Characterization of the binding of [3H]L-365,260: a new potent and selective brain cholecystokinin (CCK-B) and gastrin receptor antagonist radioligand

[3H]L-365,260, [(3R-(+)-2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4- benzodiazepin-3-yl)-N'-(3-methylphenyl)urea], a new potent and selective nonpeptide brain cholecystokinin (CCK-B) and gastrin receptor antagonist, bound saturably and reversibly to guinea pig brain membranes. Scatchard analysis indicated a single class of high affinity (Kd = 2.3 nM) binding sites. The binding of [3H]L-365,260 was stereospecific, because unlabeled L-365,260 (an R-enantiomer) was approximately 100 times more potent than its S-enantiomer in displacing binding. The relative potencies of various CCK/gastrin-related peptides and nonpeptide peripheral CCK-A antagonists in displacing [3H]L-365,260 brain binding correlated with their potencies in displacing the binding of 125I-CCK to brain receptors but not their potencies in displacing the peripherally selective CCK-A ligand [3H]L-364,718 from pancreatic receptors. The regional distribution of [3H]L-365,260 binding in various brain areas correlated with 125I-CCK binding. Specific [3H]L-365,260 binding to guinea pig brain membranes was reduced by omission of NaCl but was not affected by omission of MgCl2 or addition of guanosine 5'-(beta-gamma-imido)triphosphate or various pharmacological agents known to interact with other common peptide and nonpeptide receptor systems. [3H]L-365,260 also bound in a specific manner to guinea pig gastric glands but only negligibly to guinea pig or rat pancreas. The binding of [3H]L-365,260 to gastric glands was inhibited by CCK/gastrin antagonists with potencies similar to those for inhibition of 125I-gastrin binding in this tissue. Collectively, the data indicates that [3H]L-365,260 represents a new potent nonpeptide antagonist radioligand suitable for the study of brain CCK-B and gastrin receptors.     

Characterization of the binding of [3H]-(+/-)-L-364,718: a new potent, nonpeptide cholecystokinin antagonist radioligand selective for peripheral receptors

[3H]-(+/-)-L-364,718 a new, potent and selective nonpeptide peripheral cholecystokinin (CCK) antagonist bound saturably and reversibly to rat pancreatic membranes. The radioligand recognized a single class of binding sites with a high affinity (Kd = 0.23 nM). The binding of [3H]-(+/-)-L-364,718 was stereospecific in that the more biologically active (-)-enantiomer demonstrated greater potency than the (+)-enantiomer. The rank order of potency of various CCK agonists and antagonists in displacing [3H]-(+/-)-L-364,718 correlated with their ability to displace [125I]CCK-8 and their known pharmacological activities in peripheral tissues. However, the absolute potencies of agonists were greater in displacing [125I]CCK-8 than [3H]-(+/-)-L-364,718. As described for other physiologically relevant receptor systems, the potency for displacement of [3H]-(+/-)-L-364,718 binding by CCK agonists, but not antagonists, was reduced by guanosine 5'-(beta, gamma-imido)triphosphate and NaCl and enhanced by MgCl2. [3H]-(+/-)-L-364,718 also demonstrated specific binding to bovine gall bladder tissue but not guinea pig brain or gastric glands, consistent with its selectivity as a peripheral CCK antagonist. [3H]-(+/-)-L-364,718 binding to pancreatic membranes was not affected by various pharmacological agents known to interact with other common peptide and nonpeptide receptor systems. These data indicate that [3H]-(+/-)-L-364,718 represents a new potent nonpeptide antagonist radioligand for the study of peripheral CCK receptors which may allow differentiation of agonist and antagonist interactions.     

Angiotensin II receptor subtypes and biological responses in the adrenal cortex and medulla.

Angiotensin II (AII) receptor subtypes and their potential coupling mechanisms were studied using recently developed peptide and nonpeptide antagonists in rat and bovine adrenal zona glomerulosa cells, as well as in membranes prepared from rat and bovine adrenal cortex and medulla. Comparison of the potencies of these novel antagonists to displace 125I-[Sar1,Ile8]AII from its binding sites revealed two distinct AII binding sites in membranes prepared from rat adrenal capsules (zona glomerulosa) and from rat adrenal inner zones containing the medulla. About 85% of the binding sites of the glomerulosa zone and 30% of those of the inner zones were of the AT1 subtype, with relative affinities for the nonpeptide antagonists Dup 753 and PD 123177 and the peptide antagonist CGP 42112A in the order of Dup 753 much greater than CGP 42112A greater than PD 123177. In contrast, the relative binding potencies for the other (AT2) population of binding sites were CGP 42112A greater than PD 123177 much greater than Dup 753. Neither AII nor its peptide antagonist [Sar1,Ile8]AII could distinguish between the two sets of binding sites. The effects of the new antagonists on functional responses of rat adrenal glomerulosa cells demonstrated that both AII-stimulated aldosterone production and the AII-induced inhibition of adrenocorticotropic hormone-stimulated cAMP formation were mediated by the AT1 receptor subtype. In bovine adrenals, only AT1 receptors were detected in membranes prepared from the cortex and the medulla, as well as in cultured glomerulosa cells. The relative inhibitory potency of Dup 753 was lower by an order of magnitude at bovine than at rat AT1 receptors. The inhibition of AII-induced aldosterone production by the various antagonists was closely correlated with their inhibitory potencies on 125I-[Sar1,Ile8]AII binding to bovine glomerulosa cells. These data suggest that the known effects of AII in adrenal glomerulosa cells are mediated through the AT1 receptor subtype and that the distribution and/or specificity of the AT2 receptors shows marked species variations.     

L-163,491 is a partial angiotensin AT(1) receptor agonist in the hindquarters vascular bed of the cat

Responses to the nonpeptide angiotensin II agonist 5, 7-Dimethyl-2-ethyl-3-[[2'-([butyloxycarbonyl) aminosulfonyl]-5'-(3-methyoxybenzyl)-[1, 1'-biphenyl]-4-yl]methyl]-3H-imidazo[4,5-b]pyridine (L-163,491) were investigated and compared with responses to angiotensin II, angiotensin IV and norepinephrine in the hindquarters vascular bed of the cat under constant-flow conditions. Injections of L-163,491 into the hindquarter perfusion circuit caused dose-related increases in hindquarters perfusion pressure. In relative terms, angiotensin II was more potent than norepinephrine, which was more potent than angiotensin IV and L-163,491 in increasing hindlimb vascular resistance. The slope of the dose-response curve for L-163,491 was flat, while the apparent affinity of the compound for angiotensin AT(1) receptors was slightly greater than angiotensin IV. Responses to L-163,491 were inhibited by the angiotensin AT(1) receptor antagonist DuP 532 (2-propyl-4-pentafluoroethyl-1-[2'-(1H-tetrazol-5-yl)bipheny l-4-yl)me thyl]imidazole-5-carboxylic acid) and were not altered by the angiotensin AT(2) receptor antagonist PD123,319 (S(+)-1-[[4-(Dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl+ ++) -4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditribluoroacetate). However, the increase in hindlimb perfusion pressure in response to angiotensin II and angiotensin IV was significantly decreased following injection of L-163,491. These data suggest that the nonpeptide angiotensin analog L-163,491 has partial agonist activity, which is dependent on the stimulation of angiotensin AT(1) receptors in the hindquarters vascular bed of the cat.     

Binding of the antagonist [3H]candesartan to angiotensin II AT1 receptor-transfected [correction of tranfected] Chinese hamster ovary cells

Binding of the non-peptide angiotensin II AT1 antagonist [3H](2-ethoxy-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]- H-benzimidazoline-7-carboxylic acid ([3H]candesartan) to human angiotensin II AT1 receptor-transfected Chinese hamster ovary (CHO-AT1) cells was inhibited to the same extent by angiotensin II and non-peptide angiotensin II AT1 antagonists. No binding was observed in control CHO-K1 cells. Dissociation was slow (k(-1) = 0.0010+/-0.0001 min(-1)) after removal of the free [3H]candesartan but increased 5-fold upon addition of supramaximal concentrations of angiotensin II AT1 antagonists. Angiotensin II responses recovered equally slow from candesartan-pretreatment. When washed and further incubated, these angiotensin II responses also recovered more rapidly in the presence of 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphen yl-4-yl)methyl]imidazole (losartan), indicating that unlabelled ligands prevented reassociation. [3 H]candesartan saturation binding experiments required a long time to reach equilibrium. Therefore, the equilibrium dissociation constant (Kd = 51+/-8 pM) was calculated from the association and dissociation rate constants. Our findings indicate that the insurmountable nature of candesartan in functional studies is related to its slow dissociation from the receptor.     

The angiotensin AT(1) receptor antagonist irbesartan has near-peptide affinity and potently blocks receptor signaling

The angiotensin II type 1 (AT(1)) receptor plays a pivotal role in the regulation of blood pressure and electrolyte balance, and is involved in the control of specific ingestive behaviours. Irbesartan (SR 47436/BMS 186295) is a recently developed angiotensin AT(1) receptor antagonist, chemically described as 2-butyl-3-([2'-?1H-tetrazol-5-yl?biphenyl-4-yl]methyl)-1, 3-diazaspiro (4,4)non-1-en-4-one. Irbesartan displays higher affinity for its target receptor than other similar antagonists. In radioligand binding assays performed on membranes from WB-Fischer 344 (WB) rat liver epithelial cells, irbesartan was able to displace [125I]angiotensin II with a K(i) of 4.05 nM as compared to losartan (DuP 753) and tasosartan (WAY 126756), which had K(i) values of 25.2 nM and 46.6 nM, respectively. Similarly, in functional assays, irbesartan exhibited the highest functional potency to block angiotensin II-induced inositol trisphosphate (IP(3)) turnover. The improved affinity of irbesartan for the angiotensin AT(1) receptor does not coincide with a concomitant increase in affinity for the angiotensin AT(2) receptor, as irbesartan and losartan exhibited the same low potency to displace [125I]angiotensin II in radioligand binding assays performed on membranes from PC-12w cells. In binding assays performed on peripheral tissues in rat, irbesartan bound to the angiotensin AT(1) receptor expressed in liver, adrenal, kidney and pituitary with an overall affinity closely approaching that of the high affinity peptidic antagonist [Sar(1), Ile(8)]angiotensin II. Due to the higher affinity of irbesartan over other similar antagonists for the angiotensin AT(1) receptor in many tissues and its greater potency to block receptor activation, irbesartan may be quite useful in the study of the angiotensin AT(1) receptor and its role in controlling ingestive behaviours and, furthermore, shows great potential to improve the treatment of hypertension and other cardiovascular disease states.     

Characterization of the binding of a potent, selective, radioiodinated antagonist to the human neurokinin-1 receptor.

We have synthesized a potent, selective, radioiodinated antagonist of the human neurokinin-1 (NK1) receptor and have characterized its binding to the cloned receptor expressed in Chinese hamster ovary cells. (cis)-2-(Diphenylmethyl)-N-[(2-iodophenyl)-methyl]-1- azabicyclo[2.2.2]octan-3-amine (L-703606) inhibits binding of 125I-Tyr8-substance P to the human NK1 receptor with an IC50 of 2 nM. This compound is a competitive antagonist of substance P-induced inositol phosphate generation, with a Kb of 29 nM. [125I]L-703606 binds to a single class of high affinity binding sites in human NK1/Chinese hamster ovary cell membranes (Kd = 0.3 nM). Substance P inhibits the binding of [125I]L-703606 to 65% of the NK1 receptor sites with a Kd of 0.04 +/- 0.03 nM and to the remaining 35% of the sites with a Kd of 1.5 +/- 0.7 nM. Addition of the nonhydrolyzable GTP analog guanylyl-5'-(beta, gamma-imido)diphosphate [Gpp(NH)p] shifts greater than 90% of the binding sites to the lower affinity state. In addition, Gpp(NH)p markedly alters the dissociation of substance P from the NK1 receptor by increasing the number of sites in the low affinity, rapidly dissociating state. However, Gpp(NH)p does not affect the rate of dissociation of [125I]L-703606. These data suggest that the pharmacological properties of [125I]L-703606 binding to the human NK1 receptor are similar to those of antagonists of nonpeptide guanine nucleotide-binding protein-coupled receptors and that this ligand will be useful for the biochemical and pharmacological characterization of the human NK1 receptor.     

Receptor binding interactions of the angiotensin II antagonist, 125I-[sarcosine1,leucine8]angiotensin II, with mammalian brain and peripheral tissues

Sarcosine1,leucine8-angiotensin II ([Sar1,Leu8]AII), an angiotensin II antagonist, binds saturably, reversibly and with high affinity (KD's of 0.03-22 nM) to calf cerebellar cortex, bovine adrenal cortex and rabbit uterine membranes. The peptide specificity of 125I[Sar1,Leu8]AII binding to brain, adrenal cortex and uterus is consistent with the labeling of physiologically relevant angiotensin receptors. A detailed study of the binding potencies of 28 angiotensin peptide analogues reveals: (1) very significant correlations between peptide binding potencies at 125I-AII compared to 125I-[Sar1,Leu8]AII binding sites, (2) many similarities between brain and uterine receptor sites and marked differences between these two tissue receptors compared to adrenal cortical receptor sites, and (3) correlations among peptide physiological potencies (AII-contracted rabbit aortic strip) and receptor binding potencies in all three tissues labeled with either 125I-AII or 125I-[Sar1,Leu8]AII. The correlations are much better for adrenal cortex than for brain or uterus, suggesting that adrenal cortical angiotensin receptors are similar to aorta angiotensin receptors.     

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.     

Evidence for the AT1 subtype of the angiotensin II receptor in the rat submandibular gland

We have characterized angiotensin II (Ang II) receptor subtypes on rat submandibular gland membranes using a radioligand binding assay. [3H]Ang II binding to the membrane fractions exhibited both high (Kd =0.08 nm, Bmax =2.19 fmol/mg protein) and low (Kd =4.19 nm, Bmax = 13.7 fmol/mg protein) affinity. Ang 11, Ang III and saralasin completely displaced the [3H]Ang II binding, whereas CV-11974, an AT1 receptor antagonist and PD123319, an AT2 receptor antagonist maximally displaced up to approximately 87 and 13% of the total binding, respectively. [3H]DuP753 binding to the membrane fractions exhibited a single population of binding site with a Kd of 4.22 nM and Bmax of 3.77 pmol/mg protein. Ang II, Ang III and CV-11974 completely displaced the [3H]DuP753 binding with slope factors near unity, but PD123319 did not. These findings suggest that rat submandibular gland membranes contain predominantly the AT1 receptor subtype.     

3H-DOB (4-bromo-2,5-dimethoxyphenylisopropylamine) labels a guanyl nucleotide-sensitive state of cortical 5-HT2 receptors

3H-(+/-)-4-Bromo-2,5-dimethoxyphenylisopropylamine (3H-DOB), a putative agonist radioligand, was synthesized and used to label 5-HT2 receptors in a particulate fraction prepared from rat frontal cortex tissue homogenates. The specific binding (defined by the difference in 3H-DOB binding in the presence and absence of 10(-6) M cinanserin, a potent and specific 5-HT2 antagonist) displayed high affinity (KD = 4.1 X 10(-10) M) and saturability with a Bmax of 17.9 fmol/mg of protein. The distribution of specific 3H-DOB binding in nine brain regions correlated closely with the distribution of 3H-ketanserin (an antagonist radioligand)-labeled 5-HT2 receptors. Competition studies in frontal cortex homogenates using a variety of compounds revealed a distinct 5-HT2 receptor pharmacology. A series of 5-HT2 antagonists exhibited high affinities in competition studies for specific 3H-DOB binding. The absolute potencies of these antagonists as well as their order of potencies closely correlated with their potencies in competing for 3H-ketanserin-labeled brain 5-HT2 receptors. A series of 5-HT2 agonists also exhibited high affinities in competition studies for specific 3H-DOB binding. Although the order of potencies of these agonists was similar to their order in competing for 3H-ketanserin-labeled brain 5-HT2 receptors, the agonists displayed 10-100-fold higher affinities for the 3H-DOB-labeled sites than for the 3H-ketanserin-labeled sites. The level of specific 3H-DOB binding in the frontal cortex homogenates was approximately 5% of the levels of 3H-ketanserin-labeled 5-HT2 receptors (358 fmol/mg of protein). Taken together, these results indicate that 3H-DOB labels a subset of brain 5-HT2 receptors that has high affinity for agonists as well as antagonists); 3H-ketanserin appears to label both subsets of brain 5-HT2 receptors. Antagonists apparently do not discriminate between these two subsets of 5-HT2 receptors. 3H-DOB specific binding to 5-HT2 receptors was potently inhibited by guanosine 5'-(beta, gamma-imido)triphosphate and guanosine 5'-O-(3-thio)triphosphate (nonhydrolyzable derivatives of GTP) with IC50 values of 42 and 21 nM, respectively, whereas adenosine 5'-(beta, gamma-imido)triphosphate and adenosine 5'-O-(3-thio)triphosphate (nonhydrolyzable derivatives of ATP) had no effect. In summary, 3H-DOB specific binding displays the pharmacological characteristics of a 5-HT2 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Clonidine-displacing substance from bovine brain binds to imidazoline receptors and releases catecholamines in adrenal chromaffin cells.

Identification of nonadrenergic binding sites for clonidine and related imidazolines in brain and peripheral tissues and partial purification of an endogenous ligand for these sites have led to the postulation of a novel transmitter/receptor system. The receptors seem to be present in adrenal medulla and to regulate chromaffin cell function. The present study was undertaken to test the ability of the putative endogenous ligand clonidine-displacing substance (CDS) to displace [3H]idazoxan binding to adrenal chromaffin cell membranes and to release catecholamines from cultured chromaffin cells. CDS potently displaces [3H]idazoxan binding to chromaffin cell membranes, with an IC50 of 5 units. The displacement of [3H]idazoxan binding by CDS was not modified by guanosine 5'-(beta, gamma-imido)triphosphate, suggesting that the imidazoline binding sites may not be GTP-binding protein-coupled receptors. CDS produced a large release of catecholamines from chromaffin cells, and the release was partially blocked by cobalt, a calcium channel blocker. The calcium-dependent release reached a plateau above 5 units of CDS, with a maximal response at 15 min. It is concluded that endogenous CDS, prepared from brain, regulates the secretion of catecholamines from adrenal chromaffin cells, probably by activating imidazole receptors.     

DuP 753 can antagonize the effects of angiotensin II in rat liver

Results obtained with the use of nonpeptide angiotensin II receptor antagonists have suggested the presence of multiple subtypes of angiotensin II receptors in rat adrenal gland. However, the effects of nonpeptide antagonists on second messenger production by angiotensin II have not been investigated. In rat liver, angiotensin II can both activate phospholipase C, generating inositol polyphosphates and raising internal calcium, and inhibit adenylate cyclase. DuP 753 and PD123177, two nonpeptide angiotensin II antagonists, were used to characterize the receptor population in rat liver and to investigate the possibility that different angiotensin II receptor subtypes couple to different second messenger pathways. DuP 753 could completely antagonize the binding of angiotensin II in rat liver membranes, with a K1 of 9.3 x 10(-9) M. PD123177 had no effect on the binding of angiotensin II in rat liver at concentrations between 1 x 10(-9) M and 3 x 10(-5) M, in contrast to its ability to inhibit angiotensin II binding in rat adrenal. At a concentration of 10(-5) M, DuP 753 could inhibit increases in internal free calcium, could prevent production of inositol polyphosphates, and could attenuate inhibition of adenylate cyclase produced by angiotensin II. PD123177 at concentrations between 1 x 10(-9) M and 3 x 10(-5) M was ineffective in all of these assays. The results indicate that DuP 753 can displace the binding of angiotensin II at all receptor sites in rat liver and that this drug can attenuate both of the second messenger events produced by the angiotensin II receptor.     

Cardiovascular effects of L-158,809, a new angiotensin type 1 receptor antagonist,assessed using the halothane-anesthetized in vivo canine model

L-158,809 is a new angiotensin II type 1 receptor antagonist. We simultaneously assessed its antagonistic potency and cardiovascular effects with the halothane-anesthetized in vivo canine model (n = 5). L-158,809 was intravenously infused over 10 min at escalating doses of 0.03, 0.3 and 3 mg/kg. Angiotensin II (0.1 microg/kg, i.v.)-induced vasopressor and negative inotropic responses were significantly suppressed from the low dose L-158,809. Meanwhile, L-158,809 did not affect any of the cardiovascular parameters except that QTc was slightly shortened after the high dose administration. These results support the previous in vitro knowledge that L-158,809 is a highly selective angiotensin II receptor antagonist.     

Pharmacologic characterization of the oxytocin receptor in human uterine smooth muscle cells

[(3)H]-oxytocin was used to characterize the oxytocin receptor found in human uterine smooth muscle cells (USMC). Specific binding of [(3)H]-oxytocin to USMC plasma membranes was dependent upon time, temperature and membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high-affinity binding sites with an apparent equilibrium dissociation constant (K(d)) of 0.76 nM and a maximum receptor density (B(max)) of 153 fmol mg(-1) protein. The Hill coefficient (n(H)) did not differ significantly from unity, suggesting binding to homogenous, non-interacting receptor populations. Competitive inhibition of [(3)H]-oxytocin binding showed that oxytocin and vasopressin (AVP) receptor agonists and antagonists displaced [(3)H]-oxytocin in a concentration-dependent manner. The order of potencies for peptide agonists and antagonists was: oxytocin>[Asu(1,6)]-oxytocin>AVP= atosiban>d(CH(2))(5)Tyr(Me)AVP>[Thr(4),Gly(7)]-oxytocin>dDAVP, and for nonpeptide antagonists was: L-371257>YM087>SR 49059>OPC-21268>SR 121463A>OPC-31260. Oxytocin significantly induced concentration-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and hyperplasia in USMC. The oxytocin receptor antagonists, atosiban and L-371257, potently and concentration-dependently inhibited oxytocin-induced [Ca(2+)](i) increase and hyperplasia. In contrast, the V(1A) receptor selective antagonist, SR 49059, and the V(2) receptor selective antagonist, SR 121463A, did not potently inhibit oxytocin-induced [Ca(2+)](i) increase and hyperplasia. The potency order of antagonists in inhibiting oxytocin-induced [Ca(2+)](i) increase and hyperplasia was similar to that observed in radioligand binding assays. In conclusion, these data provide evidence that the high-affinity [(3)H]-oxytocin binding site found in human USMC is a functional oxytocin receptor coupled to [Ca(2+)](i) increase and cell growth. Thus human USMC may prove to be a valuable tool in further investigation of the physiologic and pathophysiologic roles of oxytocin in the uterus. British Journal of Pharmacology (2000) 129, 131 - 139     

Effect of saponin and filipin on antagonist binding to AT 1 receptors in intact cells

In the present study, [ 3H ]-candesartan binding experiments were performed on intact Chinese Hamster Ovary cells transfected with the human AT1 receptor (CHO-AT1 cells). Cells were pre-treated with 0.01mg/ml saponin or filipin. Both pre-treatments resulted in an increased dissociation rate and decreased affinity of the insurmountable non-peptide antagonist [3H ]-candesartan. A similar decrease in affinity was observed for the peptide antagonist Sar1-Ile8 angiotensin II and for other non-peptide antagonists, irrespectively of their degree of insurmountability. A similar discrepancy in [ 3H ]-candesartan binding was earlier observed when comparing intact CHO-AT1 cells and membrane preparations thereof. This similarity is further highlighted by the observations that saponin or filipin no longer affect [ 3H ]-candesartan binding to CHO-AT1 cell membranes and that both agents permeabilise the CHO-AT1 cells. This suggests that the intracellular composition and/or organisation of living cells play an active role with regard to antagonist-AT1 receptor interactions.     

Characterization of angiotensin II antagonism displayed by Ib, a novel nonpeptide angiotensin AT(1) receptor antagonist

The pharmacologic profile of Ib, 5-n-butyl-4-{4-[2-(1H-tetrazole-5-yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6-dichloridephenyl)-3H-1,2,4-triazol-3-one, a novel nonpeptide angiotensin AT(1) receptor antagonist, was investigated by receptor-binding studies, functional in vitro assays with rabbit and rat aorta, and in vivo experiments in rats. Ib inhibited [(125)I] angiotensin II binding to AT(1) receptors in rat liver membranes (K(i)=2.5+/-0.5 nM) and did not interact with AT(2) receptors in bovine cerebellar membranes. In functional studies with rat and rabbit aorta, Ib inhibited the contractile response to angiotensin II (pD(2)' value: 7.43 and 7.29, respectively) with a significant reduction in the maximum. In pithed rats, Ib inhibited the angiotensin II induced pressor response in a dose-related manner. After intravenous administration, Ib produced a dose-dependent antihypertensive effects in spontaneously hypertensive rats and renal hypertensive rats. These results suggest that Ib is a potent angiotensin AT(1) selective receptor antagonist with a mode of insurmountable antagonism.     

Dopamine D2 receptor binding in adrenal medulla: characterization using [3H]spiperone

The possibility that dopamine may function as a neuromodulator or neurotransmitter in the adrenal gland, and not merely serve as a precursor to the catecholamines, has been suggested. If this hypothesis is correct, receptors for dopamine should be identifiable in the adrenal. The present work demonstrates the existence of a high-affinity receptor in adrenal medulla using [3H]spiperone as the radioligand to label the receptors. [3H]Spiperone bound rapidly, reversibly, and with high affinity to bovine adrenal medullary membranes. Scatchard analysis yielded a Kd of 0.09 nM and a Bmax of 51 fmol/mg protein. In competition binding experiments, dopaminergic antagonists were at least 100 times more potent in displacing [3H]spiperone from its binding sites than adrenergic or serotonergic receptor antagonists. Similarly, agonists at the dopamine receptor more readily competed for [3H]spiperone binding than other receptor agonist drugs tested. Furthermore, D2 selective antagonists and agonists were much more potent than D1 receptor ligands. These results suggest that [3H]spiperone may bind to a high-affinity D2 dopamine receptor in adrenal medulla.     

Two distinct angiotensin II receptor binding sites in rat adrenal revealed by new selective nonpeptide ligands

The nonpeptide angiotensin II antagonists Dup-89 and WL-19 displaced specific 125I-angiotensin II binding in rat whole adrenal in a clearly biphasic manner, indicating the presence of high (nanomolar) and low (micromolar) affinity sites, each representing approximately 50% of the total maximal number of binding sites. Displacement studies using sufficient concentrations of either antagonist to prevent binding to its respective high affinity site revealed that the high affinity binding sites for Dup-89 and WL-19 were distinct and corresponded to the low affinity site of the other. Both binding sites were also present in the adrenal capsule (cortex) and adrenal decapsulated (medulla) tissue. The two 125I-angiotensin II binding sites were also differentiated by their sensitivity to dithiothreitol and the relative affinities of angiotensin II, angiotensin III, and their respective Sar1,Ile8- and Ile7-substituted antagonist analogs. Only Dup-89 (KB = 13 nM) was effective in antagonizing angiotensin II-stimulated aldosterone release from dispersed adrenal capsular cells, indicating that this functional response is mediated by an action upon the 125I-angiotensin II binding site at which Dup-89 has high affinity. Collectively, the data provide additional strong support for the presence of two distinct angiotensin II receptor subtypes in the rat adrenal.     

Tight binding of the angiotensin AT(1) receptor antagonist

Angiotensin II induces angiotensin AT(1) receptor internalization via Clathrin coated pits formation. We investigated whether insurmountable inhibition by the non-peptide antagonist 2-ethoxy-1-[(2'-(1H-tetrazol-5-yl) biphenyl-4-yl) methyl]-1H-benzimidazoline-7-carboxylic acid (candesartan) was related to receptor internalization. Mild acid treatment can discriminate between internalized and cell surface bound [(3)H]angiotensin II. In contrast, it provides no information about the subcellular localization of bound [(3)H]candesartan since this binding is acid resistant. The internalization of [(3)H]angiotensin II is rapidly inhibited in the presence of 0.4 M sucrose. Yet, no such rapid effect was noticed for [(3)H]candesartan. [(3)H]candesartan displays insurmountable/long lasting binding to the vast majority of both wild type and L(314) truncated rat angiotensin AT(1A) receptors with impaired receptor internalization. In agreement with previously published AT(1) angiotensin receptor visualization experiments, the present data suggest that non-peptide antagonist-angiotensin AT(1) receptor complexes remain at the cell surface. Insurmountable antagonism of candesartan is therefore independent from receptor internalization via clathrin-coated pits.