Abstract
The mechanisms of lung fibrosis caused by bleomycin (BL) and other fibrogenic agents are not clearly understood. Our previous studies demonstrated that the platelet-activating factor (PAF) antagonist WEB2086 reduced lung fibrosis induced by BL and amiodarone in hamsters, suggesting a critical role for PAF and/or PAF receptors in this pathogenic process. In the present study, the PAF receptors in the lung and the functional activity of PAF receptors in the alveolar macrophages from BL (7.5 U/kg, intratracheally)-treated hamsters were investigated. The PAF receptor binding, measured by a [3H]WEB2086 binding assay in lung homogenates, was significantly increased at all times after BL treatment, compared with saline-treated control hamsters. At 3 days after BL treatment, the PAF receptor density (B max = 202.4 fmol/mg protein, with K d = 41 nM) was increased over control (B max = 116.9 fmol/mg protein, with K d = 45.3 nM). Most importantly, the functional activities of PAF receptors in alveolar macrophages, as determined by PAF-induced elevation of cytosolic Ca++ (both by mobilization of Ca++ stores and by Ca++ influx), were significantly higher in the BL-treated animals than in the saline control. The EC50 of PAF to increase internal Ca++ release was 5-fold less in BL-treated lungs than in control. The Ca++ signaling could not be stimulated by lyso-PAF (inactive PAF) but was inhibited by the PAF antagonists WEB2086 (at 100 nM) and L659,989, in a dose-dependent fashion, suggesting the involvement of specific receptors for PAF. The cells from BL-treated hamster lung required much higher concentrations of the antagonists, with increases in the IC50 values of 14-fold for WEB2086 and 63-fold for L659,989 over control. These results indicated that PAF receptors were functionally up-regulated in the lungs after BL treatment in vivo, and this may be an important mechanism, at least in part, for BL-induced lung injury. These findings also explain the antifibrotic effect of the PAF receptor antagonist WEB2086 in the BL-hamster model of lung fibrosis, as reported in our earlier paper.
Footnotes
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Send reprint requests to: S. N. Giri, Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, Davis, CA 95616.
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↵1 This work was presented in part at the American Lung Association/American Thoracic Society International Conference, New Orleans, LA, May 13, 1996, and published as an abstract (Am. J. Respir. Crit. Care Med. 153: A248, 1996). This work was supported by National Heart, Lung and Blood Institute Grants HL27354 and R01-HL56262 (S.N.G.) and a research award from the University of California, Davis (J.C.).
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↵2 Current address: Stanford University, School of Medicine and VA Medical Center, GRECC 182B, 3801 Miranda Avenue, Palo Alto, CA 94304.
- Abbreviations:
- BL
- bleomycin
- BSA
- bovine serum albumin
- [Ca++]i
- intracellular calcium concentration
- HBSS
- Hanks’ balanced salt solution
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- IL
- interleukin
- MAP
- mitogen-activated protein
- PAF
- platelet-activating factor
- TGF
- transforming growth factor
- TNF
- tumor necrosis factor
- Received June 10, 1996.
- Accepted November 12, 1996.
- The American Society for Pharmacology and Experimental Therapeutics
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