This investigation focuses on transcription factor involvement in blood-brain barrier (BBB) endothelial cell-induced alterations under conditions of hypoxia and post-hypoxia/reoxygenation (H/R), using established in vivo/ex vivo and in vitro BBB models. Protein/DNA array analyses revealed a correlation in key transcription factor activation during hypoxia and H/R, including NFkappaB and hypoxia-inducible factor (HIF)1. Electrophoretic mobility shift assays confirmed NFkappaB and HIF1 binding activity ex vivo and in vitro, under conditions of hypoxia and H/R. Hypoxia- and H/R-treated BBB endothelium showed increased HIF1alpha protein expression in both cytoplasmic and nuclear fractions, in ex vivo and in vitro models. Co-immunoprecipitation of HIF1alpha and HIF1beta was shown in the nuclear fraction under conditions of hypoxia and H/R in both models. Hypoxia- and H/R-treated BBB endothelium showed increased expression of NFkappaB-p65 protein in both cytoplasmic and nuclear fractions. Co-immunoprecipitation of NFkappaB-p65 with NFkappaB-p50 was shown in the nuclear fraction under conditions of hypoxia and H/R in the ex vivo model, and after H/R in the in vitro model. These data offer novel avenues in which to alter and/or investigate BBB activity across model systems and to further our understanding of upstream regulators during hypoxia and H/R.
The blood-brain barrier (BBB) is a physical and metabolic barrier that separates the central nervous system from the peripheral circulation. Central nervous system drug delivery across the BBB is challenging, primarily because of the physical restriction of paracellular diffusion between the endothelial cells that comprise the microvessels of the BBB and the activity of efflux transporters that quickly expel back into the capillary lumen a wide variety of xenobiotics. Therapeutic manipulation of protein trafficking is emerging as a novel means of modulating protein function, and in this minireview, the targeting of the trafficking of 2 key BBB proteins, P-glycoprotein and occludin, is presented as a novel, reversible means of optimizing central nervous system drug delivery.
The blood-brain barrier (BBB) is critical to the health of the central nervous system. The BBB is formed primarily by the presence of tight junctions (TJ) between cerebral microvessel endothelial cells. In light of the known effects of nicotine on endothelial cell biology, the specific effects of nicotine on the in vivo BBB were examined. Using in situ brain perfusion, it was found that continuous administration of nicotine (4.5 mg free base x kg(-1) x day(-1)) for 1 and 7 days led to increased permeability of the BBB to [14C]-sucrose without significant changes in its initial volume of distribution. The expression and distribution of the TJ-associated proteins actin, occludin, claudin-1, -3, and -5, and ZO-1 and -2 were analyzed by Western blot and immunofluorescence microscopy. Though no changes in total protein expression were observed, nicotine treatment was associated with altered cellular distribution of ZO-1 and diminished junctional immunoreactivity of claudin-3. It is proposed that nicotine leads to changes in BBB permeability via the modulation of TJ proteins.
Peptide-based drug development is a rapidly growing field within pharmaceutical research. Nevertheless, peptides have found limited clinical use due to several physiological and pathological factors. Pluronic block copolymers represent a growing technology with the potential to enhance efficacy of peptide therapeutics. This investigation assesses Pluronic P85 (P85) and its potential to enhance opioid peptide analgesia. Two opioid peptides, [D-Pen(2),D-Pen(5)]-enkephalin (DPDPE) and biphalin, were examined as to the benefits of P85 coadministration, above (1.0%) and below (0.01%) the critical micelle concentration, with morphine as a nonpeptide control. P85 was examined in vitro to assess blood-brain barrier uptake in association with P-glycoprotein effect, DPDPE and morphine being P-glycoprotein substrates. P85 coadministration with DPDPE and biphalin showed increased (p 0.01) analgesia with both 0.01 and 1.0% P85. Morphine showed increased (p 0.01) analgesia with 0.01% P85 only. This increase in analgesia is due to both an increase in peak effect, as well as a prolongation of effect. P85 increased cellular uptake of (125)I-DPDPE and [(3)H]morphine at 0.01% (p 0.01) and 1.0% (p 0.01 and p 0.05, respectively). Cyclosporin-A coadministration with (125)I-DPDPE and [(3)H]morphine increased cellular uptake (p 0.01 and p 0.05, respectively). (125)I-DPDPE and [(3)H]morphine coadministered with 0.01% P85 and cyclosporin-A increased cellular uptake compared with control (p 0.01) and compared with cyclosporin-A coadministration without P85 (p 0.01 and p 0.05, respectively). This indicates that, in addition to P-gp inhibition, 0.01% P85 increased (125)I-DPDPE and [(3)H]morphine uptake. In our examination, we determined that P85 enhanced the analgesic profile of biphalin, DPDPE, and morphine, both above and below the critical micelle concentration.
