Endothelial cell phospholipid distribution and phospholipase activity during acute and chronic hypoxia

Abstract

We have previously reported alterations in cyclooxygenase metabolism in cultured aortic and pulmonary arterial endothelial cells exposed to acute and chronic hypoxia. These alterations depended on the duration and degree of the hypoxic exposure, on the vascular bed from which the endothelial cells were derived, and possibly on the availability of arachidonic acid secondary to modifications in metabolic substrate, membrane phospholipids, and/or membrane phospholipase activity. To investigate this last point further, we have compared plasma membrane phospholipid distribution and phospholipase activity in cultured aortic and pulmonary arterial endothelial cells exposed to both acute and chronic hypoxia, using two different precursors (acetic acid and arachidonic acid) and three different membrane preparations (cell homogenates, partially purified plasma membranes, and highly purified plasma membranes). We found that exposure to acute and chronic hypoxia has profound and complicated effects on endothelial cell phospholipid composition and phospholipase activity and that these effects depend on the origin of the endothelial cells and the duration of hypoxia. Furthermore, we found that the alterations in endothelial cell phospholipid distribution in response to hypoxia depend on the purity of the plasma membrane preparation and the metabolic precursor used to study phospholipid metabolism. Finally, these studies suggested that alterations in phospholipids during hypoxia occurred to a greater extent in compartments of endothelial cells other than the plasma membranes and that the well-recognized tolerance of endothelial cells to hypoxia may be due, in part, to preservation of the integrity of their plasma membranes during exposure to acute and chronic hypoxia.

Endothelial cell tolerance to hypoxia. Potential role of purine nucleotide phosphates.

“…Similar to previous reports, PAEC cultured long-term in 3% oxygen proliferated, retained the characteristics associated with cultured endothelial cells, and demonstrated no evidence of cellular injury (9)(10)(11)16). …”

“…In addition, this is the initial study to suggest that metabolic arrest, an increasingly reported mechanism for developing hypoxia tolerance in invertebrates and lower vertebrates, may also occur in some mammalian cells. Although further studies will be necessary to confirm these suggestions and to determine whether this preservation of high energy phosphates is sufficient, in and of itself, to confer hypoxia tolerance, it is not improbable that other measures, such as the lack of plasma membrane hydrolysis ( 16) and the upregulation of a specific set of stress proteins (1 1, 21, 42), may also contribute to the ability of PAEC to tolerate acute and chronic decreases in ambient oxygen concentration.…”

“…The sealed chambers for hypoxic incubations were from Billups-Rothenburg (Del Mar, CA Cell culture. Bovine PAEC were obtained as previously described (9)(10)(11)16). Endothelial cells were harvested by lightly scraping the luminal surface of longitudinally opened pulmonary arteries; the cells were suspended in MEM containing 15% heat inactivated bovine calf serum and initially seeded in 25-cm2 flasks or 60-mm plastic dishes.…”

“…Endothelial and epithelial cell monolayers exposed to hypoxic conditions were assessed for injury by phase microscopic appearance, adherent cell counts, trypan blue exclusion, and chromium-51 release (9,16,20,21 (Table I). In contrast, exposure to longer periods of hypoxia (18 h) modified the uptake and incorporation of radiolabeled adenosine or guanosine in both endothelial cells and renal tubular epithelial cells; however, these alterations were different depending on the cell type (Table I).…”

“…Although little is known about the mechanisms by which endothelial cells, particularly PAEC, maintain cellular and functional integrity under these physiological and pathological conditions, several recent studies have linked their hypoxia tolerance, in part, to the ability to preserve plasma membrane integrity (16) and to underlying modes of energy metabolism (17)(18)(19). The latter studies have shown that endothelial cells are predominantly governed by glycolytic metabolism and possess a large capacity to increase this metabolism.…”