Na+ pump alpha 2-subunit expression modulates Ca2+ signaling

The role of the Na⁺ pump₂-subunit in Ca²⁺ signaling was examined inprimary cultured astrocytes from wild-type(₂^+/+ = WT) mouse fetuses and thosewith a null mutation in one [₂^+/ = heterozygote (Het)] or both [₂^/ = knockout (KO)] ₂ genes. Na⁺ pump catalytic() subunit expression was measured by immunoblot; cytosol[Na⁺] ([Na⁺]_cyt) and[Ca²⁺] ([Ca²⁺]_cyt) weremeasured with sodium-binding benzofuran isophthalate and fura 2 byusing digital imaging. Astrocytes express Na⁺ pumpswith both ₁- (80% of total ) and₂- (20% of total ) subunits. Het astrocytesexpress 50% of normal ₂; those from KO express none.Expression of ₁ is normal in both Het and KO cells.Resting [Na⁺]_cyt = 6.5 mM in WT, 6.8 mMin Het (P > 0.05 vs. WT), and 8.0 mM in KO cells(P < 0.001); 500 nM ouabain (inhibits only₂) equalized [Na⁺]_cyt at 8 mMin all three cell types. Resting[Ca²⁺]_cyt = 132 nM in WT, 162 nM in Het,and 196 nM in KO cells (both P < 0.001 vs. WT).Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER)Ca²⁺ pumps and unloads the ER, induces transient (inCa²⁺-free media) or sustained (in Ca²⁺-repletemedia) elevation of [Ca²⁺]_cyt. TheseCa²⁺ responses to 10 µM CPA were augmented in Het as wellas KO cells. When CPA was applied in Ca²⁺-free media, thereintroduction of Ca²⁺ induced significantly largertransient rises in [Ca²⁺]_cyt (due toCa²⁺ entry through store-operated channels) in Het and KOcells than in WT cells. These results correlate with published evidencethat ₂ Na⁺ pumps andNa⁺/Ca²⁺ exchangers are confined to plasmamembrane microdomains that overlie the ER. The data suggest thatselective reduction of ₂ Na⁺ pump activitycan elevate local [Na⁺] and, viaNa⁺/Ca²⁺ exchange, [Ca²⁺] in thetiny volume of cytosol between the plasma membrane and ER. This, inturn, augments adjacent ER Ca²⁺ stores and therebyamplifies Ca²⁺ signaling without elevating bulk[Na⁺]_cyt.

     

Synthesis and characterization of dual-wavelength Cl--sensitive fluorescent indicators for ratio imaging

The fluorescence of quinolinium-basedCl indicators such as6-methoxy-N-(3-sulfopropyl)quinolinium(SPQ) is quenched by Cl bya collisional mechanism without change in spectral shape. A series of"chimeric" dual-wavelengthCl indicators weresynthesized by conjugatingCl-sensitive and-insensitive chromophores with spacers. The SPQ chromophore(N-substituted 6-methoxyquinolinium; MQ) was selected as theCl-sensitive moiety[excitation wavelength(_ex) 350 nm, emission wavelength (_em) 450 nm]. N-substituted 6-aminoquinolinium (AQ) waschosen as theCl-insensitive moietybecause of its different spectral characteristics (_ex 380 nm,_em 546 nm), insensitivity toCl, positive charge (tominimize quenching by chromophore stacking/electron transfer), andreducibility (for noninvasive cell loading). The dual-wavelengthindicators were stable and nontoxic in cells and were distributeduniformly in cytoplasm, with occasional staining of the nucleus. Thebrightest and mostCl-sensitive indicatorswere -MQ-'-dimethyl-AQ-xylene dichloride andtrans-1,2-bis(4-[1-'-MQ-1'-'-dimethyl-AQ-xylyl]-pyridinium)ethylene (bis-DMXPQ). At 365-nm excitation, emission maxima were at 450 nm(Cl sensitive; Stern-Volmerconstants 82 and 98 M¹)and 565 nm (Clinsensitive). Cystic fibrosis transmembrane conductanceregulator-expressing Swiss 3T3 fibroblasts were labeled with bis-DMXPQby hypotonic shock or were labeled with its uncharged reduced form(octahydro-bis-DMXPQ) by brief incubation (20 µM, 10 min). Changes inCl concentration inresponse to Cl/nitrateexchange were recorded by emission ratio imaging (450/565 nm) at 365-nmexcitation wavelength. These results establish a first-generation setof chimeric bisquinoliniumCl indicators forratiometric measurement ofCl concentration.     

Distinct Ca2+- and cAMP-dependent anion conductances in the apical membrane of polarized T84 cells

Monolayers of the human colonic epithelial cell line T84 exhibitelectrogenic Cl secretionin response to the Ca²⁺ agonistthapsigargin and to the cAMP agonist forskolin. To evaluate directlythe regulation of apical Clconductance by these two agonists, we have utilized amphotericin B topermeabilize selectively the basolateral membranes of T84 cellmonolayers. We find that apical anion conductance is stimulated by bothforskolin and thapsigargin but that these conductances aredifferentially sensitive to the anion channel blocker DIDS. DIDSinhibits thapsigargin-stimulated responses completely but forskolinresponses only partially. Furthermore, the apical membrane anionconductances elicited by these two agonists differ in anion selectivity(for thapsigargin, I > Cl; for forskolin,Cl > I). However, theDIDS-sensitive component of the forskolin-induced conductance responseexhibits anion selectivity similar to that induced by thapsigargin(I > Cl). Thusforskolin-induced apical anion conductance comprises at least twocomponents, one of which has features in common with that elicited bythapsigargin.

     

Human trabecular meshwork cell volume regulation

The volume ofcertain subpopulations of trabecular meshwork (TM) cells may modifyoutflow resistance of aqueous humor, thereby altering intraocularpressure. This study examines the contribution thatNa⁺/H⁺, Cl/HCOexchange, and K⁺-Cl efflux mechanisms have onthe volume of TM cells. Volume, Cl currents, andintracellular Ca²⁺ activity of cultured human TM cells werestudied with calcein fluorescence, whole cell patch clamping, and fura2 fluorescence, respectively. At physiological bicarbonateconcentration, the selective Na⁺/H⁺ antiportinhibitor dimethylamiloride reduced isotonic cell volume. Hypotonicitytriggered a regulatory volume decrease (RVD), which could be inhibitedby the Cl channel blocker5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), the K⁺channel blockers Ba²⁺ and tetraethylammonium, and theK⁺-Cl symport blocker[(dihydroindenyl)oxy]alkanoic acid. The fluid uptake mechanism inisotonic conditions was dependent on bicarbonate; at physiologicallevels, the Na⁺/H⁺ exchange inhibitordimethylamiloride reduced cell volume, whereas at low levels theNa⁺-K⁺-2Cl symport inhibitorbumetanide had the predominant effect. Patch-clamp measurements showedthat hypotonicity activated an outwardly rectifying, NPPB-sensitiveCl channel displaying the permeability rankingCl > methylsulfonate > aspartate.2,3-Butanedione 2-monoxime antagonized actomyosin activity and bothincreased baseline [Ca²⁺] and abolishedswelling-activated increase in [Ca²⁺], but it did notaffect RVD. Results indicate that human TM cells display aCa²⁺-independent RVD and that volume is regulated byswelling-activated K⁺ and Cl channels,Na⁺/H⁺ antiports, and possiblyK⁺-Cl symports in addition toNa⁺-K⁺-2Cl symports.

     

Astrocytes from Na(+)-K(+)-Cl(-) cotransporter-null mice exhibit absence of swelling and decrease in EAA release

We reported previously that inhibition ofNa⁺-K⁺-Cl cotransporter isoform 1 (NKCC1) by bumetanide abolishes high extracellular K⁺concentration ([K⁺]_o)-induced swelling andintracellular Cl accumulation in rat cortical astrocytes.In this report, we extended our study by using cortical astrocytes fromNKCC1-deficient (NKCC1^/) mice. NKCC1 protein andactivity were absent in NKCC1^/ astrocytes.[K⁺]_o of 75 mM increased NKCC1 activityapproximately fourfold in NKCC1^+/+ cells (P < 0.05) but had no effect in NKCC1^/ astrocytes.Intracellular Cl was increased by 70% inNKCC1^+/+ astrocytes under 75 mM[K⁺]_o (P < 0.05) butremained unchanged in NKCC1^/ astrocytes. Baselineintracellular Na⁺ concentration([Na⁺]_i) in NKCC1^+/+ astrocyteswas 19.0 ± 0.5 mM, compared with 16.9 ± 0.3 mM[Na⁺]_i in NKCC1^/ astrocytes(P < 0.05). Relative cell volume ofNKCC1^+/+ astrocytes increased by 13 ± 2% in 75 mM[K⁺]_o, compared with a value of 1.0 ± 0.5% in NKCC1^/ astrocytes (P < 0.05).Regulatory volume increase after hypertonic shrinkage was completelyimpaired in NKCC1^/ astrocytes.High-[K⁺]_o-induced ¹⁴C-labeledD-aspartate release was reduced by ~30% inNKCC1^/ astrocytes. Our study suggests that stimulationof NKCC1 is required for high-[K⁺]_o-inducedswelling, which contributes to glutamate release from astrocytes underhigh [K⁺]_o.

     

Differences in regulation of Ca2+-activated Cl- channels in colonic and parotid secretory cells

We investigatedthe regulation of Ca²⁺-activatedCl channels in cells fromthe human colonic cell line T84 and acinar cells from rat parotidglands. The participation of multifunctional Ca²⁺- and calmodulin-dependentprotein kinase (CaM kinase) II in the activation of these channels wasstudied using selective inhibitors of calmodulin and CaM kinase II.Ca²⁺-dependentCl currents were recordedusing the whole cell patch-clamp technique. Direct inhibition of CaMkinase II by 40 µM peptide 281-302 or by 10 µM KN-62, anotherCaM kinase inhibitor, did not block the Cl current in parotidacinar cells, whereas in T84 cells KN-62 markedly inhibited theCa²⁺-dependentCl current. We also usedthe calmodulin-binding domain peptide 290-309 (0.5 µM), whichcompetitively inhibits the activation of CaM kinase II. This peptidereduced the Cl current inT84 cells by ~70% but was without effect on the channels in parotidacinar cells. We conclude that theCa²⁺-dependentCl channels in T84 cellsare activated by CaM kinase II but that the channels in parotid acinarcells must be regulated by a fundamentally differentCa²⁺-dependent mechanism that doesnot utilize CaM kinase II or any calmodulin-dependent process.

     

Intracellular signaling mechanisms of interleukin-1beta in synovial fibroblasts

The possibility that membrane depolarization of synovialfibroblasts caused by interleukin-1 (IL-1) was mediated byprotein kinase C (PKC) and Ca²⁺influx was studied using inhibitor and activator analysis. The effectof IL-1 was blocked by bisindolylmaleimide I, an inhibitor of PKC,and by the Ca²⁺ channel blockersnifedipine and verapamil. In other experiments, PKC was activated usingphorbol 12-myristate 13-acetate, andCa²⁺ influx was increased by meansof a Ca²⁺ ionophore. Simultaneousapplication of phorbol ester andCa²⁺ ionophore in the absence ofIL-1 mimicked the depolarization caused by IL-1. The results wereconsistent with the hypothesis that, under the conditions studied,activation of PKC and Ca²⁺ influxare necessary and sufficient processes in the transduction of IL-1by synovial cells leading to membrane depolarization. Theessential role of protein phosphorylation andCa²⁺ influx in the earlyelectrophysiological response of synovial fibroblasts to IL-1 wastherefore established. The role of IL-1-induced depolarization inregulating protein expression by the cells remains to be determined,but the results reported here, taken together with observations thatprotein phosphorylation and Ca²⁺influx also mediate the effect of IL-1 on protease production (1, 2), suggest that electrophysiological changes are actually part of thepathway for expression of proteases in response to IL-1.

     

ANG II controls Na+-K+(NH+4)-2Cl- cotransport via 20-HETE and PKC in medullary thick ascending limb

Cell pH was monitored in medullary thick ascending limbs todetermine effects of ANG II onNa⁺-K⁺(NH⁺₄)-2Clcotransport. ANG II at 10¹⁶to 10¹² M inhibited30-50% (P < 0.005),but higher ANG II concentrations were stimulatory compared with the10¹² M ANG II levelcotransport activity; eventually,10⁶ M ANG II stimulated34% cotransport activity (P < 0.003). Inhibition by 10¹²M ANG II was abolished by phospholipase C (PLC), diacylglycerol lipase,or cytochrome P-450-dependentmonooxygenase blockade; 10¹² M ANG II had no effectadditive to inhibition by 20-hydroxyeicosatetranoic acid (20-HETE).Stimulation by 10⁶ M ANG IIwas abolished by PLC and protein kinase C (PKC) blockade and waspartially suppressed when the rise in cytosolicCa²⁺ was prevented. All ANG IIeffects were abolished by DUP-753 (losartan) but not by PD-123319. Thus10¹² M ANG II inhibitsvia 20-HETE, whereas 5 × 10¹¹ M ANG II stimulatesvia PKCNa⁺-K⁺(NH⁺₄)-2Clcotransport; all ANG II effects involveAT₁ receptors and PLC activation.

     

PGE(2), Ca(2+), and cAMP mediate ATP activation of Cl(-) channels in pigmented ciliary epithelial cells

Purines regulate intraocular pressure. Adenosine activatesCl channels of nonpigmented ciliary epithelial cellsfacing the aqueous humor, enhancing secretion. Tamoxifen and ATPsynergistically activate Cl channels of pigmented ciliaryepithelial (PE) cells facing the stroma, potentially reducing netsecretion. The actions of nucleotides alone on Cl channelactivity of bovine PE cells were studied by electronic cell sorting,patch clamping, and luciferin/luciferase ATP assay. Clchannels were activated by ATP > UTP, ADP, and UDP, but not by 2-methylthio-ATP, all at 100 µM. UTP triggered ATP release. The second messengers Ca²⁺, prostaglandin (PG)E₂,and cAMP activated Cl channels without enhancing effectsof 100 µM ATP. Buffering intracellular Ca²⁺activity with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acidor blocking PGE₂ formation with indomethacininhibited ATP-triggered channel activation. The Rp stereoisomerof 8-bromoadenosine 3',5'-cyclic monophosphothioate inhibited proteinkinase A activity but mimicked 8-bromoadenosine 3',5'-cyclicmonophosphate. We conclude that nucleotides can act at >1 P2Yreceptor to trigger a sequential cascade involving Ca²⁺,PGE₂, and cAMP. cAMP acts directly on Clchannels of PE cells, increasing stromal release and potentially reducing net aqueous humor formation and intraocular pressure.

     

Myogenic NOS in canine lower esophageal sphincter: enzyme activation, substrate recycling, and product actions

Depolarization elicited outwardK⁺ currents from canine loweresophageal sphincter (LES) muscle cells, primarily through iberiotoxin (IbTX)- and tetraethylammonium-sensitiveCa²⁺-dependentK⁺ channels. Current magnitudesvaried with pipette Ca²⁺concentration (EC₅₀ = 108.5 nM).N^G-nitro-L-arginine(L-NNA,10⁴ M), IbTX(10⁸ M), or bufferingintracellular Ca²⁺ to 8 nMdecreased outward currents >80%. Sodium nitroprusside (NaNP,10⁴ M) restoredL-NNA-inhibited or lowintracellular Ca²⁺ concentration(not IbTX)-inhibited currents.L-NNA or IbTXapplication depolarized LES cells from 43 to 35 mV. NaNPrestored the membrane potential to 46 mV afterL-NNA but not after IbTXapplication. Nifedipine (30 µM) reduced outward currents andabolished or reduced L-NNA orNaNP effects, respectively. Immunocytochemistry revealed the presenceof both argininosuccinate synthetase and argininosuccinate lyase in LESmuscle cells. L-Citrulline, likeL-arginine, reversed L-NNA inhibition of outwardcurrents; only L-argininereversed inhibition of outward currents by an antibody toargininosuccinate synthetase. Therefore, endogenous nitric oxideproduction, activated by Ca²⁺entrance involving L-typeCa²⁺ channels, may continuouslyenhance outward currents to modulate LES muscle cell membrane potentialand excitability.

     

Peroxynitrite inhibits amiloride-sensitive Na+ currents in Xenopus oocytes expressing alpha beta gamma -rENaC

We examined the effect of peroxynitrite(ONOO) on the cloned ratepithelial Na⁺ channel(-rENaC) expressed in Xenopusoocytes. 3-Morpholinosydnonimine (SIN-1) was used to concurrentlygenerate nitric oxide (· NO) and superoxide(O₂ ·), which react toform ONOO, a species knownto promote protein nitration and oxidation. Under control conditions,oocytes displayed an amiloride-sensitive whole cell conductance of 7.4 ± 2.8 (SE) µS. When incubated at 18°C with SIN-1 (1 mM) for 2 h (final ONOO concentration = 10 µM), the amiloride-sensitive conductance was reduced to0.8 ± 0.5 µS. To evaluate whether the observed inhibition was due to ONOO, as opposedto · NO, we also exposed oocytes to SIN-1 in the presence ofurate (500 µM), a scavenger ofONOO and superoxidedismutase, which scavengesO₂ ·, converting SIN-1from an ONOO to an· NO donor. Under these conditions, conductance values remained at control levels following SIN-1 treatment.Tetranitromethane, an agent that oxidizes sulfhydryl groups at pH6, also inhibited the amiloride-sensitive conductance. These datasuggest that oxidation of critical sulfhydryl groups within rENaC byONOO directly inhibitschannel activity.

     

Na+-K+-Cl- cotransport in human fibroblasts is inhibited by cytomegalovirus infection

We examined the effects of human cytomegalovirus (HCMV)infection on theNa⁺-K⁺-Clcotransporter (NKCC) in a human fibroblast cell line. Using the Cl-sensitive dye MQAE, weshowed that the mock-infected MRC-5 cells express a functional NKCC.1) IntracellularCl concentration([Cl]_i)was significantly reduced from 53.4 ± 3.4 mM to 35.1 ± 3.6 mMfollowing bumetanide treatment. 2)Net Cl efflux caused byreplacement of external Clwith gluconate was bumetanide sensitive.3) InCl-depleted mock-infectedcells, the Cl reuptake rate(in HCO₃-free media) was reduced inthe absence of external Na⁺ and bytreatment with bumetanide. After HCMV infection, we found that although[Cl]_iincreased progressively [24 h postexposure (PE), 65.2 ± 4.5 mM; 72 h PE, 80.4 ± 5.0 mM], the bumetanide andNa⁺ sensitivities of[Cl]_iand net Cl uptake and losswere reduced by 24 h PE and abolished by 72 h PE. Western blots usingthe NKCC-specific monoclonal antibody T4 showed an approximatelyninefold decrease in the amount of NKCC protein after 72 h ofinfection. Thus HCMV infection resulted in the abolition of NKCCfunction coincident with the severe reduction in the amount of NKCCprotein expressed.

     

Long-wavelength iodide-sensitive fluorescent indicators for measurement of functional CFTR expression in cells

Limitations of available indicators [such as6-methoxy-N-(3-sulfopropyl)quinolinium(SPQ)] for measurement of intracellular Cl are their relatively dimfluorescence and need for ultraviolet excitation. A series oflong-wavelength polar fluorophores was screened to identify compoundswith Cl and/orI sensitivity, brightfluorescence, low toxicity, uniform loading of cytoplasm with minimalleakage, and chemical stability in cells. The best compound found was7-(-D-ribofuranosylamino)-pyrido[2,1-h]-pteridin-11-ium-5-olate (LZQ). LZQ is brightly fluorescent with excitation andemission maxima at 400-470 and 490-560 nm, molar extinction11,100 M¹ · cm¹(424 nm), and quantum yield 0.53. LZQ fluorescence is quenched byI by a collisionalmechanism (Stern-Volmer constant 60 M¹) and is not affectedby other halides, nitrate, cations, or pH changes (pH 5-8). AfterLZQ loading into cytoplasm by hypotonic shock or overnight incubation,LZQ remained trapped in cells (leakage <3%/h). LZQ stained cytoplasmuniformly, remained chemically inert, did not bind to cytoplasmiccomponents, and was photobleached by <1% during 1 h of continuousillumination. Cytoplasmic LZQ fluorescence was quenched selectively byI (50% quenching at 38 mMI). LZQ was used tomeasure forskolin-stimulatedI/ClandI/NO₃exchange in cystic fibrosis transmembrane conductance regulator(CFTR)-expressing cell lines by fluorescence microscopy and microplatereader instrumentation using 96-well plates. The substantially improvedoptical and cellular properties of LZQ over existing indicators shouldpermit the quantitative analysis of CFTR function in gene deliverytrials and high-throughput screening of compounds for correction of thecystic fibrosis phenotype.

     

CFTR induces the expression of DRA along with Cl(-)/HCO(3)(-) exchange activity in tracheal epithelial cells

Thickening of airway mucus and lungdysfunction in cystic fibrosis (CF) results, at least in part, fromabnormal secretion of Cl and HCO₃across the tracheal epithelium. The mechanism of the defect in HCO₃ secretion is ill defined; however, a lack ofapical Cl/HCO₃ exchange may exist inCF. To test this hypothesis, we examined the expression ofCl/HCO₃ exchangers in trachealepithelial cells exhibiting physiological features prototypical ofcystic fibrosis [CFT-1 cells, lacking a functional cystic fibrosistransmembrane conductance regulator (CFTR)] or normal trachea (CFT-1cells transfected with functional wild-type CFTR, termed CFT-WT). Cellswere grown on coverslips and were loaded with the pH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, andintracellular pH was monitored. Cl/HCO₃exchange activity increased by ~300% in cells transfected with functional CFTR, with activities increasing from 0.034 pH/min in CFT-1cells to 0.11 in CFT-WT cells (P < 0.001, n = 8). This activity was significantly inhibited byDIDS. The mRNA expression of the ubiquitous basolateral AE-2Cl/HCO₃ exchanger remained unchanged.However, mRNA encoding DRA, recently shown to be aCl/HCO₃ exchanger (Melvin JE, Park K,Richardson L, Schultheis PJ, and Shull GE. J Biol Chem 274:22855-22861, 1999.) was abundantly expressed in cells expressingfunctional CFTR but not in cells that lacked CFTR or that expressedmutant CFTR. In conclusion, CFTR induces the mRNA expression of"downregulated in adenoma" (DRA) and, as a result, upregulates theapical Cl/HCO₃ exchanger activity intracheal cells. We propose that the tracheal HCO₃secretion defect in patients with CF is partly due to thedownregulation of the apical Cl/HCO₃exchange activity mediated by DRA.

     

Purification and reconstitution of an outwardly rectified Cl- channel from tracheal epithelia

We reported the identification of three outwardly rectifiedCl channel (ORCC) candidateproteins (115, 85, and 52 kDa) from bovine tracheal epithelia. We haveraised polyclonal antibodies against these isolated proteins.Incorporation into planar lipid bilayers of material partly purifiedfrom bovine tracheal apical membranes with one of these antibodies as aligand (anti-p115) resulted in the incorporation of an ORCC identicalin biophysical characteristics to one we previously described. Wedeveloped a new purification procedure to increase the yield and purityof this polypeptide. The purification scheme that gave the best results in terms of overall protein yield and purity was a combination ofanion- and cation-exchange chromatography followed byimmunopurification. By use of this purification scheme, 7 µg of the115-kDa protein were purified from 20 mg of tracheal apical membraneproteins. Incorporation of this highly purified material into planarlipid bilayers revealed a DIDS-inhibitable channel with the following properties: linear conductance of 87 ± 9 pS in symmetricalCl solutions, halideselectivity sequence of I > Cl > Br, and lack of sensitivityto protein kinase A, Ca²⁺, ordithiothreitol. Using anti-G_iantibodies to precipitate G_iprotein(s) from the partly purified preparations, we demonstrated thatthe loss of rectification of the ORCC was due to uncoupling ofG_i protein(s) from the ORCCprotein and that the 115-kDa polypeptide is an ORCC.

     

Low extracellular Ca(2+) activates a transient Cl(-) current in chicken ovarian granulosa cells

The effects of low Ca²⁺ on ion currents in hen ovariangranulosa cells were examined. A fast activating and inactivatingtransient outward current (TOC) and a slowly activating outward current (SOC) could be observed. In the presence of normal Ca²⁺concentration (2.5 mM) and with a holding potential of 80 mV, SOC wasactivated in all cells with command pulses more positive than 20 mV.In 2.5 mM Ca²⁺, TOC appeared in 10% of cells at thecommand pulse of +80 mV and in 60-85% of cells at +100 to +120mV. In low-Ca²⁺ solution and command potential of +80 mV(holding potential of 80 mV), the amplitude of TOC was enhanced incells that expressed it in normal Ca²⁺, and TOC appeared in43% of the cells that did not express it initially in normalCa²⁺. At both normal and low Ca²⁺ levels, TOCdecreased as the holding potential became more positive. TOC wasreduced in Cl-deficient solution and in the presence of5-nitro-2-(3-phenylpropylamino)benzoic acid, a Cl channelblocker. These findings suggest that chicken granulosa cells express aCa²⁺-inactivated TOC carried by Cl. Thiscurrent may serve as a signal for some of the reduced metabolic functions of granulosa cells associated with Ca²⁺ deficiency.

     

Contribution of chloride channels to volume regulation of cortical astrocytes

The objective of this study was todetermine the relative contribution of Cl channels tovolume regulation of cultured rat cortical astrocytes after hypotoniccell swelling. Using a Coulter counter, we showed that corticalastrocytes regulate their cell volume by ~60% within 45 min afterhypotonic challenge. This volume regulation was supported whenCl was replaced with Br,NO, methanesulfonate, oracetate but was inhibited when Cl wasreplaced with isethionate or gluconate.Additionally, substitution of Cl with Icompletely blocked volume regulation. Volume regulation was unaffected by furosemide or bumetanide, blockers of KCl transport, but was inhibited by Cl channel blockers, including5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and niflumicacid. Surprisingly, the combination of Cd²⁺ with NPPB,DIDS, or niflumic acid inhibited regulation to a greater extent thanany of these drugs alone. Volume regulation did not differ amongastrocytes cultured from different brain regions, as cerebellar andhippocampal astrocytes exhibited behavior identical to that of corticalastrocytes. These data suggest that Cl flux through ionchannels rather than transporters is essential for volume regulation ofcultured astrocytes in response to hypotonic challenge.

     

Nerve growth factor regulates HCO-3 absorption in thick ascending limb: modifying effects of vasopressin

Growth factorsstimulateNa⁺/H⁺exchange activity in many cell types but their effects on acidsecretion via this mechanism in renal tubules are poorly understood. Weexamined the regulation of HCO₃absorption by nerve growth factor (NGF) in the rat medullary thickascending limb (MTAL), which absorbs HCO₃via apical membraneNa⁺/H⁺exchange. MTAL were perfused in vitro with 25 mMHCO₃ solutions (pH 7.4; 290 mosmol/kgH₂O). Addition of 0.7 nMNGF to the bath decreased HCO₃absorption from 13.1 ± 1.1 to 9.6 ± 0.8 pmol · min¹ · mm¹(P < 0.001). In contrast, with10¹⁰ M arginine vasopressin(AVP) in the bath, addition of NGF to the bath increasedHCO₃ absorption from 8.0 ± 1.6 to12.5 ± 1.3 pmol · min¹ · mm¹(P < 0.01). Both effects of NGF wereblocked by genistein, consistent with the involvement of tyrosinekinase pathways. However, the AVP-dependent stimulation requiredactivation of protein kinase C (PKC), whereas the inhibition was PKCindependent, indicating that the NGF-induced signaling pathways leadingto inhibition and stimulation of HCO₃absorption are distinct. Hypertonicity blocked the inhibition but notthe AVP-dependent stimulation, suggesting that hypertonicity and NGFmay inhibit HCO₃ absorption via acommon mechanism. These data demonstrate that NGF inhibitsHCO₃ absorption in the MTAL underbasal conditions but stimulates HCO₃ absorption in the presence of AVP, effects that are mediated through distinct signal transduction pathways. They also show that AVP is acritical determinant of the response of the MTAL to growth factorstimulation and suggest that NGF can either inhibit or stimulateapical Na⁺/H⁺ exchange activitydepending on its interactions with other regulatory factors. Locallyproduced growth factors such as NGF may play a role in regulating renaltubule HCO₃ absorption.

     

Calcium dependence of C-type natriuretic peptide-formed fast K+ channel

The lipid bilayertechnique was used to characterize theCa²⁺ dependence of a fastK⁺ channel formed by a synthetic17-amino acid segment [OaCNP-39-(1-17)] ofa 39-amino acid C-type natriuretic peptide (OaCNP-39) found in platypus (Ornithorhynchusanatinus) venom (OaV). TheOaCNP-39-(1-17)-formed K⁺ channel was reversiblydependent on1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-buffered cis (cytoplasmic)Ca²⁺ concentration([Ca²⁺]_cis).The channel was fully active when[Ca²⁺]_ciswas >10⁴ M andtrans (luminal)Ca²⁺ concentration was 1.0 mM, butnot at low[Ca²⁺]_cis.The open probability of single channels increased from zero at1 × 10⁶ McisCa²⁺ to 0.73 ± 0.17 (n = 22) at10³ McisCa²⁺. Channel openings to themaximum conductance of 38 pS were rapidly and reversibly activated when[Ca²⁺]_cis,but not transCa²⁺ concentration(n = 5), was increased to >5 × 10⁴ M(n = 14). Channel openings to thesubmaximal conductance of 10.5 pS were dominant at5 × 10⁴ MCa²⁺.K⁺ channels did not open whencisMg²⁺ orSr²⁺ concentrations were increasedfrom zero to 10³ M or when[Ca²⁺]_ciswas maintained at 10⁶ M(n = 3 and 2). The Hill coefficientand the inhibition constant were 1 and 0.8 × 10⁴ McisCa²⁺, respectively. Thisdependence of the channel on high[Ca²⁺]_cissuggests that it may become active under1) physiological conditions whereCa²⁺ levels are high, e.g., duringcardiac and skeletal muscle contractions, and2) pathological conditions that leadto a Ca²⁺ overload, e.g., ischemicheart and muscle fatigue. The channel could modify a cascade ofphysiological functions that are dependent on theCa²⁺-activatedK⁺ channels, e.g., vasodilationand salt secretion.

     

AVP V1 receptor-mediated decrease in Cl- efflux and increase in dark cell number in choroid plexus epithelium

The cerebrospinalfluid (CSF)-generating choroid plexus (CP) has manyV₁ binding sites for argininevasopressin (AVP). AVP decreases CSF formation rate and choroidal bloodflow, but little is known about how AVP alters ion transport across theblood-CSF barrier. Adult rat lateral ventricle CP was loaded with³⁶Cl,exposed to AVP for 20 min, and then placed in isotope-free artificial CSF to measure release of³⁶Cl.Effect of AVP at 10¹² to10⁷ M on theCl efflux rate coefficient(in s¹) was quantified.Maximal inhibition (by 20%) ofCl extrusion at10⁹ M AVP was prevented bythe V₁ receptor antagonist[-mercapto-,-cyclopentamethyleneproprionyl¹,O-Me-Tyr²,Arg⁸]vasopressin.AVP also increased by more than twofold the number of dark and possiblydehydrated but otherwise morphologically normal choroid epithelialcells in adult CP. The V₁ receptorantagonist prevented this AVP-induced increment in dark cell frequency.In infant rats (1 wk) with incomplete CSF secretory ability,10⁹ M AVP altered neitherCl efflux nor dark cellfrequency. The ability of AVP to elicit functional and structuralchanges in adult, but not infant, CP epithelium is discussed in regardto ion transport, CSF secretion, intracranial pressure, and hydrocephalus.