GLIA 60:53–68 (2012)
Glucose Increases Intracellular Free Ca21 in Tanycytes
via ATP Released Through Connexin 43 Hemichannels
1
2
JUAN A. ORELLANA,1 PABLO J. SAEZ,
CHRISTIAN CORTES-CAMPOS,
ROBERTO J. ELIZONDO,2
KENJI F. SHOJI,1 SUSANA CONTRERAS-DUARTE,1 VANIA FIGUEROA,1 VICTORIA VELARDE,1
1,4
JEAN X. JIANG,3 FRANCISCO NUALART,2 JUAN C. SAEZ,
AND MAR
IA A. GARC
IA2*
1
Departamento de Fisiologıa, Pontificia Universidad Cat
olica de Chile, Santiago, Chile
2
Departamento de Biologıa Celular, Universidad de Concepci
on, Concepci
on, Chile
3
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas
4
Instituto Milenio, Centro Interdisciplinario de Neurociencias de Valparaıso, Valparaıso, Chile
KEY WORDS
glucosensing; hypothalamus; glucokinase; connexons
ABSTRACT
The ventromedial hypothalamus is involved in regulating
feeding and satiety behavior, and its neurons interact with
specialized ependymal-glial cells, termed tanycytes. The latter express glucose-sensing proteins, including glucose
transporter 2, glucokinase, and ATP-sensitive K1 (KATP)
channels, suggesting their involvement in hypothalamic
glucosensing. Here, the transduction mechanism involved
in the glucose-induced rise of intracellular free Ca21 concentration ([Ca21]i) in cultured b-tanycytes was examined.
Fura-2AM time-lapse fluorescence images revealed that
glucose increases the intracellular Ca21 signal in a concentration-dependent manner. Glucose transportation, primarily via glucose transporters, and metabolism via anaerobic
glycolysis increased connexin 43 (Cx43) hemichannel activity, evaluated by ethidium uptake and whole cell patch
clamp recordings, through a KATP channel-dependent pathway. Consequently, ATP export to the extracellular milieu
was enhanced, resulting in activation of purinergic P2Y1
receptors followed by inositol trisphosphate receptor activation and Ca21 release from intracellular stores. The present
study identifies the mechanism by which glucose increases
[Ca21]i in tanycytes. It also establishes that Cx43 hemichannels can be rapidly activated under physiological conditions by the sequential activation of glucosensing proteins
in normal tanycytes. V 2011 Wiley Periodicals, Inc.
C
INTRODUCTION
The ventromedial hypothalamus (VMH) is involved in
regulating feeding and satiety behaviors through their
capacity to detect changes in glucose concentrations
(Levin et al., 2004). The arcuate nucleus (AN) and the
ventromedial nucleus (VMN) form the VMH; their neurons are in close contact with highly elongated ependymal-glial cells known as tanycytes (Akmayev and Popov,
1977; Chauvet et al., 1995; Flament-Durand and Brion,
1985). Tanycytes are the main glial cells present in the
basal hypothalamus (Garcıa et al., 2001, 2003; Millan et
al., 2010) and are classified into four types, according to
their localization in the III–V ventricle and biochemical
and molecular properties: a1, a2, b1, and b2 (Akmayev
and Fidelina, 1974; Akmayev and Popov, 1977). a1- and
C 2011
V
Wiley Periodicals, Inc.
a2-tanycytes are localized beside the VMN, while b1tanycytes are localized within the lower lateral wall of
the third ventricle, contacting neurons through cell processes in the AN as well as capillaries in the hypothalamus (Garcıa et al., 2001). b2-tanycytes are found in the
floor of third ventricle lining the median eminence
(Garcıa et al., 2001).
Both a and b tanycytes express several glucose-sensing molecules, including glucose transporter 2 (GLUT2)
and ATP-sensitive K1 (KATP) channels, suggesting their
possible involvement in hypothalamus-mediated glucosensing (Alvarez et al., 1996; Garcıa et al., 2003; Millan
et al., 2010; Navarro et al., 1996). Notably, unlike atanycytes, high expression of GLUT2 and glucokinase
(GK) in the proximal pole has been observed in b1-tanycytes in vivo (Garcıa et al., 2003; Millan et al., 2010).
The specific localization of b1 tanycytes in direct contact
with cerebral spinal fluid and their prominent GLUT2/
GK expression strongly support the idea that these cells
have a high glucose uptake capacity. In fact, it has been
proposed that b1-tanycytes could uptake and metabolize
glucose to lactate through the glycolytic pathway, and
subsequently export lactate to neurons of the AN
through monocarboxylate transporters (MCTs) 1 and/or
4 (Cort
es-Campos et al., 2011; Garcıa et al., 2003; Millan
et al., 2010).
In support of the putative brain glucosensor role of
tanycytes, it was recently demonstrated that extracellular glucose increases the intracellular free Ca21 concentration ([Ca21]i) in a1- and a2-tanycytes (Frayling et al.,
2011). Although expression of glucosensing proteins by
different tanycyte subtypes is well-established, the
Additional Supporting Information may be found in the online version of this article.
Grant sponsor: CONICYT; Grant number: 24080055; Grant sponsor: FONDECYT; Grant number: 1100705; Grant sponsor: FONDECYT; Grant numbers:
1111033, 1100396; Grant sponsor: FONDEF; Grant number: DO7I1086; Grant
sponsor: Anillo; Grant number: ACT-71; Grant sponsor: NIH; Grant number:
ARO46798; Grant sponsor: Welch Foundation; Grant number: AQ-1507.
*Correspondence to: Marıa A. Garcıa, Departamento de Biologıa Celular, Universidad de Concepci
on, Concepci
on, Chile. E-mail: [email protected] or Juan A. Orellana, Departamento de Fisiologıa, Pontificia Universidad Cat
olica de Chile, Alameda 340, Santiago, Chile. E-mail: [email protected]
Received 10 June 2011; Accepted 31 August 2011
DOI 10.1002/glia.21246
Published online 10 October 2011 in Wiley Online Library (wileyonlinelibrary.
com).
54
ORELLANA ET AL.
mechanism by which these cells exhibit rises in [Ca21]i
upon exposure to extracellular glucose is not completely
understood (Dale, 2011; Frayling et al., 2011). Recently,
it was proposed that Ca21 signaling could be mediated
by hemichannels (Schalper et al., 2010) and the hemichannel activity can be modulated by the intracellular
free Ca21 concentration ([Ca21]i) (De Vuyst et al., 2009;
Schalper et al., 2008b). Hemichannels are formed by the
oligomerization of six protein subunits, termed connexins (Cxs), which are a highly conserved protein family
encoded by 21 genes in humans. In addition, members
of a recently described three-member glycoprotein family
unrelated to Cxs, termed pannexins (Panxs), can also
form hemichannels in the cell membrane of vertebrates
(Orellana et al., 2009).
The use of exogenous expression systems has permitted the study of electrophysiological (S
aez et al., 2005)
and qualitative and quantitative permeability (Orellana
et al., 2011a) properties of some hemichannels as well as
mechanisms that control their functional activity. Under
appropriate experimental conditions, physiologically relevant quantities of signaling molecules (e.g., ATP, glutamate, NAD1, and PGE2) are released via hemichannels
to the extracellular milieu (Schalper et al., 2008a). Since
hemichannels are believed to play relevant roles in
intercellular signaling, the aim of the present study was
to address the mechanism behind tanycyte glucosensing
and determine whether it is associated with hemichannel-dependent changes in [Ca21]i. Here, the [Ca21]i of
cultured tanycytes increased in response to glucose. This
response was mediated by a transduction process involving the canonical glucosensing pathway and rapid activation of Cx43 hemichannels.
MATERIALS AND METHODS
Reagents and Antibodies
A chemiluminescence detection kit was purchased
from GE Healthcare (Aulnais-sous-Bois, France). Antirabbit IgG antibodies conjugated to horseradish peroxidase (HRP) was purchased from Pierce (Rockford, IL).
Gap26 and 10panx1 mimetic peptides were obtained
from NeoMPS, SA (Strasbourg, France). HEPES, minimal essential medium (MEM), DNAse I, poly-L-lysine,
water (W3500), La31, ethidium (Etd) bromide, thapsigargin (TG), xestospongin C (XeC), BAPTA AM, glibenclamide, xestospongin B (XeB), 4,6,-O-ethylidene-D-glucose
(ETDG), alloxan, diazoxide (diazox), cytochalisin B
(Cyto-B), and probenecid (Prob) were purchased from
Sigma-Aldrich (St. Louis, MO). Fetal calf serum and fetal bovine serum (FBS) were obtained from Hyclone
(Logan, UT). Penicillin, streptomycin, 2-deoxyglucose (2DOG), 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2deoxyglucose (2-NBDG), goat anti-mouse Alexa Fluor
488 and goat anti-mouse Alexa Fluor 555, and TOPRO-3
were obtained from Invitrogen (Carlsbad, CA). 2-Deoxy3
3
D-[1,2-(N) H]glucose (2-[H ]DOG) was obtained from
DuPont-NEN (Boston, MA). Normal goat serum was
purchased from Zymed (San Francisco, CA). Cx43E2, a
GLIA
Cx43 hemichannel antibody (Siller-Jackson et al., 2008)
was made available by Dr. Jean X. Jiang (University of
Texas Health Science Center).
Animals
All animals were handled in strict accordance with
the Animal Welfare Assurance (permit number
2010101A), and all animal work was approved by the
appropriate Ethics and Animal Care and Use Committee
of the University of Concepci
on, Chile. Male adult
Sprague–Dawley rats were used for the experiments.
Animals were kept in a 12-h light/12-h dark cycle with
food and water ad libitum. Experiments in cultured cells
were performed mainly at Departamento de Fisiologıa,
Pontificia Universidad Cat
olica de Chile (PUCC), using
protocols approved by the Ethic and Biosecurity Committee of PUCC.
Cell Cultures
Cultures of hypothalamic glial cells from 1-day postnatal brains were prepared following the method
described previously (Garcıa et al., 2003). After decapitation, brains were removed and the hypothalamic area
was dissected to obtain a region close to the ependymal
layer. The dissection was carried out from tissues
immersed in dissection buffer containing 10 mM HEPES
(pH 5 7.4, 340 mOsm/L). Samples were incubated with
0.25% trypsin-0.2% EDTA (w/v) for 20 min at 37°C, and
then it was transferred to planting medium containing
MEM (Invitrogen) with 10% (v/v) FBS (Thermo Fisher
Scientific, Waltham, MA) and 2 mg/mL DNAse I (SigmaAldrich). Cells were seeded at 1.2 3 105 cells/cm2 in culture dishes coated with 0.2 mg/mL poly-L-lysine (SigmaAldrich). After 2 h, the culture medium was changed to
MEM supplemented with 10% FBS, 2 mM L-glutamine,
100 U/mL penicillin, 100 lg/mL streptomycin, and 2.5
lg/mL fungizone (Thermo Fisher Scientific). Mixed
hypothalamic cultures were prepared using the same
protocol described above; however, they were cultured in
Neurobasal medium supplemented with B27 (Invitrogen). Astroglial cultures were prepared from rat brain
cortex and cultured in MEM supplemented with 10%
FBS. Cells were cultured in the same dish for 3 weeks,
and the medium was changed every 2 days. The purity
of cell cultures was evaluated using molecular markers
for several cell types (Fig. 1). For all experiments, chemicals and saline solutions were prepared in ultra pure
H2O.
Intracellular Calcium Imaging
Cells plated on glass coverslips were loaded with 5 lM
Fura-2-AM in DMEM without serum for 45 min at 37°C,
and then washed three times in Locke’s solution (154
mM NaCl, 5.4 mM KCl, 2.3 mM CaCl2, and 5 mM
GLUCOSENSING VIA HEMICHANNELS IN TANYCYTES
55
403 water immersion objective. Changes were monitored using an imaging system equipped with a Retga
1300I fast-cooled monochromatic digital camera (12-bit)
(Qimaging, Burnaby, BC, Canada), monochromator for
fluorophore excitation, and METAFLUOR software (Universal Imaging, Downingtown, PA) for image acquisition
and analysis. Analysis involved determination of pixels
assigned to each cell. The average pixel value allocated
to each cell was obtained with excitation at each wavelength and corrected for background. Because of the low
excitation intensity, no bleaching was observed even
when cells were illuminated for a few minutes. The ratio
was obtained after dividing the 340-nm by the 380-nm
fluorescence image on a pixel-by-pixel base (R 5 F340nm/
F380nm).
Immunofluorescence and Confocal Microscopy
Cells grown on poly-L-lysine-coated glass coverslips in
24-well plates were fixed with 4% paraformaldehyde in
phosphate-buffered saline (PBS) for 30 min, washed
with Tris-HCl buffer (pH 5 7.8), and incubated in wash
buffer containing 1% bovine serum albumin and 0.2%
Triton X-100 for 5 min at room temperature. Samples
were then incubated with the following primary antibodies overnight at room temperature: rabbit anti-Cx43
(1:200) and mouse anti-vimentin (1:200, DAKO, Campintene, CA), rabbit anti-GFAP (1:200, DAKO), mouse antiMAP2 (1:50, Chemicon Temecula, CA), mouse anti-b3
tubulin (1:1,000, Promega, USA), rabbit anti-GLUT1
and GLUT2 (1:100, Alpha Diagnostic International, San
Antonio, TX), chicken anti-MCT1 (1:100, Millipore,
Temecula, CA), rabbit anti-MCT4 (1:20, Millipore), rabbit anti-von Willebrand factor (VWF, 1:300, Sigma) or
anti-Kir1.6 (1:200, Santa Cruz Biotechnology, CA). Samples were then incubated with alexa fluor 488 or alexa
fluor 555-labeled secondary antibodies and counterstained with the DNA stain, TOPRO-3 (1:1,000, Invitrogen). Preparations were analyzed using confocal laser
microscopy (D-Eclipse C1 Nikon, Tokyo, Japan).
Dye Uptake and Time-Lapse
Fluorescence Imaging
Fig. 1. Immunocytochemistry characterization of cultured tanycytes.
Tanycytes obtained from rat hypothalamus at 1-day postnatal were cultured for 3 weeks. (A–E) Representative confocal images depicting
vimentin (A, green), GFAP (B, green), MAP2 (C, green), bIII-tubulin
(D, green), Kir6.1 (E, green) in rat tanycytes under control conditions.
(F) MAP2 (red) and vimentin (green) staining in mixed hypothalamic
cultures of tanycytes and neurons. In blue are shown nuclei stained
with TOPRO-3. (G) Quantification of immunopositive expression normalized to total cells of vimentin, Kir6.1, GLUT2, GK, MCT1, MCT4,
GFAP, bIII-tubulin, MAP2, and Von Willebrand factor (VWF) in tanycytes under control conditions. Scale bar 5 80 lm.
HEPES, pH 5 7.4) followed by a de-esterification period
of 15 min at 37°C. The experimental protocol for [Ca21]i
imaging involved data acquisition every 5 s (emission at
510 nm) at 340- and 380-nm excitation wavelengths
using an Olympus BX 51W1I upright microscope with a
For time-lapse fluorescence imaging, cells plated on
glass coverslips were washed twice in PBS solution, pH
5 7.4, and then bathed with Locke’s solution containing
5 lM Etd. In some experiments, cells were bathed in a
divalent cation-free solution (DCFS) [in (mM): NaCl
(148), KCl (5), MgCl2 (1), HEPES (5), EGTA (10), pH 5
7.4; at 37°C] containing 5 lM Etd. Cells on glass coverslips were placed on the microscope stage and data were
acquired using the same microscope and camera
described for intracellular calcium imaging. Regions of
interest were placed over random cells and background
was subtracted. Fluorescence was recorded every 30 s.
To test for changes in slope, regression lines were fitted
to points before and after various treatments using the
GLIA
56
ORELLANA ET AL.
Excel program and mean value of slopes were compared
using GraphPad Prism software (GraphPad Software,
San Diego, CA).
Electrophysiology
Cells platted on glass coverslips were transferred to
an experimental chamber mounted on the stage of an
inverted microscope (Olympus IX-51; Olympus Optical).
For whole-cell voltage clamp experiments, the bath solution contained 140 mM NaCl, 5.4 mM KCl, 1 mM
MgCl2, 1.8 mM CaCl2, 2 mM BaCl2, and 10 mM HEPES,
pH 5 7.4. The pipette solution contained 130 mM CsCl,
10 mM AspNa, 0.26 mM CaCl2, 1 mM MgCl2, 2 mM
EGTA, 7 mM TEA-Cl, and 5 mM HEPES, pH 5 7.2.
Patch pipettes were made from borosilicate glass capillaries using a flaming/brown micropipette puller (P-87;
Sutter Instruments, Union City, CA). The tip resistance
was 10–15 MX when filled with pipette solution. Currents were filtered at 1 kHz and sampled at 5 kHz, and
records were filtered with a digital low pass filter of 0.5
kHz. Data acquisition and analysis were performed
using pClamp 9 (Molecular Devices, Novato, CA).
was added followed by incubation for 30 min at 4°C.
Cells were washed three times with ice-cold saline containing 15 mM glycine (pH 5 8.0) to block unreacted biotin. The cells were harvested and incubated with an
excess of immobilized NeutrAvidin (1 mL of NeutrAvidin
per 3 mg of biotinylated protein) for 1 h at 4°C after
which 1 mL of wash buffer (saline solution, pH 5 7.2
containing 0.1% SDS and 1% Nonidet P-40) was added.
The mixture was centrifuged for 2 min at 14,000 rpm at
4°C. The supernatant was removed and discarded, and
the pellet was resuspended in 40 lL of saline solution,
pH 5 2.8, and containing 0.1 M glycine to release the
proteins from the biotin. After the mixture was centrifuged at 14,000 rpm for 2 min at 4°C, the supernatant
was collected, and the pH was adjusted immediately by
adding 10 lL of 1 M Tris, pH 5 7.5. Relative protein levels were measured using Western blot analysis as
described above. Resulting immunoblot signals were
scanned, and the densitometric analysis was performed
with the Scion Image software. Densitometric arbitrary
units were normalized to the signal obtained from total
protein measured with Ponceau red.
Glucose Transport Assay
Western Blot Analysis
Cultures were rinsed twice with PBS (pH 5 7.4) and
harvested by scraping with a rubber policeman in icecold PBS containing protease and phosphatase inhibitors
(1 mM orthovanadate, 10 mM a-glycerophosphate) and a
complete miniprotease inhibitor (Roche Diagnostics, San
Francisco, CA). Proteins were measured in aliquots of
cell lysates with the Bio-Rad protein assay (Bio- Rad,
Richmond, CA). Pelleted cells were resuspended in 40
lL of the protease and phosphatase inhibitor solution,
placed on ice, and lysed by sonication (Ultrasonic cell
disrupter, Microson, Ultrasons, Annemasse, France) after which samples were stored at 280°C or analyzed by
immunoblotting. Aliquots of cell lysates or biotinylated
cell surface proteins were resuspended in 13 Laemli’s
sample buffer, boiled for 5 min, separated on 8% SDSPAGE and electro-transferred to nitrocellulose sheets.
Nonspecific protein binding was blocked by incubation of
nitrocellulose sheets in PBS-BLOTTO (5% nonfat milk
in PBS). After 30 min, blots were incubated with primary antibody for 1 h at room temperature or overnight
at 4°C, followed by four 15-min PBS washes. Blots were
incubated with goat anti-rabbit antibody conjugated to
HRP. Immunoreactivity was detected by enhanced chemiluminescence detection using the SuperSignal kit
(Pierce) according to the manufacturer’s instructions.
For 2-[H3]DOG uptake assays, cells were grown in 12well plates to a density of 1 3 105 cells per well. Cultures were carefully selected under the microscope to
ensure that only plates showing uniformly grown cells
were used. Cells were washed with incubation buffer (10
mM HEPES, 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2,
and 0.8 mM MgCl2) and incubated in this buffer for 30
min at room temperature. Uptake assays were performed in 500 lL of incubation buffer containing 0.2
mM 2-DOG and 3 lCi 2-[H3]DOG (30.6 Ci/mmol).
Uptake was stopped by washing the cells with ice-cold
PBS. Cells were lyzed in 0.5 mL of lysis buffer (10 mM
Tris-HCl, pH 5 8.0 and 0.2% SDS), and the incorporated
radioactivity was assayed by liquid scintillation counting. Wherever appropriate, competitors and inhibitors
were added to the uptake assays or preincubated with
the cells.
For 2-NBDG uptake assays, cells were grown on glass
coverslips. Tanycytes bathed in Locke’s solution were
observed with the same microscope and camera used for
dye uptake experiments (see above). 2-NBDG (100 lM)
was exited at 488 nm, and the emission was filtered at
505–550 nm (Porras et al., 2004). In each experiment,
the resulting fluorescence was measured with Metafluor
software (Universal Imaging, Downingtown, PA), and for
each value, the background value was subtracted.
Measurement of Extracellular ATP Concentration
Cell Surface Biotinylation and Quantitation
Cells cultured on 90-mm dishes were washed three
times with ice-cold Hank’s saline solution (pH 5 8.0),
and 3 mL of sulfo-NHS-SS-biotin solution (0.5 mg/mL)
GLIA
Tanycytes were grown (80% confluence) in 6-well
plates for 15 days after which they were incubated in
Locke’s solution at 37°C for 30 min. Cells were then
treated for 10 min with 10 mM glucose, and ATP con-
GLUCOSENSING VIA HEMICHANNELS IN TANYCYTES
centration in the extracellular solution was measured
using a luciferin/luciferase bioluminescence assay kit
(Sigma-Aldrich). Baseline measurements were performed on separate cultures using standard Locke’s solution. The amount of ATP in each sample was calculated
from standard curves and normalized for the protein
concentration using the BCA assay obtained from
Pierce.
Data Analysis and Statistics
For each data group, results were expressed as mean
6 standard error, and n refers to the number of independent experiments. For statistical analysis, each
treatment was compared with its respective control, and
significance was determined using a one-way ANOVA
followed, in case of significance, by a Tukey posthoc test.
For multiple group treatments, significance was determined using a two-way ANOVA followed in case of significance by a Bonferroni posthoc test.
RESULTS
Glucose Increases the Intracellular Free Ca21
Signal in Cultured Rat Tanycytes Via GLUTs,
Glucokinase, and KATP Channels
As described previously (Garcıa et al., 2003), immunohistochemical analysis revealed that cultures enriched
in differentiated hypothalamic tanycytes exhibit an
intense reactivity of vimentin, Kir6.1, and GLUT2, but
not GFAP, MAP2, bIII-tubulin, and VWF, ruling out
contamination with other hypothalamic cell types
(Fig. 1A–G). In contrast, mixed hypothalamic cultures
were highly immunopositive for MAP2 and vimentin
(Fig. 1F), whereas cortical astroglial cultures exhibited a
strong GFAP, and low vimentin reactivity (data not
shown). Most tanycytes (>90%) were highly reactive to
anti-GK, -MCT1, and -MCT4 antibodies (Fig. 1G), suggesting a high enrichment in b1-tanycytes, since b1- but
not a-tanycytes express GK, MCT1, and MCT4 in vivo
(Cort
es-Campos et al., 2011; Millan et al., 2010).
Tanycytes have been proposed to mediate, at least in
part, the mechanism by which the hypothalamus detects
changes in extracellular glucose concentrations (Millan
et al., 2010). Supporting this idea, acute in situ application of glucose increases the [Ca21]i in a-tanycytes
(Frayling et al., 2011). However, the mechanism underlying this phenomenon remains to be elucidated. Thus,
we investigated whether canonical glucosensing molecules used by specialized cells, such as pancreatic bcells, were involved in this process and if the activation
of hemichannels could contribute to the glucose-induced
changes in [Ca21]i. As indicated by time-lapse measurements of Fura-2 ratio (340/380) (from now and on called
Ca21 signal), cultured tanycytes maintained in saline
containing 2 mM glucose showed a low basal Fura-2 fluorescent ratio (Fig. 2A–E). However, exposure to 10 mM
glucose induced a rapid, strong and transient increase
57
in Fura-2 fluorescent ratio; peaking at 630% as compared with basal levels (Fig. 2A,D,E). In addition, a
delay (67.6 6 7.8 s; n 5 9) between glucose addition and
increase in Ca21signal was evident (vertical gray line in
Fig. 2A), suggesting that the glucose-induced rise in
Ca21signal might require activation of a signal transduction mechanism. Moreover, increasing glucose concentrations induced a proportional rise in Ca21signal,
approaching a plateau at about 20 mM glucose; peaking
at 815% as compared with basal levels (Fig. 2D). No
changes in Ca21signal were observed upon treatment
with 10 mM sucrose or mannitol ruling out the possibility of an osmolarity-induced effect (not shown).
Because glucosensing by specialized cells, such as pancreatic b-cells, involves GLUT2, GK, and KATP channels
(Schuit et al., 2001), the effect of inhibitors of these proteins on glucose-induced rise in Ca21signal of tanycytes
was assessed. Two GLUT inhibitors, 100 lM cytochalasin B (Cyto-B) or 100 mM ETDG (Barros et al., 2009),
greatly reduced the glucose-induced increase in
Ca21signal from 630 to 234% and 257%, respectively (Fig. 2E). Moreover, cytochalasin E did not affect
the glucose-induced rise in Ca21signal (not shown), ruling out unspecific effects of Cyto-B on actin polymerization. These results suggest that the glucose-induced rise
in Ca21signal is triggered by glucose entry primarily via
GLUTs; a small amount of glucose enters through
another pathway not yet identified (see below). In addition, pretreatment with alloxan (500 lM), a GK inhibitor, completely inhibited the glucose-induced increase in
Ca21signal in tanycytes from 630 to 109% (Fig.
2B,E). To investigate if the glucose-induced rise in
Ca21signal depends on ATP generated from anaerobic
glycolysis and/or oxidative phosphorylation, tanycytes
were treated with iodoacetic acid (IA) or antimycin A
(AA), blockers of each metabolic pathway, respectively.
Pretreatment with IA (1 mM) reduced the glucoseinduced rise in Ca21signal from 630 to 107%. However, no significant reduction was observed using AA
(200 nM; 612.1% 6 105.2%; Fig. 2E), suggesting that anaerobic glycolysis is the main source of ATP required for
the glucose-induced rise in Ca21signal. In pancreatic bcells, the rise in [Ca21]i depends on closure of KATP
channels induced by an increase in intracellular ATP
concentration (Schuit et al., 2001). Therefore, diazoxide
(1 lM, Diazox), an activator of KATP channels, was used
to examine the possible involvement of a similar mechanism in the glucose-induced rise in Ca21signal observed
in glucose-treated tanycytes. Pretreatment with Diazox
for 10 min drastically reduced the glucose-induced rise
in Ca21signal from 630 to 98% (Fig. 2C,E).
Rat Tanycytes Exhibit Functional Hemichannels
in Their Surface and Express Cx43 In Vitro
The rise in Ca21 signal induced by glucose might
result from opening of a Ca21 permeable cell membrane
pathway and/or Ca21 release from intracellular stores.
In glial cells the main connexin expressed is Cx43
GLIA
58
ORELLANA ET AL.
Fig. 2. Glucose-induced increase in intracellular free Ca21 signal
occurs by a glucokinase- and KATP channel-dependent pathway in rat
tancytes. (A–C) Representative plots of relative changes in Ca21signal
(340/380 nm) over time in cultured rat tanycytes subjected to changes
in glucose concentrations (horizontal bars, from 2 to 10 mM glucose)
under control conditions (A), in presence of 500 lM alloxan, a glucokinase inhibitor (B) or in presence of 1 lM diazoxide (C), a KATP channel
activator. In each panel, three photomicrographs of time-lapse images
show changes in Fura-2 ratio (pseudo-colored scale). The delay between
the addition of glucose and the increase in Fura-2 ratio is represented
by the vertical gray line. (D) Averaged data normalized to control
GLIA
(dashed line) of maximal Fura-2 fluorescence intensity during the peak
in tanycytes exposed to increasing glucose concentration. (E) Averaged
data normalized to control (dashed line) of maximal Fura-2 fluorescence
intensity during the peak in tanycytes exposed to 10 mM glucose alone
or in combination with the following blockers: 100 lM Cyt B, 100 mM
4,6,-O-ethylidene-D-glucose (ETDG), 500 lM alloxan, 1 mM iodoacetate
(IA), 200 nM AA (AA), and 1 lM diazoxide (Diazox). ***P < 0.001,
effect of 10 mM glucose compared with control; #P < 0.05, ##P < 0.005,
effect of blockers compared with glucose treatment. Averaged data were
obtained from at least five independent experiments.
GLUCOSENSING VIA HEMICHANNELS IN TANYCYTES
59
P < 0.05, effect of DCFS conditions compared with blockers. Averaged
data were obtained from at least five independent experiments. Scale
bar 5 30 lm. (E) Representative confocal image depicting vimentin
(red) and Cx43 (green) immunolabeling of cultured rat tanycytes under
control conditions. In blue are shown nuclei stained with TOPRO-3.
Scale bar 5 15 lm. (F) Representative plot of relative changes in Ca21
signal over time in cultured tanycytes subjected to changes in glucose
concentrations (horizontal bars, from 2 to 10 mM glucose) in absence of
extracellular Ca21 (Ca21-free) (G) Averaged data normalized to control
(dashed line) of maximal Fura-2 fluorescence intensity during the peak
in tanycytes exposed to 10 mM glucose alone or in combination with
the following blockers: 200 lM 10panx1, 500 lM probenecid (Prob), 200
lM Gap26, 1:500 Cx43E2 antibody and in absence of extracellular Ca21.
All blockers were applied 10 min before treatment with glucose. #P <
0.005, effect of blockers compared with glucose treatment. Averaged
data were obtained from at least five independent experiments.
Fig. 3. Rat tanycytes exhibit functional Cx43 hemichannels that are
involved in the glucose-induced increase in intracellular Ca21signal.
(A,B) Fluorescence micrographs of Etd uptake (10-min exposure) in
tanycytes under control conditions (A) and then exposed for 10 min to a
divalent cation (Ca21/Mg21)-free solution (DCFS) (B). (C) Time-lapse
measurement of Etd uptake in rat tanycytes under control conditions
(basal) or exposed to divalent cation free solution (DCFS). La31 (200
lM), a connexin hemichannel blocker, applied at 7 min of Etd uptake
measurements reduced the Etd uptake to values even lower than that
recorded under basal condition. (D) Averaged data normalized to control (dashed line) of Etd uptake rate by tanycytes treated with the following Cx43 hemichannel blockers co-applied during dye uptake recording: 200 lM La31, 200 lM Gap26, and 1:500 Cx43E2; or with the following Panx1 hemichannel blockers: 200 lM 10panx1 or 500 lM
probenecid (Prob). Also, it is shown the Etd uptake rate induced by
DCFS conditions alone or plus the anti-Cx43 hemichannel blocker,
Cx43E2. *** P < 0.001, ** P < 0.005, treatments compared with control;
#
(Giaume and Theis, 2010), which has been recently
shown to be Ca21 permeable (Schalper et al., 2010).
Therefore, we studied if rat tanycytes express functional
Cx43 hemichannels in their surface. Similar to cortical
astrocytes (Orellana et al., 2010), tanycytes cultured
under control conditions exhibited a low Etd uptake rate
(0.25 6 0.02 AU/min; Fig. 3A,C). The basal Etd uptake in
tanycytes under control condition was partially reduced to
about 50% of the basal level by La31 (200 lM), a general
blocker of connexin hemichannels, Gap26 (200 lM), and
the Cx43E2 antibody (1:500) (Fig. 3D), two Cx43 hemichannel blockers, suggesting the presence of a basal Cx43 hemichannel activity. However, the Panx1 hemichannel blockers, probenecid (Prob, 500 lM) or 10panx1 (200 lM), did
not significantly alter the basal Etd uptake (97.3% 6
22.3% and 102.4% 6 6.2%, respectively), suggesting that if
tanycytes express Panx1 hemichannels, they are not active
under basal conditions (Fig. 3D). To address whether both
Panx1 hemichannel blockers effectively inhibit Panx1 hem-
ichannels under our conditions, we treated neurons with
glutamate for 1 h, a known condition that opens Panx1
hemichannels in these cells (Orellana et al., 2011b). Both
blockers inhibited the increase in the glutamate-induced
Etd uptake mediated by Panx1 hemichannels (not shown).
The presence of connexin hemichannels in cultured tanycytes was supported by their activation in cells exposed to a
DCFS, which increases the open probability of connexin but
not Panx1 hemichannels (S
aez et al., 2005). Under these
conditions, a high Etd uptake rate (0.56 6 0.01 AU/min)
was found compared with control (224.3% 6 36.3%) that
was almost completely blocked by Cx43E2 antibody (61.3%
6 12.1%; Fig. 3B–D), indicating that most if not all functional hemichannels are formed by Cx43.
The presence of functional Cx43 hemichannels was also
in agreement with the immunofluorescence detection of
Cx43 in cultured tanycytes (Fig. 3E); Cx43 was primarily
localized at cell–cell interfaces, which may correspond with
gap junctions (Fig. 3E). Moreover, all cultures showed veGLIA
60
ORELLANA ET AL.
sicular Cx43 reactivity that most likely corresponds to intracellular Cx43 trafficking (Fig. 3E). Since a very small
percent (10%) of total Cx43 is known to form hemichannels at the cell surface of cortical astrocytes (Retamal et
al., 2006), it is likely that Cx43 hemichannels also correspond to a small percent of the total Cx43 protein making
difficult their detection by immunofluorescence. In spite,
we detected Cx43 at the cellular surface using the Cx43E2
antibody in nonpermeabilized cells (Supp. Info. Fig. 1A). In
permeabilized cells, Cx43 reactivity was also detected in
vesicle-like structures located at the cell interior in addition
to the labeling detected at the cell periphery (Supp. Info.
Fig. 1B). We also detected Cx43 hemichannels at the cellular surface using biotinylation of surface proteins and
whole cell patch clamp (see below).
The Glucose-Induced Increase in Intracellular
Free Ca21 Signal Is Mediated Mainly by Ca21
Release from Intracellular Stores and Not by Ca21
Influx Via Hemichannels
The possible role of Cx43 hemichannels in the glucoseinduced rise in Ca21 signal was studied using specific
blockers of Cx43 hemichannels, the Cx43E2 antibody
(Orellana et al., 2011c; Siller-Jackson et al., 2008), and
the mimetic peptide Gap26 with an amino acid sequence
identical to the second loop of Cx43 (Evans et al., 2006).
Cx43E2 (1:500) and Gap26 (200 lM) reduced almost completely the glucose-induced increase in Ca21 signal in
tanycytes to 113 and 107%, respectively (Fig. 3G).
However, pretreatment with the preimmune antibodies
(1:500) did not affect the glucose-induced increase in
Ca21 signal, supporting the specificity of Cx43E2 (not
shown). In contrast, 10panx1 (200 lM) and probenecid
(200 lM), both Panx1 hemichannel blockers (Pelegrin
and Surprenant, 2006; Silverman et al., 2008), did not
reduce the glucose-induced increase in Ca21 signal (Fig.
3G), ruling out the possible involvement of Panx1 hemichannels in the glucose-induced rise in Ca21 signal. In
spite of the strong inhibition of the glucose-induced rise
in Ca21 signal induced by Cx43E2 and Gap26, the absence of extracellular Ca21 did not significantly reduce
this response (from 615%, in the presence of extracellular Ca21 to 590% in Ca21-free; Fig. 3F,G), suggesting
that in tanicytes exposed to glucose, Cx43 hemichannels
do not allow a relevant Ca21 inflow and thus, their role
in the observed glucose-induced rise in Ca21 signal could
be indirect. In addition, the lack of effect of extracellular
Ca21-free solution on the glucose-induced rise in Ca21
signal suggested strongly the involvement of Ca21
release from intracellular reservoirs instead of Ca21
influx from the extracellular medium.
Glucose Enhances the Cx43 Hemichannel Activity
Via a Glucokinase/KATP Channel-Dependent
Pathway in Rat Tanycytes
To elucidate the involvement of Cx43 hemichannels in
the glucose-induced rise in Ca21signal, the effect of gluGLIA
cose on the hemichannel activity was assessed. Under
control conditions (2 mM glucose), tanycytes exhibited
low Etd uptake (Fig. 4A,D), which prominently
increased upon treatment with 10 mM glucose (from
0.25 6 0.02 to 0.57 6 0.02 AU/min, n 5 6) (Fig. 4B,D,F).
An important difference with the glucose-induced rise in
Ca21 signal, which showed a delay of about 1 min (Fig.
4A,B), was that the delay of the glucose-induced rise in
Etd uptake was <20 s (Fig. 4D). Moreover, as in the
case of the Ca21 signal changes, the Etd uptake increase
was glucose concentration-dependent, approaching a plateau at 40 mM glucose (425.4% 6 182.3%, compared
with basal; Fig. 4E). In these cells, no concurrent
changes in intercellular communication mediated by gap
junctions, evaluated by the intercellular transfer of
microinjected Lucifer yellow, was observed (Supp. Info.
Fig. 2A–D).
To evaluate if the glucosensing proteins, GLUTs, GK,
and KATP channels, are involved in the glucose-induced
Etd uptake, specific drugs against each of these molecular components of the glucosensor transduction system
were employed. As compared with control, 100 lM CytoB, 100 mM ETDG, 500 lM alloxan, 1 mM IA, and 1 lM
Diazox reduced the glucose-induced Etd uptake from
211 to 138, 141, 103, 105, and 109%, respectively (Fig. 4F). However, 200 nM AA did not affect it
(207.2% 6 19.4%, n 5 4; Fig. 4F). Therefore, the glucose-induced Etd uptake requires the functional participation of GLUTs, GK, and glycolysis-derived ATP to
block KATP channels. Relevant to this interpretation is
that inhibitors of these glucose-sensing proteins did not
alter the DCFS-induced Etd uptake (Supp. Info. Fig. 3),
indicating that they act as glucosensing modulators and
not as hemichannel blockers.
Glucose Increases the Macroscopic Cx43
Hemichannel Current, but Does Not Alter the
Unitary Conductance or the Surface Cx43 Levels
in Tanycytes
To identify the pathway through which glucose
increases the membrane permeability of tanycytes,
whole-cell voltage-clamp studies were undertaken, and
the macroscopic membrane current (total current measured in the absence of other active membrane channels)
and the presence of hemichannel unitary events applying voltage ramp protocols from 280 to 180 mV were
assessed (Fig. 5A).
In control tanycytes, unitary current events were not
detected in 17 cells (not shown); however, six cells
showed a few small unitary transitions at negative
potentials (Fig. 5B). In contrast, in tanycytes treated
with 10 mM glucose for 3 min the total current was
much bigger than in control cells, and numerous unitary
current events were detected at either negative (at 260
mV from 15 to 197 pA, n 5 12 cells; Fig. 5B,D) or
positive potentials (at 160 mV from 14 to 205 pA, n
5 12 cells; Fig. 5B,D). In most cells (n 5 18), unitary
current events were recorded, and point-by-point conver-
GLUCOSENSING VIA HEMICHANNELS IN TANYCYTES
61
Fig. 4. Increased Etd uptake induced by glucose is mediated by a
glucokinase/KATP channel-dependent pathway in cultured rat tanycytes.
(A–C) Representative immunofluorescence images depicting vimentin
(white) labeling and Edt (red) nuclei-staining from dye uptake experiments (10-min exposure to dye) in cultures of tanycytes under control
conditions (A) or treated with 10 mM glucose for 5 min alone (B) or glucose plus 1:500 Cx43E2, an anti-Cx43 antibody that blocks Cx43 hemichannel (C). (D) Time-lapse measurement of Etd uptake in rat tanycytes treated with 2, 10, or 20 mM glucose. La31 (200 lM) applied at
10 min of Etd uptake measurement inhibited dye uptake. (E) Average
of Etd uptake rate normalized to control (dashed line) in tanycytes
exposed to increasing concentrations of glucose. * P < 0.001, treatments
compared with control. (F) Averaged Etd uptake rate normalized to
control (dashed line) by tanycytes treated with 10 mM glucose alone or
in combination with the following blockers: 100 lM cytochalasin B
(Cyt B), 100 mM ETDG, 500 lM alloxan, 1 mM iodocetic acid (IA), 200
nM antimycin A (AA), 1 lM diazoxide (Diazox), 200 lM La31, 200 lM
Gap26, 1:500 Cx43E2, 200 lM 10panx1, or 500 lM Prob. ** P < 0.005,
10 mM glucose treatment compared with control; # P < 0.05, ## P <
0.005, effect of 10 mM glucose treatment compared with blockers. Averaged data were obtained from at least four independent experiments.
Scale bar 5 15 lm.
sion of current to conductance values revealed single
channels of 218 6 6 pS (see inset in Fig. 5B). This value
is close to the expected unitary conductance (220 pS)
of Cx43 hemichannels (Contreras et al., 2003). To elucidate the involvement of such hemichannels in the glucose-induced currents, tanycytes were treated acutely
with Cx43E2 (1:500). This Cx43 hemichannel blocker
reduced almost completely the rise in macroscopic current (at 260 mV from 197 to 27 pA and at 160 mV
from 205 to 25 pA, n 5 12) and the appearance of
discrete unitary current transitions induced by glucose
(Fig. 5B,D), indicating that glucose induces activation of
Cx43 hemichannels. Importantly, the reversal potential
of all the above mentioned currents was close to 0 mV.
However, the glucose-induced currents presented a reversal potential close 15 mV (Fig. 5B). Moreover, 10
mM glucose increased the frequency of unitary currents
of Cx43 hemichannels with conductance around 220
pS (Supp. Info. Fig. 4A–D), whereas did not significantly
alter the decay constant (s) of open time compared with
control (3.7 6 0.7 and 5.2 6 0.6 ms, respectively; Supp.
Info. Fig. 4E,F). Furthermore, 10 mM glucose increased
GLIA
62
ORELLANA ET AL.
the mean open time compared with controls conditions
(0.7 6 0.1 and 0.20 6 0.03 ms, respectively).
The increase in Cx43 hemichannel activity might be
due to an increase in open probability per hemichannel
or/and increase in the number of hemichannels present
in the cell membrane. Previous studies have associated
hemichannel-mediated dye uptake with increased surface levels of hemichannels (Orellana et al., 2010).
Therefore, the effect of glucose on the total and surface
levels of Cx43 in tanycytes was evaluated. Neither total
nor surface levels of Cx43 were affected by treatment
with 10 mM glucose for 5 min (Fig. 5C,E, respectively, n
5 3). Also, neither 500 lM alloxan nor 1 lM diazox in
combination with glucose affected total Cx43 or surface
levels in tanycytes (Fig. 5C,E, respectively, n 5 3).
Importantly, 10 mM glucose might decrease the total
nonphosphorylated Cx43 and increase the total phosphorylated form of Cx43 (Fig. 5C); however, total levels of
Cx43 were not significantly affected (Fig. 5E).
Tanycytes Are Permeable to Glucose Via
GLUTs and Cx43 Hemichannels
Since neither the rise in Ca21 signal nor the increase
in Etd uptake induced by glucose were completely inhibited with GLUT blockers (Figs. 2F and 4F) and because
other glial cells also present glucose uptake through
Cx43 hemichannels (Retamal et al. 2007), the role of
these channels as an alternative pathway for glucose
entry into tanycytes was explored. Specifically, uptake of
2-DOG and 2-NBDG in tanycytes under control conditions or bathed in DCFS to enhance the open probability
of Cx43 hemichannels (S
aez et al., 2005) was measured.
After 1 min, 2-DOG uptake close to 20–30 nmoles per
million of cells (22.1 6 8 nmol/106 cells; Fig. 6A) and low
2-NBDG uptake (37.4 6 7.3 AU, n 5 6) was observed
under control conditions (Fig. 6B). Uptake of 2-DOG and
2-NBDG was drastically reduced by 100 mM ETDG
(6 nmol/106 cells and 9 AU, respectively, n 5 5) or
100 lM Cyto-B (7 nmol/106 cells and 7 AU, respectively, n 5 5), whereas Cx43E2 (1:500) only partially
inhibited 2-DOG and 2-NBDG uptake (15 nmol/106
cells and 22 AU, respectively, n 5 3; Fig. 6). Notably,
co-application of Cyto-B and Cx43E2 almost completely
blocked the uptake of 2-DOG and 2-NBDG (0.8 nmol/
106 cells and 0.8 AU, respectively, n 5 3; Fig. 6B).
As predicted, tanycytes bathed in DCFS exhibited
higher 2-DOG (38 nmol/106 cells, n 5 5) and 2-NBDG
(66 AU, n 5 5) uptake than tanycytes under control
conditions (Fig. 6A,B). Under these conditions, uptake of
2-DOG or 2-NBDG was more prominently inhibited by
Cx43E2 (14 nmol/106 cells and 24 AU, respectively,
n 5 3), than by ETDG (22 nmol/106 cells and 31 AU,
Fig. 5. Glucose increases Cx43 hemichannel currents, but does not
affect the surface Cx43 levels in cultured rat tanycytes. (A) Voltage
ramps from 280 to 180 mV, 5 s in duration, were applied. Each ramp
was initiated and finished by a transition from 0 to 280 and 180 to 0
mV, respectively. Membrane current was measured in a whole-cell voltage-clamp configuration using low-density cultures of rat tanycytes. (B)
Voltage ramp from 280 to 180 mV, 5 s duration in tanycytes under
control conditions (black line) or treated for 3 min with 10 mM glucose
alone (magenta line) or 10 mM glucose plus 1:500 Cx43E2 (green line).
A record section at about 160 mV the current trace was point-by-point
converted in conductance values showing unitary events of about 220
pS. (C) Cultured tanycytes were treated for 5 min with 10 mM glucose.
Total (left panel) and surface (right panel) levels of Cx43 in tanycytes
under control conditions (lane 1) or treated for 5 min with 10 mM glucose alone (lane 2), 10 mM glucose plus 500 lM alloxan (lane 3) or plus
1 lM diazoxide (Diazox) (lane 4). The Cx43 phosphorylated (P1-P2) and
nonphosphorylated (NP) forms are indicated in the left. Total levels of
each analyzed protein were normalized according to the levels of atubulin detected in each lane. Surface levels of each analyzed protein
were normalized according to the total protein loaded as revealed by
staining with Ponceau red (PR) in each lane. Similar observations were
made in two other independent experiments. (D) Averaged data on current events at 160 (white bars) or 260 mV (black bars) in tanycytes
under control conditions, exposed to 10 mM glucose alone or 10 mM
glucose plus Cx43E2. * P < 0.001, glucose treatment compared with control; # P < 0.001, inhibitory effect of blocker on the glucose-induced
response. Averaged data were obtained from three independent experiments in which at least eight cells were analyzed per treatment. (E)
Quantification of total (white bars) and surface (black bars) levels of
Cx43 normalized to control (dashed line) in tanycytes treated for 5 min
with 10 mM glucose alone, plus 500 lM alloxan or 1 lM Diazox. Averaged data were obtained from three independent experiments.
GLIA
GLUCOSENSING VIA HEMICHANNELS IN TANYCYTES
Fig. 6. Glucose uptake occurs via GLUTs and Cx43 hemichannels in
rat tanycytes. Averaged data at 1 min of 2-DOG (A) and 2-NBDG (B)
uptake in tanycytes under control conditions or treated with 100 mM
ETDG, 100 lM cytochalasin B (Cyto-B), 1:500 Cx43E2 or 1:500 Cx43E2
plus 100 lM Cyto-B. In addition, 2-DOG and 2-NBDG uptake by tanycytes exposed to DCFS conditions for 1 min alone or plus 100 mM
(ETDG), 100 lM Cyto-B, 1:500 Cx43E2 or 1:500 Cx43E2 plus 100 lM
Cyto-B was analyzed. *** P < 0.001, ** P < 0.005, * P < 0.05; treatments compared with control; # P < 0.05, treatments compared with
DCFS conditions. Averaged data were obtained from at least four independent experiments.
respectively, n 5 5) or Cyto-B (24 nmol/106 cells and
27 AU, respectively, n 5 5; Fig. 6). Furthermore, in
cells treated with Cyto-B plus Cx43E2 the uptake of 2DOG and 2-NBDG was almost completely blocked (1
nmol/106 cells and 0.7 AU, respectively, n 5 3).
The Glucose-Induced Increase in Intracellular
Free Ca21 Signal Occurs Via ATP Released
Through Cx43 Hemichannels
Because in parallel experiments the onset of glucoseinduced Etd uptake occurred before (Fig. 4D) than the
rise in Ca21 signal (Fig. 2A), simultaneous measure-
63
ments of Etd uptake and Ca21signal in tanycytes
exposed to glucose were performed to better elucidate
the onset of these sequential changes. In these studies,
the Etd uptake induced by 10 mM glucose occurred at
12 s (Fig. 7A–H,I, n 5 8), whereas the glucose-induced
rise in Ca21 signal was observed at 67 s (Fig. 7A–H,I,
n 5 9). Because the glucose-induced changes did not
depend on the extracellular [Ca21] (Fig. 2B,F), the above
results suggest that opening of Cx43 hemichannels
results in the extracellular release of a molecule that
reaches a concentration sufficient to trigger Ca21 release
from intracellular stores.
Previous reports have demonstrated that Cx43 hemichannels can mediate the release of ATP (Kang et al.,
2008). Because ATP can induce Ca21 release from intracellular stores via P2Y receptors, ATP release by tanycytes in response to glucose was examined. The increase
in Ca21 signal induced by glucose was prevented by 10
U/mL apyrase, a phosphatase that degrades ATP, and
200 lM suramine, an inhibitor of both P2Y and P2X
receptors, peaking at 135 or 126%, respectively (Fig.
7J). However, no differences were observed using 200
lM oATP, a general P2X receptor blocker, or 10 lM brilliant blue G (BBG), a P2X7 receptor blocker (610 or
624%, respectively; Fig. 7J). These results indicated
that ATP and P2Y receptors are crucial for glucoseinduced increases in Ca21 signal. In support of this
interpretation, MRS2179, a P2Y1 receptor blocker at concentrations up to 30 lM, inhibited the glucose-induced
rise in Ca21 signal (129.2% 6 17.3%; Fig. 7J). Moreover,
2 lM thapsigargin, a compound that depletes the intracellular Ca21 stores, or 5 lM xestospongin C (XeC) and
5 lM xestospongin B (XeB), both IP3 receptor blockers,
inhibited the rise in Ca21 signal induced by glucose
(158, 125, and 120%, respectively; Fig. 7J), suggesting the involvement of IP3-mediated Ca21 release.
Then, the effect of glucose on ATP release via Cx43
hemichannels and glucosensing signaling was examined.
The extracellular medium of tanycytes treated with 10
mM glucose exhibited higher ATP levels than that of
control tanycytes (from 1 pmol/106 cells to 45 pmol/
3106 cells, n 5 6; Fig. 7K). The increase in extracellular
ATP concentration induced by glucose was prevented by
100 lM Cyto-B, 500 lM alloxan, 1 mM IA, 1 lM Diazox,
and Cx43E2 (7, 1, 0.9, 0.2, and 0.2 pmol/106,
respectively, n 5 6; Fig. 7K).
The effect of ATP itself on the Ca21 signal in tanycytes was also examined; 100 lM ATP induced a rise in
Ca21 signal similar to that observed with 10 mM glucose
(682%, n 5 3; Fig. 8A,B). Moreover, the ATP-induced
rise in Ca21 signal was inhibited by 200 lM suramine
(133%, n 5 3), 2 lM thapsigargin (147%, n 5 3), 5
lM XeC (156%, n 5 3), and 5 lM XeB (130%, n 5 3),
but not by 200 lM oATP or 10 lM BBG (671 and
656%, respectively, n 5 3; Fig. 8B). Thus, the results
support the hypothesis that proposes extracellular ATP
as mediator of the glucose-induced rise in [Ca21]i.
Because of the complexity behind the activation of
Cx43 hemichannels of this system, we investigated
whether an IP3 receptor-mediated Ca21 signal is needed
GLIA
64
ORELLANA ET AL.
Fig. 7. Glucose increases the intracellular free Ca21 signal in tanycytes by ATP released via Cx43 hemichannels. (A–H) Representative
fluorescence micrographs of simultaneous time-lapse imaging showing
changes in Fura-2 ratio (A–D, pseudo-colored scale) and Etd uptake
(E–H, red) in tanycytes treated with 10 mM glucose for 0 (A and E), 48
(B and F), 50 (C and G), and 200 s (D and H). (I) Simultaneous plots of
relative changes in [Ca21]i and Etd uptake over time of cells 1 (red), 2
(green), 3 (yellow), and 4 (magenta) depicted in panel A. The delay
between the increase in Etd uptake and Fura-2 ratio is indicated by
the vertical dashed line. (J) Averaged data normalized to control
(dashed line) of maximal Fura-2 fluorescence intensity during the peak
in tanycytes exposed to 10 mM glucose alone or in combination with
the followed blockers: 10 U/mL apyrase (Apyr), 200 lM oxidized ATP
(oATP), 10 lM (BBG), 200 lM suramin (Sur), 10 lM MRS2179, 2 lM
thapsigargin (TG), 5 lM xestospongin C (XeC), and 5 lM xestospongin
(XeB). ** P < 0.005, 10 mM glucose treatment compared with blockers.
(K) Averaged values of ATP released by tanycytes under control conditions or treated with 10 mM glucose alone or in combination with the following blockers: 100 lM cytochalisin B (Cyt B), 500 lM alloxan, 1 mM
IA, 1 lM diazoxide (Diazox), 200 lM 10panx1, 500 lM probenecid (Prob),
200 lM La31, 200 lM Gap26 or 1:500 Cx43E2. *** P < 0.001, 10 mM
glucose treatment compared with control; ## P < 0.005, effect of 10 mM
glucose treatment compared with blockers. Averaged data were obtained
from at least four independent experiments. Scale bar 5 35 lm.
to promote Cx43 hemichannel opening. XeC and XeB
did not modify the glucose-induced hemichannel activity
(Supp. Info. Fig. 5). Similar results were obtained in
tanycytes loaded with BAPTA (Supp. Info. Fig. 5), indicating that cytoplasmic Ca21 does not mediate the activation of Cx43 hemichannel opening. In contrast, as it
was shown before, KATP channel inhibition was necessary for the glucose-induced hemichannel activity.
However, glibenclamide, an inhibitor of KATP channels,
by itself did not induce hemichannel opening (Supp.
Info. Fig. 5), suggesting that an additional mechanism
activated almost simultaneously, and not yet identified,
is necessary for Cx43 hemichannel activation.
GLIA
DISCUSSION
The present study demonstrates that tanycytes possess a rapid, glucose-activated signal transduction path-
GLUCOSENSING VIA HEMICHANNELS IN TANYCYTES
Fig. 8. ATP increases the intracellular free Ca21 signal in tanycytes
via P2Y receptors. (A) Representative plots of relative changes in Ca21
signal over time in cultured rat tanycytes subjected to 100 lM ATP
under control conditions or pretreated with MRS21. (B) Averaged data
normalized to control (dashed line) of maximal Fura-2 fluorescence intensity during the peak in tanycytes exposed to 100-lM ATP alone or
in combination with the followed blockers: 200 lM oxidized ATP
(oATP), 10 lM brilliant blue G (BBG), 200 lM suramine (Sur), 10 lM
MRS2179, 2 lM thapsigargin (TG), 5 lM xestospongin (XeC), and 5 lM
xestospongin (XeB). * P < 0.001, 100 lM ATP treatment compared with
control; # P < 0.001, 100 lM ATP treatment compared with blockers.
way, which includes GLUTs, GK, anaerobic glycolysisderived ATP, KATP channels, Cx43 hemichannels, extracellular ATP, P2Y receptors, and IP3 activated intracellular Ca21 stores.
Recently, glucose and nonmetabolizable analogs of glucose were demonstrated to increase the [Ca21]i in hypothalamic slices, specifically in a tanycytes located in the
most dorsal region of the hypothalamus (Frayling et al.,
2011), suggesting that the pancreatic b-cell paradigm
does not apply to these cells. However, in the present
study, glucose increased the [Ca21]i in a concentrationdependent manner in cultured b-tanycytes by a process
in which glucose uptake mainly via GLUT is critical as
Cyto-B and ETDG strongly inhibited this response. This
is in agreement with the reported expression of GLUT1
65
and the glucosensing transporter, GLUT2, by b1-tanycytes (Garcıa et al., 2003). Because the glucose-induced
rise in Ca21 signal was prevented by a GK blocker as
well as a KATP channel activator, these two proteins may
play an essential role in tanycyte glucosensing, which is
also consistent with the high in vivo expression of these
proteins by these cells (Marty et al., 2007; Thomzig et
al., 2001). In addition, in vivo pharmacological and molecular inhibition of GK or KATP channels impairs feeding behavior in rodents (Miki et al., 2001; Sanders et al.,
2004). The difference between the results of the present
study and those of Frayling et al. (2011) could be
explained by the type of tanycyte population used and
the model (ex vivo versus in vitro). Frayling et al. (2011)
used a-tanycytes from brain slices, whereas our cultures
were highly enriched in b1-tanycytes, which express the
same enzymes and transporters detected in situ (e.g.,
GLUT1, GLUT2, MCT1, MCT4, GK; Cort
es-Campos et
al., 2011; Garcıa et al. 2001, 2003; Millan et al., 2010).
In this regard, tanycytes may sense glucose by more
than one mechanism, which is determined by the predominant type of tanycyte.
In the present study, glucose-induced Etd uptake was
not completely inhibited when GLUTs were blocked,
suggesting the contribution of another source of glucose
transport/diffusion through the cell membrane. Accordingly, the glucose uptake was completely inhibited when
both GLUTs and connexin hemichannels were blocked,
suggesting that under control conditions, Cx43 hemichannels participate in glucose diffusion toward the
cytoplasm of tanycytes to a minor extent despite the
increase in Cx43 hemichannel activity, while GLUTs are
the main protagonist in this transport.
Intracellular ATP generated by anaerobic glycolysis
but not produced by oxidative phosphorylation was
required for the glucose-induced rise in Ca21signal since
the effect of glucose was prevented by IA and not by AA.
This finding is similar to that reported for pancreatic bcells (Mertz et al., 1996). The high glycolytic activity of
tanycytes has been previously demonstrated; they
release lactate at physiological concentrations of glucose,
which is inhibited by classical MCT inhibitors (Cort
esCampos et al., 2011). In pancreatic b-cells, the Ca21
influx proceeds through voltage-dependent Ca21 channels and is critical for glucosensing (Schuit et al., 2001).
However, data from the present study indicate that
extracellular Ca21 influx is not involved in this response
in tanycytes, since the glucose-induced rise in Ca21 signal was observed even in the absence of extracellular
Ca21, indicating the involvement of intracellular Ca21
stores. Nevertheless, the glucose-induced rise in Ca21
signal was not detected after Cx43 hemichannel blockade, implying that they are involved in changing the
[Ca21]i by allowing release of a paracrine factor likely to
be ATP. In agreement with this interpretation, an
increase in Cx43 hemichannel activity was observed in
tanycytes treated with glucose and the increase in Ca21
signal and Etd uptake were completely inhibited by several Cx43 hemichannel blockers (e.g., La31, Gap26, and
Cx43E2). In contrast, Panx1 hemichannel blockers were
GLIA
66
ORELLANA ET AL.
without effect, indicating for the first time that tanycytes exhibit functional Cx43 hemichannels, which is
consistent with the high Cx43 immunoreactivity
detected in these cells. In support of the idea that Cx43
hemichannels are involved in glucosensing, patch clamp
experiments revealed an increase in macroscopic membrane current induced by glucose. This is most likely
due to opening of Cx43 hemichannels already present in
the cell surface due to the following: (1) it was completely inhibited by Cx43E2, a specific blocker of Cx43
hemichannels (Orellana et al., 2011c; Siller-Jackson et
al., 2008); (2) the total surface levels of Cx43 hemichannels were not affected by glucose; and (3) the unitary
events were of 220 pS, characteristics that correspond
to that of Cx43 hemichannel (Contreras et al., 2003).
Thus, it is likely that the glucose-induced increase in
total current was due to an increase in open probability
of each Cx43 hemichannel already present at the cell
surface and not to recruitment of more hemichannels at
the cell surface or increase in unitary hemichannel
conductance. Unlike cortical astrocytes that express
hemichannels formed by Panx1 (Iglesias et al., 2009;
Iwabuchi and Kawahara, 2011) or Cx43 (Orellana
et al., 2011c; Retamal et al., 2007), cultured tanycytes
seems to express mainly hemichannels formed by
Cx43. The slightly positive reversal potential observed
in tanicytes treated with glucose might reflect either a
change Cx43 hemichannel selectivity or the presence
of a more selective membrane channel simultaneously
activated by glucose and co-recorded with Cx43
hemichannels.
Since the glucose-induced hemichannel activity was
inhibited by blockers of the canonical glucosensing pathway (e.g., GLUTs, GK, and KATP channels), activation of
Cx43 hemichannels likely occurs downstream of the glucosensing molecules (Fig. 9). A puzzling aspect is how
KATP channel inhibition leads to increased Cx43 hemichannel activity. One possibility is that it might result
from membrane depolarization as consequence of KATP
channel inhibition by intracellular ATP. In agreement
with this possibility, Cx43 hemichannels expressed in
HeLa cells are activated by positive potential (Contreras
et al., 2003). However, in glucose-treated tanycytes, the
activity of Cx43 hemichannels was also high at negative
potentials, suggesting that membrane depolarization is
not the only possible mechanism involved in enhancing
Cx43 hemichannel activity. In support to this notion,
membrane depolarization under control conditions did
not enhance the membrane current mediated by Cx43
hemichannels. Moreover, glibenclamide by itself did not
induce hemichannel opening, suggesting that both glucose or its derivates (e.g., ATP) and inhibition of KATP
channels are necessary for Cx43 hemichannel activation.
Among the putative mechanism, it is possible that
glucose or its derivates activate kinases/phosphatases
regulating the phosphorylation state of Cx43 hemichannels, which are known to affect the gating mechanism
of these channels (S
aez et al., 2010). However, further
studies are required to examine this and other alternative possibilities to identify the mechanism by
GLIA
Fig. 9. Model of glucosensing mechanism in tanycytes. Upon a rise
in extracellular glucose concentration, glucose enters (1) mainly
through GLUTs and to a minor extent via Cx43 hemichannels, leading
to glucokinase-dependent phosphorylation of glucose (2). The ATP generated during glycolysis (3) via processing of glucose-derived substrates
causes closure of KATP channels (4). This event promotes by an
unknown mechanism the opening of Cx43 hemichannels (5). The ATP
released via Cx43 hemichannels (6) activates P2Y receptors (7) and
induces formation of cytoplasmic inositol (1,4,5)-trisphosphate (IP3),
which promotes the release of Ca21 stored in the endoplasmic reticulum (8), raising the [Ca21]i (9).
which Cx43 hemichannels are activated by glucose in
tanycytes.
Acute glucose increases the release of ATP in tanycytes (Frayling et al., 2011). Accordingly, the present
study shows that the extracellular ATP concentration is
enhanced in tanycyte cultures treated with glucose and
most likely is the consequence of more ATP released via
Cx43 hemichannels, because inhibitors of Cx43 hemichannels and not of Panx1 hemichannels prevented the
glucose-induced increase in extracellular ATP levels.
This increase in extracellular ATP concentration was
also prevented by MRS2179, thapsigargin and xestospongin C, revealing the involvement of the metabotropic P2Y1 receptors, IP3 receptors and intracellular
Ca21 stores, respectively. Supporting the possible regulatory role of ATP in feeding behavior, intracerebroventricular infusion of this molecule stimulates neurons localized in the lateral hypothalamic area (Wollmann et al.,
2005), dorsomedial hypothalamic nucleus (Matsumoto
et al., 2004), and VMN (Sorimachi et al., 2001). Moreover, extracellular ATP and possibly ADP may regulate
food intake via activation of P2Y1 receptors (Kittner et
al., 2006). ATP release by tanycytes may modulate neuronal activity in hypothalamic areas associated with
feeding behavior, including the AN and VMN, which are
in close contact with these cells. If so, the activation of
purinergic P2Y receptors might be turned off in part by
diffusion of ATP/ADP to distal regions as well as by
GLUCOSENSING VIA HEMICHANNELS IN TANYCYTES
desensitization of P2Y1 receptors (Choi et al., 2008) and
degradation of extracellular ATP/ADP by extracellular
phosphatases in the extracellular compartment of tanycytes (Firth and Bock, 1976).
Future in vivo studies will be required to determine
whether tanycytes could sense extracellular changes in
glucose concentration and transmit them to neurons via
Ca21 waves and/or the release of paracrine factors (e.g.,
ATP). The present work shows the involvement of several
glucosensing proteins in the glucose-induced rise in
[Ca21]i on b1-tanycytes (Fig. 9), which might contribute
to the knowledge on the glucosensing mechanism in the
brain and may open novel pharmacological strategies for
therapeutic treatment of feeding behavior disorders.
REFERENCES
Akmayev IG, Fidelina OV. 1974. Morphological aspects of the hypothalamic-hypophyseal system. V. The tanycytes: Their relation to the
hypophyseal adrenocorticotrophic function. An enzyme-histochemical
study. Cell Tissue Res 152:403–410.
Akmayev IG, Popov AP. 1977. Morphological aspects of the hypothalamic-hypophyseal system. VII. The tanycytes: Their relation to the
hypophyseal adrenocorticotrophic function. An ultrastructural study.
Cell Tissue Res 180:263–282.
Alvarez E, Roncero I, Chowen JA, Thorens B, Blazquez E. 1996.
Expression of the glucagon-like peptide-1 receptor gene in rat brain.
J Neurochem 66:920–927.
Barros LF, Bittner CX, Loaiza A, Ruminot I, Larenas V, Moldenhauer
H, Oyarzun C, Alvarez M. 2009. Kinetic validation of 6-NBDG as a
probe for the glucose transporter GLUT1 in astrocytes. J Neurochem
109 (Suppl 1):94–100.
Chauvet N, Parmentier ML, Alonso G. 1995. Transected axons of adult
hypothalamo-neurohypophysial neurons regenerate along tanycytic
processes. J Neurosci Res 41:129–144.
Choi RC, Simon J, Tsim KW, Barnard EA. 2008. Constitutive and agonist-induced dimerizations of the P2Y1 receptor: Relationship to
internalization and scaffolding. J Biol Chem 283:11050–11063.
Contreras JE, S
aez JC, Bukauskas FF, Bennett MV. 2003. Gating and
regulation of connexin 43 (Cx43) hemichannels. Proc Natl Acad Sci
USA 100:11388–11393.
Cort
es-Campos C, Elizondo R, Llanos P, Elizondo R, Uranga RM, Nualart F, Garcıa MA. 2011. MCT expression and lactate influx/efflux in
tanycytes involved in glia-neuron metabolic interaction. PLoS One
6:e16411.
Dale N. 2011. Purinergic signaling in hypothalamic tanycytes: Potential
roles in chemosensing. Semin Cell Dev Biol 22:237–244.
De Vuyst E, Wang N, Decrock E, De Bock M, Vinken M, Van Moorhem
M, Lai C, Culot M, Rogiers V, Cecchelli R, et al. 2009. Ca(21) regulation of connexin 43 hemichannels in C6 glioma and glial cells. Cell
Calcium 46:176–187.
Evans WH, De Vuyst E, Leybaert L. 2006. The gap junction cellular
internet: Connexin hemichannels enter the signalling limelight. Biochem J 397:1–14.
Firth JA, Bock R. 1976. Distribution and properties of an adenosine triphosphatase in the tanycyte ependyma of the IIIrd ventricle of the
rat. Histochemistry 47:145–157.
Flament-Durand J, Brion JP. 1985. Tanycytes: Morphology and functions: A review. Int Rev Cytol 96:121–155.
Frayling C, Britton R, Dale N. 2011. ATP-mediated glucosensing by
hypothalamic tanycytes. J Physiol 589 (Part 9):2275–2286.
Garcıa MA, Carrasco M, Godoy A, Reinicke K, Montecinos VP, Aguayo
LG, Tapia JC, Vera JC, Nualart F. 2001. Elevated expression of glucose transporter-1 in hypothalamic ependymal cells not involved in
the formation of the brain-cerebrospinal fluid barrier. J Cell Biochem
80:491–503.
Garcıa MA, Millan C, Balmaceda-Aguilera C, Castro T, Pastor P, Montecinos H, Reinicke K, Zuniga F, Vera JC, Onate SA, et al. 2003. Hypothalamic ependymal-glial cells express the glucose transporter GLUT2, a
protein involved in glucose sensing. J Neurochem 86:709–724.
Giaume C, Theis M. 2010. Pharmacological and genetic approaches to
study connexin-mediated channels in glial cells of the central nervous
system. Brain Res Rev 63:160–176.
67
Iglesias R, Dahl G, Qiu F, Spray DC, Scemes E. 2009. Pannexin 1:
the molecular substrate of astrocyte ‘‘hemichannels’’. J Neurosci
29:7092–7097.
Iwabuchi S, Kawahara K. 2011. Functional significance of the negativefeedback regulation of ATP release via pannexin-1 hemichannels
under ischemic stress in astrocytes. Neurochem Int 58:376–384
Kang J, Kang N, Lovatt D, Torres A, Zhao Z, Lin J, Nedergaard M.
2008. Connexin 43 hemichannels are permeable to ATP. J Neurosci
28:4702–4711.
Kittner H, Franke H, Harsch JI, El-Ashmawy IM, Seidel B, Krugel U,
Illes P. 2006. Enhanced food intake after stimulation of hypothalamic
P2Y1 receptors in rats: Modulation of feeding behaviour by extracellular nucleotides. Eur J Neurosci 24:2049–2056.
Levin BE, Routh VH, Kang L, Sanders NM, Dunn-Meynell AA. 2004.
Neuronal glucosensing: What do we know after 50 years? Diabetes
53:2521–2528.
Marty N, Dallaporta M, Thorens B. 2007. Brain glucose sensing, counterregulation, and energy homeostasis. Physiology (Bethesda) 22:241–251.
Matsumoto N, Sorimachi M, Akaike N. 2004. Excitatory effects of ATP
on rat dorsomedial hypothalamic neurons. Brain Res 1009:234–237.
Mertz RJ, Worley JF, Spencer B, Johnson JH, Dukes ID. 1996. Activation of stimulus-secretion coupling in pancreatic b-cells by specific
products of glucose metabolism. Evidence for privileged signaling by
glycolysis. J Biol Chem 271:4838–4845.
Miki T, Liss B, Minami K, Shiuchi T, Saraya A, Kashima Y, Horiuchi
M, Ashcroft F, Minokoshi Y, Roeper J, et al. 2001. ATP-sensitive K1
channels in the hypothalamus are essential for the maintenance of
glucose homeostasis. Nat Neurosci 4:507–512.
Millan C, Martınez F, Cortes-Campos C, Lizama I, Ya~
nez MJ, Llanos P,
Reinicke K, Rodrıguez F, Peruzzo B, Nualart F, et al. 2010. Glial glucokinase expression in adult and post-natal development of the hypothalamic region. ASN Neuro 2:e00035.
Navarro M, Rodriquez de Fonseca F, Alvarez E, Chowen JA, Zueco JA,
Gomez R, Eng J, Blazquez E. 1996. Colocalization of glucagon-like
peptide-1 (GLP-1) receptors, glucose transporter GLUT-2, and glucokinase mRNAs in rat hypothalamic cells: Evidence for a role of GLP1 receptor agonists as an inhibitory signal for food and water intake.
J Neurochem 67:1982–1991.
Orellana JA, Dıaz E, Schalper KA, Vargas AA, Bennett MV, S
aez JC.
2011a. Cation permeation through connexin 43 hemichannels is cooperative, competitive and saturable with parameters depending on the
permeant species. Biochem Biophys Res Commun 409:603–609.
Orellana JA, Froger N, Ezan P, Jiang JX, Bennett MV, Naus CC,
Giaume C, S
aez JC. 2011b. ATP and glutamate released via astroglial
connexin 43 hemichannels mediate neuronal death through activation of pannexin 1 hemichannels. J Neurochem 118:826–884.
Orellana JA, Hern
andez DE, Ezan P, Velarde V, Bennett MV, Giaume C,
S
aez JC. 2010. Hypoxia in high glucose followed by reoxygenation in
normal glucose reduces the viability of cortical astrocytes through
increased permeability of connexin 43 hemichannels. Glia 58:329–343.
Orellana JA, S
aez PJ, Shoji KF, Schalper KA, Palacios-Prado N,
Velarde V, Giaume C, Bennett MV, S
aez JC. 2009. Modulation of
brain hemichannels and gap junction channels by pro-inflammatory
agents and their possible role in neurodegeneration. Antioxid Redox
Signal 11:369–399.
Orellana JA, Shoji KF, Abudara V, Ezan P, Amigou E, S
aez PJ, Jiang
JX, Naus CC, S
aez JC, Giaume C. 2011c. Amyloid b-induced death in
neurons involves glial and neuronal hemichannels. J Neurosci
31:4962–4977.
Pelegrin P, Surprenant A. 2006. Pannexin-1 mediates large pore formation and interleukin-1b release by the ATP-gated P2X7 receptor.
EMBO J 25:5071–5082.
Porras OH, Loaiza A, Barros LF. 2004. Glutamate mediates acute glucose transport inhibition in hippocampal neurons. J Neurosci
24:9669–9673.
Retamal MA, Cort
es CJ, Reuss L, Bennett MV, S
aez JC. 2006. S-nitrosylation and permeation through connexin 43 hemichannels in astrocytes: Induction by oxidant stress and reversal by reducing agents.
Proc Natl Acad Sci USA 103:4475–4480.
Retamal MA, Froger N, Palacios-Prado N, Ezan P, S
aez PJ, S
aez JC,
Giaume C. 2007. Cx43 hemichannels and gap junction channels in
astrocytes are regulated oppositely by proinflammatory cytokines
released from activated microglia. J Neurosci 27:13781–13792.
S
aez JC, Retamal MA, Basilio D, Bukauskas FF, Bennett MV. 2005.
Connexin-based gap junction hemichannels: Gating mechanisms. Biochim Biophys Acta 1711:215–224.
S
aez JC, Schalper KA, Retamal MA, Orellana JA, Shoji KF, Bennett
MV. 2010. Cell membrane permeabilization via connexin hemichannels in living and dying cells. Exp Cell Res 316:2377–2389.
Sanders NM, Dunn-Meynell AA, Levin BE. 2004. Third ventricular
alloxan reversibly impairs glucose counterregulatory responses.
Diabetes 53:1230–1236.
GLIA
68
ORELLANA ET AL.
Schalper KA, Palacios-Prado N, Orellana JA, S
aez JC. 2008a. Currently
used methods for identification and characterization of hemichannels.
Cell Commun Adhes 15:207–218.
Schalper KA, Palacios-Prado N, Retamal MA, Shoji KF, Martinez AD,
S
aez JC. 2008b. Connexin hemichannel composition determines the
FGF-1-induced membrane permeability and free [Ca21]i responses.
Mol Biol Cell 19:3501–3513.
Schalper KA, S
anchez HA, Lee SC, Altenberg GA, Nathanson MH,
S
aez JC. 2010. Connexin43 hemichannels mediate the Ca21 influx
induced by extracellular alkalinization. Am J Physiol Cell Physiol
299:C1504–C1515.
Schuit FC, Huypens P, Heimberg H, Pipeleers DG. 2001. Glucose sensing
in pancreatic b-cells: A model for the study of other glucose-regulated
cells in gut, pancreas, and hypothalamus. Diabetes 50:1–11.
Siller-Jackson AJ, Burra S, Gu S, Xia X, Bonewald LF, Sprague E,
Jiang JX. 2008. Adaptation of connexin 43-hemichannel prosta-
GLIA
glandin release to mechanical loading. J Biol Chem 283:26374–
26382.
Silverman W, Locovei S, Dahl GP. 2008. Probenecid, a gout remedy,
inhibits pannexin 1 channels. Am J Physiol Cell Physiol 295:C761–
C767.
Sorimachi M, Ishibashi H, Moritoyo T, Akaike N. 2001. Excitatory
effect of ATP on acutely dissociated ventromedial hypothalamic neurons of the rat. Neuroscience 105:393–401.
Thomzig A, Wenzel M, Karschin C, Eaton MJ, Skatchkov SN, Karschin
A, Veh RW. 2001. Kir6.1 is the principal pore-forming subunit of
astrocyte but not neuronal plasma membrane K-ATP channels. Mol
Cell Neurosci 18:671–690.
Wollmann G, Acuna-Goycolea C, van den Pol AN. 2005. Direct excitation of hypocretin/orexin cells by extracellular ATP at P2X receptors.
J Neurophysiol 94:2195–2206.