Olov Sterner

TRPA1 mediates spinal antinociception induced by acetaminophen and the cannabinoid Delta(9)-tetrahydrocannabiorcol

TRPA1 is a unique sensor of noxious stimuli and, hence, a potential drug target for analgesics. Here we show that the antinociceptive effects of spinal and systemic administration of acetaminophen (paracetamol) are lost in Trpa1(-/-) mice. The electrophilic metabolites N-acetyl-p-benzoquinoneimine and p-benzoquinone, but not acetaminophen itself, activate mouse and human TRPA1. These metabolites also activate native TRPA1 and, as a consequence, reduce voltage-gated calcium and sodium currents in primary sensory neurons. The N-acetyl-p-benzoquinoneimine metabolite L-cysteinyl-S-acetaminophen was detected in the mouse spinal cord after systemic acetaminophen administration. In the hot-plate test, intrathecal administration of N-acetyl-p-benzoquinoneimine, p-benzoquinone and the electrophilic TRPA1 activator cinnamaldehyde produced antinociception that was lost in Trpa1(-/-) mice. Intrathecal injection of a non-electrophilic cannabinoid, Δ(9)-tetrahydrocannabiorcol, also produced TRPA1-dependent antinociception in this test. Our study provides a molecular mechanism for the antinociceptive effect of acetaminophen and discloses spinal TRPA1 activation as a potential pharmacological strategy to alleviate pain.

Endogenous unsaturated C18 N-acylethanolamines are vanilloid receptor (TRPV1) agonists.

The endogenous C18 N-acylethanolamines (NAEs) N-linolenoylethanolamine (18:3 NAE), N-linoleoylethanolamine (18:2 NAE), N-oleoylethanolamine (18:1 NAE), and N-stearoylethanolamine (18:0 NAE) are structurally related to the endocannabinoid anandamide (20:4 NAE), but these lipids are poor ligands at cannabinoid CB1 receptors. Anandamide is also an activator of the transient receptor potential (TRP) vanilloid 1 (TRPV1) on primary sensory neurons. Here we show that C18 NAEs are present in rat sensory ganglia and vascular tissue. With the exception of 18:3 NAE in rat sensory ganglia, the levels of C18 NAEs are equal to or substantially exceed those of anandamide. At submicromolar concentrations, 18:3 NAE, 18:2 NAE, and 18:1 NAE, but not 18:0 NAE and oleic acid, activate native rTRPV1 on perivascular sensory nerves. 18:1 NAE does not activate these nerves in TRPV1 gene knock-out mice. Only the unsaturated C18 NAEs elicit whole cell currents and fluorometric calcium responses in HEK293 cells expressing hTRPV1. Molecular modeling revealed a low energy cluster of U-shaped unsaturated NAE conformers, sharing several pharmacophoric elements with capsaicin. Furthermore, one of the two major low energy conformational families of anandamide also overlaps with the cannabinoid CB1 receptor ligand HU210, which is in line with anandamide being a dual activator of TRPV1 and the cannabinoid CB1 receptor. This study shows that several endogenous non-cannabinoid NAEs, many of which are more abundant than anandamide in rat tissues, activate TRPV1 and thus may play a role as endogenous TRPV1 modulators.

Novel natural vanilloid receptor agonists: new therapeutic targets for drug development

The discovery that compounds lacking a recognizable vanillyl-like motif might act as vanilloids has given new impetus to a search for novel vanilloid receptor agonists and antagonists in compound libraries. The availability of cell lines transfected with a cloned human vanilloid receptor will further expedite this search. In this article, the pharmacological properties of unsaturated dialdehydes and triprenyl phenols that represent two newly discovered chemical classes of vanilloids will be discussed. The existence of vanilloid receptors in several brain nuclei as well as in non-neuronal tissues predicts novel, innovative therapeutic indications for vanilloids. However, these findings also suggest that vanilloids might cause side-effects. An exploration of the uses of unsaturated dialdehydes in indigenous medicine might help identify new therapeutic targets for vanilloids and avoid unwanted actions.

Assays of the biological activities of guaiane sesquiterpenoids isolated from the fruit bodies of edible lactarius species

Three sesquiterpenoids that are found in the edible mushrooms Lactarius deliciosus, L. deterrimus and L. sanguifluus, have been assayed for biological activity. The compounds tested were a stearic acid ester of a sesquiterpene (I) and a sesquiterpene aldehyde (lactaroviolin, II) and alcohol (deterrol, III). The assays used were for mutagenic activity in the Ames Salmonella assay, for antimicrobial activity against bacterial fungi and algae, for cytotoxicity against Ehrlich ascitic tumour cells and L 1210 cells, and for phytotoxic activity against Lepidium sativum and Seteria italica. All three compounds showed weak mutagenic activity in the Ames assay. Two compounds (II and III) were found to have moderate cytotoxic activity and one (III) exhibited weak antibacterial activity. No compound revealed phytotoxic, algicidal or antifungal activity.

Furanocoumarins with affinity to brain benzodiazepine receptors in vitro.

Eight furanocoumarins were isolated from a methanol extract of dried roots of Angelica dahurica. One of these, phellopterin, strongly (IC50 = 0.36 microM) inhibits the binding of [3H]diazepam to central nervous system benzodiazepine receptors in vitro, while the others, despite their structural similarities with phellopterin, are considerably less active.

Characterisation of the differential interference effects of two boreal dwarf shrub species

Abstract Our purpose was to characterize the relative competitive and phytotoxic potential of two closely related dwarf-shrub species, Empetrum nigrum and E. hermaphroditum, which form clones in a mosaic pattern in post-fire successions of the boreal forest of northern Sweden. We determined morphological and growth parameters of both species, performed bioassays and chemical analysis, and established field experiments to explore possible differing interference effects on trees by the two species. Both Empetrum species had very similar morphological and growth characteristics. E. hermaphroditum exerted considerably greater negative effects than E. nigrum against Pinus sylvestris and Populus tremula seed and seedlings. These negative effects were related to the different substitution of a bibenzyl in the two species. The effect on seed germination of the two bibenzyls isolated from E. nigrum and E. hermaphroditum was compared with that of other simple phenolics; the latter were found to be inactive, indicating a specific phytotoxic component in the bibenzyls. P. sylvestris seeds planted into clones of both species in the field revealed that E. hermaphroditum had much stronger inhibitory effects than did E. nigrum and the addition of activated carbon partially reversed these effects. Seed germination, biomass and survival of P. sylvestris after four seasons were significantly lower in E. hermaphroditum- than in E. nigrum-dominated plots. We conclude that while both Empetrum species are superficially very similar morphologically, they have vastly different effects on tree seed germination, seedling establishment and growth. These effects appear to be due, at least in part, to the different chemical profile of the two species.

Metabolism of the polycyclic aromatic hydrocarbon pyrene by Aspergillus niger SK 9317

The metabolism of pyrene, a polycyclic aromatic hydrocarbon consisting of four rings, by Aspergillus niger SK 9317 was investigated. The metabolites formed were isolated and identified as 1-hydroxypyrene, 1,6- and 1,8-pyrenequinone, 1,6- and 1,8-dihydroxypyrene, 1-pyrenyl sulphate and 1-hydroxy-8-pyrenyl sulphate. This is the first report of 1-hydroxy-8-pyrenyl as a metabolite in the microbial metabolism of pyrene. The results suggest that A. niger metabolizes pyrene by cytochrome P-450 monooxygenase enzyme systems.

Galiellalactone Inhibits Stem Cell-Like ALDH-Positive Prostate Cancer Cells

Galiellalactone is a potent and specific inhibitor of STAT3 signaling which has been shown to possess growth inhibitory effects on prostate cancer cells expressing active STAT3. In this study we aimed to investigate the effect of galiellalactone on prostate cancer stem cell-like cells. We explored the expression of aldehyde dehydrogenase (ALDH) as a marker for cancer stem cell-like cells in different human prostate cancer cell lines and the effects of galiellalactone on ALDH expressing (ALDH+) prostate cancer cells. ALDH+ subpopulations were detected and isolated from the human prostate cancer cell lines DU145 and long-term IL-6 stimulated LNCaP cells using ALDEFLUOR® assay and flow cytometry. In contrast to ALDH− cells, ALDH+ prostate cancer cells showed cancer stem cell-like characteristics such as increased self-renewing and colony forming capacity and tumorigenicity. In addition, ALDH+ cells showed an increased expression of putative prostate cancer stem cell markers (CD44 and integrin α2β1). Furthermore, ALDH+ cells expressed phosphorylated STAT3. Galiellalactone treatment decreased the proportion of ALDH+ prostate cancer cells and induced apoptosis of ALDH+ cells. The gene expression of ALDH1A1 was downregulated in vivo in galiellalactone treated DU145 xenografts. These findings emphasize that targeting the STAT3 pathway in prostate cancer cells, including prostate cancer stem cell-like cells, is a promising therapeutic approach and that galiellalactone is an interesting compound for the development of future prostate cancer drugs.

Dialdehyde sesquiterpenes and other terpenoids as vanilloids

Selected naturally occurring unsaturated dialdehyde sesquiterpenes and related bioactive terpenoids were assayed for vanilloid-like activity. Out of the 25 compounds tested, eight inhibited completely the specific binding of [3H]resiniferatoxin by rat spinal cord membranes: binding affinities ranged from 0.6 microM for cinnamodial to 19.0 microM for hebelomic acid F. These values were comparable to the binding affinity of capsaicin (2.7 microM). With the exception of four ligands, compounds that inhibited resiniferatoxin binding to rat spinal cord membranes were also pungent on the human tongue where they showed cross-tachyphylaxis with capsaicin. As expected from their reactive nature, these compounds possess additional sites of action, as reflected in the complex behavior of the stimulation of calcium influx by cinnamodial and cinnamosmolide at high concentrations. This observation might explain the unexpectedly weak membrane depolarization by cinnamodial compared to capsaicin. We conclude that a range of sesquiterpene dialdehydes and related terpenoids, both pungent and non-pungent, may function as vanilloids. These compounds may represent a new chemical lead for the development of vanilloid drugs, structurally unrelated to either capsaicin or resiniferatoxin.

The stimulation of capsaicin-sensitive neurones in a vanilloid receptor-mediated fashion by pungent terpenoids possessing an unsaturated 1,4-dialdehyde moiety.

1 The irritant fungal terpenoid isovelleral caused protective eye‐wiping movements in the rat upon intraocular instillation and showed cross‐tachyphylaxis with capsaicin, the pungent principle in hot pepper. 2 Isovelleral induced a dose‐dependent calcium uptake by rat dorsal root ganglion neurones cultured in vitro with an EC50 of 95 nM, which was fully inhibited by the competitive vanilloid receptor antagonist capsazepine. 3 Isovelleral inhibited specific binding of [3H]‐resiniferatoxin (RTX), an ultrapotent capsaicin analogue, to rat trigeminal ganglion or spinal cord preparations with an IC50 of 5.2 μm; in experiments in which the concentration of [3H]‐RTX was varied, isovelleral changed both the apparent affinity (from 16 pM to 37 pM) and the co‐operativity index (from 2.1 to 1.5), but not the Bmax. 4 The affinity of isovelleral for inducing calcium uptake or inhibiting RTX binding was in very good agreement with the threshold dose (2.2. nmol) at which it provoked pungency on the human tongue. 5 For a series of 14 terpenoids with an unsaturated 1,4‐dialdehyde, a good correlation was found between pungency on the human tongue and affinity for vanilloid receptors on the rat spinal cord. 6 The results suggest that isovelleral‐like compounds produce their irritant effect by interacting with vanilloid receptors on capsaicin‐sensitive sensory neurones. Since these pungent diterpenes are structurally distinct from the known classes of vanilloids, these data provide new insights into structure‐activity relations and may afford new opportunities for the development of drugs targeting capsaicin‐sensitive pathways.