Rosa Guarneri

Neurosteroidogenesis in Rat Retinas

Abstract: Neurosteroids (steroids synthesized in the CNS) function by modulating neurotransmission. To establish an experimental model for investigation of neurosteroid synthesis and regulation, independent of blood‐borne steroids, we examined the steroidogenic activity of isolated rat retinas. We identified progesterone, pregnenolone, dehydroepiandrosterone, desoxycorticosterone, 3α,5α‐tetrahydrodesoxycorticosterone, 3α‐hydroxy‐5α‐dihydroprogesterone, 17‐hydroxyprogesterone, and 17‐hydroxypregnenolone together with their esterified forms. As pregnenolone is the precursor of all steroids, its formation was studied in detail as an index of a steroid‐synthesizing tissue. Pregnenolone was identified further by gas chromatography coupled to mass spectrometry, and its in vitro synthesis was inhibited by lovastatin, an inhibitor of mevalonolactone and cholesterol biosynthesis. We then examined pregnenolone synthesis in the presence of mevalonolactone as a precursor of sterol formation together with lovastatin, which reduces endogenous mevalonolactone synthesis, as well as with inhibitors of pregnenolone metabolism. The incorporation of mevalonolactone into pregnenolone and its sulfate ester was time‐ and concentration‐dependent and blocked by aminoglutethimide, a competitive inhibitor of cytochrome P450 side‐chain cleavage (P450scc) enzyme. Immunocytochemical studies with a specific antibody to P450scc revealed a primary localization of the enzyme at the retinal ganglion cell layer. A less pronounced immunostaining was also seen at cells of the inner nuclear layer. Compounds known to stimulate cyclic AMP content also stimulated pregnenolone formation by rat retinas. These results demonstrate that rat retinas synthesize steroids and, for the first time, they reveal the steroidogenic ability of neuronal cells. We propose rat retinas as an in vitro model system to study neurosteroidogenesis in the CNS.

Pregnenolone sulfate, a naturally occurring excitotoxin involved in delayed retinal cell death.

Abstract: The present study was designed to investigate the neurosteroid pregnenolone sulfate (PS), known for its ability to modulate NMDA receptors and interfere with acute excitotoxicity, in delayed retinal cell death. Three hours after exposure of the isolated and intact retina to a 30‐min PS pulse, DNA fragmentation as assessed by genomic DNA gel electrophoresis and a modified in situ terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end‐labeling (TUNEL) method appeared concurrently with an increase in superoxide dismutase (SOD) activity and thiobarbituric acid‐reactive substances (TBARS) levels. At 7 h, the increased amount of DNA laddering was accompanied by a higher number of TUNEL‐positive cells in the inner nuclear and ganglion cell layers. Necrotic signs were characterized by DNA smear migration, lactate dehydrogenase (LDH) release, and damage mainly in the inner nuclear layer. PS‐induced delayed cell death was markedly reduced by the NMDA receptor antagonists 4‐(3‐phosphonopropyl)‐2‐piperazinecarboxylic acid and 3α‐hydroxy‐5β‐pregnan‐20‐one sulfate but completely blocked after concomitant addition of the non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione. Steroids with antioxidant properties (progesterone, dehydroepiandrosterone and its sulfate ester, and 17β‐estradiol) differently prevented PS‐induced delayed cell death. Cycloheximide treatment protected against DNA fragmentation and LDH release but failed to prevent the rise in SOD activity and TBARS level. We conclude that a brief PS pulse causes delayed cell death in a slowly evolving apoptotic fashion characterized by a cycloheximide‐sensitive death program downstream of reactive oxygen species generation and lipid peroxidation, turning into secondary necrosis in a retinal cell subset.

Interaction of uridine with GABA binding sites in cerebellar membranes of the rat.

The effect of uridine, a postulated anticonvulsant agent, on GABA receptors has been investigated. Uridine inhibits [3H]GABA binding to rat cerebellar bufferwashed membranes. Pretreatment of the membranes with Triton X-100 increases the effect of uridine on GABA-binding. The Scatchard analysis reveals that both high and low affinities of GABA for its receptors are affected by 1 mM uridine, while the apparent number of binding sites remains unchanged. The ability of uridine to interact competitively with GABA binding sites, also examined by the Lineweaver-Burk analysis, suggests a possible mechanism of action of this anticonvulsant agent, so including it among those compounds characterized by a GABAergic agonist activity.

Nicotinamide adenine dinucleotides in the developing rat brain

Summary Measurements of oxidized and reduced nicotinamide adenine dinucleotides (NAD, NADH, NADP, NADPH) in the developing rat brain have been carried out at various time intervals after birth. The concentration of the two oxidized coenzymes (μg/g wet weight) increased in the developing brain, while the level of the reduced coenzymes underwent a progressive decline. The relevance of these results to the understanding of the metabolic organization of the nervous tissue is briefly discussed.

Lateral differences in GABA binding sites in rat brain

An asymmetric distribution of GABA binding sites was found in the cerebral cortex, hippocampus, cerebellar hemispheres, striatum, and thalamus. Higher levels of [3H]GABA binding were observed in the left-side of most brain areas and in a greater percentage of adult rats, but the opposite asymmetry was found in the thalamus. A similar left-right difference in cerebral hemispheres was also found in five day-old rats, suggesting the genetic predetermination of asymmetry.

LACTATE DEHYDROGENASE ISOENZYMES IN THE NERVOUS TISSUE. V. THE EFFECT OF X- RAYS ON THE ENZYME OF THE DEVELOPING AND ADULT RAT BRAIN

DEVELOPING neurons and proliferating glial cells are highly susceptible to ionizing radiations (BAILEY, 1962). The brain of the new-born rat would appear, therefore, to be an exceptional target to ionizing radiations. The immaturity of rat brain at birth is reflected in three main morphogenetic processes: (i) the development of dendrites and axons from neurons which have attained their final number; (ii) the active proliferation of glial cells; (iii) the myelinisation (SUGITA, 19 17). Parallel to these phenomena are several biochemical changes among which the molecular evolution of lactate dehydrogenase (LDH) has been recently investigated in this laboratory (BONAVITA, PONTE and AMORE, 1962, 19646). The present article describes an attempt to modify the maturation curve of LDH by ionizing radiations. It has been supposed that the block of glial proliferation (BAILEY, 1962) would have been responsible for an apparent acceleration of the maturation process previously reported. The experimental evidence has been in keeping with the hypothesis. It has been found, however, that there is also a delayed effect of radiation on some kinetic properties of the enzyme.

LACTATE‐DEHYDROGENASE ISOENZYMES IN NERVOUS TISSUE—III

THE BIOCHEMICAL properties of single nervous structures have not been the topic of many systematic investigations. With reference to the enzyme composition, regional neurochemistry was first studied by Lowry and coworkers who also collected data on LDHt (STROMINGER and LOWRY, 1955; ROBERTS et al., 1958). Using a different approach, BAILEY and HEALD (1961) have recently shown startling differences in the protein composition of various nervous structures by means of starch-gel electrophoresis of tissue extracts. According to these authors (BAILEY and HEALD, 1961), cytoplasmic proteins exhibit electrophoretic patterns which are peculiar to the species and to the brain structure. Clearly, differences in the electrophoretic patterns given by the pool of cytoplasmic proteins do not imply functional differences at a biochemical level per se, although the hypothesis is quite likely to be true. BONAVITA and GUARNERI (1962) have already reported significant catalytic differences among LDH isoenzymes from ox brain as separated by agar-gel electrophoresis according to GRABAR and WILLIAMS (1955). Though the electrophoretic migration on agar gel causes great conformational changes of ox brain isoenzymes, satisfactory evidence has been obtained to suggest that LDH isoenzymes of mammalian brain are catalytically different also in their native condition. Experiments with pyridine analogues of DPN support this view (FLEXNER et al., 1960; BONAVITA and GUARNERI, 1962). Starting from this observation, the possibility of a differential isoenzymic composition of various brain structures has now been examined. This paper summarizes electrophoretic and catalytic findings concerning ox brain LDH’s. Regional differences in the distribution of single isoenzymes and in the kinetic properties of unresolved LDH are not so great as those observed in the comparative analysis of vertebrates (BONAVITA and GUARNERJ, 1963). Regional data, however, are certainly much more relevant than comparative observations to the molecular basis of systemic nervous pathology.

Lactic dehydrogenase isozymes in the nervous tissue I. The reaction of isozymes with diphosphopyridine nucleotide analogues and their inhibition by sodium metabisulfite

Abstract 1. 1. Brain l (+)lactic acid dehydrogenases from several species have been submitted to agar-gel electrophoresis. When the pH is 7.6, the beef-brain enzyme, which has been studied most extensively, may be resolved into three main components. 2. 2. Some kinetic properties of three l (+)lactic acid dehydrogenase isozymes from beef brain have been investigated by using several DPN analogues and the determination of other kinetic parameters ( K m for sodium pyruvate, excess substrate inhibition, inhibition by sodium metabisulfite). 3. 3. Sodium metabisulfite has been found to be a powerful competitive inhibitor of brain l (+)lactic acid dehydrogenase. Resolved isozymes are less sensitive to the inhibitor than the native enzyme. pH-activity curves in the presence of sodium metabisulfite show definite differences among the multiple forms of l (+)lactic acid dehydrogenase. 4. 4. Experiments with the pyridine analogues of DPN and sodium metabisulfite, and also the analysis of the enzyme affinity for pyruvate, have shown that the electrophoretic migration on agar gel causes a conformational change of the single l (+)lactic acid dehydrogenases. The nature of this modification has been discussed.

NEUROPHYSIOLOGICAL AND NEUROCHEMICAL STUDIES WITH THE ISONICOTINOYLHYDRAZONE OF PYRIDOXAL 5-PHOSPHATE.

SOME five years age we reported that pyridoxal5-phosphate (PALP) can be replaced by its isonicotinoylhydrazone (PALPINH) in the reactivation of partially resolved PALP-dependent enzymes, such as aspartate aminotransferase and L-arginine carboxy-lyase (BONAVITA and SCARDI, 1958, 1959). In the attempt to offer an explanation of this finding, two alternative hypotheses were formulated: (i) PALPINH could interact with the apoprotein through a displacement reaction of INH in order to permit the binding of the aldehyde group of PALP to the enzyme; (ii) PALPINH could bind to the enzyme protein without preliminary breakdown, and a subsequent displacement reaction would occur between the aminoacid substrate and INH, thus forming the enzyme-coenzyme-substrate complex. Similar evidence of an increase in enzyme activity when PALPINH or other hydrazones are added to PALP-dependent enzymes has been provided by GONNARD and co-workers (GONNARD and NGUYEN-PHILIPPON, 1959~ ; GONNARD and NCUYENCHI, 19596; GONNARD and FENARD, 1962). It has also been shown for brain glutamate carboxy-lyase (GDC), and this has a most immediate bearing on the question as to whether the biochemical mechanism of isoniazid convulsions can be visualized as a consequence of GDC inhibition following the in vivo formation of PALPINH (KILLAM and BAIN, 1957; KILLAM, DASCUPTA and KILLAM, 1960). Since the possible activation of PALP-dependent enzymes by PALPINH is in apparent contrast with the hypothesis of GDC inhibition by a direct block of the coenzyme, it has been suggested more recently that PALPINH, formed in vivo after isoniazid injection (BAIN and WILLIAMS, 1960), has the main effect of inhibiting pyridoxal (PAL) phosphorylation (BONAVITA, 1961 ; MEDINA, BRAYMER and REEVES, 1962). This article describes neurophysiological and neurochemical experiments with PALPINH injected intravenously or intraperitoneally to intact o r curarized cats. Evidence is described favouring the conclusion that PALPINH is a powerful convulsant agent whose enzyme target is not unique. Though the injection of the compound may not duplicate the conditions of its in vivo formation, it has been felt that some relevant data could be obtained following this experimental design. PYRIDOXAL 5-PHOSPHATE*

The effect of anoxia on aspartate aminotransferase of the new-born rat

Abstract 7-day old rats have been exposed to anoxia for different time intervals, and a reversible shift of the prevailing pyridoxamine form toward the pyridoxal form of aspartate aminotransferase has been measured in brain and heart. Both tissues have also exhibited a parallel increase of dinitrophenylhydrazine-reacting compounds, thus suggesting a relationship between the elevation of keto acids and the increase of the pyridoxal form of the enzyme.