Characterization of the High Affinity [3H]Nociceptin Binding Site in Membrane Preparations of Rat Heart: Correlations with the Non-opioid Dynorphin Binding Site

The binding parameters of [3H]nociceptin were examined in membrane preparations of rat heart and compared with those of [3H]dynorphin A-(1-13) ([3H]Dyn A-(1-13)). Scatchard analysis of [3H]nociceptin binding revealed the presence of two distinct sites: a high affinity (Kd: 583 n ) low capacity (Bmax: 132 pmol/mg protein) site and a low affinity (Kd: 10 316 n ) high capacity (1552 pmol/mg protein) site. Dyn A and related peptides were potent competitors of the binding to the high affinity site with the following rank order of potencyα-neo-endorphin>Dyn A-(2-13)=Dyn A-(3-13)>Dyn A-(5-13)>Dyn A-(1-13)>Dyn A>Dyn B>Dyn A-(6-10)>>Dyn A-(1-8). Nociceptin was 6.7 times less potent than Dyn A with a Kiof 4.8μ as compared with 0.72μ for Dyn A. The order of potency of the various peptides in inhibiting [3H]nociceptin binding correlated well (r=0.93) with their ability to compete with the binding of [3H]Dyn A-(1-13) (Dumont and Lemaire, 1993). In addition, the high affinity [3H]nociceptin and non-opioid [3H]Dyn A-(1-13) sites were both sensitive to NaCl (120 m ) and the phospholipase C (PLC) inhibitors, U-73122 and neomycin (100μ ). The binding activities were less affected by the weak PLC inhibitor, U-73343, and no effect was observed with the non-hydrolysable GTP analogs, Gpp(NH)p and GTP-γ-S. Nociceptin (1–50μ ) was also shown to inhibit the uptake of [3H]noradrenaline ( [3H]NA) by cardiac synaptosomal preparations. In spontaneously hypertensive rats (SHR), the potency of nociceptin in inhibiting [3H]NA uptake was increased by 1.6-fold as compared with Wistar Kyoto (WKY) control rats and such effect was accompanied by comparable increased levels of cardiac ORL1mRNA and [3H]nociceptin high affinity sites. These changes correlated well with the previously observed increased levels of non-opioid cardiac [3H]Dyn A-(1-13) sites in SHR (1.3 times as compared with WKY) and increased potency of Dyn A-(1-13) in inhibiting [3H]NA uptake by cardiac synaptosomes in SHR (2.2-fold as compared with WKY) (Dumont and Lemaire, 1995). The results demonstrate that in rat heart the characteristics of the high affinity, low capacity [3H]nociceptin binding site are similar to those of the non-opioid Dyn binding site. The stimulation of this site by nociceptin, Dyn A or related peptides is more likely to produce a modulation of PLC activity and [3H]NA uptake and may participate to the pathophysiology of hypertension.