Abstract
Adenosine 3′,5′-cyclic monophosphate (cAMP) is an important second messenger involved in cholinergic transmission. The aims of this study were to characterize the calcium channels associated with cyclic AMP-mediated acetylcholine release and Ca++ efflux in ileal myenteric plexus. We also examined if this process can be inhibited by agents such as opioids that inhibit N-type calcium channels via a pertussis toxin-sensitive G protein. Application of a cell permeant analogue, 8-bromoadenosine cyclic AMP (8Br-cAMP) (1 mM), and an activator of the adenylyl cyclase system, forskolin (0.1 mM), in a superfusion system resulted in both Ca++ efflux and3H-acetylcholine (ACh) release from the dispersed myenteric ganglia. A preferential N-type Ca++ channel blocker, ω-Conotoxin GVIA (ω-CgTx, 10-100 nM), significantly inhibited 3H-ACh release stimulated by 8Br-cAMP. 10 nM ω-CgTx also totally inhibited 8Br-cAMP-induced Ca++efflux, whereas the L-type Ca++ channel blocker, nifedipine (1 μM), and the T-type Ca++ channel blocker, nickel (100 μM), both had no effects on the action of 8Br-cAMP.3H-ACh release during 0.1 mM forskolin stimulation was inhibited by pretreatment with a kappa receptor agonist, U50488H at 1 to 100 nM. In addition, U50488H significantly inhibited3H-Ach release and Ca++ efflux elicited by 8Br-cAMP. Inhibition of 3H-ACh release by U50488H was reversed by 3 hr pretreatment with 300 ng/ml pertussis toxin. These results suggest that, in the myenteric plexus, cyclic AMP-stimulated Ca++ efflux and Ach release were mediated by N-type calcium channels. This process may be inhibited by activation of thekappa opioid receptor through pertussis toxin-sensitive G protein(s).
Footnotes
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Send reprint requests to: Dr. Chung Owyang, 3912 Taubman Center, Box 0362, The University of Michigan Medical Center, Ann Arbor, MI 48109-0362.
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↵1 This work was supported in parts by U.S. Public Health Service Grants DK-32830 and P30 DK 34933 from the National Institute of Diabetes, Digestive and Kidney Disease.
- Abbreviations:
- ACh
- acetylcholine
- 8Br-cAMP
- 8-bromoadenosine 3′, 5′-cyclic monophosphate
- CCK
- cholesystokinin
- PTX
- pertussis toxin
- VIP
- vasoactive intestinal polypeptide
- ω-CgTx
- ω-Conotoxin GIVA
- [Ca2+]i
- intracellular free calcium concentration
- AMP
- adenosine monophosphate
- Received July 3, 1996.
- Accepted March 27, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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