Screening and bioassay of Sympatholytics by Dr. Magdy M. Awny Lecture 4

Antagonist of adrenergic activity Sympatholytics = Antagonist of adrenergic activity Drugs that interfere with the activity of the sympathetic N.S They are classified into: 1-Adrenoreceptors blockers: α and β blockers…..Examples? 2-Adrenergic neuron blockers: They block the nerve ending by different mechanisms: - ↓NE synthesis e.g. α - methyldopa - ↓NE storage e.g. reserpine - ↓ NE release e.g. guanethidine 3-Ganglionic blockers: - Competitive e.g. hexamethonium - Depolarizing e.g. large doses of nicotine, TMA

A- Screening of svmpatholvtics 1. Cat nictitating membrane I- Anesthetized cat II- Un-anesthetized cat In conscious cat, the membrane is contracted to the eye corner; adrenergic antagonists relax the membrane causing it to cover part or the entire eyeball. Relaxation of the membrane with changes in eye pupil diameter can be Used to differentiate between Sympatholytics. a- α1 blockers or ANBs….Relaxation of the membrane, miosis (without mydriasis) b- Ganglionic blockers… Relaxation of the membrane, passive mydriasis and loss of light reflex. C- Reserpine….Relax membrane but slowly after repeated administration (depletion of NE store) NB:- Muscarinic blockers (atropine)……..passive mydriasis, loss of light reflex without relaxation of the membrane.

II- Anaesthetized cat nictitating membrane Cervical symp. nerve Nictitating membrane the DRUG may be Pre post R ES NE triad control contraction of the membrane Pre post NE Pre post R ES NE Ganglionic blocker Only affect pre ES + Adrenergic neuron blocker ↓pre, post ES & potentiate Exog NE Pre post R ES NE Pre post R ES NE Alph-1 receptor blocker ↓pre, post ES & Exog NE

2- Cardiovascular test α blocker adrenaline reversal Inhibit (abolish) response to NE & C O E α≠   Β blockers block the hypotensive or cardio stimulant action of isoprenaline ANB Triphasic response in BP ↓NE reflex mech ↓not abolish vasopressor effect of CO ↑NE By inhibition of uptake ↑vasopressor effect of injected E,NE due to receptor supersensitivity GB Inhibit response to carotid occlusion & Vagal stimulation Prolonged fall in BP Reserpine pretreatment Inhibit the hypertensive effect of tyramine due to depletion of NE stores as a result of inhibition of reuptake so degraded by MAO Potentiation of response to E, NE when taken after reserpine? due to supersensitivity of receptor resulting from chemical denervation produced by reserpine

3- Finkleman's preparation A nerve smooth muscle preparation formed of piece of intestine with attached mesentery & sympathetic nerve Muscle is suspended in tyrode solution at 37 ͦc & adrenergic nerve is electrically stimulated Up on ES & injection of exogenous E, the response will be →relaxation

Uses:- to differentiate between. ANBs & adrenergic receptor blocker (α &β≠) If the drug inhibit the effect of ES only → ANB If the drug inhibit the effect of ES & exog. E →receptor blocker( α &β≠). To differentiate between α &β ≠ use the selective agonist →NE or Isoprenaline If Unkn inhibit relaxant effect of the NE → α blocker If Unkn inhibit the relaxant effect of the Isoprenaline →β blockers

control ANBs Adrenergic receptor blocker NE α β ES Exog. E ES Exog. E

Special tests for α blockers I-Cat nictitating membrane B- Un anesthetized cat: relaxation of membrane, narrowing of palpebral opening & no mydriasis A- Anesthetized cat: inhibition of the response to pre, post ganglionic ES & exog. NE II-Cardiovascular test: α blockers Inhibit response to NE, C.O Reverse Vasopressor effect of epinephrine [adrenaline reversal?] III- Isolated organ preparations: α-blockers inhibit the E or NE induced contraction of isolated organ preparations containing α receptors (e.g., vas deferens, seminal vesicle, aortic strip & spleen)

Special tests for β blockers 1-langendorff’s preparation β-blockers abolish catecholamine induced +ve inotropic and chronotropic effects on the isolated perfused mammalian heart 2-Ethylnorepinephrine reversal • EthylNE stimulates α & β receptor with predominant β stimulant effect • Injection of ethylNE into anaesthetized cat →↓in BP (β2) & tachycardia (β1) • Pretreatment with β-blocker will produce ↑ in BP (α-effect) & bradycardia (≠β1) 3-Non pregnant rat uterus in oestrus B-blocker Pretreatment with B blocker inhibit the relaxant action of isoprenaline on rat uterus in estrus ISOP ISOP

Special tests for ganglionic blockers I-Antagonism of nicotine induced convulsions • A suitable convulsive dose of nicotine is determined in groups of mice • The GB under test is injected to a group of mice before the administration of convulsive dose of nicotine • Protection against nicotine-induced convulsions indicates a ganglion blocker • The % of mice protected in each group is determined & the ED50 is calculated • Compare ED50% of S,T [quantal Assay] II-Trendlenburg preparation (peristaltic reflex) GBs block parasympathetic ganglia, abolish peristaltic reflex [local reflex (auerbach’s ‘myenteric’ plexus) i.e. not involve spinal cord] of piece of intestine stimulated by distending the lumen.

Auerbach’s (myenteric) plexus The enteric nervous system [Auerbach’s & meissner’s (submucosal) plexus] The ENS is a division of the ANS, the other divisions being the sympathetic and parasympathetic, with which it has extensive connections. It control motor activity, secretion, absorption, blood flow, and interaction with other organs such as the gallbladder or pancreas the myenteric plexus exists between the longitudinal and circular layers of muscularis externa in the gastrointestinal tract to controls GIT motility. originates in the medulla oblongata then vagus carries the axons to their location in GIT Myenteric plexus provides motor innervation to both layers of the muscular layer, having both parasympathetic and sympathetic input, whereas the submucous plexus has only parasympathetic fibers and provides secretomotor innervation to the mucosa nearest the lumen of the gut.

II-Trendlenburg preparation