Elsevier

Peptides

Volume 22, Issue 12, December 2001, Pages 2015-2020
Peptides

MDR1 P-glycoprotein transports endogenous opioid peptides

https://doi.org/10.1016/S0196-9781(01)00564-2Get rights and content

Abstract

MDR1 P-glycoprotein is generally regarded as an efflux pump for amphipathic toxic compounds. The question remains, however, whether certain endogenous compounds are also substrates for this transporter. Certain peptides have been shown to interact with MDR1 Pgp as well and we have therefore investigated whether endogenous bioactive peptides are substrates. We demonstrate here that the synthetic μ-opioid peptide DAMGO is a good substrate for MDR1 Pgp. In view of its low interaction with the membrane it is an attractive ligand for measurement of MDR1 Pgp-mediated transport activity in membrane vesicles. Various linear peptides with amidated C-termini were found to inhibit MDR1 Pgp-mediated DAMGO transport. This group includes endogenous opioid peptides such as adrenorphin and endomorphin 1 and 2, as well as the neurokinin, Substance P. The latter bioactive peptides have a relatively high affinity for the transporter. Transport of endomorphin 1 and 2 could be directly demonstrated by the uptake of the radiolabeled opioid peptides in membrane vesicles from MDR1-transfected cells with a Km of 15 and 12 μM, respectively. This opens the possibility that MDR1 Pgp is involved in the elimination and/or tissue distribution of these bioactive peptides.

Introduction

MDR1 P-glycoprotein has been characterized as an efflux pump for amphipathic drugs. It confers drug resistance against a host of uncharged and positively charged amphipathic compounds, such as alkaloids, steroids, anthracyclines, epipodophyllotoxins and short chain phospholipid analogues. Several peptides were also shown to interact with Pgp [7]. These are mainly cyclic amphipathic ionophores, such as valinomycin and gramicidin, but also linear peptides such as leupeptin. Sharom et al. [8] screened a large panel of cyclic and linear peptides for their ability to inhibit Pgp-mediated transport. They observed Dm values (concentration of half-maximal inhibition) which ranged from 130 μM for the uncharged hydrophobic peptide, N-acetyl-leucyl-leucyl-norleucinal, to 0.7 μM for cyclosporine A. The transport of a peptide has been directly measured in membrane vesicles only in one study. Sharom et al. [9] observed ATP-dependent uptake of 125I-labeled NAc-LLY-amide in membrane vesicles from multidrug resistant cells and this transport was absent from the parental cells; half-maximal transport was reached at 100 μM of this peptide.

The secretion of xenobiotic amphipaths by MDR1 Pgp represents an important physiological barrier against toxic compounds. The question has, however, always remained whether certain endogenous compounds are also substrate for Pgp. Thus far, the steroids, aldosterone, corticosterone and cortisol, are the only endogenous compounds that were shown to be transported by MDR1 P-glycoprotein [10]. The absence of a distinct phenotype with respect to these hormones in Mdr1a/b double knockout mice suggests, but does not prove, that class I P-glycoproteins do not play a crucial role in the homeostatic balance of these hormones [6]. Sarkadi et al. [5] showed that the synthetic μ-selective opioid DAMGO (Tyr-D-Ala-Gly-N-Methyl-Phe-Gly-ol) induces Pgp-mediated ATPase activity. In addition, Chen and Pollack [1] showed recently, that clearance of another δ-selective, synthetic cyclic opioid peptide DPDPE ([D-penicillamine [2], [5]enkephalin) is impaired in Mdr1a−/− mice, and that these animals are hypersensitive to DPDPE-induced analgesia, suggesting that this Mdr1a Pgp plays a role in the disposition of this peptide and in the diminution of its action. The transport of endogenous, bioactive peptides, including opioids has not been studied, however. This prompted us to investigate the transport of a number of opioid peptides including the endogenous enkephalins, endomorphins 1 and 2 and adrenorphin. The enkephalins have highest specificity for the δ-receptor [3] while the newly discovered endomorphins [11] are potent and highly selective ligands for the μ-receptor. The μ-selective opioids generally have an amidated C-terminus and therefore lack a negative charge. Since the constituent amino acids are generally uncharged, these ligands are cationic amphipathic peptides, which renders them potential substrates for MDR1 Pgp. In the present study we show that endogenous amidated opioid peptides, like endomorphin 1 and 2 and adrenorphin, are transported by MDR1 P-glycoprotein.

Section snippets

Cell culture and preparation of crude plasma membranes vesicles

For transport studies we used the non-small cell lung cancer cell line SW1573/S1, (further described as S1) and its MDR1 transfectant, S1 1.1 [2]. The cells were grown in DMEM + 10% fetal calf serum (FCS) and harvested by incubation with trypsin. After washing with Hank’s balanced salt solution (HBSS) containing 5% FCS, the cells were washed 3 times with HBSS and centrifuged. Subsequently, a crude plasma membrane vesicle suspension was prepared as described in [4]. Briefly, the cells were

Results

Because it was shown that DAMGO induces ATPase activity in membranes from cells that overexpress MDR1 P-glycoprotein [5], this compound was taken as a starting point for the measurement of transport of opioid peptides. A crude plasma membrane fraction was prepared from parental SW1573/S1 cells (further designated as S1) and its subline transfected with MDR1 (further designated as S1 1.1, for more information see ref. 2). Fig. 1A shows that the transfected cells contain a clear band at 170 kDa

Discussion

In the present report we show that the opoid peptide DAMGO is transported by MDR1 Pgp and has an affinity for the transporter that is comparable with other established substrates such as doxorubicin. A considerable advantage of DAMGO as a ligand for MDR Pgp transport assays with plasma membrane vesicles is that it displays a very low extent of aspecific membrane binding. By virtue of their hydrophobic character most other Pgp substrates display a much higher extent of non-specific binding to

Acknowledgements

The authors thank Dr. Alfred Schinkel for critically reading the manuscript.

References (11)

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