C

Aa

b

a

ARRAA

KSACAATRM

1

ithvatoCsstCtbtd

PT

0d

Cell Calcium 51 (2012) 253– 259

Contents lists available at SciVerse ScienceDirect

Cell Calcium

j ourna l ho me page: www.elsev ier .com/ locate /ceca

a2+ signaling and exocytosis in pituitary corticotropes

my Tsea,b,∗, Andy K. Leea,b, Frederick W. Tsea,b

Department of Pharmacology, University of Alberta, Edmonton, Alberta, CanadaCentre for Neuroscience, University of Alberta, Edmonton, Alberta, Canada

r t i c l e i n f o

rticle history:eceived 3 October 2011eceived in revised form 8 December 2011ccepted 10 December 2011vailable online 5 January 2012

eywords:tressdrenocorticotropic hormoneorticotrophin-releasing hormone

a b s t r a c t

The secretion of adrenocorticotrophin (ACTH) from corticotropes is a key component in the endocrineresponse to stress. The resting potential of corticotropes is set by the basal activities of TWIK-relatedK+ (TREK)-1 channel. Corticotrophin-releasing hormone (CRH), the major ACTH secretagogue, closesthe background TREK-1 channels via the cAMP-dependent pathway, resulting in depolarization and asustained rise in cytosolic [Ca2+] ([Ca2+]i). By contrast, arginine vasopressin and norepinephrine evokeCa2+ release from the inositol trisphosphate (IP3)-sensitive store, resulting in the activation of smallconductance Ca2+-activated K+ channels and hyperpolarization. Following [Ca2+]i rise, cytosolicCa2+ is taken into the mitochondria via the uniporter. Mitochondrial inhibition slows the decay of theCa2+ signal and enhances the depolarization-triggered exocytotic response. Both voltage-gated Ca2+

2+ 2+

rginine vasopressinrachidonic acidREK-1 channelseadily releasable poolitochondria

channel activation and intracellular Ca release generate spatial Ca gradients near the exocytic sitessuch that the local [Ca2+] is ∼3-fold higher than the average [Ca2+]i. The stimulation of mitochondrialmetabolism during the agonist-induced Ca2+ signal and the robust endocytosis following stimulatedexocytosis enable corticotropes to maintain sustained secretion during the diurnal ACTH surge.Arachidonic acid (AA) which is generated during CRH stimulation activates TREK-1 channels and causeshyperpolarization. Thus, corticotropes may regulate ACTH release via an autocrine feedback mechanism.

. Introduction

Corticotropes in the anterior pituitary gland are key componentsn the hypothalamic-pituitary-adrenal (HPA) axis that mediateshe endocrine response to stress. Upon stress, the hypothalamicormones, corticotrophin-releasing hormone (CRH) and arginineasopressin (AVP) act synergistically to stimulate the release ofdrenocorticotrophin (ACTH) from corticotropes [1]. ACTH secre-ion can also be stimulated by oxytocin (OT), angiotensin II (Ang II)r norepinephrine (NE) [2]. OT exhibits a synergistic effect on theRH-induced ACTH secretion but an additive effect on the AVP-timulated ACTH secretion [3,4]. While these ACTH secretagogueshare the common feature of triggering [Ca2+]i rise in corticotropes,hey elicit distinct patterns of Ca2+ signal. Moreover, the action ofRH on the electrical excitability of corticotropes was opposite tohat of AVP or NE. CRH evoked depolarization via the closure of

ackground TREK-1 K+ channels [5]. The CRH-evoked depolariza-ion was accompanied by a sustained [Ca2+]i elevation which wasue to extracellular Ca2+ entry via voltage-gated Ca2+ channels

∗ Corresponding author at: 9-70 Medical Sciences Building, Department ofharmacology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.el.: +1 780 492 5796; fax: +1 780 492 4325.

E-mail address: amy.tse@ualberta.ca (A. Tse).

143-4160/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.ceca.2011.12.007

© 2011 Elsevier Ltd. All rights reserved.

(VGCC) [6]. By contrast, both AVP and NE evoked intracellularCa2+ release which activated the small-conductance Ca2+-activatedK+ (SK) channels, resulting in hyperpolarization [7,8]. AVP trig-gered a “transient and plateau” pattern of Ca2+ signal [7] but NEtriggered [Ca2+]i oscillations [8]. The utilization of a repertoireof intracellular signaling mechanisms and ion channels to elicitdistinct patterns of Ca2+ signals may reflect an important role ofcorticotropes in integrating various signals from hypothalamichormones and neurotransmitters during the stress response.

An important feature about corticotropes is that these cells haveto handle stimulus–secretion coupling of long durations. Duringthe diurnal ACTH surge, elevation of plasma ACTH can persist for∼1 h [9,10]. Sustained ACTH secretion also occurs during chronicor uncontrolled stress. The ability of corticotropes to secrete overa long period of time is supported by the observation that theCRH-evoked ACTH release from rat anterior pituitary cell culturescould be maintained at the same level for hours [11]. Since ACTHsecretion from corticotropes is dependent on [Ca2+]i rise, it isconceivable that in corticotropes, [Ca2+]i elevation is maintainedduring prolonged ACTH secretion. In order to meet their physiolog-ical or pathophysiological demand on secretion, corticotropes have

adaptive mechanisms to regulate Ca2+ signaling and exocytosis.These are highlighted by our findings that cytosolic Ca2+ wastaken up into the mitochondria following agonist stimulation [12]and that corticotropes exhibited robust endocytosis following

2 lcium 51 (2012) 253– 259

samacd

CAmown

2

icpcccwiaraobfeat(rf

toTcAtrrrr[IsFrmdcsf

3

3

m

Fig. 1. CRH evoked similar response in GFP-labeled and wild-type mouse corti-cotropes. (A) Application of CRH (20 nM) caused depolarization and [Ca2+]i elevationin a GFP-labeled corticotrope (obtained from a POMC-eGFP mouse). (B) The actionof CRH (20 nM) on a corticotrope (identified by reverse hemolytic plaque assay)obtained from a wild-type C57BL/6J mouse. Membrane potential was measured

54 A. Tse et al. / Cell Ca

timulated exocytosis [13]. Mitochondrial Ca2+ uptake duringgonist stimulation can activate mitochondrial metabolism toatch the increase in metabolic demand imposed by the secretory

ctivity of the cell. Meanwhile, the robust endocytosis allowsorticotropes to maintain continuous cycles of exo–endocytosisuring sustained ACTH secretion.

Here, we shall first summarize our findings on how thea2+ signal in identified corticotropes is shaped by differentCTH secretagogues (CRH, AVP and NE) and various Ca2+ transportechanisms. This will be followed by a description on the influence

f Ca2+ signal on exocytosis and endocytosis in corticotropes. Lastly,e shall discuss the role of arachidonic acid (AA) as an intrinsicegative regulator of stimulus–secretion coupling in corticotropes.

. Identification of corticotropes

Corticotropes comprise ∼10% of the cells in the anterior pitu-tary gland. Due to the difficulty involved in identifying individualorticotropes among a heterogeneous population of anteriorituitary cells, many studies on the stimulus–secretion coupling oforticotropes employed ACTH-secreting human pituitary adenomaells [14,15], mouse At-T20 tumor cells [16,17] or enriched ratorticotropes [18–21] as models. In some studies, rat corticotropesere identified by morphology and then verified with ACTH

mmunocytochemistry [3,15,22]. The reverse hemolytic plaquessay was employed to study the secretion of ACTH from individualat corticotropes when stimulated by CRH or AVP [23,24]. In thisssay, each pituitary cell was initially surrounded by a monolayerf sheep erythrocytes that were pre-conjugated with ACTH anti-ody. When stimulated with ACTH secretagogues, ACTH releasedrom individual corticotropes bound to antibodies on adjacentrythrocytes and sensitized the erythrocytes to subsequent lysis by

complement reaction. Thus, individual corticotropes can be iden-ified by the presence of a surrounding zone of lysed erythrocytesa plaque). In our studies of rat corticotropes, we employed theeverse hemolytic plaque assay to identify individual corticotropesor [Ca2+]i and electrophysiological recordings [6–8,12,13,25].

Recently, we employed a transgenic mouse strain that expressedhe enhanced green fluorescent protein (eGFP) driven by the pro-piomelanocortin (POMC) promoter (POMC-eGFP mice) [26].he POMC promoter sequence targets eGFP expression in allell types that normally express POMC gene products, includingCTH. Therefore, the ACTH-containing pituitary corticotropes in

he POMC-eGFP mice express GFP fluorescence. As shown in ourecent study [5], ∼73% of the GFP-labeled mouse pituitary cellseleased sufficient amount of ACTH that could be detected by theeverse hemolytic plaque assay. Similar to our previous report inat corticotropes [6], CRH evoked depolarization and a sustainedCa2+]i elevation in GFP-labeled mouse corticotropes (Fig. 1A).mportantly, the actions of CRH on GFP-labeled corticotropes wereimilar to those of wild-type mouse corticotropes. As shown inig. 1B, application of CRH to a corticotrope (identified by theeverse hemolytic plaque assay) isolated from wild-type C57BL/6Jice (same background as the POMC-eGFP mice) also caused

epolarization and elevation of [Ca2+]i. Thus, GFP-labeled mouseorticotropes have normal electrophysiological (e.g. Fig. 1) andecretory responses to CRH [5] and can serve as a valuable modelor physiological studies of primary corticotropes.

. Ca2+ signals induced by ACTH secretagogues

.1. Actions of CRH

The resting membrane potential of rat or mouse corticotropeseasured with the perforated-patch clamp or whole-cell patch

simultaneously with [Ca2+]i (indo-1 fluorometry). The whole-cell pipette contained100 �M indo-1.

clamp technique (with a pipette solution of pH 7.4) ranged from∼−48 to ∼−56 mV [5,6]. In contrast to previous studies in enrichedrat corticotropes, we rarely detected any spontaneous firing ofaction potentials in identified rat or mouse corticotropes [5,6].Until recently, the ion channel that sets the resting potential ofcorticotropes has remained elusive. In a recent study, we found thatthe resting potential of corticotropes was determined by the activ-ities of the background, TREK-1 K+ channels [5]. TREK-1 channelis a member of the TREK family which belongs to the tandempore class of K+ (K2P) channels. TREK-1 channels are insensitiveto TEA, constitutively active at basal condition and can be openedby a number of stimuli, including membrane stretch, intracellularacidosis and AA [27]. Note that unlike the inwardly rectifying K+

channels, TREK-1 channels are outwardly rectifying and can openover the entire range of potentials (e.g. −100 to +80 mV) [27]. Formouse corticotropes, the resting potential was ∼−48 mV whenmeasured with a whole-cell pipette solution of pH 7.4 [5]. Applica-tion of fluoxetine (50 �M), a selective inhibitor of TREK-1 channels,depolarized mouse corticotropes by ∼23 mV [5]. Conversely, theactivation of TREK-1 channels by intracellular acidosis (via record-ing with a whole-cell pipette solution of pH 7.0) hyperpolarized themembrane potential of corticotropes to ∼−82 mV, near the Nernstpotential of K+.

In corticotropes, the type of channel that is the target ofCRH regulation has been controversial. In mouse AtT-20 cells,CRH inhibited the large conductance Ca2+–K+ (BK) channels [16].

However, we found that in mouse corticotropes, the Ca2+-activatedK+ current could be completely inhibited by apamin, an inhibitorof SK channels, indicating the presence of SK but not BK channels

lcium 51 (2012) 253– 259 255

(CccTti[rldancoAmrkopcmt

teisrvcatToVotVcVcr−a

3

ooACciiiocTontCs

Fig. 2. Actions of the various ACTH secretagogues on corticotropes. (A) AVP (100 nM)evoked a biphasic pattern of Ca2+ signal in a rat corticotrope. The cell was whole-cellvoltage clamped at −60 mV in a Ca2+-free bath solution (that contained 1 mM EGTA).(B) NE (1 �M) evoked [Ca2+]i oscillations in a rat corticotrope. The cell was voltageclamped at −60 mV. (C) Summary of the actions of CRH, AVP and NE. The closure

A. Tse et al. / Cell Ca

our unpublished observation). In enriched rat corticotropes,RH was reported to reduce an inwardly rectifying K+

urrent by ∼25% [21]. By contrast, our study in identified ratorticotropes shows that CRH largely suppressed a backgroundEA-insensitive K+ current at the entire range of potentials (−100o +80 mV), indicating that the CRH-sensitive current was not annward rectifier [6]. In our recent study of mouse corticotropes5], we identified TREK-1 channel as the primary target of CRHegulation. CRH reduced the TREK-1 current (elicited by intracel-ular acidification) in mouse corticotropes and resulted in strongepolarization [5]. The depolarizing action of CRH was largelyttenuated in the presence of fluoxetine, further supporting theotion that CRH acted on TREK-1 channels. Consistent with theoupling of CRH receptors to adenylate cyclase, the action of CRHn corticotropes is mediated via the elevation of cellular cAMP.pplication of the cell-permeable cAMP analog, 8-CPT-cAMP toouse corticotropes attenuated the TREK-1 current at a wide-

ange of potentials [5]. Intracellular application of the proteininase A (PKA) inhibitor, Rp-cAMPS blocked the effect of CRHn the membrane potential of rat corticotropes [6]. Thus, theroperties of the CRH-sensitive background K+ current in ratorticotropes closely resemble those of the TREK-1 current inouse corticotropes, indicating that TREK-1 current is the primary

arget of CRH regulation in both rat and mouse corticotropes.In female rat corticotropes, CRH increased the frequency of Ca2+

ransients in cells that exhibited spontaneous Ca2+ transients andvoked Ca2+ transients in cells that were silent [15]. By contrast,n our study of male rat corticotropes, the resting [Ca2+]i wastable and CRH triggered a plateau depolarization (to ∼−30 mV),esulting in a persistent activation of extracellular Ca2+ entryia VGCC and a sustained [Ca2+]i rise [6]. We found that in ratorticotropes, the threshold for VGCC activation was at ∼−40 mVnd measurement of the steady-state inactivation of VGCC showedhat ∼80% of VGCC was available for activation at ∼−30 mV [6].he CRH-mediated Ca2+ signal was abolished by VGCC inhibitorr by voltage clamping cells to negative potentials to preventGCC activation [6]. Thus, in corticotropes, CRH causes the closuref background TREK-1 channels via a cAMP/PKA pathway andhe resultant depolarization activates extracellular Ca2+ entry viaGCC (summarized in Fig. 2C). In AtT-20 cells, CRH as well as cAMPaused [Ca2+]i elevation [28] and cAMP was reported to enhanceGCC [29]. However, we found that in both rat [6] and mouseorticotropes (unpublished observation), CRH caused a small andeversible inhibition of VGCC at potentials more positive than20 mV. Thus, the CRH-mediated elevation of cAMP did not haveny enhancing effect on the VGCC of primary corticotropes.

.2. Actions of AVP and NE

In contrast to CRH, AVP stimulation evoked a biphasic patternf Ca2+ signal in rat [7] and mouse corticotropes (our unpublishedbservation). Both the “transient and plateau” phases of theVP-induced Ca2+ signal persisted in the absence of extracellulara2+ (e.g. Fig. 2A) or when individual corticotropes were voltagelamped at −90 mV to prevent VGCC activation [7], suggesting thatntracellular Ca2+ release contributed to both phases of the AVP-nduced Ca2+ signal. Consistent with the notion that AVP triggeredntracellular Ca2+ release from the IP3-sensitive store via activationf the phospholipase C pathway, intracellular dialysis of heparin (aompetitive blocker of IP3 receptors) prevented the action of AVP.he AVP-induced [Ca2+]i rise was accompanied by the activationf SK channels which in turn caused hyperpolarization. Simulta-

eous measurements of [Ca2+]i and membrane potential showedhat both the “transient and plateau” phases of the AVP-induceda2+ signal were accompanied by hyperpolarization [7], furtherupporting the notion that the plateau phase of the Ca2+ signal was

of the TREK-1 channels by CRH (via the cAMP/PKA pathway) caused depolarizationand activation of VGCC. AVP and NE stimulated Ca2+ release from the IP3-sensitivestore and caused hyperpolarization via the activation of SK channels.

independent of VGCC activation. Note that it has been reported thatin a fraction of rat corticotropes, AVP stimulated a transient Ca2+

signal that was dependent on voltage-gated Ca2+ entry [22]. It isunclear whether the heterogeneous AVP response reflects differentpopulations of rat corticotropes. Indeed, several populations ofrat corticotropes with different sensitivities to CRH and AVP havebeen detected by the reverse hemolytic plaque assays [24].

A biphasic pattern of Ca2+ signal associated with intracellularCa2+ release was also observed in corticotropes stimulated withOT [3] or Ang II (our unpublished observation). Although NE alsostimulated intracellular Ca2+ release from the IP3-sensitive store inrat corticotropes [8], the NE-induced Ca2+ signal comprised slowoscillations (e.g. Fig. 2B). The frequency of the NE-induced [Ca2+]ioscillations ranged from 0.02 to 0.08 Hz, much slower than the[Ca2+]i oscillations induced by gonadotrophin-releasing hormone(GnRH) in male rat pituitary gonadotropes (0.2–0.3 Hz) [30,31]or ovariectomized female rat gonadotropes (0.1–0.5 Hz) [32,33].Interestingly, intracellular dialysis of IP3 triggered [Ca2+]i oscil-lations in gonadotropes [30] but evoked the biphasic pattern ofCa2+ signal in corticotropes [8]. The action of NE was mediated via�-adrenergic receptors and the NE-induced [Ca2+]i oscillationswere accompanied by periodic activation of SK current [8]. Thus,AVP as well as NE evoked hyperpolarization in corticotropes, aneffect opposite to that of CRH (summarized in Fig. 2C).

4. Dominant role of mitochondria in the shaping of Ca2+

signal

As described above, ACTH secretagogues can triggerextracellular Ca2+ entry via VGCC or release Ca2+ from the

2 lcium 51 (2012) 253– 259

IegCal1skotoCp(SiccCmmmd[mttCtccoir

rcttmdcjtA

5

5

at“iromwtetaa

Fig. 3. The releasable pools of granules in rat corticotropes. (A) Flash photolysis ofcaged Ca2+ triggered two kinetically distinct components of Cm increase (reflectingexocytosis). The fast exocytic component terminated within 100 ms after the UVflash and the slow component reached a plateau in ∼5 s after the UV flash. (B) Sizeof the releasable pools of granules estimated from the amplitude of the fast andslow components of �Cm. The amplitude of the slow component was estimatedfrom �Cm at 1 and 5 s after the UV flash. Data averaged from 15 cells. (C) Size of the

56 A. Tse et al. / Cell Ca

P3-sensitive store. Following the termination of VGCC-mediatedxtracellular Ca2+ entry or intracellular Ca2+ release, [Ca2+]i decaysradually to the basal level, reflecting the clearance of cytosolica2+ by various Ca2+ transport mechanisms. In rat corticotropes,fter a depolarization-triggered [Ca2+]i rise (to several micromo-ars), the time course of the decay of the Ca2+ signal (recorded with00 �M indo-1 in the whole-cell pipette) could be described by aingle exponential function with a time constant of 5–6 s [12]. Thisinetics of cytosolic Ca2+ clearance is severalfold slower than thatf rat pancreatic � cells (time constant of 1–2 s; recorded underhe same experimental condition) [34]. It is also different fromur previous study on rat gonadotropes, in which the decay of thea2+ signal (>0.5 �M) was biphasic [35]. In both gonadotropes andancreatic � cells, the sarco-endoplasmic reticulum Ca2+-ATPaseSERCA) pump plays a major role in cytosolic Ca2+ clearance.ERCA pump inhibition slowed the rate of cytosolic Ca2+ clearancen gonadotropes or pancreatic � cells by ∼3-fold [34,35]. Byontrast, SERCA pump inhibition slowed the rate of cytosolic Ca2+

learance in corticotropes by only ∼1.2-fold [12]. In corticotropes,a2+ uptake via the mitochondrial uniporter is the dominantechanism of cytosolic Ca2+ clearance. Our study shows thatitochondrial uncouplers (CCCP and cyanide) or an inhibitor ofitochondrial uniporter (ruthenium red) slowed the decay of the

epolarization-triggered Ca2+ signal in corticotropes by ∼3-fold12]. Inhibition of the Na+/Ca2+ exchanger (NCX) or the plasma

embrane Ca2+ ATPase (PMCA) had little effect on the decay ofhe Ca2+ signal in corticotropes [12]. This result differed fromhat of AtT-20 cells, in which PMCA and NCX were the dominanta2+ clearance mechanisms [36]. Importantly, the duration ofhe Ca2+ signal strongly influenced the exocytotic response inorticotropes. Simultaneous measurements of [Ca2+]i and exo-ytosis (capacitance measurement) indicated that the slowingf the depolarization-triggered Ca2+ signal by mitochondrialnhibitor was accompanied by an enhancement of the exocytoticesponse [12].

Measurement of mitochondrial Ca2+ signal with rhod-2, alsoevealed that depolarization elevated mitochondrial [Ca2+] inorticotropes [12]. Since the CRH-mediated Ca2+ signal is dueo depolarization and VGCC activation, the uptake of Ca2+ intohe mitochondria during CRH stimulation can, in turn, activate

itochondrial metabolism to match the increase in metabolicemand imposed by the secretory activities of the cell. Thus, inorticotropes, the activation of mitochondrial metabolism in con-unction with a slower rate of cytosolic Ca2+ clearance may enablehese cells to maintain a sustained [Ca2+]i rise and a prolongedCTH release during diurnal ACTH surge or chronic stress.

. Exocytosis and endocytosis

.1. Exocytosis triggered via flash photolysis of caged Ca2+

In most endocrine cells, only a small number of granulesre ready to undergo exocytosis (“docked” and “primed”) whenriggered by a rise in [Ca2+]i and this pool of granules is namedthe readily releasable pool”. The readily releasable pool replen-shes from another pool of “docked” granules which have slowerelease kinetics (the “slowly releasable pool”) [37,38]. Most ofur knowledge of the releasable pools of granules came fromeasurements of changes in cell membrane capacitance (�Cm)hen [Ca2+]i was elevated rapidly and uniformly to a high concen-

ration (tens of micromolars) via flash photolysis of caged Ca2+. For

xample, in bovine chromaffin cells, elevation of [Ca2+]i to ∼25 �Mriggered two phases of Cm increase: a fast component (also calledn exocytic burst) that occurred within 1 s after the UV flash and

slower component with a time constant of several seconds [39].

releasable pools of granules when normalized to individual cell size. The amplitudeof the fast and slow components from each cell was normalized as the % increase ofits initial cell membrane capacitance.

The exocytic burst could be fitted with double exponentials (withtime constants of ∼60 and ∼300 ms) and the amplitudes of thedouble exponentials were interpreted as the size of the readilyreleasable pool and the slowly releasable pool, respectively [37,38].In rat corticotropes, rapid elevation of [Ca2+]i to ∼20–30 �M (viaflash photolysis of caged Ca2+) also evoked a fast Cm increasewhich terminated within 100 ms after the UV flash (e.g. Fig. 3A).This fast component could be described by a single exponentialwith an average time constant of 70 ± 7 ms (n = 12), reflecting thedepletion of the readily releasable pool of granules. On average,the depletion of the readily releasable pool of granules in ratcorticotropes increased Cm by 122 ± 9 fF (Fig. 3B), corresponding toan increase of 2.8 ± 0.1% in cell surface area (Fig. 3C) [8]. Note thatthis value is ∼50% smaller than that detected in our previous studyin gonadotropes where the depletion of the readily releasable poolof granules increased Cm by ∼270 fF (∼4.7% increase in cell surfacearea) [40]. In contrast to chromaffin cells [39], the fast Cm increasein corticotropes was often followed by a rapid decrease in cellmembrane capacitance (e.g. Fig. 3A), reflecting “fast” endocytosis(described later in Section 5.3). The “fast” endocytosis was followedby a slow component of Cm increase that had an average time con-stant of 3.4 ± 0.3 s (n = 15) and typically reached a plateau within5–8 s after the UV flash (Fig. 3A). The amplitude of the slow com-ponent measured at 1 or 5 s after the UV flash was 224 ± 40 fF and872 ± 111 fF, respectively (Fig. 3B), corresponding to 5.0 ± 0.8% and

20 ± 2% increase in cell surface area (Fig. 3C). Note that the increasein Cm (∼224 fF) at 1 s after the UV flash in corticotropes is compa-rable to the ∼300 fF increase that was attributed to the size of theslowly releasable pool of granules in chromaffin cells [38,39]. Thus,

A. Tse et al. / Cell Calcium

Fig. 4. The Ca2+-dependence of the rate of exocytosis from the readily releasablepool of granules in rat corticotropes. (A) A train of depolarization (5 voltage stepsfrom −70 mV to +10 mV; 250 ms in duration) evoked [Ca2+]i rise and exocytosis.Following exocytosis, Cm decreased (reflecting endocytosis) with a time constantof ∼7 s. (B) Flash photolysis of caged IP3 triggered [Ca2+]i rise and exocytosis. Cm

returned to the basal level with a time constant of ∼6 s. (C) The rate of exocytosisversus [Ca2+] when [Ca2+] was elevated via depolarization, flash photolysis of cagedI

M

ia

ffleoswecoa∼

5i

umyeuoo

2+

i i

P3, dialysis of Ca2+-buffered solution or flash photolysis of caged Ca2+.

odified from Fig. 8 of Tse and Lee [25].

t is likely that the slower component of exocytosis in corticotropeslso reflects release of granules from the slowly releasable pool.

We measured the Ca2+-dependence of the rate of exocytosisrom the readily releasable pool by varying the intensity of the UVash to elevate [Ca2+]i uniformly to different levels [25]. In somexperiments, [Ca2+]i was elevated to 2–3 �M via whole-cell dialysisf a Ca2+–HEDTA mixture. As shown in Fig. 4C, the rate of exocyto-is of the readily releasable granules in rat corticotropes increasedith [Ca2+]i. The data could be described by a least square fit to a Hill

quation if the binding of three Ca2+ ions is required to activate exo-ytosis (Fig. 4C). The rate of exocytosis was half-maximal at [Ca2+]if 19 ± 2 �M and saturated at 2140 ± 325 fF/s [25]. These valuesre comparable to those obtained from gonadotropes (16 �M and5000 fF/s) using identical experimental procedures [40].

.2. Exocytosis triggered by VGCC-mediated Ca2+ entry andntracellular Ca2+ release

Among the ACTH secretagogues, CRH is most potent in stim-lating ACTH secretion [11]. In many endocrine cells, includingelanotropes [41] and pancreatic � cells [42], intracellular dial-

sis of cAMP was reported to enhance depolarization-triggered

xocytosis. Since the cAMP level in corticotropes was elevatedpon CRH stimulation, we tested whether the potent action of CRHn ACTH secretion was related to a cAMP-mediated enhancementf the exocytotic response. However, we found that the inclusion

51 (2012) 253– 259 257

of cAMP in the whole-cell pipette did not cause any enhance-ment in the amount of depolarization-triggered exocytosis whencompared to controls at similar [Ca2+]i (1–4 �M) or any accelerationin the recovery of the exocytic response [25]. Thus, in corticotropes,cAMP does not enhance the depolarization-triggered exocytosis.

Since CRH triggered extracellular Ca2+ entry via VGCC and AVPtriggered intracellular Ca2+ release, one possible explanation for themore potent action of CRH was that VGCC-mediated Ca2+ entry gen-erated a spatial Ca2+ gradient near the exocytic sites. We tested thispossibility by comparing the rate of exocytosis at different [Ca2+]iwhen [Ca2+]i in individual rat corticotrope was uniformly elevatedvia flash photolysis of caged Ca2+ (e.g. Fig. 3A) with that triggered viaactivation of VGCC (e.g. Fig. 4A) or flash photolysis of caged IP3 (e.g.Fig. 4B) [25]. As shown in Fig. 4C, when [Ca2+]i was elevated via flashphotolysis of caged Ca2+, the rate of exocytosis reached 100 fF/s at[Ca2+]i of 7.5 �M. By contrast, during depolarization-triggered exo-cytosis, the same rate of exocytosis was reached at [Ca2+]i of 3 �M.This finding suggests that VGCC-mediated Ca2+ entry generateda spatial Ca2+ gradient near the exocytic sites, such that the local[Ca2+] was ∼2.5-fold higher than the mean cytosolic [Ca2+]. Whenexocytosis was triggered via flash photolysis of caged IP3, the rateof exocytosis reached 100 fF/s at [Ca2+]i of 2 �M (Fig. 4C). Thus,Ca2+ release from the IP3-sensitive store also generated a spatialCa2+ gradient comparable to that occurred during VGCC activation.Moreover, when compared at similar [Ca2+]i, the amplitude of theexocytotic response triggered by depolarization was similar tothat triggered by flash photolysis of caged IP3 [25]. Since VGCC-mediated Ca2+ entry and intracellular Ca2+ release have similarefficacy in triggering exocytosis in corticotropes, it is unlikely thatthe spatial Ca2+ gradient generated during VGCC activation canaccount for the potent action of CRH on ACTH secretion.

Note that for gonadotropes which utilize Ca2+ release from theIP3-sensitive store as the major trigger for exocytosis [43], we haveshown that GnRH stimulation generated a spatial Ca2+ gradientwhich was ∼5-fold higher than the mean cytosolic [Ca2+] (c.f. ∼3-fold in corticotropes) [40]. The smaller Ca2+-gradient generated byintracellular Ca2+ release in corticotropes may be due to a smallerCa2+ reserve or a lower abundance of IP3 stores and/or IP3 receptorexpression (thus less co-localization with the exocytic sites). In cor-ticotropes [25], both the peak amplitude (ranged from 0.6 to 3 �M)and the rate of [Ca2+]i rise (∼5 �M/s) triggered by flash photolysis ofcaged IP3 were smaller than those of gonadotropes (peak amplitudeof 3–4 �M and a rate of [Ca2+]i rise of ∼20 �M/s) [40]. Consis-tent with a weaker exocytotic response in corticotropes whenstimulated by agonist that triggered intracellular Ca2+ release,application of AVP or NE increased Cm by ∼50–60 fF [7,8], releasingonly ∼50% of the readily releasable pool of granules. By contrast,the first two cycles of [Ca2+]i oscillation induced by GnRH typicallyexhausted the readily releasable pool in gonadotropes [40,43].

5.3. Close coupling of endocytosis to exocytosis

As shown in Fig. 3A, the fast exocytic burst triggered via flashphotolysis of caged Ca2+ was followed by a “fast” form of endo-cytosis that retrieved essentially all the excess membrane withinhundreds of milliseconds. “Fast” endocytosis occurred predomi-nantly at very high [Ca2+]i and was frequently observed when flashphotolysis of caged Ca2+ elevated [Ca2+]i > 13 �M [13]. It was alsodetected in ∼30% of the exo/endocytotic events when depolariza-tion elevated [Ca2+]i > 3.5 �M [13]. By contrast, a “slow” form ofendocytosis was typically detected in corticotropes at the physio-logical range of [Ca2+]i. As shown in the examples in Fig. 4A and B,

exocytosis triggered by depolarization or Ca release from the IP3-sensitive store was followed by a “slow” endocytosis that retrievedthe membrane with a time constant of several seconds. The amountof membrane retrieved by “slow” endocytosis was typically slightly

2 lcium

l[[eeib(fiha[ac

6

rrawftTbtlvoaaacd(1e1CedccTiar

7

msnsAoIfuscta

[

[

[

[

[

[

[

58 A. Tse et al. / Cell Ca

arger (∼20%) than the increase in cell membrane during exocytosis13]. As shown in Fig. 4A and B, “slow” endocytosis continued afterCa2+]i had returned to the basal level, indicating that once “slow”ndocytosis was triggered, its completion did not require [Ca2+]ilevation. On average, the time constant of the “slow” endocytosisn corticotropes was ∼6 s and this value was not strongly affectedy the amplitude of the depolarization-triggered [Ca2+]i riseranged from 0.5 �M to several micromolars) [13]. Overall, ourndings suggest that the Ca2+ sensor for “slow” endocytosis has aigh Ca2+ affinity (∼500 nM as estimated by our simulation) whichccounted for the continuation of endocytosis at resting [Ca2+]i13]. Thus, in corticotropes, the tight coupling between exocytosisnd endocytosis may be important for maintaining the continuousycles of exo–endocytosis during sustained ACTH secretion.

. AA as an intrinsic negative feedback regulator

Upon stimulation with CRH or AVP, AA was generated in ante-ior pituitary cells [44,45]. Since both CRH and AVP trigger [Ca2+]iise in corticotropes, it is likely that the agonist-induced [Ca2+]i risectivates the Ca2+-dependent form of cytosolic phospholipase A2hich in turn cleaves membrane phospholipids to generate AA. We

ound that exogenous application of AA (5 or 10 �M) to mouse cor-icotropes hyperpolarized the membrane potential to ∼−80 mV [5].he AA-mediated hyperpolarization was due to the activation of aackground K+ current. Inhibition of AA metabolism with eicosate-raynoic acid (a non-metabolizable analog of AA that is a blocker ofipoxygenase, cycloxygenase and P450 pathways [46]) did not pre-ent the activation of K+ current by AA, suggesting a direct actionf AA on the K+ channels [5]. In the literature, AA was reported toctivate the TREK family of K+ channels, including TREK-1, TREK-2nd TWIK-related AA activated K+ (TRAAK) channels [47]. The AA-ctivated K+ current in corticotropes was reduced by fluoxetine,hlorpromazine, extracellular acidification, diphenylbutylpiperi-ine antipsychotics and the membrane permeable cAMP analog8CPT-cAMP), indicating that the current was mediated via TREK-

channels [5]. Thus, AA and CRH have opposing actions on thexcitability of corticotropes via a reciprocal regulation of the TREK-

channels. The concentration of AA near the corticotropes duringRH stimulation is unclear but the plasma concentration of AA isstimated to be at ∼2–10 �M [48]. Therefore, it is conceivable thaturing a prolonged CRH stimulation (e.g. due to chronic stress), theoncentration of AA near the plasma membrane of corticotropesould reach several micromolars, resulting in the activation ofREK-1 channels and hyperpolarization. This will limit the depolar-zation evoked by CRH, resulting in a reduction in VGCC activationnd ACTH release. Thus, AA may be an intrinsic negative feedbackegulator to prevent excessive ACTH release during chronic stress.

. Future perspectives

By employing identified rat and mouse corticotropes, we haveade significant progress in understanding how different ACTH

ecretagogues and Ca2+ transport mechanisms shape the Ca2+ sig-al and the impact of the Ca2+ signal on exo–endocytosis. However,everal major issues remain unresolved. For example, CRH andVP act synergistically to stimulate ACTH secretion but they havepposing actions on the electrical excitability of the corticotropes.mportantly, glucocorticoid acts on corticotropes to exert negativeeedback regulation on CRH-mediated ACTH secretion but thenderlying mechanism is not completely understood. Previous

tudies in AtT-20 cells [16,49] and rat pituitary cells [50] have impli-ated the involvement of BK channels, the A-type K+ channels orhe ether-a-go-go-related gene (erg) K+ channels in the inhibitoryction of glucocorticoid on corticotropes. With our recent discovery

[

51 (2012) 253– 259

that TREK-1 channel is the major determinant of resting potentialas well as the target of CRH regulation in corticotropes [5], it will beimportant to address whether TREK-1 channel is also affected byglucocorticoid. As discussed in the article of Dr. S. Stojilkovic, in thisissue, the channels that determine the resting potential of differenttypes of pituitary cells remain elusive. Since TREK-1 channels arestrongly regulated by phosphorylations [51], it can be a potentialtarget for many G-protein coupled receptors in the anterior pitu-itary gland. Thus, it is possible that TREK channels also contribute tothe regulation of resting potential in other types of pituitary cells.

Conflict of interest statement

We (Amy Tse, Andy Lee and Frederick Tse) have no con-flict of interest with other people or organizations that couldinappropriately influence (bias) our work within 3 years ofbeginning the work submitted.

Acknowledgment

This work was supported by research grants from the CanadianInstitute of Health Research.

References

[1] F.A. Antoni, Vasopressinergic control of pituitary adrenocorticotropin secretioncomes of age, Front. Neuroendocrinol. 14 (1993) 76–122.

[2] F.A. Antoni, Hypothalamic control of adrenocorticotropin secretion: advancessince the discovery of 41-residue corticotropin-releasing factor, Endocr. Rev. 7(1986) 351–378.

[3] H. Link, G. Dayanithi, K.J. Fohr, M. Gratzl, Oxytocin at physiological concen-trations evokes adrenocorticotropin (ACTH) release from corticotrophs byincreasing intracellular free calcium mobilized mainly from intracellular stores.Oxytocin displays synergistic or additive effects on ACTH-releasing factor orarginine vasopressin-induced ACTH secretion, respectively, Endocrinology 130(1992) 2183–2191.

[4] C. Viero, I. Shibuya, N. Kitamura, et al., Oxytocin: crossing the bridge betweenbasic science and pharmacotherapy, CNS Neurosci. Ther. 16 (2010) e138–e156(Review).

[5] A.K. Lee, J.L. Smart, M. Rubinstein, M.J. Low, A. Tse, Reciprocal regulationof TREK-1 channels by arachidonic acid and CRH in mouse corticotropes,Endocrinology 152 (2011) 1901–1910.

[6] A.K. Lee, A. Tse, Mechanism underlying corticotropin-releasing hormone (CRH)triggered cytosolic Ca2+ rise in identified rat corticotrophs, J. Physiol. 504 (Pt 2)(1997) 367–378.

[7] A. Tse, A.K. Lee, Arginine vasopressin triggers intracellular calcium release,a calcium-activated potassium current and exocytosis in identified rat corti-cotropes, Endocrinology 139 (1998) 2246–2252.

[8] A. Tse, F.W. Tse, alpha-Adrenergic stimulation of cytosolic Ca2+ oscillations andexocytosis in identified rat corticotrophs, J. Physiol. 512 (Pt 2) (1998) 385–393.

[9] M. Carnes, S. Lent, J. Feyzi, D. Hazel, Plasma adrenocorticotropic hormone in therat demonstrates three different rhythms within 24 h, Neuroendocrinology 50(1989) 17–25.

10] M. Carnes, S.J. Lent, S. Erisman, C. Barksdale, J. Feyzi, Immunoneutralization ofcorticotropin-releasing hormone prevents the diurnal surge of ACTH, Life Sci.45 (1989) 1049–1056.

11] J.G. Won, D.N. Orth, Roles of intracellular and extracellular calcium in the kineticprofile of adrenocorticotropin secretion by perifused rat anterior pituitarycells. I. Corticotropin-releasing factor stimulation, Endocrinology 126 (1990)849–857.

12] A.K. Lee, A. Tse, Dominant role of mitochondria in calcium homeostasis of singlerat pituitary corticotropes, Endocrinology 146 (2005) 4985–4993.

13] A.K. Lee, A. Tse, Endocytosis in identified rat corticotrophs, J. Physiol. 533 (2001)389–405.

14] J.B. Corcuff, N.C. Guerineau, A. Tabarin, P. Mollard, Tumor-related selection ofcalcium signals in vasopressin-stimulated human adenomatous corticotrophs,Am. J. Physiol. 269 (1995) E451–E457.

15] N. Guerineau, J.B. Corcuff, A. Tabarin, P. Mollard, Spontaneous andcorticotropin-releasing factor-induced cytosolic calcium transients in corti-cotrophs, Endocrinology 129 (1991) 409–420.

16] M.J. Shipston, J.S. Kelly, F.A. Antoni, Glucocorticoids block protein kinase Ainhibition of calcium-activated potassium channels, J. Biol. Chem. 271 (1996)

9197–9200.

17] E. Yamamori, Y. Iwasaki, Y. Oki, et al., Possible involvement of ryanodinereceptor-mediated intracellular calcium release in the effect of corticotropin-releasing factor on adrenocorticotropin secretion, Endocrinology 145 (2004)36–38.

lcium

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

A. Tse et al. / Cell Ca

18] Y.A. Kuryshev, G.V. Childs, A.K. Ritchie, Corticotropin-releasing hormone stim-ulation of Ca2+ entry in corticotropes is partially dependent on protein kinaseA, Endocrinology 136 (1995) 3925–3935.

19] Y.A. Kuryshev, G.V. Childs, A.K. Ritchie, Three high threshold calcium channelsubtypes in rat corticotropes, Endocrinology 136 (1995) 3916–3924.

20] Y.A. Kuryshev, G.V. Childs, A.K. Ritchie, Corticotropin-releasing hormone stim-ulates Ca2+ entry through L- and P-type Ca2+ channels in rat corticotropes,Endocrinology 137 (1996) 2269–2277.

21] Y.A. Kuryshev, L. Haak, G.V. Childs, A.K. Ritchie, Corticotropin releasing hor-mone inhibits an inwardly rectifying potassium current in rat corticotropes,J. Physiol. 502 (Pt 2) (1997) 265–279.

22] J.B. Corcuff, N.C. Guerineau, P. Mariot, B.T. Lussier, P. Mollard, Multiple cytoso-lic calcium signals and membrane electrical events evoked in single argininevasopressin-stimulated corticotrophs, J. Biol. Chem. 268 (1993) 22313–22321.

23] B.J. Canny, L.G. Jia, D.A. Leong, Corticotropin-releasing factor, but not argi-nine vasopressin, stimulates concentration-dependent increases in ACTHsecretion from a single corticotrope. Implications for intracellular signals instimulus–secretion coupling, J. Biol. Chem. 267 (1992) 8325–8329.

24] L.G. Jia, B.J. Canny, D.N. Orth, D.A. Leong, Distinct classes of corticotropes medi-ate corticotropin-releasing hormone- and arginine vasopressin-stimulatedadrenocorticotropin release, Endocrinology 128 (1991) 197–203.

25] A. Tse, A.K. Lee, Voltage-gated Ca2+ channels and intracellular Ca2+ release reg-ulate exocytosis in identified rat corticotrophs, J. Physiol. 528 (Pt 1) (2000)79–90.

26] M.A. Cowley, J.L. Smart, M. Rubinstein, et al., Leptin activates anorexigenicPOMC neurons through a neural network in the arcuate nucleus, Nature 411(2001) 480–484.

27] A. Dedman, R. Sharif-Naeini, J.H. Folgering, F. Duprat, A. Patel, E. Honore, Themechano-gated K(2P) channel TREK-1, Eur. Biophys. J. 38 (2009) 293–303.

28] S. Guild, T. Reisine, Molecular mechanisms of corticotropin-releasing factorstimulation of calcium mobilization and adrenocorticotropin release fromanterior pituitary tumor cells, J. Pharmacol. Exp. Ther. 241 (1987) 125–130.

29] A. Luini, D. Lewis, S. Guild, D. Corda, J. Axelrod, Hormone secretagogues increasecytosolic calcium by increasing cAMP in corticotropin-secreting cells, Proc.Natl. Acad. Sci. U.S.A. 82 (1985) 8034–8038.

30] A. Tse, B. Hille, GnRH-induced Ca2+ oscillations and rhythmic hyperpolariza-tions of pituitary gonadotropes, Science 255 (1992) 462–464.

31] A. Tse, F.W. Tse, B. Hille, Modulation of Ca2+ oscillation and apamin-sensitive,Ca2+-activated K+ current in rat gonadotropes, Pflugers Arch. 430 (1995)645–652.

32] N.C. Guerineau, R. Bouali-Benazzouz, J.B. Corcuff, M.C. Audy, M. Bonnin,

P. Mollard, Transient but not oscillating component of the calcium mobiliz-ing response to gonadotropin-releasing hormone depends on calcium influx inpituitary gonadotrophs, Cell Calcium 13 (1992) 521–529.

33] T. Iida, S.S. Stojilkovic, S. Izumi, K.J. Catt, Spontaneous and agonist-induced cal-cium oscillations in pituitary gonadotrophs, Mol. Endocrinol. 5 (1991) 949–958.

[

[

51 (2012) 253– 259 259

34] E. Hughes, A.K. Lee, A. Tse, Dominant role of sarcoendoplasmic reticulum Ca2+-ATPase pump in Ca2+ homeostasis and exocytosis in rat pancreatic {beta}-cells,Endocrinology 147 (2006) 1396–1407.

35] A. Tse, F.W. Tse, B. Hille, Calcium homeostasis in identified rat gonadotrophs,J. Physiol. 477 (Pt 3) (1994) 511–525.

36] J.F. Fiekers, The contributions of plasma membrane Na+–Ca2+-exchange and theCa2+-ATPase to the regulation of cytosolic calcium ([Ca2+]i) in a clonal pituitarycell line (AtT-20) of mouse corticotropes, Life Sci. 70 (2001) 681–698.

37] J.B. Sorensen, G. Nagy, F. Varoqueaux, et al., Differential control of the releasablevesicle pools by SNAP-25 splice variants and SNAP-23, Cell 114 (2003) 75–86.

38] D.R. Stevens, C. Schirra, U. Becherer, J. Rettig, Vesicle pools: lessons from adrenalchromaffin cells, Front. Synaptic Neurosci. 3 (2011) 2.

39] T. Xu, T. Binz, H. Niemann, E. Neher, Multiple kinetic components of exocytosisdistinguished by neurotoxin sensitivity, Nat. Neurosci. 1 (1998) 192–200.

40] F.W. Tse, A. Tse, B. Hille, H. Horstmann, W. Almers, Local Ca2+ release frominternal stores controls exocytosis in pituitary gonadotrophs, Neuron 18 (1997)121–132.

41] A.K. Lee, Dopamine (D2) receptor regulation of intracellular calcium and mem-brane capacitance changes in rat melanotrophs, J. Physiol. 495 (Pt 3) (1996)627–640.

42] C. Ammala, F.M. Ashcroft, P. Rorsman, Calcium-independent potentiationof insulin release by cyclic AMP in single beta-cells, Nature 363 (1993)356–358.

43] A. Tse, F.W. Tse, W. Almers, B. Hille, Rhythmic exocytosis stimulated byGnRH-induced calcium oscillations in rat gonadotropes, Science 260 (1993)82–84.

44] A.B. Abou-Samra, K.J. Catt, G. Aguilera, Role of arachidonic acid in the regu-lation of adrenocorticotropin release from rat anterior pituitary cell cultures,Endocrinology 119 (1986) 1427–1431.

45] J.G. Won, D.N. Orth, Role of lipoxygenase metabolites of arachidonic acid in theregulation of adrenocorticotropin secretion by perifused rat anterior pituitarycells, Endocrinology 135 (1994) 1496–1503.

46] L.D. Tobias, J.G. Hamilton, The effect of 5,8,11,14-eicosatetraynoic acid on lipidmetabolism, Lipids 14 (1979) 181–193.

47] A.J. Patel, E. Honore, Properties and modulation of mammalian 2P domain K+

channels, Trends Neurosci. 24 (2001) 339–346.48] H. Meves, Arachidonic acid and ion channels: an update, Br. J. Pharmacol. 155

(2008) 4–16.49] A.J. Pennington, J.S. Kelly, F.A. Antoni, Selective enhancement of an A type potas-

sium current by dexamethasone in a corticotroph cell line, J. Neuroendocrinol.6 (1994) 305–315.

50] M. Yamashita, Y. Oki, K. Iino, et al., The role of ether-a-go-go-related geneK(+) channels in glucocorticoid inhibition of adrenocorticotropin release byrat pituitary cells, Regul. Pept. 152 (2009) 73–78.

51] P. Enyedi, G. Czirjak, Molecular background of leak K+ currents: two-poredomain potassium channels, Physiol. Rev. 90 (2010) 559–605.