Interactions of dynorphin A-(1-13) and nociceptin with cardiac D2 binding sites: Inhibition of ischemia-evoked release of noradrenaline from synaptosomal-mitochondrial fractions

The effect of dynorphin A (Dyn A)-related peptides and nociceptin on the binding of the D2 receptor antagonist, [H-3]raclopride, was examined in membrane preparations of rat heart. Non-linear regression saturation binding analysis of [H-3]raclopride binding revealed the presence of a single high-affinity binding site with a K-d of 4.1 nM and a B-max of 220 fmol/mg protein. The D2 stereospecificity of [H-3]raclopride binding was demonstrated by competition experiments using two enantiomer pairs of antagonists. (+)-Butaclamol (IC50: 8.0 nM) and (-)-sulpiride (IC50: 112.3 nM) were 27 000 and 24 times more potent than (-)-butaclamol (IC50: >100 mu M) and (+)-sulpiride (IC50: 2666 nM), respectively. Nociceptin and Dyn A-(1-13) were also potent inhibitors of the binding of [H-3]raclopride with shallow inhibition curves that fitted best with two sites model. Their order of potency on the low affinity site [alpha-Neo-endorphin>nociceptin>Dyn A-(2-13)>Dyn A-(1-13)>Dyn B>Dyn A-(6-10)] correlated well with their ability to inhibit the binding of [H-3]nociceptin (r=0.82). The indirect nature of the inhibitory effects of the peptides on the D2 receptor was demonstrated by their inability to inhibit [H-3]raclopride binding to a membrane preparation (Sf9 cells transfected with the human D2(long) receptor) that does not contain the ORL1 receptor and the lack of effect of raclopride (0.1 nM-10 mu M) on both [H-3]nociceptin and [H-3]Dyn A-(1-13) binding. Isolated cardiac mitochondrial-synaptosomal fractions submitted to ischemic conditions (1 mM iodoacetate +2 mM NaCN, 5 min at 37 degrees C) released 10.9% of their content in preloaded [H-3]noradrenaline ([H-3]NA). Dyn A-(1-13) (10 mu M), nociceptin (10 mu M) and the selective D2 receptor agonist, quinpirole (10 mu M) were potent blockers of the release of [H-3]NA evoked by the ischemic conditions. The inhibitory effect of Dyn A-(1-13), nociceptin and quinpirole were antagonized by the selective D2 receptor antagonist, raclopride (10 mu M); whereas naloxone, at a concentration (1 mu M) known to affect the ORL1 receptor, blocked the effects of the peptides but not those of quinpirole. The results demonstrate the presence of D2 receptors in rat heart and suggest that Dyn A-(1-13) and nociceptin modulate ischemia-induced NA release by a mechanism that involves the participation of both ORL1 and D2 receptors. (C) 2000 Academic Press.