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RESEARCH ARTICLE

Purinergic control of AMPK activation by ATP releasedthrough connexin 43 hemichannels – pivotal roles inhemichannel-mediated cell injury

Yuan Chi1, Kun Gao1, Kai Li1,2, Shotaro Nakajima1, Satoru Kira2, Masayuki Takeda2 and Jian Yao1,*

ABSTRACT

Connexin hemichannels regulate many cell functions. However, the

molecular mechanisms involved remain elusive. Hemichannel

opening causes loss of ATP, we therefore speculated a potential

role for AMPK in the biological actions of hemichannels. Activation of

hemichannels by removal of extracellular Ca2+ led to an efflux of ATP

and a weak activation of AMPK. Unexpectedly, dysfunction of

hemichannels markedly potentiated AMPK activation, which was

reproduced by promotion of extracellular ATP degradation or

inhibition of P2 purinoceptors but counteracted by exogenous ATP.

Further analysis revealed that ATP induced a purinoceptor-

dependent activation of Akt and mTOR. Suppression of Akt or

mTOR augmented AMPK activation, whereas activation of Akt by

transfection of cells with myristoylated Akt, a constitutively active form

of Akt, abolished AMPK activation. In a pathological model of

hemichannel opening triggered by Cd2+, disclosure of hemichannels

similarly enhanced AMPK activity, which protected cells from Cd2+-

induced cell injury through suppression of mTOR. In summary, our

data point to a channel-mediated mechanism for the regulation of

AMPK through a purinergic signaling pathway. Furthermore, we

define AMPK as a pivotal molecule that underlies the regulatory

effects of hemichannels on cell survival.

KEYWORDS: Hemichannels, ATP, Purinergic signaling, AMPK, Cell

survival

INTRODUCTIONATP is the energy source for cell function and metabolic

activities, and is mainly produced in mitochondria. ATP is also

constantly secreted into the extracellular milieu, through several

different routes, where it functions as a paracrine and/or autocrine

mediator to regulate a wide range of cell functions (Baroja-Mazo

et al., 2013; Gordon, 1986; Lazarowski, 2012; Schwiebert and

Zsembery, 2003; Wang et al., 2013). Most of the actions of the

extracellular ATP are mediated through its binding to purinergic

receptors.

AMP-activated protein kinase (AMPK) is one of the major

kinases in the control of ATP homeostasis. It is ubiquitously

expressed in all cells and acts as an ultrasensitive sensor of

cellular energy. It is activated by an increase in the cellular AMP to

ATP ratio, which commonly occurs during metabolic stressesthat either interfere with ATP production or accelerateATP consumption. Once activated, AMPK switches off ATP-consuming pathways and switches on ATP-producing pathways;

thus, maintaining levels of cellular ATP (Hardie, 2011; Mihaylovaand Shaw, 2011). Although alterations in the level of intracellularATP are usually the result of changes in ATP generation or

consumption, it can also be caused by certain pathophysiologicalsituations that lead to loss of ATP into the extracellular space(Baroja-Mazo et al., 2013; Wang et al., 2013). Because of the

barrier created by the cell membrane, it is unclear whether or notthe extracellular ATP also influences AMPK activity.

Gap junctions are intercellular channels that permit the directexchange of ions, secondary messengers and small signalingmolecules between neighboring cells. Each gap junction channelis composed of two hemichannels that reside in the plasma

membrane of two closely apposed cells. Connexin (Cx) proteinsform gap junctions and, to date, more than 20 different connexinmolecules have been identified. Among them, Cx43 has been

extensively investigated because of its ubiquitous expression ina variety of cell types. Intercellular communication via gapjunctions is thought to play an important role in the regulation of

cell functions – including cell proliferation, migration,differentiation and survival (Saez et al., 2003; Yao et al., 2009;Yao et al., 2007). The majority of the biological effects of gap

junctions are mediated by the direct transmission of signalingmolecules among neighboring cells. Nonjunctional connexinhemichannels also participate in the regulation of cell signalingand cell behaviors through the release of the mediators ATP,

NAD+ or glutamate (Baroja-Mazo et al., 2013; Evans et al., 2006;Saez et al., 2003; Wang et al., 2013).

The activation of hemichannels has been reported in a varietyof pathological situations and has been shown to influence cellresponses to various stimuli (Fang et al., 2011; Ramachandran

et al., 2007; Thompson et al., 2006). The molecular mechanismsunderlying the effects of hemichannels on various cell functionsare still poorly understood. Several studies have pointed to a role

for ATP that has been released into the extracellular space.Indeed, hemichannel-derived ATP has been implicated in thetransmission of the intercellular Ca2+ signal and in the control ofcell growth, migration and survival (Anselmi et al., 2008; Fang

et al., 2011; Pearson et al., 2005; Stout et al., 2002; Zhao et al.,2005). Given that the efflux of ATP through hemichannels leadsto an altered intracellular level of ATP, we speculated that

hemichannel opening might affect the activation of AMPK andsubsequently lead to altered cell behaviors. Therefore, wesought to address this hypothesis and, here, we present our

results showing the existence of the previously unrecognized

1Department of Molecular Signaling, Interdisciplinary Graduate School ofMedicine and Engineering, University of Yamanashi, Chuo, Yamanashi 409-3898,Japan. 2Department of Urology, Interdisciplinary Graduate School of Medicineand Engineering, University of Yamanashi, Chuo, Yamanashi 409-3898, Japan.

*Author for correspondence (yao@yamanashi.ac.jp)

Received 25 July 2013; Accepted 9 January 2014

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 1487–1499 doi:10.1242/jcs.139089

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channel-mediated regulation of AMPK. Furthermore, wecharacterize AMPK as a pivotal molecule involved in the

biological actions of hemichannels on cell survival.

RESULTSCa2+ depletion activates Cx43 hemichannels and causesextracellular release of ATPWe have used a well-documented model of hemichannel openingthat is initiated by the removal of extracellular Ca2+ (Quist et al.,

2000; Stout et al., 2002). As shown in Fig. 1, the removal ofextracellular Ca2+ initiated an exchange of small molecules acrossthe plasma membrane, as indicated by the influx of the small-

molecular-mass fluorescent dyes Lucifer Yellow and ethidiumbromide, and the efflux of ATP (Fig. 1A–C). This effect was rapid asit was observable immediately after Ca2+ removal and was not

associated with an increased release of lactate dehydrogenase (LDH)(data not shown). Inhibition of hemichannels with the hemichannelblockers heptanol or lindane, or downregulation of Cx43 withspecific siRNA largely prevented the diffusion of the small

molecules (Fig. 1D–F). These results indicate that Ca2+

deprivation activates Cx43 hemichannels and induces extracellularrelease of ATP (Li et al., 2013; Muller et al., 2002; Stout et al., 2002).

Blockade of Cx43 hemichannels augments activation ofAMPKGiven that AMPK activity is inversely correlated with the levelsof intracellular ATP (Hahn-Windgassen et al., 2005; Mihaylovaand Shaw, 2011), we speculated that hemichannel-mediated loss

of ATP to the extracellular space would affect AMPK activation.Therefore, we examined the level of AMPK phosphorylation atthreonine residue 172. The level of phosphorylation at this site iscorrelated with AMPK activity (Towler and Hardie, 2007).

Removing extracellular Ca2+ induced a weak activation ofAMPK, as reflected by the appearance of a phosphorylatedband of AMPK (Fig. 2A). Contrary to our initial expectation,

blockade of the release ATP to the extracellular space with thehemichannel blockers heptanol or lindane markedly potentiatedAMPK activation (Fig. 2A,B). Treatment of cells with lanthanum

(La3+) or gadolinium (Gd3+), two nonselective cation channelblockers that also inhibit hemichannels (Saez et al., 2005), alsoproduced a similar potentiating effect (Fig. 2C).

To confirm the above finding, we modified the expression of

Cx43 by treating NRK cells with siRNA against Cx43, which isthe only connexin molecule expressed in NRK cells (Yao et al.,2010), or by transfection of gap-junction-deficient LLC-PK1 cells

with a vector encoding wild-type Cx43 (Fang et al., 2011). Asshown in Fig. 2D,E, downregulation of Cx43 in NRK cells byusing specific siRNA markedly augmented AMPK activation

upon removal of extracellular Ca2+. On the contrary, forcedexpression of Cx43 in gap-junction-deficient LLC-PK1 cellsgreatly suppressed AMPK activation, as compared with control

cells that had been transfected with a vector encoding a non-functional Cx43 mutant (Fig. 2F) (Fang et al., 2011). Similar tothe effects in NRK cells, the gap junction blockers heptanol andlindane also potentiated AMPK activation in LLC-PK1 cells

expressing wild-type EGFP–Cx43 (data not shown). Thus, theseresults indicate the existence of a mechanism for the suppressionof AMPK that is mediated by Cx43 hemichannels.

Extracellular ATP suppresses AMPK activationBecause ATP is one of the major determinants of AMPK activity

(Hahn-Windgassen et al., 2005; Mihaylova and Shaw, 2011), we

examined the possible role of ATP. Therefore, we inhibited ATPproduction by treating cells with antimycin (an inhibitor of

mitochondrial electron transport) and iodoacetic acid (an inhibitorof glycolysis). As expected, these chemicals caused a completedepletion of intracellular ATP (Fig. 3A). Consequently, theextracellular efflux of ATP that was triggered by removal of

extracellular Ca2+ was also abolished (Fig. 3B). The depletion ofATP was associated with a markedly increased level of AMPKphosphorylation (Fig. 3C). The potentiating effect of heptanol on

AMPK activation diminished in the absence of ATP. Theseresults suggest that extracellular ATP is important for theregulatory effects of hemichannels on AMPK activation.

To establish the role of the extracellular ATP, we examined theinfluence of ATP-degrading enzymes or exogenously added ATPon AMPK activation. As shown in Fig. 3D, the promotion of ATP

degradation with Apyrase augmented AMPK activation, whereassupplementing with ATP counteracted the effect of hemichannelblockers on AMPK activation (Fig. 3E,F). These results indicatethat hemichannel-derived ATP suppresses AMPK activation.

Most of the cellular actions of extracellular ATP are mediatedthrough its binding to P2 purinoceptors (Baroja-Mazo et al., 2013;Wang et al., 2013). At present, two classes of purinoceptors have

been identified (P2X and P2Y) and both are expressed in renaltubular cells (Praetorius and Leipziger, 2010). We thereforeexamined the possible involvement of purinergic signaling in

the suppression of AMPK activation. Suramin and PPADS,two effective antagonists of P2 purinoceptors (Charlton et al.,1996), markedly potentiated AMPK activation, indicating an

involvement of purinergic signaling in suppression of AMPKactivation (Fig. 3G,H).

Exogenous ATP suppresses AMPK activation throughpurinergic activation of AktTo investigate the signaling molecules downstream of P2purinoceptors, we focused on Akt (AKT1), which is a reported

negative regulator of AMPK (Hahn-Windgassen et al., 2005; Liuet al., 2012; Soltys et al., 2006). Removal of extracellular Ca2+

triggered a rapid activation of Akt that was largely blocked by the

P2 receptor antagonists suramin and PPADS (Fig. 4A).Consistent with this, exogenous ATP induced a concentration-dependent activation of Akt (Fig. 4B). Intriguingly, this effect ofATP was more robust under Ca2+-free conditions.

To establish the role of Akt in hemichannel- and/orextracellular-ATP-mediated suppression of AMPK, weexamined AMPK activation after inhibition of Akt and its

upstream regulator phosphoinositide 3-kinase (PI3K). As shownin Fig. 4C–D, the PI3K inhibitor LY294002 and the Akt inhibitorAkti1/2 effectively abrogated the activation of Akt that was

triggered by Ca2+ deprivation and both significantly potentiatedAMPK activation.

To establish the role of Akt further, we looked at the effects of

transfecting cells with a myristoylated (constitutively active)form of Akt (Johno et al., 2012). Cells expressing constitutivelyactive Akt had a higher basal level of Akt phosphorylation and alower level of AMPK activation (Fig. 4E–G). These observations,

thus, support an involvement of Akt in hemichannel-mediatedsuppression of AMPK.

As inactivation of the LKB1 kinase (STK11), which normally

phosphorylates AMPK, is one of the reported mechanismsunderlying the inhibitory effect of Akt on AMPK activity (Liuet al., 2012; Soltys et al., 2006), we examined LKB1 activation

under Ca2+-free conditions and its regulation by the P2

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purinoceptor pathway. Removal of extracellular Ca2+, indeed,

caused an elevation in the level of LKB1 phosphorylation, whichwas suppressed by the addition of exogenous ATP (Fig. 5A). Bycontrast, the promotion of ATP degradation with Apyrase orblockade of P2 purinoceptors with PPADS greatly potentiated

LKB1 activation (Fig. 5B). A similar potentiating effect was alsoachieved by blockade of the PI3K–Akt pathway with specific

inhibitors (Fig. 5C,D). These results, thus, indicate a P2-

purinoceptor-mediated suppression of LKB1 under Ca2+-freeconditions.

Activation of mammalian target of rapamycin (mTOR) hasbeen reported to be an alternative mechanism by which Akt

exerts its suppressive effect on AMPK activity (Hahn-Windgassen et al., 2005). Therefore, we assessed the possible

Fig. 1. Removal of extracellular Ca2+ activates Cx43 hemichannels. (A,B) Removal of extracellular Ca2+ leads to increased cellular uptake of LuciferYellow and ethidium bromide. NRK cells were pre-treated with or without 3 mM heptanol or 100 mM lindane for 30 min. Cells were then exposed to eithernormal or Ca2+-free culture medium that contained 0.1% Lucifer Yellow or 10 mM ethidium bromide for an additional 15 min. The uptake of Lucifer Yellow andethidium bromide was photographed. (C,D) Ca2+ depletion triggers ATP release. NRK cells were exposed to Ca2+-free culture medium for the indicated timeintervals (C) or for 5 min (D). Cell supernatants were collected the ATP concentration was quantified. Results are expressed as relative light units (RLU;mean6s.e.m., n53). *P,0.01 compared with control. (E,F) Downregulation of Cx43 with specific siRNA prevents ATP efflux and ethidium bromide uptakecaused by removal of extracellular Ca2+. NRK cells were treated with either control siRNA or siRNA against Cx43 for 48 h followed by exposure to Ca2+-freeculture medium. ATP release (E) and ethidium bromide uptake (F) was then assayed as described in Materials and Methods. The cellular lysates were alsosubjected to analysis of Cx43 by western blot to verify the efficacy of Cx43 siRNA in the downregulation of Cx43 (E, insert). Note the obvious reduction in theamount of Cx43 in cells treated with siRNA against Cx43.

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involvement of this mechanism. Ca2+ depletion, indeed, inducedan Akt-dependent activation of mTOR that was reflected

by the increased phosphorylation of p70S6K (RPS6KB1), adownstream target of mTOR, and the prevention ofphosphorylation upon treatment with the Akt inhibitor Akti1/2

(Fig. 5E,F). Treatment of cells with rapamycin, an inhibitorof mTOR, increased AMPK activation (Fig. 5E,G). Theseobservations, thus, indicate an involvement of mTOR in Akt-mediated suppression of AMPK.

Hemichannel-mediated suppression of AMPK contributes toCd2+-induced cell injuryWe have recently reported that Cd2+ activates Cx43 hemichannelsand causes an extracellular accumulation of ATP in LLC-PK1cells that express EGFP–Cx43 (Fang et al., 2011; Li et al., 2013).

Furthermore, we suggested that there is a role for hemichannels inCd2+-induced cell injury (Fang et al., 2011). Using this model, weconfirmed our findings and explored the possible participation ofthe signaling cascades in Cd2+-induced cell injury.

Fig. 2. Cx43 hemichannels regulate AMPK activation. (A–C) Dysfunction of hemichannels potentiates AMPK activation. NRK cells were pre-treated with orwithout 3 mM heptanol, 100 mM lindane, 500 mM La3+ or 500 mMGd3+ for 30 min before exposure to Ca2+-free culture medium for the indicated time (A) or 5 min(C). Cellular protein was extracted and subjected to western blot analysis for phosphorylated AMPK (pAMPKa) and its downstream target (phosphorylated)acetyl-CoA carboxylase (p-ACC). The equal loading of protein in each lane was verified by probing the blots with antibodies against AMPKa or b-actin.(B) Analysis of the phosphorylated AMPKa by using densitometry is shown in A. The results are representative of at least three separate experiments andexpressed as optical density (OD) (mean6s.e.m., n53) *P,0.01 compared with the respective timepoint zero control. (D) Augmentation of Ca2+-depletion-induced AMPKa activation by Cx43-specific siRNA (siCx43). Cells were transfected with siRNA against Cx43 or control siRNA (siControl) for 48 h. Cells werethen exposed to Ca2+-free culture medium for 5 min or left untreated. Cellular proteins were extracted and subjected to western blot analysis forphosphorylated AMPKa or Cx43. Equal loading of protein in each lane was verified by probing the blot with an antibody against b-actin. (E) Analysis ofphosphorylated AMPKa by using densitometry is shown in D. Results are representative of three separate experiments. *P,0.01 compared with cells that hadbeen treated with the control siRNA under Ca2+-free conditions. (F) Difference in AMPK activation between LLC-PK1 cells that were transfected with a vectorencoding mutant or wild-type EGFP–Cx43. Cells were exposed to Ca2+-free medium for the indicated time intervals.

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Consistent with our previous reports (Fang et al., 2011; Liet al., 2013), Cd2+ caused release of ATP into the extracellularspace in LLC-PK1 cells expressing EGFP–Cx43, which was

significantly prevented by the hemichannel blockers heptanol

and lindane (Fig. 6A), indicating an involvement ofhemichannels. Similar to the model of Ca2+ depletion,hemichannel opening that was triggered by Cd2+ was also

associated with a modest activation of AMPK, which could also

Fig. 3. Extracellular ATP suppresses AMPK activation. (A,B) The effect of the inhibition of ATP production on AMPK activation. NRK cells were pre-treatedwith 10 ng/ml antimycin A plus 0.5 mM iodoacetic acid for 20 min with or without exposure to Ca2+-free culture medium for an additional 5 min. The celllysates (A) and culture supernatants (B) were collected to assay for the release of ATP. (C) Cells were treated as above in the presence or absence of 3 mMheptanol. Cellular proteins were extracted and subjected to western blot for analysis of phosphorylated AMPKa. (D) Promotion of AMPK activation byApyrase. Cells were exposed to Ca2+-free culture medium in the presence or absence of 7.5 U/ml of Apyrase for the indicated time. (E,F) Suppression ofAMPK activation by exogenous ATP. Cells were exposed to Ca2+-free culture medium in the presence or absence of 3 mM heptanol (E) or 100 mM lindane(F) with or without the addition of 100 mM ATP for 5 min. (G,H) Involvement of purinoceptors in the suppression of AMPK activation. (G) Cells were pre-treatedwith or without 300 mM suramin or 10 mM PPADS for 30 min before exposure to Ca2+-free culture medium for an additional 5 min. Cellular proteins wereextracted and subjected to western blot analysis for phosphorylation of AMPKa. Results are representative of at least three separate experiments. (H) Analysisof the phosphorylated AMPKa by using densitometry is shown in G. Results are expressed as OD (mean6s.e.m., n53). *P,0.01 compared with the Ca2+-free control.

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be potentiated by hemichannel blockers and a P2 purinoceptorantagonist, suramin (Fig. 6B–D). These observations, thus,provide additional evidence to support an involvement of

hemichannel-derived ATP in the suppression of AMPKactivation.

Several recent studies have demonstrated that Cd2+-induced

cell death is through the activation of mTOR (Chen et al., 2011;Xu et al., 2011). Given that AMPK negatively regulates mTOR(Viollet et al., 2010), we speculated that hemichannel-mediatedregulation of AMPK might be implicated in Cd2+-induced cell

death. To test this hypothesis, we first confirmed that Cd2+

induced activation of mTOR in our experimental settings.Exposure of LLC-PK1 cells that expressed EGFP–Cx43 to Cd2+

caused a time-dependent phosphorylation of p70S6K, indicativeof mTOR activation (Fig. 7A). We then examined whethermTOR was affected by AMPK and hemichannels. Activation of

AMPK, by using aminoimidazole carboxamide ribonucleotide(AICAR), and inhibition of hemichannels with heptanol bothsuppressed mTOR activation, as indicated by the decreased

phosphorylation of p70S6K (Fig. 7B,C). Consistent with thereported mutual inhibition of mTOR and AMPK, there was aninverse correlation between the phosphorylated levels of p70S6K

and AMPK. In contrast to the decreased mTOR activity uponactivation of AMPK (Fig. 7B), suppression of mTOR withrapamycin enhanced AMPK activation (Fig. 7C). Finally, weasked whether suppression of mTOR attenuated cell injury. The

change in cell shape and the cell death induced by Cd2+ weremarkedly prevented by the mTOR inhibitor rapamycin, the AMPKactivators AICAR or metformin, and the hemichannel blockers

heptanol or lindane (Fig. 7D,E). Collectively, these observationssupport the hypothesis that involvement of hemichannel-mediatedregulation of AMPK is involved in Cd2+-induced cell death.

Fig. 4. Akt mediates extracellular-ATP-induced suppression ofAMPK. (A) Suppression of low-Ca2+-induced activation of Akt bypurinoceptor antagonists. NRK cellswere pre-treated with or without300 mM suramin or 10 mM PPADSfor 30 min before exposure to Ca2+-free culture medium for an additional5 min. Cellular proteins wereextracted and subjected to analysisby western blot for phosphorylatedAkt (p-Akt). (B) Induction of Aktactivation by extracellular ATP. Cellswere stimulated with the indicatedconcentrations of ATP in culturemedium with or without 1.8 mM Ca2+

for 5 min. (C,D) The effect ofinhibition of PI3K and Akt on AMPKactivation. Cells were pre-treatedwith or without 10 mM LY294002 or10 mM Akti1/2 for 30 min beforeexposure to Ca2+-free medium for anadditional 5 min. Phosphorylation ofAkt and AMPK was evaluated bywestern blot analysis. (D) Analysis ofthe phosphorylated AMPKa by usingdensitometry is shown in C. Resultswere expressed as OD (mean6

s.e.m., n53). *P,0.01 comparedwith the Ca2+-free control.(E) Expression of a myristoylatedconstitutively active form of Akt (myr-Akt) in low-Ca2+-induced activation ofAMPK. Cells were transfected witheither a myristoylated form of Akt orcontrol vector and exposed to eithernormal or Ca2+-free culture mediumfor 5 min. Cellular proteins wereextracted and subjected to analysisby western blot for phosphorylatedAkt and AMPK. (F,G) Analysis of thephosphorylated AKT (F) and AMPKa(G) by using densitometry is shownin E. Results were expressed as OD(mean6 s.e.m., n53). *P,0.01compared with the respective control.

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DISCUSSIONIn this study, we reveal a previously unrecognized channel-

mediated regulation of AMPK through a purinergic signalingpathway. Furthermore, we define AMPK as a pivotal moleculethat underlies the action of hemichannels on cell survival.

Activation of hemichannels has been reported in variouspathophysiological conditions, including connexin mutations,depolarization of the membrane potential, hypoxia and changesin intra- and extracellular Ca2+, as well as cellular redox status

(Anselmi et al., 2008; Saez et al., 2010). In this study,hemichannels were activated by removal of extracellular Ca2+.The opening of hemichannels under Ca2+-free conditions has

been demonstrated at both structural and functional levels. Usingan atomic force microscope, Muller and colleagues (Muller et al.,2002) observed that low extracellular Ca2+ increased the outer

pore diameter of hemichannels. Consistently, many investigators

have found increased hemichannel permeability (Li et al., 2013;Stout et al., 2002). Here, we detected an increased exchange of

small molecules across the cell membrane that was disrupted byblockade of Cx43-forming channels, indicating an involvement ofhemichannels.

In this investigation, we characterized AMPK as a novel targetof extracellular ATP. This is shown by the fact that the preventionof ATP release through blockade of hemichannels, the promotionof ATP degradation with Apyrase or the disruption of purinergic

signaling by using receptor antagonists all potentiated AMPKactivation. Furthermore, exogenous ATP suppressed AMPKactivation that had been initiated by removal of extracellular

Ca2+. ATP also suppressed AMPK activation triggered bythe AMPK activator metformin (data not shown) (Shackelfordand Shaw, 2009; Shaw, 2009). As an ultrasensitive sensor

that monitors cellular energy status and maintains energy

Fig. 5. ATP-mediated regulation of LKB1and mTOR activation. (A) Suppression ofLKB1 phosphorylation by ATP. NRK cellswere exposed to the indicated concentrationof ATP in culture medium with or without1.8 mM Ca2+ for 5 min. Cellular proteinswere extracted and subjected to westernblot analysis for phosphorylated LKB1 (p-LKB1). (B) Promotion of LKB1phosphorylation by Apyrase and PPADS.Cells were exposed to culture medium withor without 1.8 mM Ca2+ in the presence orabsence of 7.5 U/ml Apyrase or 10 mMPPADS for 5 min. (C) Promotion of LKB1phosphorylation by inhibition of PI3K andAkt. Cells were pre-treated with or without10 mM LY294002 or 10 mM Akti1/2 for30 min before being exposed to Ca2+-freemedium for an additional 5 min. Cellularproteins were extracted and subjected towestern blot analysis for phosphorylatedLKB1 and Akt. (D) Analysis ofphosphorylated LKB1 by using densitometryis shown in C. Results were expressed asOD (mean6s.e.m., n53). *P,0.01compared with Ca2+-free control. (E) Akt-mediated activation of mTOR and itsrelationship with AMPK. Cells were pre-treated with or without 10 mM LY294002 or10 mM Akti1/2 for 30 min before beingexposed to Ca2+-free medium for anadditional 5 min. Cellular proteins wereextracted and subjected to western blotanalysis for phosphorylation of p70S6K (p-P70S6K) and AMPK. (F,G) Analysis of thephosphorylated 70S6K and AMPK by usingdensitometry is shown in E. Results wereexpressed as OD (mean6s.e.m., n53).*P,0.01 compared with the Ca2+-freecontrol.

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homeostasis, AMPK is under the negative-feedback control ofATP (Hardie, 2011; Mihaylova and Shaw, 2011). In this context,suppression of AMPK by extracellular ATP could be animportant and integral part of the multifaceted regulation of

AMPK by ATP.In the presence of the P2 purinoceptor antagonists suramin and

PPADS, the hemichannel-mediated suppression of AMPK was

abrogated, indicating the involvement of purinergic signaling.Binding of ATP to P2 receptors activates several downstreamsignaling pathways. Here, it is most probable that signaling is

mediated by the P2Y receptor signal transduction pathway. AsG-protein-coupled receptors, P2Y family members activatephospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-

bisphosphate to 1,2-diacylglycerol and inositol triphosphate,subsequently causing the release of intracellular Ca2+ and theactivation of protein kinase C (PKC), as well as downstreamsignaling cascades such as those mediated by mitogen-activated

protein kinase and PI3K (Franke et al., 2009; Thelen andDidichenko, 1997). In support of an involvement of this pathway,we observed that the PLC inhibitor U73122 potentiated, whereas

PLC activator m-3M3FBS suppressed AMPK activation(supplementary material Fig. S1). Furthermore, previousstudies have demonstrated an involvement of this pathway in

the induction of the intracellular release of Ca2+ and theregulation of cell proliferation by hemichannel-derived ATP(Belliveau et al., 2006; Weissman et al., 2004). Reports have alsoimplicated G-protein-coupled receptors in growth-factor-induced

suppression of AMPK (Ning et al., 2011). Recently, more G-protein-coupled purinergic receptors have been reported toactivate the PI3K signaling pathway. These receptors include

Gq-coupled P2RY1, P2RY2, P2RY4, P2RY6 and P2RY11 andGi-coupled P2RY12, P2RY13 and P2RY14 (Burnstock, 2006;Inoue, 2006). The possible implication of these receptors in the

mediation of ATP-induced suppression of AMPK cannot beexcluded. More detailed analysis of the expression profile andfunctional roles of these purinergic receptors in NRK cells is

required in the future.Our results identify Akt as a key molecule that mediates the

suppressive effect of hemichannels and ATP on AMPKactivation. This is shown by the fact that blockade of Akt or its

upstream kinase potentiated AMPK activation, whereasoverexpression of Akt suppressed it. The involvement of Akt inthis study is not surprising because Akt has been reported to

repress AMPK activities through several pathways (Ning et al.,2011). Akt regulates energy homeostasis by increasing glycolysisand oxidative phosphorylation, causing an increased level of

Fig. 6. Potentiation of Cd2+-induced AMPK activation by hemichannel blockers. (A) Cd2+-induced ATP release in LLC-PK1 cells expressing EGFP–Cx43,and its prevention by heptanol and lindane. Cells were pre-treated with or without 3 mM heptanol or 100 mM lindane for 30 min before being exposed to30 mM CdCl2 for the indicated timepoints. The ATP concentration in the culture medium was measured, as described in Materials and Methods. The dataare expressed as RLU (mean6s.e.m., n53, #P,0.01 compared with basal release at timepoint zero; *P,0.01 versus Cd2+ alone). (B) LLC-PK1 cells expressingEGFP–Cx43 were pre-treated with or without 3 mM heptanol or 100 mM lindane for 30 min before being exposed to 30 mM Cd2+ for an additional 6 h.Cellular protein was extracted and subjected to analysis by western blot for phosphorylated AMPK. (C) Analysis of the phosphorylated AMPK by usingdensitometry is shown in B. Results were expressed as OD (mean6s.e.m., n53). *P,0.01 compared with Cd2+ alone. (D) LLC-PK1 cells expressing EGFP–Cx43 were pre-treated with or without 500 mM Gd3+ or 300 mM suramin for 30 min before exposure to 30 mM Cd2+ for an additional 6 h. Cellular protein wasextracted and subjected to western blot analysis for phosphorylated AMPK.

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intracellular ATP and a concomitant reduction in the AMP to

ATP ratio (Hahn-Windgassen et al., 2005). Akt activates mTORthrough phosphorylating TSC2 and inhibiting TSC1–TSC2 dimerformation (Hahn-Windgassen et al., 2005). Akt also suppresses

AMPK activation by inhibiting AMPK kinases, such as LKB1(Liu et al., 2012; Soltys et al., 2006). In addition, Akt affectsAMPK activity by phosphorylating AMPK itself at serine residue

485 (Ning et al., 2011). Consistent with the previous reports, we

observed that inhibition of Akt enhanced LKB1 phosphorylationbut suppressed mTOR activities. In addition, transfection of cellswith constitutively active Akt reduced the AMPK activation that

was caused by the exposure of cells to several different stimuli,including low glucose, Hank’s balanced salt solution (HBSS) andhypoxia (supplementary material Fig. S2). Thus, activation of the

Fig. 7. Hemichannel-mediated suppression of AMPK contributes to Cd2+-initiated cell injury. (A) Induction of p70S6K phosphorylation by Cd2+. LLC-PK1cells expressing EGFP–Cx43 were exposed to 30 mM Cd2+ for the indicated time. Cellular lysates were subjected to western blot analysis for phosphorylatedlevels of p70S6K. (B,C) Regulation of mTOR by an AMPK activator and a hemichannel blocker. Cells expressing EGFP–Cx43 were pre-treated with or without1 mM AICAR (B), 3 mM heptanol or 100 nM rapamycin (C) for 30 min before being exposed to 30 mM CdCl2 for an additional 6 h. Cellular lysates weresubjected to western blot analysis for phosphorylated levels of p70S6K and AMPK. Note the inverse correlation between the phosphorylated level of p70S6Kand AMPK. (D,E) The effect of an mTOR inhibitor, AMPK activators and hemichannel blockers on Cd2+-induced cell injury. Cells were pre-treated with or without100 nM rapamycin, 1 mM AICAR, 2 mM metformin, 3 mM heptanol or 100 mM lindane for 30 min before being exposed to 50 mM CdCl2 for an additional 6 h(D) or 9 h (E). The cell morphology was photographed and cellular viability was determined by WST assay. The lower panel of D is the enlarged imagescorresponding to boxed regions in the middle panel. Note the obviously reduced number of the round and loosely attached cells in samples treated with themTOR inhibitor, AMPK activators and hemichannel blockers as compared with cells treated with Cd2+ alone (arrows). The data in E were expressed as apercentage of an untreated control (mean6s.e.m., n53). *P,0.01 compared with Cd2+ alone.

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PI3K–Akt signaling pathway is responsible for the suppressiveeffects of extracellular ATP on AMPK activity.

The activity of AMPK is modulated both by the cellular AMPto ATP ratio and upstream kinases, such as LKB1 and CAMKKb(also known as CAMKK2) (Hardie, 2011; Hurley et al., 2005;Mihaylova and Shaw, 2011); however, the question arises as to

how AMPK is activated under Ca2+-free conditions. Preventionof the loss of ATP, by blocking hemichannels, enhanced AMPKactivation, suggesting that AMPK activation is not due to the loss

of ATP through hemichannels. It is also unlikely that the changein hemichannel activities affected the cellular AMP to ATP ratiobecause both AMP and ATP are small molecules that can freely

pass through hemichannels. One recent report has indicated thatCa2+ is an indispensable factor for the maintenance of the normalfunction of mitochondria; removal of extracellular Ca2+ interferes

with mitochondrial biogenesis and activates AMPK (Cardenaset al., 2010; Green and Wang, 2010). In line with this report,we detected a time-dependent decrease in the total amount ofATP following removal of extracellular Ca2+ (supplementary

material Fig. S3), which could be one of the mechanisms thatcontributes to the observed AMPK activation. We also foundthat Ca2+ deprivation was associated with an increase in LKB1

phosphorylation, the extent of which was equivalent to that ofAMPK and subject to the regulation of purinergic signaling.Thus, activation of LKB1 could be crucially involved in

AMPK activation. Consistent with this notion, we observedthat exogenous ATP strongly suppressed metformin-inducedAMPK activation (data not shown), which activates AMPK

through a LKB1-dependent mechanism (Towler and Hardie,2007). LKB1 was also activated by Cd2+, which was potentiatedby hemichannel blockers (data not shown). CAMKKb is lesslikely to be involved in our experimental setting because previous

studies have shown that activation of CAMKKb requires thepresence of extracellular Ca2+ and that depletion of extracellularCa2+ abolished AMPK activation induced by CAMKKb activators

(Chi et al., 2011; Ghislat et al., 2012).ATP is released into the extracellular space in response to

various stimuli through several different routes (Gordon, 1986;

Lazarowski, 2012; Schwiebert and Zsembery, 2003). In additionto hemichannels, ATP is released by ATP-binding cassetteproteins, exocytosis, anion channels, pannexin channels and theP2X7 receptor. It is conceivable that the observed effects of the

released ATP on AMPK could be independent of the type ofstimulant or route of secretion. In this context, our findings couldhave a wide application. It is especially noteworthy that channels

composed of pannexin subunits have been reported to share manyproperties with connexin channels and have been shown to play akey role in many pathological situations (Qu et al., 2011;

Silverman et al., 2009). However, a role for pannexin in this studyis unlikely because NRK cells have been previously reported tobe pannexin deficient (Penuela et al., 2007). Indeed, blockade of

pannexin channels with a specific blocker, probenecid, did notaffect AMPK activation (supplementary material Fig. S4).

Of note, the treatment of cells with hemichannel blockers orsiRNA against Cx43 failed to completely abolish the release of

ATP caused by Ca2+ deprivation. These observations imply thatremoval of extracellular Ca2+ also activates other ATP-releasingpathways. The exact properties of these pathways remain to be

characterized. Intriguingly, the extracellular ATP that is releasedfollowing cell exposure to Ca2+-free medium did not accumulate.Instead, it decreased in a time-dependent manner after reaching a

peak within 5 min. Further analysis of hemichannel activity using

ethidium bromide uptake revealed that the reduction of ATP wasnot associated with a corresponding decrease in the intracellular

influx of ethidium bromide (supplementary material Fig. S5),suggesting that hemichannels are still active at these latertimepoints. The decrease in the level of ATP could be attributedto the rapid degradation of ATP by extracellular nucleotidases

(Komlosi et al., 2005).It is also worth mentioning that both heptanol and lindane

potentiated the low-Ca2+-initiated AMPK activation; however,

the increase in AMPK phosphorylation did not appear to beassociated with ATP release upon treatment with lindane. Incontrast with heptanol, the timecourse over which the lindane-

induced AMPK phosphorylation decreased did not correlate withthat of the reduction in the release of ATP. In fact, there was astatistically significant difference between the potentiation of

AMPK activation by heptanol and lindane at the 30-mintimepoint. Additional experiments revealed that heptanol andlindane additively stimulated AMPK activation without furthersuppressing ATP release (supplementary material Fig. S6); thus it

is probable that lindane potentiates AMPK activation throughother mechanisms, additional to its inhibition of hemichannels.Heptanol and lindane are known to inhibit connexin channels

through completely different mechanisms. Heptanol blocksconnexin channel formation through decreasing the openprobability of the channels (Takens-Kwak et al., 1992), whereas

lindane works by regulating multiple intracellular signalingmolecules, including PKC, ERK (also known as Mapk1), PLC,cyclic AMP, arachidonic acid and glutathione (Caruso et al.,

2005; Criswell and Loch-Caruso, 1999; Krieger and Loch-Caruso, 2001; Mograbi et al., 2003; Wang and Loch-Caruso,2002). The sustained activation of AMPK by lindane might berelated to its actions on these signaling molecules. It might also

be related to the reported actions of lindane on mitochondrialdisorder and oxidative stress (Bagchi and Stohs, 1993; Benarbiaet al., 2013), both of which are known to be able to activate

AMPK (Cardenas et al., 2010; Pung et al., 2013). Thisunexpected action of lindane on AMPK should not greatly affectour conclusion because a similar potentiating effect on AMPK

activation was achieved by downregulation of Cx43 with siRNAor by using other hemichannel blockers.

The importance of our findings is further exemplified by thepivotal role of the signaling cascade in Cd2+-induced cell death.

As a negative regulator of mTOR, AMPK has been previouslyreported to be able to inhibit Cd2+-induced generation of reactiveoxygen species, mTOR activation and cell death (Chen et al.,

2011). In this context, suppression of AMPK by hemichannel-initiated activation of purinergic signaling could be an importantmechanism underlying Cd2+-induced cell death.

These data reveal a previously unrecognized hemichannel-mediated regulation of AMPK and implication of the mechanismsin the biological action of hemichannels. Our findings, thus,

provide novel and important mechanistic insights into the functionsof extracellular ATP, as well as channels that release ATP.

MATERIALS AND METHODSCellsNormal rat kidney (NRK) renal epithelial cell line NRK-E52 and porcine

kidney epithelial cell line LLC-PK1 cells were purchased from the

American Type Culture Collection (Rockville, MD). Mesangial cells

were cultured as previously described (Johno et al., 2012). Cells were

maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco-BRL,

Gaithersburg, MD) supplemented with 5% fetal bovine serum (FBS). For

comparison of cell responses between normal Ca2+ and Ca2+-free

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conditions, cells were exposed to Ca2+-free DMEM (Gibco-BRL,

catalogue number 21068) with or without supplementation of 1.8 mM

Ca2+.

ReagentsFBS, trypsin with EDTA, antibiotics, cadmium chloride (CdCl2), heptanol,

lindane, Lucifer Yellow, ethidium bromide, lanthanum chloride (La3+),

gadolinium chloride (Gd3+), suramin, pyridoxalphosphate-6-azophenyl-

29,49-disulfonic acid (PPADS) and all other chemicals were obtained from

Sigma (Tokyo, Japan). Antibodies against AMPK, Akt and b-actin were

obtained from Cell Signaling (Beverly, MA).

Dye uptake assayThe presence of functional hemichannels was evaluated by the cellular

uptake of Lucifer Yellow or ethidium bromide, as described previously

(Fang et al., 2011; Garre et al., 2010). NRK cells were exposed to Ca2+-

free medium in the presence or absence of 0.1% Lucifer Yellow or

10 mM ethidium bromide for 15 min. The cells were then rinsed

and fixed with 3% paraformaldehyde. Immunofluorescence images

were captured using a CCD camera attached to an Olympus BX50

microscope.

Western blot analysisTotal cellular protein was extracted by suspending the pre-washed cells

in sodium dodecyl sulphate (SDS) lysis buffer (62.5 mM Tris-HCl, 2%

SDS, 10% glycerol) together with freshly added proteinase inhibitor

cocktail (Nacalai tesque, Kyoto, Japan). Lysates were incubated on ice

for 30 min with intermittent mixing and then centrifuged at 12,000 r.p.m.

for 10 min at 4 C in an Eppendorf centrifuge 5415R with a F45-24-11

rotor. The supernatant was recovered and the protein concentration was

determined using the Micro BCA Protein Assay Kit (Pierce, Rockford,

IL).

Western blot was performed using the enhanced chemiluminescence

system (Chi et al., 2011; Fang et al., 2011). Briefly, extracted cellular

proteins were separated by 10% SDS polyacrylamide gels and

electrotransferred onto polyvinylidine difluoride membranes. After

blocking with 3% bovine serum albumin in PBS, the membranes were

incubated with primary antibody for 1.5 h at room temperature or at 4 C

overnight. After washing, the membranes were probed with horseradish-

peroxidase-conjugated anti-rabbit IgG (Cell Signaling, Beverly,

MA), and the bands were visualized by using the enhanced

chemiluminescence system (Amersham Biosciences, Buckinghamshire,

UK). The chemiluminescent signal was captured with a Fujifilm

luminescent image LAS-1000 analyzer (Fujifilm, Tokyo, Japan) and

quantified with the ImageJ software (http://rsb.info.nih.gov/ij). The

results of quantification were expressed as optical density (OD). To

confirm equal loading of proteins, the membranes were stripped with

62.5 mM Tris-HCl (pH 6.8) containing 2% SDS and 100 mM 2-

mercaptoethanol for 30 min at 60 C and probed for total AMPK protein

or b-actin.

siRNA transfectionNRK cells were transiently transfected with siRNA specifically targeting

Cx43 (Mm_Gja1_2 HP siRNA, catalogue number SI00191625, Qiagen,

Japan) or a negative control siRNA (AllStars Negative Control siRNA) at

a final concentration of 20 nM using HiPerfect transfection reagent for

48 h (Chi et al., 2011; Fang et al., 2011). Cells were then either left

untreated or exposed to Ca2+-free medium for the indicated intervals.

Cellular lysates were harvested and assayed for Cx43 expression and

AMPK phosphorylation using western blot analysis.

Cell transfectionVectors encoding wild-type Cx43 tagged with EGFP (wild-type Cx43-

pEGFP1) or mutant Cx43 tagged with EGFP (mutant Cx43-pEGFP1)

were a kind gift from Oyamada (Department of Pathology, Kyoto

Prefectural University of Medicine, Kyoto, Japan). These vectors were

transfected into LLC-PK1 cells by using Lipofectamine Plus reagent

(Invitrogen, Carlsbad) according to the manufacturer’s instructions (Fang

et al., 2011; Li et al., 2013). Clones with high levels of GFP fluorescence

were used for this study, which were selected by using the fluorescence

microscopy. The mutant Cx43-pEGFP1 vector, which lacks 24 bases that

correspond to amino acid residues 130 to 137 of rat Cx43 (D130–137

Cx43), was designed according to the initial report of Krutovskikh et al.

(Krutovskikh et al., 1998; Oyamada et al., 2002). Previous studies have

demonstrated that cells carrying the mutant vector cannot form gap

junctions that are capable of intercellular communication (Krutovskikh

et al., 1998; Oyamada et al., 2002). Stable transfection of mesangial cells

with myristoylated Akt was performed, as previously reported (Johno

et al., 2012). pcDNA3-myrHA-Akt1 encoding constitutively active Akt

was kindly provided by Kenneth Walsh (Boston University School of

Medicine, Boston, MA). Mock-transfected cells were also established by

stable transfection with pcDNA3.1 (Invitrogen, Life Technologies,

Carlsbad, CA).

LDH release assayCytotoxicity was evaluated by the release of LDH using an LDH

cytotoxicity detection kit (Takara Bio, Otsu, Shiga, Japan), as described

previously (Fang et al., 2011). Briefly, cells in a 96-well culture plate

were incubated with various chemicals for the described time intervals.

Culture medium was collected and measured for LDH activity. LDH

release was calculated and expressed as percentage of total release.

Culture medium was used as a background control, whereas cells treated

with 2% Triton X-100 were used as 100% release.

WST-Formazan assayThe cell viability was assessed by formazan assay using Cell Counting

Kit-8 (Dojindo Laboratory, Kumamoto, Japan), as described previously

(Fang et al., 2011).

ATP measurementATP was measured using a luciferin and luciferase bioluminescence

assay kit (Molecular Probes). The intensity of the chemiluminescent

signal was determined by using a luminometer (Gene Light 55;

Microtech Nition, Chiba, Japan), as described previously (Fang et al.,

2011; Yao et al., 2003).

Statistical analysisValues are expressed as mean6s.e.m. Comparison of two populations

was performed by using Student’s t-test. For multiple comparisons, one

way analysis of variance (ANOVA), followed by Dunett’s test, was

employed. Both analyses were performed by using the SigmaStat

statistical software (Jandel Scientific, CA). P,0.05 was considered to be

a statistically significant difference.

AcknowledgementsWe thank Hisashi Johno for providing the glomerular mesangial cells. We thankMasanori Kitamura and Hironori Kato (Department of Molecular Signaling,University of Yamanashi, Japan) for antibodies, technical support and advice onthe experiments.

Competing interestsThe authors declare no competing interests.

Author contributionsY.C. performed research and wrote the manuscript; K.G. and K.L. providedexperimental assistance; M.T. and S.K. provided crucial reagents, technicalassistance and intellectual input; J.Y. designed the study and wrote themanuscript.

FundingThis work was supported by Grants-in-Aid for Scientific Research from theMinistry of Education, Culture, Sports, Science and Technology, Japan [grantnumbers 17659255 and 20590953 to J.Y.].

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.139089/-/DC1

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