INTRODUCTION

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RELEASE OF CA²⁺ from intracellular stores is a widespread component in several signaling pathways (3). It is well known that inositol-1,4,5-tris-phosphate (IP₃) triggers Ca²⁺ release from intracellular stores (3); however, cells possess other intracellular Ca²⁺ releasing systems, including the so-called Ca²⁺-induced Ca²⁺ release system, mediated by the ryanodine receptor/channel (RyR) (13). Recently it was found that the endogenous nucleotide cADP-ribose (cADPR) is a potent activator of the RyR (15, 19). Current experimental evidence indicates that cADPR binds on its specific receptor, which is associated with the RyR, and triggers Ca²⁺ release through the RyR (15). Biosynthesis of cADPR from -NAD is catalyzed by ADP-ribosyl cyclase, and cADPR is hydrolyzed by the cADPR hydrolyzed to ADP-ribose (ADPR) (15, 19). Enzymes of cADPR metabolism are ubiquitous in mammalian cells and tissues (14, 15, 19, 23). Recently, cADPR has been proposed to be the second messenger in several intracellular signaling pathways (15, 19, 20).

Volatile anesthetics have multiple actions on intracellular Ca²⁺ homeostasis (1, 4, 29), including activation of the RyR and sensitization of this channel to pharmacological agonists such as caffeine and ryanodine (1, 26, 27). Indeed, dysfunction of the skeletal muscle RyR in response to volatile anesthetics is a key point in the pathogenesis of malignant hyperthermia (MH) (24). In addition to its role in the pathogenesis of MH, the RyR appears to be widespread and may have important roles in several cellular processes including neural function, cardiac muscle contraction, and insulin secretion (13, 15, 19). Furthermore, volatile anesthetics have complex effects on RyR function even in cells from animals not susceptible to MH (2, 11, 12, 16, 17). However, the effect of volatile anesthetics on the newly discovered cADPR signaling system has not been described to the present. I explored the effect of volatile anesthetics on cADPR-induced Ca²⁺ release in the sea urchin egg homogenate bioassay. The sea urchin egg homogenate is a very well-established model for the study of intracellular Ca²⁺ homeostasis (5-9, 22, 28). In addition, halothane-induced intracellular Ca²⁺ release, inhibited by dantrolene, has been described in this model (18). Furthermore, the cADPR-induced Ca²⁺ release system has been very well characterized in this preparation and has several similarities with mammalian models (5-9, 22, 28).

In this work I found that halothane can potentiate the cADPR-induced Ca²⁺ release through the RyR. In contrast, isoflurane and sevoflurane had no effect on cADPR-modulated Ca²⁺ release. I propose that modulation of the cADPR signaling system by halothane may be an important component of the complex effect of this volatile anesthetic on intracellular Ca²⁺ homeostasis.

	MATERIALS AND METHODS

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Tissue extracts. Tissues were harvested from adult male Sprague-Dawley rats (200-250 g body wt) killed under pentobarbital sodium anesthesia. Heart, liver, spleen, and lung were quickly dissected, chilled, and minced in ice-cold solution containing 0.25 M sucrose, 20 mM Tris-HCl (pH 7.2), and 20 µg/ml leupeptin. Tissues were homogenized (1:4 wt/vol) in a Dounce homogenizer using four to five strokes and centrifuged at 4,000 rpm for 10 min at 4°C. The supernatants, denoted further as extract, were collected and used for determination of the ADPR cyclase activity.

Sea urchin egg homogenates. Homogenates from Lytechinus pictus egg were prepared as described previously with minor modifications (6). Eggs were obtained by injection of 0.5 M KCl into the coelomic cavity and shed in artificial seawater. The jelly coats were washed from the eggs by several passages through 80-µm mesh silk. The eggs were then washed once in artificial seawater, twice in Ca²⁺-free seawater containing 1 mM EGTA, twice in Ca²⁺-free water without EGTA, and once in a homogenizing buffer containing 250 mM N-methyl glucamine, 250 mM potassium gluconate, 20 mM HEPES buffer (pH 7.2), 1 mM MgCl₂, 2 U/ml creatine kinase, 4 mM phosphocreatine, 1 mM ATP, 25 µg/ml leupeptin, 20 µg/ml aprotinin, and 100 µg/ml soybean trypsin inhibitor. A 25% suspension was prepared by homogenization with four to five strokes in a Dounce homogenizer with a type A pestle, and the homogenate was then centrifuged for 10-12 s at 13,000 g at 4°C. The supernatant was collected and stored in 1-ml aliquots at 70°C. Frozen homogenates were thawed in a 17°C water bath and diluted to 1.25% with an intracellular media (IM) solution containing 250 mM N-methyl glucamine, 250 mM potassium gluconate, 20 mM HEPES buffer (pH 7.2), 1 mM MgCl₂, 2 U/ml creatine kinase, 4 mM phosphocreatine, 1 mM ATP, 3 µg/ml oligomycin, and 3 µg/ml antimycin. After incubation at 17°C for 3 h, 3 µM fluo-3 was added. Fluo-3 fluorescence was monitored at 490 nm excitation and 535 nm emission in a 250-µl cuvette at 17°C with a circulation water bath and continuously mixed with a magnetic stirring bar, using a Hitachi spectrofluorometer (F-2000). The addition of stock solutions of various substances did not exceed 2% of the homogenate volume in the cuvette.

Ca²+ release assay. For the Ca²⁺-release experiments, sea urchin egg homogenates were loaded with Ca²⁺ for 3 h as described above.

Binding of cADPR. Binding of cADPR was performed as described previously (7), using [³H]cADPR. Sea urchin egg homogenates (2 mg/ml) were diluted in IM with addition of 1 mM EGTA and were incubated with different concentrations of [³H]cADPR (100 dpm/fmol) for 10 min on ice. After incubation, the mixture (100 µg of protein) was filtered through prewashed fiberglass (GFB, Whatman) under vacuum and rapidly washed twice with 2 ml of ice-cold IM. Radioactivity retained on the filter was determined by use of standard scintillation-counting techniques.

Preparation of nucleotides. cADPR was synthesized by Aplysia ADP-ribosyl cyclase, using homogenized Aplysia ovotestes as described previously (6). The reaction was stopped by acetone precipitation as described above for synthesis of cADPR. Neither the spontaneous hydrolysis of cADPR nor the hydrolysis catalyzed by the sea urchin egg homogenate was changed by halothane (data not shown). After acetone precipitation, the nucleotides were purified by HPLC anion exchange chromatography using AG MP-1 (Bio-Rad) resin packed into a 1 × 10 cm column. The nucleotides were eluted with a nonlinear gradient of 150 mM trifluoroacetic acid and water and were monitored by ultraviolet absorption at 250 nm. Purified cADPR was evaporated to dryness in a SpeedVac concentrator. cADPR used in all experiments was at least 99% pure as determined by HPLC.

ADPR cyclase activity. ADPR cyclase activity was determined by conversion of NAD analog nicotinamide guanine dinucleotide to the fluorescent product cGDP ribose (cGDPR) (14, 23). Tissue extracts or purified Aplysia ADP-ribosyl cyclase were incubated in a thermostated 250-µl cuvette, the contents of which were continuously mixed with a magnetic stirring bar at 30°C with 0.4 mM nicotinamide guanine dinucleotide, and generation of cGDPR was continuously monitored by using a Hitachi spectrofluorometer (F-2000) at 310 nm excitation and 410 nm emission. The ADPR cyclase activity was then calculated by using a standard curve of authentic cGDPR, and the specific activity was expressed as nanomoles of cGDPR per milligram of protein per minute.

Materials. L. pictus and Aplysia california were obtained from Marinus (Long Beach, CA). Fluo-3 was purchased from Molecular Probes; IP₃, ryanodine, oligomycin, and antimycin were from Calbiochem. All other reagents, of the highest purity grade available, were supplied from Sigma Chemical (St. Louis, MO).

	RESULTS

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Activation of RyR by cADPR. Sea urchin egg homogenates supplemented with an ATP-regenerative system sequester added Ca²⁺ into vesicular stores in an ATP-dependent manner and release Ca²⁺ in response to nanomolar concentrations of either cADPR or IP₃ (data not shown). Such homogenates display homologous desensitization to sequential additions of saturating concentrations of agents that induce Ca²⁺ release through the same channel (5-9, 28). Indeed, Ca²⁺ release induced by cADPR causes homologous desensitization of the homogenate to ryanodine but not to IP₃ (5-9, 28); furthermore, the reverse was also true: Ca²⁺ release induced by ryanodine caused homologous desensitization to cADPR but had no effect on IP₃-induced Ca²⁺ release (5-9, 28). In addition, cADPR-induced Ca²⁺ release was inhibited by several inhibitors of the RyR such as spermine, ruthenium red, and the specific cADPR inhibitor 8-Br-cADPR (5-9, 28). However, Ca²⁺ release induced by cADPR was not inhibited by 1 mg/ml heparin, a specific antagonist of the IP₃ channel (6). These observations confirmed the evidence that cADPR activates Ca²⁺ release through the RyR (5-9, 28).

Effect of halothane on RyR. I investigated the effect of halothane on the ryanodine channel by using pharmacological agonists of this channel. Fig. 1 demonstrates the effect of halothane on ryanodine-induced Ca²⁺ release. Addition of 800 µM halothane did not produce any significant Ca²⁺ release by itself; however, it sensitized the Ca²⁺ release system to ryanodine (Fig. 1) and caffeine (data not shown). As shown in Fig. 1, preincubation of the sea urchin egg homogenates with halothane decreases the half-maximal concentration of ryanodine needed to induce Ca²⁺ release about three times.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Dose dependence of ryanodine. Experiment was carried out as described in MATERIALS AND METHODS. Ca2+ release induced by different concentrations of ryanodine was tested in the absence () or presence () of 800 µM halothane. Data are representative of 3 independent experiments.

Effect of volatile anesthetics on cADPR induced Ca²+ release. As shown in Figs. 2 and 3, cADPR-induced Ca²⁺ release was potentiated by halothane. As observed with Ca²⁺ release induced by ryanodine, the half-maximal concentration of cADPR was decreased more than threefold by pretreatment of the homogenates with 800 µM halothane (Fig. 3), although the maximum Ca²⁺ release response to cADPR was not enhanced by halothane. Thus halothane increased the apparent affinity of the Ca²⁺-induced Ca²⁺ release to stimulation by cADPR. However, I also observed that halothane had no effect on [³H]cADPR binding (Fig. 4), which suggests that halothane does not interact directly with the cADPR receptor. As previously discussed, 800 µM halothane did not produce Ca²⁺ release by itself (Fig. 2); however, when homogenates were pretreated with subthreshold concentrations of cADPR, halothane could promote a significant amount of Ca²⁺ release (Fig. 2). The Ca²⁺ release induced by halothane in the presence of subthreshold concentrations of cADPR was abolished by treatment of the homogenate with ruthenium red, Mg²⁺, and 8-Br-cADPR (Table 1). On the other hand, the Ca²⁺ release induced by halothane was not inhibited by 1 mg/ml heparin, an IP₃ antagonist (Table 1). I also observed that 800 µM halothane had no effect on uptake rate and steady-state Ca²⁺ levels in the sea urchin egg homogenates; the rate of Ca²⁺ uptake in the absence and in the presence of 800 µM halothane was 2.7 ± 0.2 and 2.5 ± 0.17 nmol/min, respectively. Furthermore, the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin was not able to potentiate Ca²⁺ induced by agonists of the RyR (data not shown) (22). These observations further support the hypothesis that halothane at the concentration tested sensitizes the RyR.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Effect of halothane (Halo) on cADP-ribose (cADPR)-induced Ca2+ release. Ca2+ release in 1.25% sea urchin egg homogenates was monitored by fluo-3 as Ca2+ indicator. Arrows indicate addition of 10 nM cADPR or 800 µM halothane.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Dose dependence of cADPR. Experiment was carried out as described in MATERIALS AND METHODS. Ca2+ release induced by different concentrations of the cADPR was tested in the absence () or presence () of 800 µM halothane. Data are representative of 3 independent experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   cADPR binding. [3H]cADPR binding to sea urchin egg homogenates was carried out as described in MATERIALS AND METHODS. Dose dependence of cADPR binding was tested in the absence () or presence () of 800 µM halothane. Data are representative of 3 independent experiments.

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Effect of volatile anesthetics on cADPR-induced Ca2+ release. Net rates of Ca2+ release induced by 10 nM cADPR in homogenates preincubated with no further addition (control), 800 µM halothane (halo), 1,200 µM isoflurane (isof), or 2,000 µM sevoflurane (sevo) for 20 s are shown. Data represent means ± SE for 3 experiments.

Effect of volatile anesthetics on ADPR cyclase activity. cADPR is biosynthesized by the reaction catalyzed by ADPR cyclase (14, 15, 19, 20, 23). Several types of ADPR cyclases were isolated and characterized in detail (20). In invertebrates, this includes a 29-kDa soluble cyclase from the ovotestis of the mollusk Aplysia that has very high metabolic activity (15, 19). In vertebrate tissue, the most extensively studied ADPR cyclase is the so-called membrane-bound CD38 cyclase (20). I tested the effect of volatile anesthetics on the ADPR cyclase from Aplysia and several mammalian tissues including heart, lung, spleen, and liver. I found no effect of volatile anesthetics on the activity of these enzymes (Fig. 6 and data not shown). In contrast, as previously described, the activity of the cyclase was completely inhibited by addition of nicotinamide.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Effect of volatile anesthetics on ADP-ribose (ADPR) cyclase activity. ADPR cyclase was determined as described in MATERIALS AND METHODS in several rat tissues and in Aplysia ovotestis cyclase. Results with liver homogenates are shown. Initial rates of cGDPR formation were measured in homogenates preincubated with no further addition (control), 2 mM halo, 2 mM isof, 2 mM sevo; or 2 mM nicotinamide for 40 s. Data represent means ± SE for 3 experiments. Similar results were obtained with all the other tissues tested, namely, lung, spleen, heart, and purified Aplysia ovotestes cyclase.

	DISCUSSION

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Oscillation of intracellular Ca²⁺ release has been shown to be associated with addition of volatile anesthetics (1, 4, 29). However, the precise mechanism by which volatile anesthetics regulate intracellular Ca²⁺ is not completely understood. Effects of these compounds over the RyR and IP₃ channel have been proposed before (1, 4, 24, 26, 27, 29). It was shown that halothane could increase the open state of the RyR (1, 4, 26, 27). Furthermore, it was demonstrated that halothane could induce Ca²⁺ release from intracellular sarcoplasmic reticulum via the RyR (1, 4, 24, 26, 27, 29). All these observations are consistent with the effect of halothane on the RyR observed in the present work.

The major finding of the present study is the interaction of halothane with cADPR. Recently, cADPR has been proposed to be the endogenous regulator of the RyR and to serve as a second messenger in several signaling pathways (15, 19, 20). This observation led me to explore the effect of volatile anesthetics on the cADPR system. I used sea urchin egg homogenate as a model to study intracellular Ca²⁺ release. This experimental model has been extensively studied, and the RyR receptor found in this system resembles the mammalian RyR (5-9, 14, 15, 19, 20, 22, 23, 28).

The effect of halothane on the RyR appears to be mediated by sensitization of the Ca²⁺ channel to its agonists. First, halothane by itself did not release Ca²⁺ in my experiments. However, it could sensitize the RyR to several nonphysiological and physiological agonists including cADPR. The effect of halothane is not mediated by inhibition of the Ca²⁺ uptake, because halothane had no effect on the rate of Ca²⁺ uptake in my preparation. Furthermore, thapsigargin, a potent inhibitor of the Ca²⁺-ATPase, had no effect on the Ca²⁺ release induced by agonists of the RyR (data not shown) (8, 22).

Halothane had no effect on the binding of cADPR, indicating that this volatile anesthetic does not interact with the cADPR binding protein. Indeed, it has been previously proposed that cADPR does not bind directly to the RyR but to an accessory protein that interacts with the RyR (15, 19, 20). In mammalian cells, the FK506 binding protein (FKBP) has been proposed to be the cADPR binding protein (25). Noguchi et al. (25) showed that cADPR binding to FKBP induces dissociation of this protein from the RyR, increasing the channel open probability. I have previously shown that dissociation of the FKBP from the RyR increases the sensitivity of the channel to halothane (10). These observations taken together may indicate that cADPR increases the halothane-induced Ca²⁺ release by promoting dissociation of the FKBP from the RyR.

I also explored the effect of volatile anesthetics on the activity of the enzymes that synthesize cADPR. I found no effect of halothane, isoflurane, and sevoflurane on the activity of the ADPR-cyclase from several tissues. It has been previously observed that the activity of the ADPR cyclase can be activated by interaction with the trimeric G proteins (21). It is possible that in vivo volatile anesthetics may modulate the synthesis of cADPR by regulating the interaction between cyclase and G proteins. In analogy, it has been described that halothane can inhibit carbachol-induced synthesis of IP₃ by its effect on G proteins (29).

In conclusion, I present for the first time evidence that halothane can interact with the new second messenger system modulated by cADPR. It is possible that the effect of halothane on cADPR may play an important role in the complex effect of volatile anesthetics on intracellular Ca²⁺ homeostasis.