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.     

Detection of Mls-antigens in various mouse strains using a new method

Mice of strains CBA and BALB/c, when injected with lymphocytes from theH-2-compatible Mls-antigen-incompatible strains C3H and DBA/2, respectively, develop a reduced lymphocyte reactivity against cells of the injected strains as measured in the mixed lymphocyte culture (MLC). The mechanism of the development of a depression of the MLC response against Mlsantigens is unknown. In this investigation we have tested the MLC response of lymphocytes from CBA mice preinjected with C3H lymphocytes against cells from 12 different strains. It was observed that the response decreased against cells from strains C3H, AKR, and A/Sn. Infusion of CBA mice with AKR lymphocytes decreased their MLC response against the same three strains. In contrast, infusion of CBA mice with A/Sn lymphocytes reduced their MLC responses against strains C3H, DBA/2, and the congenic strains A/Sn, A.SW, A.CA, and A.BY. BALB/c mice which were infused with DBA/2 lymphocytes developed reduced responses against DBA/2, C3H, and AKR. On the basis of these results we propose that mice of our strains C3H and AKR possess a common Mls-antigen which is strongly stimulatory, and that DBA/2 mice possess a second Mls-antigen which is also strongly stimulatory. The congenic strains A/Sn, A.SW, A.CA, and A.BY, which have differentH-2 complexes, possess a third Mls-antigen which is less stimulatory. The Mls-antigens of the strains listed above seem to exhibit extensive immunological crossreactivity.     

Nonradioactive methods for the assay of phosphoinositide 3-kinases and phosphoinositide phosphatases and selective detection of signaling lipids in cell and tissue extracts

We describe a novel approach to quantitation of phosphoinositides in cell extracts and in vitro enzyme-catalyzed reactions using suitably tagged and/or labeled pleckstrin homology (PH) domains as probes. Stable complexes were formed between the biotinylated target lipid and an appropriate PH domain, and phosphoinositides present in samples were detected by their ability to compete for binding to the PH domain. Complexes were detected using AlphaScreen technology or time-resolved FRET. The assay procedure was validated using recombinant PI 3-kinase gamma with diC8PtdIns(4,5)P(2) as substrate and general receptor for phosphoinositides-1 (GRP1) PH domain as a PtdIns(3,4,5)P(3)-specific probe. This PI 3-kinase assay was robust, was suitable for high-throughput screening platforms, and delivered expected IC(50) values for reference compounds. The approach is adaptable to a wide range of enzymes as demonstrated by assays of the tumor suppressor protein, PTEN, a phosphoinositide 3-phosphatase, which was measured using the same reagents but with diC8PtdIns(3,4,5)P(3) as substrate. PtdIns(3,4,5)P(3) present in lipid extracts of Swiss 3T3 and HL60 cells stimulated with platelet-derived growth factor and fMLP, respectively, was also detectable at picomole sensitivity. The versatility and general utility of this approach were demonstrated by exchanging the GRP1 PH domain for that of TAPP1 (which binds PtdIns(3,4)P(2) and not PtdIns(3,4,5)P(3)). This system was used to monitor the accumulation of PtdIns(3,4)P(2) in Swiss 3T3 cells exposed to an oxidative stress. It is therefore proposed that similar procedures should be capable of measuring any known phosphoinositide present in cell and tissue extracts or produced in kinase and phosphatase assays by using one of several well-characterized protein domains with appropriate phosphoinositide-binding specificity.     

Role of calpain in human sperm activated by progesterone for fertilization

We evaluated the activation of mu-calpain in progesterone-activated human sperm. Semen collected from fertile donors with informed consent was liquefied and subjected to percoll gradient centrifugation. After exposure to different concentrations of progesterone, the samples were used for immunostaining, SDS-PAGE and Western blot analysis. An increase of the intracellular free calcium concentration in the sperm following the addition of progesterone was observed using fura-2 AM. Immunostaining using an antibody against active mu-calpain produced 6 distinct staining patterns: (1) the acrosome, (2) an equatorial segment, (3) the whole head, (4) the neck, (5) the neck and tail or (6) unstained sperm. After addition of progesterone, the predominant type changed from the neck type (90%) to the neck and tail type (79%). Western blot analysis using a pro-mu-calpain and a mu-calpain domain III antibody revealed autodigestion of mu-calpain, indicating activation by progesterone. Using calpain-specific inhibitors it was shown that calpain activation contributes to sperm motility as well as to the acrosome reaction. These results suggest the possibility that activation of mu-calpain in human sperm by progesterone plays an important role in fertilization.     

Correlation between caspase-3 activation and three different markers of DNA damage in neonatal cerebral hypoxia-ischemia

Caspase-3 has been identified as a key protease that, by targeting a limited number of proteins, can disrupt essential homeostatic processes and initiate an orderly disassembly of cells, including degradation of genomic DNA. We demonstrate the usefulness of an antibody specific for activated caspase-3 in a model of neonatal rat hypoxia-ischemia (Hl) and correlate the spatial and temporal activation of caspase-3 with three different markers of DNA damage and with the loss of a neuronal marker [microtubule-associated protein 2 (MAP 2)]. An oligonucleotide hairpin probe (HPP) with one base overhang in the 3' end displayed a close colocalization with caspase-3 activation at 3 h post-Hl, whereas terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) appeared later (24 h post-Hl). A monoclonal antibody against single-stranded DNA appeared to stain an entirely different population of cells, not positive for active caspase-3, HPP, or TUNEL at this time point. After 24 h of reperfusion, however, when cellular injury is extensive, all markers stained a large number of cells with a high degree of colocalization, and all markers delineated regions with loss of MAP 2. We conclude that the HPP shows the best correlation with pathological caspase-3 activation in this model.     

Death effector activation in the subventricular zone subsequent to perinatal hypoxia/ischemia

Perinatal hypoxia/ischemia (H/I) is the leading cause of neurological injury resulting from birth complications and pre-maturity. Our studies have demonstrated that this injury depletes the subventricular zone (SVZ) of progenitors. In this study, we sought to reveal which cell death pathways are activated within these progenitors after H/I. We found that calpain activity is detected as early as 4 h of reperfusion and is sustained for 48 h, while caspase 3 activation does not occur until 8 h and peaks at 24 h post-insult. Activated calpains and caspase 3 co-localized within precursors situated in the lateral aspects of the SVZ (which coincides with progenitor cell death), whereas neither enzyme was activated in the medial SVZ (which harbors the neural stem cells that are resilient to this insult). These studies reveal targets for neuroprotective agents to protect precursors from cell death towards the goal of restoring normal brain development after H/I.     

Progenitor cell injury after irradiation to the developing brain can be modulated by mild hypothermia or hyperthermia

Ionizing radiation induced acute cell death in the dentate gyrus subgranular zone (SGZ) and the subventricular zone (SVZ). Hypomyelination was also observed. The effects of mild hypothermia and hyperthermia for 4 h after irradiation (IR) were studied in postnatal day 9 rats. One hemisphere was irradiated with a single dose of 8 Gy and animals were randomized to normothermia (rectal temperature 36 degrees C for 4 h), hypothermia (32 degrees C for 4 h) or hyperthermia (39 degrees C for 4 h). Cellular injury, e.g. chromatin condensation and nitrotyrosine formation, appeared to proceed faster when the body temperature was higher. Caspase-3 activation was more pronounced in the hyperthermia group and nuclear translocation of p53 was less pronounced in the hypothermia group 6 h after IR. In the SVZ the loss of nestin-positive progenitors was more pronounced (48%) and the size was smaller (45%) in the hyperthermia group 7 days post-IR. Myelination was not different after hypo- or hyperthermia. This is the first report to demonstrate that hypothermia may be beneficial and that hyperthermia may aggravate the adverse side-effects after radiation therapy to the developing brain.     

Decreased oxidative stress during glycolytic inhibition enables maintenance of ATP production and astrocytic survival

Depressed energy metabolism and oxidative stress are common features in many pathological situations in the brain, including stroke. In order to investigate astrocytic responses to such stress, we induced metabolic depression in cultured rat astrocytes. Iodoacetate (IA), an inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used and resulted in a rapid inhibition of GAPDH activity. After 1h of GAPDH inhibition the ATP levels started to decrease and were completely abolished at 4h. In parallel, the activity of reactive oxygen species (ROS) was significantly increased, followed by extensive cell death involving flipping of phosphatidylserine and translocation of apoptosis-inducing factor, but not caspase-3 activation. When IA was combined with azide, a respiratory chain complex IV inhibitor, the ATP levels decreased immediately. Interestingly, with azide present, the ROS activity remained low and the astrocytes remained viable even at very low ATP levels. Addition of exogenous ROS-scavengers prevented the IA-induced ROS activity, the ATP levels were maintained and cell death was prevented. Similar protection could be obtained when astrocytes, prior to addition of IA, were incubated with substances known to activate the nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated endogenous antioxidant system. When IA was washed out, after a relatively moderate ATP depression, massive cell death occurred. This was efficiently prevented by addition of azide or ROS scavengers during the IA treatment or by pre-activation of the Nrf2 system. Our results demonstrate that astrocytes in culture can endure and recover from glycolytic inhibition if the ROS activity remained at a low level and suggest that oxidative stress can be an important component for astrocytic cell death following metabolic stress.