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(285) Phosphatidylethanolamine-binding protein regulates Mu opioid receptor induced βarrestin2 recruitment and downstream signaling
Abstracts
The Journal of Pain
S47
haplotype from block 2 was associated with a 5.94 decrease in CPI (p=0.001). Data from this study strongly implicated significant influences of ADRB2 polymorphisms and haplotypes on chronic pain in SCD.
induced barr2 recruitment and attendant opioid induced side effects. We will continue to study the role of PEBP in pain and opioid response in vivo, and are developing in vitro drug discovery screens with PEBP as a target.
D06 Intracellular Signal Transduction
D07 Ion Channels - Sodium, Potassium, Calcium, Chloride
(284) Persistent electrical activity in primary nociceptors after spinal cord injury is maintained by scaffolded adenylyl cyclase and protein kinase A and is associated with altered adenylyl cyclase regulation A Bavencoffe, Y Li, Z Wu, Q Yang, J Herrera, E Kennedy, E Walters, and C Dessauer; University of Texas Medical School at Houston, Houston, TX Little is known about intracellular signaling mechanisms that persistently excite neurons in pain pathways. We found recently that persistent spontaneous activity (SA) generated in the cell bodies of primary nociceptors within dorsal root ganglia (DRG) makes major contributions to chronic reflex hypersensitivity and to spontaneous pain in a rat model of thoracic spinal cord injury (SCI). The occurrence of SCI-induced SA in a large fraction of DRG neurons at and below the injury level for months after SCI, and the persistence of this SA long after dissociation of the neurons provide a special opportunity to define intrinsic cell signaling mechanisms that chronically drive SA at the first stage in pain pathways. We have found that SCI-induced SA generated in small dissociated DRG neurons requires continuing activity of adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA), as well as a scaffolded complex containing AC5/6, A-kinase anchoring protein 150 (AKAP150), and PKA. SCI caused a small but significant increase in the expression of AKAP150 but not other AKAPs. DRG membranes isolated from SCI animals revealed a novel alteration in the regulation of adenylyl cyclase (AC) activity. Membrane AC activity stimulated by added Ca2+-calmodulin increased modestly, while the inhibition of G(alpha)s-stimulated AC activity by G(alpha)i showed an unexpected and dramatic decrease after SCI. Localized enhancement of the activity of AC within scaffolded complexes containing PKA is likely to contribute to chronic pathophysiological consequences of SCI, including pain, that are promoted by persistent hyperactivity in DRG neurons.
(285) Phosphatidylethanolamine-binding protein regulates Mu opioid receptor induced barrestin2 recruitment and downstream signaling K Edwards, J Bogusz, J LaVigne, K Pangilinan, and J Streicher; University of Arizona, Tucson, AZ
Opioid drugs are one of the few efficacious options available for moderate to severe chronic pain, but are limited by serious side effects. Recent efforts have focused on developing functionally selective ligands for the mu opioid receptor (MOR) which will activate desired pathways or not activate deleterious pathways in order to decrease side effects. However, few targets are available for this approach. To find new targets for functionally selective drug discovery, we analyzed proteomics databases available in the literature to find candidate proteins that are regulated in the brain by chronic opioid treatment. One such candidate, phosphatidylethanolamine-binding protein (PEBP), is a cytoplasmic protein that has been shown to regulate GPCR signaling through both the Raf/MEK/ERK pathway and G protein-coupled receptor kinase 2 (GRK2), a major feedback inhibitor of GPCRs which has been implicated in a number of neurological disorders including chronic pain. The expression of PEBP was also found to be regulated by chronic morphine treatment in a proteomic study of rodent brain, suggesting that PEBP may regulate MOR signaling, however PEBP has not yet been associated with MOR signaling. We have knocked down PEBP in MOR- expressing HEK, U2OS, N2a and SH-SY5Y cells using specific siRNA to determine changes in either MOR-induced ERK signaling or barrestin2 (barr2) recruitment and receptor internalization. PEBP knockdown does not change MOR agonist induced ERK signaling efficacy or potency, but barr2 recruitment, MOR internalization, and chronic morphine desensitization potency is increased. The barr2 recruitment potency finding was confirmed using the PEBP small molecular inhibitor locostatin. This suggests a role for PEBP in regulating MOR
(286) Human tongue squamous carcinoma sensitizes tongue primary afferents in male and female mice N Scheff, J McRae, Z Conley, and B Schmidt; New York University, New York, NY
Tongue cancer patients report severe mechanically-induced pain. Tongue cancer pain is hypothesized to be due to tumor proliferation, perineural invasion, and/or secretion of pro-nociceptive mediators into the cancer microenvironment. Recent evidence shows that cytokines and chemokines produced by tongue cancer, including ET-1, NGF, ATP, and proteases, can sensitize and activate primary afferent neurons innervating the cancer microenvironment and cause pain. To elucidate the underlying mechanism that may contribute to tongue cancer pain, electrophysiology parameters of acutely dissociated retrogradely labeled tongue afferents from C57Bl/6 mice were analyzed with whole cell patch clamp recordings. Passive and active electrophysiological properties were measured from both male and female mice at baseline and following application of supernatant from the following cell lines: tongue squamous carcinoma cell line (HSC3), normal oral keratinocyte cell line (NoK), and malignant melanoma cell line (SKMEL28), which is known to be non-painful in patients. HSC3 supernatant, but not supernatant from NoK or SKMEL28, resulted in depolarization of the resting membrane potential, a decrease in input resistance, and produced an increase in excitability as measured by a significant hyperpolarization of the action potential (AP) threshold and decrease in rheobase. We also used ramp-current injections to monitor changes in current threshold, voltage threshold, and accommodation. These data suggest that mediators released from the tongue cancer cells alone, without the impact of tumor growth and perineural involvement can directly sensitize the tongue primary afferents and result in pain. Supernatant from the benign counterpart of HSC3 (i.e., NoK) and malignant melanoma did not result in an increase in neuronal excitability, consistent with the lack of reported pain in normal oral mucosa and malignant melanoma. Studies are underway to identify the components of HSC3 supernatant responsible for generating the sensitivity. This work is supported by the IASP 2014 John J. Bonica Trainee Fellowship.
(287) Anoctamin-1 Cl- channel in nociception: inhibition by T16 [inh]-A01 and activation by an N-aroylaminothiazole and capsaicin B Bessac and F Deba; Texas A&M Health Science Center Rangel College of Pharmacy, Kingsville, TX
Anoctamin 1 (ANO1 or TMEM16A) Ca2+ gated Cl channels of nociceptor sensory neurons are emerging as important molecular components of peripheral pain transduction. At physiological intracellular Cl concentrations ([Cl ]i) sensory neuronal Cl channels, such as ANO1, are excitatory. ANO1 channels are directly activated by an N-aroylaminothiazole (E-act). At physiological [Cl ]i (40mM), E-act (10mM) direct activation of ANO1 increased current sizes (in voltage-clamp) and action potential firing (in currentclamp) recorded using whole-cell electrophysiology of mouse dorsal root ganglia (DRG) sensory neurons. Nocifensive responses of mice hind paws were also dramatically induced by subcutaneous injections of E-act (5mM), which was attenuated by co-injection of ANO1-inhibitor T16A[inh]-A01 (1.3mM). ANO1 channels are also activated by intracellular Ca2+ ([Ca2+]i) from sensory neuronal TRPV1 (transient-receptor-potential vallinoid 1) ion channels and other noxicant receptors. At physiological [Cl ]i, capsaicin (15mM)-induced action potential firing was diminished by co-application with ANO1-inhibitor T16A[inh]-A01 (20mM) To not disrupt TRPV1 carried-Ca2+ activation of ANO1 in DRG neurons. (To not disrupt TRPV1 carried-Ca2+ activation of ANO1 in DRG neurons, ANO1 modulation of capsaicin-induced action potentials was
The Journal of Pain
S47
haplotype from block 2 was associated with a 5.94 decrease in CPI (p=0.001). Data from this study strongly implicated significant influences of ADRB2 polymorphisms and haplotypes on chronic pain in SCD.
induced barr2 recruitment and attendant opioid induced side effects. We will continue to study the role of PEBP in pain and opioid response in vivo, and are developing in vitro drug discovery screens with PEBP as a target.
D06 Intracellular Signal Transduction
D07 Ion Channels - Sodium, Potassium, Calcium, Chloride
(284) Persistent electrical activity in primary nociceptors after spinal cord injury is maintained by scaffolded adenylyl cyclase and protein kinase A and is associated with altered adenylyl cyclase regulation A Bavencoffe, Y Li, Z Wu, Q Yang, J Herrera, E Kennedy, E Walters, and C Dessauer; University of Texas Medical School at Houston, Houston, TX Little is known about intracellular signaling mechanisms that persistently excite neurons in pain pathways. We found recently that persistent spontaneous activity (SA) generated in the cell bodies of primary nociceptors within dorsal root ganglia (DRG) makes major contributions to chronic reflex hypersensitivity and to spontaneous pain in a rat model of thoracic spinal cord injury (SCI). The occurrence of SCI-induced SA in a large fraction of DRG neurons at and below the injury level for months after SCI, and the persistence of this SA long after dissociation of the neurons provide a special opportunity to define intrinsic cell signaling mechanisms that chronically drive SA at the first stage in pain pathways. We have found that SCI-induced SA generated in small dissociated DRG neurons requires continuing activity of adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA), as well as a scaffolded complex containing AC5/6, A-kinase anchoring protein 150 (AKAP150), and PKA. SCI caused a small but significant increase in the expression of AKAP150 but not other AKAPs. DRG membranes isolated from SCI animals revealed a novel alteration in the regulation of adenylyl cyclase (AC) activity. Membrane AC activity stimulated by added Ca2+-calmodulin increased modestly, while the inhibition of G(alpha)s-stimulated AC activity by G(alpha)i showed an unexpected and dramatic decrease after SCI. Localized enhancement of the activity of AC within scaffolded complexes containing PKA is likely to contribute to chronic pathophysiological consequences of SCI, including pain, that are promoted by persistent hyperactivity in DRG neurons.
(285) Phosphatidylethanolamine-binding protein regulates Mu opioid receptor induced barrestin2 recruitment and downstream signaling K Edwards, J Bogusz, J LaVigne, K Pangilinan, and J Streicher; University of Arizona, Tucson, AZ
Opioid drugs are one of the few efficacious options available for moderate to severe chronic pain, but are limited by serious side effects. Recent efforts have focused on developing functionally selective ligands for the mu opioid receptor (MOR) which will activate desired pathways or not activate deleterious pathways in order to decrease side effects. However, few targets are available for this approach. To find new targets for functionally selective drug discovery, we analyzed proteomics databases available in the literature to find candidate proteins that are regulated in the brain by chronic opioid treatment. One such candidate, phosphatidylethanolamine-binding protein (PEBP), is a cytoplasmic protein that has been shown to regulate GPCR signaling through both the Raf/MEK/ERK pathway and G protein-coupled receptor kinase 2 (GRK2), a major feedback inhibitor of GPCRs which has been implicated in a number of neurological disorders including chronic pain. The expression of PEBP was also found to be regulated by chronic morphine treatment in a proteomic study of rodent brain, suggesting that PEBP may regulate MOR signaling, however PEBP has not yet been associated with MOR signaling. We have knocked down PEBP in MOR- expressing HEK, U2OS, N2a and SH-SY5Y cells using specific siRNA to determine changes in either MOR-induced ERK signaling or barrestin2 (barr2) recruitment and receptor internalization. PEBP knockdown does not change MOR agonist induced ERK signaling efficacy or potency, but barr2 recruitment, MOR internalization, and chronic morphine desensitization potency is increased. The barr2 recruitment potency finding was confirmed using the PEBP small molecular inhibitor locostatin. This suggests a role for PEBP in regulating MOR
(286) Human tongue squamous carcinoma sensitizes tongue primary afferents in male and female mice N Scheff, J McRae, Z Conley, and B Schmidt; New York University, New York, NY
Tongue cancer patients report severe mechanically-induced pain. Tongue cancer pain is hypothesized to be due to tumor proliferation, perineural invasion, and/or secretion of pro-nociceptive mediators into the cancer microenvironment. Recent evidence shows that cytokines and chemokines produced by tongue cancer, including ET-1, NGF, ATP, and proteases, can sensitize and activate primary afferent neurons innervating the cancer microenvironment and cause pain. To elucidate the underlying mechanism that may contribute to tongue cancer pain, electrophysiology parameters of acutely dissociated retrogradely labeled tongue afferents from C57Bl/6 mice were analyzed with whole cell patch clamp recordings. Passive and active electrophysiological properties were measured from both male and female mice at baseline and following application of supernatant from the following cell lines: tongue squamous carcinoma cell line (HSC3), normal oral keratinocyte cell line (NoK), and malignant melanoma cell line (SKMEL28), which is known to be non-painful in patients. HSC3 supernatant, but not supernatant from NoK or SKMEL28, resulted in depolarization of the resting membrane potential, a decrease in input resistance, and produced an increase in excitability as measured by a significant hyperpolarization of the action potential (AP) threshold and decrease in rheobase. We also used ramp-current injections to monitor changes in current threshold, voltage threshold, and accommodation. These data suggest that mediators released from the tongue cancer cells alone, without the impact of tumor growth and perineural involvement can directly sensitize the tongue primary afferents and result in pain. Supernatant from the benign counterpart of HSC3 (i.e., NoK) and malignant melanoma did not result in an increase in neuronal excitability, consistent with the lack of reported pain in normal oral mucosa and malignant melanoma. Studies are underway to identify the components of HSC3 supernatant responsible for generating the sensitivity. This work is supported by the IASP 2014 John J. Bonica Trainee Fellowship.
(287) Anoctamin-1 Cl- channel in nociception: inhibition by T16 [inh]-A01 and activation by an N-aroylaminothiazole and capsaicin B Bessac and F Deba; Texas A&M Health Science Center Rangel College of Pharmacy, Kingsville, TX
Anoctamin 1 (ANO1 or TMEM16A) Ca2+ gated Cl channels of nociceptor sensory neurons are emerging as important molecular components of peripheral pain transduction. At physiological intracellular Cl concentrations ([Cl ]i) sensory neuronal Cl channels, such as ANO1, are excitatory. ANO1 channels are directly activated by an N-aroylaminothiazole (E-act). At physiological [Cl ]i (40mM), E-act (10mM) direct activation of ANO1 increased current sizes (in voltage-clamp) and action potential firing (in currentclamp) recorded using whole-cell electrophysiology of mouse dorsal root ganglia (DRG) sensory neurons. Nocifensive responses of mice hind paws were also dramatically induced by subcutaneous injections of E-act (5mM), which was attenuated by co-injection of ANO1-inhibitor T16A[inh]-A01 (1.3mM). ANO1 channels are also activated by intracellular Ca2+ ([Ca2+]i) from sensory neuronal TRPV1 (transient-receptor-potential vallinoid 1) ion channels and other noxicant receptors. At physiological [Cl ]i, capsaicin (15mM)-induced action potential firing was diminished by co-application with ANO1-inhibitor T16A[inh]-A01 (20mM) To not disrupt TRPV1 carried-Ca2+ activation of ANO1 in DRG neurons. (To not disrupt TRPV1 carried-Ca2+ activation of ANO1 in DRG neurons, ANO1 modulation of capsaicin-induced action potentials was