Brain Prostaglandin Research Articles

Primary Structure of Rat Brain Prostaglandin D Synthetase Deduced from cDNA Sequence

The amino acid sequence of rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi, O. (1985) J. Biol. Chem. 260, 12410-12415) was determined by a combination of cDNA and protein sequencing. cDNA clones specific for this enzyme were isolated from a lambda gt11 rat brain cDNA expression library. Nucleotide sequence analyses of cloned cDNA inserts revealed that this enzyme consisted of a 564- or 549-base pair open reading frame coding for a 188- or 183-amino acid polypeptide with a Mr of 21,232 or 20,749 starting at the first or second ATG. About 60% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of tryptic peptides of the purified enzyme. The recognition sequence for N-glycosylation was seen at two positions of amino acid residues 51-53 (-Asn-Ser-Ser-) and 78-80 (-Asn-Leu-Thr-) counted from the first Met. Both sites were considered to be glycosylated with carbohydrate chains of Mr 3,000, since two smaller proteins with Mr 23,000 and 20,000 were found during deglycosylation of the purified enzyme (Mr 26,000) with N-glycanase. The prostaglandin D synthetase activity was detected in fusion proteins obtained from lysogens with recombinants coding from 34 and 19 nucleotides upstream and 47 and 77 downstream from the first ATG, indicating that the glycosyl chain and about 20 amino acid residues of N terminus were not essential for the enzyme activity. The amino acid composition of the purified enzyme indicated that about 20 residues of hydrophobic amino acids of the N terminus are post-translationally deleted, probably as a signal peptide. These results, together with the immunocytochemical localization of this enzyme to rough-surfaced endoplasmic reticulum and other nuclear membrane of oligodendrocytes (Urade, Y., Fujimoto, N., Kaneko, T., Konishi, A., Mizuno, N., and Hayaishi, O. (1987) J. Biol. Chem. 262, 15132-15136) suggest that this enzyme is a membrane-associated protein.

Increased Plasma PGE2, 6-Keto-PGF1α, and 12-HETE Levels Following Experimental Concussive Brain Injury

Previous investigations have shown that brain prostaglandin levels are transiently elevated following experimental fluid percussion brain injury. Associated with these increased prostaglandin levels there is free radical production and abnormalities in cerebral arteriolar function. The purpose of this study was to determine whether experimental fluid percussion brain injury in cats is associated with increased systemic levels of prostaglandins and the lipoxygenase product, 12-HETE. Blood samples were collected before and at various periods of time after 2.7 atm of fluid percussion brain injury was produced in adult cats. Prostaglandin and 12-HETE analysis was performed by radioimmunoassay after extraction of the plasma samples. The control levels for 6-keto-PGF1 alpha, PGE2, and 12-HETE were 477 +/- 42, 2,372 +/- 431, and 13,328 +/- 1,769 pg/ml, respectively. Following injury all three eicosanoids reached peak plasma levels by 1-5 min after injury. The percentile increases for all eicosanoids were similar and increased from 70 to 110%. The increases were sustained at up to 30 min postinjury and by 1 h after injury were at control levels. As in previous studies, hypertension following injury was maximal by 1 min postinjury and blood pressure had returned to near normal levels by 5 min postinjury. These studies demonstrate prolonged systemic increases in eicosanoids following injury. Since free radical production and vascular damage occur concomitantly with eicosanoid production, the prolonged increases in these products suggest that there is an attainable therapeutic window following injury during which administration of free radical scavengers may decrease radical damage and reduce the consequences of injury.

Inhibition of prostaglandin synthesis in brain of rat by dexamethasone: Lack of effect of dexamethasone phosphate ester and various hormonal steroids

The present study was designed in order to characterize the inhibitory effect of dexamethasone upon the synthesis of prostaglandins (PG) in the brain of the rat. Rats were treated with dexamethasone (20 mg/kg b.w.) and sacrificed 0–76 hr after administration of the drug. The rate of synthesis and release of PGE 2 was followed by 1 hr of incubation of slices of cortex taken from these rats, in Krebs-Ringer solution. A significant inhibition occurred at 8 hr and maximal inhibition (45%) was attained at 16 hr after injection. A gradual increase in the rate of synthesis up to control values occurred between 24 and 76 hr. A dose-response study, at the range of 2–40 mg/kg, showed that a significant decrease was noted at 6 mg/kg and it was maximal (45% inhibition) at 20 and 40 mg/kg. Administration of dexamethasone-sodium-phosphate, as well as other synthetic glucocorticoids and various steroidal hormones (20 μM), failed to inhibit the biosynthesis of prostaglandins under the same experimental conditions. The effect of dexamethasone and dexamethasone phosphate on synthesis of PGE 2 was also studied under in vitro conditions at 5 and 20μM. When slices of cortex from intact rats were incubated for 1 or 2hr in the presence of either dexamethasone or dexamethasone phosphate only dexamethasone was effective in inhibiting the synthesis of PGE 2. The present results demonstrate that the inhibition of the synthesis of prostaglandins in brain by dexamethasone is both time- and dose-dependent. The lack of effect of closely related glucocorticoids demonstrate that the effect is highly specific to dexamethasone.

Interaction Between Bombesin and Brain Prostaglandins in the Control of Gastric Secretion

Interaction Between Bombesin and Brain Prostaglandins in the Control of Gastric Secretion

Effects of cerebral ischemia and reperfusion on prostanoid accumulation in unanesthetized and pentobarbital-treated gerbils.

Cerebral ischemia was induced in unanesthetized gerbils using bilateral carotid artery ligations. The effects of 20 min of global ischemia on the concentrations of prostaglandin F2 alpha (PGF2 alpha), PGE2, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and thromboxane B2 were determined after 0-24 h of reperfusion. Ischemia had little effect on eicosanoid production, but significant increases were observed by 5 min of reperfusion, with maximal levels reached by 15 min of reperfusion. PGF2 alpha was the most concentrated prostaglandin in postischemic brain, whereas PGE2 was most concentrated in control cerebra. Pretreatment with anesthetic doses of pentobarbital supported increased accumulation of PGF2 alpha in postischemic cerebra, increased accumulation of 6-keto-PGF1 alpha during the ischemic episode, and decreased accumulation of PGE2 at 120 min of reperfusion. It appears that the protective effects of barbiturate anesthesia are not expressed by the reduced accumulation of the above eicosanoids.

Prostaglandins and control of breathing in newborn piglets.

To investigate the possible role of prostaglandins in regulation of postnatal breathing, phrenic neural activity (PMO) was recorded as an index of breathing in 42 anesthetized, paralyzed piglets less than 30 days of age (weight 2.4 +/- 0.2 kg, age 9.9 +/- 1.5 days) who were mechanically ventilated with 100% O2 at a fixed tidal volume (8-10 ml/kg). End-tidal CO2 was held constant by an electronic servocontroller which adjusted ventilator rate; ventilator rate was monitored as an index of CO2 production. Rectal temperature was maintained at 39.0 +/- 0.2 degrees C. The effects on PMO of intravenous and brain ventricular injections of NaCl and agents active in the prostaglandin cascade were compared. Intravenous (0.25-1.0 mg/kg, n = 9) and brain (5-33 micrograms/kg, n = 6) indomethacin, a cyclooxygenase inhibitor, doubled PMO within 30 min. Intravenous (1-10 micrograms/kg, n = 6) and brain (1-40 micrograms/kg, n = 6) prostaglandin E1 inhibited PMO by one-half at 10 and 30 min.

Eicosanoids in human ventricular cerebrospinal fluid following severe brain injury

Recent evidence has shown that a variety of prostaglandins and leukotrienes can be produced in brain tissue after injury in animals. It has also been speculated that increases in brain prostaglandins occur in humans following injury. Ventricular cerebrospinal fluid (CSF) samples have been obtained from children with static lesions (controls) as well as children with acute brain injury and eicosanoids measured by immunologic techniques. Metabolites of prostacyclin (6-keto-PGF 1a) and thromboxane A 2 (thromboxane B 2) were the major eicosanoids found in CSF, and levels of these compounds were increased 3–10 times in acutely injured patients. Prostaglandin E 2 was also found in lower amounts, although in one case its level was very high. Prostaglandin D 2 was also present, but in low amounts. No leukotrienes were found in CSF samples that were purified by HPLC prior to immunoassay. Elevated levels of hydroxyeicosatetraenoic acids (HETEs) were observed in those samples stored frozen, but these metabolites were most probably due to autooxidation of archidonic acid in CSF. Arachidonic acid concentration in CSF was typically found to be in the range of 10–200 ng/ml, but was found to be 5–10 fold higher in one severely injured patient. Thus, elevated free arachidonic acid and various oxygenated metabolites were observed in CSF following brain injury.

Brain growth during ethanol-induced hypoplasia

The inhibition of fetal brain growth resulting from in utero ethanol exposure may impair central nervous system (CNS) development and thereby result in mental retardation. Studies of ethanol-induced brain hypoplasia using chick embryos have shown that the early development of the chick is significantly growth inhibited by a single dose of ethanol (1.0 g/ kg) given at the start of incubation (day 0). However, this level of ethanol exposure has been reported to have no effect on chick weight measured at hatching, suggesting that the weights of ethanol-treated chicks were regained during their development. The present experiments were undertaken to determine the biochemical changes associated with the varying growth rates believed to occur in the alcohol-treated embryos. The results indicated that between days 5 and 8 of development, the rates of DNA and protein synthesis (measured as radioactive thymidine and leucine incorporation, respectively) were inhibited by ethanol. The growth inhibition was highly correlated with blood alcohol content and there were associated increases in brain prostaglandin E (PGE) levels relative to vehicle-treated embryos. Further, there was a significant, inverse correlation between brain cyclic AMP content and individual brain weight. By day 10, the ethanoltreated embryos remained smaller than controls but their rates of DNA and protein synthesis were comparable to those of control animals. The normal rates of synthesis observed on day 10 appeared to correlate with clearance of the ethanol dose and with restoration of normal brain levels of PGE relative to 10-day vehicle-dosed embryos.

Brain-type prostaglandin D synthetase occurs in the rat cochlea.

Prostaglandin D synthetase [(5Z, 13E)-(15S)-9 alpha, 11 alpha-epidioxy-15-hydroxyprosta-5,13-dienoate D-isomerase, EC 5.3.99.2] activity was found in the high-speed (100,000 x g, 1 hr) supernatant of the homogenate of the cochlea of adult rats. The specific activity (14.0 nmol/min per mg of protein) was 6- to 7-fold higher than that in the central nervous system. The enzyme showed full activity with 1 mM glutathione, 1 mM 2-mercaptoethanol, or 0.5 mM dithiothreitol and was almost completely inhibited by 1 mM 1-chloro-2,4-dinitrobenzene. The Km value for prostaglandin H2 was about 20 microM. These catalytic properties are the same as those of rat brain prostaglandin D synthetase but different from those of rat spleen prostaglandin D synthetase. The activity decreased to less than 20% of its initial level after incubation with excess amounts of a polyclonal or a monoclonal antibody against the brain enzyme, but the activity remained unchanged with a polyclonal antibody against the spleen enzyme, indicating that the brain-type enzyme synthesizes prostaglandin D2 in the cochlea. When cryosections of 5-week-old (adult) rat cochleas were stained by an immunoperoxidase method with antibodies against the brain enzyme, the immunoreactivity was found in inner and outer hair cells, Claudius' cells, Deiters' cells, marginal cells, basal cells, and cells of Reissner's membrane. In 8-day-old rats, the immunoreactivity was found in all of these cell types except hair cells. The immunoreactivity in hair cells was found in only one specimen from 9-day-old animals, and no immunoreactivity was found in spiral ganglion cells at any of the ages examined. These findings indicate that prostaglandin D2 is produced by the brain-type synthetase in the indicated types of cochlear cells.

Interaction between bombesin and brain prostaglandins in the control of gastric secretion : A. Guglietta, B.J. Irons, and L.H. Lazarus. National Institute of Environmental Health Sciences, LBNT, Research Triangle Park, NC 27709, USA and Institute of Medical Pharmacology III, Universita di Roma, “La Sapienza” 00185 Rome, Italy

Interaction between bombesin and brain prostaglandins in the control of gastric secretion : A. Guglietta, B.J. Irons, and L.H. Lazarus. National Institute of Environmental Health Sciences, LBNT, Research Triangle Park, NC 27709, USA and Institute of Medical Pharmacology III, Universita di Roma, “La Sapienza” 00185 Rome, Italy

Biochemical and immunological characterization of rat spleen prostaglandin D synthetase.

Rat spleen prostaglandin D synthetase (Christ-Hazelhof, E., and Nugteren, D. H. (1979) Biochim. Biophys. Acta 572, 43-51) is very similar to rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi O. (1985) J. Biol. Chem. 260, 12410-12415) as judged by their pI (4.7-5.2), Mr (26,000-27,000), and self-inactivation during the isomerase reaction from prostaglandin H2 to prostaglandin D2. However, the amino acid compositions of these two enzymes were quite different. Furthermore, the spleen enzyme was associated with the glutathione S-transferase activity, differing from the brain enzyme. The synthetase and transferase activities of the spleen enzyme showed almost identical pH and glutathione dependencies, the optimum pH = 8.0 and Km for glutathione = 300 microM. The Km values for prostaglandin H2 and 1-chloro-2,4-dinitrobenzene (a substrate for the transferase) were about 200 microM and 5 mM, respectively. The synthetase activity was dose-dependently inhibited by 1-chloro-2,4-dinitrobenzene (IC50: approximately 5 mM) and more strongly by nonsubstrate ligands, such as bilirubin and indocyanine green (IC50: 150 and 2 microM, respectively). Both the synthetase and transferase activities of the purified enzyme dose-dependently decreased and showed identical immunotitration curves by incubation with antibody against this enzyme, but remained unchanged when treated with antibody against the brain enzyme. The antibody specific for the spleen enzyme absorbed almost all of the synthetase activity and about 10% of the transferase activity in the spleen, but not the transferase activity in the liver, heart, and testis. These results show that the two types of prostaglandin D synthetase are similar but different enzymes and that the spleen enzyme is a unique glutathione S-transferase differing from other isozymes and their subunits reported previously.

Carrageenan-induced brain inflammation. Characterization of the model.

Administration of the mucopolysaccharide, carrageenan (CAR), into the hind paw of the rat or mouse induces a local inflammation characterized by increased arachidonic acid metabolism, increased vascular permeability, edema, and neutrophil extravasation. Carrageenan-induced hind-paw inflammation is inhibited by prostaglandin synthesis inhibitors, and this assay predicts the clinical success of anti-inflammatory agents in reducing peripheral inflammation. The purpose of this study was to determine if intraventricular injection of CAR would induce brain inflammation similar to that evoked by CAR in peripheral tissues. The present study demonstrates that CAR injection into the ventricles of the mouse brain does in fact induce an inflammatory response very similar to that caused by injection of CAR into the peripheral tissues. The brain response to CAR was dose-dependent, with the maximum increase in cerebrovascular permeability to iodine-125-labeled human serum albumin and percent brain water occurring after injection of 50 micrograms CAR. As is seen in CAR-induced inflammation of the hind paw, the maximum increase in brain vascular permeability occurred 4 hours after CAR injection. Histological analysis of brains 4 hours after CAR administration showed global neutrophil extravasation into the subarachnoid space and evidence of focal neuronal swelling. Methotrexate-induced neutropenia, however, failed to diminish the permeability response to CAR. Gas chromatographic and mass spectrometric measurements of brain prostaglandins 4 hours after CAR injection revealed a significantly increased level of 6-keto-prostaglandin F1 alpha. These results indicate that a significant increase in prostacyclin, the pro-inflammatory arachidonic acid metabolite, during CAR-induced brain inflammation is likely. These studies suggest that CAR-induced brain inflammation may be a useful model on which to test the efficacy of anti-inflammatory agents in the brain, as well as providing information concerning the mediators and mechanisms by which the brain may sustain inflammatory injury.

Time course of ethanol's effects on brain prostaglandins in LS and SS mice

Prostaglandin synthesis inhibitors antagonize behavioral responses to alcohols. Recent work has shown that ethanol increases brain prostaglandin (PG) levels. The study reported here examined the tine course for ethanol-stimulated brain PGE and PGF production in Long Sleep and Short Sleep mice, animals bred selectively for high vs. low acute response to ethanol. Increases in brain PGE levels correlated highly with the absorption phase but only partially with the elimination phase of ethanol. PGF levels correlated significantly with blood ethanol levels across the entire three hour period. These results, plus sex and genotype interactions, provide further evidence to support the hypothesis that ethanol produces its intoxicating effects to a significant degree through a prostaglandin mediated mechanism.

Role of brain prostaglandins in the control of vasopressin secretion in the conscious rat.

Administration of 10 micrograms prostaglandin D2 (PGD2), the primary PG identified in the rat brain, into the lateral cerebral ventricle of conscious rats resulted in a significant elevation in the plasma vasopressin (AVP) concentration, without a change in mean arterial blood pressure or heart rate. The central administration of indomethacin (100 micrograms) resulted in a significant attenuation of the AVP response to a peripheral osmotic stimulus (iv 2.5 M NaCl; 100 microliters/kg X min for 30 min), but had little effect on the AVP response to hemorrhage (two successive 10% reductions in the estimated blood volume). Administration of another PG synthetase inhibitor, meclofenamate (100 micrograms, into the lateral cerebral ventricle), resulted in a significant attenuation of the AVP response to both the osmotic stimulus and hemorrhage. It is concluded that brain PGs play a central role in the control of AVP secretion.

Adaptation to cold antagonizes neurotensin-induced hypothermia in mice

Intracerebrally-administered neurotensin produces a marked hypothermia in a variety of mammals. In this study, prior adaptation to a cold environment was found to significantly antagonize the hypothermia produced by intracisternally-administered neurotensin in mice. This antagonism required both previous exposure to cold ambient temperatures and cold exposure immediately prior to, or simultaneously with, neurotensin administration. The antagonism of neurotensin-induced hypothermia by prior cold-adaptation was blocked by indomethacin, but not by acetylsalicylic acid, suggesting that brain prostaglandin synthesis may be essential for this newly-discovered phenomenon.

Involvement of GTP-regulatory protein in brain prostaglandin E 2 receptor and separation of the two components

The specific binding protein for prostaglandin (PG) E 2 solubilized from porcine brain was sensitive to guanine nucleotides. GTP inhibited the association and enhanced the dissociation of the specific [ 3H]PGE 2 binding. Scatchard analyses showed that GTP (10 μM) decreased the binding affinity more than 3-fold without major change in the number of binding site. Gel filtration separated the binding site from GTP-regulatory component (N). The separated binding protein had a reduced affinity to PGE 2 and lost its sensitivity to GTP. The addition of the separated N restored its responsiveness to GTP, and also increased the binding affinity to the original level. These results provide direct evidence for the molecular interaction between the PGE 2 binding protein and N in the brain.

Role of putative neurotransmitters in bradykinin-induced catalepsy in the rat.

Intracerebroventricular administration of bradykinin produced a dose-related cataleptic response in rats. Bradykinin-induced catalepsy was significantly attenuated following pretreatment with pharmacologic agents that decrease central prostaglandin, serotonin, and acetylcholine activity, as well as by the opioid receptor antagonist naloxone. Conversely, pharmacologic treatments that enhance central catecholamine levels and, specifically, central dopaminergic activity also inhibited bradykinin-induced catalepsy. The prostaglandin precursor, arachidonic acid, and prostaglandin E2, as well as met- and leu-enkephalin showed a synergistic effect with bradykinin catalepsy. Much evidence indicates that several actions of bradykinin, in the central nervous system and the periphery, are likely to be prostaglandin mediated. Further, the recent report from this laboratory that centrally administered bradykinin specifically augments rat brain prostaglandin E2 levels, together with the proposed role of central prostaglandins as modulators of central synaptic transmission, suggests that bradykinin-induced catalepsy is mediated and modulated through PGE effects on serotonergic, cholinergic, and dopaminergic neurotransmitter systems. The study also indicates that endogenous opioid peptides may be involved in braydkinin catalepsy.

Effect of AD-1590, a non-steroidal anti-inflammatory agent with a potent antipyretic activity, on in vitro prostaglandin generation in rabbit brain.

The inhibitory activity of 2-(8-methyl-10,11-dihydro-11-oxodibenz[b,f]oxepin-2-yl) propionic acid (AD-1590) against prostaglandin (PG) generation in the rabbit brain was compared with that of indomethacin in vitro. AD-1590 inhibited PGE2 generation by hypothalamus microsomes at concentrations of 0.1 micrograms/ml or more, its IC50 value being 0.258 micrograms/ml. On the other hand, PGE2 generation by cerebral cortex slices was suppressed by about 10 times lower concentrations of AD-1590 in comparison with that by hypothalamus microsomes; its IC50 value was 0.0292 micrograms/ml. The potency of AD-1590 was 3.4 and 2.6 times that of indomethacin in the hypothalamus microsomes and cerebral cortex slices, respectively. The inhibitory activity of AD-1590 against PGE2 generation by hypothalamus microsomes was similar to the result with renal medulla microsomes; the ratio of renal medulla to hypothalamus IC50 values of AD-1590 (0.496) was nearly equal to that of indomethacin (0.384) and aspirin (0.452). The present studies confirmed that AD-1590 inhibited PGE2 generation within the hypothalamic region. However, these results suggest that the inhibitory potency of AD-1590 against brain PG synthetase cannot explain the 20-50 times more potent antipyretic activity than that of indomethacin, previously reported. The mode of potent antipyretic action of AD-1590 is discussed.

Delta-9-tetrahydrocannabinol (THC) increases brain prostaglandins in the rat.

Intraperitoneal administration of 9-trans-delta-tetrahydrocannabinol (THC), the major psychoactive component of cannabis, in doses of 0.5, 1.0 and 2.0 mg/kg, produced a dose-related increase in the brain concentrations of prostaglandin (PG) E2 and PGF2 alpha in male rats 4 h after THC administration, as assessed by radioimmunoassay. A time-course investigation indicated that THC (2 mg/kg, IP) induced maximal increases in rat brain concentration of both PGs 2 and 4 h after administration; PG levels declined appreciably by 8 h and were normal by 24 h. A time-course study on the hexobarbitone (100 mg/kg, IP)-induced hypnosis potentiating effect of THC (2.0 mg/kg, IP) in male rats revealed that this pharmacological action of the cannabinoid correlated well with the time-course of the THC-induced increase in rat brain PG concentrations. The present study lends support to earlier reports contending that PGs may mediate some of the central actions of THC.

Purification and characterization of rat brain prostaglandin D synthetase.

Prostaglandin D synthetase was purified 2,600-fold from rat brain to apparent homogeneity, as judged by polyacrylamide gel electrophoresis and ultracentrifugation. The purified enzyme was a monomeric protein with a molecular weight of 27,000 +/- 1,000. The pI value, sedimentation coefficient, and partial specific volume were 4.6, 4.1 s, and 0.73 ml/g, respectively. The enzyme was stable between pH 4 and 11 at the temperature lower than 25 degrees C and resistant to a heat treatment under alkaline conditions (pH 8-11). About 50% of the activity was detected after a heat treatment at 100 degrees C for 5 min at pH 10. However, the enzyme was readily inactivated by the isomerase reaction of prostaglandin H2 to prostaglandin D2. The enzyme required sulfhydryl compounds such as dithiothreitol, glutathione, beta-mercaptoethanol, cysteine, and cysteamine for the reaction, but stoichiometric oxidation of these sulfhydryl compounds was not observed. The optimum pH, Km value for prostaglandin H2, and the turnover number were 9.5, 14 microM, and 170 min-1, respectively. The antibody was raised against the purified enzyme in a rabbit, which showed only one positive band in immunoblotting after gel electrophoresis of crude extracts of the brain at the same position as that of the purified enzyme. More than 90% of the prostaglandin D synthetase activity in the brain was absorbed by an excess amount of the antibody, indicating that our preparation is a major component of the enzyme responsible for the biosynthesis of prostaglandin D2 in the brain.