EFFECT OF THE NONPEPTIDE BLOCKER (+ -) CP-96,345 ON THE CELLULAR MECHANISM INVOLVED IN THE RESPONSE TO NK1 RECEPTOR STIMULATION IN HUMAN SKIN FIBROBLASTS/

The effect of the selective non-peptide antagonist for NK1 receptors ( +/-)CP 96,345 on cellular transduction mechanisms elicited by the NK1 selective agonist [Sar(9)]-substance P-sulfone ([Sar(9)]-SP) was inves tigated in a stabilized culture of human skin fibroblasts (HF) and com pared to the effects of two peptide antagonists, FK 888 and GR 82, 334 . The exposure of the cells to [Sar(9)]-SP (100 nM) produced an early increase in inositol 1,4,5-trisphosphate (IP3) level, which peaked aft er 6 s, and a later rise in cellular inositol 1-phosphate (IP1) conten t which reached the maximum level in 15 min. The cAMP level was not si gnificantly modified. The increase in IP1 was greatly reduced, at appr oximately the same extent by the 10 min pretreatment with a concentrat ion of (+/-)CP 96,345 (100 nM) 10 times smaller than that of FK 888 an d GR 82,334 (1 mu M). The cytosolic Ca2+ mobilization in response to t he NK1 agonist was monitored both by spectrofluorimetric and single-ce ll image analysis determinations on adherent cells loaded with the Ca2 +-sensitive fluorescent indicators Fura-2/AM and Indo-1, respectively. [Sar(9)]-SP (100 nM) produced a rapid increase in the intracellular C a2+ level in Fura-2/AM loaded cells. Cytosolic Ca2+ mobilization, meas ured by single-cell image analysis, indicated a concentration-dependen t increase in both the ratio and in the number of cells responding to [Sar(9)]SP. Either the non-peptide or the peptide selective NK1 recept or antagonists inhibited the increase in Ca2+ level in both the assays . In the spectrofluorimetric experiments the antagonizing effects of ( +/-)CP 96,345 (1-100 nM), FK 888 (10 nM-1 mu M) and GR 82,334 (10 nM-1 mu M) were concentration-dependent. Moreover, the non-peptide antagon ist was more potent than the two peptide antagonists, producing an 82. 5% inhibition of Ca2+ mobilization at a concentration (10 nM) at which FK 888 and GR 82,334 decreased the response by only 62.3 and 60%, res pectively. Stimulation of phosphatidylinositol turnover and calcium mo bilization were also induced by 10 nM bradykinin; these effects were i nfluenced neither by the previous administration of the NK1 receptor a gonist nor by the three antagonists tested. These results demonstrate that the cellular transduction mechanisms induced in human skin fibrob lasts by NK1 receptor stimulation are specifically and effectively ant agonized by (+/-)CP 96,345, and that this non-peptide antagonist is mo re potent than the two peptide antagonists tested.