Berger P, Tunon-De-Lara J M, Savineau J P, Marthan R
Laboratoire de Physiologie Cellulaire Respiratoire, Inserm E9937, Université Bordeaux 2, 33076 Bordeaux, France.
J Appl Physiol (1985). 2001 Aug;91(2):995-1003. doi: 10.1152/jappl.2001.91.2.995.
Tryptase, the major mast cell product, is considered to play an important role in airway inflammation and hyperresponsiveness. Tryptase produces different, sometimes opposite, effects on airway responsiveness (bronchoprotection and/or airway contraction). This study was designed to examine the effect of human lung tryptase and activation of protease-activated receptor (PAR)-2 by synthetic activated peptide (AP) SLIGKV-NH(2) on Ca(2+) signaling in human airway smooth muscle (HASM) cells. Immunocytochemistry revealed that PAR-2 was expressed by HASM cells. Tryptase (7.5--30 mU/ml) induced a concentration-dependent transient relative rise in cytoplasmic Ca(2+) concentration (Ca(2+)) that reached 207 +/- 32 nM (n = 10) measured by indo 1 spectrofluorometry. The protease inhibitors leupeptin or benzamidine (100 microM) abolished tryptase-induced Ca(2+) increase. Activation of PAR-2 by AP (1-100 microM) also induced a concentration-dependent transient rise in Ca(2+), whereas the reverse peptide produced no effect. There was a homologous desensitization of the Ca(2+) response on repeated stimulation with tryptase or AP. U-73122, a specific phospholipase C (PLC) antagonist, xestospongin, an inositol trisphosphate (IP(3))-receptor antagonist, or thapsigargin, a sarcoplamic Ca(2+)-ATPase inhibitor, abolished tryptase-induced Ca(2+) response, whereas Ca(2+) removal, in the additional presence of EGTA, had no effect. Calphostin C, a protein kinase C inhibitor, increased PAR-2 Ca(2+) response. Our results indicate that tryptase activates a Ca(2+) response, which appears as PAR-2 mediated in HASM cells. Signal transduction implicates the intracellular Ca(2+) store via PLC activation and thus via the IP(3) pathway. This study provides evidence that tryptase, which is increasingly recognized as an important mediator in airway inflammation and hyperresponsiveness, is also a potent direct agonist at the site of airway smooth muscle.
类胰蛋白酶是肥大细胞的主要产物，被认为在气道炎症和高反应性中起重要作用。类胰蛋白酶对气道反应性产生不同的、有时甚至相反的影响（支气管保护作用和/或气道收缩）。本研究旨在检测人肺类胰蛋白酶以及合成活化肽（AP）SLIGKV-NH₂对蛋白酶激活受体（PAR）-2的激活作用对人气道平滑肌（HASM）细胞内Ca²⁺信号的影响。免疫细胞化学显示PAR-2在HASM细胞中表达。类胰蛋白酶（7.5 - 30 mU/ml）可诱导细胞质Ca²⁺浓度（[Ca²⁺]i）呈浓度依赖性短暂相对升高，通过indo 1荧光分光光度法测得其达到207±32 nM（n = 10）。蛋白酶抑制剂亮抑酶肽或苯甲脒（100 μM）可消除类胰蛋白酶诱导的[Ca²⁺]i升高。AP（1 - 100 μM）激活PAR-2也可诱导[Ca²⁺]i呈浓度依赖性短暂升高，而反向肽则无作用。用类胰蛋白酶或AP重复刺激时，[Ca²⁺]i反应存在同源脱敏现象。特异性磷脂酶C（PLC）拮抗剂U-73122、肌醇三磷酸（IP₃）受体拮抗剂西司他汀或肌浆网Ca²⁺-ATP酶抑制剂毒胡萝卜素可消除类胰蛋白酶诱导的[Ca²⁺]i反应，而在额外添加乙二醇双四乙酸（EGTA）的情况下去除Ca²⁺则无影响。蛋白激酶C抑制剂钙泊三醇C可增强PAR-2的[Ca²⁺]i反应。我们的结果表明，类胰蛋白酶激活了[Ca²⁺]i反应，这在HASM细胞中似乎是由PAR-2介导的。信号转导涉及通过PLC激活从而通过IP₃途径的细胞内Ca²⁺储存。本研究提供了证据，表明类胰蛋白酶越来越被认为是气道炎症和高反应性中的一种重要介质，它在气道平滑肌部位也是一种有效的直接激动剂。
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