The Use Of Histamine In Hypersensitivity Biology Essay

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The aim of the experiments was to investigate the drug/receptor occupancy theory and also show that the similar effects of histamine could be produced by carbachol through acetylcholine receptors after inhibition of histamine receptors. The selection of the ileum was advantageous because the tissue could be used over and over before damage and also the abundance of histamine in the gut (Ganellin and Parsons, 2006). The choice of antagonist was influenced by the generalised view that the histamine inducing mediators were of the H1 type (Zseli, Zappia, Molina and Bertaccini, 1979) who cited the earlier works of Ash and Schild (1966). Further works by Hill et al. in 1977 cited by Zseli et al. (1979) showed 3H-mepyramine as an antagonist at histamine receptors.

The drugs used were mepyramine (a histamine antagonist), carbachol (a muscarinic agonist). Bovine serum albumen (BSA) and egg albumen were introduced in the second part of the investigation to provide a challenge for the mast cells in the sensitisation.

For both parts of the experiment, the tissue setup was the same. A piece of ileum, approximately 3cm long was arranged in a bath containing Tyrode's solution with oxygen bubbling through. The ileum was tied on both ends using reef notes and one end was tied to a transducer arm to record contraction responses and the other end was held firmly by the tissue holder ensuring it was completely immersed in the solution. The bath containg the tissue was in a water bath set at 35°C.

For the first part, dilutions of 10-3M to 10-6M histamine were made from a stock of 10-2M and 10-4M to 10-6M of mepyramine. Responses to volumes of 0.1ml and 0.3ml histamine starting with the lowest concentration were obtained, whilst adhering to etiquette of washing out the tissue before the next volume or concentration. Two washes were carried out, 15 seconds apart. The recorder arm was started 80 seconds prior to the next addition.

Once the maximum response had been obtained from the histamine from the trace, the mepyramine was added to a bath containing twice the amount of histamine that produced half the response on its own and labelled 2x and the addition stopped once the response caused in the presence of the mepyramine was less than that of x. A concentration/antagonist graph was plotted in order to work out the pA2 value, which would give an indication of how strong an antagonist the mepyramine was.

The second part was carried out in three stages: one control and two sensitised experiments. Dilutions of 10-4M histamine, 10-4M mepyramine and 10-4M carbachol were made.

Control

Graded responses to 0.3ml, 0.6ml and 0.9ml of histamine were obtained, followed by an addition of 0.2ml of mepyramine then 0.4ml of histamine. Finally, 0.4ml of carbachol was added, and the response noted.

Sensitised 1

0.2ml, 0.4ml and 0.6ml of histamine and carbachol were used to obtain graded responses after washing the tissue from the control experiment. 1ml if the egg albumen was added followed by 0.4ml of histamine. This was to see if the egg would prevent a contraction. 0.2ml of mepyramine was added and 30 seconds later 0.4ml of histamine. Finally 0.4ml of carbachol was added. All the responses were recorded on the trace for later analysis.

Sensitised 2

Once more, graded responses to histamine were obtained from the washed tissue. 0.2ml of mepyramine was added followed by 0.4ml of histamine 30 seconds later. 1ml of egg albumen was added after 0.2ml of mepyramine was added and finally 0.4m of carbachol was added.

RESULTS

The contraction responses were read of the trace diagram by counting the number of squares to the maximum point before line levelled off.

Figure 1- Concentration/ % response (see graph attached)

Table 1- Responses to Histamine and Mepyramine to determine pA2 value

 

Concn. (M)

Vol. Added (ml)

bath concn (M)

log bath concn 

response (mm)

response %

10(-6)

0.1

0

0

0

0

 

10(-6)

0.3

0

0

0

0

 

10(-5)

0.1

0

0

0

0

histamine

10(-5)

0.3

0

0

0

0

 

10(-4)

0.1

2 x 10(-7)

-6.7

0

0

 

10(-4)

0.3

6 x 10(-7)

-6.2

2

14

 

10(-3)

0.1

2 x 10(-6)

-5.7

7

50

 

10(-3)

0.3

6 x 10(-6)

-5.2

14

100

 

 

0.1

2 x 10(-9)

-8.7

15

100.0

 

0.2

4 x 10(-9)

-8.4

13

86.7

mepyramine

10(-6)

0.3

6 x 10(-9)

-8.2

11

73.3

and 2x

0.4

8 x 10(-9)

-8

10

66.7

 

0.5

10 x 10(-9)

-8

9

60.0

 

 

0.6

12 x 10(-9)

-7.9

8

53.3

The value for x was chosen at half the maximum response, which was 0.1ml. this was doubled to have 2x.

Trace diagrams - attached

Trace diagrams for various responses of ileum (not to scale, for illustration purposes only)

Control responses

"page 1" Graded responses to histamine (used 0.9ml l0-4M and 0.3 and 0.6ml of l0-3M)

"page 2" Response to 0.4ml of 10-4M carbachol

Response 1ml of the bovine serum albumen solution

Response 1ml of the egg albumen solution

0.2ml of l0-4M mepyramine followed after 30sec by 0.4 ml of l0-4M histamine

Response to 0.4 ml 10-4M carbachol

"Page 1" shows the graded responses to histamine

"Page 2" shows the responses when mepyramine and carbachol were added

Sensitised Experiment 1 (note the trace goes the opposite way to normal (i.e. down =response)

Histamine (0.2 and 0.4 ml of l0-4M)

0.4ml of 10-4M Carbachol

1ml of the bovine serum albumen solution

1ml of the egg albumen solution

Part B

1ml of the egg albumen solution without washing add 0.4 ml of l0-4M histamine (the peak is the response to histamine not the 2nd application of egg albumen)

0.2ml of l0-4M mepyramine followed after 30sec by 0.4 ml of l0-4M histamine (missing)

0.4 ml of 10-4M carbachol

Part B

Other groups results (for missing data on mepyramine and histamine)

Histamine (0.2 and 0.4 ml of l0-4M)

0.4ml of 10-4M Carbachol (missing)

1ml of the bovine serum albumen solution (missing)

1ml of the egg albumen solution

1ml of the egg albumen solution without washing add 0.4 ml of l0-4M histamine (the peak is the response to histamine not the 2nd application of egg albumen)

0.2ml of l0-4M mepyramine followed after 30sec by 0.4 ml of l0-4M histamine (the bit missing in the data set above)

0.4 ml of 10-4M carbachol

Sensitised Experiment 2

Part A Histamine (0.2, 0.4 and 0.3 ml of l0-4M)

Part B 0.8 ml of 10-4M Carbachol

1ml of the bovine serum albumen solution

0.2ml of 10 -4M mepyramine followed after 30sec by 1ml of egg albumen

Part C 0.2ml of 10-4M mepyramine followed after 30sec by 0.4 ml of 10-4M histamine

0.4 ml of l0-4M carbachol

Part A

Response stops here

Part B

Part C (below)

Response in the presence of histamine, mepyramine and carbachol.

Only carbachol produced a response.

DISCUSSION

From the first experiment, it was seen that the response gradually reduced as the concentration of the mepyramine increased (Figure 1) suggesting that the tissue was obeying the drug/receptor occupancy theory where mepyramine was acting on H1 histamine receptors (Zseli et al. 1979). The relatively high pA2 value was an indication of how strong the antagonism was since the concentration of mepyramine at 10-6M was lower than that of the histamine at 10-3M.

The control experiment demonstrated that histamine produced a response in the absence of its antagonist mepyramine and so did the carbachol even when the mepyramine was present. This could have been due to existence of other receptors which responded to the carbachol which Foster (1991) suggests could be the same as those used by acetylcholine, since both are muscarinic agonists.

The first sensitised experiment further supported the hypothesis that the response to carbachol could have been caused by receptors of a different nature because the histamine would not cause a contraction in the presence of mepyramine whereas the carbachol did. Upon addition of the BSA and the egg albumen, there were still contractions from histamine which were stopped by addition of mepyramine (from trace c in experiment 2) yet again showing that mepyramine was very competitive.

CONCLUSION

Carbachol acts through acetylcholine receptors and mepyramine is a very active antagonist which even at low concentration will have an inhibitory effect on the histamine receptors in the ileum.

REFERENCES AND ACKNOWLEDGEMENT

Many thanks to N0169942 for brilliant teamwork.