Mechanisms of modulation of brain microvascular endothelial cells function by thrombin.

Brain microvascular endothelial cells are a critical component of the blood-brain barrier. They form a tight monolayer which is essential for maintaining the brain homeostasis. Blood-derived proteases such as thrombin may enter the brain during pathological conditions like trauma, stroke, and inflammation and further disrupts the permeability of the blood-brain barrier, via incompletely characterized mechanisms. We examined the underlying mechanisms evoked by thrombin in rat brain microvascular endothelial cells (RBMVEC). Our results indicate that thrombin, acting on protease-activated receptor 1 (PAR1) increases cytosolic C

Effects of Platelet-Activating Factor on Brain Microvascular Endothelial Cells.

Platelet-activating factor (PAF) is a potent phospholipid mediator that exerts various pathophysiological effects by interacting with a G protein-coupled receptor. PAF has been reported to increase the permeability of the blood-brain barrier (BBB) via incompletely characterized mechanisms. We investigated the effect of PAF on rat brain microvascular endothelial cells (RBMVEC), a critical component of the BBB. PAF produced a dose-dependent increase in cytosolic Ca2+ concentration; the effect was prevented by the PAF receptor antagonist, WEB2086. The effect of PAF on cytosolic Ca2+ was abolished in Ca2+-free saline or in the presence of L-type voltage-gated Ca2+ channel inhibitor, nifedipine, indicating that Ca2+ influx is critical for PAF-induced increase in cytosolic Ca2+. PAF produced RBMVEC depolarization; the effect was inhibited by WEB2086. In cells loaded with [(4-amino-5-methylamino-2\u27,7\u27-difluoro-fluorescein)diacetate] (DAF-FM), a nitric oxide (NO)-sensitive fluorescent dye, PAF increased the NO level; the effect was prevented by WEB2086, nifedipine or by l-NAME, an inhibitor of NO synthase. Immunocytochemistry studies indicate that PAF reduced the immunostaining of ZO-1, a tight junction-associated protein, increased F-actin fibers, and produced intercellular gaps. PAF produced a decrease in RBMVEC monolayer electrical resistance assessed with Electric Cell-Substrate Impedance Sensing (ECIS), indicative of a disruption of endothelial barrier function. In vivo studies indicate that PAF increased the BBB permeability, assessed with sodium fluorescein and Evans Blue methods, via PAF receptor-dependent mechanisms, consequent to Ca2+ influx and increased NO levels. Our studies reveal that PAF alters the BBB permeability by multiple mechanisms, which may be relevant for central nervous system (CNS) inflammatory disorders

Evidence for role of acid-sensing ion channels in nucleus ambiguus neurons: essential differences in anesthetized versus awake rats.

Acid-sensing ion channels (ASIC) are widely expressed in several brain regions including medulla; their role in physiology and pathophysiology is incompletely understood. We examined the effect of acidic pH of 6.2 on the medullary neurons involved in parasympathetic cardiac control. Our results indicate that retrogradely labeled cardiac vagal neurons of nucleus ambiguus are depolarized by acidic pH. In addition, acidic saline of pH 6.2 increases cytosolic Ca(2+) concentration by promoting Ca(2+) influx in nucleus ambiguus neurons. In vivo studies indicate that microinjection of acidic artificial cerebrospinal fluid (pH 6.2) into the nucleus ambiguus decreases the heart rate in conscious rats, whereas it has no effect in anesthetized animals. Pretreatment with either amiloride or benzamil, two widely used ASIC blockers, abolishes both the in vitro and in vivo effects elicited by pH 6.2. Our findings support a critical role for ASIC in modulation of cardiac vagal tone and provide a potential mechanism for acidosis-induced bradycardia, while identifying important differences in the response to acidic pH between anesthetized and conscious rats

Choline-Sigma-1R as an Additional Mechanism for Potentiation of Orexin by Cocaine

Orexin A, an endogenous peptide involved in several functions including reward, acts via activation of orexin receptors OX1 and OX2, Gq-coupled GPCRs. We examined the effect of a selective OX1 agonist, OXA (17-33) on cytosolic calcium concentration, [Ca2+ ]i, in neurons of nucleus accumbens, an important area in the reward circuit. OXA (17-33) increased [Ca2+ ]i in a dose-dependent manner; the effect was prevented by SB-334867, a selective OX1 receptors antagonist. In Ca2+-free saline, the OXA (17-33)-induced increase in [Ca2+ ]i was not affected by pretreatment with bafilomycin A1, an endo-lysosomal calcium disrupter, but was blocked by 2-APB and xestospongin C, antagonists of inositol-1,4,5-trisphosphate (IP3 ) receptors. Pretreatment with VU0155056, PLD inhibitor, or BD-1047 and NE-100, Sigma-1R antagonists, reduced the [Ca2+ ]i response elicited by OXA (17-33). Cocaine potentiated the increase in [Ca2+ ]i by OXA (17-33); the potentiation was abolished by Sigma-1R antagonists. Our results support an additional signaling mechanism for orexin A-OX1 via choline-Sigma-1R and a critical role for Sigma-1R in the cocaine–orexin A interaction in nucleus accumbens neurons

Nesfatin-1 activates cardiac vagal neurons of nucleus ambiguus and elicits bradycardia in conscious rats.

Nesfatin-1, a peptide whose receptor is yet to be identified, has been involved in the modulation of feeding, stress, and metabolic responses. More recently, increasing evidence supports a modulatory role for nesfatin-1 in autonomic and cardiovascular activity. This study was undertaken to test if the expression of nesfatin-1 in the nucleus ambiguus, a key site for parasympathetic cardiac control, may be correlated with a functional role. As we have previously demonstrated that nesfatin-1 elicits Ca²⁺ signaling in hypothalamic neurons, we first assessed the effect of this peptide on cytosolic Ca²⁺ in cardiac pre-ganglionic neurons of nucleus ambiguus. We provide evidence that nesfatin-1 increases cytosolic Ca²⁺ concentration via a Gi/o-coupled mechanism. The nesfatin-1-induced Ca²⁺ rise is critically dependent on Ca²⁺ influx via P/Q-type voltage-activated Ca²⁺ channels. Repeated administration of nesfatin-1 leads to tachyphylaxis. Furthermore, nesfatin-1 produces a dose-dependent depolarization of cardiac vagal neurons via a Gi/o-coupled mechanism. In vivo studies, using telemetric and tail-cuff monitoring of heart rate and blood pressure, indicate that microinjection of nesfatin-1 into the nucleus ambiguus produces bradycardia not accompanied by a change in blood pressure in conscious rats. Taken together, our results identify for the first time that nesfatin-1 decreases heart rate by activating cardiac vagal neurons of nucleus ambiguus. Our results indicate that nesfatin-1, one of the most potent feeding peptides, increases cytosolic Ca²⁺ by promoting Ca²⁺ influx via P/Q channels and depolarizes nucleus ambiguus neurons; both effects are Gi/o-mediated. In vivo studies indicate that microinjection of nesfatin-1 into nucleus ambiguus produces bradycardia in conscious rats. This is the first report that nesfatin-1 increases the parasympathetic cardiac tone

Nicotinic Acid Adenine Dinucleotide Phosphate Potentiates Neurite Outgrowth

Ca2+ regulates a spectrum of cellular processes including many aspects of neuronal function. Ca2+-sensitive events such as neurite extension and axonal guidance are driven by Ca2+ signals that are precisely organized in both time and space. These complex cues result from both Ca2+ influx across the plasma membrane and the mobilization of intracellular Ca2+ stores. In the present study, using rat cortical neurons, we have examined the effects of the novel intracellular Ca 2+-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) on neurite length and cytosolic Ca2+ levels. We show that NAADP potentiates neurite extension in response to serum and nerve growth factor and stimulates increases in cytosolic Ca2+ from bafilomycin- sensitive Ca2+ stores. Simultaneous blockade of inositol trisphosphate and ryanodine receptors abolished the effects of NAADP on neurite length and reduced the magnitude of NAADP-mediated Ca2+ signals. This is the first report demonstrating functional NAADP receptors in a mammalian neuron. Interplay between NAADP receptors and more established intracellular Ca2+ channels may therefore play important signaling roles in the nervous system

Blood-Brain Barrier Disruption Mediated by FFA1 Receptor-Evidence Using Miniscope

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), obtained from diet and dietary supplements, have been tested in clinical trials for the prevention or treatment of several diseases. n-3 PUFAs exert their effects by activation of free fatty acid (FFA) receptors. FFA1 receptor, expressed in the pancreas and brain, is activated by medium- to long-chain fatty acids. Despite some beneficial effects on cognition, the effects of n-3 PUFAs on the blood-brain barrier (BBB) are not clearly understood. We examined the effects of FFA1 activation on BBB permeability in vitro, using rat brain microvascular endothelial cells (RBMVEC), and in vivo, by assessing Evans Blue extravasation and by performing live imaging of brain microcirculation in adult rats. AMG837, a synthetic FFA1 agonist, produced a dose-dependent decrease in RBMVEC monolayer resistance assessed with Electric Cell-Substrate Impedance Sensing (ECIS); the effect was attenuated by the FFA1 antagonist, GW1100. Immunofluorescence studies revealed that AMG837 produced a disruption in tight and adherens junction proteins. AMG837 increased Evans Blue content in the rat brain in a dose-dependent manner. Live imaging studies of rat brain microcirculation with miniaturized fluorescence microscopy (miniscope) showed that AMG837 increased extravasation of sodium fluorescein. Taken together, our results demonstrate that FFA1 receptor activation reduced RBMVEC barrier function and produced a transient increase in BBB permeability

1-Chromonyl-5-Imidazolylpentadienone Demonstrates Anti-Cancer Action against TNBC and Exhibits Synergism with Paclitaxel

Curcumin has been well studied for its anti-oxidant, anti-inflammatory, and anti-cancer action. Its potential as a therapy is limited due to its low bioavailability and rapid metabolism. To overcome these challenges, investigators are developing curcumin analogs, nanoparticle formulations, and combining curcumin with other compounds or dietary components. In the present study, we used a 1-chromonyl-5-imidazolylpentadienone named KY-20-22 that contains both the pharmacophore of curcumin and 1,4 benzopyrone (chromone) moiety typical for flavonoids, and also included specific moieties to enhance the bioavailability. When we tested the in vitro effect of KY-20-22 in triple-negative breast cancer (TNBC) cell lines, we found that it decreased the cell survival and colony formation of MDA-MB-231 and MDA-MB-468 cells. An increase in mitochondrial reactive oxygen species was also observed in TNBC cells exposed to KY-20-22. Furthermore, KY-20-22 decreased epithelial-mesenchymal formation (EMT) as evidenced by the modulation of the EMT markers E-cadherin and N-cadherin. Based on the fact that KY-20-22 regulates interleukin-6, a cytokine involved in chemotherapy resistance, we combined it with paclitaxel and found that it synergistically induced anti-proliferative action in TNBC cells. The results from this study suggested that 1-chromonyl-5-imidazolylpentadienone KY-20-22 exhibited anti-cancer action in MDA-MB-231 and MDA-MB-468 cells. Future studies are required to evaluate the anti-cancer ability and bioavailability of KY-20-22 in the TNBC animal model

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger in muscarinic receptor-induced contraction of guinea pig trachea

Nicotinic acid adenine dinucleotide phosphate (NAADP) is increasingly being demonstrated to be involved in calcium signaling in many cell types and species. Although it has been shown to play a role in smooth muscle cell contraction in several tissues, nothing is known about its possible role in tracheal smooth muscle, a muscle type that is clinically relevant to asthma. To determine whether NAADP functions as a second messenger in tracheal smooth muscle contraction, we used the criteria set out by Sutherland for a molecule to be designated a second messenger. We report that NAADP satisfies all five criteria as follows. First, the NAADP antagonist Ned-19 inhibited contractions in tracheal rings and calcium increases in isolated smooth muscle cells induced by the muscarinic agonist carbachol. Second, NAADP increased cytosolic calcium in isolated cells when microinjected and was blocked by Ned-19. Third, tracheal homogenates could synthesize NAADP by base exchange from exogenous NADP and nicotinic acid and metabolize exogenous NAADP to nicotinic acid adenine dinucleotide by a 2'-phosphatase. Fourth, carbachol induced a rapid and transient increase in endogenous NAADP levels. Fifth, tracheal homogenates contained NAADP-binding sites of high affinity. Taken together, these data demonstrate that NAADP functions as a second messenger in tracheal smooth muscle, and therefore, steps in the NAADP signaling pathway might provide possible new drug targets