Abstract
The different platelet-activating factor (PAF) receptor subtypes were identified in alveolar macrophages of hamster and guinea pig, based on the distinct characteristics of PAF-induced Ca++ responses and PAF antagonist potencies to these responses. PAF, but not lyso-PAF (inactive PAF), induced Ca++ release from intracellular Ca++ stores and the influx of extracellular Ca++ in a dose-dependent manner in both hamster and guinea pig alveolar macrophages. The potency for PAF-stimulated Ca++ release, however, was significantly different between the two species with EC50 values being 30- and 50-fold higher in Ca++ release and Ca++ influx responses in guinea pig than hamster, respectively. In addition, there were distinct differences in Ca++ influx characteristics between the two species; guinea pig macrophages exhibiting a rapid Ca++ extrusion and high sensitivity to thapsigargin (depletion of intracellular Ca++ store). The PAF-induced Ca++ response was sensitive to G-protein inhibitor pertussis toxin in hamster but not in guinea pig, suggesting the coupling of different types of G-proteins to PAF receptors. Pretreatment of macrophages with tyrosine kinase inhibitor, herbimycin A, caused a dose-dependent decrease in PAF-induced Ca++response in guinea pig but surprisingly an increased response in hamster. These observations suggest the possibility of a dual mechanism, for G-protein and tyrosine kinase, in PAF-induced phospholipase C activation of macrophages from both species and thus Ca++ signaling in response to PAF-mediated receptor signal transduction cascade. The PAF-induced Ca++ response was desensitized by repetitive stimulation with PAF or pretreatment with protein kinase C activator, mitogen-activated protein kinase, which had a slightly greater potency in guinea pig than hamster. Importantly, three structurally distinct PAF antagonists, WEB2086, L659,989 and CL184005, blocked PAF-induced Ca++ responses in a dose-dependent manner with a markedly different potencies between the two species. The IC50 values for inhibiting PAF-induced Ca++ release were 2.5- (WEB2086), 650- (L659,989) and 120- (CL184005) fold less in hamster than in guinea pig. The relative potencies of these PAF antagonists in hamster macrophages were L659,989 > CL184005 > WEB2086. However, in guinea pig these three antagonists showed roughly the same potency. Interestingly, the opposite inhibitory effects of these antagonists on PAF-induced Ca++ influx were found in the two species, in which the IC50 were 15- (WEB2086) and 5- (CL184005) fold greater in hamster than in guinea pig but no difference in the IC50value of L659,989 between the two species. Pretreatment of macrophages from both species with these antagonists had no effect on ATP-induced Ca++ response, suggesting that the antagonism is specific to PAF receptors. Based on our data, it was concluded that the alveolar macrophages isolated from the bronchoalveolar lavage of hamsters contain a distinct subtype PAF receptor that differs from that of guinea pigs in modulating a different signal transduction pathway.
Footnotes
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Send reprint requests to: Shri N Giri, Professor and Chair, Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616.
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↵1 This work was supported by Grants NHLBI HL27354 and R01 HL-56262.
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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:
- PAF
- platelet activating factor
- EC50
- concentration that produces 50% of maximal response
- IC50
- concentration which inhibits 50% of response
- PKC
- protein kinase C
- PT
- pertussis toxin
- TK
- tyrosine kinase
- IP3
- inositol 1, 4, 5 triphosphate
- PLC
- phospholipase C
- HBSS
- Hanks’ balanced salt solution
- PMA
- phorbol 12-myristate 13-acetate
- MAP
- mitogen activated protein kinase
- BSA
- bovine serum albumin
- Received December 30, 1996.
- Accepted February 25, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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