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. 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.

How Ca 2؉ oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca 2؉ oscillations report signal strength via frequency, whereas Ca 2؉ spike amplitude and wave velocity remain constant. IP 3 uncaging also triggers oscillatory Ca 2؉ release, but, in contrast to hormones, Ca 2؉ spike amplitude, width, and wave velocity were dependent on [IP 3 ] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP 3 uncaging are driven by the biphasic regulation of the IP 3 receptor by Ca 2؉ , and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca 2؉ did not perturb Ca 2؉ oscillations elicited by IP 3 uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca 2؉ influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca 2؉ oscillations but had no effect on Ca 2؉ increases induced by uncaging IP 3 . Importantly, PKC activation and inhibition differentially affected Ca 2؉ spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP 3 metabolism to attenuate IP 3 levels and suppress the generation of Ca 2؉ oscillations. Inhibition of PKC relieves negative feedback regulation of IP 3 accumulation and, thereby, shifts Ca 2؉ oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca 2؉ wave velocity, whereas responses to IP 3 uncaging are enhanced. The ability to assess Ca 2؉ responses in the absence of PLC activity indicates that IP 3 receptor modulation by PKC regulates Ca 2؉ release and wave velocity.
Calcium oscillations and waves generated by the activation of PLC-linked 2 GPCRs regulate a multitude of mechanisms from gene transcription to secretion (1)(2)(3). In many cell types, including hepatocytes, stimulus strength is encoded by the frequency of Ca 2ϩ oscillations, with interspike intervals ranging from Ͼ250 s at low hormone concentrations to Ͻ30 s when challenged with higher hormone levels (1, 4 -6). These Ca 2ϩ signals are generated by PLC-mediated hydrolysis of phosphatidylinositol bisphosphate (PIP 2 ) to yield IP 3 and the subsequent activation of IP 3 R Ca 2ϩ release channels in the ER (7). However, the mechanisms driving the subsequent repetitive Ca 2ϩ 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 [Ca 2ϩ ] on IP 3 R gating (13)(14)(15)(16), i.e. Ca 2ϩ -induced Ca 2ϩ release (CICR), or whetherregenerativePLCactivationand/orcyclicalproteinphosphorylation events are also required (17)(18)(19). We have demonstrated recently that intracellular buffering of IP 3 , using a recombinant protein containing the ligand binding domain of rat IP 3 R type I, results in an inhibition of Ca 2ϩ oscillations, a decrease in the rates of Ca 2ϩ rise, and a slowing of Ca 2ϩ wave propagation speed (17,20). These data demonstrate that IP 3 levels dynamically regulate Ca 2ϩ oscillations, providing evidence that cross-coupling between IP 3 and Ca 2ϩ is required to maintain hormone-induced Ca 2ϩ oscillations in non-excitable cells such as hepatocytes.
The Ca 2ϩ oscillation frequency increases with agonist concentration in hepatocytes (1,5,6), but the individual Ca 2ϩ spikes have a constant amplitude and rate of rise and propagate as intracellular Ca 2ϩ waves at a constant velocity independent of agonist dose. Nevertheless, the falling phase of the [Ca 2ϩ ] spikes shows greater diversity (1,6), and different agonists can give characteristically distinct shapes of [Ca 2ϩ ] 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 Ca 2ϩ elevation, in addition to spike frequency, has been demonstrated to regulate transcription (21)(22)(23). It is therefore important to determine not only how PLC-dependent Ca 2ϩ oscillations are generated but also how spike and wave kinetics are further modulated to account for the versatility of Ca 2ϩ signaling.
Long interspike periods between Ca 2ϩ transients at low hormone doses and the broad dynamic range of frequency modulation suggest that Ca 2ϩ interspike periods and spike kinetics are dynamically controlled by feedback loops that regulate IP 3 generation and metabolism as well as IP 3 R function (17,20). PLC-dependent signal transduction activates PKC (24), which, in turn, has the potential to phosphorylate and regulate multiple proteins in the Ca 2ϩ signaling cascade, including GPCRs (25,26), PLC (27), IP 3 R (28, 29), and IP 3 kinase (30). Importantly, concurrent with Ca 2ϩ 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 Ca 2ϩ oscillation kinetics (33,34).
In this study, we compared Ca 2ϩ signals elicited by hormone and photorelease of caged IP 3 and examined how the ensuing Ca 2ϩ oscillations are regulated in response to each stimulus. Our data reveal that Ca 2ϩ oscillations elicited by direct release of caged IP 3 are graded, with the transient amplitude, frequency, and wave velocity dependent on the amount of IP 3 released. Moreover, these Ca 2ϩ responses were independent of PLC activity, indicating that IP 3 uncaging generates Ca 2ϩ oscillations solely through CICR. This is in contrast to hormoneinduced Ca 2ϩ oscillations, which depend on IP 3 oscillations cross-coupled with Ca 2ϩ spiking (17,20) (i.e. regenerative PLC activation) and have characteristic spike properties independent of agonist dose. Therefore, uncaging of IP 3 provides a tool to assess modulators of Ca 2ϩ transients in the absence of PLC activity and other hormone-dependent signaling cascades. We show that Ca 2ϩ oscillations elicited by IP 3 uncaging persist in the absence of extracellular Ca 2ϩ , demonstrating that reloading of ER Ca 2ϩ stores does not entrain these periodic Ca 2ϩ signals. Modulation of Ca 2ϩ 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 Ca 2ϩ oscillations but via different mechanisms. Activation of PKC inhibited regenerative IP 3 generation by the GPCR/PLC, whereas inhibition of PKC relieved this negative feedback, allowing more prolonged and sustained IP 3 generation and, therefore, Ca 2ϩ release. By contrast, CICR oscillations elicited by uncaging IP 3 were potentiated by PKC activation. Furthermore, PKC down-regulation decreased Ca 2ϩ wave velocity in agonist-stimulated cells, whereas it actually increased Ca 2ϩ wave velocity after direct IP 3 release. These data demonstrate that PKC activity regulates IP 3 levels via effects on GPCR coupling, PLC activity, and/or IP 3 metabolism while also effecting IP 3 R sensitivity to regulate Ca 2ϩ spike frequency, width, and Ca 2ϩ 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.
Hormone-induced PLC Activity and IP 3 Detection-Intracellular IP 3 levels or PLC activity were measured using FRETbased 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 ␦4 YFP and PLC ␦4 CFPPH domains (cDNA was a gift from Dr. Balla, National Institutes of Health), which yield a FRET signal while bound to membrane PIP 2 that declines as PIP 2 is hydrolyzed. IP 3 measurements were determined with the IP 3 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 Ϯ 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 Ϯ 30 nm (CFP) or 535 Ϯ 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 ␦4 YFP and PLC ␦4 CFP PH domains were corrected for YFP bleach using linear regression analysis.
Photorelease of Caged IP 3 -Overnight cultured hepatocytes were loaded in HEPES-buffered physiological saline solution with the membrane-permeant form of caged IP 3 (2 M; D-2, 3-O-isopropylidene-6-O-(2-nitro-4,5-dimethoxy)benzyl-myoinositol 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 M). 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 IP 3 was achieved by light pulses from a nitrogen-charged UV laser (Photon Technology International). The cell-permeant caged IP 3 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 IP 3 is metabolized at a slower rate, in the order of minutes, compared with natural IP 3 , 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.
[ 3 H]Inositol Phosphate Accumulation-Total [ 3 H]inositol phosphate accumulation was determined as described previously (31). In brief, primary hepatocytes were labeled overnight with 2.5 Ci ml Ϫ1 myo-[ 3 H]inositol (American Radiolabeled Chemicals, Inc.) in 6-well plates. In some studies, cultures were treated overnight with phorbol-12-myristate-13acetate (PMA) or 4␣-PMA (1 M) to assess the effect of downregulating 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 M) to assess the acute effects of PKC activation and inhibition, respectively. Incubations were terminated by addition of ice-cold tricholoroacetic acid. Water soluble [H 3 ]inositol-containing components were extracted by addition of tri-n-octylamine:1,1,2-trichlorofluoroethane (1:1 ratio). [H 3 ]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. IP 3 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 inhouse 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.

Photorelease of Caged IP 3 Elicits Ca 2ϩ Oscillations in
Hepatocytes-Photorelease of caged IP 3 in hepatocytes induced cytosolic Ca 2ϩ increases, with Ca 2ϩ oscillations observed in many cells (Fig. 1A). Similar to hormone-induced Ca 2ϩ oscillations (1,9), the frequency and number of cells responding to IP 3 uncaging increased with stimulus strength, as determined by the number of UV flashes, and tended toward sustained Ca 2ϩ increases with the strongest stimulation ( Fig. 1, B and C). A single pulse from the UV laser resulted in Ca 2ϩ responses in only 22.5 Ϯ 10.2% of cells. Incrementally increasing the number of UV flashes (applied as a rapid burst) increased the percentage of cells responding (2ϫ UV, 44.2 Ϯ 14.8%; 3ϫ UV, 84 Ϯ 9.2%; 4ϫ UV, 86.7 Ϯ 8.6%; mean Ϯ S.E. from Ͼ100 cells in three independent experiments). In addition, increasing the number of UV flashes shifted the Ca 2ϩ signature from predominantly no response and single Ca 2ϩ transients at low illumination toward oscillatory and sustained (peak/plateau) Ca 2ϩ increases at higher illumination, presumably reflecting increased levels of IP 3 (Fig. 1C). Therefore, the Ca 2ϩ signals induced by uncaging IP 3 appear to mimic hormone-induced Ca 2ϩ responses, the proportion of responsive cells, and the oscillatory and saturated Ca 2ϩ responses increasing with stimulus strength.
A characteristic of hormone-induced Ca 2ϩ oscillations is that although the frequency increases with agonist dose, Ca 2ϩ 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 IP 3 resulted in Ca 2ϩ peak heights and widths that increased with stimulus strength (Fig. 1, D and E). These data are the mean Ϯ S.E. calculated from the average of the first three Ca 2ϩ transients from cells in which oscillations were observed at each level of UV exposure. Of particular note, the mean duration of Ca 2ϩ spikes elicited by a single UV flash was 3.8 Ϯ 0.24 s at half-peak height, which is much shorter than hormone-stimulated Ca 2ϩ transients measured in this work (Figs. 4 and 7) and previous studies (1,38). Broader Ca 2ϩ spike widths were achieved with multiple UV flashes (11.5 Ϯ 2.2 s at half-peak height for 3ϫ UV flashes), but this is still a shorter duration than the Ca 2ϩ spike widths of Ͼ20 s typically observed with hormone stimulation. The velocity of Ca 2ϩ waves elicited by IP 3 uncaging was also dependent on the number of UV flashes (Fig. 1F). Ca 2ϩ waves induced by hormones propagate at 15-25 m/s independent of hormone dose (5), whereas Ca 2ϩ waves induced by IP 3 uncaging rose from 8.2 m/s Ϯ 0.7 at 1ϫ UV to 23.9 m/s Ϯ 4.8 with 3ϫ UV.
IP 3 -induced Ca 2ϩ Oscillations Do Not Require PLC Activity-To address whether IP 3 regeneration through Ca 2ϩ activation of PLC is required to elicit Ca 2ϩ oscillations in response to photolysis of IP 3 , we performed experiments in the presence of the aminosteroid PLC inhibitor U71322. Hepatocytes were loaded with caged IP 3 and concurrently treated for 75 min with 20 M U73122, the inactive analogue U73433, or vehicle (dimethyl sulfoxide). The cells were first exposed to a rapid train of 4ϫ UV flashes to uncage IP 3 , followed by 10 nM vasopressin (Fig. 2, A and B). The percentage of cells eliciting a Ca 2ϩ increase and oscillatory Ca 2ϩ 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 Ca 2ϩ increases in all cells (higher concentrations perturbed Ca 2ϩ release by thapsigargin, indicating offtarget effects). Nevertheless, a significant inhibition of vasopressin-induced Ca 2ϩ transients was observed after U73122 treatment. U73122 reduced the percentage of cells responding to hormone stimulation from 81 Ϯ 6 to 33 Ϯ 9 and the percentage of cells displaying Ca 2ϩ oscillations from 60 Ϯ 5 to 22 Ϯ 6. By contrast, the Ca 2ϩ increases and oscillatory responses elicited by photolysis of caged IP 3 were not significantly different between treatment groups (Fig. 2, C and D).
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 IP 3 . 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 IP 3 or the proportion of cells displaying oscillatory changes in cytosolic Ca 2ϩ (Fig. 2, E and F). Taken together, these data indicate that positive feedback of Ca 2ϩ on PLC does not contribute to the Ca 2ϩ signals elicited by uncaging IP 3 , and, when IP 3 is sufficiently elevated, Ca 2ϩ oscillations are driven primarily by CICR at the IP 3 R.
Plasma Membrane Ca 2ϩ Entry Is Not a Requirement for IP 3driven Ca 2ϩ Oscillations-Store-operated Ca 2ϩ entry plays a fundamental role in maintaining Ca 2ϩ homeostasis and to FIGURE 1. Photorelease of caged IP 3 elicits Ca 2؉ oscillations in primary rat hepatocytes. Isolated hepatocytes were cultured overnight and then loaded with caged IP 3 and Fluo-4. A, representative trace showing cytosolic Ca 2ϩ responses to photolysis of caged IP 3 . 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 Ca 2ϩ responses observed after each train of UV pulses: no response, single spike, oscillations, or a sustained Ca 2ϩ increase (peak/plateau). Data shown are mean Ϯ S.E. from Ն100 cells from five independent experiments. D and E, summary of Ca 2ϩ transient amplitude (D) and Ca 2ϩ 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 Ϯ S.E. of the first three Ca 2ϩ transients for each individual cell (n ϭ 15). F, Ca 2ϩ wave propagation rates as a function of number of UV flashes. Data are mean Ϯ S.E. from cells in which Ca 2ϩ waves were observed after one, two, and three UV flashes (n ϭ 13). *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001; Student's t test.
replete internal Ca 2ϩ stores when cells respond to Ca 2ϩ -mobilizing hormones (39). However, there is a continuing debate regarding the importance of store-operated Ca 2ϩ entry and Ca 2ϩ store load in the generation and feedback control of hormone-stimulated Ca 2ϩ oscillations (40 -42). To determine whether extracellular Ca 2ϩ entry regulates IP 3 R activation or sensitivity to IP 3 , we assessed Ca 2ϩ signals elicited by photorelease of caged IP 3 in the absence and presence of extracellular Ca 2ϩ . Hepatocytes were maintained in either HEPES-buffered physiological saline solution containing 2 mM CaCl 2 or switched to Ca 2ϩ -free buffer 5-10 min prior to uncaging IP 3 with 3ϫ UV flashes (Fig. 3). A somewhat higher proportion of cells did not respond to photorelease of caged IP 3 in Ca 2ϩ -free (37.3 Ϯ 0.7%) compared with Ca 2ϩ -replete conditions (19.3 Ϯ 3.1%) (Fig. 3C), which may reflect an effect of partial Ca 2ϩ store depletion. Nevertheless, Ca 2ϩ oscillations were still observed in response to IP 3 uncaging in the presence or absence of extracellular Ca 2ϩ (Fig. 3, A and B), with no impact on oscillation frequency over a 5-min period (Fig. 3D). These data indicate that plasma membrane Ca 2ϩ entry is not a requirement to sustain repetitive Ca 2ϩ release from the ER. However, the width of the Ca 2ϩ spike, measured at half-peak height for the first three  JULY 24, 2015 • VOLUME 290 • NUMBER 30

JOURNAL OF BIOLOGICAL CHEMISTRY 18523
Ca 2ϩ transients, was decreased significantly in the absence of extracellular Ca 2ϩ (5.5 Ϯ 0.2 s compared with 9.3 Ϯ 0.4 s in the presence of extracellular Ca 2ϩ ). Therefore, Ca 2ϩ entry and ER Ca 2ϩ load may contribute to IP 3 R-induced Ca 2ϩ transients by prolonging spike duration (Fig. 3E).
PKC Down-regulation Perturbs Negative Feedback Inhibition of Ca 2ϩ Mobilization-Previous studies have highlighted the complexity of PKC regulation of hormone-induced Ca 2ϩ 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 M PMA or the inactive analogue 4␣-PMA. A comparison of responses in control 4␣-PMA-treated and PMA-treated hepatocytes revealed a dramatic shift in the type of Ca 2ϩ responses elicited by phenylephrine (20 M). Under control conditions, 65 Ϯ 6.5% of cells responded with an oscillatory Ca 2ϩ signature (Fig. 4A shows a representative trace of a Ca 2ϩ response in control cells), whereas PKC-DR resulted in predominately sustained Ca 2ϩ rises in 73 Ϯ 3% of cells (see Fig.  4B for a representative trace) compared with only 11 Ϯ 0.6% of sustained responses in control cells. In PKC-DR cells, only 16.2 Ϯ 2.2% responded with oscillatory Ca 2ϩ signatures (Fig. 4C shows a representative trace of Ca 2ϩ 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 Ca 2ϩ oscillations were observed displayed responses characteristi-cally different from the stereotypic Ca 2ϩ oscillations in control cells. The oscillation frequency was reduced, and the spike widths were very substantially prolonged. The individual Ca 2ϩ spike widths measured at half-peak height in phenylephrinestimulated control cells were very consistent, with a mean value of 24 Ϯ 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 Ϯ 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, PKC␣, and novel PKC⑀ (phorbol ester-activated PKC isoenzymes in hepatocytes) without affecting the atypical isoform PKC1 (Fig. 4F).
PKC phosphorylation of the plasma membrane Ca 2ϩ pump and of Orai channels has been reported (43,44), indicating that PKC activity may regulate plasma membrane Ca 2ϩ efflux and/or entry. Indeed, Orai1 has been shown to be basally phosphorylated by PKC, and inhibition of PKC leads to enhanced Ca 2ϩ entry (44). To determine whether PKC-DR affects Ca 2ϩ transport across the plasma membrane in hepatocytes, we measured Ca 2ϩ influx and efflux rates in cells treated overnight with PMA or 4␣-PMA (Fig. 5). ER Ca 2ϩ stores were depleted with thapsigargin (Fig. 5, A-C) or ATP (to assess agonist-dependent effects on Ca 2ϩ influx) (Fig. 5, D--F) in the absence of extracellular Ca 2ϩ to induce Store-operated Ca 2ϩ entry pathways. The PKC-DR protocol did not affect the rates of Ca 2ϩ influx upon Ca 2ϩ readdition (Fig. 5, B and E) or the rates of plasma membrane Ca 2ϩ pump-mediated Ca 2ϩ efflux from the cells after removal of extracellular Ca 2ϩ (Fig. 5, C and F). These data suggest that PKC activity does not play a major role in regulating Ca 2ϩ entry or Ca 2ϩ extrusion at the plasma membrane in hepatocytes.
Negative feedback regulation by PKC on both GPCRs and PLC isoenzymes has been implicated in the regulation of Ca 2ϩ oscillations (27,31,45). Therefore, we assessed the effect of PKC-DR on hormone-stimulated PLC activity and IP 3 generation in hepatocytes using FRET-based molecular indicators. To monitor PLC activity, CFP and YFP proteins conjugated to PLC␦4PH domain were coexpressed. Hormone-stimulated PIP 2 breakdown leads to a decrease in FRET between the CFP and YFP moieties, as described previously for CFP and YFP PLC␦1PH (46). The pleckstrin homology (PH) domain of PLC␦4 has a lower affinity for IP 3 compared with PLC␦1, providing a more selective readout of PLC activity (PIP 2 hydrolysis) over intracellular [IP 3 ]. Dynamic changes in cytosolic [IP 3 ] were determined with the IRIS-1 molecular probe containing a mutated version of the ligand binding domain of IP 3 R type 1 flanked by CFP and Venus (20,36). PLC activity elicited by agonist stimulation (ATP, 200 M) was potentiated in PKC-DR cells more than 2-fold compared with control 4␣-PMA-treated cells (Fig. 6A). Similar effects on ATP-induced IP 3 increases were also observed (Fig. 6B). Therefore, loss of PKC enhances PLC activity and increases the overall level of cellular [IP 3 ], resulting in sustained and prolonged Ca 2ϩ responses. These data indicate that negative feedback inhibition of IP 3 generation by PKC is a key element in shaping agonist-induced Ca 2ϩ oscillations.
Acute Effect of PKC Activation and Inhibition on Hormoneevoked Ca 2ϩ Signaling-In view of results with PKC-DR, we investigated the effects of acute activation or inhibition of PKC on Ca 2ϩ signals evoked by hormone. Hepatocytes were treated with phenylephrine at a dose that elicited repetitive Ca 2ϩ oscillations (1-20 M), and then the acute effects of PMA (1 nM) or BIM (5 M) on the Ca 2ϩ response was determined in each cell. Changes in Ca 2ϩ oscillation frequency and Ca 2ϩ spike width were calculated in hepatocytes that displayed continuous Ca 2ϩ 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 Ϯ 5% of the cell population and terminated the response in the remaining 68 Ϯ 5%. This negative regulatory effect of PKC activation is consistent with the enhanced PLC/ IP 3 and Ca 2ϩ responses observed in PKC-DR cells described above. However, counterintuitively, inhibition of PKC with BIM also decreased Ca 2ϩ oscillation frequency. Following BIM treatment, the Ca 2ϩ oscillation frequency was reduced in a majority of cells (63 Ϯ 11% of cells, Fig. 7B, top panel), and there was a complete loss of Ca 2ϩ oscillations in a smaller proportion of cells (37 Ϯ 11% of cells, Fig. 7B, bottom panel).
Although the effects of PMA and BIM on the frequency of agonist-induced Ca 2ϩ 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 Ca 2ϩ oscillation frequency (Fig. 7C) but dramatically prolonged the duration of the Ca 2ϩ spikes (Fig. 7D). Furthermore, comparison of the effects of PMA and BIM revealed qualitative differences with respect to the termination of the Ca 2ϩ oscillations. PKC activation with PMA caused an abrupt termination of the response or one to three blunted Ca 2ϩ spikes prior to cessation, as shown in Fig. 7A, bottom panel. The termination of Ca 2ϩ oscillations following PKC inhibition with BIM was quite different. There was a final sustained or peak/plateau Ca 2ϩ 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 Ca 2ϩ signaling observed with PKC-DR. There is a broadening of the Ca 2ϩ oscillations and shift from oscillatory to sustained Ca 2ϩ signals (compare Fig. 7B with Fig. 4). Moreover, the apparent reduction in Ca 2ϩ oscillation frequency with BIM can  (2 mM) to initiate Ca 2ϩ entry. Where indicated, the buffer was switch to Ca 2ϩ -free medium plus 5 mM BAPTA to stop Ca 2ϩ influx and measure the rates of Ca 2ϩ efflux from the cell. Ca 2ϩ 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).
be ascribed to the prolongation of the Ca 2ϩ spike widths because the interspike interval actually decreased from 56.7 Ϯ 5.7 s to 39.6 Ϯ 4.6 s (p Ͻ 0.01) after BIM addition. The decreased Ca 2ϩ oscillation frequency and complete termination of Ca 2ϩ 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 [ 3 H]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 Ca 2ϩ and IP 3 imaging data, indicating that elimination of PKC activity results in elevated IP 3 generation because of loss of negative feedback. At the single cell level, acute PMA treatment reduced Ca 2ϩ oscillation frequency (Fig.  7C), but there was no comparable effect on [ 3 H]inositol phosphate accumulation (Fig. 7E). This finding may reflect multiple opposing effects of PKC, including negative regulation of PLC activation and positive regulation of IP 3 5-phosphatase (49), because the assay measures total inositol phosphate formation in the presence of Li ϩ . This assay was used because the low sensitivity of the [ 3 H]inositol labeling approach in hepatocytes precludes measurement of individual IP 3 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 IP 3 metabolism, leading to sustained or significantly broader Ca 2ϩ transients. Consistent with this, acute activation of PKC blunts hormone-induced Ca 2ϩ responses because of activation of these negative feedback loops. Therefore, PKC acts at multiple targets to modulate the frequency and shape of hormone-induced Ca 2ϩ oscillations.
Effects of PKC on Ca 2ϩ Oscillations Induced by Uncaging IP 3 -To further elucidate targets of PKC, we examined the effect of PKC-DR and acute activation or inhibition of PKC on Ca 2ϩ oscillations triggered by uncaging IP 3 . Significantly, comparison of IP 3 -induced Ca 2ϩ transients in control 4␣-PMAtreated cells and PKC-DR cells (representative traces are shown in Fig. 8A) revealed no differences in the Ca 2ϩ signals elicited by increasing exposure to UV. PKC-DR had no effect on the proportion of cells responding (Fig. 8B) or the type of Ca 2ϩ signature observed (Fig. 8C). Furthermore, no signifi- cant effects were observed on Ca 2ϩ oscillation frequency (Fig. 8D) or Ca 2ϩ spike width (Fig. 8E). Therefore, elimination of phorbol ester-sensitive PKC activity through PKC down-regulation does not affect IP 3 R function in the absence of hormone.
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 IP 3 (Fig.  8G) or the Ca 2ϩ spike width for oscillations resulting from IP 3 uncaging (data not shown). However, PKC activation with PMA causes a 2-fold increase in the Ca 2ϩ oscillation frequency elicited by photo-released IP 3 , from 1.1 Ϯ 0.26 spikes min Ϫ1 in control cells to 2.35 Ϯ 0.30 min Ϫ1 in PMA-treated cells (Fig.  8H). This is in clear contrast to the inhibitory effect of PMA to reduce the frequency of phenylephrine-induced Ca 2ϩ oscillations (Fig. 7C). This result suggests that there is a direct modulation of IP 3 Rs by PKC, which enhances channel activity and excitability. With global activation of PKC by PMA, the nega- The effect of drug treatment on phenylephrine-induced Ca 2ϩ oscillation frequency (C) and Ca 2ϩ spike width at half-peak height (D) are summarized. The frequencies of agonist-induced Ca 2ϩ oscillations were calculated from 5-min periods in the absence or presence of the drugs. Ca 2ϩ 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 Ϯ S.E. from Ն15 cells from three independent experiments. **, p Ͻ 0.01; ***, p Ͻ 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 Ϯ S.E. from three independent experiments. *, p Ͻ 0.05; **, p Ͻ 0.01; analysis of variance. tive feedback mediated by PLC inhibition presumably predominates during hormone stimulation, whereas the positive feedback effect on IP 3 -induced Ca 2ϩ release becomes apparent during IP 3 uncaging when PLC is not activated. Therefore, under physiological conditions, activation of PKC by specific hormone receptors may differentially target negative feedback regulation of IP 3 generation (or degradation) and positive feedback on Ca 2ϩ release to shape the resulting Ca 2ϩ transients.
PKC Activity Modulates Ca 2ϩ Wave Velocity in Response to Both Hormone and Photorelease of Caged IP 3 -In the liver, hepatocyte and whole organ function is regulated not only by Ca 2ϩ spike frequency but by Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ wave propagation is driven by a saltatory CICR processes (5,54). However, we reported recently that the cytosolic expression of an intracellular IP 3 buffer slows Ca 2ϩ wave velocity in a stimulus strength-dependent fashion (20). Those findings are consistent with a role for regeneration of IP 3 via positive feedback of Ca 2ϩ on PLC, either globally or locally, which yields a crosscoupling between IP 3 and Ca 2ϩ that maximizes the CICR process, leading to stereotypic waves of IP 3 R activation.
In this study, we examined whether PKC has the potential to regulate Ca 2ϩ wave propagation rates in addition to Ca 2ϩ oscillation frequency and kinetics. First we examined the effect of PKC activation and down-regulation on intracellular Ca 2ϩ waves elicited by IP 3 uncaging. As shown in Fig. 9A, acute activation of PKC with PMA led to a 2-fold increase in the rates of Ca 2ϩ waves elicited by photorelease of IP 3   reflects the potentiation of IP 3 R activity that also manifests as an increased Ca 2ϩ oscillation frequency during IP 3 uncaging (Fig. 8F). As with IP 3 -induced Ca 2ϩ oscillations, the effect of PKC down-regulation on Ca 2ϩ wave propagation was not significant (Fig. 9B), presumably because there is little basal activity even without PKC-DR in the absence of hormone stimulation.
The effect of PKC on hormone-induced Ca 2ϩ waves is more complex because it affects both IP 3 generation and IP 3 R function. In phenylephrine-stimulated hepatocytes, acute activation of PKC with PMA caused a decrease in Ca 2ϩ wave velocity from a control rate of 19.4 Ϯ 2.0 m/s to 11.2 Ϯ 1.3 m/s (Fig.  9C). The negative effect of acute PMA on Ca 2ϩ wave velocity presumably results from suppression of IP 3 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 Ca 2ϩ oscillations (Fig. 7). However, despite the fact that PKC-DR prevents this inhibitory effect on PLC activation and greatly enhances IP 3 generation, its predominant effect at the level of Ca 2ϩ waves was also to slow the rate of propagation. The wave propagation rate in control 4␣-PMA treated cells was 14.7 Ϯ 1.4 m/s, and this decreased to 8.8 Ϯ 0.8 m/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 IP 3 R excitability. This provides further evidence that hormone-activated PKC positively regulates Ca 2ϩ release and wave propagation by enhancing IP 3 R 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 IP 3 R) and negative feedback (IP 3 generation and metabolism) mechanisms that regulate Ca 2ϩ spike width, oscillation frequency, and wave velocity.

Discussion
IP 3 -dependent Ca 2ϩ oscillations and waves are a major class of Ca 2ϩ signals, and understanding the mechanisms that drive the oscillatory behavior and shape the kinetics of individual Ca 2ϩ spikes is key to elucidating how Ca 2ϩ -regulated targets are modulated. There is a substantial stochastic component to IP 3 R-dependent Ca 2ϩ oscillations (55), but the Ca 2ϩ 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 Ca 2ϩ signaling machinery beyond IP 3 R isoform expression and subcellular distribution. A combination of modeling and experimental data demonstrate that hormone-induced Ca 2ϩ oscillations in hepatocytes depend on positive feedback of Ca 2ϩ on PLC and consequent cross-coupling of Ca 2ϩ and IP 3 oscillations (17,20).
In this study, we characterized Ca 2ϩ responses induced by IP 3 uncaging in hepatocytes and show that, in the absence of a GPCR ligand, Ca 2ϩ 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 Ca 2ϩ oscillations elicited by direct release of IP 3 . Second, graded steps of IP 3 uncaging with increasing numbers of UV flashes in the absence of hormone resulted in stimulus strength-dependent effects on Ca 2ϩ spike amplitude, width, and wave velocity. This is in clear contrast to the constant Ca 2ϩ spike amplitude and kinetic properties for GPCR-dependent Ca 2ϩ oscillations and waves over a wide range of hormone doses (1,5,6,9,56). Third, Ca 2ϩ spike widths elicited by caged IP 3 were of substantially shorter duration than hormone-induced Ca 2ϩ oscillations reported in this study and previously (1,9). Significantly, these data demonstrate that, although Ca 2ϩ oscillations can be generated by CICR at the IP 3 R independent of PLC activity, this is not sufficient to recapitulate the oscillatory Ca 2ϩ signals elicited by hormones. Regenerative PLC activation and cyclical fluctuations in IP 3 levels are essential features of hormone-generated baseline-separated Ca 2ϩ oscillations. The ability to compare IP 3 uncaging with GPCR-generated Ca 2ϩ signals has enabled us to further dissect how Ca 2ϩ oscillations are shaped and regulated.
It has been suggested that hormone-induced Ca 2ϩ oscillations may rely in part on positive Ca 2ϩ feedback regulation of the Ca 2ϩ sensitive, but hormone-insensitive, PLC isoforms, i.e. ␦ and (57,58). However, we found that global Ca 2ϩ increases induced by photolysis of caged IP 3 do not increase PLC activity in hepatocytes, even though this causes similar [Ca 2ϩ ] i increases to those observed with hormone, in a range (0.1-10 M) sufficient to activate PLC␦ 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 PLC␤ isoforms.
We found that photorelease of IP 3 caused Ca 2ϩ oscillations with similar frequency in the presence or absence of extracellular Ca 2ϩ . This provides evidence that plasma membrane Ca 2ϩ entry pathways and the associated refilling of intracellular Ca 2ϩ stores is not an intrinsic component of IP 3 -dependent Ca 2ϩ oscillations in hepatocytes (39). In addition, these data suggest that the Ca 2ϩ filling state of the ER does not determine Ca 2ϩ oscillation frequency in hepatocytes. Nevertheless, we observed a reduction in Ca 2ϩ spike width in the absence of Ca 2ϩ entry, providing evidence that store-operated Ca 2ϩ entry can play a role in shaping Ca 2ϩ transients.
PKC isoenzymes are key mediators of GPCR/PLC signaling, acting to decode complex spatiotemporal Ca 2ϩ changes and regulate cell function (31,32). However, because many of the proteins involved in generating Ca 2ϩ signals are also PKC substrates, this family of enzymes may also dynamically regulate Ca 2ϩ signaling (25,29). Indeed, multiple and sometimes opposing effects of PKC on PLC, IP 3 , and Ca 2ϩ release are highlighted in this study, revealing targets both upstream and downstream of IP 3 generation. Down-regulation of phorbol ester-sensitive PKC isoforms had the most dramatic effect on the hormoneinduced Ca 2ϩ oscillations, potentiating PLC activity and the intracellular levels of IP 3 and Ca 2ϩ .
The effects of acute PKC inhibition with BIM were similar to PKC-DR, evoking broader Ca 2ϩ spike widths and maximal Ca 2ϩ responses in the presence of hormone. By contrast, inhibition or elimination of PKC activity had no effect on the Ca 2ϩ responses elicited by direct photorelease of caged IP 3 . These data demonstrate a fundamental role of PKC in the termination of Ca 2ϩ transients via negative feedback regulation of IP 3 levels. Indeed, differences in the declining phase of each Ca 2ϩ spike during Ca 2ϩ 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 Ca 2ϩ oscillation frequency and spike width in the presence of hormone, whereas the frequency of oscillations after direct release of IP 3 was increased. These data can be explained by dual opposing actions of PKC to suppress IP 3 generation while enhancing IP 3 R activity. Most interesting is our observation that PKC down-regulation decreases Ca 2ϩ wave velocity in the presence of hormone, despite increasing IP 3 generation. Although these results may appear contradictory, they can also be explained by the dual actions of PKC to inhibit IP 3 generation and enhance IP 3 -induced Ca 2ϩ release. Specifically, even though PKC-DR suppresses the negative feedbacks that limit IP 3 generation, allowing for more prolonged Ca 2ϩ release in response to hormone, PKC-DR also eliminates the positive actions of PKC to enhance IP 3 R excit-ability and, thereby, slows Ca 2ϩ wave propagation. This is supported by the very different effects of PKC-DR and PMA on the velocity of Ca 2ϩ waves initiated by photorelease of caged IP 3 . Therefore, PKC-DR has no effect on IP 3 -induced Ca 2ϩ waves because there is no role for negative feedback of PKC on IP 3 generation and no sensitization of the IP 3 R (this would require PLC activation and diacylglycerol generation). Similarly, PMA dramatically enhances IP 3 -induced Ca 2ϩ waves because it directly sensitizes the IP 3 R but has no negative feedback effect on IP 3 generation. This modulation of Ca 2ϩ wave propagation rates by PKC action on IP 3 R sensitivity provides an important, hitherto unrecognized, level of regulation of intracellular Ca 2ϩ signaling.
Taken together, the data presented here show that PKC regulates multiple and sometimes counteracting steps in the IP 3dependent Ca 2ϩ signaling pathway. Our data identify a number of potential PKC targets capable of Ca 2ϩ signal modulation, but further work is required to elucidate which PKC isoforms regulate each target and whether these are cell type/receptorspecific. 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 IP 3 , Ca 2ϩ oscillations and waves can be generated in hepatocytes simply by biphasic regulation of the IP 3 R by Ca 2ϩ . However, at physiologically relevant hormone levels, Ca 2ϩ oscillations depend on positive feedback of Ca 2ϩ on PLC␤ and are driven by cross-coupling between Ca 2ϩ and IP 3 , and these elements of the Ca 2ϩ 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 Ca 2ϩ oscillation kinetics, wave propagation rates, and the balance between positive and negative feedback mechanisms.