title: A novel nonopioid action of enkephalins: competitive inhibition of the mammalian brain high affinity L-proline transporter.
abstract: The high affinity L-proline transporter (PROT) is a member of the family of Na+ (and Cl-)-dependent plasma membrane transport proteins that comprises transporters for several neurotransmitters, osmolytes, and metabolites. The brain-specific expression of PROT in a subset of putative glutamatergic pathways implies a specialized function for this novel transporter and its presumed natural substrate L-proline in excitatory synaptic transmission. However, definitive studies of the physiological role(s) of high affinity L-proline uptake have been precluded by the lack of specific uptake inhibitors. Here, we report that Leu- and Met-enkephalin and their des-tyrosyl derivatives potently and selectively inhibited high affinity L-proline uptake in rat hippocampal synaptosomes and in PROT-transfected HeLa cells. High concentrations of the opiate receptor antagonist naltrexone did not block the inhibitory actions of these peptides, arguing against an involvement of opioid receptors. Des-tyrosyl-Leu-enkephalin elevated the apparent K(m) of L-proline transport in transfected HeLa cells without altering the V(max). PROT-transfected HeLa cells did not accumulate [3H]Leu-enkephalin above background levels, demonstrating that enkephalins are not substrates for PROT. These findings indicate that enkephalins competitively inhibit mammalian brain PROT through a direct interaction with the transporter protein at or near the L-proline binding site. The high potency and specificity of des-tyrosyl-Leu-enkephalin make this compound a useful tool for elucidating the structure-function properties and physiological role(s) of PROT.
authors.affiliation: Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, USA. rtf@galactose.mc.duke.edu
authors.forename: R T
authors.initials: RT
authors.lastname: Fremeau
authors.forename: M
authors.initials: M
authors.lastname: Velaz-Faircloth
authors.forename: J W
authors.initials: JW
authors.lastname: Miller
authors.forename: V A
authors.initials: VA
authors.lastname: Henzi
authors.forename: S M
authors.initials: SM
authors.lastname: Cohen
authors.forename: J V
authors.initials: JV
authors.lastname: Nadler
authors.forename: S
authors.initials: S
authors.lastname: Shafqat
authors.forename: R D
authors.initials: RD
authors.lastname: Blakely
authors.forename: B
authors.initials: B
authors.lastname: Domin
mesh_terms: D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D001921:Brain / Q000187:drug effects*; D004745:Enkephalins / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D008297:Male; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors*; D009271:Naltrexone / Q000494:pharmacology; D011392:Proline / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013329:Structure-Activity Relationship
chemical_list: D026921:Amino Acid Transport Systems, Neutral; D004745:Enkephalins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D009271:Naltrexone; C033082:proline transporter; D011392:Proline