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 hydrolyse the membrane phospholipid into diglycerol (DAG) and IP3. Both IP3 and DAG are secondary messengers, DAG activates protein kinase C (PKC), this in turn acts as a catalyst in phosphorylation of certain transcription factors and enzymes, thereby facilitating or inhibiting them.(smyth et.al,1985).
IP3 is released into the cytosol where it binds specific receptors in the membrane of the endoplasmic reticulum (ER) which causes stored calcium to be released.

METHODS
To carry out this experiment, two stock solutions of 10mM histamine one containing 2mM of CaCl2 ad the other 0.1M of EGTA. Using serial dilutions of 1 in 10 to make up concentrations of histamine from concentrations 10-4 to 10-8 .For the 24 well cultured plates provided.
The drugs were added to the cells in the wells at 900µl of Calcium containing HHB in wells 1-12 and 900µl of calcium free HHB in wells 13-24. The plate was incubated for 30minutes at 37 degrees Celsius and then 100µl of histamine was added to each well, a further incubation period of 40minutes at the same temperature was observed. After the period of incubation the reaction was stopped by adding 900µl of termination mix.

After a week of having the cells in the freezer, the samples were prepared for column chromatography this was done by setting up a rack containing 24 scintillation vial inserts numbered 1-24, 4.2ml 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 absence of calcium respectively. Also, from the mean accumulation of IP3, there is a clear increase in IP3 production in the presence of calcium.
This suggests that calcium might be significant in the accumulation of IP3. This could be by stimulating PLC (phospholipase C) to produce IP3 and not just histamine (Horowitz, et.al).
A study of PLC in living cells in terms of activation, inhibition, calcium requirement and regulation suggest that calcium is necessary for PLC activation. PLC catalyses the hydrolysis of PIP2 to IP3 and DAG.
PLC can be activated by G-Protein coupled receptors which in this case is the histamine receptors. Stimulation of GPCR in cells usually leads to intracellular calcium being released, this is mediated by IP3 receptors. This action can be categorised into two phases. The first phase is when calcium is released from the endoplasmic reticulum via the IP3 receptors and the second phase is the depletion of stored calcium which results from IP3 receptor activation signal influx of extracellular calcium. This is done through membrane calcium channels known as store-operated calcium channel entry (SOCE) (MONTANO, et.al 2015). SOCE is reversible process that terminates in response to calcium store refilling. A recent study has shown that during cell division, Store-Operated calcium channel (SOCE) depletion is suppressed due to STMIM1 phosphorylation, upregulation and rearrangement into near plasma membrane. SOCE is important for maintaining adequate stores that facilitate the calcium oscilltions and also calcium entry through plasma membrane channels which can provide calcium signals that specifically couple to downstream effector pathways (SMYTH, et.al 2010).
Stoare-Operated calcium channel entry also shows that downward signalling pathways will be active except when calcium signals are generated through high concentrations of agonist; in this case Histamine which induces calcium store depletion or a low concentration of histamine which causes calcium release. A recently published article by Di Capite ET AL suggests that SOCE is the primary source of signalling calcium.
Referring back to the results, IP3 accumulation could have been higher in the presence of calcium due to the aforementioned mechanism of PLC activation and SOCE. Calcium stimulating PLC leads to a further hydrolysis of IP3 and DAG as opposed to where the cells are absent of calcium (Aquiler et.al 1986).