mGluR5 from Primary Sensory Neurons Promotes Opioid-Induced Hyperalgesia and Tolerance by Interacting with and Potentiating Synaptic NMDA Receptors.

Aberrant activation of presynaptic NMDARs in the spinal dorsal horn is integral to opioid-induced hyperalgesia and analgesic tolerance. However, the signaling mechanisms responsible for opioid-induced NMDAR hyperactivity remain poorly identified. Here, we show that repeated treatment with morphine or fentanyl reduced monomeric mGluR5 protein levels in the dorsal root ganglion (DRG) but increased levels of mGluR5 monomers and homodimers in the spinal cord in mice and rats of both sexes. Coimmunoprecipitation analysis revealed that monomeric and dimeric mGluR5 in the spinal cord, but not monomeric mGluR5 in the DRG, directly interacted with GluN1. By contrast, mGluR5 did not interact with µ-opioid receptors in the DRG or spinal cord. Repeated morphine treatment markedly increased the mGluR5-GluN1 interaction and protein levels of mGluR5 and GluN1 in spinal synaptosomes. The mGluR5 antagonist MPEP reversed morphine treatment-augmented mGluR5-GluN1 interactions, GluN1 synaptic expression, and dorsal root-evoked monosynaptic EPSCs of dorsal horn neurons. Furthermore, CRISPR-Cas9-induced conditional mGluR5 knockdown in DRG neurons normalized mGluR5 levels in spinal synaptosomes and NMDAR-mediated EPSCs of dorsal horn neurons increased by morphine treatment. Correspondingly, intrathecal injection of MPEP or conditional mGluR5 knockdown in DRG neurons not only potentiated the acute analgesic effect of morphine but also attenuated morphine treatment-induced hyperalgesia and tolerance. Together, our findings suggest that opioid treatment promotes mGluR5 trafficking from primary sensory neurons to the spinal dorsal horn. Through dimerization and direct interaction with NMDARs, presynaptic mGluR5 potentiates and/or stabilizes NMDAR synaptic expression and activity at primary afferent central terminals, thereby maintaining opioid-induced hyperalgesia and tolerance.SIGNIFICANCE STATEMENT Opioids are essential analgesics for managing severe pain caused by cancer, surgery, and tissue injury. However, these drugs paradoxically induce pain hypersensitivity and tolerance, which can cause rapid dose escalation and even overdose mortality. This study demonstrates, for the first time, that opioids promote trafficking of mGluR5, a G protein-coupled glutamate receptor, from peripheral sensory neurons to the spinal cord; there, mGluR5 proteins dimerize and physically interact with NMDARs to augment their synaptic expression and activity. Through dynamic interactions, the two distinct glutamate receptors mutually amplify and sustain nociceptive input from peripheral sensory neurons to the spinal cord. Thus, inhibiting mGluR5 activity or disrupting mGluR5-NMDAR interactions could reduce opioid-induced hyperalgesia and tolerance and potentiate opioid analgesic efficacy.

Surgical smoke: A hidden killer in the operating room.

Surgical smoke is a byproduct of aerosols containing several components produced by energy equipment. The characteristics of surgical smoke components produced by different types of tissues or using different kinds of energy devices vary. For example, the average diameter of smoke particles produced by electrocautery is smaller, and the possibility of viable cells and pathogens in surgical smoke produced by an ultrasonic knife is higher. According to the characteristics of its composition, surgical smoke may be an important risk factor affecting the health and safety of operating room staff and patients. The use of surgical masks, suction devices and portable smoke evacuation systems can reduce this risk to some extent. However, most operating room staff members do not implement corresponding measures to protect themselves. In this paper, the characteristics of surgical smoke and the research progress in protective measures are briefly reviewed.

HDAC2 in Primary Sensory Neurons Constitutively Restrains Chronic Pain by Repressing α2δ-1 Expression and Associated NMDA Receptor Activity.

α2δ-1 (encoded by the Cacna2d1 gene) is a newly discovered NMDA receptor-interacting protein and is the therapeutic target of gabapentinoids (e.g., gabapentin and pregabalin) frequently used for treating patients with neuropathic pain. Nerve injury causes sustained α2δ-1 upregulation in the dorsal root ganglion (DRG), which promotes NMDA receptor synaptic trafficking and activation in the spinal dorsal horn, a hallmark of chronic neuropathic pain. However, little is known about how nerve injury initiates and maintains the high expression level of α2δ-1 to sustain chronic pain. Here, we show that nerve injury caused histone hyperacetylation and diminished enrichment of histone deacetylase-2 (HDAC2), but not HDAC3, at the Cacna2d1 promoter in the DRG. Strikingly, Hdac2 knockdown or conditional knockout in DRG neurons in male and female mice consistently induced long-lasting mechanical pain hypersensitivity, which was readily reversed by blocking NMDA receptors, inhibiting α2δ-1 with gabapentin or disrupting the α2δ-1-NMDA receptor interaction at the spinal cord level. Hdac2 deletion in DRG neurons increased histone acetylation levels at the Cacna2d1 promoter, upregulated α2δ-1 in the DRG, and potentiated α2δ-1-dependent NMDA receptor activity at primary afferent central terminals in the spinal dorsal horn. Correspondingly, Hdac2 knockdown-induced pain hypersensitivity was blunted in Cacna2d1 knockout mice. Thus, our findings reveal that HDAC2 functions as a pivotal transcriptional repressor of neuropathic pain via constitutively suppressing α2δ-1 expression and ensuing presynaptic NMDA receptor activity in the spinal cord. HDAC2 enrichment levels at the Cacna2d1 promoter in DRG neurons constitute a unique epigenetic mechanism that governs acute-to-chronic pain transition.SIGNIFICANCE STATEMENT Excess α2δ-1 proteins produced after nerve injury directly interact with glutamate NMDA receptors to potentiate synaptic NMDA receptor activity in the spinal cord, a prominent mechanism of nerve pain. Because α2δ-1 upregulation after nerve injury is long lasting, gabapentinoids relieve pain symptoms only temporarily. Our study demonstrates for the first time the unexpected role of intrinsic HDAC2 activity at the α2δ-1 gene promoter in limiting α2δ-1 gene transcription, NMDA receptor-dependent synaptic plasticity, and chronic pain development after nerve injury. These findings challenge the prevailing view about the role of general HDAC activity in promoting chronic pain. Restoring the repressive HDAC2 function and/or reducing histone acetylation at the α2δ-1 gene promoter in primary sensory neurons could lead to long-lasting relief of nerve pain.

Protein Kinase C-Mediated Phosphorylation and α2δ-1 Interdependently Regulate NMDA Receptor Trafficking and Activity.

N-methyl-d-aspartate receptors (NMDARs) are important for synaptic plasticity associated with many physiological functions and neurologic disorders. Protein kinase C (PKC) activation increases the phosphorylation and activity of NMDARs, and α2δ-1 is a critical NMDAR-interacting protein and controls synaptic trafficking of NMDARs. In this study, we determined the relative roles of PKC and α2δ-1 in the control of NMDAR activity. We found that α2δ-1 coexpression significantly increased NMDAR activity in HEK293 cells transfected with GluN1/GluN2A or GluN1/GluN2B. PKC activation with phorbol 12-myristate 13-acetate (PMA) increased receptor activity only in cells coexpressing GluN1/GluN2A and α2δ-1. Remarkably, PKC inhibition with GÓ§6983 abolished α2δ-1-coexpression-induced potentiation of NMDAR activity in cells transfected with GluN1/GluN2A or GluN1/GluN2B. Treatment with PMA increased the α2δ-1-GluN1 interaction and promoted α2δ-1 and GluN1 cell surface trafficking. PMA also significantly increased NMDAR activity of spinal dorsal horn neurons and the amount of α2δ-1-bound GluN1 protein complexes in spinal cord synaptosomes in wild-type mice, but not in α2δ-1 knockout mice. Furthermore, inhibiting α2δ-1 with pregabalin or disrupting the α2δ-1-NMDAR interaction with the α2δ-1 C-terminus peptide abolished the potentiating effect of PMA on NMDAR activity. Additionally, using quantitative phosphoproteomics and mutagenesis analyses, we identified S929 on GluN2A and S1413 (S1415 in humans) on GluN2B as the phosphorylation sites responsible for NMDAR potentiation by PKC and α2δ-1. Together, our findings demonstrate the interdependence of α2δ-1 and PKC phosphorylation in regulating NMDAR trafficking and activity. The phosphorylation-dependent, dynamic α2δ-1-NMDAR interaction constitutes an important molecular mechanism of synaptic plasticity.SIGNIFICANCE STATEMENT A major challenge in studies of protein phosphorylation is to define the functional significance of each phosphorylation event and determine how various signaling pathways are coordinated in response to neuronal activity to shape synaptic plasticity. PKC phosphorylates transporters, ion channels, and G-protein-coupled receptors in signal transduction. In this study, we showed that α2δ-1 is indispensable for PKC-activation-induced surface and synaptic trafficking of NMDARs, whereas the α2δ-1-NMDAR interaction is controlled by PKC-induced phosphorylation. Our findings reveal that α2δ-1 mainly functions as a phospho-binding protein in the control of NMDAR trafficking and activity. This information provides new mechanistic insight into the reciprocal roles of PKC-mediated phosphorylation and α2δ-1 in regulating NMDARs and in the therapeutic actions of gabapentinoids.

α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly.

Many neurological disorders show an increased prevalence of GluA2-lacking, Ca2+-permeable AMPA receptors (CP-AMPARs), which dramatically alters synaptic function. However, the molecular mechanism underlying this distinct synaptic plasticity remains enigmatic. Here, we show that nerve injury potentiates postsynaptic, but not presynaptic, CP-AMPARs in the spinal dorsal horn via α2δ-1. Overexpressing α2δ-1, previously regarded as a Ca2+ channel subunit, augments CP-AMPAR levels at the cell surface and synapse. Mechanistically, α2δ-1 physically interacts with both GluA1 and GluA2 via its C terminus, inhibits the GluA1/GluA2 heteromeric assembly, and increases GluA2 retention in the endoplasmic reticulum. Consequently, α2δ-1 diminishes the availability and synaptic expression of GluA1/GluA2 heterotetramers in the spinal cord in neuropathic pain. Inhibiting α2δ-1 with gabapentin or disrupting the α2δ-1-AMPAR complex fully restores the intracellular assembly and synaptic dominance of heteromeric GluA1/GluA2 receptors. Thus, α2δ-1 is a pivotal AMPAR-interacting protein that controls the subunit composition and Ca2+ permeability of postsynaptic AMPARs.

Deficient LRRC8A-dependent volume-regulated anion channel activity is associated with male infertility in mice.

Ion channel-controlled cell volume regulation is of fundamental significance to the physiological function of sperm. In addition to volume regulation, LRRC8A-dependent volume-regulated anion channel (VRAC) activity is involved in cell cycle progression, insulin signaling, and cisplatin resistance. Nevertheless, the contribution of LRRC8A and its dependent VRAC activity in the germ cell lineage remain unknown. By utilizing a spontaneous Lrrc8a mouse mutation (c.1325delTG, p.F443*) and genetically engineered mouse models, we demonstrate that LRRC8A-dependent VRAC activity is essential for male germ cell development and fertility. Lrrc8a-null male germ cells undergo progressive degeneration independent of the apoptotic pathway during postnatal testicular development. Lrrc8a-deficient mouse sperm exhibit multiple morphological abnormalities of the flagella (MMAF), a feature commonly observed in the sperm of infertile human patients. Importantly, we identified a human patient with a rare LRRC8A hypomorphic mutation (c.1634G>A, p.Arg545His) possibly linked to Sertoli cell-only syndrome (SCOS), a male sterility disorder characterized by the loss of germ cells. Thus, LRRC8A is a critical factor required for germ cell development and volume regulation in the mouse, and it might serve as a novel diagnostic and therapeutic target for SCOS patients.

The α2δ-1-NMDA Receptor Complex Is Critically Involved in Neuropathic Pain Development and Gabapentin Therapeutic Actions.

α2δ-1, commonly known as a voltage-activated Ca2+ channel subunit, is a binding site of gabapentinoids used to treat neuropathic pain and epilepsy. However, it is unclear how α2δ-1 contributes to neuropathic pain and gabapentinoid actions. Here, we show that Cacna2d1 overexpression potentiates presynaptic and postsynaptic NMDAR activity of spinal dorsal horn neurons to cause pain hypersensitivity. Conversely, Cacna2d1 knockdown or ablation normalizes synaptic NMDAR activity increased by nerve injury. α2δ-1 forms a heteromeric complex with NMDARs in rodent and human spinal cords. The α2δ-1-NMDAR interaction predominantly occurs through the C terminus of α2δ-1 and promotes surface trafficking and synaptic targeting of NMDARs. Gabapentin or an α2δ-1 C terminus-interfering peptide normalizes NMDAR synaptic targeting and activity increased by nerve injury. Thus, α2δ-1 is an NMDAR-interacting protein that increases NMDAR synaptic delivery in neuropathic pain. Gabapentinoids reduce neuropathic pain by inhibiting forward trafficking of α2δ-1-NMDAR complexes.

Molecular Basis of Regulating High Voltage-Activated Calcium Channels by S-Nitrosylation.

Nitric oxide (NO) is involved in a variety of physiological processes, such as vasoregulation and neurotransmission, and has a complex role in the regulation of pain transduction and synaptic transmission. We have shown previously that NO inhibits high voltage-activated Ca(2+) channels in primary sensory neurons and excitatory synaptic transmission in the spinal dorsal horn. However, the molecular mechanism involved in this inhibitory action remains unclear. In this study, we investigated the role of S-nitrosylation in the NO regulation of high voltage-activated Ca(2+) channels. The NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) rapidly reduced N-type currents when Cav2.2 was coexpressed with the Cavß1 or Cavß3 subunits in HEK293 cells. In contrast, SNAP only slightly inhibited P/Q-type and L-type currents reconstituted with various Cavß subunits. SNAP caused a depolarizing shift in voltage-dependent N-type channel activation, but it had no effect on Cav2.2 protein levels on the membrane surface. The inhibitory effect of SNAP on N-type currents was blocked by the sulfhydryl-specific modifying reagent methanethiosulfonate ethylammonium. Furthermore, the consensus motifs of S-nitrosylation were much more abundant in Cav2.2 than in Cav1.2 and Cav2.1. Site-directed mutagenesis studies showed that Cys-805, Cys-930, and Cys-1045 in the II-III intracellular loop, Cys-1835 and Cys-2145 in the C terminus of Cav2.2, and Cys-346 in the Cavß3 subunit were nitrosylation sites mediating NO sensitivity of N-type channels. Our findings demonstrate that the consensus motifs of S-nitrosylation in cytoplasmically accessible sites are critically involved in post-translational regulation of N-type Ca(2+) channels by NO. S-Nitrosylation mediates the feedback regulation of N-type channels by NO.

Stromal interaction molecule 1 (STIM1) and Orai1 mediate histamine-evoked calcium entry and nuclear factor of activated T-cells (NFAT) signaling in human umbilical vein endothelial cells.

Histamine is an important immunomodulator involved in allergic reactions and inflammatory responses. In endothelial cells, histamine induces Ca(2+) mobilization by releasing Ca(2+) from the endoplasmic reticulum and eliciting Ca(2+) entry across the plasma membrane. Herein, we show that histamine-evoked Ca(2+) entry in human umbilical vein endothelial cells (HUVECs) is sensitive to blockers of Ca(2+) release-activated Ca(2+) (CRAC) channels. RNA interference against STIM1 or Orai1, the activating subunit and the pore-forming subunit of CRAC channels, respectively, abolishes this histamine-evoked Ca(2+) entry. Furthermore, overexpression of dominant-negative CRAC channel subunits inhibits while co-expression of both STIM1 and Orai1 enhances histamine-induced Ca(2+) influx. Interestingly, gene silencing of STIM1 or Orai1 also interrupts the activation of calcineurin/nuclear factor of activated T-cells (NFAT) pathway and the production of interleukin 8 triggered by histamine in HUVECs. Collectively, these results suggest a central role of STIM1 and Orai1 in mediating Ca(2+) mobilization linked to inflammatory signaling of endothelial cells upon histamine stimulation.

GDF15 regulates Kv2.1-mediated outward K+ current through the Akt/mTOR signalling pathway in rat cerebellar granule cells.

GDF15 (growth/differentiation factor 15), a novel member of the TGFß (transforming growth factor ß) superfamily, plays critical roles in the central and peripheral nervous systems, but the signal transduction pathways and receptor subtypes involved are not well understood. In the present paper, we report that GDF15 specifically increases the IK (delayed-rectifier outward K+ current) in rat CGNs (cerebellar granule neurons) in time- and concentration-dependent manners. The GDF15-induced amplification of the IK is mediated by the increased expression and reduced lysosome-dependent degradation of the Kv2.1 protein, the main α-subunit of the IK channel. Exposure of CGNs to GDF15 markedly induced the phosphorylation of ERK (extracellular-signal-regulated kinase), Akt and mTOR (mammalian target of rapamycin), but the GDF15-induced IK densities and increased expression of Kv2.1 were attenuated only by Akt and mTOR, and not ERK, inhibitors. Pharmacological inhibition of the Src-mediated phosphorylation of TGFßR2 (TGFß receptor 2), not TGFßR1, abrogated the effect of GDF15 on IK amplification and Kv2.1 induction. Immunoprecipitation assays showed that GDF15 increased the tyrosine phosphorylation of TGFßRII in the CGN lysate. The results of the present study reveal a novel regulation of Kv2.1 by GDF15 mediated through the TGFßRII-activated Akt/mTOR pathway, which is a previously uncharacterized Smad-independent mechanism of GDF15 signalling.

Differential roles of the C and N termini of Orai1 protein in interacting with stromal interaction molecule 1 (STIM1) for Ca2+ release-activated Ca2+ (CRAC) channel activation.

The entry of extracellular Ca(2+), which is mediated by Ca(2+) release-activated Ca(2+) (CRAC) channels, is essential for T cell activation and the normal functioning of other immune cells. Although the molecular components of CRAC channels, the Orai1 pore-forming subunit and the STIM1-activating subunit have been recently identified, the gating mechanism by which Orai1 channels conduct Ca(2+) entry upon Orai1-STIM1 interaction following Ca(2+) store release remains elusive. Herein, we show that C-terminal truncations or point mutations prevented Orai1 from binding to STIM1 and subsequent channel opening. In contrast, an Orai1 mutant with an N-terminal truncation interacted with but failed to be activated by STIM1. Moreover, Orai1 channels with C-terminal disruption, but not N-terminal truncation, could be gated by fused functional domains of STIM1. Interestingly, the channel activities of Orai1 mutants carrying either an N-terminal or a C-terminal truncation were restored by a methionine mutation at the putative gating hinge, the conserved Gly-98 site in the first transmembrane segment (TM1) of Orai1. Collectively, these results support a stepwise gating mechanism of STIM1-operated Orai1 channels; the initial binding between STIM1 and the C terminus of Orai1 docks STIM1 onto the N terminus of Orai1 to initiate conformational changes of the pore-lining TM1 helix of Orai1, leading to the opening of the channel.

STIM1 restores coronary endothelial function in type 1 diabetic mice.

RATIONALE: The endoplasmic reticulum (ER) is a major intracellular Ca(2+) store in endothelial cells (ECs). The Ca(2+) concentration in the ER greatly contributes to the generation of Ca(2+) signals that regulate endothelial functions. Many proteins, including stromal interaction molecule 1/2 (STIM1/2), Orai1/2/3, and sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 3 (SERCA3), are involved in the ER Ca(2+) refilling after store depletion in ECs. OBJECTIVE: This study is designed to examine the role of Ca(2+) in the ER in coronary endothelial dysfunction in diabetes. METHODS AND RESULTS: Mouse coronary ECs (MCECs) isolated from diabetic mice exhibited (1) a significant decrease in the Ca(2+) mobilization from the ER when the cells were treated by SERCA inhibitor, and (2) significant downregulation of STIM1 and SERCA3 protein expression in comparison to the controls. Overexpression of STIM1 restored (1) the increase in cytosolic Ca(2+) concentration due to Ca(2+) leak from the ER in diabetic MCECs, (2) the Ca(2+) concentration in the ER, and (3) endothelium-dependent relaxation that was attenuated in diabetic coronary arteries. CONCLUSIONS: Impaired ER Ca(2+) refilling in diabetic MCECs, due to the decrease in STIM1 protein expression, attenuates endothelium-dependent relaxation in diabetic coronary arteries, while STIM1 overexpression has a beneficial and therapeutic effect on coronary endothelial dysfunction in diabetes.

Cholesterol enhances neuron susceptibility to apoptotic stimuli via cAMP/PKA/CREB-dependent up-regulation of Kv2.1.

Cholesterol is a major component of membrane lipid rafts. It is more abundant in the brain than in other tissues and plays a critical role in maintaining brain function. We report here that a significant enhancement in apoptosis in rat cerebellar granule neurons (CGNs) was observed upon incubation with 5mM K(+) /serum free (LK-S) medium. Cholesterol enrichment further potentiated CGN apoptosis incubated under LK-S medium. On the contrary, cholesterol depletion using methyl-beta-cyclodextrin protected the CGNs from apoptosis induced by LK-S treatment. Cholesterol enrichment, however, did not induce apoptosis in CGNs that have been incubated with 25mM K(+) /serum medium. Mechanistically, increased I(K) currents and DNA fragmentation were found in CGNs incubated in LK-S, which was further potentiated in the presence of cholesterol. Cholesterol-treated CGNs also exhibited increased cAMP levels and up-regulation of Kv2.1 expression. Increased levels of activated form of PKA and phospho-CREB further supported activation of the cAMP/PKA pathway upon treatment of CGNs with cholesterol-containing LK-S medium. Conversely, inhibition of PKA or small G protein Gs abolished the increase in I(K) current and the potentiation of Kv2.1 expression, leading to reduced susceptibility of CGNs to LK-S and cholesterol-induced apoptosis. Our results demonstrate that the elevation of membrane cholesterol enhances CGN susceptibility to apoptotic stimuli via cAMP/PKA/CREB-dependent up-regulation of Kv2.1. Our data provide new evidence for the role of cholesterol in eliciting neuronal cell death.

TGF-ß1 enhances Kv2.1 potassium channel protein expression and promotes maturation of cerebellar granule neurons.

Members of the transforming growth factor-ß (TGF-ß) family of cytokines are involved in diverse physiological processes. Although TGF-ß is known to play multiple roles in the mammalian central nervous system (CNS), its role in neuronal development has not been explored. We have studied the effects of TGF-ß1 on the electrophysiological properties and maturation of rat primary cerebellar granule neurons (CGNs). We report that incubation with TGF-ß1 increased delayed rectifier potassium current (I(K) ) amplitudes in a dose- and time-dependent manner, but did not affect the kinetic properties of the channel. Exposure to TGF-ß1 (20 ng/ml) for 36 h led to a 37.2% increase in I(K) amplitudes. There was no significant change in mRNA levels for the key Kv2.1 channel protein, but translation blockade abolished the increase in protein levels and channel activity, arguing that TGF-ß1 increases I(K) amplitudes by upregulating translation of the Kv2.1 channel protein. Although TGF-ß1 treatment did not affect the activity of protein kinase A (PKA), and constitutive activation of PKA with forskolin failed to increase I(K) amplitudes, inhibition of PKA prevented channel upregulation, demonstrating that basal PKA activity is required for TGF-ß1 stimulation of I(K) channel activity. TGF-ß1 also promoted the expression of the γ-aminobutyric acid (GABA(A) ) receptor α6 subunit, a marker of mature CGNs, and calcium influx during depolarizing stimuli was reduced by TGF-ß1. The effects of TGF-ß1 were only observed during a narrow developmental time-window, and were lost as CGNs matured. These findings suggest that TGF-ß1 upregulates K(+) channel expression and I(K) currents and thereby promotes CGN maturation.

Amoxapine inhibits delayed outward rectifier K(+) currents in cerebellar granule cells via dopamine receptor and protein kinase A activation.

BACKGROUND: Although tricyclic antidepressants amoxapine is proposed to target 5-HT and D2 receptors, very few studies have addressed the effect of amoxapine on molecular and cellular mechanisms via receptor pathways. In this study, we test the effect of amoxapine on rat cerebellar granule neurons (CGNs) to address this possibility. METHODS: CGNs cell culture, whole-cell current recording using a patch-clamp technique, western blot and non-radioactive detection analysis of phosphorylated protein kinase A (PKA) were used. RESULTS: Amoxapine inhibits delayed rectifier potassium (I(K)) current in a dose-dependent manner and modulates inactivation properties in CGNs. Those effects were not eliminated by preincubation with 5-HT or 5-HT receptor antagonists, but abolished by dopamine and D1/D5 receptor antagonists. Application of GTPγ-S and inhibitor of the Gs signalling cascade abolished the amoxapine-induced effect on I(K). The application of forskolin or dibutyryl-cAMP mimicked the inhibitory effect of amoxapine on I(K). Western blotting for phosphorylated PKA revealed that amoxapine significantly increased the intracellular levels of phosphorylated PKA, a marker of PKA activation. CONCLUSION: Amoxapine inhibits I(K) currents in rat CGNs via cAMP/PKA-dependent pathways, as in mouse cortical neurons we reported earlier, but that involves D1-like receptors instead of 5-HT receptors.

Arachidonic acid modulates Na+ currents by non-metabolic and metabolic pathways in rat cerebellar granule cells.

AA (arachidonic acid), which possesses both neurotoxic and neurotrophic activities, has been implicated as a messenger in both physiological and pathophysiological processes. In the present study, we investigated the effects of both extracellular and intracellular application of AA on the activity of Na(V) (voltage-gated Na(+) channels) in rat cerebellar GCs (granule cells). The extracellular application of AA inhibited the resultant I(Na) (Na(V) current), wherein the current-voltage curve shifted to a negative voltage direction. Because this effect could be reproduced by treating the GCs with ETYA (eicosa-5,8,11,14-tetraynoic acid) or a membrane-impermeable analogue of AA, AA-CoA (arachidonoyl coenzyme A), we inferred that AA itself exerted the observed modulatory effects on I(Na). In contrast, intracellular AA significantly augmented the elicited I(Na) peak when the same protocol that was used for extracellular AA was followed. The observed I(Na) increase that was induced by intracellular AA was mimicked by the AA cyclo-oxygenase metabolite PGE(2) (prostaglandin E(2)), but not by ETYA. Furthermore, cyclo-oxygenase inhibitors decreased I(Na) and quenched AA-induced channel activation, indicating that the effect of intracellular AA on Na(V) was possibly mediated through AA metabolites. In addition, the PGE2-induced activation of I(Na) was mimicked by cAMP and quenched by a PKA (protein kinase A) inhibitor, a G(s) inhibitor and EP (E-series of prostaglandin) receptor antagonists. The results of the present study suggest that extracellular AA modulates Na(V) channel activity in rat cerebellar GCs without metabolic conversion, whereas intracellular AA augments the I(Na) by PGE(2)-mediated activation of cAMP/PKA pathways. These observations may explain the dual character of AA in neuronal pathogenesis.

Kv 1.1 is associated with neuronal apoptosis and modulated by protein kinase C in the rat cerebellar granule cell.

Previously, we reported that apoptosis of cerebellar granular neurons induced by low-K+ and serum-free (LK-S) was associated with an increase in the A-type K+ channel current (I(A)), and an elevated expression of main alpha-subunit of the I(A) channel, which is known as Kv4.2 and Kv4.3. Here, we show, as assessed by quantitative RT-PCR and whole-cell recording, that besides Kv4.2 and Kv4.3, Kv1.1 is very important for I(A) channel. The expression of Kv1.1 was elevated in the apoptotic neurons, while silencing Kv1.1 expression by siRNA reduced the I(A) amplitude of the apoptotic neuron, and increased neuron viability. Inhibiting Kv1.1 current by dendrotoxin-K evoked a similar effect of reduction of I(A) amplitude and protection of neurons. Applying a protein kinase C (PKC) activator, phorbol ester acetate A (PMA) mimicked the LK-S-induced neuronal apoptotic effect, enhanced the I(A) amplitude and reduced the granule cell viability. The PKC inhibitor, bisindolylmaleimide I and Gö6976 protected the cell against apoptosis induced by LK-S. After silencing the Kv1.1 gene, the effect of PMA on the residual K+ current was reduced significantly. Quantitative RT-PCR and Western immunoblot techniques revealed that LK-S treatment and PMA increased the level of the expression of Kv1.1, in contrast, bisindolylmaleimide I inhibited Kv1.1 expression. In addition, the activation of the PKC isoform was identified in apoptotic neurons. We thus conclude that in the rat cerebellar granule cell, the I(A) channel associated with apoptotic neurons is encoded mainly by the Kv1.1 gene, and that the PKC pathway promotes neuronal apoptosis by a brief modulation of the I(A) amplitude and a permanent increase in the levels of expression of the Kv1.1 alpha-subunit.