Tiotropium increases cytosolic muscarinic M3 receptors and acetylated H3 histone proteins in induced sputum cells of COPD patients.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is characterized by irreversible progressive airflow limitation related to tobacco smoking. This limitation is caused by chronic inflammation of the airways and lung parenchyma and is associated with increased activity of parasympathetic system. The most effective bronchodilators in COPD are muscarinic receptor antagonists (MRA), which reverse, at least in part, compromised respiratory function. MRA also contribute to control inflammatory processes via interactions with inflammatory signaling molecules. The use of the long-acting cholinolytic bronchodilatator - tiotropium, with high affinity to M3 receptors, is suggested as a first line maintenance treatment in COPD patients. MATERIAL AND METHODS: In this study we assessed M3 receptor protein expression in induced sputum of 27 stable COPD patients before and after therapy consisting of 18 µg once daily tiotropium for 12 weeks. Lung function tests including spirometry, lung volumes, and DLCO were performed before and after therapy in all COPD patients. The patients were subjected to the sputum induction procedures before and after therapy. Sputum cells were isolated, sample-specific cell profiles were characterized, and the cells were processed to isolate pure cytosolic fractions. Cytosolic M3 protein and HDAC2 levels and nuclear acetylated histone H3 (AcH3) expression was quantified using specific antibodies against human proteins and Western blot with enhanced luminescence detection. RESULTS: Therapy significantly increased the mean forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) volume (P<0.05). The mean expression of M3 protein was higher by 37% after therapy (P<0.05), HDAC2 expression was not altered, while AcH3 level was increased by about 90% (P<0.01), compared with the corresponding data before therapy. HDAC2 expression before therapy was positively correlated with AcH3 expression (r = 0.74), while after therapy no correlation was detected. FEV1, FCV, and cytosolic M3 protein expression did not correlate with other biochemical parameters tested. CONCLUSIONS: Twelve weeks of tiotropium therapy in COPD patients improves clinical indices of lung function and involves alterations in sputum cell chromatin acetylation and also increased cholinergic M3 receptor internalization.

Nuclear HSP90 and HSP70 in COPD patients treated with formoterol or formoterol and corticosteroids.

OBJECTIVE: Heat shock proteins assist cellular protein folding and are required for the normal activity of steroid receptors. In this study we assessed nuclear HSP90 and HSP70 proteins and mRNA levels in cells isolated from induced sputum of chronic obstructive pulmonary disease patients treated for 4 weeks with formoterol (F) or formoterol+budesonide (F/ICS). METHODS: Nuclear heat shock protein levels were assessed by Western blot and specific mRNAs were quantified in cell lysates using qRT-PCR. RESULTS: Both HSP90 and HSP70 protein levels were higher in the F/ICS-treated patients in comparison with the F-treated group (by 31%, P<0.05 and 28%, P<0.05, respectively), while specific mRNAs were lowered. HSP86/HSP89 and D6S182/HSP90-BETA were repressed by about 40% (P<0.05) while HSP70-1/HSP70-1A, HSP70-1B/HSP70-2, and HSP70-HSC54/HSC70 were repressed by 47% (P<0.01), 57% (P<0.01) and 65% (P<0.01), respectively. CONCLUSIONS: It is possible that increased nuclear heat shock proteins may play a role in the attenuation of the response to glucocorticoids in COPD patients.