2+ Signaling mediated Ca 2+ cADP- -Ribose Ribose- -mediated Ca - - PowerPoint PPT Presentation

cADP cADP-

• Ribose

Ribose-

• mediated Ca

mediated Ca2+

2+ Signaling

Signaling in Morphine in Morphine-

• induced Tolerance

induced Tolerance

Mark Bradham Brewster

• Dept. of Pharmacology & Toxicology

Virginia Commonwealth University

Background: Periaqueductal Grey (PAG) Background: Periaqueductal Grey (PAG)

Efferent nociceptive trunk between brain and spinal chord Opiates acting on PAG induce analgesic effects by

inhibiting nociception

http://www.colorado.edu/epob/epob3730rlynch/image/figure12-6.jpg

Periaqueductal Grey Periaqueductal Grey

http://www.neuroanatomy.wisc.edu/virtualbrain/BrainStem/24PAG.html

Background: Known Effect of Morphine on PAG Background: Known Effect of Morphine on PAG

Morphine increases intracellular Ca2+ in PAG Increased intracellular Ca2+ invokes analgesia Analgesia caused by PAG inhibition of pain

signal transmission

Role of Role of cADPR cADPR in Morphine in Morphine-

• Induced Tolerance

Induced Tolerance

cADPR cADPR RYR RYR

(-)

ER ER Ca Ca2+

2+

(+)

Neuron firing NT Release

OP agonist OP agonist

cAMP PKA

(-)

NAD NAD

Ni Ni

C D 3 8 G G

R R (-)

Oxotremerin Depolarization

(+) Nicotinamide CD 38-/-

Ca Ca2+

2+

Analgesia Analgesia

Hypothesis Hypothesis

cADPR-mediated Ca2+ 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)

Fluorescence Intensity 0.0 0.4 0.8 1.2 1.6 2.0 40 80 120 80 (ug) PAG Kidney

Western Blot Analysis of CD38 in PAG Western Blot Analysis of CD38 in PAG

45kDa- CD38 PAG Kidney 40 80 120 ug ug ug

A B

Gene Expression Level of CD38 in PAG Gene Expression Level of CD38 in PAG

A

Cortex CD38 Relative Gene Expression Level in Mouse Cortex and PAG Brain Tissue

0.0 0.1 0.2 0.3 0.4 0.5

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

CD38 CD38

HYDROLYSIS

β β-

• NAD

NAD+

+

β β-

• NGD

NGD+

+

cGDP cGDP-

• R

R ADP ADP-

• R

R cADP cADP-

• R

R

Measurement of CD38 Cylase Activity in Measurement of CD38 Cylase Activity in Mouse PAG Membrane Mouse PAG Membrane

CYCLIZATION

GDP GDP-

• R

R

CD38 is functioning in Mouse PAG Membrane CD38 is functioning in Mouse PAG Membrane

A: Standard B: 40ug PAG

PAG Concentration (ug/uL) 0ug 20ug 40ug 60ug Conversion Rate (nmol/min) 10 20 30 40

C

cGDPR cGDPR NGD+

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

CTRL MOR NXN Conversion Rate (nmol/mg) 200 400 600 800

C A B

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)