Neuropeptide Y (NPY) metabolism by endopeptidase-2 hinders characterization of NPY receptors in rat kidney.
1. Despite the observation of pharmacological responses to neuropeptide Y (NPY) in mammalian kidneys, there are species differences in the ease with which specific NPY binding sites can be demonstrated; we have investigated whether this can be explained by differential metabolism of NPY by a membrane-bound peptidase. 2. NPY receptors were identified on cell membranes isolated from the rabbit kidney (KD = 97 +/- 16 pM, Bmax = 290 +/- 30 fmol mg-1 protein), and this preparation did not degrade [125I]-NPY. However, a similar preparation of cell membranes from the rat kidney exhibited a much lower apparent receptor affinity (IC50 approximately 30 nM); these membranes rapidly degraded [125I]-NPY to fragments which did not bind NPY receptors in either tissue. 3. [125I]-NPY binding sites were revealed in the rat kidney when degradation was inhibited by insulin B chain. Chelating agents also inhibited degradation, but interfered with receptor binding. Binding sites could not be demonstrated in sections of rat kidney, even in the presence of insulin B chain. 4. The difference in degradative activity between rat and rabbit renal cell membranes, inhibition of degradation by chelating agents and insulin B chain, and insensitivity to phosphoramidon suggest that the enzyme responsible was endopeptidase-2, and this was confirmed by comparing the hydrolysis of [125I]-NPY by purified enzyme with rat renal tissue. Activity of this enzyme explains the difficulties encountered demonstrating receptors in the rat kidney. 5. Renal cell membranes from the mouse digested [125I]-NPY in a similar manner and this may be due to the closely related enzyme, meprin. NPY degradation has not previously been reported. The results suggest that NPY should be added to the list of peptides sensitive to these enzymes.
Price
J S
JS
Clinical Pharmacology Unit, University of Cambridge Clinical School, Addenbrooke's Hospital.
Kenny
A J
AJ
Huskisson
N S
NS
Brown
M J
MJ
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism*; D008297:Male; D008666:Metalloendopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009478:Neuropeptide Y / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D011863:Radioimmunoassay; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter / Q000378:metabolism*
D009478:Neuropeptide Y; D010446:Peptide Fragments; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter; D008666:Metalloendopeptidases; C053833:meprin A