Monoacylglycerols Activate TRPV1 – A Link between Phospholipase C and TRPV1

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H₁ receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H₁ receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous “entourage” compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.     

Cannabidiol inhibits the skeletal muscle Nav1.4 by blocking its pore and by altering membrane elasticity

Cannabidiol (CBD) is the primary nonpsychotropic phytocannabinoid found in Cannabis sativa, which has been proposed to be therapeutic against many conditions, including muscle spasms. Among its putative targets are voltage-gated sodium channels (Navs), which have been implicated in many conditions. We investigated the effects of CBD on Nav1.4, the skeletal muscle Nav subtype. We explored direct effects, involving physical block of the Nav pore, as well as indirect effects, involving modulation of membrane elasticity that contributes to Nav inhibition. MD simulations revealed CBD’s localization inside the membrane and effects on bilayer properties. Nuclear magnetic resonance (NMR) confirmed these results, showing CBD localizing below membrane headgroups. To determine the functional implications of these findings, we used a gramicidin-based fluorescence assay to show that CBD alters membrane elasticity or thickness, which could alter Nav function through bilayer-mediated regulation. Site-directed mutagenesis in the vicinity of the Nav1.4 pore revealed that removing the local anesthetic binding site with F1586A reduces the block of I_Na by CBD. Altering the fenestrations in the bilayer-spanning domain with Nav1.4-WWWW blocked CBD access from the membrane into the Nav1.4 pore (as judged by MD). The stabilization of inactivation, however, persisted in WWWW, which we ascribe to CBD-induced changes in membrane elasticity. To investigate the potential therapeutic value of CBD against Nav1.4 channelopathies, we used a pathogenic Nav1.4 variant, P1158S, which causes myotonia and periodic paralysis. CBD reduces excitability in both wild-type and the P1158S variant. Our in vitro and in silico results suggest that CBD may have therapeutic value against Nav1.4 hyperexcitability.     

The Effect of Antimicrobial Peptide Temporin-Ra on Cell Viability and Gene Expression of Pro-inflammatory Factors in A549 Cell Line

Antimicrobial peptide Temporin-Ra was isolated from the skin secretions of marsh frog Rana ridibunda. Temporin-Ra was chemically synthesized and purified using RP-HPLC technique. The cytotoxicity of peptide on lung airway epithelial cell line (A549) and peripheral blood mononuclear cells (PBMC) was studied by MTT assay. Furthermore, the effect of Temporin-Ra on the expression of pro-inflammatory factors such as IL-1β and IL-8 in A549 cell line was evaluated at peptide concentrations of 15, 30 and 50 μg/mL for 6, 12 and 24 h using semi-quantitative RT-PCR and real-time PCR methods. The result of our experiments revealed that Temporin-Ra decreased cell viability about 3–13 % in A549 cells and 4–6 % in PBMC cells. Moreover, Temporin-Ra induced the mRNA expression of IL-1β and IL-8 genes in a dose- and time-dependent manner. According to our results, maximum IL-8 mRNA expression was observed after a 24-h treatment of cancer cells with 50 μg/mL peptide with approximately 18-fold increase in expression. The least expression level of IL-1β was observed after 6-h of incubation in the presence of 15 μg/mL peptide with 2.5-fold increase in expression whereas the most expression level was obtained following 24 h-treatment with 50 μg/mL peptide with 26-fold increase in mRNA expression. Eventually, the present study highlights the role of Temporin-Ra as a potent antimicrobial peptide in the activation and maintenance of inflammatory processes.     

Brachytherapy with gamma radiation of a coronary artery for in-stent restenosis may induce the regression of in-stent restenosis of an adjacent coronary artery without angioplasty. First case report and review of the literature

Here, we present a case of a 63-year-old male who presented with in-stent restenosis of two coronary arteries simultaneously (mid circumflex and proximal ramus). After the brachytherapy of the circumflex artery for in-stent restenosis, the patient refused the staged procedure for the ramus in-stent restenosis. After approximately 2 years, the patient underwent coronary angiography for recurrent chest pain. Surprisingly, the proximal ramus stent showed marked regression of in-stent restenosis. We hypothesized that the gamma brachytherapy of the circumflex artery could have induced the regression of in-stent restenosis of the adjacent ramus artery due to the deep tissue penetration of gamma radiation. Based on our observation, we believe that in the treatment of in-stent restenosis of a coronary artery, the initial balloon angioplasty may not be as important as the radiation itself. This observation warrants further study to evaluate the effect of external or internal radiation on in-stent restenosis without balloon angioplasty. If our hypothesis is confirmed, the treatment of in-stent restenosis with external radiation could substantially simplify the treatment of this disease. This case report follows a brief review of the literature.     

Antisense-inhibition of ADP-glucose pyrophosphorylase in Vicia narbonensis seeds increases soluble sugars and leads to higher water and nitrogen uptake

We previously reported on Vicia narbonensis seeds with largely decreased alpha- D-glucose-1-phosphate adenyltransferase (AGP; EC 2.7.7.27) due to antisense inhibition [H. Weber et al. (2000) Plant J 24:33-43]. In an extended biochemical analysis we show here that in transgenic seeds both AGP activity and ADP-glucose levels were strongly decreased but starch was only moderately reduced and contained less amylose. The flux control coefficient of AGP to starch accumulation was as low as 0.08, i.e. AGP exerts low control on starch biosynthesis in Vicia seeds. Mature cotyledons of antisense seeds had increased contents of lipids, nitrogen and sulfur. The protein content was higher due, in particular, to increased sulfur-rich albumins. Globulin fractions of storage proteins had a lower ratio of legumin to vicilin. Isolated cotyledons partitioned less [14C]sucrose into starch and more into soluble sugars with no change in the protein fraction. Respiration of isolated cotyledons and activities of the major glycolytic and carbohydrate-metabolizing enzymes were not affected. Sucrose and the hexose-phosphate pool were increased but UDP-glucose, 3-phosphoglyceric acid, phospho enolpyruvate, pyruvate, ATP and ADP were unchanged or even lower, indicating that carbon partitioning changed from starch to sucrose without affecting the glycolytic and respiratory pathways. Soluble compounds were increased but osmolality remained unchanged, indicating compensatory water influx resulting in higher water contents. Developmental patterns of water and nitrogen accumulation suggest a coupled uptake of amino acids and water into cotyledons. We conclude that, due to higher water uptake, transgenic cotyledons take up more amino acids, which become available for protein biosynthesis leading to a higher protein content. Obviously, a substantial part of amino acid uptake into Vicia seeds occurs passively and is osmotically controlled and driven by water influx.     

Transgenic tobacco plants expressing antisense ferredoxin-NADP(H) reductase transcripts display increased susceptibility to photo-oxidative damage

Ferredoxin-NADP(H) reductase (FNR) catalyses the final step of the photosynthetic electron transport in chloroplasts. Using an antisense RNA strategy to reduce expression of this flavoenzyme in transgenic tobacco plants, it has been demonstrated that FNR mediates a rate-limiting step of photosynthesis under both limiting and saturating light conditions. Here, we show that these FNR-deficient plants are abnormally prone to photo-oxidative injury. When grown under autotrophic conditions for 3 weeks, specimens with 20-40% extant reductase undergo leaf bleaching, lipid peroxidation and membrane damage. The magnitude of the effect was proportional to the light intensity and to the extent of FNR depletion, and was accompanied by morphological changes involving accumulation of aberrant plastids with defective thylakoid stacking. Damage was initially confined to chloroplast membranes, whereas Rubisco and other stromal proteins began to decline only after several weeks of autotrophic growth, paralleled by partial recovery of NADPH levels. Exposure of the transgenic plants to moderately high irradiation resulted in rapid loss of photosynthetic capacity and accumulation of singlet oxygen in leaves. The collected results suggest that the extensive photo-oxidative damage sustained by plants impaired in FNR expression was caused by singlet oxygen building up to toxic levels in these tissues, as a direct consequence of the over-reduction of the electron transport chain in FNR-deficient chloroplasts.     

Normoxic polymer gel dosimetry using less toxic monomer of N-isopropyl acrylamide and X-ray computed tomography for radiation therapy applications

Background

Polymer gel dosimetry has been used extensively in radiation therapy for its capability in depicting a three dimensional view of absorbed dose distribution. However, more studies are required to find less toxic and more efficient polymers for application in radiotherapy dosimetry.

Aim

The purpose of this work was to evaluate the N-isopropyl acrylamide (NIPAM) gel dosimetric characteristics and optimize the protocol for X-ray computed tomography (CT) imaging of gel dosimeters for radiation therapy application.

Material and methods

A polymer gel dosimeter based on NIPAM monomer was prepared and irradiated with ⁶⁰Co photons. The CT number changes following irradiation were extracted from CT images obtained with different sets of imaging parameters.

Results

The results showed the dose sensitivity of ΔN_CT (H) = 0.282 ± 0.018 (H Gy⁻¹) for NIPAM gel dosimeter. The optimized set of imaging exposure parameters was 120 kV_p and 200 mA with the 10 mm slice thickness. Results of the depth dose measurement with gel dosimeter showed a great discrepancy with the actual depth dose data.

Conclusion

According to the current study, NIPAM-based gel dosimetry with X-ray CT imaging needs more technical development and formulation refinement to be used for radiation therapy application.     

The nucleotide exchange factor Ric-8A is a chaperone for the conformationally dynamic nucleotide-free state of Gαi1

Heterotrimeric G protein α subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of Gα, catalyzed by guanine nucleotide exchange factors (GEFs). In addition to transmembrane G protein-coupled receptors (GPCRs), which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class Gα subunits. Ric-8A binds to Gα?GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free Gαi1 is stable, but dissociates upon binding of GTP to Gαi1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from Gαi1, experiments were conducted to characterize the physical state of nucleotide-free Gαi1 (hereafter referred to as Gαi1[ ]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free Gαi1 is more accessible to trypsinolysis than Gαi1?GDP, but less so than Gαi1[ ] alone. The TROSY-HSQC spectrum of [(15)N]Gαi1[ ] bound to Ric-8A shows considerable loss of peak intensity relative to that of [(15)N]Gαi1?GDP. Hydrogen-deuterium exchange in Gαi1[ ] bound to Ric-8A is 1.5-fold more extensive than in Gαi1?GDP. Differential scanning calorimetry shows that both Ric-8A and Gαi1?GDP undergo cooperative, irreversible unfolding transitions at 47° and 52°, respectively, while nucleotide-free Gαi1 shows a broad, weak transition near 35°. The unfolding transition for Ric-8A:Gαi1[ ] is complex, with a broad transition that peaks at 50°, suggesting that both Ric-8A and Gαi1[ ] are stabilized within the complex, relative to their respective free states. The C-terminus of Gαi1 is shown to be a critical binding element for Ric-8A, as is also the case for GPCRs, suggesting that the two types of GEF might promote nucleotide exchange by similar mechanisms, by acting as chaperones for the unstable and dynamic nucleotide-free state of Gα.