Differential Modulation of Sodium- and Chloride-Dependent Opioid Peptide Transport System by Small Nonopioid Peptides and Free Amino Acids

Abstract

We recently identified a novel opioid peptide transport system in the retinal pigment epithelium that transports opioid peptides by a Na ϩ /Cl Ϫ -dependent process. Here we describe a similar transport system expressed in SK-N-SH cells (a human neuronal cell line) and show for the first time that the activity of the transport system is modulated differentially by lysine and small nonopioid peptides. The transport process in SK-N-SH cells, monitored with deltorphin II as the substrate, is Na ϩ /Cl Ϫ -dependent and interacts with several opioid peptides, consisting of 5 to 13 amino acids. The activity of this transport system is markedly stimulated by specific dipeptides and tripeptides, with significant stimulation observable at low micromolar concentrations. The ion dependence, Na ϩ /Cl Ϫ -activation kinetics, and opioid peptide selectivity of the transport system, however, remain unchanged. The stimulation by the modulatory peptides is associated with an increase in maximal velocity with no change in substrate affinity of the system. Amino acids have no or little effect on the transport system, with the exception of lysine. This cationic amino acid inhibits the transport system, with significant inhibition occurring at physiologic concentrations of the amino acid. The inhibitory effect is primarily associated with a decrease in the maximal velocity of the transport system with little change in substrate affinity. Methyl and ethyl esters of lysine retain the inhibitory potency, but most other structural analogs have no effect. The differential modulation of the transport system by lysine and specific small peptides has important implications in the biology and pharmacology of opioid peptides.

Evidence for two different broad-specificity oligopeptide transporters in intestinal cell line Caco-2 and colonic cell line CCD841

“…Currently, the only feature that distinguishes between SOPT1 and SOPT2 is the differential effects of dipeptides and tripeptides. SOPT1 is stimulated by several dipeptides and tripeptides, whereas SOPT2 is inhibited by the same small peptides (1,2,24,30). None of these peptides, however, is a transportable substrate for SOPT1 and SOPT2.…”

“…Accordingly, we named these transporters sodium-coupled oligopeptide transporter (SOPT)1 and SOPT2 (1,2). SOPT1 and SOPT2 transport a variety of endogenous opioid peptides, exogenous peptides such as Tat 47-57 [a fragment of the Tat protein encoded by human immunodeficiency virus (HIV)-1], and synthetic opioid peptides (1,2,17,24). Currently, the only feature that distinguishes between SOPT1 and SOPT2 is the differential effects of dipeptides and tripeptides.…”

“…Recent studies from our laboratory (1,2,12,17,24,30) have identified two new peptide transporters at the functional level in the human retinal pigment epithelial cell line ARPE-19, the rabbit conjunctival cell line CJVE, and the human neuronal cell line SK-N-SH. These transporters are distinct from PEPT1 and PEPT2.…”

Antiapoptotic Properties of α-Crystallin–Derived Peptide Chaperones and Characterization of Their Uptake Transporters in Human RPE Cells

“…Both transporters have overlapping substrate specificity and can recognize an array of both endogenous as well as synthetic peptides regardless of their amino acid sequence. 14,[16][17][18] Substrate selectivity studies have revealed that SOPT1 and SOPT2 accept oligopeptides consisting of five or more amino acids. 14,[16][17][18] We wished to study whether a-crystallin mini-chaperones elicit antiapoptotic properties that were shown previously to be associated with parent proteins.…”

“…14,[16][17][18] Substrate selectivity studies have revealed that SOPT1 and SOPT2 accept oligopeptides consisting of five or more amino acids. 14,[16][17][18] We wished to study whether a-crystallin mini-chaperones elicit antiapoptotic properties that were shown previously to be associated with parent proteins. Secondly, we also wished to determine the mechanisms of uptake of these a-crystallin mini-chaperones in human RPE cells and to characterize the putative oligopeptide transporter(s).…”