I do not believe that the compounds are membrane permeable. Actually they do not need to enter the cell to fonction, the PE receptor are G-protein coupled receptors expressed in the cell membrane

I once contacted Cayman to ask about membrane permeability of severel EP receptor agonists and they informed me that this information was not available. 

PGE2 is membrane permeable (althoug it diffuses slowly and in cells that synthesize PGE2 there is a transporter for PGE2) and since most selective agonists are structural analogs of PGE2, I think they are membrane permeable to some degree, but it would take them a long time to accumulate in the cell.

I find that when I wash out EP receptor agonists (R-butaprost and CAY10580) after 40 min stimulation they instantly stop signalling, which seems to indicate that they stay extracelllular and receptor signalling occurs from the cytosolic membrane. So for acute studies, it seems fair to assume that they stay extracellular, but it may be different during long-term treatment.

Thank you for your response, Emma. I thought they might. That is an interesting idea to see if washing the cells changes the responses. How did you determine, whether they stopped signalling? measuring cAMP? We are using the compounds for early and late timepoints, so potentially problematic...

For butaprost, I measured cAMP by adding IBMX right after agonist removal and then measured cAMP after an incubation time in IBMX (this can be necessary for technical reasons). cAMP was the same after washout as control levels measured in the same way (but greatly increased during stimulation). I have also tried the same method with other compounds, e.g. forskolin, where cAMP is still increased after washout!

For the EP4 agonist, this did not increase cAMP, but the effect of the agonist decreased rapidly after removal.

I once discussed with a chemist whether PGE2/other compounds could be tagged for direct visualization, but then the tagging could of course affect the membrane permeability, so I abandoned the idea... But another method could be to look at proteins that are modified rapidly, e.g. by phosphorylation/dephosphorylation, if you know of such!

Emma's suggestion is a viable way to accomplish what you are looking for. You should, however be aware that different cells respond differently to the substances you are using. Also you should bear in mind that mechanical force (eg. centrifugation) may modulate cellular cNMP levels, protein phosphorylation etc.

Actually I did not get the point what you are expecting regarding prostanoid uptake by the cells. If it is because of unexpected dose-response effects you should also take effects of the biomatrix (whatever your cell medium contains) into account.

Addition of unselective PDE inhibitors (like IBMX) is sometimes problematic as they may cause significant cNMP increases simply by preventing cNMP degradation.

Quantification of protein phosphorylation as a read out for cNMP increase is mostly more reliable and robust than determination of cNMP as we have shown with a solid phase phosphorylation assay.

It is very interesting that EP2 and EP4 agonists had a different effect on cAMP - I was always convinced they would be working through the same pathway.

I think protein phosphorylation might be a good option in our case, as we might already have some antibodies to measure this.

Joerg: We were interested in protanoid uptake, because it was previously shown that functional EP3 and EP4 can also be localised on the nuclear membrane (Bhattacharya et al., 1999), and that thus, the agonists might be able to mimic Cox-derived PGE2 responses.