Uwe R. Juergens

Muscarinic receptors mediate stimulation of collagen synthesis in human lung fibroblasts

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 [3H]-proline into cellular proteins was determined as measure of collagen synthesis. In MRC-5 cells, the muscarinic agonist carbachol enhanced [3H]-proline incorporation in a concentration-dependent manner (effective concentration of 50%: 220 nM, increase at 10 µM by 40–55%, in a different series of experiments). Likewise, 10 µM oxotremorine caused an increase of ∼65%. For comparison, transforming growth factor-β1 (5 ng·mL−1) caused an increase of ∼80%. Effects of carbachol on total [3H]-proline incorporation and collagenase-sensitive [3H]-proline fraction were similar. The effect of 10 µM 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 [3H]-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.

Role of Epac1 in mediating anti-proliferative effects of prostanoid EP2 receptors and cAMP in human lung fibroblasts

In lung fibroblasts, proliferation is inhibited by activation of EP2 prostanoid receptors which are known to couple to adenylyl cyclase. Beside the classic target of cAMP, protein kinase A (PKA), alternative cAMP effectors have been identified, among them Epac (exchange protein activated by cAMP). The present study aimed to illuminate transduction pathways mediating the anti-proliferative effects of EP2 receptors in lung fibroblasts. Proliferative activity of human lung fibroblasts was determined by measuring [3H]-thymidine incorporation. The selective EP2 receptor agonist butaprost inhibited [3H]-thymidine incorporation by 75%, an effect mimicked by forskolin, the phosphodiesterase inhibitor IBMX, the stable cAMP analogues dibutyryl-cAMP and bromo-cAMP, as well as by the Epac selective cAMP analogues 8-pCPT-2′-O-Me-cAMP and Sp-8-pCPT-2′-O-Me-cAMPS, whereas the PKA selective agonist 6-Bnz-cAMP was inactive. The PKA inhibitor Rp-8-Br-cAMPS inhibited butaprost-induced phosphorylation of CREB (cAMP response element-binding protein), but did not affect butaprost-induced inhibition of [3H]-thymidine incorporation. Partial knockdown of Epac1 by specific siRNA transfection resulted in a marked attenuation of the inhibitory potency of butaprost, whereas transfection of Epac2 siRNA or non-silencing siRNA did not affect the effectiveness of butaprost to inhibit [3H]-thymidine incorporation. In conclusion, Epac1 rather than the classic cAMP effector PKA is a crucial element in the signal transduction pathway mediating anti-proliferative effects of EP2 receptor activation.

New Evidence of H1-Receptor Independent COX-2 Inhibition by Fexofenadine HCl in vitro

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.

Reproterol--A monomolecular combination of orciprenaline and theophylline: novel aspects of its mode of action in asthma.

Background and Objective: Reproterol is a monomolecular combination of orciprenaline and theophylline used as β-adrenergic agonist to induce bronchodilation in bronchial asthma. Since the mechanism of action of reproterol has not been investigated so far, its potential anti-inflammatory activity in asthma remains still unknown. Therefore, we have studied in vitro whether the theophylline component of the reproterol molecule might enhance the stimulatory effect of the β-adrenoceptor on cAMP production resulting in suppression of inflammatory mediator production. Methods: The effects of reproterol, orciprenaline and theophylline (10–9–10–5 M) on spontaneous cAMP (5 × 104 cells/30 min)- and on LPS (10 μg/ml)-stimulated LTB4 production (105 cells/4 h) were determined in normal monocytes in vitro. Results: Production of cAMP (n = 9) was significantly augmented in a dose-dependent manner by orciprenaline (30 ± 8%) and theophylline (28 ± 10%), but mostly by reproterol (127 ± 8%) at 10–5 M. Despite incubation with propranolol, significant stimulation of cAMP production was notable following reproterol therapy. Production of LTB4 was significantly inhibited by reproterol (–48 ± 14%) and less by theophylline (–28 ± 10%), but was stimulated by orciprenaline (+20 ± 8%) at 10–5 M. Conclusion: We conclude that reproterol exerts a strong stimulatory effect on monocyte cAMP production and a suppressive effect on LTB4 production possibly due to a synergistic mode of action on adenylate cyclase activity and inhibition of phosphodiesterases. More clinical studies in bronchial asthma will be needed to determine whether these results may translate into clinically relevant effects.