1PHM142 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 acidsiii. Nutritional approach to prevent heart diseaseiv. COX Inhibitors (NSAIDs) (antiplatelet drugs)

B) Endothelial cell functions: Prostacyclin and plasminogen

activator formationRole of endothelial cells in regulating blood pressure:

- Angiotensin and EDRFNitric oxide formation, signaling and toxicity:

- Use of Nitric Oxide generators: (vasodilator / antihypertensive drugs)

2

A) Platelet Function

3

Plateletst1/2 = 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) granules contain clotting factors and PDGF (platelet derived growth factor) 80% ATP from glycolysis and 20% from mitochondria

4PLATELETSa) 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 PlateletsImages From: http://www-personal.engin.umich.edu/~tkinzer/

5

2. Mechanism of platelet aggregation to form a clot

ADPreleasedfrom plateletgranule

receptor

Ca2+ influxin other platelets

MembranePhospholipaseA2 activated

Phospholipid

Arachidonate

cyclooxygenaseacetylates

Aspirin(drug)

PGH2

Thromboxane A2 (TxA2)

Diacylglycerol (DAG)+ inositol triphosphate (IP3)

PhosphatidylinositolMembrane

Phospholipase C

collagenreceptor

COLLAGEN

TxA2 synthetase

Dazoxiben(drug)

receptor

Ca2+ InfluxContraction

of microtubules

Granulerelease

PLATELETAGGREGATION

2O2

6

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.

3. Fibrin clot formed by zymogen activation cascadeINTRINSIC PATHWAY

Traps aggregatedplatelets

7Anticoagulant 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).

O

O

CH3

CH3

H

6

Vitamin K

OO

OH

C

O O

OH

H

H

O

OO

OH

CH

CH3

Dicoumarol- Spoiled sweet clover causes fatal hemorrhagic disease in cattle.

Warfarin for thrombosis but high CYP2C9 polymorphism.Drug name Coumadin (rat poison)

Anticoagulants Drugs – Vit. K antagonists

2-3 days before it works don’t admin. again for 3d

8

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.

9

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.

10

Biochemical Structure of Typical Fatty Acids Incorporated Into Membrane

Phospholipids

PGE1

PGE2

PGE3

11

2. Overview of Cyclooxygenase (COX) PathwaysCellular Phospholipid

Free Arachidonic Acid

Prostaglandin H

Prostaglandins

Phospholipase A2

PG H Synthase

ProstacyclinSynthase

ENDOTHELIAL CELL

Prostacyclins

TX Synthase

Thromboxanes

PLATELET

PG Synthases

12Conversion of Arachidonic Acid to PGD2, PGF2 and PGE2

Prostaglandin synthase consists of cyclooxygenase (COX) and peroxidase

COOHO

O

COOH

OOH

O

O

COOH

OH

OH

O

COOH

OH

OH

OH

COOH

OH

OH

O

COOH

OH

Arachidonate

CYCLOOXYGENASE

2O2

AspirinIndomethacin

Ibuprofen

PGG2

PEROXIDASE

Drugs, Carcinogens

Oxidised (Toxic)*

*

PGH2

PGE2

PGF2

PGD2

ISOMERASE

ISOMERASE

REDUCTASE

Cyclooxygenase

_

Peroxidase

Isomerase

Reductase

Isomerase

* NSAID now used as prophylaxis to prevent colon carcinogenesis

Toxin prev. by NSAIDs e.g. Colon cancer

NSAIDs

13

3. Prostaglandin and Prostacyclin and Heart Disease

O

O

COOH

OHPGH2 Prostaglandin H2

Endothelial Cells

COOH

OH

O

PGI2 ProstacyclinCOOH

OH

O

O

Platelets

Prevents Platelet Aggregation

TXA2 Thromboxane A2 COOH

OH

O

Causes Platelet Aggregation

TXB2 Thromboxane B2

H2O

OH

HO

OH

14

Induction andInhibition ofPlatelet Aggregation

15c) Good and bad fatty acid nutrition

1 Linoleate (18C) (2 double bonds)

-Linolenate (18C)(3 double bonds)

+2C

PGE1, PGF1, TXA,

LTAT, PGI1

2. Arachidonate(meat; 4 double bonds 20C)

PGE2, PGF2,TXA2, LTA2,PGI2

3.fatty acidsa-linolenate(vegetables3 double bonds)

eicosapentaenoicacid (fish; 20C5 double bonds)

PGE3, PGF3,

PGI3

TXA3

PGI3 >> PGI2

prevents plateletaggregationinduced by TXA2

very effectively

poor at causingplatelet aggregation(Inuit have noheart disease)

Clinical Science 107,1-11(2004) omega-3 fatty acid nutrition

Platelet aggregation & CV risk

-3-3 fatty acids2. -6 Arachidonate (meat; 4 double bonds 20C)

PGI2

16

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) 18:3• Eicosapentanoic acid 20:5• Docosahexanoic acid 22:6

fish

17

UnstauratedFatty AcidFormation

-Linolenic acid (LNA)

Stearidonic Acid (SDA)

Eicosatetraenoic acid

Eicosapentaenoic Acid

(EPA)

Series 3 Prostaglandins

Series 1 Prostaglandins

Series 2 Prostaglandins

Arachidonic Acid

Dihomogamma Linolenic acid

(DGLA)

-Linolenic Acid (GLA)

Linoleic Acid (LA)

Delta-6- Desaturase

Elongase

Delta-5- Desaturase

Essential Fatty Acids From the diet

OMEGA-3 OMEGA-6

18:3 18:2

CH3 CH3CH3 CH3

18

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)

d) Antiplatelet drugs Cyclooxygenase (COX) and COX inhibitors (NSAIDs)

Colon cancer prevented by low daily doses of aspirin or COX inhib.

• 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)

19

20NSAIDSCOX-1 inhibitors interaction with the COX enzyme- GI toxicityAs PGE protects intestinal mucosa

1) Aspirin - irreversible - acetylates Ser 530, prevents arachidonic acid binding2) 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

21

B) Role of endothelial cells in regulating blood pressure

andNitric oxide formation, signaling

and toxicity

22

Cell Surface Activity (exoenzymes)1) Angiotensin converting enzyme (inhibited by specific dipeptides) e.g.

Captopril*25-50mg (but agranulocytosis risk, cough), Enalapril*1-20 mg. Ramipril 2.5-20 mg (10mg useful for preventing cardiovascular/stroke in diabetics and antihypertensive.

Function of Endothelial cells (line vessel walls)

Angiotensin converting enzyme formation and action

Angiotensinogen (411 aa) synthesised in the liver Plasma

renin (synthesised in kidney, a protease)

Angiotensin I in plasma (10aa)

2aa

angiotensin converting enzyme(endothelial cells) (therefore a dipeptidase)

ACE inhibitors

Angiotensin II in plasma (8 aa)

Vasoconstrictor

blood pressure

23

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

Endothelial cells (line vessel walls)

2) ATPase and 5’-nucleotidase degrade ATP and ADP

3) Inactivate prostaglandins E and F and leukotrienes C4 and D4

Cell Surface Activ. – (Con’t)

24

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)

Plasminogen incorporated into

clot fibrin

Plasmin(a serine protease)

Fibrin

Plasminogen Activator*(a serine protease)Endothelial cell

CO

Dissolve fibrinTherefore activator

Fibrin-boundplasmin

Fibrinolysis

CO?

25

6) Stroke therapy (con’t)

Drug companies are using recombinant DNA technology to makePLASMINOGEN ACTIVATORS to reduce myocardial damagefollowing acute coronary thrombosis (but x 10 expensive and nobetter/safer.

Cigarette smoke CO damages endothelial cell

Prevent clotting e.g., stored blood

1. Complex Ca2+ with citrate or oxalate2. Heparin* helps remove thrombin (produced by mast cells)

26Endothelial cells (con’t)

7) PROSTACYCLIN* synthesis and release (resting state) (PGI2) prevents platelet aggregation

phospholipidarachidonic

acid PGG2 PGI2

phospholipase A2PGI2 synthetaseprostaglandin

synthetase(cyclooxygenase)

Therefore endothelial cells function to prevent thrombosisProstacyclin synthase inhib. by COX2 inhibitor e.g. rofecoxib.

8) EDRF (i.e. nitric oxide, a vasodilator) formed from arginine

Plasma Membrane Activity

278) EDRF - Endothelial Derived Relaxing Factor

i.e. acetylcholinerelaxes vascular smooth muscle only if endothelial cells are present.

ENDOTHELIAL derivedRELAXING FACTOR:- unstable andinactivated by oxygenand hemoglobin only a local hormone - indentified as nitric oxide!Involved in regulation of the microcirculation.

Endothelial cell regulation of blood pressurei.e. effect on smooth muscle cells

28Nitric 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

H2N CH C

CH2

OH

O

CH2

CH2

NH

C

NH2

NH

H2N CH C

CH2

OH

O

CH2

CH2

NH

C

NH2

N

H2N CH C

CH2

OH

O

CH2

CH2

NH

C

NH2

O

NOS NOS

OH

NADPH NADP+

O2 H2O

NADPH NADP+1/21/2

O2 H2O

Analogous to"P450 + reductase"

L-Arginine N-OH Arginine L-Citrulline

+ NO

29

4)

30

CH2ONO2

CHONO2

CH2ONO2

LiverNADPH microsomes

via reduced P450

NO2reduced by mitochondria NO

CH2OH

CHONO2

CH2ONO2

CH2ONO2

CH2OH

CH2ONO2

Glycerol

glucuronidatedurine

CO2

GSH GSHtransferase GSNO GSH

GSSG

NO

Vascular smooth musclecells and endothelial cells

Drugs that act by Generating Nitric Oxide (NO.)1) NITROGLYCERIN - antianginal, antihypertensive, vasodilator (Baxter, Parke Davis)

Adverse effect: HEADACHE unless tolerantTolerance 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

31

2) “AMYL” NITRITE (i.e. isopentyl nitrite) - potent vasodilators which are inhaled (Roberts)

+ GSH transferase (GST 4-4)

CHCH2CH2H3C

H3C

ON O mitochondria

or P450 reductase-ONO

CHCH2CH2OHH3C

H3C

NOAmyl nitrite

GSHGSNO

GSH or reducingsystem

Nitrite anionradical

32

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 met- hemoglobin (Poison control centre) and then Fe3+ of methemoglobin complexes cyanide.

333) NITROPRUSSIDE (NIPRIDE) - Roche ANTIHYPERTENSIVE

For treatment of hypertensive emergencies - infusionwith sterile 5% dextrose. Controlling the rate of NPinfusion allows very rapid decrease of blood pressureduring surgery without overshoot. Also used in treat-ment of chronic hypertension and management ofmyocardial infaction. Adverse effects: cyanide poisoning in rhodanese deficient patients, methemo-globinemia. Biochem. Pharmacol. 42, 5157-65 (1991).

Vas

cula

r ce

ll G

SH

Fe

NONC

NC CN

CN

CN

Fe

NONC

NC CN

CNFe

NONC

NC CN

CN

NO

Cyanide

GS GSSG

REDUCTION by oxyhemoglobinin red blood cells

Mitochondria orNADPH P450

reductase

Fe

NONC

NC CN

CN

CN

SG

O2 O2

34

35

ACTION AND ROLE OF NITRIC OXIDE VERSUS cAMP/Ca2+

Protein Kinase A

Glycogen Phosphorylase

Glucose-1-Phosphate

GlycolysisMitochondria

ATP

MUSCLE CONTRACTION

Endothelial Cells NO

Smooth Muscle Cells

Guanylate cyclase

cGMP Activates cGMP-dependent Protein kinase

Phosphoryln. OfCa2+ regulatory proteins

incr. Ca 2+ in muscle cell

MUSCLE RELAXATION

Protein Kinase A (Active)

Phosphorylase Kinase

cAMP

Ca2+

Protein Kinase A

Glycogen

36

cAMP

37Formation of IP3 and DAG from PIP2 and their signaling Functions