Role of K opioid receptors in modulating cholinergic twitches in the circular muscle of guinea‐pig colon

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 μ m ), tetrodotoxin (0.3 μ m ) or ω‐conotoxin GVIA (0.1 μ m ). 2 Various opioid receptor agonist concentration‐dependently inhibited twitches with the following rank order of potency (EC 50 values in brackets): U 50488 (0.31 nM)>dermorphin (4.3 nM) = dynorphin A (1–13) (6.2 nM)>[D‐Ala 2 , N‐MePhe 4 , Gly 5 ‐ol]‐enkephalin (DAMGO, 33.5 nM) = [D‐Ala 2 , D‐Leu 5 ]‐enkephalin (DADLE, 60 nM)>[D‐Pen 2 , D‐Pen 5 ]‐enkepahlin (DPDPE, 1144 nM). 3 Peptidase inhibitors (captopril, thiorphan and bestatin, 1 μ m 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 EC 50 of 0.35 nM, comparable to that of the selective K 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 μ m atropine. 5 Twitches suppression induced by dynorphin A (1–13) (30 nM) was partly reversed (46 ± 8%, n = 6) by naloxone (0.3 μ m ). 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 (p K 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 μ m ) and N G ‐nitro‐L‐arginine (30 μ m ) 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 μ m ), 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, K opioid receptors, activated by sub‐nanomolar concentrations of dynorphin A (1–13), mediate a powerful and pure prejunctional inhibition of acetylcholine release.