Dysregulation of blood-brain barrier (BBB) transport function is thought to exacerbate neuronal damage in acute ischemic stroke. The purpose of this study was to clarify the characteristics of pannexin (Px) and/or connexin (Cx) hemichannel(s)-mediated transport of organic anions and cations in human BBB endothelial cell line hCMEC/D3 and to identify inhibitors of hemichannel opening in hCMEC/D3 cells in the absence of extracellular Ca2+, a condition mimicking acute ischemic stroke. In the absence of extracellular Ca2+, the cells showed increased uptake and efflux transport of organic ionic fluorescent dyes. Classic hemichannel inhibitors markedly inhibited the enhanced uptake and efflux. Quantitative targeted absolute proteomics confirmed Px1 and Cx43 protein expression in plasma membrane of hCMEC/D3 cells. Knockdown of Px1 and Cx43 with the small interfering RNAs significantly inhibited the enhanced uptake and efflux of organic anionic and cationic fluorescent dyes. Clinically used cilnidipine and progesterone, which have neuroprotective effects in animal ischemia models, were identified as inhibitors of hemichannel opening. These findings suggest that altered transport dynamics at the human BBB in the absence of extracellular Ca2+ is at least partly attributable to opening of Px1 and Cx43 hemichannels. Therefore, we speculate that Px1 and Cx43 may be potential drug targets to ameliorate BBB transport dysregulation during acute ischemia.
The blood-brain barrier (BBB), which consists of brain capillary endothelial cells linked by complex tight junctions, strictly regulates transcellular influx and efflux transport of organic ions, including nutrients and xenobiotics, by means of polarized expression of numerous transporters and receptors (Tachikawa et al., 2014). It has been proposed that dysregulation of BBB transport function exacerbates neuronal damage in the acute stage of ischemic stroke. Therefore, identifying targets for intervention to minimize BBB disruption and to rapidly protect BBB transport function would be helpful in the development of therapeutic strategies to minimize neuronal injury. Since the paracellular permeability of the BBB is not affected up to at least 40 minutes in the acute stage in rat models of brain ischemia (Fang et al., 2013), it seems unlikely that the BBB disruption could be fully explained in terms of impairment of the tight junctions. Instead, it seems probable that dysregulation of the cellular transport function in brain capillary endothelial cells plays a key role in neuronal damage in the early acute stage.
There is considerable evidence that pannexin (Px) and connexin (Cx) hemichannels exhibit stimulus-dependent opening and mediate influx and/or efflux transport of organic ions, including fluorescent dyes and endogenous signaling molecules, such as ATP and glutamate (Bargiotas et al., 2009; MacVicar and Thompson, 2010; Bosco et al., 2011). Thompson et al. (2006) proposed that the opening of Px1 in pyramidal neurons under ischemia-like oxygen/glucose deprivation conditions is a major contributor to the increased influx and efflux transport of sulforhodamine 101 (SR-101) and calcein, respectively. It has also been shown that: 1) Cx43 and Cx37 proteins are expressed in rat brain capillary endothelial RBE4 cells (De Bock et al., 2011), 2) mouse inner retina capillary endothelial cells show luminal localization of Px1 (Shestopalov and Panchin, 2008), and 3) the Cx43- and Cx37-mediated influx of propidium iodide (PI) is increased in RBE4 cells in the absence of extracellular Ca2+ (De Bock et al., 2011, 2012), which reflect the conditions in acute brain ischemia (Silver and Erecinska, 1990). These lines of evidence prompted us to hypothesize that specific types of hemichannels play key roles in the changes of cellular transport function at the BBB in the absence of extracellular Ca2+.
Liquid chromatography–tandem mass spectrometry–based quantitative targeted absolute proteomics (QTAP) has revealed distinct interspecies differences in the protein expression profiles of transporters in isolated brain microvessels from humans and rodents (Kamiie et al., 2008; Uchida et al., 2011; Hoshi et al., 2013). This highlighted the importance of using human in vitro BBB models. The human brain microvessel endothelial cell line (hCMEC/D3; Weksler et al., 2005) is thought to retain most of the transport characteristics of human BBB, given the consistency of expression levels of transporters and receptors between hCMEC/D3 cells and isolated human brain microvessels (Ohtsuki et al., 2013). Thus, the hCMEC/D3 cell line might be a suitable model to pursue the molecular mechanism(s) of changes in human BBB transport function under pathologic conditions.
Therefore, the purpose of the present study was to clarify the characteristics of Px and/or Cx hemichannel(s)-mediated transport of organic anions and cations at the human BBB and to identify drugs that inhibit hemichannel opening in the absence of extracellular Ca2+ by using hCMEC/D3 cells and QTAP technology.
Materials and Methods
SR-101, Lucifer yellow (LY), fluorescein isothiocyanate (FITC), PI, carbenoxolone (CBX), 18β-glycyrrhetinic acid (GA), and 2-aminoethoxydiphenyl borate (2-APB) were purchased from Sigma-Aldrich (St. Louis, MO). YoPro-1 (4-[(3-methyl-1,3-benzoxazol-2(3H)-ylidene)methyl]-1-[3-(trimethylammonio)propyl]quinolinium diiodide) and INI-0602 [3-(((3S,4aR,6aR,6bS,8aS,11S,12aR,14aR,14bS)-11-carboxy-4,4,6a,8a,11,14b-heptamethyl-14-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydro-picen-3-yloxy)carbonyl)-1-methylpyridinium iodide] were obtained from Life Technologies (Carlsbad, CA) and Wako Pure Chemical Industries (Osaka, Japan), respectively. Calcein-AM and Fluo-3 were purchased from Dojindo Laboratories (Kumamoto, Japan). The mimetic peptides for Px1 (10Panx; WRQAAFVDSY) and Cx43 (Gap27; SRPTEKTIFII), and the peptides with scrambled sequences of Px1scr (10Panxscr; FSVYWAQADR) and Cx43scr (Gap27scr; REKIITSFIPT) were purchased from AnaSpec, Inc. (Fremont, CA). The other mimetic peptides for Cx31 and Cx45 (Cx31 peptide 1; VCYDNYFPISNIR, Cx31 peptide 2; ARPTEKKIFTY, Cx45 peptide 1; QVHPFYVCSRLPCPHK, Cx45 peptide 2; VCYDAFAPLSHVR and Cx45 peptide 3; SRPTEKTIFLL) were synthesized on synthetic resins. They were purified by reversed-phase high-performance liquid chromatography (COSMOSIL 5C18-MS-II column, 10-150 mm; Nakalai Tesque, Kyoto, Japan). The isolated peptides were identified with a Bruker Daltonics Autoflex Speed LRF mass spectrometer (Bruker Daltonics, Billerica, MA). All other chemicals were commercial products of analytical grade.
Mass Spectrometry Data for the Mimetic Peptides.
Cx31 peptide 1: mass spectrometry (MS) [matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF)]: m/z calculated (calcd) for C75H110O21N19S [M+H]+ 1645.89; found 1645.98. Cx31 peptide 2: MS (MALDI-TOF): m/z calcd for C65H104O17N17 [M+H]+ 1395.89; found 1395.83. Cx45 peptide 1: MS (MALDI-TOF): m/z calcd for C89H135O20N16S2 [M+H]+ 1953.37; found 1954.47. Cx45 peptide 2: MS (MALDI-TOF): m/z calcd for C69H104O18N19S [M+H]+ 1519.78; found 1519.84. Cx45 peptide 3: MS (MALDI-TOF): m/z calcd for C62H105O17N16 [M+H]+ 1346.62 found 1346.76.
hCMEC/D3 Cell Culture.
hCMEC/D3 cells were seeded at 1.5 × 106 cells onto a 10-cm dish coated with rat tail collagen type I. The cells were cultured in EBM-2 medium (Takara Bio, Shiga, Japan) supplemented with 2.5% fetal bovine serum, 0.025% vascular endothelial growth factor, 0.025% R3-insulin–like growth factor, 0.025% human epidermal growth factor, 0.01% hydrocortisone, 5 μg/ml basic fibroblast growth factor, 10 mM HEPES, and 1% penicillin–streptomycin in an atmosphere of 95% air and 5% CO2 at 37°C for 3−4 days for routine culture. The passage number was kept below 35. For fluorescent dye uptake and efflux studies, hCMEC/D3 cells were seeded at a density of 1 × 105 cells/well onto the collagen type I–coated 24-well plate (Asahi Techno Glass, Tokyo, Japan) and cultured for 6–7 days. For SR-101 uptake study in a Transwell system, hCMEC/D3 cells were seeded at a density of 5 × 104 cells/cm2 on a 12-mm Transwell with a 0.4-μm pore polycarbonate membrane insert (Corning, Tewksbury, MA) coated with rat tail collagen type I. Transport activity was examined 7 days after seeding.
Fluorescent Dye Uptake and Efflux Studies Using hCMEC/D3 Cells on a 24-Well Plate.
hCMEC/D3 cells cultured on the 24-well plate were washed with normal extracellular fluid (ECF) buffer (122 mM NaCl, 25 mM NaHCO3, 3 mM KCl, 0.4 mM K2HPO4, 10 mM glucose, 1.4 mM CaCl2, 1.2 mM MgSO4, and 10 mM HEPES, pH 7.4) and preincubated at 37°C for 5 minutes in normal ECF buffer. To examine inhibition of fluorescent dye uptake by mimetic peptides, a peptide was added to the culture medium 30 minutes prior to washing the cells. The peptide was also added for the subsequent preincubation with normal ECF buffer. Uptake was initiated by applying 200 μl of normal ECF buffer or the Ca2+-free ECF buffer containing a fluorescent dye at 37°C, in the presence or absence of various compounds. The Ca2+-free ECF buffer was prepared by removing CaCl2 from the normal ECF buffer. For calcein efflux study, hCMEC/D3 cells cultured on the 24-well plate were incubated with 0.5 μM calcein acetoxymethyl ester for 30 minutes at 37°C in normal ECF buffer. The cells were then incubated with 200 μl of normal ECF buffer or Ca2+-free ECF buffer in the presence or absence of various compounds. The concentration of fluorescent dye was determined on the basis of the minimal concentration at which the fluorescence could be detected within the range of the standard curves at the shortest incubation time period and under inhibited conditions. The incubation times of SR-101 and PI were within the region of linear time–dependent increase. At the designated times, the uptake was terminated by removing the solution, and the cells were washed in ice-cold normal ECF buffer. The cells were then homogenized in distilled water using a sonicator. The protein content of the cell homogenate was determined by the Lowry method with bovine serum albumin as a standard. The homogenate was centrifuged at 21,600g for 5 minutes at 4°C. Cell-associated fluorescence and fluorescence in the medium were measured with a fluorescence detector (Fluoroskan Ascent FL; Thermo Fisher Scientific, Waltham, MA). The accumulation of fluorescent dye in hCMEC/D3 cells was expressed as the cell-to-medium ratio (μl/mg of protein), calculated by dividing the cellular uptake amount (mol/mg of protein) by the test substrate concentration in the uptake medium (moles per liter). The extracellular free calcium concentration was measured using Fluo-3, a calcium indicator, according to the protocol reported previously (Tepikin et al., 1992).
SR-101 Uptake Study Using hCMEC/D3 Cells on a Transwell System.
Both sides of the chamber were washed with normal ECF buffer and preincubated at 37°C for 5 minutes in normal ECF buffer. Uptake was initiated by applying 500 μl normal ECF buffer in the upper chamber and 1.5 ml Ca2+-free ECF buffer containing SR-101 at 37°C in the bottom chamber, in the presence or absence of CBX at a 1 mM concentration. At 60 minutes, uptake was terminated by removing the solution, and both sides of the chamber were washed in ice-cold normal ECF buffer. The cells left on the membranes were homogenized in distilled water using a sonicator. The accumulation of SR-101 in hCMEC/D3 cells was expressed as the cell-to-medium ratio (μl/mg of protein).
Quantification of Protein Expression Levels in hCMEC/D3 Cells Using QTAP.
Plasma and crude membrane fractions of hCMEC/D3 cells were prepared as previously reported (Ohtsuki et al., 2013). QTAP analysis was performed with an electrospray ionization–triple quadruple mass spectrometer (QTRAP5500; AB SCIEX, Framingham, MA) coupled with an LC system (ekspert MicroLC 200; Eksigent Technologies, Dublin, CA). Selection of target peptides to be quantified (Supplemental Table 1), preparation of trypsin- and lysylendopeptidase-treated samples, LC separation of the peptides, and detection/quantification of the target peptides by multiplexed selected/multiple reaction monitoring analysis were carried out according to our previously reported method (Ohtsuki et al., 2013). If a positive peak was detected at only two or fewer selected/multiple reaction monitoring transitions, the protein expression level was defined as under the limit of quantification.
Px1 and Cx43 Small Interfering RNA Transfection into hCMEC/D3 Cells.
Double-stranded small interfering RNAs (siRNAs) targeted to human Px1 and Cx43 mRNAs were purchased from Ambion (Austin, TX) and Qiagen (Hilden, Germany), respectively. The siRNA sequences containing 3′-dTdT extensions were 5′-GCA UCA AAU CAG GGA UCC UTT-3′ for Px1 and 5′-GCU UAG AGU GGA CUA UUA ATT-3′ for Cx43. Stealth RNAi negative control duplexes used as a negative control was purchased from Life Technologies (Grand Island, NY). hCMEC/D3 cells were plated on a rat-tail collagen type I–coated 24-well plate at 5 × 104 cells/well, and grown for 24 hours at 37°C. Px1- and Cx43-specific siRNAs and negative control siRNAs at the final concentration of 25 nM were then transfected using Lipofectamine RNAi MAX and Opti-MEM medium (Life Technologies). Transport activity was examined 3 days after transfection.
Quantitative Real-Time Polymerase Chain Reaction Analysis.
Quantitative real-time polymerase chain reaction (PCR) was performed using the Applied Biosystems 7300 Real Time PCR System (Applied Biosystems, Foster City, CA) with SYBR Premix Ex Taq II (Tli RNaseH Plus; Takara Bio Inc., Otsu, Japan) and gene-specific primers. Conditions were: 95°C for 10 seconds, and 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds, 95°C for 15 seconds, 60°C for 1 minute, 95°C for 15 seconds, and 60°C for 15 seconds. The sequences of the specific primers were as follows. For glyceraldehyde 3-phosphate dehydrogenase (GenBank accession number: M17851), the sense sequence was 5′-ATGGGGAAGGTGAAGGTCG-3′ and the antisense sequence was 5′-GAGGTCAATGAAGGGGTCAT-3′. For Cx43 (NM_000165), the sense sequence was 5′-GGAATGCAAGAGAGGTTGAAAG-3′ and the antisense sequence was 5′-GGCATTTGGAGAAACTGGTAGA-3′. For Px1 (NM_015368), the sense sequence was 5′-CTGTGGACAAGATGGTCACG-3′ and the antisense sequence was 5′-CAGCAGGATGTAGGGGAAAA-3′. A standard curve was generated for each run using different amounts of plasmid containing the target gene. This allowed quantification of the initial copy number of the target mRNA in the samples despite the different PCR conditions of the target genes. Each mRNA expression level was normalized with respect to glyceraldehyde 3-phosphate dehydrogenase mRNA expression.
Unless otherwise indicated, all data are expressed as mean ± S.E.M. The kinetic parameters are presented as the mean ± S.D. Statistical analysis was performed with an unpaired, two-tailed Student’s t test, taking P < 0.05 as the criterion of a significant difference between two group means. One-way analysis of variance followed by the modified Fisher’s least-squares difference method was used to assess the statistical significance of differences among means of more than two groups.
The kinetic parameters for SR-101 and PI uptake by hCMEC/D3 cells were obtained from eqs. 1 and 2, respectively:(1)(2)Values of half-maximal effective Ca2+ concentration (EC50) were calculated according to eq. 3:(3)where V is the initial velocity of uptake, Vmax is the maximum uptake velocity, Vmin is the minimum uptake velocity, S is the substrate concentration in the medium, Km is the Michaelis-Menten constant, Kns is the nonsaturable uptake clearance, and [Ca2+] is the extracellular concentration of Ca2+. Curve fitting was carried out by iterative nonlinear least-squares regression analysis with the program MULTI (Yamaoka et al., 1986).
Effect of Change in Extracellular Ca2+ Concentration on Uptake of Fluorescent Dyes by hCMEC/D3 Cells.
Cellular uptake of SR-101, an anionic fluorescent dye, and PI, a cationic fluorescent dye, increased linearly up to 45 and 20 minutes, respectively, in the absence of extracellular Ca2+ (Fig. 1, A and B). The magnitude of the increase in uptake was inversely dependent on the extracellular Ca2+ concentration, with EC50 values of 22.0 ± 9.3 μM (SR-101) and 0.795 ± 0.409 μM (PI) (Fig. 1, C and D). The extracellular Ca2+ concentration in the buffer was estimated to be 1.00 ± 0.17 μM and 1.78 ± 0.11 μM at 20 and 45 minutes, respectively, after initiation of calcium-free Ca2+ conditions. Considering that uptakes of SR-101 and PI were both significantly enhanced at extracellular Ca2+ concentrations of 1–2 μM (Fig. 1, C and D), the difference in the time frames (20 and 45 minutes) may not significantly influence the observed transport activity. As shown in Supplemental Fig. 1, the cellular uptake of other anionic fluorescent dyes (LY and FITC) and another cationic fluorescent dye (YoPro-1) increased time dependently in an extracellular Ca2+ concentration–dependent manner, with EC50 values of 253 ± 110 μM (LY), 21.0 ± 7.4 μM (FITC), and 25.9 ± 5.4 μM (YoPro-1).
The SR-101 uptake in the absence of extracellular Ca2+ consisted of a single saturable component with a Km value of 23.5 ± 2.7 μM and a Vmax of 194 ± 16 pmol/(mg of protein⋅min) (Fig. 2, A and B). On the other hand, PI uptake showed both a saturable component and a nonsaturable component. The apparent Km, Vmax, and Kns values were 24.0 ± 5.7 μM, 121 ± 23 pmol/(mg of protein⋅min), and 0.26 ± 0.08 μl/(mg of protein⋅min), respectively (Fig. 2, C and D). These results indicate that SR-101 and PI are predominantly taken up via a carrier-mediated transport system in hCMEC/D3 cells in the absence of extracellular Ca2+.
Figure 3 shows the effect of inhibitors (CBX, GA, and INI-0602) and substrates (ATP and glutamate) of Px and/or Cx hemichannels and gap junctions on the uptake of SR-101 and PI in the absence of extracellular Ca2+. CBX and GA each significantly inhibited the uptake of SR-101 and PI by more than 30%, respectively, except in the case of SR-101 uptake in the presence of 10 μM CBX. ATP and glutamate significantly inhibited SR-101 and PI uptake. In contrast, an in vivo BBB-permeable analog of GA INI-0602, which has been reported to inhibit Cx32-mediated glutamate release from microglial cells (Takeuchi et al., 2011), increased SR-101 and PI uptake. Addition of 1.4 mM CaCl2 in the Ca2+-free ECF buffer significantly reduced the enhanced uptake of SR-101 and PI. The effect of adding extracellular Ca2+ showed modest variability within the range of 60–80% reduction throughout the experiments.
When hCMEC/D3 cells that had been preloaded with calcein acetoxymethyl ester were exposed to Ca2+-free ECF buffer for 30 minutes, calcein release from the cells was several times greater than that in normal ECF buffer and was time-dependent (Fig. 4A). The efflux was significantly inhibited in the presence of either CBX or 2-APB at the concentration of 100 μM and in the presence of 1.4 mM CaCl2 (Fig. 4B).
Expression of Hemichannel Subtypes in Plasma Membrane of hCMEC/D3 Cells.
As shown in Table 1, Px1 and Cx43 were detected at protein expression levels of 3.63 ± 0.54 and 4.33 ± 0.61 fmol/mg of protein, respectively, in the plasma membrane fraction of hCMEC/D3 cells by QTAP. In contrast, Cx31, Cx32, and Cx45 were below the detection limit. These results indicate that Px1 and Cx43 are localized on the plasma membrane of hCMEC/D3 cells. The expression levels of Px1 and Cx43 were comparable to that of the major BBB transporter, multidrug resistance protein 1 (MDR1), and were approximately 10- to 20-fold smaller than those of glucose transporter 1 and Na+/K+-ATPase.
Contribution of Px1 and Cx43 to Enhanced Transport Function of hCMEC/D3 Cells in the Absence of Extracellular Ca2+.
Treatment of hCMEC/D3 cells with Px1 siRNA or Cx43 siRNA significantly decreased the transcript levels of Px1 and Cx43, respectively (Fig. 5A). QTAP analysis confirmed that the protein level of Cx43 was decreased after treatment with Cx43 siRNA (1.08 ± 0.64 fmol/μg of protein for the negative control siRNA versus under the limit of quantification <0.275 fmol/μg of protein for Cx43 siRNA treatment). Either Px1 siRNA or Cx43 siRNA treatment significantly reduced the increase of both SR-101 and PI uptake in the absence of extracellular Ca2+ (Fig. 5, B and C). Combined treatment with Px1 and Cx43 siRNAs further decreased the SR-101 uptake (Fig. 5B). The inhibition ratio (approximately 40–50%; Fig. 3) of broad-spectrum inhibitors of Pxs and Cxs (CBX and GA) at the concentration of 100 μM seems to be comparable to that (approximately 40–70%; Fig. 5, B and C) of Px1 and Cx43 siRNA treatment. Although the affinities of CBX and GA for Px1- and Cx43-mediated SR-101 and PI uptake remain to be determined, it is seems reasonable to consider that Px1 and Cx43 make a major contribution to the enhanced transport function of hCMEC/D3 cells in the absence of extracellular Ca2+. Furthermore, the enhancement of calcein efflux in hCMEC/D3 cells was significantly reduced by either Px1 siRNA or Cx43 siRNA (Fig. 5D). Indeed, the calcein accumulation in the cells was significantly increased, whereas the calcein release from the cells was decreased by either Px1 siRNA or Cx43 siRNA.
The effect of mimetic peptides of Px1, Cx43, Cx31, and Cx45 was also studied. The selected mimetic peptides were partial peptide sequences located in the extracellular loop of human Px1, Cx43, Cx31, and Cx45. These mimetic peptides are thought to disturb the assembly of functional hemichannel hexamer and thereby to inhibit the transport activity (Evans et al., 2006). Mimetic peptides of Px1 and Cx43 attenuated the increase of PI uptake, whereas scrambled versions of the Px1 and Cx43 mimetic peptides, as well as mimetic peptides of Cx31 and Cx45, were ineffective (Fig. 6).
SR-101 Uptake on the Basal Membrane of hCMEC/D3 Cells Using a Transwell System.
As shown in Fig. 7, CBX significantly inhibited the basal Transwell compartment-to-cell transport of SR-101 under Ca2+-free conditions in the bottom chamber. This result supports the idea that the hemichannel(s) would be involved in SR-101 uptake in the absence of Ca2+ at the basal side of hCMEC/D3 cells.
Inhibitory Effect of Various Compounds on the Increased Uptake of SR-101 and PI by hCMEC/D3 Cells in the Absence of Extracellular Ca2+.
Table 2 summarizes the inhibition profiles of classic hemichannels/gap junction inhibitors, some clinically approved drugs, and CBX-related compounds on hemichannel opening–enhanced transport in hCMEC/D3 cells. Cilnidipine, a dual L/N-type Ca2+ channel blocker and progesterone, a neurosteroid, each significantly reduced the increase of both SR-101 and PI uptake by hCMEC/D3 cells. Among the classic hemichannel/gap junctions inhibitors, 2-APB, an inhibitor of Cx gap junctions (Bai et al., 2006), reduced both SR-101 and PI uptake, whereas flufenamic acid, an inhibitor of Px1 (Iglesias et al., 2008) and Cx43 (Stout et al., 2002) and Brilliant Blue G, an inhibitor of Px1 (Qiu and Dahl, 2009), inhibited SR-101 uptake. Among clinically approved drugs that have been reported to have neuroprotective effects in humans or animal models, fluvastatin, citostasol, and FK506 inhibited only SR-101 uptake, and pravastatin and edaravone had no significant effect. Among the Ca2+ channel blockers, nifedipine, nicardipine, and nimodipine significantly reduced only SR-101 uptake, whereas amlodipine, diltiazem, and verapamil had no inhibitory effect on either SR-101 or PI uptake. Among the CBX-related compounds, estrone, hydrocortisone, and dexamethasone had a little or no significant effect on SR-101 or PI uptake.
Our present findings indicate that extracellular Ca2+-dependent transport function involves Px1 and Cx43 hemichannels in human brain microvessel endothelial cells, and opening of these hemichannels is likely to be a contributor to dysregulation of BBB transport.
hCMEC/D3 cells exhibited an increase in the uptake of organic ionic fluorescent dyes in response to a decrease of extracellular Ca2+ (Fig. 1; Supplemental Fig. 1). This is consistent with the reported characteristics of Px1 (Poornima et al., 2012) and Cx43 (De Bock et al., 2011). Considering that the extracellular Ca2+ concentration in the brain interstitial fluid (ISF) falls during ischemic stroke (Silver and Erecinska, 1990), it seems reasonable to consider that decrease of extracellular Ca2+ concentration plays a key role as a trigger of the opening of Px1 and Cx43 and the consequent increase of membrane permeability in brain capillary endothelial cells. Poornima et al. (2012) have proposed a model in which Px1 associates with P2X7 receptor (P2X7R) to form a large calcein dye–permeable pore upon Px1-mediated ATP release under conditions of extracellular Ca2+ deprivation. Since hCMEC/D3 cells express several types of P2X receptors, including P2X7R, at the transcriptional level (Bintig et al., 2012), it is probable that the association of Px1 and P2X7R plays a role in promoting formation of a fluorescent dye–permeable pore in hCMEC/D3 cells. Furthermore, oxygen/glucose deprivation also stimulates Px1 opening (Thompson et al., 2006). Since brain capillary endothelial cells face both the circulating blood and the brain ISF, not only reduced extracellular Ca2+ concentration in the brain ISF but also oxygen/glucose deprivation owing to decreased blood flow would stimulate the transport functions of Px1 and Cx43.
Because the present study evaluated the transport function by means of uptake analyses using hCMEC/D3 cells seeded on collagen-coated plates, the polarization of hCMEC/D3 cells needs to be discussed. Tai et al. (2009) have reported that MDR1 is localized on not only the apical plasma membrane but also the basolateral membrane in hCMEC/D3 cells grown on Transwell inserts, and the ratio of apical to basolateral membrane localization was approximately 2.8:1. Considering that MDR1 exhibits predominant luminal membrane localization in human brain endothelial cells in vivo (Cordon-Cardo et al., 1989), hCMEC/D3 cells may not be completely polarized. Although the polarization of the localizations of Px1, Cx43, P2X receptors, and ISF Ca2+-sensing machinery need further investigation, the uptake by hCMEC/D3 cells grown on collagen-coated plates may at least partly reflect substrate movement across the basal plasma membrane as well as the apical membrane. In the present study, we found that CBX, an inhibitor of classic hemichannels/gap junctions, significantly inhibited the basal Transwell compartment-to-cell transport of SR-101 in the absence of Ca2+ at the basal side of hCMEC/D3 cells (Fig. 7). These results suggest that hemichannel(s) would be involved in the basal membrane transport activity of brain capillary endothelial cells under the low Ca2+ conditions existing in the brain ISF.
Fluorescent dyes, such as LY, FITC, PI, and calcein, exhibit low membrane permeability in hCMEC/D3 cells under normal conditions (Figs. 1 and 4; Supplemental Fig. 1). On the other hand, Px1 and Cx43 are involved in the increased uptake of SR-101 and PI, most probably via the saturable transport component, under extracellular Ca2+-free conditions (Fig. 2). Furthermore, the process of PI uptake by hCMEC/D3 cells is composed of passive diffusion as well as a saturable component. These results suggest that Px1 and Cx43 would have an impact on carrier-mediated transport at the BBB under pathologic conditions, although further study would be needed to examine the involvement of Px1 and Cx43 in the non–carrier-mediated pathways.
The results of QTAP, siRNA knock down, and mimetic peptide inhibition studies (Figs. 5 and 6; Table 1) supported the existence of marked interspecies differences in Px1 expression in brain endothelial cells between human and rat (De Bock et al., 2011). It has been reported that the rat brain capillary endothelial RBE4 cells do not show protein expression of Px1 (De Bock et al., 2011). In contrast, our QTAP data indicate that the expression level of Px1 in plasma membrane fraction of hCMEC/D3 cells is quite similar to that of Cx43 (Table 1). We also found that combined treatment with Px1 and Cx43 siRNAs resulted in the greatest inhibitory effect on SR-101 uptake by hCMEC/D3 cells in the absence of extracellular Ca2+ (Fig. 5). These results suggest that extracellular Ca2+-dependent transport dynamics in hCMEC/D3 cells is determined at least in part by the interplay between Px1 and Cx43. Since association between different subtypes of Cx to form hemichannels has been reported (Koval et al., 2014), one possible explanation of our findings is formation of a Px1–Cx43 heterohexamer on the plasma membrane. Another possibility is that Px1 and Cx43 function in distinct localizations in brain capillary endothelial cells, e.g., Px1 on the luminal and/or abluminal membrane and Cx43 mainly in the gap junction regions. This is similar to the model proposed by Gaete et al. (2014). In support of this notion, Px1 is localized on the plasma membrane of mouse inner retinal endothelial cells (Shestopalov and Panchin, 2008). Several lines of evidence confirm the belief that the gap junction formed by Cx hexamers is a determinant of the tightness of tight junctions and is associated with oscillations of intracellular Ca2+ concentration (De Bock et al., 2014). Thus, connexins influence paracellular permeability (De Bock et al., 2011, 2012), and therefore, interfering with endothelial connexins may be a novel approach to limit BBB permeability increase.
Several reports have provided evidence that blockade of hemichannels can suppress neuronal pathologic processes in animal models: 1) Px1 and Px2 double-knockout mice exhibit a better functional outcome and smaller infarcts than wild-type mice when subjected to ischemic stroke (Bargiotas et al., 2012), and 2) a blocker of gap junction hemichannels, the BBB-permeable CBX-dihydropyridine conjugate INI-0602, prevented neuronal damage by inhibiting Cx32-mediated glutamate release in microglia (Takeuchi et al., 2011). Our present data showed that a dual L- and N-type Ca2+ channel blocker, cilnidipine, and a neurosteroid, progesterone, each significantly inhibited both SR-101 and PI uptake by hCMEC/D3 cells in the absence of extracellular Ca2+ (Table 2). Therefore, drugs that block hemichannels opening at the BBB might be effective in ameliorating ischemic brain damage. This idea is supported by the previous findings that cilnidipine and progesterone reduce infarct volume in a focal brain ischemia model (Takahara et al., 2004; Ishrat et al., 2009). It has been reported that progesterone given soon after ischemic stroke protects the functional and structural integrity of the BBB by inhibiting expression of key inflammatory cytokines and matrix metalloproteinases that are involved in proteolytic degradation of the vascular basement membrane (Ishrat et al., 2010). However, these actions of progesterone do not seem sufficient to account fully for the BBB protective effect. In this regard, our present results suggest an additional mechanism through which cilnidipine and progesterone may exert a neuroprotective effect, i.e., by inhibiting the opening of Px1 and/or Cx43 hemichannels in the acute phase of ischemic stroke. Thompson et al. (2006) have reported that opening of Px1 expressed in pyramidal neurons is a key contributor to the increased plasma membrane permeability leading to ischemic insult. It has also been proposed that opening of Cx43 expressed on the surface of astrocytes is involved in the marked increase of permeability during hypoxia and ischemia (Retamal et al., 2006). Thus, it seems plausible that the neuroprotective effects of these compounds are attributable to interaction not only with BBB hemichannels but also with hemichannels in neurons and astrocytes.
It has been widely accepted that hemichannels transport inorganic and organic ions with a molecular mass of less than 1 kDa in a nonspecific manner (Simpson et al., 1977; Li et al., 2012). However, we found different inhibition spectra for uptake of the organic anion SR-101 and the organic cation PI by hCMEC/D3 cells in the absence of extracellular Ca2+ (Table 2), suggesting that Px1 and Cx43 show substrate specificity. It will be intriguing to see whether the putative differences in affinity and substrate specificity between Px1 and Cx43 mean that the two hemichannels have distinct pathophysiological roles.
In conclusion, our results suggest that Px1 and Cx43 hemichannels in human brain capillary endothelial cells may be a primary cause of disruption of the highly regulated BBB transport system.
The authors thank A. Niitomi and N. Handa for secretarial assistance.
Participated in research design: Kaneko, Tachikawa, Hosoya, Terasaki.
Conducted experiments: Kaneko, Tachikawa, Akaogi, Ishibashi.
Contributed new reagents or analytic tools: Fujimoto, Tachikawa, Ishibashi, Ohtsuki, Couraud, Uchida.
Performed data analysis: Kaneko, Tachikawa, Akaogi, Ishibashi, Terasaki.
Wrote or contributed to the writing of the manuscript: Kaneko, Tachikawa, Akaogi, Fujimoto, Hosoya, Terasaki.
- Received September 29, 2014.
- Accepted February 9, 2015.
This study was supported in part by four Grants-in-Aid from the Japanese Society for the Promotion of Science (JSPS) for Young Scientists (A) [KAKENHI: 23790170] and for Scientific Research (A) [KAKENHI: 24249011]; and by the JSPS and Centre National de la Recherche Scientifique (CNRS) under the Japan-France Basic Scientific Cooperation Program. T.T. and S.O. are full professors at Tohoku University and Kumamoto University, respectively, and are also directors of Proteomedix Frontiers Co. Ltd. This study was not supported by Proteomedix Frontiers Co. Ltd., and their positions at Proteomedix Frontiers Co. Ltd. did not affect the design of the study, the collection, analysis, and interpretation of the data, the writing of the manuscript, or the decision to publish, and does not present any financial conflicts. The remaining authors declare no competing financial interests.
- 2-Aminoethoxydiphenyl borate
- blood-brain barrier
- extracellular fluid
- fluorescein isothiocyanate
- 18β-glycyrrhetinic acid
- hCMEC/D3 cell
- human cerebral microvascular endothelial cell line
- 3-(((3S,4aR,6aR,6bS,8aS,11S,12aR,14aR,14bS)-11-carboxy-4,4,6a,8a,11,14b-heptamethyl-14-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydro-picen-3-yloxy)carbonyl)-1-methylpyridinium iodide
- interstitial fluid
- Lucifer yellow
- matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
- multidrug resistance protein 1
- mass spectrometry
- P2X7 receptor
- polymerase chain reaction
- propidium iodide
- quantitative targeted absolute proteomics
- small interfering RNA
- sulforhodamine 101
- 4-[(3-methyl-1,3-benzoxazol-2(3H)-ylidene)methyl]-1-[3-(trimethylammonio)propyl]quinolinium diiodide
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics