Chlorpromazine inhibits store-operated calcium entry and subsequent noradrenaline secretion in PC12 cells

Abstract

1. The effect of chlorpromazine on the store-operated Ca2+ entry activated via the phospholipase C signalling pathway was investigated in PC12 cells. 2. Chlorpromazine inhibited the sustained increase after the initial peak in the intracellular Ca2+ concentration produced by bradykinin while having no effect on the initial transient response. The inhibition was lowered by the removal of extracellular free Ca2+. However, chlorpromazine did not inhibit bradykinin-induced inositol 1,4,5-trisphosphate production. 3. Chlorpromazine inhibited the bradykinin-induced noradrenaline secretion in a concentration-dependent manner (IC(50): 24+/-5 microM, n=3). 4. To test for a direct effect of chlorpromazine on store-operated Ca2+ entry, thapsigargin, an inhibitor of microsomal Ca(2+)-ATPase, was used to induce store-operated Ca2+ entry in PC12 cells. Chlorpromazine reduced the thapsigargin-induced sustained Ca2+ level (IC(50): 24+/-2 microM, n=3), and the inhibition also occluded the inhibitory action of 1-[-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenyl]-1H-imidazole hydrochloride (SK&F96365). 5. The results suggest that chlorpromazine negatively modulates the store-operated Ca2+ entry activated subsequent to PLC activation.

Figure 1

Effect of chlorpromazine on bradykinin-induced [Ca²⁺]_i rise in PC12 cells. (A) Fura-2-loaded cells were challenged with 300 nM bradykinin (BK) in the presence (solid trace) or absence (hatched trace) of 30 μM chlorpromazine (CPZ). Typical Ca²⁺ traces obtained in more than five separate experiments are presented. The results were reproducible. (B) [Ca²⁺]_i levels at the time of the bradykinin treatment (2 s marked as a) and after 90 s (marked as b) in the data of A were quantitatively analysed. The levels of [Ca²⁺]_i are depicted as % of the bradykinin-induced [Ca²⁺]_i rise without chlorpromazine treatment. Each point was obtained and represents the mean±s.e.mean from triplicate experiments. *P<0.01. (C) The same experiments as in A were performed in the absence of extracellular free Ca²⁺. After the challenge with bradykinin, 2.2 mM CaCl₂ (Ca²⁺) was added. Typical Ca²⁺ traces obtained in more than five separate experiments are presented. The results were reproducible. (D) The [Ca²⁺]_i level after the bradykinin treatment (Release, marked as a) and after the CaCl₂ treatment (Influx, marked as b) in the data of C were quantitatively analysed. The levels of the [Ca²⁺]_i are depicted as % of the bradykinin-induced [Ca²⁺]_i rise without chlorpromazine treatment. Each point was obtained from triplicate experiments and represents the mean±s.e.mean. ^*P<0.01.

Figure 2

Inhibitory effect of chlorpromazine on the bradykinin-induced noradrenaline secretion of PC12 cells. [³H]-noradrenaline-loaded PC12 cells were treated with 300 nM bradykinin for 10 min in the presence of the indicated concentrations of chlorpromazine. The secretion of [³H]-noradrenaline induced by bradykinin in the absence of chlorpromazine is also presented. Three separate experiments were done, and each point represents a mean±s.e.mean value. The results were reproducible.

Figure 3

Effect of chlorpromazine on thapsigargin-induced store-operated Ca²⁺ entry (SOCE) in PC12 cells. (A) Fura-2-loaded cells were treated with the indicated concentrations of chlorpromazine (CPZ) after an incubation with 1 μM thapsigargin (TG). (B) Concentration-dependency of the chlorpromazine effect on thapsigargin-induced SOCE. Cells were treated with various concentrations of chlorpromazine after an incubation with 1 μM thapsigargin. Inhibition of [Ca²⁺]_i rise was calculated with reference to the [Ca²⁺]_i level at the point a, b, and c in A using the equation (b – c)/(b – a)×100. Data are depicted as % of the thapsigargin-induced Ca²⁺ level rise without chlorpromazine treatment. Each point was obtained from triplicate experiments and represents the mean±s.e.mean. The results were reproducible.

Figure 4

Effect of chlorpromazine on bradykinin- and thapsigargin-induced Mn²⁺ influx into PC12 cells. Mn²⁺-induced fura-2 fluorescence quenching was recorded for fura-2-loaded cells preincubated with 500 μM Mn²⁺ and drug addition at the indicated times (arrow). The influx of Mn²⁺ was measured as described in the Methods. The data represent fluorescence intensities at 360 nm (F₃₆₀). (A) Bradykinin-induced Mn²⁺ influx was monitored with the stimulation with 300 nM bradykinin (BK) in the presence or absence of 50 μM chlorpromazine (CPZ). The trace without stimuli was depicted as dotted trace (Control). (B) The times (Δt) during the changes in fluorescence (from 1.6 to 1.4, arbitrary units) were quantitatively analysed using the results in A. Each point was obtained from triplicate experiments and represents the mean±s.e.mean. ^*P<0.01. (C) Thapsigargin-induced Mn²⁺ influx was monitored with or without the stimulation with 1 μM thapsigargin (TG) in the presence or absence of 50 μM chlorpromazine (CPZ). The trace without stimuli was depicted as dotted trace (Control). They are representative of four independent experiments. The results were reproducible. (D) The times (Δt) during the changes in fluorescence (from 1.6 to 1.4, arbitrary units) were quantitatively analysed using the results in C. Each point was obtained from triplicate experiments and represents the mean±s.e.mean. ^*P<0.01.

Figure 5

Effect of SK&F96365 on the inhibition of the thapsigargin-induced SOCE by chlorpromazine. (A) Fura-2-loaded PC12 cells were treated with 1 μM thapsigargin (TG), then challenged with 50 μM chlorpromazine (CPZ) in the presence of 20 μM SK&F96365 (SKF). (B) The [Ca²⁺]_i level at point a, b, and c were quantitatively analysed using the results in A. Each point was obtained from triplicate experiments and represents the mean±s.e.mean. No statistical significance was evident between the data of b and c. (C) Cells were treated with 1 μM thapsigargin (TG), then challenged with 20 μM SK&F96365 (SKF) in the presence of 50 μM chlorpromazine (CPZ). The data are representative of more than four independent experiments. The results were reproducible. (D) The [Ca²⁺]_i level at point a, b, and c were quantitatively analysed using the results in C. Each point was obtained from triplicate experiments and represents the mean±s.e.mean. No statistical significance could be seen between the data of b and c.