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

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-ß1 (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 Ca²âº 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 Ca²âº levels in CF; however, after preincubation for 1h with DAKD and re-stimulation with the same agonist, we found a low increase in intracellular Ca²âº levels. Finally, DAKD increased intracellular Ca²âº 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.

Differential participation of angiotensin II type 1 and 2 receptors in the regulation of cardiac cell death triggered by angiotensin II.

BACKGROUND: The Angiotensin II (Ang II) type 1 (AT(1)R) and type 2 (AT(2)R) receptors are increased in the heart following myocardial infarction and dilated cardiomyopathy, yet their contribution at a cellular level to compensation and/or failure remains controversial. METHODS: We ectopically expressed AT(1)R and AT(2)R in cultured adult rat cardiomyocytes and cardiac fibroblasts to investigate Ang II-mediated cardiomyocyte hypertrophy and cardiac cell viability. RESULTS: In adult rat cardiomyocytes, Ang II did not induce hypertrophy via the AT(1)R, and no effect of Ang II on cell viability was observed following AT(1)R or AT(2)R expression. In adult rat cardiac fibroblasts, Ang II stimulated cell death by apoptosis via the AT(1)R (but not the AT(2)R), which required the presence of extracellular calcium, and induced a rapid dissipation of mitochondrial membrane potential, which was significant from 8 h. CONCLUSIONS: We conclude that Ang II/AT(1)R triggers apoptosis in adult rat cardiac fibroblasts, which is dependent on Ca2+ influx.