Modulation of neurosteroid synthesis/accumulation by L-ascorbic acid in rat brain tissue: inhibition by selected serotonin antagonists.

We have investigated the possibility that the synthesis/accumulation of neurosteroids, i.e., brain-produced steroids putatively endowed with modulatory actions in the CNS, is regulated by monoaminergic receptor-mediated mechanisms. In minces of rat brain cortex, L-ascorbic acid concentration-dependently (0.07-1.0 mM) increases the levels of pregnenolone, allotetrahydrodeoxycorticosterone, and dehydroepiandrosterone. This effect of L-ascorbic acid is region-dependent: in hippocampus, progesterone and allopregnanolone are also increased, whereas dehydroepiandrosterone is unchanged, and in corpus striatum only progesterone is increased significantly. 5-Hydroxytryptamine (10 microM), 1-(3-chlorophenyl)piperazine (1.0 microM), and 5-methoxytryptamine (0.4 microM) mimic the effect of L-ascorbic acid, whereas a pretreatment with p-chlorophenylalanine (400 mg/kg i.p., 2 days) reduces the amplitude of the L-ascorbic acid effect on brain cortical neurosteroids. The effect of L-ascorbic acid is blocked by the nonselective serotonin antagonists methiothepin, clozapine, methysergide, and pizotifen, but not mesulergine, spiperone, MDL 72222, and DL-propranolol, nor by the catecholaminergic receptor antagonists prazosin and S(-)-sulpiride. L-Ascorbic acid is not additive with dibutyryl-cyclic AMP and, furthermore, the inhibition of adenylate cyclase by MDL 12330A, but not of phospholipase C by U-73122, markedly attenuates the L-ascorbic acid-induced increase of pregnenolone in rat brain cortical minces. Together these data suggest that L-ascorbic acid plays a role in the modulation of neurosteroidogenesis, presumably by favoring the activation of the purported serotonin type 6 receptor by endogenous serotonin.

The effects of inhibitors of GABAergic transmission and stress on brain and plasma allopregnanolone concentrations.

1. This study was undertaken to investigate the relationship between a reduction in brain GABAA receptor function and the cerebro-cortical content of 3 alpha-hydroxy-5 alpha-pregnan-20 one (allopregnanolone, AP), a potent endogenous positive modulator of 7-aminobutyric acid (GABA) action at GABAA receptors, with anticonflict and anticonvulsant effects in rodents. 2. An acute depletion of the cerebral content of GABA or an attenuation of GABAA receptor-mediated transmission by systemic injections of isoniazid (375 mg kg-1, s.c.) or FG 7142 (15 mg kg-1, i.p.) induced a transient increase in the cerebro-cortical and plasma concentrations of AP in handling-habituated (not stressed) rats. 3. Two stress paradigms, handling in naive rats and mild foot shock in handling-habituated rats, that reduce central GABAergic tone mimicked the effects of isoniazid and FG 7142 on cortical AP content; foot shock in handling-habituated rats, but not handling in naive animals, also increased plasma AP. Isoniazid, FG 7142, and foot shock also each increased the concentrations of the AP precursors, pregnenolone and progesterone, in both brain and plasma of handling-habituated rats, whereas handling in naive rats increased the concentrations of these steroids only in brain. 4. Pretreatment of handling-habituated rats with the anxiolytic beta-carboline derivative abecarnil, a positive allosteric modulator of GABAA receptors, which per se failed to affect the AP concentration in brain or plasma, prevented the increase in brain and plasma AP induced by foot shock or isoniazid. 5. In adrenalectomized and castrated rats foot shock or isoniazid failed to increase AP both in brain cortex and plasma. 6. These observations indicate that inhibition of GABAergic transmission, induced by foot shock or pharmacological manipulations, results in an increase in the concentrations of AP in brain and plasma, possibly via a modulation of hypothalamic-pituitary-adrenal (HPA) axis. 7. Given that AP enhances GABAA receptor function with high efficacy and potency, an increase in brain AP concentration may be important in the fine tuning of the GABA-mediated inhibitory transmission in the central nervous system.

Functional correlation between allopregnanolone and [35S]-TBPS binding in the brain of rats exposed to isoniazid, pentylenetetrazol or stress.

1. The relation between changes in the cerebral cortical concentration of allopregnanolone and gamma-aminobutyric acid (GABA) type A receptor function after intracerebroventricular injection of this neurosteroid was investigated in male rats. 2. Intracerebroventricular administration of allopregnanolone (1.25 to 15 micrograms) produced a maximal increase (100 fold at the highest dose) in cortical allopregnanolone concentration within 5 min; the concentration remained significantly increased at 15 and 30 min, before returning to control values by 60 min. 3. The same treatment induced a rapid and dose-dependent decrease in the binding of t-[35S]-butylbicyclophosphorothionate ([35S]-TBPS) to cerebral cortical membranes measured ex vivo, an effect mimicked by the benzodiazepine midazolam but not by the 3 beta-hydroxyepimer of allopregnanolone. The time course of changes in [35S]-TBPS binding paralleled that of brain allopregnanolone concentration. 4. In a dose-dependent manner, allopregnanolone both delayed the onset of convulsions and inhibited the increase in [35S]-TBPS binding to cortical membranes induced by isoniazid. The potency of allopregnanolone in inhibiting [35S]-TBPS binding in isoniazid-treated rats was approximately four times that in control animals. 5. The ability of allopregnanolone to decrease [35S]-TBPS binding in isoniazid-treated rats also correlated with its anticonvulsant activity against pentylenetetrazol-induced seizures as well as its inhibitory effect on the increase in [35S]-TBPS binding induced by foot shock. 6. The results indicate that the in vivo administration of allopregnanolone enhances the function of GABAA receptors in rat cerebral cortex and antagonizes the inhibitory action of stress and drugs that reduce GABAergic transmission.

Stress-induced increase in brain neuroactive steroids: antagonism by abecarnil.

Acute foot shock stress elicits a selective and time-dependent increase of neuroactive steroid (pregnenolone, progesterone, allotetrahydrodeoxycorticosterone) concentrations in rat brain cortex, accompanied by a marked increase of plasma corticosterone. The brain cortical neuroactive steroid levels peaked between 10 and 30 min poststress and returned to control values by 2 h. Abecarnil (0.3 mg/kg), i.p.), a beta-carboline derivative with anxiolytic properties, completely antagonized the effect of foot shock on brain cortical neuroactive steroids. A single administration of the anxiogenic beta-carboline FG 7142 (15 mg/kg, i.p.), in contrast, mimicked the effect of foot shock. These data support the hypothesis for the existence of a functional relationship between brain neuroactive steroid concentrations and GABAA receptor function/emotional state of the animal.

Time-dependent changes in rat brain neuroactive steroid concentrations and GABAA receptor function after acute stress.

The time courses of changes in rat brain neuroactive steroid concentrations and gamma-aminobutyric acid type A (GABAA) receptor function elicited by acute stress were investigated in animals exposed to CO2 for 1 min, a treatment known to induce stress in rats and panic attacks in humans. Inhalation of CO2 induced increases in cerebral cortical steroid concentrations, the time dependence of which varied with the steroid examined. Thus, progesterone and deoxycorticosterone showed maximal increases (10- and 4-fold, respectively) 10 min after CO2 inhalation and had returned to basal values by 30 and 60 min, respectively. In contrast, pregnenolone and 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone) concentrations showed maximal increases (+174 and + 200%, respectively) at 30 min, were still higher than control at 60 min and returned to control values 120 min after stress. Inhalation of CO2 also resulted in increases in plasma steroid concentrations, most of which peaked at 30 min and had returned to control values by 60 min. A parallel analysis of the stress-induced changes in GABAA receptor function, assessed either biochemically by t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to cerebral cortical membranes or behaviorally by the punished responding score in Vogel's test, showed that the effects of CO2 inhalation on both parameters were maximal (+51 and -40%, respectively) after 10 min; the behavioral reaction returned to normal after 60 min, whereas [35S]TBPS binding had returned to control values 120 min after stress. The results show that: (a) the maximal increase in the brain concentrations of allopregnanolone, a potent and efficacious positive modulator of GABAA receptors, occurred at a time (30 min) when both conflict behavior and [35S]TBPS binding begun to decrease, and (b) both allopregnanolone concentrations and [35S]TBPS binding had returned to control values 120 min after CO2 inhalation. The data are thus consistent with a physiological role of neuroactive steroids in restoring GABAergic tone after stress.

Isoniazid-induced inhibition of GABAergic transmission enhances neurosteroid content in the rat brain.

Isoniazid (375 mg/kg, s.c.), a drug that decreases GABAA receptor-mediated transmission, elicited a time-dependent increase of neuroactive steroid (pregnenolone, progesterone and allotetrahydrodeoxycorticosterone) concentrations in rat brain and plasma. This treatment also time-dependently increased the plasma concentration of corticosterone. Brain and plasma neuroactive steroid levels peaked between 40 and 120 min after isoniazid administration, respectively, and returned to control values by 5 hr. Acute foot shock stress mimicked the effect of isoniazid by increasing in a time-dependent manner the same neuroactive steroids both in brain and plasma. Abecarnil (0.3 mg/kg, i.p.), a beta-carboline derivative with anxiolytic properties, antagonized the effect of both isoniazid and foot shock on brain and plasma neuroactive steroids and on plasma corticosterone level. These data indicate that an inhibition of central GABAergic transmission enhances the concentrations of THDOC and its precursors pregnenolone and progesterone in the rat brain and plasma as well as the plasma levels of corticosterone. This finding suggests that GABA exerts a tonic inhibitory action on the mechanisms involved in the regulation of the synthesis and release of these neuroactive steroids in the central nervous system and plasma.

Modulatory mechanisms of cyclic AMP-stimulated steroid content in rat brain cortex.

The modulation of cyclic AMP dependent neurosteroidogenesis was studied in minces prepared from the cerebral cortex of adult rat. Forskolin or dibutyryl-cyclic AMP enhanced pregnenolone and progesterone production in a time and dose-dependent manner. The forskolin effect was mimicked by the cyclic AMP phosphodiesterase inhibitor isobutyl-methyl-xanthine, but not by the adenylate cyclase inactive forskolin analogue 1,9,dideoxy-forskolin. 4'-Chloro-diazepam, a high affinity ligand for the mitochondrial diazepam binding inhibitor (DBI) receptor, also elicited a time dependent increase in steroidogenesis. The forskolin and the 4'-chloro-diazepam stimulated pregnenolone increase was prevented by preexposing the rat brain cortical minces to 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinoline carboxamide (PK 11195), a high affinity ligand for the mitochondrial DBI receptor endowed with antagonistic properties. The protein synthesis inhibitor cycloheximide prevented the forskolin and 4'-chloro-diazepam stimulation of pregnenolone formation. In brain cortical minces of adrenalectomised/orchiectomised rats dibutyryl-cyclic AMP increased both pregnenolone and progesterone formation, while forskolin only increased progesterone. These data show that cyclic AMP enhances brain steroidogenesis by acting on a labile protein substrate which interacts with the mitochondrial DBI receptor.

Neurosteroids in the brain of handling-habituated and naive rats: effect of CO2 inhalation.

In rats habituated to the manipulation that precedes killing (handling-habituated) the cerebral cortical concentrations of pregnenolone and progesterone were significantly lower (-57% and -69%, respectively) than in naive animals. An acute stress, induced by CO2 inhalation, elicited a marked increase in the concentrations of pregnenolone, progesterone and deoxycorticosterone in the brain cortex and hippocampus of handling-habituated rats. An accepted stress, such as foot shock, also enhanced the brain cortical levels of pregnenolone, progesterone and deoxycorticosterone in handling-habituated rats. These data show that the rat brain cortical and hippocampal steroid content is related to the 'emotional state' of the animal.

Cyclic AMP-dependent increase of steroidogenesis in brain cortical minces.

Rat brain cortical minces were incubated with forskolin and dibutyryl-cyclic AMP for 60 and 30 min, respectively. The concentrations of pregnenolone, progesterone and desoxycorticosterone in this preparation were significantly increased by both substances. The results indicate that, in brain tissue, steroidogenesis appears to be regulated by receptor transduction mechanisms that operate through adenylate cyclase.

In vitro interactions of opioid peptides with phospholipids. IV. Methionine enkephalin versus leucine enkephalin.

The interaction of methionine and leucine enkephalin with phosphatidylserine and phosphatidylcholine was studied by optical spectroscopy techniques. The data reported indicate that with both peptides the binding is controlled by ionic parameters. They also indicate that the differences in the binding behavior of the two peptides induced by changing these parameters are minor. Non-ionic interactions are also important in the binding phenomena, but the above observations hold in this case as well. Finally, the tridimensional structure of both enkephalins appears to be modified in the presence of phospholipids. Moreover, the changes induced by these lipids appear to differ from one peptide to the other.

Hydrolysis and binding of leucine enkephalin to lymphomic and erythroleukaemic cell lines.

Hydrolysis and binding of labelled leucine enkephalin have been measured in the presence of cell lines of lymphoid and erythroid origin. The radioactive label was found to be associated to all lines studied. In the presence of these cells, enkephalin is rapidly hydrolyzed, forming three tyrosine-containing peptides: Tyr, Tyr-Gly and Tyr-Gly-Gly. Conversely, the presence of selective protease inhibitors reduces both enkephalin degradation and binding. Data obtained suggest the involvement in enkephalin hydrolysis of aminopeptidases, dypeptidylaminopeptidases and dypeptidylcarboxypeptidases. In addition, they suggest that the radioactive label associated to cells can be related to the peptides formed by the enzyme degradation of enkephalin and not to the intact pentapeptide.

Hydrolysis and protection from hydrolysis of enkephalins in human plasma.

Seven groups of enkephalin-degrading enzymes and three groups of inhibitors active on these enzymes were separated from human plasma. The activity of the enzymes in hydrolyzing enkephalins and of the inhibitors in protecting enkephalins from proteolysis was measured. Results obtained with the endogenous inhibitors were compared to those relative to synthetic inhibitors. Data obtained indicate that all enkephalin-degrading enzymes found in plasma are significantly inhibited by the endogenous substances present in this tissue. The inhibition of the different classes of plasma enzymes by two of the three groups of endogenous substances is quite uniform, while one group of inhibitors appears specific to dipeptidylpeptidases. Results obtained are discussed in terms of the functional role of the inhibitory substances and of the possible pharmacological implication of their presence in human plasma.

Enkephalin binding systems in human plasma. III: Comparative protection of different peptides.

We have investigated the hydrolysis and protection from hydrolysis of several peptides by plasma enzymes and by the plasma components previously described as inhibitors of enkephalins' hydrolysis. The results shown indicate that all the peptides actually hydrolyzed are also partially protected from hydrolysis by the enkephalin-protecting substances. Protection is fairly uniform for all the peptides tested, but considerably higher in the case of leu- and met-enkephalin, suggesting a partial specificity of the protecting substances towards opioid peptides.

Enkephalin activity on antigen-induced proliferation of human peripheral blood mononucleate cells.

The effect of the two opioid pentapeptides met- and leu-enkephalin and of the tetrapeptide TYR-GLY-GLY-PHE on the stimulated proliferation of human peripheral blood mononucleate cells (PBMC) is described. Cell populations obtained from different donors were induced to proliferate with a Candida antigen. In the presence of the antigen, met-enkephalin and TYR-GLY-GLY-PHE had a concentration-dependent diphasic effect. The proliferation of PBMC was stimulated at high peptide concentration, while it was inhibited at the lower concentrations used. The effect of leu-enkephalin appears to be rather ambiguous, and directed towards the inhibition rather than the stimulation of PBMC proliferation.

Mechanisms protecting plasma peptides from enzyme hydrolysis: a comparative study.

1. The role of the enkephalin-protecting plasma substances in the protection of non-opioid peptides from enzyme hydrolysis has been studied in laboratory animals and in man. 2. The results obtained indicate that all the peptides hydrolyzed by the plasma enzymes are also protected from the hydrolysis by the enkephalin-protecting substances. 3. The protection is fairly uniform in all the species and for all the peptides examined. However, in the human species the protection of leucine enkephalin is considerably higher than the average. These results are discussed in terms of a possible differential inhibition of the different plasma aminopeptidases.

In vitro interaction of opioid peptides with phospholipids. III. The role of lipid.

The binding of tritiated Leu-enkephalin to phosphatidylserine and phosphatidylcholine vesicles, both unmodified and modified by the incorporation of free fatty acid, has been studied by steric exclusion chromatography, ultraviolet difference spectroscopy and fluorescence anisotropy. The results obtained tend to confirm that both ionic and hydrophobic interactions are important in the binding phenomena. On the other hand, it seems likely that steric factors play a very limited role in the recognition of the phospholipid by the opioid peptide. Finally, these results confirm the existence of three complexes of different size, as already demonstrated. But, unlike the previously presented results, they stress the importance of the larger of the three complexes formed through binding.

Effect of different parameters on the binding of two anti-inflammatory drugs to human serum albumin.

The binding to human serum albumin of two anti-inflammatory drugs, indomethacin and indoprofen, has been studied by chromatographic and spectroscopic techniques. The results shown indicate that the binding of both drugs--but more notably of indoprofen--is very sensitive to variations of the environmental conditions. The binding is also dependent upon limited modifications in the tertiary structure of the protein. The evidences shown tend to indicate that these two phenomena are related, and that the binding is permitted under conditions of a relatively open structure of the protein molecule.

Control mechanisms of peripheral enkephalin hydrolysis in mammalian plasma.

The protection of the adrenal-released enkephalins from enzyme hydrolysis by endogenous plasma components was studied in laboratory animals and in man. The results indicate that mechanisms active in protecting leu-enkephalin from hydrolysis are present in the plasma of all species examined. The protection seems to be due to two groups of substances, possibly of peptidic nature. The amount of protection given by these substances seems to be sufficient to play a significant role in controlling the physiological levels of leu-enkephalin released into the bloodstream.

Mechanisms of leu-enkephalin hydrolysis in human plasma.

The present work describes the kinetics of enkephalin hydrolysis by plasma enzymes and the fragmentation pattern of both the parent peptide and of the first hydrolysis by-products. The degradation kinetics were followed by positive identification of the hydrolysis fragments by chromatographic methods, by amino acid analysis and by scintillation counting of tritium-labeled enkephalin. In addition, the results presented confirm the role of the low molecular weight plasma components in the control of the hydrolysis of the peripherally-released enkephalins.