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.
