Neuropathic pain is a debilitating clinical problem and difficult to treat. Nerve injury causes a long-lasting reduction in K+ channel expression in the dorsal root ganglion (DRG), but little is known about the epigenetic mechanisms involved. Here we show that nerve injury increased H3K9me2 occupancy at Kcna4, Kcnd2, Kcnq2 and Kcnma1 promoters but did not affect DNA methylation levels of these genes in DRGs. Nerve injury increased activity of G9a, histone deacetylases and EZH2, but only G9a inhibition consistently restored K+ channel expression. Selective G9a knockout in DRG neurons completely blocked K+ channel silencing and chronic pain development after nerve injury. Remarkably, RNA sequencing analysis revealed that G9a inhibition not only reactivated 40 of 42 silenced K+ channel genes but also normalized 638 genes down- or up-regulated by nerve injury. Thus G9a plays a dominant role in transcriptional repression of K+ channels and in acute-to-chronic pain transition after nerve injury.
Calcium influx through voltage-activated Ca2؉ channels (VACCs) plays a critical role in neurotransmission. Capsaicin application inhibits VACCs and desensitizes nociceptors. In this study, we determined the signaling mechanisms of the inhibitory effect of capsaicin on VACCs in primary sensory neurons. Whole-cell voltage clamp recordings were performed in acutely isolated rat dorsal root ganglion neurons. Capsaicin caused a profound decrease in the Ca TRPV11 is a nonselective cation channel with high Ca 2ϩ permeability and is the molecular target of capsaicin, the main pungent ingredient in chili peppers. The TRPV1 channel is expressed in subsets of primary sensory neurons and nerve terminals and plays an essential role in detecting noxious heat and several other nociceptive stimuli (1, 2). Capsaicin can excite nociceptive sensory neurons and produce transient pain in animals and humans (3, 4). Paradoxically, exposure to capsaicin desensitizes nociceptive sensory neurons and results in long lasting pain relief. For example, topical or local application of capsaicin is effective in treating many acute and chronic pain syndromes in patients (5-7). Capsaicin also causes an unexplained synaptic transmission block in the spinal cord dorsal horn (8).The voltage-activated Ca 2ϩ channels (VACCs) play a critical role in signal transduction, synaptic neurotransmitter release, and nociceptive transmission (9 -11). VACCs also are an important molecular target of many analgesic drugs such as opioids (12, 13). Interestingly, capsaicin causes a profound inhibition of VACC currents in dorsal root ganglion (DRG) neurons (14). However, the cellular and signaling mechanisms of the capsaicin effect on VACCs remain poorly understood.Protein kinases and phosphatases are key enzymes in signal transduction pathways for a wide range of cellular processes. The enzymatic addition or removal of phosphate esters on serine and threonine hydroxyls alters the activity of many proteins that are essential to the characteristic structure and function of neurons. An important mechanism regulating VACC function is through phosphorylation by protein kinases and phosphatases (15)(16)(17)(18). We now show that a Ca 2ϩ -dependent serine/threonine phosphatase, calcineurin (protein phosphatase 2B), is critically involved in down-regulation of high voltage-activated Ca 2ϩ channels (HVACCs) by capsaicin in native DRG neurons. Furthermore, the basal intracellular Ca 2ϩ level and endogenous protein phosphatases tonically modulate the HVACC current in DRG neurons. These findings are not only important to our understanding of the functional interaction between TRPV1 and HVACCs in primary nociceptors but are also significant to our understanding of the Ca 2ϩ -dependent feedback regulation of neuronal Ca 2ϩ channels in general. MATERIALS AND METHODSIsolation of DRG Neurons-All procedures conformed to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of the P...
Macrophage-derived foam cell formation elicited by oxidized low-density lipoprotein (oxLDL) is the hallmark of early atherogenesis. Detection of foam cell formation is conventionally practiced by Oil Red O (ORO) staining of lipid-laden macrophages. Other methods include 1,1'-dioctadecyl-3,3,3'3'-tetra-methylindocyanide percholorate (DiI)-labeled oxLDL (DiI-oxLDL) uptake and Nile Red staining. The purpose of the present study is to report an optimized method for assessing foam cell formation in cultured macrophages by ORO staining and DiI-oxLDL uptake. After incubation with oxLDL (50 μg/ml) for 24 h, the macrophages were fixed, stained with ORO for just 1 min, pronounced lipid droplets were clearly observed in more than 90% of the macrophages. To test the in vivo applicability of this method, lesions (or foam cells) of cryosections of aortic sinus or primary mouse peritoneal macrophages from ApoE deficient mice fed a high cholesterol diet were successfully stained. In another set of experiments, treatment of macrophages with DiI-oxLDL (10 μg/ml) for 4 h resulted in significant increase in oxLDL uptake in macrophages as demonstrated by confocol microscopy and flow cytometry. We conclude that the optimized ORO staining and fluorescent labeled oxLDL uptake techniques are very useful for assessing intracellular lipid accumulation in macrophages that are simpler and more rapid than currently used methods.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.