Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

Role of Receptors and Ion Channels inNeuropathic PainManju Bhat, Ph.D.THE BHATLABORATORYRESEARCH FELLOWSSeok Kon Kim, M.D.Hongyu Zhang, M.D.RESEARCH TECHNOLOGISTKristen Yankura, B.S.COLLABORATORSLiliana Berti-Mattera, Ph.D. 1Derek S. Damron, Ph.D. 2Salim M. Hayek, M.D., Ph.D. 3Anoopa Kumar, Ph.D. 1Minh Lam, Ph.D. 1Ram Nagaraj, Ph.D. 1Anna-Liisa Nieminen, Ph.D. 1Andrea Romani, M.D., Ph.D. 1Basil D. Roufogalis, Ph.D. 4Hiroshi Takeshima, Ph.D. 51Case Western Reserve Univ.,Cleveland, OH2Ctr. for Anesthesiology Res.,CCF3Dept. of Pain Management,CCF4Tohoku Univ., Sendai, Japan5Univ. of Sydney, AustraliaAccording to the World Health Organization,90% of all illnesses are associatedwith pain, and patients with chronic painuse health services up to five times as frequentlyas the rest of the population. Treating symptomaticpain is challenging because of its subjectivenature, yet adverse side effects of existing painmedications demand further research to developnew protocols for treating different types of pain.Neuropathic conditions, including peripheralneuropathy, are the most commonly encounteredgroup of chronic complications associated withdiabetes. The actual prevalence of diabeticneuropathy is not known, but 60-70% of diabeticpatients develop neuropathy at some stage. It isestimated that by 2010, over 220 million peopleworldwide will suffer from these diseases. It isimportant to understand the underlying pathophysiologyto identify novel therapeutics forneuropathic and other related pain. Significantprogress has recently been made in understandingthe neurobiology of pain, with the identificationof new receptors, ion channels and signalingmolecules that may serve as potential therapeutictargets. However, the molecular mechanism(s)of the function and regulation of these cellulartargets are not clearly understood.Our research focus is to understand thefunction and regulation of cell surface receptorsinvolved in nociception, such as opioid andopioid- like receptors (m, d, k and nociceptin/orphanin FQ) and capsaicin receptors and toelucidate the pathophysiologic role of calcium(Ca 2+ ) as an intracellular second messenger in thetransduction and modulation of nociceptivesignals. Considerable evidence exists for theinvolvement of Ca 2+ in the transmission ofnociceptive signals within the peripheral andcentral nervous systems. For example, agents thatincrease the concentration of intracellular Ca 2+decrease the analgesic potency of opioid drugs,whereas calcium chelators and calcium channelantagonists potentiate opioid-induced antinociception.Furthermore, altered intracellularCa 2+ homeostasis is thought to be one of thereasons for decreased anti-nociceptive response toopioid receptor agonists in diabetic neuropathy.We are investigating sensory neuronal Ca 2+signaling mechanism(s) and the role of Ca 2+ in thepathophysiology of diabetic neuropathy. Insensory neurons from dorsal root ganglia (DRG),activation of Ca 2+ release from the endoplasmicreticulum via ryanodine receptors (RyR) triggersCa 2+ influx through the plasma membrane via amechanism called capacitative Ca 2+ entry (CCE).Current research efforts are aimed at characterizingthe molecular properties and regulation ofRyR and proteins involved in CCE pathway.Preliminary results indicate that this functionalinteraction between different Ca 2+ transportpathways is altered in diabetes. The role of Ca 2+and Ca 2+ channels in the function and regulationof nociceptive receptors is also being investigated.Molecular and cellular techniques are beingused to investigate the signal transductionmechanism(s) involved in such disease models asneuropathic pain, including diabetic neuropathy.The function and regulation of receptors and ionchannels involved in nociception are beingstudied using both freshly isolated DRG neuronsand immortalized sensory neurons in culture.Ion-selective fluorescent dyes and confocalmicroscopy are used to examine the changes inintracellular ionic fluxes. Electrophysiologicaltechniques (patch-clamp and planar lipid bilayerreconstitution) will be used to examine singleion-channelfunction and its regulation byreceptors and other modulators. Our long-termgoal is to pursue the molecular findings in animalmodels and eventually patients, with the hope ofidentifying potential therapeutic targets that mayhelp in developing “mechanism based” treatmentparadigms.Chipuk, J.E., Bhat, M., Hsing, A.Y., Ma, J., and D. Danielpour (2001) Bcl-xL blocks transforming growthfactor-beta 1-induced apoptosis by inhibiting cytochrome c release and not by directly antagonizingApaf-1-dependent caspase activation in prostate epithelial cells. J. Biol. Chem. 276:26614-26621.Bhat, M.B., and J. Ma (2002) The transmembrane segment of ryanodine receptor contains an intracellularmembrane retention signal for Ca 2+ release channel. J. Biol. Chem. 277:8597-8601.Paul-Pletzer, K., Yamamoto, T., Bhat, M.B., Ma, J., Ikemoto, N., Jimenez, L.S., Morimoto, H., Williams,P.G., and J. Parness (2002) Identification of a dantrolene-binding sequence on the skeletal muscle ryanodinereceptor. J. Biol. Chem. 277:34918-34923.Shin, D.W., Pan, Z., Bandyopadhyay, A., Bhat, M.B., Kim, D.H., and J. Ma (2002) Ca 2+ -dependent interactionbetween FKBP12 and calcineurin regulates activity of the Ca 2+ release channel in skeletal muscle.Biophys. J. 83:2539-49.Shin, D.W., Pan, Z., Kim, E.K., Lee, J.M., Bhat, M.B., Parness, J., Kim do H, and J. Ma (2003) A retrogradesignal from calsequestrin for the regulation of store-operated Ca 2+ entry in skeletal muscle. J.Biol. Chem. 278:3286-3292.154