NORADRENERGIC NEUROEFFECTOR TRANSMISSION AS A TARGET OF DRUG ACTION. 1/3/2019

OVERVIEW The peripheral noradrenergic neuron and the structures that it innervates are important targets for drug action, both as objects for investigation in their own right and as points of attack for many clinically useful drugs. 1/3/2019

Learning Objectives At the end of the session students must be able to understand and describe: the physiology and function of noradrenergic neurons The properties of adrenoceptors (receptors on which noradrenaline and adrenaline act) and various classes of drugs that affect them Synthesis of noradrenaline Drugs that affect noradrenergic transmission 1/3/2019

CATECHOLAMINES Are compounds containing a catechol moiety (a benzene ring with two adjacent hydroxyl groups) and an amine side chain. Pharmacologically, the most important ones are:- Noradrenaline (norepinephrine), a transmitter released by sympathetic nerve terminals Adrenaline (epinephrine), a hormone secreted by the adrenal medulla 1/3/2019

CATECHOLAMINES… Dopamine, the metabolic precursor of noradrenaline and adrenaline, also a transmitter/neuromodulator in the central nervous system. Isoprenaline (also known as isoproterenol), a synthetic derivative of noradrenaline, not present in the body 1/3/2019

Classification of adrenoceptors Main pharmacological classification into - and - subtypes, based originally on order of potency among agonists, later on selective antagonists. There are two main - adrenoceptor subtypes which are : -1- and 2- (each divided into three further subtypes) 1/3/2019

Classification of Adrenoceptors (cont.) There are three - adrenoceptor subtypes: -1, 2, 3 All adrenoceptors belong to the superfamily of G-protein-coupled receptors. Each of these receptor classes is associated with a specific second messenger system: 1/3/2019

Classification of Adrenoceptors (cont.) 1- adrenoceptors activate phospholipase C, thus producing inositol triphosphate and diacylglycerol as second messengers 2 –adrenoceptors inhibit adenylate cyclase and thus decrease CAMP ( cyclic 3’5’-adenosine monophosphate) formation All types of -adrenoceptors stimulate adenylate cyclase 1/3/2019

Classification of adrenoceptors (cont) The main effects of receptor activation are: -1-adrenoceptors: vasoconstriction, relaxation of g.i.t smooth muscle, salivary secretion and hepatic glycogenolysis 2-adrenoceptors: inhibition of transmitter release (including noradrenaline and acetylcholine release from autonomic nerves), platelet aggregation, contraction of vascular smooth muscle, inhibition of insulin release 1/3/2019

Classification of adrenoceptors (cont.) 1- adrenoceptors: increased cardiac rate and force 2-adrenoceptors: bronchodilatation, vasodilatation, relaxation of visceral smooth muscle, hepatic glycogenolysis and muscle tremor 3-adrenoceptors: lipolysis 1/3/2019

Physiology of Noradrenergic transmission Noradrenergic neurons in the periphery are postganglionic sympathetic neurons; their cell bodies lie in sympathetic ganglia. Have long axons that end in a series of varicosities strung along the branching terminal network. These varicosities contain numerous synaptic vesicles, which are the sites of synthesis and release of NA and of co released mediators such as ATP (adenosine triphosphate) and neuropeptide Y. 1/3/2019

Physiology of Noradrenergic transmission (cont) NA is highly concentrated in these varicosities and released by exocytosis In most peripheral tissues, and also in the brain, the tissue content of NA closely parallels the density of sympathetic innervation. With the exception of adrenal medulla, sympathetic nerve terminals account for all NA content of peripheral tissues Heart, spleen, vas deferens and some blood vessels are rich in NA (5-50nmol/g of tissue) 1/3/2019

SYNTHESIS OF NORADRENALINE The biosynthesis of noradrenaline may be regarded as the first stage in the process of noradrenergic transmission. The starting material is dietary L-phenylalanine. This amino acid is actively absorbed from the gut and oxidized in the liver by phenylalanine hydroxylase to form L-tyrosine, which circulates in the bloodstream. 1/3/2019

The membrane Na+/K+ATPase then actively pumps the Na+ outwards. Like other neutral amino acids entering mammalian cells, it is co-transported across the cytoplasmic membrane of noradrenergic neurons with Na+. The membrane Na+/K+ATPase then actively pumps the Na+ outwards. Within the cytoplasm of the noradrenergic neurone, L-tyrosine is hydroxylated to form L-dihydroxyphenylalanine (L-dopa, levodopa). 1/3/2019

This is an example of feedback ( product ) inhibition. This reaction is catalysed by tyrosine hydroxylase and is the rate –limiting step in the biosynthesis of noradrenaline. The activity of tyrosine hydroxylase is governed by the cytoplasmic concentration of NA, a large NA concentration inhibiting enzyme activity. This is an example of feedback ( product ) inhibition. 1/3/2019

Tyrosine hydroxylase exhibits some substrate specificity but is susceptible to inhibition by metirosine (-methyltyrosine ). This inhibits tyrosine hydroxylase and reduces catecholamine synthesis. It is useful in limiting the catecholamine output of rare tumor of the adrenal medullary chromaffin cells ( phaeochromocytoma ), both preoperatively or as a long term therapy in inoperable cases. 1/3/2019

Aromatic l-amino acid decarboxylase is a cytoplasmic enzyme of low substrate specificity that converts l-dopa to dopamine. Levodopa is useful to replace a functional deficiency of dopamine in the treatment of Parkinson’s disease. Aromatic l-amino acid decarboxylase can be inhibited by benserazide and by carbidopa. 1/3/2019

These agents are hydrophilic analoques of l-dopa and they do not enter the brain. They can therefore produce an inhibition selectively restricted to peripherally located enzyme. This property makes the utilization of levodopa more efficient in central than peripheral dopaminergic and noradrenergic neurons 1/3/2019

Hence, unwanted peripheral effect of cardiac tachy-dysrhythmias, due to excessive catecholamine production, and the dose of levodopa needed for the treatment of Parkinson’s disease are reduced. 1/3/2019

Dopamine, synthesized within the neuronal cytoplasm, is actively transported into the transmitter storage vesicles of the axon terminals. There, it is oxidized by dopamine -hydroxylase ( an enzyme of low substrate specificity ) to form NA. 1/3/2019

STORAGE OF NORADRENALINE IN VESICLES Endogenous noradrenaline is stored in membrane- limited vesicles, which are formed in the neuronal cell body and transported to the varicosities of the axon terminal by axoplasmic flow. Within the vesicles, noradrenaline is weakly complexed with ATP and a soluble protein called CHROMOGRANIN. 1/3/2019

The retention of NA inside vesicles results from its affinity for the vesicular contents and from the continued operation of the amine uptake process in the vesicle membrane ( a process requiring energy from the breakdown of ATP by Mg2+- dependent ATPase) 1/3/2019

DRUGS THAT INTERFERE WITH THE VESICULAR RETENTION OF NORADRENALINE Reserpine and Tetrabenazine inhibit the amine uptake process in the vesicle membrane and thereby allow the leakage of NA into cytoplasm where it is largely metabolized by neuronal monoamine oxidase ( MAO ). Further more , since vesicular dopamine uptake is inhibited , NA synthesis is impaired. 1/3/2019

For these 2 reasons the storage vesicles become depleted of NA (chromaffin cells of the adrenal medulla and noradrenergic, dopaminergic and 5-Hydroxytryptaminergic (5-HT) neurons within the CNS are also susceptible to this action of Reserpine). NA depletion induced by Reserpine or Tetrabenazine is accelerated by action potential activity in the neurone. 1/3/2019

Noradrenergic neuroeffector transmission fails when the noradrenaline content is reduced to approximately 25% of normal. When large doses of Reserpine are used, recovery of neurone function depends upon the synthesis of new vesicles and their transport to axon terminals ( approx.10 days). 1/3/2019

Pretreatment with Reserpine: 1) Abolishes the effects of sympathetic noradrenergic neurone activity. 2) Abolishes the effects of agents that cause the release of NA from axon terminals- the indirectly acting sympathomimetic agents. 1/3/2019

3) Does not reduce responses of effector cells to exogenous noradrenaline or to other agonists acting on adrenoceptors. 4) Has similar effects on noradrenergic, dopaminergic and 5-HT neurotransmission in the CNS. 1/3/2019

Reserpine was formerly useful in the treatment of severe hypertension. Part of the hypotensive action of Reserpine results from the impairment of mono-aminergic transmission in BP control centers of the CNS. It is now rarely used because it can induce severe ( suicidal ) depression. 1/3/2019

Tetrabenazine is of value in Huntington’s chorea and related disorders of movement, presumably because its action depletes the trasmitter stores of central dopaminergic neurons The usefulness of Tetrabenazine, like Reserpine, is limited by development of severe depression. NB: Huntington’s chorea ( a hereditary disease where there is a progressive impairment of motor coordination with grimacing, distorted speech and bizarre movements of the limbs conveying a dance-like gait (hence chorea). 1/3/2019

Drugs That Compete With NA for Vesicular Storage Certain drugs on gaining access to the neuronal cytoplasm, can compete with dopamine or NA for uptake into the vesicles. They may then stoichiometrically displace NA from its storage site. Drugs in this category include -methyldopamine formed from methyldopa and certain indirectly sympathomimetic agents (Amphetamine, Tyramine). 1/3/2019

As a consequence of NA displacement: 1) Less NA is available for release during neuroeffector transmission. 2) The displacing drug may be released in place of NA during neuroeffector transmission. 3) The response of the effector cell to the displacing drug may result from the pharmacological effects of displaced NA. 1/3/2019

Methyldopa is a substrate for aromatic l-amino acid decarboxylase and hence can be converted to -methyldopamine. -methyldopamine is not the substrate for neuronal MAO (because it carries -methyl substituent, so it compete very successfully with dopamine for transport into the storage vesicles. Vesicular dopamine  hydroxylase then oxidizes -methyldopamine to yield -methylnoradrenaline. 1/3/2019

This substance function as a false transmitter since it can be stored in the vesicles and is subsequently released into the junctional cleft on arrival of the nerve action potential. -Methylnoradrenaline is approximately equipotent with NA in evoking a response from effector cells in the periphery by agonist action at postjunctional 1-adrenoceptors but is more potent than NA at prejunctional 2 adrenoceptors. 1/3/2019

Pretreatment with methyldopa It reduces neuroeffector transmission by reducing the amount of neuro-transmitter released. Pretreatment with methyldopa 1)Reduces the effects of noradrenergic neurone activity, and of dopaminergic neurone activity in the CNS. 2)Reduces the effects of agents that cause the release of NA from the axon terminals – the indirectly acting sympathomimetic agents. 1/3/2019

3)Does not reduce the responses of effector cells to exogenous NA or other agonist acting on adrenoceptors. Methyldopa is useful in the treatment of moderate to severe hypertension in whom antagonists at -adrenoceptors are contraindicated and in pregnancy. 1/3/2019

Its main site of action in reducing cardiac output and peripheral resistance seems to be the central noradrenergic neurons involved in the control of BP. Unwanted effects include drowsiness, depression and retention of salt and water by the kidney. 1/3/2019

NORADRENERGIC NEURONE BLOCKING AGENTS The noradrenergic neurone blocking agents (e.g. guanethidine ) selectively impairs transmission at noradrenergic neuroeffector junctions. These agents show weak local anaesthetic activity on non- noradrenergic neurons but are selectively accumulated by noradrenergic neurons by the same mechanism that transport NA into cell- the neuronal uptake pump. 1/3/2019

Thus the noradrenergic neurone blocking agents are accumulated within noradrenergic neurons to a concentration sufficiently large to exert a local anaesthetic effect. NA release is abolished mainly by the prevention of nerve action potential conduction in terminal neuronal branches. 1/3/2019

In addition, these drugs cause depletion of stored NA and may interfere with exocytosis. Large doses of Guanethidine may cause structural damage in noradrenergic nerve terminals, so that the axon partly and temporarily dies back towards the cell body. 1/3/2019

Noradrenergic neurone blocking agents (cont.) The noradrenergic neurone blocking agents: Prevent The effects of noradrenergic neurone activity Prevent the effects of agents that cause the release of NA from axon terminals-the indirectly acting sympathomimetic agents 1/3/2019

Do not reduce the effects of exogenous NA or other agonists at adrenoceptors. Indeed, such an agent is potentiated if it is a substrate for the neuronal noradrenaline uptake process 1/3/2019

The selectivity of noradrenergic neurone blocking agents and their ability to modify the actions of indirectly acting sympathomimetic agents and some agonists acting on adrenoceptors all depend on their being substrate for neuronal NA uptake process. The actions of these agents are impaired by other drugs that either compete for uptake into neurone ( tyramine ) or block the uptake process ( cocaine, imipramine ). 1/3/2019

Guanethidine eye drops (formulated with adrenaline) can reduce intaocular pressure in chronic, open-angle glaucoma. The noradrenergic neurone blocking agents were sometimes useful in the treatment of severe hypertension that was resistant to other drugs. The wide range of adverse effects included postural and exercise hypotension , retention of salt and water by kidney, diarrhoea and failure of ejaculation. 1/3/2019

Noradrenaline Release from Axon Terminals The release of NA from noradrenergic axons is comparable with the release of acetylcholine from acetylcholinergic axons. In the absence of action potential traffic in noradrenergic nerves, the random migration of storage vesicles to the cell surface occasionally results in exocytosis 1/3/2019

Noradrenaline Release Although the amount of NA released is small, it can still influence the membrane of the postjunctional cell if the cleft is narrow. E.g. spontaneous postjunctional potentials are seen in some noradrenergically innervated smooth muscles 1/3/2019

Noradrenaline Release Reserpine, by depleting the vesicles of stored NA prevents both the spontaneous and action potential-evoked release of NA When an action potential invades the varicosities, membrane permeability changes occur. Na+, Cl- and Ca++ enter the axon terminals and K+ emerges. N-type voltage sensitive Ca++ channels are activated 1/3/2019

Noradrenaline Release The influx of Ca++ triggers many storage vesicles to release NA, ATP, chromogranin and dopamine  -hydroxylase into the extracellular space (exocytosis) and this NA diffuses down its concentration gradient to stimulate adrenoceptors on the effector cell surface 1/3/2019

Noradrenaline Release The empty vesicles are probably retained within the cell and subsequently refilled with transmitter. Since transmitter release by this mechanism requires the nerve action potential, it is prevented by noradrenergic neurone blocking agents. 1/3/2019

Noradrenaline Release Since transmitter release by this mechanism requires the influx of Ca++, release is reduced if the extracellular environment is deficient in this ion or contains a large concentration of Mg++ After treatment of tissues with methyldopa, neuronal action potentials release less NA but this is accompanied by the release of -methylnoradrenaline (false transmission) from the terminals of noradrenergic axons 1/3/2019

INDIRECTLY ACTING SYMPATHOMIMETIC AGENTS The effects of activation of NA neurons are called ( rather imprecisely ) sympathomimetic effects. Hence an agonist at adrenoceptors could be called a directly acting sympathomimetic agent. An indirectly acting sympathomimetic agent does not itself activate adrenoceptors. 1/3/2019

It evokes sympathomimetic effects either (nicotine, tyramine ) by promoting the release of neuronal NA or ( cocaine ) by preventing the inactivation of NA. 1/3/2019

AGONIST AT THE NICOTINIC Ach RECEPTORS Acetylcholine and Nicotine cause action potentials to be generated in noradrenergic neurons and so evoke release of neural NA , thereby indirectly inducing sympathomimetic effects. 1/3/2019

TYRAMINE-LIKE INDIRECTLY ACTING SYMPATHOMIMETIC AGENTS Certain chemical modification of the NA molecule ( loss of catechol-OH; loss of the -OH group; methylation of the -C atom) yield agents that are sympathomimetic despite being unable themselves to activate adrenoceptors. Examples include tyramine and amphetamine. 1/3/2019

These drugs do act as substrate for NA uptake process in the neuronal membrane and gain access to the neuronal cytoplasm by that route. In addition, amphetamine is sufficiently lipid soluble to enter the neuronal by diffusion across the neuronal membrane. From the cytoplasm they are then transported into the transmitter storage vesicles where they stoichiometrically (molecule by molecule) displace NA 1/3/2019

Tyramine is a substrate and amphetamine an inhibitor of MAO. A large proportion of the displaced NA escapes from the neurone and subsequently activate post-junctional adrenoceptors because the MAO associated with mitochondria, which would normally break down excess cytoplasmic NA, is occupied by a competing molecule. Tyramine is a substrate and amphetamine an inhibitor of MAO. 1/3/2019

NA released evoked by tyramine-like agents does not require the discharge of neuronal action potentials. The properties of tyramine like group of indirectly acting sympathomimetic agents are compared with those of NA and adrenaline and the physiological agonists acting on adrenoceptors. 1/3/2019

INHIBITORS OF THE NEURONAL UPTAKE OF NA. Cocaine and Imipramine inhibit the neuronal uptake of NA. Endogenous NA ( spontaneously released or released by neuronal action potentials ), therefore accumulates in the junctional cleft and activates adrenoceptors. 1/3/2019

Cocaine and Imipramine are sufficiently lipid soluble to be able to penetrate into the CNS. Their central effects are, in part, consequences of inhibition of neuronal uptake of NA there 1/3/2019

SUMMARY Most postganglionic sympathetic neurones are noradrenergic, as are some CNS neurones. The synthesis of dopamine, noradrenaline and adrenaline can be modified by drugs acting as substrates (e.g. levodopa, methyldopa) or inhibitors of the synthetic enzymes. 1/3/2019

SUMMARY A non-physiological substrate leads to the synthesis, storage and release of a false transmitter. Drugs are available that disrupt the vesicular storage of NA- their effects are widespread Drugs are available that prevent the propagation of action potentials through noradrenergic nerve terminals-their effects are widespread 1/3/2019

SUMMARY A Drug that activates adrenoceptors indirectly does so, not by combining with them itself but by increasing the local concentration of the physiological agonists, either by causing their release or reducing their inactivation 1/3/2019

SUMMARY Tyramine-like indirectly acting sympathomimetic agents displace NA from the neuronal storage granules so that it leaves the nerve endings and produces noradrenergic effects 1/3/2019

TO CONTINUE…! 1/3/2019