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.

Beta-Adrenoceptor antagonists

রাজশাহী বিশ্ববিদ্যালয়ের ফার্মেসী বিভাগের বি. ফার্ম (সম্মান) তৃতীয় বর্ষের সিলেবাস অনুযায়ী প্রণিতঃ

Syllabus
Drug Acting on ANS:
a) (i) Parasympathomimetic agents: Acetyl choline, Methacoline, Carbachol. (ii) Sympathomimetic drugs: Epinephrine, norepinephrine. (iii) Anticholinesterase agents: Physostigmine, Edrophonium. Organophosphorous compounds.
b) (i) Antimuscarinic Agents or Atropine Drugs: atropine sulfate, scopolamine hydrobromide, homatropine hydrobromide. (ii) Drugs inhibiting adrenergic nerves and structures innervated by them, Adrenergic blocking agents.
c) Ganglion Stimulating and Blocking Agents.

β-Adrenoceptor antagonists
Drugs in this category share the common feature of antagonizing the effects of catecholamines at β adrenoceptors. Beta-blocking drugs occupy β receptors and competitively reduce receptor occupancy by catecholamines and other β agonists. Most β-blocking drugs in clinical use are pure antagonists; ie, the occupancy of a β receptor by such a drug causes no activation of the receptor. However, some are partial agonists; ie, they cause partial activation of the receptor, albeit less than that caused by the full agonists epinephrine and isoproterenol. Partial agonists inhibit the activation of β receptors in the presence of high catecholamine concentrations but moderately activate the receptors in the absence of endogenous agonists. Another major difference among the many β-receptor-blocking drugs concerns their relative affinities for β1 and β2 receptors. Some of these antagonists have a higher affinity for β1 than for β2 receptors, and this selectivity may have important clinical implications. Since none of the clinically available β receptor antagonists are absolutely specific for β1 receptors, the selectivity is dose-related, ie, it tends to diminish at higher drug concentrations.
Other major differences among β antagonists relate to their pharmacokinetic characteristics and local anesthetic membrane-stabilizing effects.
All the clinically available β-blockers are competitive antagonists. Nonselective β-blockers act at both β1 and β2 receptors, whereas cardioselective β-antagonists primarily block β1 receptors. These drugs also differ in intrinsic sympathomimetic activity, in central nervous system (CNS) effects, and in pharmacokinetics. Although all β-blockers lower blood pressure in hypertension, they do not induce postural hypotension because the α-adrenoceptors remain functional; therefore, normal sympathetic control of the vasculature is maintained. β-blockers are also effective in treating angina, cardiac arrhythmias, myocardial infarction, and glaucoma, as well as serving in the prophylaxis of migraine headaches.

SAR
The O-CH2 group between aromatic ring and the ethylamino side chain is responsible for the antagonistic property.
Replacement of catechol hydroxyl group with chlorine or phenyl ring retains the beta blocking activity.
N,N- di substitution decrease beta blocking activity. Activity is maintained when phenylethyl, hydroxyl phenyl ethyl or methoxy phenyl ethyl groups are added to amine as a part of molecule.
The two carbon side chain is essential for the activity.
Nitrogen atom should be of secondary amine for optimum beta blocking activity.
The carbon side chain having hydroxyl group must be S- configuration for optimum affinity to beta receptor.(Ex- Levobunolol, Timolol)
The aryloxy propanolamines are more potent than aryl ethanolamines.
Replacement of ethereal oxygen in aryloxy propanolamines with S, CH2 or N-CH3 is decreased the beta blocking activity.
The most effective substituents at amino group is isopropyl and tertiary butyl group.
The aromatic portion of the molecules could be varied with good activity.
Converting the aromatic portion to phenanthrene or anthracene decrease the activity.
Cyclic alkyl substituents are better than corresponding open chain substituents at nitrogen atom of amine.
Alpha methyl group at side chain decrease activity.

PROPRANOLOL: A NONSELECTIVE β-ANTAGONIST
Propranolol is the prototype β-adrenergic antagonist and blocks both β1 and β2 receptors. Sustained release preparations for once-a-day dosing are available.
Propranolol was the first β-blocker shown to be effective in hypertension and ischemic heart disease. It is now clear that all β-adrenoceptor-blocking agents are very useful for lowering blood pressure in mild to moderate hypertension. In severe hypertension, β-blockers are especially useful in preventing the reflex tachycardia that often results from treatment with direct vasodilators. Beta blockers have been shown to reduce mortality in patients with heart failure, and they are particularly advantageous for treating hypertension in that population.

Mechanism & Sites of Action
Propranolol's efficacy in treating hypertension as well as most of its toxic effects result from nonselective β-blockade. Propranolol decreases blood pressure primarily as a result of a decrease in cardiac output. Other β-blockers may decrease cardiac output or decrease peripheral vascular resistance to various degrees, depending on cardioselectivity and partial agonist activities.
Beta-blockade in brain, kidney, and peripheral adrenergic neurons has been proposed as contributing to the antihypertensive effect observed with β-receptor blockers. In spite of conflicting evidence, the brain appears unlikely to be the primary site of the hypotensive action of these drugs, because some β-blockers that do not readily cross the blood-brain barrier (eg, nadolol) are nonetheless effective antihypertensive agents.
Propranolol inhibits the stimulation of renin production by catecholamines (mediated by β1-receptors). It is likely that propranolol's effect is due in part to depression of the renin-angiotensinaldosterone system. Although most effective in patients with high plasma renin activity, propranolol also reduces blood pressure in hypertensive patients with normal or even low renin activity. Beta blockers might also act on peripheral presynaptic β-adrenoceptors to reduce sympathetic vasoconstrictor nerve activity. In mild to moderate hypertension, propranolol produces a significant reduction in blood pressure without prominent postural hypotension.

Pharmacokinetics
Most of the drugs in this class are well absorbed after oral administration; peak concentrations occur 1–3 hours after ingestion. Sustained-release preparations of propranolol and metoprolol are available.
Propranolol undergoes extensive hepatic (first-pass) metabolism; its bioavailability is relatively low 30 (dose dependent), elimination half-life 3.5–6 hours. The elimination of drugs such as propranolol may be prolonged in the presence of liver disease, diminished hepatic blood flow, or hepatic enzyme inhibition.
The β-antagonists are rapidly distributed and have large volumes of distribution. Propranolol is quite lipophilic and readily cross the blood-brain barrier. Most antagonists have half-lives in the range of 3–10 hours. Propranolol and metoprolol are extensively metabolized in the liver, with little unchanged drug appearing in the urine.

Dosage
Resting bradycardia and a reduction in the heart rate during exercise are indicators of propranolol's β-blocking effect. Measures of these responses may be used as guides in regulating dosage. Propranolol can be administered once or twice daily.

Toxicity
The principal toxicities of propranolol result from blockade of cardiac, vascular, or bronchial β-receptors. The most important of these predictable extensions of the β-blocking action occur in patients with bradycardia or cardiac conduction disease, asthma, peripheral vascular insufficiency, and diabetes. When propranolol is discontinued after prolonged regular use, some patients experience a withdrawal syndrome, manifested by nervousness, tachycardia, increased intensity of angina, or increase of blood pressure. Myocardial infarction has been reported in a few patients. Although the incidence of these complications is probably low, propranolol should not be discontinued abruptly. The withdrawal syndrome may involve up-regulation or supersensitivity of β-adrenoceptors.

Pharmacological Actions
Cardiovascular: Propranolol diminishes cardiac output, having both negative inotropic and chronotropic effects. It directly depresses sino-auricular and atrioventricular activity. The resulting bradycardia usually limits the dose of the drug. Cardiac output, work, and oxygen consumption are decreased by blockade of β1 receptors; these effects are useful in the treatment of angina. The β-blockers are effective in attenuating supraventricular cardiac arrhythmias but are generally not effective against ventricular arrhythmias (except those induced by exercise).
Peripheral vasoconstriction: Blockade of β receptors prevents β2-mediated vasodilation. The reduction in cardiac output leads to decreased blood pressure. This hypotension triggers a reflex peripheral vasoconstriction, which is reflected in reduced blood flow to the periphery. On balance, there is a gradual reduction of both systolic and diastolic blood pressures in hypertensive patients. No postural hypotension occurs, since the β-adrenergic receptors that control vascular resistance are unaffected.
Bronchoconstriction: Blocking β1 and β2 receptors in the lungs of susceptible patients causes contraction of the bronchiolar smooth muscle. This can precipitate a respiratory crisis in patients with chronic obstructive pulmonary disease or asthma. β-Blockers are thus contraindicted in patients with asthma.
Increased Na+ retention: Reduced blood pressure causes a decrease in renal perfusion, resulting in an increase in Na+ retention and plasma volume. In some cases this compensatory response tends to elevate the blood pressure. For these patients, β-blockers are often combined with a diuretic to prevent Na+ retention.
Disturbances in glucose metabolism: β blockade leads to decreased glycogenolysis and decreased glucagon secretion. Therefore, if an insulin-dependent diabetic is to be given propranolol, very careful monitoring of blood glucose is essential, since pronounced hypoglycemia may occur after insulin injection. β -Blockers also attenuate the normal physiologic response to hypoglycemia.