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Drug Receptor Occupation Theory Experiment

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.

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.

Effect of Temperature on Plant Physiology | Experiment

The physiological processes of many organisms are sensitive to temperature. In order to see this effect of temperature, we examined the heart rate of a Daphnia magna over a range of different temperatures. Being an ectothermic animal, the Daphnia’s body temperature is dependent on water temperature. It was hypothesized that since most physiological processes are faster at higher temperatures, the Daphnia’s heart rate will be faster at higher temperatures and slower at low temperatures. This was, in fact, true and a pattern was evident which showed that heart rate increased as temperature increased. The Q10 was high at higher temperatures which show elevated sensitivity at higher temperatures. Clearly, Daphnia have an optimal temperature range outside which they do not function to their full potential. A Daphnia’s heart rate, then, was proved to be dependent on temperature.
Introduction Daphnia magna is a widespread freshwater zooplankton. Since Daphnia are ectothermic animals, their body temperature fluctuates with environmental temperature. Hence, these animals are ideal to study the effects of temperature. Most such animals function well at certain specific temperatures. They have an optimal temperature range, outside which they are unable to perform physiological processes effectively (Lamkemeyer et al. 2003). It is believed that most physiological processes take place more rapidly at higher temperatures and that changes in temperature can influence physiological rates (Ziarek et al. 2010). In order to investigate this, we questioned whether the heart rate of a Daphnia is different at different temperatures. Q10, which is the temperature sensitivity of a reaction, was a useful tool. We hypothesized that the Daphnia will have different heart rates at different temperatures and hence that temperature will affect heart rate. It was also hypothesized that Q10 will differ at different temperatures. This hypothesis was tested by exposing the Daphnia to different water temperatures, letting it equilibrate to the water temperature and counting its heart beat in a systematic way. Since most physiological processes increase at higher temperatures, we predicted that if the temperature is higher (close to 35°C) then the heart rate of the Daphnia will be faster and if the temperature is low (close to 5°C) then it would be slower. In addition, we predicted that Q10 will be higher at low temperatures and lower at high temperatures. In view of the fact that Daphnia had an optimal temperature range, it would be understandable if the Daphnia was more sensitive to temperatures outside this range and consequently reacted by altering its heart rate.
Methods A Daphnia was placed on a small smear of Vaseline on the bottom of a culture dish (Olaveson and Rush 2011). Aged water at room temperature was added to the dish. Five minutes were allowed for the Daphnia to adjust to the water temperature and the temperature of the water was measured and recorded. Under a dissecting microscope, the Daphnia was placed and the 4X lens were used to locate the heart and count the heartbeats. The number of beats was counted over a 10 second period which was followed by a 10 second pause in counting and then 10 seconds of counting again. In order to get 9 measurements of the heart rate, this pattern was repeated for 3 minutes. Then, ice and water were mixed in a beaker to make a water mixture between 5°C to 10°C. To replace the tap water in the culture dish with chilled water, a Pasteur pipette was used. Five minutes were allowed for the Daphnia to reach equilibrium and then the heart beat was counted to obtain 9 measures of heart rate (heartbeats/ 10 seconds). The values were recorded. The temperature was then increased in 5°C increments till 35°C and heart rate was measured at each point. Small amounts of the colder water were replaced with the warmer water (obtained from a water bath) till the desired temperature had been reached. Five minutes were always allowed for equilibration and using the same method, 9 measures of heart rate were recorded. The 9 estimates of heart rate taken at each temperature were used to find the average heart rate at each temperature. These values were entered into an excel document by all students and later used for analysis.
Results Statistical analysis and data processing shed light upon the effect of temperature on the heart rate of a Daphnia. The Statistical t test analysis proved that the Ho could be rejected for all the three tests proving that temperature does have a significant effect on the heart rate of a Daphnia. The Q10 as well as the average heart rates at different temperatures provided evidence that supported the hypothesis that temperature would affect Daphnia heart rate too.
At the temperature interval of 4°C to 14°C, the Q10 was found to be 1.31 (Table 1). Although this was not the highest Q10 value and hence not the most sensitive temperature interval, a decrease in heart rate was evident at the lower temperature of 4°C compared to other higher temperatures (figure 1). The heart rate at 4°C was found to be 106.74 beats per minute where as the heart rate at 14°C was 140.10 beats per minute. The significant decrease in heart rate at 4°C compared to heart rate at the ambient temperature (24°C) was supported by the t test analysis (sample t statistic: 14.3938; critical t statistic:1.978; df:136; p = 0.05). The temperature interval from 14°C to 24°C showed increased sensitivity (Q10:1.40). This indicated the increase in heart rate at 24°C compared to lower temperatures (figure 1) and was supported by the t test analysis as the Ho (hypothesis that no change in heart rate would be evident) was rejected (t statistic: 8.6519; critical t statistic:1.978; df:136; p = 0.05). During the temperature interval from 24°C to 34°C, the highest Q10 was noted (table 1).

This sensitivity to high temperatures was obvious when heart rates at the two temperatures were compared (heart rate at 24°C: 196.32 beats/min; at 34°C: 277.92 beats/ min). The H0 was hence rejected (t statistic: 9.7792; critical t statistic: 1.978; df: 136; p = 0.05).
All the three tests provided evidence that suggested that temperature had an effect on the Daphnia’s heart rate. At higher temperatures, the heart rate was faster and at lower temperatures, it was slower. Generally, as temperature increased so did the Daphnia’s heart rate (figure 1).
Discussion All organisms have an optimum temperature range over which they function best. Consequently, at certain temperatures, the physiological processes of a Daphnia magna are at its utmost potential. Some hypothesized that Daphnia optimize their fitness by allocating the time spent in the different habitats depending on the temperature gradient (Kessler