I am not exactly familiar how ryanodine preparation is done, but I assumed I can perfuse it onto the cells during calcium transient measurement? But since its target is an intracellular component, maybe that will not work too? I am also confused because it is available as a 1 mg vial (Sigma), so if I want to use micromolar concentrations, I will have to consume all at once? I would really appreciate any tips on this. Thank you!
Dr. Acsai,
Thank you very much for your answer! It seems that using ryanodine is very expensive? I can only make 100 mL of ryanodine solution if I wish to use 100 uM, am i right? Have you ever done an experiment using ryanodine? I plan on using this on cardiomyocytes, and I am also considering other RyR2 blockers (dantroline, FLA 365, ruthenium red) but still confused with which one to use.
Hi,
Ryanodine penetrates intact cells and affects RyRs. It has been shown to both inhibit RyR activation and to lock them in an open sub-conductance state, depending on the concentration applied. We have used ryanodine many times to fully block RyR activity. We found that it works quite slowly when perfused on cells, but gives a rapid block if a co-application of caffeine is used. This is most probably related to the use-dependent action of ryanodine, whereby it binds to activated channels. Caffeine rapidly activates the RyRs and allows ryanodine to bind and inhibit further opening.
We never had any effect of ruthenium red on intact cells.
Best,
Martin
Ryanodine gets into the cell easily. In my experience with cardiac tissue preparations, 20 uM ryanodine inhibits calcium release from the sarcoplasmic reticulum within 2 minutes @ 23'C. Ryanodine is expensive. The cheapest is offered by Abcam. I use this and it works fine. hope this helps.
Dr. Logantha,
Thank you for recommending a cheaper option! The price and amount of ryanodine is really what's keeping me from purchasing it but it seems it's the best option too. I just got confused when my professor told me that it should be in the intracellular solution. So I assumed she meant that it should be incubated, since that is what I have seen with the previous references I have found. But I will use it with Normal Tyrode so maybe there's no problem. Thanks again!
Dr. Bootman,
Great point on the use of caffeine, thanks for mentioning that! If it's not too much to ask, would you happen to have that study on your page? I will try to look for it. Thanks again!
Hi Julius,
Using caffeine at the same time may cut down the time you need to perfuse with the ryanodine solution, and therefore reduce costs. Testing the cells with a pulse of caffeine after ryanodine treatment is a good way of ensuring that the RyRs are actually inhibited.
We used caffeine + ryanodine co-application a lot in this study:
Article: Quantal Ca2+ mobilization by ryanodine receptors is due to all-or-none release from functionally discrete intracellular stores. Biochemical Journal 09/1994; 301 ( Pt 3):879-83.
It wasn't a study on cardiac myocytes, but it shows the kind of protocol we used, and it works similarly on cardiac myocytes.
Best of success with your experiments,
Martin
Yes you can perfuse it. What is missing from your enquiry is what is the scientific question you are asking. This will determine what additional agents you could use as well as whether this is best done with tissue, whole myocytes, or organelles.
Professor Jones,
Thank you for your response! As for your question, and I apologize for that, I am curious as to how the ryanodine is able to permeate the cell membrane to act upon the RyR? That is why I was concerned whether perfusion would be a right way of applying ryanodine. Also, would this method (measuring calcium transient attributes of RyR2-inhibited cardiomyocytes) an efficient protocol for the assessment of RyR2 function?
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