Expression, localization, and functional evaluation of CFTR in bovine corneal endothelial cells

HCO-dependentfluid secretion by the corneal endothelium controls corneal hydrationand maintains corneal transparency. Recently, it has been shown thatmRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the corneal endothelium; however, protein expression, functional localization, and a possible role in HCO transport have not been reported. Immunoblotting for CFTR showed asingle band at ~170 kDa for both freshly isolated and primary cultures of bovine corneal endothelial cells. Indirectimmunofluorescence confocal microscopy indicated that CFTR locates tothe apical membrane. Relative changes in apical and basolateralchloride permeability were estimated by measuring the rate offluorescence quenching of the halide-sensitive indicator6-methoxy-N-ethylquinolinium iodide during Clinflux in the absence and presence of forskolin (FSK). Apical andbasolateral Cl permeability increased 10- and 3-fold,respectively, in the presence of 50 µM FSK. FSK-activated apicalchloride permeability was unaffected by H₂DIDs (250 µM);however, 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB; 50 µM) and glibenclamide (100 µM) inhibited activated Clfluxes by 45% and 30%, respectively. FSK-activated basolateral Cl permeability was insensitive to NPPB, glibenclamide,or furosemide but was inhibited 80% by H₂DIDS.HCO permeability was estimated by measuring changesin intracellular pH in response to quickly lowering bath[HCO]. FSK (50 µM) increased apicalHCO permeability by twofold, which was inhibited42% by NPPB and 65% by glibenclamide. BasolateralHCO permeability was unaffected by FSK. Genistein(50 µM) significantly increased apical HCO andCl permeability by 1.8- and 16-fold, respectively. When50 µM genistein was combined with 50 µM FSK, there was no furtherincrease in Cl permeability; however,HCO permeability was reduced to the control level.In summary, we conclude that CFTR is present in the apical membrane ofbovine corneal endothelium and could contribute to transendothelialCl and HCO transport. Furthermore,there is a cAMP-activated Cl pathway on the basolateralmembrane that is not CFTR.

     

Nitric oxide inhibits superoxide production by inflammatory polymorphonuclear leukocytes

Nitric oxide(NO ·) has a complex role in the inflammatory response. Inthis study, we modified the levels of endogenous NO · in vivoin an acute model of inflammation and evaluated the interactionsbetween NO · and superoxide anion() produced bypolymorphonuclear leukocytes (PMNs) accumulated in the inflamed area.We injected phosphate-buffered saline (control group), 6 µmol ofL-N⁵-(1-iminoethyl)ornithine(L-NIO group), or 6 µmol ofL-arginine (L-arginine group) into thegranuloma pouch induced by carrageenan in rats. plus (indicative of NO · generation) was 188 nmol in the exudate of the control group, but itdecreased in the L-NIO group(P < 0.05) and increased in theL-arginine group(P < 0.05). When PMNsfrom treated rats were incubated in vitro, the productionof superoxide anion () decreased by ~46% in theL-arginine group. Furthermore, was inhibited in PMNs whenL-arginine was addedto the incubation medium before phorbol 12-myristate 13-acetatestimulation but not when added simultaneously. Our results suggest aprotective role for NO · in inflammation, through theinactivation of NADPH oxidase and the consequent impairment of production for cell-mediatedinjury.

     

Effect of ventilation on vascular permeability and cyclic nucleotide concentrations in ischemic sheep lungs

Ventilation during ischemia attenuatesischemia-reperfusion lung injury, but the mechanism is unknown.Increasing tissue cyclic nucleotide levels has been shown to attenuatelung ischemia-reperfusion injury. We hypothesized thatventilation prevented increased pulmonary vascular permeability duringischemia by increasing lung cyclic nucleotide concentrations.To test this hypothesis, we measured vascular permeability and cGMP andcAMP concentrations in ischemic (75 min) sheep lungs that wereventilated (12 ml/kg tidal volume) or statically inflated with the samepositive end-expiratory pressure (5 Torr). The reflection coefficientfor albumin (_alb) was 0.54 ± 0.07 and 0.74 ± 0.02 (SE) in nonventilated and ventilatedlungs, respectively (n = 5, P < 0.05). Filtration coefficientsand capillary blood gas tensions were not different. The effect ofventilation was not mediated by cyclic compression of alveolarcapillaries, because negative-pressure ventilation(n = 4) also was protective (_alb = 0.78 ± 0.09). Thefinal cGMP concentration was less in nonventilated than in ventilatedlungs (0.02 ± 0.02 and 0.49 ± 0.18 nmol/g blood-free dry wt,respectively, n = 5, P < 0.05). cAMP concentrations werenot different between groups or over time. Sodium nitroprussideincreased cGMP (1.97 ± 0.35 nmol/g blood-free dry wt) and_alb (0.81 ± 0.09) innonventilated lungs (n = 5, P < 0.05). Isoproterenol increasedcAMP in nonventilated lungs (n = 4, P < 0.05) but had no effect on_alb. The nitric oxide synthaseinhibitor N^G-nitro-L-arginine methylester had no effect on lung cGMP (n = 9) or _alb(n = 16) in ventilated lungs but didincrease pulmonary vascular resistance threefold(P < 0.05) in perfused sheep lungs (n = 3). These results suggest thatventilation during ischemia prevented an increase in pulmonaryvascular protein permeability, possibly through maintenance of lungcGMP by a nitric oxide-independent mechanism.

     

CFTR modulates programmed cell death by decreasing intracellular pH in Chinese hamster lung fibroblasts

To study the potentialinfluence of cystic fibrosis conductance regulator (CFTR) onintracellular pH regulation during apoptosis induction, we usedPS120 Chinese hamster lung fibroblasts devoid of theNa⁺/H⁺ exchanger (NHE1 isoform) transfectedwith constructs, allowing the expression of CFTR and/or NHE1. Kineticsof lovastatin-induced apoptosis were measured by orceinstaining, double staining with Hoechst-33258, propidium iodide, DNAfragmentation, and annexin V labeling. In PS120 control cells, thepercentage of apoptotic cells after 40 h of lovastatintreatment was 23 ± 3%, whereas in PS120 CFTR-transfected cells,this percentage was 40 ± 4%. In PS120 NHE1 cells, thetransfection with CFTR did not modify the percentage of apoptoticcells after 40 h (control: 19 ± 3%, n = 8;CFTR: 17 ± 1%, n = 8), indicating that blockingintracellular acidification by overexpressing theNa⁺/H⁺ exchanger inhibited the enhancement ofapoptosis induced by CFTR. In all cell lines, the initial pHvalues were identical (pH = 7.46 ± 0.04, n = 9), and treatment with lovastatin led to intracellular acidification.However, the pH value after 40 h was lower in PS120 CFTR-transfected cells (pH = 6.85 ± 0.02, n = 10) than in PS120 cells (pH = 7.15 ± 0.03, n = 10). To further investigate the origin of thisincreased intracellular acidification observed in CFTR-transfected cells, the activity of the DIDS-inhibitableCl/HCO exchanger was studied.8-Bromoadenosine 3',5'-cyclic monophosphate incubation resulted inCl/HCO exchanger activation in PS120 CFTR-transfected cells but had no effect on PS120 cells. Together, ourresults suggest that CFTR can enhance apoptosis in Chinese hamster lung fibroblasts, probably due to the modulation of the Cl/HCO exchanger, resulting in a more efficient intracellular acidification.

     

Effect of reactive oxygen species on NH4+ permeation in Xenopus laevis oocytes

To investigate theeffects of reactive oxygen species (ROS) on NHpermeation in Xenopus laevis oocytes, we used intracellulardouble-barreled microelectrodes to monitor the changes in membranepotential (V_m) and intracellular pH(pH_i) induced by a 20 mM NH₄Cl-containingsolution. Under control conditions, NH₄Cl exposure induceda large membrane depolarization (to V_m = 4.0 ± 1.5 mV; n = 21) and intracellularacidification [reaching a change in pH_i(pH_i) of 0.59 ± 0.06 pH units in 12 min]; theinitial rate of cell acidification (dpH_i/dt) was0.06 ± 0.01 pH units/min. Incubation of the oocytes in thepresence of H₂O₂ or -amyloid protein had nomarked effect on the NH₄Cl-induced pH_i. Bycontrast, in the presence of photoactivated rose bengal (RB),tert-butyl-hydroxyperoxide (t-BHP), orxanthine/xanthine oxidase (X/XO), the same experimental maneuverinduced significantly greater pH_i anddpH_i/dt. These increases in pH_iand dpH_i/dt were prevented by the ROS scavengershistidine and desferrioxamine, suggesting involvement of the reactivespecies ¹gO₂ and ·OH. Using thevoltage-clamp technique to identify the mechanism underlying theROS-measured effects, we found that RB induced a large increase in theoocyte membrane conductance (G_m). ThisRB-induced G_m increase was prevented by 1 mMdiphenylamine-2-carboxylate (DPC) and by a low Na⁺concentration in the bath. We conclude that RB, t-BHP, andX/XO enhance NH influx into the oocyte via activationof a DPC-sensitive nonselective cation conductance pathway.

     

Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart

Isolated rat heart perfused with 1.5-7.5µM NO solutions or bradykinin, which activates endothelial NOsynthase, showed a dose-dependent decrease in myocardial O₂uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 µM NO, n = 18,P < 0.05) and to 1.2 ± 0.1 µM O₂ · min¹ · gtissue¹ (10 µM bradykinin, n = 10,P < 0.05). Perfused NO concentrations correlated with aninduced release of hydrogen peroxide (H₂O₂) inthe effluent (r = 0.99, P < 0.01). NO markedlydecreased the O₂ uptake of isolated rat heart mitochondria(50% inhibition at 0.4 µM NO, r = 0.99,P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b andcytochrome c, which accounts for the effects in O₂uptake and H₂O₂ release. Most NO was bound tomyoglobin; this fact is consistent with NO steady-state concentrationsof 0.1-0.3 µM, which affect mitochondria. In the intact heart,finely adjusted NO concentrations regulate mitochondrial O₂uptake and superoxide anion production (reflected byH₂O₂), which in turn contributes to thephysiological clearance of NO through peroxynitrite formation.

     

Regional deposition of inhaled particles in human lungs: comparison between men and women

We measured detailed regional depositionpatterns of inhaled particles in healthy adult male(n = 11; 25 ± 4 yr of age) and female (n = 11; 25 ± 3 yr of age)subjects by means of a serial bolus aerosol delivery technique formonodisperse fine [particle diameter(D_p) = 1 µm] and coarse aerosols(D_p = 3 and 5 µm). The bolus aerosol (40 ml half-width) was delivered to a specificvolumetric depth (Vp) of the lung ranging from 100 to 500 ml with a50-ml increment, and local deposition fraction (LDF) was assessed for each of the 10 local volumetric regions. In all subjects, the deposition distribution pattern was very uneven with respect to Vp,showing characteristic unimodal curves with respect to particle sizeand flow rate. However, the unevenness was more pronounced in women.LDF tended to be greater in all regions of the lung in women than inmen for D_p = 1 µm. For D_p = 3 and 5 µm, LDF showed a marked enhancement in the shallow region of Vp  200 ml in women compared with men(P < 0.05). LDF in women wascomparable to or smaller than those of men in deep lung regions of Vp > 200 ml. Total lung deposition was comparable between men and womenfor fine particles but was consistently greater in women than men forcoarse particles regardless of flow rates used: the difference rangedfrom 9 to 31% and was greater with higher flow rates(P < 0.05). The results indicatethat 1) particledeposition characteristics differ between healthy men and women undercontrolled breathing conditions and2) deposition in women is greaterthan that in men.

     

Regulation of glutamate transport and transport proteins in a placental cell line

We utilized HRP.1 cells derived from midgestation ratplacental labyrinth to determine that the primary pathway for glutamate uptake is via system X, a Na⁺-dependenttransport system. Kinetic parameters of system X activity were similar to those previously determined in rat and humanplacental membrane vesicle preparations. Amino acid depletion caused asignificant upregulation of system X activity at 6, 24, and 48 h. This increase was reversed by the addition ofglutamate and aspartate but not by the addition of -(methylamino)isobutyric acid. Immunoblot analysis of the three transport proteins previously associated with systemX activity indicated a trend toward an increase inGLT1, EAAC1, and GLAST1 immunoreactive protein contents by 48 h;cell surface expression of the same was enhanced by 24 h.Inhibition analysis suggested key roles for EAAC1 and GLAST1 in basalanionic amino acid transfer, with an enhanced role for GLT1 underconditions of amino acid depletion. In summary, amino acid availabilityas well as intracellular metabolism regulate anionic amino acid uptake into this placental cell line.

     

Role of inhibition of nitric oxide production in monocrotaline-induced pulmonary hypertension

Mathew, Rajamma, Elizabeth S. Gloster, T. Sundararajan, Carl I. Thompson, Guillermo A. Zeballos, andMichael H. Gewitz. Role of inhibition of nitric oxide productionin monocrotaline-induced pulmonary hypertension. J. Appl. Physiol. 82(5): 1493-1498, 1997.Monocrotaline (MCT)-induced pulmonary hypertension (PH) isassociated with impaired endothelium-dependent nitric oxide(NO)-mediated relaxation. To examine the role of NO in PH,Sprague-Dawley rats were given a single subcutaneous injection ofnormal saline [control (C)], 80 mg/kg MCT, or the same doseof MCT and a continuous subcutaneous infusion of 2 mg · kg¹ · day¹of molsidomine, a NO prodrug (MCT+MD). Two weeks later, plasma NO₃ levels, pulmonary arterialpressure (Ppa), ratio of right-to-left ventricular weights (RV/LV) toassess right ventricular hypertrophy, and pulmonary histology wereevaluated. The plasma NO₃ level inthe MCT group was reduced to 9.2 ± 1.5 µM(n = 12) vs. C level of 17.7 ± 1.8 µM (n = 8; P < 0.02). In the MCT+MD group,plasma NO₃ level was 12.3 ± 2.0 µM (n = 8). Ppa and RV/LV in theMCT group were increased compared with C [Ppa, 34 ± 3.4 mmHg(n = 6) vs. 19 ± 0.8 mmHg(n = 8) and 0.41 ± 0.01 (n = 9) vs. 0.25 ± 0.008 (n = 8), respectively;P < 0.001]. In the MCT+MDgroup, Ppa and RV/LV were not different when compared with C [19 ± 0.5 mmHg (n = 5) and 0.27 ± 0.01 (n = 9), respectively;P < 0.001 vs. MCT]. Medial wall thickness of lung vessels in the MCT group was increased comparedwith C [31 ± 1.5% (n = 9)vs. 13 ± 0.66% (n = 9);P < 0.001], and MDpartially prevented MCT-induced pulmonary vascular remodeling [22 ± 1.2% (n = 11);P < 0.001 vs. MCT and C].These results indicate that a defect in the availability of bioactive NO may play an important role in the pathogenesis of MCT-induced PH.

     

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.

     

Activity and protein localization of multiple glutamate transporters in gestation day 14vs. day 20rat placenta

Concentrative absorption of glutamate by the developing placentais critical for proper fetal development. The expression of GLAST1,GLT1, EAAC1, and EAAT4, known to be capable ofD-aspartate-inhibitable andNa⁺-coupled glutamate transport(system ), was evaluated inday 14 vs. day20 rat chorioallantoic placenta. Steady-state mRNAlevels were greater at day 20 for alltransporters. Immunohistochemistry determined that the expression ofGLAST1, GLT1, and EAAC1 was greater throughout the day20 placenta and was asymmetric with respect to cellularlocalization. EAAT4 protein was not detected. System activity was responsible formost of the Na⁺-dependentglutamate uptake and was greater in day20 than in day 14 apical and basal membrane subdomains of the labyrinthsyncytiotrophoblast. Greater quantities of EAAC1 and GLAST1 proteinwere identified on day 20, andquantities were greater in basal than in apical membranes. GLT1expression, unchanged in apical membranes, was decreased in basalmembranes. These data correlate transporter mRNA and protein contentwith transport activity and demonstrate an increasing capacity forglutamate absorption by the developing placenta.

     

Thromboxane A2 mediates increased pulmonary microvascular permeability after intestinal reperfusion

Turnage, Richard H., John L. LaNoue, Kevin M. Kadesky, YanMeng, and Stuart I. Myers. ThromboxaneA₂ mediates increased pulmonarymicrovascular permeability after intestinal reperfusion. J. Appl. Physiol. 82(2): 592-598, 1997.This study examines the hypothesis that intestinal reperfusion(IR)-induced pulmonary thromboxane A₂(TxA₂) release increases localmicrovascular permeability and induces pulmonary vasoconstriction.Sprague-Dawley rats underwent 120 min of intestinal ischemia and 60 minof IR. Sham-operated animals (Sham) served as controls. After IR orSham, the pulmonary vessels were cannulated, and the lungs wereperfused in vitro with Krebs buffer. Microvascular permeability wasquantitated by determining the filtration coefficient(K_f),and pulmonary arterial (Ppa), venous (Ppv), and capillary (Ppc)pressures were measured to calculate vascular resistance (Rt). Afterbaseline measurements, imidazole(TxA₂ synthase inhibitor) orSQ-29,548 (TxA₂-receptorantagonist) was added to the perfusate; thenK_f, Ppa, Ppv, and Ppc were again measured. TheK_fof lungs from IR animals was four times greater than that of Sham(P = 0.001), and Rt was 63% greaterin the injured group (P = 0.01). Pc of IR lungs was twice that of controls (5.4 ± 1.0 vs. 2.83 ± 0.3 mmHg, IR vs. Sham, respectively; P < 0.05). Imidazole or SQ-29,548 returnedK_fto baseline measurements (P < 0.05)and reduced Rt by 23 and 17%, respectively(P < 0.05). IR-induced increases in Pc were only slightly reduced by 500 µg/ml imidazole (14%;P = 0.05) but unaffected by lowerdoses of imidazole (5 or 50 µg/ml) or SQ-29,548. These data suggestthat IR-induced pulmonary edema is caused by both increasedmicrovascular permeability and increased hydrostatic pressure and thatthese changes are due, at least in part, to the ongoing release ofTxA₂.

     

Protein kinase C reduces the KCa current of rat tail artery smooth muscle cells

The hypothesis that protein kinase C (PKC) isable to regulate the whole cell Ca-activated K(K_Ca) current independently of PKC effects on local Ca release events was tested using the patch-clamp technique and freshly isolated rat tail artery smooth muscle cells dialyzed with a strongly buffered low-Ca solution. The active diacylglycerol analog1,2-dioctanoyl-sn-glycerol (DOG) at 10 µM attenuated the current-voltage(I-V)relationship of the K_Ca current significantly and reduced the K_Cacurrent at +70 mV by 70 ± 4% (n = 14). In contrast, 10 µM DOG after pretreatment of the cells with 1 µM calphostin C or 1 µM PKC inhibitor peptide, selective PKCinhibitors, and 10 µM1,3-dioctanoyl-sn-glycerol, aninactive diacylglycerol analog, did not significantly alter theK_Ca current. Furthermore, thecatalytic subunit of PKC (PKC_C)at 0.1 U/ml attenuated theI-Vrelationship of the K_Ca currentsignificantly, reduced the K_Cacurrent at +70 mV by 44 ± 3% (n = 17), and inhibited the activity of singleK_Ca channels at 0 mV by 79 ± 9% (n = 6). In contrast, 0.1 U/mlheat-inactivated PKC_C did notsignificantly alter the K_Cacurrent or the activity of singleK_Ca channels. Thus these resultssuggest that PKC is able to considerably attenuate theK_Ca current of freshly isolatedrat tail artery smooth muscle cells independently of effects of PKC onlocal Ca release events, most likely by a direct effect on theK_Ca channel.     

Atrial natriuretic peptide levels and plasma volume contraction in acute alveolar hypoxia

Albert, T. S. E., V. L. Tucker, and E. M. Renkin.Atrial natriuretic peptide levels and plasma volume contraction in acute alveolar hypoxia. J. Appl.Physiol. 82(1): 102-110, 1997.Arterial oxygentensions (Pa_O2), atrial natriureticpeptide (ANP) concentrations, and circulating plasma volumes (PV) weremeasured in anesthetized rats ventilated with room air or 15, 10, or8% O₂(n = 5-7). After 10 min ofventilation, Pa_O2 values were 80 ± 3, 46 ± 1, 32 ± 1, and 35 ± 1 Torrand plasma immunoreactive ANP (irANP) levels were 211 ± 29, 229 ± 28, 911 ± 205, and 4,374 ± 961 pg/ml, respectively. AtPa_O2 40 Torr, irANP responses weremore closely related to inspiredO₂(P = 0.014) than toPa_O2 (P = 0.168). PV was 36.3 ± 0.5 µl/g in controls but 8.5 and9.9% lower (P  0.05) for10 and 8% O₂, respectively.Proportional increases in hematocrit were observed in animals withreduced PV; however, plasma protein concentrations were not differentfrom control. Between 10 and 50 min of hypoxia, small increases (+40%)in irANP occurred in 15% O₂;however, there was no further change in PV, hematocrit, plasma protein,or irANP levels in the lower O₂groups. Urine output tended to fall during hypoxia but was notsignificantly different among groups. These findings are compatiblewith a role for ANP in mediating PV contraction during acute alveolarhypoxia.

     

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.

     

A method for isolating adult and neonatal airway smooth muscle cells and measuring shortening velocity

Methods are described for isolating smooth muscle cells from thetracheae of adult and neonatal sheep and measuring the single-cell shortening velocity. Isolated cells were elongated,Ca²⁺ tolerant, and contractedrapidly and substantially when exposed to cholinergic agonists, KCl,serotonin, or caffeine. Adult cells were longer and widerthan preterm cells. Mean cell length in 1.6 mMCaCl₂ was 194 ± 57 (SD) µm(n = 66) for adult cells and 93 ± 32 µm (n = 20) for preterm cells(P < 0.05). Mean cell width at thewidest point of the adult cells was 8.2 ± 1.8 µm(n = 66) and 5.2 ± 1.5 µm(n = 20) for preterm cells(P < 0.05). Cells were loaded into aperfusion dish maintained at 35°C and exposed to agonists, andcontractions were videotaped. Cell lengths were measured from 30 videoframes and plotted as a function of time. Nonlinear fitting of celllength to an exponential model gave shortening velocities faster thanmost of those reported for airway smooth muscle tissues. For a sampleof 10 adult and 10 preterm cells stimulated with 100 µM carbachol,mean (± SD) shortening velocity of the preterm cells was notdifferent from that of the adult cells (0.64 ± 0.30 vs. 0.54 ± 0.27 s¹, respectively), butpreterm cells shortened more than adult cells (68 ± 12 vs. 55 ± 11% of starting length, respectively;P < 0.05). The preparative andanalytic methods described here are widely applicable to other smoothmuscles and will allow contraction to be studied quantitatively at thesingle-cell level.

     

Protective effects of intravascular pressure and nitric oxide in ischemic lung injury

Cessation of bloodflow during ischemia will decrease both distending and shearforces exerted on endothelium and may worsen ischemic lung injury bydecreasing production of nitric oxide (NO), which influences vascularbarrier function. We hypothesized that increased intravascular pressure(Piv) during ventilated ischemia might maintain NO productionby increasing endothelial stretch or shear forces, thereby attenuatingischemic lung injury. Injury was assessed by measuring the filtrationcoefficient(K_f) and theosmotic reflection coefficient for albumin(_alb) after 3 h of ventilated(95% O₂-5%CO₂; expiratory pressure 3 mmHg) ischemia. Lungs were flushed with physiological salt solution, and then Piv was adjusted to achieve High Piv (mean 6.7 ± 0.4 mmHg, n = 15) or Low Piv (mean0.83 ± 0.4 mmHg, n = 10).N^G-nitro-L-arginine methyl ester(L-NAME;10⁵ M,n = 10),N^G-nitro-D-argininemethyl ester (D-NAME;10⁵ M,n = 11), orL-NAME(10⁵M)+L-arginine (5 × 10⁴ M,n = 6) was added at the start ofischemia in three additional groups of lungs with High Piv.High Piv attenuated ischemic injury compared with Low Piv(_alb 0.67 ± 0.04 vs. 0.35 ± 0.04, P < 0.05). Theprotective effect of High Piv was abolished byL-NAME(_alb 0.37 ± 0.04, P < 0.05) but not byD-NAME(_alb 0.63 ± 0.07). The effects of L-NAME were overcomeby an excess of L-arginine(_alb 0.56 ± 0.05, P < 0.05).K_f did not differsignificantly among groups. These results suggest that Piv modulatesischemia-induced barrier dysfunction in the lung, and theseeffects may be mediated by NO.

     

Interaction between airway edema and lung inflation on responsiveness of individual airways in vivo

Brown, Robert H., Wayne Mitzner, and Elizabeth M. Wagner.Interaction between airway edema and lung inflation onresponsiveness of individual airways in vivo. J. Appl.Physiol. 83(2): 366-370, 1997.Inflammatorychanges and airway wall thickening are suggested to cause increasedairway responsiveness in patients with asthma. In fivesheep, the dose-response relationships of individual airways weremeasured at different lung volumes to methacholine (MCh) before andafter wall thickening caused by the inflammatory mediator bradykininvia the bronchial artery. At 4 cmH₂O transpulmonary pressure(Ptp), 5 µg/ml MCh constricted the airways to a maximum of 18 ± 3%. At 30 cmH₂O Ptp, MCh resultedin less constriction (to 31 ± 5%). Bradykinin increased airwaywall area at 4 and 30 cmH₂O Ptp(159 ± 6 and 152 ± 4%, respectively;P < 0.0001). At 4 cmH₂O Ptp, bradykinin decreasedairway luminal area (13 ± 2%; P < 0.01), and the dose-response curve was significantly lower (P = 0.02). At 30 cmH₂O, postbradykinin, the maximalairway narrowing was not significantly different (26 ± 5%;P = 0.76). Bradykinin produced substantial airway wall thickening and slight potentiation ofthe MCh-induced airway constriction at low lung volume. At high lung volume, bradykinin increased wall thickness but had no effecton the MCh-induced airway constriction. We conclude that inflammatoryfluid leakage in the airways cannot be a primary cause of airwayhyperresponsiveness.

     

Airway surface fluid volume and Cl content in cystic fibrosis and normal bronchial xenografts

We describe theuse of an in vivo human bronchial xenograft model of cystic fibrosis(CF) and non-CF airways to investigate pathophysiological alterationsin airway surface fluid (ASF) volume (V_s) and Cl content.V_s was calculated based on thedilution of an impermeable marker,[³H]inulin, duringharvesting of ASF from xenografts with an isosmotic Cl-free solution.These calculations demonstrated thatV_s in CF xenographs (28 ± 3.0 µl/cm²;n = 17) was significantly less thanthat of non-CF xenografts (35 ± 2.4 µl/cm²;n = 30). The Cl concentration of ASF([Cl]_s) wasdetermined using a solid-state AgCl electrode and adjusted for dilutionduring harvesting using the impermeable[³H]inulin marker.Cumulative results demonstrate small but significant elevations(P < 0.045) in[Cl]_s in CF (125 ± 4 mM; n = 27) compared with non-CF(114 ± 4 mM; n = 48) xenografts.To investigate potential mechanisms by which CF airways may facilitatea higher level of fluid absorption yet retain slightly elevated levelsof Cl, we sought to evaluate the capacity of CF and non-CF airways toabsorb both ²²Na and³⁶Cl. Two consistent findings wereevident from these studies. First, in both CF and non-CF xenografts,²²Na and³⁶Cl were always absorbed in anequal molar ratio. Second, CF xenografts hyperabsorbed (~1.5-foldhigher) both ²²Na and³⁶Cl compared with non-CFxenografts. These results substantiate previously documented findingsof elevated Na absorption in CF airways and also suggest that theslightly elevated[Cl]_s found in thisstudy of CF xenograft epithelia does not occur through a mechanism ofdecreased apical permeability to Cl.     

Rabbit conjunctival epithelium transports fluid, and P2Y22 receptor agonists stimulate Cl{-} and fluid secretion

Rabbit conjunctival epithelium exhibits UTP-dependentCl secretion into the tears. We investigated whetherfluid secretion also takes place. Short-circuit current(I_sc) was 14.9 ± 1.4 µA/cm²(n = 16). Four P2Y₂ purinergic receptoragonists [UTP and the novel compounds INS365, INS306, and INS440(Inspire Pharmaceuticals)] added apically (10 µM) resulted intemporary (~30 min) I_sc increases (88%, 66%,57%, and 28%, respectively; n = 4 each). Importantly, the conjunctiva transported fluid from serosa to mucosa at a rate of6.5 ± 0.7 µl · h¹ · cm² (range2.1-15.3, n = 20). Fluid transport was stimulatedby mucosal additions of 10 µM: 1) UTP, from 7.4 ± 2.3 to 10.7 ± 3.3 µl · h¹ · cm²,n = 5; and 2) INS365, from 6.3 ± 1.0 to 9.8 ± 2.5 µl · h¹ · cm²,n = 5. Fluid transport was abolished by 1 mMouabain (n = 5) and was drastically inhibited by 300 µM quinidine (from 6.4 ± 1.2 to 3.6 ± 1.0 µl · h¹ · cm²,n = 4). We conclude that this epithelium secretes fluidactively and that P2Y₂ agonists stimulate bothCl and fluid secretions.