Peptidases and smooth muscle cell angiotensin II receptor pharmacology.
Angiotensin (A) II receptors on rat aortic smooth muscle (RASM) cell membranes were characterized using the radioligand [125I][Sar1Ile8]AII ([125I]SI-AII). Angiotensin I, AII, and AIII inhibited specific [125I]SI-AII binding, and their rank order of potencies, and Ki values (nM) were: AII (3.7) > AI (32.5) > or = AIII (54.0), which differed from that observed for rat adrenal cortex: AII (0.85) > AIII (3.3) >> AI (100). Similar results were observed for RASM membranes in the presence of guanine nucleotides, and for intact cells in the absence or presence of an internalization inhibitor. Lowering the incubation temperature from 37 degrees C to 4 degrees C, or inclusion of PMSF (1 mM), and preparing membranes in the presence of EGTA (1 mM) altered the rank order of potencies and Ki values (nM) of the angiotensin peptides to: AII (1.1) > AIII (7.0) >> AI (144). [125I]Angiotensin I was metabolized completely over the course of 90 min to small (<tetrapeptide) fragments as measured by HPLC. There was no evidence for formation of AII or AIII from AI, which would have explained the unusually high potency of AI. [125I]Angiotensin I metabolism could be attenuated by inhibitors of serine proteases PMSF, aprotinin, and chymostatin. The beneficial effects of PMSF and EGTA suggested that serine protease(s) and metalloproteases contribute to the observed anomalous pharmacological characteristics of AI and AIII, respectively. The RASM cell membranes contained a homogeneous population of binding sites for losartan, and its Ki value differed in the absence (50 nM) or presence (16 nM) of protease inhibitors, which suggests that the receptor may also be a target for these peptidases.(ABSTRACT TRUNCATED AT 250 WORDS)
Cohen
R B
RB
Department of Cardiovascular Biochemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08643-4000.
Webb
M L
ML
Dickinson
K E
KE
D000302:Adrenal Cortex / Q000378:metabolism; D000803:Angiotensin I / Q000378:metabolism; D000804:Angiotensin II / Q000378:metabolism; D000805:Angiotensin III / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D010450:Endopeptidases / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011945:Receptors, Angiotensin / Q000187:drug effects / Q000378:metabolism*; D012504:Saralasin / Q000378:metabolism
D011480:Protease Inhibitors; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D000805:Angiotensin III; D000803:Angiotensin I; D010450:Endopeptidases; D012504:Saralasin