Tyramine, ␤-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 and͞or 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.

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.

Norepinephrine contributes to antinociceptive, sedative, and sympatholytic responses in vivo, and ␣ 2 adrenergic receptor (␣ 2 AR) agonists are used clinically to mimic these effects. Lack of subtype-specific agonists has prevented elucidation of the role that each ␣ 2 AR subtype (␣ 2A , ␣ 2B , and ␣ 2C ) plays in these central effects. ␣ 2 -adrenergic receptors (␣ 2 ARs) present in the central nervous system (CNS) respond to norepinephrine (NE) and epinephrine and mediate sympatholytic, sedative-hypnotic, analgesic, anesthetic-sparing, hypotensive, and anxiolytic responses (1). Many of these responses are therapeutically useful and are exploited clinically, for example, during anesthesia and to attenuate the symptoms of opioid withdrawal (2). Three ␣ 2 AR subtypes have been revealed by pharmacological (␣ 2A AR, ␣ 2B AR, and ␣ 2C AR) and molecular cloning (␣ 2a AR, ␣ 2b AR, and ␣ 2c AR) strategies (3), and all couple, via pertussis toxin-sensitive G i ͞G o proteins, to attenuation of adenylyl cyclase, suppression of voltage-gated Ca 2ϩ channels, and activation of inwardly rectifying K ϩ channels (4).Multiple experimental limitations have precluded clarifying the involvement of each ␣ 2 AR subtype in catecholaminemediated physiological responses in the CNS. Subtype-specific ␣ 2 AR agonists and antagonists are not available (5); even when subtype selectivity has been noted in vitro, varying and unknown in vivo bioavailability precludes confident correlation of the administered dose with the amount of drug at the receptor site. Previous studies to explore ␣ 2 AR involvement in various responses have used prazosin to block catecholamine responses mediated by ␣ 1 adrenergic receptors (␣ 1 AR); however, it is now known that the ␣ 2B AR and ␣ 2C AR subtypes also are blocked by prazosin (5), thus confounding the interpretations of these earlier studies. In addition, because ␣ 1 AR can functionally antagonize ␣ 2 AR-mediated responses in some settings, ␣ 2 AR responses in the presence of prazosin (added to block ␣ 1 AR, ␣ 2B AR, and ␣ 2C AR) may reflect the disturbance of the balance between the functionally antagonistic ␣ 2 AR and ␣ 1 AR systems rather than provide insights concerning the role of the ␣ 2A AR subtype. Consequently, we manipulated the mouse genome to provide definitive evidence regarding the role of the ␣ 2A AR subtype in CNS responses.We used the ''hit and run'' targeting variant of homologous recombination (6, 7) to substitute a subtle mutation of the ␣ 2a AR, D79N into the mouse genome as a tool to explore the role of the ␣ 2a AR in vivo (8). The aspartate residue at position 79 (D79) is highly conserved in a topologically identical position in the second transmembrane span in a large subset of G protein-coupled receptors (9). Mutation of this residue has been shown to eliminate allosteric regulation of receptor binding by monovalent cations (10-13) and to perturb receptor-G protein-effector coupling (14-17) in heterologous expression systems. Thus, the animals expressing the D79N ␣ 2a AR provi...

1 The subtype and anatomical localization of P-adrenoceptors mediating facilitation of stimulusinduced overflow of noradrenaline ('prejunctional P-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 P-adrenoceptor agonist, resulted in a dose-dependent facilitation of stimulus-induced neurotransmitter overflow. This response was inhibited by propranolol, the f,-and P2-adrenoceptor antagonist, with a pA2 of 9.20 suggesting that the prejunctional P-adrenoceptors are not of the P3-subtype. 3 The rank order of potency and potency ratios of P-adrenoceptor agonists at renal prejunctional P-adrenoceptors (EC50 for agonist/EC50 for (-)-isoprenaline) were: (-)-isoprenaline (1) >procaterol (2) > salbutamol (3) >adrenaline (10) > (+)-isoprenaline (25). However, dobutamine, the Pf-adrenoceptor agonist, failed to enhance stimulus-induced overflow of noradrenaline. These results are indicative of the presence of P2-adrenoceptors as prejunctional ,-adrenoceptors. 4 Facilitation elicited by (-)-isoprenaline and procaterol, the selective P2-adrenoceptor agonist, was inhibited by ICI 118,551, the selective P2-adrenoceptor antagonist, with pKb values of 9.20 and 9.35, respectively at renal prejunctional P-adrenoceptors. Similarly, the pKb values of metoprolol, the selective fi-adrenoceptor antagonist, at renal prejunctional P-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 P2-adrenoceptors as prejunctional P-adrenoceptors. 5 Radio-ligand binding analysis of renal P-adrenoceptors revealed the prevalence of the fi,-subtype as compared to the P2-subtype (63% vs 37%). However, surgical denervation of the rat kidney, resulting in more than 90% reduction in renal noradrenaline content, selectively reduced the P2-adrenoceptor population by 80%, implying the presence of P2-adrenoceptors on renal sympathetic nerve terminals.6 Autoradiographic analysis demonstrated the presence of P,-adrenoceptors on cortical structures such as glomeruli and tubules. fi2-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 P2-adrenoceptors associated with the intraparenchymal branches, without affecting the P-adrenoceptor populations at other sites. These results support the notion that the P2-subtype is present on renal sympathetic nerve terminals and demonstrate that these prejunctional P2-adrenoceptors are associated with the renal vasculature ...

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