Meliton Angelo Y, Muñoz Nilda M, Liu Jie, Lambertino Anissa T, Boetticher Evan, Myo Saori, Myou Shigeharu, Zhu Xiangdong, Johnson Malcolm, Leff Alan R
Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, IL 60637, USA.
J Allergy Clin Immunol. 2003 Aug;112(2):404-10. doi: 10.1067/mai.2003.1637.
Prior investigations have demonstrated that beta(2)-adrenoceptor stimulation is ineffective in inhibiting synthesis of eicosanoids in human eosinophils. This effect has been postulated to relate to density or structural differences in the beta(2)-adrenoceptor or its coupled G-protein. However, recent reports indicate that cAMP-specific PDE4 activity in eosinophils is 10-fold that of other inflammatory cells. We postulated that selective blockade of PDE4 in eosinophils would unmask the inhibitory effect of beta(2)-adrenoceptor stimulation and that this inhibition would result from decreased phosphor-ylation of cytosolic group IV-PLA(2) (gIV-PLA(2)).
To determine (a) whether PDE4 inhibition alone with rolipram blocked secretions of arachidonic acid (AA) and leukotriene C(4) (LTC(4)) caused by activation of eosinophils with formyl-met-leu-phe plus cytochalasin B (FMLP/B), (b) to determine if PDE4 inhibition plus beta(2)-adrenoceptor agonist act additively to augment endogenous cAMP concentration, and (c) to determine the mechanism by which additive inhibition of AA and LTC(4) synthesis is regulated by cAMP.
Human eosinophils were pretreated with buffer, salmeterol or rolipram (singly or combination) before FMLP/B activation. Release of AA and LTC(4), intracellular cAMP concentration, and phosphorylation and activation of gIV-PLA(2) were determined.
Rolipram unmasked the inhibitory effect of beta(2)-adrenoceptor stimulation with salmeterol and significantly attenuated the stimulated release of AA and subsequent LTC(4). Inhibition corresponded to increased cAMP production caused by rolipram alone or rolipram plus salmeterol and blocked proportionately the phosphorylation and activation of gIV-PLA(2) in FMLP/B-activated eosinophils.
Inhibition of PDE4 by rolipram unmasks beta(2)-adrenergic blockade of LTC(4) synthesis caused by FMLP/B.
先前的研究表明，β₂ - 肾上腺素能受体刺激在抑制人类嗜酸性粒细胞中类花生酸的合成方面无效。据推测，这种效应与β₂ - 肾上腺素能受体或其偶联的G蛋白的密度或结构差异有关。然而，最近的报告表明，嗜酸性粒细胞中cAMP特异性磷酸二酯酶4（PDE4）的活性是其他炎症细胞的10倍。我们推测，选择性阻断嗜酸性粒细胞中的PDE4将揭示β₂ - 肾上腺素能受体刺激的抑制作用，并且这种抑制将源于细胞溶质IV型磷脂酶A₂（gIV - PLA₂）磷酸化的减少。
确定（a）单独使用咯利普兰抑制PDE4是否能阻断由甲酰 - 甲硫 - 亮 - 苯丙氨酸加细胞松弛素B（FMLP/B）激活嗜酸性粒细胞所引起的花生四烯酸（AA）和白三烯C₄（LTC₄）的分泌，（b）确定PDE4抑制加β₂ - 肾上腺素能受体激动剂是否具有相加作用以增加内源性cAMP浓度，以及（c）确定cAMP调节AA和LTC₄合成相加抑制的机制。
在FMLP/B激活之前，用人嗜酸性粒细胞分别用缓冲液、沙美特罗或咯利普兰（单独或联合使用）进行预处理。测定AA和LTC₄的释放、细胞内cAMP浓度以及gIV - PLA₂的磷酸化和激活情况。
咯利普兰揭示了沙美特罗对β₂ - 肾上腺素能受体刺激的抑制作用，并显著减弱了AA和随后LTC₄的刺激释放。抑制作用与咯利普兰单独或咯利普兰加沙美特罗引起的cAMP产生增加相对应，并相应地阻断了FMLP/B激活的嗜酸性粒细胞中gIV - PLA₂的磷酸化和激活。
咯利普兰对PDE4的抑制揭示了FMLP/B引起的LTC₄合成的β₂ - 肾上腺素能阻断作用。
