Altered pH(i) regulation and Na(+)/HCO3(-) transporter activity in choroid plexus of cilia-defective Tg737(orpk) mutant mouse

Tg737^orpk mice have defects in cilia assembly and develop hydrocephalus in the perinatal period of life. Hydrocephalus is progressive and is thought to be initiated by abnormal ion and water transport across the choroid plexus epithelium. The pathology is further aggravated by the slow and disorganized beating of motile cilia on ependymal cells that contribute to decreased cerebrospinal fluid movement through the ventricles. Previously, we demonstrated that the hydrocephalus phenotype is associated with a marked increase in intracellular cAMP levels in choroid plexus epithelium, which is known to have regulatory effects on ion and fluid movement in many secretory epithelia. To evaluate whether the hydrocephalus in Tg737^orpk mutants is associated with defects in ion transport, we compared the steady-state pH_i and Na⁺-dependent transport activities of isolated choroid plexus epithelium tissue from Tg737^orpk mutant and wild-type mice. The data indicate that Tg737^orpk mutant choroid plexus epithelium have lower pH_i and higher Na⁺-dependent HCO₃^– transport activity compared with wild-type choroid plexus epithelium. In addition, wild-type choroid plexus epithelium could be converted to a mutant phenotype with regard to the activity of Na⁺-dependent HCO₃^– transport by addition of dibutyryl-cAMP and mutant choroid plexus epithelium toward the wild-type phenotype by inhibiting PKA activity with H-89. Together, these data suggest that cilia have an important role in regulating normal physiology of choroid plexus epithelium and that ciliary dysfunction in Tg737^orpk mutants disrupts a signaling pathway leading to elevated intracellular cAMP levels and aberrant regulation of pH_i and ion transport activity. cAMP; ion transport     

Membrane cholesterol extraction decreases Na+ transport in A6 renal epithelia

In this study, we have investigated the dependence of Na⁺ transport regulation on membrane cholesterol content in A6 renal epithelia. We continuously monitored short-circuit current (I_sc), transepithelial conductance (G_T), and transepithelial capacitance (C_T) to evaluate the effects of cholesterol extraction from the apical and basolateral membranes in steady-state conditions and during activation with hyposmotic shock, oxytocin, and adenosine. Cholesterol extraction was achieved by perfusing the epithelia with methyl--cyclodextrin (mCD) for 1 h. In steady-state conditions, apical membrane cholesterol extraction did not significantly affect the electrophysiological parameters; in contrast, marked reductions were observed during basolateral mCD treatment. However, apical mCD application hampered the responses of I_sc and G_T to hypotonicity, oxytocin, and adenosine. Analysis of the blocker-induced fluctuation in I_sc demonstrated that apical mCD treatment decreased the epithelial Na⁺ channel (ENaC) open probability (P_o) in the steady state as well as after activation of Na⁺ transport by adenosine, whereas the density of conducting channels was not significantly changed as confirmed by C_T measurements. Na⁺ transport activation by hypotonicity was abolished during basolateral mCD treatment as a result of reduced Na⁺/K⁺ pump activity. On the basis of the findings in this study, we conclude that basolateral membrane cholesterol extraction reduces Na⁺/K⁺ pump activity, whereas the reduced cholesterol content of the apical membranes affects the activation of Na⁺ transport by reducing ENaC P_o. epithelial Na⁺ channel; Na⁺-K⁺-ATPase activity; short-circuit current; methyl--cyclodextrin; channel open probability     

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.

     

Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals

Renal epithelial cells release ATP constitutively under basal conditions and release higher quantities of purine nucleotide in response to stimuli. ATP filtered at the glomerulus, secreted by epithelial cells along the nephron, and released serosally by macula densa cells for feedback signaling to afferent arterioles within the glomerulus has important physiological signaling roles within kidneys. In autosomal recessive polycystic kidney disease (ARPKD) mice and humans, collecting duct epithelial cells lack an apical central cilium or express dysfunctional proteins within that monocilium. Collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpk ( Tg737 ) ) mouse model of ARPKD lack a well-formed apical central cilium, thought to be a sensory organelle. We compared these cells grown as polarized cell monolayers on permeable supports to the same cells where the apical monocilium was genetically rescued with the wild-type Tg737 gene that encodes Polaris, a protein essential to cilia formation. Constitutive ATP release under basal conditions was low and not different in mutant versus rescued monolayers. However, genetically rescued principal cell monolayers released ATP three- to fivefold more robustly in response to ionomycin. Principal cell monolayers with fully formed apical monocilia responded three- to fivefold greater to hypotonicity than mutant monolayers lacking monocilia. In support of the idea that monocilia are sensory organelles, intentionally harsh pipetting of medium directly onto the center of the monolayer induced ATP release in genetically rescued monolayers that possessed apical monocilia. Mechanical stimulation was much less effective, however, on mutant orpk collecting duct principal cell monolayers that lacked apical central monocilia. Our data also show that an increase in cytosolic free Ca(2+) primes the ATP pool that is released in response to mechanical stimuli. It also appears that hypotonic cell swelling and mechanical pipetting stimuli trigger release of a common ATP pool. Cilium-competent monolayers responded to flow with an increase in cell Ca(2+) derived from both extracellular and intracellular stores. This flow-induced Ca(2+) signal was less robust in cilium-deficient monolayers. Flow-induced Ca(2+) signals in both preparations were attenuated by extracellular gadolinium and by extracellular apyrase, an ATPase/ADPase. Taken together, these data suggest that apical monocilia are sensory organelles and that their presence in the apical membrane facilitates the formation of a mature ATP secretion apparatus responsive to chemical, osmotic, and mechanical stimuli. The cilium and autocrine ATP signaling appear to work in concert to control cell Ca(2+). Loss of a cilium-dedicated autocrine purinergic signaling system may be a critical underlying etiology for ARPKD and may lead to disinhibition and/or upregulation of multiple sodium (Na(+)) absorptive mechanisms and a resultant severe hypertensive phenotype in ARPKD and, possibly, other diseases.     

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     

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     

Apical electrolyte concentration modulates barrier function and tight junction protein localization in bovine mammary epithelium

In vitro mammary epithelial cell models typically fail to form a consistently tight barrier that can effectively separate blood from milk. Our hypothesis was that mammary epithelial barrier function would be affected by changes in luminal ion concentration and inflammatory cytokines. Bovine mammary epithelial (BME-UV cell line) cells were grown to confluence on permeable supports with a standard basolateral medium and either high-electrolyte (H-elec) or low-electrolyte (L-elec) apical medium for 14 days. Apical media were changed to/from H-elec medium at predetermined times prior to assay. Transepithelial electrical resistance (R_te) was highest in monolayers continuously exposed to apical L-elec. A time-dependent decline in R_te began within 24 h of H-elec medium exposure. Change from H-elec medium to L-elec medium time-dependently increased R_te. Permeation by FITC-conjugated dextran was elevated across monolayers exposed to H-elec, suggesting compromise of a paracellular pathway. Significant alteration in occludin distribution was evident, concomitant with the changes in R_te, although total occludin was unchanged. Neither substitution of Na⁺ with N-methyl-D-glucosamine (NMDG⁺) nor pharmacological inhibition of transcellular Na⁺ transport pathways abrogated the effects of apical H-elec medium on R_te. Tumor necrosis factor alpha, but not interleukin-1 nor interleukin-6, in the apical compartment caused a significant decrease in R_te within 8 h. These results indicate that mammary epithelium is a dynamic barrier whose cell-cell contacts are acutely modulated by cytokines and luminal electrolyte environment. Results not only demonstrate that BME-UV cells are a model system representative of mammary epithelium but also provide critical information that can be applied to other mammary model systems to improve their physiological relevance. transepithelial electrical resistance; apical cation concentration; paracellular permeability; mastitis; inflammatory cytokines; occludin     

Ovine male genital duct epithelial cells differentiate in vitro and express functional CFTR and ENaC

To investigate the biology of the malegenital duct epithelium, we have established cell cultures from theovine vas deferens and epididymis epithelium. These cells develop tightjunctions, high transepithelial electrical resistance, and alumen-negative transepithelial potential difference as a sign of activetransepithelial ion transport. In epididymis cultures the equivalentshort-circuit current (I_sc) averaged 20.8 ± 0.7 µA/cm² (n = 150) and was partially inhibited byapical application of amiloride with an inhibitor concentration of 0.64 µM. In vas deferens cultures, I_sc averaged 14.4 ± 1.1 µA/cm² (n = 18) and was also inhibited byapical application of amiloride with a half-maximal inhibitorconcentration (K_i) of 0.68 µM. The remainingamiloride-insensitive I_sc component in epididymisand vas deferens cells was partially inhibited by apical application ofthe Cl channel blocker diphenylamine-2-carboxylicacid (1 mM). It was largely dependent on extracellularCl and, to a lesser extent, on extracellularHCO₃. It was further stimulated bybasolateral application of forskolin (10⁵ M), which increasedI_sc by 3.1 ± 0.3 µA/cm² (n=65) in epididymis and 0.9 ± 0.1 µA/cm² (n =11) in vas deferens. These findings suggest that cultured ovine vasdeferens and epididymis cells absorb Na⁺ viaamiloride-sensitive epithelial Na⁺ channels (ENaC) andsecrete Cl and HCO₃via apical cystic fibrosis transmembrane conductance regulator (CFTR)Cl channels. This interpretation is supported byRT-PCR data showing that vas deferens and epididymis cells express CFTRand ENaC mRNA.

     

Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport

We report, for the epithelialNa⁺ channel (ENaC) in A6 cells,the modulation by cell pH (pH_c)of the transepithelial Na⁺ current(I_Na), thecurrent through the individual Na⁺channel (i), the openNa⁺ channel density(N_o), and thekinetic parameters of the relationship betweenI_Na and theapical Na⁺ concentration. Thei andN_o were evaluatedfrom the Lorentzian I_Na noise inducedby the apical Na⁺ channel blocker6-chloro-3,5-diaminopyrazine-2-carboxamide.pH_c shifts were induced, understrict and volume-controlled experimental conditions, byapical/basolateral NH₄Cl pulses orbasolateral arrest of theNa⁺/H⁺exchanger (Na⁺ removal; block byethylisopropylamiloride) and were measured with the pH-sensitive probe2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Thechanges in pH_c were positivelycorrelated to changes inI_Na and theapically dominated transepithelial conductance. The sole pH_c-sensitive parameter underlyingI_Na wasN_o. Only thesaturation value of theI_Na kinetics wassubject to changes in pH_c.pH_c-dependent changes inN_o may be causedby influencingP_o, the ENaC openprobability, or/and the total channel number,N_T = N_o/P_o.

     

Regulation of the epithelial Na(+) channel by extracellular acidification

The effect of extracellular acidification wastested on the native epithelial Na⁺ channel (ENaC) in A6epithelia and on the cloned ENaC expressed in Xenopusoocytes. Channel activity was determined utilizing blocker-inducedfluctuation analysis in A6 epithelia and dual electrode voltage clampin oocytes. In A6 cells, a decrease of extracellular pH(pH_o) from 7.4 to 6.4 caused a slow stimulation of theamiloride-sensitive short-circuit current (I_Na)by 68.4 ± 11% (n = 9) at 60 min. This increaseof I_Na was attributed to an increase of openchannel and total channel (N_T) densities. Similar changes were observed with pH_o 5.4. The effects ofpH_o were blocked by buffering intracellularCa²⁺ with 5 µM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Inoocytes, pH_o 6.4 elicited a small transient increase of theslope conductance of the cloned ENaC (11.4 ± 2.2% at 2 min)followed by a decrease to 83.7 ± 11.7% of control at 60 min (n = 6). Thus small decreases of pH_ostimulate the native ENaC by increasing N_T butdo not appreciably affect ENaC expressed in Xenopus oocytes.These effects are distinct from those observed with decreasingintracellular pH with permeant buffers that are known to inhibit ENaC.

     

Dependence of Na+-K+ pump current-voltage relationship on intracellular Na+, K+, and Cs+ in rabbit cardiac myocytes

To examine effects of cytosolicNa⁺, K⁺, and Cs⁺ on the voltagedependence of the Na⁺-K⁺ pump, we measuredNa⁺-K⁺ pump current (I_p)of ventricular myocytes voltage-clamped at potentials(V_m) from 100 to +60 mV. Superfusates weredesigned to eliminate voltage dependence at extracellular pump sites.The cytosolic compartment of myocytes was perfused with patch pipette solutions with a Na⁺ concentration ([Na]_pip)of 80 mM and a K⁺ concentration from 0 to 80 mM or withsolutions containing Na⁺ in concentrations from 0.1 to 100 mM and K⁺ in a concentration of either 0 or 80 mM. When[Na]_pip was 80 mM, K⁺ in pipette solutionshad a voltage-dependent inhibitory effect on I_pand induced a negative slope of theI_p-V_m relationship. Cs⁺ in pipette solutions had an effect onI_p qualitatively similar to that ofK⁺. Increases in I_p with increasesin [Na]_pip were voltage dependent. The dielectriccoefficient derived from[Na]_pip-I_p relationships at thedifferent test potentials was 0.15 when pipette solutions included 80 mM K⁺ and 0.06 when pipette solutions were K⁺ free.

     

SGK1 activates Na+-K+-ATPase in amphibian renal epithelial cells

Serum- and glucocorticoid-induced kinase 1 (SGK1) is thought to be an important regulator of Na⁺ reabsorption in the kidney. It has been proposed that SGK1 mediates the effects of aldosterone on transepithelial Na⁺ transport. Previous studies have shown that SGK1 increases Na⁺ transport and epithelial Na⁺ channel (ENaC) activity in the apical membrane of renal epithelial cells. SGK1 has also been implicated in the modulation of Na⁺-K⁺-ATPase activity, the transporter responsible for basolateral Na⁺ efflux, although this observation has not been confirmed in renal epithelial cells. We examined Na⁺-K⁺-ATPase function in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible promoter. The results showed that expression of a constitutively active mutant of SGK1 (SGK1^T_S425D) increased the transport activity of Na⁺-K⁺-ATPase 2.5-fold. The increase in activity was a direct consequence of activation of the pump itself. The onset of Na⁺-K⁺-ATPase activation was observed between 6 and 24 h after induction of SGK1 expression, a delay that is significantly longer than that required for activation of ENaC in the same cell line (1 h). SGK1 and aldosterone stimulated the Na⁺ pump synergistically, indicating that the pathways mediated by these molecules operate independently. This observation was confirmed by demonstrating that aldosterone, but not SGK1^T_S425D, induced an 2.5-fold increase in total protein and plasma membrane Na⁺-K⁺-ATPase ₁-subunit abundance. We conclude that aldosterone increases the abundance of Na⁺-K⁺-ATPase, whereas SGK1 may activate existing pumps in the membrane in response to chronic or slowly acting stimuli. sodium transport; serum- and glucocorticoid-induced kinase; A6 cells; sodium pump     

K+ channel KVLQT1 located in the basolateral membrane of distal colonic epithelium is not essential for activating Cl- secretion

The cellular mechanism for Cl^– and K⁺ secretion in the colonic epithelium requires K⁺ channels in the basolateral and apical membranes. Colonic mucosa from guinea pig and rat were fixed, sectioned, and then probed with antibodies to the K⁺ channel proteins K_VLQT1 (Kcnq1) and minK-related peptide 2 (MiRP2, Kcne3). Immunofluorescence labeling for Kcnq1 was most prominent in the lateral membrane of crypt cells in rat colon. The guinea pig distal colon had distinct lateral membrane immunoreactivity for Kcnq1 in crypt and surface cells. In addition, Kcne3, an auxiliary subunit for Kcnq1, was detected in the lateral membrane of crypt and surface cells in guinea pig distal colon. Transepithelial short-circuit current (I_sc) and transepithelial conductance (G_t) were measured for colonic mucosa during secretory activation by epinephrine (EPI), prostaglandin E₂ (PGE₂), and carbachol (CCh). HMR1556 (10 µM), an inhibitor of Kcnq1 channels (Gerlach U, Brendel J, Lang HJ, Paulus EF, Weidmann K, Brüggemann A, Busch A, Suessbrich H, Bleich M, and Greger R. J Med Chem 44: 3831–3837, 2001), partially (50%) inhibited Cl^– secretory I_sc and G_t activated by PGE₂ and CCh in rat colon with an IC₅₀ of 55 nM, but in guinea pig distal colon Cl^– secretory I_sc and G_t were unaltered. EPI-activated K⁺-secretory I_sc and G_t also were essentially unaltered by HMR1556 in both rat and guinea pig colon. Although immunofluorescence labeling with a Kcnq1 antibody supported the basolateral membrane presence in colonic epithelium of the guinea pig as well as the rat, the Kcnq1 K⁺ channel is not an essential component for producing Cl^– secretion. Other K⁺ channels present in the basolateral membrane presumably must also contribute directly to the K⁺ conductance necessary for K⁺ exit during activation of Cl^– secretion in the colonic mucosa. HMR1556; K⁺ secretion; epinephrine; prostaglandin E₂; cholinergic     

UTP inhibits Na+ absorption in wild-type and Delta F508 CFTR-expressing human bronchial epithelia

Ca²⁺-mediated agonists,including UTP, are being developed for therapeutic use in cysticfibrosis (CF) based on their ability to modulate alternativeCl conductances. As CF isalso characterized by hyperabsorption ofNa⁺, we determined the effect ofmucosal UTP on transepithelial Na⁺transport in primary cultures of human bronchial epithelia (HBE). Insymmetrical NaCl, UTP induced an initial increase in short-circuit current (I_sc)followed by a sustained inhibition. To differentiate between effects onNa⁺ absorption andCl secretion,I_sc was measuredin the absence of mucosal and serosal Cl(I_Na). Again,mucosal UTP induced an initial increase and then a sustained decreasethat reduced amiloride-sensitiveI_Na by 73%. TheCa²⁺-dependent agonists histamine,bradykinin, serosal UTP, and thapsigargin similarly induced sustainedinhibition (62-84%) ofI_Na. Mucosal UTPinduced similar sustained inhibition (half-maximal inhibitory concentration 296 nM) ofI_Na in primarycultures of human CF airway homozygous for the F508 mutation.BAPTA-AM blunted UTP-dependent inhibition ofI_Na, butinhibitors of protein kinase C (PKC) and phospholipaseA₂ had no effect. Indeed, directactivation of PKC by phorbol 12-myristate 13-acetate failed to inhibitNa⁺ absorption. Apyrase, a tri-and diphosphatase, did not reverse inhibitory effects of UTP onI_Na, suggesting along-term inhibitory effect of UTP that is independent of receptoroccupancy. After establishment of a mucosa-to-serosaK⁺ concentration gradient andpermeabilization of the mucosal membrane with nystatin, mucosal UTPinduced an initial increase in K⁺current followed by a sustained inhibition. We conclude that increasingcellular Ca²⁺ induces a long-terminhibition of transepithelial Na⁺transport across normal and CF HBE at least partly due todownregulation of a basolateral membraneK⁺ conductance. Thus UTP may havea dual therapeutic effect in CF airway:1) stimulation of aCl secretory response and2) inhibition ofNa⁺ transport.     

Adrenergic stimulation of Na+ transport across alveolar epithelial cells involves activation of apical Cl- channels

Alveolar epithelial cells were isolated from adultSprague-Dawley rats and grown to confluence on membrane filters. Mostof the basal short-circuit current(I_sc; 60%) wasinhibited by amiloride (IC₅₀ 0.96 µM) or benzamil (IC₅₀ 0.5 µM).Basolateral addition of terbutaline (2 µM) produced a rapid decreasein I_sc, followed by a slow recovery back to its initial amplitude. WhenCl was replaced withmethanesulfonic acid, the basalI_sc was reduced and the response to terbutaline was inhibited. In permeabilized monolayer experiments, both terbutaline and amiloride produced sustained decreases in current. The current-voltage relationship of the terbutaline-sensitive current had a reversal potential of28 mV. Increasing Cl concentration in thebasolateral solution shifted the reversal potential to more depolarizedvoltages. These results were consistent with the existence of aterbutaline-activated Cl conductance in the apicalmembrane. Terbutaline did not increase the amiloride-sensitiveNa⁺ conductance. We conclude that -adrenergicstimulation of adult alveolar epithelial cells results in an increasein apical Cl permeability and thatamiloride-sensitive Na⁺ channels are not directly affectedby this stimulation.

     

Distinct K+ conductive pathways are required for Cl- and K+ secretion across distal colonic epithelium

Secretion of Cl^– and K⁺ in the colonic epithelium operates through a cellular mechanism requiring K⁺ channels in the basolateral and apical membranes. Transepithelial current [short-circuit current (I_sc)] and conductance (G_t) were measured for isolated distal colonic mucosa during secretory activation by epinephrine (Epi) or PGE₂ and synergistically by PGE₂ and carbachol (PGE₂ + CCh). TRAM-34 at 0.5 µM, an inhibitor of K_Ca3.1 (IK, Kcnn4) K⁺ channels (H. Wulff, M. J. Miller, W. Hänsel, S. Grissmer, M. D. Cahalan, and K. G. Chandy. Proc Natl Acad Sci USA 97: 8151–8156, 2000), did not alter secretory I_sc or G_t in guinea pig or rat colon. The presence of K_Ca3.1 in the mucosa was confirmed by immunoblot and immunofluorescence detection. At 100 µM, TRAM-34 inhibited I_sc and G_t activated by Epi (4%), PGE₂ (30%) and PGE₂ + CCh (60%). The IC₅₀ of 4.0 µM implicated involvement of K⁺ channels other than K_Ca3.1. The secretory responses augmented by the K⁺ channel opener 1-EBIO were inhibited only at a high concentration of TRAM-34, suggesting further that K_Ca3.1 was not involved. Sensitivity of the synergistic response (PGE₂ + CCh) to a high concentration TRAM-34 supported a requirement for multiple K⁺ conductive pathways in secretion. Clofilium (100 µM), a quaternary ammonium, inhibited Cl^– secretory I_sc and G_t activated by PGE₂ (20%) but not K⁺ secretion activated by Epi. Thus Cl^– secretion activated by physiological secretagogues occurred without apparent activity of K_Ca3.1 channels but was dependent on other types of K⁺ channels sensitive to high concentrations of TRAM-34 and/or clofilium. epinephrine; prostaglandin E₂; cholinergic; Kcnn4; TRAM-34; clofilium     

sgk: an essential convergence point for peptide and steroid hormone regulation of ENaC-mediated Na+ transport

To studythe role of sgk (serum, glucocorticoid-induced kinase) inhormonal regulation of Na⁺ transport mediated by theepithelial Na⁺ channel (ENaC), clonal cell lines stablyexpressing human sgk, an S422A sgk mutant, or aD222A sgk mutant were created in the background of the A6model renal epithelial cell line. Expression of normal sgkresults in a 3.5-fold enhancement of basal transport and potentiationof the natriferic response to antidiuretic hormone (ADH). Transfectionof a S422A mutant form of sgk, which cannot bephosphorylated by phosphatidylinositol-dependent kinase (PDK)-2, results in a cell line that is indistinguishable from the parent linein basal and hormone-stimulated Na⁺ transport. The D222Asgk mutant, which lacks kinase activity, functions as adominant-negative mutant inhibiting basal as well as peptide- andsteroid hormone-stimulated Na⁺ transport. Thussgk activity is necessary for ENaC-mediated Na⁺transport. Phosphorylation and activation by PDK-2 are necessary forsgk stimulation of ENaC. Expression of normal sgkover endogenous levels results in a potentiated natriferic response toADH, suggesting that the enzyme is a rate-limiting step for the hormoneresponse. In contrast, sgk does not appear to be therate-limiting step for the cellular response to aldosterone or insulin.

     

Genistein stimulates electrogenic Cl(-) secretion in mouse jejunum

We used the short-circuit current (I_sc) technique to investigate the effects of the isoflavone genistein on the electrogenic Cl^– secretion of the mouse jejunum. Genistein stimulated a sustained increase in I_sc that was dose dependent. Bumetanide inhibited 76 ± 5% of the genistein-stimulated I_sc consistent with activation of Cl^– secretion. Genistein failed to stimulate I_sc following maximal activation of the cAMP pathway by forskolin. In addition, forskolin had a reduced effect on I_sc of the mouse jejunum in the presence of genistein. Glibenclamide, a blocker of CFTR, eliminated the genistein-stimulated increase of I_sc and reduced the forskolin-activated I_sc. Clotrimazole, a Ca²⁺-activated K⁺ channel blocker, failed to reduce the genistein-stimulated I_sc. Vanadate, a blocker of tyrosine-dependent phosphatases, reduced the genistein-activated I_sc. Tyrphostin A23, a tyrosine kinase inhibitor, reduced basal I_sc, after which genistein failed to stimulate I_sc. These data suggest that genistein activated a sustained Cl^– secretory response of the mouse jejunum and that the effect of genistein was via a tyrosine-dependent phosphorylation pathway. 1-ethyl-2-benzimidazolone; vanadate; tyrphostin A23; cantharidic acid; phosphatase     

Role of SGK1 in nitric oxide inhibition of ENaC in Na+-transporting epithelia

Several studies have shown that nitric oxide (NO) inhibits Na⁺ transport in renal and alveolar monolayers. However, the mechanisms by which NO alters epithelial Na⁺ channel (ENaC) activity is unclear. Therefore, we examined the effect of applying the NO donor drug L-propanamine 3,2-hydroxy-2-nitroso-1-propylhidrazino (PAPA-NONOate) to cultured renal epithelial cells. A6 and M1 cells were maintained on permeable supports in medium containing 1.5 µM dexamethasone and 10% bovine serum. After 1.5 µM PAPA-NONOate was applied, amiloride-sensitive short-circuit current measurements decreased 29% in A6 cells and 44% in M1 cells. This differed significantly from the 3% and 19% decreases in A6 and M1 cells, respectively, treated with control donor compound (P < 0.0005). Subsequent application of PAPA-NONOate to amiloride-treated control (no NONOate) A6 and M1 cells did not further decrease transepithelial current. In single-channel patch-clamp studies, NONOate significantly decreased ENaC open probability (P_o) from 0.186 ± 0.043 to 0.045 ± 0.009 (n = 7; P < 0.05) without changing the unitary current. We also showed that aldosterone significantly decreased NO production in primary cultures of alveolar type II (ATII) epithelial cells. Because inducible nitric oxide synthase (iNOS) coimmunoprecipitated with the serum- and glucocorticoid-inducible kinase (SGK1) and both proteins colocalized in the cytoplasm (as shown in our studies in mouse ATII cells), SGK1 may also be important in regulating NO production in the alveolar epithelium. Our study also identified iNOS as a novel SGK1 phosphorylated protein (at S733 and S903 residues in miNOS) suggesting that one way in which SGK1 could increase Na⁺ transport is by altering iNOS production of NO. aldosterone; epithelial sodium channel; serum- and glycocorticoid-inducible kinase     

K+ transport and capacitance of the basolateral membrane of the larval frog skin

Skin from larval bullfrogs was mounted in an Ussing-type chamberin which the apical surface was bathed with a Ringer solution containing 115 mM K⁺ and thebasolateral surface was bathed with a Ringer solution containing 115 mMNa⁺. Ion transport was measured asthe short-circuit current(I_sc) with alow-noise voltage clamp, and skin resistance(R_m) wasmeasured by applying a direct current voltage pulse. Membrane impedance was calculated by applying a voltage signal consisting of 53 sine wavesto the command stage of the voltage clamp. From the ratio of theFourier-transformed voltage and current signals, it was possible tocalculate the resistance and capacitance of the apical and basolateralmembranes of the epithelium(R_a andR_b,C_a and C_b,respectively). With as the anion,R_m decreasedrapidly within 5 min following the addition of 150 U/ml nystatin to theapical solution, whereasI_sc increasedfrom 0.66 to 52.03 µA/cm² over a60-min period. These results indicate that nystatin becomes rapidlyincorporated into the apical membrane and that the increase inbasolateral K⁺ permeabilityrequires a more prolonged time course. Intermediate levels ofI_sc were obtainedby adding 50, 100, and 150 U/ml nystatin to the apical solution. Thisproduced a progressive decrease in R_a andR_b whileC_a andC_b remainedconstant. With Cl as theanion, I_sc valuesincreased from 2.03 to 89.57 µA/cm² following treatment with150 U/ml nystatin, whereas with gluconate as the anionI_sc was onlyincreased from 0.63 to 11.64 µA/cm². This suggests that theincrease in basolateral K⁺permeability produced by nystatin treatment, in the presence of morepermeable anions, is due to swelling of the epithelial cells of thetissue rather than the gradient for apicalK⁺ entry. Finally,C_b was notdifferent among skins exposed toCl,, or gluconate, despite the largedifferences inI_sc, nor didinhibition of I_scby treatment with hyperosmotic dextrose cause significant changes inC_b. These resultssupport the hypothesis that increases in cell volume activateK⁺ channels that are alreadypresent in the basolateral membrane of epithelial cells.