Neutral Sphingomyelinase Inhibitor Research Articles

Promotion of endocytosis efficiency through an ATP-independent mechanism at rat calyx of Held terminals.

At rat calyx of Held terminals, ATP was required not only for slow endocytosis, but also for rapid phase of compensatory endocytosis. An ATP-independent form of endocytosis was recruited to accelerate membrane retrieval at increased activity and temperature. ATP-independent endocytosis primarily involved retrieval of pre-existing membrane, which depended on Ca2+ and the activity of neutral sphingomyelinase but not clathrin-coated pit maturation. ATP-independent endocytosis represents a non-canonical mechanism that can efficiently retrieve membrane at physiological conditions without competing for the limited ATP at elevated neuronal activity. Neurotransmission relies on membrane endocytosis to maintain vesicle supply and membrane stability. Endocytosis has been generally recognized as a major ATP-dependent function, which efficiently retrieves more membrane at elevated neuronal activity when ATP consumption within nerve terminals increases drastically. This paradox raises the interesting question of whether increased activity recruits ATP-independent mechanism(s) to accelerate endocytosis at the same time as preserving ATP availability for other tasks. To address this issue, we studied ATP requirement in three typical forms of endocytosis at rat calyx of Held terminals by whole-cell membrane capacitance measurements. At room temperature, blocking ATP hydrolysis effectively abolished slow endocytosis and rapid endocytosis but only partially inhibited excess endocytosis following intense stimulation. The ATP-independent endocytosis occurred at calyces from postnatal days 8-15, suggesting its existence before and after hearing onset. This endocytosis was not affected by a reduction of exocytosis using the light chain of botulinum toxin C, nor by block of clathrin-coat maturation. It was abolished by EGTA, which preferentially blocked endocytosis of retrievable membrane pre-existing at the surface, and was impaired by oxidation of cholesterol and inhibition of neutral sphingomyelinase. ATP-independent endocytosis became more significant at 34-35°C, and recovered membrane by an amount that, on average, was close to exocytosis. The results of the present study suggest that activity and temperature recruit ATP-independent endocytosis of pre-existing membrane (in addition to ATP-dependent endocytosis) to efficiently retrieve membrane at nerve terminals. This less understood endocytosis represents a non-canonical mechanism regulated by lipids such as cholesterol and sphingomyelinase.

P 037 - Effect of Neutral Sphingomyelinase Inhibition on ER Stress and Apoptosis in Liver Ischemia–Reperfusion Injury

This study determined the role of selective neutral sphingomyelinase (N-SMase) inhibition on endoplasmic reticulum (ER) stress and apoptotic markers in a rat model of liver ischemia reperfusion (IR) injury. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60 min, followed by 60 min reperfusion. Sphingmyelin and ceramide levels in liver tissue were determined by tandem mass spectrometry. Spingomyelin levels were significantly increased in all IR groups compared to controls. Treatment with a specific N-SMase inhibitor significantly decreased all measured ceramides in IR injury. A significant increase was observed in ER stress markers C/EBP-homologous protein (CHOP) and 78 kDa glucose-regulated protein (GRP78) in IR injury, which was not significantly altered by N-SMase inhibition. Inhibition of N-SMase caused a significant reduction in phospho-NF-kB levels, hepatic TUNEL staining, cytosolic cytochrome c and caspase-3, -8 and -9 activities which were significantly increased in IR injury. Data herein confirm the role of ceramide in increased apoptotic cell death and highlight the protective effect of N-SMase inhibition in down-regulation of apoptotic stimuli responses occurring in hepatic IR injury.

Neutral sphingomyelinase inhibition alleviates apoptosis, but not ER stress, in liver ischemia–reperfusion injury

Previous studies have revealed the activation of neutral sphingomyelinase (N-SMase)/ceramide pathway in hepatic tissue following warm liver ischemia reperfusion (IR) injury. Excessive ceramide accumulation is known to potentiate apoptotic stimuli and a link between apoptosis and endoplasmic reticulum (ER) stress has been established in hepatic IR injury. Thus, this study determined the role of selective N-SMase inhibition on ER stress and apoptotic markers in a rat model of liver IR injury. Selective N-SMase inhibitor was administered via intraperitoneal injections. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60 min, followed by 60 min reperfusion. Levels of sphingmyelin and ceramide in liver tissue were determined by an optimized multiple reactions monitoring (MRM) method using ultrafast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Spingomyelin levels were significantly increased in all IR groups compared with controls. Treatment with a specific N-SMase inhibitor significantly decreased all measured ceramides in IR injury. A significant increase was observed in ER stress markers C/EBP-homologous protein (CHOP) and 78 kDa glucose-regulated protein (GRP78) in IR injury, which was not significantly altered by N-SMase inhibition. Inhibition of N-SMase caused a significant reduction in phospho-NF-kB levels, hepatic TUNEL staining, cytosolic cytochrome c, and caspase-3, -8, and -9 activities which were significantly increased in IR injury. Data herein confirm the role of ceramide in increased apoptotic cell death and highlight the protective effect of N-SMase inhibition in down-regulation of apoptotic stimuli responses occurring in hepatic IR injury.

Macrophages transfer antigens to dendritic cells by releasing exosomes containing dead-cell-associated antigens partially through a ceramide-dependent pathway to enhance CD4(+) T-cell responses.

Defects in rapid clearance of apoptotic cells lead to an accumulation of dead cells (late apoptotic or secondary necrotic cells), which results in an aberrant immune response. However, little is known about whether and how macrophages (Mφs) cooperate with dendritic cells (DCs) in the presentation of dead-cell-associated antigens in this process. By transferring high numbers of dead cells to mimic a failure of apoptotic cell clearance in vivo, we found that Mφs and neutrophils were the predominant phagocytes in the uptake of dead cells in the spleen. Moreover, both Mφs and DCs were required for an optimal CD4(+) T-cell response triggered by dead-cell-associated antigens. Importantly, although Mφs alone had a poor capacity for antigen presentation, they could transfer phagocytosed antigens to DCs for potent antigen presentation to enhance T-cell responses. Finally, we found that exosomes released from Mφs acted as a transmitter to convey antigens to DCs partially in a ceramide-dependent manner, since treatment with the neutral sphingomyelinase inhibitor GW4869 and spiroepoxide resulted in a significant reduction of T-cell proliferation in vitro and in vivo. These findings point to a novel pathway of cross-talk between Mφs and DCs, which will be helpful to explain possible mechanisms for autoimmune diseases characterized by increased rates of apoptosis.

Sensing of Porcine Reproductive and Respiratory Syndrome Virus-Infected Macrophages by Plasmacytoid Dendritic Cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) represents a macrophage (MØ)-tropic virus which is unable to induce interferon (IFN) type I in its target cells. Nevertheless, infected pigs show a short but prominent systemic IFN alpha (IFN-α) response. A possible explanation for this discrepancy is the ability of plasmacytoid dendritic cells (pDC) to produce IFN-α in response to free PRRSV virions, independent of infection. Here, we show that the highly pathogenic PRRSV genotype 1 strain Lena is unique in not inducing IFN-α production in pDC, contrasting with systemic IFN-α responses found in infected pigs. We also demonstrate efficient pDC stimulation by PRRSV Lena-infected MØ, resulting in a higher IFN-α production than direct stimulation of pDC by PRRSV virions. This response was strain-independent, required integrin-mediated intercellular contact, intact actin filaments in the MØ and was partially inhibited by an inhibitor of neutral sphingomyelinase. Although infected MØ-derived exosomes stimulated pDC, an efficient delivery of the stimulatory component was dependent on a tight contact between pDC and the infected cells. In conclusion, with this mechanism the immune system can efficiently sense PRRSV, resulting in production of considerable quantities of IFN-α. This is adding complexity to the immunopathogenesis of PRRSV infections, as IFN-α should alert the immune system and initiate the induction of adaptive immune responses, a process known to be inefficient during infection of pigs.

Autophagy Controls Exosomal Release of Prions and Lateral Prion Infection

Prion diseases are fatal transmissible neurodegenerative disorders that affect both human and animals. They are characterized by conformational conversion of the normal cellular prion protein (PrPc) into a disease associated pathological isoform (PrPSc). It has been shown that PrPSc can infect recipient cells when it is packaged into endosomal derived nanoparticles called exosomes. Exosomes are present in all biological fluids such as blood, urine, milk, and saliva. Exosome secretion is under the influence of the autophagic system, a basic cellular degradation machinery. Autophagy stimulation can inhibit exosomal release, whereas inhibition of autophagy seems to enhance exosomal release. In this paradigm, our work investigates the effect of autophagy modulation on exosomal release of prions and how this interplay impacts prion infection. We successfully isolated and characterized exosomes from a prion‐infected neuronal cell line (ScN2a) using differential ultracentrifugation technique. The isolated exosomes contained PrPSc as shown by immunoblot. Treatment of ScN2a cells with the neutral sphingomyelinase inhibitor GW4869 resulted in a block of the exosomal release and reduced PrPSc levels in exosomes. When we stimulated autophagy in ScN2a cells using rapamycin, a well‐known autophagy stimulator that inhibits the mammalian target of Rapamycin (mTOR) pathway, we observed a strong inhibition of exosomal release and decreased levels of PrPSc in exosomes compared to vehicle treated cells. To further assess the impact of autophagy on exosomal prion release, we knocked‐out Atg5 (a main player in the autophagic machinery) in N2a cells using the CRISPR‐Cas9 system. CRISPR‐Cas9 is the clustered, regularly interspaced, short palindromic repeats (CRISPR)‐associated protein (Cas) system which involves RNA–guided site specific DNA double strand cleavages. Non‐homologous end joining is used by cells for repair of double strand breaks and may result in functional gene knock‐out by introduction of insertions and deletions. Using this technology we generated various N2a cell clones with functional knockouts in different exons of the Atg5 gene, as verified by sequencing and immunoblot analysis. Upon stably infecting these Atg5 knock‐out cells with prions, we found a highly increased release of exosomes and exosome‐associated PrPSc compared to wild type cells. Taken together, our data shows that autophagy modulation can control lateral prion infection by interfering with exosomal release of PrPSc. Our present work correlates these findings to outcomes of prion infection in recipient cells and in mouse animal models. Overall, our study describes a novel interplay of basic cellular machinery which affects the live cycle of prions. This new understanding will result in novel targets for therapy against prion diseases and protein misfolding disorders.Support or Funding InformationThe Natural Sciences and Engineering Research Council of Canada (NSERC) Alberta Prion Research Institute (APRI) National Institute of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) Alberta Innovates Health Solutions (AIHS)

Inhibition of neutral sphingomyelinase decreases elevated levels of nitrative and oxidative stress markers in liver ischemia–reperfusion injury

Oxidative stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS)-2 have been shown in the pathogenesis of liver ischemia–reperfusion (IR) injury. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression therefore this study determined the role of selective N-SMase inhibition on nitrative and oxidative stress markers following liver IR injury. Selective N-SMase inhibitor was administered via intraperitoneal injections. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60 min, followed by 60 min reperfusion. Nitrative and oxidative stress markers were determined by evaluating NOS2 expression, protein nitration, nitrite/nitrate levels, 4-hydroxynonenal (HNE) formation, protein carbonyl levels and xanthine oxidase/xanthine dehydrogenase (XO/XDH) activity. Levels of sphingmyelin and ceramide in liver tissue were determined by an optimized multiple reaction monitoring method using ultra-fast liquid chromatography coupled with tandem mass spectrometry (MS/MS). Spingomyelin levels were significantly increased in all IR groups compared to controls. Treatment with a specific N-SMase inhibitor significantly decreased all measured ceramides in IR injury. NOS2 expression, nitrite/nitrate levels and protein nitration were significantly greater in IR injury and decreased with N-SMase inhibition. Treatment with a selective N-SMase inhibitor significantly decreased HNE formation, protein carbonyl levels and the hepatic conversion of XO. Data confirm the role of nitrative and oxidative injury in IR and highlight the protective effect of selective N-SMase inhibition. Future studies evaluating agents blocking N-SMase activity can facilitate the development of treatment strategies to alleviate oxidative injury in liver I/R injury.

Direct regulation of IGF-binding protein 1 promoter by interleukin-1β via an insulin- and FoxO-1-independent mechanism.

The level of insulin-like growth factor-binding protein 1 (IGFBP1), a liver-produced serum protein that regulates insulin-like growth factor-I bioactivity, glucose homeostasis, and tissue regeneration, increases during inflammation. This manuscript describes a novel pathway for the regulation of hepatic IGFBP1 mRNA and protein levels by interleukin (IL)-1β. Experiments with the luciferase reporter system show that IL-1β stimulates transcriptional activity from the 1-kb promoter region of IGFBP1. Although IL-1β stimulation suppresses the insulin activation of protein kinase B, the major upstream regulator of IGFBP1 mRNA transcription, the induction of IGFBP1 by IL-1β did not require an intact insulin response element. Furthermore, neither overexpression nor silencing of FoxO-1 had any effect on the IL-1β-induced increase in IGFBP1 mRNA levels and promoter activity. However, inhibition of the ERK MAP kinases effectively prevented the IL-1β effects. Inhibition of neutral sphingomyelinase, a key player in the IL-1β signaling cascade that acts upstream of ERK, also suppressed the IL-1β effects, while increasing the ceramide, through the addition of C2-ceramide or via treatment with exogenous sphingomyelinase, was sufficient to induce IGFBP1 promoter-driven luciferase activity. Studies in primary rat hepatocytes where the levels of neutral sphingomyelinase were either elevated or suppressed using adenoviral constructs affirmed the key role of neutral sphingomyelinase and ceramide (exerted likely through ERK activation) in the IL-1β-induced IGFBP1 production. Finally, the IL-1β effects on IGFBP1 mRNA production and protein secretion could be abolished by the addition of insulin, either at very late time points or at very high doses.

Interleukin-1β activates an Src family kinase to stimulate the plasma membrane Ca2+ pump in hippocampal neurons.

The plasma membrane Ca(2+) ATPase (PMCA) plays a major role in clearing Ca(2+) from the neuronal cytoplasm. The cytoplasmic Ca(2+) clearance rate affects neuronal excitability, synaptic plasticity, and neurotransmission. Here, we examined the modulation of PMCA activity by PTKs in hippocampal neurons. PMCA-mediated Ca(2+) clearance slowed in the presence of pyrazolopyrimidine 2, an inhibitor of Src family kinases (SFKs), and accelerated in the presence of C2-ceramide, an activator of PTKs. Ca(2+) clearance kinetics were attenuated in cells expressing a dominant-negative Src mutant, suggesting that the pump is tonically stimulated by a PTK. Tonic stimulation was reduced in hippocampal neurons expressing short hairpin (sh)RNA directed to mRNA for Yes. shRNA-mediated knockdown of PMCA isoform 1 (PMCA1) removed tonic stimulation of Ca(2+) clearance, indicating that the kinase stimulates PMCA1. IL-1β accelerated Ca(2+) clearance in a manner blocked by an IL-1β receptor antagonist or by an inhibitor of neutral sphingomyelinase, the enzyme that produces ceramide. Thus IL-1β activates an SFK to stimulate the plasma membrane Ca(2+) pump, decreasing the duration of Ca(2+) transients in hippocampal neurons.

Inhibition of neutral sphingomyelinase protects mice against systemic tuberculosis

Inhibition of neutral sphingomyelinase protects mice against systemic tuberculosis

Inhibition of neutral sphingomyelinase protects mice against systemic tuberculosis.

Tuberculosis is one of the most serious infectious diseases worldwide. The initial pulmonal localization of the pathogens often develops into systemic infection with high lethality. We investigated the role of the mammalian neutral sphingomyelinase (Nsm)/ceramide system in systemic infection of mice and murine macrophages with Mycobacterium bovis Bacillus Calmette-Guerin (BCG). Our results demonstrate that BCG infection of RAW cells, a macrophage cell line, results in rapid activation of Nsm but not of acid sphingomyelinase (Asm). Activation of Nsm is associated with a massive release of superoxide. Genetic knock-down of Nsm in RAW cells prevented superoxide production upon BCG infection. Superoxide suppressed autophagy in BCG-infected macrophages in vitro and in vivo: Knock-down of Nsm or inhibition of superoxide restored autophagy in macrophages and increased killing of intracellular bacteria upon BCG infection. Most importantly, autophagy was also massively increased in Nsm-heterozygous mice, protecting these mice from systemic BCG infections, granuloma development, and chronic infections of liver and spleen. These findings indicate that the Nsm/ceramide system plays a role in protecting mice against systemic tuberculosis by preventing superoxide-mediated inhibition of autophagy.

Glutaminase-containing microvesicles from HIV-1-infected macrophages and immune-activated microglia induce neurotoxicity.

BackgroundHIV-1-infected and/or immune-activated microglia and macrophages are pivotal in the pathogenesis of HIV-1-associated neurocognitive disorders (HAND). Glutaminase, a metabolic enzyme that facilitates glutamate generation, is upregulated and may play a pathogenic role in HAND. Our previous studies have demonstrated that glutaminase is released to the extracellular fluid during HIV-1 infection and neuroinflammation. However, key molecular mechanisms that regulate glutaminase release remain unknown. Recent advances in understanding intercellular trafficking have identified microvesicles (MVs) as a novel means of shedding cellular contents. We posit that during HIV-1 infection and immune activation, microvesicles may mediate glutaminase release, generating excessive and neurotoxic levels of glutamate.ResultsMVs isolated through differential centrifugation from cell-free supernatants of monocyte-derived macrophages (MDM) and BV2 microglia cell lines were first confirmed in electron microscopy and immunoblotting. As expected, we found elevated number of MVs, glutaminase immunoreactivities, as well as glutaminase enzyme activity in the supernatants of HIV-1 infected MDM and lipopolysaccharide (LPS)-activated microglia when compared with controls. The elevated glutaminase was blocked by GW4869, a neutral sphingomyelinase inhibitor known to inhibit MVs release, suggesting a critical role of MVs in mediating glutaminase release. More importantly, MVs from HIV-1-infected MDM and LPS-activated microglia induced significant neuronal injury in rat cortical neuron cultures. The MV neurotoxicity was blocked by a glutaminase inhibitor or GW4869, suggesting that the neurotoxic potential of HIV-1-infected MDM and LPS-activated microglia is dependent on the glutaminase-containing MVs.ConclusionsThese findings support MVs as a potential pathway/mechanism of excessive glutamate generation and neurotoxicity in HAND and therefore MVs may serve as a novel therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-015-0058-z) contains supplementary material, which is available to authorized users.

TNFα triggers release of extracellular vesicles containing TNFR1 and TRADD, which can modulate TNFα responses of the parental cells

Tumor necrosis factor-α (TNFα)-induced reactions are effective to maintain homeostasis; however, excessive responses play progressive roles in the pathogenesis of various chronic inflammatory diseases. We demonstrate that TNFα triggered the release of its receptor TNFR1 as a content of extracellular vesicles (EVs) from the human bronchial epithelial cell, BEAS-2b. The TNFR1 cytoplasmic domain binding partner, TNFR-associated death domain (TRADD), was released by TNFα treatment along with TNFR1. TNFα-triggered release of EVs was decreased in the presence of amitriptyline, an inhibitor of acid sphingomyelinase (A-SMase), or of GW4869, an inhibitor of neutral sphingomyelinase (N-SMase), indicating that EVs containing TNFR1 and TRADD are released through A-SMase and N-SMase dependent manners. From sucrose density gradient analysis, each sphingomyelinase is involved in the generation of distinct populations of EVs. Inhibition of A-SMase or N-SMase resulted in significantly increased responses to TNFα in parental cells. Given that TRADD serves as a platform for the assembly of subsequent signaling molecules, the TNFα triggered release of TNFR1 and TRADD might be an effective strategy for down regulation of the TNFα responses of parental cells.

New oleyl glycoside as anti-cancer agent that targets on neutral sphingomyelinase

We designed and synthesized two anomeric oleyl glucosaminides as anti-cancer agents where the presence of a trifluoroacetyl group close to the anomeric center makes them resistant to hydrolysis by hexosaminidases. The oleyl glycosides share key structural features with synthetic and natural oleyl derivatives that have been reported to exhibit anti-cancer properties. While both glycosides showed antiproliferative activity on cancer cell lines, only the α-anomer caused endoplasmic reticulum (ER) stress and cell death on C6 glioma cells. Analysis of sphingolipids and glycosphingolipds in cells treated with the glycosides showed that the α-anomer caused a drastic accumulation of ceramide and glucosylceramide and reduction of lactosylceramide and GM3 ganglioside at concentrations above a threshold of 20μM. In order to understand how ceramide levels increase in response to α-glycoside treatment, further investigations were done using specific inhibitors of sphingolipid metabolic pathways. The pretreatment with 3-O-methylsphingomyelin (a neutral sphingomyelinase inhibitor) restored sphingomyelin levels together with the lactosylceramide and GM3 ganglioside levels and prevented the ER stress and cell death caused by the α-glycoside. The results indicated that the activation of neutral sphingomyelinase is the main cause of the alterations in sphingolipids that eventually lead to cell death. The new oleyl glycoside targets a key enzyme in sphingolipid metabolism with potential applications in cancer therapy.

KRAS-dependent sorting of miRNA to exosomes.

Mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor microenvironment. To test whether exosomal RNAs also contribute to changes in gene expression in recipient cells, and whether mutant KRAS might regulate the composition of secreted microRNAs (miRNAs), we compared small RNAs of cells and matched exosomes from isogenic CRC cell lines differing only in KRAS status. We show that exosomal profiles are distinct from cellular profiles, and mutant exosomes cluster separately from wild-type KRAS exosomes. miR-10b was selectively increased in wild-type exosomes, while miR-100 was increased in mutant exosomes. Neutral sphingomyelinase inhibition caused accumulation of miR-100 only in mutant cells, suggesting KRAS-dependent miRNA export. In Transwell co-culture experiments, mutant donor cells conferred miR-100-mediated target repression in wild-type-recipient cells. These findings suggest that extracellular miRNAs can function in target cells and uncover a potential new mode of action for mutant KRAS in CRC.

Guggulsterone and bexarotene induce secretion of exosome-associated breast cancer resistance protein and reduce doxorubicin resistance in MDA-MB-231 cells.

Many breast cancer cells acquire multidrug resistance (MDR) mediated by ABC transporters such as breast cancer resistance protein (BCRP/ABCG2). Here we show that incubation of human breast cancer MDA-MB-231 cells with farnesoid X receptor antagonist guggulsterone (gug) and retinoid X receptor agonist bexarotene (bex) elevated ceramide, a sphingolipid known to induce exosome secretion. The gug+bex combination reduced cellular levels of BCRP to 20% of control cells by inducing its association and secretion with exosomes. Exogenous C6 ceramide also induced secretion of BCRP-associated exosomes, while siRNA-mediated knockdown or GW4869-mediated inhibition of neutral sphingomyelinase 2 (nSMase2), an enzyme generating ceramide, restored cellular BCRP. Immunocytochemistry showed that ceramide elevation and concurrent loss of cellular BCRP was prominent in Aldefluor-labeled breast cancer stem-like cells. These cells no longer excluded the BCRP substrate Hoechst 33342 and showed caspase activation and apoptosis induction. Consistent with reduced BCRP, ABC transporter assays showed that gug+bex increased doxorubicin retention and that the combination of gug+bex with doxorubicin enhanced cell death by more than fivefold. Taken together, our results suggest a novel mechanism by which ceramide induces BCRP secretion and reduces MDR, which may be useful as adjuvant drug treatment for sensitizing breast cancer cells and cancer stem cells to chemotherapy.

CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction

Hypoxic pulmonary vasoconstriction (HPV) optimizes pulmonary ventilation-perfusion matching in regional hypoxia, but promotes pulmonary hypertension in global hypoxia. Ventilation-perfusion mismatch is a major cause of hypoxemia in cystic fibrosis. We hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) may be critical in HPV, potentially by modulating the response to sphingolipids as mediators of HPV. HPV and ventilation-perfusion mismatch were analyzed in isolated mouse lungs or in vivo. Ca(2+) mobilization and transient receptor potential canonical 6 (TRPC6) translocation were studied in human pulmonary (PASMCs) or coronary (CASMCs) artery smooth muscle cells. CFTR inhibition or deficiency diminished HPV and aggravated ventilation-perfusion mismatch. In PASMCs, hypoxia caused CFTR to interact with TRPC6, whereas CFTR inhibition attenuated hypoxia-induced TRPC6 translocation to caveolae and Ca(2+) mobilization. Ca(2+) mobilization by sphingosine-1-phosphate (S1P) was also attenuated by CFTR inhibition in PASMCs, but amplified in CASMCs. Inhibition of neutral sphingomyelinase (nSMase) blocked HPV, whereas exogenous nSMase caused TRPC6 translocation and vasoconstriction that were blocked by CFTR inhibition. nSMase- and hypoxia-induced vasoconstriction, yet not TRPC6 translocation, were blocked by inhibition or deficiency of sphingosine kinase 1 (SphK1) or antagonism of S1P receptors 2 and 4 (S1P2/4). S1P and nSMase had synergistic effects on pulmonary vasoconstriction that involved TRPC6, phospholipase C, and rho kinase. Our findings demonstrate a central role of CFTR and sphingolipids in HPV. Upon hypoxia, nSMase triggers TRPC6 translocation, which requires its interaction with CFTR. Concomitant SphK1-dependent formation of S1P and activation of S1P2/4 result in phospholipase C-mediated TRPC6 and rho kinase activation, which conjointly trigger vasoconstriction.

Inhibition of neutral sphingomyelinase decreases elevated levels of inducible nitric oxide synthase and apoptotic cell death in ocular hypertensive rats

Endoplasmic reticulum (ER) stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of neuronal retinal cell death in ocular hypertension. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression, hence this study determined the role of selective neutral sphingomyelinase (N-SMase) inhibition on retinal NOS2 levels, ER stress, apoptosis and visual evoked potentials (VEPs) in a rat model of elevated intraocular pressure (EIOP). NOS2 expression and retinal protein nitration were significantly greater in EIOP and significantly decreased with N-SMase inhibition. A significant increase was observed in retinal ER stress markers pPERK, CHOP and GRP78 in EIOP, which were not significantly altered by N-SMase inhibition. Retinal TUNEL staining showed increased apoptosis in all EIOP groups; however N-SMase inhibition significantly decreased the percent of apoptotic cells in EIOP. Caspase-3, -8 and -9 activities were significantly increased in EIOP and returned to baseline levels following N-SMase inhibition. Latencies of all VEP components were significantly prolonged in EIOP and shortened following N-SMase inhibition. Data confirm the role of nitrative injury in EIOP and highlight the protective effect of N-SMase inhibition in EIOP via down-regulation of NOS2 levels and nitrative stress.

Exosome-mediated extracellular release of polyadenylate-binding protein 1 in human metastatic duodenal cancer cells.

Exosomes are small vesicles secreted from cells that transport their embedded molecules through bidirectional exocytosis- and endocytosis-like pathways. Expression patterns of exosomal molecules such as proteins and RNAs can be indicative of cell type since their signature is thought to be unique among cells. Using human primary (AZ-521) and metastatic (AZ-P7a) duodenal cancer cell lines, we conducted a comparative exosomal proteome analysis to identify proteins with metastatic marker potential. As determined by LC-MS/MS and Western blot analyses, polyadenylate-binding protein 1 (PABP1) was found to be predominantly abundant in AZ-P7a exosomes. The amount of exosomal PABP1 in AZ-P7a cells increased by treating the cells with inhibitors for the classical ER/Golgi secretory pathway (brefeldin A and monensin) and the ubiquitin-proteasome pathway (MG-132 and PYR-41). Treatment of AZ-P7a cells with the neutral sphingomyelinase inhibitor GW4869, which suppresses exosome release, not only reduced the amount of exosomal PABP1 but also produced PABP1-immunoreactive products cleaved via a proteolysis-like process. Taken together, these results suggest that AZ-P7a cells do not tolerate intracellular PABP1 accumulation and are thus exported into the extracellular milieu by the exosome-mediated pathway. In addition, PABP1 has a potential use as a biomarker for metastatic duodenal cancer.

Multiple sphingolipid abnormalities following cerebral microendothelial hypoxia.

Hypoxia has been previously shown to inhibit the dihydroceramide (DHC) desaturase, leading to the accumulation of DHC. In this study, we used metabolic labeling with [3H]-palmitate, HPLC/MS/MS analysis, and specific inhibitors to show numerous sphingolipid changes after oxygen deprivation in cerebral microendothelial cells. The increased DHC, particularly long-chain forms, was observed in both whole cells and detergent-resistant membranes. This was reversed by reoxygenation and blocked by the de novo sphingolipid synthesis inhibitor myriocin, but not by the neutral sphingomyelinase inhibitor GW-4869. Furthermore, oxygen deprivation of microendothelial cells increased levels of dihydro-sphingosine (DH-Sph), DH-sphingosine1-phosphate (DH-S1P), DH-sphingomyelin (DH-SM), DH-glucosylceramide (DH-GlcCer), and S1P levels. In vitro assays revealed no changes in the activity of sphingomyelinases or sphingomyelin synthase, but resulted in reduced S1P lyase activity and 40% increase in glucosylceramide synthase (GCS) activity, which was reversed by reoxygenation. Inhibition of the de novo sphingolipid pathway (myriocin) or GCS (EtPoD4) induced endothelial barrier dysfunction and increased caspase 3-mediated cell death in response to hypoxia. Our findings suggest that hypoxia induces synthesis of S1P and multiple dihydro-sphingolipids, including DHC, DH-SM, DH-GlcCer, DH-Sph and DH-S1P, which may be involved in ameliorating the effects of stroke . Progressive hypoxia leads to the accumulation of several dihydrosphingolipids in cerebral microendothelial cells. Hypoxia also increases sphingosine-1-phosphate and the activity of glucosylceramide (Glc-Cer) synthase. These changes reverse by inhibiting the de novo sphingolipid synthesis, which worsens hypoxia-induced endothelial barrier dysfunction and apoptosis, suggesting that the identified sphingolipids may be vasculoprotective.