title: Differential regulation of collagen secretion by kinin receptors in cardiac fibroblast and myofibroblast.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Centro de Estudios Moleculares de la Célula, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Chile.
authors.forename: Mabel
authors.initials: M
authors.lastname: Catalán
authors.forename: Christian
authors.initials: C
authors.lastname: Smolic
authors.forename: Ariel
authors.initials: A
authors.lastname: Contreras
authors.forename: Pedro
authors.initials: P
authors.lastname: Ayala
authors.forename: Ivonne
authors.initials: I
authors.lastname: Olmedo
authors.forename: Miguel
authors.initials: M
authors.lastname: Copaja
authors.forename: Pía
authors.initials: P
authors.lastname: Boza
authors.forename: Raúl
authors.initials: R
authors.lastname: Vivar
authors.forename: Yennifer
authors.initials: Y
authors.lastname: Avalos
authors.forename: Sergio
authors.initials: S
authors.lastname: Lavandero
authors.forename: Victoria
authors.initials: V
authors.lastname: Velarde
authors.forename: Guillermo
authors.initials: G
authors.lastname: Díaz-Araya
mesh_terms: D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D003094:Collagen / Q000378:metabolism*; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism*; D007150:Immunohistochemistry; D007609:Kallidin / Q000031:analogs & derivatives / Q000494:pharmacology; D007705:Kinins / Q000378:metabolism; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism*; D058628:Myofibroblasts / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D043783:Receptor, Bradykinin B1 / Q000819:agonists / Q000378:metabolism; D043782:Receptor, Bradykinin B2 / Q000819:agonists / Q000378:metabolism; D018002:Receptors, Bradykinin / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
chemical_list: D016861:Cyclooxygenase Inhibitors; D007705:Kinins; D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D007609:Kallidin; C052787:kallidin, des-Arg(10)-; D003094:Collagen; D019001:Nitric Oxide Synthase; D011451:Prostaglandin-Endoperoxide Synthases; D002118:Calcium