The thermo- and pain-sensitive Transient Receptor Potential Melastatin 3 and 8 (TRPM3 and TRPM8) ion channels are functionally associated in the lipid rafts of the plasma membrane. We have already described that cholesterol and sphingomyelin depletion, or inhibition of sphingolipid biosynthesis decreased the TRPM8 but not the TRPM3 channel opening on cultured sensory neurons. We aimed to test the effects of lipid raft disruptors on channel activation on TRPM3- and TRPM8-expressing HEK293T cells in vitro, as well as their potential analgesic actions in TRPM3 and TRPM8 channel activation involving acute pain models in mice. CHO cell viability was examined after lipid raft disruptor treatments and their effects on channel activation on channel expressing HEK293T cells by measurement of cytoplasmic Ca2+ concentration were monitored. The effects of treatments were investigated in Pregnenolone-Sulphate-CIM-0216-evoked and icilin-induced acute nocifensive pain models in mice. Cholesterol depletion decreased CHO cell viability. Sphingomyelinase and methyl-beta-cyclodextrin reduced the duration of icilin-evoked nocifensive behavior, while lipid raft disruptors did not inhibit the activity of recombinant TRPM3 and TRPM8. We conclude that depletion of sphingomyelin or cholesterol from rafts can modulate the function of native TRPM8 receptors. Furthermore, sphingolipid cleavage provided superiority over cholesterol depletion, and this method can open novel possibilities in the management of different pain conditions.
- MeSH
- analgetika farmakologie terapeutické užití MeSH
- beta-cyklodextriny * farmakologie MeSH
- bolest chemicky indukované farmakoterapie metabolismus MeSH
- CHO buňky MeSH
- cholesterol metabolismus MeSH
- Cricetulus MeSH
- HEK293 buňky MeSH
- kationtové kanály TRPM * metabolismus genetika MeSH
- lidé MeSH
- membránové mikrodomény metabolismus účinky léků MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- pregnenolon farmakologie MeSH
- pyrimidinony farmakologie MeSH
- sfingomyelinfosfodiesterasa * metabolismus farmakologie MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Sphingomyelinase D (SMase D), the main toxic component of Loxosceles venom, has a well-documented role on dermonecrotic lesion triggered by envenomation with these species; however, the intracellular mechanisms involved in this event are still poorly known. Through differential transcriptomics of human keratinocytes treated with L. laeta or L. intermedia SMases D, we identified 323 DEGs, common to both treatments, as well as upregulation of molecules involved in the IL-1 and ErbB signaling. Since these pathways are related to inflammation and wound healing, respectively, we investigated the relative expression of some molecules related to these pathways by RT-qPCR and observed different expression profiles over time. Although, after 24 h of treatment, both SMases D induced similar modulation of these pathways in keratinocytes, L. intermedia SMase D induced earlier modulation compared to L. laeta SMase D treatment. Positive expression correlations of the molecules involved in the IL-1 signaling were also observed after SMases D treatment, confirming their inflammatory action. In addition, we detected higher relative expression of the inhibitor of the ErbB signaling pathway, ERRFI1, and positive correlations between this molecule and pro-inflammatory mediators after SMases D treatment. Thus, herein, we describe the cell pathways related to the exacerbation of inflammation and to the failure of the wound healing, highlighting the contribution of the IL-1 signaling pathway and the ERRFI1 for the development of cutaneous loxoscelism.
- MeSH
- erbB receptory metabolismus MeSH
- fosfodiesterasy toxicita MeSH
- interleukin-1 metabolismus MeSH
- kousnutí pavoukem patologie MeSH
- lidé MeSH
- pavoučí jedy * toxicita MeSH
- pavouci chemie metabolismus MeSH
- sfingomyelinfosfodiesterasa * metabolismus MeSH
- signální transdukce MeSH
- zánět MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Neutral sphingomyelinase 2 (nSMase2) catalyzes the cleavage of sphingomyelin to phosphorylcholine and ceramide, an essential step in the formation and release of exosomes from cells that is critical for intracellular communication. Chronic increase of brain nSMase2 activity and related exosome release have been implicated in various pathological processes, including the progression of Alzheimer's disease (AD), making nSMase2 a viable therapeutic target. Recently, we identified phenyl (R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate 1 (PDDC), the first nSMase2 inhibitor that possesses both favorable pharmacodynamics and pharmacokinetic (PK) parameters, including substantial oral bioavailability, brain penetration, and significant inhibition of exosome release from the brain in vivo. Herein we demonstrate the efficacy of 1 (PDDC) in a mouse model of AD and detail extensive structure-activity relationship (SAR) studies with 70 analogues, unveiling several that exert similar or higher activity against nSMase2 with favorable pharmacokinetic properties.
- MeSH
- Alzheimerova nemoc farmakoterapie patologie MeSH
- exozómy metabolismus MeSH
- inhibitory enzymů chemie metabolismus farmakologie terapeutické užití MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- mozek metabolismus MeSH
- myši transgenní MeSH
- myši MeSH
- pyridaziny chemie metabolismus terapeutické užití MeSH
- sfingomyelinfosfodiesterasa antagonisté a inhibitory metabolismus farmakologie MeSH
- tělesná hmotnost účinky léků MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Acid sphingomyelinase deficiency (ASMD), due to mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene, is divided into infantile neurovisceral ASMD (Niemann-Pick type A), chronic neurovisceral ASMD (intermediate form, Niemann-Pick type A/B) and chronic visceral ASMD (Niemann-Pick type B). We conducted a long-term observational, single-center study including 16 patients with chronic visceral ASMD. RESULTS: 12 patients were diagnosed in childhood and 4 others in adulthood, the oldest at the age of 50. The mean time of follow-up was approximately 10 years (range: 6 months - 36 years). Splenomegaly was noted in all patients at diagnosis. Hepatomegaly was observed in 88% of patients. Moderately elevated (several-fold above the upper limit of normal values) serum transaminases were noted in 38% of patients. Cherry-red spots were found in five Gypsy children from one family and also in one adult Polish patient, a heterozygote for p.delR610 mutation. Dyslipidemia was noted in 50% of patients. Interstitial lung disease was diagnosed in 44% of patients. Plasmatic lysosphingomyelin (SPC) was elevated in all the patients except one with p.V36A homozygosity and a very mild phenotype also presenting with elevated plasmatic SPC-509 but normal chitotriosidase activity. The most common variant of SMPD1 gene was p.G166R. We found a previously unreported variant in exon 2 (c.491G > T, p.G164 V) in one patient. CONCLUSIONS: Chronic visceral ASMD could constitute a slowly progressing disease with a relatively good outcome. The combined measurement of lysosphingomyelin (SPC) and lysospingomyelin-509 (SPC-509) is an essential method for the assessment of ASMD course.
- MeSH
- dítě MeSH
- dospělí MeSH
- exony genetika MeSH
- hexosaminidasy genetika metabolismus MeSH
- homozygot MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mutace genetika MeSH
- následné studie MeSH
- Niemannova-Pickova nemoc typu A genetika metabolismus MeSH
- předškolní dítě MeSH
- sfingomyelinfosfodiesterasa genetika metabolismus MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Polsko MeSH
SCOPE: The major alimentary sources for the plasma membrane lipid sphingomyelin (SM) are dairy products, eggs, and meat. We recently reported that the SM metabolite ceramide induces cathepsin D mediated apoptosis in murine intestinal epithelial cells (IECs) and increases inflammation in acute colitis. We investigated the impact of SM and phosphatidylcholine on apoptosis in human IECs and point out BH3-interacting death agonist (BID) as link between cathepsin D and apoptosis. METHODS AND RESULTS: HT-29 and isolated human IECs were stimulated with SM or phosphatidylcholine. SM treatment resulted in increased apoptosis. Phosphatidylcholine showed contrary effects. Western revealed higher amounts of cathepsin D and BID activation upon lipid stimulation. Western blotting revealed BID activation through SM in both an induced and a spontaneous mouse model of colitis. CONCLUSION: Dietary phospholipids may induce or abolish apoptosis in IECs and seem to play a role in the pathogenesis of inflammatory bowel diseases. This nutritional factor might be considered when evaluating the pathogenesis of inflammatory bowel diseases. Effects of SMase- and SM treatment on inflammation in dextran sulfate sodium induced animal models of colitis and in vitro experiments are discussed as controversial. Variable sources of SM, feeding techniques, and mouse strains might play a role.
- MeSH
- adhezní spoje MeSH
- apoptóza * účinky léků MeSH
- buněčná smrt účinky léků MeSH
- buňky HT-29 účinky léků MeSH
- ceramidy metabolismus MeSH
- epitelové buňky * patologie účinky léků MeSH
- fosfatidylcholiny farmakologie metabolismus MeSH
- kathepsin D metabolismus MeSH
- kolitida metabolismus patologie MeSH
- kultivované buňky MeSH
- lidé MeSH
- liposomy farmakologie MeSH
- myši inbrední C57BL MeSH
- potravní doplňky MeSH
- protein Bid metabolismus MeSH
- sfingomyelinfosfodiesterasa metabolismus MeSH
- sfingomyeliny farmakologie metabolismus MeSH
- střeva * cytologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
Tumour necrosis factor (TNF) related apoptosis inducing ligand (TRAIL), a membrane-bound ligand from the TNF family, has attracted significant attention due to its rather specific and effective ability to induce apoptotic death in various types of cancer cells via binding to and activating its pro-apoptotic death receptors. However, a significant number of primary cancer cells often develop resistance to TRAIL treatment, and the signalling platform behind this phenomenon is not fully understood. Upon blocking endosomal acidification by the vacuolar ATPase (V-ATPase) inhibitors bafilomycin A1 (BafA1) or concanamycin A, we observed a significantly reduced initial sensitivity of several, mainly colorectal, tumour cell lines to TRAIL-induced apoptosis. In cells pretreated with these inhibitors, the TRAIL-induced processing of caspase-8 and the aggregation and trafficking of the TRAIL receptor complexes were temporarily attenuated. Nuclear factor κB or mitogen activated protein/stress kinase signalling from the activated TRAIL receptors remained unchanged, and neither possible lysosomal permeabilization nor acid sphingomyelinase was involved in this process. The cell surface expression of TRAIL receptors and their TRAIL-induced internalization were not affected by V-ATPase inhibitors. The inhibitory effect of BafA1, however, was blunted by knockdown of the caspase-8 inhibitor cFLIP. Altogether, the data obtained provide the first evidence that endosomal acidification could represent an important regulatory node in the proximal part of TRAIL-induced pro-apoptotic signalling.
- MeSH
- aktivace enzymů MeSH
- antitumorózní látky farmakologie MeSH
- apoptóza MeSH
- down regulace MeSH
- endozomy metabolismus MeSH
- FLIP (buněčný) metabolismus MeSH
- kaspasa 8 metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- makrolidy farmakologie MeSH
- nádorové buněčné linie MeSH
- protein TRAIL farmakologie MeSH
- sfingolipidy fyziologie MeSH
- sfingomyelinfosfodiesterasa metabolismus MeSH
- signální adaptorové proteiny receptorové domény smrti metabolismus MeSH
- signální transdukce účinky léků MeSH
- TRAIL receptory metabolismus MeSH
- transport proteinů MeSH
- vakuolární protonové ATPasy antagonisté a inhibitory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- dítě MeSH
- dospělí MeSH
- fenotyp MeSH
- fibroblasty cytologie fyziologie MeSH
- finanční podpora výzkumu jako téma MeSH
- genotyp MeSH
- hydrolýza MeSH
- játra metabolismus MeSH
- kojenec MeSH
- kůže cytologie MeSH
- lidé MeSH
- missense mutace MeSH
- mladiství MeSH
- mutageneze cílená MeSH
- Niemannova-Pickova nemoc genetika mortalita patofyziologie MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- posunová mutace MeSH
- předškolní dítě MeSH
- prevalence MeSH
- sfingomyelinfosfodiesterasa genetika metabolismus MeSH
- stupeň závažnosti nemoci MeSH
- transformované buněčné linie MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Geografické názvy
- Česká republika MeSH
- Slovenská republika MeSH
