Potentiation of capsaicin receptor activity by metabotropic ATP receptors as a possible mechanism for ATP-evoked pain and hyperalgesia.

The capsaicin (vanilloid) receptor, VR1, is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. It has been proposed that ATP, released from different cell types, initiates the sensation of pain by acting predominantly on nociceptive ionotropic purinoceptors located on sensory nerve terminals. In this study, we examined the effects of extracellular ATP on VR1. In cells expressing VR1, ATP increased the currents evoked by capsaicin or protons through activation of metabotropic P2Y(1) receptors in a protein kinase C-dependent pathway. The involvement of G(q/11)-coupled metabotropic receptors in the potentiation of VR1 response was confirmed in cells expressing both VR1 and M1 muscarinic acetylcholine receptors. In the presence of ATP, the temperature threshold for VR1 activation was reduced from 42 degrees C to 35 degrees C, such that normally nonpainful thermal stimuli (i.e., normal body temperature) were capable of activating VR1. This represents a novel mechanism through which the large amounts of ATP released from damaged cells in response to tissue trauma might trigger the sensation of pain.

Glutamate receptors: brain function and signal transduction.

Glutamate receptors are important in neural plasticity, neural development and neurodegeneration. N-methyl-d-aspartate (NMDA) receptors and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors act as glutamate-gated cation channels, whereas metabotropic receptors (mGluRs) modulate the production of second messengers via G proteins. Molecular studies from our and other laboratories indicated that NMDA receptors and mGluRs exist as multiple subunits (NMDAR1 and NMDAR2A-2D) and multiple subtypes (mGluR1-mGluR8). In light of the molecular diversity of glutamate receptors, we explored the function and intracellular signaling mechanisms of different members of glutamate receptors. In the visual system, retinal bipolar cells receive glutamate transmission from photoreceptors and contribute to segregating visual signals into ON and OFF pathways. The molecularly cloned mGluR6 is restrictedly expressed at the postsynaptic site of ON-bipolar cells in both rod and cone systems. Gene targeting of mGluR6 results in a loss of ON responses without changing OFF responses and severely impairs detecting visual contrasts. Since AMPA receptors mediate OFF responses in OFF-bipolar cells, two distinct types of glutamate receptors effectively operate for ON and OFF responses. mGluR1 and mGluR5 are both coupled to inositol triphosphate (IP3)/calcium signal transduction with an identical agonist selectivity. Single-cell intracellular calcium ([Ca2+]i) recordings indicated that glutamate evokes a non-oscillatory and oscillatory [Ca2+]i response in mGluR1-expressing and mGluR5-expressing cells, respectively. This difference results from a single amino acid substitution, aspartate of mGluR1 or threonine of mGluR5, at the G protein-interacting carboxy-terminal domains. Protein kinase C phosphorylation of the threonine of mGluR5 is responsible for inducing [Ca2+]i oscillations in mGluR5-expressing cells and cultured glial cells. Thus, the two closely related mGluR subtypes mediate diverging intracellular signaling in glutamate transmission.

The mGluR6 5' upstream transgene sequence directs a cell-specific and developmentally regulated expression in retinal rod and ON-type cone bipolar cells.

We generated transgenic mice, using 9.5 kilobase pairs of the 5' upstream sequence from the mouse metabotropic glutamate receptor subtype 6 (mGluR6) gene fused to the beta-galactosidase (lacZ) reporter gene, and investigated the promoter function of the cell-specific and developmentally regulated expression of mGluR6. Most of the independent transgenic lines commonly showed the lacZ expression in the defined cell layers of the retina, and four transgenic lines were characterized in detail for cell-specific lacZ expression patterns by X-gal staining and lacZ immunostaining. The lacZ-expressing retinal cells were classified into two cell types. One cell type was identified as rod bipolar cells on the basis of colocalization of protein kinase C (PKC) immunoreactivity and morphological criteria. The other cell type was PKC-immunonegative and resided at the cell layers corresponding precisely to ON-type cone bipolar cells. The latter bipolar cells were found to exist as a large cell population comparable to rod bipolar cells. This observation was confirmed by coimmunostaining of dissociated retinal cells with the lacZ and PKC antibodies. The ontogeny analysis indicated that the lacZ expression completely agrees with a temporal expression pattern of mGluR6 during retinal development. This study demonstrates that the mGluR6 5' upstream genomic sequence is capable of directing a cell-specific and developmentally regulated expression of mGluR6 in ON-type bipolar cells and supports the view that mGluR6 is responsible for ON responses in both the rod and cone systems.

Phenotype of mice lacking functional Deleted in colorectal cancer (Dcc) gene.

The DCC (Deleted in colorectal cancer) gene was first identified as a candidate for a tumour-suppressor gene on human chromosome 18q. More recently, in vitro studies in rodents have provided evidence that DCC might function as a receptor for the axonal chemoattractant netrin-1. Inactivation of the murine Dcc gene caused defects in axonal projections that are similar to those observed in netrin-1-deficient mice but did not affect growth, differentiation, morphogenesis or tumorigenesis in mouse intestine. These observations fail to support a tumour-suppressor function for Dcc, but are consistent with the hypothesis that DCC is a component of a receptor for netrin-1.

Vertebrate homologues of C. elegans UNC-5 are candidate netrin receptors.

In the developing nervous system, migrating cells and axons are guided to their targets by cues in the extracellular environment. The netrins are a family of phylogenetically conserved guidance cues that can function as diffusible attractants and repellents for different classes of cells and axons. In vertebrates, insects and nematodes, members of the DCC subfamily of the immunoglobulin superfamily have been implicated as receptors that are involved in migration towards netrin sources. The mechanisms that direct migration away from netrin sources (presumed repulsions) are less well understood. In Caenorhabditis elegans, the transmembrane protein UNC-5 (ref. 14) has been implicated in these responses, as loss of unc-5 function causes migration defects and ectopic expression of unc-5 in some neurons can redirect their axons away from a netrin source. Whether UNC-5 is a netrin receptor or simply an accessory to such a receptor has not, however, been defined. We now report the identification of two vertebrate homologues of UNC-5 which, with UNC-5 and the product of the mouse rostral cerebellar malformation gene (rcm), define a new subfamily of the immunoglobulin superfamily, and whose messenger RNAs show prominent expression in various classes of differentiating neurons. We provide evidence that these two UNC-5 homologues, as well as the rcm gene product, are netrin-binding proteins, supporting the hypothesis that UNC-5 and its relatives are netrin receptors.

Deleted in Colorectal Cancer (DCC) encodes a netrin receptor.

The guidance of developing axons in the nervous system is mediated partly by diffusible chemoattractants secreted by axonal target cells. Netrins are chemoattractants for commissural axons in the vertebrate spinal cord, but the mechanisms through which they produce their effects are unknown. We show that Deleted in Colorectal Cancer (DCC), a transmembrane protein of the immunoglobulin superfamily, is expressed on spinal commissural axons and possesses netrin-1-binding activity. Moreover, an antibody to DCC selectively blocks the netrin-1-dependent outgrowth of commissural axons in vitro. These results indicate that DCC is a receptor or a component of a receptor that mediates the effects of netrin-1 on commissural axons, and they complement genetic evidence for interactions between DCC and netrin homologs in C. elegans and Drosophila.

Characterization of excitatory amino acid neurotoxicity in N-methyl-D-aspartate receptor-deficient mouse cortical neuronal cells.

Roles and mechanisms of N-methyl-D-aspartate (NMDA) receptors in glutamate neurotoxicity were investigated in cultures of NMDA receptor-deficient cortical neuronal cells. Mutant mice lacking a functional NMDA receptor were generated by gene targeting of the NR1 NMDA receptor subunit. Cortical neuronal cells prepared from wild-type NR1+/+, heterozygous NR1+/- and homozygous mutant NR1-/- mice at 15-17 days of gestation grew indistinguishably from each other. Brief exposures (5 min) of both NR1+/+ and NR1+/- neuronal cells to glutamate or NMDA, but not kainate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), resulted in widespread neuronal degeneration by the following day. In contrast, neither glutamate nor NMDA treatment caused neuronal degeneration in NR1-/- cells, indicating that NMDA receptors are responsible for rapidly triggered glutamate neurotoxicity. The above four compounds were all effective in inducing the death of NR1+/+ and NR1+/- neuronal cells after prolonged exposure (20-24 h). However, NMDA had no neurotoxic effects on NR1-/- cells, although the other three compounds wer neurotoxic with potencies comparable to those for NR1+/+ and NR1+/- cells. The AMPA and kainate receptors are thus sufficient for inducing slowly triggered glutamate neurotoxicity. Brief exposure of a mixed population of NR1+/+ and NR1-/- neuronal cells to NMDA selectively killed the NMDA receptor-expressing cells without any appreciable effects on neighbouring NMDA receptor-deficient cells. This finding further supports a direct and indispensable role for NMDA receptors in NMDA-evoked neuronal cell death.

Specific deficit of the ON response in visual transmission by targeted disruption of the mGluR6 gene.

Taking advantage of the restricted expression of metabotropic glutamate receptor subtype 6 (mGluR6) in retinal ON bipolar cells, we generated knockout mice lacking mGluR6 expression. The homozygous mutant mice showed a loss of ON responses but unchanged OFF responses to light. The mutant mice displayed no obvious changes in retinal cell organization nor in the projection of optic fibers to the brain. Furthermore, the mGluR6-deficient mice showed visual behavioral responses to light stimulation as examined by shuttle box avoidance behavior experiments using light exposure as a conditioned stimulus. The results demonstrate that mGluR6 is essential in synaptic transmission to the ON bipolar cell and that the OFF response provides an important means for transmitting visual information.

Ultrastructure and microvasculature of the parotid duct in the cat.

Ultrastructure and microvascular pattern of the parotid duct (main excretory duct) in the cat were investigated by TEM and SEM. The duct was lined by a pseudostratified epithelium which was surrounded by a double-layered vascular sheath. Three kinds of principal cells were observed in this epithelium--tall columnar, light and basal cells. The tall columnar cells did not bear basement membrane specialization (infoldings) but well-developed lateral interdigitations. Microvilli were present on their luminal surface. Basal infoldings and lateral interdigitations did not develop in the light cells. Basal cells were located on the basement membrane of the epithelium. The basal surface of the duct epithelium was studded with half-desmosomes. The external sheath of the double-layered vascular sheath was composed of arterioles and venules, and the inner sheath was formed by a capillary network immediately beneath the epithelium. Meshes of the capillary network in the collecting ducts and the region of origin appeared round and in the superficial and deep regions became elongated along the duct axis. It can be concluded that the parotid duct in the cat not only transports the saliva into the oral cavity, but also actively builds up the primary saliva to the final saliva, owing to absorptive and secretory functions which were made by an important contribution of the double-layered vascular sheath surrounding the whole length of the duct.

Molecular diversity of glutamate receptors and their physiological functions.

Glutamate receptors play an important role in many integrative brain functions and in neuronal development. We report the molecular diversity of NMDA receptors and metabotropic glutamate receptors on the basis of our studies of molecular cloning and characterization of the diverse members of these receptors. The NMDA receptors consist of two distinct types of subunits. NMDAR1 possesses all properties characteristic of the NMDA receptor-channel complex, whereas the four NMDAR2 subunits, termed NMDAR2A-2D, show no channel activity but potentiate the NMDAR1 activity and confer functional variability by different heteromeric formations. The NMDA receptor subunits are considerably divergent from the other ligand-gated ion channels, and the structural architecture of these subunits remains elusive. The mGluRs form a family of at least seven different subtypes termed mGluR1-mGluR7. These receptor subtypes have, seven transmembrane segments and possess a large extracellular domain at their N-terminal regions. The seven mGluR subtypes are classified into three subgroups according to their sequence similarities, signal transduction mechanisms and agonist selectivities: mGluR1/mGluR5, mGluR2/mGluR3 and mGluR4/mGluR6/mGluR7. On the basis of our knowledge of the molecular diversity of the NMDA receptors and mGluRs, we have studied the physiological roles of individual receptor subunits or subtypes. We have shown that K(+)-induced depolarization or NMDA treatment in primary cultures of neonatal cerebellar granule cells induces the functional NMDA receptor and specifically up-regulates NMDAR2A mRNA among the multiple NMDA receptor subunits through the increase in resting intracellular Ca2+ concentrations. Our study demonstrates that the regulation of the specific NMDA receptor subunit mRNA governs the NMDA receptor induction that is thought to play an important role in granule cell survival and death. Analysis of an agonist selectivity and an expression pattern of mGluR6 has indicated that mGluR6 is responsible for synaptic neurotransmission from photoreceptor cells to ON-bipolar cells in the visual system. We have also investigated the function of mGluR2 in granule cells of the accessory olfactory bulb by combining immunoelectron-microscopic analysis with slice-patch recordings on the basis of the identification of a new agonist selective for this receptor subtype. Our results demonstrate that mGluR2 is present at the presynaptic site of granule cells and modulates inhibitory GABA transmission from granule cells to mitral cells. This finding indicates that the mGluR2 activation relieves excited mitral cells from GABA inhibition but maintains the lateral inhibition of unexcited mitral cells, thus resulting in enhancement of the signal-to-noise ratio between the excited mitral cells and their neighboring unexcited mitral cells.

Gene mapping of NMDA receptors and metabotropic glutamate receptors in the rat (Rattus norvegicus).

Five N-methyl-D-aspartate receptor subunit genes and six metabotropic glutamate receptor subtype genes have been assigned to particular rat chromosomes by using a rat x mouse somatic cell hybrid clone panel. N-Methyl-D-aspartate receptor subunit genes (gene symbol, GRIN) GRIN1, GRIN2A, GRIN2B, GRIN2C, and GRIN2D have been assigned to chromosomes (Chr) 3, 10, 4, 10, and 1, respectively. Metabotropic glutamate receptor subtype genes (gene symbol, GRM) GRM1, GRM2, GRM3, GRM4, GRM5, and GRM6 have been assigned to Chr 1, 8, 4, 20, 1, and 10, respectively. In addition, GRIN2A and GRM6 loci were successfully localized on Chr 10 linkage maps by linkage analyses. The genetic distances between loci in cM (+/- SD) are as follows: GRIN2A-28.6(+/- 7.0)-RR24-23.3(+/- 6.4)-MYHSE, from a linkage analysis using the (SHR x WTC)F1 x WTC cross, and RR24-4.2(+/- 2.9)-GRM6-4.2(+/- 2.9)-MMYHSE-2.1(+/- 2.1)-ASGR, SHBG-27.1(+/- 6.4)-PPY, from a linkage analysis using the (ZI x TM)F1 x ZI cross.

Molecular characterization of NMDA and metabotropic glutamate receptors.

Our molecular studies have revealed the existence of a large number of different subunits or subtypes for the NMDA and metabotropic glutamate receptors. The individual receptors show functional variabilities and distinct expression patterns in the CNS. The NMDA receptors belong to the ligand-gated ion channel family and consist of a key subunit NMDAR1 and four accessory subunits NMDAR2A-NMDAR2D. The combination of NMDAR1 and NMDAR2 in heteromeric configurations potentiates glutamate response and produces a functional variability. All the NMDAR subunits have an asparagine residue at the corresponding position of the second transmembrane segments, and these residues are thought to be responsible for controlling Ca2+ permeation and the channel blockade by Mg2+ and cationic channel blockers. Individual NMDAR subunit mRNAs are different in their expression patterns during development and in the adult brain. The mGluR family consists of at least six different subtypes. These subtypes are divided into three subgroups according to their sequence similarities, signal transduction mechanisms, and pharmacological properties. Although their physiological roles largely remain to be elucidated, the retinal L-AP4-sensitive mGluR may have a specific function that mediates excitatory neurotransmission in the visual system. It is thus undoubtedly important to investigate specific functions of different combinations of the NMDA receptor subunits and different subtypes of mGluRs and to explore the molecular mechanisms of glutamate receptor-mediated neuronal plasticity and neurotoxicity.

Role of the large extracellular domain of metabotropic glutamate receptors in agonist selectivity determination.

Metabotropic glutamate receptors consist of at least six different subtypes termed mGluR1-mGluR6. They belong to the family of G protein-coupled receptors and commonly possess an unusually large extracellular domain preceding the seven transmembrane segments. mGluR1 and mGluR2 show similar affinities for L-glutamate but distinct patterns in their responsiveness to quisqualate and trans-1-amino-1,3-cyclopentane-dicarboxylate (tACPD). To assign structural determinants for the different agonist selectivities, we constructed a series of chimeric receptors at the extracellular domains of mGluR1 and mGluR2 and determined their agonist selectivities by measuring their electrophysiological responses to L-glutamate, quisqualate, and tACPD in Xenopus oocytes. Replacement of the extracellular domain up to about one-half of the amino-terminal extracellular domain of mGluR1 with the corresponding portion of mGluR2 generated a pattern of the agonist selectivity characteristic of mGluR2. The acquirement of this property in agonist selectivity was further indicated by the selective responses of these chimeric receptors to an mGluR2-specific agonist, (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine. This investigation demonstrates that the extracellular domain of mGluR is critical in determining agonist selectivity and that the mode of determination of agonist selectivity of mGluR is different from that of other G protein-coupled receptors for small molecule transmitters.

Signal transduction, pharmacological properties, and expression patterns of two rat metabotropic glutamate receptors, mGluR3 and mGluR4.

The metabotropic glutamate receptors are coupled to intracellular signal transduction via G-proteins and consist of a family of at least five different subtypes, termed mGluR1-mGluR5. We studied the signal transduction mechanism and pharmacological characteristics of the rat mGluR3 and mGluR4 subtypes in Chinese hamster ovary cells permanently expressing the cloned receptors. Both mGluR3 and mGluR4 inhibit the forskolin-stimulated accumulation of intracellular cAMP formation in response to agonist interaction. Consistent with the high degree of sequence similarity to mGluR2, mGluR3 closely resembles mGluR2 in its agonist selectivity; the potency rank order of agonists is L-glutamate > trans-1-aminocyclopentane-1,3-dicarboxylate > ibotenate > quisqualate. mGluR4 is totally different in its agonist specificity from any other member of the metabotropic receptors. This receptor potently reacts with L-2-amino-4-phosphonobutyrate (L-AP4) in a stereo-selective manner and moderately responds to L-serine-O-phosphate. mGluR4 thus corresponds well to the putative L-AP4 receptor characterized from brain preparations. Blot and in situ hybridization analyses indicated that both mRNAs are widely distributed in the rat brain. mGluR3 mRNA is highly expressed in neuronal cells of the cerebral cortex and the caudate-putamen, and in granule cells of the hippocampal dentate gyrus. The expression pattern of mGluR4 mRNA is more restricted, and this expression is prominent in the cerebellum, olfactory bulb, and thalamus. Furthermore, the mGluR3 mRNA, unlike the other mRNAs for the metabotropic receptors, is highly expressed in glial cells throughout the brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning and chromosomal localization of the key subunit of the human N-methyl-D-aspartate receptor.

A complementary DNA encoding the key subunit of the human N-methyl-D-aspartate (NMDA) receptor (NMDAR1) has been cloned using a probe derived from the rat NMDAR1 cDNA. The cDNA encodes a 938-amino acid protein, which shows 99% amino acid homology with the rat counterpart. Of the 7 of 938 amino acids which are different, three occur in the region of the signal peptide and the others in the extracellular amino-terminal domain preceding the 4 putative transmembrane segments. Expression in Xenopus oocytes demonstrated that the single protein encoded by the cloned cDNA possesses the electrophysiological and pharmacological properties characteristic of the NMDA receptor, including Ca2+ permeability, voltage-dependent Mg2+ block, and inhibition by selective antagonists such as Zn2+ and channel blockers. The high evolutionary conservation in the structure and properties of NMDAR1 argues strongly for the importance of this receptor in functions of glutamate neurotransmission. RNA blot analysis showed abundant expression of mRNA whose size is about 4.5 and 4.8 kilonucleotides. The human gene encoding the NMDAR1 subunit has been mapped to chromosome 9q34.3 by the analyses of blot hybridization of a DNA panel of human/hamster somatic cell hybrids and fluorescence in situ hybridization of human chromosomes.

Molecular characterization of the family of the N-methyl-D-aspartate receptor subunits.

cDNA clones for four different N-methyl-D-aspartate (NMDA) receptor subunits (NMDAR2A-NMDAR2D) were isolated through polymerase chain reactions followed by molecular screening of a rat brain cDNA library. These subunits are only about 15% identical with the key subunit of the NMDA receptor (NMDAR1) but are highly homologous (approximately 50% homology) with one another. They also commonly possess large hydrophilic domains at both amino- and carboxyl-terminal sides of the four putative transmembrane segments. NMDAR2A and NMDAR2C expressed individually in Xenopus oocytes showed no electrophysiological response to agonists. However, these subunits in combined expression with NMDAR1 markedly potentiated the NMDAR1 activity and produced functional variability in the affinity of agonists, the effectiveness of antagonists, and the sensitivity to Mg2+ blockade. Thus, NMDAR1 is essential for the function of the NMDA receptor, and multiple NMDAR2 subunits potentiate and differentiate the function of the NMDA receptor by forming different heteromeric configurations with NMDAR1. Northern blotting and in situ hybridization analyses revealed that the expressions of individual mRNAs for the NMDAR2 subunits overlap in some brain regions but are also specialized in many other regions. This investigation demonstrates the anatomical and functional differences of the NMDAR2 subunits, which provide the molecular basis for the functional diversity of the NMDA receptor.

Alteration of Ca2+ permeability and sensitivity to Mg2+ and channel blockers by a single amino acid substitution in the N-methyl-D-aspartate receptor.

The N-methyl-D-aspartate (NMDA) receptor plays an important role in glutamate-mediated neuronal plasticity and neurotoxicity in the central nervous system. This receptor is composed of a fundamental subunit (NMDAR1) and its potentiating subunits (NMDAR2A-NMDAR2D). The NMDA receptor is distinct from other glutamate receptor channels because of its high Ca2+ permeability and inhibition by selective cationic channel blockers such as Mg2+, Zn2+, and MK-801. In this study, we investigated the structural features that control Ca2+ permeation and channel blockade of the NMDA receptor by in vitro mutagenesis and expression in Xenopus oocytes. We constructed a series of mutations with single amino acid substitutions in the second transmembrane segment of NMDAR1 and examined channel properties of the resultant mutants in combined expression with the NMDAR2A subunit. Substitution of the asparagine with either glutamine or arginine altered both the Ca2+ permeability and the sensitivity to blockades by Mg2+ and MK-801. These mutations also reduced the inhibitory effects of Zn2+ and an antidepressant, desipramine. Based on these results, we concluded that an asparagine ring formed in the central part of the channel-forming second transmembrane segments plays a critical role in determining the Ca2+ permeability and the inhibition of open channel blockers.