- Email: [email protected]
(289) Angiotensin II causes mechanical hypersensitivity via macrophage-derived ROS production and activation of TRPA1
S48
Abstracts
The Journal of Pain
measured by perforated-patch (Amphotericin B) current-clamp technique). Under conditions of low [Cl ]i (10mM), ANO1 actions were reversed. Specifically, E-act did not trigger action potentials; however, capsaicin-induced action potential firing was inhibited by co-application of E-act, but was unaffected by co-application of T16A[inh]-A01. Nocifensive responses of mice hind paws were dramatically induced by subcutaneous injections of E-actcapsaicin (50mM). The nocifensive responses were attenuated by co-injection with T16A[inh]-A01 (1.3mM). In summary, an ANO1-activator induced [Cl ]i-dependent sensory neuronal action potentials and mouse nocifensive behaviors; thus, direct ANO1 activation can induce pain perception. ANO1-inhibition attenuated capsaicintriggering of action potentials and capsaicin-induced nocifensive behaviors. The results indicate that sensory neuronal ANO1 facilitates TRPV1 triggering of action potentials, resulting in enhanced nociception and inhibition of ANO1 could be a novel means of inducing analgesia.
trols. The ROS scavenger N-acetylcysteine attenuated this ROS production and abolished Ang II-induced mechanical hypersensitivity, indicating that ROS production is a necessary downstream component of these effects. We also show that cutaneous macrophages are a likely source of Ang II-induced ROS, since cultured macrophages exhibit enhanced ROS production in response to Ang II in vitro, and inducible depletion of macrophages in vivo also abolishes the effects of Ang II. The mechanical hypersensitivity induced by Ang II could also be attenuated by co-injection of a TRPA1 antagonist, suggesting that ROS may converge on TRPA1 to elicit mechanical hypersensitivity. Furthermore, TRPA1-dependent calcium flux in cultured mouse DRG neurons was enhanced by Ang II, but only when macrophages were also present in the culture. Together, these data suggest crosstalk between sensory neurons and macrophages mediates the mechanical hypersensitivity induced by Ang II.
(288) Potential use of site directed RNA editing to treat chronic pain
D12 Neurotrophins
L Fernandez-Alacid and J Rosenthal; Institute of Neurobiology San Juan, Puerto Rico, Puerto Rico
Chronic pain is a common medical disorder. A general strategy for reducing pain is to reduce excitability in relevant fibers. Voltagegated ion channels are critical components for excitability. Na+ channels activate the action potential and K+ channels turn it off; therefore, one strategy for reducing excitability would be to convert some Na+ channels into channels selective for both K+ and Na+. The DEKA motif is an important determinant for selectivity in Na+ channels. Studies have shown that when the DEKA’s lysine is converted to arginine the channels become permeable to K+ as well. However, when it is changed to other residues, they become permeable to Ca2+. How might we convert the lysine selectively to arginine? RNA editing by adenosine deamination is a natural process where specific adenosines within mRNAs are converted to inosine, a biological mimic for guanosine. Our group has developed a system for directing RNA editing to adenosines of our choosing. Our method (site-directed RNA editing; SDRE), uses an RNA guide to direct the catalytic domain of ADAR, an RNA editing enzyme. We decided to use SDRE to convert K1237 (AAG) of Nav1.4 to arginine (AGG), but not to glutamate (GAG) or glycine (GGG). As a first step, we designed a specific guide oligonucleotide that could accomplish this in vitro under optimal conditions. However, at higher enzyme concentrations some glutamates and glycines were generated as well. Because Nav1.4 K1237E and K1237G have not been tested, we verified that these mutations render the channel permeable to both K+ and Ca2+ in HEK293T cells using patch-clamp. Ongoing experiments are testing our ability to edit K1237 to K1237R in cellula. This work was supported by NINDS 1R0111223855, NINDS 1R01NS64259, NIGMS P20GM103642, and NIMHD 8G12MD007600.
D08 Ion Channels - TRPs (289) Angiotensin II causes mechanical hypersensitivity via macrophage-derived ROS production and activation of TRPA1
(290) Toll-like receptor 2 regulates nerve growth factor through NF-kappaB and MAPK signaling in human intervertebral discs E Krock, B Currie, D Rosenzweig, M Weber, J Ouellet, L Stone, and L Haglund; McGill University/Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada
Chronic low back pain is a leading cause of disability with an estimated socioeconomic cost of $100-300 billion per year in the United States. Intervertebral disc degeneration is the most common cause of chronic low back pain, but the pain mechanisms are poorly understood. Nerve growth factor (NGF) is a key protein in chronic low back pain associated with disc degeneration. However, NGF regulation in pathogenic connective tissues, like the disc, is poorly understood. Toll-like receptors (TLR) are pattern recognition receptors classically associated with innate immunity, but more recently were found to be activated by endogenous alarmins such as fragmented extra-cellular matrix protein found in degenerating discs. We investigated if TLR activation can regulate NGF synthesis and determined the signaling mechanisms involved. Nucleus pulposus and annulus fibrosus cells isolated from non-degenerate human discs were treated with peptidoglycan (PGN, TLR2 agonist), lipopolysaccharide (LPS, TLR4 agonist) and IL-1b (positive control). Both TLR2 and TLR4 agonists increased NGF and brain-derived neurotrophic factor (BDNF) gene expression. Only TLR2 activation increased NGF protein secretion. BDNF protein levels were below the ELISA detection limit. TLR2 activation of cell signaling pathways was investigated using phosphorylation specific antibodies. NF-kB, ERK1/2, and p38, but not JNK all showed increased signaling activity following TLR2 activation. Small molecule inhibition studies found NF-kB, and to a lesser extent p38 inhibition decreased NGF synthesis, whereas ERK1/2 inhibition had no effect. Taken together these results identify a novel mechanism of NGF regulation in pathogenic connective tissues, which may be distinct from physiological NGF synthesis in the central nervous system. Furthermore, TLR2 and NF-kB signaling increase cytokines and catabolic proteases that contribute to disc degeneration. Therefore, therapeutically targeting TLR2 or NF-kB may both attenuate chronic pain and slow the degenerative progress in vivo.
A Shepherd and D Mohapatra; Washington University School of Medicine, Saint Louis, MO
Angiotensin II (Ang II) was initially described for its vasoconstrictive properties and accordant effects on blood pressure. Pro-inflammatory effects of Ang II have been reported on leukocytes, endothelial cells and vascular smooth muscle. Furthermore, agents that decrease Ang II signaling (by targeting either angiotensin converting enzyme or AT1/AT2 receptors) have been proposed to have analgesic effects. However, the mechanisms responsible for any pro-algesic effect of Ang II remain unclear. We showed that intraplantar injection of Ang II caused dosedependent mechanical hypersensitivity, without any significant change in thermal sensitivity. This mechanical hypersensitivity was absent in AT2R-null animals and could also be blocked by the AT2R-specific antagonist PD123319. Consistent with a role for AT2R signaling, production of reactive oxygen species (ROS) was elevated at the site of Ang II injection, relative to saline con-
D13 Peptides (291) Complement-mediated effects of the VGF peptide TLQP-21 J Cook, K Kitto, M Riedl, C Fairbanks, and L Vulchanova; University of Minnesota, Minneapolis, MN
VGF is a granin-related neuropeptide precursor that is cleaved into a number of bioactive peptides. VGF expression is rapidly and robustly increased in dorsal root ganglion and dorsal horn neurons following peripheral nerve injury. We have demonstrated that the VGF-derived peptide TLQP-21 contributes to both the development and maintenance of hypersensitivity after peripheral nerve injury and inflammation, and that intrathecal administration of TLQP-21 results in thermal hyperalgesia. The receptor for TLQP-21 was found to be the complement 3a
Abstracts
The Journal of Pain
measured by perforated-patch (Amphotericin B) current-clamp technique). Under conditions of low [Cl ]i (10mM), ANO1 actions were reversed. Specifically, E-act did not trigger action potentials; however, capsaicin-induced action potential firing was inhibited by co-application of E-act, but was unaffected by co-application of T16A[inh]-A01. Nocifensive responses of mice hind paws were dramatically induced by subcutaneous injections of E-actcapsaicin (50mM). The nocifensive responses were attenuated by co-injection with T16A[inh]-A01 (1.3mM). In summary, an ANO1-activator induced [Cl ]i-dependent sensory neuronal action potentials and mouse nocifensive behaviors; thus, direct ANO1 activation can induce pain perception. ANO1-inhibition attenuated capsaicintriggering of action potentials and capsaicin-induced nocifensive behaviors. The results indicate that sensory neuronal ANO1 facilitates TRPV1 triggering of action potentials, resulting in enhanced nociception and inhibition of ANO1 could be a novel means of inducing analgesia.
trols. The ROS scavenger N-acetylcysteine attenuated this ROS production and abolished Ang II-induced mechanical hypersensitivity, indicating that ROS production is a necessary downstream component of these effects. We also show that cutaneous macrophages are a likely source of Ang II-induced ROS, since cultured macrophages exhibit enhanced ROS production in response to Ang II in vitro, and inducible depletion of macrophages in vivo also abolishes the effects of Ang II. The mechanical hypersensitivity induced by Ang II could also be attenuated by co-injection of a TRPA1 antagonist, suggesting that ROS may converge on TRPA1 to elicit mechanical hypersensitivity. Furthermore, TRPA1-dependent calcium flux in cultured mouse DRG neurons was enhanced by Ang II, but only when macrophages were also present in the culture. Together, these data suggest crosstalk between sensory neurons and macrophages mediates the mechanical hypersensitivity induced by Ang II.
(288) Potential use of site directed RNA editing to treat chronic pain
D12 Neurotrophins
L Fernandez-Alacid and J Rosenthal; Institute of Neurobiology San Juan, Puerto Rico, Puerto Rico
Chronic pain is a common medical disorder. A general strategy for reducing pain is to reduce excitability in relevant fibers. Voltagegated ion channels are critical components for excitability. Na+ channels activate the action potential and K+ channels turn it off; therefore, one strategy for reducing excitability would be to convert some Na+ channels into channels selective for both K+ and Na+. The DEKA motif is an important determinant for selectivity in Na+ channels. Studies have shown that when the DEKA’s lysine is converted to arginine the channels become permeable to K+ as well. However, when it is changed to other residues, they become permeable to Ca2+. How might we convert the lysine selectively to arginine? RNA editing by adenosine deamination is a natural process where specific adenosines within mRNAs are converted to inosine, a biological mimic for guanosine. Our group has developed a system for directing RNA editing to adenosines of our choosing. Our method (site-directed RNA editing; SDRE), uses an RNA guide to direct the catalytic domain of ADAR, an RNA editing enzyme. We decided to use SDRE to convert K1237 (AAG) of Nav1.4 to arginine (AGG), but not to glutamate (GAG) or glycine (GGG). As a first step, we designed a specific guide oligonucleotide that could accomplish this in vitro under optimal conditions. However, at higher enzyme concentrations some glutamates and glycines were generated as well. Because Nav1.4 K1237E and K1237G have not been tested, we verified that these mutations render the channel permeable to both K+ and Ca2+ in HEK293T cells using patch-clamp. Ongoing experiments are testing our ability to edit K1237 to K1237R in cellula. This work was supported by NINDS 1R0111223855, NINDS 1R01NS64259, NIGMS P20GM103642, and NIMHD 8G12MD007600.
D08 Ion Channels - TRPs (289) Angiotensin II causes mechanical hypersensitivity via macrophage-derived ROS production and activation of TRPA1
(290) Toll-like receptor 2 regulates nerve growth factor through NF-kappaB and MAPK signaling in human intervertebral discs E Krock, B Currie, D Rosenzweig, M Weber, J Ouellet, L Stone, and L Haglund; McGill University/Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada
Chronic low back pain is a leading cause of disability with an estimated socioeconomic cost of $100-300 billion per year in the United States. Intervertebral disc degeneration is the most common cause of chronic low back pain, but the pain mechanisms are poorly understood. Nerve growth factor (NGF) is a key protein in chronic low back pain associated with disc degeneration. However, NGF regulation in pathogenic connective tissues, like the disc, is poorly understood. Toll-like receptors (TLR) are pattern recognition receptors classically associated with innate immunity, but more recently were found to be activated by endogenous alarmins such as fragmented extra-cellular matrix protein found in degenerating discs. We investigated if TLR activation can regulate NGF synthesis and determined the signaling mechanisms involved. Nucleus pulposus and annulus fibrosus cells isolated from non-degenerate human discs were treated with peptidoglycan (PGN, TLR2 agonist), lipopolysaccharide (LPS, TLR4 agonist) and IL-1b (positive control). Both TLR2 and TLR4 agonists increased NGF and brain-derived neurotrophic factor (BDNF) gene expression. Only TLR2 activation increased NGF protein secretion. BDNF protein levels were below the ELISA detection limit. TLR2 activation of cell signaling pathways was investigated using phosphorylation specific antibodies. NF-kB, ERK1/2, and p38, but not JNK all showed increased signaling activity following TLR2 activation. Small molecule inhibition studies found NF-kB, and to a lesser extent p38 inhibition decreased NGF synthesis, whereas ERK1/2 inhibition had no effect. Taken together these results identify a novel mechanism of NGF regulation in pathogenic connective tissues, which may be distinct from physiological NGF synthesis in the central nervous system. Furthermore, TLR2 and NF-kB signaling increase cytokines and catabolic proteases that contribute to disc degeneration. Therefore, therapeutically targeting TLR2 or NF-kB may both attenuate chronic pain and slow the degenerative progress in vivo.
A Shepherd and D Mohapatra; Washington University School of Medicine, Saint Louis, MO
Angiotensin II (Ang II) was initially described for its vasoconstrictive properties and accordant effects on blood pressure. Pro-inflammatory effects of Ang II have been reported on leukocytes, endothelial cells and vascular smooth muscle. Furthermore, agents that decrease Ang II signaling (by targeting either angiotensin converting enzyme or AT1/AT2 receptors) have been proposed to have analgesic effects. However, the mechanisms responsible for any pro-algesic effect of Ang II remain unclear. We showed that intraplantar injection of Ang II caused dosedependent mechanical hypersensitivity, without any significant change in thermal sensitivity. This mechanical hypersensitivity was absent in AT2R-null animals and could also be blocked by the AT2R-specific antagonist PD123319. Consistent with a role for AT2R signaling, production of reactive oxygen species (ROS) was elevated at the site of Ang II injection, relative to saline con-
D13 Peptides (291) Complement-mediated effects of the VGF peptide TLQP-21 J Cook, K Kitto, M Riedl, C Fairbanks, and L Vulchanova; University of Minnesota, Minneapolis, MN
VGF is a granin-related neuropeptide precursor that is cleaved into a number of bioactive peptides. VGF expression is rapidly and robustly increased in dorsal root ganglion and dorsal horn neurons following peripheral nerve injury. We have demonstrated that the VGF-derived peptide TLQP-21 contributes to both the development and maintenance of hypersensitivity after peripheral nerve injury and inflammation, and that intrathecal administration of TLQP-21 results in thermal hyperalgesia. The receptor for TLQP-21 was found to be the complement 3a