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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 10Contains 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 PAGAntinociceptive tolerance may result from perpetual action of opiates on PAGMorphine causes increase in intracellular [Ca+] in the PAG in chronic morphine treatment (CMT) mice
Role of NAD(P)H Oxidase in Morphine Induced Tolerance
NAD(P)H NAD(P)
H+
H
2O2
eextracellular
cytoplasm
membrane P22
P67 P47
Morphine(-)
NA +
H+
+
P22
P67
NO
(-O2
._
ONOO -
Rac
gP91
?
?R
+
analgesia NAD(P)H NAD(P)
H+
H
2O2
eextracellular
cytoplasm
membrane P22
P67 P47
Morphine(-)
NA +
H+
+
P22
P67
NO
(-O2
._
ONOO -
Rac
gP91
?
?R
+
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
β -actin
gp91phox
M V M VPAG Cortex
58kDa
45kDa
PAG Cortex0.0
0.6
1.2
1.8
2.4
3.0 VehicleMorphine
gp91phox proteinexpression
(Ratio to β−action)
Western Blot Analysis of the p47 subunit of NAD(P)H Oxidase in PAG
47kDa
45kDaM MV V
PAG Cortex
p47phox
β-actin
0.0
0.6
1.2
1.8
2.4
3.0 VehicleMorphine
PAG Cortex
p47ph
oxpr
o tei
n e
xpre
ssio
n(R
a tio
to β
−ac
t in)
Gene Expression Level of the NOX-2 subunit of NAD(P)H Oxidase in PAG
PAG0
1
2
3
4
5 VehicleMorphine
Cortex
Expression of gp91phox mRNA
(Tn )
Gene Expression Level of the p47 subunit of NAD(P)H Oxidase in PAG
PAG0
5
10
15
20
25VehicleMorphine
Cortex
Expression of p47phox mRNA
(Tn)
Protocol3 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 antibodyQualitatively analyzed results by taking pictures of images obtained by microscope
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
PAG Cortex
p47
Expr
essi
on
A
Neg
ativ
e C
ontro
l
B
p47
Expr
essi
onN
egat
ive
Con
trol PAG Cortex
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.
PAG Cortex
NO
X-2
Exp
ress
ion
A
Neg
ativ
e C
ontro
l
B
Neg
ativ
e C
ontro
l PAG Cortex
NO
X-2
Exp
ress
ion
Results
ConclusionNAD(P)H oxidase is present in the PAG of mice brain tissue
Future DirectionPerform ESR to detect the levels of superoxide in the PAGPerform HPLC to assess the functioning of NAD(P)H Oxidase in the PAG
Figure 3: Expression of the NOX-1 antigen in the cortex and medulla of rat kidney tissue. A) 400X magnification. B) 1000X magnification
Results
Cortex Medulla
NO
X-1
Exp
ress
ion
A
Neg
ativ
e C
ontro
l
B
Neg
ativ
e C
ontro
l Cortex Medulla
NO
X-1
Exp
ress
ion
Figure 4: Expression of the NOX-1 antigen in the cortex and medulla of mouse kidney tissue. A) 400X magnification. B) 1000X magnification
Results
B
Neg
ativ
e C
ontro
l Cortex Medulla
NO
X-1
Exp
ress
ion
Cortex Medulla
NO
X-1
Exp
ress
ion
A
Neg
ativ
e C
ontro
l
Figure 5: Expression of the NOX-2 antigen in the cortex and medulla of rat kidney tissue. A) 400X magnification. B) 1000X magnification
Results
Cortex Medulla
NO
X-2
Exp
ress
ion
A
Neg
ativ
e C
ontro
l
B
Neg
ativ
e C
ontro
l Cortex Medulla
NO
X-2
Exp
ress
ion
Figure 6: Expression of the NOX-2 antigen in the cortex and medulla of mouse kidney tissue. A) 400X magnification. B) 1000X magnification
Results
B
Neg
ativ
e C
ontro
l Cortex Medulla
NO
X-2
Exp
ress
ion
Cortex Medulla
NO
X-2
Exp
ress
ion
A
Neg
ativ
e C
ontro
l
Figure 7: Expression of the NOX-3 antigen in the cortex and medulla of rat kidney tissue. A) 400X magnification. B) 1000X magnification
Results
Cortex Medulla
NO
X-3
Exp
ress
ion
A
Neg
ativ
e C
ontro
l
B
Neg
ativ
e C
ontro
l Cortex Medulla
NO
X-3
Exp
ress
ion
Figure 8: Expression of the NOX-3 antigen in the cortex and medulla of mouse kidney tissue. A) 400X magnification. B) 1000X magnification
B
Neg
ativ
e C
ontro
l Cortex Medulla
NO
X-3
Exp
ress
ion
Cortex Medulla
NO
X-3
Exp
ress
ion
A
Neg
ativ
e C
ontro
l
Results
Figure 9: Expression of the NOX-4 antigen in the cortex and medulla of rat kidney tissue. A) 400X magnification. B) 1000X magnification
Results
Cortex Medulla
NO
X-4
Exp
ress
ion
A
Neg
ativ
e C
ontro
l
B
Neg
ativ
e C
ontro
l Cortex Medulla
NO
X-4
Exp
ress
ion
Figure 10: Expression of the NOX-4 antigen in the cortex and medulla of mouse kidney tissue. A) 400X magnification. B) 1000X magnification
Results
B
Neg
ativ
e C
ontro
l Cortex Medulla
NO
X-4
Exp
ress
ion
Cortex Medulla
NO
X-4
Exp
ress
ion
A
Neg
ativ
e C
ontro
l
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. DeweyProgram for Summer Research Experience of Undergraduates in Pharmacology & Toxicology
BibliographyBagley, 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 AntinociceptiveTolerance. (2005).Periaqueductal Gray. http://www.neuroanatomy.wisc.edu/virtualbrain/BrainStem/24PAG.html. (2006).The Mouse Brain Library. http://www.mbl.org. (2005).