2+ Signaling mediated Ca 2+ cADP- -Ribose Ribose- -mediated Ca Signaling cADP in Morphine- -induced Tolerance induced Tolerance in Morphine Mark Bradham Brewster Dept. of Pharmacology & Toxicology Virginia Commonwealth University
Background: Periaqueductal Grey (PAG) Background: Periaqueductal Grey (PAG) Periaqueductal Grey Periaqueductal Grey http://www.colorado.edu/epob/epob3730rlynch/image/figure12-6.jpg http://www.neuroanatomy.wisc.edu/virtualbrain/BrainStem/24PAG.html � Efferent nociceptive trunk between brain and spinal chord � Opiates acting on PAG induce analgesic effects by inhibiting nociception
Background: Known Effect of Morphine on PAG Background: Known Effect of Morphine on PAG � Morphine increases intracellular Ca 2+ in PAG � Increased intracellular Ca 2+ invokes analgesia � Analgesia caused by PAG inhibition of pain signal transmission
Role of cADPR cADPR in Morphine in Morphine- -Induced Tolerance Induced Tolerance Role of Oxotremerin Depolarization Nicotinamide CD 38-/- (+) OP agonist OP agonist (-) cAMP R R cADPR cADPR G G PKA C D 3 8 (-) Ni Ni NAD NAD (+) Neuron firing NT Release Analgesia Analgesia Ca 2+ 2+ Ca RYR RYR (-) Ca 2+ 2+ Ca ER ER
Hypothesis Hypothesis � cADPR-mediated Ca 2+ signaling plays an integral role in morphine-induced tolerance
Questions Questions � Is CD38 present in PAG? (WB, RT-PCR) � Is CD38 functioning in PAG? (HPLC)
Western Blot Analysis of CD38 in PAG Western Blot Analysis of CD38 in PAG A PAG Kidney 45kDa- CD38 40 ug 80 ug 120 ug B 2.0 1.6 Fluorescence Intensity 1.2 0.8 0.4 0.0 80 (ug) 40 80 120 Kidney PAG
Gene Expression Level of CD38 in PAG Gene Expression Level of CD38 in PAG A in Mouse Cortex and PAG Brain Tissue CD38 Relative Gene Expression Level 0.5 0.4 0.3 0.2 0.1 0.0 Cortex
Measurement of CD38 Cylase Activity in Measurement of CD38 Cylase Activity in Mouse PAG Membrane Mouse PAG Membrane � CD38 is bifunctional enzyme as cylase and hydrolase � Post-cyclization cADPR is further reduced to ADP-ribose � cGDPR production is used to calculate cyclase activity β - β + -NAD NAD + cADP- -R R ADP- -R R cADP ADP CYCLIZATION HYDROLYSIS CD38 CD38 β - β GDP- -R R GDP + -NGD NGD + cGDP- -R R cGDP
CD38 is functioning in Mouse PAG Membrane CD38 is functioning in Mouse PAG Membrane A: Standard cGDPR C 40 Conversion Rate 30 (nmol/min) B: 40ug PAG 20 10 cGDPR 0 0ug 20ug 40ug 60ug NGD+ PAG Concentration (ug/uL)
Protocol: Protocol: � 3 groups of mice: Untreated, Morphine treated, Naloxone treated (n=5 in each) � PAG homogenates were incubated with 1mM β -NGD+ for 2 hours at 37C � Measured cGDPR production by HPLC analysis.
Effect of Morphine on CD38 Activity in Effect of Morphine on CD38 Activity in Mouse PAG Mouse PAG A C 800 Conversion Rate 600 (nmol/mg) 400 B 200 0 CTRL MOR NXN
Summary Summary � CD38 present in PAG � Higher levels of CD38 present in cortex than PAG, but higher CD38 activity in PAG than cortex � CD38 activity in PAG when incubated with NGD � Morphine increases enzymatic activity of CD38 in PAG
Future Direction Future Direction � 1. Ca2+ Assay: Study effects of different pharmcological interventions on morphine-induced Ca2+ increase � 2. Study the effects of these inhibitors on morphine tolerence
Acknowledgements Acknowledgements � Pin-Lan Li, M.D. Ph.D. � Andy Y Zhang � Fan Zhang, MD, PhD � Li Chen � Thomas Lee � William L Dewey, PhD � Labs of Dr. Li and Dr. Dewey � Summer Research Program for Undergraduate Students
Bibliography Bibliography � De Flora, A., et al. Autocrine and Paracrine Calcium Signaling by the CD38/NAD + /Cyclic ADP-Ribose System. Ann. N.Y. Acad. Sci. 1028 : 176-191(2004) � Ceni, C., et al. CD38-dependent ADP-ribosyl cyclase activity in developing and adult mouse brain. Biochem. J. 370 : 175-183(2003) � Verderio, C., et al. Evidence of a role for cyclic ADP-ribose in calcium signalling and neurotransmitter release in cultured astrocytes. 78 : 646- 657(2001)
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