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Item Bone pain induced by multiple myeloma is reduced by targeting V-ATPase and ASIC3(AACR Publications, 2017-03-15) Hiasa, Masahiro; Okui, Tatsuo; Allette, Yohance M; Ripsch, Matthew S; Sun-Wada, Ge-Hong; Wakabayashi, Hiroki; Roodman, G David; White, Fletcher A.; Yoneda, Toshiyuki; Medicine, School of MedicineMultiple myeloma (MM) patients experience severe bone pain (MMBP) that is undertreated and poorly understood. In this study, we studied MMBP in an intratibial mouse xenograft model which employs JJN3 human MM cells. In this model, mice develop MMBP associated in bone with increased sprouting of calcitonin gene-related peptide-positive (CGRP+) sensory nerves and in dorsal root ganglia (DRG) with upregulation of phosphorylated ERK1/2 (pERK1/2) and pCREB, two molecular indicators of neuron excitation. We found that JJN3 cells expressed a vacuolar proton pump (V-ATPase) that induced an acidic bone microenvironment. Inhibition of JJN3-colonized bone acidification by a single injection of the selective V-ATPase inhibitor, bafilomycin A1, decreased MMBP, CGRP+ SN sprouting, and pERK1/2 and pCREB expression in DRG. CGRP+ sensory nerves also expressed increased levels of the acid-sensing nociceptor ASIC3. Notably, a single injection of the selective ASIC3 antagonist APETx2 dramatically reduced MMBP in the model. Mechanistic investigations in primary DRG neurons co-cultured with JJN3 cells showed increased neurite outgrowth and excitation inhibited by bafilomycin A1 or APETx2. Further, combining APETx2 with bafilomycin A1 reduced MMBP to a greater extent than either agent alone. Lastly, combining bafilomycin A1 with the osteoclast inhibitor zoledronic acid was sufficient to ameliorate MMBP which was refractory to zoledronic acid. Overall, our results show that osteoclasts and MM cooperate to induce an acidic bone microenvironment that evokes MMBP as a result of the excitation of ASIC3-activated sensory neurons. Further, they present a mechanistic rationale for targeting ASIC3 on neurons along with the MM-induced acidic bone microenvironment as a strategy to relieve MMBP in patients.Item Cellular Mechanisms Mediating the Actions of Nerve Growth Factor in Sensory Neurons(2007-08-08T15:24:37Z) Park, Kellie Adrienne; Vasko, Michael R.Nerve growth factor (NGF) is a neurotrophin upregulated with injury and inflammation. Peripheral administration of NGF causes hyperalgesia and allodynia in animals. Blocking NGF signaling reverses these effects. At the cellular level, chronic exposure of sensory neurons to NGF enhances expression the neurotransmitter, calcitonin gene-related peptide (CGRP). Acute exposure to NGF increases capsaicin-evoked CGRP release from sensory neurons in culture. Thus, NGF increases peptide release from neurons by: (1) increasing expression of peptides, and/or (2) altering their sensitivity. The increase in peptide outflow by either mechanism could contribute to development of hyperalgesia and allodynia. The signaling cascades mediating the actions of NGF in sensory neurons are unclear. Therefore, experiments were designed to determine which pathways regulate changes in iCGRP content and evoked release from primary sensory neurons in culture. The Ras/MEK/ERK cascade was identified as a possible regulator of iCGRP expression in response to NGF. To test this pathway, it was manipulated in neurons by (1) expression of dominant negative or constitutively active isoforms of Ras, (2) farnesyltransferase inhibition, (3) manipulation of the RasGAP, synGAP, and (4) blocking MEK activity. When the pathway was blocked, the NGF-induced increase in iCGRP expression was attenuated. When the Ras pathway was activated, iCGRP expression increased. These data indicate that Ras, and downstream signaling kinases, MEK and ERK, regulate the NGF-induced increases in CGRP in sensory neurons. To determine which pathway(s) regulate the increase in capsaicin-evoked iCGRP release upon brief exposure to NGF, the Ras/MEK/ERK pathway was manipulated as described above, and pharmacological inhibitors of the PI3 kinase, PLC, and Src kinase pathways were used. There were no differences observed in NGF-sensitization when the Ras and PI3 kinase pathways were inhibited, suggesting these two pathways were not involved. However, when the Src kinase inhibitor PP2 was used, the NGF-induced increase in release was completely blocked. Furthermore, the PKC inhibitor, BIM, also inhibited the sensitization by NGF. This data indicate Src and PKC regulate of sensitivity of sensory neurons in response to brief exposure to NGF. Thus, there is differential regulation of iCGRP content and evoked release from sensory neurons in response to NGF.Item Effects of 12, 13 dibutyrate (PDBu) on resting and capsaicin-stimulated glutamate release in adult rat dorsal root neurons in culture(2003-07-31) Richard, Joyce; Richter, Judith; Agbor-Baiyee, WilliamThis investigation explored the role of PKC as an activator of VR1 through the use of capsaicin, a natural ligand for VR1, and the PKC activating phorbol ester 12,13 dibutyrate (PDBu). Direct effects of 30nM, 300nM, and 3000nM capsaicin and 0nM, 0.3nM, 1.0nM, 3.0nM, 10nM, and 30nM PDBu on glutamate release from adult rat dorsal root ganglia (DRGs) were examined. Based on those observations, the concentration of capsaicin causing the least glutamate release and the concentrations of PDBu that did not elicit significant glutamate release were selected to study effects of capsaicin on same cell type acutely sensitized with the selected concentrations of PDBu. It was found that glutamate released from combinations of 30nM capsaicin and 1.0nM PDBu (p<0.036) as well as 30nM capsaicin and 3.0nM PDBu (p<0.01) were statistically significant from the stimulation control—which was 30nM capsaicin alone.Item SCHWANN CELLS MODULATE THE RELEASE OF CALCITONIN GENE-RELATED PEPTIDE FROM SENSORY NEURONS(Office of the Vice Chancellor for Research, 2012-04-13) Meadows, Rena M.; Hingtgen, Cynthia M.An alteration in the interaction between Schwann cells and sensory neurons may be involved in inflammatory neuropathies associated with altered sensation and pain. The release of the peptide transmitter, calcitonin gene-related peptide (CGRP), is one method to monitor the sensitivity of a subclass of primary sensory neurons involved in pain signaling. We utilized an in vitro assay to investigate the interaction between Schwann cells and sensory neurons in an inflammatory state. Schwann cells and sensory neurons were isolated from adult mouse sciatic nerve and dorsal root ganglia, respectively, and maintained in culture. Schwann cells were exposed to HEPES buffer containing the inflammatory mediators histamine, prostaglandin E2, bradykinin, and serotonin (all 10-5 M), potassium (7 mM), and at pH 7.0 for 10 minutes. After this activation, the Schwann cells were incubated in HEPES buffer alone for 1 hour. This Schwann cell-conditioned buffer (SCCB) was collected and sensory neurons were exposed to three consecutive 10 minute incubations in HEPES buffer alone or SCCB. The amount of CGRP released during each of these incubations was measured using radioimmunoassay. Incubation with SCCB elicited a seven-fold increase in the release of CGRP compared to neurons exposed to HEPES buffer alone. The release of CGRP elicited by SCCB was abolished when neurons were exposed to SCCB containing no added calcium. After treatment with the inflammatory mediators detailed above for 10 minutes, Schwann cell lysates showed a significant decrease in six cytokines, while SCCB demonstrated an increase in interleukin-6 (IL-6) as measured by a cytokine array panel. These results suggest that during inflammation, Schwann cells release substances, which directly stimulate sensory neurons, as measured by an increase in CGRP release. These findings reinforce the importance of identifying the mechanisms underlying the interaction between Schwann cells and sensory neurons to discover novel therapeutics for treating inflammatory pain.Item Sphingosine 1-phosphate enhances excitability of sensory neurons through sphingosine 1-phosphate receptors 1 and/or 3(2014) Li, Chao; Vasko, Michael R.; Cummins, Theodore R.; Hudmon, Andrew; Nicol, Grant D.; Quilliam, Lawrence A.Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that has proven to be an important signaling molecule both as an extracellular primary messenger and as an intracellular second messenger. Extracellular S1P acts through a family of five S1P receptors, S1PR1-5, all of which are G protein-coupled receptors associated with different G proteins. Previous work from our laboratory shows that externally applied S1P increases the excitability of small-diameter sensory neurons by enhancing the action potential firing. The increased neuronal excitability is mediated primarily, but not exclusively, through S1PR1. This raises the question as to which other S1PRs mediate the enhanced excitability in sensory neurons. To address this question, the expression of different S1PR subtypes in small-diameter sensory neurons was examined by single-cell quantitative PCR. The results show that sensory neurons express the mRNAs for all five S1PRs, with S1PR1 mRNA level significantly greater than the other subtypes. To investigate the functional contribution of other S1PRs in augmenting excitability, sensory neurons were treated with a pool of three individual siRNAs targeted to S1PR1, R2 and R3. This treatment prevented S1P from augmenting excitability, indicating that S1PR1, R2 and/or R3 are essential in mediating S1P-induced sensitization. To study the role of S1PR2 in S1P-induced sensitization, JTE-013, a selective antagonist at S1PR2, was used. Surprisingly, JTE-013 by itself enhanced neuronal excitability. Alternatively, sensory neurons were pretreated with FTY720, which is an agonist at S1PR1/R3/R4/R5 and presumably downregulates these receptors. FTY720 pretreatment prevented S1P from increasing neuronal excitability, suggesting that S1PR2 does not mediate the S1P-induced sensitization. To test the hypothesis that S1PR1 and R3 mediate S1P-induced sensitization, sensory neurons were pretreated with specific antagonists for S1PR1 and R3, or with siRNAs targeted to S1PR1 and R3. Both treatments blocked the capacity of S1P to enhance neuronal excitability. Therefore my results demonstrate that the enhanced excitability produced by S1P is mediated by S1PR1 and/or S1PR3. Additionally, my results indicate that S1P/S1PR1 elevates neuronal excitability through the activation of mitogen-activated protein kinase kinase. The data from antagonism at S1PR1 to regulate neuronal excitability provides insight into the importance of S1P/S1PR1 axis in modulating pain signal transduction.