[PPT] - Internalization, Dimerization, and Activation of CD38 during mNOX PowerPoint Presentation

SLIDE 1

Internalization, Dimerization, and Activation

f CD38 during mNOX Activation:

O2

and Ca2+ Signaling in Coronary Arterial Smooth Muscle

Molly Hilliker Department of Pharmacology & Toxicology Virginia Commonwealth University Medical College of Virginia Campus

SLIDE 2

Redox Signaling under Physiological Conditions

Redox-mediated signaling is emerging as a fundamental regulatory mechanism

in cell biology.

Many cellular proteins, such as transcription factors, receptors, enzymes and ion

channels are sensitive to reactive oxygen species (ROS).

SLIDE 3

Modified based on www-dsv.cea.fr/thema/bbsi/vengl/taoxyd.htm NADH NAD+

NAD(P)H NAD(P)H Oxidase Oxidase: A Major Enzyme Mediating O : A Major Enzyme Mediating O2

2 . .-

Production and

Production and Redox Redox Signaling in Vessels Signaling in Vessels

SLIDE 4

cADP cADP-

R

R-

mediated Ca

mediated Ca2+

2+Mobilization in the

Mobilization in the Control of Vascular Tone Control of Vascular Tone

Vascular Vascular Tone Tone RYR RYR cADP cADP-

R or NAADP

R or NAADP

SR SR

Ca Ca2+

2+

Ach Ach OXO OXO

Membrane Membrane c y c l a s e

? ?

NAD(P) NAD(P) NAD NAD

R R Ni Ni

NO NO EET EET

SLIDE 5

Linkage of NAD(P)H Linkage of NAD(P)H Oxidase Oxidase-

Mediated

Mediated Redox Redox Signaling to cADP Signaling to cADP-

R

R-

Ca

Ca2+

2+ Signaling

Signaling

Vascular Vascular Tone Tone RYR RYR cADP cADP-

R or NAADP

R or NAADP

SR SR

Ca Ca2+

2+

Ach Ach OXO OXO

Membrane Membrane c y c l a s e

? ?

NAD(P) NAD(P)

R R Ni Ni

NO NO EET EET

Vascular Vascular Tone Tone RYR RYR cADP cADP-

R or NAADP

R or NAADP

SR SR

Ca Ca2+

2+

Ach Ach OXO OXO

Membrane Membrane c y c l a s e

? ?

NAD(P) NAD(P)

R R Ni Ni

NO NO EET EET

RYR RYR cADP cADP-

R or NAADP

R or NAADP

SR SR

Ca Ca2+

2+

Ach Ach OXO OXO

Membrane Membrane c y c l a s e

? ?

NAD(P) NAD(P)

R R Ni Ni

NO NO EET EET

NAD(P)H Oxidase-Mediated Redox Signaling cADP-Ribose-Mediated Ca2+ Signaling

NADH NAD+

NAD

SLIDE 6

Revised Hypothesis: Revised Hypothesis: Redox Redox Amplification of Ca Amplification of Ca2+

2+ Signaling

Signaling

NO

cADPR

M e m b r a n e

H+ NADH NAD+ 1

Em G

KCl K OXO 5-HT VP

O

2

+

O

2 .-

CD38 (-)

NO NO

M e m b r a n e

NADH 1

Em G

KCl K OXO 5-HT VP

e- O

2

+

CD38

O

2 .-

2

Ca2+

ONOO ¯

CD38 CD38 (-) NOX-C NOX-C

RyR NADH

(+) (+ )

NADH

(+) (+ )

RyR

SR SR

O

2 .-

O

2 .-

NOX

C

NOX

C

O

2 .-

O

2 .-

3

O

2 .-

O

2 .-

O

2 .-

O

2 .-

O

2 .-

O

2 .-

SLIDE 7

Revised Hypothesis: Revised Hypothesis: Redox Redox Amplification of Ca Amplification of Ca2+

2+ Signaling

Signaling

NO

cADPR

M e m b r a n e

H+ NADH NAD+ 1

Em G

KCl K OXO 5-HT VP

O

2

+

O

2 .-

CD38 (-)

NO NO

M e m b r a n e

NADH 1

Em G

KCl K OXO 5-HT VP

e- O

2

+

CD38

O

2 .-

2

Ca2+

ONOO ¯

CD38 CD38 (-) NOX-C NOX-C

RyR NADH

(+) (+ )

NADH

(+) (+ )

RyR

SR SR

O

2 .-

O

2 .-

NOX

C

NOX

C

O

2 .-

O

2 .-

3

O

2 .-

O

2 .-

O

2 .-

O

2 .-

O

2 .-

O

2 .-

Contraction

SLIDE 8

How do we test our hypothesis?

SLIDE 9

Experimental Design

Coronary arterial myocytes (CAMs) from freshly dissected bovine

coronary arteries (BCA) were homogenized and ultracenterfuged into cytosol and microsome.

mNOX was stimulated by incubating the BCA microsome at 370C at

differing times, to find an optimal peak in incubation time for stimulation, in different agonists such as oxotremorine (OXO) and later xanthine/xanthine oxidase (X/XO).

In separate groups, NOX inhibitors DPI, apocynin (Apo), and SOD,

along vehicle were incubated at 370C for differing times to find an

ptimal peak in incubation time for inhibition.
These samples are then detected by HPLC (Fluorescence)

SLIDE 10

Anticipated Results

Increased O2
production and cGDPR expression in the BCA

microsome with the addition of the agonist OXO in comparison with control homogenate.

Decreased O2
production and cGDPR expression with the

addition of NOX inhibitors DPI and Apo to OXO treated homogenase in comparison to addition of OXO alone.

SLIDE 11

HPLC High Performance Liquid Chromatography

HPLC detectors pass a beam of light

through a column effluent as the fluid passes through a low-volume flow cell. Variations in light intensity are recorded and a chromatograph is generated.

HPLC detectors use several detection
methods. Ultraviolet (UV) detectors

measure the ability of a sample to absorb light at one or more

wavelengths. Light scattering detectors

nebulize the effluent, vaporize the solvent, and then detect droplets in a light scattering cell.

The fluorescence detector is one of the

most sensitive LC detectors and for this reason is often used for trace analysis.

SLIDE 12

HPLC High Performance Liquid Chromatography

Utilizes special instruments

designed to separate, quantify and analyze components of a chemical mixture.

Samples of interest are introduced

to a solvent flow path; carried through a column packed with specialized materials for component separation; and component data is obtained through the combination of a detection mechanism coupled with a data recording system.

SLIDE 13

In this chromatogram, there are five types of molecules. The molecules at the peak labeled “A” are probably the smallest molecules, because they took the shortest time amount of time to go through the HPLC. The molecules at the peak labeled “E” are probably the largest. The amount of time it takes for a molecule to run through the HPLC is called its retention time. These peaks are differentiated by retention time along the X-axis and standards are run to establish an expected retention time for each molecule.

HPLC High Performance Liquid Chromatography

SLIDE 14

cGDPR Production after Incubation

The first part of the protocol

was finding optimal incubation times for the agonists being added and whether to use microsome or cytosol

Microsome was found to be

much more efficient

Optimal incubation times for

NGD (1uM) and OXO(50uM) were found to be 5mins and 15min, respectively.

From previous protocol, 15mins

was determined to be the

ptimal incubation time for

DPI(50uM) and Apo(100uM).

2 4 6 8 10 12 14 16 18 CM CC OM OC 10 20 30 40 50 60 NGD 1min 3min 5min 10min 15min Ctrl OXO 0.0 5.0 10.0 15.0 20.0 25.0 30.0 CM CC OM OC OXO 15min OXO 30min OXO 60min OXO 15min OXO 30min

SLIDE 15

cGDPR Production after Incubation

After a total of 5 experiments

with 2 samples each, the inhibitors consistently raised the expression of cGDPR.

Even with differing

concentrations DPI and Apo the data remained consistent

2 4 6 8 10 12 14 Homo OXO DPI APO DPI + OXO APO + OXO 2 4 6 8 10 12 14 Homo OXO DPI APO DPI 25uM OXO DPI 50uM OXO DPI 100uM OXO APO 50uM OXO APO 100uM OXO APO 200uM OXO

SLIDE 16

BCA microsome was then

incubated with SOD and X/XO

While there were SOD

problems, the results found with X/XO were very interesting

5 10 15 20 25 30 35 40 Homo SOD cat X/XO SOD X/XO OXO SOD OXO X/XO DPI

cGDPR Production after Incubation

SLIDE 17

Xanthine/xanthine oxidase was

causing significant extra peaks

Incubated alone, xanthine did

not do much

X/O however created large

peaks

When combined together, some
f these peaks were inhibited,

which would be interesting to investigate further

cGDPR Production after Incubation

SLIDE 18

Conclusions

Oxotremorine was found to increase the expression of cGDPR

when homogenate was incubated at 370C for 15mins.

DPI and Apo were not found to inhibit the expression of cGDPR

when combined with OXO. There may be different pathways involved or possibly toxicity effects.

The results from X/XO yielded results that may prove interesting

in further studies

SLIDE 19

Acknowledgment

The SPUR Program

Dr. Pin-Lan Li
Dr. Dewey
Dr. Forrest Smith

Ningjun Vicky Steven Max Kelly Fan & Others