Limited utilization of exogenous arachidonic acid by the prostaglandin cyclooxygenase in gastric mucosa: the role of protein binding, glutathione peroxidase, and hydrogen peroxides.
We investigated the utilization of exogenous 14C-labelled arachidonic acid by the cyclooxygenase system of the gastric mucosa and its alteration by cytosolic factors, protein binding, glutathione peroxidase (GSH-Px), and hydrogen peroxides. Total prostaglandin (PG) synthesis from gastric microsomes was reduced in a dose- dependent manner to 12% and 68% of controls by increasing amounts of the 105,000g supernatant or albumin (8mg protein/ml), respectively (p less than 0.01). The inhibitory cytosolic factor was heat labile, protease sensitive, and was retained by a 300,000 Dalton ultrafiltration membrane. Thus, it was likely a protein. Other possible inhibitory mechanisms like protease- or heme-induced destabilization of the cyclooxygenase, haptoglobin-mediated inhibition, or self-inactivation by endogenous substrate were excluded. N-ethylmaleimide (NEM), an agent that alkylates sulfhydryl-groups thereby inhibiting GSH-Px, abolished the inhibitory effect of cytosol in a dose-dependent fashion. In contrast to their inhibition of prostaglandin synthesis, the binding of arachidonic acid by albumin or cytosolic proteins accounted to 75% and 19% under comparable conditions, respectively, however, cytosolic fatty acid binding was unaffected by NEM. Thus, it was concluded that the inhibitory effect of cytosol, in contrast to albumin, was mediated by a sulfhydryl-depending process, probably a GSH-Px. This conclusion was supported by a qualitatively comparable inhibition by a purified GSH-Px from bovine erythrocytes. The inhibitory action of cytosolic proteins was reduced significantly by increasing concentrations or repeated application of arachidonic acid; therefore, cytosolic GSH-Px was likely to affect substrate utilization by the microsomal PGH synthase through reduction of activating substrate peroxides. Similarly, the in vitro formation of cyclooxygenase products by mucosal homogenate or gastric microsomes in the absence of cytosol was limited at substrate concentrations below 80 microM, despite sufficient nonesterified arachidonic acid remaining in the incubate. This limitation was mediated only partially by self-inactivation of the prostaglandin cyclooxygenase. Neither N-ethylmaleimide nor repeated application of hydrogen peroxides increased substrate utilization by isolated microsomes, excluding contamination by GSH-Px or simply a lack of hydrogen peroxides as possible mechanisms for the limited utilization. From these results, a special role of substrate-linked lipid peroxides in the activation of mucosal prostaglandin synthesis is proposed. The reduction of these peroxides by glutathione dependent or independent peroxidases, e.g. the PGH synthase-linked hydroperoxidase activity itself, could explain the reduced utilization of nonesterified arachidonic acid by the gastric mucosa.