IP3-Dependent Ca2+ Oscillations Switch into a Dual Oscillator Mechanism in the Presence of PLC-Linked Hormones

Abstract

Ca²⁺ oscillations that depend on inositol-1,4,5-trisphosphate (IP₃) have been ascribed to biphasic Ca²⁺ regulation of the IP₃ receptor (IP₃R) or feedback mechanisms controlling IP₃ levels in different cell types. IP₃ uncaging in hepatocytes elicits Ca²⁺ transients that are often localized at the subcellular level and increase in magnitude with stimulus strength. However, this does not reproduce the broad baseline-separated global Ca²⁺ oscillations elicited by vasopressin. Addition of hormone to cells activated by IP₃ uncaging initiates a qualitative transition from high-frequency spatially disorganized Ca²⁺ transients, to low-frequency, oscillatory Ca²⁺ waves that propagate throughout the cell. A mathematical model with dual coupled oscillators that integrates Ca²⁺-induced Ca²⁺ release at the IP₃R and mutual feedback mechanisms of cross-coupling between Ca²⁺ and IP₃ reproduces this behavior. Thus, multiple Ca²⁺ oscillation modes can coexist in the same cell, and hormonal stimulation can switch from the simpler to the more complex to yield robust signaling.

Keywords: Cell Biology; Mathematical Biosciences; Specialized Functions of Cells.

Graphical abstract

Figure 1

Effect of Incremental Flash Photolysis of Caged IP₃ on [Ca²⁺]_c Oscillation Properties Isolated hepatocytes were transfected with GCaMP3, cultured overnight, and then loaded with caged IP₃ (2 μM; 1 h). (A) Representative trace showing cytosolic Ca²⁺ responses to photolysis of caged IP₃ elicited by rapid trains of 1, 2, 3, and 4 UV pulses (arrows). After a recovery period, the same cells were stimulated with 5 nM vasopressin (VP). (B–E) Summary data for the effect of increasing UV stimulation compared with VP for [Ca²⁺]_c spike amplitude (B), peak width measured as full width at half maximum (FWHM) (C), rate of rise (D), and interspike interval (ISI) (E). Data are mean ± SEM of the second [Ca²⁺] transient for amplitude, width, and rate of rise, and 3 consecutive oscillations for ISI after UV pulse or 5 nM VP addition (11 cells from 6 independent experiments).

Figure 2

Buffering of Intracellular IP₃ Has No Effect on Ca²⁺ Oscillations Elicited by Slow Release of Caged IP₃ (A–D) Hepatocytes cotransfected with RGECO1 and either GFP (A) or GFP-LBD (B) were loaded with caged IP₃ (2 μM; 1 h). The gray area shows the duration of slow IP₃ uncaging elicited by low-intensity UV illumination (50-ms exposures from xenon lamp at 2 Hz). Expression of GFP-LBD had no effect on the peak width (FWHM) or ISI (C) or the rate of Ca²⁺ rise (D). Data are mean ± SEM of 10 cells from 4 independent experiments.

Figure 3

Spatial Properties of [Ca²⁺]_c Responses Elicited by Flash Photolysis of caged IP₃ Isolated hepatocytes were transfected with GCaMP3, cultured overnight, and then loaded with caged IP₃ (2 μM; 1 h). (A) Single video frames showing focal [Ca²⁺]_c release events. Scale bar, 10 μM. (B and C) Representative traces of changes in [Ca²⁺]_c at the local puff site (red) and distal pole (blue) of two individual hepatocytes during IP₃ uncaging. (D) Interpuff interval (IPI, red) and interspike interval (ISI, blue) during the 3 UV pulse response from (C). (E) Ordinal plot of the Ca²⁺ response pattern for cells with increasing UV pulse density (bursts of 1–4 flashes, as indicated). Response patterns of increasing strength are classified as follows: no response, local Ca²⁺ transients at a discrete puff site, partial propagation where Ca²⁺ waves only propagated across the cell intermittently, propagation where Ca²⁺ waves consistently propagated across the cell, and global for whole-cell Ca²⁺ responses that were not spatially resolved (summary of 59 cells from 6 independent experiments).

Figure 4

Effect of IP₃ Uncaging on [Ca²⁺]_c Responses Elicited by Vasopressin (A–D) Isolated hepatocytes were transfected with GCaMP3, cultured overnight and then loaded with caged IP₃ (2 μM; 1h). Hepatocytes were stimulated with vasopressin (VP) and then IP₃ uncaging was achieved by a brief (1-s) pulse of 340-nm light from the microscope fluorescence illuminator Xeon lamp. Representative traces showing (A) a slight slowing of Ca²⁺ oscillation frequency or (B) the temporary arrest of oscillations. Summary data showing a decrease in oscillation frequency (C) and an increase in peak width (D) after the IP₃ uncaging event (data are mean ± SEM from ≥26 individual hepatocytes from 4 independent experiments, ∗∗p < 0.01, ∗∗∗p < 0.001 paired Student's t test). (E) Representative trace of hepatocyte stimulated with VP and then IP₃ uncaging with 3 UV flashes from UV laser.

Figure 5

Fast [Ca²⁺]_c Oscillations Elicited by IP₃ Uncaging Transition to Broad Baseline-Separate [Ca²⁺]_c Oscillations in the Presence of Hormone Isolated hepatocytes were transfected with GCaMP3, cultured overnight, and then loaded with caged IP₃ (2 μM; 1 h). Rapid trains of 1, 2, 3, or 4 UV pulses were applied as indicated (arrows) followed by the addition of VP (5 nM). (A and B) Representative traces showing cytosolic [Ca²⁺]_c oscillations at puff site (red) and distal pole (blue) of two cells in response to IP₃ uncaging and VP (see also Videos S1 and S2). (C) Mathematical model combining class 1 and class 2 oscillation mechanisms predicts the observed behavior in this paradigm (red line [Ca²⁺]_c, green line [IP₃]). (D) IPI and ISI of cell shown in (B) in response to UV pulses and VP (transition period between UV and VP responses not shown). (E–G) Comparison of ISI (E), spike width (FWHM) (F), and rate of rise (G) at the puff site and distal pole of hepatocytes. (H) Comparison of [Ca²⁺]_c wave propagation rate elicited by UV and VP. Data are mean ± SEM of 10 cells from 5 independent experiments. ∗∗p < 0.01, paired Student's t test.