Metabotropic, but not allosteric, effects of neurosteroids on GABAergic inhibition depend on the phosphorylation of GABAA receptors.

Neuroactive steroids (NASs) are synthesized within the brain and exert profound effects on behavior. These effects are primarily believed to arise from the activities of NASs as positive allosteric modulators (PAMs) of the GABA-type A receptor (GABAAR). NASs also activate a family of G protein-coupled receptors known as membrane progesterone receptors (mPRs). Here, using surface-biotinylation assays and electrophysiology techniques, we examined mPRs' role in mediating the effects of NAS on the efficacy of GABAergic inhibition. Selective mPR activation enhanced phosphorylation of Ser-408 and Ser-409 (Ser-408/9) within the GABAAR ß3 subunit, which depended on the activity of cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC). mPR activation did not directly modify GABAAR activity and had no acute effects on phasic or tonic inhibition. Instead, mPR activation induced a sustained elevation in tonic current, which was blocked by PKA and PKC inhibition. Substitution of Ser-408/9 to alanine residues also prevented the effects of mPR activation on tonic current. Furthermore, this substitution abolished the effects of sustained NAS exposure on tonic inhibition. Interestingly, the allosteric effects of NAS on GABAergic inhibition were independent of Ser-408/9 in the ß3 subunit. Additionally, although allosteric effects of NAS on GABAergic inhibition were sensitive to a recently developed "NAS antagonist," the sustained effects of NAS on tonic inhibition were not. We conclude that metabotropic effects of NAS on GABAergic inhibition are mediated by mPR-dependent modulation of GABAAR phosphorylation. We propose that this mechanism may contribute to the varying behavioral effects of NAS.

Compromising the phosphodependent regulation of the GABAAR ß3 subunit reproduces the core phenotypes of autism spectrum disorders.

Alterations in the efficacy of neuronal inhibition mediated by GABAA receptors (GABAARs) containing ß3 subunits are continually implicated in autism spectrum disorders (ASDs). In vitro, the plasma membrane stability of GABAARs is potentiated via phosphorylation of serine residues 408 and 409 (S408/9) in the ß3 subunit, an effect that is mimicked by their mutation to alanines. To assess if modifications in ß3 subunit expression contribute to ASDs, we have created a mouse in which S408/9 have been mutated to alanines (S408/9A). S408/9A homozygotes exhibited increased phasic, but decreased tonic, inhibition, events that correlated with alterations in the membrane stability and synaptic accumulation of the receptor subtypes that mediate these distinct forms of inhibition. S408/9A mice exhibited alterations in dendritic spine structure, increased repetitive behavior, and decreased social interaction, hallmarks of ASDs. ASDs are frequently comorbid with epilepsy, and consistent with this comorbidity, S408/9A mice exhibited a marked increase in sensitivity to seizures induced by the convulsant kainic acid. To assess the relevance of our studies using S408/9A mice for the pathophysiology of ASDs, we measured S408/9 phosphorylation in Fmr1 KO mice, a model of fragile X syndrome, the most common monogenetic cause of ASDs. Phosphorylation of S408/9 was selectively and significantly enhanced in Fmr1 KO mice. Collectively, our results suggest that alterations in phosphorylation and/or activity of ß3-containing GABAARs may directly contribute to the pathophysiology of ASDs.

Proteomic Characterization of Inhibitory Synapses Using a Novel pHluorin-tagged γ-Aminobutyric Acid Receptor, Type A (GABAA), α2 Subunit Knock-in Mouse.

The accumulation of γ-aminobutyric acid receptors (GABAARs) at the appropriate postsynaptic sites is critical for determining the efficacy of fast inhibitory neurotransmission. Although we know that the majority of synaptic GABAAR subtypes are assembled from α1-3, ß, and γ2 subunits, our understanding of how neurons facilitate their targeting to and stabilization at inhibitory synapses is rudimentary. To address these issues, we have created knock-in mice in which the pH-sensitive green fluorescent protein (GFP) and the Myc epitope were introduced to the extracellular domain of the mature receptor α2 subunit (pHα2). Using immunoaffinity purification and mass spectroscopy, we identified a stable complex of 174 proteins that were associated with pHα2, including other GABAAR subunits, and previously identified receptor-associated proteins such as gephyrin and collybistin. 149 of these proteins were novel GABAAR binding partners and included G-protein-coupled receptors and ion channel subunits, proteins that regulate trafficking and degradation, regulators of protein phosphorylation, GTPases, and a number of proteins that regulate their activity. Notably, members of the postsynaptic density family of proteins that are critical components of excitatory synapses were not associated with GABAARs. Crucially, we demonstrated for a subset of these novel proteins (including cullin1, ephexin, potassium channel tetramerization domain containing protein 12, mitofusin2, metabotropic glutamate receptor 5, p21-activated kinase 7, and Ras-related protein 5A) bind directly to the intracellular domains of GABAARs, validating our proteomic analysis. Thus, our experiments illustrate the complexity of the GABAAR proteome and enhance our understanding of the mechanisms neurons use to construct inhibitory synapses.

Neurosteroids promote phosphorylation and membrane insertion of extrasynaptic GABAA receptors.

Neurosteroids are synthesized within the brain and act as endogenous anxiolytic, anticonvulsant, hypnotic, and sedative agents, actions that are principally mediated via their ability to potentiate phasic and tonic inhibitory neurotransmission mediated by γ-aminobutyric acid type A receptors (GABAARs). Although neurosteroids are accepted allosteric modulators of GABAARs, here we reveal they exert sustained effects on GABAergic inhibition by selectively enhancing the trafficking of GABAARs that mediate tonic inhibition. We demonstrate that neurosteroids potentiate the protein kinase C-dependent phosphorylation of S443 within α4 subunits, a component of GABAAR subtypes that mediate tonic inhibition in many brain regions. This process enhances insertion of α4 subunit-containing GABAAR subtypes into the membrane, resulting in a selective and sustained elevation in the efficacy of tonic inhibition. Therefore, the ability of neurosteroids to modulate the phosphorylation and membrane insertion of α4 subunit-containing GABAARs may underlie the profound effects these endogenous signaling molecules have on neuronal excitability and behavior.

The Role of Cell-Penetrating Peptide and Transferrin on Enhanced Delivery of Drug to Brain.

The challenge of effectively delivering therapeutic agents to brain has led to an entire field of active research devoted to overcome the blood brain barrier (BBB) and efficiently deliver drugs to brain. This review focusses on exploring the facets of a novel platform designed for the delivery of drugs to brain. The platform was constructed based on the hypothesis that a combination of receptor-targeting agent, like transferrin protein, and a cell-penetrating peptide (CPP) will enhance the delivery of associated therapeutic cargo across the BBB. The combination of these two agents in a delivery vehicle has shown significantly improved (p < 0.05) translocation of small molecules and genes into brain as compared to the vehicle with only receptor-targeting agents. The comprehensive details of the uptake mechanisms and properties of various CPPs are illustrated here. The application of this technology, in conjunction with nanotechnology, can potentially open new horizons for the treatment of central nervous system disorders.

Maternal nutrient restriction during pregnancy impairs an endothelium-derived hyperpolarizing factor-like pathway in sheep fetal coronary arteries.

The mechanisms underlying developmental programming are poorly understood but may be associated with adaptations by the fetus in response to changes in the maternal environment during pregnancy. We hypothesized that maternal nutrient restriction during pregnancy alters vasodilator responses in fetal coronary arteries. Pregnant ewes were fed a control [100% U.S. National Research Council (NRC)] or nutrient-restricted (60% NRC) diet from days 50 to 130 of gestation (term = 145 days); fetal tissues were collected at day 130. In coronary arteries isolated from control fetal lambs, relaxation to bradykinin was unaffected by nitro-l-arginine (NLA). Iberiotoxin or contraction with KCl abolished the NLA-resistant response to bradykinin. In fetal coronary arteries from nutrient-restricted ewes, relaxation to bradykinin was fully suppressed by NLA. Large-conductance, calcium-activated potassium channel (BKCa) currents did not differ in coronary smooth muscle cells from control and nutrient-restricted animals. The BKCa openers, BMS 191011 and NS1619, and 14,15-epoxyeicosatrienoic acid [a putative endothelium-derived hyperpolarizing factor (EDHF)] each caused fetal coronary artery relaxation and BKCa current activation that was unaffected by maternal nutrient restriction. Expression of BKCa-channel subunits did not differ in fetal coronary arteries from control or undernourished ewes. The results indicate that maternal undernutrition during pregnancy results in loss of the EDHF-like pathway in fetal coronary arteries in response to bradykinin, an effect that cannot be explained by a decreased number or activity of BKCa channels or by decreased sensitivity to mediators that activate BKCa channels in vascular smooth muscle cells. Under these conditions, bradykinin-induced relaxation is completely dependent on nitric oxide, which may represent an adaptive response to compensate for the absence of the EDHF-like pathway.

Influence of short-chain cell-penetrating peptides on transport of doxorubicin encapsulating receptor-targeted liposomes across brain endothelial barrier.

PURPOSE: To investigate the influence of different cell penetrating peptides (CPPs-TAT, Penetratin and Mastoparan), on the transport of doxorubicin encapsulating transferrin (Tf)-liposomes across brain endothelial barrier, in vitro and in vivo. METHODS: The cellular uptake of dual-functionalized, (Tf-CPP), liposomes into various tumor cells was assessed using HPLC. The transport of liposomes was also measured across a robust 3D brain tumor model constructed using chitosan-PLGA scaffolds. The growth of tumor cells was monitored using H&E staining and the fully grown tumor scaffolds were visualized using SEM. The tumor scaffolds were combined with the culture inserts carrying tightly packed brain endothelial cells. The in vitro and in vivo transport of drug (using Tf-CPP-liposomes) across the brain endothelial barrier was determined by extraction of the drug from cells and tissues followed by analysis using HPLC. RESULTS: The results demonstrated improved delivery of doxorubicin using dual-functionalized liposomes versus the single ligand or unmodified liposomes. Among different Tf-CPP-liposomes, the Tf-Penetratin liposomes showed efficient cellular transport of the encapsulated drug (approximately 90-98%) and maximum translocation of the drug across the brain endothelial barrier (approximately 15% across in vitro and 4% across in vivo BBB). The Tf-Penetratin and Tf-TAT liposomes demonstrated excellent cellular biocompatibility and no hemolytic activity upto 200nM phospholipid concentration. CONCLUSIONS: The Tf-CPP liposomes showed efficient translocation of the anticancer drug across the brain endothelial barrier. In addition, an absolute and robust in vitro brain tumor model was successfully constructed to overcome the practical intricacies of developing a successful in vivo orthotopic brain tumor model.

Angiotensin-(1-7) attenuates the chronotropic response to angiotensin II via stimulation of PTEN in the spontaneously hypertensive rat neurons.

Several studies have focused on the beneficial effects of peripheral angiotensin-(1-7) [Ang-(1-7)] in the regulation of cardiovascular function, showing its counterregulatory effect against the actions of angiotensin II (ANG II). However, its actions in the central nervous system are not completely understood. In the present study, we investigated the intracellular mechanisms underlying the action of ANG-(1-7) using the patch-clamp technique in neurons cultured from the hypothalamus of neonatal spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Superfusion of neurons with ANG II (100 nM) significantly increased neuronal firing in both strains of rats, and this chronotropic effect of ANG II was significantly enhanced in prehypertensive SHR neurons compared with WKY rat neurons. The enhanced chronotropic effect of ANG II was attenuated by a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, LY 294002 (10 µM). Superfusion of neurons with ANG-(1-7) (100 nM) did not alter the neuronal firing rate in either SHR or WKY neurons; however, it significantly attenuated the chronotropic action of ANG II exclusively in prehypertensive SHR neurons. This counterregulatory effect of ANG-(1-7) on ANG II action in prehypertensive SHR neurons was attenuated by cotreatment with either A-779, a Mas receptor antagonist, or bisperoxovanadium, a phosphatase and tensin homologue deleted on chromosome ten (PTEN) inhibitor. In addition, incubation of WKY and prehypertensive SHR neurons with ANG-(1-7) significantly increased PTEN activity. The data demonstrate that ANG-(1-7) counterregulates the chronotropic action of ANG II via a PTEN-dependent signaling pathway in prehypertensive SHR neurons.

Invertible micellar polymer assemblies for delivery of poorly water-soluble drugs.

Strategically designed amphiphilic invertible polymers (AIPs) are capable of (i) self-assembling into invertible micellar assemblies (IMAs) in response to changes in polarity of environment, polymer concentration, and structure, (ii) accommodating (solubilizing) substances that are otherwise insoluble in water, and (iii) inverting their molecular conformation in response to changes in the polarity of the local environment. The unique ability of AIPs to invert the molecular conformation depending on the polarity of the environment can be a decisive factor in establishing the novel stimuli-responsive mechanism of solubilized drug release that is induced just in response to a change in the polarity of the environment. The IMA capability to solubilize lipophilic drugs and deliver and release the cargo molecules by conformational inversion of polymer macromolecules in response to a change of the polarity of the environment was demonstrated by loading IMA with a phytochemical drug, curcumin. It was demonstrated that four sets of micellar vehicles based on different AIPs were capable of delivering the curcumin from water to an organic medium (1-octanol) by means of unique mechanism: AIP conformational inversion in response to changing polarity from polar to nonpolar. The IMAs are shown to be nontoxic against human cells up to a concentration of 10 mg/L. On the other hand, the curcumin-loaded IMAs are cytotoxic to breast carcinoma cells at this concentration, which confirms the potential of IMA-based vehicles in controlled delivery of poorly water-soluble drug candidates and release by means of this novel stimuli-responsive mechanism.

Pressor effect of apelin-13 in the rostral ventrolateral medulla: role of NAD(P)H oxidase-derived superoxide.

Microinjection of apelin-13 into the rostral ventrolateral medulla (RVLM) in the brainstem increases blood pressure in rats. In the present study, we tested the hypotheses that apelin-13 directly stimulates neuronal activity in neurons cultured from the brainstem and that NAD(P)H oxidase-derived reactive oxygen species are involved in this action of apelin-13. Microinjection of apelin-13 into the RVLM resulted in increases in arterial pressure and in renal sympathetic nerve activity in Sprague-Dawley rats. The pressor effect of apelin-13 was attenuated by the specific NAD(P)H-oxidase inhibitor gp91ds-tat. In neurons cultured from the ventral brainstem, spontaneous action potentials were recorded using current-clamp recording. Superfusion of neurons with apelin-13 (100 nM) increased the neuronal firing rate from 0.79 ± 0.14 to 1.45 ± 0.26 Hz (n = 7, P < 0.01) in angiotensin II receptor-like 1-positive neurons, identified with single-cell reverse transcriptase-polymerase chain reaction. Neither the angiotensin II type 1 receptor antagonist losartan nor the angiotensin II type 2 receptor antagonist 1-[[4-(dimethylamino)-3-methylphenyl[methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditrifluoroacetate (PD123319) altered the positive chronotropic effect of apelin-13. Pretreatment of cells with either the reactive oxygen species scavenger superoxide dismutase [polyethylene glycol-superoxide dismutase (PEG-SOD), 25 U/ml] or with gp91ds-tat significantly attenuated the chronotropic action of apelin-13. PEG-SOD and gp91ds-tat alone had no effect on basal neuronal firing. In addition, apelin-13 significantly increased NAD(P)H oxidase activity and elevated intracellular superoxide levels in neuronal cultures. The superoxide generator xanthine-xanthine oxidase also increased neuronal activity in neurons, mimicking the neuronal response to apelin-13. These observations provide the first evidence that apelin-13 directly increases neuronal activity via stimulation of NAD(P)H oxidase-derived superoxide, a cellular signaling mechanism that may be involved in the pressor effect of apelin-13 in the RVLM.

Preclinical characterization of zuranolone (SAGE-217), a selective neuroactive steroid GABAA receptor positive allosteric modulator.

Zuranolone (SAGE-217) is a novel, synthetic, clinical stage neuroactive steroid GABAA receptor positive allosteric modulator designed with the pharmacokinetic properties to support oral daily dosing. In vitro, zuranolone enhanced GABAA receptor current at nine unique human recombinant receptor subtypes, including representative receptors for both synaptic (γ subunit-containing) and extrasynaptic (δ subunit-containing) configurations. At a representative synaptic subunit configuration, α1ß2γ2, zuranolone potentiated GABA currents synergistically with the benzodiazepine diazepam, consistent with the non-competitive activity and distinct binding sites of the two classes of compounds at synaptic receptors. In a brain slice preparation, zuranolone produced a sustained increase in GABA currents consistent with metabotropic trafficking of GABAA receptors to the cell surface. In vivo, zuranolone exhibited potent activity, indicating its ability to modulate GABAA receptors in the central nervous system after oral dosing by protecting against chemo-convulsant seizures in a mouse model and enhancing electroencephalogram ß-frequency power in rats. Together, these data establish zuranolone as a potent and efficacious neuroactive steroid GABAA receptor positive allosteric modulator with drug-like properties and CNS exposure in preclinical models. Recent clinical data support the therapeutic promise of neuroactive steroid GABAA receptor positive modulators for treating mood disorders; brexanolone is the first therapeutic approved specifically for the treatment of postpartum depression. Zuranolone is currently under clinical investigation for the treatment of major depressive episodes in major depressive disorder, postpartum depression, and bipolar depression.

Neuroactive Steroids Reverse Tonic Inhibitory Deficits in Fragile X Syndrome Mouse Model.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. A reduction in neuronal inhibition mediated by γ-aminobutyric acid type A receptors (GABAARs) has been implicated in the pathophysiology of FXS. Neuroactive steroids (NASs) are known allosteric modulators of GABAAR channel function, but recent studies from our laboratory have revealed that NASs also exert persistent metabotropic effects on the efficacy of tonic inhibition by increasing the protein kinase C (PKC)-mediated phosphorylation of the α4 and ß3 subunits which increase the membrane expression and boosts tonic inhibition. We have assessed the GABAergic signaling in the hippocampus of fragile X mental retardation protein (FMRP) knock-out (Fmr1 KO) mouse. The GABAergic tonic current in dentate gyrus granule cells (DGGCs) from 3- to 5-week-old (p21-35) Fmr1 KO mice was significantly reduced compared to WT mice. Additionally, spontaneous inhibitory post synaptic inhibitory current (sIPSC) amplitudes were increased in DGGCs from Fmr1 KO mice. While sIPSCs decay in both genotypes was prolonged by the prototypic benzodiazepine diazepam, those in Frm1-KO mice were selectively potentiated by RO15-4513. Consistent with this altered pharmacology, modifications in the expression levels and phosphorylation of receptor GABAAR subtypes that mediate tonic inhibition were seen in Fmr1 KO mice. Significantly, exposure to NASs induced a sustained elevation in tonic current in Fmr1 KO mice which was prevented with PKC inhibition. Likewise, exposure reduced elevated membrane excitability seen in the mutant mice. Collectively, our results suggest that NAS act to reverse the deficits of tonic inhibition seen in FXS, and thereby reduce aberrant neuronal hyperexcitability seen in this disorder.

Nanotechnology: Revolutionizing the Science of Drug Delivery.

Growing interest in the field of nanotechnology has led to its emergence in the field of medicine too. Nanomedicines encompass the various medical tools, diagnostic agents and the drug delivery vehicles being evolved with the advancements in the aura of nanotechnology. This review emphasizes on providing a cursory literature on the past events that led to the procession of nanomedicines, various novel drug delivery systems describing their structural features along with the pros and cons associated with them and the nanodrugs that made a move to the clinical practice. It also focuses on the need of the novel drug delivery systems and the challenges faced by the conventional drug delivery systems.

Developmental seizures and mortality result from reducing GABAA receptor α2-subunit interaction with collybistin.

Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2-1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.

Endogenous and synthetic neuroactive steroids evoke sustained increases in the efficacy of GABAergic inhibition via a protein kinase C-dependent mechanism.

The neuroactive steroid (NAS) tetrahydrodeoxycorticosterone (THDOC) increases protein kinase C (PKC) mediated phosphorylation of extrasynaptic GABAA receptor (GABAAR) subunits leading to increased surface expression of α4/ß3 subunit-containing extrasynaptic GABAARs, leading to a sustained increase in GABAAR tonic current density. Whether other naturally occurring and synthetic NASs share both an allosteric and metabotropic action on GABAARs is unknown. Here, we examine the allosteric and metabotropic properties of allopregnanolone (ALLO), and synthetic NASs SGE-516 and ganaxolone. ALLO, SGE-516, and ganaxolone all allosterically enhanced prototypical synaptic and extrasynaptic recombinant GABAARs. In dentate gyrus granule cells (DGGCs) all three NASs, when applied acutely, allosterically enhanced tonic and phasic GABAergic currents. In separate experiments, slices were exposed to NASs for 15 min, and then transferred to a steroid naïve recording chamber followed by ≥ 30 min wash before tonic currents were measured. A sustained increase in tonic current was observed following exposure to ALLO, or SGE-516 and was prevented by inhibiting PKC with GF 109203X. No increase in tonic current was observed with exposure to ganaxolone. In agreement with the observations of an increased tonic current, the NASs ALLO and SGE-516 increased the phosphorylation and surface expression of the ß3 subunit-containing GABAARs. Our studies demonstrate that neuroactive steroids have differential abilities to induce sustained increases in the efficacy of tonic inhibition by promoting GABAAR phosphorylation and membrane trafficking dependent on PKC activity.

Genetic Deletion of the Clathrin Adaptor GGA3 Reduces Anxiety and Alters GABAergic Transmission.

Golgi-localized γ-ear-containing ARF binding protein 3 (GGA3) is a monomeric clathrin adaptor that has been shown to regulate the trafficking of the Beta-site APP-cleaving enzyme (BACE1), which is required for production of the Alzheimer's disease (AD)-associated amyloid ßpeptide. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that depletion of GGA3 results in increased BACE1 levels and activity owing to impaired lysosomal trafficking and degradation. We further demonstrated the role of GGA3 in the regulation of BACE1 in vivo by showing that BACE1 levels are increased in the brain of GGA3 null mice. We report here that GGA3 deletion results in novelty-induced hyperactivity and decreased anxiety-like behaviors. Given the pivotal role of GABAergic transmission in the regulation of anxiety-like behaviors, we performed electrophysiological recordings in hippocampal slices and found increased phasic and decreased tonic inhibition in the dentate gyrus granule cells (DGGC). Moreover, we found that the number of inhibitory synapses is increased in the dentate gyrus of GGA3 null mice in further support of the electrophysiological data. Thus, the increased GABAergic transmission is a leading candidate mechanism underlying the reduced anxiety-like behaviors observed in GGA3 null mice. All together these findings suggest that GGA3 plays a key role in GABAergic transmission. Since BACE1 levels are elevated in the brain of GGA3 null mice, it is possible that at least some of these phenotypes are a consequence of increased processing of BACE1 substrates.

Apelin-13 inhibits large-conductance Ca2+-activated K+ channels in cerebral artery smooth muscle cells via a PI3-kinase dependent mechanism.

Apelin-13 causes vasoconstriction by acting directly on APJ receptors in vascular smooth muscle (VSM) cells; however, the ionic mechanisms underlying this action at the cellular level remain unclear. Large-conductance Ca(2+)-activated K(+) (BKCa) channels in VSM cells are critical regulators of membrane potential and vascular tone. In the present study, we examined the effect of apelin-13 on BK(Ca) channel activity in VSM cells, freshly isolated from rat middle cerebral arteries. In whole-cell patch clamp mode, apelin-13 (0.001-1 µM) caused concentration-dependent inhibition of BK(Ca) in VSM cells. Apelin-13 (0.1 µM) significantly decreased BK(Ca) current density from 71.25 ± 8.14 pA/pF to 44.52 ± 7.10 pA/pF (n=14 cells, P<0.05). This inhibitory effect of apelin-13 was confirmed by single channel recording in cell-attached patches, in which extracellular application of apelin-13 (0.1 µM) decreased the open-state probability (NPo) of BK(Ca) channels in freshly isolated VSM cells. However, in inside-out patches, extracellular application of apelin-13 (0.1 µM) did not alter the NPo of BK(Ca) channels, suggesting that the inhibitory effect of apelin-13 on BKCa is not mediated by a direct action on BK(Ca). In whole cell patches, pretreatment of VSM cells with LY-294002, a PI3-kinase inhibitor, markedly attenuated the apelin-13-induced decrease in BK(Ca current density. In addition, treatment of arteries with apelin-13 (0.1 µM) significantly increased the ratio of phosphorylated-Akt/total Akt, indicating that apelin-13 significantly increases PI3-kinase activity. Taken together, the data suggest that apelin-13 inhibits BK(Ca) channel via a PI3-kinase-dependent signaling pathway in cerebral artery VSM cells, which may contribute to its regulatory action in the control of vascular tone.

Cell penetrating peptide tethered bi-ligand liposomes for delivery to brain in vivo: Biodistribution and transfection.

Targeted nano-particulate systems hold extraordinary potential for delivery of therapeutics across blood brain barrier (BBB). In this work, we investigated the potential of novel bi-ligand (transferrin-poly-l-arginine) liposomal vector for delivery of desired gene to brain, in vivo. The in vivo evaluation of the delivery vectors is essential for clinical translation. We followed an innovative approach of combining transferrin receptor targeting with enhanced cell penetration to design liposomal vectors for improving the transport of molecules into brain. The biodistribution profile of 1, 1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine iodide(DiR)-labeled liposomes was evaluated in adult rats after single intravenous injection at dose of 15.2µmoles of phospholipids/kg body weight. We demonstrated that bi-ligand liposomes accumulated in rat brain at significantly (p<0.05) higher concentrations as compared to the single-ligand (transferrin) or plain liposomes. In addition, the bi-ligand liposomes resulted in increased expression of ß-galactosidase(ß-gal) plasmid in rat brain tissue in comparison to the single-ligand liposomes. Histological examination of the transfected tissues did not show any signs of tissue necrosis or inflammation. Hemolysis assay further authenticated the biocompatibility of bi-ligand liposomes in blood up to 600 nmoles of phospholipids/1.4×10(7) erythrocytes. The findings of this study provide important and detailed information regarding the distribution of bi-ligand liposomes in vivo and accentuate their ability to demonstrate improved brain penetration and transfection potential over single-ligand liposomes.

Grafting of cell-penetrating peptide to receptor-targeted liposomes improves their transfection efficiency and transport across blood-brain barrier model.

We report bifunctional liposomal delivery system, combining transferrin (Tf)-mediated receptor targeting and poly-L-arginine (PR)-facilitated cell penetration, which overcomes the drawback of saturation of delivery. PR was conjugated to the distal end of distearoyl phosphoethanolamine-polyethylene glycol (PEG) 2000 and was incorporated with other phospholipids in chloroform/methanol (2:1) to form PR liposomes using thin-film hydration technique. Tf-PEG phospholipid micelles were incorporated into PR liposomes using postinsertion technique to form Tf-PR liposomes. The bifunctional liposomes demonstrated significantly (p < 0.05) higher cellular uptake by brain endothelial cells (bEnd.3) and about eightfold higher transfection in primary culture of glial cells as compared with the Tf liposomes. Cell viabilities of Tf-conjugated and bifunctional liposomes were not markedly different; however, transport across in vitro blood-brain barrier model improved considerably after dual modification. The study underlines the potential of bifunctional liposomes as high-efficiency and low-toxicity gene delivery system for the treatment of central nervous system disorders.