Gαi/o-dependent Ca(2+) mobilization and Gαq-dependent PKCα regulation of Ca(2+)-sensing receptor-mediated responses in N18TG2 neuroblastoma cells

Abstract

A functional Ca(2+)-sensing receptor (CaS) is expressed endogenously in mouse N18TG2 neuroblastoma cells, and sequence analysis of the cDNA indicates high homology with both rat and human parathyroid CaS cDNAs. The CaS in N18TG2 cells appears as a single immunoreactive protein band at about 150 kDa on Western blots, consistent with native CaS from dorsal root ganglia. Both wild type (WT) and Gαq antisense knock-down (KD) cells responded to Ca(2+) and calindol, a positive allosteric modulator of the CaS, with a transient increase in intracellular Ca(2+) concentration ([Ca(2+)]i), which was larger in the Gαq KD cells. Stimulation with 1 mM extracellular Ca(2+) (Ca(2+)e) increased [Ca(2+)]i in N18TG2 Gαq KD compared to WT cells. Ca(2+) mobilization was dependent on pertussis toxin-sensitive Gαi/o proteins and reduced by 30 μM 2-amino-ethyldiphenyl borate and 50 μM nifedipine to the same plateau levels in both cell types. Membrane-associated PKCα and p-PKCα increased with increasing [Ca(2+)]e in WT cells, but decreased in Gαq KD cells. Treatment of cells with 1 μM Gӧ 6976, a Ca(2+)-specific PKC inhibitor reduced Ca(2+) mobilization and membrane-associated PKCα and p-PKCα in both cell types. The results indicate that the CaS-mediated increase in [Ca(2+)]i in N18TG2 cells is dependent on Gαi/o proteins via inositol-1,4,5-triphosphate (IP3) channels and store-operated Ca(2+) entry channels, whereas modulation of CaS responses involving PKCα phosphorylation and translocation to the plasma membrane occurs via a Gαq mechanism.

Keywords: Ca(2+) mobilization; CaS responses; Gα(i/o); Gα(q) antisense knock-down; N18TG2 cells; Neuronal CaS; Protein kinase C.

Figure 1. Expression analysis of CaS in N18TG2 neuroblastoma cells

A. Total RNA was extracted from cells and subjected to RT-PCR with primers specific for the full length dorsal root ganglia CaS and analyzed on agarose gels followed by staining with ethidium bromide. M, 1 kB DNA ladder; WT, Wild type N18TG2 cells; Gα_q KD, N18TG2 cells in which Gα_q protein was knocked down by anti-sense nucleotide expression. B. Western blot analysis of plasma membrane proteins from (i) N18TG2 cells, and (ii) total proteins from DRG and parathyroid (PT) isolated from rats with the polyclonal anti-CaS antibody and horseradish peroxidase (HRP)-conjugated IgG and developed with ECL. The CaS protein from N18TG2 cells, DRG and PT migrated as single bands of ≈ 140 kDa. C. Western blot analysis showing reduced expression of Gα_q N18TG2 cells. Data are expressed as means ± SEM (n = 4 experiments).

Figure 2. Pharmacologic properties of the CaS-mediated Ca²⁺ mobilization in N18TG2 WT and Gα_q KD cells

Cells grown on glass cover slips were loaded with 5 μM Fura-2/AM and responses to 1 mM Ca²⁺ and calindol were determined by microfluorimetry. A. Responses of N18TG2 WT and N18TG2 Gα_q KD cells to 1 mM Ca²⁺ are shown. After the experimental treatment, the maximum fluorescence in response to 10 μM ionomycin was determined, followed by addition of 20 mM EGTA to determine the Ca²⁺-free minimum fluorescence. B. A histogram showing changes in the peak heights of [Ca²⁺]_i in cells following stimulation. Data are expressed as mean ± SEM (n = 4 experiments). C.-E. Responses to 5 μM and 10 μM calindol determined by microfluorimetry. C. N18TG2 WT cells, D. N18TG2 Gα_q KD cells. E. Histogram showing changes in the peak heights of [Ca²⁺]_i in cells following stimulation with calindol. Data are expressed as means ± SEM (n = 5 experiments). *Significantly different from WT cells (p < 0. 05).

Figure 2. Pharmacologic properties of the CaS-mediated Ca²⁺ mobilization in N18TG2 WT and Gα_q KD cells

Cells grown on glass cover slips were loaded with 5 μM Fura-2/AM and responses to 1 mM Ca²⁺ and calindol were determined by microfluorimetry. A. Responses of N18TG2 WT and N18TG2 Gα_q KD cells to 1 mM Ca²⁺ are shown. After the experimental treatment, the maximum fluorescence in response to 10 μM ionomycin was determined, followed by addition of 20 mM EGTA to determine the Ca²⁺-free minimum fluorescence. B. A histogram showing changes in the peak heights of [Ca²⁺]_i in cells following stimulation. Data are expressed as mean ± SEM (n = 4 experiments). C.-E. Responses to 5 μM and 10 μM calindol determined by microfluorimetry. C. N18TG2 WT cells, D. N18TG2 Gα_q KD cells. E. Histogram showing changes in the peak heights of [Ca²⁺]_i in cells following stimulation with calindol. Data are expressed as means ± SEM (n = 5 experiments). *Significantly different from WT cells (p < 0. 05).

Figure 3. Effect of blocking Gα_i/o with PTX on CaS-mediated mobilization of Ca²⁺ in N18TG2 WT and Gα_q KD cells

Cells, grown on glass cover slips and treated with PTX (50 ng/ml) or vehicle for 16 hr. were loaded with 5 μM Fura-2/AM for 30 min before measurement of responses to 1 mM Ca²⁺ (A-C) and 5 μM calindol (D, E). A. N18TG2 WT cells, B. N18TG2 Gα_q KD cells, C. Histogram showing changes in the peak height of [Ca²⁺]_i following stimulation with 1 mM Ca²⁺. Responses of cells to 5 μM calindol following PTX treatment; D. N18TG2 WT cells; E. N18TG2 Gα_q KD cells. Data are expressed as means ± SEM (n = 4-6 experiments). *Significantly different from controls (p < 0.05).

Figure 3. Effect of blocking Gα_i/o with PTX on CaS-mediated mobilization of Ca²⁺ in N18TG2 WT and Gα_q KD cells

Cells, grown on glass cover slips and treated with PTX (50 ng/ml) or vehicle for 16 hr. were loaded with 5 μM Fura-2/AM for 30 min before measurement of responses to 1 mM Ca²⁺ (A-C) and 5 μM calindol (D, E). A. N18TG2 WT cells, B. N18TG2 Gα_q KD cells, C. Histogram showing changes in the peak height of [Ca²⁺]_i following stimulation with 1 mM Ca²⁺. Responses of cells to 5 μM calindol following PTX treatment; D. N18TG2 WT cells; E. N18TG2 Gα_q KD cells. Data are expressed as means ± SEM (n = 4-6 experiments). *Significantly different from controls (p < 0.05).

Figure 4. Effect of Ca²⁺ channel blockers on CaS-mediated Ca²⁺ bilization in WT and Gα_q KD N18TG2 cells

Responses of N18TG2 WT (A, D) and N18TG2 Gα_q KD (B, E) cells loaded with 5 μM Fura-2/AM were pre-incubated with 30 μM 2-APB (A.- C.) or 50 μM nifedipine (D.-F.) for 20 min in PSS containing 0.25 mM Ca²⁺ before measurement of responses to 1 mM Ca²⁺. Tracings are representative of 4 separate experiments carried out under similar conditions. C, F. Histograms showing changes in the peak heights of [Ca²⁺]_i in cells following stimulation with 1 mM Ca²⁺. Data are expressed as means ± SEM (n = 4 experiments). *Significantly different from controls (p < 0.05).

Figure 4. Effect of Ca²⁺ channel blockers on CaS-mediated Ca²⁺ bilization in WT and Gα_q KD N18TG2 cells

Responses of N18TG2 WT (A, D) and N18TG2 Gα_q KD (B, E) cells loaded with 5 μM Fura-2/AM were pre-incubated with 30 μM 2-APB (A.- C.) or 50 μM nifedipine (D.-F.) for 20 min in PSS containing 0.25 mM Ca²⁺ before measurement of responses to 1 mM Ca²⁺. Tracings are representative of 4 separate experiments carried out under similar conditions. C, F. Histograms showing changes in the peak heights of [Ca²⁺]_i in cells following stimulation with 1 mM Ca²⁺. Data are expressed as means ± SEM (n = 4 experiments). *Significantly different from controls (p < 0.05).

Figure 5. Expression of multiple isoforms of PKC in N18TG2 WT, Gα_q KD, and PTX-treated N18TG2 cells

Gene expression profiles of PKC isoforms from N18TG2 cells, as indicated, were determined using a Qiagen qPCR gene expression array. ΔΔCT values were calculated, converted to the antilog and normalized to N18TG2 WT PKCα as 100. Data are means (± SEM) from three separate experiments compared by 2-way ANOVA, which indicated significant differences between cell types/treatment groups as well as for the indicated PKC isoforms. Differences due to cell type/treatment determined by a Bonferroni post-hoc test are indicated. The average Coefficient of Variation (n = 3 gene expression arrays) of the CT values for the data shown was 0.038. *Significantly different from WT (p<0.05).

Figure 6. Effect of activation of PKC with PMA on CaS-mediated Ca²⁺ mobilization in WT and Gα_q KD N18TG2 cells

N18TG2 WT (A) and N18TG2 Gα_q KD (B) cells loaded with 5 μM Fura-2/AM were pre-incubated with 100 nM PMA for 20 min before measurement of responses to 1 mM Ca²⁺ Tracings are representative of 4 separate experiments. C. Histogram showing changes in the peak heights of [Ca²⁺]_i (means ± SEM) in cells following stimulation with 1 mM Ca²⁺ . Data are expressed as mean ± SEM (n = 4 experiments). *Significantly different from control (p < 0.05).

Figure 7. Effect of PKC inhibition on Ca²⁺ mobilization by calindol in N18TG2 WT and Gα_q KD cells

N18TG2 WT (A) and N18TG2 Gα_q KD (B) cells loaded with 5 μM Fura-2/AM were pre-incubated with 100 nM or 1 μM of the Ca²⁺-specific PKC inhibitor, Gö 6976 and responses to 5 μM calindol determined. Tracings are representative of separate experiments; insets are histograms showing changes in the peak heights of [Ca²⁺]_i in cells following stimulation. Data are expressed as means ± SEM (n = 4-9 experiments). *Significantly different from control and 100 nM calindol (p < 0.05).

Figure 8. [Ca²⁺]_e-dependent membrane translocation of PKCα in N18TG2 cells

Cells were stimulated with the indicated [Ca²⁺]_e for 30 min and isolated plasma membrane and supernatant fractions (100 μg/lane) analyze;d by SDS-PAGE followed by blotting with polyclonal PKCα antibody. β-Actin was used as the loading control. A. N18TG2 WT cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. B. N18TG2 Gα_q KD cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. C. N18TG2 WT cells: i) Western blot; ii) Histogram showing densitometric analysis of p-PKCα bands. D. N18TG2 Gα_q KD cells: i) Western blot; ii) Histogram showing densitometric analysis of p-PKCα bands. Bands were normalized to β-actin (loading control) and calculated as means (± SEM). Blots shown are representative of 4 separate experiments carried out under similar conditions. *Significantly higher than basal (0.25 mM Ca²⁺; p < 0.05).

Figure 8. [Ca²⁺]_e-dependent membrane translocation of PKCα in N18TG2 cells

Cells were stimulated with the indicated [Ca²⁺]_e for 30 min and isolated plasma membrane and supernatant fractions (100 μg/lane) analyze;d by SDS-PAGE followed by blotting with polyclonal PKCα antibody. β-Actin was used as the loading control. A. N18TG2 WT cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. B. N18TG2 Gα_q KD cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. C. N18TG2 WT cells: i) Western blot; ii) Histogram showing densitometric analysis of p-PKCα bands. D. N18TG2 Gα_q KD cells: i) Western blot; ii) Histogram showing densitometric analysis of p-PKCα bands. Bands were normalized to β-actin (loading control) and calculated as means (± SEM). Blots shown are representative of 4 separate experiments carried out under similar conditions. *Significantly higher than basal (0.25 mM Ca²⁺; p < 0.05).

Figure 8. [Ca²⁺]_e-dependent membrane translocation of PKCα in N18TG2 cells

Cells were stimulated with the indicated [Ca²⁺]_e for 30 min and isolated plasma membrane and supernatant fractions (100 μg/lane) analyze;d by SDS-PAGE followed by blotting with polyclonal PKCα antibody. β-Actin was used as the loading control. A. N18TG2 WT cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. B. N18TG2 Gα_q KD cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. C. N18TG2 WT cells: i) Western blot; ii) Histogram showing densitometric analysis of p-PKCα bands. D. N18TG2 Gα_q KD cells: i) Western blot; ii) Histogram showing densitometric analysis of p-PKCα bands. Bands were normalized to β-actin (loading control) and calculated as means (± SEM). Blots shown are representative of 4 separate experiments carried out under similar conditions. *Significantly higher than basal (0.25 mM Ca²⁺; p < 0.05).

Figure 8. [Ca²⁺]_e-dependent membrane translocation of PKCα in N18TG2 cells

Cells were stimulated with the indicated [Ca²⁺]_e for 30 min and isolated plasma membrane and supernatant fractions (100 μg/lane) analyze;d by SDS-PAGE followed by blotting with polyclonal PKCα antibody. β-Actin was used as the loading control. A. N18TG2 WT cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. B. N18TG2 Gα_q KD cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. C. N18TG2 WT cells: i) Western blot; ii) Histogram showing densitometric analysis of p-PKCα bands. D. N18TG2 Gα_q KD cells: i) Western blot; ii) Histogram showing densitometric analysis of p-PKCα bands. Bands were normalized to β-actin (loading control) and calculated as means (± SEM). Blots shown are representative of 4 separate experiments carried out under similar conditions. *Significantly higher than basal (0.25 mM Ca²⁺; p < 0.05).

Figure 9. Effect of PKC inhibition on [Ca²⁺]_e-dependent translocation of PKCα in N18TG2 cells

Cells were stimulated with the indicated [Ca²⁺]_e in the presence of 1 μM Gö 6976 for 30 min and isolated plasma membrane and supernatant fractions (100 μg/lane) analyzed by SDS-PAGE followed by blotting with polyclonal PKCα or p-PKCα antibodies. β-Actin was used as the loading control. A. N18TG2 WT cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. B. N18TG2 Gα_q KD cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. C. N18TG2 WT cells: i) Western blot; ii) Histogram showing densitometric analysis of p-PKCα bands. D. N18TG2 Gα_q KD cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. Bands were normalized to β-actin (loading control) and calculated as the means (± SEM). Blots shown are representative of 4 separate experiments carried out under similar conditions.

Figure 9. Effect of PKC inhibition on [Ca²⁺]_e-dependent translocation of PKCα in N18TG2 cells

Cells were stimulated with the indicated [Ca²⁺]_e in the presence of 1 μM Gö 6976 for 30 min and isolated plasma membrane and supernatant fractions (100 μg/lane) analyzed by SDS-PAGE followed by blotting with polyclonal PKCα or p-PKCα antibodies. β-Actin was used as the loading control. A. N18TG2 WT cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. B. N18TG2 Gα_q KD cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. C. N18TG2 WT cells: i) Western blot; ii) Histogram showing densitometric analysis of p-PKCα bands. D. N18TG2 Gα_q KD cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. Bands were normalized to β-actin (loading control) and calculated as the means (± SEM). Blots shown are representative of 4 separate experiments carried out under similar conditions.

Figure 9. Effect of PKC inhibition on [Ca²⁺]_e-dependent translocation of PKCα in N18TG2 cells

Cells were stimulated with the indicated [Ca²⁺]_e in the presence of 1 μM Gö 6976 for 30 min and isolated plasma membrane and supernatant fractions (100 μg/lane) analyzed by SDS-PAGE followed by blotting with polyclonal PKCα or p-PKCα antibodies. β-Actin was used as the loading control. A. N18TG2 WT cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. B. N18TG2 Gα_q KD cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. C. N18TG2 WT cells: i) Western blot; ii) Histogram showing densitometric analysis of p-PKCα bands. D. N18TG2 Gα_q KD cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. Bands were normalized to β-actin (loading control) and calculated as the means (± SEM). Blots shown are representative of 4 separate experiments carried out under similar conditions.

Figure 9. Effect of PKC inhibition on [Ca²⁺]_e-dependent translocation of PKCα in N18TG2 cells

Cells were stimulated with the indicated [Ca²⁺]_e in the presence of 1 μM Gö 6976 for 30 min and isolated plasma membrane and supernatant fractions (100 μg/lane) analyzed by SDS-PAGE followed by blotting with polyclonal PKCα or p-PKCα antibodies. β-Actin was used as the loading control. A. N18TG2 WT cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. B. N18TG2 Gα_q KD cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. C. N18TG2 WT cells: i) Western blot; ii) Histogram showing densitometric analysis of p-PKCα bands. D. N18TG2 Gα_q KD cells: i) Western blot; ii) Histogram showing densitometric analysis of PKCα bands. Bands were normalized to β-actin (loading control) and calculated as the means (± SEM). Blots shown are representative of 4 separate experiments carried out under similar conditions.