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Background and purpose Add-on therapies with the monoterpene 1,8-cineole were shown to be effective in controlled studies, on asthma and chronic obstructive pulmonary diseases (COPD) We hypothesized, that 1,8-cineole (C) can improve steroid effects when combined with guideline medications (budesonide (BUD), formoterol (F)) for asthma and COPD. Methods Peripheral blood monocytes of 12 healthy volunteers were isolated and stimulated (10 5 cells/ml) with LPS (10 μg/ml, 20 h) alone or in the presence of C, BUD, BUD + F and C+(BUD + F) in therapeutically relevant concentrations. Inflammatory mediators (IL-1β, IL-6, IL-8, TNF-α) were determined in supernatants by ELISA. Results 1,8-cineole (0.15–1.5 μM) concentration dependently inhibited the LPS induced cytokine releases (IL-6 > IL1β > IL-8 ≥ TNF-α) up to 100%; BUD inhibited the cytokine releases in lower concentrations (10 −9 –10 −5 M) and in a different sequence (IL-1β > TNF-α = IL-6 = IL-8) up to 80%. Relevant airway concentrations of 1,8-cineole (2 × 10 −6 M) increased the inhibitory effects of BUD (10 −9 M ≤ 28%). 1,8-cineole in concentrations ≥4 × 10 −6 M inhibited already alone releases of all cytokines between 80 and 100% without additional effects of budesonide. BUD (10- 11 M–10 −9 M) + F (10 −9 M) inhibited dose-dependently TNF-α and IL-1β releases up to 59 and 40%, respectively. Co-incubation with 1,8-cineole (4 × 10 −6 M) enhanced the inhibition of TNF-α releases between 15 and 33% and the one of IL-1β releases between 14 and 44.5% (p Conclusions These results suggest a new mode of action of 1,8-cineole as non-steroidal, anti-inflammatory agent. Its potential to suppress airway inflammation and to improve the efficacy of inhaled steroids in COPD and asthma should be clinically further investigated.

Background and Purpose Artificial agonists may have higher efficacy for receptor activation than the physiological agonist. Until now, such ‘superagonism’ has rarely been reported for GPCRs. Iperoxo is an extremely potent muscarinic receptor agonist. We hypothesized that iperoxo is a ‘superagonist’. Experimental Approach Signalling of iperoxo and newly synthesized structural analogues was compared with that of ACh at label-free M2 muscarinic receptors applying whole cell dynamic mass redistribution, measurement of G-protein activation, evaluation of cell surface agonist binding and computation of operational efficacies. Key Results In CHO-hM2 cells, iperoxo significantly exceeds ACh in Gi/Gs signalling competence. In the orthosteric loss-of-function mutant M2-Y1043.33A, the maximum effect of iperoxo is hardly compromised in contrast to ACh. ‘Superagonism’ is preserved in the physiological cellular context of MRC-5 human lung fibroblasts. Structure–signalling relationships including iperoxo derivatives with either modified positively charged head group or altered tail suggest that ‘superagonism’ of iperoxo is mechanistically based on parallel activation of the receptor protein via two orthosteric interaction points. Conclusion and Implications Supraphysiological agonist efficacy at muscarinic M2 ACh receptors is demonstrated for the first time. In addition, a possible underlying molecular mechanism of GPCR ‘superagonism’ is provided. We suggest that iperoxo-like orthosteric GPCR activation is a new avenue towards a novel class of receptor activators. Linked Article This article is commented on by Langmead and Christopoulos, pp. 353–356 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12142

Asthma and chronic obstructive pulmonary disease (COPD) are obstructive lung diseases characterized by airway obstruction, airway inflammation and airway remodelling. Next to inflammatory cells and airway epithelial cells, airway mesenchymal cells, including airway smooth muscle cells and (myo)fibroblasts, substantially contribute to disease features by the release of inflammatory mediators, smooth musde contraction, extracellular matrix deposition and structural changes in the airways. Current pharmacological treatment of both diseases intends to target the dynamic features of the endogenous intracellular suppressor cyclic AMP (cAMP). This review will summarize our current knowledge on cAMP and will emphasize on key discoveries and paradigm shifts reflecting the complex spatio-temporal nature of compartmentalized cAMP signalling networks in health and disease. As airway fibroblasts and airway smooth muscle cells are recognized as central players in the development and progression of asthma and COPD, we will focus on the role of cAMP signalling in their function in relation to airway function and plasticity. We will recapture on the recent identification of cAMP-sensing multi-protein complexes maintained by cAMP effectors, including A-kinase anchoring proteins (AKAPs), proteins kinase A (PICA), exchange protein directly activated by cAMP (Epac), CAMP-elevating seven-transmembrane (7TM) receptors and phospho-diesterases (PDEs) and we will report on findings indicating that the pertubation of compartmentalized cAMP signalling correlates with the pathopysiology of obstructive lung diseases. Future challenges include studies on CAMP dynamics and compartmentalization in the lung and the development of novel drugs targeting these systems for therapeutic interventions in chronic obstructive inflammatory diseases. (C) 2012 Elsevier Inc. All rights reserved.

Background: ET-1 receptor antagonists are essential elements in the treatment of PAH suggesting a role of ET-1 in PAH pathogenesis. Since LFb are endowed with an endothelinergic system which drives pro-fibrotic processes (Ahmedat et al. Br. J. Pharmacol. (2013) 168: 471), ET-1 expression in LFb from patients with severe PAH was studied. Methods: LFb from patients with PAH obtained after lung transplantation, commercially available LFb from caucasian healthy donors (phLFb) (PromoCell) and MRC-5 cells (human LFb cell line) were cultured for 24 h in absence or presence of cycloheximide (CHX) and/or TGF-s 1 . Expression of ppET-1 mRNA was determined by qPCR. mRNA levels are expressed as n-fold over detection limit (20 PCR cycles over GAPDH; 2 20 -Δ Ct ). Results: ppET-1 mRNA levels in 1 st passage PAH LFb (94.495±27.947, mean±SEM) were markedly higher than in those phLFb (1.174±259) or MRC-5 cells (1.174±259). ppET-1 expression appears to be under inhibitory control of short-living regulatory peptides which can be removed by CHX (Ahmedat et al.). CHX enhanced ppET-1 mRNA in phLFb and MRC-5 cells by 4.284±996 and 2.794±373%, resp., but not in PAH LFb (118±13% of contols). TGF-β 1 (1ng/ml) increased ppET-1 mRNA in phLFb and MRC-5 cells by 1585±603 and 953±184% resp., but not in PAH LFb (107±18% of controls). During passaging of PAH LFb ppET-1 mRNA increased, 5fold in 3 rd passage. Conclusions: Loss of inhibitory control of ET-1 expression in PAH LFb resulting in marked over-expression of ET-1 may be involved in the pathogenesis of PAH. Epigenetic changes which appear to vanish during cell passaging could be an underlying mechanism.

Drug discovery strives for selective ligands to achieve targeted modulation of tissue function. Here we introduce engineered context-sensitive agonism as a postreceptor mechanism for tissue-selective drug action through a G protein-coupled receptor. Acetylcholine M2-receptor activation is known to mediate, among other actions, potentially dangerous slowing of the heart rate. This unwanted side effect is one of the main reasons that limit clinical application of muscarinic agonists. Herein we show that dualsteric (orthosteric/allosteric) agonists induce less cardiac depression ex vivo and in vivo than conventional full agonists. Exploration of the underlying mechanism in living cells employing cellular dynamic mass redistribution identified context-sensitive agonism of these dualsteric agonists. They translate elevation of intracellular cAMP into a switch from full to partial agonism. Designed context-sensitive agonism opens an avenue toward postreceptor pharmacologic selectivity, which even works in target tissues operated by the same subtype of pharmacologic receptor.

Dipeptidyl peptidase 4 (DPP4) is the target of the gliptins, a recent class of oral antidiabetics. DPP4 (also called CD26) was previously characterized in immune cells but also has important metabolic functions which are not yet fully understood. Thus, we investigated the function of DPP4 in human white preadipocytes and adipocytes. We found that both cell types express DPP4 in high amounts; DPP4 release markedly increased during differentiation. In preadipocytes, lentiviral DPP4 knockdown caused significant changes in gene expression as determined by whole-genome DNA-array analysis. Metabolic genes were increased, e.g. PDK4 18-fold and PPARγC1α (=PGC1α) 6-fold and proliferation-related genes were decreased (e.g. FGF7 5-fold). These effects, contributing to differentiation, were not inhibited by the PPARγ antagonist T0070907. Vice versa, the PPARγ agonist pioglitazone induced a different set of genes (mainly FABP4). DPP4 knockdown also affected growth factor signaling and, accordingly, retarded preadipocyte proliferation. In particular, basal and insulin-induced ERK activation (but not Akt activation) was markedly diminished (by around 60%). This indicates that DPP4 knockdown contributes to adipocyte maturation by mimicking growth factor withdrawal, an early step in fat cell differentiation. In mature adipocytes, DPP4 becomes liberated so that adipose tissue may constitute a relevant source of circulating DPP4.

Background and Purpose Since endothelin (ET) may act as pro-fibrotic mediator, expression and release of ET isoforms, their receptors and potential pro-fibrotic ET effects were studied in human lung fibroblasts. Experimental Approach MRC-5 and primary human lung fibroblasts (phLFb) were cultured. Expression of prepro-ET isoforms was determined by qPCR and release of ET-1 by elisa. ET receptor function was analysed by real-time measurement of dynamic mass redistribution (DMR). Incorporation of [3H]-thymidine was determined as measure of proliferation and that of [3H]-proline for collagen synthesis. Phospho-p42/44 MAP kinase was determined by Western blot. Key Results ET-1 is the predominant ET in human lung fibroblasts (hLF), and TGF-β caused a further, selective and sustained up-regulation of ET-1 resulting in increased extracellular ET-1 accumulation. hLFb express mRNA encoding ET-A and ET-B receptors. Expression of both receptors was confirmed at protein level. ET-1 induced marked DMR signals, an effect that involved ET-A and ET-B receptors. Stimulatory effects of ET-1 on hLFb proliferation and collagen synthesis were mediated exclusively via ET-A receptors. ET-1, again via ET-A receptors, induced rapid activation of ERK MAPK, shown to be a crucial cellular signal in ET-1-induced collagen synthesis. ET-1-induced activation of ERK and collagen synthesis was, in contrast to corresponding effect of a muscarinic agonist, largely insensitive to pertussis toxin. Conclusions and Implications hLFb are endowed with all elements necessary to build a functional autocrine/paracrine endothelinergic system, which appears to drive pro-fibrotic airway and lung remodelling processes, effects for which only ET-A, but not ET-B receptors appear to be of significance.

Oenema TA, Smit M, Smedinga L, Racke K, Halayko AJ, Meurs H, Gosens R. Muscarinic receptor stimulation augments TGF-beta(1)-induced contractile protein expression by airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 303: L589-L597, 2012. First published August 3, 2012; doi:10.1152/ajplung.00400.2011.-Acetylcholine (ACh) is the primary parasympathetic neurotransmitter in the airways. Recently, it was established that ACh, via muscarinic receptors, regulates airway remodeling in animal models of asthma and chronic obstructive pulmonary disease (COPD). The mechanisms involved are not well understood. Here, we investigated the functional interaction between muscarinic receptor stimulation and transforming growth factor (TGF)-beta(1) on the expression of contractile proteins in human airway smooth muscle (ASM) cells. ASM cells expressing functional muscarinic M-2 and M-3 receptors were stimulated with methacholine (MCh), TGF-beta(1), or their combination for up to 7 days. Western blot analysis revealed a strong induction of sm-alpha-actin and calponin by TGF-beta(1), which was increased by MCh in ASM cells. Immunocytochemistry confirmed these results and revealed that the presence of MCh augmented the formation of sm-alpha-actin stress fibers by TGF-beta(1). MCh did not augment TGF-beta(1)-induced gene transcription of contractile phenotype markers. Rather, translational processes were involved in the augmentation of TGF-beta(1)-induced contractile protein expression by muscarinic receptor stimulation, including phosphorylation of glycogen synthase kinase-3 beta and 4E-binding protein 1, which was enhanced by MCh. In conclusion, muscarinic receptor stimulation augments functional effects of TGF-beta(1) in human ASM cells on cellular processes that underpin ASM remodeling in asthma and COPD.

Aims The present study aimed to explore possible effects of endothelin-1 (ET-1) on s 2 -adrenoceptor gene transcription in human lung fibroblasts. Main methods MRC-5 human lung fibroblasts were cultured in absence or presence of test substances, followed by s 2 -adrenoceptor mRNA determination by quantitative real time PCR. Key findings ET-1 caused a marked and rapid in onset (1 hr) increase in β 2 -adrenoceptor mRNA, an effect additive to that of short time (1 hr) exposure to the β 2 -adrenoceptor agonist olodaterol. The stimulatory effect of ET-1 on β 2 -adrenoceptor mRNA was prevented by the non-selective ET-A/ET-B receptor antagonist bosentan, indicating that it was mediated via specific ET receptors. In the presence of actinomycin D the effect of ET-1 was prevented indicating that ET-1 acts via increased transcription of the β 2 -adrenoceptor gene. ET-1-induced up-regulation of β 2 -adrenoceptor mRNA was also seen in the presence of cycloheximide excluding indirect effects via up-regulation of other regulatory proteins. Conclusions ET-1 can up-regulate β-adrenoceptor gene transcription in human lung fibroblasts.

Human lung fibroblasts are a potential source of endothelin-1 (ET-1), a pro-fibrotic mediator. The present study explored possible muscarinic and β-adrenergic modulations of ET-1 expression in human lung fibroblasts. MRC-5 human lung fibroblasts were cultured. Expression of prepro-endothelin-1 (ppET-1) mRNA was determined by quantitative real time PCR. [(3)H]-Proline incorporation was determined as measure of collagen synthesis. The muscarinic agonist oxotremorine induced, in a tiotropium-sensitive manner, a three-fold increase in ppET-1 mRNA. The β(2)-adrenoceptor agonist olodaterol caused a reduction of ppET-1 mRNA by 45%. Olodaterol also opposed the stimulatory effect of oxotremorine. The effect of olodaterol was mimicked by the protein kinase A agonist 6-Bnz-cAMP, whereas the Epac (exchange protein activated by cAMP) agonist 8-CPT-2'-O-Me-cAMP was less effective. Transforming growth factor-β(1) (TGF-β, 0.3 and 1 ng/ml) induced a three- and eight-fold increase in pp-ET-1 mRNA, respectively. Olodaterol opposed the effect of 0.3, but not that of 1 ng/ml TGF-β. Likewise, 6-Bnz-cAMP opposed the effect of 0.3, but not that of 1 ng/ml TGF-β. TGF-β inhibited β(2)-adrenoceptor mRNA expression, maximally by 90%. Muscarinic agonist-induced stimulation of [(3)H]-proline incorporation was attenuated by the endothelin ET1 receptor antagonist bosentan. In conclusion, ET-1 expression in human lung fibroblasts is regulated by stimulatory muscarinic receptors and inhibitory β(2)-adrenoceptors. Since muscarinic up-regulation of ET-1 contributes to pro-fibrotic effects of muscarinic stimuli, inhibition of ET-1 expression could contribute to long-term beneficial effects of long-acting β(2)-adrenoceptor agonists and long-acting muscarinic antagonists. However, excessive exposure to TGF-β results in loss of β-adrenoceptor expression and function of its down-stream signaling.

Tiotropium is commonly used in the treatment of chronic obstructive pulmonary disease. Although largely considered to be a long-acting bronchodilator, its demonstrated efficacy in reducing the frequency of exacerbations and preliminary evidence from early studies indicating that it might slow the rate of decline in lung function suggested mechanisms of action in addition to simple bronchodilation. This hypothesis was examined in the recently published UPLIFT study and, although spirometric and other clinical benefits of tiotropium treatment extended to four years, the rate of decline in lung function did not appear to be reduced by the addition of tiotropium in this study. This article summarizes data from a variety of investigations that provide insights into possible mechanisms to account for the effects of tiotropium. The report summarizes the discussion on basic and clinical research in this field. (c) 2009 Elsevier Ltd. All rights reserved.

Clinical observations indicate that in chronic obstructive pulmonary disease patients, the long-acting muscarinic antagonist tiotropium delays decline in airway function, suggesting that cholinergic mechanisms contribute to long-term structural changes. Human lung fibroblasts express muscarinic receptors and the present study aimed to explore their role in controlling collagen synthesis. MRC-5, HEL-299 and primary human lung fibroblasts (phLFb) were cultured. Incorporation of [(3)H]-proline into cellular proteins was determined as measure of collagen synthesis. In MRC-5 cells, the muscarinic agonist carbachol enhanced [(3)H]-proline incorporation in a concentration-dependent manner (effective concentration of 50%: 220 nM, increase at 10 microM by 40-55%, in a different series of experiments). Likewise, 10 microM oxotremorine caused an increase of approximately 65%. For comparison, transforming growth factor-beta1 (5 ng x mL(-1)) caused an increase of approximately 80%. Effects of carbachol on total [(3)H]-proline incorporation and collagenase-sensitive [(3)H]-proline fraction were similar. The effect of 10 microM carbachol was inhibited by tiotropium (inhibitory concentration of 50%: 110 pM), prevented by pertussis toxin and the mitogen-activated protein kinase inhibitor, PD 98059. Muscarinic agonists also enhanced [(3)H]-proline incorporation in a tiotropium-sensitive manner in HEL-299 cells and phLFb. In human lung fibroblasts, muscarinic receptors exert stimulatory effects on collagen synthesis. Prolonged blockade of muscarinic-induced collagen synthesis may contribute to reported beneficial long-term effects of anticholinergics in chronic obstructive pulmonary disease.

Insulin plays an important role as a growth factor and its contribution to tumor proliferation is intensely discussed. It acts via the cognate insulin receptor (IR) but can also activate the IGF1 receptor (IGF1R). Apart from increasing proliferation, insulin might have additional effects in lung cancer. Therefore, we investigated insulin action and effects of IR knockdown (KD) in three (NCI-H292, NCI-H226 and NCI-H460) independent non-small cell lung cancer (NSCLC) cell lines. All lung cancer lines studied were found to express IR, albeit with marked differences in the ratio of the two variants IR-A and IR-B. Insulin activated the classical signaling pathway with IR autophosphorylation and Akt phosphorylation. Moreover, activation of MAPK was observed in H292 cells, accompanied by enhanced proliferation. Lentiviral shRNA IR KD caused strong decrease in survival of all three lines, indicating that the effects of insulin in lung cancer go beyond enhancing proliferation. Unspecific effects were ruled out by employing further shRNAs and different insulin-responsive cells (human pre-adipocytes) for comparison. Caspase assays demonstrated that IR KD strongly induced apoptosis in these lung cancer cells, providing the physiological basis of the rapid cell loss. In search for the underlying mechanism, we analyzed alterations in the gene expression profile in response to IR KD. A strong induction of certain cytokines (e.g. IL20 and tumour necrosis factor) became obvious and it turned out that these cytokines trigger apoptosis in the NSCLC cells tested. This indicates a novel role of IR in tumor cell survival via suppression of pro-apoptotic cytokines.

Airway remodeling is a structural alteration associated with chronic inflammatory and obstructive airway diseases, wherein fibroblasts are crucially involved. The present study investigates whether lung fibroblast proliferation is influenced by muscarinic mechanisms. For this purpose, expression of muscarinic receptors in MRC-5 human lung fibroblasts was characterized by semiquantitative RT-PCR, and the effects of muscarinic agonists and antagonists on ((3)H)-thymidine incorporation as a measure of proliferative activity were studied under different culture conditions. MRC-5 fibroblasts express mRNA encoding different subtypes of muscarinic receptors (M(2)M(3)M(4), traces for M(5) and no M(1)). Expression of M(2) and M(3) receptors was confirmed at the protein level by immunoblot analysis. Under different culture conditions, carbachol (up to 10 microM) or oxotremorine (10 microM) stimulated ((3)H)-thymidine incorporation, with maximum increases between about 40 and 100%. The stimulatory effect of 10 microM carbachol was prevented by pretreatment with pertussis toxin and antagonized in a concentration-dependent manner by the muscarinic receptor antagonists tiotropium, AQ-RA 741, AF-DX 384, 4-diphenylacetoxy-N-methylpiperidine methoiodide, himbacine, p-fluorohexahydrosiladifenidol, and pirenzepine, with concentrations producing 50% inhibition of 14 pM, 24, 64, 127, 187, 452 nM, and 1.5 microM, respectively. Primary human lung fibroblasts were also found to express mRNA for muscarinic receptors (M(2)M(1)M(3), traces for M(4) and no M(5)), and showed a pertussis toxin-sensitive proliferative response to muscarinic receptor stimulation. In conclusion, proliferation of human lung fibroblasts can be stimulated by activation of muscarinic receptors with a pharmacologic profile correlating best to M(2) receptors.

In the respiratory tract acetylcholine is neurotransmitter in ganglia and postganglionic parasympathetic nerves, but in addition is paracrine mediator released from various non-neuronal cells. Almost every cell type present in the respiratory tract expresses nicotinic and muscarinic receptors and therefore appears to be a target for acetylcholine. The present review describes the mechanisms of synthesis and release of acetylcholine from neuronal and non-neuronal cells and the differential control mechanisms. The different cholinoceptors, multiple nicotinic and muscarinic receptors and their signalling are outlined and their involvement in the modulation of the function of various target cells, smooth muscles, nerves, surface epithelial, secretory cells, fibroblasts and inflammatory cells is discussed in detail.

Background/Aims: Fexofenadine HCl (FEX) has previously been shown to have anti-inflammatory properties in relieving nasal congestion in allergic rhinitis. The objective of this study was to further elucidate the mechanism of action behind the anti-inflammatory properties of FEX in addition to its H1-receptor antagonism. Methods: The effects of two antihistamines, FEX and loratadine (LOR), were investigated on cyclooxygenase (COX)-1 and -2 enzymesin vitro. FEX (10–9–10–3 mol/l) and LOR (10–9–10–4 mol/l) were incubated with arachidonic acid in a COX screening assay with either ovine COX-1 or COX-2 or human COX-2. COX-2 enzyme inhibitory activity for the antihistamines was compared with the known selective COX-2 inhibitor DuP-679. Results: High concentrations of FEX (10–3 mol/l) significantly inhibited arachidonic acid-mediated ovine COX-1 activity, but low concentrations had no effect. Low concentrations of FEX (10–8 mol/l) inhibited ovine COX-2 activity, and this inhibition decreased with increasing concentrations. The inhibition of COX-2 activity by FEX was similar to that seen with the selective COX-2 inhibitor, DuP-679. Conversely, LOR inhibited COX-1 activity at low concentrations (10–8 mol/l), but had little inhibitory effect on COX-1 at high concentrations. LOR (10–5 mol/l) markedly stimulated COX-2 activity. Conclusion: FEX showed selective arachidonic acid-mediated COX-2 inhibitory enzyme activity, which differed markedly from the COX inhibitory enzyme activity of LOR. This selective COX-2 inhibitor activity by FEX may contribute to its anti-inflammatory properties in relieving nasal congestion in allergic rhinitis.

Airway nitric oxide concentrations are reduced in cystic fibrosis (CF). Arginases compete for L-arginine, the substrate of nitric oxide synthesis.We hypothesized that increased arginase activity may be one factor contributing to nitric oxide deficiency in CF.We therefore studied sputum arginase activity, exhaled nitric oxide, and pulmonary function in patients with cystic fibrosis.Mean (+/- SEM) sputum arginase activity was significantly higher in patients admitted for pulmonary exacerbation compared with patients with stable disease (1.032 +/- 0.148 vs. 0.370 +/- 0.091 U/mg protein, p = 0.004). Fourteen days of intravenous antibiotic treatment resulted in significantly decreased sputum arginase activity in all patients (p = 0.0002). However, arginase activity was still significantly (p = 0.0001) higher in CF sputum after treatment for exacerbation compared with induced sputum from healthy control subjects (0.026 +/- 0.006 U/mg protein). Negative correlations were found for sputum arginase activity at admission with FEV1 (r = -0.41, p = 0.01), as well as changes in arginase activity with percent change in FEV1 during antibiotic therapy (r = -0.4, p0.01) in CF. Exhaled nitric oxide in CF was positively correlated to FEV1 (r = 0.34, p = 0.007), and in patients admitted for pulmonary exacerbation negatively correlated to sputum arginase activity (r = -0.45, p = 0.03).These data suggest that increased sputum arginase activity contributes to nitric oxide deficiency in CF lung disease and may be relevant in the pathogenesis of CF airway disease.

The relief of nasal congestion with the antihistamine fexofenadine in seasonal allergic rhinitis is thought to be due to its additional anti-inflammatory properties. The objective of this study was to evaluate the in vitro effects of fexofenadine on stimulated arachidonic acid metabolism. Human monocytes, isolated from blood and donated by 5 healthy volunteers, were either incubated for 20 h with 10 µg/ml lipopolysaccharide, with and without fexofenadine (10–8–10–3 mol/l, n = 8–19), or were incubated for 20 h, with and without fexofenadine, and then stimulated with 0.5 mg/ml zymosan for 2 h. Leukotriene B4 (LTB4), LTC4, LTD4 and LTE4, prostaglandin E2 (PGE2) and F2α(PGF2α) production was determined by enzyme immunoassay. Zymosan-stimulated production of LTC4, LTD4 and LTE4 was significantly inhibited by clinically relevant concentrations of fexofenadine HCl: 10–7mol/l (22% inhibition vs. control, p = 0.008) and 10–6 mol/l (24% inhibition vs. control, p = 0.020). Higher concentrations of fexofenadine (10–4 and 10–3 mol/l) inhibited LTB4 generation. Lipopolysaccharide-stimulated production of PGE2 was significantly inhibited by fexofenadine HCI 10–6 mol/l (26% inhibition, p = 0.035) and 10–5 mol/l (40% inhibition, p = 0.001). Higher concentrations of fexofenadine HCI (10–4 and 10–3 mol/l) significantly inhibited PGF2α production by 50% (p = 0.026) and 63% (p = 0.001), respectively. Fexofenadine, at both clinically relevant and higher concentrations, inhibits LTC4, LTD4, LTE4 and PGE2 in cultured human monocytes. These additional anti-inflammatory properties may underlie the relief of nasal congestion observed in clinical studies.

Arginase appears to play a significant role in the pathogenesis of inflammatory and obstructive airway diseases by interfering with NO synthesis (hyperreactivity) and by providing substrate for collagen synthesis (remodelling). IL-4 and IL-13 are key proinflammatory cytokines in asthma, and their effects on arginase in rat primary airway fibroblasts in culture were studied. Airway fibroblasts showed significant arginase activity, which was higher when the culture medium contained 10% fetal calf serum (FCS) (20 mU/mg protein) compared to 5% FCS (6 mU/mg protein). In presence of 10% FCS addition of IL-4 or IL-13 (10 ng/ml each) for 20 h or 40 h caused an increase in arginase activity by 76% and 160% (IL-4) and by 134% and 213% (IL-13), respectively. Using RT-PCR mRNA for arginase I was clearly detectable with 30 PCR cycles, whereas mRNA for arginase II was hardly detectable with 35 PCR cycles. IL-4 and IL-13 caused a clear increase in the mRNA of both arginase isoenzymes. Dexamethasone (1 microM) did not affect basal arginase activity, but largely opposed the stimulatory effects of IL-4 and IL-13. In conclusion, IL-4 and IL-13 can cause a marked up-regulation of arginase activity in rat airway fibroblasts and these effects can be inhibited by glucocorticoids.

By screening nucleotide databases, sequences containing the complete genes of the human cationic amino acid transporters (hCATs) 1, 2 and 4 were identified. Analysis of the genomic organization revealed that hCAT-2 consists of 12 translated exons and most likely of 2 untranslated exons. The splice variants hCAT-2A and hCAT-2B use exon 7 and 6, respectively. The hCAT-2 gene structure is closely related to the structure of hCAT-1, suggesting that they belong to a common gene family. hCAT-4 consists of only 4 translated exons and 3 short introns. Exons of identical size and highly homologous to exon 3 of hCAT-4 are present in hCAT-1 and hCAT-2.

It was tested whether the inducible nitric oxide synthase (iNOS) pathway might be involved in lipopolysaccharides-(LPS)-induced up-regulation of L-arginine transport in rat alveolar macrophages (AM). AM were cultured in absence or presence of LPS. Nitrite accumulation was determined in culture media and cells were used to study [3H]-L-arginine uptake or to isolate RNA for RT - PCR. Culture in presence of LPS (1 microg ml(-1), 20 h) caused 11 fold increase of nitrite accumulation and 2.5 fold increase of [3H]-L-arginine uptake. The inducible NO synthase (iNOS) inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) present alone during culture had only marginal effects on [3H]-L-arginine uptake. However, AMT present during culture additionally to LPS, suppressed LPS-induced nitrite accumulation and LPS-stimulated [3H]-L-arginine uptake in the same concentration-dependent manner. AMT present only for the last 30 min of the culture period had similar effects on [3H]-L-arginine uptake. AMT present only during the uptake period also inhibited LPS-stimulated [3H]-L-arginine uptake, but with lower potency. The inhibitory effect of AMT could not be opposed by the NO releasing compound DETA NONOate. LPS caused an up-regulation of the mRNA for the cationic amino acid transporter CAT-2B, and this effect was not affected by AMT. AMT (100 microM) did not affect L-arginine transport studied by electrophysiological techniques in Xenopus laevis oocytes expressing either the human cationic amino acid transporter hCAT-1 or hCAT-2B. In conclusion, iNOS inhibition in rat AM abolished LPS-activated L-arginine uptake. This effect appears to be caused by reduced flow of L-arginine through the iNOS pathway.

As arginase by limiting nitric oxide (NO) synthesis may play a role in airway hyperresponsiveness and glucocorticoids are known to induce the expression of arginase I in hepatic cells, glucocorticoid effects on arginase in alveolar macrophages (AMΦ) were studied. Rat AMΦ were cultured in absence or presence of test substances. Thereafter, nitrite accumulation, arginase activity, and the expression pattern of inducible NO synthase, arginase I and II mRNA (RT – PCR) and proteins (immunoblotting) were determined. Lipopolyssacharides (LPS, 20 h) caused an about 2 fold increase in arginase activity, whereas interferon-γ (IFN-γ), like LPS a strong inducer of NO synthesis, had no effect. Dexamethasone decreased arginase activity by about 25% and prevented the LPS-induced increase. Mifepristone (RU-486) as partial glucocorticoid receptor agonist inhibited LPS-induced increase by 45% and antagonized the inhibitory effect of dexamethasone. Two different inhibitors of the NF-κB-pathway also prevented LPS-induced increase in arginase activity. Rat AMΦ expressed mRNA and protein of arginase I and II, but arginase I expression was stronger. Arginase I mRNA and protein was not affected by IFN-γ, but increased by LPS and this effect was prevented by dexamethasone. Both, LPS and IFN-γ enhanced the levels of arginase II mRNA and protein, effects also inhibited by dexamethasone. As IFN-γ did not affect total arginase activity, arginase II may represent only a minor fraction of total arginase activity. In rat AMΦ glucocorticoids inhibit LPS-induced up-regulation of arginase activity, an effect which may contribute to the beneficial effects of glucocorticoids in the treatment of inflammatory airway diseases. British Journal of Pharmacology (2001) 132, 1349–1357; doi:10.1038/sj.bjp.0703951

The role of lipid mediators derived from membrane glycerophospholipids and sphingolipids as intracellular messenger has been studied intensively during the last two decades, but with the recent discovery of high affinity G-protein coupled receptors for the lysophospholipids lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), increasing attention has been paid to the role of these lipid mediators as extracellular mediators. This review will summarize the biosynthesis and metabolism of lysophospholipids and describe the family of endothelial differentiation gene (EDG) receptors as high affinity receptors for lysophospholipids. Furthermore, an overview of the numerous biological effects of lysophospholipids which might be mediated by EDG receptors will be given together with an outlook on the potential role of such mechanisms in pulmonary physiology and pathophysiology.

Alveolar macrophages (AMsmall ef, Cyrillic) express considerable arginase activity which can be modulated by various mediators. As inhibitors of phosphodiesterase (PDE) play an increasing role in the treatment of chronic inflammatory and obstructive airway disease, we tested whether PDE inhibitors affect arginase activity in AMsmall ef, Cyrillic. Isolated rabbit AMsmall ef, Cyrillic were cultured for 20 h in the absence or presence of bacterial lipopolysaccharides (LPS) and/or different test substances. Thereafter arginase activity was determined by measuring the formation of [(3)H]-L-ornithine during 1 h incubation with [(3)H]-L-arginine. Lipopolysaccharide-enhanced (0. 01-5 microg/ml) maximal arginase activity by about 2.5-fold. The non-selective PDE inhibitor IBMX and the PDE4 selective inhibitor rolipram (each up to 30 microM) caused a 2.4-fold increase in arginase activity, and these effects were additive to those of LPS. The PDE3-selective inhibitor siguazodan had only marginal effects. Forskolin (10 microM) also enhanced arginase activity in the absence and presence of LPS. The effect of forskolin was almost prevented by cycloheximide (30 microM) and largely attenuated by the protein kinase A inhibitor KT 5720 (300 nM). In conclusion, inhibition of the cAMP-specific PDE4, like direct activation of adenylyl cyclase, causes an up-regulation of arginase activity in rabbit AMsmall ef, Cyrillic.

Eosinophil-derived cationic proteins play an essential role in the pathogenesis of bronchial asthma. We tested whether cationic proteins interfere with the cationic amino-acid transport in alveolar macrophages (AMPhi) and tracheal epithelial cells, and whether L-arginine-dependent pathways were affected. The effect of cationic polypeptides on cellular uptake of [(3)H]-L-arginine, nitrite accumulation, and the turnover of [(3)H]-L-arginine by nitric oxide (NO) synthase and arginase (formation of [(3)H]-L-citrulline and [(3)H]-L-ornithine, respectively) were studied. Poly-L-arginine reduced [(3)H]-L-arginine uptake in rat AMPhi and tracheal epithelial cells in a concentration-dependent manner (at 300 microgram/ml by 70%). Poly-L-lysine, protamine, and major basic protein (each up to 300 microgram/ml) tested in rat AMPhi inhibited [(3)H]-L-arginine uptake by 35 to 50%. During 6 h incubation in amino acid-free Krebs solution, rat AMPhi, precultured in the absence or presence of LPS (1 microgram/ml), accumulated 1.4 and 3.5 nmol/10(6) cells nitrite, respectively. Addition of 100 microM L-arginine increased nitrite accumulation by 70 and 400% in control and lipopolysaccharide-treated AMPhi, respectively. Nitrite accumulation in the presence of L-arginine was reduced by poly-L-arginine and poly-L-lysine (100 and 300 microgram/ml) by 60 to 85% and 20 to 30%, respectively. Poly-L-arginine, but not poly-L-lysine, inhibited nitrite accumulation already in the absence of extracellular L-arginine. Poly-L-arginine (300 microgram/ml) inhibited [(3)H]-L-citrulline formation by AMPhi stronger than that of [(3)H]-L-ornithine. We conclude that cationic proteins can inhibit cellular transport of L-arginine and this can limit NO synthesis. Poly-L-arginine inhibits L-arginine uptake more effectively than other cationic proteins and exerts additional direct inhibitory effects on NO synthesis.

In the present study it was tested whether rat alveolar macrophages (AMphi) convert L-citrulline to L-arginine to maintain nitric oxide (NO) synthesis under conditions of limited availability of L-arginine. Rat AMphi (0.5 x 10(6) cells/well, cultured for 20 h in the absence or presence of 1 microg/ml lipopolysaccharides, LPS), were incubated for 6 h in amino acid-free Krebs solution and nitrite accumulation was determined as a measure of NO synthesis. After culture in the absence of LPS, nitrite in the incubation media was at the detection limit, independent of the addition of L-arginine or L-citrulline. AMphi, cultured in the presence of LPS, produced about 4 nmol per 10(6) cells and 6 h nitrite, and L-arginine enhanced nitrite accumulation in a concentration-dependent manner, maximally about threefold (EC50: 55 microM). In LPS-treated AMphi L-citrulline (up to 10 mM) failed to enhance nitrite accumulation, but rather inhibited it by about 50% in the presence of 100 microM L-arginine, i.e. when NO synthesis was enhanced. L-Arginine in the culture medium was 3H-labelled and its metabolism analysed by HPLC. In medium of AMphi exposed to LPS [3H]-L-arginine was reduced by about 60% after a 20-h culture period and this was almost balanced by an almost equal increase in [3H]-L-citrulline and [3H]-L-ornithine, i.e. L-arginine was markedly consumed. When [14C]-L-citrulline was added to the culture medium of AMphi, no significant formation of [14C]-L-arginine could be detected. On the other hand, argininosuccinate synthetase mRNA (by RT-PCR) and protein (by Western blot) was marginally detectable in control AMphi, but clearly induced after exposure to LPS. Finally, L-citrulline was shown to inhibit L-arginine uptake in a concentration dependent manner, by about 50% at 10 mM. In conclusion, although the expression of argininosuccinate synthetase in rat AMphi can be induced by LPS, AMphi appear not to be able to recycle significant amounts of L-citrulline to L-arginine to maintain sustained NO synthesis. On the contrary, at high concentrations L-citrulline can reduce NO synthesis, and this effect appears to be caused by inhibitory effects on L-arginine uptake.

1 The release of neuronal [3H]acetylcholine (ACh) from isolated human bronchi after labelling with [3H]choline was measured to investigate the effects of prostanoids. 2 A first period of electrical field stimulation (S1) caused a [3H]ACh release of 320±70 and 200±40 Becquerel (Bq) g−1 in epithelium-denuded and epithelium-containing bronchi respectively (P>0.05). Subsequent periods of electrical stimulation (Sn, n=2, 3, and 4) released less [3H]ACh, i.e. decreasing Sn/S1 values were obtained (0.76±0.09, 0.68±0.07 and 0.40±0.04, respectively). 3 Cumulative concentrations (1–1000 nM) of EP-receptor agonists like prostaglandin E2, nocloprost, and sulprostone (EP1 and EP3 selective) inhibited evoked [3H]ACh release in a concentration dependent manner with IC50 values between 4–14 nM and maximal inhibition of about 70%. 4 The inhibition of evoked [3H]ACh release by prostaglandin E2, nocloprost and sulprostone was not affected by the DP-, EP1- and EP2-receptor antagonist AH6809 at a concentration of 3 μM, i.e. a 3–30 times greater concentration than its affinity (pA2 values) at the respective receptors. 5 Circaprost (IP-receptor agonist; 1–100 nM), iloprost (IP- and EP1-receptor agonist; 10-1000 nM) and U-46619 (TP-receptor agonist; 100–1000 nM) did not significantly affect [3H]ACh release. 6 Blockade of cyclooxygenase by 3 μM indomethacin did not significantly modulate evoked [3H]ACh release in epithelium-containing and epithelium-denuded bronchi. Likewise, the combined cyclo- and lipoxygenase inhibitor BW-755C (20 μM) did not affect evoked [3H]ACh release. 7 In conclusion, applied prostanoids appear to inhibit [3H]ACh release in epithelium-denuded human bronchi under the present in vitro conditions, most likely via prejunctional prostanoid receptors of the EP3 subtype. British Journal of Pharmacology (1998) 125, 271–276; doi:10.1038/sj.bjp.0702057

1 The role of protein kinase C in controlling L-arginine transport in alveolar macrophages was investigated. 2 L-[3H]Arginine uptake in rabbit alveolar macrophages declined by 80 % after 20 h in culture. 4β-Phorbol 12-myristate 13-acetate (PMA), but not 4α-phorbol 12-myristate 13-acetate (α-PMA), present during 20 h culture, enhanced L-[3H]arginine uptake more than 10-fold. Staurosporine and chelerythrine opposed this effect. 3 L-[3H]Arginine uptake was saturable and blockable by L-lysine. After PMA treatment Vmax was increased more than 5-fold and Km was reduced from 0.65 to 0.32 mM. 4 Time course experiments showed that PMA increased L-[3H]arginine uptake almost maximally within 2 h. This short-term effect was not affected by cycloheximide or actinomycin D. 5 L-[3H]Arginine uptake and its stimulation by PMA was also observed in sodium-free medium. 6 L-Leucine (0.1 mM) inhibited L-[3H]arginine uptake by 50 % in sodium-containing medium, but not in sodium-free medium. At 1 mM, L-leucine caused significant inhibition in sodium-free medium also. L-Leucine showed similar effects on PMA-treated cells. 7 N-Ethylmaleimide (200 μm, 10 min) reduced L-[3H]arginine uptake by 70 % in control cells, but had no effect on PMA-treated (20 or 2 h) cells. 8 In alveolar macrophages, multiple transport systems are involved in L-arginine uptake, which is markedly stimulated by protein kinase C, probably by modulation of the activity of already expressed cationic amino acid transporters.

The cholinergic system in rat and human airways and the effects of glucocorticoids were investigated by assay of choline acetyltransferase activity, by high-pressure liquid chromatography measurement of acetylcholine, and by anti-choline acetyltransferase immunocyto-/histochemistry. Human bronchi were obtained at surgery from patients with lung cancer. Group 1 patients did not suffer from additional lung diseases and had not been treated with glucocorticoids. Group 2 patients, who suffered in addition to lung cancer from chronic obstructive bronchitis, had been treated for at least 6 weeks before surgery with four puffs of flusinolid daily. Isolated bronchial epithelial cells as well as intact surface epithelium of human bronchi expressed choline acetyltransferase immunoreactivity and choline acetyltransferase enzyme activity (3 +/- 1 nmol/mg protein per h). Ciliated epithelial cells showed strong choline acetyltransferase immunoreactivity at the basal body and the roolet of cilia. Surface epithelium in group 1 and 2 bronchi contained 23 +/- 6 (n = 14) and 1.8 +/- 0.3 pmol/g acetylcholine) (n = 7, P0.001), respectively, whereas the transmural acetylcholine content did not differ significantly between both groups. The amount of choline acetyltransferase immunoreactivity appeared similar in the surface epithelium of both groups. In an animal (rat) study the effects of oral dexamethasone (3 mg/day, 1 week) on choline acetyltransferase activity and acetylcholine levels were investigated. Dexamethasone treatment reduced epithelial acetylcholine in the airways and small intestine by about 80% and inhibited epithelial choline acetyltransferase activity. In conclusion, epithelial cells of human airways possess components of the cholinergic system, i.e., contain the synthesizing enzyme choline acetyltransferase and store acetylcholine. The data obtained from the animal study indicate that glucocorticoids can inhibit epithelial acetylcholine.

Human bronchi were incubated in organ baths to measure histamine release. The calcium ionophore A23187 (10 mumol/L; 1 min) stimulated histamine release by 148 +/- 28% (n = 11) above the prestimulation level but was ineffective in epithelium-denuded bronchi. Neither bradykinin (0.1 mumol/L) nor compound 48/80 (10 micrograms/ml) triggered the release of histamine from epithelium-intact bronchi. Acetylcholine did not affect spontaneous histamine release (about 2 nmol/g x 5 min) but inhibited A23187-evoked histamine release in an atropine-sensitive manner. Already a concentration as low as 0.1 nmol/L acetylcholine was effective, the maximal inhibition (by 89%) occurred at 100 nmol/L, whereas a concentration of 10 mumol/L acetylcholine was ineffective. Oxotremorine (1 nmol/L), a stable agonist at muscarinic receptors, suppressed stimulated histamine release completely. Physostigmine (0.1 mumol/L), an acetylcholinesterase inhibitor, reduced A23187-evoked histamine release by 58%. Antihuman IgE antibody stimulated histamine release by 127 +/- 30% (n = 6) above the prestimulation level. Acetylcholine (100 nmol/L) inhibited also the immunologically evoked histamine release by 70%. In conclusion, the present experiments provide a model to characterize mast cells that are localized in or close to the airway surface epithelium. Acetylcholine via muscarinic receptors strongly inhibits the releasability of these mucosal mast cells being among the first cells to interact with inhaled antigens and environmental agents. The inhibitory action of physostigmine indicates the involvement of endogenous, probably non-neuronal acetylcholine expressed in airway epithelial cells.

1. Alveolar macrophages (AM phi) exhibit arginase activity and may, in addition, express an inducible form of nitric oxide (NO) synthase (iNOS). Both pathways may compete for the substrate. L-arginine. The present study tested whether two recently described potent inhibitors of liver arginase (N omega-hydroxy-D,L-indospicine and 4-hydroxyamidino-D,L-phenylalanine) might also inhibit arginase in AM phi and whether inhibition of arginase might affect L-arginine utilization by iNOS. 2. AM phi obtained by broncho-alveolar lavage of rat and rabbit isolated lungs were disseminated (2.5 or 3 x 10(6) cells per well) and allowed to adhere for 2 h. Thereafter, they were either used to study [3H]-L-arginine uptake (37 kBq, 0.1 microM, 2 min) or cultured for 20 h in the absence or presence of bacterial lipopolysaccharide (LPS). Cultured AM phi were incubated for 1 h with [3H]-L-arginine (37 kBq, 0.1 microM) and the accumulation of [3H]-L-citrulline (NOS activity) and [3H]-L-ornithine (arginase activity) was determined. 3. During 1 h incubation of rabbit AM phi with [3H]-L-arginine, no [3H]-L-citrulline, but significant amounts of [3H]-L-ornithine (150 d.p.m x 1000) were formed. N omega-hydroxy-D,L-indospicine and 4-hydroxyamidino-D,L-phenylalanine, present during incubation, concentration-dependently reduced [3H]-L-ornithine formation (IC50: 2 and 45 microM, respectively). 4. N omega-hydroxy-D,L-indospicine (up to 100 microM) had no effect on [3H]-L-arginine uptake into rabbit AM phi, whereas 4-hydroxyamidino-D,L-phenylalanine caused a concentration-dependent inhibition (IC50: 300 microM). 5. Rat AM phi, cultured in the absence of LPS, formed significant amounts of [3H]-L-citrulline and [3H]-L-ornithine (133 and 212 d.p.m x 1000, respectively) when incubated for 1 h with [3H]-L-arginine. When AM phi had been cultured in the presence of 0.1 or 1 microgram ml-1 LPS, the formation of [3H]-L-citrulline was enhanced by 37 +/- 8.3 and 99 +/- 12% and that of [3H]-L-ornithine reduced by 21 +/- 8.7 and 70 +/- 2.5%, respectively. 6. In rat AM phi, cultured in the absence or presence of LPS, N omega-hydroxy-D,L-indospicine (10 and 30 microM) greatly reduced formation of [3H]-L-ornithine (by 80-95%) and this was accompanied by increased formation of [3H]-L-citrulline. However, only 20-30% of the [3H]-L-arginine not metabolized to [3H]-L-ornithine after inhibition of arginase was metabolized to [3H]-L-citrulline, when the AM phi had been cultured in the absence of LPS (i.e. low level of iNOS). On the other hand, when the AM phi had been cultured in the presence of LPS (i.e. high level of iNOS), all the [3H]-L-arginine not metabolized by the inhibited arginase was metabolized to [3H]-L-citrulline. 7. In conclusion, N omega-hydroxy-D,L-indospicine is a potent and specific inhibitor of arginase in AM phi. In cells in which, in addition to arginase, iNOS is expressed, inhibition of arginase can cause a shift of L-arginine metabolism to the NOS pathway. However, the extent of this shift appears to depend in a complex manner on the level of iNOS.

Effects of endogenous nitric oxide (NO) on the release of mediators of the lipoxygenase and cyclo-oxygenase pathway from rat alveolar macrophages were studied. Alveolar macrophages, freshly isolated or after 18-h culture, were incubated in (amino acid-free) Krebs medium and labelled with [3H]arachidonic acid. The release of [3H]leukotriene B4 and [3H]prostanoids (separated by high performance liquid chromatography) was determined. A 23187 was used as stimulus, as rising intracellular Ca2+ activates directly the phospholipase A2 and lipoxygenase pathway. A 23187 (10 microM) enhanced [3H]leukotriene B4 release from freshly prepared alveolar macrophages about 65-fold, but only 5- to 6-fold from cultured alveolar macrophages. Evoked [3H]leukotriene B4 release and spontaneous [3H]prostanoid release were inhibited when L-arginine (300 microM) was added to the Krebs incubation medium of alveolar macrophages, in which marked NO synthase had been induced by culture with lipopolysaccharides (10 microg/ml). Inhibitory effects of L-arginine were prevented by N(G)-monomethyl-L-arginine (L-NMMA, 100 microM). Inhibition of NO synthase during the culture period by L-NMMA (culture medium, in contrast to Krebs medium, already contains the substrate of NO synthase, L-arginine), resulted in attenuation of the 'culture-dependent' decline of the evoked release of [3H]leukotriene B4 and allowed lipopolysaccharides to cause an increase in spontaneous [3H]prostanoid release (i.e., to induce cyclo-oxygenase activity). In conclusion, in rat alveolar macrophages, endogenous NO appears to inhibit the release of mediators of the cyclo-oxygenase and lipoxygenase pathway through multiple sites of action.

1. Isolated tracheae of newborn rabbits were incubated in vitro and the outflow of 5-hydroxytryptamine (5-HT) was determined by h.p.l.c. with electrochemical detection. Evidence has previously been provided that this 5-HT outflow derives from neuroendocrine epithelial (NEE) cells of the airway mucosa. 2. Phenylephrine (1, 10 and 30 microM) enhanced the outflow of 5-HT by 80, 290 and 205%, respectively. 5-HT outflow evoked by 10 microM phenylephrine was not affected by the presence of the neurotoxin tetrodotoxin (1 microM). 3. Rauwolscine, ARC 239 (an alpha(2B)-adrenoceptor preferring antagonist), yohimbine and prazosin antagonized the effect of 10 microM phenylephrine in a concentration-dependent manner with IC50 values of 150, 295, 300 and 1,700 nM, respectively. Comparison of the ratios (between all antagonists) of the present IC50 values with the corresponding ratios of Ki values obtained in binding studies for the alpha(2A)-, alpha(2B)-, alpha(2C)- and alpha(2D)-adrenoceptor subtypes strongly suggests the involvement of an alpha(2B)-receptor. 4. 5-HT outflow evoked by 10 microM phenylephrine was inhibited by 65% in the presence of 1 microM forskolin and abolished in the presence of 10 microM forskolin. 5. 5-HT outflow evoked by 10 microM phenylephrine was inhibited by about 45 and 70% in the presence of 0.1 and 1 microM isoprenaline, respectively. The inhibitory effect of 1 microM isoprenaline was only marginally antagonized by 1 microM, but blocked by 10 microM propranolol. 6. 5-HT outflow was not affected by the muscarine receptor agonist oxotremorine (10 microM), but was enhanced by 175% by 100 microM nicotine. The effect of nicotine was blocked by 100 microM hexamethonium and prevented by 1 microM tetrodotoxin or 1 microM yohimbine. 7. In conclusion, 5-HT release from NEE cells of the rabbit trachea is stimulated via alpha-adrenoceptors most likely of the alpha(2B)-subtype localized directly at the NEE cells. Activation of beta-adrenoceptors as well as direct activation of adenylyl cyclase by forskolin exert inhibitory effects on 5-HT release. Activation of nicotinic, but not of muscarinic receptors, also evokes the release of 5-HT. However, the effect of nicotine appears to be mediated indirectly via the release of noradrenaline.

Stored endogenous acetylcholine (ACh) and in vitro synthesis of [3H]ACh were measured in isolated, mucosa-intact and mucosa-denuded airways of rat, guinea pig, and humans. In addition, choline acetyltransferase (ChAT) activity and ACh content were measured in freshly isolated airway mucosa as well as in cultured epithelial cells of rat tracheas. Rat tracheas stored 25 nmol/g ACh, whereas guinea pig tracheas and human bronchi contained only 2-3 nmol/g ACh. When incubated with [3H]choline, the isolated airways of rat, guinea pig, and human synthesized significant amounts of [3H]ACh. In guinea pig and human airways, removal of the mucosa affected neither stored ACh nor in vitro synthesis of [3H]ACh. In rat tracheas, however, removal of the mucosa resulted in a 50% reduction of stored ACh. Freshly isolated mucosa wiped off from the luminal surface of rat tracheas contained large amounts of ACh (6.5 nmol/g airway), whereas in human mucosa (central bronchi) only small amounts of ACh were found. In enzymatically isolated mucosal cells of rat tracheas, a considerable ChAT activity (21 nmol.mg protein 1.h-1) was detected, blockable by bromoacetylcholine. Enzymatically isolated human mucosa contained a rather low ChAT-like activity (0.5 nmol.mg protein 1.h-1), not sensitive to bromoacetylcholine. In cultured epithelial cells of rat tracheas (4th-6th passage), neither ChAT activity nor ACh was detected. The large airways of rat, guinea pig, and humans contain considerable amounts of ACh, supporting histological evidence of a dense cholinergic innervation, particularly of rat tracheas. The mucosa of rat tracheas synthesizes and stores large amounts of ACh, whereas the low ChAT activity in human mucosa argues against the presence of cholinergic neurons able to synthesize and store ACh.

Large amounts of 5-HT are present in the mammalian intestine where the amine is concentrated in the enterochromaffin cells (ECs) of the mucosa. ECs have the enzymes to synthesize 5-HT, are endowed with a specific, imipramine-sensitive 5-HT uptake mechanism and can store 5-HT in specific secretory vesicles. ECs can secrete 5-HT in a calcium-dependent manner. In particular, calcium influx through voltage-regulated channels and receptor-mediated liberation of intracellular calcium can evoke 5-HT release. 5-HT secretion from ECs occurs predominantly at the interstitial side and is controlled by a complex pattern of receptor-mediated mechanisms. Stimulatory receptors (beta-adrenoceptors, muscarine, nicotine and 5-HT3 receptors) and inhibitory receptors (alpha 2-adrenoceptors, histamine H3, GABAA- and GABAB-, A2 and P2y alpha purine and 5-HT4 receptors as well as receptors for vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase stimulating peptide (PACAP) and somatostatin) have been shown to be involved in the control of 5-HT release from the ECs.

Receptor-mediated regulation of acetylcholine release in the airways, particularly in humans, remains unclear. In the present study, the tissue content of acetylcholine and release of [3H]acetylcholine were measured in freshly dissected human bronchi obtained at thoracotomy. Large (main and lobar bronchi) and small (segmental and subsegmental bronchi) airways contained considerable amounts of endogenous acetylcholine (300 +/- 50 pmol/100 mg wet weight), whereas significantly less was found in lung parenchyma (60 +/- 30 pmol/100 mg). Isolated small bronchi incubated in an organ bath with the precursor [3H]choline synthesized significant amounts of [3H]acetylcholine (26,000 +/- 4,000 dpm/100 mg). Subsequent transmural stimulation (four 20 s trains at 15 Hz) of radiolabeled bronchi caused an enhanced tritium outflow that was abolished by removal of extracellular calcium or by tetrodotoxin. HPLC analysis of the medium collected before, during, and after transmural stimulation showed that the electrically stimulated tritium outflow represented exclusively [3H]acetylcholine, whereas the outflow of [3H]choline and [3H]phosphorylcholine was not affected by electrical stimulation. Oxotremorine (0.1 and 1 mumol/L) inhibited evoked [3H]acetylcholine release in a concentration-related manner, whereas atropine (0.03 mumol/L) enhanced evoked [3H]acetylcholine release. Inactivation of cyclooxygenase activity by 3 mumol/L of indomethacin did not impair the inhibitory effect of 0.1 or 1 mumol/L of oxotremorine. In conclusion, the present experiments indicate a considerable cholinergic innervation of human large and small airways.

The hypothesis was investigated that the nitric oxide (NO) synthase intermediate, NG-hydroxy-l-arginine (HOArg), is an arginase inhibitor in rabbit or rat alveolar macrophages. Exogenously applied HOArg strongly inhibited the arginase activity present in these cells (IC50 ≥ 15 μM), and attenuated l-[3H]arginine transport (IC50 ≥ 500 μM) in rabbit alveolar macrophages. Moreover, up to 37 μM HOArg were detected in the conditioned medium, but not in the lysate, of rat alveolar macrophages exposed to bacterial lipopolysaccharide for 18 h. HOArg may thus be a potent endogenous arginase inhibitor in these cells which increases the availability of l-arginine for NO biosynthesis.

Human lung fibroblasts are a potential source of endothelin-1 (ET-1), a pro-fibrotic mediator. The present study explored possible muscarinic and β-adrenergic modulations of ET-1 expression in human lung fibroblasts. MRC-5 human lung fibroblasts were cultured. Expression of prepro-endothelin-1 (ppET-1) mRNA was determined by quantitative real time PCR. [(3)H]-Proline incorporation was determined as measure of collagen synthesis. The muscarinic agonist oxotremorine induced, in a tiotropium-sensitive manner, a three-fold increase in ppET-1 mRNA. The β(2)-adrenoceptor agonist olodaterol caused a reduction of ppET-1 mRNA by 45%. Olodaterol also opposed the stimulatory effect of oxotremorine. The effect of olodaterol was mimicked by the protein kinase A agonist 6-Bnz-cAMP, whereas the Epac (exchange protein activated by cAMP) agonist 8-CPT-2'-O-Me-cAMP was less effective. Transforming growth factor-β(1) (TGF-β, 0.3 and 1 ng/ml) induced a three- and eight-fold increase in pp-ET-1 mRNA, respectively. Olodaterol opposed the effect of 0.3, but not that of 1 ng/ml TGF-β. Likewise, 6-Bnz-cAMP opposed the effect of 0.3, but not that of 1 ng/ml TGF-β. TGF-β inhibited β(2)-adrenoceptor mRNA expression, maximally by 90%. Muscarinic agonist-induced stimulation of [(3)H]-proline incorporation was attenuated by the endothelin ET1 receptor antagonist bosentan. In conclusion, ET-1 expression in human lung fibroblasts is regulated by stimulatory muscarinic receptors and inhibitory β(2)-adrenoceptors. Since muscarinic up-regulation of ET-1 contributes to pro-fibrotic effects of muscarinic stimuli, inhibition of ET-1 expression could contribute to long-term beneficial effects of long-acting β(2)-adrenoceptor agonists and long-acting muscarinic antagonists. However, excessive exposure to TGF-β results in loss of β-adrenoceptor expression and function of its down-stream signaling.

The present study aimed to explore possible effects of endothelin-1 (ET-1) on ß(2)-adrenoceptor gene transcription in human lung fibroblasts.MRC-5 human lung fibroblasts were cultured in absence or presence of test substances, followed by ß(2)-adrenoceptor mRNA determination by quantitative real time PCR.ET-1 caused a marked and rapid in onset (1 hr) increase in β(2)-adrenoceptor mRNA, an effect additive to that of short time (1 hr) exposure to the β(2)-adrenoceptor agonist olodaterol. The stimulatory effect of ET-1 on β(2)-adrenoceptor mRNA was prevented by the non-selective ET-A/ET-B receptor antagonist bosentan, indicating that it was mediated via specific ET receptors. In the presence of actinomycin D the effect of ET-1 was prevented indicating that ET-1 acts via increased transcription of the β(2)-adrenoceptor gene. ET-1-induced up-regulation of β(2)-adrenoceptor mRNA was also seen in the presence of cycloheximide excluding indirect effects via up-regulation of other regulatory proteins.ET-1 can up-regulate β-adrenoceptor gene transcription in human lung fibroblasts.

Fibrosis is part of airway remodelling observed in bronchial asthma and COPD. Pro-fibrotic activity of lung fibroblasts may be suppressed by beta-adrenoceptor activation. We aimed, first, to characterise the expression pattern of beta-adrenoceptor subtypes in human lung fibroblasts and, second, to probe beta-adrenoceptor signalling with an emphasis on anti-fibrotic actions. Using reverse transcription PCR, messenger RNA (mRNA) encoding beta(2)-adrenoceptors was detected in MRC-5, HEL-299 and primary human lung fibroblasts, whereas transcripts for beta(1)- and beta(3)-adrenoceptors were not found. Real-time measurement of dynamic mass redistribution in MRC-5 cells revealed beta-agonist-induced G(s)-signalling. Proliferation of MRC-5 cells (determined by [H-3]-thymidine incorporation) was significantly inhibited by beta-agonists including the beta(2)-selective agonist formoterol (-logIC(50), 10.2) and olodaterol (-logIC(50), 10.6). Formoterol's effect was insensitive to beta(1)-antagonism (GCP 20712, 3 mu M), but sensitive to beta(2)-antagonism (ICI 118,551; apparent, pA (2), 9.6). Collagen synthesis in MRC-5 cells (determined by [H-3]-proline incorporation) was inhibited by beta-agonists including formoterol (-logIC(50), 10.0) and olodaterol (-logIC(50), 10.3) in a beta(2)-blocker-sensitive manner. alpha-Smooth muscle actin, a marker of myo-fibroblast differentiation, was down-regulated at the mRNA and the protein level by about 50% following 24 and 48 h exposure to 1 nM formoterol, a maximally active concentration. In conclusion, human lung fibroblasts exclusively express beta(2)-adrenoceptors and these mediate inhibition of various markers of pro-fibrotic cellular activity. Under clinical conditions, anti-fibrotic actions may accompany the therapeutic effect of long-term beta(2)-agonist treatment of bronchial asthma and COPD.

The release of endogenous noradrenaline from rat isolated tracheae was evoked by electrical field stimulation (3 Hz, 540 pulses) in the presence of yohimbine, desipramine and tyrosine. The muscarine receptor agonist oxotremorine concentration-dependently inhibited the evoked release of noradrenaline by 95% at 1 μmol/l, EC50 values in two series of experiments 41 and 57 nmol/l, respectively. The effect of oxotremorine was antagonized by the non-selective muscarine receptor antagonist scopolamine (10–1000 nmol/l) in a manner suggesting a simple competitive interaction (slope of Schild plot −0.94; pA2 value 8.88). However, the M2 selective muscarine receptor antagonist methoctramine (0.1–10 μmol/l) affected the action of oxotremorine in a manner suggesting a complex interaction (slope of Schild plot −0.47). Addition of indomethacin (3 μmol/l) caused an increase of the evoked release of noradrenaline by 45% and low concentrations of oxotremorine (0.01 and 0.1 μmol/l, but not 1 μmol/l) became less effective resulting in a slight shift to the right of the concentration response curve (EC50 169 nmol/l). Moreover, in the presence of indomethacin methoctramine (0.1–10 μmol/l) antagonized the effects of oxotremorine in a manner suggesting a simple competitive interaction (slope of Schild plot −0.93, pA2 value 7.61). In the presence of indomethacin, the concentration response curve of oxotremorine was only slightly shifted to the right in the presence of the M1 receptor selective antagonist pirenzepine (1 μmol/l, −log KB 6.1) and not significantly affected by the M3 receptor selective antagonist pfluoro-hexahydrosiladifenidol (1 μmol/l). In conclusion, the release of noradrenaline in the rat trachea is inhibited via presynaptic muscarine heteroreceptors of the M2 subtype. In addition, activation of muscarine receptors affects the release of noradrenaline also indirectly, probably via the release of inhibitory prostanoids, and this involves muscarine receptors which are pharmacologically different from those at the sympathetic nerve terminals.

Insulin has been approved for inhaled application, but safety concerns remain, because of un-physiologically high insulin concentrations in the lung. Since insulin may act as growth factor, possible proliferative effects of insulin, insulin analogues and insulin-like growth factor-1 (IGF-1) on human lung fibroblasts were studied. As measure of proliferation [(3)H]-thymidine incorporation was studied in HEL-299, MRC-5, IMR-90 and primary human lung fibroblasts. In all cells, mRNA encoding IGF-1 receptors and two variants of insulin receptors was detected. Insulin and IGF-1 stimulated [(3)H]-thymidine incorporation in all cells. Comparison of the concentration-dependent effects in HEL-299 cells showed that IGF-1 and insulin glargine were more potent (EC(50), 3 and 6 nM) and more effective (maximum increase, by 135-150%) than insulin and insulin detemir (EC(50), 22 and 110 nM; maximum increase: by 80%). Proliferative effects of IGF-1 and insulin were inhibited to the same extent by an antibody (1H7) directed against the IGF-1 receptor α-subunit. Insulin-induced stimulation of [(3)H]-thymidine incorporation was reduced by 83% after siRNA-mediated down-regulation of IGF-1 receptor by about 75%, but not affected by a similar down-regulation of the insulin receptor. Insulin and IGF-1 caused rapid up-regulation of the early genes FOS, EGR-1 and EGR-2 as well as of the gene coding for IGF-1. In conclusion, in human lung fibroblasts insulin exerts marked proliferative effects and the pharmacological profile of this response as well as specific receptor knock-down experiments suggest mediation via IGF-1 receptors. The risk of unwanted structural lung alterations by long-term inhalative application of insulin should be considered.

Inhaled insulin may contribute to bronchial carcinoma due to IGF-I receptor activation by high local concentrations. Therefore, effects of insulin and IGF-I on human bronchial carcinoma cells (H292) and normal bronchial epithelium cells (HBE) were studied. TGF-β was included since it also influences carcinoma progression. H292 and HBE cells expressed both the insulin receptor and the IGF-I receptor; in H292 cells an additional, shorter, splicing variant (IR-A) of the insulin receptor was present. Insulin receptor expression was around four to five times higher in H292 than in HBE cells at mRNA and protein levels. Insulin and TGF-β exerted contrary actions on proliferation and gene expression in H292 cells. Genes regulated by insulin, IGF-I, and TGF-β were linked to inflammation, cell adhesion, muscle contraction and differentiation. Insulin and IGF-I also suppressed DNA repair genes. EC 50 for insulin-induced proliferation was around 5 nM in H292 and around 30 nM HBE cells. The EC 50 values for gene expression ranged from 9 to 90 nM in both cell types, dependent on the gene studied. In H292 cells, the proliferative response was much stronger if TGF-β was present. In HBE cells this interaction of insulin and TGF-β was not observed, and changes in gene expression were mostly lower by at least 10-fold as compared to H292. All in all, the insulin effects in H292 were generally much stronger than in HBE cells and – with regard to proliferation – occurred at lower concentrations. Thus, insulin will hardly induce cancer from normal bronchial cells but may favour progression of pre-existing tumours.