Evidence for effective structure-based neuromodulatory effects of new analogues of neurosteroid allopregnanolone.

The neurosteroid allopregnanolone (AP) modulates neuroendocrine/neurobiological processes, including hypothalamic-pituitary-adrenocortical activities, pain, anxiety, neurogenesis and neuroprotection. These observations raised the hope of developing AP-based therapies against neuroendocrine and/or neurodegenerative disorders. However, the pleiotropic actions of AP, particularly its cell-proliferation-promoting effects, hamper the development of selective/targeted therapies. For example, although AP-induced neurogenesis may serve to compensate neuronal loss in degenerative brains, AP-evoked cell-proliferation is contraindicated for steroid-sensitive cancer patients. To foster progress, we synthesised 4 novel AP analogues of neurosteroids (ANS) designated BR053 (12-oxo-epi-AP), BR297 (O-allyl-epi-AP), BR351 (O-allyl-AP) and BR338 (12-oxo-AP). First, because AP is well-known as allosteric modulator of GABAA receptors (GABAA-R), we used the electrophysiological patch-clamp technique to determine the structure-activity relationship of our ANS on GABAA-activated current in NCB20 cells expressing functional GABAA-R. We found that the addition of 12-oxo-group did not significantly change the respective positive or negative allosteric effects of 3α-AP or 3ß-(epi)-AP analogues. Importantly, substitution of the 3α-hydroxyl-group by 3α-O-allyl highly modified the ANS activities. Unlike AP, BR351 induced a long-lasting desensitisation/inhibition of GABAA-R. Interestingly, replacement of the 3ß-hydroxyl by 3ß-O-allyl (BR297) completely reversed the activity from negative to positive allosteric action. In a second step, we compared the actions of AP and ANS on SH-SY5Y neuronal cell viability/proliferation using MTT-reduction assays. Different dose-response curves were demonstrated for AP and the ANS. By contrast to AP, BR297 was totally devoid of cell-proliferative effect. Finally, we compared AP and ANS abilities to protect against oxidative stress-induced neuronal death pivotally involved in neurodegenerative diseases. Both BR351 and BR297 had notable advantages over AP in protecting SH-SY5Y cells against oxidative stress-induced death. Thus, BR297 appears to be a potent neuroprotective compound devoid of cell-proliferative activity. Altogether, our results suggest promising perspectives for the development of neurosteroid-based selective and effective strategies against neuroendocrine and/or neurodegenerative disorders.

Behavioral and electromyographic assessment of oxaliplatin-induced motor dysfunctions: Evidence for a therapeutic effect of allopregnanolone.

The antineoplastic oxaliplatin (OXAL) is pivotal for metastatic cancer treatments. However, OXAL evokes sensory and motor side-effects including pain, muscle weakness, motor nerve fiber dysfunctions/neuropathies that significantly impact patients' lives. Therefore, preclinical investigations are struggling to characterize effective analgesics against OXAL-induced painful/sensory symptoms but surprisingly, OXAL-evoked motor dysfunctions received little attention although these neurological symptoms are also disabling for patients. Here, we validated a rat model of OXAL-induced motor neuropathy by using (i) behavioral methods as the wire suspension and balance beam tests to assess muscle weakness and (ii) electrophysiological techniques to record the gastrocnemius electromyography (EMG). The conductance velocity of motor fibers was reduced and compound muscle action potential (CMAP) duration increased in OXAL-treated rats, leading to CMAP dispersion with no modification of the area under the curve, reflecting a heterogeneous demyelination of motor fibers. Functional motor unit analysis revealed a 50 % decrease of their estimated number which was compensated by a motor unit size increase. OXAL-induced motor weakness appeared as a combined consequence of motor fiber demyelination and motor axonopathy. Because we previously observed that allopregnanolone (AP) counteracted OXAL-evoked painful/sensory symptoms, we evaluated its action against OXAL-induced motor neurological dysfunctions. AP treatment successfully corrected motor behaviors, conductance velocity, CMAP duration, motor unit number (MUN) and motor unit size altered by OXAL-chemotherapy. These results, which are the first to show that AP efficiently rescues OXAL-induced motor neuropathy, consolidate the idea that AP-based therapy may be relevant for the treatment of both sensory and motor peripheral neuropathies.

Gamma-hydroxybutyrate, acting through an anti-apoptotic mechanism, protects native and amyloid-precursor-protein-transfected neuroblastoma cells against oxidative stress-induced death.

Clinical observations suggested that gamma-hydroxybutyrate (GHB) protects nerve cells against death but the direct proofs are missing. Here, we combined several approaches to investigate GHB capacity to protect human neuroblastoma SH-SY5Y cells against hydrogen peroxide (H2O2)-induced death. To increase the patho-physiological relevancy of our study, we used native SH-SY5Y cells and SH-SY5Y cells stably transfected with the wild-type amyloid-precursor-protein (APPwt) or control-vector-pCEP4. Trypan Blue exclusion and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium-bromide) assays combined with pharmacological analyses showed that H2O2 reduced native and genetically modified cell viability and APPwt-transfected cells were the most vulnerable. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and activated caspase-3 staining assessed by flow cytometry revealed a basally elevated apoptotic signal in APPwt-transfected cells. Reverse-transcription, real-time quantitative polymerase chain reaction (qPCR) and Western blotting showed that mRNA and protein basal ratios of apoptotic modulators Bax/Bcl-2 were also high in APPwt-transfected cells. GHB efficiently and dose-dependently rescued native and genetically modified cells from H2O2-induced death. Interestingly, GHB, which strongly decreased elevated basal levels of TUNEL-staining, activated caspase 3-labeling and Bax/Bcl-2 in APPwt-transfected cells, also counteracted H2O2-evoked increased apoptotic markers in native and genetically modified SH-SY5Y cells. Since GHB did not promote cell proliferation, anti-apoptotic action through the down-regulation of Bax/Bcl-2 ratios and/or caspase 3 activity appears as a critical mechanism involved in GHB-induced protection of SH-SY5Y cells against APPwt-overexpression- or H2O2-evoked death. Altogether, these results, providing multi-parametric evidence for the existence of neuroprotective action of GHB, also open interesting perspectives for the development of GHB analog-based strategies against neurodegeneration or nerve cell death.

Potential role of allopregnanolone for a safe and effective therapy of neuropathic pain.

Because the treatment and management of neuropathic pain are extremely complicated, the characterization of novel analgesics and neuroprotectors with safe toxicological profiles is a crucial need to develop efficient therapies. Several investigations revealed that the natural neurosteroid allopregnanolone (AP) exerts analgesic, neuroprotective, antidepressant and anxiolytic effects. These effects result from AP ability to modulate GABA(A), glycine, L- and T-type calcium channels. It has been shown that AP treatment induced beneficial actions in humans and animal models with no toxic side effects. In particular, a multi-parametric analysis revealed that AP efficiently counteracted chemotherapy-evoked neuropathic pain in rats. It has also been demonstrated that the modulation of AP-producing enzyme, 3α-hydroxysteroid oxido-reductase (3α-HSOR), in the spinal cord regulates thermal and mechanical pain thresholds of peripheral nerve injured neuropathic rats. The painful symptoms were exacerbated by intrathecal injections of provera (pharmacological inhibitor of 3α-HSOR) which decreased AP production in the spinal cord. By contrast, the enhancement of AP concentration in the intrathecal space induced analgesia and suppression of neuropathic symptoms. Moreover, in vivo siRNA-knockdown of 3α-HSOR expression in healthy rat dorsal root ganglia increased thermal and mechanical pain perceptions while AP evoked a potent antinociceptive action. In humans, blood levels of AP were inversely associated with low back and chest pain. Furthermore, oral administration of AP analogs induced antinociception. Altogether, these data indicate that AP, which possesses a high therapeutic potential and a good toxicological profile, may be used to develop effective and safe strategies against chronic neuropathic pain.

The small GTPase RhoA regulates the expression and function of the sodium channel Nav1.5 in breast cancer cells.

Voltage-gated Na+ channels (VGSCs) are highly expressed in several types of carcinomas including breast, prostate and lung cancers as well as in mesothelioma and cervical cancers. Although the VGSCs activity is considered crucial for the potentiation of cancer cell migration and invasion, the mechanisms responsible for their functional expression and regulation in cancer cells remain unclear. In the present study, the role of the small GTPase RhoA in the regulation of expression and function of the Nav1.5 channel in the breast cancer cell lines MDA-MB 231 and MCF-7 was investigated. RhoA silencing significantly reduced both Nav1.5 channel expression and sodium current indicating that RhoA exerts a stimulatory effect on the synthesis of an active form of Nav1.5 channel in cancer cells. The inhibition of Nav1.5 expression dramatically reduced both cell invasion and proliferation. In addition, a decrease of RhoA protein levels induced by Nav1.5 silencing was observed. Altogether, these findings revealed: i) the key role of the small GTPase RhoA in upregulation of Nav1.5 channel expression and tumor aggressiveness, and ii) the existence of a positive feedback of Nav1.5 channels on RhoA protein levels.

Milestones on Steroids and the Nervous System: 10 years of basic and translational research.

During the last 10 years, the conference on 'Steroids and Nervous System' held in Torino (Italy) has been an important international point of discussion for scientists involved in this exciting and expanding research field. The present review aims to recapitulate the main topics that have been presented through the various meetings. Two broad areas have been explored: the impact of gonadal hormones on brain circuits and behaviour, as well as the mechanism of action of neuroactive steroids. Relationships among steroids, brain and behaviour, the sexual differentiation of the brain and the impact of gonadal hormones, the interactions of exogenous steroidal molecules (endocrine disrupters) with neural circuits and behaviour, and how gonadal steroids modulate the behaviour of gonadotrophin-releasing hormone neurones, have been the topics of several lectures and symposia during this series of meetings. At the same time, many contributions have been dedicated to the biosynthetic pathways, the physiopathological relevance of neurosteroids, the demonstration of the cellular localisation of different enzymes involved in neurosteroidogenesis, the mechanisms by which steroids may exert some of their effects, both the classical and nonclassical actions of different steroids, the role of neuroactive steroids on neurodegeneration, neuroprotection, and the response of the neural tissue to injury. In these 10 years, this field has significantly advanced and neuroactive steroids have emerged as new potential therapeutic tools to counteract neurodegenerative events.

Calcium and cAMP signaling induced by gamma-hydroxybutyrate receptor(s) stimulation in NCB-20 neurons.

The NCB-20 neurohybridoma cells differentiated with dibutyryl-cyclic-AMP represent an interesting model to study several components of the gamma-hydroxybutyrate (GHB) system in brain. In particular, an active Na(+)-dependent uptake and a depolarization-evoked release of GHB is expressed by these cells, together with high affinity specific binding sites for this substance. However, only little is known about cellular mechanisms following GHB receptor(s) stimulation in these neurons. Electrophysiological data indicate that GHB can differently affect Ca(2+) currents. L-type calcium channels were typically inhibited by GHB when NCB-20 cells were depolarized. In contrast, when NCB-20 cells were at resting potential, GHB induced a specific Ca(2+) entry through T-type calcium channels. In this study, we investigated the effect induced on cytosolic free Ca(2+) level and cAMP production by GHB receptor(s) stimulated with micromolar concentrations of GHB or structural analogues of GHB. Ca(2+) movements studied by cellular imaging were dose-dependently increased but disappeared for GHB concentrations >25 microM. In addition, nanomolar doses of GHB inhibited forskolin-stimulated adenylate cyclase. This effect was also rapidly desensitized at higher GHB concentrations. Acting as an antagonist, NCS-382 decreased GHB receptor(s) mediated cAMP and calcium signals. The agonist NCS-356 mimicked GHB effects which were not affected by the GABA(B) receptor antagonist CGP-55-845. Our results reveal the occurrence of Ca(2+)-dependent adenylate cyclase inhibition in NCB-20 neurons after GHB receptor(s) stimulation by GHB concentrations <50 microM. Above this dose, GHB effects were inactivated. In addition, at GHB concentrations exceeding 50 microM, GTP-gammaS binding was also reduced, confirming the desensitization of GHB receptor(s). Taken together, these results support the existence in NCB-20 neurons of GHB receptors belonging to GPCR family that may recruit various G protein subtypes.

Endogenous steroid production in the spinal cord and potential involvement in neuropathic pain modulation.

It has recently been demonstrated that the spinal cord (SC) is an active production center of neuroactive steroids including pregnenolone, dehydroepiandrosterone, progesterone and allopregnanolone. Indeed, anatomical, cellular and biochemical investigations have shown that the SC dorsal horn (DH), a pivotal structure in nociception, contains various active steroidogenic enzymes such as cytochrome P450side-chain-cleavage, cytochrome P450c17, 3beta-hydroxysteroid dehydrogenase, 5alpha-reductase and 3alpha-hydroxysteroid oxido-reductase. Reviewed here are several data obtained with in vitro and vivo experiments showing that endogenous steroids synthesized in the SC are involved in the modulation of nociceptive mechanisms. Various approaches were used as the real-time polymerase chain reaction after reverse transcription to determine the effects of neuropathic pain on the expression of genes encoding steroidogenic enzymes in the DH. Combination of the pulse-chase technique with high performance liquid chromatography and continuous flow scintillation detection allowed investigations of the impact of noxious signals on the activity of steroid-producing enzymes in the SC in vitro. Radioimmunological analyses of spinal tissue extracts contributed to determine the link between the painful state and endogenous steroid secretion in the SC in vivo. Finally, the physiological relevance of the modification of endogenous steroid formation in the SC during painful situation was discussed.

Regulation of neurosteroid allopregnanolone biosynthesis in the rat spinal cord by glycine and the alkaloidal analogs strychnine and gelsemine.

The neurosteroid allopregnanolone (3alpha,5alpha-THP) is well characterized as a potentially therapeutic molecule which exerts important neurobiological actions including neuroprotective, antidepressant, anxiolytic, anesthetic and analgesic effects. We have recently observed that neurons and glial cells of the rat spinal cord (SC) contain various key steroidogenic enzymes such as 5alpha-reductase and 3alpha-hydroxysteroid oxido-reductase which are crucial for 3alpha,5alpha-THP biosynthesis. Furthermore, we demonstrated that the rat SC actively produces 3alpha,5alpha-THP. As the key factors regulating neurosteroid production by nerve cells are unknown and because glycine is one of the pivotal inhibitory neurotransmitters in the SC, we investigated glycine effects on 3alpha,5alpha-THP biosynthesis in the rat SC. Glycine markedly stimulated [(3)H]-progesterone conversion into [(3)H]3alpha,5alpha-THP by SC slices. The alkaloid strychnine, well-known as a glycine receptor (Gly-R) antagonist, blocked glycine stimulatory effect on 3alpha,5alpha-THP formation. Gelsemine, another alkaloid containing the same functional groups as strychnine, increased 3alpha,5alpha-THP synthesis. The stimulatory effects of glycine and gelsemine on 3alpha,5alpha-THP production were additive when the two drugs were combined. These results demonstrate that glycine and gelsemine, acting via Gly-R, upregulate 3alpha,5alpha-THP biosynthesis in the SC. The data also revealed a structure-activity relationship of the analogs strychnine and gelsemine on neurosteroidogenesis. Possibilities are opened for glycinergic agents and gelsemine utilization to stimulate selectively 3alpha,5alpha-THP biosynthetic pathways in diseases evoked by a decreased neurosteroidogenic activity of nerve cells.

Selective regulation of neurosteroid biosynthesis in human neuroblastoma cells under hydrogen peroxide-induced oxidative stress condition.

Neurosteroid biosynthesis is demonstrated in many species but key factors interacting with neurosteroidogenesis under pathophysiological conditions are unknown. Hydrogen peroxide (H(2)O(2))-induced oxidative stress is an etiological factor involved in several disorders. We hypothesized that, if neurosteroidogenesis is a pivotal mechanism for nerve cell protection or viability, it might be selectively regulated under oxidative stress condition. To check our hypothesis, we investigated H(2)O(2) effects on neurosteroidogenesis in human neuroblastoma SH-SY5Y cells. Pulse-chase, high performance liquid chromatography and flow-scintillation analyses showed that, along neurosteroidogenic pathways converting pregnenolone into various neurosteroids, only estradiol synthesis selectively decreased in SH-SY5Y cells after H(2)O(2)-treatment. Testosterone conversion into estradiol was also inhibited by H(2)O(2). Real-time reverse transcription-polymerase chain reaction revealed aromatase gene repression in SH-SY5Y cells 12 h after the oxidative stress onset. Consistently, viability assays showed that chronic inhibition of aromatase activity by letrozole killed neuroblastoma cells. A 12-h pretreatment of SH-SY5Y cells with estradiol was protective against H(2)O(2)-induced death. In addition, estradiol was also capable of rescuing markedly neuroblastoma cells from letrozole-evoked death. Altogether, these results suggest that endogenous estradiol formation is pivotal for SH-SY5Y cell viability. Serum deprivation-evoked stress, which also killed SH-SY5Y cells, unaffected neurosteroidogenesis, indicating that inhibitory effect on neuroprotective-neurosteroid estradiol biosynthesis is specific for H(2)O(2)-induced stress. Selective targeting of neurosteroidogenic pathways may therefore constitute an interesting strategy against H(2)O(2)-evoked neurodegenerative processes.

Neuroactive steroids: state of the art and new perspectives.

Neuroactive steroids include synthetic steroidal compounds and endogenous steroids, produced by endocrine glands (hormonal steroids) or the nervous tissue (neurosteroids), which regulate neural functions. These steroids bind to nuclear receptors or act through the activation of membrane-associated signaling pathways to modulate various important processes including the development of the nervous system, neural plasticity and the adaptive responses of neurons and glial cells under pathological conditions. Reviewed and updated in the present paper are the pleiotropic and protective abilities of neuroactive steroids. The fundamental evidence and knowledge gained constitute a profound background that offers interesting possibilities for developing effective strategies against several disorders of the nervous system.

Seasonal variation of the impact of a stressful procedure on open field behaviour and blood corticosterone in laboratory mice.

Behavioural and hormonal seasonal changes are well documented in various vertebrate species living in their natural environment but circannual variations that may occur in laboratory animals reared in standard conditions are poorly investigated. This study shows that, in laboratory mice, the effects of stress on behavioural inhibition, investigatory behaviour and blood concentration of corticosterone are seasonally dependent. No consistency was observed between the reactivity of biological structures controlling the hormonal response to stress and the behavioural activities investigated at every period of the year. During the spring time, stress, which elicited a decrease of investigatory behaviour (estimated by the walking time in an open field), increased behavioural inhibition (estimated by the percentage of walking in the central area of the open field) as well as the blood corticosterone concentration in laboratory mice. In autumn, stress had no significant effect on behaviour despite the great hormonal concentration increase. The results reveal that, at certain period of the year, a stressful procedure is unable to affect behavioural parameters in laboratory mice which were maintained in constant 12-h dark/12-h light cycle. The report constitutes a novel piece of information suggesting a potential role of the endogenous biological clock in the modulation of stress response in mammals.

Effect of streptozotocin-induced diabetes on the gene expression and biological activity of 3beta-hydroxysteroid dehydrogenase in the rat spinal cord.

Abnormal secretion of steroids by the adrenals and gonads is one of the disturbances occurring in diabetics but the impact of diabetes on steroid formation in the nervous system has never been studied. However, it is well known that numerous actions of peripheral steroids on the nervous system require their conversion into neuroactive metabolites within the neural tissue. As this in situ steroid synthesis/metabolism is crucial for the control of several neurobiological functions, we investigated the effects of streptozotocin-induced diabetes on the gene expression and activity of 3beta-hydroxysteroid dehydrogenase in the spinal cord, a pivotal structure involved in sensorimotor and neurovegetative mechanisms. 3beta-Hydroxysteroid dehydrogenase is a key enzyme which participates to the biosynthesis of all classes of steroids by converting delta5-3beta-hydroxysteroids such as pregnenolone and dehydroepiandrosterone into delta4-3-ketosteroids as progesterone and androstenedione, respectively. Reverse transcription coupled with quantitative real-time polymerase chain reaction revealed that 3beta-hydroxysteroid dehydrogenase gene was over-expressed in the spinal cord of streptozotocin-treated rats compared with controls. Pulse-chase experiments combined with high performance liquid chromatography and continuous flow detection of newly-synthesized steroids showed an increase of 3beta-hydroxysteroid dehydrogenase activity responsible for a hyper-production of progesterone in the spinal cord of diabetic rats. This up-regulation of progesterone biosynthesis was concomitant with a decrease of its transformation into tetrahydroprogesterone, a process which facilitated progesterone accumulation in the spinal cord of streptozotocin-treated rats. Since progesterone is a potent neuroprotective steroid, increase of its production appeared as an endogenous molecular and biochemical mechanism triggered by spinal nerve cells to cope with degenerative effects of streptozotocin-induced diabetes. Our results constitute the first direct evidence showing an impact of diabetes on steroid biosynthetic and metabolic pathways in the nervous system. The data open new perspectives for the modulation of deleterious effects of diabetes by neuroprotective steroids.

Impact of neuropathic pain on the gene expression and activity of cytochrome P450side-chain-cleavage in sensory neural networks.

Development of efficient therapy against chronic and stubborn pains requires fundamental identification of adequate cellular and molecular targets. This study combined cellular, molecular and biochemical approaches to investigate the gene expression and enzymatic activity of cytochrome P450side-chain-cleavage (P450scc) in spinal neural networks under normal and neuropathic pain states. P450scc is the key onset enzyme for steroidogenesis in endocrine glands and for neurosteroid biosynthesis in nerve cells. The P450scc gene was over-expressed in spinal and supra-spinal networks during neuropathic pain provoked by sciatic nerve ligature. Plasticity was observed in P450scc cellular distribution in pain circuits and its activity also increased inducing in vivo, hyper-secretion of pregnenolone and allopregnanolone which strongly stimulates type A receptors for g-aminobutyric acid, a pivotal neurotransmitter involved in pain modulation. These results, by establishing a direct link between neuropathic pain and neuroactive steroid formation in the nervous system, open new perspectives for chronic-pain modulation by endogenous neurosteroids.

Anatomical and biochemical evidence for the synthesis of unconjugated and sulfated neurosteroids in amphibians.

Various studies have shown that, in mammals, neurons and glial cells are capable of synthesizing bioactive steroids, or neurosteroids, which regulate the activity of the central nervous system (CNS). However, although steroid hormones are involved in the regulation of behavioral and neuroendocrine processes in amphibians, neurosteroid biosynthesis has never been studied in the CNS of non-mammalian vertebrates. Reviewed here are several data sets concerning the production of unconjugated and sulfated neurosteroids in amphibians. These data were obtained by investigating the immunohistochemical localization and activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and hydroxysteroid sulfotransferase (HST), in the frog brain. Numerous 3beta-HSD-immunoreactive neurons were detected in the anterior preoptic area, nucleus of the periventricular organ, posterior tuberculum, ventral and dorsal hypothalamic nuclei. 17beta-HSD-like immunoreactivity was found in ependymal gliocytes bordering the lateral ventricles of the telencephalon. Two populations of HST-immunoreactive neurons were localized in the anterior preoptic area and the dorsal magnocellular nucleus of the hypothalamus. High amounts of progesterone (PROG), 17-hydroxyprogesterone (17OH-PROG), testosterone (T) and dehydroepiandrosterone sulfate (DHEAS) were measured in the frog brain by combining HPLC analysis of tissue extracts with radioimmunoassay detection. Incubation of telencephalic or hypothalamic explants with tritiated pregnenolone ([3H]PREG) yielded the synthesis of various metabolites including PROG, 17OH-PROG, DHEA and T. Incorporation of [35S]3'-phosphoadenosine 5'-phosphosulfate ([35S]PAPS) and [3H]PREG or [3H]DHEA into frog brain homogenates led to the formation of [3H,35S]pregnenolone sulfate ([3H,35S]PREGS) or [3H,35S]DHEAS, respectively. Altogether, these results demonstrate that the process of neurosteroid biosynthesis occurs in amphibians as previously seen in mammals.

Immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase in the brain of the African lungfish Protopterus annectens.

The localization of the enzymes responsible for the biosynthesis of neurosteroids in the brain of dipnoans has not yet been determined. In the present study, we investigated the immunohistochemical distribution of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 5 alpha-reductase (5 alpha-R) in the brain and pituitary of the African lungfish Protopterus annectens by using antibodies raised against type I human 3 beta-HSD and type I human 5 alpha-R. The 3 beta-HSD and 5 alpha-R immunoreactivities were detected in cell bodies and fibers located in the same areas of the lungfish brain, namely, in the pallium, thalamus, hypothalamus, tectum, and periaqueductal gray. Identification of astrocytes, oligodendrocytes, and neurons with antisera against glial fibrillary acidic protein, galactocerebroside and neurofilaments revealed that, in the lungfish brain, 3 beta-HSD immunolabeling is expressed exclusively by neurons, whereas the 5 alpha-R-immunoreactive material is contained in both neurons and glial cells. In the pituitary gland, 3 beta-HSD- and 5 alpha-R-like immunoreactivity was localized in both the pars distalis and the pars intermedia. The present study provides the first immunocytochemical mapping of two key steroidogenic enzymes in the brain and pituitary of a lungfish. These data strongly suggest that neurosteroid biosynthesis occurs in the brain of fishes, as previously shown for amphibians, birds, and mammals.

In vivo regulation of vasomotricity by nitric oxide and prostanoids during gestation.

Pharmacological studies using the Doppler technique revealed that pregnancy decreases the systemic blood pressure and enhances uterine blood velocity in rats. The reactivity of the uterine artery to alpha-adrenoceptor and muscarinic receptor agonists was higher than that of systemic arteries. Sodium nitroprusside increased uterine arterial blood velocity slightly during gestation and markedly in non-pregnant rats. N(G)-L-Arginine methyl ester (L-NAME) decreased the uterine blood velocity mainly in gravid animals. The effect of diclofenac on uterine blood velocity was also more pronounced during pregnancy. The actions of sodium nitroprusside, L-NAME and diclofenac on systemic blood pressure were similar in pregnant and virgin rats. Altogether, these results indicate that pregnancy enhances nitric oxide (NO) and vasodilatory prostanoid production in the uterine vascular muscle which becomes less sensitive to exogenous NO. The uterine vasodilated status appears to be determined by conjugated actions of endothelial NO and vasodilator prostanoids of which the synthesis and the effects are weakly modified in systemic arteries during gestation.

The octadecaneuropeptide ODN stimulates neurosteroid biosynthesis through activation of central-type benzodiazepine receptors.

Neurosteroids may play a major role in the regulation of various neurophysiological and behavioural processes. However, while the biochemical pathways involved in the synthesis of neuroactive steroids in the central nervous system are now elucidated, the mechanisms controlling the activity of neurosteroid-producing cells remain almost completely unknown. In the present study, we have investigated the effect of the octadecaneuropeptide (ODN), an endogenous ligand of benzodiazepine receptors, in the control of steroid biosynthesis in the frog hypothalamus. Glial cells containing ODN-like immunoreactivity were found to send their thick processes in the close vicinity of neurones expressing the steroidogenic enzyme 3 beta-hydroxysteroid dehydrogenase. Exposure of frog hypothalamic explants to graded concentrations of ODN (10(-10)-10(-5) M) produced a dose-dependent increase in the conversion of tritiated pregnenolone into various radioactive steroids, including 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone and dihydrotestosterone. The ODN-induced stimulation of neurosteroid biosynthesis was mimicked by the central-type benzodiazepine receptor (CBR) inverse agonists methyl beta-carboline-3-carboxylate (beta-CCM) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). The stimulatory effects of ODN, beta-CCM and DMCM on steroid formation was markedly reduced by the CBR antagonist flumazenil. The ODN-evoked stimulation of neurosteroid production was also significantly attenuated by GABA. Collectively, these data indicate that the endozepine ODN, released by glial cell processes in the vicinity of 3 beta-hydroxysteroid dehydrogenase-containing neurones, stimulates the biosynthesis of neurosteroids through activation of central-type benzodiazepines receptors.

The triakontatetraneuropeptide (TTN) stimulates thymidine incorporation in rat astrocytes through peripheral-type benzodiazepine receptors.

Astrocytes and astrocytoma cells actively express the diazepam-binding inhibitor (DBI) gene, suggesting that DBI-processing products may regulate glial cell activity. In the present study, we have investigated the possible effect of one of the DBI-derived peptides, the triakontatetraneuropeptide (TTN), on [(3)H]thymidine incorporation in cultured rat astrocytes. Reversed-phase HPLC analysis of incubation media indicated that TTN is the major form of DBI-derived peptides released by cultured astrocytes. At very low concentrations (10(-14)-10(-11) M), TTN induced a dose-dependent increase in [(3)H]thymidine incorporation, whereas at higher concentrations (10(-10)-10(-5) M) the effect of TTN gradually declined. In the same range of concentrations, the specific peripheral-type benzodiazepine receptor (PBR) agonist Ro 5-4864 mimicked the bell-shaped stimulatory effect of TTN on [(3)H]thymidine incorporation. The PBR antagonist PK11195 (10(-6) M) suppressed the stimulatory action of both TTN and Ro 5-4864 on [(3)H]thymidine incorporation, whereas the central-type benzodiazepine receptor antagonist flumazenil (10(-6) M) had no effect. The present study demonstrates that the endozepine TTN stimulates DNA synthesis in rat glial cells through activation of PBRs. These data strongly suggest that TTN exerts an autocrine/paracrine stimulatory effect on glial cell proliferation.