Blockade of mGluR1 receptor results in analgesia and disruption of motor and cognitive performances: effects of A-841720, a novel non-competitive mGluR1 receptor antagonist.

BACKGROUND AND PURPOSE: To further assess the clinical potential of the blockade of metabotropic glutamate receptors (mGluR1) for the treatment of pain. EXPERIMENTAL APPROACH: We characterized the effects of A-841720, a novel, potent and non-competitive mGluR1 antagonist in models of pain and of motor and cognitive function. KEY RESULTS: At recombinant human and native rat mGluR1 receptors, A-841720 inhibited agonist-induced calcium mobilization, with IC50 values of 10.7+/-3.9 and 1.0 +/- 0.2 nM, respectively, while showing selectivity over other mGluR receptors, in addition to other neurotransmitter receptors, ion channels, and transporters. Intraperitoneal injection of A-841720 potently reduced complete Freund's adjuvant-induced inflammatory pain (ED50 = 23 micromol kg(-1)) and monoiodoacetate-induced joint pain (ED50 = 43 micromol kg(-1)). A-841720 also decreased mechanical allodynia observed in both the sciatic nerve chronic constriction injury and L5-L6 spinal nerve ligation (SNL) models of neuropathic pain (ED50 = 28 and 27 micromol kg(-1), respectively). Electrophysiological studies demonstrated that systemic administration of A-841720 in SNL animals significantly reduced evoked firing in spinal wide dynamic range neurons. Significant motor side effects were observed at analgesic doses and A-841720 also impaired cognitive function in the Y-maze and the Water Maze tests. CONCLUSIONS AND IMPLICATIONS: The analgesic effects of a selective mGluR1 receptor antagonist are associated with motor and cognitive side effects. The lack of separation between efficacy and side effects in pre-clinical models indicates that mGluR1 antagonism may not provide an adequate therapeutic window for the development of such antagonists as novel analgesic agents in humans.

Two olfactory marker proteins in Xenopus laevis.

Mature olfactory receptor neurons of mammals are characterized by the expression of the highly conserved olfactory marker protein (OMP) encoded by single copy genes. In Xenopus laevis, two homologous genes encoding olfactory marker proteins have been identified that share a sequence identity with mammalian OMPs of about 50%. Sequence comparison revealed significant variability in the N-terminus and C-terminus regions; in contrast, two internal domains were highly conserved between amphibian and mammalian OMPs, suggesting some functional relevance. The two OMP subtypes were regionally expressed in the olfactory nasal epithelium of Xenopus. XOMP1 transcripts were more abundant in the lateral diverticulum and XOMP2 in the medial diverticulum. The lateral location of XOMP1 and medial location of XOMP2 correspond to the suggested locations of olfactory receptor neurons responsive to water-borne and air-borne odorants, respectively.

Identification of a nonmammalian Golf subtype: functional role in olfactory signaling of airborne odorants in Xenopus laevis.

Attempts to identify the Galpha subtypes in the two compartments of the olfactory system from Xenopus, which are supposed to be specialized for detecting aquatic and volatile odorous compounds, revealed that a Galpha(o1) subtype is characteristic for the "water nose," the lateral diverticulum, whereas a novel Galpha(s) subtype predominates in the "air nose," the medial diverticulum. The newly identified Galpha(s)-type is more closely related to Galpha(olf) of rat and human than to the known Galpha(s)-isoform of Xenopus; it is therefore considered the first identified nonmammalian Galpha(olf) subtype. Sequence comparison of Galpha(olf) from amphibia and mammals revealed a particular conservation within the alpha-helical domains, which are supposed to control the GDP/GTP-exchange rate. The selective expression of different Galpha subtypes in the two anatomically separated and functionally specialized nasal compartments parallels the expression of distinct classes of olfactory receptors. Moreover, biochemical analysis revealed that stimulation with appropriate odorous compounds elicits the formation of inositol trisphosphate in the lateral diverticulum. In contrast, cyclic adenosine monophosphate signals were induced in the medial diverticulum, and this response appears to be mediated by the novel Galpha(olf) subtype. The data indicate that olfactory sensory neurons in each of the nasal cavities are equipped not only with defined sets of receptor types but also with a distinct molecular machinery for the chemo-electrical transduction process.

A ß-amyloid oligomer directly modulates P/Q-type calcium currents in Xenopus oocytes.

BACKGROUND AND PURPOSE: ß-amyloid (Aß) oligomers have been implicated in the early pathophysiology of Alzheimer's disease (AD). While the precise nature of the molecular target has not been fully revealed, a number of studies have indicated that Aß oligomers modulate neuron-specific ion channels. We recently provided evidence that Aß oligomers suppress isolated P/Q-type calcium currents in cultured nerve cells. Using a heterologous expression system, we aimed to prove a direct effect on the membrane channel mediating such current. EXPERIMENTAL APPROACH: The effects of a synthetically generated Aß oligomer, Aß globulomer, were investigated on P/Q-type currents recorded from Xenopus laevis oocytes expressing the full P/Q-type calcium channel or the pore-forming subunit only. We also examined the effects of Aß globulomer on recombinant NMDA receptor currents. Finally, we compared the modulation by Aß globulomer with that induced by a synthetic monomeric Aß. KEY RESULTS: Aß globulomer directly and dose-dependently modulated P/Q-type calcium channels. A leftward shift of the current-voltage curve indicated that the threshold for channel opening was reduced. The effect of Aß globulomer was also present when only the α1A subunit of the normally tripartite channel was expressed. In contrast, the monomeric Aß had no effect on P/Q current. Also globulomer Aß had no effect on glutamate-induced NMDA currents. CONCLUSIONS AND IMPLICATIONS: The α1A subunit of the P/Q-type calcium channel is directly modulated by oligomeric Aß. Threshold reduction as well as an increase in current at synaptic terminals may facilitate vesicle release and could trigger excitotoxic events in the brains of patients with AD.

Characteristic features and ligand specificity of the two olfactory receptor classes from Xenopus laevis.

Amphibia have two classes of olfactory receptors (ORs), class I (fish-like receptors) and class II (mammalian-like receptors). These two receptor classes correspond to the two classes identified in other vertebrates, and amphibians thus provide a unique opportunity to compare olfactory receptors of both classes in one animal species, without the constraints of evolutionary distance between different vertebrate orders, such as fish and mammals. We therefore identified the complete open reading frames of class I and class II ORs in Xenopus laevis. In addition to allowing a representative comparison of the deduced amino acid sequences between both receptor classes, we were also able to perform differential functional analysis. These studies revealed distinct class-specific motifs, particularly in the extracellular loops 2 and 3, which might be of importance for the interaction with odorants, as well as in the intracellular loops 2 and 3, which might be responsible for interactions with specific G-proteins. The results of functional expression studies in Xenopus oocytes, comparing distinct receptor types, support the idea that class I receptors are activated by water-soluble odorants, whereas class II receptors are activated by volatile compounds.

Cloning and functional expression of GABA(B) receptors from Drosophila.

The neurotransmitter GABA (gamma-aminobutyric acid) functions as the major inhibitory neurotransmitter in the central nervous system of vertebrates and invertebrates. In vertebrates GABA signals both through ionotropic receptors (GABA(A), GABA(C)), which induce fast synaptic inhibitory responses, and through metabotropic receptors (GABA(B)), which play a fundamental role in the reduction of presynaptic transmitter release and postsynaptic inhibitory potentials. Whilst GABA(A) and GABA(C) receptors have been cloned from vertebrates as well as invertebrates, GABA(B) receptors have only been identified in vertebrate species to date, although indirect evidence suggests their existence in arthropods, too. Here we report the cloning of three putative invertebrate GABA(B) receptor subtypes (D-GABA(B)R1, R2 and R3) isolated from Drosophila melanogaster. Whilst D-GABA(B)R1 and R2 show high sequence identity to mammalian GABA(B)R1 and R2, respectively, the receptor D-GABA(B)R3 seems to be an insect-specific subtype with no known mammalian counterpart so far. All three D-GABA(B)R subtypes are expressed in the embryonic central nervous system. In situ hybridization of Drosophila melanogaster embryos shows that two of the D-GABA(B)Rs (D-GABA(B)R1 and R2) are expressed in similar regions, suggesting a coexpression of the two receptors, whilst the third D-GABA(B)R (D-GABA(B)R3) displays a unique expression pattern. In agreement with these results we have only been able to functionally characterize D-GABA(B)R1 and R2 when the two subtypes are coexpressed either in Xenopus laevis oocytes or mammalian cell lines, whilst D-GABA(B)R3 was inactive in any combination. The pharmacology of the coexpressed D-GABA(B)R1/2 receptor was different from the mammalian GABA(B)Rs: e.g. baclofen, an agonist of mammalian GABA(B)Rs, showed no effect.

Expression of olfactory receptors during development in Xenopus laevis.

A coordinated expression of tissue- and cell-specific genes during development is required to establish the complex functional organization of the vertebrate olfactory system. Owing to the unique features of its olfactory system and the well-characterized phases of its development, Xenopus laevis was chosen as a model organism to study the onset and the temporal and spatial patterns of expression of olfactory-specific genes. Using RT-PCR and in situ hybridization, it was found that expression of Xenopus olfactory marker protein and of class I receptors, which are thought to be responsible for the perception of water-soluble odorants, was detectable as early as stage 32, less than 2 days after fertilization. In contrast, expression of class II receptors, which are thought to recognize airborne odours, was not detected before stage 49, approximately 12 days after fertilization. The results indicate that the expression of olfactory receptors and marker protein is governed by temporally regulated cues during development.