NAME: Charles Poon

T.A.: Paige Ruiz and Jennifer Lee

LAB # 4

MUSCARINIC & ADRENERGIC RECEPTORS

IN CARDIAC MUSCLE

MCDB 126/226AL

PHARMACOLOGY LABORATORY

IntroductionThe purpose of this lab is to test the effects of 11 different drugs on the

muscarinic M2, adrenergic α1, and adrenergic β1 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

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. β1-adrenergic receptors predominate in the cardiac tissue and are primarily responsible for modulated cardiac function. Agonism of β1 receptors activates the G protein coupled receptor (GPCR) Gs pathway. Gsα 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 α1 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 α1 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 Giβγ 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

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 β1 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 β adrenergic receptors than α It has the same effects as E and NE and similarly increases force and rate of contractions. In terms of affinity for the β1 receptor, isoproterenol has the highest, followed by E, and lastly NE.

Propranolol is a non-selective competitive antagonist of the catecholamines on the β-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 β1 adrenergic receptor.

Phenoxybenzamine is a non-specific, irreversible α adrenergic antagonist that acts on the sympathetic nervous system to inhibit agonism of α1 receptors.

Each receptor the drugs affected influenced the Chronotrophy, the Inotrophy,and the Dromotrophy. Chronotrophy (heart rate) is increased primarily throughsympathetic β1 activation and secondarily by α1 activation (5). A decrease in chronotrophy can be attributed to muscarinic M2 activation. Force of heart contraction (inotrophy) is increased by β1 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 heart’s 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).

Methods

Table 1: Drug Dilution TableDrug [Stock]

(M)[Bench]

(M)Dilution Factor

Vdrug

(mL)Vringer

(mL)Vdose

(mL)[Bath]

(M)

Acetylcholine5E-3 1E-3 5 1 41E-3 1E-4 10 0.5 4.51E-4 1E-5 10 0.5 4.5 0.2 1E-71E-5 1E-6 10 0.5 4.5 0.2 1E-8

Methacholine 5E-3 1E-3 5 1 41E-3 1E-4 10 0.5 4.5 0.2 1E-6

TMA 5E-1 1E-1 5 1 4 0.2 1E-3Physostigmine 5E-3 1E-3 5 1 4 0.2 1E-5

Atropine 5E-3 1E-3 5 1 41E-3 1E-4 10 0.5 4.5 0.4 2E-6

Epinephrine 5E-3 1E-3 5 1 4 0.2 1E-5Norepinephrine 5E-3 1E-3 5 1 4 0.2 1E-5Isoproterenol 2E-2 2E-3 10 0.5 4.5

2E-3 2E-4 10 0.5 4.5 0.4 4E-6Propanolol 5E-3 1E-3 5 1 4

1E-3 1E-4 10 0.5 4.5 0.2 1E-6Phenoxybenzamine 1E-3 1E-4 10 0.5 4.5

1E-4 1E-5 10 0.5 4.5 0.4 2E-7Verapamil 5E-3 5E-4 10 0.5 4.5

5E-4 5E-5 10 0.5 4.5 0.4 1E-6

Table 1 Sample Calculations:

Acetylcholine dilution Factor: [ Stock ][Bench ]

= 1 E−4 M1 E−5 M

= 10

Acetylcholine Vdrug: C1V1 = C2V2

(1E-4 M)(V1) = (1E-5 M)(5 mL)V1 = 0.5 mLAcetylcholine Vringer: 5 mL –Vdrug

5 mL – 0.5 mL = 4.5 mLAcetylcholine VDose: C1V1 = C2V2

(1E-5 M)(V1) = (1E-7 M)(20 mL)V1 = 0.2 mL

Table 2: Drug administration cycle

Drug administered Bath concentration Additional instructionsNotes:

Allow 1-1.5 minutes of drug exposure before washing. Wait 1-2 minutes to return to control levels after washing Record control contractions at least 1 minute before next drug dose. Do not allow heart to stop beating when administering parasympathetic drugs

Acetylcholine 1E-7 MMethacholine 1E-6 MTetramethylammonium (TMA) 1E-3 MPhysostigmine 1E-5 M Without washing; after two minutes dose

nextAcetylcholine 1E-5 M before beat stops, add nextAtropine 4E-6 M allow to act for two minutes before washEpinephrine 1E-5 MNorepinephrine 1E-5 MIsoproterenol 4E-6 MPropanolol 1E-6 M After two minutes without washing dose

next drugEpinephrine 1E-5 MPropanolol 1E-6 M After two minutes without washing dose

next drugNorepinephrine 1E-5 MPropanolol 1E-6 M After two minutes without washing dose

next drugIsoproterenol 4E-6 MPhenoxybenzamine 2E-7 M After three minutes without washing dose

next drugEpinephrine 1E-5 MPhenoxybenzamine 2E-7 M After three minutes without washing dose

next drugNorepinephrine 1E-5 MPhenoxybenzamine 2E-7 M After three minutes without washing dose

next drugIsoproterenol 4E-6 MVerapamil 1E-6 M

Procedure:First, vials were labeled with drug names

and corresponding bench concentrationsto keep all materials organized. The drug dilutions were prepared accordingly using Krebs Ringer as a buffer (Table 1). The Atropine vials were kept under foil to prevent light degradation. The prepared dilutions were then set in an ice bath.

Isolation of auricles took place in aerated ringer solution. During removal of excess fat and connective tissue, one of the auricles was cut in half. Metal instruments should not have come in

Figure 1: Sutured heart Double knot

on auricle

contact with the auricles due to suppression of contractile activity. The tips of the auricles were sutured with a double knot, and ventricular tissue was then cut off (Figure 1). One strand of suture was looped to the hook inside the tissue bath, and the heart was left to beat and acclimate for 15 minutes (Figure 2). The Krebs ringer bath temperature was set at 290C, and the tissue was aerated gently with 95% O2 and 5% CO2.

The PolyVIEW amplifier was calibrated to 0.00 Volts (balance) and 1.00 Volts (gain). The chart speed on the recorder was set to 5 mm/sec.

Acetylcholine (ACh) 1E-7 M was administered for only 35 seconds before wash to avoid heart arrest. Methacholine 1E-6M was administered for only 30 seconds before wash to avoid heart arrest. Tetramethylammonium 1E-3 M was administered for only 41 seconds before wash to avoid heart arrest. Physostigmine 1E-5 M was administered for

90 seconds; without washing Acetylcholine 1E-8 M was only administered for 30 seconds before the addition of atropine 2E-6M. Contraction forces of 0.1 g during the ACh dose made it hard to determine when a flatline would occur so atropine was dosed after 30 seconds to be safe and prevent heart arrest. Data was recorded for two minutes before washing. Epinephrine 1E-5 M was dosed for 91 seconds before wash. Norepinephrine 1E-5 M was dosed for 100 seconds before wash. Isoproterenol 4E-6 M was dosed for 110 seconds before wash.

After seeing no response from norepinephrine (NE) and isoproterenol (ISO), the group repeated the NE 1E-5 M and ISO 4E-6 M doses an additional two times with the allowance of two minutes of drug exposure for each dose. Lack of responses from the attempts led to the instruction

of Paige Ruiz to continue the experiment with the remaining doses.

Propranolol 1E-6 M was dosed for 95 seconds, without washing 1E-5 M Epinephrinewas dosed for 65 seconds before wash. Propranolol 1E-6 M was dosed for 120 seconds, without washing NE 1E-5 M was dosed for 75 seconds before wash. Propranolol 1E-6 M was dosed for 120 seconds, without washing ISO 4E-6 M was dosed for 85 seconds before wash.

Phenoxybenzamine 2E-7 M was dosed for 120 seconds, without washing Epinephrine 1E-5 M was dosed 100 seconds before wash. Phenoxybenzamine 2E-7 M was dosed for 130 seconds, without washing NE 1E-5 M was dosed for 100 seconds before wash. Phenoxybenzamine 2E-7 M was dosed for 95 seconds, without washing isoproterenol 4E-6 M for 140 seconds before wash. Lastly, verapamil 1E-6 M was dosed for 130 seconds before wash. At least 1 minute of control contractions were established after washes before administering next drug dose. All data was recorded on Polyview.

Figure 2: Tissue in bath Tissue in

bath set at 29oC and gently aerated with 95% O2 and 5% CO2

Strand of suture looped to hook

Figure 3: Polygraph - Tissue apparatus

Results

Table 3: Group 3 + Group 4 Inotrophy/Chronotrophy responses and percent changes

Drug [Bath] M Inotrophy % Change Chronotrphy % ChangeGroup 3 Group 4 Group 3 Group 4

Acetylcholine 1E-7 -50 -46 0 -16Methacholine 1E-6 -65 -80 -13 0

Tetramethylammonium 1E-3 -40 -75 0 -17Physostigmine 1E-5 0 -14

-27 -45Acetylcholine 1E-8 0 -64Atropine 4E-6 -25 -86

Epinephrine 1E-5 +71 +182 +50 +39Norepinephrine 1E-5 +33 +80 0 +18Isoproterenol 4E-6 -13 +55 +6 +33Propanolol 1E-6 0 0

Tyrode Ringer reservoir

Amplifier

CO2/O2 controller

Transducer

PolyVIEW Grass Polygraph Recorder

Drain

0 0Epinephrine 1E-5 -14 -7Propanolol 1E-6 0 -14

-17 +4Norepinephrine 1E-5 0 +25Propanolol 1E-6 0 -17

+10 +18Isoproterenol 4E-6 +14 +117Phenoxybenzamine 2E-7 -17 -10

-20 -4Epinephrine 1E-5 0 +30Phenoxybenzamine 2E-7 -14 -29

-50 -4Norepinephrine 1E-5 -25 +29Phenoxybenzamine 2E-7 -17 0

+50 +13Isoproterenol 4E-6 +50 +240Verapamil 1E-6 -29 0 0 0

Note: Group 4 data was obtained from Cameron Noorbakhsh

Drug [Bath] M Time of administratio

n (Hr:Min:Sec)

Target Nervous System

Mechanism of

Action

Target Receptor(s)

Expected response Observed responseInotroph

yChronotrophy Inotrophy Chronotrophy

Acetylcholine 1E-7 00:04:00 P A N / M2 Decrease Decrease Decrease DecreaseMethacholine 1E-6 00:08:00 P A M2 Decrease Decrease Decrease NCTetramethylammonium

1E-3 00:13:00 P A N Decrease Decrease Decrease DecreaseP PA M2

Physostigmine 1E-5 00:18:00 P AChE Inhibitor

AChE NC * NC * Decrease Decrease

Acetylcholine 1E-8 00:20:00 P A N / M2 Decrease Decrease Decrease DecreaseAtropine 4E-6 00:21:30 P Ant M2 Increase Increase Decrease DecreaseEpinephrine 1E-5 00:43:00 S A α1 / β1 Increase Increase Increase IncreaseNorepinephrine 1E-5 00:58:00 S A α1 / β1 Increase Increase Increase IncreaseIsoproterenol 4E-6 01:06:00 S A α1 / β1 Increase Increase Increase IncreasePropanolol 1E-6 01:18:00 S Ant β1 NC

IncreaseNC

NCEpinephrine 1E-5 01:20:00 S A α1 / β1 Increase DecreasePropanolol 1E-6 01:30:00 S Ant β1 NC

IncreaseDecrease

IncreaseNorepinephrine 1E-5 01:32:00 S A α1 / β1 Increase IncreasePropanolol 1E-6 01:37:00 S Ant β1 NC

IncreaseDecrease

IncreaseIsoproterenol 4E-6 01:39:00 S A α1 / β1 Increase IncreasePhenoxybenzamine 2E-7 01:48:00 S Ant α1 NC

IncreaseDecrease

DecreaseEpinephrine 1E-5 01:51:00 S A α1 / β1 Increase IncreasePhenoxybenzamine 2E-7 01:57:00 S Ant α1 NC

IncreaseDecrease

DecreaseNorepinephrine 1E-5 02:00:00 S A α1 / β1 Increase IncreasePhenoxybenzamine 2E-7 02:06:00 S Ant α1 NC

IncreaseNC

IncreaseIsoproterenol 4E-6 02:09:00 S A α1 / β1 Increase IncreaseVerapamil 1E-6 02:19:00 P Channel

blockerL-type Ca2+

channel NC NC NC NCKey

S = SympatheticP = ParasympatheticAChE = AcetylcholinesteraseNC = No Change

A = AgonistAnt = AntagonistPA = Partial agonist* = or small decrease

N = Nicotinic receptorM2 = Muscarinic M2 receptorα1 = Adrenergic α1 receptorβ1 = Adrenergic β1 receptor

Note:Data in merged cells represent data for the combined doses of drugs aligned in the same rowsThe data in this table was acquired from Group #4

Table 4: Group 4 drug summary

Table 5: Group 4 Inotrophy / Chronotrophy responses and percent changesDrug [Bath] M Time of

administration (Hr:Min:Sec)

Inotrophy ChronotrophyControl

Response (g)Drug

Response (g)Percent

Change (%)Control Rate

(BPM)Response

Rate (BPM)Percent

Change (%)Acetylcholine 1E-7 00:04:00 0.65 0.35 -46 114 96 -16Methacholine 1E-6 00:08:00 0.50 0.10 -80 102 102 0Tetramethylammonium 1E-3 00:13:00 0.40 0.10 -75 102 84 -17Physostigmine 1E-5 00:18:00

0.700.60 -14

120 66 -45Acetylcholine 1E-8 00:20:00 0.25 -64Atropine 4E-6 00:21:30 0.10 -86Epinephrine 1E-5 00:43:00 0.55 1.55 +182 108 150 +39Norepinephrine 1E-5 00:58:00 0.50 0.90 +80 120 142 +18Isoproterenol 4E-6 01:06:00 0.50 1.10 +55 126 168 +33Propanolol 1E-6 01:18:00

0.800.80 0

174 174 0Epinephrine 1E-5 01:20:00 0.75 -7Propanolol 1E-6 01:30:00

0.400.35 -14

138 144 +4Norepinephrine 1E-5 01:32:00 0.50 +25Propanolol 1E-6 01:37:00

0.300.25 -17

132 156 +18Isoproterenol 4E-6 01:39:00 0.65 +117Phenoxybenzamine 2E-7 01:48:00

0.500.45 -10

150 144 -4Epinephrine 1E-5 01:51:00 0.65 +30Phenoxybenzamine 2E-7 01:57:00

0.350.25 -29

144 138 -4Norepinephrine 1E-5 02:00:00 0.45 +29Phenoxybenzamine 2E-7 02:06:00

0.250.25 0

138 156 +13Isoproterenol 4E-6 02:09:00 0.85 +240Verapamil 1E-6 02:19:00 0.50 0.50 0 150 150 0Key:BPM = Beats Per MinuteNote:Data in merged cells represent data for the combined doses of drugs aligned in the same rowsThe data in this table was acquired from Group #4

Table 5 Sample CalculationsInotrophy Chronotrophy

Control Response = Average control height – Average baseline

Control Response (Acetylcholine 1E-7 M) = 0.40 – (-0.25)

= 0.65 g

Control Rate (BPM) = ¿of beats

10 seconds∗6

Control Rate (Acetylcholine 1E-7 M) = 19 beats

10 seconds∗6

= 114 BPM

Drug Response = Average drug height – Average baseline

Acetylcholine 1E-7 M Response = 0.1- (-0.25)

= 0.35 g

Response Rate (BPM) = ¿of beats

10 seconds∗6

Response Rate (Acetylcholine 1E-7 M) = 16 beats

10 seconds∗6

= 96 BPM

Percent Change = Drug−Control

Control∗100

Acetylcholine 1E-7 M percent change = 0.35−0.65

0.65∗100

= -46%

Percent Change (BPM) = Drug BPM−Control BPM

Control BPM∗6

Percent Change (Acetylcholine 1E-7 M) = 96−114

114∗100

= -16%

Note:All calculations for percent change have been rounded to the nearest percentBPM = Beats per minute

Table 6: Group 3 drug summaryDrug [Bath] M Time of

administration (Hr:Min:Sec)

Target Nervous System

Mechanism of Action

Target Receptor(s)

Expected response Observed responseInotrophy Chronotrophy Inotrophy Chronotrophy

Acetylcholine 1E-7 00:04:00 P A N / M2 Decrease Decrease Decrease NCMethacholine 1E-6 00:08:00 P A M2 Decrease Decrease Decrease DecreaseTetramethylammonium 1E-3 00:13:00 P A N Decrease Decrease Decrease NC

P PA M2

Physostigmine 1E-5 00:18:00 P AChE inhibitor

AChE NC * NC * NC

DecreaseAcetylcholine 1E-8 00:20:00 P A N / M2 Decrease Decrease NCAtropine 4E-6 00:21:30 P Ant M2 Increase Increase DecreaseEpinephrine 1E-5 00:43:00 S A α1 / β1 Increase Increase Increase IncreaseNorepinephrine 1E-5 00:58:00 S A α1 / β1 Increase Increase Increase NCIsoproterenol 4E-6 01:06:00 S A α1 / β1 Increase Increase Decrease IncreasePropanolol 1E-6 01:18:00 S Ant β1 NC

IncreaseNC

NCEpinephrine 1E-5 01:20:00 S A α1 / β1 Increase DecreasePropanolol 1E-6 01:30:00 S Ant β1 NC

IncreaseNC

DecreaseNorepinephrine 1E-5 01:32:00 S A α1 / β1 Increase NCPropanolol 1E-6 01:37:00 S Ant β1 NC

IncreaseNC

IncreaseIsoproterenol 4E-6 01:39:00 S A α1 / β1 Increase IncreasePhenoxybenzamine 2E-7 01:48:00 S Ant α1 NC

IncreaseDecrease

DecreaseEpinephrine 1E-5 01:51:00 S A α1 / β1 Increase NCPhenoxybenzamine 2E-7 01:57:00 S Ant α1 NC

IncreaseDecrease Decrease

Norepinephrine 1E-5 02:00:00 S A α1 / β1 Increase DecreasePhenoxybenzamine 2E-7 02:06:00 S Ant α1 NC

IncreaseDecrease

IncreaseIsoproterenol 4E-6 02:09:00 S A α1 / β1 Increase IncreaseVerapamil 1E-6 02:19:00 P Channel

blockerL-type Ca2+

channelNC NC Decrease NC

KeyS = SympatheticP = ParasympatheticAChE = AcetylcholinesteraseNC = No Change

A = AgonistAnt = AntagonistPA = Partial agonist* = or small decrease

N = Nicotinic receptorM2 = Muscarinic M2 receptorα1 = Adrenergic α1 receptorβ1 = Adrenergic β1 receptor

Note:

Data in merged cells represent data for the combined doses of drugs aligned in the same rows

Table 7: Group 3 Inotrophy / Chronotrophy responses and percent changesDrug [Bath] M Time of

administration (Hr:Min:Sec)

Inotrophy ChronotrophyControl

Response (g)Drug

Response (g)Percent

Change (%)Control Rate

(BPM)Response

Rate (BPM)Percent

Change (%)Acetylcholine 1E-7 00:04:00 0.30 0.15 -50 90 90 0Methacholine 1E-6 00:08:00 0.20 0.07 -65 96 84 -13Tetramethylammonium 1E-3 00:13:00 0.05 0.03 -40 66 66 0Physostigmine 1E-5 00:18:00

0.100.10 0

66 48 -27Acetylcholine 1E-8 00:20:00 0.10 0Atropine 4E-6 00:21:30 0.075 -25Epinephrine 1E-5 00:43:00 0.10 0.35 +71 54 108 +50Norepinephrine 1E-5 00:58:00 0.15 0.20 +33 120 120 0Isoproterenol 4E-6 01:06:00 0.34 0.30 -13 108 114 +6Propanolol 1E-6 01:18:00

0.350.35 0

72 72 0Epinephrine 1E-5 01:20:00 0.30 -14Propanolol 1E-6 01:30:00

0.350.35 0

72 60 -17Norepinephrine 1E-5 01:32:00 0.35 0Propanolol 1E-6 01:37:00

0.350.35 0

60 66 +10Isoproterenol 4E-6 01:39:00 0.40 +14Phenoxybenzamine 2E-7 01:48:00

0.300.25 -17

60 48 -20Epinephrine 1E-5 01:51:00 0.30 0Phenoxybenzamine 2E-7 01:57:00

0.400.35 -14

72 36 -50Norepinephrine 1E-5 02:00:00 0.30 -25Phenoxybenzamine 2E-7 02:06:00

0.300.25 -17

60 90 +50Isoproterenol 4E-6 02:09:00 0.45 +50Verapamil 1E-6 02:19:00 0.30 0.25 -29 72 72 0Key:BPM = Beats Per MinuteNote:Data in merged cells represent data for the combined doses of drugs aligned in the same rowsThe data in this table was acquired from Group #4

Table 7 Sample CalculationsInotrophy Chronotrophy

Control Response = Average control height – Average baseline

Control Response (Acetylcholine 1E-7 M) = 0.35 – (0.05)

= 0.30 g

Control Rate (BPM) = ¿of beats

10 seconds∗6

Control Rate (Acetylcholine 1E-7 M) = 15 beats

10 seconds∗6

= 90 BPM

Drug Response = Average drug height – Average baseline

OR

Drug Response = Average drug height – Average drug baseline

Acetylcholine 1E-7 M Response = 0.15 – 0

= 0.15 g

Response Rate (BPM) = ¿of beats

10 seconds∗6

Response Rate (Acetylcholine 1E-7 M) = 15 beats

10 seconds∗6

= 90 BPM

Percent Change = Drug−Control

Control∗100

Acetylcholine 1E-7 M percent change = 0.15−0.30

0.30∗100

= -50%

Percent Change (BPM) = Drug BPM−Control BPM

Control BPM∗6

Percent Change (Acetylcholine 1E-7 M) = 90−90

90∗100

= 0%

Note:All calculations for percent change have been rounded to the nearest percentBPM = Beats per minute

Drug Structures

Acetylcholine Methacholine Tetramethylammonium

Physostigmine Atropine Epinephrine

Norepinephrine Isoproterenol Propanolol

Phenoxybenzamine Verapamil

Discussion:The purpose of this experiment was to test the effects of 11 different drugs on

the muscarinic M2, adrenergic α1, and adrenergic β1 receptors within cardiac muscle of the guinea pig auricles. Acetylcholine (ACh), methacholine, atropine, tetramethylammonium (TMA), physostigmine, epinephrine (Epi), norepinephrine (NE), isoproterenol(ISO), propranolol, phenoxybenzamine(PBZ), and verapamil were administered to test their affects on inotrophy, chronotrophy, and dromotrophy.

The ventricles were removed from the heart, because they have their own pacemaker cells which would have made it difficult to record the mixed signals from both auricles and ventricles on the PolyVIEW system. Two action potentials are responsible for cardiac muscle contractions. The autorhythmic cardiac cells (pacemaker cells) initiate the action potential which propagates to myocardial pumping cells (non-pacemaker). The autorhythmic cardiac cells are responsible for chronotrophy (heart rate). These cells operate in a rhythmic fashion and set the rate of heart contractions. Na + ions leak in through the F-type channels as Ca2+ ions flow in through T-type channels to cause a steady, threshold-graded depolarization (5). Next, rapid opening of voltage-gated Ca2+channels causes the rapid depolarization phase. Reopening of potassium in addition to closing of calcium channels cause repolarization.

Myocardial pumping cells responsible for ionotrophy (contraction force) 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 closing and L-type calcium channels opening. Repolarization (phase 3) occurs when L-type calcium channels close and slow potassium channels open. The cell then returns to resting potential (phase 4).

Data used in this discussion is from group 4. Acetylcholine is a parasympathetic neurotransmitter that acts as a nicotinic and muscarinic M2 agonist mainly located in the atria (5). Agonism of M2 muscarinic receptors activates the Gi GPCR pathway to inhibit AC. The Giβγ subunit causes K+ channels to open (causes hyperpolarization) and inhibits L-type Ca2+ channels from opening (inhibition of depolarization). The ion fluctuation of increased potassium ions and decreased calcium ions causes a hyperpolarization of non-pacemaker cells should decrease force of contractions. The opening of K+ channels in pacemaker cells results in hyperpolarization and prevents the cells from reaching the action potential as frequently.

Acetylcholine 1E-7 M was first dosed. Inotrophy decreased by 46% and chronotrophy decreased by 16% as expected.

Administration of methacholine 1E-6 M caused a decrease of 80% for inotrophy and 0% change for chronotrophy. A decrease in chronotrophy was expected because 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 receptors which activates the Gi GPCR pathway to inhibits AC. The Giβγ subunit cause K+ channels to open (causes hyperpolarization) and inhibits L-type Ca2+ channels from opening (inhibition of depolarization). Methacholine should have elicited a decrease in force and rate of contractions; however, it is a possibility that while the M2 receptors in the non-pacemaker cells were agonized, the M2 receptors in the pacemaker cells were desensitized from the Ach 1E-7 M dose. The main

target of ACh is the pacemaker cell. It is possible that methacholine has a higher affinity for M2

receptors in non-pacemaker cells. A dose of tetramethylammonium 1E-3 M caused a decrease of 75% for inotrophy and

decrease of 17% for chronotrophy as expected because TMA is a nicotinic receptor agonist and partial muscarinic agonist whose downstream effect activates the M2 muscarinic receptors in cardiac cells. TMA enhances the release of ACh at post-ganglionic synapses which would target both pacemaker and non-pacemaker cells. Agonism of M2 muscarinic receptors activates the Gi GPCR pathway to inhibit AC. The Giβγ subunit causes K+ channels to open (causes hyperpolarization) and inhibits L-type Ca2+ channels from opening (inhibition of depolarization). TMA should cause parasympathetic effects, eliciting a decrease in force and rate of contractions.

Physostigmine is a reversible inhibitor of acetylcholinesterase (AChE) and should prevent acetylcholine from being metabolized to acetic acid and choline. Acetylcholine left unmetabolized will activate the M2 receptor and cause a decrease in force and rate of contractions. Dosed alone, physostigmine should not cause any changes to chronotrophy or inotrophy because it does not agonize any receptors. A dose of Physostigmine 1E-5 M elicited a decrease of 14% for inotrophy. Physostigmine may have caused the small decrease in inotrophy and chronotrophy prior to the addition of ACh because there is still some blood in the tissue. Blood contains pseudocholinesterase which hydrolyzes ACh in a fashion similar to AChE. The ACh naturally released from parasympathetic nerves affecting the SA node and atrial muscle would be protected from AChE via inhibition by physostigmine. This would explain the small decrease in inotrophy and chronotrophy before ACh is even added.

Acetylcholine 1E-8 M dosed after physostigmine 1E-5 M, caused an expected inotrophic decrease of 64%. ACh was protected from pseudocholinesterases in the blood. In comparison to ACh 1E-7 M which elicited an inotrophic decrease of 46%, ACh 1E-8 M elicited a greater inotrophic decrease with a smaller dose. This occurred because the physostigmine inhibited AChE and enhanced the effect of ACh by preventing hydrolysis

Atropine 4E-6 M dosed after ACh 1E-8 M caused an inotrophic decrease of 86% and chronotrophic decrease of 45%. However, the addition of atropine was expected to bring inotrophic and chronotrophic responses back to control responses because atropine is a competitive antagonist of muscarinic M2 receptors in the parasympathetic nervous system (1). Atropine should have returned the force and rate of contractions back to a baseline state following muscarinic receptor agonism. This deviation from the expected response can be attributed to administration of ACh (1E-5 M) before atropine (4E-6 M). If the receptors were already occupied by ACh, atropine would be unable to block the effects of ACh. The 90 second administration time of ACh before atropine must have been ample time for ACh to occupy all of the M2 receptors before atropine had a chance to compete for the binding site.

A dose of epinephrine 1E-5 M caused an inotrophic increase of 182% and chronotrophic increase of 39% as expected because Epi is a nonselective agonist of all adrenergic receptors. Agonism of α1 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. Epinephrine primarily acts as an agonist of the β1 receptor to increase the force and rate of contractions. The primary target of epinephrine is the pacemaker cells. Agonism of β1 receptors activates the G protein coupled receptor (GPCR) Gs pathway. Gsα 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.

A dose of NE 1E-5 M caused an inotrophic increase of 80% and chronotrophic increase of 18% as expected because Norepinephrine (NE) is also a nonselective agonist of all adrenergic receptors and shares the same mechanism of action as epinephrine in its function to increase force and rate of contraction. NE is only slightly less potent than epinephrine.

Isoproterenol is another catecholamine which functions as an agonist of all adrenergic

receptors but has a higher affinity for β than α. ISO shares the same mechanism of action as epinephrine and NE in its function to increase force and rate of contractions. ISO is known to have the highest affinity for the β1 receptor out of the three catecholamines (5) and was expected to elicit the greatest chronotrophic and inotrophic effect. However, a dose of isoproterenol 4E-6 M only elicited an inotrophic increase of 55% and chronotrophic increase of 33%. These values are significantly less than the responses elicited by NE and E. This is not surprising because the dose of isoproterenol 4E-6 M was the third consecutive drug dose targeted at the adrenergic receptors. Other studies have discovered that desensitization of the heart to isoproterenol was associated with a reduction in both sensitivity and maximal response of adenylate cyclase to activation by isoproterenol. A decrease in the number of β -adrenergic receptors in the desensitized hearts was also noted (7). Receptor desensitization may have occurred and was the reason why the ISO dose elicited smaller responses than NE and E.

The next six doses involved propanolol, a competitive antagonist of β receptors, and the three catecholamines (ISO, E, NE). In theory, propanolol should not elicit any effects on its own but should antagonize the effects of isoproterenol the most, followed by epinephrine, and lastly norepinephrine. Propanolol 1E-6 M was administered, causing no inotrophic change, as expected. Epinephrine 1E-5 M dosed afterwards elicited an inotrophic decrease of 7% and no chronotrophic change. The 7% decrease in inotrophy may have been due to antagonism of the catecholamines released from sympathetic nerves at atrial muscle, and the 0% change for chronotrophy indicates propanolol’s complete block of epinephrine when compared to the previous epinephrine dose at 00:43:00 (hour:min:sec) which caused a inotrophic increase of 182% and chronotrophic increase of 39%. However, a slight increase in heart rate and force of contractions. The lack of response is most likely due to a combination of receptor desensitization from the previous catecholamine doses and the antagonistic effect of propranolol.

Another dose of propanolol 1E-6 M was administered, causing an inotrophic decrease of 14 percent. This slight decrease may be due to antagonism of the neurotransmitters released from sympathetic nerves at the atrial muscle. Antagonism of the catecholamines released from sympathetic nerves at the atrial muscle would inhibit contractile force (5). The following dose of Norepinephrine 1E-5 M caused an inotrophic increase of 25% and chronotrophic increase of 4%. The previous NE dose at 00:58:00 (hours:min:sec) elicited an inotrophic increase of 80% and chronotrophic increase of 18%. As expected, propanolol exerted less of an inhibition on norepinephrine than on epinephrine.

The final dose of propanolol 1E-6 M was administered and caused an inotrophic decrease of 17%. Once again this was likely due to antagonism of the catecholamines released from sympathetic nerves at the atrial muscle. A dose of Isoproterenol 4E-6 M caused an inotrophic increase of 117% and chronotrophic increase of 18%. In comparison to the isoproterenol dose at 01:06:00 (hour:min:sec) which elicited an inotrophic increase of 55% and chronotrophic increase of 33%, the inotrophic increase of 117% was the highest seen so far with the use of isoproterenol

and may have been due to the consistent use of propanolol in the last few doses, which allowed the resensitization of the β receptors in non-pacemaker cells. The inhibition of inotrophy (increase of 18% versus increase of 55%) agreed with the expected response.

The next six doses involved pheonoxybenzamine, a non-specific, irreversible α adrenergic antagonist and the three catecholamines (ISO, E, NE). Propanolol should have the greater inhibitory effects on epinephrine and norepinephrine since ISO has a higher affinity for β receptors. Propanolol should have no effect on chronotrophy and inotrophy when administered alone since it is an antagonist, which is unable to elicit biological responses. The combined dose of phenoxybenzamine and agonist should elicit greater responses than propanolol and agonist because the majority of cardiac output is determined by β receptors.

The first dose of phenoxybenzamine 2E-7 M caused an inotrophic decrease of 10%. This may be attributed to antagonism of catecholamines released from sympathetic nerves at the atrial muscle. The following dose of epinephrine 1E-5 M caused an inotrophic increase of 30% and chronotrophic decrease of 4%. In comparison to the combined dose of propanolol (1E-6 M) and epinephrine (1E-5 M) which elicited an inotrophic decrease of 7% and no change in chronotrophy. The dose of phenoxybenzamine and epinephrine elicited a greater inotrophic response, as expected. The decrease in chronotrophy may be attributed to β receptor desensitization in the pacemaker cell.

The second dose of phenoxybenzamine 2E-7 M caused an inotrophic decrease of 29% which may be attributed to antagonism of catecholamines released from sympathetic nerves at the atrial muscle. A dose of norepinephrine 1E-5 M caused an inotrophic increase of 29% and chronotrophic decrease of 4%. In comparison to the combined dose of propanolol (1E-6 M) and NE (1E-5 M) which elicited an inotrophic increase of 25% and chronotrophic increase of 4%, the dose of phenoxybenzamine and NE elicited a greater inotrophic response, as expected. The decrease in chronotrophy may be attributed to β receptor desensitization in the pacemaker cell.

The third dose of phenoxybenzamine 2E-7 M caused no change in inotrophy, as expected. The following dose of isoproterenol 4E-6 M caused an inotrophic increase of 240% and chronotrophic increase of 13%. In comparison to the combined dose of propanolol (1E-6 M) and ISO (4E-6 M) which elicited an inotrophic increase of 117% and chronotrophic increase of 18%, the dose of phenoxybenzamine and NE elicited a greater inotrophic response, as expected. However, the chronotrophic increase elicited from the combined dose of phenoxybenzamine and ISO should have been greater than the chronotrophic increase elicited from the combine dose of propanolol and ISO. This deviation may be due to β receptor desensitization in the pacemaker cell.

Analysis of the control responses recorded for propanolol doses and phenoxybenzamine doses showed a trend. The first dose of propanolol had a control response of 0.8 g, the second dose had a control response of 0.4 g, and the third dose had a control response of 0.3 g. Consecutive doses of propanolol inhibited the control responses most likely through antagonism of catecholamines released by sympathetic nerves. Upon the switch to phenoxybenzamine, the control response increased to 0.5g. The rise in control response is likely due to the change of antagonist. Propanolol inhibits the β receptors which are the main effectors of cardiac output, while pheonoxybenzamine inhibited α receptors. The final two doses of phenoxybenzmine lowered the control responses to 0.35 g and 0.25 g which indicates antagonism of catecholamines released by sympathetic nerves. This data indicates that α receptors may play a larger role in sympathetic heart function than anticipated.

The final drug dosed in this experiment was verapamil 1E-6 M. Verapamil is an L-type Calcium ion channel blocker that slows atrioventricular conduction, by prolonging the effective refractory period in the AV node. This decrease in conduction velocity leads to a decrease in heart rate (2). However, if enough of the ventricles were removed, there should be no effect on the tissue. Verapamil 1E-6 M caused no change in inotrophy and chronotrophy as expected.

Group 3’s data was skewed due to the accidental cut of the auricle and a hole in the atria muscle tissue. Contractile activity of the cardiac cells was suppressed due to contact with metal scissors. Group 3’s control and response heights were consistently lower than Group 4’s. Furthermore, the hole in the atria most likely created a physical gap between the SA node (pacemaker cells) and the rest of the cardiac tissue, preventing proper cell communication. In a normal heart, when the SA node fails, other heart tissues will set the new pace (9). The SA node depolarizes 60-100 times per minute and the AV node depolarizes 40-60 times per minute in the human heart. Group 4’s control rate of beats per minute (BPM) ranged from 102 – 174 BPM. Group 3’s control rate of BPM ranged from 54 – 120 BPM. Clearly, Group 3’s pace was slower than group 4’s pace and the non-cardiac tissue must have utilized the AV node’s pace in response to the loss of communication with the SA node.

Acetylcholine 1E-7 M elicited an inotrophic decrease of 50% and no change in chronotrophy. Chronotrophy and inotrophy responses should have both decreased as seen in group 4’s data which recorded an inotrophic decrease of 50% and chronotrophic decrease of 16%. Group 3’s inotrophic decrease of 50% matched with expected responses because contractile force is modulated via activation of M2 receptors. Agonism of M2 muscarinic receptors activates the Gi GPCR pathway to inhibit AC. The Giβγ subunit causes K+ channels to open (causes hyperpolarization) and inhibits L-type Ca2+ channels from opening (inhibition of depolarization). The ion fluctuation of increased potassium ions and decreased calcium ions causes a hyperpolarization of non-pacemaker cells should decrease force of contractions. The lack of chronotrophic response did not match expected responses. The main target of ACh are the pacemaker cells (5), so while ACh may have agonized the M2 receptors in the SA node, the decrease in pace was most likely not transduced to the rest of the atria tissue due to poor communication between the AV node and non-pacemaker cells as a result of the hole.

Methacholine 1E-6 M elicited an inotrophic decrease of 65% and chronotrophic decrease of 13%, as expected because 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 receptors which activates the Gi GPCR pathway to inhibits AC. The Giβγ subunit cause K+ channels to open (causes hyperpolarization) and inhibits L-type Ca2+ channels from opening (inhibition of depolarization), resulting in a decrease of rate and force.

Tetramethylammonium 1E-3 M elicited an inotrophic decrease of 40% and no change in chronotrophy. Chronotrophy and inotrophy responses should have both decreased as seen in group 4’s data, which recorded an inotrophic decrease of 75% and chronotrophic decrease of 17%, because TMA is a nicotinic receptor agonist and partial muscarinic agonist whose downstream effect activates the M2 muscarinic receptors in cardiac cells. TMA enhances the release of ACh at post-ganglionic synapses which would target both pacemaker and non-pacemaker cells. Agonism of M2 muscarinic receptors activates the Gi GPCR pathway to inhibit AC. The Giβγ subunit causes K+ channels to open (causes hyperpolarization) and inhibits L-type Ca2+ channels from opening (inhibition of depolarization). TMA should cause parasympathetic effects, eliciting a decrease in force and rate of contractions. The inotrophic decrease of 40% was

expected, but the lack of chronotrophic response did not match expected responses. The main target of methacholine is similar to ACh. While methacholine may have agonized the M2 receptors in the SA node, the decrease in pace was most likely not transduced to the rest of the atria tissue due to poor communication between the AV node and non-pacemaker cells as a result of the hole.

Physostigmine 1E-5 M elicited no change in inotrophy, as expected. Physostigmine is an AChE inhibitor and does not agonize any receptors.

The next dose of Acetylcholine 1E-8 M elicited no change in inotrophy. This dose of ACh should have elicited a decrease in inotrophy as seen in group 4’s data, which recorded an inotrophic decrease of 64%. This could be due to receptor desensitization from the previous ACh 1E-7 M, methacholine 1E-6 M, and TMA 1E-3 M doses.

Atropine 4E-6 M dosed after ACh 1E-5 M elicited an inotrophic decrease of 25% and chronotrophic decrease of 27%. The addition of atropine was expected to bring inotrophic and chronotrophic responses back up to control responses because atropine is a competitive antagonist of muscarinic M2 receptors in the parasympathetic nervous system (1). Atropine should bring the force and rate of contractions back to a baseline state following muscarinic receptor agonism. Compared to group 4’s data which recorded an inotrophic decrease of 86% and chronotrophic decrease of 45%, group 3’s data was a better match to the expected response. However, group 3 administered ACh for 30 seconds before the addition of atropine while group 4 administered ACh for 90 seconds. The difference in administration time may have been the reason why atropine had more of an antagonistic effect on ACh in group 3. It is possible that ACh was not given enough time to occupy all of the M2 receptors before atropine began to compete for the binding site.

Epinephrine 1E-5 M elicited an inotrophic increase of 71% and chronotrophic increase of 50%, as expected because epinephrine is a nonselective agonist of all adrenergic receptors. Agonism of α1 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. Agonism of β1 receptors activates the G protein coupled receptor (GPCR) Gs pathway. Gsα 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. Compared to group 4’s data which recorded an inotrophic increase of 182% and chronotrophic increase of 39%, the observed responses from group 3 matched expected responses as well as group 4’s responses.

Norepinephrine 1E-5 M elicited an inotrophic increase of 33% and no change in chrontrophy. Chronotrophy and inotrophy responses should have both increased as seen in group 4’s data which recorded an inotrophic increase of 80% and chronotrophic increase of 18%. Group 3’s inotrophic increase of 33% matched with expected responses since NE shares the same mechanism of action as epinephrine in its function to increase force and rate of contraction. The lack of chronotrophic response did not match expected responses. The main target of NE are the α1 and β1 receptors and in the SA node and the increase in pace was most likely not transduced to the rest of the atria tissue due to poor communication between the AV node and non-pacemaker cells as a result of the hole.

Isoproterenol 4E-6 M elicited an inotrophic decrease of 13% and chronotrophic increase of 6% Chronotrophy and inotrophy responses should have both increased as seen in group 4’s

data which recorded an inotrophic increase of 55% and chronotrophic increase of 33%. ISO is another catecholamine which functions as an agonist of all adrenergic receptors but has a higher affinity for β than α. ISO shares the same mechanism of action as epinephrine and NE in its function to increase force and rate of contractions. ISO is known to have the highest affinity for the β1 receptor out of the three catecholamines (5) and was expected to elicit the greatest chronotrophic and inotrophic effect. The decrease in inotrophy and small increase in chronotrophy was most likely due to receptor desensitization from the previous epinephrine 1E-5 M and NE 1E-5 M doses and poor communication between the AV node and non-pacemaker cells as a result of the hole.

Propanolol 1E-6 M elicited no change in inotrophy and chronotrophy. Group 4’s data recorded no change in inotrophy and chronotrophy as well. This was expected because propanolol is a competitive antagonist of β receptors, and antagonists are unable to elicit biological responses.The following dose of Epinephrine 1E-5 M elicited an inotrophic decrease of 13% and no chronotrophic change. One would expect to see a slight increase in heart rate and force of contractions; however, group 4’s data also recorded an inotrophic decrease of 7% and no chronotrophic change. The lack of a positive inotrophic response was most likely due to a combination of receptor desensitization from the previous catecholamine doses and the antagonistic effect of propanolol. Other studies have discovered that desensitization of the heart to isoproterenol was associated with a reduction in both sensitivity and maximal response of adenylate cyclase to activation by isoproterenol (7). It was also possible that the increase in pace was most likely not transduced to the rest of the atria tissue due to poor communication between the AV node and non-pacemaker cells as a result of the hole.

Another dose of propanolol 1E-6 M elicited no change in inotrophy and chronotrophy again, as expected. Norepinephrine 1E-5 M dose caused no change in inotrophy and a chronotrophic decrease of 17%. Chronotrophy and inotrophy responses should have both increased as seen in group 4’s data which recorded an inotrophic increase of 25% and chronotrophic increase of 4%. The lack of a positive inotrophic response was most likely due to a combination of receptor desensitization from the previous catecholamine doses and the antagonistic effect of propanolol. It was also possible that the increase in pace was most likely not transduced to the rest of the atria tissue due to poor communication between the AV node and non-pacemaker cells as a result of the hole.

The final dose of propanolol 1E-6 M was administered and elicited no change in inotrophy and chronotrophy as expected. A dose of Isoproterenol 4E-6 M caused an inotrophic increase of 14% and chronotrophic increase of 10%. Chronotrophy and inotrophy responses should have both increased the most in comparison to the previous propanolol and agonist dose cycles. Group 4’s data recorded an inotrophic increase of 117% and chronotrophic increase of 18%. The responses recorded from group 3 agreed with the predicted response as well as group 4’s responses.

The first dose of phenoxybenzamine 2E-7 M caused an inotrophic decrease of 17%. Group 4 recorded a 10% decrease which is fairly similar to group 3’s data. There should have been little decrease to no change for inotrophy. The decreases may be attributed to antagonism of catecholamines released from sympathetic nerves at the atrial muscle. The following dose of epinephrine 1E-5 M caused no change in inotrophy and a chronotrophic decrease of 20%. Chronotrophy and inotrophy responses should have both increased. Group 4’s data, recorded an inotrophic increase of 30% and a chronotrophic decrease of 4%. The lack of a positive inotrophic response in group 3 was most likely due to a combination of receptor desensitization from the

previous catecholamine doses and the antagonistic effect of phenoxybenzamine. The negative chronotropic response seen in group 3 and group 4’s data may be attributed to β receptor desensitization in the pacemaker cell. For group 3, it is possible that the increase in pace was most likely not transduced to the rest of the atria tissue due to poor communication between the AV node and non-pacemaker cells as a result of the hole.

The second dose of phenoxybenzamine 2E-7 M caused an inotrophic decrease of 14%. Group 4 recorded an inotrophic decrease of 29%. There should have been little decrease to no change for inotrophy. The decreases may be attributed to antagonism of catecholamines released from sympathetic nerves at the atrial muscle. A dose of norepinephrine 1E-5 M caused an inotrophic decrease of 25% and chronotrophic decrease of 4%. Chronotrophy and inotrophy responses should have both increased. Group 4’s data, recorded an inotrophic increase of 29% and a chronotrophic decrease of 4%. The lack of a positive inotrophic response in group 3 was most likely due to a combination of receptor desensitization from the previous catecholamine doses and the antagonistic effect of phenoxybenzamine. The negative chronotropic response seen in group 3 and group 4’s data may be attributed to β receptor desensitization in the pacemaker cell. For group 3, it is possible that the increase in pace was most likely not transduced to the rest of the atria tissue due to poor communication between the AV node and non-pacemaker cells as a result of the hole.

The third dose of phenoxybenzamine 2E-7 M caused an inotrophic decrease of 17%. Group 4 recorded no inotrophic change. There should have been little decrease to no change for inotrophy. The decrease in inotrophy in group 3 may be attributed to antagonism of catecholamines released from sympathetic nerves at the atrial muscle. A dose of isoproterenol 4E-6 M caused an inotrophic increase of 50% and chronotrophic increase of 50%. %. Chronotrophy and inotrophy responses should have both increased. Group 4’s data, recorded an inotrophic increase of 240% and a chronotrophic increase of 13%. The responses recorded from group 3 agreed with the predicted responses.

The final drug dosed in this experiment was verapamil 1E-6 M. Verapamil is an L-type Calcium ion channel blocker that slows atrioventricular conduction, by prolonging the effective refractory period in the AV node. This decrease in conduction velocity leads to a decrease in heart rate (2). Verapamil 1E-6 M caused an inotrophic decrease of 29% and no change in chronotrophy. Group 4’s data recorded no change in inotrophy and chronotrophy. If enough of the ventricles were removed, there should have been no effect on chronotrophy and inotrophy. The decrease of inotrophy in group 3 may have been due to the depression of atrial fibers (5). As expected, there was no change in chronotrophy in group 3 and group 4.

In conclusion, the data obtained from group 3 did not provide a clear correlation between the biological responses that should have been elicited by different drugs targeted at either the sympathetic or parasympathetic nervous system. Group 4’s data was more useful in verifying predicted responses of the different drugs affecting receptors within the auricles of a guinea pig heart. A more careful approach at preparing the tissue would have prevented the source of error for group 3.

References:1. Elsevier (2011). Rang and Dale’s Pharmacology 7e. Retrieved October 31, 2012,

from:www.studentconsult.com2. Woolsey J et al. (January 2006). “DrugBank: A Comprehensive Resource for in

silico Drug Discovery and Exploration”. Retrieved October 31, 2012,from: Nucleic Acids Jan, 34 (Database issue):D668-72. http://drugbank.ca/

3. The Franklin Institute (2011). “Structure of the Human Heart.” Retrieved October31, 2012, from: http://www.fi.edu/learn/heart/structure/structure.html

4. Lamba, Sumant and Abraham, William T (2000). “Alteration in Adrenergic Receptor Signaling in Heart Failure.” Retrieved October 31, 2012, from: Heart Failure Reviews, 5, 7-16.

5. Lecture slides6. B. Ringdahl (1986). “Dissociation constants and relative efficacies of acetylcholine, (+)-

and (-)-methacholine at muscarinic receptors in the guinea-pig ileum”. Retrieved November 4, 2012, from: Br J Pharmacol. ; 89(1): 7–13.

7. Hoffman and Lefkowitz (1982). “Cardiac adrenergic receptors.” Retrieved November 4, 2012, from: Annu. Rev. Physiol. 1982.44:475-482.

8. Basics of Cardiac Arrythmias. Molson Medical Informatics project. 03 January, 2000 Retrieved November 4, 2012 <http://sprojects.mmi.mcgill.ca/cardiophysio/AnatomySAnode.htm>

9. The children’s heart institute. Abdallah, Hassan. Retrieved November 4, 2012 <http://www.childrensheartinstitute.org/educate/heartwrk/elechhse.htm>