The CCK(2) receptor antagonist, YF476, inhibits Mastomys ECL cell hyperplasia and gastric carcinoid tumor development.

YF476 is a potent and highly selective cholecystokin 2 (CCK(2)) receptor antagonist of the benzodiazepine class. It inhibits gastric neuroendocrine enterochromaffin-like (ECL) cell secretion, proliferation and spontaneous formation of gastric neuroendocrine tumors (carcinoids) in cotton rats. The Mastomys rodent species exhibits a genetic predisposition to gastric ECL neuroendocrine tumor formation which can be accelerated by acid suppression and induction of hypergastrinemia. In this respect, it mimics the human condition of atrophic gastritis, hypergastrinemia and gastric carcinoid development. We investigated whether YF476 could inhibit acid suppression-induced ECL cell hyperplasia and neoplasia in this model. In addition, we examined whether YF476 could reverse established ECL cell hyperplasia and neoplasia. Targeting the CCK(2) receptor during Loxtidine-induced hypergastrinemia resulted in a reduction in ECL cell secretion (plasma and mucosal histamine, and histidine decarboxylase (HDC) transcripts, p<0.05) and proliferation (numbers of HDC-positive cells, connective tissue growth factor (CTGF) and cyclin D1 transcription). This was associated with a decrease in ECL cell hyperplasia and a 60% reduction in gastric ECL cell microcarcinoid (tumors <0.3mm in size) formation. YF476 inhibited ECL cell neoplasia (gastric carcinoid) in animals with hyperplasia, inhibited the formation of ECL cell tumors when co-administered with Loxtidine and reversed the growth and developement of gastric ECL cell carcinoids in long-term acid suppressed Mastomys. Variable importance analysis using a logistic multinomial regression model indicated the effects of YF476 were specific to the ECL cell and alterations in ECL cell function reflected inhibition of transcripts for HDC, Chromogranin A (CgA), CCK(2) and the autocrine growth factor, CTGF. We conclude that specifically targeting the CCK(2) receptor inhibits gastrin-mediated ECL cell secretion and ECL cell proliferation and tumor development in vivo.

A comparison of the effects of gastrin, somatostatin and dopamine receptor ligands on rat gastric enterochromaffin-like cell secretion and proliferation.

Gastrin regulates ECL cell histamine release and is a critical determinant of acid secretion. ECL cell secretion and proliferation is inhibited by gastrin antagonists and somatostatin but little is known about the role of dopamine agonists in this process. Since the ECL cell exhibits all three classes of receptor we evaluated and compared the effects of the gastrin receptor antagonist, (YF476), lanreotide (SST agonist) and novel dopaminergic agents (BIM53061 and BIM27A760) on ECL cell histamine secretion and proliferation. Highly enriched (>98%) ECL cell preparations prepared from rat gastric mucosa using a FACS approach were studied. Real-time PCR confirmed presence of the CCK2, SS2 and SS5 and D1 receptors on ECL cells. YF476 inhibited histamine secretion and proliferation with IC(50)s of 1.25 nM and 1.3 x 10(-11) M respectively, values 10-1000x more potent than L365,260. Lanreotide inhibited secretion and proliferation (2.2 nM, 1.9 x 10(-10) M) and increased YF476-inhibited proliferation a further 5-fold. The dopamine agonist, BIM53061, inhibited gastrin-mediated ECL cell secretion and proliferation (17 nM, 6 x 10(-10) M) as did the novel dopamine/somatostatin chimera BIM23A760 (22 nM, 4.9 x 10(-10) M). Our studies demonstrate that the gastrin receptor antagonist, YF476, is the most potent inhibitor of ECL cell histamine secretion and proliferation. Lanreotide, a dopamine agonist and a dopamine/somatostatin chimera inhibited ECL cell function but were 10-1000x less potent than YF476. Agents that selectively target the CCK2 receptor may provide alternative therapeutic strategies for gastrin-mediated gastrointestinal cell secretion and proliferation such as evident in the hypergastrinemic gastric carcinoids associated with low acid states.

Histamine H3-receptor agonists and imidazole-based H3-receptor antagonists can be thermodynamically discriminated.

BACKGROUND AND PURPOSE: Studies suggest that measurement of thermodynamic parameters can allow discrimination of agonists and antagonists. Here we investigate whether agonists and antagonists can be thermodynamically discriminated at histamine H(3) receptors. EXPERIMENTAL APPROACH: The pK(L) of the antagonist radioligand, [(3)H]-clobenpropit, in guinea-pig cortex membranes was estimated at 4, 12, 21 and 30 degrees C in 20 mM HEPES-NaOH buffer (buffer A), or buffer A containing 300 mM CaCl(2), (buffer A(Ca)). pK(I)' values for ligands with varying intrinsic activity were determined in buffer A and A(Ca) at 4, 12, 21 and 30 degrees C. KEY RESULTS: In buffer A, the pK(L) of [(3)H]-clobenpropit increased with decreasing temperature while it did not change in buffer A(Ca). The Bmax was not affected by temperature or buffer and n (H) values were not different from unity. In buffer A, pK(I)' values for agonists remained unchanged or decreased with decreasing temperature, while antagonist pK(I) values increased with decreasing temperature; agonist binding was entropy-driven while antagonist binding was enthalpy and entropy-driven. In buffer A(Ca), temperature had no effect on antagonist and agonist pK(I) values; both agonist and antagonist binding were enthalpy and entropy-driven. CONCLUSIONS AND IMPLICATIONS: The binding of H(3)-receptor agonists and antagonists can be thermodynamically discriminated under conditions where agonist pK(I)' values are over-estimated (pK(I)' not = pK(app)). However, under conditions when agonist pK(I) approximately pK(app), the thermodynamics underlying the binding of agonists are not different to those of antagonists.

Correlation of apparent affinity values from H3-receptor binding assays with apparent affinity (pKapp) and intrinsic activity (alpha) from functional bioassays.

BACKGROUND AND PURPOSE: Agonist apparent affinities (pK(I)') in histamine H(3)-receptor binding assays were higher than expected from apparent affinity values (pK(app)) estimated in bioassay. Here, we investigate whether the degree of pK(I)' overestimation is related to agonist intrinsic efficacy, by studying the effect of buffer composition on the pK(I)' of ligands with varying intrinsic activity. EXPERIMENTAL APPROACH: In the guinea-pig ileum bioassay, intrinsic activity (alpha) was determined from the maximal inhibition of the contraction produced by increasing agonist concentration. pK(app) values were estimated using the method of Furchgott. The pK(L) of [(3)H]clobenpropit in guinea-pig cerebral cortex was estimated by saturation analysis in 20 mM HEPES-NaOH buffer (buffer B(0,0,0)), or buffer B(0,0,0) containing 70 mM CaCl(2), 100 mM NaCl and 100 mM KCl (buffer B(0.07,0.1,0.1)). PK(I) values were determined in competition studies in both buffers. KEY RESULTS: [(3)H]clobenpropit saturation isotherms had n (H) values of unity in both buffers. In buffer B(0.07,0.1,0.1), agonist pK(I)' values were closer to pK(app) values than in buffer B(0,0,0) but were associated with n (H) values <1. A two-site analysis of agonist data in buffer B(0.07, 0.1, 0.1) provided a better fit than a one-site fit and low affinity values (pK(IL)) were comparable to pK(app). Differences between the pK(I)' in buffer B(0,0,0) and pK(IL) values in buffer B(0.07,0.1,0.1) (DeltapK) were correlated with alpha. CONCLUSIONS AND IMPLICATIONS: H(3)-receptor binding assays conducted in buffer B(0,0,0) and buffer B(0.07,0.1,0.1) can provide a measure of ligand affinity (pK(app)) and intrinsic efficacy. The assay predicts that some ligands previously classified as H(3)-receptor antagonists may possess residual intrinsic efficacy.

Unexpected relationship between plasma protein binding and the pharmacodynamics of 2-NAP, a CCK1-receptor antagonist.

UNLABELLED: WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT?: * Two chemically diverse CCK1 receptor antagonists have been shown clinically to inhibit CCK-evoked contraction of human gallbladder [2, 3]. These studies have not examined the relationship between plasma concentration and effect, the latter usually considered to be predictive from the free drug concentration [8]. * We wanted to examine our novel CCK1 receptor antagonist in this validated model and also to explore its PK-PD relationship. WHAT THIS STUDY ADDS: * 2-NAP inhibited CCK-evoked human gallbladder contraction in vivo but at a plasma free concentration that was, in theory, too low to have achieved adequate CCK1 receptor occupancy. * The study serves as a caveat to the assumption that free plasma concentration can be used to predict pharmacological effect. AIMS: To study the pharmacokinetics and pharmacodynamics of 2-NAP (2-naphthalenesulfonyl-L-aspartyl-(2-phenethyl)amide), a selective CCK1 receptor antagonist in healthy volunteers. METHODS: 2-NAP was given to 12 healthy male volunteers in an ascending dose, safety and PK phase 1a study by 1 h i.v. infusion (0.6-9.6 mg kg(-1) h(-1)). A further 12 healthy male volunteers received i.v. CCK-8S (6.25 pmol kg(-1) h(-1)) to produce gallbladder contraction, measured by ultrasound recordings of gallbladder volume, and the effect of concurrent i.v. 2-NAP administration was studied. Plasma protein binding in vitro and ex vivo was measured by ultrafiltration and by equilibrium dialysis. RESULTS: 2-NAP was generally well tolerated, displayed linear pharmacokinetics and a very high degree of plasma protein binding (99.9%). A 105 min i.v. CCK-8S infusion induced a reduction in gallbladder volume of 14.9 (+/-7.0) ml during placebo co-infusion and this was reduced to 2.4 (+/-5.9) ml when 2-NAP was co-infused with CCK-8S (P = 0.00024, paired t-test, mean change 12.5 ml; 95% CI For mean 7.4, 18.3 ml). This extent of inhibition was consistent with a 2-NAP total plasma concentration of 36 microm, but when protein binding corrections were made, the 'free concentration' of 2-NAP was only 0.04 microm, a value much less than the average equilibrium dissociation constant of 2-NAP for human CCK1 receptors ( approximately 0.7 microm). CONCLUSIONS: The pharmacological effect of a drug is usually considered to be determined by its free concentration. However, the complete inhibition of CCK-8S-evoked gallbladder contraction by a free plasma concentration of 0.04 microm 2-NAP was much greater than would have been predicted from simple drug-receptor occupancy theory and cautions against the general use of free concentration of drug for predicting pharmacological effect.

Isolation of BVDV from bovine serum by EMEA/CVMP and 9 CFR: a comparison.

Data derived from both experience and validation exercises on the behaviour and properties of BVDV is presented as applied to recovery of the virus from bovine serum used in the industrial production of animal biologicals. Licensed products both in the EU and the U.S. must use ingredients shown to be free of this agent and both regulatory agencies have published regulations governing this topic (EU: EMEA/CVMP and US: 9 CFR). The two systems differ in some specifics covering cell cultures used, timing of testing within the assays and the use of multiple approaches to the isolation of this virus. It was found that selected cell lines instead of primary cells could be used for BVDV isolation. FA tests, however, needed to be performed weekly for maximum sensitivity instead of once at the end of the test. BVDV interference assay (comparative titration) was found to be an unreliable test for the detection of non-CPE BVDV in cell culture. BVDV neutralization assays, while desirable from an informational standpoint, were determined to be unnecessary in serum that was to be adequately irradiated. Ultracentrifuge concentration of BVDV from serum was found to be a satisfactory method of increasing the sensitivity of assays for this agent, although quantification of the virus was found unnecessary in serum to be irradiated. Recommendations for the harmonization of 9 CFR and EMEA/CVMP into one assay are given.

Cardiac-specific overexpression of human beta2 adrenoceptors in mice exposes coupling to both Gs and Gi proteins.

1. Left atrial strips from transgenic (TG4) mice with cardiac-specific overexpression ( approximately 200-fold) of the beta(2) adrenoceptor (beta(2)AR) were isolated, and their isometric force of contraction (F(c)) in response to electrical stimulation was measured. 2. The betaAR agonist isoprenaline elicited negative inotropic responses in all left atrial strips; in 6/11 preparations, it also had a small positive inotropic effect. This 'up-phase' was observed from 0.1 to 10 nM, with the 'down-phase' occurring at higher concentrations. Both phases were mediated by beta(2)AR, as shown by their sensitivity to the beta(2)AR antagonist ICI-118,551 (100 nM; pA(2) 8.60+/-0.07, 8.45+/-0.19, for 'up-phase' and 'down-phase,' respectively), but not the beta(1)AR antagonist CGP-20712A (100 nM). Conversely, nontransgenic littermate preparations responded to isoprenaline treatment solely by an increase in F(c), which was beta(1)AR-mediated. 3. Pretreatment of left atrial strips with either 10 nM isoprenaline or 1 mM 8-bromo-cAMP significantly attenuated the TG4 'up-phase', while having no effect on either the TG4 'down-phase' or the littermate controls' responses. B. pertussis toxin treatment of the animals prevented isoprenaline's negative inotropic effects in TG4 preparations, but had no effect in littermate controls. 4. The findings imply that the responses of TG4 left atrium to isoprenaline are because of beta(2)AR coupling to G(s) and G(i) proteins, consistent with the model of Daaka et al., in which protein kinase A phosphorylation of the beta(2)AR causes a switch from G(s) to G(i) protein coupling.

Development of peptide 3D structure mimetics: rational design of novel peptoid cholecystokinin receptor antagonists.

The two hormones cholecystokinin and gastrin share the same C-terminal sequence of amino acids, namely Gly(29)-Trp(30)-Met(31)-Asp(32)-Phe(33)-NH(2). Nevertheless, this congruence has not precluded using this structure to develop selective ligands for either CCK(1) or CCK(2) receptors. Manipulation of the hydrophobic residues at positions 31 and 33 gave a series of CCK(1) tripeptide antagonists, typified by N-t-BOC-Trp-2-Nal-Asp-2-(phenyl)ethylamide (pK(B) 6.8 +/- 0.3). Molecular modeling was used to identify the bioactive conformation of these CCK(1)-selective compounds and prompted the design of new peptoid structures. We aimed to maintain the conformation of the parent series by exploiting patterns of hydrogen-bonding and pi-stacking interactions present in the original molecule, rather than introducing additional covalent bonds. The prototype, N-(succinyl-D-Asp-2-phenylethylamido)-L-Trp-2-(2-naphthyl)ethylami de, was a potent and selective CCK(1) antagonist (pK(B) 7.2 +/- 0.3). Furthermore, the new series showed patterns of biological activity that mirrored those of the parent tripeptides. These compounds contain elements of both peptide primary and secondary structure and represent a novel approach to designing peptidomimetics. Interesting results were obtained from comparing models of a representative tripeptide CCK(1) antagonist with a conformation of CCK(30)(-)(33) that others have proposed to be responsible for its activity at the CCK(2) receptor. The results suggest that CCK(1) and CCK(2) receptors recognize enatiomeric dispositions of the Trp(30) indole, Asp(32) carboxylic acid, and C-terminal phenyl groups arrayed about a common backbone configuration. This "functional chirality" may underpin the mechanism by which these closely related receptor systems bind CCK(30)(-)(33) and explain patterns of selectivity observed with optical isomers of a number of peptoid and nonpeptide ligands.

Negative inotropic effects of isoprenaline on isolated left atrial assays from aged transgenic mice with cardiac over-expression of human beta(2)-adrenoceptors.

The action of isoprenaline has been evaluated in an isolated, left atrial assay, from aged transgenic mice with cardiac-specific over-expression of the beta(2)-adrenoceptor. In the assay, isoprenaline produced a negative inotropic concentration-response curve that was not altered by incubation with CGP-20712A (1 microM), a beta(1)-adrenoceptor antagonist. However, after incubation with ICI-118,551 (300 nM), a selective beta(2)-adrenoceptor antagonist, isoprenaline produced a positive inotropic concentration-effect curve that was located to the left of the negative inotropic curve. This suggests that the negative inotropic effect was mediated by a homogenous population of negatively-coupled beta(2)-adrenoceptors. In the presence of CGP-20712A (300 nM), the positive curve was shifted to the right, suggesting that the positive inotropic effect was mediated, at least in part, by beta(1)-adrenoceptors. These results differ substantially from those previously obtained in young transgenic mice. An outline of an explanatory model, based on a concept of over-expressed receptors 'stealing' G-proteins, is suggested.

Characterization of the binding of [3H]-clobenpropit to histamine H3-receptors in guinea-pig cerebral cortex membranes.

1 We have investigated the binding of a novel histamine H3-receptor antagonist radioligand, [3H]- clobenpropit ([3H]-VUF9153), to guinea-pig cerebral cortex membranes. 2 Saturation isotherms for [3H]-clobenpropit appeared biphasic. Scatchard plots were curvilinear and Hill plot slopes were significantly less than unity (0.63+/-0.03; n = 12+/-s.e.mean). The radioligand appeared to label two sites in guinea-pig cerebral cortex membranes with apparent affinities (pKD') of 10.91+/-0.12 (Bmax = 5.34+/-0.85 fmol mg(-1) original wet weight) and 9.17+/-0.16 (Bmax = 23.20+/-6.70 fmol mg(-1)). 3 In the presence of metyrapone (3 mM) or sodium chloride (100 mM), [3H]-clobenpropit appeared to label a homogeneous receptor population (Bmax=3.41+/-0.46 fmol mg-1 and 3.49+/-0.44 fmol mg(-1), pKD' = 10.59+/-0.17 and 10.77+/-0.02, respectively). Scatchard plots were linear and Hill slopes were not significantly different from unity (0.91+/-0.04 and 0.99+/-0.02, respectively). Granisetron (1 microM), rilmenidine (3 microM), idazoxan (0.3 microM), pentazocine (3 microM) and 1,3-di-(2-tolyl)guanidine (0.3 microM) had no effect on the binding of [3H]-clobenpropit. 4 The specific binding of [3H]-clobenpropit appeared to reach equilibrium after 25 min at 21+/-3 degrees C and remained constant for >180 min. The estimated pKD' (10.27+/-0.27; n = 3+/-s.e.mean) was not significantly different from that estimated by saturation analysis in the presence of metyrapone. 5 A series of histamine H3-receptor ligands expressed affinity values for sites labelled with [3H]-clobenpropit which were not significantly different from those estimated when [3H]-R-alpha-MH was used to label histamine H3-receptors in guinea-pig cerebral cortex membranes.

Evidence that histamine homologues discriminate between H3-receptors in guinea-pig cerebral cortex and ileum longitudinal muscle myenteric plexus.

1. The binding of the selective histamine H3-receptor agonist ([3H]-R-alpha-methylhistamine) to sites in guinea-pig cerebral cortex and ileum longitudinal muscle myenteric plexus has been characterized and a comparison made of the apparent affinities of a series of H3-receptor ligands. 2. Saturation analysis suggested that [3H]-R-alpha-methylhistamine labelled a homogeneous population of histamine H3-receptors in guinea-pig cerebral cortex (pKD=9.91+/-0. 07; nH=1.07+/-0.03; n=5) and ileum longitudinal muscle myenteric plexus (pKD=9.75+/-0.21; nH=0.97+/-0.02; n=5). There was no significant difference in the estimated affinity of [3H]-R-alpha-methylhistamine in the two tissues. The cerebral cortex had a significantly higher receptor density (3.91+/-0.37 fmol mg-1 tissue) than the ileum longitudinal muscle myenteric plexus (0. 39+/-0.11 fmol mg-1). 3. Overall, the apparent affinities of compounds, classified as H3-receptor ligands, in cerebral cortex and ileum longitudinal muscle myenteric plexus were well correlated (r=0. 91, P<0.0001) and consistent with the cerebral cortex and ileum longitudinal muscle myenteric plexus expressing histamine H3-receptor population(s) that are pharmacologically indistinguishable by the majority of histamine H3-receptor ligands. However, it was evident that the homologues of histamine within this group of compounds could discriminate between the receptor populations in the two tissues. Thus, the estimated affinity of five imidazole unbranched alkylamines (histamine, homohistamine, VUF4701, VUF4732 and impentamine) were significantly higher in the guinea-pig cerebral cortex than in the ileum longitudinal muscle myenteric plexus assay.

Analysis of the behaviour of selected CCKB/gastrin receptor antagonists in radioligand binding assays performed in mouse and rat cerebral cortex.

1. The previously described complex behaviour of the CCKB/gastrin receptor antagonist, L-365,260, in radioligand binding assays could be explained by a variable population of two binding sites. We have investigated whether other CCKB/gastrin receptor ligands (PD134,308, PD140,376, YM022 and JB93182) can distinguish between these sites. 2. In the mouse cortex assay, Hill slopes were not different from unity and the ligand pKI values did not differ when either [125I]-BH-CCK-8S or [3H]-PD140,376 was used as label as expected for a single site (G2). 3. In the rat cortex, where previous analysis of replicate (n=48) L-365,260 data indicated the presence of two CCKB/gastrin sites (G1 and G2), the competition data for PD134,308, PD140,376, YM022 and JB93182 could be explained by a homogeneous population of CCKB/gastrin sites because the Hill slope estimates were not significantly different from unity. However, the estimated affinity values for JB93182 and YM022 were significantly higher and that for PD134,308 was significantly lower than those obtained in the mouse cortex when the same radioligand was used. In view of our previous data obtained with L-365,260, the rat cortex data were also interpreted using a two-site model. In this analysis, SR27897 expressed approximately 9 fold, PD134,308 approximately 13 fold and PD140,376 approximately 11 fold selectivity for the G2 site. In contrast, JB93182 expressed approximately 23 fold and YM022 approximately 4 fold selectivity for the G1 site. If the two-site interpretation of the data is valid then, because of its reverse selectivity to L-365,260, JB93182 has been identified as a compound which if radiolabelled could provide a test of this receptor subdivision.

Characterization of the binding of a novel radioligand to CCKB/gastrin receptors in membranes from rat cerebral cortex.

1. We have investigated the binding of a novel radiolabelled CCKB/gastrin receptor ligand, [3H]-JB93182 (5[[[(1S)-[[(3,5-dicarboxyphenyl)amino]carbonyl]-2-phenylethyla mino]-carbonyl]-6-[[(1-adamantylmethyl) amino]carbonyl]-indole), to sites in rat cortex membranes. 2. The [3H]-JB93182 was 97% radiochemically pure as assessed by reverse-phase HPLC (RP-HPLC) and was not degraded by incubation (150 min) with rat cortex membranes. 3. Saturation analysis indicated that [3H]-JB93182 labelled a homogeneous population of receptors in rat cortex membranes (pKD=9.48+/-0.08, Bmax=3.61+/-0.65 pmol g(-1) tissue, nH=0.97+/-0.02, n=5). The pKD was not significantly different when estimated by association-dissociation analysis (pKD=9.73+/-0.11; n=10). 4. In competition studies, the low affinity of the CCKA receptor antagonists, L-364,718; SR27897 and 2-NAP, suggest that, under the assay conditions employed, [3H]-JB93182 (0.3 nM) does not label CCKA receptors in the rat cortex. 5. The affinity estimates obtained for reference CCKB/gastrin receptor antagonists were indistinguishable from one of the affinity values obtained when a two site model was used to interpret [125I]-BH-CCK8S competition curves obtained in the same tissue (Harper et al., 1999). 6. This study provides further evidence for the existence of two CCKB/gastrin sites in rat cortex. [3H]-JB93182 appears to label selectively sites previously designated as gastrin-G1 and therefore it may be a useful compound for the further discrimination and characterization of these putative receptor subtypes.

Breast disease: dynamic spiral MR imaging.

PURPOSE: To compare various subjective, empiric, and pharmacokinetic methods for interpreting findings at dynamic magnetic resonance (MR) imaging of the breast. MATERIALS AND METHODS: Dynamic spiral breast MR imaging was performed in 52 women suspected of having or with known breast disease. Gadolinium-enhanced images were obtained at 12 locations through the whole breast every 7.8 seconds for 8.5 minutes after bolus injection of contrast material. Time-signal intensity curves from regions of interest corresponding to 57 pathologically proved lesions were analyzed by means of a two-compartment pharmacokinetic model, and the diagnostic performance of various parameters was analyzed. RESULTS: Findings included invasive carcinoma in 17 patients, isolated ductal carcinoma in situ (DCIS) in six, and benign lesions in 34. Although some overlap between carcinomas and benign diagnoses was noted for all parameters, receiver operating characteristic analysis indicated that the exchange rate constant had the greatest overall ability to discriminate benign and malignant disease. The elimination rate constant and washout were the most specific parameters. The exchange rate constant, wash-in, and extrapolation point were the most sensitive parameters. DCIS was not consistently distinguished from benign disease with any method. CONCLUSION: Dynamic spiral breast MR imaging proved an excellent method with which to collect contrast enhancement data rapidly enough that accurate comparisons can be made between many analytic methods.

Pharmacological classification of adenosine receptors in the sinoatrial and atrioventricular nodes of the guinea-pig.

1. The effects of seven agonist and three antagonist adenosine receptor ligands were compared on the guinea-pig sinoatrial (SA) node (isolated right atrium) and atrioventricular (AV) node (perfused whole heart). Single agonist concentration-effect curves were obtained to 5'-N-ethylcarboxamidoadenosine (NECA), R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA), N6-cyclohexyladenosine (CHA), 2-chloroadenosine (CADO),),S(+)-N6-(2-phenylisopropyl)adenosine (L-PIA), 2-phenylaminoadenosine (CV 1808) and N6-aminoadenosine (MeAdo). Adenosine and/or NECA curves were obtained in the absence and presence of the antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 9-chloro-2 (2-furanyl)-5,6-dihydro-1,2,4-triazolo[1,5-c]quinazolin-5-imine (CGS15943) and N6-(endonorbornan-2-yl)-9-methyladenine (N-0861). 2. A formal comparison of the agonist and antagonist potency data was made by fitting the data to a straight line using a least squares procedure based on principal components analysis to account for the variance on both axes. The antagonist affinity estimates made on the two assays did not deviate significantly from the line of identity. 3. The agonist p[A]50 data obtained on the two assays did not deviate from the line of identity, indicating that there were no significant differences in potencies between the two assays. The p[A]50 ratio of R-PIA and S-PIA was 1.24+/-0.09 in the SA node and 1.36+/-0.11 in the AV node, indicating no difference in the stereoselectivity of the PIA isomers between the two tissues. 4. The agonist potency and antagonist affinity data obtained are consistent with previous findings showing that the AV and SA node data are pharmacologically indistinguishable and belong to the adenosine A1-receptor class. No evidence for the reported A3-receptor was found.