Dopamine and its receptor

Dopamine and its receptor

The neurotransmitter dopamine is the primary endogenous ligand for dopamine receptors. Dopamine receptors are a class of metabotropic G protein-coupled receptors that are prominent in the vertebrate central nervous system (CNS).

Biosynthesis of Dopamine

Dopamine, a catecholamine, is synthesized in the terminals of dopaminergic neurons from tyrosine, which is transported across the blood-brain barrier by an active process. The rate-limiting step in the synthesis of dopamine is the conversion of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA), catalyzed by the enzyme tyrosine hydroxylase. L-DOPA is converted rapidly to dopamine by aromatic L-amino acid decarboxylase.

Fig. 1 Dopaminergic terminal.

Location

CNS- Assays of distinct regions of the CNS eventually revealed that the distributions of dopamine and norepinephrine are markedly different. In fact, more than half the CNS content of catecholamine is dopamine and extremely large amounts are found in the basal ganglia, the nucleus accumbens, the olfactory tubercle, the central nucleus of the amygdala, the median eminence, and restricted fields of the frontal cortex.

Cardio-pulmonary- In humans, the pulmonary artery expresses D1, D2, D4, and D5 and receptor subtypes, which may account for vasorelaxive effects of dopamine in the blood. In rats, D1-like receptors are present on the smooth muscle of the blood vessels in most major organs.

D4 receptors have been identified in the atria of rat and human hearts. Dopamine increases myocardial contractility and cardiac output, without changing heart rate, by signaling through dopamine receptors.

Renal- Dopamine receptors are present along the nephron in the kidney, with proximal tubule epithelial cells showing the highest density. In rats, D1-like receptors are present on the juxtaglomerular apparatus and on renal tubules, while D2-like receptors are present on the renal tubules, glomeruli, postganglionic sympathetic nerve terminals, and zona glomerulosa cells of the renal cortex. Dopamine signaling affects diuresis and natriuresis.

Storage and release

In dopaminergic nerve terminals, dopamine is taken up into vesicles by a transporter protein; this process is blocked by reserpine, which leads to depletion of dopamine. Release of dopamine from nerve terminals occurs through exocytosis of presynaptic vesicles, a process that is triggered by depolarization leading to entry of Ca2+. Release, triggered by depolarization and entry of Ca2+, allows dopamine to act on postsynaptic dopamine receptors (DAR).

Metabolism

Once dopamine is in the synaptic cleft, its actions may be terminated by reuptake through a membrane carrier protein, a process antagonized by drugs such as cocaine. Alternatively, dopamine can be degraded by the sequential actions of monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) to yield two metabolic products, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxyphenylacetic acid (HVA). In human beings, HVA is the primary product of the metabolism of dopamine

Dopamine receptors

Dopamine receptors have key roles in many processes, including the control of motivation, learning, and fine motor movement, as well as modulation of neuroendocrine signaling. Abnormal dopamine receptor signaling and dopaminergic nerve function is implicated in several neuropsychiatric disorders. Thus, dopamine receptors are common neurologic drug targets.

Antipsychotics- dopamine receptor antagonists while psychostimulants- indirect agonists of dopamine receptors. At the cellular level, the actions of dopamine depend on receptor subtype expression.

The dopamine receptors share several structural features, including the presence of seven alpha-helical segments capable of spanning the cell membrane. This structure identifies the dopamine receptors as members of the larger superfamily of seven-transmembrane-region receptor proteins. All members of this superfamily act through guanine nucleotide-binding proteins.

The actions of dopamine in the brain are mediated by a family of dopamine receptor proteins. Two types of dopamine receptors were identified in the mammalian brain using pharmacological techniques:

D1 receptors family, which stimulate the synthesis of the intracellular second messenger cyclic AMP, and

D2 receptors family, which inhibit cyclic AMP synthesis.

The five dopamine receptors can be divided into two groups on the basis of their pharmacological and structural properties (Fig. 2).

SNpc, substantia nigra pars compacta; cAMP-cyclic AMP; psi-voltage.

Fig. 2 Distribution and characteristics of dopamine receptors.

D1-like family (excitatory)

The D1 and D5 proteins have a long intracellular carboxy-terminal tail and are members of the pharmacologically defined D1 class. Activation of the D1-like family receptors is coupled to the G protein Gαs, which subsequently activates adenylyl cyclase, increasing the intracellular concentration of the second messenger Cyclic adenosine monophosphate (cAMP). Increased cAMP in neurons is typically excitatory and can induce an action potential by modulating the activity of ion channels.

D2-like family (inhibitory)

The D2, D3, and D4 receptors share a large third intracellular loop and are of the D2 class. D2-like activation is coupled to the G protein Gαi, which subsequently increased phosphodiesterase activity. Phosphodiesterases break down cAMP, producing an inhibitory effect in neurons. They decrease cyclic AMP formation and modulate K+ and Ca2+ currents.

D1-like receptor agonists

Fenoldopam, Piribedil, Ibopamine, SKF 3893, Apomorphine

Therapeutic uses of D1-like receptor agonists

* Decreases peripheral resistance

* Inducing lowering of arterial blood pressure-increases in heart rate and increases in sympathetic tone

* Increases in activity of the rennin-aldosterone system

D2-like receptor agonists

Bromocriptine, Pergolid, Lisuride, Guinpirole, Carmoxirole

Therapeutic uses of D2-like receptor agonists

* Used for treating Parkinson’s disease

* Inhibits prolactin release (which decreases tumor size)

D1-like receptor antagonists

SCH23390, Clozapine (used for treating schizophrenia)

D2-like receptor antagonists

Metoclopramid, Domperidone (Gastric Motility Disorders), Sulpiride, Haloperidol,

Dopamine receptors in disease

Dysfunction of dopaminergic neurotransmission in the CNS has been implicated in a variety of neuropsychiatric disorders, including Tourette's syndrome (inherited neurological disorders- tic disorder (involuntary movement), Parkinson's disease, schizophrenia, Attention-deficit hyperactivity disorder (ADHD), and drug and alcohol dependence.

Attention-deficit hyperactivity disorder

Dopamine receptors have been recognized as important components in the etiology of ADHD for many years. Drugs used to treat ADHD, including methylphenidate and amphetamine, have significant effects on dopamine signaling in the brain. Studies of gene association have implicated several genes within dopamine signaling pathways; in particular, the D4.7 variant of D4 has been consistently shown to be more frequent in ADHD patients. The D4.7 allele has suppressed gene expression compared to other variants.

Drug abuse

Dopamine is the primary neurotransmitter involved in the reward pathways in the brain. Thus, drugs that increase dopamine signaling may produce euphoric effects. Cocaine and methamphetamine—two examples of such drugs—alter the functionality of the dopamine transporter (DAT), the protein responsible for removing dopamine from the neural synapse. When DAT activity is blocked, the synapse floods with dopamine and increases dopaminergic signaling. When this occurs, particularly in the nucleus accumbens, increased D1 and D2 receptor signaling mediates the "rewarding" stimulus of drug intake.

Schizophrenia

While there is evidence that the dopamine system is involved in schizophrenia, the theory that hyperactive dopaminergic signal transduction induces the disease is controversial. Psychostimulants, such as amphetamine and cocaine, induce dramatic changes in dopamine signaling; large doses and prolonged usage can induce symptoms that resemble schizophrenia. Additionally, many antipsychotic drugs target dopamine receptors, especially D2 receptors.

Genetic hypertension

Dopamine receptor mutations can cause genetic hypertension in humans. This can occur in animal models and humans with defective dopamine receptor activity, particularly D1.

Gamma amino butyric acid (GABA) and its receptor

GABA

GABA is the major inhibitory amino acid transmitter of the mammalian central nervous system and it is present in some 40% of all neurones.

Synthesis, storage and function

Gamma amino butyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system. It is found in almost every region of brain, and is formed through the activity of the enzyme glutamic acid decarboxylase (GAD).

GAD catalyzes the formation of GABA from glutamic acid. The synthesis of GABA is linked to the Kreb's cycle. GAD requires vitamin B6 (pyridoxal phosphate) as a cofactor, which can be used to regulate the levels of GABA. GABA is destroyed by a transamination reaction, in which the amino group is transferred to alpha-oxoglutaric acid (to yield glutamate), with the production of succinic semialdehyde, and then succinic acid. The reaction is catalysed by GABA transaminase.

Vigabatrine, a GABA agonist, is used to treat epilepsy by inhibiting GABA transaminase.

GABA-ergic neurons and astrocytes take up GABA by specific transporters, and it is this, rather than GABA transaminase, that removes the GABA after it has been released. GABA transport is inhibited by Guvacine and Nipecotic acid.

Compounds such as the competitive GAD inhibitor allylglycine, inhibit GABA formation and cause convulsions due to the lack of GABA activity.

Sodium valproate (or valproic acid) on the other hand, blocks GABA transaminase activity, thereby elevating GABA levels, and thus alleviating seizures. Sodium valproate is useful in the treatment of epilepsy and bipolar mood disorders. Another strategy is to block GABA-transaminase with g-acetylenic GABA, thereby increasing the concentration of GABA at the synapse.

GABA receptors

Structure and Pharmacology

The GABA receptors are a class of receptors that respond to the neurotransmitter gamma aminobutyric acid (GABA), the chief inhibitory neurotransmitter in the vertebrate central nervous system. There are three classes of GABA receptors: GABAA, GABAB, and GABAС.

GABAA and GABAС receptors are ligand-gated ion channels (also known as ionotropic receptors), whereas GABAB receptors are G protein-coupled receptors (also known as metabotropic receptors).

Ligand-gated ion channels

GABAA

It has long been recognized that the fast response of neurons to GABA that is blocked by bicuculline and picrotoxin is due to direct activation of an anion channel.

This channel was subsequently termed the GABAA receptor. Fast-responding GABA receptors are members of family of Cys-loop ligand-gated ion channels. Members of this superfamily, which includes nicotinic acetylcholine receptors, GABAA and GABAС receptors, glycine and 5-HT3 receptors, possess a characteristic loop formed by a disulphide bond between two cysteine residues.

The GABAA receptor consists of three separate subunits with an alpha2, beta2, gamma arrangement similar to that found for neuronal nicotinic receptors. The ligand binding site is located at the interface between the alpha and beta subunits. The benzodiazepine binding site is located at a similar level at the interface between the alpha and gamma subunits. Each subunit is comprised of four hydrophobic sequences which span the membrane, with a large extracellular amino terminus containing the binding site and a carboxyl terminus located intracellularly.

Electrophysiological studies demonstrated that the activation of the receptor resulted in increased chloride conductance of the cell membrane with the concentration-response curve exhibiting positive cooperativity, consistent with the presence of at least two agonist binding sites on the receptor molecule. The agonist induced current decreased on continued exposure to high agonist concentrations suggesting that these receptors undergo desensitisation.

GABAC

In addition to the GABAA receptors there is a distinct class of ligand gated ion channels that are activated by GABA; referred to as the GABAC receptor. The natural agonist GABA is about an order of magnitude more potent at the GABA receptors than at the most common of the GABA receptors. The GABA receptors are activated by cis-aminocrotonic acid (CACA), which is not recognised by either the GABAA or GABAC receptors, suggesting that they recognise the partially folded conformation of GABA.

GABAC receptors are not blocked by bicuculline and do not recognise the benzodiazepines, barbiturates or the neuroactive steroids but, like GABAA receptors are blocked by picrotoxin, while 1,2,5,6-tetrahydropyridine-4-yl methyl phosphinic acid appears to inhibit GABAC receptors selectively.

However, molecular cloning studies have revealed that this pharmacological profile is remarkably similar to that exhibited by the rho-subunits when expressed ectopically. Two homologous subunits, rho1 and rho2, have been identified in man and these can be expressed as homomers or heteromers, but do not co-assemble with any of the GABA receptor subunits.

Common characteristics

In ionotropic GABAA and GABAС receptors, binding of GABA molecules to their binding sites in the extracellular part of receptor triggers opening of a chloride ion-selective pore.

Opening of a chloride conductance drives the membrane potential towards the reversal potential of the Cl¯ ion which is about –80 mV in neurons, inhibiting the firing of new action potentials.

However, there are numerous reports on GABAA receptors, which are actually excitatory. This phenomenon is due to increased intracellular concentration of Cl¯ ions either during development of the nervous system or in certain cell populations.

After this period of development, a Chloride pump is up-regulated and inserted into the cell membrane, pumping Cl- ions into the extracellular space of the cell. Further openings via GABA binding to the receptor then produce inhibitory responses. Over-excitation of this receptor induces receptor remodeling and the eventual invagination of the GABA receptor. As a result, further GABA binding becomes inhibited and IPSPs are no longer relevant.

G protein coupled receptor: GABAB

A slow response to GABA is mediated by GABAB receptors, originally defined on the basis of pharmacological properties.

In studies focused on the control of neurotransmitter release, it was noted that a GABA receptor was responsible for modulating evoked release in a variety of isolated tissue preparations. This ability of GABA to inhibit neurotransmitter release from these preparations was not blocked by bicuculline, was not mimicked by isoguvacine, and was not dependent on Cl¯, all of which are characteristic of the GABAA receptor. The most striking discovery was the finding that baclofen (β-parachlorophenyl GABA), a clinically employed spasmolytic mimicked, in a stereoselective manner, the effect of GABA.

Later ligand-binding studies provided direct evidence of binding sites for baclofen on central neuronal membranes. cDNA cloning confirmed that the GABAB receptor belongs to the family of G-protein coupled receptors. Additional information on GABAB receptors has been reviewed elsewhere.

Drugs acting on GABA receptors

Below is the table showing the effector pathway, agonists and antagonists of GABA receptors.

GABAA

GABA binds to GABAA receptors in the extended form as demonstrated by the activity of trans 4-aminocrotonoic acid and the lower activity of cis 4-aminocrotonoic acid.

Muscimol is a naturally occurring compound isolated from Amanita muscaria and acts as a GABAA agonist. The isoxazole ring also is found in the GABAA agonist tetrahydroisoxazolopyridinol (THIP).

Bicucullin acts as a direct antagonist at the GABAA receptor as does SR 42641.

GABAB

Baclofen is direct agonist at GABAB receptors, which are coupled to G proteins. GABAB receptors may regulate Ca2+ and K+ influx through the Gi/o family of G proteins and act presynaptically to inhibit the release of excitatory amino acids such as glutamate. Baclofen is orally active as a muscle relaxant and has been used in the treatment of rigidity and spasticity of cerebral palsy. Phaclofen is a weak partial agonist, while saclofen is an antagonist at GABAB receptors.

Anxiolytics: Benzodiazepines

Benzodiazepines represent a class of compounds collectively referred to as anxiolytics. Benzodiazepines modulate the binding of GABA to the GABAA receptor. Benzodiazepines increase the binding of GABA to GABAA receptors and promote Cl- influx.

All the overt effects of the benzodiazepines: sedative, anxiolytic, anticonvulsant, muscle relaxant and amnesic, are produced via the GABAA receptors.

Diazepam (Valium) is one of the most widely prescribed sedatives on the market.

Benzodiazepines exhibit anxiolytic activity due to their ability to promote GABA binding. They act as indirect agonists. Several ligands can block the actions of benzodiazepines, including flumazenil, a benzodiazepine antagonist.

Anticonvulsant drugs

Anticonvulsants are used to treat the various forms of epilepsy, which is characterized by excessive neuronal firing in the cortical and temporal lobe regions of the brain. Electroencephalograms (EEG) can pick up the rhythmic discharge of neurons from electrodes placed on the scalp. Rhythmic discharges of spikes and slow waves characterize the EEG during seizures.

The benzodiazepines have an anticonvulsant action in addition to the anxiolytic activity. Recent work has helped distinguish between the two roles for the benzodiazepines. Picrotoxin is a convulsant which interacts with the GABA receptor complex and blocks the Cl- ionophore.

Diphenylhydantoin (phenytoin) is useful in the treatment of epilepsy. Phenytoin stabilizes the neuron against the excitation associated with seizure activity, without producing sedation.

Beta-adrenoceptors

রাজশাহী বিশ্ববিদ্যালয়ের ফার্মেসী বিভাগের এম. ফার্ম সিলেবাস অনুযায়ী প্রণীতঃ
Syllabus- Molecular and cellular mechanisms of 1) Glutamate receptors, 2) GABA and its receptors, 3) Catecholamine receptors ( alpha- and beta-adrenoceptors, dopamine receptors), 4) Acetylcholine receptors (nicotinic and muscarinic receptors), 5) Opioid receptors.

β-Adrenoceptor Subtypes

The β -adrenoceptors were initially divided into β1 and β2-adrenoceptors defined in terms of agonist potencies, β1-adrenoceptors demonstrated equal affinity for adrenaline and nor- adrenaline while β2-adrenoceptors displayed a higher selectivity for nor-adrenaline than for adrenaline. The discovery of these receptor subtypes led to the development of selective agonists and antagonists for each subtype.

The story does not end there; further experimentation using β-antagonists exposed another receptor subtype which appeared to be insensitive to typical β-adrenoceptor antagonists this was classified as β3-adrenoceptor. More recent pharmacological evidence is now emerging in support of a further receptor subtype β4-adrenoceptor, although as yet there are no selective compounds for this particular subtype.

Characteristics of β-adrenoceptors

β1 adrenoceptors

All β1 adrenoceptors mediate responses to noradrenaline released from the sympathetic nerve terminals and to circulating adrenaline. β1-adrenoceptors were originally defined based on their rank order of potency of these endogenous agonists and by the synthetic agonist isoprenaline. Isoprenaline has shown to be the most potent of the three agonists with adrenaline and noradrenaline showing equal affinity for this particular receptor.

Subsequently, synthetic antagonists have been identified for β1 receptors these include the non-selective propranolol as well as the more selective compounds like metoprolol, practolol, atenolol and betaxolol. The selective β1-adrenoceptor agonists so far identified (denopamine, xamoterol and Ro 363) have limited utility as pharmacological tools because of low selectivity and/or efficacy.

β1-Adrenoceptor Location and Function

β1-adrenoceptors are largely postsynaptic and are located mainly in the heart but are also found in platelets, the salivary glands and the non-sphincter part of the gastrointestinal tract (GIT). They can however be found presynaptically. Activation causes an increase in the rate and contractile force of the heart, relaxation of the non-sphincter part of the GIT, aggregation of platelets and amylase secretion from the salivary glands. Presynaptically, their activation causes an increase in noradrenaline release.

The major ligands used for the autoradiographic localisation of β-adrenoceptor subtypes are [125I] cyanopindolol, [3H] dihydroalprenolol and [125I] pindolol. Of these [125I] cyanopindolol is the most commonly used because of its high affinity, ease of preparation, relatively short exposure time, and selectivity for β-adrenoceptors.

Catecholamines acting on β1 adrenoceptors mediate a number of tissue responses including increases in cardiac rate and force of contraction, stimulation of renin secretion, relaxation of coronary arteries and relaxation of gastrointestinal smooth muscle.

Transduction Mechanisms

All known adrenoceptors are coupled to their effector systems by guanine nucleotide binding proteins (G proteins). β1 adrenoceptors are positively coupled to the membrane bound enzyme adenylate cyclase via activation of Gs G-protein. Activation of adenylate cyclase produces alterations in cellular activity by increasing intracellular levels of cAMP. In the myocardial cell increases in cAMP cause more L-type voltage sensitive Ca2+ channels to open in response to depolarisation. Calcium then enters the cell by this route with each action potential and causes an immediate rise in the concentration of intracellular calcium.

Raised levels of intracellular calcium stimulate further calcium release from the sarcoplasmic reticulum (see diagram above). Activation of the contractile machinery is due partly to this influx of calcium but mainly to the secondary release from the sarcoplasmic reticulum.

Both the heart rate (chronotropic effect) and the force of contraction (inotropic effect) are increased, resulting in a markedly increased cardiac output and cardiac oxygen consumption. The cardiac efficiency is reduced. Catecholamines can also cause disturbance of the cardiac rhythm, cumulating in ventricular fibrillation.

β2 adrenoceptors

β2 adrenoceptors are mainly associated with causing relaxation of smooth muscle in a variety of tissues (see diagram below). The β2 subtype has a high affinity for the endogenous agonist adrenaline. Synthetic β2 agonists include terbutaline, salbutamol, salmeterol and zinterol, all of which have proved therapeutically useful in the treatment of asthma. Selective β2 antagonist have also been identified these include butoxamine and ICI118 551.

Diagram adapted from Pharmacology (4th edition) Rang Dale and Ritter

β2-Adrenoceptor Location and Function

β2-receptors are also mainly postsynaptic and are located on a number of tissues including blood vessels, bronchi, GIT, skeletal muscle, liver and mast cell. Activation results in vasodilatation, bronchodilation, relaxation of the GIT, glycogenolysis in the liver, tremor in skeletal muscle and inhibition of histamine release from mast cells.

The location of β2 adrenoceptors can be identified using the radiolabelled ligand [3H] dihydroalprenolol or [125I] iodopindolol and its analogues.

β2 adrenoceptors have been cloned and expressed in a variety of species. Cloning was first achieved by screening hamster genomic libraries with oligonucleotides complimentary to peptide fragments of purified hamster lung β2 adrenoceptors. This yielded a clone that when expressed had functional and radioligand-binding characteristics consistent with those of the β2 adrenoceptors. Furthermore there appears to be only minor species differences between these clones, with approximately 87 to 93% overall amino acid identity.

Catecholamines acting on β2 adrenoceptors mediate a number of tissue responses. Bronchial smooth muscle is strongly dilated by activation of β2 receptors. Uterine smooth muscle responds in a similar way and β2 agonist are frequently used to delay premature labour. Skeletal muscle is also affected by adrenaline acting on β2 receptors though the effect is far less dramatic than that on the heart. The twitch tension of fast contracting fibres (white muscle) is increased by adrenaline, particularly if the muscle is fatigued, whereas the twitch of slow (red) muscle is reduced.

In man adrenaline and other β2 agonists cause a marked tremor, this is demonstrated in the shakiness that accompanies fear and excitement or the excessive use of β2 agonists (treatment of asthma being an example of this). β2 agonists also cause long term changes in expression of the sarcoplasmic reticulum proteins that control contraction kinetics and thereby increase the rate and force of contraction of skeletal muscle. Clenbuterol is a β2 agonist, which acts in this way and has been used illegally by "sportsmen" to enhance performance.

Histamine release is also inhibited by catecholamines acting apparently at β2 receptors on mast cells and the release of glucose from the liver is also controlled by β2 receptors leading to glycogenolysis.

Transduction Mechanisms

β2 adrenoceptors are also positively coupled to the membrane bound enzyme adenylate cyclase via activation of Gs G-protein. β2 adrenoceptor activation causes relaxation of smooth muscle through the activation of adenylate cyclase. Stimulation of adenylate cyclase produces alterations in cellular activity by increasing levels of cAMP. The resulting increased levels of cAMP activate protein kinase A, which phosphorylates and inactivates myocin light chain kinase (MLCK), the contractile machinery of smooth muscle.

β3 adrenoceptors

The β3 adrenoceptor has a profile quite distinct from that of β1 and β2 adrenergic receptors. Rodents, humans and other mammals share many of the characteristic β3 properties, although observable species-species differences have been identified. The naturally occurring variant of the human β3 receptor was correlated with hereditary obesity in Pima Indians, in Japanese individuals and in Western obese patients. Weight gain was also observed in female mice whose β3 gene had been disrupted.

These examples provided a picture of the important role of the β3 receptor in the regulation of lipid metabolism and as a possible target for drugs to treat certain forms of obesity.

β3-Adrenoceptor Location and Function

β3-adreceptors are expressed predominately in adipose tissue. Activation is proposed to be involved with noradrenaline induced changes in energy metabolism via lipolysis and thermogenesis.

Early antagonist/agonist studies of β adrenergic receptors suggested the existence of atypical responses clearly different from effects mediated by the β1 and β2 adrenoceptors. The new agonist BR37344 stimulated lipolysis in rodent brown adipocytes even in the presence of the antagonist propranolol. This and other discoveries led to the notion that additional receptors existed in fat, in the gut, in the heart and possibly in skeletal muscle. Cloning, sequencing and expression of the β3 gene in various species has now served to confirm the existence of this receptor.

The β3 adrenoceptor is composed of a single 408 amino acid residue peptide chain that belongs to the family of G-protein coupled receptors. The G-protein receptors are characterised by seven hydrophobic stretches of about 22 to 28 residues forming seven transmembrane segments. The transmembrane (TM) regions are linked with three intracellular and three extracellular loops. The amino acid (N) terminal of these receptors is located extracellularly and is glycosylated. The carboxy (C) terminal is intracellular and the case of the β3 receptors does not have any phosphorylation sites.

Transduction Mechanisms

For all β-adrenoceptors transduction is via G-proteins coupled to the intracellular second messenger adenylate cyclase. All β-receptors are positively coupled to adenylate cyclase via activation of Gs G-protein. However, activation of the β2 and β3-adrenoceptors results in stimulation or stimulation and inhibition of adenylate cyclase. Activation of the β1 and β4 receptor results in an increase in the formation of cAMP and the subsequent stimulation of cAMP-dependent protein kinase.

Comparing β3 with β3 receptors of other species revealed a high degree of sequence homology approximately 80-90% between human, bovine, rodent and canine. The human, monkey and bovine β3 receptors are closer to each other than any of the rodents sequences, in particular in transmembrane segment one.

Molecular Biology of β-adrenoceptors

Extensive studies on the molecular features of the β-adrenoceptors have been carried out and the β-adrenoceptor is now among the best defined of the G-protein-linked superfamily of receptors.

The human β-adrenoceptors (β1, β2 and β3) are derived from at least three distinct genes and comprise of 477, 413 and 408 amino acids respectively. All three β-adrenoceptors show high amino acid sequence homology for each receptor between species. For example the β1 receptor of the mouse and rat are 90% homologous to the human β1. Similarly the mouse β2 receptor is 95% homologous to the human β2. However there are marked sequence differences between the subtypes. The β1 shows 48.9% homology with β2 and 50.7% homology with β3, whereas β2 show 45.5% homology with the β3.

The G-protein superfamilies are characterised by their seven hydrophobic transmembrane spanning domains. Three extracellular and three cytoplasmic (intracellular) loops, with a glycosylated extracellular amine (N) terminus and cytoplasmic carboxy (C) terminus link the transmembrane domains. The seven transmembrane domains arrange in the membrane to form a "pocket".

A great deal of the research done to determine the functional domains of the β-adrenoceptors has been carried out on the β2 receptor. Many of these studies have involved the preparation and expression of site directed mutations (deletion or substitution) of particular amino acids and also the synthesis of chimeric receptors.

β1

Three amino acids-Leu110, Thr117 and Val120 in TM2 of the β1-AR were identified as major determinants for β1-AR selective agonists. TM4 is responsible for β1-selective binding of noradrenaline.

β2

Important residues involved in ligand binding also include Asp79, 113, and 130. Asp79 appears to be essential for the binding of high affinity agonist but is not required for the binding of antagonists. Asp113 located in the third transmembrane domain is vital for binding of both agonist and antagonist in the β2 receptor. Substitution of this residue with a Glu results in a marked decrease in the ability of the receptor to couple to adenylate cyclase. Substitution of Asp130 increases the affinity of the receptor for the agonist but reduces the ability of the receptor to couple to the G-protein.

Other important amino acids associated with binding include Ser203, 204 & 207 in TM5; Tyr326 and Phe289, 290 alteration of these residues by substitution resulted in an increased affinity for agonist with no effect on antagonist binding. Ser204 forms hydrogen bonds with the meta hydroxy group of catecholamines; similarly Ser207 interacts with the para hydroxy group group of catecholamines.

Chimeric receptor studies revealed that transmembrane domains six and seven were important in determining the specificity of antagonist binding. Asn293 in TM6 interacts with the β-OH group of β-AR ligands and is responsible for stereoselectivity.

Residues of the third intracellular loop are involved in the coupling of the receptor to the Gs-protein, where the deletion of amino acids 222-229 and 239-272 caused complete loss of coupling to adenylate cyclase.

β3

Computer modelling has defined an image of the β3 ligand-binding site. At least four of the seven transmembrane domains are essential for ligand binding (see diagram below). The amino acids that are involved were identified by site-directed mutagenesis and photoaffinty labelling these are Asp117 in TM3, a residue found to be essential for binding all biogenic amines. Ser169 in TM4, is thought to form a hydrogen bonds with the hydroxyl of the ethanolamine side-chain. Ser209 and Ser212 in TM5, also located in many biogenic amine receptors, are thought to form hydrogen bonds with the hydroxyls of the catechol side chain. Also Phe309 in TM6, involved in hydrophobic interactions with the aromatic ring of catecholamines. Two of the three TM domains are involved in Gs activation, TM2, which contain Asp83, and TM7, which contains Tyr336.

Attempts to explain why several β2 antagonists behave as β3 agonist have not yet been successful. β3-adrenergic receptors appear to contain less bulky residues, and could therefore accommodate the larger β1/β2 antagonists. Mutagenesis has been used to substitute the small Gly residue with the larger Phe residue at position 53 in the β3 receptor; this change however was not enough to convert the β3 agonist to an antagonist.

The Gs interaction site on β3 is situated in the intracellular region, mainly the membrane proximal regions of the second and third (i2, i3) intracellular loops and the carboxy-terminal domains. Several reports propose that the β3 receptor may be coupled to more than one second messenger system. It has been shown that β3 receptors in rat adipocytes interact with both Gs and Gi proteins. The β3 adrenergic receptor in the septum of the human heart has also been reported to link with the Gi protein, resulting in a negative inotropic effect. This is in contrast to previous observations that always associated β3-adrenoceptors with positive inotropism in the heart.

All of the β-adrenoceptors have a glycosylated extracellular site on the N terminus; these sites are not associated with binding or signal transduction however it is thought that they are important for insertion of the receptor into the membrane. β adrenoceptors also contain a number of Cys residues (Cys106, 184, 190 & 191) which are thought to form disulphide bonds between the hydrophilic extracellular loops, which stabilise the receptor conformation.

β4-adrenergic receptor

β4-receptors are localised in cardiac tissue with activation causing an increase in heart rate and force.

Clinical Uses

Adrenergic drugs are used in the treatment of a wide range of medical conditions. Including the use of β2-receptor selective agonists in the treatment of asthma and other related bronchospastic conditions examples of these drugs include salbutamol and salmeterol. Beta-blocker drugs are commonly used in the treatment of angina pectoris, cardiac arrhythmia and for the long-term treatment of patients who survive myocardial infarction. β-receptor antagonists have also been used as anti-hypertensive for a number of years. Beta -blockers have also proven useful in the treatment of conditions such as migraine, anxiety disorders, hyperthyroidism, alcohol withdrawal and when applied topically are useful in the treatment of glaucoma and ocular hypertension.

Alpha1- and alpha2- adrenergic receptor

রাজশাহী বিশ্ববিদ্যালয়ের ফার্মেসী বিভাগের এম. ফার্ম সিলেবাস অনুযায়ী প্রণীতঃ

Syllabus- Molecular and cellular mechanisms of 1) Glutamate receptors, 2) GABA and its receptors, 3) Catecholamine receptors ( alpha- and beta-adrenoceptors, dopamine receptors), 4) Acetylcholine receptors (nicotinic and muscarinic receptors), 5) Opioid receptors.

Alpha- adrenoceptor Subtypes

With the aid of pharmacological and molecular biological techniques the alpha-adrenoceptor subtypes were determined. alpha-adrenoceptors exist on peripheral sympathetic nerve terminals and are divided into two subtypes alpha1, and alpha2. These subtypes were at first classified by their anatomical location; alpha1 is found mostly postsynaptically, whilst alpha2 although typically sited presynaptically, can also occur postsynaptically. These initial subtypes were further divided into alpha1a, alpha1b, and alpha1d; and alpha2a, alpha2b, alpha2c, and alpha2d. This knowledge has led to the development of selective agonists and antagonists for each subtype.

alpha1-adrenergic receptor

alpha1-adrenoceptors are of particular interest therapeutically because of their important role in the control of blood pressure. All alpha-adrenoceptors consist of single polypeptide chains with 7 membrane spanning domains, and are members of the G-protein coupled receptor superfamily.

Classification

There are three subtypes of the alpha1 receptor: alpha1A, alpha1B, and alpha1D. Uppercase letters are used to denote 'functionally defined' subtypes while lowercase is used to denote 'molecularly defined' subtypes. Most tissues express mixtures of the three subtypes, but the relative expression levels have been found to be different in different reports. These subtypes appear to coexist in different densities and ratios, and in most cases responses to alpha1-adrenoceptor selective agonists are probably due to activation of more than one subtype.

Alpha-Adrenoceptor Location and Function:

Alpha1- adrenoceptors are found throughout the body, they are found in the brain where their functional role is not yet clear, they also play critical roles elsewhere in controlling contraction and growth of smooth and cardiac muscle. alpha1-adrenoceptors are found in both the central and peripheral nervous system.

>In the Central Nervous System they are found mostly postsynaptically and have an excitatory function.

>Peripherally they are responsible for contraction and are situated on vascular and on non-vascular smooth muscle. Alpha1-adrenoceptors on vascular smooth muscle are located intrasynaptically and function in response to neurotransmitter release. For non-vascular smooth muscle they can be found on the liver, where they cause hepatic glycogenolysis and potassium release. On the heart they mediate a positive inotropic effect. Cause relaxation of GI smooth muscle and decrease salivary secretion.

Drug affinity and selectivity

The affinities and selectivities of drugs for alpha1- adrenoceptor subtypes have been determined primarily by competition for radioligand binding to heterologously expressed recombinant subtypes. Most antagonists show little or no selectivity between the three known alpha1-adrenoceptor subtypes. However, a variety of drugs, including prazosin which has selectivity for alpha1A, with varying degrees of selectivity have been found. Studies suggest that noradrenaline and adrenaline activate all three alpha1-adrenoceptor subtypes with similar potencies, and that synthetic agonists show significant selectivity between the subtypes.

Functional domains on alpha1

Investigative research into the function of the various domains and/or amino acid residues of the adrenoceptors has produced a number of findings. The aspartate in the third transmembrane domain and the two serines in the fifth transmembrane domain that are conserved in all catecholamine receptors probably interacts with the protonated amine and two hydroxyls of the catecholamines. Using selective alpha1A agonists (e.g. oxymetazoline) Hwa et al (1995) used site directed mutagenesis to identify critical residues in alpha1A- and alpha1B-adrenoceptors that are responsible for apparent differences in agonist binding potency. The results showed that conversion of alanine to valine in the fifth transmembrane domain and leucine to methionine in the sixth transmembrane domain of the alpha1B subtype increased this receptors affinity towards the selective alpha1A agonists until its affinity became similar to that of the alpha1A subtype. These two residues are therefore critical in subtype selective agonist binding, and may interact structurally within the receptor. Other studies suggest that the fifth transmembrane domain and a portion of the second extracellular loop are critically important in subtype selective antagonist binding. This kind of evidence suggests that alpha1-adrenoceptor antagonists may bind near the surface of the receptor, rather than deep within the transmembrane domains like the agonists.

Transduction Mechanisms

All alpha-adrenoceptors use G-proteins as their transduction mechanism. Differences occur in the type of G-protein the receptors are coupled to. alpha1-adrenoceptors are coupled through the Gp/Gq mechanism, whereas alpha2-adrenoceptors are coupled through Gi/Go. Gp/Gq activates phospholipase C that phosphorylates phosphatidyl inositol to produce inositol triphosphate, and diacylglycerol. These compounds act as second messengers and cause release of calcium from intracellular stores in the sarcoplasmic reticulum, and activation of calcium channels respectively. They produce their effects by the release of calcium.

Alpha-adrenoceptors are G-protein coupled receptors. The alpha1 class of adrenoceptors belong to the Gq/11 type of G-protein. An agonist acting at the alpha1-adrenoceptor binding site causes Gq/11 to activate phospholipase C dependent hydrolysis of phosphotidyl inositol 4,5, biphosphate. The conversion of this compound by phospholipase C results in the generation of 1) Inositol triphosphate, and 2) Diacyl glycerol (DAG). 1) Inositol triphosphate acts to release calcium from intracellular stores in the sarcoplasmic reticulum. 2) Diacyl glycerol synergises with calcium to activate protein kinase C which phosphorylates specific target proteins in the cell to change their function.

Alpha1 adrenoceptors have been implicated in other signalling pathways including; calcium influx, arachadonic acid release, and mitogenic activity. alpha1-adrenoceptors may couple directly to activation of calcium channels in certain cells. Activation of alpha1-adrenoceptors leads to potentiation of a calcium current in a protein kinase C dependent manner.

Phospholipase A2 is an enzyme responsible for the release of arachidonic acid from phospholipids. alpha1B and alpha1D adrenoceptors have been shown to couple to phospholipase A2 and cause the activation of this enzyme through a pertussis toxin - sensitive pathway in CHO cells.

Mitogenic activity refers to cell growth and the mechanisms underlying it. G-protein coupled receptors, including alpha1-adrenoceptors have been shown to have mitogenic activity through mitogen activated protein kinase pathways.

 Alpha2-adrenergic receptor


Classification
There are at least 3 different subtypes of the alpha2-adrenoceptor within a species: alpha2A-, alpha2B- and alpha2C-adrenoceptors. Alpha2-adrenoceptors are usually found presynaptically. Presynaptic alpha2 receptors inhibit the release of noradrenaline and thus serve as an important receptor in the negative feedback control of noradrenaline release. Postsynaptic alpha2 receptors are also found.

Alpha2-Adrenoceptor Location and Function
Alpha2-adrenoceptors: are found in both the central and peripheral nervous system. They are found both pre- and postsynaptically and serve to produce inhibitory functions.

-Presynaptic alpha2 receptors inhibit the release of noradrenaline and thus serve as an important receptor in the negative feedback control of noradrenaline release.
-Postsynaptic alpha2 receptors are located on liver cells, platelets, and the smooth muscle of blood vessels. Activation of these receptors causes platelet aggregation, and blood vessel constriction.

Sympathetic nerves are present at the adventitial-medial border of arteries and increase of noradrenaline at these sites causes constriction of the arteries. alpha2-adrenoceptor agonists as well as alpha1-adrenoceptor antagonists are therefore used for the treatment of hypertension. Blockade of presynaptic alpha2-adrenoceptors enhances the overflow of noradrenaline from sympathetic nerves and potentiates the response to sympathetic stimulation. This can be a problem when trying to functionally study innervated alpha2-adrenoceptors because alpha2-adrenoceptor antagonists, by inhibiting pre-junctional alpha2-receptors, also increase neurotransmitter release and thereby mask any contribution made by post-junctional alpha2-adrenoceptors.

Functional domains on alpha2
Alpha2-adrenoceptors are of comparable size to the beta-adrenoceptors but differ in structure from alpha1 and beta- by having relatively short amino and carboxyl termini, and by possessing a very long third intracellular loop. A few amino acid residues appear to be critical for agonist or antagonist binding. For example, if Phe412 of the alpha2A is mutated to asparagine, the affinity for several alpha2-adrenoceptor antagonists is reduced by several orders of magnitude. An aspartic acid in transmembrane helix 3 has been found to be neccessary for specific binding of ligands to alpha2-adrenoceptors. This was shown to by inducing a mutation in which Asp113 was substituted by asparagine, this resulted in the elimination of specific binding of [3H]yohimbine to the alpha2-adrenoceptor. Analysis with photoaffinity probes has shown that partial agonists and antagonist ligands bind to an amino acid within the fourth transmembrane-spanning helix, although the precise location of the attachment could not be determined.

Transduction Mechanisms
All alpha-adrenoceptors use G-proteins as their transduction mechanism. Differences occur in the type of G-protein the receptors are coupled to. alpha1-adrenoceptors are coupled through the Gp/Gq mechanism, whereas alpha2-adrenoceptors are coupled through Gi/Go. The alpha2-adrenoceptor G-protein, Gi/Go, has been shown to be negatively coupled to adenylate cyclase and so reduces the formation of cyclic AMP which leads to a decreased influx of calcium during the action potential - the ion responsible for transmitter release. Therefore lowered levels of calcium will correspondingly lead to a decrease in transmitter release. The alpha2-adrenoceptors belong to the Gi type of G-protein which acts to inhibit adenyl cyclase the enzyme responsible for synthesising the second messenger molecule cAMP from ATP. cAMP acts by activating protein kinases which catalyse the phosphorylation of serine and threonine residues in different cellular proteins, using ATP as the source of the phosphate groups. This mechanism acts to regulate cellular functions. The cellular functions cAMP can regulate include: cell division and cell differentiation, ion transport, ion channel function which leads to changes in electrical excitability, the contractile proteins in smooth muscle, and regulation of enzymes involved in energy metabolism.

The activation of an alpha2-adrenoceptor by agonist causes the alpha2-adrenoceptor to interact with a Gi type of G protein which inhibits the action of adenyl cyclase and thus the actions of cAMP.

Clinical Uses
The clinical uses of adrenergic compounds are vast. The treatment of many medical conditions can be attributed to the action of drugs acting on adrenergic receptors. alpha-adrenoceptor ligands can be used in the treatment of hypertension. Drugs such as indoramin and prazosin are alpha1-adrenoceptor antagonists and have antihypertensive effects, as is clonidine an alpha2 adrenoceptor agonist. alpha1-adrenoceptor antagonists are also employed in the control of benign prostatic hypertrophy. However there can be cardiovascular side effects associated with alpha1 block. Alpha2-adrenoceptor agonists such as clonidine are often used as an adjunct to general anaesthetics.

Nitric Oxide as Vasodilator

রাজশাহী বিশ্ববিদ্যালয়ের ফার্মেসী বিভাগের এম. ফার্ম সিলেবাস অনুযায়ী প্রণীতঃ

Syllabus- Vasodilators: Nitric oxide - Biosynthesis of nitric oxide and its control, Degradation and carriage of nitric oxide, Effects of nitric oxide, Therapeutic use of nitric oxide and nitric oxide donors, Inhibition of nitric oxide, Clinical conditions in which nitric oxide may play a part.

Introduction
Nitric oxide (NO) plays a critical role in various bodily functions, including the vasodilatation of smooth muscle, neurotransmission, regulation of wound healing, and nonspecific immune responses to infection, host defense, and cytotoxicity.
NO is a soluble gas that is produced not only by the endothelial cells, but also by macrophages and specific neurons in the brain. Because the half-life of NO is only a matter of seconds, the gas acts only on cells in close proximity to where it is produced.
NO is synthesized from L-arginine, molecular O2, and nicotinamide adenine dinucleotide phosphate, and other cofactors by the enzyme nitric oxide synthase (NOS). Then, NO induces the guanosine monophosphate (GMP), which initiates a series of intracellular events, leading to response such as vasodilatation.
Discovery of endogenous Nitric OxideIn 1980, Furchgott and Zawadzki discovered that endothelial cells stimulated by acetylcholine released a vasodilator. Initially named endothelium-derived relaxing factor, its real nature was established several years later, and the molecule was identified as nitric oxide. In 1977, Murad’s laboratory has reported that “nitrovasodilators” such as nitroglycerin and nitroprusside caused smooth muscle relaxation via generation of NO that activated soluble guanylyl cyclase and increased the concentration of cyclic guanosine monophosphate (cGMP) in tissues.
Biosynthesis of NO and its controlAfter it was recognized that this diatomic gas performs crucial functions in a wide array of physiological processes, including signal transduction and the immune response, the hunt for its biological source was on, and it was soon discovered that NO was synthesized from the amino acid L-arginine by an enzyme that, predictably, was described nitric-oxide synthase (NOS). This enzyme turned out to be a real gem for structural chemists, enzymologists, and pharmacologists alike. In its active center, NOS contains a heme of the same type as found in cytochrome P450 (P450).
NOS is a modular enzyme that consists of an N-terminal oxygenase and a C-terminal reductase domain. Catalysis takes place at a cysteinyl sulfur-coordinated b-type heme in the oxygenase domain. The heme iron binds O2 as a sixth ligand in the distal pocket, which also serves as the site for substrate binding. In close proximity, (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) is bound as an additional cofactor. The required electrons are provided by the reductase domain, which shuttles electrons from NADPH to the heme via two flavin cofactors, one FAD moiety that accepts electrons two at a time from NADPH, and one FMN moiety that transfers them one at a time to the heme.
The two domains are separated by a short amino acid sequence that must bind calmodulin to enable interdomain electron transfer. NOS is only active as a homodimer, because electron transfer can only occur from the reductase domain of one subunit to the oxygenase domain of the second subunit.
The stability of the dimer is enhanced by a zinc ion that is coordinated to four cysteinyl sulfurs in the dimer interface. A schematic illustration of NOS structure and cofactor content is presented in Fig.

Fig. Schematic presentation of the NOS homodimer including all cofactors and the electron transfer pathway. Note the presence of calmodulin, BH4, and Zn2+ as additional cofactors in NOS.
Mammalian NOS comes in three isoforms. Neuronal and endothelial NOS (nNOS and eNOS) are constitutively expressed, eNOS being also in cardiac myocytes, renal mesangial cells, osteoblasts and osteocytes and small amounts in platelets, and their activity is under strict regulatory control in keeping with their role in signal transduction. Both constitutive enzymes are sensitive to the calcium ion concentration, because calmodulin binds to these isoforms only in the presence of Ca2+.
In contrast to the constitutive isoforms, the inducible isoform (iNOS) is only expressed in response to cytokines in macrophages and Kupffer cells, neutrophils, fibroblasts, vascular smooth muscle and endothelial cells. The affinity of iNOS for calmodulin is so much higher than that of eNOS and nNOS that it binds calmodulin in the virtual absence of free Ca2+. As a result, iNOS lacks the tight control characteristic of the constitutive isoforms and is able to stir up large quantities of NO for an extended period, in line with its function in the immune response.
Synthesis
Endogenous NOEndogenous NO is synthesized by NO synthase (NOS), which catalyzes NO synthesis by combining O2 with L-arginine in two distinct cycles with N-hydroxy-L-arginine (NHA) as an intermediate product that is processed to citrulline and NO without being released from the enzyme. The reaction requires nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, and tetrahydrobiopterin as cofactors. Both cycles consume one molecule of O2 and both require the input of exogenous electrons- two in the first and one in the second cycle- that are furnished by NADPH.

In vivo synthesis of NO occurs in the lungs in the vascular endothelium, epithelial cells, nerve cells, smooth muscle cells, and inflammatory cells such as macrophages. In addition, the cells of the upper airways, especially in the nose and paranasal sinuses, generate large amounts of NO, which is inhaled whenever the patient inspires through the nose.
ControlRate of production of NO is determined by the activity of the enzyme rather than by substrate availability. Nevertheless, very high doses of L-arginine are able to restore endothelial NO biosynthesis in some pathological states (eg. Hypercholesterolemia), in which endothelial function is impaired.
The activity of the constitutive isoforms of NOS is controlled by intracellular Ca-calmodulin. The most important stimuli controlling endothelial NO synthesis in resistance vessels under physiological conditions are mechanical and shear stress, Ca++-ionophore, polycations and receptor mediated vasodilator including acetylcholine, bradykinin etc. Occupation of which increases Ca++ ion concentration, thereby stimulating endothelial NO synthesis.
Several drugs with principal actions on other tissues (eg. Propofol, an intravenous anesthetic agent and nebivolol, a beta-adrenoceptor antagonist) also release NO from endothelium.
The activity of iNOS is independent of [Ca++]. Though iNOS contains a binding site for Ca-calmodulin, the very high affinity of their site for its ligand means that iNOS is activated even at the low values of [Ca++] present under resting condition. The enzyme is induced by bacterial lipopolysaccharide (LPS) and/or cytokines synthesized in response to LPS.
Degradation and carriage of Nitric Oxide
a) NO reacts with oxygen to form N2O4, which combines with water to produce a mixture of nitrite and nitrate anions. Nitrite ions are oxidized to nitrate by oxyhemoglobin.

2NO+O2 leads to N2O4
N2O4+H2O leads to NO3-+NO2-+2H+
NO2-+HbO leads to NO3- +Hb

b) NO reacts with O2- to form peroxynitrite (ONOO-), which can further form its acid form, peroxynitrous acid (ONOOH), a very unstable and reactive oxidizing species. ONOO- has a high affinity for sulphydryl groups and thus inactivates several key sulphydryl bearing enzymes.
Carriage
a) NO diffuses freely across cell membranes, accounting adequately for its local actions on vascular smooth muscle or on monocytes or platelets adhering to the endothelium. The possibility that analogous carrier mechanisms (eg., cysteine and/or –SH containing proteins) operate in mammals and allow NO to act at a distance from its site of biosynthesis. When NO diffuses from endothelium into the blood, it reacts rapidly with haem, which has an affinity for NO>10,000 times greater than for O2. In the absence of oxygen, NO bound to hemoglobin is relatively stable but in the presence of oxygen, NO is immediately converted to nitrate and the haem iron oxidized to methaemoglobin.
Glutathione also reacts with NO under physiologic conditions to generate S-nitroglutathione, a more stable form of NO. Nitroglutathione may serve as an endogenous long-lived adduct or carrier of nitric oxide.
Effects of NO- NO has been involved in many physiological and pathophysiological processes.
Vascular effects: NO has a role in vasodilatation, and, because of the rapid combination of NO with hemoglobin contained in red blood cells, it can be rapidly inactivated, thereby limiting vasodilatation to pulmonary vessels.
Shear stress or receptor activation of vascular endothelium by bradykinin or acetylcholine results in an influx of calcium. The consequent increase in intracellular calcium stimulates the constitutive NOS. The NO formed from L-arginine by this enzyme diffuses to nearby smooth-muscle cells, in which it stimulates the soluble guanylate cyclase (sGC), resulting in enhanced synthesis of cyclic GMP (cGMP) from guanosine triphosphate. This increase in cGMP in the smooth-muscle cells leads to their relaxation.

Pulmonary effects: Researchers have postulated that because glyceryl trinitrate and sodium nitroprusside relax the airway smooth muscle in vitro, NO may be expected to act as a bronchodilator, as has been shown in animal models.
NO has several potentially beneficial effects on pulmonary function by maintaining low pulmonary arterial pressures and sustaining normal vascular permeability.
In addition to its effect on the pulmonary vasculature, NO has some antibacterial actions provided through formation of reactive nitrogen oxides like peroxynitrite. NO also modulates ciliary beat frequency and can inhibit or stimulate mucus secretion.
Inhaled NO (iNO) has been considered for a long time to be a selective pulmonary vasodilator that has no clinically significant effect on blood pressure and cardiac output. Its selective action results from the fixation of iNO to the heme moiety of the hemoglobin molecule after passing through the pulmonary vessel wall. NO is then oxidized to NO2 and NO3. Hemoglobin is transformed to methemoglobin, which is secondarily reduced to hemoglobin by methemoglobin reductase. Although iNO has no systemic hemodynamic effects, it does have extra-pulmonary activity.
Effects of NO on coagulation: NO interferes with platelet and leukocyte functions, fibrinolysis, restenosis, and reperfusion injury by inhibiting expression of adhesion molecules at leukocyte surfaces and by activating sGC, which lead to rapid increase in platelet cGMP and inhibition of platelet aggregation. NO also inhibits vascular smooth muscle cell proliferation, leading to decreased neointimal hyperplasia.
Effects on inflammation: NO plays an important role in vascular function during inflammatory responses. NO is a potent vasodilator. In addition to vascular smooth muscle relaxation, NO plays other important roles in inflammation. It reduces platelet aggregation and adhesion, inhibits several features of mast cell-induced inflammation, and serves as a regulator of leukocyte recruitment. Blocking NO production under normal conditions promotes leukocyte rolling and adhesion in postcapillary venules, and delivery of exogenous NO reduces leukocyte recruitment in acute inflammatory processes. Thus, the overproduction of NO from iNOS is a compensatory mechanism that decreases leukocyte recruitment in inflammatory responses.
Effects on infection: NO also acts in the host’s response to infection. NO antimicrobial activity includes the following: (1) reactive species derived from NO synthase possess antimicrobial activity and (2) interactions occur between NO and reactive oxygen species, leading to formation of multiple antimicrobial metabolites (peroxy nitrate, 5-nitrosothiols, and nitrogen dioxide), each able to damage microbial DNA protein and lipids.
High levels of NO production by a variety of cells appear to limit replication of bacteria, helminthes, protozoa, and viruses, at the risk of potential inflammatory damage to host cells and tissues. Experimental evidence is accumulating that indicates NO has antimicrobial activity against a growing list of organisms. In vitro studies have shown that oxides of nitrogen inhibit the growth of or kill a number of fungi, parasites, helminthes, protozoa, yeasts, mycobacteria, and bacteria. NO may play a role in killing tumor cells and in halting viral replication. Strong evidence exists that NO has a static effect on bacterial growth, and recent in vitro studies using NO donor compounds have demonstrated that NO may even be bactericidal.
Role of NO in Immunity: Researchers have shown that resistance to cancer can be enhanced in a nonspecific way by bacterial products. This nonspecific immunity is associated with the induction of NOS. If this is the case, NO-dependent, nonspecific immunity is a general phenomenon involving the reticuloendothelial system, as well as nonreticuloendothelial cells such as vascular smooth muscle, hepatocytes, and the vascular endothelium, in all of which the inducible NOS has been detected. The liver and the lung in NO-dependent nonspecific immunity play crucial roles because both organs are placed in the circulation to serve as immunologic filters. Furthermore, NO has been hypothesized to play a suppressor role in allograft rejection.
Other effects of NO: Inhaled NO increases urine output without changing systemic hemodynamic parameters in pigs, rats, and humans.

Conclusions
NO is an important mediator in host defense mechanisms as well as in homeostatic processes. Future challenges for researchers are to learn more about the beneficial and harmful effects of NO and infection and how to selectively inhibit excessive NO production or to use NO-releasing drugs to treat infection and to avoid toxic effects against nontarget host cells.

Therapeutic use of NO and NO donors
Though inhalation of high concentration of NO causes acute pulmonary oedema and methaemoglobinemia but at 5-300 ppm, NO inhibits bronchospasm in guinea pigs. But main action of inhaled NO is pulmonary vasodilatation. Two distinctive features make this action potentially therapeutically important.
>first, it is limited to the pulmonary circulation
>second, since NO is administered in inspired air, it acts preferentially on ventilated alveoli.
These properties have raised hopes that inhaled NO may be therapeutically useful in disorders such as adult respiratory distress syndrome.
Intrapulmonary shunting (i.e., pulmonary arterial blood entering the pulmonary vein without passing through capillaries in contact with ventilated alveoli) resulting in arterial hypoxemia. Inhaled NO cause vasodilatation especially in ventilated alveoli and thus reduce shunting.
NO donors: There is considerable interest in the potential for selectivity of these agents, for instance glyceryl trinitrate is more potent on vascular smooth muscle than on platelets whereas s-nitroso glutathione selectively inhibits platelet junctions.
Clinical use of glyceryl trinitrates are-
> used sublingually for rapid antianginal effect
> isosorbide mononitrate used orally for prophylaxis and more sustained effect
> organic nitrates are used to reduce cardiac pre-load in patients with heart failure, especially those unable to take angiotensin-converting enzyme inhibitors.

Inhibition of NO
There are many potential mechanisms by which drugs can inhibit NO synthesis or action.
a) The most common useful strategy remains the use of arginine analogues. Several such compounds-
-N-monomethyl-L-arginine, -N-nitro-L-arginine methyl ester
b) Glucocorticoids inhibit biosynthesis of inducible (but not constitutive) NOS.
c) An endogenous protein inhibitor of nNOS (termed PIN) which works by destabilizing the NOS dimer.
d) Assymetric dimethyl arginine has been detected in human urine, raising the possibility that they influence the L-arginine/NO pathway under pathological conditions.
 e) N-iminoethyl-L-ornithine is a potent and irreversible inhibitor of iNOS in activated macrophages, 7-nitroindazole inhibits mouse cerebral NOS.

Clinical conditions in which NO play a part
The wide distribution of NOS and diverse actions of NO have suggested that abnormalities in this pathway could be involved in the pathophysiology of numerous clinical disorders. Either increased or reduced production could play a part in disease states and hypotheses abound.
Evidence is harder to come by but has been sought using various indirect approach including-
a) Analysis of nitrate or cGMP in urine
b) Measurement of vasoconstrictor effects of NOS inhibitors
c) Comparison of vascular responses to endothelium-dependent agonists (e.g.-Ach) with endothelium-independent agonists that work through the same effector mechanism.
d) Measurement of the dilator response to increased blood flow in the brachial artery, which is partly NO-mediated.
e) Study of histochemical appearances and pharmacological responses of tissue in vitro.

Some postulated phalogical roles of excessive or reduced NO production and summarized below- iNOS
a) NO is of benefit in host defence early in the sequence of sepsis by contributing to microbial killing, subsequent excessive NO production can cause harmful hypotension. Chronic low-grade endotoxaemia occurs in patients with cirrhosis of the liver, many of whom are systemically vasodilated. Urinary excretion of cGMP is increased in such patients and the vasodilatation may be caused by induction of NOS leading to increased vascular NO synthesis.
Constitutive NOS isoforms-
eNOS
a) There is suggestive evidence of reduced NO biosynthesis in patients with hypercholesterolaemia and some other disorders that predispose to atheromatous vascular disease including cigarette smoking and diabetes mellitus.
b) An inhibitor of NOS markedly potentiates atherogenesis without increasing blood pressure or influencing plasma lipid concentrations.
nNOS
a) Excessive NMDA receptor activation contributes to several forms of neurological damage. nNOS is absent in pyloric tissue from babies with idiopathic hypertrophic pyloric stenosis.