CFTR involvement in nasal potential differences in mice and pigs studied using a thiazolidinone CFTR inhibitor

Nasal potential difference (PD) measurements have been used to demonstrate defective CFTR function in cystic fibrosis (CF) and to evaluate potential CF therapies. We used the selective thiazolidinone CFTR inhibitor CFTR(inh)-172 to define the involvement of CFTR in nasal PD changes in mice and pigs. In normal mice infused intranasally with a physiological saline solution containing amiloride, nasal PD was -4.7 +/- 0.7 mV, hyperpolarizing by 15 +/- 1 mV after a low-Cl- solution, and a further 3.9 +/- 0.5 mV after forskolin. CFTR(inh)-172 produced 1.1 +/- 0.9- and 4.3 +/- 0.7-mV depolarizations when added after low Cl- and forskolin, respectively. Systemically administered CFTR(inh)-172 reduced the forskolin-induced hyperpolarization from 4.7 +/- 0.4 to 0.9 +/- 0.1 mV but did not reduce the low Cl(-)-induced hyperpolarization. Nasal PD was -12 +/- 1 mV in CF mice after amiloride, changing by <0.5 mV after low Cl- or forskolin. In pigs, nasal PD was -14 +/- 3 mV after amiloride, hyperpolarizing by 13 +/- 2 mV after low Cl- and a further 9 +/- 1 mV after forskolin. CFTR(inh)-172 and glibenclamide did not affect nasal PD in pigs. Our results suggest that cAMP-dependent nasal PDs in mice primarily involve CFTR-mediated Cl- conductance, whereas cAMP-independent PDs are produced by a different, but CFTR-dependent, Cl- channel. In pigs, CFTR may not be responsible for Cl- channel-dependent nasal PDs. These results have important implications for interpreting nasal PDs in terms of CFTR function in animal models of CFTR activation and inhibition.     

Electrolyte transport in the epithelium of pulmonary segments of normal and cystic fibrosis lung.

The epithelium of pulmonary segments from trachea to aveoli actively transports electrolytes and allows osmotic movement of water to maintain the ionic environment in the airway lumen. Models of airway absorption and secretion depict the operation of transporters localized to apical or basolateral membrane. In many epithelia, a variety of electrolyte transporters operate in different combinations to produce absorption or secretion. This also applies to pulmonary epithelium of the large airways (trachea, main-stem bronchi), bronchioles, and alveoli. Na+ absorption occurs in all three pulmonary segments but by different transporters: apical Na+ channels in large airways and bronchioles; Na+/H+ exchange and Na+ channels in adult alveoli. The Na+ channels in each pulmonary segment share a sensitivity to amiloride, a potent inhibitory of epithelial Na+ channels. Fetal alveoli display spontaneous Cl- secretion, as do the large airways of some mammals, such as dog and bovine trachea. Cl- channels differ in conductance properties and in regulation by intracellular second messengers, osmolarity, and voltage mediate stimulated Cl- secretion. Electroneutral carriers, such as NaCl(K) cotransport, Cl-/HCO3- exchange, and Na+/HCO3- exchange, operate in large airways and alveoli during absorption and secretion. Abnormal ion transport in airways of cystic fibrosis (CF) patients is manifest as a reduced Cl- conductance and increased Na+ conductance. Isolation of the CF gene and identification of its product CFTR now allow investigations into the basic defect. Intrinsic to these investigations is the development of systems to study the function of CFTR and its relation to electrolyte transporters and their regulation.     

Modeling the Current-Voltage Characteristics of Charophyte Membranes. II. The Effect of Salinity on Membranes of Lamprothamnium papulosum

Lamprothamnium is a salt-tolerant charophyte that inhabits a broad range of saline environments. The electrical characteristics of Lamprothamnium cell membranes were modeled in environments of different salinity: full seawater (SW), 0.5 SW, 0.4 SW, and 0.2 SW. The cells were voltage-clamped to obtain the I/V (current-voltage) and G/V (conductance-voltage) profiles of the cell membranes. Cells growing at the different salinities exhibited one of three types of I/V profiles (states): pump-, background- and K(+)-states. This study concentrates on the pump- and background-states. Curved (pump-dominated) I/V characteristics were found in cells with resting membrane PDs (potential differences) of -219 +/- 12 mV (in 0.2 SW: 6 cells, 16 profiles), -161 +/- 12 mV (in 0.4 SW: 6 cells, 7 profiles), -151 +/- 12 mV (in 0.5 SW: 6 cells, 12 profiles) and -137 +/- 12 mV (in full SW: 8 cells, 13 profiles). The linear I/V characteristics of the background-state were found in cells with resting PDs of -107 +/- 12 mV (in 0.4 SW: 7 cells, 12 profiles), -108 +/- 12 mV (in 0.5 SW: 7 cells, 10 profiles) and -104 +/- 12 mV (in full SW: 3 cells, 5 profiles). The resting conductance (G) of the cells progressively increased with salinity, from 0.5 S x m(-2) (in 0.2 SW) to 22.0 S x m(-2) (in full SW). The pump peak conductance only rose from 2 S x m(-2) (0.2 SW) to 5 S x m(-2) (full SW), accounting for the increasingly depolarized resting PD observed in cells in more saline media. Upon exposure to hypertonic medium, both the pump and an inward K+ rectifier were stimulated. The modeling of the I/V profiles identified the inward K+ rectifier as an early electrical response to hypertonic challenge.     

Membrane Potentials in the Xylem in Roots of Intact Plants

The membrane potential differences (PDs) of root cells of intact,illuminated Trifolium repens L. and Lolium perenne L. have beenmeasured. In T. repens the PDs were the same for all cell typesexcept for the xylem vessels, which were more positive, andfor some cells immediately adjacent to the xylem vessels whichwere 10 mV more negative. The mean PD for all cells was emdash164.6 ± 0.6 mV and the mean for cells adjacent to thexylem vessels with elevated PDs was 178.4 ± 2.4 mV. Whenthe electrode tip was in a xylem vessel a low but stable PD(mean = emdash 89.9 mV) was recorded. The results for L. perennewere similar except that there were no cells with elevated PDsadjacent to the xylem vessels. An inhibitor of ion transport from the root to the shoot, p-fluorophenylalanine(p-FPA), caused a depolarization of 10 mV in the cell PDs butin the xylem vessels the depolarization was 50 mV. The possibility that the elevated PDs of cells adjacent to thexylem vessels are related to the transport of ions into thevessels is discussed.     

Determining deposition sites of inhaled lung particles and their effect on clearance

An essential component of lung defense is clearance of particulates and infectious vectors from the mucus membrane of the tracheobronchial tree and the alveolar regions of the lung. To partition clearance between these areas we determined the bronchial branching pattern, the anatomical sites of particle deposition, and subsequent clearance in the same animal. Using a 2.85-microns particle tagged with 57Co for inhalation and deposition in the sheep lung, we followed clearance via a series of computer-stored gamma-scintillation lung images. The same sheep was reinhaled, and the particle distributions for both inhalations were compared. After the animals were killed, the bronchial branching pattern and length of the bronchial tree were documented. The number of particles depositing in all bronchi down to 1 mm diam was determined by scintillation counting, and the number in respiratory bronchioles and alveoli was microscopically counted. We conclude that particles deposited in bronchi greater than or equal to 1 mm diam clear in 2-4 h postdeposition. Bronchi distal to 1-mm-diam bronchi and alveoli clear evenly over 72 h, and the number of particles equal to the tracheobronchial deposition cleared after 45 h.     

Alveolar transepithelial potential difference and ion transport in adult rat lung

The complex morphology of the mammalian lung complicates characterization of solute transport across the intact alveolar epithelium. We impaled the subpleural alveolar epithelium with microelectrodes and measured the transepithelial potential difference (PD) of the liquid-filled vascular-perfused left lobe of the rat lung. When the air space was filled entirely with Krebs-Ringer-bicarbonate, the PD was 4.7 mV (lumen negative). The PD was not affected significantly by agents that modify either Na+ or Cl- transport, but replacement of luminal Cl- with gluconate resulted in a fourfold hyperpolarization, a response also noted for large airways. When the airways were blocked by an immiscible nonconducting fluorocarbon, basal PD was not different from unblocked lobes (4.0 mV) but was inhibited 73% by luminal amiloride. Cl(-)-free Krebs-Ringer-bicarbonate blocked in the alveoli with fluorocarbon did not induce hyperpolarization. This result suggests that 1) Cl- permselectivity of the alveolar epithelium is less than that of large airway epithelium and 2) airway PD dominates the voltage across the liquid-filled lung, even when measurements are made from alveoli. When airways are blocked by fluorocarbon, the PD across the alveolar epithelium is largely dependent on Na+ flow through a path with amiloride-sensitive channels.     

Bioelectric properties and ion flow across excised human bronchi

Bioelectric properties and ion transport of excised human segmental/subsegmental bronchi were measured in specimens from 40 patients. Transepithelial electric potential difference (PD), short-circuit current (Isc), and conductance (G), averaged 5.8 mV (lumen negative), 51 microA X cm-2, and 9 mS X cm-2, respectively. Na+ was absorbed from lumen to interstitium under open- and short-circuit conditions. Cl- flows were symmetrical under short-circuit conditions. Isc was abolished by 10(-4) M ouabain. Amiloride inhibited Isc (the concentration necessary to achieve 50% of the maximal effect = 7 X 10(-7) M) and abolished net Na+ transport. PD and Isc were not reduced to zero by amiloride because a net Cl- secretion was induced that reflected a reduction in Cl- flow in the absorptive direction (Jm----sCl-). Acetylcholine (10(-4) M) induced an electrically silent, matched flow of Na+ (1.7 mueq X cm-1 X h-1) and Cl- (1.9 mueq X cm-12 X h-1) toward the lumen. This response was blocked by atropine. Phenylephrine (10(-5) M) did not affect bioelectric properties or unidirectional ion flows, whereas isoproterenol (10(-5) M) induced a small increase in Isc (10%) without changing net ion flows significantly. We conclude that 1) Na+ absorption is the major active ion transport across excised human bronchi, 2) Na+ absorption is both amiloride and ouabain sensitive, 3) Cl- secretion can be induced by inhibition of the entry of luminal Na+ into the epithelia, and 4) cholinergic more than adrenergic agents modulate basal ion flow, probably by affecting gland output.     

Ion transport by sheep distal airways in a miniature chamber

Ion transport and the electric profile of distal airways of sheep lungs were studied in a miniature polypropylene chamber with a 1-mm aperture. Small airways with an inner diameter < 1 mm were isolated, opened longitudinally, and then mounted as a flat sheet onto the 1-mm aperture where it was glued and secured with an O-ring. Both sides of the tissue were bathed with identical physiological solutions at 37 degrees C and oxygenated. Pooled data from 27 distal airways showed an inner airway diameter of 854 +/- 22 (SE) microm and a transepithelial potential difference (PD) of 1.86 +/- 0.29 mV, lumen negative. Short-circuit current (I(sc)) was 25 +/- 3.5 microA/cm(2), tissue resistance was 96 +/- 14 Omega, and conductance was 15.2 +/- 1.7 mS/cm(2). At baseline, amiloride-sensitive Na transport accounted for 51% of I(sc) (change in I(sc) = 9.7 +/- 2.6 microA/cm(2); n = 8 airways), corresponding to 0.36 microeq. cm(-2). h(-1). Treatment with 0.1 mM bumetanide did not reduce the I(sc) (n = 5 airways). Exposure to 1 microM Ca ionophore A-23187 raised the I(sc) by 9 microA/cm(2) (47%; P < 0.03; n = 6 airways). The latter effect was blunted by bumetanide. Carbachol at 1 microM provoked a biphasic response, an initial rapid rise in I(sc) followed by a decline (n = 3 airways). There was no significant increase in PD or I(sc) in response to isoproterenol or dibutyryl cAMP. The data suggest that Na absorption constitutes at least 50% of baseline transport activity. Cl or other anion secretion such as HCO(3) appears to be present and could be stimulated by raising intracellular Ca.     

In vivo characterization of the transitional bronchioles by aerosol-derived airway morphometry.

Effective airway dimensions (EADs) were determined in vivo by aerosol-derived airway morphometry as a function of volumetric lung depth (VLD) to identify and characterize, noninvasively, the caliber of the transitional bronchiole region of the human lung and to compare the EADs by age, gender, and disease. By logarithmically plotting EAD vs. VLD, two distinct regions of the lung emerged that were identified by characteristic line slopes. The intersection of proximal and distal segments was defined as VLD(trans) and associated EAD(trans). In our normal subjects (n = 20), VLD(trans) [345 +/- 83 (SD) ml] correlated significantly with anatomic dead space (224 +/- 34 ml) and end of phase II of single-breath nitrogen washout (360 +/- 53 ml). The corresponding EAD(trans) was 0.42 +/- 0. 07 mm, in agreement with other ex vivo measurements of the transitional bronchioles. VLD(trans) was smaller (216 +/- 64 ml) and EAD(trans) was larger (0.83 +/- 0.04 mm) in our patients with chronic obstructive pulmonary disease (n = 13). VLD(trans) increased with age for children (age 8-18 yr; P = 0.006, n = 26) and with total lung capacity for age 8-81 yr (P < 0.001, n = 61). This study extends the usefulness of aerosol-derived airway morphometry to in vivo measurements of the transitional bronchioles.     

Bioelectric properties and ion transport across excised canine fetal and neonatal airways

Knowledge of liquid secretion by fetal lung stems from studies of sheep. We extended these studies to dogs and examined the persistence of the fetal pattern of airway epithelial permeability and ion transport in the neonatal animal. Plasma and lung liquid from fetal dogs were analyzed for Na+, K+, Cl-, and HCO3-. Only the Cl- concentration of fetal lung liquid (129 meq/l) was significantly different from that of fetal plasma (111 meq/l). Segments of trachea from fetal and neonatal (less than 1, 7-10, and 21-46 days after birth) dogs were excised and mounted in flux chambers. The transepithelial potential difference (PD) of all tissues was oriented lumen negative (9.8-14.8 mV). Under short-circuit conditions, unidirectional Na+ flows were symmetrical. Cl- was secreted, and the secretion was equivalent to short-circuit current (Isc). Cl- secretion persisted under open-circuit conditions. Lobar bronchi from 21- to 46-day neonates absorbed Na+ (1.9 mueq.cm-2.h-1), but unidirectional flows of Cl- were symmetrical. Amiloride (10(-4) M) reduced Isc of neonatal bronchi by 47% but did not affect fetal bronchi. Isoproterenol increased Isc of both fetal (33%) and neonatal (40%) bronchi. These responses suggest that fetal bronchi do not absorb Na+ but can be stimulated to secrete Cl-. We conclude that Cl- secretion by epithelium of large airways may contribute to fetal lung liquid production, but it is unlikely that the tracheal epithelium is involved in fluid absorption at birth. Whereas fetal bronchi appear to secrete Cl-, neonatal bronchi absorb Na+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Airway narrowing in excised canine lungs measured by high-resolution computed tomography.

The exact site of airway narrowing in asthma and chronic obstructive pulmonary disease is unknown. High-resolution computed tomography (HRCT) is a sensitive noninvasive imaging technique that can be used to measure airway dimensions. After determining the optimal computed tomographic parameters using a phantom, we measured lobe volume and airway dimensions of isolated canine lung lobes at a transpulmonary pressure of 25 cmH2O. These measurements were repeated after deflation and administration of aerosolized saline and carbachol (256 mg/ml). Lobe volume decreased with all treatments. The maximal lobar volume change was 26% at 6 cmH2O after carbachol. Average airway lumen area decreased with all treatments. After carbachol, at transpulmonary pressures of 25, 15, 10, 8, and 6 cmH2O, lumen area decreased by 7.3 +/- 4.1, 62.0 +/- 4.9, 77.5 +/- 3.0, 31.9 +/- 9.0, and 95.2 +/- 1.0% (SE), respectively. When the airways were divided into four categories on the basis of initial lumen diameter (less than 2, 2-4, 4-6, and greater than 6 mm), the greatest decreases in luminal area after carbachol were seen in intermediate-sized airways (2-4 mm, 56 +/- 4%; 4-6 mm, 59 +/- 3%). HRCT can be used to make accurate measurements of airway dimensions and airway narrowing in excised lungs. HRCT may allow measurement of airway wall thickness and determination of the site of airway narrowing in asthma.     

Secretion of acid and base equivalents by intact distal airways

Secretion of HCO(3)(-) by airway submucosal glands is essential for normal liquid and mucus secretion. Because the liquid bathing the airway surface (ASL) is acidic, it has been proposed that the surface epithelium may acidify HCO(3)(-)-rich glandular fluid. The aim of this study was to investigate the mechanisms by which intact distal bronchi, which contain both surface and glandular epithelium, modify pH of luminal fluid. Distal bronchi were isolated from pig lungs, cannulated in a bath containing HCO(3)(-)-buffered solution, and perfused continually with an aliquot of similar, lightly buffered solution (LBS) in which NaCl replaced NaHCO(3)(-) (pH 7 with NaOH). The pH of this circulating LBS initially acidified (by 0.053 +/- 0.0053 pH units) and transepithelial potential difference (PD) depolarized. The magnitude of acidification was increased when pH(LBS) was higher. This acidification was unaffected by luminal dimethylamiloride (DMA, 100 microM) but was inhibited by 100 nM bafilomycin A(1) (by 76 +/- 13%), suggesting involvement of vacuolar-H(+) ATPase. Addition of ACh (10 microM) evoked alkalinization of luminal LBS and hyperpolarization of transepithelial PD. The alkalinization was inhibited in HCO(3)(-)-free solutions containing acetazolamide (1 mM) and by DMA and was enhanced by bumetanide (100 microM), an inhibitor of Cl(-) secretion. The hyperpolarization was unaffected by these maneuvers. The anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate (300 microM) and combined treatment with DMA and bumetanide blocked both the alkalinization and hyperpolarization responses to ACh. These results are consistent with earlier studies showing that ACh evokes glandular secretion of HCO(3)(-) and Cl(-). Isolated distal airways thus secrete both acid and base equivalents.     

Predominant constitutive CFTR conductance in small airways

Background

The pathological hallmarks of chronic obstructive pulmonary disease (COPD) are inflammation of the small airways (bronchiolitis) and destruction of lung parenchyma (emphysema). These forms of disease arise from chronic prolonged infections, which are usually never present in the normal lung. Despite the fact that primary hygiene and defense of the airways presumably requires a well controlled fluid environment on the surface of the bronchiolar airway, very little is known of the fluid and electrolyte transport properties of airways of less than a few mm diameter.

Methods

We introduce a novel approach to examine some of these properties in a preparation of minimally traumatized porcine bronchioles of about 1 mm diameter by microperfusing the intact bronchiole.

Results

In bilateral isotonic NaCl Ringer solutions, the spontaneous transepithelial potential (TEP; lumen to bath) of the bronchiole was small (mean ± sem: -3 ± 1 mV; n = 25), but when gluconate replaced luminal Cl^-, the bionic Cl^- diffusion potentials (-58 ± 3 mV; n = 25) were as large as -90 mV. TEP diffusion potentials from 2:1 NaCl dilution showed that epithelial Cl^- permeability was at least 5 times greater than Na⁺ permeability. The anion selectivity sequence was similar to that of CFTR. The bionic TEP became more electronegative with stimulation by luminal forskolin (5 μM)+IBMX (100 μM), ATP (100 μM), or adenosine (100 μM), but not by ionomycin. The TEP was partially inhibited by NPPB (100 μM), GlyH-101* (5–50 μM), and CFTR_Inh-172* (5 μM). RT-PCR gave identifying products for CFTR, α-, β-, and γ-ENaC and NKCC1. Antibodies to CFTR localized specifically to the epithelial cells lining the lumen of the small airways.

Conclusion

These results indicate that the small airway of the pig is characterized by a constitutively active Cl^- conductance that is most likely due to CFTR.     

Studies on the endocrinology of parturition: relative steroidogenesis in coexisting genetically dissimilar ovine fetuses, concomitant with the temporal patterns of maternal C18 and C19 steroids and prostaglandin F2 alpha release

Ovine embryos collected from two breeds of ewes possessing different gestational periods [Finnish Landrace (Finn), 145.5 +/- 1.4 days; Rambouillet (Ra), 150.7 +/- 1.3 days] were transferred to common, randomly selected recipients. Fetal plasma samples were collected from mixed-breed (MB, one Finn and one Ra; n = 6) and single-breed (SB, single Finn, n = 3; single Ra, n = 3) chronic fetal preparations during the last trimester. In the MB pregnancy, the Ra sibling had temporal patterns of androstenedione similar to those observed in the Finn co-twin; however, these particular changes were greatly accelerated in comparison to the same day of gestation in SB Ra fetuses. Similarly, temporal patterns of change in unconjugated estrone, estrone sulfate, and 17 beta-estradiol were accelerated by as much as 4.6 to 5 days in Ra fetuses of MB pregnancies when compared to Ra fetuses in the SB pregnancies (P less than 0.001), with no difference noted between Ra and Finn co-twins. Maternal progesterone concentrations changed from 8 to 2 ng/ml, androstenedione from 200 to 400 pg/ml, estrone from 65 to 250 pg/ml, and 15-keto-13,14-dihydroprostaglandin F2 alpha from 200 pg/ml to 6000 pg/ml over the last 4 or 5 days of gestation. Chronic fetal manipulation accelerated parturition, with mean gestational lengths in MB, SB Finn, and SB Ra pregnancies being 141.5 +/- 0.7, 141.7 +/- 0.7, and 149.3 +/- 0.9 days, respectively. In this study fetal endocrine changes were largely controlled in the MB pregnancy by the fetal co-twin exhibiting a shorter gestational period.     

Distribution of chymase-containing mast cells in human bronchi.

Mast cell chymase stimulates secretion from cultured airway gland serous cells and hydrolyzes bronchoactive peptides in vitro. To explore the likelihood of these interactions occurring in situ, we examined the distribution and concentration of chymase-containing mast cells near glands and smooth muscle of major human bronchi from eight individuals without known airway disease. Total airway mast cells and the subset of mast cells containing chymase were detected by staining for methylene blue metachromasia and chloroacetate esterase activity, respectively. The percentage of chymase-containing mast cells was found to differ strikingly among bronchial tissue compartments. Near glands, for example, the concentration of chymase-positive mast cells (640 +/- 120 cells/mm3) was 73 +/- 9% that of total mast cells (910 +/- 130 cells/mm3), whereas in smooth muscle the concentration of chymase-positive mast cells (450 +/- 200 cells/mm3) was only 14 +/- 4% that of total mast cells (2920 +/- 620 cells/mm3). Of all chymase-containing mast cells in the airway subepithelium, 30 +/- 4% were located within 20 microns of submucosal glands. Although the percentage of chymase-containing cells varied, the absolute concentration of chymase-containing mast cells was similar in all compartments. These results reveal a differential distribution of mast cell subpopulations in human airway and suggest that mast cells containing chymase are near gland and smooth muscle targets.     

Effect of C-fiber-mediated, ozone-induced rapid shallow breathing on airway epithelial injury in rats.

We examined the relationship between C-fiber-mediated, ozone-induced rapid shallow breathing and airway epithelial cell injury at different airway sites within the lower respiratory tract of conscious Wistar rats (n = 24). We combined an acute 8-h ozone inhalation with vagal perineural capsaicin treatment, a selective C-fiber conduction block, and 5-bromo-2'-deoxyuridine (BrdU) labeling as an index of epithelial injury. Vehicle-treated rats that inhaled ozone developed a rapid shallow breathing pattern during ozone inhalation, whereas the capsaicin-treated rats that inhaled ozone showed no changes in respiratory frequency. In vehicle-treated, ozone-exposed rats that developed rapid shallow breathing, a progressive increase in BrdU-labeling density (no. of BrdU-labeled cells/mm(2) airway) was observed starting at the bifurcation of the left main stem bronchi (central airway) and going down either a short or long airway path. In vehicle-treated, ozone-exposed rats, terminal bronchioles supplied by short and long airway paths had a similar degree of BrdU-labeling density that was significantly (P < 0.05) greater than the BrdU-labeling density of the proximal airways that supply them. In contrast, the attenuation of rapid shallow breathing produced by capsaicin treatment resulted in a significantly reduced BrdU-labeling density in the terminal bronchioles supplied by short airway paths compared with the terminal bronchioles supplied by long airway paths. Our data indicate that ozone-induced rapid shallow breathing protects large conducting airways while producing a more even distribution of injury to terminal bronchioles.     

Acid secretion and proton conductance in human airway epithelium

Acid secretion and proton conductive pathways across primary human airway surface epithelial cultures were investigated with the pH stat method in Ussing chambers and by single cell patch clamping. Cultures showed a basal proton secretion of 0.17 +/- 0.04 micromol.h(-1).cm(-2), and mucosal pH equilibrated at 6.85 +/- 0.26. Addition of histamine or ATP to the mucosal medium increased proton secretion by 0.27 +/- 0.09 and 0.24 +/- 0.09 micromol.h(-1).cm(-2), respectively. Addition of mast cells to the mucosal medium of airway cultures similarly activated proton secretion. Stimulated proton secretion was similar in cultures bathed mucosally with either NaCl Ringer or ion-free mannitol solutions. Proton secretion was potently blocked by mucosal ZnCl(2) and was unaffected by mucosal bafilomycin A(1), Sch-28080, or ouabain. Mucosal amiloride blocked proton secretion in tissues that showed large amiloride-sensitive potentials. Proton secretion was sensitive to the application of transepithelial current and showed outward rectification. In whole cell patch-clamp recordings a strongly outward-rectifying, zinc-sensitive, depolarization-activated proton conductance was identified with an average chord conductance of 9.2 +/- 3.8 pS/pF (at 0 mV and a pH 5.3-to-pH 7.3 gradient). We suggest that inflammatory processes activate proton secretion by the airway epithelium and acidify the airway surface liquid.     

Effects of extracellular nucleotides on electrical properties of subconfluent Madin Darby canine kidney cells

ATP and ADP but not AMP lead to sustained hyperpolarization of Madin Darby canine kidney (MDCK) cells. The present study has been performed to test for an influence of other nucleotides on the potential difference across the cell membrane (PD) in subconfluent MDCK cells. PD has been continuously monitored with conventional microelectrodes during rapid exchange of extracellular fluid. Application of 1 mumol/1 UTP leads to a rapid (less than 2 s) hyperpolarization of the cell membrane by -17.0 +/- 0.4 mV (from -50.1 +/- 0.6 mV), a reduction of cell membrane resistance and an increase of the sensitivity of PD to alterations of extracellular potassium. The concentration needed for half maximal effect of UTP is approximately equal to 0.2 mumol/1. ITP is similarly effective, whereas UDP, GTP and GDP are less effective. Up to 1 mmol/1 UMP, GMP, TTP or CTP do not significantly alter PD. In calcium-free extracellular fluid the hyperpolarizing effect of UTP is blunted (-11.6 +/- 2.3 mV) and only transient. In conclusion, UTP similar to purine triphosphates hyperpolarizes MDCK cells by increasing the potassium conductance. The activation of potassium channels requires calcium, which is apparently recruited from both intra- and extracellular sources.     

VIP and PHM and their role in nonadrenergic inhibitory responses in isolated human airways

There is increasing evidence in many species that vasoactive intestinal peptide (VIP) may be a neurotransmitter in nonadrenergic inhibitory nerves. We have studied the effect of electrical field stimulation (EFS), exogenous VIP, and isoproterenol (Iso) on human airways in vitro. We have also studied a related peptide, peptide histidine methionine (PHM), which coexists with VIP in human airway nerves, and in separate experiments studied fragments of the VIP amino acid sequence (VIP1-10 and VIP16-28) for agonist and antagonist activity. Human airways were obtained at thoracotomy and studied in an organ bath. In bronchi EFS gave an inhibitory response that was unaltered by 10(-6) M propranolol but was blocked by tetrodotoxin, whereas in bronchioles there was little or no nonadrenergic inhibitory response. VIP, PHM, and Iso all caused dose-dependent relaxation of bronchi, VIP and PHM being approximately 50-fold more potent than Iso. VIP, but not Iso, mimicked the time course of nonadrenergic inhibitory nerve stimulation. In contrast bronchioles relaxed to Iso but not to VIP or PHM. Neither propranolol nor indomethacin altered the relaxant effects of VIP or PHM, suggesting a direct effect of these peptides on airway smooth muscle. Neither of the VIP fragments showed either agonist or antagonist activity. We conclude that VIP and PHM are more potent bronchodilators of human bronchi than Iso and that the association between the relaxant effects of these peptides and nonadrenergic inhibitory responses suggests that they may be possible neurotransmitters of nonadrenergic inhibitory nerves in human airways.     

Effect of resting smooth muscle length on contractile response in resistance airways

We studied the effect of resting smooth muscle length on the contractile response of the major resistance airways (generations 0-5) in 18 mongrel dogs in vivo using tantalum bronchography. Dose-response curves to 10(-10) to 10(-7) mol/kg methacholine (MCh) were generated [at functional residual capacity (FRC)] by repeated intravenous bolus administration using tantalum bronchography after each dose. Airway constriction varied substantially with dose-equivalent stimulation and varied sequentially from trachea (8.8 +/- 2.2% change in airway diam) to fifth-generation bronchus (49.8 +/- 3.0%; P less than 0.001). Length-tension curves were generated for each airway to determine the airway diameter (i.e., resting in situ smooth muscle length) at which maximal constriction was elicited using bolus intravenous injection of 10(-8) mol/kg MCh. A Frank-Starling relationship was obtained for each airway; the transpulmonary pressure at which maximal constriction was elicited increased progressively from 2.50 +/- 1.12 cmH2O for trachea (approximately FRC) to 18.3 +/- 1.05 cmH2O for fifth-generation airways (approximately 50% TLC) (P less than 0.001). A similar relationship was obtained when change in airway diameter was plotted as a function of airway radius. We demonstrate substantial heterogeneity in the lung volumes at which maximal constriction is elicited and in distribution of parasympathomimetic constriction within the first few generations of resistance bronchi. Our data also suggest that lung hyperinflation may lead to augmented airway contractile responses by shifting resting smooth muscle length toward optimum resting smooth muscle length.