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37Ca2+ Signaling

the cytosol from intracellular stores[12]. It was established many years ago that the initial secretory response to stimulation with either ACh or CCK is independent of extracellular Ca2+[13], whereas sustained secretion is acutely dependent on external Ca2+ (Fig.4.1c). This is explained by the limited capacity of the intracellular Ca2+ stores and the fact that release of Ca2+ from stores into the cytosol inevitably activates Ca2+ pumps in the plasma membrane extruding Ca2+, so that after a shorter or longer period of stimulation (depending on the intensity of stimulation) the contents of the intracellular Ca2+ stores have been exported to the extracellular solution [14]. A reduction of [Ca2+] in the intracellular stores activates a process known as storeoperated Ca2+ entry. A signal is transmitted from the stores to the plasma membrane activating special Ca2+ channels (store-

operated channels) that allow Ca2+

entry[15]. It is this Ca

entry process that sustains the secretory response during prolonged stimulation, after the stores have been emptied.

Ca2+ Signaling

It is well established that stimulation of acinar cells with either ACh or CCK elicits a rise in [Ca2+]i (Fig.4.2). At low, physiologically relevant, concentrations of neurotransmitter or hormone, the typical Ca2+ signal pattern consists of repetitive [Ca2+]i spikes confined to the apical (granular) pole, as originally shown in 1993 [17]. Increasing the stimulating agonist concentration causes Ca2+ signal globalization, a process whereby a local Ca2+ signal initiated in the apical pole spreads as a wave from the apex to the base of the cell (Fig.4.2).

ACh

140

120

100

100 nM

[Ca

1µM

]

Apical

Basal

50 µm

Mit

(d)

Acinar lumen

Figure4.2 Ca2+ signaling and organelle distribution in the intact mouse pancreas. (a) Merged confocal images showing distribution of

specific fluorescent markers for zymogen granules (ZG– red), nuclei (N– blue), and mitochondria (Mit– green). The optical slice goes through three cells (nuclei). The ZG are seen distributed around the lumen and are surrounded by mitochondria. Mitochondria are also located around the nuclei and close to the plasma membrane. (b) Confocal image of larger part of the pancreas showing many acinar units. One cell is highlighted by white dashed lines and in this cell apical (red) and basal (blue) regions of interest are signposted. The traces shown in (c) are from these two regions. (c) ACh- elicited cytosolic Ca

spikes are seen exclusively in the apical pole. When the ACh concentration is increased to 1 μM, there is a rise in [Ca2+]i in both the apical and basal regions. (d) Fluorescent images showing (upper row) a single local apical Ca (c))and (lower row) the initial Ca signposted in (c)). Source: Adapted from [16]/Elsevier/CC BY-4.0.

Transmitted

Global

wave generation following the increase in ACh concentration (numbers again refer to time points

Local 1

Fluorescence intensity

Local (1–3)

signals. At the low ACh concentration of 100 nM, repetitive

Global (4–7)

spike (numbers refer to time points in

50 sec

Physiology ofAcinar Cell Secretion

Mitochondria

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Organelles Important for Ca2+ Homeostasis

Early work in the 1970s on Ca2+ transport in exocrine glands indicated that ACh evokes Ca2+ signals in acinar cells by causing release of Ca2+ from the endoplasmic reticulum (ER)[7] but, for many years, the link between ACh occupation of muscarinic receptors on the cell surface and the outflow of Ca2+ from the ER was obscure. In 1983, Irene Schulz and Michael Berridge discovered that the intracellular water- soluble messenger inositol 1,4,5- trisphosphate (IP3), generated inside the cell by receptor- activated phospholipase C action on a membrane phospholipid, phosphatidylinositol 4,5- bisphosphate (PIP2), releases Ca2+ from the ER in permeabilized pancreatic acinar cells[18]. All subsequent work on many different cell types confirmed the generality of the concept that hormonerelease is mediated principally via IP3- evoked Ca2+ release from the ER [7,19]. Although the original discovery of

or neurotransmitter- elicited intracellular Ca2+

Ca pump

Ca -activated Cl

channel

- evoked Ca2+ release was made on pancreatic acinar

cells[18], there are difficulties in applying this concept to these particular cells. The problem is that the physiologically relevant Ca2+ signals occur specifically in the apical granular pole (see Fig.4.2), which contains mostly ZG and little ER. This difficulty was finally overcome by the results of the so- called Ca2+ tunnel experiments, in which it could be shown that Ca2+ taken up at the base of the cell into the ER could diffuse easily in the ER lumen and reach the apex via thin ER extensions penetrating deeply into the granular area between the ZGs (see Fig.4.3). Upon stimulation, Ca2+ is released primarily from the ER elements in the apical pole due to the high concentration of ER Ca2+ release channels specifically in this part of the cell (Fig.4.3)[20,22].

It was initially a surprise that cytosolic Ca2+ signals initiated in the apical pole could remain local in such a relatively small cell (~20 μm diameter). This could not be easily understood before it was discovered that the mitochondria in the acinar cell are distributed in a very specific manner [23]. The mitochondria are primarily

Lumen

Exocytosis

Granules

Lumenally connected ER

Nucleus

Cytosol

Figure4.3 Organelle distribution and Ca2+ transport events in acinar cell. The main part of the figure shows a model cell with the

distribution of organelles and Ca

−

current I

(autofluorescence). It is seen that ACh evokes a rapid rise in

, mitochondrial Ca2+ concentration ([Ca2+]m— measured by Rhod- 2 fluorescence— and concentration of NADH

Cl Ca,

transport pathways signposted. Insert (in red frame) shows triple measurements of Ca2+- activated

Basolateral

pool

Base

SOC

delay, by an increase in the NADH concentration signifying activation of mitochondrial metabolism and therefore ATP production.

Cytosol

NADH

RyR

300pA

120

F/Fo (%)

100

ACh

0 100 200

Time (s)

SERCA

, which is followed immediately by a rise in [Ca2+]m and, after a small

Cl Ca,

–

Cl , Ca

[Ca2+]

NADH

Rhod-2

250

200

150

F/Fo (%)

100

Source:Adapted from [21] / John Wiley and Sons and [20] / With permission of Elsevier.

localized in a belt surrounding the ZG, separating the

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apical granular pole from the rest of the cell (see Figs4.2 and4.3). Due to their ability to take up Ca2+, the mitochondria function as a Ca2+ diffusion barrier, effectively acting as a firewall preventing the spread of cytosolic Ca2+ signals from the apical pole into the basal part of the cell containing the nucleus (Fig.4.3). The nucleus is well protected against Ca2+ signal invasion from the apical pole, since there is an additional mitochondrial belt surrounding the nucleus (Fig.4.2). Finally, there is a concentration of mitochondria just beneath the plasma membrane (Fig. 4.2). The general concept that has emerged from studies of Ca2+ transport in the cytosol, ER, and mitochondria is that Ca2+ moves easily in the ER lumen, but with much more difficulty in the cytosol, due to the barriers created by the mitochondria[7,24].

The fact that the physiologically most important Ca

signals occur in the apical granular area has also prompted interest in the possibility that Ca2+ could be released from ZG and other acid pools in the apical pole. In studies on isolated ZG, it was shown that both IP3 and another Ca2+- releasing messenger, cyclic ADP- ribose (cADPR, derived from NAD) can liberate Ca2+ stored in this organelle (see Fig.4.4). It has been clear for a long time that ACh causes intracellular Ca2+ release via generation of IP3, acting primarily on the ER. It is now equally clear that CCK releases Ca2+ via generation of NAADP [7,26,27] and that NAADP primarily acts on acid stores (Fig. 4.4a). This may explain the somewhat different Ca2+ signal patterns that can be generated by CCK and ACh[28].

Mechanisms of Ca2+ Signal Generation

Figure4.4 illustrates some of the most important steps. As already mentioned, there are two major signal transduction pathways, one initiated by hormonal (CCK) stimulation and the other by nervous (ACh) stimulation. CCK acts on high- affinity CCK1 receptors in the basolateral plasma membrane [29,30], whereas ACh acts on muscarinic M3 receptors, which are also localized predominantly in the basolateral membrane[16]. With state- of- the- art imaging technology, it is now possible to visualize some of the most important signal transduction steps.

Figure4.5 demonstrates the ACh- elicited breakdown of PIP2 in the basolateral membrane and the appearance of the water- soluble Ca2+- releasing messenger IP3 in the cytosol. The enzyme responsible for PIP2 breakdown, phospholipase C, can in some cases be Ca2+ activated. However, the experimental result shown in Fig.4.5 demonstrates that, at least in the pancreatic acinar cell, the disappearance of PIP2 from the plasma membrane and the appearance of IP3 in the cytosol are not secondary to

signal generation, since a directly generated Ca2+ signal (via uncaging of Ca2+ in the cytosol) does not induce these effects, whereas ACh does.

Direct infusion of IP3 into isolated cells elicits repetitive cytosolic Ca2+ spikes confined to the apical granular pole (see Fig. 4.6), in this way mimicking the effect of externally applied ACh (see Fig.4.2). The importance of functional IP3 receptors (IP3R) for ACh- elicited Ca2+ signal generation and secretion in pancreatic acinar cells has been demonstrated very clearly by knockout experiments, in which it was shown that knockout of either type 2 or type 3 IP3R had very little effect, whereas double knockout of both these receptors abolished AChelicited Ca2+ signal generation as well as secretion[34]. This directly confirms earlier data in which it was shown that intracellular infusion of the IP3R antagonist heparin abolished both IP

- and ACh- elicited Ca2+ spiking[35].

NAADP is a real intracellular messenger for CCKinduced activation of pancreatic acinar cells. Work from Galione’s group in Oxford shows that physiologic CCK concentrations (1–10 pmol/L) evoke clear and dosedependent increases in the cellular NAADP concentration. This effect is specific for CCK, since ACh has no effect on the NAADP level[26].

Intracellular infusion of NAADP, even at concentrations much lower (nanomolar) than those needed to obtain effects of IP3 or cADPR, elicits repetitive cytosolic Ca2+ spikes in the apical pole that look very similar to those generated by IP3 and cADPR[32]. The NAADP receptor has the interesting property that it can be inactivated by relatively high (micromolar) intracellular NAADP concentrations. Using such selective inhibition of the NAADP receptor, it has been shown that Ca2+ spiking evoked by physiologic CCK concentrations (<10 pmol/L) is blocked by a high intracellular NAADP concentration. This blocking effect is specific for the CCK response, since AChelicited Ca2+ spiking is unaffected[36]. Furthermore, the NAADP antagonist Ned- 19 blocks CCK- elicited Ca2+ spiking, but has no effect on the action of ACh[27].

Although ACh and CCK initiate Ca2+ signaling via different pathways (see Fig.4.4a), these ultimately converge and the measurable local apical Ca2+ transients are due to positive and negative feedback interactions between IP3R and RyR on the ER (Fig.4.4a). These Ca2+- mediated positive and negative interactions are functionally important. For example, a Ca2+ signal initiated by an increase in the intracellular IP3 concentration will subsequently activate RyR, inducing further Ca2+ release. This positive feed- forward effect explains the rising phase of the cytosolic Ca2+ spike. However, at a higher level of [Ca2+]i, a further [Ca2+]i rise inhibits opening of both IP3R and RyR. This explains the falling phase of the spike [7,37]. It has recently become clear that there is another class of channel involved in intracellular Ca2+

39Mechanisms of Ca2+ Signal Generation

Physiology ofAcinar Cell Secretion

(a)

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(b)

10 µm

ACh

M3R

Gαq

PLC

Mitochondrial belt

ACh

[Ca2+]

+/–

IP3R

+/–

RyR

Mitochondrial belt

ACh

ARC

cADPR NAADP

CCK1R

Acid stores

(c)

CCK

10 µm

RyRTPC

(d)

Cytosol

SP:

ZGs

Matrix

Hleak

HCa

RyR

cADPR

NAADP

transporter

Vacuolar

H -ATPase

Figure4.4 Ca2+ transport and signaling events in acinar cell. (a) Two receptor pathways are shown. ACh binding to muscarinic M3

receptors activates, via interaction with a classical trimeric G- protein, phospholipase C (PLC) generating the messenger IP diacyl glycerol— not shown in diagram). CCK interaction with CCK1 receptors results in activation, via ADP- ribosyl cyclase (ARC), of two separate messengers, namely NAADP and cADPR. NAADP releases Ca causes Ca the ER. Source: Adapted from[7]. (b) Confocal fluorescent images illustrating changes in organellar [Ca left image shows the high resting [Ca has been reduced markedly (shift from warm (red) to cold (green) color) and the perigranular mitochondrial belt is now clearly seen (yellow). This indicates that Ca complete loss of Ca

release from both acid stores and the ER via ryanodine receptors (RyR). cADPR releases Ca2+ via RyR from both acid stores and

] in the ER (mostly in the basal (left) part of the cell). After maximal ACh stimulation, [Ca2+] in the ER

from the ER and the still elevated [Ca2+] in the perigranular mitochondria. (c) Confocal image showing the

lost from the ER has been taken up in part by the mitochondria. The third image shows the almost

distribution of fluorescent thapsigargin (white), a very specific marker for the ER Ca only through one nucleus– N). It is seen that by far the highest ER Ca important to note that there are some light elements in the darker granular (secretory pole– SP) areas signifying ER elements with Ca pumps also in this part of the cell. (d) Schematic drawing of Ca

primarily from acid stores, via two pore channels (TPC). It also

] following ACh stimulation. The

pump density is in the basolateral parts of the cell, but it is

, H+, and K+ transports across the ZG membrane. Source: [25] / With

pump. The optical slice goes through two cells (but

(as well as

permission of Elsevier.

release, namely the so- called two- pore channel (TPC) (Fig.4.4a). In the pancreatic acinar cells, these channels

of Ca2+ from the ER and the acid stores occurring via

RyR[7,27] (Fig.4.4a). are important specifically for the Ca2+ release evoked by CCK, which is mediated by NAADP. The sequence of events with regard to CCK- elicited Ca2+ signal genera-

Ca2+ Entry and Exit

tion is likely to be an initial release of a very small (trigger) amount of Ca2+ from endosomes/lysosomes, which is then amplified by a much more substantial liberation

Although the primary event responsible for activation of

secretion by ACh or CCK is intracellular Ca2+ release, it

PIP2 in plasma membrane

IP3 in cytosol

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41Ca2+ Entry and Exit

Unstimulated Ca

Figure4.5 Acetylcholine (ACh)- induced breakdown of phosphatidylinositol 4,5- bisphosphate (PIP2) in the plasma membrane (PM) and

generation of inositol 1,4,5- trisphosphate (IP with high affinity to both PIP the presence of PIP does not cause any reduction in the PIP that seen after Ca appearance of fluorescence in the cytosol indicating appearance of IP

at this site. (2) Generation of a substantial rise in [Ca2+]i by photolytic release of Ca2+ into the cytosol from caged Ca2+

uncaging, but in this case there is loss of GFP fluorescence from the basolateral membrane, signifying loss of PIP2, and

and IP3. (1) Before stimulation, the main GFP fluorescence is seen in the basolateral membrane, indicating

uncaged

Ratio [GFP] cyt/PM

10 s

uncaging

) in the cytosol (cyt). The green fluorescent protein (GFP)- linked PH domain of PLCδ1 binds

concentration in the membrane. (3) ACh (1 μmol/L) causes a rise in [Ca2+]i of similar magnitude to

. Source: Adapted from [31].

[Ca2+]i

ACh

∆ ratio

0.2

1.2

1.0

ACh

is also very important for both physiology and pathology to consider the overall cellular Ca2+ homeostasis, that is, Ca2+ entry and exit. All cells have to be protected against cellular Ca2+ overload, since it is well established that this causes cell death [38]. The plasma membrane must therefore be relatively impermeable to Ca

and there must be mechanisms for cellular Ca2+ extrusion. Ca2+ entry and exit across the plasma membrane of pancreatic acinar cells is controlled by specific transport mechanisms. Unlike many electrically excitable cells (e.g., cardiac cells), the acinar cells do not possess functional Na Ca2+ exchangers, so that the only mechanism for extruding Ca2+ across the plasma membrane is via the plasma membrane Ca2+- activated ATPase (PMCA) [25]. This pump is activated by increases in [Ca2+]i above the basal level of 0.1 μmol/L, but has limited capacity. Interestingly, this pump is not uniformly distributed over the plasma membrane, but is specifically concentrated in the apical plasma membrane and therefore extrudes Ca2+ principally into the acinar lumen (see Fig.4.7). The concentration of the pump in the apical membrane is functionally

important, since the principal intracellular Ca sites are located in the apical pole (Figs4.2 and4.6), but carries the risk that Ca2+ overload, due to inappropriate Ca2+ entry across the basal membrane in pathologic conditions, cannot be dealt with adequately[25].

Physiologic stimulation of acinar cells does not primarily increase the permeability of the plasma membrane for Ca

, but after depletion of the ER Ca2+ store there is specific opening of the so- called store- operated Ca2+ channels in the basolateral membrane. This can

most easily be visualized by measuring the uptake of

entering the base of the cell into the peripheral mitochondria situated immediately beneath the plasma membrane, as shown in Fig.4.7. In these types of experiments, the ER Ca2+ store is depleted by poisoning the

pump in the ER very specifically with thapsigargin in the absence of external Ca2+. Thereafter, Ca2+ is readmitted to the external solution and an increase in the Ca2+ concentration of those mitochondria situated very close to the plasma membrane can be visualized directly (Fig.4.7). The nature of the store- operated Ca2+ channels

release

Physiology ofAcinar Cell Secretion

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0.4 nA

10 s

IP3 in pipette

–

, Ca

IP3 in Pipette

activated

Cell

Between spikes Apical Ca

–

conductance

Secretion

(exocytosis)

3nS

800fF

500 ms

spike

Figure4.6 IP3- elicited local apical Ca2+ spikes and exocytotic secretion. The main part of the figure shows the result from a patch clamp

experiment with internal acinar cell perfusion. The trace shows the repetitive spikes of Ca IP

infusion (10 μM). The images below illustrate the experimental configuration and the distribution of the elevated [Ca2+]i during the

height of a spike. It can be clearly seen that the Ca frame) shows correlation between a single apical Ca and the exocytotic response recorded as an increase in membrane capacitance (ΔC). It is seen that the increase in Cl sensitive indicator of [Ca returns to the inter- spike level. Source: Adapted from [33] / With permission of Elsevier.

]i) slightly precedes the rise in capacitance and that the secretory response is completed just before [Ca2+]i

in the pancreatic acinar cells has been clarified by patch clamp studies, in which it has been possible to record

signal occurs in the apical granular pole. Source: Adapted from[32]. The insert (in red

spike (during IP3 infusion), recorded here as an increase in Cl− conductance (ΔG),

extrusion stops and dangerous Ca resulting in necrosis[7,46].

- dependent Cl− current elicited by intracellular

−

conductance (a

overload occurs,

directly the tiny Ca2+ currents flowing across the basolateral membrane upon depletion of the ER Ca2+ store. The biophysical properties of this current shows that the channels belong to the very Ca

- selective CRAC (Ca2+

Ca2+- Mediated Control of Enzyme Secretion

release activated Ca2+) channel type [41], which is also present in various immune cells [15]. These channels, including those in the acinar cells as well as other pancreatic cell types, can be blocked by very specific CRAC channel inhibitors[7,41–45].

During sustained stimulation with either ACh or CCK, one can usually observe a plateau of elevated [Ca2+]i, which represents a delicate balance of Ca2+ entry through store- operated Ca

exit mainly through Ca2+ pumps located in the api-

channels in the basal membrane and

cal plasma membrane [24]. Ca2+ extrusion is energydependent. Therefore, if intracellular ATP levels fall during pathologic conditions, for example when cells are exposed to nonoxidative alcohol metabolites[46], Ca2+

It has been known for many years that intracellular Ca2+ is the main acute regulator of exocytosis [47]. In experiments on the isolated perfused pancreas, it has been demonstrated that during sustained stimulation with high concentrations of ACh or CCK, both fluid and enzyme secretion are acutely dependent on the presence of external Ca2+ (see Fig.4.1). During this phase of the secretory response, the ER will have been partly depleted of Ca

and Ca

therefore has to be supplied by entry from the external solution through store- operated Ca2+ channels as described earlier. However, during physiologic stimulation, with low concentrations of CCK or ACh, there is not a sustained elevated [Ca2+]i but rather a series of

050 100 150 200

(a)

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Apical Ca2+ exit Basal Ca2+ entry

(b)

(c)

43Ca2+- Mediated Control of Fluid Secretion

10 µm

(e)(d)

(g)

Figure4.7 Overall Ca2+ homeostasis: Ca2+ entry and exit. The left part illustrates an experiment in which [Ca2+] is measured outside an

isolated acinar cell by using a Ca mobility. The morphology of the cell, with clear identification of the granular apical (Ap) pole is shown in (a). (b) to (i) are fluorescent images (taken at 3It is clear that the Ca rise in [Ca measured with a fluorescent probe and traces from three regions of interest (red, black, and green) are shown. The cell was initially poisoned with thapsigargin in the absence of external Ca 10 region of interest, very close to the basal plasma membrane. The image marked with a red arrow shows the distribution of the elevated [Ca a region very close to the plasma membrane. The EM picture shows a mitochondrion (Mit) situated very close to the plasma membrane (PM). Source: Adapted from [39] and [40].

mM Ca

] at the time indicated by a similar red arrow above the fluorescence traces. Clearly the elevation of [Ca2+]m has essentially occurred in

s intervals) showing the distribution of the extracellular [Ca

extrusion from the cell occurs predominantly across the apical membrane. The right part of the figure illustrates the

] of mitochondria close to the basal plasma membrane during store- operated Ca2+ entry. Mitochondrial [Ca2+] ([Ca2+]m) was

, Ca2+ was readmitted to the external solution and it is seen that there was a marked rise in [Ca2+]m particularly in the red

- sensitive fluorescent indicator linked to high molecular weight dextran, thereby limiting the indicator

(f)

(i)(h)

to deplete the ER of Ca2+. During the time period indicated by the bar labeled

10 µm

F/Fo

2.4

2.2

2.0

1.8

1.6

1.4

1.2

1.0

0.8

[Ca

] rise immediately following stimulation with ACh (10 μM).

]

10 mM Ca

Mit

sec

short- lasting Ca2+ spikes localized specifically in the critical apical region (Figs4.2 and4.6). These spikes are essentially independent of external Ca2+ and are due to repetitive release of small amounts of Ca2+ from the ER[7,48].

Can the short- lasting local Ca2+ spikes, evoked by low agonist concentrations or direct intracellular messenger infusion, control secretion? The most sensitive method for evaluating exocytotic secretion is measurement of membrane capacitance. When granules fuse with the plasma membrane, the surface area of the plasma membrane increases, but only transiently since the additional membrane inserted is subsequently removed by the process of endocytosis. As shown in Fig.4.6 (inset), there is indeed a transient increase in the pancreatic acinar membrane capacitance during an individual short- lasting

spike, which can most easily be recorded by electro-

physiologic methods. Results, such as those recorded in Fig.4.6, demonstrate clearly the very fine control exerted by the local apical [Ca

Ca2+- Mediated Control of Fluid Secretion

How is acinar fluid secretion regulated? The generally accepted model for isotonic fluid secretion by exocrine glands is illustrated in Fig. 4.1a. The principal step for activation of fluid secretion is Ca Cl− channels, which are specifically located in the apical plasma membrane[49]. This will cause Cl− exit into the acinar lumen. The increased lumen negativity will attract cations and the principal extracellular cation, Na+, will move through the very leaky junctions between the acinar

]i on exocytotic secretion.

- activated opening of

Physiology ofAcinar Cell Secretion

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cells, which are named (inappropriately!) tight junctions. These junctions sit very close to the apical membrane and separate the luminal fluid compartment from the basal and interstitial compartments, and also separate the very different properties of the apical and basolateral membranes (Fig.4.1a). NaCl in the acinar lumen will osmotically attract water, which can pass through both the cell membranes (via aquaporins) and the tight junctions.

The principal activating step of acinar fluid secretion, namely the exit of Cl− from the cell interior to the lumen through Ca2+- activated Cl− channels, only occurs if there is an electrochemical gradient favoring transport in this direction. The intracellular Cl− concentration must be held above thermodynamic equilibrium and a Cl−accumulating mechanism is therefore needed. As shown in Fig. 4.1a, a Na+/K+/2Cl− cotransporter is situated in the basolateral membrane (for graphical convenience shown in Fig.4.1a only in the basal membrane). Energy for this process comes from the transmembrane Na+ gradient established by the Na+/K+ pump, which is also situated in the basolateral membrane. Increased Cl− secretion requires stimulation of the Na+ pump, which occurs via the increased intracellular Na+ concentration mediated by enhanced turnover of the Na+/K+/2Cl− cotransporter. This in turn requires additional K+ cycling across the plasma membrane, which is mediated by Ca2+ activation of specific K+ channels situated in the basolateral membrane (Fig.4.1a).

As seen in Fig.4.1a, it is the concerted Ca2+ activation of Cl− and K+ channels that controls the fluid secretion process. It is important to understand that the whole of the basolateral membrane is uniform with regard to distribution of surface membrane ion channels. Thus, the Ca2+- activated K+ channels are found not only in thebasal membrane as illustrated in Fig.4.1a, but also in the lateral membranes, up to the tight junctions. Therefore, local apical Ca2+ signals will be able to activate both Cl− channels in the apical membrane and K+ channels in the part of the lateral membrane close to the tight junctions. A limited amount of fluid secretion can proceed without special activation of K

channels, since rodent pancreatic acinar cells lack Ca2+- activated K+ channels [12]. If the resting K+ permeability is sufficiently high, recirculation of K+ can still occur. However, human pancreatic acinar cells, like all salivary and lacrimal glands in all species so far studied, do possess very sensitive Ca2+- activated K+ channels, which undoubtedly contribute to the fine regulation of human acinar fluid secretion[50]. In the human acinar cells, these Cl

−

and K+ channels will be activated by the short- lasting repetitive local Ca2+ signals in the secretory granule region, which have been directly demonstrated in response to stimulation with either ACh or physiologic concentrations (low pM) of CCK[51].

Dangers of Ca2+ Signaling

As described in this chapter, local Ca2+ signaling elicited by physiologically relevant agonist concentrations is a remarkably precise mechanism for fine regulation of pancreatic acinar secretion (Fig.4.6). These Ca2+ signals also control the production of ATP (Fig.4.3), which is required to fuel both fluid and enzyme secretion. However, Ca2+ signaling carries a risk of cellular Ca2+ overload, which has the capacity to kill cells[7]. This occurs when acinar cells are hyperstimulated with, for example, CCK. In this situation Ca2+ spiking is replaced by a sustained elevated [Ca2+]i which, by a still poorly understood mechanism, activates the digestive proteases inside the cells [25]. The sustained elevated [Ca2+]i is due to open CRAC channels and can be markedly reduced by CRAC channel inhibitors[7,41–43]. A sustained elevated [Ca lasting Ca2+ spikes, does not stimulate mitochondrial ATP production, but inhibits it[52]. The combination of a global and sustained high [Ca2+]i with a low cytosolic ATP level is lethal[7,53].

Intracellular digestive enzyme activation is a hallmark of pancreatitis, which is mostly related to biliary disease or excessive alcohol intake. Both bile- related and alcohol- related acute pancreatitis are due to cytosolic and mitochondrial Ca2+ overload, which have been brought about by excessive opening of CRAC channels in the plasma membrane triggered by excessive release of Ca2+ from intracellular stores[7]. Both bile acids and the combination of ethanol and fatty acids (generating fatty acid ethyl esters) have the capacity to elicit excessive release of Ca2+ from intracellular stores[7]. The cytosolic Ca2+ overload leads to mitochondrial Ca2+ overload, which causes opening of the so- called mitochondrial permeability transition pore (MPTP) and this depolarizes the inner mitochondrial membrane resulting in loss of ATP production[7,46,52]. The dangerous effects of bile acids or fatty acid ethyl esters can be markedly inhibited by reducing the intracellular Ca

release via caffeine inhibition of IP3 receptors[35,54], by reducing Ca2+ inflow via CRAC channels using specific CRAC channel inhibitors[7,41–43] or by inhibiting the MPTP [55]. The pancreatic acinar cell lives dangerously. It very effectively employs Ca2+ signaling as a finely coordinated mechanism for regulation of both fluid and enzyme secretion, but excessive intracellular Ca2+ release or Ca2+ entry has the capacity to cause necrosis.

The Ca2+- mediated toxic actions on the acinar cells, initiated by bile acids or the combination of ethanol and fatty acids, are markedly amplified by interactions with the neighboring stellate cells as well as with the

]i, unlike repetitive short-

Nucleus

cell

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Acinar cell

Overload

CRAC

Channels

45Dangers of Ca2+ Signaling

Stellate

CRAC

Channels

receptor

BradykininKallikrein

Trypsin

P2Y1

ATP

ADP

P2Y13

ATP

Necrosis

CRAC

Channels

IL, TNF-α

Acute

pancreatitis

Macrophage

Figure4.8 Ca2+- dependent interactions, between acinar cells, neighboring stellate cells and resident macrophages in the acinar

environment, drive the development of acute pancreatitis. Inhibition of Ca (denoted by red asterisks) would be a rational therapy against acute pancreatitis. For details, see text. Source: Adapted from[7].

influx in these three cell types through CRAC channels

macrophages that invade the pancreatic tissue in the early stages of acute pancreatitis (Fig.4.8). Loss of kallikrein from the acinar cells that are most sensitive to the toxic actions will lead to generation of bradykinin (BK), which then acts on type 2 BK receptors on stellate cells, releasing Ca2+ from internal stores that in turn opens CRAC channels. The generation of Ca2+ signals in the stellate cells activates nitric oxide formation[6], which, via an unknown mechanism, promotes necrosis of the acinar cells, starting a vicious circle (Fig. 4.8). ATP and ADP released from dying acinar cells act on purinergic receptors on macrophages generating Ca2+

signals in these cells due to activation of CRAC channels[44]. This stimulates secretion of many cytokines, including interleukins and TNF- α, which in turn causes further necrosis of the acinar cells (Fig.4.8). It is therefore these necrotic amplification loops, dependent on Ca2+ influx through CRAC channels that drive the development of acute pancreatitis (Fig.4.8). The therapeutic and preventive effects of small molecule CRAC channel inhibitors[41–43] are therefore not only due to reduction of Ca2+ influx into acinar cells, but also due to inhibition of CRAC channel opening in the neighboring stellate cells and macrophages[4,7,44,45].

Physiology ofAcinar Cell Secretion

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