Nonsteroidal Anti-inflammatory Drugs (NSAIDs)

Nonsteroidal anti-inflammatory drugs, usually abbreviated as NSAIDs with analgesic and antipyretic (fever-reducing) effects and which have, in higher doses, anti-inflammatory effects.

The term "Nonsteroidal" is used to distinguish these drugs from steroids, which, among a broad range of other effects, have a similar eicosanoid-depressing, anti-inflammatory action. As analgesics, NSAIDs are unusual in that they are non-narcotic.

Types of NSAIDs

There are two main types of NSAIDs: nonselective and selective.

Nonselective NSAIDs

Nonselective NSAIDs inhibit the enzymes found in the stomach, blood platelets, and blood vessels (COX-1) as well as the enzymes found at sites of inflammation (COX-2) to a similar degree. Nonselective NSAIDs include drugs such as aspirin, ibuprofen, naproxen and dichlorofenac.

Selective NSAIDs09:20:44

Selective NSAIDs (also called COX-2 inhibitors) inhibit the COX enzyme found at sites of inflammation (COX-2) more than the type of enzyme normally found in the stomach, blood platelets, and blood vessels (COX-1). Celecoxib is a selective NSAID

CLASSIFICATION OF NSAIDs

1) NONSELECTIVE IRREVERSIBLE COX INHIBITORS

                   (a) Salicylates           :          Aspirin (Acetyl-salicylic acid)

                                                                 Sodium salicylate

                                                                Methyl salicylate

                                                                 Salicylic acid

                        Others                  :          Olsalazine

2) NONSELECTIVE REVERSIBLE COX INHIBITORS

                   (a) Indole derivatives    :    Indomethacin

                                                                 Sulindac

                   (b)Propionic acid derivatives: Ibuprofen

                                                                       Ketoprofen

                                                                       Flurbiprofen

                                                                       Naproxen

                     (c)Aryl acetic acid derivatives: diclorofenac

                                                                           Aceclofenac

                     (d) Anthracitic acid: Mefenamic acid

                                                           Flufenamic acid

                      (e) Pyrazolone derivatives: Phenyl butanone

                                                                        Oxiphenbutazone

                      (f) Oxicam derivatives    : Tenoxicam

                                                                  Pyroxicam

                      (g) pyrrole-pirole derivatives: ketorolac

                                                                           Tolmethin

                                                                          Oxaprozin

3) SELECTIVE COX-2 INHIBITORS:

                                                 Celecoxib

                                                 Rofecoxib

                                                 Valdexoxib

4) WEAK INHIBITOR OF COX1&COX2

                                                          Nimusalide

5) PREFERENTIAL COX 2 INHIBITORS

                                                         Meloxicam

                                                          Ethodolac

                                                           Nabumethone

6) COX 3 INHIBITOR /REVERSIBLE INHIBITOR OF COX1

                                                           Paracetamol

                                                            Methamizol

7) OTHER NON STEROIDAL DRUGS

                                           Nefopam

3) MORE COX-2 SELECTIVE INHIBITORS

                                             Nimusalide

                                              Etodolak

                                              Meloxicam

                                              Nabumethone

4) COX-2 SELECTIVE INHIBITORS

                                            Celecoxib

                                             Etorcoxib

                                           Valdecoxib

General Mechanism of action

Most NSAIDs act as nonselective inhibitors of the enzyme cyclooxygenase (COX), inhibiting both the cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) iso enzymes. COX catalyzes the formation of prostaglandins and thromboxane from Arachidonic acid (itself derived from the cellular phospholipid bilayer by phospholipase A₂). Prostaglandins act (among other things) as messenger molecules in the process of inflammation. This mechanism of action was elucidated by John Vane (1927–2004), who later received a Nobel Prize for his work (see Mechanism of action of aspirin

                                       Tissue Injury

                            ↓

           Phospholipids in cell membrane

                          ↓PHOSPHOLIPASE A₂

                               Arachidonic acid

   NSAIDS INHIBITORS (-) ↓cox

                                                 PGG₂    

                          ↓cox

                                    PGH₂      

                  ↙      ↓      ↘ 

      ProstaglandinE₁      prostacyclin     ThrombaxaneA₂

      PGD₂, PGE2, PGF2                 PGI₂                               TXA₂, TXB2

                

Cox-1&cox2

Cox-1 

·         Present in most tissues.

·         In the GIT, it maintains the normal lining of the stomach.

·         Involved in kidney and platelet aggregation.

Cox-2

·         Present in macrophages and monocytes.

·         Inducible.

·         Responsible for pain and inflammation.

NON SELECIVE IRREVERSIBLE INHIBITORS OF COX

ASPIRIN

Analgesia

→These are the salts or esters of salicylic acid.

           →Salicylic acid itself is a strong irritant.

           →It is one of the oldest analgesic-anti-inflammatory drugs and is still widely used.

           →It is rapidly converted in the body to salicylic acid which is responsible for most of the actions.

  Mechanism of action

·         NSAIDS inhibits cyclooxygenase which is responsible for the synthesis of prostaglandin and thromboxane

·         It also inhibits platelet aggregation

Pharmacological actions

1. Analgesic action

→Aspirin is a weaker analgesic than morphine.

→These are effective only in dull- aching pain of low intensity.

→They do not relieve severe pain like visceral pain.

→They act by preventing the integration of pain sensation in the thalamus But they do not alter the                                         emotional reaction to pain.

2. Anti-pyretic effect

Salicylates do not lower normal body temperature.

Only the elevated temperature is lowered.

Mechanism

Fever is caused by elevated levels of prostaglandin E2, which   alters the firing rate of neurons with in the hypothalamus that control thermoregulation. Antipyretics work by inhibiting the enzyme COX, which causes the general inhibition of prostanoid biosynthesis (PGE2) within the hypothalamus.PGE2 signals to the hypothalamus to increase the body's thermal set point. Ibuprofen has been shown to be more effective as an antipyretic than acetaminophen. Arachidonic acid is the precursor substrate for cyclooxygenase leading to the production of prostaglandins F, D & E. This is reset for a lower temperature by salicylates

The salicylates produce sweating which also lowers body temperature

3. Anti- inflammatory action

→Aspirin exert the anti-inflammatory action at high doses 3- 6 grams /day

→Signs of inflammation like pain, tenderness, swelling, vasodilatation, and leukocyte infiltration are suppressed.

→Aspirin inhibits cyclooxygenase activity, it diminishes the formation of prostaglandins and modulates those aspects of inflammation in which prostaglandins act as mediators.

→Aspirin inhibits inflammation in arthritis.

4. on respiration

→Salicylates stimulate respiration

→The stimulation is depend on the dose

→Salicylates stimulates respiration directly by stimulating the respiratory Centre

5. Cardiovascular system

No effect at normal dose.

Large doses increase cardiac output to meet increased peripheral O₂ demand and cause direct vasodilatation.

Toxic doses produce paralysis of vasomotor Centre and BP may fall.

6. GI Tract

Salicylates produce nausea and vomiting due to direct stimulation chemoreceptor trigger zone.

Salicylates can also cause gastric ulceration and hemorrhage.

7. Anti- rheumatic effect

Salicylates have powerful anti-rheumatic effect

This effect is produced by reducing pain and inflammation of the joints

8. Blood

Salicylates lower the erythrocyte sedimentation rate (ESR) which is high in rheumatic fever

They also decrease prothrombin level of plasma.

9. Uricosuric effect

Low doses ( 1 or 2/ g day ) may decrease the urate excretion and increase plasma urea concentration

Intermediate doses ( 2 or 3 g / day ) do not alter urate excretion.

Large doses (over 5 g / day) induce urocosuric effect and lower plasma urate levels 

10. Metabolic effects

Salicylates produce uncoupling of oxidative phosphorylation

They produce hyperglycemia and glycosuria

They inhibit the synthesis but enhance the breakdown of fatty acids

Pharmacokinetics

Aspirin is taken through oral administration they are are absorbed from the stomach and small intestine

Adverse reactions

nausea, vomiting , diarrhea , ulceration perforation ,hemorrhage, skin rashes, agranulocytosis, thrombocytopenia , plastic anemia,  headache, difficulty in hearing, drowsiness, lethargy and confusion

Therapeutic Uses

1. It is used as analgesic for light and moderate pain --- 0.3 - 0.6 g - 3 times

2. used as anti-pyretic in fever ---   0.3 - 0.6 g - 3 times

3. Used as anti – inflammatory   & anti-rheumatic & ---4 – 6 g or 75– 100   mg/kg/day in divided doses

4. Anti- platelet effect - 75 – 300 mg / day

5. Used in the treatment of the gout

6. Used in the closure of ductus artereosus

İİ. Non selective reversible inhibitor of cox

Indomethacin

It has anti-inflammatory, analgesic, antipyretic and antigout actions.

It relieves pain and reduces temperature in febrile patients.

Reduces pain and joint swelling in rheumatoid arthritis but does not modify progress.

Mechanism of action

It is a portent inhibitor of cyclooxygenase thus reducing prostaglandin synthesis

Pharmacokinetics

Orally administered, well absorbed through liver, 90% bound to plasma proteins & half-life is 4 – 6 hours

Adverse effects

Adverse effects are high.

Gastrointestinal irritation with nausea, GI bleeding, vomiting, diarrhea and peptic ulcers can occur

Hypersensitivity reactions like skin rashes, leucopenia and asthma in aspirin sensitive individuals

Drug interactions

Indomethacin blunts the diuretic action of furosemide and the anti-hypertensive action of thiazides,  beta blockers and ACE inhibitors by causing salt and water retention

Dose

25 -30 mg

Uses

Rheumatoid arthritis

Gout

Ibuprofen

It is introduced in 1969. It is propionic acid derivatives better tolerated than aspirin.

Its analgesic activity is independent of anti- inflammatory activity and has both central and peripheral effect.

Temperature is reduced in febrile patients

Mechanism of action

It is a potent inhibitor of the enzyme cyclooxygenase resulting in the blockage of prostaglandin synthesis

It also prevents formation of thromboxane A₂ by platelet aggregation

It exhibits anti- inflammatory, analgesic and antipyretic activities

All have similar pharmacodynamics properties but differ considerably in potency and to some extent in duration of action

Analgesic, antipyretic and anti-inflammatory efficacy is slightly lower than aspirin

It is 99% bound to plasma proteins

Adverse effects

Nausea , Vomiting, Gastric discomfort, CNS effects, Hypersensitivity reactions.

Dose

400- 800 mg

Uses: 1. It has analgesic and antipyretic activity

              2. It is used in the treatment of gout

              3. Surgical removal of impacted tooth – a combination of ibuprofen with

                     a muscle relaxant like  chlorzoxaxone is recommended

               4. It is a drug of choice in rheumatoid arthritis because of lesser adverse effects

SELECTIVE COX-2 INHIBITORS

Celecoxib

This is highly selective inhibitor of cox2 enzyme .it is more selective towards cox-2 than the cox-1.

It does not have any inhibitory effect against TXA_{2 at} therapeutic doses

Pharmacokinetics

Orally administered excretion via renal and rectal route

Pharmacological actions

Anti-inflammatory, analgesic, antipyretic, antiplatelet action

Adverse effects

Skin rashes, hypersensitivity, ulceration, hemorrhage, diarrhea, dyspepsia, gastric discomfort,

 Mild hypertension, edema

Contraindications

Celecoxib is contraindicated in patients prone to cardiovascular or cerebrovascular disease.

Therapeutic use

·         Used in rheumatoid arthritis.

·         Used in treatment of osteoarthritis.

COX 3 INHIBITOR /REVERSIBLE INHIBITOR OF COX1

Paracetamol

It is a Para-amino phenol derivative

It has analgesic and antipyretic effects like salicylates

Paracetamol, a metabolite of phenacetin is found to be safer and effective

It has analgesic, good antipyretic and weak anti- inflammatory properties

Due to weak PG inhibitory activity in the periphery, it has poor anti-inflammatory actions

Paracetamol is active on cyclooxygenase in the brain which accounts for its antipyretic action

In presence of peroxides present at the site of inflammation, it has poor ability  

            To inhibit cyclooxygenase

It does not stimulate respiration

It has no action on acid- base balance, cellular metabolism, cardiovascular system and platelet function

It does not produce gastrointestinal irritation and uricosuric effect

It is analgesic and antipyretic of choice especially in patients in whom salicylates or other NSAID are contraindicated

Mechanism of action

Paracetamol exhibits analgesic action by peripheral blockage of pain impulse generation.

It produces analgesic and antipyretic action by inhibiting the action of endogenous pyrogen on the hypothalamic heat regulating centers.

Its weak anti-inflammatory activity is related to inhibition of prostaglandin synthesis in the CNS.

ADME

Rapid absorption through oral administration.  30% protein binding

It is metabolized by the hepatic microtonal enzymes in liver.

Plasma half-life is 2-3 hrs. 

Effects after an oral dose last 3-5 hrs.

It is mainly excreted I urine as conjugation products of glucuronic and sulphuricacids

The ability of the infant liver for glucuronidation of Paracetamol is poor and this results in enhanced toxicity of the drug in neonates

Adverse effects

·         In antipyretic doses, Paracetamol is safe and well – tolerated

·         Nausea and rashes may occur

Dose

0.5     :- 1 g every 4-6 hrs.

Chile 6 – 12 yrs : – 250 – 500 mg every 4-6 hrs

1– 5 years: - 120 – 250 mg every 4 – 6 hrs.

Less than 3 months 10 mg / kg body weight every 4 – 6 hrs.

Maximum dose for adult: – 4 g daily 

Maximum dose for a child: - 4 doses in 24 hrs.

Acute Paracetamol poisoning

When large doses are taken, acute Paracetamol poisoning results.

Children’s are more susceptible because the ability of their liver to metabolite Paracetamol is poor.

10 – 15 grams in adults cause serious toxicity.

Symptoms

                   Nausea,

                   Vomiting,

                   Paracetamol is hepatotoxic and causes severe hepatic damage.

                   Hepatic lesions are reversible when promptly treated.

Mechanism

Small portion of Paracetamol is metabolized to toxic compound – N-acetyl – benzoquinone- imine which is inactivated generally by binding with glutathione in the liver

But when large doses of Paracetamol are taken, larger amounts of the toxic compound are fumed and glutathione in the liver is not sufficient to inactivate it

As a result the toxic metabolite now binds to hepatic proteins resulting in hepatic necrosis

Chronic alcoholics and infants are more likely to develop hepatotoxicity

Paracetamol can also cause nephrotoxicity which may result in acute renal failure in some.

Uses

·         Paracetamol is prescribed in head ache, tooth ache, backache, myalgia etc.

·         Excellent antipyretic.

·         Used in children without any risk.

·         Drug of choice in osteoarthritis.

ANOVA TEST

ANOVA (Analysis of variance test)

Groups	Control	Group A	Group 11	Group 111	Group  1v	Total
	17	19	18	20	24
	21	22	16	23	28
	19	25	17	25	29
	11	18	13	20	25
Observations	4	4	4	4	4	20(n)
Sigma X	68	84	64	88	106	410  (T)
Mean	17	21	16	22	26.5
Sigma X 2	1212	1794	1038	1954	2826	8824
(Signa X)2 /n	1156	1764	1024	1936	2809	8689

Correction factor   Cf    = T²/N   =  (410) 2  

                                                          ------------    =  8405

                                                              20  

Total sum of squares    =  sigma X²   -  c.f  =  8824 – 8405  = 419

B/N groups sum of squares  = ( sigma X²)/n)   -  c.f  =  8689 – 8405  = 284

                                   

Within groups  =  Total sum of square  - between groups sum of squares

                            =  419 – 284  =  135

Degree of freedom

B/n  groups  =  number of groups  - 1   ,  5-1 =4

Total degree of freedom  =  Total observation of all groups – 1

                                                       20 – 1 = 19

Error (df )  Total df  - between groups df  = 19-4 =15

Mean square(b/n groups)  =   B/n groups sum of squares/degree of

                                                                                                        freedom           

                                                  = 284/4 = 71

                                           

Mean square within groups   = 135/15    =  9

F  =  Between mean squares / within groups mean square

     =   71/9  =  7.89

Referring to F – ration table for (4,15) degrees of freedom we get for F = 7.89 , p greater than  0.01 , hence there is a significant difference between groups

For finding out the differences b/n the groups, error mean square Anova is made use by applying dunnets t test

T test formula

Where s2 is the error mean square obtained from Anova

Dunnets  t test to determine effect of drug against control group

Statistic	A	B	C	D
t	1.886	0.472	2.358	4.481

D.F   = 15

*P less than  0.05  ,          ***P less than 0.001

Therefore drug C and drug D  differ significantly from control , but drug D is highly effective

 Tukeys test

Steps

1. Calculate an analysis of variance (e.g., One-way between-subjects ANOVA).
2. Select two means and note the relevant variables (Means, Mean Square Within, and number per condition/group)
3. Calculate Tukey's test for each mean comparison
4. Check to see if Tukey's score is statistically significant with Tukey's probability/critical value table taking into account appropriate df_within and number of treatments.

Problem: Susan Sound predicts that students will learn most effectively with a constant background sound, as opposed to an unpredictable sound or no sound at all. She randomly divides twenty-four students into three groups of eight. All students study a passage of text for 30 minutes. Those in group 1 study with background sound at a constant volume in the background. Those in group 2 study with noise that changes volume periodically. Those in group 3 study with no sound at all. After studying, all students take a 10 point multiple choice test over the material. She begins by conducting a One-way, between-subjects Analysis of Variance. She finds a significant F score. The relevant variables from her ANOVA table are:
MS_within =4.18; M₁ =6; M₂ =4; M₃ =3; df_within = 21; n = 8

EXAMPLES OF THE NULL HYPOTHESIS

A researcher may postulate a hypothesis:

H₁: Tomato plants exhibit a higher rate of growth when planted in compost rather than in soil.

And a null hypothesis:

H₀: Tomato plants do not exhibit a higher rate of growth when planted in compost rather than soil.

It is important to carefully select the wording of the null, and ensure that it is as specific as possible. For example, the researcher might postulate a null hypothesis:

H₀: Tomato plants show no difference in growth rates when planted in compost rather than soil.

There is a major flaw with this null hypothesis. If the plants actually grow more slowly in compost than in soil, an impasse is reached. H₁ is not supported, but neither is H₀, because there is a difference in growth rates.

If the null is rejected, with no alternative, the experiment may be invalid. This is the reason why science uses a battery of deductive and inductive processes to ensure that there are no flaws in the hypotheses.

Many scientists neglect the null, assuming that it is merely the opposite of the alternative, but it is good practice to spend a little time creating a sound hypothesis. It is not possible to change any hypothesis retrospectively, including H₀.

SIGNIFICANCE TESTS

If significance tests generate 95% or 99% likelihood that the results do not fit the null hypothesis, then it is rejected, in favor of the alternative.

Otherwise, the null is accepted. These are the only correct assumptions, and it is incorrect to reject, or accept, H₁.

Accepting the null hypothesis does not mean that it is true. It is still a hypothesis, and must conform to the principle of falsifiability, in the same way that rejecting the null does not prove the alternative. 

PERCEIVED PROBLEMS WITH THE NULL

The major problem with the null hypothesis is that many researchers, and reviewers, see accepting the null as a failure of the experiment. This is very poor science, as accepting or rejecting any hypothesis is a positive result.

Even if the null is not refuted, the world of science has learned something new. Strictly speaking, the term ‘failure’, should only apply to errors in the experimental design, or incorrect initial assumptions. 

DEVELOPMENT OF THE NULL

The Flat Earth model was common in ancient times, such as in the civilizations of the Bronze Age or Iron Age. This may be thought of as the null hypothesis, H₀, at the time.

H₀: World is Flat

Many of the Ancient Greek philosophers assumed that the sun, moon and other objects in the universe circled around the Earth. Hellenistic astronomy established the spherical shape of the earth around 300 BC.

H₀: The Geocentric Model: Earth is the centre of the Universe and it is Spherical

Copernicus had an alternative hypothesis, H₁ that the world actually circled around the sun, thus being the center of the universe. Eventually, people got convinced and accepted it as the null, H₀.

H₀: The Heliocentric Model: Sun is the centre of the universe

Later someone proposed an alternative hypothesis that the sun itself also circled around the something within the galaxy, thus creating a new null hypothesis. This is how research works - the null hypothesis gets closer to the reality each time, even if it isn't correct, it is better than the last null hypothesis.

Gluconeogenesis

TRANSDUCTION MECHANISMS

Type 1: ligand-gated ion channels:- (also known as ionotropic receptors). These are membrane proteins with a similar structure to other ion channels, and incorporate a ligand-binding (receptor) site, usually in the extracellular domain. Typically, these are the receptors on which fast neurotransmitters act. Examples include the nicotinic acetylcholine receptor (nAChR); GABA_A receptor  and glutamate receptors of the NMDA, AMPA and kainate types

The nicotinic acetylcholine receptor, the first to be cloned, It is assembled from

four different types of subunit, termed α, β, γ and δ.The pentameric structure

(α₂, β, γ, δ) possesses two acetylcholine binding sites, each lying at the interface

between one of the two α subunits and its neighbour. Both must bind

acetylcholine molecules in order for the receptor to be activated.

THE GATING MECHANISM

Receptors of this type control the fastest synaptic events in the nervous system, in which a neurotransmitter acts on the postsynaptic membrane of a nerve or muscle cell and transiently increases its permeability to particular ions. Most excitatory neurotransmitters, such as acetylcholine at the neuromuscular  Junction or glutamate in the central nervous system, cause an increase in Na+ and K+s permeability. This results in a net inward current carried mainly by Na+, which depolarises the cell and increases the probability that it will generate an action potential. The action of the transmitter reaches a peak in a fraction of a millisecond,and usually decays within a few milliseconds. The sheer speed of this response implies that the coupling between the receptor and the ionic channel is a direct one, and the molecular structure of the receptor-channel complex agrees with this.In contrast to other receptor families, no intermediate  biochemical steps are involved in the transduction process. Type 2: G-protein-coupled receptors (GPCRs):- These are also known as metabotropic receptors or 7-transmembrane-spanning (heptahelical) receptors. They are membrane receptors that are coupled to intracellular effector systems via a G-protein (see below). They constitute the largest family,5 and include receptors for many hormones and slow transmitters, for example the muscarinic acetylcholine receptor (mAChR), adrenergic receptors and chemokine receptors G-proteins and their role G-proteins comprise a family of membrane-resident proteins whose function is to recognise activated GPCRs and pass on the message to the effector systems that generate a cellular response They are the go-between proteins, but were actually called G-proteins because of their interaction with the guanine nucleotides, GTP and GDP. G-proteins consist of three subunits: α, β and γ. Guanine nucleotides bind to the α subunit, which has enzymic activity, catalysing the conversion of GTP to GDP. The β and γ subunits remain together as a βγ complex. G-proteins appear to be freely diffusible in the plane of the membrane, so a single pool of G-protein in a cell can interact with several different receptors and effectors.In the 'resting' state, the G-protein exists as an unattached αβγ trimer, with GDP occupying the site on the α subunit. When a GPCR is activated by an agonist molecule, a conformational change occurs, involving the cytoplasmic domain of the receptor, causing it to acquire high affinity for αβγ. Association of αβγ with the receptor causes the bound GDP to dissociate and to be replaced with GTP (GDP-GTP exchange), which in turn causes dissociation of the G-protein trimer, releasing α-GTP and βγ subunits; these are the 'active' forms of the G-protein, which diffuse in the membrane and can associate with various enzymes and ion channels, causing activation of the target. Signalling is terminated when the hydrolysis of GTP to GDP occurs through the GTPase activity of the α subunit. The resulting α-GDP then dissociates from the effector, and reunites with βγ, completing the cycle. TARGETS FOR G-PROTEINS The main targets for G-proteins, through which GPCRs control different aspects of cell function cAMP is a nucleotide synthesised within the cell from ATP by the action of a membrane-bound enzyme, adenylyl cyclase. It is produced continuously and inactivated by hydrolysis to 5´-AMP, by the action of a family of enzymes known as phosphodiesterases (PDEs). Many different drugs, hormones and neurotransmitters act on GPCRs and produce their effects by increasing or decreasing the catalytic activity of adenylyl cyclase, thus raising or lowering the concentration of cAMP within the cell. Cyclic AMP regulates many aspects of cellular function including, for example, enzymes involved in energy metabolism, cell division and cell differentiation, ion transport, ion channels, and the contractile proteins in smooth muscle. These varied effects are, however, all brought about by a common mechanism, namely the activation of protein kinases by cAMP. Protein kinases regulate the function of many different cellular proteins by controlling protein phosphorylation. Examples 1.       Increased cAMP production in response to β-adrenoceptor activation affects enzymes involved in glycogen and fat metabolism in liver, fat and muscle cells. The result is a coordinated response in which stored energy in the form of glycogen and fat is made available as glucose to fuel muscle contraction. 2.       Other examples of regulation by cAMP-dependent protein kinases include the increased activity of voltage-activated calcium channels in heart muscle cells. Phosphorylation of these channels increases the amount of Ca2+ entering the cell during the action potential, and thus increases the force of contraction of the heart. 3.       In smooth muscle, cAMP-dependent protein kinase phosphorylates (thereby inactivating) another enzyme, myosin-light-chain kinase, which is required for contraction. This accounts for the smooth muscle relaxation produced by many drugs that increase cAMP production in smooth muscle 4.       include certain types of mAChR (e.g. the M2 receptor of cardiac muscle; see, α2-adrenoceptors in smooth muscle, and opioid receptors The phospholipase C/inositol phosphate system The phosphoinositide system, an important intracellular second messenger system. PIP2 is the substrate for a membrane-bound enzyme, phospholipase Cβ (PLCβ), which splits it into DAG and inositol (1,4,5) trisphosphate (IP3), both of which function as second messengers. The activation of PLCβ by various agonists is mediated through a G-protein. After cleavage of PIP2, the status quo is restored. DAG being phosphorylated to form phosphatidic acid (PA), while the IP3 is dephosphorylated and then recoupled with PA to form PIP2 once again. Inositol phosphates and intracellular calcium Inositol (1,4,5) trisphosphate is a water-soluble mediator that is released into the cytosol and acts on a specific receptor-the IP3 receptor-which is a ligand-gated calcium channel present on the membrane of the endoplasmic reticulum. The main role of IP3, is to control the release of Ca2+ from intracellular stores. Because many drug and hormone effects involve intracellular Ca2+, this pathway is particularly important. IP3 is converted inside the cell to the (1,3,4,5) tetraphosphate, IP4, by a specific kinase. The exact role of IP4 remains unclear, but there is evidence that it too is involved in Ca2+ signalling. One possibility is that it facilitates Ca2+ entry through the plasma membrane, thus avoiding depletion of the intracellular stores as a result of the action of IP3. Diacylglycerol and protein kinase C Diacylglycerol is produced as well as IP3 whenever receptor-induced PI hydrolysis occurs. The main effect of DAG is to activate a membrane-bound protein kinase, protein kinase C (PKC), which catalyses the phosphorylation of a variety of intracellular proteins. DAG, unlike the inositol phosphates, is highly lipophilic and remains within the membrane. It binds to a specific site on the PKC molecule, which migrates from the cytosol to the cell membrane in the presence of DAG, thereby becoming activated. Ion channels as targets for G-proteins G-protein-coupled receptors can control ion channel function directly by mechanisms that do not involve second messengers such as cAMP or inositol phosphates. This was first shown for cardiac muscle, but it now appears that direct G-protein-channel interaction may be quite general . In cardiac muscle, for example, mAChRs are known to enhance K+ permeability (thus hyperpolarising the cells and inhibiting electrical activity. Similar mechanisms operate in neurons, where many inhibitory drugs such as opiate analgesics reduce excitability by opening potassium channels. Type 3: kinase-linked and related receptors:- This is a large and heterogeneous group of membrane receptors responding mainly to protein mediators. They comprise an extracellular ligand-binding domain linked to an intracellular domain by a single transmembrane helix. In many cases, the intracellular domain is enzymic in nature (with protein kinase or guanylyl cyclase activity). Type 3 receptors include those for insulin and for various cytokines and growth factors the receptor for atrial natriuretic factor (ANF), is the main example of the guanylyl cyclase type. The two kinds are very similar structurally, even though their transduction mechanisms differ. KINASE-LINKED AND RELATED RECEPTORS These membrane receptors are quite different in structure and function from either the ligand-gated channels or the GPCRs. They mediate the actions of a wide variety of protein mediators, including growth factors and cytokines, and hormones such as insulin and leptin, whose effects are exerted mainly at the level of gene transcription. They play a major role in controlling cell division, growth, differentiation, inflammation, tissue repair, apoptosis and immune responses, The main types are as follow Receptor tyrosine kinases (RTKs). These receptors have the basic structure, incorporating a tyrosine kinase moiety in the intracellular region. They include receptors for many growth factors, such as epidermal growth factor and nerve growth factor, and also the group of Toll-like receptors that recognise bacterial lipopolysaccarides and play an important role in the body's reaction to infection. The insulin receptor also belongs to the RTK class, although it has a more complex dimeric structure. Serine/threonine kinases. This smaller class is similar in structure to RTKs but phosphorylate serine and/or threonine residues rather than tyrosine. The main example is the receptor for transforming growth factor (TGF). Cytokine receptors. These receptors lack intrinsic enzyme activity. When occupied, they associate with, and activate, a cytosolic tyrosine kinase, such as Jak (the Janus kinase) or other kinases. Ligands for these receptors include cytokines such as interferons and colony-stimulating factors involved in immunological responses. Guanylyl cyclase-linked receptors. These are similar in structure to RTKs, but the enzymic moiety is guanylyl cyclase and they exert their effects by stimulating cGMP formation. The main example is the receptor for ANF. In many cases, ligand binding to the receptor leads to dimerisation. The association of the two intracellular kinase domains allows a mutual autophosphorylation of intracellular tyrosine residues to occur. The phosphorylated tyrosine residues then serve as high-affinity docking sites for other intracellular proteins that form the next stage in the signal transduction cascade. One important group of such 'adapter' proteins is known as the SH2 domain proteins (standing for Src homology, because it was first identified in the Src oncogene product).       Two well-defined signal transduction pathways are summarised in The Ras/Raf pathway mediates the effect of many growth factors and mitogens. Ras, which is a proto-oncogene product, functions like a G-protein, and conveys the signal (by GDP/GTP exchange) from the SH2 domain protein, Grb, which is phosphorylated by the RTK. Activation of Ras in turn activates Raf, which is the first of a sequence of three serine/threonine kinases, each of which phosphorylates, and activates, the next in line. The last of these, mitogen-activated protein (MAP) kinase, phosphorylates one or more transcription factors that initiate gene expression, resulting in a variety of cellular responses, including cell division. Many SH2 domain proteins are enzymes, such as protein kinases or phospholipases. Some growth factors activate a specific subtype of phospholipase C (PLCγ), thereby causing phospholipid breakdown, IP3 formation and Ca2+ release. Other SH2-containing proteins couple phosphotyrosine-containing proteins with a variety of other functional proteins, including many that are involved in the control of cell division and differentiation. The end result is to activate or inhibit, by phosphorylation, a variety of transcription factors that migrate to the nucleus and suppress or induce the expression of particular genes.       A second pathway, the Jak/Stat pathway is involved in responses to many cytokines. Dimerisation of these receptors occurs when the cytokine binds, and this attracts a cytosolic tyrosine kinase unit (Jak) to associate with, and phosphorylate, the receptor dimer. Jaks belong to a family of proteins, different members having specificity for different cytokine receptors. Among the targets for phosphorylation by Jak are a family of transcription factors (Stats). These are SH2 domain proteins that bind to the phosphotyrosine groups on the receptor-Jak complex, and are themselves phosphorylated. Thus activated, Stat migrates to the nucleus and activates gene expression.       The membrane-bound form of guanylyl cyclase, the enzyme responsible for generating the second messenger cGMP in response to the binding of peptides such as atrial natriuretic peptide, resembles the tyrosine kinase family and is activated in a similar way by dimerisation when the agonist is bound. Type 4: nuclear receptors:- These are receptors that regulate gene transcription. The term nuclear receptors is something of a misnomer, because some are actually located in the cytosol and migrate to the nuclear compartment when a ligand is present. They include receptors for steroid hormones, thyroid hormone, and other agents such as retinoic acid and vitamin D. NUCLEAR RECEPTORS Receptors for steroid hormones such as oestrogen and the glucocorticoids were present in the cytoplasm of cells and translocated into the nucleus after binding with their steroid partner. Other hormones, such as the thyroid hormone T3 and the fat-soluble vitamins D and A (retinoic acid) and their derivatives that regulate growth and development, were found to act in a similar fashion.