Causalgia, pathological pain, and adrenergic receptors

Control of expression of molecular receptors for chemical messengers and modulation of these receptors’ activity are now established as ways to alter cellular reaction. This paper extends these mechanisms to the arena of pathological pain by presenting the hypothesis that increased expression of α-adrenergic receptors in primary afferent neurons is part of the etiology of pain in classical causalgia. It is argued that partial denervation by lesion of peripheral nerve or by tissue destruction induces a change in peripheral nociceptors, making them excitable by sympathetic activity and adrenergic substances. This excitation is mediated by α-adrenergic receptors and has a time course reminiscent of experimental denervation supersensitivity. The change in neuronal phenotype is demonstrable after lesions of mixed nerves or of the sympathetic postganglionic supply. Similar partial denervations also produce a substantial increase in the number of dorsal root ganglion neurons evidencing the presence of α-adrenergic receptors. The hypothesis proposes the increased presence of α-adrenergic receptors in primary afferent neurons to result from an altered gene expression triggered by cytokines/growth factors produced by disconnection of peripheral nerve fibers from their cell bodies. These additional adrenergic receptors are suggested to make nociceptors and other primary afferent neurons excitable by local or circulating norepinephrine and epinephrine. For central pathways, the adrenergic excitation would be equivalent to that produced by noxious events and would consequently evoke pain. In support, evidence is cited for a form of denervation supersensitivity in causalgia and for increased expression of human α-adrenergic receptors after loss of sympathetic activity.

Src, a molecular switch governing gain control of synaptic transmission mediated by N-methyl-d-aspartate receptors

The N-methyl-d-aspartate (NMDA) receptor is a principal subtype of glutamate receptor mediating fast excitatory transmission at synapses in the dorsal horn of the spinal cord and other regions of the central nervous system. NMDA receptors are crucial for the lasting enhancement of synaptic transmission that occurs both physiologically and in pathological conditions such as chronic pain. Over the past several years, evidence has accumulated indicating that the activity of NMDA receptors is regulated by the protein tyrosine kinase, Src. Recently it has been discovered that, by means of up-regulating NMDA receptor function, activation of Src mediates the induction of the lasting enhancement of excitatory transmission known as long-term potentiation in the CA1 region of the hippocampus. Also, Src has been found to amplify the up-regulation of NMDA receptor function that is produced by raising the intracellular concentration of sodium. Sodium concentration increases in neuronal dendrites during high levels of firing activity, which is precisely when Src becomes activated. Therefore, we propose that the boost in NMDA receptor function produced by the coincidence of activating Src and raising intracellular sodium may be important in physiological and pathophysiological enhancement of excitatory transmission in the dorsal horn of the spinal cord and elsewhere in the central nervous system.

GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors.

The yeast two-hybrid system was used to isolate a clone from a 17-day-old mouse embryo cDNA library that codes for a novel 812-aa long protein fragment, glucocorticoid receptor-interacting protein 1 (GRIP1), that can interact with the hormone binding domain (HBD) of the glucocorticoid receptor. In the yeast two-hybrid system and in vitro, GRIP1 interacted with the HBDs of the glucocorticoid, estrogen, and androgen receptors in a hormone-regulated manner. When fused to the DNA binding domain of a heterologous protein, the GRIP1 fragment activated a reporter gene containing a suitable enhancer site in yeast cells and in mammalian cells, indicating that GRIP1 contains a transcriptional activation domain. Overexpression of the GRIP1 fragment in mammalian cells interfered with hormone-regulated expression of mouse mammary tumor virus-chloramphenicol acetyltransferase gene and constitutive expression of cytomegalovirus-beta-galactosidase reporter gene, but not constitutive expression from a tRNA gene promoter. This selective squelching activity suggests that GRIM can interact with an essential component of the RNA polymerase II transcription machinery. Finally, while a steroid receptor HBD fused with a GAL4 DNA binding domain did not, by itself, activate transcription of a reporter gene in yeast, coexpression of this fusion protein with GRIP1 strongly activated the reporter gene. Thus, in yeast, GRIP1 can serve as a coactivator, potentiating the transactivation functions in steroid receptor HBDs, possibly by acting as a bridge between HBDs of the receptors and the basal transcription machinery.

Low ethanol concentrations enhance GABAergic inhibitory postsynaptic potentials in hippocampal pyramidal neurons only after block of GABAB receptors.

Despite considerable evidence that ethanol can enhance chloride flux through the gamma-aminobutyric acid type A (GABA/A/) receptor-channel complex in several central neuron types, the effect of ethanol on hippocampal GABAergic systems is still controversial. Therefore, we have reevaluated this interaction in hippocampal pyramidal neurons subjected to local monosynaptic activation combined with pharmacological isolation of the various components of excitatory and inhibitory synaptic potentials, using intracellular current- and voltage-clamp recording methods in the hippocampal slice. In accord with our previous findings, we found that ethanol had little effect on compound inhibitory postsynaptic potentials/currents (IPSP/Cs) containing both GABA/A/ and GABA/B/ components. However, after selective pharmacological blockade of the GABA/B/ component of the IPSP (GABA/B/-IPSP/C) by CGP-35348, low concentrations of ethanol (22-66 mM) markedly enhanced the peak amplitude, and especially the area, of the GABA/A/ component (GABA/A/-IPSP/C) in most CA1 pyramidal neurons. Ethanol had no significant effect on the peak amplitude or area of the pharmacologically isolated GABA/B/-inhibitory postsynaptic current (IPSC). These results provide new data showing that activation of GABAB receptors can obscure ethanol enhancement of GABA/A/ receptor function in hippocampus and suggest that similar methods of pharmacological isolation might be applied to other brain regions showing negative or mixed ethanol-GABA interactions.

Identification of a Drosophila G protein alpha subunit (dGq alpha-3) expressed in chemosensory cells and central neurons.

We have identified another Drosophila GTP-binding protein (G protein) alpha subunit, dGq alpha-3. Transcripts encoding dGq alpha-3 are derived from alternative splicing of the dGq alpha locus previously shown to encode two visual-system-specific transcripts [Lee, Y.-J., Dobbs, M.B., Verardi, M.L. & Hyde, D.R. (1990) Neuron 5, 889-898]. Immunolocalization studies using dGq alpha-3 isoform-specific antibodies and LacZ fusion genes show that dGq alpha-3 is expressed in chemosensory cells of the olfactory and taste structures, including a subset of olfactory and gustatory neurons, and in cells of the central nervous system, including neurons in the lamina ganglionaris. These data are consistent with a variety of roles for dGq alpha-3, including mediating a subset of olfactory and gustatory responses in Drosophila, and supports the idea that some chemosensory responses use G protein-coupled receptors and the second messenger inositol 1,4,5-trisphosphate.

Benzodiazepine-insensitive mice generated by targeted disruption of the gamma 2 subunit gene of gamma-aminobutyric acid type A receptors.

Vigilance, anxiety, epileptic activity, and muscle tone can be modulated by drugs acting at the benzodiazepine (BZ) site of gamma-aminobutyric acid type A (GABAA) receptors. In vivo, BZ sites are potential targets for endogenous ligands regulating the corresponding central nervous system states. To assess the physiological relevance of BZ sites, mice were generated containing GABAA receptors devoid of BZ sites. Following targeted disruption of the gamma 2 subunit gene, 94% of the BZ sites were absent in brain of neonatal mice, while the number of GABA sites was only slightly reduced. Except for the gamma 2 subunit, the level of expression and the regional and cellular distribution of the major GABAA receptor subunits were unaltered. The single channel main conductance level and the Hill coefficient were reduced to values consistent with recombinant GABAA receptors composed of alpha and beta subunits. The GABA response was potentiated by pentobarbital but not by flunitrazepam. Diazepam was inactive behaviorally. Thus, the gamma 2 subunit is dispensable for the assembly of functional GABAA receptors but is required for normal channel conductance and the formation of BZ sites in vivo. BZ sites are not essential for embryonic development, as suggested by the normal body weight and histology of newborn mice. Postnatally, however, the reduced GABAA receptor function is associated with retarded growth, sensorimotor dysfunction, and drastically reduced life-span. The lack of postnatal GABAA receptor regulation by endogenous ligands of BZ sites might contribute to this phenotype.

kappa-Opioid receptor in humans: cDNA and genomic cloning, chromosomal assignment, functional expression, pharmacology, and expression pattern in the central nervous system.

Using the mouse delta-opioid receptor cDNA as a probe, we have isolated genomic clones encoding the human mu- and kappa-opioid receptor genes. Their organization appears similar to that of the human delta receptor gene, with exon-intron boundaries located after putative transmembrane domains 1 and 4. The kappa gene was mapped at position q11-12 in human chromosome 8. A full-length cDNA encoding the human kappa-opioid receptor has been isolated. The cloned receptor expressed in COS cells presents a typical kappa 1 pharmacological profile and is negatively coupled to adenylate cyclase. The expression of kappa-opioid receptor mRNA in human brain, as estimated by reverse transcription-polymerase chain reaction, is consistent with the involvement of kappa-opioid receptors in pain perception, neuroendocrine physiology, affective behavior, and cognition. In situ hybridization studies performed on human fetal spinal cord demonstrate the presence of the transcript specifically in lamina II of the dorsal horn. Some divergences in structural, pharmacological, and anatomical properties are noted between the cloned human and rodent receptors.

Determination of alpha-conotoxin binding modes on neuronal nicotinic acetylcholine receptors

alpha-Conotoxins, from cone snails, and alpha-neurotoxins, from snakes, are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) that have overlapping binding sites in the ACh binding pocket. These disulphide-rich peptides are used extensively as tools to localize and pharmacologically characterize specific nAChRs subtypes. Recently, a homology model based on the high-resolution structure of an ACh binding protein (AChBP) allowed the three-fingered alpha-neurotoxins to be docked onto the alpha7 nAChR. To investigate if alpha-conotoxins interact with the nAChR in a similar manner, we built homology models of human alpha7 and alpha3beta2 nAChRs, and performed docking simulations of alpha-conotoxins ImI, PnIB, PnIA and MII using the program GOLD. Docking revealed that alpha-conotoxins have a different mode of interaction compared with alpha-neurotoxins, with surprisingly few nAChR residues in common between their overlapping binding sites. These docking experiments show that Imi and PnIB bind to the ACh binding pocket via a small cavity located above the beta9/beta10 hairpin of the (+)alpha7 nAChR subunit. Interestingly, PnIB, PnIA and MII were found to bind in a similar location on alpha7 or alpha3beta2 receptors mostly through hydrophobic interactions, while ImI bound further from the ACh binding pocket, mostly through electrostatic interactions. These findings, which distinguish alpha-conotoxin and alpha-neurotoxin binding modes, have implications for the rational design of selective nAChR antagonists. Copyright (C) 2004 John Wiley Sons, Ltd.

Adenosine triphosphate acts as both a competitive antagonist and a positive allosteric modulator at recombinant N-methyl-D-aspartate receptors

ATP and glutamate are fast excitatory neurotransmitters in the central nervous system acting primarily on ionotropic P2X and glutamate [N-methyl-D-aspartate (NMDA) and non-NMDA] receptors, respectively. Both neurotransmitters regulate synaptic plasticity and long-term potentiation in hippocampal neurons. NMDA receptors are responsible primarily for the modulatory action of glutamate, but the mechanism underlying the modulatory effect of ATP remains uncertain. In the present study, the effect of ATP on recombinant NR1a + 2A, NR1a + 2B, and NR1a + 2C NMDA receptors expressed in Xenopus laevis oocytes was investigated. ATP inhibited NR1a + 2A and NR1a + 2B receptor currents evoked by low concentrations of glutamate but potentiated currents evoked by saturating glutamate concentrations. In contrast, ATP potentiated NR1a + 2C receptor currents evoked by nonsaturating glutamate concentrations. ATP shifted the glutamate concentration-response curve to the right, indicating a competitive interaction at the agonist binding site. ATP inhibition and potentiation of glutamate-evoked currents was voltage-independent, indicating that ATP acts outside the membrane electric field. Other nucleotides, including ADP, GTP, CTP, and UTP, inhibited glutamate-evoked currents with different potencies, revealing that the inhibition is dependent on both the phosphate chain and nucleotide ring structure. At high concentrations, glutamate outcompetes ATP at the agonist binding site, revealing a potentiation of the current. This effect must be caused by ATP binding at a separate site, where it acts as a positive allosteric modulator of channel gating. A simple model of the NMDA receptor, with ATP acting both as a competitive antagonist at the glutamate binding site and as a positive allosteric modulator at a separate site, reproduced the main features of the data.

Differential involvement of rat medial prefrontal cortex dopamine receptors in modulation of hypothalamic-pituitary-adrenal axis responses to different stressors

Recent investigations have implicated the medial prefrontal cortex (mPFC) in modulation of subcortical pathways that contribute to the generation of behavioural, autonomic and endocrine responses to stress. However, little is known of the mechanisms involved. One of the key neurotransmitters involved in mPFC function is dopamine, and we therefore aimed, in this investigation, to examine the role of mPFC dopamine in response to stress in Wistar rats. In this regard, we infused dopamine antagonists SCH23390 or sulpiride into the mPFC via retrodialysis. We then examined changes in numbers of cells expressing the c-fos immediate-early gene protein product, Fos, in subcortical neuronal populations associated with regulation of hypothalamic-pituitary-adrenal (HPA) axis stress responses in response to either of two stressors; systemic injection of interleukin-1beta, or air puff. The D-1 antagonist, SCH23390, and the D-2 antagonist, sulpiride, both attenuated expression of Fos in the medial parvocellular hypothalamic paraventricular nucleus (mpPVN) corticotropin-releasing factor cells at the apex of the HPA axis, as well as in most extra-hypothalamic brain regions examined in response to interleukin-1beta. By contrast, SCH23390 failed to affect Fos expression in response to air puff in any brain region examined, while sulpiride resulted in an attenuation of the air puff-induced response in only the mpPVN and the bed nucleus of the stria terminalis. These results indicate that the mPFC differentially processes the response to different stressors and that the two types of dopamine receptor may have different roles.

Pharmacological evaluation of an [I-123] labelled imidazopyridine-3-acetamide for the study of benzodiazepine receptors

In vitro binding of the iodinated imidazopyri dine, N',N'-dimethyl-6-methyl-(4'-[I-123]iodophenyl)imidazo[1,2-a]pyridine-3-acetamide [I-123]IZOL to benzodiazepine binding sites on brain cortex, adrenal and kidney membranes is reported. Saturation experiments showed that [I-123]IZOL, bound to a single class of binding site (n(H)=0.99) on adrenal and kidney mitochondrial membranes with a moderate affinity (K-d=30 nM). The density of binding sites was 22 +/- 6 and 1.2 +/- 0.4 pmol/mg protein on adrenal and kidney membranes, respectively. No specific binding was observed in mitochondrial-synaptosomal membranes of brain cortex. In biodistribution studies in rats, the highest uptake of [I-123]IZOL was found 30 min post injection in adrenals (7.5% ID/g), followed by heart, kidney, lung (1% ID/g) and brain (0.12% ID/g), consistent with the distribution of peripheral benzodiazepine binding sites. Pre-administration of unlabelled IZOL and the specific PBBS drugs, PK 11195 and Ro 5-4864 significantly reduced the uptake of [I-123]IZOL by 30% (p < 0.05) in olfactory bulbs and by 51-86% (p < 0.01) in kidney, lungs, heart and adrenals, while it increased by 30% to 50% (p < 0.01) in the rest of the brain and the blood. Diazepam, a mixed CBR-PBBS drug, inhibited the uptake in kidney, lungs, heart, adrenals and olfactory bulbs by 32% to 44% (p < 0.01) but with no effect on brain uptake and in blood concentration. Flumazenil, a central benzodiazepine drug and haloperidol (dopamine antagonist/sigma receptor drug) displayed no effect in [I-123]IZOL in peripheral organs and in the brain. [I-123]IZOL may deserve further development for imaging selectively peripheral benzodiazepine binding sites. (c) 2006 Elsevier Inc. All rights reserved.

Synaptic activation of transient receptor potential channels by metabotropic glutamate receptors in the lateral amygdala

Classical mammalian transient receptor potential channels form non-selective cation channels that open in response to activation of phospholipase C-coupled metabotropic receptors, and are thought to play a key role in calcium homeostasis in non-excitable cells. Within the nervous system transient receptor potential channels are widely distributed but their physiological roles are not well understood. Here we show that in the rat lateral amygdala transient receptor potential channels mediate an excitatory synaptic response to glutamate. Activation of group l etabotropic glutamate receptors on pyramidal neurons in the lateral amygdala with either exogenous or synaptically released glutamate evokes an inward current at negative potentials with a current voltage relationship showing a region of negative slope and steep outward rectification. This current is blocked by inhibiting G protein function with GTP-beta-S, by inhibiting phospholipase C or by infusing transient receptor potential antibodies into lateral amygdala pyramidal neurons. Using RT-PCR and Western blotting we show that transient receptor potential 1, transient receptor potential 4 and transient receptor potential 5 are present in the lateral amygdala. Single cell PCR confirms the presence of transient receptor potential 1 and transient receptor potential 5 in pyramidal neurons and we show by co-immunoprecipitation that transient receptor potential 1 and transient receptor potential 5 co-assemble as a heteromultimers in the amygdala. These results show that in lateral amygdala pyramidal neurons synaptically released glutamate activates transient receptor potential channels, which we propose are likely to be heteromultimeric channels containing transient receptor potential 1 and transient receptor potential 5/transient receptor potential 4. (c) 2005 Published by Elsevier Ltd on behalf of IBRO.