Basal Efflux Research Articles

The effects of in vitro vitamin D treatment on glycolytic reprogramming of bone marrow-derived dendritic cells from Ldlr knock-out mouse

Dendritic cells (DCs) undergo glycolytic reprogramming, a metabolic conversion process essential for their activation. Vitamin D has been reported to affect the function of DCs, but studies in metabolic diseases are insufficient. This study investigates the effects of in vitro 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) treatment on glycolytic reprogramming of bone marrow-derived dendritic cells (BMDCs) from control, obese, and atherosclerosis mice. Six-week-old male C57BL/6J mice were fed a control diet (CON) or a Western diet (WD), and B6.129S7-Ldlrtm1Her/J mice were fed a Western diet (LDLR−/−) for 16 weeks. BMDCs were cultured in a medium containing 1,25(OH)2D3 (10 nM) for 7 days and stimulated with lipopolysaccharide (LPS, 50 ng/mL) for 24 h. In mature BMDCs, 1,25(OH)2D3 treatment decreased basal and compensatory glycolytic proton efflux rates (glycoPER), the expression of surface markers related to immune function of DCs (MHC class II, CD80, and CD86), and IL-12p70 production. In addition, mTORC1 activation and nitric oxide (NO) production were suppressed by 1,25(OH)2D3 treatment in mature BMDCs. The effect of 1,25(OH)2D3 treatment on IL-12p70 production and mTORC1 activity in the LDLR−/− group was greater than in the CON group. These findings suggest that vitamin D can affect the metabolic environment of BMDCs by regulating glycolytic reprogramming as well as by inducing tolerogenic phenotypes of DCs.

Thymoquinone’ potent impairment of multidrug-resistant Staphylococcus aureus NorA efflux pump activity

The drug efflux pump is a crucial mechanism implicated in resistance to multiple antimicrobials. Thymoquinone (TQ) has evidently demonstrated multiple activities, antibacterial being the most effective. Knowledge about TQ activity against multidrug-resistant Staphylococcus aureus is very scarce. Therefore, the present study was conducted to investigate TQ resistance modulation in ciprofloxacin (CIP) and doxycycline (DO) multidrug-resistant S. aureus. Forty-seven samples were collected from different sources, and S. aureus was isolated and identified. Then, S. aureus resistance profiles to antimicrobials, N. sativa essential oil, and TQ; the correlation between TQ-MIC readings and disc diffusion; cartwheel and ethidium bromide (EtBr) accumulation assays; and norA gene expression were all described within silico molecular docking for TQ interactions with norA efflux pump protein. TQ-MICs ranged from 5–320 µg/ml. TQ down-regulated norA gene expression, resulting in a drop in efflux pump activity of 77.5–90.6% in the examined strains, comparable to that observed with verapamil. Exposure of S. aureus strains to CIP and DO raises the initial basal efflux pumping expression to 34.2 and 22.9 times, respectively. This induced efflux pumping overexpression was substantially reduced by 97.7% when TQ was combined with CIP or DO. There was a significant reduction of MICs of CIP and DO MICs by 2–15 and 2–4 folds, respectively, after treatment with 0.5XMIC-TQ in resistance modulation assays. These results refer to TQ ligand inhibitory interactions with NorA protein in molecular docking. Interpretations of inhibition zone diameters (IZDs) of disc diffusion and TQ-MICs exhibit independence of MICs from IZDs, as indicated by invalid linear regression analysis. TQ significantly reduced efflux pumping S. aureus induced by CIP and DO, but further investigations are needed to improve TQ-pharmacokinetics to restore CIP and DO activity and suppress fluoroquinolone and doxycycline-resistant S. aureus selection in clinical and animal settings.

Repetitive mild traumatic brain injury impairs norepinephrine system function and psychostimulant responsivity

Traumatic brain injury (TBI) is a complex pathophysiological process that results in a variety of neurotransmitter, behavioral, and cognitive deficits. The locus coeruleus-norepinephrine (LC-NE) system is a critical regulator of arousal levels and higher executive processes affected by TBI including attention, working memory, and decision making. LC-NE axon injury and impaired signaling within the prefrontal cortex (PFC) is a potential contributor to the neuropsychiatric symptoms after single, moderate to severe TBI. The majority of TBIs are mild, yet long-term cognitive deficits and increased susceptibility for further injury can accumulate after each repetitive mild TBI. As a potential treatment for restoring cognitive function and daytime sleepiness after injury psychostimulants, including methylphenidate (MPH) that increase levels of NE within the PFC, are being prescribed “off-label”. The impact of mild and repetitive mild TBI on the LC-NE system remains limited. Therefore, we determined the extent of LC-NE and arousal dysfunction and response to therapeutic doses of MPH in rats following experimentally induced single and repetitive mild TBI. Microdialysis measures of basal NE efflux from the medial PFC and arousal measures were significantly lower after repetitive mild TBI. Females showed higher baseline PFC-NE efflux than males following single and repetitive mild TBI. In response to MPH challenge, males exhibited a blunted PFC-NE response and persistent arousal levels following repetitive mild TBI. These results provide critical insight into the role of catecholamine system dysfunction associated with cognitive deficits following repeated injury, outcome differences between sex/gender, and lack of success of MPH as an adjunctive therapy to improve cognitive function following injury.

Assessing belowground carbon storage after converting a temperate permanent grassland into a bamboo (Phyllostachys) plantation

AbstractBamboo (Phyllostachys sp.) is considered a sustainable resource that can replace fossil fuel‐based products. Its additional ability to sequester organic carbon in the soil (SOC) makes it a promising nature‐based solution for combating climate change. However, bamboo's soil C storage potential may vary considerably between species or growing conditions and needs to be better quantified, especially in temperate climates where data are lacking. In the present research, the SOC dynamics of plots converted from grassland to plantations of three bamboo species (i.e. Phyllostachys nigra, Phyllostachys aurea and Phyllostachys aureosulcata), planted 12 years ago on podzol (World Reference Base classification) in the Belgian Campine region, have been studied. Soil and root samples were taken until a depth of 40 cm using a 10 cm interval. Besides, the total belowground C stability (mgCO2‐C g−1 C h−1) was assessed by measuring during 3 months the carbon dioxide (CO2) efflux relative to the belowground C stock. Based on an equivalent soil mass, only P. aureosulcata, the species with the highest culm basal area, had a significant (p < .001) SOC increase of 5.0 kg C m−2 (relative increase of +94%) as compared with grassland. Considering the sum of C stocks in the soil, roots and leaf litter, all bamboo species showed significant (p < .001) C storage, i.e. +3.6 kg C m−2 (+64%), +5.3 kg C m−2 (+94%) and +8.6 kg C m−2 (+151%) for P. nigra, P. aurea and P. aureosulcata, respectively. In addition, bamboo's relative basal CO2 efflux (0.007, 0.006 and 0.008 mgCO2‐C g−1 C h−1, respectively) was remarkably lower than in the grassland (0.012 mgCO2‐C g−1 C h−1), though it was only significant for P. aurea. This study highlights that converting temperate permanent grassland into Phyllostachys bamboo plantation can result in net and rapid organic C storage by increasing the total belowground C stability and C input. Further research regarding the net CO2 balance of bamboo‐derived products is still required to fully assess its climate change mitigation potential.

Differential patterns of basal and naloxone-evoked dopamine efflux in the rat dorsal and ventral striatum following prolonged-intermittent exposure to morphine.

Hypodopaminergia in the ventral striatum is a putative neurobiological correlate of withdrawal in opioid-dependent individuals. This perspective stands in contrast to brain imaging studies with chronic opioid users showing that naloxone-enhanced dopamine (DA) release in the dorsal striatum is positively correlated with withdrawal aversion. Here, we examined regional differences in striatal DA function associated with opioid withdrawal in rats exposed to intermittent morphine injections for 31 days. Basal concentrations of DA were reduced (i.e., indicating a hypodopaminergic state) in the ventral striatum on Day 10 of morphine exposure, whereas a more prolonged period of morphine treatment was required to reveal hypodopaminergia in the dorsal striatum on Day 31. The ventral striatum consistently exhibited naloxone-induced transient reductions in DA below the hypodopaminergic basal levels, whereas morphine enhanced DA efflux. In the dorsal striatum, DA responsivity to naloxone shifted from a significant decrease on Day 10 to a notable increase above hypodopaminergic basal levels on Day 31, corroborating the findings in the human dorsal striatum. Unexpectedly, the magnitude of morphine-evoked increases in DA efflux on Day 31 was significantly blunted relative to values on Day 10. These findings indicate that prolonged-intermittent access to morphine results in a sustained hypodopaminergic state as reflected in basal levels in the striatum, which is accompanied by regional differences in DA responsivity to naloxone and morphine. Overall, our findings suggest that prolonging the duration of morphine exposure to 31 days is sufficient to reveal neuroadaptations that may underlie the transition from initial drug exposure to opioid dependence.

Contribution of efflux and mutations in fluoroquinolone susceptibility in MDR enterobacterial isolates: a quantitative and molecular study.

The emergence of MDR strains is a public health problem in the management of associated infections. Several resistance mechanisms are present, and antibiotic efflux is often found at the same time as enzyme resistance and/or target mutations. However, in the laboratory routinely, only the latter two are identified and the prevalence of antibiotic expulsion is underestimated, causing a misinterpretation of the bacterial resistance phenotype. The development of a diagnostic system to quantify the efflux routinely would thus improve the management of patients. A quantitative technique based on detection of clinically used fluoroquinolones was investigated in Enterobacteriaceae clinical strains with a high or basal efflux activity. The detail of efflux involvement was studied from MIC determination and antibiotic accumulation inside bacteria. WGS was carried out on selected strains to determine the genetic background associated with efflux expression. Only 1 Klebsiella pneumoniae isolate exhibited a lack of efflux whereas 13 isolates had a basal efflux and 8 presented efflux pump overexpression. The antibiotic accumulation evidenced the efficacy of the efflux mechanism in strains, and the contribution of dynamic expulsion versus target mutations in fluoroquinolone susceptibility. We confirmed that phenylalanine arginine β-naphthylamide is not a reliable marker of efflux due to the affinity of the AcrB efflux pump for different substrates. We have developed an accumulation test that can be used efficiently on clinical isolates collected by the biological laboratory. The experimental conditions and protocols ensure a robust assay that with improvements in practice, expertise and equipment could be transferred to the hospital laboratory to diagnose the contribution of efflux in Gram-negative bacteria.

Adolescence as a critical period for nandrolone-induced muscular strength in relation to abuse liability, alone and in conjunction with morphine, using accumbal dopamine efflux in freely moving rats.

Nandrolone, an anabolic androgenic steroid, is included in the prohibited list of the World Anti-Doping Agency. Drugs of abuse activate brain dopamine neurons and nandrolone has been suspected of inducing dependence. Accordingly, possible critical periods for the effects of nandrolone on muscular strength and dopaminergic activity have been investigated, including the effects of chronically administered nandrolone alone and on morphine-induced increases in dopamine efflux in the nucleus accumbens. Six- or 10-week-old male Sprague-Dawley rats were used. Treatment with nandrolone was initiated in adolescent (6-week-old) and young adult (10-week-old) rats. Nandrolone (5.0 mg/kg s.c.) or sesame oil vehicle was given once daily, on six consecutive days per week, for 3 weeks and then once per day for 4 consecutive days. Nandrolone enhanced the developmental increase in grip strength of 6- but not 10-week-old rats, without altering the developmental increase in body weight of either age group. Using in vivo microdialysis in freely moving 6-week-old rats given nandrolone for 4 weeks, basal accumbal dopamine efflux was unaltered, while the increase in dopamine efflux induced by acute administration of morphine (1.0 mg/kg s.c.) was reduced. The present study provides in vivo evidence that adolescence constitutes a critical period during which repeated administration of nandrolone enhances increases in muscular strength without influencing increases in body weight. Though repeated administration of nandrolone during this period of adolescence did not stimulate in vivo mesolimbic dopaminergic activity, it disrupted stimulation by an opioid, the drug class that is most commonly coabused with nandrolone.

Endothelial Pannexin1‐purinergic signaling pulmonary hypertension

Endothelial cell (EC) Ca2+ signals dilate small pulmonary arteries (PAs) and maintain a low resting pulmonary arterial pressure (PAP). Impairment of endothelial Ca2+ signaling mechanisms is a crucial contributor to elevated PAP in pulmonary hypertension (PH). Studies in systemic arteries show that flow/shear stress activates endothelial Ca2+ signals to cause vasodilation. Although PAs are a “high‐flow” vascular bed, flow‐induced endothelial Ca2+ signaling mechanisms have not been investigated in PH. In this regard, we recently showed that Ca2+ influx through endothelial transient receptor potential vanilloid 4 (TRPV4EC) ion channels dilates PAs and lowers resting PAP (Daneva et al., PNAS, 2021). Moreover, flow/shear stress activates the efflux of adenosine triphosphate (ATP) through endothelial Pannexin 1 (Panx1EC) in PAs. Panx1EC‐effluxed ATP, in turn, stimulates endothelial purinergic P2Y2 receptor (P2Y2REC) to increase TRPV4EC channels activity (Daneva et al., eLife, 2021). I hypothesized that impaired Panx1EC—P2Y2REC—TRPV4EC signaling axis reduces flow‐induced dilation of PAs and elevates PAP in PH. Confocal Ca2+ imaging studies in fluo‐4‐loaded en face PAs (~ 50 mm) showed that the baseline and agonist (GSK1016790A or GSK101)‐induced activity of TRPV4EC sparklets (individual Ca2+ influx signals through TRPV4EC channels) is reduced in the mice exposed to Sugen 5416 + chronic hypoxia (Su+CH; 3 weeks; 10% O2) compared to the normoxic control mice, indicating a decrease in TRPV4EC channel activity in PH. The expression of TRPV4 channels at the mRNA level, however, was not altered in Su+CH mice. The activation of TRPV4EC sparklets by exogenous ATP (1 mM) was also reduced in PAs from Su+CH mice, suggesting an impairment of ATP‐P2Y2REC‐TRPV4EC channel signaling axis in PH. Accordingly, ATP‐induced, endothelium‐dependent dilation was absent in PAs from Su+CH mice. We previously showed that basal ATP efflux in PAs predominantly occurs through endothelial Panx1, and flow/shear stress increases ATP efflux entirely through the activation of endothelial Panx1. Bioluminescence measurements of extracellular ATP concentration showed lower baseline ATP levels in PAs from Su+CH mice than control mice, suggesting a decrease in ATP efflux in PH. Importantly, flow/shear stress (4 – 15 dynes/cm2) increased the efflux of ATP from normal mice, but not in PAs from Su+CH mice, indicating that flow/shear stress‐Panx1EC signaling is also impaired in PH. Together, these data support the idea that flow‐Panx1EC and P2Y2REC‐TRPV4EC signaling are impaired in PH. Targeting individual elements of this pathway may provide novel therapeutic options in PH and other pulmonary vascular disorders characterized by endothelial dysfunction. Future studies will assess the individual signaling linkages of the flow‐Panx1EC‐P2Y2REC‐TRPV4EC signaling axis in PH.

Effects of pre-eclampsia on HDL-mediated cholesterol efflux capacity after pregnancy

Background and aimsPreeclampsia (PE) is associated with life-long increased risk of cardiovascular disease. One of the main protective functions of high-density lipoprotein (HDL) is its role in reverse cholesterol transport. HDL-mediated cholesterol efflux capacity (CEC) is decreased during pregnancy in women with PE. Whether this persists postpartum is unknown. MethodsBasal and transporter-specific CEC were determined 6 months postpartum in women who had a normotensive (n = 44) or a PE (n = 42) pregnancy. CEC was also measured in 23 normotensive and 20 PE women for whom samples were collected 24 months postpartum. Basal, ATP-binding cassette transporter-A1 (ABCA1)- and -G1 (ABCG1)-specific CEC were primarily determined using Chinese hamster ovary cells stably expressing human ABCA1 or ABCG1, and were also assessed using a J774 mouse macrophage cell line. ResultsABCA1-specific CEC was significantly lower in women who had PE 6 months postpartum (0.57 ± 0.1 vs 0.53 ± 0.08; p < 0.05), whilst basal and ABCG1-specific efflux were not significantly different. cAMP-specific CEC in J774 cells was also lower 6 months after PE (0.85 ± 0.21 vs 0.75 ± 0.25, p < 0.05). Although apoA-I, apoE, plasminogen and PON-1 levels were not significantly different in women who had PE compared with controls, ABCA1 efflux did correlate with apoA-l, HDL-C and apoE levels after a normal, and with apoA-l and HDL-C levels after a PE pregnancy. ABCA1-specific efflux decreased in all women between 6 and 24 months postpartum, by 11 ± 1.6% in women who had a normotensive pregnancy and 9 ± 1.3% in women who had PE. After adjustment for apoA-I levels, there was no significant difference in ABCA1-specific efflux between the groups at 6 months postpartum and in normotensive women over time, but remained significantly different between 6 and 24 months in women who had PE. ConclusionsABCA1-mediated CEC is impaired 6 months postpartum after a PE pregnancy and decreases thereafter in both normotensive and PE pregnancies. ABCA1-mediated efflux is dynamic after pregnancy but is unlikely to explain the long-term increased CVD risk in women with PE.

In vivo microdialysis reveals that blockade of accumbal orexin OX2 but not OX1 receptors enhances dopamine efflux in the nucleus accumbens of freely moving rats.

The nucleus accumbens contain orexinergic neural inputs and orexin OX1 - and OX2 -receptors. Behavioural studies suggest that accumbal orexin receptors modulate accumbal dopaminergic activity-dependent locomotion in rats. We studied the effects of intra-accumbal injection of orexin receptor ligands on accumbal extracellular dopamine levels in freely moving rats, using in vivo microdialysis and analysed the roles of OX1 - and OX2 -receptors in the regulation of basal accumbal dopamine efflux. The orexin receptor ligands were applied intra-accumbally though a microinjection needle attached with a dialysis probe. Neither the nonselective OX1 - and OX2 -receptor agonist orexin-A nor the preferential OX2 -receptor agonist orexin-B (500.0pg and 5.0ng) altered accumbal dopamine levels. The nonselective OX1 - and OX2 -receptor antagonist MK-4305 (suvorexant, 500.0pg, 2.5 and 5.0ng) enhanced dopamine efflux. A 2-h tetrodotoxin infusion into nucleus accumbens through the probe or co-administration of orexin-A (500.0pg) strongly inhibited MK-4305 (5.0ng)-induced accumbal dopamine efflux. The selective OX2 -receptor antagonist EMPA (90.0 and 900.0pg, 9.0ng) increased dopamine efflux. Intra-accumbal infusion of tetrodotoxin abolished EMPA (9.0ng)-induced dopamine efflux. The selective OX1 -receptor antagonist SB-334867 (10.0 and 20.0ng) failed to alter dopamine efflux. Co-administration of orexin-B (500.0pg) inhibited both EMPA (9.0ng)- and MK-4305 (5.0ng)-induced dopamine efflux. Intraperitoneal injection of MK-4305 (10.0mg/kg) did not affect accumbal dopamine efflux. The present study provides in vivo neuropharmacological evidence that accumbal OX2 - but not OX1 -receptors exert inhibitory regulation of basal accumbal dopamine efflux and that blockade of accumbal OX2 -receptors enhances dopamine efflux in nucleus accumbens of freely moving rats.

Hyperglycemia-Induced Metabolic Reprogramming Mediates a Proatherogenic Phenotype in Healthy Human Monocytes

Introduction: Poor glycemic control is considered an important contributor to cardiovascular disease in patients with diabetes. Episodic hyperglycemia as a surrogate for glycemic variability promotes monocyte adhesion and increases the prevalence of proinflammatory monocytes within atherosclerotic plaques of patients with diabetes. We previously found that acute hyperglycemia-induced a pro-inflammatory phenotype and promoted the development of foamy monocytes by increasing total cholesterol deposition, cholesterol ester, and free cholesterol content by enhancing oxidized LDL uptake. However, the mechanism by which acute hyperglycemia induces monocyte cholesterol deposition and inflammation remains unknown. Methods: Monocytes isolated from healthy individuals (age range 20–40; n=5) were cultured in low (5mM) or high (16.7mM) glucose conditions with or without a glycolysis inhibitor (2-deoxyglucose, 2DG, 5 mM) or an endoplasmic reticulum stress inhibitor (4-phenylbutyric acid, PBA; 20mM) for 6 hrs. After treatment, cytokine release, oxidized LDL uptake, and metabolic assays using Seahorse Technology were performed. Results: Healthy human monocytes exposed under high glucose conditions showed a pro-atherosclerotic phenotype with higher levels of the pro-inflammatory cytokines, TNFα(median of differences 6.34 pg/ml, p=0.002) and IL1β(12.04 pg/ml, p=0.003), and increased oxidized LDL uptake (5062ug Dil-Ox LDL/mg, p=0.001). Furthermore, hyperglycemia resulted in higher levels of glycolysis (basal glycolysis 12.94 pmol/min, p=0.01; basal proton efflux rate 15.5 pmol/min, p=0.03) and mitochondrial respiration (percentage of respiratory capacity 16pmol/min p=0.04), suggesting a significant alteration in the metabolic programming of these monocytes. Treatment with 2-DG or PBA attenuated the pro-atherosclerotic phenotype induced by hyperglycemia, promoting a reduction of cytokine release, a reduction of oxidized LDL uptake, and near normalization of the glycolic rate and mitochondrial respiration, stabilizing cellular bioenergetics. Conclusions: Altogether, our results suggest that monocyte ER stress in response to acute hyperglycemia promotes a hypermetabolic state characterized by a proinflammatory and proatherogenic monocyte phenotype. Therefore, acute hyperglycemia is a potential mechanism promoting atherosclerosis in patients with type 2 diabetes.

Investigation of the roles of orexin receptor subtypes in regulation of basal dopamine efflux in the nucleus accumbens of freely moving rats

Investigation of the roles of orexin receptor subtypes in regulation of basal dopamine efflux in the nucleus accumbens of freely moving rats

XPR1 Mediates the Pancreatic β-Cell Phosphate Flush.

Glucose-stimulated insulin secretion is the hallmark of the pancreatic β-cell, a critical player in the regulation of blood glucose concentration. In 1974, the remarkable observation was made that an efflux of intracellular inorganic phosphate (Pi) accompanied the events of stimulated insulin secretion. The mechanism behind this “phosphate flush,” its association with insulin secretion, and its regulation have since then remained a mystery. We recapitulated the phosphate flush in the MIN6m9 β-cell line and pseudoislets. We demonstrated that knockdown of XPR1, a phosphate transporter present in MIN6m9 cells and pancreatic islets, prevented this flush. Concomitantly, XPR1 silencing led to intracellular Pi accumulation and a potential impact on Ca2+ signaling. XPR1 knockdown slightly blunted first-phase glucose-stimulated insulin secretion in MIN6m9 cells, but had no significant impact on pseudoislet secretion. In keeping with other cell types, basal Pi efflux was stimulated by inositol pyrophosphates, and basal intracellular Pi accumulated following knockdown of inositol hexakisphosphate kinases. However, the glucose-driven phosphate flush occurred despite inositol pyrophosphate depletion. Finally, while it is unlikely that XPR1 directly affects exocytosis, it may protect Ca2+ signaling. Thus, we have revealed XPR1 as the missing mediator of the phosphate flush, shedding light on a 45-year-old mystery.

Slc43a3 is a regulator of free fatty acid flux

Adipocytes take up long chain FAs through diffusion and protein-mediated transport, whereas FA efflux is considered to occur by diffusion. To identify potential membrane proteins that are involved in regulating FA flux in adipocytes, the expression levels of 55 membrane transporters without known function were screened in subcutaneous adipose samples from obese patients before and after bariatric surgery using branched DNA methodology. Among the 33 solute carrier (SLC) transporter family members screened, the expression of 14 members showed significant changes before and after bariatric surgery. One of them, Slc43a3, increased about 2.5-fold after bariatric surgery. Further investigation demonstrated that Slc43a3 is highly expressed in murine adipose tissue and induced during adipocyte differentiation in primary preadipocytes and in OP9 cells. Knockdown of Slc43a3 with siRNA in differentiated OP9 adipocytes reduced both basal and forskolin-stimulated FA efflux, while also increasing FA uptake and lipid droplet accumulation. In contrast, overexpression of Slc43a3 decreased FA uptake in differentiated OP9 cells and resulted in decreased lipid droplet accumulation. Therefore, Slc43a3 seems to regulate FA flux in adipocytes, functioning as a positive regulator of FA efflux and as a negative regulator of FA uptake.

Energy Deficit Phenotype of Cerebral Microvascular Network in the Aged Brains

ObjectiveAging is associated with an increased risk of neurodegenerative diseases and stroke. Impaired microvascular function was proposed to be the connecting link, but the underlying mechanisms are unclear. Microvasculature is critical for cerebrovascular blood flow, neurovascular coupling, and nutrient transport, and blood‐brain‐barrier. Maintaining these energy‐demanding functions by cerebral microvasculature needs optimal functioning of energy pathways like glycolysis and mitochondrial respiration. Characterization of cellular energy metabolism in the young and aged microvasculature was essential to understand the aging‐induced cerebrovascular pathologies.MethodsWe isolated the microvessels from the brains from young (3 months) and aged mice (24 months). Microvessels were isolated by a combination of filtration (40μm – 300μm) and gradient centrifugation. Microvessels were plated on Seahorse XFe24 culture plates. Basal and compensatory glycolysis, and proton efflux rate (PER) were measured from extracellular acidification rates (ECAR) using glycolysis rate kit. Similarly, mitochondrial respiratory parameters were determined by the measurements from the oxygen consumption rate (OCR), and the fuel dependency of the microvasculature was measured using mitofuel kit.ResultsAged brain microvasculature showed 26% (73.87±7.6 vs. 100.1±2.9 picomoles of H+/min/μg protein, p&lt;0.01, n=7–9 mice/phenotype) decrease in basal glycolysis when compared with the microvessels from the young mouse brain. Compensatory glycolysis is decreased by 28.3% (121.0±7.0 vs 168.9±13.4 picomoles of H+/min/μg protein, p&lt;0.004, n=7–9 mice/phenotype) in the aged mouse brain microvessels compared to the young mouse brain vessels, whereas the basal proton efflux is decreased by 35.3% (105.2±6.3 vs 162.8±5.9 picomoles of H+/min/μg protein, p&lt;0.0001, n=7–9 mice/phenotype). Microvessels from the aged mouse brain had 36% lower mitochondrial respiration compared to the young mouse brain microvasculature (64.0±10.4 vs 100.0±7.6 picomoles of O2/min/μg protein, p&lt;0.01, n=7–9 mice/phenotype). Glucose (65.6±0.2%, n=4 mice) is the primary fuel preference by the mitochondria in the young mouse brain microvessels followed by glutamine (32.9±0.5%) and fatty acids (27.9±3.0%). Aged microvasculature exhibited unaltered fuel preferences concerning glucose and fatty acids, whereas glutamine utilization is increased by 58% (54.1±2.9% vs 34.3% vs 3.57, n=3 mice) when compared to the young mouse brain microvessels.ConclusionsWe conclude that aged brain microvasculature exhibits compromised energy metabolism including both glycolysis and the mitochondrial respiration. We further conclude that mitochondrial utilization of glutamine as energy fuel is increased in the aged brain microvasculature. The energy deficit phenotype of the brain microcirculatory network may contribute to the aging‐associated cerebrovascular complications.Support or Funding InformationNational Institute of Health: National Institute of General Medical Sciences and National Institute of Neurological Disorders and Stroke (Katakam: R01NS094834) and National Institute on Aging (Mostany: AG047296).

In vitro Antisickling and Sub Chronic Toxicity Studies of the Ethanol Extract of Cnidoscolus aconitifolius in Wistar Rats

Objective: The current study investigated the mechanism(s) of action of the antisickling property of Cnidiscolus aconitifolius ethanol extract and determined its histopathological effects on the liver, kidney and spleen of Wistar rats. Methods: The antisickling properties of C. aconitifolius (CA) ethanol extract was tested at different concentrations, using the inhibitory and reversal models. Its effect on the density and mean cell volume of Hb S red blood cells was evaluated using the discontinuous density gradient assay. Ciklavit®, an antisickling nutritional supplement was employed as positive while phosphate buffered saline (pH 7.0) was the negative control. Changes in percentage Red Cell Count (RBC) and Mean Cell Volume (MCV) in each density fraction were determined. Quantification of extracellular potassium leakage of RBC before and after incubation with CA extract was also conducted. CA was evaluated for its acute toxicity (LD50) while sub-acute toxicity was evaluated by oral administration to Wistar rats for 28 days at 125, 250 and 500 mg/kg body weight. Rats were anaesthetized on the 29th day and blood collected through cardiac puncture for hematological analysis. The liver, kidney and spleen of the rats were harvested for histopathological examinations. Results: The results showed that the percentage of dense cells in blood treated with CA was reduced significantly (p<0.05) compared to positive control. CA increased MCV of cells and inhibited basal potassium efflux with a rightward shift in the dose-response curve. The median lethal dose (LD50) of CA was 3808 mg/kg; neither mortality nor structural or histological alterations were observed in the liver, kidney and spleen at the administered dose. The extract of CA enhanced erythropoiesis. Conclusion: C. aconitifolius extract prevented potassium efflux and reduced red cell density in vitro. The CA crude extract was safe for consumption and increased the production of red blood cells in vivo.

Abstract 2623: Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death

Abstract Pediatric brain cancer medulloblastoma standard-of-care results in numerous comorbidities. Medulloblastoma is comprised of four molecular subgroups. Group 3 subgroup patients have the highest relapse rates and 20% survival after standard-of-care. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABAAR). We are advancing a therapeutic approach for group 3 based on GABAAR modulation using benzodiazepine-derivatives. We performed analysis of GABR and MYC expression in medulloblastoma tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional ‘druggable’ GABAAR in group 3 cells. Analysis of expression of 763 medulloblastoma tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABAAR subunits α5, β3 and γ2 and 3. There are ~1000 functional α5-GABAARs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2x109 ions/sec. Benzodiazepines, designed to prefer α5-GABAAR, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine (‘KRM-II-08’) binds to the α5-GABAAR (0.8 μM EC50) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter (BAD) protein localization. GABR expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABAAR is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis. Citation Format: Laura Kallay, Havva Keskin, Alexandra Ross, Manali Rupji, Olivia A. Moody, Xin Wang, Guanguan Li, Taukir Ahmed, Farjana Rashid, Michael Rajesh Stephen, Kirsten A. Cottrill, Austin Nuckois, Maxwell Xu, Deborah E. Martinson, Frank Tranghese, Yanxin Pei, James M. Cook, Jeanne Kowalski, Michael D. Taylor, Andrew Jenkins, Daniel Pomeranz Krummel, Soma Sengupta. Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2623.

Mutational effects of human dopamine transporter at tyrosine88, lysine92, and histidine547 on basal and HIV-1 Tat-inhibited dopamine transport

Dysregulation of dopaminergic system induced by HIV-1 Tat protein-mediated direct inhibition of the dopamine transporter (DAT) has been implicated as a mediating factor of HIV-1 associated neurocognitive disorders. We have reported that single point mutations on human DAT (hDAT) at tyrosine88 (Y88F), lysine92 (K92M), and histidine547 (H547A) differentially regulate basal dopamine uptake but diminish Tat-induced inhibition of dopamine uptake by changing dopamine transport process. This study evaluated the effects of double (Y88F/H547A) and triple (Y88F/K92M/H547A) mutations on basal dopamine uptake, Tat-induced inhibition of DAT function, and dynamic transport process. Compared to wild-type hDAT, the Vmax values of [3H]Dopamine uptake were increased by 96% in Y88F/H547A but decreased by 97% in Y88F/K92M/H547A. [3H]WIN35,428 binding sites were not altered in Y88F/H547A but decreased in Y88F/K92M/H547A. Y88F/H547A mutant attenuated Tat-induced inhibition of dopamine uptake observed in wild-type hDAT. Y88F/H547A displayed an attenuation of zinc-augmented [3H]WIN35,428 binding, increased basal dopamine efflux, and reduced amphetamine-induced dopamine efflux, indicating this mutant alters transporter conformational transitions. These findings further demonstrate that both tyrosine88 and histidine547 on hDAT play a key role in stabilizing basal dopamine transport and Tat-DAT integration. This study provides mechanistic insights into developing small molecules to block multiple sites in DAT for Tat binding.

Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death

PurposePediatric brain cancer medulloblastoma (MB) standard-of-care results in numerous comorbidities. MB is comprised of distinct molecular subgroups. Group 3 molecular subgroup patients have the highest relapse rates and after standard-of-care have a 20% survival. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABAAR). We are advancing a therapeutic approach for group 3 based on GABAAR modulation using benzodiazepine-derivatives.MethodsWe performed analysis of GABR and MYC expression in MB tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional ‘druggable’ GABAAR in group 3 cells.ResultsAnalysis of expression of 763 MB tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABAAR subunits α5, β3 and γ2 and 3. There are ~ 1000 functional α5-GABAARs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2 × 109 ions/s. Benzodiazepines, designed to prefer α5-GABAAR, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine (‘KRM-II-08’) binds to the α5-GABAAR (0.8 µM EC50) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter protein localization.ConclusionGABRA5 expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABAAR is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis.

Relation of High-Density Lipoprotein Charge Heterogeneity, Cholesterol Efflux Capacity, and the Expression of High-Density Lipoprotein-Related Genes in Mononuclear Cells to the HDL-Cholesterol Level.

The heterogeneity and content of human plasma high-density lipoprotein (HDL) related to their atheroprotective properties determined by various molecular and cellular mechanisms still remain to be completely clarified. For 29 atherosclerosis-free male subjects, we studied the relationship of plasma lipid levels and the content of apolipoprotein A-I (apoA-I)-containing HDL with preβ-electrophoretic mobility, the efficiency of BODIPY-cholesterol efflux from RAW 264.7 macrophages to apolipoprotein B (apoB)-deficient plasma, and the expression level of 22 genes related to HDL metabolism in mononuclear cells. A significant decrease in the absolute content of apoA-I in preβ-HDL was found in subjects with hypoalphalipoproteinemia compared with the subjects with hyperalphalipoproteinemia. The preβ-to-α-ratio of the apoA-I content was constant within the HDL-cholesterol (HDL-C) range 0.59 to 2.24 mM. However, this ratio was significantly increased with an increase in the plasma triacylglycerol (TAG) content from 0.59 to 3.42 mM. A correlation of the level of preβ-HDL with the basal and ABCA1-mediated efflux of cholesterol is shown. The transcript levels for six HDL-metabolizing genes (LDLR, LCAT, ABCA1, SCARB1, ZDHHC8, and BMP1) were decreased, while the transcript level of APOA1 gene was increased in mononuclear cells of subjects with hyperalphalipoproteinemia as compared with subjects with hypoalphalipoproteinemia. A reduction of the intracellular cholesterol level and inhibition of the expression of cholesterol transporters by nascent HDL in mononuclear cells from subjects with hyperalphalipoproteinemia are suggested. Hyperalphalipoproteinemia can be a driving force of the decreased flux of cholesteryl ester to the liver and the increased TAG hydrolysis. The atheroprotective effect of preβ-HDL in hypertriglyceridemia is proposed.