The repertoire of olfactory C family G protein-coupled receptors in zebrafish: candidate chemosensory receptors for amino acids.

BACKGROUND: Vertebrate odorant receptors comprise at least three types of G protein-coupled receptors (GPCRs): the OR, V1R, and V2R/V2R-like receptors, the latter group belonging to the C family of GPCRs. These receptor families are thought to receive chemosensory information from a wide spectrum of odorant and pheromonal cues that influence critical animal behaviors such as feeding, reproduction and other social interactions. RESULTS: Using genome database mining and other informatics approaches, we identified and characterized the repertoire of 54 intact "V2R-like" olfactory C family GPCRs in the zebrafish. Phylogenetic analysis - which also included a set of 34 C family GPCRs from fugu - places the fish olfactory receptors in three major groups, which are related to but clearly distinct from other C family GPCRs, including the calcium sensing receptor, metabotropic glutamate receptors, GABA-B receptor, T1R taste receptors, and the major group of V2R vomeronasal receptor families. Interestingly, an analysis of sequence conservation and selective pressure in the zebrafish receptors revealed the retention of a conserved sequence motif previously shown to be required for ligand binding in other amino acid receptors. CONCLUSION: Based on our findings, we propose that the repertoire of zebrafish olfactory C family GPCRs has evolved to allow the detection and discrimination of a spectrum of amino acid and/or amino acid-based compounds, which are potent olfactory cues in fish. Furthermore, as the major groups of fish receptors and mammalian V2R receptors appear to have diverged significantly from a common ancestral gene(s), these receptors likely mediate chemosensation of different classes of chemical structures by their respective organisms.

High-affinity interaction of (des-Tyrosyl)dynorphin A(2-17) with NMDA receptors.

The opioid peptide dynorphin A elicits non-opioid receptor-mediated, neurotoxic response in vivo, which is blocked by pretreatment with MK-801, a noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist. In the present study, we examined the possible direct interaction of dynorphin A on the NMDAR. A nonopioid dynorphin A analog, (125)I-(des-tyrosyl) dynorphin A(2-17), was used in radioligand binding analysis on rat cortical brain membranes. This radioligand exhibited a saturable, specific binding at high affinity with a K(d) value of 9.4+/-1.6 nM and maximal binding of 2.4+/-0.6 pmol/mg protein. This binding site was associated with the NMDAR complex because it was modulated by a number of NMDAR ligands. Transient expression of the rat NR1a/NR2A complex in human embryonic kidney 293 cells confirmed a coexpression of (125)I-(des-tyrosyl) dynorphin A(2-17), [(3)H]CGP39,653, and [(3)H]MK-801 binding. These data provide direct evidence of the presence of a high-affinity binding site for dynorphin A on the NMDAR. The modulatory effect of the various NMDAR-selective ligands on dynorphin A binding suggests that dynorphin A may bind preferentially to the closed/desensitized state of the NMDAR. The physiological role of dynorphin A binding to the NMDAR remains to be established.

The non-NMDA subtype of excitatory amino acid receptor plays the major role in control of cardiovascular function by the subretrofacial nucleus in cats.

Recent studies have reported that microinjection of kynurenic acid (KYN 12.5 nmol), the nonselective Excitatory Amino acid (EAA) antagonist, into the rostral ventrolateral medulla of the cat decreases arterial blood pressure (BP) and inferior cardiac sympathetic nerve discharge. The purpose of our study was to confirm this finding and determine the subtypes of EAA receptor(s) responsible for mediating this effect. This was done by microinjecting various EAA antagonists bilaterally into the SRFN of chloralose-anesthetized animals while monitoring BP and HR. KYN (12.5 nmol; N = 5) produced a decrease in mean BP (31 +/- 9 mmHg, P < .05) with no significant change in HR. To determine the subtype of EAA receptor responsible for eliciting tonic sympathetic outflow from the SRFN, specific antagonists of N-methyl-D-aspartate (NMDA) and non-NMDA EAA receptors were tested. The NMDA receptor antagonist 3-(RS)-Carboxypiperazin-4-yl)-proyl- 1-phosphonic acid (CPP-2.25 nmol; N = 3) microinjected into the SRFN produced a small but significant decrease in BP (-13 +/- 1 mmHg; P < .05). This effect of CPP was significantly less than that seen with KYN. Two antagonists of the non-NMDA subtype of EAA receptor, 6-cyano-7-nitroquinoxaline-2,3-dione (0.05 nmol; N = 4) and gamma-D-glutamylaminomethyl sulphonic acid (2.5 nmol; N = 4), were microinjected into the SRFN. Both of these drugs produced decreases in BP (-29 +/- 4 and -23 +/- 3 mmHg, respectively; P < 0.05) similar to that observed with KYN. No significant changes in HR were noted with CPP, 6 cyano-7-nitroquinoxaline-2,3-dione or gamma-G-glutamylamino-methylsulfonate. These data indicate that a non-NMDA EAA receptor plays the major role in control of cardiovascular function by the SRFN.

Influence of excitatory amino acid receptor subtypes on the electrophysiological activity of dopaminergic and nondopaminergic neurons in rat substantia nigra.

In vivo electrophysiological recording methods were used to evaluate the effects of selective and nonselective agonists for excitatory amino acid (EAA) receptor subtypes on the activity of dopaminergic (DA) and nondopaminergic (non-DA) neurons in the substantia nigra of chloral hydrate-anesthetized rats. Microiontophoretic administration of (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), 2-carboxy-4-(1-methyl-ethenyl)-3-pyrrolidinacetate (kainate), N-methyl-D-aspartate (NMDA) and glutamate excited neurons with an apparent rank order of potency of AMPA = kainate = glutamate > NMDA on DA neurons, and AMPA = kainate > glutamate = NMDA on non-DA neurons. These agonists also changed the firing pattern of DA neurons, which displayed an increase in burst-firing and a reduction in the regularity of the firing pattern. Regularity of firing was indexed by the variation coefficient of a sample of interspike intervals. The apparent potencies of the four agonists to increase burst-firing and variation coefficient were similar to their potencies to increase neuronal firing rate. Blockade of NMDA receptor function by coiontophoresis of 5R,10S-(+/-)-5-methyl-10,11-dihydro-5H-dibenzo[and]cyclohepten-5,1 0-imine hydrogen maleate (MK-801), a selective noncompetitive NMDA antagonist, did not alter kainate-induced changes in firing rate and firing pattern, which indicated that kainate-induced increases in burst-firing were not dependent on concomitant NMDA receptor activation by endogenous excitatory amino acid.(ABSTRACT TRUNCATED AT 250 WORDS)

New mechanisms of excitatory transmitter neurotoxicity.

Accumulating evidence suggests that either excessive activation or suppression of either ionotropic or metabotropic EAA receptors can have neurotoxic consequences, and a variety of different mechanisms may be involved. The major excitatory neurotransmitters in the mammalian CNS-glutamate (Glu) and acetylcholine (ACh)--have vitally important beneficial functions, but also harbor treacherous neurotoxic potential which, as will be described in this review, can be expressed as classical excitotoxicity or in several other ways that have yet to be studied in detail.

[Receptive mechanisms of excitatory amino acid signals].

Excitatory amino acid (EAA) receptors are nowadays divided into 2 major categories according to their signal transduction systems. A metabotropic type of EAA receptors is linked to hydrolysis of peculiar phospholipids or formation of cyclic nucleotides, while an ionotropic type is associated with an ion channel permeable to particular cations. The ionotropic receptors are further classified on the basis of differential sensitivity to excitement by the exogenous agonist N-methyl-D-aspartic acid (NMDA). Ionotropic receptors insensitive to NMDA are distinguishable by preference to excitation by the other exogenous agonist alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic or kainic acid. These ionotropic receptors have been analyzed by conventional ligand binding techniques which often meet with critical methodological pitfalls and artifacts. In this review, therefore, our data obtained using accurate and reproducible receptor binding and gel-shift assays will be outlined with respect to signal transduction mediated by EAA from cell membranes to nuclei. Pharmacological evaluation is also discussed on the search for and the development of drugs useful for therapy and treatment of a variety of neurodegenerative disorders associated with dysfunction of EAA receptors.

Excitatory amino acid neurotransmission in superior laryngeal nerve-evoked inspiratory termination.

Superior laryngeal nerve (SLN) stimulation elicits a transient inhibition of inspiration (single shocks) or inspiratory termination (stimulus trains). The neural pathways mediating these responses are unknown, but the medial nucleus tractus solitarius (mNTS) has been implicated in the termination reflex. This study tested the hypothesis that SLN-evoked inspiratory termination requires excitatory amino acid (EAA) neurotransmission in medial aspects of the NTS. Experiments were conducted in decerebrate, vagotomized, and paralyzed adult cats. Inspiratory motor output was recorded from the phrenic nerve. After control responses to SLN stimulation were recorded, a unilateral injection of the EAA antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 mM) was made into the mNTS. The transient inhibitions were not altered by DNQX. Inspiratory termination elicited by stimulation of the SLN contralateral to the injection persisted after DNQX (n = 4). Stimulation of the ipsilateral SLN no longer elicited termination (5 of 9 animals) or did so only at greatly elevated thresholds (4 of 9). We conclude that EAA neurotransmission in the mNTS is not required in the transient reflex but is necessary for the production of the SLN-evoked inspiratory termination.