Pharmacodynamics at receptor level

2. MECHANISM OF DRUG
ACTION
DR PRANESH PAWASKAR
FirstYear Resident
Dept. of Pharmacology
L.T.M.M.C. Sion, Mumbai
400022
Date: 08/10/2016
2

3. OVERVIEW
• INTRODUCTION.
• PHARMACODYNAMIC CONCEPTS.
• QUANTITATIVE ACTION OF DRUG INTERACTIONS WITH
RECEPTORS.
• PHARMACODYNAMICAL VARIABILITY.
• MECHANISMS OF ACTION.
• RECEPTOR DESENSITISATION AND REGULATION OF
RECEPTORS.
• PHARMACODYNAMIC INTERACTIONS IN A MULTICELLULAR
CONTEXT. 3

4. INTRODUCTION
4
PHARMACOLOGY
PHARMACOKINETICS
PHARMACODYNAMICS

5. INTRODUCTION
• PHARMACOKINETICS : Understanding the Absorption,
Distribution, Metabolism and Elimination.
• PHARMACODYNAMICS : Study of the biochemical and
physiological effects of drugs and their mechanism of action.
5

6. WHY TO STUDY PHARMACODYNAMICS?
• Basis for the rational therapeutic use drug.
• Design of new and superior therapeutic agent.
• Effect of drug on body.
• In contrast _ _ _ _
• Many adverse effects and events of drugs and drug
toxicities can be anticipated by understanding Drug’s
Mechanism Of Action.
• Safety and Success.
6

7. PHARMACODYNAMIC CONCEPTS
• Effects of most drugs = macromolecular components ; Drug
Receptor or DrugTarget = cellular macromolecule or
macromolecular complex.
• Alter the rate or magnitude of an intrinsic cellular response.
• Receptors located on = surface of cells, nucleus.
• Acceptors = interact with acceptors = alter the pharmacokinetics.
• Receptors for hormones, growth factors, transcription factors, and
neurotransmitters; the enzymes of crucial metabolic or regulatory
pathways = Proteins ; Others like DNA = Cancer
chemotherapeutics. 7

8. PHYSIOLOGICAL RECEPTORS
• Majority drug receptor = endogenous regulatory ligand proteins =
physiological receptors = great selectivity
• Drugs that bind to physiological receptors and mimic the regulatory
effects of the endogenous signalling compounds are termed
Agonists.
• If the drug binds to the same recognition site as the endogenous
agonist = Orthosteric site = Primary Agonist.
• Allosteric (Allotopic) Agonists bind to a different region on the
receptor referred to as an allosteric or Allotopic site.
• Drugs that block or reduce the action of an agonist are termed
Antagonists. 8

10. • Competition with an agonist for the same or overlapping site =
Syntopic
• Competition with an agonist for the other sites on the receptor =
Allosteric
• Competition with an agonist by combining with agonist = Chemical
• In-Directly inhibiting functions of agonist = Functional
• Agents that are only partly as effective as agonists regardless of the
concentration employed are termed Partial Agonists.
• Many receptors show constitutive activity in the absence of a
regulatory ligand ;drugs that stabilize such receptors in an inactive
conformation are termed Inverse Agonists. 10

11. 11

12. DRUG SPECIFICITY
• Strength of reversible interaction between drug and receptor =
Dissociation constant.
• Chemical structure of drug determines affinity, intrinsic activity
and drug’s specificity.
• Drug acting on a single receptor expressed on limited no. of cells
exhibit high specificity = Ranitidine (H2). And vice-versa.
• Numerous examples of drugs having discrete action e.g. (Digoxin
– NaK ATPase), (methotrexate-dihydrofolate reductase),
(lidocaine- peripheral nerves,heart,CNS), (immunosuppressive
drug- opportunistic infection), (Furosemide- muscle cramps,
arrhythmias)
12

13. • Broad specificity = clinical utility more = adverse effects more
• E.g. Amiodarone (cardiac arrhythmia) (Na,Cl,K Beta) = Thyroid
hormone (structural similarity)
• Sterioisomerism (Sotalol- d/k blocker and l/beta agonist) other
drug Labetolol
• Chronic administration = Down Regulation/ Desensitization = e.g.
Nitroglycerin = also known as Tachyphylaxis.
• Differential tolerance development = Opioid to analgesia but not
Respiratory depression
13

14. • No receptor mechanism for = Aluminium hydroxide and
Magnesium hydroxide, Mannitol act by colligative property,
Cholestyramine resins acts by decreasing dietary cholesterol
absorption.
• Antibiotics mostly act by inhibiting receptor or enzyme specific to
pathogen not host.
• Antibiotics such as Penicillin inhibit a key enzyme required for
synthesis of bacterial cell walls, an enzyme not present in humans
or animals.
• Mutation of the target receptor, increased expression of enzymes
that degrade or increase efflux of the drug from the infective
agent, and development of alternative biochemical pathways.
14

15. STRUCTURE-ACTIVITY RELATIONSHIPS
AND DRUG DESIGN
• Detail knowledge of a drugs molecular target can inform about
development of new drugs efficacy and toxicity.
• Sequencing entire human genome identified novel receptors for
them but ligands for them not known. (Orphan receptors)
• Orphan receptors are still found in G Protein Coupled Receptors and
Nuclear Hormone Receptor Families.
• Transgenic animal model helps in predicting Agonism OrAntagonism
by genetically altering the receptors mechanism and function
15

16. • Even minor changes in structure can bring major differences in
drug pharmacological activities
• This stringent nature of structure to bind to its receptor can be
illustrated by capacity of receptors to interact selectively with
optical isomers.
• dl-Hyoscyamine and its Atropinic effects.
• Minor modifications in structure have profound effect even on
pharmacokinetics e.g. PO4 ester at N3 in Phenytoin makes more
soluble
16

17. • Advances in molecular modelling of organic compounds and
methods for drug target discovery and biochemical
measurements of primary actions of drugs at their receptor have
enriched quantitation of structure activity relationship and its use
in drug design.
• Drugs binding to selectively mutated receptors improves affinity
and selectivity of drug.
• X-ray crystallography helps designing ligands and molecular
basis of drug resistance ( BCR-ABL and Imatinib like inhibitors)
17

18. QUANTITATIVE ASPECTS OF DRUG
INTERACTIONS WITH RECEPTORS
• Basic currency of receptor pharmacology is dose response or
concentration curve.
• Concentration of drug that produces 50% of the maximal response
quantifies drug activity and is referred to as the EC50.
18

19. AFFINITY, EFFICACY AND POTENCY
• Drug-receptor interaction is characterized by binding of drug to
receptor and generation of a response.
• Drug or ligand is denoted as L and the inactive receptor as R.The
first reaction, the reversible formation of the ligand-receptor
complex LR, is governed by the chemical property of Affinity.
• Concentration of ligand-receptor complex [LR] is equal to the
product of k+1[L][R], the rate of formation of the bi-molecular
complex LR, minus the product k–1[LR], the rate dissociation of LR
into L and R.
19

20. • The Equilibrium Dissociation Constant (KD) is then described by
ratio of the Off And On rate constants (k–1/k+1).
• The Affinity Constant or Equilibrium AssociationConstant (KA) is
the reciprocal of the Equilibrium Dissociation Constant (i.e., KA =
1/KD)
• Thus a high-affinity drug has a low KD and will bind a greater
number of a particular receptor at a low concentration than a low-
affinity drug.
• Note that this relationship describes only receptor occupancy, not
the eventual response that is often amplified by the cell.
• Many signaling systems reach a full biological response with only a
fraction of receptors occupied (described later)
20

21. • The relative Potency of two agonists (Drug X, red line; DrugY,
purple line) obtained in the same tissue is a function of their relative
affinities and intrinsic efficacies.
• The EC50 of Drug X occurs at a concentration that is one-tenth the
EC50 of DrugY.Thus, Drug X is more potent than DrugY.
21

22. QUANTIFYING AGONISM AND
ANTAGONISM
• Measuring agonist potency by comparison of EC50 values is one
method of measuring the capability of different agonists to induce
a response in a test system and for predicting comparable activity
in another.
• Another method of estimating agonist activity is to compare
maximal asymptotes in systems where the agonists do not produce
maximal response.
22

23. • Characteristic patterns of antagonism are associated with certain
mechanisms of blockade of receptors.
• One is straight forward Competitive Antagonism, whereby a drug
with affinity for a receptor but lacking intrinsic efficacy competes
with the agonist for the primary binding site on the receptor.
23

24. • If the antagonist binds to the same site as the agonist but does so
irreversibly or Pseudo-Irreversibly (slow dissociation but no
covalent bond), it causes a shift of the dose-response curve to the
right, with further depression of the maximal response.
24

25. • Allosteric effects occur when an Allosteric ligand I or P binds to a
different site on the receptor to either inhibit (I) the response (see
panel C) or potentiate (P) the response (see panel D).This effect is
saturable; inhibition or potentiation reaches a limiting value when
the allosteric site is fully occupied.
25

26. PHARMACODYNAMICAL
VARIABILITY
• Individuals vary in the magnitude of their response to the same
concentration of a single drug or to similar drugs, and a given
individual may not always respond in the same way to the same
drug concentration.
• Attempts have been made to define and measure individual
"Sensitivity" (Or "Resistance") to drugs in the clinical setting, and
progress has been made in understanding some of the
determinants of sensitivity to drugs that act at specific receptors.
• Drug responsiveness may change because of disease or because
of previous drug administration.
• Receptors are dynamic, and their concentration and function may
be up- or down-regulated by endogenous and exogenous factors.

27. FACTORS MODIFYING DRUG ACTION
27

28. PHARMACOGENETICS
• Pharmacogenetics refers to the genetic and genomic variations
that give rise to variability in both pharmacokinetic and
pharmacodynamic aspects of drug therapy.
• Inter-individual variability of responsiveness to many drugs. e.g.
Warfarin.
• Nearly 60% of the variability is due to genetic variation in the
primary metabolizing enzyme (CYP2C9) and in the drug's receptor,
Vitamin K Epoxide Reductase Complex, subunit 1 (VKORC1).
Polymorphisms in CYP2C9 (especially homozygosity in the allele)
increase sensitivity towards warfarin, whereas coding region
polymorphisms in VKORC1 result in a warfarin-resistant
phenotype.
• FDA recommended that pharmacogenetics be used to optimize
warfarin dosing, but did not provide specific protocol.
28

29. MECHANISMS OF ACTION
Receptors that Affect Concentrations of Endogenous Ligands:-
• Many drugs act on endogenous ligands like neurotransmitters,
hormones and alter their synthesis storage release and transport.
• Many examples of drugs that act on neuroeffector junctions by
altering neurotransmitter synthesis, storage of neurotransmitter in
vesicles, release of neurotransmitters into the synaptic cleft, and
subsequent removal of the neurotransmitter from the synaptic
cleft.
• E.g. Alpha-Methyltyrosine (inhibits synthesis of norepinephrine
(NE)), Cocaine (blocks NE reuptake), Amphetamine (promotes NE
release), and Selegeline (inhibits NE breakdown).
29

30. Receptors that Regulate the Ionic Milieu-
• Some drugs act by affecting the ionic millieu of blood, urine, and
the GI tract.
• Receptors in this case are ion pumps and transporters.
• Drug effects on many of these receptors can have effects
throughout the body due to changes in blood electrolytes and pH.
• e.g., Furosemide, Chlorothiazide, Amiloride act by directly
affecting ion pumps and transporters in epithelial cells of the
nephron that increase the movement of Na+ into the urine.
• Another therapeutically important target is the H+,K+-ATPase
(Proton Pump) of gastric parietal cells like Esmoprazole (90%).
30

31. Cellular Pathways Activated by Physiological Receptors –
1) SignalTransduction Pathways –
• Physiological receptors have at least two major functions, ligand
binding and message propagation (i.e., signaling).
• So there is existence of at least two functional domains within the
receptor: A Ligand-binding Domain And An Effector Domain.
• Many drugs target the extracelluar ligand-binding domain of
physiological receptors. E.g. Beta Blockers.
• However, drugs can affect the receptor by targeting either domain,
as in the case of Cetuximab – extra cellular domain, Geftinib
Erlotinib on intracellular domain.
31

32. • Regulatory actions of a receptor may be exerted directly on its
cellular target called as Transducers.
• The receptor, its cellular target, and any intermediary molecules
are referred to as a receptor-effector system or signal transduction
pathway.
• Frequently ultimate physiological target is an Enzyme, Ion
Channel, OrTransport ProteinThat Creates, Moves, Or Degrades a
small molecule.Termed as Second Messenger.
32

33. 2) Signal integration and amplification –
• Receptors and their associated effector and transducer proteins
also act as integrators of information as they coordinate signals
from multiple ligands with each other and with the differentiated
activity of the target cell.
• For example, signal transduction systems regulated by changes in
Cyclic AMP (cAMP) and Intracellular Ca2+ are integrated in many
excitable tissues.
• In cardiac myocytes, an increase in cellular cAMP caused by
activation of adrenergic receptors enhances cardiac contractility by
augmenting the rate and amount of Ca2+ delivered to the
contractile apparatus; thus, cAMP and Ca2+ are positive contractile
signals in cardiac myocytes.
33

34. • Another important property of physiological receptors is their
capacity to significantly Amplify a physiological signal.
• Neurotransmitters, hormones, and other extracellular ligands are
often present at the ligand-binding domain of a receptor in very
low concentrations .(Nano Moles).
• Effector domain contains enzymes and enzyme cascades to
catalytically amplify the intended signal.
• The ability of virtually all receptors to amplify physiological signals
makes them excellent targets for natural ligands and drugs.
• E.g. binding of a single photon to cis-retinal in the photoreceptor
Rhodpsin is eventually amplified ~1 x 106-fold.
• A single steroid hormone molecule binding to its receptor initiates
the transcription of many copies of specific mRNAs.
34

35. STRUCTURAL AND FUNCTIONAL FAMILIES
OF PHYSIOLOGICAL RECEPTORS
35

36. G PROTEIN–COUPLED RECEPTORS
(GPCRS)
• A bundle of Seven Alfa-helices.
• Over 800 GPCRs that make up the third largest family of genes.
• Half of these GPCRs dedicated to sensory perception (Smell,Taste,
AndVision).
• Remaining receptors regulate an impressive number of
physiological functions including Nerve Activity,Tension Of
Smooth Muscle, Metabolism, Rate And Force Of Cardiac
Contraction, AndThe Secretion of most glands.
• ligands for GPCRs are – Ach, NE, Eicosanides, Peptide Hormones,
GABA.
• GPCRs are important regulators of CNS and Autonomic nervous
system. 36

37. • Because of their number and physiological importance, GPCRs are
the targets for many drugs; perhaps half of all non-antibiotic
prescription drugs act at these receptors.
37

38. Receptor Subtypes –
• The Alfa 1, Alfa 2, and Beta adrenergic receptors differ from each
other both in ligand selectivity and in coupling to G proteins.
• The Beta 1, 2, and 3 adrenergic receptor subtypes exhibit
differences in both tissue distribution and regulation by
phosphorylation by G–protein receptor kinases (GRKs) and PKA.
• Pharmacological differences among receptor subtypes are
exploited therapeutically through the development and use of
receptor-selective drugs.
• E.g. Beta 2 Agonist Salbutamol for Broncho-dilatation.To
minimise side effects of Beta 1 effects on heart.
38

39. Receptor Dimerization –
• GPCRs undergo both homo- and Heterodimerization and possibly
Oligomerization.
• Opioid receptors can exist as Homodimers of mu or delta
receptors, or as mu-delta Heterodimers with distinctly different
Pharmacodynamic properties than either Homodimer.
• Dimerization also may permit binding of receptors to other
regulatory proteins such as transcription factors.
39

40. G PROTEINS
• GPCRs couple to a family of heterotrimeric GTP-binding
regulatory proteins termed G proteins.
• G–protein-regulated effectors include enzymes such as Adenylyl
Cyclase, Phospholipase C, Cyclic GMP, Phosphodiesterase (PDE6),
And Membrane Ion Channels selective forCa2+And K+.
• The G protein family is comprised of 23 subunits and 4 families
Gs, Gi, Gq, and G12/13.

41. G PROTEIN ACTIVATION
• When an Agonist binds to a GPCR, there is a conformational
change in the receptor that is transmitted from the ligand-binding
pocket to the second and third intracellular loops of the receptor
which couple to the G protein.
• Conformational change causes the Alfa subunit to exchange its
bound GDP for GTP.
• Binding of GTP activates the Alfa subunit and causes it to release
both the Beta-Gamma dimer.
• Beta-Gamma heterodimer become active signaling molecule.
• Following activation of one G protein, the receptor is freed to
interact with other G proteins, the active, GTP-bound form binds to
and regulates effectors such as adenylyl cyclase (via Gs alfa) or
phospholipase C Beta (via Gq Alfa ).
41

42. 42

43. SECOND MESSENGERS
Cyclic Amp –
• Cyclic AMP is synthesized by Adenylyl Cyclase under the control of
many GPCR.
• Stimulation is mediated by the Gs-Alfa subunit, inhibition by the
Gi-Alfa subunit.
• Membrane-boundACs exhibit basal enzymatic activity that is
modulated by binding of GTP-liganded Alfa subunits of the
stimulatory and inhibitory G proteins.
• Cyclic AMP generated by Adenylyl Cyclases has three major targets
in most cells, the cyclic AMP dependent protein kinase (PKA),
cAMP-regulated guanine nucleotide exchange factors termed
EPACs (exchange factors directly activated by cAMP), and via PKA
phosphorylation.
43

44. PKA-
• Target of Cyclic AMP is the PKA Holoenzyme have two catalytic
(c) subunits reversibly bound to a Regulatory subunit (R) to form
a Heterotetramer (R2C2).
• At low concentrations of Cyclic AMP the R subunits inhibit C
subunits thus the holoenzyme is inactive.
• When Adenyl Cyclase is activated cAMP concentration is
increased causing C subunit activation.
• The active C subunit phosphorylase Serine AndThreonine
residues on specific protein substrates. Present in metabolic
enzymes, transport proteins and numerous regulatory protein.

45. PKG –
• Stimulation of receptors that raise Intracellular Cyclic GMP
concentrations leads to the activation of the cyclic dependent
protein kinase (PKG) that are PKG specific.
• The catalytic domain and cyclic nucleotide binding domains of
PKG are expressed as a single polypeptide and forms PKG
holoenzyme
• Pharmacologically important effects of elevated cyclic GMP
include modulation of platelet activation and relaxation of
smooth muscles

46. PDE –
• Cyclic Nucleotide Phosphodiesterases form another family of
important signalling proteins whose activities are regulated via
the rate of gene transcription as well as by second messengers.
• PDEs Mainly PDE3 are drug targets for treatment of diseases
such as Asthma, CardioVascular Diseases Such As Heart Failure,
Atherosclerotic Coronary and Peripheral Arterial Disease and
neurological disorders.
• PDE5 inhibitors are used in treating COPD and Erectile
Dysfunction. Inhibition of PDE5 causes accumulation of cGMP in
cells of smooth muscles of corps cavernosum thereby enhancing
its relaxation.

47. OTHER SECOND MESSENGERS
• Ca can enter the cell through Ca channels in the plasma
membrane or be released by hormones or growth factors from
intracellular stores
• The basal Ca level in cell is maintained by membrane Ca pumps
which extrude Ca into extracellular space and sarcoplasmic
reticulum.
• Hormones and growth factors release Ca from its intra cellular
storage via a signalling pathway that begins with activation of
Phospholipase C
• Phospholipase C has two primary forms, PLC beta and PLC
gamma.
• GPCR activate PLC by activating G protein alfa subunit.

48. • PLC isoforms are activated by tyrosine phosphorylation.
• Growth factor receptors such as epidermal growth factor
receptor EGFR are receptor tyrosine kinase RTK.
• This RTK Auto phosphorylate upon binding their cognate growth
factor.

49. ION CHANNELS
• The lipid bilayer of the plasma membrane is impermeable to
Anions And Cations
• To establish and maintain the electrochemical gradients required
to maintain a membrane potential, all cells express ion
transporters for Na, K, Ca, Cl.
• E.g. Na-K-ATPase pump
• Passive ion fluxes down cellular electrochemical gradients are
regulated by a large family of ion channels
• Humans express ~232 distinct ion channels to precisely regulate
the flow of Na, K, Ca, Cl across cell membrane
• These proteins are important target for drug actions

50. • This diverse family of ion channels can be divided into sub
families depending upon mechanisms of their channels.
• They can also be classified as voltage activated, ligand activated,
store activated, stretch activated and temperature activated.

51. Voltage Gated Channels –
• Humans express multiple isoforms of voltage gated channels for
Na, K, Ca, Cl ions.
• In nerves and muscle cells, voltage gated Na channels are
responsible for the generation of robust action potentials.
• These sodium channels are composed of three subunits, a pore
forming alpha subunit and two regulatory beta subunits.
• The alfa subunit is a 260 kDa protein containing four domains
that form a Na ion selective pore.
• The voltage activated Na channels in pain neurons are targets for
local anaesthetics such as Lidocaine andTetracaine which block
pore, inhibit depolarisation and thus block the sensation of pain.

53. • They are also the targets of the naturally occurring MarineToxins,
Tetrodotoxin and Saxitoxin.
• These are also important targets of many drugs used to treat
Cardiac Arrhythmias.

54. Ligand gated channels –
• These channels activated by binding of a ligand to a specific site
in the channel protein have a diverse architecture and set of
ligands.
• Activation of these channels is responsible for the majority of
synaptic transmission by neurons both in the CNS and periphery.
• There are a variety of more specialised ion channels that are
activated by intra cellular small molecules and structurally
distinct from conventional ligand gated ion channels.
• Formally members of Kv family such as hyperpolarisation and
CMP gated (HCN) channel expressed in heart.

56. • Cyclic Nucleotide-Gated channels (CNG) important for vision.
• Ion channels also include the IP3 sensitive Ca channel responsible
for release of Ca from ER and the Sulphonyl urea receptor SUR1
that associates with Kir6.2 channel to regulate the ATP
dependent K channel in pancreatic beta cells.
• K ATP channel is the target of oral hypoglycaemic drugs such as
Sulphonylurea and Meglitinide.
• Other specialised channels include the 5HT3 regulated channel
expressed on vagal nerves that stimulates emesis.
• Ondensetron is an important antagonist of the 5HT3 gated
channel used to inhibit emesis caused by drugs or disease.

57. TPR channels –
• The Transient Receptor Potential (TRP) channels comprise a
superfamily of ubiquitously expressed ion channels that is
remarkable in its diversity and domain structure.
• Are not presently targets of approved drugs.
• Significant interest in developing drugs that can alter the function
of these ion channels.
• Their roles in various sensory phenomena such as Pain,
Temperature, Osmolarity,Touch, Olfaction,Vision, and Hearing.
• Most can be activated by multiple mechanisms.
• Mutations inTRP channels are known to cause several disease
including Hypomagnesemia And Hypocalcemia, and various
Renal Disorders And Neurodegenerative Diseases.

58. TRANSMEMBRANE RECEPTORS LINKED
TO INTRACELLULAR ENZYMES
• Mammalian cells express a diverse group of physiological
membrane receptors with extracellular ligand-binding domains
and an intrinsic enzymatic activity on the cytoplasmic surface of
the cell.
• These molecules include the ReceptorTyrosine Kinases (RTKs)
such as the Epidermal Growth Factor (EGF) and Insulin
Receptors, which contain intrinsic tyrosine kinases in the
cytoplasmic domain.
• Tyrosine Kinase-associated receptors without enzymatic activity,
such as the receptors for Gamma-interferon, which recruit the
cytoplasmic Janus tyrosine kinases (JAKs).

59. • Receptor Serine-Threonine Kinases such as the TGF- Receptor.
• Receptors linked to other enzyme activities such as the receptors
for Natriuretic Peptides, which have a cytoplasmic guanylate
cyclase activity that produces a soluble second messenger, cyclic
GMP.
• Receptors responsible for innate immunity, theToll-like receptors
and those for tumor necrosis factors (TNF-Alfa) , have many
features in common with the JAK-STAT receptors.

60. ReceptorTyrosine Kinases-
• The receptor tyrosine kinases include receptors for hormones
such as Insulin, for multiple growth factors such EGF, platelet-
derived growth factor (PDGF), nerve growth factor (NGF),
fibroblast growth factor (FGF), vascular endothelial growth factor
(VEGF), and Ephrins.
• Activation of growth factor receptors leads to cell survival, cell
proliferation, and differentiation. Activation of the Ephrin
receptors leads to neuronal angiogenesis, axonal migration, and
guidance.

62. JAK-STAT Receptor Pathway -
• Cells express a family of receptors for cytokines such as -
Interferon And Hormones like Growth Hormone And Prolactin,
which signal to the nucleus by a more direct manner than the
receptor tyrosine kinases.
• Upon the dimerization induced by ligand binding, JAKs
phosphorylate other proteins termed signal transducers and
activators of transcription (STATs), which translocate to the
nucleus and regulate transcription.
• There are four JAKs and six STATs in mammals.
• Prolactin appears to use JAK1, JAK2, and STAT5 to stimulate milk
production.

64. Receptor Serine-Threonine Kinases -
• Protein ligands such as TGF-Beta activate a family of receptors
that are analogous to the ReceptorTyrosine Kinases.
• In the basal state, these proteins exist as monomers; upon
binding an agonist ligand, they dimerize, leading to
phosphorylation of the kinase domain of the type I monomer,
which activates the receptor.
• Receptor then phosphorylates a gene regulatory protein termed
a Smad.
• There are multiple Smads in cells.
• Regulates genes leading to MorphogenesisAnd Transformation.

65. Toll-like Receptors -
• Signaling related to the innate immune system is carried out by a
family of over ten single membrane-spanning receptors termed
Toll-like receptors (TLR).
• Highly expressed in Hematopoeitic Cells.
• In a single polypeptide chain, these receptors contain a large
extracellular ligand-binding domain, a short membrane-spanning
domain.
• Ligands forTLR are comprised of a multitude of pathogen
products including Lipids, Peptidoglycans, Lipo-peptides, And
Viruses.
• Activation of these receptors produces an Inflammatory
Response.

66. TNF-alfa Receptors -
• The mechanism of action of tumor necrosis factor alfa (TNF-alfa )
signaling to the NF-kappaB transcription factors is very similar to
that used by Toll-like receptors.
• Receptor has no enzymatic activity.
• TNF receptor is another single membrane-spanning receptor with
an extracellular ligand-binding domain, a transmembrane
domain, and a cytoplasmic domain termed the Death Domain.

67. RECEPTORS THAT STIMULATE
SYNTHESIS OF CYCLIC GMP
• The signalling pathways that regulate the synthesis of cyclic GMP
in cells include hormonal regulation of Transmembrane
Guanylate Cyclases.
• Such as the atrial natriuretic peptide receptor (ANP)
• Activation of soluble forms of guanylate cyclase by nitric oxide
(NO).
• Effects of cyclic GMP are carried out by multiple isoforms of PKG,
cyclic GMP-gated ion channels, and cyclic GMP-modulated
Phosphodiesterases.

68. Natriuretic Peptide Receptors -
• Membrane receptors with intrinsic enzymatic activity includes
the receptors for three small peptide ligands released from cells
in cardiac tissues and the vascular system.
• Atrial Natriuretic Peptide (ANP), Brain Natriuretic Peptide (BNP),
C-type Natriuretic Peptide (CNP).
• BNP, CNP is not stored, its synthesis and release are increased by
growth factors and sheer stress on vascular endothelial cells.
• The major physiological effects of these hormones are to
Decrease Blood Pressure (ANP, BNP), to reduce Cardiac
HypertrophyAnd Fibrosis (BNP), and to Stimulate Long Bone
Growth (CNP).

70. • The ANP receptor (NPR-A) is the molecule that responds to ANP
and BNP.
• ANP and BNP play a role in maintaining the normal state of the
cardiovascular system as NPR-A knockout mice have
hypertension and cardiac hypertrophy.
• A synthetic BNP Agonist, Nesiritide, is used for treatment of
Acute Decompensated Heart Failure.
• The NPR-B receptor widely expressed but prominent in bone,
brain, kidney, lung, liver, and cardiac and vascular smooth muscle.

71. NO Synthase And Soluble Guanylate Cyclase –
• Nitric oxide (NO) is a unique signal.
• A very labile gas produced locally in cells by the enzyme Nitric
Oxide Synthase (NOS).
• Resulting NO is able to markedly stimulate the soluble form of
guanylate cyclase to produce cyclic GMP.
• Three forms of nitric oxide synthase, neuronal NOS (nNOS or
NOS1), endothelial NOS (eNOS or NOS3), and inducible NOS
(iNOS or NOS2).
• They are found in Myocytes,Vascular Smooth Muscle Cells,
Endothelial Cells, Hematopoietic Cells, And Platelets.

72. NUCLEAR HORMONE RECEPTORS AND
TRANSCRIPTION FACTORS
• In humans, nuclear hormone receptors comprise a superfamily of
48 receptors that respond to a diverse set of ligands.
• The receptor proteins are transcription factors able to regulate
the expression of genes controlling numerous physiological
processes such as reproduction, development, and metabolism.
• Well-known members of the family include receptors for
circulating steroid hormones such as Androgens, Estrogens,
Glucocorticoids,Thyroid Hormone, and Vitamin D.

74. • Examples include Retinoic Acid Receptor (RXR); the Liver X
Receptor (LXR —the ligand is 22-OH cholesterol); the Farnesoid X
Receptor (FXR—the ligand is chenodeoxycholic acid); and the
Peroxisome Proliferator-activated Receptors (PPARs alfa, beta ,
and gamma ; 15 Deoxy Prostaglandin J2 is one possible ligand for
beta PPAR ; the Cholesterol-lowering Fibrates bind to and
regulate PPAR gamma ).

75. APOPTOSIS
• The maintenance of many organs requires the continuous renewal of
cells.
• Mucosal cells lining the intestine and a variety of cells in the immune
system includingT-cells and Neutrophils.
• The process by which cells are genetically programmed for death is
termed Apoptosis.
• Apoptosis is a highly regulated program of biochemical reactions that
leads to Cell Rounding, Shrinking Of The Cytoplasm, Condensation Of
The Nucleus And Nuclear Material, And Changes InThe Cell
Membrane that eventually lead to presentation of phosphatidylserine
on the outer surface of the cell.

76. • Understanding the pathways regulating apoptosis is important
because apoptosis plays an important role in normal cells and
because alterations in apoptotic pathways are implicated in a
variety of diseases such as Cancer,And Neurodegenerative And
Autoimmune Diseases.
• Thus, maintaining or restoring normal apoptotic pathways is the
goal of major drug development efforts to treat diseases that
involve dysregulated apoptotic pathways and selectively
stimulating apoptotic pathways could be useful in removing
unwanted cells.

77. • Two major signaling pathways induce Apoptosis. Apoptosis can
be initiated by external signals that have features in common
with those used by ligands such as TNF- alfa or by an internal
pathway activated by DNA damage, improperly folded proteins,
or withdrawal of cell survival factors.
• The apoptotic program is carried out by a large family of
Cysteine-proteases termed Caspases.
• The Caspases are highly specific cytoplasmic proteases that are
inactive in normal cells but become activated by apoptotic
signals.

79. RECEPTOR DESENSITIZATION AND
REGULATION OF RECEPTORS
• Receptors are subject to many regulatory and haemostatic
controls.
• These controls include regulation of the synthesis and
degradation of the receptor, covalent modification, association
with other regulatory proteins, and relocalization within the cell.
• Receptors are almost always subject to feedback regulation by
their own signalling.
• Continued stimulation of cells with agonists generally results in a
state of Desensitization (also referred to as Adaptation,
Refractoriness, or Down-regulation)

80. • The effect that follows continued or subsequent exposure to the
same concentration of drug is diminished. This phenomenon,
called Tachyphylaxis.
• Tachyphylaxis, occurs rapidly and is important therapeutically.
• An example is attenuated response to the repeated use of beta
adrenergic receptor agonists as bronchodilators for the
treatment of asthma.
• Desensitization can result from temporary inaccessibility of the
receptor to agonist or from fewer receptors being synthesized
and available at the cell surface (e.g., Down-regulation of
receptor number).
• Phosphorylation of GPCR receptors by specific GPCR kinases
(GRKs) plays a key role in triggering rapid desensitization.

81. • The - Arrestins recruit proteins, such as PDE4, that limit cyclic
AMP signaling, and Clathrin and 2-Adaptin, that promote
sequestration of receptor from the membrane (Internalization).
• Conversely, Supersensitivity to Agonists also frequently follows
chronic reduction of receptor stimulation. Such situations can
result
• e.g., following withdrawal from prolonged receptor
blockade.(e.g., the long-term administration of Beta Adrenergic
Receptor Antagonists such as Metoprolol)
• or in the case where chronic denervation of a preganglionic fiber
induces an increase in neurotransmitter release per pulse,
indicating postganglionic neuronal Supersensitivity.

83. Diseases Resulting From Receptor Malfunction -
• Alteration in receptors and their immediate signaling effectors
can be the cause of disease.
• Deficiencies in widely employed signaling pathways have broad
effects, as are seen in myasthenia gravis and some forms of
insulin-resistant diabetes mellitus, which result from
autoimmune depletion of nicotinic cholinergic receptors. Or
Insulin receptors.
• Among the most significant events is the appearance of aberrant
receptors as products of oncogenes that transform otherwise
normal cells into malignant cells.Virtually any type of signaling
system may have oncogenic potential.

84. PHARMACODYNAMIC INTERACTIONS IN A
MULTICELLULAR CONTEXT
• It is instructive to examine the pharmacodynamic interactions of
physiological ligands and drugs that can occur in the context of a
pathophysiological setting.
• Consider the vascular wall of an arteriole.
• Several cell types interact at this site, including vascular smooth
muscle cells (SMCs), endothelial cells (ECs), platelets, and
postganglionic sympathetic neurons.
• A variety of physiological receptors and ligands are represented,
including ligands that cause SMCs to contract (Angiotensin II
[AngII], Norepinephrine [NE]) and relax (Nitric Oxide [NO], B-
type Natriuretic Peptide [BNP], and Epinephrine), as well as
ligands that alter SMC gene expression (platelet-derived growth
factor [PDGF], AngII, NE, and Eicosanoids).

86. • Ang-II has both acute and chronic effects on SMC. Interaction of
Ang-II with AT1 receptors (AT1-R) causes the formation of the
second messenger IP3 causing release of Ca from SR leading to
smooth muscle contraction.
• NE binds 1 adrenergic receptors that couple to the Gq-PLC-IP3
pathway, causing an increase in intracellular Ca2+ and, as a result,
contraction, an effect that is additive to that of Ang-II.
• NO is formed in ECs by the action of two NO synthase enzymes,
eNOS and iNOS.The NO formed in ECs diffuses into SMCs, and
activates the soluble form of Guanylate Cyclase (sGC), which
catalyzes the formation of cyclic GMP from GTP.The increase in
cyclic GMP activates PKG, which phosphorylates protein
substrates in SMCs that reduce intracellular concentrations of
Ca2+

87. • Intracellular concentrations of cyclic GMP are also increased by
activation of the transmembrane BNP receptor (BNP-R), whose
guanylate cyclase activity is increased when BNP binds. BNP is
released from cardiac muscle in response to increased filling
pressures.
• Beta 1 antagonists reduce secretion of renin (the rate-limiting
first step in Ang-II synthesis)
• A direct renin inhibitor (Aliskiren) to block the rate-limiting step
in Ang-II production
• Angiotensin-converting enzyme (ACE) inhibitors (e.g., Enalapril)
to reduce the concentrations of circulating Ang-II
• AT1 receptor blockers (e.g., Losartan) to blockAng-II binding to
AT1 receptors on SMCs

88. • Alfa 1 adrenergic blockers to block NE binding to SMCs.
• Sodium Nitroprusside to increase the quantities of NO produced.
• Ca2+ channel blocker (e.g., Nifedipine) to block Ca2+ entry into
SMCs.
• Thus, the choices and mechanisms are complex, and the
appropriate therapy in a given patient depends on many
considerations, including the diagnosed causes of hypertension in
the patient, possible side effects of the drug, efficacy in a given
patient, and cost.

89. REFERENCES
• Gough DJ, Levy DE, Johnstone RW, Clarke CJ. IFN- signaling-does it
mean JAK-STAT? Cytokine Growth Factor Rev, 2008, 19:383–394.
[PMID: 18929502]
• Hayden MS, Ghosh S. Shared principles in NF B signaling. Cell, 2008,
132:344–362. [PMID: 18267068]
• JeglaTJ, Zmasek CM, Batalov S, Nayak SK. Evolution of the human ion
channel set. Comb Chem HighThroughput Screen, 2009, 12:2–23.
[PMID: 19149488]
• KataokaT. Chemical biology of inflammatory cytokine signaling. J
Antibiot (Tokyo), 2009
• KleinTE, Altman RB, Eriksson N, et al. Estimation of the warfarin dose
with clinical and pharmacogenetic data. N EnglJ Med, 2009, 360:753–
764. [PMID: 19228618]
• McEwan IJ. Nuclear receptors: One big family. Methods Mol Biol, 2009,
505:3–18. [PMID: 19117136]
89

90. REFERENCES
• Baillie GS. Compartmentalized signalling: Spatial regulation of cAMP
by the action of compartmentalized phosphodiesterases. FEBS J,
2009, 276:1790–1799. [PMID: 19243430]
• Carnegie GK, Means CK, Scott JD. A-kinase anchoring proteins: From
protein complexes to physiology and disease. IUBMB Life, 2009,
61:394–406. [PMID: 19319965]
• Fahrner M, Muik M, Derler I, et al. Mechanistic view on domains
mediating stim1-orai coupling. Immunol Rev, 2009,231:99–112.
[PMID: 19754892]
• Ferguson KM. Structure-based view of epidermal growth factor
receptor regulation. Annu Rev Biophys, 2008, 37:353–373. [PMID:
18573086]
• Ghavami S, Hashemi M, Ande SR, et al.Apoptosis and cancer:
Mutations within caspase genes. J Med Genet, 2009, 46:497–510.
[PMID: 19505876]
• Ghosh S, Hayden MS. New regulators of NF B in inflammation. Nat
Rev Immunol, 2008, 8:837–848. [PMID:18927578]
90

91. REFERENCES
• Tsai EJ, Kass DA. Cyclic GMP signaling in cardiovascular
pathophysiology and therapeutics. PharmacolTher, 2009, 122:216–238.
[PMID: 19306895]
• Wahl-Schott C, Biel M. HCN channels: Structure, cellular regulation and
physiological function. Cell Mol Life Sci,2009, 66:470–494. [PMID:
18953682]
• Wang X, Lupardus P, Laporte SL, Garcia KC. Structural biology of shared
cytokine receptors. Annu Rev Immunol, 2009, 27:29–60. [PMID:
19449536]
• Wilson NS, DixitV, Ashkenazi A. Death receptor signal transducers:
Nodes of coordination in immune signalling networks. Nat Immunol,
2009, 10:348–355. [PMID: 19295631]
• Venkatachalam K, Montell C.TRP Channels. Annu Rev Biochem, 2007,
76:387–417. [PMID: 17579562]
• Ross EM, WilkieTM. GTPase-activating proteins for heterotrimeric G
proteins: Regulators of G protein signaling(RGS) and RGS-like proteins.
Annu Rev Biochem, 2000, 69:795–827. [PMID: 10966476]
91

92. REFERENCES
• Potter LR,Yoder AR, Flora DR, et al. Natriuretic peptides:Their
structures, receptors, physiologic functions and therapeutic
applications. Handb Exp Pharmacol, 2009, 341–366.
• Prakriya M.The molecular physiology of CRAC channels. Immunol Rev,
2009, 231:88–98. [PMID: 19754891]
• Purves D, Augustine GJ, Fitzpatrick D, et al. Channels and transporters.
In, Neuroscience. Sinauer, Sunderland, MA, 2008, pp. 61–84.
• RongY, Distelhorst CW. Bcl-2 protein family members:Versatile
regulators of calcium signaling in cell survival and apoptosis. Annu Rev
Physiol, 2008, 70:73–91. [PMID: 17680735]
• Skaug B, Jiang X, Chen ZJ.The role of ubiquitin in NF B regulatory
pathways. Annu Rev Biochem, 2009, 78: 769–796. [PMID: 19489733]
• Taylor SS, Kim C, Cheng CY, et al. Signaling through cAMP and cAMP-
dependent protein kinase: Diverse strategies for drug design. Biochim
Biophys Acta, 2008, 1784:16–26. [PMID: 17996741]
92