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.

     

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.

     

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.

     

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.     

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.

     

Proton secretion in the male reproductive tract: involvement of Cl--independent HCO-3 transport

The lumen of theepididymis is the site where spermatozoa undergo their final maturationand acquire the capacity to become motile. An acidic luminal fluid isrequired for the maintenance of sperm quiescence and for the preventionof premature activation of acrosomal enzymes during their storage inthe cauda epididymis and vas deferens. We have previously demonstratedthat a vacuolar H⁺-ATPase[proton pump (PP)] is present in the apical pole of apical and narrow cells in the caput epididymis and of clear cells in thecorpus and cauda epididymis and that this PP is responsible for themajority of proton secretion in the proximal vas deferens. We now showthat PP-rich cells in the vas deferens express a high level of carbonicanhydrase type II (CAII) and that acetazolamide markedly inhibits therate of proton secretion by 46.2 ± 6.1%. The rate ofacidification was independent ofCl and was stronglyinhibited by SITS under both normal andCl-free conditions (50.6 ± 5.0 and 57.5 ± 6.0%, respectively). In the presence ofCl,diphenylamine-2-carboxylate (DPC) had no effect, whereas SITS inhibitedproton secretion by 63.7 ± 11.3% when applied together with DPC. In Cl-freesolution, DPC markedly inhibited proton efflux by 45.1 ± 7.6%,SITS produced an additional inhibition of 18.2 ± 6.6%, and bafilomycin had no additive effect. In conclusion, we propose that CAIIplays a major role in proton secretion by the proximal vas deferens.Acidification does not require the presence ofCl, but DPC-sensitiveCl channels mightcontribute to basolateral extrusion ofHCO₃ underCl-free conditions. Theinhibition by SITS observed under both normal andCl-free conditionsindicates that aCl/HCO₃exchanger is not involved and that an alternativeHCO₃ transporter participates in proton secretion in the proximal vas deferens.

     

Ouabain and substrate affinities of human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells

HumanNa⁺-K⁺-ATPase₁₁,₂₁, and ₃₁heterodimers were expressed individually in yeast, and ouabainbinding and ATP hydrolysis were measured in membrane fractions. Theouabain equilibrium dissociation constant was 13-17 nM for₁₁ and ₃₁at 37°C and 32 nM for ₂₁, indicatingthat the human -subunit isoforms have a similar high affinity forcardiac glycosides. K_0.5 values for antagonism of ouabain binding by K⁺ were ranked in order as follows:₂ (6.3 ± 2.4 mM) > ₃(1.6 ± 0.5 mM)  ₁ (0.9 ± 0.6 mM),and K_0.5 values for Na⁺ antagonismof ouabain binding to all heterodimers were 9.5-13.8 mM. Themolecular turnover for ATP hydrolysis by₁₁ (6,652 min¹) was abouttwice as high as that by ₃₁ (3,145 min¹). These properties of the human heterodimersexpressed in yeast are in good agreement with properties of the humanNa⁺-K⁺-ATPase expressed in Xenopusoocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-DHorisberger, L Lelievie, and K Geering. J Biol Chem275: 1976-1986, 2000). In contrast to Na⁺ pumpsexpressed in Xenopus oocytes, the₂₁ complex in yeast membranes wassignificantly less stable than ₁₁ or₃₁, resulting in a lower functionalexpression level. The ₂₁ complex was also more easily denatured by SDS than was the₁₁ or the₃₁ complex.

     

Malignant gliomas display altered pH regulation by NHE1 compared with nontransformed astrocytes

Malignantgliomas exhibit alkaline intracellular pH (pH_i) and acidicextracellular pH (pH_e) compared with nontransformedastrocytes, despite increased metabolic H⁺ production. Theacidic pH_e limits the availability ofHCO₃, thereby reducing both passiveand dynamic HCO₃-dependent buffering.This implies that gliomas are dependent upon dynamic HCO₃-independent H⁺buffering pathways such as the type 1 Na⁺/H⁺exchanger (NHE1). In this study, four rapidly proliferating gliomas exhibited significantly more alkaline steady-state pH_i(pH_i = 7.31-7.48) than normal astrocytes(pH_i = 6.98), and increased rates of recovery fromacidification, under nominallyCO₂/HCO₃-free conditions.Inhibition of NHE1 in the absence ofCO₂/HCO₃ resulted inpronounced acidification of gliomas, whereas normal astrocytes wereunaffected. When suspended inCO₂/HCO₃ medium astrocytepH_i increased, yet glioma pH_i unexpectedlyacidified, suggesting the presence of anHCO₃-dependent acid loadingpathway. Nucleotide sequencing of NHE1 cDNA from the gliomasdemonstrated that genetic alterations were not responsible for thisaltered NHE1 function. The data suggest that NHE1 activity issignificantly elevated in gliomas and may provide a useful target forthe development of tumor-selective therapies.

     

CFTR upregulates the expression of the basolateral Na+-K+-2Cl- cotransporter in cultured pancreatic duct cells

The purpose ofthe current experiments was 1) toassess basolateralNa⁺-K⁺-2Clcotransporter (NKCC1) expression and2) to ascertain the role of cysticfibrosis transmembrane conductance regulator (CFTR) in the regulationof this transporter in a prototypical pancreatic duct epithelial cellline. Previously validated human pancreatic duct celllines (CFPAC-1), which exhibit physiological features prototypical ofcystic fibrosis, and normal pancreatic duct epithelia (stablerecombinant CFTR-bearing CFPAC-1 cells, termed CFPAC-WT) were grown toconfluence before molecular and functional studies. High-stringencyNorthern blot hybridization, utilizing specific cDNA probes, confirmedthat NKCC1 was expressed in both cell lines and its mRNA levels weretwofold higher in CFPAC-WT cells than in CFPAC-1 cells(P < 0.01, n = 3).Na⁺-K⁺-2Clcotransporter activity, assayed as the bumetanide-sensitive, Na⁺- andCl-dependentNH⁺₄ entry into the cell (withNH⁺₄ acting as a substitute forK⁺), increased by ~115% inCFPAC-WT cells compared with CFPAC-1 cells(P < 0.01, n = 6). Reducing the intracellularCl by incubating the cellsin a Cl-free mediumincreasedNa⁺-K⁺-2Clcotransporter activity by twofold (P < 0.01, n = 4) only in CFPAC-WT cells. We concluded that NKCC1 is expressed in pancreatic duct cellsand mediates the entry ofCl. NKCC1 activity isenhanced in the presence of an inwardCl gradient. The resultsfurther indicate that the presence of functional CFTR enhances theexpression of NKCC1. We speculate that CFTR regulates this process in aCl-dependent manner.

     

Similarity of A(3)-adenosine and swelling-activated Cl(-) channels in nonpigmented ciliary epithelial cells

Chloride release from nonpigmented ciliary epithelial (NPE)cells is a final step in forming aqueous humor, and adenosine stimulates Cl transport by these cells. Whole cell patchclamping of cultured human NPE cells indicated that theA₃-selective agonist1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl--D-ribofuranuronamide (IB-MECA) stimulated currents (I_IB-MECA) by~90% at +80 mV. Partial replacement of external Clwith aspartate reduced outward currents and shifted the reversal potential (V_rev) from 23 ± 2 mV to0.0 ± 0.7 mV. Nitrate substitution had little effect. Perfusionwith the Cl channel blockers5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acidinhibited the currents. Partial Cl replacement withaspartate and NO₃, and perfusion with NPPB, hadsimilar effects on the swelling-activated whole cell currents(I_Swell). Partial cyclamate substitution for external Cl inhibited inward and outward currents of bothI_IB-MECA and I_Swell. Bothsets of currents also showed outward rectification and inactivation atlarge depolarizing potentials. The results are consistent with theconcept that A₃-subtype adenosine agonists and swellingactivate a common population of Cl channels.

     

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.

     

PKA and arachidonic acid activation of human recombinant ClC-2 chloride channels

An HEK-293 cell line stably expressing the humanrecombinant ClC-2 Cl channel was used in patch-clampstudies to study its regulation. The relative permeabilityP_x/P_Cl calculated fromreversal potentials was I > Cl = NO₃ = SCNBr. Theabsolute permeability calculated from conductance ratios wasCl = Br = NO₃  SCN > I. The channel was activatedby cAMP-dependent protein kinase (PKA), reduced extracellular pH, oleicacid (C:18 cis9), elaidic acid (C:18trans9), arachidonic acid (AA; C:20cis5,8,11,14), and by inhibitors of AA metabolism,5,8,11,14-eicosatetraynoic acid (ETYA; C:20trans5,8,11,14),-methyl-4-(2-methylpropyl)benzeneacetic acid (ibuprofen), and2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2Cl channels were activated by a combination of forskolinplus IBMX and were inhibited by the cell-permeant myristoylated PKAinhibitor (mPKI). Channel activation by reduction of bath pH wasincreased by PKA and prevented by mPKI. AA activation of the ClC-2Cl channel was not inhibited by mPKI or staurosporine andwas therefore independent of PKA or protein kinase C activation.

     

Serotonin-elicited inhibition of Cl(-) secretion in the rabbit conjunctival epithelium

The effects of serotonin[5-hydroxytryptamine (5-HT)] on the transepithelial electricalproperties of the short-circuited rabbit conjunctiva were examined.With this epithelium, the short-circuit current(I_sc) measures Cl secretion plusan amiloride-resistant Na⁺ absorptive process. Apicaladdition of 5-HT (10 µM) elicited a prompt I_screduction from 14.2 ± 1.2 to 10.9 ± 1.2 µA/cm² and increased transepithelial resistance from0.89 ± 0.05 to 1.03 ± 0.06 k · cm²(means ± SE, n = 21, P < 0.05).Similar changes were obtained with conjunctivae bathed withoutNa⁺ in the apical bath, as well as with conjunctivaepreexposed to bumetanide with the Cl-dependentI_sc sustained by the parallel activities ofbasolateral Na⁺/H⁺ andCl/HCO exchangers. In contrast, the5-HT-evoked effects were attenuated by the absence of Cl(I_sc = 0.5 ± 0.2, n = 5), suggesting that reduced Clconductance(s) is an effect of 5-HT exposure. In amphotericin B-treatedconjunctiva and in the presence of a transepithelial K⁺gradient, 5-HT addition reduced K⁺ diffusion across thepreparation by 13% and increased transepithelial resistance by 4%(n = 6, P < 0.05), indicating that aninhibition in K⁺ conductance(s) was also detectable.Significant electrical responses also occurred under physiologicalconditions when 5-HT was introduced to epithelia pretreated withadrenergic agonists or protein kinase C, phospholipase C,phosphodiesterase, or adenylyl cyclase inhibitors or after perturbationof Ca²⁺ homeostasis. Briefly, the conjunctiva harbors theonly known Cl-secreting epithelium in which 5-HT evokesCl transport inhibition; receptor subtype and signaltransduction mechanism were not determined.

     

Cloning and characterization of a human electrogenic Na+-HCO-3 cotransporter isoform (hhNBC)

Our group recentlycloned the electrogenicNa⁺-HCO₃cotransporter (NBC) from salamander kidney and later from mammaliankidney. Here we report cloning an NBC isoform (hhNBC) from a humanheart cDNA library. hhNBC is identical to human renal NBC (hkNBC),except for the amino terminus, where the first 85 amino acids in hhNBCreplace the first 41 amino acids of hkNBC. About 50% of the amino acidresidues in this unique amino terminus are charged, compared with~22% for the corresponding 41 residues in hkNBC. Northern blotanalysis, with the use of the unique 5' fragment of hhNBC as aprobe, shows strong expression in pancreas and expression in heart andbrain, although at much lower levels. InXenopus oocytes expressing hhNBC,adding 1.5% CO₂/10 mMHCO₃ hyperpolarizes the membrane andcauses a rapid fall in intracellular pH(pH_i), followed by apH_i recovery. Subsequent removalof Na⁺ causes a depolarization anda reduced rate of pH_i recovery.Removal of Cl from the bathdoes not affect the pH_i recovery.The stilbene derivative DIDS (200 µM) greatly reduces thehyperpolarization caused by addingCO₂/HCO₃.In oocytes expressing hkNBC, the effects of addingCO₂/HCO₃and then removing Na⁺ were similarto those observed in oocytes expressing hhNBC. We conclude that hhNBCis an electrogenicNa⁺-HCO₃cotransporter and that hkNBC is also electrogenic.     

Regulation of ATP-sensitive K(+) channels by protein kinase C in murine colonic myocytes

We investigated the regulation ofATP-sensitive K⁺ (K_ATP) currents in murinecolonic myocytes with patch-clamp techniques. Pinacidil(10⁵ M) activated inward currents in the presence of highexternal K⁺ (90 mM) at a holding potential of 80 mV indialyzed cells. Glibenclamide (10⁵ M) suppressedpinacidil-activated current. Phorbol 12,13-dibutyrate (PDBu; 2 × 10⁷ M) inhibited pinacidil-activated current.4--Phorbol ester (5 × 10⁷ M), an inactive formof PDBu, had no effect on pinacidil-activated current. In cell-attachedpatches, the open probability of K_ATP channels wasincreased by pinacidil, and PDBu suppressed openings ofK_ATP channels. When cells were pretreated withchelerythrine (10⁶ M) or calphostin C (10⁷M), inhibition of the pinacidil-activated whole cell currents by PDBuwas significantly reduced. In cells studied with the perforated patchtechnique, PDBu also inhibited pinacidil-activated current, and thisinhibition was reduced by chelerythrine (10⁶ M).Acetylcholine (ACh; 10⁵ M) inhibited pinacidil-activatedcurrents, and preincubation of cells with calphostin C(10⁷ M) decreased the effect of ACh. Cells dialyzed withprotein kinase C -isoform (PKC) antibody had normal responses topinacidil, but the effects of PDBu and ACh on K_ATP wereblocked in these cells. Immunofluorescence and Western blots showedexpression of PKC in intact muscles and isolated smooth muscle cellsof the murine proximal colon. These data suggest that PKC regulates K_ATP in colonic muscle cells and that the effects of ACh onK_ATP are largely mediated by PKC. PKC appears to be themajor isozyme that regulates K_ATP in murine colonic myocytes.

     

Osmotic regulation of intestinal epithelial Na+-K+-Cl- cotransport: role of Cl- and F-actin

Previous data indicate that adenosine 3',5'-cyclicmonophosphate activates the epithelial basolateralNa⁺-K⁺-Clcotransporter in microfilament-dependent fashion in part by direct action but also in response to apicalCl loss (due to cellshrinkage or decreased intracellularCl). To further addressthe actin dependence ofNa⁺-K⁺-Clcotransport, human epithelial T84 monolayers were exposed to anisotonicity, and isotopic flux analysis was performed.Na⁺-K⁺-Clcotransport was activated by hypertonicity induced by added mannitol but not added NaCl. Cotransport was also markedly activated by hypotonic stress, a response that appeared to be due in part to reduction of extracellularCl concentration and alsoto activation of K⁺ andCl efflux pathways.Stabilization of actin with phalloidin blunted cotransporter activationby hypotonicity and abolished hypotonic activation ofK⁺ andCl efflux. However,phalloidin did not prevent activation of cotransport by hypertonicityor isosmotic reduction of extracellularCl. Conversely, hypertonicbut not hypotonic activation was attenuated by the microfilamentdisassembler cytochalasin D. The results emphasize the complexinterrelationship among intracellularCl activity, cell volume,and the actin cytoskeleton in the regulation of epithelialCl transport.

     

Ca2+-activated Cl- current in cultured myenteric neurons from murine proximal colon

Whole cell patch-clamprecordings were made from cultured myenteric neurons taken from murineproximal colon. The micropipette contained Cs⁺ to removeK⁺ currents. Depolarization elicited a slowly activatingtime-dependent outward current (I_tdo), whereasrepolarization was followed by a slowly deactivating tail current(I_tail). I_tdo andI_tail were present in ~70% of neurons. Weidentified these currents as Cl currents(I_Cl), because changing the transmembraneCl gradient altered the measured reversal potential(E_rev) of both I_tdo andI_tail with that for I_tailshifted close to the calculated Cl equilibrium potential(E_Cl). I_Cl areCa²⁺-activated Cl current[I_Cl(Ca)] because they were Ca²⁺dependent. E_Cl, which was measured from theE_rev of I_Cl(Ca) using agramicidin perforated patch, was 33 mV. This value is more positivethan the resting membrane potential (56.3 ± 2.7 mV), suggestingmyenteric neurons accumulate intracellular Cl.-Conotoxin GIVA [0.3 µM; N-type Ca²⁺ channelblocker] and niflumic acid [10 µM; knownI_Cl(Ca) blocker], decreased theI_Cl(Ca). In conclusion, these neurons haveI_Cl(Ca) that are activated by Ca²⁺entry through N-type Ca²⁺ channels. These currents likelyregulate postspike frequency adaptation.

     

Inhibition of proximal tubular fluid absorption by nitric oxide and atrial natriuretic peptide in rat kidney

Atrial natriuretic factor (ANF) and nitric oxide (NO) stimulateproduction of guanosine 3',5'-cyclic monophosphate (cGMP) and are natriuretic. Split-drop micropuncture was performed on anesthetized rats to determine the effects of ANF and the NO donor sodium nitroprusside (SNP) on proximal tubular fluid absorption rate(J_va). Comparedwith control solutions, SNP(10⁴ M) decreasedJ_va by 23% whenadministered luminally and by 35% when added to the peritubularperfusate. Stimulation of fluid uptake by luminal angiotensin II (ANGII; 10⁹ M) was abolished bySNP (10⁴ and10⁶ M). In proximal tubulesuspensions, ANF (10⁶ M)increased cGMP concentration to 143%, whereas SNP(10⁶,10⁵,10⁴,10³ M) raised cGMP to 231, 594, 687, and 880%, respectively.S-nitroso-N-acetylpenicillamine (SNAP) also raised cGMP concentrations with similar dose-response relations. These studies demonstrate inhibition by luminal and peritubular NO of basal and ANG II-stimulated proximal fluid absorption in vivo. The ability of SNP to inhibit basal fluid uptake whereas ANFonly affected ANG II-stimulated transport may be because of productionof higher concentrations of cGMP by SNP.

     

Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells

We investigated theeffects of epidermal growth factor (EGF) on activeNa⁺ absorption by alveolarepithelium. Rat alveolar epithelial cells (AEC) were isolated andcultivated in serum-free medium on tissue culture-treated polycarbonatefilters. mRNA for rat epithelial Na⁺ channel (rENaC) -, -,and -subunits and Na⁺ pump₁- and₁-subunits were detected inday 4 monolayers by Northern analysisand were unchanged in abundance in day5 monolayers in the absence of EGF. Monolayerscultivated in the presence of EGF (20 ng/ml) for 24 h fromday 4 to day5 showed an increase in both₁ and₁Na⁺ pump subunit mRNA but noincrease in rENaC subunit mRNA. EGF-treated monolayers showed parallelincreases in Na⁺ pump₁- and₁-subunit protein by immunoblotrelative to untreated monolayers. Fixed AEC monolayers demonstratedpredominantly membrane-associated immunofluorescent labeling withanti-Na⁺ pump₁- and₁-subunit antibodies, withincreased intensity of cell labeling for both subunits seen at 24 hfollowing exposure to EGF. These changes inNa⁺ pump mRNA and protein precededa delayed (>12 h) increase in short-current circuit (measure ofactive transepithelial Na⁺transport) across monolayers treated with EGF compared with untreated monolayers. We conclude that EGF increases activeNa⁺ resorption across AECmonolayers primarily via direct effects onNa⁺ pump subunit mRNA expressionand protein synthesis, leading to increased numbers of functionalNa⁺ pumps in the basolateralmembranes.

     

Relationship between intracellular pH and chloride in Xenopus oocytes expressing the chloride channel ClC-0

During maturation of oocytes,Cl conductance (G_Cl) oscillatesand intracellular pH (pH_i) increases. ElevatingpH_i permits the protein synthesis essential to maturation.To examine whether changes in G_Cl andpH_i are coupled, the Cl channel ClC-0 washeterologously expressed. Overexpressing ClC-0 elevatespH_i, decreases intracellular Cl concentration([Cl]_i), and reduces volume. Acuteacidification with butyrate does not activate acid extrusion inClC-0-expressing or control oocytes. The ClC-0-induced pH_ichange increases after overnight incubation at extracellular pH 8.5 butis unaltered after incubation at extracellular pH 6.5. Membranedepolarization did not change pH_i. In contrast, hyperpolarization elevates pH_i. Thus neither membranedepolarization nor acute activation of acid extrusion accounts for theClC-0-dependent alkalinization. Overnight incubation in lowextracellular Cl concentration increases pH_iand decreases [Cl]_i in control and ClC-0expressing oocytes, with the effect greater in the latter. Incubationin hypotonic, low extracellular Cl solutions preventedpH_i elevation, although the decrease in[Cl]_i persisted. Taken together, ourobservations suggest that KCl loss leads to oocyte shrinkage, whichtransiently activates acid extrusion. In conclusion, expressing ClC-0in oocytes increases pH_i and decreases[Cl]_i. These parameters are coupled viashrinkage activation of proton extrusion. Normal, cyclical changes ofoocyte G_Cl may exert an effect onpH_i via shrinkage, thus inducing meiotic maturation.