Tryptase

Cardiac fibroblasts were obtained from adult male Sprague Dawleys rats (8 weeks old, n=4), spontaneously hypertensive rats (SHR; 12 weeks old, n=4) and Wistar Kyoto rats (WKY; 12 weeks old, n=4). All studies conformed to the principles of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the University of South Carolina Institution’s Animal Care and Use Committee. Anesthesia at the experimental end point was affected by sodium pentobarbital (50 mg/kg, IP). Briefly, hearts were minced and digested with Liberase 3 (Roche) and fibroblasts purified by selective attachment to plastic culture ware. Fibroblasts were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% neonatal bovine serum (NBS), and 5% fetal calf serum (FCS) with replacement of media every other day. All fibroblasts were used on the second passage to minimize changes in phenotype associated with culture.^{7 (link)} Prior to experimentation, one million fibroblasts were seeded on 100 mm plastic culture plates and allowed to adhere to the plate for 24 hours in DMEM with 10% NBS and 5% FCS. Next, cells were rinsed with Moscona’s Salt Solution and the media replaced with serum–free DMEM-F12 media for 24 hours. For the studies assessing collagen production, fibroblasts were either untreated, or treated with tryptase (1000 mU, Promega) dissolved in DMEM with 1.5% FBS for 24 or 72 hours. Additionally, tryptase treated fibroblast were pretreated for one hour with the PAR-2 peptide antagonist (FSLLRY, 10 μM, Peptides International) or the mitogen activated protein kinase kinase (MEK)1/2 inhibitor (PD98059, 10 μM, Cayman Chemical). PD98059 was used since MEK1/2 is immediately upstream of ERK1/2, thus, inhibiting its effects. For MAPK activation experiments, fibroblasts were incubated with tryptase, tryptase in the presence of FSLLRY or PD98059 for 10 minutes. At the completion of the experiments, fibroblasts and media were collected and snap frozen.