metabolic inhibition increases activity of connexin-32 hemichannels permeable to ca2+ in transfected...

doi:10.1152/ajpcell.00200.2009 297:665-678, 2009. First published Jul 8, 2009;Am J Physiol Cell Physiol

Helmuth A. Sánchez, Juan A. Orellana, Vytas K. Verselis and Juan C. Sáez cells hemichannels permeable to Ca2+ in transfected HeLa Metabolic inhibition increases activity of connexin-32

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Metabolic inhibition increases activity of connexin-32 hemichannelspermeable to Ca2� in transfected HeLa cells

Helmuth A. Sanchez,1,2 Juan A. Orellana,1 Vytas K. Verselis,2 and Juan C. Saez1,3

1Departamento Ciencias Fisiologicas, Pontificia Universidad Catolica de Chile, Santiago, Chile; 2Dominick P. PurpuraDepartment of Neurosciences, Albert Einstein College of Medicine, Bronx, New York; and 3Nucleo de Inmunologıa eInmunoterapia, Santiago, Chile

Submitted 6 May 2009; accepted in final form 1 July 2009

Sanchez HA, Orellana JA, Verselis VK, Saez JC. Metabolic inhi-bition increases activity of connexin-32 hemichannels permeable to Ca2�

in transfected HeLa cells. Am J Physiol Cell Physiol 297: C665–C678,2009. First published July 8, 2009; doi:10.1152/ajpcell.00200.2009.—Numerous cell types express functional connexin (Cx) hemichannels(HCs), and membrane depolarization and/or exposure to a divalentcation-free bathing solution (DCFS) have been shown to promote HCopening. However, little is known about conditions that can promoteHC opening in the absence of strong depolarization and when extra-cellular divalent cation concentrations remain at physiological levels.Here the effects of metabolic inhibition (MI), an in vitro model ofischemia, on the activity of mouse Cx32 HCs were examined. In HeLacells stably transfected with mouse Cx32 (HeLa-Cx32), MI inducedan increase in cellular permeability to ethidium (Etd). The increase inEtd uptake was directly related to an increase in levels of Cx32 HCspresent at the cell surface. Moreover, MI increased membrane cur-rents in HeLa-Cx32 cells. Underlying these currents were channelsexhibiting a unitary conductance of �90 pS, consistent with Cx32HCs. These currents and Etd uptake were blocked by HC inhibitors.The increase in Cx32 HC activity was preceded by a rapid reductionin mitochondrial membrane potential and a rise in free intracellularCa2� concentration ([Ca2�]i). The increase in free [Ca2�]i wasprevented by HC blockade or exposure to extracellular DCFS and wasvirtually absent in parental HeLa cells. Moreover, inhibition of Cx32HCs expressed by HeLa cells in low-confluence cultures drasticallyreduced cell death induced by oxygen-glucose deprivation, which is amore physiological model of ischemia-reperfusion. Thus HC blockadecould reduce the increase in free [Ca2�]i and cell death induced byischemia-like conditions in cells expressing Cx32 HCs.

ischemia; connexons; calcium; cell death

CONNEXIN (Cx) hemichannels (HCs) are integral membraneproteins that originally were thought of only as precursors togap junction (cell-cell) channels. However, in the last severalyears, it has become clear that Cx HCs can function in theplasma membrane in the absence of docking (6, 14, 18, 21). CxHCs are hexamers of Cx subunits and constitute large ionchannels permeable to inorganic ions and signaling molecules.In the human and mouse genomes 21 and 20 Cxs, respectively,have been identified (13). Cx HCs are highly regulated, and itis becoming apparent that their opening can occur underphysiological and pathophysiological conditions (33).

Induction of cell death triggered by the opening of the CxHCs was originally observed in Xenopus laevis oocytes in-jected with rat Cx46 mRNA (34). Subsequently, it was re-

ported that Cx43 HCs opened in ventricular myocytes (23),astrocytes (8, 27), and epithelial cells of the proximal renaltubule (47) when cells were subjected to metabolic inhibition(MI). Opening of Cx HCs could contribute to the loss of ionicgradients and metabolites across the membrane, which couldaccelerate cell death. Accordingly, HeLa cells transfected with themouse Cx43 gene (HeLa-Cx43) exhibit functional Cx43 HCs andare more susceptible to cell death when subjected to MI thanparental HeLa cells (HeLa-p) (10). We have also shown (8) thattreatment with gap junction channel inhibitors blocks uptake ofthe permeability tracers Lucifer yellow and ethidium (Etd). How-ever, it is unknown whether HCs formed by Cxs other than Cx43and Cx46 act as mediators of cell death. The exogenous expres-sion of Cx32 in the C6 cell line (derived from a rat astroglioma)placed in medium with a low Ca2� concentration correlated withincreased release of extracellular ATP to the medium, suggestingthat Cx32 HCs are permeable to the nucleotide (3, 11). However,it remains unknown whether Cx32 HCs open under similarconditions.

The present study examines whether MI, a condition thatmimics an ischemic episode, affects the activity of Cx32 HCs. InHeLa-Cx32 but not HeLa-p cells, MI induced cellular permeabi-lization and was accompanied by an increase in membrane cur-rent. These MI-induced effects could be blocked with HC inhib-itors. Instances in which single-channel activity was visible re-vealed a unitary conductance of �90 pS, in agreement with thatexpected of a Cx32 HC. Activity of Cx32 HCs was directlyrelated to increased levels of Cx32 HCs present at the cell surface.The rise in HC activity was preceded by a small, but rapidincrease in intracellular Ca2� concentration ([Ca2�]i), which wasdue to influx from the extracellular medium via basally activeCx32 HCs. This rise in [Ca2�]i, in turn, increased surface expres-sion of HCs, leading to increased Ca2� influx. Oleamide, a HCblocker, considerably increased cell viability in HeLa-Cx32 underoxygen-glucose deprivation (OGD).

METHODS

Cell cultures. HeLa cell cultures transfected with mouse Cx32(HeLa-Cx32) and parental HeLa (HeLa-p) cells were kindly providedby Dr. Klaus Willecke (University of Bonn, Bonn, Germany). Previ-ously, our laboratory tested the null expression of Cx32 and other Cxsin HeLa-p cells (43). Cells were cultured in DMEM-10% fetal bovineserum (GIBCO, Invitrogen), 100 U/ml penicillin, 100 �g/ml strepto-mycin sulfate, and 1 �g/ml puromycin, the latter as a selector oftransfected cells. The cells were seeded at a density of 50,000cells/plate with 60-mm-diameter glass coverslips (no. 1, HirschmannLaborgerate, Eberstadt, Germany) and were used 24 h later to maxi-mize the number of noncontacting cells, generally reaching �5%confluence (low-confluence cultures). Alternatively, the cells wereseeded at a density of 500,000 cells per 60-mm-diameter dish with

Address for reprint requests and other correspondence: J. C. Saez, Depar-tamento de Ciencias Fisiologicas, Facultad de Ciencias Biologicas, PontificiaUniversidad Catolica de Chile, Alameda 340, Santiago, Chile (e-mail: jsaez@bio.puc.cl).

Am J Physiol Cell Physiol 297: C665–C678, 2009.First published July 8, 2009; doi:10.1152/ajpcell.00200.2009.

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glass coverslips, fed 24 h later, and used 48 h after seeding. In thiscondition, the cells generally reached 50–60% confluence (subcon-fluent cultures). Because untransfected HeLa-p cells and HeLa-p cellstransfected with the plasmid used to express Cxs showed similarresponses as described previously (39), we used untransfected HeLa-pcells as controls in our experiments. Cultures of HeLa-Cx32 at the twolevels of confluence used gave similar Cx32 levels (not shown) andthus were considered equivalent for the purposes described in thepresent work.

Metabolic inhibition and removal of oxygen and glucose. Cellcultures were subjected to MI with chemical ischemia or applicationof compounds that block the pathways of ATP production. We usediodoacetic acid (0.3 mM), an inhibitor of the enzyme glyceraldehyde-3-phosphate dehydrogenase, and antimycin A (10 ng/ml, from a 2.5mg/ml stock solution), an inhibitor of complex III of the mitochon-drial respiratory chain (8, 19, 23). Both inhibitors (Sigma, St. Louis,MO) were dissolved in saline and applied to cultures.

In the OGD model, cell cultures were kept in saline solutionwithout glucose, used in dye uptake assays (see below). Subsequently,the cultures were placed in a chamber with a controlled seal (Billups-Rothenberg, Del Mar) that had two valves communicating with theoutside. One of these introduced nitrogen to 100%, maintaining aconstant flow for 3 min. After different time periods, up to 6 h, thechamber was opened and the cultures were exposed to a salinesolution with glucose (5 mM) in the presence of ambient oxygen.Some coverslips were subjected to dye uptake assays (see below), andothers were exposed for 2 min to a saline solution with glucose (5mM) and dextran conjugated with rhodamine (Dex-Rd, mol mass 10kDa, 10 �M) and then rinsed twice carefully; the uptake incidencewas used as an index of cell death.

Immunofluorescence. The subcellular localization of Cxs was as-sessed by indirect immunofluorescence. HeLa cells seeded on glasscoverslips (no. 1) were fixed and permeabilized with 70% ethanol at�20°C for 20 min and then incubated with blocking solution [ratserum, 50% in sterile PBS plus 0.05% (wt/vol) bovine serum albumin]for 30 min. Subsequently, the samples were incubated at 4°C over-night with an appropriate dilution of primary antibody (72-F, mono-clonal anti-Cx32 antibody) dissolved in blocking solution. After sixwashes of 5 min each in PBS, the samples were incubated with asecondary fluorescein thiocyanate-conjugated antibody dissolved inblocking solution for 30 min in the dark at room temperature. After asecond round of six washes, each 5 min in PBS, the coverslips werewashed in distilled water, dried by runoff, and mounted with Fluoro-mount G or settlement (DABCO) for inspection under a OlympusBX51WI immersion microscope equipped with an image acquisitionsystem.

Dye uptake assay. Dye uptake was measured with ethidium bromide(Etd) as previously described (8, 37, 43). Briefly, coverslips with sub-confluent HeLa cell cultures were transferred to a 30-mm dish coatedwith a thin layer of Vaseline to promote adhesion. Immediately, aphosphate-free buffer solution (in mM: 140 NaCl, 5.4 KCl, 1.8 CaCl2,and 10 HEPES, pH 7.4, containing 5 �M Etd) was exchanged for theculture medium. Recordings were made with an Olympus BX51WImicroscope (Melville) equipped with an image acquisition system(Q-IMAGING 32-0086B-125 Retiga1300i, QImaging, Surrey, BC,Canada). Results were analyzed with MetaFluo (Universal Images,Molecular Devices) and transferred to a spreadsheet (Microsoft Excel)for plotting. For analysis of nonconfluent cultures, we calculated theaverage fluorescence obtained from a region of interest (ROI) whoseshape corresponded approximately to the shape of the cell. For eachvalue, the mean fluorescence obtained from a ROI located immedi-ately adjacent to the cell and the same distance from the center of thefield optical region was used as background. For analysis of subcon-fluent cultures, we calculated the average fluorescence obtained froma ROI whose shape corresponded approximately to the shape of thesubconfluent cell, but the background fluorescence was taken as theaverage fluorescence obtained from five ROIs in various locations

within the optical field that were devoid of cells. In cultures under MI,ROIs were adjusted at each time of data acquisition if they showednoticeable changes in volume.

Some of the uptake assays were conducted with DCFS (Ca2� andMg2�). To accomplish this, the corresponding salts were not includedin the stock solution, and ethylene glycol-bis(�-aminoethyl ether)-N,N,N�,N�-tetraacetic acid (EGTA, 5 mM Titriplex-VI, Merck) wasadded. In some assays, cells were preincubated for 1 h with the Ca2�

chelator BAPTA-AM (5 �M).Biotinylation of plasma membrane proteins. We used the protocol

described by Musil and Goodenough (30, 31) and modified byRetamal et al. (37) and Schalper et al. (43). Briefly, subconfluent cellcultures (90-mm-diameter dishes) were washed three times withHanks’ phosphate buffer solution (HPBS, pH 8.0) and cooled on ice.Three milliliters of HPBS containing 0.5 mg/ml sulfo-NHS SS-biotin(Pierce, Rockford, IL) was added and incubated for 30 min at 4°C.The cultures were then washed three times with HPBS containing 15mM glycine (pH 8.0) to neutralize the excess free biotin. Cells wereharvested with a scraper (rubber policeman) in the same way assamples prepared for immunotransfer (described below). After theamount of protein was determined in each sample volumes containingthe same amount of protein were taken, and to each was added anexcess of NeutrAvidin (Pierce) (1 ml of NeutrAvidin per 3 g biotin-ylated protein, according to manufacturer’s instructions). The mixturewas incubated for 1 h at 4°C. One milliliter of wash buffer was added(HPBS pH 7.2 � 0.1% SDS and 1% Nonidet P-40) and centrifugedfor 2 min at 14,000 rpm and 4°C, and the supernatant was discarded.The NeutrAvidin pellet was washed three times with the same buffer.After the last wash, 40 ml of HPBS containing 0.1 M glycine (pH 2.8)was added to the pellet to release proteins bound to biotin. The pelletwas resuspended and spun at 14,000 rpm for 2 min at 4°C. Thesupernatant was transferred to an Eppendorf tube (1.5 ml), and the pHwas adjusted by adding 10 �l of 1 M Tris buffer pH 7.5 and 40 �l ofLaemmli buffer. The relative levels of Cx32 present in each samplewere measured by immunoblotting as described below. The density ofthe bands was measured, and the results are expressed in arbitraryunits (AU). The relative levels were compared with the levels ofimmunoreactivity present in samples containing known concentra-tions of total protein derived from sister cultures.

Western blot analysis. Cell cultures were washed once with PBSsolution (pH 7.4), and the cells were harvested with a scraper in 1 mlof a mixture of protease inhibitors (200 mg/ml soybean trypsininhibitor, 1 mg/ml benzamidine, 1 mg/ml ε-aminocaproic acid, and 2mM PMSF) and phosphatases (20 mM Na4P2O7 and 100 mM NaF).The suspension was centrifuged at 5,000 rpm for 1 min at 4°C. The pelletwas resuspended in 50 ml of solution containing protease inhibitors andsonicated on ice (Ultrasonic disrupted cell, MICROSON). Protein levelswere measured in aliquots of cell lysates with the Bio-Rad assay(Bio-Rad Laboratories, Hercules, CA). The samples were resuspendedin Laemmli buffer and immediately loaded onto a 8% acrylamide gel.After electrophoresis, proteins were electrotransferred to a nitrocellu-lose membrane. The amount of protein loaded per wheal was checkedagain by staining the blots with Ponceau red, followed by washes withdistilled water. Nonspecific protein binding was blocked by incuba-tion of the nitrocellulose membrane in PBS-BLOTTO (5% skim milkin PBS solution) for 40 min. The membrane was then incubatedovernight with the primary antibody previously described (Ref. 39,monoclonal anti-Cx32 antibody 72-F), followed by several washeswith PBS, and incubated with antibody conjugated with horseradishperoxidase (Pierce). The immuno-enhanced chemiluminescence wasdetected with the SuperSignal kit (Pierce) according to manufacturer’sinstructions. The resulting immunoblots were scanned, and densito-metric analyses were performed with Scion IMAGE software.

Estimate of intracellular free Ca2� concentration. We estimatedthe free [Ca2�]i with the Ca2� indicator fura-2 (Molecular Probes,Eugene, OR) as described by Schalper et al. (43). In brief, cells wereincubated in DMEM (serum free) culture medium containing 5 �M of

C666 ISCHEMIA AND CONNEXONS PERMEABLE TO Ca2�

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the membrane-permeant AM ester form of fura-2 (fura-2 AM) dis-solved in DMSO for 1 h. The cells were subsequently washedthoroughly with buffer and imaged. Fluorescence was measured atexcitation wavelengths of 340 and 380 nm. The emission wavelengthwas 510 nm. Images were acquired at a rate of 1/min and analyzedwith MetaFluo software (Universal Images, Molecular Devices).Ca2� was estimated from the ratio of fluorescence (340/380) asdescribed previously (43).

Estimate of mitochondrial membrane potential. We used a modi-fication of the method described by Ankarcrona et al. (1) using theprobe JC-1 (5,5�,6,6�-tetrachloro-l,l�,3,3�-tetraethylbenzimidazolcar-bocyanine iodide, Molecular Probes). Cells were incubated with 0.5mg/ml JC-1 for 10 min. The cells were washed thoroughly with bufferand imaged. JC-1 is a liposoluble cation that enters the cell, where itincorporates into organelles that exhibit negative membrane poten-tials. JC-1 was detected in two forms. The multimeric form is specificto mitochondria because of the hyperpolarized potential of the mito-chondrial matrix (�m) and detected at a wavelength of 590 nm (480nm excitation). A monomer form is not associated with mitochondriaand is detected at 527 nm (480 nm excitation). The 590-to-527fluorescence ratio is directly proportional to �m and can be used as anindex of �m (1, 22, 26). Alternating images of JC-1 and fura-2fluorescence were acquired in the same cells at a rate of one imageevery 5 min.

Electrophysiology. To examine opening of HCs in nonconfluentcells electrophysiologically, whole cell patch recordings were em-

ployed (3, 23), as modified by Contreras et al. (9). Nonconfluentcultures were used to record isolated cells. Briefly, cells were grownon coverslips, which were transferred to a recording chamber (SA-0LY/2, Warner) mounted on an Olympus IX70 inverted microscope(Melville). The chamber was superfused with a modified Krebs-Ringer solution (in mM: 140 NaCl, 1 MgCl2, 5.4 KCl, 1.8 CaCl2 and10 HEPES, pH 7.3). The pipette solution contained (in mM) 10 Naaspartate, 1 MgCl2, 130 KCl, 0.26 CaCl2, 2 EGTA, 5 TEA Cl, and 5HEPES, pH 7.3. In MI experiments, KCl was replaced by an equimo-lar amount of CsCl in the extracellular solution, and 2 mM BaCl2 wasadded to suppress Ca2�-activated K� currents. The intracellularsolution also was changed to (in mM) 130 CsCl, 10 Na aspartate, 0.1EGTA, 7 TEA Cl, 1 MgCl2, and 10 HEPES, pH 7.3. Recordings wereobtained with an Axopatch 200B amplifier (Axon Instruments) andacquired with an AT-MIO-16X board (National Instruments, Austin,TX) and our own acquisition software (written by Dr. E. B. Trexler).

Statistical analysis. For each group of data, the results are ex-pressed as means SE, and n is the number of independent experi-ments or the number of cells, as indicated. For statistical analysis oftwo data sets, we used a t-test. Three or more data sets were comparedwith each other by one-way analysis of variance (ANOVA) followedby a Newman-Keuls posttest. To compare data sets obtained fromdifferent groups of cells (HeLa-p vs. HeLa-Cx32) and under the samecondition (control or MI), we used a one-way ANOVA followed by aNewman-Keuls posttest. Differences were considered significant at

Fig. 1. Metabolic inhibition enhances ethidium (Etd) uptake in HeLa cells transfected with connexin32 (HeLa-Cx32) but not in HeLa-parental (HeLa-p) cells.Fluorescent and corresponding bright-field images of cells exposed to 100 �M Etd for 2 min are shown. A: HeLa-p and HeLa-Cx32 cells under control conditions(Ctrl) or after 60 min of metabolic inhibition (MI-60min). B: representative plot of intensity of Etd fluorescence [in arbitrary units (AU)] over time. At the startof the experiment, 5 �M Etd was added to the bath. At �20 min, cells were exposed to MI, and at �70 min 100 �M La3� was applied. Each point is theaverage SE fluorescence of 9 cells. Gray lines correspond to extrapolations of the linear regressions computed for each condition; m, rate of Etd uptake. Cand D: rates of Etd uptake (5 �M) measured in low-confluence and subconfluent cultures of HeLa-p and HeLa-Cx32, respectively. Shown are rates of Etd uptakeunder control conditions (CTRL) or under MI, MI � 100 �M La3� (La3�), or MI � 200 �M oleamide (OLE). Number of experiments is indicated in eachcolumn. Each experiment monitored fluorescence in 15 (C) and 25 (D) cells. Each bar indicates mean SE. *P � 0.05, **P � 0.01, ***P � 0.001, 1-wayANOVA with Newman-Keuls posttest.

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P � 0.05. The analyses were performed with GraphPad Prism 5software for Windows (1992–2007, GraphPad Software).

RESULTS

Metabolic inhibition induces permeabilization of Cx32 butnot parental HeLa cells. MI has been used as a simple modelof ischemia by several groups, including ours (8, 23, 37, 47).Although MI occurs in normoxia, it shares several character-istics with hypoxia-reoxygenation, including the decline inATP levels and the generation of free radicals (8, 37). In the

time period analyzed (�90 min) in this study, MI did not affectthe permeability of HeLa-p cells to Etd (Fig. 1). Basal uptakeof Etd in HeLa-Cx32 and HeLa-p cells was similar, but MIinduced chemically with iodoacetic acid and antimycin A (seeMETHODS) increased Etd uptake (�100%) only in HeLa-Cx32cells. In HeLa-p cells, Etd uptake was not affected by La3�

(not shown), whereas in HeLa-Cx32 cells under MI, uptakewas reduced by La3� to values comparable to those of HeLa-Cx32 under basal conditions treated with La3� (Fig. 1, B–D).In low-confluence or subconfluent cultures, the increase in the

Fig. 2. Metabolic inhibition increases the number ofhemichannels (HCs) at the surface of HeLa-Cx32 cells.HeLa-Cx32 cells were exposed to MI for �65 min inthe presence of 10 �M Etd, and images were taken toevaluate dye uptake rate. The cells were then fixed andprocessed for Cx32 detection by immunofluorescence.A, left: Etd uptake induced by MI. Right: Cx32 immu-noreactivity (Cx32-IR) in the same field shown on left.B: time lapse record showing Etd uptake over time inthe cells (1–7) indicated in A. C: correlation analysis ofrate of Etd uptake and level of Cx32-IR in cells underMI. Each point corresponds to Etd uptake and immu-nofluorescence measured in a single cell in a total of 3separate experiments. D, left: immunoblot of Cx32present in an aliquot of total rat liver homogenate (L; 50�g of proteins) and at the cellular surface of subconflu-ent HeLa-Cx32 cells under control conditions (Ctrl) orexposed to MI for 15 or 60 min. In the last 3 lanes,surface proteins were biotinylated, precipitated, andsubjected to immunoblot for Cx32. Right: samples oftotal HeLa-p homogenate and serial dilutions of totalHeLa-Cx32 cell homogenates were analyzed by immu-noblotting. L, aliquot of total liver homogenate used asa marker of the electrophoretic migration of the Cx32monomer (27 kDa); dim indicates the mobility of theCx32 dimer in the lanes loaded with a total liverhomogenate aliquot for both panels. E: estimatedamount of Cx32-forming HCs expressed as % of totalamount of Cx32. Regression line was obtained fromknown quantities of proteins indicated in D. Percentagesof Cx32-forming HCs under control conditions (Ctrl) orunder MI for 60 min (MI-60) are indicated by dottedlines. F: results from 4 separate experiments. Eachvalue is the average SE. **P � 0.01, t-test.

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Etd uptake induced by MI was similar and completely blockedby application of La3� (100 �M) or oleamide (200 �M) (Fig.1, C and D), which are known HC blockers (8, 23, 25),suggesting that the increase in Etd uptake was mediated byCx32 HCs. In this study, Etd uptake that was resistant to La3�

was considered to be mediated by a process unrelated to CxHCs; we did not undertake studies to identify this process.

Increase in membrane permeability induced by MI is di-rectly related to expression of Cx32 and levels of Cx32 HCs atcell surface. The increase in Etd uptake induced by MI corre-lated well (r2 0.813) with an increase in overall Cx32immunoreactivity detected by immunofluorescence, suggestingthat MI may increase the number of HCs in the plasmamembrane (Fig. 2, A–C). To demonstrate biochemically thatHeLa-Cx32 cells contain Cx32 protein in the plasma mem-brane, we biotinylated cell surface proteins, followed by pre-cipitation and immunoblotting. We found that HeLa-Cx32cells contain Cx32 on the surface, that levels increased approx-imately twofold during the first 15 min of exposure to MI (Fig.2D), and that increased levels remained high for 45–60 min,paralleling the magnitude and time course of the increase inEtd uptake (Fig. 2, E and F). Furthermore, by comparing the

levels of biotinylated protein with the total amount of protein,we found that �4% and �8% of Cx32 form HCs in resting andmetabolically inhibited cells, respectively (Fig. 2E).

Inhibition of P2x or TRPV1 channels does not preventMI-induced permeabilization of HeLa-Cx32 cells. During ische-mia, mechanisms that lead to cell depolarization and cell deathare activated, including the activation of purinergic P2x recep-tors (16) and transient receptor potential (TRP)V channels(28). Therefore, it has been suggested that these other channelscould mediate the uptake of fluorescent tracers (2, 32, 45).Thus MI experiments were conducted in the presence of 300�M oxidized ATP (oATP), a P2x channel blocker, or 10 �Mcapsazepine, a TRPV1 channel blocker. We found that neithercompound applied �10 min after the addition of the metabolicinhibitors could reverse the increase in Etd uptake (Fig. 3),indicating that P2x and TRPV1 channels do not mediate theincrease in Etd uptake induced by MI.

MI increases macroscopic membrane current but does notaffect unitary conductance of Cx32 HCs. Gomez-Hernandezet al. (17) recorded unitary currents of Cx32 HCs expressed inXenopus oocytes, which in the absence of added extracellulardivalent cations exhibited a unitary conductance of �87 pS. To

Fig. 3. Etd uptake induced by MI is not blocked by either oxidized ATP or capsazepine in HeLa-Cx32 cells. A and C: representative plots obtained from timelapse experiments of Etd uptake in HeLa-Cx32 cells. Cells were maintained under control conditions for the first 15 min and then treated to induce MI. OxidizedATP (oATP, 300 �M; A) or capsazepine (CPZ, 10 �M; C) was added �20 min after initiation of MI. Each point corresponds to the average SE of 16 (A)and 9 (C) cells. B and D: mean rates of Etd uptake obtained from experiments illustrated in A and C, respectively (n 3 experiments). *P � 0.05, **P � 0.01,1-way ANOVA with Newman-Keuls posttest.

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assess Cx32 HC activity during MI, whole cell patch record-ings were obtained in HeLa cells. A larger macroscopic current(�2-fold) was present in HeLa-Cx32 cells compared withHeLa-p cells, particularly at positive membrane potentials (Fig. 4,A and B). Currents were larger still when cells were exposed toMI. Unitary current events were only occasionally observednear the resting membrane potential but were more evident ondepolarization to positive potentials. Current transitions had amean value of 95 21 pS (n 219 events) under controlconditions and 95 20 pS (n 75 events) under MI (Fig. 4Band Fig. 5). These conductance values were independent of theapplied voltage indicative of a lack of significant open channelrectification. We also applied voltage ramps from �80 to �80

mV (8 s) and found transitions of 98 21 pS (n 103 events)under control conditions and 95 18 pS (n 158 events)under MI. Both macroscopic currents (Fig. 4, C and D) and theassociated unitary transitions (not shown) were inhibited by me-floquine or La3� under control and MI conditions, indicating thatthere is basal Cx32 HC activity that increases during MI.

Both reduction in �m and rise in [Ca2�]i precede increasein membrane permeability of HeLa-Cx32 cells under MI. Innormal cells [Ca2�]i is closely regulated, and mitochondrialdysfunction has been attributed as an important cause of therise in [Ca2�]i observed in ischemia-reperfusion (15). Thus wemonitored �m and found that MI induced a rapid fall in �m

that was similar in HeLa-p and HeLa-Cx32 cells, indicating

Fig. 4. HeLa-Cx32 cells exhibit macroscopic currents attributable to Cx32 HCs. Low-confluence cell cultures were used to record from single cells. Currentswere obtained with whole cell patch-clamp recordings under control conditions (CTRL, black lines) and after exposure to MI (gray lines). Examples of membranecurrents (I) recorded in HeLa-p cells (A) and in HeLa-Cx32 cells (B) are shown. The voltage (V) protocol (shown above current records) consisted of rampsapplied from �80 to �80 mV, preceded by a �20 mV prepulse. HeLa-Cx32 cells showed larger currents after MI, particularly at positive voltages. C and D: HCblockers reduce currents attributable to Cx32 HCs under control conditions and MI. Current responses were evoked by applying voltage ramps as described inA and B. Shown is an example with mefloquine (MFQ, 15 �M), which reduced currents in a control HeLa-Cx32 cell (C, gray line), and La3� (100 �M), whichcompletely abolished the large increase in current observed after 60 min under MI (D, gray line).

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that MI-induced changes in �m are independent of Cx expres-sion (Fig. 6A). However, in HeLa-Cx32 cells, the fall in �m wasaccompanied by a concomitant increase in [Ca2�]i (Fig. 6B). Toassess the possible relationship between the increase in [Ca2�]i

and HC activity during MI, Etd uptake and changes in [Ca2�]i

were measured simultaneously. The increase in [Ca2�]i during MIin HeLa-Cx32 cells was found to precede the increase in Etduptake by �10 min (Fig. 6C), suggesting that HCs are present atthe cell surface before the increase in Etd uptake (Figs. 1 and 6)and the increase in HC levels at the cell surface (Fig. 2) weresufficient to produce an increase in [Ca2�]i of �50%.

MI elevates [Ca2�]i of HeLa cells in a Cx32-dependentmanner. It has been suggested that [Ca2�]i is a key factor thatcontrols the levels of Cx43 HCs in HeLa cells (43). As

previously indicated, a rapid increase in [Ca2�]i occurred afterapplication of metabolic inhibitors (Fig. 6, B and C). Theincrease in [Ca2�]i did not occur in HeLa-p cells, indicating thatthe rise in [Ca2�]i was linked to Cx32 expression (Fig. 7A). Toidentify the source of Ca2� entry, HC blockers La3� and18�-glycyrrhetinic acid (BGA) were added 10 min beforeinitiation of MI in HeLa-Cx32 cells. Both La3� and BGAprevented the increase in [Ca2�]i (Fig. 7B), suggesting thatCa2� enters the cells from the extracellular medium throughbasally active Cx32 HCs present in the surface membrane.

MI-induced increase in membrane permeability of HeLa-Cx32 cells requires rise in [Ca2�]i. To ascertain whether theincrease in Etd uptake required an increase in [Ca2�]i, cellswere preloaded with BAPTA-AM, a chelator of intracellular

Fig. 5. MI enhances the activity but not the unitary conductance of Cx32-HCs. Membrane currents were measured in a whole cell voltage-clamp recordingconfiguration using low-density cultures of HeLa-Cx32 cells under control conditions (CTRL) or under MI. Recordings were obtained �40 min after transferof cells on coverslips from culture medium to the recording extracellular solution. A: representative current traces elicited by 10-s voltages steps from �80 to�80 mV, in 20-mV increments. B: views of unitary current events recorded at �60 and �60 mV taken from the current trace shown in A under control conditionsand MI, respectively. Despite a high level of background noise in the whole cell configuration, unitary current transitions were identifiable and were convertedon a point-by-point basis to conductance values (C) as indicated in the expanded region of the records. Unitary conductances of transitions recorded under controlor MI conditions were �90 pS. Open (O) and closed (C) hemichannel states are indicated.

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free Ca2�. In the presence of BAPTA, the increase in Etduptake induced by MI was completely abolished (Fig. 8A).Moreover, about two-thirds of the remaining Etd uptake wassensitive to La3�, suggesting that most of it was mediated bybasally active Cx32 HCs (Fig. 8A). To assess whether anincrease in [Ca2�]i is sufficient to enhance Etd uptake, HeLa-Cx32 were treated with 5 �M 4Br-A-23187, a Ca2� ionophore,and Etd uptake was evaluated over time. After an �10-min lagperiod, Etd uptake increased and remained high throughout the�50-min recording. Subsequent application of 100 �M La3�

reduced the rate of Etd uptake to �0.1 AU/min (Fig. 8B).Notably, this value was comparable to that measured undercontrol conditions in HeLa-p or HeLa-Cx32 cells (compareFig. 8C with Fig. 1, C or D) or under MI in HeLa-Cx32 cellspreloaded with BAPTA, suggesting that the increase in mem-brane permeability observed after 4Br-A-23187 was mediatedby Cx32 HCs.

It should be noted that cells treated only with DMSO, thevehicle used to dissolve BAPTA-AM and 4Br-A-23187, showeda higher baseline uptake than cells under control conditions [0.4AU/min (Fig. 8A) compared with 0.2 AU/min (Fig. 1, C and D,and Fig. 3, B and D)]. This could be explained by the fact thatDMSO has been shown to increase [Ca2�]i (42), which couldincrease the activity of HCs without inducing MI. However, onexposure to MI an even greater increase in uptake occurred,concomitant with a larger increase in [Ca2�]i (Fig. 8A).

Increase in membrane permeability induced by MI can bepotentiated by low extracellular concentration of divalentcations. Exposure to DCFS has been shown to increase theactivity of Cx32 HCs (17). To assess whether Cx32 HCs ofcells under MI are sensitive to extracellular divalent cations,Etd uptake was measured in HeLa-Cx32 cells under MI andwas followed by exposure to a DCFS. After �10 min of MI,Etd uptake increased approximately twofold. Subsequent ex-posure to a DCFS produced a further increase of �40% thatoccurred rapidly, with little delay (Fig. 9, A and B). Thedifferent time courses of the these responses suggests that MIincreases HC activity by increasing the levels of HCs in the cellsurface, whereas removal of divalent cations increases HCactivity by a different mechanism, most likely by enhancingthe open probability of existing HCs, as suggested by Gomez-Hernandez et al. (17). When we reversed the order of these twotreatments, i.e., removed divalent cations first and then exposedcells to MI while maintaining low divalent cations, we ob-served that Etd uptake rose rapidly on removal of extracellulardivalent cations, but subsequent exposure to MI did not pro-duce a further increase in Etd uptake (Fig. 9, C and D).Treatment with La3� reduced Etd uptake induced by either MIfollowed by DCFS or DCFS followed by MI, indicating thatunder both conditions Etd uptake was mediated by Cx32 HCs(Fig. 9). When DCFS followed MI, the lack of a significant risein [Ca2�]i was consistent with removal of the source of Ca2�,namely, the extracellular solution. Thus, the lack of effect ofMI on the Etd uptake in cells bathed in DCFS (Fig. 9D) can beexplained by the absence of Ca2� influx that is required toinduce Cx32 HC activity when cells are exposed to MI (Fig.9D). As can be seen in Fig. 9, C and D, MI exposure followingDCFS actually caused a small decrease in Etd uptake. Anti-mycin A, which we used as a metabolic inhibitor in thesestudies, has been proposed to be a gap junction blocker (27,36). However, acute application of antimycin A, at the con-

Fig. 6. MI increases intracellular Ca2� concentration ([Ca2�]i), which isinversely related to the decrease of mitochondrial membrane potential (�m)and precedes the increase in membrane permeability to Etd. A: representativeplots of the fluorescence ratio (590 nm/527 nm) with the probe JC-1 obtainedfrom HeLa-p and HeLa-Cx32 cells. This ratio reflects �m; a decrease in thisratio indicates a fall in �m. Dashed vertical line indicates the start of MI.B: time-lapse experiment showing changes in JC-1 and fura-2 fluorescenceratios to simultaneously monitor changes in �m and [Ca2�]i over time. Cellswere maintained under control conditions for 20 min and then exposed to MI.Total recording time was 80 min. Dashed vertical line indicates the start of MI.C: time-lapse experiment showing Etd uptake simultaneously with changes in[Ca2�]i. As in B, MI was applied after 20 min in control conditions. Firstdashed vertical line indicates the start of MI, and second dashed vertical lineindicates the time at which there was a change in the rate of Etd uptake afterMI. Solid gray line is an extrapolation of the basal rate of Etd uptake undercontrol conditions. Data points in B and C represent average SE fluorescenceratios obtained from 10 cells.

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centration used to induce MI in this study, caused only a smallinhibition of Cx32 HCs evidenced by a small reduction in Etduptake in HeLa-Cx32 cells that was not statistically significant(Supplemental Fig. S1).1 However, the tendency to inhibitCx32 HCs might explain the smaller than expected (�1.4-fold)increase in Etd uptake caused by DCFS in cells under MI (Fig.9B) compared with the �2.8-fold increase induced undercontrol conditions (Fig. 9D).

Inhibition of Cx32 HCs reduces susceptibility of HeLa-Cx32cells exposed to oxygen-glucose deprivation. In HeLa-Cx43and rat cortical astrocytes, death induced by MI was shown tobe accelerated by enhanced Cx43 HC activity (8, 10). More-over, OGD was shown to increase the membrane permeabilityof endothelial cells as well as cortical astrocytes, which expressCx43 HCs (33). We found that exposure to 6-h OGD followedby 1 h of reoxygenation caused a increase in the rate of Etduptake in cultures of HeLa-Cx32 but not HeLa-p cells [0.47 0.04 (n 6) in HeLa-Cx32 vs. 0.21 (n 1) in HeLa-p; eachis average of Etd uptake rate for 15 cells; data not shown].Therefore, we tested whether OGD followed by reoxygenationin glucose-containing saline induced death of HeLa-Cx32cells. Exposure to 6-h hypoxia followed by 0-h reoxygenationincreased the number of dead cells in HeLa-Cx32 but notHeLa-p cultures (Fig. 10). The increase in cell death was evenmore pronounced after 4-h reoxygenation, at which time thepercentage of dead cells was �40% in HeLa-Cx32 and �12%in HeLa-p cultures, suggesting that Cx32 HCs reduce cellviability. Four hours of reoxygenation drastically reduced theviability of HeLa-Cx32 cells but caused only a small increasein cell death in cultures of HeLa-p cells (Fig. 10). Moreover,oleamide applied 2 h before reoxygenation completely pre-vented the increase in cell death observed in HeLa-Cx32cultures but did not significantly affect cell viability in HeLa-pcultures (Fig. 10).

DISCUSSION

In this work we show that MI, a simple model for in vitroischemia-reperfusion, increases the activity of Cx32 HCs. Inaddition, we found that Cx32 HCs appear to be permeable toCa2� and that inhibitors of Cx32 HCs enhance the viability ofcells subjected to OGD. Therefore, we suggest that ischemia-like conditions increase the activity of Cx32 HCs, which serveas a pathway for Ca2� entry, thereby contributing to acceler-ation of cell death.

Under basal conditions, which includes physiological con-centrations of extracellular divalent cations, Cx32 HCs ap-peared to remain predominantly closed because the rate of Etduptake was low and not significantly reduced by La3�. Insupport of this, we rarely observed unitary events with con-ductance values corresponding to Cx32 HCs, particularly athyperpolarized resting membrane potentials. Similar resultswere reported for Cx32 HCs comprised of human Cx32, whichis highly homologous to mouse Cx32 (17). When we exposedHeLa-Cx32 cells to MI, cellular permeabilization mediated byCx32 HCs increased despite the presence of extracellulardivalent cations. This conclusion is supported by the followingfindings: 1) MI did not permeabilize HeLa-p cells, 2) thecellular levels of Cx32 protein and rate of Etd uptake in cellsunder MI showed a close positive correlation, 3) the MI-induced increase in Etd uptake was insensitive to blockers ofP2x and TRPV1 channels, two other putative transmembranepathways, 4) under MI, large unitary events consistent withthose of Cx32 HCs were recorded in HeLa-Cx32 but notHeLa-p cells, 5) the rise in Cx32 levels at the cell surface(�2-fold) was similar to the rise in Etd uptake and the increasein macroscopic membrane currents, and 6) the rise in free[Ca2�]i occurred in HeLa-Cx32 but not HeLa-p cells and wassensitive to HC blockers. Although macroscopic currents inHeLa-Cx32 cells under MI tended to increase on depolariza-tion, consistent with opening of Cx32 HC, we often observedthat macroscopic currents at hyperpolarized membrane poten-1 The online version of this article contains supplemental material.

Fig. 7. Increase in [Ca2�]i occurs with metabolic inhibition in HeLa-Cx32 but not HeLa-p cells and is prevented by HC blockers. A: representative plots ofrelative changes in [Ca2�]i over time during MI in HeLa-p cells (n 11) and HeLa-Cx32 cells (n 15). B: time course of relative change in [Ca2�]i inHeLa-Cx32 cells exposed to MI in the absence or presence of blockers. MI was induced after 20 min. HC blockers La3� (100 �M) and 18�-glycyrrhetinic acid(BGA, 35 �M) were applied 10 min before induction of MI and maintained during the experiment. Each point represents the mean SE of 8 (CTRL�MI),10 (CTRL�La3��MI), or 15 (CTRL�BGA�MI) cells.

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tials were increased compared with control HeLa-p cells (spe-cifically during ramp protocols; Fig. 4D). These were alwaysinhibited by La3�, whereas control cells displaying comparableleak currents were not. Thus, although Cx32 HCs tend to openwith depolarization, they do function at negative voltages.

The amount of Cx32-forming HCs was �4% in cells underresting condition and was �8% after 60 min of MI. In ratcortical astrocytes, which express only Cx43 HCs, the amountof surface HCs was also reported to be low (�15%) undercontrol conditions and during MI increased to about twofold(37). The increase in Cx32 HC levels in the plasma membranedetected by biotinylation of surface proteins could have re-sulted from a reduced rate of internalization and/or enhancedinsertion. Further studies will be required to elucidate whichmechanism(s) is at play. The increase in Cx32 HC levels at thecell surface alone can account for the observed proportionalincrease in Etd uptake and in membrane currents, assuming nodifference in the properties of the individual HCs under controland MI conditions. It is possible that the conductance andpermeability properties of Cx32 HCs could have changed,perhaps as a consequence of posttranslational modification ofCx32-forming HCs during MI. However, this possibility isunlikely, given that the unitary conductance of Cx32 HCs incells under MI was identical to that in cells under controlconditions (�90 pS). Moreover, the presence of unitary eventscharacteristic of Cx32 HCs and their sensitivity to La3�, whichdoes not block Pnx1 HCs (35), further supports our contentionthat Etd uptake was mediated by Cx32 HCs. We did observethat the increase in the rate of Etd uptake of HeLa-Cx32 cellsexposed to extracellular DCFS under MI was lower (1.4-fold)than the 2.8-fold increase observed when HeLa-Cx32 cellswere exposed to DCFS without MI. Although this inconsis-tency might be the result of differences in Cx HCs undercontrol conditions and under MI, it could also be the result ofa nonlinear relationship between levels of surface HCs and thefraction of HCs sensitive to DCFS. There is also an inhibitoryeffect of the metabolic inhibitor used, antimycin A. Nonethe-less, further studies are needed to explain this finding.

During MI, we observed a decrease in the mitochondrialpotential �m, which could explain the sharp decline in intra-cellular ATP levels in cells under MI (8). It has been proposedthat dysfunctional mitochondria release Ca2� (48). Thus MIinduced by antimycin A, which blocks the respiratory chain,could produce a rise in [Ca2�]i via mechanisms related tomitochondrial dysfunction. Accordingly, we found that thedecrease in �m was accompanied by an increase in [Ca2�]i.Additionally, the reduction in ATP levels, which could drasti-cally reduce Ca2� pump activity, could also contribute to thenet rise in [Ca2�]i (46, 48). The lack of ATP can also inhibitthe exchange of Na� and K� through ATPase, which can leadto cell depolarization. Therefore, activation of voltage-depen-dent Ca2� channels might occur, and they may serve as apathway for Ca2� influx from the extracellular medium. How-ever, we found that [Ca2�]i increased in HeLa-Cx32 cells, butnot HeLa-p cells, and that the increase was prevented byinhibition of Cx32 HCs. Thus, although mechanisms related toCa2� extrusion from the cell interior and release from intra-cellular stores may contribute to the sustained rise in [Ca2�]i

[as suggested by the very small increase in [Ca2�]i induced byMI in HeLa-p (Fig. 7A) or in HeLa Cx32 under MI in theabsence of external Ca2� (Fig. 9B)], our findings indicate that

Fig. 8. BAPTA-AM, a chelator of intracellular free Ca2�, prevents theincrease in Etd uptake induced by MI, whereas 4Br-A-23187, a Ca2� iono-phore, increases Etd uptake in HeLa-Cx32 cells. A: rates of Etd uptakemeasured in subconfluent HeLa-Cx32 cell cultures preincubated for 1 h withDMSO [0.01% (vol/vol), Pre-veh] or with BAPTA-AM (5 �M, Pre-BAPTA-AM). Each bar is the average SE of 5 experiments. In each experiment, 20cells were analyzed. At the end of each experiment 100 �M La3� (MI�La3�)was added. *P � 0.05, **P � 0.01, ***P � 0.001, 1-way ANOVA withNewman-Keuls posttest. B: representative plot of Etd uptake over time ob-tained from HeLa-Cx32 cells in a subconfluent culture. Cells were exposed to4-Br-A-23187 (5 �M) (indicated by vertical dashed line) and then to La3�

(100 �M) as indicated. C: rates of Etd uptake; each bar is the average SEof 10 cells. ***P � 0.001, 1-way ANOVA with Newman-Keuls posttest.

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in HeLa-Cx32 cells under MI the main source of the increasein [Ca2�]i is Ca2� influx through HCs. Under resting condi-tions Ca2� influx is low, consistent with a low level of Cx32HC activity and because the ATP-dependent extrusion mech-anisms are functioning fully. Under MI, Ca2� influx is in-creased because of an increase in HC activity and possiblybecause the extrusion mechanisms become partly compro-mised. During MI, we observed that [Ca2�]i reached a newsteady state in �20 min, which might be explained by theinvolvement of Ca2� binding sites (e.g., proteins and othermolecules) with lower affinities compared with those operatingunder normal conditions and the possible existence of a lowerCa2� level of extrusion activity maintained by the lower ATPlevels. Clearly, in other cell types under MI additional mem-brane channels, including TRP channels, P2x, and NMDAreceptors, could contribute to the Ca2� influx (4, 12, 28, 41).

However, in the HeLa cells used in this study, Cx32 HCs arethe main route of Ca2� entry. It is important to emphasize thatlevels of Cx32 HCs rise in the first 15 min of MI (Fig. 2D), just5–10 min before the establishment of a new steady state of[Ca2�]i (Fig. 6C and 7), suggesting that Ca2� influx into thecell is mediated by increasing HC numbers. After a criticaltime, the amount of Cx32 HCs is enough to induce a visiblechange in the rate of Etd uptake (Fig. 6C). Experiments withhigher time resolution are needed to clarify this point. Cx32HCs also might play a relevant role in several cell types thatexpress Cx32 endogenously such as oligodendrocytes, hepato-cytes, and lacrimal cells (40).

What could be the mechanism by which elevated [Ca2�]i

increases Cx32 HC activity? One possibility involves proteinkinases. It was reported that the activity of p38 MAP kinase isnecessary for increased activity of Cx43 HCs induced by proin-

Fig. 9. MI induces Ca2� entry from the extracellular space but does not induce opening of all Cx32 HCs. A: plots of Etd uptake and changes in [Ca2�]i

simultaneously in response to MI followed by exposure to a divalent cation-free solution (DCFS) under MI. La3� (100 �M) was applied after exposure to DCFS.B: plots of Etd uptake and changes in [Ca2�]i simultaneously in response to DCFS followed by exposure to MI in DFCS. La3� (100 �M) was applied afterexposure to MI. C: average rates of Etd uptake obtained under MI alone, MI followed by DCFS, and after addition of La3�. Each bar represents the average SE of 8 experiments. D: average rates of Etd uptake rates obtained on exposure to DCFS alone, DCFS followed by MI, and after addition of La3�. Each barrepresents the average SE of 10 experiments. Each experiment in B and D includes analysis of 15 cells. *P � 0.05, **P � 0.01, 1-way ANOVA withNewman-Keuls posttest. &P � 0.05 with respect to control.

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flammatory cytokines (38) and by FGF-1 or 4Br-A-23187 (43). Inthis work, preincubation with SB-203519 (an inhibitor of the p38MAP kinase) partially prevented the increase in Etd uptakeinduced by MI (Supplemental Fig. S2), indicating that despite afall in ATP levels during MI, p38 MAP kinase activity is neededto induce increased activity of Cx32 HCs, possibly by a Ca2�-dependent pathway (11, 43). The activity of p38 MAP kinasecould be increased by [Ca2�]i increase (29) or as consequence ofoxidant stress (20), which also occurs in cells under metabolicinhibition (37). It remains unknown whether p38 MAP kinaseinduces covalent changes in Cx32-forming HCs or whether itaffects the vesicle trafficking pathway leading to an increase in thenumber of Cx32 HCs at the cell surface.

Cx32 forms poorly selective channels permeable to nega-tively charged molecules such as ATP (net charge: �3; Refs.3, 7) as well as positively charged molecules, such as Etd (netcharge: �1) and propidium (net charge: �2, Ref. 11). Thus itis conceivable that Cx32 HCs could be permeable to divalentcations such as Ca2�. Although extracellular Ca2� regulatesCx32 HCs (17), the sensitivity is such that substantial HCactivity can remain at physiological Ca2� concentrations,thereby allowing for Ca2� flux.

Agents that elevate [Ca2�]i have been proposed to favorATP release to the extracellular environment through Cx32HCs (11). This increase in ATP release might result from therise in levels of surface Cx32 HCs and/or changes in biophys-

ical properties of Cx32 HCs leading to higher ATP permeabil-ity. Here we show that BAPTA-AM, an intracellular Ca2�

chelator, drastically reduced MI-induced Etd uptake. This isconsistent with the previously proposed importance of thissecond messenger in regulating activity of Cx32 HCs (11).Recently, it was shown that HeLa cells transfected with Cx43or Cx45 show an increase in the levels of HCs in the cellsurface after treatment with a Ca2� ionophore (43). A similarmechanism might explain the increase in activity of Cx32 HCs(evaluated with Etd uptake) in response to 4Br-A-23187 sincethe response was similar to that induced by MI, where levels ofCx32 HCs increased about twofold.

What other factors could increase the activity of HCs duringischemia? As mentioned above, the levels of intracellular ATPdrop sharply, and this occurs during the first 15 min of MI (8,10), which is before a rise in Etd uptake takes place. ATP alsodrops during ischemia in various tissues, producing a lack ofsubstrate for protein kinases. Protein subunits of HCs includingCx32 and Cx43 are phosphoproteins (39, 44), and the openprobability of at least Cx43 HCs increases on dephosphoryla-tion (5, 24). A similar mechanism might affect Cx32 HCs, butfurther studies will be required for its demonstration.

The protective effect of oleamide on the survival of cellsexposed to OGD followed by reoxygenation reveals an impor-tant role of HCs in the process of cell death. Cell death isinduced by the insult (OGD and reoxygenation), and an in-

Fig. 10. Necrosis induced by oxygen-glucose depri-vation (OGD) in HeLa-Cx32 cells is prevented byoleamide. A: cultures of HeLa-p or HeLa-Cx32 cellswere subjected to 6 h of OGD in the absence orpresence of oleamide (Ole, 200 �M) applied 2 hbefore restoration of oxygen and glucose. Subse-quently, cultures were exposed to solutions withglucose in the presence of ambient oxygen (OG) andincubated with dextran conjugated with rhodamine(Dex-Rd, 10 �M) for 5 min at time zero (0 h) or 4 hlater (4 h) in the absence or presence of Ole (200�M). The number of dead cells was quantified.Shown are examples of fluorescent (top) and corre-sponding bright-field (bottom) images taken underthe conditions indicated. Cultures were kept undercontrol conditions {saline solutions with glucose inpresence of ambient oxygen for 6 h [CTRL (6h)] or10 h [CTRL (10h)]}. B: % of cells stained withDex-Rd (Dx-R, 10 �M) obtained in dissociatedcultures of HeLa-p or HeLa-Cx32 cells. Each col-umn represents the mean SE for the number offields indicated, obtained from 5 independent exper-iments. **P � 0.01, ***P � 0.001, 1-way ANOVAwith Newman-Keuls posttest.

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creased Cx32 HC activity could enhance the cells’ suscepti-bility to the insult by disruption of the electrochemical gradi-ents across the plasma membrane. Therefore, inhibition ofCx32 HCs in tissues expressing Cx32 that are affected byischemia-reperfusion might increase cell viability. Inhibition ofCx32 HCs should prevent or reduce the influx of Ca2�, whichactivates intracellular pathways related to cell death includingproteases, lipases, and nucleases (1, 4).

In conclusion, our results indicate that HeLa-Cx32 cellspresent functional Cx32 HCs under resting conditions, butduring MI HC activity increases mainly because of an increasein HCs at the cell surface. This change in the number offunctional HCs increases the permeability of the cell mem-brane and could accelerate cell death processes induced byepisodes of hypoxia-reoxygenation.

ACKNOWLEDGMENTS

Data presented in this work are from a thesis presented as partial fulfillmentof the requirement for the degree of Doctor in Biological Sciences (H. A.Sanchez) in the Pontificia Universidad Catolica de Chile.

GRANTS

This work was supported by FONDECYT Grant 1070591, FONDEF GrantD07I1086, and Nucleo Milenio de Inmunologıa e Inmunoterapia PO4/03-F (toJ. C. Saez) and National Institute of General Medical Sciences Grant GM-54179 (to V. K. Verselis). H. A. Sanchez received a Comision Nacional deInvestigacion Cientıfica y Tecnologica fellowship for graduate studies.

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