My cells do not seem to respond to a concentration as high as 1 mM theophylline.
Theophylline should be able to get into your cells. What cells are you using and what are you dissolving your theophylline in? What are you measuring that indicates to you that it is not working? Have you tried other non specific PDE inbitors like IBMX?
I'm using Leishmania. I first was dissolving theophylline in 1 M NH4OH but I found that the NH4OH control I was performing was cytotoxic itself. Hence I shifted to dissolving theophylline in water (giving me a stock conc of ~20 mM). Incubating my cells with the same (final conc 1 mM) did not lead to any change in the cell's morphology or motility. Yes I have IBMX in mind. Haven't tried it yet though.
Some possible explanations for your negative result, from someone who has not worked with Leishmania or phosphodiesterases:
(1) theophylline is not an inhibitor of phosphodiesterases of Leishmania.
(2) inhibition of phosphodiesterases has no effect on Leishmania cells in culture.
(3) theophylline is converted into something else that doesn't inhibit phosphodiesterases when it gets into Leishmania.
(4) Leishmania is impermeable to theophylline.
(5) Leishmania expresses efflux pumps that keep theophylline from accumulating in the cells.
(6) Theophylline gets into the cells but is in a separate subcellular compartment from the phosphodiesterases.
Hi Aakash,
My experience with PDE inhibitors is in the context of increasing cAMP and thereby increaing insulin secretion. We routinely use IBMX to augment insulin secretion in preifusion experiments. I have no experience with Leishmania and do not know if it is reasonable to expect changes in morphology or motility. Is it possible that the theophylline is doing its thing, but your parameters are not sensitive enough to see a change? Can you measure cAMP or cGMP? Try increasing the dose?
Good luck!
IBMX does enter Leishmania, but is only effective in growth inhibition in the millimolar range:
http://www.biomedcentral.com/1471-2180/6/25
Other compounds are more potent, but have other effects on these parasites, e.g. inhibition of purine transport. There is a strucutre of IBMX bound to one Leishmania PDE, see review by Seebeck:
http://www.ncbi.nlm.nih.gov/pubmed/21859303
I would expect that theophylline will certainly enter leishmania cells, like any other cell.
Thanks @All!
I did some reading and found out that theophylline is a potent PDE only at a much higher concentrations for Leishmania sp. Need to perform a few experiments to validate the same.
IBMX is however a more potent Leishmania specific PDE inhibitor with an IC50 of approx 2 mM.
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