To study the effects of K+, Ca2+, acetylcholine and adrenaline on frog’s heart

Heart is supplied by autonomic nervous system. Adrenaline acts as agonist. It acts on β-receptors and increases heart rate and amplitude. Acetylcholine acts on muscarinic receptors as an agonist and decreases the heart rate and amplitude. Excess concentration of KCl stops the heart beat during diastolic phase. Ca2+ excess concentration stops the heart beat during systolic phase. K+ and Ca2+act on cardiac muscle through non-receptor mechanism of action.

Requirements

Equipment and other items required.

Kymograph, strling heart lever, L-stand T-rod, X-blocks, Syme’s cannula, screw clip, mariotte bottle, rubber tubes, tuberculine syringe, 26 no. needle, surgical instrument box.

Animal required

Frog.

Physiological salt solutions required

Frog ringer solution.

Drug solution required

1. Adrenaline hydrochloride 10µg/ml in distill water.
2. Acetylcholine hydrochloride 10µg/ml in distill water.
3. Potassium chloride 4% in distill water.
4. Calcium chloride 4% in distill water.

Procedure

1. Set up the assembly for above mentioned experiment.
2. Pith the frog by passing the needle through the occipito-atlantic junction between the brain and spinal cord. The stretching out of limbs indicates that the pithing is proper.
3. Place the frog in a tray with ventral side facing up.
4. Make an incision on the skin longitudinally and the expose the rectus abdominal muscle.
5. Make an incision around the rectus muscle without damaging the anterior abdominal vein.
6. Expose the heart after cutting the sternum.
7. Remove the pericardial membrane.
8. Tie one side of the aorta.
9. Put a not around the inferior vena cava then make a small cut for cannulation.
10. After cannulation with Syme’s cannula, cut the other side of aorta and isolate the heart from the body and perfuse with frog ringer solution. Adjust the flow of the frog ringer solution through the horizontal arm of the syme’s cannula.
11. Place a heart-clip on the apex of the heart and connect it to a staling lever.
12. Record the normal heart beat on the paper of the drum.
13. Inject 0.05-0.1ml of adrenaline solution into Syme’s cannula. Immediately switch on the kymograph and record the effect of adrenaline for 2 minutes period. After 2 minutes switch off the kymograph till the heat beat and amplitude comes to the normal. Observe the onset and duration of action of adrenaline.
14. Inject 0.05-0.1ml of acetylcholine solution into Syme’s cannula. Immediately switch on the kymograph and record the effect of acetylcholine for 2 minutes period. After 2 minutes switch off the kymograph till the heat beat and amplitude comes to the normal. Observe the onset and duration of action of acetylcholine.
15. Inject the 0.1 ml KCl solution into Syme’s cannula. Immediately switch on the kymograph and record the effect of KCl for 2 minutes period. After 2 minutes switch off the kymograph till the heat beat and amplitude comes to the normal. Observe the onset and duration of action of KCl.
16. Inject the 0.1 to 0.4 ml CaCl2 solution into Syme’s cannula. Immediately switch on the kymograph and record the effect of CaCl2 for 3 minutes period. After 3 minutes switch off the kymograph till the heat beat and amplitude comes to the normal. Observe the onset and duration of action of CaCl2.

Procedure

Observation and conclusion

1. Adrenaline  increases  the  heart  rate  and  amplitude.  Heart  contains  β-receptors.
2. Adrenaline stimulates β-receptors and increases the heart rate and amplitude. Based upon this experimental observation, adrenaline 1 in 10,000 solution is recommended in a dose of 10 ml by i.v. infusion for cardiac arrest. Drugs which block β-receptors (propranolol, atenolol etc) are clinically used in the hypertension and tachycardia.
3. Acetylcholine decreases the heart rate and amplitude. This effect is similar to the effect produced by vagus nerve stimulation. This effect is mediated through muscarinic receptors. Hence, muscarinic blockers (atropine, belladonna extract) are used to reduce vagal tone and muscarinic actions.
4. Potassium excess concentration decreases the heart rate and amplitude.
5. Calcium concentration in excess stop the heart beat during systolic phase, an effect similar to digoxin poisoning. Therefore, EDTA (ethylene diamine tetra-aceticacid) is used in digitalis poisoning. Calcium channel blockers like verapamil, diltiazem, amlodipine etc. are used as antihypertensive agents.