Illuminating the life of GPCRs

The investigation of biological systems highly depends on the possibilities that allow scientists to visualize and quantify biomolecules and their related activities in real-time and non-invasively. G-protein coupled receptors represent a family of very dynamic and highly regulated transmembrane proteins that are involved in various important physiological processes. Since their localization is not confined to the cell surface they have been a very attractive "moving target" and the understanding of their intracellular pathways as well as the identified protein-protein-interactions has had implications for therapeutic interventions. Recent and ongoing advances in both the establishment of a variety of labeling methods and the improvement of measuring and analyzing instrumentation, have made fluorescence techniques to an indispensable tool for GPCR imaging. The illumination of their complex life cycle, which includes receptor biosynthesis, membrane targeting, ligand binding, signaling, internalization, recycling and degradation, will provide new insights into the relationship between spatial receptor distribution and function. This review covers the existing technologies to track GPCRs in living cells. Fluorescent ligands, antibodies, auto-fluorescent proteins as well as the evolving technologies for chemical labeling with peptide- and protein-tags are described and their major applications concerning the GPCR life cycle are presented

[Nphe(1)]nociceptin-(1-13)NH2 selectively antagonizes nociceptin effects in the rabbit isolated ileum

When suspended in vitro in isolated organ baths, segments of the rabbit ileum show a fairly strong and stable spontaneous activity, which derives from the continuous release of acetylcholine and the activation of muscarinic receptors, since the activity is completely eliminated by atropine. Dynorphin A pEC50: 8.6"0.07., neuropeptide Y and its congener human pancreatic polypeptide pEC50: 9.40"0.10., and nociceptin pEC50: 8.08"0.12. dose-dependently inhibit the spontaneous activity through the activation of receptors, which are specifically antagonised respectively by naloxone p A2: 7.17"0.12., 2-naphtalen-1-ylamino.-3-phenylpropionitrile JCF 104; p A2: 5.80"0.10., and wNphe1xnociceptin-1–13.NH2 p A2: 6.17"0.19.. This last compound, a selective nociceptin-receptor OP4. antagonist, inhibits the effect of nociceptin in a competitive manner, as demonstrated by Schild analysis. wNphe1xnociceptin-1–13.NH2 also antagonizes the effects of other OP4 receptor ligands such as the full agonist, nociceptin-1–13.-NH2, and the partial agonists, wPhe1cCH2–NH.Gly 2 xnociceptin-1–13.-NH2 intrinsic activity a E.s0.5. and Ac-RYYWK-NH2 a Es0.5., with p A2 values ranged from 5.8 to 6.2. These results indicate that the functional site mediating the inhibitory effect of nociceptin in the rabbit ileum, is pharmacologically identical to the OP4 sites of other species mouse, rat, guinea pig, man., since the potencies p A2 values. of the pure and competitive antagonist wNphe1xnociceptin-1–13.NH2 is very similar to the values obtained in the other species. Moreover, the rabbit ileum is one of the few isolated organs that allow classifying compounds, which interact with OP4 receptors as full agonists, partial agonists, or pure antagonists

[Nphe(1)]nociceptin-(1-13)NH2 selectively antagonizes nociceptin effects in the rabbit isolated ileum

When suspended in vitro in isolated organ baths, segments of the rabbit ileum show a fairly strong and stable spontaneous activity, which derives from the continuous release of acetylcholine and the activation of muscarinic receptors, since the activity is completely eliminated by atropine. Dynorphin A pEC50: 8.6"0.07., neuropeptide Y and its congener human pancreatic polypeptide pEC50: 9.40"0.10., and nociceptin pEC50: 8.08"0.12. dose-dependently inhibit the spontaneous activity through the activation of receptors, which are specifically antagonised respectively by naloxone p A2: 7.17"0.12., 2-naphtalen-1-ylamino.-3-phenylpropionitrile JCF 104; p A2: 5.80"0.10., and wNphe1xnociceptin-1\u201313.NH2 p A2: 6.17"0.19.. This last compound, a selective nociceptin-receptor OP4. antagonist, inhibits the effect of nociceptin in a competitive manner, as demonstrated by Schild analysis. wNphe1xnociceptin-1\u201313.NH2 also antagonizes the effects of other OP4 receptor ligands such as the full agonist, nociceptin-1\u201313.-NH2, and the partial agonists, wPhe1cCH2\u2013NH.Gly 2 xnociceptin-1\u201313.-NH2 intrinsic activity a E.s0.5. and Ac-RYYWK-NH2 a Es0.5., with p A2 values ranged from 5.8 to 6.2. These results indicate that the functional site mediating the inhibitory effect of nociceptin in the rabbit ileum, is pharmacologically identical to the OP4 sites of other species mouse, rat, guinea pig, man., since the potencies p A2 values. of the pure and competitive antagonist wNphe1xnociceptin-1\u201313.NH2 is very similar to the values obtained in the other species. Moreover, the rabbit ileum is one of the few isolated organs that allow classifying compounds, which interact with OP4 receptors as full agonists, partial agonists, or pure antagonists

In vivo characterization of B-2 receptors mediating hypotension in anesthetized rabbits and guinea pigs

With the discovery of suitable pharmacologic tools for B 2 receptor characterization, it has been demonstrated in vitro that the pharmacological spectrum of this receptor type obtained in various organs (e.g. intestine, vessels, urogenital tract) remains the same within the species but may show marked differences among species (e.g. the rabbit, the guinea pig) (Regoli et al., 1993; Regoli et al., 1994). Thus, orders of potency of agonists in rabbit and guinea pig tissues are opposite in that [Hyp3]BK is approximately 50- 100 times more potent than [AibT]BK in the rabbit and inversely, the latter compound is 2-10 times more active than [Hyp3]BK in the guinea pig. Furthermore, competitive antagonists, such as DArg[Hyp3,d-PheV,LeuS]BK and WIN 64338 (a nonpeptide compound), have also shown differences in their ability to block bradykinin responses in these two species while HOE 140, a non-competitive and long-acting antagonist, shows equipotent activities on both. Based on these results, we have suggested that B 2 receptors may be pharmacologically subject to interspecies variability. The present study was designed to find out if results obtained in vitro can be reproduced in vivo by measuring pharmacological parameters (namely EDs0 for agonists and ICso for antagonists) on kinin-induced blood pressure changes in the rabbit and the guinea pig

Kinin receptors in the diabetic mouse

It has been proposed that kinins are important inflammatory mediators involved in the pathogenesis of several diseases. In the present study, we attempted to determine the effects of kinins in a type I diabetic mouse model, using in vitro assays. Injection of streptozotocin (STZ) to the C57BL/Ks mdb mice causes an insulitis (inflammation of Langerhans islets) that leads to the diabetic condition. Ten days following the STZ treatment, the mice showed increased glycemia. We examined the effect of kinins and other agents (substance P, neurokinin A, acetylcholine) on the stomach fundus and urinary bladder of control and diabetic mice. Our results show that the sensitivity of the stomach fundus to bradykinin (BK) and desArg9BK (DBK), but not to other contractile agents, was substantially increased in the tissues of diabetic mice. The maximal contractions induced by BK and DBK were increased 1.5- to 2-fold in the stomachs from diabetic mice compared with those from normal mice. BK induced similar maximal contractions of urinary bladder strips from normal or STZ-treated mice, while DBK did not show any effect on this preparation. Interestingly, the apparent affinities of all agonists are similar in the two groups, normal and diabetic. These results suggest that B, and B, receptors are overexpressed in the stomach fundus but not in the urinary bladder of diabetic mice

Pharmacological Assays for Investigating the NOP Receptor.

The nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) is a G protein-coupled receptor involved in the regulation of several physiological functions and pathological conditions. Thus, researchers from academia and industry are pursuing NOP to discover and study novel pharmacological entities. In a multidisciplinary effort of pharmacologists, medicinal chemists, and molecular and structural biologists the mechanisms of NOP activation and inhibition have been, at least partially, disentangled. Here, we review the in vitro methodologies employed, which have contributed to our understanding of this target. We hope this chapter guides the reader through the mostly established assay platforms to investigate NOP pharmacology, and gives some hints taking advantage from what has already illuminated the function of other GPCRs. We analyzed the pharmacological results obtained with a large panel of NOP ligands investigated in several assays including receptor binding, stimulation of GTPγS binding, decrease of cAMP levels, calcium flux stimulation via chimeric G proteins, NOP/G protein and NOP/β-arrestin interaction, label-free assays such as dynamic mass redistribution, and bioassays such as the electrically stimulated mouse vas deferens