Characterization of the high affinity [H-3]nociceptin binding site in membrane preparations of rat heart: Correlations with the non-opioid dynorphin binding site

The binding parameters of [H-3]nociceptin were examined in membrane prepara tions of rat heart and compared with those of [H-3]dynorphin A-(1-13) ([H-3 ]Dyn A-(1-13)). Scatchard analysis of [3H]nociceptin binding revealed the p resence of two distinct sites: a high affinity (K-d: 583 nM) low capacity ( B-max: 132 pmol/mg protein) site and a low affinity (Kd: 10 316 nM) high ca pacity (1552 pmol/mg protein) site. Dyn A and related peptides were potent competitors of the binding to the high affinity site with the following ran k order of potency: alpha-neo-endorphin>Dyn A-(2-13)=Dyn A-(3-13)>D37n 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 IC, of 4.8 mu M as compared with 0.72 mu M 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 compe te with the binding of [3H]Dyn A-(1-13) (Dumont and Lemaire, 1993). In addi tion, the high affinity [3H]nociceptin and non-opioid [H-3]Dyn A-(1-13) sit es were both sensitive to NaCl (120 mM) and the phospholipase C (PLC) inhib itors, U-73122 and neomycin (100 mu M). The binding activities were less af fected by the weak PLC inhibitor, U-73343, and no effect was observed with the non-hydrolysable GTP analogs, Gpp(NH)p and GTP-gamma-S. Nociceptin (1-5 0 mu M) was also shown to inhibit the uptake of [H-3]noradrenaline ( [H-3]N A) by cardiac synaptosomal preparations. In spontaneously hypertensive rats (SHR), the potency of nociceptin in inhibiting [H-3]NA uptake was increase d by 1.6-fold as compared with Wistar Kyoto (WRY) control rats and such eff ect was accompanied by comparable increased levels of cardiac ORL1 mRNA and [H-3]nociceptin high affinity sites, These changes correlated well with th e previously observed increased levels of non-opioid cardiac [H-3]Dyn A-(1- 13) sites in SHR (1.3 times as compared with (WKY) and increased potency of Dyn A-(1-13) in inhibiting [H-3]NA uptake by cardiac synaptosomes in SHR ( 2.2-fold asl compared with WKY) (Dumont and Lemaire, 1995). The results dem onstrate that in rat heart the characteristics of the high affinity, low ca pacity [H-3]nociceptin binding site are similar to those of the non-opioid Dyn binding site. The stimulation of this site by nociceptin, Dyn A or rela ted peptides is more likely to produce a modulation of PLC activity and [H- 3]NA uptake and may participate to the pathophysiology of hypertension. (C) 1998 Academic Press.