Expression, regulation, and function of P2X4 purinergic receptor in human cervical epithelial cells

Micromolarconcentrations of ATP stimulate biphasic change in transepithelialconductance across CaSki cultures on filters, an acute transientincrease (phase I response; triggered by P2Y₂ receptor and mediated by calcium mobilization-dependent cell volume decrease) followed by a slower decrease in permeability (phase II response). Phase II response is mediated byaugmented calcium influx and protein kinase C-dependent increase intight junctional resistance. The objective of the study was todetermine the role of P2X₄ receptor as a mediator ofphase II response. Human cervical epithelial cells expressP2X₄ receptor mRNA (1.4-, 2.2-, and 4.4-kb isoforms byNorthern blot analysis) and P2X₄ protein. Depletion ofvitamin A reversibly downregulated P2X₄ receptor mRNA andprotein and ATP-induced calcium influx. Depletion of vitamin Aabrogated phase II response, and the effect could bepartially reversed only with retinoic acid receptor (RAR)-selectiveretinoids but not retinoid X receptor (RXR) agonists. Depletion ofvitamin A also abrogated protein kinase C increase in tight junctionalresistance, and the effect could not be reversed with retinoids.Depletion of vitamin A also abrogated phase I increase inpermeability and reversibly downregulated P2Y₂ receptormRNA and ATP-induced calcium mobilization. However, in contrast tophase II response, both RAR and RXR agonists could fullyreverse those effects. These results suggest that phase IIresponse is mediated by a P2X₄ receptor mechanism.

     

Desensitization of P2Y2 receptor-activated transepithelial anion secretion

Desensitization ofP2Y₂ receptor-activated anionsecretion may limit the usefulness of extracellular nucleotides insecretagogue therapy of epithelial diseases, e.g., cystic fibrosis(CF). To investigate the desensitization process for endogenousP2Y₂ receptors, freshly excised orcultured murine gallbladder epithelia (MGEP) were mounted in Ussingchambers to measure short-circuit current (I_sc), an indexof electrogenic anion secretion. Luminal treatment with nucleotidereceptor agonists increased theI_sc with apotency profile of ATP = UTP > 2-methylthioATP >>,-methylene-ATP. RT-PCR revealed the expression ofP2Y₂ receptor mRNA in the MGEPcells. The desensitization of anion secretion required a 10-minpreincubation with the P2Y₂receptor agonist UTP and increased in aconcentration-dependent manner(IC₅₀  10⁶ M). Approximately 40%of the anion secretory response was unaffected by maximal desensitizingconcentrations of UTP. Recovery from UTP-induced desensitization wasrapid (<10 min) at preincubation concentrations less than theEC₅₀ (1.9 × 10⁶ M) but requiredprogressively longer time periods at greater concentrations.UTP-induced total inositol phosphate production and intracellularCa²⁺ mobilization desensitizedwith a concentration dependence similar to that of anion secretion. Incontrast, maximal anion secretion induced byCa²⁺ ionophore ionomycin wasunaffected by preincubation with a desensitizing concentration of UTP.It was concluded that 1)desensitization of transepithelial anion secretion stimulated by theP2Y₂ receptor agonist UTP is timeand concentration dependent; 2)recovery from desensitization is prolonged (>90 min) at UTPconcentrations >10⁵ M;and 3) UTP-induced desensitizationoccurs before the operation of the anion secretory mechanism.     

Retinoids modulate P2U purinergic receptor-mediated changes in transcervical paracellular permeability

In human cervical cells, extracellular ATP induces an acutedecrease in the resistance of the lateral intercellular space, thephase I response, followed by adelayed increase in tight junctional resistance, thephase II response. These responses depend on vitamin A because incubation of cells in retinoid-free medium(RFM) abolished both responses. Treatment with retinoic acid restoredthe phase I response in full, but theamplitude of the phase II response wasrestored only partly. Shorter incubations and lower concentrations ofretinoic acid [half-maximal effective concentration(K_1/2) = 0.1 µM] were required for restoring the phaseI response than were required for reversing thephase II response(K_1/2 = 1 µM).The phase I response could be restoredby ligands that bind to either retinoic acid receptors (RARs) orretinoid X receptors, but only RAR agonists had an effect onphase II response. RFM had no effecton decreases in resistance induced by ionomycin, but it attenuatedphase II-like increases in resistanceinduced by KCl or by1,2-dioctanoyl-sn-diglycerol (diC8).Actinomycin D blocked phase IIresponse but not phase I response orthe responses to ionomycin, KCl, or diC8. These results suggest thatretinoids act on cervical cells via distinct retinoid receptormechanisms and modulate phase I andphase II changes in resistance byregulating distinct signal mechanisms.

     

Stimulation of cardiac L-type calcium channels by extracellular ATP

The co-release of ATP with norepinephrine from sympatheticnerve terminals in the heart may augment adrenergic stimulation ofcardiac Ca²⁺ channel activity. To test for a possibledirect effect of extracellular ATP on L-type Ca²⁺ channels,single channels were reconstituted from porcine sarcolemma into planarlipid bilayers so that intracellular signaling pathways could becontrolled. Extracellular ATP (2-100 µM) increased the openprobability of the reconstituted channels, with a maximal increase of~2.6-fold and an EC₅₀ of 3.9 µM. The increase in open probability was due to an increase in channel availability and adecrease in channel inactivation rate. Other nucleotides displayed arank order of effectiveness of ATP > ,-methylene-ATP > 2-methylthio-ATP > UTP > adenosine5'-O-(3-thiotriphosphate) >> ADP; adenosine had no effect.Several antagonists of P2 receptors had no impact on the ATP-dependentincrease in open probability, indicating that receptor activation wasnot required. These results suggest that extracellular ATP and othernucleotides can stimulate the activity of cardiac L-typeCa²⁺ channels via a direct interaction with the channels.

     

DCEBIO stimulates Cl- secretion in the mouse jejunum

We investigated the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one(DCEBIO) on the Cl^– secretory response of the mouse jejunum using the Ussing short-circuit current (I_sc) technique. DCEBIO stimulated a concentration-dependent, sustained increase in I_sc (EC₅₀ 41 ± 1 µM). Pretreating tissues with 0.25 µM forskolin reduced the concentration-dependent increase in I_sc by DCEBIO and increased the EC₅₀ (53 ± 5 µM). Bumetanide blocked (82 ± 5%) the DCEBIO-stimulated I_sc consistent with Cl^– secretion. DCEBIO was a more potent stimulator of Cl^– secretion than its parent molecule, 1-ethyl-2-benzimidazolinone. Glibenclamide or NPPB reduced the DCEBIO-stimulated I_sc by >80% indicating the participation of CFTR in the DCEBIO-stimulated I_sc response. Clotrimazole reduced DCEBIO-stimulated I_sc by 67 ± 15%, suggesting the participation of the intermediate conductance Ca²⁺-activated K⁺ channel (IK_Ca) in the DCEBIO-activated I_sc response. In the presence of maximum forskolin (10 µM), the DCEBIO response was reduced and biphasic, reaching a peak response of the change in I_sc of 43 ± 5 µA/cm² and then falling to a steady-state response of 17 ± 10 µA/cm² compared with DCEBIO control tissues (61 ± 6 µA/cm²). The forskolin-stimulated I_sc in the presence of DCEBIO was reduced compared with forskolin control tissues. Similar results were observed with DCEBIO and 8-BrcAMP where adenylate cyclase was bypassed. H89, a PKA inhibitor, reduced the DCEBIO-activated I_sc, providing evidence that DCEBIO increased Cl^– secretion via a cAMP/PKA-dependent manner. These data suggest that DCEBIO stimulates Cl^– secretion of the mouse jejunum and that DCEBIO targets components of the Cl^– secretory mechanism. 1-ethyl-2-benzimidazolinone; forskolin; glibenclamide; clotrimazole; H89     

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.

     

Ca2+ depolarizes adrenal cortical cells through selective inhibition of an ATP-activated K+ current

Bovine adrenalzona fasciculata cells (AZF) express a noninactivatingK⁺ current(I_AC) whoseinhibition by adrenocorticotropic hormone and ANG II may be coupled tomembrane depolarization andCa²⁺-dependentcortisol secretion. We studiedI_ACinhibition byCa²⁺ and theCa²⁺ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette)Ca²⁺concentration([Ca²⁺]_i)buffered to 0.02 µM,I_AC reachedmaximum current density of 25.0 ± 5.1 pA/pF(n = 16); raising[Ca²⁺]_ito 2.0 µM reduced it 76%. In inside-out patches, elevated[Ca²⁺]_idramatically reducedI_AC channelactivity. Ionomycin inhibited I_AC by 88 ± 4% (n = 14) without altering rapidlyinactivating A-type K⁺ current.Inhibition of I_ACby ionomycin was unaltered by adding calmodulin inhibitory peptide tothe pipette or replacing ATP with its nonhydrolyzable analog5'-adenylylimidodiphosphate.I_AC inhibition byionomycin was associated with membrane depolarization. When[Ca²⁺]_iwas buffered to 0.02 µM with 2 and 11 mM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ionomycin inhibitedI_AC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively (n = 4). ANG II inhibitedI_AC significantlymore effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising[Ca²⁺]_iinhibits I_ACthrough a mechanism not requiring calmodulin or protein kinases,suggesting direct interaction withI_AC channels. ANGII may inhibitI_AC anddepolarize AZF by activating parallel signaling pathways, one of whichuses Ca²⁺ asa mediator.

     

Ca2+ influx inhibits voltage-dependent and augments Ca2+-dependent K+ currents in arterial myocytes

These experiments were performed to determine the effects ofreducing Ca²⁺ influx(Ca_in) onK⁺ currents(I_K) inmyocytes from rat small mesenteric arteries by1) adding externalCd²⁺ or2) lowering externalCa²⁺ to 0.2 mM. When measured froma holding potential (HP) of 20 mV(I_K20),decreasing Ca_in decreasedI_K at voltageswhere it was active (>0 mV). When measured from a HP of 60 mV(I_K60),decreasing Ca_in increasedI_K at voltagesbetween 30 and +20 mV but decreased I_K at voltagesabove +40 mV. Difference currents(I_K) weredetermined by digital subtraction of currents recorded under controlconditions from those obtained whenCa_in was decreased. At testvoltages up to 0 mV,I_K60 exhibitedkinetics similar to controlI_K60, with rapidactivation to a peak followed by slow inactivation. At 0 mV, peakI_K60 averaged75 ± 13 pA (n = 8) withCd²⁺ and 120 ± 20 pA(n = 9) with lowCa²⁺ concentration. At testvoltages from 0 to +60 mV,I_K60 always had an early positive peak phase, but its apparent "inactivation" increased with voltage and its steady value became negative above +20mV. At +60 mV, the initial peakI_K60 averaged115 ± 18 pA with Cd²⁺ and 187 ± 34 pA with low Ca²⁺. With 10 mM pipette BAPTA, Cd²⁺ produced asmall inhibition ofI_K20 but stillincreased I_K60 between 30 and +10 mV. InCa²⁺-free external solution,Cd²⁺ only decreased bothI_K20 andI_K60. In thepresence of iberiotoxin (100 nM) to inhibitCa²⁺-activatedK⁺ channels(K_Ca),Cd²⁺ increasedI_K60 at allvoltages positive to 30 mV while BAY K 8644 (1 µM) decreasedI_K60. Theseresults suggest that Ca_in, through L-type Ca²⁺ channels and perhapsother pathways, increases K_Ca(i.e., I_K20) and decreases voltage-dependent K⁺currents in this tissue. This effect could contribute to membrane depolarization and force maintenance.

     

Calcium influx through If channels in rat ventricular myocytes

The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac (I_f)/neuronal (I_h) time- and voltage-dependent inward cation current channels, are conventionally considered as monovalent-selective channels. Recently we discovered that calcium ions can permeate through HCN4 and I_h channels in neurons. This raises the possibility of Ca²⁺ permeation in I_f, the I_h counterpart in cardiac myocytes, because of their structural homology. We performed simultaneous measurement of fura-2 Ca²⁺ signals and whole cell currents produced by HCN2 and HCN4 channels (the 2 cardiac isoforms present in ventricles) expressed in HEK293 cells and by I_f in rat ventricular myocytes. We observed Ca²⁺ influx when HCN/I_f channels were activated. Ca²⁺ influx was increased with stronger hyperpolarization or longer pulse duration. Cesium, an I_f channel blocker, inhibited I_f and Ca²⁺ influx at the same time. Quantitative analysis revealed that Ca²⁺ flux contributed to 0.5% of current produced by the HCN2 channel or I_f. The associated increase in Ca²⁺ influx was also observed in spontaneously hypertensive rat (SHR) myocytes in which I_f current density is higher than that of normotensive rat ventricle. In the absence of EGTA (a Ca²⁺ chelator), preactivation of I_f channels significantly reduced the action potential duration, and the effect was blocked by another selective I_f channel blocker, ZD-7288. In the presence of EGTA, however, preactivation of I_f channels had no effects on action potential duration. Our data extend our previous discovery of Ca²⁺ influx in I_h channels in neurons to I_f channels in cardiac myocytes. calcium ion flux; hyperpolarization-activated, cyclic nucleotide-gated/cardiac time- and volume-dependent cation current channels     

Pharmacological modulation of ion transport across wild-type and DeltaF508 CFTR-expressing human bronchial epithelia

Forskolin,UTP, 1-ethyl-2-benzimidazolinone (1-EBIO), NS004, 8-methoxypsoralen(Methoxsalen; 8-MOP), and genistein were evaluated for theireffects on ion transport across primary cultures of human bronchialepithelium (HBE) expressing wild-type (wt HBE) and F508(F-HBE) cystic fibrosis transmembrane conductance regulator. In wtHBE, the baseline short-circuit current (I_sc)averaged 27.0 ± 0.6 µA/cm² (n = 350). Amiloride reduced this I_sc by 13.5 ± 0.5 µA/cm² (n = 317). In F-HBE,baseline I_sc was 33.8 ± 1.2 µA/cm² (n = 200), and amiloride reducedthis by 29.6 ± 1.5 µA/cm² (n = 116), demonstrating the characteristic hyperabsorption of Na⁺ associated with cystic fibrosis (CF). In wt HBE,subsequent to amiloride, forskolin induced a sustained,bumetanide-sensitive I_sc(I_sc = 8.4 ± 0.8 µA/cm²; n = 119). Addition ofacetazolamide, 5-(N-ethyl-N-isopropyl)-amiloride, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid further reduced I_sc, suggesting forskolin also stimulatesHCO₃ secretion. This was confirmed by ionsubstitution studies. The forskolin-induced I_scwas inhibited by 293B, Ba²⁺, clofilium, and quinine,whereas charybdotoxin was without effect. In F-HBE the forskolinI_sc response was reduced to 1.2 ± 0.3 µA/cm² (n = 30). In wt HBE, mucosal UTPinduced a transient increase in I_sc ( I_sc = 15.5 ± 1.1 µA/cm²;n = 44) followed by a sustained plateau, whereas inF-HBE the increase in I_sc was reduced to5.8 ± 0.7 µA/cm² (n = 13). In wtHBE, 1-EBIO, NS004, 8-MOP, and genistein increased I_sc by 11.6 ± 0.9 (n = 20), 10.8 ± 1.7 (n = 18), 10.0 ± 1.6 (n = 5), and 7.9 ± 0.8 µA/cm²(n = 17), respectively. In F-HBE, 1-EBIO, NS004, and8-MOP failed to stimulate Cl secretion. However, additionof NS004 subsequent to forskolin induced a sustained Clsecretory response (2.1 ± 0.3 µA/cm²,n = 21). In F-HBE, genistein alone stimulatedCl secretion (2.5 ± 0.5 µA/cm²,n = 11). After incubation of F-HBE at 26°C for24 h, the responses to 1-EBIO, NS004, and genistein were allpotentiated. 1-EBIO and genistein increased Na⁺ absorptionacross F-HBE, whereas NS004 and 8-MOP had no effect. Finally,Ca²⁺-, but not cAMP-mediated agonists, stimulatedK⁺ secretion across both wt HBE and F-HBE in aglibenclamide-dependent fashion. Our results demonstrate thatpharmacological agents directed at both basolateral K⁺ andapical Cl conductances directly modulate Clsecretion across HBE, indicating they may be useful in ameliorating theion transport defect associated with CF.

     

K+ current inhibition by amphiphilic fatty acid metabolites in rat ventricular myocytes

Fatty acid metabolites accumulate in the heart underpathophysiological conditions that affect -oxidation and can elicit marked electrophysiological changes that are arrhythmogenic. The purpose of the present study was to determine the impact of amphiphilic fatty acid metabolites on K⁺currents that control cardiac refractoriness and excitability. Transient outward(I_to) andinward rectifier(I_K1)K⁺ currents were recorded by thewhole cell voltage-clamp technique in rat ventricular myocytes, and theeffects of two major fatty acid metabolites were examined:palmitoylcarnitine and palmitoyl-coenzyme A (palmitoyl-CoA).Palmitoylcarnitine (0.5-10 µM) caused a concentration-dependent decrease in I_todensity in myocytes internally dialyzed with the amphiphile; 10 µMreduced mean I_todensity at +60 mV by 62% compared with control(P < 0.05). In contrast, externalpalmitoylcarnitine at the same concentrations had no effect, nor didinternal dialysis significantly alterI_K1. Dialysiswith palmitoyl-CoA (1-10 µM) produced a smaller decrease inI_to densitycompared with that produced by palmitoylcarnitine; 10 µM reduced meanI_to density at+60 mV by 37% compared with control(P < 0.05). Both metabolites delayedrecovery of I_tofrom inactivation but did not affect voltage-dependent properties.Moreover, the effects of palmitoylcarnitine were relatively specific,as neither palmitate (10 µM) nor carnitine (10 µM) alone significantly influencedI_to when added tothe pipette solution. These data therefore suggest that amphiphilicfatty acid metabolites downregulateI_to channels by amechanism confined to the cytoplasmic side of the membrane. Thisdecrease in cardiac K⁺ channelactivity may delay repolarization under pathophysiological conditionsin which amphiphile accumulation is postulated to occur, such asdiabetes mellitus or myocardial infarction.

     

Genistein activates CFTR-mediated Cl(-) secretion in the murine trachea and colon

The action of the isoflavonegenistein on the cystic fibrosis transmembrane conductance regulator(CFTR) has been studied in many cell systems but not in intact murinetissues. We have investigated the action of genistein on murine tissuesfrom normal and cystic fibrosis (CF) mice. Genistein increased theshort-circuit current (I_sc) in tracheal(16.4 ± 2.8 µA/cm²) and colonic (40.0 ± 4.4 µA/cm²) epithelia of wild-type mice. This increase wasinhibited by furosemide, diphenylamine-2-carboxylate, andglibenclamide, but not by DIDS. In contrast, genistein produced nosignificant change in the I_sc of the trachealepithelium (0.9 ± 1.1 µA/cm²) and decreased theI_sc of colons from CF null (13.1 ± 2.3 µA/cm²) and F508 mice (10.3 ± 1.3 µA/cm²). Delivery of a human CFTRcDNA-liposome complex to the airways of CF null mice restored thegenistein response in the tracheas to wild-type levels. Tracheas fromF508 mice were also studied: 46% of trachea showed no response togenistein, whereas 54% gave an increase in I_scsimilar to that in wild type. We conclude that genistein activatesCFTR-mediated Cl secretion in the murine trachea anddistal colon.

     

Activation of calcium release assessed by calcium release-induced inactivation of calcium current in rat cardiac myocytes

In mammalian cardiac myocytes, calcium released into the dyadic space rapidly inactivates calcium current (I_Ca). We used this Ca²⁺ release-dependent inactivation (RDI) of I_Ca as a local probe of sarcoplasmic reticulum Ca²⁺ release activation. In whole cell patch-clamped rat ventricular myocytes, Ca²⁺ entry induced by short prepulses from —50 mV to positive voltages caused suppression of peak I_Ca during a test pulse. The negative correlation between peak I_Ca suppression and I_Ca inactivation during the test pulse indicated that RDI evoked by the prepulse affected only calcium channels in those dyads in which calcium release was activated. Ca²⁺ ions injected during the prepulse and during the subsequent tail current suppressed peak I_Ca in the test pulse to a different extent. Quantitative analysis indicated that equal Ca²⁺ charge was 3.5 times less effective in inducing release when entering during the prepulse than when entering during the tail. Tail Ca²⁺ charge injected by the first voltage-dependent calcium channel (DHPR) openings was three times less effective than that injected by DHPR reopenings. These findings suggest that calcium release activation can be profoundly influenced by the recent history of L-type Ca²⁺ channel activity due to potentiation of ryanodine receptors (RyRs) by previous calcium influx. This conclusion was confirmed at the level of single RyRs in planar lipid bilayers: using flash photolysis of the calcium cage NP-EGTA to generate two sequential calcium stimuli, we showed that RyR activation in response to the second stimulus was four times higher than that in response to the first stimulus. excitation-contraction coupling     

Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells

Patch-clamping and cell imageanalysis techniques were used to study the expression of thevolume-activated Cl current,I_Cl(vol), and regulatory volume decrease (RVD)capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated aCl current with a linear conductance, negligibletime-dependent inactivation, and a reversal potential close to theCl equilibrium potential. The sequence of anionpermeability was I > Br > Cl > gluconate. The Cl channelblockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),and ATP inhibited I_Cl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by adouble chemical-block (thymidine and hydroxyurea) technique. Theexpression of I_Cl(vol) was cell cycle dependent,being high in G₁ phase, downregulated in S phase, butincreasing again in M phase. Hypotonic solution activated RVD, whichwas cell cycle dependent and inhibited by the Cl channelblockers NPPB, tamoxifen, and ATP. The expression of I_Cl(vol) was closely correlated with the RVDcapacity in the cell cycle, suggesting a functional relationship.Inhibition of I_Cl(vol) by NPPB (100 µM)arrested cells in G₀/G₁. The data also suggest that expression of I_Cl(vol) and RVD capacity areactively modulated during the cell cycle. The volume-activatedCl current associated with RVD may therefore play animportant role during the cell cycle progress.

     

p38 mitogen-activated protein kinase inhibits calcium-dependent chloride secretion in T84 colonic epithelial cells

We havepreviously shown that Ca²⁺-dependent Clsecretion across intestinal epithelial cells is limited by a signalingpathway involving transactivation of the epidermal growth factorreceptor (EGFR) and activation of ERK mitogen-activated protein kinase (MAPK). Here, we have investigated a possible role for p38 MAPK inregulation of Ca²⁺-dependent Cl secretion.Western blot analysis of T₈₄ colonic epithelial cells revealed that the muscarinic agonist carbachol (CCh; 100 µM)stimulated phosphorylation and activation of p38 MAPK. The p38inhibitor SB-203580 (10 µM) potentiated and prolonged short-circuitcurrent (I_sc) responses to CCh acrossvoltage-clamped T₈₄ cells to 157.4 ± 6.9% of thosein control cells (n = 21; P < 0.001).CCh-induced p38 phosphorylation was attenuated by the EGFR inhibitortyrphostin AG-1478 (0.1 nM-10 µM) and by the Src family kinaseinhibitor PP2 (20 nM-2 µM). The effects of CCh on p38phosphorylation were mimicked by thapsigargin (TG; 2 µM), whichspecifically elevates intracellular Ca²⁺, and wereabolished by the Ca²⁺ chelator BAPTA-AM (20 µM), implyinga role for intracellular Ca²⁺ in mediating p38 activation.SB-203580 (10 µM) potentiated I_sc responses toTG to 172.4 ± 18.1% of those in control cells (n = 18; P < 0.001). When cells were pretreated withSB-203580 and PD-98059 to simultaneously inhibit p38 and ERK MAPKs,respectively, I_sc responses to TG and CCh weresignificantly greater than those observed with either inhibitor alone.We conclude that Ca²⁺-dependent agonists stimulate p38 MAPKin T₈₄ cells by a mechanism involving intracellularCa²⁺, Src family kinases, and the EGFR. CCh-stimulated p38activation constitutes a similar, but distinct and complementary,antisecretory signaling pathway to that of ERK MAPK.

     

Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]o regulation

The neuronal K-Cl cotransporter isoform (KCC2) was functionallyexpressed in human embryonic kidney (HEK-293) cell lines. Two stablytransfected HEK-293 cell lines were prepared: one expressing anepitope-tagged KCC2 (KCC2-22T) and another expressing theunaltered KCC2 (KCC2-9). The KCC2-22T cells produced aglycoprotein of ~150 kDa that was absent from HEK-293 control cells.The ⁸⁶Rb influx in both cell lineswas significantly greater than untransfected control HEK-293 cells. TheKCC2-9 cells displayed a constitutively active⁸⁶Rb influx that could beincreased further by 1 mMN-ethylmaleimide (NEM) but not by cellswelling. Both furosemide [inhibition constant (K_i) ~25µM] and bumetanide (K_i~55 µM) inhibited the NEM-stimulated ⁸⁶Rb influx in the KCC2-9cells. This diuretic-sensitive⁸⁶Rb influx in theKCC2-9 cells, operationally defined as KCC2 mediated, required external Clbut not external Na⁺ and exhibiteda high apparent affinity for externalRb⁺(K⁺)[Michaelis constant(K_m) = 5.2 ± 0.9 (SE) mM; n = 5] but alow apparent affinity for externalCl(K_m >50 mM). Onthe basis of thermodynamic considerations as well as the unique kineticproperties of the KCC2 isoform, it is hypothesized that KCC2 may servea dual function in neurons: 1) themaintenance of low intracellularCl concentration so as toallow Cl influx vialigand-gated Cl channelsand 2) the buffering of externalK⁺ concentration([K⁺]_o) in the brain.

     

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.

     

Volume-regulated chloride conductance in the LNCaP human prostate cancer cell line

Patch-clamp recordings were used to study ioncurrents induced by cell swelling caused by hypotonicity in humanprostate cancer epithelial cells, LNCaP. The reversal potential of the swelling-evoked current suggested that Cl was the primarycharge carrier (termed I_Cl,swell). Theselectivity sequence of the underlying volume-regulated anion channels(VRACs) for different anions wasBrI > Cl > F > methanesulfonate glutamate, with relativepermeability numbers of 1.26, 1.20, 1.0, 0.77, 0.49, and 0.036, respectively. The current-voltage patterns of the whole cell currentsas well as single-channel currents showed moderate outwardrectification. Unitary VRAC conductance was determined at 9.6 ± 1.8 pS. Conventional Cl channel blockers5-nitro-2-(3-phenylpropylamino)benzoic acid (100 µM) and DIDS (100 µM) inhibited whole cell I_Cl,swell in a voltage-dependent manner, with the block decreasing from 39.6 ± 9.7% and 71.0 ± 11.0% at +50 mV to 26.2 ± 7.2% and14.5 ± 6.6% at 100 mV, respectively. Verapamil (50 µM), astandard Ca²⁺ antagonist and P-glycoprotein functioninhibitor, depressed the current by a maximum of 15%. Protein tyrosinekinase inhibitors downregulated I_Cl,swell(genistein with an IC₅₀ of 2.6 µM and lavendustin A by60 ± 14% at 1 µM). The protein tyrosine phosphatase inhibitorsodium orthovanadate (500 µM) stimulatedI_Cl,swell by 54 ± 11%. We conclude thatVRACs in human prostate cancer epithelial cells are modulated viaprotein tyrosine phosphorylation.

     

Changes in intracellular Ca2+ concentration induced by L-type Ca2+ channel current in guinea pig gastric myocytes

We investigatedthe relationship between voltage-operatedCa²⁺ channel current and thecorresponding intracellular Ca²⁺concentration([Ca²⁺]_i)change (Ca²⁺ transient) in guineapig gastric myocytes. Fluorescence microspectroscopy was combined withconventional whole cell patch-clamp technique, and fura 2 (80 µM) wasadded to CsCl-rich pipette solution. Step depolarization to 0 mVinduced inward Ca²⁺ current(I_Ca) andconcomitantly raised[Ca²⁺]_i.Both responses were suppressed by nicardipine, an L-typeCa²⁺ channel blocker, and thevoltage dependence of Ca²⁺transient was similar to the current-voltage relation ofI_Ca. When pulseduration was increased by up to 900 ms, peakCa²⁺ transient increased andreached a steady state when stimulation was for longer. The calculatedfast Ca²⁺ buffering capacity(B value), determined as the ratio ofthe time integral ofI_Ca divided bythe amplitude of Ca²⁺ transient,was not significantly increased after depletion of Ca²⁺ stores by the cyclicapplication of caffeine (10 mM) in the presence of ryanodine (4 µM).The addition of cyclopiazonic acid (CPA, 10 µM), a sarco(endo)plasmicreticulum Ca²⁺-ATPase inhibitor,decreased B value by ~20% in areversible manner. When KCl pipette solution was used,Ca²⁺-activatedK⁺ current[I_K(Ca)]was also recorded during step depolarization. CPA sensitivelysuppressed the initial peak and oscillations of I_K(Ca) withirregular effects on Ca²⁺transients. The above results suggest that, in guinea pig gastric myocyte, Ca²⁺ transient is tightlycoupled to I_Caduring depolarization, and global[Ca²⁺]_iis not significantly affected byCa²⁺-inducedCa²⁺ release from sarcoplasmicreticulum during depolarization.

     

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