Proceedings of The Physiological Society
University College Dublin (2009) Proc Physiol Soc 15, C55
Relationship Between Glutathione Redox State And The Vasoconstrictor Response To Hypoxia In Rat Pulmonary Arteries
J. Prieto-Lloret1, J. P. Ward1, P. I. Aaronson1
1. Asthma, Allergy and Lung Biology, King's College London, London, United Kingdom.
Hypoxia elicits constriction of pulmonary arteries (hypoxic pulmonary vasoconstriction, HPV) but dilation of systemic arteries. It is proposed that hypoxia-induced changes in the pulmonary artery smooth muscle cell redox state and/or reactive oxygen species trigger HPV, although there is controversy as to whether this involves a net oxidation or reduction. Glutathione is the major thiol-disulfide redox buffer of the cell, so measurement of GSH and GSSG levels should provide an indication of overall cytoplasmic redox state. We measured the effect of hypoxia on GSH and GSSG in rat small intrapulmonary arteries (IPA) and aorta, using the approach of Griffith (1980), and investigated the effect of agents which should cause cell oxidation on HPV and GSH/GSSG ratio. The redox potential (expressed in mV) of the GSH-GSSG couple was calculated using the Nernst equation. Under normoxic conditions, the GSH-GSSG redox potential was -176±4mV (n=18) in IPA and -150mV (n=16) in aorta. After 45 min of hypoxia imposed by gassing the solution with 0, 1 and 2% O2, the redox potential in IPA was decreased to -159±6mV (n=14, p<0.05), -162±4mV (n=20, p<0.05) and -174±4mV (n=10, ns), respectively. Conversely, the redox potential of aorta after 45 min of hypoxia was -142±11 mV (n=12), -155±3mV (n=12) and -171±4mV (n=10, p<0.01), in 0, 1 and 2% O2, respectively. Thus, hypoxia tended to oxidize IPA. Glutathione reductase (GR) converts GSSG to GSH, whilst oxidizing NADPH to NADP+; NADPH is reformed from NADP+ by glucose-6-phosphate dehydrogenase. The conversion of GSH back to GSSG is mediated by glutathione peroxidase (GPx); peroxide is reduced to water in a coupled reaction. Carmustine (100μM), a GR blocker, completely abolished the sustained phase of HPV (n=8), and shifted the cell redox potential during normoxia from -172±3mV (n=8) to -154±6mV (n=6). Conversely, mercaptosuccinate (MS; 100μM), a blocker of GPx, increased the sustained phase of HPV by 47±13% (n=8, p<0.05). Neither carmustine nor MS affected the intitial transient phase of HPV. Ebselen (30μM), a GPx mimic, attenuated both transient (44±9%, n=3) and sustained phases (39±18%, n=3) of HPV. Dehydroepiandrosterone (100μM), which blocks glucose-6-phosphate dehydrogenase, shifted the normoxic cell redox potential to -154±6mV (n=6, P<0.05 vs control) and blocked sustained HPV by 43±13% (n=3). Diethylmaleate (1 mM), which depletes glutathione, shifted the cell redox potential to -105±6mV (n=3), and blocked sustained HPV by 36±6% (n=6). In summary, hypoxia caused an oxidizing shift in the redox state in intact IPA. Moreover, several substances which reduce total glutathione levels in tissue and caused cell oxidation consistently inhibited sustained HPV.
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