Inhibitor Experiment 2
The procedure for the second inhibitor experiment was the same as for the first one, however the inhibitors themselves were different. In this second experiment, the inhibitors used were 3 x 10-6 M Xestospongin C, 10-5 M Nifedipine, and 1 mM EGTA.
Results
Time Course Experiment
The experiment was undertaken in order to determine whether GHS functions via the cAMP secondary messenger system. There were eight samples tested for each of the GH-producers – four to measure the cAMP levels and four to measure the GH levels. The averages and standard deviations were then calculated to be used in calculations and graphs. The results of Fig 1 depict the rapid cAMP growth period of the GHRH between the 5 and 20 minute marks. The GHS did not show any significant cAMP growth at any time. The results of Fig 2 show the increase in GH production of both the GHRH and GHS over the time period. Both produced the hormone at approximately the same rate, with an increase in production between the 5 and 20 minute marks. (Therefore, the GHS is not related to the cAMP)
Inhibitor Experiment 1
This experiment was performed in order to determine which inhibitor(s) had the greatest effect on the GH-production of each stimulator. The negative control for this experiment was using no treatment on the cell cultures to measure the level of GH-production with no stimulator present. The positive control was to using each stimulator by itself, in order to measure the GH-production of the cells under optimal conditions. As shown in figure 3, the H89 inhibitor had the greatest influence on GH-production for the GHRH stimulator, with a 418.75 ng/ml difference between the positive control and the inhibitor. There was however, a significantly higher production of GH with the H89 than with the negative control. Figure 3 also demonstrated that the LY333531 had the greatest effect on the GHS GH-production, with a difference of 355.5 ng/ml between the positive control and the inhibitor. Again though, compared to the negative control the LY333531 still produced significantly more GH. In each case, the other inhibitors had no significant effect on the production of GH compared to the positive control.
Inhibitor Experiment 2
The purpose of this experiment was essentially the same as the previous one: to determine which inhibitor(s) had the biggest effect on the production of GH in each of the two stimulators (using different inhibitors, of course). The positive and negative controls were also the same as the previous experiment, namely the presence or absence of the stimulators alone. As shown in figure 4, the solution that was the most successful at inhibiting GH-production for the GHRH was the EGTA, with a difference of 456.75 ng/ml between the inhibitor and the control. The levels of GH production were actually lower than the negative control in this case. The Nifedipine also had a large effect of the GH-production levels of the GHRH, with a difference of 393.75. For the GHS stimulator, figure 4 shows that the Xestospongin C made the biggest difference at 207.75 ng/ml, with the EGTA following at 182 ng/ml difference. The Xestospongin C had no significant effect on the GHRH GH-production, nor did the Nifedipine on the GHS GH-production.
Discussion
The first experiment showed us that while there is definitely a correlation between the levels of cAMP produced and the amount of GH produced for the GHRH stimulator; there is no such correlation for the GHS. In other words, the action mechanism by which the GHS stimulates the pituitary cells to produce GH does not involve cAMP or its secondary messenger system. It therefore must stimulate the cells by some other means.
The first of the inhibitory experiments demonstrated the necessity of the protein kinase A (PKA) in the secondary messenger system of the GHRH. Essentially, the GHRH stimulates the receptors of the pituitary cells, resulting in an increase in the concentration of cAMP. This in turn triggers a rapid increase in the PKA levels, which bind to the cAMP. The rapid activation of this enzyme results in an increase in calcium levels inside the cell, and therefore also of GH-production. When the H89 inhibits the PKA activation, the cAMP does not bind to the enzyme. Calcium therefore does not flow into the cell, and the production of GH is significantly reduced. However, since the production of GH was not completely stopped, the H89 is not a perfect inhibitor. Since neither PKC nor protein synthesis have anything to do with the secondary messenger system of the GHRH, they have no effect on the GH-production. The second inhibitory experiment further demonstrated the need for calcium to be present in the cell to promote GH production. When the two inhibitors used to block the flow of calcium were used, the levels of GH dropped significantly. In this case, however, the EGTA did work perfectly as an inhibitor, allowing less calcium to enter the cell than even the negative control.
In terms of the GHS, the first inhibitory experiment showed us the necessity of the protein kinase C (PKC) in the IP3/DAG pathway system. The second experiment demonstrated the importance of the inositol triphosphate receptors and the calcium in the process. Essentially, the pathway begins with the GHS binding to the receptors of the pituitary cells. The phospholipase C on the plasma membrane of the cell is activated, releasing the IP3 into the cell and the DAG further on the membrane. The IP3 opens the calcium channels in the membrane of the ER, allowing the calcium to flow into the cell. This rise in calcium levels causes the PKC to travel to and bind with the plasma membrane, where it is activated by the DAG. Once it is activated, the PKC can produce the GH. When the LY333513 inhibited the PKC activity of the cell, the DAG could not be activated, and therefore the production of GH was significantly reduced. Neither the H89 nor the Cyclohexamide had an effect on the GHS system as neither protein synthesis nor PKA are required in this pathway. The LY333513 inhibition was not perfect compared to the negative control, however it was more efficient than the PKA inhibition of the GHRH stimulator. The Xestospongin and the EGTA both effected to the production of GH, but not to the extent that the LY333513 did. This is perhaps reflective of the relative importance of calcium and PKC in the process, as well as to the minimal amount of calcium already present in the cytosol.
Lindsay Cornelson
Student# 992363660
Lab section P101A
Writing Project 2:
Scientific Writing
References
Lodish, H., Berk, A., Matsudaira, P., Kaiser, C.A., Kreiger, M., Scott, M., Zipurksy, S. L., Darnell, J. 2003. Molecular Cell Biology 5.0, W.H. Freeman and Company, New York, pp 541, 563
Way, H.M., Kazan, Z., Mitter, N., Goulter, K.C., Birch, R.G., and Manners, J.M. 2002. Constitutive expression of a phenylalanine ammonia-lyase gene from Stylosanthes humilis in transgenic tobacco leads to enhanced disease resistance but impaired plant growth [Electronic Version]. Physiological and Molecular Plant Pathology 60:275-282
