610 Medizin und Gesundheit
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Alkylglycerol monooxygenase (AGMO) is a tetrahydrobiopterin (BH4)-dependent enzyme with major expression in the liver and white adipose tissue that cleaves alkyl ether glycerolipids. The present study describes the disclosure and biological characterization of a candidate compound (Cp6), which inhibits AGMO with an IC50 of 30–100 µM and 5–20-fold preference of AGMO relative to other BH4-dependent enzymes, i.e., phenylalanine-hydroxylase and nitric oxide synthase. The viability and metabolic activity of mouse 3T3-L1 fibroblasts, HepG2 human hepatocytes and mouse RAW264.7 macrophages were not affected up to 10-fold of the IC50. However, Cp6 reversibly inhibited the differentiation of 3T3-L1 cells towards adipocytes, in which AGMO expression was upregulated upon differentiation. Cp6 reduced the accumulation of lipid droplets in adipocytes upon differentiation and in HepG2 cells exposed to free fatty acids. Cp6 also inhibited IL-4-driven differentiation of RAW264.7 macrophages towards M2-like macrophages, which serve as adipocyte progenitors in adipose tissue. Collectively, the data suggest that pharmacologic AGMO inhibition may affect lipid storage.
Alcoholism is one of the leading and increasingly prevalent reasons of liver associated morbidity and mortality worldwide. Alcoholic hepatitis (AH) constitutes a severe disease with currently no satisfying treatment options. Lipoxin A4 (LXA4), a 15-lipoxygenase (ALOX15)-dependent lipid mediator involved in resolution of inflammation, showed promising pre-clinical results in the therapy of several inflammatory diseases. Since inflammation is a main driver of disease progression in alcoholic hepatitis, we investigated the impact of endogenous ALOX15-dependent lipid mediators and exogenously applied LXA4 on AH development. A mouse model for alcoholic steatohepatitis (NIAAA model) was tested in Alox12/15+/+ and Alox12/15−/− mice, with or without supplementation of LXA4. Absence of Alox12/15 aggravated parameters of liver disease, increased hepatic immune cell infiltration in AH, and elevated systemic neutrophils as a marker for systemic inflammation. Interestingly, i.p. injections of LXA4 significantly lowered transaminase levels only in Alox12/15−/− mice and reduced hepatic immune cell infiltration as well as systemic inflammatory cytokine expression in both genotypes, even though steatosis progressed. Thus, while LXA4 injection attenuated selected parameters of disease progression in Alox12/15−/− mice, its beneficial impact on immunity was also apparent in Alox12/15+/+ mice. In conclusion, pro-resolving lipid mediators may be beneficial to reduce inflammation in alcoholic hepatitis.
Background: Caloric restriction is associated with broad therapeutic potential in various diseases and an increase in health and life span. In this study, we assessed the impact of caloric restriction on acute and inflammatory nociception in mice, which were either fed ad libitum or subjected to caloric restriction with 80% of the daily average for two weeks.
Results: The behavioral tests revealed that inflammatory nociception in the formalin test and in zymosan-induced mechanical hypersensitivity were significantly decreased when mice underwent caloric restriction. As potential mediators of the diet-induced antinociception, we assessed genes typically induced by inflammatory stimuli, AMP-activated kinase, and the endocannabinoid system which have all already been associated with nociceptive responses. Zymosan-induced inflammatory markers such as COX-2, TNFα, IL-1β, and c-fos in the spinal cord were not altered by caloric restriction. In contrast, AMPKα2 knock-out mice showed significant differences in comparison to C57BL/6 mice and their respective wild type littermates by missing the antinociceptive effects after caloric restriction. Endocannabinoid levels of anandamide and 2-arachidonyl glyceroldetermined in serum by LC-MS/MS were not affected by either caloric restriction alone or in combination with zymosan treatment. However, cannabinoid receptor type 1 expression in the spinal cord, which was not altered by caloric restriction in control mice, was significantly increased after caloric restriction in zymosan-induced paw inflammation. Since increased cannabinoid receptor type 1 signaling might influence AMP-activated kinase activity, we analyzed effects of anandamide on AMP-activated kinase in cell culture and observed a significant activation of AMP-activated kinase. Thus, endocannabionoid-induced AMP-activated kinase activation might be involved in antinociceptive effects after caloric restriction.
Conclusion: Our data suggest that caloric restriction has an impact on inflammatory nociception which might involve AMP-activated kinase activation and an increased activity of the endogenous endocannabinoid system by caloric restriction-induced cannabinoid receptor type 1 upregulation.
Macrophages respond to the Th2 cytokine IL-4 with elevated expression of arachidonate 15-lipoxygenase (ALOX15). Although IL-4 signaling elicits anti-inflammatory responses, 15-lipoxygenase may either support or inhibit inflammatory processes in a context-dependent manner. AMP-activated protein kinase (AMPK) is a metabolic sensor/regulator that supports an anti-inflammatory macrophage phenotype. How AMPK activation is linked to IL-4-elicited gene signatures remains unexplored. Using primary human macrophages stimulated with IL-4, we observed elevated ALOX15 mRNA and protein expression, which was attenuated by AMPK activation. AMPK activators, e.g. phenformin and aminoimidazole-4-carboxamide 1-β-d-ribofuranoside inhibited IL-4-evoked activation of STAT3 while leaving activation of STAT6 and induction of typical IL-4-responsive genes intact. In addition, phenformin prevented IL-4-induced association of STAT6 and Lys-9 acetylation of histone H3 at the ALOX15 promoter. Activating AMPK abolished cellular production of 15-lipoxygenase arachidonic acid metabolites in IL-4-stimulated macrophages, which was mimicked by ALOX15 knockdown. Finally, pretreatment of macrophages with IL-4 for 48 h increased the mRNA expression of the proinflammatory cytokines IL-6, IL-12, CXCL9, and CXCL10 induced by subsequent stimulation with lipopolysaccharide. This response was attenuated by inhibition of ALOX15 or activation of AMPK during incubation with IL-4. In conclusion, limiting ALOX15 expression by AMPK may promote an anti-inflammatory phenotype of IL-4-stimulated human macrophages.
FTY720 is a novel immunosuppressive drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. In its phosphorylated form FTY720 is a potent S1P receptor agonist. Recently it was also shown that FTY720 can reduce prostaglandin synthesis through the direct inhibition of the cytosolic phospholipase A2 (cPLA2). Since prostaglandins are important mediators of nociception, we studied the effects of FTY720 in different models of nociception. We found that intraperitoneal administration of FTY720 reduced dose-dependently the nociceptive behaviour of rats in the formalin assay. Although the antinociceptive doses of FTY720 were too low to alter the lymphocyte count, prostanoid concentrations in the plasma were dramatically reduced. Surprisingly, intrathecally administered FTY720 reduced the nociceptive behaviour in the formalin assay without altering spinal prostaglandin synthesis, indicating that additional antinociceptive mechanisms beside the inhibition of prostaglandin synthesis are involved. Accordingly, FTY720 reduced also the nociceptive behaviour in the spared nerve injury model for neuropathic pain which does not depend on prostaglandin synthesis. In this model the antinociceptive effect of FTY720 was similar to gabapentin, a commonly used drug to treat neuropathic pain. Taken together we show for the first time that FTY720 possesses antinociceptive properties and that FTY720 reduces nociceptive behaviour during neuropathic pain.
The low-threshold L-type calcium channel Cav1.3 accelerates the pacemaker rate in the heart, but its functional role for the extended dynamic range of neuronal firing is still unresolved. Here, we show that Cav1.3 calcium channels act as unexpectedly simple, full-range linear amplifiers of firing rates for lateral dopamine substantia nigra (DA SN) neurons in mice. This means that they boost in vitro or in vivo firing frequencies between 2 and 50 hertz by about 30%. Furthermore, we demonstrate that clinically relevant, low nanomolar concentrations of the L-type channel inhibitor isradipine selectively reduce the in vivo firing activity of these nigrostriatal DA SN neurons at therapeutic plasma concentrations. Thus, our study identifies the pacemaker function of neuronal Cav1.3 channels and provides direct evidence that repurposing dihydropyridines such as isradipine is feasible to selectively modulate the in vivo activity of highly vulnerable DA SN subpopulations in Parkinson's disease.
Oral presentation from 4th International Conference of cGMP Generators, Effectors and Therapeutic Implications ; Regensburg, Germany. 19–21 June 2009 Background: An exaggerated pain sensitivity is the dominant feature of inflammatory and neuropathic pain both in the clinical setting and in experimental animal models. It manifests as pain in response to normally innocuous stimuli (allodynia), increased response to noxious stimuli (hyperalgesia) or spontaneous pain, and can persist long after the initial injury is resolved. Research over the last decades has revealed that several signaling pathways in the spinal cord essentially contribute to the pain sensitization. To test the contribution of cGMP produced by NO-sensitive guanylyl cyclase (NO-GC) to pain sensitization, we investigated the localization of NO-GC in the spinal cord and in dorsal root ganglia, and we characterized the nociceptive behavior of mice deficient in NO-GC (GC-KO mice). Results: We show that NO-GC (β1 subunit) is distinctly expressed in neurons of the mouse spinal cord, while its distribution in dorsal root ganglia is restricted to non-neuronal cells. GC-KO mice exhibited a considerably reduced nociceptive behavior in models of inflammatory or neuropathic pain, but their responses to acute pain were not impaired. Moreover, GC-KO mice failed to develop pain sensitization induced by spinal administration of drugs releasing NO. Surprisingly, during spinal nociceptive processing cGMP produced by NO-GC may activate signaling pathways different from cGMP-dependent protein kinase I (cGKI), while cGKI can be activated by natriuretic peptide receptor-B (NPR-B) dependent cGMP production. Conclusion: Taken together, our results provide evidence that NO-GC has a dominant role in the development of exaggerated pain sensitivity during inflammatory and neuropathic pain. Furthermore, beside the NO-mediated cGMP synthesis, cGMP produced by NPR-B contributes to pain sensitization by activation of cGKI.
Blood-pressure-lowering drugs are proposed to foster SARS-CoV-2 infection by pharmacological upregulation of angiotensin-converting enzyme 2 (ACE2), the binding partner of the virus spike (S) protein, located on the surface of the host cells. Conversely, it is postulated that angiotensin–renin system antagonists may prevent lung damage caused by SARS-CoV-2 infection, by reducing angiotensin II levels, which can induce permeability of lung endothelial barrier via its interaction with the AT1 receptor (AT1R). Methods: We have investigated the influence of the ACE inhibitors (lisinopril, captopril) and the AT1 antagonists (telmisartan, olmesartan) on the level of ACE2 mRNA and protein expression as well as their influence on the cytopathic effect of SARS-CoV-2 and on the cell barrier integrity in a Caco-2 cell model. Results: The drugs revealed no effect on ACE2 mRNA and protein expression. ACE inhibitors and AT1R antagonist olmesartan did not influence the infection rate of SARS-CoV-2 and were unable to prevent the SARS-CoV-2-induced cell barrier disturbance. A concentration of 25 µg/mL telmisartan significantly reduced the virus replication rate. Conclusion: ACE inhibitors and AT1R antagonist showed neither beneficial nor detrimental effects on SARS-CoV-2-infection and cell barrier integrity in vitro at pharmacologically relevant concentrations.
Consequences of a human TRPA1 genetic variant on the perception of nociceptive and olfactory stimuli
(2014)
Background: TRPA1 ion channels are involved in nociception and are also excited by pungent odorous substances. Based on reported associations of TRPA1 genetics with increased sensitivity to thermal pain stimuli, we therefore hypothesized that this association also exists for increased olfactory sensitivity.
Methods: Olfactory function and nociception was compared between carriers (n = 38) and non-carriers (n = 43) of TRPA1 variant rs11988795 G.A, a variant known to enhance cold pain perception. Olfactory function was quantified by assessing the odor threshold, odor discrimination and odor identification, and by applying 200-ms pulses of H2S intranasal. Nociception was assessed by measuring pain thresholds to experimental nociceptive stimuli (blunt pressure, electrical stimuli, cold and heat stimuli, and 200-ms intranasal pulses of CO2).
Results: Among the 11 subjects with moderate hyposmia, carriers of the minor A allele (n = 2) were underrepresented (34 carriers among the 70 normosmic subjects; p = 0.049). Moreover, carriers of the A allele discriminated odors significantly better than non-carriers (13.161.5 versus 12.361.6 correct discriminations) and indicated a higher intensity of the H2S stimuli (29.2613.2 versus 21612.8 mm VAS, p = 0.006), which, however, could not be excluded to have involved a trigeminal component during stimulation. Finally, the increased sensitivity to thermal pain could be reproduced.
Conclusions: The findings are in line with a previous association of a human TRPA1 variant with nociceptive parameters and extend the association to the perception of odorants. However, this addresses mainly those stimulants that involve a trigeminal component whereas a pure olfactory effect may remain disputable. Nevertheless, findings suggest that future TRPA1 modulating drugs may modify the perception of odorants.
Consequences of altered eicosanoid patterns for nociceptive processing in mPGES-1-deficient mice
(2007)
Cyclooxygenase-2 (COX-2)-dependent prostaglandin (PG) E2 synthesis in the spinal cord plays a major role in the development of inflammatory hyperalgesia and allodynia. Microsomal PGE2 synthase-1 (mPGES-1) isomerizes COX-2-derived PGH2 to PGE2. Here, we evaluated the effect of mPGES-1-deficiency on the noci-ceptive behavior in various models of nociception that depend on PGE2 synthesis. Surprisingly, in the COX-2-dependent zymosan-evoked hyperalgesia model, the nociceptive behavior was not reduced in mPGES-1-deficient mice despite a marked decrease of the spinal PGE2 synthesis. Similarly, the nociceptive behavior was unaltered in mPGES-1-deficient mice in the formalin test. Importantly, spinal cords and primary spinal cord cells derived from mPGES-1-deficient mice showed a redirection of the PGE2 synthesis to PGD2, PGF2α and 6-keto-PGF1α (stable metabolite of PGI2). Since the latter prostaglandins serve also as mediators of noci-ception they may compensate the loss of PGE2 synthesis in mPGES-1-deficient mice.