Chen J, Giri S N
Department of Molecular Biosciences, University of California, Davis 95616, USA.
J Pharmacol Exp Ther. 1997 Jun;281(3):1047-58.
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 IC50 value 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.
基于血小板活化因子（PAF）诱导的钙离子反应特性以及PAF拮抗剂对这些反应的效力，在仓鼠和豚鼠的肺泡巨噬细胞中鉴定出了不同的PAF受体亚型。在仓鼠和豚鼠的肺泡巨噬细胞中，PAF能以剂量依赖的方式诱导细胞内钙离子库释放钙离子以及细胞外钙离子内流，而溶血PAF（无活性PAF）则无此作用。然而，两种动物中PAF刺激钙离子释放的效力存在显著差异，豚鼠在钙离子释放和钙离子内流反应中的半数有效浓度（EC50）值分别比仓鼠高30倍和50倍。此外，两种动物在钙离子内流特性上也存在明显差异；豚鼠巨噬细胞表现出快速的钙离子外排以及对毒胡萝卜素（耗尽细胞内钙离子库）的高敏感性。PAF诱导的钙离子反应在仓鼠中对G蛋白抑制剂百日咳毒素敏感，而在豚鼠中则不敏感，这表明不同类型的G蛋白与PAF受体偶联。用酪氨酸激酶抑制剂赫曲霉素A预处理巨噬细胞，会使豚鼠中PAF诱导的钙离子反应呈剂量依赖性降低，但令人惊讶的是，在仓鼠中反应却增强。这些观察结果表明，在两种动物的巨噬细胞中，PAF诱导的磷脂酶C活化以及因此产生的对PAF介导的受体信号转导级联反应的钙离子信号传导可能存在G蛋白和酪氨酸激酶的双重机制。用PAF重复刺激或用蛋白激酶C激活剂、丝裂原活化蛋白激酶预处理会使PAF诱导的钙离子反应脱敏，豚鼠中的效力略高于仓鼠。重要的是，三种结构不同的PAF拮抗剂，WEB2086、L659,989和CL184005，能以剂量依赖的方式阻断PAF诱导的钙离子反应，两种动物之间的效力明显不同。在仓鼠中，抑制PAF诱导的钙离子释放的半数抑制浓度（IC50）值比豚鼠低2.5倍（WEB2086）、650倍（L659,989）和120倍（CL184005）。这些PAF拮抗剂在仓鼠巨噬细胞中的相对效力为L659,989 > CL184005 > WEB2086。然而，在豚鼠中这三种拮抗剂的效力大致相同。有趣的是，在两种动物中发现这些拮抗剂对PAF诱导的钙离子内流有相反的抑制作用，仓鼠中WEB2086和CL184005的IC50值分别比豚鼠高15倍和5倍，而L659,989的IC50值在两种动物之间没有差异。用这些拮抗剂预处理两种动物的巨噬细胞对ATP诱导的钙离子反应没有影响，这表明这种拮抗作用对PAF受体具有特异性。根据我们的数据得出结论，从仓鼠支气管肺泡灌洗中分离出的肺泡巨噬细胞含有一种独特的PAF受体亚型，在调节不同的信号转导途径方面与豚鼠的不同。
