Pharmacodynamics: Mechanism of Drug Action; Receptor Pharmacology

Chapter 4

Pharmacodynamics: Mechanism of Drug Action; Receptor Pharmacology

Pharmacodynamics is the study of drug effects. It attempts to elucidate the complete action-effect sequence and the dose-effect relationship. Modification of the action of one drug by another drug is also an aspect of pharmacodynamics.

PRINCIPLES OF DRUG ACTION

Drugs (except those gene based) do not impart new functions to any system, organ or cell; they only alter the pace of ongoing activity. The basic types of drug action can be broadly classed as:

1. Stimulation

It refers to selective enhancement of the level of activity of specialized cells, e.g. adrenaline stimulates heart, pilocarpine stimulates salivary glands. However, excessive stimulation is often followed by depression of that function, e.g. high dose of picrotoxin, a central nervous system (CNS) stimulant, produces convulsions followed by coma and respiratory depression.

2. Depression

It means selective diminution of activity of specialized cells, e.g. barbiturates depress CNS, quinidine depresses heart. Certain drugs stimulate one type of cells but depress the other, e.g. acetylcholine stimulates intestinal smooth muscle but depresses SA node in heart. Thus, most drugs cannot be simply classed as stimulants or depressants.

3. Irritation

This connotes a nonselective, often noxious effect and is particularly applied to less specialized cells (epithelium, connective tissue). Mild irritation may stimulate associated function, e.g. bitters increase salivary and gastric secretion, counterirritants increase blood flow to the site. But strong irritation results in inflammation, corrosion, necrosis and morphological damage. This may result in diminution or loss of function.

4. Replacement

This refers to the use of natural metabolites, hormones or their congeners in deficiency states, e.g. levodopa in parkinsonism, insulin in diabetes mellitus, iron in anemia.

5. Cytotoxic action

Selective cytotoxic action for invading parasites or cancer cells, attenuating them without significantly affecting the host cells is utilized for cure/palliation of infections and neoplasms, e.g. penicillin, chloroquine, zidovudine, cyclophosphamide, etc.

MECHANISM OF DRUG ACTION

• Only a handful of drugs act by virtue of their simple physical or chemical property; examples are:

• Bulk laxatives (ispaghula)—physical mass

• Dimethicone, petroleum jelly—physical form, opacity.

• Para amino benzoic acid—absorption of UV rays

• Activated charcoal—adsorptive property 

• Mannitol, mag. sulfate—osmotic activity 

• 131I and other radioisotopes—radioactivity

• Antacids—neutralization of gastric HCl

• Pot. permanganate—oxidizing property 

• Chelating agents (EDTA, dimercaprol)—chelation of heavy metals. 

• Cholestyramine—sequestration of cholesterol in the gut 

• Mesne Scavenging of varicotic reactive metabolites of cyclophosphamide

• Majority of drugs produce their effects by interacting with a discrete target biomolecule, which usually is a protein. Such mechanism confers selectivity of action to the drug. Functional proteins that are targets of drug action can be grouped into four major categories, viz. enzymes, ion channels, transporters and receptors (See Fig. 4.1). However, a few drugs do act on other proteins (e.g. colchicine, vinca alkaloids, taxes bind to the structural protein tubulin) or on nucleic acids (alkylating agents).

I. ENZYMES

Almost all biological reactions are carried out under catalytic influence of enzymes; hence, enzymes are a very important target of drug action. Drugs can either increase or decrease the rate of enzymatically mediated reactions. However, in physiological systems enzyme activities are often optimally set. Thus, stimulation of enzymes by drugs, that are truly foreign substances, is unusual. Enzyme stimulation is relevant to some natural metabolites only, e.g. pyridoxine acts as a cofactor and increases decarboxylase activity. Several enzymes are stimulated through receptors and second messengers, e.g. adrenaline stimulates hepatic glycogen phosphorylase through β receptors and cyclic AMP. Stimulation of an enzyme increases its affinity for the substrate so that rate constant (kM) of the reaction is lowered (Fig. 4.2)

Apparent increase in enzyme activity can also occur by enzyme induction, i.e. synthesis of more enzyme protein. This cannot be called stimulation because the kM does not change. Many drugs induce microsomal enzymes (see p. 27). Inhibition of enzymes is a common mode of drug action.

A. Nonspecific inhibition

Many chemicals and drugs are capable of denaturing proteins. They alter the tertiary structure of any enzyme with which they come in contact and thus inhibit it. Heavy metal salts, strong acids and alkalis, alcohol, formaldehyde, phenol inhibit enzymes nonspecifically. Such inhibitors are too damaging to be used systemically.

B. Specific inhibition

Many drugs inhibit a particular enzyme without affecting others. Such inhibition is either competitive or noncompetitive.

• Competitive (equilibrium type) The drug being structurally similar competes with the normal substrate for the catalytic binding site of the enzyme so that the product is not formed, or a nonfunctional product is formed (Fig. 4.1A), and a new equilibrium is achieved in the presence of the drug. Such inhibitors increase the Kem but the Vmax remains unchanged (Fig. 4.2), i.e. higher concentration of the substrate is required to achieve ½ maximal reaction velocity, but if substrate concentration is sufficiently increased, it can displace the inhibitor and the same maximal reaction velocity can be attained.

• Physostigmine and neostigmine compete with acetylcholine for cholinesterase. 

• Sulfonamides compete with PABA for bacterial folate synthetase.

• Moclobemide competes with catecholamines for monoamine oxidase-A (MAO-A). 

• Captopril competes with angiotensin 1 for angiotensin converting enzyme (ACE). 

• Finasteride competes with testosterone for 5α-reductase 

• Letrozole competes with androstenedione and testosterone for the aromatase enzyme. 

• Allopurinol competes with hypoxanthine for xanthine oxidase; is itself oxidized to alloxanthins (a non competitive inhibitor).

• Carbidopa and methyldopa compete with levodopa for dopa decarboxylase.

• A nonequilibrium type of enzyme inhibition can also occur with drugs which react with the same catalytic site of the enzyme but either form strong covalent bonds or have such high affinity for the enzyme that the normal substrate is not able to displace the inhibitor, e.g. 

• Organophosphates react covalently with the enteritic site of the enzyme cholinesterase. 

• Methotrexate has 50,000 times higher affinity for dihydrofolate reductase than the normal substrate DHFA. In these situations, Kim is increased, and Vmax is reduced.

• Noncompetitive The inhibitor reacts with an adjacent site and not with the catalytic site but alters the enzyme in such a way that it loses its catalytic property. Thus, kM is unchanged, but Vmax is reduced. Examples are given in the box.

II. ION CHANNELS

• Proteins which act as ion selective channels participate in transmembrane signaling and regulate intracellular ionic composition. This makes them a common target of drug action (Fig. 4.1B). Drugs can affect ion channels either through specific receptors (ligand gated ion channels, G-protein operated ion channels, see Fig. 4.4 and p. 48), or by directly binding to the channel and affecting ion movement through it, e.g. local an aesthetics which physically obstruct voltage sensitive Na+ channels (See Ch 26). In addition, certain drugs modulate.

• Quinidine blocks myocardial Na+ channels.

• Dovetailed and amiodarone block myocardial delayed rectifier K+ channel.

• Nifedipine blocks L-type of voltage sensitive Ca2+ channel. 

• Nicorandil opens ATP-sensitive K+ channels. 

• Sulfonylurea hypoglycemic's inhibit pancreatic ATP-sensitive K+ channels. 

• Amiloride inhibits renal epithelial Na+ channels. 

• Phenytoin modulates (prolongs the inactivated state of) voltage sensitive neuronal Na+ channel.

• Ethosuximide inhibits T-type of Ca2+ channels in thalamic neurons.

III. TRANSPORTERS

• Several substrates are translocated across membranes by binding to specific transpor(carriers) which either facilitate diffusion in the direction of the concentration gradient or pump the metabolite/ion against the concentration gradient using metabolic energy (see p. 13–15; Fig. 2.5). Many drugs produce their action by directly interacting with the solute carrier (SLC) class of transporter proteins to inhibit the ongoing physiological transport of the metabolite/ion (Fig. 4.1C). Examples are:

• Desipramine and cocaine block neuronal reuptake of noradrenaline by interacting with norepinephrine transporter (NET). 

• Fluoxetine (and other SSRIs) inhibit neuronal reuptake of 5-HT by interacting with serotonin transporter (SERT).

•  Amphetamines selectively block dopamine reuptake in brain neurons by dopamine transporter (DAT).

• Reserpine blocks the granular reuptake of noradrenaline and 5-HT by the vesicular amine transporter. 

• Hemisodium blocks choline uptake into cholinergic neurons and depletes acetylcholine. 

• The anticonvulsant tiagabine acts by inhibiting reuptake of GABA into brain neurons by GABA transporter GAT 1. 

• Furosemide inhibits the Na+K+2Cl¯ cotransporter in the ascending limb of loop of Henle. Hydrochlorothiazide inhibits the Na+Cl¯ symporter in the early distal tubule. 

• Probenecid inhibits active transport of organic acids (uric acid, penicillin) in renal tubules by interacting with organic anion transporter (OAT). 

IV. RECEPTOR

• The largest number of drugs do not bind directly to the effectors, viz. enzymes, channels, transporters, structural proteins, template biomolecules, etc. but act through specific regulatory macromolecules which control the above listed effectors. These regulatory macromolecules or the sites on them which bind and interact with the drug are called ‘receptors’. 

• Receptor: It is defined as a macromolecule or binding site located on the surface or inside the effector cell that serves to recognize the signal molecule/drug and initiate the response to it, but itself has no other function.

• Though, in a broad sense all types of target biomolecule's, including the effectors (enzymes, channels, transporters, etc.) with which a drug can bind to produce its action have been denoted as ‘receptors’ by some authors, such designation tends to steal the specific meaning of this important term. If so applied, xanthine oxidase would be the ‘receptor’ for allopurinol, L-type Ca2+ channel would be the ‘receptor’ for nifedipine, serotonin transporter (SERT) would be the ‘receptor’ for fluoxetine; a connotation not in consonance with the general understanding of the term. It is therefore better to reserve the term ‘receptor’ for purely regulatory macromolecules which combine with and mediate the action of signal molecules including drugs. The following terms are used in describing drug-receptor interaction:

• Agonist An agent which activates a receptor to produce an effect similar to that of the physiological signal molecule.

• Inverse agonist An agent which activates a receptor to produce an effect in the opposite direction to that of the agonist.

• Antagonist An agent which prevents the action of an agonist on a receptor or the subsequent response, but does not have any effect of its own.

• Partial agonist An agent which activates a receptor to produce submaximal effect but antagonizes the action of a full agonist.

• Ligand (Latin: ligare—to bind) Any molecule which attaches selectively to particular receptors or sites. The term only indicates affinity or binding without regard to functional change: agonists and competitive antagonists are both ligands of the same receptor. The overall scheme of drug action through receptors is depicted in Fig. 4.1D

• Basic evidence for drug action through receptors

• Many drugs exhibit structural specificity of action, i.e. specific chemical configuration is associated with a particular action, e.g. isopropyl substitution on the ethylamine side chain of sympathetic drugs produces compounds with marked cardiac and bronchial activity—most β adrenergic agonists and antagonists have this substitution. A 3 carbon interneurons separation in the side chain of phenothiazines.

• results in antidopaminergic-antipsychotic compounds, whereas 2 carbon separation produces anticholinergic antihistaminic compounds. Further, chiral drugs show stereospecificity in action, e.g. levo noradrenaline is 10 times more potent than dextrose noradrenaline; d-propranolol is about 100 times less potent in blocking β receptors than the l-isomer, but both are equipotent local anesthetics. Thus, the cell must have some mechanism to recognize a particular chemical configuration and three-dimensional structure.
• Competitive antagonism is seen between specific agonists and antagonists. Langley in 1878 was so impressed by the mutual antagonism among two alkaloids pilocarpine and atropine that he proposed that both reacted with the same ‘receptive substance’ on the cell. Ehrlich (1900) observed quantitative neutralization between toxins and antitoxins and designated ‘receptor’ to be the anchoring group of the protoplasmic molecule for the administered compound.

• It was calculated by Clark that adrenaline and acetylcholine produce their maximal effect on frog’s heart by occupying only 1/6000th of the cardiac cell surface— thus, special regions of reactivity to such drugs must be present on the cell.

Receptor occupation theory

After studying quantitative aspects of drug action, Clark (1937) propounded a theory of drug action based on occupation of receptors by specific drugs and that the pace of a cellular function can be altered by interaction of these receptors with drugs which, in fact, are small molecular ligands. He perceived the interaction between the two molecular species, viz. drug (D ) and receptor (R) to be governed by the law of mass action, and the effect (E) to be a direct function of the drug-receptor complex (DR) formed:

Subsequently, it has been realized that occupation of the receptor is essential but not itself sufficient to elicit a response; the agonist must also be able to activate (induce a conformational change in) the receptor. The ability to bind with the receptor designated as affinity, and the capacity to induce a functional change in the receptor designated as intrinsic activity (IA) or efficacy are independent properties. Competitive antagonists occupy the receptor but do not activate it. Moreover, certain drugs are partial agonists which occupy and submaxim ally activate the receptor. An all or none action is not a must at the receptor. A theoretical quantity(S) denoting strength of stimulus imparted to the cell was interposed in the Clark’s equation:

• Depending on the agonist, DR could generate a stronger or weaker S, probably as a function of the conformational change brought about by the agonist in the receptor. Accordingly:

• Agonists have both affinity and maximal intrinsic activity (IA = 1), e.g. adrenaline, histamine, morphine.

• Competitive antagonists have affinity but no intrinsic activity (IA = 0), e.g. propranolol, atropine, chlorpheniramine, naloxone.

• Partial agonists have affinity and submaximal intrinsic activity (IA between 0 and 1), e.g. dichloroisoproterenol (on β adrenergic receptor), pentazocine (on μ opioid receptor).

• Inverse agonists have affinity but intrinsic activity with a minus sign (IA between 0 and –1), e.g. DMCM (on benzodiazepine receptor). It has also been demonstrated that many full agonists can produce maximal response even while occupying <1% of the available receptors large receptor reserve exists in their case, or a number of spare receptors are present.

• The two-state receptor model

• A very attractive alternative model for explaining the action of agonists, antagonists, partial agonists and inverse agonists has been proposed. The receptor is believed to exist in two interchangeable states: Ra (active) and Ri (inactive) which are in equilibrium. In the case of majority of receptors, the Ri state is favored at equilibrium—no/very weak signal is generated in the absence of the agonist—the receptor exhibits no constitutive activation (Fig. 4.3I). The agonist (A) binds preferentially to the Ra conformation and shifts the equilibrium → Ra predominates, and a response is generated (Fig. 4.3II) depending on

• the concentration of A. The competitive antagonist.

• binds to Ra and Ri with equal affinity → the equilibrium is not altered → no response is generated (Fig. 4.3 III), and when the agonist is applied fewer Ra are available to bind it— response to agonist is decreased. If an agonist has only slightly greater affinity for Ra than for Ri, the equilibrium is only modestly shifted towards Ra (Fig. 4.3 IV) even at saturating concentrations → a submaximal response is produced, and the drug is called a partial agonist 
• The inverse agonist 

• has high affinity for the Ri state (Fig. 4.3V), therefore it can produce an opposite response, provided the resting equilibrium was in Favour of the Ra state. Certain receptors (mainly G-protein coupled ones) such as benzodiazepine, histamine H2, angiotensin AT1, adrenergic β1 and cannabinoid receptors exhibit constitutive activation, i.e. an appreciable intensity signal is generated even in the basal state (no agonist present). In their case the inverse agonist stabilizes the receptor in the inactive conformation resulting in an opposite response. Only few inverse agonists are known at present, but as more receptors with constitutive activation are found, more inverse agonists are likely to be discovered.

• This model has gained wide acceptance because it provides an explanation for the phenomenon of positive cooperativity often seen with neurotransmitters and is supported by studies of conformational mutants of the receptor with altered equilibrium.

• Nature of receptors

• Receptors are regulatory macromolecules, mostly proteins, though nucleic acids may also serve as receptors. They are no longer hypothetical. Hundreds of receptor proteins have been isolated, purified, cloned and their primary amino acid (AA) sequence has been worked out. Molecular cloning has also helped in obtaining the receptor protein in larger quantity to study its structure and properties, and in subclassifying receptors. The cell surface receptors with their coupling and effector proteins are considered to be floating in a sea of membrane lipids; the folding, orientation and topography of the system being determined by interactions between the lipophilic and hydrophilic domains of the peptide chains with solvent molecules (water on one side and lipids on the other). Nonpolar portions of the AA chain tend to bury within the membrane, while polar groups tend to come out in the aqueous medium. In such a delicately balanced system, it is not difficult to visualize that a small molecular ligand binding to one site in the receptor molecule could be capable of tripping the balance (by altering distribution of charges, etc.) and bringing about conformational changes at distant sites. Each of the four major families of receptors (described later) have a well-defined common structural motif, while the individual receptors differ in the details of amino acid sequencing, length of intra/extracellular loops, etc. Majority of receptor molecules are made up of several non-identical subunits (heteropoly Meric), and agonist binding has been shown to bring about changes in their quaternary structure or relative alignment of the subunits, e.g. on activation the subunits of nicotinic receptor move apart opening a centrally located cation channel. Radioligand binding studies have helped in characterizing and classifying receptors even when they have been dissociated from the effector system.

• Many drugs act upon physiological receptors which mediate responses to transmitters, hormones, autacoids and other endogenous signal molecules; examples are cholinergic, adrenergic, histaminergic, steroid, leukotriene, insulin and other receptors. In addition, now some truly drug receptors have been described for which there are no known physiological ligands, e.g. benzodiazepine receptor, sulfonylurea receptor, cannabinoid receptor.

• Receptor subtypes

• The delineation of multiple types and subtypes of receptors for signal molecules has played an important role in the development of a number of targeted and more selective drugs. Even at an early stage of evolution of receptor pharmacology, it was observed that actions of acetylcholine could be grouped into ‘muscarinic’ and ‘nicotinic’ depending upon whether they were mimicked by the then known alkaloids muscarine or nicotine. Accordingly, they were said to be mediated by two types of cholinergic receptors, viz. muscarinic (M) or nicotinic (N); a concept strengthened by the finding that muscarinic actions were blocked by atropine, while nicotinic actions were blocked by curare. In a landmark study, Ahlquist (1948) divided adrenergic receptors into ‘α’ and ‘β’ on the basis of two distinct rankorder of potencies of adrenergic agonists. These receptors have now been further subdivided (M1, M2 ….M5), (NM, NN) (α1, α2) (β1, β2, β3). Multiple subtypes of receptors for practically all transmitters, autacoids, hormones, etc. are now known and have paved the way for introduction of numerous clinically superior drugs. In many cases, receptor classification has provided sound explanation for differences observed in the actions of closely related drugs. 

• The following criteria have been utilized in classifying receptors:

• Pharmacological criteria Classification is based on relative potencies of selective agonists and antagonists. This is the classical and oldest approach with direct clinical bearing; was used in delineating M and N cholinergic, α and β adrenergic, H1 and H2 histaminergic receptors, etc. 

• Tissue distribution The relative organ/tissue distribution is the basis for designating the subtype, e.g. the cardiac β adrenergic receptors as β1, while bronchial as β2. This division was confirmed by selective agonists and antagonists as well as by molecular cloning.

• Ligand binding Measurement of specific binding of high affinity radio-labelled ligand to cellular fragments (usually membranes) in vitro, and its displacement by various selective agonists/antagonists is used to delineate receptor subtypes. Multiple 5-HT receptors were distinguished by this approach. Autoradiography has helped in mapping distribution of receptor subtypes in the brain and other organs.

• Transducer pathway Receptor subtypes may be distinguished by the mechanism through which their activation is linked to the response, e.g. M cholinergic receptor acts through G-proteins, while N cholinergic receptor gates influx of Na+ ions; α adrenergic receptor.

• Molecular cloning The receptor protein is cloned and its detailed amino acid sequence as well as three-dimensional structure is worked out. Subtypes are designated on the basis of sequence homology. This approach has in the recent years resulted in a flood of receptor subtypes and several isoforms (which do not differ in ligand selectivity) of each subtype. The functional significance of many of these subtypes/ isoforms is dubious. Even receptors without known ligands (orphan receptors) have been described.

• Application of so many approaches has thrown up several detailed, confusing and often conflicting classifications of receptors. However, a consensus receptor classification is now decided on a continuing basis by an expert group of the International Union of Pharmacological Sciences (IUPHAR).

• Silent receptors These are sites which bind specific drugs but no pharmacological response is elicited. They are better called drug acceptors or sites of loss, e.g. plasma proteins which have binding sites for many drugs. To avoid confusion, the term receptor should be restricted to those regulatory binding sites which are capable of generating a response.

ACTION-EFFECT SEQUENCE

• Drug action’ and ‘drug effect’ are often loosely used interchangeably but are not synonymous.

• Drug action It is the initial combination of the drug with its receptor resulting in a conformational change in the latter (in case of agonists), or prevention of conformational change through exclusion of the agonist (in case of antagonists).

• Drug effect It is the ultimate change in biological function brought about as a consequence of drug action, through a series of intermediate steps (transducer).

• Receptors subserve two essential functions, viz, recognition of the specific ligand molecule and transduction of the signal into a response. Accordingly, the receptor molecule has a ligand binding domain (spatially and energetically suitable for binding the specific ligand) and an effector domain (Fig. 4.4) which undergoes a functional conformational change. These domains have.

•  now actually been identified in some receptors. The perturbation in the receptor molecule is variously translated into the response. The sequential relationship between drug action, transducer and drug effect can be seen in Fig. 4.1D and 4.6.

TRANSDUCER MECHANISMS

• Considerable progress has been made in the understanding of transducer mechanisms which in most instances have been found to be highly complex multistep processes that provide for amplification and integration of concurrently received extra- and intra-cellular signals at each step. Because only a handful of transducer pathways are shared by a large number of receptors, the cell is able to generate an integrated response reflecting the sum total of diverse signal input. The transducer mechanisms can be grouped into 4 major categories. Receptors falling in one category have also been found to possess considerable structural homology, and belong to one super family of receptors.

1. G-protein coupled receptors (GPCR)

• These are a large family of cell membrane receptors which are linked to the effector (enzyme/ channel/carrier protein) through one or more GTP-activated proteins (G-proteins) for response effectuation. All such receptors have a common pattern of structural organization (Fig. 4.5). The molecule has 7 α-helical membrane spanning hydrophobic amino acid (AA) segments which run into 3 extracellular and 3 intracellular loops. The agonist binding site is located somewhere between the helices on the extracellular face, while another recognition site formed by cytosolic segments binds the coupling G-protein. The Gproteins float in the membrane with their exposed

• domain lying in the cytosol, and are heterotrimeric in composition (α, β and γ subunits). In the inactive state GDP is bound to their exposed domain; activation through the receptor leads to displacement of GDP by GTP. The active αsubunit carrying GTP dissociates from the other two subunits and either activates or inhibits the effector. The βγ subunits have also been shown to modulate certain effectors like receptor operated K+ channels, adenylyl cyclase (AC) and phospholipase C.

• A number of G proteins distinguished by their α subunits have been described. The important ones with their action on the effector are: Gs : Adenylyl cyclase ↑, Ca2+ channel ↑ Gi : Adenylyl cyclase ↓, K+ channel ↑ Go : Ca2+ channel ↓ Gq : Phospholipase C ↑ G13 : Na+/H+ exchange ↑

• In addition Gn, Gk, Gt and Golf have been distinguished. A limited number of G-proteins are shared between different receptors and one receptor can utilize more than one G-protein (agonist pleiotropy), e.g. the following couplers have been associated with different receptors

• Receptor Coupler Muscarinic Gi, Go, Gq Dopamine D2 Gi, Go β-adrenergic Gs, Gi α1-adrenergic Gq α2-adrenergic Gi, Gs, Go GABAB Gi, Go 5-HT Gi, Gq, Gs, Gk

• In addition, a receptor can utilize different biochemical pathways in different tissues. The α-subunit has GTPase activity: the bound GTP is slowly hydrolyses to GDP: the α-subunit then dissociates from the effector to rejoin its other subunits, but not before the effector has been activated/inhibited for a few seconds and the signal has been amplified. The onset time of response through this type of receptors is also in seconds. There are three major effector pathways (Table 4.1) through which GPCRs function.\

• Adenylyl cyclase: cAMP pathway

•  Activation of AC results in intracellular accumulation of second messenger cAMP (Fig. 4.6) which functions mainly through cAMP-dependent protein kinase (PKA). The PKA phosphorylates and alters the function of many enzymes, ion channels, transporters and structural proteins to manifest as increased contractility/impulse generation (heart), relaxation (smooth muscle), glycogenolysis, lipolysis, inhibition of secretion/mediator release, modulation of junctional transmission, hormone synthesis, etc. In addition, cAMP directly opens a specific type of membrane Ca2+ channel called cyclic nucleotide gated channel (CNG) in the heart, brain and kidney. Responses opposite to the above are produced when AC is inhibited through inhibitory Gi-protein.

• Phospholipase C: IP3-DAG pathway Activation of phospholipase C (PLc) hydrolyses the membrane phospholipid phosphatidyl inositol 4, 5-bisphosphate (PIP2) to generate the second messenger's inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). The IP3 mobiliser Ca2+ from intracellular organellar depots and DAG enhances protein kinase C (PKc) activation by Ca2+ (Fig. 4.7). Cytosolic Ca2+ (third messenger in this.

• setting) is a highly versatile regulator acting through calmodulin (CAM), Kc and other effectors—mediates/modulates contraction, secretion/transmitter release, eicosanoid synthesis, neuronal excitability, intracellular movements, membrane function, metabolism, cell proliferation, etc. Like AC, the Plc. can also be inhibited through inhibitory G-protein when directionally opposite responses would be expected.

• Intracellular Ca2+ release has been found to occur in waves (Ca2+ mediated Ca2+ release from successive pools facilitated by inositol 1, 3, 4, 5-tetrakisphosphate—IP4) and exhibits a variety of agonist and concentration dependent oscillatory patterns. The activation of different effectors may depend on the amplitude and pattern of these oscillations. Thus, the same intracellular messenger can trigger different responses depending on the nature and strength of the extracellular signal.

• Channel regulation 

The activated Proteins' can also open or close ionic channels

• specific for Ca2+, K+ or Na+, without the intervention of any second messenger like cAMP or IP3, and bring about hyperpolarization/depolarization/ changes in intracellular Ca2+. The Gs opens Ca2+ channels in myocardium and skeletal muscles, while Gi and Go open K+ channels in heart and smooth muscle as well as close neuronal Ca2+ channels. Physiological responses like changes in inotropy, chronotropy, transmitter release, neuronal activity and smooth muscle relaxation follow. Receptors found to regulate ionic channels through G-proteins are listed in Table 4.1.

• Receptors with intrinsic ion channel

• These cell surface receptors, also called ligand gated ion channels, enclose ion selective channels (for Na+, K+, Ca2+ or Cl¯) within their molecules. Agonist binding opens the channel (Fig. 4.4) and causes depolarization/hyperpolarization/ changes in cytosolic ionic composition, depending on the ion that flows through. The nicotinic cholinergic, GABA-A, glycine (inhibitory), excitatory AA (kainite, NMDA or N-methyl-aspartate, Quisqualis) and 5HT3 receptors fall in this category.

• The receptor is usually a pentameric protein; all subunits, in addition to large intra- and extracellular segments, generally have four membrane spanning domains in each of which the AA chain traverses the width of the membrane six times. The subunits are thought to be arranged round the channel like a rosette and the α subunits usually bear the agonist binding sites.

• Certain receptor-operated (or ligand-gated) ion channels also have secondary ligands which bind to an allosteric site and modulate the gating of the channel by the primary ligand, e.g. the benzodiazepine receptor modulates GABAA gated Schanel. Thus, in these receptors, agonists directly operate ion channels, without the intervention of any coupling protein or second messenger. The onset and offset of responses through this class of receptors is the fastest (in milliseconds).

3. Enzyme-linked receptors

• This class of receptors have a subunit with enzymatic property or bind a JAK (Janus-Kinase) enzyme on activation. The agonist binding site and the catalytic site lie respectively on the outer and inner face of the plasma membrane (Fig. 4.8). These two domains are interconnected through a single transmembrane stretch of peptide chain. There are two major subgroups of such receptors. 

• Those that have intrinsic enzymatic activity.

• Those that lack intrinsic enzymatic activity, but bind a JAK-STAT kinase on activation

• Intrinsic enzyme receptors The intracellular domain is either a protein kinase or guanylyl cyclase

• In most cases the protein kinase specifically phosphorylates tyrosine residues on substrate proteins (Fig. 4.8A), e.g. insulin, epidermal growth factor (EGF), nerve growth factor (NGF) receptors, but in few it is a serine or threonine protein kinase. In the monomeric state, the kinase remains inactive. Agonist binding induces dimerization of receptor molecules and activates the kinase to autophosphorylation tyrosine residues on each other, increasing their affinity for binding substrate proteins and carrying forward the cascade of tyrosine phosphorylation's. Intracellular events are triggered by phosphorylation of relevant proteins, many of which carry a SH2 domain. A general feature of this class of receptors

• is that their dimerization also promotes receptor internalization, degradation in lysosomes and down regulation. The enzyme can also be guanylyl cyclase (GC), as in the case of atrial natriuretic peptide (ANP). Agonist activation of the receptor generates cGMP in the cytosol as a second messenger which in turn activates cGMP-dependent protein kinase and modulates cellular activity.

• JAK-STAT-kinase binding receptors These receptors differ in not having any intrinsic catalytic domain. Agonist induced dimerization alters the intracellular domain conformation to increase its affinity for a cytosolic tyrosine protein kinase JAK. On binding, JAK gets activated and phosphorylates tyrosine residues of the receptor, which now binds another free moving protein STAT (signal transducer and activator of transcription) which is also phosphorylated by JAK. Pairs of phosphorylated STAT dimerize and translocate to the nucleus to regulate gene transcription resulting in a biological response. Many cytokines, growth hormone, interferons, etc. act through this type of receptor.

• The enzyme-linked receptors transduce responses in a matter of few minutes to a few hour.

• Receptors regulating gene expression (Transcription factors)