1 Department of Medical Cell Biology, Uppsala University, Biomedical Centre, Box 571, SE-75123 Uppsala, Sweden
2 Department of Biophysics, National T. Shevchenko University of Kiev, Kiev, Ukraine
* Author for correspondence (e-mail: anders.tengholm{at}medcellbiol.uu.se)
Accepted 7 July 2005
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Summary |
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Key words: Phospholipase C, Ca2+, Pancreatic ß-cell, Evanescent wave microscopy, Green fluorescent protein, Store-operated Ca2+ entry
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Introduction |
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In pancreatic ß-cells, PLC mediates the potentiating action on insulin secretion of many hormones and neurotransmitters. For example, it is well established that cholinergic stimulation of insulin secretion is associated with accumulation of IP3 and diacylglycerol (Gilon and Henquin, 2001). This effect is due to activation of muscarinic M3 receptors, which, as in other tissues, are believed to stimulate PLC-ß via the Gq family of heterotrimeric G proteins. It was recognized early on that phospholipid hydrolysis and inositol phosphate production after cholinergic stimulation was larger in ß-cells maintained in Ca2+-containing medium than in Ca2+-deficient medium (Best, 1986
; Biden et al., 1987
; Garcia et al., 1988
). This phenomenon is poorly understood but may be explained by Ca2+-mediated activation of PLC (Biden et al., 1987
). Analyses of [Ca2+]i responses in ß-cells have demonstrated that stimulation with the muscarinic-receptor agonist carbachol is associated with a biphasic increase of [Ca2+]i with a rapid peak followed by a sustained plateau, sometimes with superimposed oscillations (Gylfe, 1991
; Liu and Gylfe, 1997
). Whereas the first phase reflects rapid IP3-mediated mobilization of intracellular Ca2+, the second phase depends on Ca2+ influx through store-operated channels in the plasma membrane (Liu and Gylfe, 1997
). As all PLC isoforms require Ca2+, it is possible that such receptor-induced Ca2+ signals result in feedback activation of the enzyme.
Owing to difficulties in measuring PLC activity in individual cells, little is known about how physiological changes of [Ca2+]i influence the activity of the lipase. Most studies of PLC have employed radiotracer techniques in populations of cells, but with the advent of phosphoinositide-specific fluorescent biosensors, it has become possible to measure the enzyme activity in individual living cells (Stauffer et al., 1998; Varnai and Balla, 1998
). The most commonly used single-cell biosensor for PLC activity is the pleckstrin homology (PH) domain from PLC
1 fused to the green fluorescent protein (PHPLC
-GFP), which binds PIP2 and IP3 with high affinity and specificity (Stauffer et al., 1998
; Varnai and Balla, 1998
). In unstimulated cells, the construct is therefore located mainly to the plasma membrane. Upon PIP2 hydrolysis and formation of IP3, PHPLC
-GFP dissociates from the membrane and binds to IP3 in the cytoplasm. This PHPLC
-GFP translocation can be used as an indicator of PLC activity. Using an evanescent wave microscopy approach for simultaneous measurements of PLC activity and [Ca2+]i, we recently demonstrated that PLC activity in the electrically excitable insulin-secreting ß-cell is tightly controlled by [Ca2+]i elevations that result from voltage-dependent Ca2+ entry (Thore et al., 2004
). In the present paper, we test the hypothesis that elevations of [Ca2+]i following receptor stimulation result in positive feedback activation of PLC. After stimulation of endogenous muscarinic receptors in insulin-secreting cells, two distinct phases of PLC activation were resolved: an initial transient phase that is amplified by mobilization of intracellular Ca2+; and a second sustained phase, which is dependent on Ca2+ entry through store-operated channels in the plasma membrane. Moreover, activation of PLC by Ca2+ mobilized from intracellular stores was found to occur in primary mouse pancreatic ß-cells after stimulation with the insulinotropic hormone glucagon.
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Materials and Methods |
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Cell culture and transfection
Insulin-secreting MIN-6 ß-cells (passage 16-30) (Miyazaki et al., 1990) were cultured at 37°C in a humidified atmosphere containing 5% CO2 in DMEM containing 25 mM glucose and supplemented with 15% foetal calf serum, 2 mM glutamine, 70 µM 2-mercaptoethanol, 100 units/ml penicillin and 100 µg/ml streptomycin. After plating onto 25 mm coverslips at a density of 1.5x105/ml, the cells were transiently transfected with 2 µg of plasmid DNA with Lipofectamine 2000 (Invitrogen) in a 1:2.5 DNA:lipid ratio (according to the manufacturer's protocol) and further cultured for 24-48 hours.
Mouse pancreatic ß-cells were obtained from collagenase-isolated islets of Langerhans from ob/ob mice. Free cells were prepared by shaking the islets in a Ca2+-deficient medium (Lernmark, 1974). The cells were then suspended in RPMI 1640 medium supplemented with 10% foetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin and 30 µg/ml gentamycin, and allowed to attach to 25 mm coverslips for 1-3 days in culture at 37°C in a humidified atmosphere of 5% CO2. The ob/ob mouse islets contain more than 90% ß-cells (Hellman, 1965
), which respond normally to glucose and to other regulators of insulin release (Hahn et al., 1974
). Transfection of the primary mouse ß-cells was performed by electroporation of in vitro transcribed mRNA, as outlined below.
In vitro transcription and processing of mRNA
As an alternative to conventional plasmid and viral methods commonly applied to transfect pancreatic ß-cells, we used an mRNA transfection technique (Yokoe and Meyer, 1996) to express PHPLC
fused to yellow fluorescent protein (YFP) in the ob/ob mouse ß-cells. First, we generated the vector pCS2-PHPLC
-YFP. The PH domain from PHPLC
-GFP was PCR amplified using primers 5'-TTTTGGATCCACCATGGGCCTACAGGATGATGAGGA-3' (forward) and 5'-TTTTTCTAGAGCCTGGATGTTGAGCTCCTTCAG-3' (reverse) containing BamHI and XbaI restriction sites, respectively (underlined). The product was subsequently ligated into the corresponding sites of the transcription vector pCS2-YFP (Tengholm and Meyer, 2002
). The plasmid was linearized with NotI and subsequent in vitro transcription with SP6 RNA polymerase and poly-A tail addition were performed according to the manufacturers' protocol using commercial kits [mMESSAGE mMACHINE, and Poly(A) Tailing kit, respectively, Ambion Europe]. After purification of the mRNA by column chromatography (RNeasy, Qiagen), the eluent was dried and the mRNA dissolved at 2 µg/µl in phosphate-buffered saline (PBS; pH 7.00).
Electroporation
Electroporation of the adherent mouse pancreatic ß-cells was performed using a custom-built small-volume electroporator (Teruel and Meyer, 1997). After replacement of the medium with electroporation buffer (PBS supplemented with 20 mM glucose at pH 7.00), 1-2 µl of the 2 µg/µl mRNA sample was applied to the
15 µl electroporation chamber. Electroporation was performed at 220 V/cm, using three voltage pulses, each 30 mseconds and 40 seconds apart. After transfection, the electroporation buffer was replaced with RPMI 1640 medium and the cells were kept in culture for 10-20 hours to allow for expression of PHPLC
-YFP.
Fluorescence microscopy
Before experiments, the cells were transferred to a buffer containing 125 mM NaCl, 4.8 mM KCl, 1.28 mM CaCl2, 1.2 mM MgCl2, 3 mM glucose and 25 mM HEPES with pH adjusted to 7.40 with NaOH. Where indicated, transfected cells were loaded with the Ca2+ indicator Fura Red by a 40 minute incubation at 37°C with 10 µM of its acethoxymethyl ester. The coverslips were used as exchangeable bottoms of a 50 µl open chamber and superfused with buffer at a rate of 0.3 ml/minute. All experiments were performed at 37°C.
GFP, YFP and Fura Red fluorescence was measured using an evanescent wave microscopy setup built around an Eclipse TE2000 microscope (Nikon) as previously described (Thore et al., 2004). The 488 nm beam of an argon ion laser (Creative Laser Production, Munich, Germany) was homogenized, expanded and refocused onto the periphery of the back focal plane of a 60x 1.45-NA objective (Nikon) to achieve total internal reflection at the interface between the coverslip and the adherent cells. The fluorescence excited by the evanescent field was detected using an IEEE1394 Orca-ER camera (Hamamatsu) controlled by MetaMorph or MetaFluor software (Universal Imaging). Selection of emission wavelength was made with interference (525/25 nm for GFP; 550/30 for YFP) and long-pass (>630 nm for Fura Red) filters (Chroma Technology) mounted in a Lambda 10-2 filter wheel (Sutter Instruments) capable of changing positions within 60 mseconds. Images (or image pairs) were acquired every 5 seconds, except for in the experiments in Fig. 4, where image pairs were acquired at
1.5 Hz in the data-streaming mode of MetaFluor. To minimize exposure of the cells to the potentially harmful laser light, the beam was blocked by an electronic shutter (Sutter Instruments) between image captures.
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Results |
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To verify the role of store-operated Ca2+ entry for activation of PLC, we compared the influence of extracellular Ca2+ on membrane PHPLC-GFP fluorescence under control conditions and after activating this Ca2+ influx pathway by depletion of the intracellular Ca2+ stores. Under control conditions, Ca2+ removal with addition of 2 mM EGTA and the following reintroduction of 1.28 and 2.56 mM of the ion resulted in less than 2% changes in PHPLC
-GFP fluorescence (n=9; Fig. 2G). Subsequent activation of the store-operated pathway by addition of 100 µM of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) inhibitor CPA was without significant effect on PHPLC
1-GFP fluorescence in Ca2+-deficient medium. By contrast, there was an immediate translocation of PHPLC
-GFP upon reintroduction of 1.28 mM Ca2+ to the medium (5.4±0.7% loss of fluorescence, n=9, P<0.01 for difference from Ca2+ addition before CPA; Fig. 2G). No further change of fluorescence was observed when raising Ca2+ to 2.56 mM. The Ca2+ effect was specific, as there was no change in the fluorescence in control cells expressing cytoplasmic or membrane-targeted GFP alone (data not shown). Taken together, the results indicate that sustained PLC activation depends on Ca2+ influx through store-operated channels.
PLC activation involves positive feedback from intracellular Ca2+ mobilization
In view of the potent effect of Ca2+ influx on PLC activity, we investigated the influence of Ca2+ mobilized from intracellular stores on receptor-triggered PLC activity. Simultaneous recording of PLC activity with PHPLC-GFP and [Ca2+]i with Fura Red demonstrated that the transient PLC activation induced by carbachol in Ca2+-deficient medium containing EGTA was associated with a rapid and pronounced spike of [Ca2+]i (Fig. 3A). Increasing the Ca2+ buffering capacity of the cytoplasm by loading the cells with 1 mM of the acetoxymethyl ester of the Ca2+ chelator BAPTA resulted in altered response patterns for both [Ca2+]i and PHPLC
-GFP translocation (Fig. 3B). The [Ca2+]i response had a lower amplitude and longer duration in the BAPTA-loaded cells (Fig. 3B,E), and similar differences were observed for PHPLC
-GFP translocation (Fig. 3B,D), indicating that the [Ca2+]i response is important for PLC activation kinetics. Further support for this idea was obtained from experiments in which intracellular Ca2+ stores had been depleted by SERCA inhibition with 100 µM CPA (Fig. 3C-E) or 1 µM thapsigargin (Fig. 3D,E). Accordingly, both agents not only abolished the [Ca2+]i elevation induced by carbachol, but also strongly suppressed the PHPLC
-GFP translocation (Fig. 3C-E). These findings indicate that PLC activation in response to muscarinic-receptor stimulation is enhanced by positive feedback from Ca2+ that is mobilized from intracellular stores.
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Discussion |
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We now extend these findings by showing that also PLC activity triggered by a muscarinic-receptor agonist is tightly regulated by Ca2+. Our data indicate that Ca2+ exerts pronounced amplification of both the initial and sustained receptor-triggered PLC activity. Whereas the initial activation of the enzyme in response to carbachol was unaffected by omission of extracellular Ca2+, it was markedly suppressed after depletion of Ca2+ from the ER. The latter effect is probably explained by the failure of carbachol to mobilize Ca2+ from intracellular stores. A similar effect was thus observed when the [Ca2+]i elevation in response to carbachol was prevented by increasing the cytoplasmic Ca2+ buffering capacity with BAPTA. Depletion of ER Ca2+ has previously been found to suppress 1B-adrenoceptor-mediated oscillations of IP3 in CHO cells, indicating a role for Ca2+ feedback on PLC for periodic generation of IP3 (Young et al., 2003
). Direct support for the idea that Ca2+ mobilization from intracellular stores enhances PLC activity was now provided by the finding in primary mouse pancreatic ß-cells that cAMP-sensitized intracellular Ca2+ mobilization is rapidly followed by activation of PLC. It is important to note that the glucagon receptor does not activate PLC in ß-cells (S.T. and A.T., unpublished) and that the pronounced transients of [Ca2+]i seen in this cell type after stimulation with cAMP-elevating agents is due to Ca2+-induced Ca2+ release via PKA-mediated sensitization of IP3 receptors (Liu et al., 1996
; Dyachok and Gylfe, 2004
).
Whereas the initial PLC activation was amplified by intracellular Ca2+ mobilization, the second sustained phase of PLC activity after receptor stimulation crucially depended on Ca2+ influx from the extracellular medium. Although carbachol under some conditions may depolarize the ß-cell sufficiently to reach the activation threshold for voltage-dependent Ca2+ entry (Gilon and Henquin, 2001), the involvement of such a mechanism is not required, as neither hyperpolarization with diazoxide nor direct inhibition of the Ca2+ channels with nifedipine had any effect on the sustained PLC activity. Instead, the PLC activity was suppressed by La3+ and commonly used inhibitors of store-operated Ca2+ channels.
None of the currently available inhibitors of the store-operated Ca2+ influx pathway is entirely specific. Although 2-APB was originally described as a membrane-permeable IP3-receptor inhibitor (Maruyama et al., 1997), later studies have shown that this effect is weak and that, instead, 2-APB is a reliable inhibitor of store-operated Ca2+ entry in various types of cells, including pancreatic ß-cells (Bootman et al., 2002
; Dyachok and Gylfe, 2001
). If the currently observed effect of 2-APB on PHPLC
-GFP fluorescence were due to inhibition of IP3 receptors, the early and late phase of the carbachol response should have been affected equally. However, 2-APB only inhibited the late sustained PLC activation upon carbachol-mediated stimulation, consistent with a negligible effect on IP3 receptors. The inhibition of PLC activity with unrelated blockers of store-operated channels and the stimulation obtained after activation of store-operated Ca2+ entry together support the conclusion that this pathway is involved in receptor-triggered PLC activation in insulin-secreting cells. In this context, it is worth noting that an inhibitory effect of 2-APB on acetylcholine-induced IP3 production in pancreatic acinar cells was attributed to a novel unknown action of this drug (Wu et al., 2004
). In view of the present data, this observation may represent suppression of IP3 production after inhibition of store-operated Ca2+ entry.
The maintenance of PLC activity by store-operated Ca2+ entry can explain early observations that carbachol-stimulated inositol phosphate production is larger in ß-cells maintained in Ca2+-containing than in Ca2+-deficient medium (Best, 1986; Biden et al., 1987
; Garcia et al., 1988
). Regulation of receptor-triggered PLC activity by Ca2+ entry has been described also in other types of cells. Using CHO cells expressing heterologous G-protein-coupled receptors, Nash et al. (Nash et al., 2001
; Nash et al., 2002
) have reported that Ca2+ influx stimulates PLC activity triggered by muscarinic M3 receptors,
1B adrenoceptors and mGlu1a metabotropic glutamate receptors, but inhibits that triggered by mGlu5 receptors. Receptor-activated Ca2+ entry has also been reported to promote PLC activity in bradykinin-stimulated PC12 cells (Kim et al., 1999
), in B-cell receptor-ligated DT40 B lymphocytes (Nishida et al., 2003
) and in rabbit gastric smooth muscle cells stimulated with some Gi/o-coupled receptor agonists (Murthy et al., 2004
). These findings suggest that store-operated Ca2+ entry in various types of cells, including insulin-secreting ß-cells, serves to amplify Ca2+ signalling not only by elevating [Ca2+]i and replenishing intracellular stores, but also by directly stimulating PLC.
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It still remains to be clarified which PLC isoforms account for the different phases of receptor activation. All PLC isoforms depend on Ca2+ for activity (Rhee, 2001) and PLC
has been suggested to be most sensitive (Allen et al., 1997
). The store-operated Ca2+ entry following bradykinin-induced PLCß activation in PC12 cells (Kim et al., 1999
) and that induced by Gi/o-coupled receptor agonists in smooth muscle cells (Murthy et al., 2004
) were thus found to stimulate PLC
1, whereas B-cell receptor signalling in DT40 lymphocytes is amplified by Ca2+-mediated activation of PLC
2 (Nishida et al., 2003
). When overexpressed in MIN6 ß-cells, the PLC-ß1 and -
1 isoforms were reported to be equally sensitive to elevation of [Ca2+]i (Ishihara et al., 1999
), while the overexpressed ß1, but not the
1, isoform was stimulated by [Ca2+]i elevation in insulin-secreting RINm5F cells (Kelley et al., 2001
).
In summary, we demonstrate that activation of PLC by endogenous muscarinic receptors in electrically excitable insulin-secreting ß-cells is enhanced by positive feedback from Ca2+ entering the cytoplasm from intracellular Ca2+ stores and via store-operated channels in the plasma membrane (Fig. 5). Agonist binding to the G-protein-coupled receptor leads to partial activation of PLC and production of sufficient amounts of IP3 to trigger Ca2+ release from the ER. The resulting elevation of [Ca2+]i leads to marked amplification of PLC activity with further IP3 production and elevation of [Ca2+]i. The reduction of Ca2+ in the ER leads to opening of store-operated channels in the plasma membrane with entry of Ca2+, which also stimulates PLC and serves to maintain enzyme activity during sustained stimulation. Finally, we have shown that [Ca2+]i spikes occurring as a result of PKA-mediated sensitization of IP3 receptors, induce transient activation of PLC in primary mouse pancreatic ß-cells. Thus, amplification of receptor signalling by feedback activation of PLC is involved in the physiological regulation of insulin secretion by hormones and neurotransmitters.
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Acknowledgments |
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References |
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