CHARACTERIZATION OF THE ANGIOTENSIN-II AT(1) 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) AT(1) 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 AT(1) subtype and (ii) molecular cloning and biochemical studies have provided evidence for the existence of different AT(1) 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 [[H-3]-thymidine incorporation with an EC(50) of 1.41 +/- 0.51 nM. Maximal stimulation was observed in the presence of 100 nM AII and corresponded to 271 +/- 40% of basal [H-3]-thymidine incorporation. 3 To characterize the AII AT(1) 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 [[H-3]-thymidine incorporation in VSMC was antagonized by the non-selective AT(1)/AT(2), receptor antagonist, [Sar(1),Ile(8)]-AII (IC50 = 5.6 nM), by the AT(1A)/AT(1B) receptor antagonist, losartan (IC50 = 10.5 nM) and the AT(1) receptor antagonist, L-158809 (IC50= 0.20 nM). The selective AT(2) receptor ligand, CGP 421121A, antagonized AII-induced [H-3]-thymidine incorporation with an IC50 of 6.3 +/- 1.3 mu M while the AT(2)/AT(1B), receptor antagonist, PD 123319, was found to be almost inactive (IC50> 10 mu M). 4 Under the same experimental conditions, angiotensin III (AIII) was found to be at least 50 times less potent than AII with an apparent EC(50) of 81.6 +/- 7.7 nM. At the highest concentration tested (10 mu M), the effect of AIII corresponded to 327 +/- 61% of basal [H-3]-thymidine incorporation. 5 These results confirm that AII can stimulate DNA synthesis in VSMC through an AT(1) receptor. Furthermore, the pharmacological characterization of this AT(1), receptor is compatible with the AT(1A) receptor subtype recently described on cultured mesangial cells since (i) the AT(1A)/AT(1B) receptor antagonist losartan is active at nanomolar concentrations, (ii) micromolar concentrations of the AT(2)/ AT(1B) receptor antagonist PD 123319 are ineffective at antagonizing the AII-induced [H-3]-thymidine incorporation and (iii) AII is at least 50 times more potent than AIII in stimulating DNA synthesis.