FASEB journal : official publication of the Federation of American Societies for Experimental Biology
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Inflammatory pain represents an important unmet clinical need with important socioeconomic implications. Ceramide, a potent proinflammatory sphingolipid, has been shown to elicit mechanical hyperalgesia, but the mechanisms remain largely unknown. We now demonstrate that, in addition to mechanical hyperalgesia, intraplantar injection of ceramide (10 μg) led to the development of thermal hyperalgesia that was dependent on induction of the inducible cyclooxygenase (COX-2) and subsequent increase of prostaglandin E(2) (PGE(2)). ⋯ Our results further revealed that COX-2 induction is regulated by NF-κB and p38 kinase activation, since intraplantar injection of SC-514 (0.1-1 μg) or SB 203580 (1-10 μg), well-characterized inhibitors of NF-κB and p38 kinase activation, respectively, blocked COX-2 induction and increased formation of PGE(2) and thermal hyperalgesia in a dose-dependent manner. Moreover, activation of NF-κB was dependent on upstream activation of p38 MAPK, since SB 203580 (10 μg) blocked p65 phosphorylation, whereas p38 kinase phosphorylation was unaffected by NF-κB inhibition by SC-514 (1 μg). Our findings not only provide mechanistic insight into the signaling pathways engaged by ceramide in the development of hyperalgesia, but also provide a potential pharmacological basis for developing inhibitors targeting the ceramide metabolic-to-COX-2 pathway as novel analgesics.
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Airway surface liquid (ASL) volume depletion and mucus accumulation occur in cystic fibrosis (CF). The ASL comprises a superficial mucus layer (ML) overlying a periciliary fluid layer (PCL) that contacts surface epithelial cells. We measured viscosity of the ML and PCL from the diffusion of FITC-dextran dissolved in the ASL of unperturbed, well-differentiated primary cultures of human bronchial epithelia grown at an air-liquid interface. ⋯ Relative viscosities (with saline=1) were in the range 7-10 in the non-CF ML and PCL, and 25-30 in both ML and PCL in CF, and greatly reduced by amiloride treatment or mucin washout. These data indicate that the CF airway surface epithelium, even without hyperviscous secretions from submucosal glands, produces an intrinsically hyperviscous PCL and ML, which likely contributes to CF lung disease by impairment of mucociliary clearance. Our results challenge the view that the PCL is a relatively watery, nonviscous fluid layer in contact with a more viscous ML, and offer an explanation for CF lung disease in the gland-free lower airways.
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We have recently shown that antibody-induced blockade of C5a, C5a receptors, or IL-17A greatly reduced the harmful outcomes of sepsis. In the current study, normal cardiomyocytes from young (300 g) male Sprague-Dawley rats responded in vitro to C5a (ED(50)=55 nM) with release of IL-6 and TNFα, peaking between 2 to 8 h. Neutralizing antibodies to mouse C5a or IL-17A (ED(50)=40 μg for each, based on improved survival) reduced spontaneous in vitro release of cardiosuppressive cytokines and chemokines in cardiomyocytes obtained from mice with polymicrobial sepsis. ⋯ Cardiomyocytes from septic C5aR(-/-) or C5L2(-/-) mice did not show spontaneous in vitro release of cytokines and chemokines. These data suggest that cardiomyocytes from septic mice release suppressive cytokines in a C5a-, C5aR-, and IL-17A-dependent manner, followed by mediator reactivity with receptors on cardiomyocytes, resulting in defective contractility and relaxation. These data may be relevant to a strategy for the treatment of heart dysfunction developing during sepsis.
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Nowadays, the emerging role of amyloid-β peptide (Aβ) oligomers in Alzheimer's disease (AD) is widely accepted, putting aside the old idea that fibrils are the primary entities responsible for the onset of the disease. Besides, carrying the E4 isoform of apolipoprotein E (apoE) represents the highest risk of developing AD. Nevertheless, the involvement of apoE4 in AD remains confusing. ⋯ ApoE4 would bind and block Aβ in its oligomeric conformation, inhibiting further formation of less toxic fibrillar forms of Aβ. While previous studies mostly correlated E4 with fibrils, our report underlines a link between apoE4 and Aβ oligomers and therefore reconciles apoE4 with the new amyloid cascade hypothesis. Our observations suggest that apoE4 strongly stabilizes Aβ oligomers, the pathological species responsible for Alzheimer's disease.
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In this study, the principles of surface sensing of translation (SUnSET) were used to develop a nonradioactive method for ex vivo and in vivo measurements of protein synthesis (PS). Compared with controls, we first demonstrate excellent agreement between SUnSET and a [(3)H]phenylalanine method when detecting synergist ablation-induced increases in skeletal muscle PS ex vivo. We then show that SUnSET can detect the same synergist ablation-induced increase in PS when used in vivo (IV-SUnSET). ⋯ Furthermore, transfection with Ras homolog enriched in brain (Rheb) revealed that a PKB-independent activation of mammalian target of rapamycin is also sufficient to induce an increase in skeletal muscle PS. Finally, IV-IHC-SUnSET exposed the existence of fiber type-dependent differences in skeletal muscle PS, with PS in type 2B and 2X fibers being significantly lower than that in type 2A fibers within the same muscle. Thus, our nonradioactive method allowed us to accurately visualize and quantify PS under various ex vivo and in vivo conditions and revealed novel insights into the regulation of PS in skeletal muscle.