Enhanced Expression And Activity of NAD(P)H Oxidase in Mouse - PowerPoint PPT Presentation

Enhanced Expression And Activity of NAD(P)H Oxidase in Mouse Periaqueductal Gray Tissue During Morphine Antinociceptive Tolerance Department of Pharmacology & Toxicology Virginia Commonwealth University Emily C. Wright

Background: Periaqueductal Gray (PAG) � Area surrounding cerebral aqueduct in brain stem levels 9 and 10 � Contains receptors for opiate peptides which can eliminate the perception of pain

Background: Known Effect of Morphine on PAG � Pain reduction takes place when opiates turn on inhibitory neurons in PAG � Antinociceptive tolerance may result from perpetual action of opiates on PAG � Morphine causes increase in intracellular [Ca+] in the PAG in chronic morphine treatment (CMT) mice

Role of NAD(P)H Oxidase in Morphine Induced Tolerance 2O 2O ? ? 2 2 extracellular extracellular + + NO NO H H e e O 2 O 2 ._ ._ Morphine Morphine ONOO - ONOO - ( ( ( ( - - - - ) ) R R P22 P22 P22 P22 membrane membrane Rac Rac + + ? ? gP91 gP91 P47 P47 P67 P67 P67 P67 cytoplasm cytoplasm + + H H + + H H + + NAD(P)H NAD(P)H NA NAD(P) NA NAD(P) analgesia analgesia

Question � Is NAD(P)H oxidase (subunits p47 and NOX-2) present in the PAG? -Approach: Immunohistochemistry ( process used to localize proteins in cells of tissue sections )

Hypothesis � NAD(P)H oxidase plays an important role in morphine- induced tolerance.

Western Blot Analysis of the NOX-2 subunit of NAD(P)H Oxidase in PAG 58kDa gp91 phox β -actin 45kDa M V M V PAG Cortex 3.0 Vehicle Morphine 2.4 gp91 phox protein expression 1.8 (Ratio to β− action) 1.2 0.6 0.0 PAG Cortex

Western Blot Analysis of the p47 subunit of NAD(P)H Oxidase in PAG p47 phox 47kDa β -actin 45kDa M V V M Cortex PAG 3.0 Vehicle p47 phox protein expression Morphine 2.4 (Ratio to β − actin) 1.8 1.2 0.6 0.0 Cortex PAG

Gene Expression Level of the NOX-2 subunit of NAD(P)H Oxidase in PAG 5 Vehicle Morphine 4 3 Expression of gp91 phox mRNA (T n ) 2 1 0 Cortex PAG

Gene Expression Level of the p47 subunit of NAD(P)H Oxidase in PAG 25 Vehicle Morphine 20 15 Expression of p47phox mRNA (Tn) 10 5 0 Cortex PAG

Protocol � 3 groups of mice: naïve, placebo pellet, and morphine pellet (morphine tolerant) � Performed a two-day immunohisto- chemistry protocol that included over-night incubation with the primary antibody � Qualitatively analyzed results by taking pictures of images obtained by microscope

Results A B PAG Cortex PAG Cortex Negative Control Negative Control p47 Expression p47 Expression Figure 1: Expression of the p47 antigen in the periaqueductal gray and cortex of placebo pellet mouse brain tissue. A) 400X magnification. B) 1000X magnification.

Results A B PAG Cortex PAG Cortex Negative Control Negative Control NOX-2 Expression NOX-2 Expression Figure 2: Expression of the NOX-2 antigen in the periaqueductal gray and cortex of placebo pellet mouse brain tissue. A) 400X magnification. B) 1000X magnification.

Conclusion � NAD(P)H oxidase is present in the PAG of mice brain tissue

Future Direction � Perform ESR to detect the levels of superoxide in the PAG � Perform HPLC to assess the functioning of NAD(P)H Oxidase in the PAG

Results A B Medulla Cortex Cortex Medulla Negative Control Negative Control NOX-1 Expression NOX-1 Expression Figure 3: Expression of the NOX-1 antigen in the cortex and medulla of rat kidney tissue. A) 400X magnification. B) 1000X magnification

Results A B Cortex Medulla Cortex Medulla Negative Control Negative Control NOX-1 Expression NOX-1 Expression Figure 4: Expression of the NOX-1 antigen in the cortex and medulla of mouse kidney tissue. A) 400X magnification. B) 1000X magnification

Results A B Cortex Medulla Cortex Medulla Negative Control Negative Control NOX-2 Expression NOX-2 Expression Figure 5: Expression of the NOX-2 antigen in the cortex and medulla of rat kidney tissue. A) 400X magnification. B) 1000X magnification

Results A B Cortex Medulla Cortex Medulla Negative Control Negative Control NOX-2 Expression NOX-2 Expression Figure 6: Expression of the NOX-2 antigen in the cortex and medulla of mouse kidney tissue. A) 400X magnification. B) 1000X magnification

Results A B Cortex Medulla Cortex Medulla Negative Control Negative Control NOX-3 Expression NOX-3 Expression Figure 7: Expression of the NOX-3 antigen in the cortex and medulla of rat kidney tissue. A) 400X magnification. B) 1000X magnification

Results A B Cortex Medulla Cortex Medulla Negative Control Negative Control NOX-3 Expression NOX-3 Expression Figure 8: Expression of the NOX-3 antigen in the cortex and medulla of mouse kidney tissue. A) 400X magnification. B) 1000X magnification

Results A B Cortex Medulla Cortex Medulla Negative Control Negative Control NOX-4 Expression NOX-4 Expression Figure 9: Expression of the NOX-4 antigen in the cortex and medulla of rat kidney tissue. A) 400X magnification. B) 1000X magnification

Results A B Cortex Medulla Cortex Medulla Negative Control Negative Control NOX-4 Expression NOX-4 Expression Figure 10: Expression of the NOX-4 antigen in the cortex and medulla of mouse kidney tissue. A) 400X magnification. B) 1000X magnification

Conclusion � There are some differences between rat and mouse kidney tissue in their expression of the NOX isoforms

Future Direction � Positive controls for NOX-3 and NOX-4 antigens in mice and rat kidney tissue

Acknowledgements � Dr. Pin-Lan Li, M.D., Ph.D. � Dr. William Dewey, Ph.D. � Labs of Dr. Li and Dr. Dewey � Program for Summer Research Experience of Undergraduates in Pharmacology & Toxicology

Bibliography Bagley, E. E., et al. Opioid tolerance in periaqueductal gray neurons � isolated from mice chronically treated with morphine. (2005). Li, C., et al. Enhanced Expression and Activity of NAD(P)H Oxidase in � Mouse Periaqueductal Gray Neurons During Morphine Antinociceptive Tolerance. (2005). Periaqueductal Gray. � http://www.neuroanatomy.wisc.edu/virtualbrain/BrainStem/24PAG.html. (2006). The Mouse Brain Library. http://www.mbl.org. (2005). �