It's hard to address that without knowing a bit more about your experimental design.  Is this in hippocampal brain slices?  Are you co-applying the agonist/antagonist or pre-applying one or the other?  There could be differences in equilibrium between the two compounds (in terms of penetration into the tissue and access to receptor).  

Yes. Hippocampal brain slices. I preincubate with the antagonist for > 30 mins and take baseline. Then apply agonist prepared in the antagonist solution. So antagonist is always present during recording.

It sounds like they must be binding at different sites---maybe the antagonist site is not accessible until the agonist binds, so it isn't efficiently blocking the initial response, but becomes more effective following receptor activation.  Either that, or the irreversibility is due to some secondary mechanism (cAMP, etc) that is blocked by the antagonist.

What happens if you apply the antagonist at varying times after the agonist?  In other words, can it always reverse the agonist effect, even if the agonist was applied for a long time?  Also, have you varied agonist concentrations?

Thanks Alex. I haven't tried applying antagonist after the agonist, but its worth looking at. I have attached an example recording. Maybe you can give me more insight after looking at it.

So that recording sort of looks like a 'chemical LTP'---the initial early increase in the response has not reached a stable plateau when you stop the agonist application, but continues to increase to a stable state after ~40min. The antagonist is clearly only blocking the later phase. So it could be that if you have a receptor coupled to cAMP, that you are elevating cAMP levels and producing a form of LTP, and the antagonist is only blocking this latter effect.

Given the slow onset, however, I also wonder---have you tested other drugs in your system?  Do you usually have slow onset to peak drug effects, or is this just a property of this particular agonist? 

Is your agonist able to act on different receptors of a family? If yes, another possibility to explain your results is that the agonist is acting on two (or more) different receptors, and only one of them, the involved in the late, sustained phase of the agonist-response, is affected by the antagonist. The inhibition of the antagonist-sensitive receptor permits a more rapid activation of the other(s), antagonist-insensitive receptor(s) by the agonist. But this would only works if you are studying an agonist that is able to act by activating different receptors, as it would be the case, for example, of endogenous opioids, tachykinins..... I believe that the better would be to follow the advice of Alexander, i.e., to assay different agonists, to see if you find a similar response with all of them, and to assay different antagonists, alone and together, to analyze the effects on the agonist response.

Thank you Alex and Luz!

Alex: You're right, we think its chemical LTP. I have done some occlusion experiments and this increase does occlude TBS-induced LTP and vice-versa. Also about slow onset, since I apply all drugs through an infusion line, it takes about 5-7 minutes for the drug solution to even reach the chamber. Similarly for the washout solution. I observe the same for other drug classes as well.

Luz: The agonist concentration that i have used in the experiment I showed above is high enough that we think it acts on multiple receptor subtypes. That is why we wanted to use antagonists to figure out which receptor was responsible for the increase it induced. So if what you're suggesting were true, you mean the early and late-phase effect may be through different receptor types? I think that sounds like a possibility. The other receptor-subtype antagonist blocks both initial and late-phase.

Yes Nisha, if you are studying an agonist acting on a multi-receptor system, the early and late phases may be mediated through different receptor types, even each phase may be the result of the activation of different subtypes. If this is the case in your experiments, the blockage of one receptor type may cause an additional increase (or decrease, if different receptor types mediate opposite responses) in the response of the other, non-blocked, receptor types, as you are changing the delicate equilibrium between them. Your additional experiments with other agonists and antagonists would help you to clarify this!!

If you name the drugs I may look into any covert partial agonist or antagonist effects that as referred earlier maybe the reason.