Effect of ozone treatment on airway reactivity and epithelium-derived relaxing factor in guinea pigs

Ozone (O(3)) is toxic to respiratory epithelium and causes airway inflammation and hyperreactivity. To evaluate the role of the epithelium in the development of hyperreactivity, we examined in guinea pigs the effects of inhaled O(3) (3 ppm for 1 h; 0-24 h after exposure) on 1) reactivity to inhaled methacholine (MCh), 2) reactivity of the isolated, perfused trachea (IPT) to MCh, 3) epithelium-derived relaxing factor (EpDRF)-mediated relaxations of IPT induced by mucosal hyperosmolar solutions, 4) neurogenic contraction and relaxation responses, 5) transepithelial potential difference, and 6) microscopic analysis of nitrotyrosine immunofluorescence, substance P fiber density, and tracheal morphology. At 0 h, O(3) caused hyperreactivity to inhaled MCh and mucosally but not serosally applied MCh in IPT (only in the presence of the epithelium) and a decrease in transepithelial potential difference. Inhibition of EpDRF-induced relaxation responses occurred at 2 h. All of these changes returned to control by 12 to 18 h. O(3) had no effect on neurogenic responses. Nitrotyrosine immunofluorescence appeared in the trachea at 0 h in detached epithelial cell ghosts and in intrapulmonary airways by 6 h. Substance P fiber density was elevated in smooth muscle at 0 and 18 h but not in epithelium or lamina propria of intrapulmonary and extrapulmonary bronchi. Loss of cilia and mucosubstances in the mucosa occurred at 0 h; the epithelium became markedly attenuated over 12 to 24 h. A reversible increase in epithelial permeability and a decrease in EpDRF production may contribute to O(3)-induced hyperreactivity to MCh.     

Hyperosmolarity-induced dilation and epithelial bioelectric responses of guinea pig trachea in vitro: role of kinase signaling

Exercise-induced airway obstruction is thought to involve evaporative water loss and hyperosmolarity of the airway surface liquid. Hyperosmolar challenge of the epithelium of isolated, perfused guinea pig trachea rapidly alters transepithelial potential difference (V(t)), and it elicits smooth muscle relaxation mediated by epithelium-derived relaxing factor (EpDRF). In many cell types, protein kinases mediate responses to hyperosmolarity and regulatory volume increase. In this study, inhibitors were used to investigate the involvement of kinases and phosphatases in bioelectric responses of epithelium to hyperosmolarity and their possible relationship to EpDRF-mediated relaxation. After contraction of the perfused trachea with extraluminal methacholine, D-mannitol applied intraluminally (< or = 80 mosM) increased V(t) and elicited dilation of the smooth muscle with a similar concentration-dependence; higher concentrations decreased V(t). In tracheas exposed to 30 mosM D-mannitol (approximately EC(50)), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB 203580) and SKF 86002 [6-(4-fluorophenyl)-2,3-dihydro-5-(4-pyridyl)imidazo[2,1-b]thiazole] (p38 inhibitors) potentiated the dilation, whereas SP 600125 [anthra[1,9-cd]pyrazol-6(2H)-one-1,9-pyrazoloanthrone] and dicumarol [c-Jun NH(2)-terminal kinase (JNK) inhibitors], chelerythrine [nonselective protein kinase C (PKC) inhibitor], and NaAsO(2) (mitogen-activated protein kinase stress inducer) and Na(3)VO(4) (protein tyrosine phosphatase inhibitor) inhibited the hyperpolarization. Large increases in the phosphorylation of p38 and JNK occurred at concentrations higher than those needed to elicit functional responses. The phosphatidylinositol 3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002) and Na(3)VO(4) did not affect the V(t) responses, but they inhibited methacholine-induced constriction; SP 600125 and dicumarol potentiated, and chelerythrine inhibited, methacholine-induced epithelial hyperpolarization. These results suggest that JNK, PKC, and phosphatase(s) are involved in hyperosmolarity-induced hyperpolarization of the tracheal epithelium but that p38 is involved in EpDRF-mediated relaxation.     

Changes in smooth muscle tone during osmotic challenge in relation to epithelial bioelectric events in guinea pig isolated trachea

The relationship between epithelial bioelectric events and epithelium-dependent relaxant and contractile responses of airway smooth muscle in response to hyperosmolar and hypo-osmolar solutions was investigated in guinea pig isolated trachea. Tracheae were perfused with normal or nonisosmotic modified Krebs-Henseleit solution while simultaneously monitoring transepithelial potential difference (VT) and contractile and relaxant responses of the muscle. Baseline VT was -10.1 to -13.3 mV (distal and proximal ends, respectively). Intraluminal amiloride (10(-4) M) induced a 3.7-mV depolarization, verifying that the VT was of epithelial origin. Extraluminal methacholine (3 x 10(-7) M; EC50) caused hyperpolarization and smooth muscle contraction; intraluminal methacholine had very little effect. Increasing intraluminal bath osmolarity via addition of 240 mOsM NaCl or KCl caused an immediate and prolonged depolarization and epithelium-dependent relaxation. Increasing intraluminal bath osmolarity with sucrose evoked similar responses, except that an immediate, transient hyperpolarization and contraction preceded the depolarization and relaxation. Increasing extraluminal bath osmolarity with 240 mOsM NaCl induced depolarization and a longer lasting epithelium-dependent relaxation, whereas extraluminally added 240 mOsM KCl induced a complex smooth muscle response (i.e., transient relaxation followed by contraction), which was accompanied by prolonged depolarization. Intraluminal hypo-osmolarity produced a transient hyperpolarization followed by depolarization along with contraction of the smooth muscle. Bioelectric responses always preceded smooth muscle responses. These results suggest that bioelectric events in the epithelium triggered by nonisosmotic solutions are associated with epithelium-dependent responses in tracheal smooth muscle.     

Hyperosmolar solution effects in guinea pig airways. I. Mechanical responses to relative changes in osmolarity

In the guinea pig isolated perfused trachea contracted with serosal methacholine (MCh), increasing the osmolarity of the mucosal bathing solution elicits relaxation of smooth muscle mediated by epithelium-derived relaxing factor (EpDRF). The present study was undertaken to determine whether a specific modality of the hyperosmolar stimulus induced the relaxation response. Mucosal hyperosmolar challenge with D-mannitol, N-methyl-D-glucamine (NMDG)-chloride, NMDG-gluconate (NMDG-Glu), or urea elicited relaxation with equal potency. In contrast, hyperosmolar solutions at the serosal surface induced diverse, osmolyte-specific responses. In tracheae contracted with MCh, abrupt replacement of the mucosal modified Krebs-Henseleit solution (MKHS) with isosmolar osmolyte solutions to stimulate cell shrinkage elicited five discrete response patterns related to the membrane permeance of the solute, but increasing the osmolarity of the isosmolar solution via the further addition of the same solute always induced relaxation. Similarly, perfusion of the lumen with water induced a transient contraction, but subsequent addition of MKHS, or isosmolar D-mannitol, urea, NMDG-Glu, NaCl, or KCl induced relaxation. Subsequent hyperosmolar addition of the same osmolyte-evoked relaxation. Compatible osmolytes had no effect on smooth muscle tone and did not affect responses to hyperosmolar challenge. The results suggest that the airway epithelium acts as an osmolarity sensor, which communicates with airway smooth muscle through EpDRF. The mechanical responses of the smooth muscle resulting from changes in the osmotic environment are associated with discrete modalities of the osmolar stimulus, including membrane reflection of the particles, incremental change in osmolarity and directionality, but not cell shrinkage.     

Hyperosmolar solution effects in guinea pig airways. III. Studies on the identity of epithelium-derived relaxing factor in isolated perfused trachea using pharmacological agents

Hyperosmolar challenge of airway epithelium stimulates the release of epithelium-derived relaxing factor (EpDRF), but the identity of EpDRF is not known. We examined the effects of pharmacological agents on relaxant responses of methacholine (3 x 10(-7) M)-contracted guinea pig perfused trachea to mucosal hyperosmolar challenge using D-mannitol. Responses were inhibited by gossypol (5 x 10(-6) M), an agent with diverse actions, by the carbon monoxide (CO) scavenger hemoglobin (10(-6) M), and by the heme oxygenase (HO) inhibitor zinc (II) protoporphyrin IX (10(-4) M). The HO inhibitor chromium (III) mesoporphyrin IX (10(-4) M) was not inhibitory, and the HO activator heme-L-lysinate (3 x 10(-4) M) did not evoke relaxant responses. The CO donor tricarbonyldichlororuthenium (II) dimer (2.2 x 10(-4) M) elicited small relaxation responses. Other agents without an effect on responses included: apyrase, adenosine, 6-anilino-5,8-quinolinequinone (LY83583), proadifen, (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK 571), diphenhydramine, glibenclamide, HgCl2, tetrodotoxin, nystatin, alpha-hemolysin, 8-bromoguanosine 3',5'-cyclic monophosphothioate, Rp-isomer, 12-O-tetradecanoylphorbol-13-acetate, cholera toxin, pertussis toxin, thapsigargin, nifedipine, Ca(2+)-free mucosal solution, hydrocortisone, and epidermal growth factor. Cytoskeleton inhibitors, includingerythro-9-(2-hydroxyl-3-nonyl)adenine, colchicine, nocodazole, latrunculin B, and cytochalasins B and D, had no effect on relaxation responses. The results suggest provisionally that a portion of EpDRF activity may be due to CO and that the release of EpDRF does not involve cytoskeletal reorganization.     

Luminal vasopressin modulates transport in the rabbit cortical collecting duct.

We explored the action of luminal AVP in rabbit CCD perfused in vitro at 37 degrees C. Nanomolar concentrations of luminal AVP induced a sustained hyperpolarization of transepithelial voltage (Vt) in contrast to a transient hyperpolarization caused by basolateral AVP. 10 microM basolateral ouabain abolished the latter but not the former change in Vt. Despite a sustained hyperpolarization (from -20.7 +/- 2.9 to -34.1 +/- 4.7 mV; P less than 0.01), 10 nM luminal AVP only slightly altered net Na+ and K+ fluxes (7.6% stimulation and no significant change, respectively). Instead, luminal AVP appeared to modulate an acetazolamide-sensitive electrogenic ion transport because 200 microM basolateral acetazolamide suppressed the luminal AVP-induced hyperpolarization (percentage of Vt from -50.4 +/- 10.8 to -5.1 +/- 1.4; P less than 0.005). In terms of water transport, 10 nM luminal AVP did not change hydraulic conductivity (Lp, x 10(-7) cm/atm per s) (from 3.9 +/- 0.8 to 5.0 +/- 1.2), but suppressed the increase in Lp induced by 20 pM basolateral AVP (134.9 +/- 19.2 vs. 204.3 +/- 21.1 in control; P less than 0.05). These findings demonstrate distinct luminal action of AVP, suggesting amphilateral regulation of epithelial transport by AVP in the CCD.     

Perfusion of isolated tubules of the shark rectal gland. Electrical characteristics and response to hormones.

Both the mammalian thick ascending limb of Henle's loop and the shark rectal gland actively transport Cl against an electrochemical gradient by mechanisms involving hormone-sensitive NaCl transport. In contrast to mammalian renal tubules, individual tubules of the shark rectal gland previously have not been perfused in vitro. Using a combination of renal slice and microdissection techniques we were able to isolate and perfuse single rectal gland tubules without the use of enzyme treatment. Single tubules consistently generated lumen-negative transepithelial voltages (Vt) of -1.8 mV when perfused and bathed with identical shark Ringer's solution. The addition of cyclic AMP, vasoactive intestinal peptide (VIP), and adenosine to the bath increased Vt to -7.5, -9.0, and -4.3 mV, respectively (all P less than 0.02 compared with paired controls). Each stimulation could be reversed by addition by furosemide to the bath. The adenosine response was inhibited by theophylline, a specific inhibitor of adenosine receptors. The tubules had a low transepithelial electrical resistance of 12-26 omega X cm2 and exhibited a transepithelial permselectivity for small cations. These results indicate that tubules of the rectal gland can be perfused in vitro and have receptors for VIP and adenosine. Cyclic AMP and secretagogues hyperpolarize the membrane consistent with electrogenic chloride transport, and these effects are reversed by furosemide, an inhibitor of coupled sodium-potassium-chloride co-transport. The response of Vt to cyclic AMP and furosemide, the transepithelial electrical resistance, and the cation selective permeability of tubules are remarkably similar to measurements in perfused mammalian thick ascending limbs.     

Influence of epithelium on the reactivity of guinea pig isolated, perfused trachea to bronchoactive drugs.

The mechanisms by which the epithelium affects reactivity of guinea pig trachealis to agonists were examined using the isolated, perfused trachea preparation. Contractile agonists (acetylcholine, methacholine, carbachol or histamine) were more potent when applied to the serosal (extraluminal, EL) surface compared to the mucosal (intraluminal, IL) surface, and the IL maximum responses to these agents were smaller. In epithelium-denuded tracheae, IL reactivity to the agonists was increased to the EL level. Physostigmine (10(-7) M) increased the EL and IL potency of acetylcholine to that of carbachol (+/- epithelium), and elevated the IL acetylcholine maximum response (+ epithelium); the relative role of epithelial acetylcholinesterase could not be defined. Indomethacin (3 x 10(-6) M) increased, in an epithelium-dependent manner, the IL acetylcholine, carbachol and histamine maximum responses to the EL level. Phentolamine plus propranolol (both 10(-6) M) potentiated the IL maximum response to methacholine, Isoproterenol also was more potent extraluminally than intraluminally, and the EL and IL maximum responses were similar. IL isoproterenol reactivity was elevated to the EL level in rubbed tracheae. Corticosterone (5 x 10(-5) M) potentiated EL and IL responses to isoproterenol (+/- epithelium); the relative role of epithelial extraneuronal uptake could not be delineated. The epithelium reduces reactivity to mucosally applied drugs by acting as a diffusion barrier. In addition, responses to mucosally administered contractile agonists are inhibited by a physiological antagonism caused by modulatory prostanoids, catecholamines and, possibly, epithelium-derived relaxing factor.     

Hyperosmolar solution effects in guinea pig airways. II. Epithelial bioelectric responses to relative changes in osmolarity

Osmotic challenge of airways alters the bioelectric properties of the airway epithelium and induces the release of factors that modulate smooth muscle tone. Recent studies in our laboratory suggested that methacholine-contracted airways relax in response to incremental increases in osmolarity, rather than from cell shrinkage or absolute solute concentration. In the present study, guinea pig tracheae were mounted in Ussing chambers to elucidate the bioelectric effects of challenge of the epithelium with hyperosmolar and isosmolar solutions. Transepithelial short-circuit current (Isc) across tracheae stimulated with basolateral methacholine was inhibited by apical amiloride, apical 5-nitro-2-(3-phenylpropylamino)benzoic acid, basolateral bumetanide, basolateral ouabain, and Cl(-)-free solution, but not by basolateral iberiotoxin. Apical hyperosmolar challenge with NaCl variably decreased or increased Isc, but D-mannitol (D-M) always inhibited Isc; bumetanide attenuated decreases in Isc. The effects of the transport blockers depended upon whether Isc was initially decreased or increased. Unique concentration-dependent changes in Isc and transepithelial resistance (Rt) were observed when ionic (NaCl and KCl), nonionic impermeant (D-M and sucrose), and nonionic permeant (urea) osmolytes were added to the apical and basolateral baths. At concentrations that doubled the osmolarity of the apical bath, D-M, urea, and N-methyl-D-glucamine-gluconate (NMDG-Glu) decreased Isc. Apical isosmolar NMDG-Glu solution decreased Isc, and additional NMDG-Glu caused a further decrease in Isc. Inclusion of one permeant ion, either Na+,K+, or Cl-, reversed the response to apical isosmolar and hyperosmolar solutions. Thus, bioelectric responses of the airway epithelium to hyperosmolar solution are induced by incremental increases in osmolarity.     

Role of airway epithelium on the reactivity of smooth muscle from guinea pigs sensitized to ovalbumin by inhalatory method

It has been demonstrated that the lack of epithelium in healthy airways from different species modifies the reactivity of tracheobronchial smooth muscle. In the present work we have investigated the effect of airway epithelium removal in tracheal chains from guinea pigs sensitized to ovalbumin by inhalatory route and unsensitized animals. We found that in both groups the mechanical removal of airway epithelium produced an increase in the tracheal sensitivity and maximum contractile response to histamine. The greatest responses were observed in epithelium-lacking sensitized tracheas. These findings, at least in vitro, support the idea that the damage of respiratory epithelium plays an important role in modulating the airway reactivity and this is more noteworthy in tracheal chains from previously sensitized guinea pigs.     

Effects of L-arginine and phosphodiesterase-5 inhibitor, sildenafil, on inflammation and airway responsiveness of sensitized BP2 mice

Nitric oxide (NO) levels are elevated in the exhaled breath of asthmatic patients and NO is considered as a biomarker of airway inflammation. However, the functions of NO in the airways are not completely understood. L-arginine, as the substrate of NO synthases, is the precursor of NO which stimulates guanylate cyclase and leads to the formation of cyclic GMP (cGMP). Sildenafil, a phosphodiestérase-5 (PDE-5) inhibitor, prevents the degradation of cGMP. In this study the effects of L-arginine and sildenafil treatment, alone or in combination, were evaluated in ovalbumin-sensitized BP2 mice. These effects concerning the airway responsiveness to inhaled methacholine (MCh) were evaluated by whole-body plethysmography (WBP), the inflammatory response evaluated by bronchoalveolar lavage fluid (BALF) analyses and lung tissue biopsies (eosinophilic inflammation associated with lung remodelling), and NO metabolite measurements (by Griess reaction) in BALF. Ovalbumin sensitization induced: (a) an inflammatory reaction with eosinophil and neutrophil influx in BALF and lung; and (b) an increased bronchial responsiveness to MCh. L-arginine treatment [50 mg/kg intraperitoneally (i.p.), for 7 days] increased the relative amount of eosinophils and neutrophils in BALF, had a tendency to increase the airway responsiveness to inhaled MCh and increased the NO metabolite level in BAL. Sildenafil treatment (20 mg/kg i.p. for 7 days) did not affect the airway responsiveness to MCh and had a lower effect compared with L-arginine on inflammatory reactions. The combination of the two treatments resulted in a dramatic enhancement of the airway responsiveness to inhaled MCh. The relative amount of eosinophils was increased and lung histology showed obvious worsened tissular lesions such as epithelial shedding and hypertrophy, hyperplasia of smooth muscle cells, and fibrosis. These findings are consistent with the notion that NO production plays a role in the development of airway inflammation and hyperresponsiveness of sensitized mice and highlighted the potential risk of the L-arginine dietary complement or PDE5 treatment in asthmatic patients.     

Histamine-induced intracellular free Ca++, inositol phosphates and electrical changes in murine N1E-115 neuroblastoma cells

Apparent intracellular free Ca++ concentration [(Ca++]i) was measured in differentiated N1E-115 neuroblastoma by microinjecting cells with aequorin (estimated intracellular concentration, 4 microM) and measuring light emission. Histamine produced a transient, dose-dependent increase in [Ca++]i. Pyrilamine blocked completely the response to histamine whereas cimetidine had no effect. Omitting Ca++ from the external medium reversibly blocked the response. As well as a rise in [Ca++]i, histamine caused a concomitant cell hyperpolarization that was not blocked by ouabain, low Cl-, tetraethylammonium chloride/tetradotoxin or metiamide but was blocked by apamin and pyrilamine. A secondary small depolarization caused by histamine was also blocked by apamin but not by ouabain, low Cl- or tetraethylammonium chloride/tetrodotoxin. Direct iontophoretic injection of Ca++ into cells caused only hyperpolarization. Injection of inositol 1,4,5-trisphosphate [IP3(1,4,5)] caused an increase in [Ca++]i and rapid hyperpolarization. Inositol 1,3,4-trisphosphate [IP3(1,3,4)] caused an increase in [Ca++]i, rapid hyperpolarization and a slower depolarization. Repeated injections of IP3(1,3,4) led to a diminished [Ca++]i response and decreased hyperpolarization but had no effect on depolarization. Inositol 1,3,4,5-tetrakisphosphate was without effect on [Ca++]i or on cellular membrane potential. The results suggest that histamine causes an H1 receptor-dependent increase in [Ca++]i, probably by the increased entry of extracellular Ca++, although there may be a contribution from intracellular Ca++ released by IP3(1,4,5). The increase in [Ca++]i activates K+ channels leading to cell hyperpolarization. IP3(1,3,4) formed from inositol 1,3,4,5-tetrakisphosphate, which is itself a product of IP3(1,4,5), causes a slower depolarization by a mechanism that does not involve Na+ channels or an increase in [Ca++]i.     

Pseudotyped human lentiviral vector-mediated gene transfer to airway epithelia in vivo

We used a replication defective human lentiviral (HIV) vector encoding the lacZ cDNA and pseudotyped with the vesicular stomatitis virus (VSV) glycoprotein (G) to evaluate the utility of this vector system in airway epithelia. In initial studies, apical application of vector to polarized well differentiated human airway epithelial cell cultures produced minimal levels of transgene expression whereas basolateral application of vector enhanced levels of transduction approximately 30-fold. Direct in vivo delivery of HIV vectors to the nasal epithelium and tracheas of mice failed to mediate gene transfer, but injury with sulfur dioxide (SO2) before vector delivery enhanced gene transfer efficiency to the nasal epithelium of both mice and rats. SO2 injury also enhanced HIV vector-mediated gene transfer to the tracheas of rodents. These data suggest that SO2 injury increases access of vector to basal cells and/or the basolateral membrane of airway surface epithelial cells. Quantification of gene transfer efficiency in murine tracheas demonstrated that transduction was more efficient when vector was delivered on the day of exposure (7.0%, n = 4) than when vector was delivered on the day after SO2 exposure (1.7%, n = 4).     

Optical imaging of subacute airway remodeling and adipose stem cell engraftment after airway injury

Acquired airway injury is frequently caused by endotracheal intubations, long-term tracheostomies, trauma, airway burns, and some systemic diseases. An effective and less invasive technique for both the early assessment and the early interventional treatment of acquired airway stenosis is therefore needed. Optical coherence tomography (OCT) has been proposed to have unique potential for early monitoring from the proliferative epithelium to the cartilage in acute airway injury. Additionally, stem cell therapy using adipose stem cells is being investigated as an option for early interventional treatment in airway and lung injury. Over the past decade, it has become possible to monitor the level of injury using OCT and to track the engraftment of stem cells using stem cell imaging in regenerative tissue. The purpose of this study was to assess the engraftment of exogenous adipose stem cells in injured tracheal epithelium with fluorescent microscopy and to detect and monitor the degree of airway injury in the same tracheal epithelium with OCT. OCT detected thickening of both the epithelium and basement membrane after tracheal scraping. The engraftment of adipose stem cells was successfully detected by fluorescent staining in the regenerative epithelium of injured tracheas. OCT has the potential to be a high-resolution imaging modality capable of detecting airway injury in combination with stem cell imaging in the same tracheal mucosa.OCIS codes: (170.6935) Tissue characterization, (170.3880) Medical and biological imaging, (170.1610) Clinical applications, (170.4500) Optical coherence tomography     

Transepithelial water permeability in microperfused distal airways. Evidence for channel-mediated water transport.

Water movement across the airway epithelium is important for regulation of the volume and composition of airspace fluid. A novel approach is reported here to measure osmotic and diffusional water permeability in intact airways. Small airways (100-200 microns diameter, 1-2 mm length) from guinea pig lung were microdissected and perfused in vitro using concentric glass holding and perfusion pipettes. For measurement of osmotic water permeability (Pf), the airway lumen was perfused wit PBS (300 mOsM) containing a membrane impermeable fluorophore, fluorescein sulfonate (FS), and the airway was bathed in solutions of specified osmolalities. Pf determination was based on the changes in FS fluorescence at the distal end of the airway resulting from transepithelial water transport. Pf was 4-5 x 10(-3) cm/s at 23 degrees C and independent of lumen flow rate (10-100 nl/min) and the magnitude and direction of the osmotic gradient (bath osmolality 50-600 mOsM). Temperature dependence measurements gave an activation energy of 4.4 kcal/mol (15-37 degrees C). Pf was not altered by 0.3 mM HgCl2 or 50 microM forskolin, but was increased to 31 x 10(-3) cm/s by 100 micrograms/ml amphotericin B, indicating that osmosis is not limited by unstirred layers. Diffusional water permeability (Pd) was measured by H2O/D2O (deuterium oxide) exchange using the H2O/D2O-sensitive fluorescent probe aminonapthelane trisulfonic acid in the lumen. Measured Pd was 3-6 x 10(-6) cm/s at 23 degrees C, indicating significant restriction to water diffusion by unstirred layers. Antibody localization of water channels showed strong expression of the mercurial-insensitive water channel (AQP-4) at the basolateral membrane of airway epithelial cells. These results provide functional evidence that water movement across the distal airway epithelium is mediated by water channels.     

Osmotic regulation of airway reactivity by epithelium

Inhalation of nonisotonic solutions can elicit pulmonary obstruction in asthmatic airways. We evaluated the hypothesis that the respiratory epithelium is involved in responses of the airways to nonisotonic solutions using the guinea pig isolated, perfused trachea preparation to restrict applied agents to the mucosal (intraluminal) or serosal (extraluminal) surface of the airway. In methacholine-contracted tracheae, intraluminally applied NaCl or KCl equipotently caused relaxation that was unaffected by the cyclo-oxygenase inhibitor, indomethacin, but was attenuated by removal of the epithelium and Na+ and Cl- channel blockers. Na+-K+-2Cl- cotransporter and nitric oxide synthase blockers caused a slight inhibition of relaxation, whereas Na+,K+-pump inhibition produced a small potentiation. Intraluminal hyperosmolar KCl and NaCl inhibited contractions in response to intra- or extraluminally applied methacholine, as well as neurogenic cholinergic contractions elicited with electric field stimulation (+/- indomethacin). Extraluminally applied NaCl and KCl elicited epithelium-dependent relaxation (which for KCl was followed by contraction). In contrast to the effects of hyperosmolarity, intraluminal hypo-osmolarity caused papaverine-inhibitable contractions (+/- epithelium). These findings suggest that the epithelium is an osmotic sensor which, through the release of epithelium-derived relaxing factor, can regulate airway diameter by modulating smooth muscle responsiveness and excitatory neurotransmission.     

Signature current of SO2-induced bronchitis in rabbit.

To investigate abnormalities of airway epithelial ion transport underlying chronic inflammatory airway diseases, we performed electrophysiological, histological, and molecular biological experiments using rabbits exposed to SO2 as a model of bronchitis. By comparison with control, the SO2-exposed trachea exhibited decreased short circuit current (Isc) and conductance associated with increased potential difference. In normal trachea, apical ATP induced a transient Isc activation followed by a suppression, whereas the bronchitis model exhibited a prolonged activation without suppression. This pathological ATP response was abolished by diphenylamine 2-carboxylate or Cl--free bath solution. A significant increase in net Cl- flux toward the lumen was observed after ATP in our bronchitis model. Isoproterenol or adenosine evoked a sustained Isc increase in SO2-exposed, but not in normal, tracheas. The Northern blot analysis showed a strong expression of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in SO2-exposed epithelium. The immunohistochemical study revealed a positive label of CFTR on cells located luminally only in SO2-exposed rabbits. We concluded that the prolonged ATP response in our bronchitis model was of a superimposed normal and adenosine-activated current. The latter current was also activated by isoproterenol and appeared as a signature current for the bronchitis model airway. This was likely mediated by CFTR expressed in the course of chronic inflammation.     

慢性乙醇摄取对急性肺损伤大鼠气道上皮细胞间连接蛋白occludin和E-cadherin及屏障功能的影响

目的 观察慢性乙醇摄取及内毒素处理对大鼠气道上皮屏障功能及紧密连接(TJ)特征性蛋白occludin和黏附连接(AJ)蛋白E-cadherin的影响.方法 将40只SD大鼠随机均分为对照组、慢性乙醇摄取组(乙醇组)、内毒素处理组(LPS组)、慢性乙醇摄取合并内毒素处理组(乙醇+LPS组).利用荧光示踪剂异硫氰酸荧光素标记的右旋糖酐(FD4)测定支气管肺泡上皮的通透性;免疫荧光共聚焦显微镜下观察大鼠气道上皮occludin和E-cadherin蛋白分布及表达;蛋白质免疫印迹法(Western blotting)和逆转录-聚合酶链反应(RT-PCR)测定肺组织中occludin和E-cadherin的蛋白及mRNA表达;并观察肺组织病理学改变.结果 乙醇组及LPS组支气管肺泡上皮通透性均较对照组明显增高(P均＜0.05);乙醇+LPS组支气管肺泡上皮的通透性进一步增高(P＜0.01).occludin和E-cadherin蛋白在对照组大鼠气道上皮呈连续、均匀的胞膜及胞质中表达;在乙醇组、LPS组胞膜呈部分断裂、不连续的表达,且胞膜和胞质的表达下降;在乙醇+LPS组的表达显著下降,且胞膜表达呈明显的断裂甚至消失.Western blotting和RT-PCR显示,乙醇组和LPS组肺组织中occludin和E-cadherin的蛋白及mRNA表达均较对照组明显下降(P均＜0.05);乙醇+LPS组中蛋白及mRNA表达下降最为明显,与其余各组比较差异均有统计学意义(P均＜0.01).结论 慢性乙醇摄取通过降低TJ蛋白occludin和AJ蛋白E-cadherin的蛋白及mRNA表达水平,并干扰各蛋白在胞膜上的定位,最终导致气道上皮屏障功能受损,加重内毒素诱导的急性肺损伤.     

Activation of Ca2+-dependent K+ channels in human B lymphocytes by anti-immunoglobulin.

Many mammalian cell types exhibit Ca2+-dependent K+ channels, and activation of these channels by increasing intracellular calcium generally leads to a hyperpolarization of the plasma membrane. Their presence in B lymphocytes is as yet uncertain. Crosslinking Ig on the surface of B lymphocytes is known to increase the level of free cytoplasmic calcium ([Ca2+]i). However, rather than hyperpolarization, a depolarization has been reported to occur after treatment of B lymphocytes with anti-Ig. To determine if Ca2+-dependent K+ channels are present in B lymphocytes, and to examine the relationship between intracellular free calcium and membrane potential, we monitored [Ca2+]i by means of indo-1 and transmembrane potential using bis(1,3-diethylthiobarbituric)trimethine oxonol in human tonsillar B cells activated by anti-IgM. Treatment with anti-IgM induced a biphasic increase in [Ca2+]i and a simultaneous hyperpolarization. A similar hyperpolarization was induced by ionomycin, a Ca2+ ionophore. Delaying the development of the [Ca2+]i response by increasing the cytoplasmic Ca2+-buffering power delayed the hyperpolarization. Conversely, eliminating the sustained phase of the [Ca2+]i response by omission of external Ca2+ abolished the prolonged hyperpolarization. In fact, a sizable Na+-dependent depolarization was unmasked. This study demonstrates that in human B lymphocytes, Ca2+-dependent K+ channels can be activated by crosslinking of surface IgM. Moreover, it is likely that, by analogy with voltage-sensitive Ca2+ channels, Na+ can permeate through these ligand-gated Ca2+ "channels" in the absence of extracellular Ca2+.     

Delivery of an adenovirus vector in a calcium phosphate coprecipitate enhances the therapeutic index of gene transfer to airway epithelia

Gene transfer could provide a novel treatment for cystic fibrosis. However, current vectors, including recombinant adenoviruses, are relatively inefficient at gene transfer to airway epithelia. We have found that delivering adenovirus in a calcium phosphate coprecipitate (Ad:CaPi coprecipitates) enhanced the efficiency of gene transfer to airway epithelia in vitro and in vivo. However, the potential for injury to the epithelium was not evaluated. In NIH 3T3 cells treated with Ad:CaPi coprecipitates, we found that a 30-min exposure, which was sufficient for maximal transgene expression, produced no toxicity; whereas some other transfection reagents induced significant toxicity. Moreover, when Ad:CaPi coprecipitates were applied to the apical surface of differentiated airway epithelia in vitro, they did not reduce transepithelial resistance, even after prolonged incubation. Delivery of Ad:CaPi coprecipitates to mouse lung induced an inflammatory response, but it was not substantially different from that following administration of adenovirus alone. Thus, Ad:CaPi coprecipitates significantly enhance gene transfer to differentiated human airway epithelia in vitro and to mouse lung in vivo without increasing toxicity or the inflammatory response. Thus, CaPi coprecipitates may enhance the therapeutic index of adenovirus-based gene transfer vectors.