Simulating the influence of plasma protein on measured receptor affinity in biochemical assays reveals the utility of Schild analysis for estimating compound affinity for plasma proteins.

BACKGROUND AND PURPOSE: Plasma protein binding (PPB) influences the free fraction of drug available to bind to its target and is therefore an important consideration in drug discovery. While traditional methods for assessing PPB (e.g. rapid equilibrium dialysis) are suitable for comparing compounds with relatively weak PPB, they are not able to accurately discriminate between highly bound compounds (typically >99.5%). The aim of the present work was to use mathematical modelling to explore the potential utility of receptor binding and cellular functional assays to estimate the affinity of compounds for plasma proteins. Plasma proteins are routinely added to in vitro assays, so a secondary goal was to investigate the effect of plasma proteins on observed ligand-receptor interactions. EXPERIMENTAL APPROACH: Using the principle of conservation of mass and the law of mass action, a cubic equation was derived describing the ligand-receptor complex [LR] in the presence of plasma protein at equilibrium. KEY RESULTS: The model demonstrates the profound influence of PPB on in vitro assays and identifies the utility of Schild analysis, which is usually applied to determine receptor-antagonist affinities, for calculating affinity at plasma proteins (termed KP ). We have also extended this analysis to functional effects using operational modelling and demonstrate that these approaches can also be applied to cell-based assay systems. CONCLUSIONS AND IMPLICATIONS: These mathematical models can potentially be used in conjunction with experimental data to estimate drug-plasma protein affinities in the earliest phases of drug discovery programmes.

Potent and efficacious inhibition of CXCR2 signaling by biparatopic nanobodies combining two distinct modes of action.

Chemokines and chemokine receptors are key modulators in inflammatory diseases and malignancies. Here, we describe the identification and pharmacologic characterization of nanobodies selectively blocking CXCR2, the most promiscuous of all chemokine receptors. Two classes of selective monovalent nanobodies were identified, and detailed epitope mapping showed that these bind to distinct, nonoverlapping epitopes on the CXCR2 receptor. The N-terminal-binding or class 1 monovalent nanobodies possessed potencies in the single-digit nanomolar range but lacked complete efficacy at high agonist concentrations. In contrast, the extracellular loop-binding or class 2 monovalent nanobodies were of lower potency but were more efficacious and competitively inhibited the CXCR2-mediated functional response in both recombinant and neutrophil in vitro assays. In addition to blocking CXCR2 signaling mediated by CXCL1 (growth-related oncogene α) and CXCL8 (interleukin-8), both classes of nanobodies displayed inverse agonist behavior. Bivalent and biparatopic nanobodies were generated, respectively combining nanobodies from the same or different classes via glycine/serine linkers. Interestingly, receptor mutation and competition studies demonstrated that the biparatopic nanobodies were able to avidly bind epitopes within one or across two CXCR2 receptor molecules. Most importantly, the biparatopic nanobodies were superior over their monovalent and bivalent counterparts in terms of potency and efficacy.

Detailed analysis of biased histamine H4 receptor signalling by JNJ 7777120 analogues.

BACKGROUND AND PURPOSE: The histamine H4 receptor, originally thought to signal merely through Gαi proteins, has recently been shown to also recruit and signal via ß-arrestin2. Following the discovery that the reference antagonist indolecarboxamide JNJ 7777120 appears to be a partial agonist in ß-arrestin2 recruitment, we have identified additional biased hH4R ligands that preferentially couple to Gαi or ß-arrestin2 proteins. In this study, we explored ligand and receptor regions that are important for biased hH4R signalling. EXPERIMENTAL APPROACH: We evaluated a series of 48 indolecarboxamides with subtle structural differences for their ability to induce hH4R-mediated Gαi protein signalling or ß-arrestin2 recruitment. Subsequently, a Fingerprints for Ligands and Proteins three-dimensional quantitative structure-activity relationship analysis correlated intrinsic activity values with structural ligand requirements. Moreover, a hH4R homology model was used to identify receptor regions important for biased hH4R signalling. KEY RESULTS: One indolecarboxamide (75) with a nitro substituent on position R7 of the aromatic ring displayed an equal preference for the Gαi and ß-arrestin2 pathway and was classified as unbiased hH4R ligand. The other 47 indolecarboxamides were ß-arrestin2-biased agonists. Intrinsic activities of the unbiased as well as ß-arrestin2-biased indolecarboxamides to induce ß-arrestin2 recruitment could be correlated with different ligand features and hH4R regions. CONCLUSION AND IMPLICATIONS: Small structural modifications resulted in diverse intrinsic activities for unbiased (75) and ß-arrestin2-biased indolecarboxamides. Analysis of ligand and receptor features revealed efficacy hotspots responsible for biased-ß-arrestin2 recruitment. This knowledge is useful for the design of hH4R ligands with biased intrinsic activities and aids our understanding of the mechanism of H4R activation.

Reassessment of the pharmacology of Sphingosine-1-phosphate S1P3 receptor ligands using the DiscoveRx PathHunter™ and Ca2+ release functional assays.

BACKGROUND AND PURPOSE: DiscoverRx's PathHunter™ assay measures GPCR agonist potency, via the recruitment of ß-arrestin, independent of the subtype of G(α) protein activated. This assay is frequently used in drug discovery although little is known about the agonist pharmacology generated. Here we have compared agonist potency, efficacy and affinity values obtained in PathHunter™ assays with those from more established radioligand binding and functional techniques. EXPERIMENTAL APPROACH: Using cells expressing the human sphingosine-1-phosphate S1P(3) receptor at four different densities, we compared pharmacological affinity and efficacy values of four structurally distinct ligands - FTY720-P, VPC24191, CYM5442 and the endogenous agonist S1P - obtained from competition binding, functional Ca(2+) release and PathHunter™ assays. KEY RESULTS: The pK(i) values for S1P were significantly different (9.34 ± 0.10 and 8.92 ± 0.15) in clones expressing different receptor levels using the binding assay. In the PathHunter™ and Ca(2+) assays, S1P and CYM5442 were full agonists, FTY720-P was a partial agonist, while the efficacy of VPC24191 could not be detected in PathHunter™ assays. VPC23019, previously described as a S1P(1/3) receptor antagonist, behaved as an S1P(3) receptor partial agonist in the Ca(2+) release assay. CONCLUSIONS AND IMPLICATIONS: Comparison of data from the PathHunter™ assay with binding and functional Ca(2+) assays suggest that PathHunter™ assays measured a different agonist-bound receptor conformation. While this assay has great utility in drug discovery, care must be taken as high-efficacy, low-affinity agonist compounds would not be detected. Therefore highly amplified, more traditional assays are necessary to identify agonists with low efficacy.

A common intracellular allosteric binding site for antagonists of the CXCR2 receptor.

BACKGROUND AND PURPOSE: We have previously shown that SB265610 (1-(2-bromo-phenyl)-3-(7-cyano-3H-benzotriazol-4-yl)-urea) behaves as an allosteric, inverse agonist at the C-X-C chemokine (CXCR)2 receptor. The aim of this study was to determine whether SB265610, in addition to two other known antagonists, bind to either of the two putative, topographically distinct, allosteric binding sites previously reported in the Literature. EXPERIMENTAL APPROACH: Ten single point mutations were introduced into the CXCR2 receptor using site-directed mutagenesis. Three CXCR2 antagonists were investigated, SB265610, Pteridone-1 (2-(2,3 difluoro-benzylsulphanyl)-4-((R)-2-hydroxy-1-methyl-ethylamino)-8H-pteridin-7-one) and Sch527123 (2-hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methyl-furan-2-yl)-propyl]amino]-3,4-dioxo-cyclobut-1enylamino}-benzamide), and the effect of these mutations on their binding affinity and ability to inhibit interleukin-8-stimulated binding of [(35)S]GTPgammaS was examined. KEY RESULTS: Seven of the nine mutations introduced into the C-terminal domain and intracellular loops of the receptor produced a significant reduction in affinity at least one of the antagonists tested. Of those seven mutations, three produced a significant reduction in the affinity of all three antagonists, namely K320A, Y314A and D84N. In all but one mutation, the changes observed on antagonist affinity were matched with effects on inhibition of interleukin-8-stimulated [(35)S]GTPgammaS binding. CONCLUSIONS AND IMPLICATIONS: These antagonists bind to a common intracellular, allosteric, binding site of the CXCR2 receptor, which has been further delineated. As many of these mutations are close to the site of G protein coupling or to a region of the receptor that is responsible for the transduction of the activation signal, our results suggest a molecular mechanism for the inhibition of receptor activation.

Pharmacogenetic characterization of indacaterol, a novel beta 2-adrenoceptor agonist.

BACKGROUND AND PURPOSE: Indacaterol is a novel beta(2)-adrenoceptor agonist in development for the treatment of chronic obstructive pulmonary disease. The aim of this study was to investigate the comparative pharmacology of indacaterol in recombinant cells expressing the common polymorphic variants of the human beta(2)-adrenoceptor and in human primary airway smooth muscle (ASM) cells. EXPERIMENTAL APPROACH: Chinese hamster ovarian-K1 cell lines expressing high and low levels of the common human beta(2)-adrenoceptor variants were generated [Gly16-Glu27-Val34-Thr164(GEVT), RQVT, GQVT] and also the rare GQVI variant. Human primary ASM cells were isolated from explants of trachealis muscle. Adenosine-3',5'-cyclic-monophosphate production was used as an outcome measure. KEY RESULTS: In both the low- and high-expression recombinant GEVT 'wild type' cell lines indacaterol is a high-efficacy agonist. Salmeterol and formoterol were identified as low- and high-efficacy agonists, respectively, and showed similar potencies to indacaterol irrespective of the beta(2)-adrenoceptor genotype. The I164 variant cell line was associated with a reduced capacity to generate adenosine-3',5'-cyclic-monophosphate in response to beta(2)-adrenoceptor agonist. In the human primary ASM cells indacaterol gave a maximal response intermediate between that of salmeterol and formoterol. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that indacaterol is a high-efficacy agonist in recombinant cell systems but acts with lower efficacy in human primary ASM cells. No marked genotype-dependent effects were observed for common variants; however, changes in I164 receptor activity were identified, which were dependent on the level of expression of beta(2)-adrenoceptors.

SB265610 is an allosteric, inverse agonist at the human CXCR2 receptor.

BACKGROUND AND PURPOSE: In several previous studies, the C-X-C chemokine receptor (CXCR)2 antagonist 1-(2-bromo-phenyl)-3-(7-cyano-3H-benzotriazol-4-yl)-urea (SB265610) has been described as binding competitively with the endogenous agonist. This is in contrast to many other chemokine receptor antagonists, where the mechanism of antagonism has been described as allosteric. EXPERIMENTAL APPROACH: To determine whether it displays a unique mechanism among the chemokine receptor antagonists, the mode of action of SB265610 was investigated at the CXCR2 receptor using radioligand and [(35)S]-GTPgammaS binding approaches in addition to chemotaxis of human neutrophils. KEY RESULTS: In equilibrium saturation binding studies, SB265610 depressed the maximal binding of [(125)I]-interleukin-8 ([(125)I]-IL-8) without affecting the K(d). In contrast, IL-8 was unable to prevent binding of [(3)H]-SB265610. Kinetic binding experiments demonstrated that this was not an artefact of irreversible or slowly reversible binding. In functional experiments, SB265610 caused a rightward shift of the concentration-response curves to IL-8 and growth-related oncogene alpha, but also a reduction in maximal response elicited by each agonist. Fitting these data to an operational allosteric ternary complex model suggested that, once bound, SB265610 completely blocks receptor activation. SB265610 also inhibited basal [(35)S]-GTPgammaS binding in this preparation. CONCLUSIONS AND IMPLICATIONS: Taken together, these data suggest that SB265610 behaves as an allosteric inverse agonist at the CXCR2 receptor, binding at a region distinct from the agonist binding site to prevent receptor activation, possibly by interfering with G protein coupling.

Analysis of second messenger pathways stimulated by different chemokines acting at the chemokine receptor CCR5.

The chemokine receptor, CCR5, responds to several chemokines leading to changes in activity in several signalling pathways. Here, we investigated the ability of different chemokines to provide differential activation of pathways. The effects of five CC chemokines acting at CCR5 were investigated for their ability to inhibit forskolin-stimulated 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation and to stimulate Ca(2+) mobilisation in Chinese hamster ovary (CHO) cells expressing CCR5. Macrophage inflammatory protein 1alpha (D26A) (MIP-1alpha (D26A), CCL3 (D26A)), regulated on activation, normal T-cell expressed and secreted (RANTES, CCL5), MIP-1beta (CCL4) and monocyte chemoattractant protein 2 (MCP-2, CCL8) were able to inhibit forskolin-stimulated cAMP accumulation, whilst MCP-4 (CCL13) could not elicit a response. CCL3 (D26A), CCL4, CCL5, CCL8 and CCL13 were able to stimulate Ca(2+) mobilisation through CCR5, although CCL3 (D26A) and CCL5 exhibited biphasic concentration-response curves. The Ca(2+) responses induced by CCL4, CCL5, CCL8 and CCL13 were abolished by pertussis toxin, whereas the response to CCL3 (D26A) was only partially inhibited by pertussis toxin, indicating G(i/o)-independent signalling induced by this chemokine. Although the rank order of potency of chemokines was similar between the two assays, certain chemokines displayed different pharmacological profiles in cAMP inhibition and Ca(2+) mobilisation assays. For instance, whilst CCL13 could not inhibit forskolin-stimulated cAMP accumulation, this chemokine was able to induce Ca(2+) mobilisation via CCR5. It is concluded that different chemokines acting at CCR5 can induce different pharmacological responses, which may account for the broad spectrum of chemokines that can act at CCR5.

Expression and functional analysis of chemokine receptors in human peripheral blood leukocyte populations.

Emerging evidence indicates that chemokine receptor expression patterns are critical in determining the spectrum of action of the chemokines. We have analysed the expression patterns of 17 chemokine receptors and two orphan chemokine receptor-like genes in various freshly prepared human peripheral blood leucocyte populations, including neutrophils, lymphocytes, and naïve and differentiated monocytes using real-time quantitative polymerase chain reaction (TaqMan). This is the first comprehensive study of chemokine receptor expression in such a wide variety of cell types. Human peripheral blood leukocyte populations were found to express a wide range of chemokine receptors that varies depending on cell type and differentiation state. Novel expression patterns of certain chemokine receptors were seen during our analysis. For example, the orphan chemokine receptor HCR was expressed at very high levels by both primary neutrophils and primary monocytes, and was further upregulated on neutrophil activation and during monocyte to macrophage differentiation. When neutrophil calcium transients were measured in response to a panel of 30 different chemokines the results clearly correlated with the chemokine receptor expression profile. For example strong calcium responses were seen in neutrophils following stimulation with the CXCR1 and CXCR2 ligands, interleukin (IL-)8, GCP-2 and Gro-beta. These data have implications for the study of the functional responses of leukocytes to external stimuli and will aid in our understanding of general leukocyte biology.

Evidence that P2Y4 nucleotide receptors are involved in the regulation of rat aortic smooth muscle cells by UTP and ATP.

1. Previous studies have shown that ATP and UTP are able to stimulate phospholipase C (PLC) and proliferation in cultured aortic smooth muscle cells. Here we set out to characterize the receptor responsible, and investigate a possible role for p42 and p44 mitogen activated protein kinase (MAPK) in the proliferative response. 2. The phospholipase C response of spontaneously hypertensive rat (SHR) derived aortic smooth muscle cells in culture showed that the response to ATP was partial compared to the response to UTP. 3. Further studies characterized the responses of the SHR derived cells. UTP was the only full agonist with the SHR cells; UDP gave a partial response while ADP, 2-methythio-ATP and alpha,beta-methylene ATP were essentially ineffective. The response to UDP was almost lost in the presence of hexokinase, consistent with this being due to extracellular conversion to UTP. These observations are inconsistent with the response being mediated by either P2Y1 or P2Y6 receptors. 4. When increasing concentrations of ATP were present with a maximally effective concentration of UTP, the size of the response diminished, consistent with UTP and ATP acting at a single population of receptors for which ATP was a partial agonist. This is inconsistent with a response mainly at P2Y2 receptors. 5. 1321N1 cells transfected with human P2Y4 receptors gave a similar agonist response profile, with ATP being partial compared to UTP, loss of response to UDP with hexokinase treatment, and with the response to UTP diminishing in the presence of increasing concentrations of ATP. 6. Use of the reverse transcriptase-polymerase chain reaction confirmed the presence of mRNA encoding P2Y4 receptors in SHR derived vascular smooth muscle cells. Transcripts for P2Y2, P2Y4 and P2Y6 receptors, but not P2Y1 receptors, were detected. 7. Stimulation of SHR derived cells with UTP enhanced the tyrosine phosphorylation of both p42 and p44 MAPK, and the incorporation of [3H]-thymidine into DNA. Both these responses were diminished in the presence of an inhibitor of activation of MAPK. 8 These results lead to the conclusion that in SHR derived cultured aortic smooth muscle cells, PLC responses to extracellular UTP and ATP are predominantly at P2Y4 receptors, and suggest that these receptors are coupled to mitogenesis via p42/p44 MAPK.

Enhancement of the response to purinergic agonists in P2Y1 transfected 1321N1 cells by antagonists suramin and PPADS.

1. We have previously shown that both suramin and pyridoxal-phosphate-6-azophenyl-2',4' disulphonic acid (PPADS) act as antagonists at transfected P2Y1 receptors. Here we show that under certain experimental conditions these two P2 antagonists can enhance the response to agonists acting at these receptors. 2. The expression of either P2Y1 or P2Y2 receptors in 1321N1 human astrocytoma cells results, on a change of medium, in an elevation of basal (no added agonist) accumulation of [3H]-inositol(poly)phosphates([3H]-InsPx) compared to cells not expressing these receptors. This elevation is much greater in P2Y1 transfectants than in P2Y, transfectants. 3. Both PPADS and suramin reduced this basal level of [3H]-InsPx accumulation in the P2Y1 expressing cells. 4. When a protocol was used which required changing the culture medium, antagonists were added at a concentration which reduced the basal accumulation by about 50%, there was a significant stimulation in response to increasing concentrations of 2-methylthioadenosine 5'-triphosphate (2MeSATP), in the absence of antagonists there was no significant effect of the agonist. 5. However, when 2MeSATP was added in the absence of a change of medium and with no antagonist present, there was a several fold increase in [3H]-InsPx accumulation. These results show that a release of endogenous agonist activity (possibly ATP/ADP) from the P2Y1 expressing cells can create conditions in which a response to an agonist such as 2MeSATP can only be seen in the presence of a competitive antagonist.

Cloned and transfected P2Y4 receptors: characterization of a suramin and PPADS-insensitive response to UTP.

The P2Y family of receptors are G protein-coupled receptors for ATP, ADP, UTP and UDP. Recently several members of this family have been cloned, including the P2Y4, which is activated by UTP but not by ATP. In the present report, using receptors stably transfected into 1321N1 cells, we show that suramin acts as an antagonist at cloned P2Y1 and (less potently) P2Y2 receptors, but not at the cloned P2Y4 receptor. Furthermore, PPADS (pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid), a potent antagonist at the P2Y1 receptor, is a relatively inneffective antagonist at the cloned P2Y4 receptor. This work moves us closer to the goal of classifying the native P2Y receptors on the basis of agonist and antagonist profiles.

PPADS and suramin as antagonists at cloned P2Y- and P2U-purinoceptors.

1. The effect of suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on the stimulation of phospholipase C in 1321N1 cells transfected with the human P2U-purinoceptor (h-P2U-1321N1 cells) or with the turkey P2Y-purinoceptor (t-P2Y-1321N1 cells) was investigated. 2-Methylthioadenosine triphosphate (2MeSATP) was used as the agonist at t-P2Y-1321N1 cells and uridine triphosphate (UTP) at h-P2U-1321N1 cells. 2. Suramin caused a parallel shift to the right of the concentration-response curves for 2MeSATP in the t-P2Y-1321N1 cells, yielding a Schild plot with a slope of 1.16 +/- 0.08 and a pA2 value of 5.77 +/- 0.11. 3. Suramin also caused a shift to the right of concentration-response curves for UTP in the h-P2U-1321N1 cells, and on Schild plots gave a slope different from unity (1.57 +/- 0.19) and an apparent pA2 value of 4.32 +/- 0.13. Suramin was therefore a less potent antagonist at the P2U-purinoceptor than the P2Y-purinoceptor. 4. In the presence of the ectonucleotidase inhibitor, ARL 67156 (6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP) there was no significant difference in the EC50 or shapes of curves with either cell type, and no difference in pA2 values for suramin. 5. PPADS caused an increase in the EC50 for 2MeSATP in the t-P2Y-1321N1 cells. The Schild plot had a slope different from unity (0.55 +/- 0.15) and an X-intercept corresponding to an apparent pA2 of 5.98 +/- 0.65. 6. PPADS up to 30 microM had no effect on the concentration-response curve for UTP with the h-P2U-1321N1 cells. 7. In conclusion, suramin and PPADS show clear differences in their action at the 2 receptor types, in each case being substantially more effective as an antagonist at the P2Y-purinoceptor than at the P2U-purinoceptor. Ectonucleotidase breakdown had little influence on the nature of the responses at the two receptor types, or in their differential sensitivity to suramin.

Responses of the longitudinal muscle and the muscularis mucosae of the rat duodenum to adenine and uracil nucleotides.

1. Previous studies have shown that the rat duodenum contains P1 and P2Y purinoceptors via which it relaxes to adenosine and adenosine 5'-triphosphate (ATP) respectively. It has also been shown to contract to uridine 5'-triphosphate (UTP) and adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), and based on their differential inhibition by the P2 antagonist suramin it has been suggested that they act via two separate receptors. In addition, the rat duodenum has been shown to dephosphorylate ATP rapidly via ectonucleotidases and adenosine deaminase. In this study the responses of two preparations from the rat duodenum, the longitudinal muscle and the muscularis mucosae, were investigated using a series of nucleotides and suramin. 2. 2-Methylthioadenosine 5'-triphosphate (2-MeSATP), ATP, ATP-gamma-S and adenosine 5'-alpha,beta-methylene-triphosphonate (AMPCPP) each relaxed the longitudinal muscle, with an agonist potency order of 2-MeSATP > ATP = ATP-gamma-S > AMPCPP, while UTP and uridine 5'-diphosphate (UDP) were not observed to elicit relaxation. This indicates the presence of a relaxant P2Y-purinoceptor on the longitudinal muscle. The longitudinal muscle did not contract to any of the agonists at concentrations of 300 microM, apart from ATP-gamma-S which caused very weak contractions. 3. ATP-gamma-S, adenosine 5'-methylenediphosphonate (AMPCP), AMPCPP, ATP, UTP, adenosine 5'-diphosphate (ADP), UDP and 2-MeSATP each contracted the muscularis mucosae with an agonist potency order of ATP-gamma-S > or = AMPCP > or = AMPCPP = ATP = UTP = ADP = UDP >> 2-MeSATP, although maximal responses were not obtained at concentrations of 300 microM. The muscularis mucosae did not relax to any of the agonists at concentrations of 300 microM. 4. Suramin (1 mM) inhibited relaxations induced by ATP on the longitudinal muscle, shifting the relaxation concentration-response curve to the right. This further supports the presence of a P2Y-purinoceptor on this muscle layer. Suramin (1 mM) inhibited contractions induced by AMPCPP, but not those induced by ATP, UTP or ATP-gamma-S, in the muscularis mucosae. Desensitization of the muscularis mucosae was seen with AMPCPP, but not with UTP or ATP-gamma-S, and no cross-desensitization between AMPCPP and UTP or ATP-gamma-S was observed. This suggests there are two receptors which mediate contraction on the rat duodenum muscularis mucosae, one suramin-sensitive and the other suramin-insensitive. 5. ATP was rapidly degraded by the muscularis mucosae to ADP, adenosine 5'-monophosphate (AMP) and inosine, with no adenosine being detected. A similar rate of degradation was seen for UTP with UDP, uridine 5'-monophosphate (UMP) and uridine being formed and for 2-MeSATP with 2-methylthioadenosine 5'-diphosphate (2-MeSADP), 2-methylthioadenosine 5'-monophosphate (2-MeSAMP) and 2-methylthioadenosine being formed. AMPCPP and ATP-gamma-S were both degraded more slowly, AMPCPP being degraded to AMPCP, and ATP-gamma-S to ADP, AMP and inosine. Suramin (1 mM), did not significantly affect the rate and pattern of degradation of these nucleotides, apart from AMPCPP which was degraded slightly more slowly in the presence of suramin. 6. These results show that there is a P2Y-purinoceptor which mediates relaxation in the rat duodenum longitudinal muscle. They also show that there is a contraction-mediating suramin-sensitive receptor on the rat duodenum muscularis mucosae which is desensitized by AMPCPP, and thus is probably of the P2X subtype. In addition, there is a contraction-mediating suramin-insensitive receptor on the rat duodenum muscularis mucosae which is not desensitized by UTP or ATP-gamma-S, and at which ATP and UTP show equal potency, and is thus probably of the P2U subtype. In addition, the rat duodenum muscularis mucosae contains ectonucleotidases and adenosine deaminase, which rapidly degrade nucleotides, although the inhibition by suramin of this deg