Oxidative activation of guinea pig splenic cell guanylate cyclase activity by dehydroascorbate, ascorbate, fatty acid hydroperoxides and prostaglandin endoperoxides

The modulation of cyclic GMP metabolism by oxidative-reductive related processes was studied in intact guinea pig splenic cells and with respect to the activities of soluble and particulate guanylate cyclase. In intact cells the oxidant, dehydroascorbic acid (DHA), produced time and concentration dependent increases in cyclic GMP steady state levels. The thiol reductants, cysteine or DTT, decreased cellular cGMP levels 50 to 70%. Exposure of cells to sulfhydryl reactive reagents such as N-ethylmaleimide (NEM), p-hydroxymercuribenzoate, or 5,5′ dithiobis- [2-nitrobenzoic acid] or to the transition metal chelator, 4,7 -diphenyl-1,10 phenanthroline also caused striking lowering of cGMP levels. DHA-induced elevation of cGMP in intact cells persisted after removal of the oxidant and could be reversed upon reduction by DTT. Depression of cGMP steady state levels was induced by DTT which was also shown to persist after removal of the reductant and to be reversed by exposure of the cells to the oxidant, DHA. The effects produced by DHA, DTT, and NEM on soluble and particulate fractions of guanylate cyclase from these cells corresponded to the alterations each produced on cGMP steady state levels in the intact cells. DHA (0.1 to 10 mm) activated while DTT (0.1 to 5 mm) or NEM (0.5 to 5 mm) inhibited guanylate cyclase activity. DHA activation of the soluble enzyme was rapid and additive with the slower activation that occurred spontaneously in air; DTT could prevent or reverse DHA as well as spontaneous air activation. Activation or inhibition by DHA or DTT, respectively, of the particulate enzyme persisted after removal of the effectors but the stable effect produced by the oxidant could be reversed by the reductant and vice versa. Low concentrations of NEM (0.05–0.1 mm) which inhibited basal guanylate cyclase activity minimally (20–25%) completely abolished sensitivity to DHA-activation. Cells exposed to DHA, resuspended in DHA-free media and lysed, exhibited 3 to 4 fold enhanced activity of guanylate cyclase which was restored to the lower basal level of activity upon addition of DTT during the course of the cyclase reaction. Ascorbic acid has similar effects on cGMP metabolism in intact cells including the effect to produce a stable enhancement of guanylate cyclase activity which persists after cell disruption and is suppressed by DTT. However, unlike DHA, ascorbate has no effect on guanylate cyclase activity after cell disruption. Fatty acid hydroperoxides 12-OOH-20:4 (HPETE), 15-OOH-20:4 13-OOH-18:2, and PGE2-OOH and the prostaglandin endoperoxides PGG2 and PGH2 were also shown to be effective activators of soluble (but not particulate) splenic cell guanylate cyclase activity. The hydroxy analogues of these fatty acids and the prostaglandins PGE2 and PGF2 α as well as arachidonic, linoleic, oleic and stearic acids had little or no effect. The stimulatory effects of the lipid hydroperoxides and endoperoxides were demonstrable in air or an argon atmosphere and were prevented and reversed by thiol reductants. Stimulation by both DHA and PGG2 exhibited additivity with respect to promoting the rate at which activation proceeded to achieve a state of maximal catalysis. These results indicate that the soluble guanylate cyclase from splenic cells exhibits regulatory sites for hydrophobic and hydrophilic oxidants which can activate the enzyme by promoting a sulfhydryl-disulfide interconversion at these sites. It is suggested that the action of certain hormones and other membrane active substances may be linked to guanylate cyclase through specific redox systems. The components comprising the link, which remain to be identified, may differ depending upon the cell-type or specific effector at the cell plasma membrane.