Abstract
Airway submucosal gland cell (SMGC) secretions are under the control of various neurotransmitters and hormones. Interactions between different pathways, such as those mediated by cAMP and Ca2+, in controlling mucus or electrolyte secretions are not well understood. Prostaglandin E2 (PGE2) or forskolin has been shown to enhance acetylcholine (ACh)-induced short circuit current (ISC) in SMGC mucous cell monolayers. We show that PGE2, by activating cAMP-dependent protein kinase A (PKA), enhanced ACh-induced, Ca2+-mediated current and changes in [Ca2+]i in mucous cells. PGE2 pretreatment sensitized ACh-induced ISC (ΔISC) by activating endoprostanoid (EP2) receptors. PKA inhibitors 14-22 amide PKI (PKI) and Rp-diastereomer (Rp) of cAMPs prevented the effect of PGE2. Removing external Ca2+ or pretreatment with the Ca2+ entry blocker, SKF96365 [1-[β-(3-(4-methoxyphenyl) propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy] ethyl] imidazole], shifted the concentration-response relationships for ACh to the right but did not abolish PGE2-induced sensitization of the ACh response. An inositol 1,4,5-trisphosphate (IP3) receptor antagonist and Ca2+ entry blocker, 2-aminoethoxydiphenyl borate, abolished the ACh-induced response. Charybdotoxin, but not iberiotoxin (IbTX), inhibited the ACh-induced ΔISC. Clotrimazole, but not IbTX, inhibited the ACh-induced serosal K+ current. Under whole-cell patch clamp, ACh-induced K+ and Cl– currents were coincident with increases in [Ca2+]i in single mucous cells. PGE2 or forskolin pretreatment did not induce current or [Ca2+]i changes but enhanced ACh-induced currents, membrane hyperpolarization, and [Ca2+]i changes. Intra-cellular dialysis with the PKA-catalytic subunit enhanced ACh-induced whole-cell current as well. These findings demonstrate that PGE2, via EP2 receptors and the cAMP/PKA pathway, activates Ca2+ entry-independent mechanisms, possibly by increasing IP3-mediated Ca2+ release, resulting in the sensitization of ACh-induced currents.
Footnotes
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This study was supported in part by a grant from the American Heart Association (to J.M.F.).
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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.107.120154.
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ABBREVIATIONS: SMGC, submucosal gland cells; 2-APB, 2-aminoethoxydiphenyl borate; ACh, acetylcholine; PGE2, prostaglandin E2; AH6809, 6-isopropoxy-9-oxoxanthene-2-carboxylic acid; ANOVA, analysis of variance; BK, large conductance, calcium-activated K+ channels; CaCC, Ca2+-activated Cl– channels; CFTR, cystic fibrosis transmembrane conductance regulator; ChTX, charybdotoxin; DMSO, dimethyl sulfoxide; EP, endoprostanoid; IbTX, iberiotoxin; IK, intermediate conductance calcium-activated K+ channel; ISC, short circuit current; PG, prostaglandin; PKA, cAMP-dependent protein kinase A; PKI, PKA inhibitor, 14-22 amide; SC19220, 8-chloro-dibenzo[b,f][1,4]oxazepine-10(11H)-carboxylic acid, 2-acetylhydrazide; IP3, inositol 1,4,5-trisphosphate; SKF96365, 1-[β-(3-(4-methoxyphenyl) propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy] ethyl] imidazole; VIP, vasoactive intestinal peptide; Rp, Rp-diastereomer; EGF, epidermal growth factor; AG1478, 4-(3-chloroanillino)-6,7-dimethoxyquinazoline.
- Received January 19, 2007.
- Accepted May 3, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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