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Biochemical and Biophysical Research Communications 259, 656–660 (1999)

Article ID bbrc.1999.0818, available online at http://www.idealibrary.com on

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illisecond Analyses of Ca Initiation Sites Evoked byuscarinic Receptor Stimulation in Exocrine Acinar Cells

aruo Takemura,*,1 Shohei Yamashina,† and Akihisa Segawa†Department of Pharmacology, Sapporo Medical University, South 1, West 17, Sapporo 060-8556; andDepartment of Anatomy, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan

eceived May 5, 1999

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High speed laser confocal microscopy (8 ms/image)as applied to the dissociated parotid acini as a model

o study Ca21 signaling mechanisms in non-excitablexocrine secretory cells. Immunofluorescence micros-opy showed the localization of IP3 receptor type 2 alonghe apical membrane region. Muscarinic stimulationith carbachol evoked a rise in [Ca21]i that was initiated

rom apical region and propagated into basal region asa21 waves. This was most clearly observed when extra-ellular Ca21 was omitted. Carbachol also triggered thebrupt increase of [Ca21]i simultaneously at both basalnd apical regions in many acini. Within an acinus, eachell responded synchronously. The present results sug-est that one Ca21 initiation site in the rat parotid acinarell is apical region, corresponding to the localization ofP3 receptors. Another Ca21 initiation site is basal re-ion, which seems to be related to Ca21 entry from ex-racellular medium and/or Ca21 release from basallyocated organelles such as nuclei and endoplasmic retic-lum. © 1999 Academic Press

Exocrine acinar cells, including salivary acinar cells,re structurally and functionally polarized with theasal region and apical (luminal) region (1, 2). Theeceptors for secretagogues are localized in the basalegion whereas the apical region has a high density ofecretory granules which ultimately are released byxocytosis at the luminal plasma membrane. Manytudies showed that the increase in cytoplasmic freealcium ([Ca21]i) initiated at the apical region andropagated to the basal region (3–6). Accordingly, ino-itol 1,4,5-trisphosphate (IP3) receptors are localized inhe apical membrane region in pancreatic and subman-ibular acinar cells (7, 8). In contrast, Dissing and hisollaborators (9–11) reported that muscarinic stimula-ion results in an increase in [Ca21]i simultaneouslyoth at baso-lateral and apical regions in parotid, lac-

1 To whom correspondence should be addressed. Fax: 81-11-612-861. E-mail: takemura@sapmed.ac.jp.

656006-291X/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

f Ca21 signals in exocrine acinar cells are still obscure.ccording to the reports, the increase in [Ca21]i accom-lishes within a few second after onset of Ca21 initia-ion. Such changes take place in the thick acinus (ap-roximately 30–50 mm in diameter), which hampershe clear imaging of Ca21 by the overlapping out-of-ocus noise. The structural integrity of acinus wouldave deep biological significance to perform normalxocrine function; if it is destroyed the secretory func-ion of acinar cells reduces greatly (1). These particularharacteristics in exocrine glands make it quite diffi-ult for conventional approaches to obtain accuratenformation on Ca21 signalling under the physiologicalondition. Here we used high speed confocal laser scan-ing microscope, which allows rapid acquisition of sec-ioned images non-destructively from thick specimensore than the video-rate (33 ms/image), to study cal-

ium signalling mechanisms in rat parotid acinar cellsocated in the intact acini, in situ. Parotid acinar cellsave an IP3-mediated calcium signalling system driveny muscarinic receptor stimulation (12). Ca21 waves (6)nd Ca21 oscillations (13) are constantly observed anduggested to be related to IP3-sensitive intracellulara21 stores, yet direct evidence to support this conceptas been lacking. The present results showed unex-ectedly rapid changes of [Ca21]i, of the order of milli-econd, to occur in parotid acinar cells in response tohe secretagogue stimulation although exocrine acinarells are recognized as non-excitable cells.

ATERIALS AND METHODS

Immunofluorescence microscopy of IP3 receptor. Pieces of parotidlands were fixed with 4% paraformaldehyde for one hour, and cryo-ections (8 mm) were treated with mouse monoclonal antibody againstP3 receptor type 2 (KM1083) raised against the synthetic peptideorresponding to the COOH-terminal regions of the human IP3 receptors described previously (14). After washing, the sections were treatedith FITC-labeled anti-mouse IgG and observed under the epifluores-

ence microscope Zeiss Axioplan (Carl Zeiss, Germany).

Isolation of acini. The isolated parotid acini were prepared fromale Sprague-Dawley rats (4–5 week-old) as described previously

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1, 2). Briefly, the parotid gland was removed, trimmed of the con-ective tissue and cut into small pieces. They were digested withollagenase (2 mg/ml) in Dulbecco’s modified Eagle medium (DMEM)ontaining 25 mM Hepes, pH 7.4 for 30 min at 37 6 °C with constanthaking and gentle pipetting. The isolated acini were incubated withuo-3/AM (10 mM) for 20 min in DMEM at room temperature. Afterashing, the acini were resuspended in Krebs-Ringer-Hepes me-ium (KRH) containing 0.2% bovine serum albumin. The composi-ion of KRH is as follows: 120 mM NaCl, 5.4 mM KCl, 1.0 mM CaCl2,.8 mM MgCl2, 11.1 mM glucose, 20 mM Hepes, (pH 7.4). For omis-ion of extracellular Ca21, Ca21-free KRH containing 0.2 mM EGTAas used.

Observation of the intracellular Ca21 signals by confocal lasercanning microscopy. Fluo-3-loaded acini were placed onto a cover-lip and viewed with the inverted light microscope (Nikon Diaphot00, objective 340, n.a. 1.3, Nikon, Tokyo, Japan) equipped withigh speed confocal laser scanning microscope (Oz with InterVersionoftware, Noran Instruments Inc., Middleton, WI). For muscariniceceptor stimulation, drops of KRH containing carbachol (10 mM)armed at 37 6 °C were added directly onto the cells (15, 16). The

ntracellular Ca21 signals from acini were measured according to theethod of Guerineau et al. (17). Confocal images were taken every 8s. An Ar/Kr laser was used to excite the fluo-3 at 488 nm, and

mission signals were collected through a 515-nm barrier filter. Thebtained images were pseudocoloured; cool colours represent lowuorescence intensity whereas warm colours correspond to highuorescence intensity.

ESULTS

Localization of IP3 receptors. The localization of IP3

eceptor in the rat parotid acinar cells was studied bymmunofluorescence using monoclonal antibodygainst IP3 receptor type 2 (14). As shown in Fig. 1,ositive fluorescence was observed along the apico-ateral region of acinar cells. Simultaneous F-actintaining with rhodamine-phalloidin revealed the colo-alized staining pattern (data not shown), indicatinghat the area showing positive fluorescence is the in-ercellular canaliculi, a specialized lumen structureadiating from the center of acini (1, 18). Thus theositive structure for IP3 receptor type 2 is consideredo localize along, or close to, the apical (luminal)

FIG. 1. Immunofluorescence localization of IP3 receptor in the rositive immunofluorescence can be seen along the intercellular can

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lasma membrane. The polarized pattern of IP3 recep-or observed in the parotid acinar cell was quite similaro that in the submandibular gland acinar cells (2, 8).

Millisecond analyses of Ca21 by high speed confocalicroscopy. Figure 2 shows the changes of fluores-

ent profiles in fluo-3-loaded parotid acinar cells follow-ng carbachol stimulation. High speed confocal imag-ng taken every 8 ms clearly showed two patterns ofuorescent changes occurring in the stimulated cellsFigs. 2A and 2B). In one type (Fig. 2A), an increase ofuorescent intensity initiated at the center of acini andpread out to basal region. This pattern of fluorescenthange was prominently observed in the absence ofxtracellular Ca21. In another type (Fig. 2B), fluores-ent intensity elevated from almost whole cytoplasmicrea of acinar cells.Spatiotemporal fluorescent changes in acinar cells

hown in Fig. 2 was analyzed in more detail, by theuantitative measurement of fluorescent intensity inhe small areas plotted on the basal and apical regionsf the acini (Fig. 3). In cells shown in Figs. 3A and 3Bapical type), [Ca21]i in the apical region exhibitedteep rise for the initial 200 ms, then the rise in [Ca21]i

ecomes slow and reached a plateau by 1 s. [Ca21]i inhe basal region increased slowly and steadily, andnally reached its plateau level by 1 s. In Figs. 3C andD (whole cell type), the rise in [Ca21]i initiated fromoth the basal and apical region. After an abrupt in-rease during the initial 100 ms, [Ca21]i elevated grad-ally and reached a plateau by 600–800 ms. Althoughhe pattern of [Ca21]i differed in the apico-basal direc-ion, cells in either type exhibited the synchronizedime course of fluorescent changes within the acini.

ISCUSSION

IP3 receptors and Ca21 signalling in exocrine glands.olecular cloning studies have shown that there are

hree types of IP3 receptor derived from distinct genes

parotid acinar cells. (A) Nomarsky image. (B) Fluorescence image.culi (arrows). Bar, 10 mm.

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FIG. 2. Ca21 signals in the dissociated rat parotid acini stimulated by carbachol shown by high speed confocal microscopy. Fluorescencehanges of fluo-3-loaded cells were imaged every 8 ms in the absence (A) and presence (B) of extracellular Ca21. Cells which exhibited thearliest response are shown. Images are displayed from the top left to the right bottom. Final panel (right bottom) in (A) and (B) shows theuorescent image 1 s after stimulation with carbachol. Bar, 10 mm.

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19). Nathanson et al. (7) reported that IP3 receptorype 3 is localized in the Ca21 wave trigger zone ofancreatic acinar cells. In addition, all three types ofP3 receptors are observed in the apical region in pan-reatic acini (7, 8) and submandibular acinar and ductells (8). We have recently shown that IP3 receptor typelocalized in the apical region of submandibular duct

ells and was correlated to the initiation sites of Ca21

pikes (2). In the present study, IP3 receptor type 2 wasocalized in the apical membrane region of parotid aci-ar cells (Fig. 1), from where Ca21 waves was initiatedven in the absence of extracellular Ca21 (Fig. 2A).pical Ca21 signalling stimulated by carbachol in thebsence of extracellular Ca21 has also been observed inhe dispersed parotid acinar cells (6). Gromada et al.10) have reported that IP3 is released to the cytosolithin the first second after stimulation in the same

ell system. It is, thus, likely that IP3-mediated Ca21

ignalling mechanisms is located in the apical mem-rane area in the rat parotid acinar cells.IP3-sensitive intracellular Ca21 pools have been

hown to associate with secretory granules in subman-

FIG. 3. Quantitative analyses of the fluorescent changes in the aasal and apical regions of the acini, respectively. Bar, 10 mm.

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ibular (20) and pancreatic (21) acinar cells. However,he present study failed to demonstrate the positivemmuno-signals of IP3 receptor type 2 in the secretoryranule area. Yule et al. (22) argued whether zymogenranules are physiologically relevant Ca21 pools, basedn the observation that zymogen granules do not ex-ress IP3 receptors in pancreatic acinar cells. It isherefore needed to clarify whether IP3 receptors otherhan type 2 are present on secretory granules in pa-otid acinar cells, and if so, whether they are capable ofeleasing Ca21 from secretory granules under the phys-ological condition.

Mechanisms responsible for basal Ca21 signalling.Ca21]i in basal region was low and increased by mus-arinic stimulation, if not all, in the absence of extra-ellular Ca21 (Figs. 2A and 3B). This indicates thata21 initiation sites correlate with calcium entry from

he extracellular Ca21 into the cells. The fundamentalechanism why so rapid Ca21 response occurs is un-

nown. Voltage-gated Ca21 channels, responsible forhe rapid Ca21 response in excitable cells such as neu-

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ng the exocrine acinar cells (23). Receptor-mediateda21 channels in relation with capacitative Ca21 entry,hich exists in parotid acinar cells (24), might be op-rative; however, Huang and Putney (25) have recentlyhowed that a significant delay is observed between thenitiation of Ca21 depletion of IP3-sensitive pools andhe activation of a release-activated Ca21 current.

Besides, major organelles located in the basal cyto-lasm of parotid acinar cells are nuclei and rough endo-lasmic reticulum (ER) (1). ER is one of well-recognizeda21 pools. The outer membrane of nuclear envelope is

ontinuous with the ER membrane and has been sug-ested to possess ER properties (26, 27). In our observa-ion, transmitted light images showed the correlationetween basal Ca21 initiation sites with the nuclei andough ER (data not shown). Thus, other sites involved ina21 initiation seem to be related with Ca21 release fromasally-located organelles such as nuclei and ER.

Intercellular communication. One striking observa-ion in the present study was the synchronized calciumesponse occurring between cells that constitute the aci-us. In some acini, we found Ca21 oscillations in theuclear region, which also occurred synchronously evenhough the distance of nuclei were more than 25 mmpart (unpublished observation). This is an apparent con-rast to that observed in the duct, in which calcium re-ponse started non-synchronously from “pioneer cells”nd spread to neighboring cells (2). Intercellular commu-ication of Ca21 between cells in the acini must have an

mportant role in Ca21 signaling. Immunohistochemicaltudies on gap junctional channel proteins, connexins,evealed the presence of positive reactions between thecinar, but not duct, cells (28, 29). Gap junctional com-unication modulates Ca21 oscillations and enzyme se-

retion in pancreatic acini (30). Pretreatment of cells withctanol, a gap junction inhibitor, abolished an elevation ofCa21]i induced by carbachol in parotid acini as a wholeunpublished observation).

CKNOWLEDGMENTS

We are grateful for the gift of antibody against IP3 receptor fromrs. T. Sugiyama and K. Mikoshiba. We also thank Ms. M. Miya-awa and M. Ino for the image analysis and technical support ofsing a confocal laser microscopic system (Oz).

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