András Seregi

Are cerebral prostanoids of astroglial origin? Studies on the prostanoid forming system in developing rat brain and primary cultures of rat astrocytes

Prostanoid forming capacity in vitro and convulsion-induced prostanoid formation in vivo were studied in the developing rat brain. For comparison, prostanoid synthesis in homogenates of primary astrocyte cultures of different ages was also examined. There was no significant prostanoid production in homogenates from primary astrocyte cultures prepared one week after cultivation. Two-week-old astrocyte cultures possessed a prostanoid synthesizing system of high specific activity. The relative proportions of the products were similar to those obtained in brain homogenates of adult rats, prostaglandin D2 (PGD2) being the major product. Prostanoid forming capacity of brain homogenates was low at birth, increased during development and nearly reached adult values by day 21. Generalized convulsions could be evoked by pentylenetetrazol (PTZ) irrespective of age, but convulsion-induced prostanoid formation characteristic of adult rodents did not take place before the third week of postnatal life. The close similarities between the characteristic features of prostanoid synthesis in both brain and astroglial homogenates, together with the coincidence during brain development of the expression of cerebral prostanoid synthesis with the appearance of mature astrocytes suggest that astrocytes are an important source of brain prostanoids.

Receptor independent stimulatory effect of noradrenaline on Na,K-ATPase in rat brain homogenate: Role of lipid peroxidation

The effect of different adrenoceptor agonists on Na,K-ATPase activity and lipid peroxidation of rat brain homogenate was studied. Drugs which enhanced Na,K-ATPase activity--noradrenaline, adrenaline and oxymethazoline--were found to inhibit endogenous membrane lipid peroxidation. Other drugs--phenylephrine, xylazine and clonidine--which did not cause any change in the enzyme activity did not influence lipid peroxidation either. No increase of Na,K-ATPase activity by noradrenaline could be detected after preincubation of the homogenate for 5 min at 37 degrees. During this time endogenous lipid peroxidation of considerable extent could be observed. It is concluded that there is no correlation between the adrenoceptor agonist feature of noradrenaline and its stimulatory effect on Na,K-ATPase activity of rat brain homogenate. However, it seems likely that in rat brain homogenate the increase of Na,K-ATPase activity and inhibition of endogenous lipid peroxidation by noradrenaline are related.

Lipid peroxidation as the cause of the ascorbic acid induced decrease of adenosine triphosphatase activities of rat brain microsomes and its inhibition by biogenic amines and psychotropic drugs

Abstract The inhibitory effect of ascorbic acid on microsomal Na + , K + -ATPase and Mg 2+ -ATPase activities of rat brain and the ability of several mediator substances and of many drugs acting on the nervous system to antagonize this inhibition was studied. The maximal effect of ascorbic acid on ATPase activities was completely antagonized by catecholamines, apomorphine, oxypertine, reserpine, tetrabenazine, phenothiazines (chlorpromazine and promethazine) and yohimbine at a concentration of 10 −4 M or below. Apomorphine proved to be the most effective compound, fully antagonizing the effect of ascorbic acid at a concentration of 10 −6 M. A partial inhibition of the effect of ascorbic acid was induced by 10 −4 M serotonin, desipramine, imipramine and LSD. During the incubation of the microsomes for ATPase activity determinations in the presence of ascorbic acid, a significant amount of lipid peroxide was formed. Compounds which antagonized the effect of ascorbic acid on the ATPase activities inhibited at the same concentrations the lipid peroxide formation. The well-known inhibitors of lipid peroxidation eliminated the effect of ascorbic acid on the ATPase activities. It has been established that the inhibition of ATPase activities by ascorbic acid is a consequence of lipid peroxidation. The mechanism of action of the antagonizing compounds is discussed.

Protective role of brain ascorbic acid content against lipid peroxidation.

The high ascorbic acid concentration in the brain inhibits lipid peroxidation induced by various agents in rat brain microsomes. The physiological importance of the fact is discussed.

Effects of biogenic amines and psychotropic drugs on endogenous prostaglandin biosynthesis in the rat brain homogenates

Abstract Phenylalkylamine and indolalkylamine derivatives, as well as several drugs acting on the central nervous system, were tested for their effects on endogenous prostaglandin, (PG) biosynthesis in the rat brain homogenates. In the particulate suspension obtained by the removal of the soluble fraction from the rat brain homogenates PG-biosynthesis could be stimulated by noradrenaline. dopamine, adrenaline, serotonin, tryptamine and to a slight extent by tyramine. Isoprenaline, DOPA. α-methyl noradrenaline, α-methyl dopamine, α-methyl tryptamine and 5-hydroxy tryptophan were ineffective. PG-biosynthesis stimulated by catecholamines or indolalkylamines responsively could be inhibited by compounds with monoamine oxidase blocking properties. In the total rat brain homogenates another type of PG-biosynthesis could be demonstrated in the absence of catecholamine or indolalkylamine that could not, or but to a slight extent, be inhibited by monoamine oxidase blocking agents. Apomorphine, oxypertine, α-methyl noradrenaline, promethazine, DOPA, reserpine, chlorpromazine, desipramine, yohimbine and tetrabenazine inhibited this type of PG-biosynthesis, though they failed to influence PG-formation stimulated by catecholamine or indolalkylamine. A correlation could be established between the PG-formation inhibitory and lipid peroxidation antagonizing effects of these compounds. Non-steroidal anti-inflammatory agents, such as indomethacin, acetylsalicylic acid and dipyrone, inhibited both types of PG-biosynthesis. The results permit the conclusion that psychotropic drugs exert their effects on endogenous PG biosynthesis in the rat brain homogenates by inhibiting various activation processes.

On the mechanism of the involvement of monoamine oxidase in catecholamine-stimulated prostaglandin biosynthesis in particulate fraction of rat brain homogenates: Role of hydrogen peroxide

Abstract: The mechanism of involvement of monoamine oxidase (MAO) in catecholamine‐stimulated prostaglandin (PG) biosynthesis was studied in the particulate fraction of rat brain homogenates. High concentrations of either noradrenaline (NA) or dopamine (DA) stimulated effectively PGF2α formation. The same amount of 2‐phenylethylamine (PEA) acted similarly, provided that it was administered together with a catecholamine analogue or metabolite possessing the 3,4‐dihydroxyphenyl nucleus–3, 4‐dihydroxyphenylalanine (DOPA), 3,4‐dihydroxyphenylacetic acid (DOPAC), 3,4‐dihydroxyphenyl‐glycol (DOPEG), 3,4‐dihydroxyphenylacetaldehyde (DOPAL), or α‐methylnoradrenaline (α‐met‐NA)–or with SnCl2. In the absence of PEA, these compounds were ineffective with regard to stimulation of PGF2α formation. Catalase, pargyline, or indomethacin abolished completely PGF2α formation elicited either by catecholamines or by PEA plus a 3,4‐dihydroxyphenyl compound or SnCl2. With regard to the stimulation of PGF2α formation in the presence of α‐met‐NA, PEA could be replaced by H2O2, generated by the glucose oxidase(GOD)‐glucose system. The effect of H2O2 was inhibited by indomethacin or catalase, but pargyline was ineffective. It is assumed that catecholamines play a dual role in the activation of PG biosynthesis in brain tissue. During the enzymatic decomposition of catecholamines MAO produces H2O2, which stimulates endoperoxide synthesis. Simultaneously, catecholamines as hydrogen donors promote the nonenzymatic transformation of endoperoxides into PGF2α. The possible physiological importance of these findings is discussed.

Evidence for the Localization of Hydrogen Peroxide‐Stimulated Cyclooxygenase Activity in Rat Brain Mitochondria: A Possible Coupling with Monoamine Oxidase

Abstract: The distribution of basal and of H2 O2‐ stimulated cyclooxygenase activity in the primary fractions of rat brain homogenates and in the subfractions of crude mitochondrial fraction was studied. For comparison, the localization of H2 O2‐ generating monoamine oxidase (MAO) as well as that of the mitochondrial marker succinate dehydrogenase (SDH) was also examined. H2 O2 was generated by MAO using 5 × 10−4M noradrenaline (NA) or 2 × 10−4M 2‐phenylethylamine (PEA) as substrates, or by 25 μg glucose oxidase (GOD) per ml in the presence of 1 mM glucose. For nonstimulated (basal) cyclooxygenase, the relative specific activity (RSA) was high in microsomes (1.79) and in the free mitochondria‐containing subfraction of the crude mitochondrial fraction (1.94). Parallel distribution of MAO and H2 O2‐ stimulated cyclooxygenase was observed in all fractions studied in the presence of NA. The highest RSA was found in the purified mitochondria for both enzymes (1.85 for MAO and 1.97 for H2 O2‐ stimulated cyclooxygenase). The enrichment of SDH (RSA = 2.21) indicated a high concentration of mitochondria in this fraction. The same distribution of H2 O2‐ stimulated cyclooxygenase was obtained when, instead of the MAO‐NA system, hydrogen peroxide was generated by GOD in the presence of glucose. H2 O2 generated by deamination of NA or PEA by MAO, or during the enzymatic oxydation of glucose by GOD, caused a threefold increase in mitochondrial endoperoxide formation. Indomethacin (2 × 104M), catalase (50 μg/ml) and pargyline (2 × 10−4M) eliminated the MAO‐dependent mitochondrial synthesis of PG endoperoxides. The GOD‐dependent cyclooxygenase activity of this fraction was abolished by in domethacin or catalase, but not by pargyline. The results show the existence of a mitochondrial cyclooxygenase in brain tissue. The enzyme is sensitive to H2 O2 and produces prostaglandin endoperoxides from an endogenous source of arachidonic acid. The identical localization of H2 O2‐ producing MAO and H2 O2‐ sensitive cyclooxygenase suggests a possible coupling between monoamine and arachidonic acid metabolism.

Characterization of Seizure-Induced Cysteinyl-Leukotriene Formation in Brain Tissue of Convulsion-Prone Gerbils

Abstract: Tonic‐clonic seizures elicited in convulsionprone gerbils resulted in a large increase in immunoreactive prostaglandin (PG) F2α and in a smaller increase in immunoreactive leukotriene (LT) C4‐like material in brain tissue. Brain tissue contents of both eicosanoids were found to reach a maximum at 6 min after the onset of seizures and were still elevated at 54 min after the beginning of convulsions. By reversed phase HPLC the immunoreactive LTC4‐like material was identified as LTC4 and LTD4 at 6 min after the onset of convulsions, whereas at 54 min after the onset, transformation of LTD4 to LTE4 could be detected as well. In gerbils showing only weak seizure activity a small increase in PGF2α but no increase in immunoreactive LTC4‐like material could be detected at 6 min after the onset of convulsions. Pretreatment with indomethacin abolished the formation of PGF2α but significantly enhanced the biosynthesis of immunoreactive LTC4‐like material at 18 min after the beginning of seizures. The results demonstrate formation of cysteinyl‐LT following tonicclonic convulsions in spontaneously convulsing gerbils which could be enhanced by inhibition of the cyclooxygenase pathway of arachidonic acid metabolism. Since cysteinyl‐LT have potent biological actions in various organs this finding warrants further investigations on the potential role of cysteinyl‐LT in the CNS.

Prostanoid formation in primary astroglial cell cultures: Ca2+-dependency and stimulation by a 23187, melittin and phospholipases A2 and C

Prostaglandin (PG) and thromboxane B(2) (TXB(2)) biosynthesis was studied in cultured astrocytes from neonatal rat brain hemispheres. After two weeks of cultivation, prostanoids were formed with the spectrum: PGD(2) > TXB(2) > PGF(2?) > PGE(2), as measured by specific radioimmunoassays. Under basal conditions PGD(2) biosynthesis (9.55 ng/mg protein/15 min) was in the same order of magnitude as the sum of the other prostanoids. The formation of prostanoids was stimulated in a concentration dependent manner (up to 6-10 fold) by the calcium ionophore A 23187 (0.01-10 ?M) as well as by melittin (0.01-5 ?g/ml), phospholipase A(2) (10-40 U/ml) and phospholipase C (0.01-1 U/ml). Basal and evoked PG and TXB(2) biosynthesis depended on the availability of Ca(2+), as demonstrated in Ca(2+) free incubation medium containing Na(2)EDTA (1 ?M), or with verapamil (100 ?M) and 3,4,5-trimethoxybenzoic acid-8-(diethylamino)-octylester-HCl (TMB-8, 1-100 ?M). Indomethacin (10 ?M), mepacrine (100 ?M) and p-bromophenacylbromide (50 ? M) inhibited basal and evoked PG formation. Thin-layer chromatography (TLC) detection after incubation of the cells with [(3)H]arachidonic acid (1 ?Ci/ml, for 60 min) confirmed the results obtained by radioimmunoassay. Incubation of [(3)H]arachidonic acid labelled cells with inonophore or phospholipases, followed by lipid extraction and TLC, showed that A 23187 liberated [(3)H]arachidonic acid predominantly from phosphatidylethanolamine, whereas phospholipase A(2) and C reduced mainly the labelling of the phosphatidyl-inositol/-choline fraction. Potassium depolarization of the cells did not enhance prostanoid formation. Similarly, drugs with affinity to ?- or ?-adrenoceptors, or to dopamine-, 5-hydroxytryptamine-, muscarine-, histamine-, glutamate-, aspartate-, GABA, adenosine- and opioid-receptors failed to stimulate prostanoid biosynthesis. Also compounds like angiotensin, bradykinin and thrombin were ineffective in this respect. In conclusion, our results confirm that cultured astrocytes possess the complete pattern of enzymes necessary for prostanoid formation and hence might play a crucial role in brain prostanoid biosynthesis. Stimulation of prostanoid biosynthesis involves Ca(2+)-dependent activation of phospholipase A(2), cyclooxygenase reaction and further PG metabolism. However, the endogenous stimulus for enhanced prostanoid synthesis in the brain still has to be established.

Multiple pertussis toxin substrates as candidates for regulatory G proteins of adenylate cyclase coupled to the somatostatin receptor in primary rat astrocytes.

The involvement of G proteins in receptor mediated astroglial cAMP formation was studied. Isoproterenol or prostaglandin E2 stimulated adenylate cyclase of primary astroglial cells was inhibited by somatostatin. Preincubation, of cells with increasing concentrations of islet activating protein (IAP) diminished somatostatin inhibition of adenylate cyclase. At an IAP concentration of 50 ng/ml somatostatin inhibition was completely abolished. Studies on IAP catalyzed32P-ADP-ribosylation of astroglial cell particulate material revealed an incorporation of radiolabel into three polypeptides in the molecular weight range of 41,000–39,000 Dalton. Pretreatment of intact cells with IAP reduced radiolabeling of this molecular species in a concentration dependent manner. No further radiolabeling above background level was detectable after pretreatment of cultures with 10 ng IAP/ml or more. At present, the occurrence of at least three IAP substrates (G proteins) does not permit an identification of the somatostatin receptor coupled G protein. Rather, the finding reveals that astrocytes are endowed with multiple variants of GTP binding proteins likely to be coupled to different receptors.