Novel endogenous N-acyl amides activate TRPV1-4 receptors, BV-2 microglia, and are regulated in brain in an acute model of inflammation.

A family of endogenous lipids, structurally analogous to the endogenous cannabinoid, N-arachidonoyl ethanolamine (Anandamide), and called N-acyl amides have emerged as a family of biologically active compounds at TRP receptors. N-acyl amides are constructed from an acyl group and an amine via an amide bond. This same structure can be modified by changing either the fatty acid or the amide to form potentially hundreds of lipids. More than 70 N-acyl amides have been identified in nature. We have ongoing studies aimed at isolating and characterizing additional members of the family of N-acyl amides in both central and peripheral tissues in mammalian systems. Here, using a unique in-house library of over 70 N-acyl amides we tested the following three hypotheses: (1) Additional N-acyl amides will have activity at TRPV1-4, (2) Acute peripheral injury will drive changes in CNS levels of N-acyl amides, and (3) N-acyl amides will regulate calcium in CNS-derived microglia. Through these studies, we have identified 20 novel N-acyl amides that collectively activate (stimulating or inhibiting) TRPV1-4. Using lipid extraction and HPLC coupled to tandem mass spectrometry we showed that levels of at least 10 of these N-acyl amides that activate TRPVs are regulated in brain after intraplantar carrageenan injection. We then screened the BV2 microglial cell line for activity with this N-acyl amide library and found overlap with TRPV receptor activity as well as additional activators of calcium mobilization from these lipids. Together these data provide new insight into the family of N-acyl amides and their roles as signaling molecules at ion channels, in microglia, and in the brain in the context of inflammation.

Bisphenol A activates BK channels through effects on α and ß1 subunits.

We demonstrated previously that BK (K(Ca)1.1) channel activity (NP(o)) increases in response to bisphenol A (BPA). Moreover, BK channels containing regulatory ß1 subunits were more sensitive to the stimulatory effect of BPA. How BPA increases BK channel NPo remains mostly unknown. Estradiol activates BK channels by binding to an extracellular site, but neither the existence nor location of a BPA binding site has been demonstrated. We tested the hypothesis that an extracellular binding site is responsible for activation of BK channels by BPA. We synthesized membrane-impermeant BPA-monosulfate (BPA-MS) and used patch clamp electrophysiology to study channels composed of α or α + ß1 subunits in cell-attached (C-A), whole-cell (W-C), and inside-out (I-O) patches. In C-A patches, bath application of BPA-MS (100 µM) had no effect on the NP(o) of BK channels, regardless of their subunit composition. Importantly, however, subsequent addition of membrane-permeant BPA (100 µM) increased the NP(o) of both α and α + ß1 channels in C-A patches. The C-A data indicate that in order to alter BK channel NP(o), BPA must interact with the channel itself (or some closely associated partner) and diffusible messengers are not involved. In W-C patches, 100 µM BPA-MS activated current in cells expressingα subunits, whereas cells expressing α + ß1 subunits responded similarly to a log-order lower concentration (10 µM). The W-C data suggest that an extracellular activation site exists, but do not eliminate the possibility that an intracellular site may also be present. In I-O patches, where the cytoplasmic face was exposed to the bath, BPA-MS had no effect on the NP(o) of BK α subunits, but BPA increased it. BPA-MS increased the NP(o) of α + ß1 channels in I-O patches, but not as much as BPA. We conclude that BPA activates BK α via an extracellular site and that BPA-sensitivity is increased by the ß1 subunit, which may also constitute part of an intracellular binding site.

Targeted lipidomics in Drosophila melanogaster identifies novel 2-monoacylglycerols and N-acyl amides.

Lipid metabolism is critical to coordinate organ development and physiology in response to tissue-autonomous signals and environmental cues. Changes to the availability and signaling of lipid mediators can limit competitiveness, adaptation to environmental stressors, and augment pathological processes. Two classes of lipids, the N-acyl amides and the 2-acyl glycerols, have emerged as important signaling molecules in a wide range of species with important signaling properties, though most of what is known about their cellular functions is from mammalian models. Therefore, expanding available knowledge on the repertoire of these lipids in invertebrates will provide additional avenues of research aimed at elucidating biosynthetic, metabolic, and signaling properties of these molecules. Drosophila melanogaster is a commonly used organism to study intercellular communication, including the functions of bioactive lipids. However, limited information is available on the molecular identity of lipids with putative biological activities in Drosophila. Here, we used a targeted lipidomics approach to identify putative signaling lipids in third instar Drosophila larvae, possessing particularly large lipid mass in their fat body. We identified 2-linoleoyl glycerol, 2-oleoyl glycerol, and 45 N-acyl amides in larval tissues, and validated our findings by the comparative analysis of Oregon-RS, Canton-S and w1118 strains. Data here suggest that Drosophila represent another model system to use for the study of 2-acyl glycerol and N-acyl amide signaling.

CP47,497-C8 and JWH073, commonly found in 'Spice' herbal blends, are potent and efficacious CB(1) cannabinoid receptor agonists.

'Spice' is an herbal blend that has been reported to produce cannabis-like effects when smoked and is marketed as an alternative to marijuana. Synthetic additives have been identified in numerous 'Spice' preparations from different sources. Common among many of the preparations were the compounds JWH018 and a dimethyloctyl variant of CP47,497 (CP47,497-C8) and, more recently JWH073. The synaptic effects of each of these compounds were uncharacterized. We previously reported that JWH018 is a potent and efficacious CB(1) cannabinoid receptor agonist. In this study we have examined the abilities of CP47,497-C8 and JWH073 to inhibit neurotransmission in cultured autaptic hippocampal neurons. Each inhibited EPSCs with an efficacy and potency similar to JWH018. We also analyzed these compounds' effects on promoting internalization of CB(1) receptors in HEK293 cells stably expressing CB(1) receptors. Similar to our neurotransmission data, CP47,497-C8 internalized CB(1) in a fashion indistinguishable from JWH018. However, JWH073 was less potent and produced slower internalization than JWH018 and CP47,497-C8. It appears that 'Spice' contains a number of cannabinoid receptor agonists that activate CB(1) receptors to inhibit synaptic transmission with similar potencies and efficacies. It is highly probable that the cannabis-like effects of 'Spice' are due to the presence of these and analogous synthetic additives acting on CB(1) receptors.

Tyrosine sulfation influences the chemokine binding selectivity of peptides derived from chemokine receptor CCR3.

The interactions of chemokines with their G protein-coupled receptors play critical roles in the control of leukocyte trafficking in normal homeostasis and in inflammatory responses. Tyrosine sulfation is a common post-translational modification in the amino-terminal regions of chemokine receptors. However, tyrosine sulfation of chemokine receptors is commonly incomplete or heterogeneous. To investigate the possibility that differential sulfation of two adjacent tyrosine residues could bias the responses of chemokine receptor CCR3 to different chemokines, we have studied the binding of three chemokines (eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26) to an N-terminal CCR3-derived peptide in each of its four possible sulfation states. Whereas the nonsulfated peptide binds to the three chemokines with approximately equal affinity, sulfation of Tyr-16 gives rise to 9-16-fold selectivity for eotaxin-1 over the other two chemokines. Subsequent sulfation of Tyr-17 contributes additively to the affinity for eotaxin-1 and eotaxin-2 but cooperatively to the affinity for eotaxin-3. The doubly sulfated peptide selectively binds to both eotaxin-1 and eotaxin-3 approximately 10-fold more tightly than to eotaxin-2. Nuclear magnetic resonance chemical shift mapping indicates that these variations in affinity probably result from only subtle differences in the chemokine surfaces interacting with these receptor peptides. These data support the proposal that variations in sulfation states or levels may regulate the responsiveness of chemokine receptors to their cognate chemokines.

Regulation of chemokine recognition by site-specific tyrosine sulfation of receptor peptides.

Sulfation of tyrosine is a common posttranslational modification of secreted proteins that influences numerous physiological and pathological processes. Studies of tyrosine sulfation have been hindered by the difficulty of introducing sulfate groups at specific positions of peptides and proteins. Here we report a general strategy for synthesis of peptides containing sulfotyrosine at one or more specific position(s). The approach provides a substantial improvement in both yield and convenience over existing methods. Using synthetic sulfopeptides derived from the chemokine receptor CCR3, we demonstrate that sulfation enhances affinity for the chemokine eotaxin by approximately 7-fold or more than 28-fold, depending on which of two adjacent tyrosine residues is sulfated. The synthetic methodology will substantially enhance efforts to understand the functional and structural consequences of protein tyrosine sulfation.

Microsomal omega-hydroxylated metabolites of N-arachidonoyl dopamine are active at recombinant human TRPV1 receptors.

N-Arachidonoyl dopamine (NADA) is an endogenous lipid that modulates signal transduction in neuronal and immune pathways. NADA activates the non-selective cation channel, transient receptor potential vanilloid type 1 (TRPV(1)) and cannabinoid receptor 1. That NADA is comprised of an arachidonic acid (AA) backbone suggests that it may be metabolized through many of the enzymes that act upon AA such as the other AA-derived signaling lipids, the endogenous cannabinoids. To investigate the metabolism of NADA through the cytochrome P450 (CYP450) metabolic pathway, we studied the in vitro rat liver microsomal production of hydroxylated metabolites and their activity at recombinant human TRPV(1) receptors. We showed that following microsomal activation in the presence of NADA, omega and (omega-1) hydroxylated metabolites (19- and 20-HETE-DA) were formed. These metabolites were active at recombinant human TRPV(1) receptors, inducing a dose-dependent calcium influx. Both metabolites exhibited lower potency compared to NADA. We conclude that CYP450 enzymes are capable of metabolizing this signaling lipid forming a larger family of potential neuromodulators.

A role for sulfation-desulfation in the uptake of bisphenol a into breast tumor cells.

Bisphenol A (BPA) is a widely used plasticizer whose estrogenic properties may impact hormone-responsive disorders and fetal development. In vivo, BPA appears to have greater activity than is suggested by its estrogen receptor (ER) binding affinity. This may be a result of BPA sulfation/desulfation providing a pathway for selective uptake into hormone-responsive cells. BPA is a substrate for estrogen sulfotransferase, and bisphenol A sulfate (BPAS) and disulfate are substrates for estrone sulfatase. Although the sulfated xenobiotics bind poorly to the ER, both stimulated the growth of receptor-positive breast tumor cells. Treatment of MCF-7 cells with BPAS leads to desulfation and uptake of BPA. No BPAS is found inside the cells. These findings suggest a mechanism for the selective uptake of BPA into cells expressing estrone sulfatase. Therefore, sulfation may increase the estrogenic potential of xenobiotics.

A comprehensive approach to the synthesis of sulfate esters.

A comprehensive approach to the synthesis of sulfate esters was developed. This approach permits the direct and high-yielding synthesis of protected sulfate monoesters. Subsequent deblocking to reveal sulfate monoesters is accomplished in near-quantitative yield. The exceptionally stable neopentyl protecting group and the labile isobutyl protecting group were utilized in the synthesis of aromatic and aliphatic sulfate monoesters. Strategies for tuning protecting group reactivity were also explored and developed.

Facile deprotection of O-Cbz-protected nucleosides by hydrogenolysis: an alternative to O-benzyl ether-protected nucleosides.

[reaction: see text] Because of side-reactions encountered during hydrogenolysis, benzyl ethers are usually not an effective protecting group for nucleosides. Benzyloxycarbamates provide an alternative to traditional benzyl ethers for protection of nucleoside hydroxyl groups, as they are much more labile to hydrogenolysis. Deprotection conditions using transfer hydrogenolysis are described that avoid the reduction of the pyrimidine nucleobase during deblocking of O-Cbz-protected nucleosides. Additionally, an experiment is described that suggests the nucleobase component of a nucleoside is responsible for the sluggish hydrogenolysis of nucleosides.

Overview of the synthesis of nucleoside phosphates and polyphosphates.

This overview summarizes methodology used for the synthesis of nucleoside mono-, di-, and triphosphates. Selected techniques such as the Mitsunobu reaction, displacement reactions involving nucleoside 5'-tosylates, "anion-exchange" techniques, and phosphoramidite and phosphoramidate methodologies are highlighted. The chemistry of phosphorylation is detailed with respect to advantages and limitations under various conditions. Applicability of the methods toward the synthesis of analogs such as imidophosphates, phosphorothioates, and radiolabeled nucleotides is also addressed.

34S isotope effect on sulfate ester hydrolysis: mechanistic implications.

In this communication, we report the first determination of 34S kinetic isotope effects (KIEs) for the hydrolysis of sulfate monoesters. The method involves the conversion of the inorganic sulfate, acquired at partial extent of reaction, to SO2, followed by isotope ratio determination by mass spectrometry. The KIEs determined for p-nitrophenyl sulfate and p-acetylphenyl sulfate are 1.0154 (+/-0.0002) and 1.0172 (+/-0.0003), respectively. These results, together with previous peripheral 18O KIE values, are inconsistent with an associative mechanism. The isotope effect method we report should also prove useful for studying the mechanism of other sulfuryl group transfers, including sulfatase and sulfotransferase reactions, as well as sulfate hydrolyses under other conditions.

Cerium(III) chloride-mediated reactions of sulfonamide dianions.

Presented here is the first report on the ability of cerium(III) chloride to mediate high-yielding and, oftentimes, highly diastereoselective additions of N-benzyl-alpha, N-dilithio methanesulfonamide to aldehydes and ketones of biological importance. Smooth addition was effected to base-sensitive substrates such as Fmoc-protected alaninal, citral, 5-cholesten-3-one, uridine 5'-aldehyde, 3'-ketouridine, and 3'-ketothymidine. The reaction was chemoselective for aldehydes in the presence of nitriles. Acetoxy groups are labile and thus not suitable protecting groups for alcohols under these conditions. N-Benzyl-alpha, N-dilithio methanesulfonamide was found to be of sufficient basicity to cause enolate formation with sensitive substrates, such as 1-phenylacetone. However, the addition of cerium(III) chloride mediated the basicity of the dianion and suppressed enolate formation in these cases. Further, cerium(III) has general utility for the addition of various N-aliphatic/aromatic methanesulfonamide dianions to 3'-ketouridine.