PHM226 3) Cells Utilizing Oxygen to Form Lipid Regulators and Nitric Oxide

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PHM226 3) Cells Utilizing Oxygen to Form Lipid
Regulators and Nitric Oxide

• Platelet function
• Blood clotting and stroke (anticoagulant drugs)
• Prostaglandin and thromboxane formation from
  polyunsaturated fatty acids
• Nutritional approach to prevent heart disease
• COX Inhibitors (NSAIDs) (antiplatelet drugs)
• Endothelial cell functions Prostacyclin and
  plasminogen activator formation
• Role of endothelial cells in regulating blood
  pressure
• - Angiotensin and EDRF
• Nitric oxide formation, signaling and toxicity
• - Use of Nitric Oxide generators
  (vasodilator / antihypertensive drugs)

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A) Platelet Function
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Platelets
t1/2 4 days Fragments of Megakaryocytes of
bone marrow Contain glycogen granules
Mitochondria
Lysosomes No nucleus, DNA,
protein synthesis. 1) dense granules ADP, ATP,
serotonin (5HT) 2) a granules contain clotting
factors and PDGF (platelet derived growth factor)

80 ATP from glycolysis and 20 from mitochondria
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PLATELETS
a) Function is to plug blood leaks (blood
clotting) 1. Activated by collagen (damaged
vascular surface) or thrombin which binds to
receptors. Platelet disc shape changes to sphere
i.e. swell, form pseudopods, become sticky and
attach to collagen
Resting platelets
Activated Platelets
Images From http//www-personal.engin.umich.edu/
tkinzer/
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2. Mechanism of platelet aggregation to form a
clot
Phospholipid
ADP released from platelet granule
receptor
Membrane Phospholipase A2 activated
Ca2 influx in other platelets
Aspirin (drug)
Arachidonate
acetylates
cyclooxygenase
2O2
Diacylglycerol (DAG) inositol triphosphate (IP3)
PLATELET AGGREGATION
PGH2
TxA2 synthetase
Dazoxiben (drug)
Granule release
Thromboxane A2 (TxA2)
Phosphatidylinositol Membrane
Phospholipase C
receptor
collagen receptor
Ca2 Influx
Contraction of microtubules
COLLAGEN
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3. Fibrin clot formed by zymogen activation
cascade
INTRINSIC PATHWAY

• Modified from Fig. 10.37, Stryer (5th ed).
• A fibrin clot (blood clot) is formed by the
  interplay of the intrinsic, extrinsic, and final
  common pathways.
• Intrinsic pathway
• initiated when factor XII is activated by
  contact with abnormal surfaces due to injury.
• Extrinsic pathway
• triggered by trauma, which activates factor VII
  which releases tissue factor.
• a indicates activated form of clotting factor.

Traps aggregated platelets
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Anticoagulant drugs (stroke treatment prevents
new clots) .

• Vitamin K is essential for prothrombin synthesis
  (and other clotting factors).
• Abnormal prothrombin is formed (does not bind
  Ca2) in the absence of Vit. K or in the presence
  of Vit. K antagonists (see below).

Anticoagulants Drugs Vit. K antagonists
Vitamin K
Dicoumarol - Spoiled sweet clover causes fatal
hemorrhagic disease in cattle.
Warfarin for thrombosis but high CYP2C9
polymorphism. Drug name Coumadin (rat poison)
2-3 days before it works ? dont admin. again for
3d
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4. Platelets Repair Broken Blood Vessel
a. Platelets secrete platelet derived growth
factor (PDGF) a growth factor ?migration and
division of vascular endothelial cells,
smooth muscle cells, fibroblasts ? ?REPAIR
OF DAMAGED VASCULAR WALLS b. MEMBRANE ACTIN
AND MYOSIN CONTRACT? platelet attached to
fibrin are retracted ? CLOT RETRACTS ?edges of
broken blood vessel are pulled together
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b) Prostaglandin and thromboxane synthesis
stages1. Membrane Phospholipid Containing a
Glycerol Backbone and a Fatty Acid At the First
Position, Arachidonic Acid at the Second Position
and a Polar Phopholipid Moiety at the Third
Position.
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Biochemical Structure of Typical Fatty Acids
Incorporated Into MembranePhospholipids
??
PGE1
PGE2
PGE3
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2. Overview of Cyclooxygenase (COX) Pathways
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Conversion of Arachidonic Acid to PGD2, PGF2a
and PGE2
Toxin prev. by NSAIDs e.g. Colon cancer
NSAIDs
Prostaglandin synthase consists of cyclooxygenase
(COX) and peroxidase
NSAID now used as prophylaxis to prevent colon
carcinogenesis
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3. Prostaglandin and Prostacyclin and Heart
Disease
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Induction and Inhibition of Platelet Aggregation
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c) Good and bad fatty acid nutrition
-3
-3
fatty acids
2. ?-6 Arachidonate (meat 4 double bonds 20C)
PGI2
Platelet aggregation
Clinical Science 107,1-11(2004) omega-3 fatty
acid nutrition
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Omega-3 fatty acid therapy

• Decrease risk of heart disease and stroke
• Augment cancer therapy (J. Nutr. 132 (11 suppl)
  3508S-3512S) and prevents breast cancer
• Inflammatory bowel disease
• Psoriasis
• Depression
• SOURCES OF OMEGA-3 FATTY ACID
• ?-linolenic acid (flaxseed) 183
• Eicosapentanoic acid 205
• Docosahexanoic acid 226

fish
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OMEGA-3
OMEGA-6
Unstaurated Fatty Acid Formation
182
183
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d) Antiplatelet drugs Cyclooxygenase (COX) and
COX inhibitors (NSAIDs)
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NSAIDS COX-1 inhibitors interaction with the COX
enzyme- GI toxicity As PGE protects intestinal
mucosa 1) Aspirin - irreversible
- acetylates Ser 530, prevents arachidonic acid
binding 2) Mefenamate, Ibuprofen - reversible,
competitive with fatty acid
binding 3) Flurbiprofen
- slow binding (salt bridge) competitive
inhibition by binding in the hydrophobic
channel Indomethacin - non-selective
- binds deepest in hydrophobic
channel COX-2 inhibitors much less GI
toxicity 4) Vioxx 7 - Merck - for rheumatoid
arthritis, osteoarthritis, Alzheimers,
chronic inflammation (WITHDRAWN 2004) heart
attack/ stroke ?). Celecoxib (celebrex) 20 -
Searle/Pfizer
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B) Role of endothelial cells in regulating blood
pressureandNitric oxide formation, signaling
and toxicity
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Function of Endothelial cells (line vessel
walls)
Cell Surface Activity (exoenzymes) 1) Angiotensin
converting enzyme (inhibited by specific
dipeptides) e.g. Captopril25-50mg(but
agranulocytosis risk), Enalapril1-20 mg.
Ramipril 2.5-20 mg (10mg useful for preventing
cardiovascular/stroke in diabetics (DIABHCAR
trial) and antihypertensive.
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Endothelial cells (line vessel walls)
Cell Surface Activ. (Cont)
2) ATPase and 5-nucleotidase degrade ATP and ADP
3) Inactivate prostaglandins E and F and
leukotrienes C4 and D4
Intracellular Activity 4) Monoamine oxidase
? plasma norepinephrine or serotonin 5)
Thromboplastin synthesis and secretion (activated
state) initiate blood clotting (extrinsic
pathway) 6) Synthesis and secrete plasminogen
activators (resting state) initiates clot
fibrinolysis
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6) Synthesis and Secretion of Plasminogen
Activators (Resting State) initiate Clot
Formation (treated with PA clot busters)
CO
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6) Stroke therapy (cont)
Drug companies are using recombinant DNA
technology to make PLASMINOGEN ACTIVATORS to
reduce myocardial damage following acute coronary
thrombosis (but x 10 expensive and
no better/safer. Cigarette smoke ? CO ? damages
endothelial cell Prevent clotting e.g., stored
blood 1. Complex Ca2 with citrate or oxalate 2.
Heparin helps remove thrombin (produced by mast
cells)
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Endothelial cells (cont)
Plasma Membrane Activity
7) PROSTACYCLIN synthesis and release (resting
state) (PGI2) prevents platelet aggregation
Therefore endothelial cells function to prevent
thrombosis Prostacyclin synthase inhibitor
(camonogrel dazoxiben) prevents ischemic diabetic
retinopathy. 8) EDRF (i.e. nitric oxide, a
vasodilator) formed from arginine
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8) EDRF - Endothelial Derived Relaxing Factor
Endothelial cell regulation of blood
pressure i.e. effect on smooth muscle cells
i.e. acetylcholine relaxes vascular smooth
muscle only ifendothelial cells are present.
ENDOTHELIAL RELAXING FACTOR - unstable
and inactivated by oxygen and hemoglobin ? only
a local hormone - indentified as nitric
oxide! Involved in regulation of the
microcirculation.
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Nitric Oxide Synthases (NOS)
Arginine (100 uM in blood, 2 mM in endothelial
cells) - synthesized from citrulline or enters
via transporter (regulated by cytokines) Km 5
uM
Science 278, 425-431 (1997). 2.6A? X-ray
crystallography of NOS
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4)
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Drugs that act by Generating Nitric Oxide (NO.)
1) NITROGLYCERIN - antianginal, antihypertensive,
vasodilator
(Baxter, Parke Davis)
Adverse effect HEADACHE unless
tolerant Tolerance because vascular ALDH in
mitochondria reduces NG but is inactivated.by
peroxynitrite formed when NO reacts with O2??
(formed when NO inhibits respiratory chain)
(J.Clin.Invest.113,482-9(2004)Ann Rev
Pharmac.Toxicol 44,67-85(2004). Tolerance
reversed with antioxidants,thiols,hydralazine,argi
nine,ACE inhibitors,diuretics
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2) AMYL NITRITE (i.e. isopentyl nitrite) -
potent vasodilators which are inhaled
(Roberts)
GSH transferase (GST 4-4)
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Amyl nitrite (cont)
A) Prescription drug (except 1960-1969) In
glass ampules enclosed in mesh - crushed in the
fingers popping sound. Poppers -
inhaled to relieve angina. B) Recreational
inhalant (locker room) - Butyl nitrite
aphrodisiac used for enhancing sexual pleasure
as nitrite induces vasodilation of the rectal
mucosal muscles. Pharmacotherapy (1984) 4,
284-91. but causes acute hemolysis if G6PDH
deficient. May deplete cells of the immune
system (wrongly thought to cause Kaposis
sarcoma) J.of Toxicol-Clin
Toxicol.42,313-6(2004)J. Neuroimmunol.83, 157-61
(98) C) Cyanide antidote - nitrite oxidises
oxyhemoglobin to met- hemoglobin (Poison
control centre) and then Fe3 of methemoglobin
complexes cyanide.
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3) NITROPRUSSIDE (NIPRIDE) - Roche
ANTIHYPERTENSIVE
Vascular cell GSH
For treatment of hypertensive emergencies -
infusion with sterile 5 dextrose. Controlling
the rate of NP infusion allows very rapid
decrease of blood pressure during surgery without
overshoot. Also used in treat- ment of chronic
hypertension and management of myocardial
infaction. Adverse effects cyanide poisoning in
rhodanese deficient patients, methemo- globinemia.
Biochem. Pharmacol. 42, 5157-65 (1991).
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ACTION AND ROLE OF NITRIC OXIDE VERSUS cAMP/Ca2
Protein Kinase A
Endothelial Cells NO
Protein Kinase A
cAMP
Protein Kinase A (Active)
Smooth Muscle Cells
Guanylate cyclase
Phosphorylase Kinase
Ca2
Glycogen Phosphorylase
Activates cGMP-dependent Protein kinase
cGMP
Glycogen
Glucose-1-Phosphate
Glycolysis Mitochondria
Phosphoryln. Of Ca2 regulatory proteins
incr. Ca 2 in muscle cell
ATP
MUSCLE RELAXATION
MUSCLE CONTRACTION
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cAMP
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Formation of IP3 and DAG from PIP2 and their
signaling Functions