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J. Gierow, M. C. Edman, S. K. Carlsson; Both Adenosine A2 Receptors Are Present in the Rabbit Lacrimal Gland. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2449.
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Studies in our laboartory have previously shown the presence of the adenosine A1 receptor (Edman et al., Exp. Eye Res, in press, 2007), and indicated the presence of the A2a in the rabbit lacrimal gland (Invest. Opthalmol. Vis. Sci. 47, E-abstract 1943, 2006). However, farmacological and immunological studies could not exclude the A2b receptor. Therefore, it was of interest to further investigate the A2a and A2b receptors in the lacrimal gland by using tools of molecular biology.
Rabbit lacrimal gland acinar cells were prepared according to our standard procedure (Andersson et al., Exp. Eye Res. 83, 543-553, 2006) yielding single cells that were placed in a serum-free culture medium on standard culture plates for two days, and then rinsed briefly, incubated w./w.o. secretagogues and/or antagonists as indicated. Secretion was determined enzymatically as secreted beta-hexosaminidase activity. RNA was isolated from tissue which had been snap-frozen in liquid N2, and receptor mRNA presence was determined by RT-RCR using primers designed from multiple alignments (A2a) of sequences from other species or an already reported sequence (A2b).
A two-fold increase of beta-hexosaminidase secretion was observed when cells were stimulated with adenosine or CPCA (A2a and A2b agonist), and an approximative five-fold increase when stimulated with carbachol. Combination of either one with carbachol, resulted in an approximate 10-fold, synergistic increase of secretion over basal. Blocking the receptors with SCH 58261 (A2a receptor antagonist) or PSB 1115 (A2b antagonist) lead to a secretion reduced by at least 50 %, compared to in the absence of antagonist, thus suggesting a possible role for both receptors in the synergy between CPCA and carbachol. The A2a and A2b receptors were detected as single bands on an agarose gel by RT-PCR. Sequencing of the A2a band showed an 88 %, 90 % and 87 % similarity with human, horse and dog A2a receptor, respectively. The sequenced A2b band revealed a 100 % similarity with the previously reported rabbit A2b receptor.
Both the A2a and the A2b receptors are present in the rabbit lacrimal gland, and both can participate in the adenosine-carbachol synergy.
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