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Endomorphin-1 and endomorphin-2 are partial agonists at the human μ-opioid receptor

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Abstract

Recently two tetrapeptide ligands that bind preferentially to the μ-opioid receptor were identified and named endomorphin-1 and endomorphin-2. We examined the ability of these peptides to stimulate G protein activation in human μ-opioid receptor transfected B82 fibroblasts as measured by [35S]GTPγS binding to cell membranes. Both endomorphin-1 and -2 act as partial agonists in this assay system compared with the μ-selective agonist [d-Ala2,N-Me-Phe4, Gly-ol5]enkephalin (DAMGO). In addition, endomorphins demonstrate efficacy similar to morphine. These findings demonstrate that endomorphin peptides have similar activity at the μ-opioid receptor as morphine and suggest that these peptides have the potential to modulate neuronal activity in vivo.

Introduction

Over 20 years ago, Kosterlitz and his coworkers began to investigate the purpose of opioid receptors in vivo (Hughes et al., 1975; Lord et al., 1977). These investigators reasoned that since opioid receptors exist, there must also be endogenous ligands that interact with these receptors. Concurrently with this work it was demonstrated that there are three different opioid receptors, namely the δ-, κ- and μ-opioid receptors. These receptors have been renamed as OP1, OP2 and OP3 receptors, respectively, based on the order in which these G protein coupled receptors were cloned (Dhawan et al., 1996). Endogenous peptide ligands that activate opioid receptors have previously been identified (Brownstein, 1993; Hughes et al., 1975); however, a new class of μ-opioid receptor-selective peptides have only recently been isolated from the mammalian brain (Zadina et al., 1997). These peptides, Tyr-Pro-Trp-Phe-NH2 and Tyr-Pro-Phe-Phe-NH2, are known as endomorphin-1 and endomorphin-2, respectively. Endomorphin-1 is 4000- and 15 000-fold selective for μ-opioid receptors as compared to δ- and κ-opioid receptors, respectively, whereas endomorphin-2 is >13 000- and >7500-fold selective, respectively. Both peptides mediate analgesia in mice after intracerebroventricular injection with similar potency to morphine (Zadina et al., 1997) as well as mediate analgesia after intrathecal injection (Stone et al., 1997). These peptides also have been demonstrated to have hypotensive activity in rabbits (Champion et al., 1997).

The EC50 (potency) of a drug in a functional assay is dictated by: (a) the affinity of drug for receptor; (b) the receptor density in a test tissue or cell; and (c) the capacity of the drug bound receptor to initiate a functional response (Ruffolo, 1982). In contrast, efficacy is a measure of the coupling efficiency of drug-bound receptors to activate a functional response. Efficacy is calculated utilizing the potency value in a functional assay and the inhibition constant (Ki) determined from the displacement of radiolabeled antagonist from μ-opioid receptors by drug (Ehlert, 1985). While observed efficacy values are dependent on the receptor density in the test tissue (Furchgott, 1966), we believe that efficacy values better characterize the ability of drugs to stimulate a functional response as compared to potency values. In this report we have determined the efficacy of the newly discovered endogenous μ-opioid receptor agonists endomorphin-1 and endomorphin-2 to modulate the first step in opioid-mediated signal transduction, namely G protein activation.

Section snippets

Preparation of membranes from human μ-opioid receptor transfected B82 fibroblasts

Stably transfected B82 fibroblasts, expressing the human μ-opioid receptor at 151 fmol/mg membrane protein, were previously prepared (Knapp et al., 1995). The cell line was grown in 162 cm2 culture flasks in Dulbecco's Modified Eagles Medium: Nutrient Mixture F12 with 5% fetal calf serum, 5% newborn calf serum, G418 (500 μg/ml), penicillin (100 U/ml) and streptomycin (100 μg/ml). Growth medium was removed when the cells were 80% confluent and cells were removed from the plate by a 5-min

Results

We initially examined G protein activation stimulated by the μ-opioid receptor-selective agonists DAMGO, morphine, endomorphin-1 and endomorphin-2 as measured by [35S]GTPγS binding to cell membranes (Fig. 1A). The rank order for maximal stimulation (intrinsic activity) was DAMGO>endomorphin-2≈endomorphin-1=morphine. The intrinsic activity of DAMGO was significantly greater than that of the other μ-opioid receptor selective agonists. Differences in intrinsic activity between endomorphin-1,

Discussion

Efficacy values describe the relationship between drug occupancy of receptors and the stimulation of a functional response. When comparing efficacy values for two drugs that bind to the same receptor, the drug with the higher efficacy value will occupy a smaller fraction of available receptor to mediate a given level of functional response. When efficacy is calculated according to the methods utilized in this paper, full agonists have efficacies of one or greater. When observed efficacy values

Acknowledgements

This research was supported in part by grants from the Arizona Disease Commission and the National Institute on Drug Abuse.

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