Ca(2)(+)i signalling is a key regulatory mechanism in sperm function. In mammalian sperm the Ca(2)(+)-permeable plasma membrane ion channel CatSper is central to Ca(2)(+)i signalling, but there is ...good evidence that Ca(2)(+) stored in intracellular organelles is also functionally important. Here we briefly review the current understanding of the diversity of Ca(2)(+) stores and the mechanisms for the regulation of their activity. We then consider the evidence for the involvement of these stores in Ca(2)(+)i signalling in mammalian (primarily human) sperm, the agonists that may activate these stores and their role in control of sperm function. Finally we consider the evidence that membrane Ca(2)(+) channels and stored Ca(2)(+) may play discrete roles in the regulation of sperm activities and propose a mechanism by which these different components of the sperm Ca(2)(+)-signalling apparatus may interact to generate complex and spatially diverse Ca(2)(+)i signals.
Brominated flame retardants (BFRs) are chemicals commonly used to reduce the flammability of consumer products and are considered pollutants since they have become widely dispersed throughout the ...environment and have also been shown to bio-accumulate within animals and man. This study investigated the cytotoxicity of some of the most commonly used groups of BFRs on SH-SY5Y human neuroblastoma cells. The results showed that of the BFRs tested, hexabromocyclododecane (HBCD), tetrabromobisphenol-A (TBBPA) and decabromodiphenyl ether (DBPE), all are cytotoxic at low micromolar concentrations (LC(50) being 2.7 ± 0.7 µM, 15 ± 4 µM and 28 ± 7 µM, respectively). They induced cell death, at least in part, by apoptosis through activation of caspases. They also increased intracellular Ca(2+) levels and reactive-oxygen-species within these neuronal cells. Furthermore, these BFRs also caused rapid depolarization of the mitochondria and cytochrome c release in these neuronal cells. Elevated intracellular Ca(2+) levels appear to occur through a mechanism involving microsomal Ca(2+)-ATPase inhibition and this maybe responsible for Ca(2+)-induced mitochondrial dysfunction. In addition, µM levels of these BFRs caused β-amyloid peptide (Aβ-42) processing and release from these cells with a few hours of exposure. These results therefore shows that these pollutants are both neurotoxic and amyloidogenic in-vitro.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A diversity of SERCA Ca2+ pump inhibitors Michelangeli, Francesco; East, J Malcolm
Biochemical Society transactions,
06/2011, Letnik:
39, Številka:
3
Journal Article
Recenzirano
The SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) is probably the most extensively studied membrane protein transporter. There is a vast array of diverse inhibitors for the Ca2+ pump, and ...many have proved significant in helping to elucidate both the mechanism of transport and gaining conformational structures. Some SERCA inhibitors such as thapsigargin have been used extensively as pharmacological tools to probe the roles of Ca2+ stores in Ca2+ signalling processes. Furthermore, some inhibitors have been implicated in the cause of diseases associated with endocrine disruption by environmental pollutants, whereas others are being developed as potential anticancer agents. The present review therefore aims to highlight some of the wide range of chemically diverse inhibitors that are known, their mechanisms of action and their binding location on the Ca2+ ATPase. Additionally, some ideas for the future development of more useful isoform-specific inhibitors and anticancer drugs are presented.
•HBCD is the most potent BFR tested to inhibit SERCA Ca2+ pumps.•HBCD is the most potent at inducing death of SH-SY5Y cells.•For a range of BFRs there is a correlation between cell death & SERCA ...inhibition.•HBCD inhibits ATP binding and the E2 to E1 transition of SERCA.
Hexabromocyclododecane (HBCD) is a widely utilised brominated flame retardant (BFR). It has been shown to bio-accumulate within organisms, including man, and possibly cause neurological disorders. The acute neurotoxicity of HBCD, and six other unrelated BFRs, were assessed in SH-SY5Y human neuroblastoma cells by 24h viability assays and HBCD proved to be the most lethal (LC50, 3μM). In addition, the effects of these BFRs were also assessed for their potency at inhibiting the sarcoplasmic–endoplasmic reticulum Ca2+ ATPase (SERCA) derived from the SH-SY5Y cells and again HBCD was the most potent (IC50, 2.7μM). The data for the other BFRs tested showed a direct correlation (coefficient 0.94) between the potencies of inducing cell death and inhibiting the Ca2+ ATPase, indicating that SERCA is likely to be the molecular target for acute toxicity. Mechanistic studies of HBCD on the Ca2+ ATPase suggest that it affects ATP binding, phosphorylation as well as the E2 to E1 transition step.
Background: Ca2+ signals, elicited by cues from the oocyte and female tract, regulate human sperm behavior.
Results: CatSper channel activation (flagellum) and Ca2+ store mobilization (neck) caused ...similar Ca2+i elevation but induced functionally different behaviors.
Conclusion: Sperm motility pattern is determined by the site of Ca2+ mobilization.
Significance: Selection of Ca2+ signaling components and/or regulation of their availability for activation controls human sperm behavior.
Ca2+i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca2+ signaling pathway used. Activation of CatSper (by raising pHi or stimulating with progesterone) caused sustained Ca2+i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 μm thimerosal to mobilize stored Ca2+ caused sustained Ca2+i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pHi and mobilized Ca2+ stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca2+-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the Ca2+i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca2+ at the sperm neck can be mobilized by Ca2+-induced Ca2+ release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca2+ store, which may be regulated by capacitation and NO from the cumulus.
Drug resistance in cancer has been classified as innate resistance or acquired resistance, which were characterized by apoptotic defects and ABC transporters overexpression respectively. Therefore, ...to preclude or reverse these resistance mechanisms could be a promising strategy to improve chemotherapeutic outcomes. In this study, a natural product from Osage Orange, pomiferin, was identified as a novel autophagy activator that circumvents innate resistance by triggering autophagic cell death via SERCA inhibition and activation of the CaMKKβ-AMPK-mTOR signaling cascade. In addition, pomiferin also directly inhibited the P-gp (MDR1/ABCB1) efflux and reversed acquired resistance by potentiating the accumulation and efficacy of the chemotherapeutic agent, cisplatin. In vivo study demonstrated that pomiferin triggered calcium-mediated tumor suppression and exhibited an anti-metastatic effect in the LLC-1 lung cancer-bearing mouse model. Moreover, as an adjuvant, pomiferin potentiated the anti-tumor effect of the chemotherapeutic agent, cisplatin, in RM-1 drug-resistant prostate cancer-bearing mouse model by specially attenuating ABCB1-mediated drug efflux, but not ABCC5, thereby promoting the accumulation of cisplatin in tumors. Collectively, pomiferin may serve as a novel effective agent for circumventing drug resistance in clinical applications.
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Schematic diagram of the proposed molecular targets and mechanisms of pomiferin in induction of autophagic cell death and inhibition of P-glycoprotein.
Flavonoids are group of plant‐derived hydroxylated polycyclic molecules found in fruit and vegetables. They are known to bio‐accumulate within humans and are considered to have beneficial health ...effects, including cancer chemoprotection. One mechanism proposed to explain this is that they are able to induce apoptosis in cancer cells by inhibiting a variety of kinases and also the Ca2+ ATPase. An investigation was undertaken with respect to the mechanism of inhibition for three flavonoids: quercetin, galangin and 3,6 dihydroxyflavone (3,6‐DHF). Each inhibited the Ca2+ ATPase with Ki values of 8.7, 10.3 and 5.4 μm, respectively, showing cooperative inhibition with n ~ 2. Given their similar structures, the flavonoids showed several differences in their mechanisms of inhibition. All three flavonoids stabilized the ATPase in the E1 conformation and reduced 32P‐ATP binding. However, both galangin and 3,6‐DHF increased the affinity of Ca2+ for the ATPase by decreasing the Ca2+‐dissociation rate constant, whereas quercetin had little effect. Ca2+‐induced changes in tryptophan fluorescence levels were reduced in the presence of 3,6‐DHF and galangin (but not with quercetin), indicating that Ca2+‐associated changes within the transmembrane helices are altered. Both galangin and quercetin reduced the rates of ATP‐dependent phosphorylation and dephosphorylation, whereas 3,6‐DHF did not. Modelling studies suggest that flavonoids could potentially bind to two sites: one directly where nucleotides bind within ATP binding site and the other at a site close by. We hypothesize that interactions of these two neighbouring sites may account for both the cooperative inhibition and the multimode mechanisms of action seen with related flavonoids.
Flavonoids are cancer chemo‐protective agents that target the Ca2+ ATPase. Here, we show that flavonoids cause cooperative inhibition (n~2) and that related flavonoids inhibit different steps, including; ATP binding, phosphorylation and the E2/ E1 transition. From enzymatic and modeling studies, we postulate that flavonoids are likely to bind to two sites which are located close together within the ATP binding domain.
Endogenous molecules, such as heat shock proteins (HSP), can function as danger signals when released into the extracellular environment in response to cell stress, where they elicit an immune ...response such as cytokine secretion. There has also been some suggestion that contamination of exogenous HSPs with lipopolysaccharide (LPS) may be responsible for these effects. This study investigates the effects of exogenous HSPA1A and HSPB1 on the activation of immune cells and the resulting secretion of cytokines, which are involved in inflammatory responses. To address whether exogenous HSPs can directly activate cytokine secretion, naïve U937 cells, differentiated U937 cells and peripheral blood mononuclear cells (PBMCs) were treated with either exogenously applied HSPA1A or HSPB1 and then secreted IL‐1β, TNF‐α and IL‐10 were measured by ELISA. Both HSPs were able to induce a dose‐dependent increase in IL‐10 secretion from naïve U937 cells and dose‐dependent IL‐1β, TNF‐α and IL‐10 secretion were also observed in differentiated U937 cells and PBMCs. We also observed that CD14 affects the secretion levels of IL‐1β, TNF‐α and IL‐10 from cells in response to exogenous HSP treatment. In addition, HSPA1A and HSPB1 were shown to interact with CD14, CD36 and CD11b extracellular receptor proteins. Several approaches used in this study indicate that HSP‐induced cytokine secretion is largely independent of any contaminating LPS in the samples.
Cell stress activates the release of endogenous molecules such as heat shock proteins (HSPs) into the extracellular environment. Here, we investigated cytokine release in naïve, differentiated U937 cells and peripheral blood mononuclear cells following exposure to HSPs. Both HSPA1A and HSPB1 activated cytokine secretion, and data suggests CD14, CD36 and CD11b may play a major role in signalling.
Abstract
Resistance of cancer cells to chemotherapy is a significant clinical concern and mechanisms regulating cell death in cancer therapy, including apoptosis, autophagy or necrosis, have been ...extensively investigated over the last decade. Accordingly, the identification of medicinal compounds against chemoresistant cancer cells
via
new mechanism of action is highly desired. Autophagy is important in inducing cell death or survival in cancer therapy. Recently, novel autophagy activators isolated from natural products were shown to induce autophagic cell death in apoptosis-resistant cancer cells in a calcium-dependent manner. Therefore, enhancement of autophagy may serve as additional therapeutic strategy against these resistant cancers. By computational docking analysis, biochemical assays, and advanced live-cell imaging, we identified that neferine, a natural alkaloid from
Nelumbo nucifera
, induces autophagy by activating the ryanodine receptor and calcium release. With well-known apoptotic agents, such as staurosporine, taxol, doxorubicin, cisplatin and etoposide, utilized as controls, neferine was shown to induce autophagic cell death in a panel of cancer cells, including apoptosis-defective and -resistant cancer cells or isogenic cancer cells,
via
calcium mobilization through the activation of ryanodine receptor and Ulk-1-PERK and AMPK-mTOR signaling cascades. Taken together, this study provides insights into the cytotoxic mechanism of neferine-induced autophagy through ryanodine receptor activation in resistant cancers.
Intracellular Ca²⁺ stores play a central role in the regulation of cellular Ca²⁺i and the generation of complex Ca²⁺ signals such as oscillations and waves. Ca²⁺ signalling is of particular ...significance in sperm cells, where it is a central regulator in many key activities (including capacitation, hyperactivation, chemotaxis and acrosome reaction) yet mature sperm lack endoplasmic reticulum and several other organelles that serve as Ca²⁺ stores in somatic cells. Here, we review i) the evidence for the expression in sperm of the molecular components (pumps and channels) which are functionally significant in the activity of Ca²⁺ stores of somatic cells and ii) the evidence for the existence of functional Ca²⁺ stores in sperm. This evidence supports the existence of at least two storage organelles in mammalian sperm, one in the acrosomal region and another in the region of the sperm neck and midpiece. We then go on to discuss the probable identity of these organelles and their discrete functions: regulation by the acrosome of its own secretion and regulation by membranous organelles at the sperm neck (and possibly by the mitochondria) of flagellar activity and hyperactivation. Finally, we consider the ability of the sperm discretely to control mobilisation of these stores and the functional interaction of stored Ca²⁺ at the sperm neck/midpiece with CatSper channels in the principal piece in regulation of the activities of mammalian sperm.
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