Expression of functional type 1 protease-activated thrombin receptors by mouse primary palatal mesenchymal cells in vitro

Development of the primary palate involves a series of processes including cell growth, differentiation, and morphogenesis. To study the molecular and cellular processes during mouse primary palatogenesis, mesenchymal cells w ere isolated from the primary palate of BALB/cBy embryos (day-11, hour 20). Most of the primary palatal mesenchymal (PPM) cells were morphologically s imilar to fibroblasts. The population doubling time was about 36 h. At conc entrations of 5 and 10 unit/ml, alpha-thrombin significantly stimulated the proliferation of these palatal cells by 2- to 2.4-fold compared to untreat ed controls over a 72 hour incubation period. Reverse transcriptase-polymer ase chain reaction using primers based on the mouse type 1 protease-activat ed thrombin receptor (PARI) detected PARI mRNA in the PPM cells, the authen ticity of which was confirmed by partial DNA sequencing. Blocking of the al pha-thrombin proteolytic site with the highly specific inhibitor D-phenylal anyl-prolyl-arginyl chloromethyl ketone significantly suppressed the mitoge nic effect of thrombin on the PPM cells by 71%. These results suggest that PAR1 is present on PPM cells in the mouse embryo and that serine protease a ctivity is important for the receptor activation. (C) 2000 Elsevier Science Ltd. All rights reserved.