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

Bradykinin has multiple effects on differentiated NG108–15 neuroblastoma×glioma cells: it increases Ins(1,4,5)P 3 production and intracellular Ca 2+ concentration [Ca 2+ ] i , evokes a Ca 2+ 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. Preincubation with 1 or 5 μ m U73122 for 15 min partly suppressed Ins(1,4,5)P 3 generation and the increase in [Ca 2+ ] i induced by 1 μ m bradykinin. U73122 10 μ m caused total and irreversible inhibition. The inactive analogue U73343 was without effect. Resting levels of Ins(1,4,5)P 3 were not affected. However, resting [Ca 2+ ] i was increased by 10 μ m U73122, but not by U73343. Individual cells responded to 10 μ m U73122 with a small increase in [Ca 2+ ] i , followed in some cells by a large further rise. Pretreatment of whole‐cell clamped cells with 1 μ 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 pretreatment with 5 μ m U73122 was required. Again, U73343 was without effect. U73122 and U73343 at concentrations of 5–10 μ m 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. M current ( I M ) measured with 1 s pulses, was also decreased by 5–10 μ m 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. Preincubation with 1 or 3 m m neomycin for 15 min did not affect Ins(1,4,5)P 3 generation and the increase in [Ca 2+ ] i induced by bradykinin. Pretreatment with 3 m m neomycin for about 20 min diminished the bradykinin‐induced I K(Ca) to a fifth of its normal size. 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 Ca 2+ from intracellular stores. (d) Externally applied neomycin is not an effective inhibitor of PLC‐mediated signalling pathways in NG108–15 cells.British Journal of Pharmacology (1997) 120 , 841–850; doi: 10.1038/sj.bjp.0700991