I am testing the effects of inorganic phosphate on the force-pCa relationship in mammalian skinned fibres at room temperature. I have lovely force-pCa relationships, but 20 mM phosphate has no effect on force or the Ca50, which seems ridiculous given the overwhelming evidence that it reduces force and reduces Ca2+ sensitivity. I'm currently using rabbit psoas skinned in glycerol solution.
I have some things to try:
1) Adding phosphocreatine to the solutions in case the PO4 produced by crossbridge cycling is high and the diffusion rate so low that the effects I think I should be seeing are masked.
2) Secondary skinning with triton-x to further dissolve the sarcolemma.
3) I'm using really old samples as practice tissue which could be problematic.
4) The only other thing I can think of is that I have completely bungled the recipes for the solutions. We have a set of standard pCa solutions which work great. To make a 20 mM PO4 solution, I added 20 mM KH2PO4 and reduced the concentration of K-Proprionate to match the ionic strength of the two solution types. pH 7.0.
Has anyone encountered this before or have any other suggestions.
I think the problem is in activation at high (room) temperature. Check what's happening to sarcomere length, how well are they ordered when activated. What is maximum active tension the fibres develope? We studied effect of added Pi (10 mM) in sarcomere controlled T-jump experiments (~20 y ago) and found that it suppress tension by ~20-25% at low temperature (5-10 oC) and accelerated T-jump induced transient. At 30-35oC these effects disappeared completely. Sarcomere disordering can also mask the effects of added Pi.
Hello Ian,
As hinted by Sergey above, you are dealing with / thinking about interesting but complex problems, temp, Pi, Ca etc,
Might be worth checking /reading some old papers referred to in Methods and control expts in
Journal of Physiology (2001), 536.3, pp.879–891 (Coupland et al paper).
Hope it helps, regards,
KW,
Thank you very much for the advice, Kuda and Sergey.
I will look into sarcomere disordering - with the age of the samples I was testing, sarcomere disorder is a real concern. I was not aware of the interaction with phosphate. I have fresh sample to test now.
The Coupland paper was an interesting read; particularly the methods section and the control experiments it outlines. I'm considering using a phosphate mop system to ensure phosphate contamination is minimal.
Best regards,
Ian
To follow this up: there was a problem with the recipes. I was treating the KH2PO4 as a strong acid instead of a weak acid, so the ionic strengths of the solutions were different between the PO4 and control solutions. Everything looks to be working properly now. Thanks again for the helpful suggestions.
Ian
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