Cellular consequences of the expression of Alzheimer's disease-causing presenilin 1 mutations in human neuroblastoma (SH-SY5Y) cells.

Mutations in the presenilin 1 (PS1) gene lead to early-onset Alzheimer's disease with the S170F mutation causing the earliest reported age of onset. Expression of this, and other PS1 mutations, in SH-SY5Y cells resulted in significant loss of cellular viability compared to control cells. Basal Ca2+ concentrations in PS1 mutants were never lower than controls and prolonged incubation in Ca2+ -free solutions did not deplete Ca2+ stores, demonstrating there was no difference in Ca2+ leak from endoplasmic reticulum (ER) stores in PS1 mutants. Peak muscarine-evoked rises of [Ca2+]i were variable, but the integrals were not significantly different, suggesting, while kinetics of Ca2+ store release might be affected in PS1 mutants, store size was similar. However, when Ca2+ -ATPase activity was irreversibly inhibited with thapsigargin, the S170F and ΔE9 cells showed larger capacitative calcium entry indicating a direct effect on Ca2+ influx pathways. There was no significant effect of any of the mutations on mitochondrial respiration. Amyloid ß(Aß(1-40)) secretion was reduced, and Aß(1-42) secretion increased in the S170F cells resulting in a very large increase in the Aß42/40 ratio. This, rather than any potential disruption of ER Ca2+ stores, is likely to explain the extreme pathology of this mutant.

Carbon monoxide inhibits L-type Ca2+ channels via redox modulation of key cysteine residues by mitochondrial reactive oxygen species.

Conditions of stress, such as myocardial infarction, stimulate up-regulation of heme oxygenase (HO-1) to provide cardioprotection. Here, we show that CO, a product of heme catabolism by HO-1, directly inhibits native rat cardiomyocyte L-type Ca2+ currents and the recombinant alpha1C subunit of the human cardiac L-type Ca2+ channel. CO (applied via a recognized CO donor molecule or as the dissolved gas) caused reversible, voltage-independent channel inhibition, which was dependent on the presence of a spliced insert in the cytoplasmic C-terminal region of the channel. Sequential molecular dissection and point mutagenesis identified three key cysteine residues within the proximal 31 amino acids of the splice insert required for CO sensitivity. CO-mediated inhibition was independent of nitric oxide and protein kinase G but was prevented by antioxidants and the reducing agent, dithiothreitol. Inhibition of NADPH oxidase and xanthine oxidase did not affect the inhibitory actions of CO. Instead, inhibitors of complex III (but not complex I) of the mitochondrial electron transport chain and a mitochondrially targeted antioxidant (Mito Q) fully prevented the effects of CO. Our data indicate that the cardioprotective effects of HO-1 activity may be attributable to an inhibitory action of CO on cardiac L-type Ca2+ channels. Inhibition arises from the ability of CO to promote generation of reactive oxygen species from complex III of mitochondria. This in turn leads to redox modulation of any or all of three critical cysteine residues in the channel's cytoplasmic C-terminal tail, resulting in channel inhibition.

Hypoxic remodelling of Ca(2+) signalling in proliferating human arterial smooth muscle.

Ca(2+) homeostasis in proliferating smooth muscle (SM) cells strongly influences neointima formation, which can cause failure of coronary artery bypass surgery. During surgical procedures and subsequent revascularization, SM cells are also exposed to a period of hypoxia. Problems with bypass surgery in general involve neointima formation which is in turn dependent on SM proliferation and migration. Here, we have directly monitored [Ca(2+)](i) fluorimetrically in proliferating internal mammary artery (IMA) SM cells, and investigated how this is modulated by chronic hypoxia (CH; 24 h, 2.5% O(2)). IMA is the most successful replacement conduit vessel in bypass grafts. Basal [Ca(2+)](i) was unaffected by CH, but removal of extracellular Ca(2+) evoked far smaller reductions in [Ca(2+)](i) than were seen in normoxic cells. Voltage-gated Ca(2+) entry was suppressed in CH cells, and this was attributable to activation of the transcriptional regulator, hypoxia inducible factor. Furthermore, the relative contributions to voltage-gated Ca(2+) entry of L- and T-type Ca(2+) channels was markedly altered, with T-type channels becoming functionally more important in CH cells. Agonist-evoked mobilization of Ca(2+) from intracellular stores was not affected by CH, whilst subsequent capacitative Ca(2+) entry was modestly suppressed. Our data provide novel observations of the remodelling of Ca(2+) homeostasis by CH in IMASM cells which may contribute to their superior patency as coronary bypass grafts.

Differential involvement of TNF alpha in hypoxic suppression of astrocyte glutamate transporters.

Transporter-mediated glutamate uptake is a principal function of astrocytes. Our previous studies have shown that this process is compromised under hypoxic conditions through the NF-kappaB mediated inhibition of expression of the glutamate transporters EAAT-1 and EAAT-2. Here, we demonstrate that identical conditions of hypoxia (1% O(2), 24 h) lead to a dramatic increase in TNFalpha production from astrocytes without altering their viability. This hypoxia-evoked production of TNFalpha was prevented in the presence of any of three mechanistically distinct NF-kappaB inhibitors. Exogenous application of TNFalpha was without effect on EAAT-1 expression as determined by Western blotting, but mimicked the effects of hypoxia to suppress expression of EAAT-2. Furthermore thalidomide, which prevents TNFalpha production, was without effect on hypoxic suppression of EAAT-1 but prevented hypoxic suppression of EAAT-2. These data indicate that regulation of glutamate transporter expression in astrocytes by hypoxia is subtype specific. Regulation of both EAAT-1 and EAAT-2 is mediated by NF-kappaB, and this transcriptional regulator is also required for increased production of TNFalpha. However, while TNFalpha is essential for hypoxic suppression of EAAT-2, hypoxic modulation of EAAT-1 expression is unaffected by this cytokine.

H2O 2-stimulated Ca2+ influx via TRPM2 is not the sole determinant of subsequent cell death.

Activation of transient receptor potential melastatin 2 (TRPM2), a non-selective, Ca(2+)-permeable cation channel, is implicated in cell death. Channel opening is stimulated by oxidative stress, a feature of numerous disease states. The wide expression profile of TRPM2 renders it a potentially significant therapeutic target in a variety of pathological settings including cardiovascular and neurodegenerative diseases. HEK293 cells transfected with human TRPM2 (HEK293/hTRPM2) were more vulnerable to H(2)O(2)-mediated cell death than untransfected controls in which H(2)O(2)-stimulated Ca(2+) influx was absent. Flufenamic acid partially reduced Ca(2+) influx in response to H(2)O(2) but had no effect on viability. N-(p-Amylcinnamoyl) anthranilic acid substantially attenuated Ca(2+) influx but did not alter viability. Poly(adenosine diphosphate ribose) polymerase inhibitors (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide, 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone and nicotinamide) reduced Ca(2+) influx and provided a degree of protection but also had some protective effects in untransfected controls. These data suggest H(2)O(2) triggers cell death in HEK293/hTRPM2 cells by a mechanism that is in part Ca(2+) independent, as blockade of channel opening (evidenced by suppression of Ca(2+) influx) did not correlate well with protection from cell death. Determining the underlying mechanisms of TRPM2 activation is pertinent in elucidating the relevance of this channel as a therapeutic target in neurodegenerative diseases and other pathologies associated with Ca(2+) dysregulation and oxidative stress.

Hypoxic remodelling of Ca2+signalling in SH-SY5Y cells: influence of glutathione.

Prolonged hypoxia alters various cellular processes, including Ca2+ signalling. As these effects can be prevented by antioxidants, we examined the role of glutathione, the major intracellular redox buffer, in modulation of Ca2+ signalling in the human neuroblastoma SH-SY5Y by hypoxia. Rises of [Ca2+]i evoked by bradykinin, and subsequent capacitative Ca2+ entry, were enhanced by prior hypoxia (1% O2, 24 h) without effect on reduced glutathione levels. Glutathione depletion reversed the effects of chronic hypoxia, but did not affect normoxically cultured cells. Elevation of glutathione levels also prevented the effects of hypoxia, but restored such effects in glutathione-depleted cells. Glutathione is therefore required for hypoxia to modify Ca2+ signalling, but its role is more complex than simple buffering of reactive oxygen species.

Agonist-induced calcium and oxidative stress responses in endothelial cells.

[Ca(2+)](i) (cytosolic [Ca(2+)]) and OS (oxidative stress) were measured simultaneously in calf pulmonary artery endothelial cells using fura-2 and carboxy-2',7'-dichlorodihydrofluorescein. ATP stimulated a [Ca(2+)](i) increase that was followed a few seconds later by an increase in OS. Pre-exposure to 5 microM H(2)O(2) potentiated these responses to ATP. Elevating or removing extracellular Ca(2+) increased or reduced the [Ca(2+)](i) response to ATP and caused parallel changes in the OS response, suggesting that this response was a consequence of the [Ca(2+)](i) response. Inhibition of mitochondria with rotenone or antimycin A affected the responses but not in a manner that allowed a simple interpretation of the role of mitochondria. These data show an intimate connection between [Ca(2+)](i) and OS that can be modulated by low levels of exogenously applied OS, allowing the possibility of positive feedback.