Abstract
We examined the effect of substances released by swine alveolar macrophages (AMs) on ionic currents in airway submucosal gland cells (SGCs). AMs obtained by lavage were activated by 24-h zymosan exposure (0.1 mg/ml). Supernatant was collected and used to stimulate short-circuit current changes (ΔISC) in SGC monolayers in Ussing chambers. Dexamethasone (1 μM) or indomethacin (5 μM) during zymosan exposure of AMs reduced or abolished the supernatant-induced ΔISC. Zymosan exposure induced a 5-fold increase in cyclooxygenase (COX)-2 but not COX-1 protein levels in AMs. Prostaglandin E2 (PGE2) concentration in the supernatant from zymosan-activated AMs was 550 ± 10 nM (n = 3) compared with 28 ± 3 nM for unstimulated AMs (n = 3). PGE2, applied serosally, induced ΔISC with an EC50 of 15.5 ± 1.3 nM (n = 4) and 3.6 ± 1.8 μM (n = 3) when applied apically. Four types of endoprostanoid receptors (EP1–4) were detected in SGCs using Western blot. PGE2-induced ΔISC were inhibited by AH6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) but not by SC19220 (8-chloro-dibenzo[b,f][1,4]oxazepine-10(11H)-carboxylic acid, 2-acetylhydrazide), suggesting that endoprostanoid (EP)2 but not EP1 receptors were activated by PGE2. Pretreatment of SGCs with supernatant from zymosan-activated AMs, PGE2, or forskolin enhanced the sensitivity to acetylcholine (ACh)-induced ΔISC. PGE2-induced ΔISC were blocked by charybdotoxin (ChTX), chromanol 293B, or glibenclamide. ACh-induced ΔISC were only blocked by ChTX or glibenclamide. None of these blockers altered PGE2 pretreatment-induced sensitization of ACh-induced ΔISC. These results demonstrate that prostanoids released from activated AMs directly increase cystic fibrosis transmembrane conductance regulator and K+ channel activity. ACh-induced ΔISC are also enhanced due to enhanced activation of Ca2+-activated K+ channels (KCa).
Footnotes
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.105.088542.
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ABBREVIATIONS: SGC, submucosal gland cell; AM, alveolar macrophage; ISC, short circuit current; ACh, acetylcholine; PG, prostaglandin; COX, cyclooxygenase; CFTR, cystic fibrosis transmembrane conductance regulator; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; DPC, diphenylamine-2-carboxylate; DIDS, disodium 4,4′-diisothiocyanatostilbene-2,2′-disulfonate; TBS, Tris-buffered saline; TBST, TBS/Tween 20; EP, endoprostanoid; AH6809, 6-isopropoxy-9-oxoxanthene-2-carboxylic acid; SC19220, 8-chloro-dibenzo[b,f][1,4]oxazepine-10(11H)-carboxylic acid, 2-acetylhydrazide; ChTX, charybdotoxin; ANOVA, analysis of variance; Mϕ, macrophage; PKA, cAMP-dependent protein kinase A.
- Received April 27, 2005.
- Accepted July 26, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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