Study of dose response curve of acetylcholine on frog’s rectus abdominis muscle

BACKGROUND

Dose response curves (DRC) demonstrate graded responses to drugs or agonists where an increase in response is recorded with a subsequent increase in the dose or the drug concentration. The DRC is sigmoid or S-shaped.

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The first portion (25% of graph) of that curve has poor discrimination between the doses whereas the middle part of the curve shows greater sensitivity to different concentrations, and the responses of increasing drug concentrations are linearly differentiated. The last portion of the curve (plateau) shows the ceiling effect where no more increase in the response is seen with further increase in the dose.

Sometimes cumulative DRCs are employed for the study. It is achieved by increasing the drug concentration in the organ bath without washing the preceding doses. This technique is simple and less time consuming. It is generally employed in those preparations where contractions of the tissue and the relaxations are relatively slow. However, this method is not suited for drugs showing faded phenomenon.¹

When doses of the drugs are increased by geometric progression (logarithmic intervals) and the response is plotted against log doses, the relationship is called log-dose response curve.

The logarithmic transformation offers some advantages:

1. The linear section of sigmoid curve becomes straight.
2. Comparison of two DRCs much simpler.
3. Large dose ranges can be plotted which is otherwise difficult in DRCs.
4. The error is distributed all through the graph, independent of doses.

The analysis of DRC indicates:

1. Relative potential of the agonist, when the curve is more towards the left, it indicates that the drug is more effective. The vice versa is also true.
2. The slope of the response curve points out the error and reliability (precision) of the bioassay. Steeper the slope more precision is the assay and the vice versa is also true.

Rectus abdominis muscle of the frog is the voluntary skeletal muscle that produces a slower contraction in response to the drug, Acetylcholine (ACh). Isolated rectus abdominis preparation is an isolated preparation of tissue which is widely used for nicotinic effect study of ACh as it operates on nicotinic ACh mechanism at neuro muscular junctions.²

Cholinergic drug like Ach stimulates the nicotinic receptors and produces skeletal musclecontraction. The response of these drugs on nicotinic receptors is blocked by neuromuscular blocking drug.

Concentration of an agonist that produces 50% of its maximal effect is known as EC₅₀. pD₂ is the negative logarithm to base 10 of EC₅₀ of an agonist.³ It points to the affinity or the strength of the agonist to bind to the receptor.

Aim of this experimental work is to record the DRC of Acetylcholine by using rectus abdominis muscle preparation of frog.

REQUIREMENTS

Animal:                             Frog

Physiological solution:     Frog ringer solution

Chemicals:                        Acetylcholine in different concentrations (1:10000 i.e. 100 µg/ml), Fixing solution

Instruments:                     Kymograph, Student organ bath, Aerator, Insulin syringe to inject drugs in small fractions, Dissecting board and various dissecting instruments, Simple straw lever and stand, Pipette, Stop watch

Miscellaneous:                  Kymograph paper, plasticin, clips, and thread.

PROCEDURE FOR DOSE RESPONSE CURVE

Dissection procedure

Pith the frog and lay out on the frog dissection board. Cut the skin of the wall of anterior abdominal by a midline incision which is prolonged laterally onto the frontal aspects of the limbs. This shows the flat whitish muscle of the anterior abdominal wall from the forg’s pubic origin to sternal insertion.

Place the cuttings separately on each rectus abdominis from the nearby muscle and from its fellow.Remove the recti and place in Frog’s Ringer solution in a dish with continuous aeration. Clean and trim one of thecutting muscle to the preferred size and mount the tissue in a student’s organ bath into Frog’s Ringers solution at room temp and aerated with oxygen.

An organ tube capacity of 5 ml is sufficient but in this case solution of drugs must be so made that when a dose is added, a change of volume should not occur more than 15% in ringer solution. For recording purpose, use an isotonic lever with a sideway writing point which will be tangential to the smoked drum and also be balanced with a tension of 2.5 gm with an extra load of 1 gm on the long arm.

Later it serves to bring back the lever towards baseline since the muscle will be contracted, usually it does not relax quickly even on washing out of the drug.

The muscle should be kept for relaxation for 30-45 min by applying load using plasticin (as mention above) on the long side arm of the lever that must be equidistant from fulcrum even after loading new, the lever is balanced.

Experimental procedure

Take the base-line at a distance of approximately 2-3 cms lower from one third of the drum. Stop the drum. Then again start the drum and add 0.1 ml of drug solution then record the response of the drug for 90 sec. switch off the drum and start washing the muscle tissue with Frog’s ringer solution 2-3 times. There make a gap of least 30 sec to 60 sec, between each washing (total period 6-7 min).

Record the responses at increasing doses (0.1, 0.2, 0.4, 0.8, 1 and 1.2 ml) of Ach or till the maximum response. The maximum response is achieved if one gets the same or slightly less response with higher concentration. This is known as ceiling effect of the drug and the dose is called ceiling dose.

Properly label the graph, write the name, date and fix the tracing with the help of fixing solution. Measure the height of the drug response in mm and draw a dose response graph by taking log dose on X-axis and % response on Y-axis.

Describe the shape and various parts of the graph.

Precautions

1. The level of ringer solution of the organ bath should remain constant throughout the experiment by marking the level with elastic ring/marker.
2. The thread of the rectus muscle should not come in contact with the wall of the organ tube.
3. A steady and constant stream of air/O₂ should be passed in the bath to avoid tissue destruction.
4. 6-7 min cycle should be followed rigidly
5. Drum speed should be slow and the lever should be placed tangential to the drum.
6. The no. of washing and the interval between them should be kept constant.
7. If the desired response is not obtained with 0.1 ml of ACh, then the dose should be increased.

Observation table.

Sl.no	Dose (ug/ml)	Response (mm)	Log dose	Percent response (%)
–	–	–	–	–

CONCLUSION

Nicotinic receptors are present in skeletal muscles, where, ACh produces contraction of the muscle through the stimulation of nicotinic receptors.

REFERENCES

1. Kulkarni SK. Practical pharmacology and Clinical pharmacy. 1^st ed. Vallabh Prakashan, New Delhi; 2009:99-102.
2. Ronald J. T & Leonard S.J. The dose response relation in pharmacology. 1^st ed. Springer-verlag, New York; 1979: 163.
3. Neubig RR, Spedding M, Kenakin T, Christopoulos A. International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. XXXVIII. Update on Terms and Symbols in Quantitative Pharmacology. Pharmacol Rev. 2003;55:597–606.

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