Endothelin ET(B) receptors show different binding profiles in intact cells and cell membrane preparations.

We examined the affinity of endothelin-1, endothelin-3 and four endothelin receptor ligands, BQ788 (cis-2,6-dimethylpiperidinocarbonyl-gamma-methyl-Leu-D-Trp(1-CO 2CH3-D-Nle-ONa), SB-209670 ((+)-(1S,2R,3S)-3-(2-carboxymethoxy-4-methoxyphenyl)-1-(3,4-methylenedio xyphenyl)-5-(prop-1-yloxy)indane-2-carboxylic acid), IRL-1620 (succinyl-[Glu9,Ala11,15]endothelin-1(8-21)), and L-749329 (3',4'-methylenedioxy-1-(2-propyl-4-carboxyphenoxy)-N-(4-isopropyl -phenylsulfonyl)-benzene acetamide), for endothelin ET(B) receptors in human and rat heart cells. The affinities of these ligands showed good correlation between both types of living cells and between their membrane preparations (r = 0.861, P < 0.001), but less significant correlation between each of the living cells and its respective membrane preparation (r = 0.569, 0.02 < P < 0.05). These results suggest that there is no species difference in the affinities of these ligands and that destruction of the intact cell membrane structure may lead to changes in binding properties of the endothelin ET(B) receptor.

Binding characterization of [3H]S-0139, an antagonist of the endothelin ET(A) receptor subtype.

S-0139 (27-O-3-[2-(3-carboxy-acryloylamino)-5-hydroxyphenyl]-acryloylo xy myricerone, sodium salt) is a highly specific nonpeptide endothelin ET(A) receptor antagonist. The binding of [3H]S-0139 was compared to that of [125I]endothelin-1 to characterize the binding of the antagonist in porcine aortic smooth muscle membranes. Scatchard analysis revealed a single class of [3H]S-0139 binding sites with a Kd value of 0.61 +/- 0.10 nM and a Bmax of 0.72 +/- 0.16 pmol/mg protein. These sites were saturable and reversible. [125I]Endothelin-1 also showed binding with high affinity (Kd = 0.12 +/- 0.02 nM) to a homogeneous population of binding sites, whose Bmax (0.71 +/- 0.20 pmol/mg protein) was almost the same as that for [3H]S-0139. In both cases, the binding could be displaced by known endothelin receptor ligands and their IC50 values in each case showed a very close correlation (r = 0.986). The potency of seven endothelin receptor antagonists to displace [3H]S-0139 binding also correlated highly to the potency for inhibiting the endothelin-1-induced increase in cytosolic Ca2+ concentration (r = 0.949). Myriceric acid A showed a more potent functional activity than expected from its binding affinity, but this seemed to result from the different assay conditions, such as incubation time. Together, the results suggest that S-0139 labels only endothelin ET(A) receptor binding sites in porcine aortic smooth muscle.

A long-term receptor stimulation is requisite for angiotensin II-dependent DNA synthesis in vascular smooth muscle cells from spontaneously hypertensive rats.

Angiotensin II stimulates DNA synthesis in aortic smooth muscle cells prepared from spontaneously hypertensive rats, with maximal levels detected 20 h after stimulation. Angiotensin II receptor antagonists inhibited the angiotensin II-induced DNA synthesis. In particular, the noncompetitive antagonist 2-ethoxy-1-[[2'(1 H-tetrazol-5-yl) biphenyl-4-yl]methyl]-1 H-benzimidazole-7-carboxylic acid (CV11974) was more effective than expected from its affinity for the angiotensin II receptor and its potency for inhibiting angiotensin II-induced increase in cytosolic free Ca2+ concentration 2-n-Butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1 H-tetrazol-5-yl)biphenyl-4-yl) methyl]imidazole, potassium salt (losartan), one of the antagonists, inhibited angiotensin II-induced DNA synthesis by 92% and 79%, even when added 2 and 4 h after angiotensin II stimulation, respectively. Angiotensin II also increases the mRNA of platelet-derived growth factor-A chain and basic fibroblast growth factor. The increase was observed within 4 h after angiotensin II stimulation. In this case, the addition of losartan at 4 h after angiotensin II stimulation hardly influenced the time course of the mRNA level of growth factors. Also, conditioned media of cells stimulated with angiotensin II did not influence DNA synthesis in the presence of CV11974. These results suggest that sustained receptor stimulation with angiotensin II is required for DNA synthesis in addition to the early intracellular signaling following phospholipase C activation in a manner independent of the induction of growth factors such as platelet-derived growth factor-AA and basic fibroblast growth factor.

Kinetic studies on the interaction of nonlabeled antagonists with the angiotensin II receptor.

Angiotensin AT1 receptor antagonists are divided into two types, surmountable and insurmountable, based on the way they inhibit angiotensin II-induced vasoconstriction. To elucidate what causes the difference, we studied how antagonists associate with and dissociate from AT1 receptor sites in rat liver membranes. Three antagonists, 6-propyl-7-oxo-4[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-4,7- dihydropyrazolo[1,5-a]pyrimidine-3-carboxylic acid (SRL1080227), 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H- benzimidazole-7-carboxylic acid (CV-11974) and 2-butyl-3-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-3H-imidazo- [4,5-b]pyridine (FK739), showed competitive antagonism when they were added simultaneously with [125I]angiotensin II, but CV11974 and SRL1080227 showed apparently noncompetitive antagonism when membranes were preincubated with each antagonist. The longer the preincubation time with CV11974 or SRL1080227 was, the more effectively the antagonist inhibited [125I]angiotensin II binding, while the inhibition by FK739 did not change with the preincubation time. To estimate their dissociation rate from the receptor binding site, we studied [125I]angiotensin II binding to membranes which had been preincubated with each antagonist and washed twice. Membranes pretreated with FK739 completely recovered the ability to bind [125I]angiotensin II with a period of 60 min, while membranes preincubated with CV11974 did not read this level of recovery. [125I]angiotensin II binding to membranes preincubated with SRL1080227 increased gradually, but did not reach the control level during the experiment. The kinetic properties of SRL1080227, CV11974 and FK739 were consistent with their characteristic modes in inhibiting angiotensin II-induced contraction of isolated rabbit aorta and decreasing blood pressure of spontaneously hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning and characterization of the angiotensin receptor subtype in porcine aortic smooth muscle.

A cDNA encoding porcine aortic smooth muscle angiotensin II (AII) receptor has been isolated using the homology screening approach and sequenced. Specific binding of [125I]AII was found in COS-7 cells transfected with the cDNA (Kd = 0.37 nM, Bmax = 1 approximately 3 x 10(4)/cell) and was displaced with unlabeled AII-related peptides and DUP753 in the order of [Sar1,Ile8]AII = AII > des-Asp1-[Ile8]AII = DUP753 > angiotensin I = angiotensin III. EXP655 had no effect on [125I]AII binding. COS-7 cells transfected with the cDNA responded to AII by only a small increase in the concentration of intracellular free Ca2+. However, electrophysiological study of the receptor expressed in Xenopus laevis oocytes provided strong evidence that it could functionally couple to a second messenger system leading to the mobilization of intracellular stores of Ca2+. Northern blot analysis in cultured porcine aortic smooth muscle cells demonstrated that the expression of this gene varies with the culture media. These results indicate that the cDNA encodes the functional and regulated AT1 subtype of AII receptors.

Possible implication of peptidase activity in different potency of angiotensins II and III for displacing [125I]angiotensin II binding in pig aorta.

A single class of [125I]angiotensin II ([125I]AII) binding sites was found in porcine aortic smooth muscle membranes. Des-Asp1-AII (AIII) and des-Asp1-[Ile8]AII were 20 times less potent than AII or [Sar1,Ile8]AII to displace [125I]AII binding. In contrast, AII and AIII equipotently induced an increase in cytosolic free Ca2+ concentration in cultured porcine aortic smooth muscle cells. Des-Asp1-[Ile8]AII and [Sar1,Ile8]AII equipotently inhibited the increase induced by either AII or AIII. In order to explain this discrepancy, we studied [125I]AIII binding. In the presence of amastatin or a potent inhibitor of aminopeptidase M, [125I]AII binding remained stable for 90 min, but [125I]AIII binding decreased gradually after the peak at 30 min. The decrease was completely blocked by the presence of amastatin and phenylmethylsulfonylfluoride. Consequently, AIII was as potent as AII to displace [125I]AII binding in the presence of these two protease inhibitors. These results suggest that the lower potency of AIII to displace [125I]AII binding is related primarily to AIII-specific degradation by proteases.