Division of Physiology & Pharmacology, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK.
Br J Pharmacol. 2009 Dec;158(7):1695-704. doi: 10.1111/j.1476-5381.2009.00415.x.
Here we have examined the effects of the novel peptide antagonist N-[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indol-5-yl]aminocarbonyl]-glycinyl-L-lysinyl-L-phenylalanyl-N-benzhydrylamide (K-14585) on proteinase-activated receptor (PAR)(2)-mediated intracellular signalling events.
Using NCTC2544 cells expressing PAR(2), we assessed the effects of K-14585 on PAR(2)-mediated [(3)H] inositol phosphate accumulation, MAP kinase activation, p65 NFkappaB phosphorylation and DNA binding and IL-8 production.
Pretreatment with K-14585 (5 microM) inhibited [(3)H] inositol phosphate levels stimulated by PAR(2)-activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV-OH) in PAR(2)-expressing NCTC2544 cells. K-14585 pretreatment did not influence PAR(2)-mediated extracellular regulated kinase activation but inhibited p38 MAP kinase phosphorylation. At a higher concentration (30 microM), K-14585 alone stimulated p38 MAP kinase activation. These effects were replicated in EAhy926 cells, endogenously expressing PAR(2), but not in parental or PAR(4)-expressing NCTC2544 cells, suggesting these effects were PAR(2)-dependent. SLIGKV-mediated stimulation of p38 MAP kinase phosphorylation was substantially reduced by the G(q/11) inhibitor YM-254890, without affecting K-14585-mediated phosphorylation. Pretreatment with K-14585 inhibited PAR(2)-mediated p65 NFkappaB phosphorylation and NFkappaB-DNA binding. K-14585 (30 microM) alone stimulated comparable NFkappaB reporter activity to SLIGKV-OH. K-14585 inhibited SLIGKV-stimulated IL-8 production, but given alone increased IL-8. While SLIGKV-induced IL-8 formation was reduced by both SB203580 and YM-254890, the response to K-14585 was sensitive to SB203580 but not YM-254890.
These data reveal that K-14585 has a duality of action functioning both as an antagonist and agonist due to either partial agonist actions or possible agonist-directed signalling. The data also suggest two modes of p38 MAP kinase activation emanating from PAR(2), one G(q/11)-dependent and the other G(q/11)-independent.
在这里，我们研究了新型肽拮抗剂 N-[1-(2,6-二氯苯基)甲基]-3-(1-吡咯烷基甲基)-1H-吲哚-5-基]氨基甲酰基]-甘氨酰-L-赖氨酸-L-苯丙氨酸-N-苯并二氢吡喃-4-基酰胺（K-14585）对蛋白酶激活受体（PAR）（2）介导的细胞内信号转导事件的影响。
使用表达 PAR（2）的 NCTC2544 细胞，我们评估了 K-14585 对 PAR（2）激活肽 Ser-Leu-Ile-Gly-Lys-Val（SLIGKV-OH）刺激的 [(3)H]肌醇磷酸盐积累、MAP 激酶激活、p65 NFkappaB 磷酸化和 DNA 结合以及 IL-8 产生的影响。
K-14585（5 μM）预处理抑制了 PAR（2）表达的 NCTC2544 细胞中由 PAR（2）激活肽 Ser-Leu-Ile-Gly-Lys-Val（SLIGKV-OH）刺激的 [(3)H]肌醇磷酸盐水平。K-14585 预处理不影响 PAR（2）介导的细胞外调节激酶激活，但抑制 p38 MAP 激酶磷酸化。在更高浓度（30 μM）下，K-14585 本身即可刺激 p38 MAP 激酶的激活。这些作用在内源性表达 PAR（2）的 EAhy926 细胞中得到复制，但在亲本或 PAR（4）表达的 NCTC2544 细胞中未得到复制，表明这些作用是 PAR（2）依赖性的。SLIGKV 介导的 p38 MAP 激酶磷酸化的刺激被 G(q/11)抑制剂 YM-254890 大大减弱，而不影响 K-14585 介导的磷酸化。K-14585 预处理抑制了 PAR（2）介导的 p65 NFkappaB 磷酸化和 NFkappaB-DNA 结合。K-14585（30 μM）本身即可刺激与 SLIGKV-OH 相当的 NFkappaB 报告基因活性。K-14585 抑制 SLIGKV 刺激的 IL-8 产生，但单独给予 K-14585 则增加了 IL-8。虽然 SLIGKV 诱导的 IL-8 形成被 SB203580 和 YM-254890 均减少，但对 K-14585 的反应对 SB203580 敏感，而对 YM-254890 不敏感。
这些数据表明，K-14585 具有双重作用，既可以作为拮抗剂，也可以作为激动剂，这是由于部分激动剂作用或可能的激动剂导向信号转导。数据还表明，PAR（2）产生两种 p38 MAP 激酶激活模式，一种依赖于 G(q/11)，另一种不依赖于 G(q/11)。
