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 bi nding of the D2 receptor antagonist, [H-3]raclopride, was examined in membr ane preparations of rat heart. Non-linear regression saturation binding ana lysis of [H-3]raclopride binding revealed the presence of a single high-aff inity 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 co mpetition experiments using two enantiomer pairs of antagonists. (+)-Butacl amol (IC50: 8.0 nM) and (-)-sulpiride (IC50: 112.3 nM) were 27 000 and 24 t imes more potent than (-)-butaclamol (IC50: >100 mu M) and (+)-sulpiride (I C50: 2666 nM), respectively. Nociceptin and Dyn A-(1-13) were also potent i nhibitors 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 af finity 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 inh ibit [H-3]raclopride binding to a membrane preparation (Sf9 cells transfect ed with the human D2(long) receptor) that does not contain the ORL1 recepto r and the lack of effect of raclopride (0.1 nM-10 mu M) on both [H-3]nocice ptin and [H-3]Dyn A-(1-13) binding. Isolated cardiac mitochondrial-synaptos omal fractions submitted to ischemic conditions (1 mM iodoacetate +2 mM NaC N, 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 blocker s of the release of [H-3]NA evoked by the ischemic conditions. The inhibito ry effect of Dyn A-(1-13), nociceptin and quinpirole were antagonized by th e 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 demonstra te 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 inv olves the participation of both ORL1 and D2 receptors. (C) 2000 Academic Pr ess.