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.     

Localization of ornithine decarboxylase in the chick embryo during organogenesis

Summary The localization of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis and thus in cell growth, was determined in the 4.5-day-old chick embryo, using two independent methods of analysis. ODC protein was identified by indirect immunofluorescence with a monospecific ODC antibody, and catalytically active ODC was identified by autoradiography with -(5-³H) difluoromethylornithine. Both methods revealed a basically similar distribution of ODC within the embryo. Among the organs, the brain exhibited the highest ODC levels. ODC levels were also high in spinal cord, mesonephric tubules and heart. Similar levels, but confined to limited areas, were found in liver tissue, head mesenchyme, and the oral and pharyngeal regions. Organs that exhibited high ODC levels are all engaged in rapid growth, as well as in extensive tissue remodeling and differentiation.     

A functional analysis of the repeated methionine initiator tRNA genes (IMT) in yeast

Summary Standard laboratory yeast strains have from four to five genes encoding the methionine initiator tRNA (IMT). Strain S288C has four IMT genes with identical coding sequences that are colinear with the RNA sequence of tRNA _I ^Met . Each of the four IMT genes from strain S288C is located on a different chromosome. A fifth IMT gene with the same coding sequence is present in strain A364A but not in S288C. By making combinations of null alleles in strain S288C, we show that each of the four IMT genes is functional and that tRNA _I ^Met is not limiting in yeast strains with three or more intact genes. Strains containing a single IMT2, 3 or 4 gene grow only after amplification of the remaining IMT gene. Strains with only the IMT1 gene intact are viable but grow extremely slowly; normal growth is restored by the addition of another IMT gene by transformation, providing a direct test for IMT function.Abbreviations IMT and imt (imt=initiator methionine tRNA), designate the genotype of the wild-type and the mutant alleles respectively, of the initiator methionine transfer RNA gene - met-tRNA _I ^Met methionylated initiator methionine transfer RNA - eIF-2 eukaryotic initiation factor two - GTP guanosine 5-triphosphate The calculation of Td values (the temperature at which half of the duplex is dissociated) for oligonucleotides used as probes in hybridizations was based on the assumption that the increase in Td value was 4° C for each G:C base pair and 2° C for each A:T base pair (Wallace et al. 1981)     

The seed dimorphism of Spergularia marina in relation to dispersal by wind and water

Summary The seeds of the halophyte Spergularia marina differ both within and between individuals in that they either possess or lack a membranaceous border. This paper presents a morphological study of the length, weight and area of the seed types, and their dispersal characteristics under experimental conditions of wind and water dispersal. The winged seeds are shown to be larger both by length and by weight. Their rate of descent increases with wing loading. If the wing is lacking, however, the rate of descent increases with weight only. The distance of dispersal is equal for both seed types except at low wind speeds, when the winged seeds disperse farther. If the seed wing is removed, the excised seeds have shorter dispersal distances. When dispersed by water, a difference in the distance seeds are dispersed can only be detected in the presence of vegetation. The winged seeds are more frequently trapped in the vegetation as compared to the unwinged seeds. The hypothesis that the seed dimorphism is an adaptation for differential dispersal distances is discussed.     

Morphological changes in the junctional complex of cells in the arachnoid layer of the rat after cold injury

Summary The junctional complexes of cells in the outer arachnoid layer overlying the cerebral cortex of 2-week-old rats were examined with freeze-fracture electron microscopy up to 60 min after transcranial cold injury to the dorsal surface of the brain. Within 30 min after injury, areas of gap and tight junctions with morphological features characteristic of junction formation and/or junction disruption were found scattered among normal junctional complexes in some arachnoid cells. Within 60 min after injury, tight junctions with features typical of less leaky zonulae occludentes were present in all arachnoid cells examined. These morphological features include increases in the number of tight junctional strands and the number of strand-to-strand anatomoses. Gap junctions were interspersed among the tight junctional strands, and many were completely encircled by the strands. The increase in the number and complexity of the tight junctional strands in response to brain injury may be the morphological basis for the maintenance of the cerebrospinal fluid-blood dural barrier.This study was supported by the National Institute of Neurological and Communicative Disorders and Stroke Grant NS20590. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the DoD or the USUHS. The experiments reported herein were conducted according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, DHEW Pub. No. (NIH) 78-23     

Regulation of rat liver microsomal glutathione S-transferase activity by thiol/disulfide exchange

The regulation of purified glutathione S-transferase from rat liver microsomes was studied by examining the effects of various sulfhydryl reagents on enzyme activity with 1-chloro-2,4-dinitrobenzene as the substrate. Diamide (4 mM), cystamine (5 mM), and N-ethylmaleimide (1 mM) increased the microsomal glutathione S-transferase activity by 3-, 2-, and 10-fold, respectively, in absence of glutathione; glutathione disulfide had no effect. In presence of glutathione, microsomal glutathione S-transferase activity was increased 10-fold by diamide (0.5 mM), but the activation of the transferase by N-ethylmaleimide or cystamine was only slightly affected by presence of glutathione. The activation of microsomal glutathione S-transferase by diamide or cystamine was reversed by the addition of dithiothreitol. Glutathione disulfide increased microsomal glutathione S-transferase activity only when membrane-bound enzyme was used. These results indicate that microsomal glutathione S-transferase activity may be regulated by reversible thiol/disulfide exchange and that mixed disulfide formation of the microsomal glutathione S-transferase with glutathione disulfide may be catalyzed enzymatically in vivo.     

Iodine binding and peroxidase activity in the endostyle of Salpa fusiformis,Thalia democratica,Dolioletta gegenbauri and Doliolum nationalis (Tunicata,Thaliacea)

Summary The protothyroid region in the endostyles of four species of tunicates was examined by means of autoradiography and cytochemistry, at both the light and electronmicroscopic levels. To reveal the primary binding site for iodine, autoradiography was carried out on endostylar tissue from animals that had been incubated with high activity ¹²⁵I^- over a short period of time. The specific iodine binding enzyme, a peroxidase, was traced by its reaction with DAB. In accordance with previous findings, the iodinebinding cells proved to be the same as those containing the peroxidase. There were also strong indications of a secondary uptake of iodinated compounds and subsequent release into the body fluid. Together with the ultrastructural features, the data provided strong evidence indicating that these cells constitute a protothyroid region, which partly functions as an endocrine organ, possibly homologous with the vertebrate thyroid gland. Since the number of zones varied between the species, the numeration of the protothyroid region also varied. However, in all the examined endostyles, the protothyroid region was seen to be situated dorsolaterally to the glandular regions of the endostyle concerned with food capture.     

Microbial communities in the saturated groundwater environment II: Diversity of bacterial communities in a Pleistocene sand aquifer and their in vitro activities

Bacterial cell numbers obtained from 103 water and sediment samples from a Pleistocene sandy aquifer in the Lower Rhine region (Bocholt, FRG) were determinated on P-agar and by direct count. Below 5 m under the surface, colony-forming unit (cfu) numbers in water samples were less than 100/ml, and in many cases less than 50/ml. In sediment samples, they were 10- to 100-fold higher (10²–10⁴ cfu/g dry wt), but changing markedly between different depths. Direct cell counts yielded numbers two to three orders of magnitude higher.About 2,700 strains of bacteria from 60 samples were isolated randomly and characterized by morphological and physiological properties. Of all the isolates, 71.6% were gram-negative, and 52.2% were gram-negative straight rods. Water communities, with one exception, had low proportions of gram-positive bacteria (<11%), whereas in all but one of the sediment communities percentages of gram-positive isolates were three- to sevenfold higher (35–43%). Water and sediment communities, as well as communities from different sampling sites and communities from different depths of the same sampling site, differed in their qualitative and quantitative morphotype composition and physiological capabilities.The in vitro activities of strains within a single community were quite different, indicating that each community is composed of many diverse bacteria, several having extremely different capabilities. Thus, each community has its own specific activity pattern. Gram-positive bacteria showed on an average lower total activities than did gram-negative bacteria. Grampositive bacteria as well as gram-negative bacteria from sediment had higher values of in vitro activities than the corresponding groups isolated from water. Many water and sediment bacteria preferred the same substrates which were utilized at high rates. However, there were differences in the degradation of the various other substrates present, and each community showed preferences for particular substrates, which they degraded best.The results of cell morphology and physiology studies indicated that all eight characterized communities were very different from one another and very diversely structured.     

Kinetics of multiproduct acidogenic and solventogenic batch fermentations

Summary Large amounts of data indicated that most of the metabolic processes of the acidogenic (acid producing) and the solventogenic (solvent producing) fermentations were regulated by product accumulation. A simple unstructured model simulated microbial growth, product formation and substrate utilization in six different fermentations, where five different microorganisms produced various combinations of ten different products. Specific growth rates of these microorganisms decreased proportionally with overall product accumulation. The products were excreted in non-growth associated pattern. Excretion of some of these products were inhibited by the overall product accumulation similarly as the microbial growth. A substrate consumption model which considered the biomass and individually all the products as separate substrate sinks simulated the data satisfactorily.