Activation of phospholipase C increases intramembrane electric fields in N1E-115 neuroblastoma cells

Abstract

We imaged the intramembrane potential (a combination of transmembrane, surface, and dipole potential) on N1E-115 neuroblastoma cells with a voltage-sensitive dye. After activation of the B(2) bradykinin receptor, the electric field sensed by the dye increased by an amount equivalent to a depolarization of 83 mV. The increase in intramembrane potential was blocked by the phospholipase C (PLC) inhibitors U-73122 and neomycin, and was invariably accompanied by a transient rise of [Ca(2+)](i). A depolarized inner surface potential, as the membrane loses negative charges via phosphatidylinositol 4,5-bisphosphate (PIP(2)) hydrolysis, and an increase in the dipole potential, as PIP(2) is hydrolyzed to 1,2-diacylglycerol (DAG), can each account for a small portion of the change in intramembrane potential. The primary contribution to the measured change in intramembrane potential may arise from an increased dipole potential, as DAG molecules are generated from hydrolysis of other phospholipids. We found bradykinin produced an inhibition of a M-type voltage-dependent K(+) current (I(K(M))). This inhibition was also blocked by the PLC inhibitors and had similar kinetics as the bradykinin-induced modulation of intramembrane potential. Our results suggest that the change in the local intramembrane potential induced by bradykinin may play a role in mediating the I(K(M)) inhibition.

FIGURE 1

Fluorescence ratios of N1E-115 neuroblastoma cells in response to bradykinin. At time = 0, 1 μM bradykinin or EBSS was externally applied to the bathing medium of a cell stained with di-8-ANEPPS. The 440 nm/530 nm fluorescence ratios were collected with a cooled CCD camera. The relative change in ratio (left Y axis) and the calculated change in intramembrane potential as an equivalent of transmembrane potential (right Y axis) versus time were plotted. Each point is the mean ± SE of bradykinin-treated (filled circle, n = 11) or EBSS-treated cells (empty circle, n = 5).

FIGURE 2

Bradykinin increases the intramembrane potential along the surface of a single N1E-115 neuroblastoma cells. (A) The intramembrane potential along the surface of the cell, measured by the pseudocolored 440 nm/530 nm fluorescence ratios, increases to a peak at 160 s and decays to baseline in the continued presence of bradykinin. (B) The 440 nm/530 nm fluorescence ratio of the whole cell (^*) and of high ratio area (framed area in panel A, +) versus time. (C) The relative change in ratio (left Y axis) and the calculated change in intramembrane potential as an equivalent of transmembrane potential (right Y axis) for the whole cell (^*) and the high ratio area (filled +) versus time.

FIGURE 3

Effect of bradykinin (1 μM, added at arrow) on M-like current in N1E-115 neuroblastoma cell. (A) Membrane potential change induced by bradykinin (1 μM) in an untreated cell (panel a) and U-73122 pretreated cell (panel b). Membrane potential was recorded by whole-cell current clamp, and membrane resistance was monitored by the voltage response to brief hyperpolarizing current injections (0.15 nA). Average resting membrane potential of N1E-115 cells is −37.0 ± 2.2 mV (n = 9) for untreated cells, −30.7 ± 2.4 mV (n = 8) for U-73122 pretreated cells, and −29.2 ± 6.6 mV (n = 5) for neomycin. (c) The change in membrane potential, expressed as the measured membrane potential minus the baseline membrane potential, for the cell in panel A, a (open circle), and for the cell in panel A, b (closed circle), versus time are plotted. Bradykinin (1 μM) was added at time 0. (d) The PLC inhibitors blocks BK-induced membrane depolarization. Mean ± SE modulation of transmembrane potential by bradykinin (1 μM), control vehicle (EBSS) (n = 9), or bradykinin after pretreatment with U-73122 (n = 8) and neomycin (n = 5) are plotted. Bradykinin induced significant (P < 0.01, ^**) membrane depolarization in unpretreated cells, which was abolished by pretreatment with U-73122 and neomycin. (B) Leak-subtracted current responses of another voltage-clamped cell. The cell was clamped at −30 mV and stepped to −70 mV and 0 mV for 1 s before (panel a) and after (panel b) addition of bradykinin (1 μM). The graph in panel B, c shows the absolute current level attained at the end of each voltage step (ordinates, nA) plotted against the command potential (abscissae, mV) before (open circle) and after (closed circle) addition of bradykinin.

FIGURE 4

Effects of bradykinin on modulating intracellular Ca and intramembrane potentials in the responding N1E-115 cells. At time = 0, 1 μM bradykinin was externally applied to the bathing medium of the cells stained with both Calcium Green-1-AM (CG-1) and di-8-ANEPPS. Calcium Green fluorescence (dashed curve) and fluorescence ratios of di-8-ANEPPS (solid curve) recorded simultaneously were plotted versus time. Each data point represents the mean ± SE of the eight cells that showed significant response to bradykinin.

FIGURE 5

Correlation between the relative change in intramembrane potential and the increase in Calcium Green fluorescence induced by bradykinin. The relative change in Calcium Green fluorescence at 10 s was plotted versus the relative change in the 440 nm/530 nm fluorescence ratio of di-8-ANEPPS at 150 s after the addition of bradykinin (1 μM). A correlation coefficient (r) of 0.70 was obtained by correlation analysis (P < 0.05). Data were obtained from 14 cells that were double stained with di-8-ANEPPS and Calcium Green-1-AM and then treated with 1 μM bradykinin at time = 0.

FIGURE 6

The PLC inhibitors block BK-induced increase in intramembrane potential. Mean ± SE modulation of di-8-ANEPPS fluorescence ratio and corresponding intramembrane potential by bradykinin (1 μM) in neuroblastoma cells treated with EBSS (n = 15), U-73343 (n = 11), U-73122 (n = 10), or neomycin (n = 8) is plotted. The BK-mediated increase in ratio is significantly lower in cells treated with U-73122 and neomycin than in control EBSS treated cells (P < 0.01, ^**).

FIGURE 7

DAG induces wavelength shifts revealed by normalized difference spectra of PS lipid vesicles containing 0.2 μM di-4-ANEPPS. The plot represents the difference in the normalized excitation spectrum of pure PS vesicles and vesicles composed of 9:1 PS:DAG; the emission wavelength is 610 nm.