Receptor- and calcium-dependent induced inositol 1,4,5-trisphosphate increases in PC12h cells as shown by fluorescence resonance energy transfer imaging Mitsuhiro Morita1,2, Fumito Yoshiki1, Akira Nakane1, Yoshiumi Okubo1 and Yoshihisa Kudo1 Laboratory of Cellular Neurobiology, School of Life Science, Tokyo University of Pharmacy and Life Science, Japan Department of Neurosurgery, University of New Mexico, Albuquerque, NM, USA Keywords calcium; fluorescent resonance energy transfer; inositol 1,4,5-trisphosphate; muscarinic acetylcholine receptor; phospholipase C Correspondence M Morita, Laboratory of Cellular Neurobiology, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1, Horinouchi, Hachioji, 192-0392, Tokyo, Japan Fax: +81 426 76 8841 Tel: +81 426 76 8963 E-mail: moritam@ls.toyaku.ac.jp (Received 15 May 2007, revised August 2007, accepted August 2007) doi:10.1111/j.1742-4658.2007.06035.x The production and further metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] require several calcium-dependent enzymes, but little is known about subsequent calcium-dependent changes in cellular Ins(1,4,5)P3 To study the calcium dependence of muscarinic acetylcholine receptor-induced Ins(1,4,5)P3 increases in PC12h cells, we utilized an Ins(1,4,5)P3 imaging system based on fluorescence resonance energy transfer and using green fluorescent protein variants fused with the pleckstrin homology domain of phospholipase C-d1 The intracellular calcium concentration, monitored by calcium imaging, was adjusted by thapsigargin pretreatment or alterations in extracellular calcium concentration, enabling rapid receptor-independent changes in calcium concentration via storeoperated calcium influx We found that Ins(1,4,5)P3 production was increased by a combination of receptor- and calcium-dependent components, rather than by calcium alone The level of Ins(1,4,5)P3 induced by the receptor was found to be half that induced by the combined receptor and calcium components Increases in calcium levels prior to receptor activation did not affect the subsequent receptor-induced Ins(1,4,5)P3 increase, indicating that calcium does not influence Ins(1,4,5)P3 production without receptor activation Removal of both the receptor agonists and calcium rapidly restored calcium and Ins(1,4,5)P3 levels, whereas removal of calcium alone restored calcium to its basal concentration Similar calciumdependent increases in Ins(1,4,5)P3 were also observed in Chinese hamster ovary cells expressing m1 muscarinic acetylcholine receptor, indicating that the observed calcium dependence is common to Ins(1,4,5)P3 production To our knowledge, our results are the first showing receptor- and calciumdependent components within cellular Ins(1,4,5)P3 Phosphatidylinositol hydrolysis and subsequent increases in intracellular calcium, activated by G-protein-coupled receptors or receptor tyrosine kinases, are important regulators of various cellular functions [1,2] The initial step in receptor-mediated phosphatidylinositol (PtdIns) metabolism involves the activation of phospholipase C (PLC), which in turn hydrolyzes PtdIns When the substrate is phosphatidylinositol Abbreviations BSS, basal salt saline; CCh, carbachol; CFP, cyan fluoresent protein; CHO, Chinese hamster ovary cells; FRET, fluorescent resonance energy transfer; GFP, green fluorescent protein; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; mAChR, muscarinic acetylcholine receptor; MDCK cells, Madin–Darby canine kidney cells; PHD, pleckstrin homology domain; PtdIns, phosphatidylinositol; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; SOC, store-operated calcium entry; Tg, thapsigargin; YFP, yellow fluoresent protein FEBS Journal 274 (2007) 5147–5157 ª 2007 The Authors Journal compilation ª 2007 FEBS 5147 Calcium-dependent Ins(1,4,5)P3 metabolism in PC12h cells 4,5-bisphosphate [PtdIns(4,5)P2], one of the hydrolysis products, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], can induce the release of calcium from intracellular calcium stores via the Ins(1,4,5)P3 receptor, a mechanism referred to as Ins(1,4,5)P3-induced calcium release [3] The pattern of calcium increase via Ins(1,4,5)P3-induced calcium release has been shown to be diverse, namely transient, sustained and oscillatory [4] In some cases, the intracellular Ins(1,4,5)P3 concentration oscillates simultaneously with the calcium concentration [2,5] Some PLCs contain calcium-binding domains [6], and other Ins(1,4,5)P3-metabolizing enzymes, including inositol polyphosphate 3-kinases and inositol polyphosphate 5-phosphatases, are regulated by calcium [7] Together, these findings suggest a complicated mutual regulation between Ins(1,4,5)P3 metabolism and Ins(1,4,5)P3-induced calcium release, which is thought to contribute to the overall finetuning of cellular functions Biochemical assays of PtdIns metabolism, using radiolabeled compounds with restricted spatial and temporal resolution, differ from cellular responses under physiological conditions, because radiolabeling usually requires lithium to enhance the accumulation of PtdIns metabolism products [8] Alternatively, imaging assays, using green fluorescent protein (GFP) variants fused with either the C1 domain of protein kinase C or the pleckstrin homology domain (PHD) of PLCd can assess PtdIns metabolism at a temporal resolution similar to that of calcium imaging [5] These fusion proteins bind to the products of PtdIns metabolism and change their intracellular localization Recently, a fluorescence resonance energy transfer (FRET)-based assay of the redistribution of PHD fusion proteins from plasma membrane to cytosol was found to be a more reliable and quantitative method for measuring increases in cellular Ins(1,4,5)P3[9] PHD binds to PtdIns(4,5)P2, and more preferentially to Ins(1,4,5)P3, at a 20-fold higher affinity [5] In addition, the PHD fusion proteins of the cyan and yellow variants of GFP (cyan fluoresent protein CFP–PHD and yellow fluoresent protein YFP–PHD) bound to PtdIns(4,5)P2 before stimulation, and were localized close to the plasma membrane As the hydrolysis of PtdIns proceeds, the subsequently formed PtdIns(4,5)P2 is converted to Ins(1,4,5)P3, and the fusion proteins essentially lose their ability to interact with each other, becoming redistributed throughout the cytosol while bound to Ins(1,4,5)P3 This loss of interaction between the fusion proteins is reflected in the change in their respective fluorescent intensities, as monitored by FRET 5148 M Morita et al In this study, we used FRET-based Ins(1,4,5)P3 imaging to monitor the response of the rat pheochromocytoma cell line (PC12h) towards muscarinic acetylcholine receptor (mAChR) activation Using this system, we quantitatively assessed the interaction between intracellular calcium and Ins(1,4,5)P3 metabolism Furthermore, we compared these results with those obtained from Chinese hamster ovary (CHO) cells expressing m1 mAChR to determine the variation between the two cell types Results FRET-based imaging of Ins(1,4,5)P3 metabolism in PC12h cells PHD proteins fused with the cyan and yellow variants of GFP (CFP–PHD and YFP–PHD, respectively) were coexpressed in PC12h cells, and their interaction was analyzed by measuring changes in fluorescence intensity Increases in cellular calcium and Ins(1,4,5)P3 were induced using carbachol (CCh; 100 lm), which specifically activates mAChR and induces Ins(1,4,5)P3 production in PC12 cells, even at 500 lm[10] Following CCh stimulation, the fluorescence intensity of the FRET donor (CFP–PHD) increased, whereas the intensity of the corresponding acceptor (YFP–PHD) decreased, as expected from dissociation of the exchanging partners, causing a subsequent decrease in the overall YFP ⁄ CFP ratio (Fig 1) After complete removal of CCh, fluorescence intensities returned to their original values over several minutes Fig Ins(1,4,5)P3 FRET-based imaging applied to PC12h cells in response to mAChR activation (A) Representative response of PC12h cells expressing CFP–PHD and YFP–PHD to CCh (100 lM, min, bar) The fluorescence ratio (EYFP ⁄ ECFP) (red line) was calculated from the corresponding fluorescence changes in CFP (cyan line) and YFP (yellow line), which are shown in normalized form (F ⁄ Fo) FEBS Journal 274 (2007) 5147–5157 ª 2007 The Authors Journal compilation ª 2007 FEBS M Morita et al Calcium dependence of Ins(1,4,5)P3 metabolism in PC12h cells To determine whether Ins(1,4,5)P3 metabolism in PC12h cells responding to mAChR activation is dependent on calcium, we stimulated cells in normal (2 mm Ca2+) or Ca2+-free extracellular solution [Ca2+-free basal salt saline (BSS)] In the presence of mm extracellular calcium, mAChR activation induced both a transient and a sustained increase in intracellular calcium, as measured by Fura ⁄ AM-based calcium imaging, together with a sustained increase in Ins(1,4,5)P3, which peaked later than calcium (Fig 2Aa) By contrast, when Ca2+-free BSS was Fig Calcium dependence of cellular calcium and Ins(1,4,5)P3 levels in PC12h cells Cellular calcium (upper traces) and Ins(1,4,5)P3 (lower traces) were measured in PC12h cells containing Fura ⁄ AM or expressing CFP–PHD and YFP–PHD, respectively (A) Effects of extracellular calcium on responses induced by CCh (100 lM, bars) Responses are shown as mean ± SEM (a) Normal BSS (2 mM Ca2+; n ¼ 7); (b) Ca2+-free BSS (Ca2+-free; n ¼ 7) (B) Effects of cellular calcium on Ins(1,4,5)P3 levels Representative changes of (a) a cell pretreated with BAPTA-AM (10 lM, 30 min) and exposed to CCh (100 lM, bar); (b) a cell depolarized with KCl (30 mM, bar); and (c) a cell responding to SOC entries and mAChR activation In (c), the cells were pretreated with Tg (1 lM, min) in Ca2+-free BSS prior to imaging, SOC entries were induced after substitution of Ca2+-free BSS (white bar) with normal BSS (bold bar), and stimulation with CCh (100 lM, bar) Scale bars: horizontal (30 s); vertical (0.1) for DRatio (340 ⁄ 380) and (0.1) for DRatio (YFP ⁄ CFP) Calcium-dependent Ins(1,4,5)P3 metabolism in PC12h cells used, only a transient calcium increase was observed, and the level of Ins(1,4,5)P3 production was reduced (Fig 2Ab) This initial transient calcium increase was insensitive to extracellular calcium, a finding similar to that of other cells exposed to a variety of agonists [11] These findings indicate that transient calcium increases result from the release of Ca2+ from intracellular stores, whereas subsequent sustained calcium increases reflect calcium entry, including the so-called storeoperated calcium (SOC), which is activated by depletion of the calcium store Because use of Ca2+-free BSS effectively prevented any sustained calcium increase, as well as reducing the production of Ins(1,4,5)P3 to 63.2 ± 15.8% (mean ± SEM, n ¼ 7, P < 0.01, t-test) of the peak amplitude in normal medium, a significant proportion of the Ins(1,4,5)P3 increase can be regarded as dependent on intracellular calcium The calcium dependence of this increase in Ins(1,4,5)P3 was further examined using other stimulatory conditions To prevent intracellular calcium increases triggered by CCh while still maintaining normal extracellular calcium, cells were loaded with the calcium chelator BAPTA-AM (10 lm, 30 min) We found that CCh did not trigger any detectable increase in calcium, as measured with Fura ⁄ AM, a high-affinity indicator, but an increase in Ins(1,4,5)P3 was observed, albeit at a reduced level, 68.4 ± 13.2% (n ¼ 4, P < 0.05, t-test) of the peak amplitude observed in the absence of treatment (Fig 2Ba) Although depolarization with 30 mm KCl induced a receptor-independent increase in calcium, it did not induce an increase in Ins(1,4,5)P3 (Fig 2Bb) Pretreatment with the sarco-endoplasmic calcium ATPase inhibitor, thapsigargin (Tg), which depletes calcium stores and induces SOC, and variations in extracellular calcium concentration induced a similar increase in calcium via SOC, but had no effect on Ins(1,4,5)P3 production By contrast, mAChR activation after the calcium increase induced an increase in Ins(1,4,5)P3 (Fig 2Bc), but less than that observed in the absence of treatment (65.2 ± 18.8%; n ¼ 5, P < 0.05, t-test), even in the presence of both receptor activation and calcium increase (Fig 2Bc) These findings suggest that calcium becomes less effective in enhancing Ins(1,4,5)P3 production if its level is increased prior to receptor stimulation Although the effect of calcium increases prior to receptor stimulation was not studied further, it is likely that the same effect may account for the finding that the total Ins(1,4,5)P3 increase induced by receptor activation and calcium entry was slightly larger in the absence than in the presence of preceding calcium treatment (Fig 3) Basal calcium FEBS Journal 274 (2007) 5147–5157 ª 2007 The Authors Journal compilation ª 2007 FEBS 5149 Calcium-dependent Ins(1,4,5)P3 metabolism in PC12h cells M Morita et al Fluctuations in the basal Ins(1,4,5)P3 level were larger than those of calcium, presumably because of fluctuations in transfection efficiency of CFP–PHD and YFP–PHD expression vectors Taken together, these results suggest that increased calcium alone is not sufficient to increase Ins(1,4,5)P3 in PC12 cells Moreover, the requirement for receptor activation suggests that calcium has a modulatory effect on Ins(1,4,5)P3 production once it has been induced by receptor activation Calcium-dependent enhancement of receptorinduced Ins(1,4,5)P3 production in PC12h cells Fig Effect of calcium release and calcium entry on cellular Ins(1,4,5)P3 levels in PC12h cells (A) Representative changes in cellular calcium (upper trace) and Ins(1,4,5)P3 (lower trace) levels Cells were stimulated with CCh (100 lM, bars) in Ca2+-free BSS (white bars), which was replaced with normal BSS (bold bars) Where indicated, cells were pretreated without (a) and with (b) Tg (1 lM, min) in Ca2+-free BSS Scale bars: horizontal (30 s); vertical (0.1) for DRatio (340 ⁄ 380) and (0.1) for DRatio (YFP ⁄ CFP) (B) Effect of Tg pretreatment on the total amplitude of Ins(1,4,5)P3 increases in response to mAChR activation, in the absence (white bars) and presence (hatched bars) of extracellular calcium Results were calculated from the traces as indicated in (A), with (Tg, n ¼ 16) and without (–, n ¼ 20) Tg pretreatment Each bar represents the mean ± SEM for the ratio of YFP ⁄ CFP, represented by differences between the peak and basal values, prior to CCh stimulation (DR) and Ins(1,4,5)P3 levels in all experiments in Fig were within 11.2 and 28.5%, respectively, of their mean values in containing mm calcium (Fig 2Aa); these basal levels were not altered significantly by pretreatment with calcium-free medium, BAPTA-AM and Tg 5150 To investigate the modulatory effect of calcium on receptor-induced Ins(1,4,5)P3 production, mAChRs were activated with CCh in Ca2+-free BSS (Fig 2Ab); the extracellular solution was replaced with normal BSS containing CCh, which induced a second calcium increase and a further increase in Ins(1,4,5)P3 (Fig 3Aa) Both of these calcium increases were accompanied by increased Ins(1,4,5)P3, indicating that calcium can enhance receptor-induced Ins(1,4,5)P3 increases The peak Ins(1,4,5)P3 increases were similar for both mAChR-activated and reintroduced extracellular calcium, indicating that calcium entry supplemented a reduction in Ins(1,4,5)P3 production caused by the restoration of intracellular calcium To separate the calcium-dependent component of Ins(1,4,5)P3 induction, PC12h cells were pretreated with Tg in Ca2+-free BSS (1 lm, min) to prevent the possible release of calcium, and were subjected to the same stimulation protocol We found that Tg pretreatment successfully prevented the release of calcium induced by mAChR activation, but did not inhibit the calcium entry caused by re-addition of extracellular calcium (Fig 3Ab) The receptor-induced Ins(1,4,5)P3 increase was smaller in the absence of calcium release than in its presence [compare initial Ins(1,4,5)P3 peaks in Fig 3Aa,b] The reintroduction of extracellular calcium induced a larger Ins(1,4,5)P3 increase when the cells had been pretreated with Tg Figure 3B summarizes the Ins(1,4,5)P3 increases induced by receptor activation and the reintroduction of calcium, as well as the effects of Tg on the components of these responses The peak amplitudes were calculated as the difference between signals before and after mAChR activation (Fig 3B) For cells not pretreated with Tg, the receptor-induced Ins(1,4,5)P3 increase was 79.6 ± 7.4% of that induced by reintroduced calcium, whereas for the pretreated cells it was, 39.8 ± 3.0%, which was significantly lower (P < 0.01, t-test) than for untreated cells The total Ins(1,4,5)P3 increase induced by the FEBS Journal 274 (2007) 5147–5157 ª 2007 The Authors Journal compilation ª 2007 FEBS M Morita et al reintroduction of calcium in Tg-pretreated cells was slightly, but significantly, larger than that in untreated cells (P < 0.05, t-test), whereas the receptor-induced portion of the Ins(1,4,5)P3 increase was significantly smaller in Tg-pretreated than in untreated cells (P < 0.01, t-test) These results strongly suggest that calcium modulates the receptor-induced Ins(1,4,5)P3 production in PC12h cells The calcium dependence of increased Ins(1,4,5)P3 production was further investigated by more accurately controlling the amount of intracellular calcium, because we had found that the total amounts of calcium differed for released calcium and calcium entry (Fig 4) When extracellular calcium was reintroduced to Tg-pretreated PC12h cells, the calcium increase was uniform and not affected by mAChR activation (Fig 4, also see Fig 5) By contrast to our previous results (Fig 3Ab), in which cells were also treated with Tg, receptor activation accompanied by calcium entry enhanced Ins(1,4,5)P3 production (Fig 4A) Although removal of extracellular calcium restored the intracellular calcium concentration, the increase in Ins(1,4,5)P3 was sustained In addition, the subsequent increase in calcium was less effective at enhancing the increase initial receptor-activated Ins(1,4,5)P3 Although similar to our previous results (Fig 3Aa), these findings show more clearly that the presence of calcium initially enhanced receptor-induced Ins(1,4,5)P3 By contrast to the immediate restoration of intracellular calcium, the removal of extracellular calcium led to a gradual decrease in Ins(1,4,5)P3 Because the rates of Ins(1,4,5)P3 and calcium restoration were similar following the termination of receptor activation caused by the removal CCH, these findings suggest that PC12h cells can clear Ins(1,4,5)P3 and ⁄ or re-synthesize PtdIns(4,5)P2 as rapidly as they clear calcium This result could be observed with our imaging system, which uses FRET to measure the rapid translocation of fluorescent proteins Because our results suggested that mAChR induces Ins(1,4,5)P3 production even after the removal of calcium entry, we compared the averaged time-dependent restoration plots for Ins(1,4,5)P3 and calcium following removal of extracellular calcium and removal of both calcium and carbachol (Fig 4Ba,b) Although the removal of calcium after had little effect on the level of receptor- and calcium-induced Ins(1,4,5)P3, the removal of both calcium and CCh reduced the receptor- and calcium-induced Ins(1,4,5)P3 to 31% By contrast, the time-dependent restoration of intracellular calcium was similar under both conditions The gradual loss of Ins(1,4,5)P3 production may have been due to the retention of calcium in some Calcium-dependent Ins(1,4,5)P3 metabolism in PC12h cells Fig Calcium and Ins(1,4,5)P3 restoration rates following the removal of calcium entry or calcium entry and receptor activation (A) Representative changes in cellular calcium (upper trace) and Ins(1,4,5)P3 (lower trace) levels Cells were pretreated with Tg (1 lM,5 min) in Ca2+-free BSS and stimulated with CCh (100 lM, bar) in Ca2+-free BSS (white bars) or normal BSS (bold bars) mAChR activation (1 min) in the presence of extracellular calcium was followed by its removal (1 min) and reintroduction (1 min) Scale bars: horizontal (30 s); vertical (0.05) for DRatio (340 ⁄ 380) and (0.1) for DRatio(YFP ⁄ CFP) (B) Time-dependent restoration of cellular calcium and Ins(1,4,5)P3 levels following removal of calcium or calcium and CCh The average changes in Ins(1,4,5)P3 (a, n ¼ 4) and intracellular calcium (b, n ¼ 14) are expressed as mean ± SEM All traces were calculated from the response to the stimulatory paradigm, as shown in (A) The black and red lines represent the changes occurring after flushing the calcium and calcium ⁄ CCh mixture, respectively The flushing process was initiated at the point indicated by the arrows Bar ¼ 10 s Ins(1,4,5)P3 metabolism-related process, which includes calcium-dependent enzymes, even after intracellular calcium restoration To address this, we assayed the FEBS Journal 274 (2007) 5147–5157 ª 2007 The Authors Journal compilation ª 2007 FEBS 5151 Calcium-dependent Ins(1,4,5)P3 metabolism in PC12h cells Fig Effect of prior calcium levels on cellular Ins(1,4,5)P3 production in PC12h cells Representative changes in cellular calcium (upper trace) and Ins(1,4,5)P3 (lower trace) levels In all experiments, cells were pretreated with Tg (1 lM,5 min) in Ca2+-free BSS and stimulated with CCh (100 lM, bars) in Ca2+-free BSS (white bars) and later in normal BSS (bold bar) (A) mAChR activation (1 min) in the absence of extracellular calcium followed by its re-introduction (1 min); (B) addition (1 min) and removal (1 min) of extracellular calcium, followed by mAChR activation (1 min) in the absence of extracellular calcium and its reintroduction (1 min) Scale bars: horizontal (30 s); vertical (0.05) for DRatio(340 ⁄ 380) and (0.1) for DRatio(YFP ⁄ CFP) effects of increased calcium prior to receptor activation by comparing the receptor- and calcium-induced Ins(1,4,5)P3 increases in Tg-pretreated cells in the presence (Fig 5A) and absence (Fig 5B) of preceding calcium entry (1 min), which had been removed prior to receptor activation Because a similar level of Ins(1,4,5)P3 had been induced under both conditions, the preceding calcium increase had no effect on either receptor- or calcium-induced Ins(1,4,5)P3, suggesting that the calcium-dependent process in Ins(1,4,5)P3 metabolism is unable to retain calcium without receptor activation Therefore, in the absence of receptor activation, Ins(1,4,5)P3 metabolism is likely either insensitive to calcium or incapable of retaining it By contrast, once the calcium-dependent process has been activated by both receptor and calcium, the effects of calcium are retained, even after the calcium is removed Calcium-dependent enhancement of receptorinduced Ins(1,4,5)P3 increases in CHO cells expressing m1 mAChR PC12h cells express m1 mAChR and CCh-induced increases in calcium and investigate the cell-type dependence production, we assayed receptor- and 5152 utilize it in the Ins(1,4,5)P3 To of Ins(1,4,5)P3 calcium-induced M Morita et al Ins(1,4,5)P3 increases in CHO cells that express m1 mAChR (CHO-m1 cells), using the same stimulatory conditions as described above (Fig 3) Because original CHO cells, which not express m1 mAChR, lack both calcium and Ins(1,4,5)P3 responses to CCh [13], any responses observed were likely due to mAChR We found that mAChR activation, coupled with the reintroduction of extracellular calcium, induced both the release and entry of calcium, as well as increasing Ins(1,4,5)P3 levels (Fig 6Aa) Furthermore, although Tg pretreatment effectively inhibited the additional release of calcium, it had no effect on calcium entry Although the lack of calcium release essentially reduced the degree of receptor-induced Ins(1,4,5)P3, it enhanced Ins(1,4,5)P3 induced by the reintroduction of extracellular calcium (Fig 6Ab) The receptor-induced level of Ins(1,4,5)P3 in cells not pretreated with Tg was 120 ± 12.8% of the Ins(1,4,5)P3 level induced by the reintroduction of extracellular calcium (Fig 6B) By contrast, in cells pretreated with Tg, the level of receptor-induced Ins(1,4,5)P3 was 46.3 ± 4.1%, because Tg prevented any further release of calcium The Ins(1,4,5)P3 increases induced by the reintroduction of extracellular calcium were about the same for cells pretreated with Tg (DR; 0.49 ± 0.06) and those without (DR; 0.49 ± 0.05) These results were similar to those obtained for PC12h cells, indicating that the Ins(1,4,5)P3 metabolism of both cell types (PC12h and CHO) exhibits similar calcium dependence Discussion We have shown that a FRET-based Ins(1,4,5)P3 imaging system can be used to monitor the level of Ins(1,4,5)P3 production in mAChR-activated PC12h cells, with the main focus centered on the effects of intracellular calcium changes We found that removal of extracellular calcium had no effect on mAChR-induced calcium release, although it prevented subsequent calcium entry, thus reducing the overall production of Ins(1,4,5)P3 By contrast, calcium increases induced by depolarization or SOC without mAChR activation did not increase Ins(1,4,5)P3 production, indicating that Ins(1,4,5)P3 increases are likely to be modulated, but not activated, by calcium The effect of calcium on Ins(1,4,5)P3 production was further investigated by separately analyzing receptor- and calcium-induced Ins(1,4,5)P3 increases The level of receptor-induced Ins(1,4,5)P3 was about half that induced by the combination of receptor and calcium, and was further enhanced by the subsequent increase in intracellular calcium levels The increase in calcium levels just prior FEBS Journal 274 (2007) 5147–5157 ª 2007 The Authors Journal compilation ª 2007 FEBS M Morita et al Fig Effect of calcium release and calcium entry on cellular Ins(1,4,5)P3 in CHO cells expressing an m1 receptor (A) Representative changes in cellular calcium (upper trace) and Ins(1,4,5)P3 (lower trace) levels Cells were stimulated with CCh (100 lM, bars) in Ca2+-free BSS (white bar), later replaced by normal BSS (bold bar) Where indicated, cells were incubated in the absence (a) or presence (b) of Tg pretreatment (1 lM, min) in Ca2+-free BSS Scale bars: horizontal (30 s); vertical (0.1) for DRatio (340 ⁄ 380) and (0.1) for DRatio (YFP ⁄ CFP) (B) Effect of Tg pretreatment on the total amplitude of Ins(1,4,5)P3 response to mAChR activation, in the absence (white bar) or presence (hatched bar) of extracellular calcium Values were calculated from the traces in (A) with (Tg, n ¼ 14), and without (–, n ¼ 19) Tg pretreatment Each bar represents the mean ± SEM for the ratio of YFP ⁄ CFP, using the differences between peak and basal values prior to CCh stimulation (DR) Calcium-dependent Ins(1,4,5)P3 metabolism in PC12h cells to receptor activation did not affect subsequent receptor- and calcium-induced Ins(1,4,5)P3, suggesting that calcium has no effect on Ins(1,4,5)P3 production in the absence of mAChR preactivation Although removal of receptor agonists and calcium from the medium promoted the rapid recovery of both calcium and Ins(1,4,5)P3 levels, the removal of calcium alone led to the recovery of calcium, but had little or no effect on Ins(1,4,5)P3 production These findings were confirmed by assaying the receptor- and calcium-induced effects on Ins(1,4,5)P3 production in CHO cells expressing m1 mAChR Imaging of Ins(1,4,5)P3 with fluorescently labeled PHD fusion proteins relies on the cellular localization of fluorescent fusion proteins by confocal microscopy [5,14] and measurement of the interaction between the fusion proteins using FRET, as shown here [9] Both methods depend on the redistribution of fusion proteins from the plasma membrane to the cytosol In our earlier study on PC12h cells, in which we used confocal microscopy, mAChR activation resulted in the production of a redistribution peak, which was significantly delayed relative to the increase in calcium levels [15] By contrast, when we used FRET, we observed that calcium increase and Ins(1,4,5)P3 production occurred within a similar time frame FRET is considered the more accurate measure of time dependence, because the interaction between fusion proteins changes almost instantaneously following the disruption of PtdIns(4,5)P2 binding Therefore, the delay observed in our previous study indicates that the release of fusion proteins from the plasma membrane and their migration to the cytosol requires several tens of seconds A similar, but shorter, delay has been reported in CHO cells, suggesting that the diffusion constant of the fusion proteins, or the spatial organization between the plasma membranes and confocal planes, varies among cell types We could not determine whether the redistribution of fluorescently labeled PHD fusion proteins from the plasma membrane to the cytosol results from an increase in Ins(1,4,5)P3 or a decrease in PtdIns(4,5)P2 Because PHD from PLCd1 has a 20-fold greater affinity for Ins(1,4,5)P3 than for PtdIns(4,5)P2[5], however, the fluorescently labeled PHD fusion proteins would likely favor Ins(1,4,5)P3 binding, thus moving from the plasma membrane to the cytosol immediately following an increase in Ins(1,4,5)P3 This has been observed in Madin–Darby canine kidney (MDCK) cells, in which Ins(1,4,5)P3 injection caused the redistribution of fusion proteins, regardless of calcium production [5] Moreover, the introduction of inositol 5-phosphatase, which hydrolyzes Ins(1,4,5)P3, inhibited any further FEBS Journal 274 (2007) 5147–5157 ª 2007 The Authors Journal compilation ª 2007 FEBS 5153 Calcium-dependent Ins(1,4,5)P3 metabolism in PC12h cells redistribution of the fusion proteins [5] By contrast, others have failed to observe the Ins(1,4,5)P3-dependent redistribution of fusion proteins [9] For example, in N1E-115 cells, weak photoactivation of caged Ins(1,4,5)P3 has been found to induce a total release of calcium, although uncaged Ins(1,4,5)P3 was not sufficient to promote the redistribution of fusion proteins [9] In addition, using radiolabeled inositol in adrenal glomerular cells, enhanced accumulation of Ins(1,4,5)P3, but not the redistribution of fusion proteins, was observed in the presence of an inositol 3-kinase inhibitor (Sr3+) [9] A recent study, testing the response of N1E-115 cells to bradykinin, suggested that the redistribution of fusion protein is reflected by the combination of increases and decreases in Ins(1,4,5)P3 and PtdIns(4,5)P2 [16] To address the CCh-induced FRET signal changes in PC12h cells to increased Ins(1,4,5)P3 or to decreased PtdIns(4,5)P2, we tested the effects of inositol 5-phosphatase expression, which had been utilized to attribute PHD fusion protein translocation to Ins(1,4,5)P3 increase in previous studies [5,17], on CCh-induced calcium release and Ins(1,4,5)P3 metabolism We found that this enzyme significantly, but partially suppressed these responses (Fig S1) The partial inhibition of calcium release suggests that this enzyme does not possess sufficient activity for complete hydrolysis of the Ins(1,4,5)P3 resulting from CCh stimulation, and we therefore could not use this enzyme to determine the process underlying the fluorescence changes These fluorescence changes, however, were completely abolished in of 36 cells expressing inositol 5-phosphatase, but in of 15 cells not expressing this enzyme, indicating that these fluorescence changes were likely due to an increase in Ins(1,4,5)P3 production The effect of calcium on Ins(1,4,5)P3 production has been described in cell types other than PC12h, by measuring the translocation of GFP and PHD fusion protein In cerebellar Purkinje cells, increased intracellular calcium induced a more efficient increase in Ins(1,4,5)P3 than did the activation of the group I metabotropic glutamate receptor [17] In bovine adrenal glomerular cells, both intracellular calcium and receptor induced an increase in Ins(1,4,5)P3[18] In both MDCK and CHO cells, the apparently synchronized oscillation of intracellular calcium and Ins(1,4,5)P3 following receptor activation may be due to the calcium dependence of Ins(1,4,5)P3 production [5,19] That is, positive and negative calcium feedback in Ins(1,4,5)P3 metabolism may induce Ins(1,4,5)P3 oscillation, which in turn promotes calcium oscillation By contrast, the PC12 cell line is believed to be incapa5154 M Morita et al ble of generating an oscillatory calcium response Several of our results, however, are in apparent disagreement with this oscillation hypothesis For example, we found similar calcium-dependent Ins(1,4,5)P3 increases in PC12h and CHO cells, and calcium was retained in Ins(1,4,5)P3 metabolismrelated enzymes even after the restoration of calcium levels In HEK293 cells, mAChR-induced calcium oscillation was not accompanied by Ins(1,4,5)P3 oscillation, suggesting that calcium oscillation may not always require Ins(1,4,5)P3 oscillation [20] To determine the physiological role of the calcium dependence of Ins(1,4,5)P3 metabolism, a more detailed analysis of the effects of calcium and the cross-talk between other signaling systems (e.g protein kinase C) is required Although calcium has been found to affect receptor desensitization processes via G-protein receptor kinase or PKC [21,22], it is less likely that calcium increases Ins(1,4,5)P3 metabolism by inhibiting receptor desensitization, inasmuch as the reduction in Ins(1,4,5)P3 metabolism by receptor desensitization during CCh stimulation was almost negligible It is therefore likely that the effect of calcium on Ins(1,4,5)P3 production may be a reflection of the calcium dependence of PLC Other enzymes involved in Ins(1,4,5)P3 metabolism, such as inositol polyphosphate 3-kinase and inositol polyphosphate 5-phosphatase, are calcium dependent, but their activation by calcium is expected to promote Ins(1,4,5)P3 degradation, thus reducing the amount of cellular Ins(1,4,5)P3 The calciumdependent activation of these enzymes may explain the reduced cellular Ins(1,4,5)P3 content in MDCK cells observed at higher extracellular calcium concentrations [5] Four PLC enzyme subtypes, designated b, c, d and e, have been identified to date, with all, except for e, possessing a calcium-binding domain and requiring calcium for proper activation [6] Although PC12 cells express PLCb, -c and -d[23], there is little evidence of PLCe expression in this cell line PLCe is usually expressed in neurons [24], and PC12 cells exhibit considerable neuronal character [25], suggesting that PLCe can be expressed in PC12h cells Calcium-dependent PLC isozymes are inactive in these cells at calcium concentrations below 100 nm, but become active following physiological activation [26] Therefore, the calcium-independent component of the mAChR-induced Ins(1,4,5)P3 increase, which was approximately half the total Ins(1,4,5)P3 increase, may reflect the activity of calcium-independent PLCe This PLC subtype is activated by mAChR via a small G-protein Rho [27], with a similar mechanism hypothesized in PC12h cells The calcium-dependent FEBS Journal 274 (2007) 5147–5157 ª 2007 The Authors Journal compilation ª 2007 FEBS M Morita et al activation of receptor-induced Ins(1,4,5)P3 increases we observed in PC12h cells is reflected in the corresponding activation of calcium-dependent PLCs, particularly PLCb and -d, with PLCb activated by Gq in a calcium-dependent manner [28] and purified PLCd activated by calcium alone [26] Thus, although PC12 cells express significant amounts of PLCd, an increase in intracellular calcium in the absence of receptor activation would induce Ins(1,4,5)P3 production only when PLCd is overexpressed [29] The lack of activity of endogenous PLCd in PC12 cells and other cell types has yet to be established, but changes in cellular PtdIns(4,5)P2 content may provide one possible explanation For example, the ratio of PtdIns to PtdIns(4,5)P2 (100 : 2.8) in NIE-115 cells has been reported to decrease almost immediately (within 10 s) after bradykinin stimulation [16] Because the binding of PHD to PtdIns(4,5)P2 is essential for PLCd activation [26], the low PtdIns(4,5)P2 content prior to receptor activation may lead to inhibition of this enzyme Recently, a Gq-coupled calcium-sensing receptor was shown to induce PtdIns(4,5)P2 production through the utilization of a small G-protein [30], suggesting that this receptor activates Gq-dependent PLCb and calcium-dependent PLCd via PtdIns(4,5)P2 production If mAChR-activation in PC12h cells follows a similar pathway, receptor-induced PtdIns(4,5) P2 may provide the PtdIns(4,5)P2 necessary for the calcium-dependent activation of PLCd The Ins(1,4,5)P3 imaging method utilized here reveals some interesting quantitative and time-dependent properties of Ins(1,4,5)P3 metabolism Because PtdIns metabolism is a fundamental mechanism that controls several major cellular processes, with varying dynamics, this method, coupled with the overexpression of enzymes involved in Ins(1,4,5)P3 metabolism or their suppression by RNA interference, may lead to a greater understanding of the molecular mechanisms involved in many cellular functions Experimental procedures Recombinant DNA The expression vectors for CFP–PHD and YFP–PHD were constructed as described for the GFP fusion protein [15], using an expression vector containing the SRa promoter [31] Cell culture and transfection PC12h cells were seeded on 12-mm diameter polyethylene– imine precoated cover slips (1 lgỈmL)1) in Dulbecco’s Calcium-dependent Ins(1,4,5)P3 metabolism in PC12h cells modified Eagle’s medium-high (Asahi Technoglass, Funabashi, Japan) containing 5% horse serum (Gibco BRL, Gaithersburg, MD) and 5% semifetal bovine serum (Mitsubishi Kagaku, Tokyo, Japan) The CFP–PHD and YFP–PHD expression vectors were transfected using TransFast (Promega, Madison, WI) CHO cells expressing m1 mAChR, the gift of T Haga (Gakushuin University, Tokyo, Japan), were seeded on 12-mm cover slips in Nutrient Mixture (Ham) F-12 containing 10% fetal bovine serum (Equitech-Bio, Ingram, TX), whereas the CFP–PHD and YFP–PHD expression vectors were transfected using LipofectAMINE2000 (Gibco BRL) All cells were imaged for 48–72 h Imaging Extracellular BSSs were used in all physiological experiments (normal BSS; 130 mm NaCl, 5.4 mm KCl, 5.5 mm glucose, mm CaCl2, mm MgCl2, 20 mm Hepes, pH 7.4) Calcium-free extracellular solution (Ca2+-free BSS) lacked CaCl2 and contained 0.5 mm EGTA To load the calcium indicator, the cells were incubated for 45 at 30 °C in normal BSS containing Fura ⁄ AM (7.5 lm; Dojin-kagaku, Kumamoto, Japan), washed three times, and incubated at room temperature for 20 For calcium imaging, sulfinpyrazone (100 lm) was added to normal BSS at each step after repeated washing Fluorescence images in Figs 1–4 and were obtained using an E600FN upright microscope (Nikon, Tokyo, Japan) equipped with a Polychrome IV, high-speed tunable scanning monochromatic light source (T.I.L.L Photonics GmbH, Grafelng, ă Germany) and a C6790 CCD camera (Hamamatsu Photonics, Hamamatsu, Japan), while the images in Figs and were obtained using an IX70 inverted microscope, fitted with an OSP-EXA filter exchanger (Olympus, Tokyo, Japan) and a C6790 CCD camera The image data were analyzed using aquacosmos software (Hamamatsu Photonics) For FRET imaging, the fluorescence was split by a W-View dichroic mirror system (Hamamatsu Photonics) equipped with a dichroic mirror (510LP) and the barrier filters 480DF30 and 535DF25 for YFP–PHD and CFP–PHD, respectively Calcium and Ins(1,4,5)P3 increases were expressed as changes in the ratio of fluorescence intensities (DR) For calcium imaging, the fluorescence ratio was calculated by dividing the fluorescence intensity obtained at 510 nm after excitation at 340 nm by the intensity at 510 nm after excitation at 380 nm For Ins(1,4,5)P3 imaging, the fluorescence ratio was calculated by dividing the fluorescence intensity of YFP–PHD (535 nm) by the intensity of CFP–PHD (489 nm), both of which were obtained by excitation at 430 nm The fluorescence ratio of the FRET response is larger in Figs 3–6 than Figs 1–2, because of the different neutral density filter configuration for excitation, and each of YFP–PHD and CFP–PHD FEBS Journal 274 (2007) 5147–5157 ª 2007 The Authors Journal compilation ª 2007 FEBS 5155 Calcium-dependent Ins(1,4,5)P3 metabolism in PC12h cells M Morita et al Materials CCh was purchased from Wako Chemicals (Tokyo, Japan) All other chemicals were purchased from Sigma (St Louis, MO) 11 12 Acknowledgements This work was supported by Grant-in-Aid 10214204 for Scientific Research on Priority Areas (B) on ‘Regulation of Neural Transduction by Glial Cells’, Grant-in-Aid 15082101 for Scientific Research on Priority Areas on ‘Elucidation of Glia–Neuron Network-Mediated Information Processing Systems’ and Grant-in-Aid 14780582 for Young Scientists on ‘Application of an Insect Receptor to the Investigation of Neuronal Networks’ all from the Ministry of Education, Science and Culture in Japan We thank Ms Hiromi Yanaka and Ms Keiko Suzuki for their excellent secretarial assistance We also thank Dr John A Conner of the University of New Mexico for scientific criticism of the manuscript 13 14 15 References Berridge MJ, Lipp P & Bootman MD (2000) The versatility and universality of calcium signalling Nat Rev Mol Cell Biol 1, 11–21 Carafoli E (2002) Calcium signaling: a tale for all seasons Proc Natl Acad Sci USA 99, 1115–1122 Irvine RF & Schell MJ (2001) Back in the water: the return of the inositol phosphates Nat Rev Mol Cell Biol 2, 327–338 Fewtrell C (1993) Ca2+ oscillations in non-excitable cells Annu Rev Physiol 55, 427–454 Hirose K, Kadowaki S, Tanabe M, Takeshima H & Iino M (1999) Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies complex Ca2+ mobilization patterns Science 284, 1527–1530 Rhee SG (2001) Regulation of phosphoinositide-specific phospholipase C Annu Rev Biochem 70, 281–312 Woodring PJ & Garrison JC (1997) Expression, purification, and regulation of two isoforms of the inositol 1,4,5-trisphosphate 3-kinase J Biol Chem 272, 30447– 30454 Berridge MJ, Downes CP & Hanley MR (1982) Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands Biochem J 206, 587–595 van der Wal J, Habets R, Varnai P, Balla T & Jalink K (2001) Monitoring agonist-induced phospholipase C activation in live cells by fluorescence resonance energy transfer J Biol Chem 276, 15337–15344 10 Vicentini LM, Ambrosini A, Di Virgilio F, Pozzan T & Meldolesi J (1985) Muscarinic receptor-induced phos- 5156 16 17 18 19 20 21 22 23 phoinositide hydrolysis at resting cytosolic Ca2+ concentration in PC12 cells J Cell Biol 100, 1330–1333 Bennett DL, Bootman MD, Berridge MJ & Cheek TR (1998) Ca2+ entry into PC12 cells initiated by ryanodine receptors or inositol 1,4,5-trisphosphate receptors Biochem J 329, 349–357 Ebihara T & Saffen D (1997) Muscarinic acetylcholine receptor-mediated induction of zif268 mRNA in PC12D cells requires protein kinase C and the influx of extracellular calcium J Neurochem 68, 1001–1010 Burford NT, Tobin AB & Nahorski SR (1995) Differential coupling of m1, m2 and m3 muscarinic receptor subtypes to inositol 1,4,5-trisphosphate and adenosine 3¢,5¢-cyclic monophosphate accumulation in Chinese hamster ovary cells J Pharmacol Exp Ther 274, 134–142 Varnai P & Balla T (1998) Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools J Cell Biol 143, 501–510 Morita M, Yoshiki F & Kudo Y (2003) Simultaneous imaging of phosphatidyl inositol metabolism and Ca(2+) levels in PC12h cells Biochem Biophys Res Commun 308, 673–678 Xu C, Watras J & Loew LM (2003) Kinetic analysis of receptor-activated phosphoinositide turnover J Cell Biol 161, 779–791 Okubo Y, Kakizawa S, Hirose K & Iino M (2001) Visualization of IP(3) dynamics reveals a novel AMPA receptor-triggered IP(3) production pathway mediated by voltage-dependent Ca(2+) influx in Purkinje cells Neuron 32, 113–122 Balla T, Nakanishi S & Catt KJ (1994) Cation sensitivity of inositol 1,4,5-trisphosphate production and metabolism in agonist-stimulated adrenal glomerulosa cells J Biol Chem 269, 16101–16107 Young KW, Nash MS, Challiss RA & Nahorski SR (2003) Role of Ca2+ feedback on single cell inositol 1,4,5-trisphosphate oscillations mediated by G-proteincoupled receptors J Biol Chem 278, 20753–20760 Sinnecker D & Schaefer M (2004) Real-time analysis of phospholipase C activity during different patterns of receptor-induced Ca2+ responses in HEK293 cells Cell Calcium 35, 29–38 Haga K, Kameyama K, Haga T, Kikkawa U, Shiozaki K & Uchiyama H (1996) Phosphorylation of human m1 muscarinic acetylcholine receptors by G protein-coupled receptor kinase and protein kinase C J Biol Chem 271, 2776–2782 Pitcher JA, Freedman NJ & Lefkowitz RJ (1998) G protein-coupled receptor kinases Annu Rev Biochem 67, 653–692 Mazzoni M, Bertagnolo V, Neri LM, Carini C, Marchisio M, Milani D, Manzoli FA & Capitani S FEBS Journal 274 (2007) 5147–5157 ª 2007 The Authors Journal compilation ª 2007 FEBS M Morita et al 24 25 26 27 28 29 (1992) Discrete subcellular localization of phosphoinositidase C beta, gamma and delta in PC12 rat pheochromocytoma cells Biochem Biophys Res Commun 187, 114–120 Wu D, Tadano M, Edamatsu H, Masago-Toda M, Yamawaki-Kataoka Y, Terashima T, Mizoguchi A, Minami Y, Satoh T & Kataoka T (2003) Neuronal lineage-specific induction of phospholipase Cepsilon expression in the developing mouse brain Eur J Neurosci 17, 1571–1580 Pollock JD, Krempin M & Rudy B (1990) Differential effects of NGF, FGF, EGF, cAMP, and dexamethasone on neurite outgrowth and sodium channel expression in PC12 cells J Neurosci 10, 2626–2637 Allen V, Swigart P, Cheung R, Cockcroft S & Katan M (1997) Regulation of inositol lipid-specific phospholipase Cdelta by changes in Ca2+ ion concentrations Biochem J 327, 545–552 Evellin S, Nolte J, Tysack K, vom Dorp F, Thiel M, Weernink PA, Jakobs KH, Webb EJ, Lomasney JW & Schmidt M (2002) Stimulation of phospholipase C-epsilon by the M3 muscarinic acetylcholine receptor mediated by cyclic AMP and the GTPase Rap2B J Biol Chem 277, 16805–16813 Park D, Jhon DY, Kriz R, Knopf J & Rhee SG (1992) Cloning, sequencing, expression, and Gq-independent activation of phospholipase C-beta J Biol Chem 267, 16048–16055 Kim YH, Park TJ, Lee YH, Baek KJ, Suh PG, Ryu SH & Kim KT (1999) Phospholipase C-delta1 is activated Calcium-dependent Ins(1,4,5)P3 metabolism in PC12h cells by capacitative calcium entry that follows phospholipase C-beta activation upon bradykinin stimulation J Biol Chem 274, 26127–26134 30 Huang C, Handlogten ME & Miller RT (2002) Parallel activation of phosphatidylinositol 4-kinase and phospholipase C by the extracellular calcium-sensing receptor J Biol Chem 277, 20293–20300 31 Takebe Y, Seiki M, Fujisawa J, Hoy P, Yokota K, Arai K, Yoshida M & Arai N (1988) SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type long terminal repeat Mol Cell Biol 8, 466–472 Supplementary material The following supplementary material is available online: Fig S1 Effect of INPP5A expression on the levels of calcium and Ins(1,4,5)P3 in PC12h cells following mAChR activation This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 5147–5157 ª 2007 The Authors Journal compilation ª 2007 FEBS 5157 ... were coexpressed in PC12h cells, and their interaction was analyzed by measuring changes in fluorescence intensity Increases in cellular calcium and Ins(1,4,5)P3 were induced using carbachol (CCh;... of these calcium increases were accompanied by increased Ins(1,4,5)P3, indicating that calcium can enhance receptor -induced Ins(1,4,5)P3 increases The peak Ins(1,4,5)P3 increases were similar... Ins(1,4,5)P3 increases The level of receptor -induced Ins(1,4,5)P3 was about half that induced by the combination of receptor and calcium, and was further enhanced by the subsequent increase in