Sarah K. Inglis

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

In porcine bronchi, inhibition of both Cl- and[Formula: see text] 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 [Formula: see text] transport inhibitors [1 mM acetazolamide, 1 mM DIDS, and 0.1 mM dimethylamiloride (DMA)] was used to block carbonic anhydrase, Cl-/[Formula: see text]exchange, and Na+/H+exchange, respectively. The aim of the present study was to obtain a better understanding of the mechanism of ACh-induced[Formula: see text] 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 [Formula: see text] transport.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.

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

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). Apical ATP (100 μM) 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. 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. 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. Basolateral isoprenaline (10 μM) 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 β‐adrenoceptors thus allow their respective agonists to exert mutually opposing effects on ISC.

Regulation of intracellular pH in Calu-3 human airway cells

The Calu‐3 human cell line exhibits features of submucosal gland serous cells and secretes HCO3−. The aim of this study was to identify the HCO3− transporters present in these cells by studying their role in the regulation of intracellular pH (pHi). Calu‐3 cells were grown on coverslips, loaded with the pH‐sensitive fluorescent dye BCECF, and their fluorescence intensity monitored as an indication of pHi. Cells were acidified with NH4Cl (25 mm, 1 min) and pHi recovery recorded. In the absence of HCO3−, initial recovery was 0.208 ± 0.016 pH units min−1(n = 37). This was almost abolished by removal of extracellular Na+ and by amiloride (1 mm), consistent with the activity of a Na+‐H+ exchanger (NHE). In the presence of HCO3− and CO2, recovery (0.156 ± 0.018 pH units min−1) was abolished (reduced by 91.8 ± 6.7 %, n = 7) by removal of Na+ but only attenuated (by 63.3 ± 5.8 %, n = 9) by amiloride. 4,4‐Dinitrostilbene‐2,2‐disulfonic acid (DNDS) inhibited recovery by 45.8 ± 5.0 % (n = 7). The amiloride‐insensitive recovery was insensitive to changes in membrane potential, as confirmed by direct microelectrode measurements, brought about by changing extracellular [K+] in the presence of either valinomycin or the K+ channel opener 1‐EBIO. In addition, forskolin (10 μm), which activates the cystic fibrosis transmembrane conductance regulator Cl− conductance in these cells and depolarises the cell membrane, had no effect on recovery. Removal of extracellular Cl− trebled pHi recovery rates, suggesting that an electroneutral, DNDS‐sensitive, Cl−‐HCO3− exchanger together with a NHE may be involved in pHi regulation and HCO3− secretion in these cells. RT‐PCR detected the expression of the electrogenic Na+‐HCO3− cotransporter NBC1 and the Cl−‐HCO3− exchanger (AE2) but not the electroneutral Na+‐HCO3− cotransporter NBCn1.

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, ISC) was monitored and effects of nucleotides upon ISC were studied. Mucosal UTP (100 μM) evoked a transient rise in ISC that was followed by a sustained fall below basal ISC maintained for 30 min. Mucosal ATP (100 μM) also stimulated a transient rise in ISC but in contrast to UTP did not inhibit basal ISC. Submucosal UTP and ATP both transiently increased ISC. 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 ISC. The UTP‐induced rise was reduced by pretreatment with either bumetanide (100 μM), diphenylamin‐2‐carboxylic acid (DPC, 1 mM), or Cl− and HCO3−‐free solution whilst the fall was abolished by amiloride pretreatment. Thapsigargin (0.3 μM) abolished the UTP‐induced increase in ISC but not the subsequent decrease. Staurosporine (0.1 μM) inhibited basal ISC and blocked UTP‐induced inhibition of ISC. 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 P2Y2 receptors and an increase in [Ca2+]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.

O2 can raise fetal pneumocyte Na+ conductance without affecting ENaC mRNA abundance

In fetal pneumocytes, increasing P(O(2)) can raise apical Na(+) conductance (G(Na(+))) and increase the abundance of epithelial Na(+) channel subunit (alpha-, beta-, and gamma-ENaC) mRNA, suggesting that the rise in G(Na(+)), which may be important to the perinatal maturation of the lung, reflects O(2)-evoked ENaC gene expression. However, we now show that physiologically relevant increases in P(O(2)) do not affect alpha-, beta-, and gamma-ENaC mRNA abundance in pneumocytes maintained (approximately 48 h) in hormone-free medium or in medium supplemented with dexamethasone and tri-iodothyronine, although the response does persist in cells maintained in medium containing a complex mixture of hormones/growth factors. However, parallel electrometric studies revealed clear increases in G(Na(+)) under all tested conditions and so it is now clear that O(2)-evoked increases in G(Na(+)) can occur without corresponding increases in ENaC mRNA abundance. It is therefore unlikely that this rise in G(Na(+)) is secondary to O(2)-evoked ENaC gene expression.

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

Isolated rat fetal distal lung epithelial (FDLE) cells were cultured (≈48 h) on permeable supports in medium devoid of hormones and growth factors whilst PO2 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 (ISC) attributable to spontaneous Na+ absorption. Cells at neonatal PO2 generated larger currents than did cells at fetal PO2, indicating that this Na+ transport process is oxygen sensitive. Irrespective of PO2, isoprenaline failed to elicit a discernible change in ISC, demonstrating that β‐adrenoceptor agonists do not stimulate Na+ transport under these conditions. However, isoprenaline did elicit cAMP accumulation in these cells, indicating that functionally coupled β‐adrenoceptors are present. Further experiments showed that isoprenaline did increase ISC in cells treated (24 h) with a combination of tri‐iodothyronine (T3, 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 membranes Na+ conductance (GNa), whereas isoprenaline‐evoked changes in apical Cl− conductance (GCl) can occur in both control and hormone‐treated cells. Irrespective of their hormonal status, FDLE cells thus express β‐adrenoceptors that are functionally coupled to adenylate cyclase, and allow β‐adrenoceptor agonists to modulate the apical membranes anion conductance. However, T3 and dexamethasone are needed if these receptors are to exert control over GNa. These hormones may thus play an important role in the functional maturation of the lung by allowing β‐adrenoceptor‐mediated control over epithelial Na+ channels in the apical plasma membrane.

Effects of peroxisome proliferator‐activated receptor γ agonists on Na+ transport and activity of the kinase SGK1 in epithelial cells from lung and kidney

Background and purpose:  Peroxisome proliferator‐activated receptor γ (PPARγ) agonists, such as rosiglitazone and pioglitazone, sensitize cells to insulin, and are therefore used to treat type 2 diabetes. However, in some patients, these drugs induce oedema, and the present study tests the hypothesis that this side effect reflects serum and glucocorticoid‐inducible kinase 1 (SGK1)‐dependent enhancement of epithelia Na+ absorption.

Adenosine-evoked Na+ transport in human airway epithelial cells

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.

Reports of Perceived Adverse Events of Stimulant Medication on Cognition, Motivation, and Mood: Qualitative Investigation and the Generation of Items for the Medication and Cognition Rating Scale

Abstract Objective: There is no questionnaire to specifically monitor perceived adverse events of methylphenidate (MPH) on cognition, motivation, and mood. The current study therefore had two goals. First, to harvest accounts of such putative events from transcripts of interviews in samples enriched for such potential experiences. Second, to use the derived data to generate items for a new questionnaire that can be used for monitoring such events in medication trials or routine clinical care. Methods: Following a literature search aimed at identifying associations between MPH and cognition and/or motivation, a qualitative semistructured interview was designed to focus specifically on the domains of cognition (i.e., reasoning, depth/breadth of thinking, intellectual capacity, and creativity) and motivation (i.e., drive, effort, and attitudes toward rewards/incentives). Interviews were conducted with 45 participants drawn from the following four groups: (a) clinicians, child and adolescent psychiatrists, and pediatricians specializing in attention-deficit/hyperactivity disorder (ADHD) (n = 15); (2) teachers, with experience of teaching at least 10 medicated children with ADHD (n = 10); (3) parents of children with ADHD (n = 8) treated with MPH; and (4) adolescents/adults with ADHD (n = 12). Purposeful sampling was used to selectively recruit ADHD participants whose histories suggested a degree of vulnerability to MPH adverse events. Data were analyzed using a deductive approach to content analysis. Results: While we probed purposefully for cognitive and motivational adverse events, a third domain, related to mood, emerged from the reports. Therefore, three domains, each with a number of subdomains, were identified from the interview accounts: (i) Cognition (six subdomains; attention/concentration, changes in thinking, reduced creativity, sensory overload, memory, slower processing speed); (ii) motivation (four subdomains; loss of intrinsic motivation for goal-directed activities, external locus of control, lack of effort/engagement in daily tasks, increased focus on incentives); and (iii) mood (three subdomains; dampening of spontaneity/flat affect, mood dysregulation, increased anxiety/edginess). On the basis of these reports, 34 items were specified and incorporated into a prototype questionnaire, which was piloted and refined on the basis of field-testing. Conclusions: Items were identified that capture potential/perceived cognitive, motivational, and mood-related adverse events of MPH. The items generated will allow us to further develop and psychometrically examine their prevalence, and the extent to which they are associated with medication adherence, treatment outcome, impairment, and other reported adverse events (e.g., loss of appetite/cardiovascular effects).