Effects of PLC-β1 or PLC-β4 overexpression on Ca²⁺ and IP₃ dynamics evoked by histamine stimulation.

<p>(A) Western blotting analyses of cell lysates prepared from HeLa cells transfected with PLC-β1-IRES-mRFP (lane 2) or PLC-β4-IRES-mRFP (lane 4). Non-transfected cells were used as controls (lanes 1 and 3). (B) Representative traces of Indo-5F signal changes (F/F₀; top) and IRIS-1 signal changes (ΔR/R₀; bottom) in transfected cells stimulated with 3 µM histamine. The plasmid DNAs used to transfect the cells are shown on the left. The horizontal broken lines indicate the baseline levels of IRIS-1 and Indo-5F signals. The vertical broken lines indicate the onsets of stimulation. (B–D) Histograms for the inverse time constants for exponential decay of the Ca²⁺ oscillation amplitude (B), Ca²⁺ oscillation frequencies (C), and integrated IP₃ signals (D) in cells expressing mRFP (top row), PLC-β1 and mRFP (second row), and PLC-β4 and mRFP (third row). The means ± SD are shown at the bottom. The numbers of cells measured are shown in parentheses. Statistical analyses were performed by one-way ANOVA followed by Scheffe’s multiple comparison test. **P<0.01, vs. the values in IRES-mRFP transfected cells.</p

A possible mechanism that underlies the generation of cell-specific patterns of histamine-induced Ca²⁺ signals.

<p>The diagrams show the typical IP₃ and Ca²⁺ dynamics observed in HeLa cells stimulated with histamine. Low-frequency sustained Ca²⁺ oscillations tend to be observed in cells in which PLC-β1 is dominant (left), while high-frequency damped oscillations tend to be observed in cells in which PLC-β4 is dominant (right).</p

Classification of cells depending on the time constants of peak amplitude decay of Ca²⁺ spikes.

<p>Representative traces of Indo-5F signal changes (F/F₀; top) and IRIS-1 signal changes (ΔR/R₀; bottom) in cells showing sustained Ca²⁺ oscillations (S-cell) (A) and damped oscillations (D-cell) (B). Three different concentrations of histamine (1, 3, and 10 µM) were sequentially applied to the same cells with an interval of 20 min. (C) Relationships between the histamine concentrations and the inverse time constants of Ca²⁺ amplitude decay in S-cells (red) and D-cells (blue). (D) Relationships between the histamine concentrations and the Ca²⁺ oscillation frequencies observed in S-cells (red) and D-cells (blue). (E–G) Comparisons of the Ca²⁺ oscillation frequencies between S-cells (red) and D-cells (blue). The histamine concentrations were 1 µM (E), 3 µM (F), and 10 µM (G). (H–J) Comparisons of integrated IRIS-1 signals between S-cells (red) and D-cells (blue). The histamine concentrations were 1 µM (H), 3 µM (I), and 10 µM (J).</p

Identification of PLC isozymes involved in IP₃ generation in HeLa cells stimulated with histamine.

<p>(A) Representative traces of IRIS-1 signal changes (ΔR/R₀; top) and Indo-5F signal changes (F/F₀; bottom) observed in thapsigargin-treated HeLa cells. The horizontal broken lines indicate the baseline levels. (B) Traces of the mean ± SD IRIS-1 signal changes (ΔR/R₀) observed in thapsigargin-treated HeLa cells after addition of 3 µM histamine (blue circles) or 3 µM histamine plus 2 mM Ca²⁺ (green circles). (C–F) Effects of PLC isozyme knockdown on the IP₃ increase evoked by 3 µM histamine alone (C), the IP₃ increase evoked by 2 mM Ca²⁺ alone (D), the first component of the IP₃ increase evoked by 3 µM histamine plus 2 mM Ca²⁺, and the second component of the IP₃ increase evoked by 3 µM histamine plus 2 mM Ca²⁺. Data are shown as means ± SD. The numbers of cells measured are shown in parentheses. Statistical analyses were performed by one-way ANOVA followed by Scheffe’s multiple comparison test. *P<0.05, **P<0.01, vs. the values in control siRNA-transfected cells.</p

siRNA sequences.

a<p>The GC% column indicates the GC content of each siRNA sequence.</p>b<p>The numbers in the position column denote where the sequence is located in the mRNA of the target gene (counted from the first nucleotide of the start codon) of human origin.</p

Effects of PLC-β1 or PLC-β4 knockdown on Ca²⁺ and IP₃ dynamics evoked by histamine stimulation.

<p>(A) Representative traces of Indo-5F signal changes (F/F₀; top) and IRIS-1 signal changes (ΔR/R₀; bottom) in siRNA-treated cells stimulated with 3 µM histamine. The siRNAs used are shown on the left. The horizontal broken lines indicate the baseline levels of the IRIS-1 and Indo-5F signals. The vertical broken lines indicate the onsets of stimulation. Stacked histograms of the inverse time constants for exponential decay of the Ca²⁺ oscillation amplitude (B), Ca²⁺ oscillation frequencies (C), and integrated IP₃ signals (D) of cells treated with control siRNA (top row), PLCβ1KD siRNAs (second row), and PLCβ4KD siRNAs (third row). The results for the two siRNAs are shown in different colors. The means ± SD are shown at the bottom. The numbers of cells measured are shown in parentheses. Statistical analyses were performed by one-way ANOVA followed by Scheffe’s multiple comparison test. **P<0.01, vs. the values in control siRNA mGC-transfected cells.</p

Specific primer sequences for RT-PCR analyses.

a<p>F and R denote foreword and reverse primers, respectively.</p

Reproducible cell-specific patterns in Ca²⁺ and IP₃ responses in HeLa cells stimulated with histamine.

<p>(A) Venus fluorescence image of IRIS-1-expressing HeLa cells before histamine stimulation. Bar, 20 µm. (B) Changes in 460–510 nm emission of Indo-5F signals (F/F_0, where F₀ is the basal level of F; top) and ECFP/Venus emission ratio of IRIS-1 signals (ΔR/R₀; ΔR was defined as R – R₀, where R₀ is the basal level of R; bottom) after repeated additions of 3 µM histamine with a 20-min interval in two different cells within the same field of view shown in (A). The horizontal broken lines indicate the baseline levels of the IRIS-1 and Indo-5F signals. The vertical broken lines indicate the onsets of stimulation.</p