Morphological and functional analysis of protease-activated receptor 2 on intracellular calcium dynamics in rat parotid gland acinar cells

蛋白水解酶激活受体(PARs)是一类新的跨膜区G蛋白偶联受体，可被蛋白水解酶切割激活。最近的研究报道，PARs存在于各种细胞中，并与许多重要功能的调节密切相关。研究了刺激PARS是否在腮腺引起反应的问题，特别是PARS刺激过程中细胞内钙的动态变化。在本研究中，PAR2基因在腮腺中表达较强。在腮腺腺泡细胞中，PAR2激活肽(PAR2-AP)SLIGRL-NH_2可引起[Ca^&lt；2&gt；]_i的升高，清除细胞外钙和应用钙通道阻断剂均不能抑制PAR2-AP引起的[Ca^&lt；2&gt；]_i升高。对PAR-2激活的反应主要是由细胞内钙库的钙动员引起的。一氧化氮合酶抑制剂L-NAME可部分抑制该肽诱导的钙离子释放和内流。NO供体GEA 3162模拟PAR2激活非获能钙内流(NCCE)的作用，而不是8-bromo-cGMP。KN93和W7(钙调素抑制剂)均完全阻断细胞内钙离子释放和细胞外钙离子内流。丁卡因和DHAB部分阻断了这些增加。这些结果表明，PAR2-AP激活NCCE通路。我们认为，PAR-2在腮腺的作用依赖于CaMKII，并且PAR2-AP激活兰尼定样受体。

Protease-activated receptors(PARs) represent a novel class seven transmembrane domain G-protein coupled receptors, which are activated by proteolytic cleavage. Recent studies have reported that PARs are present in a variety of cells and have been prominently implicated in the regulation of a number of vital functions. The issue of whether the stimulation of PARs induces responses in parotid glands was examined ; with special reference to intracellular Ca^<2+>([Ca^<2+>]_i) dynamics during PARs stimulation. In the present study, PAR2 mRNA was expressed strongly in the parotid glands. In parotid acinar cells, PAR2-activating peptide(PAR2-AP), SLIGRL-NH_2, induced an increase in[Ca^<2+>]_i. Both removing of extracellular Ca^<2+> and using of Ca^<2+> channel blockers did not inhibit the PAR2-AP-induced[Ca^<2+>]_i increase. The response to PAR-2 activation was mainly caused by Ca^<2+> mobilization from intracellular Ca^<2+> stores. This peptide induced Ca^<2+> release and entry were partially inhibited by the nitric oxide synthase(NOS) inhibitor, L-NAME. The NO donor, GEA 3162, but not 8-bromo-cGMP, mimicked the effects of PAR2 in activating non capacitative calcium entry(NCCE). Both KN93(a CAM kinase II inhibitor) and W7(a calmodulin inhibitor) completely blocked a Ca^<2+> release from intracellular Ca2+store and a Ca^<2+> influx from extracellular spaces. Tetracaine and DHAB partially blocked these increases. These results indicate that PAR2-AP activates NCCE pathway. And we proposed that an effect of PAR-2 in parotid gland is dependent on CAMKII and a PAR2-AP activates the ryanodine-like receptors.