I want to determine the role of intestinal nor-epinephrine on pathogen survival and replication in an impaired immunological background. For that I wanted to first use propranolol, as shown in previous publications. However, none of these papers mention anything about how to test that the propranolol solution you use is effective. Any easy and not too expensive suggestion? Thanks for your help.
Isa
Am I understanding correctly? Are you testing the effects of b-adrenergic receptor blockers on the pathogen as apposed to mammalian cells? i.e. there are no mammalian cells in your system?
That's correct. To explain a bit more what I am trying to do: I have intestinal samples from different models, one being impaired in many intestinal barrier functions. This model is more prone to infection by food-borne pathogens than their immuno-competent counterparts. When I use a filtrate of intestinal content from this mice to test the growth rate of bacteria in vitro from both model, I see a faster growth rate in both filtrates than in any lab medium (it is also greater in filtrate from my immuno-deficient model than in that from the normal model). As serum and intestinal norepinephrine (NE) for example has been described by Mark Lyte and others since then as a growth promoting factor on bacteria, I was wondering if i) the faster growth rate I see is linked to NE or not by using Propranolol as a b-adrenergic blocker as shown in in vitro assays by Freestone, Haigh and Lyte (2007, BMC Microbiol) and then see whether there is a difference in NE levels between immunocompromised and competent models. However, I can't find any information was to what I could use in vitro to test that indeed my stock solution of propranolol is working, hence my very poorly explained post on Research gate. I hope this makes sense. Thanks for your help
Isa
That sounds like a very interesting study and experiment. I've not done it myself, but I have looked a little into b-adrenergic receptors for my work. I've attached a link to one of the papers I found referring to expression and function in lung epithelial cells. They used cAMP in cell lines. Might be useful.
http://www.ncbi.nlm.nih.gov/pubmed?term=Expression%20of%20functional%20B2%20adrenergic%20receptors%20in%20the%20lung%20epithelial%20cell%20lines%2016HBE14%20Calu3%20and%20A549
Thanks a lot for your time and suggestion Keith. I could indeed test my stock solution on mammalian cells and look at cyclic AMP production.
Good luck with your research
Isa
I agree with Keith....As beta adrenergic stimulates Adenilate ciclase, measuring the levels of cAMP must be useful.
Thanks Jean. An alternative for me would be also simply to test the blocking effect of Beta adrenergic blockers on the growth promoting effect of purified norepinephrine, as described by Lyte et al.
Isa
Please consider my suggestions only as spontaneous brain storming suggestions - not as a valid expert advice (since this is not my research area). 1. I remember a standard experiment in medical school (25 years ago), where we used freshly isolated frog intestine (maybe 1-3cm long part) put some little weight on it and measured the change in length after adding different mimetika and antagonists... acould be re-used fter rinsing... 2. I helped supervising physics students to work with beating mouse cardiomyocytes, which for example beat under the light microscope with 60/min, after adding b-mimetika doubled the frequency, which could be reduced or prevented with b-blocker... (Markus Pax, Janosch Rieger, Robert H. Eibl, et al. 2005). I think there are many other ways to check for a functionl check of your blocking solution of b-blocker.
Article Measurements of fast fluctuation of viscoelastic properties ...
Dear Robert
I must admit that I have enjoyed reading your suggestions as these were not at all what I had in mind. But it is very refreshing to get other people's opinions when they come from different research background with different expertise. I have since found in vitro methods to test if my solutions of B-blockers were active, but would like to thank you a lot for your time and advice.
Isa
Hi Isabelle, did you try to use binding experiments using whole cells or membranes from cells expressing beta-adrenergic receptors?
For me is the simply way to check the specifity of your preparations and/or your solutions.
Cheers,
Natalio
Dear Isa
I have to admit that I really enjoyed reading your answer as well. It was refreshing getting such a nice update. Thank you.
Robert
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