[³H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂] ([³H)CTOP), a potent and highly selective peptide for mu opioid receptors in rat brain

The cyclic, conformationally restricted octapeptide [³H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂] ([³H)CTOP) was synthesized and its binding to mu opioid receptors was characterized in rat brain membrane preparations. Association rates (k₊₁) of 1.25 x 10⁸ M^-1 and 2.49 x 10⁸ M^-1 min^-1 at 25 and 37°C, respectively, were obtained, whereas dissociation rates (k_-1) at the same temperatures were 1.93 x 10^-2 min^-1 and 1.03 x 10^-1 min^-1 at 25 and 37°C, respectively. Saturation isotherms of [³H]CTOP binding to rat brain membranes gave apparent K(d) values of 0.16 and 0.41 nM at 25 and 37°C, respectively. Maximal number of binding sites in rat brain membranes were found to be 94 and 81 fmol/mg of protein at 25 and 37°C, respectively. [³H]CTOP binding over a concentration range of 0.1 to 10 nM was best fit by a one site model consistent with binding to a single site. The general effect of different metal ions and guanyl-5'-yl-imidodiphosphate on [³H]CTOP binding was to reduce its affinity. High concentrations (100 mM) of sodium also produced a reduction of the apparent mu receptor density. Utilizing the delta opioid receptor specific peptide [³H]-[D-Pen², D-Pen⁵]enkephalin, CTOP appeared to be about 2000-fold more specific for mu vs. delta opioid receptor than naloxone. Specific [³H]CTOP binding was inhibited by a large number of opioid or opiate ligands. Putative mu opioid receptor ligands such as naltrexone, naloxone, CTOP and Tyr-D-Ala-Gly-N-MePhe-Gly-ol inhibited specific [³H]CTOP binding with high affinity. Other ligands, including delta and kappa opioids, somatostatin, substance P, alpha and beta adrenergic, dopaminergic as well as cholinergic ligands were much less potent or were ineffective. The density of [³H]CTOP binding sites in different regions of the central nervous system is consistent with reports of the regional distribution of mu receptors in the rat brain. These data show that [³H]CTOP is a potent and selective ligand and may be useful for further characterization of mu opioid receptors.