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Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Campus Gasthuisberg O/N, Leuven, Belgium
Abstract
We have described previously a novel Ca2+-induced Ca2+-release (CICR) mechanism in permeabilized A7r5 cells (embryonic rat aorta) and 16HBE14o-cells (human bronchial mucosa) cells (J Biol Chem 278:27548eC27555, 2003). This CICR mechanism was activated upon the elevation of the free cytosolic calcium concentration [Ca2+]c and was not inhibited by pharmacological inhibitors of the inositol-1,4,5-trisphosphate (IP3) receptor nor of the ryanodine receptor. This CICR mechanism was inhibited by calmodulin (CaM)1234, a Ca2+-insensitive CaM mutant, and by different members of the superfamily of CaM-like Ca2+-binding proteins. Here, we present evidence that the CICR mechanism that is expressed in A7r5 and 16HBE14o-cells is strongly activated by suramin and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). We found several indications that both activation mechanisms are indeed two different modes of the same release system. Suramin/DIDS-induced Ca2+ release was only detected in cells that displayed the CICR mechanism, and cell types that do not express this type of CICR mechanism did not exhibit suramin/DIDS-induced Ca2+ release. Furthermore, we show that the suramin-stimulated Ca2+ release is regulated by Ca2+ and CaM in a similar way as the previously described CICR mechanism. The pharmacological characterization of the suramin/DIDS-induced Ca2+ release further confirms its properties as a novel CaM-regulated Ca2+-release mechanism. We also investigated the effects of disulfonated stilbene derivatives on IP3-induced Ca2+ release and found, in contrast to the effect on CICR, a strong inhibition by DIDS and 4'-acetoamido-4'-isothiocyanostilbene-2',2'-disulfonic acid.
Changes in cytosolic free Ca2+ concentration ([Ca2+]c) mediate a variety of cellular processes, ranging from fertilization to cell death. The endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR) serve as the main sources of releasable Ca2+ for cytosolic cellular signaling. Calcium pumps of the sarco(endo)-plasmic-reticulum Ca2+-ATPase family import Ca2+ into the organelle lumen. Two families of intracellular Ca2+-release channels are primarily responsible for the release, the inositol-1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR) (Berridge et al., 2003).
Recent studies have emphasized the role of novel types of intracellular Ca2+-release channels possibly playing an important role in intracellular Ca2+ signaling. Wissing et al. (2002) identified a novel Ca2+-induced Ca2+-release (CICR) mechanism in permeabilized hepatocytes that responded to modest increases in [Ca2+]c. A CICR atypical of the SR type was found in mouse pancreatic -cells (Beauvois et al., 2004). Polycystin-2, the product of the gene mutated in type-2 autosomal dominant polycystic kidney disease and a prototypical member of a subfamily of the transient receptor potential channel superfamily (TRP), is expressed abundantly in the ER. It was shown recently that polycystin-2 expressed in the ER of epithelial cells is a Ca2+-activated channel that is permeable for divalent cations. Increased levels of intracellular Ca2+ activated polycystin-2-mediated release of Ca2+ from intracellular stores (Koulen et al., 2002). Moreover, there are indications that some bona fide plasmalemmal Ca2+-permeable TRP channels (e.g., TRPV1 and TRPM8) also reside in intracellular membranes where they may function as Ca2+-release channels (Turner et al., 2003; Zhang and Barritt, 2004). In a previous study, we described a novel CICR mechanism in permeabilized A7r5 cells, a permanent cell line derived from embryonic rat aorta (Nadif Kasri et al., 2003). This CICR mechanism was activated upon the elevation of the [Ca2+]c and was not inhibited by pharmacological inhibition of the IP3R or of the RyR. Moreover, we found that this CICR mechanism could be inhibited by CaM1234, a Ca2+-insensitive CaM mutant, and by different members of the superfamily of CaM-like Ca2+-binding proteins. Our data suggested that the CICR mechanism described here may represent a novel type of Ca2+-release channel, which is silent at low [Ca2+]c because of inhibition by bound apocalmodulin and which becomes activated by the Ca2+-dependent interaction with CaM.
Suramin and disulfonic stilbene derivatives have been extensively used as pharmacological probes to study the transport kinetics and molecular structures of a wide range of membrane transporters. An excellent example of this includes the structure of the ATP-binding site of the sarco(endo)plasmic-reticulum Ca2+-ATPase (Hua and Inesi, 1997). 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) has been widely used to understand the role and mechanism of various ion transport processes in the muscle sarcolemma and the SR (Cabantchik and Greger, 1992). 4'-acetoamido-4'-isothiocyanostilbene-2',2'-disulfonic acid (SITS) is commonly used for the study of anion transporters, and like DIDS it possesses structural similarities to suramin. Suramin (1,3,5-naphthylenetrisulfonic acid) is a trypanoside that acts as an ATP antagonist for P2-purinoceptors (Hoyle et al., 1990). On the RyR it acts as a strong activator, and regulates the RyR via a binding site that is distinct from its adenine nucleotide binding site (Emmick et al., 1994). Suramin has also been postulated to act on the RyR via binding to the CaM-binding site (Klinger et al., 1999). Suramin was found to bind directly to CaM-binding sites on the RyR and the IP3R (Klinger et al., 1999; Nadif Kasri et al., 2004). Although suramin and disulfonic stilbene derivatives have been extensively used to study the RyR, data on the effects on IP3Rs or IP3-induced Ca2+ release (IICR) are scarce. Previous results, however, have shown that endogenous sulfonate derivatives can regulate IP3R function (Watras et al., 2000). In this study, therefore, we have used suramin and stilbene derivatives to further characterize the previously detected CICR mechanism, and we also investigated the effects of these compounds on IICR.
In a first part, we present evidence that the CICR mechanism that is expressed in A7r5 and 16HBE14o-cells is strongly activated by suramin and DIDS. Other cell types that do not express this type of CICR mechanism did not exhibit any suramin/DIDS-induced Ca2+ release. Furthermore, we show that this suramin-stimulated Ca2+ release is regulated in a similar way as the previously described CICR mechanism (Nadif Kasri et al., 2003). The pharmacological characterization further confirms the properties of this CICR mechanism as a novel CaM-regulated Ca2+-release mechanism. We also investigated the effects on IICR and found, in contrast to the effect on CICR, a strong inhibition by DIDS and SITS.
Materials and Methods
Materials. Suramin was purchased from Sigma-Aldrich (Bornem, Belgium). DIDS, SITS, and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) were purchased from Molecular Probes (Leiden, The Netherlands).
45Ca2+ Fluxes. A7r5 cells, which are derived from embryonic rat aorta smooth muscle cells, were obtained from the American Type Culture Collection (Manassas, VA) (CRL 1444). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 3.8 mM L-glutamine, 0.9% (v/v) nonessential amino acids, 85 IU/ml penicillin, 85 e/ml streptomycin, and 20 mM HEPES, pH 7.4. For 16HBE14o-(human bronchial mucosa) cells, a mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium was used, and for LLC-PK1 cells minimal essential medium- was used. L15 cells were obtained by stable exogenous expression of IP3R1 in Lvec cells, whereas Lvec cells represent the control cells expressing the empty vector (Miyawaki et al., 1990; Mackrill et al., 1996). 45Ca2+ fluxes were performed on saponin-permeabilized cells. The cells were seeded in 12-well clusters (Costar, Cambridge, MA) at a density of approximately 4 x 104 cmeC2. Experiments were carried out on confluent monolayers of cells (3 x 105 cells/well) between the seventh and ninth day after plating. Cells were permeabilized by incubating them for 10 min with a solution containing 120 mM KCl, 30 mM imidazole-HCl, pH 6.8, 2 mM MgCl2, 1 mM ATP, 1 mM EGTA, and 20 e/ml saponin at 25°C. The nonmitochondrial Ca2+ stores were loaded for 45 min at 25°C in 120 mM KCl, 30 mM imidazole-HCl, pH 6.8, 5 mM MgCl2, 5 mM ATP, 0.44 mM EGTA, 10 mM NaN3, and 150 nM free 45Ca2+ (28 e藽i/ml). The cells were then washed twice with 1 ml of efflux medium containing 120 mM KCl, 30 mM imidazole-HCl, pH 6.8, 1 mM EGTA, and 10 e thapsigargin (TG). TG was added to block the ER Ca2+ pumps during subsequent additions of Ca2+. The efflux medium was replaced every 2 min, and the efflux was performed at 25°C. The additions of 40Ca2+, IP3, suramin, and stilbene derivatives are indicated on the figures (arrow). Free [Ca2+] was calculated by the Cabuf program (ftp://ftp.cc.kuleuven.ac.be/pub/droogmans/cabuf.zip) and based on the stability constants as published previously (Fabiato and Fabiato, 1979). At the end of the experiment, the 45Ca2+ remaining in the stores was released by incubation with 1 ml of a 2% sodium dodecyl sulfate solution for 30 min. Ca2+ release in some experiments is plotted as the fractional loss (i.e., the amount of Ca2+ released in 2 min divided by the total store Ca2+ content at that time). The latter value was calculated by summing in retrograde order the amount of tracer remaining in the cells at the end of the efflux and the amounts of tracer collected during the successive time intervals.
CaM-Sepharose Pull-Down Assay. CaM-Sepharose 4B (50 e) (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK) was incubated with 500 ng of recombinant IP3R1 purified from Sf9 cells for 2 h at 4°C in incubation buffer [i.e., one part Tris-buffered saline (20 mM Tris-HCl, pH 7.2, and 150 mM NaCl) mixed with one part bacterial ProFound lysis buffer containing a Tris-buffered solution of 75 mM NaCl with 1% of a nonionic detergent (according to the manufacturer's protocol; Pierce Chemical, Rockford, IL), and supplemented with 1 mM -mercaptoethanol]. Unbound protein was removed by washing the Sepharose beads four times with 500 e of the incubation buffer. Bound IP3R1 protein was eluted by incubating the beads with LDS (Invitrogen, Carlsbad, CA) for 10 min at 70°C, and the beads were removed by centrifugation at 20,000g for 1 min. All samples were separated on NuPAGE 3 to 8% Tris-acetate SDS-polyacrylamide gel electrophoresis gels and analyzed by Western blotting, using a rabbit polyclonal antibody against IP3R1 (Rbt03) as the primary antibody (Parys et al., 1995)
[3H]IP3-Binding Experiments. Binding studies were performed as described previously. [3H]IP3 binding was performed at 0°C in 100 e of binding buffer containing 50 mM Tris-HCl, pH 7.0, 1 mM EGTA, 10 mM -mercaptoethanol, and 10 nM [3H]IP3. Nonspecific binding was determined in the presence of 12.5 e unlabeled IP3. After 30 min of incubation, the samples were rapidly filtered through glass-fiber filters. The amount of Sf9 microsomes expressing IP3R1 ranged between 100 and 150 e. Statistical analysis was performed using the paired Student's t test. Values were considered significantly different when P < 0.05.
Expression and Purification of Recombinant Proteins. Recombinant CaM and CaM1234 were expressed and purified as described previously (Sienaert et al., 2002).
Expression and Purification of IP3R1 from Sf9 Cells. Production of recombinant viruses, expression and purification of recombinant IP3R1 proteins in Sf9 insect cells, and preparation of the microsomes were as described previously (Sipma et al., 1999; Vermassen et al., 2004).
Results
Suramin Stimulates Ca2+ Release from Intracellular Stores. In A7r5 cells, Ca2+ release from intracellular stores, mainly from the ER, occurs to a large extent via production of the second messenger IP3. In the permeabilized cell system, a maximal effective dose of IP3 (100 e) can release up to 95% of the intracellular Ca2+ content (Missiaen et al., 1990). We previously identified a CICR mechanism that contributed significantly to the Ca2+ release from the ER (40%) (Nadif Kasri et al., 2003). This CICR mechanism was found to be highly regulated by CaM. Because suramin has already been used extensively to study intracellular Ca2+-release channels and has been reported to bind directly on CaM-binding sites (Klinger et al., 1999, 2001), we tested the effects of suramin on intracellular Ca2+ release in A7r5 cells. The nonmitochondrial stores of permeabilized A7r5 cells were loaded to steady state with 45Ca2+ and then incubated in a nonlabeled efflux medium containing 10 e thapsigargin. The loss of Ca2+ from the stores under these conditions is plotted as ER Ca2+ content as a function of time (Fig. 1A). After 10 min of efflux, the cells were challenged with 100 e suramin during 2 min, as indicated by the arrow. Suramin (100 e) strongly increased the rate of Ca2+ release (Fig. 1A). In these conditions, the amount of the stored Ca2+ that could be released by suramin was 33% (Fig. 1B). The total amount of releasable Ca2+ was measured by treating the cells with 5 e ionophore A23187 (triangles). A dose-response curve is shown in Fig. 1B. Suramin stimulated Ca2+ release from the intracellular stores with an EC50 of 93 ± 9 e, and the activation curve had a Hill coefficient of 1.0 ± 0.1. A saturating concentration of suramin was found to release up to 70% of the stored Ca2+ in A7r5 cells.
Suramin-Induced Ca2+ Release Is Expressed in the Same Cell Types as the CICR Mechanism. Next, we investigated whether this suramin-induced Ca2+ release could be observed in other cell types that express the CICR mechanism described previously in A7r5 cells. Therefore, we performed the same experiments in 16HBE14o-cells, in which a similar CICR mechanism is expressed, and in COS-1, LLC-PK1, L15, and Lvec cells, in which we could not detect this mechanism (Nadif Kasri et al., 2003). Suramin-induced Ca2+ release was only observed in 16HBE14o-cells and not in the other cell types (Fig. 2). Suramin maximally released 40% of the stored Ca2+ with an EC50 of 104 ± 8 e in 16HBE14o-cells, which is identical to that observed in A7r5 cells. These data indicate that the Ca2+ release induced by suramin is not a property of all cells and is therefore not caused by a nonspecific leak. Hence, these data suggest that suramin-induced Ca2+ release is a property of cells that express the previously described CICR mechanism.
Suramin-Induced Ca2+ Release Is Neither IP3R- nor RyR-Mediated. The two major classes of intracellular Ca2+-release channels are the IP3Rs and the RyRs. In A7r5 cells, only IP3R1 (73%) and IP3R3 (26%) are expressed (De Smedt et al., 1994). No evidence has been found for a functional activity of the RyR in A7r5 cells, as there was no measurable Ca2+ release by caffeine or cyclic ADP-ribose (Missiaen et al., 1990).
Because suramin was shown to potently activate several types of ion channels (Hill et al., 2004, and references therein), we wanted to investigate the possibility that suramin acted directly on the IP3R. Heparin and xestospongin C (XeC) are often used as antagonists of the IP3R. In Fig. 3A, we show that these compounds did not affect the fractional loss induced by 100 e suramin, strongly suggesting that the IP3R was not involved in this mechanism. Although there is no evidence for a functional RyR in A7r5 cells, we also used an antagonist of the RyR to exclude any role of the RyR in this suramin-induced Ca2+ release. Figure 3B illustrates that 100 e ruthenium red (RuRed) had no effect on the fractional loss induced by 100 e suramin.
Ca2+ release stimulated by sphingosine-1-phosphate (S1P) (Pyne and Pyne, 2000) and NAADP (Genazzani and Galione, 1996) has been observed in a number of cell types. However, it is unlikely that one of these mechanisms is activated by suramin in A7r5 cells because no S1P- or NAADP-stimulated Ca2+ release was observed in A7r5 cells under our assay conditions (data not shown).
Characteristics of the Observed Suramin-Induced Ca2+ Release. Fig. 4A illustrates that the suramin-induced Ca2+ release was controlled by the level of store loading. Ca2+ stores from permeabilized A7r5 cells loaded to steady state with 45Ca2+ were incubated in Ca2+-free efflux medium and their Ca2+ content plotted as a function of time. Suramin (100 e) was added either after 2 min (, full stores) or after 20 min (, less filled stores). The relative amount of Ca2+ released by suramin from fully loaded stores was higher than the release from less filled stores. The Ca2+ content of the cells induced by suramin after 2 min was depleted more extensively than the Ca2+ content of cells that were induced after 20 min. These results indicate that the suramin-induced Ca2+ release was controlled by the luminal [Ca2+]. In this respect, the suramin-induced Ca2+ release shows the same dependence on the luminal Ca2+ content as described for both the CICR (Nadif Kasri et al., 2003) or for the IICR mechanism in those cells (Missiaen et al., 1992).
We have shown previously that the CICR mechanism in A7r5 cells originated from the IP3-sensitive and TG-sensitive stores. We investigated whether the suramin-induced Ca2+ release described here was originating from the same IP3- and TG-sensitive stores. Permeabilized cells were loaded with 45Ca2+ in the presence or absence of a saturating dose of IP3 (300 e) or TG (10 e). Efflux was then performed in Ca2+-free medium (EGTA). After 10 min, cells were incubated for 2 min with 100 e suramin. No Ca2+ release was observed in cells that were loaded in the presence of IP3 or TG (data not shown). This finding suggests that suramin can only release Ca2+ from the IP3-sensitive and TG-sensitive stores.
To confirm that CICR and suramin-induced Ca2+ release originate from the same stores, we first challenged cells with 300 e suramin and subsequently activated CICR by addition of 3 e free 40Ca2+. Cells exposed with suramin did not show subsequent CICR, in contrast to nontreated cells (Fig. 4B). From these experiments we conclude that CICR and suramin-induced Ca2+ release originated from the same Ca2+ stores in A7r5 cells.
In the next step, we have measured the effect of the free [Ca2+] on the suramin-induced Ca2+ release. When the Ca2+ release induced by 100 e suramin was plotted as a function of the free [Ca2+]c, we observed a Ca2+-dependent component with an EC50 of 900 ± 46 nM free Ca2+ (Fig. 4C). This Ca2+ dependence had a similar EC50 as that previously measured for activation of the CICR mechanism (700 nM free Ca2+) (Nadif Kasri et al., 2003). We also observed that suramin-induced Ca2+ release and CICR were only partially additive. This could suggest that suramin and Ca2+ activate the same mechanism and that Ca2+ is also a modulator of the suramin-induced Ca2+ release.
In contrast to the CICR mechanism, Ca2+ release induced by suramin was not potentiated by ATP (data not shown). Suramin was identified previously as an ATP antagonist for P2-purinoceptors and RyRs (Hoyle et al., 1990; Mallard et al., 1992; Emmick et al., 1994). Therefore, we can hypothesize that the lack of activation by ATP could be explained by binding of suramin to a ATP-binding site.
An important characteristic of CICR described previously was the role of CaM as a Ca2+ sensor (Nadif Kasri et al., 2003). We showed that when cells were incubated with CaM1234, CICR was completely blocked. Further characterization revealed that CaM acted as a Ca2+ sensor for this CICR mechanism and that CaM1234 had dominant negative properties. Assuming that suramin activates the same CICR mechanism, CaM1234 should also block suramin-induced Ca2+ release. Indeed, when 10 e CaM1234 was added to permeabilized cells, suramin-induced Ca2+ release was nearly completely inhibited (89 ± 4%). This was not the case when CaM itself was added (Fig. 5). Thus, suramin-induced Ca2+ release shows remarkable similarities with the CICR mechanism in A7r5 cells with respect to the Ca2+ and CaM dependence. Suramin-induced Ca2+ release, however, does also occur in the complete absence of Ca2+ (EGTA), which suggests that suramin is an independent activator and not merely a regulator of the CICR mechanism.
Stilbene Derivatives Induce Ca2+ Release in A7r5 Cells. DIDS and SITS (Fig. 6A) are normally regarded as chemical probes for the study of anion transporters. However, because they possess structural similarities to suramin, they have also been used as a ligand for purinoceptors and RyRs. We have tested the effects of those derivatives on intracellular Ca2+ release in A7r5 cells. When applied to permeabilized cells, 100 e DIDS induced a Ca2+ release (18 ± 3%) comparable with the activation of suramin, whereas SITS and DNDS were not effective (Fig. 6B). A dose-response curve of the activation by DIDS revealed that the EC50 for DIDS activation was higher than for suramin (Fig. 6C). Similar to the suramin-induced Ca2+ release, CaM 1234 inhibited Ca2+ release induced by DIDS by 83 ± 3% (data not shown), suggesting that both suramin and DIDS activate the same Ca2+-release mechanism.
Stilbene Derivatives Inhibit IICR. Although stilbene derivatives have been extensively used to study several types of ion channels, no data are available for IP3Rs. We have therefore investigated the effects of DIDS, SITS, and DNDS on IICR. To prevent any interference with the previously described suramin/DIDS-induced Ca2+-release mechanism, we used permeabilized L15 and Lvec fibroblast cells, which do not express CICR and in which suramin did not promote Ca2+ release (Fig. 2). Western blots indicated a 3:1 ratio for IP3R1/IP3R3 for L15 and the reverse ratio for Lvec cells (data not shown). This comparison was made because differences between both IP3R isoforms may yield different results, as was shown for RyR isoforms (Sitsapesan, 1999; O'Neill et al., 2003; Hill et al., 2004).
In permeabilized L15 or Lvec cells, the addition of 200 nM IP3 to the efflux medium induced Ca2+ release from the nonmitochondrial internal stores. IICR was inhibited in a concentration-dependent way by both DIDS and SITS, whereas DNDS did not have any effect. Because the inhibition occurred to the same extent in both cell lines, it can be concluded that these effects are not isoform specific. Dose-response curves for DIDS and SITS inhibition in L15 cells are presented in Fig. 7A. With 200 nM IP3 in L15 cells, DIDS half-maximally inhibited IICR at a concentration of 0.7 ± 0.02 e and SITS at a concentration of 9.3 ± 0.47 e. As indicated in Fig. 7B, the blocking effect of DIDS or SITS was essentially irreversible. Permeabilized L15 cells were preincubated with 100 e DIDS or SITS for 6 min followed by a washout period of 4 min before activation with 200 nM IP3. The decrease of IICR was compared with cells that were incubated with 100 e DIDS or SITS for 12 min with no washout period before activation with 200 nM IP3. Cells that were treated for 6 min showed a similar decrease in IICR compared with cells that were incubated for 12 min. DIDS completely inhibited IICR, whereas SITS inhibited IICR by 74 ± 4% (Fig. 7B). We conclude that washing away the stilbene derivatives did not reverse the inhibition of IICR. Irreversible binding of stilbene derivatives was also observed for the interaction with the RyR (O'Neill et al., 2003) and may involve isothiocyanate groups on DIDS and SITS.
For suramin, we have previously shown that it decreased the apparent sensitivity of the IP3R for IP3, presumably by binding to the N-terminal CaM-binding sites of the receptor (Nadif Kasri et al., 2004). Therefore, we performed IP3-binding experiments to see whether DIDS, SITS, or DNDS interfered in a similar way with the IP3R. IP3-binding measurements were performed in the presence of 50 e stilbene derivative on microsomes from Sf9 cells expressing IP3R1. In contrast with suramin, DIDS, SITS, and DNDS had no effect on IP3 binding (Fig. 7C).
Another property of the suramin interaction was the interference with the CaM-binding sites of the IP3R. We previously showed that suramin interacted with the two different CaM-binding sites on the IP3R1 (Nadif Kasri et al., 2004). Pull-down experiments with CaM-Sepharose 4B were performed in the presence of 1 e suramin, DIDS, SITS, or DNDS. Suramin inhibited the binding of IP3R1 to the CaM-Sepharose 4B by 58 ± 5% (Fig. 7D). In contrast, none of the other tested stilbene reagents showed a significant interference. Higher concentrations of DIDS, SITS, and DNDS (100 e) also did not result in an inhibition of the binding of IP3R1 to the beads (data not shown). From this part, we conclude that DIDS and SITS are both potent inhibitors of IICR. Although both compounds are structurally related to suramin, the inhibition of IICR occurs via a different mechanism.
Discussion
The present data clearly show that suramin and disulfonic stilbene derivatives potently induce Ca2+ release from the ER in A7r5 and 16HBE14o-cells (Fig. 1). The properties of this suramin-induced Ca2+ release closely resemble those of the recently identified novel CICR mechanism (Nadif Kasri et al., 2003).
Disulfonic stilbene derivatives are known to affect ion channels and transporters by both reversible and nonreversible mechanisms. Kawasaki and Kasai (1989) were the first to report that DIDS and SITS activated RyR1 from rabbit skeletal muscle in SR vesicles and lipid bilayers. They found that these compounds locked RyRs in an open state. Irreversible effects are related to reactive isothiocyanate groups that form covalent bonds with a variety of amino acid residues by reacting with NH2 groups on lysine residues, OH-groups on serine residues, and aromatic groups on tyrosine and cysteine residues. DIDS has two isothiocyanate groups, whereas SITS has only one and DNDS lacks reactive isothiocyanate groups (Fig. 6A). It is therefore not surprising that DIDS, but not SITS or DNDS, induced Ca2+ release in A7r5 cells similarly to suramin. An increase in intracellular Ca2+ release by DIDS has also been shown for rat pulmonary artery smooth muscle cells (Cruickshank et al., 2003). This suggests that such an increase in Ca2+ release by disulfonic stilbene derivatives not only occurs in cultured cells but also in some primary cells.
There are two main families of intracellular Ca2+-release channels, RyRs and IP3Rs. However, there is growing evidence that other, not yet identified, intracellular Ca2+-release pathways could also play important roles. This was already proposed for the release induced by S1P (Pyne and Pyne, 2000) and NAADP (Genazzani and Galione, 1996), but the molecular identity of both release channels is largely unknown. Besides the "classic" Ca2+-release pathways, a poorly understood Ca2+ leak from the ER has also been postulated, but its molecular nature is also still unknown (Camello et al., 2002), although it has recently been proposed to be a property of a hyperphosphorylated IP3R1 (Oakes et al., 2005) or a ribosome-translocon complex (Lomax et al., 2002). Our data lend support to the idea that additional intracellular Ca2+-release pathways may be present in some cell types. We showed evidence that suramin activates a release pathway very similar to a CICR mechanism described previously in A7r5 and 16HBE14o-cells (Nadif Kasri et al., 2003). Such a CICR mechanism was already found in hepatocytes (Wissing et al., 2002) and pancreatic -cells (Beauvois et al., 2004).
The exact mechanism by which suramin and DIDS activate Ca2+ release remains largely unknown. We found that CaM plays an important role in the regulation of suramin-induced Ca2+ release. We previously proposed that the CICR mechanism in A7r5 cells was activated upon binding of Ca2+ to CaM. Dissociation or relocalization of CaM would lead to activation of CICR. Because suramin has been shown to interact directly with the CaM-binding sites on the IP3R and RyR (Klinger et al., 2001; Papineni et al., 2002; Nadif Kasri et al., 2004), suramin might induce Ca2+ release by replacing endogenous CaM. However, DIDS, which also stimulates Ca2+ release, does not interact in the same way with CaM-binding sites. Suramin was also shown to interact with ATP-binding sites on purinoceptors (Hoyle et al., 1990) and RyRs (Emmick et al., 1994). Therefore, it is possible that suramin activates Ca2+ release through direct binding to a regulatory ATP-binding site. This could explain why ATP did not further enhance suramin-induced Ca2+ release. However, when comparing with the RyR, we can expect binding of suramin and DIDS to be more complex. It was demonstrated that suramin exerts a triphasic effect on the open probability of the RyR, indicating the presence of high-, intermediate- and low-affinity suramin-binding sites (Hill et al., 2004). The most probable mechanism for the activation of Ca2+ release in A7r5 cells could therefore also involve multiple binding sites for suramin and DIDS on an as yet unidentified protein. Some of these interaction sites are supposedly irreversible. Within the time resolution of our experiments, we could observe that CICR was faster than the suramin-induced Ca2+ release (Fig. 2). This could indicate that the suramin interaction might be more complex compared with the Ca2+ effects. Ca2+ could act directly on a binding site on the protein, whereas the suramin interaction could involve a more complex interference with other modulators such as CaM or ATP. Concerning the Ca2+ dependence of the suramin-induced Ca2+ release, we concluded that suramin can release an important part of the stored Ca2+ in the absence of free Ca2+, although we also observed a modulation of the suramin-induced Ca2+ release by [Ca2+]c. Because to a large extent, the suramin- and Ca2+-induced Ca2+ release was not additive, this would suggest that suramin and Ca2+ could act on the same release mechanism and that Ca2+ can also modulate the suramin-induced Ca2+ release.
We also found that DIDS and SITS are potent blockers of IICR (Fig. 7), similar to suramin (Nadif Kasri et al., 2004). However, we found that DIDS and SITS act differently on the IP3R. Whereas suramin decreased the affinity for IP3 by binding to CaM-binding sites, neither DIDS nor SITS affected IP3 binding to IP3R1 (Fig. 7C). Moreover, there was no effect on the binding of CaM to the IP3R1 (Fig. 7D). This suggests that DIDS and SITS may interact in a more complex way with IP3Rs and that they could inactivate the IP3R by irreversible binding. It has also been reported that endogenous sulfonates can play an important role in regulating IICR in cerebellum. An endogenous IP3R inhibitor was identified and was found to be a sulfonated compound (Watras et al., 2000). The presence of an endogenous inhibitor in neuronal tissues may be important for extending the dynamic range of IICR. It is possible that suramin, DIDS, or SITS could work in a similar way on the IP3R.
We showed that stilbene derivatives may have activatory or inhibitory effects, depending on which Ca2+-release mechanism they work on. Therefore, caution must be taken in using these pharmacological reagents to study the properties of the intracellular Ca2+-release mechanisms.
In summary, in this study, we confirmed the presence in A7r5 and 16HBE14o-cells of an as-yet-unidentified intracellular Ca2+-release pathway that is clearly independent from the IP3R and RyR. We have shown here that this pathway can be activated in both cell lines by the pharmacological agents suramin and DIDS. In contrast, the frequently used stilbene derivatives DIDS and SITS were also found to be potent inhibitors of the IP3R in the cells. This clearly shows the promiscuous effect of these compounds on different types of Ca2+ channels.
Acknowledgements
We thank Lea Bauwens, Marina Crabbee. and Silvia Vangeel for technical assistance. The mammalian CaM cDNA was kindly provided by Dr. Z. Grabarek (Boston Biomed Research Institute, Watertown, MA) and the rat cDNA for CaM1234 was kindly provided by Dr. J. Adelman (Oregon Health and Science University, Portland, OR). We thank Dr. D. C. Gruenert (University of Vermont, Colchester, VT) for the supply of 16HBE14o-cells. We thank Dr. K. Mikoshiba (University of Tokyo, Tokyo, Japan) for the supply of L15 and Lvec cells.
doi:10.1124/mol.105.013045.
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