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J. Biol. Chem., Vol. 281, Issue 50, 38430-38439, December 15, 2006

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Novel Functions of Small Conductance Ca²⁺-activated K⁺ Channel in Enhanced Cell Proliferation by ATP in Brain Endothelial Cells^*

Daiju Yamazaki, Mineyoshi Aoyama, Susumu Ohya, Katsuhiko Muraki^¶, Kiyofumi Asai, and Yuji Imaizumi¹

From the Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabedori, Mizuho-ku, Nagoya 467-8603, the Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-ku, Nagoya 467-8601, and the ^¶Cell Signaling & Ion Channel Research Group, Cellular Pharmacology, School of Pharmacy, Aichi Gakvin University, Chikusaku Nagoya 464-8650, Japan

Received for publication, April 24, 2006 , and in revised form, September 11, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Brain capillary endothelial cells (BCECs) form the blood-brain barrier (BBB), which is essential for maintaining homeostasis of the brain. Net cellular turnover, which results from the balance between cell death and proliferation, is important in maintaining BBB homeostasis. Here we report a novel mechanism that underlies ATP-induced cell proliferation in t-BBEC 117, a cell line derived from bovine brain endothelial cells. Application of 0.1-30 µM ATP to t-BBEC 117 concentration-dependently increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) in two phases: an initial transient phase and a later and smaller sustained one. These two phases of [Ca²⁺]_i rise were mainly due to Ca²⁺ release and sustained Ca²⁺ influx, respectively. The pretreatment with apamin, a selective blocker of small conductance Ca²⁺-activated K⁺ channels (SK), significantly reduced both the [Ca²⁺]_i increase and K⁺ current induced by ATP. Transcripts corresponding to P2Yx, SK2, and transient receptor potential channels were detected in t-BBEC 117. Knock down of SK2 protein, which was the predominant Ca²⁺-activated K⁺ channel expressed in t-BBEC 117, by siRNA significantly reduced both the sustained phase of the [Ca²⁺]_i rise and the K⁺ current induced by ATP. Cell proliferation was increased significantly by the presence of the stable ATP analogue ATPS. This effect was blunted by UCL1684, a synthesized SK blocker. In conclusion, in brain endothelial cells ATP-induced [Ca²⁺]_i rise activates SK2 current, and the subsequent membrane hyperpolarization enhances Ca²⁺ entry presumably through transient receptor potential channels. This positive feedback mechanism can account for the augmented cell proliferation by ATP.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Under physiological conditions, the blood-brain barrier (BBB)² restricts movement of substances from the circulation, blocks the invasion of noxious matters to the brain, and maintains the homeostasis of the brain. Brain capillary endothelial cells (BCECs) form the BBB whose functional characteristics include the presence of intercellular tight junctions and a relating low transcellular vesicular transport induced by surrounding astrocytes (1). In the BBB cell homeostasis requires a delicate balance between formation of new cells by proliferation and their elimination by cell death.

Cell proliferation is stimulated by ATP in a variety of cells (2, 3). There is a line of evidence for a critical role of intracellular Ca²⁺ activity in the regulation of cell proliferation (4-6). ATP is known to increase intracellular Ca²⁺ concentration ([Ca²⁺]_i) via activation of metabotropic P2Y receptors (7), which in turn can trigger Ca²⁺ entry (8). A variety of K⁺ channels have also been implicated in the regulation of cell proliferation (9). In peripheral endothelial cells, ion channels such as Ca²⁺-activaed K⁺ channels (K_Ca) (10, 11), inward rectifier K⁺ channels (K_ir2.x) (12, 13) and transient receptor potential (TRP) channels (14, 15) have been reported to be present, and some of their functions have been identified. In contrast, electrophysiological characteristics, including ion channel expression and their functions in BCECs, have not been studied extensively. Only the accumulation of information about transporters such as P-glycoprotein in BCECs has been recently well accelerated (16). Experimental difficulties in preparing freshly isolated BCECs and identifying BCECs among various types of cell are major reasons why the electrophysiology of BCECs using single cell preparation and patch clamp methods has progressed rather slowly, despite its importance (17).

In this study, we have used t-BBEC 117, an immortalized bovine BCEC line, which was established by transfection of SV40 large T antigen and then by isolating a single clone. In vitro culture of these cells has been accomplished, and the BBB phenotypes have been confirmed based on the following criteria: (i) spindle-shape morphology, (ii) rapid uptake of acetylated-LDL, (iii) formation of tight junction-like structures, (iv) high alkaline phosphatase activity, and (v) expression of multidrug resistance and glucose transporter-1 mRNA (18). We have analyzed the functional and molecular expression of ion channels and purinoceptors in t-BBEC 117. Our findings revealed a novel mechanism of positive feedback in the regulation of [Ca²⁺]_i by ATP. This involves the activation of two types of ion channels, Ca²⁺-activated K⁺ channels (K_Cas) and Ca²⁺-permeable channels, which are, presumably, TRP channels. We therefore postulate that ATP, which is released into in the cleft between BCEC and astrocytes, regulates and sustains cell proliferation of BCECs by the positive feedback mechanism.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Cultures—An immortalized endothelial cell line, t-BBEC 117, was established from primary cultured bovine brain endothelial cells, using the transfection of SV40 large T antigen expressing vector as described previously (18). t-BBEC 117 were maintained at 37 °C, 5% CO₂ in low glucose (1000 mg/liter) Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Sigma), 100 units/ml penicillin (Meiji Seika Kaisha, Ltd., Tokyo, Japan), and 0.2 mg/ml streptomycin (Meiji Seika Kaisha, Ltd.).

RT-PCR—Total RNA was prepared using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Reverse transcription (RT) was carried out using random hexamers and SuperScript II RNase H^- reverse transcriptase (Invitrogen). The resultant cDNA was subjected to PCR-based amplification. Our oligonucleotide primers for the K⁺ channels and -actin, and for the purinoceptors, are shown in supplemental Tables S1 and S2, respectively. The reactions for K⁺ channels and -actin contained 1 µl of RT reaction product as template DNA and were carried out for 35 cycles, using a 95 °C, 45-s denaturing step; a 56 °C, 30-s annealing step; and a 72 °C, 60-s extension step. The reaction for purinoceptors contained 1 µl of RT reaction product as template DNA and was carried out for 35 cycles, using a 95 °C, 15-s denaturing step and a 60 °C, 60-s annealing and extension step. The PCR products were visualized by ethydium bromide staining following separation on 2% agarose gels. Amplified products were sequenced using BigDye terminator v3 kit (Applied Biosystems) with an ABI PRIZM 3100 genetic analyzer (Applied Biosystems).

Western Blot Analysis—Cells cultured under the specified conditions were harvested into Ca²⁺- and Mg²⁺-free phosphate-buffered saline and centrifuged for 10 min at 800 x g at 4 °C. Cell pellets were suspended in 100 µl of Tris-buffered saline (TBS, 20 mM Tris-HCl buffer, pH 7.6, 137 mM NaCl) containing 100 µM phenylmethanesulfonyl fluoride, 10 µM pepstatin A, 10 µM leupeptin, 2 mM EDTA. The quantity of protein was measured using a BCA protein assay regent kit (Pierce). 30-µg aliquots from protein sample were separated by 12% SDS-PAGE and transferred to a Clear Blot Membrane-P (ATTO, Tokyo, Japan). The membrane filters were blocked for 1 h with 5% skim milk in TBS-T (20 mM Tris-HCl buffer, pH 7.6, 137 mM NaCl, 0.1% Tween 20), and then incubated overnight at 4 °C with the following antisera: SK1 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), SK2 (1:500, Alomone Laboratories, Jerusalem, Israel), or SK3 (1:200, Alomone Laboratories). After washing the membranes for 15 min with TBS-T, they were incubated for 1 h with anti-rabbit IgG (GE Healthcare Bio-Sciences Corp.,) or anti-goat IgG (Santa Cruz Biotechnology) coupled to horseradish peroxidase diluted 1:4000 in TBS-T. Bands were visualized using an ECL detection system (GE Healthcare Bio-Sciences Corp.).

Measurement of [Ca²⁺]_i—t-BBEC 117 was incubated with 10 µM fura-2 acetoxymethyl ester (Fura-2 AM, Molecular Probes Inc., Eugene, OR) in normal HEPES solution for 30 min at room temperature. Fura-2 fluorescent signals was measured using the ARUGAS/HiSCA imaging system (Hamamatsu Photonics, Hamamatsu, Japan). The frequency of image acquisition was selected to be one image every 1 s for [Ca²⁺]_i measurement. In some experiments, the ratios of fluorescence intensity were transformed into [Ca²⁺]_i using Equation 1,

(Eq. 1)

where R is the fluorescence ratio 340 nm/380 nm, R_min and R_max are the fluorescence ratios determined by addition of 1 mM EGTA and 2.2 mM Ca²⁺, respectively, after the permeabilization of cells with 10 µM ionomycin, and B is the averaged fluorescence proportionality coefficients obtained at 380 nm under R_min and R_max conditions.

Electrophysiological Recordings—Whole cell membrane currents were applied to single t-BBEC 117 with patch pipettes using a CEZ-2400 (Nihon Koden, Tokyo, Japan) amplifier. The resistance of microelectrodes filled with pipette solution was 3-5 M. Membrane currents were stored and analyzed as described previously (10, 19). Briefly, membrane currents were monitored on a storage oscilloscope (VC-6041, Hitachi, Tokyo, Japan) and stored on videotape after being digitized by a PCM-recording system (modified to acquire a DC signal, PCM 501ES, SONY, Tokyo, Japan). The data on the tape were later loaded into a computer (IBM-AT compatible) through an analog-digital converter (DT2801A; Data Translation, Marlboro, MA). Data acquisition and analysis for whole cell current were carried out using AQ/Cell-soft, developed in the laboratory of Dr. Wayne Giles (University of Calgary). In some experiments, ramp waveforms were applied as a voltage clamp command using a multipulse generator (FS-1915, NF Electronics, Tokyo, Japan). All currents recorded were filtered at 3 kHz (four-pole Bessel filter, NF Electronics).

Membrane Potential Measurements Using a Voltage-sensitive Fluorescent Dye—The measurement of membrane potential changes by DiBAC₄(3), which is a bisbarbituric acid oxonol dye with excitation maximal at 490 nm, was performed as described previously (20). Before the fluorescence measurements, cells were incubated with 100 nM DiBAC₄(3) in HEPES-buffered solution for 30 min at room temperature. Experiments were carried out in the constant presence of DiBAC₄(3). Hyperpolarization results in the extrusion of the dye from cells and a subsequent decrease in fluorescence intensity. The decrease in fluorescence intensity by 1% corresponded to 0.5 mV hyperpolarization in the membrane potential range of 20 and 70 mV (20). Data collection and analyses were performed using ARGUS-HiSCA. The sampling interval of DiBAC₄(3) fluorescence measurements was 5 s.

Specific Knockdown of SK2 by RNA Interference—The sequences of short interference RNA (siRNA) used in this study, selected according to the criteria suggested previously (21), were as follows: 5'-GGUACCAUGAUCAACAGGATT-3' for sense strand, and 5'-GCAUACGACUUCAGAGGAATT-3' for antisense strand. These oligonucleotides were annealed and labeled by Cy5. The siRNA target site for SK2 was 5'-GGUACCAUGAUCAACAGGA-3' (nucleotides 1004-1022). The cells in a 35-mm dish were washed with 2 ml of the fresh culture medium 30 min prior to transfection. 25 µl of siRNA solution (1 µM) and 5 µl of LipofectAMINE2000 (Invitrogen) were each mixed with 250 µl of Opti-MEM. The two mixtures were combined and incubated for 20 min at room temperature for complex formation. The entire mixture was added to the cells, resulting in a final concentration of 10 nM for the siRNA. The cells were incubated for 5-8 h in a CO₂ chamber, washed once, and supplied with 2 ml of the fresh culture medium.

MTT Assay—Cell viability was monitored by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma) assay as described (22). MTT was dissolved in phosphate-buffered saline at 5 mg/ml and filtered to sterilize and remove a small amount of insoluble residue present in some batches of MTT. Cells (4 x 10³ cells/well) were seeded onto 96-well plates and incubated with test substances for a selected time at 37 °C in 5% CO₂. At the time points indicated, stock MTT solution (10 µl per 100-µl medium) was added to all wells of an assay plate, and the plates were incubated at 37 °C for 4 h. After 4 h, 20% w/v SDS (50% N,N-dimethyl formamide and demineralized water) was added to all wells, and the plates were incubated at 37 °C for 6-8 h. These plates were then analyzed following absorption on a Multiscan JX (Ver1.1 system; Thermo Labsystems), using a test wavelength of 595 nm and a reference wavelength of 650 nm. Unless specified in the text, no mixing was performed, and no medium was removed prior to the addition of any ingredient.

[³H]Thymidine Incorporation—Cells (5 x 10³ cells/well) were seeded onto 96-well plates and incubated with test substances for a selected period at 37 °C in 5% CO₂. Cells were labeled with 20 nCi/well of [³H]thymidine (24 h, 37 °C) and then trypsinized for isolation after the washout of ³H. Cells were harvested on glass fiber filters by using a multiple cell harvester (Labo mash). Filters were air-dried and placed in scintillation vials containing 2 ml of ACSII (GE Healthcare Bioscience) scintillation fluid. The cell-associated ³H radioactivity was determined on a model 5801 liquid scintillation counter (Beckman).

Solutions—For electrophysiology bath solutions, we used normal HEPES solution, of the following composition, in mM: NaCl 137, KCl 5.9, CaCl₂ 2.2, MgCl₂ 1.2, glucose 14, HEPES 10, NaOH to pH 7.4. For Ca²⁺ imaging, we used normal HEPES solution and Ca²⁺-free HEPES solution, of the following composition, in mM: NaCl 137, KCl 5.9, MgCl₂ 1.2, glucose 14, HEPES 10, EGTA 5, NaOH to pH 7.4. For DiBAC₄(3) fluorescence imaging, we used 140 mM K⁺ solution, of the following composition, in mM: NaCl 2.9, KCl 140, CaCl₂ 2.2, MgCl₂ 1.2, glucose 14, HEPES 10, NaOH to pH 7.4. All whole cell patch clamp pipettes contained, in mM: KCl 140, MgCl₂ 4, ATP-Na₂ 5, EGTA 5, CaCl₂ 4.3, HEPES 10, KOH to pH7.2 (pCa 6.0) and KCl 22, potassium aspartate 118, MgCl₂ 4, ATP-Na₂ 5, EGTA 0.05, HEPES 10, KOH to pH 7.2.

Statistics—Pooled data were expressed as mean ± S.E. Statistical significance was examined using the paired or unpaired Student's t test for two groups, respectively, and Scheffe's multiple comparisons for three groups. In all figures, ^* and #, and ^** and ^##, indicate statistical significance at p values of 0.05 and 0.01, respectively.

Drugs and Chemical Agents—The following compounds were used in this study: apamin, ATP, UDP, ATPS, thapsigargin, adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS), UCL1684, U0126, Go6983, and PD98059 (Sigma); barium chloride (Ba²⁺) and lanthanum chloride (La³⁺) (Wako, Osaka, Japan); DCEBIO (Tocris); UTP (Yamasa shouyu, Chiba, Tokyo); ADP (Oriental Yeast Co., Ltd., Tokyo, Japan); SK&F 96365 (Funakoshi, Tokyo, Japan); and L-NAME (Dojindo, Osaka, Japan). The solvents (distilled water and Me₂SO) had no effect on [Ca²⁺]_i, membrane current, and membrane potential, when the corresponding amount was applied by superfusion.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Identification of Purinoceptor Subtypes in t-BBEC 117—Application of 10 µM ATP to t-BBEC 117 consistently resulted in a marked rise of [Ca²⁺]_i (Fig. 1A, panel a). This ATP-induced [Ca²⁺]_i rise (805.0 ± 102.2 nM in the control) was almost abolished by pretreatment with 5 µM thapsigargin (TG) (33.8 ± 7.9 nM), suggesting that it is due to Ca²⁺ release from intracellular storage sites (Fig. 1A, panels a and b). To determine subtype of purinoceptor expressed in t-BBEC 117, RT-PCR analysis was performed using primers for 16 subtypes of P2XR and P2YR. Transcripts of P2X₁, P2X₄, P2X₆, P2X₇, P2Y₁, P2Y₂, and P2Y₁₂ were detected (Fig. 1B). Based on these results, the purinoceptor subtypes functionally expressed in t-BBEC 117 are considered to be P2Y₁, P2Y₂, and/or P2Y₁₂, because P2YRs are known to couple with G_q/11 proteins and inositol 1,4,5-trisphosphate signal transduction pathways to induce Ca²⁺ release from endoplasmic reticulum. -actin primers were used to confirm that the products generated were representative of RNA (542 bp) and not contaminated with genomic DNA (intron containing 767-bp band), because these primers were designed to span an intron as well as three exons (data not shown).

To examine further the subtypes of purinoceptor in t-BBEC 117, the potency of P2R agonists in eliciting these [Ca²⁺]_i responses were compared. ATP, ADP, UTP, UDP, and ATPS at concentrations from 0.1 to 100 µM were applied consecutively. The dose-response curves are shown in Fig. 1C. The 50% effective concentrations (EC₅₀) were (in µM); ATP: 3.0 ± 0.7, ADP: 0.82 ± 0.03, UTP: 0.93 ± 0.05, UDP: 36.5 ± 1.67, and ATPS: 6.2 ± 0.4. The order of the potency was: ADP = UTP > ATP > ATPS > UDP. This pattern of results suggests the involvement of P2YR in this response (7). The contribution of P2Y₁ for this Ca²⁺ response was examined using ADP and A3P5PS, because these compounds are specific agonist and antagonist for P2Y₁, respectively (23). The ADP-induced Ca²⁺ response was markedly suppressed by 10 µM A3P5PS (Fig. 1D). Taken together, these results strongly suggested that P2Y₁ is mainly responsible for the [Ca²⁺]_i response to ATP in t-BBEC 117.

View larger version (35K): [in this window] [in a new window]   FIGURE 1. Identification of purinoceptor subtypes in t-BBEC 117. A, ATP-induced [Ca2+]i changes and their susceptibility to thapsigargin (TG) were examined in t-BBEC 117. a, ATP (10 µM) was applied twice. The second ATP application was preceded by treatment with 5 µM TG. Note that the response to the second ATP application was almost abolished by TG treatment. b, summarized data obtained from experiments shown in a. Data were obtained from 20 cells. **, p < 0.01 versus ATP alone. B, expression of purinoceptor subtypes in t-BBEC 117 was examined by RT-PCRs (35 cycles) using 16 pair primers. BB and 117 denote bovine brain and t-BBEC 117, respectively. C, [Ca2+]i changes due to P2R stimulation were characterized pharmacologically by using five agonists: ATP, ADP, UTP, UDP, and ATPS. Data are summarized and illustrated as concentration-response relationships for five agonists in terms of evoked changes in [Ca2+]i. The increase in [Ca2+]i at each concentration of ATP (), ADP (), UTP (•), UDP (), or ATPS () was normalized by the maximum [Ca2+]i increase. Data were obtained from 81, 52, 75, 51, and 67 cells for each agonist. D, responses to ADP are blocked by the P2Y1 receptor antagonist, A3P5PS. a, typical recordings of [Ca2+]i changes in response to 1 µM ADP in the presence and absence of 10 µM A3P5PS. b, summarized data obtained from typical experiments shown in a. Values in the presence of 10 µM A3P5PS or after washout have been normalized by the [Ca2+]i response to the first ADP application in each preparation. Data were obtained from 58 cells (n) and from 6 dishes (N). **, p < 0.01.

The Ca²⁺ entry through cation channels, presumably TRP channels, is often not voltage-dependent. Instead it is driven by membrane potential gradient (24). Accordingly a membrane hyperpolarization by activation of K⁺ channel can enhance Ca²⁺ entry through non-voltage-dependent cation channels (24). To examine the possibility that the Ca²⁺ rise due to the initial release activates Ca²⁺-activated K⁺ (K_Ca) channels, these effects of specific blockers of K_Ca on the [Ca²⁺]_i were examined. Iberiotoxin and TRAM-34, selective blockers of large and intermediate conductance K_Ca (BK and IK) channels, respectively, were not effective (not shown). In contrast apamin, a blocker of small conductance K_Ca (SK) channels significantly reduced the [Ca²⁺]_i rise (Fig. 2C). Apamin significantly reduced the relative value of [Ca²⁺]_i from 58.5 ± 1.6% to 31.0 ± 1.2%. These important new findings suggest the possibility that the activation of SK2 can enhance Ca²⁺ entry and form part of a positive feedback mechanism for the regulation of [Ca²⁺]_i in t-BBEC 117.

View larger version (35K): [in this window] [in a new window]   FIGURE 2. Expression of non-selective cation channels in t-BBEC 117. A, panel a, typical recordings of [Ca2+]i changes in responses to 10 µM ATP applied consecutively three times. b, in the second application of ATP, Ca2+ in the external solution was omitted (Ca2+-free). c, summarized data from experiments shown in a and b. Each [Ca2+]i signal was integrated, and this value corresponding to the first ATP application was taken as 100%. The baseline for integrates of [Ca2+]i is indicated by the dotted line. Data were obtained from 106 and 86 cells in control and Ca2+-free conditions, respectively. **, p < 0.01 versus control. B, effects of 10 µM La3+ (a) and Gd3+ (b) on [Ca2+]i rise induced by 10 µM ATP. c, the results shown in a and b have been expressed in the same manner as in A, except that the response to the second ATP application was taken as 100% and the third response was evaluated. Effects of 10 µM SK&F 96365 was also obtained in this manner. Data were obtained from 37-46 cells. **, p < 0.01 versus control. C, effects of 100 nM apamin on [Ca2+]i response to ATP. The second ATP application was made either in the absence (a) or presence (b) of apamin. c, results are expressed in the same manner as in A. Data were obtained from 106 and 82 cells. **, p < 0.01 versus control.

View larger version (37K): [in this window] [in a new window]   FIGURE 3. Activation of Ca2+-activated K+ current or membrane hyperpolarization by ATP. A, panel a, effects of 10 µM ATP on membrane current were examined under whole cell voltage clamp in the absence and presence of 10 nM apamin. Ramp voltage clamp waveform from -120 to +60 mV were applied once every 10 s (holding potential, -40 mV). The inward current amplitude measured at -120 mV was plotted against time. b, the relationship between current density and voltage for the ATP-activated current component was obtained by subtraction of currents in the absence (i) and presence (ii) of ATP. The I-V relationships in the absence and presence of apamin were measured at the time shown in A, panel a by two pairs of black and gray dots. c, the relationship between current density and voltage of apamin-sensitive component was obtained by subtraction using the data shown in b at membrane potential from -120 mV to +60 mV, in 20-mV steps. Data were obtained from six cells. B, measurement of changes in DiBAC4(3) fluorescence in response to apamin. a, fluorescence intensity of DiBAC4(3) was measured from clusters of t-BBEC 117 in the continuous presence of 100 nM DiBAC4(3) after loading for 30 min. The fluorescence intensity measured at 1 min before the application of 10 µM ATP was taken as 1.0. b, summarized values of DiBAC4(3) fluorescence intensity in the control, in the presence of 10 µM ATP, and in the simultaneous presence of 10 µM ATP and 100 nM apamin are illustrated from the experiments shown in a. Data were obtained from six dishes. **, p < 0.01. C, panel a, measurement of changes in DiBAC4(3) fluorescence in response to 10 µM DCEBIO, and addition of 10 nM apamin. b, summarized data obtained from eight dishes. *, p < 0.05.

Molecular Identification of SK2 Channel and Effects of SK2 Channel Knockdown by siRNA in t-BBEC 117—To identify the molecular basis of K_Ca channels in t-BBEC 117, mRNA expression of five types of K_Ca channel -subunits; BK, SK1, SK2, SK3, and IK (SK4), were examined by RT-PCR. Expression of BK and IK were barely detectable (Fig. 4A). In contrast, the expression of SK1, SK2, and SK3 could be detected unambiguously. The sequence analysis of PCR products for SK1 and SK2 revealed that the sequences of bovine SK1 and SK2 are highly homologous to those of human and mouse, and variants with a 3-amino acid insertion were found both in SK1 and SK2 (data not shown). Those sequences were submitted to the DNA Data Bank of Japan (DDBJ), EMBL, and GenBank^TM as the accession number AB176709 [GenBank] for bovine SK1 and AB114474 [GenBank] for bovine SK2.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Molecular expression of KCa in t-BBEC 117 and effects of siRNA to knock-down SK2. A, RT-PCR (35 cycles) analysis was performed using six pair primers for Ca2+-activated K+ channels and -actin. B, Western blot analysis for identification of SK channel proteins in t-BBEC 117 and hSK channels (SK1-SK3) in HEK293 cells transfected with corresponding cDNAs, serves as the positive control. C, effects of SK2 knockdown on the relationship between current density and voltage of apamin-sensitive currents examined using voltage clamp waveform from -140 to +60 mV from Vh of -40 mV. a, the relationship between current density and voltage of apamin-sensitive component was obtained by subtraction of these digital records in the same manner as shown in Fig. 3A (panel c). b, the summarized values of apamin-sensitive current density at -140 mV. Data were obtained from seven cells. **, p < 0.01 versus siRNA-SK2. D, effects of SK2 knockdown on ATP-induced [Ca2+]i rise. a, [Ca2+]i records obtained from cells treated with siRNA-SK2 and siRNA-random are superimposed. b, summarized data of [Ca2+]i, which were measured at the time indicated by an arrow in D, panel a. Data were obtained from 37 and 31 cells in siRNA-SK2 and -random, respectively. **, p < 0.01 versus siRNA-SK2.

To obtain direct evidence for the hypothesis that SK2 channel activation can contribute to ATP-induced [Ca²⁺]_i rise, and that this process forms part of a positive feedback mechanism, including the enhancement of Ca²⁺ entry by membrane hyperpolarization, we next employed an siRNA gene-silencing approach to suppress selectively the functional SK2 expression. The I-V relationship of the current component, which was activated by ATP and susceptible to 100 nM apamin, was measured with a whole cell patch clamp method in t-BBEC 117 (Fig. 4C, panel a). To compare SK2 currents quantitatively, [Ca²⁺]_i in the pipette solution and presumably in the cytosol was maintained at 1 µM with Ca²⁺-EGTA buffer in the pipette-filling solution. In t-BBEC 117 transfected with SK2-silencing siRNA (siRNA-SK2), the membrane current elicited by the voltage clamp waveform ramp was markedly reduced in comparison with those treated with random sequence of siRNA (siRNA-random). Again, the apamin-sensitive current in siRNA-random had the reversal potential of -63.0 ± 6.6 mV (n = 7). Fig. 4C (panel b) shows the change () in the current density at -140 mV in the cells transfected with siRNA-SK2 or -random (-0.22 ± 0.19 and -2.06 ± 0.33 pA/picofarad, respectively). Specific loss of SK2 current by the treatment with siRNA-SK2 was also confirmed in HEK293 cells stably expressing SK2 (data not shown). Fig. 4D (panel a) shows the traces of [Ca²⁺]_i response to ATP in the cells transfected with siRNA-SK2 or -random. The sustained phase of [Ca²⁺]_i was markedly reduced by SK2-specific knockdown. The concentration of [Ca²⁺]_i at the time to withdrawal of ATP (arrowhead in Fig. 4D, panel a) is summarized in Fig. 4C (panel b): 144.1 ± 11.2 nM in siRNA-SK2 and 261.2 ± 25.2 nM in siRNA-random. These results indicate that SK2 channels can contribute to sustained phase of Ca²⁺ entry induced by ATP stimulation and that SK2 channels play a key role in the positive feedback mechanism in Ca²⁺ regulation.

Contribution of SK2 Channels to the Enhancement of Cell Proliferation and [³H]Thymidine Incorporation by ATP—In HaCaT keratinocytes, it has been reported that cell proliferation is enhanced by the activation of metabotropic P2Y receptors and the subsequent increase in [Ca²⁺]_i (2). In the next series of experiments, we examined whether the sustained [Ca²⁺]_i rise in response to ATP enhances cell proliferation of t-BBEC 117. The MTT assay, based on the principle that the tetrazolium ring is cleaved in active mitochondria, was used. ATPS, a non-hydrolyzed ATP analogue, UCL1684, a nonpeptide SK channel blocker (26), and La³⁺, a TRP channel blocker, were used to stimulate P2YRs, to block SK2, and to inhibit Ca²⁺ entry, respectively. In Fig. 5A (panel a), cells were cultivated for 0, 24, 48, or 72 h in the absence or presence of 100 µM ATPS, and the cell growth was expressed as relative value based on time 0 data (see "Experimental Procedures"). The cell proliferation for 24 and 48 h was significantly increased by the presence of ATPS. The cell proliferation for 24, 48, and 72 h was enhanced by incubation with ATPS to 220, 190, and 170% of the control, respectively. Fig. 5A (panel b) shows that ATPS in the concentration range (10-100 µM) was applied just after the start of cell culture for 48 h. These results show that ATPS promoted cell proliferation in a dose-dependent manner (control, 1.499 ± 0.052; 10 µM ATPS, 1.523 ± 0.206; 30 µM ATPS, 1.586 ± 0.176; and 100 µM ATPS; 1.937 ± 0.164). This enhancement of cell proliferation by 100 µM ATPS was significant. Addition of 10 µM La³⁺ reduced this 100 µM ATPS-induced enhancement (Fig. 5A, panel c). Addition of 100 nM UCL1684 and a mixture with 10 µM La³⁺ abolished the ATPS-induced enhancement (control, 1.499 ± 0.052; ATPS, 1.937 ± 0.164; ATPS plus La³⁺, 1.592 ± 0.069; ATPS plus UCL1684, 1.575 ± 0.073; mixture, 1.545 ± 0.079). Only UCL1684 or La³⁺ without ATPS did not change the cell proliferation (data not shown).

View larger version (38K): [in this window] [in a new window]   FIGURE 5. Effects of inhibitor on cell proliferation and [3H]thymidine incorporation promoted by ATPS. A, panel a, the proliferation of t-BBEC 117 was increased by ATPS in a time-dependent manner. Cell growth was measured with the MTT method. These values were normalized to data obtained at the start of cell culture (1.0). Data were obtained from four sample wells. *, p < 0.05 versus control. b, proliferation of t-BBEC 117 was increased by ATPS in a dose-dependent manner. Cells were cultivated for 48 h in the absence (Control) or presence of 10, 30, and 100 µM ATPS. Data were obtained from four sample wells. *, p < 0.05 versus control. c, effects of 10 µM La3+, 100 nM UCL1684, and both of these agents on ATPS-evoked cell proliferation measured after the treatment with ATPS for 48 h. Data were obtained from four separate wells. *, p < 0.05 versus control; # and ##, p < 0.05, 0.01 versus ATPS. B, panel a, the [3H]thymidine incorporation was enhanced by 100 µM ATPS in a time-dependent manner in t-BBEC 117. Data were obtained from four separate wells. *, p < 0.05 versus control. b, effects of La3+, UCL1684, and both together as a mixture on ATPS-induced enhancement of [3H]thymidine incorporation measured after 48-h treatment. c, effects of 300 µM L-NAME and 1 µM Go6983 on ATPS-induced enhancement of [3H]thymidine incorporation measured after 48-h treatment. d, effects of 100 nM U0126 and 1 µM PD98059 on ATPS-induced [3H]thymidine incorporation measured after 48-h treatment. Data were obtained from five separate wells. **, p < 0.01 versus control; # and ##, p < 0.05 and 0.01 versus ATPS.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
BCECs form an essential component of the BBB. They exhibit distinct characteristics that differ from peripheral endothelial cells. These include the presence of intercellular tight junctions with very low transcellular vesicular transport activity by surrounding astrocytes (17). Recently, evidence has been reported showing that several endogenous substances, including ATP, function as signal transmitters in the intercellular information transmission pathway between neurons, glial cells, and presumably BCECs (27, 28). Signals are transmitted between BCECs and astrocytes by substances such as intracellular inositol 1,4,5-trisphosphate (through gap junctions) and by extracellular ATP, which has been released into the intercellular clefts (29, 30). In t-BBEC 117, the activation of purinoceptors resulted in a [Ca²⁺]_i rise, due to the release from endoplasmic reticulum and a later Ca²⁺ influx. This second response was elicited as the consequence of the entry from store-operated Ca²⁺ channel (SOC), as has been reported previously from studies on human bronchial epithelial cells (8). Based on our RT-PCR analysis, the strong suppression of ATP-induced [Ca²⁺]_i rise by pretreatment with TG, and the pharmacological characterization using agonists and antagonist, the predominant purinoceptors in t-BBEC 117 are P2Y₁ and/or P2Y₂. Although P2XR transcripts were also detected by RT-PCR, their contribution to ATP-induced [Ca²⁺]_i rise is rather unlikely based on these results.

It has been shown that ATP promotes proliferation in several types of cells (2, 3). This occurs via P2YR activation and both Ca²⁺ release and influx. The increase in [Ca²⁺]_i has been suggested to be the central signal pathway for the promotion of cell proliferation. However, the specific mechanism underlying ATP-promoted cell proliferation has not been determined. On the other hand, the evidence for a significant role of K⁺ channels in cell proliferation has been obtained with various types of cells, particularly in non-excitable cells. In these preparations, a membrane hyperpolarization due to K⁺ channel activation enhances Ca²⁺ influx through voltage-independent Ca²⁺-permeable channels (4). As an example, the activation of SK4 channels is essential for the differentiation of T-lymphocytes in immune response (31) and the proliferation of vascular smooth muscle cells under pathophysiological conditions (32, 33).

Our results clearly show that SK2 is predominantly expressed, compared with other selected K⁺ channels (SK1-3, IK, and BK channels) in t-BBEC 117. Although the expression of BK channels and its function in regulation of the permeability of blood-brain tumor barrier has been reported (34), expression and functions of K⁺ channels in BCECs has not been reported previously. The knock down of SK2 channels by siRNA markedly reduced apamin-sensitive current and decreased the sustained phase of [Ca²⁺]_i rise induced by ATP. In contrast, this maneuver did not affect the transient phase of [Ca²⁺]_i rise. One of the most important results in this study is that the application of 100 nM UCL1684, a stable and specific blocker of SK1-3, almost completely removed the ATPS-induced enhancement of proliferation (Fig. 5A) and [³H]thymidine incorporation (Fig. 5B) in t-BBEC 117. To explain the causal relationship between these two observations, we prepared the novel hypothesis that the activation of SK2 channels (due to Ca²⁺ release by inositol 1,4,5-trisphosphate formation via G-protein-coupled receptor stimulation) plays an obligatory role for the secondary Ca²⁺ influx through voltage-independent Ca²⁺-permeable channels. These form a novel positive feedback mechanism for [Ca²⁺]_i regulation triggered by ATP. Because it has been suggested that the constitutive release of ATP from astrocytes keeps the concentration of ATP at 10-100 µM in the cleft between astrocytes and BCECs (35), this positive feedback mechanism may be functional even under physiological conditions. In any case the observed ATP-induced promotion of cell proliferation is attributable to this novel mechanism.

The Ca²⁺ entry pathway triggered by ATP application is most likely the non-selective cation channel, because the reversal potential of ATP-induced current in the presence of apamin was -4 mV (Fig. 3A, panel b). This non-selective cation channel corresponds to a so-called "receptor-operated Ca²⁺-permeable channel" and/or "store-operated" (SOC) ones (36). Because TG almost completely abolished the ATP-induced [Ca²⁺]_i rise, it is likely that ATP-induced Ca²⁺ release increased the activity of SOC channels and that membrane hyperpolarization by SK2 channel activation increased the driving force of Ca²⁺ entry through the channels. The possibility that P2Y receptors themselves can contribute to the Ca²⁺ entry as receptor-operated channels is unlikely as discussed above.

TRP channels have been reported to be the pathway responsible for operating Ca²⁺ influx in non-excitable cells, in which voltage-dependent Ca²⁺ channels are not expressed (15, 24, 37, 38). In t-BBEC 117, we detected neither voltage-dependent Ca²⁺ channel currents nor [Ca²⁺]_i rise blocked by nicardipine (data not shown). Based on the RT-PCR analysis (supplemental Fig. S1), t-BBEC 117 expressed transcripts of TRPC1, TRPC3, and TRPC5. Any of these could be possible molecular candidates for SOC channels (36, 39, 40). Although TRPC5 homotetramer channels are activated by Gd³⁺ in 10 µM (41), ATP-induced [Ca²⁺]_i rise in t-BBEC 117 was blocked by 10 µM Gd³⁺. In addition, we could not detect the TRPC5 protein in t-BBEC 117 (data not shown). Moreover, the lack of effects to SK&F96365 is similar to SOC in cerebral arteriole smooth muscle cells, where TRPC1 is suggested to be the responsible molecule (42). In contrast, it has been shown that TRPC3 expressed in HEK293 cells is strongly blocked by 10 µM SK&F96365 (43). The possibility that TRPM7, which was also detected as a transcript in t-BBEC 117 by RT-PCR, contributes to the Ca²⁺ entry cannot be ruled out completely, although so far no literature suggests that TRPM7 works as the SOC (40). We suggest that the TRP channel subtype, which is responsible for Ca²⁺ entry during application of ATP in t-BBEC 117, is possibly the TRPC1 and/or the complex of TRPC1 and TRPC3. However, the molecular basis for the Ca²⁺ entry channel remains to be determined.

It has been reported that the mitogenic effect of UTP is mediated through a P2Y₂ receptor that involves the activation of Ras/Raf/MEK/MAPK pathway associated with cell proliferation in cultured C₆ glioma cells (44). UTP-induced MAPK activation is modulated by Ca²⁺, protein kinase C, and tyrosine kinase. It has been also reported that eNOS is involved in thymidine incorporation and cell proliferation in human umbilical vein endothelial cells (45). Based on the experiments using pharmacological tools such as L-NAME as an eNOS inhibitor, Go6983 as a protein kinase C inhibitor, U0126 and PD98059 as MEK1/2 inhibitors, it can be suggested that the signal pathway of ATP-induced increase in DNA synthesis in t-BBEC 117 following the rise of [Ca²⁺]_i is also mediated by eNOS, MEK1/2, and MAPK, whereas protein kinase C activation may not be involved in the pathway.

In conclusion, SK2 channels have an obligatory role in a novel positive feedback mechanism, which is responsible for the sustained [Ca²⁺]_i rise in response to P2Y_1/2 activation. The activation of SK2 channels hyperpolarized the endothelial cells and hence increased the driving force for Ca²⁺ entry through TRPC in t-BBEC 117. The sustained [Ca²⁺]_i rise is responsible for the promotion of cell proliferation by ATP. This ATP-induced feedback mechanism via SK2 activation may contribute to turnover of BCECs to maintenance of BBB function under physiological conditions.

	FOOTNOTES

 
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB114474 [GenBank] .

^* This work was supported by a grant-in-aid for Scientific Research (B) from the Japan Society for the Promotion of Science and by a grant-in-aid for Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation (to Y. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and Tables S1 and S2.

¹ To whom correspondence should be addressed. Tel./Fax: 81-52-836-3431; E-mail: yimaizum{at}phar.nagoya-cu.ac.jp.

² The abbreviations used are: BBB, blood-brain barrier; BCEC, brain capillary endothelial cell; [Ca²⁺]_i, intracellular Ca²⁺ concentration; K_Ca, Ca²⁺-activated K⁺ channel; SK2, small conductance Ca²⁺-activated K⁺ channel type 2; TRP, transient receptor potential; K_ir2.x, inward rectifier K⁺ channels; TG, thapsigargin; A3P5PS, adenosine 3'-phosphate 5'-phosphosulfate; DCEBIO, 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one; ATPS, adenosine 5'-(3-thiotriphosphate) tetralithium salt; UCL1684, 6,10-diazo-(3-thiotriphosphate)3(1,3),8(1,4)-dibenzena-1,5(1,4)-diquinolinacyclodecaphane; DiBAC₄(3), bis-(1,3-dibutylbarbituric acid) trimethine oxonol; siRNA, short interference RNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MEK1/2, MAPK kinase 1/2; MAPK, mitogen-activated protein kinase; nt, nucleotide(s); RT, reverse transcription; eNOS, endothelial nitric-oxide synthase; BK, large conductance K_Ca; IK, intermediate conductance K_Ca; SOC, store-operated channel; L-NAME, N_w-nitro-L-arginine methyl ester hydrochloride.

	ACKNOWLEDGMENTS

 
We thank Manami Yamamoto for technical assistance and Dr. Wayne R. Giles (University of California, San Diego, CA) for providing data acquisition and analysis programs and for reading the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Abbott, N. J. (2002) J. Anat. 200, 629-638[CrossRef][Medline] [Order article via Infotrieve]
2. Lee, W. K., Choi, S. W., Lee, H. R., Lee, E. J., Lee, K. H., and Kim, H. O. (2001) J. Dermatol Sci. 25, 97-105[CrossRef][Medline] [Order article via Infotrieve]
3. Franke, H., and Illes, P. (2006) Pharmacol. Ther. 109, 297-324[CrossRef][Medline] [Order article via Infotrieve]
4. Parekh, A. B., and Penner, R. (1997) Physiol. Rev. 77, 901-930[Abstract/Free Full Text]
5. Berridge, M. J., Bootman, M. D., and Lipp, P. (1998) Nature 395, 645-648[CrossRef][Medline] [Order article via Infotrieve]
6. Berridge, M. J., Lipp, P., and Bootman, M. D. (2000) Nat. Rev. Mol. Cell. Biol. 1, 11-21[CrossRef][Medline] [Order article via Infotrieve]
7. King, B. F., Townsend-Nicholson, A., and Burnstock, G. (1998) Trends Pharmacol Sci. 19, 506-514[CrossRef][Medline] [Order article via Infotrieve]
8. Bahra, P., Mesher, J., Li, S., Poll, C. T., and Danahay, H. (2004) Br. J. Pharmacol. 143, 91-98[CrossRef][Medline] [Order article via Infotrieve]
9. Lang, F., Foller, M., Lang, K. S., Lang, P. A., Ritter, M., Gulbins, E., Vereninov, A., and Huber, S. M. (2005) J. Membr. Biol. 205, 147-157[CrossRef][Medline] [Order article via Infotrieve]
10. Muraki, K., Imaizumi, Y., Ohya, S., Sato, K., Takii, T., Onozaki, K., and Watanabe, M. (1997) Biochem. Biophys. Res. Commun. 236, 340-343[CrossRef][Medline] [Order article via Infotrieve]
11. Sollini, M., Frieden, M., and Beny, J. L. (2002) Br. J. Pharmacol. 136, 1201-1209[CrossRef][Medline] [Order article via Infotrieve]
12. Forsyth, S. E., Hoger, A., and Hoger, J. H. (1997) FEBS Lett. 409, 277-282[CrossRef][Medline] [Order article via Infotrieve]
13. Yang, D., MacCallum, D. K., Ernst, S. A., and Hughes, B. A. (2003) Invest. Ophthalmol. Vis. Sci. 44, 3511-3519[Abstract/Free Full Text]
14. Muraki, K., and Imaizumi, Y. (2001) J. Physiol. 537, 431-441[Abstract/Free Full Text]
15. Antoniotti, S., Lovisolo, D., Fiorio Pla, A., and Munaron, L. (2002) FEBS Lett. 510, 189-195[CrossRef][Medline] [Order article via Infotrieve]
16. Hosoya, K., Ohtsuki, S., and Terasaki, T. (2002) Int. J. Pharm. 248, 15-29[CrossRef][Medline] [Order article via Infotrieve]
17. Abbott, N. J., Ronnback, L., and Hansson E. (2006) Nat. Rev. Neurosci. 7, 41-53[CrossRef][Medline] [Order article via Infotrieve]
18. Sobue, K., Yamamoto, N., Yoneda, K., Hodgson, M. E., Yamashiro, K., Tsuruoka, N., Tsuda, T., Katsuya, H., Miura, Y., Asai, K., and Kato, T. (1999) Neurosci. Res. 35, 155-164[CrossRef][Medline] [Order article via Infotrieve]
19. Imaizumi, Y., Muraki, K., and Watanabe, M. (1989) J. Physiol. 411, 131-159[Abstract/Free Full Text]
20. Yamada, A., Gaja, N., Ohya, S., Muraki, K., Narita, H., Ohwada, T., and Imaizumi, Y. (2001) Jpn. J. Pharmacol. 86, 342-350[CrossRef][Medline] [Order article via Infotrieve]
21. Elbashi, R. S. M., Harbort, H. J., Webe, R. K., and Tusch, L. T. (2002) Methods 26, 199-213[CrossRef][Medline] [Order article via Infotrieve]
22. Hansen, M. B., Nielsen, S. E., and Berg, K. (1989) J. Immunol. Methods 119, 203-210[CrossRef][Medline] [Order article via Infotrieve]
23. Takasaki, J., Kamohara, M., Saito, T., Matsumoto, M., Matsumoto, S., Ohishi, T., Soga, T., Matsushime, H., and Furuichi, K. (2001) Mol. Pharmacol. 60, 432-439[Abstract/Free Full Text]
24. Nilius, B., and Droogmans, G. (2001) Physiol. Rev. 81, 1415-1459[Abstract/Free Full Text]
25. Sailer, C. A., Hu, H., Kaufmann, W. A., Trieb, M., Schwarzer, C., Storm J. F., and Knaus, H. G. (2002) J. Neurosci. 22, 9698-9707[Abstract/Free Full Text]
26. Malik-Hall, M., Ganellin, C. R., Galanakis, D., and Jenkinson, D. H. (2000) Br. J. Pharmacol. 129, 1431-1438[CrossRef][Medline] [Order article via Infotrieve]
27. Haydon, P. G. (2001) Nat. Rev. Neurosci. 2, 185-193[CrossRef][Medline] [Order article via Infotrieve]
28. Hansson, E., and Ronnback, L. (2003) FASEB J. 17, 341-348[Abstract/Free Full Text]
29. Paemeleire, K., and Leybaert, L. (2000) J. Neurotrauma 17, 345-358[Medline] [Order article via Infotrieve]
30. Braet, K., Paemeleire, K., D'Herde, K., Sanderson, M. J., and Leybaert, L. (2001) Eur. J. Neurosci. 13, 79-91[CrossRef][Medline] [Order article via Infotrieve]
31. Ghanshani, S., Wulff, H., Miller, M. J., Rohm, H., Neben, A., Gutman, G. A., Cahalan, M. D., and Chandy, K. G. (2000) J. Biol. Chem. 275, 37137-37149[Abstract/Free Full Text]
32. Cheong, A., Bingham, A. J., Li, J., Kumar, B., Sukumar, P., Munsch, C., Buckley, N. J., Neylon, C. B., Porter, K. E., Beech, D. J., and Wood, I. C. (2005) Mol. Cell 20, 45-52[CrossRef][Medline] [Order article via Infotrieve]
33. Srivastava, S., Li, Z., Lin, L., Liu, G., Ko, K., Coetzee, W. A., and Skolnik, E. Y. (2005) Mol. Cell. Biol. 25, 3630-3638[Abstract/Free Full Text]
34. Ningaraj, N. S., Rao, M., Hashizume, K., Asotra, K., and Black, K. L. (2002) J. Pharmacol. Exp. Ther. 301, 838-851[Abstract/Free Full Text]
35. Dubyak, G. R., and el-Moatassim, C. (1993) Am. J. Physiol. 265, C577-C606
36. Beech, D. J., Muraki, K., and Flemming, R. (2004) J. Physiol. 559, 685-706[Abstract/Free Full Text]
37. Kamouchi, M., Philipp, S., Flockerzi, V., Wissenbach, U., Mamin, A., Raeymaekers, L., Eggermont, J., Droogmans, G., and Nilius, B. (1999) J. Physiol. 518, 345-358[Abstract/Free Full Text]
38. Beech, D. J. (2005) Pflugers Arch. 451, 53-60[CrossRef][Medline] [Order article via Infotrieve]
39. Nilius, B., Viana, F., and Droogmans, G. (1997) Annu. Rev. Physiol. 59, 145-170[CrossRef][Medline] [Order article via Infotrieve]
40. Parekh, A. B., and Putney, J. W., Jr. (2005) Physiol. Rev. 85, 757-810[Abstract/Free Full Text]
41. Xu, S. Z., Zeng, F., Boulay, G., Grimm, C., Harteneck, C., and Beech, D. J. (2005) Br. J. Pharmacol. 145, 405-414[CrossRef][Medline] [Order article via Infotrieve]
42. Flemming, R., Xu S. Z., and Beech, D. J. (2003) Br. J. Pharmacol. 139, 955-965[CrossRef][Medline] [Order article via Infotrieve]
43. Zhu, X., Jiang, M., and Birnbaumer, L. (1998) J. Biol. Chem. 273, 133-142[Abstract/Free Full Text]
44. Tu, M. T., Luo, S. F., Wang, C. C., Chien, C. S., Chiu, C. T., Lin, C. C., and Yang, C. M. (2000) Br. J. Pharmacol. 129, 1481-1489[CrossRef][Medline] [Order article via Infotrieve]
45. Rojas, S., Rojas, R., Lamperti, L., Casanello, P., and Sobrevia L. (2003) Exp. Physiol. 88, 209-219[Abstract]

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