LPA receptor expression in the central nervous system in health and following injury

Lysophosphatidic acid (LPA) is released from platelets following injury and also plays a role in neural development but little is known about its effects in the adult central nervous system (CNS). We have examined the expression of LPA receptors 1-3 (LPA_1–3) in intact mouse spinal cord and cortical tissues and following injury. In intact and injured tissues, LPA₁ was expressed by ependymal cells in the central canal of the spinal cord and was upregulated in reactive astrocytes following spinal cord injury. LPA₂ showed low expression in intact CNS tissue, on grey matter astrocytes in spinal cord and in ependymal cells lining the lateral ventricle. Following injury, its expression was upregulated on astrocytes in both cortex and spinal cord. LPA₃ showed low expression in intact CNS tissue, viz. on cortical neurons and motor neurons in the spinal cord, and was upregulated on neurons in both regions after injury. Therefore, LPA_1–3 are differentially expressed in the CNS and their expression is upregulated in response to injury. LPA release following CNS injury may have different consequences for each cell type because of this differential expression in the adult nervous system.     

Complex N-Glycosylation Stabilizes Surface Expression of Transient Receptor Potential Melastatin 4b Protein

N-Glycosylation is important for the function and regulation of ion channels. We examined the role of N-glycosylation of transient receptor potential melastatin (Trpm) 4b, a membrane glycoprotein that regulates calcium influx. Trpm4b was expressed in vivo in all rat tissues examined. In each tissue, Trpm4b had a different molecular mass, between ∼129 and ∼141 kDa, but all reverted to ∼120 kDa following treatment with peptide:N-glycosidase F, consistent with N-glycosylation being the principal form of post-translational modification of Trpm4b in vivo. In six stable isogenic cell lines that express different levels of Trpm4b, two forms were found, high mannose, core-glycosylated and complex, highly glycosylated (HG), with HG-Trpm4b comprising 85% of the total Trpm4b expressed. For both forms, surface expression was directly proportional to the total Trpm4b expressed. Complex N-glycosylation doubled the percentage of Trpm4b at the surface, compared with high mannose N-glycosylation. Mutation of the single N-glycosylation consensus sequence at Asn-988 (Trpm4b-N988Q), located near the pore-forming loop between transmembrane helices 5 and 6, prevented glycosylation, but did not prevent surface expression, impair formation of functional membrane channels, or alter channel conductance. In transfection experiments, the time courses for appearance of HG-Trpm4b and Trpm4b-N988Q on the surface were similar. In experiments with cycloheximide inhibition of protein synthesis, the time course for disappearance of HG-Trpm4b from the surface was much slower than that for Trpm4b-N988Q. We conclude that N-glycosylation is not required for surface expression or channel function, but that complex N-glycosylation plays a crucial role in stabilizing surface expression of Trpm4b.     

On potential interactions between non-selective cation channel TRPM4 and sulfonylurea receptor SUR1

The sulfonylurea receptor SUR1 associates with Kir6.2 or Kir6.1 to form K(ATP) channels, which link metabolism to excitability in multiple cell types. The strong physical coupling of SUR1 with Kir6 subunits appears exclusive, but recent studies argue that SUR1 also modulates TRPM4, a member of the transient receptor potential family of non-selective cation channels. It has been reported that, following stroke, brain, or spinal cord injury, SUR1 is increased in neurovascular cells at the site of injury. This is accompanied by up-regulation of a non-selective cation conductance with TRPM4-like properties and apparently sensitive to sulfonylureas, leading to the postulation that post-traumatic non-selective cation currents are determined by TRPM4/SUR1 channels. To investigate the mechanistic hypothesis for the coupling between TRPM4 and SUR1, we performed electrophysiological and FRET studies in COSm6 cells expressing TRPM4 channels with or without SUR1. TRPM4-mediated currents were Ca(2+)-activated, voltage-dependent, underwent desensitization, and were inhibited by ATP but were insensitive to glibenclamide and tolbutamide. These properties were not affected by cotransfection with SUR1. When the same SUR1 was cotransfected with Kir6.2, functional K(ATP) channels were formed. In cells cotransfected with Kir6.2, SUR1, and TRPM4, we measured K(ATP)-mediated K(+) currents and Ca(2+)-activated, sulfonylurea-insensitive Na(+) currents in the same patch, further showing that SUR1 controls K(ATP) channel activity but not TRPM4 channels. FRET signal between fluorophore-tagged TRPM4 subunits was similar to that between Kir6.2 and SUR1, whereas there was no detectable FRET efficiency between TRPM4 and SUR1. Our data suggest that functional or structural association of TRPM4 and SUR1 is unlikely.     

Transient Receptor Potential Melastatin 1: A Hair Cell Transduction Channel Candidate

Sound and head movements are perceived through sensory hair cells in the inner ear. Mounting evidence indicates that this process is initiated by the opening of mechanically sensitive calcium-permeable channels, also referred to as the mechanoelectrical transducer (MET) channels, reported to be around the tips of all but the tallest stereocilia. However, the identity of MET channel remains elusive. Literature suggests that the MET channel is a non-selective cation channel with a high Ca²⁺ permeability and ∼100 picosiemens conductance. These characteristics make members of the transient receptor potential (TRP) superfamily likely candidates for this role. One of these candidates is the transient receptor potential melastatin 1 protein (TRPM1), which is expressed in various cells types within the cochlea of the mouse including the hair cells. Recent studies demonstrate that mutations in the TRPM1 gene underlie the inherited retinal disease complete congenital stationary night blindness in humans and depolarizing bipolar cell dysfunction in the mouse retina, but auditory function was not assessed. Here we investigate the role of Trpm1 in hearing and as a possible hair cell MET channel using mice homozygous for the null allele of Trpm1 (Trpm1^−/−) or a missense mutation in the pore domain of TRPM1 (Trpm1^{tvrm27/tvrm27}). Hearing thresholds were evaluated in adult (4–5 months old) mice with auditory-evoked brain stem responses. Our data shows no statistically significant difference in hearing thresholds in Trpm1^−/− or Trpm1^{tvrm27/tvrm27} mutants compared to littermate controls. Further, none of the mutant mice showed any sign of balance disorder, such as head bobbing or circling. These data suggest that TRPM1 is not essential for development of hearing or balance and it is unlikely that TRPM1 is a component of the hair cell MET channel.     

Convergence of Melatonin and Serotonin (5-HT) Signaling at MT2/5-HT2C Receptor Heteromers

Inasmuch as the neurohormone melatonin is synthetically derived from serotonin (5-HT), a close interrelationship between both has long been suspected. The present study reveals a hitherto unrecognized cross-talk mediated via physical association of melatonin MT₂ and 5-HT_2C receptors into functional heteromers. This is of particular interest in light of the “synergistic” melatonin agonist/5-HT_2C antagonist profile of the novel antidepressant agomelatine. A suite of co-immunoprecipitation, bioluminescence resonance energy transfer, and pharmacological techniques was exploited to demonstrate formation of functional MT₂ and 5-HT_2C receptor heteromers both in transfected cells and in human cortex and hippocampus. MT₂/5-HT_2C heteromers amplified the 5-HT-mediated G_q/phospholipase C response and triggered melatonin-induced unidirectional transactivation of the 5-HT_2C protomer of MT₂/5-HT_2C heteromers. Pharmacological studies revealed distinct functional properties for agomelatine, which shows “biased signaling.” These observations demonstrate the existence of functionally unique MT₂/5-HT_2C heteromers and suggest that the antidepressant agomelatine has a distinctive profile at these sites potentially involved in its therapeutic effects on major depression and generalized anxiety disorder. Finally, MT₂/5-HT_2C heteromers provide a new strategy for the discovery of novel agents for the treatment of psychiatric disorders.     

Localisation of Formyl-Peptide Receptor 2 in the Rat Central Nervous System and Its Role in Axonal and Dendritic Outgrowth

Arachidonic acid and docosahexaenoic acid (DHA) released by the action of phospholipases A₂ (PLA₂) on membrane phospholipids may be metabolized by lipoxygenases to the anti-inflammatory mediators lipoxin A4 (LXA4) and resolvin D1 (RvD1), and these can bind to a common receptor, formyl-peptide receptor 2 (FPR2). The contribution of this receptor to axonal or dendritic outgrowth is unknown. The present study was carried out to elucidate the distribution of FPR2 in the rat CNS and its role in outgrowth of neuronal processes. FPR2 mRNA expression was greatest in the brainstem, followed by the spinal cord, thalamus/hypothalamus, cerebral neocortex, hippocampus, cerebellum and striatum. The brainstem and spinal cord also contained high levels of FPR2 protein. The cerebral neocortex was moderately immunolabelled for FPR2, with staining mostly present as puncta in the neuropil. Dentate granule neurons and their axons (mossy fibres) in the hippocampus were very densely labelled. The cerebellar cortex was lightly stained, but the deep cerebellar nuclei, inferior olivary nucleus, vestibular nuclei, spinal trigeminal nucleus and dorsal horn of the spinal cord were densely labelled. Electron microscopy of the prefrontal cortex showed FPR2 immunolabel mostly in immature axon terminals or ‘pre-terminals’, that did not form synapses with dendrites. Treatment of primary hippocampal neurons with the FPR2 inhibitors, PBP10 or WRW4, resulted in reduced lengths of axons and dendrites. The CNS distribution of FPR2 suggests important functions in learning and memory, balance and nociception. This might be due to an effect of FPR2 in mediating arachidonic acid/LXA4 or DHA/RvD1-induced axonal or dendritic outgrowth.     

The role of Transient Receptor Potential Vanilloid 1 (TRPV1) channels in pancreatitis

Premature activation of digestive enzymes within the pancreas which leads to autodigestion of the gland is an early step in the pathogenesis of pancreatitis. Pancreatic injury is followed by other manifestations of inflammation including plasma extravasation, edema, and neutrophil infiltration which constitute the features of pancreatitis. Recent studies indicate that neural innervation of the pancreas may play an important role in the initiation and maintenance of the inflammatory response to injury. The pancreas is innervated by vagal, sympathetic and parasympathetic neurons, as well as sensory neurons. Activation of pancreatic primary sensory neurons causes the release of inflammatory neuropeptides both in the spinal cord to signal pain and in the pancreas itself where they produce plasma extravasation and neutrophil infiltration. Recent studies indicate that primary sensory neurons of the pancreas express transient receptor potential V1 (TRPV1) channels whose activation induces pancreatic inflammation. Moreover, blockade of these TRP channels significantly ameliorates experimental pancreatitis. This review describes our current understanding of the role of TRPV1 channels in pancreatitis and illustrates how this mechanism might be used to direct future treatments of pancreatic diseases.     

Functional Interaction between Angiotensin II Receptor Type 1 and Chemokine (C-C Motif) Receptor 2 with Implications for Chronic Kidney Disease

Understanding functional interactions between G protein-coupled receptors is of great physiological and pathophysiological importance. Heteromerization provides one important potential mechanism for such interaction between different signalling pathways via macromolecular complex formation. Previous studies suggested a functional interplay between angiotensin II receptor type 1 (AT₁) and Chemokine (C-C motif) Receptor 2 (CCR2). However the molecular mechanisms are not understood. We investigated AT₁-CCR2 functional interaction in vitro using bioluminescence resonance energy transfer in HEK293 cells and in vivo using subtotal-nephrectomized rats as a well-established model for chronic kidney disease. Our data revealed functional heteromers of these receptors resulting in CCR2-Gαi1 coupling being sensitive to AT₁ activation, as well as apparent enhanced β-arrestin2 recruitment with agonist co-stimulation that is synergistically reversed by combined antagonist treatment. Moreover, we present in vivo findings where combined treatment with AT₁- and CCR2-selective inhibitors was synergistically beneficial in terms of decreasing proteinuria, reducing podocyte loss and preventing renal injury independent of blood pressure in the subtotal-nephrectomized rat model. Our findings further support a role for G protein-coupled receptor functional heteromerization in pathophysiology and provide insights into previous observations indicating the importance of AT₁-CCR2 functional interaction in inflammation, renal and hypertensive disorders.     

Central Administration of C-X-C Chemokine Receptor Type 4 Antagonist Alleviates the Development and Maintenance of Peripheral Neuropathic Pain in Mice

Aim

To explore the roles of C-X-C chemokine receptor type 4 (CXCR4) in spinal processing of neuropathic pain at the central nervous system (CNS).

Methods

Peripheral neuropathic pain (PNP) induced by partial sciatic nerve ligation (pSNL) model was assessed in mice. Effects of a single intrathecal (central) administration of AMD3100 (intrathecal AMD3100), a CXCR4 antagonist, on pain behavior and pain-related spinal pathways and molecules in the L3-L5 spinal cord segment was studied compare to saline treatment.

Results

Rotarod test showed that intrathecal AMD3100 did not impair mice motor function. In pSNL-induced mice, intrathecal AMD3100 delayed the development of mechanical allodynia and reversed the established mechanical allodynia in a dose-dependent way. Moreover, intrathecal AMD3100 downregulated the activation of JNK1 and p38 pathways and the protein expression of p65 as assessed by western blotting. Real-time PCR test also demonstrated that substance P mRNA was decreased, while adrenomedullin and intercellular adhesion molecule mRNA was increased following AMD3100 treatment.

Conclusion

Our results suggest that central (spinal) CXCR4 is involved in the development and maintenance of PNP and the regulation of multiple spinal molecular events under pain condition, implicating that CXCR4 would potentially be a therapeutic target for chronic neuropathic pain.     

The Interaction Affinity between Vascular Cell Adhesion Molecule-1 (VCAM-1) and Very Late Antigen-4 (VLA-4) Analyzed by Quantitative FRET

Very late antigen-4 (VLA-4), a member of integrin superfamily, interacts with its major counter ligand vascular cell adhesion molecule-1 (VCAM-1) and plays an important role in leukocyte adhesion to vascular endothelium and immunological synapse formation. However, irregular expressions of these proteins may also lead to several autoimmune diseases and metastasis cancer. Thus, quantifying the interaction affinity of the VCAM-1/VLA-4 interaction is of fundamental importance in further understanding the nature of this interaction and drug discovery. In this study, we report an ‘in solution’ steady state organic fluorophore based quantitative fluorescence resonance energy transfer (FRET) assay to quantify this interaction in terms of the dissociation constant (K_d). We have used, in our FRET assay, the Alexa Fluor 488-VLA-4 conjugate as the donor, and Alexa Fluor 546-VCAM-1 as the acceptor. From the FRET signal analysis, K_d of this interaction was determined to be 41.82 ± 2.36 nM. To further confirm our estimation, we have employed surface plasmon resonance (SPR) technique to obtain K_d = 39.60 ± 1.78 nM, which is in good agreement with the result obtained by FRET. This is the first reported work which applies organic fluorophore based ‘in solution’ simple quantitative FRET assay to obtain the dissociation constant of the VCAM-1/VLA-4 interaction, and is also the first quantification of this interaction. Moreover, the value of K_d can serve as an indicator of abnormal protein-protein interactions; hence, this assay can potentially be further developed into a drug screening platform of VLA-4/VCAM-1 as well as other protein-ligand interactions.     

NK1 Receptor Blockade Is Ineffective in Improving Outcome following a Balloon Compression Model of Spinal Cord Injury

The neuropeptide substance P (SP) is a well-known mediator of neurogenic inflammation following a variety of CNS disorders. Indeed, inhibition of SP through antagonism of its receptor, the tachykinin NK1 receptor, has been shown to be beneficial following both traumatic brain injury and stroke. Such studies demonstrated that administration of an NK1 receptor antagonist reduced blood-brain-barrier permeability, edema development and improved functional outcome. Furthermore, our recent studies have demonstrated a potential role for SP in mediating neurogenic inflammation following traumatic spinal cord injury (SCI). Accordingly, the present study investigates whether inhibition of SP may similarly play a neuroprotective role following traumatic SCI. A closed balloon compression injury was induced at T10 in New Zealand White rabbits. At 30 minutes post-injury an NK1 receptor antagonist was administered intravenously. Animals were thereafter assessed for blood spinal cord barrier (BSCB) permeability, spinal water content (edema), intrathecal pressure (ITP), and histological and functional outcome from 5 hours to 2 weeks post-SCI. Administration of an NK1 receptor antagonist was not effective in reducing BSCB permeability, edema, ITP, or functional deficits following SCI. We conclude that SP mediated neurogenic inflammation does not seem to play a major role in BSCB disruption, edema development and consequential tissue damage seen in acute traumatic SCI. Rather it is likely that the severe primary insult and subsequent hemorrhage may be the key contributing factors to ongoing SCI injury.     

Receptor heteromerization expands the repertoire of cannabinoid signaling in rodent neurons

A fundamental question in G protein coupled receptor biology is how a single ligand acting at a specific receptor is able to induce a range of signaling that results in a variety of physiological responses. We focused on Type 1 cannabinoid receptor (CB₁R) as a model GPCR involved in a variety of processes spanning from analgesia and euphoria to neuronal development, survival and differentiation. We examined receptor dimerization as a possible mechanism underlying expanded signaling responses by a single ligand and focused on interactions between CB₁R and delta opioid receptor (DOR). Using co-immunoprecipitation assays as well as analysis of changes in receptor subcellular localization upon co-expression, we show that CB₁R and DOR form receptor heteromers. We find that heteromerization affects receptor signaling since the potency of the CB₁R ligand to stimulate G-protein activity is increased in the absence of DOR, suggesting that the decrease in CB₁R activity in the presence of DOR could, at least in part, be due to heteromerization. We also find that the decrease in activity is associated with enhanced PLC-dependent recruitment of arrestin3 to the CB₁R-DOR complex, suggesting that interaction with DOR enhances arrestin-mediated CB₁R desensitization. Additionally, presence of DOR facilitates signaling via a new CB₁R-mediated anti-apoptotic pathway leading to enhanced neuronal survival. Taken together, these results support a role for CB₁R-DOR heteromerization in diversification of endocannabinoid signaling and highlight the importance of heteromer-directed signal trafficking in enhancing the repertoire of GPCR signaling.     

Dopamine D2 and D4 receptor heteromerization and its allosteric receptor-receptor interactions

Dopamine D₂ and D₄ receptors partially codistribute in the dorsal striatum and appear to play a fundamental role in complex behaviors and motor function. The discovery of D₂R–D_4.xR (D_4.2R, D_4.4R or D_4.7R) heteromers has been made in cellular models using co-immunoprecipitation, in situ Proximity Ligation Assays and BRET¹ techniques with the D₂R and D_4.7R receptors being the least effective in forming heteromers. Allosteric receptor–receptor interactions in D₂R–D_4.2R and D₂R–D_4.4 R heteromers were observed using the MAPK assays indicating the existence of an enhancing allosteric receptor–receptor interaction in the corresponding heteromers between the two orthosteric binding sites. The bioinformatic predictions suggest the existence of a basic set of common triplets (ALQ and LRA) in the two participating receptors that may contribute to the receptor–receptor interaction interfaces.     

Increase of annexin 1 immunoreactivity in spinal cord of newborn opossum (Monodelphis domestica) at the time when regeneration after injury stops being possible

Annexins constitute a family of proteins that associate reversibly with cell membranes in a calcium dependent manner. We have studied the distribution of annexin 1, which is known to mediate anti-inflammatory actions of glucocorticoids, and which is upregulated after spinal cord injury, in newborn and adult South American opossum (Monodelphis domestica) spinal cord. We show the increase in the annexin 1 immunoreactivity in spinal cords of neonatal opossums over the critical period when regeneration after injury ceases to be possible. We further show the restricted and specific sites at which it is detected in adult opossum cerebellum and hippocampus. Since the procedures used in immunochemistry of annexin in CNS have in the past yielded conflicting results, different procedures were tested and shown to be reliable. As a control, annexin 1 distribution was surveyed in kidney.     

Homomeric and heteromeric assembly of KCNQ (Kv7) K+ channels assayed by total internal reflection fluorescence/fluorescence resonance energy transfer and patch clamp analysis

M-type K(+) channels, consisting of KCNQ1-5 (Kv7.1-7.5) subunits, form a variety of homomeric and heteromeric channels. Whereas all the subunits can assemble into homomeric channels, the ability of the subunits to assemble into heteromultimers is highly variable. KCNQ3 is widely thought to co-assemble with several other KCNQ subtypes, whereas KCNQ1 and KCNQ2 do not. However, the existence of other subunit assemblies is not well studied. To systematically explore the heteromeric assembly of KCNQ channels in individual living cells, we performed fluorescence resonance energy transfer (FRET) between cyan fluorescent protein- and yellow fluorescent protein-tagged KCNQ subunits expressed in Chinese hamster ovary cells under total internal reflection fluorescence microscopy in which excitation light only penetrates several hundred nanometers into the cell, thus isolating membrane events. We found significant FRET between homomeric subunits as expected from their functional expression in heterologous expression systems. Also as expected from previous work, robust FRET was observed between KCNQ2 and KCNQ3. KCNQ3 and KCNQ4 also showed substantial FRET as did KCNQ4 and KCNQ5. To determine functional assembly of KCNQ4/KCNQ5 heteromers, we performed two types of experiments. In the first, we constructed a mutant tetraethylammonium ion-sensitive KCNQ4 subunit and tested its assembly with KCNQ5 by patch clamp analysis of the tetraethylammonium ion sensitivity of the resulting current; however, those data were not conclusive. In the second, we co-expressed a KCNQ4 (G285S) pore mutant with KCNQ5 and found the former to act as a dominant negative, suggesting co-assembly of the two types of subunits. These data confirm that among the allowed assembly conformations are KCNQ3/4 and KCNQ4/5 heteromers.     

Nuclear translocation of PKM2 modulates astrocyte proliferation via p27 and β-catenin pathway after spinal cord injury

Aberrant functionality of the cell cycle has been implicated in the pathology of traumatic SCI. Although it has been reported that the expressions of various cell cycle related proteins were altered significantly following SCI, detailed information on the subject remains largely unclear. The embryonic pyruvate kinase M2 (PKM2) is an important metabolic kinase in aerobic glycolysis or the warburg effect, however, its functions in central nervous system (CNS) injury remains elusive. Here we demonstrate that PKM2 was not only significantly upregulated by western blot and immunohistochemistry but certain traumatic stimuli also induced translocation of PKM2 into the nucleus in astrocytes following spinal cord injury (SCI). Furthermore, the expression levels and localization of p-β-catenin, p27, cyclin D1 and PCNA were correlated with PKM2 after SCI. In vitro, we also found that PKM2 co-immunoprecipitation with p-β-catenin and p27 respectively. Knockdown of PKM2 apparently decreased the level of PCNA, cyclinD1, p27 in primary astrocyte cells. Taken together, our findings indicate that nuclear translocation of PKM2 promotes astrocytes proliferation after SCI through modulating cell cycle signaling. These discoveries firstly uncovered the role of PKM2 in spinal cord injury and provided a potential therapeutic target for CNS injury and repair.     

Changes in expression of ciliary neurotrophic factor (CNTF) and CNTF-receptor α after spinal cord injury

To determine if ciliary neurotrophic factor (CNTF) is involved in the response to spinal cord injury, we studied changes in the expression of CNTF and that of its receptor, CNTF-receptor α (CNTFRα), in the rat spinal cord after a unilateral spinal cord hemisection. Using in situ hybridization, we found that CNTFRα mRNA levels in spinal cord motoneurons increased dramatically by 1 day after hemisecting the spinal cord at T2. This increase in expression was present only in motoneurons caudal, but not rostral, to the lesion. In addition, we detected increased levels of CNTF mRNA in the spinal cord white matter, also by 1 day following injury. Unlike CNTFRα, however, the increase in CNTF mRNA was evident both rostral and caudal to the lesion. Levels of both CNTF and CNTFRα mRNA declined between 1 and 5 days, and by 10 days they were not significantly different from normal animals. These findings suggest that CNTF may play a local role in the response to spinal cord injury. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 251–261, 1997.     

Lubiprostone activates CFTR, but not ClC-2, via the prostaglandin receptor (EP4)

The goal of this study was to determine the mechanism of lubiprostone activation of epithelial chloride transport. Lubiprostone is a bicyclic fatty acid approved for the treatment of constipation [1]. There is uncertainty, however, as to how lubiprostone increases epithelial chloride transport. Direct stimulation of ClC-2 and CFTR chloride channels as well as stimulation of these channels via the EP₄ receptor has been described [2], [3], [4] and [5]. To better define this mechanism, two-electrode voltage clamp was used to assay Xenopus oocytes expressing ClC-2, with or without co-expression of the EP₄ receptor or β adrenergic receptor (βAR), for changes in conductance elicited by lubiprostone. Oocytes co-expressing CFTR and either βAR or the EP₄ receptor were also studied. In oocytes co-expressing ClC-2 and βAR conductance was stimulated by hyperpolarization and acidic pH (pH = 6), but there was no response to the β adrenergic agonist, isoproterenol. Oocytes expressing ClC-2 only or co-expressing ClC-2 and EP₄ did not respond to the presence of 0.1, 1, or 10 μM lubiprostone in the superperfusate. Oocytes co-expressing CFTR and βAR did not respond to hyperpolarization, acidic pH, or 1 μM lubiprostone. However, conductance was elevated by isoproterenol and inhibited by CFTR_inh172. Co-expression of CFTR and EP₄ resulted in lubiprostone-stimulated conductance, which was also sensitive to CFTR_inh172. The EC₅₀ for lubiprostone mediated CFTR activation was ∼10 nM. These results demonstrate no direct action of lubiprostone on either ClC-2 or CFTR channels expressed in oocytes. However, the results confirm that CFTR can be activated by lubiprostone via the EP₄ receptor in oocytes.     

HMGB1 Protein Does Not Mediate the Inflammatory Response in Spontaneous Spinal Cord Regeneration: A HINT FOR CNS REGENERATION*

Uncontrolled, excessive inflammation contributes to the secondary tissue damage of traumatic spinal cord, and HMGB1 is highlighted for initiation of a vicious self-propagating inflammatory circle by release from necrotic cells or immune cells. Several regenerative-competent vertebrates have evolved to circumvent the second damages during the spontaneous spinal cord regeneration with an unknown HMGB1 regulatory mechanism. By genomic surveys, we have revealed that two paralogs of HMGB1 are broadly retained from fish in the phylogeny. However, their spatial-temporal expression and effects, as shown in lowest amniote gecko, were tightly controlled in order that limited inflammation was produced in spontaneous regeneration. Two paralogs from gecko HMGB1 (gHMGB1) yielded distinct injury and infectious responses, with gHMGB1b significantly up-regulated in the injured cord. The intracellular gHMGB1b induced less release of inflammatory cytokines than gHMGB1a in macrophages, and the effects could be shifted by exchanging one amino acid in the inflammatory domain. Both intracellular proteins were able to mediate neuronal programmed apoptosis, which has been indicated to produce negligible inflammatory responses. In vivo studies demonstrated that the extracellular proteins could not trigger a cascade of the inflammatory cytokines in the injured spinal cord. Signal transduction analysis found that gHMGB1 proteins could not bind with cell surface receptors TLR2 and TLR4 to activate inflammatory signaling pathway. However, they were able to interact with the receptor for advanced glycation end products to potentiate oligodendrocyte migration by activation of both NFκB and Rac1/Cdc42 signaling. Our results reveal that HMGB1 does not mediate the inflammatory response in spontaneous spinal cord regeneration, but it promotes CNS regeneration.     

Interactions between Intracellular Domains as Key Determinants of the Quaternary Structure and Function of Receptor Heteromers

G protein-coupled receptor (GPCR) heteromers are macromolecular complexes with unique functional properties different from those of its individual protomers. Little is known about what determines the quaternary structure of GPCR heteromers resulting in their unique functional properties. In this study, using resonance energy transfer techniques in experiments with mutated receptors, we provide for the first time clear evidence for a key role of intracellular domains in the determination of the quaternary structure of GPCR heteromers between adenosine A_2A, cannabinoid CB₁, and dopamine D₂ receptors. In these interactions, arginine-rich epitopes form salt bridges with phosphorylated serine or threonine residues from CK1/2 consensus sites. Each receptor (A_2A, CB₁, and D₂) was found to include two evolutionarily conserved intracellular domains to establish selective electrostatic interactions with intracellular domains of the other two receptors, indicating that these particular electrostatic interactions constitute a general mechanism for receptor heteromerization. Mutation experiments indicated that the interactions of the intracellular domains of the CB₁ receptor with A_2A and D₂ receptors are fundamental for the correct formation of the quaternary structure needed for the function (MAPK signaling) of the A_2A-CB₁-D₂ receptor heteromers. Analysis of MAPK signaling in striatal slices of CB₁ receptor KO mice and wild-type littermates supported the existence of A₁-CB₁-D₂ receptor heteromer in the brain. These findings allowed us to propose the first molecular model of the quaternary structure of a receptor heteromultimer.