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.