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Elucidation of the ryanodine-sensitive Ca2+ release mechanism of rat pancreatic acinar cells: modulation by cyclic ADP-ribose and FK506
Biochimica et Biophysica Acta 1693 (2004) 159 – 166 www.bba-direct.com
Elucidation of the ryanodine-sensitive Ca2+ release mechanism of rat pancreatic acinar cells: modulation by cyclic ADP-ribose and FK506 Terutaka Ozawa * Department of Physiology, Graduate School of Medicine, Tohoku University, Seiryo-machi 2-1, Aoba-ku, Sendai 980-8575, Japan Received 18 August 2003; received in revised form 1 March 2004; accepted 16 April 2004 Available online 6 May 2004
Abstract The effects of cyclic ADP-ribose (cADPR) and the immunosuppressant drug FK506 on microsomal Ca2 + release through a ryanodinesensitive mechanism were investigated in rat pancreatic acinar cells. After a steady state of 45Ca2 + uptake into the microsomal vesicles, ryanodine or caffeine was added. Preincubation of the vesicles with cADPR (0.5 AM) shifted the dose – response curve of ryanodine- or caffeine-induced 45Ca2 + release from the vesicles to the left. Preincubation with cADPR shifted the dose – response curve of the FK506induced 45Ca2 + release upward. Preincubation with FK506 (3 AM) shifted the dose – response curve of the ryanodine- or caffeine-induced 45 Ca2 + release to the left by the same extent as that in the case of cADPR. FK506 shifted the dose – response curve of the cADPR-induced 45 Ca2 + release upward. The presence of both cADPR and FK506 enhanced the ryanodine (30 AM)- or caffeine (10 mM)-induced 45Ca2 + release by the same extent as that in the case of cADPR alone or FK506 alone. These results indicate that cADPR and FK506 modulate the ryanodine-sensitive Ca2 + release mechanism of rat pancreatic acinar cells by increasing the ryanodine or caffeine sensitivity to the mechanism. In addition, there is a possibility that the mechanisms of modulation by cADPR and FK506 are the same. D 2004 Elsevier B.V. All rights reserved. Keywords: Caffeine; Cyclic ADP-ribose; FK506; Ryanodine; Ryanodine receptor; Pancreatic acinar cell
1. Introduction In excitable cells, the ryanodine-sensitive Ca2 + release mechanism (ryanodine receptor: RyR), which is activated by ryanodine, caffeine or Ca2 +, has been well characterized, and the channel protein has been purified [1,2] and cloned [3,4]. The ryanodine-sensitive mechanism in nonexcitable cells such as liver cells and exocrine cells has also been characterized [5– 11]. The endogenous NAD+ metabolite cyclic ADP-ribose (cADPR), which is thought to be an intracellular messenger in addition to inositol 1,4,5-trisphosphate (IP3) [12], induces Ca2 + release from RyRs in sea urchin eggs [13,14], cardiac muscle cells [15], brain cells [16] and pancreatic h cells Abbreviations: cADPR, cyclic ADP-ribose; ER, endoplasmic reticulum; FKBP, FK506-binding protein; IP3, inositol 1,4,5-trisphosphate; SR, sarcoplasmic reticulum; RyR, ryanodine receptor * Fax: +81-22-717-8098. E-mail address: [email protected] (T. Ozawa). 0167-4889/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2004.04.003
[17]. It is also known that a low concentration of cADPR can modulate the RyR [8,18]. Two specific cADPR-binding proteins have been identified in sea urchin eggs [19]. However, it was suggested from the measurement of molecular mass that these proteins are different from RyR proteins. Little is known about the action of cADPR on the Ca2 + channel. The immunosuppressant drug FK506 has been shown to modulate RyRs [20,21]. In the skeletal muscle sarcoplasmic reticulum (SR), the RyR is known to be tightly associated with a 12-kDa binding protein [22,23]. The 12-kDa binding protein is a cytosolic receptor for FK506 and is referred to as FK506-binding protein (FKBP12). In association with RyR, FKBP12 has been shown to stabilize the closed conformation of the Ca2 + release channel [20], and FK506 has been shown to promote dissociation of FKBP12 from the RyR complex [20]. Compared with control SR vesicles, FKBP12deficient SR vesicles have been shown to increase caffeine sensitivity to caffeine-induced Ca2 + release [20] and to increase open probability and mean open times for single
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channel recordings of the receptor [24]. In cardiac muscle cells, FKBP12.6 binds to the RyR [25,26]. It has been suggested that cADPR can dissociate FKBP12.6 from the RyR in rat pancreatic islets [27]. Although FK506 has been reported to affect [3H]ryanodine binding to the RyR in nonexcitable cells [28,29], details of the FK506-dependent mechanism, including the presence of FKBP, are unclear. It has been demonstrated that the ryanodine-sensitive Ca2 + release mechanism, which is activated by Ca2 +, caffeine, ryanodine or cADPR, operates in the endoplasmic reticulum (ER) of rat pancreatic acinar cells [7,10,30]. In the present study, modulatory effects of cADPR and FK506 on the ryanodine-sensitive Ca2 + channel in rat pancreatic acinar cells were investigated by using isolated microsomal vesicles preloaded with 45Ca2 +. It was found that cADPR and FK506 shift the dose –response curve of ryanodine- or caffeine-induced 45Ca2 + release from pancreatic microsomal vesicles to the left and increase ryanodine or caffeine sensitivity to the RyR.
incubation buffer containing: 155 mM KCl, 5 mM HEPES, 0.15 mM CaCl2 (corresponding to 2 AM free Ca2 + concentration), 1.0 mM EDTA, 3.57 mM MgCl2 (corresponding to 1.0 mM free Mg2 + concentration), 10 mM NaN3, 5 AM oligomycin, 5 Ag/ml antimycin A, 10 mM creatine phosphate, 8U/ml creatine kinase, and 1 ACi/ml of 45CaCl2, adjusted with Tris/HCl to pH 7.0 at 25 jC. 45Ca2 + uptake into the vesicles was initiated by the addition of K2ATP at a
2. Materials and methods 2.1. Materials Creatine kinase and trypsin inhibitor were obtained from Boehringer Mannheim (Mannheim, Germany). Adenosine trisphosphate dipotassium salt (K2ATP), creatine phosphate disodium salt and benzamidine were purchased from Sigma Chemical (St. Louis, MO, USA). FK506 was kindly provided by Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan). cADPR was obtained from Wako Pure Chemical (Osaka, Japan). Ryanodine was obtained from Calbiochem (La Jolla, CA, USA). 45CaCl2 (14 –15 Ci/g) was purchased from New England Nuclear (Boston, MA, USA). 2.2. Preparation of microsomal vesicles Pancreatic microsomal vesicles were prepared as described previously [7,31,32]. Briefly, isolated acinar cells from male Wistar rats (180 –200 g) were homogenized in an ice-cold ‘‘mannitol buffer’’ containing: 290 mM mannitol, 10 mM KCl, 5 mM HEPES, 1 mM MgCl2, 1 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride, and 20 Ag/ml of trypsin inhibitor, adjusted with Tris to pH 7.0. After centrifugation of the cell homogenate at 11,000 g for 15 min, the ‘‘fluffy layer’’ on top of the pellet, which is enriched by about twofold in the ER [31], was collected. The microsomal vesicles were kept frozen in liquid nitrogen until use. The protein concentration was measured by the method of Bradford [33] using bovine serum albumin as a standard. 2.3. Measurement of
45
Ca2+ uptake
Isolated membrane vesicles were preincubated for 15 min at a protein concentration of 1.0 mg/ml in 0.6 ml of an
Fig. 1. Effect of cADPR (0.5 AM) on ryanodine-induced 45Ca2 + release from pancreatic microsomes. (A) Typical curves for ryanodine (10 AM)induced 45Ca2 + release from microsomal vesicles in the absence and presence of cADPR (0.5 AM). The vesicles (1 mg/ml) were preincubated in 0.6 ml of a KCl buffer containing 45CaCl2 (1 ACi/ml) for 15 min. 45Ca2 + uptake was initiated by the addition of 2 mM K2ATP (at 0 min). Ryanodine (Ry., 10 AM) was added to the vesicles as indicated in the absence (Control; o) and presence (.) of cADPR. cADPR or the same volume of H2O as a control was added at the beginning of the preincubation. (B) Dose – response curves of ryanodine (0.1 – 500 AM)-induced 45Ca2 + release from the vesicles in the absence (Control; o) and presence (.) of cADPR (0.5 AM). The vertical line represents the ratio of released 45Ca2 + over a 6-min period after the addition of ryanodine to the 45Ca2 + that had been taken up by an ATP-dependent mechanism. Each point is the mean F S.E. from 5 to 10 experiments of the type shown in (A). Note that cADPR shifted the dose – response curve of the ryanodine-induced 45Ca2 + release to the left. Asterisks indicate the level of significant difference compared to the control without cADPR at the corresponding ryanodine concentration. *P < 0.05, **P < 0.01, ***P < 0.001.
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final concentration of 2 mM. After a steady state of 45Ca2 + uptake had been reached, ryanodine, caffeine, cADPR or FK506 was added to the medium. The drug-induced 45Ca2 + release from the vesicles in the absence and that in the presence of either cADPR or FK506 were compared. FK506 was dissolved in DMSO and was added from the stock solution in a volume not exceeding 0.5%. For a control, the vesicles were preincubated with the same volume of a solvent (H2O or DMSO) instead of cADPR or FK506, respectively. At indicated times, 45Ca2 + content of membrane vesicles was determined by a rapid filtration technique as described previously [8]. The radioactivity was counted in a liquid scintillation counter (LS6500, Beckman). Values are presented as means F S.E. Statistical analysis was performed using Student’s t-test.
3. Results 3.1. Modulation of the ryanodine-sensitive mechanism by cADPR A steady state of 45Ca2 + uptake into pancreatic microsomal vesicles was reached about 20 min after the addition of ATP (Fig. 1A). The amount of 45Ca2 + taken up into the microsomal vesicles over a period of 20 min after the addition of ATP was 4.3 F 0.1 nmol/mg protein (n = 173). Ryanodine (10 AM) was added to the vesicles after a steady state of ATP-dependent 45Ca2 + uptake had been reached, and the effect of cADPR (0.5 AM) on ryanodine (10 AM)Fig. 3. Effect of cADPR (0.5 AM) on FK506-induced 45Ca2 + release from pancreatic microsomes. (A) Typical curves for FK506 (3 AM)-induced 45 Ca2 + release from the microsomal vesicles in the absence and presence of cADPR (0.5 AM). The experimental procedures were the same as those described in the legend to Fig. 1A except for the addition of FK506. FK506 (3 AM) was added to the vesicles as indicated in the absence (Control; o) and presence (.) of cADPR. (B) Dose – response curves of FK506 (0.1 – 10 AM)-induced 45Ca2 + release from the vesicles in the absence (o) and presence (.) of cADPR (0.5 AM). The vertical line represents the ratio of released 45Ca2 + as described in the legend to Fig. 1B. Each point is the mean F S.E. from 4 to 11 experiments of the type shown in (A). Note that cADPR shifted the dose – response curve of the FK506-induced 45Ca2 + release upward. Asterisks indicate the level of significant difference compared to the control without cADPR at the corresponding FK506 concentration. **P < 0.01, ***P < 0.001. Lineweaver – Burk plot analysis of the FK506-induced 45Ca2 + release (inset) yielded Km values of 0.47 and 0.70 AM and Vmax values of 8.1% and 14.4% in the absence (o) and presence (.) of cADPR, respectively. Fig. 2. Dose – response curves of caffeine (1 – 30 mM)-induced 45Ca2 + release from pancreatic microsomes in the absence (Control; o) and presence (.) of cADPR (0.5 AM). The experimental procedures were the same as those described in the legend to Fig. 1 except for the addition of caffeine. The vertical line represents the ratio of released 45Ca2 + as described in the legend to Fig. 1B. Each point is the mean F S.E. from 4 to 12 experiments. Note that cADPR shifted the dose – response curve of the caffeine-induced 45Ca2 + release to the left. Asterisks indicate the level of significant difference compared to the control without cADPR at the corresponding caffeine concentration. *P < 0.05, **P < 0.01, ***P < 0.001.
induced 45Ca2 + release from the vesicles was investigated. Pretreatment of the vesicles with cADPR did not affect the ATP-dependent 45Ca2 + uptake before the addition of ryanodine (Fig. 1A) but significantly increased the ryanodine (10 AM)-induced 45Ca2 + release (Fig. 1A). The effect of cADPR (0.5 AM) on the 45Ca2 + release induced by ryanodine (0.1 –500 AM) is shown at each ryanodine concentration in Fig. 1B. Ryanodine was effective at 0.3 AM in the presence of cADPR, and the ryanodine-induced release at
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release from the vesicles was also investigated. When FK506 (3 AM) was added to the vesicles in a steady state of the ATP-dependent 45Ca2 + uptake, about 5% of the 45 Ca2 + that had been taken up by ATP was released from the vesicles (Fig. 3A). The presence of cADPR (0.5 AM) significantly enhanced the FK506 (3 AM)-induced 45Ca2 + release (Fig. 3A). The effect of cADPR (0.5 AM) on the FK506 (0.1 – 10 AM)-induced 45Ca2 + release is shown at each FK506 concentration in Fig. 3B. cADPR shifted the dose – response curve of the FK506-induced 45Ca2 + release upward. Lineweaver –Burk plot evaluation of the release (Fig. 3B, inset) showed Km values of 0.47 and 0.70 AM and Vmax values of 8.1% and 14.4% in the absence and presence of cADPR, respectively. 3.2. Modulation of the ryanodine-sensitive mechanism by FK506 The effect of FK506 on ryanodine-induced 45Ca2 + release from the vesicles was investigated. Preincubation of the vesicles with FK506 (3 AM) did not affect the ATPdependent 45Ca2 + uptake (Fig. 4A) but significantly increased the ryanodine (10 AM)-induced 45Ca2 + release (Fig. 4A). The effect of FK506 (3 AM) on the ryanodine (0.1 – 500 AM)-induced 45Ca2 + release is shown in Fig. 4B. In the presence of FK506, ryanodine was effective at 0.3 AM as it was in the presence of cADPR (cf. Figs. 1B and 4B). FK506 significantly ( P < 0.05– 0.001) increased the ryanodine-induced release at concentrations ranging from 1 to 100 AM (Fig. 4B) and shifted the dose – response curve of the Fig. 4. Effect of FK506 (3 AM) on ryanodine-induced 45Ca2 + release from pancreatic microsomes. (A) Typical curves for ryanodine (10 AM)-induced 45 Ca2 + release from the microsomal vesicles in the absence and presence of FK506 (3 AM). The experimental procedures were the same as those described in the legend to Fig. 1A except for the preincubation with FK506. Ryanodine (Ry., 10 AM) was added to the vesicles as indicated in the absence (Control; o) and presence (.) of FK506. FK506 or the same volume of DMSO as a control was added at the beginning of the preincubation. (B) Dose – response curves of ryanodine (0.1 – 500 AM)induced 45Ca2 + release from the vesicles in the absence (Control; o) and presence (.) of FK506 (3 AM). The vertical line represents the ratio of released 45Ca2 + as described in the legend to Fig. 1B. Each point is the mean F S.E. from 5 to 9 experiments of the type shown in (A). Note that FK506 shifted the dose – response curve of the ryanodine-induced 45Ca2 + release to the left. Asterisks indicate the level of significant difference compared to the control without FK506 at the corresponding ryanodine concentration. *P < 0.05, **P < 0.01, ***P < 0.001.
concentrations ranging from 1 to 100 AM was significantly ( P < 0.05 – 0.001) stimulated by the presence of cADPR (Fig. 1B). cADPR shifted the dose –response curve of the ryanodine-induced 45Ca2 + release to the left (Fig. 1B). The effect of cADPR (0.5 AM) on caffeine (1 –30 mM)-induced 45 Ca2 + release from the vesicles was also investigated. cADPR significantly ( P < 0.05 –0.001) increased the release at lower concentrations (1 –10 mM) of caffeine and shifted the dose – response curve of the caffeine-induced 45Ca2 + release to the left (Fig. 2). The effect of FK506 on 45Ca2 +
Fig. 5. Dose – response curves of caffeine (1 – 30 mM)-induced 45Ca2 + release from pancreatic microsomes in the absence (Control; o) and presence (.) of FK506 (3 AM). The experimental procedures were the same as those described in the legend to Fig. 4 except for the addition of caffeine. The vertical line represents the ratio of released 45Ca2 + as described in the legend to Fig. 1B. Each point is the mean F S.E. from 4 to 12 experiments. Note that FK506 shifted the dose – response curve of the caffeine-induced 45 Ca2 + release to the left. Asterisks indicate the level of significant difference compared to the control without FK506 at the corresponding caffeine concentration. *P < 0.05, **P < 0.01, ***P < 0.001.
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ryanodine-induced 45Ca2 + release to the left by the same extent as that in the case of cADPR (cf. Figs. 1B and 4B). The effect of FK506 (3 AM) on caffeine (1 – 30 mM)induced 45Ca2 + release from the vesicles was also investigated. FK506 significantly ( P < 0.05 –0.001) enhanced the release at lower concentrations (1– 10 mM) of caffeine (Fig. 5) and shifted the dose – response curve of the caffeineinduced 45Ca2 + release to the left by the same extent as that in the case of cADPR (cf. Figs. 2 and 5). The effect of FK506 (3 AM) on the 45Ca2 + release induced by cADPR (0.5 –10 AM) was also investigated. The presence of FK506 shifted the dose – response curve of the cADPR-induced 45 Ca2 + release upward (Fig. 6). Lineweaver – Burk plot analysis of the release (Fig. 6, inset) showed that FK506
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increased the Vmax value from 8.7% to 16.4% without affecting the Km value (1.7 AM). 3.3. Modulation of the ryanodine-sensitive mechanism by both cADPR and FK506 To determine whether the stimulatory effect on ryanodine- or caffeine-induced 45Ca2 + release from the vesicles by cADPR and that by FK506 are additive, the ryanodine (30 AM)- or caffeine (10 mM)-induced 45Ca2 + release was investigated in the presence of both cADPR (0.5 AM) and FK506 (3 AM). The presence of both cADPR and FK506 enhanced the ryanodine (30 AM)- or caffeine (10 mM)induced 45Ca2 + release by the same extent as that in the case
Fig. 6. Dose – response curves of cADPR (0.5 – 10 AM)-induced 45Ca2 + release from pancreatic microsomes in the absence (o) and presence (.) of FK506 (3 AM). The experimental procedures were the same as those described in the legend to Fig. 4 except for the addition of cADPR. The vertical line represents the ratio of released 45Ca2 + over an 11-min period after the addition of cADPR to the 45Ca2 + that had been taken up by ATP. Each point is the mean F S.E. from 4 to 9 experiments. Note that FK506 shifted the dose – response curve of the cADPR-induced 45Ca2 + release upward. Asterisks indicate the level of significant difference compared to the control without FK506 at the corresponding cADPR concentration. *P < 0.05, **P < 0.01. Lineweaver – Burk plot analysis of the cADPR-induced 45Ca2 + release (inset) yielded Km values of 1.7 and 1.7 AM and Vmax values of 8.7% and 16.4% in the absence (o) and presence (.) of FK506, respectively.
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Fig. 7. Effects of cADPR (0.5 AM), FK506 (3 AM) and cADPR + FK506 on 45 Ca2 + release from pancreatic microsomes induced by ryanodine (30 AM) or caffeine (10 mM). The experimental procedures were the same as those described in the legends to (Figs. 1, 2, 4 and 5). cADPR, FK506 and the same volume of solvents (H2O/DMSO) as a control were added at the beginning of the preincubation. The 45Ca2 + release induced by ryanodine or caffeine in the absence (Control) and that in the presence of cADPR alone, FK506 alone or both cADPR and FK506 were compared. The vertical line represents the ratio of released 45Ca2 + as described in the legend to Fig. 1B. Each value is the mean F S.E. from four paired experiments. Note that the ryanodine- or caffeine-induced 45Ca2 + release in the presence of both cADPR and FK506 was nearly the same as that in the presence of either cADPR or FK506.
of cADPR alone or FK506 alone (Fig. 7). Since the stimulatory effects on the release by cADPR and by FK506 were not additive, it is possible that cADPR and FK506 modulate the ryanodine-sensitive Ca2 + channel by the same mechanism.
4. Discussion In the present study, it was demonstrated that cADPR and FK506 modulate the ryanodine-sensitive Ca2 + channels of rat pancreatic acinar cells by increasing the ryanodine or caffeine sensitivity to the channel (Figs. 1, 2, 4 and 5). In addition, it is possible that the mechanisms of modulation by cADPR and by FK506 are the same, since cADPR shifted the dose – response curve of the ryanodine- or caffeine-induced 45Ca2 + release to the left by the same extent as that in the case of FK506 (cf. Figs. 1B and 4B or cf. Figs. 2 and 5, respectively), and the stimulatory effects on ryanodine (30 AM)- or caffeine (10 mM)-induced 45Ca2 + release by cADPR and by FK506 were not additive (Fig. 7). To the author’s knowledge, this is the first report of FK506 sensitivity to Ca2 + release from microsomes prepared from nonexcitable tissues. cADPR not only activates the ryanodine-sensitive Ca2 + channel [13 –17] but also modulates the Ca2 + channel by increasing the Ca2 + or caffeine sensitivity in sea urchin eggs [18] and rat parotid glands [8]. In the modulator model
proposed by Lee et al. [34], cADPR modulates the Ca2 + or caffeine effect on the RyR at an endogenous level. The cADPR concentration (0.5 AM) used for the modulation of ryanodine- or caffeine-induced 45Ca2 + release from rat pancreatic microsomal vesicles is nearly the same as that reported to be endogenous levels (0.2 – 0.68 AM cADPR) in the rat brain, heart and liver tissues [35]. Regarding the action of cADPR on the receptor, two specific cADPRbinding proteins in sea urchin eggs have been identified [19]. Molecular masses of the proteins (140 and 100 kDa) are less than that of known RyR proteins (380 kDa). This finding suggests that cADPR receptors are unknown variants of RyR proteins or RyR accessory proteins. FK506 is known to enhance Ca2 + release through the RyR in skeletal muscle [20,21]. FK506 dissociates FKBP12 from the RyR – FKBP12 complex [20]. By the removal of this accessory protein, the RyR exhibits subconductance states [21], and the Ca2 + or caffeine sensitivity of the channel is enhanced [20,24]. The FK506 concentration (3 AM) used for the modulation of the ryanodine-sensitive mechanism in rat pancreatic acinar cells (Figs. 4 – 6) is nearly the same as that required to dissociate FKBP12 from the RyR complex in skeletal muscle [20]. Type 2 (cardiac type) RyR associates FKBP12.6 as an accessory protein [25 – 27]. Since a type 2 RyR isoform has been identified in rat pancreatic acinar cells [36,37], FKBP12.6 may be involved in the modulation by FK506 of Ca2 + release through the RyR of pancreatic acinar cells. It has been found that cADPR can bind to FKBP12.6 and dissociate FKBP12.6 from pancreatic islet microsomes [27]. An antibody against FKBP12.6 has been shown to inhibit activation of the ryanodine-sensitive Ca2 + release channel induced not only by FK506 but also by cADPR in reconstituted vesicles of coronary arterial smooth muscle cells [38]. These findings suggest that cADPR can dissociate FKBP12.6 from the RyR – FKBP12.6 complex to activate or modulate the Ca2 + channel. Since cADPR can play the same role in the modulation of Ca2 + release through the RyR in rat pancreatic acinar cells as FK506 can (Figs. 1, 2, 4 and 5), it is possible that cADPR modulates the RyR by dissociating FKBP from the RyR. If cADPR modulates the RyR by the same mechanism as that by which FK506 modulates the RyR, this can explain why the stimulatory effect on ryanodine- or caffeine-induced 45Ca2 + release from the vesicles by cADPR and that by FK506 were not additive (Fig. 7). However, it is doubtful whether the cADPR-induced 45 Ca2 + release following pretreatment of the vesicles with FK506 shown in Fig. 6 is due to the dissociation of FKBP since it is thought that FKBP had been removed from the RyR by FK506 before the addition of cADPR. cADPRinduced responses caused by the dissociation of FKBP should be reduced in the presence of FK506. Actually, pretreatment with FK506 abolished the cADPR-induced Ca2 + release from pancreatic islet microsomes [27] and inhibited the cADPR-induced increase in open probability
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of the ryanodine-sensitive Ca2 + channel in coronary arterial smooth muscle cells [38], while the presence of FK506 in rat pancreatic acinar cells stimulated the cADPR-induced 45 Ca2 + release from the vesicles (Fig. 6). Binding of 32Plabeled cADPR to sea urchin egg homogenates was not affected by the presence of FK506 (30 AM) [39]. This finding suggests that cADPR-binding proteins of sea urchin eggs are not FKBPs. An FKBP-independent mechanism may be involved in the cADPR-induced 45Ca2 + release shown in Fig. 6. The FK506-induced 45Ca2 + release following pretreatment with cADPR shown in Fig. 3 may also not be due to the dissociation of FKBP as in the case of the results shown in Fig. 6. FK506 is a compound with a macrocyclic lactone ring structure. It has been shown that rapamycin and ivermectin, macrocyclic lactone derivatives, increased the open probability of FKBP12-stripped RyRs in skeletal muscle [40,41]. This finding suggests that the compounds activate the RyR by a mechanism other than dissociation of FKBP. Avermectin B1a, another macrocyclic lactone compound, is known to bind to the GABA receptoractivated Cl channel [42,43]. The FK506-induced 45Ca2 + release from the vesicles shown in Fig. 3 may be explained by a direct activation of the RyR by FK506. Since the cADPR- and FK506-induced 45Ca2 + release from the vesicles was stimulated by the presence of FK506 and cADPR, respectively (Figs. 3 and 6), the release induced by cADPR or FK506 may be modulated by the dissociation of FKBP from the RyR. Further studies are needed to clarify what subtype of FKBP is involved in modulation of the RyR in rat pancreatic acinar cells and to obtain detailed information on the actions of cADPR and FK506 on the RyR.
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Acknowledgements I am grateful to Fujisawa Pharmaceutical Co., Ltd. for providing FK506.
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Elucidation of the ryanodine-sensitive Ca2+ release mechanism of rat pancreatic acinar cells: modulation by cyclic ADP-ribose and FK506 Terutaka Ozawa * Department of Physiology, Graduate School of Medicine, Tohoku University, Seiryo-machi 2-1, Aoba-ku, Sendai 980-8575, Japan Received 18 August 2003; received in revised form 1 March 2004; accepted 16 April 2004 Available online 6 May 2004
Abstract The effects of cyclic ADP-ribose (cADPR) and the immunosuppressant drug FK506 on microsomal Ca2 + release through a ryanodinesensitive mechanism were investigated in rat pancreatic acinar cells. After a steady state of 45Ca2 + uptake into the microsomal vesicles, ryanodine or caffeine was added. Preincubation of the vesicles with cADPR (0.5 AM) shifted the dose – response curve of ryanodine- or caffeine-induced 45Ca2 + release from the vesicles to the left. Preincubation with cADPR shifted the dose – response curve of the FK506induced 45Ca2 + release upward. Preincubation with FK506 (3 AM) shifted the dose – response curve of the ryanodine- or caffeine-induced 45 Ca2 + release to the left by the same extent as that in the case of cADPR. FK506 shifted the dose – response curve of the cADPR-induced 45 Ca2 + release upward. The presence of both cADPR and FK506 enhanced the ryanodine (30 AM)- or caffeine (10 mM)-induced 45Ca2 + release by the same extent as that in the case of cADPR alone or FK506 alone. These results indicate that cADPR and FK506 modulate the ryanodine-sensitive Ca2 + release mechanism of rat pancreatic acinar cells by increasing the ryanodine or caffeine sensitivity to the mechanism. In addition, there is a possibility that the mechanisms of modulation by cADPR and FK506 are the same. D 2004 Elsevier B.V. All rights reserved. Keywords: Caffeine; Cyclic ADP-ribose; FK506; Ryanodine; Ryanodine receptor; Pancreatic acinar cell
1. Introduction In excitable cells, the ryanodine-sensitive Ca2 + release mechanism (ryanodine receptor: RyR), which is activated by ryanodine, caffeine or Ca2 +, has been well characterized, and the channel protein has been purified [1,2] and cloned [3,4]. The ryanodine-sensitive mechanism in nonexcitable cells such as liver cells and exocrine cells has also been characterized [5– 11]. The endogenous NAD+ metabolite cyclic ADP-ribose (cADPR), which is thought to be an intracellular messenger in addition to inositol 1,4,5-trisphosphate (IP3) [12], induces Ca2 + release from RyRs in sea urchin eggs [13,14], cardiac muscle cells [15], brain cells [16] and pancreatic h cells Abbreviations: cADPR, cyclic ADP-ribose; ER, endoplasmic reticulum; FKBP, FK506-binding protein; IP3, inositol 1,4,5-trisphosphate; SR, sarcoplasmic reticulum; RyR, ryanodine receptor * Fax: +81-22-717-8098. E-mail address: [email protected] (T. Ozawa). 0167-4889/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2004.04.003
[17]. It is also known that a low concentration of cADPR can modulate the RyR [8,18]. Two specific cADPR-binding proteins have been identified in sea urchin eggs [19]. However, it was suggested from the measurement of molecular mass that these proteins are different from RyR proteins. Little is known about the action of cADPR on the Ca2 + channel. The immunosuppressant drug FK506 has been shown to modulate RyRs [20,21]. In the skeletal muscle sarcoplasmic reticulum (SR), the RyR is known to be tightly associated with a 12-kDa binding protein [22,23]. The 12-kDa binding protein is a cytosolic receptor for FK506 and is referred to as FK506-binding protein (FKBP12). In association with RyR, FKBP12 has been shown to stabilize the closed conformation of the Ca2 + release channel [20], and FK506 has been shown to promote dissociation of FKBP12 from the RyR complex [20]. Compared with control SR vesicles, FKBP12deficient SR vesicles have been shown to increase caffeine sensitivity to caffeine-induced Ca2 + release [20] and to increase open probability and mean open times for single
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channel recordings of the receptor [24]. In cardiac muscle cells, FKBP12.6 binds to the RyR [25,26]. It has been suggested that cADPR can dissociate FKBP12.6 from the RyR in rat pancreatic islets [27]. Although FK506 has been reported to affect [3H]ryanodine binding to the RyR in nonexcitable cells [28,29], details of the FK506-dependent mechanism, including the presence of FKBP, are unclear. It has been demonstrated that the ryanodine-sensitive Ca2 + release mechanism, which is activated by Ca2 +, caffeine, ryanodine or cADPR, operates in the endoplasmic reticulum (ER) of rat pancreatic acinar cells [7,10,30]. In the present study, modulatory effects of cADPR and FK506 on the ryanodine-sensitive Ca2 + channel in rat pancreatic acinar cells were investigated by using isolated microsomal vesicles preloaded with 45Ca2 +. It was found that cADPR and FK506 shift the dose –response curve of ryanodine- or caffeine-induced 45Ca2 + release from pancreatic microsomal vesicles to the left and increase ryanodine or caffeine sensitivity to the RyR.
incubation buffer containing: 155 mM KCl, 5 mM HEPES, 0.15 mM CaCl2 (corresponding to 2 AM free Ca2 + concentration), 1.0 mM EDTA, 3.57 mM MgCl2 (corresponding to 1.0 mM free Mg2 + concentration), 10 mM NaN3, 5 AM oligomycin, 5 Ag/ml antimycin A, 10 mM creatine phosphate, 8U/ml creatine kinase, and 1 ACi/ml of 45CaCl2, adjusted with Tris/HCl to pH 7.0 at 25 jC. 45Ca2 + uptake into the vesicles was initiated by the addition of K2ATP at a
2. Materials and methods 2.1. Materials Creatine kinase and trypsin inhibitor were obtained from Boehringer Mannheim (Mannheim, Germany). Adenosine trisphosphate dipotassium salt (K2ATP), creatine phosphate disodium salt and benzamidine were purchased from Sigma Chemical (St. Louis, MO, USA). FK506 was kindly provided by Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan). cADPR was obtained from Wako Pure Chemical (Osaka, Japan). Ryanodine was obtained from Calbiochem (La Jolla, CA, USA). 45CaCl2 (14 –15 Ci/g) was purchased from New England Nuclear (Boston, MA, USA). 2.2. Preparation of microsomal vesicles Pancreatic microsomal vesicles were prepared as described previously [7,31,32]. Briefly, isolated acinar cells from male Wistar rats (180 –200 g) were homogenized in an ice-cold ‘‘mannitol buffer’’ containing: 290 mM mannitol, 10 mM KCl, 5 mM HEPES, 1 mM MgCl2, 1 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride, and 20 Ag/ml of trypsin inhibitor, adjusted with Tris to pH 7.0. After centrifugation of the cell homogenate at 11,000 g for 15 min, the ‘‘fluffy layer’’ on top of the pellet, which is enriched by about twofold in the ER [31], was collected. The microsomal vesicles were kept frozen in liquid nitrogen until use. The protein concentration was measured by the method of Bradford [33] using bovine serum albumin as a standard. 2.3. Measurement of
45
Ca2+ uptake
Isolated membrane vesicles were preincubated for 15 min at a protein concentration of 1.0 mg/ml in 0.6 ml of an
Fig. 1. Effect of cADPR (0.5 AM) on ryanodine-induced 45Ca2 + release from pancreatic microsomes. (A) Typical curves for ryanodine (10 AM)induced 45Ca2 + release from microsomal vesicles in the absence and presence of cADPR (0.5 AM). The vesicles (1 mg/ml) were preincubated in 0.6 ml of a KCl buffer containing 45CaCl2 (1 ACi/ml) for 15 min. 45Ca2 + uptake was initiated by the addition of 2 mM K2ATP (at 0 min). Ryanodine (Ry., 10 AM) was added to the vesicles as indicated in the absence (Control; o) and presence (.) of cADPR. cADPR or the same volume of H2O as a control was added at the beginning of the preincubation. (B) Dose – response curves of ryanodine (0.1 – 500 AM)-induced 45Ca2 + release from the vesicles in the absence (Control; o) and presence (.) of cADPR (0.5 AM). The vertical line represents the ratio of released 45Ca2 + over a 6-min period after the addition of ryanodine to the 45Ca2 + that had been taken up by an ATP-dependent mechanism. Each point is the mean F S.E. from 5 to 10 experiments of the type shown in (A). Note that cADPR shifted the dose – response curve of the ryanodine-induced 45Ca2 + release to the left. Asterisks indicate the level of significant difference compared to the control without cADPR at the corresponding ryanodine concentration. *P < 0.05, **P < 0.01, ***P < 0.001.
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final concentration of 2 mM. After a steady state of 45Ca2 + uptake had been reached, ryanodine, caffeine, cADPR or FK506 was added to the medium. The drug-induced 45Ca2 + release from the vesicles in the absence and that in the presence of either cADPR or FK506 were compared. FK506 was dissolved in DMSO and was added from the stock solution in a volume not exceeding 0.5%. For a control, the vesicles were preincubated with the same volume of a solvent (H2O or DMSO) instead of cADPR or FK506, respectively. At indicated times, 45Ca2 + content of membrane vesicles was determined by a rapid filtration technique as described previously [8]. The radioactivity was counted in a liquid scintillation counter (LS6500, Beckman). Values are presented as means F S.E. Statistical analysis was performed using Student’s t-test.
3. Results 3.1. Modulation of the ryanodine-sensitive mechanism by cADPR A steady state of 45Ca2 + uptake into pancreatic microsomal vesicles was reached about 20 min after the addition of ATP (Fig. 1A). The amount of 45Ca2 + taken up into the microsomal vesicles over a period of 20 min after the addition of ATP was 4.3 F 0.1 nmol/mg protein (n = 173). Ryanodine (10 AM) was added to the vesicles after a steady state of ATP-dependent 45Ca2 + uptake had been reached, and the effect of cADPR (0.5 AM) on ryanodine (10 AM)Fig. 3. Effect of cADPR (0.5 AM) on FK506-induced 45Ca2 + release from pancreatic microsomes. (A) Typical curves for FK506 (3 AM)-induced 45 Ca2 + release from the microsomal vesicles in the absence and presence of cADPR (0.5 AM). The experimental procedures were the same as those described in the legend to Fig. 1A except for the addition of FK506. FK506 (3 AM) was added to the vesicles as indicated in the absence (Control; o) and presence (.) of cADPR. (B) Dose – response curves of FK506 (0.1 – 10 AM)-induced 45Ca2 + release from the vesicles in the absence (o) and presence (.) of cADPR (0.5 AM). The vertical line represents the ratio of released 45Ca2 + as described in the legend to Fig. 1B. Each point is the mean F S.E. from 4 to 11 experiments of the type shown in (A). Note that cADPR shifted the dose – response curve of the FK506-induced 45Ca2 + release upward. Asterisks indicate the level of significant difference compared to the control without cADPR at the corresponding FK506 concentration. **P < 0.01, ***P < 0.001. Lineweaver – Burk plot analysis of the FK506-induced 45Ca2 + release (inset) yielded Km values of 0.47 and 0.70 AM and Vmax values of 8.1% and 14.4% in the absence (o) and presence (.) of cADPR, respectively. Fig. 2. Dose – response curves of caffeine (1 – 30 mM)-induced 45Ca2 + release from pancreatic microsomes in the absence (Control; o) and presence (.) of cADPR (0.5 AM). The experimental procedures were the same as those described in the legend to Fig. 1 except for the addition of caffeine. The vertical line represents the ratio of released 45Ca2 + as described in the legend to Fig. 1B. Each point is the mean F S.E. from 4 to 12 experiments. Note that cADPR shifted the dose – response curve of the caffeine-induced 45Ca2 + release to the left. Asterisks indicate the level of significant difference compared to the control without cADPR at the corresponding caffeine concentration. *P < 0.05, **P < 0.01, ***P < 0.001.
induced 45Ca2 + release from the vesicles was investigated. Pretreatment of the vesicles with cADPR did not affect the ATP-dependent 45Ca2 + uptake before the addition of ryanodine (Fig. 1A) but significantly increased the ryanodine (10 AM)-induced 45Ca2 + release (Fig. 1A). The effect of cADPR (0.5 AM) on the 45Ca2 + release induced by ryanodine (0.1 –500 AM) is shown at each ryanodine concentration in Fig. 1B. Ryanodine was effective at 0.3 AM in the presence of cADPR, and the ryanodine-induced release at
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release from the vesicles was also investigated. When FK506 (3 AM) was added to the vesicles in a steady state of the ATP-dependent 45Ca2 + uptake, about 5% of the 45 Ca2 + that had been taken up by ATP was released from the vesicles (Fig. 3A). The presence of cADPR (0.5 AM) significantly enhanced the FK506 (3 AM)-induced 45Ca2 + release (Fig. 3A). The effect of cADPR (0.5 AM) on the FK506 (0.1 – 10 AM)-induced 45Ca2 + release is shown at each FK506 concentration in Fig. 3B. cADPR shifted the dose – response curve of the FK506-induced 45Ca2 + release upward. Lineweaver –Burk plot evaluation of the release (Fig. 3B, inset) showed Km values of 0.47 and 0.70 AM and Vmax values of 8.1% and 14.4% in the absence and presence of cADPR, respectively. 3.2. Modulation of the ryanodine-sensitive mechanism by FK506 The effect of FK506 on ryanodine-induced 45Ca2 + release from the vesicles was investigated. Preincubation of the vesicles with FK506 (3 AM) did not affect the ATPdependent 45Ca2 + uptake (Fig. 4A) but significantly increased the ryanodine (10 AM)-induced 45Ca2 + release (Fig. 4A). The effect of FK506 (3 AM) on the ryanodine (0.1 – 500 AM)-induced 45Ca2 + release is shown in Fig. 4B. In the presence of FK506, ryanodine was effective at 0.3 AM as it was in the presence of cADPR (cf. Figs. 1B and 4B). FK506 significantly ( P < 0.05– 0.001) increased the ryanodine-induced release at concentrations ranging from 1 to 100 AM (Fig. 4B) and shifted the dose – response curve of the Fig. 4. Effect of FK506 (3 AM) on ryanodine-induced 45Ca2 + release from pancreatic microsomes. (A) Typical curves for ryanodine (10 AM)-induced 45 Ca2 + release from the microsomal vesicles in the absence and presence of FK506 (3 AM). The experimental procedures were the same as those described in the legend to Fig. 1A except for the preincubation with FK506. Ryanodine (Ry., 10 AM) was added to the vesicles as indicated in the absence (Control; o) and presence (.) of FK506. FK506 or the same volume of DMSO as a control was added at the beginning of the preincubation. (B) Dose – response curves of ryanodine (0.1 – 500 AM)induced 45Ca2 + release from the vesicles in the absence (Control; o) and presence (.) of FK506 (3 AM). The vertical line represents the ratio of released 45Ca2 + as described in the legend to Fig. 1B. Each point is the mean F S.E. from 5 to 9 experiments of the type shown in (A). Note that FK506 shifted the dose – response curve of the ryanodine-induced 45Ca2 + release to the left. Asterisks indicate the level of significant difference compared to the control without FK506 at the corresponding ryanodine concentration. *P < 0.05, **P < 0.01, ***P < 0.001.
concentrations ranging from 1 to 100 AM was significantly ( P < 0.05 – 0.001) stimulated by the presence of cADPR (Fig. 1B). cADPR shifted the dose –response curve of the ryanodine-induced 45Ca2 + release to the left (Fig. 1B). The effect of cADPR (0.5 AM) on caffeine (1 –30 mM)-induced 45 Ca2 + release from the vesicles was also investigated. cADPR significantly ( P < 0.05 –0.001) increased the release at lower concentrations (1 –10 mM) of caffeine and shifted the dose – response curve of the caffeine-induced 45Ca2 + release to the left (Fig. 2). The effect of FK506 on 45Ca2 +
Fig. 5. Dose – response curves of caffeine (1 – 30 mM)-induced 45Ca2 + release from pancreatic microsomes in the absence (Control; o) and presence (.) of FK506 (3 AM). The experimental procedures were the same as those described in the legend to Fig. 4 except for the addition of caffeine. The vertical line represents the ratio of released 45Ca2 + as described in the legend to Fig. 1B. Each point is the mean F S.E. from 4 to 12 experiments. Note that FK506 shifted the dose – response curve of the caffeine-induced 45 Ca2 + release to the left. Asterisks indicate the level of significant difference compared to the control without FK506 at the corresponding caffeine concentration. *P < 0.05, **P < 0.01, ***P < 0.001.
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ryanodine-induced 45Ca2 + release to the left by the same extent as that in the case of cADPR (cf. Figs. 1B and 4B). The effect of FK506 (3 AM) on caffeine (1 – 30 mM)induced 45Ca2 + release from the vesicles was also investigated. FK506 significantly ( P < 0.05 –0.001) enhanced the release at lower concentrations (1– 10 mM) of caffeine (Fig. 5) and shifted the dose – response curve of the caffeineinduced 45Ca2 + release to the left by the same extent as that in the case of cADPR (cf. Figs. 2 and 5). The effect of FK506 (3 AM) on the 45Ca2 + release induced by cADPR (0.5 –10 AM) was also investigated. The presence of FK506 shifted the dose – response curve of the cADPR-induced 45 Ca2 + release upward (Fig. 6). Lineweaver – Burk plot analysis of the release (Fig. 6, inset) showed that FK506
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increased the Vmax value from 8.7% to 16.4% without affecting the Km value (1.7 AM). 3.3. Modulation of the ryanodine-sensitive mechanism by both cADPR and FK506 To determine whether the stimulatory effect on ryanodine- or caffeine-induced 45Ca2 + release from the vesicles by cADPR and that by FK506 are additive, the ryanodine (30 AM)- or caffeine (10 mM)-induced 45Ca2 + release was investigated in the presence of both cADPR (0.5 AM) and FK506 (3 AM). The presence of both cADPR and FK506 enhanced the ryanodine (30 AM)- or caffeine (10 mM)induced 45Ca2 + release by the same extent as that in the case
Fig. 6. Dose – response curves of cADPR (0.5 – 10 AM)-induced 45Ca2 + release from pancreatic microsomes in the absence (o) and presence (.) of FK506 (3 AM). The experimental procedures were the same as those described in the legend to Fig. 4 except for the addition of cADPR. The vertical line represents the ratio of released 45Ca2 + over an 11-min period after the addition of cADPR to the 45Ca2 + that had been taken up by ATP. Each point is the mean F S.E. from 4 to 9 experiments. Note that FK506 shifted the dose – response curve of the cADPR-induced 45Ca2 + release upward. Asterisks indicate the level of significant difference compared to the control without FK506 at the corresponding cADPR concentration. *P < 0.05, **P < 0.01. Lineweaver – Burk plot analysis of the cADPR-induced 45Ca2 + release (inset) yielded Km values of 1.7 and 1.7 AM and Vmax values of 8.7% and 16.4% in the absence (o) and presence (.) of FK506, respectively.
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Fig. 7. Effects of cADPR (0.5 AM), FK506 (3 AM) and cADPR + FK506 on 45 Ca2 + release from pancreatic microsomes induced by ryanodine (30 AM) or caffeine (10 mM). The experimental procedures were the same as those described in the legends to (Figs. 1, 2, 4 and 5). cADPR, FK506 and the same volume of solvents (H2O/DMSO) as a control were added at the beginning of the preincubation. The 45Ca2 + release induced by ryanodine or caffeine in the absence (Control) and that in the presence of cADPR alone, FK506 alone or both cADPR and FK506 were compared. The vertical line represents the ratio of released 45Ca2 + as described in the legend to Fig. 1B. Each value is the mean F S.E. from four paired experiments. Note that the ryanodine- or caffeine-induced 45Ca2 + release in the presence of both cADPR and FK506 was nearly the same as that in the presence of either cADPR or FK506.
of cADPR alone or FK506 alone (Fig. 7). Since the stimulatory effects on the release by cADPR and by FK506 were not additive, it is possible that cADPR and FK506 modulate the ryanodine-sensitive Ca2 + channel by the same mechanism.
4. Discussion In the present study, it was demonstrated that cADPR and FK506 modulate the ryanodine-sensitive Ca2 + channels of rat pancreatic acinar cells by increasing the ryanodine or caffeine sensitivity to the channel (Figs. 1, 2, 4 and 5). In addition, it is possible that the mechanisms of modulation by cADPR and by FK506 are the same, since cADPR shifted the dose – response curve of the ryanodine- or caffeine-induced 45Ca2 + release to the left by the same extent as that in the case of FK506 (cf. Figs. 1B and 4B or cf. Figs. 2 and 5, respectively), and the stimulatory effects on ryanodine (30 AM)- or caffeine (10 mM)-induced 45Ca2 + release by cADPR and by FK506 were not additive (Fig. 7). To the author’s knowledge, this is the first report of FK506 sensitivity to Ca2 + release from microsomes prepared from nonexcitable tissues. cADPR not only activates the ryanodine-sensitive Ca2 + channel [13 –17] but also modulates the Ca2 + channel by increasing the Ca2 + or caffeine sensitivity in sea urchin eggs [18] and rat parotid glands [8]. In the modulator model
proposed by Lee et al. [34], cADPR modulates the Ca2 + or caffeine effect on the RyR at an endogenous level. The cADPR concentration (0.5 AM) used for the modulation of ryanodine- or caffeine-induced 45Ca2 + release from rat pancreatic microsomal vesicles is nearly the same as that reported to be endogenous levels (0.2 – 0.68 AM cADPR) in the rat brain, heart and liver tissues [35]. Regarding the action of cADPR on the receptor, two specific cADPRbinding proteins in sea urchin eggs have been identified [19]. Molecular masses of the proteins (140 and 100 kDa) are less than that of known RyR proteins (380 kDa). This finding suggests that cADPR receptors are unknown variants of RyR proteins or RyR accessory proteins. FK506 is known to enhance Ca2 + release through the RyR in skeletal muscle [20,21]. FK506 dissociates FKBP12 from the RyR – FKBP12 complex [20]. By the removal of this accessory protein, the RyR exhibits subconductance states [21], and the Ca2 + or caffeine sensitivity of the channel is enhanced [20,24]. The FK506 concentration (3 AM) used for the modulation of the ryanodine-sensitive mechanism in rat pancreatic acinar cells (Figs. 4 – 6) is nearly the same as that required to dissociate FKBP12 from the RyR complex in skeletal muscle [20]. Type 2 (cardiac type) RyR associates FKBP12.6 as an accessory protein [25 – 27]. Since a type 2 RyR isoform has been identified in rat pancreatic acinar cells [36,37], FKBP12.6 may be involved in the modulation by FK506 of Ca2 + release through the RyR of pancreatic acinar cells. It has been found that cADPR can bind to FKBP12.6 and dissociate FKBP12.6 from pancreatic islet microsomes [27]. An antibody against FKBP12.6 has been shown to inhibit activation of the ryanodine-sensitive Ca2 + release channel induced not only by FK506 but also by cADPR in reconstituted vesicles of coronary arterial smooth muscle cells [38]. These findings suggest that cADPR can dissociate FKBP12.6 from the RyR – FKBP12.6 complex to activate or modulate the Ca2 + channel. Since cADPR can play the same role in the modulation of Ca2 + release through the RyR in rat pancreatic acinar cells as FK506 can (Figs. 1, 2, 4 and 5), it is possible that cADPR modulates the RyR by dissociating FKBP from the RyR. If cADPR modulates the RyR by the same mechanism as that by which FK506 modulates the RyR, this can explain why the stimulatory effect on ryanodine- or caffeine-induced 45Ca2 + release from the vesicles by cADPR and that by FK506 were not additive (Fig. 7). However, it is doubtful whether the cADPR-induced 45 Ca2 + release following pretreatment of the vesicles with FK506 shown in Fig. 6 is due to the dissociation of FKBP since it is thought that FKBP had been removed from the RyR by FK506 before the addition of cADPR. cADPRinduced responses caused by the dissociation of FKBP should be reduced in the presence of FK506. Actually, pretreatment with FK506 abolished the cADPR-induced Ca2 + release from pancreatic islet microsomes [27] and inhibited the cADPR-induced increase in open probability
T. Ozawa / Biochimica et Biophysica Acta 1693 (2004) 159–166
of the ryanodine-sensitive Ca2 + channel in coronary arterial smooth muscle cells [38], while the presence of FK506 in rat pancreatic acinar cells stimulated the cADPR-induced 45 Ca2 + release from the vesicles (Fig. 6). Binding of 32Plabeled cADPR to sea urchin egg homogenates was not affected by the presence of FK506 (30 AM) [39]. This finding suggests that cADPR-binding proteins of sea urchin eggs are not FKBPs. An FKBP-independent mechanism may be involved in the cADPR-induced 45Ca2 + release shown in Fig. 6. The FK506-induced 45Ca2 + release following pretreatment with cADPR shown in Fig. 3 may also not be due to the dissociation of FKBP as in the case of the results shown in Fig. 6. FK506 is a compound with a macrocyclic lactone ring structure. It has been shown that rapamycin and ivermectin, macrocyclic lactone derivatives, increased the open probability of FKBP12-stripped RyRs in skeletal muscle [40,41]. This finding suggests that the compounds activate the RyR by a mechanism other than dissociation of FKBP. Avermectin B1a, another macrocyclic lactone compound, is known to bind to the GABA receptoractivated Cl channel [42,43]. The FK506-induced 45Ca2 + release from the vesicles shown in Fig. 3 may be explained by a direct activation of the RyR by FK506. Since the cADPR- and FK506-induced 45Ca2 + release from the vesicles was stimulated by the presence of FK506 and cADPR, respectively (Figs. 3 and 6), the release induced by cADPR or FK506 may be modulated by the dissociation of FKBP from the RyR. Further studies are needed to clarify what subtype of FKBP is involved in modulation of the RyR in rat pancreatic acinar cells and to obtain detailed information on the actions of cADPR and FK506 on the RyR.
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Acknowledgements I am grateful to Fujisawa Pharmaceutical Co., Ltd. for providing FK506.
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