amastatin and kyotorphin

Neuropeptide inactivation is generally thought to occur via peptidase-mediated degradation. However, a recent study found increased analgesia after L-kyotorphin (L-Tyr-L-Arg; L-KTP) administration in mice lacking an oligopeptide transporter, PEPT2. The current study examines the role of PEPT2 in L-KTP uptake by astrocytes and compares it to astrocytic L-KTP degradation. L-[(3)H]KTP uptake was measured in primary cultures of neonatal astrocytes from rats and from Pept2(+/+) and Pept2(-/-) mice. Uptake was further characterized using potential inhibitors. L-[(3)H]KTP degradation was examined in primary astrocyte cultures from Pept2(-/-) mice by following the formation of L-[(3)H]tyrosine. The uptake of L-[(3)H]KTP in both rat and Pept2(+/+) mouse neonatal astrocytes was inhibited by known PEPT2 inhibitors. L-[(3)H]KTP uptake was also reduced in Pept2(-/-) astrocytes as compared to those from Pept2(+/+) mice. Kinetic analysis indicated the presence of a high affinity (K(m) approximately 50 microM) transporter for L-[(3)H]KTP, identified as Pept2, and a low affinity transporter (K(m) approximately 3-4 mM), inhibited by amastatin, bestatin and tyrosine. Astrocytes also degraded L-KTP through a low affinity peptidase (K(m) approximately 2 mM). Astrocytic clearance of L-KTP occurs via both peptidase activity and transport. These processes occur at similar rates and may be linked. This supports the contention that oligopeptide transport may have an impact on the extracellular clearance (and potentially activity) of certain neuropeptides.

Topics: Animals; Animals, Newborn; Arginine; Astrocytes; Brain; Cefadroxil; Cells, Cultured; Dose-Response Relationship, Drug; Endorphins; Glycylglycine; Mannitol; Mice; Mice, Inbred C57BL; Mice, Knockout; Peptides; Protein Transport; Rats; Rats, Sprague-Dawley; Statistics, Nonparametric; Symporters; Tritium; Tyrosine

The neuropeptide kyotorphin (Tyr-Arg) was degraded by rat brain synaptosomes via a synaptic membrane-bound peptidase which was inhibited by bestatin but not by amastatin. The Km for kyotorphin was 8 x 10(-6) M and the Ki for bestatin was 1 x 10(-7) M. The kyotorphin-degrading enzyme was distinguished from at least one other dipeptide-hydrolyzing activity in synaptosomes which was inhibited by both bestatin and amastatin. Gel permeation chromatography of detergent-extracted synaptosomes resulted in the separation of the dipeptide-hydrolyzing activities. A single kyotorphin-degrading enzyme peak was observed which had a M(r) = 52,000. The activity peak could degrade other dipeptides including Phe-Arg, a synaptic membrane-generated metabolic of bradykinin. The kyotorphin-degrading enzyme appears to be novel and can be distinguished from other known dipeptidases on the basis of substrate specificity, subcellular localization, and inhibition profile.

Topics: Animals; Anti-Bacterial Agents; Brain; Chromatography, Gel; Detergents; Dipeptidases; Endorphins; Leucine; Oligopeptides; Peptides; Rats; Rats, Inbred Strains; Substrate Specificity; Synaptosomes