Osamu Hayaishi

Nociceptive effects induced by intrathecal administration of prostaglandin D2, E2, or F2α to conscious mice

The effects of intrathecal administration of prostaglandins on pain responses in conscious mice were evaluated by using hot plate and acetic acid writhing tests. Prostaglandin D2 (0.5-3 ng/mouse) had a hyperalgesic action on the response to a hot plate during a 3-60 min period after injection. Prostaglandin E2 showed a hyperalgesic effect at doses of 1 pg to 10 ng/mouse, but the effect lasted shorter (3-30 min) than that of prostaglandin D2. Similar results were obtained by acetic acid writhing tests. The hyperalgesic effect of prostaglandin D2 was blocked by simultaneous injection of a substance P antagonist (greater than or equal to 100 ng) but not by AH6809, a prostanoid EP1-receptor antagonist. Conversely, prostaglandin E2-induced hyperalgesia was blocked by AH6809 (greater than or equal to 500 ng) but not by the substance P antagonist. Prostaglandin F2 alpha had little effect on pain responses. These results demonstrate that both prostaglandin D2 and prostaglandin E2 exert hyperalgesia in the spinal cord, but in different ways.

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.

Awaking effect of prostaglandin E2 in freely moving rats

The awaking effect of prostaglandin (PG) E2 was further examined in a long-term bioassay system. PGE2 in saline solution was infused between 11.00 and 17.00 h at 0.1, 1, 10, and 100 pmol/min (infusion volume 10 microliters/h) into the third cerebral ventricle of freely moving rats. These rats were otherwise infused with saline continuously and exhibited a circadian cycle, spending 70% of the daytime and 37% of the night in sleep. In the rats that received PGE2 infusion at 1, 10, and 100 pmol/min, slow wave sleep (SWS) decreased to 84%, 69% and 71% and paradoxical sleep (PS) to 85%, 37% and 40% of the paired controls. Thus, the effect of PGE2 was not specific to either SWS or PS. No effects were observed in the rats that received PGE2 at 0.1 pmol/min. After PGE2 infusion at 10 and 100 pmol/min, marked rebounds of both SWS and PS occurred during the night. SWS reduction by PGE2 was due to the shortened duration of SWS episodes, while SWS increase in the rebound phase was due to the increased number of episodes. PS reduction was due to both the shortened duration and decreased number of PS episodes and PS rebound was due to both the prolonged duration and increased number of episodes. The circadian sleep-wake cycle returned to the baseline on the first or second recovery day after PGE2 infusion. Sleep reduction by PGE2 was accompanied by elevation of the brain temperature and rebound increase of sleep occurred with the fall of the brain temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of prostaglandin D2 and its analogue, BW245C, on intraocular pressure in humans.

The effects of topically applied prostaglandin (PG) D2 and BW245C, a potent PGD2 agonist, on intraocular pressure (IOP) were studied in normotensive human volunteers. Doses of 5 and 10 μg PGD2 induced a mean reduction in IOP of 0.8 and 1 mmHg, respectively. At a dose of 50 μg, hypotension was preceded by initial hypertension (4 mmHg at 0.5 h) and the magnitude of the mean 101? reduction during the hypotensive phase was 1.1 mmHg. The application of BW245C (2.5 μg) induced an IOP change similar to that observed following treatment with 50 μg PGD2. Side effects caused by these compounds included conjunctival hyperemia, itching, and foreign-body and mild burning sensations. However, miosis and signs of intraocular inflammation were not observed. These results indicate that although PGD2 and BW245C are effective in reducing human IOP, their clinical usefulness as anti-glaucoma drugs may be limited by the extraocular side effects.

Decrease in tetrahydrobiopterin content and neurotransmitter amine biosynthesis in rat brain by an inhibitor of guanosine triphosphate cyclohydrolase.

To investigate the regulatory role of tetrahydrobiopterin in neurotransmitter amine biosynthesis, 2,4-diamino-6-hydroxypyrimidine, a potent inhibitor of guanosine triphosphate cyclohydrolase which is a rate-limiting enzyme of tetrahydrobiopterin biosynthesis, was administered intraperitoneally to weanling rats. Four h after 4 injections at 4-h intervals, the biopterin contents in plasma and liver were reduced to the level of 9 and 3.5%, respectively, of those in the control group injected with saline; while the contents in the whole brain, neocortex + striatum, diencephalon, and brainstem were 34, 50, 33 and 28%, respectively, of the control level. When in vivo tyrosine and tryptophan hydroxylase activities were measured over a 30-min period after the inhibition of aromatic amino acid decarboxylase, the accumulation of dihydroxyphenylalanine was reduced to 74, 77, 67 and 69% of the control in the whole brain, neocortex + striatum, diencephalon, and brainstem, respectively; and the accumulation of 5-hydroxytryptophan, to 71, 74, 66 and 65% of the control, respectively. On the other hand, 5-hydroxytryptamine and 5-hydroxyindole acetic acid contents were not altered in any brain regions, although norepinephrine and dopamine contents were reduced to approximately 70% of the control in the brainstem and the contents of dopamine metabolites were significantly decreased in the diencephalon and brainstem. Plasma phenylalanine level was significantly elevated, while the plasma tyrosine level was reduced, compared with the control level of these amino acids. These results indicate that the drug-treated rats could be an animal model for tetrahydrobiopterin-deficient disease involving neurological disorder.

Changes of enzymes involved in prostaglandin metabolism and prostaglandin binding proteins in rat brain during development and aging.

Abstract: In the developing rat brain, the enzymatic formation of prostaglandin D2 from prostaglandin H2 increased 60‐fold from day 12 of gestation to birth. The activity still rose gradually to the highest level (90 nmol/min/g wet tissue) at day 7 after birth. The activities of prostaglandin E2 and F2α synthetases in rat brain were highest at gestational age 19 days (30 nmol/min/g wet tissue) and at gestational age 14 days (15 nmol/min/g wet tissue), respectively. The specific activity of NADP‐dependent 15‐hydroxy‐prostaglandin D2 dehydrogenase in rat brain was highest at the earliest gestational age we examined (day 12 of gestation), The specific bindings of prostaglandin D2 and E2 to the crude mitochondrial fraction of rat brain were observed from day 16 of gestation and increased to day 7 after birth. Although the activities of the enzymes responsible for prostaglandin metabolism were unchanged postmaturationally, the maximal concentrations of the binding sites on the synaptic membrane for both prostaglandins D2 and E2 decreased with constant affinity to less than one‐sixth with age from 1 week to 24 months after birth. These results indicate that prostaglandins may play important roles during maturation and aging in rat brain.

Effect of topical prostaglandin D2 on the aqueous humor dynamics in rabbits.

Effects of topically applied prostaglandin (PG) D2 on the aqueous outflow facility, uveoscleral flow, and aqueous flow rate were studied in rabbits to reveal its intraocular pressure (IOP)-reducing mechanism. The outflow facility after PGD2 application (50 μg) as measured by 2-min tonography was 0.19 μl/min/mm Hg and did not differ from that before the application (0.22) or from that of the control contralateral eye (0.21). Direct measurement of the uveoscleral flow by perfusion of the anterior chamber for 30 min with fluorescein isothiocyanate-labeled dextran solution showed the flow rate to be about 0.15 μl/min in indomethacin-treated (10 mg/kg, i.p.) rabbits. In these animals, PGD2 (50 μg) was ineffective in changing the flow rate, while PGF2α (50 μg) significantly increased the flow by about 35%. Indirect assessment of the effects of PGs on this flow system was made using the uveoscleral flow-antagonizing ability of topical pilocarpine (1.5 mg). IOP reduction by PGD2 (50 μg) was only slightly inhibited by pilocarpine, while that by PGF2α (10 μg) was markedly reduced. Aqueous flow rate measured fluorophotometrically was about 3.5 μl/min in normal eyes. After PGD2 (50 μg) the flow rate was significantly reduced to 3.0 μl/min. The magnitude of this reduction was estimated to be enough to account for the IOP reduction after application of 50 μg of PGD2. These results indicate that IOP reduction caused by topical PGD2 application is due mostly to the inhibition of aqueous flow rate.

Muramyl dipeptide-elicited production of PGD2 from astrocytes in culture

We used primary cultures of rat brain astroglial cells in order to investigate the interrelationship between PGD2 and other sleep-promoting substances such as muramyl dipeptide (MDP), lipopolysaccharide (LPS), delta-sleep-inducing peptide (DSIP), uridine, and interleukin 1 (IL-1). A large amount of PGD2 was released into the culture medium by stimulation with MDP, LPS, and IL-1 but DSIP and uridine failed to stimulate such release. These results suggest that PGD2 may be part of the series of biochemical steps involved in induction of sleep by MDP, LPS, and IL-1.

Late‐Phase Accumulation of Inositol Phosphates Stimulated by Prostaglandins D2 and F2α in Neuroblastoma × Glioma Hybrid NG108‐15 Cells

Abstract: The accumulation of inositol phosphates (IPs) in response to prostaglandins (PGs) was studied in NG108‐15 cells preincubated with myo‐[3H]inositol. As a positive control, bradykinin caused accumulation of IPs transiently at an early phase (within 1 min) and continuously during a late phase (15‐60 min) of incubation in the cells. PGD2 and PGF2adid not significantly cause the accumulation of IPs at an early phase but significantly stimulated inositol bisphosphate (IP2) and inositol monophosphate (IP1) formation at a late phase of incubation. The maximum stimulation was obtained at <10−7M concentrations of these PGs. the levels being three‐and twofold for IP2 and IP1 respectively. 9α, 11 β‐PGF2 has a slight effect but PGE2 and the metabolites of PGD2 and PGF2α have no effect up to 10−6M. The effects of PGD2 and PGF2α were not additive, but the effect of each PG was additive to that of bradykinin at a late phase of incubation.

Alteration in morphology and induction of glutamine synthetase in rat glioma C6BU-1 cells cultured with prostaglandin D2-supplemented media

We evaluated the effects of prostaglandins (PGs) on rat glioma C6BU-1 cells by supplementing the culture media with PGs. In the medium containing PGD(2) (15 or 20 ?M), the glial cells showed altered morphology from an elongated fibroblastic form to a spreading multipolar one within 24 h, and their growth rate was suppressed to half of that of control cultures. In these cultures, the specific activity of glutamine synthetase (GS) increased approximately twofold within 48 h in comparison to the value for vehicle-treated controls. Simultaneous treatment with actinomycin D or cycloheximide completely blocked the PGD(2)-elicited increase in GS specific activity, suggesting that the increase was due to de novo synthesis of the enzyme. PGD(2)-like prostanoids such as PGD(1) and 9-deoxy-?(9), ?(12)-13,14-dihydro-PGD(2) (?(12)-PGJ(2)), when added to the culture medium, mimicked the actions of PGD(2) on the C6BU-I cells, though their effective concentrations were not necessarily identical. PGs of the E- and F-series had almost no discernible effect on the glioma. These results might imply a possibility that PGD(2) plays a regulatory effect in growth and/or differentiation of rat glioma C6BU-1 cells.