Transcript III: Cells Utilizing Oxygen to Form Lipid Regulators and

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PHM142
3) Cells Utilizing Oxygen to Form Lipid Regulators and Nitric Oxide
A) Platelet function:
i. Blood clotting and stroke (anticoagulant drugs)
ii. Prostaglandin and thromboxane formation from
polyunsaturated fatty acids
iii. Nutritional approach to prevent heart disease
iv. COX Inhibitors (NSAIDs) (antiplatelet drugs)
B) 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
Ca2+
influx
in other platelets
Diacylglycerol (DAG)
+ inositol triphosphate
(IP3)
Membrane
Phospholipase
A2 activated
Aspirin
Arachidonate
(drug)
acetylates
cyclooxygenase
2O2
PLATELET
AGGREGATION
PGH2
Dazoxiben
(drug)
Phosphatidylinositol
Membrane
Phospholipase C
Granule
release
Thromboxane A2 (TxA2)
receptor
collagen
receptor
COLLAGEN
TxA2
synthetase
Contraction
of microtubules
Ca2+ Influx
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) 7
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• 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
O
O
CH3
O
OH
H
C
H
O
CH3
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H
OH
O
O
Warfarin for thrombosis but high
CYP2C9 polymorphism.
Drug name Coumadin (rat poison)
2-3 days before it works 
don’t admin. again for 3d
Dicoumarol
- Spoiled sweet clover
causes fatal
hemorrhagic disease in
cattle.
O
O
H
C
OH
O
CH3
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
c. Platelets collected and stored for use in surgeries , transplants and
cancer therapy to stop bleeding.
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b) Prostaglandin and thromboxane synthesis stages
1. 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 Membrane
Phospholipids

PGE1
PGE2
PGE3
2. Overview of Cyclooxygenase (COX) Pathways
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Conversion of Arachidonic Acid to PGD2, PGF2a
and PGE2
2O2
O
COOH
COOH
Cyclooxygenase
CYCLOOXYGENASE
Arachidonate
O
PGG2
*
_
Aspirin
Indomethacin
Ibuprofen
Drugs, Carcinogens
Peroxidase
PEROXIDASE
*
O
Isomerase
ISOMERASE
COOH
O
PGH2
O
COOH
OH
PGE2
Oxidised (Toxic)
Toxin prev. by NSAIDs
e.g. Colon cancer
NSAIDs
Prostaglandin synthase
consists of cyclooxygenase
(COX) and peroxidase
OH
OOH
OH
Isomerase
ISOMERASE
OH
COOH
Reductase
REDUCTASE
OH
O
COOH
OH
OH
PGF2a
OH
PGD2
* 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
c) Good and bad fatty acid nutrition
1 Linoleate (18C)
(2 double bonds)
2. Arachidonate
2. -6
Arachidonate
(meat;
4 double
(meat; 4 double
bonds
20C)
bonds
20C)
-Linolenate (18C)
(3 double bonds)
PGE2, PGF2,
TXA2, LTA2, PGI2
PGI2
+2C
3.fatty
acids
-3-3fatty
acids
a-linolenate
(vegetables
3 double bonds)
eicosapentaenoic
acid (fish; 20C
5 double bonds)
PGE1, PGF1a, TXA,
LTAT, PGI1
Platelet aggregation & CV risk
poor at causing
platelet aggregation
(Inuit have no
heart disease)
PGE3, PGF3a,
PGI3
TXA3
PGI3 >> PGI2
prevents platelet
aggregation
induced by TXA2
very effectively
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:
• a-linolenic acid (flaxseed) 18:3
• Eicosapentanoic acid
20:5
fish
• Docosahexanoic acid
22:6
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OMEGA-3
H3C
Unstaurated
Fatty Acid
Formation
CH3
Essential Fatty Acids
From the diet
OMEGA-6
H3C
CH3
Linoleic Acid
(LA) 18:2
a-Linolenic acid
(LNA)18:3
Delta-6Desaturase
Stearidonic Acid
(SDA)
-Linolenic Acid
(GLA)
Elongase
Eicosatetraenoic
acid
Dihomogamma
Linolenic acid
(DGLA)
Delta-5Desaturase
Eicosapentaenoic
Acid
(EPA)
Series 3
Prostaglandins
Arachidonic
Acid
Series 1
Prostaglandins
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Series 2
Prostaglandins
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d) Antiplatelet drugs
Cyclooxygenase (COX) and COX inhibitors (NSAIDs)
COX-1 (ALL CELLS)
COX-2 (INFLAMMATORY CELLS)
Physiological Function – all cells
- PG’s cytoprotective (stomach, kidney)
- TXA platelet (clotting function)
Inflammatory cells (also brain)
- mediate inflammation and pain
- inducible by cytokines, mitogens,
endotoxins (contributes to rheumatoid
arthritis)
- promote colon cancer by preventing
apoptosis
- causes premature labour
Inhibitors
- aspirin, NSAIDS, antithrombosis drugs
but cause gastric/renal lesions
Inhibitors
- selective NSAIDS e.g. nimesulide
- chemoprevention/colon cancer
- antipyretic (knockout mice – infertile
females, no intestinal polyps)
Colon cancer prevented by low daily
doses of aspirin or COX inhib.
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• Aspirin inhibits COXI which catalyse the peroxidase
activation of environmental arylamines to human
carcinogens e.g. benzidine,naphthylamine.
Azoxymethane rodent colon carcinogen.
• COX2 activity & extracellular matrix
metalloproteinases is induced by inflammation or
colorectal cancer CRC.
• Inhibitors of these enzymes decrease CRC
metastasis Diseases Colon Rectum 51,342-347(2008)
NSAIDS
COX-1 inhibitors interaction with the COX enzyme- GI toxicity
As PGE protects intestinal mucosa
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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
(incr. risk of hypertension,congestive heart failure unlike Celecoxib)
COX-2 inhibitors – much less GI toxicity
4) Vioxx 7% - Merck - for rheumatoid arthritis, osteoarthritis,
Alzheimer’s, chronic inflammation (WITHDRAWN 2004) heart attack/
stroke ?). Celecoxib (celebrex) 20% -Rofecoxib incr. cardiorenal edema
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B) Role of endothelial cells in
regulating blood pressure
and
Nitric oxide formation, signaling
and toxicity
Function of Endothelial cells (line vessel walls)
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Cell Surface Activity (exoenzymes)
1) Angiotensin converting enzyme (inhibited by specific dipeptides) e.g.
Captopril*25-50mg (but agranulocytosis risk, cough), Enalapril*120 mg. Ramipril 2.5-20 mg (10mg useful for preventing
cardiovascular/stroke in diabetics and antihypertensive.
Angiotensin converting enzyme formation and action
Angiotensinogen (411 aa) synthesised in the liver
Plasma
renin (synthesised in
kidney, a protease)
ACE inhibitors
Angiotensin II in plasma (8 aa)
angiotensin converting enzyme
(endothelial cells)
(therefore a dipeptidase)
Angiotensin I in plasma (10aa)
2aa
Vasoconstrictor
blood pressure
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Endothelial cells (line vessel walls)
Cell Surface Activ. – (Con’t)
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 (reversed by clot
busters” e.g.plasmin & plasminogen activator)
Stroke Therapy (Dissolve clots)
Fibrin
Plasminogen
incorporated into
clot fibrin
Plasmin
(a serine protease)
Plasminogen Activator*
(a serine protease)
Endothelial cell
CO
CO?
Dissolve fibrin
Therefore activator
Fibrin-bound
plasmin
Fibrinolysis
6) Stroke therapy (con’t)
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 (con’t)
Plasma Membrane Activity
7) PROSTACYCLIN* synthesis and release (resting state) (PGI2)
prevents platelet aggregation
arachidonic
phospholipid
PGG2
PGI2
acid
phospholipase A2
prostaglandin
synthetase
(cyclooxygenase)
PGI2 synthetase
Therefore endothelial cells function to prevent thrombosis
Prostacyclin synthase inhib. by COX2 inhibitor e.g. rofecoxib.
8) EDRF (i.e. nitric oxide, a vasodilator) formed from arginine
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
if endothelial cells
are present.
ENDOTHELIAL derived
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)
O
O
H 2N
CH
C
H2N
OH
CH
C
O
OH
H 2N
CH
CH2
CH2
CH2
OH
CH2
1
NADP+
NADPH
C
/
NADPH
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NADP+
/
2
CH2
CH2
NO
+
CH2
NOS
O2
NH
C
H2O
-
A
"
A
n
P
r
a
4
g
l
5
iN
CH2
NOS
O2
NH
NH
NH2
L
CH2
C
N
NH2
o
0
n-
H2O
NH
OH
C
g
iO
o
+
nH
u
e
s
r
LA
O
e
t
d
-r
Cg
NH2
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
o
u
ii
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4)
Drugs that act by Generating Nitric Oxide
(NO.)
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1) NITROGLYCERIN - antianginal, antihypertensive, vasodilator
(Baxter, Parke Davis)
r
e
d
u
c
e
d
NO2
NO
CH2 ONO2
N
CHONO2
v
L
A
i
i
D
a
CH2 ONO2
v
P
e 2 OH
r
CH
H
m
CHONO2
r
e
d
CH2 ONO2
CH2 ONO2
i
c
CH2 OH
u
c
e
CH2 ONO2
g
G
V
c
a
e
s
l
St
Hr
c
u
l
s
r
d
l
o
G
s
l
u
c2
CO
i
u
r
G
S
H
aG
nS Gs N Sf O He
r
a
s
e
l
a
r
s
m
o
o
t
G
S
S
G
a
n
d
e
n
d
o
t
NO
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,arginine,ACE inhibitors,diuretics
P
h
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2) “AMYL” NITRITE (i.e. isopentyl nitrite) - potent vasodilators which
are inhaled (Roberts)
H3C
CHCH2CH2OH
H3C
m
N O
H3C
H3C
O
CHCH2CH2
A
m
G
o
y
it
r
l
S
+ GSH transferase
(GST 4-4)
G
n
H
o
c
h
o
-ONO
P
4
5
0
N
it
r
it
rr
ait
de
ic
ti
n
d
r
N
s
O
S
y
a
H
s
o
t
e
a
e
l
NO
S
G
r
e
r
m
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Amyl nitrite (con’t)
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 Kaposi’s sarcoma)
J.of Toxicol-Clin Toxicol.42,313-6(2004);J. Neuroimmunol.83, 157-61 (98)
C) Cyanide antidote - nitrite oxidises oxyhemoglobin to methemoglobin (Poison control centre) and then Fe3+ of methemoglobin
complexes cyanide.
3) NITROPRUSSIDE (NIPRIDE) - Roche ANTIHYPERTENSIVE
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NO
REDUCTI ON by oxyhe m ogl obi n
i n r e d bl ood ce l l s
NC
Fe
CN
Vascular cell GSH
NC
NO
CN
NC
CN
NC
M i t ochondr i a or
NADPH P450
r e duc t a s e
NC
NC
NO
Fe
CN
NC
CN
NC
NO
Fe
CN
CN
O2
O2
SG
Cyani de
NO
Fe
CN
CN
CN
GS
GSSG
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 treatment of chronic hypertension and management of
myocardial infaction. Adverse effects: cyanide
poisoning in rhodanese deficient patients, methemoglobinemia. Biochem. Pharmacol. 42, 5157-65 (1991).
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ACTION AND ROLE OF NITRIC OXIDE VERSUS cAMP/Ca2+
Protein Kinase A
cAMP
Protein Kinase A
Protein Kinase A
(Active)
Phosphorylase Kinase
Ca2+
Glycogen
Glycogen
Phosphorylase
Endothelial Cells
NO
Smooth
Muscle
Cells
Guanylate
cyclase
Activates
cGMP-dependent
Protein kinase
cGMP
Phosphoryln. Of
Ca2+ regulatory
proteins
incr. Ca 2+
Glucose-1-Phosphate
Glycolysis
Mitochondria
ATP
MUSCLE CONTRACTION
in muscle cell
MUSCLE
RELAXATION
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cAMP
Formation of IP3 and DAG from PIP2 and
their signaling Functions
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