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Chronic inflammation is a hallmark of HIV infection. Eicosanoids reflect inflammation, oxidant stress, and vascular health and vary by sex and metabolic parameters. Raltegravir (RAL) is an HIV-1 integrase inhibitor that may have limited metabolic effects. We assessed urinary F2-isoprostanes (F2-IsoPs), prostaglandin E2 (PGE-M), prostacyclin (PGI-M), and thromboxane B2 (TxB2) in HIV-infected women switching to RAL-containing antiretroviral therapy (ART). Thirty-seven women (RAL = 17; PI/NNRTI = 20) with a median age of 43 years and BMI 32 kg/m(2) completed week 24. TxB2 increased in the RAL versus PI/NNRTI arm (+0.09 versus -0.02; P = 0.06). Baseline PGI-M was lower in the RAL arm (P = 0.005); no other between-arm cross-sectional differences were observed. In the PI/NNRTI arm, 24-week visceral adipose tissue change correlated with PGI-M (rho = 0.45; P = 0.04) and TxB2 (rho = 0.44; P = 0.005) changes, with a trend seen for PGE-M (rho = 0.41; P = 0.07). In an adjusted model, age ≥ 50 years (N = 8) was associated with increased PGE-M (P = 0.04). In this randomized trial, a switch to RAL did not significantly affect urinary eicosanoids over 24 weeks. In women continuing PI/NNRTI, increased visceral adipose tissue correlated with increased PGI-M and PGE-M. Older age (≥ 50) was associated with increased PGE-M. Relationships between aging, adiposity, ART, and eicosanoids during HIV-infection require further study.
BACKGROUND - Antiretroviral drugs vary in their central nervous system penetration, with better penetration possibly conferring neurocognitive benefit during human immunodeficiency virus (HIV) therapy. The efflux transporter gene ABCB1 is expressed in the blood-brain barrier, and an ABCB1 variant (3435C → T) has been reported to affect ABCB1 expression. The integrase inhibitor raltegravir is a substrate for ABCB1. We examined whether ABCB1 3435C → T affects raltegravir disposition into cerebrospinal fluid (CSF), and explored associations with polymorphisms in other membrane transporter genes expressed in the blood-brain barrier.
METHODS - Forty healthy, HIV-negative adults of European descent (20 homozygous for ABCB1 3435 C/C, 20 homozygous for 3435 T/T, each group divided equally between males and females) were given raltegravir 400 mg twice daily for 7 days. With the final dose, plasma was collected for pharmacokinetic analysis at 9 timepoints over 12 hours, and CSF collected 4 hours post dose.
RESULTS - The 4-hour CSF concentration correlated more strongly with 2-hour (r(2)=0.76, P=1.12 x 10(-11)) than 4-hour (r(2)=0.47, P=6.89 x 10(-6)) single timepoint plasma concentration, and correlated strongly with partial plasma area-under-the-curve values (AUC0-4h r(2)=0.86, P=5.15 x 10(-16)). There was no significant association between ABCB1 3435C → T and ratios of CSF-to-plasma AUC or concentration (p>0.05 for each comparison). In exploratory analyses, CSF-to-plasma ratios were not associated with 276 polymorphisms across 16 membrane transporter genes.
CONCLUSIONS - Among HIV-negative adults, CSF raltegravir concentrations do not differ by ABCB1 3435C → T genotype but strongly correlate with plasma exposure.
TRIAL REGISTRATION - ClinicalTrials.gov NCT00729924 http://clinicaltrials.gov/show/NCT00729924.
Ethanol-induced damage in the developing hippocampus may result in cognitive deficits such as those observed in fetal alcohol spectrum disorder (FASD). Cognitive deficits in FASD are partially mediated by alterations in glutamatergic synaptic transmission. Recently, we reported that synaptic transmission mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is impaired following fetal ethanol exposure. This finding led us to develop a rational approach for the treatment of alcohol-related cognitive deficits using aniracetam, an allosteric AMPAR modulator. In the present study, 28 to 34-day-old rats exposed to ethanol in utero were treated with aniracetam, and subsequently exhibited persistent improvement in mEPSC amplitude, frequency, and decay time. Furthermore, these animals expressed positive changes in synaptic single channel properties, suggesting that aniracetam ameliorates prenatal ethanol-induced deficits through modifications at the single channel level. Specifically, single channel open probability, conductance, mean open and closed times, and the number and burst duration were positively affected. Our findings emphasize the utility of compounds which slow the rate of deactivation and desensitization of AMPARs such as aniracetam.
Aniracetam is a nootropic compound and an allosteric modulator of AMPA receptors (AMPARs) which mediate synaptic mechanisms of learning and memory. Here we analyzed impairments in AMPAR-mediated synaptic transmission caused by moderate prenatal ethanol exposure and investigated the effects of postnatal aniracetam treatment on these abnormalities. Pregnant Sprague-Dawley rats were gavaged with ethanol or isocaloric sucrose throughout pregnancy, and subsequently the offspring were treated with aniracetam on postnatal days (PND) 18 to 27. Hippocampal slices prepared from these pups on PND 28 to 34 were used for the whole-cell patch-clamp recordings of AMPAR-mediated spontaneous and miniature excitatory postsynaptic currents in CA1 pyramidal cells. Our results indicate that moderate ethanol exposure during pregnancy results in impaired hippocampal AMPAR-mediated neurotransmission, and critically timed aniracetam treatment can abrogate this deficiency. These results highlight the possibility that aniracetam treatment can restore synaptic transmission and ameliorate cognitive deficits associated with the fetal alcohol syndrome.
Mitochondrial Ca2+ (mCa2+) handling is an important regulator of liver cell function that controls events ranging from cellular respiration and signal transduction to apoptosis. Cytosolic Ca2+ enters mitochondria through the ruthenium red-sensitive mCa2+ uniporter, but the mechanisms governing uniporter activity are unknown. Activation of many Ca2+ channels in the cell membrane requires PLC. This activation commonly occurs through phosphitidylinositol-4,5-biphosphate (PIP2) hydrolysis and the production of the second messengers inositol 1,4,5-trisphosphate [I(1,4,5)P3] and 1,2-diacylglycerol (DAG). PIP2 was recently identified in mitochondria. We hypothesized that PLC exists in liver mitochondria and regulates mCa2+ uptake through the uniporter. Western blot analysis with anti-PLC antibodies demonstrated the presence of PLC-delta1 in pure preparations of mitochondrial membranes isolated from rat liver. In addition, the selective PLC inhibitor U-73122 dose-dependently blocked mCa2+ uptake when whole mitochondria were incubated at 37 degrees C with 45Ca2+. Increasing extra mCa2+ concentration significantly stimulated mCa2+ uptake, and U-73122 inhibited this effect. Spermine, a uniporter agonist, significantly increased mCa2+ uptake, whereas U-73122 dose-dependently blocked this effect. The inactive analog of U-73122, U-73343, did not affect mCa2+ uptake in any experimental condition. Membrane-permeable I(1,4,5)P3 receptor antagonists 2-aminoethoxydiphenylborate and xestospongin C also inhibited mCa2+ uptake. Although extra mitochondrial I(1,4,5)P3 had no effect on mCa2+ uptake, membrane-permeable DAG analogs 1-oleoyl-2-acetyl-sn-glycerol and DAG-lactone, which inhibit PLC activity, dose-dependently inhibited mCa2+ uptake. These data indicate that PLC-delta1 exists in liver mitochondria and is involved in regulating mCa2+ uptake through the uniporter.
PURPOSE - The role of adenosine receptor (AdoR) antagonists in human retinal endothelial cell function in vitro has previously been determined. In this study, efficacy of AdoR antagonist administration in reducing retinal neovascularization was examined in a mouse pup model of oxygen-induced retinopathy.
METHODS - A previously described model of oxygen-induced retinal neovascularization in newborn mouse pups was used to examine the effect of various AdoR antagonists on neovascularization. The nonselective AdoR antagonist xanthine amine congener (XAC), the A(2A)-selective antagonist ZM241385, the A(2B)-selective antagonists 3-N-propylxanthine (enprofylline) and 3-isobutyl-8-pyrrolidinoxanthine (IPDX), and the A(1)-selective antagonist cyclopentyl-1,3-dipropylxanthine (CPX) were used. After the hyperoxia exposure the animals received daily intraperitoneal injections of pharmacologically relevant doses of AdoR antagonists for 5 days. Control animals received vehicle (0.1% dimethyl sulfoxide [DMSO]) alone. The animals were then killed and perfused with fluorescein-dextran. Wholemounts of retinas from one eye were prepared and examined, whereas the retinas of the contralateral eye were embedded, sectioned, and stained for counting neovascular nuclei extending beyond the internal limiting membrane into the vitreous.
RESULTS - Angiography of wholemount retinas showed reduction of neovascular tufts in animals treated with selective A(2B) AdoR antagonists. Quantification of the extraretinal neovascular nuclei showed that only animals treated with XAC, enprofylline, or IPDX showed a significant reduction in retinal neovascularization. By contrast, neither CPX nor ZM241385 had an effect on neovascularization.
CONCLUSIONS - The A(2B)-selective AdoR antagonists inhibited oxygen-induced retinal neovascularization in vivo and may provide a basis for developing pharmacologic therapies for the treatment of proliferative retinopathies.
The antiasthmatic drug enprofylline was the first known selective, though not potent, A(2B) antagonist. On the basis of structure-activity relationships (SARs) of xanthine derivatives, we designed a novel selective adenosine A(2B) receptor antagonist, 3-isobutyl-8-pyrrolidinoxanthine (IPDX), with potency greater than that of enprofylline. IPDX displaced [3H]ZM241385 ([3H]4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a]-[1,3,5]triazin-5-ylamino]ethyl)phenol) from human A(2B) adenosine receptors with a K(i) value of 470 +/- 2 nM and inhibited A(2B)-dependent cyclic AMP (cAMP) accumulation in human erythroleukemia (HEL) cells with a K(B) value of 625 +/- 71 nM. We found that IPDX was more selective than enprofylline toward human A(2B) receptors. It was 38-, 55-, and 82-fold more selective for human A(2B) than for human A(1) (K(i) value of 24 +/- 8 microM), human A(2A) (K(B) value of 36 +/- 8 microM), and human A(3) (K(i) value of 53 +/- 10 microM) adenosine receptors, respectively. IPDX inhibited NECA (5'-N-ethylcarboxamidoadenosine)-induced interleukin-8 secretion in human mast cells (HMC-1) with a potency close to that determined for A(2B)-mediated cAMP accumulation in HEL cells, thus confirming the role of A(2B) adenosine receptors in mediating human mast cell activation. Since adenosine triggers bronchoconstriction in asthmatic patients through human mast cell activation, IPDX may become a basis for the development of new antiasthmatic drugs with improved properties compared with those of enprofylline. Our data demonstrate that IPDX can be used as a tool to differentiate between A(2B) and other adenosine receptor-mediated responses.
In an attempt to improve behavioral memory, we devised a strategy to amplify the signal-to-noise ratio of the cAMP pathway, which plays a central role in hippocampal synaptic plasticity and behavioral memory. Multiple high-frequency trains of electrical stimulation induce long-lasting long-term potentiation, a form of synaptic strengthening in hippocampus that is greater in both magnitude and persistence than the short-lasting long-term potentiation generated by a single tetanic train. Studies using pharmacological inhibitors and genetic manipulations have shown that this difference in response depends on the activity of cAMP-dependent protein kinase A. Genetic studies have also indicated that protein kinase A and one of its target transcription factors, cAMP response element binding protein, are important in memory in vivo. These findings suggested that amplification of signals through the cAMP pathway might lower the threshold for generating long-lasting long-term potentiation and increase behavioral memory. We therefore examined the biochemical, physiological, and behavioral effects in mice of partial inhibition of a hippocampal cAMP phosphodiesterase. Concentrations of a type IV-specific phosphodiesterase inhibitor, rolipram, which had no significant effect on basal cAMP concentration, increased the cAMP response of hippocampal slices to stimulation with forskolin and induced persistent long-term potentiation in CA1 after a single tetanic train. In both young and aged mice, rolipram treatment before training increased long- but not short-term retention in freezing to context, a hippocampus-dependent memory task.