Peptidases of the peripheral chemoreceptors: biochemical, immunological, in vitro hydrolytic studies and electron microscopic analysis of neutral endopeptidase-like activity of the carotid body.
The purposes of the present study are to identify and characterize the major peptidase(s) that may be involved in the inactivation of neuropeptides in the mammalian carotid body. Measurements of a number of peptidase activities in the cell-free extract of the cat carotid body using specific substrates and inhibitors indicated that the previously identified neutral endopeptidase (NEP)-like activity [Kumar et al., Brain Res., 517 (1990) 341-343] is the major peptidase in the chemoreceptor tissue. The NEP-like activity of the carotid body was further characterized using a monoclonal antibody to human neutral endopeptidase, EC 3.4.24.11. Immune blot analysis indicated strong immunoreactivity toward the cat and calf carotid bodies but a weak cross-reactivity with the rabbit carotid body. Furthermore, western blot analysis of the cat carotid body extract revealed the presence of a major 97-kDa protein and a minor 200-kDa protein. The 97-kDa NEP form of the carotid body was comparable to EC 3.4.24.11 and was consistent with its reported molecular weight suggesting NEP-like activity of the carotid body is structurally similar to the neutral endopeptidase, EC 3.4.24.11. In order to assess whether NEP is the primary peptide degrading activity in the cat carotid body in vitro hydrolysis studies using substance P (SP) as a model peptide were performed. HPLC analysis showed that SP is hydrolyzed maximally at pH 7.0 by carotid body peptidases with the formation of SP(1-7) and SP(1-8) as stable intermediates. Inhibitors specific to NEP also inhibited the SP-hydrolyzing activity of the carotid body. Analyses of the cell-free extracts showed the occurrence of both NEP and SP-hydrolyzing activities in the rabbit and rat carotid bodies although at 2- and 4-fold lower levels respectively than that observed in the cat carotid body. Immunoelectron microscopy showed that NEP-specific immunoreactivity is associated with the intercellular region between the type I cells and cell clusters of the carotid body. Taken together, the results from this investigation demonstrate that neutral endopeptidase (EC 3.4.24.11) is one of the major endopeptidases which mediates the degradation and inactivation of neuropeptides in the carotid body.
Kumar
G K
GK
Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106-4935, USA. kgk@po.cwru.edu
D000818:Animals; D015153:Blotting, Western; D002344:Carotid Body / Q000187:drug effects / Q000201:enzymology*; D002415:Cats; D002417:Cattle; D002628:Chemoreceptor Cells / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism*; D005260:Female; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015151:Immunoblotting; D066298:In Vitro Techniques; D008297:Male; D008854:Microscopy, Electron; D015260:Neprilysin / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D013373:Substance P / Q000378:metabolism
D011480:Protease Inhibitors; D013373:Substance P; D010450:Endopeptidases; D015260:Neprilysin