Progesterone, 5α-pregnane-3,20-dione and 3α-hydroxy-5α-pregnane-20-one in specific regions of the human female brain in different endocrine states

Post-mortem concentrations of progesterone, 5α-pregnane-3,20-dione (5α-DHP) and 3α-hydroxy-5α-pregnane-20-one (allopregnanolone) were measured in 17 brain areas and serum in five fertile and five postmenopausal women. Steroid concentrations were measured with radioimmunoassay after extraction of brain tissue with ethanol and purification with celite chromatography. There were regional differences in brain concentrations of all three steroids. The highest progesterone levels were noted in the amygdala, cerebellum and hypothalamus and the highest levels of 5α-DHP and allopregnanolone were seen in the substantia nigra and basal hypothalamus. Brain concentrations of all three steroids were significantly higher in the fertile women in luteal phase compared to their postmenopausal controls (P<0.01). In general, the study showed that there is a variation in brain concentrations depending on ovarian steroid production, indicating that the secretion pattern during the menstrual cycle is reflected in the brain. However, regional differences in brain steroid levels imply local mechanisms for steroid uptake and binding as well. Investigations of gonadal steroid distributions in the human brain might be of importance considering the actions of these steroids in the central nervous system. Such studies could provide information about physiological mechanisms, such as the ovulation, and also form a baseline for comparative studies of normal and pathological conditions involving steroids, for instance, catamenial epilepsy and the premenstrual tension syndrome.     

Synthesis, metabolism and levels of the neuroactive steroid, 3α-hydroxy-4-pregnen-20-one (3αHP), in rat pituitaries

The neuroactive steroid, 3α-hydroxy-4-pregnen-20-one (3αHP), is a metabolite of progesterone and a precursor of 3α-hydroxy-5α-pregnan-20-one (5αP3α; allopregnanolone). In addition to analgesic and anxiolytic effects by interaction with the GABA_A receptor complex, 3αHP regulates pituitary FSH secretion by rapid non-genomic interaction with the Ca²⁺-driven cell signaling mechanisms. Since gonadectomy and adrenalectomy do not result in elimination of 3αHP, and since there is the possibility of paracrine and/or autocrine regulation of FSH release, the capacity of pituitary cells to regulate levels (by synthesis, metabolism, and storage) of 3αHP was examined. Anterior pituitaries from random cycling female rats were incubated, either as fragments or as cultured cells, for 1, 4 or 8 h with ³H- or ¹⁴C-labeled progesterone. The steroid metabolites were identified by thin-layer chromatography, autoradiography, high pressure liquid chromatography (HPLC), derivatization and GC/MS. Pituitary cells actively converted progesterone to 3αHP along with 5αP3α, 5α-pregnane-3,20-dione, 20α-hydroxy-5α-pregnan-3-one, 3β-hydroxy-5α-pregnan-20-one, 5α-pregnane-3α(β), 20α-diols, 20α-hydroxy-4-pregnen-3-one, and 4-pregnene-3α(β), 20α-diols. The results indicate the presence of the following steroidogenic enzymes in anterior pituitary cells: 3α-hydroxysteroid oxidoreductase (3α-HSO), 20α-HSO, 3β-HSO, and 5α-reductase. The activities of 5α-reductase and 3α-HSO were approximately equal and greatly exceeded those of the other enzymes. After 8 h of incubation with 100 ng progesterone per pituitary, about 20% of the progesterone was metabolized and 3.18 ng of 3αHP had been formed. The accumulation of 3αHP increased approximately linearly with the time of incubation. Metabolism studies using [1,2,6,7-³H]3αHP showed that pituitary cells convert about 29% and 8% of the 3αHP to progesterone and 5αP3α, respectively, in 2 h. Specific radioimmunoassays determined 11.6 and 7.5 ng of 3αHP per pituitary, respectively, in 25- and 40-day-old non-cycling female rats; these concentrations of 3αHP were about 2–3-fold greater than those of progesterone in the same pituitaries. In older (80–100 days old) cycling rats, the levels of 3αHP were about 9.4 and 18.6 ng/pituitary at 13.00 h and 22.00 h, respectively, on the day of proestrus, while the concomitant circulating levels were 13.7 and 5.4 ng/ml. The results indicate a marked capacity of rat pituitary cells to synthesize the neuroactive and FSH regulating steroid, 3αHP, from progesterone, and in turn to metabolize 3αHP to the neurosteroid, allopregnanolone, and to progesterone. The studies suggest cyclic biosynthetic and metabolic pathways for 3αHP and other steroids in the pituitary. They also indicate that the regulation of FSH secretion by 3αHP may be (in part, or in whole) via paracrine or autocrine mechanisms.     

Effects of diet and obesity on brain α1- and α2-noradrenergic receptors in the rat

The chronic feeding of a sweetened condensed milk/corn oil diet (CM diet) to adult male rats produced significant increases in body weight and levels of plasma insulin in 34% of the rats fed this diet with respect to chow-fed controls. Levels of alpha 1-noradrenergic receptor binding were lower (32%) in the hypothalamic ventromedial nucleus (VMN) of only those rats which became obese (DIO rats) with respect to both chow-fed controls and those rats which resisted the development of obesity on the CM diet (DR rats). Also, alpha 1-noradrenergic binding was inversely proportional to body weight gain in the VMN (r = -0.831). alpha 2-Noradrenergic receptors were 30-37% lower in both the DIO and DR rats in the dorsomedial nucleus and dorsal area of the hypothalamus, and the medial dorsal area and nucleus reuniens of the thalamus. The similar decreases in alpha 2-noradrenergic receptors in both the DIO and DR rats in these areas suggested that dietary factors alone were responsible for these changes. There were no significant differences from chow-fed rats for hypothalamic dopamine (D2) or beta-noradrenergic (beta 1- and beta 2-) receptors in either DR or DIO rats. These results indicate that VMN alpha 1-noradrenergic receptors co-vary with body weight and implicate a role for alpha 1-receptors in the VMN in the central neuronal regulation of body weight.     

Changes in brain α-adrenoceptors with increasing age in rabbits

The effects of aging on rabbit brain alpha-adrenoceptor number was studied using radioligand binding techniques. The ligands [3H]prazosin and [3H]clonidine were used to examine binding to alpha 1 and alpha 2-adrenoceptors, respectively, in forebrain and hindbrain membranes from rabbits aged from 1 month to 3 years. Changes in specific [3H]clonidine binding were compared to changes in central alpha 2-adrenoceptor function as measured by the fall in blood pressure after intracisternal administration of clonidine. Both alpha 1- and alpha 2-adrenoceptor number decreased with age in the rabbit brain. The maximum fall in specific [3H]prazosin binding occurred in animals aged between 3 and 6 months. [3H]clonidine binding in forebrain was also reduced in 6-month-old animals but [3H]clonidine binding in hindbrain was only reduced in animals over 2 years old. Despite the reduction in [3H]clonidine binding no decrease in the in vivo response to centrally administered clonidine was observed in 2-year-old animals when compared to 3-month-old rabbits.     

Reduced antiheparin effect of desialyzed α1-acid glycoprotein

α₁-Acid glycoprotein (α₁-acid GP) of human plasma counteracts the heparin-accelerated, antithrombin III-mediated inactivation of thrombin and factor Xa. In some preparations of α₁-acid GP which had a reduced antiheparin effect, less than normal amounts of sialic acid were found. No antiheparin activity remained after almost complete desialization of the purified glycoprotein with insolubilized neuraminidase. The significance of the sialyl residues for the antiheparin effect of α₁-acid GP is discussed.     

Idazoxan and brain α2-adrenoceptors in the rabbit

The effect of intravenous infusion of idazoxan on the depressor response to intracisternal clonidine 1 microgram/kg and on [3H]yohimbine binding in the fore- and hindbrain of the rabbit was examined. Idazoxan was infused either acutely (30 min) or chronically (5 days) at doses of 0.56 or 1.1 mg/h. Idazoxan 1.1 mg/h reduced the fall in blood pressure after clonidine. This attenuation of the depressor response was observed in the groups that were given the higher dose of idazoxan both acutely and chronically. The extent of attenuation was not modified by the duration of treatment. The low dose of idazoxan given acutely had no significant effect on the response to clonidine but the chronically infused group showed an enhanced response. A significant increase in the number of [3H]yohimbine binding sites (83%) was observed in the forebrain after 5 days infusion of 1.1 mg/h idazoxan with no change in the hindbrain. The lower dose of infusion did not cause any significant change in [3H]yohimbine binding in either brain region. Thus it appears that the susceptibility of the alpha 2-adrenoceptor binding sites to up-regulation by idazoxan may depend on the brain region observed.     

The subunits of α2-macroglobulin receptor/low density lipoprotein receptor-related protein, native and transformed α2-macroglobulin and interleukin 6 in Alzheimer's disease

To explore the role of α₂-macroglobulin receptor/low density lipoprotein receptor-related protein (α₂M-R/LRP) and its ligands in the pathogenesis of Alzheimer's disease (AD), antibodies were raised against its α- and β-subunits and their expression pattern in the CNS in AD and control cases was correlated with that of native and transformed α₂-macroglobulin (α₂M) and interleukin 6 (IL-6). The transmembranous β-subunit of α₂M-R/LRP and transformed α₂M were found in plaque cores in AD. Extramembranous α-subunit and native α₂M immunoreactivities were localized in activated plaque-associated astrocytes and extracellulary in plaques. IL-6 immunostaining was associated with neurofibrillary changes, and was also found extracellularly in the center of plaques and in microglial cells. Our finding that plaque cores contain a second transmembranous protein fragment, the β-subunit of α₂M-R/LRP, suggests ongoing membrane-protein degradation. By altering clearance and scavenger-like functions, fragmentation and breakdown of α₂M-R/LRP may have an important role in extracellular amyloid deposition and the formation of neurofibrillary tangles in AD.     

The AMPA antagonist NBQX protects thalamic reticular neurons from degeneration following cardiac arrest in rats

Thalamic reticular (RT) neurons are selectively vulnerable to degeneration following global ischemia. The degenerative mechanism is thought to involve an excitotoxic component, mediated in part by sustained post-ischemia activation of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) type excitatory amino acid (EAA) receptors. In order to test this hypothesis, the selective competitive AMPA type EAA antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxalinedione) was administered at 30 mg/kg to rats 1, 3, and 6 h after resuscitation from 10 min cardiac arrest. NBQX treatment resulted in a 2-fold increase of spared RT neurons, from a mean density of 3.6 ± 0.8 × 10³ neurons/mm³ in cardiac arrest cases to 7.4 ± 1.1 × 10³ neurons/mm³ in the NBQX treated group, which represents sparing of 41.7% of the normal population of RT neurons, and protection of 26.9% of vulnerable RT neurons. Neurons within the central core of the RT manifest both a higher degree of vulnerability to ischemic degeneration, >92% loss, and a higher sensitivity to sparing following NBQX administration, 460% increased sparing, than neuronal sub-populations in the medial or lateral13 of the RT. Protection by post-arrest administration of NBQX suggests that sustained post-arrest stimulation of AMPA receptors is an important component in the process of ischemic degeneration of RT neurons.     

Regulation of central α-adrenoceptors by serotoninergic denervation

α₁- and α₂-adrenoceptors were assessed by binding studies using [³H]prazosin and [³H]p-aminoclonidine as ligands in membrane preparations from the cortex, hippocampus and hypothalamus of rats, 3 weeks after intracerebroventricular injection of the neurotoxin 5,7-dihydroxytryptamine. Cortical α₁ and hippocampal α₂ adrenoceptors were significantly increased. Treatment also affected the affinity of cortical α₂ adrenoceptors. These results suggest a heterologous, region-specific regulation of both subtypes of central α-adrenergic receptors by serotonin.     

Interferon gamma up-regulates α2 macroglobulin expression in human astrocytoma cells

An established human astrocytoma cell line (T67) was shown to constitutively produce the proteinase inhibitor α₂macroglobulin (α₂M). Interferon gamma (IFNγ), a potent immunoregulatory lymphokine, was able to increase the synthesis of α₂M by these cells, as measured by ELISA on cell supernatants. The α₂M induction was also observed in other human glioma cell lines (T70 and ADF) and in human fetal astrocyte cultures following IFNγ treatment. In T67 cells this effect was dose-dependent and the maximum (2.7-fold increase) was obtained with 2000 U/ml of IFNγ. A corresponding enhanced α₂M mRNA accumulation was demonstrated by PCR and Northern blot techniques. Our results suggest an important role of α₂M during inflammatory and immune processes in the CNS. An increased release of α₂M following IFNγ stimulation may allow the removal of the bulk of proteases released at the site of inflammation, strengthening at the same time the antigen presentation processes.     

Behavior and binding: Correlations between α1-adrenergic stimulation of acoustic startle and α1-adrenoceptor occupancy and number in rat lumbar spinal cord

The relationship between alterations in α₁-adrenoceptors and behavioral effects of α₁-adrenergic agonists were investigated in a localized region of the rat central nervous system. Direct infusion of the α₁-adrenergic agonists,d-amphetamine or phenylephrine. into the subarachnoid space of the lumbar cord (intrathecal administration) increased the amplitude of the acoustic startle reflex, The magnitude of this behavioral facilitation correlated highly with the degree of α₁-adrenoceptor occupation measured by [³H]prazosin binding in lumbar spinal tissue. Using an in vitro estimate of receptor occupation, maximal potentiation of startle occurred following approximately 30% occupation of the receptors, using eitherd-amphetamine or phenylephrine. Intrathecal administration of 6-OHDA produced a 95% decrease in spinal norepinephrine and markedly enhanced the behavioral response to intrathecal phenylephrine as well as the number of α₁-adrenoceptors. The correlation between the time course of the behavioral and binding changes was 0.99. No change in receptor affinity (K_D) or receptor occupation by phenylephrine was found after 6-OHDA. The data indicate that receptor binding parameters do have predictive value for behavior, especially if localized regions of the nervous system, critical to the behavior, are analyzed.     

Tritium-sensitive film autoradiography of guinea pig brain α2-noradrenergic receptors

Tritium-sensitive film autoradiography was used to determine the distribution of α-noradrenergic receptors (i.e. [³H] p-aminoclonidine binding sites) in guinea pig forebrain. α₂-Receptors are heterogeneously distributed throughout the forebrain. Many limbic system structures, such as bed nucleus of stria terminalis, medial preoptic area, medial amygdaloid nucleus and lacunosum molecular layer in hippocampus were heavily labeled. We did not quantify receptor density in areas containing principally white matter but the optical density in those areas was similar to film background suggesting a very low receptor density. Low receptor concentrations were also found in areas that do not contain a high percentage of white matter, such as lateral septum and ventromedial hypothalamic nucleus.     

Serotonergic agents modulate antidepressant-like effect of the neurosteroid 3α-hydroxy-5α-pregnan-20-one in mice

The present study demonstrated the antidepressant-like effect of neurosteroid 3α-hydroxy-5α-pregnan-20-one (3α, 5α THP) in mouse forced swim test of depression and its modulation by different serotonergic agents. Pretreatment with the selective serotonin reuptake inhibitor, fluoxetine (5 mg/kg, i.p.), the 5-HT releaser, fenfluramine (10 mg/kg, i.p.), the 5-HT_1A receptor agonist, 8-OH-DPAT (0.1 mg/kg, s.c.), the 5-HT_1B/1C receptor agonist, TFMPP (4 mg/kg, s.c.) and the 5-HT_2A/1C receptor agonist, DOI (2 mg/kg, s.c.) potentiated the antidepressant-like effect of 3α, 5α THP. At these doses the serotonergic agents per se did not modify the duration of immobility. However, fluoxetine (20 mg/kg, i.p.), fenfluramine (20 mg/kg, i.p.) or imipramine (5 or 20 mg/kg, i.p.) not only reduced immobility but also enhanced the antidepressant-like effect of 3α, 5α THP. Such a potentiating effect of the 5-HT_1A or the 5-HT_2A/1C receptor agonist was not antagonized by the sub-effective dose (0.1 mg/kg, s.c.) of their respective antagonists p-MPPI or ketanserin. Pretreatment with p-CPA (300×3 mg/kg, i.p.), a depleter of 5-HT neuronal store failed to block the influence of fluoxetine and fenfluramine on antidepressant-like effect of 3α, 5α THP. The accelerated effect of 3α, 5α THP in presence of serotonergic agents was antagonized by the GABA_A receptor antagonist, bicuculline (1 mg/kg, i.p.) or the 3α-hydroxysteroid oxidoreductase enzyme inhibitor, indomethacin (5 mg/kg, i.p.). These findings for the first time demonstrate that serotonergic agents potentiate the antidepressant-like action of 3α, 5α THP, by enhancing the GABAergic tone as a likely consequence of increased brain content of this neurosteroid.     

NMDA receptors mediate heat shock protein induction in the mouse brain following administration of the ibotenic acid analogue AMAA

Expression of inducible heat shock protein-70 (HSP-70) and hsp-70 mRNA were studied in the adult mouse brain following systemic administration of the ibotenic acid analogue (±)-2-amino-3-hydroxy-5-methyl-4-isoxazoleacetic acid (AMAA), which is a potent N-methyl- d-aspartate (NMDA) agonist. At the dose of 20 mg/kg, AMAA produced excitatory behaviours in adult mice but overt convulsions were not seen. This treatment did not result in any detectable morphological brain damage at 4 days following administration. At 2.5 h and 5 h following treatment induction of hsp-70 mRNA expression was found in the pyramidal cell layers of CA1 and, to a lesser extent, CA3 fields of hippocampal Ammon's horn, amygdala, olfactory lobes, tenia tecta, hypothalamic nuclei and a superficial layer of cingulate, frontal and retrosplenial cortices. The presence of HSP-70 was detected by immunohistochemistry at 24 h following drug administration in those regions previously showing hsp-70 mRNA induction. AMAA-induced hsp-70 mRNA expression was prevented by pre-treatment with the non-competitive NMDA antagonist MK-801. These results suggest that NMDA receptors are involved in the stress response induced by AMAA.     

Comparison of the binding of nicotinic agonists to receptors from human and rat cerebral cortex and from chick brain (α4β2) transfected into mouse fibroblasts with ion channel activity

(−)-Nicotine, cytisine and carbachol evoked⁸⁶Rb efflux from mouse fibroblasts stably transfected with α₄β₂ chick brain nicotinic subunits. This response to (−)-nicotine was inhibited by mecamylamine and dihydro-β-erythroidine and was mirrored by a rise in intracellular Ca²⁺ measured by microspectrofluorimetry. Lobeline and isoarecolone methiodide evoked no significant⁸⁶Rb from cells and unlike the above agonists displayed significantly different IC₅₀ values for the displacement of [³H]nicotine from mammalian (rat and human cerebral cortex) and transfected fibroblast membranes.     

Specific increase of hypothalamic α1-adrenoceptors in spontaneously hypertensive rats: Effect of hypotensive drug treatment

To study potential central adrenoceptor alterations in the hypertension, we have determined α₁, α₂ and β-adrenoceptors using [³H]WB4101, [³H]yohimbine and [³H]DHA in the brain regions of spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP) and renal hypertensive rats. There was a significant increase in specific [³H]WB4101 binding only in the hypothalamus of SHR and SHRSP at 16–24 weeks of age compared to that of age-matched Wistar-Kyoto rats (WKY). Scatchard analysis revealed a 28–33% increase in the B_max value for hypothalamic [³H]WB4101 binding without a change in the K_d value, suggesting a change in the receptor density. An increased density of α₁-adrenoceptors was consistently observed in the prehypertensive (5 weeks) and developmental (10 weeks) stages of spontaneous hypertension. In contrast, there was no α₁-adrenoceptor alteration in the hypothalamus of rats with renal hypertension. The receptor alteration in the SHRSP hypothalamus was not abolished by a chronic hypotensive treatment which prevented the development of hypertension, thereby suggesting that an increased density of the α₁-adrenoceptors in spontaneous hypertension does not occur secondarily to the elevation of blood pressure. The SHRSP hypothalamus showed significantly lowered levels of noradrenaline. There was no change in specific binding of [³H]yohimbine and [³H]DHA in the brain regions of SHRSP, except the brainstem which showed a significant decrease in the [³H]yohimbine binding. Thus, the present study suggests an important role for hypothalamic α₁-adrenoceptors in the pathogenesis of spontaneous hypertension.     

Autoradiographic studies of central α2A- and α2C-adrenoceptors in the rat using [H]MK912 and subtype-selective drugs

In the present study we examined the distribution of α_2A- and α_2C-adrenoceptors in tissue slices from the rat cervical spinal cord and from brain slices collected at the level of the striatum. To differentiate between α_2A- and α_2C-adrenoceptors, the slices were incubated with [³H]MK912 in the presence of graded concentrations of the α_2A-selective drug, BRL44408, or the α_2C-selective drug, spiroxatrine. Computer analysis of the autoradiograms indicated that 0.4 nM [³H]MK912 plus 185 nM BRL44408 selectively labeled α_2C-adrenoceptors, while 0.4 nM [³H]MK912 plus 220 nM spiroxatrine selectively labeled α_2A-adrenoceptors. Using this approach, α_2C-adrenoceptors were detected in the striatum, while α_2A-adrenoceptors predominated in the cortical layers 1–4, the spinal cord distal dorsal horn, the septum and the endopiriform nucleus.     

The influence of denervation on β-amyloid protein precursor and α1-antichymotrypsin in mouse skeletal muscle

A serpin, α₁-antichymotrypsin (α₁-ACT), and Kunitz inhibitor containing forms of the β-amyloid precursor protein (βAPP) may be important components of the balance between serine proteases and inhibitors in the nervous system. In the current report we studied whether axotomy affected the localization of βAPP and α₁-ACT in adult mouse muscle. Immunocytochemical experiments indicated that βAPP was present in normal muscle both at neuromuscular junctions and within intramuscular nerves. α₁-ACT was also present at neuromuscular junctions, on the perineurium of nerves and endothelial cell surfaces. Following axotomy, both βAPP and α₁-ACT disappeared from intramuscular nerves simultaneously. However, at the neuromuscular junction α₁-ACT decreased more rapidly with βAPP lingering before disappearing.