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J. Gierow, M. Edman, D. Delbro; Presence of an A2a Adenosine Receptor in the Rabbit Lacrimal Gland . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1943.
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Our laboratory has previously shown that the adenosine A1–receptor is present in the lacrimal gland, using pharmacological techniques, immunochemistry and molecular biology. (Invest. Opthalmol. Vis. Sci. 44: a2512, 2003; Invest. Ophthalmol. Vis. Sci.45, p3860). However, a specific A1 antagonist did not block the secretion stimulated by adenosine or an A1 specific agonist by more than 50%. The aim of the present study is, therefore, to study the presence of other adenosine receptors and their signalling pathways.
Rabbit lacrimal gland acinar cells were prepared according to our standard procedure (Am. J. Physiol. (Cell Physiology) 271: C1685, 1996; Glycobiol. 15: 211, 2005) yielding single cells that were placed in a serum–free culture medium on standard culture plates coated with Matrigel for two days. The cultured cells were rinsed briefly and secretion was measured after a pre–incubation for 30 min at 37 C, with buffer alone, followed by incubation for an additional 30 min w./w.o. secretagogues and/or inhibitors as indicated. Basal secretion and regulated secretion was determined enzymatically as secreted beta–hexosaminidase activity. The lacrimal gland was also subjected to immunochemistry to confirm the presence of adenosine receptors.
Secretion was stimulated 2–fold by cyclopropylcarboxamidoadenosine(CPCA – an A2a agonist). Synergistic effects similar to those obtained with adenosine and CPA, an A1 specific agonist, were observed when combined with carbachol (a cholinergic agonist). These effects were blocked by an antagonists against the A2a–receptor (SCH 58261). The use of 2–APB, an inhibitor of IP3–mediated Ca2+ release, completely blocked the carbachol and CPCA stimulated release, whereas no significant effects were observed using chelerythrine, an inhibitor of protein kinase C. Immunohistochemistry confirmed the presence of the A2a–receptor.
Our results indicate that an A2a adenosine receptor is present in the rabbit lacrimal gland and that Ca2+ is an important mediator of the response to carbachol and purinergic stimulation and this is now subject to further studies.
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