The CA1 and CA3 regions of the hippocampus markedly differ in their susceptibility to hypoxia in general, and more particularly to the intermittent hypoxia that characterizes sleep apnea. Proteomic approaches were used to identify proteins differentially expressed in the CA1 and CA3 regions of the rat hippocampus and to assess changes in protein expression following a 6‐h exposure to intermittent hypoxia (IH). Ninety‐nine proteins were identified, and 15 were differentially expressed in the CA1 and the CA3 regions. Following IH, 32 proteins in the CA1 region and only 7 proteins in the more resistant CA3 area were up‐regulated. Hypoxia‐regulated proteins in the CA1 region included structural proteins, proteins related to apoptosis, primarily chaperone proteins, and proteins involved in cellular metabolic pathways. We conclude that IH‐mediated CA1 injury results from complex interactions between pathways involving increased metabolism, induction of stress‐induced proteins and apoptosis, and, ultimately, disruption of structural proteins and cell integrity. These findings provide initial insights into mechanisms underlying differences in susceptibility to hypoxia in neural tissue, and may allow for future delineation of interventional strategies aiming to enhance neuronal adaptation to IH.

In neurons, hypoxia activates intracellular death‐related pathways, yet the antiapoptotic mechanisms triggered by hypoxia remain unclear. In RN46A neuronal cells, minimum media growth conditions induced cell death as early as 12 h after the cells were placed in these conditions (i.e., after removal of B‐27 supplement). However, apoptosis occurred in hypoxia (1% O ₂ ) only after 48 h, and in fact hypoxia reduced the apoptosis associated with trophic factor withdrawal. Furthermore, hypoxia induced time‐dependent increases in expression of platelet‐derived growth factor (PDGF) B mRNA and protein, as well as PDGF‐β receptor phosphorylation. Although exogenous PDGF‐BB induced only transient Akt activation, hypoxia triggered persistent activation of Akt for up to 24 h. Inhibition of phosphatidylinositol 3‐kinase (PI3K) or of PDGF‐β receptor phosphorylation abrogated both hypoxia‐induced and exogenous PDGF‐BB‐induced Akt phosphorylation, and it completely abolished hypoxia‐induced protection from media supplement deprivation, which suggests that the long‐lasting activation of Akt during hypoxia and the prosurvival induction were due to endogenously generated PDGF‐BB. Furthermore, these inhibitors decreased hypoxia‐inducible factor 1α (HIF‐1α) DNA binding, which suggests that the PDGF/PDGF‐β receptor/Akt pathway induces downstream HIF‐1α gene transcription. We conclude that in RN46A neuronal cells, hypoxia activates an autocrine‐paracrine antiapoptotic mechanism that involves up‐regulation of PDGF‐B and PDGF‐β receptor‐dependent activation of the PI3K/Akt signaling pathway to induce downstream transcription of survival genes.

Episodic hypoxia, a characteristic feature of obstructive sleep apnea, induces cellular changes and apoptosis in brain regions associated with neurocognitive function. To investigate whether mild, intermittent hypoxia would induce more extensive neuronal damage than would a similar degree of sustained hypoxia, rat pheochromocytoma PC-12 neuronal cells were subjected to either sustained (5% O₂) or intermittent (alternating 5% O₂ 35 min, 21% O₂ 25 min) hypoxia for 2 or 4 days. Quantitative assessment of apoptosis showed that while mild sustained hypoxia did not significantly increase cell apoptosis at 2 days (1.31 ± 0.29-fold, n = 8; P = NS), a significant increase in apoptosis occurred after 4 days (2.25 ± 0.4-fold, n = 8; P < 0.002), without increased caspase activation. Furthermore, caspase inhibition with the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) did not modify sustained hypoxia-induced apoptosis. In contrast, mild, intermittent hypoxia induced significant increases in apoptosis at 2 days (3.72 ± 1.43-fold, n = 8; P < 0.03) and at 4 days (4.57 ± 0.82-fold, n = 8; P < 0.001) that was associated with enhanced caspase activity and attenuated by Z-VAD-FMK pretreatment. We conclude that intermittent hypoxia induces an earlier and more extensive apoptotic response than sustained hypoxia and that this response is at least partially dependent on caspase-mediated pathways. In contrast, caspases do not seem to play a role in sustained hypoxia-induced apoptosis. These findings suggest that different signaling pathways are involved in sustained and intermittent hypoxia-induced cell injury and may contribute to the understanding of differential brain susceptibility to sustained and intermittent hypoxia.

Tyrosine hydroxylase, a hypoxia-regulated gene, may be involved in tissue adaptation to hypoxia. Intermittent hypoxia, a characteristic feature of sleep apnea, leads to significant memory deficits, as well as to cortex and hippocampal apoptosis that are absent after sustained hypoxia. To examine the hypothesis that sustained and intermittent hypoxia induce different catecholaminergic responses, changes in tyrosine hydroxylase mRNA, protein expression, and activity were compared in various brain regions of male rats exposed for 6 h, 1 day, 3 days, and 7 days to sustained hypoxia (10% O₂), intermittent hypoxia (alternating room air and 10% O₂), or normoxia. Tyrosine hydroxylase activity, measured at 7 days, increased in the cortex as follows: sustained > intermittent > normoxia. Furthermore, activity decreased in the brain stem and was unchanged in other brain regions of sustained hypoxia-exposed rats, as well as in all regions from animals exposed to intermittent hypoxia, suggesting stimulus-specific and heterotopic catecholamine regulation. In the cortex, tyrosine hydroxylase mRNA expression was increased, whereas protein expression remained unchanged. In addition, significant differences in the time course of cortical Ser⁴⁰tyrosine hydroxylase phosphorylation were present in the cortex, suggesting that intermittent and sustained hypoxia-induced enzymatic activity differences are related to different phosphorylation patterns. We conclude that long-term hypoxia induces site-specific changes in tyrosine hydroxylase activity and that intermittent hypoxia elicits reduced tyrosine hydroxylase recruitment and phosphorylation compared with sustained hypoxia. Such changes may not only account for differences in enzyme activity but also suggest that, with differential regional brain susceptibility to hypoxia, recruitment of different mechanisms in response to hypoxia will elicit region-specific modulation of catecholamine response.

Alveolar macrophages play a critical role in silica-induced lung fibrosis. Silica exposure induces tumor necrosis factor (TNF)- α release and nuclear factor (NF)- κ B activation, and apoptotic mechanisms have been implicated in silica-induced pathogenesis. To characterize potential relationships between these signaling events, we studied their induction in two murine macrophage cell lines. The RAW 264.7 macrophage cell line was more sensitive, and the IC-21 macrophage cell line more tolerant to silica exposure (0.2 or 1 mg/ml for 6 h) as evidenced by significantly higher apoptotic responses in RAW 264.7 (P < 0.05). RAW 264.7 macrophages exhibited enhanced TNF- α production and NF- κ B activation in response to silica, whereas IC-21 macrophages did not produce TNF- α in response to silica and did not induce NF- κ B nuclear binding. Inhibition of NF- κ B in RAW 264.7 cells with BAY11–7082 significantly increased apoptosis while inhibiting TNF- α release. In addition, TNF- α and NF- κ B activation, but not apoptosis, were induced by lipopolysaccharide (LPS) in both cell lines, and NF- κ B inhibition reduced LPS-induced TNF- α release. These data suggest that TNF- α induction is dependent on NF- κ B activation in both cell lines. However, silica can induce apoptosis in murine macrophages, independently of TNF- α stimulation, as in IC-21 macrophages. Furthermore, NF- κ B activation in macrophages may play dual roles, both pro- and antiapoptotic during silica injury.