Modelling asthma in macaques: longitudinal changes in cellular and molecular markers.

The aim of the present study was to determine whether systemic sensitisation and chronic aeroallergen challenge in macaques replicate the classical and emerging immunology and molecular pathology of human asthma. Macaques were immunised and periodically challenged over 2 yrs with house dust mite allergen. At key time-points, serum, bronchoalveolar lavage (BAL) and bronchial biopsies were assayed for genes, proteins and lymphocyte subpopulations relevant to clinical asthma. Immunisation and periodic airway challenge induced changes in immunoglobulin E, airway physiology and eosinophilia consistent with chronic, dual-phase asthma. Sensitisation increased interleukin (IL)-1ß and -6 concentrations in serum, and IL-13 expression in BAL cells. Airway challenge increased: early expression of IL-5, -6, -13 and -19, and eotaxin; and variable late-phase expression of IL-4, -5 and -13, and thymus- and activation-regulated chemokine in BAL cells. CD4+ lymphocytes comprised 30% of the CD3+ cells in BAL, increasing to 50% in the late phase. Natural killer T-cells represented <3% of the CD3+ cells. Corticosteroid treatment reduced serum histamine levels, percentage of CD4+ cells and monocyte-derived chemokine expression, while increasing CD3+ and CD8+ cells in BAL. Sensitisation and periodic aeroallergen challenge of cynomolgus macaques results in physiological, cellular, molecular and protein phenotypes, and therapeutic responses observed in human asthma, providing a model system useful in target and biomarker discovery, and translational asthma research.

Ragweed-induced expression of GATA-3, IL-4, and IL-5 by eosinophils in the lungs of allergic C57BL/6J mice.

Allergen-induced recruitment of T lymphocytes and eosinophils to the airways is associated with increased expression of the transcription factor GATA-3. In this study, the relationship between airway inflammation and GATA-3 expression in the lungs was investigated using ragweed-sensitized C57BL/6J mice. Intratracheal ragweed challenge increased both the number of GATA-3-expressing cells in the perivascular and peribronchial regions and the amount of expression per cell. Interleukin (IL)-4 and IL-5 levels in bronchoalveolar lavage fluid were upregulated in parallel with GATA-3 expression. GATA-3 mRNA and protein colocalized to eosinophils. Eosinophils isolated from the lungs and stimulated with phorbol 12-myristate 13-acetate and/or A-23187 released IL-5. The release was inhibited by actinomycin D, which indicates that de novo synthesis of the cytokine was involved. Western blot analysis of proteins from isolated eosinophils demonstrated expression of the p50 subunit of nuclear factor-kappaB, a transcription factor that is implicated in control of GATA-3 expression. These data provide evidence that allergen challenge increases GATA-3 and proinflammatory cytokine expression by pulmonary eosinophils, which could provide positive feedback for the inflammatory response.

Th1 adjuvant N-acetyl-D-glucosamine polymer up-regulates Th1 immunity but down-regulates Th2 immunity against a mycobacterial protein (MPB-59) in interleukin-10-knockout and wild-type mice.

Treatment of mice with heat-killed (HK) Mycobacterium bovis BCG or 1- to 10-microm chitin particles (nonantigenic N-acetyl-D-glucosamine polymers) is known to induce innate immune responses, including gamma interferon (IFN-gamma) production, which plays a Th1 adjuvant role. However, HK BCG further induces prostaglandin E2-releasing spleen macrophages (Mphi) (PGE2-Mphi), which potentially inhibit Th1 adjuvant activities. We found that chitin particles did not induce PGE2-Mphi formation. To further assess whether chitin has Th1 adjuvant effects, interleukin-10 (IL-10)-knockout (KO) mice and their wild-type (WT, C57BL/6) controls were immunized with a 30-kDa MPB-59 mycobacterial protein mixed with chitin. Immunization with MPB-59 alone induced Th2 responses, characterized by increases in total serum immunoglobulin E (IgE) and specific serum IgG1 levels and spleen Th2 cells producing IL-4, IL-5, and IL-10. No IFN-gamma-producing spleen Th1 cells, specific serum IgG2a, or delayed-type hypersensitivity (DTH) footpad reactions were detected. On the other hand, chitin-MPB-59 immunization significantly increased spleen Th1 responses, DTH reaction, and serum IgG2a levels along with decreases of Th2 responses. The magnitude of these Th1 adjuvant effects was greater in IL-10-KO mice than in WT mice. In contrast, immunization with HK BCG-MPB-59 showed little or no Th1 adjuvant effect. These data indicate that chitin has a unique Th1 adjuvant effect on the development of Th1 immunity against a mycobacterial antigen. IL-10 down-regulates the adjuvant effect of chitin.

IL-10 gene knockout attenuates allergen-induced airway hyperresponsiveness in C57BL/6 mice.

Intratracheal administration of interleukin-10 (IL-10) has been reported to inhibit allergic inflammation but augment airway hyperresponsiveness (AHR). In the present study, airway and smooth muscle responsiveness to methacholine (MCh) were compared in wild-type (WT) and IL-10-deficient (IL-10-KO) mice to investigate the role of endogenous IL-10 in AHR development. Naive WT and IL-10-KO mice exhibited similar dose-dependent increases in airway resistance (Raw) to intravenous MCh. Sensitization and challenge with ragweed (RW) induced a twofold increase in responsiveness to intravenous MCh in WT mice, but hyperresponsiveness was not observed in similarly treated IL-10-KO mice. Likewise, tracheal rings from RW-sensitized and -challenged WT mice exhibited a fourfold greater responsiveness to MCh than IL-10-KO tracheal preparations. Measurements of airway constriction by whole body plethysmography further supported the Raw and tracheal ring data (i.e., AHR was not observed in the absence of IL-10). Interestingly, factors previously implicated in the development of AHR, including IL-4, IL-5, IL-13, IgA, IgG1, IgE, eosinophilia, and lymphocyte recruitment to the airways, were upregulated in the IL-10-KO mice. Treatment with recombinant murine IL-10 at the time of allergen challenge reduced the magnitude of inflammation but reinstated AHR development in IL-10-KO mice. Adoptive transfer of mononuclear splenocytes to IL-10-sufficient severe combined immunodeficient mice indicated that lymphocytes were an important source of the IL-10 impacting AHR development. These results provide evidence that IL-10 expression promotes the development of allergen-induced smooth muscle hyperresponsiveness.

Mucosal IL-12 is more effective than systemic IL-12 in augmenting IFN-gamma expression and inhibiting allergic lung eosinophilia in murine lungs.

The relative efficacy of mucosal (intratracheal) and systemic (intraperitoneal) delivery of interleukin (IL)-12 was evaluated in a mouse model of allergic lung eosinophilia. Mucosal administration of IL-12 achieved 100- to 600-fold higher bronchoalveolar lavage (BAL) levels of IL-12, but 2- to 10-fold lower serum levels compared to systemic administration. Whereas both mucosal and systemic IL-12 inhibited BAL eosinophil recruitment at high doses (100-1000 ng), only mucosal IL-12 was effective at low doses (1-10 ng). Mucosal, but not systemic, administration of 1000 ng of IL-12 increased interferon (IFN)-gamma expression in BAL cells. In a model of ongoing eosinophilic inflammation, when mucosal or systemic IL-12 doses were initiated prior to peak eosinophilia, further eosinophil recruitment was inhibited. However, when IL-12 treatment was initiated after peak eosinophil recruitment occurred, recovery from eosinophilic inflammation was not facilitated. Our findings are the first to demonstrate that locally administered IL-12 inhibits eosinophil recruitment at 100-fold lower doses than systemic IL-12. The most likely mechanism of this enhanced inhibitory activity is a sustained increase in lung levels of IL-12 that augments IFN-gamma production from BAL cells. We suggest that future studies should evaluate the efficacy of low doses of nebulized IL-12 in inhibiting eosinophilic lung inflammation in asthma.

Mucosal IL-12 inhibits airway reactivity to methacholine and respiratory failure in murine asthma.

The worldwide incidence, prevalence, and fatality rates from asthma are increasing despite currently available therapeutic modalities. Systemic administration of interleukin (IL)-12 has been shown to inhibit airway reactivity in murine models of asthma, but the required dosage is high and may be toxic. This study tested the hypothesis that IL-12 administered directly into the lungs is more effective in inhibiting airway reactivity than systemically administered IL-12, allowing lower doses to be used. A low dose (10 ng) of IL-12 was delivered either intratracheally (mucosal delivery) or intraperitoneally (systemic delivery) at the time of ragweed (RW) challenge in mice sensitized to RW. Basal airway resistance and airway reactivity to methacholine were measured 3 days after RW challenge. Compared to phosphate-buffered saline (PBS) challenge of RW sensitized mice, RW challenge increased basal resistance and the slope of the methacholine dose-response curve. Methacholine challenge of RW-challenged mice also induced premature respiratory failure (respiratory rate < 150/min, tidal volume < 0.15 mL) in some animals. Administration of mucosal or systemic IL-12 at the time of RW challenge decreased basal airway resistance. However, only mucosal IL-12 decreased airway reactivity and inhibited respiratory failure during methacholine challenge. These findings indicate that mucosal delivery of a low dose of IL-12 is more effective than systemic IL-12 in inhibiting airway reactivity and respiratory failure in a mouse model of asthma.

IL-10 reduces Th2 cytokine production and eosinophilia but augments airway reactivity in allergic mice.

We investigated the effects of interleukin (IL)-10 administration on allergen-induced Th2 cytokine production, eosinophilic inflammation, and airway reactivity. Mice were sensitized by intraperitoneal injection of ragweed (RW) adsorbed to Alum and challenged by intratracheal instillation of the allergen. Sensitization and challenge with RW increased concentrations of IL-10 in bronchoalveolar lavage (BAL) fluid from undetectable levels to 60 pg/ml over 72 h. Intratracheal instillation of 25 ng of recombinant murine IL-10 at the time of RW challenge further elevated BAL fluid IL-10 concentration to 440 pg/ml but decreased BAL fluid IL-4, IL-5, and interferon-gamma levels by 40-85% and eosinophil numbers by 70% (P < 0.0001). Unexpectedly, the same IL-10 treatment increased airway reactivity to methacholine in spontaneously breathing mice that had been sensitized and challenged with RW (P < 0.001). IL-10 treatment in naive animals or RW-sensitized mice challenged with PBS failed to increase airway reactivity, demonstrating that IL-10 induces an increase in airway reactivity only when it is administered in conjunction with allergic sensitization and challenge. The results demonstrate that IL-10 reduces Th2 cytokine levels and eosinophilic inflammation but augments airway hyperreactivity. Thus, despite its potent anti-inflammatory activity, IL-10 could contribute to the decline in pulmonary function observed in asthma.

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.

Immunoregulatory roles of IL-10 in innate immunity: IL-10 inhibits macrophage production of IFN-gamma-inducing factors but enhances NK cell production of IFN-gamma.

In our study of the immunoregulatory roles of IL-10 in innate immunity, nonantigenic phagocytosable chitin particles were administered i.v. to IL-10-deficient (knockout (KO)) mice or KO mice pretreated with anti-NK1.1 or anti-IFN-gamma Abs. The results established that chitin treatment of KO mice increased superoxide anion release from alveolar macrophages (Mphi) to a level much higher than that in wild-type (WT) mice. The results also suggested that the NK cell is the source of IFN-gamma that is primarily responsible for this alveolar Mphi priming. To further study the roles of IL-10-inhibiting chitin-induced IFN-gamma production, we used spleen cell cultures. The experiments showed that IL-12, IL-18, and TNF-alpha, which were produced by chitin-stimulated Mphi, contributed to the IFN-gamma-inducing activity of chitin. Our results established that exogenous IL-10 inhibited chitin-induced IFN-gamma production in spleen cell cultures from both KO and WT mice. Exogenous IL-10 also inhibited IL-12 and TNF-alpha production by chitin-stimulated Mphi. Exogenous IL-10 decreased IL-12- or IL-18-induced IFN-gamma levels in KO but not in WT NK cell cultures. However, exogenous IL-10 enhanced IFN-gamma levels when NK cells were stimulated simultaneously with both IL-12 and IL-18 in KO and WT cultures. Our in vitro data indicate that IL-10 has differential effects on chitin-induced IFN-gamma production. However, the inhibitory effects of endogenous IL-10 appear to be dominant in the chitin-induced alveolar Mphi priming response in vivo.

Characterization of endothelin release from guinea-pig tracheal epithelium: influence of proinflammatory mediators including major basic protein.

The characteristics of endothelin (ET) release from guinea-pig tracheal epithelium were investigated, including examination of the effects of several pro-inflammatory mediators. In confluent cultured guinea-pig tracheal epithelial cells (GPTECs) there was a time-dependent basal release of immunoreactive ET (ir-ET) from 4-48 h. Basal ir-ET release from GPTECs was unaffected by the peptidase inhibitors, thiorphan (10 microM), benzamidine (1 mM), pepstatin-A (30 microM), aprotinin (1 microgram/ml), bacitracin (20 micrograms/ml) or leupetin (50 microM), but was inhibited by phosphoramidon, the neutral metalloprotease inhibitor (IC50 = 16.8 microM), or the calcium chelator, EGTA (10 mM). There was little ir-ET release 1 day after placing GPTECs in culture, although appreciable release (> 10-fold higher) was detected on days 5 and 7. No significant release of ir-ET was demonstrated from intact guinea-pig trachea. Human thrombin (0.1-10 U/ml), LPS (0.3-10 ng/ml) and the phorbol ester, phorbol 12-myristate-13-acetate (0.1 nM-1 microM), significantly increased ir-ET release, whereas TNF-alpha (0.1-10 ng/ml), RANTES (0.1-100 nM), IL-1 (0.01-10 ng/ml), bradykinin (1 nM-10 microM), CGRP (0.01 nM-1 microM), PDGF (0.1-3 ng/ml), Sar9, Met(O2)11-Sub P, Nle10-NKA 4-10 and senktide (selective NK-1, NK-2 and NK-3 receptor agonists, respectively; 1 nM-10 microM), LTD4 (1 nM-10 microM) or major basic protein (10 nM-1 microM) were without stimulatory effect. The results indicate that the enzyme responsible for the basal release of ET from cultured GPTECs is a Ca(2+)-dependent, phosphoramidon-sensitive, neutral metalloprotease. Furthermore, normally there is minimal ET release from guinea-pig airway epithelium but this can be increased markedly by culturing the cells to confluence, and by select pro-inflammatory mediators.

Differential effects of polycationic protein on Cl- secretory and Na+ absorptive airways.

Effects of cationic proteins on electrolyte transport depend on the specific channels and electrochemical driving forces expressed by epithelia. The bioelectric responses of canine tracheal and bronchial epithelia (CTE and CBE, respectively) to a polycationic protein, protamine, were therefore compared. CTE exhibited a brief transient inhibition of shortcircuit current (I(SC)) followed by a prolonged increase of 18 microA/cm2. The apical membrane transiently hyperpolarized and then depolarized by 11 mV. The increase in I(SC) was inhibited by bumetanide. Adenosine 3',5'-cyclic monophosphate, ionomycin, and thapsigargin attenuated the response whereas indomethacin or hypotonic solution had no effect, indicating that latent cystic fibrosis transmembrane regulator Cl- channels were activated. CBE preparations exhibited a 4-microA/cm2 decrease in I(SC), 2 mV hyperpolarization of the apical membrane, and an increase in fractional resistance of the apical membrane on exposure to protamine. These results were consistent with inhibition of the Na+ conductance in the apical membrane of CBE and confirmed that polycationic proteins exert differential effects on Cl- secretory and Na+ absorptive airways.

CFTR-like chloride channels in non-ciliated bronchiolar epithelial (Clara) cells.

Distal airways are believed to be a major site of disease in cystic fibrosis. The product of the CF gene, CFTR, is expressed in non-ciliated bronchiolar epithelial (Clara) cells. Clara cells in primary culture are capable of Cl- secretion which can be stimulated by cAMP and extracellular nucleotides. We used the patch clamp technique to look for Cl- channels in the apical membrane of rabbit Clara cells. In cell-attached patches, we recorded Cl- channels with a conductance for outward currents of 7.5 +/- 0.4 pS (n = 10) and for inward currents of 3.2 +/- 0.5 pS (n = 10); these channels typically exhibited slow kinetics and were not inhibited by the Cl- channel blockers NPPB and DIDS. Channel activity was not noticeably dependent on pipette potential. Addition of chlorophenylthio-cyclic AMP (cpt-cAMP) to the bath increased the percentage of cell-attached patches with active channels (33.8% vs 56.7%; p< 0.05) and the channel open probability (0.49 +/- 0.03 vs 0.84 +/- 0.02; p< 0.05). Extracellular ATP increased the percentage of cell-attached patches with active channels (28.7% to 50.0%; p< 0.05) but had no significant effect on the channel open probability (0.62 +/- 0.07 vs 0.60 +/- 0.06). In conclusion, rabbit non-ciliated bronchiolar epithelial cells express low-conductance Cl- channels that share many similarities with the CFTR-related Cl- channel and are regulated by cAMP and extracellular ATP.

Activation of water-responsive laryngeal afferents: role of epithelial ion transport.

The role of epithelial ion transport in the activation of water-responsive laryngeal afferent was investigated in anesthetized, spontaneously breathing cats. Single-fiber recordings from the peripheral cut-end of the superior laryngeal nerve were carried out to identify water-responsive laryngeal afferent. Substitution of chloride ions (Cl-) of the Krebs solution with gluconate activated the water-responsive endings when the gluconate concentration was > or = 50 mM. Amiloride (10(-4), 10(-3) and 10(-2) M), an inhibitor of epithelial sodium channels, reduced the water-responsiveness of these afferents, whereas EIPA (5 x 10(-5) M), an amiloride analogue which inhibits Na+/H+ exchange, had no effect. Both ouabain (10(-4) M), an inhibitor of Na+/K+ ATPase, and bumetanide (10(-4) M), an inhibitor of Na(+)-K(+)-2Cl- cotransport, reduced the water response, but no significant reduction in the response was observed with DIDS and DPC, two chloride channel inhibitors. These findings suggest that the epithelium modulates the water-responsiveness of laryngeal afferent but is not the primary determinant of the response.

Bioelectric response of human nasal epithelial cells to polycationic protein.

Polycationic proteins alter electrolyte transport by epithelium and endothelium, and in asthma are thought to disrupt the airway epithelium and contribute to hyperresponsiveness and airway plugging. In the present study, we used primary cultures of human nasal epithelial cells to investigate the response of respiratory tract epithelium to luminal presentation of a polycationic protein, protamine. Protamine (100 micrograms/ml) in the apical bathing solution had no significant effect on basal transepithelial resistance (Rt) but decreased short-circuit current (Isc) and hyperpolarized the apical membrane, indicating that Na+ absorption had been inhibited. Pretreating with amiloride inverted the response to protamine, resulting in an increase in Isc, depolarization of the apical membrane, and decrease in the fractional resistance of the apical membrane (fRa). The increase in Isc was inhibited by pretreatment with bumetanide. These results indicated that protamine augmented amiloride-induced Cl- secretion. Induction of Cl- secretion by bathing the apical surface in 3 mM Cl(-)-Ringer solution similarly resulted in protamine-induced depolarization of the apical membrane. Heparin precipitated protamine from solution and reversed the Isc responses. In summary, low concentrations of polycationic protein can alter electrolyte transport by human airway epithelium without desquamation, and the response is dependent on the secretory state of the tissue.

Beta-adrenergic regulation of Cl- and HCO3- secretion by Clara cells.

Previous studies on beta-adrenergic agonist regulation of ion transport in distal airways yielded discordant results. The present study was performed to further investigate this process in isolated bronchiolar epithelial cells and resolve the discrepancies. Epithelial enriched in rabbit nonciliated bronchiolar epithelial (Clara) cells responded to isoproterenol with a biphasic increase in transepithelial short circuit current (Isc) and decrease in transepithelial resistance (Rt). The first phase of the Isc response consisted of a transient, 11 microA/cm2 increase in current that was inhibited by HCO3(-)-free bathing solutions, but was not inhibited by amiloride, bumetanide, or Cl(-)-free bathing solutions. The ED50 for isoproterenol stimulation of the initial peak was 81 pM. The second phase was a prolonged, 27 microA/cm2 elevation in Isc. Amiloride in the apical bath inhibited basal Isc and the prolonged change in Isc induced by isoproterenol. Bumetanide in the basolateral bath and bilateral Cl(-)-free bathing solutions likewise inhibited the plateau phase of the isoproterenol response, and the inhibition was accentuated in the presence of amiloride. HCO3(-)-free bathing solutions did not inhibit the plateau phase. The ED50 for isoproterenol stimulation of the plateau phase was 6.3 nM. The bioelectric response to isoproterenol was mimicked by isobutylmethylxanthine (IBMX) and, to a lesser degree, by dibutyryl-cAMP. Culturing the cells in medium containing cholera toxin completely inhibited the bioelectric response, yet the preparations continued to respond to isoproterenol with an increase in cAMP production. These results indicated that beta-adrenergic stimulation of Clara cells induced electrogenic transepithelial secretion of Cl- and HCO3- and resolved discrepancies between previous studies.

Purinergic regulation of ion transport across nonciliated bronchiolar epithelial (Clara) cells.

Previous studies demonstrated that elevation of intracellular calcium concentration ([Ca2+]i) increased electrogenic anion transport by bronchiolar epithelia. Extracellular nucleotides were shown to elevate [Ca2+]i and transepithelial short-circuit current (Isc) in proximal airways epithelia. In this study purine and pyrimidine nucleotides were investigated for their ability to regulate ion transport by rabbit nonciliated bronchiolar epithelial (Clara) cells in culture. ATP in the apical bath induced a concentration-dependent transient increase in [Ca2+]i and Isc. Mean effective doses (ED50) of the responses were 10(-7) M and 10(-6) M, respectively. Transepithelial resistance (Rt) decreased. The peak changes in Isc and Rt were 7.8 +/- 1.2 microA/cm2 and -59 +/- 14 omega.cm2 (n = 26, basal Isc = 47.4 +/- 4.3 microA/cm2 and Rt = 428 +/- 40 omega.cm2). Some preparations exhibited a small residual increase in Isc after the initial response, but the change was not statistically significant (delta Isc = 1.7 +/- 1.2 microA/cm2, n = 18). Addition of ATP to the basolateral bath had no detectable effects. Purinoceptor agonists were used to characterize the receptors mediating the change in Isc. UTP and ATP gamma S increased Isc and inhibited subsequent stimulation by ATP. ADP, ADP beta S, 2-methylthio-ATP, and alpha, beta-methylene-ATP had negligible effects on the peak delta Isc and subsequent stimulation by ATP. The ionic mechanism underlying the ATP-induced increase in Isc was investigated with the use of specific ion-transport inhibitors and by ion substitution.(ABSTRACT TRUNCATED AT 250 WORDS)

Epithelial modulation of afferent nerve endings: differential effects of amiloride on afferent subtypes.

Mechanisms underlying the differing chemosensitivity of laryngeal afferents have not been defined. The role of airway epithelium in transducing the chemical stimuli to neural signals was investigated by using Na(+)- and Cl(-)-channel inhibitors in anesthetized spontaneously breathing cats. Single-fiber action potentials were recorded from the peripheral cut end of the superior laryngeal nerve. Luminal application of amiloride (10(-4) M), an inhibitor of epithelial Na+ channels, reduced the responsiveness of non-respiratory-modulated endings (n = 25) to distilled water (65.76 +/- 5.77 vs. 50.67 +/- 5.13 Hz; P < 0.01). Water responsiveness of these endings was unaffected by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and diphenylamine-2-carboxylate, two Cl(-)-channel blockers. Respiratory-modulated endings (water responsive, n = 8; water nonresponsive, n = 9) were unaffected by Na(+)- and Cl(-)-channel blockers. These results suggest that epithelial Na+ channels play a role in the modulation of non-respiratory-modulated laryngeal endings. The lack of an effect by amiloride on other subtypes may be due to differences in location or intrinsic properties of nerve endings. Cl- channels do not appear to play an important role in the modulation of laryngeal afferents targeted in this study.

Sodium- and chloride-conductive pathways in cultured mouse tracheal epithelium.

The utility of a transgenic murine model of cystic fibrosis (CF) lung disease will likely depend on whether the mouse's proximal airway epithelium is characterized by Na(+)- and Cl(-)-conductive pathways comparable to those found in human airways. Therefore, the electrophysiological properties of primary cultures of mouse tracheal epithelium (MTE) were investigated using double-barreled, Cl(-)-selective microelectrodes. Epithelial cells isolated from freshly excised mouse tracheae formed confluent polarized monolayers on permeable collagen supports and developed significant transepithelial potential differences (approximately -10 mV) within 5-6 days postseeding. Under basal conditions, the MTE monolayers had an equivalent short-circuit current (Ieq) of -21.1 +/- 2.1 microA/cm2 and a transepithelial resistance of 424 +/- 49 omega.cm2. Intracellular measurements indicated that the apical (Va) and basolateral (Vb) membrane potential differences were -16.9 +/- 1.5 and -25.4 +/- 1.5 mV, respectively; apical membrane fractional resistance was 0.36 +/- 0.03; and intracellular Cl- activity was 56.1 +/- 2.3 mM. The presence of an apical Na+ conductance was demonstrated by luminal amiloride application (10(-4)M), which decreased Ieq, hyperpolarized Va, and increased the fractional resistance of the apical membrane. The presence of an apical Cl- conductance was demonstrated by substitution of Cl- with gluconate in the luminal bath, which decreased intracellular Cl- activity and increased the fractional resistance of the apical membrane. Luminal application of ATP (10(-4) M was also found to increase the rate of Cl- secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular Ca2+ and regulation of ion transport across rabbit Clara cells.

We investigated whether Ca2+ was involved in regulation of ion transport across rabbit distal airway epithelial cells by studying the effects that elevation of intracellular Ca2+ (Cai) had on the bioelectric properties of nonciliated bronchiolar (Clara) cell epithelia in culture. Exposure of Clara cells to 5 x 10(-7) M ionomycin increased Cai concentration and transepithelial short-circuit current (Isc). Changing extracellular Ca2+ concentration in the presence of ionomycin demonstrated that changes in Isc paralleled changes in Cai. Another ionophore, 4-bromo-A23187, also increased Cai and Isc. Ionomycin-induced changes in Isc were insensitive to amiloride and were inhibited greater than 50% by pretreating the cells with bumetanide or substituting gluconate for Cl- in the bathing solution. Bradykinin and carbachol, which increased Cai and caused an increase in Isc across tracheal cell cultures, had no effect on Cai or Isc in Clara cell preparations. These results support the hypothesis that changes in Cai are linked to regulation of Cl- secretion across bronchiolar epithelial cells, but physiological regulators of Cai in Clara cells remain to be defined.