Effect Of Pregnenolone Sulfate Research Articles

Neuroprotection by the synthetic neurosteroid enantiomers ent-PREGS and ent-DHEAS against Aβ25–35 peptide-induced toxicity in vitro and in vivo in mice

Pregnenolone sulfate (PREGS) and dehydroepiandrosterone sulphate (DHEAS) are pro-amnesic, anti-amnesic and neuroprotective steroids in rodents. In Alzheimer's disease (AD) patient's brains, their low concentrations are correlated with high levels of Aβ and tau proteins. The unnatural enantiomer ent-PREGS enhanced memory in rodents. We investigated here whether ent-PREGS and ent-DHEAS could be neuroprotective in AD models. The effects of PREGS, ent-PREGS, DHEAS and ent-DHEAS against Aβ25-35 peptide-induced toxicity were examined in vitro on B104 neuroblastoma cells and in vivo in mice. B104 cells pretreated with the steroids before Aβ25-35 were analysed by flow cytometry measuring cell viability and death processes. Mice injected intracerebroventricularly with Aβ25-35 and the steroids were analysed for their memory abilities. Additionally, lipid peroxidation levels in the hippocampus were measured. ent-PREGS and PREGS significantly attenuated the Aβ25-35-induced decrease in cell viability. Both steroids prevented the Aβ25-35-induced increase in late apoptotic cells. PREGS further attenuated the ratio of necrotic cells. ent-DHEAS and DHEAS significantly reduced the Aβ25-35-induced toxicity and prevented the cells from entering late apoptosis and necrosis. All steroids stimulated neurite outgrowth per se and prevented the Aβ25-35-induced decrease. In vivo, ent-PREGS and ent-DHEAS significantly attenuated the Aβ25-35-induced decrease in memory (spontaneous alternation and passive avoidance) and an increase in lipid peroxidation levels. In contrast to the natural steroids, both enantiomers prevented amnesia when injected 6 h before Aβ25-35 in contrast to the natural steroids. The unnatural steroids ent-PREGS and ent-DHEAS are potent neuroprotective agents and could be effective therapeutical tools in AD.

Is connexin36 critical for GABAergic hypersynchronization in the hippocampus?

Synchronous bursting of cortical GABAergic interneurons is important in epilepsies associated with excitatory GABAergic signalling. If electrical coupling was critical for the generation of this pathological activity, then the development of selective blockers of connexin36-based interneuronal gap junctions could be of therapeutic value. We have addressed this issue in the 4-aminopyridine model of epilepsy in vitro by comparing GABAergic epileptiform currents and their sensitivity to gap junction blockers in wild-type vs. connexin36 knockout mice. Although electrical coupling was abolished in stratum lacunosum-moleculare interneurons from knockout animals, epileptiform currents were not eliminated. Furthermore, epileptiform currents propagated similarly across hippocampal layers in the two genotypic groups. Blockade of electrical coupling with carbenoxolone suppressed amplitude, frequency and half-width of the epileptiform currents both in wild-type and in knockout animals, whereas mefloquine had no effects. Carbenoxolone also depressed responses to exogenous and synaptic GABA application onto interneurons. We conclude that, in the 4-aminopyridine model of epilepsy in vitro, connexin36 is not critical for the generation of epileptiform discharges in GABAergic networks and that the observed antiepileptic effects of carbenoxolone are likely to be due to blockade of GABAA receptors and not of connexin36-based gap junctions. Lastly, because of its chemical structure and its effects on amplitude and kinetics of GABAergic currents, we tested the hypothesis that carbenoxolone acted via specific sites on GABAA receptors, such as the one mediating the effects of the neurosteroid pregnenolone sulfate, or the allosteric regulatory site of benzodiazepines/β-carbolines. Our results suggest that neither of these is involved.

Presynaptic Modulation of Synaptic Transmission by Pregnenolone Sulfate as Studied by Optical Recordings

The effects of pregnenolone sulfate (PREGS), a putative neurosteroid, on the transmission of perforant path-granule cell synapses were investigated with an optical recording technique in rat hippocampal slices stained with voltage-sensitive dyes. Application of PREGS to the bath solution resulted in an acute augmentation of EPSP in a dose-dependent manner. The PREGS effect was dependent on the extracellular Ca(2+) concentration ([Ca(2+)](o)), but independent of NMDA receptor activation. PREGS caused a decrease in paired-pulse facilitation, which implies that PREGS positively modulates presynaptic neurotransmitter releases. Firmer support for this mechanism was that PREGS augmented the synaptically induced glial depolarization (SIGD) that reflects the activity of electrogenic glutamate transporters in glial cells during the uptake of released glutamate. The selective alpha7nAChR antagonist alpha-BGT or MLA prevented the SIGD increase by PREGS. Furthermore DMXB, a selective alpha7nAChR agonist, mimicked the PREGS effect on SIGD and antagonized the effect of PREGS. The presynaptic effect of PREGS was partially attenuated by the L-type Ca(2+) channel (VGCC) blocker nifedipine. Based on these findings, we proposed a novel mechanism underlying the facilitated synaptic transmission by PREGS: this neurosteroid sensitizes presynaptic alpha7nAChR that is followed by an activation of L-type VGCC to increase the presynaptic glutamate release.

Neurosteroid enhances glutamate release in rat prelimbic cortex via activation of α1-adrenergic and σ1 receptors

The present paper studied the effect and mechanism of neurosteroid pregnenolone sulfate (PREGS) on spontaneous glutamate release using electrophysiological and biochemical methods combined with a pharmacological approach. The results suggested that PREGS had a selective enhancing effect on spontaneous glutamate release in the prelimbic cortex and the hippocampus but not in the striatum. The effect of PREGS in the prelimbic cortex appeared to be via modulation of alpha1-adrenergic and sigma1 receptors, but in the hippocampus it might be dependent on sigma1 receptors only. The activation of alpha1-adrenergic receptors synergized sigma1 receptor activation in the prelimbic cortex. Intracellular calcium released from the endoplasmic reticulum, protein kinase C, adenylyl cyclase and protein kinase A played a key role in the effect of PREGS. Intracellular calcium, protein kinase C and adenylyl cyclase might be upstream events in the activation of protein kinase A after PREGS.

Mechanism underlying inhibition of the capsaicin receptor-mediated current by pregnenolone sulfate in rat dorsal root ganglion neurons

The effect of pregnenolone sulfate (PS) on the capsaicin receptor-mediated current was studied in acutely dissociated rat dorsal root ganglion neurons using the whole-cell voltage-clamp technique. PS acted extracellularly to inhibit the capsaicin-induced current in a not competitive but concentration-dependent manner, with an EC 50 of 13 μM and a maximal inhibition of 65%. Furthermore, antagonism of the capsaicin response by PS is neither voltage- nor agonist-dependent, indicating that PS did not act as an open-channel blocker. The inhibitory action of PS on the capsaicin-induced current may provide a basis for reducing capsaicin receptor-mediated nociception.

Inhibition by pregnenolone sulfate of nicotinic acetylcholine response in adrenal chromaffin cells

To evaluate whether pregnenolone sulfate, an abundant neurosteroid in the brain, modulates nicotinic receptor-mediated responses, the effect of pregnenolone sulfate on acetylcholine-induced catecholamine secretion was investigated in cultured bovine adrenal chromaffin cells. Pregnenolone sulfate inhibited acetylcholine-induced catecholamine secretion (IC 50: 27 μM). In addition, pregnenolone sulfate inhibited acetylcholine-induced Na + (IC 50: 12 μM) and Ca 2+ (IC 50: 20 μM) influxes. However, pregnenolone sulfate did not inhibit either catecholamine secretion or Ca 2+ influx stimulated by high K +. Binding of [ 3H]nicotine to nicotinic receptors was not altered by pregnenolone sulfate. The inhibitory effect on the acetylcholine-induced secretion was insurmountable by increasing acetylcholine concentrations, but was enhanced by decreasing external Na + concentrations. These results suggest strongly that pregnenolone sulfate noncompetitively inhibits nicotinic receptor-operated ion channels, thereby suppressing Na + influx through the channels and, consequently, attenuates both Ca 2+ influx and catecholamine secretion. Our results further indicate that pregnenolone sulfate may modulate nicotinic receptor-mediated responses in the brain.

Non-linear effects in the retention of an avoidance task induced by anabolic steroids

The results of the study reported in Brain Research in 1995 by Isaacson et al. [Isaacson, R.L., Varner, J.A., Baars, J.-M., de Wied, D., 1995. The effects of pregnenolone sulfate and ethylestrenol on retention of a passive avoidance task. Brain Res. 689, 79–84] have been re-examined with special emphasis placed on the distributions of latencies found in the passive avoidance task using rats. This study used two retention tests, one 24 h after training the other at 48 h after training. In the first experiment in that study a range of doses of two anabolic steroids, pregnenolone sulfate and ethylestrenol, were given s.c. just after the footshock training trial. In experiment 2 a similar range of doses of both steroids was given to the rats 1 h before the first retention test. Placing emphasis on the distributions rather than measures of central tendencies revealed that, in contrast to the vehicle treated animals, the anabolic steroid treated animals exhibited bimodal distributions of response latencies. These differences between control and hormone treated animals were observed in both experiments. The new information was interpreted in terms of non-linear dynamics including some aspects of Chaos theory.

POTENTIATION OF MUSCIMOL-INDUCED LONG-TERM DEPRESSION BY BENZODIAZEPINES AND PREVENTION OR REVERSAL BY PREGNENOLONE SULFATE

We have recently reported a new protocol for inducing long-term depression through activation of GABAAreceptors in the hippocampal slices. This long-term depression is reversed by bicuculline and potentiated by neurosteroids such as alphaxalone. It was also shown that glutamate receptor activity or extracellular calcium are not involved in the induction of this type of long-term depression. The present study investigated the possible relation between muscimol-induced long-term depression and barbiturates/benzodiazepine-induced amnesia and attempts to determine the possible effect of pregnenolone sulfate on muscimol-induced long-term depression. Extracellular recordings were made in the CA1 pyramidal cell layer of rat hippocampal slices following orthodromic stimulation of Schaffer collateral fibres in stratum radiatum (0.01 Hz). It was observed that pentobarbital, benzodiazepines and pregnanolone at concentrations that did not have any effect themselves on the population spike, potentiate the ability of muscimol to induce long-term depression. In addition to this, the long-term depression was either blocked or reversed by pregnenolone sulfate at concentrations (10 μM) where pregnenolone sulfate did not induce any multiple burst or increase of spike size. The results suggest that the potentiation of this type of long-term depression by benzodiazepines and barbiturates can explain the main adverse effect of these drugs, amnesia and cognitive impairment. Moreover, the prevention or reversal of this type of long-term depression by pregnenolone sulfate, may suggest a clinical application of this agent in the management of amnesia or dementia.

The neurosteroid pregnenolone sulfate increases cortical acetylcholine release: a microdialysis study in freely moving rats.

The effects of pregnenolone sulfate (Preg-S) administrations (0, 12, 48, 96, and 192 nmol intracerebroventricularly) on acetylcholine (ACh) release in the frontal cortex and dorsal striatum were investigated by on-line microdialysis in freely moving rats. Following Preg-S administration, extracellular ACh levels in the frontal cortex increased in a dose-dependent manner, whereas no change was observed in the striatum. The highest doses (96 and 192 nmol) induced a threefold increase above control values of ACh release, the intermediate dose of 48 nmol led to a twofold increase, whereas after the dose of 12 nmol, the levels of ACh were not different from those observed after vehicle injection. The increase in cortical ACh reached a maximum 30 min after administration for all the active doses. Taken together, these results suggest that Preg-S interacts with the cortical cholinergic system, which may account, at least in part, for the promnesic and/or antiamnesic properties of this neurosteroid.

The effects of pregnenolone sulfate and ethylestrenol on retention of a passive avoidance task

Two experiments using male rats evaluated the effects of a range of doses of the neurosteroid, pregnenolone sulfate (PS), or of the synthetic neurosteroid, ethylestrenol (E), on the retention of a passive avoidance task. The steroids either were given immediately after the training trial or 1 h before the first retention test. Retention tests were given both 24 h and 48 h after acquisition. In both experiments, separate groups of animals were trained under low or moderate footshock conditions. At all doses tested both PS and E improved retention under the low footshock conditions. In groups trained with the higher footshock, the steroid-treated groups performed no better than the vehicle controls. Indeed, there were suggestions that some doses impaired retention. These results seem best understood as an induction of bimodality or ‘turbulence’ in behavior as used in Chaos theory rather than a shift in an inverted U-shaped retention function. In the second experiment in which the steroids were given before retention testing, they were generally without effect.

The effect of pregnenolone sulfate on uterine contractility

The role of myometrial progesterone block in the maintenance of pregnancy was tested in 12 women in labor by infusing pregnenolone sulfate, the immediate precursor of placental progesterone. Uterine contractions were recorded through a catheter inserted transparietally into the amniotic cavity. The intensity, frequency, and activity of contractions during a 90 minute control period (considered as 100 per cent) were compared with those during 45 minutes' infusion of pregnenolone sulfate (3 mg. per kilogram body weight). Uterine activity decreased significantly to a mean of 67 per cent and frequency of contractions to 75 per cent whereas the intensity decreased only 91 per cent of the preinfusion values. During the 90 minutes following the infusion, uterine activity started to recover. Plasma progesterone level which was 13.8 μg per cent during the control period did not change during pregnenolone infusion (13.3 μg per cent). This suggests that reduced uterine contractility cannot be caused by increased placental progesterone output. Plasma pregnenolone sulfate level rose from 21.05 to 1,333.39 μg per cent. This high level might exert a pharmacologic depressive effect on the myometrium. The therapeutic application of pregnenolone sulfate is now under study.