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A notice for coffee drinkers
- Gabriele Baier (3 December 2003)
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Gabriele Baier, a.Univ.Prof Institute for med.Chemistry and Biochemistry,University of Innsbruck, Fritz Pregl Str.3, A-6020 Inns
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In the interesting Editorial: Tobacco, coffee, and Parkinson`s disease (BMJ 2003; 326: 561-562), Christopher Martyn and Chris Gale discussed the potential improving effects of tobacco and coffee for Parkinson`s disease patients. Authors proposed that the neuroprotective effects of caffeine may be due to its ability to block adenosine A2A receptors that are concentrated in the dopamine rich areas of the brain. In particular A1 and A2A subtypes of adenosine receptors are significantly blocked at concentrations of caffeine achieved with single servings of brewed coffee (100-200 mg). With our contribution we would like to report that at higher concentrations caffeine might be overcome by unwanted side- effects of an adenosine receptor blockade. For a demonstration we have incubated PC12 cells with caffeine (10µM). After double staining nuclei with the intercalating dyes Hoechst and propidium iodide, cell death was analyzed by fluorescence microscopy. In cell cultures which were incubated with caffeine we observed cells in early apoptosis (blue fragmentated nuclei) as well as in late apoptosis and necrosis (red cells, arrow) (Fig.1b). In addition, apoptotic nuclei were quantified by FACS scan analysis. Results supported the microscopic data as described above, and showed that an elevated concentration of cells (1.6 fold of the control) underwent cell death by apoptosis (Fig.1 c). To analyze whether the effect of caffeine was due to an inhibition of the adenosine receptor, we added adenosine to our cultures and observed a significant rescue of cell death (82%) (Fig.1d). Since PC12 mainly express the A2A and A2B adenosine receptors, it may be assumed that apoptosis in our system was initialized by the downregulation of the cAMP –mediated signalling-cascade. Based upon these results we want to underscore the importance of the interaction of adenosine receptors with its agonist (for a review see: 2), and indicate that for us coffee drinkers the outcome of coffee consumption not exclusively depends on the brand of coffee used but also on the amount of caffeine we consume. Since apoptotic cell death is apparently involved in the loss of dopaminergic neurons in Parkinson's patients, the side-effects of caffeine may additionally speed this process. Valerie Podhraski
References 1 C.Martyn and C.Gale., BMJ 2003; 326: 561. 2 B.B. Fredholm et al., Pharmacol Rev. 1999; 51: 83.
1x105 non-differentiated PC12 cells/ml were plated on collagen-S for the experiment. Cells were allowed to rest for 24 hours in full medium (containg 5% fetal bovine serum and 10 % horse serum, Penc/Strep and Glutamine) and for another 24 hours in medium with low serum (1% fetal bovine serum and 0.5% horse serum, Penc/Strep and Glutamine). Hence cells were incubated with a) low serum medium (control) or b) pretreated for 30 minutes with caffeine (8-(3-chlorostyryl) caffeine (CSC, 10µM) and then incubated with low serum medium for 20-24 hours. At the end of the experiment apoptotic cells were characterized by double staining with Hoechst 33342 and propidium iodide on a Zeiss Axioplan Fluorescence Microscope (a and b). Double stained cells (red, arrow) are dead (late apoptosis and necrosis) cells. For quantification isolated nuclei were characterized by 'morphology' (forward scatter (FCS) versus side scatter (SSC) and quantitation of the intranuclear contents of fluorescing DNA by propidium iodide staining and FACS analysis (c and d). To verify the specificity of caffeine effects, adenosine (500µM, Ad) was added to cultures (d). Values are representative of the means and the S.E.M. (n=4). Competing interests: None declared |
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