Effects of inhibitors of the lipo-oxygenase family of enzymes on the store-operated calcium current I(CRAC) in rat basophilic leukaemia cells

Abstract

In non-excitable cells, the major Ca2+ entry pathway is the store-operated pathway in which emptying of intracellular Ca2+ stores activates Ca2+ channels in the plasma membrane. In many cell types, store-operated influx gives rise to a Ca2+-selective current called I(CRAC) (Ca2+ release-activated Ca2+ current). Using both the whole-cell patch clamp technique to measure I(CRAC) directly and fluorescent Ca2+ imaging, we have examined the role of the lipo-oxygenase pathway in the activation of store-operated Ca2+ entry in the RBL-1 rat basophilic leukaemia cell-line. Pretreatment with a variety of structurally distinct lipo-oxygenase inhibitors all reduced the extent of I(CRAC), whereas inhibition of the cyclo-oxygenase enzymes was without effect. The inhibition was still seen in the presence of the broad protein kinase blocker staurosporine, or when Na+ was used as the charge carrier through CRAC channels. The lipo-oxygenase blockers released Ca2+ from intracellular stores but this was not associated with subsequent Ca2+ entry. Lipo-oxygenase blockers also reduced both the amount of Ca2+ that could subsequently be released by the combination of thapsigargin and ionomycin in Ca2+-free solution and the Ca2+ influx component that occurred when external Ca2+ was re-admitted. The inhibitors were much less effective if applied after I(CRAC) had been activated. This inhibition of I(CRAC) could not be rescued by dialysis with 5(S)-hydroxyperoxyeicosa-6E,8Z,11Z,14Z,tetraenoic acid (5-HPETE), the first product of the 5-lipo-oxygenase pathway. Our findings indicate that exposure to pharmacological tools that inhibit the lipo-oxygenase enzymes all decrease the extent of activation of the current. Our results raise the possibility that a lipo-oxygenase might be involved in the activation of I(CRAC). Alternative explanations are also discussed.

Figure 1. The lipo-oxygenase inhibitor NDGA interferes with the activity of I_CRAC

A, the development of I_CRAC (measured at −80 mV from the voltage ramps) in control conditions (trace i, open circles) and then following pre-incubation (> 15 min) with different concentrations of NDGA. B, I–V relationships taken when the currents had reached steady state (80–100 s). Stores were emptied by dialysis with InsP₃+ 10 mm EGTA. C, the relationship between the concentration of NDGA and inhibition of I_CRAC is shown. D, the delay before I_CRAC activates versus concentration of NDGA. E, time to peak versus NDGA concentration. In this, and all subsequent figures, the data points are means ± s.e.m.

Figure 2. Structurally distinct lipo-oxygenase blockers all reduce the extent of I_CRAC

A, the effect of various inhibitors on the activation of I_CRAC. Ctrl (open circles) denotes control (0.1% DMSO-treated cells), Indo (filled circles) denotes indomethacin (50 μm). B, the histogram summarizes mean data from several cells. Gossy denotes gossypol, Keto denotes ketoconazole. 20 and 50 Indo represent 20 and 50 μm Indo, respectively. All inhibitors were tested at a concentration of 20 μm (except gossypol which was used at 5 μm), and pre-incubated for at least 15 min. *P < 0.05 and ^**P < 0.01. C, amplitude of I_CRAC against activation time constant. D, amplitude of I_CRAC against time to peak. E, table summarizing the percentage block of I_CRAC for the various inhibitors together with reported IC₅₀ values for the different lipo-oxygenases, taken from the literature. Data for Ndga are from Salari et al. (1984) and Hope et al. (1983); data for Indo are from Salari et al. (1984) and Shen & Winter (1977); data for Etya are from Salari et al. (1984), Taylor et al. (1985) and Bokoch & Reed, 1981; data for Gossy were from Hamasaki & Tai (1985); and data for Cdc were from Cho et al. (1991).

Figure 3. Lipo-oxygenase inhibition of I_CRAC persists in the presence of thapsigargin, ATP and staurosporine

A, the size of I_CRAC in control cells, in those pre-exposed to 20 μm NDGA, and then after either 2 mm Mg-ATP or 2 μm thapsigargin have been included in the pipette solution following pre-exposure to NDGA. B, the size of I_CRAC in control cells, after exposure to 20 μm CDC, and then after inclusion of either Mg-ATP or thapsigargin in the pipette solution following CDC pretreatment. Also included are data taken from cells first exposed to staurosporine and then CDC. Note that CDC still reduces I_CRAC even in the presence of the kinase blocker. Each bar is the mean of 4–7 cells.

Figure 4. NDGA reduces store-operated Ca²⁺ influx to thapsigargin and ionomycin

A, Ca²⁺ release by the combination of ionomycin (100 nm) and thapsigargin (2 μm) is followed by store-operated Ca²⁺ influx when external Ca²⁺ is readmitted. B, pretreatment with NDGA (> 20 min) abolishes Ca²⁺ release to ionomycin/thapsigargin and subsequent Ca²⁺ entry is substantially reduced. C, pooled data for the extent of Ca²⁺ release for control (i.e. where thapsigargin and ionomycin were applied) cells and for those pre-exposed to NDGA. D, the extent of store-operated Ca²⁺ entry for control cells and after pre-treatment with NDGA. Included in the bar chart are the results from cell pre-exposed to thapsigargin for 15 min, perfused with Ca²⁺-free solution, and then external Ca²⁺ was re-admitted (see text for details). E, the time to peak of the store-operated Ca²⁺ entry signal for the different conditions indicated. The Thap pre-treatment data are discussed in a later section.

Figure 5. NDGA releases Ca²⁺ in a concentration-dependent manner

Aa, the time course of Ca²⁺ release from a cell exposed to 5 μm NDGA. b depicts a similar experiment but now in the presence of external Ca²⁺. B, time course of the Ca²⁺ response in a cell in which stores were fully depleted (combination of ionomycin and thapsigargin) in the presence of external Ca²⁺. C, dose-response curve to NDGA in the absence (open circles) and presence (filled circles) of external Ca²⁺. Included in the graph are the responses to ionomycin+ thapsigargin in the absence (open triangle) and presence (filled triangle) of external Ca²⁺. These latter two points are included in the graph for comparative purposes, but the cells were not exposed to NDGA.

Figure 6. Effects of other lipo-oxygenase blockers on Ca²⁺ release

A, ETYA (20 μm) evoked a small Ca²⁺ increase both in the absence (a) and presence (b) of external Ca²⁺. The integrated signals were not significantly different (c). B, CDC and gossypol both caused a fall in the fluorescence ratio, due to a decrease in the 356 nm signal.

Figure 7. Pretreatment with thapsigargin does not prevent I_CRAC from developing

A, recordings taken from a control cell and from a cell that had been exposed to 2 μm thapsigargin in 10 mm external Ca²⁺ for 20 min. B, size of I_CRAC against half-time for control and thapsigargin-treated cells. C, size of the current against the time to peak for the two conditions.

Figure 8. Pretreatment with either NDGA or CDC suppresses the Na⁺ current through CRAC channels in divalent ion-free external solution

Aa, comparison of the time course of the monovalent Na⁺ current for a control cell and that for a cell which had been pre-incubated with 20 μm NDGA for around 15 min. b, the ramp I–V relationships are shown for the cells in a, taken at 60 s for both conditions. c, mean data from four control cells and four cells pre-exposed to NDGA. Ba, time course of a control cell and a cell pre-incubated with 20 μm CDC. b, corresponding ramp I–V. c, mean data from four control cells and four cells pre-exposed to CDC prior to break-in. Data have been normalized to the amplitude of the controls.

Figure 9. Effect of application of the lipo-oxygenase inhibitors after I_CRAC had developed

A, the effects of applying 50 μm NDGA after I_CRAC had developed in two cells. Ramp I–V relations are shown on the right. B, the effects of applying ETYA either before (filled circles) or after (open circles) I_CRAC had activated. C, comparison of the ability of CDC to interfere with I_CRAC when applied before (filled circles) or after (open circles) I_CRAC had activated. D, summary of the effects of the three inhibitors on I_CRAC. The filled columns represent the inhibition seen when cells were pre-incubated with each drug and the open columns reflect the inhibition seen following exposure to each drug after I_CRAC had developed. For cells pre-exposed to inhibitors, recordings were normalized to controls taken from the same preparations in the absence of inhibitors. For those experiments where drugs were applied after I_CRAC had developed, the steady-state level in the presence of inhibitors was normalized to the peak amplitude reached before the inhibitors were applied. These latter currents were not significantly different from the controls used for normalizing the currents obtained following pretreatment with the lipo-oxygenase blockers. Each column represents the mean of 6–11 cells. In all cases, inhibition was significantly less if the drug was applied after store depletion than before.

Figure 10. Effects of 5-HPETE on I_CRAC

A, representative whole-cell recording from a cell dialysed with 5-HPETE in 120 nm buffered Ca²⁺. I_CRAC did not activate. After 180 s, thapsigargin was applied and I_CRAC subsequently developed. B, size of I_CRAC following thapsigargin application in control cells, and in those dialysed with 5-HPETE. There was no significant difference. C, control recording (filled circles), recording taken after pre-exposure to CDC (diamonds) and recording after pre-exposure to CDC but with 5-HPETE in the pipette solution. The recording pipette contained InsP₃+ 10 mm EGTA. Data have been normalized to control recordings. D, pooled data indicating that the reduction in I_CRAC amplitude following pretreatment with CDC cannot be rescued by inclusion of 5-HPETE in the pipette.