SGK1 activity in Na+ absorbing airway epithelial cells monitored by assaying NDRG1-Thr346/356/366 phosphorylation.

Studies of HeLa cells and serum- and glucocorticoid-regulated kinase 1 (SGK1) knockout mice identified threonine residues in the n-myc downstream-regulated gene 1 protein (NDRG1-Thr(346/356/366)) that are phosphorylated by SGK1 but not by related kinases (Murray et al., Biochem J 385:1-12, 2005). We have, therefore, monitored the phosphorylation of NDRG1-Thr(346/356/366) in order to explore the changes in SGK1 activity associated with the induction and regulation of the glucocorticoid-dependent Na(+) conductance (G (Na)) in human airway epithelial cells. Transient expression of active (SGK1-S422D) and inactive (SGK1-K127A) SGK1 mutants confirmed that activating SGK1 stimulates NDRG1-Thr(346/356/366) phosphorylation. Although G (Na) is negligible in hormone-deprived cells, these cells displayed basal SGK1 activity that was sensitive to LY294002, an inhibitor of 3-phosphatidylinositol phosphate kinase (PI3K). Dexamethasone (0.2 muM) acutely activated SGK1 and the peak of this response (2-3 h) coincided with the induction of G (Na), and both responses were PI3K-dependent. While these data suggest that SGK1 might mediate the rise in G (Na), transient expression of the inactive SGK1-K127A mutant did not affect the hormonal induction of G (Na) but did suppress the activation of SGK1. Dexamethasone-treated cells grown on permeable supports formed confluent epithelial sheets that generated short circuit current due to electrogenic Na(+) absorption. Forskolin and insulin both stimulated this current and the response to insulin, but not forskolin, was LY294002-sensitive and associated with the activation of SGK1. While these data suggest that SGK1 is involved in the control of G (Na), its role may be minor, which could explain why sgk1 knockout has different effects upon different tissues.

Adenosine-evoked Na+ transport in human airway epithelial cells.

BACKGROUND AND PURPOSE: Absorptive epithelia express apical receptors that allow nucleotides to inhibit Na(+) transport but ATP unexpectedly stimulated this process in an absorptive cell line derived from human bronchiolar epithelium (H441 cells) whilst UTP consistently caused inhibition. We have therefore examined the pharmacological basis of this anomalous effect of ATP. EXPERIMENTAL APPROACH: H441 cells were grown on membranes and the short circuit current (I(SC)) measured in Ussing chambers. In some experiments, [Ca(2+)](i) was measured fluorimetrically using Fura -2. mRNAs for adenosine receptors were determined by the polymerase chain reaction (PCR). KEY RESULTS: Cross desensitization experiments showed that the inhibitory response to UTP was abolished by prior exposure to ATP whilst the stimulatory response to ATP persisted in UTP-pre-stimulated cells. Apical adenosine evoked an increase in I(SC) and this response resembled the stimulatory component of the response to ATP, and could be mimicked by adenosine receptor agonists. Pre-stimulation with adenosine abolished the stimulatory component of the response to ATP. mRNA encoding A(1), A(2A) and A(2B) receptor subtypes, but not the A(3) subtype, was detected in H441 cells and adenosine receptor antagonists could abolish the ATP-evoked stimulation of Na(+) absorption. CONCLUSIONS AND IMPLICATIONS: The ATP-induced stimulation of Na(+) absorption seems to be mediated via A(2A/B) receptors activated by adenosine produced from the extracellular hydrolysis of ATP. The present data thus provide the first description of adenosine-evoked Na(+) transport in airway epithelial cells and reveal a previously undocumented aspect of the control of this physiologically important ion transport process.

Prospective observational study protocol to investigate long-term adverse effects of methylphenidate in children and adolescents with ADHD: the Attention Deficit Hyperactivity Disorder Drugs Use Chronic Effects (ADDUCE) study.

INTRODUCTION: Methylphenidate is the most frequently used medication for the treatment of attention-deficit/hyperactivity disorder (ADHD) in Europe. Following concerns about its safety, the European Commission called for research into the long-term effects of methylphenidate on children and adolescents with ADHD. The Attention Deficit Hyperactivity Disorder Drugs Use Chronic Effects (ADDUCE) research programme was designed to address this call. At the heart of this programme is a 2-year longitudinal naturalistic pharmacovigilance study being conducted in 27 European sites. METHODS AND ANALYSIS: 3 cohorts of children and adolescents (aged 6-17) living in the UK, Germany, Italy and Hungary are being recruited:Group 1 (Medicated ADHD): 800 ADHD medication-naive children and adolescents with a clinical diagnosis of ADHD about to start methylphenidate treatment for the first time.Group 2 (Unmedicated ADHD): 400 children and adolescents with a clinical diagnosis of ADHD who have never been treated with ADHD medication and have no intention of beginning medication.Group 3 (Non-ADHD): 400 children and adolescents without ADHD who are siblings of individuals in either group 1 or 2.All participants will be assessed 5 times during their 2-year follow-up period for growth and development, psychiatric, neurological and cardiovascular health. The primary outcome measure will be the height velocity SD score. ETHICS AND DISSEMINATION: Ethical approval for the study has been granted by the East of Scotland Research Ethics Service. Following this approval, patient information leaflets and consent forms were translated as necessary and submissions made by lead sites in each of the other 3 countries to their own ethics committees. Following ethical approval in each country, local ethical permissions at each site were sought and obtained as needed. The study's website (http://www.adhd-adduce.org/page/view/2/Home) provides information for researchers, participants and the general public. TRIAL REGISTRATION NUMBER: NCT01470261.

Cystic fibrosis and airway submucosal glands.

The chronic pulmonary infections and inflammation associated with cystic fibrosis (CF) are responsible for almost all the morbidity and mortality of this disease. Our understanding of the mechanisms that underlie the very early stages of CF lung disease, that result directly from mutations in the CF gene, is relatively poor. However, the demonstration that the predominant sites of expression of the CF gene in normal lungs are the submucosal glands, together with the histological observations showing that hyperplasia of these glands and mucin occlusion of the gland ducts are the earliest signs of disease in the CF lung, suggest that malfunction of the submucosal glands may be an important factor contributing to the early pathophysiology of CF lung disease. This review describes the function of submucosal glands in normal lungs, and the way in which their function may be disrupted in CF and may thus contribute to the early stages of CF lung disease.

Differential effects of UTP and ATP on ion transport in porcine tracheal epithelium.

Isolated segments of porcine tracheal epithelium were mounted in Ussing chambers, current required to maintain transepithelial potential difference at 0 mV (short circuit current, I(SC)) was monitored and effects of nucleotides upon I(SC) were studied. Mucosal UTP (100 microM) evoked a transient rise in I(SC) that was followed by a sustained fall below basal I(SC) maintained for 30 min. Mucosal ATP (100 microM) also stimulated a transient rise in I(SC) but in contrast to UTP did not inhibit basal I(SC). Submucosal UTP and ATP both transiently increased I(SC). UTP-prestimulated epithelia were refractory to ATP but prestimulation with ATP did not abolish the response to UTP. The epithelia thus appear to express two populations of apical receptors allowing nucleotides to modulate I(SC). The UTP-induced rise was reduced by pretreatment with either bumetanide (100 microM), diphenylamin-2-carboxylic acid (DPC, 1 mM), or Cl(-) and HCO(3)(-)-free solution whilst the fall was abolished by amiloride pretreatment. Thapsigargin (0.3 microM) abolished the UTP-induced increase in I(SC) but not the subsequent decrease. Staurosporine (0.1 microM) inhibited basal I(SC) and blocked UTP-induced inhibition of I(SC). Inhibitors of either protein kinase C (PKC) (D-erythro sphingosine) or PKA (H89) had no effect. This study suggests that UTP stimulates Cl(-) secretion and inhibits basal Na(+) absorption. ATP has a similar stimulatory effect, which may be mediated by activation of P2Y(2) receptors and an increase in [Ca(2+)](in), but no inhibitory effect, which is likely mediated by activation of a pyrimidine receptor and possible inhibition of a protein kinase other than PKC or PKA.

Culture substrate-specific expression of P2Y2 receptors in distal lung epithelial cells isolated from foetal rats.

ATP and UTP did not evoke [Ca2+]i signals in rat foetal lung epithelial cells grown on glass but elicited clear responses in cells grown into functionally polarised epithelia on permeable supports. Moreover, P2Y2 receptor mRNA could not be detected in cells on glass by the polymerase chain reaction but this mRNA species was clearly expressed by polarised cells. P2Y2 receptor expression thus appears to be a feature of the polarised phenotype.

P2Y2 receptor-mediated inhibition of ion transport in distal lung epithelial cells.

1 Rat foetal distal lung epithelial cells were plated onto permeable supports where they became integrated into epithelial sheets that spontaneously generated short circuit current (ISC). 2 Apical ATP (100 microM) evoked a transient fall in ISC that was followed by a rise to a clear peak which, in turn, was succeeded by a slowly developing decline to a value below control. Apical UTP evoked an essentially identical response. 3 UDP and ADP were ineffective whilst ATP had no effect when added to the basolateral solution. These effects thus appear to be mediated by apical P2Y2 receptors. 4 The rising phase of the responses to ATP/UTP was selectively inhibited by anion transport inhibitors but persisted in the presence of amiloride, which abolished the inhibitory effects of both nucleotides. Thus, apical nucleotides appear to evoke a transient stimulation of anion secretion and sustained inhibition of Na+ absorption. 5 Basolateral isoprenaline (10 microM) elicited a rise in ISC but subsequent addition of apical ATP reversed this effect. Conversely, isoprenaline restored ISC to its basal level following stimulation with ATP. Apical P2Y2 receptors and basolateral beta-adrenoceptors thus allow their respective agonists to exert mutually opposing effects on ISC.

Differential effects of furosemide on porcine bronchial arterial and airway smooth muscle.

Furosemide attenuates airway obstruction in asthmatic subjects when administered as an aerosol pretreatment. This protective effect of furosemide could be related to relaxation of bronchial smooth muscle or to increased bronchial blood flow. To determine whether furosemide dilates bronchial smooth muscle, isometric contractile responses in distal bronchi from young pigs were studied. In bronchial smooth muscle rings that were precontracted with 10(-5) M acetylcholine, significant relaxation occurred with 10(-8) to 3 x 10(-6) M isoproterenol but not with 10(-8) to 10(-3) M furosemide. In contrast, bronchial arteries that were precontracted with either 10(-4) M norepinephrine or 10(-8) M vasopressin significantly relaxed in response to 10(-4) to 3 x 10(-3) M and 10(-3) to 3 x 10(-3) M furosemide, respectively. We conclude that furosemide, under the described experimental conditions, relaxes airway vascular smooth muscle but not bronchial smooth muscle. These results are consistent with previous suggestions that inhaled furosemide increases blood flow to airway tissues (Gilbert IA, Lenner KA, Nelson JA, Wolin AD, and Fouke JM. J Appl Physiol 76: 409-415, 1994).

Expression of intermediate-conductance, Ca2+-activated K+ channel (KCNN4) in H441 human distal airway epithelial cells.

Electrophysiological studies of H441 human distal airway epithelial cells showed that thapsigargin caused a Ca(2+)-dependent increase in membrane conductance (G(Tot)) and hyperpolarization of membrane potential (V(m)). These effects reflected a rapid rise in cellular K(+) conductance (G(K)) and a slow fall in amiloride-sensitive Na(+) conductance (G(Na)). The increase in G(Tot) was antagonized by Ba(2+), a nonselective K(+) channel blocker, and abolished by clotrimazole, a KCNN4 inhibitor, but unaffected by other selective K(+) channel blockers. Moreover, 1-ethyl-2-benzimidazolinone (1-EBIO), which is known to activate KCNN4, increased G(K) with no effect on G(Na). RT-PCR-based analyses confirmed expression of mRNA encoding KCNN4 and suggested that two related K(+) channels (KCNN1 and KCNMA1) were absent. Subsequent studies showed that 1-EBIO stimulates Na(+) transport in polarized monolayers without affecting intracellular Ca(2+) concentration ([Ca(2+)](i)), suggesting that the activity of KCNN4 might influence the rate of Na(+) absorption by contributing to G(K). Transient expression of KCNN4 cloned from H441 cells conferred a Ca(2+)- and 1-EBIO-sensitive K(+) conductance on Chinese hamster ovary cells, but this channel was inactive when [Ca(2+)](i) was <0.2 microM. Subsequent studies of amiloride-treated H441 cells showed that clotrimazole had no effect on V(m) despite clear depolarizations in response to increased extracellular K(+) concentration ([K(+)](o)). These findings thus indicate that KCNN4 does not contribute to V(m) in unstimulated cells. The present data thus establish that H441 cells express KCNN4 and highlight the importance of G(K) to the control of Na(+) absorption, but, because KCNN4 is quiescent in resting cells, this channel cannot contribute to resting G(K) or influence basal Na(+) absorption.

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.

Effect of anion secretion inhibitors on mucin content of airway submucosal gland ducts.

In porcine bronchi, inhibition of both Cl- and HCO3- transport is required to block the anion secretion response to ACh and to cause mucus accumulation within ACh-treated submucosal gland ducts [S. K. Inglis, M. R. Corboz, A. E. Taylor, and S. T. Ballard. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L372-L377, 1997]. In this previous study, a combination of three potential HCO3- transport inhibitors [1 mM acetazolamide, 1 mM DIDS, and 0.1 mM dimethylamiloride (DMA)] was used to block carbonic anhydrase, Cl-/HCO3- exchange, and Na+/H+ exchange, respectively. The aim of the present study was to obtain a better understanding of the mechanism of ACh-induced HCO3- secretion in airway glands by determining which of the three inhibitors, in combination with bumetanide, is required to block anion secretion and so cause ductal mucin accumulation. Gland duct mucin content was measured in distal bronchi isolated from domestic pigs. Addition of either bumetanide alone, bumetanide plus acetazolamide, or bumetanide plus DIDS had no significant effect on ACh-induced mean gland duct mucin content. In contrast, glands treated with bumetanide plus DMA as well as glands treated with all four anion transport blockers were almost completely occluded with mucin after the addition of ACh. These data suggest that mucin is cleared from the ducts of bronchial submucosal glands by liquid generated from Cl(-)- and DMA-sensitive HCO3- transport.

Hormonal modulation of Na(+) transport in rat fetal distal lung epithelial cells.

Isolated rat fetal distal lung epithelial (FDLE) cells were cultured (approximately 48 h) on permeable supports in medium devoid of hormones and growth factors whilst P(O2) was maintained at the level found in either the fetal (23 mmHg) or the postnatal (100 mmHg) alveolar regions. The cells became incorporated into epithelial layers that generated a basal short-circuit current (I(SC)) attributable to spontaneous Na(+) absorption. Cells at neonatal P(O2) generated larger currents than did cells at fetal P(O2), indicating that this Na(+) transport process is oxygen sensitive. Irrespective of P(O2), isoprenaline failed to elicit a discernible change in I(SC), demonstrating that beta-adrenoceptor agonists do not stimulate Na(+) transport under these conditions. However, isoprenaline did elicit cAMP accumulation in these cells, indicating that functionally coupled beta-adrenoceptors are present. Further experiments showed that isoprenaline did increase I(SC) in cells treated (24 h) with a combination of tri-iodothyronine (T(3), 10 nM) and dexamethasone (200 nM). Studies of basolaterally permeabilised cells showed that these hormones are essential for the isoprenaline-evoked increase in the apical membrane's Na(+) conductance (G(Na)), whereas isoprenaline-evoked changes in apical Cl(-) conductance (G(Cl)) can occur in both control and hormone-treated cells. Irrespective of their hormonal status, FDLE cells thus express beta-adrenoceptors that are functionally coupled to adenylate cyclase, and allow beta-adrenoceptor agonists to modulate the apical membrane's anion conductance. However, T(3) and dexamethasone are needed if these receptors are to exert control over G(Na). These hormones may thus play an important role in the functional maturation of the lung by allowing beta-adrenoceptor-mediated control over epithelial Na(+) channels in the apical plasma membrane.

In situ visualization of bronchial submucosal glands and their secretory response to acetylcholine.

Airway submucosal glands secrete both macromolecules and liquid, yet the mechanisms by which these substances are secreted are not well understood. In this study, a video microscope was used to directly visualize the submucosal glands in isolated porcine distal bronchi and to observe their responses to acetylcholine (ACh), a glandular secretagogue. Submucosal glands were classified as either "antral," "linear," or "convoluted" glands based on the morphology of their terminal collecting ducts. Because antral duct glands were most easily visualized, the response to ACh was studied in detail in this gland type. Within 5-10 s after addition of 10 microM ACh, the cross-sectional area of the gland duct openings to the airway surface increased severalfold but returned to pre-ACh dimensions within 1 min. Between 30 s and 10 min after ACh addition, spherical particles (1-10 microm) entered the antral ducts from distal acini and exited through the duct openings to the airway surface. Some of the particles were retained within the antral duct where they were kept in constant motion by the action of cilia present within the antral duct. The particles, which are likely to contain the macromolecular secretory products of mucous and/or serous cells, maintained their spherical shape within the gland duct, suggesting that the secretion product was membrane bound. To our knowledge, these studies provide the first description of airway submucosal gland secretion as viewed in situ.

Effect of anion transport inhibition on mucus secretion by airway submucosal glands.

To model the airway glandular defect in cystic fibrosis (CF), the effect of anion secretion blockers on submucosal gland mucus secretion was investigated. Porcine distal bronchi were isolated, pretreated with a Cl- secretion blocker (bumetanide) and/or a combination of blockers to inhibit HCO3- secretion (dimethylamiloride, acetazolamide, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), and then treated with acetylcholine (ACh), a glandular liquid and mucus secretagogue. Bronchi were then fixed, sectioned, and stained for mucins. Each gland duct was ranked for mucin content from zero (no mucin) to five (duct completely occluded with mucin). Untreated bronchi, bronchi treated only with ACh, and ACh-treated bronchi that received either bumetanide or the HCO3- secretion blockers all exhibited low gland duct mucin content (1.18 +/- 0.34, 0.59 +/- 0.07, 0.65 +/- 0.03, and 0.83 +/- 0.11, respectively). However, pretreatment with both Cl- and HCO3- secretion blockers before ACh addition resulted in substantial and significant ductal mucus accumulation (3.57 +/- 0.22). In situ videomicroscopy studies of intact airways confirmed these results. Thus inhibition of the anion (and presumably liquid) secretion response to ACh leads to mucus obstruction of submucosal gland ducts that resembles the early pathological changes observed in CF.

Tracheal microvascular responses to beta-adrenergic stimulation in anesthetized rats.

Previous study of adrenergic control of the tracheal vasculature in rats (1) demonstrated that beta-adrenergic blockade heightened arteriolar and large venular contractile responses to norepinephrine, a nonselective alpha-adrenergic agonist. The present study was undertaken to confirm the presence of functional beta-adrenergic receptors and to determine which beta-adrenergic receptor subtypes mediate vasodilation in this tissue. Tracheal adventitial arterioles (12.0 to 47.0 micro m initial diameter, n=39) and venules (48.0 to 98.5 micrometers initial diameter, n=44) were observed through a video microscope, and vessel diameters were measured. Vessels were preconstricted with 10(-4) M phenylephrine (PHE), a selective alpha 1-adrenergic agonist, to achieve sufficient tone for measurement of dilation responses. When vessels were treated only with PHE, arterioles and venules constricted to 55.9% and 67.6% of their initial diameter, respectively, after 15 min of suffusion. When preconstricted vessels were treated with the nonselective beta-adrenergic agonist isoproterenol (10(-5) M), both arterioles and venules significantly dilated from 63.4% to 82.9% and from 71.5% to 81.3% of their initial diameters. At high concentration (10(-5) M), the putative beta 2-adrenergic agonist terbutaline also caused preconstricted arterioles and venules to significantly dilate from 70.8% to 79.8% and from 71.5% to 83.4% of their initial diameters. The selective beta 1-adrenergic antagonist atenolol (10(-6) M) did not affect terbutaline-induced dilation in preconstricted arterioles, but greatly attenuated dilation in preconstricted venules. From these data, we conclude that beta 2-adrenergic receptors are present in and mediate dilation of tracheal arterioles, and also, that the dilation in large tracheal venules is mediated in large part through beta 1-adrenergic receptors.

Tracheal microvascular responses to inhibition of nitric oxide synthesis in anesthetized rats.

We previously demonstrated that rat tracheal arterioles and large venules constrict in response to alpha-adrenergic agonists and dilate in response to beta-adrenergic agonists. To further investigate the vasodilatory mechanisms in these vessels, we hypothesized that the vascular tone in the tracheal microcirculation is regulated in part by endogenously released nitric oxide (NO). To test this hypothesis, rat tracheal microvessels were visualized in vivo with a video microscope. The change in diameter af adventitial arteriolar (14.5 to 42.0 microm initial diameter, n = 41) and large venular (50.0 to 100.0 microm initial diameter, n = 41) microvessels following the suffusion of vasoactive drugs was measured with video calipers. Significant constriction was observed in arterioles (to 70.0% of initial diameter) and large venules (to 76.6% of initial diameter) after 20 min of suffusion with L-NAME, an inhibitor of NO synthesis. This constriction was in large part reversed by L-arginine, a biochemical precursor of NO, in arterioles (to 93.2% of initial diameter) and in venules (to 90.8% of initial diameter). The combination of prazosin, a selective alpha1-adrenergic antagonist, and yohimbine, a selective alpha2-adrenergic antagonist, also reduced L-NAME-induced constriction in arterioles (to 87.9% of initial diameter) and in venules (to 85.2% of initial diameter). L-arginine or the combination of prazosin and yohimbine alone did not affect the diameter of tracheal microvessels. These data suggest that NO exerts an important influence on tracheal microvascular tone in rats, and may attenuate alpha-adrenergic constriction in these vessels.

Dilatory effect of furosemide on rat tracheal arterioles and venules.

Furosemide pretreatment greatly reduces the severity of an asthmatic response to several types of bronchoconstrictor challenge. Indirect evidence suggests that furosemide exerts its protective effects by dilating the airway vasculature during thermal stress. To test the hypothesis that furosemide dilates airway microvessels, the tracheas of anesthetized rats were surgically exposed and continuously suffused with Krebs Ringer bicarbonate warmed to 37 degrees C. Tracheal adventitial arterioles (13.0 to 41.0 microns initial diameter, n = 47) and venules (50.0 to 99.0 microns initial diameter, n = 46) were visualized with a videomicroscope, and vessel diameters were measured using videocalipers. When vessels were preconstricted with 10(-4) M phenylephrine, a selective alpha 1-adrenergic agonist, and then treated with 10(-4) M furosemide, significant (p < 0.05) dilation was observed in both arterioles (from 64.6 to 79.5% of their initial diameter) and venules (from 52.1 to 65.4% of their initial diameter). When vessels were preconstricted with 10(-4) phenylephrine, after pretreatment with the cyclooxygenase inhibitor indomethacin (5.0 mg/kg), 10(-4) M furosemide significantly dilated arterioles (from 77.5 to 93.0% of their initial diameter) and venules (from 58.5 to 80.1% of their initial diameter). In vessels preconstricted with 10(-3) M L-NAME, an inhibitor of nitric oxide synthesis, addition of 10(-4) M furosemide to the suffusion still caused significant dilation in arterioles, from 77.4 to 88.8% of their initial diameter, and in venules from 79.5 to 86.7% of their initial diameter. These data confirm that furosemide, when applied topically, dilates tracheal arterioles and venules by cyclooxygenase- and nitric oxide-independent mechanisms.

Effect of luminal nucleotides on Cl- secretion and Na+ absorption in distal bronchi.

P2Y receptor agonists stimulate Cl- secretion across both normal and cystic fibrosis (CF) airway epithelia, and therefore have potential for use in the treatment of CF. Although CF pathology is manifest primarily in the distal airways, most studies of P2Y-receptor-mediated airway epithelial ion transport have used cells cultured from proximal regions. Here we report the results of studies of P2Y-receptor-mediated ion transport in distal bronchi isolated from porcine lungs, cannulated and perfused. A luminal microelectrode was used to record transepithelial potential difference (PD) and cable analysis was applied to determine resistance (Rt) and equivalent short-circuit current (I(SC)). Luminal UTP (100 micromol/l) transiently hyperpolarized PD (from -5.8+/-0.3 to -6.5+/-0.4 mV) and increased I(SC) (from 47+/-6 to 55+/-8 microA cm(-2)) before inhibiting PD to below the pre-UTP level (-5.0+/-0.4 mV). The decline was attenuated by pretreatment with amiloride, and additional treatment with bumetanide inhibited the initial hyperpolarization, suggesting that UTP stimulates Cl- secretion and inhibits Na+ absorption across distal bronchi. Luminal addition of P2Y1 [ADP, 2-methylthio-ATP (2MeSATP)] and P2Y6 (UDP) receptor agonists had no effect on ion transport. Pretreatment with thapsigargin (0.3 micromol/l) did not prevent the UTP-induced increase in PD and I(SC) but attenuated the secondary fall in PD. Pretreatment with BAPTA/AM (50 micromol/l), however, had no effect on the response to UTP. Additional studies of isolated bronchial epithelial cells using Fura-2 showed that UTP increases [Ca2+]in, and this increase is blocked by pretreatment with thapsigargin. These results suggest that in intact distal bronchi luminal UTP stimulates Cl- secretion by a Ca2+-independent mechanism and inhibits Na+ absorption by a Ca2+-dependent mechanism. Both effects are likely to favour increased hydration of the airway surface, and may therefore be beneficial in CF.

Regulation of ion transport across porcine distal bronchi.

Distal airways comprise the vast majority of total human airway surface area, yet little is known about transepithelial ion transport in these tissues. Pathways that regulate ion transport in porcine small bronchi (3.62 +/- 0.04 mm outer diameter) were studied by measuring the effects of secretogogues that stimulate Cl- secretion in proximal airways. Resting potential difference (PD), shortcircuit current (ISC), and resistance (Rt) across the distal bronchi were 3.4 +/- 0.1 mV (lumen negative), 40.8 +/- 1.7 microA/cm2, and 92.1 +/- 5.0 omega.cm2, respectively. Isoproterenol (10 microM), acetylcholine (ACh; 10 microM), or ATP (100 microM) induced immediate significant (P < 0.05) increases in PD and ISC. The Ca2+ ionophore A23187 (1 microM) induced a slow increase in PD that was accompanied by a significant 2.4-fold increase in Rt and no change in ISC. The responses to isoproterenol, ACh, and ATP were maintained in the presence of 10 microM amiloride. Pretreatment with 10 microM bumetanide to block Cl- secretion inhibited responses to isoproterenol and ATP but not to ACh. The ACh effect was inhibited only after both Cl- and HCO3- secretion were blocked. These data suggest that isoproterenol, ACh, and ATP stimulate anion secretion. Isoproterenol and ATP specifically stimulate Cl- secretion, whereas ACh can stimulate both Cl- and HCO3- secretion. A23187 has no effect on active transepithelial ion transport but increases barrier resistance in intact distal bronchial epithelium.

Inhibition of airway liquid secretion and its effect on the physical properties of airway mucus.

The combination of both Cl- and HCO3- secretion inhibitors causes an accumulation of mucins within the submucosal gland ducts of acetylcholine (ACh)-treated bronchi [S. K. Inglis, M. R. Corboz, A. E. Taylor, and S. T. Ballard. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L372-L377, 1997], suggesting indirectly that these agents block airway gland liquid secretion. The present study tested the hypotheses that ACh-stimulated liquid secretion is driven by Cl- and HCO3- secretion and that inhibition of this process leads to secretion of a dehydrated mucus with altered rheological properties. Excised distal bronchi from pigs were pretreated with either a combination of Cl- and HCO3- secretion inhibitors (bumetanide, acetazolamide, dimethylamiloride, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) or the dimethyl sulfoxide vehicle and were then treated with ACh to induce secretion. The rate of mucus liquid secretion was substantially reduced when the airways were pretreated with the anion secretion inhibitors. Mucus liquid from inhibitor-pretreated airways contained almost threefold more nonvolatile solids than the control liquid. Rheological analysis revealed that mucus liquid from inhibitor-pretreated airways expressed a significantly greater log G* (rigidity factor), whereas tangent delta (recoil factor) was significantly reduced. These results suggest that 1) ACh-induced liquid secretion in bronchi is driven by both Cl- and HCO3- secretion and 2) inhibition of ACh-induced liquid secretion results in the secretion of mucus with a reduced water content and altered rheological properties.