increase in transepithelial Na+ current. Increased current was abolished by the nonmetabolized AA analog 5,8,11,14-eicosatetraynoic acid (ETYA), suggesting that AA, rather than one of its metabolic products, affected current. In single channel studies, ETYA produced a decrease in ENaC open probability. This suggests that cPLA2 is tonically active in A6 cells and that the end effect of liberated AA at the apical membrane is to reduce Na+ transport via actions on ENaC. In contrast, aristolochic acid applied basolaterally inhibited current, and the effect was not reversed by ETYA. Basolateral application of the cyclooxygenase inhibitor ibuprofen also inhibited current. Both effects were reversed by prostaglandin E2 (PGE2). This suggests that cPLA2 activity and free AA, which is metabolized to PGE2, are necessary to support transport. This study supports the fine-tuning of Na+ transport and reabsorption through the regulation of free AA and AA metabolism.     

Neutrophil elastase activates near-silent epithelial Na+ channels and increases airway epithelial Na+ transport

Neutrophil elastase is a serine protease that is abundant in the airways of individuals with cystic fibrosis (CF), a genetic disease manifested by excessive airway Na(+) absorption and consequent depletion of the airway surface liquid layer. Although endogenous epithelium-derived serine proteases regulate epithelial Na(+) transport, the effects of neutrophil elastase on epithelial Na(+) transport and epithelial Na(+) channel (ENaC) activity are unknown. Low micromolar concentrations of human neutrophil elastase (hNE) applied to the apical surface of a human bronchial cell line (16HBE14o-/beta gamma) increased Na(+) transport about twofold. Similar effects were observed with trypsin, also a serine protease. Proteolytic inhibitors of hNE or trypsin selectively abolished the enzyme-induced increase of epithelial Na(+) transport. At the level of the single channel, submicromolar concentrations of hNE increased activity of near-silent ENaC approximately 108-fold in patches from NIH-3T3 cells expressing rat alpha-, beta-, and gamma-ENaC subunits. However, no enzyme effects were observed on basally active ENaCs. Trypsin exposure following hNE revealed no additional increase in amiloride-sensitive short-circuit current or in ENaC activity, suggesting these enzymes share a common mode of action for increasing Na(+) transport, likely through proteolytic activation of ENaC. The hNE-induced increase of near-silent ENaC activity in CF airways could contribute to Na(+) hyperabsorption, reduced airway surface liquid height, and dehydrated mucus culminating in inefficient mucociliary clearance.     

Characterization of the Na+-dependent Mg2+ transport in sheep ruminal epithelial cells

This study examines the routes by which Mg2+ leaves cultured ovine ruminal epithelial cells (REC). Mg2+-loaded (6 mM) REC were incubated in completely Mg2+-free solutions with varying Na+ concentrations, and the Mg2+ extrusion rate was calculated from the increase of the Mg2+ concentration in the incubation medium determined with the aid of the fluorescent probe mag-fura 2 (Na+ salt). In other experiments, REC were also studied for the intracellular free Mg2+ concentration ([Mg2+]i; using mag-fura 2), the intracellular Na+ concentration (using Na+-binding benzofuran isophthalate), the intracellular cAMP concentration ([cAMP]i; using an enzyme-linked immunoassay), and Na+/Mg2+ exchanger existence [using a monoclonal antibody (mAb) raised against the porcine red blood cell Na+/Mg2+ exchanger]. Mg2+-loaded REC show a Mg2+ efflux that was strictly dependent on extracellular Na+. The Mg2+ extrusion rate increased from 0.018+/-0.009 in a Na+-free medium to 0.73+/-0.3 mM.l cells-1.min-1 in a 145 mM Na+ medium and relates to extracellular Na+ concentration ([Na+]e) according to a typical saturation kinetic (Km value for [Na+]e=24 mM; maximal velocity=11 mM.l cells-1.min-1). Mg2+ efflux was reduced by imipramine (48%) and increased after application of dibutyryl-cAMP (55%) or PGE2 (17%). These effects are completely abolished in Na+-free media. Furthermore, an elevation of [cAMP]i led to an [Mg2+]i decrease that amounted to 375+/-105 microM. The anti-Na+/Mg2+ exchanger mAb inhibits Mg2+ extrusion; moreover, it detects a specific 70-kDa immunoreactive band in protein lysates of ovine REC. The data clearly demonstrate that a Na+/Mg2+ exchanger is existent in the cell membrane of REC. The transport protein is the main pathway (97%) for Mg2+ extrusion and can be assumed to play a considerable role in the process of Mg2+ absorption as well as the maintenance of the cellular Mg2+ homeodynamics.     

Extracellular Zn2+ activates epithelial Na+ channels by eliminating Na+ self-inhibition

Inhibition of epithelial Na(+) channel (ENaC) activity by high concentrations of extracellular Na(+) is referred to as Na(+) self-inhibition. We investigated the effects of external Zn(2+) on whole cell Na(+) currents and on the Na(+) self-inhibition response in Xenopus oocytes expressing mouse alphabetagamma ENaC. Na(+) self-inhibition was examined by analyzing inward current decay from a peak current to a steady-state current following a fast switching of a low Na(+) (1 mm) bath solution to a high Na(+) (110 mm) solution. Our results indicate that external Zn(2+) rapidly and reversibly activates ENaC in a dose-dependent manner with an estimated EC(50) of 2 microm. External Zn(2+) in the high Na(+) bath also prevents or reverses Na(+) self-inhibition with similar affinity. Zn(2+) activation is dependent on extracellular Na(+) concentration and is absent in ENaCs containing gammaH239 mutations that eliminate Na(+) self-inhibition and in alphaS580Cbetagamma following covalent modification by a sulfhydryl-reactive reagent that locks the channels in a fully open state. In contrast, external Ni(2+) inhibition of ENaC currents appears to be additive to Na(+) self-inhibition when Ni(2+) is present in the high Na(+) bath. Pretreatment of oocytes with Ni(2+) in a low Na(+) bath also prevents the current decay following a switch to a high Na(+) bath but rendered the currents below the control steady-state level measured in the absence of Ni(2+) pretreatment. Our results suggest that external Zn(2+) activates ENaC by relieving the channel from Na(+) self-inhibition, and that external Ni(2+) mimics or masks Na(+) self-inhibition.     

Structural and functional organization of Na+ transport in epithelial systems. I. Epithelial Na+ channels

The review summarizes recent data on the structural and functional organization and regulation mechanisms of Na+ transport in epithelial systems. The review is focused on the structure, function, regulation and pathology of epithelial Na+ channels, which are critical for Na+ homeostasis maintenance and blood pressure control.     

pH alterations "reset" Ca2+ sensitivity of brain Na+ channel 2, a degenerin/epithelial Na+ ion channel, in planar lipid bilayers

Members of the degenerin/epithelial Na(+) channel superfamily of ion channels subserve many functions, ranging from whole body sodium handling to mechanoelectrical transduction. We studied brain Na(+) channel 2 (BNaC-2) in planar lipid bilayers to examine its single channel properties and regulation by Ca(2+). Upon incorporation of vesicles made from membranes of oocytes expressing either wild-type (WT) BNaC-2 or BNaC-2 with a gain-of-function (GF) point mutation (G433F), functional channels with different properties were obtained. WT BNaC-2 resided in a closed state with short openings, whereas GF BNaC-2 was constitutively activated; a decrease in the pH in the trans compartment of the bilayer activated WT BNaC-2 and decreased its permeability for Na(+) over K(+). Moreover, these maneuvers made the WT channel more resistant to amiloride. In contrast, GF BNaC-2 did not respond to a decrease in pH, and its amiloride sensitivity and selectivity for Na(+) over K(+) were unaffected by this pH change. Buffering the bathing solutions with EGTA to reduce the free [Ca(2+)] to <10 nm increased WT single channel open probability 10-fold, but not that of GF BNaC-2. Ca(2+) blocked both WT and GF BNaC-2 in a dose- and voltage-dependent fashion; single channel conductances were unchanged. A drop in pH reduced the ability of Ca(2+) to inhibit these channels. These results show that BNaC-2 is an amiloride-sensitive sodium channel and suggest that pH activation of these channels could be, in part, a consequence of H(+) "interference" with channel regulation by Ca(2+).     

Comparative study on sodium transport and Na+,K+-ATPase activity in Caco-2 and rat jejunal epithelial cells: effects of dopamine

The present study reports on the effects of dopamine on sodium transepithelial transport and Na+,K+-ATPase activity in Caco-2 cells, a human epithelial intestinal cell line which undergoes enterocyte differentiation in culture, and jejunal epithelial cells from 20 day old Wistar rats. Addition of amphotericin B to the mucosal side stimulated Isc in a concentration dependent manner (Caco-2 cells, EC50=0.9 [0.5, 1.7] microM; rat jejunum, EC50=7.4 [0.8; 70.1] microM). The presence of 1 microM dopamine did not change the effect of amphotericin B in Caco-2 cells, but produced a significant (P<0.05) decrease in the maximal effect of amphotericin B in the rat jejunum. Dopamine (1 microM), added to the serosal side, did not change the Isc profile in Caco-2 cells, but produced a significant increase in the rat jejunum. This effect was antagonized by SKF 83566 (1 microM), but not S-sulpiride (1 microM), and was mimicked by SKF 38393 (10 nM), but not by quinerolane (10 nM). Basal Na+,K+-ATPase activity (in nmol Pi mg protein(-1) min(-1)) in Caco-2 cells (49.5+/-0.2) was similar to that observed in isolated rat jejunal epithelial cells (52.3+/-3.4). Dopamine (1 microM) significantly (P<0.05) decreased Na+,K+-ATPase activity in rat jejunal epithelial cells, but failed to inhibit Na+,K+-ATPase in Caco-2 cells. This effect of dopamine was antagonized by SKF 83566 (1 microM), but not S-sulpiride (1 microM), and was mimicked by SKF 38393 (10 nM), but not by quinerolane (10 nM). The specific binding of [3H]-Sch 23390 to the rat intestinal mucosa was saturable with an apparent dissociation constant (KD) of 2.4 (0.4; 4.5) nM and maximum receptor density of 259.8+/-32.6 fmol/mg protein. No significant specific binding of [3H]-Sch 23390 was observed in membranes from Caco-2 cells. In conclusion, the results obtained show that D1-like receptor mediated effects of dopamine in the rat jejunum on sodium absorption are absent in Caco-2 cells, most probably because this cell line does not express D1-like dopamine receptors, which ultimately are responsible for the inhibitory effect of the amine upon intestinal Na+,K+-ATPase.     

Regulation of the epithelial Na+ channel by intracellular Na+

The hypothesis that the intracellularNa⁺ concentration([Na⁺]_i)is a regulator of the epithelialNa⁺ channel (ENaC) was tested withthe Xenopus oocyte expression systemby utilizing a dual-electrode voltage clamp.[Na⁺]_iaveraged 48.1 ± 2.2 meq (n = 27)and was estimated from the amiloride-sensitive reversal potential.[Na⁺]_iwas increased by direct injection of 27.6 nl of 0.25 or 0.5 MNa₂SO₄.Within minutes of injection,[Na⁺]_istabilized and remained elevated at 97.8 ± 6.5 meq(n = 9) and 64.9 ± 4.4 (n = 5) meq 30 min after theinitial injection of 0.5 and 0.25 MNa₂SO₄,respectively. This increase of[Na⁺]_icaused a biphasic inhibition of ENaC currents. In oocytes injected with0.5 MNa₂SO₄(n = 9), a rapid decrease of inwardamiloride-sensitive slope conductance(g_Na) to 0.681 ± 0.030 of control within the first 3 min and a secondary, slowerdecrease to 0.304 ± 0.043 of control at 30 min were observed.Similar but smaller inhibitions were also observed with the injectionof 0.25 MNa₂SO₄.Injection of isotonicK₂SO₄(70 mM) or isotonicK₂SO₄made hypertonic with sucrose (70 mMK₂SO₄-1.2M sucrose) was without effect. Injection of a 0.5 M concentration ofeitherK₂SO₄,N-methyl-D-glucamine (NMDG) sulfate, or 0.75 M NMDG gluconate resulted in a much smaller initial inhibition (<14%) and little or no secondary decrease. Thusincreases of[Na⁺]_ihave multiple specific inhibitory effects on ENaC that can betemporally separated into a rapid phase that was complete within 2-3 min and a delayed slow phase that was observed between 5 and 30 min.

     

Na+ and K+ transport at basolateral membranes of epithelial cells. III. Voltage independence of basolateral membrane Na+ efflux

Na+ efflux across basolateral membranes of isolated epithelia of frog skin was tested for voltage sensitivity. The intracellular Na+ transport pool was loaded with 24Na from the apical solution and the rate of isotope appearance in the basolateral solution (JNa23) was measured at timed intervals of 30 s. Basolateral membrane voltage was depolarized by either 50 mM K+, 5 mM Ba++, or 80 mM NH+4. Whereas within 30 s ouabain caused inhibition of JNa23, depolarization of Vb by 30-60 mV caused no significant change of JNa23. Thus, both pump-mediated and leak Na+ effluxes were voltage independent. Although the pumps are electrogenic, pump-mediated Na+ efflux is voltage independent, perhaps because of a nonlinear relationship between pump current and transmembrane voltage. Voltage independence of the leak Na+ efflux confirms a previous suggestion (Cox and Helman, 1983. American Journal of Physiology. 245:F312-F321) that basolateral membrane Na+ leak fluxes are electroneutral.     

Na+ and K+ transport at basolateral membranes of epithelial cells. I. Stoichiometry of the Na,K-ATPase

The stoichiometry of pump-mediated Na/K exchange was studied in isolated epithelial sheets of frog skin. 42K influx across basolateral membranes was measured with tissues in a steady state and incubated in either beakers or in chambers. The short-circuit current provided estimates of Na+ influx at the apical membranes of the cells. 42K influx of tissues bathed in Cl- or SO4-Ringer solution averaged approximately 8 microA/cm2. Ouabain inhibited 94% of the 42K influx. Furosemide was without effect on pre-ouabain-treated tissues but inhibited a ouabain-induced and Cl--dependent component of 42K influx. After taking into account the contribution of the Na+ load to the pump by way of basolateral membrane recycling of Na+, the stoichiometry was found to increase from approximately 2 to 6 as the pump-mediated Na+ transport rate increased from 10 to 70 microA/cm2. Extrapolation of the data to low rates of Na+ transport (less than 10 microA/cm2) indicated that the stoichiometry would be in the vicinity of 3:2. As pump-mediated K+ influx saturates with increasing rates of Na+ transport, Na+ efflux cannot be obligatorily coupled to K+ influx at all rates of transepithelial Na+ transport. These results are similar to those of Mullins and Brinley (1969. Journal of General Physiology. 53:504-740) in studies of the squid axon.     

Na+ transport in plants

The ability of plants to grow in high NaCl concentrations is associated with the ability of the plants to transport, compartmentalize, extrude, and mobilize Na(+) ions. While the influx and efflux at the roots establish the steady state rate of entry of Na(+) into the plant, the compartmentation of Na(+) into the cell vacuoles and the radial transport of Na(+) to the stele and its loading into the xylem establish the homeostatic control of Na(+) in the cytosol of the root cells. Removal of Na(+) from the transpirational stream, its distribution within the plant and its progressive accumulation in the leaf vacuoles, will determine the ability to deal with the toxic effects of Na(+). The aim of this review is to highlight and discuss the recent progress in understanding of Na(+) transport in plants.     

Regulation by Na+ and Ca2+ of renal epithelial Na+ channels reconstituted into planar lipid bilayers

Purified bovine renal epithelial Na+ channels when reconstituted into planar lipid bilayers displayed a specific orientation when the membrane was clamped to -40 mV (cis-side) during incorporation. The trans-facing portion of the channel was extracellular (i.e., amiloride- sensitive), whereas the cis-facing side was intracellular (i.e., protein kinase A-sensitive). Single channels had a main state unitary conductance of 40 pS and displayed two subconductive states each of 12- 13 pS, or one of 12-13 pS and the second of 24-26 pS. Elevation of the [Na+] gradient from the trans-side increased single-channel open probability (Po) only when the cis-side was bathed with a solution containing low [Na+] (< 30 mM) and 10-100 microM [Ca2+]. Under these conditions, Po saturated with increasing [Na+]trans. Buffering of the cis compartment [Ca2+] to nearly zero (< 1 nM) with 10 mM EGTA increased the initial level of channel activity (Po = 0.12 +/- 0.02 vs 0.02 +/- 0.01 in control), but markedly reduced the influence of both cis- and trans-[Na+] on Po. Elevating [Ca2+]cis at constant [Na+] resulted in inhibition of channel activity with an apparent [KiCa2+] of 10-100 microM. Protein kinase C-induced phosphorylation shifted the dependence of channel Po on [Ca2+]cis to 1-3 microM at stationary [Na+]. The direct modulation of single-channel Po by Na+ and Ca2+ demonstrates that the gating of amiloride-sensitive Na2+ channels is indeed dependent upon the specific ionic environment surrounding the channels.