THE EFFECT OF THROMBIN AND SERINE PROTEASES ON INTRACELLULAR CA2-MUSCLE CELLS( IN RAT AORTIC SMOOTH)

Cultures of vascular smooth muscle cells (VSMC) are commonly used to s tudy the events and defects found in hypertension and atherosclerosis. In particular Ca2+ homeostasis in cellular signalling has been the fo cus of extensive research. Since trypsin has been shown to mobilise Ca 2+ in some cell types, we have investigated its effect on various aspe cts of Ca2+ homeostasis in rat aortic smooth muscle cells (RASMC). The effects of trypsin, alpha-chymotrypsin and elastase (other serine pro teases) on intracellular Ca2+ in cultured aortic cells isolated from W istar rats have been investigated. Trypsin (24 mu g/ml) elicits intrac ellular Ca2+ mobilisation, after which cells become nonresponsive to t hrombin Ca2+ mobilisation but retain responsiveness to Angiotensin II (AII). alpha-Chymotrypsin (24 mu g/m) inhibits the thrombin Ca2+ mobil ising response, without itself initiating a Ca2+ transient or affectin g AII Ca2+ mobilisation. Elastase (24 mu g/ml) was not effective in mo bilising intracellular Ca2+ or inhibiting the thrombin response. We ha ve also observed diminished thrombin Ca2+ mobilisation responses betwe en cells in suspension and cell monolayers, which appeared to be unrel ated to proteolysis but due to morphological changes of the cells. Our results suggest that trypsin acts on the thrombin receptor via a spec ific proteolysis mechanism to mobilise intracellular Ca2+([Ca2+](i)) i n RASMC. The amount of Ca2+ released by thrombin or trypsin is depende nt on the morphology of the cell and the state of the tethered ligand of the thrombin receptor exposed by the protease.