IL-1β increases intracellular calcium through an IL-1 type 1 receptor mediated mechanism in C6 astrocytic cells

Interleukin-1β (IL-1β) is a cytokine that regulates a variety of biological processes. In addition to its traditional role in the immune system, IL-1β plays an integral role in neural-immune and developmental processes in the nervous system. The pleiotropic ability of IL-1β may be due to the activation of different signal transduction mechanisms in specific cell types or under certain cellular conditions. We have previously demonstrated that IL-1β regulates healing and repair in the developing, mammalian nervous system. In the damaged perinatal mouse brain, IL-1β is expressed in astrocytes that change from a stellate to a spindle-shaped morphology. The spindle-shaped astrocytes enclose the wound, separating the healthy from damaged neural tissue. The shape change and subsequent repair processes are IL-1β activity-dependent, acting through the IL-1 type 1 receptor (IL-1R1), as co-application of the IL-1type 1 receptor antagonist protein (IL-1ra) blocks IL-1β induced effects. In the C6 astrocytic cell line, IL-1β induced similar shape changes and upregulated expression of the cytoskeletal protein, glial fibrillary acidic protein (GFAP). Since cytoskeletal changes, as well as specific signal transduction mechanisms, are associated with increases in intracellular calcium ([Ca²⁺]_i), studies were carried out to determine if increases in [Ca²⁺]_i induced by IL-1β occurred through activation of the IL-1R1 in C6 cells. Cells were treated with IL-1β and/or IL-1ra, followed by measurement of relative changes in [Ca²⁺]_i using fura-2 fluorescence imaging methods. IL-1β increased [Ca²⁺]_i levels in a dose and time dependent manner. Treatment with IL-1ra blocked IL-1β induced increases in [Ca²⁺]_i, indicating that IL-1β acts through the IL-1R1. Immunocytochemistry experiments showed that untreated C6 cells normally express IL-1β, IL-1ra, and IL-1R1. Thus, IL-1 system molecules may play a role in normal C6 astrocyte physiology.     

Interleukin-1 beta, but not IL-1 alpha, mediates nerve growth factor secretion from rat astrocytes via type I IL-1 receptor.

In astrocytes, nerve growth factor (NGF) synthesis and secretion is stimulated by the cytokine interleukin-1 beta (IL-1 beta). In the present study, the role of IL-1 receptor binding sites in the regulation of NGF release was evaluated by determining the pharmacological properties of astroglially localized IL-1 receptors, and, by comparing the effects of both the agonists (IL-1 alpha and IL-1 beta) and the antagonist (IL-1ra)-members of the IL-1 family on NGF secretion from rat neonatal cortical astrocytes in primary culture. Using receptor-binding studies, binding of [(125)I] IL-1 beta to cultured astrocytes was saturable and of high affinity. Mean values for the K(D) and B(max) were calculated to be 60.7+/-7.4 pM and 2.5+/-0.1 fmol mg(-1) protein, respectively. The binding was rapid and readily reversible. IL-1 receptor agonists IL-1 alpha (K(i) of 341.1 pM) and IL-1 beta (K(i) 59.9 pM), as well as the antagonist IL-1ra (K(i) 257.6 pM), displaced specific [(125)I] IL-1 beta binding from cultured astrocytes in a monophasic manner. Anti-IL-1RI antibody completely blocked specific [(125)I] IL-1 beta binding while anti-IL-1RII antibody had no inhibitory effect. Exposure of cultured astrocytes to IL-1 alpha and IL-1 beta revealed the functional difference between the agonists in influencing NGF release. In contrast to IL-1 beta (10 U/ml), which caused a 3-fold increase in NGF secretion compared to control cells, IL-1 alpha by itself had no stimulatory action on NGF release. The simultaneous application of IL-1 alpha and IL-1 beta elicited no additive response. IL-1ra had no effect on basal NGF release but dose-dependently inhibited the stimulatory response induced by IL-1 beta. We concluded that IL-1 beta-induced NGF secretion from cultured rat cortical astrocytes is mediated by functional type I IL-1 receptors, whereas IL-1 alpha and IL-1ra, in spite of their affinity for IL-1RI, have no effect on NGF secretion from these cells. Type II IL-1R is not present on rat neonatal cortical astrocytes.     

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.     

Intercellular adhesion molecule-1 expression induced by interleukin (IL)-1β or an IL-1β fragment is blocked by an IL-1 receptor antagonist and a soluble IL-1 receptor

The effects of a recombinant human interleukin-1 (IL-1) receptor antagonist (IL-1ra) and a recombinant human soluble IL-1 receptor (sIL-1R) on cytokine-induced intercellular adhesion molecule-1 (ICAM-1) expression in a human glioblastoma cell line and a neuroblastoma cell line were determined. Cells were incubated with IL-1β, tumor necrosis factor (TNF)α and interferon (IFN)γ. Cells were also tested under identical conditions with an IL-1β synthetic peptide fragment (IL-1β_208–240) previously shown to possess biological activity. IL-1β, TNFα and IFNγ potentiated ICAM-1 expression in both cell lines in a dose-related manner. The IL-1β_208–240 fragments, corresponding to the rabbit, rat and human sequences, enhanced ICAM-1 expression in glioblastoma cells at high doses. ICAM-1 expression induced by IL-1β, rabbit IL-1β_208–240 and human IL-1β_208–240 was blocked by the IL-1ra, while TNFα- and IFNγ-induced ICAM-1 expression were not. ICAM-1 expression induced by IL-1β and human IL-1β_208–240 was also blocked by the sIL-1R. Our findings suggest that IL1β_208–240 acts as an IL-1β agonist in enhancing ICAM-1 expression in vitro and that this effect is receptor-mediated.     

α1-Adrenergic receptors in the brain: characterization in astrocytic glial cultures and comparison with neuronal cultures

Binding of [¹²⁵I]HEAT to membranes prepared from primary cultures of astrocytic glial cells was time-dependent and 70–85% specific. Various adrenergic agonists and antagonists competed for [¹²⁵I]HEAT binding to the potencies of prazosin >, yohimbine , clonidine, norepinephrine (NE), and propranolol. Scatchard analysis showedB_max of 209 fmol/mg protein and a K_d of 184 pM for [¹²⁵I]HEAT binding by astrocytic glial membranes. Pretreatment of astrocytes with NE resulted in a dose-dependent downregulation of [¹²⁵I]HEAT binding sites with a maximal response observed after 8 h at 100 μM NE. Removal of NE from cultures after pretreatment resulted in a time- and protein synthesis-dependent recovery of binding sites to control levels within 120 h. Incubation of astrocytic glial cultures with NE stimulated phosphoinositide (PI) hydrolysis in a time- and dose-dependent manner with a maximal stimulation of 2-fold observed in 60 min by 100 μM NE.Clonidine expressed differential effects on α₁-adrenergic receptors of the neuronal and astrocytic glial cultures. Pretreatment with 10 μM clonidine caused a 40% decrease in the B_max of [¹²⁵I]HEAT binding without influencing the K_d value in neuronal cultures. This downregulatory effect of clonidine was associated with a reduction in the ability of NE to stimulate PI hydrolysis in clonidine pretreated cells. In contrast to neuronal cultures, clonidine neither downregulated [¹²⁵I]HEAT binding sites nor stimulated PI hydrolysis in glial cultures.     

The antihyperalgesic activity of a selective P2X7 receptor antagonist, A-839977, is lost in IL-1αβ knockout mice

The pro-inflammatory cytokine interleukin-1β (IL-1β) has been implicated in both inflammatory processes and nociceptive neurotransmission. Activation of P2X7 receptors is the mechanism by which ATP stimulates the rapid maturation and release of IL-1β from macrophages and microglial cells. Recently, selective P2X7 receptor antagonists have been shown to reduce inflammatory and neuropathic pain in animal models. However, the mechanisms underlying these analgesic effects are unknown. The present studies characterize the pharmacology and antinociceptive effects of a structurally novel P2X7 antagonist. A-839977 potently (IC₅₀ = 20–150 nM) blocked BzATP-evoked calcium influx at recombinant human, rat and mouse P2X7 receptors. A-839977 also potently blocked agonist-evoked YO-PRO uptake and IL-1β release from differentiated human THP-1 cells. Systemic administration of A-839977 dose-dependently reduced thermal hyperalgesia produced by intraplantar administration of complete Freund's adjuvant (CFA) (ED₅₀ = 100 μmol/kg, i.p.) in rats. A-839977 also produced robust antihyperalgesia in the CFA model of inflammatory pain in wild-type mice (ED₅₀ = 40 μmol/kg, i.p.), but the antihyperalgesic effects of A-839977 were completely absent in IL-1αβ knockout mice. These data demonstrate that selective blockade of P2X7 receptors in vivo produces significant antinociception in animal models of inflammatory pain and suggest that the antihyperalgesic effects of P2X7 receptor blockade in an inflammatory pain model in mice are mediated by blocking the release of IL-1β.     

IL-1 and anti-inflammatory drugs modulate A β cytotoxicity in PC12 cells

We studied the effect of the inflammatory cytokine interleukin-1β (IL-1) and several anti-inflammatory drugs on amyloid β-peptide (A β) cytotoxicity. Incubation of PC12 cells with 10⁻⁶ M Aβ₁₋₄₂ for 24 h reduced viabilty to 36%; coincubation with 10⁻⁷ M IL-1 further reduced viability to 13% of baseline. With preincubation of PC12 cells with the drugs indomethacin, dexamethasone and chloroquine, reduction in viabilty was limited to 51%, 48% and 44% respectively, compared to 32% with A β alone. These experiments support further study of anti-inflammatory therapy in Alzheimer's disease.     

Release of β-lipotropin and β-endorphin from rat hypothalami in vitro

Hypothalamic tissue extracts of rats were chromatographed and β-endorphin immunoreactivity (β-End_i) was measured. The two major peaks of β-End_i co-eluted with β-lipotropin (β-LPH) and β-End respectively. Hypophysectomy caused a local decrease of β-LPH and β-End concentrations in the mediobasal hypothalamus. During superfusion of hypothalamic tissue blocks in vitro, membrane depolarization by electric stimulation or 45 mM K⁺ induced a Ca²⁺-dependent release of both β-LPH and β-End.     

Subtype-specificity of the presynaptic α2-adrenoceptors modulating hippocampal norepinephrine release in rat

In vivo brain microdialysis and high-performance liquid chromatography with electrochemical detection were used to study the effect of different selective α₂-antagonists on hippocampal norepinephrine (NE) release in freely moving awake rat. Systemic administration (0.5 mg/kg i.p.) of either the α_2AD-antagonist BRL 44408 or the α-_2BC-antagonist ARC 239 did not significantly change the basal release of NE. At a higher dose (5 mg/kg i.p.) ARC 239 was still ineffective, whereas BRL 44408 caused a significant increase of the extracellular level of NE. Similar results were obtained from in vitro perfusion experiments. Rat hippocampal slices were loaded with [³H]NE and the electrical stimulation-evoked release of [³H]NE was determined. The α₂-antagonists were applied in a concentration range of 10⁻⁸ to 10⁻⁶ M. ARC 239 was ineffective, whereas BRL 44408 significantly increased the electrically induced release of [³H]NE. In agreement with the data of microdialysis and perfusion experiments. BRL 44408 displaced [³H]yohimbine from hippocampal and cortical membranes of rat brain with high affinity whereas ARC 239 was less effective. The pK_i values of eight different α₂-adrenergic compounds showed a very good correlation (r = 0.98, slope = 1.11 P < 0.0001) in hippocampus and frontal cortex where the α₂-adrenoceptors have been characterized as α_2D-subtype. Our data indicate that hippocampal NE release in rat is regulated by α_2D-adrenoceptors, a species variation of the human α_2A-subtype.     

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.     

Characterization of [I]HEAT binding to α1-receptors in human brain: assessment in aging and Alzheimer's disease

The binding of [¹²⁵I]2-(β-4-hydroxyphenylethylamino-ethyltetralone ([¹²⁵I]HEAT), an α₁-adrenergic receptor antagonist, to human brain membranes was characterized and the binding assessed in tissue from subjects with Alzheimer's disease (AD) and aging controls. Under Na⁺-K⁺ phosphate buffer conditions, [¹²⁵I]HEAT bound to a single class of binding sites in prefrontal cortex (Brodmann area 10) with a K_d of about 120 pM. High binding capacities of [¹²⁵I]HEAT were evident in the hippocampus and neocortex but were low in subcortical areas and cerebral microvessels comparable to the regional distribution of [³H]prazosin binding reported previously. Displacement of [¹²⁵I]HEAT by various adrenergic drugs was consistent with its binding to α₁-adrenergic receptors. The specific binding was not affected by postmortem delay between death and freezing of tissue at autopsy. There was no correlation of [¹²⁵I]HEAT binding with age of subjects. In AD subjects, the binding was significantly decreased in prefrontal cortex by about 25% but not changed in hippocampus, putamen or cerebellum compared to age-matched controls. The reduced binding of [¹²⁵I]HEAT in prefrontal cortex may reflect a region-specific change in α₁-adrenergic receptors associated with neuronal loss in AD.     

α2-Adrenergic receptor activation inhibits calcitonin gene-related peptide expression in cultured dorsal root ganglia neurons

Calcitonin gene-related peptide (CGRP), a potent vasodilator, is produced in dorsal root ganglia (DRG) neurons which extend nerves peripherally to blood vessels and centrally to the spinal cord. We previously reported that neuronal CGRP expression is significantly reduced in the spontaneously hypertensive rat (SHR) which could contribute to the elevated BP. Other studies suggest that the enhanced activity of the sympathetic nervous system in the SHR may mediate, at least in part, this reduction in neuronal CGRP expression via activation of α₂-adrenoreceptors (α₂-AR) on DRG neurons. To test this hypothesis in vitro we employed primary cultures of adult rat DRG neurons. Neuronal cultures were initially exposed (24 h) to either the α₂-AR agonist UK 14,304 (10⁻⁶ M) or vehicle; however, no changes in CGRP mRNA content or immunoreactive CGRP (iCGRP) release were observed. Using the rationale that in vivo DRG neurons receive a continuous supply of target tissue derived nerve growth factor (NGF), which stimulates CGRP synthesis, the cultured neurons were treated (24 h) with either vehicle, NGF (25 ng/ml) alone, or NGF plus UK. NGF treatment increased CGRP mRNA accumulation 5.5±0.9-fold (p<0.001) and iCGRP release 2.9±0.4-fold (p<0.001) over control levels. The stimulatory effects of NGF were markedly attenuated, but not abolished, by UK (NGF+UK vs. control, CGRP mRNA, 2.9±0.4-fold, p<0.05; iCGRP, 1.7±0.2-fold, p<0.05). These values were also significant (p<0.05) when compared to NGF treatment alone. Experiments performed using the α₂-antagonist yohimbine confirmed that the effects of UK were mediated by the α₂-AR. These results, therefore, demonstrate that α₂-AR activation attenuates the stimulatory effects of NGF on CGRP expression in DRG neurons.     

Increased density of M1 receptors in the hippocampus of juvenile rats chronically deprived of NGF

Binding studies were used to assess the changes in affinity and/or number of M1 muscarinic receptors in hippocampi from juvenile rats chronically deprived of NGF. NGF deprivation was obtained by implanting into right ventricle at postnatal day 2 (P2) hybrydoma cells secreting high levels of monoclonal antibodies against NGF (αD11). Parenteral myeloma cells (P3U) were used as controls. Competition experiments were used to characterise the [³H]-PNZ binding sites in membrane preparations of hippocampi from rats sacrificed at P15. [³H]-PNZ bound M1 receptors both in P3U and αD11 group as shown by displacing potency order of antagonists: TLZ=4-DAMP>PNZ>p-F-HHSiD>MTC. The deprivation of NGF for two weeks significantly increased the number of M1 receptors without changing the K_i values of antagonists with exception of methoctramine which showed an increase in affinity in αD11 group. Similar changes in binding parameters were already observed after the first week of anti-NGF treatment. In contrast, a treatment for a week with implant at postnatal day 15 failed to produce any changes in M1 binding parameters. These results provide further physiological evidence for developmentally regulated modulatory role of NGF in the cholinergic function in the hippocampus.     

Signalling events regulating the retrograde axonal transport of I−βNerve growth factor in vivo

The molecular mechanisms regulating the retrograde axonal transport of nerve growth factor (NGF) are currently unknown. This study identifies some of the signalling events involved. The phosphoinositide 3-kinase (PI3-kinase) inhibitor wortmannin (1 nmol/eye) irreversibly inhibits the amount of ¹²⁵I−βNGF retrogradely transported in both sensory and sympathetic neurons. Another PI3-kinase inhibitor LY294002 (100 nmol/eye) also inhibited ¹²⁵I−βNGF retrograde transport in sensory neurons. The pp70^S6K inhibitor rapamycin (1 μmol/eye) had the same effect, inhibiting ¹²⁵I−βNGF transport only in sensory neurons. The cPLA₂ inhibitor AACOCF₃ (10 nmol/eye) had no effect on ¹²⁵I−βNGF transport in either sensory or sympathetic neurons. The TrkA receptor tyrosine kinase inhibitor AG-879 (10 nmol/eye) reduced ¹²⁵I−βNGF transport by approximately 50% in both sensory and sympathetic neurons. Cytochalasin D (2 nmol/eye), a disruptor of actin filaments and the dynein ATPase inhibitor erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) both inhibited ¹²⁵I−βNGF retrograde transport. These results demonstrate that in vivo TrkA tyrosine kinase activity, actin filaments and dynein are involved in the retrograde transport of NGF. In addition, different PI3-kinase isoforms may be recruited within different neuronal populations to regulate the retrograde transport of NGF. Potentially, these isoforms could activate alternative signalling pathways, such as pp70^S6K in sensory neurons.     

Induction of nitric oxide synthase activity in human astrocytes by interleukin-1β and interferon-γ

Nitric oxide (NO) is a short-lived, diffusible molecule that has a variety of biological activities including vasorelaxation, neurotransmission, and cytotoxicity. In the central nervous system, a constitutive form of nitric oxide synthase (NOS) has been localized in a subset of neurons and in endothelial cells. In addition, both constitutive and LPS-inducible NOS has been demonstrated in rat astrocytes and microglia in vitro. In this report, we present evidence for the production of NO, as measured by the production of nitrite, in highly enriched human fetal astrocyte cultures stimulated with IL-1β. The production of nitrite paralleled the induction of NADPH diaphorase enzyme activity in the perikarya of the majority of stimulated astrocytes. The IL-1β-induced nitrite production by astrocytes was markedly enhanced when cells were co-stimulated with IFN-γ or TNF-α (IFN-γ > TNF-α); LPS had no effect used as a single agent or in combination with other cytokines. N^GMMA and N^G-nitro-arginine, competitive inhibitors of NOS, diminished the accumulation of nitrite, but calmodulin antagonists (trifluoperazine, W-5 and W-7) had little or no inhibitory effect. Human fetal microglia, in contrast to astrocytes, failed to secrete significant amounts of nitrite in response to various stimuli. The results demonstrate the presence of an inducible form of NOS in human fetal astrocytes; human microglia, in turn, may control astrocyte NO production by providing IL-1β as an activating signal.     

Modulation of rat brain α- and β-adrenergic receptor populations by lesions of the dorsal noradrenergic bundle

Bilateral lesion of the ascending noradrenergic fibers in the dorsal bundle of adult Wistar rats with 4 μg 6-hydroxydopamine caused extensive depletion of norepinephrine in all forebrain areas, but led to a 54% increase in norepinephrine levels in the cerebellum. β-Adrenergic receptor binding of [³Hdihydroalprenolol was significantly increased in all forebrain areas depleted of norepinephrine except hypothalamus. The increase in [³Hdihydroalprenolol binding was due to 62% and 34% increases in the number of β-receptor sites in the frontal cerebral cortex and hippocampus respectively. Binding of [³HWB-4101 toα₁-adrenergic receptors after dorsal bundle lesion was augmented generally to a lesser extent than β-receptor binding, with significantly increased numbers of sites only in the frontal cortex (74%), thalamus (20%) and septum. Bothα₁-andβ-receptor binding sites were reduced in number by 25–28% in the cerebellum of dorsal bundle-lesioned rats, whereas intraventricular administration of 6-hydroxydopamine to adult rats, which depletes norepinephrine in the cerebellum by 96%, increased cerebellarα₁-andβ-receptor binding by 33–40%. Binding of [³Hclonidine to forebrainα₂-adrenergic receptors was significantly elevated in the frontal cortex, but reduced in the amygdala and septum, after dorsal bundle lesion.     

Inhibition of interleukin-1β and prostaglandin E2 thermogenesis by glycyl-glutamine, a pro-opiomelanocortin-derived peptide

Interleukin-1β (IL-1β) and other cytokines produce fever by stimulating prostaglandin E₂ (PGE₂) synthesis in thermoregulatory regions of the preoptic area and anterior hypothalamus (POA/AH). Prostaglandin E₂ is thought to raise body temperature, at least in part, by stimulating β-endorphin release from pro-opiomelanocortin neurons that innervate the POA/AH. In this study, we investigated whether glycyl-glutamine (β-endorphin_30–31), an inhibitory dipeptide synthesized from β-endorphin post-translationally, inhibits IL-1β and PGE₂-induced hyperthermia. Hyperthermic sites were identified by microinjecting PGE₂ (3 fmol/1 μl) into the medial preoptic area (mPOA) of conscious, unrestrained rats. Interleukin-1β (1 U) injection into the same PGE₂ responsive thermogenic sites in the mPOA elicited a prolonged rise in colonic temperature (T_c) (+1.02±0.06°C) that persisted for at least 2 h. Glycyl-glutamine (3 nmol) co-injection into the mPOA inhibited IL-1β thermogenesis completely (T_c=−0.18±0.22°C). Glycyl-glutamine had no effect on body temperature when given alone to normothermic rats. Co-injection of individual amino acids, glycine and glutamine (3 nmol each amino acid), failed to influence IL-1β-induced thermogenesis, which indicates that Gly-Gln hydrolysis does not explain its inhibitory activity. Glycyl-glutamine (3 nmol) also prevented the rise in body temperature produced by PGE₂ (PGE₂=0.89±0.05°C; PGE₂ plus Gly-Gln=−0.16±0.14°C), consistent with evidence that PGE₂ mediates IL-1β-induced fever. These findings demonstrate that Gly-Gln inhibits the thermogenic response to endogenous pyrogens.     

Age-related changes of noradrenergic-NPY interaction in rat brain: norepinephrine, NPY levels and α-adrenoceptors

Noradrenergic-neuropeptide Y interaction, which is implicated in different physiological functions, was studied in senescent rats. Norepinephrine (NE) and neuropeptide Y (NPY) levels were measured in brainstem and hypothalamus, and α-adrenergic binding was investigated in brainstem in young (4 months) and old (34 months) Wistar rats. NE concentration was the same in senescent rats, whether NPY concentration was decreased both in brainstem and hypothalamus compared to levels in young rats. [³H]prazosin binding to α₁-adrenoceptors was not modified, but [³H]rauwolscine binding to α₂-adrenoceptors was altered with age. In fact, the density of α₂-adrenoceptors(B_max) was lower, while the binding affinity(K_d) was increased in old compared to young rats. These results suggest that the decrease of NPY levels could be one of the possible reasons for changes in [³H]rauwolscine binding to α₂-adrenoceptors in old rats. The G-protein-adenylate cyclase system, which is impaired in senescent rats, could be involved in the disorganization of noradrenergic-NPY interaction.     

Dual effects of interleukin-1β on N-methyl- -aspartate-induced retinal neuronal death in rat eyes

In this study we determine if interleukin-1β (IL-1β) modulates N-methyl- -aspartate (NMDA)-induced retinal damage. Sprague–Dawley rats were anesthetized with inhalation of halothane, after which a single injection of 5 μl of IL-1β (0.1 to 10 ng/eye) (and/or IL-1 receptor antagonist (IL-1ra)) for experimental eyes was administered. Two days later (or simultaneously), NMDA (20 nmol) was injected into the vitreous space. One week later, each eye was enucleated and transverse sections were subjected to morphometric analysis. Enzyme-linked immunosorbent assay (ELISA) was conducted for the determination of IL-1β levels in retina. Immunohistochemical and immunoblot studies were also performed. In eyes that received an intravitreal injection of IL-1β (0.1 to 10 ng/eye), significant thinning of the inner plexiform layer (IPL) was observed (P<0.05). Immunohistochemical and ELISA studies demonstrated upregulated expression of IL-1β in retinas that had undergone NMDA injection. Treatment with 10 ng of IL-1ra induced a protective effect against NMDA-induced retinal damage. Pretreatment with IL-1β induced a significant protective effect on NMDA-induced retinal damage. Our studies suggest that IL-1β induces neuronal cell death directly, as shown by the protective effects of IL-1ra, but has a protective effect on NMDA-induced retinal damage indirectly after an incubation time of at least 2 days.