THE EFFECTS OF BRADYKININ ON K-15 CELLS TREATED WITH U73122, A PHOSPHOLIPASE-C INHIBITOR, OR NEOMYCIN( CURRENTS IN NG108)

1 Bradykinin has multiple effects on differentiated NG108-15 neuroblas toma x glioma cells: it increases Ins(1,4,5)P-3 production and intrace llular Ca2+ concentration [Ca2+](i), evokes a Ca2+ activated K+ curren t (I-K(Ca)) and inhibits M current (I-M). We studied the effect of the aminosteroid U73122 and the antibiotic neomycin, both putative blocke rs of phospholipase C (PLC), on these four bradykinin effects. 2 Prein cubation with 1 or 5 mu M U73122 for 15 min partly suppressed Ins(1,4, 5)P-3 generation and the increase in [Ca2+](i) induced by 1 mu M brady kinin. U73122 10 mu M caused total and irreversible inhibition. The in active analogue U73343 was without effect. 3 Resting levels of Ins(1,4 ,5)P-3 were not affected. However, resting [Ca2+](i) was increased by 10 mu M U73122, but not by U73343. Individual cells responded to 10 mu M U73122 With a small increase in [Ca2+](i), followed in some cells b y a large further rise. 4 Pretreatment of whole-cell clamped cells wit h 1 mu M U73122 for 30 min reduced the bradykinin-induced I-K(Ca) to a fifth of its normal size. To suppress it totally, a 7-12 min pretreat ment with 5 mu M U73122 was required. Again, U73343 was without effect . 5 U73122 and U73343 at concentrations of 5-10 mu M irreversibly decr eased the holding current (I-h) which at a holding potential of -30 or -20 mV mainly flows through open M channels. The decrease was often p receded by a transient increase. 6 M current (I-M) measured with 1 s p ulses, was also decreased by 5-10 mu M U73122 and U73343, but short ap plications of U73122 could cause a small increase. The bradykinin-indu ced inhibition of I-M was not affected by U73122. 7 Preincubation with 1 or 3 mM neomycin for 15 min did not affect Ins(1,4,5)P-3 generation and the increase in [Ca2+](i) induced by bradykinin. Pretreatment wit h 3 mM neomycin for about 20 min diminished the bradykinin-induced I-K (Ca) to a fifth of its normal size. 8 The four main conclusions drawn from the results are: (a) U73122 suppresses bradykinin-induced PLC act ivation and I-K(Ca), but not I-M inhibition. (b) This indicates that t he transient outward current I-K(Ca), but not the decrease of I-M in r esponse to bradykinin, is mediated by PLC. (c) U73122 itself inhibits I-M and mobilizes Ca2+ from intracellular stores. (d) Externally appli ed neomycin is not an effective inhibitor of PLC-mediated signalling p athways in NG108-15 cells.