The effects of 100% wild blueberry (Vaccinium angustifolium) juice consumption on cardiometablic biomarkers: a randomized, placebo-controlled, crossover trial in adults with increased risk for type 2 diabetes.

BACKGROUND: Wild blueberries have a high content of polyphenols, but there is limited data evaluating their health benefits in adults at risk for type 2 diabetes. The objective of the study was to investigate whether consumption of 100% wild blueberry juice improves cardiometabolic biomarkers associated with type 2 diabetes risk. METHODS: A single-blind, randomized, placebo-controlled, crossover design trial was conducted in which adults (women, n = 19, ages 39-64 y) at risk for type 2 diabetes consumed 240 mL of wild blueberry juice or a placebo beverage as part of their free-living diet for 7 days. Blood was collected to determine various biomarkers such as fasting plasma glucose, fasting serum insulin, surrogate markers of insulin sensitivity, triglycerides, inflammation (interleukin-6, interleukin-10, high-sensitivity C-reactive protein, tumor necrosis factor-alpha, serum amyloid A), adhesion molecules (soluble intercellular adhesion molecule-1, soluble vascular adhesion molecule-1), oxidative stress (LDL-oxidation, total 8-isoprostanes), and nitric oxide. Endothelial function and blood pressure were also assessed. RESULTS: Wild blueberry juice consumption for 7 days produced no significant changes in glucose, insulin, insulin sensitivity, triglycerides, inflammatory markers, adhesion molecules, oxidative stress, endothelial function or blood pressure. However, wild blueberry juice consumption showed a trend for lowering systolic blood pressure: 120.8 ± 2.2 mmHg in the placebo group vs 116.0 ± 2.2 mmHg in the blueberry juice group (P = 0.088). Serum concentrations of nitrates and nitrites, an index of nitric oxide production, increased from 2.9 ± 0.4 µM after placebo drink to 4.1 ± 0.4 µM after drinking wild blueberry juice (P = 0.039). CONCLUSIONS: Short-term consumption of wild blueberry juice may promote cardioprotective effects, by improving systolic blood pressure, possibly through nitric oxide production, in adults at risk for type 2 diabetes. This outcome warrants longer-term human studies of blueberries, including defined amounts of either the whole fruit or juice, to clarify whether polyphenol-rich foods can be efficacious for improving cardiometabolic biomarkers in adults at risk for type 2 diabetes. TRIAL REGISTRATION: NCT02139878, clinicaltrials.gov; date of registration: May 4, 2014.

Antioxidants and free radical scavengers for the treatment of stroke, traumatic brain injury and aging.

The overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is a common underlying mechanism of many neuropathologies, as they have been shown to damage various cellular components, including proteins, lipids and DNA. Free radicals, especially superoxide (O(2)*-), and non-radicals, such as hydrogen peroxide (H(2)O(2)), can be generated in quantities large enough to overwhelm endogenous protective enzyme systems, such as superoxide dismutase (SOD) and reduced glutathione (GSH). Here we review the mechanisms of ROS and RNS production, and their roles in ischemia, traumatic brain injury and aging. In particular, we discuss several acute and chronic pharmacological therapies that have been extensively studied in order to reduce ROS/RNS loads in cells and the subsequent oxidative stress, so-called "free-radical scavengers." Although the overall aim has been to counteract the detrimental effects of ROS/RNS in these pathologies, success has been limited, especially in human clinical studies. This review highlights some of the recent successes and failures in animal and human studies by attempting to link a compound's chemical structure with its efficacy as a free radical scavenger. In particular, we demonstrate how antioxidants derived from natural products, as well as long-term dietary alterations, may prove to be effective scavengers of ROS and RNS.

Feeding rats diets enriched in lowbush blueberries for six weeks decreases ischemia-induced brain damage.

Oxidative stress is an important element in the etiology of ischemic stroke. Lowbush blueberries (Vaccinium angustifolium Aiton) have a high antioxidant capacity and thus we determined whether consumption of lowbush blueberries would protect neurons from stroke-induced damage. Rats were fed AIN-93G diets containing 0 or 14.3% blueberries (g fresh weight/100 g feed) for 6 weeks. Stroke was then simulated by ligation of the left common carotid artery (ischemia), followed by hypoxia. One week later, plasma and urine were collected, and neuronal damage in the hippocampus was determined histologically. In control rats, hypoxia-ischemia resulted in 40 +/- 2% loss of neurons in the hippocampus of the left cerebral hemisphere, as compared to the right hemisphere. Rats on blueberry-supplemented diets lost only 17 +/- 2% of neurons in the ischemic hippocampus. Neuroprotection was observed in the CA1 and CA2 regions, but not CA3 region, of the hippocampus. The blueberry diet had no detectable effects on the plasma or urine oxygen radical absorbance capacity (ORAC) or plasma lipids. We conclude that consumption of lowbush blueberries by rats confers protection to the brain against damage from ischemia, suggesting that inclusion of blueberries in the diet may improve ischemic stroke outcomes.

Selective effect of malathion on blood coagulation versus locomotor activity.

The objective of this study was to compare the neurotoxic effects of acute malathion exposure with the potential blood coagulation effects. We administered various doses of malathion to Wistar rats by i.p. injection, and then we assessed the effects on blood coagulation screening tests and activity in the open field locomotor test. The activated partial thromboplastin time (APTT), but not the prothrombin time (PT), was significantly prolonged by up to 6.6 seconds (38% of the preinjection value) after the administration of malathion (3-75 mg/kg; 1-25% of the LD50). This effect was observed 5 minutes after injection and persisted for at least 3 hours. In contrast, no effect was observed on the general locomotor activity, movement, rearing, or stereotopy during 3 hours after the i.p. administration of the same doses of malathion. Our data indicate that malathion affects blood clotting before the nervous system (locomotor) function.

Adenosine A1 receptor activation preferentially protects cultured cerebellar neurons versus astrocytes against hypoxia-induced death.

Administration of adenosine A1 receptor agonists in vivo is neuroprotective in various stroke models. Experiments using either mixed cultures of neurons and astrocytes or brain slices, in which several cell types are present, have demonstrated that activation of A1 receptors also id protective against hypoxia and/or hypoglycemia in vitro. In this study, we have examined the effect of the A1 agonist cyclopentyladenosine (CPA) on cellular damage, measured by efflux of lactate dehydrogenase (LDH), in highly enriched primary cultures of either neurons of astrocytes exposed to different metabolic insults. CPA reduced neuronal LDH release induced by a combination of hypoxia and substrate deprivation ("simulated ischemia"; IC50 = 28 nM) of by hypoxia alone (IC50 = 170 nM). In contrast, CPA had no effect on neuronal damage induced by substrate deprivation alone, not did it affect ischemic death to astrocytes. The neuroprotective effect of CPA during simulated ischemia and hypoxia were reversed by the A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). These data demonstrate that activation of an adenosine A1 receptor on neurons, but not astrocytes, is protective against cellular damage of death induced specifically by hypoxia as opposed to other metabolic insults such as hypoglycemia.

Mechanisms that result in damage during and following cerebral ischemia.

The destructive mechanisms associated with stroke are initiated by activation of glutamate receptors resulting in elevated intracellular Ca2+ and reactive oxygen species (ROS) formation. Three major approaches have been investigated to ameliorate ischemia-induced brain damage: (i) interfering with the excitatory action of glutamate; (ii) preventing intracellular accumulation of Ca2+; and (iii) preventing the destructive actions of reactive oxygen species (ROS). Interference with glutamate action can be achieved by: (i) facilitating mechanisms that maintain membrane potentials; (ii) blocking glutamate receptors; and (iii) inhibiting transmitter glutamate synthesis. Prevention of intracellular Ca2+ accumulation may be achieved by: (i) blocking Ca2+ channels; and (ii) facilitating endogenous Ca2+ homeostatic mechanisms. Destructive actions of ROS can be minimized by: (i) administration of ROS-scavenging drugs; (ii) upregulating endogenous ROS-scavenging mechanisms; and (iii) preventing leukocyte invasion of the affected brain tissue. Current therapies that have arisen out of animal experimentation have not met expectations due, mainly to actions of the drugs outside the lesion site. For future research, we suggest: (i) exploring the ability of compromised blood-brain barrier to specifically target therapeutic drugs to the site of lesion; (ii) preventing inflammation by preventing leukocyte infiltration; (iii) identifying signal transduction mechanisms that upregulate neuronal Ca2+ homeostatic mechanisms; and (iv) identifying means that will upregulate endogenous ROS-scavenging mechanisms. Past success in reducing the incidence of stroke has been due, to a great extent, to changes to lifestyle behavioural patterns. We predict that future success in decreasing the morbidity associated with stroke will, to a certain extent, also be due to long-term behavioural changes. It seems possible that simple dietary changes may enable the CNS to be better able to cope with ischemic insults by augmenting ROS-scavenging mechanisms, down-regulating pro-inflammatory responses and increasing Ca(2+)-homeostatic mechanisms.

Neuroprotective effects of adenosine in cerebral ischemia: window of opportunity.

The inhibitory neuromodulator adenosine is neuroprotective against damage induced by cerebral ischemia. Its vasodilator effects add to its suitability as a possible anti-stroke agent, but also account for unwanted side effects following systemic administration of adenosine receptor agonists. ATP breakdown during ischemia produces adenosine which effluxes out of the neuron. This review will focus on endogenously produced adenosine and its subsequent protection against ischemia-induced neuronal damage in some stroke models, but will also highlight possible disadvantages to increasing adenosine concentrations. In the advantages column, therapeutic benefits have been obtained by enhancing synaptic concentrations of endogenous adenosine using the adenosine uptake inhibitor propentofylline, but not dipyridamole. There is an emerging role for endogenous adenosine in preventing delayed cell death, e.g. following hypoxic pre-conditioning. One of the cons associated with enhancing the synaptic concentration of adenosine is the appearance of adenosine receptor desensitization over time. Thus, there is a therapeutic window of opportunity during which activation of an adenosine A1 receptor is beneficial to an ischemic neuron.

Adenosine release and uptake in cerebellar granule neurons both occur via an equilibrative nucleoside carrier that is modulated by G proteins.

These is debate about the mechanisms mediating adenosine release from neurons. In this study, the release of adenosine evoked by depolarizing cultured cerebellar granule neurons with 50 mM K+ was inhibited by 49 +/- 7% in Ca2+-free medium. The remaining release was blocked by dipyridamole (IC50 = 6.4 x 10(-8) M) and nitrobenzylthioinosine (IC50 = 3.6 x 10(-8) M), inhibitors of adenosine uptake. Ca2+-dependent release was reduced by 78 +/- 9% following a 21-h pretreatment of the cells with pertussis toxin, which ADP-ribosylates Gi/Go G proteins, thereby preventing their dissociation. The nucleoside transporter-mediated component of K+-induced adenosine release also was inhibited by 62 +/- 8% by pertussis toxin and was potentiated by 78 +/- 11% following cholera toxin treatment, which permanently activates Gs. Uptake of [3H]adenosine into cultured cerebellar granule neurons over a 10-min period was not dependent on extracellular Na+ but was reduced by dipyridamole (IC50 = 3.2 x 10(-8) M) and nitrobenzylthioinosine (IC50 = 2.6 x 10(-8) M). Thus, adenosine uptake likely occurs via the same transporter mediating Ca2+-independent adenosine release. Adenosine uptake was potentiated by cholera toxin pretreatment (152 +/- 15% of control), but pertussis toxin had no statistically significant effect. It is possible that Gs, Gi/Go, or free Gbetagamma dimer modulate the equilibrative, inhibitor-sensitive nucleoside carrier to enhance adenosine transport.

Cellular mechanisms involved in brain ischemia.

Cellular mechanisms, both destructive and protective, that are associated with cerebral ischemia are reviewed in this paper. Central to understanding the evolution of stroke are the concepts of ischemic core and surrounding penumbral region damage, delayed neuronal death, and neuronal rescue. The role of spreading depression in the evolution of subsequent ATP depletion, ion shifts, glutamate release, activation of glutamate receptors, intracellular Ca2+ changes, and generation of reactive oxygen species in the penumbra in relationship to neuronal and glial cell damage are discussed. We conclude that the most fruitful areas for future stroke research include traditional approaches as well as novel approaches. Traditional approaches include stroke prevention and examination of the effects of combinations of proven and promising effective therapeutic interventions. Novel approaches include delineating mechanisms whereby growth factors and compounds such as deprenyl and staurosporine afford neuroprotection, ultimately leading to direct manipulation of the signal transduction pathways that lead to neuronal dysfunction and death. This includes determining which genes are activated and repressed in specific response to hypoxia-ischemia and determining how such alterations in gene expression affect survival and function of neurons. We also suggest that advantage be taken of the blood-brain barrier compromise during stroke in designing neuroprotective therapies.

Adenosine A1 agonists and the Ca2+ channel agonist bay K 8644 produce a synergistic stimulation of the GTPase activity of Go in rat frontal cortical membranes.

The identity and role of G proteins in coupling adenosine receptors to effectors have been studied to a limited degree. We have identified the G proteins whose GTPase activity is stimulated by adenosine receptor agonists in neuronal membranes. (R)-Phenylisopropyladenosine, 2-chloroadenosine, and N-ethylcarboxamideadenosine produced a concentration-dependent stimulation of GTPase. At 10(-5) M, the increase above basal GTPase in frontal cortex was 25 +/- 4, 20 +/- 3, and 8 +/- 1%, respectively, and in the cerebellum 55 +/- 2, 41 +/- 4, and 22 +/- 2%, respectively. The effects of (R)-phenylisopropyladenosine and 2-chloroadenosine were inhibited by (1) A1 antagonists (76-96% reduction), (2) pretreatment with pertussis toxin (90-100% reduction), and (3) antibodies raised against the alpha-subunit of Gi and G(o) (55-57% reduction by each), suggesting that A1 receptors interact equally with Gi and G(o). (R)-Phenylisopropyladenosine increased the binding of a nonhydrolyzable analogue of GTP to membranes in a pertussis toxin-sensitive manner, indicative of activation of Gi or G(o). Previously, (+/-)-Bay K 8644 enhanced GTP hydrolysis by G(o) but not Gi. Now we report a profound synergistic stimulation of GTPase in the presence of (R)-phenylisopropyladenosine and (+/-)-Bay K 8644 (10(-7) to 10(-5) M). (+/-)-Bay K 8644 had no effect on nucleotide exchange and, thus, cannot activate G(o). It appears that a positive cooperative stimulation of G(o) occurs when it is first activated by A1 receptors and subsequently interacts with the L-type Ca2+ channel.

Morphine-evoked release of adenosine from the spinal cord occurs via a nucleoside carrier with differential sensitivity to dipyridamole and nitrobenzylthioinosine.

We have investigated the potential role of a bi-directional nucleoside carrier in the release of endogenous adenosine from spinal cord synaptosomes by examining the effects of dipyridamole and nitrobenzylthioinosine (NBI) on evoked release of adenosine. When 40 pmol adenosine were added to synaptosomes, only 70 +/- 2% was recovered, suggesting 30% uptake of adenosine. Dipyridamole (0.1-10 microM) reduced this uptake and also increased basal adenosine release, probably due to inhibition of the re-uptake of adenosine derived from released nucleotide. In contrast, NBI (0.1-10 microM) had no effect on either uptake of added adenosine or on basal release of adenosine. Addition of K+ (24 mM) and morphine (10 microM) produced a 50-60% increase in the release of adenosine, and this was reduced 35-98% by both dipyridamole and NBI (0.01-10 microM). Dipyridamole (0.01-1 microM) had no effect on the release of nucleotides (detected as adenosine) induced by noradrenaline, 5-hydroxytryptamine (5-HT) and capsaicin (50 microM each), although 10 microM dipyridamole significantly reduced release evoked by noradrenaline and 5-HT. This latter effect of dipyridamole was determined not to be due to inhibition of ATP release when measured directly. Within the spinal cord, there is a removal system for adenosine which is dipyridamole-sensitive but NBI-insensitive. Release of adenosine, but not nucleotides, appears to occur via this carrier system. The inhibition of release by NBI, but its lack of effect on uptake, suggests the involvement of heterogeneous carrier molecules in adenosine uptake and release from the spinal cord.

Pertussis toxin treatment increases glutamate release and dihydropyridine binding sites in cultured rat cerebellar granule neurons.

This study was designed to examine the ability of pertussis toxin to block various responses due to (-)-baclofen in cultured cerebellar granule neurons of the rat. Treatment with pertussis toxin for 3 h markedly reduced the ability of (-)-baclofen to stimulate GTPase in membranes, and its ability to inhibit forskolin-stimulated adenylyl cyclase in intact cells, whereas the ability of (-)-baclofen to inhibit glutamate release was not affected at 3 h, but was abolished after 16 and 48 h treatment with pertussis toxin. The amount of ADP-ribosylation of Gi/Go due to pertussis toxin in intact cells correlated well with the former two effects, but not with the prevention of the ability of baclofen to inhibit glutamate release. Pertussis toxin treatment for up to 48 h did not significantly affect the levels of Gs, Gi and Go in membranes from granule neurons determined by immunoblotting. Pertussis toxin treatment for 16 or 48 h but not 3 h increased the total amount of stimulated release of glutamate by about 40% under normal conditions, and by 84% under depolarizing conditions. In parallel experiments it was observed that pertussis toxin treatment for 16 h increased the number of dihydropyridine binding sites by about 90% on intact granule neurons. Whole-cell calcium channel currents, recorded under several conditions in the cells, were not increased in amplitude by pertussis toxin treatment for up to 48 h, although the ability of baclofen to inhibit calcium channel currents was blocked by pertussis toxin. These results indicate that the pertussis toxin-induced increase in glutamate release may be due to an increase in dihydropyridine binding sites, possibly localized to the presynaptic terminals.

1,4-Dihydropyridines modulate GTP hydrolysis by Go in neuronal membranes.

Several lines of evidence suggest that L-type Ca2+ channels (1,4-dihydropyridine receptors) are modulated by GTP-binding proteins. We have further examined this interaction by measuring the effect of 1,4-dihydropyridines on GTPase activity in brain membranes. Dihydropyridine agonists significantly increased GTPase, reflected by an increase in the maximal rate of GTP hydrolysis, without affecting the affinity for GTP or the binding of a non-hydrolysable analogue of GTP. The stimulating effect on GTPase was abolished by antisera raised against Go alpha but not Gi alpha. L-type Ca2+ channels may act as endogenous GTPase activating proteins (GAPs) to stimulate GTP hydrolysis by Go.

Actions of arginine polyamine on voltage and ligand-activated whole cell currents recorded from cultured neurones.

1. Toxins from invertebrates have proved useful tools for investigation of the properties of ion channels. In this study we describe the actions of arginine polyamine which is believed to be a close analogue of FTX, a polyamine isolated from the American funnel web spider, Agelenopsis aperta. 2. Voltage-activated Ca2+ currents and Ca(2+)-dependent Cl- currents recorded from rat cultured dorsal root ganglion neurones were reversibly inhibited by arginine polyamine (AP; 0.001 to 100 microM). Low voltage-activated T-type Ca2+ currents were significantly more sensitive to AP than high voltage-activated Ca2+ currents. The IC50 values for the actions of AP on low and high voltage-activated Ca2+ currents were 10 nM and 3 microM respectively. AP was equally effective in inhibiting high voltage-activated currents carried by Ba2+, Sr2+ or Ca2+. However, AP-induced inhibition of Ca2+ currents was attenuated by increasing the extracellular Ca2+ concentration from 2 mM to 10 mM. 3. The actions of AP on a Ca(2+)-independent K+ current were more complex, 1 microM AP enhanced this current but 10 microM AP had a dual action, initially enhancing but then inhibiting the K+ current. 4. gamma-Aminobutyric acid-activated Cl- currents were also reversibly inhibited by 1 to 10 microM AP. In contrast N-methyl-D-aspartate currents recorded from rat cultured cerebellar neurones were greatly enhanced by 10 microM AP. 5. We conclude that at a concentration of 10 nM, AP is a selective inhibitor of low threshold T-type voltage-activated Ca2+ currents. However, at higher concentrations 1-10 microM AP interacts with ion channels or other membrane constituents to produce a variety of actions on both voltage and ligand gated ion channels.

Intracerebroventricular morphine releases adenosine and adenosine 3',5'-cyclic monophosphate from the spinal cord via a serotonergic mechanism.

Intracerebroventricular (i.c.v.) administration of morphine produces antinociception which is antagonized by intrathecal (i.t.) injection of adenosine receptor antagonists, suggesting that adenosine release from the spinal cord may partially mediate antinociception produced by supraspinal morphine. In the present study we have examined this hypothesis directly. Administration of 40 nmol of morphine i.c.v. acutely to rats increased tail-flick and hot-plate nociceptive latencies. This antinociception was antagonized by i.t. theophylline (12 nmol) and 8-phenyltheophylline (1.2 and 12 nmol), but was potentiated by a high dose of theophylline (120 nmol). The same dose of i.c.v. morphine increased the release of adenosine into i.t. perfusates by 40 to 60% above basal release values, and also released a nucleotide which was identified as cyclic AMP by using high performance liquid chromatography. This release of adenosine and cyclic AMP was reduced after i.t. pretreatment with 5,7-dihydroxytryptamine (100 micrograms) but not 6-hydroxydopamine (100 micrograms). Spinal release of purines induced by i.c.v. morphine also was reduced by i.t. perfusion with 50 microM methysergide whereas 50 microM phentolamine had no effect. These data suggest that i.c.v. morphine activates descending serotonergic pathways to release purines from the spinal cord, and that such release is secondary to release of 5-hydroxytryptamine. Extracellular adenosine may contribute significantly to antinociception produced by supraspinal morphine.

5-Hydroxytryptamine releases adenosine and cyclic AMP from primary afferent nerve terminals in the spinal cord in vivo.

5-Hydroxytryptamine (5-HT) releases a purine nucleotide, which is subsequently converted to adenosine, from primary afferent nerve terminals in vitro. This release may mediate spinal antinociception by 5-HT. In the present study, we have investigated whether release also occurs from the spinal cord in vivo using an intrathecal perfusion system in rats. Adenosine was quantitated using high performance liquid chromatography (HPLC) with fluorescence detection. Following perfusion of the spinal cord with 50 and 500 microM 5-HT, a 35-50% increase in the release of endogenous adenosine was observed. This release was completely blocked by 50 microM methysergide, and by intrathecal injection with 100 micrograms capsaicin 5-8 days prior to release experiments. Intrathecal perfusion with 50 and 500 microM 5-HT also released a nucleotide which eluted from the HPLC column at a retention time identical to that of cyclic AMP standards, and was reduced following incubation with cyclic AMP phosphodiesterase. This release of cyclic AMP also was eliminated following intrathecal pretreatment with capsaicin. In contrast to 5-HT, noradrenaline (NA, 500 microM and 5 mM) did not release adenosine or cyclic AMP from the intact spinal cord. These data demonstrate that release of nucleotide, probably cyclic AMP, and subsequent metabolism to adenosine, can be induced by 5-HT but not NA in vivo. This strengthens the hypothesis that release of adenosine from the spinal cord may mediate antinociception by intrathecal 5-HT but not NA.

Adenosine release may mediate spinal analgesia by morphine.

Spinal analgesia produced by morphine is blocked by methylxanthine adenosine receptor antagonists. In biochemical studies, morphine releases adenosine from spinal cord synaptosomes prepared from the dorsal spinal cord, as well as from the intact spinal cord in vivo. Adenosine release is reduced by intrathecal and neonatal pretreatment with capsaicin but not by intrathecal pretreatment with 6-hydroxydopamine or 5,7-dihydroxytryptamine, indicating that adenosine originates from small-diameter primary afferent neurons but not descending monoaminergic pathways. In this Viewpoint Jana Sawynok and colleagues review the evidence supporting the hypothesis that the spinal analgesic action of morphine is due to the release of adenosine from primary afferent nerve terminals and subsequent activation of A1 and A2 adenosine receptors.