Summary
Zinc concentrations in pelagic surface waters are within the range that limits growth in marine phytoplankton cultures. However, the influence of zinc on marine primary production and ...phytoplankton communities is not straightforward due to largely uncharacterized abilities for some phytoplankton to access zinc species that may not be universally bioavailable and substitute zinc with cobalt or cadmium. We used a quantitative proteomic approach to investigate these strategies and other responses to zinc limitation in the coccolithophore Emiliania huxleyi, a dominant species in low zinc waters. Zinc limitation resulted in the upregulation of metal transport proteins (ZIP, TroA‐like) and COG0523 metallochaperones. Some proteins were uniquely sensitive to growth under replete zinc, substitution of zinc with cobalt, or enhancement of growth with cadmium, and may be useful as biomarkers of zinc stress or substitution in situ. Several proteins specifically upregulated under cobalt‐supported or cadmium‐enhanced growth appear to reflect stress responses, despite titration of these metals to optimal nutritive levels. Relief from zinc limitation by zinc or cadmium resulted in increased expression of a δ‐carbonic anhydrase. Our inability to detect metal binding enzymes that are specifically induced under cobalt‐ or cadmium‐supported growth suggests cambialism is important for zinc substitution in E. huxleyi.
We measured the capacity for luxury iron (Fe) uptake and ferritin abundance in the marine cyanobacterium Prochlorococcus marinus (strain MED4) acclimated to various Fe concentrations. We also ...followed changes in ferritin abundance and physiological measurements after abruptly changing Fe availability by adding Fe-EDTA or desferrioxamine B (DFB) to cultures. Fe quotas (normalized to carbon) increased from 46 to 112 Fe:C (μmol:mol) in cultures grown at Fe′ (the summed concentrations of all Fe species not complexed with EDTA) between 21 and 180 pM. No further increase in Fe:C was observed at 684 pM Fe′. Growth rates and variable fluorescence did not vary significantly in cultures acclimated to Fe′ values ranging from 15 to 679 pM, demonstrating that Prochlorococcus can store ~ 2.4-fold more Fe than it requires for maximum growth. Ferritin abundance increased from below detection at 15 pM Fe′ to maximum values at 175 to 679 pM Fe′. Ferritin abundance, growth, and variable fluorescence decreased after the addition of DFB, and ferritin abundance increased upon addition of Fe-EDTA. These results further support the protein's role in Fe storage in Prochlorococcus. This capacity for luxury Fe uptake may help Prochlorococcus survive periods of low Fe availability in the open ocean, and the recycling of ferritin-stored Fe may be an important component of the marine iron cycle.
Based on both binding and functional data, this study introduces SR 144528 as the first, highly potent, selective and orally active antagonist for the CB2 receptor. This compound which displays ...subnanomolar affinity (Ki = 0.6 nM) for both the rat spleen and cloned human CB2 receptors has a 700-fold lower affinity (Ki = 400 nM) for both the rat brain and cloned human CB1 receptors. Furthermore it shows no affinity for any of the more than 70 receptors, ion channels or enzymes investigated (IC50 > 10 microM). In vitro, SR 144528 antagonizes the inhibitory effects of the cannabinoid receptor agonist CP 55,940 on forskolin-stimulated adenylyl cyclase activity in cell lines permanently expressing the h CB2 receptor (EC50 = 10 nM) but not in cells expressing the h CB1 (no effect at 10 microM). Furthermore, SR 144528 is able to selectively block the mitogen-activated protein kinase activity induced by CP 55,940 in cell lines expressing h CB2 (IC50 = 39 nM) whereas in cells expressing h CB1 an IC50 value of more than 1 microM is found. In addition, SR 144528 is shown to antagonize the stimulating effects of CP 55,940 on human tonsillar B-cell activation evoked by cross-linking of surface Igs (IC50 = 20 nM). In vivo, after oral administration SR 144528 totally displaced the ex vivo 3H-CP 55,940 binding to mouse spleen membranes (ED50 = 0.35 mg/kg) with a long duration of action. In contrast, after the oral route it does not interact with the cannabinoid receptor expressed in the mouse brain (CB1). It is expected that SR 144528 will provide a powerful tool to investigate the in vivo functions of the cannabinoid system in the immune response.
SR141716A is the first selective and orally active antagonist of the brain cannabinoid receptor. This compound displays nanomolar affinity for the central cannabinoid receptor but is not active on ...the peripheral cannabinoid receptor. In vitro, SR141716A antagonises the inhibitory effects of cannabinoid receptor agonists on both mouse vas deferens contractions and adenylyl cyclase activity in rat brain membranes. After intraperitoneal or oral administration SR141716A antagonises classical pharmacological and behavioural effects of cannabinoid receptor agonists. This compound should prove to be a powerful tool for investigating the in vivo functions of the anandamide/cannabinoid system.
The cDNA sequences encoding the central cannabinoid receptor, CB1, are known for two species, rat and human. However, little information concerning the flanking, noncoding regions is presently ...available. We have isolated two overlapping clones from a human lung cDNA library with CB1 cDNA inserts. One of these, cann7, contains a short stretch of the CB1 coding region and 4 kilobase pairs (kb) of the 3'-untranslated region (UTR), including two polyadenylation signals. The other, cann6, is identical to cann7 upstream from the first polyadenylation signal, and in addition, it contains the whole coding region and extends for 1.8 kb into the 5'-UTR. Comparison of cann6 with the published sequence (Gérard, C. M., Mollereau, C., Vassart, G., and Parmentier, M. (1991) Biochem. J. 279, 129-134) shows the coding regions to be identical, but reveals important differences in the flanking regions. Notably, the cann6 sequence appears to be that of an immature transcript, containing 1.8 kb of an intronic sequence in the 5'-UTR. In addition, polymerase chain reaction amplification of the CB1 coding region in the IM-9 cell line cDNA resulted in two fragments, one containing the whole CB1 coding region and the second lacking a 167-base pair intron within the sequence encoding the amino-terminal tail of the receptor. This alternatively spliced form would translate to an NH2-terminal modified isoform (CB1A) of the receptor, shorter than CB1 by 61 amino acids. In addition, the first 28 amino acids of the putative truncated receptor are completely different from those of CB1, containing more hydrophobic residues. Rat CB1 mRNA is similarly alternatively spliced. A study of the distribution of the human CB1 and CB1A mRNAs by reverse transcription-polymerase chain reaction analysis showed the presence of both CB1 and CB1A throughout the brain and in all the peripheral tissues examined, with CB1A being present in amounts of up to 20% of CB1.
The peripheral benzodiazepine receptor, implicated in the transport of cholesterol from the outer to the inner mitochondrial membrane, is predicted by hydropathy analysis to feature five ...membrane-spanning domains, with the amino terminus within the mitochondrial periplasm and the carboxyl terminus in the external cytoplasm. We have tested these structural predictions directly by immunodetection of c-Myc-tagged peripheral benzodiazepine receptor on intact yeast mitochondria and by specific labeling in yeast membranes of cysteine residues introduced by site-directed mutagenesis. The combined results support the model originally proposed with some minor but important modifications. The theoretical model predicted relatively short α-helical domains, only long enough to span a phospholipid monolayer, whereas the results presented here would support a model with extended α-helices sufficiently long to span an entire membrane bilayer, with concomitant shorter loop and tail regions.
We have investigated whether there are cannabinoid CB2 receptors that can mediate cannabinoid-induced inhibition of electrically evoked contractions in the mouse vas deferens or guinea-pig myenteric ...plexus-longitudinal muscle preparation. Our results showed that mouse vas deferens and guinea-pig whole gut contain cannabinoid CB1 and CB2-like mRNA whereas the myenteric plexus preparation seemed to contain only cannabinoid CB1 mRNA. JWH-015 (1-propyl-2-methyl-3-( -naphthoyl)indole) and JWH-051 (1-deoxy-11-hydroxy-delta8-tetrahydrocannabinol-dimethylheptyl+ ++), which have higher affinities for CB2 than CB1 cannabinoid binding sites, inhibited electrically evoked contractions of both tissues in a concentration related manner. This inhibition was attenuated by 31.62 nM of the cannabinoid CB1 receptor selective antagonist SR141716A N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide hydrochloride only in the myenteric plexus preparation. Vasa deferentia from delta9-tetrahydrocannabinol-pretreated mice (20 mg/kg i.p. once daily for two days) showed reduced sensitivity to JWH-015 and JWH-051. The results suggest that these compounds exert their inhibitory effects through cannabinoid CB1 receptors in the myenteric plexus preparation, but mainly through CB2-like cannabinoid receptors in the vas deferens.
It has long been established that the cannabinoid CB1 receptor transduces signals through a pertussis toxin-sensitive Gi/Go inhibitory pathway. Although there have been reports that the cannabinoid ...CB1 receptor can also mediate an increase in cyclic AMP levels, in most cases the presence of an adenylyl cyclase costimulant or the use of very high amounts of agonist was necessary. Here, we present evidence for dual coupling of the cannabinoid CB receptor to the classical pathway and to a pertussis toxin-insensitive adenylyl cyclase stimulatory pathway initiated with low quantities of agonist in the absence of any costimulant. Treatment of Chinese hamster ovary (CHO) cells expressing the cannabinoid CB1 receptor with the cannabinoid CP 55,940, {(-)-cis-3-2-hydroxy-4-(1,1-dimethylheptyl)phenyl-trans-4-(3-hyd roxypropyl) cyclohexan-1-ol} resulted in cyclic AMP accumulation in a dose-response manner, an accumulation blocked by the cannabinoid CB1 receptor-specific antagonist SR 141716A, {N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide hydrochloride}. In CHO cells coexpressing the cannabinoid CB1 receptor and a cyclic AMP response element (CRE)-luciferase reporter gene system, CP 55,940 induced luciferase expression by a pathway blocked by the protein kinase A inhibitor N-2-(p-bromocinnamylamino)ethyl-5-isoquinolinesulfonamide hydrochloride (H-89). Under the same conditions the peripheral cannabinoid CB2 receptor proved to be incapable of inducing cAMP accumulation or luciferase activity. This incapacity allowed us to study the luciferase activation mediated by CB /CB2 chimeric constructs, from which we determined that the first and second internal loop regions of the cannabinoid CB1 receptor were involved in transducing the pathway leading to luciferase gene expression.
The recent isolation and cloning of the G protein-coupled central cannabinoid receptor (CB1) from brain tissue has provided a molecular basis to elucidate how cannabinoid compounds may mediate their ...psychoactive effects. Here we report the high expression of cannabinoid receptors in human astrocytoma tumors of different grades, in the astrocytoma cell lines U373 MG and GL-15, as well as in normal astrocytes. From an analysis of the coupling mechanisms of functional CB1 receptors in U373 MG, we show that, in addition to the inhibition of adenylyl cyclase, activation by the cannabinoid agonist CP-55940 induces the expression of the immediate-early gene krox-24, also known as NGFI-A, zif/268, egr-1, and TIS8. The amount of Krox-24 protein and the level of Krox-24 DNA binding activity, as measured by Western blot and electrophoretic mobility shift assay, respectively, were also increased by the addition of CP-55940. These effects were blocked by incubation with pertussis toxin but not by treatment with hydrolysis-resistant cAMP analogues, suggesting that the transduction pathway between the cannabinoid receptor and krox-24 involves a pertussis toxin-sensitive GTP-binding protein and is independent of cAMP metabolism. The specific involvement of CB1 in Krox-24 induction was demonstrated in Chinese hamster ovary cells transfected with the human CB1 receptor and also in experiments using the CB1-selective cannabinoid antagonist SR 141716A.
Two proteins with seven transmembrane‐spanning domains typical of guanosine‐nucleotide‐binding‐protein‐coupled receptors have been identified as cannabinoid receptors; the central cannabinoid ...receptor, CB1, and the peripheral cannabinoid receptor, CB2, initially described in rat brain and spleen, respectively. Here, we report the distribution patterns for both CB1 and CB2 transcripts in human immune cells and in several human tissues, as analysed using a highly sensitive and quantitative PCR‐based method. CB1 was mainly expressed in the central nervous system and, to a lower extent, in several peripheral tissues such as adrenal gland, heart, lung, prostate, uterus, ovary, testis, bone marrow, thymus and tonsils. In contrast, the CB2 gene, which is not expressed in the brain, was particularly abundant in immune tissues, with an expression level 10–100‐fold higher than that of CB1. Although CB2 mRNA was also detected in some other peripheral tissues, its level remained very low. In spleen and tonsils, the CB2 mRNA content was equivalent to that of CB1 mRNA in the central nervous system. Among the main human blood cell subpopulations, the distribution pattern of the CB2 mRNA displayed important variations. The rank order of CB2 mRNA levels in these cells was B‐cells > natural killer cells ≫ monocytes > polymorphonuclear neutrophil cells > T8 cells > T4 cells. The same rank order was also established in human cell lines belonging to the myeloid, monocytic and lymphoid lineages. The prevailing expression of the CB2 gene in immune tissues was confirmed by Northern‐blot analysis. In addition, the expression of the CB2 protein was demonstrated by an immunohistological analysis performed on tonsil sections using specific anti‐(human CB2) IgG; this experiment showed that CB2 expression was restricted to B‐lymphocyte‐enriched areas of the mantle of secondary lymphoid follicles. These results suggest that (a) CB1 and CB2 can be considered as tissue‐selective antigens of the central nervous system and immune system, respectively, and (b) cannabinoids may exert specific receptor‐mediated actions on the immune system through the CB2 receptor.
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