No foskolin->cAMP response in HEK parentals...normal?

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klw
klw's picture
No foskolin->cAMP response in HEK parentals...normal?

Does anyone know the reason why forskolin would NOT stimulate increases in cAMP in HEK parental cells?  Is this expected?  If so, could you explain as I believed that forskolin should increase cAMP by directly stimulating adenylate cyclase (and should not need the presence of a GPCR)?
In recent experiments, I've been working with Gi-coupled GPCRs transfected into HEK cells.  In these experiments, you typically use forksolin to stimulate CAMP and then you use your GPCR agonists to inhibit the cAMP.  So far, those experiments have worked very well.
However, I recently tried to use forskolin in the parental HEK cells (with no GPCR) as a control and I got NO stimulation of cAMP.  I was a little confused by this as I believe forskolin should increase cAMP by directly stimulating adenylate cyclase (and should not need the presence of a GPCR).
I've repeated both of these experiments with the same results so I know it isn't my forskolin stock.  And the cells are identical...only one is transfected with a GPCR and the other is not.
The amount of forskolin I use in the transfected cells (where I DO see an effect) is anywhere between 0.1 uM and 10 uM.  The amount of forskolin I use in the non-transfected cells (where I do NOT see an effect) is anywhere between 1 nM and 100 uM.
Thanks for any help you can provide.

The FFM
The FFM's picture
What assay are you using to

What assay are you using to quantify the cAMP responses?
Are you using ELISAs, HitHunter and CatchPoint cAMP assay kits or a live cell CNGC-based cAMP measurement?

klw
klw's picture
TheFFM wrote:

TheFFM wrote:

What assay are you using to quantify the cAMP responses?
Are you using ELISAs, HitHunter and CatchPoint cAMP assay kits or a live cell CNGC-based cAMP measurement?

 
Cisbio HTRF cAMP dynamic 2 kits.
http://www.htrf.com/products/gpcr/camp/
My assays use 20,000 cells in half area 96-well plates.  GPCR transfected HEKs have a great forskolin response.  Parentals have no response.
Just saw this in my searches: http://endo.endojournals.org/cgi/reprint/135/4/1713.pdf where Figure 2 shows a cAMP response in HEK cells at 5-10 uM.  Hmmmm....
 

Bluejay
Bluejay's picture
People also use prostaglandin

People also use prostaglandin or isoproteranol as positive controls for cAMP assays since PGE2or4 and B-adrenergic receptors are often present.  I've tested many different cell lines and not had any that lack a forskolin response.