Decreased
oxygen delivery to cells (
hypoxia) is prevalent in a number of important diseases. Little is known about mechanisms of
oxygen sensing at the cellular level or about whether functional correlates of
oxygen sensing exist. In this study, we examined the impact of
hypoxia on stimulated epithelial ion transport function. T84 cells, a model of intestinal epithelia, were grown on permeable supports, exposed to
hypoxia (range 1-21% O2) for periods of time between 0 and 72 h and assessed for stimulated ion transport.
Hypoxia evoked a specific decrease in
cyclic nucleotide-stimulated (cAMP and cGMP) but not Ca++-stimulated ion transport. 86Rb (K+ tracer) uptake and 125I (Cl- tracer) efflux were reduced in hypoxic cells by >50% and >40%, respectively, fluid movement was reduced by
hypoxia (>50% decrease) and reoxygenation resulted in partial recovery of the ion transport responses. Stimulated and basal levels of both cAMP and cGMP were decreased in response to
hypoxia, although intracellular
ATP levels were unaltered under similar conditions. Exogenous addition of
cobalt,
nickel or
manganese, all of which compete for
oxygen binding on
heme-containing
proteins, mimicked
hypoxia. Because
guanylate cyclase is a
heme protein, we measured the influence of
cobalt on activity of
guanylate cyclase in purified plasma membrane preparations and found
cobalt to inhibit stimulated cGMP levels in this cell-free system. Finally, pharmacological lowering of intracellular cGMP (using
LY83583) resulted in decreased cAMP-stimulated Cl- secretion, and direct elevation of cGMP (using 8-bromo-cGMP or dibutyryl-cGMP) restored this
hypoxia-induced activity. We conclude that a potential
oxygen-sensing mechanism of epithelial cells involves the cooperation of
heme-containing
proteins such as
guanylate cyclase and that biochemical cross-talk between cAMP- and cGMP-stimulated pathways may be important in such responses.