RNA interference targeted to multiple P2X receptor subtypes attenuates zinc-induced calcium entry

A postulated therapeutic avenue in cystic fibrosis (CF) is activation of Ca²⁺-dependent Cl^– channels via stimulation of Ca²⁺ entry from extracellular solutions independent of CFTR functional status. We have shown that extracellular zinc and ATP induce a sustained increase in cytosolic Ca²⁺ in human airway epithelial cells that translates into stimulation of sustained secretory Cl^– transport in non-CF and CF human and mouse airway epithelial cells, cell monolayers, and nasal mucosa. On the basis of these studies, the Ca²⁺ entry channels most likely involved were P2X purinergic receptor channels. In the present study, molecular and biochemical data show coexpression of P2X₄, P2X₅, and P2X₆ subtypes in non-CF (16HBE14o^–) and CF (IB3-1) human bronchial epithelial cells. Other P2X receptor Ca²⁺ entry channel subtypes are expressed rarely or not at all in airway epithelia, epithelial cell models from other CF-relevant tissues, or vascular endothelia. Novel transient lipid transfection-mediated delivery of small interference RNA fragments specific to P2X₄ and P2X₆ (but not P2X₅) into IB3-1 CF human airway epithelial cells inhibited extracellular zinc- and ATP-induced Ca²⁺ entry markedly in fura-2 Ca²⁺ measurements and "knocked down" protein by >65%. These data suggest that multiple P2X receptor Ca²⁺ entry channel subtypes are expressed in airway epithelia. P2X₄ and P2X₆ may coassemble on the airway surface as targets for possible therapeutics for CF independent of CFTR genotype. purinergic receptors; zinc receptors; airway epithelia; cystic fibrosis; therapy     

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.     

Regional deposition and retention of particles in shallow, inhaled boluses: effect of lung volume

The regionaldeposition of particles in boluses delivered to shallow lung depths andtheir subsequent retention in the airways may depend on the lung volumeat which the boluses are delivered. To evaluate the effectof end-inspiratory lung volume on aerosol bolus delivery, we hadhealthy subjects inhale radiolabeled, monodisperse aerosol(^99mTc-iron oxide, 3.5-µm massmedian aerodynamic diameter) boluses (40 ml) to a volumetric frontdepth of 70 ml into the lung at lung volumes of 50, 70, and 85% oftotal lung capacity (TLC) end inhalation. By gamma camera analysis, wefound significantly greater deposition in the left (L) vs. right (R)lungs at the 70 and 85% TLC end inhalation; ratio of deposition in Lto R lung, normalized to L-to-R ratio of lung volume (mean L/R), was1.60 ± 0.45 (SD) and 1.96 ± 0.72, respectively(P < 0.001 for comparison to 1.0) for posterior images. However, at 50% TLC, L/R was 1.23 ± 0.37, not significantly different from 1.0. These data suggest that the L andR lungs may be expanding nonuniformly at higher lung volumes. On theother hand, subsequent retention of deposited particles at 2 and 24 hpostdeposition was independent of L/R at the various lung volumes. Thusasymmetric bolus ventilation for these very shallow boluses does notlead to significant increases in peripheral alveolar deposition. Thesedata may prove useful for 1)designing aerosol delivery techniques to target bronchial airways and2) understanding airway retention ofinhaled particles.

     

Interleukin-8: novel roles in human airway smooth muscle cell contraction and migration

In patients with cystic fibrosis (CF) and asthma, elevated levels of interleukin-8 (IL-8) are found in the airways. IL-8 is a CXC chemokine that is a chemoattractant for neutrophils through CXCR1 and CXCR2 G protein-coupled receptors. We hypothesized that IL-8 acts directly on airway smooth muscle cells (ASMC) in a way that may contribute to the enhanced airway responsiveness and airway remodeling observed in CF and asthma. The aim of this study was to determine whether human ASMC (HASMC) express functional IL-8 receptors (CXCR1 and CXCR2) linked to cell contraction and migration. Experiments were conducted on cells harvested from human lung specimens. Real-time PCR and fluorescence-activated cell sorting analysis showed that HASMC expressed mRNA and protein for both CXCR1 and CXCR2. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) increased from 115 to 170 nM in response to IL-8 (100 nM) and decreased after inhibition of phospholipase C (PLC) with U-73122. On blocking the receptors with specific neutralizing antibodies, changes in [Ca²⁺]_i were abrogated. IL-8 also contracted the HASMC, decreasing the length of cells by 15%, and induced a 2.5-fold increase in migration. These results indicate that HASMC constitutively express functional CXCR1 and CXCR2 that mediate IL-8-triggered Ca²⁺ release, contraction, and migration. These data suggest a potential role for IL-8 in causing abnormal airway structure and function in asthma and CF. chemokines; lung; signal transduction     

Regional clearance of solute from peripheral airway epithelia: recovery after sublobar exposure to ozone

The influence oflocal exposure to ozone (O₃) onrespiratory epithelial permeability of sublobar lung segments wasstudied by using aerosolized^99mTc-diethylenetriaminepentaacetic acid (DTPA; mol wt, 492). Two bronchoscopes were insertedthrough an endotracheal tube in anesthetized, mechanically ventilated,mixed breed dogs and were wedged into sublobar bronchi located in theright and left lower lobes, respectively. Segments were ventilated viathe bronchoscope with 5% CO₂ inair delivered at 200 ml/min, and an aerosol of^99mTc-DTPA was generated anddelivered through the scope and into the sublobar segment over a 30-speriod. Clearance of ^99mTc-DTPAwas measured simultaneously from right and left lower lung segments atbaseline and 1, 7, and 14 days after a 6-h sublobar exposure tofiltered air or 400 parts per billionO₃.O₃ treatment significantlydecreased the clearance halftime(t₅₀) of^99mTc-DTPA by 50% from thebaseline mean of 32.3 to 16.0 min at 1 day postexposure. After 7 daysof recovery, t₅₀was still reduced by 28.8%; however, by 14 days postexposure,clearance of ^99mTc-DTPA hadrecovered, and thet₅₀ had a meanvalue of 30.0 min. ^99mTc-DTPAclearance was not altered by exposure to filtered air, andt₅₀ values werecomparable to baseline at 1, 7, and 14 days postexposure. These resultsreveal that a single local exposure toO₃ increases transepithelialclearance, but only for epithelia directly exposed toO₃, and that 7-14 days ofrecovery are required before permeability to small-molecular-weightsolutes returns to normal.     

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

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

     

Skeletal muscle chemoreflex and pHi in exercise ventilatory control

Oelberg, David A., Allison B. Evans, Mirko I. Hrovat, PaulP. Pappagianopoulos, Samuel Patz, and David M. Systrom. Skeletal muscle chemoreflex and pH_i inexercise ventilatory control. J. Appl.Physiol. 84(2): 676-682, 1998.To determinewhether skeletal muscle hydrogen ion mediates ventilatory drive inhumans during exercise, 12 healthy subjects performed three bouts ofisotonic submaximal quadriceps exercise on each of 2 days in a 1.5-Tmagnet for ³¹P-magnetic resonancespectroscopy(³¹P-MRS). Bilaterallower extremity positive pressure cuffs were inflated to 45 Torr duringexercise (BLPP_ex) or recovery(BLPP_rec) in a randomized orderto accentuate a muscle chemoreflex. Simultaneous measurements were madeof breath-by-breath expired gases and minute ventilation, arterializedvenous blood, and by ³¹P-MRS ofthe vastus medialis, acquired from the average of 12 radio-frequencypulses at a repetition time of 2.5 s. WithBLPP_ex, end-exercise minuteventilation was higher (53.3 ± 3.8 vs. 37.3 ± 2.2 l/min;P < 0.0001), arterializedPCO₂ lower (33 ± 1 vs. 36 ± 1 Torr; P = 0.0009), and quadricepsintracellular pH (pH_i) more acid (6.44 ± 0.07 vs. 6.62 ± 0.07; P = 0.004), compared withBLPP_rec. Bloodlactate was modestly increased withBLPP_ex but without a change inarterialized pH. For each subject, pH_i was linearly relatedto minute ventilation during exercise but not to arterialized pH. Thesedata suggest that skeletal muscle hydrogen ion contributes to theexercise ventilatory response.

     

Assessment of regional deposition of inhaled particles in human lungs by serial bolus delivery method

Kim, Chong S., S. C. Hu, P. DeWitt, and T. R. Gerrity.Assessment of regional deposition of inhaled particles in human lungs by serial bolus delivery method. J. Appl.Physiol. 81(5): 2203-2213, 1996.Detailedregional deposition of inhaled particles was investigated in youngadults (n = 11) by use of aserial bolus aerosol delivery technique. A small bolus (45 mlhalf-width) of monodisperse aerosols [1-, 3-, and5-µm particle diameter(D_p)] wasdelivered sequentially to a specific volumetric depth of the lung(100-500 ml in 50-ml increments), while the subject inhaled cleanair via a laser aerosol photometer (25-ml dead volume) with a constantflow rate ( = 150, 250, and 500 ml/s) andexhaled with the same without a pause to theresidual volume. Deposition efficiency (LDE) and deposition fraction in10 local volumetric regions and total deposition fraction of the lungwere obtained. LDE increased monotonically with increasing lung depthfor all three D_p.LDE was greater with smaller values in all lungregions. Deposition was distributed fairly evenly throughout the lungregions with a tendency for an enhancement in the distal lung regions for D_p = 1 µm.Deposition distribution was highly uneven forD_p = 3 and 5 µm, and the region of the peak deposition shifted toward the proximalregions with increasingD_p. Surface dosewas 1-5 times greater in the small airway regions and 2-17times greater in the large airway regions than in the alveolar regions.The results suggest that local or regional enhancement of deposition occurs in healthy subjects and that the local enhancement can be animportant factor in health risk assessment of inhaled particles.

     

Elastic moduli of excised constricted rat lungs

When airways constrict, the surrounding parenchyma undergoesstretch and distortion. Because of the mechanical interdependence between airways and parenchyma, the material properties of the parenchyma are important factors that modulate the degree ofbronchoconstriction. The purpose of this study was to investigate theeffect of changes in transpulmonary pressure (Ptp) and inducedconstriction on parenchymal bulk (k)and shear (µ) moduli. In excised rat lungs, pressure was measured atthe airway opening, and pressure-volume curves were obtained byimposing step decreases in volume with a calibrated syringe from totallung inflation. Calculation was made ofk during small-volume oscillations (1 Hz). Absolute lung volume at 0 cmH₂O Ptp was obtained bysaline displacement. To calculate µ, a lung-indentation test wasperformed. The lung surface was deformed with a cylindrical punch(diameter = 0.45 cm) in 0.25-mm increments, and the force required toeffect this displacement was measured by a weight balance. Measurementsof k and µ were obtained at 4 and 10 cmH₂O Ptp, and again at 4 cmH₂O Ptp, after delivery ofmethacholine aerosol (100 mg/ml) into the trachea. Values ofk and µ in rat lungs were similar tothose reported in other species. In addition, k and µ were dependent on Ptp. Afterinduced constriction, k and µ increased significantly. That k and µ can increase after induced constriction has important implicationsvis a vis the factors modulating airway narrowing.

     

Role of protein phosphatases in the activation of CFTR (ABCC7) by genistein and bromotetramisole

Genistein and bromotetramisole(Br-t) strongly activate cystic fibrosis transmembrane conductanceregulator (CFTR; ABCC7) chloride channels on Chinese hamster ovarycells and human airway epithelial cells. We have examined the possiblerole of phosphatases in stimulation by these drugs using patch-clampand biochemical methods. Genistein inhibited the spontaneous rundown ofchannel activity that occurs after membrane patches are excised fromcAMP-stimulated cells but had no effect on purified protein phosphatasetype 1 (PP1), PP2A, PP2B, PP2C, or endogenous phosphatases when assayed as [³²P]PO₄ release from prelabeled casein,recombinant GST-R domain fusion protein, or immunoprecipitatedfull-length CFTR. Br-t also slowed rundown of CFTR channels, but, inmarked contrast to genistein, it did inhibit all four proteinphosphatases tested. Half-maximal inhibition of PP2A and PP2C wasobserved with 0.5 and 1.5 mM Br-t, respectively. Protein phosphataseswere also sensitive to (+)-p-Br-t, a stereoisomer of Br-tthat does not inhibit alkaline phosphatases. Br-t appeared to actexclusively through phosphatases since it did not affect CFTR channelsin patches that had low apparent endogenous phosphatase activity (i.e.,those lacking spontaneous rundown). We conclude that genistein and Br-tact through different mechanisms. Genistein stimulates CFTR withoutinhibiting phosphatases, whereas Br-t acts by inhibiting amembrane-associated protein phosphatase (probably PP2C) that presumablyallows basal phosphorylation to accumulate.

     

Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein

The patch-clamp technique was used to investigate the effects ofthe isoflavone genistein on disease-causing mutations (G551D andF508) of the cystic fibrosis transmembrane conductance regulator (CFTR). In HeLa cells recombinantly expressing thetrafficking-competent G551D-CFTR, the forskolin-stimulated Cl currentswere small, and average open probability of G551D-CFTR wasP_o = 0.047 ± 0.019. Addition of genistein activated Cl currents~10-fold, and the P_o of G551D-CFTRincreased to 0.49 ± 0.12, which is aP_o similar towild-type CFTR. In cystic fibrosis (CF) epithelial cells homozygous forthe trafficking-impaired F508 mutation, forskolin and genistein activated Cl currents only after 4-phenylbutyrate treatment. These datasuggested that genistein activated CFTR mutants that were present inthe cell membrane. Therefore, we tested the effects of genistein in CFpatients with the G551D mutation in nasal potential difference (PD)measurements in vivo. The perfusion of the nasal mucosa of G551D CFpatients with isoproterenol had no effect; however, genisteinstimulated Cl-dependent nasal PD by, on average, 2.4 ± 0.6 mV, which corresponds to 16.9% of the responses (to -adrenergicstimulation) found in healthy subjects.

     

Desensitization of P2Y2 receptor-activated transepithelial anion secretion

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

Cytosolic potassium controls CFTR deactivation in human sweat duct

Absorptive epithelial cells must admit large quantities of salt (NaCl) during the transport process. How these cells avoid swelling to protect functional integrity in the face of massive salt influx is a fundamental, unresolved problem. A special preparation of the human sweat duct provides critical insights into this crucial issue. We now show that negative feedback control of apical salt influx by regulating the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel activity is key to this protection. As part of this control process, we report a new physiological role of K⁺ in intracellular signaling and provide the first direct evidence of acute in vivo regulation of CFTR dephosphorylation activity. We show that cytosolic K⁺ concentration ([K⁺]_c) declines as a function of increasing cellular NaCl content at the onset of absorptive activity. Declining [K⁺]_c cause parallel deactivation of CFTR by dephosphorylation, thereby limiting apical influx of Cl^– (and its co-ion Na⁺) until [K⁺]_c is stabilized. We surmise that [K⁺]_c stabilizes when Na⁺ influx decreases to a level equal to its efflux through the basolateral Na⁺-K⁺ pump thereby preventing disruptive changes in cell volume. electrolytes; phosphatases; protein kinase A; cystic fibrosis transmembrane conductance regulator; epithelial Na⁺ channel     

Hypotonicity activates a lanthanide-sensitive pathway for K+ release in A6 epithelia

The nature of the pathway forK⁺ release activated duringregulatory volume decrease (RVD) in A6 epithelia was investigated bymeasuring cell thickness (T_c) asan index of cell volume and by probingK⁺ efflux with⁸⁶Rb as tracer forK⁺(R_Rb). Cell swelling was inducedby sudden reduction of basolateral osmolality (from 260 to 140 mosmol/kgH₂O). Experiments wereperformed in the absence of Na⁺transport. Apical R_Rb wasnegligible in iso- and hyposmotic conditions. On the other hand,osmotic shock increased basolateralR_Rb(R^bl_Rb) rapidly, reaching a maximum 7 minafter the peak in T_c. Quinine (0.5 mM) completely inhibited RVD and R^bl_Rb.Also verapamil (0.2 mM) impeded volume recovery considerably; lidocaine(0.2 mM) did not exert a noticeable effect. TheK⁺ channel blockerBa²⁺ (30 mM) delayed RVD but couldnot prevent complete volume recovery. Cs⁺ inhibited RVD noticeably atconcentrations <40 mM. With large Cs⁺ concentrations (>40 mM), theinitial osmometric swelling was followed by a gradual increase ofT_c, suggesting activation of Cs⁺ influx. Chronic exposure ofthe basolateral surface to 0.5 mM La³⁺ orGd³⁺ completely abolished RVD andR^bl_Rb. Acute administration oflanthanides at the time of osmolality decrease did not affect theinitial phase of RVD and reduced R^bl_Rbonly slightly. Apical Gd³⁺ exertedan inhibitory effect on RVD and R^bl_Rb.The effect of Gd³⁺ shouldtherefore be localized at an intracellular site. The role ofCa²⁺ entry could be excluded byfailure of extracellular Ca²⁺removal to inhibit volume recovery. In contrast to lanthanides, chronically and acutely administeredMg²⁺ (0.5 mM) inhibited RVD andR^bl_Rb by ~50%. These data suggest thatK⁺ excretion during RVD occursthrough a rather poorly selective pathway that does not seem to bedirectly activated by membrane stretch.

     

Gadolinium prevents high airway pressure-induced permeability increases in isolated rat lungs

To determine theinitial signaling event in the vascular permeability increase afterhigh airway pressure injury, we compared groups of lungs ventilated atdifferent peak inflation pressures (PIPs) with (gadolinium group) andwithout (control group) infusion of 20 µM gadolinium chloride, aninhibitor of endothelial stretch-activated cationchannels. Microvascular permeability was assessed by using the capillary filtration coefficient(K_fc), ameasure of capillary hydraulic conductivity.K_fc was measuredafter ventilation for 30-min periods with 7, 20, and 30 cmH₂O PIP with 3 cmH₂O positive end-expiratorypressure and with 35 cmH₂O PIPwith 8 cmH₂O positive end-expiratory pressure. In control lungs,K_fc increasedsignificantly to 1.8 and 3.7 times baseline after 30 and 35 cmH₂O PIP, respectively. In thegadolinium group,K_fc was unchangedfrom baseline (0.060 ± 0.010 ml · min¹ · cmH₂O¹ · 100 g¹) after any PIPventilation period. Pulmonary vascular resistance increasedsignificantly from baseline in both groups before the lastK_fc measurementbut was not different between groups. These results suggest thatmicrovascular permeability is actively modulated by a cellular responseto mechanical injury and that stretch-activated cation channels mayinitiate this response through increases in intracellular calciumconcentration.

     

cAMP-independent phosphorylation activation of CFTR by G proteins in native human sweat duct

It is generally believed thatcAMP-dependent phosphorylation is the principle mechanism foractivating cystic fibrosis transmembrane conductance regulator (CFTR)Cl channels. However, we showed that activating Gproteins in the sweat duct stimulated CFTR Cl conductance(G_Cl) in the presence of ATP alone without cAMP. The objective of this study was to test whether the G protein stimulation of CFTR G_Cl is independent ofprotein kinase A. We activated G proteins and monitored CFTRG_Cl in basolaterally permeabilized sweat duct.Activating G proteins with guanosine5'-O-(3-thiotriphosphate) (10-100 µM) stimulated CFTRG_Cl in the presence of 5 mM ATP alone withoutcAMP. G protein activation of CFTR G_Cl requiredMg²⁺ and ATP hydrolysis (5'-adenylylimidodiphosphate couldnot substitute for ATP). G protein activation of CFTRG_Cl was 1) sensitive to inhibition bythe kinase inhibitor staurosporine (1 µM), indicating that theactivation process requires phosphorylation; 2) insensitive to the adenylate cyclase (AC) inhibitors 2',5'-dideoxyadenosine (1 mM)and SQ-22536 (100 µM); and 3) independent ofCa²⁺, suggesting that Ca²⁺-dependent proteinkinase C and Ca²⁺/calmodulin-dependent kinase(s) are notinvolved in the activation process. Activating AC with10⁶ M forskolin plus 10⁶ M IBMX (in thepresence of 5 mM ATP) did not activate CFTR, indicating that cAMPcannot accumulate sufficiently to activate CFTR in permeabilized cells.We concluded that heterotrimeric G proteins activate CFTR G_Cl endogenously via a cAMP-independent pathwayin this native absorptive epithelium.

     

Ventilation distribution during histamine provocation

Verbanck, S., D. Schuermans, A. Van Muylem, M. Paiva, M. Noppen, and W. Vincken. Ventilation distribution during histamine provocation. J. Appl. Physiol. 83(6):1907-1916, 1997.We investigated ventilation inhomogeneity duringprovocation with inhaled histamine in 20 asymptomatic nonsmokingsubjects. We used N₂multiple-breath washout (MBW) to deriveparameters S_condand S_acin as ameasurement of ventilation inhomogeneity in conductive and acinar zonesof the lungs, respectively. A 20% decrease of forced expiratory volume in 1 s (FEV₁) was used todistinguish responders from nonresponders. In the responder group,average FEV₁ decreased by 26%,whereas S_condincreased by 390% with no significant change inS_acin. In thenonresponder group, FEV₁ decreasedby 11%, whereasS_cond increased by 198% with no significantS_acin change.Despite the absence of change inS_acin duringprovocation, baselineS_acin wassignificantly larger in the responder vs. the nonresponder group. Themain findings of our study are that during provocation largeventilation inhomogeneities occur, that the small airways affected bythe provocation process are situated proximal to the acinar zone wherethe diffusion front stands, and that, in addition to overall decreasein airway caliber, there is inhomogeneous narrowing of parallelairways.

     

Letters to the Editor

The following is the abstract of the article discussed in thesubsequent letter:

Verbanck, S., D. Schuermans, A. Van Muylem, M. Paiva, M. Noppen, and W. Vincken. Ventilation distribution duringhistamine provocation. J. Appl. Physiol.83(6):1907-1916, 1997.We investigated ventilation inhomogeneityduring provocation with inhaled histamine in 20 asymptomatic nonsmokingsubjects. We used N₂ multiple-breath washout (MBW) toderive parameters S_cond andS_acin as a measurement of ventilationinhomogeneity in conductive and acinar zones of the lungs,respectively. A 20% decrease of forced expiratory volume in 1 s(FEV₁) was used to distinguish responders fromnonresponders. In the responder group, average FEV₁decreased by 26%, whereas S_cond increased by390% with no significant change in S_acin. In the nonresponder group, FEV₁ decreased by 11%, whereasS_cond increased by 198% with no significantS_acin change. Despite the absence of change inS_acin during provocation, baselineS_acin was significantly larger in the respondervs. the nonresponder group. The main findings of our study are thatduring provocation large ventilation inhomogeneities occur, that thesmall airways affected by the provocation process are situated proximalto the acinar zone where the diffusion front stands, and that, inaddition to overall decrease in airway caliber, there is inhomogeneousnarrowing of parallel airways.

     

Lung resistance and elastance in spontaneously breathing preterm infants: effects of breathing pattern and demographics

Reported values of lung resistance(RL) and elastance (EL) in spontaneouslybreathing preterm neonates vary widely. We hypothesized that thisvariability in lung properties can be largely explained by both inter-and intrasubject variability in breathing pattern and demographics.Thirty-three neonates receiving nasal continuous positive airwaypressure [weight 606-1,792 g, gestational age (GA) of25-33 wk, 2-49 days old] were studied. Transpulmonary pressure was measured by esophageal manometry and airway flow by facemask pneumotachography. Breath-to-breath changes in RL andEL in each infant were estimated by Fourier analysis ofimpedance (Z) and by multiple linear regression (MLR).RL_MLR (RL_MLR = 0.85 × RL_Z 0.43; r² = 0.95) and EL_MLR(EL_MLR = 0.97 × EL_Z + 8.4; r² = 0.98) werehighly correlated to RL_Z andEL_Z, respectively. Both RL(mean ± SD; RL_Z = 70 ± 38, RL_MLR = 59 ± 36 cmH₂O · s · l¹)and EL (EL_Z = 434 ± 212, EL_MLR = 436 ± 210 cmH₂O/l)exhibited wide intra- and intersubject variability.Regardless of computation method, RL was found to decreaseas a function of weight, age, respiratory rate (RR), and tidal volume(VT) whereas it increased as a function ofRR · VT and inspiratory-to-expiratorytime ratio (TI/TE). EL decreasedwith increasing weight, age, VT and female gender andincreased as RR and TI/TE increased. Weconclude that accounting for the effects of breathing patternvariability and demographic parameters on estimates of RLand EL is essential if they are to be of clinical value.Multivariate statistical models of RL and ELmay facilitate the interpretation of lung mechanics measurements inspontaneously breathing infants.

     

Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation

We have designed and synthesizedbenzo[c]quinolizinium derivatives and evaluated their effects on theactivity of G551D cystic fibrosis transmembrane conductance regulator(CFTR) expressed in Chinese hamster ovary and Fisher ratthyroid cells. We demonstrated, using iodide efflux, whole cell patchclamp, and short-circuit recordings, that5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91)restored the activity of G551D CFTR (EC₅₀ = 85 µM)and activated CFTR in Calu-3 cells (EC₅₀ = 47 µM).MPB-91 has no effect on the ATPase activity of wild-type and G551DNBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylatekinase activities of purified NBD2. The activation of CFTR by MPB-91 isindependent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTRhaving 10 mutated protein kinase A sites (10SA-CFTR). The newpharmacological agent MPB-91 may be an important candidate drug toameliorate the ion transport defect associated with CF and to point outa new pathway to modulate CFTR activity.