Alpha-Thrombin stimulates contraction of human bronchial rings by activation of protease-activated receptors.

Abstract

In a variety of diseases, inflammation causes microvascular leakage and activates thrombin. Evidence suggests that thrombin increases cytosolic calcium and stimulates human airway smooth muscle (ASM) cell proliferation. The receptor subtypes, however, that mediate the effects of thrombin on ASM cell growth or calcium mobilization remain unknown. In this study, we postulate that thrombin, which activates specific protease-activated receptors (PARs), also stimulates contraction of isolated human bronchial rings. With the use of intact human bronchial rings, alpha-thrombin (1-20 U/ml) increased bronchial tone to 19 +/- 3% of basal tone (P = 0.008; n = 5 experiments) and represents 20 +/- 8% of the maximum carbachol response. The EC(50) for thrombin-induced force generation was 12.2 U/ml (95% confidence interval 9.9-15.3 U/ml) and was not altered in bronchial rings that had the epithelium removed. In parallel experiments, a specific thrombin receptor-activating peptide (TRAP-14; 0.1-100 micromol/l) increased isometric tension to levels (14 +/- 2%; P = 0.0005; n = 5 experiments) comparable to those rings stimulated with thrombin. To characterize the receptors that mediate thrombin effects on human ASM, the expression of PARs in cultured human ASM cells was analyzed by RT-PCR analysis with specific primers for PARs. In these cells, PAR1 (thrombin receptor), PAR2, and PAR3 were expressed at comparable levels. In other experiments using immunocytochemical staining with specific antibodies to PAR1 and PAR2, we showed that ASM in bronchial rings and cultured ASM cells express PAR1 and PAR2 proteins. Taken together, these studies suggest that alpha-thrombin, in a receptor-specific and dose-dependent manner, induces contraction of bronchial rings in vitro. In addition, cultured human ASM cells express mRNA of PAR1, PAR2, and PAR3 and express PAR1 and PAR2 protein. Further studies are needed to determine whether alpha-thrombin plays a role in stimulating bronchoconstriction in inflammatory airway diseases such as asthma and bronchiolitis obliterans.