Introduction

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Elevation of intracellular Ca²⁺ concentration by release from intracellular stores regulates a number of important physiological events, including hormone secretion, muscle contraction, and fertilization (Clapham, 1995). The wide variety of responses initiated by this process is consistent with the existence of multiple Ca²⁺ mobilization pathways, each with a different pattern of release. Ca²⁺ mobilization has been extensively studied in the sea urchin egg, since this has multiple Ca²⁺ release pathways that are amenable to detailed analysis (for review, see Lee, 1997). In particular, the sea urchin egg homogenate system provides an excellent system to characterize Ca²⁺ release mechanisms due to its reliability and high signal-to-noise ratio. Importantly, the intracellular stores of sea urchin eggs contain all three major pathways for Ca²⁺ release found in mammalian cells, involving the endogenous messengers inositol 1,4,5-trisphosphate (IP₃), cyclic adenosine diphosphate ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP) (Clapper and Lee, 1985; Lee et al., 1989; Lee and Aarhus, 1995). Significantly, both cADPR and NAADP were originally discovered in the sea urchin egg.

IP₃-mediated Ca²⁺ release occurs via activation of the IP₃ receptor and is characterized pharmacologically by competitive inhibition by heparin (Clapham, 1995). Considerable evidence suggests that cADPR is the endogenous modulator of the ryanodine receptor (RyR)-mediated Ca²⁺-induced Ca²⁺ release (CICR) system (McPherson and Campbell, 1993). Both caffeine and ryanodine, pharmacological activators of CICR in muscle, display cross-desensitization with cADPR (Galione et al., 1991). In addition, pharmacological blockers of CICR, such as procaine and ruthenium red, specifically inhibit cADPR-sensitive Ca²⁺ release, indicating that cADPR acts on the same Ca²⁺ release system (Galione et al., 1993). This is further supported by the finding that low concentrations of cADPR that are not sufficient to induce Ca²⁺ release can greatly potentiate the effect of caffeine (Lee, 1993). Finally, a more direct demonstration that cADPR acts through the CICR system comes from evidence that cADPR can potentiate Ca²⁺ release induced by the divalent cations Ca²⁺ and Sr²⁺(Lee, 1993).

The mechanism of NAADP-mediated Ca²⁺ release from intracellular stores is yet to be clearly defined (Genazzani and Galione, 1997), although its pharmacology is distinct from that of IP₃- and cADPR-induced calcium release (Genazzani et al., 1997). In particular NAADP-induced release has been shown to be blocked noncompetitively by both L-type Ca²⁺ channel antagonists and the L-type Ca²⁺ channel agonist BAY K 8644, whereas IP₃- and cADPR-induced Ca²⁺ release are insensitive to these agents (Genazzani et al., 1997). This presumably reflects an underlying homology between the NAADP-activated channel and L-type Ca²⁺ channels. In addition, NAADP exhibits unique inactivation properties, whereby low concentrations of this molecule desensitize any further response of the system to the same agent (Aarhus et al., 1996; Genazzani et al., 1996). Finally, in contrast to both IP₃ and cADPR, which operate on thapsigargin-sensitive endoplasmic reticular stores, NAADP has been shown to access Ca²⁺ from a thapsigargin-insensitive pool (Genazzani and Galione, 1996).

Aside from caffeine and ryanodine, a limited number of other pharmacological agonists of RyRs are available. Most notably, bromoeudistomin D and 9-methyl-7-bromoeudistomin, derivatives of eudistomin D isolated from the Caribbean tunicate Eudistoma olivaceum, have been reported to activate ryanodine-sensitive CICR (Nakamura et al., 1986; Seino et al., 1991), sharing the same binding site as caffeine (Fang et al., 1993). Recently, 2-hydroxycarbazole, a structurally related compound based on a carbazole ring rather than a -carboline ring, has been reported to potently release Ca²⁺ in cardiac and skeletal muscle microsomes in a manner sensitive to the CICR blocker ruthenium red and the local anesthetic tetracaine (Tovey et al., 1998). In contrast to known modulators of this receptor, however, 2-hydroxycarbazole was found not to effect binding of the plant alkaloid ryanodine to the RyR (Tovey et al., 1998).

Here we undertake an analysis of the activity of 2-hydroxycarbazole in sea-urchin egg homogenate and provide evidence that it activates Ca²⁺ release in a manner pharmacologically distinct from the RyR/cADPR release mechanism as well as that of IP₃ and NAADP.

	Experimental Procedures

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Materials. Fluo-3 (pentapotassium salt) was from Molecular Probes (Eugene, OR), 2-hydroxycarbazole was from Aldrich Chemical (Milwaukee, WI), and all other chemicals were from Sigma (Poole, Dorset, UK). Concentrated stock solutions of 2-hydroxycarbazole were prepared in dimethyl sulfoxide (DMSO) and kept in the dark. DMSO alone at the volume used as the vehicle (1%) did not affect Ca²⁺ release from sea urchin egg homogenates.

Collection of Eggs. Lytechinus pictus sea urchins were obtained from Marinus Inc. (Long Beach, CA). Eggs were obtained by stimulating ovulation of female sea urchins with an intracoelomic injection of KCl. Jelly was removed by filtration through 90-µm nylon mesh and eggs were then washed twice in artificial sea water (435 mM NaCl, 15 mM MgSO₄, 11 mM CaCl₂, 10 mM KCl, 2.5 mM NaHCO₃, and 1.0 mM EDTA at pH 8.0).

Ca²⁺ Release Assay and Calibration. Homogenates of sea urchin eggs were prepared as described previously (Dargie et al., 1990). Ca²⁺ loading of intracellular stores was achieved by incubating for 3 h at 17°C in an "intracellular medium" consisting of 250 mM potassium gluconate, 250 mM N-methylglucamine, 20 mM HEPES, and 1 mM MgCl₂, pH 7.2. Additions of 1 mM ATP, 10 U ml¹ creatine kinase and 10 mM phosphocreatine were made to achieve an ATP-regenerating system, plus 25 µg ml¹ leupeptin, 10 µg ml¹ aprotinin, and 50 µg ml¹ soya bean trypsin inhibitor, as protease inhibitors, and 1 µg ml¹ oligomycin, 1 µg ml¹ antimycin, and 1 mM sodium azide, as mitochondrial inhibitor. Free ionized Ca²⁺ was measured with fluo-3 (3 µM) at 17°C, using 500 µl of homogenate in a PerkinElmer LS-50B fluorometer at 490-nm excitation and 535-nm emission. Additions were made in 5-µl volumes and changes in relative fluorescence units measured. In the absence of fluo-3, background fluorescence was not detected and consequently no correction for background fluorescence was required. The peak value, rather than the area under the curve, was measured for consistency with previous reports of IP₃, cADPR, and NAADP activity in sea urchin egg homogenate) (Clapper and Lee, 1985; Lee et al., 1989; Lee and Aarhus, 1995). Changes in fluorescence were calibrated to known Ca²⁺ additions using separate samples of the same homogenate. These were aliquoted after addition of fluo-3 to ensure equal indicator concentration in all the samples. Amount resequestered Ca²⁺ was determined by measuring the difference between basal Ca²⁺ levels (prior to agonist addition) and the steady-state Ca²⁺ level following resequestration of Ca²⁺ (following agonist addition). This value was then expressed as a percentage of total Ca²⁺ release by agonist. For figures a nonlinear scale was used to express amount of Ca²⁺, since this value was derived empirically. Control values for each drug treatment were obtained from separate aliquots of the same homogenate, rather than from the same samples prior to test administration.

[³H]Ryanodine Binding Assay. Sea urchin egg homogenates (2.5% v/v) were incubated with various concentrations of [³H]ryanodine (85 Ci/mmol) in intracellular medium for 16 to 18 h at room temperature either in the absence or presence of 500 µM 2-hydroxycarbazole. Reactions were terminated by rapid filtration through Whatman GF-B filters and bound radiolabel quantified by liquid scintillation counting. Nonspecific binding (typically 70-90% of total binding) was determined in the presence of 50 µM unlabeled ryanodine. The final concentration of vehicle (DMSO) was 1% (v/v).

Statistical Analysis. Data are expressed as mean ± S.E. of n values. Where appropriate statistical analysis was carried out with analysis of variance (ANOVA) and a post hoc Fisher's least-significant difference test.

	Results

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2-Hydroxycarbazole-Induced Ca²⁺ Release in Egg Homogenate. Figure 1A shows that 2-hydroxycarbazole potently released Ca²⁺ from egg homogenate in a concentration-dependent manner (EC₅₀ of approximately 200 µM and maximal release at approximately 500 µM). 2-Hydroxycarbazole-induced release appeared to be biphasic in nature, consisting of a short fast phase followed by a prolonged slow phase that eventually reached plateau. For all concentrations, released Ca²⁺ was resequestered into stores as determined by return to near basal fluo-3 fluorescence. For example, following addition of 500 µM 2-hydroxycarbazole to homogenate, 86.3 ± 4.4% (n = 3) of the released Ca²⁺ was resequestered. This compares to 79.4 ± 1.7 (n = 3) for 2 µM IP₃. No further resequestration of Ca²⁺ occurred 60 min after addition of 2-hydroxycarbazole to egg homogenates. Thus, in agreement with Tovey et al. (1998) this compound does not appear to inhibit the microsomal Ca²⁺-ATPase pump. Following resequestration of Ca²⁺ after treatment of homogenates with 500 µM 2-hydroxycarbazole, ionomycin (10 µM)-mediated release was 92.4 ± 3.6% (n = 4) of that in control homogenates. This further suggests that 2-hydroxycarbazole is not mediating a nonspecific effect on store loading. Changes in fluorescence upon addition of known amounts of Ca²⁺ were also unaltered following the reuptake that ensues after 2-hydroxycarbazole-induced release (data not shown), suggesting that 2-hydroxycarbazole does not interfere with changes in fluo-3 fluorescence.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Characterization of the Ca2+ mobilizing activity of 2-hydroxycarbazole in egg homogenate. Free ionized Ca2+ was measured with 3 µM fluo-3 (490-nm excitation and 535-nm emission) at 17°C, using 500 µl of homogenate. Additions were made in 5-µl volumes and changes in relative fluorescence units calibrated to known Ca2+ additions using separate samples of the same homogenate. A, representative fluorometric traces (each from six to nine determinations) of 2-hydroxycarbazole-induced Ca2+ release in sea urchin egg homogenates, illustrating concentration dependence of release. B, representative fluorometric trace of 2-hydroxycarbazole (500 µM)-induced release Ca2+ release. Following resequestration of Ca2+ the homogenate was desensitized to further additions of 2-hydroxycarbazole (500 µM). C, comparison of Ca2+ release by maximal concentrations of 2-hydroxycarbazole (500 µM), IP3 (1 µM), cADPR (100 nM), caffeine (10 mM), and NAADP (100 nM). Ca2+ release, calibrated from the peak increase in fluo-3 fluorescence, is expressed as a percentage of total release by the ionophore ionomycin (10 µM). Values are expressed as mean ± standard error of six to nine determinations. ANOVA, followed by Fisher's least-significant difference test was performed on the data. The least-significant difference (P < 0.01) = 9.17, as indicated by the inset bar.

Pharmacological Comparison of 2-Hydroxycarbazole-Evoked Release to That Induced by cADPR, IP₃, and NAADP. To characterize 2-hydroxycarbazole-mediated release its pharmacology was compared with that of cADPR-, IP₃-, and NAADP-induced Ca²⁺ release (Table 1). Many compounds are known to inhibit CICR (Palade et al., 1989; McPherson and Campbell, 1993). Tetracaine, ruthenium red, and Mg²⁺ are representative inhibitors of CICR and the effect of these agents on 2-hydroxycarbazole-induced release from the egg homogenate was examined. Figure 2A shows that whereas responses to cADPR and caffeine were inhibited by ruthenium red, that of 2-hydroxycarbazole together with IP₃ and NAADP was unaffected. Similarly, tetracaine inhibited both cADPR and caffeine but did not significantly inhibit the response to 2-hydroxycarbazole or IP₃ (Fig. 2B). However, as shown in Fig. 2B, tetracaine strongly inhibited Ca²⁺ release by NAADP. Mg²⁺ (10 mM) was found to strongly reduce Ca²⁺ release by cADPR and caffeine but had no effect on release by 2-hydroxycarbazole, IP₃, or NAADP (Fig. 2C).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effects of CICR inhibitors ruthenium red (50 µM) (A), tetracaine (500 µM) (B), and 10 mM Mg2+ (C) on Ca2+ release by 2-hydroxycarbazole (200 µM), IP3 (1 µM), cADPR (100 nM), caffeine (10 mM), and NAADP (100 nM). Homogenates were pretreated for 2 min with the respective inhibitor and then challenged with agonist. Control values for each drug treatment were obtained from separate aliquots of the same homogenate. Free ionized Ca2+ was measured with 3 µM fluo-3 (490-nm excitation and 535-nm emission) at 17°C, using 500 µl of homogenate. Additions were made in 5-µl volumes and changes in relative fluorescence units calibrated to known Ca2+ additions using separate samples of the same homogenate. Values are expressed as mean ± standard error of four to six determinations. *, statistical significance against control (100%) as determined by ANOVA with subsequent separation means with Fisher's least-significant difference test, P < 0.001.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effect of the competitive IP3 antagonist heparin (500 µg ml1) on Ca2+ release by 2-hydroxycarbazole (200 µM), IP3 (1 µM), cADPR (100 nM), caffeine (10 mM), and NAADP (100 nM). Homogenates were pretreated for 2 min with heparin and then challenged with agonist. Free ionized Ca2+ was measured with 3 µM fluo-3 (490-nm excitation and 535-nm emission) at 17°C, using 500 µl of homogenate. Additions were made in 5-µl volumes and changes in relative fluorescence units calibrated to known Ca2+ additions. Values are expressed as mean ± standard error of four to six determinations. *, statistical significance against control (100%) as determined by ANOVA with subsequent separation means with Fisher's least-significant difference test, P < 0.001.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effects of NAADP-sensitive release mechanism antagonists nifedipine (100 µM) (A) and BAY K 8644 (100 µM) (B) on Ca2+ release by 2-hydroxycarbazole (200 µM), IP3 (1 µM), cADPR (100 nM), caffeine (10 mM), and NAADP (100 nM). Homogenates were pretreated for 2 min with the respective inhibitor and then challenged with agonist. Free ionized Ca2+ was measured with 3 µM fluo-3 (490-nm excitation and 535-nm emission) at 17°C, using 500 µl of homogenate. Additions were made in 5-µl volumes and changes in relative fluorescence units calibrated to known Ca2+ additions. Values are expressed as mean ± standard error of four to six determinations. *, statistical significance against control (100%) as determined by ANOVA with subsequent separation means with Fisher's least-significant difference test, P < 0.001.

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2-Hydroxycarbazole-Mediated Release in Homogenates, Refractory to cADPR, IP₃, and NAADP. Following return to basal free Ca²⁺ levels after treatment with cADPR, homogenates were desensitized to subsequent additions of cADPR, as well as caffeine and ryanodine (Fig. 5D). However, pretreatment with cADPR did not desensitize the homogenate to 2-hydroxycarbazole (200 µM) (Fig. 5B). Likewise, after recovery from IP₃- and NAADP-mediated calcium release, homogenates were desensitized to subsequent additions of IP₃ and NAADP, respectively, but 2-hydroxycarbazole (200 µM) could still trigger large Ca²⁺ release (Fig. 5, A and C). The response to 200 µM 2-hydroxycarbazole after recovery from IP₃-, cADPR-, or NAADP-induced release was 93.3 ± 8.5% (n = 9), 98.9 ± 4.0% (n = 9), and 92.7 ± 7.6% (n = 9), respectively, compared with 2-hydroxycarbazole alone. Conversely, after treatment with 2-hydroxycarbazole (200 µM), NAADP, IP₃, and cADPR still induced significant Ca²⁺ release, with release being 97.1 ± 2.6% (n = 4), 93.0 ± 2.1% (n = 4), and 94.7 ± 1.6% (n = 4), respectively, of that found in separate aliquots of the same homogenate in the absence of 2-hydroxycarbazole.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   2-Hydroxycarbazole-mediated release in homogenates refractory to IP3, cADPR/ryanodine, and NAADP. Free ionized Ca2+ was measured with 3 µM fluo-3 (490-nm excitation and 535-nm emission) at 17°C, using 500 µl of homogenate. Additions were made in 5-µl volumes and changes in relative fluorescence units calibrated to known Ca2+ additions. Representative traces (of six to nine determinations) illustrating Ca2+ release by the approximate EC50 concentration of 2-hydroxycarbazole (200 µM), after return to basal calcium levels following addition of 1 µM IP3 (A), 500 nM cADPR (B), or 100 nM NAADP (C). Representative traces (of three determinations) illustrating desensitization of homogenate to caffeine (10 mM) and ryanodine (600 µM) following treatment with cADPR (500 nM) (D).

Caffeine-Mediated Inhibition of 2-Hydroxycarbazole Activity. The interaction between 2-hydroxycarbazole and caffeine was investigated. Caffeine (10 mM) was added to homogenate causing Ca²⁺ release and Ca²⁺ levels were allowed to return to basal levels. Subsequent addition of 200 µM 2-hydroxycarbazole failed to elicit Ca²⁺ release (Fig. 6A). To investigate whether the interaction between caffeine and 2-hydroxycarbazole was competitive in nature, the concentration of 2-hydroxycarbazole was increased in an attempt to recover Ca²⁺ release. Indeed, it was found that following treatment with caffeine (10 mM) the concentration-response curve of 2-hydroxycarbazole had shifted to the right with proportionate increase of both half-maximal and maximal release, activated by 500 µM and 1 mM 2-hydroxycarbazole, respectively (Fig. 6B). Maximal levels of Ca²⁺ release in the presence of caffeine constituted 82.5 ± 6.0% of control. In contrast to caffeine, following treatment with a supramaximal concentration of cADPR (1 µM) the concentration-response curve of 2-hydroxycarbazole was unaltered (approximate EC₅₀ of 200 µM and maximal release at 500 µM). These results suggest that caffeine is acting predominantly by competitively excluding 2-hydroxycarbazole from its binding site(s). However, the decreased maximal response (i.e., 82.5 ± 6.0% of control) may also indicate a minor noncompetitive inhibition by caffeine. Caffeine was also found to mediate an inhibitory effect on IP₃-mediated release, as observed in other systems, for example, Xenopus laevis oocytes (Berridge, 1991; Parker and Ivorra, 1991). Indeed, following addition of 10 mM caffeine and subsequent reuptake of released Ca²⁺, the amount of Ca²⁺ released by 1 µM IP₃ was reduced to 55 ± 3.1% (n = 3) of control. In contrast, caffeine did not alter the response of homogenate to NAADP (n = 3).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Caffeine-mediated inhibition of 2-hydroxycarbazole activity. Free ionized Ca2+ was measured with 3 µM fluo-3 (490-nm excitation and 535-nm emission) at 17°C, using 500 µl of homogenate. Additions were made in 5-µl volumes and changes in relative fluorescence units calibrated to known Ca2+ additions. A, representative fluorometric trace of 2-hydroxycarbazole (200 µM)-induced Ca2+ release following treatment with caffeine. B, concentration-response curve of 2-hydroxycarbazole in the presence and absence of 10 mM caffeine. These data are presented as the percentage of maximal Ca2+ release by control 2-hydroxycarbazole. Values are expressed as mean ± standard error of six to nine determinations.

2-Hydroxycarbazole-Mediated Ca²⁺ Release from a Thapsigargin-Insensitive Pool. The Ca²⁺ pools released by 2-hydroxycarbazole were investigated. This required the use of the potent and selective sarco(endo)plasmic reticulum Ca²⁺ pump inhibitor thapsigargin. Treatment of homogenate with a supramaximal concentration (10 µM) of this agent led to the Ca²⁺ level slowly rising to plateau value after which no resequestration was observed (Fig. 7). Subsequent Ca²⁺ release by cADPR (500 nM) and IP₃ (1 µM) was reduced to 14.5 ± 2.6% (n = 5) and 10.9 ± 1.0% (n = 4), respectively, of that in the absence of thapsigargin, whereas that by NAADP (500 nM) was not significantly affected [96.1 ± 5.5% (n = 5) of control]. Pretreatment with thapsigargin completely abolished release by caffeine (10 mM), however, release by 2-hydroxycarbazole (500 µM) was only reduced to approximately 64.7 ± 5.3% (n = 6) of control (Fig. 7). Therefore, in contrast to IP₃, cADPR and caffeine 2-hydroxycarbazole still activate substantial Ca²⁺ release after depletion of thapsigargin-sensitive stores.

View larger version (9K): [in this window] [in a new window]   Fig. 7.   2-hydroxycarbazole-mediated Ca2+ release from a thapsigargin-insensitive pool. Representative fluorometric trace of Ca2+ release by a maximal concentration of 2-hydroxycarbazole (500 µM) and caffeine (10 mM) after addition of 10 µM thapsigargin. Once thapsigargin-induced Ca2+ release reached a plateau, agonists were applied. Note that the time scale is different in the two parts of the figure to accentuate the different response of homogenate to caffeine and 2-hydroxycarbazole. Free ionized Ca2+ was measured with 3 µM fluo-3 (490-nm excitation and 535-nm emission) at 17°C, using 500 µl of homogenate. Additions were made in 5-µl volumes and changes in relative fluorescence units calibrated to known Ca2+ additions.

Effect of 2-Hydroxycarbazole on [³H]Ryanodine Binding to Sea Urchin Egg Homogenate. The plant alkaloid ryanodine binds preferentially to the open state of the RyR channel and its binding is enhanced by other modulators that induce channel opening. Consequently, measurement of [³H]ryanodine binding provides an extremely useful probe for activators of the RyR. As shown in Fig. 8, 2-hydroxycarbazole failed to enhance [³H]ryanodine binding to sea urchin egg homogenate. This is in contrast to caffeine (5 mM), which under identical experimental conditions enhanced ryanodine binding by approximately 2-fold (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Concentration-response curves of [3H]ryanodine binding to sea urchin egg homogenate in the presence () and absence () of 500 µM 2-hydroxycarbazole. Sea urchin egg homogenates (2.5% v/v) were incubated with [3H]ryanodine (85 Ci/mmol) for 16 to 18 h after which specific binding was determined in the presence of 50 µM unlabeled ryanodine. Values are expressed as mean ± standard error of three determinations.

	Discussion

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The intracellular stores of sea urchin eggs contain the three major pathways for Ca²⁺ mobilization found in mammalian cells, involving the endogenous messengers IP₃, cADPR, and NAADP, and thus provide a model system to characterize Ca²⁺ release mechanisms. In this study we demonstrate that 2-hydroxycarbazole specifically mobilizes Ca²⁺ in sea urchin egg homogenate with a similar potency to that previously reported by Tovey et al. (1998) in rat cardiac and skeletal muscle microsomes (EC₅₀ of approximately 200 µM). As in these systems, this compound was markedly more potent than caffeine, which induces half-maximal release in egg homogenate at concentrations between 3 and 5 mM (Galione et al., 1991). Furthermore, total Ca²⁺ release in sea urchin egg by maximal concentrations of 2-hydroxycarbazole was approximately 3 times greater than that induced by maximal caffeine. The occurrence of apparent desensitization together with resequestration of released calcium into stores confirms that 2-hydroxycarbazole is activating a specific Ca²⁺ release pathway rather than inducing nonspecific leakage.

Interestingly, the evidence presented in this study suggests that 2-hydroxycarbazole acts to modulate a Ca²⁺ release mechanism with distinct pharmacological properties to those previously reported in the sea urchin egg. In contrast to cADPR and caffeine, 2-hydroxycarbazole-activated Ca²⁺ release was insensitive to blockers of ryanodine-sensitive CICR tetracaine, ruthenium red, or Mg²⁺ (Palade et al., 1989; McPherson and Campbell, 1993). Also, nonactivating concentrations of cADPR, which strongly potentiate release by pharmacological modulators of CICR, for example, caffeine (Lee, 1993), had no effect on release by submaximal concentrations of 2-hydroxycarbazole. More direct evidence that the effect of 2-hydroxycarbazole is independent of CICR was its failure to potentiate Ca²⁺ release by the divalent cation strontium. Similarly, 2-hydroxycarbazole-mediated release was not amenable to pharmacological blockers of IP₃ or NAADP-activated release. Cross-desensitization studies confirmed that this compound is activating a distinct Ca²⁺ release mechanism to cADPR, as well as IP₃ and NAADP, since the response to 2-hydroxycarbazole was unaltered in homogenates refractory to each of these modulators. A further finding of the present study was that caffeine strongly inhibits release by 2-hydroxycarbazole. The interaction of caffeine with 2-hydroxycarbazole is of interest since 2-hydroxycarbazole is structurally related to 9-methyl-7-bromoeudistomin, a compound that occupies the putative caffeine-binding site on the RyR (Fang et al., 1993). Inhibition of 2-hydroxycarbazole activity by caffeine was found not to operate through RyR desensitization, since desensitization of the RyR with either ryanodine or cADPR did not preclude release by 2-hydroxycarbazole. Instead, the nature of caffeine-mediated inhibition was found to be predominantly competitive, as demonstrated by the restoration of near-maximal response by increasing 2-hydroxycarbazole concentration. Caffeine has previously been reported to inhibit IP₃-mediated release, for example, from X. laevis oocytes (Berridge, 1991; Parker and Ivorra, 1991) and rat cerebellar microsomes (Brown et al., 1992), and such an effect was also observed in the sea urchin egg.

The operation of a non-CICR mechanism in sea urchin egg is in variance to the proposed mechanism for 2-hydroxycarbazole-mediated release in cardiac and skeletal muscle, in which the response is blocked by the RyR inhibitors tetracaine and ruthenium red (Tovey et al., 1998). This might suggest activation of the RyR in a novel manner, independent of the CICR mechanism. Indeed, procaine, an analog of tetracaine, binds to the site that influences the Ca²⁺ sensitivity of the Ca²⁺ regulatory site and Mg²⁺ inhibits the Ca²⁺-gated open state of the channels by direct competition with Ca²⁺ regulatory site (Pessah et al., 1987). Ruthenium red, having a large positive charge (+6) has been reported to bind to the Ca²⁺ binding site of sarcoplasmic reticulum (Corbalan-Garcia et al., 1992), suggesting that the ruthenium red binding site is the Ca²⁺-binding site regulating CICR. Thus, inhibitors such as ruthenium red, tetracaine, and Mg²⁺ interfere with Ca²⁺ activation of the RyR and in turn might only inhibit agents such as cADPR and caffeine that modulate this process. Alternatively, the operation of a non-CICR mechanism by 2-hydroxycarbazole could suggest the activation of a Ca²⁺ release pathway in sea urchin egg, distinct from the RyR. In turn, this might suggest that the effects of 2-hydroxycarbazole in striated muscle are due to CICR secondary to 2-hydroxycarbazole-induced release from a different store, rather than direct activation of the RyR.

Analysis of the Ca²⁺ stores accessed by 2-hydroxycarbazole together with its effect on [³H]ryanodine binding provides further evidence for the operation of an entirely novel Ca²⁺ mobilization pathway. It has been recognized that the Ca²⁺ release mechanisms operating in the egg homogenate system can be dissected on the basis of sensitivity to the Ca²⁺-ATPase inhibitor thapsigargin (Genazzani and Galione, 1996). In agreement with Genazzani and Galione (1996), IP₃ and cADPR were shown to selectively release Ca²⁺ from thapsigargin-sensitive pools, whereas the Ca²⁺ release mechanism operated by NAADP acted on thapsigargin-insensitive stores. In contrast to caffeine, 2-hydroxycarbazole mediated significant release following thapsigargin treatment. Differential sensitivity to thapsigargin between 2-hydroxycarbazole and IP₃/cADPR suggests that the 2-hydroxycarbazole-mediated release mechanism is independent of those induced by these low molecular weight messengers. Moreover, the abolition of caffeine-induced Ca²⁺ release following the emptying of thapsigargin-sensitive pools is consistent with the RyR being predominantly located on thapsigargin-sensitive stores. This in turn implies that 2-hydroxycarbazole is activating a non-RyR release mechanism. Direct evidence for this is the inability of 2-hydroxycarbazole to potentiate [³H]ryanodine binding. Indeed, since ryanodine binds preferentially to the open state of the RyR channel, modulators that induce channel opening, for example, caffeine, serve to enhance binding. Thus, [³H]ryanodine binding studies provide a probe for activators of the RyR and the inability of 2-hydroxycarbazole to potentiate binding in sea urchin egg as well as mammalian tissue (Tovey et al., 1998), is inconsistent with the operation of a RyR mechanism.

To conclude, the data presented in the current study demonstrate that 2-hydroxycarbazole activates intracellular Ca²⁺ release in a manner distinct from the RyR/cADPR release mechanism, as well as that of IP₃ and NAADP. The sea urchin egg is already known to possess considerable redundancy in Ca²⁺ release and the existence of an additional Ca²⁺ release pathway would serve to further underlie the complexity of Ca²⁺ mobilization in this system. It is likely that the different release pathways act to coordinate the Ca²⁺ signaling phenotype during fertilization and we can speculate that if the release pathway modulated by 2-hydroxycarbazole is via a novel Ca²⁺ release channel then this, too, may participate in the regulation of this process.