Trace amines: identification of a family of mammalian G protein-coupled receptors.

Tyramine, beta-phenylethylamine, tryptamine, and octopamine are biogenic amines present in trace levels in mammalian nervous systems. Although some "trace amines" have clearly defined roles as neurotransmitters in invertebrates, the extent to which they function as true neurotransmitters in vertebrates has remained speculative. Using a degenerate PCR approach, we have identified 15 G protein-coupled receptors (GPCR) from human and rodent tissues. Together with the orphan receptor PNR, these receptors form a subfamily of rhodopsin GPCRs distinct from, but related to the classical biogenic amine receptors. We have demonstrated that two of these receptors bind and/or are activated by trace amines. The cloning of mammalian GPCRs for trace amines supports a role for trace amines as neurotransmitters in vertebrates. Three of the four human receptors from this family are present in the amygdala, possibly linking trace amine receptors to affective disorders. The identification of this family of receptors should rekindle the investigation of the roles of trace amines in mammalian nervous systems and may potentially lead to the development of novel therapeutics for a variety of indications.

Identification and characterization of two G protein-coupled receptors for neuropeptide FF.

The central nervous system octapeptide, neuropeptide FF (NPFF), is believed to play a role in pain modulation and opiate tolerance. Two G protein-coupled receptors, NPFF1 and NPFF2, were isolated from human and rat central nervous system tissues. NPFF specifically bound to NPFF1 (K(d) = 1.13 nm) and NPFF2 (K(d) = 0.37 nm), and both receptors were activated by NPFF in a variety of heterologous expression systems. The localization of mRNA and binding sites of these receptors in the dorsal horn of the spinal cord, the lateral hypothalamus, the spinal trigeminal nuclei, and the thalamic nuclei supports a role for NPFF in pain modulation. Among the receptors with the highest amino acid sequence homology to NPFF1 and NPFF2 are members of the orexin, NPY, and cholecystokinin families, which have been implicated in feeding. These similarities together with the finding that BIBP3226, an anorexigenic Y1 receptor ligand, also binds to NPFF1 suggest a potential role for NPFF1 in feeding. The identification of NPFF1 and NPFF2 will help delineate their roles in these and other physiological functions.

Substitution of a mutant alpha2a-adrenergic receptor via "hit and run" gene targeting reveals the role of this subtype in sedative, analgesic, and anesthetic-sparing responses in vivo.

Norepinephrine contributes to antinociceptive, sedative, and sympatholytic responses in vivo, and alpha2 adrenergic receptor (alpha2AR) agonists are used clinically to mimic these effects. Lack of subtype-specific agonists has prevented elucidation of the role that each alpha2AR subtype (alpha2A, alpha2B, and alpha2C) plays in these central effects. Here we demonstrate that alpha2AR agonist-elicited sedative, anesthetic-sparing, and analgesic responses are lost in a mouse line expressing a subtly mutated alpha2AAR, D79N alpha2AAR, created by two-step homologous recombination. These functional changes are accompanied by failure of the D79N alpha2AAR to inhibit voltage-gated Ca2+ currents and spontaneous neuronal firing, a measure of K+ current activation. These results provide definitive evidence that the alpha2AAR subtype is the primary mediator of clinically important central actions of alpha2AR agonists and suggest that the D79N alpha2AAR mouse may serve as a model for exploring other possible alpha2AAR functions in vivo.

Genetic evidence for involvement of multiple effector systems in alpha 2A-adrenergic receptor inhibition of stimulus-secretion coupling.

The alpha 2A-adrenergic receptor (alpha 2AAR), via its interaction with the pertussis toxin-sensitive Gi/G(o) class of G proteins, modulates multiple effector systems, including inhibition of adenylyl cyclase and Ca2+ channels and activation of K+ channels. Mutation of a membrane-embedded aspartate residue, highly conserved among G protein-coupled receptors, in the alpha 2AAR to asparagine (D79N alpha 2AAR) results in selective uncoupling of the receptor to K+ currents but retention of inhibition of cAMP production and of voltage-sensitive Ca2+ currents when expressed in AtT20 anterior pituitary cells in culture. It is known that attenuation of cAMP synthesis alone cannot account for alpha 2AAR suppression of stimulus-secretion coupling; thus, the D79N alpha 2AAR provides a unique tool with which to assess the relative contribution of K+ current activation and Ca2+ current suppression in mediating the cellular responses of alpha 2AAR. The wild-type alpha 2AAR suppresses basal and secretagogue-evoked adrenocorticotropic hormone (ACTH) release in a manner indistinguishable from response to the endogenous somatostatin receptor. In contrast, the D79N alpha 2AAR does not attenuate basal ACTH release and is only partially effective in suppressing ACTH secretion evoked by the secretagogue isoproterenol. Regulation of ACTH release evoked by 8-bromo-cAMP, which bypasses receptor regulation of cAMP synthesis, suggests that attenuation of cAMP production, although not sufficient for inhibition of ACTH secretion, nevertheless participates in a functionally relevant manner. Taken together, the present findings indicate that alpha 2AAR-mediated suppression of neuropeptide secretion requires concomitant regulation of K+ and Ca2+ currents in parallel with attenuation of cAMP production.

Pharmacological characterization and anatomical localization of prejunctional beta-adrenoceptors in the rat kidney.

1. The subtype and anatomical localization of beta-adrenoceptors mediating facilitation of stimulus-induced overflow of noradrenaline ('prejunctional beta-adrenoceptors') are not conclusively known to date. The present study was undertaken to characterize these receptors by use of pharmacological methods as well as to define their localization (prejunctional or postjunctional) with radio-ligand binding and autoradiography techniques combined with surgical denervation of the sympathetic innervation to the rat kidney. 2. Exposure of the kidney to (-)-isoprenaline, the nonselective beta-adrenoceptor agonist, resulted in a dose-dependent facilitation of stimulus-induced neurotransmitter overflow. This response was inhibited by propranolol, the beta 1- and beta 2-adrenoceptor antagonist, with a pA2 of 9.20 suggesting that the prejunctional beta-adrenoceptors are not of the beta 3-subtype. 3. The rank order of potency and potency ratios of beta-adrenoceptor agonists at renal prejunctional beta-adrenoceptors (EC50 for agonist/EC50 for (-)-isoprenaline) were: (-)-isoprenaline (1) > procaterol (2) > salbutamol (3) > adrenaline (10) > (+)-isoprenaline (25). However, dobutamine, the beta 1-adrenoceptor agonist, failed to enhance stimulus-induced overflow of noradrenaline. These results are indicative of the presence of beta 2-adrenoceptors as prejunctional beta-adrenoceptors. 4. Facilitation elicited by (-)-isoprenaline and procaterol, the selective beta 2-adrenoceptor agonist, was inhibited by ICI 118,551, the selective beta 2-adrenoceptor antagonist, with pKb values of 9.20 and 9.35, respectively at renal prejunctional beta-adrenoceptors. Similarly, the pKb values of metoprolol, the selective beta 1-adrenoceptor antagonist, at renal prejunctional beta-adrenoceptors were determined to be 6.25 and 6.18 against (-)-isoprenaline and procaterol, respectively. These results suggest the presence of a homogeneous population of beta 2-adrenoceptors as prejunctional beta-adrenoceptors. 5. Radio-ligand binding analysis of renal beta-adrenoceptors revealed the prevalence of the beta 1-subtype as compared to the beta 2-subtype (63% vs 37%). However, surgical denervation of the rat kidney, resulting in more than 90% reduction in renal noradrenaline content, selectively reduced the beta 2-adrenoceptor population by 80%, implying the presence of beta 2-adrenoceptors on renal sympathetic nerve terminals. 6. Autoradiographic analysis demonstrated the presence of beta 1-adrenoceptors on cortical structures such as glomeruli and tubules. beta-Adrenoceptors were found to be present on tubules (minor population), collecting tubules in outer medulla and the adventitia and adventitial-medial border of intraparenchymal branches of the renal artery. Surgical denervation of the rat kidney resulted in the disappearance of Beta2-adrenoceptors associated with the intraparenchymal branches, without affecting the Beta-adrenoceptor populations at other sites. These results support the notion that the Beta2-subtype is present on renal sympathetic nerve terminals and demonstrate that these prejunctional Beta2-adrenoceptors are associated with the renal vasculature and not with renal tubules.7. The results of the present investigation demonstrate that renal prejunctional Beta-adrenoceptors are of the Beta2-subtype in nature. These receptors are present on sympathetic nerve terminals which are associated with the renal vasculature.

Dependence of prejunctional beta-adrenoceptor facilitation of sympathetic neurotransmission on stimulus train length and agonist exposure time.

Prejunctional beta-adrenoceptors facilitate stimulus-induced neuronal norepinephrine release in the rat kidney. The mechanism(s) responsible for the bell-shaped concentration-facilitatory response curve to isoproterenol generated in the presence of alpha-adrenoceptor blockade and uptake blockade was explored. The concentration-response curve to isoproterenol generated using 30 pulses/stimulation and 20 min exposure to each agonist concentration was bell-shaped in nature, exhibiting a maximum response at 10 nM isoproterenol. Shortening the isoproterenol exposure time to 5 min for each concentration still resulted in a bell-shaped curve. Reducing the stimulus train length from 30 pulses to 10 pulses/stimulation failed to change the shape of the curve but produced a greater maximum response. However, shortening the isoproterenol exposure time to 2 min (10 pulses/stimulation) resulted in a curve which was not bell-shaped. These results suggest that prejunctional beta-adrenoceptors are susceptible to rapid desensitization upon prolonged agonist exposure and that an inverse relationship exists between stimulus train length and prejunctional beta-adrenoceptor-mediated facilitation.