A. R. Whorton

Nitric oxide stimulates Nrf2 nuclear translocation in vascular endothelium.

Vascular endothelial cells respond to nitric oxide by activating MAPK pathways and upregulating stress-activated proteins such as gamma-glutamylcysteine synthetase (gamma-GCS) and heme oxygenase-1 (HO-1). Since consensus sequences for the antioxidant response element (ARE) are found in the promoters of the gamma-GCS and HO-1 genes, we examined nuclear translocation of Nrf2, a CNC-bZIP protein which binds to and activates the ARE. We found a dramatic increase in Nrf2 nuclear translocation 1-8h following the nitric oxide donor spermine NONOate. Translocation was inhibited by pretreatment of cells with N-acetylcysteine suggesting involvement of an oxidative mechanism in this response. Translocation was also blocked by PD 98059 and SB 203580, inhibitors of ERK and p38 pathways, respectively. In addition to effects on Nrf2 subcellular localization, spermine NONOate increased Nrf2 protein levels by a mechanism which was inhibited by PD 98059. Pretreatment with N-acetylcysteine, PD 98059, and SB 203580 decreased HO-1 upregulation in spermine NONOate-treated cells. These results suggest that ERK and p38 pathways may regulate nitric oxide-mediated adaptive responses in vascular endothelium via translocation of Nrf2 and activation of the ARE.

Nitric oxide production is enhanced in rat brain before oxygen-induced convulsions.

Central nervous system oxygen toxicity (CNS O2 toxicity) is preceded by release of hyperoxic vasoconstriction, which increases regional cerebral blood flow (rCBF). These increases in rCBF precede the onset of O2-induced convulsions. We have tested the hypothesis that hyperbaric oxygen (HBO2) stimulates NO* production in the brain that leads to hyperemia and anticipates electrical signs of neurotoxicity. We measured rCBF and EEG responses in rats exposed at 4 to 6 atmospheres (ATA) of HBO2 and correlated them with brain interstitial NO* metabolites (NO(x)) as an index of NO* production. During exposures to hyperbaric oxygen rCBF decreased at 4 ATA, decreased for the initial 30 min at 5 ATA then gradually increased, and increased within 30 min at 6 ATA. Changes in rCBF correlated positively with NO(x) production; increases in rCBF during HBO2 exposure were associated with large increases in NO(x) at 5 and 6 ATA and always preceded EEG discharges as a sign of CNS O2 toxicity. In rats pretreated with L-NAME, rCBF remained maximally decreased throughout 75 min of HBO2 at 4, 5 and 6 ATA. These data provide the first direct evidence that increased NO* production during prolonged HBO2 exposure is responsible for escape from hyperoxic vasoconstriction. The finding suggests that NO* overproduction initiates CNS O2 toxicity by increasing rCBF, which allows excessive O2 to be delivered to the brain.

Short-term regulation of Na+/K+ adenosine triphosphatase by recombinant human serotonin 5-HT1A receptor expressed in HeLa cells.

Agonist occupancy of the cloned human serotonin (5-HT)1A receptor expressed in HeLa cells stimulates Na+/K+ ATPase activity as assessed by rubidium uptake. The purpose of the study was to determine which of the receptor-associated signaling mechanisms was responsible for this effect. 5-HT stimulated Na+/K+ ATPase 38% at 2 mM extracellular potassium, an effect characterized by a decrease in apparent K0.5 from 2.8 +/- 0.3 to 1.8 +/- 0.3 mM potassium without a significant change in apparent Vmax. The EC50 for the transport effect was approximately 3 microM 5-HT. The response was pertussis toxin-sensitive but did not involve inhibition of adenylate cyclase, as stimulation of Na+/K+ ATPase by 5-HT was observed in the presence of excess dibutyryl cAMP. Protein kinase C was not required for the response since short-term incubation with the phorbol esters phorbol 12 myristate, 13 acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) did not mimic the 5-HT effect. Moreover, 5-HT increased Na+/K+ ATPase activity after inactivation of protein kinase C by overnight incubation with PMA. 5-HT and the sesquiterpene lactone thapsigargin increased cytosolic calcium in this cell model, and the EC50 for 5-HT corresponded with that for stimulation of Na+/K+ ATPase. Both thapsigargin and A23187, a calcium ionophore, also increased Na+/K+ ATPase activity in a dose-responsive fashion. The response to 5-HT, thapsigargin, and A23187 was blocked by conditions that removed the cytosolic calcium response. By two-dimensional gel electrophoresis, we established evidence for a calcium-sensitive but protein kinase C-independent signaling pathway. We conclude that the 5-HT1A receptor, which we have previously shown to stimulate phosphate uptake via protein kinase C, stimulates Na+/K+ ATPase via a calcium-dependent mechanism. This provides evidence for regulation of two separate transport processes by a single receptor subtype via different signaling mechanisms.

Tunicamycin increases intracellular calcium levels in bovine aortic endothelial cells

Tunicamycin is a nucleoside antibiotic that inhibits protein glycosylation and palmitoylation. The therapeutic use of tunicamycin is limited in animals because of its toxic effects, particularly in cerebral vasculature. Tunicamycin decreases palmitoylation of the endothelial isoform of nitric oxide synthase, stimulates nitric oxide synthesis, and increases the concentration of intracellular calcium ([Ca2+]i) in bovine aortic endothelial cells (B. J. Buckley and A. R. Whorton. FASEB J. 11: A110, 1997). In the present study, we investigated the mechanism by which tunicamycin alters [Ca2+]i using the Ca2+-sensitive dye fura 2. We found that tunicamycin increased [Ca2+]i without increasing levels of inositol phosphates. When cells were incubated in the absence of extracellular Ca2+, [Ca2+]i rapidly rose in response to tunicamycin, although a full response was not achieved. The pool of intracellular Ca2+ mobilized by tunicamycin overlapped with that mobilized by thapsigargin. Extracellular nickel blocked a full response to tunicamycin when cells were incubated in the presence of extracellular Ca2+. The effects of tunicamycin on [Ca2+]i were partially reversed by washing out the drug, and the remainder of the response was inhibited by removing extracellular Ca2+. These results indicate that tunicamycin mobilizes Ca2+ from intracellular stores in a manner independent of phospholipase C activation and increases the influx of Ca2+ across the plasma membrane.Tunicamycin is a nucleoside antibiotic that inhibits protein glycosylation and palmitoylation. The therapeutic use of tunicamycin is limited in animals because of its toxic effects, particularly in cerebral vasculature. Tunicamycin decreases palmitoylation of the endothelial isoform of nitric oxide synthase, stimulates nitric oxide synthesis, and increases the concentration of intracellular calcium ([Ca2+]i) in bovine aortic endothelial cells (B. J. Buckley and A. R. Whorton. FASEB J. 11: A110, 1997). In the present study, we investigated the mechanism by which tunicamycin alters [Ca2+]iusing the Ca2+-sensitive dye fura 2. We found that tunicamycin increased [Ca2+]iwithout increasing levels of inositol phosphates. When cells were incubated in the absence of extracellular Ca2+, [Ca2+]irapidly rose in response to tunicamycin, although a full response was not achieved. The pool of intracellular Ca2+ mobilized by tunicamycin overlapped with that mobilized by thapsigargin. Extracellular nickel blocked a full response to tunicamycin when cells were incubated in the presence of extracellular Ca2+. The effects of tunicamycin on [Ca2+]iwere partially reversed by washing out the drug, and the remainder of the response was inhibited by removing extracellular Ca2+. These results indicate that tunicamycin mobilizes Ca2+ from intracellular stores in a manner independent of phospholipase C activation and increases the influx of Ca2+ across the plasma membrane.

A cystine-cysteine shuttle mediated by xCT facilitates cellular responses to S-nitrosoalbumin.

We have shown previously that extracellular cysteine is necessary for cellular responses to S-nitrosoalbumin. In this study we have investigated mechanisms involved in accumulation of extracellular cysteine outside vascular smooth muscle cells and characterized the role of cystine-cysteine release in transfer of nitric oxide (NO)-bioactivity. Incubation of cells with cystine led to cystine uptake, reduction, and cysteine release. The process was inhibitable by extracellular glutamate, suggesting a role for system x(c)(-) amino acid transporters. Smooth muscle cells express this transporter constitutively and induction of the light chain component (xCT) by either diethyl maleate or 3-morpholino-sydnonimine (SIN-1) led to glutamate-inhibitable cystine uptake and an increased rate of cysteine release from cells. Likewise, overexpression of xCT in smooth muscle cells or endothelial cells led to glutamate-inhibitable cysteine release. The resulting extracellular cysteine was found to be required for transfer of NO from extracellular S-nitrosothiols into cells via system L transporters leading to formation of cellular S-nitrosothiols. Cysteine release coupled to cystine uptake was also found to be required for cellular responses to S-nitrosoalbumin and facilitated S-nitrosoalbumin-mediated inhibition of epidermal growth factor signaling. These data show that xCT expression can constitute a cystine-cysteine shuttle whereby cystine uptake drives cysteine release. Furthermore, we show that extracellular cysteine provided by this shuttle mechanism is necessary for transfer of NO equivalents and cellular responses to S-nitrosoablumin.