Hideya Hayashi

A superfamily of NADPH-dependent reductases in eukaryotes and prokaryotes

Aldose reductase (AR) is implicated in some of the disabling complications of diabetes, including neuropathy, retinopathy and cataracts. Our studies are aimed at further clarifying the role of AR in diabetes and facilitating the design of new classes of potent, specific AR inhibitors by gaining an understanding of the protein structure of AR. To this end, we have determined the complete protein sequence of rat lens AR using cDNA analysis and primer extension of mRNA. By comparing protein sequences, we have found that the structural relatedness (41% to 57%) among the vertebrate proteins, aldose reductase, aldehyde reductase, prostaglandin F synthase and the frog lens protein rho-crystallin can now be extended to prokaryotes by the inclusion of Corynebacterium 2,5-diketo-D-gluconate reductase. This more distantly related protein shares 30-40% identity with the vertebrate enzymes. Sequence alignments reveal that 18% of the amino acids are completely conserved in all members of the superfamily, many of them in clusters, suggesting that they mark important structural features such as the nucleotide binding site and substrate binding site. rho-Crystallin, which is structurally related to this superfamily of NADPH-dependent reductases, does not appear to reduce PGH2, PGD2, xylose or glyceraldehyde to any appreciable extent. It does, however, bind NADPH.

Transport of prostaglandin D2 into brain

We examined the brain uptake of prostaglandin D2 after its i.v. administration to mice. The determination of this prostaglandin in tissues was performed by a sensitive and rapid radioimmunoassay after purification of the prostaglandin by high-performance liquid chromatography. When 1.0 mg/kg of prostaglandin D2 was injected, it was detectable in the brain 30 s after its administration (44 ng/g brain, about 0.08% of the administered dose). Brain uptake of prostaglandin D2 was dose-dependent over the dose range from 0.1 to 1.0 mg/kg and its half-life in the brain and blood was 1.1 and 0.9 min, respectively. The brain/blood ratio of the prostaglandin D2 concentration was 0.03 at 30 s and gradually increased to 0.2 during the first 15 min. These results suggest that prostaglandin D2 is taken up intact by the brain and disappears rapidly. Studies on the metabolic fate of tritium-labeled prostaglandin D2 revealed that it was a major component at 1 min, was metabolized to a number of hydrophilic and hydrophobic substances in the brain, whereas it was metabolized to mainly hydrophilic substances in the blood at 6 and 15 min, and the total radioactivities were cleared from the brain and blood with half-lives of 1.6 and 1.5 min, respectively.

Basal level of prostaglandin D2 in rat brain by a solid-phase enzyme immunoassay

A solid-phase enzyme immunoassay for prostaglandin D2 (PGD2) was developed in which PGD2 was labeled with horseradish peroxidase. After competitive binding to the immobilized antibody between enzyme-labeled and free PGD2, the activity of the enzyme bound to the antibody was assayed fluorometrically using 3-(p-hydroxyphenyl)-propionic acid and hydrogen peroxide as substrates. The procedure allowed determinations of 3-100 pg for PGD2. The IC50 value for PGD2 in the solid-phase enzyme immunoassay was about 25 pg and the sensitivity was improved about 10 times compared to those in radioimmunoassay and in solution-phase enzyme immunoassay. The solid-phase enzyme immunoassay was applied to the measurement of PGD2 content in rat brain and thereby an octadecylsilyl silica cartridge and a reversed-phase HPLC were sequentially used for sample preparations. Heads were immediately frozen in liquid nitrogen after decapitation to avoid a postmortem formation of PGD2. PGD2 contents measured by solid-phase enzyme immunoassay correlated well with the values obtained by radioimmunoassay (r = 0.966) after raising its contents by intravenous administration of PGD2. The in vivo level of PGD2 in rat brain was extremely low but determined to be 0.11 +/- 0.03 ng/g tissue (mean +/- S.E.M.) with this enzyme immunoassay. The result was equal to the value extrapolated to zero time from the postmortem change.

Synergistic Effect of Prostaglandin E2 and Ouabain on Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells

Abstract: We recently reported that prostaglandin E2 (PGE2) stimulated phosphoinositide metabolism in cultured bovine adrenal chromaffin cells and that PGE2 and ouabain, an inhibitor of Na+, K+‐ATPase, synergistically induced a gradual secretion of catecholamines from the cells. The effect on catecholamine release was specific for prostaglandin E1 (PGE1) and PGE2 among prostaglandins tested (E1= E2 > F2α > D2). The release evoked by PGE2 plus ouabain was greatly reduced in Na+‐depleted medium and not observed in Ca2+‐free medium. Here we examined the synergistic effect of PGE2 and ouabain on the release with specific reference to ion fluxes. Regardless of the presence of PGE2, ouabain stimulated the release in a dose‐dependent manner with half‐maximal stimulation at 1 μM, and omission of K+ from the medium, a condition which suppresses the Na+, K+‐ATPase activity, also enhanced the release from chromaffin cells exposed to PGE2. Ouabain induced a continuous accumulation of 22Na+ and 45Ca2+, as well as secretion of catecholamines. Although PGE2 itself showed hardly any effects on these cellular responses, PGE2 potentiated all of them induced by ouabain. The time course of catecholamine release was correlated with that of accumulation of 45Ca2+ rather than with that of 22Na+. The release evoked by PGE2 and ouabain was inhibited in a dose‐dependent manner by amiloride and the analogue ethylisopropylamiloride, inhibitors of the Na+,H+‐antiport, but not by the Na+‐channel inhibitor tetrodotoxin nor by the nicotinic receptor antagonist hexamethonium. Ethylisopropylamiloride at 1 μM inhibited PGE2‐enhanced accumulation of 22Na+ and 45Ca2+ and release of catecholamine by 40, 83, and 71%, respectively. Activation of the Na+,H+‐antiport by elevation of the extracellular pH from 6.6 to 8.0 increased the release of catecholamines linearly. Furthermore, PGE2 induced a sustained increase in intracellular pH by about 0.1 pH unit above the resting value, which was abolished by amiloride or in Na+‐free medium. These results taken together indicate that PGE2 activates the Na+,H+‐antiport by stimulating phosphoinositide metabolism and that the increase in intracellular Na+ by both inhibition of Na+, K+‐ATPase and activation of Na+,H+‐antiport may lead to the redistribution of Ca2+, which is the initial trigger of catecholamine release.

Enzymatic formation of prostaglandin F2α in human brain

Prostaglandin (PG)E2 9-ketoreductase, which catalyzes the conversion of PGE2 to PGF2α, was purified from human brain to apparent homogeneity. The molecular weight, isoelectric point, optimum pH, Km value for PGE2, and turnover number were 34,000, 8.2, 6.5–7.5, 1.0 mM, and 7.6 min−1, respectively. Among PGs tested, the enzyme also catalyzed the reduction of other PGs such as PGA2, PGE1, and 13,14-dihydro-15-keto PGF2α, but not that of PGD2, 11β-PGE2, PGH2, PGJ2, or Δ12-PGJ2. The reaction product formed from PGE2 was identified as PGF2α, by TLC combined with HPLC. This enzyme, as is the case for carbonyl reductase, was NADPH-dependent, preferred carbonyl compounds such as 9,10-phenanthrenequinone and menadione as substrates, and was sensitive to indomethacin, ethacrynic acid, and Cibacron blue 3G-A. The reduction of PGE2 was competitively inhibited by 9,10-phenanthrenequinone, which is a good substrate of this enzyme, indicating that the enzyme catalyzed the reduction of both substrates at the same active site. These results suggest that PGE2 9-ketoreductase, which belongs to the family of carbonyl reductases, contributes to the enzymatic formation of PGF2α in human brain.

The Role and New Action Mechanism of Prostaglandin E2 in Neurotransmission

Prostaglandin E, (PGE,) is known to inhibit the release of norepinephrine from sympathetic nerve terminals in response to acetylcholine. Since acetylcholine stimulates the formation of PGs, Hedqvist proposed that PGE, could act as a negative feedback inhibitor of adrenergic transmission. A survey of the distribution of [3H]PGE2 binding activity in various tissues from several species revealed a highly specific activity in the bovine adrenal medulla.* Chromaffin cells of the adrenal medulla synthesize, store, and secrete catecholamines, and the secretory response is elicited via nicotinic cholinergic receptors, Therefore, chromaffin cells are considered as a model system of sympathetic nerve cells, and they have offered insights into the workings of neurotransmission. In this study, we examined the effect of PGE, on catecholamine release from cultured bovine chromaffin cells. FIGURE 1 shows the time course of catecholamine release induced by various stimuli. Neither 1 KM PGE, nor 100 pM ouabain, an inhibitor of Na, K+-ATPase, has hardly any effect by itself on catecholamine release over the basal level. However, PGE2 and ouabain together enhanced the release drastically. The time course of catecholamine release was slow at the onset, and progressive at least until 30 min. Incubation of chromaffin cells with 10 pM nicotine induced a rapid and transient secrekion of catecholamines, 11% of the total being released from the cells during a 10-min period. The difference in the rate and magnitude of the secretory response to PGE2 plus ouabain and nicotine indicates that these two stimuli are acting by different mechanisms.