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激肽受体对心肌成纤维细胞和肌成纤维细胞胶原分泌的差异调节。

Differential regulation of collagen secretion by kinin receptors in cardiac fibroblast and myofibroblast.

机构信息

Centro de Estudios Moleculares de la Célula, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Chile.

出版信息

Toxicol Appl Pharmacol. 2012 Jun 15;261(3):300-8. doi: 10.1016/j.taap.2012.04.013. Epub 2012 Apr 18.

DOI:10.1016/j.taap.2012.04.013

PMID:22554775

Abstract

UNLABELLED

Kinins mediate their cellular effects through B1 (B1R) and B2 (B2R) receptors, and the activation of B2R reduces collagen synthesis in cardiac fibroblasts (CF). However, the question of whether B1R and/or B2R have a role in cardiac myofibroblasts remains unanswered.

METHODS

CF were isolated from neonate rats and myofibroblasts were generated by an 84 h treatment with TGF-β1 (CMF). B1R was evaluated by western blot, immunocytochemistry and radioligand assay; B2R, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and cyclooxygenases 1 and 2 (COX-1, and COX-2) were evaluated by western blot; intracellular Ca⁺² levels were evaluated with Fluo-4AM; collagen secretion was measured in the culture media using the picrosirius red assay kit.

RESULTS

B2R, iNOS, COX-1 and low levels of B1R but not eNOS, were detected by western blot in CF. Also, B1R, B2R, and COX-2 but not iNOS, eNOS or COX-1, were detected by western blot in CMF. By immunocytochemistry, our results showed lower intracellular B1R levels in CF and higher B1R levels in CMF, mainly localized on the cell membrane. Additionally, we found B1R only in CMF cellular membrane through radioligand displacement assay. Bradykinin (BK) B2R agonist increased intracellular Ca²⁺ levels and reduced collagen secretion both in CF and CMF. These effects were blocked by HOE-140, and inhibited by L-NAME, 1400 W and indomethacin. Des-Arg-kallidin (DAKD) B1R agonist did not increase intracellular Ca²⁺ levels in CF; however, after preincubation for 1h with DAKD and re-stimulation with the same agonist, we found a low increase in intracellular Ca²⁺ levels. Finally, DAKD increased intracellular Ca²⁺ levels and decreased collagen secretion in CMF, being this effect blocked by the B1R antagonist des-Arg9-Leu8-kallidin and indomethacin, but not by L-NAME or 1400 W.

CONCLUSION

B1R, B2R, iNOS and COX-1 were expressed differently between CF and CMF, and collagen secretion was regulated differentially by kinin receptor agonists in cultured CF and CMF.

摘要

未加说明

激肽通过 B1（B1R）和 B2（B2R）受体发挥其细胞作用，B2R 的激活可减少心肌成纤维细胞（CF）中胶原的合成。然而，B1R 和/或 B2R 是否在心肌成纤维细胞中起作用仍未得到解答。

方法

从新生大鼠中分离 CF，并通过 TGF-β1（CMF）处理 84 小时生成肌成纤维细胞。通过 Western blot、免疫细胞化学和放射性配体测定评估 B1R；通过 Western blot 评估 B2R、诱导型一氧化氮合酶（iNOS）、内皮型一氧化氮合酶（eNOS）和环氧化酶 1 和 2（COX-1 和 COX-2）；通过 Fluo-4AM 评估细胞内 Ca²⁺水平；使用 picrosirius 红试剂盒在培养物中测量胶原分泌。

结果

Western blot 在 CF 中检测到 B2R、iNOS、COX-1 和低水平的 B1R，但未检测到 eNOS。此外，Western blot 在 CMF 中检测到 B1R、B2R 和 COX-2，但未检测到 iNOS、eNOS 或 COX-1。免疫细胞化学结果显示 CF 中的细胞内 B1R 水平较低，而 CMF 中的 B1R 水平较高，主要定位于细胞膜上。此外，通过放射性配体置换测定，我们发现 B1R 仅存在于 CMF 细胞膜上。缓激肽（BK）B2R 激动剂增加 CF 和 CMF 中的细胞内 Ca²⁺水平并减少胶原分泌。这些作用被 HOE-140 阻断，并被 L-NAME、1400 W 和吲哚美辛抑制。去精氨酸-缓激肽（DAKD）B1R 激动剂未增加 CF 中的细胞内 Ca²⁺水平；然而，在与 DAKD 孵育 1 小时并再次用相同的激动剂刺激后，我们发现细胞内 Ca²⁺水平略有增加。最后，DAKD 增加了 CMF 中的细胞内 Ca²⁺水平并减少了胶原分泌，该作用被 B1R 拮抗剂去精氨酸 9-亮氨酸-缓激肽和吲哚美辛阻断，但不受 L-NAME 或 1400 W 的影响。