A long line of experimental evidence indicates that endogenous cannabinoid mechanisms play important roles in nociceptive information processing in various areas of the nervous system including the spinal cord. Although it is extensively documented that the cannabinoid-1 receptor (CB(1)-R) is strongly expressed in the superficial spinal dorsal horn, its cellular distribution is poorly defined, hampering our interpretation of the effect of cannabinoids on pain processing spinal neural circuits. Thus, we investigated the cellular distribution of CB(1)-Rs in laminae I and II of the rodent spinal dorsal horn with immunocytochemical methods. Axonal varicosities revealed a strong immunoreactivity for CB(1)-R, but no CB(1)-R expression was observed on dendrites and perikarya of neurons. Investigating the co-localization of CB(1)-R with markers of peptidergic and non-peptidergic primary afferents, and axon terminals of putative glutamatergic and GABAergic spinal neurons we found that nearly half of the peptidergic (immunoreactive for calcitonin gene-related peptide) and more than 20% of the non-peptidergic (binding isolectin B4) nociceptive primary afferents, more than one-third and approximately 20% of the axon terminals of putative glutamatergic (immunoreactive for vesicular glutamate transporter 2) and GABAergic (immunoreactive for glutamic acid decarboxylase; GAD65 and/or GAD67) spinal interneurons, respectively, were positively stained for CB(1)-R. In addition to axon terminals, almost half of the astrocytic (immunoreactive for glial fibrillary acidic protein) and nearly 80% of microglial (immunoreactive for CD11b) profiles were also immunolabeled for CB(1)-R. The findings suggest that the activity-dependent release of endogenous cannabinoids activates a complex signaling mechanism in pain processing spinal neural circuits into which both neurons and glial cells may contribute.
Accumulating evidence supports the role of astrocytes in endocannabinoid mediated modulation of neural activity. It has been reported that some astrocytes express the cannabinoid type 1 receptor (CB1-R), the activation of which is leading to Ca2+ mobilization from internal stores and a consecutive release of glutamate. It has also been documented that astrocytes have the potential to produce the endocannabinoid 2-arachidonoylglycerol, one of the best known CB1-R agonist. However, no relationship between CB1-R activation and 2-arachidonoylglycerol production has ever been demonstrated. Here we show that rat spinal astrocytes co-express CB1-Rs and the 2-arachidonoylglycerol synthesizing enzyme, diacylglycerol lipase-alpha in close vicinity to each other. We also demonstrate that activation of CB1-Rs induces a substantial elevation of intracellular Ca2+ concentration in astrocytes. Finally, we provide evidence that the evoked Ca2+ transients lead to the production of 2-arachidonoylglycerol in cultured astrocytes. The results provide evidence for a novel cannabinoid induced endocannabinoid release mechanism in astrocytes which broadens the bidirectional signaling repertoire between astrocytes and neurons.
Dendritic cells (DCs) respond to changes in their lipid environment by altering gene expression and immunophenotype. Some of these alterations are mediated via the nuclear receptor superfamily. However, little is known about the contribution of liver X receptor (LXR) to DC biology. In this study, we present a systematic analysis of LXR, activated by synthetic ligands or naturally occurring oxysterols in developing human monocyte-derived DCs. We found that LXRs are present and can be activated throughout DC differentiation in monocyte- and blood-derived DCs. Administration of LXR-specific natural or synthetic activators induced target gene expression accompanied by increased expression of DC maturation markers, such as CD80 and CD86. In mature DCs, LXR activation augmented the production of inflammatory cytokines IL-12, TNF-α, IL-6, and IL-8 and resulted in an increased capacity to activate CD4+ T cell proliferation upon ligation with TLR4 or TLR3 ligands. These effects appear to be underpinned by prolonged NF-κB signaling. Supporting such an inflammatory role, we found that LXR positive DCs are present in reactive lymph nodes in vivo. We propose that activation of LXR represents a novel lipid-signaling paradigm that alters the inflammatory response of human DCs.
Several new therapeutic options emerged recently to treat metastatic melanoma; however, the high frequency of intrinsic and acquired resistance among patients shows a need for new therapeutic options. Previously, we identified the plasma membrane Ca2+ ATPase 4b (PMCA4b) as a metastasis suppressor in BRAF-mutant melanomas and found that mutant BRAF inhibition increased the expression of the pump, which then inhibited the migratory and metastatic capability of the cells. Earlier it was also demonstrated that histone deacetylase inhibitors (HDACis) upregulated PMCA4b expression in gastric, colon, and breast cancer cells. In this study, we treated one BRAF wild-type and two BRAF-mutant melanoma cell lines with the HDACis, SAHA and valproic acid, either alone, or in combination with the BRAF inhibitor, vemurafenib. We found that HDACi treatment strongly increased the expression of PMCA4b in all cell lines irrespective of their BRAF mutational status, and this effect was independent of ERK activity. Furthermore, HDAC inhibition also enhanced the abundance of the housekeeping isoform PMCA1. Combination of HDACis with vemurafenib, however, did not have any additive effects on either PMCA isoform. We demonstrated that the HDACi-induced increase in PMCA abundance was coupled to an enhanced [Ca2+]i clearance rate and also strongly inhibited both the random and directional movements of A375 cells. The primary role of PMCA4b in these characteristic changes was demonstrated by treatment with the PMCA4-specific inhibitor, caloxin 1c2, which was able to restore the slower Ca2+ clearance rate and higher motility of the cells. While HDAC treatment inhibited cell motility, it decreased only modestly the ratio of proliferative cells and cell viability. Our results show that in melanoma cells the expression of both PMCA4b and PMCA1 is under epigenetic control and the elevation of PMCA4b expression either by HDACi treatment or by the decreased activation of the BRAF-MEK-ERK pathway can inhibit the migratory capacity of the highly motile A375 cells.
We hypothesized that increasing chick plasma testosterone concentrations, transmitted from the mothers via their eggs, enhances survival of their offspring and that the fitness of the young, depending on the maternal hormones, is influenced by parental quality. To test our hypotheses we distinguished the broods of white storks Ciconia ciconia L. where chicks died and those where all chicks survived. We analysed the plasma testosterone concentrations in the chicks, the ability of the chicks to be first to receive food and the mass of chicks before fledging in relation to their hatching order and recorded the body mass of parents and food mass delivered by them. Female storks used the asymmetries in testosterone concentrations within a brood to control brood size and adjusted the number of young hatched to match the parental ability to rear offspring. Females of poor condition altered the testosterone concentrations to produce large differences between the chicks: The first‐hatched chicks, which had high plasma testosterone levels, responded faster to the feeding parent and received more food than did their younger siblings. One or two later‐hatched chicks, which had lower testosterone levels, died in these broods. Females in good condition produced small differences in testosterone concentrations between the chicks and all chicks survived in their brood. Chicks that were raised by the females of poor condition in reduced broods were heavier than chicks that were raised by females of good condition in broods where all chicks survived. We suggest that the control of brood size by testosterone concentration, transmitted by the mother to the chicks, is a hormonal means of condition‐dependent reproductive strategy in the white stork.
The pedunculopontine nucleus (PPN) is known as the cholinergic part of the reticular activating system (RAS) and it plays an important role in transitions of slow-wave sleep to REM sleep and wakefulness. Although both exogenous and endocannabinoids affect sleep, the mechanism of endocannabinoid neuromodulation has not been characterized at cellular level in the PPN. In this paper, we demonstrate that both neurons and glial cells from the PPN respond to cannabinoid type 1 (CB1) receptor agonists. The neuronal response can be depolarization or hyperpolarization, while astrocytes exhibit more frequent calcium waves. All these effects are absent in CB1 gene-deficient mice. Blockade of the fast synaptic neurotransmission or neuronal action potential firing does not change the effect on the neuronal membrane potential significantly, while inhibition of astrocytic calcium waves by thapsigargin diminishes the response. Inhibition of group I metabotropic glutamate receptors (mGluRs) abolishes hyperpolarization, whereas blockade of group II mGluRs prevents depolarization. Initially active neurons and glial cells display weaker responses partially due to the increased endocannabinoid tone in their environment. Taken together, we propose that cannabinoid receptor stimulation modulates PPN neuronal activity in the following manner: active neurons may elicit calcium waves in astrocytes via endogenous CB1 receptor agonists. Astrocytes in turn release glutamate that activates different metabotropic glutamate receptors of neurons and modulate PPN neuronal activity.
It is generally accepted that the endocannabinoid system plays important roles in spinal pain processing. Although it is documented that cannabinoid-1 receptors are strongly expressed in the superficial spinal dorsal horn, the cellular distribution of enzymes that can synthesize endocannabinoid ligands is less well studied. Thus, using immunocytochemical methods at the light and electron microscopic levels, we investigated the distribution of diacylglycerol lipase-alpha (DGLα) and N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), enzymes synthesizing the endocannabinoid ligands, 2-arachidonoylglycerol (2-AG) and anandamide, respectively. Positive labeling was revealed only occasionally in axon terminals, but dendrites displayed strong immunoreactivity for both enzymes. However, the dendritic localization of DGLα and NAPE-PLD showed a remarkably different distribution. DGLα immunolabeling in dentrites was always revealed at membrane compartments in close vicinity to synapses. In contrast to this, dendritic NAPE-PLD labeling was never observed in association with synaptic contacts. In addition to dendrites, a substantial proportion of astrocytic (immunoreactive for GFAP) and microglial (immunoreactive for CD11b) profiles were also immunolabeled for both DGLα and NAPE-PLD. Glial processes immunostained for DGLα were frequently found near to synapses in which the postsynaptic dendrite was immunoreactive for DGLα, whereas NAPE-PLD immunoreactivity on glial profiles at the vicinity of synapses was only occasionally observed. Our results suggest that both neurons and glial cells can synthesize and release 2-AG and anandamide in the superficial spinal dorsal horn. 2-AG can primarily be released by postsynaptic dendrites and glial processes adjacent to synapses, whereas anandamide can predominantly be released from non-synaptic dendritic and glial compartments.
Endocannabinoids are pleiotropic lipid messengers that play pro-homeostatic role in cellular physiology by strongly influencing intracellular Ca 2+ concentration through the activation of cannabinoid receptors. One of the best-known endocannabinoid '2-AG' is chemically unstable in aqueous solutions, thus its molecular rearrangement, resulting in the formation of 1-AG, may influence 2-AG-mediated signaling depending on the relative concentration and potency of the two isomers. To predict whether this molecular rearrangement may be relevant in physiological processes and in experiments with 2-AG, here we studied if isomerization of 2-AG has an impact on 2-AG-induced, CB1-mediated Ca 2+ signaling in vitro. We found that the isomerization-dependent drop in effective 2-AG concentration caused only a weak diminution of Ca 2+ signaling in CB1 transfected COS7 cells. We also found that 1-AG induces Ca 2+ transients through the activation of CB1, but its working concentration is threefold higher than that of 2-AG. Decreasing the concentration of 2-AG in parallel to the prevention of 1-AG formation by rapid preparation of 2-AG solutions, caused a significant diminution of Ca 2+ signals. However, various mixtures of the two isomers in a fix total concentration -mimicking the process of isomerization over time -attenuated the drop in 2-AG potency, resulting in a minor decrease in CB1 mediated Ca 2+ transients. Our results indicate that release of 2-AG into aqueous medium is accompanied by its isomerization, resulting in a drop of 2-AG concentration and simultaneous formation of the similarly bioactive isomer 1-AG. Thus, the relative concentration of the two isomers with different potency and efficacy may influence CB1 activation and the consequent biological responses. In addition, our results suggest that 1-AG may play role in stabilizing the strength of cannabinoid signal in case of prolonged 2-AG dependent cannabinoid mechanisms.
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