Differential Regulation of Multiple Steps in Inositol 1,4,5-Trisphosphate Signaling by Protein Kinase C Shapes Hormone-stimulated Ca²⁺ Oscillations

Background: The effects of inositol 1,4,5-trisphosphate (IP₃)-linked hormones are determined by the frequency, amplitude, and duration of Ca²⁺ oscillations.

Results: Comparison of IP₃ uncaging and hormone stimulation showed that PKC has distinct effects on IP₃ formation, metabolism, IP₃ receptor function, and Ca²⁺ wave propagation.

Conclusion: PKC modulates Ca²⁺ oscillation frequency, duration, and wave velocity.

Significance: PKC feedback shapes Ca²⁺ oscillations and provides signal versatility.

Abstract

How Ca²⁺ oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca²⁺ oscillations report signal strength via frequency, whereas Ca²⁺ spike amplitude and wave velocity remain constant. IP₃ uncaging also triggers oscillatory Ca²⁺ release, but, in contrast to hormones, Ca²⁺ spike amplitude, width, and wave velocity were dependent on [IP₃] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP₃ uncaging are driven by the biphasic regulation of the IP₃ receptor by Ca²⁺, and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca²⁺ did not perturb Ca²⁺ oscillations elicited by IP₃ uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca²⁺ influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca²⁺ oscillations but had no effect on Ca²⁺ increases induced by uncaging IP₃. Importantly, PKC activation and inhibition differentially affected Ca²⁺ spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP₃ metabolism to attenuate IP₃ levels and suppress the generation of Ca²⁺ oscillations. Inhibition of PKC relieves negative feedback regulation of IP₃ accumulation and, thereby, shifts Ca²⁺ oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca²⁺ wave velocity, whereas responses to IP₃ uncaging are enhanced. The ability to assess Ca²⁺ responses in the absence of PLC activity indicates that IP₃ receptor modulation by PKC regulates Ca²⁺ release and wave velocity.

Introduction

Calcium oscillations and waves generated by the activation of PLC-linked² GPCRs regulate a multitude of mechanisms from gene transcription to secretion (1–3). In many cell types, including hepatocytes, stimulus strength is encoded by the frequency of Ca²⁺ oscillations, with interspike intervals ranging from >250 s at low hormone concentrations to <30 s when challenged with higher hormone levels (1, 4–6). These Ca²⁺ signals are generated by PLC-mediated hydrolysis of phosphatidylinositol bisphosphate (PIP₂) to yield IP₃ and the subsequent activation of IP₃R Ca²⁺ release channels in the ER (7). However, the mechanisms driving the subsequent repetitive Ca²⁺ oscillations have yet to be fully resolved (8–12). Many studies have aimed to determine whether these oscillations arise solely because of the biphasic effects of cytosolic [Ca²⁺] on IP₃R gating (13–16), i.e. Ca²⁺-induced Ca²⁺ release (CICR), or whether regenerative PLC activation and/or cyclical protein phosphorylation events are also required (17–19). We have demonstrated recently that intracellular buffering of IP₃, using a recombinant protein containing the ligand binding domain of rat IP₃R type I, results in an inhibition of Ca²⁺ oscillations, a decrease in the rates of Ca²⁺ rise, and a slowing of Ca²⁺ wave propagation speed (17, 20). These data demonstrate that IP₃ levels dynamically regulate Ca²⁺ oscillations, providing evidence that cross-coupling between IP₃ and Ca²⁺ is required to maintain hormone-induced Ca²⁺ oscillations in non-excitable cells such as hepatocytes.

The Ca²⁺ oscillation frequency increases with agonist concentration in hepatocytes (1, 5, 6), but the individual Ca²⁺ spikes have a constant amplitude and rate of rise and propagate as intracellular Ca²⁺ waves at a constant velocity independent of agonist dose. Nevertheless, the falling phase of the [Ca²⁺] spikes shows greater diversity (1, 6), and different agonists can give characteristically distinct shapes of [Ca²⁺] spikes that vary only in the decay phase, even when observed in the same individual cell (2, 17, 19, 20). Moreover, the duration of cytosolic Ca²⁺ elevation, in addition to spike frequency, has been demonstrated to regulate transcription (21–23). It is therefore important to determine not only how PLC-dependent Ca²⁺ oscillations are generated but also how spike and wave kinetics are further modulated to account for the versatility of Ca²⁺ signaling.

Long interspike periods between Ca²⁺ transients at low hormone doses and the broad dynamic range of frequency modulation suggest that Ca²⁺ interspike periods and spike kinetics are dynamically controlled by feedback loops that regulate IP₃ generation and metabolism as well as IP₃R function (17, 20). PLC-dependent signal transduction activates PKC (24), which, in turn, has the potential to phosphorylate and regulate multiple proteins in the Ca²⁺ signaling cascade, including GPCRs (25, 26), PLC (27), IP₃R (28, 29), and IP₃ kinase (30). Importantly, concurrent with Ca²⁺ oscillations, repetitive translocation of PKC isoforms, both conventional and novel, to the plasma membrane have been reported (31, 32), indicating cyclic activation of these enzymes. Furthermore, previous studies in hepatocytes have shown that both activation and inhibition of PKC can affect hormone-induced Ca²⁺ oscillation kinetics (33, 34).

In this study, we compared Ca²⁺ signals elicited by hormone and photorelease of caged IP₃ and examined how the ensuing Ca²⁺ oscillations are regulated in response to each stimulus. Our data reveal that Ca²⁺ oscillations elicited by direct release of caged IP₃ are graded, with the transient amplitude, frequency, and wave velocity dependent on the amount of IP₃ released. Moreover, these Ca²⁺ responses were independent of PLC activity, indicating that IP₃ uncaging generates Ca²⁺ oscillations solely through CICR. This is in contrast to hormone-induced Ca²⁺ oscillations, which depend on IP₃ oscillations cross-coupled with Ca²⁺ spiking (17, 20) (i.e. regenerative PLC activation) and have characteristic spike properties independent of agonist dose. Therefore, uncaging of IP₃ provides a tool to assess modulators of Ca²⁺ transients in the absence of PLC activity and other hormone-dependent signaling cascades. We show that Ca²⁺ oscillations elicited by IP₃ uncaging persist in the absence of extracellular Ca²⁺, demonstrating that reloading of ER Ca²⁺ stores does not entrain these periodic Ca²⁺ signals. Modulation of Ca²⁺ signaling by PKC was assessed in both PLC- and CICR-dependent paradigms. Paradoxically, both activation and inhibition of PKC decreased the frequency of hormone-induced Ca²⁺ oscillations but via different mechanisms. Activation of PKC inhibited regenerative IP₃ generation by the GPCR/PLC, whereas inhibition of PKC relieved this negative feedback, allowing more prolonged and sustained IP₃ generation and, therefore, Ca²⁺ release. By contrast, CICR oscillations elicited by uncaging IP₃ were potentiated by PKC activation. Furthermore, PKC down-regulation decreased Ca²⁺ wave velocity in agonist-stimulated cells, whereas it actually increased Ca²⁺ wave velocity after direct IP₃ release. These data demonstrate that PKC activity regulates IP₃ levels via effects on GPCR coupling, PLC activity, and/or IP₃ metabolism while also effecting IP₃R sensitivity to regulate Ca²⁺ spike frequency, width, and Ca²⁺ wave velocity.

Experimental Procedures

Primary Cell Culture

Isolated hepatocytes were prepared by collagenase perfusion of livers obtained from male Sprague-Dawley rats. Cells were maintained in Williams E medium for 2–6 h for experiments using freshly isolated cells or 16–24 h for experiments using overnight cultured cells, as described previously (1, 4). Animal studies were approved by the Institutional Animal Care and Use Committee at Rutgers, New Jersey Medical School.

Cytosolic Ca²⁺ Measurements in Response to Hormones

Calcium imaging experiments were performed in HEPES-buffered physiological saline solution comprised of 25 mm HEPES (pH 7.4), 121 mm NaCl, 5 mm NaHCO₃, 10 mm glucose, 4.7 mm KCl, 1.2 mm KH₂PO₄, 1.2 mm MgSO₄, 2.0 mm CaCl₂, and 0.25% (w/v) fatty acid-free BSA and supplemented with the organic anion transport inhibitors sulfobromophthalein (100 μm) or probenecid (200 μm). Hepatocytes were loaded with Fura-2 by incubation with 2–5 μm Fura-2/AM and Pluronic acid® F-127 (0.02% v/v) for 20–40 min. Cells were transferred to a thermostatically regulated microscope chamber (37 °C). Fura-2 fluorescence images (excitation, 340 and 380 nm, emission 420–600 nm) were acquired at 1.5- to 3-s intervals with a cooled charge-coupled device camera coupled to an epifluorescent microscope, as described previously (35).

Hormone-induced PLC Activity and IP₃ Detection

Intracellular IP₃ levels or PLC activity were measured using FRET-based genetically engineered probes. Isolated hepatocytes were transfected by electroporation with the Amaxa rat/mouse hepatocyte nucleofector kit according to the instructions of the manufacturer (Lonza). PLC activity was determined by cotransfection with PLC_δ4YFP and PLC_δ4CFPPH domains (cDNA was a gift from Dr. Balla, National Institutes of Health), which yield a FRET signal while bound to membrane PIP₂ that declines as PIP₂ is hydrolyzed. IP₃ measurements were determined with the IP₃ sensor IRIS-1. IRIS-1 cDNA was a gift from Dr. Mikoshiba (RIKEN Brain Science Institute, Japan) (36). FRET images were acquired at 3-s intervals by illumination with 436 ± 20 nm using a 455-nm-long band pass dichroic filter. FRET donor and acceptor fluorescence images were separated with a 505-nm-long band pass dichroic mirror and directed to 480 ± 30 nm (CFP) or 535 ± 40 nm (YFP/Venus) emission filters using an image beamsplitter (Optical Insights^TM). The FRET ratio was calculated on a cell-by-cell basis and averaged from all expressing cells in the microscope field. FRET signal changes between PLC_δ4YFP and PLC_δ4CFP PH domains were corrected for YFP bleach using linear regression analysis.

Photorelease of Caged IP₃

Overnight cultured hepatocytes were loaded in HEPES-buffered physiological saline solution with the membrane-permeant form of caged IP₃ (2 μm; d-2, 3-O-isopropylidene-6-O-(2-nitro-4,5-dimethoxy)benzyl-myo-inositol 1,4,5-trisphosphate-hexakis(propionoxymethyl) ester; Sichem GmbH) for 45 min at room temperature, followed by 30-min loading with the calcium indicator dye Fluo-4/AM (5 μm). Cells were transferred to the microscope chamber of a spinning disc confocal microscope. Fluo-4 images (excitation, 488 nm; emission, 510-nm-long band pass filter) were acquired at 10 Hz. Photorelease of caged IP₃ was achieved by light pulses from a nitrogen-charged UV laser (Photon Technology International). The cell-permeant caged IP₃ is synthesized with the 2- and 3-hydroxyl groups of myo-inositol protected by an isopropylidene group to ensure that the phosphate groups remain in the 1,4 and 5 positions (37). Of note, when released from the cage, this modified form of IP₃ is metabolized at a slower rate, in the order of minutes, compared with natural IP₃, which is metabolized in seconds (37). Cell viability was assessed by the addition of maximal hormone concentrations at the end of each experiment. Only cells responsive to hormone stimulation are included in the presented data.

Western Blotting

Hepatocyte lysates were prepared in a buffer comprised of 150 mm NaCl, 10 mm Tris-HCl, 1 mm EDTA, and 0.2 mm PMSF supplemented with 1% (w/v) SDS, 0.5% (v/v) Nonidet P-40, 10 μg/ml aprotinin, and 1 μg/ml leupeptin (pH 7.4). Lysates were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to a PVDF membrane. Membranes were blocked for 1 h in Tris-buffered saline (pH 7.5) containing 5% (w/v) nonfat dry milk and 0.1% (v/v) Tween 20. The membranes were incubated with anti-PKCα, (Cell Signaling Technology), anti-PKCϵ, and anti-PKCζ (Santa Cruz Biotechnology) overnight at 4 °C. The protein loading control was determined by stripping PVDF membranes and reprobing with anti-α-tubulin (Cell Signaling Technology).

[³H]Inositol Phosphate Accumulation

Total [³H]inositol phosphate accumulation was determined as described previously (31). In brief, primary hepatocytes were labeled overnight with 2.5 μCi ml⁻¹ myo-[³H]inositol (American Radiolabeled Chemicals, Inc.) in 6-well plates. In some studies, cultures were treated overnight with phorbol-12-myristate-13-acetate (PMA) or 4α-PMA (1 μm) to assess the effect of down-regulating PKC. Cultures were washed with HEPES-buffered physiological saline solution and incubated for 20 min at 37 °C, followed by an additional 10-min treatment with 10 mm LiCl to block inositol monophosphatase activity. Cells were treated with 100 nm vasopressin for 15 min in the absence or presence of PMA (1 nm) or bisindolylmaleimide I (BIM, 5 μm) to assess the acute effects of PKC activation and inhibition, respectively. Incubations were terminated by addition of ice-cold tricholoroacetic acid. Water soluble [H³]inositol-containing components were extracted by addition of tri-n-octylamine:1,1,2-trichlorofluoroethane (1:1 ratio). [H³]inositol phosphates were separated by ion exchange chromatography using Dowex resin in the formate form. Lower-order inositols and glycerophospholipids were removed by elution with 0.4 m ammonium formate/0.1 M formic acid. IP₃ and higher-order inositols were then eluted with 1.2 m ammonium formate/0.1 M formic acid. Ultima-Flo (PerkinElmer Life Sciences) was added to the eluate, and disintegrations per minute was determined using liquid scintillation counting. Data are expressed as a -fold increase over basal (inositol phosphate turnover levels in the absence of hormone).

Data Analysis

Image analysis was performed using in-house customized software and ImageJ (National Institutes of Health). Graph plotting and data analysis were performed with GraphPad Prism software. Statistical analysis was performed using Student's t test or one-way analysis of variance where indicated.

Results

Photorelease of Caged IP₃ Elicits Ca²⁺ Oscillations in Hepatocytes

Photorelease of caged IP₃ in hepatocytes induced cytosolic Ca²⁺ increases, with Ca²⁺ oscillations observed in many cells (Fig. 1A). Similar to hormone-induced Ca²⁺ oscillations (1, 9), the frequency and number of cells responding to IP₃ uncaging increased with stimulus strength, as determined by the number of UV flashes, and tended toward sustained Ca²⁺ increases with the strongest stimulation (Fig. 1, B and C). A single pulse from the UV laser resulted in Ca²⁺ responses in only 22.5 ± 10.2% of cells. Incrementally increasing the number of UV flashes (applied as a rapid burst) increased the percentage of cells responding (2× UV, 44.2 ± 14.8%; 3× UV, 84 ± 9.2%; 4× UV, 86.7 ± 8.6%; mean ± S.E. from >100 cells in three independent experiments). In addition, increasing the number of UV flashes shifted the Ca²⁺ signature from predominantly no response and single Ca²⁺ transients at low illumination toward oscillatory and sustained (peak/plateau) Ca²⁺ increases at higher illumination, presumably reflecting increased levels of IP₃ (Fig. 1C). Therefore, the Ca²⁺ signals induced by uncaging IP₃ appear to mimic hormone-induced Ca²⁺ responses, the proportion of responsive cells, and the oscillatory and saturated Ca²⁺ responses increasing with stimulus strength.

FIGURE 1.

Photorelease of caged IP₃ elicits Ca²⁺ oscillations in primary rat hepatocytes. Isolated hepatocytes were cultured overnight and then loaded with caged IP₃ and Fluo-4. A, representative trace showing cytosolic Ca²⁺ responses to photolysis of caged IP₃. Rapid trains of one, two, three, or four UV pulses were applied as indicated (arrows). B, the percentage of cells responding to one to four UV flash events. C, comparison of the types of Ca²⁺ responses observed after each train of UV pulses: no response, single spike, oscillations, or a sustained Ca²⁺ increase (peak/plateau). Data shown are mean ± S.E. from ≥100 cells from five independent experiments. D and E, summary of Ca²⁺ transient amplitude (D) and Ca²⁺ spike width measured at half-peak height (E) in cells in which oscillations were observed after one, two, and three UV flashes. Data are mean ± S.E. of the first three Ca²⁺ transients for each individual cell (n = 15). F, Ca²⁺ wave propagation rates as a function of number of UV flashes. Data are mean ± S.E. from cells in which Ca²⁺ waves were observed after one, two, and three UV flashes (n = 13). *, p < 0.05; **, p < 0.01; ***, p < 0.001; Student's t test.

A characteristic of hormone-induced Ca²⁺ oscillations is that although the frequency increases with agonist dose, Ca²⁺ spike kinetics, including amplitude, rate of rise, and peak width are constant for all agonist doses (1, 5, 6, 9). By contrast, photorelease of caged IP₃ resulted in Ca²⁺ peak heights and widths that increased with stimulus strength (Fig. 1, D and E). These data are the mean ± S.E. calculated from the average of the first three Ca²⁺ transients from cells in which oscillations were observed at each level of UV exposure. Of particular note, the mean duration of Ca²⁺ spikes elicited by a single UV flash was 3.8 ± 0.24 s at half-peak height, which is much shorter than hormone-stimulated Ca²⁺ transients measured in this work (Figs. 4 and 7) and previous studies (1, 38). Broader Ca²⁺ spike widths were achieved with multiple UV flashes (11.5 ± 2.2 s at half-peak height for 3× UV flashes), but this is still a shorter duration than the Ca²⁺ spike widths of >20 s typically observed with hormone stimulation. The velocity of Ca²⁺ waves elicited by IP₃ uncaging was also dependent on the number of UV flashes (Fig. 1F). Ca²⁺ waves induced by hormones propagate at 15–25 μm/s independent of hormone dose (5), whereas Ca²⁺ waves induced by IP₃ uncaging rose from 8.2 μm/s ± 0.7 at 1× UV to 23.9 μm/s ± 4.8 with 3× UV.

FIGURE 4.

Down-regulation of PKC enhances hormone-stimulated Ca²⁺ signals in cultured hepatocytes. Cultured hepatocytes were treated overnight with the inactive analogue 4α-PMA (1 μm, control) or PMA (1 μm, PKC-DR) to down-regulate classical and novel PKC isoforms. Cells were loaded with Fura-2 and then stimulated with phenylephrine (PE, 20 μm). A—C, typical agonist-induced Ca²⁺ responses are shown for control (A) and PKC-DR (B and C) cells. D and E, summary data showing the effects of PKC down-regulation on the type of Ca²⁺ responses and the width of the Ca²⁺ spikes induced by PE stimulation. Data are mean ± S.E. from ≥50 cells from three independent experiments. **, p < 0.01; ***, p < 0.001; Student's t test. F, Western blots showing PKCα, PKCϵ, and PKCζ protein levels in control and PKC-DR hepatocyte lysates. Levels of α-tubulin are shown as loading controls.

FIGURE 7.

Effects of acute activation and inhibition of PKC on hormone-induced Ca²⁺ oscillations. The effects of PMA (1 nm) and BIM (5 μm) on phenylephrine (PE)-induced Ca²⁺ oscillations were examined in hepatocytes cultured for 1 h. Representative traces of PMA- (A) and BIM-treated (B) hepatocytes show either a decrease in oscillation frequency (top panel) or suppression of oscillations (bottom panel). The effect of drug treatment on phenylephrine-induced Ca²⁺ oscillation frequency (C) and Ca²⁺ spike width at half-peak height (D) are summarized. The frequencies of agonist-induced Ca²⁺ oscillations were calculated from 5-min periods in the absence or presence of the drugs. Ca²⁺ spike widths were calculated from the three oscillations prior to and after drug treatment. The drugs were present at least 1 min prior to carrying out the analysis. Data are mean ± S.E. from ≥15 cells from three independent experiments. **, p < 0.01; ***, p < 0.001; Student's t test. E, the effect of acute activation or inhibition of PKC and PKC-DR on total inositol phosphate production in response to 100 nm vasopressin (VP) stimulation for 15 min. Data are expressed as -fold increase over basal and are the mean ± S.E. from three independent experiments. *, p < 0.05; **, p < 0.01; analysis of variance.

IP₃-induced Ca²⁺ Oscillations Do Not Require PLC Activity

To address whether IP₃ regeneration through Ca²⁺ activation of PLC is required to elicit Ca²⁺ oscillations in response to photolysis of IP₃, we performed experiments in the presence of the aminosteroid PLC inhibitor U71322. Hepatocytes were loaded with caged IP₃ and concurrently treated for 75 min with 20 μm U73122, the inactive analogue U73433, or vehicle (dimethyl sulfoxide). The cells were first exposed to a rapid train of 4× UV flashes to uncage IP₃, followed by 10 nm vasopressin (Fig. 2, A and B). The percentage of cells eliciting a Ca²⁺ increase and oscillatory Ca²⁺ responses to each stimulus are summarized in Fig. 2, C and D. In our hands, non-toxic concentrations of U73122 were insufficient to completely block hormone-induced Ca²⁺ increases in all cells (higher concentrations perturbed Ca²⁺ release by thapsigargin, indicating off-target effects). Nevertheless, a significant inhibition of vasopressin-induced Ca²⁺ transients was observed after U73122 treatment. U73122 reduced the percentage of cells responding to hormone stimulation from 81 ± 6 to 33 ± 9 and the percentage of cells displaying Ca²⁺ oscillations from 60 ± 5 to 22 ± 6. By contrast, the Ca²⁺ increases and oscillatory responses elicited by photolysis of caged IP₃ were not significantly different between treatment groups (Fig. 2, C and D).

FIGURE 2.

IP₃-induced Ca²⁺ oscillations do not depend upon the activation of PLC. Hepatocytes cultured overnight and loaded with caged IP₃ and Fluo-4 were treated with the PLC inhibitor U73122, the inactive analogue U74344 (20 μm for each drug, 75 min), or vehicle (dimethyl sulfoxide (DMSO), 0.1% v/v). Cells were then stimulated with UV illumination followed by 3 nm vasopressin (VP) as indicated. A and B, representative Ca²⁺ responses in dimethyl sulfoxide-treated (A) or U73122-treated (B) cells are shown. C and D, the effect of PLC inhibition on the percentage of responsive (C) and oscillating cells (D) to each stimulus are summarized. Data are representative of ≥60 cells from four independent experiments. E and F, summary of the effect of apyrase (30 units/ml, 5 min) on the proportion of cells responding (E) or the percentage of cells giving oscillatory Ca²⁺ responses (F). Data are mean ± S.E. of 45 cells from three independent experiments. *, p < 0.05; **, p < 0.01; Student's t test.

We also considered the possibility that ATP released from the hepatocytes in culture might act in a paracrine fashion to cause tonic subthreshold activation of PLC, the activity of which could be amplified upon direct photorelease of IP₃. Pretreatment of hepatocytes with 30 units/ml (5 min) of apyrase to hydrolyze extracellular ATP was without effect on the number of cells responding to photolysis of caged IP₃ or the proportion of cells displaying oscillatory changes in cytosolic Ca²⁺ (Fig. 2, E and F). Taken together, these data indicate that positive feedback of Ca²⁺ on PLC does not contribute to the Ca²⁺ signals elicited by uncaging IP₃, and, when IP₃ is sufficiently elevated, Ca²⁺ oscillations are driven primarily by CICR at the IP₃R.

Plasma Membrane Ca²⁺ Entry Is Not a Requirement for IP₃-driven Ca²⁺ Oscillations

Store-operated Ca²⁺ entry plays a fundamental role in maintaining Ca²⁺ homeostasis and to replete internal Ca²⁺ stores when cells respond to Ca²⁺-mobilizing hormones (39). However, there is a continuing debate regarding the importance of store-operated Ca²⁺ entry and Ca²⁺ store load in the generation and feedback control of hormone-stimulated Ca²⁺ oscillations (40–42). To determine whether extracellular Ca²⁺ entry regulates IP₃R activation or sensitivity to IP₃, we assessed Ca²⁺ signals elicited by photorelease of caged IP₃ in the absence and presence of extracellular Ca²⁺. Hepatocytes were maintained in either HEPES-buffered physiological saline solution containing 2 mm CaCl₂ or switched to Ca²⁺-free buffer 5–10 min prior to uncaging IP₃ with 3× UV flashes (Fig. 3). A somewhat higher proportion of cells did not respond to photorelease of caged IP₃ in Ca²⁺-free (37.3 ± 0.7%) compared with Ca²⁺-replete conditions (19.3 ± 3.1%) (Fig. 3C), which may reflect an effect of partial Ca²⁺ store depletion. Nevertheless, Ca²⁺ oscillations were still observed in response to IP₃ uncaging in the presence or absence of extracellular Ca²⁺ (Fig. 3, A and B), with no impact on oscillation frequency over a 5-min period (Fig. 3D). These data indicate that plasma membrane Ca²⁺ entry is not a requirement to sustain repetitive Ca²⁺ release from the ER. However, the width of the Ca²⁺ spike, measured at half-peak height for the first three Ca²⁺ transients, was decreased significantly in the absence of extracellular Ca²⁺ (5.5 ± 0.2 s compared with 9.3 ± 0.4 s in the presence of extracellular Ca²⁺). Therefore, Ca²⁺ entry and ER Ca²⁺ load may contribute to IP₃R-induced Ca²⁺ transients by prolonging spike duration (Fig. 3E).

FIGURE 3.

Extracellular Ca²⁺ is not required for IP₃-induced Ca²⁺ oscillations. Isolated hepatocytes loaded with caged IP₃ and Fluo-4 were stimulated with 3 UV flashes (UV). A and B, representative traces of Ca²⁺ responses are shown in the presence (A) and absence (B) of extracellular Ca²⁺. C–E, summary of the Ca²⁺ signatures observed (C), oscillation frequency (D), and Ca²⁺ peak width at half-height (E) in the presence and absence of extracellular Ca²⁺. Data are mean ± S.E. of 25 cells from two independent experiments. *, p < 0.05, Student's t test.

PKC Down-regulation Perturbs Negative Feedback Inhibition of Ca²⁺ Mobilization

Previous studies have highlighted the complexity of PKC regulation of hormone-induced Ca²⁺ oscillations in hepatocytes, reporting that both activators and inhibitors of PKC suppressed the responses to phenylephrine (33, 34). We examined the effect of chronic down-regulation of conventional and novel PKCs (PKC-DR) by overnight treatment (16–24 h) with 1 μm PMA or the inactive analogue 4α-PMA. A comparison of responses in control 4α-PMA-treated and PMA-treated hepatocytes revealed a dramatic shift in the type of Ca²⁺ responses elicited by phenylephrine (20 μm). Under control conditions, 65 ± 6.5% of cells responded with an oscillatory Ca²⁺ signature (Fig. 4A shows a representative trace of a Ca²⁺ response in control cells), whereas PKC-DR resulted in predominately sustained Ca²⁺ rises in 73 ± 3% of cells (see Fig. 4B for a representative trace) compared with only 11 ± 0.6% of sustained responses in control cells. In PKC-DR cells, only 16.2 ± 2.2% responded with oscillatory Ca²⁺ signatures (Fig. 4C shows a representative trace of Ca²⁺ oscillations after PKC-DR). The proportion of cells responding with oscillatory or sustained responses for each treatment group is summarized in Fig. 4D. The small population of PKC-DR cells in which Ca²⁺ oscillations were observed displayed responses characteristically different from the stereotypic Ca²⁺ oscillations in control cells. The oscillation frequency was reduced, and the spike widths were very substantially prolonged. The individual Ca²⁺ spike widths measured at half-peak height in phenylephrine-stimulated control cells were very consistent, with a mean value of 24 ± 0.6 s, whereas the spike durations in PKC-DR cells were almost 3-fold longer, with a width at half-peak height of 68 ± 3.3 s (Fig. 4E). We confirmed by Western blotting that treating cells overnight with phorbol ester leads to the down-regulation and degradation of the conventional, PKCα, and novel PKCϵ (phorbol ester-activated PKC isoenzymes in hepatocytes) without affecting the atypical isoform PKCζ1 (Fig. 4F).

PKC phosphorylation of the plasma membrane Ca²⁺ pump and of Orai channels has been reported (43, 44), indicating that PKC activity may regulate plasma membrane Ca²⁺ efflux and/or entry. Indeed, Orai1 has been shown to be basally phosphorylated by PKC, and inhibition of PKC leads to enhanced Ca²⁺ entry (44). To determine whether PKC-DR affects Ca²⁺ transport across the plasma membrane in hepatocytes, we measured Ca²⁺ influx and efflux rates in cells treated overnight with PMA or 4α-PMA (Fig. 5). ER Ca²⁺ stores were depleted with thapsigargin (Fig. 5, A–C) or ATP (to assess agonist-dependent effects on Ca²⁺ influx) (Fig. 5, D--F) in the absence of extracellular Ca²⁺ to induce Store-operated Ca²⁺ entry pathways. The PKC-DR protocol did not affect the rates of Ca²⁺ influx upon Ca²⁺ readdition (Fig. 5, B and E) or the rates of plasma membrane Ca²⁺ pump-mediated Ca²⁺ efflux from the cells after removal of extracellular Ca²⁺ (Fig. 5, C and F). These data suggest that PKC activity does not play a major role in regulating Ca²⁺ entry or Ca²⁺ extrusion at the plasma membrane in hepatocytes.

FIGURE 5.

Measurement of Ca²⁺ influx and efflux rates in control and PKC-DR hepatocytes. Hepatocytes were treated overnight with 4α-PMA (1 μm, Control) or PMA (1 μm, PKC-DR) and then loaded with Fura-2. Cultures were washed into Ca²⁺-free buffer prior to data acquisition. A—F, internal Ca²⁺ stores were depleted with thapsigargin (Thaps, 4 μm) (A–C) or by stimulation with the purinergic agonist ATP (200 μm) (D–F), followed by repletion of extracellular Ca²⁺ (2 mm) to initiate Ca²⁺ entry. Where indicated, the buffer was switch to Ca²⁺-free medium plus 5 mm BAPTA to stop Ca²⁺ influx and measure the rates of Ca²⁺ efflux from the cell. Ca²⁺ influx (B and E) and efflux (C and F) were plotted, and exponential rate constants (tau) were calculated using non-linear regression analysis. Similar results were obtained when the initial rates were measured (data not shown).

Negative feedback regulation by PKC on both GPCRs and PLC isoenzymes has been implicated in the regulation of Ca²⁺ oscillations (27, 31, 45). Therefore, we assessed the effect of PKC-DR on hormone-stimulated PLC activity and IP₃ generation in hepatocytes using FRET-based molecular indicators. To monitor PLC activity, CFP and YFP proteins conjugated to PLCδ4PH domain were coexpressed. Hormone-stimulated PIP₂ breakdown leads to a decrease in FRET between the CFP and YFP moieties, as described previously for CFP and YFP PLCδ1PH (46). The pleckstrin homology (PH) domain of PLCδ4 has a lower affinity for IP₃ compared with PLCδ1, providing a more selective readout of PLC activity (PIP₂ hydrolysis) over intracellular [IP₃]. Dynamic changes in cytosolic [IP₃] were determined with the IRIS-1 molecular probe containing a mutated version of the ligand binding domain of IP₃R type 1 flanked by CFP and Venus (20, 36). PLC activity elicited by agonist stimulation (ATP, 200 μm) was potentiated in PKC-DR cells more than 2-fold compared with control 4α-PMA-treated cells (Fig. 6A). Similar effects on ATP-induced IP₃ increases were also observed (Fig. 6B). Therefore, loss of PKC enhances PLC activity and increases the overall level of cellular [IP₃], resulting in sustained and prolonged Ca²⁺ responses. These data indicate that negative feedback inhibition of IP₃ generation by PKC is a key element in shaping agonist-induced Ca²⁺ oscillations.

FIGURE 6.

Down-regulation of PKC potentiates hormone-stimulated PLC activity and IP₃ levels. Hepatocytes were transfected with eCFP-PH-PLCδ4 and eYFP-PH-PLCδ4 to monitor PIP₂ levels (PLC activity) or IRIS-1 to monitor IP₃ production (see “Experimental Procedures”) and then cultured overnight in the presence of 2 μm 4α-PMA (Control) or 2 μm PMA (PKC-DR). Cells were stimulated with ATP (200 μm), and maximal FRET changes were determined. A, representative traces showing the mean PLC response in control and PKC-DR cells. Traces are averaged from 27 and 25 cells, respectively, and are normalized to the basal FRET level (mean basal FRET values were 0.85 ± 0.011 for control and 1.028 ± 0.066 for PKC-DR cells, p = 0.033, Student's t test). B, mean peak FRET change (absolute values) ± S.E. for ≥70 cells from five independent experiments. **, p < 0.01, Student's t test. C, representative experiment showing the effect of PKC down-regulation on ATP-induced increases in IP₃ levels for control and PKC-DR cells. Traces are averaged from five and four cells, respectively, and are normalized to the basal FRET level (mean basal FRET values were 0.66 ± 0.016 for control and 0.70 ± 0.013 for PKC-DR cells, p = 0.053, Student's t test). D, mean peak FRET change (absolute values) ± S.E. for ≥25 cells from 10 independent experiments. ***, p < 0.001, Student's t test.

Acute Effect of PKC Activation and Inhibition on Hormone-evoked Ca²⁺ Signaling

In view of results with PKC-DR, we investigated the effects of acute activation or inhibition of PKC on Ca²⁺ signals evoked by hormone. Hepatocytes were treated with phenylephrine at a dose that elicited repetitive Ca²⁺ oscillations (1–20 μm), and then the acute effects of PMA (1 nm) or BIM (5 μm) on the Ca²⁺ response was determined in each cell. Changes in Ca²⁺ oscillation frequency and Ca²⁺ spike width were calculated in hepatocytes that displayed continuous Ca²⁺ spiking for at least 5 min after application of drugs. Activation of PKC by PMA caused either a decrease in oscillation frequency (Fig. 7A, top panel) or a halt in oscillations (Fig. 7A, bottom panel). PMA treatment reduced the oscillation frequency in 32 ± 5% of the cell population and terminated the response in the remaining 68 ± 5%. This negative regulatory effect of PKC activation is consistent with the enhanced PLC/IP₃ and Ca²⁺ responses observed in PKC-DR cells described above. However, counterintuitively, inhibition of PKC with BIM also decreased Ca²⁺ oscillation frequency. Following BIM treatment, the Ca²⁺ oscillation frequency was reduced in a majority of cells (63 ± 11% of cells, Fig. 7B, top panel), and there was a complete loss of Ca²⁺ oscillations in a smaller proportion of cells (37 ± 11% of cells, Fig. 7B, bottom panel).

Although the effects of PMA and BIM on the frequency of agonist-induced Ca²⁺ oscillations both manifest as frequency decreases, there were quantitative and qualitative differences in the responses to activation and inhibition of PKC with these agents. PMA treatment caused a 50% reduction in oscillation frequency (Fig. 7C) but only a small change in spike width (Fig. 7D). By contrast, BIM caused a modest 20% reduction in Ca²⁺ oscillation frequency (Fig. 7C) but dramatically prolonged the duration of the Ca²⁺ spikes (Fig. 7D). Furthermore, comparison of the effects of PMA and BIM revealed qualitative differences with respect to the termination of the Ca²⁺ oscillations. PKC activation with PMA caused an abrupt termination of the response or one to three blunted Ca²⁺ spikes prior to cessation, as shown in Fig. 7A, bottom panel. The termination of Ca²⁺ oscillations following PKC inhibition with BIM was quite different. There was a final sustained or peak/plateau Ca²⁺ increase (Fig. 7B) similar to those typically observed with a maximum hormone dose (1, 47, 48). Therefore, the effects of PKC inhibition with BIM are compatible with the enhanced Ca²⁺ signaling observed with PKC-DR. There is a broadening of the Ca²⁺ oscillations and shift from oscillatory to sustained Ca²⁺ signals (compare Fig. 7B with Fig. 4). Moreover, the apparent reduction in Ca²⁺ oscillation frequency with BIM can be ascribed to the prolongation of the Ca²⁺ spike widths because the interspike interval actually decreased from 56.7 ± 5.7 s to 39.6 ± 4.6 s (p < 0.01) after BIM addition. The decreased Ca²⁺ oscillation frequency and complete termination of Ca²⁺ signals with PMA treatment is also consistent with the PKC-DR data, where negative feedback effects of PKC are ablated.

We also compared the effects of PMA, BIM, and PKC-DR on total [³H]inositol phosphate accumulation (Fig. 7E). PKC-DR and acute BIM treatment both potentiated inositol phosphate accumulation in comparison with vasopressin alone. This corroborates our single-cell Ca²⁺ and IP₃ imaging data, indicating that elimination of PKC activity results in elevated IP₃ generation because of loss of negative feedback. At the single cell level, acute PMA treatment reduced Ca²⁺ oscillation frequency (Fig. 7C), but there was no comparable effect on [³H]inositol phosphate accumulation (Fig. 7E). This finding may reflect multiple opposing effects of PKC, including negative regulation of PLC activation and positive regulation of IP₃ 5-phosphatase (49), because the assay measures total inositol phosphate formation in the presence of Li⁺. This assay was used because the low sensitivity of the [³H]inositol labeling approach in hepatocytes precludes measurement of individual IP₃ isomers. Taken together, the data described above demonstrate that acute inhibition of PKC or PKC-DR eliminates an important negative feedback pathway at the level of hormone-stimulated PLC activity and/or IP₃ metabolism, leading to sustained or significantly broader Ca²⁺ transients. Consistent with this, acute activation of PKC blunts hormone-induced Ca²⁺ responses because of activation of these negative feedback loops. Therefore, PKC acts at multiple targets to modulate the frequency and shape of hormone-induced Ca²⁺ oscillations.

Effects of PKC on Ca²⁺ Oscillations Induced by Uncaging IP₃

To further elucidate targets of PKC, we examined the effect of PKC-DR and acute activation or inhibition of PKC on Ca²⁺ oscillations triggered by uncaging IP₃. Significantly, comparison of IP₃-induced Ca²⁺ transients in control 4α-PMA-treated cells and PKC-DR cells (representative traces are shown in Fig. 8A) revealed no differences in the Ca²⁺ signals elicited by increasing exposure to UV. PKC-DR had no effect on the proportion of cells responding (Fig. 8B) or the type of Ca²⁺ signature observed (Fig. 8C). Furthermore, no significant effects were observed on Ca²⁺ oscillation frequency (Fig. 8D) or Ca²⁺ spike width (Fig. 8E). Therefore, elimination of phorbol ester-sensitive PKC activity through PKC down-regulation does not affect IP₃R function in the absence of hormone.

FIGURE 8.

Effects of PKC on IP₃ release-induced Ca²⁺ oscillations. A–E, hepatocytes were treated overnight with 4α-PMA (1 μm, Control) or PMA (1 μm, PKC-DR) then loaded with caged IP₃ and Fluo-4. Shown are representative traces of single control and PKC-DR hepatocytes stimulated with increasing UV light flashes (A), the percentage of cells responding to one, two, and three UV light flashes (B), the percentage of cells with no response, single spike, oscillations, or saturated (peak/plateau) Ca²⁺ responses (C), the oscillation frequency (D), and the spike width half-peak height (E). F—H, overnight cultured hepatocytes loaded with caged IP₃ and Fluo-4 were treated with PMA (1 μm), 4α-PMA (1 μm), or BIM (5 μm) for 5 min prior to IP₃ uncaging (F) representative traces. Data summarize (G) the percentage of cells responding to two and four UV light flashes and (H) the oscillation frequency after four UV flashes. Data are mean ± S.E. from ≥25 cells from four independent experiments. *, p < 0.05, analysis of variance.

Acute treatment of cells with PMA or BIM (representative traces are shown in Fig. 8F) was also without effect on the proportion of cells responding to photorelease of caged IP₃ (Fig. 8G) or the Ca²⁺ spike width for oscillations resulting from IP₃ uncaging (data not shown). However, PKC activation with PMA causes a 2-fold increase in the Ca²⁺ oscillation frequency elicited by photo-released IP₃, from 1.1 ± 0.26 spikes min⁻¹ in control cells to 2.35 ± 0.30 min⁻¹ in PMA-treated cells (Fig. 8H). This is in clear contrast to the inhibitory effect of PMA to reduce the frequency of phenylephrine-induced Ca²⁺ oscillations (Fig. 7C). This result suggests that there is a direct modulation of IP₃Rs by PKC, which enhances channel activity and excitability. With global activation of PKC by PMA, the negative feedback mediated by PLC inhibition presumably predominates during hormone stimulation, whereas the positive feedback effect on IP₃-induced Ca²⁺ release becomes apparent during IP₃ uncaging when PLC is not activated. Therefore, under physiological conditions, activation of PKC by specific hormone receptors may differentially target negative feedback regulation of IP₃ generation (or degradation) and positive feedback on Ca²⁺ release to shape the resulting Ca²⁺ transients.

PKC Activity Modulates Ca²⁺ Wave Velocity in Response to Both Hormone and Photorelease of Caged IP₃

In the liver, hepatocyte and whole organ function is regulated not only by Ca²⁺ spike frequency but by Ca²⁺ wave propagation across individual cells (intracellular waves) and between cells (intercellular waves) within the liver lobule (5, 50–52). The propagation rates for hormone-induced intracellular Ca²⁺ waves are fixed over a wide range of agonist doses (52, 53). This lack of dependence on stimulus strength has led to the assumption that Ca²⁺ wave propagation is driven by a saltatory CICR processes (5, 54). However, we reported recently that the cytosolic expression of an intracellular IP₃ buffer slows Ca²⁺ wave velocity in a stimulus strength-dependent fashion (20). Those findings are consistent with a role for regeneration of IP₃ via positive feedback of Ca²⁺ on PLC, either globally or locally, which yields a cross-coupling between IP₃ and Ca²⁺ that maximizes the CICR process, leading to stereotypic waves of IP₃R activation.

In this study, we examined whether PKC has the potential to regulate Ca²⁺ wave propagation rates in addition to Ca²⁺ oscillation frequency and kinetics. First we examined the effect of PKC activation and down-regulation on intracellular Ca²⁺ waves elicited by IP₃ uncaging. As shown in Fig. 9A, acute activation of PKC with PMA led to a 2-fold increase in the rates of Ca²⁺ waves elicited by photorelease of IP₃ (16 ± 3.8 μm/s in control 4α-PMA treated cells versus 35.5 ± 5.4 μm/s after PMA treatment). This enhancement of Ca²⁺ wave propagation likely reflects the potentiation of IP₃R activity that also manifests as an increased Ca²⁺ oscillation frequency during IP₃ uncaging (Fig. 8F). As with IP₃-induced Ca²⁺ oscillations, the effect of PKC down-regulation on Ca²⁺ wave propagation was not significant (Fig. 9B), presumably because there is little basal activity even without PKC-DR in the absence of hormone stimulation.

FIGURE 9.

Ca²⁺ wave velocity is regulated by PKC activity. Isolated hepatocytes were treated overnight with 4α-PMA (1 μm, Control) or PMA (1 μm, PKC-DR) or treated acutely with 1 nm PMA to assess the effect of PKC on Ca²⁺ waves initiated by phenylephrine or caged IP₃. Ca²⁺ wave propagation rates were calculated in micrometers per second by determining time at half-peak height from regions of interest from the wave initiation site and the opposite pole of the hepatocyte. A and B, the effect of acute PKC activation (1 μm PMA, A) and PKC-DR down-regulation (B) on caged IP₃ (three UV flashes) induced Ca²⁺ wave velocity (micrometers per second ± S.E. for ≥16 cells from three independent experiments). C and D, the effect of acute PKC activation (1 nm) (C) and PKC-DR (D) on Ca²⁺ wave propagation rate in response to phenylephrine. Data are mean wave velocity (micrometers per second) ± S.E. for ≥18 cells from four independent experiments and from ≥20 cells from three independent experiments, respectively. *, p < 0.05; **, p < 0.01; ***, p < 0.001; Student's t test.

The effect of PKC on hormone-induced Ca²⁺ waves is more complex because it affects both IP₃ generation and IP₃R function. In phenylephrine-stimulated hepatocytes, acute activation of PKC with PMA caused a decrease in Ca²⁺ wave velocity from a control rate of 19.4 ± 2.0 μm/s to 11.2 ± 1.3 μm/s (Fig. 9C). The negative effect of acute PMA on Ca²⁺ wave velocity presumably results from suppression of IP₃ production via enhanced negative feedback inhibition at the level of the hormone receptor or PLC. Therefore, the inhibitory effect of PMA predominates just as it does for the generation of Ca²⁺ oscillations (Fig. 7). However, despite the fact that PKC-DR prevents this inhibitory effect on PLC activation and greatly enhances IP₃ generation, its predominant effect at the level of Ca²⁺ waves was also to slow the rate of propagation. The wave propagation rate in control 4α-PMA treated cells was 14.7 ± 1.4 μm/s, and this decreased to 8.8 ± 0.8 μm/s after PKC down-regulation (Fig. 9D). Therefore, in the presence of hormone, the effects of PKC-DR are also manifest in a reduced level of IP₃R excitability. This provides further evidence that hormone-activated PKC positively regulates Ca²⁺ release and wave propagation by enhancing IP₃R function, either directly or indirectly. Taken together, these data demonstrate that PKC activation during hormone stimulation of the GPCR/PLC signaling system has positive (targeting the IP₃R) and negative feedback (IP₃ generation and metabolism) mechanisms that regulate Ca²⁺ spike width, oscillation frequency, and wave velocity.

Discussion

IP₃-dependent Ca²⁺ oscillations and waves are a major class of Ca²⁺ signals, and understanding the mechanisms that drive the oscillatory behavior and shape the kinetics of individual Ca²⁺ spikes is key to elucidating how Ca²⁺-regulated targets are modulated. There is a substantial stochastic component to IP₃R-dependent Ca²⁺ oscillations (55), but the Ca²⁺ responses to different hormones have distinct stereotypic shapes with hormone-specific kinetic properties in hepatocytes (1, 34). Therefore, there must be further deterministic regulation of the Ca²⁺ signaling machinery beyond IP₃R isoform expression and subcellular distribution. A combination of modeling and experimental data demonstrate that hormone-induced Ca²⁺ oscillations in hepatocytes depend on positive feedback of Ca²⁺ on PLC and consequent cross-coupling of Ca²⁺ and IP₃ oscillations (17, 20).

In this study, we characterized Ca²⁺ responses induced by IP₃ uncaging in hepatocytes and show that, in the absence of a GPCR ligand, Ca²⁺ oscillations are driven by CICR and do not require PLC activation. This is on the basis of a number of lines of evidence. First, PLC inhibition failed to suppress Ca²⁺ oscillations elicited by direct release of IP₃. Second, graded steps of IP₃ uncaging with increasing numbers of UV flashes in the absence of hormone resulted in stimulus strength-dependent effects on Ca²⁺ spike amplitude, width, and wave velocity. This is in clear contrast to the constant Ca²⁺ spike amplitude and kinetic properties for GPCR-dependent Ca²⁺ oscillations and waves over a wide range of hormone doses (1, 5, 6, 9, 56). Third, Ca²⁺ spike widths elicited by caged IP₃ were of substantially shorter duration than hormone-induced Ca²⁺ oscillations reported in this study and previously (1, 9). Significantly, these data demonstrate that, although Ca²⁺ oscillations can be generated by CICR at the IP₃R independent of PLC activity, this is not sufficient to recapitulate the oscillatory Ca²⁺ signals elicited by hormones. Regenerative PLC activation and cyclical fluctuations in IP₃ levels are essential features of hormone-generated baseline-separated Ca²⁺ oscillations. The ability to compare IP₃ uncaging with GPCR-generated Ca²⁺ signals has enabled us to further dissect how Ca²⁺ oscillations are shaped and regulated.

It has been suggested that hormone-induced Ca²⁺ oscillations may rely in part on positive Ca²⁺ feedback regulation of the Ca²⁺ sensitive, but hormone-insensitive, PLC isoforms, i.e. δ and η (57, 58). However, we found that global Ca²⁺ increases induced by photolysis of caged IP₃ do not increase PLC activity in hepatocytes, even though this causes similar [Ca²⁺]_i increases to those observed with hormone, in a range (0.1–10 μm) sufficient to activate PLCδ isoforms (59). On the basis of these data, we conclude that GPCR stimulation is a prerequisite for regenerative PLC activation and, presumably, depends on the PLCβ isoforms.

We found that photorelease of IP₃ caused Ca²⁺ oscillations with similar frequency in the presence or absence of extracellular Ca²⁺. This provides evidence that plasma membrane Ca²⁺ entry pathways and the associated refilling of intracellular Ca²⁺ stores is not an intrinsic component of IP₃-dependent Ca²⁺ oscillations in hepatocytes (39). In addition, these data suggest that the Ca²⁺ filling state of the ER does not determine Ca²⁺ oscillation frequency in hepatocytes. Nevertheless, we observed a reduction in Ca²⁺ spike width in the absence of Ca²⁺ entry, providing evidence that store-operated Ca²⁺ entry can play a role in shaping Ca²⁺ transients.

PKC isoenzymes are key mediators of GPCR/PLC signaling, acting to decode complex spatiotemporal Ca²⁺ changes and regulate cell function (31, 32). However, because many of the proteins involved in generating Ca²⁺ signals are also PKC substrates, this family of enzymes may also dynamically regulate Ca²⁺ signaling (25, 29). Indeed, multiple and sometimes opposing effects of PKC on PLC, IP₃, and Ca²⁺ release are highlighted in this study, revealing targets both upstream and downstream of IP₃ generation. Down-regulation of phorbol ester-sensitive PKC isoforms had the most dramatic effect on the hormone-induced Ca²⁺ oscillations, potentiating PLC activity and the intracellular levels of IP₃ and Ca²⁺.

The effects of acute PKC inhibition with BIM were similar to PKC-DR, evoking broader Ca²⁺ spike widths and maximal Ca²⁺ responses in the presence of hormone. By contrast, inhibition or elimination of PKC activity had no effect on the Ca²⁺ responses elicited by direct photorelease of caged IP₃. These data demonstrate a fundamental role of PKC in the termination of Ca²⁺ transients via negative feedback regulation of IP₃ levels. Indeed, differences in the declining phase of each Ca²⁺ spike during Ca²⁺ oscillations elicited by activation of distinct GPCRs (1, 6) may reflect differential sensitivity to PKC or specific pools of PKC associated with each hormone receptor type (61).

The effects of acute PKC activation were more complex. PMA treatment modestly decreased Ca²⁺ oscillation frequency and spike width in the presence of hormone, whereas the frequency of oscillations after direct release of IP₃ was increased. These data can be explained by dual opposing actions of PKC to suppress IP₃ generation while enhancing IP₃R activity. Most interesting is our observation that PKC down-regulation decreases Ca²⁺ wave velocity in the presence of hormone, despite increasing IP₃ generation. Although these results may appear contradictory, they can also be explained by the dual actions of PKC to inhibit IP₃ generation and enhance IP₃-induced Ca²⁺ release. Specifically, even though PKC-DR suppresses the negative feedbacks that limit IP₃ generation, allowing for more prolonged Ca²⁺ release in response to hormone, PKC-DR also eliminates the positive actions of PKC to enhance IP₃R excitability and, thereby, slows Ca²⁺ wave propagation. This is supported by the very different effects of PKC-DR and PMA on the velocity of Ca²⁺ waves initiated by photorelease of caged IP₃. Therefore, PKC-DR has no effect on IP₃-induced Ca²⁺ waves because there is no role for negative feedback of PKC on IP₃ generation and no sensitization of the IP₃R (this would require PLC activation and diacylglycerol generation). Similarly, PMA dramatically enhances IP₃-induced Ca²⁺ waves because it directly sensitizes the IP₃R but has no negative feedback effect on IP₃ generation. This modulation of Ca²⁺ wave propagation rates by PKC action on IP₃R sensitivity provides an important, hitherto unrecognized, level of regulation of intracellular Ca²⁺ signaling.

Taken together, the data presented here show that PKC regulates multiple and sometimes counteracting steps in the IP₃-dependent Ca²⁺ signaling pathway. Our data identify a number of potential PKC targets capable of Ca²⁺ signal modulation, but further work is required to elucidate which PKC isoforms regulate each target and whether these are cell type/receptor-specific. Translocation of GFP-tagged PKC isoenzymes have provided some insight into receptor-specific effects (62) or differential subcellular distributions of the enzymes upon hormone stimulation (60). However, whether the endogenous PKCs behave in a similar fashion or whether overexpressed PKC protein buffers cellular responses leave the data open to interpretation.

We conclude that, in the presence of sufficient cytosolic IP₃, Ca²⁺ oscillations and waves can be generated in hepatocytes simply by biphasic regulation of the IP₃R by Ca²⁺. However, at physiologically relevant hormone levels, Ca²⁺ oscillations depend on positive feedback of Ca²⁺ on PLCβ and are driven by cross-coupling between Ca²⁺ and IP₃, and these elements of the Ca²⁺ signaling pathway can be specifically tuned and modulated by PKC. Therefore, physiological activation and deactivation of different PKC isoforms with distinct temporal and spatial profiles has the ability to profoundly shape Ca²⁺ oscillation kinetics, wave propagation rates, and the balance between positive and negative feedback mechanisms.

Author Contributions

A. P. T., P. J. B., and L. D. G. conceived and designed the study. P. J. B. wrote the manuscript. P. J. B. and W. M. designed and performed experiments and analyzed data. All authors reviewed the results and approved the final version of the manuscript.