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

Internalization, Dimerization, and Activation of CD38 during mNOX Activation: - and Ca 2+ Signaling in Coronary Arterial Smooth Muscle O 2 Molly Hilliker Department of Pharmacology & Toxicology Virginia Commonwealth University Medical College of Virginia Campus

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).

.- - NAD(P)H Oxidase Oxidase: A Major Enzyme Mediating O : A Major Enzyme Mediating O 2 . NAD(P)H 2 Production and Redox Redox Signaling in Vessels Signaling in Vessels Production and NADH NAD + Modified based on www-dsv.cea.fr/thema/bbsi/vengl/taoxyd.htm

2+ Mobilization in the mediated Ca 2+ cADP- -R R- -mediated Ca Mobilization in the cADP Control of Vascular Tone Control of Vascular Tone Membrane Membrane Ach Ach OXO OXO cADP- -R or NAADP R or NAADP cADP R R ? ? NO NO c y c l a s e EET EET Ni Ni NAD(P) NAD(P) 2+ Ca 2+ Vascular Vascular Ca NAD NAD Tone Tone RYR RYR SR SR

Linkage of NAD(P)H Oxidase Oxidase- -Mediated Mediated Redox Redox Linkage of NAD(P)H 2+ Signaling Signaling to cADP- -R R- -Ca Ca 2+ Signaling Signaling to cADP NAD(P)H Oxidase-Mediated cADP-Ribose-Mediated Redox Signaling Ca 2+ Signaling Membrane Membrane Membrane Membrane Membrane Membrane Ach Ach Ach Ach Ach Ach OXO OXO OXO OXO OXO OXO cADP- cADP- cADP- -R or NAADP -R or NAADP -R or NAADP R or NAADP R or NAADP R or NAADP cADP cADP cADP R R R R R R ? ? ? ? ? ? NO NO NO NO NO NO c c c y y y c c c l l l a a a s s s e e e EET EET EET EET EET EET Ni Ni Ni Ni Ni Ni Vascular Vascular Vascular Vascular NAD(P) NAD(P) NAD(P) NAD(P) NAD(P) NAD(P) Ca 2+ Ca 2+ Ca 2+ 2+ 2+ 2+ Ca Ca Ca NADH Tone Tone Tone Tone NAD RYR RYR RYR RYR RYR RYR NAD + SR SR SR SR SR SR

Revised Hypothesis: Revised Hypothesis: 2+ Signaling Amplification of Ca 2+ Redox Amplification of Ca Signaling Redox ONOO ¯ ( - ) ( - ) NO NO NO .- .- O O 2 2 M M e e m m e - O O b b r r a a n n e e CD38 CD38 + + CD38 2 2 H + NOX-C NOX-C CD38 2 2 NAD + 1 1 Em Em KCl KCl cADPR OXO OXO NADH NADH 5-HT 5-HT VP VP G G K K Ca 2+ ( + ) ( + ) ( + ( + ) ) O O .- .- O O .- .- 2 2 2 2 O O .- .- O O .- .- RyR RyR 2 2 2 2 3 O O .- .- 2 2 NOX NOX -C -C NADH NADH SR SR

Revised Hypothesis: Revised Hypothesis: 2+ Signaling Amplification of Ca 2+ Redox Amplification of Ca Signaling Redox ONOO ¯ ( - ) ( - ) NO NO NO .- .- O O 2 2 M M e e m m e - O O b b r r a a n n e e CD38 CD38 + + CD38 2 2 H + NOX-C NOX-C CD38 2 2 NAD + 1 1 Em Em KCl KCl cADPR OXO OXO NADH NADH 5-HT 5-HT VP VP G G Contraction K K Ca 2+ ( + ) ( + ) ( + ( + ) ) O O .- .- O O .- .- 2 2 2 2 O O .- .- O O .- .- RyR RyR 2 2 2 2 3 O O .- .- 2 2 NOX NOX -C -C NADH NADH SR SR

How do we test our hypothesis?

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 37 0 C 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 37 0 C for differing times to find an optimal peak in incubation time for inhibition. • These samples are then detected by HPLC (Fluorescence)

Anticipated Results - production and cGDPR expression in the BCA • Increased O 2 microsome with the addition of the agonist OXO in comparison with control homogenate. • Decreased O 2 - production and cGDPR expression with the addition of NOX inhibitors DPI and Apo to OXO treated homogenase in comparison to addition of OXO alone.

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.

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.

HPLC High Performance Liquid Chromatography 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.

cGDPR Production after Incubation 18 16 • The first part of the protocol 14 was finding optimal incubation OXO 15min 12 OXO 30min 10 times for the agonists being 8 added and whether to use 6 4 microsome or cytosol 2 0 CM CC OM OC 60 • Microsome was found to be 50 much more efficient 40 Ctrl 30 OXO 20 • Optimal incubation times for 10 NGD (1uM) and OXO(50uM) 0 NGD 1min 3min 5min 15min were found to be 5mins and 10min 30.0 15min, respectively. 25.0 20.0 • From previous protocol, 15mins OXO 15min OXO 30min was determined to be the 15.0 OXO 60min optimal incubation time for 10.0 DPI(50uM) and Apo(100uM). 5.0 0.0 CM CC OM OC

cGDPR Production after Incubation 14 • After a total of 5 experiments 12 10 with 2 samples each, the 8 inhibitors consistently raised the 6 expression of cGDPR. 4 2 0 Homo OXO DPI APO DPI + OXO APO + OXO • Even with differing concentrations DPI and Apo the 14 data remained consistent 12 10 8 6 4 2 0 Homo OXO DPI DPI DPI DPI APO APO APO APO 100uM 200uM 25uM 50uM 100uM 50uM OXO OXO OXO OXO OXO OXO

cGDPR Production after Incubation • BCA microsome was then 40 incubated with SOD and 35 X/XO 30 25 20 15 • While there were SOD 10 problems, the results found 5 0 with X/XO were very Homo SOD cat X/XO SOD X/XO OXO SOD OXO X/XO DPI interesting

cGDPR Production after Incubation • 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 of these peaks were inhibited, which would be interesting to investigate further

Conclusions • Oxotremorine was found to increase the expression of cGDPR when homogenate was incubated at 37 0 C 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

Acknowledgment The SPUR Program Dr. Pin-Lan Li Dr. Dewey Dr. Forrest Smith Ningjun Vicky Steven Max Kelly Fan & Others