Characterization of the angiotensin II AT1 receptor subtype involved in DNA synthesis in cultured vascular smooth muscle cells.

1. This study was undertaken in cultured vascular smooth muscle cells to characterize the angiotensin II (AII) AT1 receptor subtype involved in DNA synthesis because (i) the AII receptor involved in vascular proliferation has previously been characterized in vitro in rat aortic cells and identified as an AT1 subtype and (ii) molecular cloning and biochemical studies have provided evidence for the existence of different AT1 receptor subtypes. 2. In cultured rat aortic vascular smooth muscle (VSMC), exposure to AII (0.1 to 100 nM) resulted in a concentration-dependent increase in [3H]-thymidine incorporation with an EC50 of 1.41 +/- 0.51 nM. Maximal stimulation was observed in the presence of 100 nM AII and corresponded to 271 +/- 40% of basal [3H]-thymidine incorporation. 3. To characterize the AII AT1 receptor subtype involved in this effect, cells were exposed to AII (3 nM) in the absence or presence of increasing concentrations of various AII receptor antagonists. The stimulatory effect of AII (3 nM) on [3H]-thymidine incorporation in VSMC was antagonized by the non-selective AT1/AT2 receptor antagonist, [Sar1, Ile8]-AII (IC50 = 5.6 nM), by the AT1A/AT1B receptor antagonist, losartan (IC50 = 10.5 nM) and the AT1 receptor antagonist, L-158809 (IC50 = 0.20 nM). The selective AT2 receptor ligand, CGP 42112A, antagonized AII-induced [3H]-thymidine incorporation with an IC50 of 6.3 +/- 1.3 microM while the AT2/AT1B receptor antagonist, PD 123319, was found to be almost inactive (IC50 > 10 microM). 4. Under the same experimental conditions, angiotensin III (AIII) was found to be at least 50 times less potent than All with an apparent EC50 of 81.6 +/- 7.7 nM. At the highest concentration tested (10 microM),the effect of AIII corresponded to 327 +/- 61% of basal [3H]-thymidine incorporation.5. These results confirm that All can stimulate DNA synthesis in VSMC through an AT, receptor.Furthermore, the pharmacological characterization of this AT1 receptor is compatible with the ATlA receptor subtype recently described on cultured mesangial cells since (i) the ATIA/ATIB receptor antagonist losartan is active at nanomolar concentrations, (ii) micromolar concentrations of the AT2/AT1B receptor antagonist PD 123319 are ineffective at antagonizing the AII-induced [3H]-thymidine incorporation and (iii) All is at least 50 times more potent than AIII in stimulating DNA synthesis.