Introduction

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The blood-brain tumor barrier (BTB), formed by brain tumor capillaries, significantly limits delivery of therapeutic drugs to brain tumors (Groothuis, 2000). During the past decade, a considerable effort has been made and various strategies have been used to deliver selectively therapeutic drugs to brain tumors and injured brain. We developed the biochemical approach to increase BTB permeability and to enhance delivery of therapeutic drugs or small- to large-sized substances to brain tumors, including contrast-enhancing agents, antitumor compounds, therapeutic proteins, and viral vectors (Inamura et al., 1994; Nakano et al., 1996; Black et al., 1997; Matsukado et al., 1998; Rainov et al., 1998; Sugita and Black, 1998), to brain tumors selectively, with little or no drug delivery to the normal brain. This drug delivery strategy exploits the responsiveness of brain tumor capillaries to intravascular infusion of low doses of vasomodulators, such as bradykinin (BK), causing BTB permeability increase via a mechanism involving BK type 2 (B2) receptors (Inamura et al., 1994), nitric oxide (NO) (Nakano et al., 1996), cGMP (Sugita and Black, 1998), and calcium-dependent potassium (K_Ca) channels (Fig. 1).

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Biochemical and cellular pathway for BTB permeability regulation by specific vasomodulators for enhanced drug delivery to brain tumors. The activators used (shown in blue) include BK (Inamura et al., 1994; Black et al., 1997), the guanylate cyclase activator YC-1, NO donors (Ningaraj et al., 2000), and KCa channel opener NS-1619 (Ningaraj et al., 2001). The inhibitors used (shown in red) include B2 receptor antagonist HOE140 (Inamura and Black, 1994), NOS inhibitor N-nitro-L-arginine methyl ester (Nakano et al., 1996), the guanylate cyclase inhibitor ODQ (Sugita and Black, 1998), the phosphodiesterase inhibitor zaprinast (Sugita and Black, 1998), and the KCa channel inhibitor IBTX (Miura et al., 1999).

BK-induced changes in BTB permeability are transient (Inamura et al., 1994; Matsukado et al., 1998) and variable (Black et al., 1997). For example, the BK analog RMP-7 elicited a varying BTB permeability increase in patients with glioblastoma multiforme (Black et al., 1997). In the subsequent study (Liu et al., 2001b), we showed varying B2 receptor expression in different types of rat brain tumors, which correlated with varying responses to BK. Moreover, refractoriness of BTB response to prolonged infusion of BK (Inamura and Black, 1994), possibly due to B2 receptor internalization (Pizard et al., 1999), prompted us to identify additional molecular targets and cellular mechanisms to achieve consistent and selective drug delivery to brain tumors.

Recent evidence suggests that K_Ca channels are present in cerebral blood vessels to regulate cerebral blood vessel tone (Kitazono et al., 1995) and, probably, BBB permeability. K_Ca channels in brain are inhibited by iberiotoxin (IBTX), a highly specific (IC₅₀ = 68 pM) inhibitor (Wanner et al., 1999). K_Ca channel activity is triggered by depolarization and enhanced by an increase in cytosolic Ca²⁺ (Faraci and Heistad, 1998). Endothelium-dependent regulation of cerebral blood vessel functions is impaired in brain tumors (Cobbs et al., 1995), which might affect tumor capillary permeability in response to vasomodulators.

Evidence is accumulating that K_Ca channels play an important role in vasodilation mediated by BK (Berg and Koteng, 1997; Sobey et al., 1997), NO-donors (Bolotina et al., 1994), cGMP (Robertson et al., 1993; Lohse et al., 1998), and soluble guanylate cyclase (sGC) activators (Koesling, 1998). BK is thought to increase intracellular calcium ([Ca²⁺]_i), which could activate K_Ca channels, and alter membrane potential in cells (Miura et al., 1999). In brain endothelial cells, BK-induced K_Ca channel activation was potentiated by NS-1619, a selective agonist (EC₅₀ = 20 nM) for K_Ca channels (Nelson and Quayle, 1995), and attenuated by IBTX (Miura et al., 1999). We performed immunoblot and immunolocalization studies in rat cerebral capillary endothelial and tumor cells to elucidate differential responses of the BTB to K_Ca channel modulators. We also investigated the functional presence of putative K_Ca channels in rat brain endothelial and tumor cells by measuring the membrane potential changes induced by K_Ca channel modulators.

Brain capillary endothelial cells form "tight junctions" that are thought to regulate, in part, the movement of molecules across the blood brain barrier (BBB) and BTB. Using transmission electron microscopy, we investigated whether BK and NS-1619 induce accelerated formation of transport vesicles in both brain tumor capillary endothelium and tumor cells. Previously, a cGMP analog, dibutyryl cGMP (db-cGMP), was shown to increase the number of transcytotic vesicles in the normal cerebral capillary endothelium, possibly by K_Ca channel modulation (Joo et al., 1983). Stewart et al. (1987), however, reported an increased density of microvessel endothelial vesicles in human brain tumors. Moreover, a direct correlation between increased BTB permeability and vesicle formation has been shown in brain tumor capillaries (Stewart, 2000). We investigated whether K_Ca channels mediate increase in BTB permeability in response to BK or NS-1619 and whether such permeability increase is by increased endothelial tight junctions or accelerated transcytotic vesicle formation in tumor capillary endothelium. Additionally, we investigated whether BK and NS-1619 enhance transendothelial vesicular transport to a tracer, horseradish peroxidase (HRP), across the luminal-abluminal axis of tumor capillary endothelium. Herein, we report that K_Ca channels mediate the effect of BK, cGMP, NO donors, and sGC activators and therefore serve as a crucial target protein in the regulation of BTB permeability.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

BK (Sigma Chemical Co., St. Louis, MO), NS-1619, IBTX, YC-1, 1H-[1,2,4]oxadiazolo[4,3-a]quinozalin-1-one (ODQ) (Sigma/RBI, Natik, MA), a membrane potential assay kit (Molecular Devices, Sunnyvale, CA), [¹⁴C]-aminoisobutyric acid ([¹⁴C]AIB, 57.6 mCi/mmol; molecular mass, 103 Da; PerkinElmer Life Sciences, Boston, MA), and NO donors (Alexis Corporation, Läufelfingen, Switzerland), 2-(N,N-diethylamino)-diazonolate-2-oxide (PAPA-NONOate), (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl) amino] (DEA-NONOate) were used for the present study.

In Vivo BBB/BTB Permeability. All animal experiments were conducted in accordance with policies set by the Institutional Animal Care and Usage Committee and NIH (Bethesda, MD) guidelines. A rat xenograft tumor model was prepared using female Wistar rats weighing 180 to 200 g for BBB/BTB permeability studies, as described earlier (Sugita and Black, 1998). In brief, rats received an intracerebral injection of 1 × 10⁵ RG2 cells in 5 µl of medium with 1.2% methyl cellulose using a Hamilton syringe. The coordinates were 5 mm lateral to the bregma and 4.5 mm deep to the basal ganglia. RG2 cells were maintained in a monolayer culture in F-12 medium with 10% calf serum. Seven days after the tumor implantation, the rats were anesthetized with an i.p. injection of ketamine/xylazine. A polyethylene catheter was inserted retrograde through the external carotid artery into the common carotid artery until the bifurcation, ipsilateral to the tumor. The pterygopalatine artery was cauterized. A femoral vein was cannulated to administer [¹⁴C]AIB. In regional permeability studies, 5 min after the start of the intracarotid infusion, 100 µCi/kg [¹⁴C]AIB in 1 ml of PBS was injected as an intravenous bolus within a 5-s period. Arterial blood pressure was monitored throughout the experimental period with a blood pressure monitor (DigiMed, Louisville, KY) using the catheter surgically inserted into right femoral artery. Hematocrit and arterial blood gases, pCO₂ and pO₂, were measured with a blood gas machine (Bayer Diagnostics, Tarrytown, NY) using blood drawn from the catheter inserted in the left femoral artery for the purpose of a continuous blood draw. Cerebral laser-Doppler flowmetry (LDF) is a semiquantitative measure of cerebral blood flow (CBF) at a given area (Watanabe et al., 2001). To determine whether infusion of vasomodulators affect CBF at the tumor area, we performed LDF using a laser-Doppler (DRT4; Moore Instruments Ltd., Devon, England) equipped with a DP3 optical (1-mm diameter) probe during a 15-min intracarotid infusion of vasomodulators in some rats (n = 3/group). A shallow indentation was made on the skull above the right brain hemisphere with a low-speed drill for placement of the single-laser fiber probe (sensitive up to 2-3 mm deep). The LDF signal was recorded continuously during intracarotid infusion of vehicle or vasomodulators. Rats with abnormal CBF, blood gases, or blood pressure were excluded from the study.

K_i Measurement. The unilateral transfer constant K_i (microliters per gram per minute) was determined for radiotracer [¹⁴C]AIB in the tumor core, tumor-adjacent brain tissue, and contralateral brain tissue. The BBB permeability constant for [¹⁴C]AIB was determined using the quantitative autoradiographic (QAR) method (Sugita and Black, 1998). The K_i, which is an initial rate for blood-to-brain transfer of the tracer, was calculated by the method of Ohno et al. (1978). An optimum dose of BK (10 µg/kg/min), which was established in earlier studies (Inamura et al., 1994; Sugita and Black, 1998), was used for K_i measurements. Additionally, in a separate QAR study, varying doses of IBTX (0-0.52 µg/kg/min) were used to establish an optimal dose, which maximally attenuates the BK (10 µg/kg/min)-induced BTB permeability increase in RG2 tumor-bearing rats. Additional experiments were performed in rats with RG2 tumor by coinfusing NS-1619, DEA-NONOate, PAPA-NONOate, or YC-1 with IBTX (0.26 µg/kg/min) to investigate whether inhibition of K_Ca channels by IBTX attenuates permeability increases induced by these vasomodulators.

Dose Response Study. To establish the optimal and safe dose that selectively increases BTB permeability without appreciably altering systemic blood pressure, varying doses (0-60 µg/kg/min) of NS-1619 were used in a separate QAR study. Similarly, optimum doses of DEA-NONOate, PAPA-NONOate, or YC-1 that did not significantly affect systemic blood pressure were determined by Evans blue delivery studies in rats with RG2 tumors.

Time Course. To determine whether vasomodulator-induced BTB permeability increase in RG2 tumor-bearing rat was transient or could be sustained over a longer period, a separate QAR study was performed. BK (10 µg/kg/min), NS-1619 (26.66 µg/kg/min), or DEA-NONOate was intracarotidly infused separately for 15-, 30-, and 60-min periods, and K_i determined as described above.

Isolation of Brain Microvessels. To demonstrate the presence of K_Ca channels in rat brain capillaries, we isolated microcapillaries from rat brain using the method of Pardridge et al. (1985), with slight modifications. In brief, cortex (approximately 10 g) obtained from 20 female Wistar rat brains was washed with DMEM to remove meninges and superficial vessels. The tissue was minced and suspended in 20 ml of DMEM, and dispase was added to a final concentration of 0.5%. The suspension was placed at 37°C with constant stirring for 2 h, and the homogenate was centrifuged (1000g at 4°C for 20 min). The pellet was resuspended in 25 ml of DMEM containing 13% Dextran (molecular mass, 70 kDa) and centrifuged at 7000g for 20 min at 4°C. The pellet was resuspended in 5 ml of DMEM and passed through 200-µm Nylon mesh to retain microvessels and red cells, which were concentrated by centrifugation at 5000g for 10 min at 4°C. The pellet was resuspended in 2 ml of DMEM, laid on the top of a pre-established 50% Percoll gradient in Medium-199 (Invitrogen, Carlsbad, CA) and 30 mM Na-HEPES, pH 7.4, and centrifuged at 1000g at 4°C for 10 min. The middle layer, rich in microvessels, was collected and washed with DMEM to remove Percoll. This process was repeated once more to separate most of the contaminating red cells. The morphology of the microvessels was verified under a light microscope and stored at 180°C in a cryopreservation solution containing physiological buffer with 14% glycerol and 1.4% sorbitol, pH 7.4, until further use.

Preparation of Capillary Endothelial Cells. To investigate the expression of K_Ca channels in rat brain endothelial (RBE) cells, we isolated endothelial cells from the brains of neonatal rats using a modified method after Estrada et al. (1990). Briefly, the brains were rinsed with PBS, minced, and cleaned of meninges and superficial vessels. The tissue was homogenized and serially sieved through 70- and 20-µm Nylon meshes. The material retained was digested overnight with 1 mg/ml collagenase (Roche Molecular Biochemicals, Indianapolis, IN) in a shaker incubator at 37°C and centrifuged (1000g for 30 min). The pellet was resuspended in trypsin-EDTA (0.05% and 0.53 mM, respectively) for 30 min at 37°C. The sample was then centrifuged at 1000g for 30 min. To suppress the growth of astrocytes, the pellet was resuspended in culture medium [minimal essential medium containing D-valine, 20% fetal calf serum, 1% penicillin-streptomycin, 0.6% L-glutamine, 0.1% endothelial cell growth supplement (Upstate Biotechnology, Lake Placid, NY), and 0.1 µM dexamethasone] and plated in collagen-coated culture flasks. Culture medium was replaced every 24 h until a confluent monolayer of endothelial cells was formed. The homogeneity (>90-95%) of endothelial cells was verified by immunostaining with v. Willebrandt factor (Factor VIII; Dako Diagnostika, Hamburg, Germany). For potentiometric assays, RBE cells were seeded in a gelatin-coated, 96-well plate to obtain a monolayer.

Immunohistochemistry. For immunohistochemical studies, rats with intracerebral RG2 tumors were perfused transcardially with 0.1 M Tris/HCl-buffered saline (TBS), and brains were fixed with 4% paraformaldehyde in TBS buffer. Serial sections (15-20 µm thick) of brain were cut on a cryostat, washed in PBS, treated with or without 0.2% Triton X-100 for 10 min, and washed three times in TBS. Nonspecific binding sites on brain sections were blocked with 3% normal goat serum in TBS for 4 h. To determine whether K_Ca channels are present on capillary endothelial and tumor cells, brain tumor sections were incubated with a polyclonal anti-K_Ca channel antibody raised against its -subunit, and anti-Factor VIII monoclonal primary antibody. The colocalization of K_Ca channels and Factor VIII was accomplished with FITC and rhodamine-conjugated secondary antibodies using fluorescence and confocal microscopes. To demonstrate the expression of K_Ca channels in different rat glioma cells, a monolayer of RG2, C6, and 9L cells fixed with 4% paraformaldehyde were processed similarly and immunolocalized with anti-K_Ca channel polyclonal primary antibody and secondary IgG conjugated to either FITC or rhodamine. In addition, immunolocalization of K_Ca channels and Factor VIII was performed in a monolayer of primary RBE cells. To investigate the differential expression of K_Ca channels by Western blot analyses, the extracts of normal brain and tumor tissues obtained from Wistar rats, which harbored intracerebral RG2 tumor for 7 to 10 days post RG2 cell implantation, were used. Immunoblot analysis was also performed on RG2 cells, RBE cells, and rat brain capillaries. The samples for Western blot analyses were prepared after three washes with PBS and lysis in lysing buffer (50 mM Tris-HCl, pH 8.0, 20 mM EDTA, 0.1% Triton X-100, and 100 mM NaCl). The protein concentration of the samples was determined using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). The samples, containing 20 µg of protein, were boiled for 5 min and subjected to electrophoresis on 8% SDS-polyacrylamide gel electrophoresis. After the transfer of protein onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA), the membrane was incubated in blocking buffer (5% nonfat dairy milk in PBS) for 2 h at ambient temperature and probed with affinity-purified polyclonal anti-K_Ca channel primary antibody (1:500) in the blocking buffer overnight at 4°C. The membrane was washed four times with 0.1% Tween-20 in PBS, probed with the secondary antibody in the blocking buffer for 1 h at ambient temperature, and washed again with 0.1% Tween-20 in PBS. The signals were detected with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ). The polyclonal primary anti-K_Ca channel antibody raised against a pore-forming 125-kDa -subunit peptide sequence (1118-1135) was a generous gift from Dr. Knaus (University of Innsbruck, Innsbruck, Austria). Anti--actin monoclonal antibody was obtained from Santa Cruz Biotech (Santa Cruz, CA).

Confocal Microscopy and Image Analysis for Visualization of K_Ca Channels. Images were captured using a Leica (Mannheim, Germany) TCS SP laser-scanning confocal microscope (inverted) equipped with argon (Ar; 488 nm) and krypton (Kr; 568 nm) lasers. FITC signals for K_Ca channels expressed on cultured rat endothelial cells, glioma cells, or RG2 tumor-bearing rat brain sections were visualized using the 488-nm Ar laser line, and TRITC signals for Factor VIII were visualized using the 568-nm Kr laser line. Fluorescence signals for FITC and TRITC were displayed individually as green and red pseudo-color projections or merged as overlay projections to visualize possible colocalization of two different antigens within specific subcellular structures. Typically, stacks of 20 to 50 optical sections (512 × 512 pixel arrays) with a z-step of 0.2 to 0.5 µm between optical sections of single- or dual-stained samples were captured using an HCX PL APO 40X oil immersion objective lens (numerical aperture, 1.25) and an optical zoom factor between 1 and 4. Laser illumination was attenuated, and the pinhole was opened (2 times Airy disc) for improved collection of fluorescence emission signals. Detector gain and offset were adjusted at the beginning of each scan. When necessary, bright field contrast images of immunostained cells and capillaries in tumor-bearing brain sections were also acquired.

K⁺ Efflux Measurement. For the purpose of demonstrating K_Ca channel activity in isolated RBE and RG2 cells (ATCC, Manassas, VA), cells were suspended in 5 ml of growth media (DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 units/ml streptomycin), and approximately 1 × 10⁴ cells/well from cell passages 2 to 5 were coated in an eight-well chamber slide. Separately, RBE and RG2 cells were allowed to grow for 24 h to attain a monolayer. The cells were washed twice with potentiometric assay buffer (20 mM HEPES, 120 mM NaCl, 2 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, and 5 mM glucose, pH 7.4) at room temperature just before the assay. The electrode was calibrated with standard concentrations (1-50 µM) of KCl. The vehicle (0.5% ethanol in PBS) or NS-1619 (0-50 µM) dissolved in vehicle was added to cells, shaken gently, and incubated for 30 min at 37°C in a CO₂ incubator. Aliquots of assay buffer, adjusted to pH 7.4 and collected at various time points, were injected into a blood-gas diagnostic unit (Bayer Diagnostics, Midfield, MA) to measure K⁺ concentration. The values are expressed in nanomoles per liter.

Membrane Potential Assay. Functional activities of putative K_Ca channels in RBE and RG2 cells were measured by detecting changes in voltage across the cell membrane. The procedure is based on the method described by Gopalakrishnan et al. (1999) with some modifications. Instead of a fluorescent membrane potential dye DiBAC₄(3), we used the membrane potential assay kit provided by manufacturers of the FLEXstation (Molecular Devices). This kit provided a fast, simple, and consistent mix-and-read procedure. In brief, RBE and RG2 cells obtained from cell passages 2 to 5 were suspended in 5 ml of growth media (DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 units/ml streptomycin). The cell density was adjusted to 1 × 10³ cells/well and coated in sterile clear bottom black 96-well plates (Corning, Inc., Acton, MA), precoated with poly L-Lysine, and allowed to achieve a monolayer within 24 h. The monolayer cells were incubated with the membrane potential assay kit reagents for 30 min and read directly by FLEXstation. The anionic potentiometric dye that traverses between cells and extracellular solution in a membrane potential-dependent manner serves as an indicator of vasomodulator-induced voltage changes across the cell membrane. To determine the optimum dose that elicits detectable voltage changes across RBE and RG2 cell membranes, dose response studies were performed with 0 to 50 µM K_Ca channel agonist, BK or NS-1619, and K_Ca channel antagonist IBTX (10 nM) using the spectrofluorometer (FLEXstation) set to the following parameters; excitation (485 nm), emission (525 nm), and emission cut-off (515 nm) wavelengths. Additionally, the effects of the optimum dose of 10 µM BK or NS-1619 and IBTX (10 nM) on K_Ca channels activity in RBE and RG2 cells were determined.

Transmission Electron Microscopy. Seven days after RG2 tumor cell implantation, rats were infused intracarotidly with PBS, BK (10 µg/kg/min in PBS + 0.5% ethanol), or NS-1619 (2 µg/kg/min in PBS + 0.5% ethanol) for 15 min. Rats were infused with 10 ml of cold PBS and perfuse-fixed with 250 ml of 1% glutaraldehyde in PBS, pH 7.4, through the heart. Tumor-bearing brains were removed, and 1-mm³ tissue pieces encompassing tumor mass, brain surrounding tumor, and normal brain were cut. These samples were immersion-fixed in 1% glutaraldehyde at 4°C for 2 h, incubated overnight in 5% sucrose in 0.1 M phosphate buffer at pH 7.4 at 4°C, postfixed in 1% osmium tetroxide for 2 h, dehydrated in ascending ethanol series, propylene oxide, and embedded in Epon. Ultrathin sections were cut from selected blocks, mounted on Formar-coated grids, and double stained with uranyl acetate and lead citrate. For ultrastructural studies on vesicular transport, HRP was used as a tracer. Five minutes after initiating intracarotid infusion of PBS, BK, or NS-1619, 200 mg/kg HRP was injected as an intravenous bolus through the femoral vein. Ten minutes after HRP injection, rats were perfuse-fixed with 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate, pH 7.4, by cardiac puncture. Forty-micrometer thick vibratome sections of tumor-bearing brain sections were incubated in 5 ml of 0.06 M Tris buffer containing 3,3'-diaminobenzidine tetrahydrochloride (0.7 mg/ml) for 30 min at room temperature, followed by a 2- to 3-min treatment in the same buffer containing hydrogen peroxide (0.2 mg/ml). Sections were postfixed in 2% osmium tetroxide in 0.1 M phosphate buffer for 2 h, HRP localized with 3,3'-diaminobenzidine-peroxide treatment and observed under a JEOL electron microscope (JEOL, Tokyo, Japan) operating at 80 kV.

Quantitative Analysis. At least 10 profiles of capillaries from each group sectioned transversely and photographed at low magnification (7200×) were evaluated for their general features. Micrographs were placed on a digitizing screen, and structural features were measured using Scan Pro 4, a computer-assisted image analysis system (Jandel Scientific, Corte Madera, CA). Abluminal and luminal circumferences, areas of endothelial cytoplasm excluding nuclei and vacuoles, and mean thickness of endothelial cytoplasm were measured and compared with those in the control group. The mean thickness of the endothelium was calculated by subtraction of the luminal radius from the abluminal radius, which were obtained from the areas encircled by luminal and abluminal circumferences, respectively. For analysis of vesicles, we selected transversely sectioned capillary profiles, which were defined as vessels having a single, nearly round endothelial cell surrounded by basement membrane. Four test zones of endothelial cytoplasm were photographed at higher magnification (29,000×). They were chosen randomly, typically at the 3, 6, 9, and 12 o'clock positions on the capillary circumference. Three vessels were sampled from each rat, and each treatment group contained 5 to 6 rats to obtain 15 to 18 capillaries per group and a total number of 60 to 72 test zones. Intracytoplasmic vesicles were recognized as bilayered circular organelles with moderate electron density. Invaginations of the luminal or abluminal membrane were counted as vesicles if their neck was narrower than their maximum diameter. The cytoplasmic areas of the test zones, excluding nuclei and vacuoles, were digitally measured on 8 × 10-inch micrographs, and vesicle density was expressed as a number per micrometer squared of cytoplasm. The cumulative area of all vesicles contained in the cytoplasm was also measured to compare it with that of the cytoplasm. The proportion of total vesicular area to the cytoplasmic area was expressed as a percentage, to derive another parameter to characterize vesicular transport.

Cleft Index. Thirty junctional profiles were randomly selected from each group. They were evaluated by measuring the total length of the junctional profile and the cumulative length and area of the junctional clefts, which were areas where the outer leaflets of opposing membranes were not fused. To quantify the degree of opening of tight junctions, we determined two indices: a cleft index and a cleft area index. A cleft index was defined as the proportion of the junctional profile that was composed of clefts. It was expressed in the percentage of the cumulated length of clefts to the total length of junctional profile as described by Stewart et al. (1987). The cleft area is a translucent zone located between two electron-dense tight junctions and regions of adjacent capillary membranes, which run between the luminal and abluminal surfaces. A cleft area index, which we derived to identify enlarged clefts, was defined as the proportion of the cumulative area of the clefts compared with the total length of the junctional profile. We tested for a significant difference in junctional cleft index among tissue types and between treatment groups.

Statistics. Results are expressed as mean ± S.D. where applicable. For all in vivo permeability studies, we used n  4 rats/group unless otherwise stated. An unpaired two-tailed Student's t test was used to compare the control and treated groups. The statistical analyses of K_i, vesicle density, vesicular area, cleft index, and cleft area index comparison among different groups, with or without drug treatment, were performed using analysis of variance followed by either unpaired parametric analysis of Student's t test or by nonparametric analysis of Mann-Whitney's U test. P < 0.05 was considered statistically significant. The effect of intracarotid infusion of BK or NS-1619 was compared with that of PBS infusions by statistical analysis of the vesicular density and proportion of cumulative vesicular area.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

K_Ca Channels Mediate BK-Induced BTB Permeability Increase. BBB and BTB permeabilities were measured by QAR of cryosections from RG2 tumor-bearing rat brains as the unidirectional transfer constant K_i (µl/g/min) following injection of the radiotracer ([¹⁴C]AIB), subsequent to intracarotid infusion of various vasomodulators. To determine whether K_Ca channels mediate BK-induced increases in BTB permeability, rats with implanted intracerebral RG2 tumors received intracarotid infusion of PBS or BK, either alone or with IBTX, or received intracarotid infusion of NS-1619, either alone or with IBTX. The delivery of Evans blue dye (molecular mass, 961 Da) to implanted tumor and normal brain served as a qualitative measure of BTB and BBB permeabilities, respectively (Fig. 2A). The dye delivery was low in control rats infused with PBS. Intracarotid infusion of BK (10 µg/kg/min) or NS-1619 (26.66 µg/kg/min) for 15 min increased BTB permeability and resulted in enhanced delivery of Evans blue to tumor tissue, without any increase of dye delivery to peritumoral and contralateral normal brain (Fig. 2A). The BK- and NS-1619-induced BTB permeability increases were attenuated when coinfused with IBTX (Fig. 2A). These results demonstrate that K_Ca channels mediate the effects of both BK and NS-1619.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Quantitative increases in BTB permeability are shown. A, coronal sections of rat brain showing effects of BK (10 µg/kg/min) and NS-1619 (26.66 µg/kg/min; for dose-dependent effects, see Fig. 3B), with or without IBTX (0.26 µg/kg/min; for dose-dependent effects, see Fig. 3A) on Evans blue delivery to intracerebral RG2 tumor. B, color-enhanced autoradiographs of coronal brain sections showing little [14C]AIB delivery to RG2 tumor in PBS-treated but significantly enhanced delivery in BK- and NS-1619-treated rats. The BK- and NS-1619- induced increases in [14C]AIB delivery are significantly diminished when IBTX is coinfused. The scale at left shows pseudo-color intensities of tissue-calibrated 14C standards from 40 to 450 nCi/g specific activities for comparison. C, the mean Ki for [14C]AIB significantly increased after intracarotid infusion of BK (n = 6) or NS-1619 (n = 6) compared with PBS controls (n = 10) and significantly attenuated by coadministration of IBTX (n = 4). D, the mean Ki values increased significantly in response to intracarotid infusion of a dose of 26.66 µg/kg/min each of DEA-NONOate (DEA-NO; n = 4) or PAPA-NONOate (PAPA-NO; n = 4) compared with PBS-treated controls (n = 6). A cotreatment with IBTX (n = 4) significantly attenuated the effect of both NO donors. E, intracarotid infusion of YC-1 (13.33 µg/kg/min, n = 4) increased BTB permeability significantly when compared with PBS group (n = 6). The YC-1-induced Ki increase was significantly attenuated by coinfusion of selective inhibitor of sGC, ODQ (10 µg/kg/min, n = 4), or IBTX (n = 4). Data in C, D, and E are presented as mean ± S.D. , P < 0.001 versus PBS group; , P < 0.01 versus agonist-treated group.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   A, varying doses of IBTX (0, 0.065, 0.13, 0.26, and 0.52 µg/kg/min) were used to determine the optimum dose of IBTX that maximally attenuates BK-induced Ki increase in rats (n = 3 each dose) with RG2 tumor. IBTX at 0.26 µg/kg/min attenuated BK-induced Ki increase maximally (>70%). B, dose-dependent studies showed that the optimal dose of NS-1619 was 26.66 µg/kg/min, which caused a significant Ki increase in rat tumors (n = 4) without affecting MAP. C, time course study shows that BK-induced Ki increase was transient, which lasted for about 15 to 20 min, and at 30 and 60 min BK-induced Ki increase was not sustained. In contrast, DEA-NONOate and NS-1619 elicited sustained increases in mean Ki values for up to 30 and 60 min, respectively. Data in A, B, and C are presented as mean ± S.D. , P < 0.001 versus PBS group; , P < 0.01 versus PBS group; , P < 0.05 versus PBS group.

K_Ca Channel Activation and BTB Permeability. Intracarotid infusion of various concentrations of NS-1619 (0-60 µg/kg/min) increased BTB permeability in implanted intracerebral RG2 tumor in a dose-dependent manner (Fig. 3B). The data were obtained with rats (n = 3) for each dose of NS-1619. The mean arterial blood pressure (MAP) was not significantly affected by intracarotid infusion of NS-1619 at lower doses in rats. At higher doses (>26.66 µg/kg/min), however, a significant drop in MAP was observed (Table 1). Hence, further studies were performed with a dose of 26.66 µg/kg/min, which did not appreciably affect arterial blood pressure. Intracarotid infusion of NS-1619 (26.66 µg/kg/min) significantly increased K_i (26.5 ± 4.0 µl/g/min; P < 0.001) for [¹⁴C]AIB in the tumor center compared with vehicle-administered controls (8.0 ± 1.5 µl/g/min) (Fig. 2C). The NS-1619-induced increase in BTB permeability was significantly inhibited (12.4 ± 3.0 µl/g/min; P < 0.01) by coadministration with IBTX at a dose of 0.26 µg/kg/min for 15 min (Fig. 2C), indicating a K_Ca channel-specific effect.

View this table: [in this window] [in a new window]   TABLE 1 Physiological measurements during BBB/BTB permeability determination The physiological parameters were determined either before or during the infusion of vasomodulators. The values are mean ± S.D. of n  4 rats, except for CBF where three representative rats per group were used. pH is arterial pH; PaO2 is arterial oxygen; PaCO2 is arterial carbon dioxide; and MAP is mean arterial blood pressure. For BTB permeability by QAR, a vasomodulator dose was selected that did not appreciably alter MAP in rats. Data of treated group were significantly different (****P < 0.001; **P < 0.01) when compared to control group.

BTB Permeability Modulation is Generally Independent of CBF. Earlier we showed that infusion of BK (10 µg/kg/min) did not alter cerebral blood volume in tumor and normal brain (Liu et al., 2001a). In the present study, using a laser-operated Doppler, we showed that intracarotid administration of 26.66 µg/kg/min of NS-1619 or DEA-NONOate or YC-1 (13.33 µg/kg/min) did not significantly affect CBF (Table 1), but BTB permeability increased in the tumor area (Fig. 2, D and E). In contrast, infusion of BK (10 µg/kg/min) significantly (P < 0.01) increased CBF compared with the vehicle-administered group. In contrast to increased BTB permeabilities, BBB permeabilities in normal contralateral brain were unaffected by intracarotid infusions of BK, NS-1619, or NS-1619 with IBTX (Fig. 2).

The NO-cGMP System Modulates BTB Permeability. To investigate whether infusion of NO donors can increase BTB permeability through activation of K_Ca channels, RG2 tumor-bearing rats received intracarotid infusion of short-acting NO donors, DEA-NONOate (t_1/2 = 2 min), or PAPA-NONOate (t_1/2 = 15 min) with or without IBTX for 15 min. BTB permeability (K_i) in the tumor center was significantly increased by DEA-NONOate (32 ± 9.0 µl/g/min; P < 0.001) as well as by PAPA-NONOate (36.4 ± 4.0 µl/g/min; P < 0.001) compared with the control group (9.0 ± 2.0 µl/g/min) (Fig. 2D). Coinfused IBTX significantly attenuated the effect of DEA-NONOate (15.3 ± 2.3 µl/g/min; P < 0.01) and PAPA-NONOate (15.0 ± 1.7 µl/g/min; P < 0.01), demonstrating that NO-sensitive K_Ca channels may mediate NO-induced increase in BTB permeability (Fig. 2D).

Time Course. Permeability studies by QAR in rats bearing RG2 tumor showed that intracarotid infusion of NS-1619 or DEA-NONOate significantly enhanced and sustained delivery of [¹⁴C]AIB in the tumor center for 30 min (P < 0.001) and 60 min (P < 0.01) infusions (Fig. 3C). In contrast, a 30- and 60-min infusion of BK failed to sustain the initial increase of K_i (P < 0.001) attained at 15 min (Fig. 3C). Hence, BK-induced K_i increase was transient, possibly due to internalization of B2 receptors.

Membrane Potential Assays. The functional activity of putative K_Ca channels in a monolayer of RBE and RG2 cells, determined by measuring K⁺ efflux using a sensitive K⁺ electrode of a blood-gas diagnostic unit, increased with the addition of NS-1619 in a dose-dependent manner (Fig. 4A). Additionally, a decrease in the membrane potential of RBE and RG2 cells in response to the addition of BK or NS-1619, and a return to resting membrane potential with IBTX was measured spectrofluorometrically using a fluorescent probe. Both BK and NS-1619 elicited the depolarization effect in RBE cells (Fig. 4B). The depolarization actions of BK and NS-1619 were highly pronounced in RG2 (Fig. 4C) compared with RBE cells (Fig. 4B). These results demonstrate K_Ca channel activation in RBE and RG2 cells in response to BK or NS-1619. In other experiments, we observed that NS-1619 and BK caused a dose-dependent decrease in K⁺ dye-specific fluorescence intensity in both RBE and RG2 cells, which was reversed by IBTX (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   K+ efflux measurements and membrane potentiometric assays reveal the presence of functional KCa channels. A, correlation of varying doses of NS-1619 with increases in K+ efflux from NS-1619-treated RBE and RG2 cells, measured using a K+ electrode. The lines through the points represent linear regression (r = 0.97). B, changes in specific fluorescence intensity in RBE cells in response to 10 µM of BK or NS-1619. C, changes in membrane potential in RG2 cells following addition of 10 µM BK or NS-1619. Dose-dependent changes in membrane potential of RBE and RG2 cells following addition of varying concentrations of NS-1619 (0-50 µM) were reproducibly observed (not shown). The decrease in fluorescence intensity corresponding to membrane potential changes is plotted on y-axis as relative fluorescence units (RFU). Addition of IBTX (10 nM) reversed the membrane potential to resting values.

Immunolocalization of K_Ca Channels. We also sought to determine if K_Ca channels are differentially and abundantly expressed on tumor cells and tumor capillaries compared with those of normal brain, which might explain the BK- or NS-1619-induced selective BTB permeability increases. Immunoblot experiments with the affinity-purified anti-K_Ca channel -subunit antibody reveal higher expression of K_Ca channel protein in brain tumor tissue compared with the normal brain tissue, as shown in Fig. 5A. This antibody also recognized K_Ca channel -subunit in RG2 cells, RBE cells, and rat brain capillaries (Fig. 5A). The NIH-1.61-aided immunoblot analyses reveal greater than 4- and 6-fold increase in K_Ca channel density in RG2 cells and RG2 tumor, respectively, compared with normal brain (Fig. 5B). The K_Ca channel protein levels in RBE cells and brain capillaries were similar to that in normal brain tissue (Fig. 5B).

View larger version (47K): [in this window] [in a new window]   Fig. 5.   A, immunoblot analysis of SDS-polyacrylamide gel electrophoresis fractionated samples (20 µg of protein/lane) reveals differential expression of KCa channel protein (~125-kDa band) immunoreactive with an anti-peptide antibody specific for KCa channels in normal rat brain tissue (lane 1), cultured RG2 cells (lane 2), RG2 tumor tissue (lane 3), primary cultures of RBE cells isolated from neonatal rat brain (lane 4), and isolated rat brain capillaries (lane 5). Also shown are the intensities of a 43 kDa -actin band in the same immunoblot to ascertain protein-loading variance. B, semiquantitative analyses of protein bands shown in A demonstrate abundant expression of KCa channels in RG2 cells and RG2 tumor tissue. The ratios of KCa channel protein/-actin for each sample were plotted along the y-axis to compare the relative expression levels of KCa channel protein in different samples.

View larger version (42K): [in this window] [in a new window]   Fig. 6.   Confocal microscopic immunolocalization of KCa channels (green; B and F) and Factor VIII (red; C and G) in endothelial cells and RG2 cells. Corresponding phase contrast images (A and E) and merged images (D and H) are also shown. The KCa channels are predominantly expressed on plasma membrane of the endothelial cell (A-D) and RG2 cell (E-H). The low-magnification image shows immunolocalization of KCa channels in cultured RG2 cells (I). The 3D digital images, reconstructed from high-magnification confocal microscopic optical sections show that KCa channels are restricted to the plasma membrane and cytoplasm of cultured RG2 cells (J), C6 (K), and 9L (L) rat glioma cells permeabilized with 0.2% Triton X-100.

View larger version (67K): [in this window] [in a new window]   Fig. 7.   Expression of KCa channel (-subunit) in rat brain harboring RG2 tumor. Immunostaining reveals greater expression of KCa channels in the tumor center (B) and in the tumor periphery (C) compared with the contralateral normal brain (A).

View larger version (80K): [in this window] [in a new window]   Fig. 8.   Immunolocalization of KCa channels and Factor VIII in rat brain tumor sections are shown. The contralateral brain area shows very few capillaries that express KCa channels (A-D). Capillaries in the tumor periphery (E-H) and in the area adjacent to the tumor (I-L) coexpress KCa channels and Factor VIII suggesting that higher expression of KCa channels occurs in the tumor capillary endothelium.

View larger version (157K): [in this window] [in a new window]   Fig. 9.   Induction of vesicular transport in tumor capillary endothelium and tumor cells in vivo. A, in PBS-infused rat, brain tumor microvessel endothelial cell shows very few vesicles (arrows). B, BK infusion caused an increased formation of vesicles (arrows) by luminal membrane invagination; these vesicles with an average diameter of 75- to 80-nm dock and fuse with the basal membrane. C, NS-1619 induces rapid formation of pinocytotic vesicles (arrows) in tumor capillary endothelial cytoplasm. D, very few HRP-laden vesicles are seen in tumor capillary endothelium of the vehicle-treated rat. E, BK or NS-1619 infusion triggers the formation of an array of HRP-laden vesicles in tumor capillary endothelium that move across the abluminal endothelium. F and G, insets are enlarged in adjacent boxes to show fusion of these HRP-laden vesicles in the form of a tube-like structure throughout the endothelium cytoplasm. H, no HRP-laden vesicles are seen in a tumor cell of a vehicle-treated rat. I and J, a number of electron-dense, HRP-laden vesicles (arrows) are seen in tumor cells, following intracarotid infusion of BK or NS-1619, and intravenous injection of HRP in rats with RG2 tumors. Ab, abluminal; BM, basal membrane; E, endothelial cytoplasm; L, luminal; PV, pinocytic vesicles (arrows).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The BTB prevents the delivery of most hydrophilic molecules and antitumor agents to brain tumor. Brain tumor capillaries, which surround the BTB, are functionally different from normal brain capillaries (Nishio et al., 1983; Pardridge et al., 1992; Asotra and Black, 2000). We exploited these differences to selectively enhance drug delivery to brain tumor tissue without affecting normal brain tissue. Here, we used vasomodulators, such as BK, NO donors, sGC activator, and NS-1619, to increase BTB permeability, which significantly enhanced tracer delivery specifically to the brain tumor.

B2 receptors mediate the effects of BK on BTB permeability resulting in enhanced drug delivery selectively to brain tumor (Matsukado et al., 1998). In our clinical trial, the BK analog RMP-7 produced variable increase in delivery of ⁶⁸Ga-EDTA to the tumor of patients with a variety of brain tumors (Black et al., 1997). This variable response to RMP-7 was possibly due to differences in B2 receptor expression in tumors of those patients. Indeed, in a subsequent study (Liu et al., 2001b), we demonstrated differential expression of B2 receptors in rat gliomas, which positively correlated with BK-mediated BTB permeability increases. These observations underscore the limitations in the use of BK for a predictable drug delivery and clinical outcome. Moreover, at high doses, BK nonselectively increases BBB permeability (Raymond et al., 1986). BK-induced BTB permeability increase is also transient, lasting only 20 min (Inamura and Black, 1994), reportedly due to B2 receptor internalization (Pizard et al., 1999) following prolonged BK infusion. In fact, BK-activated downstream signaling targets, such as endogenous NO, sGC, or cGMP, could be independently modulated to elicit a selective and enhanced BTB permeability response (Figs. 1 and 2). More importantly, the present study demonstrates that one can circumvent these signaling pathways and yet selectively increase BTB permeability by directly activating K_Ca channels (Figs. 1 and 2). Consistent with our findings, Reiser et al. (1990) showed that BK directly activates K_Ca channels in rat glioma cells. BK was also shown to activate K_Ca channels through the NO-cGMP system (Zhou et al., 1998). Furthermore, in our study IBTX attenuated BK-mediated effect on BTB permeability, which is consistent with the observation that IBTX blocks BK-induced cerebral vasodilation (Faraci and Heistad, 1998). Previously, we demonstrated that infusion of BK did not affect the cerebral blood volume in tumor and normal brain (Liu et al., 2001a). We showed that intracarotid infusion of NS-1619, DEA-NONOate, or YC-1 at concentrations used in the present study did not significantly alter CBF in the tumor as measured by laser-Doppler flowmetry (Table 1), with the exception of BK. Hence, the measured increase in K_i was not due to the possible change in vasomodulator-induced blood flow but rather due to the direct or indirect effect of these vasomodulators on K_Ca channels.

Although the vasodilatory effect of the NO-cGMP system on normal brain macrovasculature is well known, its effect on brain tumor microvasculature permeability is largely unknown. Recent evidence shows that NO activates K_Ca channels by both cGMP-dependent (Zhou et al., 1998; Lohse et al., 1998) and cGMP-independent mechanisms (Wu et al., 1995). Expression of NO synthase in cerebral endothelium and brain tumors has been demonstrated by electrophysiological and immunohistochemical studies (Kitazono et al., 1995). Consistent with our findings, Zhou et al. (1998) reported that DEA-NONOate activated K_Ca channels by modifying the -subunit of the K_Ca channels. Recently, we reported (Ningaraj et al., 2000) that K_Ca channels mediate the selective BTB permeability increase in rats induced by short-acting NO donors, DEA-NONOate, and PAPA-NONOate. Here, we present further evidence that both endogenous and exogenous NO can directly activate endogenous K_Ca channels to modulate BTB permeability. Time course study with NS-1619 and DEA-NONOate demonstrates their ability to elicit enhanced and sustained drug delivery to tumor tissue for up to 60 min (Fig. 3C), as opposed to the transient effect of BK. Furthermore, vasomodulators at the concentrations we investigated in the current study did not significantly affect physiological parameters (Table 1).

Although several studies have demonstrated the presence of K_Ca channels in cerebrovascular endothelium (Kitazono et al., 1995), none have described the occurrence of K_Ca channels (- or -subunits) either on brain or tumor capillary endothelium and their possible effect on capillary permeability. To immunolocalize K_Ca channels in rat brain sections with RG2 tumor, we used anti-K_Ca channel antibody, raised against the -subunit (Wanner et al., 1999). The immunoblot studies (Fig. 5) demonstrate the abundant expression of K_Ca channels in RG2 cells (both in vitro and in vivo) in contrast to cultured endothelial cells and normal brain tissue. However, confocal microscopic analyses reveal abundant expression of K_Ca channels (-subunits) in rat brain endothelial and tumor cells (Fig. 6), tumor tissue (Fig. 7), and tumor capillaries (Fig. 8) compared with normal brain tissue. Moreover, we demonstrated functional activity of K_Ca channels in RG2 cells by quantifying BK or NS-1619 -induced K⁺ efflux and membrane potential changes and their reversal by IBTX. In light of these results, we surmise that functional K_Ca channels are present and abundantly expressed on RG2 tumor cells and tumor capillaries compared with those of normal brain, which might explain the selective BTB permeability increases caused by BK, NO donors, YC-1, and NS-1619. Because microcapillaries in rat gliomas are biologically different from normal brain capillaries (Nishio et al., 1983), K_Ca channels expressed in tumor capillary endothelium and tumor cells may have altered sensitivity to K_Ca channel modulators. Hence, the present data on 1) increases in BTB permeability, 2) functional activity of K_Ca channels, and 3) abundance of K_Ca channels in tumor capillary endothelium and tumor cells clearly demonstrate that K_Ca channels serve as a convergence point in the mechanism that regulates vasomodulator-mediated selective increase in BTB permeability to enhance drug delivery to brain tumor.

We investigated whether increased drug transport across the BTB in response to infusion of either BK or NS-1619 occurs via tumor capillary endothelial tight junctions or through increased vesicle formation. Our results demonstrate that BK and NS-1619 induce accelerated formation of transport vesicles in both brain tumor capillary endothelium and tumor cells. These results provide evidence that vesicular transport is largely responsible for enhanced delivery of drugs across brain tumor capillaries into tumor tissue. Consistent with our observations in rat tumor microvessels, Stewart et al. (1987) reported an increased density of microvessel endothelial vesicles in human brain tumors. Moreover, a relationship between brain capillary endothelial vesicles and macromolecular permeability has been suggested (Stewart, 2000). Most importantly, our study demonstrates that rat brain tumor microvessels form an even greater number of vesicles in response to vasomodulator infusion, resulting in greater BTB permeability compared with PBS-treated controls. BK (at a low dose), which increases intracellular cGMP (Sugita and Black, 1998), however, did not increase transport vesicle formation in normal capillary endothelium in our studies (Table 2). Our results are different from a previous study (Joo et al., 1983), where db-cGMP increased the number of transcytotic vesicles in the normal cerebral capillary endothelium. This difference may relate to the high doses of db-cGMP used by Joo et al. (1983).

In summary, we have demonstrated in a rat brain tumor model that K_Ca channels are the convergence point (Figs. 1 and 2) for BTB permeability modulation by vasomodulators, such as BK, NO donors, YC-1, and NS-1619, that selectively increase permeability in brain tumor capillaries. The K_Ca channel-mediated increase in BTB permeability in response to BK or NS-1619 (Fig. 2C) correlates with 1) functional activity of K_Ca channels in RBE and RG2 cells (Fig. 4, A-C), 2) abundance of K_Ca channels in tumor cells (Figs. 5 and 6), tumor tissue (Figs. 5 and 7), and tumor capillary endothelium (Fig. 8), 3) BK- or NS-1619-induced accelerated transcytotic vesicle formation in brain tumor capillary endothelium (Fig. 9, B and C); and 4) rapid transport of HRP-laden vesicles across the BTB into tumor cells (Fig. 9, E, I, and J). Furthermore, K_Ca channel-mediated permeability increase in tumor capillaries can be sustained for a longer period with NS-1619 or DEA-NONOate (Fig. 3C). These findings support a pivotal role for K_Ca channels in BTB permeability regulation, besides their previously known role in cerebral vasodilation.

Taken together, our findings demonstrate that K_Ca channels are effective targets for BTB permeability modulation. It is conceivable that other types of potassium channels may have some role in BTB permeability regulation (Ningaraj et al., 2001), which remains to be thoroughly investigated. This study presents evidence that activation of K_Ca channels by specific agonists and agents that produce NO and cGMP in situ can sustain enhanced and selective drug delivery to brain tumors. This drug delivery system is independent of B2 receptors and circumvents the disadvantages of BK, specifically variable and refractory BTB permeability responses. In conclusion, our results further delineate the operative mechanisms that regulate BTB permeability for selective and enhanced delivery of molecules across brain tumor microvessels following biochemical modulation. This study may have significant implications for improving targeted delivery of anti-neoplastic agents to brain tumors and neuropharmaceutics to diseased brain regions, while leaving normal brain unaffected.