Interactions between neutral endopeptidase (EC 3.4.24.11) and the substance P (NK₁) receptor expressed in mammalian cells

Interactions between neutral endopeptidase-24.11 (NEP) and the substance P receptor (SPR;NK₁) were investigated by examining substance P (SP) degradation, SP binding and SP-induced Ca²⁺ mobilization in epithelial cells transfected with cDNA encoding the rat SPR and rat NEP. Expression of NEP accelerated the degradation of SP by intact epithelial cells and by membrane preparations, and degradation was reduced by the NEP inhibitor thiorphan. In cells expressing SPR alone, specific ¹²⁵I-SP binding after 20 min incubation at 37°C was 92.2±3.1% of maximal binding and was unaffected by thiorphan. Coexpression of NEP in the same cells as the SPR markedly reduced SP binding to 13.9±0.5% of maximal, and binding was increased to 82.7±2.4% of maximal with thiorphan. Coexpression of NEP in the same cells as the SPR also reduced to undetectable the increase in intracellular Ca²⁺ in response to low concentrations of SP (0.3 and 0.5 nM), and significantly reduced the response to higher concentrations (1 and 3 nM). The Ca²⁺ response was restored to control values by inhibition of NEP with thiorphan. In contrast, SP binding and SP-induced Ca²⁺ mobilization were only slightly reduced when cells expressing SPR alone were mixed with a 3- to 24-fold excess of cells expressing NEP alone. Therefore, in this system, NEP markedly down-regulates SP binding and SP-induced Ca²⁺ mobilization only when coexpressed in the same cells as the SPR.