Opposing effects of different steroid sulfates on GABAA receptor-mediated chloride uptake

Steroids with the 3alpha-hydroxy-5alpha- or 5beta-reduced configurations of the A ring interact with the gamma-aminobutyric acid (GABA) type A receptor chloride channel complex and potentiate the stimulation of Cl- uptake by GABA agonists. Conversely, the sulfate esters of 3beta-hydroxy-5-ene neurosteroids pregnenolone and dehydroepiandrosterone behave as inhibitory modulators. In the present work, steroid sulfates were tested for their ability to modulate muscimol-induced chloride ion uptake into cortical synaptoneurosomes. 3alpha-Hydroxy-5alpha-pregnan-20-one sulfate and several other 3alpha-hydroxy-steroid sulfates potentiated, whereas 3beta-hydroxy-steroid sulfates inhibited muscimol effect. It is concluded that GABA-agonistic or antagonistic properties of steroid sulfates depend on the alpha or beta orientation of the sulfate moiety linked to the A ring.     

Neurosteroids: a novel function of the brain

Neurosteroids are synthetized in the central and peripheral nervous system, particularly but not exclusively in myelinating glial cells, from cholesterol or steroidal precursors imported from peripheral sources. They include 3-hydroxy-delta 5-compounds, such as pregnenolone (PREG) and dehydroepiandrosterone (DHEA), their sulfates, and reduced metabolites such as the tetrahydroderivative of progesterone 3 alpha-hydroxy-5 alpha-pregnane-20-one (3 alpha, 5 alpha-TH PROG). These compounds can act as allosteric modulators of neurotransmitter receptors, such as GABAA, NMDA and sigma receptors. Progesterone (PROG) is also a neurosteroid, and a progesterone receptor (PROG-R) has been identified in peripheral and central glial cells. At different places in the brain, neurosteroid concentrations vary according to environmental and behavioral circumstances, such as stress, sex recognition and aggressiveness. A physiological function of neurosteroids in the central nervous system is strongly suggested by the role of hippocampal PREGS with respect to memory, observed in aging rats. In the peripheral nervous system, a role for PROG synthesized in Schwann cells has been demonstrated in the repair of myelin after cryolesion of the sciatic nerve in vivo and in cultures of dorsal root ganglia neurites. It may be important to study the effect of abnormal neurosteroid concentrations/metabolism with a view to the possible treatment of functional and trophic disturbances of the nervous system.     

Facilitation of acetylcholine release in rat frontal cortex by indeloxazine hydrochloride: involvement of endogenous serotonin and 5-HT4 receptors

Effects of indeloxazine hydrochloride, an inhibitor of serotonin (5-HT) and norepinephrine (NE) reuptake with a facilitatory effect on 5-HT release, on acetylcholine (ACh) output in frontal cortex of conscious rats were characterized using an in vivo microdialysis technique. Systemic administration of indeloxazine (3 and 10 mg/kg, i.p.) increased ACh and 5-HT output in a dose-dependent manner. Depletion of endogenous monoamines by reserpine and of 5-HT by p-chlorophenylalanine, but not that of catecholamines by alpha-methyl-p-tyrosine, significantly attenuated the facilitatory effect of indeloxazine on ACh release. When applied locally by reverse dialysis, indeloxazine (10 and 30 microM) and the selective 5-HT reuptake inhibitor citalopram (10 microM), but not the NE reuptake inhibitor maprotiline (30 microM), increased cortical ACh output. Indeloxazine (10 mg/kg)-induced increase in ACh release was significantly inhibited by local application of the 5-HT4 receptor antagonists RS23597 (50 microM) and GR113803 (1 microM), while the 5-HT1A antagonist WAY-100135 (100 microM), 5-HT1A/1B/beta-adrenoceptor antagonist (-)propranolol (150 microM), 5-HT2A/2C antagonist ritanserin (10 microM) and 5-HT3 antagonist ondansetron (10 microM) failed to significantly modify this effect. Neither depletion of monoamines nor treatment with serotonergic antagonists significantly changed the basal ACh level, indicating that endogenous monoamines do not tonically activate ACh release. These results suggest that indeloxazine-induced facilitation of ACh release in rat frontal cortex is mediated by endogenous 5-HT and involves at least in part cortical 5-HT4 receptors.     

Development and regeneration of the nervous system: a role for neurosteroids

Several steroids, termed 'neurosteroids', are synthesized from cholesterol within both the central and peripheral nervous systems. These include pregnenolone and its sulfate ester, progesterone and its 5 alpha-reduced metabolites. Dehydroepiandrosterone, mainly in its sulfated form, also remains present in the brain long after removal of the steroidogenic endocrine glands. Its biosynthesis in brain remains an open possibility, but the pathways involved are unknown. Little information is available concerning the role of neurosteroids during the maturation of the nervous system, although they are already synthesized by glial cells and by some populations of neurons during embryonic life. Cell culture experiments suggest that neurosteroids may increase the survival and differentiation of both neurons and glial cells. In the adult nervous system, neurosteroids modulate neurotransmission by acting directly on the neuronal membrane and also produce structural changes in neurons and in astrocytes. Studies of neurosteroid levels are currently conducted to examine their possible role during aging. We have recently reported that progesterone, synthesized by Schwann cells, promotes the formation of new myelin sheaths after lesion of the mouse sciatic nerve. Thus, neurosteroids may also play an important role during regeneration of the nervous system.     

Enhanced plasma DHEAS, brain acetylcholine and memory mediated by steroid sulfatase inhibition

Steroid sulfatase inhibitors can alter the metabolism of neurosteroids which modulate brain function. Administration of the non-steroidal steroid sulfatase inhibitor (p-O-sulfamoyl)-N-tetradecanoyl tyramine (DU-14) to rats for 15 days increased plasma dehydroepiandrosterone sulfate (DHEAS) concentrations by 88.2%, decreased plasma dehydroepiandrosterone (DHEA) concentrations by 84.6%, increased hippocampal acetylcholine (ACh) release determined via in vivo microdialysis by almost 3-fold, and produced a significant blockade of scopolamine-induced amnesia as measured by a passive avoidance test. These results suggest DHEAS rather than DHEA enhances brain cholinergic function and that steroid sulfatase inhibition may become an important tool for enhancing neuronal functions, such as memory, mediated by excitatory neurosteroids.     

Differential interaction between 5-HT3 receptors and GABAergic neurons inhibiting acetylcholine release in rat entorhinal cortex slices

The 5-HT3 receptor antagonists, ondansetron, MDL 72222 and granisetron (0.01-1 microM), produced a concentration-dependent increase of K+-evoked [3H]ACh efflux in slices from rat entorhinal cortex preloaded with [3H]choline. Bicuculline and flumazenil, antagonists at different sites of the GABAA receptor, also enhanced [3H]ACh efflux. While the ACh releasing effect of ondansetron was markedly potentiated, in a TTX-sensitive manner, by bicuculline, the effects of MDL 72222 and granisetron were not significantly modified. A qualitatively identical interaction was found by using flumazenil, a GABAA antagonist at the benzodiazepine recognition site, in combination with the 5-HT3 receptor antagonists. The potentiation by the GABAA antagonists of [3H]ACh efflux was also observed in a superfusion medium deficient in Cl-. The nonspecific K+-channel blockers TEA and Ba2+ also increased K+-evoked [3H]ACh efflux in this preparation but the releasing effect was not modified by bicuculline. The results support the functional interaction of ondansetron with GABAergic interneurons in the rat entorhinal cortex, GABA-independent mechanisms may however be involved in the regulation of cortical cholinergic function by other 5-HT3 receptor antagonists.     

Effects of ascorbic acid on in vitro steroidogenesis in guinea pigs

Guinea pig ovarian whole tissue homogenates were incubated with [14C]-labelled cholesterol, pregnenolone, and progesterone. Testicular homogenates were incubated with [14C]-progesterone. All incubations were carried out in the presence of 0, 0.5, 1.0, or 2.0 mM ascorbic acid. The conversion of cholesterol to pregnenolone was significantly decreased in testosterone and progesterone production. The addition of 0.5 mM ascorbic acid increased the conversion of pregnenolone to delta 4 steroids and decreased its conversion to delta 5 steroids, relative to the other ascorbic acid treatments. The conversion of progesterone to 17 A-hydroxyprogesterone was significantly decreased in the presence of 1.5 mM ascorbic acid over the O mM treatment. The data supports a general inhibitory effect of high ascorbic acid on the steroid hydroxylations, and a possible regulatory role of ascorbic acid on the conversion of pregnenolone to delta 4 and delta 5 steroids.     

Kinetic analysis of successive reactions catalyzed by bovine cytochrome p450(17alpha,lyase)

Bovine P450(17alpha,lyase) containing an additional four histidine residues at the COOH terminus was expressed in Escherichia coli and purified by one-step column chromatography using Ni-chelate resin. The membrane enzyme was incorporated into liposome membranes having similar lipid composition to that of the endoplasmic reticulum. In the presence of excess substrate, the P450-proteoliposomes metabolize pregnenolone (Delta5-steroid) to 17alpha-hydroxypregnenolone and further to dehydroepiandrosterone. The enzyme catalyzed 17alpha-hydroxylation of progesterone (Delta4-steroid) but did not form androstenedione from progesterone, although the proteoliposomes could catalyze the conversion of 17alpha-hydroxyprogesterone to androstenedione. The kinetic analysis of rapid quenching experiments showed that about 20% of the pregnenolone consumed was converted successively to dehydroepiandrosterone via a fraction of 17alpha-hydroxypregnenolone that did not dissociate from the enzyme. The rapid quenching experiments for progesterone metabolism by the proteoliposomes revealed that the dissociation rate of 17alpha-hydroxyprogesterone was 10 times faster than that of 17alpha-hydroxypregnenolone. The release of the intermediate metabolite of Delta4-steroid is sufficiently faster than the lyase reaction to prevent further reaction by the P450. It is concluded that the dissociation rates of the first hydroxylation metabolites regulate the successive reactions of P450(17alpha,lyase).     

Dehydroepiandrosterone 7alpha- and 7beta-hydroxylation in mouse brain microsomes. Effects of cytochrome P450 inhibitors and structure-specific inhibition by steroid hormones

Presently, several works question the effects of dehydroepiandrosterone (DHEA) reported in vivo and designate its 7-hydroxylated metabolites as native antiglucocorticoids and potent mediators in the triggering of immune response. Among mouse tissues and organs, and second to liver, the largest production of 7alpha-and 7beta-hydroxylated derivatives of DHEA takes place in brain microsomes. To contribute to identification of cytochromes P450 (CYPs) responsible for 7alpha- and 7beta-hydroxy-DHEA production, effects of CYP inhibitors and of several steroid hormones on DHEA 7-hydroxylation were examined. Using mouse brain microsomes as a source of enzyme, we report now that strong and smaller inhibitions of DHEA 7alpha-hydroxylation were obtained with ketoconazole and alpha-naphthoflavone, respectively, and that neither changed DHEA 7beta-hydroxylation. Metyrapone and antipyrine also inhibited 7alpha-hydroxylation, but by contrast, significantly increased 7beta-hydroxylation of DHEA. This indicated that at least, two different CYPs were responsible for 7alpha- and 7beta-hydroxylation of DHEA. Steroids sharing a 3beta-hydroxylated structure with DHEA, namely pregnenolone, 5-androstene-3beta,17beta-diol and 3beta-hydroxy-5alpha-androstan-17-one, were strong inhibitors of DHEA 7alpha-hydroxylation (non-competitive inhibition with pregnenolone, Ki=2.0 +/- 0.3 microM). In contrast, 7beta-hydroxylation yields were not decreased by the 3beta-hydroxysteroids tested. Moderate inhibition of 7alpha- and 7beta-hydroxylation was obtained with 3-oxosteroids, namely testosterone, progesterone, corticosterone and 4-androsten-3,17-dione. Taken together, these data indicate specific inhibition patterns of DHEA 7alpha- and 7beta-hydroxylation by CYP inhibitors and steroid hormones in mouse brain microsomes and may be used as criteria necessary for identification of the responsible CYP species.     

Decreased levels of a soluble form of the human adhesion receptor CD58 (LFA-3) in sera and synovial fluids of patients with rheumatoid arthritis

The mode of interaction between muramyl dipeptide (MDP), a compound with immunopharmacological activities, and 5-hydroxtryptamine (5-HT, serotonin) was studied in isolated nerve-smooth muscle preparations of the carp stomach. Application of exogenous 5-HT evoked direct smooth muscle contractions; electric neurogenic stimulation evoked twitches due to release of 5-HT from nerve endings. Contractions evoked by a high concentration of 5-HT (3-30 microM) were resistant to atropine and potentiated in the presence of MDP. Isamoltan (5-HTID antagonist) decreased the amplitude of contractions, whereas ketanserin (5-HT2 antagonist) and MDL 72,222 (5-HT3 antagonist) had no effect. The addition of low concentrations (0.1-1.5 microM) of 5-HT did not contract the preparation but caused a decrease in the amplitude of neurogenic twitches, which might be due to the presynaptic inhibition of serotonin release. This effect of 5-HT was not changed by isamoltan or ketanserin, but it was largely reduced in the presence of 5-HT3 antagonists tropisetron and MDL 72,222. This inhibitory effect of 5-HT on twitch amplitude was potentiated by MDP. The interaction of MDP with the serotonergic system thus involved not only potentiation of the postsynaptic effect of higher 5-HT concentrations, which might have been mediated via the 5-HT1 subsystem, but also presynaptic inhibition. MDP enhancement of 5-HT's inhibitory effect, mediated via 5-HT3 receptors, might represent a new feature in mutual 5-HT-MDP interactions.     

Species differences in brain adenosine A1 receptor pharmacology revealed by use of xanthine and pyrazolopyridine based antagonists

1. The pharmacological profile of adenosine A1 receptors in human, guinea-pig, rat and mouse brain membranes was characterized in a radioligand binding assay by use of the receptor selective antagonist, [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX). 2. The affinity of [3H]-DPCPX binding sites in rat cortical and hippocampal membranes was similar. Binding site affinity was higher in rat cortical membranes than in membranes prepared from guinea-pig cortex and hippocampus, mouse cortex and human cortex. pKD values (M) were 9.55, 9.44, 8.85, 8.94, 8.67, 9.39 and 8.67, respectively. The binding site density (Bmax) was lower in rat cortical membranes than in guinea-pig or human cortical membranes. 3. The rank order of potency of seven adenosine receptor agonists was identical in each species. With the exception of 5'-N-ethylcarboxamidoadenosine (NECA), agonist affinity was 3.5-26.2 fold higher in rat cortical membranes than in human and guinea-pig brain membranes; affinity in rat and mouse brain membranes was similar. While NECA exhibited 9.3 fold higher affinity in rat compared to human cortical membranes, affinity in other species was comparable. The stable GTP analogue, Gpp(NH)p (100 microM) reduced 2-chloro-N6-cyclopentyladenosine (CCPA) affinity 7-13.9 fold, whereas the affinity of DPCPX was unaffected. 4. The affinity of six xanthine-based adenosine receptor antagonists was 2.2-15.9 fold higher in rat cortical membranes compared with human or guinea-pig membranes. The rank order of potency was species-independent. In contrast, three pyrazolopyridine derivatives, (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-2-piperidine ethanol (FK453), (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-piperidin-2-yl acetic acid (FK352) and 6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-pyridazinebutyric acid (FK838) exhibited similar affinity in human, guinea-pig, rat and mouse brain membranes. pKi values (M) for [3H]-DPCPX binding sites in human cortical membranes were 9.31, 7.52 and 7.92, respectively. 5. Drug affinity for adenosine A2A receptors was determined in a [3H]-2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido ade nosine ([3H]-CGS 21680) binding assay in rat striatal membranes. The pyrazolopyridine derivatives, FK453, FK838 and FK352 exhibited pKi values (M) of 5.90, 5.92 and 4.31, respectively, compared with pKi values of 9.31, 8.18 and 7.57 determined in the [3H]-DPCPX binding assay in rat cortical membranes. These novel pyrazolopyridine derivatives therefore represent high affinity, adenosine A1 receptor selective drugs that, in contrast to xanthine based antagonists, exhibit similar affinity for [3H]-DPCPX binding sites in human, rat, mouse and guinea-pig brain membranes.     

13C-NMR spectroscopy of keratan sulphates--assignments for five sialylated pentasaccharides derived from the non-reducing chain termini of bovine articular cartilage keratan sulphate by keratanase II digestion

The effects of dexfenfluramine on the release of brain dopamine and serotonin into striatal dialysates were measured in freely moving rats. Samples collected every 20 min were assayed for dopamine and serotonin by high-performance liquid chromatography in a single run. The administration of a lower anorectic dose of dexfenfluramine (0.5 or 1.0 mg/kg intraperitoneally) significantly increased dialysate serotonin concentrations without affecting those of dopamine. A higher dexfenfluramine dose (2.5 mg/kg intraperitoneally) increased both serotonin and dopamine. The increase in dopamine could be blocked by administering the mixed-acting serotonin antagonist methiothepin (20 microM), and was reproduced by applying serotonin (3-10 microM) directly to striatal neurons. Tetrodotoxin (1 microM) added to the striatal perfusates decreased the basal release of dopamine and serotonin; it also blocked the effect of dexfenfluramine (2.5 mg/kg intraperitoneally) on dopamine release and decreased the increment in serotonin release (by approximately 70%). Amphetamine (1 mg/kg subcutaneously) or phentermine (2 mg/kg intraperitoneally) increased dialysate dopamine concentrations without affecting those of serotonin, and tetrodotoxin (1 microM) failed to block the response to amphetamine. These findings suggest that (1) lower anorectic doses of dexfenfluramine release serotonin but not dopamine, and (2) higher doses of dexfenfluramine also increase dopamine release by an indirect mechanism mediated via serotonin.     

Neuroprotective effects of NMDA receptor glycine recognition site antagonism: dependence on glycine concentration

High-affinity NMDA receptor glycine recognition site antagonists protect brain tissue from ischemic damage. The neuroprotective effect of 5-nitro-6,7-dichloro-2,3-quinoxalinedione (ACEA 1021), a selective NMDA receptor antagonist with nanomolar affinity for the glycine binding site, was examined in rat cortical mixed neuronal/glial cultures. ACEA 1021 alone did not alter spontaneous lactate dehydrogenase (LDH) release. Treatment with ACEA 1021 (0.1-10 microM) before 500 microM glutamate, 30 microM NMDA, or 300 microM kainate exposure was found to reduce LDH release in a concentration-dependent fashion. These effects were altered by adding glycine to the medium. Glycine (1 mM) partially reversed the effect of ACEA 1021 on kainate cytotoxicity. Glycine (100 microM-1 mM) completely blocked the effects of ACEA 1021 on glutamate and NMDA cytotoxicity. The glycine concentration that produced a half-maximal potentiation of excitotoxin-induced LDH release in the presence of 1.0 microM ACEA 1021 was similar for glutamate and NMDA (18 +/- 3 and 29 +/- 9 microM, respectively). ACEA 1021 also reduced kainate toxicity in cultures treated with MK-801. The effects of glycine and ACEA 1021 on glutamate-induced LDH release were consistent with a model of simple competitive interaction for the strychnine-insensitive NMDA receptor glycine recognition site, although nonspecific effects at the kainate receptor may be of lesser importance.     

Serotonin inhibits the induction of long-term potentiation in rat primary visual cortex

1. The effect of serotonin (5-hydroxytryptamine; 5-HT) on the induction of long-term potentiation (LTP) in rat visual cortex was investigated by using slice preparations in vitro. 2. Bath application of 5-HT (0.1-10 microM) did not affect the baseline synaptic potentials evoked by single-pulse test stimulation, but inhibited the induction of LTP in a concentration-dependent manner. 3. The effect of 5-HT was blocked by the 5-HT1 receptor antagonist pindolol or the 5-HT2,7 receptor antagonist ritanserin, but not by the 5-HT3,4 receptor antagonist MDL72222. 4. These results suggest that 5-HT plays a role in suppressing the induction of LTP in the rat visual cortex.     

Variation in genome organization of the plant pathogenic fungus Colletotrichum lindemuthianum

The effects of 5-HT3 receptor antagonists on ethanol intake were examined in the selectively bred alcohol-preferring P line of rats under continuous and limited access to 10% (v/v) ethanol with food and water ad lib. Single daily injections of either MDL 72222 (MDL) or ICS 205-930 (ICS) (0.01-3.0 mg/kg, SC) given 60 min before a 4-h scheduled access period for 4 consecutive days failed at all doses to alter the intake of a 10% (v/v) ethanol solution by P rats. However, multiple daily injections of either MDL (1-3 mg/kg, SC) or ICS (3.0 and 5.0 mg/kg, SC), given three times daily at 4-h intervals, significantly reduced ethanol intake under 24-h free-choice conditions on the first treatment day. Additionally, a single administration of 1.0 mg/kg MDL reduced 24-h free-choice ethanol intake by approximately 50% of control values and had no effect on 24-h saccharin intake. The effects of MDL were further examined in a 2-h schedule access paradigm in which rats received the access period at the same time every day (Fixed) or randomly during the dark cycle (Variable). Although 1.0 mg/kg MDL had little effect on ethanol drinking in the Fixed group, ethanol intake was reduced by 55% of control levels in the Variable group. Overall, the data indicate that drinking conditions influence the effectiveness of 5-HT3 antagonists to reduce ethanol consumption. Furthermore, the results suggest that conditions, associated with limited access ethanol drinking, markedly reduce the actions of 5-HT3 antagonists on ethanol intake.     

Analysis and degradation study of glyphosate and of aminomethylphosphonic acid in natural waters by means of polymeric and ion-exchange solid-phase extraction columns followed by ion chromatography-post-column derivatization with fluorescence detection

Excessive stimulation of the N-methyl-d-aspartate (NMDA)-type glutamate receptor has been implicated in the neuronal death resulting from focal hypoxia-ischemia. Certain neurosteroids, steroids synthesized de novo in the central nervous system (CNS), have been shown to modulate the action of neurotransmitters at their cellular receptors. Pregnenolone sulfate (PS) is an abundant neurosteroid that enhances the current evoked by NMDA. Using the Ca2+-sensitive fluorescent dye, Fluo-3, AM, and a trypan blue exclusion assay, we evaluated the ability of PS to modulate NMDA-induced changes in intracellular free calcium concentration ([Ca2+]i) and neuronal death in primary cultures of rat hippocampal neurons. The results demonstrate that PS potentiates NMDA-induced increases in [Ca2+]i by 150%. Further, PS exacerbates the MK-801-sensitive neuronal death produced by acute (PS EC50=37 microM) or chronic NMDA exposure, reducing the EC50 of NMDA from 13 to 4 microM under chronic exposure conditions, whereas pregnenolone is ineffective. Our results show that PS, or related sulfated neurosteroids, may play a role in the onset of excitotoxic neuronal death in vivo.     

Modulation of brainstem 5-HT1C receptors by serotonergic drugs in the rat

1. The sparse population of brainstem 5-hydroxytryptamine1C (5-HT1C) (also called 5-HT2C) receptors has received little attention despite its possible role in the serotonin syndrome and 5-HT-mediated shaking behavior. We characterized [3H]mesulergine binding in rat brainstem and, to determine if brainstem 5-HT1C sites respond to serotonergic manipulations, performed saturation studies of [3H]mesulergine binding in brainstem from rats treated chronically with 11 different 5-HT1C/2 agonists and antagonists. 2. In competition studies in vitro, the rank order of drug potency was most compatible with a 5-HT1C receptor binding site: mianserin, 5-HT, cinanserin, 1-(3-chlorophenyl)piperazine (m-CPP), 1-(2-5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), MDL 100,907, RU 24969, 5-carboxamidotryptamine (5-CT), 8-OH-DPAT, MDL 72,222. 3. Chronic treatment with the agonists quipazine and trifluoromethylphenylpiperazine (TFMPP) and the antagonists ritanserin and methiothepin significantly down-regulated brainstem 5-HT1C sites, which were 65% of [3H]mesulergine-labeled sites in brainstem. Only metergoline and ritanserin significantly increased pKD. 4. Chronic treatment in vivo with DOI, m-CPP, mianserin, methysergide, spiperone, cyproheptadine, and metergoline had no significant effect on BMAX at the dose studied. 5. These data suggest similarities in the regulation of 5-HT1C and 5-HT2 sites at which both 5-HT1C 2 agonists and antagonists also induce receptor down-regulation. 6. 5-HT1C/2 agonists and antagonists that did not down-regulate brainstem 5-HT1C sites may be more active in vivo at 5-HT2 sites, at 5-HT1C sites in other brain regions, have effects on 5-HT1C receptors not detectable at the recognition site, or differ for pharmacokinetic reasons.     

Induction of neurosteroid synthesis by NMDA receptors in isolated rat retina: a potential early event in excitotoxicity

Here we investigated the possible regulation of neurosteroidogenesis by N-methyl-D-aspartic acid (NMDA) receptor activation and addressed the hypothesis that neurosteroid synthesis may be involved in acute excitotoxicity. In the isolated retina, exposure to NMDA modified pregnenolone and pregnenolone sulphate formation. This effect was dose and time dependent, the synthesis being increased by relatively moderate NMDA doses (1-100 microM) within 30 min exposure and reduced to its control value by 60 min or by raising drug concentrations. NMDA-stimulated neurosteroid synthesis was blocked by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine hydrogen maleate (MK-801) and 3(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP), depended on extracellular calcium and reproduced by glutamate. Lactate dehydrogenase (LDH) release and morphological analysis revealed that retinal cell viability was not significantly affected after 30 min exposure to 50 microM NMDA, but severe cell damage occurred by 60 min. When the GABAA (gamma-aminobutyric acid) receptor agonist muscimol (1-1000 microM), known to activate retinal neurosteroidogenesis, was added together with NMDA, no additional increase in neurosteroid synthesis was observed, and NMDA-induced LDH release remained unchanged. However, exposure to a high concentration of muscimol alone (500 microM) provoked a similar degree of toxicity to NMDA. By contrast, bicuculline abolished the increase in neurosteroidogenesis and LDH release. Similarly, pretreatment with R (+)-p-aminoglutethimide (AMG), an inhibitor of cholesterol side-chain cleavage cytochrome P450, attenuated acute retinal cell damage. The inhibitory nature of AMG on NMDA-stimulated neurosteroidogenesis was confirmed in the observation that drug treatment reduced pregnenolone content and did not affect the bindings of [3H] MK-801 and [3H] muscimol. The results demonstrate that NMDA receptors regulate neurosteroidogenesis through a transneuronal mechanism, which implies GABAA receptor activation. The early NMDA-mediated stimulation of neurosteroid synthesis seems to play a critical role in acute excitotoxicity; consequently, its inhibition is likely to delay neuronal cell death.     

Neurosteroids modulate calcium currents in hippocampal CA1 neurons via a pertussis toxin-sensitive G-protein-coupled mechanism

The inhibition of Ca2+ channel currents by endogenous brain steroids was examined in freshly dissociated pyramidal neurons from the adult guinea pig hippocampal CA1 region. The steady-state inhibition of the peak Ca2+ channel current evoked by depolarizing steps from -80 to -10 mV occurred in a concentration-dependent manner with the following IC50 values: pregnenolone sulfate (PES), 11 nM; pregnenolone (PE), 130 nM; and allotetrahydrocorticosterone (THCC), 298 nM. THCC, PE, and PES depressed a fraction of the Ca2+ channel current with a maximal inhibition of 60% of the total current. However, substitution of an acetate group for the sulfate group on PES resulted in a complete loss of activity. Progesterone had no effect (4% inhibition at 100 microM). Intracellular dialysis of PES had no effect on the Ca2+ current; concomitant extracellular perfusion of PES showed normal inhibitory activity, suggesting that the steroid binding site can only be accessed extracellularly. Analysis of tail currents at -80 mV demonstrated that THCC and PES slowed the rate of Ca2+ current activation and deactivation with no change in the voltage dependence of activation. Inhibition of the Ca2+ channel current by THCC and PES was voltage dependent. THCC primarily inhibits the omega-conotoxin (CgTX)-sensitive or N-type Ca2+ channel current. PE was nonselective in inhibiting both the CgTX- and the nifedipine (NIF)-sensitive Ca2+ channel current. These neurosteroids had no effect on the CgTX/NIF-insensitive current. In neurons isolated from pertussis toxin (PTX)-treated animals by chronic intracerebroventricular infusion (1000 ng/24 hr for 48 hr), the Ca2+ channel current inhibition by PES, PE, and THCC was significantly diminished. Intracellular dialysis with GDP-beta-S (500 microM) also significantly diminished the neurosteroid inhibition of the Ca2+ channel current. Intracellular dialysis with the general kinase inhibitors H-7 (100 microM), staurosporine (400 nM), and a 20 amino acid protein kinase inhibitor (1 microM) also significantly prevented the THCC and PES inhibition of the Ca2+ channel current. Intracellular dialysis with the more specific inhibitors of protein kinase C (PKC), the pseudosubstrate inhibitor (PKCI 19-36) (1-2 microM) and bisindolylmaleimide (1 microM) significantly diminished the THCC and PE inhibition of the Ca2+ channel current. Rp- cAMP (100 microM), a specific inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the THCC and PE inhibition of the Ca2+ current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pregnenolone sulfate modulates NMDA receptors, inducing and potentiating acute excitotoxicity in isolated retina

Pregnenolone sulfate (PS) acts as a positive allosteric modulator of N-methyl-D-aspartate (NMDA) receptor-mediated responses. In the retina, we previously observed that the synthesis of pregnenolone and PS increases after stimulation of NMDA receptors and blockade of the synthesis reduces retinal cell death. This study was carried out to explore in the isolated and intact retina the possible role of PS in NMDA-induced excitotoxicity. Lactate dehydrogenase (LDH) measurements and morphological analysis revealed that a 90-min exogenous application of PS at 0.1-500 microM concentrations potentiated NMDA-induced cell death and at 50-500 microM concentrations caused cytotoxicity. After 45 min, either NMDA or PS caused no significant LDH release; but their co-application resulted in a high degree of toxicity. In addition, we found that a mild NMDA insult developed into serious damage when even low PS concentrations (0.1-10 microM) were used. Toxicity-inducing and -potentiating effects were specific to PS modulatory action on NMDA receptors, in that they were blocked by 4-(3-phosphonopropyl)2-piperazinecarboxylic acid (CPP) and MK-801 but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and neither dehydroepiandrosterone sulfate nor pregnenolone caused LDH release. Prevention of degenerative signs was seen in retinae pretreated with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a Cl- channel blocker, thus indicating a Na+/Cl--dependent acute mode of excitotoxic cell death responsible for PS toxicity. The positive interaction between the neurosteroid and NMDA receptors was further proved by a PS dose-dependent increase in NMDA-induced stimulation of [3H] MK-801 binding to retinal membranes. The results suggest a crucial role of PS in retinal vulnerability and propose the toxicity-potentiating effects as an important key in linking NMDA-induced endogenous synthesis to acute excitotoxicity.