Regulation of Blood-Brain Tumor Barrier Permeability by Calcium-Activated Potassium Channels

The blood-brain tumor barrier (BTB) limits the delivery of therapeutic drugs to brain tumors. We demonstrate in a rat brain tumor (RG2) model an enhanced drug delivery to brain tumor following intracarotid infusion of bradykinin (BK), nitric oxide (NO) donors, or agonists of soluble guanylate cyclase (sGC) and calcium-dependent potassium (KCa) channels. We modulated KCa channels by specific agonists and agents that produce NO and cGMP in situ to obtain sustained enhancement of selective drug delivery to brain tumors. Intracarotid infusion of BK or 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5- (trifluoromethyl)-2H-benzimidazol-2-one (NS-1619) significantly enhanced BTB permeability (Ki ) to [14C]-aminoisobutyric acid in the brain tumor area but not in normal brain tissue. The Ki increase achieved by BK, NS-1619, NO donors, or the sGC activator 3-(5- hydroxymethyl-2furyl)-1-benzylindazole (YC-1) was significantly attenuated when coinfused with a KCa channel antagonist, iberio toxin. Immunoblot and immunolocalization studies demonstrate overexpression of KCa channels in tumor cells and capillaries compared with normal brain. The potentiometric assays demonstrate the functional activity of KCa channels in rat brain endothelial and glioma cells. Additionally, we show that BK and NS-1619 significantly increased the density of transport vesicles in the cytoplasm of brain tumor capillary endothelia and tumor cells. The cleft indices and cleft area indices in rat tumor capillaries were significantly higher than in normal brain capillaries, and BK infusion did not alter these indices. These data demonstrate that the cellular mechanism for KCa channel-mediated BTB permeability increase is due to accelerated formation of pinocytotic vesicles, which can transport drugs across BTB. We conclude that KCa channels serve as a convergence point in the biochemical regulation of BTB permeability.

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 calciumdependent potassium (KCa) channels (Fig. 1)

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 KCa channels are present in cerebral blood vessels to regulate cerebral blood vessel tone (Kitazono et al., 1995) and, probably, BBB permeability. KCa channels in brain are inhibited by iberiotoxin (IBTX), a highly specific (IC50 68 pM) inhibitor (Wanner et al., 1999). KCa 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 KCa 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 KCa channels, and alter membrane potential in cells (Miura et al., 1999). In brain endothelial cells, BK-induced KCa channel activation was potentiated by NS1619, a selective agonist (EC50 20 nM) for KCa 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 KCa channel modulators. We also investigated the functional presence of putative KCa channels in rat brain endothelial and tumor cells by measuring the membrane potential changes induced by KCa 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 (dbcGMP), was shown to increase the number of transcytotic vesicles in the normal cerebral capillary endothelium, possibly by KCa 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 KCa 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 KCa 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