Changes in mitochondrial activity evoked by cholecystokinin in isolated mouse pancreatic acinar...

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Cellular Signalling 15 (2003) 1039–1048

Changes in mitochondrial activity evoked by cholecystokinin

in isolated mouse pancreatic acinar cells

Antonio Gonzalez*, Marıa P. Granados, Gines M. Salido, Jose A. Pariente

Faculty of Veterinary Sciences, Department of Physiology, University of Extremadura, Avenida Universidad s/n E-10071, Caceres, Spain

Received 23 February 2003; accepted 11 April 2003

Abstract

In the present study, we have employed confocal laser scanning microscopy to investigate the effect that stimulation of mouse pancreatic

acinar cells with the secretagogue cholecystokinin (CCK) has on mitochondrial activity. We have monitored changes in cytosolic as well as

mitochondrial Ca2 + concentrations, mitochondrial membrane potential and FAD autofluorescence by loading the cells with fluo-3, rhod-2 or

JC-1, respectively.

Our results show that stimulation of cells with cholecystokinin led to release of Ca2 + from intracellular stores that then accumulated into

mitochondria. In the presence of the hormone a depolarization of mitochondrial membrane potential was observed, which partially recovered;

in addition a transient increase in FAD autofluorescence could be observed. Similarly, treatment of cells with thapsigargin induced increases

in mitochondrial Ca2 + and FAD autofluorescence, and depolarized mitochondria. Pretreament of cells with thapsigargin blocked

cholecystokinin-evoked changes. Similar results were obtained when the cells were incubated in the presence of rotenone, which blocks the

mitochondrial electron transport chain. Our findings are consistent with changes in mitochondrial activity in response to stimulation of

pancreatic acinar cells with cholecystokinin. Following stimulation, mitochondria take up Ca2 + that could in turn activate the mitochondrial

machinery that may match the energy supply necessary for the cell function during secretion, suggesting that Ca2 + can act as a regulator of

mitochondrial activity.

D 2003 Elsevier Science Inc. All rights reserved.

Keywords: Pancreas; Cholecystokinin; Calcium; Mitochondria; FAD; Fluorescence; Confocal microscopy

1. Introduction

In pancreatic acinar cells activation of phospholipase C

(PLC)-linked receptors by agonists, such as acetylcholine

(ACh) or cholecystokinin (CCK), produces an increase in

the concentration of inositol 1,4,5-trisphosphate (IP3) in

the cytosol. IP3 in turn releases calcium (Ca2 +) from

cytoplasmic stores leading to an increase in cytosolic free

calcium concentration ([Ca2 +]c) [1]. Ca2 + signals are not

only a result of its release from intracellular stores but as

well a co-ordinate influx from the extracellular space [2] as

well as Ca2 + transport across plasma membrane [3] and

into intracellular organelles [4]. Although agonist-evoked

Ca2 + signals have been the subject of numerous studies

during the recent years, a point of the investigations is

0898-6568/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved.

doi:10.1016/S0898-6568(03)00067-6

* Corresponding author. Tel./fax: +34-927-257154.

E-mail address: [email protected] (A. Gonzalez).

actually being focused onto the role of mitochondria in

Ca2 + signalling.

Mitochondria has been described as an organelle with a

high ability to sequester Ca2 + [5]. Ca2 + uptake into mito-

chondria is mediated by a high capacity uniporter in the

inner membrane that permits transport of calcium down its

electrochemical gradient [6]. Entry of Ca2 + into mitochon-

dria can take place as well via a rapid uptake mode [7,8].

Ca2 + efflux occurs via Na+-dependent and Na+-independent

mechanisms [6]. In addition, Ca2 + efflux from mitochondria

can take place through the opening of the permeability

transition pore [6,9] as well as via a Ca2 + uniporter [10].

It has been shown in different tissues that, under phys-

iological conditions, agonist-induced increases in [Ca2 +]care accompanied by increases in mitochondrial free Ca2 +

concentration ([Ca2 +]m) [11–13]. In exocrine pancreatic

acinar cells stimulation with a maximal concentration of a

Ca2 +-mobilizing agonists leads to an increase in mitochon-

drial Ca2 + content [14–19].

A. Gonzalez et al. / Cellular Signalling 15 (2003) 1039–10481040

The activity of mitochondria in Ca2 + signalling has

been further studied and raised interesting results that

support an active role in Ca2 + handling by the cell. It

has been proposed that mitochondria are disposed in the

immediate vicinity to Ca2 + releasing channels of the

endoplasmic reticulum (ER) [20–22], mainly close to IP3receptors [13,23], as well as to the ryanodine receptor

[16,24]. Moreover, participation of mitochondria in Ca2 +-

induced Ca2 + release (CICR) mechanisms has been

described [25], which has been explained by opening of

A. Gonzalez et al. / Cellular Signalling 15 (2003) 1039–1048 1041

permeability transition pores in the inner mitochondrial

membrane [6,9] as well as via the Ca2 + uniporter [10].

Due to its Ca2 + accumulating nature, mitochondria has

been proposed to be a sink for Ca2 + that could allow

modulation of [Ca2 +]c and serve as a stimulatory meta-

bolic signal when the cell is stimulated [26]. The increase

in [Ca2 +]c, after release of Ca2 + from the ER, is followed

by an increase in [Ca2 +]m [16]; recovery of intramitochon-

drial Ca2 + signal is longer than that for the cytosol [12,15]

and the peak of [Ca2 +]m is attained later than the peak in

[Ca2 +]c [15,27].

Given that the pancreatic acinar cell presents a polarized

architecture, distribution of mitochondria within the cytosol

has been object of study. Consistent evidence exists regard-

ing its distribution surrounding the zymogen granular area,

as well as between the granules, around the nucleus and

just beneath the plasma membrane [15]. The facts presented

until recently make it interesting to the study of the

relationship of mitochondria with Ca2 + signalling. How-

ever, although most of the studies have been focused onto

the Ca2 + buffering capacity of mitochondria and modu-

lation of cytosolic signals, less attention has been paid to

the consequences of uptake of Ca2 + by the organelle on its

activity. The mitochondrial electron transport chain pro-

vides the energy which drives the diffusion of protons into

the matrix promoting ATP synthesis. Hence, whenever it is

needed, mitochondrial metabolism is adapted to the actual

energy demand of the cell [11,28,29]. Ten years ago, it

was shown that mitochondrial function was progressively

altered in anoxia in arterial chemoreceptors [30]. The

authors followed the changes in autofluorescence derived

from NAD(P)H and FAD and concluded that the observed

changes were related to activation of mitochondrial dehy-

drogenases. In another study, carried out in mouse sensory

neurons, it was suggested that changes in mitochondrial

energetics were related to Ca2 + [31]. Activation of mito-

chondrial respiratory chain and an increase in the proton

motive force by Ca2 + accumulation into the organelle

leads to an increase in the activity of mitochondrial

dehydrogenases with a net action on production of NADH

[11,32]. Recently, a correlation between NADH and Ca2 +

signals in mouse pancreatic acinar cells has been proposed

[33]. Since NADH and FAD fluoresce when excited at

Fig. 1. Cytosolic distribution of mitochondria after incubation of pancreatic

mitochondria. Employing confocal laser scanning microscopy bright fluorescent

area, although concentration near the zymogen granule area, surrounding the nuc

(A0) Cells were loaded with MitoTrackerk Green FM at room temperature. (A1)

in the plain in which image A0 was taken transforms the punctuate, mitochondria

observed at the parts of the cell where the zymogen granules (ZG) and the nucle

(B) cell pole shows points of higher intensity of fluorescence at the points at w

where no mitochondria is found. Left panels (in B0, C0 and D0) show transmitted

distribution of zymogen granules located in the luminal (secretory) cell pole. Rig

with the specific mitochondrial dyes Rhod-2 for [Ca2 +]m (B1) and JC-1 for wm

loading revealed the presence of bright structures within the cytoplasm, which d

FAD autofluorescence.

certain wavelengths, it can serve as a valuable tool to

monitor changes in mitochondrial metabolism [11,30,

31,33].

In the present study, we provide further evidences about

the implications that uptake of Ca2 + by mitochondria

presents on its metabolical activity in the exocrine pan-

creas. The results are consistent with changes in mitochon-

drial activity in response to stimulation of pancreatic acinar

cells with CCK-8. Following stimulation of the cells,

mitochondria take up Ca2 + that would in turn activate

the mitochondrial machinery that may match the energy

supply necessary for the cell function during secretion. Our

findings show that the changes in [Ca2 +]m, wm and FAD

autofluorescence depend on mobilization of Ca2 + from

intracellular stores and are independent of extracellular

Ca2 +.

2. Materials and methods

2.1. Animals and chemicals

Adult male swiss mice were used for this study. Fluo-3

AM, Rhod-2 AM, MitoTrackerk Green FM and 5,5V-6,6V-tetrachloro-1,1V,3,3V-tetraethylbenzimidazolylcarbocyanine

iodide (JC-1) were obtained from Molecular Probes

(Europe) and thapsigargin from Alomone Labs (Israel).

All other materials used were obtained from Sigma

(Spain).

2.2. Preparation of isolated pancreatic acinar cells

Mouse pancreatic acinar cells were isolated as described

previously [34]. Briefly, animals were sacrificed by cer-

vical dislocation, the pancreas was rapidly removed and

placed in a buffer of the following composition: 130 mM

NaCl, 4.7 mM KCl, 1.3 mM CaCl2, 1 mM MgCl2, 1.2

mM KH2PO4, 10 mM glucose, 0.2% (w/v) albumin,

0.01% trypsin inhibitor and 10 mM HEPES (pH 7.4

adjusted with NaOH). The pancreas was then incubated

in the presence of collagenase type V for 10 min at 37 jCin a shaking water bath. With this isolation procedure

single cells as well as small clusters consisting of 2–5

acinar cells with different fluorophores that selectively accumulate into

spots could be detected, being in principle spread all through the cytosolic

leus and beneath the basolateral plasma membrane could be observed too.

A three-dimensional projection of the spatial distribution of mitochondria

, into increased levels of fluorescence. A valley, lower fluorescence, can be

us (N) are located. (A2) A line taken from the lumen (L) towards the basal

hich mitochondria are present; fluorescence comes close to zero in areas

light photograph of pancreatic acinar cells depicting the typical polarized

ht panels show the fluorescence image of pancreatic acinar cells incubated

determinations (C1), and signals due to FAD autofluorescence (D1). Dye

epicted a similar pattern of distribution for all fluorophores as well as for

Fig. 2. CCK-8-evoked response in [Ca2 +]c and [Ca2 +]m. (A) Perfusion

pancreatic acinar cells with 10 nM CCK-8 led to a transient increase in

[Ca2 +]c followed by a progressive decrease towards the prestimulation

level. (B) Stimulation of pancreatic acinar cells by inclusion of 10 nM

CCK-8 in the perfusion medium led to an increase in [Ca2 +]m. Time course

of changes in [Ca2 +]m differed greatly from that of [Ca2 +]c; at the time

points at which [Ca2 +]c was expected to return towards the basal level

[Ca2 +]m still remained elevated, and lasted longer at this point compared to

[Ca2 +]c.


cells were obtained. All experiments were performed at

room temperature (23–25 jC) and in the presence of Ca2 +

(1.3 mM) in the extracellular medium, except those experi-

ments in the presence of thapsigargin, where no Ca2 +

present and 1 mM EGTA was added to the perfusion

medium. Cell viability was greater than 95% as assayed by

Trypan blue exclusion test.

2.3. Dye loading

Freshly isolated mouse pancreatic acinar cells were

loaded with a different fluorescent dye depending on the

parameter to be monitored, following previously established

methods [15]. For determination of [Ca2 +]c cells were

loaded with fluo-3 AM (4 AM) at room temperature (23–

25 jC) for 40 min. [Ca2 +]m was monitored following

loading of the cells with rhod-2 AM (8 AM) at 4 jC for

15 min. Changes in wm were determined after loading of

cells with JC-1 (10 Ag/ml) at 37 jC for 15 min. Loading of

cells with MitoTrackerk Green FM (100 nM) was per-

formed at room temperature (23–25 jC) for 30 min. This

dye has been widely employed as a mitochondrial marker.

Following dye loading cells were centrifuged and resus-

pended in fresh buffer. For determinations of FAD fluores-

cence no dye was employed. The oxidized form FAD is

autofluorescent [29,30] and no dye loading of the cells is

required.

After loading, cells were kept at 4 jC until use and the

experiments were performed within the next 4 h.

2.4. Confocal microscopy studies

For monitorization of fluorescence signals, aliquots of

dye-loaded cells were placed onto a coverslip attached to the

bottom of a perfusion chamber on the stage of a confocal

microscope (Nikon eclipse TE300) and continuously super-

fused with a Na–HEPES buffer containing (in mM): 140

NaCl, 4.7 KCl, 1.3 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose

(pH adjusted to 7.4). Employing a confocal laser-scanning

system (Bio-Rad, MRC 1024) fluorescence images of

256� 256 pixels with a resolution of 0.287 Am/pixel were

recorded every 4 s. Excitation lights at 488 nm from a 100-

mW Argon ion laser and 543 nm from a 25-mW helium–

neon laser were employed depending on the fluorophore

assayed. Determination of emitted fluorescence of the dyes

was performed employing different photomultipliers as

previously described [15].

For FAD autofluorescence, cells were excited at 488 nm

and emitted fluorescence was detected with a band pass

filter in a photomultiplier at 580/35 nm. Intensity of exci-

tation light had to be increased up to 10% by removal of

neutral density filters, which had to be employed for the

other dyes, and that reduced laser intensity to 0.3–1% to

diminish photobleaching. For monitorization of changes in

mitochondrial fluorescence, small bright fluorescent areas

were selected [15].

Results are expressed as the values of fluorescence

emitted at each wavelength. In the case of JC-1, a ratio-

metric dye, data are presented as emission ratios.

3. Results

3.1. Intracellular distribution of mitochondria

In our experiments, we have employed different fluores-

cent dyes that selectively accumulate into mitochondria and

report changes in physiological parameters within these

organelles. It was of interest to analyze if, following

incubation, the various fluorophores have the same distri-

bution in the cell. Initially, localization of mitochondria

within pancreatic acinar cells was assayed by incubation

of cells in the presence of MitoTrackerk Green FM, a dye

that selectively accumulates inside these organelles [35].

Under these conditions bright fluorescent spots were in

principle spread all through the cytosolic area, but often

concentrated near the zymogen granule area, between the

nucleus and beneath the basolateral plasma membrane (n = 7

Fig. 3. Effect of CCK-8 stimulation of pancreatic acinar cells on wm. (A)

Stimulation of pancreatic acinar cells with 10 nM CCK-8 led to a decrease

in fluorescence of JC-1 ratio, as an index of a depolarization of wm.

Mitochondrial depolarization recovered partially although not completely,

reaching a stable fluorescence value under the prestimulation level. (B)

Disruption of mitochondrial electron transport chain by perfusion of cells

with rotenone (10 AM) led to a decrease in wm and blocked CCK-8-induced

decrease in wm.

Fig. 4. Effect of CCK-8 stimulation of pancreatic acinar cells on FAD

autofluorescence and control experiments with FCCP. (A) Stimulation of

pancreatic acinar cells with 10 nM CCK-8 led to a transient increase in FAD

autofluorescence that later returned towards the resting levels. (B) Perfusion

of pancreatic acinar cells with FCCP (100 nM) induced as well changes in

the oxidized state of FAD that slowly returned towards the prestimulation

level. (C) To further characterize the effect of FCCP on mitochondrial

activity, JC-1-loaded cells were perfused with the protonophore. As it can

be observed, FCCP (100 nM) led to a non-reversible loss of mitochondrial

membrane potential.


exp/23 cell) (Fig. 1A). Single mitochondria could also be

found in between the zymogen granules in the luminal area.

Sometimes, it could be observed an arrangement of mito-

chondria in a belt shaped form between the secretory and the

basal cell pole. However, this was not the typical, and

therefore not the general, pattern of fluorescence distribution

observed in our experimental conditions. Distribution of

fluorescence from the other dyes employed throughout the

present study, Rhod-2 for determination of changes in

[Ca2 +]m (n = 19 exp/43 cells) and JC-1 for determination

of changes in wm (n = 42 exp/101 cell), was similar to that of

MitoTrackerk Green FM (Fig. 1B and C). The same

distribution was observed for FAD autofluorescence (Fig.

1D) (n= 58 exp/230 cell). Fig. 1A shows a representative

fluorescence image of a pancreatic acinar cell labelled with

MitoTrackerk Green FM. The image shows the typical

punctuated pattern of staining of mitochondria (n = 7 exp/23

cell). A three-dimensional representation of the spatial

distribution of mitochondria in the plain shown in Fig. 1A

can be seen in Fig. 1B. Higher levels of fluorescence

coincide with the distribution of the mitochondria whereas

a valley can be observed coinciding with the localization of

the nucleus (N) and the zymogen granules (ZG) in the basal

and luminal cell poles, respectively. The intensity of fluo-

rescence from lumen (L) to basal (B) due to accumulation of

MitoTrackerk Green FM in the mitochondria can be

observed in Fig. 1C. Lower levels of fluorescence coincide

with the area of the zymogen granules (ZG) and the nucleus

(N). A similar pattern of distribution has been observed

when the pancreatic acinar cells were loaded with the other


dyes tested in the present work, as well as for FAD

autofluorescence.

3.2. Effect of CCK-8 on cytosolic ([Ca2+]c) and mitochon-

drial ([Ca2+]m) Ca2+ concentrations

As expected, stimulation of fluo-3-loaded pancreatic

acinar cells with 10 nM CCK led to a transient increase

Fig. 5. Control experiments performed to check whether autofluorescence w

Autofluorescence recorded from one single area, considered mitochondria. Rote

should decrease the oxidative state of FAD. Pretreatment of pancreatic acinar cells

and blocked CCK-8-evoked increases. (B) Changes in autofluorescence from a wh

and changes recorded (shown in A). Changes in autofluorescence depict the sam

Changes in autofluorescence when a small area in the cytosol at the basolate

autofluorescence when a small area in the granular region was selected. (E) Change

detectable changes in autofluorescence could be detected showing therefore that the

of FAD within the mitochondria.

in fluorescence, index of an increase in [Ca2 +]c, followed

by a progressive decrease towards the resting level (n = 3

exp/12 cell) (Fig. 2A). A value of stable fluorescence, close

to prestimulation level, was reached after 5 min of stim-

ulation with CCK-8. When rhod-2-loaded cells were stimu-

lated with the hormone an increase in fluorescence could be

observed, related to increase in [Ca2 +]m, that remained

elevated longer time compared to cytosolic responses

as due to changes in the oxidized state of mitochondrial FAD. (A)

none (10 AM) inhibits mitochondrial electron transport chain and, hence,

with the mitochondrial inhibitor led to a decrease in FAD autofluorescence

ole cell in which independent fluorescent areas, mitochondria, were selected

e pattern both in single (mitochondrial) areas and in the complete cell. (C)

ral side of the cell without mitochondria was selected. (D) Changes in

s in autofluorescence when a small area in the nuclear area was selected. No

observed effects in the treatments were due to changes in the oxidized state

Fig. 6. Ca2 + dependence of changes in [Ca2 +]m, wm and FAD

autofluorescence. (A) Inclusion of thapsigargin (1 AM) in the perfusion

medium led to an increase in [Ca2 +]m. Stimulation of pancreatic acinar cells

with CCK-8 (10 nM) following thapsigargin treatment failed to induce a

further increase in [Ca2 +]m. (B) Perfusion of pancreatic acinar cells with

thapsigargin (1 AM) led to a depolarization of wm. The presence of

thapsigargin inhibited CCK-8-evoked changes in wm. (C) Inclusion of

thapsigargin (1 AM) in the perfusion medium led to a transient increase in

FAD autofluorescence. Stimulation of pancreatic acinar cells with CCK-8

(10 nM) following thapsigargin treatment failed to induce a further increase

in FAD autofluorescence. All experiments were performed in the absence of

extracellular Ca2 + (1 mM EGTA).


(n = 4 exp/8 cell/46 mit. area) (Fig. 2B). The time course of

rhod-2 reported fluorescence for [Ca2 +]m differed clearly

from that of fluo-3 for [Ca2 +]c. At the time points when

[Ca2 +]c was returning towards the prestimulation level,

[Ca2 +]m still remained elevated, returning to resting con-

centration at a later time point compared to [Ca2 +]c. A

similar pattern of Ca2 + mobilization was observed in the

absence of extracellular Ca2 +, and in the presence of 1 mM

EGTA (data not shown), showing that CCK-8-evoked

responses are due to release of Ca2 + from intracellular

stores.

3.3. Changes in mitochondrial membrane potential (wm)

When cells loaded with the mitochondria-specific volt-

age-sensitive dye JC-1 were perfused with 10 nM CCK-8

a depolarization of wm could be observed. CCK-8-evoked

depolarization of mitochondria recovered slowly, although

not completely, remaining in a value below the prestimu-

lation level. A stable value of wm was reached approx-

imately 5 min after stimulation with CCK-8 (n= 6 exp/13

cell/61 mit. area) (Fig. 3A). Disruption of the activity of

the mitochondrial electron transport chain, which main-

tains a large negative wm, would lead to loss of this

gradient and therefore to depolarization of mitochondria.

Preincubation of cells in the presence of 10 AM rotenone,

which blocks mitochondrial electron transport chain, depo-

larized mitochondria and inhibited the response induced by

CCK-8 stimulation on wm (n = 4 exp/6 cell/17 mit. area)

(Fig. 3B).

3.4. Effect of CCK-8 stimulation on FAD autofluorescence

As it has been shown, stimulation of pancreatic acinar

cells with CCK-8 leads to an increase in [Ca2 +]m and

depolarises wm. As a part of the influence of Ca2 + uptake

by mitochondria, the activity of the electron transport chain

could be modified, leading to changes in the rate of

oxidation of FAD. In our experimental conditions a transient

increase in FAD autofluorescence could be observed follow-

ing stimulation of cells with the Ca2 +-mobilizing agonist,

that later decreased progressively towards the prestimulation

level (n = 7 exp/30 cell) (Fig. 4A).

Control experiments were carried out to verify that the

observed changes in fluorescence in this set of experiments

were due to changes in oxidation of mitochondrial FAD.

Perfusion of pancreatic acinar cells with the mitochondrial

uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhy-

drazone (FCCP; 100 nM), induced changes in FAD auto-

fluorescence consisting of a transient increase that later

returned to prestimulation level (n = 3 exp/32 cell) (Fig. 4B).

We further characterized the effect of FCCP on mito-

chondrial parameters. In a set of experiments, wm was

monitored. We could observe that perfusion of pancreatic

acinar cells with the protonophore produced a rapid break-

down of wm, identified as a decrease of JC-1 fluorescence

ratio (Fig. 4C). Depolarization of mitochondria was not

reversible (n = 4 exp/6 cell/39 mit. areas).

Additional control experiments were carried out to verify

that changes in fluorescence were due to changes in mito-

chondrial FAD. The experiments were performed by perfu-

sion of cells with rotenone, an inhibitor of the mitochondrial


electron transport chain. Perfusion of cells with the mito-

chondrial inhibitor (10 AM) induced a decrease in FAD

autofluorescence. In this set of experiments, inclusion of

rotenone in the perfusion medium blocked the CCK-8-

induced increase in FAD autofluorescence (n = 6 exp/19

cell) (Fig. 5A and B).

When small areas were recorded at the basolateral pole of

the cell in the cytosol without mitochondria (n = 2 exp/2

cell/10 area), in the granular area (2 exp/2 cell/8 area) and in

the nucleus (n = 2 exp/2 cell/4 area) no changes in FAD

autofluorescence could be detected, in spite of clearly

detectable signals when mitochondria were selected (n = 2

exp/2 cell/15 mit. area) (Fig. 5).

3.5. Dependence of CCK-8-evoked responses on Ca2+

mobilization

CCK-8 could stimulate the observed changes in mito-

chondrial parameters by terms of its Ca2 +-mobilizing

nature. Therefore, we performed a series of experiments in

which intracellular stores were depleted by perfusion of

pancreatic acinar cells with thapsigargin (Tps), an inhibitor

of the sarco-endoplasmic reticulum Ca2 +-ATPase (SERCA),

in a Ca2 +-free medium (containing 1 mM EGTA). Treat-

ment of cells with 1 AM Tps in the absence of extracellular

Ca2 + induced transient increases in both [Ca2 +]m (n = 3 exp/

6 cell/42 mit. area) and FAD autofluorescence (3 exp/32

cell). A depolarization of wm in response to treatment of

cells with the SERCA inhibitor was as well observed (n = 3

exp/4 cell/38 mit. area). Following the pretreatment of cells

with Tps, stimulation of pancreatic acinar cells with CCK-8

(10 nM) failed to evoke further changes in these parameters

(100% of cells tested) (Fig. 6).

4. Discussion

In the present study, we have employed confocal laser

scanning microscopy to investigate the effect that stimula-

tion of mouse pancreatic acinar cells with the secretagogue

CCK-8 has on mitochondrial activity. The consequences of

changes in [Ca2 +]c in response to the hormone on mito-

chondrial function were assessed. We have carried out

monitorization of changes in [Ca2 +]c as well as [Ca2 +]m,

wm and FAD autofluorescence.

In our experimental conditions, bright fluorescent spots

could be detected which were in principle spread all through

the cytosolic area, but often concentrated near the zymogen

granule area, between the nucleus and beneath the baso-

lateral plasma membrane, as we have previously described

[15]. Single mitochondria could also be found in between

the zymogen granules in the luminal area. Sometimes it

could be observed an arrangement of mitochondria in a belt-

shaped form between the secretory and the basal cell pole as

it has been as well described [18,33]. The typical distribu-

tion of mitochondria that the latter group of authors has

proposed was, in our hands, more of a casual observation in

few, but not all, cells studied. Evidence for a general

widespread presence of mitochondria within the cytosol

were obtained from the experiments in which incubation

of pancreatic acinar cells were performed with different

markers that selectively accumulate into mitochondria. Dis-

tribution of fluorescence from the dyes employed through-

out the present study, rhod-2 for determination of changes in

[Ca2 +]m and JC-1 for determination of changes in wm, was

similar to that of MitoTrackerk Green FM. The same

distribution was observed for FAD autofluorescence, as it

has been as well shown for NADH [33]. Coincidence of

fluorescence signals of NADH with that of MitoTrackerkGreen FM has been proved [36].

It has been shown that stimulation of pancreatic acinar

cells with Ca2 +-mobilizing agonists leads to increases in

[Ca2 +]m [15,18]. Our data are in agreement with those

reported previously. When rhod-2-loaded cells were stimu-

lated with CCK-8 an increase in the fluorescence of rhod-2-

loaded cells could be observed. [Ca2 +]m remained elevated

longer time compared to cytosolic responses. The time

course of rhod-2 reported fluorescence for [Ca2 +]m differed

clearly from that of fluo-3 for [Ca2 +]c. At the time points

when [Ca2 +]c was returning towards the prestimulation

level, [Ca2 +]m still remained elevated, returning to resting

concentration at a later time point compared to [Ca2 +]c. A

similar pattern of Ca2 + mobilization was observed in the

absence of extracellular Ca2 +, showing that CCK-8-evoked

responses are due to release of Ca2 + from intracellular stores

(data not shown).

The uptake of Ca2 + by mitochondria depends on wm,

which is established by the electron-transport chain [31].

Therefore, disruption of wm by protonophores should be

associated to release of Ca2 + from mitochondria [15]. An

increase in [Ca2 +]m should lead to a depolarization of wm,

which can be explained by: first, cycling of Ca2 + across the

inner mitochondrial membrane and second, increase in ATP

consumption by the ATPases that transport Ca2 + out of the

cytosol, which would lead to increased flux of protons into

mitochondria for ATP synthesis through the F0�F1 ATP

synthase [31,37].

When the cells loaded with mitochondria-specific volt-

age-sensitive dye JC-1 were perfused with 10 nM CCK-8, a

depolarization of wm could be observed. CCK-8-evoked

depolarization of mitochondria recovered slowly although

not completely, remaining in a value under the prestimula-

tion level. Disruption of the activity of the mitochondrial

electron transport chain, which maintains a large negative

wm, would lead to loss of this gradient and therefore to

depolarization of mitochondria. As expected, preincubation

of cells in the presence of 10 AM rotenone, which blocks

mitochondrial electron transport chain, depolarized mito-

chondria and inhibited the response induced by CCK-8

stimulation on wm.

Depolarization of wm and the increase in [Ca2 +]m should

reflect changes in the mitochondrial redox state. This can be


monitored by following changes in the oxidized state of

NADH and FADH2. To further investigate the effect of

CCK-8 on mitochondrial activity, FAD autofluorescence

was monitored. Flavoproteins fluoresce when excited at

450 nm and resulting fluorescence can be monitored

between 510 and 590 nm. It is the oxidized form which

fluoresces under these conditions; therefore, the signal

decreases with reduction and increases with oxidation

[30]. Mitochondrial electron transport chain is associated

to oxidation of NADH and FADH2. The final step is

transfer of electrons to oxygen. A component of the chain

is a proton motive force which governs ATP production by

the F0�F1 ATP synthase located in the inner mitochon-

drial membrane. As it has been shown, stimulation of

pancreatic acinar cells with CCK-8 leads to an increase in

[Ca2 +]m and depolarizes wm. As a part of the influence of

Ca2 + uptake by mitochondria, the activity of the electron

transport chain could be modified, leading to changes in the

rate of oxidation of FAD. In our experimental conditions a

transient increase in FAD autofluorescence, i.e., the oxi-

dized form, could be observed following stimulation of

cells with the Ca2 +-mobilizing agonist. Afterwards, auto-

fluorescence decreased reflecting an increase in the reduced

form, FADH2. Depolarizations of wm reduce ATP synthesis

which, together with ATP consumption by Ca2 + extrusion

through membrane ATPases, should lead to an acceleration

of the electron transport chain for ATP synthesis. Therefore,

a rise in [Ca2 +]c, that initially depolarizes wm, should lead

to an increased state of oxidation of intramitochondrial

coenzymes (FADH2 and NADPH). An initial consumption

of reduzing equivalents, increase in FAD autofluorescence,

for ATP synthesis should be expected followed by a

stimulation of the Krebs cycle, where the reduzing equiv-

alents are produced and that later enter the electron trans-

port chain [29,33]. The latter should be reflected by a

decrease in FAD autofluorescence, as we have shown in

our study.

Control experiments were carried out to verify that the

observed changes in autofluorescence in this set of experi-

ments were due to changes in oxidation of mitochondrial

FAD, and not from signals generated in the cytosol. The

mitochondrial uncoupler FCCP increases oxygen consump-

tion by the cell, oxidizing mitochondrial NADH and FADH2

and, therefore, should increase FAD autofluorescence [30].

As expected, perfusion of pancreatic acinar cells with FCCP

led to a transient increase in FAD autofluorescence that later

returned to prestimulation level. To further characterize the

effect of the protonophore on mitochondrial activity we

tested its effect on wm. Application of protonophores to

resting pancreatic acinar cells produces a rapid breakdown

of wm that can be demonstrated by incubation of cells with

JC-1. Inclusion of FCCP in the perfusion medium was

associated with a decrease of JC-1 fluorescence ratio, which

is an index of depolarization of mitochondria.

We further verified that changes in autofluorescence were

due to changes in mitochondrial FAD, and additional con-

trol experiments were carried out by perfusion of cells with

rotenone. Rotenone, that is an inhibitor of complex I of the

electron transport chain, decreases oxygen consumption

increasing mitochondrial NADH [30,33]. Additionally, in

the presence of rotenone, a slowdown in the rate of FADH2

oxidation at complex II should occur and thus, a decrease in

FAD autofluorescence would be observed [30]. As can be

observed in our experiments, perfusion of cells with the

mitochondrial inhibitor induced a decrease in FAD auto-

fluorescence. In this set of experiments, inclusion of rote-

none in the perfusion medium blocked the CCK-8-induced

increase in FAD autofluorescence. Therefore, the observed

changes in FAD autofluorescence in our conditions, in

response to CCK-8, can be related to a stimulation of the

electron transport chain by the hormone.

It is worth noting that when small areas were recorded at

the basolateral pole of the cell in the cytosol without

mitochondria, in the granular area and in the nucleus, no

changes in FAD autofluorescence could be detected in spite

of clearly detectable signals when mitochondria were

selected. This indicates that the mitochondria are the major

contributors to the total FAD signal from whole cells, as it

has been previously described for NADH in studies carried

out in arterial chemoreceptors, where it is stated that

fluorescence from cytosolic NADH accounts for less than

20% of the total signal [30]. Handling of cells with

mitochondrial inhibitors, together with the absence of fluo-

rescence signal when small areas were selected in the

cytosol, in the granular area and in the nucleus serve as

control that shows that the monitored signals indeed ensue

from mitochondria.

To eliminate the possibility that the observed effects of

FCCP and rotenone on wm and FAD autofluorescence could

be due to uncoupling of F0�F1 ATP synthase from ATP

synthesis and, therefore, to a decrease in cellular ATP,

oligomycin was included in the perfusion medium. Oligo-

mycin hyperpolarized wm, as previously reported [29], and

did not inhibit FCCP- and rotenone-induced depolarization

(data not shown). Inclusion of oligomycin in the extracel-

lular solution did not block changes of FAD autofluores-

cence induced neither by 100 nM FCCP nor 10 AMrotenone, suggesting that the signals do not reflect a

response to reversal of the proton gradient across the inner

mitochondrial membrane and, thus, to ATP depletion (data

not shown).

It has been proposed that Ca2 + accumulation by mito-

chondria stimulates respiration as well as Ca2 +-dependent

intramitochondrial dehydrogenases [37,38], i.e., it can be

argued that changes in autofluorescence can be secondary

to changes in [Ca2 +]m [31]. In our experimental conditions,

CCK-8 could stimulate the observed changes in mitochon-

drial parameters by terms of its Ca2 +-mobilizing nature.

Therefore, we performed a series of experiments in which

intracellular Ca2 + stores were depleted by perfusion of

pancreatic acinar cells with Tps in a Ca2 +-free medium.

Tps is an inhibitor of the SERCA pump which transport


Ca2 + into the endoplasmic reticulum, and has been widely

employed to deplete cytosolic Ca2 + stores (see elsewhere).

Treatment of cells with Tps in the absence of extracellular

Ca2 + induced an increase in [Ca2 +]m and FAD autofluor-

escence. A depolarization of wm in response to treatment of

cells with thapsigargin was as well observed. These results

argue in favour of an interrelationship between the observed

variations in wm and FAD autofluorescence and the changes

in [Ca2 +]m. Following the pretreatment of cells with Tps,

stimulation of pancreatic acinar cells with CCK-8 failed to

evoke further changes in these parameters. These findings

are consistent with a dependence of changes in the mito-

chondrial parameters on Ca2 + mobilization by the agonist,

and are in agreement with previously reported data in which

changes in autofluorescence were completely abolished if

Ca2 + was excluded from the extracellular medium [30].

Furthermore, our results are consistent with an increased

cycling of Ca2 + into the mitochondria that then is associ-

ated to a depolarization of wm, stimulation of respiration

and changes in oxidized FAD [30,36]. Recently it has been

shown in pancreatic acinar cells that changes in NADH

content correlates with Ca2 + signals [33].

In conclusion, the results presented in this work are

consistent with changes in mitochondrial activity in response

to stimulation of pancreatic acinar cells with CCK-8. Fol-

lowing stimulation, mitochondria take up Ca2 + and depola-

rization of wm follows. As a consequence, Ca2 + could in turn

activate the mitochondrial mechanisms that match the energy

supply necessary for the cell function during secretion. The

changes in [Ca2 +]m, wm and FAD autofluorescence depend

on mobilization of Ca2 + from intracellular stores and are

independent of extracellular Ca2 +. Furthermore, the time

courses of changes in [Ca2 +]c, [Ca2 +]m, wm and FAD

autofluorescence are all consistent with a direct interrelation-

ship. Changes in [Ca2 +]c and, therefore, in [Ca2 +]m may

promote significant and long-lasting changes in mitochon-

drial metabolism needed for energy production and, there-

fore, for cellular economy.

Acknowledgements

This work was supported by DGI (Ministerio de Ciencia y

Tecnologıa; BFI2001-0624). Marıa P. Granados is supported

by a grant from Junta de Extremadura. The authors would like

to thank Mrs. Mercedes Gomez Blazquez for her excellent

technical support and Dr. Andreas Schmid from the II

Physiologisches Institut (Universitat des Saarlandes, Ger-

many), for his helpful critique in the elaboration of this

manuscript.

References

[1] Streb H, Irvine RF, Berridge MJ, Schulz I. Nature 1983;306:67–9.

[2] Putney JW. Cell Calcium 1988;7:1–12.

[3] Carafoli E. Physiol Rev 1991;71:129–53.

[4] Gonzalez A, Pariente JA, Salido GM, Camello PJ. Pflugers Arch

1997;434:609–14.

[5] Rizzuto R, Berbardi P, Pozzan T. J Physiol 2000;529:37–47.

[6] Gunter TE, Pfeiffer DR. Am J Physiol 1990;258:C755–86.

[7] Buntinas L, Gunter KK, Saparagna GC, Gunter TE. Biochim Biophys

Acta 2001;1504:248–61.

[8] Sparagna GC, Gunter KK, Sheu S-S, Gunter TE. J Biol Chem 1995;

270:27510–5.

[9] Ischas F, Jouaville LS, Mazat J-P. Cell 1997;89:1145–53.

[10] Montero M, Alonso MT, Albillos A, Garcıa-Sancho J, Alvarez J. Mol

Biol Cell 2001;12:63–71.

[11] Hajnoczky G, Robb-Gaspers LD, Seit MB, Thomas AP. Cell 1995;

82:415–24.

[12] Boitier E, Rea R, Duchen MR. J Cell Biol 1999;145:795–808.

[13] Simpson PB, Russel JT. J Biol Chem 1996;271:33493–501.

[14] Camello-Almaraz C, Salido GM, Pariente JA, Camello PJ. Biochem

Pharmacol 2002;63:283–92.

[15] Gonzalez A, Schulz I, Schmid A. J Biol Chem 2000;275:38680–6.

[16] Pacher P, Csordas G, Schneider TG, Hajnoczky G. J Physiol 2000;

529:553–64.

[17] Rizzuto R, Bastianutto C, Brini M, Murgia M, Pozzan T. J Cell Biol

1994;126:1183–94.

[18] Tinel H, Cancela JM, Mogami H, Gerasimenko JV, Gerasimenko OV,

Tepikin AV, et al. EMBO J 1999;18:4999–5008.

[19] Toescu EC, Lawry AM, Petersen OH, Gallacher DV. EMBO J 1992;

11:1623–9.

[20] Csordas G, Thomas AP, Hajnoczky G. EMBO J 1999;18:96–108.

[21] Jouaville LS, Ischas F, Holmuhamedov EL, Camacho P, Lechleiter

JD. Nature 1995;377:341–8.

[22] Rizzuto R, Brini M, Murgia M, Pozzan T. Science 1993;262:744–7.

[23] Hajnoczky G, Hager R, Thomas A. J Biol Chem 1999;274:

14157–62.

[24] Szalai G, Csordas G, Hantash BM, Thomas AP, Hajnoczky G. J Biol

Chem 2000;275:15305–13.

[25] Alonso MT, Barrero MJ, Michelena P, Carnicero E, Cuchillo I, Garcıa

AG, et al. J Cell Biol 1999;144:241–54.

[26] Ricken S, Leipziger J, Greger R, Nitschke R. J Biol Chem 1998;

273:34961–9.

[27] Park MK, Ashby MC, Erdemli G, Petersen OH, Tepikin AV. EMBO J

2001;20:1863–74.

[28] Denton RM, McCormack JG. FEBS Lett 1980;199:1–8.

[29] Duchen MR, Biscoe TJ. J Physiol 1992;450:33–61.

[30] Duchen MR, Biscoe TJ. J Physiol 1992;450:13–31.

[31] Duchen MR. Biochem J 1992;283:41–50.

[32] Robb-Gaspers LD, Burnett P, Rutter GA, Denton RM, Rizzuto R,

Thomas AP. EMBO J 1998;17:4987–5000.

[33] Voronina S, Sukhomlin T, Johnson PR, Erdemli G, Petersen OH,

Tepikin AV. J Physiol 2000;539:41–52.

[34] Gonzalez A, Pfeiffer F, Schmid A, Schulz I. Am J Physiol 1998;

275:C810–7.

[35] Hoth M, Fanger CM, Lewis RS. J Cell Biol 1997;137:633–48.

[36] Mironov SL, Richter DW. J Physiol 2001;533:227–36.

[37] Hansford RG. Rev Physiol, Biochem Pharmacol 1985;102:1–72.

[38] McCormack JG, Denton RM. Biochem J 1984;218:235–47.

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