Nitric Oxide in the Body

what rxn is major source?

what enzymes are responsible?

how are enzymes activated in different tissues (3)?

how do NOS antagonists affects of NO?

- The primary source of tissue NO is the enzymatic conversion of arginine to citrulline.  
- The enzymes responsible are known as NOS, nitric oxide synthases.

- Some are constitutive such as neuronal NOS (nNOS or NOS1) and endothelial NOS (eNOS or NOS3). These are activated by increases in intracellular calcium (can be hormone mediated)

- There is also an inducible NOS (iNOS or NOS2) in macrophages and other cells whose transcription is induced by inflammatory mediators (e.g. TNF- α , interferon- γ ).

- shear stress to vessel wall --> inc NO synthesis

- There are antagonists of the synthase such as L-NMMA  an arginine analog. With NOS                blocked, drugs that normally release NO from endothelium lose their dilating effect, as they would if the endothelium were damaged.

How does NO produce vasodilation?

How does the mediator it affects cause the dilation directly?

- NO acts in the vasculature by interacting with the iron in the heme moiety of soluble (cytoplasmic) guanylyl cyclase and increasing the production of cGMP.

- The cGMP via activation of a kinase(PKG) can reduce vascular tone by multiple mechanisms including decreasing cytosolic free calcium, opening Ca-activated K channels, and activation of  myosin light-chain phosphatase which dephosphorylates the myosin light chain and the muscle relaxes.

1) bind/inhibit CYP450 & cytochrome oxidase

2) thiols which are nitrosylated

3) superoxide can convert NO to peroxynitrite which can nitrate protein tyrosines (don't know effect) and cause oxidative damage to nucleic acids

Other NO effects (besides vasodilator and antithrombotic)

Immune system: mediates macrophage-induced cytoxicity

CNS: neuromodulator

Cardiac tissue: causes remodeling

1) oxyhemoglobin

2) superoxides

Therapeutic uses of NO (2)

Systemic effects?

1) Only FDA Approved use: inhalation in term and near-term infants with resp failure associated with pulmonary HTN (no clear improvement in mortality or morbidity)

2) OFF Label use: adults with acute resp distress syndrome, pulmonary HTN, and other (long-term evidence lacking)

In general, minimal systemic effects occur because NO strongly binds to oxyhemoglobin which inactivates it by sequestration and by oxidation to nitrate

Endogenous NO Modulator: Sildenafil (Viagra)

Mechanism?

1) selective inhibitor of phoshopdiesterase 5 (the main one present in the corpuse cavernosum)

2) this incresaes the t_1/2 of cGMP produced in response to NO, therefore, enhancing NO effects

Endogenous NO Modulator: Sildenafil (Viagra)

Metabolized?

Endogenous NO Modulator: Sildenafil (Viagra)

Side Effx (3)?

1) headache

2) disturbance in color vision (sildenafil has only a 10x selectivity for PDE5 vs. PDE6, both exist in the retina)

3) Abrupt loss of vision or hearing (unclear whether caused by the drug)

Endogenous NO Modulator: Sildenafil (Viagra)

Drug Interactions (2)

1) contraindicated in patients using nitrates - taken with nitrates, sildenafil can cause profound hypotension

2) contraindicated in patients taking drugs that have alpha blockading action - hypotension

Endogenous NO Modulator: Sildenafil (Viagra)

Approved uses (3)?

1) Erectile Dysfxn: PDE5 in corpus cavernosum

2) Pulmonary HTN: in pts with this condition PDE5 is inuduced in small pulm arterioles and in right ventricular myocytes (in myocytes the increased cyclic G inhibits PDE3, thus increasing contractility)

3) benign prostatic hyperplasia (tadalafil)

Endogenous NO Modulator: Sildenafil (Viagra)

Potential (not approved) Uses (2)?

1) pulmonary effx may be beneficial in heart failure

2) PDE5 blockade in cardiac myoctyes may limit hypertrophic response to pressure and beta agonism (stops remodeling?)

1) Nitroglycerin (Glyceryl trinitrate)

2) Isosorbide Dinitrate

3) Isosorbide Mononitrate

Organic Nitrate Vasodilators:

Mechanism (2)?

1) interact with some sort of tissue component known as a "nitrate receptor" (unclear) that mediates the NO or NO-like substance production

2) the released NO or NO-like substance activates guanylyl cyclase, inc cGMP conc, and thus induces smooth muscle relaxation

3) they release NO or NO equivalents on their own w/o endogenous NO or NOS. Therefore, they can even dilate vessels that lack an intact vascular endothelium.

Nitroglycerin:

Mechanism (2)?

1) Nitroglycerin is activated by the mitochondrial aldehyde dehydrogenase (ALDH2) to form NO itself (nitrite (NO2-),which is reduced to NO in the mitochondria) or some other NO-like substance. 

2) While this appears to be a therapeutically relevant pathway, it is not the only mechanism for formation of NO from nitroglycerin

Organic nitrate vasodilators:

Mechanism of activation?

Other organic nitrates such as isosorbide  dinitrate and mononitrate do not appear to be ALDH2 substrates, and may be metabolized by CYP450s, or other enzymes to release NO

Organic Nitrate Vasodilators:

What do they affect more venous vs. arterial?

Venous vasodilation > arterial (presumably due to distribution of the enzyme(s) mediating conversion. 

Organic Nitrate Vasodilators:

Low dose effects in systemic vessels (4)?

1) decreased RVEDP, LVEDP, and ventricle size

2) much smaller change in TPR (although face/neck arterioles may dilate --> flush)

3) BP may fall slightly and HR may increase slightly

4) Pulmonary vascular resistance and CO are slight reduced

5) Decreased preload cand decrease O2 demand of the heart, alleviating CHF

Organic Nitrate Vasodilators:

High dose effects in systemic vessels (2)?

1) frank venous pooling as well as arterial dilation

2) decrease in systolic and diastolic BP with reduced CO (may lead to dizziness, pallor, and compensatory symp reflexes to shore up BP & CO)

- if pt has autonomic failure and can't compensate, angina may worsen as CO and coronary flow decrease, and hypotension become severe

Organic Nitrate Vasodilators:

Effects on Coronary vessels (2-4)?

What's the main effect (1)

- may or may not be an increase in total coronary flow

1)  a redistribution of flow in the presence of partial coronary occlusion such that flow increases primarily in ischemic regions

2) endocardial vessel flow increases because of reduction in end diastolic ventricular pressure

3)  Large epicardial vessels dilate (nitroglycerin preferentially dilates vessels > 200µm in diameter) without dilation of the smaller resistance vessels that would produce coronary steal.

4) Collateral flow to ischemic regions increases.

Organic Nitrate Vasodilators:

Mechanism in patients with angina secondary to vasospasm (Prnzmetal's variant, vasospastic angina)?

Organic Nitrate Vasodilators:

Effects on Myocardial O2 Demand (1)?

How?

1) reduces O2 demand

- O2 demand is a fxn of cardia work, which a fxn of ventricular wall tension (determined by preload and to a lesser extent, afterload), heart rate, and myocardial contractility

- nitroglycerin reduces preload and afterload, therefore decreasing oxygen demand of myocardium

Organic Nitrate Vasodilators:

Effect in other smooth muscles?

Organic Nitrate Vasodilators: Tolerance

When does it happen?

How can it be prevented or reversed?

=>  Frequently repeated or continuous exposure to organic nitrates leads to a diminution of effect.

=>  Significant tolerance can be prevented by interrupting therapy for 8-12 hrs a day.

Organic Nitrate Vasodilators: Tolerance

What are some hypotheses for the mechanism (3)?

 i.  Diminished ability for enzymatic conversion of the parent drug to NO or NO-like substances

=> (Aldehyde dehydrogenase has sulfhydryls adjacent to the enzymatic site and these are postulated to be involved in the denitration of nitroglycerin. Prolonged administration of nitroglycerin in vitro decreases the activity of this enzyme, and historically administration of large quantities of reducing agents could prevent or reverse nitrate tolerance).

ii. Alteration in soluble guanylyl cyclase or subsequent step in the vasorelaxant pathway

iii. Physiological compensatory reactions that counter the nitrate effect.

Organic Nitrate Vasodilators: Kinetics

How are they metabolized?

Organic Nitrate Vasodilators: Kinetics of Nitroglycerin tablet/metered dose spray kinetics

Peak plasma?

t_1/2?

Metabolite?

Novelty of this route of administration?

1) Peak plasma: 4-7 mins

2) t_1/2: 1-3 mins

3) Dinitrate metabolite is 1/10 as potent and has a t_1/2 of 40 mins

4) this route avoids first pass metabolism by rapid absorption from mucosa

Organic Nitrate Vasodilators: Available Forms of Administration/Formulations

Nitroglycerin (6)?

Isosorbide dinitrate (2)?

Nitroglycerin: sublingual tablets, metered dose spray, IV preparation, an ointment, a transdermal disc, and a sustained release oral preparation (which has poor bioavailability)

Isosorbide dinitrate: sublingual preparation & oral

Organic Nitrate Vasodilators: Kinetics of Isorbide 5-mononitrate

t1/2?

metabolite of what?

bioavailability? why?

available formulations?

1) t 1/2 = 3-6 hrs

2) which is also a metabolite of isosorbide dinitrate

3) excellent bioavailability p.o.

4) has little first pass metabolism and thus has 

5) It is also available in an extended release preparation

Organic nitrate vasodilators: Therapeutic utility nitroglycerin

Uses (6)

 1.  Effort angina: prophylactic and to terminate attack
                  a. Relieves symptoms but does not            increase survival.

2. Variant (vasospastic) angina
3. Congestive heart failure
4. Unstable angina, myocardial infarction     
                  a. May be beneficial in reducing pain,  no   clear evidence for survival benefit
5. Control of intraoperative blood pressure
6. As ointment: moderate to severe pain associated with chronic anal fissure

Organic Nitrate Vasodilators: Adverse effects/CI and Drug interactions

Side effx (5)?

Contraindication?

Drug interaction (2)?

1. Headache, flushing, tachycardia, dizziness (postural hypotension), fainting.

2. Relative contraindication in hypertrophic cardiomyopathy (the decrease in LVEDP contributes to block of access to aortic valve).          

3. Interaction with sildenafil and other PDE5 inhibitors : see above

A nitrovasodilator, that releases NO rapidly, via poorly understood enzymatic and non-enzymatic mechanisms. 

Once released the NO mechanism of  action is identical to that described above.

1) patients with normal left ventricular function: both arterioles and venules dilate and thus nitroprusside lowers TPR and venous return (so the hypotensive effect is greater when the patient is upright) --> CO generally falls

2)  impaired left ventricular fxn: then afterload reduction predominates and CO rises.

1) tolerance is not reported with continued use

2) patients tolerant to organic nitrates may show no cross tolerance to nitroprusside

Sodium Nitroprusside: Kinetics

administration/duration of effx?

metabolites?

1) must be given IV bc effx of single injxn last only 5 mins

2) metabolism releases NO and CN!

1. Hypertensive emergencies

2. Acute exacerbation of CHF
3. For controlled hypotension in surgery
4. Post MI to decrease oxygen consumption (off label)

Sodium Nitroprusside: Adverse effx (2)?

Prevention for each?

Adverse Effx:

1) Excessive Vasodilation: closely monitor BP and use a variable infusion pump

2) CN poisoning

-usually metabolized by hepatic rhodanese to thiocyanate which is eliminated in the urine, CN poisoning can occur with high infusion rates and it may be necessary to also give thiosulfate to prevent accumulation of CN.

-Thiocyanate is also toxic and with long-term administration, especially with renal impairment, you may get toxicity including anorexia, nausea, fatigue, disorientation and psychosis. (Thiocyanate is dialysable) 

Voltage gated calcium channels consist of a large pore forming  α1 subunit  with 4 domains  each with 6 membrane spanning units(similar to Na channel) and    modulatory subunits ( α2−δ,  β, γ ). 

There are many subtypes of alpha subunits,  they differ among the various classes of Ca channels (L,N,P/Q, R,T) and even within classes.

Ca 2+ Channel Blockers: 3 Classes, 4 drugs

bold is what we have to know

 a. phenylalkylamines--verapamil
 b. benzothiazepine--diltiazem
 c. dihydropyridine--nifedipine is prototype, amlodipine, felodipine, isradipine, nicardipine, nisoldipine

Alpha subunit (the pore forming subunit) - different classes bind at different sites

L channels -  different classes have different selectivities for L calcium channels relative to other cation channels

Ca 2+ Channel Blockers:

Which blockers have more cardiac vs. vascular effx in vivo?

Ca 2+ Channel Blockers:

Which classes have Ca Channel vs. Na Channel blocking activity?

1) decrease the probability that a channel will open given level of depolarization. 

Graph 1: In the heart, modulation of L channels by beta-1 agonists shifts the curve in  the opposite direction.

[image]

Ca Channel Blockers: Effx on Vasculature

what types of channels are effected?

at higher conc?

what's the mechanism?

effx are predominantly arterial or venous?

1) Ca blockers are most potent in blocking voltage activated Ca channels (L); at higher concentrations they can also block receptor activated Ca channels and Ca release from sarcoplasmic reticulum.

2) By reducing cytoplasmic Ca, there is a reduction in Ca-calmodulin activation of myosin light chain kinase, and thus reduced phosphorylation of myosin light chain and blockade of muscle contraction.

3) The effects are predominantly arterial (including coronary arteries), not venous so afterload is significantly reduced but not preload.

Ca Fxns in the Heart (3)

 1) Ca is involved in both action potential formation and conduction (SA and AV nodes)

2 ) in maintenance of the plateau in atrial and ventricular action potential

3) in excitation-contraction coupling (via binding to troponin and thus releasing the inhibitory troponin from blocking actin-myosin interaction)

Ca Channel Blockers: Effx on the Heart of Dihydropyridines

Do they have any?

Why?

What about nifedipine?

NO! - at clinically used doses --> no effx on <3 contractility or nodal conductioin

1) somewhat more potent on vasculature then they are  on heart: at concentrations that directly affect cardiac channels, the profound arterial dilation results in compensatory sympathetic drive on the heart that virtually obliterates the direct action of the drug.

Nifedipine: In nodal tissue, in addition to the reflex response it does not show use-dependent block and does not increase the recovery time for inactivated Ca channels as do verapamil and diltiazem.

Ca Channel Blockers: Effx on the Heart of Verapamil (4)

1) increases recovery time of cardiac L channels (by selective affinity for inactivated channels --similar to mechanisms of action of local anesthetics at the sodium channel) and 
2) has rate dependent blocking effects

3) verapamil will slow AV conduction (increases the PR interval) and can have 4) negative inotropic effects: because verapamil is more potent than dihydropyridines at cardiac L channels relative to vascular channels, 

At high concentrations, verapamil also can block sodium channel

also has higher cardiac L channel potency relative to vascular potency

 Ca Channel Blockers: Net hemodynamic effects of the dihydropyridines vs. verapamil

for each of the dihydropyridines?

Nifedipine: acutely decreases BP with little venous pooling, cardiac output and HR increase. Other drugs in the class are similar.

Amlodipine: produces less reflex tachycardia, perhaps because with its plasma half-life of 1-2 days and slow absorption, there is little peak to trough variation with chronic once a day dosing.

What do the net hemodynamic effx of Ca Channel blockers depend on (3)?

1) dose, 2) cardiac status, 3) reflex responsiveness

Memorize the effx of Ca channel blockers (Nifedipine, Diltiazem, Verapamil) on

Coronary vasodilation

Peripheral vasodilation

Myocardial contractility

Cardiac output

Resting Heart Rate

AV conduction

Ca Channel Blockers: Kinetics

What is common administration and metabolism for all?

How does first pass metabolism affect F of each?

Half-lives?

IV formulations for which drugs?

Active metabolites for which drugs?

1) All are orally available and metabolized by the liver

2) verapamil 0.2- 0.3, diltiazem 0.4, nifedipine 0.3- 0.6, amlodipine 0.6-0.9

3) half-lives vary, but short acting drugs are available in sustained release preparations

4) IV prep for verapamil and diltiazem

5) verapamil and diltiazem have active metabolites (less potent)

 a. Angina - amlodipine nifedipine, diltiazem, verapamil
 b. Arrhythmias - verapamil, diltiazem
 c. Hypertension - amlodipine, nifedipine, diltiazem, verapamil
 d. Prophylaxis - pts with angiographically documented CAD without heart failure--amlodipine

 a. Hypertrophic cardiomyopathy--verapamil
b. Migraine-- verapamil
c. Raynaud's syndrome--nifedipine, diltiazem, amlodipine
d. Diabetic nephropathy--amlodipine, verapamil, diltiazem

Ca Channel Blockers:

Adverse effects (3)

Adverse effects specific to Vera/Dilt (3)

CIs for Vera/Dilt (2)

Toxicity via what mechanism for Vera/Dilt?

1. Excessive arteriolar dilation: dizziness, hypotension, headache, flushing, edema
2. increased anginal discomfort w/ dihydropyridines (bc they are short acting): in some if given without a beta  blocker, due to sympathetic reflex increasing cardiac oxygen demand-->long-acting preparations preferred

3. Interference with other calcium dependent processes: constipation, GI reflux (relaxation of lower esophageal sphincter)
4. For verapamil and diltiazem:  bradycardia, exacerbation of CHF, AV block--especially if given with a beta blocker.

Relative CIs for verapamil and diltiazem are SA or AV nodal disturbances or ventricular dysfunction.

Both are also CYP3A4 inhibitors, Verapamil, in addition blocks the P-glycoprotein transporter.

opens K channels --> hyperpolarizes smooth muscle cells --> dec Ca entry --> relaxation

A pro-drug that is metabolized by hepatic sulfotransferase to minoxidil N-O sulfate (a minor metabolite) which activates the ATP-modulated K channel  (increased ATP closes the channel) thus moving the resting potential of smooth muscle towards the K equilibrium potential (hyperpolarization) which in turn decreases voltage dependent Ca entry and thus induces relaxation.

1) inc heart rate

2) inc contractility

Its action is essentially that of an arterial vasodilator which induces sympathetic baroreceptor reflexes that increase cardiac rate and contractility.

3) inc CO

There is marked increase in cardiac output secondary to increased venous return (due to distribution of blood to regional vascular beds with fast time constant for return).

a. Retention of salt and water secondary to increased renin production Diuretics can control the fluid retention.
b. Myocardial ischemia: induced because sympathetic reflex can increase cardiac oxygen consumption MI in patients with coronary artery disease can be induced. Beta blockers can be used to control this.
c. pulmonary hypertension in patients with left ventricular hypertrophy and diastolic dysfunction (loss of ventricular compliance): Increased venous return coupled with the retention of salt and water can cause        
d. Flattened and inverted T waves secondary to the activation of cardiac K channels that shorten APD.
e. Hypertrichosis--see therapeutic utility

a. hair growth (Rogaine): Primary current use (topical preparation)

b. Severe chronic hypertension refractory to other agents and combinations: Never use it alone, always with a diuretic and beta blocker (see above)

1) inc HR and contractility: bc of powerful symp baroreflex

2) inc renin and inc salt and water retention: bc of powerful symp baroreflex

3) direct positive inotropic action

4) vascular superoxide production

Hydralazine: Kinetics

metabolism?

what variable is the main determinant of half-life?

1) Liver - N acetylation is main form of metabolism

2) hepatic blood flow: hepatic clearance is high extraction

- there are also other pathways for metabolism, so that pharmacogenetic variation in acetylator status is not the main determinant of half life

Hydralazine: Therapeutic Utility (2)

Used alone? Why?

1) hypertension developing in mid to late pregnancy

2) hypertensive emergencies in pregnancy

Not generally used alone for tx of chronic HTN because of cardiovascular reflex effx and fluid retention

Hydralazine + isosorbide dinitrate is an adjunct therapy for black CHF patients (showed to dec mortality only in black patients)

Hydralazine:

CI?

Adverse Effx (4)?

 The probability of coronary steal is so high that it is CI in patients with coronary artery disease. - Similar to minoxidil without the consequences shortened APD.

Immune rxns - drug-induced lupus erythematosis most common (generally after at least 6 months of use of higher than minimally effective doses)--ANA conversion is faster in slow acetylators, not all ANA positive patients actually develop lupus. (may also induce hemolytic anemia, glomerulonephritis, vasculitis)