I am currently studying permeability in the endothelium of vessels. I have read a paper that has shown up regulation of a water channel protein, aquaporin-1, by a hormone arginine vasopressin on trophoblast placenta cells. I hope to achieve upregulation of aquaporin-1 by arginine vasopressin on bovine aortic endothelial cells. In the paper that I am basing my experiment on, placenta cells were grown with RPMI 1640 medium supplimented with 5% FBS & antibiotics on 6 well plates at 100k cells/well for 24 h. Cells were cultured in serum free and hormone deprived RPMI for another 12 h before treatments. Treatments consisted of 0.1nM and 10nM AVP formulations in RPMI medium with 1% FBS. Controls were incubated in medium containing 1% FBS alone.
The results of this paper showed a 5 fold increase in AQP-1 level with 0.1nM AVP.
My experiment is different because I grow my BAEC cells into a monolayer and then measure hydraulic conductivity in a cell chamber to quantify permeability. I have carried out the following experiments to understand starvation's effect on permeability and integrity of the monolayer.
Grow cells plated at 128k on 12 well dish with 10% FBS in MEM. Grow for 3-4 days.
1. 0% FBS in Minimum Essential Media
2. 1% FBS in MEM
3. 0% FBS in Optimem
1. 1% FBS in MEM with 0.1 nM AVP
2. 1% FBS in MEM
I ran 3 pairs, each being starved 1 of 3 ways and then ran treatments to test the effect of AVP. My results were not only much higher then my normal baseline (5.4E-6 cm/s) but also showed a weird trend.
Starvation technique and Hydraulic Conductivity
1. 16.86 E-6 cm/s for both control and treated
2. 17.90 E-6 cm/s control 14.11 E-6 cm/s treated
3. 18.92 E-6 cm/s control 13.10 E-6 cm/s treated
so the AVP decreased the Hydraulic conductivity and overall it looks like the starvation made the junctions very loose. I plan on running a test to see the effect of starvation alone. I would like any ideas that I am not considering to make this experiment work and keep the integrity of the mono layer. I would appreciate any help.
Edited by cbraval, 21 May 2009 - 11:38 AM.