Characterization of neurotransmitter receptor-mediated information transduction

A relevant primary model system under investigation are those neurotransmitter receptors which are linked to their signal transduction pathways via guanine nucleotide binding regulatory (G) proteins with specific emphasis on opioid and nociceptin receptors. Specific G proteins have been shown to link these receptors to the activation and inhibition of various nucleotide cyclases, phospholipases, and several ion channels. In order to characterize these receptors at the biochemical and molecular levels and study their regulation, there are several major interrelated lines of research which are ongoing. These include investigations of receptor structure/function/pharmacology relationships, receptor-effector coupling mechanisms, G-protein interactions and biochemical/molecular mechanisms of receptor desensitization. Additional projects involve the molecular cloning and expression of novel receptor cDNAs and genes encoding receptors which interact with bile acid receptors, site-directed mutagenesis and receptor chimera studies, analysis and characterization of gene structure, and regulation of receptor expression in both normal and pathophysiological states.

Research description:

I) Internalization, recycling and signalling of the nociceptin receptor (hNOP) Prolonged activation of G protein-coupled receptors (GPCRs) leads to a greatly decreased sensitivity of the receptor to a subsequent agonist challenge. The molecular mechanisms that lead to agonist-dependent desensitization of GPCRs are not fully defined but several distinct events are involved, including uncoupling of receptors from their heterotrimeric G proteins and reduction in the number of receptors at the cell surface by either internalization or down-regulation which may be associated with degradation. We are investigating NC-promoted internalization of the human NOP (hNOP) receptor occurring in the neuroblastoma cell line SK-N-BE or of the cloned human receptors expressed in CHO cells by measuring the loss of binding sites for the hydrophilic ligand [3 H]-NC in viable cells and by confocal microscopy analysis. These events are related to desensitization of this receptor in intact cells by investigating different signalling pathways activated by this receptor.

II) Characterization of novel sigma ligands and role of sigma1 receptors in the regulation of autonomic functions. Sigma (s) recognition sites are a unique class of binding sites, distributed in the nervous system and in peripheral organs acting as receptors for some unidentified endogenous ligand.They bind an array of structural classes of compounds including haloperidol and (+)-benzomorphans, such as (+)-pentazocine. We are investigating their role in the regulation of autonomic functions since they may interfere with neurotransmitter release, modulating their action on innervated tissue. We found distinguishable populations of sites in the rabbit iris-ciliary body and evaluating their contribution in the regulation of intraocular pressure in the rabbit. In vivo findings are completed by in vitro analysis of their role on electrically stimulated release of [3 H]NE from postganglionic sympathetic neurons and on isoproterenol-induced cAMP accumulation in isolated iris-ciliary body. These findings may prove a contribution of sigma ligands in the control of ocular hypertension.

III)Pharmacological characterization of endomorphin derivatives as novel analgesic agents Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2 ) are peptides isolated from the mammalian brain which bind to µ-opioid receptors with high affinity and selectivity. Endomorphins, i.c.v. or i.t. administered, display antinociceptive activity and appear to relieve neuropathic pain, which is assumed to be less sensitive to traditional opioid receptor agonists. We are investigating novel endomorphin-1 analogues which are more resistant to enzymatic hydrolysis than endomorphin-1 by receptor binding and signal transduction analysis in in vitro cell models to ascertain that they behave as a µ-opioid receptor agonist. Antinociceptive activity of these compounds, administered peripherally, shows that they will be extremely useful for exploring the pharmacological profile of endomorphins in vivo and confirms the potential therapeutic interest of endomorphin derivatives as novel analgesic agents.


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