Effects of ABA on primary terpenoids and Δ<Superscript>9</Superscript>-tetrahydrocannabinol in <Emphasis Type="Italic">Cannabis sativa</Emphasis> L. at flowering stage

This work examined the effects of exogenously applied abscisic acid (ABA) on the content of chlorophyll, carotenoids, α-tocopherol, squalene, phytosterols, Δ⁹-tetrahydrocannabinol (THC) concentration, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and 1-deoxy-d-xylulose 5-phosphate synthase (DXS) activity in Cannabis sativa L. at flowering stage. Treatment with 1 and 10 mg l⁻¹ ABA significantly decreased the contents of chlorophyll, carotenoids, squalene, stigmasterol, sitosterol, and HMGR activity in female cannabis plants. ABA caused an increase in α-tocopherol content and DXS activity in leaves and THC concentration in leaves and flowers of female plants. Chlorophyll content decreased with 10 mg l⁻¹ ABA in male plants. Treatment with 1 and 10 mg l⁻¹ ABA showed a decrease in HMGR activity, squalene, stigmasterol, and sitosterol contents in leaves but an increase in THC content of leaves and flowers in male plants. The results suggest that ABA can induce biosynthesis of 2-methyl-d-erythritol-4-phosphate (MEP) pathway secondary metabolites accumulation (α-tocopherol and THC) and down regulated biosynthesis of terpenoid primary metabolites from MEP and mevalonate (MVA) pathways (chlorophyll, carotenoids, and phytosterols) in Cannabis sativa.     

Pulmonary vascular response to thrombin: Effects of thromboxane synthetase inhibition with OKY-046 and OKY-1581

We examined the effects of thromboxane synthetase inhibition with OKY-1581 and OKY-046 on pulmonary hemodynamics and lung fluid balance after thrombin-induced intravascular coagulation. Studies were made in anesthetized sheep prepared with lyng lymph fistulas. Pulmonary intravascular coagulation was induced by i.v. infusion of α-thrombin over a 15 min period. Thrombin infusion in control sheep resulted in immediate increases in pulmonary artery pressure (P ) and pulmonary vascular resistance (PVR), which associated with rapid 3-fold increase in pulmonary lymph flow (Q̇lym) and a delayed increase in lymph-to-plasma protein concentration (L/P) ratio, indicating an increase in the pulmonary microvascular permeability to proteins. Thrombin-induced intravascular coagulation alos increased arterial thromboxane B₂ (a metabolite of thromboxane A₂) and 6-keto-PGF_1α concentrations (a metabolite of prostacyclin). Both OKY-1581 and OKY-046 prevented thromboxane B₂ and 6-keto-PGF_1α generation. The initial increments in P and PVR were attenuated in both treated groups. The increases in Q̇lym were gradual in the treated groups but attained the same levels as in control group. However, the increases in Q̇lym were associated with decreases in L/P ratio. In both treated groups, the leukocyte count decreased after thrombin infusion but then increased steadily above the baseline value, whereas the leukocyte count remained depressed in the control group after thrombin. These studies indicate that a part of the initial pulmonary vasoconstrictor response to thrombin-induced intravascular coagulation is mediated by thromboxane generation. In addition, thromboxane may also contribute to the increase in lung vascular permeability to proteins that occurs after intravascular coagulation and this effect may be mediated by a thromboxane-neutrophil interaction.     

G alpha12 interaction with alphaSNAP induces VE-cadherin localization at endothelial junctions and regulates barrier function

The involvement of heterotrimeric G proteins in the regulation of adherens junction function is unclear. We identified alphaSNAP as an interactive partner of G alpha12 using yeast two-hybrid screening. Glutathione S-transferase pull-down assays showed the selective interaction of alphaSNAP with G alpha12 in COS-7 as well as in human umbilical vein endothelial cells. Using domain swapping experiments, we demonstrated that the N-terminal region of G alpha12 (1-37 amino acids) was necessary and sufficient for its interaction with alphaSNAP. G alpha13 with its N-terminal extension replaced by that of G alpha12 acquired the ability to bind to alphaSNAP, whereas G alpha12 with its N terminus replaced by that of G alpha13 lost this ability. Using four point mutants of alphaSNAP, which alter its ability to bind to the SNARE complex, we determined that the convex rather than the concave surface of alphaSNAP was involved in its interaction with G alpha12. Co-transfection of human umbilical vein endothelial cells with G alpha12 and alphaSNAP stabilized VE-cadherin at the plasma membrane, whereas down-regulation of alphaSNAP with siRNA resulted in the loss of VE-cadherin from the cell surface and, when used in conjunction with G alpha12 overexpression, decreased endothelial barrier function. Our results demonstrate a direct link between the alpha subunit of G12 and alphaSNAP, an essential component of the membrane fusion machinery, and implicate a role for this interaction in regulating the membrane localization of VE-cadherin and endothelial barrier function.     

Protein kinase Calpha phosphorylates the TRPC1 channel and regulates store-operated Ca2+ entry in endothelial cells

The TRPC1 (transient receptor potential canonical-1) channel is a constituent of the nonselective cation channel that mediates Ca2+ entry through store-operated channels (SOCs) in human endothelial cells. We investigated the role of protein kinase Calpha (PKCalpha) phosphorylation of TRPC1 in regulating the opening of SOCs. Thrombin or thapsigargin added to the external medium activated Ca2+ entry after Ca2+ store depletion, which we monitored by changes in cellular Fura 2 fluorescence. Internal application of the metabolism-resistant analog of inositol 1,4,5-trisphosphate (IP3) activated an inward cationic current within 1 min, which we recorded using the whole cell patch clamp technique. La3+ or Gd3+ abolished the current, consistent with the known properties of SOCs. Pharmacological (G?6976) or genetic (kinase-defective mutant) inhibition of PKCalpha markedly inhibited IP3-induced activation of the current. Thrombin or thapsigargin also activated La3+-sensitive Ca2+ entry in a PKCalpha-dependent manner. We determined the effects of a specific antibody directed against an extracellular epitope of TRPC1 to address the functional importance of TRPC1. External application of the antibody blocked thrombin- or IP3-induced Ca2+ entry. In addition, we showed that addithrombin or thapsigargin induced phosphorylation of TRPC1 within 1 min. Thrombin failed to induce TRPC1 phosphorylation in the absence of PKCalpha activation. Phosphorylation of TRPC1 and the resulting Ca2+ entry were essential for the increase in permeability induced by thrombin in confluent endothelial monolayers. These results demonstrate that PKCalpha phosphorylation of TRPC1 is an important determinant of Ca2+ entry in human endothelial cells.     

Lysophosphatidylcholine modulates neutrophil oxidant production through elevation of cyclic AMP

Lysophosphatidylcholine (LPC) is an oxidized phospholipid present in micromolar concentrations in blood and inflamed tissues. The effects of LPC on neutrophil functions remain incompletely understood, because conflicting reports exist for its stimulatory and inhibitory roles. We report in this study that LPC inhibits superoxide generation in fMLP- and PMA-stimulated neutrophils without affecting fMLP-induced Ca(2+) mobilization and cell viability. This effect was observed with LPC dissolved in ethanol, but not with LPC stock solutions prepared in water or in BSA-containing aqueous solution with sonication. Under the same experimental conditions, platelet-activating factor primed neutrophils for superoxide generation. The inhibitory effect of LPC was observed within 30 s after its application and was maximal at LPC concentrations between 0.1 and 1 muM. Inhibition of superoxide generation was accompanied by a 2.5-fold increase in the intracellular cAMP concentration. In addition, LPC reduced fMLP-stimulated phosphorylation of ERK and Akt and membrane translocation of p67(phox) and p47(phox). The protein kinase A inhibitors H-89 and adenosine 3'5'-cyclic monophosphorothioate Rp-isomer (Rp-cAMP) partially restored superoxide production in LPC-treated neutrophils, indicating involvement of protein kinase A in LPC-mediated inhibition. Using an ex vivo mouse lung perfusion model that measures lung weight change and capillary filtration coefficient, we found that LPC prevented lung vascular injury mediated by fMLP-activated neutrophils. Taken together, these results suggest that LPC-induced elevation of intracellular cAMP is partially responsible for its inhibition of neutrophil NADPH oxidase activation. A similar mechanism of inhibition may be used for the control of neutrophil-mediated tissue injury.     

Regulating the regulator of ROS production

Balancing inflammatory reactive oxygen species (ROS) production is essential for safely eliminating pathogenic microbes. The newly described protein Negative Regulator of ROS (NRROS) dampens ROS production by restricting NOX2 availability, and thus “cools-off” inflammation.Oxygen-based metabolism was essential for proliferation and diversification of life on Earth but imposed the need to cope with reactive oxygen species (ROS) as an unavoidable aspect of aerobic respiration. Multicellular organisms have evolved a host of mechanisms to utilize ROS to their advantage, thus employing them as mediators of cellular signaling and bactericidal agents against invading pathogens. Excessive ROS production, however, results in oxidative tissue injury, and as recently shown, may participate in the etiology of autoimmunity¹. Collateral oxidative cell damage is inseparable from the host innate immunologic responses, underscoring the necessity of regulatory mechanisms that modulate ROS production. A recent publication in Nature has identified a crucial mechanism that balances ROS production and thus modulates the inflammatory response¹. Noubade et al.¹ describe a protein Negative Regulator of ROS (NRROS, a.k.a. Lrrc33) that interacts with NOX2 (gp91phox) and affects its stability. The NRROS-bound NOX2 in the endoplasmic reticulum (ER) was targeted for proteosomal degradation, and thereby NRROS limited NOX2-derived ROS by impeding NOX2 trafficking to the phagosomal membrane. The authors showed that NRROS and p22phox competed for nascent gp91phox in the ER. Binding to p22phox stabilized gp91phox allowing subsequential trafficking and assembly of the functional NOX2 at the plasma membrane whereas binding to NRROS induced gp91phox trafficking to ER-associated protein degradation (ERAD) pathway (schematically summarized in Figure 1).Open in a separate windowFigure 1Nascent NOX2 interactions in the ER. NOX2 is a major source of superoxide radical anion during phagocytic cell activation. Interaction of nascent NOX2 with p22phox stabilizes NOX2 in the ER and allows for the maturation and trafficking of NOX2 to the plasma membrane where it becomes functional. NRROS is a newly identified NOX2-binding partner in the ER. Binding of NOX2 by NRROS accelerates NOX2 degradation, and thereby modulates ROS production by reducing NOX2 level.NOX2 is a major source of superoxide radicals produced by phagocytic cells during the oxidative burst. Thus, an important discovery made is that NRROS is downregulated in response to priming by pro-inflammatory stimuli including lipopolysaccharide and type II interferon. NRROS deficiency resulting from exposure to these stimuli caused the cells to respond to subsequent inflammatory challenges with an appropriately robust ROS burst required for killing bacteria and viruses. The kinetics of ROS production in stimulated NRROS-deficient versus wild-type cells was identical, suggesting that NRROS functions to establish the upper limit in the achievable levels of ROS production following phagocyte activation. These observations provide important insights into the widely recognized but poorly understood amplifying effect of basal inflammation on the subsequent activation of innate immune responses. The implication is that chronic exposure to low levels of pro-inflammatory mediators can exacerbate the availability of functional NOX2 by chronically repressing NRROS. In support of this idea, NRROS-knockout mice were better equipped to fight infection by L. monocytogenes and survived better in contrast to wild-type animals, consistent with the known effect of ROS in promoting pathogen killing. On the flipside, however, elimination of a central repressive mechanism limiting NOX2-derived ROS induced lethal encephalomyelitis (EAE) with rapid deterioration of the central nervous system in immunized NRROS-knockout mice¹. A possible clinically relevant interpretation of this observation is that while severe oxidative stress facilitates elimination of pathogenic organisms, it can also overwhelm the host''s ability to clear oxidized biomolecules which may trigger the onset of autoimmunity. The findings raise the intriguing question whether dysregulated ROS production during successive acute inflammatory events is conducive to the pathogenesis of autoimmune disorders. This concept also resonates with some earlier studies highlighting the effects of environment-induced chronic systemic inflammation and the propensity of mammals to develop degenerative neurological disorders^2,3. For example, it was observed that exacerbated NOX2 activity underlies microglia-mediated neurotoxicity that can lead to Parkinson''s and Alzheimer''s diseases². It was previously theorized that chronic low-grade inflammatory states promoted by exposure to environmental toxicants (air pollution, pesticides, etc.) primes microglia (the macrophages of the brain) to produce exaggerated amounts of ROS generated largely by NOX2³. As demonstrated by studies testing this hypothesis, microglia from animals exposed to diesel exhaust particles produced robust bursts of ROS when subsequently challenged with lipopolysaccharides². Levels of ROS produced by primed microglia were demonstrated to be neurotoxic to dopaminergic neurons and induce neurodegeneration in a mouse model⁴. Other studies showed that feedback signaling by NOX2-derived ROS limits TNFα and interleukin-6 expression by activated macrophages, which alleviated acute inflammatory lung injury⁵. The production of ROS by specialized systems is currently considered to be limited by cofactor and O₂ availability⁶. The identification of NRROS and its function indicates that on the contrary specific mechanisms exist to dynamically regulate the levels of ROS. Taken together, these studies suggest that budgeting ROS production is indispensable for safe interactions of the host with the environment. They also suggest that exacerbated ROS production leads to production of pseudo-antigens and oxidized biomolecules whose clearance may be the rate-limiting factor, and hence the necessity to limit their production by dampening ROS generation. This provocative hypothesis is consistent with the finding of higher levels of malonaldehyde (MDA)-adduct proteins after immunization in NRROS-deficient mice¹. Another bit of evidence supporting this idea is the finding that administration of ROS scavengers after the onset of autoimmune EAE reduced the lethality of immunized NRROS-knockout mice back to wild-type control levels¹. Although the levels of oxidized proteins were not assessed after ROS scavenger treatment, the near abrogation of autoimmune EAE by ROS scavengers administered after the onset of EAE indicates that resolution of ongoing oxidative stress (or the clearance of oxidized pseudo-antigens) is sufficient to prevent further neurologic deterioration.The role of ROS as mediators of tissue damage has been established (see reviews^7,8). To date, much less is understood about the role of ROS in regulating the initiation, intensity, localization, and resolution of the inflammatory process. As the redox sensitivity of different signaling cascades varies, the concentrations and kinetics of ROS production are likely to shape specific inflammatory programs. Dysregulated ROS generation as shown by this study¹ is likely to cause detrimental effects produced by inflammation gone awry. Addressing questions related to the integration of redox signaling in inflammation and temporal control of differential ROS fluxes will further our understanding about the role of ROS in modulating inflammation and will provide exciting opportunities for therapeutic interventions.