Currents In Dorsal Root Ganglion Neurons Research Articles

Effects of nanoparticle-encapsulated curcumin on HIV-gp120-associated neuropathic pain induced by the P2X3 receptor in dorsal root ganglia.

HIV-1 envelope glycoprotein (Glycoprotein 120, gp120) can directly stimulate primary sensory afferent neurons and cause chronic neuropathic pain. The P2X3 receptor in the dorsal root ganglia (DRG) is associated with the transmission of neuropathic pain. Curcumin isolated from the herb Curcuma rhizome has anti-inflammatory and anti-tumor effects. The water solubility, targeting and bioavailability of curcumin can be improved by nanoparticle encapsulation. In this study, we sought to explore the effects of nanoparticle-encapsulated curcumin (nano curcumin) on HIV-gp120-induced neuropathic pain mediated by the P2X3 receptor in DRG neurons. The results showed that mechanical and thermal hyperalgesia in rats treated with gp120 were increased compared to those in the control group. The expression levels of P2X3 mRNA and protein in rats treated with gp120 were higher than those in the control group. Nano curcumin treatment decreased mechanical hyperalgesia and thermal hyperalgesia and upregulated the expression levels of P2X3 mRNA and protein in rats treated with gp120. Nano curcumin treatment also reduced the ERK1/2 phosphorylation levels in gp120-treated rat DRG. In addition, P2X3 agonist α,β-methylene ATP (α,β-meATP)-induced currents in DRG neurons cultured with gp120 significantly decreased after co-treatment with nano curcumin. Therefore, nano curcumin treatment may inhibit P2X3 activation, decrease the sensitizing DRG primary afferents and relieve mechanical hyperalgesia and thermal hyperalgesia in gp120-treated rats.

The P2X7 receptor in dorsal root ganglia is involved in HIV gp120-associated neuropathic pain.

Human immunodeficiency virus (HIV)-associated neuropathic pain is common, and studies have shown that HIV envelope glycoprotein 120 (gp120) can directly stimulate primary sensory afferent neurons causing hyperalgesia. The P2X7 receptor in the dorsal root ganglia (DRG) is involved in pain transmission and is closely related to the inflammatory and immune response. In this study, we aimed to explore the role of the P2X7 receptor in gp120-induced neuropathic pain using a rat model specific for this type of pain. The results showed that mechanical hyperalgesia, thermal hyperalgesia and P2X7 expression levels were increased in rats treated with gp120. The P2X7 antagonist, brilliant blue G (BBG), decreased hyperalgesia and P2X7 expression levels in rats treated with gp120. BBG also decreased IL-1β and TNF-α receptor expression and ERK1/2 phosphorylation levels and increased IL-10 expression in the gp120-treated rat DRG. In addition, P2X7 agonist (BzATP)-activated currents in DRG neurons cultured with gp120 were larger than those in control neurons, and the inhibitory effect of BBG on BzATP-induced currents in gp120-treated DRG neurons was larger than that in control neurons. Therefore, inhibition of the P2X7 receptor in rat DRG relieved gp120-induced mechanical hyperalgesia and thermal hyperalgesia.

Inhibition of TRPM3 Ion Channels by G-Protein Beta-Gamma Subunits

Transient Receptor Potential Melastatin 3 (TRPM3) is a non-selective, Ca2+ permeable cation channel activated by heat, and chemical activators such as pregnenolone sulphate and CIM0216. Here we show that activation of receptors coupled to heterotrimeric Gi proteins inhibits TRPM3 currents in a mammalian expression system. This inhibition was alleviated by co-expression of proteins that act as sinks for the βγ subunits of heterotrimeric G-proteins (Gβγ). Co-expression of Gβ1γ2, but not a constitutively active mutant of Gαo inhibited pregnenolone sulphate-induced TRPM3 currents. TRPM3 was also inhibited by purified Gβγ proteins applied to excised inside out patches, indicating a direct effect. Baclofen and somatostatin, agonists of Gi coupled receptors, inhibited Ca2+ signals induced by pregnenolone sulphate and CIM0216 in dorsal root ganglion (DRG) neurons. The GABAB receptor agonist baclofen also inhibited CIM0216-induced currents in DRG neurons, and nocifensive responses elicited by this TRPM3 agonist in vivo. Our data show that Gi-coupled receptors regulate TRPM3 channels via direct inhibition by Gβγ.

A317491 relieved HIV gp120-associated neuropathic pain involved in P2X3 receptor in dorsal root ganglia.

Glycoprotein 120 (gp120) is an HIV envelope glycoprotein. Gp120 can directly stimulate the primary sensory afferent neurons and cause hyperalgesia. The P2X3 receptor in dorsal root ganglia (DRG) is involved in the transmission of pain. In this study, we aimed to explore the role of the P2X3 receptor in gp120-induced neuropathic pain. Our data showed that mechanical and thermal hyperalgesia in rats treated with gp120 were increased compared to those in the control group. The expression levels of the P2X3 mRNA and protein in rats treated with gp120 were higher than those in the control group. The P2X3 antagonist A317491 decreased mechanical hyperalgesia and thermal hyperalgesia and the up-regulated expression levels of P2X3 mRNA and protein in rats treated with gp120. A317491 decreased ERK1/2 phosphorylation levels in the gp120-treated rat DRG. In addition, P2X3 agonist α,β-methylene ATP (α,β-meATP)-activated currents in DRG neurons cultured with gp120 were higher than those in control neurons. The inhibitory effect of A317491 on α,βme-ATP-induced currents in DRG neurons from the gp120-treated neurons was larger than that for control neurons. Molecular docking data showed that A317491 may be acted in the gp120 protein to inhibit the gp120 initiated the P2X3 activation, decrease the sensitizing DRG primary afferents and reduce the signal transmission of neuropathic pain in gp120-treated rats. Therefore, the inhibition of the P2X3 receptor in rat DRG neurons relieved gp120-induced mechanical hyperalgesia.

TRPA1 Function in Skeletal Muscle Sensory Neurons Following Femoral Artery Occlusion

Background/Aims: Transient receptor potential channel A1 (TRPA1) is engaged in amplified autonomic responses evoked by stimulation of muscle afferent nerves in rats with experimental peripheral arterial disease. The purposes of this study were to characterize current responses induced by activation of TRPA1 in dorsal root ganglion (DRG) neurons of control limbs and limbs with femoral artery occlusion. Methods: DRG neurons from rats were labeled by injecting the fluorescence tracer DiI into the hindlimb muscles and whole-cell patch clamp experiments were performed to determine TRPA1 currents. Results: Data show that AITC (a TRPA1 agonist) from the concentrations of 50 µM to 200 µM produces a dose-dependent increase of amplitudes of inward current responses. Notably, the peak current amplitude induced by AITC is significantly larger in DRG neurons of ligated limbs than that in control limbs. AITC-induced current responses are observed in small and medium size DRG neurons, and there is no difference in size distribution of DRG neurons between control limbs and ligated limbs. However, femoral occlusion increases the percentage of the AITC-sensitive DRG neurons as compared to control. AITC-induced currents in DRG neurons are significantly attenuated by exposure to 10 µM of HC-030031, a potent and selective inhibitor of TRPA1, in both control and femoral occlusion groups. In addition, capsaicin (a TRPV1 agonist) evokes a greater increase in the amplitude of AITC-currents in DRG neurons of ligated limbs than that in control limbs. Conclusions: A greater current response with activation of TRPA1 is developed in muscle afferent nerves when hindlimb arterial blood supply is deficient under ischemic conditions; and TRPV1 is partly responsible for augmented TRPA1 responses induced by arterial occlusion.

SHANK3 Deficiency Impairs Heat Hyperalgesia and TRPV1 Signaling in Primary Sensory Neurons

Abnormal pain sensitivity is commonly associated with autism spectrum disorders (ASDs) and affects the life quality of ASD individuals. SHANK3 deficiency was implicated in ASD and pain dysregulation. Here, we report functional expression of SHANK3 in mouse dorsal root ganglion (DRG) sensory neurons and spinal cord presynaptic terminals. Homozygous and heterozygous Shank3 complete knockout (Δe4-22) results in impaired heat hyperalgesia in inflammatory and neuropathic pain. Specific deletion of Shank3 in Nav1.8-expressing sensory neurons also impairs heat hyperalgesia in homozygous and heterozygous mice. SHANK3 interacts with transient receptor potential subtype V1 (TRPV1) via Proline-rich region and regulates TRPV1 surface expression. Furthermore, capsaicin-induced spontaneous pain, inward currents in DRG neurons, and synaptic currents in spinal cord neurons are all reduced after Shank3 haploinsufficiency. Finally, partial knockdown of SHANK3 expression in human DRG neurons abrogates TRPV1 function. Our findings reveal a peripheral mechanism of SHANK3, which may underlie pain deficits in SHANK3-related ASDs.

Inhibitory effect of estrogen receptor beta on P2X3 receptors during inflammation in rats.

Estrogen receptor beta (ERβ) has been shown to play a therapeutic role in inflammatory bowel disease (IBD). However, the mechanism underlying how ERβ exerts therapeutic effects and its relationship with P2X3 receptors (P2X3R) in rats with inflammation is not known. In our study, animal behavior tests, visceromotor reflex recording, and Western blotting were used to determine whether the therapeutic effect of ERβ in rats with inflammation was related with P2X3R. In complete Freund adjuvant (CFA)-induced chronic inflammation in rats, paw withdrawal threshold was significantly decreased which were then reversed by systemic injection of ERβ agonists, DPN or ERB-041. In 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats, weight loss, higher DAI scores, increased visceromotor responses, and inflammatory responses were reversed by application of DPN or ERB-041. The higher expressions of P2X3R in dorsal root ganglia (DRG) of CFA-treated rats and those in rectocolon and DRG of TNBS-treated rats were all decreased by injection of DPN or ERB-041. DPN application also inhibited P2X3R-evoked inward currents in DRG neurons from TNBS rats. Mechanical hyperalgesia and increased P2X3 expression in ovariectomized (OVX) CFA-treated rats were reversed by estrogen replacements. Furthermore, the expressions of extracellular signal-regulated kinase (ERK) in DRG and spinal cord dorsal horn (SCDH) and c-fos in SCDH were significantly decreased after estrogen replacement compared with those of OVX rats. The ERK antagonist U0126 significantly reversed mechanical hyperalgesia in the OVX rats. These results suggest that estrogen may play an important therapeutic role in inflammation through down-regulation of P2X3R in peripheral tissues and the nervous system, probably via ERβ, suggesting a novel therapeutic strategy for clinical treatment of inflammation.

Modulation of Nav1.8 by Lysophosphatidic Acid in the Induction of Bone Cancer Pain.

Given that lysophosphatidic acid (LPA) and the tetrodotoxin-resistant sodium channel Nav1.8 are both involved in bone cancer pain, the present study was designed to investigate whether crosstalk between the LPA receptor LPA1 (also known as EDG2) and Nav1.8 in the dorsal root ganglion (DRG) contributes to the induction of bone cancer pain. We showed that the EDG2 antagonist Ki16198 blocked the mechanical allodynia induced by intrathecal LPA in naïve rats and attenuated mechanical allodynia in a rat model of bone cancer. EDG2 and Nav1.8 expression in L4-6 DRGs was upregulated following intrathecal or hindpaw injection of LPA. EDG2 and Nav1.8 expression in ipsilateral L4-6 DRGs increased with the development of bone cancer. Furthermore, we showed that EDG2 co-localized with Nav1.8 and LPA remarkably enhanced Nav1.8 currents in DRG neurons, and this was blocked by either a protein kinase C (PKC) inhibitor or a PKCε inhibitor. Overall, we demonstrated the modulation of Nav1.8 by LPA in DRG neurons, and that this probably underlies the peripheral mechanism by which bone cancer pain is induced.

Tentonin 3/TMEM150c Confers Distinct Mechanosensitive Currents in Dorsal-Root Ganglion Neurons with Proprioceptive Function

Touch sensation or proprioception requires the transduction of mechanical stimuli into electrical signals by mechanoreceptors in the periphery. These mechanoreceptors are equipped with various transducer channels. Although Piezo1 and 2 are mechanically activated (MA) channels with rapid inactivation, MA molecules with other inactivation kinetics have not been identified. Here we report that heterologously expressed Tentonin3 (TTN3)/TMEM150C isactivated by mechanical stimuli with distinctly slow inactivation kinetics. Genetic ablation of Ttn3/Tmem150c markedly reduced slowly adapting neurons in dorsal-root ganglion neurons. The MA TTN3 currents were inhibited by known blockers of mechanosensitive ion channels. Moreover, TTN3 was localized in muscle spindle afferents. Ttn3-deficient mice exhibited the loss of coordinated movements and abnormal gait. Thus, TTN3 appears to be a component of a mechanosensitive channel with a slow inactivation rate and contributes to motor coordination. Identification of this gene advances our understanding of the various types of mechanosensations, including proprioception.

Potentiation of acid-sensing ion channel activity by peripheral group I metabotropic glutamate receptor signaling

Glutamate activates peripheral group I metabotropic glutamate receptors (mGluRs) and contributes to inflammatory pain. However, it is still not clear the mechanisms are involved in group I mGluR-mediated peripheral sensitization. Herein, we report that group I mGluRs signaling sensitizes acid-sensing ion channels (ASICs) in dorsal root ganglion (DRG) neurons and contributes to acidosis-evoked pain. DHPG, a selective group I mGluR agonist, can potentiate the functional activity of ASICs, which mediated the proton-induced events. DHPG concentration-dependently increased proton-gated currents in DRG neurons. It shifted the proton concentration⿿response curve upwards, with a 47.3±7.0% increase of the maximal current response to proton. Group I mGluRs, especially mGluR5, mediated the potentiation of DHPG via an intracellular cascade. DHPG potentiation of proton-gated currents disappeared after inhibition of intracellular Gq/11 proteins, PLCβ, PKC or PICK1 signaling. Moreover, DHPG enhanced proton-evoked membrane excitability of rat DRG neurons and increased the amplitude of the depolarization and the number of spikes induced by acid stimuli. Finally, peripherally administration of DHPG dose-dependently exacerbated nociceptive responses to intraplantar injection of acetic acid in rats. Potentiation of ASIC activity by group I mGluR signaling in rat DRG neurons revealed a novel peripheral mechanism underlying group I mGluRs involvement in hyperalgesia.

Opioidergic Regulation of P2X3 Receptor-Mediated Currents in DRG Neurons

As is traditionally believed, the analgesic effects of opioids are due to their action on the CNS mechanisms. At present, however, proofs are been accumulated that activation of opioid receptors at the periphery can also result in a strong analgesic effect (especially in the case of inflammatory processes). In neurons of the dorsal root ganglia (DRG) of rats, we examined the effects of activation of opioid receptors on purinergic (P2X3) receptor-mediated ion currents and tried to identify possible intracellular signal pathways between the respective receptors. P2X3-mediated ion currents in DRG neurons were evoked by applications of αβ-Me-ATP. These currents were strongly (on average, by 74%) and reversibly inhibited after application of an endogenous opioid peptide, leu-enkephalin (L-Enk, 100 nM). A selective competitive antagonist of μ opioid receptors, CTOP, completely abolished the effect of L-Enk. We believe that a pathway mediated by phospholipase C (PLC) is involved in intracellular signaling between opioid and P2X3 receptors, and our experiments confirmed this hypothesis. A synthetic activator of PLC suppressed P2X3-mediated currents; an inhibitor of synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), wortmannin, accelerated and augmented the inhibitory effect of L-Enk on P2X3-mediated currents. Therefore, the inhibitory effect of L-Enk on the latter receptors is based on activation on PLC and hydrolysis of PIP2.

Oxytocin inhibits the activity of acid‐sensing ion channels through the vasopressin, V1A receptor in primary sensory neurons

A growing number of studies have demonstrated that oxytocin (OT) plays an analgesic role in modulation of nociception and pain. Most work to date has focused on the central mechanisms of OT analgesia, but little is known about whether peripheral mechanisms are also involved. Acid-sensing ion channels (ASICs) are distributed in peripheral sensory neurons and participate in nociception. Here, we investigated the effects of OT on the activity of ASICs in dorsal root ganglion (DRG) neurons. Electrophysiological experiments were performed on neurons from rat DRG. Nociceptive behaviour was induced by acetic acid in rats and mice lacking vasopressin, V1A receptors. OT inhibited the functional activity of native ASICs. Firstly, OT dose-dependently decreased the amplitude of ASIC currents in DRG neurons. Secondly, OT inhibition of ASIC currents was mimicked by arginine vasopressin (AVP) and completely blocked by the V1A receptor antagonist SR49059, but not by the OT receptor antagonist L-368899. Thirdly, OT altered acidosis-evoked membrane excitability of DRG neurons and significantly decreased the amplitude of the depolarization and number of action potentials induced by acid stimuli. Finally, peripherally administered OT or AVP inhibited nociceptive responses to intraplantar injection of acetic acid in rats. Both OT and AVP also induced an analgesic effect on acidosis-evoked pain in wild-type mice, but not in V1A receptor knockout mice. These results reveal a novel peripheral mechanism for the analgesic effect of OT involving the modulation of native ASICs in primary sensory neurons mediated by V1A receptors.

Tetrodotoxin-resistant sodium channels in sensory neurons generate slow resurgent currents that are enhanced by inflammatory mediators.

Resurgent sodium currents contribute to the regeneration of action potentials and enhanced neuronal excitability. Tetrodotoxin-sensitive (TTX-S) resurgent currents have been described in many different neuron populations, including cerebellar and dorsal root ganglia (DRG) neurons. In most cases, sodium channel Nav1.6 is the major contributor to these TTX-S resurgent currents. Here we report a novel TTX-resistant (TTX-R) resurgent current recorded from rat DRG neurons. The TTX-R resurgent currents are similar to classic TTX-S resurgent currents in many respects, but not all. As with TTX-S resurgent currents, they are activated by membrane repolarization, inhibited by lidocaine, and enhanced by a peptide-mimetic of the β4 sodium channel subunit intracellular domain. However, the TTX-R resurgent currents exhibit much slower kinetics, occur at more depolarized voltages, and are sensitive to the Nav1.8 blocker A803467. Moreover, coimmunoprecipitation experiments from rat DRG lysates indicate the endogenous sodium channel β4 subunits associate with Nav1.8 in DRG neurons. These results suggest that slow TTX-R resurgent currents in DRG neurons are mediated by Nav1.8 and are generated by the same mechanism underlying TTX-S resurgent currents. We also show that both TTX-S and TTX-R resurgent currents in DRG neurons are enhanced by inflammatory mediators. Furthermore, the β4 peptide increased excitability of small DRG neurons in the presence of TTX. We propose that these slow TTX-R resurgent currents contribute to the membrane excitability of nociceptive DRG neurons under normal conditions and that enhancement of both types of resurgent currents by inflammatory mediators could contribute to sensory neuronal hyperexcitability associated with inflammatory pain.

Femoral artery occlusion amplifies TRPA1 function in skeletal muscle sensory neurons (1132.5)

A prior study showed that transient receptor potential channel A1 (TRPA1) in thin‐fiber muscle afferents contributes to the amplified reflex autonomic responses observed in rats with femoral artery ligation. Thus, in this study we characterized current responses induced by activation of TRPA1 in skeletal muscle dorsal root ganglion (DRG) neurons of control limbs and limbs with 24 hrs of femoral ligation using whole‐cell patch clamp methods. Our results show that AITC (a TRPA1 agonist, 50‐200 µM) produced a dose‐dependent increase of amplitudes of inward current responses. Also, the peak current amplitude induced by AITC is significantly larger in DRG neuron of ligated limbs. There is no difference in size distribution of DRG neurons with AITC‐induced current responses between two groups. However, arterial occlusion increases the percentage of the AITC‐sensitive DRG neurons compared with control. AITC‐induced currents in DRG neurons are significantly attenuated by exposure to 10 µM of HC‐030031, a selective inhibitor of TRPA1. In addition, capsaicin (a TRPV1 agonist) evokes a greater increase in the amplitude of AITC‐currents in DRG neurons of ligated limbs. Overall, our data suggest that a greater TRPA1 function is developed in muscle afferent nerves with the hindlimb ischemia and TRPV1 is partly responsible for augmented TRPA1 responses induced by arterial occlusion.Grant Funding Source: NIH P01 HL096570 and AHA EIA 0840130N

Inhibition of acid‐sensing ion channel currents by propofol in rat dorsal root ganglion neurons

Acid-sensing ion channels (ASICs), part of the epithelial sodium channel/degenerin family, are activated by extracellular protons. The ASICs play a significant role in the acidosis-mediated perception of pain. The anaesthetic agent propofol also exerts antinociceptive effects, but the underlying mechanisms for this effect are not clear. We used whole-cell patch clamping to investigate the effect of propofol on proton-gated currents in: (i) rat dorsal root ganglion (DRG) neurons; and (ii) HEK293 cells transfected with either ASIC1a or ASIC3. Propofol inhibited the amplitude of proton-gated currents in DRG neurons, but did not change the sensitivity of ASICs to H(+). Notably, propofol altered acid-evoked excitability of rat DRG neurons and decreased the number of action potentials induced by acid stimuli. In addition, we demonstrated that propofol inhibited ASICs by directly binding with these channels in HEK293 cells. These results suggest that propofol inhibits proton-gated currents in DRG neurons and that inhibition of proton-gated currents explains, in part, the antinociceptive effects of propofol in primary afferent neurons.

(344) Tetrodotoxin-resistant sodium channels in sensory neurons generate slow resurgent currents that are enhanced by inflammatory mediators

Resurgent sodium currents contribute to the regeneration of action potentials and enhanced neuronal excitability. Tetrodotoxin-sensitive (TTX-S) resurgent currents have been described in many different neuron populations including cerebellar and dorsal root ganglion (DRG) neurons. In the current study, we found a novel TTX-resistant (TTX-R) resurgent current recorded from rat DRG neurons. The TTX-R resurgent currents are similar to classic TTX-S resurgent currents in many respects, such as that they are activated by membrane repolarization, inhibited by lidocaine, and enhanced by a peptide-mimetic of the β4 sodium channel subunit intracellular domain. However, the TTX-R resurgent currents exhibit much slower kinetics, occur at more depolarized voltages and are sensitive to the Nav1.8 blocker A803467. These results suggest slow TTX-R resurgent currents in DRG neurons are mediated by Nav1.8 and are generated by the same mechanism underlying TTX-S resurgent currents. Moreover, We found that TTX-R resurgent currents in DRG neurons are enhanced by inflammatory mediators and that the membrane excitability of small DRG neurons was enhanced by β4 peptide in the presence of TTX. We propose that these slow TTX-R resurgent currents contribute to the membrane excitability of nociceptive DRG neurons under normal conditions and that enhancement of both types of resurgent currents by inflammatory mediators could contribute to sensory neuronal hyperexcitability associated with inflammatory pain. The work was supported by a Lilly Research Award. T.R.C. was also supported in part by a National Institutes of Health grant (NS053422). Resurgent sodium currents contribute to the regeneration of action potentials and enhanced neuronal excitability. Tetrodotoxin-sensitive (TTX-S) resurgent currents have been described in many different neuron populations including cerebellar and dorsal root ganglion (DRG) neurons. In the current study, we found a novel TTX-resistant (TTX-R) resurgent current recorded from rat DRG neurons. The TTX-R resurgent currents are similar to classic TTX-S resurgent currents in many respects, such as that they are activated by membrane repolarization, inhibited by lidocaine, and enhanced by a peptide-mimetic of the β4 sodium channel subunit intracellular domain. However, the TTX-R resurgent currents exhibit much slower kinetics, occur at more depolarized voltages and are sensitive to the Nav1.8 blocker A803467. These results suggest slow TTX-R resurgent currents in DRG neurons are mediated by Nav1.8 and are generated by the same mechanism underlying TTX-S resurgent currents. Moreover, We found that TTX-R resurgent currents in DRG neurons are enhanced by inflammatory mediators and that the membrane excitability of small DRG neurons was enhanced by β4 peptide in the presence of TTX. We propose that these slow TTX-R resurgent currents contribute to the membrane excitability of nociceptive DRG neurons under normal conditions and that enhancement of both types of resurgent currents by inflammatory mediators could contribute to sensory neuronal hyperexcitability associated with inflammatory pain. The work was supported by a Lilly Research Award. T.R.C. was also supported in part by a National Institutes of Health grant (NS053422).

Puerarin blocks the signaling transmission mediated by P2X3 in SG and DRG to relieve myocardial ischemic damage

P2X₃ receptors in stellate ganglia (SG) and cervical dorsal root ganglia (DRG) neurons are involved in sympathoexcitatory reflex induced by myocardial ischemic damage. Puerarin, a major active ingredient extracted from the traditional Chinese plant medicine Ge-gen, has been widely used in treatment of myocardial and cerebral ischemia. The present study is aimed to observe the effects of puerarin on the signaling transmission mediated by P2X₃ receptor in SG and DRG after myocardial ischemic damage. Our results showed that systolic blood pressure and heart rate increased, and the expression levels of P2X₃ mRNA and protein in SG and DRG were up-regulated after myocardial ischemic damage. Puerarin reduced systolic blood pressure and heart rate, relieved pain and decreased up-regulated expression of P2X₃ mRNA and protein in SG and DRG after myocardial ischemia. Puerarin inhibited the up-regulated ATP-activated currents in DRG neurons after myocardial ischemia. Thus, puerarin can relieve myocardial ischemic damage through blocking the P2X₃ signaling transmission and then depressed the aggravated sympathoexcitatory reflex.

Artemin growth factor increases nicotinic cholinergic receptor subunit expression and activity in nociceptive sensory neurons.

BackgroundArtemin (Artn), a member of the glial cell line-derived growth factor (GDNF) family, supports the development and function of a subpopulation of peptidergic, TRPV1-positive sensory neurons. Artn (enovin, neublastin) is elevated in inflamed tissue and its injection in skin causes transient thermal hyperalgesia. A genome wide expression analysis of trigeminal ganglia of mice that overexpress Artn in the skin (ART-OE mice) showed elevation in nicotinic acetylcholine receptor (nAChR) subunits, suggesting these ion channels contribute to Artn-induced sensitivity. Here we have used gene expression, immunolabeling, patch clamp electrophysiology and behavioral testing assays to investigate the link between Artn, nicotinic subunit expression and thermal hypersensitivity.ResultsReverse transcriptase-PCR validation showed increased levels of mRNAs encoding the nAChR subunits α3 (13.3-fold), β3 (4-fold) and β4 (7.7-fold) in trigeminal ganglia and α3 (4-fold) and β4 (2.8-fold) in dorsal root ganglia (DRG) of ART-OE mice. Sensory ganglia of ART-OE mice had increased immunoreactivity for nAChRα3 and exhibited increased overlap in labeling with GFRα3-positive neurons. Patch clamp analysis of back-labeled cutaneous afferents showed that while the majority of nicotine-evoked currents in DRG neurons had biophysical and pharmacological properties of α7-subunit containing nAChRs, the Artn-induced increase in α3 and β4 subunits resulted in functional channels. Behavioral analysis of ART-OE and wildtype mice showed that Artn-induced thermal hyperalgesia can be blocked by mecamylamine or hexamethonium. Complete Freund’s adjuvant (CFA) inflammation of paw skin, which causes an increase in Artn in the skin, also increased the level of nAChR mRNAs in DRG. Finally, the increase in nAChRs transcription was not dependent on the Artn-induced increase in TRPV1 or TRPA1 in ART-OE mice since nAChRs were elevated in ganglia of TRPV1/TRPA1 double knockout mice.ConclusionsThese findings suggest that Artn regulates the expression and composition of nAChRs in GFRα3 nociceptors and that these changes contribute to the thermal hypersensitivity that develops in response to Artn injection and perhaps to inflammation.

Stomatin‐domain protein interactions with acid‐sensing ion channels modulate nociceptor mechanosensitivity

Acid-sensing ion channels (ASICs) and their interaction partners of the stomatin family have all been implicated in sensory transduction. Single gene deletion of asic3, asic2, stomatin, or stoml3 all result in deficits in the mechanosensitivity of distinct cutaneous afferents in the mouse. Here, we generated asic3−/−:stomatin−/−, asic3−/−:stoml3−/− and asic2−/−:stomatin−/− double mutant mice to characterize the functional consequences of stomatin–ASIC protein interactions on sensory afferent mechanosensitivity. The absence of ASIC3 led to a clear increase in mechanosensitivity in rapidly adapting mechanoreceptors (RAMs) and a decrease in the mechanosensitivity in both Aδ- and C-fibre nociceptors. The increased mechanosensitivity of RAMs could be accounted for by a loss of adaptation which could be mimicked by local application of APETx2 a toxin that specifically blocks ASIC3. There is a substantial loss of mechanosensitivity in stoml3−/− mice in which ∼35% of the myelinated fibres lack a mechanosensitive receptive field and this phenotype was found to be identical in asic3−/−:stoml3−/− mutant mice. However, Aδ-nociceptors showed much reduced mechanosensitivity in asic3−/−:stoml3−/− mutant mice compared to asic3−/− controls. Interestingly, in asic2−/−:stomatin−/− mutant mice many Aδ-nociceptors completely lost their mechanosensitivity which was not observed in asic2−/− or stomatin−/− mice. Examination of stomatin−/−:stoml3−/− mutant mice indicated that a stomatin/STOML3 interaction is unlikely to account for the greater Aδ-nociceptor deficits in double mutant mice. A key finding from these studies is that the loss of stomatin or STOML3 in asic3−/− or asic2−/− mutant mice markedly exacerbates deficits in the mechanosensitivity of nociceptors without affecting mechanoreceptor function.

Role of TRPM2 cation channels in dorsal root ganglion of rats after experimental spinal cord injury

We sought to determine the contribution of oxidative stress-dependent activation of TRPM2 and L-type voltage-gated Ca(2+) channels (VGCC) in dorsal root ganglion (DRG) neurons of rats after spinal cord injury (SCI). The rats were divided into 4 groups: control; sham control; SCI; and SCI+nimodipine groups. The neurons of the SCI groups were also incubated with non-specific TRPM2 channel blockers, 2-aminoethoxydiphenylborate (2-APB) and N-(p-amylcinnamoyl)anthranilic acid (ACA), before H2 O2 stimulation. The [Ca(2+) ]i concentrations were higher in the SCI group than in the control groups, although their concentrations were decreased by nimodipine and 2-APB. The H2 O2 -induced TRPM2 current densities in patch-clamp experiments were decreased by ACA and 2-APB incubation. In the nimodipine group, the TRPM2 channels of neurons were not activated by H2 O2 or cumene hydroperoxide. Increased Ca(2+) influx and currents in DRG neurons after spinal injury indicated TRPM2 and voltage-gated Ca(2+) channel activation.