High density of prostaglandin E2 binding sites in the anterior wall of the 3rd ventricle: a possible site of its hyperthermic action

Prostaglandin E₂ (PGE₂) exerts a potent hyperthermic action when injected into the preoptic-hypothalamic area (POHA) and is considered to be a central mediator of fever. To determine the exact functional sites of PGE₂, we used in vitro quantitative autoradiography of [³H]PGE₂ binding sites in the rat POHA. The highest density of [³H]PGE₂ binding was found in the regions of the anterior wall of the 3rd ventricle (A3V). Within the A3V, binding density was especially high in regions closet to the third ventricle or surrounding the organum vasculosum laminae terminalis (OVLT) but was relatively low within the OVLT itself. It seems likely that the A3V PGE₂ binding sites identified in this study are responsible for PGE₂ mediation of fever.     

Effect of edta and PGE1 on β-thromboglobulin liberation from platelets

The aim of this study was to evaluate the effect of PGE₁ and EDTA on liberation of β-thromboglobulin (βTG) from platelets in vitro. Liberation of BTG was followed in citrated blood at room temperature for 120 minutes after venesection. PGE₁ reduced βTG liberation, and maximal inhibition was attained by concentrations greater than 2 × 10⁻⁶M. EDTA induced the efflux of βTG. This EDTA-induced efflux was delayed but not prevented by PGE₁ and by citrate; it was not found at 0–4°C. Therefore the use of EDTA to prevent βTG liberation during sampling for in vitro or in vivo studies depends heavily on modifying factors such as PGE₁ and low temperature, and on the time taken to process samples. Its effectiveness must be in some doubt where the platelets may be sufficiently stimulated to overcome these modifying influences, or where handling of samples is less than optimal.     

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.     

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.     

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.     

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.     

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.     

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.     

NSAIDS inhibit the IL-1β-induced IL-6 release from human post-mortem astrocytes: the involvement of prostaglandin E2

Epidemiological studies have shown that steroidal as well as non-steroidal anti-inflammatory drugs lower the risk of developing Alzheimer's Disease (AD). A suppressive effect of these anti-inflammatory drugs on local inflammatory events in AD brains has been suggested, however the mechanisms responsible are still unknown. In this study we investigated at cellular level the influence of two anti-inflammatory drugs—dexamethasone and indomethacin—and an experimental specific cyclooxygenase-2 inhibitor, BF389, on the production of the pro-inflammatory cytokine IL-6 and the inflammatory mediator PGE₂ by human astrocytes. Two human post-mortem astrocyte cultures (A157 and A295) and astroglioma cell lines (U251 and U373 MG) were found to secrete considerable amounts of IL-6 upon stimulation with IL-1β. The glucocorticoid dexamethasone inhibited the IL-1β-activated release of IL-6 from the postmortem astrocyte cultures A157 and A295 and from the astroglioma cell lines. The non-specific cyclooxygenase inhibitor indomethacin and BF389 only suppressed the IL-6 release by post-mortem astrocyte culture A157. This post-mortem astrocyte culture was found to produce large amounts of PGE₂ upon stimulation with IL-1β, whereas in the supernatants of the postmortem astrocyte culture A295 and the astroglioma cell lines, low PGE₂ concentrations were detected. Addition of exogenous PGE₂ prevented the inhibitory effect of indomethacin and BF389 on the IL-1β-activated IL-6 release from A157 astrocytes and largely potentiated the IL-1-induced release of IL-6 from all astrocytes/astroglioma cells tested. Dexamethasone also inhibited the PGE₂ release from the astrocytes and astroglioma cells, however the inhibitory effect of dexamethasone on the IL-1β-activated IL-6 release could not be prevented by the addition of PGE₂. The observed reduction of IL-6 and/or PGE₂ from astrocytes may be involved in the mechanism underlying the beneficial effects of these drugs in AD.     

α2, α2A, α2B/2C-Adrenoceptor subtype antagonists prevent lipopolysaccharide-induced fever response in rabbits

Exogenous pyrogens, e.g., bacterial lipopolysaccharides (LPS), are thought to stimulate macrophages to release endogenous pyrogens, e.g., TNFα, IL-1 β, and IL-6, which act in the hypothalamus to produce fever. We studied the effect of different α₁⁻ and α₂-adrenoceptor subtype antagonists, applied intraperitoneally, on the febrile response induced by LPS in rabbits. Evidence was obtained that prazosin, an α₁⁻ and α_2B/2C-adrenoceptor antagonist; WB-4101, an α₁⁻ and α_2A-adrenoceptor antagonist; CH-38083, a highly selective α₂-adrenoceptor antagonist (α₂: α₁ > 2000); BRL-44408, an α_2A-adrenoceptor antagonist; and ARC-239, an α_2B/2C⁻ and also α₁-adrenoceptor antagonist, blocked the increase of colonic temperature of the rabbit produced by 2 μg/kg LPS administered intravenously without being able in themselves to affect colonic temperature. In addition, prazosin, WB-4101 and CH-38083 antagonized the fall in skin temperature that occurred at the time when the colonic temperature was rising in control animals injected with LPS. All these results suggest that norepinephrine, through stimulation of both α₁⁻andα₂⁻ (α_2A⁻andα_2B/2C⁻) adrenoceptor subtypes, is involved in producing fever in response to bacterial LPS.     

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.     

Cerebral vessels express interleukin 1β after focal cerebral ischemia

Rapid and marked increased levels of expression of interleukin 1β (IL-1β) mRNA have been detected in animal models of cerebral ischemia. However, the protein production of IL-1β and the cellular sources of IL-1β are largely undefined after cerebral ischemia. In the present study, we have measured the cellular localization of IL-1β protein in brain tissue from non-ischemic and ischemic mice using immunohistochemistry. Male C57B/6J (n=45) mice were subjected to middle cerebral artery (MCA) occlusion by a clot or a suture. The mice were sacrificed at time points spanning the period from 15 min to 24 h after onset of the MCA occlusion. Non-operated and sham-operated mice were used as control groups. A monoclonal anti-IL-1β antibody was used to detect IL-1β. In the non-operated and sham-operated mice, a few IL-1β immunoreactive cells were detected scattered throughout both hemispheres. IL-1β immunoreactive cells increased in the ischemic lesion as early as 15 min and peaked at 1 h to 2 h after MCA occlusion. IL-1β immunoreactivity was detected in the cortex of the contralateral hemisphere 1 h after ischemia. By 24 h after onset of ischemia, IL-1β immunoreactivity was mainly present adjacent to the ischemic lesion and in the non-ischemic cortex. IL-1β immunoreactivity was found on endothelial cells and microglia. This study demonstrates an early bilateral expression of IL-1β on endothelium after MCA occlusion in mice.     

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.     

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.     

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.     

Relationship of interleukin-1β and β2-microglobulin with neuropeptides in cerebrospinal fluid of patients with dementia of the Alzheimer type

We studied interleukin-1β (IL-1β), β₂-microglobulin (β₂-m, β-endorphin, substance P, neuropeptide Y and somatostatin concentrations in the cerebrospinal fluid of 13 patients with dementia of the Alzheimer type (DAT), 13 patients with multi-infarct dementia (MID) and 15 age-matched control subjects. Substance P was significantly lower in DAT than in controls (P < 0.05), as well as somatostatin in DAT as compared to both controls (P < 0.01) and MID (P < 0.05), whereas β₂-m was higher in DAT than in controls (P < 0.01). Neuropeptide Y, β-endorphin and IL-1β showed similar concentrations in the three groups studied. A significantly positive correlation was observed between IL-1β and substance P (r = 0.79, P < 0.01) and somtostatin (r = 0.75, P < 0.05) in DAT, which was not observed in MID. In addition, β₂-m showed a negative correlation with IL-1β (r = −0.73, P < 0.05) in DAT, and age correlated negatively with IL-1β in controls and MID, but positively in DAT. Therefore, these results support the idea that an altered relationship may exist in Alzheimer's disease between the nervous and immune system.     

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.