As I have already known that forskolin can stimulate adenylyl cyclase directly and NECA is a non-selective adenosine agonist which not stimulate adenylyl cyclase directly. The topic of my experiment is a small molecule which is supposed to inhibit the production of cAMP, probably target A3 adenosine receptor. My concern is that which compound is better to indicate the efficiency of my small molecule on inhibiting cAMP, forskolin or NECA. And I guess that forskolin is better because NECA is also a A3 adenosine receptor agonist which can cause competitive effect with my small molecule, and eventually reduce the cAMP evoked by NECA.
The original poster probably no longer needs this answer, but I figured I would answer anyway, in case someone else has the same question.
When looking for agonist activity at A3 AR, it makes sense to treat with forskolin, just as the original poster said. That way, you can compare the treatments to the forskolin control and see if any of the treatments have decreased cAMP relative to control (which means that A3 AR was activated).
I suppose that one could also test for antagonistic activity at A3 AR by having a forskolin control along with a forskolin+NECA control. A successful antagonist would be able to out-compete some of the NECA and would yield a higher cAMP value compared to the forskolin+NECA control. However, in our lab we have never actually done this. We have only ever tested for agonism at A3 AR.
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