Cimifugin Relieves Histamine-Independent Itch in Atopic Dermatitis via Targeting the CQ Receptor MrgprA3.

Background: We previously found that cimifugin has a potent antiallergic inflammatory effect in atopic dermatitis (AD). However, whether cimifugin has an antipruritic effect in AD was unknown. Methods: Mouse scratching behavior tests were performed to verify the proposed antipruritic effect of cimifugin on DNFB- or FITC-mediated AD. Chloroquine (CQ)- and compound 48/80-evoked acute itch models were employed to clarify the effect of cimifugin on histamine-dependent or -independent itch. Intracellular calcium changes were assessed in a primary culture of mouse dorsal root ganglia (DRG) in response to pruritogen exposure with or without cimifugin treatment, including CQ, histamine, allyl-isothiocyanate (AITC), and capsaicin. Molecular docking and microscale thermophoresis (MST) assays were performed to predict and verify the binding ability and modes between cimifugin and the CQ receptor MrgprA3, respectively. Results: We found that cimifugin attenuates itch behaviors effectively in FITC-induced AD. Notably, cimifugin significantly alleviated acute itching behaviors induced by CQ but not compound 48/80 in vivo. Moreover, cimifugin remarkably inhibited CQ-evoked calcium influx in DRG cells but had no obvious effect on histamine-induced calcium influx. Nevertheless, cimifugin did not interfere with either AITC-stimulated TRPA1 activation- or capsaicin-stimulated TRPV1 activation-mediated calcium influx in DRG cells. Molecular docking predicted that CQ and cimifugin might share similar binding abilities and binding modes with MrgprA3. MST assay confirmed cimifugin directly targeting MrgprA3. Conclusion: The present study demonstrates that cimifugin has a potent antipruritic effect in AD with a histamine-independent mechanism via targeting the CQ receptor MrgprA3. Thus, cimifugin is a promising candidate antipruritic agent for AD.

Oligodendrocyte precursor cells stop sensory axons regenerating into the spinal cord.

Primary somatosensory axons stop regenerating as they re-enter the spinal cord, resulting in incurable sensory loss. What arrests them has remained unclear. We previously showed that axons stop by forming synaptic contacts with unknown non-neuronal cells. Here, we identified these cells in adult mice as oligodendrocyte precursor cells (OPCs). We also found that only a few axons stop regenerating by forming dystrophic endings, exclusively at the CNS:peripheral nervous system (PNS) borderline where OPCs are absent. Most axons stop in contact with a dense network of OPC processes. Live imaging, immuno-electron microscopy (immuno-EM), and OPC-dorsal root ganglia (DRG) co-culture additionally suggest that axons are rapidly immobilized by forming synapses with OPCs. Genetic OPC ablation enables many axons to continue regenerating deep into the spinal cord. We propose that sensory axons stop regenerating by encountering OPCs that induce presynaptic differentiation. Our findings identify OPCs as a major regenerative barrier that prevents intraspinal restoration of sensory circuits following spinal root injury.

Toll-like receptor 4 activation enhances Orai1-mediated calcium signal promoting cytokine production in spinal astrocytes.

Toll-like receptor 4 (TLR4) has been implicated in pathological conditions including chronic pain. Activation of astrocytic TLRs leads to the synthesis of pro-inflammatory cytokines like interleukin 6 (IL-6) and tumor necrosis factor-ɑ (TNF-α), which can cause pathological inflammation and tissue damage in the central nervous system. However, the mechanisms of TLR4-mediated cytokine releases from astrocytes are incomplete understood. Our previous study has shown that Orai1, a key component of calcium release activated calcium channels (CRACs), mediates Ca2+ entry in astrocytes. How Orai1 contributes to TLR4 signaling remains unclear. Here we show that Orai1 deficiency drastically attenuated lipopolysaccharides (LPS)-induced TNF-α and IL-6 production in astrocytes. Acute LPS treatment did not induce Ca2+ response and had no effect on thapsigargin (Ca2+-ATPase inhibitor)-induced store-dependent Ca2+ entry. Inhibition or knockdown of Orai1 showed no reduction in LPS-induced p-ERK1/2, p-c-Jun N-terminal kinase, or p-p38 MAPK activation. Interestingly, Orai1 protein level was significantly increased after LPS exposure, which was blocked by inhibition of NF-κB activity. LPS significantly increased basal Ca2+ level and SOCE after exposure to astrocytes. Moreover, elevating extracellular Ca2+ concentration increased cytosolic Ca2+ level, which was almost eliminated in Orai1 KO astrocytes. Our study reports novel findings that Orai1 acts as a Ca2+ leak channel regulating the basal Ca2+ level and enhancing cytokine production in astrocytes under the inflammatory condition. These findings highlight an important role of Orai1 in astrocytic TRL4 function and may suggest that Orai1 could be a potential therapeutic target for neuroinflammatory disorders including chronic pain.

Dual loss of regulator of G protein signaling 2 and 5 exacerbates ventricular myocyte arrhythmias and disrupts the fine-tuning of Gi/o signaling.

AIMS: Cardiac contractility, essential to maintaining proper cardiac output and circulation, is regulated by G protein-coupled receptor (GPCR) signaling. Previously, the absence of regulator of G protein signaling (RGS) 2 and 5, separately, was shown to cause G protein dysregulation, contributing to modest blood pressure elevation and exaggerated cardiac hypertrophic response to pressure-overload. Whether RGS2 and 5 redundantly control G protein signaling to maintain cardiovascular homeostasis is unknown. Here we examined how the dual absence of RGS2 and 5 (Rgs2/5 dbKO) affects blood pressure and cardiac structure and function. METHODS AND RESULTS: We found that Rgs2/5 dbKO mice showed left ventricular dilatation at baseline by echocardiography. Cardiac contractile response to dobutamine stress test was sex-dependently reduced in male Rgs2/5 dbKO relative to WT mice. When subjected to surgery-induced stress, male Rgs2/5 dbKO mice had 75% mortality within 72-96 h after surgery, accompanied by elevated baseline blood pressure and decreased cardiac contractile function. At the cellular level, cardiomyocytes (CM) from Rgs2/5 dbKO mice showed augmented Ca2+ transients and increased incidence of arrhythmia without augmented contractile response to electrical field stimulation (EFS) and activation of ß-adrenergic receptors (ßAR) with isoproterenol. Dual loss of Rgs2 and 5 suppressed forskolin-induced cAMP production, which was restored by Gi/o inactivation with pertussis toxin that also reduced arrhythmogenesis during EFS or ßAR stimulation. Cardiomyocyte NCX and PMCA mRNA expression was unaffected in Rgs2/5 dbKO male mice. However, there was an exaggerated elevation of EFS-induced cytoplasmic Ca2+ in the presence of SERCA blockade with thapsigargin. CONCLUSIONS: We conclude that RGS2 and 5 promote normal ventricular rhythm by coordinating their regulatory activity towards Gi/o signaling and facilitating cardiomyocyte calcium handling.

Cytochrome P450 3A5 polymorphism affects the metabolism of sorafenib and its toxicity for hepatocellular carcinoma cells in vitro.

OBJECTIVE: Cytochrome P450 3A5 (CYP3A5) is a highly polymorphic gene and the encoded protein variants differ in catalytic activity, leading to inter-individual variation in metabolic ability. The aim of the current study was to investigate the effects of seven allelic variants on the ability of CYP3A5 to metabolize sorafenib in vitro and further explore the impacts of CYP3A5 polymorphism on the proliferation and apoptosis of hepatocellular carcinoma cell line (HepG2) induced by sorafenib. METHODS: Wild-type and variant CYP3A5 enzymes were expressed in Spodoptera frugiperda insect cells using a baculovirus dual-expression system, and protein expression was checked by western blot. The enzymes were incubated with sorafenib at 37°C for 30 min, and formation of the major metabolite sorafenib N-oxide was assayed using ultra-performance liquid chromatography and tandem mass spectrometry. Intrinsic clearance values (Vmax/Km) were calculated for each enzyme. Additionally, recombinant HepG2 cells transfecting with CYP3A5 variants were used to investigate the effects of sorafenib on the proliferation of HepG2 cells. RESULTS: Intrinsic clearance of the six variants CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 was 26.41-71.04% of the wild-type (CYP3A5*1) value. In contrast, the clearance value of the variant CYP3A5*6 was significantly higher (174.74%). Additionally, the decreased ATP levels and cell viability and the increased cell apoptosis in HepG2 cells transfected with CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 were observed, whereas, the increased ATP levels and cell viability and the reduced cell apoptosis in HepG2 cells transfected with CYP3A5*6 were also investigated when compared to CYP3A5*1. CONCLUSION: Our results suggest that CYP3A5 polymorphism influences sorafenib metabolism and pharmacotherapeutic effect in hepatic carcinomas. These data may help explain differential response to drug therapy for hepatocellular carcinoma, and they support the need for individualized treatment.

Orai1 is a crucial downstream partner of group I metabotropic glutamate receptor signaling in dorsal horn neurons.

ABSTRACT: Group I metabotropic glutamate receptors (group I mGluRs) have been implicated in several central nervous system diseases including chronic pain. It is known that activation of group I mGluRs results in the production of inositol triphosphate (IP3) and diacylglycerol that leads to activation of extracellular signal-regulated kinases (ERKs) and an increase in neuronal excitability, but how group I mGluRs mediate this process remains unclear. We previously reported that Orai1 is responsible for store-operated calcium entry and plays a key role in central sensitization. However, how Orai1 is activated under physiological conditions is unknown. Here, we tested the hypothesis that group I mGluRs recruit Orai1 as part of its downstream signaling pathway in dorsal horn neurons. We demonstrate that neurotransmitter glutamate induces STIM1 puncta formation, which is not mediated by N-Methyl-D-aspartate (NMDA) or α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Glutamate-induced Ca2+ entry in the presence of NMDA or AMPA receptor antagonists is eliminated in Orai1-deficient neurons. Dihydroxyphenylglycine (DHPG) (an agonist of group I mGluRs)-induced Ca2+ entry is abolished by Orai1 deficiency, but not affected by knocking down of transient receptor potential cation channel 1 (TRPC1) or TRPC3. Dihydroxyphenylglycine-induced activation of ERKs and modulation of neuronal excitability are abolished in cultured Orai1-deficient neurons. Moreover, DHPG-induced nociceptive behavior is markedly reduced in Orai1-deficient mice. Our findings reveal previously unknown functional coupling between Orai1 and group I mGluRs and shed light on the mechanism underlying group I mGluRs-mediated neuronal plasticity.

Cytochrome P450 3A5 polymorphism affects the metabolism of sorafenib and its toxicity for hepatocellular carcinoma cells in vitro.

OBJECTIVE: Cytochrome P450 3A5 (CYP3A5) is a highly polymorphic gene and the encoded protein variants differ in catalytic activity, leading to inter-individual variation in metabolic ability. The aim of the current study was to investigate the effects of seven allelic variants on the ability of CYP3A5 to metabolize sorafenib in vitro and further explore the impacts of CYP3A5 polymorphism on the proliferation and apoptosis of hepatocellular carcinoma cell line (HepG2) induced by sorafenib. METHODS: Wild-type and variant CYP3A5 enzymes were expressed in Spodoptera frugiperda insect cells using a baculovirus dual-expression system, and protein expression was checked by western blot. The enzymes were incubated with sorafenib at 37°C for 30 min, and formation of the major metabolite sorafenib N-oxide was assayed using ultra-performance liquid chromatography and tandem mass spectrometry. Intrinsic clearance values (Vmax/Km) were calculated for each enzyme. Additionally, recombinant HepG2 cells transfecting with CYP3A5 variants were used to investigate the effects of sorafenib on the proliferation of HepG2 cells. RESULTS: Intrinsic clearance of the six variants CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 was 26.41-71.04% of the wild-type (CYP3A5*1) value. In contrast, the clearance value of the variant CYP3A5*6 was significantly higher (174.74%). Additionally, the decreased ATP levels and cell viability and the increased cell apoptosis in HepG2 cells transfected with CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 were observed, whereas, the increased ATP levels and cell viability and the reduced cell apoptosis in HepG2 cells transfected with CYP3A5*6 were also investigated when compared to CYP3A5*1. CONCLUSION: Our results suggest that CYP3A5 polymorphism influences sorafenib metabolism and pharmacotherapeutic effect in hepatic carcinomas. These data may help explain differential response to drug therapy for hepatocellular carcinoma, and they support the need for individualized treatment.

Resveratrol inhibits oral squamous cell carcinoma cells proliferation while promoting apoptosis through inhibition of CBX7 protein.

As a natural compound, resveratrol (Res) is confirmed to be promising drug for the treatment of malignant tumors. Therefore, our study aimed to observe the impacts of Res on the proliferation and apoptosis of oral squamous cell carcinoma cells (HSC-3 cells) as well as the mechanism involving chromobox protein homolog 7 (CBX7) signal transduction. HSC-3 cells were treated with Res, Akt agonist (AL3818) and p16 inhibitor (SC79), and transfected with CBX7 mimics and inhibitor plasmids. The CCK-8 assay was used to detect cell proliferation, flow cytometry was performed to assess cell cycle and apoptosis, and cell colonies and histone DNA level were also measured. Western blot analysis was used to determine the expression levels of related proteins. HSC-3 cells showed decreased cell proliferation, colonies, BrdU-counled cells and increased apoptosis, histone DNA level, the activities of caspase-3 and caspase-9 when treated with Res. Western blot analysis revealed elevated Cle-PARP and Cle-caspase 3 expression and reduced t-PARP expression in HSC-3 cells treated with Res compared with control. AL3818 and SC79 could decrease the inhibitory effects of Res on the growth of HSC-3 cells. Furthermore, CBX7 overexpression could also partly reverse the roles of Res in the growth of HSC-3 cells, and Akt and p16 signal transduction. Our results demonstrate that Res suppresses the proliferation, and induces the apoptosis of oral squamous cell carcinoma cells through the inhibition of CBX7/Akt and the activation of p16 cascades.

Stability and Compatibility of Ramosetron with Midazolam in 0.9% Sodium Chloride Injection for Postoperative Nausea and Vomiting Administration.

BACKGROUND: Combination antiemetic therapy has become a common practice for the prevention of postoperative nausea and vomiting (PONV). The aim of the present study was to evaluate the stability and compatibility of ramosetron hydrochloride and midazolam in 0.9% sodium chloride injection when stored at 4°C and 25°C for up to 14 days. METHODS: Admixtures were assessed initially and for 14 days after preparation in polyolefin bags and glass bottles using 0.9% sodium chloride injection as the diluent and stored at 4°C or 25°C. The initial concentrations were 0.3 mg/100 mL ramosetron hydrochloride and 0.5 mg/100 mL midazolam hydrochloride. For all samples, the compatibility parameters (including precipitation, cloudiness, discoloration and pH values) were evaluated. Chemical stability was also determined using high-performance liquid chromatography (HPLC) analysis. RESULTS: After a 14-day period of storage at 4°C or 25°C, the percent of the initial concentration of ramosetron hydrochloride and midazolam hydrochloride in the various solutions were maintained at a minimum of 97%. All of the mixtures remained clear and colourless throughout the observation period, and no colour change or precipitation was observed. CONCLUSION: The results indicate that admixtures of 0.3 mg/100 mL ramosetron hydrochloride and 0.5 mg/100 mL midazolam hydrochloride in normal saline were stable for 14 days at 4°C or 25°C when packaged in polyolefin bags or glass bottles and protected from light.

Reactive oxygen species play a role in P2X7 receptor-mediated IL-6 production in spinal astrocytes.

Astrocytes mediate a remarkable variety of cellular functions, including gliotransmitter release. Under pathological conditions, high concentrations of the purinergic receptor agonist adenosine triphosphate (ATP) are released into the extracellular space leading to the activation of the purinergic P2X7 receptor, which in turn can initiate signaling cascades. It is well-established that reactive oxygen species (ROS) increase in macrophages and microglia following P2X7 receptor activation. However, direct evidence that activation of P2X7 receptor leads to ROS production in astrocytes is lacking to date. While it is known that P2X7R activation induces cytokine production, the mechanism involved in this process is unclear. In the present study, we demonstrated that P2X7 receptor activation induced ROS production in spinal astrocytes in a concentration-dependent manner. We also found that P2X7R-mediated ROS production is at least partially through NADPH oxidase. In addition, our ELISA data show that P2X7R-induced IL-6 release was dependent on NADPH oxidase-mediated production of ROS. Collectively, these results reveal that activation of the P2X7 receptor on spinal astrocytes increases ROS production through NADPH oxidase, subsequently leading to IL-6 release. Our results reveal a role of ROS in the P2X7 signaling pathway in mouse spinal cord astrocytes and may indicate a potential mechanism for the astrocytic P2X7 receptor in chronic pain.

Anti-hypertensive mechanisms of cyclic depsipeptide inhibitor ligands for Gq/11 class G proteins.

Augmented vasoconstriction is a hallmark of hypertension and is mediated partly by hyper-stimulation of G protein couple receptors (GPCRs) and downstream signaling components. Although GPCR blockade is a key component of current anti-hypertensive strategies, whether hypertension is better managed by directly targeting G proteins has not been thoroughly investigated. Here, we tested whether inhibiting Gq/11 proteins in vivo and ex vivo using natural cyclic depsipeptide, FR900359 (FR) from the ornamental plant, Ardisia crenata, and YM-254890 (YM) from Chromobacterium sp. QS3666, or it's synthetic analog, WU-07047 (WU), was sufficient to reverse hypertension in mice. All three inhibitors blocked G protein-dependent vasoconstriction, but to our surprise YM and WU and not FR inhibited K+-induced Ca2+ transients and vasoconstriction of intact vessels. However, each inhibitor blocked whole-cell L-type Ca2+ channel current in vascular smooth muscle cells. Subcutaneous injection of FR or YM (0.3 mg/kg, s.c.) in normotensive and hypertensive mice elicited bradycardia and marked blood pressure decrease, which was more severe and long lasting after the injection of FR relative to YM (FRt1/2 ≅ 12 h vs. YMt1/2 ≅ 4 h). In deoxycorticosterone acetate (DOCA)-salt hypertension mice, chronic injection of FR (0.3 mg/kg, s.c., daily for seven days) reversed hypertension (vehicle SBP: 149 ± 5 vs. FR SBP: 117 ± 7 mmHg), without any effect on heart rate. Our results together support the hypothesis that increased LTCC and Gq/11 activity is involved in the pathogenesis of hypertension, and that dual targeting of both proteins can reverse hypertension and associated cardiovascular disorders.

Identification of Novel Allosteric Modulators of Glutamate Transporter EAAT2.

Dysfunction of excitatory amino acid transporters (EAATs) has been implicated in the pathogenesis of various neurological disorders, such as stroke, brain trauma, epilepsy, and neurodegenerative diseases, among others. EAAT2 is the main subtype responsible for glutamate clearance in the brain, having a key role in regulating transmission and preventing excitotoxicity. Therefore, compounds that increase the expression or activity of EAAT2 have therapeutic potential for neuroprotection. Previous studies identified molecular determinants for EAAT2 transport stimulation in a structural domain that lies at the interface of the rigid trimerization domain and the central substrate binding transport domain. In this work, a hybrid structure based approach was applied, based on this molecular domain, to create a high-resolution pharmacophore. Subsequently, virtual screening of a library of small molecules was performed, identifying 10 hit molecules that interact at the proposed domain. Among these, three compounds were determined to be activators, four were inhibitors, and three had no effect on EAAT2-mediated transport in vitro. Further characterization of the two best ranking EAAT2 activators for efficacy, potency, and selectivity for glutamate over monoamine transporters subtypes and NMDA receptors and for efficacy in cultured astrocytes is demonstrated. Mutagenesis studies suggest that the EAAT2 activators interact with residues forming the interface between the trimerization and transport domains. These compounds enhance the glutamate translocation rate, with no effect on substrate interaction, suggesting an allosteric mechanism. The identification of these novel positive allosteric modulators of EAAT2 offers an innovative approach for the development of therapies based on glutamate transport enhancement.

Orai1 Plays a Crucial Role in Central Sensitization by Modulating Neuronal Excitability.

Pathological pain is a common and debilitating condition that is often poorly managed. Central sensitization is an important mechanism underlying pathological pain. However, candidate molecules involved in central sensitization remain unclear. Store-operated calcium channels (SOCs) mediate important calcium signals in nonexcitable and excitable cells. SOCs have been implicated in a wide variety of human pathophysiological conditions, including immunodeficiency, occlusive vascular diseases, and cancer. However, the role of SOCs in CNS disorders has been relatively unexplored. Orai1, a key component of SOCs, is expressed in the human and rodent spinal cord dorsal horn, but its functional significance in dorsal horn neurons is poorly understood. Here we sought to explore a potential role of Orai1 in the modulation of neuronal excitability and A-type potassium channels involved in pain plasticity. Using both male and female Orai1 knock-out mice, we found that activation of Orai1 increased neuronal excitability and reduced A-type potassium channels via the protein kinase C-extracellular signal-regulated protein kinase (PKC-ERK) pathway in dorsal horn neurons. Orai1 deficiency significantly decreased acute pain induced by noxious stimuli, nearly eliminated the second phase of formalin-induced nociceptive response, markedly attenuated carrageenan-induced ipsilateral pain hypersensitivity and abolished carrageenan-induced contralateral mechanical allodynia. Consistently, carrageenan-induced increase in neuronal excitability was abolished in the dorsal horn from Orai1 mutant mice. These findings uncover a novel signaling pathway involved in the pain process and central sensitization. Our study also reveals a novel link among Orai1, ERK, A-type potassium channels, and neuronal excitability.SIGNIFICANCE STATEMENT Orai1 is a key component of store-operated calcium channels (SOCs) in many cell types. It has been implicated in such pathological conditions as immunodeficiency, autoimmunity, and cancer. However, the role of Orai1 in CNS disorders remains poorly understood. The functional significance of Orai1 in neurons is elusive. Here we demonstrate that activation of Orai1 modulates neuronal excitability and Kv4-containing A-type potassium channels via the protein kinase C-extracellular signal-regulated protein kinase (PKC-ERK) pathway. Genetic knock-out of Orai1 nearly eliminates the second phase of formalin-induced pain and markedly attenuates carrageenan-induced pain hypersensitivity and neuronal excitability. These findings reveal a novel link between Orai1 and neuronal excitability and advance our understanding of central sensitization.

Orai1 and Orai3 Mediate Store-Operated Calcium Entry Contributing to Neuronal Excitability in Dorsal Root Ganglion Neurons.

Store-operated calcium channels (SOCs) are highly calcium-selective channels that mediate calcium entry in various cell types. We have previously reported that intraplantar injection of YM-58483 (a SOC inhibitor) attenuates chronic pain. A previous study has reported that the function of SOCs in dorsal root ganglia (DRG) is enhanced after nerve injury, suggesting that SOCs may play a peripheral role in chronic pain. However, the expression, functional distribution and significance of the SOC family in DRG neurons remain elusive and the key components that mediate SOC entry (SOCE) are still controversial. Here, we demonstrated that the SOC family (STIM1, STIM2, Orai1, Orai2, and Orai3) was expressed in DRGs and STIM1 was mainly present in small- and medium-sized DRG neurons. Using confocal live cell imaging, Ca2+ imaging and electrophysiology techniques, we demonstrated that depletion of the endoplasmic reticulum Ca2+ stores induced STIM1 and STIM2 translocation, and that inhibition of STIM1 or blockage of Orai channels with pharmacological tools attenuated SOCE and SOC currents. Using the small inhibitory RNA knockdown approach, we identified STIM1, STIM2, Orai1, and Orai3 as the key components of SOCs mediating SOCE in DRG neurons. Importantly, activation of SOCs by thapsigargin induced plasma membrane depolarization and increased neuronal excitability, which were completely abolished by inhibition of SOCs or double knockdown of Orai1 and Orai3. Our findings suggest that SOCs exert an excitatory action in DRG neurons and provide a potential peripheral mechanism for modulation of pain hypersensitivity by SOC inhibition.

STIMs and Orai1 regulate cytokine production in spinal astrocytes.

BACKGROUND: Our previous study demonstrated that a store-operated calcium channel (SOCC) inhibitor (YM-58483) has central analgesic effects. However, the cellular and molecular mechanisms of such effects remain to be determined. It is well-known that glial cells play important roles in central sensitization. SOC entry (SOCE) has been implicated in many cell types including cortical astrocytes. However, the role of the SOCC family in the function of astrocytes has not been determined. Here, we thoroughly investigated the expression and the functional significance of SOCCs in spinal astrocytes. METHODS: Primary cultured astrocytes were prepared from neonatal (P2-P3) CD1 mice. Expressions of mRNAs and proteins were respectively assessed by real-time PCR and Western blot analysis. SOCE was measured using a calcium imaging system. Live-cell STIM1 translocation was detected using a confocal microscope. Cytokine levels were measured by the enzyme-linked immunosorbent assay. RESULTS: We found that the SOCC family is expressed in spinal astrocytes and that depletion of calcium stores from the endoplasmic reticulum by cyclopiazonic acid (CPA) resulted in a large sustained calcium entry, which was blocked by SOCC inhibitors. Using the siRNA knockdown approach, we identified STIM1 and Orai1 as primary components of SOCCs in spinal astrocytes. We also observed thapsigargin (TG)- or CPA-induced puncta formation of STIM1 and Orai1. In addition, activation of SOCCs remarkably promoted TNF-α and IL-6 production in spinal astrocytes, which were greatly attenuated by knockdown of STIM1 or Orai1. Importantly, knockdown of STIM2 and Orai1 dramatically decreased lipopolysaccharide-induced TNF-α and IL-6 production without changing cell viability. CONCLUSIONS: This study presents the first evidence that STIM1, STIM2, and Orai1 mediate SOCE and are involved in cytokine production in spinal astrocytes. Our findings provide the basis for future assessment of SOCCs in pain and other central nervous system disorders associated with abnormal astrocyte activities.

Native store-operated calcium channels are functionally expressed in mouse spinal cord dorsal horn neurons and regulate resting calcium homeostasis.

Store-operated calcium channels (SOCs) are calcium-selective cation channels that mediate calcium entry in many different cell types. Store-operated calcium entry (SOCE) is involved in various cellular functions. Increasing evidence suggests that impairment of SOCE is responsible for numerous disorders. A previous study demonstrated that YM-58483, a potent SOC inhibitor, strongly attenuates chronic pain by systemic or intrathecal injection and completely blocks the second phase of formalin-induced spontaneous nocifensive behaviour, suggesting a potential role of SOCs in central sensitization. However, the expression of SOCs, their molecular identity and function in spinal cord dorsal horn neurons remain elusive. Here, we demonstrate that SOCs are expressed in dorsal horn neurons. Depletion of calcium stores from the endoplasmic reticulum (ER) induced large sustained calcium entry, which was blocked by SOC inhibitors, but not by voltage-gated calcium channel blockers. Depletion of ER calcium stores activated inward calcium-selective currents, which was reduced by replacing Ca(2+) with Ba(2+) and reversed by SOC inhibitors. Using the small inhibitory RNA knockdown approach, we identified both STIM1 and STIM2 as important mediators of SOCE and SOC current, and Orai1 as a key component of the Ca(2+) release-activated Ca(2+) channels in dorsal horn neurons. Knockdown of STIM1, STIM2 or Orai1 decreased resting Ca(2+) levels. We also found that activation of neurokinin 1 receptors led to SOCE and activation of SOCs produced an excitatory action in dorsal horn neurons. Our findings reveal that a novel SOC signal is present in dorsal horn neurons and may play an important role in pain transmission.

Mechanosensitive release of adenosine 5'-triphosphate through pannexin channels and mechanosensitive upregulation of pannexin channels in optic nerve head astrocytes: a mechanism for purinergic involvement in chronic strain.

As adenosine 5'-triphosphate (ATP) released from astrocytes can modulate many neural signaling systems, the triggers and pathways for this ATP release are important. Here, the ability of mechanical strain to trigger ATP release through pannexin channels and the effects of sustained strain on pannexin expression were examined in rat optic nerve head astrocytes. Astrocytes released ATP when subjected to 5% of equibiaxial strain or to hypotonic swelling. Although astrocytes expressed mRNA for pannexins 1-3, connexin 43, and VNUT, pharmacological analysis suggested a predominant role for pannexins in mechanosensitive ATP release, with Rho kinase contribution. Astrocytes from panx1(-/-) mice had reduced baseline and stimulated levels of extracellular ATP, confirming the role for pannexins. Swelling astrocytes triggered a regulatory volume decrease that was inhibited by apyrase or probenecid. The swelling-induced rise in calcium was inhibited by P2X7 receptor antagonists A438079 and AZ10606120, in addition to apyrase and carbenoxolone. Extended stretch of astrocytes in vitro upregulated expression of panx1 and panx2 mRNA. A similar upregulation was observed in vivo in optic nerve head tissue from the Tg-MYOC(Y437H) mouse model of chronic glaucoma; genes for panx1, panx2, and panx3 were increased, whereas immunohistochemistry confirmed increased expression of pannexin 1 protein. In summary, astrocytes released ATP in response to mechanical strain, with pannexin 1 the predominant efflux pathway. Sustained strain upregulated pannexins in vitro and in vivo. Together, these findings provide a mechanism by which extracellular ATP remains elevated under chronic mechanical strain, as found in the optic nerve head of patients with glaucoma.

Potent analgesic effects of a store-operated calcium channel inhibitor.

Chronic pain often accompanies immune responses and immune cells are known to be involved in chronic pain. Store-operated calcium (SOC) channels are calcium-selective cation channels and play an important role in the immune system. YM-58483, a potent SOC channel inhibitor, has been shown to inhibit cytokine production from immune cells and attenuate antigen-induced hypersensitivity reactions. Here, we report that YM-58483 has analgesic actions in chronic pain and produces antinociceptive effects in acute pain and prevents the development of chronic pain in mice. Oral administration of 10mg/kg or 30 mg/kg YM-58483 dramatically attenuated complete Freund adjuvant (CFA)-induced thermal hyperalgesia and prevented the development of thermal and mechanical hypersensitivity in a dose-dependent manner. Analgesic effects were observed when YM-58483 was administered systemically, intrathecally and intraplantarly. YM-58483 decreased spared nerve injury (SNI)-induced thermal and mechanical hypersensitivity and prevented the development of SNI-induced pain hypersensitivity. Pretreatment with YM-58483 strongly reduced both the first and second phases of formalin-induced spontaneous nocifensive behavior in a dose-dependent manner. YM-58483 produced antinociception in acute pain induced by heat or chemical or mechanical stimuli at a dose of 30 mg/kg. YM-58483 diminished CFA-induced paw edema, and reduced production of TNF-α, IL-1ß and PGE2 in the CFA-injected paw. In vitro, SOC entry in nociceptors was more robust than in nonnociceptors, and the inhibition of SOC entry by YM-58483 in nociceptors was much greater than in nonnociceptors. Our findings indicate that YM-58483 is a potent analgesic and suggest that SOC channel inhibitors may represent a novel class of therapeutics for pain.

Neurons respond directly to mechanical deformation with pannexin-mediated ATP release and autostimulation of P2X7 receptors.

Mechanical deformation produces complex effects on neuronal systems, some of which can lead to dysfunction and neuronal death. While astrocytes are known to respond to mechanical forces, it is not clear whether neurons can also respond directly. We examined mechanosensitive ATP release and the physiological response to this release in isolated retinal ganglion cells. Purified ganglion cells released ATP upon swelling. Release was blocked by carbenoxolone, probenecid or peptide (10)panx, implicating pannexin channels as conduits. Mechanical stretch of retinal ganglion cells also triggered a pannexin-dependent ATP release. Whole cell patch clamp recording demonstrated that mild swelling induced the activation of an Ohmic cation current with linear kinetics. The current was inhibited by removal of extracellular ATP with apyrase, by inhibition of the P2X(7) receptor with A438079, zinc, or AZ 10606120, and by pannexin blockers carbenoxolone and probenecid. Probenecid also inhibited the regulatory volume decrease observed after swelling isolated neurons. Together, these observations indicate mechanical strain triggers ATP release directly from retinal ganglion cells and that this released ATP autostimulates P2X(7) receptors. Since extracellular ATP levels in the retina increase with elevated intraocular pressure, and stimulation of P2X(7) receptors on retinal ganglion cells can be lethal, this autocrine response may impact ganglion cells in glaucoma. It remains to be determined whether the autocrine stimulation of purinergic receptors is a general response to a mechanical deformation in neurons, or whether preventing ATP release through pannexin channels and blocking activation of the P2X(7) receptor, is neuroprotective for stretched neurons.

Calcium signaling through CatSper channels in mammalian fertilization.

The molecular mechanisms underlying Ca(2+) entry into sperm are now much more well defined thanks to direct recordings of mature sperm cells. This article reviews the function of a sperm-specific ion channel, CatSper. CatSpers have a clearly defined function in sperm's hyperactivated motility and are essential for male fertility. We propose that physiological stimuli such as zona pellucida and cyclic nucleotides induce Ca(2+) entry through CatSper channels instead of acting on Ca(V) and CNG channels as previously thought.