We use a non-hydrolysable form of ATP to stimulate P2X receptors; ATPgammaS. There are also subtype specific agonists available that are more stable depending on which type of purinergic receptors you are interested in stimulating. Try searching tocris.com for agents that are more specific for the subtype of receptor you are interested in stimulating.

Micromolar ATP solutions are quite stable for 4-5 hours at room temperature. Some problems exist only with low-nanomolar solutions (see Khmyz V et al., 2008 for ref)

ATP activates all P2Y and P2X receptors. UTP only activates P2Y2-P2Y4-P2Y6 receptors. So, the agonist you have to use depends on the receptor subtype you have in your sample . Both ATP and UTP are metabolized very fast. You have to use non-hydrolyzing derivatives if you want to avoid that problem.

UTP is for sure also hydrolyzed as ARL can clearly increase its function response. http://www.ncbi.nlm.nih.gov/pubmed/17612577

you can use ATPgammaS, ADPbetaS, alphabetamethyleneATP as these are more stable agonists. Be aware of the relative selectivity towards the different receptors. The same is true for UTP, acts on P2Y2 and 4 while its breakdown product acts onP2Y 6, not on the other receptors.

Dear Maria Lisa,

regarding stability you could use ATP analogs that usually are more stable (e.g. 2MT- ATP).

But I would worry about stability in case you have to apply nucleotides for long time. For acute experiments if the powder or the stock solution are kept below zero, I 'd not worry.

More than stability, you should consider which P2 receptor you are interested in. UTP do not activate other than P2y2,4,6 (let's say uridine nucleotides). Enjoy your experiments,

Sergio

For stability I would make my ATP solutions fresh that morning, store on ice until ready to load them. Briefly warm them to room temperature and patch for a few hours. Longer than 4-5 hours I would make them up again fresh.

As the others have mentioned, it all depends on which receptor sub-type you are most interested in. With regards to the P2X receptors, although they are all activated by ATP at different concentrations and there are some specific agonists, I would recommend looking for a good antagonist for your sub-type (As Jonathan mentioned for the agonists, Tocris is also good for antagonists). There are many which are very specific and selective. You could apply ATP at a range of concentrations in the absence or presence of your inhibitor. For competitive inhibitors that don't require preincubation to be effective, you can set up your micro-injectors to have:

1: vehicle

2: ATP

3: Antagonist

4: ATP + Antagonist

Then you can apply 1 then 2, see the response. Then apply 3 then 4 and see if your response to ATP is deminished.

Like Jonathan suggested the use of non-hydrolysable analogues could be used and Tocris offers the best panel of purinergic tools.

I would only like to add 2 points regarding stability:

1- You should know which ecto-nucleotidases are present on your cells as they can affect purinergic signalling even when average concentration of ATP is not significantly lowered like we showed for NTPDase1 and P2X7. Often "specific agonists" are ATP- or UTP-derivatives that will be hydrolyzed to some extent. If you are aiming at P2Y2R (as your question suggest), you could use more stable molecules like ATPgammaS, UTPgammaS that can resist hydrolysis by NTPDases (not NPPs). In addition PSB1114 (like ARL67156) will probably resist to hydrolysis by NTPDases and NPPs.

2- UTP will not be hydrolyzed as efficiently by all ecto-nucleotidases that metabolize ATP, so you should expect a little more stability. However, UTP can degraded to UDP +Pi and is an excellent substrate for NTPDase1 at 37°C.

First you need to find out what types of P2Rs your cells express? Once you've know which subtypes of P2Rs it will easier to stimulate your cells using specific agonists . ATP generally activates all P2Rs. There are specific agonists for P2Rs. For example, UTP is for P2Y2 and P2Y4, BzATP is specific for P2X7 and 2MeSADP is for P2Y1.

Dear Maria,

We always kept our ATP solutions on ice and warm up fresh for experiments. The use of ATP or UTP depends on what receptors are expressed in the cells you are using. ATP is active on all P2X ion channel receptor subtypes 1 to 7, while UTP is equipotent with ATP on P2Y2 and P2Y4 G protein-coupled receptors.

I hope that this is helpful. I wish you success and pleasure in your experiments.

With best wishes,

Geoff Burnstock

I agree with Dr Burnstock. We normally make fresh solutions from powder ATP or UTP on the day of the experiment. it really depends on what receptors you are trying to activate. Atp will activate p2y and p2x1-7 but sometimes it`s d ATP concentration which determines which receptor it is activating. For more specific p2y stimulation, you car try mes-atp. and for p2x stimulation, atp sounds good. UTP only stimulates p2y2 and p2y4 receptors. Dnt worry about atp hydrolyzing because it takes time to do it and most forms available in the market are quite stable. Good luck :)

I had done some work with various purinergic analogs back in grad school and have published a summary of how ATP stimulated the MAPK pathway for enhanced neurite expression in PC12 cells. Part of that work examined what receptor system was responsible for the enhanced phenotype and so various purinergic analogs were employed to delineate which receptor system was involved, given the different sensitivities of receptors to them (e.g. one system would be most activated by ATP but the least by adenosine). I agree that non-hydrolyzable analogs are a good way to assess your system without the interference of breakdown products. Please see my articles and/or other published reviews of purinergic receptors that outline the analog-receptor activation profiles and the differences between P1/P2 and P2x (ionotropic) and P2y (metabotropic) receptor activation.

Dear Maria,

It is difficult to add a new comment after the comment from Prof. Burnstock:) As for our experience - we prepared ATP stock (10mM in water), aliquoted, froze and use those aliquots 2 weeks at least. Having the stock solution you can change "old" ATP (2-3 hours) to a fresh one in a minute.

Best

Roma

Stock solutions of ATP (or UTP) in water (no additional salts) are actually quite stable. Non-enzymatic hydrolysis becomes a significant issue only after the ATP/ UTP is added to experimental test salines that contain divalent cations. As most other commenters have noted, such working test solutions should be freshly prepared and not stored for more than a few hours. With regard to using ATP or UTP for patch clamp studies, nucleotide hydrolysis usually isn't a major issue because the small numbers of cells in the culture dish are being continuously superfused and the test nucleotides are usually applied in discrete pulses or "puffs" for only several seconds or a few minutes at most.

I agree with Dr. Dubyak and Dr. Burnstock. I use fresh ATP in water in my electrophysiological (Patch-clamp & Using chamber) experiments.

Please note that puffing ATP in water solution (not adjusted to physiological saline)onto your cells may cause unwanted acute hypo-osmotic effects.

I agree with you. We mix require amount of ATP (in H2O) with E-buffer just before application.