Abstract
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.
Footnotes
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Send reprint requests to: Sean P. Colgan, Ph.D., Center for Experimental Therapeutics & Reperfusion Injury, Brigham and Women’s Hospital, Thorn 7, 75 Francis Street, Boston, MA 02115.
- Abbreviations:
- GC
- guanylate cyclase
- AC
- adenylate cyclase
- SCC
- short circuit current
- TER
- transepithelial resistance
- IBMX
- 3-isobutyl 1 methylxanthine
- HBSS
- Hanks’ balanced salt solution
- PMSF
- phenylmethylsulfonyl fluoride
- ANOVA
- analysis of variance
- VIP
- vasoactive intestinal peptide
- 5′-AMP
- 5′-adenosine monophosphate
- STa
- E. coli heat-stable enterotoxin
- Received March 28, 1997.
- Accepted October 7, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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