Flubendiamide, a Novel Ca Channel Modulator, Reveals Evidence for Functional Cooperation between Ca Pumps and Ca Release

Flubendiamide, developed by Nihon Nohyaku Co., Ltd. (Tokyo, Japan), is a novel activator of ryanodine-sensitive calcium release channels (ryanodine receptors; RyRs), and is known to stabilize insect RyRs in an open state in a species-specific manner and to desensitize the calcium dependence of channel activity. In this study, using flubendiamide as an experimental tool, we examined an impact of functional modulation of RyR on Ca pump. Strikingly, flubendiamide induced a 4-fold stimulation of the Ca pump activity (EC50 11 nM) of an insect that resequesters Ca to intracellular stores, a greater increase than with the classical RyR modulators ryanodine and caffeine. This prominent stimulation, which implies tight functional coupling of Ca release with Ca pump, resulted in a marginal net increase in the extravesicular calcium concentration despite robust Ca release from the intracellular stores by flubendiamide. Further analysis suggested that luminal Ca is an important mediator for the functional coordination of RyRs and Ca pumps. However, kinetic factors for Ca pumps, including ATP and cytoplasmic Ca , failed to affect the Ca pump stimulation by flubendiamide. We therefore conclude that the stimulation of Ca pump by flubendiamide is mediated by the decrease in luminal calcium, which may induce calcium dissociation from the luminal Ca binding site on the Ca pump. This mechanism should play an essential role in precise control of intracellular Ca homeostasis. Pharmacological probes have gained widespread acceptance as reagents to reveal the functional consequences of intracellular calcium regulation. Many exogenous effectors modulate intracellular calcium homeostasis through effects on ryanodine-sensitive calcium release channels (ryanodine receptors; RyRs). However, their actions on RyRs are often complex, not completely clarified, or both (Zucchi and RoncaTestoni, 1997). This complexity is thought to be attributable to calcium dependence of the functional modulation, leading to apparently antithetical effects on the RyR. Such complexity is a serious obstacle to understanding the sequential effects of RyR activity in fluctuating calcium conditions. Flubendiamide, a new phthalic diamide compound (Fig. 1), stabilizes insect RyR to an open state, evoking massive calcium release from intracellular stores (EbbinghausKintscher et al., 2006). The compound possesses distinct pharmacological characteristics, mediated by a binding site different from that of ryanodine, a widely used probe for the RyR. Interestingly, flubendiamide achieved maximum activation of the RyR in the presence of a broad range of calcium concentrations, including in low calcium conditions. This characteristic might contribute to revealing the relationships between functionally linked components by minimizing the complexity in functional modulation of the RyR. Thus, this compound is expected to provide a new experimental device for exploring the impacts of RyR activity. The action of flubendiamide is highly specific to insect RyRs and results in selective toxicity in restricted taxa, including Lepidoptera (Tohnishi et al., 2005). The characteristic symptoms caused by continuous muscle contractions through straightforward activation of the insect RyR suggest a pivotal role for the RyR in insect muscle contraction. The physiological importance of RyR in insect muscle implies that basic understanding of excitation-contraction coupling, which has been postulated from evidence accumulated in comprehensive investigations (Lockyer et al., 1998; Quinn et al., 1998; Takekura and Franzini-Armstrong, 2002; VázquezMartı́nez et al., 2003), is broadly deducible in the animal kingdom, including the Insecta. However, distinct pharmaArticle, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.020339. ABBREVIATIONS: RyR, ryanodine receptor; cADPR, cyclic ADP ribose; SR, sarcoplasmic reticulum; diBr-BAPTA, 1,2-bis(2-amino-5-bromophenoxy)ethane-N,N,N ,N -tetraacetic acid; DTT, dithiothreitol; MOPS, 3-(N-morpholino)propanesulfonic acid; A23187, calcimycin. 0026-895X/06/6905-1733–1739$20.00 MOLECULAR PHARMACOLOGY Vol. 69, No. 5 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 20339/3111071 Mol Pharmacol 69:1733–1739, 2006 Printed in U.S.A. 1733 at A PE T Jornals on A ril 1, 2017 m oharm .aspeurnals.org D ow nladed from cological characteristics are seen in the functional properties of the insect RyR, despite its essential similarity to mammalian isoforms (Lehmberg and Casida, 1994; Puente et al., 2000; Scott-Ward et al., 2001). We therefore believe that the insect muscle is a unique model for understanding the physiological impact of altered RyR activity. The Ca released via the RyR during excitatory states is eventually returned to intracellular stores by the Ca pump. Functional interference with the Ca pump by the RyR is implied in the recent controversy around studies of cADPR (Lukyanenko et al., 2001a). cADPR was first reported as an endogenous RyR activator, but subsequent studies suggested RyR activation by cADPR was a result of Ca pump activation mediated by increased luminal calcium. In this study, we used a unique approach to elucidate the physiological impact of RyR activity using this new probe in a distinct RyR isoform derived from an insect. In particular, we focused our attention on SR Ca pump activity, a functionally complementary factor of the RyR. The results presented here demonstrate an intriguing relationship between the Ca pump and RyR activation by flubendiamide, mediated by luminal Ca . Materials and Methods Chemicals and Reagents. Flubendiamide (Fig. 1) was synthesized at Nihon Nohyaku Co., Ltd. The compound was dissolved in dimethyl sulfoxide for all assays (below 0.5% final concentration). [H]Ryanodine (56 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Ryanodine, ATP/Tris, thapsigargin, A23187 (calcimycin), and caffeine were purchased from Sigma-Aldrich (St. Louis, MO). DiBr-BAPTA and Rhod-2 were purchased from Invitrogen (Carlsbad, CA). All other chemicals were the best available grade. Insects. The strain of Spodoptera litura (Lepidoptera: Noctuidae) used has been maintained at Nihon Nohyaku Co., Ltd. for 15 years. Larvae of S. litura were reared on an artificial diet of Insecta LFS, purchased from Nihon Nohsan Kogyo (Yokohama, Japan). The colony was maintained at 25°C under a 16-h light/8-h dark photoperiod, with constant access to the larval diet. Preparation of Muscle Membranes. Sixth instars of S. litura were dissected under a stereoscopic microscope, and longitudinal ventral muscle tissues were collected. Collected tissues were homogenized on ice in 9 volumes (based on tissue wet weight) of the homogenization solution (250 mM sucrose, 5 mM DTT, and 10 mM Tris/HCl, pH 7.4) with a Teflon-pestle homogenizer. The homogenates obtained were centrifuged at 9000gmax (7000gavg) for 30 min at 4°C. The resulting supernatants were filtered through a cell strainer (BD Falcon, Bedford, MA) and centrifuged at 140,000gmax, (104,000gavg) for 1 h at 4°C. The resulting supernatants were discarded, and pellets were resuspended in four volumes (based on tissue wet weight) of the homogenization solution without DTT, followed by recentrifugation under the conditions described above. The resulting pellets were suspended in a half volume of the homogenization solution without DTT. The suspensions obtained were stored at 80°C until use. Calcium Release from the Muscle Membrane Preparations. Calcium release from the membrane preparation was investigated by calcium fluorimetry. The membrane preparations (40 g) described above were diluted with 315 l of assay solution (100 mM potassium phosphate buffer, pH 7.4, 3 mM MgCl2, 100 mM sucrose, 25 M CaCl2, and 5 M Rhod-2). The processes of calcium influx and efflux were monitored by the fluorescence derived from the Ca / Rhod-2 complexes using a fluorescence spectrophotometer (F-4500; Hitachi, Tokyo, Japan) with excitation at 552 nm and emission at 580 nm. The assays were performed at 22°C with sustained stirring of the assay solution. In the absence of Rhod-2, no background fluorescence was detected. The free-calcium concentrations in the assay solutions were calculated according to Berman (2000). Measurements of ATPase Activity. Membrane preparations (20 g) were suspended in 100 l of Ca -ATPase assay solution (100 mM KCl, 6 mM MgCl2, and 50 mM Tris/MOPS, pH 7.4) with various concentrations of Ca or Ca buffer (for fixing the free calcium concentration below 2.3 M), as detailed in the figure legends. After a 30-min preincubation at 25°C, reactions were initiated by the addition of ATP/Tris. Unless otherwise stated, the added ATP/Tris concentration was fixed at 1 mM. For determination of the basal activity, 0.8 mM EGTA was added to the assay solution. Inorganic phosphate liberated during the reaction was colorimetrically determined based on a previously described method (Marsh, 1959). In addition, ATPase activity was evaluated using the ATP-regenerating system according to Chu et al. (1988), with minor modification. In brief, 40 g of membrane preparation was suspended in 2 ml of an assay solution containing coupling mixture (100 mM KCl, 6 mM MgCl2, 0.05 mM CaCl2, 50 mM Tris/HCl, pH 7.4, 0.2 mM -NADH, 2 mM phosphoenolpyruvate, 4.3 units/ml pyruvate kinase, and 6.3 units/ml L-lactate dehydrogenase). The reactions were performed at 25°C with continuous stirring of the reaction mixture. Consumption of -NADH was monitored by measuring the absorbance at 340 nm with a double-beam spectrophotometer (U-3000; Hitachi). Equilibrium [H]Ryanodine Binding Assay. [H]Ryanodine was incubated at 25°C with 40 to 80 g of the membrane preparation in 0.4 ml of binding solution containing 1 M KCl, 0.3 M sucrose, and 10 mM Tris/HCl, pH 7.4, in the absence or presence of flubendiamide. After incubation for 80 min, the assay solutions were rapidly filtered through Whatman GF/F filters (Whatman, Maidstone, UK) presoaked in the binding solution. The filters were then rinsed with 20 ml of washing solution (1 M KCl, 0.3 M sucrose, and 10 mM Tris/HCl, pH 7.4). The radioactivity remaining on the filter was counted using a liquid scintillation counter (model 1409; PerkinElmer Wallac, Turku, Finland). In all assays, nonspecific binding of [H]ryanodine was determined in the presence of 10 M unlabeled ryanodine. Under the experimental conditions, specific binding was greater than 70% of the total binding of 5 nM [H]ryanodine. Each data point represents the mean of at least triplicate determinations.