Sleep-disordered breathing with recurrent apnea (periodic cessation of breathing) results in chronic intermittent hypoxia (IH), which leads to cardiovascular and respiratory pathology. Molecular mechanisms underlying IH-evoked cardio-respiratory co-morbidities have not been delineated. Mice with heterozygous deficiency of hypoxia-inducible factor 1α (HIF-1α) do not develop cardio-respiratory responses to chronic IH. HIF-1α protein expression and HIF-1 transcriptional activity are induced by IH in PC12 cells. In the present study, we investigated the signaling pathways associated with IH-evoked HIF-1α accumulation. PC12 cells were exposed to aerobic conditions (20% O 2 ) or 60 cycles of IH (30 sec at 1.5% O 2 followed by 5 min at 20% O 2 ). Our results show that IH-induced HIF-1α accumulation is due to increased generation of ROS by NADPH oxidase. We further demonstrate that ROS-dependent Ca 2+ signaling pathways involving phospholipase Cγ and protein kinase C activation are required for IH-evoked HIF-1α accumulation. IH leads to activation of mTOR and S6 kinase and rapamycin partially inhibited IHinduced HIF-1α accumulation. IH also decreased hydroxylation of HIF-1α protein and antioxidants as well as inhibitors of Ca +2 signaling prevented this response. Thus, both increased mTOR-dependent HIF-1α synthesis and decreased hydroxylase-dependent HIF-1α degradation contribute to IH-evoked HIF-1α accumulation. Following IH, HIF-1α and phosphorylated mTOR levels remained elevated during 90 min of re-oxygenation despite re-activation of prolyl hydroxylase. Rapamycin or cycloheximide, blocked increased HIF-1α levels during reoxygenation indicating that mTOR-dependent protein synthesis is required for the persistent elevation of HIF-1α levels during re-oxygenation.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity is modulated in vivo either by reaction with CO2 and Mg2+ to carbamylate a lysine residue in the catalytic site, or by the binding of inhibitors within the catalytic site. Binding of inhibitors blocks either activity or the carbamylation of the lysine residue that is essential for activity. At night, in many species, 2-carboxyarabinitol-1-phosphate (CA1P) is formed which binds tightly to Rubisco, inhibiting catalytic activity. Recent work has shown that tight-binding inhibitors can also decrease Rubisco activity in the light and contribute to the regulation of Rubisco activity. Here we determine the influence that such inhibitors of Rubisco exert on catalytic activity during drought stress. In tobacco plants, 'total Rubisco activity', i.e. the activity following pre-incubation with CO2 and Mg2+, was positively correlated with leaf relative water content. However, 'total Rubisco activity' in extracts from leaves with low water potential increased markedly when tightly bound inhibitors were removed, thus increasing the number of catalytic sites available. This suggests that in tobacco the decrease of Rubisco activity under drought stress is not primarily the result of changes in activation by CO2 and Mg2+ but due rather to the presence of tight-binding inhibitors. The amounts of inhibitor present in leaves of droughted tobacco based on the decrease in Rubisco activity per mg soluble protein were usually much greater than the amounts of the known inhibitors (CA1P and 'daytime inhibitor') that can be recovered in acid extracts. Alternative explanations for the difference between maximal and total activities are discussed.

Intermittent hypoxia (IH) occurs in many pathological conditions including recurrent apneas. Hypoxia-inducible factors (HIFs) 1 and 2 mediate transcriptional responses to low O 2 . A previous study showed that HIF-1 mediates some of the IH-evoked physiological responses. Because HIF-2␣ is an orthologue of HIF-1␣, we examined the effects of IH on HIF-2␣, the O2-regulated subunit expression, in pheochromocytoma 12 cell cultures. In contrast to the up-regulation of HIF-1␣, HIF-2␣ was down-regulated by IH. Similar down-regulation of HIF-2␣ was also seen in carotid bodies and adrenal medullae from IH-exposed rats. Inhibitors of calpain proteases (ALLM, ALLN) prevented IH-evoked degradation of HIF-2␣ whereas inhibitors of prolyl hydroxylases or proteosome were ineffective. IH activated calpain proteases and down-regulated the endogenous calpain inhibitor calpastatin. IH-evoked HIF-2␣ degradation led to inhibition of SOD2 transcription, resulting in oxidative stress. Over-expression of transcriptionally active HIF-2␣ prevented IH-evoked oxidative stress and restored SOD2 activity. Systemic treatment of IH-exposed rats with ALLM rescued HIF-2␣ degradation and restored SOD2 activity, thereby preventing oxidative stress and hypertension. These observations demonstrate that, unlike continuous hypoxia, IH leads to down-regulation of HIF-2␣ via a calpain-dependent signaling pathway and results in oxidative stress as well as autonomic morbidities.calcium signaling ͉ hypoxia inducible factors S leep-disordered breathing with recurrent apneas is a leading cause of morbidity and mortality affecting an estimated 18 million people in the United States alone (1-4). Recurrent apneas are characterized by transient, repetitive cessations of breathing (Ϸ10 sec in adults) resulting in periodic decreases in arterial blood O 2 or intermittent hypoxia (IH). Patients with recurrent apneas are at risk for developing several comorbidities including hypertension, sympathetic activation, breathing abnormalities, atherosclerosis, and stroke (4-8). Exposure of rodents to IH alone induces several co-morbidities reported in patients with recurrent apnea (9-11). However, little information is available on the molecular mechanisms underlying the morbidities associated with IH.Hypoxia-inducible factors (HIFs) mediate transcriptional responses to low O 2 (12). HIF-1 is the prototypical member of the HIF family and comprises an O 2 -regulated ␣ subunit and a constitutive ␤ subunit (13). HIF-1 transcriptional activity is induced under continuous hypoxia (CH) as a result of HIF-1␣ protein accumulation resulting from decreased O 2 -dependent proline hydroxylation, ubiquitination, and proteasomal degradation (14). Recent studies showed that IH leads to HIF-1␣ accumulation and utilizes signaling pathways distinct from those identified with CH (15). The importance of HIF-1 to IHassociated physiological and pathophysiological responses was studied in mice with heterozygous deficiency of HIF-1␣. IHevoked cardio-respiratory and metabolic morbidities were absent ...