Measurement Of Ip3 In Cultured Cells

1157 words - 5 pages

INTRODUCTION
Histamine which is an agonist induced an accumulation of inositol phosphate (IP3) in cultured cells both in the presence of calcium and in the absence of calcium. This mechanism of cellular response to the histamine receptor s a G-protein mediated activated (GTP) (Hill,1990).
This mechanism occurs when a response to a photon of light called rhodopsin causes a change in the G-protein which is associated. Transduction then occurs which makes the α subunit to replace GDP with a triphosphate GTP, this then separates from the βγ subunits. (Tuteja,2009).
Interaction of α subunit or the βγ complex with some particular isoforms of G-protein could excite phospholipase C (PLC) to ...view middle of the document...

2ml of neutralising solution was added to each vial, 800µl of sample was carefully taken from each well and added to the appropriate vial insert.
The content of the scintillation vial was inserted onto the appropriated matching column and allowed to drain through. The empty scintillation vial inserts was put in a waste bucket.

RESULTS
WELL NUMBER MEAN DPM1(IP3 accumulation) % RESPONSE LOG CONC.(histamine)
1 AND 2 486.035 26 -3
3 AND 4 1792.055 100 -4
5 AND 6 1069.510 60 -5
7 AND 8 788.220 44 -6
9 AND 10 385.480 22 -7
11 AND 12 336.775 19 -8
13 AND 14 234.774 78 -3
15 AND 16 134.421 100 -4
17 AND 18 60.896 81 -5
19 AND 20 43.367 73 -6
21 AND 22 139.051 50 -7
23 AND 24 4.533 37 -8

TABLE 1 shows the mean accumulation of IP3, percentage response ad log concentration of histamine of cells in 24 wells with 1-12 have calcium and 13-24 calcium free.
From the Table above, there is a positively significant difference in the mean IP3 accumulation between the experiment with calcium and the calcium free experiments.
The biggest difference can be seen at the highest log concentration of 10-3 Histamine, where mean DPM1 change for well with calcium is 1792.05 against that without calcium of 134.42. At concentration of 10-4, there is a difference of 259.54 in IP3 accumulation. As we look through the table there is significant difference in IP3 accumulation where calcium is absent and present

Graph A is a representation of the percentage response and histamine log concentration, with the sigmoidal curves indicating the experimental values with calcium and without calcium. There is a clear significant difference where the calcium free curve is lower down the graph.
Extrapolating the EC50. EC50 is a concentration of a drug that gives half maximum response.
EC50 of experiment with calcium is 10-6M.
EC50 of experiment without calcium is 10-4.5M.

Graph B illustrates the mean IP3 accumulation against histamine log concentration, where there is also a clear difference in the curves. This graph shows higher IP3 accumulation in presence of calcium as opposed to without calcium.
DISCUSSION
In this experiment, the histamine is the agonist and the G- Protein coupled receptors act to stimulate the production of IP3 in the presence and absence of calcium. According to the results obtained, there is a significant difference in the EC50 values of histamine in the presence and...

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