iv g. well-established second messengers camp second messenger: cyclic amp 1. camp is 2nd messenger...

IVG. Well-established Second Messengers

cAMPSecond messenger: Cyclic AMP

1. cAMP is 2nd messenger - released into cytoplasm due to 1st messenger (hormone) binding; a number of others in eukaryotic cells

2. 2nd messengers can activate many cell activities leading to large-scale, coordinated response

IVG. Well-established Second Messengers cAMP

mediates such hormonal responses as:

the mobilization of stored energy*the breakdown of carbohydrates in the liver*the breakdown of triglycerides in fat cells

increased rate and strength of heart muscle contraction

the conservation of water by the kidneyscAMP inducing agent=vasopressin

Ca++ homeostasiscAMP inducing agent = parathyroid hormone

many many other responses mediated by cAMP

Beta-adrenergic catecholamines


GTP

Agonist binding

AC

Activation of Gs and stimulation of the effector Adenylyl Cyclase (AC)

ATP

cAMP

Conversion of ATP to cyclic AMP (cAMP) by AC.

Cat

CatReg

Reg

cAMP-dependent protein kinase[CAMP kinase]

Reg= regulatory regionCat= catalytic region


GTP

Agonist binding

ACATP

cAMP

Cat

Reg

Reg

cAMP-dependent protein kinase[CAMP kinase]

cAMP

cAMP

Binding of cAMP to Reg sites releases the cat regions which can phosphorylate proteins.

SubstrateSubstratePATP

Cellular Response Cat


SubstrateP

Cellular Response

Can include any of these:

*Enzyme activation*Protein synthesis*Muscle relaxation*Nerve stimulation*Hormone secretion


When the agonist stimulus stops, the intracellular actions of cAMP are terminated by three mechanisms (1-3).

GTP

Agonist dissociates

ACATP

cAMP

GDP

GTP hydrolysis

5’-AMP

Cyclic nucleotide phosphodiesterases

X X

CAMP kinase activation is inhibitedRe-establishment of the tetramer

SubstrateP

SubstrateP

Phosphatases

Diminished cellular responseCatCat Reg

Reg

Phosphorylated substrate generated by CAMP kinase is de-phosphorylated

1 2

3


GTP

Agonist dissociates

ACATP

cAMP

GDP

GTP hydrolysis

5’-AMP

Cyclic nucleotide phosphodiesterases

X

FYICaffeineTheophyllineOther methylxanthines

Act as competitive inhibitors ofphosphodiesterases

What would you expect the effect ofcaffeine on cAMP levels to be?

How about on CAMP kinase?


Different cells express different types of substrates for CAMP kinase, which helps explain some of the tissue-specific effects:

CAMPPhosphorylase KinasePhosphorylase Kinase

ATP

P

Glucose released from glycogen

Glycogen Synthase

CAMP

ATPGlycogen Synthase

P

Inhibition of glycogen synthesis

In LiverActivated by phosphorylation

inactivated by phosphorylation


Ca++ (Calcium) and Phosphoinositides

Another well-studied 2nd messenger system involves receptor-mediated stimulation of phosphoinositide hydrolysis.

Some of the agonists, hormones and growth factors that trigger this pathway bind to G-protein coupled receptors (Gq-coupled)

Receptoracetylcholine (muscarinic)

alpha1-adrenergic

platelet activating factor

serotonin (5-HT 1C and 5-HT 2)

2nd messenger

Ca++ &phosphoinositides


Ca++ (Calcium) and PhosphoinositidesInositiol-Phosphate Pathway

A. Ligand binding activates G protein

B. G protein activates phospholipase C (PLC)

C. PLC hydrolyzes phosphatidyl inositol 4,5 bis-phosphate to diacylglycerol (DAG) and inositol 1,4,5 tris-phosphate (IP3) - both second messengers

1. IP3 goes to ER where it stimulates the - release of calcium and activates protein

kinases

2. DAG stays in membrane where it binds and activates protein kinase C (PKC)



PLC family that produces two second messengers, diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3) by hydrolyzing the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2).

GTP

Agonist binding

PLC PIP2DAG

IP3Gq proteinstimulation



IP3 goes to ER where it stimulates release of calcium activates protein kinases

GTP

Agonist binding

PLC PIP2DAG


Ca++ Ca++

Ca++Ca++



Released Ca++ binds to calmodulin.

GTP

Agonist binding

PLC PIP2DAG


Ca++ Ca++

Ca++Ca++

Ca++Ca++

Ca++ release

ER

Calmodulin

Calmodulin



Calmodulin becomes activated and stimulates signaling through calcium/ calmodulin dependent protein kinases

GTP

Agonist binding

PLC PIP2DAG

IP3Ca++ Ca++

Ca++Ca++Ca++Ca++

Ca++ release

ER

Ca++Ca++Calcium/Calmodulin-Dependent

Protein Kinase

Calmodulin



GTP

Agonist binding

PLC PIP2DAG

IP3Ca++ Ca++

Ca++Ca++Ca++Ca++

Ca++ release

ER

Ca++Ca++Calcium/Calmodulin-Dependent

Protein Kinase

InactiveActive P

Calmodulin



Ca++Ca++

Calcium/Calmodulin-Dependent Protein Kinase

Substrate Substrate

P

ATP

Cellular Response

active



GTP

Agonist binding

PLC PIP2DAG


Meanwhile, DAG stays in membrane where it binds and activates protein kinase C (PKC)

PKCinactive



GTP

Agonist binding

PLC PIP2DAG


Activated PKC will phosphorylate certain substrates involved in cellular response.

PKCactive

Substrate

Substrate

P

ATP,Ca++

Cellular Response



In addition to general calcium/calmodulin-dependent protein kinases that can phosphorylate a wide variety of substrates,

Different cell types may contain one or more specialized Calcium/calmodulin-dependent protein kinases with limited substrate specificity

(eg. myosin light chain kinase).

At least nine different types of PKC have been characterized.


Ca++ (Calcium) and PhosphoinositidesMultiple mechanisms exist to terminate signaling by this PLC pathway:

IP3 is rapidly dephosphorylated by phosphatases

DAG is either phosphorylated and converted back to into phospholipids

ordeacylated to yield arachidonic acid

Ca++ is actively removed from the cytoplasm by calcium ion pumps (into ER)

These and other nonreceptor elements of the calcium-phosphoinositide signaling pathway are now becoming targets for drug development.


cGMP

cGMP (cyclic guanosine-3’,5’-monophosphate) has established signaling roles in only a few cell types.

In intestinal mucosa and vascular smooth muscle, cGMP-based signal transduction is initiated when:

*ligand binds to extracellular domain of receptor*ligand binding stimulates intracellular guanylyl

cyclase activity*cGMP activates cGMP-dependent protein kinases

GTP cGMPGuanylyl cyclase

cGMP-dependent protein kinasecGMP-dependent protein kinase

Substrates get phosphorylated


cGMP

Increased cGMP

ANF-R

GCactivity

Atrial natriuretic factorBinds its receptor

GTP

cGMP

cGMP accumulation

Guanylyl cyclase

NOGTP

cGMP

cGMP accumulation

The lipid-soluble gas nitric oxide (NO) is released by nearbyvascular endothelial cellsAnd direct activates the enzyme.

Several vasodilator drugs mimic NO

IVH. Phosphorylation: a Common Theme

*Reversible phosphorylation is involved in almost all 2nd messenger systems.

*Phosphorylation plays a key role in every step of signaling:

Regulation of receptors (eg. autophosphoryltion; desensitization)

Regulation of kinases and kinase-substrates

modulating cellular responses

IVH. Phosphorylation: a Common Theme

Think of phosphorylation as a molecular ‘memory’

phosphorylation records the memory

dephosphorylation erases the memory, often taking longer than is required

for dissociation of ligand

Lastly,cAMP, Ca++ and other 2nd messengers can use the presence or absence of kinases or kinase substrates to produce different effects in different cell types.

V. Receptor Classes and Drug Development Receptor SubtypesEvidence for receptor subtypes arose because agonists that supposedly mimicked the same neurotransmitter had radically different postsynaptic effects at different sites.

For example, although both smooth and striated muscle contain acetylcholine receptors, nicotine exerts potent agonistic effects on striated muscle, yet is nearly ineffective on smooth muscle.

Similarly, muscarine exerts potent agonistic effects on smooth muscle, yet is much less effective on striated muscle. Thus acetylcholine receptors come in at least two varieties, nicotinic and muscarinic.

V. Receptor Classes and Drug Development The same chemical can act on completely different receptor classes:

Acetylcholineactivates nicotinic acetylcholine receptors

*Ligand-gated ion channel

activates muscarinic acetylcholine receptors*G-protein coupled receptor (Gq)

Each receptor class usually includes multiple subtypes of receptor, often with significantly different signaling or regulatory properties.

V. Receptor Classes and Drug Development The same chemical can act on completely different receptor classes:

Norepinephrineactivates many structurally-related receptors

beta-adrenergicG protein-coupled, Gs; increased heart rate

alpha1-adrenergicG protein-coupled, Gq; vasoconstriction

alpha2-adrenergicG-protein coupled, Gi; opening of K+ channels; decreased heart

rate

V. Receptor Classes and Drug Development The existence of multiple receptor classes and subtypes for the same ligand has opened up opportunities fro drug development:

Propranolol, a selective antagonist of beta-adrenergic receptors

can reduce heart rate without preventing the sympathetic nervous system from inducing

vasoconstriction(because it acts at beta-adrenergic and not

alpha)(alpha mediates vasoconstriction)

V. Receptor Classes and Drug Development

Drug selectivity may apply to structurally identical receptors expressed in different cells

for example:the drug tamoxifen acts as an

*antagonist on estrogen receptors in mammary tissue (useful as treatment in breast cancer)

*agonist on estrogen receptors in bone.(may help against osteoporosis)

*partial agonist on estrogen receptors in the uterus

(stimulates endometrial cell proliferation)


Drug selectivity may apply to structurally identical receptors expressed in different cells

WHY?

different cell types express different accessory proteins which interact with steroid receptors and change the functional effects of drug- receptor interaction.


NEW DRUG DEVELOPMENTnot confined to agents that act on receptors

clinically useful agents might be developed that act selectively on specific:

G proteins

kinases

phosphatases

or the enzymes that degrade 2nd messengers

VI. Relationship Between Drug Dose and Clinical Response

When faced with a patient who needs treatment:

*variety of possible drugs

which one will drug will produce a maximal benefit?

what kind of dosing regimen is required?

The prescriber must understand:*how drug-receptor interactions underlie the

relations between dose and response in patients

*are there known variations in responsiveness to the drug? Toxic side effects?

VIA. Dose and Response in PatientsGraded Dose-Response Curves

show effects on a continuous scale and the intensity of the effect is proportional to the dose.

(what we’ve been discussing thus far)

EFF

EC

T(%

of

maxim

um

)

Log concentration

Which is more potent?A lower dose needed to elicit 50% max response

VIA. Dose and Response in PatientsGraded Dose-Response Curves

When choosing among drugs and determining appropriate doses of drug, it is important to consider each drug’s potency and maximal efficacy.

A BPotencyrefers to the concentration EC50 or dose ED50 of drug required to produce 50% of that particular drug’s maximal effect.

Resp

onse

100%

50%

Log [Drug]

EC50 EC50

VIA. Dose and Response in Patients

A BR

esp

on

se

100%

50%

Log [Drug]

EC50 EC50

Which is more potent?C lower dose needed to elicit 50% of a particular drug’s max response 25% Potencyrefers to the concentration EC50 or dose ED50 of drug required to produce 50% of that particular drug’s maximal effect.

C

EC50

NOTE: Drug C acts as a partial agonist.

VIA. Dose and Response in PatientsEfficacy

the measure of an effect produced by a drug.

In this example, the maximal efficacy of drug C is less than the maximal efficacies of drugs A and B.

Drugs A and B have the same efficacy.

A B

Resp

on

se

100%

50%

Log [Drug]

ED50 ED50

25%

C

ED50

VIA. Dose and Response in PatientsEfficacy

depends on factors such as:

route of administration

absorption

distribution throughout the body

clearance from the blood or the site of action


Shape of Dose-Response Curves

Extremely steep dose response curves may have important clinical consequences if the upper portion of the curve represents an undesirable extent of response (eg. coma caused by a sedative-hypnotic).

Steep dose-response curves can also be produced by a receptor-effector system in which most receptors must be occupied before any effect is seen.

EFF

EC

T(S

ed

ati

on

)

Log [Drug]

More desirablesleep

coma Undesirable


Quantal Dose-Effect Curves

Graded dose-reponse curves are limited in their application to clinical decision making:

*impossible to use them if pharmacologic response is an ‘either-or’ event (a quantal event)

prevention of: convulsions, arrhythmias, death

*clinical relevance of a graded dose response curve in a single patient may be limited in its application to other patients

potential variability among patients in *severity of disease*responsiveness to drug

VIA. Dose and Response in PatientsQuantal Dose-Effect Curves

These problems may be avoided by:

determining the dose of drug required to produce an effect of specific magnitude in large numbers of patients (or animals) and then

plotting the cumulative frequency distribution of responders vs. the log dose.

Patients tend to respond to drugs in a distribution similar to a Gaussian normal curve.

Perc

en

t of

Ind

ivid

uals

Resp

on

din

gTo T

reatm

en

t (e

g.

for

head

ach

e) 100

Log [Drug]

50

ED50

Dose at which 50% of patients exhibit the specified quantal effect


Quantal Dose-Effect Curves

Quantal dose-effect curves may also be used to generate information regarding the margin of safety.

Toxic effects of a drug on humans or animals can also be assessed by plotting the cumulative frequency distribution of responders vs. the log dose.

As for the therapeutic effects, potentially toxic effects of drugs display a distribution similar to a Gaussian normal curve in people or animals.

In order for a drug to have a high margin of safety in patients or animals, therapeutic effects should be observed at lower doses than toxic effects.

VIA. Dose and Response in PatientsPerc

en

t of

Ind

ivid

uals

Resp

on

din

g 100

Log [Drug]

50

TD50

Dose at which 50% of patients exhibit a toxic effect

ED50


Cumulative percent of patients exhibiting therapeutic effect

Cumulative percent of patients exhibiting toxic effect

Quantal curve of a hypothetical drug that provides relief for headaches.

Therapeutic effects and toxic effects do not overlap


en

t of

Ind

ivid

uals

Resp

on

din

g 100

Log [Drug]

50

TD50


ED50




Quantal curve of a second drug that provides relief for headaches.

Therapeutic effects and toxic effects slightly overlap


en

t of

Ind

ivid

uals

Resp

on

din

g 100

Log [Drug]

50

TD50


ED50




Quantal curve of a third drug that provides relief for headaches.

Therapeutic effects and toxic significantly overlap


Perc

ent

of

Indiv

iduals

Resp

ondin

gNo overlap in the quantal dose-response curve is highly desired (to avoid unwanted toxic effects), but not always possible.

The margin of safety of a drug will depend on the ratio between ED50 and TD50.

100

Log [Drug]

50

TD50ED50

The therapeutic index is defined as the ratio of

TD50-------ED50

What can be said of a drug’s safety if this ratio is equal or close to 1?

VIA. Dose and Response in PatientsThe therapeutic index (TI) of a drug in humans is almost never known

most studies involving obvious toxicity are halted

toxicity studies in animals are used to estimate a drug’s therapeutic index.

In summary,

Both graded and quantal dose-effect curves provide information concerning the potency and selectivity of

drugs.

The graded dose-response curve indicates the maximal efficacy of a drug.

The quantal dose-effect curve indicates the potential variability of responsiveness among patients.

VIB. Variation in Drug Responsiveness

Individuals may vary in responsiveness to a drug.

Responses include:idiosyncratic

an unusual response very rarely observed in most patients

hypo-responsivedrug effect is smaller than expected

hyper-responsivedrug effect is larger than expected



Responses include:tolerance

responsiveness decreases as a consequence of continued drug administration

tachyphylaxisresponsiveness diminishes rapidly after

administration of the drug

When these effects occur, the dose should be modified or the drug itself changed.

NOT COVERED IN LECTURE... PLEASE REANOT COVERED IN LECTURE... PLEASE READD



FACTORS to be considered in variable drug responses:

age body size

sex disease state

simultaneous administration of other drugs

Four general mechanisms may contribute to variations in drug responsiveness.

variable drug response may be caused by more than one of these mechanisms



1. alteration in concentration of drug that reaches the receptor

*rate of drug absorption

*altered drug distribution in body compartments

*altered drug metabolizing enzymes

repeated measurements of drug concentrations in blood during the course of treatment are often helpful in dealing with the variability of clinical response caused by pharmacokinetic differences among individuals.



2. variation in concentration of an endogenous receptor ligand

example:saralasin, a weak partial agonist of angiotensin receptors

this agent will lower blood pressure in patients with hypertension and lots of angiotensin

in patients with low levels of angiotensin, this agent will elevate blood pressure



3. alterations in number or function of receptors

*increases or decreases in the number of receptor sites

likely to account for much of the variability in response to SOME drugs among individuals

(particularly drugs that act at receptors for hormones, catecholamines,

neurotransmitters)

Not rigorously established in humans.. BUT:eg. thyroid hormone increases the number

of beta- adrenergic receptors in rat heart muscle and cardiac sensitivity to catecholamines.

tachycardia has been observed in patients with overactive thyroid glands.




in some cases, the agonist can induce a ‘down-regulation’ of its own receptor

eg. receptor internalization and degradation >>> synthesis

in other cases,

an antagonist may increase the # of receptors in a cell or tissue by preventing down-regulation. When the antagonist is withdrawn, the elevated number of

receptors can produce an exaggerated response to physiological concentrations of the a agonist.




Withdrawal symptoms can often occur when administration of an agonist is discontinued.

the # of receptors which has been decreased by drug-induced down-regulation is too low for endogenous agonist to produce effective stimulation.For example,

clonidinean agonist of the alpha2-adrenergic receptor

whose activity reduces blood pressure

Can produce hypertensive crisis if withdrawn abruptly, probably because the drug down-regulates alpha2 receptors.




Various therapeutic strategies can be used to address receptor-specific changes in drug responsiveness:

*tolerance may require increasing the dose or substituting a different drug

*the down- or up- regulation of receptors may make it dangerous to discontinue certain drugs abruptly.

the patient may have to be weaned slowly from the drug and watched carefully for signs of

withdrawal.



4. changes in components of response distal to receptor

Although drugs act through receptors, drug response depends on

the functional integrity of biochemical processes in the responding cell and physiologic regulation by interacting organ systems.

CHANGES IN POSTRECEPTOR PROCESSES represent the largest and most important class of mechanisms that cause variations in drug responses.




Characteristics that may limit the clinical response:

age and general health of the patient

severity and pathophysiologic mechanism of the disease

wrong diagnosis (e.g.)congestive heart failure will not

respond to agents that increase myocardial contractility if the pathophysiologic mechanism is unrecognized stenosis of the mitral valve rather than myocardial insufficiency.




Unsatisfactory therapeutic response can often be traced to compensatory mechanisms in the patient that respond to and oppose the beneficial effects of the drug.

For example,tolerance to an antihypertensive vasodilator

agent may be due to compensatory increases in sympathetic nervous response as well as fluid retention by the kidney.

The patient in which something like this is occurring may require additional drugs to achieve a useful therapeutic response.

VII. Clinical Selectivity: Beneficial vs. Toxic Effects of Drugs

Drugs are classified according to their primary effect

BUT no drug causes only a single, specific effect!

It is more appropriate to say that drugs are selective, rather than specific, in their actions and receptor affinities.

that is, they bind one or a few types of receptor more tightly than any other receptors.


Selectivity can be measured by:*comparing binding affinities of a drug to different receptors

*comparing EC50 values for different effects of a drug

Two types of drug effects:

Beneficial (Therapeutic)

Toxic (side effect)


Beneficial and toxic effects mediated by the same receptor-effector mechanism:

Direct pharmacologic extension of the therapeutic actions

eg. bleeding caused by excess anticoagulant therapy

(the dose makes the poison)

HOW to deal with this?judicious management of dose can avoid toxicity(along with careful patient monitoring)

not administering the drug at all(use of an alternate drug)



In some instances, a drug is clearly necessary and beneficial but produces unacceptable toxicity at doses that yield benefit.(in such cases, addition of another drug may be possible)

eg. prazosin, an alpha1-adrenergic receptor

antagonist

acts on receptors in vascular smooth muscle to reduce blood pressure

as a consequence, patients may suffer postural hypotension when standing (sudden drop in BP

when standing)



as a consequence, patients may suffer postural hypotension when standing (sudden drop in BP

when standing)

Appropriate management?In addition to alpha1 receptors, BP is regulated by changes in blood volume and tone of arterial

smooth muscle.

Giving a diuretic and a vasodilator may allow the dose of prazosin to be lowered with relief of postural hypotension and continued control of blood pressure.



DRUG

Receptor

DRUG

Receptor

ToxicTherapeutic

Occur within the same tissue

eg. vascular smooth muscle; prazosin


Beneficial and toxic effects mediated by the same receptor-effector mechanism:Postural hypotension:

While at rest, quadrupeds have a distinct orthostatic advantage over bipedal humans because their blood reservoirs (mostly veins) are at a similar level as the brain and heart. In contrast, a human in the act of standing has approximately 750 mL of thoracic blood abruptly translocated downward. Standing fills venous blood reservoirs below the heart, removes venous return from the heart, and reduces cerebral perfusion because of the hydrostatic change in BP. In contrast, more than 70% of a dog's vascular capacitance is situated at or above cardiac level, and the dog's brain is at a similar level.


Beneficial and toxic effects mediated by the same receptor-effector mechanism:Postural hypotension (cont):Upright posture in humans, therefore, is a fundamental stressor. Upright posture requires rapid and effective circulatory and neurologic compensations to maintain BP, cerebral blood flow, and consciousness. Without these compensatory mechanisms, the brain's precarious position well above the neutral cardiac point (roughly at the right atrium) and the presence of large venous reservoirs below the neutral point would cause BP to decrease rapidly because of gravitational pooling of blood within the dependent veins; cerebral ischemia and loss of consciousness would follow rapidly. Once consciousness and postural tone are lost, the resultant fall would render a person recumbent, remobilizing the blood and restoring consciousness. Evolution apparently has dictated a trade-off between manual dexterity and orthostatic competence.

http://www.emedicine.com/ped/topic2860.htm


Beneficial and toxic effects mediated by identical receptors but in different tissues or by different effector pathways:

Many drugs produce their desired effects and toxic effects by acting at the same receptor

digitalis glycosides (inhibit Na+/K+ ATPase)augment cardiac contractilityBUT alsocardiac arrhythmias, g.i. effects, vision

methotrexateinhibition of dihydrofolate reductase

death of tumor cells BUT also,death of healthy cells



Therapeutic strategies to avoid these toxicities?

*drug should ALWAYS be administered at the lowest dose that produces acceptable benefit

(complete abolition of symptoms may not be achieved)*adjunctive drugs that act through different receptor mechanisms may allow lowering the dose of the first drug, decreasing its toxicity.

*specifically placing the drug in parts of the body where it will have reduced toxicity (eg. infusion of drug into a tumor)



DRUG

Receptor

DRUG

Receptor

ToxicTherapeuticTissue 1

Tissue 2

eg. digitalis; therapeutic in cardiac contractility; toxic effects in gastrointestinal tract and eye


Beneficial and toxic effects mediated by different types of receptors:

New drugs are emerging with improved receptor selectivity.

DRUG

Receptor1 DRUG

Receptor1

Response 1

Receptor2

DRUG

Receptor2

DRUGResponse 2

eg. alpha and beta-adrenergic agonists

iv g. well-established second messengers camp second messenger: cyclic amp 1. camp is 2nd messenger...

Documents

messengers campwhen

messengers campmediates

messengers ca calcium

camp levels

messengers campdifferent

cyclic amp camp

intracellular actions

protein synthesis