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Objectives: This study aimed to demonstrate how to estimate the value of health gain after patients with a multisystem disease achieve a condition-specific composite response endpoint. Methods: Data from patients treated in routine practice with an exemplar multisystem disease (systemic lupus erythematosus) were extracted from a national register (British Isles Lupus Assessment Group Biologics Register). Two bespoke composite response endpoints (Major Clinical Response and Improvement) were developed in advance of this study. Difference-in-differences regression compared health utility values (3-level version of EQ-5D; UK tariff) over 6 months for responders and nonresponders. Bootstrapped regression estimated the incremental quality-adjusted life-years (QALYs), probability of QALY gain after achieving the response criteria, and population monetary benefit of response. Results: Within the sample (n = 171), 18.2% achieved Major Clinical Response and 49.1% achieved Improvement at 6 months. Incremental health utility values were 0.0923 for Major Clinical Response and 0.0454 for Improvement. Expected incremental QALY gain at 6 months was 0.020 for Major Clinical Response and 0.012 for Improvement. Probability of QALY gain after achieving the response criteria was 77.6% for Major Clinical Response and 72.7% for Improvement. Population monetary benefit of response was £1 106 458 for Major Clinical Response and £649 134 for Improvement. Conclusions: Bespoke composite response endpoints are becoming more common to measure treatment response for multisystem diseases in trials and observational studies. Health technology assessment agencies face a growing challenge to establish whether these endpoints correspond with improved health gain. Health utility values can generate this evidence to enhance the usefulness of composite response endpoints for health technology assessment, decision making, and economic evaluation.

1. Prostaglandin E2 (PGE2) is thought to be an important inhibitory modulator of inflammatory processes in the airway. It inhibits inflammatory cell function and cholinergic neurotransmission in vitro and roles have been postulated in vivo in refractoriness and in the mechanism of action of the diuretic agent, frusemide. 2. The production of PGE2 by bovine cultured airway smooth muscle cells has been studied under a range of conditions. The effects of cyclo-oxygenase inhibitors (flurbiprofen, indomethacin, acetyl salicylic acid) on serum-induced production of PGE2 were assessed over a range of concentrations (10(-7)-10(-4) M). 3. Serum-stimulated production of PGE2 in control wells ranged from 350 to 800 ng PGE2 ml-1 in cells from different animals. All three cyclo-oxygenase inhibitors inhibited PGE2 production with an order of potency, flurbiprofen > indomethacin > acetyl salicylic acid. Log IC50 values were -6.24 for flurbiprofen, -5.23 for indomethacin and -3.50 for acetyl salicylic acid. 4. PGE2 production was stimulated by arachidonic acid (10(-5) M) or addition of the proinflammatory mediator, bradykinin (10(-8)-10(-5) M). 5. Incubation of cells for 24 h with 5 bromo deoxyuridine (BRDU) (10(-4) M) to prevent DNA synthesis did not alter PGE2 production in response to serum, suggesting that it was not a function of proliferation per se. 6. Our study suggests that airway smooth muscle may be an important source of PGE2. Production of PGE2 may be a novel feedback mechanism whereby airway smooth muscle cells can negatively modulate airways inflammation. The differing potencies of the cyclo-oxygenase inhibitors may explain the contrasting effect of these drugs in recent studies in asthma.

Inhaled furosemide has been shown to protect subjects with asthma from bronchoconstriction induced by a wide variety of stimuli, including allergen, but the mechanism of action is controversial. We have used an in vitro model of allergen-induced bronchoconstriction to examine the effects of furosemide and other ion transport inhibitors. Human bronchial rings were passively sensitized by incubation with serum from an atopic donor and were challenged with Dermatophagoides pteronyssinus. Allergen-challenged bronchial rings developed bronchoconstriction which was effectively inhibited by the cysteinyl-leukotriene antagonist ICI 198,615 (10 M) and to a lesser extent by terfenadine (0 M). Assessed over 60 min furosemide 10, 10, and 10 M inhibited contractions by a mean (95% confidence interval [CI]) 7.9% (-23.5, 39.3%, p > 0.05), 44.2% (12.9, 75.2%, p < 0.01), and 86.9% (55.5, 118.3%, p < 0.001) respectively (n = 5). The same concentrations of bumetanide inhibited contractions by 21.5% (-8.4, 51.4%, p > 0.05), 13.6% (-16.3, 43.4%, p > 0.05) and 51.6% (21.7, 81.4%, p < 0.01) respectively (n = 5). The sodium, transport inhibitor amiloride and the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) were without effect (both 10 M; n = 4). Furosemide increased PGE production by the bronchial rings by 134% (95% CI 53, 259%). Indomethacin (3 x 10 M) blocked the furosemide-induced increase in PGE production and reduced the protection afforded by 10 M furosemide against allergen- induced contractions from 67.9% to 34.7% (mean difference 33.2%; 95% CI 9.7, 56.6%; p < 0.01; n=8). PGE (10, 10, and 10 M) did not alter airway smooth muscle responsiveness to methacholine but inhibited allergen- induced contractions by 5% (-68.2, 78.3%), 14.5% (-58.8, 87.7%), and 100% (26.7, 173.2%; p < 0.02) respectively (n = 5). Thus furosemide and to a lesser extent bumetanide inhibit allergen-induced contractions of passively sensitized human bronchi. Production of PGE may play a role in the inhibitory effect of furosemide.

Pseudomonas aeruginosa forms biofilms in the cystic fibrosis lung. Quorum sensing (QS) controls biofilm maturation, immune evasion, antibiotic tolerance and virulence factor production. Garlic shows QS inhibitory activity in vitro and in animal models. We report the first clinical trial in man of a QS inhibitor.We randomized 34 patients to garlic or olive oil capsules (both 656 mg daily). Clinical outcomes and safety bloods were measured at baseline and after 8 weeks treatment. In this exploratory study, analysis was per protocol.Eight patients withdrew, leaving 26 for analysis (13 garlic). With placebo, there was a greater decline in mean (SD) percentage change from baseline FEV(1) [-3.6% (11.3)] than with garlic [-2.0% (12.3)]. This was not significant (mean difference = 1.6, 95% CI -12.7 to 15.9, P = 0.8). The mean (SD) increase in weight was greater with garlic [1.0% (2.0)] than with placebo [0.6% (2.0)]--non-significant (mean difference = 0.4%, 95% CI -1.3 to 2.0, P = 0.6). The median (range) change in clinical score with garlic was -1 (-3 to 5) and 1 (-1 to 4) with placebo (negative score means improvement). This was non-significant [median difference = -1 (-3 to 0), P = 0.16]. In the garlic group, seven patients had IV antibiotics versus five placebo. There was a highly significant correlation between plasma and sputum measurements of the QS molecule 3-oxo-C12-HSL (Pearson correlation coefficient = 0.914, P = 0.004). At the end of treatment five patients in each group had abnormal liver function or triglycerides and five garlic patients (one placebo) reported minor adverse effects.Garlic capsules were well tolerated. Although there was no significant effect of garlic compared to placebo in this pilot study, there was a suggestion of improvement with garlic which should be investigated in a larger trial.

Here we tested the effect of interleukin-1β, a pro-inflammatory cytokine, on cAMP accumulation and chloride efflux in Calu-3 airway epithelial cells in response to ligands binding to adenylyl cyclase-coupled receptors such as the β2 adrenoreceptor and EP prostanoid receptors. Interleukin-1β significantly increased isoprenaline-induced cAMP accumulation by increasing β2 adrenoreceptor numbers via a protein kinase A-dependent mechanism. In contrast, interleukin-1β significantly impaired prostaglandin E2-induced cAMP accumulation by induction of cyclo-oxygenase-2, prostaglandin E2 production, and a resulting down-regulation of adenylyl cyclase. The cAMP changes were all mirrored by alterations in chloride efflux assessed using the fluorescent chloride probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide with interleukin-1β increasing chloride efflux in response to isoprenaline and reducing the response to prostaglandin E2. Studies with glibenclamide confirmed that chloride efflux was via the cystic fibrosis transmembrane conductance regulator. Calu-3 expresses EP4 receptors, but not EP2, and receptor expression is reduced by interleukin-1β. Collectively, these results provide mechanistic insight into how interleukin-1β can differentially regulate cAMP generation and chloride efflux in response to different adenylyl cyclase-coupled ligands in the same cell. These findings have important implications for diseases involving inflammation and abnormal ion flux such as cystic fibrosis.

Prostanoids are major regulators of smooth muscle function that are generated by cyclooxygenase (COX). Here we hypothesized that cytokines and mediators that regulate the pulmonary circulation would alter COX expression and prostanoid generation in pulmonary artery smooth muscle cells. Bradykinin, transforming growth factor-β1 (TGF-β1), and interleukin-1β (IL-1β) increased inducible COX-2 expression and prostaglandin E2 (PGE2) release. Transfection studies using a COX-2 promoter construct demonstrated that all three agents acted transcriptionally. Constitutive COX-1 protein expression was unchanged. The COX inhibitor indomethacin, the COX-2 inhibitor NS-398, the protein synthesis inhibitor cycloheximide, and the glucocorticoid dexamethasone abrogated the increased PGE2levels. Dexamethasone and cycloheximide prevented COX-2 induction. Hypoxia (3% O2-5% CO2-92% N2) for 24 h selectively augmented TGF-β1-stimulated PGE2 production and COX-2 induction but had no effect alone. Prolonged hypoxic culture alone for 48 and 72 h enhanced COX-2 induction and increased PGE2. These studies show that a number of stimuli are capable of inducing COX-2 in pulmonary artery smooth muscle cells. The interaction between hypoxia and TGF-β1 may be particularly relevant to pulmonary hypertension.

Interleukin (IL)-8, the C-X-C chemokine, is a potent neutrophil chemoattractant that has been implicated in a number of inflammatory airway diseases such as cystic fibrosis. Here we tested the hypothesis that bradykinin, an inflammatory mediator and chloride secretagogue, would increase IL-8 generation in airway epithelial cells through autocrine generation of endogenous prostanoids. Bradykinin increased IL-8 generation in both a non-cystic fibrosis (A549) and cystic fibrosis epithelial cell line (CFTE29[Formula: see text]) that was inhibited by the nonselective cyclooxygenase (COX) inhibitor indomethacin and the COX-2 selective inhibitor NS-398. COX-2 was the only isoform of COX expressed in both cell lines. Furthermore, the COX substrate arachidonic acid and exogenous prostaglandin E2 both increased IL-8 release in A549 cells. These results suggest that bradykinin may contribute to neutrophilic inflammation in the airway by generation of IL-8 from airway epithelial cells. The dependence of this response on endogenous production of prostanoids by COX-2 suggests that selective COX-2 inhibitors may have a role in the treatment of airway diseases characterized by neutrophilic inflammation such as cystic fibrosis or chronic obstructive pulmonary disease.

Bronchial vascular remodeling is an important feature of the pathology of chronic asthma, but the responsible mechanisms and main sources of angiogenic factors are unclear. Here we report that human airway smooth muscle cells express vascular endothelial growth factor (VEGF)121, 165, 189, 206 splice variants and secrete VEGF protein constitutively. VEGF protein secretion was increased by the proinflammatory asthma mediator bradykinin through post-transcriptional mechanisms. Bradykinin-induced VEGF secretion was dependent on the B2 bradykinin receptor, activation of protein kinase C, and generation of endogenous prostanoids. This is the first report that bradykinin can increase VEGF secretion in any biological system and the first to show that airway smooth muscle cells produce VEGF. Our results suggest a novel role for human airway smooth muscle in contributing to bronchial mucosal angiogenesis in chronic asthma by secretion of VEGF and suggest a wider role for mesenchymal cell products in mediating angiogenesis in inflammatory and allergic diseases.

The majority of drugs, biological products, and devices used for children are done so off-label (ie, for age groups or indications that have not been approved by the Food and Drug Administration). Off-label use of drugs in neonates is particularly common, which makes caring for these children particularly difficult for physicians. The American Academy of Pediatrics (AAP) and other groups have been working actively for decades with the FDA and Congress to correct this problem. Last year, we published an overview of the problems that off-label use of drugs pose for children of all ages and discussed what was being done to correct this situation (Pediatr Infect Dis J 17:739-744, 1998).1 The purpose of this report is 0 to focus on how inadequate pediatric drug information negatively impacts the ability of physicians to safely treat diseases in children, especially neonates, and to update the reader on the status of recent legislation and regulations that have been put in place to solve this problem. Although inadequate pediatric information affects all classes of drugs, biological products, and devices, specific examples in this report focus on the use of antimicrobial agents.

1 Airway smooth muscle (ASM) in human asthma shows reduced relaxation and cyclic AMP generation in response to β-adrenoceptor agonists. IL-β attenuates cyclic AMP generation but the underlying mechanism is unclear. We have reported that IL-1β induces cyclo-oxygenase-2 (COX-2) in human ASM cells and results in a marked increase in prostanoid generation with PGE2 and PGI2 as the major products. 2 We investigated the role of COX-2 induction and prostanoid release (measured as PGE2) in IL-1β induced attenuation of cyclic AMP generation in response to the β-adrenoceptor agonist isoprenaline (ISO). 3 Pre-treatment of human ASM cells with IL-1β significantly attenuated cyclic AMP generation in response to high concentrations of ISO (1.0–10.0 μm) in a time- and concentration-dependent manner. The effect was accompanied by a high concentration of PGE2 release. The non-selective COX inhibitor indomethacin (Ind), the selective COX-2 inhibitor NS-398, the protein synthesis inhibitors cycloheximide (CHX) and actinomycin D and the steroid dexamethasone (Dex) all abolished the PGE2 release and prevented the attenuated cyclic AMP generation. 4 COX substrate arachidonic acid time- and concentration-dependently mimicked IL-1β induced attenuation and the effect was prevented by the non-selective COX inhibitors Ind and flurbiprofen, but not by NS-398, CHX and Dex. 5 In contrast to IL-1β, TNFα and IFNγ, which are ineffective in inducing COX-2 and releasing PGE2 from human ASM cells, did not affect the cyclic AMP formation. 6 Our study demonstrates that COX-2 induction and the consequent release of prostanoids plays a crucial role in IL-1β induced attenuation of human ASM cell cyclic AMP response to ISO. British Journal of Pharmacology (1998) 125, 1320–1328; doi:10.1038/sj.bjp.0702193

Guanosine 3′:5′-cyclic monophosphate (cyclic GMP) is an important second messenger mediating the effects of nitric oxide (NO) and natriuretic peptides. Cyclic GMP pathways regulate several aspects of lung pathophysiology in a number of airway cells. The regulation of this system has not been extensively studied in pulmonary epithelial tissue. We have studied the production of cyclic GMP by suspensions of ovine tracheal epithelial cells in response to activators of soluble guanylyl cyclase (sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP) and particulate guanylyl cyclase (atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and E. coli heat stable enterotoxin (STa)). Both 10−710−3m and 10−710−3m SNAP generated a concentration-dependent marked elevation in cyclic GMP production when incubated with 10−3m 3-isobutyl-1-methylxanthine (IBMX) (both greater than 25×baseline values with highest drug concentration). The increase in production of cyclic GMP in response to 10−6m SNP and 10−5m SNAP was markedly inhibited by both 5×10−5m haemoglobin (102% and 92% inhibition) and 5×10−5m methylene blue (82% and 84% inhibition). The increase in cyclic GMP in response to 10−3m SNP was measured following co-incubation with the phosphodiesterase inhibitors 10−710−3m IBMX, 10−710−4m milrinone and 10−710−4m SKF 96231. Only 10−410−3m IBMX significantly increased cyclic GMP levels. Cyclic GMP production was also significantly elevated from baseline by 10−5m ANP, 10−5m BNP, 10−5m CNP and 200 iu ml−1 of E. coli STa toxin in the presence of 10−3m IBMX. Increases with these natriuretic peptides and STa toxin were smaller in magnitude (24 fold) than those seen with SNP and SNAP. CNP was the most potent of the natriuretic peptides studied suggesting type B membrane bound guanylate cyclase is the predominant form expressed. These results suggest that ovine tracheal epithelial cells contain active guanylyl cyclases. The more marked response to SNP and SNAP than to natriuretic peptides suggests that soluble guanylyl cyclase predominates. British Journal of Pharmacology (1997) 120, 1249–1254; doi:10.1038/sj.bjp.0701040

Prostaglandin E2 (PGE2) is thought to be an important inhibitory modulator of inflammatory processes in the airway. Previous studies have shown that it is produced by bovine cultured airway smooth muscle (ASM) cells in large quantities, but its regulation by second messengers has not been studied in this tissue. To determine whether PGE2 production by ASM might be an important action of beta-adrenoceptor agonists in asthma, the regulation of PGE2 production by adenosine 3′,5′-cyclic monophosphate (cAMP) was assessed using dibutyryl cAMP (DBcAMP), forskolin, and albuterol. DBcAMP increased PGE2 production over a 24-h time course. Forskolin and albuterol both increased PGE2 production over control cells to similar levels after 24 h. Incubation of albuterol-treated cells with propranolol significantly (70%) reduced the stimulatory effect of albuterol on PGE2 production. Incubation of forskolin-treated cells with Rp-cAMP, a cAMP antagonist, inhibited the PGE2 response evoked by forskolin by 80%. Ro-20–1724, a selective inhibitor of type IV phosphodiesterase, stimulated PGE2 production (P = 0.02). Cycloheximide, a protein-synthesis inhibitor, did not inhibit the response to DBcAMP. The effects of DBcAMP were additive with the effects of bradykinin, a proinflammatory mediator known to increase PGE2 production (P < 0.05). These studies suggest that cAMP may play an important regulatory role in stimulating PGE2 production by ASM. This may be a novel beneficial action of beta-adrenoceptor agonists in asthma.

Here we tested the effect of interleukin-1beta, a pro-inflammatory cytokine, on cAMP accumulation and chloride efflux in Calu-3 airway epithelial cells in response to ligands binding to adenylyl cyclase-coupled receptors such as the beta2 adrenoreceptor and EP prostanoid receptors. Interleukin-1beta significantly increased isoprenaline-induced cAMP accumulation by increasing beta2 adrenoreceptor numbers via a protein kinase A-dependent mechanism. In contrast, interleukin-1beta significantly impaired prostaglandin E2-induced cAMP accumulation by induction of cyclo-oxygenase-2, prostaglandin E2 production, and a resulting down-regulation of adenylyl cyclase. The cAMP changes were all mirrored by alterations in chloride efflux assessed using the fluorescent chloride probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide with interleukin-1beta increasing chloride efflux in response to isoprenaline and reducing the response to prostaglandin E2. Studies with glibenclamide confirmed that chloride efflux was via the cystic fibrosis transmembrane conductance regulator. Calu-3 expresses EP4 receptors, but not EP2, and receptor expression is reduced by interleukin-1beta. Collectively, these results provide mechanistic insight into how interleukin-1beta can differentially regulate cAMP generation and chloride efflux in response to different adenylyl cyclase-coupled ligands in the same cell. These findings have important implications for diseases involving inflammation and abnormal ion flux such as cystic fibrosis.

We have recently shown that endogenous prostanoids are critical in bradykinin-stimulated interleukin (IL)-8 release from human airway smooth muscle (ASM) cells. In this study, we tested the ability of transforming growth factor (TGF)-β1 to stimulate IL-8 release, cyclooxygenase (COX)-2 expression and PGE2 generation in cultured human ASM cells and explored the role of COX products and COX-2 induction on IL-8 release. TGF-β1 stimulated IL-8 release, COX-2 induction, and PGE2 generation in a concentration- and time-dependent manner. Maximal IL-8 release was achieved with 10 ng/ml of TGF-β1 after 16 h of incubation, which was inhibited by the transcription inhibitor actinomycin D and the corticosteroid dexamethasone but was not affected by the nonselective COX inhibitor indomethacin and the selective COX-2 inhibitor NS-398 despite their inhibition on TGF-β1-induced PGE2 release. These results show for the first time that TGF-β1 stimulates IL-8 release, COX-2 induction, and PGE2 generation in human ASM cells and that PGE2 generation is not critical for TGF-β1-induced IL-8 release. These findings suggest that TGF-β1 may play an important role in the pathophysiology of asthma.

Cyclo-oxygenase (COX) inhibitors may have a role in reducing inflammation in asthma and other pulmonary diseases. COX inhibitors have different selectivities for the two COX isoenzymes (COX 1 and COX 2) which vary between purified enzyme and intact cell preparations. The relative selectivity of COX inhibitors has not been studied in human airway cells. A number of COX inhibitors in cultured human airway cells were compared which exclusively express either COX 1 (primary degree cultured human airway smooth muscle (HASM) cells) or COX 2 (A549 pulmonary epithelial cell-line) as measured by Western blotting. COX activity was assayed by prostaglandin (PG)E2 production following 30 min incubation with 5 mM arachidonic acid. COX activity in both cell types was similar; HASM cells 92.2+/-12.1 ng PGE2 x mg-1 protein, A549 cells 87.7+/-24.4 ng PGE2 mg-1 protein. In HASM cells the median inhibitory concentration (IC50) was >10-5 M for nimesulide, 3.2 x 10-6 M for N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulphonamide (NS398), 1.8 x 10-8 M for flurbiprofen, 6.7 x 10-9 M for indomethacin and >10-5 M for aspirin. In A549 cells the IC50 was 1.8 x 10-9M for nimesulide, 4.1 x 10-9 M for NS398,6.2 x 10-10 M for flurbiprofen, 1.3 x 10-8 M for indomethacin and >10-5 M for aspirin. Sodium valerate had no effect in either HASM or A549 cells. The COX 2:COX 1 selectivity ratio (COX 2 IC50/COX I IC50) was

We previously showed that cultured human airway smooth-muscle cells (HASMC) contain soluble and particulate guanylyl cyclases (GCs). We studied the desensitization of soluble and particulate GCs in HASMC. Homologous desensitization of soluble GC occurred after incubation with S-nitroso-N-acetyl pencillamine (SNAP). SNAP-dependent desensitization was blocked by hemoglobin, a nitric oxide (NO) scavenger, suggesting that it was due to NO release. Cross-desensitization between SNAP and sodium nitroprusside (SNP) and the lack of thiol reduction after SNAP or SNP treatment suggested that thiol depletion was not involved. Assays for soluble GC activity and experiments using protein synthesis inhibitors suggested that SNAP-dependent desensitization was due to reduced soluble GC. Homologous desensitization of particulate GC occurred after pretreatment with atrial natriuretic peptide (ANP) accompanied by reduced particulate GC activity. Recovery required protein synthesis, suggesting that it was due to reduction in particulate GC. Homologous desensitization to either SNAP or ANP was not altered by phosphodiesterase (PDE) inhibitors, suggesting that increased PDE activity was not involved. Cross-desensitization experiments using SNAP and ANP and experiments using zaprinast to elevate cyclic guanosine monophosphate showed no evidence of heterologous desensitization. Our results suggest that pretreatment of HASMC with SNAP or ANP causes homologous, but not heterologous, desensitization of soluble and particulate GCs, respectively.

Interleukin (IL)-1β impairs human airway smooth muscle (ASM) cell cAMP responses to isoproterenol (Iso). We investigated if bradykinin (BK) could cause a similar effect and the role of cyclooxygenase (COX) products in this event, since we have recently reported that BK, like IL-1β, also causes COX-2 induction and prostanoid release in human ASM cells. BK pretreatment significantly attenuated Iso-induced cAMP generation in a time- and concentration-dependent manner. cAMP generation by prostaglandin (PG) E2but not by forskolin was also impaired. The COX inhibitor indomethacin completely prevented the impairment, whereas the selective COX-2 inhibitors NS-398 and nimesulide, protein synthesis inhibitors cycloheximide and actinomycin D, and steroid dexamethasone were all partially effective. The impairment was mimicked by the B2agonist [Tyr(Me)8]BK, the Ca2+ionophore A-23187, and PGE2and prevented by the B2antagonist HOE-140, but anti-IL-1β serum was ineffective. The results indicate that BK impairs human ASM cell responses to Iso, and the effect is largely mediated by B2receptor-related COX product release via both COX isoforms and is independent of IL-1β.

The basolateral Na+/K+ ATPase plays a critical role in sodium reabsorption across airway epithelium. Nasal epithelium shows increased Na+/K+ ATPase activity in cystic fibrosis (CF) but Na+/K+ ATPase has not been characterized in human lung epithelium or compared in CF and normal lung. We measured 3[H] ouabain binding and Na+/K+ ATPase activity in human tracheal epithelium and compared Na/K ATPase activity in bronchial epithelium in CF and control subjects. In tracheal epithelium Na+/K+ ATPase pumps were abundant and of high 3[H] ouabain affinity (Kd 4.7 nM, Bmax 38 pmol/mg) and Na+/K+ ATPase activity was 55 +/- 8 nmol/mg protein/min. Bronchial epithelial Na+/K+ ATPase activity was twofold higher in CF patients than in controls. The increased Na+/K+ ATPase activity may contribute to the increased sodium reabsorption seen in cystic fibrosis.

Although guanosine 3′,5′-cyclic monophosphate (cGMP) acts as a relaxant second messenger, the regulation of intracellular cGMP has not been comprehensively studied in human airway smooth muscle. We studied the production of cGMP by cultured human airway smooth muscle cells (HASMC) after stimulation with activators of soluble guanylyl cyclase [sodium nitroprusside (SNP) and S-nitroso- N-acetylpenicillamine (SNAP)] and particulate guanylyl cyclase [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and Escherichia coli heat stable enterotoxin (STa)]. cGMP was measured by enzyme-linked immunosorbent assay. Both SNP (10−6to 10−3M) and SNAP (10−6to 10−3M) caused concentration-dependent elevation of cGMP in the presence of the nonselective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (10−3M), with cGMP increasing 6- and 15-fold in response to SNP and SNAP, respectively, at the highest concentration tested (10−3M). The increases in cGMP in response to SNP (5 × 10−5M) and SNAP (10−5M) were inhibited by hemoglobin (Hb; 5 × 10−5M), a nitric oxide scavenger, and methylene blue (MB; 5 × 10−4M), an inhibitor of guanylyl cyclase. cGMP accumulation after SNAP was abolished by both Hb and MB. The response to SNP was inhibited by 79% with Hb and was abolished with MB. ANP, BNP, and CNP (10−9to 10−5M) + phosphoramidon (10−6M) caused a concentration-dependent elevation in cGMP with an order of potency ANP > BNP > CNP. cGMP formation in the presence of the highest concentration of the most potent natriuretic peptide (10−5M ANP) was two- to threefold greater than with the highest concentration of SNAP. The increase in cGMP seen with natriuretic peptides was similar in the presence or absence of phosphoramidon, a neutral endopeptidase (NEP) inhibitor, suggesting that NEP is not playing a role in modulating the effect of natriuretic peptides in HASMC. STa (400 IU/ml) had no effect on cGMP levels. SNAP- and ANP-induced cGMP accumulation was increased by the selective type V PDE inhibitors SKF-96231 and zaprinast, suggesting that type V PDE is responsible for cGMP breakdown in HASMC. These results suggest that cultured HASMC contain both soluble and particulate guanylyl cyclases. The order of potency of the natriuretic peptides ANP > BNP > CNP suggests that type A particulate membrane-bound guanylate cyclase predominates.