The effects of bradykinin on K+ currents in NG108-15 cells treated with U73122, a phospholipase C inhibitor, or neomycin

Abstract

1. Bradykinin has multiple effects on differentiated NG108-15 neuroblastoma x glioma cells: it increases Ins(1,4,5)P3 production and intracellular Ca2+ concentration [Ca2+](i), evokes a Ca2+ activated K+ current (I(K(Ca)) and inhibits M current (I(M)). We studied the effect of the aminosteroid U73122 and the antibiotic neomycin, both putative blockers of phospholipase C (PLC), on these four bradykinin effects. 2. Preincubation with 1 or 5 μM U73122 for 15 min partly suppressed Ins(1,4,5)P3 generation and the increase in [Ca2+](i) induced by 1 μM bradykinin. U73122 10 μM caused total and irreversible inhibition. The inactive analogue U73343 was without effect. 3. Resting levels of Ins(1,4,5)P3 were not affected. However, resting [Ca2+](i) was increased by 10 μM U73122, but not by U73343. Individual cells responded to 10 μM U73122 with a small increase in [Ca2+](i), followed in some cells by a large further rise. 4. Pretreatment of whole-cell clamped cells with 1 μM 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 pretreatment with 5 μM U73122 was required. Again, U73343 was without effect. 5. U73122 and U73343 at concentrations of 5-10 μM irreversibly decreased 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 preceded by a transient increase. 6. M current (I(M)) measured with 1 s pulses, was also decreased by 5-10 μM U73122 and U73343, but short applications of U73122 could cause a small increase. The bradykinin-induced 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)P3 generation and the increase in [Ca2+](i) induced by bradykinin. Pretreatment with 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 activation and I(K(Ca)), but not I(M) inhibition. (b) This indicates that the transient outward current I(K(Ca)), but not the decrease of I(M), in response to bradykinin, is mediated by PLC. (c) U73122 itself inhibits I(M) and mobilizes Ca2+ from intracellular stores. (d) Externally applied neomycin is not an effective inhibitor of PLC-mediated signalling pathways in NG108-15 cells.