A2 adenosine receptors in Mongolian gerbil middle ear epithelium and their regulation of Cl− secretion

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Abstract
The present study investigates the effects of adenosine and its analogues on Cl− secretion in primary cultures of gerbil middle ear epithelium. Short‐circuit current (Isc), an index of transepithelial active transport, was measured on the same cells cultured on porous filters. Baseline Isc and transepithelial resistance were 27.0 ± 0.7 μA cm−2 and 275 ± 7 Ω cm2, respectively (n = 178). Extracellular adenosine and its analogues elicited a sustained increase in Isc when added to apical or basolateral surfaces. Both the A2A selective agonist 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamido adenosine and the A2A/A2B nonselective agonist 5'‐(N‐ethyl‐carboxamido)adenosine (NECA) increased Isc, but NECA was more effective than CGS21680. A1 selective antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine did not reduce NECA‐induced Isc. These results suggest the presence of both A2A and A2B receptors. NECA did not stimulate a rise in the intracellular Ca2+ concentration ([Ca2+]i) in single middle ear epithelial cells cultured on glass coverslips. Dibutyryl cAMP (dbcAMP) induced an initial transient increase in Isc followed by the sustained plateau. Addition of dbcAMP also caused a transient increase in [Ca2+]i. The protein kinase A inhibitor, N‐[2‐(p‐bromocinnamylamino)ethyl]‐5‐isoquinolinesulfonamide, greatly reduced the increase in the Isc responses to NECA. 1,2‐Bis‐(2‐aminophenoxy)ethane N,N,N',N'‐tetraacetic acid‐acetoxymethyl ester influenced neither the NECA‐induced increase in Isc nor the dbcAMP‐induced sustained phase of Isc, but greatly inhibited the dbcAMP‐induced transient increase in Isc. Glibenclamide, a cystic fibrosis transmembrane conductance regulator (CFTR) channel inhibitor, reduced the NECA‐induced Isc. These results indicate that extracellular adenosine and its analogues activate the cAMP‐protein kinase A system, but not intracellular Ca2+‐dependent mechanisms, leading to Cl− secretion, possibly through the CFTR Cl− channels in the cultured gerbil middle ear epithelium.