Figure 7.

Regulation of AT1R responses by CB₁R in activated HSCs. (A) eHSCs were stimulated for 10 min with 1 μM Ang II in the absence or presence of SR141716 (SR, 1 μM) and the levels of pERK and ERK examined as described in ‘Materials and methods’. Data are expressed as the mean±s.e.m. (n=3 independent experiments). ^**P<0.01. (B) eHSCs were treated with THL (1 μM, for 0, 10, or 30 min) before treatment with Ang II (1 μM). Data are expressed as the mean±s.e.m. (n=3 independent experiments). ^*P<0.05; ^**P<0.01. (C) cHSCs were stimulated for 10 min with 1 μM Ang II in the absence or presence of SR141716 (SR) and the levels of pERK and ERK examined as described in ‘Materials and methods’. (D) cHSCs pretreated with THL for 10 or 30 min were stimulated with 1 μM Ang II. Lysates were subjected to SDS–PAGE and immunoblotting. The levels of Ang II-mediated pERK normalized to total ERK are indicated. Data are expressed as the mean±s.e.m. (n=3–4 independent experiments). (E) eHSCs and cHSCs in 24-well plates were incubated with increasing concentrations of Ang II in the absence or presence of the CB₁R antagonist PF514273 (1 μM) at 37°C for 15 min in the cAMP treatment buffer (0.5 mM isobutylmethylxanthine and 10 μM forskolin in Krebs-Ringer-HEPES buffer). After terminating the reaction by heating at 95°C, cAMP concentrations were determined as described in ‘Materials and methods’. Data are expressed as the mean±s.e.m. (n=3 independent experiments). ^***P<0.001. (F) eHSCs were stimulated with Ang II (1 μM) in the absence or presence of SR141716 (SR, 1 μM) or of PD98059 (MEK inhibitor, 10 μM) for 4 h before the RNA was harvested. After reverse transcription, the number of copies of mRNA for the indicated transcripts were determined by real-time PCR. Data were normalized to GAPDH mRNA and are expressed as the mean±s.e.m. (n=3 in quadruplicate). ^*P<0.05; ^**P<0.01 (versus Ang II treatment); NS, non-significant (Ang II+PD98059 versus Ang II+SR).