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Expression and distribution of mTOR, p70S6K, and 4E-BP1 in rat dorsal root ganglion and spinal cord dorsal horn
Abstracts
S33
E20 Non-Opioid Analgesics
E22 Primary Afferents
(228) Novel small molecule alpha9 alpha10 nicotinic receptor antagonists for pain management
(230) Expression and distribution of mTOR, p70S6K, and 4E-BP1 in rat dorsal root ganglion and spinal cord dorsal horn
J Holtman, L Dwoskin, E Wala, P Crooks, and J McIntosch; University of Kentucky, Lexington, KY
Y Tao, J Xu, X Zhao, M Yaster, and S Raja; Johns Hopkins University School of Medicine, Baltimore, MD
Chronic pain is inadequately managed with currently available analgesic drug classes (opioids, NSAIDs, adjuvants). A great deal of emphasis has been placed on identifying novel molecular targets for analgesics. Potential novel targets include specific subtypes of nicotinic acetylcholine receptors (nAChRs). Efforts have focused on antagonists acting primarily on subtypes such as a4b2 and a7 nAChRs. Recent studies employing a-conotoxins have identified a novel target, the a9a10 subtype, with potential importance for pain management. Antagonism of the a9a10 nAChR by peptide a-conotoxins produced analgesia in rodent models of neuropathic and inflammatory pain. In order to evaluate non-peptide antagonists of this subtype, we have recently synthesized a series of tetrakis-, tris-, and bis-quaternary ammonium salts. Compounds from each group, GZ-5-56A, ZZ-2-04G and ZZ-1-61C, respectively, were found to block rat a9a10 nAChRs expressed in Xenopus oocytes with IC50s in a range similar to those for the analgesic a-conotoxins previously tested. These analogs had selectivity for a9a10 nAChR when compared to other nAChRs (e.g., a7). Parenteral administration of GZ-5-56A and ZZ-2-04G in a dose-related manner reversed mechanical hyperalgesia in a rodent (Sprague-Dawley male rats) model of neuropathic pain (chronic constriction nerve injury, CCI) and alleviated persistent inflammatory pain in the formalin test. Analgesia was not confounded by motor impairment (rotarod). Tolerance was not observed with repeated dosing (CCI). Neither compound was effective in the tail-flick model of acute nociception. Testing with ZZ-1-61C demonstrated analgesic activity in the formalin test following both parenteral and oral administration. These preclinical data suggest that novel quaternary ammonium salts may be potential candidates for development as analgesics with a unique mechanism of action at the a9a10 receptor. (Support: NIH U19 DA017548, RO1 MH5363, DA022091.)
Mammalian target of rapamycin (mTOR) controls protein translation and has an important role in the mechanism of pain hypersensitivity under persistent pain conditions. However, a complete understanding of its localization in pain-related regions of the nervous system is still lacking. Here, we examined the expression and distribution of mTOR and its two downstream effectors, eukaryotic initiation factor 4E-binding protein1/2 (4E-BP1/2) and p70 ribosomal S6 protein kinase (p70S6K), in two major pain-related regions, the dorsal root ganglion (DRG) and spinal cord dorsal horn. Reverse transcriptase-polymerase chain reaction showed that mTOR, 4E-BP1, and p70S6K mRNA are expressed in the DRG and dorsal horn. Western blot analysis further confirmed the existence of their protein products in these two regions. Immunohistochemistry revealed that mTOR and p70S6K were expressed exclusively in the DRG neurons. Quantitative analysis showed that approximately 26.1% of DRG neurons were positive for mTOR and 19.1% were positive for p70S6K. Most of these neurons were small—less than 600 mm2 in cross-sectional area—and some co-labeled with substance P or isolectin B4. Surprisingly, 4EBP1 was observed only in the DRG satellite glial cells. In the dorsal horn, mTOR, p70S6K, and 4E-BP1 were expressed in the neurons, but not in the astrocytes or microglia. They were distributed in the whole dorsal horn, especially in the superficial dorsal horn. The results indicate that in the DRG, cellular localization of 4E-BP1 is different from that of mTOR and p70S6K but that in the dorsal horn, the three proteins have the same localization and distribution. Our findings provide morphologic evidence to support the role of mTOR, p70S6K, and 4E-BP1 in peripheral and central mechanisms of pain hypersensitivity in persistent pain.
(229) Topical menthol and capsaicin for evaluation of small peripheral nerve fiber function
E23 Psychophysics/Hyperalgesia
K Gooden, A Tobias, J Schneider, K Nixdorf, and M Backonja; UW-Madison, Madison, WI Neuropathic pain is common in clinical practice and presents a significant diagnostic challenge, especially when functional properties of small peripheral nerve fibers need to be tested. Thermal noxious stimuli which elicit burning and cooling sensations are transduced via distinct receptors TRPV1 and TRPM8, respectively, are conveyed by small fiber afferents. TRPV1 are also activated with capsaicin and TRPM8 with menthol. The objective of this study was to establish functional response properties of these receptors and associated afferents in normal healthy subjects to application of different concentrations of capsaicin and menthol. The goal is to study 60 subjects, ages of 20-80 years; ten subjects for each decade. Only subjects who are in good health, free of neurological sensory symptoms and free of pain, are included. Capsaicin 0.025% and 0.1% and menthol 2.5% and 16% as well as inert control cream are applied in random order over an area of 1 square inch for each application, in blinded (to the subject and the examiner) fashion, for duration of 10 minutes and sensations evoked by these applications are rated by subjects. In addition quantitative sensory testing at application site is tested after cream is removed. At this time 12 subjects are enrolled and preliminary results reveal: evoked sensations from the current concentrations are described as very mild; higher concentrations of capsaicin had a significant effect on perception of hot pain and there was a trend for warm stimulation. The specificity of distinct activation of receptor and associated small peripheral nerve fibers activation produced by application of capsaicin and menthol provides a unique opportunity for testing underlying abnormalities in neuropathic pain related to small peripheral fiber function and this data from normal subjects should help in interpretation of this type of information.
(231) Spatial summation of pain: a sub-additive phenomenon A Quevedo, C Mørch, O Andersen, and R Coghill; Aalborg University, Aalborg, Denmark Spatial summation is a common feature of different sensory modalities and can be defined as the increase of perceived intensity by increasing the size of stimulated area. Although nociceptive information is able to summate, spatial summation of pain (SSP) is sub-additive in that increasing the stimulus area produces a disproportionately small increase in the perceived intensity of pain. A possible explanation for sub-additive summation may be the modulation of convergent excitatory information from peripheral areas by central inhibition. Traditionally, SSP has been induced by enlarging the stimulated area, which can involve larger portions of excitatory receptive fields but may also recruit inhibitory surround areas. To test the hypothesis that surround inhibition may limit SSP, we delivered 3 different patterns of noxious thermal stimuli to the abdomens of 15 subjects using a computer-controlled CO2-laser. Lines (5mm wide) of variable lengths (4, 8, 12cm) were compared to two different controls (one-point and two-point stimuli delivered at the same position/separation as the length of lines: 4, 8, 12cm). When compared to one-point control (VAS=2.0360.3), SSP was not evoked by 4cm (VAS=1.9260.29) and 8cm (VAS=2.160.29) lines and only was observed with 12cm lines (VAS=2.3360.29) (p<0.05). At 4cm and 8cm, two-point control stimuli (VAS= 2.3160.31 and VAS=2.3760.35 respectively) produced higher pain intensity than lines (p<0.001 and p<0.05 respectively), despite the fact that the two-point stimuli activate smaller areas. Thus, the stimulation of regions between the end-points of the lines appears to produce inhibition. This suggest that the current definition of SSP is an oversimplification of the phenomenon. Instead, results from interactions between excitatory/inhibitory mechanisms. (Supported by 2008 IASP Research Grant funded by ScanDesign by Bruun Foundation.)
S33
E20 Non-Opioid Analgesics
E22 Primary Afferents
(228) Novel small molecule alpha9 alpha10 nicotinic receptor antagonists for pain management
(230) Expression and distribution of mTOR, p70S6K, and 4E-BP1 in rat dorsal root ganglion and spinal cord dorsal horn
J Holtman, L Dwoskin, E Wala, P Crooks, and J McIntosch; University of Kentucky, Lexington, KY
Y Tao, J Xu, X Zhao, M Yaster, and S Raja; Johns Hopkins University School of Medicine, Baltimore, MD
Chronic pain is inadequately managed with currently available analgesic drug classes (opioids, NSAIDs, adjuvants). A great deal of emphasis has been placed on identifying novel molecular targets for analgesics. Potential novel targets include specific subtypes of nicotinic acetylcholine receptors (nAChRs). Efforts have focused on antagonists acting primarily on subtypes such as a4b2 and a7 nAChRs. Recent studies employing a-conotoxins have identified a novel target, the a9a10 subtype, with potential importance for pain management. Antagonism of the a9a10 nAChR by peptide a-conotoxins produced analgesia in rodent models of neuropathic and inflammatory pain. In order to evaluate non-peptide antagonists of this subtype, we have recently synthesized a series of tetrakis-, tris-, and bis-quaternary ammonium salts. Compounds from each group, GZ-5-56A, ZZ-2-04G and ZZ-1-61C, respectively, were found to block rat a9a10 nAChRs expressed in Xenopus oocytes with IC50s in a range similar to those for the analgesic a-conotoxins previously tested. These analogs had selectivity for a9a10 nAChR when compared to other nAChRs (e.g., a7). Parenteral administration of GZ-5-56A and ZZ-2-04G in a dose-related manner reversed mechanical hyperalgesia in a rodent (Sprague-Dawley male rats) model of neuropathic pain (chronic constriction nerve injury, CCI) and alleviated persistent inflammatory pain in the formalin test. Analgesia was not confounded by motor impairment (rotarod). Tolerance was not observed with repeated dosing (CCI). Neither compound was effective in the tail-flick model of acute nociception. Testing with ZZ-1-61C demonstrated analgesic activity in the formalin test following both parenteral and oral administration. These preclinical data suggest that novel quaternary ammonium salts may be potential candidates for development as analgesics with a unique mechanism of action at the a9a10 receptor. (Support: NIH U19 DA017548, RO1 MH5363, DA022091.)
Mammalian target of rapamycin (mTOR) controls protein translation and has an important role in the mechanism of pain hypersensitivity under persistent pain conditions. However, a complete understanding of its localization in pain-related regions of the nervous system is still lacking. Here, we examined the expression and distribution of mTOR and its two downstream effectors, eukaryotic initiation factor 4E-binding protein1/2 (4E-BP1/2) and p70 ribosomal S6 protein kinase (p70S6K), in two major pain-related regions, the dorsal root ganglion (DRG) and spinal cord dorsal horn. Reverse transcriptase-polymerase chain reaction showed that mTOR, 4E-BP1, and p70S6K mRNA are expressed in the DRG and dorsal horn. Western blot analysis further confirmed the existence of their protein products in these two regions. Immunohistochemistry revealed that mTOR and p70S6K were expressed exclusively in the DRG neurons. Quantitative analysis showed that approximately 26.1% of DRG neurons were positive for mTOR and 19.1% were positive for p70S6K. Most of these neurons were small—less than 600 mm2 in cross-sectional area—and some co-labeled with substance P or isolectin B4. Surprisingly, 4EBP1 was observed only in the DRG satellite glial cells. In the dorsal horn, mTOR, p70S6K, and 4E-BP1 were expressed in the neurons, but not in the astrocytes or microglia. They were distributed in the whole dorsal horn, especially in the superficial dorsal horn. The results indicate that in the DRG, cellular localization of 4E-BP1 is different from that of mTOR and p70S6K but that in the dorsal horn, the three proteins have the same localization and distribution. Our findings provide morphologic evidence to support the role of mTOR, p70S6K, and 4E-BP1 in peripheral and central mechanisms of pain hypersensitivity in persistent pain.
(229) Topical menthol and capsaicin for evaluation of small peripheral nerve fiber function
E23 Psychophysics/Hyperalgesia
K Gooden, A Tobias, J Schneider, K Nixdorf, and M Backonja; UW-Madison, Madison, WI Neuropathic pain is common in clinical practice and presents a significant diagnostic challenge, especially when functional properties of small peripheral nerve fibers need to be tested. Thermal noxious stimuli which elicit burning and cooling sensations are transduced via distinct receptors TRPV1 and TRPM8, respectively, are conveyed by small fiber afferents. TRPV1 are also activated with capsaicin and TRPM8 with menthol. The objective of this study was to establish functional response properties of these receptors and associated afferents in normal healthy subjects to application of different concentrations of capsaicin and menthol. The goal is to study 60 subjects, ages of 20-80 years; ten subjects for each decade. Only subjects who are in good health, free of neurological sensory symptoms and free of pain, are included. Capsaicin 0.025% and 0.1% and menthol 2.5% and 16% as well as inert control cream are applied in random order over an area of 1 square inch for each application, in blinded (to the subject and the examiner) fashion, for duration of 10 minutes and sensations evoked by these applications are rated by subjects. In addition quantitative sensory testing at application site is tested after cream is removed. At this time 12 subjects are enrolled and preliminary results reveal: evoked sensations from the current concentrations are described as very mild; higher concentrations of capsaicin had a significant effect on perception of hot pain and there was a trend for warm stimulation. The specificity of distinct activation of receptor and associated small peripheral nerve fibers activation produced by application of capsaicin and menthol provides a unique opportunity for testing underlying abnormalities in neuropathic pain related to small peripheral fiber function and this data from normal subjects should help in interpretation of this type of information.
(231) Spatial summation of pain: a sub-additive phenomenon A Quevedo, C Mørch, O Andersen, and R Coghill; Aalborg University, Aalborg, Denmark Spatial summation is a common feature of different sensory modalities and can be defined as the increase of perceived intensity by increasing the size of stimulated area. Although nociceptive information is able to summate, spatial summation of pain (SSP) is sub-additive in that increasing the stimulus area produces a disproportionately small increase in the perceived intensity of pain. A possible explanation for sub-additive summation may be the modulation of convergent excitatory information from peripheral areas by central inhibition. Traditionally, SSP has been induced by enlarging the stimulated area, which can involve larger portions of excitatory receptive fields but may also recruit inhibitory surround areas. To test the hypothesis that surround inhibition may limit SSP, we delivered 3 different patterns of noxious thermal stimuli to the abdomens of 15 subjects using a computer-controlled CO2-laser. Lines (5mm wide) of variable lengths (4, 8, 12cm) were compared to two different controls (one-point and two-point stimuli delivered at the same position/separation as the length of lines: 4, 8, 12cm). When compared to one-point control (VAS=2.0360.3), SSP was not evoked by 4cm (VAS=1.9260.29) and 8cm (VAS=2.160.29) lines and only was observed with 12cm lines (VAS=2.3360.29) (p<0.05). At 4cm and 8cm, two-point control stimuli (VAS= 2.3160.31 and VAS=2.3760.35 respectively) produced higher pain intensity than lines (p<0.001 and p<0.05 respectively), despite the fact that the two-point stimuli activate smaller areas. Thus, the stimulation of regions between the end-points of the lines appears to produce inhibition. This suggest that the current definition of SSP is an oversimplification of the phenomenon. Instead, results from interactions between excitatory/inhibitory mechanisms. (Supported by 2008 IASP Research Grant funded by ScanDesign by Bruun Foundation.)