Abstract
Angiotensin II has been demonstrated to act as a growth factor in rat cardiac fibroblasts. However, the signaling events that lead to fibroblast cell growth in response to angiotensin II remain to be elucidated. This study was designed to determine whether angiotensin II stimulated tyrosine phosphorylation of proteins in cardiac fibroblasts. Immunoblot analysis demonstrated rapid tyrosine phosphorylation of distinct substrates of 125, 95, 46-60, and 44 kDa in response to 10 nM angiotensin II. Tyrosine phosphorylation was maximal at 5 min and persisted for at least 180 min. Additional tyrosine-phosphorylated proteins of 185, 145, and 85 kDa were detected in response to 10 ng/ml platelet-derived growth factor BB. A cluster of 75-80-kDa proteins were phosphorylated in response to angiotensin II, phorbol ester, and platelet-derived growth factor. Angiotensin II-induced tyrosine phosphorylation was unaffected by phorbol ester-sensitive protein kinase C down-regulation and could be partially blocked by pertussis toxin pretreatment. Angiotensin II stimulation resulted in increased cytosolic tyrosine kinase activity which was recovered by immunoprecipitation. Immunoblot analysis demonstrated tyrosine phosphorylation of p44MAPK, and, in addition, we demonstrated for the first time tyrosine phosphorylation of p125FAK, p46SHC, and p56SHC in response to angiotensin II. The finding that angiotensin II and platelet-derived growth factor stimulated tyrosine phosphorylation of p46SHC and p56SHC suggested that this protein may serve as a common tyrosine kinase substrate in the mitogenic signaling cascade induced by G-protein-coupled receptors and growth factors and is consistent with the hypothesis that angiotensin II-induced tyrosine phosphorylation is involved in mitogenic signaling pathways in neonatal rat cardiac fibroblasts.
Highlights
From the Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822 and the §Department of Biology, Susquehanna University, Selinsgrove, Pennsylvania 17870
The observation that vasodilators, in contrast to and persisted for at least 180 min.Additional tyrosine- angiotensin convertingenzyme inhibitors,are ineffective in phosphorylated proteins of 185, 145, and 85 kDa were preventing interstitialremodeling suggests that AI1 promotes detected in response to 10 ng/mpl latelet-derived growth fibroblast proliferation in the heart independentlyof changes factor BB.Acluster of 75-80-kDaproteins were phosphorylated in response to angiotensin 11,phorbol ester, and platelet-derived growth factor
Phosmay serve as a common tyrosine kinase substrate in the phorylation of tyrosine residues are early ancdritical events in mitogenic signaling cascade induced by G-proteincoupled receptors and growth factors and isconsistent with the hypothesis that angiotensin II-induced tyrosine phosphorylation is involved in mitogenic signaling pathways in neonatal rat cardiac fibroblasts
Summary
Time (min) from hearts of0-3-day-old Sprague-Dawley rat pups and grown a s previously described [35].Two or three daysfollowing dispersion, cells were passagedand seeded a t a density of 104/cm2on 100-mm plates. Immunoprecipitation a n d Immunoblot Analysis-Cultures of cardiac fibroblasts were grown until subconfluent on 100-mm dishes, in-. Proteins were transferred to nitrocellulose, and blots were incubated overnight in Tris-buffered saline, TB(2S0TmM Tris base, 137mM. Blots were washed in TBS(20 mM Tris base, 137 mM NaCl, pH 7.6, and 0.1% Tween (v/v)),and immunoreactivity was determined using the enhanced chemiluminescence reaction (ECL, Amersham). Serum-starved culturesof neonatal rat cardiac fibroblasts were pCi of Y-["~PIATP (300C0Vmmol, Dupont NEN), and the protein kinaseexposed to 10 nM AII, 100 nM PDBu, or 10 ng/ml PDGF-BB for the reaction was performed for 15 min a t 3 7 "C. The reaction was terminated by addition of SDS-sample buffer. Cell lysates were separated using8% SDS-gel electrophoresis, transferred to nitrocellulose membranes, and imunoblotted with anti-P-Qr antibody. Shown are representative blots of three experiments using cells from different dispersions
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