In vitro tracheal hyperresponsiveness to muscarinic receptor stimulation by carbachol in a rat model of bleomycin-induced pulmonary fibrosis.
1 Bleomycin-induced lung injury is widely used as an experimental model to investigate the pathophysiology of pulmonary fibrosis but the alterations in the pharmacological responsiveness of airways isolated from bleomycin-exposed animals has been scarcely investigated. The aim of this study was to examine the in vitro tracheal responses to muscarinic receptor stimulation with carbachol in a rat bleomycin model. 2 Concentration-response curves to carbachol (10 nm to 0.1 mm) were obtained in tracheal rings isolated from Sprague-Dawley rats 14 days after endotracheal bleomycin or saline. The intracellular calcium signal in response to carbachol (10 microm) was measured by epifluorescence microscopy using fura-2 in primary cultures of tracheal smooth muscle cells from bleomycin- and saline-exposed rats. Circulating plasma tumour necrosis factor (TNF)-alpha/interleukin (IL)-1beta levels were measured by enzyme-linked immunosorbent assay. 3 Maximal contraction in response to carbachol was significantly greater in tracheal rings from bleomycin-exposed rats compared with controls (15.8 +/- 1.3 mN vs. 11.8 +/- 1.4 mN; n = 19, P < 0.05). 4 Carbachol (10 microm) elicited a transient increase of intracellular calcium with greater increment in tracheal smooth muscle cells from bleomycin-exposed rats compared with controls (372 +/- 42 nmvs. 176 +/- 20 nm; n = 7, P < 0.01). 5 Circulating plasma levels of TNF-alpha/IL-1beta were augmented in bleomycin-exposed rats compared with controls. Tissue incubation with TNF-alpha (100 ng ml(-1))/IL-1beta (10 ng ml(-1)) increased in vitro tracheal responsiveness to carbachol. 6 In conclusion, tracheal contraction in response to muscarinic receptor stimulation with carbachol was increased in bleomycin-exposed rats. This in vitro cholinergic hyperresponsiveness may be related to the augmented levels of inflammatory cytokines in bleomycin-exposed rats.
Barrio
J
J
Department of Pharmacology, Faculty of Medicine, University of Valencia, Av. Blasco Ibanez 15, E-46010 Valencia, Spain.
Cortijo
J
J
Milara
J
J
Mata
M
M
Guijarro
R
R
Blasco
P
P
Morcillo
E J
EJ
D000818:Animals; D001761:Bleomycin; D016535:Bronchial Hyperreactivity / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002217:Carbachol / Q000494:pharmacology*; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000097:blood / Q000494:pharmacology; D008297:Male; D018721:Muscarinic Agonists / Q000494:pharmacology*; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism; D011658:Pulmonary Fibrosis / Q000097:blood / Q000139:chemically induced / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011976:Receptors, Muscarinic / Q000187:drug effects* / Q000378:metabolism; D014132:Trachea / Q000187:drug effects* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology
D007375:Interleukin-1; D018721:Muscarinic Agonists; D011976:Receptors, Muscarinic; D014409:Tumor Necrosis Factor-alpha; D001761:Bleomycin; D002217:Carbachol