In vivo pharmacology of L-158,809, a new highly potent and selective nonpeptide angiotensin II receptor antagonist.

L-158,809 (5,7-dimethyl-2-ethyl-3-[[2'-(1H-tetrazol-5yl)[1,1']-bi- phenyl-4-yl]-methyl]-3H-imidazo[4,5-b]pyridine) is a potent, competitive and specific antagonist of AT1 subtype of angiotensin II (AII) receptors in in vitro radioligand binding and functional isolated tissue assays. The present study was carried out to characterize the in vivo pharmacology of this potent AII receptor antagonist. In conscious, normotensive and anesthetized pithed rats, L-158,809 inhibits AII (0.1 microgram/kg i.v.) elevations in blood pressure without altering pressor responses to methoxamine or arginine vasopressin. In conscious rats, the relative potencies (ED50) were 29 micrograms/kg i.v. and 23 micrograms/kg p.o. Duration of action with single i.v. or p.o. doses exceeded 6 hr in rats. In similar experiments using rhesus monkeys, the potencies of L-158,809 were 10 micrograms/kg i.v. and approximately 100 micrograms/kg p.o. In these rats and monkeys, L-158,809 was 10 to 100 times more potent than DuP-753 (losartan) and approximately 3 times more potent than the metabolite, EXP3174. AII-induced elevation of plasma aldosterone in rats was also inhibited by L-158,809. Unlike angiotensin converting enzyme inhibitors, L-158,809 did not potentiate the hypotensive responses to i.v. bradykinin. L-158,809 was antihypertensive in high renin hypertensive rats (aortic coarction) and volume-depleted rhesus monkeys. The maximum hypotensive responses with acute doses of L-158,809 were equal to those with an angiotensin converting enzyme inhibitor in these renin-dependent animal models. From these in vivo data, L-158,809 is a selective AII receptor antagonist with high potency, good p.o. absorption, long duration and antihypertensive efficacy equal to angiotensin converting enzyme inhibition after single doses.     

In vitro pharmacological characterization of UP 269-6, a novel nonpeptide angiotensin II receptor antagonist

Summary— The in vitro pharmacology of UP 269-6, a novel nonpeptide angiotensin II antagonist, was examined in radioligand binding and functional isolated tissue assays. UP 269-6 bound selectively to AT₁ receptors as evidenced by the inhibition of specific [¹²⁵I] Sar¹, Ile⁸-AII binding in rat adrenal membranes (IC₅₀ = 35.8 nM) and in cultured vascular smooth muscle cells (IC₅₀ = 23.8 nM). UP 269-6 displayed a very high selectivity for the AT₁ compared to the AT₂ receptor subtype (IC₅₀ > 10,000 nM). UP 269-6 inhibited the AII-induced contraction of isolated rabbit aortic strips. The pattern of AII antagonism suggested competitive antagonism at low concentrations (10⁻¹⁰, 3 × 10⁻¹⁰, 10⁻⁹ M) of UP 269-6 and insurmountable antagonism at higher concentrations (3 × 10⁻⁹, 10⁻⁸, 3 × 10⁻⁸ M). Based on the calculated p A₂ values, UP 269-6 (9.86 ± 0.25) was an angiotensin II receptor antagonist as potent as L-158,809 (9.82 ± 0.37) and much more potent than losartan (7.96 ± 0.38). UP 269-6 was devoid of affinity (IC₅₀ > 10,000 nM) for many other receptors, ion channels and uptake sites, demonstrating its high specificity for AII receptors. Furthermore, this compound did not affect the contractile response to KCl or phenylephrine in rabbit aorta and exhibited no effect on angiotensin converting enzyme activity. These data demonstrate that UP 269-6 is a highly potent, selective and specific AT₁ receptor antagonist.     

Nonpeptide angiotensin II receptor antagonists. III. Structure-function studies

A series of 1-benzylimidazole-5-acetate derivatives defining the critical substituents on the phenyl ring was synthesized in order to improve on the affinity of 2-butyl-4-chloro-1-(2-nitrobenzyl)imidazole-5-acetate, sodium (S-8308) for the angiotensin II (AII) receptor. The analogs, substituted with -1-(4-carboxybenzyl) (EXP6155),-1-[4-(2-carboxybenzamido)benzyl] (EXP6159) and the 5-methylacetate of EXP6159 (EXP6803), were found to inhibit the binding of [3H]AII to AII receptors in rat adrenal cortical microsomes with 9-, 35- and 107-fold higher affinity, respectively, than that of S-8308 (IC50, 15 X 10(-6) microM). Scatchard analysis of the [3H]AII binding revealed that in the presence of EXP6155 (10(-6) M), the dissociation constant for AII was increased from 1.2 to 3.9 X 10(-9) M, whereas the total number of binding sites remained unchanged, suggesting a competitive nature of antagonism. A similar order of affinity or potency (saralasin much greater than EXP6803 greater than EXP6159 greater than EXP6155 greater than S8308) was observed in various in vitro and in vivo assays: rat smooth muscle cells AII binding, 45Ca++ influx in rat aortic rings, contractile response in isolated rabbit aorta and AII-induced pressor response in anesthetized rats. Responses (45Ca++ and contraction) elicited by norepinephrine or by KCl were unaltered by these agents at concentrations of up to 10(-4) M. In addition, they exerted no direct effect on the activity of rabbit angiotensin converting enzyme and rat renin. In conscious renal artery-ligated rats, EXP6155, EXP6159 and EXP6803 were p.o. inactive, but caused a rapid decrease in mean arterial pressure when administered i.v.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization of the nonpeptide angiotensin II receptor antagonist, SK&F 108566.

The angiotensin II (AII) antagonist activity of (E)-alpha-[[2-butyl-1-[(4-carboxyphenyl)methyl]-1H-imidazol-5- yl]methylene]-2-thiophenepropanoic acid (SK&F 108566), was examined in a number of in vitro and in vivo assays. In rat and human adrenal cortical membranes, SK&F 108566 displaced specifically bound [125I]AII with IC50 of 9.2 and 3.9 nM, respectively. SK&F 108566 also inhibited [125I]AII binding to human liver membranes (IC50 = 1.7 nM) and to rat mesenteric artery membranes (IC50 = 1.5 nM). In rabbit aortic smooth muscle cells, SK&F 108566 caused a concentration-dependent inhibition of AII-induced increases in intracellular Ca++ levels. In rabbit aortic rings, SK&F 108566 produced parallel rightward shifts in the AII concentration-response curve without affecting the maximal contractile response. Schild analysis of the data yielded a KB value of 0.26 nM and a slope not different from 1, indicative of competition antagonism. SK&F 108566 had no effect on the contractile responses to KCl, norepinephrine or endothelin in rabbit aorta. In conscious normotensive rats, i.v. administration of SK&F 108566 (0.01-0.3 mg/kg) produced dose-dependent parallel shifts in the AII pressor dose-response curve. Administration of SK&F 108566 (3-10 mg/kg) intraduodenally or intragastrically to conscious normotensive rats resulted in a dose-dependent inhibition of the pressor response to AII (250 ng/kg, i.v.). At 10 mg/kg, i.d., significant inhibition of the pressor response to AII was observed for 3 hr. In this same rat model, SK&F 108566 had no effect on base-line pressure or on the pressor response to norepinephrine or vasopressin. The data demonstrate that SK&F 108566 is a potent, highly selective, competitive nonpeptide AII antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of renal angiotensin II receptors using subtype selective antagonists.

Two angiotensin II (AII) receptor subtypes, AT1 and AT2, have recently been identified based on their relative affinities for selective peptide and nonpeptide antagonists. In the present study we used various AII peptide analogs, the AT1 subtype selective antagonists, DuP 753 and SK&F 108566, and the AT2 subtype selective antagonists, WL-19 and CGP 42112A, to determine whether AII receptor subtypes exist in the kidney. In agreement with previous studies, octapeptide (Sar1,Ile8-AII) and heptapeptide (AIII and Ile8-AIII) AII analogs displaced [125I]AII bound to rat glomerular membranes with similar affinities. However, in membranes derived from cortical tubules and the outer medulla, the heptapeptide analogs were 20-fold less potent in competing with [125I]AII binding than octapeptide analogs. The AT1 subtype selective nonpeptide AII antagonists, DuP 753 and SK&F 108566, totally displaced [125I]AII binding from all three membrane preparations in a monophasic manner with IC50 values in the 5 to 30 nM range. The AT2 selective peptide antagonist, CGP 42112A, had a low affinity in AII three membranes (IC50 = 450-1050 nM), whereas the nonpeptide AT2 selective antagonist, WL-19, had no activity at concentrations up to 10 microM. Dithiothreitol and the nonhydrolyzable GTP analog, 5'-guanylyl-imidodiphosphate, inhibited AII binding to all three membrane preparations. Based on these results, we conclude that the AII receptors located on glomeruli, tubules and in the outer medulla belong to the AT1 subtype, and that the physiologically important renal actions of AII are mediated through activation of AT1 receptors.     

CGS 21680C, an A2 selective adenosine receptor agonist with preferential hypotensive activity

CGS 21680C (2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethyl-carboxamido adenosine) a 2-substituted analog of the riboside uronamide, 5'-N-ethylcarboxamido adenosine and the related analog CGS 21577 (2-phenethylamino-5'-N-ethylcarboxamido adenosine), have high in vitro affinity for brain striatal adenosine A2 receptors (IC50 values = 22 and 13 nM, respectively). Both compounds were considerably less active at A1 receptors with CGS 21577 and CGS 21680C having respective IC50 values of 0.76 and 3.1 microM. The former compound was thus 59-fold selective for A2 receptors whereas CGS 21680C was 140-fold selective. In contrast, the reference A2 selective ligand, CV 1808 (2-phenylaminoadenosine), showed only 8-fold selectivity as an A2 ligand, having an IC50 of 115 nM in the [3H]-5'N-ethylcarboxamide adenosine assay and an IC50 of 910 nM at the N6-[3H] cyclohexyladenosine site. Further examination of CGS 21680C showed that the compound was without effect on binding to 17 other putative neurotransmitter/neuromodulator sites indicating its selectivity as an adenosine receptor ligand. In an isolated perfused working rat heart model, CGS 21680C effectively increased coronary flow with an ED25 value of 1.8 nM. The corresponding value for CGS 21577 was 3 nM whereas that for CV 1808 was 110 nM. The EC25 for eliciting bradycardia for all three compounds was greater than 1000 nM. The effects of all three compounds could be reversed by treatment with the xanthine adenosine antagonist, xanthine amine congener.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of receptor cycling in the regulation of angiotensin II surface receptor number and angiotensin II uptake in rat vascular smooth muscle cells.

In vivo data on the factors controlling angiotensin II (AII) cell surface binding are conflicting. We studied the specific effects of AII on AII binding in rat mesenteric artery vascular smooth muscle cells in culture. Incubation with unlabeled AII at 21 degrees C resulted in time- and concentration-dependent decreases in AII surface binding at 4 degrees C, with a 30% reduction after exposure to 300 nM AII for 15 min. Reductions in cell surface binding were due to decrements in receptor number rather than changes in binding affinity. Loss of surface receptors was mediated by receptor internalization as maneuvers that blocked ligand internalization (cold temperature and phenylarsine oxide [PAO]) attenuated AII-induced loss of surface receptors. After removal of AII, recovery of surface binding was rapid (t1/2 = 15 min) and was mediated by reinsertion of a preexisting pool of receptors into the surface membrane rather than by new receptor synthesis. To determine the role of receptor cycling on AII-induced surface receptor loss, cells were incubated with the endosomal inhibitor chloroquine during exposure to AII at 21 degrees C. Incubation with AII plus chloroquine resulted in a 70% greater loss of surface binding than after incubation with AII alone. To determine the role of receptor cycling on uptake of ligand, cells were incubated with PAO or endosomal inhibitors during exposure to AII at 4 and 21 degrees C. Compared with buffer these agents did not alter AII uptake at 4 degrees C, but decreased uptake by 12-50% at 21 degrees C. These results indicate that after binding AII receptors cycle and that receptor cycling attenuates AII-induced losses of surface receptors and enhances ligand uptake by providing a continuous source of receptors to the cell surface.     

Cardiac angiotensin receptors: effects of selective angiotensin II receptor antagonists, DUP 753 and PD 121981, in rabbit heart.

Angiotensin II (AII) elicits a positive inotropic response in cardiac muscle preparations from several species including humans. The purpose of this study was to characterize the AII binding sites and inotropic responses in rabbit ventricle using the selective AII receptor antagonists/ligands, DuP 753 (AT1) and PD 121981 (AT2). Biphasic displacement of specific 125I-Sar1,Ile8-AII binding was observed with both DuP 753 and PD 121981, suggesting the presence of two AII binding sites. The high affinity site for DuP 753 (29 nM) was a low affinity site for PD 121981 (91 microM), and the high affinity site for PD 121981 (78 nM) was a low affinity site for DuP 753 (81 microM). Of the specific AII binding, 70% was DuP 753 (AT1)-sensitive sites. Positive inotropic responses to AII in isolated papillary muscles from rabbit heart were antagonized competitively by both DuP 753 and PD 121981. The potencies of DuP 753 (pA2 = 7.99) and PD 121981 (pA2 = 4.28) to antagonize AII inotropic responses were similar to their potencies to displace 125I-Sar1,Ile8-AII from AT1 sites. There was no apparent functional consequence of AII interaction with AT2 site. Inotropic responses to isoproterenol were unaffected by DuP 753 and PD 121981. Therefore, there are two binding sites for AII in rabbit ventricle; however, only one site, AT1, participates in the inotropic response to AII. The roles of these receptor subtypes in other cardiac responses to AII have yet to be determined. Also, DuP 753 and PD 121981 are useful tools to study these two AII binding sites in cardiac preparations.     

Valdecoxib: assessment of cyclooxygenase-2 potency and selectivity

The discovery of a second isoform of cyclooxygenase (COX) led to the search for compounds that could selectively inhibit COX-2 in humans while sparing prostaglandin formation from COX-1. Celecoxib and rofecoxib were among the molecules developed from these efforts. We report here the pharmacological properties of a third selective COX-2 inhibitor, valdecoxib, which is the most potent and in vitro selective of the marketed COX-2 inhibitors that we have studied. Recombinant human COX-1 and COX-2 were used to screen for new highly potent and in vitro selective COX-2 inhibitors and compare kinetic mechanisms of binding and enzyme inhibition with other COX inhibitors. Valdecoxib potently inhibits recombinant COX-2, with an IC(50) of 0.005 microM; this compares with IC values of 0.05 microM for celecoxib, 0.5 microM for rofecoxib, and 5 microM for etoricoxib. Unique binding interactions of valdecoxib with COX-2 translate into a fast rate of inactivation of COX-2 (110,000 M/s compared with 7000 M/s for rofecoxib and 80 M/s for etoricoxib). The overall saturation binding affinity for COX-2 of valdecoxib is 2.6 nM (compared with 1.6 nM for celecoxib, 51 nM for rofecoxib, and 260 nM for etoricoxib), with a slow off-rate (t(1/2) approximately 98 min). Valdecoxib inhibits COX-1 in a competitive fashion only at very high concentrations (IC(50) = 150 microM). Collectively, these data provide a mechanistic basis for the potency and in vitro selectivity of valdecoxib for COX-2. Valdecoxib showed similar activity in the human whole-blood COX assay (COX-2 IC(50) = 0.24 microM; COX-1 IC(50) = 21.9 microM). We also determined whether this in vitro potency and selectivity translated to significant potency in vivo. In rats, valdecoxib demonstrated marked potency in acute and chronic models of inflammation (air pouch ED(50) = 0.06 mg/kg; paw edema ED(50) = 5.9 mg/kg; adjuvant arthritis ED(50) = 0.03 mg/kg). In these same animals, COX-1 was spared at doses greater than 200 mg/kg. These data provide a basis for the observed potent anti-inflammatory activity of valdecoxib in humans.     

In vitro pharmacology of a nonpeptidic angiotensin II receptor antagonist, SC-51316.

The properties of a novel nonpeptidic angiotensin II (AII) receptor antagonist, 2,5-dibutyl-2,4-dihydro-4-([2-(1H-tetrazol-5-yl)(1,1'-biphenyl) -4'-yl]methyl)-3H-1,2,4-triazol-3-one (SC-51316), are described. SC-51316 inhibited [125I]AII binding selectively to the AT1 receptor with IC50 values of 3.6 and 5.1 nM in rat adrenal cortical and rat uterine membrane preparations, respectively. The compound was a competitive and reversible antagonist of AII-mediated contraction of rabbit aortic rings with a pA2 of 8.86. In addition, SC-51316 inhibited AII-induced aldosterone release from rat adrenal zona glomerulosa cells and blocked inhibition of renin release by AII from rat kidney slices with pA2 values of 8.62 and 8.9, respectively. The agent (0.1 mM) did not inhibit angiotensin-converting enzyme or plasma renin activity. These data demonstrate that SC-51316 is a potent AII receptor antagonist which may prove to be useful as a pharmacologic tool for studying the role of the renin-angiotensin system in cardiovascular diseases.     

Tectoridins modulate skeletal and cardiac muscle sarcoplasmic reticulum calcium-release channels

The isoflavones tectoridin (TTR) and 3'-hydroxy TTR (3'-TTR) were isolated from an Ayurvedic herbal preparation Vac? and evaluated for their affinity and effect on ryanodine receptors (RyR) using junctional sarcoplasmic reticulum vesicles (JSRVs). In [(3)H]ryanodine displacement binding affinity assays, TTR and 3'-TTR exhibited IC(50) values of 17.3 +/- 1.3 microM (K(d) = 6.7 +/- 0.4 microM) and 6.6 +/- 1.4 microM (K(d) = 2.4 +/- 0.2 microM), respectively, for fast skeletal muscle RyR (RyR1) compared with an IC(50) value for ryanodine of 6.2 +/- 0.4 nM (K(d) = 2.4 nM). TTR demonstrated a 3-fold higher affinity for cardiac RyR (RyR2) [IC(50) value of 5.2 +/- 0.6 microM (K(d) = 0.95 +/- 0.3 microM)] than for RyR1. The displacement isotherms for both TTRs paralleled that for ryanodine, consistent with the notion that all three are likely binding to similar site(s) on the receptors. Calcium efflux from and calcium influx into JSRVs were used to measure function effects of TTRs on binding to RyR. In calcium efflux assays, TTR (up to 1 mM) enhanced the release of (45)Ca(2+) from JSRVs in a concentration-dependent manner (EC(50act) of 750 microM). Higher concentrations deactivated (partially closed) RyR1. 3'-TTR had similar effects, but was approximately 2-fold more potent, exhibiting an EC(50act) value of 480 microM. Using passive calcium influx assays, TTR activated and deactivated RyR1 in a time- and concentration-dependent manner. The aglycone tectorigenin also was effective in displacing [(3)H]ryanodine from RyR1 but not from RyR2. These results demonstrate that TTRs are capable of interacting at ryanodine binding sites to differentially modulate fast skeletal and cardiac calcium-release channels.     

In vitro antagonistic properties of a new angiotensin type 1 receptor blocker, azilsartan, in receptor binding and function studies

The angiotensin II (AII) antagonistic action of azilsartan (AZL) [2-ethoxy-1-{[2'-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methyl}-1H-benzimidazole-7-carboxylic acid] was investigated in radioligand binding and function studies. AZL inhibited the specific binding of 12?I-Sar1-Ile?-AII to human angiotensin type 1 receptors with an IC?? of 2.6 nM. The inhibitory effect of AZL persisted after washout of the free compound (IC(50) value of 7.4 nM). Olmesartan, telmisartan, valsartan, and irbesartan also inhibited the specific binding with IC?? values of 6.7, 5.1, 44.9, and 15.8 nM, respectively. However, their inhibitory effects were markedly attenuated with washout (IC?? values of 242.5, 191.6, >10,000, and >10,000 nM). AZL also inhibited the accumulation of AII-induced inositol 1-phosphate (IP1) in the cell-based assay with an IC?? value of 9.2 nmol; this effect was resistant to washout (IC?? value of 81.3 nM). Olmesartan and valsartan inhibited IP1 accumulation with IC?? values of 12.2 and 59.8 nM, respectively. The activities of these compounds were markedly reduced after washout (IC?? value of 908.5 and 22,664.4 nM). AZL was defined as an inverse agonist in an experiment by using a constitutively active mutant of human angiotensin type 1 receptors. In isolated rabbit aortic strips, AZL reduced the maximal contractile response to AII with a pD'? value of 9.9. The inhibitory effects of AZL on contractile responses induced by AII persisted after the strips were washed; these inhibitory effects were more potent than those of olmesartan. These results suggest that AZL is a highly potent and slowly dissociating AII receptor blocker. Its tight receptor binding might be expected to produce potent and long-lasting antihypertensive effects in preclinical and clinical settings.     

Modulation of ligand binding to leukotriene B4 receptors on guinea pig lung membranes by sulfhydryl modifying reagents

We investigated the mechanism and modulation of 3H-labeled leukotriene B4 (LTB4) binding to guinea pig lung membranes. [3H] LTB4 bound specifically and rapidly (Kobs = 0.06 +/- 0.006 min-1) to high affinity (Kd = 0.63 +/- 0.06 nM) and saturable (Bmax = 224 +/- 16 fmol/mg) sites without cooperativity (nH = 1.05). Bound ligand was partially (70%) dissociated with excess LTB4 or the selective antagonist U-75,302. Complete dissociation could be achieved with either LTB4 or U-75,302 in combination with 10 microM GTP gamma S. A series of structural analogs and selective antagonists inhibited binding in a competitive manner with the following order: LTB4 much greater than 20-(OH)-LTB4 greater than LTB5 greater than LTB-dimethylamide = U-75,302 greater than 12(R)-hydroxy-eicosatetraenoic acid greater than 5(S),12(S)-dihydroxy,6-trans,8-cis,10-trans,14-cis-ETE greater than 12(S)-hydroxy-eicosatetraenoic acid much greater than trans-LTB4 isomers. 5(S)-hydroxy-eicosatetraenoic acid, prostaglandins, peptide-leukotrienes and platelet-activating factor (at 10 microM each) did not inhibit, providing evidence that [3H]LTB4 bound to specific receptors on guinea pig lung membranes. N-Ethylmaleimide (NEM) and p-chloromercuriphenyl sulfonic acid (PCMP) inhibited binding (IC50 = 144 +/- 66 and 4.6 +/- 1.0 microM, respectively) in an irreversible manner, as evident by an inability to overcome the inhibition by extensive washing.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel phencyclidine analog interacts selectively with mu opioid receptors

A new potent analgesic drug, 1-(1-phenylcyclohexyl)-4-phenyl-4-piperidinol (PCP-4-Ph-4-OH), derived from phencyclidine was tested for its interactions with different types of opioid receptors. The antinociceptive effect of PCP-4-Ph-4-OH in the mouse writhing test (ED50 = 0.3 mg/kg) is reversed by low doses of naloxone (pA2 = 6.98). The potency of PCP-4-Ph-OH in the inhibition of the electrically induced contractions of the guinea-pig ileum (IC50 = 17 nM) is 8-fold higher than that in the mouse vas deferens preparation (IC50 = 130 nM). The concentration of naloxone required to double the IC50 (Ke) of PCP-4-Ph-4-OH is 1.5 to 1.9 nM in both preparations. In opioid radioreceptor assays, PCP-4-Ph-4-OH displays 60- to-300 fold higher affinity for the [3H] dihydromorphine (mu) and D-[3H]Ala2-MePhe-Gly-ol5-enkephalin (mu) binding sites than for D-[3H]Ala2-D-Leu5-enkephalin (delta) sites in rat brain and [3H]bremazocine (kappa) sites in guinea-pig cerebellar membrane preparations. These results suggest that PCP-4-Ph-4-OH interacts with high affinity and selectivity with mu opioid receptors.     

Biochemical and pharmacological properties of SR 49059, a new, potent, nonpeptide antagonist of rat and human vasopressin V1a receptors.

SR 49059, a new potent and selective orally active, nonpeptide vasopressin (AVP) antagonist has been characterized in several in vitro and in vivo models. SR 49059 showed high affinity for V1a receptors from rat liver (Ki = 1.6 +/- 0.2) and human platelets, adrenals, and myometrium (Ki ranging from 1.1 to 6.3 nM). The previously described nonpeptide V1 antagonist, OPC-21268, was almost inactive in human tissues at concentrations up to 100 microM. SR 49059 exhibited much lower affinity (two orders of magnitude or more) for AVP V2 (bovine and human), V1b (human), and oxytocin (rat and human) receptors and had no measurable affinity for a great number of other receptors. In vitro, AVP-induced contraction of rat caudal artery was competitively antagonized by SR 49059 (pA2 = 9.42). Furthermore, SR 49059 inhibited AVP-induced human platelet aggregation with an IC50 value of 3.7 +/- 0.4 nM, while OPC-21268 was inactive up to 20 microM. In vivo, SR 49059 inhibited the pressor response to exogenous AVP in pithed rats (intravenous) and in conscious normotensive rats (intravenous and per os) with a long duration of action (> 8 h at 10 mg/kg p.o). In all the biological assays used, SR 49059 was devoid of any intrinsic agonistic activity. Thus, SR 49059 is the most potent and selective nonpeptide AVP V1a antagonist described so far, with marked affinity, selectivity, and efficacy toward both animal and human receptors. With this original profile, SR 49059 constitutes a powerful tool for exploring the therapeutical usefulness of a selective V1a antagonist.     

Characterization of the antagonist activity of the PAF antagonists RP 59227 and WEB 2086 on elicited guinea pig peritoneal macrophages. Evidence for variable affinities and kinetics.

The pharmacological selectivity and affinity of the two PAF antagonists RP 59227 and WEB 2086 for macrophage platelet activating factor (PAF) receptors have been investigated. PAF produced a dose-related increase in superoxide generation from elicited guinea pig peritoneal macrophages (IC50 41 +/- 16 nM, n = 9). After a 5 min preincubation time RP 59227 (0.1, 1.0 and 10 microM) produced a dose-related rightward displacement of the PAF dose-response curve, the Schild plot gave a pA2 of 7.39 +/- 0.07 (n = 5) with a slope of 1.17 +/- 0.11, indicating competitive antagonism. WEB 2086 over the same concentration range was without effect at the lower two concentrations, but at 10 microM, the displacement of the PAF dose-response curve was similar to that obtained with 10 microM RP 59227. Increasing the preincubation time to 30 min increased the affinity of RP 59227 (apparent pA2 8.76 +/- 0.28, n = 5). This was associated with a reduction in the slope of the Schild plot to 0.67 +/- 0.11, suggesting noncompetitive kinetics. The affinity of WEB 2086 could not be determined, but the antagonism appeared noncompetitive in nature. The results indicate that PAF antagonists can show variable affinities for macrophage PAF receptors depending on the experimental conditions, as well as competitive or noncompetitive kinetics depending on the contact time.     

Neuromedin B binds with high affinity, elevates cytosolic calcium and stimulates the growth of small-cell lung cancer cell lines.

Previously, gastrin-releasing peptide (GRP) receptors were identified on small-cell lung cancer (SCLC) cells and GRP functioned as a SCLC autocrine growth factor. Here the effects of neuromedin B (NMB) on SCLC cells were investigated. [125I-Tyr0]NMB bound with high affinity to three of seven SCLC cell lines examined. [125I-Tyr0]NMB bound to SCLC cell line NCI-H209 and NCI-H345 in a time-dependent and reversible manner. [125I-Tyr0]NMB bound with high affinity (Kd = 1 nM) to a single class of sites (Bmax = 800/cell). Specific [125I-Tyr0]NMB binding was inhibited with high affinity by NMB (IC50 = 1 nM) and moderate affinity by bombesin, GRP and [D-Arg1, D-Pro2, D-Trp7,9, Leu11]substance P ([APTTL]SP) but not GRP1-16 (IC50 = 50, 100, 1,000 and > 10,000 nM, respectively). In Fura 2 AM loaded NCI-H345 cells, NMB elevated cytosolic calcium in a concentration-dependent manner. NMB (10 nM) elevated the cytosolic calcium from 150 to 180 nM and calcium was released from intracellular pools. The increase in cytosolic calcium caused by 10 nM NMB was reversed by 1 microM [APTTL]SP but not 1 microM [D-Phe6]bombesin6-13methylester, a GRP receptor antagonist. Also, NMB stimulated the clonal growth of NCI-H209 and NCI-H345 in a concentration-dependent manner. The increase in the clonal growth caused by NMB was reversed by 1 microM [APTTL]SP. These data suggest that NMB receptors may regulate the proliferation of some SCLC cells.     

Muscarinic cholinergic receptor subtype on frog esophageal peptic cells: binding and secretion studies

The muscarinic receptors coupled to pepsinogen secretion on isolated frog esophageal peptic cells have been characterized using functional and radioligand binding techniques. N-[3H]methylscopolamine [( 3H]NMS) binding to intact cells was complex and indicative of a high affinity, low capacity site and a high capacity uptake site. Binding to the high capacity site was inhibited by atropine with high affinity (IC50, 3 nM) and by imipramine and propranolol with IC50 values of 70 and 270 nM, respectively. After inhibition of uptake by 30 microM propranolol, [3H]NMS bound to a single population of high affinity sites (KD, 125 +/- 16 pM), which exhibited binding site maximum of 2.1 fmol/10(6) cells, equivalent to 1260 sites/cell. Binding to these sites was reversible, stereoselective and inhibited by muscarinic receptor agonists with an order of potency: oxotremorine greater than acetylcholine greater than carbachol greater than bethanechol and by antagonists with an order of potency:atropine greater than 4-diphenylacetoxy-N-methylpiperidine methobromide greater than pirenzepine greater than AF-DX 116 (11-2[2-[[diethylamino) methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]-benzodiazepine-6-one). Pepsinogen secretion was stimulated by the agonists with an order of potency: acetylcholine greater than or equal to carbachol greater than oxotremorine greater than bethanechol. Atropine, pirenzepine and AF-DX 116 competitively inhibited carbachol-stimulated pepsinogen secretion with pA2 values of 9.58, 7.37 and 6.68, respectively, which correlated with their log (inhibition constants) for receptor binding. By contrast, agonists with significant efficacy exhibited EC50 values which were 20 to 90 times lower than their inhibition constants for binding which suggests the possibility of "spare" muscarinic receptors. Our findings indicate that functional muscarinic receptors on peptic cells exhibit similar characteristics to the high affinity sites labeled by [3H]NMS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytoskeleton-dependent endocytosis is required for apical type 1 angiotensin II receptor-mediated phospholipase C activation in cultured rat proximal tubule cells.

Renal proximal tubule sodium reabsorption is enhanced by apical or basolateral angiotensin II (AII). Although AII activates phospholipase C (PLC) in other tissues, AII coupling to PLC on either apical or basolateral surfaces of proximal tubule cells is unclear. To determine if AII causes PLC activation, and the differences between apical and basolateral AII receptor function, receptors were unilaterally activated in rat proximal tubule cells cultured on permeable, collagen-coated supports. Apical AII incubation resulted in concentration- and time-dependent inositol trisphosphate (IP3) formation. Basolateral AII caused greater IP3 responses. Apical AII-induced IP3 generation was inhibited by DuP 753, suggesting that the type 1 AII receptor subtype mediated proximal tubule PLC activation. Apical AII signaling did not result from paracellular ligand leak to basolateral receptors since AII-induced PLC activation occurred when basolateral AII receptors were occupied by Sar-Leu AII or DuP 753. Inhibition of endocytosis with phenylarsine oxide prevented apical (but not basolateral) AII-induced IP3 formation. Cytoskeletal disruption with colchicine or cytochalasin D also prevented apical AII-induced IP3 generation. These results demonstrate that in cultured rat proximal tubule cells, AII is coupled to PLC via type 1 AII receptors and cytoskeleton-dependent endocytosis is required for apical (but not basolateral) AII receptor-mediated PLC activation.     

Biochemical characterization of the triazoloquinazoline, CGS 15943, a novel, non-xanthine adenosine antagonist

CGS 15943A, a triazoloquinazoline, is a potent and selective adenosine receptor antagonist as assessed by its effects on radioligand binding and adenosine-stimulated adenylate cyclase activity in guinea pig synaptoneurosomes. At the adenosine A-1 receptor labeled with [3H]cyclohexyladenosine, CGS 15943A had an IC50 value of 20 nM. At the striatal A-2 receptor labeled with [3H]5'-N-ethylcarboxamidoadenosine in the presence of a low concentration of cyclopentyladenosine to block A-1 receptors labeled by this nonselective adenosine agonist, CGS 15943A had an IC50 value of 3 nM, indicating that the compound had some degree of selectivity for the A-2 receptor. Analysis of the effect of the compound on the saturation isotherms for each of the receptors indicated that it was a competitive antagonist at the brain A-1 receptor but that it was noncompetitive at the striatal A-2 receptor. CGS 15943A was a potent adenosine antagonist in the adenosine-stimulated adenylate cyclase system in guinea pig synaptoneurosomes, where the compound was found to have an IC50 value of 30 to 70 nM against the increase in cyclic AMP evoked by 5 microM adenosine. CGS 15943A had no effect on the binding of [3H]nitrobenzylthioinosine, a ligand thought to bind to adenosine uptake sites, and, at a concentration of 10 microM, had no effect on beef heart type III phosphodiesterase activity.(ABSTRACT TRUNCATED AT 250 WORDS)