REGULAR PAPER J. Physiol. Sci. Vol. 56, No. 6; Dec. 2006; pp. 415–423 Online Nov. 3, 2006; doi:10.2170/physiolsci.RP006406

Organelles Containing Trisphosphate Receptor Type 2 in Adrenal Medullary Cells

Yutaka ENDO1, Keita HARADA1, Naoji FUJISHIRO1, Hisasachi FUNAHASHI2, Seiji SHIODA2, Glenn D. PRESTWICH3, Katsuhiko MIKOSHIBA4, and Masumi INOUE1 1Department of and System Physiology, University of Occupational and Environmental Health School of Medicine, Kitakyushu, 807-8555 Japan; 2Department of Anatomy, Showa University School of Medicine, Tokyo, 142-8555 Japan; 3Department of Medical Chemistry, University of Utah, 419 Wakara Way, Suite 205, Salt Lake City, UT 84108, USA; and 4Division of Molecular Neurobiology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan

Abstract: To identify which contained inositol tris- bution of the (ER) identified by an anti- (InsP3) receptor type 2 (InsP3R2) in adrenal medul- Ab, and a prior application of thapsigargin significantly lary (AM) cells, immunocytochemical and biochemical studies eliminated BODIPY-FL-thapsigargin bindings, suggesting that were performed on AM cells of several species. InsP3R2-like im- BODIPY-FL-thapsigargin bindings were mediated by thapsigar- munoreactive materials produced by two different anti-InsP3R2 gin, but not the fluorescence molecule. The anti-InsP3R2 Ab that antibodies (Abs) (Chemicon and Sigma) were distributed in rat produced stainings consistent with BODIPY-FL-InsP3 bindings AM cells in agreement with BODIPY-FL-InsP3 binding sites. For recognized a protein with about 250 kDa. A fractional analysis of two other Abs (KM1083 and Santa Cruz), some of the anti- bovine adrenal medullae revealed that the 250 kDa InsP3R2 InsP3R2 immunoreactive materials were stained with an anti- was detected in a crude membrane fraction, but not in a secreto- dopamine-β-hydroxylase Ab, but not by BODIPY-FL-InsP3. BO- ry granule fraction. The results suggest that the InsP3R2 was DIPY-FL-thapsigargin binding sites were consistent with a distri- present in the ER, but not in secretory granules in AM cells.

Key words: receptor, ER, secretory granule, chromaffin cell.

It is generally believed that the endoplasmic reticulum in secretory granules, the cell will require special trans- (ER) functions as an intracellular Ca2+ store site [1–4]. In port and anchoring systems for the granules to maintain goblet cells, however, secretory granules were reported to them [14]. release Ca2+ in response to inositol 1,4,5-trisphosphate Our immunocytochemical study [15] using an antibody

(InsP3) [5], whereas in submandibular acinar cells InsP3 against calnexin, an integral protein of the ER membrane, receptors (InsP3Rs) were shown to be present in vesicles, revealed that in rat AM cells the ER is distributed not only but not in the rough ER [6]. Moreover, Yoo and his col- in the vicinity of the nucleus, but also underneath the plas- leagues reported that all three subtypes of InsP3Rs resided ma membrane. Therefore, InsP3Rs below the plasma in secretory granules as well as the ER in bovine adrenal membrane are not necessarily located in secretory gran- medullary (AM) cells [7–9]. From a functional point of ules, but they are possibly present in the peripheral ER view, the presence of InsP3Rs in secretory granules is [16]. In pancreatic acinar cells, InsP3Rs in the apical pole readily explained. If InsP3Rs are located in secretory gran- were reported to be present in secretory granules [17, 18]. ules underlying the plasma membrane, a stimulation of re- However, later studies suggested that the InsP3-induced ceptors associated with C would induce an Ca2+ release from a crude granule fraction might have increase in [Ca2+] near the plasma membrane. This would come from the ER contaminated in the fraction [19, 20]. efficiently facilitate the fusion of the secretory granule These results led us to investigate the notion that the membrane with the plasma membrane and result in secre- InsP3Rs at the cell periphery of AM cells may not be tion [10]. However, secretory granules are organelles to- present in secretory granules. In the present experiment, tally different from the ER, having a unique set of proteins we examine this possibility by combining immunocy- [11, 12]. Furthermore, the pH in the secretory granules is tochemistry with biochemical methods. more acidic than that in the ER [13]. If InsP3Rs are present

Received on Jun 7, 2006; accepted on Nov 1, 2006; released online on Nov 3, 2006; doi:10.2170/physiolsci.RP006406 Correspondence should be addressed to Masumi Inoue, Department of Cell and System Physiology, University of Occupational and Environmental Health School of Medicine, Kitakyushu, 807-8555 Japan. Tel: +81-93-691-7235, Fax: +81-93-602-9883, E-mail: [email protected]

The Journal of Physiological Sciences Vol. 56, No. 6, 2006 415 Y. ENDO et al.

were visible with an illumination of 365 nm and emission METHODS above 397 nm. Fluorescence was observed through the Immunocytochemistry. Unless specified, immunocy- whole cell with a full width at a half-maximum of 0.7 µm, tochemistry was performed in dissociated rat AM cells. and the colocalization of InsP3R2-like immunoreactivity Male rats and guinea pigs were killed by cervical disloca- with fluorescent agent binding was analyzed at a section tion, and the adrenal glands were excised. Adrenal medul- where both fluorescences were strongest. Whole-cell im- lae were cut into three to six pieces and incubated for 30 ages were acquired with an illumination of the 488 nm la- min with 0.25% collagenase dissolved in Ca2+-deficient ser and an emission of all wavelengths, and the cell shape saline (137 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl2, 0.53 or edge was determined by looking at staining intensity mM NaH2PO4, 5 mM D-glucose, 5 mM Hepes, and 4 mM above background levels. A superimposition of cell shape NaOH, pH 7.4). After incubation, the tissues were washed and fluorescence images was made with the use of Photo- in Ca2+-deficient saline, and placed in a dish, then dissoci- shop software (version 6.0). To examine the specificity for ated using fine needles. The dissociated AM cells were the immunoreaction, the preparation was treated with a fixed in 2% paraformaldehyde-containing phosphate- nonimmune serum instead of a primary Ab, and almost no buffered saline (PBS: 145 mM NaCl, 8 mM Na2HPO4, 2 immunoreaction was observed under the same conditions mM NaH2PO4) for 2 h at 4°C, then treated in 5% goat or as those used for the primary Ab. rabbit serum containing PBS with 0.3% Triton X for 30 Immunoblot. Rat adrenal medullae were homogenized min. For indirect immunofluorescence studies, the cells in a Tris buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl) were treated with a rabbit anti-InsP3R type 2 (InsP3R2) Ab containing a inhibitor cocktail (Calbiochem). produced by Sigma (at a dilution of 1:100) overnight, rab- Homogenates were spun down at 2,000 × g for 10 min at bit anti-InsP3R2 Ab by Chemicon (1:10) overnight, goat 4°C, and the postnuclear supernatants were then mixed anti-InsP3R2 Ab by Santa Cruz (1:100) for one or two with equal volumes of a twofold concentrated Laemmli days, or mouse anti-InsP3R2 Ab KM1083 [21] (1:1,000) sample buffer. The crude membrane fraction was obtained overnight, and/or with a rabbit anti-dopamine-β-hydroxy- with a centrifugation of the postnuclear supernatant at lase (DβH) Ab (Chemicon) (1:500) overnight. The AM 100,000 × g for 1 h. The resulting pellet was dissolved in cells were also treated with a rabbit anti-calnexin Ab the sample buffer. Since catecholamines interfere with a (Stressgen) (1:400) overnight. The immunoreactions were BCA protein assay kit (Pierce), protein concentrations in visualized with respective secondary Abs conjugated with samples were measured after the proteins were precipitat- Alexa 488 or 546 (Molecular Probe) (1:300). In the case ed by acetone and resuspended in the sample buffer. Just of Alexa 488 (FITC-like fluorescence), immunoreactivity before electrophoresis, 5% (v/v) 2-mercaptoethanol and was observed with an illumination of a 488 nm laser and 1% (w/v) bromophenol blue were added to the sample, an emission of 510–525 nm, whereas in the case of Alexa and the proteins were separated by 10% or 7.5% (w/v) 546 (rhodamine-like fluorescence), it was observed with SDS-PAGE, then transferred to a PVDF membrane. The an illumination of a 543 nm laser and emission above 560 membrane was blocked with 5% (w/v) fat-free powdered nm. Cross-talk between FITC- and rhodamine-like fluo- milk dissolved in the Tris buffer with 0.1% Tween 20 and rescence in double stainings was assessed in experiments then incubated with primary Abs. The immunoreaction where FITC- or rhodamine-like fluorescence was ob- was detected by incubating the membrane with respective served after treatment with one secondary Ab coupled secondary Abs linked to horseradish peroxidase and then with Alexa 546 or 488, respectively. In our experimental with ECL-Plus (Amersham). conditions, there was no significant cross-talk between Fractionation study. Bovine secretory granules were FITC- and rhodamine-like fluorescence. To identify or- isolated, as described previously [22]. Briefly, bovine ad- ganelles stained with the anti-InsP3R2 Abs, cells with im- renal medullae were homogenized in SME buffer (300 munoreactions were immersed in PBS containing 30 µM mM sucrose, 10 mM MOPS, and 5 mM EDTA), filtered BODIPY-FL-InsP3 [16], 1 µM ER Tracker (Molecular through one layer of fine mesh cotton gauge. Following Probe), or 4.4 µM 3,3´-dihexyloxacarbocyanine iodide centrifugation at 1,000 × g for 15 min, the postnuclear su- (DIO) (Molecular Probe) whereas to study the relation be- pernatant was spun at 25,000 × g for 20 min to obtain a tween the ER and Ca2+ store sites, the cells treated with crude granule pellet and a postgranule supernatant. The the anti-calnexin Ab were placed in 0.5 µM BODIPY-FL- pellet was resuspended in SME buffer and layered over thapsigargin (Molecular Probe). The immunoreactivity step gradients of Percoll (60%, 40%, and 20%). The gradi- was observed using a confocal laser scanning microscope ents were spun at 10,000 × g for 30 min. The material (Zeiss LSM 410) with X63 objective lenses (a numerical banded at the 40% to 60% interface was resuspended in aperture of 1.25 or 1.4). The binding of ligands conjugated SME buffer, then centrifugated at 25,000 × g for 20 min. with BODIPY moiety (fluorescent ligands) and DIO was This procedure was twice repeated to remove Percoll. The visualized with an illumination of a 488 nm laser and an resulting pellet was dissolved in the sample buffer, result- emission of 510–525 nm, whereas ER Tracker bindings ing in a secretory granule fraction. On the other hand, the

416 The Journal of Physiological Sciences Vol. 56, No. 6, 2006 and Muscle Atrophy postgranule supernatant was spun at 100,000 × g for 1 h, RESULTS and the resulting pellet was dissolved in the sample buffer, the solution was designated a crude membrane fraction. Immunocytochemistry for InsP3Rs Immunogold electron microscopy. Female guinea-pigs We previously showed that rat AM cells had mainly the were deeply anesthetized with sodium pentobarbital (50 InsP3R2 isoform [23]. Thus to identify intracellular or- mg/kg, i.p.) and perfused through the ascending aorta with ganelles having InsP3R2, an immunostaining of InsP3R2 50 ml of saline, then with 250–300 ml of PBS containing was compared with the binding of BODIPY-FL-InsP3, 4% paraformaldehyde and 0.1% glutaraldehyde for 30 which was shown to represent the ER [15]. Figure 1 min. The adrenal glands were removed immediately and shows immunostainings obtained with anti-InsP3R2 Abs postfixed in the same fixative for 1 day at 4°C. After being produced by Chemicon, Sigma, and Santa Cruz (SC) and washed with PBS, the fixed tissues were trimmed to with KM1083, monoclonal anti-InsP3R2 Ab. The Chemi- smaller pieces containing the medulla, incubated for 2 con anti-InsP3R2 Ab-immunoreactive materials, which days in 30% sucrose in PBS, and incubated for 3 days in were observed as rhodamine-like fluorescence, were dis- cold PBS with 1.84 M sucrose and 20% polyvinyl pyrroli- tributed both in the vicinity of the nucleus and at the cell done. The tissues were cut into very small pieces and rap- periphery in dissociated AM cells (Fig. 1A). In all 24 cells idly frozen in liquid nitrogen. Frozen ultrathin sections examined, this distribution of immunoreactivity was near-

(150 nm thick) were made with a Lica Ultracut S/FCS ly coincident with the binding of BODIPY-InsP3 (Fig. (Austria). The sections were picked up on a formvar/car- 1B), which was visible as FITC-like fluorescence. On the bon-coated nickel grid, then incubated overnight with the other hand, the Sigma Ab produced not only immunoreac-

Santa Cruz anti-InsP3R2 Ab diluted at 1:100 in the PBS tive materials in the vicinity of the nucleus and the plasma with 2% gelatin and 10 mM glycine. The sections were membrane, but also dotlike materials in the nucleus (Fig. washed five times with PBS containing 0.5% gelatin and 1C). The distribution of the former generally agreed with

10 mM glycine, then incubated for 2 h with 10 nm colloi- that of fluorescent InsP3 bindings in 13 cells, whereas the dal gold-labeled rabbit anti-goat IgG Ab (2 µg/ml) (Brit- latter lacked fluorescent InsP3 bindings (Fig. 1D). ish Biocell International). After being washed again, the The distribution of materials immunoreactive to sections were postfixed in 0.1% glutaraldehyde, stained KM1083 that were present in a punctate fashion in the vi- with 2% uranyl acetate, embedded in polyvinyl alcohol, cinity of the nucleus and the plasma membrane nearly and observed with a Jeol JEM-1200 EXII electron micro- overlapped with BODIPY-FL-InsP3 binding in 2 of 9 cells scope (Japan). examined, whereas in the remaining 7, strips at the cell pe- riphery were additionally stained with KM1083, and this

Fig. 1. Immunostaining of rat adre- Chemicon Ab Fluo IP3Sigma Ab Fluo IP3 nal medullary cells for InsP3R2. A, C, E, and G represent confocal im- A BCD ages of adrenal medullary (AM) cells stained with an anti-InsP3R2 Ab produced by Chemicon, one by Sigma, KM1083, and one by Santa * Cruz (SC), respectively, whereas * B, D, F, and H are those of BO-

DIPY-FL-InsP3 bindings in the same level as A, C, E, and G, re- spectively. AM cells were treated KM1083 Fluo IP3 SC Ab Fluo IP3 for one or two days with anti- EFG H InsP3R2 Abs, then incubated with an Alexa 546-conjugated second- ary Ab. After a washout of the sec- ondary Ab, the cells were im- * * mersed in 30 µM BODIPY-FL-

InsP3-containing PBS. Immunore- actions and fluorescent InsP3 bind- ings were observed as rhodamine- 5 Pm * and FITC-like fluorescence, re- spectively. The sites at the cell pe- riphery indicated by the closed ar- rowheads were stained with both Abs and fluorescent InsP3. The open arrowheads in C indicate nonspecific immunoreactions. The strip indicated by the arrow was immunoreactive to KM1083, but lacked fluorescent InsP3 binding. Asterisks represent the nucleus in this and the following figures. The calibration of 5 µm applies from A to H.

The Journal of Physiological Sciences Vol. 56, No. 6, 2006 417 Y. ENDO et al.

Fig. 2. Presence of InsP R2-like immu- 3 ER Tracker KM1083 noreactivity in the endoplasmic reticulum A B near the nucleus. A and B represent confo- cal images of rat AM cells stained with ER Tracker and KM1083 at the same level, re- spectively. ER Tracker binding and immu- * noreaction to KM1083 were visualized with an illumination of a 365 nm laser and emis- * sion above 397 nm and with an illumination * of a 488 nm and emission of 510–525 nm, respectively. C and D show confocal imag- DIO Sigma Ab es of a rat AM cell stained with DIO and the C D E Sigma anti-InsP3R2 Ab, respectively. DIO binding and immunoreaction to the Sigma Ab were visible as FITC- and rhodamine- like fluorescence, respectively. The images * were a superimposition of cell profile (gray) and DIO binding (blue) or immunoreaction to the Sigma Ab (green) (see METHODS for details). E represents a superimposition of DIO KM1083 D and sites (red) with both DIO binding and FG H immunoreaction to the Sigma Ab. F and G show confocal images of a rat AM cell stained with DIO and KM1083 at the same level. DIO binding and immunoreaction to 5 Pm KM1083 were visible as FITC- and * rhodamine-like fluorescence, respectively. The images were a superimposition of cell profile (gray) and DIO binding (blue) or im- munoreaction to KM1083 (green). H represents a superimposition of G and sites (red) with both DIO binding and immunoreac- tion to KM1083. The sites indicated by the arrowheads were stained with both DIO and KM1083, whereas the strip indicated by the arrow was labeled by KM1083, but not by DIO. The calibration of 5 µm applies from A to H.

striplike immunoreactivity was devoid of fluorescent materials immunoreactive to anti-InsP3R2 Abs, whose InsP3 binding (Fig. 1, E and F). On the other hand, the SC distribution agrees with BODIPY-FL-InsP3 bindings, re- anti-InsP3R2 Ab produced immunostaining patterns that side in the ER. were entirely different from BODIPY-FL-InsP3 bindings in 12 cells (Fig. 1, G and H). The materials immunoreac- InsP3R2-like immunoreactive materials and tive to the Ab were mainly confined to the cell periphery, secretory granules which showed no fluorescent InsP3 binding. Taken togeth- Our observation that striplike immunoreactive materi- er, the materials immunoreactive to so-called anti- als to KM1083 at the cell periphery lacked fluorescent

InsP3R2 Abs consisted of two components: one set of ma- InsP3-binding activity raised the possibility that they were terials distributed in a punctate fashion coincided with flu- located in organelles other than the ER. This possibility orescent InsP3 bindings in the , whereas the oth- was confirmed in Fig. 2, F, G, and H, which revealed that er set present as a strip at the cell periphery lacked them. the KM1083-immunoreactive strip along the cell periph- ery was not stained with DIO either. This finding was con- ER markers and anti-InsP3R2-like immunoreactive sistently observed in 7 cells examined. These two lines of materials evidence indicate that part of KM1083-immunoractive

Since InsP3R2 is generally thought to be present in the materials present at the cell periphery were located in or- ER, we performed double stainings with anti-InsP3R2 ganelles other than the ER, most probably secretory gran- Abs and ER markers, ER Tracker, and DIO [24]. The dis- ules. This possibility was investigated with a double stain- tribution of KM1083-immunoreactive materials in the vi- ing. As shown in Fig. 3, A, B, and C, the region labeled by cinity of the nucleus agreed with that of ER Tracker bind- an Ab raised against DβH that resides in secretory gran- ings in 11 cells (Fig. 2, A and B). Similarly, materials ules [25] overlapped minimally to that of fluorescent immunoreactive to the Sigma Ab in the cytoplasm coin- InsP3 binding in 8 cells, suggesting that the anti-DβH Ab cided with DIO bindings in 7 cells, whereas immunoreac- had not labeled the ER. Most of the KM1083-immunore- tive materials present in the nucleus had no DIO binding active area along the cell periphery was stained with the activity (Fig. 2, C, D, and E). These results indicate that anti-DβH Ab in 7 cells (Fig. 3, D, E, and F). Similarly,

418 The Journal of Physiological Sciences Vol. 56, No. 6, 2006 Estrogen and Muscle Atrophy

DEH Fluo IP3 Fig. 3. Materials immu- ABC noractive to the Santa J Cruz Ab and KM1083 are present in secretory granules. A and B show confocal images of a rat AM cell stained with an anti-DβH Ab and BO- DIPY-FL-InsP , respec- DEH KM1083 3 tively. C represents a su- D EF perimposition of A and B,

and fluorescent InsP3 binding sites with DβH- like immunoreactivity (IR) were expressed in red. D and E show confo- cal images of rat AM DEH SC Ab cells stained with the anti-DβH Ab and GHI KM1083, respectively. F 1 Pm represents a superimpo- sition of E and sites (brown) with immunore- actions to both the Abs. G and H show confocal 5 Pm images of a guinea pig AM cell with the anti-DβH

Ab and SC anti-InsP3R2 Ab, respectively. I represents a superimposition of H and sites (brown) with immunoreactions to both the Abs. J shows an immu- noelectron micrograph of guinea-pig AM cells treated with the SC anti-InsP3R2 Ab (see METHODS for details). Arrowheads indi- cate deposits of gold in secretory granules. The insertion is 3× enlargement of the square indicated. The calibration of 5 µm ap- plies from A to I.

Fig. 4. Immunoblots of rat adrenal Chemicon KM1083 Santa Cruz medullae with various anti-InsP3R2 Abs. Immunoblots of homogenate HSC HSC HSC (H), supernatant (S), and crude membrane fraction (C) of rat adre- 250 250 250 nal medulla for InsP R2. The PVDF 3 150 150 150 membranes were treated overnight 100 100 with the Chemicon Ab (at a dilution 100 75 75 of 1:100), KM1083 (1:4,000), and 75 50 Santa Cruz Ab (1:2,000). The same 50 amounts of proteins (5 and 15 µg) 50 37 were loaded in each fraction. The 37 37 exposure time for the detection of bands was adjusted so that the kDa kDa kDa

InsP3R2 levels detected with the Chemicon Ab were comparable to those with KM1083. These immunoblots represent 3 to 4 experiments for the Abs. The post- nuclear homogenate (H) was spun at 100,000 × g for 1 h, and the resulting supernatant and pellet were designated as superna- tant (S) and a crude membrane fraction (C) (see METHODS for details). most of the SC Ab-immunoreactive area was stained with Immunoblot the anti-DβH Ab in 7 cells (Fig. 3, G, H, and I). These im- The foregoing results raised the possibility that the ER munocytochemical findings indicated that most strips and secretory granules were immunoreactive to putative stained with KM 1083 and the SC Ab at the cell periphery anti-InsP3R2 Abs, as shown in bovine AM cells [9]. How- were secretory granules. This notion was further con- ever, this complexity might be ascribed to heterogeneous firmed at the electron microscopic level. Figure 3 J clearly immunoreactivities of the Abs used. Thus we investigated revealed that immunoreaction with the SC Ab selectively the specificity of Abs using immunoblotting. The Chemi- deposited gold particles in secretory granules. con Ab specifically recognized a protein of about 250 kDa

The Journal of Physiological Sciences Vol. 56, No. 6, 2006 419 Y. ENDO et al.

Fig. 5. The distribution of thapsigargin bind- Thap + Fluo Thap Calnexin-like IR ing sites agrees with that of the endoplasmic ABC P < 0.001 reticulum recognized by the anti-calnexin an- 100 tibody. A and B represent confocal images of a rat AM cell stained with 0.5 µM BODIPY-FL- 80 thapsigargin in the presence of 10 µM thapsi- 60 gargin and with an anti-calnexin Ab at the same level, respectively. After treatment with 40 Intensity (au) the anti-calnexin Ab and then a secondary 20 Ab, the cell was exposed to 0.5 µM BODIPY- FL-thapsigargin in the presence of 10 µM -+ thapsigargin. BODIPY fluorescence and cal- thapsigargin nexin-like IR were visible as FITC- and Fluo Thap Calnexin-like IR rhodamine-like fluorescence, respectively. C DE summarizes the fluorescence levels of BO- DIPY-FL-thapsigargin bindings in the pres- ence and absence of thapsigargin. The fluo- rescence intensity of BODIPY-FL- thapsigargin bindings was expressed in arbi- trary units (au). The means and s.e.m. are shown (see RESULTS for details). The statisti- cal significance was determined with a Stu- dent’s t-test. D and E represent confocal im- ages of a rat AM cell stained with BODIPY- FL-thapsigargin (Fluo Thap) and anti-calnexin Ab at the same level, respectively. BODIPY fluorescence and calnexin-like IR were visible as FITC- and rhodamine-like fluorescence, respectively. in a homogenate and a crude membrane fraction of the rat ABDEH Calnexin adrenal medulla and not in a supernatant fraction (Fig. 4), HOSGCM HO SG CM indicating that the Ab was specific for InsP R2. On the 3 250 250 other hand, the SC Ab did not detect the 250 kDa protein, 150 150 although it recognized bands with lower molecular 100 100 weights. KM1083 mainly recognized the 250 kDa protein, 75 75 but it also recognized bands with lower molecular weights 50 50 in the homogenate, supernatant, and crude membrane 37 37 fraction. The results indicated that the Chemicon Ab was kDa kDa most selective for InsP3R2 among the three Abs, at least in the rat. C IP3R2 Distribution of fluorescent thapsigargin bindings HO SG CM Our previous study indicated that Ca2+ store sites sensi- 250 tive to muscarinic receptor stimulation in the rat AM cells 150 were depleted by thapsigargin [23], a specific inhibitor of 100 sarcoplasmic and/or endoplasmic reticulum Ca2+ (SER- 75 CA) pumps [26], and Ca2+ mobilization in response to kDa muscarinic receptor stimulation in AM cells was most Fig. 6. Fractional analysis of InsP R2 distribution in bovine likely mediated by InsP [27, 28]. These results suggest 3 3 adrenal medullae. A and B represent immunoblots of homo- that SERCA pumps are responsible for Ca2+ uptake into genate (HO), highly purified secretory granule (SG), and 2+ the InsP3-sensitive Ca store sites. SERCA pumps are crude membrane fraction (CM) for DβH and calnexin, respec- firmly established to be present in the SR in the muscles tively. Note that SG and CM fractions were rich in DβH and [2–4], but in other cells [24, 29] they were also very likely calnexin contents, respectively. C shows an immunoblot of to reside in other organelles, such as the Golgi. Thus we HO, SG, and CM for InsP3R2. InsP3R2 was detected in HO studied whether SERCA pumps were located in the ER. and CM, but not in SG with the Chemicon Ab. In A and B, 25 To explore the distribution of SERCA pumps, rat AM µg proteins were loaded in each lane, whereas in C 15 µg cells were exposed to BODIPY-FL-thapsigargin, and the were loaded. binding was visualized as FITC-like fluorescence. Figure 5 shows that BODIPY-FL-thapsigargin binding was not When fluorescent intensities of FITC-like fluorescence at due to the fluorescent moiety, but to thapsigargin itself. 3 to 9 sites per cell, which corresponded to those with cal-

420 The Journal of Physiological Sciences Vol. 56, No. 6, 2006 Estrogen and Muscle Atrophy

nexin-like immunoreactivity visible as rhodamine-like minus of the rat InsP3R2. The BODIPY-FL-InsP3 binding fluorescence, were measured in 6 cells with and without a sites below the plasma membrane as well as in the vicinity 10 min prior exposure to 20 µM thapsigargin (Fig. 5, A of the nucleus in chromaffin cells were previously shown and B), mean intensity per pixel (pixel size = 0.079 µm × to be identical with sites stained with an anti-calnexin Ab 0.079 µm) was significantly diminished by the prior expo- [15], whereas materials immunoreactive to the Sigma Ab sure (Fig. 5C). In 13 cells, the distribution of fluorescent were distributed similarly to DIO binding sites, except for thapsigargin binding (Fig. 5, D and E) overlapped with the nucleus. These previous and present results indicate that of the ER, as identified by immunostaining for caln- that central and peripheral sites with InsP3R2-like immu- exin, an integral protein of the ER. noreacitivity that agree with BODIPY-FL-InsP3-binding sites represented the ER. On the other hand, the SC anti- Fractionation study InsP3R2 Ab, which was raised in goats against a peptide The presence of InsP3R2 in secretory granules was fur- mapping at the carboxy terminal of human InsP3R2, ther studied by the fractionation of a postnuclear homoge- stained strips along the cell periphery, which were labeled nate of the bovine adrenal medulla. A highly purified with an anti-DβH Ab, but not BODIPY-FL-InsP3, indicat- secretory granule (SG) fraction, which was obtained in ing that the immunoreactive materials to the SC Ab were two steps using centrifugation with a Percoll gradient, was present in secretory granules. This notion was confirmed rich in DβH and had almost no calnexin, whereas a crude at the electron microscopic level. The immunoreactive membrane (CM) fraction, which was obtained with cen- properties of KM1083, which was also raised against the trifugation of a postgranular supernatant, was rich in caln- carboxy terminal of human InsP3R2 [21], were between exin, but lacked DβH (Fig. 6, A and B). Figure 6 C reveals those of the rabbit Abs and the goat Ab. Most of these that InsP3R2 was detected in the CM fraction, but not in mAb-labeled materials in the vicinity of the cell mem- the SG fraction, suggesting that InsP3R2 is present in the brane were stained with the anti-DβH Ab, but not with ER, but not in secretory granules. BODIPY-FL-InsP3, whereas the mAb-sensitive area near the nucleus was stained with the latter and ER Tracker, which selectively bind to the ER. In summary, the four DISCUSSION Abs could be divided into two groups: one , including the Secretory granules, such as chromaffin [30, 31] and zy- Sigma and Chemicon Abs, did not stain strips at the cell mogen granules [32] in pancreatic acinar cells, are known periphery; the other, comprising the SC Ab and KM1083, to contain large amounts of Ca2+ ions. The physiological did stain them. 2+ 2+ significance of these intragranular Ca ions ([Ca ]g), al- The immunocytochemical studies with the various Abs though suggested to be involved in secretion [7], still re- in the present and previous experiments [23] might sug- mains obscure. Chromaffin and zymogen granules were gest that InsP3R2 resides not only in the ER, but also in 2+ shown to release Ca ions in response to InsP3 [8, 17], secretory granules, as proposed in bovine AM cells [7]. and InsP3Rs were localized in chromaffin granules by the However, we think that this possibility is not feasible. use of immunogold electron microscopy [9]. Consistent First, the Chemicon Ab specifically recognized InsP3R2 with the notion that secretory granules are dynamic Ca2+ of about 250 kDa in rat adrenal medullae. On the other stores, the measurement of [Ca2+] in -containing hand, KM1083 in the second group mainly recognized the granules of MIN6 cells with a vesicle-associated mem- 250 kDa protein, but it also recognized proteins with low- brane protein aequorin chimera revealed a dramatic de- er molecular weights whereas the SC Ab scarcely detected 2+ crease in [Ca ]g in response to caffeine and cyclic ADP the InsP3R2. Thus the Chemicon Ab appeared to be most ribose [11]. However, the use of a similar approach to specific for the InsP3R2 at least in the rat. This Ab showed 2+ measure [Ca ]g in neurosecretory PC12 cells revealed a staining similar to BODIPY-FL-InsP3-binding sites in 2+ that [Ca ]g was altered with a change in intracellular pH the vicinity of the nucleus and , but it pro- [33], but it did not noticeably respond to InsP3 or cyclic duced no striplike stainings at the cell periphery. ADP ribose [12]. Furthermore, the presence of InsP3Rs in Our notion that the InsP3R2 is present in the ER, but zymogen granules has been disputed [19, 20]. not in secretory granules, was further supported by frac-

In the present experiment, four different Abs were used tionation studies revealing that the 250 kDa InsP3R2 was to elucidate organelles having InsP3R2 in rat AM cells. recovered in a CM fraction, but not in a highly purified SG The Sigma Ab was produced in rabbits with the synthetic fraction. Furthermore, BODIPY-FL-thapsigargin -binding peptide corresponding to residues 317–334 of sites coincided with those labeled with the anti-calnexin the rat InsP3R2. The materials immunoreactive to the Ab Ab. We [23] and others [34] reported that thapsigargin de- in the vicinity of the nucleus and cell membrane bound to pleted Ca2+ ions from store sites, from which Ca2+ mobili- the same regions as BODIPY-FL-InsP3 did. A similar re- zation occurred in response to InsP3-generating receptor sult was obtained with the Chemicon Ab, which was made agonists. Thus the coincidence of BODIPY-FL-thapsigar- in rabbits with a peptide corresponding to the carboxy ter- gin-binding sites with calnexin-like immunoreactive sites

The Journal of Physiological Sciences Vol. 56, No. 6, 2006 421 Y. ENDO et al. is consistent with the notion that the ER alone is responsi- 10. Tse FW, Tse A, Hille B, Horstmann H, Almers W. Local Ca2+ release from internal ble for InsP -sensitive store sites in chromaffin cells. stores controls exocytosis in pituitary gonadotrophs. Neuron. 1997;18:121-32. 3 11. Mitchell KJ, Pinton P, Varadi A, Tacchetti C, Ainscow EK, Pozzan T, Rizzuto R, These immunocytochemical and biochemical studies sug- Rutter GA. Dense core secretory vesicles revealed as a dynamic Ca2+ store in neuroendocrine cells with a vesicle-associated membrane protein aequorin gest that InsP3R2 is distributed in the ER, but not in secre- tory granules in rat adrenal chromaffin cells. This notion chimaera. J Cell Biol. 2001;155:41-51. 12. Pouli AE, Karagenc N, Wasmeier C, Hutton JC, Bright N, Arden S, Schofield JG, compares with electron microscopic studies of the cere- Rutter GA. A phogrin-aequorin chimaera to image free Ca2+ in the vicinity of bellum, which revealed that immunoreactive products to secretory granules. Biochem J. 1998;330:1399-404. an anti-InsP R1 Ab were present in the ER, but not in oth- 13. Schoonderwoert VThG, Martens GJM. Proton pumping in the secretory pathway. 3 J Membr Biol. 2001;182:159-69. er organelles, such as the Golgi complex or synaptic vesi- 14. Parker AKT, Gergely FV, Taylor CW. Targeting of inositol 1,4,5-trisphosphate cles [35, 36]. receptors to the endoplasmic reticulum by multiple signals within their Yoo’s group recently extended immunogold electron transmembrane domains. J Biol Chem. 2004;279:23797-805. 15. Lin H, Ozaki S, Fujishiro N, Takeda K, Imanaga I, Prestwich GD, Inoue M. microscopic studies to show that InsP3R-like immunore- Subunit composition and role of Na+,K+-ATPases in adrenal chromaffin cells. J active materials were distributed not only in the ER and Physiol. 2005;564:161-72. secretory granules, but also in the plasma membrane, mi- 16. Inoue M, Sakamoto Y, Fujishiro N, Imanaga I, Ozaki S, Prestwich GD, Warashina A. Homogeneous Ca2+ stores in rat adrenal chromaffin cells. Cell . 2003; tochondria, and nuclear envelope [37]. Furthermore, the 33:19-26. nucleoplasm was also shown to have InsP3R-like prod- 17. Gerasimenko OV, Gerasimenko JV, Belan PV, Petersen OH. Inositol ucts, as was noted with the Sigma anti-InsP R2 Ab. Thus trisphosphate and cyclic ADP-ribose-mediated release of Ca2+ from single 3 isolated pancreatic zymogen granules. Cell. 1996;84:473-80. according to a series of papers by Yoo and his colleagues 18. Petersen OH. Can Ca2+ be released from secretory granules or synaptic [9, 37], InsP3Rs turn out to be present almost everywhere vesicles?. Trends Neurosci. 1996;19:411-3. in bovine chromaffin cells. This apparent complexity of 19. Nezu A, Tanimura A, Morita T, Irie K, Yajima T, Tojyo Y. Evidence that zymogen 2+ InsP R distributions, in our opinion, may at least in part be granules do not function as an intracellular Ca store for the generation of the 3 Ca2+ signal in rat parotid acinar cells. Biochem J. 2002;363:59-66. due to nonspecific binding. There was a similar controver- 20. Yule DI, Ernst SA, Ohnishi H, Wojcikiewicz RJH. Evidence that zymogen granules are not a physiologically relevant calcium pool. Defining the distribution sy about whether InsP3Rs were present in secretory gran- ules in pancreatic β cells or not. The InsP R type 3 was re- of inositol 1,4,5-trisphosphate receptors in pancreatic acinar cells. J Biol Chem. 3 1997;272: 9093-8. ported to be present in insulin-containing secretory 21. Sugiyama T, Furuya A, Monkawa T, Yamamoto-Hino M, Satoh S, Ohmori K, granules [38, 39]. However, its presence in insulin gran- Miyawaki A, Hanai N, Mikoshiba K, Hasegawa M. Monoclonal antibodies ules was contradicted because the Ab that was used distinctively recognizing the subtypes of inositol 1,4,5-trisphosphate receptor: application to the studies on inflammatory cells. FEBS Lett. 1994;354:149-54. (AB3Ab) cross-reacted with rat insulin [40]. Thus to elu- 22. Meyer DI, Burger MM. Isolation of a protein from the plasma membrane of cidate which organelles contain target proteins, we would adrenal medulla which binds to secretory vesicles. J Biol Chem. 1979;254:9854- need to perform immunocytochemical studies with multi- 9. 23. Inoue M, Lin H, Imanaga I, Ogawa K, Warashina A. InsP3 receptor type 2 and ple Abs, which were raised against different epitopes. oscillatory and monophasic Ca2+ transients in rat adrenal chromaffin cells. Cell Calcium. 2004;35:59-70. This work was supported in part by a Grant-in-Aid from the Japan Society 24. Hauser K, Pavlovic N, Klauke N, Geissinger D, Plattner H. Green fluorescent for the Promotion of Science (13670050 to M. I.) and by a grant from the 2+ National Institutes of Health (NS29632 to G. D. P.). Thanks are also due to protein-tagged sarco(endo)plasmic reticulum Ca -ATPase overexpression in Paramecium cells: isoforms, subcellular localization, biogenesis of cortical S Ozaki for preparing the BODIPY-FL-InsP3 and to Hai Lin for a preliminary experiment. calcium stores and functional aspects. Mol Microbiol. 2000;37:773-87. 25. Winkler H, Apps DK, Fiseher-Colbrie R. The molecular function of adrenal chromaffin granules: established facts and unresolved topics. Neuroscience. REFERENCES 1986;18:261-90. 26. Lytton J, Westlin M, Hanley MR. Thapsigargin inhibits the sarcoplasmic or 1. Berridge MJ. Inositol trisphosphate and calcium signalling. Nature. 1993;361: endoplasmic reticulum Ca-ATPase family of calcium pumps, J Biol Chem.1991; 315-25. 266:17067-71. 2. Blaustein MP, Golovina VA. Structural complexity and functional diversity of 27. Inoue M, Sakamoto Y, Imanaga I. is involved in endoplasmic reticulum Ca2+ stores. Trends Neurosci. 2001;24:602-8. production of Ca2+-dependent currents, but not non-selective cation currents, by 3. Meldolesi J, Pozzan T. The endoplasmic reticulum Ca2+ store: a view from the muscarine in chromaffin cells. Eur J Pharmacol. 1995;276:123-9. lumen. Trends Biochem Sci. 1998;23:10-4. 28. Ohta T, Asano T, Ito S, Kitamura N, Nakazato Y. Characteristics of cytosolic Ca2+ 4. Pozzan T, Rizzuto R, Volpe P, Meldolesi J. Molecular and cellular physiology of elevation induced by muscarinic receptor activation in single adrenal chromaffin intracellular calcium stores. Physiol Rev. 1994;74:595-636. cells of the guinea pig. Cell Calcium. 1996;20:303-14. 5. Nguyen T, Chin W-C, Verdugo P. Role of Ca2+/K+ ion exchange in intracellular 29. Lin P, Yao Y, Hofmeister R, Tsien RY, Farquhar MG. Overexpression of CALNUC storage and release of Ca2+. Nature. 1998;395:908-12. (Nucleobindin) increases agonist and thapsigargin releasable Ca2+ storage in 6. Yamamoto-Hino M, Miyawaki A, Segawa A, Adachi E, Yamashina S, Fujimoto T, the Golgi. J Cell Biol. 1999;145:279-89. Sugiyama T, Furuichi T, Hasegawa M, Mikoshiba K. Apical vesicles bearing 30. Fasolato C, Zottini M, Clementi E, Zacchetti D, Meldolesi J, Pozzan T. inositol 1,4,5-trisphosphate receptors in the Ca2+ initiation site of ductal Intracellular Ca2+ pools in PC12 cells. Three intracellular pools are distinguished epithelium of submandibular gland. J Cell Biol.1998;141:135-42. by their turnover and mechanisms of Ca2+ accumulation, storage, and release. J 7. Yoo SH. Coupling of the IP3 receptor/Ca2+ channel with Ca2+ storage proteins Biol Chem. 1991;266:20159-67. chromogranins A and B in secretory granules. Trends Neurosci. 2000;23:424-8. 31. Winkler H, Westhead E. The molecular organization of adrenal chromaffin 8. Yoo SH, Albanesi JP. Inositol 1,4,5-trisphosphate-triggered Ca2+ release from granules. Neuroscience. 1980;5:1803-23. bovine adrenal medullary secretory vesicles. J Biol Chem.1990;265:13446-8. 32. Clemente F, Meldolesi J. Calcium and pancreatic secretion. I. Subcellular 9. Yoo SH, Oh YS, Kang MK, Huh YH, So SH, Park HS, Park HY. Localization of distribution of calcium and magnesium in the exocrine of the guinea three types of the inositol 1,4,5-trisphosphate receptor/ Ca2+ channel in the pig. J Cell Biol. 1975;65:88-102. secretory granules and coupling with the Ca2+ storage proteins chromogranins A 33. Moreno A, Lobatón CD, Santodomingo J, Vay L, Hernández-SanMiguel E, and B. J Biol Chem. 2001;276:45806-12. Rizzuto R, Montero M, Alvarez J. Calcium dynamics in catecholamine-

422 The Journal of Physiological Sciences Vol. 56, No. 6, 2006 Estrogen and Muscle Atrophy

containing secretory vesicles. Cell Calcium. 2005;37:555-64. trisphosphate receptor isoforms in adrenal chromaffin cells. FEBS Lett. 34. Poulsen JCJ, Caspersen C, Mathiasen D, East JM, Tunwell REA, Lai FA, Maeda 2005;579:2597-603. N, Mikoshiba K, Treiman M. Thapsigargin-sensitive Ca2+-ATPases account for 38. Blondel O, Bell GI, Moody M, Miller RJ, Gibbons SJ. Creation of an inositol 1,4,5- Ca2+ uptake to inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive Ca2+ trisphosphate-sensitive Ca2+ store in secretory granules of insulin-producing stores in adrenal chromaffin cells. Biochem J. 1995;307:749-58. cells. J Biol Chem. 1994;269:27167-70. 35. Satoh T, Ross CA, Villa A, Supattapone S, Pozzan T, Snyder SH, Meldolesi J. 39. Blondel O, Moody MM, Depaoli AM, Sharp AH, Ross CA, Swift H, Bell GI. The inositol 1,4,5,-trisphosphate receptor in cerebellar Purkinje cells: Localization of inositol trisphosphate receptor subtype 3 to insulin and quantitative immunogold labeling reveals concentration in an ER somatostatin secretory granules and regulation of expression in islets and subcompartment. J Cell Biol. 1990;111:615-24. insulinoma cells. Proc Natl Acad Sci USA. 1994;91:7777-81. 36. Volpe P, Villa A, Damiani E, Sharp AH, Podini P, Snyder SH, Meldolesi J. 40. Ravazzola M, Halban PA, Orci L. Inositol 1,4,5-trisphosphate receptor subtype 3 Heterogeneity of microsomal Ca2+ stores in chicken Purkinje neurons. EMBO J. in pancreatic islet cell secretory granules revisited. Proc Natl Acad Sci USA. 1991;10:3183-9. 1996;93: 2745-8. 37. Huh YH, Yoo JA, Bahk SJ, Yoo SH. Distribution profile of inositol 1,4,5-

The Journal of Physiological Sciences Vol. 56, No. 6, 2006 423