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* School of Biomedical Sciences, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham, UK
1Correspondence: E-mail: steve.alexander{at}nottingham.ac.uk
The comments from Dieterich et al. on our recent publication in The FASEB Journal (1)
is welcomed, as we have yet to determine a precise mechanism for the effects which we reported. However, their interpretation of the data glosses over some key points.
Addition of ADP (or ATP) causes relaxation of isolated coronary artery smooth muscle via indirect activation of A2A adenosine receptors. This may come about through sequential metabolism of ADP to adenosine (Fig. 1
, favored by Dieterich et al.) or through release of intracellular adenosine (our hypothesis). We agree that conventional nucleoside transporters are not involved in the release phenomenon, but rather in uptake/re-uptake of adenosine in this tissue [Figure 5 of our manuscript indicates the involvement of ENT1 transporters, since potentiation of ADP-evoked responses occurs (1)
].
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Previous studies making use of cultured smooth muscle cells from Dieterich et al. (2)
provide evidence for 5'-nucleotidase (5'NT; Fig. 1
) activity playing a prominent role in extracellular adenosine generation, making use of the best characterized inhibitor of 5'NT (
ß-methyleneADP). It may be an experiment worth conducting, but we did not feel a need to use ß-methyleneADP in our isolated tissue experiments, since we showed that a stable ADP analogue (ADPßS) elicits relaxation via A2A adenosine receptors in this tissue, which is not consistent with a mechanism involving breakdown of ADP to adenosine (see below). We used a number of alternative potential inhibitors of nucleotidase, including 1 mM CTP or TTP or 30 µM dATP, all of which were without significant effect.
A central issue not addressed by Dieterich et al. is presented in Figure 4A (1)
, which shows (at t=7.5') a peak of [3H]-adenosine present in suprafusate from [3H]-adenine-labeled tissues, which was only observed in the presence of ADP plus U46619, not with U46619 alone. While this peak is small in comparison to peaks for hypoxanthine and adenine/inosine, it would be our contention that the time- and ADP-dependent increase in extracellular radioactivity [Fig. 4A (1)
] is reflective of [3H]-adenosine efflux. The larger peak of adenine/inosine, we contend, is the result of an active ecto-deaminase metabolism leading to accumulation of inosine.
We feel the likelihood that a P2 receptor is involved in coronary artery smooth muscle is slight. Space didn’t allow inclusion of the data in the original submission, but responses to ADPßS appear to be mediated through exactly the same mechanism as responses to ADP. Thus, responses to 100 µM ADPßS (86±9% relaxation) are unaffected by either 1 µM MRS2179 (83±9%) or 10 µM PPADS (78±11%), but are completely blocked by 1 µM ZM241385 (–6±8%) (n=6–7). Since ADPßS is poorly hydrolyzed, the metabolic route of ADPase and 5'NT action (Fig. 1)
is unlikely.
In summary, while we have, as yet, no molecular evidence for a candidate NTXchange protein which would allow the efflux of adenosine in response to extracellular adenine nucleotides (Fig. 1)
, we feel that this possibility is better supported by the data than the metabolic route.
FOOTNOTES
The opinions expressed in editorials, essays, letters to the editor, and other articles comprising the Up Front section are those of the authors and do not necessarily reflect the opinions of FASEB or its constituent societies. The FASEB Journal welcomes all points of view and many voices. We look forward to hearing these in the form of op-ed pieces and/or letters from its readers addressed to journals{at}faseb.org.
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