Binding and intracellular degradation of atrial natriuretic factor by cultured vascular smooth muscle cells

Abstract

Binding studies were performed on vascular smooth muscle cells (VSMC) from the rat aorta, using 125I-atrial natriuretic factor (Ser99-Tyr126) (ANF (Ser99-Tyr126] as the ligand. Kinetic studies at 37 degrees C indicated a rapid onset of binding with a maximum total binding of 25% being reached by 60 min. Crosslinking experiments demonstrated that ANF bound to a 120 kDa and a 60 kDa protein with the former dissociating into the 60 kDa species in presence of beta-mercaptoethanol. Of the total radioactivity bound, 15% represented internalized material. Analysis of the medium after different incubation periods revealed a 42% degradation of 125I-ANF by 120 min. At 4 degrees C, no internalization of 125I-ANF was observed. However, surface binding occurred, albeit at a much slower rate, and not reaching a maximum even at the end of 3 h. No degraded material was detected in the extracellular medium even after a 2-h incubation. Chloroquine (100 microM) and monensin (10 microM) significantly increased the cell-associated radioactivity, causing a 2- to 3-fold elevation of internalized material and a 1.5- to 2-fold rise in the surface-bound ligand. Both lysosomotropic agents also inhibited ANF degradation by 70-80%. Kinetic of the intracellular labeled material was analyzed: within 5-10 min it reaches a maximum level and it decreases rapidly. In presence of monensin the intracellular signal was amplified and the decay was minimized. The intracellular material was found to be mostly bound to a 60 kDa protein. These studies suggest an intracellular degradation of ANF, probably in the lysosomal compartment, following receptor-mediated endocytosis.