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  2. 2. Introduction The purpose of this lab is to test the effects of 11 different drugs on the muscarinic M2, adrenergic 1, and adrenergic 1 receptors found in the Guinea Pig auricles in vitro. More specifically, the experiment focuses on the changes in force of muscle contraction and beats per minute in comparison to control contractions with each drug administration. The isolated auricle tissue was held in a solution of Krebs ringer at 29oC, and aerated with 95% O2 and 5% CO2. Krebs ringer is a physiological salt solution designed to mimic a normal biological environment to keep the tissue alive and enable contractions to occur in a normal fashion in vitro. The heart is muscular organ enclosed in the pericardium and epicardium with watery fluid serving as a lubricant between the two fibrous sacs (5). The auricles are located on the anterior surfaces of the outer-walls of the myocardium. The auricles control the volume and velocity of blood entering the heart through the use of cardiac muscle. The inner surfaces of the cardiac chambers are lined by a thin layer of endothelial cells (5). Blood flow through the heart enters the superior vena cava and travels through the right atrium, atrioventricular valve, right ventricle, aorta, pulmonary artery, and out the left atrium. The atria consists of the right atrium (receives deoxygenated blood from veins) which pumps blood to the right ventricle to be oxygenated in the lungs, and the left atrium, which takes the oxygenated blood and sends it to the left ventricle, where it is pumped through the arteries to the rest of the body (3). The interventricular septum separates the right and left ventricles. Cardiac muscle cells are striated like skeletal muscle, but are shorter and branch out. Heartbeat is coordinated though the depolarization of the plasma membrane and approximately 1% of cardiac cells are dedicated to heart excitation (pacemaker cells). The conducting system initiates the heart beat and spreads the impulse throughout the heart and consists of the SA node, AV node, bundle of his, and purkinje fibers. Intercalated disks (gap junctions) connect the muscle cells and allow the rapid spread of action potentials (AP) from one cell to another which is needed for synchronized contraction. The Sinoatrial (SA) node consists of pacemaker cells which initiates the impulse. The AV node slowly transmits (0.1 secs) the action potential from the atria to the ventricles so that the atria is given a chance to completely contract before ventricular contraction (5). Bundle of His in the interventricular septum propagates the AP to the purkinje fibers (large conducting cells) which distributes the impulse through the ventricles (5). Heart action potentials are the result of different ion channels opening and closing in an orderly fashion. Autorhythmic cardiac cells and myocardial pumping cells exhibit distinctly different action potential curves on a graph plotting time against membrane potential (mV). Autorhythmic cells depolarize via sodium ions leaking in through the funny channels and calcium ions moving in through T channels (5). Following depolarization, rapid opening of voltage-gated L-type calcium channels initiate the rapid depolarization phase. Reopening of potassium channels and closing of calcium channels cause repolarization (5). Myocardial pumping cells have five action potential phases. Phase 0 involves the opening of voltage gated sodium channels and closure of potassium channels for rapid depolarization. Partial repolarization (phase 1) is due to sodium channels closing and fast potassium channels opening. The plateau (phase 2) is due to fast potassium channels
  3. 3. closing and L-type calcium channels opening. Repolarization (phase 3) occurs when L- type calcium channels close and slow potassium channels open. Finally, the cell is returned to resting potential (phase 4). Cardiac muscle contraction initiates when the AP from an adjacent cell enters and causes the voltage gated calcium channel in the T-tubule to open. Calcium ions rush into the cell to bind ryanodine receptor-channels to induce calcium ion release from the sarcoplasmic reticulum. The local release of calcium ions causes a Ca2+ spark to create a Ca2+ signal (5). The calcium ions lastly bind troponin to initiate contraction. The Heart has three main receptors that will respond to the various drugs. 1- adrenergic receptors predominate in the cardiac tissue and are primarily responsible for modulated cardiac function. Agonism of 1 receptors activates the G protein coupled receptor (GPCR) Gs pathway. Gs activates adenylyl cyclase (AC) to catalyze the conversion of ATP to cAMP which in turn binds to Protein Kinase A (PKA) and activates it to phosphorylate the L-type calcium channel. The influx of Ca2+ ions causes depolarization and initiates the action potential which leads to an increase in heart rate and force of contractions. Adrenergic 1 receptors have been associated with activation of Ca2+ channels and inhibition of K+ channels which both increase heart rate and force of contractions (4). Agonism of 1 receptors activate the GQ pathway in which phospholipase C cleaves Phosphatidylinositold (4,5) bisphosphate (PIP2) to form diacylglycerol (DAG) and inositol triphosphate (IP3). IP3 causes a rise in calcium ion concentration which elicits the Ca2+ signal to bind troponin and cause contraction. Muscarinic M2 receptors activate the Gi GPCR pathway which inhibits AC. The Gi subunit causes K+ channels to open (causes hyperpolarization) and inhibits L-type Ca2+ channels from opening (inhibition of depolarization). The drugs used for this experiment were: Acetylcholine (ACh), Methacholine, Atropine, Tetramethylammonium (TMA), Physostigmine, Epinephrine, Norepinephrine, Isoproterenol, Propranolol, Phenoxybenzamine, and Verapamil. Acetylcholine is a parasympathetic neurotransmitter that acts as an nicotinic and muscarinic M2 agonist in the heart. The ion fluctuation of increased potassium ions and decreased calcium ions causes a hyperpolarization of the cardiac cell membrane, therefore causing a decrease in force and rate of contractions. Methacholine is a non-selective muscarinic receptor agonist in the parasympathetic nervous system that is more resistant to actions of acetylcholinesterase. The ester agonizes the M2 muscarinic receptor and causes a decrease in force and rate of contractions. Atropine is a competitive antagonist of muscarinic M2 receptors in the parasympathetic nervous system (1). This should bring the rate of contractions back to a baseline state following muscarinic receptor agonism. Tetramethylammonium (TMA) is a nicotinic receptor agonist and partial muscarinic agonist whose downstream effect activates the M2 muscarinic receptors in cardiac cells. This causes agonistic parasympathetic effects for a decrease in force and rate of contractions. Physostigmine is a reversible inhibitor of acetylcholinesterase and should prevent acetylcholine from being metabolized to acetic acid and choline. Acetylcholine left
  4. 4. unmetabolized will activate the M2 receptor and cause a decrease in force and rate of contractions. Verapamil is an L-type Calcium ion channel blocker that slows atrioventricular conduction from the AV node. This decrease in conduction velocity leads to a decrease in heart rate (2). However, if enough ventricle has been removed, there should be no effect on the tissue. Epinephrine is a nonselective agonist of all adrenergic receptors, but primarily agonizes the 1 receptor to increase the force and rate of contractions. Norepinephrine (NE) acts similarly to Epinephrine, and causes an increase in force and rate of contractions but is less potent. Isoproterenol is an agonist with a higher affinity for adrenergic receptors than It has the same effects as E and NE and similarly increases force and rate of contractions. In terms of affinity for the 1 receptor, isoproterenol has the highest, followed by E, and lastly NE. Propranolol is a non-selective competitive antagonist of the catecholamines on the -adrenoreceptors (2). This reduces the rate and force of contractions caused by norepinephrine, epinephrine, and isoproterenol by competing for the common binding site on the 1 adrenergic receptor. Phenoxybenzamine is a non-specific, irreversible adrenergic antagonist that acts on the sympathetic nervous system to inhibit agonism of 1 receptors. Each receptor the drugs affected influenced the Chronotrophy, the Inotrophy, and the Dromotrophy. Chronotrophy (heart rate) is increased primarily through sympathetic 1 activation and secondarily by 1 activation (5). A decrease in chronotrophy can be attributed to muscarinic M2 activation. Force of heart contraction (inotrophy) is increased by 1 activation, and decreased by M2 muscarinic receptor activation (5). Ca2+ ion concentration is the effector of inotrophy. Ca2+ ion influx during action potentials, Ca2+ ion release from the sarcoplasmic reticulum, and sensitization of troponin C to Ca2+ ions all increase inotrophy. The hearts conduction velocity (dromotrophy), is decreased by M2 muscarinic activation. AV conduction velocity is primarily affected by this receptor. Sympathetic stimulation increases conduction velocity throughout the heart, while parasympathetic stimulation inhibits the rate of action potential spread through the AV node and atria (5).
  5. 5. Methods Table 1: Drug Dilution Table Drug [Stock] (M) [Bench] (M) Dilution Factor Vdrug (mL) Vringer (mL) Vdose (mL) [Bath] (M) Acetylcholine 5E-3 1E-3 5 1 4 1E-3 1E-4 10 0.5 4.5 1E-4 1E-5 10 0.5 4.5 0.2 1E-7 1E-5 1E-6 10 0.5 4.5 0.2 1E-8 Methacholine 5E-3 1E-3 5 1 4 1E-3 1E-4 10 0.5 4.5 0.2 1E-6 TMA 5E-1 1E-1 5 1 4 0.2 1E-3 Physostigmine 5E-3 1E-3 5 1 4 0.2 1E-5 Atropine 5E-3 1