Purification of endothelin from a conditioned medium of cardiac fibroblastic cells using beating rate assay of myocytes cultured in a serum free medium

A conditioned medium from cardiac fibroblastic cells stimulated the beating of quiescent cardiac myocytes cultured in a serum-free medium, The aim of this study was to isolate and characterize the myocyte beat-stimulating activity of the conditioned medium of fibroblastic cells. Cardiac myocytes and fibroblastic cells were isolated individually from neonatal rats. The fibroblastic cells were grown in a growth medium until they became confluent, then serum-free conditioned medium was obtained from them. For the beating-rate assay, the cardiac myocytes were cultured in a completely serum-free medium. The beat-stimulating factor of myocytes in the conditioned medium was purified by reverse-phase liquid chromatographies and gel filtration, and was characterized by measuring the molecular weight of the activity and a pharmacological antagonistic study. The beat-stimulating activity in the conditioned medium was purified into two active fractions, Both of the activities have a molecular weight of 2.5 kDa, and the activities were abolished similarly by FR139317, an endothelin type-A receptor antagonist. These results indicate that cardiac fibroblastic cells secrete endothelin and that this may contribute in part to the functional abnormalities of the heart in patients with myocardial fibrosis, (C) 1997 Academic Press Limited.