Suramin and Disulfonated Stilbene Derivatives Stimulate the Ca -Induced Ca -Release Mechanism in A7r5 Cells

We have described previously a novel Ca -induced Ca release (CICR) mechanism in permeabilized A7r5 cells (embryonic rat aorta) and 16HBE14ocells (human bronchial mucosa) cells (J Biol Chem 278:27548–27555, 2003). This CICR mechanism was activated upon the elevation of the free cytosolic calcium concentration [Ca ]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 Ca 2 -insensitive CaM mutant, and by different members of the superfamily of CaMlike Ca -binding proteins. Here, we present evidence that the CICR mechanism that is expressed in A7r5 and 16HBE14ocells 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 Ca 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 Ca release. Furthermore, we show that the suramin-stimulated Ca release is regulated by Ca and CaM in a similar way as the previously described CICR mechanism. The pharmacological characterization of the suramin/DIDS-induced Ca release further confirms its properties as a novel CaM-regulated Ca release mechanism. We also investigated the effects of disulfonated stilbene derivatives on IP3-induced Ca 2 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 Ca concentration ([Ca ]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 Ca for cytosolic cellular signaling. Calcium pumps of the sarco(endo)plasmic-reticulum Ca -ATPase family import Ca into the organelle lumen. Two families of intracellular Ca -release channels are primarily responsible for the release, the inositol1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR) (Berridge et al., 2003). Recent studies have emphasized the role of novel types of intracellular Ca -release channels possibly playing an important role in intracellular Ca signaling. Wissing et al. (2002) identified a novel Ca -induced Ca -release (CICR) mechanism in permeabilized hepatocytes that responded to modest increases in [Ca ]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 Ca -activated channel that is permeable for divalent cations. Increased levels of intracellular Ca activated polycystin-2-mediated release of Ca from intracellular stores (Koulen et al., 2002). Moreover, This work was supported by grants G.0210.03 (to H.D.S. and J.B.P.) and G.O206.01 (to L.M.) of the Fund for Scientific Research Flanders (Belgium) and by grant GOA2004/07 from the Concerted Actions of the Katholieke Universiteit Leuven (to L.M., H.D.S., G.C., and J.B.P.). G.B. is a postdoctoral fellow of the Fund for Scientific Research Flanders (Belgium). We are also grateful for the support by the Interuniversity Poles of Attraction ProgrammeBelgian State, Prime Minister’s Office, Federal Office for Scientific, Technical, and Cultural Affairs, IUAP P5/05. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.013045. ABBREVIATIONS: [Ca ]c, cytosolic free calcium concentration; ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; IP3R, inositol-1,4,5trisphosphate receptor; RyR, ryanodine receptor; CICR, calcium-induced calcium release; TRP, transient receptor potential; CaM, calmodulin; DIDS, 4,4 -diisothiocyanatostilbene-2,2 -disulfonic acid; SITS, 4 -acetoamido-4 -isothiocyanostilbene-2 ,2 -disulfonic acid; IICR, inositol-1,4,5trisphosphate-induced Ca release; DNDS, 4,4 -dinitrostilbene-2,2 -disulfonic acid; TG, thapsigargin; IP3, inositol-1,4,5-trisphosphate; XeC, xestospongin C; RuRed, ruthenium red; S1P, sphingosine-1-phosphate. 0026-895X/05/6801-241–250$20.00 MOLECULAR PHARMACOLOGY Vol. 68, No. 1 Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics 13045/3040762 Mol Pharmacol 68:241–250, 2005 Printed in U.S.A. 241 at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from there are indications that some bona fide plasmalemmal Ca -permeable TRP channels (e.g., TRPV1 and TRPM8) also reside in intracellular membranes where they may function as Ca -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 [Ca ]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 Ca 2 insensitive CaM mutant, and by different members of the superfamily of CaM-like Ca -binding proteins. Our data suggested that the CICR mechanism described here may represent a novel type of Ca -release channel, which is silent at low [Ca ]c because of inhibition by bound apocalmodulin and which becomes activated by the Ca -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 Ca -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 -acetoamido4 -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 Ca 2 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 16HBE14ocells 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 Ca release. Furthermore, we show that this suramin-stimulated Ca 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 Ca -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). Ca 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 g/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 mediumwas 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). Ca fluxes were performed on saponin-permeabilized cells. The cells were seeded in 12-well clusters (Costar, Cambridge, MA) at a density of approximately 4 10 cm . Experiments were carried out on confluent monolayers of cells (3 10 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 g/ml saponin at 25°C. The nonmitochondrial Ca 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 Ca (28 Ci/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 M thapsigargin (TG). TG was added to block the ER Ca pumps during subsequent additions of Ca . The efflux medium was replaced every 2 min, and the efflux was performed at 25°C. The additions of Ca , IP3, suramin, and stilbene derivatives are indicated on the figures (arrow). Free [Ca ] 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 Ca remaining in the stores was released by incubation with 1 ml of a 2% sodium dodecyl sulfate solution for 30 min. Ca release in some experiments is plotted as the fractional loss (i.e., the amount of Ca released in 2 min divided by the total store Ca 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 l) (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 Trisbuffered saline (20 mM Tris-HCl, pH 7.2, and 150 mM NaCl) mixed with one part bacterial ProFound lysis buffer containing a Trisbuffered 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 l 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) [H]IP3-Binding Experiments. Binding studies were performed as described previously. [H]IP3 binding was performed at 0°C in 100 l of binding buffer containing 50 mM Tris-HCl, pH 7.0, 1 mM 242 Kasri et al. at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from EGTA, 10 mM -mercaptoethanol, and 10 nM [H]IP3. Nonspecific binding was determined in the presence of 12.5 M 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 g. 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).