MECHANISMS REGULATING THE RESPONSIVENESS OF CELLS TO SUBSTANCE-P - CELL-SURFACE DEGRADATION OF SUBSTANCE-P AND NK-1 RECEPTOR ENDOCYTOSIS

Substance P (SP) degradation by the cell-surface enzyme neutral endope ptidase (NEP), and endocytosis of the NK-1 receptor (NK-1R) are potent ial mechanisms that limit the responsiveness of cells to SP. These mec hanisms were studied in epithelial cells (KNRK) transfected with cDNA encoding an epitope labeled Flag-NK-1R or NEP. In cells expressing NK- 1R alone, specific saturable I-125-SP binding at 37 degrees C was 28% +/- 3% of total counts. Go-expression of NEP with the NK-1R reduced bi nding to 4 +/- 0.3% of total counts. Addition of the NEP inhibitor thi orphan restored binding to control levels. In cells expressing NK-1R a lone, SP (1 nM) stimulated an increase in [Ca2+](i) of 163 +/- 16 nM. Co-expression of NEP reduced the [Ca2+](i) response to 52 +/- 13 nM, a nd this response was restored to control levels by thiorphan. Antibodi es to the NK-1R and rhodamine labeled SP were used to observe endocyto sis in KNRK cells expressing the NK-1R alone. When cells were incubate d with SP for 60 min at 4 degrees C, NK-1R and rhodamine SP were confi ned to the plasma membrane. After 3 and 10 min at 37 degrees C, there was a reduction in cell-surface staining and NK-1R and rhodamine-SP we re localized in intracellular vesicles. Binding experiments with I-125 -SP confirmed the rapid internalization of peptide. Therefore, the NK- 1R and SP are internalized within minutes of binding. Thus, ligand deg radation and receptor endocytosis limit the cellular response to SP.