Distinction between surmountable and insurmountable selective AT1 receptor antagonists by use of CHO-K1 cells expressing human angiotensin II AT1 receptors

1. CHO-K1 cells that were stably transfected with the gene for the human AT1 receptor (CHO-AT1 cells) were used for pharmacological studies of non-peptide AT1 receptor antagonists. 2. In the presence of 10 mM LiCl, angiotensin II caused a concentration-dependent and long-lasting increase of inositol phosphates accumulation with an EC50 of 3.4 nM. No angiotensin II responses are seen in wild-type CHO-K1 cells. 3. [3H]-Angiotensin II bound to cell surface AT1 receptors (dissociates under mild acidic conditions) and is subject to rapid internalization. 4. Non-peptide selective AT1 antagonists inhibited the angiotensin II (0.1 microM) induced IP accumulation and the binding of [3H]-angiotensin II (1 nM) with the potency order: candesartan > EXP3174 > irbesartan > losartan. Their potencies are lower in the presence of bovine serum albumin. 5. Preincubation with the insurmountable antagonist candesartan decreased the maximal angiotensin II induced inositol phosphate accumulation up to 94% and, concomitantly, decreased the maximal binding capacity of the cell surface receptors. These inhibitory effects were half-maximal for 0.6 nM candesartan and were attenuated by simultaneous preincubation with 1 microM losartan indicating a syntopic action of both antagonists. 6. Losartan caused a parallel rightward shift of the angiotensin II concentration-response curves and did not affect the maximal binding capacity. EXP3174 (the active metabolite of losartan) and irbesartan showed a mixed-type behavior in both functional and binding studies. 7. Reversal of the inhibitory effect was slower for candesartan as compared with EXP3174 and irbesartan and it was almost instantaneous for losartan, suggesting that the insurmountable nature of selective AT1 receptor antagonists in functional studies was related to their long-lasting inhibition.     

Different types of receptor interaction of peptide and nonpeptide angiotensin II antagonists revealed by receptor binding and functional studies.

The pharmacological effects of angiotensin II (AII) are potently inhibited by several peptide and recently synthesized nonpeptide AII receptor antagonists. The interaction of sarcosine1, isoleucine8-AII (sarile), sarcosine1,O-methyltyrosine4-AII (sarmesin), and the nonpeptide AII antagonists 2-n-butyl-4-chloro-5- hydroxymethyl-1-[(2'-(1H-tetrazole-5-yl)biphenyl-4-yl)- methyl]imidazole (DuP 753, Losartan potassium) and its metabolite 2-n-butyl-4-chloro-1-[(2'-(1H-tetrazole-5-yl)biphenyl-4-yl)methyl]imidaz ole - 5-carboxylic acid (EXP3174) with AII binding sites was investigated in radioligand binding and functional studies. Sarile, sarmesin, DuP 753, and EXP3174 inhibited 125I-AII binding to rat lung tissue, with Ki values of 3.5, 16.1, 23.7, and 10.4 nM, respectively. The Hill coefficients of all displacement curves, except for sarile (nH, 1.45), were not significantly different from unity. In functional experiments using rabbit aorta, sarmesin and DuP 753 competitively inhibited the contractile response to AII, with pA2 values of 6.75 and 8.01, respectively. Sarile, in contrast, revealed noncompetitive antagonism, i.e., the maximum contractile force and the slope of the concentration-contractile force curve were significantly and concentration-dependently depressed. The concentration-contractile response curve for AII was shifted to the right in a parallel fashion in the presence of EXP3174 (3 nM to 1 microM); however, the maximum contractile force was significantly decreased, by 24%. The marked noncompetitive antagonism of sarile (3 nM) was reversed in the presence of increasing concentrations of sarmesin (30 nM to 30 microM) or DuP 753 (10 nM to 1 microM), whereas in the presence of increasing concentrations of EXP3174 (3-300 nM) a 25% depression in maximum contractile force persisted. Moreover, the reduction of the maximum contractile force by EXP3174 (10 nM) was concentration-dependently restored in the presence of increasing concentrations of DuP 753 (10 nM to 1 microM), indicating interaction with the same binding site. Whereas sarile (0.3-10 nM) did not affect the 125I-AII binding capacity in radioligand saturation experiments, a 54% reduction of Bmax was observed in the presence of 100 nM EXP3174. The data provide evidence that all antagonists inhibit the functional response to AII by interacting with a common binding site at the receptor. The noncompetitive behavior of sarile seems to be due to slow dissociation from this receptor site. An additional mechanism must be postulated for EXP3174. An allosteric interaction with the receptor, as suggested by the reduction in Bmax, may be, at least in part, responsible for the nonclassical antagonism of this compound.     

Insurmountable angiotensin AT1 receptor antagonists: the role of tight antagonist binding.

Angiotensin II increased the inositol phosphates production (EC50 = 3.4+/-0.7 nM) in Chinese hamster ovary (CHO) cells expressing the cloned human angiotensin AT1 receptor (CHO-AT1 cells). Coincubation with angiotensin AT1 receptor antagonists produced parallel rightward shifts of the concentration-response curve without affecting the maximal response. The potency order is 2-ethoxy-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]-1H-benz imidazoline-7-carboxylic acid (candesartan) > 2-n-butyl-4-chloro-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]i midazole-5-carboxylic acid (EXP3174) > 2-n-butyl-4-spirocyclopentane-1-[(2'-(1H-tetrazol-5-yl)biphe nyl-4-yl)methyl]2-imidazolin-5-one (irbesartan)> of 2-n-butyl-4-chloro-5-hydroxymethyl-1-(2'-(1H-tetrazol-5-yl)bipheny l-4-yl)methyl]imidazole (losartan). Additionally, preincubation with these antagonists depressed the maximal response, i.e., 95%, 70%, 30% of the control response for candesartan, EXP3174 and irbesartan and not detectable for losartan. Increasing the antagonist concentration or prolonging the preincubation time did not affect this depression. Furthermore, these values remained constant for candesartan and EXP3174, when the angiotensin II incubation time varied between 1 and 5 min. Our data indicate that antagonist-receptor complexes are divided into a fast reversible/surmountable population and a tight binding/insurmountable population at the very onset of the incubation with angiotensin II.     

Antagonist interaction with endogenous AT(1) receptors in human cell lines

Using Chinese Hamster Ovary cells expressing human AT(1) receptors cells (CHO-hAT(1)), it was previously shown that insurmountable inhibition of the angiotensin II response by non-peptide antagonists is related to the duration of their receptor occupancy. In the present study it was shown that these antagonists displayed similar binding characteristics to endogenously expressed AT(1) receptors in human adrenal cortex cells (NCI-h295) and renal vascular smooth muscle cells (HVSMC). Competition binding studies with [(3)H]candesartan for NCI-h295 cells, with [(125)I]Sar(1)-Ile(8) angiotensin II for HVSMC and with both radioligands for CHO-hAT(1) cells displayed the same potency order for unlabelled antagonists: candesartan>EXP3174>irbesartan>losartan. The AT(2) receptor antagonist PD123319 displayed low potency in all instances. The apparent half-lives of the antagonist-AT(1) receptor complexes in NCI-h295 cells and HVSMC were comparable to those obtained under identical conditions with CHO-hAT(1) cells. Angiotensin II increased the inositol phosphate accumulation dose dependently with half-maximal response at 17.4+/-1.6nM for NCI-h295 cells and 4.5+/-0.8nM for HVSMC. Pre-incubation of the cells with losartan only produced concentration-dependent rightward shifts of the angiotensin II concentration-response curve. The maximal response was decreased by 85-92% with candesartan, 70-88% with EXP3174 and 60% with irbesartan. The similar binding and inhibitory properties of these antagonists among the investigated cell types validates the use of CHO-hAT(1) cells for investigating pharmacological properties of human AT(1) receptors.     

Functional interaction between losartan and central tachykinin NK3 receptors in the conscious rat.

1. The cardiovascular and behavioural effects elicted by the intracerebroventricular (i.c.v.) injection of substance P (SP), neurokinin A (NKA), [MePhe7]neurokinin B ([MePhe7]NKB) or angiotensin II (AII) in the conscious rat were assessed before and 5 min after i.c.v. pretreatment with antagonists selective for angiotensin AT1 (losartan and its active metabolite EXP 3174), angiotensin AT2 (PD 123,319) or tachykinin NK3 (R 486) receptors. 2. I.c.v. administration of 25 pmol AII evoked an increase in mean arterial blood pressure (MAP) and water intake behaviour, accompanied by a transient bradycardia, whereas 25 pmol [MePhe7]NKB caused a transient increase in MAP and heart rate (HR) concurrently with marked wet dog shake behaviour. At the same dose, SP and NKA were more potent than [MePhe7]NKB in increasing MAP and HR, but did not produce water intake or wet dog shake behaviours. 3. Losartan (650 pmol, i.c.v.) reduced significantly the cardiovascular and behavioural responses to AII or [MePhe7]NKB, but not to SP or NKA. While 65 pmol losartan was inactive, 260 pmol inhibited selectively the central effects of AII. Whereas EXP 3174 (6.5 nmol) blocked both AII and [MePhe7]NKB-mediated responses, the dose of 650 pmol blocked only the responses to AII. 4. The central responses to AII and [MePhe7]NKB were not affected by PD 123,319 (650 pmol). On the other hand, the [MePhe7]NKB-induced central effects were significnatly reduced by R 486 (650 pmol). The NK3-selective antagonist had no effect against AII. 5. This study provides functional evidence, to support earlier binding data, that losartan (and to some extent its active metabolite EXP 3174) interact with the tachykinin NK3 receptor in rat brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

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).(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional comparison of the antagonistic properties of some Angiotensin II type 1 receptor blockers on the contraction elicited by Angiotensin II and thromboxane A2 on human saphenous veins

We previously described on human vascular preparations that, in addition to its antagonistic properties on Angiotensin II type 1 (AT1) receptor, losartan could also inhibit the contraction elicited by the stable thromboxane A2 mimetic U46619. The present study was designed (1) to investigate, in human vascular preparations (the saphenous veins) whether these antagonistic properties on thromboxane A2/prostaglandin H2 (TP) receptor were shared by some other AT1 receptor antagonists (irbesartan and valsartan) and the active metabolite of losartan EXP3174, and (2) to compare their antagonistic properties on TP receptors to their antagonistic properties on AT1 receptors. In the presence of indomethacin (10 microM) and Nomega-nitro-L-arginine (100 microM), irbesartan, valsartan, and EXP3174 induced a rightward shift of U46619- and angiotensin II-induced contraction. The inhibitory effect of irbesartan, valsartan, and EXP3174 on U46619-induced contraction was significant from 100 microM while their inhibitory effect on the contraction elicited by angiotensin II was significant from 1 nM. With regard to the plasma therapeutic concentrations of irbesartan, valsartan, and EXP3174, these data suggest that TP receptor blockade does not account for the antihypertensive effects of these AT1 receptor blockers.     

Effects of angiotensin II receptor blockade on proximal fluid uptake in the rat kidney

Angiotensin II has a well described dose-dependent biphasic action on proximal tubule fluid uptake, although the concentration and effect of endogenous luminal angiotensin II remain controversial. Shrinking split-droplet micropuncture was used to examine the fluid uptake in response to the luminal application of three AT1 antagonists (losartan, EXP3174, candesartan). Addition of losartan at 10(-8) M decreased fluid uptake rate (Jva) by 17.5+/-2.2% (P<0.05). Luminal addition of EXP3174 at concentrations between 10(-9)-10(-5) M caused a dose-dependent decrease in fluid uptake, with a maximum decrease of 41.0+/-9.5% (P<0.01) at 10(-6) M. Candesartan also decreased fluid uptake, by 21.9+/-4.9% (P<0.05) at 10(-8) M and 23.6+/-5.5% (P<0.05) at 10(-5) M. All three antagonists at a low concentration (10(-8) M) decreased fluid uptake. EXP3174 and candesartan at a higher concentration (10(-5) M) also decreased fluid uptake in contrast to the previously reported effect of losartan. We conclude that the endogenous concentration of antiotensin II in the proximal luminal fluid is low and exerts a stimulatory effect on fluid absorption. Losartan at concentrations greater than 10(-6) M may have a non-selective action on fluid uptake.     

Inhibitory effect of KT3-671, a non-peptide angiotensin subtype 1 receptor antagonist, on sympathetic neurotransmission in isolated rabbit aorta.

Effect of KT3-671 on the sympathetic neurotransmission in isolated rabbit aorta was studied and compared with those of losartan and its active metabolite, EXP3174. Angiotensin (Ang) II (30 n m) produced approximately 1.7-fold increase in the transmural nerve stimulation (TNS)-evoked tritium overflow in the aorta preloaded with [(3)H]noradrenaline. KT3-671 (1 microm) by itself did not alter the TNS-evoked tritium overflow but it (0.1-1 microm) concentration-dependently inhibited the enhancing effect of Ang II on the TNS-evoked tritium overflow. Both losartan (1 and 3 microm) and EXP3174 (0.03-0.3 microm) also inhibited the Ang II effect. KT3-671 was approximately 8.6 and 0.3 times more potent than losartan and EXP3174, respectively, in inhibiting the Ang II response. This is consistent with the previous results showing the relative potency of the three antagonists to block AT(1)receptors. None of Ang II, KT3-671, losartan and EXP3174 affected significantly the spontaneous tritium outflow. These results suggest that KT3-671 as well as losartan and EXP3174 may inhibit vascular sympathetic neurotransmission by blocking presynaptic Ang II subtype 1 receptors, which appears to contribute partly to its antihypertensive action. 2000 Academic Press@p$hr Copyright 2000 Academic Press.     

Interactions of ligands at angiotensin II-receptors and imidazoline receptors

Ligands for angiotensin II-(AT)-receptors and imidazoline receptors have structural similarities and influence blood pressure via various mechanisms. The goal of this study was to study the specificity of various ligands by displacement experiments. Antazoline, cimetidine, clonidine, efaroxan, guanabenz, guanethidine, idazoxan, moxonidine and rilmenidine up to a concentration of 100 microM failed to displace the specific binding of [125I]Sar1,Ile8 angiotensin II at the AT1-receptor characterized by losartan (IC50 = 26 +/- 12 nM) in liver homogenate. The same substances up to 100 microM produced no reduction of specific [125I]Sar1,Ile8 angiotensin II binding to the AT2-receptor of phaeochromocytoma cell membranes characterized by PD123319 (IC50 = 20 +/- 5 nM). Displacement experiments at the imidazoline I1-receptors were performed on bovine adrenal medulla membranes using [3H]clonidine after characterization by the I1-ligand clonidine (IC50 = 459 +/- 13 nM) and the I2-ligand idazoxan (IC50 = 3.29 +/- 0.88 microM). The investigated AT-receptor ligands angiotensin II, losartan, EXP 3174 and PD123319 revealed no displacement of [3H]clonidine up to a concentration of 100 microM. The I2-receptor in liver homogenate was characterized by displacement of [3H]idazoxan by cold idazoxan and clonidine (IC50 = 0.37 +/- 0.17 and 68 +/- 31 microM, respectively). The investigated AT-receptor ligands angiotensin II, losartan and PD123319 failed to displace [3H]idazoxan specifically up to 100 microM. Hence, the tested substances showed no cross-reactivity at the corresponding AT- and I-receptors up to 100 microM, a concentration markedly higher than the plasma concentrations achieved after therapeutic application.     

Norepinephrine release and ventricular fibrillation in myocardial ischemia/reperfusion: roles of angiotensin and bradykinin

Exogenous bradykinin (BK), acting at B2-receptors, enhances norepinephrine (NE) release and exacerbates arrhythmias (VF) in myocardial ischemia/reperfusion. Inhibition of BK formation (with serine proteinase inhibitors) alleviates NE release and VF, whereas prevention of BK degradation (with kininase inhibitors) potentiates them. Yet serine proteinase and kininase inhibitors also prevent the formation of angiotensin (AII), a potent NE-release enhancer. Thus we assessed the respective contribution of AII and BK to NE release and VF by using selective B2- and AT1-receptor antagonists. Isolated guinea pig hearts were subjected to 10- and 20-min global ischemia and 45-min reperfusion. NE overflow (pmol/g) was approximately 8 (exocytotic) and approximately 750 (carrier mediated). VF, associated with carrier-mediated NE release, lasted approximately 2 min. The B2-receptor antagonist Hoe 140 (30 nM) affected neither NE overflow nor VF. In contrast, the AT1-receptor antagonist EXP3174 (100 nM) markedly reduced exocytotic and carrier-mediated NE release and shortened VF. When EXP3174 was combined with Hoe 140, NE overflow and VF were decreased even further. Thus in myocardial ischemia, local AII production contributes to NE release and VF via AT1-receptors. Although BK production increases in myocardial ischemia, the effects of BK on adrenergic nerve terminals are uncovered only when BK half-life is prolonged and/or when AII effects are suppressed.     

Reversible and syntopic interaction between angiotensin receptor antagonists on Chinese hamster ovary cells expressing human angiotensin II type 1 receptors

Evidence for a competitive type of interaction between angiotensin II type 1 (AT(1)) antagonists on Chinese hamster ovary cells expressing the human AT(1) receptor (CHO-AT(1)) was obtained by analyzing the binding of [(3)H]-2-ethoxy-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]-1H- ben zimidazoline-7-carboxylic acid ([(3)H]candesartan) and by measuring the AT-induced production of inositol phosphates. The AT(1) antagonists candesartan, 2-n-butyl-4-chloro-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]+ ++imid azole-5-carboxylic acid (EXP3174), or 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)bip hen yl- 4-yl)methyl]imidazole (losartan) produced a concentration-dependent increase in the apparent K(d) values of [(3)H]candesartan in saturation binding experiments, while the B(max) values were unchanged. Furthermore, the dissociation rate of the radioligand initiated by 1 microM unlabelled candesartan was not changed in the presence of 10 microM losartan, 10 microM EXP3174, or 10 microM irbesartan (2-n-butyl-4-spirocyclopentane-1-[(2'-(1H-tetrazol-5-yl)b iph enyl-4-yl) methyl]2-imidazolin-5-one)). Preincubation of the CHO-AT(1) cells with candesartan, EXP3174, and irbesartan caused a reduction in the maximal AT-induced inositol mono-, bis-, and trisphosphate production. This insurmountable effect was reversed in the presence of 1 microM losartan. In line with this finding, the insurmountable antagonist concentration-inhibition curves at 10 microM AT were shifted to the right in the presence of losartan. For candesartan this effect was concentration-dependent, yielding a pK(B) value for losartan of 7.7, which is similar to the pK(B) from previously obtained AT concentration-response curves. Finally, the dissociation rate of candesartan, EXP3174, irbesartan, and losartan was determined by measuring the recovery of AT responses after antagonist pretreatment and washing of the cells with medium containing 1 microM losartan to prevent re-association of the insurmountable antagonists. In addition, similar kinetic data were obtained from the slowing of the [(3)H]candesartan association rate to antagonist preincubated cells.     

Inhibition of the haemodynamic effects of angiotensin II in conscious rats by AT2-receptor antagonists given after the AT1-receptor antagonist, EXP 3174.

1. Conscious, Long Evans rats (n = 10), chronically instrumented for the measurement of regional haemodynamics, were studied on 3 consecutive experimental days to assess responses to angiotensin II (AII) (125 pmol kg-1, i.v.) and noradrenaline (1 nmol kg-1, i.v.) in the absence and presence of the AT2-receptor antagonist, PD 123319 (10 mg kg-1, i.v.) (day 1), the AT1-receptor antagonist, EXP 3174 (1 mg kg-1, i.v.) (day 2), and PD 123319 (10 mg kg-1, i.v.) given 24 h after EXP 3174 (day 3). 2. In naive rats (day 1), PD 123319 did not antagonize the haemodynamic effects of AII or noradrenaline. EXP 3174 (day 2) caused a marked, prolonged blockade of the haemodynamic effects of AII but not those of noradrenaline. Twenty four h after administration of EXP 3174 (day 3) there was still significant attenuation of the haemodynamic effects of AII. However, administration of PD 123319 at this time caused a further inhibition (lasting 1 h) of the effects of AII but not those of noradrenaline. 3. An identical 3 day protocol was used in a separate group of rats (n = 6) in which the AT2-receptor antagonist, PD 123177, was given instead of PD 123319, and the results were essentially the same, i.e., PD 123177 significantly attenuated the haemodynamic effects of AII but only when given 24 h after EXP 3174.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonpeptide angiotensin II receptor antagonists. Studies with DuP 753 and EXP3174 in dogs.

DuP 753 (or EXP3174) and PD123177 are nonpeptide angiotensin (AII)-specific ligands, which show high affinities for two AII receptor subtypes, i.e. AT1 and AT2 sites, respectively. In furosemide-treated conscious dogs with high renin, DuP 753 and EXP3714, but not PD123177, were as effective as captopril in lowering blood pressure. Both DuP 753 and EXP3174 exhibited selective vascular antagonism of AII. In conscious dogs with normal renin, DuP 753, but not captopril or EXP3174, caused a dose-dependent but transient decrease in blood pressure. In anesthetized dogs, DuP 753 and captopril caused similar renal vasodilatation and natriuresis. The renal hemodynamic effects of DuP 753 and captopril were more pronounced in dogs with sodium depletion. These results suggest that the AT1 receptor mediates the pressor and renal effects of AII in dogs. The acute transient hypotensive effect of DuP 753 in normal-renin conscious dogs is probably unrelated to AII antagonism.     

Characterization of the angiotensin II receptor expressed by the human hepatoma cell line, PLC-PRF-5.

Radioligand binding studies were undertaken to establish the expression of angiotensin II (AII) receptors on the human hepatoma cell line, PLC-PRF-5. Cell membranes were shown to express a large number of AII receptors with high and low affinity binding sites having Bmax values of 1269 +/- 365 and 4190 +/- 1055 fmol/mg protein and affinities (Kd) of 2.0 +/- 0.3 nM and 8.7 +/- 0.4 nM, respectively. In intact cells a single class of AII binding site was seen with an affinity (Kd) of 6.7 +/- 1 nM and a Bmax value of 315 +/- 32 fmol/mg. In both membranes and intact cells AII, AIII and the selective angiotensin AT1 receptor antagonist, DuP 753, all had a high affinity for the receptor (Ki values in the nanomolar range), but the selective angiotensin AT2 ligands, PD 123177 and p-aminophenylalanine6 AII, had low affinity (Ki values in the micromolar range). These results indicate that the PLC-PRF-5 cells express the angiotensin AT1 receptor subtype. This was further supported by the demonstration of the sensitivity of the receptor to dithiothreitol (DTT). Pretreatment of membranes with DTT reduced [3H]AII binding in a concentration-dependent manner with an IC50 of 4.2 +/- 0.9 mM. The coupling of the AT1 receptor to signal transduction pathways was investigated. In intact cells AII (100 nM) evoked an increase in intracellular calcium ([Ca2+]i). This increase in [Ca2+]i was unaffected by PD 123177 (100 microM) but was abolished by DuP 753 (100 microM). Furthermore, AII (100 nM) did not inhibit forskolin (0.1-10 microM) stimulated cyclic AMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacology of LR-B/081, a new highly potent, selective and orally active, nonpeptide angiotensin II AT1 receptor antagonist.

1. The pharmacological profile of LR-B/081, (methyl 2-[[4-butyl-2-methyl- 6-oxo-5-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-1(6H)- pyrimidinyl]methyl]-3-thiophenecarboxylate), a novel antagonist at the angiotensin II (AII) AT1-receptor, was studied in vitro and in vivo. 2. In rabbit aortic strips incubated with LR-B/081 (1-1,000 nM), the concentration-response curve to AII was displaced to the right in a nonparallel fashion and the maximal contraction was progressively reduced, indicating that the compound is an insurmountable antagonist in this preparation (apparent pKB = 9.50 +/- 0.23). However, the interaction of LR-B/081 with AII receptors was found to be reversible, since the maximal response to AII was restored by coincubation with losartan, a surmountable AII AT1-antagonist. Contractions elicited by KCl or phenylephrine were not affected by 10 microM LR-B/081. 3. In rat isolated perfused kidney, LR-B/081 and losartan antagonized the AII-induced vasoconstriction [IC50 (95% confidence limits) = 17(13-24) and 39(32-54) nM, respectively]. The LR-B/081 antagonism was incompletely reversed by excess AII, while losartan was fully displaced. The IC50 values of LR-B/081 and losartan obtained against vasoconstriction induced by endothelin-1 and noradrenaline were two orders of magnitude higher. 4. In pithed rats, the intravenous administration of LR-B/081 (0.2-2 mumol kg-1) dose-dependently shifted to the right in a nonparallel fashion the dose-pressor response curve to AII. The maximal pressor response to AII was reduced by LR-B/081 in a dose-dependent fashion.(ABSTRACT TRUNCATED AT 250 WORDS)     

A two-state receptor model for the interaction between angiotensin II type 1 receptors and non-peptide antagonists

The interaction between non-peptide antagonists and the human angiotensin II type 1 (AT1) receptor in CHO-K1 cells was investigated by incubating the cells with antagonist, followed by a brief exposure to angiotensin II and measurement of the resulting inositol phosphate accumulation. The experimental data, expressed either as angiotensin II concentration-response curves or as antagonist concentration-inhibition curves, were in good agreement with computer-generated data according to a single-state model for the surmountable antagonist losartan and according to a two-step, two-state receptor model for the insurmountable antagonists candesartan, EXP3174, and irbesartan. Experimental and computer-generated data concerning the simultaneous exposure of the receptors to EXP3174 and losartan indicated that losartan produced a concentration-dependent restoration of the maximal response (angiotensin II concentration-response curves) as well as a rightward shift of the insurmountable portion of the EXP3174 inhibition curves, thus counteracting the higher-affinity EXP3174 binding. In conclusion, these findings provide further support for the concept that insurmountable and surmountable AT1 antagonists are mutually competitive and that insurmountable antagonist-receptor complexes may adopt different states.     

Implication of phospholipase C in the steroidogenic action of angiotensin II

Angiotensin II (AII) is a major regulator of aldosterone synthesis and secretion by the adrenal zona glomerulosa. Although it has been suggested by many authors that AII acts by increasing the turnover of inositol-lipids, these studies were mainly focussed on the identity and on the kinetics of appearance of inositol phosphates. The purpose of the present study was to establish a relationship between phospholipase C activation and steroidogenesis in the adrenal cortex. A primary culture of bovine adrenal glomerulosa cells was used. Dose-response curves for receptor occupation, inositol phosphate production and aldosterone secretion were made under the same experimental conditions, on the third day of culture. 125I-[Sar1, Val5, D-Phe8]AII binding to glomerulosa cells was progressively inhibited by increasing concentrations of AII up to 30 nM. Scatchard analyses showed a Kd of 1.9 +/- 1.1 nM and a maximal binding capacity of 49,000 +/- 4,500 receptors/cell (six experiments). Dose-response curves for AII-induced inositol 1,4,5-trisphosphate production showed an EC50 of 0.5 +/- 0.1 nM (five experiments). The threshold dose for AII-induced inositol phosphates was around 0.1 nM and the maximal effect was obtained with 30 nM AII. The AII-stimulated steroidogenesis occurred at a threshold dose around 0.03 nM and the maximal effect was obtained with 10 nM AII with an EC50 of 0.5 +/- 0.1 nM (five experiments). These results support previous suggestions that phospholipase C is involved in the steroidogenic action of angiotensin II.     

Functional and binding studies of insurmountable antagonism of 606A, a novel AT1-receptor antagonist in rabbit tissues.

Angiotensin AT1-receptor antagonists can be classified into two types, surmountable and insurmountable ones, based on the way they inhibit angiotensin II (AII)-induced vasoconstriction. To elucidate the causes of the difference, we studied how several antagonists associate with and dissociate from AT1-receptor sites by using rabbit adrenal cortical membrane. Four antagonists, 606A, EXP3174, CV11974, and E4177, showed equipotent competitive antagonism when they were added simultaneous with [125I]-AII in binding experiments. However, in AII-induced contraction studies with rabbit aorta, 606A, EXP3174, and CV11974 inhibited the contraction noncompetitively, whereas E4177 inhibited competitively. The longer the pretreatment period with EXP3174 or CV11974, the more effectively the antagonists suppressed AII-induced contraction. However, the suppression of contraction by 606A and E4177 changed little with the length of the pretreatment period. AII-induced contraction of 606A- or E4177-treated aorta recovered easily by washout, but that of CV11974-treated aorta was hard to recover by washout. These results obtained in the aorta were consistent with their characteristics observed in the AII binding study in the rabbit adrenal cortical membrane in cases of EXP3174 and CV11974. The differences between association rate with and dissociation rate from the AT1 receptor of E4177 and 606A were slight, in spite of the clear difference between their action in the contraction study. Because of the variations observed with the four compounds, mechanisms of insurmountable antagonism may not be uniform among AT1-receptor antagonists.     

Pharmacological profile of GR117289 in vitro: a novel, potent and specific non-peptide angiotensin AT1 receptor antagonist.

1. This paper describes the effects of GR117289 (1-[[3-bromo-2-[2-(1H-tetrazol-5-yl)phenyl]-5-benzo-furanyl]methyl ]-2-butyl-4-chloro-1H-imidazole-5-carboxylic acid) at angiotensin receptors and binding sites in rabbit aorta, rat liver and bovine cerebellum preparations in vitro. 2. In rabbit isolated aortic strips, GR117289 (0.3, 1 and 3 nM) caused a concentration-related, insurmountable suppression of the concentration-response curve to angiotensin II (AII). When the contact time was increased, a greater degree of antagonism of AII was observed, suggesting that GR117289 is slow to reach equilibrium. A pKB of 9.8 +/- 0.1 was calculated for GR117289 after 3 h incubation. GR117289 (1 microM) did not affect contractile responses to phenylephrine or 5-hydroxytryptamine (5-HT) in the rabbit aorta. 3. GR117289 (1 nM) alone caused a marked suppression and a slight rightward displacement of the AII concentration-response curve. Co-incubation with the competitive, surmountable AT1 receptor antagonist, losartan (10 nM, 100 nM and 1 microM), resulted in a concentration-related upward and rightward displacement of the concentration-response curve to subsequently administered AII. In separate experiments in which preparations were pre-incubated with GR117289 (1 nM), subsequent addition of losartan (1 microM) for 2, 15 or 45 min caused a further, but similar, rightward displacement of the concentration-response curve to subsequently administered AII with a time-dependent increase in the maximum response.(ABSTRACT TRUNCATED AT 250 WORDS)