Abstract
In PC Cl3 cells, a continuous, fully differentiated rat thyroid cell line, P2Y2 purinoceptor activation provoked a transient increase of [Ca2+]i, followed by a decreasing sustained phase. The α and β1 protein kinase C (PKC) inhibitor Gö6976 decreased the rate of decrement to the basal [Ca2+]i level and increased the peak of Ca2+ entry of the P2Y2-provoked Ca2+transients. These effects of Gö 6976 were not caused by an increased permeability of the plasma membrane, since the Mn2+ and Ba2+ uptake were not changed by Gö 6976. Similarly, the Na+/Ca2+ exchanger was not implicated, since the rate of decrement to the basal [Ca2+]i level was equally decreased in physiological and Na+-free buffers, in the presence of Gö 6976. On the contrary, the activity of the sarcoplasmic–endoplasmic reticulum Ca2+ATPase (SERCA) 2b was profoundly affected by Gö 6976 since the drug was able to completely inhibit the stimulation of the SERCA 2b activity elicited by P2-purinergic agonists. Finally, the PKC activator phorbol myristate acetate had effects opposite to Gö 6976, in that it markedly increased the rate of decrement to the basal [Ca2+]i level after P2Y2 stimulation and also increased the activity of SERCA 2b. These results suggest that SERCA 2b plays a role in regulating the sustained phase of Ca2+ transients caused by P2Y2 stimulation.
Introduction
P2-purinergic agonists have important biological functions on several tissues, due to the wide distribution of their receptors and their ubiquitous nature, since they derive from the cytosol of damaged cells or exocytotic vesicles and/or granules contained in many types of secretory cells (Dubyak & el-Moatassim 1993). There are two families of P2-purinergic receptors: the P2X ligand-gated ionotropic channel family and the P2Y metabotropic G-protein-coupled receptor family (Vassort 2001). Most P2Y receptors are coupled to phospholipase Cβ (PLCβ) and their engagement by ligands causes phosphoinositide hydrolysis, raise of the [Ca2+]i and protein kinase C (PKC) activation (Vassort 2001). They mediate a vast variety of effects ranging from regulation of epithelial transport (Bucheimer & Linden 2004) to platelet aggregation (Hechler et al. 2005).
The signal transmission pathway and physiological effects of P2-purinergic agonists have been studied in the thyrocyte, an epithelial cell. P2-purinergic agonists act on thyroid cells through a P2Y2 receptor, via PLC stimulation, causing PKC activation and a Ca2+ response composed of Ca2+ store depletion, capacitative Ca2+ entry and L-type voltage-dependent Ca2+ channels activation (Marsigliante et al. 2002). The activation of this signalling cascade stimulates H2O2 production (Bjorkman & Ekholm 1992) and iodide efflux (Okajima et al. 1988), two important events in the biological function of thyroid gland, namely thyroglobulin (Tg) iodination and subsequent T3/T4 synthesis. However, we have previously shown that P2-purinergic agonists also stimulate Tg secretion, another crucial event for Tg iodination and thyroid hormone production (Di Jeso et al. 1993). Therefore, P2 purinergic effectively stimulates many important steps of thyroid hormonogenesis.
It has also been shown that even an exclusive Ca2+ response, elicited in thyroid cells by the Ca2+ ionophore A23187, is able to induce H2O2 production and iodide efflux (Weiss et al. 1984, Bjorkman & Ekholm 1988), suggesting that the Ca2+ branch of the signalling of P2-purinergic receptor agonists plays an important role in the regulation of thyroid hormonogenesis. In addition, a Ca2+ response could have more general effects, ranging from proliferation to apoptosis (Berridge 2004). In all cells, a Ca2+ response is ended by the contribution of several Ca2+-extruding proteins, i.e. the Na+/Ca2+ exchanger and the sarcoplasmic–endoplasmic reticulum Ca2+ ATPase (SERCA) and plasma-membrane Ca2+ATPase (PMCA; Lytton et al. 1991). We have recently characterized the presence and regulation of the SERCA in thyroid (Pacifico et al. 2003, Ulianich et al. 2004) and demonstrated that P2Y2 receptor stimulation activates several signal transmission pathways, mainly PKCs, extracellular signal-regulated kinases (ERKs) and fos (Elia et al. 2005).
In this study, our aim has been to determine if and how the Ca2+ response elicited in thyroid cells by P2-purinergic agonists is regulated. We have focussed our attention on the activation of the PKC pathway by P2-purinergic agonists by studying if PKC modulates the dynamics of the P2-purinergic-induced Ca2+ response.
Materials and Methods
Fetal bovine serum (FBS) and glutamine were from Euroclone (Paignton, Devon, UK). Fura 2-AM, thapsigargin and pluronic F-127 were from Molecular Probes (Eugene, OR, USA). Hydrocortisone, transferrin, l-glycyl-histidyl-lysine and somatostatin were from ICN Biomedicals (Costa Mesa, CA, USA). Coon’s modified Ham’s F12 medium, BSA, Gö 6976, GF109203X and other reagents were from Sigma-Aldrich Co.
Cell culture
Several different batches of the PC Cl3 cells were used in the experiments and the cells were grown for approximately 40 passages. No difference was observed in the responsiveness of the cells to ATP and UTP during those passages. PC Cl3 rat thyroid cells were grown as previously reported (Pacifico et al. 2003, Ulianich et al. 2004, Elia et al. 2005). Fresh medium with 0.5% FBS was always added 24 h prior to an experiment.
Determination of [Ca2+]i
The medium was aspirated and the cells were harvested with 0.05% trypsin–EDTA solution. After washing the cells three times by pelleting (100 g for 5 min), the cells (4 × 106) were incubated with 5 μM fura-2-AM and 0.2% pluronic F-127 for 45 min at 37 °C with continuous shaking (100 cycles/ min). Following the loading period, the cells were washed twice with a modified Krebs–Ringer buffer in which the bicarbonate was replaced by 20 mM Hepes (pH 7.4), incubated again for at least 10 min at room temperature to facilitate hydrolysis of the esterified probe and washed once again. The cells were resuspended in 2 ml of the same buffer containing 0.1% BSA and 20 μl cell suspension was added to a 2 ml fluorescence cuvette kept at 37 °C, and stirred throughout the experiment. The fluorescence intensity was measured with a JASCO FP 750 fluorimeter (Jasco Corporation, Hachioji, Tokyo, Japan). The excitation wavelengths were 340 and 380 nm and the emission was measured at 510 nm. The maximal fluorescence was determined at the end of the assay by adding 20 μl, 10% SDS and the minimal fluorescence by adding 20 μl, 0.5 M EGTA solution (pH 9.0). The cytoplasmic Ca2+ concentration at time t was calculated using the software of the fluorimeter and assuming a Kd for the fura-2–Ca2+ complex of 224 nM, according to the Grynkiewicz equation (Grynkiewicz et al. 1985)
where F denotes the time-course of the fluorescence at 510 nm after dual excitation at 340/380 nm and F380, the fluorescence at 510 nm after excitation at 380 nm.
Ba2+ rendered fura-2 fluorescence spectra similar to Ca2+ with a distinct increase and an accompanying decrease in fluorescence intensity at excitation wavelengths near 340 and 380 nm respectively, whereas Mn2+ strongly quenched the fura-2 fluorescence at 360 nm. The results are expressed as relative fluorescence quenching with respect to the maximal quenching induced by the addition of digitonin (80 μg/ml).
Assay of Ca2+-dependent ATPase activity of SERCAs
We previously developed a method to determine the SERCA activity (Pacifico et al. 2003) based on the use of the specific SERCAs-inhibitor thapsigargin (Lytton et al. 1992), which is sensitive enough to be used not only on purified microsomal fractions but also on total cellular lysates. In this study, we used total cellular lysates to analyze the regulated expression of SERCA 2b in thyroid, prepared as previously reported (Ulianich et al. 2004). ATPase activities were determined at 37 °C (Ottolenghi 1975) by measuring inorganic phosphate (Pi) production by a modification of the method of Fiske and Subbarrow (Higgins 1987). This method is based on the reaction between phosphate and molybdate to give the yellow molybdate phosphoric acid, which contains a molybdate, Mo (VI), which is then reduced to Mo (V) present in a blue-coloured heteropolyacid compound. This blue compound is directly measured by reading the absorbance at 700 nm. Typically, 100 μg total cell lysates were incubated in 25 mM KMOPS, 100 mM KCl, 5 mM MgCl2, 4 mM ATP, 0.11 mM EGTA, 107 μM CaCl2 (pH 7.0), [Ca2+]free 5 μM, for 10 min at 37 °C. The free Ca2+ concentration has been calculated using the CHELATOR program (implemented in Turbo Pascal 5.5 for IBM PC; Pacifico et al. 2003). The phosphate produced in the presence or absence of 25 nM thapsigargin was determined directly by reading the absorbance at 700 nm of a standard curve. Activity in the presence of 25 nM thapsigargin (Lytton et al. 1992) was subtracted. Negative controls for each experimental condition were set by boiling cell lysates for 15 min before starting the reactions in the presence and absence of 25 nM thapsigargin (the activity of the negative controls was always less than 5% of the activity of the samples). When the assay was performed on purified microsomal fractions (Pacifico et al. 2003), the activities in the presence of EGTA and thapsigargin were comparable and inclusion in the buffer of sodium azide or p-trifluoromethoxyphenylhydrazone, potent inhibitors of mitochondrial Ca2+ uptake, did not modify the results. Reactions were linear with respect to time and protein concentrations.
Statistical analysis
Experimental points represent the mean ± s.d. of three to six replicates. Statistical analysis was carried out using Student’s t-test for unpaired samples and ANOVA. When indicated, post hoc tests (Bonferroni and Dunn) were also performed. A P value of <0.05 was considered to be statistically significant.
Results
Effect of PKC inhibitors on [Ca2+]i in PC Cl3 cells stimulated by UTP/ATP
To assess the involvement of PKC in the modulation of the ATP/UTP-induced [Ca2+]i transients (Marsigliante et al. 2002), the calcium-dependent PKC inhibitor Gö 6976 and the calcium-dependent and -independent PKC inhibitor GF109203X were used (Martiny-Baron et al. 1993, Qatsha et al. 1993). In the absence of PKC inhibitors, the addition of ATP or UTP evoked an early peak rise in [Ca2+]i with maximal increases occurring at approximately 30 s, followed by a progressively decreasing level (over 6 min) to the initial, pre-stimulated level (Fig. 1A). Pre-incubation of cells with both Gö 6976 and GF109203X (10 and 1000 nM for 15 min) had no effects on the early peak rise in [Ca2+]i, but changed the rate of decrement to the basal [Ca2+]i level significantly (Fig. 1A and B). More precisely, these inhibitors increased the Δ[Ca2+]i after 4 min from approximately 130 to 400 nM, thus retaining the time needed to reach the pre-stimulated [Ca2+]i level.
To measure Ca2+ release from the intracellular stores, cells were stimulated with UTP/ATP in the absence of extracellular Ca2+, using calcium-free buffers, which were previously passed through a Chelex 100 column. Subsequently, 2 mM Ca2+was added to the medium to allow Ca2+influx to occur. As shown in Fig. 1C and D, when PC Cl3 cells were stimulated with 100 μM UTP/ATP in Ca2+-free medium, there was no significant difference in Ca2+ release from the intracellular stores, but large differences occurred in Ca2+ influx between cells incubated or not with PKC inhibitors. Precisely, the peakof extracellular Ca2+ entry in the presence of PKC inhibitors increased from 300 ± 20 to 500 ± 25 nM.
Effect of UTP/ATP on Mn2+ quenching and Ba2+ uptake in PC Cl3 cells
To test whether the changes of the Ca2+ response caused by UTP and ATP were due to modulation of the plasma membrane permeability, we measured Mn2+and Ba2+influx after the addition of ATP and UTP. Mn2+and Ba2+are good Ca2+-entry tracers, since they are not pumped out of the cell (Hallam et al. 1988, Merritt & Hallam 1988, Suh et al. 1996). Mn2+ uptake was estimated by the quenching of fura-2 fluorescence when excited at 360 nm, which is an isosbestic wavelength and is insensitive to variations in Ca2+ concentration. Ba2+uptake was estimated by the increase in the fura-2 fluorescence ratio when excited at 340 and 380 nm. Figure 2A shows the fluorescence quenching by Mn2+influx when cells were stimulated with 100 μM UTP/ATP. The presence or absence of PKC inhibitors Gö 6976 did not change the Mn2+ influx (Fig. 2C and E). This result was also supported by the Ba2+ uptake. To measure Ba2+ influx, cells were stimulated with UTP/ATP in the absence of external Ca2+. When the Ba2+ was added to the medium, it caused an increase in the fluorescence intensity reflecting Ba2+ uptake (Fig. 2B). The influx of Ba2+ elicited by ATP and UTP was similar in the presence or absence of Gö 6976 (Fig. 2D and F). The same results were obtained with the use of GF109203X (data not shown). These results suggest that the UTP/ATP-activated PKCs modulate the activity of one or more Ca2+transporters without interfering with the plasmalemma Ca2+permeability.
To study the involvement of a Na+/Ca2+exchanger in the PKC-controlled [Ca2+]i changes, the effects of PKC inhibitors on the ATP/UTP-evoked Ca2+ changes were investigated using buffers made at 0 and 140 mM Na+. The replacement of extracellular Na+ with choline significantly increased the resting levels of [Ca2+]i in fura-2-loaded cells (from 69.5 ± 5.3 to 92 ± 7 nM, Student’s t-test, P<0.01, n = 4). The effects of ATP/UTP were significantly dependent on the Na+ concentration, since in the absence of Na+, the early peak rise in Δ[Ca2+]i changed from 421 ± 30 to 528 ± 28 (ANOVA; P<0.01, n = 4), while the Δ[Ca2+]i (Δ[Ca 2+]i represents the difference between the pre-stimulatory and the transient levels of [Ca2+]i) after 4 min did not change (Fig. 3A and B).
Both Gö 6976 and GF109203X pre-incubations (10 nM for 15 min) had no effects on the early peak rise in [Ca2+]i evoked by ATP/UTP regardless of the Na+ concentration, but significantly changed the Δ[Ca2+]i to the same rate of decrement to the basal [Ca2+]i level in both physiological and Na+-free buffers (Fig. 3A and B), suggesting that Gö 6976 and GF109203X have no effects on the Na+/Ca2+ exchanger activity.
P2-purinergic agonists stimulate the activity of SERCA 2b via a PKC α and β1 activation
As shown above, the plasma membrane channels and the Na+/Ca2+exchanger were not involved in the PKC-mediated regulation of the [Ca2+]i-sustained phase following the UTP/ATP stimulation. Therefore, we investigated if the SERCA, that actively pump Ca2+from the cytosol to the ER lumen and, as such, should be able to modulate the Ca2+ response, are actually involved in this regulation.
As a first step, we studied if SERCA activity is UTP/ATP responsive. With this aim, we employed a very sensitive and reproducible method previously developed by the authors (Pacifico et al. 2003). Furthermore, since thyroid and PC Cl3 cells express almost exclusively SERCA 2b (Pacifico et al. 2003), the measured activity reflects that of this SERCA isoform.
PC Cl3 cells were left untreated or exposed for the indicated times to 100 μM UTP and Ca2+-ATPase activity was measured. As shown in Fig. 4A, UTP stimulated SERCA 2b activity by about 50%. The increase was evident at 10 min (the shortest time investigated) and was sustained until 30 min (the longest time studied). As expected, very similar results were obtained with 100 μM ATP (Fig. 4B), as both are ligands of the P2Y2 receptors (Sak & Webb 2002).
Therefore, SERCA 2b activity is stimulated by P2Y2 receptor occupancy, strongly suggesting its role in the PKC-mediated regulation of the [Ca2+]i-sustained phase following a P2-purinergic stimulation. Thus, we tested if UTP/ATP stimulates SERCA 2b activity via PKC. PC Cl3 cells were stimulated by UTP/ATP in the absence or presence of the α and the β1 PKC inhibitor Gö 6976, added to cells 30 min before UTP/ATP. As shown in Fig. 5, 10 and 1000 nM Gö 6976 completelyabolished the UTP/ATP stimulation of SERCA 2b activity, implying SERCA 2b activity as a determinant of the [Ca2+]i-plateau phase following UTP/ATP stimulation. The broader PKC inhibitor, GF109203X, gave results highly similar to Gö 6976 (not shown), indicating a specific role of α and β1 PKC in mediating the UTP/ATP effect on SERCA 2b.
Effects of phorbol myristate acetate (PMA) on the [Ca2+]i response to UTP/ATP in PC Cl3 cells
To further examine the role of PKC in the regulation of [Ca2+]i, the effect of PMA on the ATP/UTP-induced changes in [Ca2+]i was studied. As shown in Fig. 6A and B, pre-incubation of PC Cl3 cells with 100 nM PMA for 10 min, followed by 100 μM ATP/UTP stimulation, markedly increased the rate of decrement to the basal [Ca2+]i level from 80 ± 6 nM [Ca2+]i/min in the absence of PMA to 160 ± 22 nM [Ca2+]i/min in the presence of 100 nM PMA (n = 4).
The Ca2+ release from the intracellular stores and the extracellular Ca2+ entry was assessed in PC Cl3 cells pre-incubated with 100 nM PMA. PC Cl3 cells were stimulated with UTP/ATP in the absence of extracellular Ca2+ and 2 mM Ca2+ was added to the medium subsequently. As shown in Fig. 6C and D, there was a significant decrease in extracellular Ca2+ entry in PMA-treated cells from 300 ± 6 to 210 ± 15 nM.
Since PMA has an opposite effect with respect to PKC inhibitors on the rate of decrement to the basal [Ca2+]i level following UTP/ATP stimulation, we verified if PMA, at the same concentrations used in the [Ca2+]i experiments, stimulated SERCA 2b activity. PC Cl3 cells were starved as described above and stimulated by 100 nM PMA for the indicated times. As shown in Fig. 7A, PMA caused a significant increase in the SERCA 2b activity at 10 and 30 min. Moreover, we intentionally extended the incubation time with PMA to 3 h, to try to show a biphasic effect of PMA, indicative of an early stimulation and a following downregulation of PKC activity, as reported in many systems (Cabot et al. 1989, Thompson et al. 1997, Hsu et al. 1998, Komati et al. 2004) and also in differentiated thyroid cells in continuous culture (Gupta et al. 1995). As shown in Fig. 7A, long treatments of PC Cl3 cells with 100 nM PMA reveal inhibition of SERCA 2b activity, strongly suggesting that 100 nM PMA provoked an early stimulation and a late downregulation of PKC. Furthermore, a low dose of PMA was stimulatory at all times investigated (Fig. 7B).
These results further strengthened the SERCA 2b role in the [Ca2+]i-sustained phase following a P2-purinergic stimulation.
Discussion
Thyroid cells in primary (Raspe et al. 1991a,b) and continuous culture (Okajima et al. 1989) respond to P2-purinergic agonists through P2Y receptors. In the PC Cl3 cell line, P2Y2 receptor stimulation activates PLC, which in turn, hydrolyzes phosphatidylinositol 4,5-bisphosphate to diacyl-glycerol (DAG) and inositol 1,4,5-trisphosphate (InsP3). InsP3 mobilizes Ca2+ from InsP3-sensitive intracellular stores, which is followed by a Ca2+ capacitative influx, while DAG activates PKCs (Marsigliante et al. 2002, Elia et al. 2005). Data showed here suggest that this signal transmission pathway has an internal regulatory loop that, through PKC and SERCA 2b, regulates the rate of decrement to the basal [Ca2+]i level. Precisely, the present results suggest a role for conventional PKCs in thyroid cells, i.e. the control of intracellular Ca2+ homeostasis after purinergic stimulation, based, at least in part, upon the increase in SERCA 2b activity, which may be of physiological importance. We base our conclusion on the following observations obtained after conventional PKC inhibition: first, the P2Y2-evoked plateau level in [Ca2+]i increased; second, no effects on the P2Y2-evoked entry of both manganese and barium in the cells was noticed; and third, the P2Y2-stimulated SERCA 2b activity decreased to the basal, unstimulated, level.
We show that an inhibition of PKCs decreased the rate of ecovery to basal [Ca2+]i level following a Ca2+ peak evoked by P2Y2 receptor activation. Hence, PKCs promote the recovery phase of the Ca2+ transient (Fig. 1). As shown previously, P2Y2 activation provoked in PC Cl3 cells a cytosol-to-membrane translocation of PKC-α, -βI and -ε; of these, the novel PKC-ε does not seem to be involved in the modulation of Ca2+transient shown here, since inhibition of Ca2+-dependent or both Ca2+-dependent and -independent PKCs gave similar results. Altogether, PKC-ε was shown to be implicated in the P2Y2 activation of the mitogen-activated protein kinase (MAPK)/ERK pathway (Elia et al. 2005). In theory, the modulation of the sustained phase of the Ca2+ transient could be achieved by several mechanisms, such as changes in plasma membrane permeability to Ca2+, activity of SERCA or plasma membrane Ca2+ ATPase (PMCA), or of the Na+/Ca2+ exchanger. We have excluded the possibility that PKC regulates plasma membrane Ca2+ permeability since Mn2+ and Ba2+ cell entry (both entering the cytosol from the extracellular space through the same pathways as Ca2+) were unaffected by Gö 6976 (Fig. 2).
As far as the Na+/Ca2+ exchange is concerned, it is evident that the Ca2+ response driven by UTP/ATP stimulation also resulted in the involvement of this antiporter, which appeared to be operative in control conditions (unstimulated cells). In fact, the replacement of extracellular Na+ with choline significantly increased resting levels of [Ca2+]i, and the ATP-induced transient increase in [Ca2+]i also increased (Fig. 3). PKC has no effect on Na+/Ca2+ exchanger activity since Gö 6976 has the same effect on the sustained phase of the Ca2+-transient elicited by ATP/UTP in both physiological and Na+-free buffers (Fig. 3).
On the other hand, we have shown that P2Y2 agonists stimulate SERCAs activity and that Gö 6976 was able to completely prevent such activation (Figs 4 and 5). Therefore, the same PKC isoforms, αand β1, were involved in the regulation of the plateau phase of the P2Y2-evoked Ca2+ transient and in the purinergic-stimulated SERCA activity. This strongly suggests that, indeed, SERCAs are involved, at least in part, in the regulation of the Ca2+transient in thyroid cells.
We have also shown that PMA increases the rate of recovery to basal [Ca2+]i level following a Ca2+ peak evoked by P2Y2 receptor activation (Fig. 6). At the same time, PMA stimulates SERCA 2b activity (Fig. 7A). Therefore, PMA acts in an exactly oppositeway with respect to Gö 6976. A biphasic effect of a high dose of PMA and an exclusive stimulatory effect of a low dose of PMA on SERCA activity was evident (Fig. 7A and B). This suggests that the predominant effect of PMA, under our experimental conditions, is the stimulation of PKC, since it is well documented that low PMA doses correlate with PKC membrane translocation, while high doses correlate with PKC downregulation in a variety of systems (Cabot et al. 1989, Thompson et al. 1997, Hsu et al. 1998, Komati et al. 2004) and also in thyroid cells in culture (Gupta et al. 1995).
Obviously, Ca2+ is actively extruded out of the cell through PMCA, the involvement of which, in these phenomena, was not investigated at this stage.
These results also extend our previous findings on expression and regulation of SERCAs in thyroid cells. We have shown that thyroid expresses the 2b form of SERCAs exclusively and that, in thyroid, its expression and activity is regulated by neoplastic transformation (Pacifico et al. 2003) and thyroid-stimulating hormone through the cAMP–PKA pathway (Ulianich et al. 2004). Here, it is shown that SERCA 2b activity is stimulated by P2-purinergic agonists through the PLC–PKC pathway. These results strengthen the concept that SERCA 2b, in highly differentiated cells, such as thyroid (Pacifico et al. 2003) or enamel cells (Franklin et al. 2001), is regulated by various signal transduction pathways. This new standpoint extends our understanding of SERCA 2b function, considered until recently housekeeping at variance with that of SERCA 3 that varies with differentiation in several systems (Papp et al. 1993, Gelebart et al. 2002).
In the thyroid, the physiological effects of P2-purinergic receptors stimulation are remarkable. They stimulate H2O2 production (Bjorkman & Ekholm 1992) and iodide efflux (Okajima et al. 1988), two necessary events for Tg iodination. In addition, we have previously shown that P2-purinergic agonists also stimulate the secretion of Tg, which constitutes the molecular site of synthesis of thyroid hormones (Di Jeso et al. 1993). Since Tg must be secreted in order to be iodinated, its secretion constitutes another crucial event for thyroid hormone production. Therefore, P2-purinergic stimulation of thyroid cells could be very important for hormonogenesis. Of the two branches of the signal transduction pathway elicited by P2-purinergic agonists, the PKC branch and the Ca2+-response branch, the latter seems to be more important than the former for the stimulation of H2O2 production and iodide efflux (Weiss et al. 1984, Bjorkman & Ekholm 1988). It is conceivable that the internal loop of the P2-purinergic-signalling cascade described here could have some effect in the regulation of these physiological responses. In fact, it is well known that the spatiotemporal aspects of Ca2+signalling are as important as its amplitude (Berridge 1993).
In addition, a Ca2+ response could have general effects on cells, such as regulation of proliferation and apoptosis (Berridge 2004). Studies are in progress in our laboratories to understand if the Ca2+response elicited by P2Y2 receptor-signalling cascade and its regulation shown here, plays a role in crucial cellular responses, such as proliferation and apoptosis, particularly in PC Cl3 cells transformed by several oncogenes that are available in our laboratories.
(A) Effect of ATP/UTP on Ca2+ mobilization in PC Cl3 cells in the absence (▴) or presence of Gö 6976 10 nM (▪) and 1000 nM (*). (B) [Ca2+]i values obtained from A after 30 s (early peak rise) and 4 min stimulation with ATP/UTP. The data are means ± s.d. of four different experiments run in triplicate. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. a vs b P<0.001; a vs c P<0.05. (C) Extracellular Ca2+entry into PC Cl3 cells pre-incubated (10, ▪and 1000 nM, *) or not (▴) with Gö 6976. In nominal Ca2+-free medium, 100 μM ATP/UTP was added followed by 2 mM CaCl2 after 260 s. (D) [Ca2+]i values obtained from C representing the mobilization of internal calcium (peak) and the extracellular Ca2+ entry (+CaCl2) after stimulation with ATP/UTP in the presence or absence of Gö 6976. The data are means ± s.d. of four different experiments run in triplicate. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. a vs b P<0.01; a vs c P<0.001. Arrow indicates time point at which ATP was added. Results shown are representative of at least four experiments.
Citation: Journal of Endocrinology 190, 3; 10.1677/joe.1.06455
(A, C, E) Cells were kept in nominally Ca2+-free medium, stimulated with 100 μM UTP/ATP and cation entry was initiated by addition of MnCl2. Mn 2+ entry was measured by the quenching of fluorescence with an excitation wavelength of 360 nm and an emission wavelength of 510 nm. The curves represent fluorescence relative to total quenching attained by the addition of digitonin to saturate fura-2 with the indicated quenching cation. Since 2 mM Mn2+ was applied to the medium (arrow), it entered cells with the same velocity, regardless of the presence of Gö 6976. (B, D, F) UTP/ATP-induced entry of Ba2+ in PC Cl3 cells. Cells, in nominally Ca2+-free medium, were first stimulated with 100 μM ATP/UTP in the presence or absence of Gö 6976 and cation entry was initiated by cumulative addition of Ba2+ at the final concentrations of 2, 5 and 10 mM. Arrows indicate the time point at which cations were added. Each tracing is representative of five separate experiments.
Citation: Journal of Endocrinology 190, 3; 10.1677/joe.1.06455
(A) Effects of UTP/ATP on Ca2+influx using buffers with Na+concentrations of 0 and 140 nM in cells preincubated (C and D) or not (A and B) with 10 nM Gö 6976. Results shown are representative of four experiments. (B) [Ca2+]i values obtained from A after 30 s (early peak rise) and 4-min stimulation with ATP/UTP in normal and Na+-free buffers in the absence of Gö 6976. The data are means ± s.d. of four different experiments run in triplicate. a vs b P<0.0001; a vs c P<0.01; c vs d P<0.001. (C) Effects of UTP/ATP on Ca2+influx using buffers with Na+concentrations of 0 and 140 nM in cells preincubated with 10 nM Gö 6976. (D) [Ca2+]i values obtained from C after 30 s (early peak rise) and 4-min stimulation with ATP/UTP in normal and Na+-free buffers in the presence of Gö 6976. The data are means ± s.d. of four different experiments run in triplicate. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests.
Citation: Journal of Endocrinology 190, 3; 10.1677/joe.1.06455
The effects of (A) UTP and (B) ATP on the activity of SERCA 2b in PC Cl3 cells. Cells were incubated with a single dose of 100 μM UTP/ATP for 10 or 30 min and activity was assayed. The data are means ± s.d. of six different experiments run in triplicate and are presented as nmol Pi/h per mg protein. ANOVA for (A) and (B), P<0.0001. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests.
Citation: Journal of Endocrinology 190, 3; 10.1677/joe.1.06455
The calcium-dependent PKC inhibitor Gö 6976 prevents UTP/ATP-induced SERCA activity but does not affect basal activity. PC Cl3 cells were treated with 10 and 1000 nM Gö 6976 and then with either control medium or medium containing 100 μM UTP/ATP for 10 min and activity was assayed. The data are means ± s.d. of six different experiments run in triplicate and are presented as nmol Pi/h per mg protein. ANOVA for (A) and (B), P<0.0001. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests.
Citation: Journal of Endocrinology 190, 3; 10.1677/joe.1.06455
(A) Effect of ATP/UTP on Ca2+mobilization in PC Cl3 cells pre-incubated (▴) or not (▪) with 100 nM PMA. (B) [Ca2+]i values obtained from A after 30 s (early peak rise) and 4-min stimulation with ATP/UTP. The data are means ± s.d. of four different experiments run in triplicate. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. (C) Extracellular Ca2+ entry into PC Cl3 cells pre-incubated (▴) or not (▪) with 100 μM PMA. In nominal Ca2+-free medium, 100 μ M UTP/ATP was added followed by 2 mM CaCl2 after 260 s. (D) [Ca2+]i values obtained from C representing the mobilization of internal calcium (peak) and the extracellular Ca2+ entry (+CaCl2) after stimulation with ATP/UTP in the presence or absence of PMA. The data are means ± s.d. of four different experiments run in triplicate. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. Arrow indicates the time point at which ATP was added. Results shown are representative of at least four experiments.
Citation: Journal of Endocrinology 190, 3; 10.1677/joe.1.06455
The stimulation of an cells with the PKC activator phorbol 12-myristate acetate (100 and 10 nM) induced an increase in SERCA 2b activity in PC Cl3 cells. Cells were incubated with a single dose of UTP/ATP (A) 100 nM or (B) 10 nM for the times indicated and activity was assayed. Results are means ± s.d. of three different experiments run in triplicate and are presented as nmol Pi/h per mg protein. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. P<0.0001.
Citation: Journal of Endocrinology 190, 3; 10.1677/joe.1.06455
The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
References
Berridge MJ 1993 Cell signalling. A tale of two messengers. Nature 365 388–389.
Berridge MJ 2004 Calcium signal transduction and cellular control mechanisms. Biochimica et Biophysica Acta 1742 3–7.
Bjorkman U & Ekholm R 1988 Accelerated exocytosis and H2O2 generation in isolated thyroid follicles enhance protein iodination. Endocrinology 122 488–494.
Bjorkman U & Ekholm R 1992 Hydrogen peroxide generation and its regulation in FRTL-5 and porcine thyroid. Endocrinology 130 393–399.
Bucheimer RE & Linden J 2004 Purinergic regulation of epithelial transport. Journal of Physiology 555 311–321.
Cabot MC, Welsh CJ, Zhang ZC & Cao HT 1989 Evidence for a protein kinase C-directed mechanism in the phorbol diester-induced phospho-lipase D pathway of diacylglycerol generation from phosphatidylcholine. FEBS Letters 245 85–90.
Di Jeso B, Laviola L, Liguoro D, Formisano S & Consiglio E 1993 P2 purinergic agonists and 12-O-tetradecanoylphorbol-13-acetate, as well as protein kinase A activators, stimulate thyroglobulin secretion in FRTL-5 cells. Biochemical and Biophysical Research Communications 191 385–391.
Dubyak GR & el-Moatassim C 1993 Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. American Journal of Physiology 265 C577–C606.
Elia MG, Muscella A, Romano S, Greco S, Di Jeso B, Verri T, Storelli C & Marsigliante S 2005 Effects of extracellular nucleotides in the thyroid: P2Y2 receptor-mediated ERK1/2 activation and c-Fos induction in PC Cl3 cells. Cellular Signalling 17 739–749.
Franklin IK, Winz RA & Hubbard MJ 2001 Endoplasmic reticulum Ca2+-ATPase pump is up regulated in calcium-transporting dental enamel cells: a non-housekeeping role for SERCA 2b. Biochemical Journal 358 217–224.
Gelebart P, Kovacs T, Brouland JP, Van Gorp R, Grossmann J, Rivard N, Panis Y, Martin V, Bredoux R, Enouf J et al.2002 Expression of endomembrane calcium pumps in colon and gastric cancer cells. Induction of SERCA 3 expression during differentiation. Journal of Biological Chemistry 277 26310–26320.
Grynkiewicz G, Poenie M & Tsien RY 1985 A new generation of Ca2+ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260 3440–3450.
Gupta S, Gomez-Munoz A, Matowe WC, Brindley DN & Ginsberg J 1995 Thyroid-stimulating hormone activates phospholipase D in FRTL-5 thyroid cells via stimulation of protein kinase C. Endocrinology 136 3794–3799.
Hallam TJ, Jacob R & Merritt JE 1988 Evidence that agonists stimulate bivalent-cation influx into human endothelial cells. Biochemical Journal 255 179–184.
Hechler B, Cattaneo M & Gachet C 2005 The P2 receptors in platelet function. Seminars in Thrombosis Hemostasis 31 150–161.
Higgins A 1987 Separation and analysis of membrane lipid components. In Biological Membranes, A Practical Approach, pp 103–137. Eds JBC Findlay & WH Evans. Oxford, Washington, DC: IRL Press.
Hsu SL, Chou YH, Yin SC & Liu JY 1998 Differential effects of phorbol ester on growth and protein kinase C isoenzyme regulation in human hepatoma Hep3B cells. Biochemical Journal 333 57–64.
Komati H, Minasi A, Naro F, Lagarde M, Prigent AF, Adamo S & Nemoz G 2004 Phorbol ester-induced differentiation of L6 myogenic cells involves phospholipase D activation. FEBS Letters 577 409–414.
Lytton J, Westlin M & Hanley MR 1991 Thapsigargin inhibits the sarcoplasmic and endoplasmic reticulum Ca-ATPase family of calcium pumps. Journal of Biological Chemistry 266 17067–17071.
Lytton J, Westlin M, Burk SE, Shull GE & MacLennan DH 1992 Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps. Journal of Biological Chemistry 267 14483–14489.
Marsigliante S, Elia MG, Di Jeso B, Greco S, Muscella A & Storelli C 2002 Increase of [Ca2+]i via activation of ATP receptors in PC-Cl3 rat thyroid cell line. Cellular Signalling 14 61–67.
Martiny-Baron G, Kazanietz MG, Mischak H, Blumbergs PM, Kochs G, Hug H, Marmè D & Schachtele C 1993 Selective inhibition of protein kinase C isozymes by the indolocarbazole GO 6976. Journal of Biological Chemistry 268 9194–9197.
Merritt JE & Hallam TJ 1988 Platelets and parotid acinar cells have different mechanisms for agonist-stimulated divalent cation entry. Journal of Biological Chemistry 263 6161–6164.
Okajima F, Sho K & Kondo Y 1988 Inhibition by islet-activating protein, pertussis toxin, of P2-purinergic receptor-mediated iodide efflux and phosphoinositide turnover in FRTL-5 cells. Endocrinology 123 1035–1043.
Okajima F, Sato K & Kondo Y 1989 P2-purinergic agonists activate phospholipase C in a guanine nucleotide- and Ca2+-dependent manner in FRTL-5 thyroid cell membranes. FEBS Letters 253 132–136.
Ottolenghi P 1975 The reversible delipidation of a solubilized sodium-plus-potassium-ion-dependent adenosine triphosphatase from the salt gland of the spiny dogfish. Biochemical Journal 151 61–66.
Pacifico F, Ulianich L, De Micheli S, Treglia S, Leonardi A, Vito P, Formisano S, Consiglio E & Di Jeso B 2003 The expression of the sarco/endoplasmic reticulum Ca2+-ATPases in thyroid and its down-regulation following neoplastic transformation. Journal of Molecular Endocrinology 30 399–409.
Papp B, Corvazier E, Magnier C, Kovacs T, Bourdeau N, Levy-Toledano S, Bredoux R, Levy B, Poitevin P & Lompre AM 1993 Spontaneously hypertensive rats and platelet Ca2+-ATPases: specific up-regulation of the 97 kDa isoform. Biochemical Journal 295 685–690.
Qatsha KA, Rudolph C, Marme D, Schachtele C & May WS 1993 Go 6976, a selective inhibitor of protein kinase C, is a potent antagonist of human immunodeficiency virus 1 induction from latent/low-level-producing reservoir cells in vitro. PNAS 15 4674–4678.
Raspe E, Laurent E, Andry G & Dumont JE 1991a ATP, bradykinin, TRH and TSH activate the Ca2+-phosphatidylinositol cascade of human thyrocytes in primary culture. Molecular and Cellular Endocrinology 81 175–183.
Raspe E, Laurent E, Corvilain B, Verjans B, Erneux C & Dumont JE 1991b Control of the intracellular Ca2+-concentration and the inositol phosphate accumulation in dog thyrocyte primary culture: evidence for different kinetics of Ca2+-phosphatidylinositol cascade activation and for involvement in the regulation of H2O2 production. Journal of Cellular Physiology 146 242–250.
Sak K & Webb TE 2002 Retrospective of recombinant P2Y receptor subtypes and their pharmacology. Archives of Biochemistry and Biophysics 397 131–136.
Suh BC, Song SK, Kim YK & Kim KT 1996 Induction of cytosolic Ca2+ elevation mediated by Mas-7 occurs through membrane pore formation. Journal of Biological Chemistry 271 32753–32759.
Thompson MG, Mackie SC, Thom A & Palmer RM 1997 Regulation of phospholipase D in L6 skeletal muscle myoblasts. Role of protein kinase c and relationship to protein synthesis. Journal of Biological Chemistry 272 10910–10916.
Ulianich L, Secondo A, De Micheli S, Treglia S, Pacifico F, Liguoro D, Moscato F, Marsigliante S, Annunziato L, Formisano S et al.2004 TSH/cAMP up-regulate sarco/endoplasmic reticulum Ca2+-ATPases expression and activity in PC Cl3 thyroid. European Journal of Endocrinology 150 851–861.
Vassort G 2001 Adenosine 5′-triphosphate: a P2-purinergic agonist in the myocardium. Physiological Review 81 767–806.
Weiss SJ, Philp NJ & Grollman EF 1984 Effect of thyrotropin on iodide efflux in FRTL-5 cells mediated by Ca2+. Endocrinology 114 1108–1113.
