Role of κ opioid receptors in modulating cholinergic twitches in the circular muscle of guinea-pig colon

Abstract

1 Single pulse electrical field stimulation (EFS, 0.5 ms pulse width, 60 V at a frequency of 0.05 Hz) induced twitch contractions of mucosa-free circular muscle strips from the guinea-pig proximal colon which were abolished by atropine (0.3 μM), tetrodotoxin (0.3 μM) or ω-conotoxin GVIA (0.1 μM). 2 Various opioid receptor agonist concentration-dependently inhibited twitches with the following rank order of potency (EC50 values in brackets): U 50488 (0.31 nM)> dermorphin (4.3 nM) = dynorphin A (1-13) (6.2 nM) > [D-Ala2, N-MePhe4, Gly5-ol]-enkephalin (DAMGO, 33.5 nM) = [D-Ala2, D-Leu5]-enkephalin (DADLE, 60 nM) > [D-Pen2, D-Pen5]-enkepahlin (DPDPE, 1144 nM). 3 Peptidase inhibitors (captopril, thiorphan and bestatin, 1 μM each) did not modify the amplitude of twitches. In the presence of peptidase inhibitors the concentration-response curve to dynorphin A (1-13) was displaced to the left to yield an EC50 of 0.35 nM, comparable to that of the selective κ receptor agonist, U50488. The curves to the other opioid receptor agonist were unaffected by peptidase inhibitors. 4 DPDPE, DADLE, dermorphin and DAMGO consistently induced a concentration-unrelated transient increase in basal tone and a small and transient facilitation of twitches before development of their inhibitory effect. These transient excitatory effects were not observed upon application of dynorphin A (1-13) or U 50488. The contraction produced by DPDPE (30 nM) was largely inhibited (> 80%) by 1 μM atropine. 5 Twitches suppression induced by dynorphin A (1-13) (30 nM) was partly reversed (46 ± 8%, n = 6) by naloxone (0.3 μM). The potent and selective K opioid receptor antagonist nor-binaltorphimine (Nor-BNI, 3-100 nM)) did not affect the amplitude of twitches and potently antagonized (pK(B) 9.83 ± 0.09, n = 10) the inhibitory effect of dynorphin. 6 Naloxone (1-300 nM) concentration-dependently depressed the cholinergic twitches: this depressant effect was largely counteracted in the presence of apamin (0.1 μM) and N(G)-nitro-L-arginine (30 μM) which potentiated cholinergic twitches on their own. 7 Dynorphin A (1-13) (10 nM, n = 6) did not affect the contractile response to exogenous acetylcholine (1 μM), indicating that depression of evoked twitches occurs prejunctionally. 8 We conclude that multiple opioid receptors modulate cholinergic twitches in the circular muscle of guinea-pig proximal colon. While μ and δ opioid receptor agonists produced mixed excitatory and inhibitory effects, κ opioid receptors, activated by sub-nanomolar concentrations of dynorphin A (1-13), mediate a powerful and pure prejunctional inhibition of acetylcholine release.