Characterization of the phosphorylated state of protein 4.2 from a patient partially deficient in protein 4.2

These studies compare the protein 4.2 found in a patient with osmotically fragile, spherocytic erythrocytes to the normal protein 4.2. The patient protein 4.2 is present in the erythrocyte ghost membranes as a doublet of 74 and 72 KDa at a concentration less than 1% of normal. The patient protein 4.2 becomes highly phosphorylated in the presence of Zn⁺⁺ and is phosphorylated, relative to the amount of protein present, to a greater extent than the normal 72 KDa protein 4.2. These studies indicate that both the patient and the normal protein 4.2 usually exists in a highly phosphorylated state. The phosphorylation sites on the patient protein 4.2 appear to be more readily cycled than on the normal protein 4.2. V8 protease generates similar phosphopeptides in both the normal and patient protein 4.2 except for an extra 11 KDa phosphopeptide generated from the 74 KDa form of the protein.     

Differential effects of zinc on hyperpolarizing and depolarizing GABAA synaptic potentials in hippocampal slice cultures

We have examined the changes in GABA_A-mediated synaptic potentials recorded from CA3 pyramidal neurons in hippocampal slice cultures following application of zinc (Zn²⁺). Unlike 4-AP, Zn²⁺ did not enhance fast hyperpolarizing potentials but primarily enhanced depolarizing GABA_A potentials. Zn²⁺ did not alter the postsynaptic response of pyramidal neurons to pressure applied GABA, consistent with previous reports that Zn²⁺ enhances the release of GABA from presynaptic terminals. To examine the role of local circuitry in the production of Zn²⁺ responses, we recorded from cultures maintained for 7–10 days following removal of the dentate and hilus to allow complete degeneration of the mossy fibers (DGX cultures). Zn²⁺ produced giant depolarizing potentials (GDPs) in DGX cultures that were identical to those in intact cultures. In contrast, the 4-AP response was dramatically altered in DGX cultures. In DGX cultures, Zn²⁺ co-applied with 4-AP appeared to inhibit the production of fast hyperpolarizing GABA_A synaptic potentials produced by 4-AP alone. This inhibition of fast hyperpolarizing potentials suggests that Zn²⁺ may reduce the release of GABA onto pyramidal cell somata. These observations suggest that Zn²⁺ enhances GABA release from local circuit neurons that synapse onto pyramidal cell dendrites, and inhibits GABA release onto pyramidal cell somata.     

Effect of temperature, pH and ionic strength and composition on fibrin network structure and its development

Networks were developed in fibrinogen solution under pathophysiological conditions of clotting. Mass-length ratio (a measure of fibre thickness) was derived independently from the turbidity ω_T) and from the permeability (μ_p) of the network. Kinetics of network growth were investigated turbidimetrically. Physiological concentrations of Ca⁺⁺ and Mg⁺⁺ increased μ_T while those of K⁺, Na⁺, Cl-, HCO₃, H₂P0₄ and S0₄- had no effect. As pH and ionic strength were increased stepwise within the pathophysiological range, network development was delayed. Under these conditions the turbidity curves did not cross and both μ_T and μ_p were progressively decreased. When temperature was lowered, although network growth was delayed, the turbidy curves crossed: the equilibrium turbidity was higher at lower temperatures. It appears that while pH and ionic strength affect network structure by influencing fibrin assembly, lowering the temperature influences both the rate of fibrin monomer generation and fibrin assembly.     

Development of GABAA receptors on medial septum/diagonal band (MS/DB) neurons after postnatal ethanol exposure

The impact of ‘binge-like' ethanol exposure on postnatal days (PD) 4–9 was examined on development of γ-aminobutyric acid type A receptors (GABA_AR) during the first month of life in the rat. Whole-cell patch-clamp recordings in acutely isolated medial septum/diagonal band (MS/DB) neurons were used to define effects of rapidly applied ethanol and other allosteric modulators on bicuculline-sensitive GABA currents. Three age groups were examined including ‘pups' (PD 4–10), ‘juveniles' (PD 11–16) and ‘young adults' (PD 25–35). In untreated neurons, maximum responses to GABA and the apparent GABA EC₅₀ increased 2-fold during the first month of life. Potentiation of GABA responses by pentobarbital, midazolam, and loreclezole all increased with age, while Zn²⁺ inhibition declined. Initial inhibition by ethanol switched to potentiation of GABA responses during this time. In vivo, binge-like ethanol treatment (4.5 g kg⁻¹ day⁻¹ divided into two doses, 2 h apart on PD 4–9) reduced both the GABA maximal response and GABA EC₅₀ measured on PD 11–16. These measures returned to control levels by PD 25–35. After binge-like postnatal ethanol exposure, age-dependent loss of Zn²⁺ inhibition of GABA responses was increased, while potentiating actions of in vitro ethanol were blocked. GABA_AR modulation by other drugs was unaffected. These data suggest that early postnatal ethanol exposure disrupts the expected developmental pattern of GABA_AR function in MS/DB neurons, an action that could contribute to neurobehavioral deficits associated with the fetal alcohol syndrome. Whether these changes are due to cellular damage, delayed gene expression or post-translational modification needs to be determined.     

Purification and activation of human factor X: Cooperative effect of Ca on the activation reaction

Human Factor X has been purified 6000 fold by a combination of DEAE-Sephadex, heparin-Sepharose and hydroxylapatite chromatography. The preparation is virtually homogeneous with a monomeric molecular weight of 75,000 as judged by polyacrylamide gel electrophoresis. The activation of Factor X by the coagulant protein of Russell's viper venom has been investigated utilizing Bz---Ile---Glu---Gly---Arg---p---nitroanilide (S-2222). Human Factor Xa hydrolyzes S-2222 with a Km of 0.6 mM. The coagulant protein has a Km for human Factor X of 0.01 μM, 10-fold lower than that of bovine Factor X. Ca⁺⁺ increases the Vmax of the activation reaction and exhibits a cooperative effect. Hill plots demonstrate that there are at least three cooperative sites for Ca⁺⁺ binding. These studies indicate that Ca⁺⁺ binding to human Factor X induces a conformation change which increases the rate of the cleavage of the peptide bond required for activation.     

A natural scavenger of peroxynitrites, rosmarinic acid, protects against impairment of memory induced by Abeta(25-35)

Peroxynitrite (ONOO⁻)-mediated damage is regarded to be responsible for the cognitive dysfunction induced by amyloid beta protein (Aβ) in Alzheimer's disease (AD). In the present study, we examined the protective effects of rosmarinic acid (RA), a natural scavenger of ONOO⁻, on the memory impairment in a mouse model induced by acute i.c.v. injection of Aβ_25–35. Mice daily received i.p. several doses of RA after the injection of Aβ_25–35. RA prevented the memory impairments induced by Aβ_25–35 in the Y maze test and novel object recognition task. RA, at the effective lowest dose (0.25 mg/kg), prevented Aβ_25–35-induced nitration of proteins, an indirect indicator of ONOO⁻ damage, in the hippocampus. At this dose, RA also prevented nitration of proteins and impairment of recognition memory induced by ONOO⁻-i.c.v.-injection. Co-injection of the non-memory-impairing dose of ONOO⁻ with Aβ_25–35 blocked the protective effects of RA (0.25 mg/kg). These results demonstrated that the memory protective effects of RA in the neurotoxicity of Aβ_25–35 is due to its scavenging of ONOO⁻, and that daily consumption of RA may protect against memory impairments observed in AD.     

Protein kinase activities associated with ribosomes of developing rat brain. Identification of eukaryotic initiation factor 2 kinases

Protein kinases associated with ribosomes in the brains of suckling (4–10 days) and adult (2 months) rats were extracted from ribosomal fraction with 0.5 M KCl. The different protein kinase activities were characterized by their ability to phosphorylate three exogenous substrates: casein, histone IIs and histone IIIs in the presence of different modulators. Ribosomal salt wash fractions contain a high casein kinase activity which was partially inhibited by heparin and stimulated by calmodulin in the presence of Ca²⁺, indicating the presence of casein kinase I and II and calcium/calmodulindependent kinases. Cyclic AMP and cyclic GMP-dependent kinases and protein kinase C (calcium/phospholipids-dependent kinase) were also present. No differences were found in the casein kinase activities of suckling and adult animals, but histone kinase activities were higher in adult than in suckling animals.

To identify initiation factor 2 kinases, purified factor from adult brains was used as a protein marker. In addition to the phosphorylation of both factor subunits and β by casein kinase I or II, an increased phosphorylation was detected of a subunit in the presence of cyclic AMP, and β subunit, in the presence of Ca²⁺/calmodulin or Ca²⁺/phospholipids.

Present results reinforce our hypothesis that, as occurs in other eukaryotic cells, the decreased rate of protein synthesis during brain development may be regulated by phosphorylation of initiation factor 2.     

Microtubules have special physical associations with smooth endoplasmic reticula and mitochondria in axons

Ultrastructural morphometry was used to document the non-random spatial distributions of organelles within the compact myelinated region of avian oculomotor axons. These regions contain large numbers of loosely packed neurofilaments (NFs) (241/μm²) and only a relatively small number of microtubules (MTs) (4/μm²), mitochondria (0.6/μm²), and smooth endoplasmic reticulum (SER) (1.6/μm²). Random co-occurrences between the relatively sparsely distributed MTs, mitochondria, and SER are probably infrequent in these axons. The actual co-occurrences of MTs, mitochondria, and SER with MTs were counted and compared to the co-occurrences expected in a random Poisson distribution. At long distances (200 nm), the co-occurrences were random. At shorter distances (40 nm and less), MTs were still randomly associated with other MTs. However, at these shorter distances, the spatial associations of mitochondria with MTs and of SER with MTs were not random; such preferential stable associations may be produced by specific MT associated cross-bridging proteins. In axons, MTs tend to be clustered together, giving the appearance of MT bundles. We propose that the MT-MT bundling is an indirect result of MT concentration along the continuous intra-axonal SER network, to which the MTs are apparently tied directly by dynamic molecular cross-bridges.     

Reduced functions of intracellular Ca in aggregation, secretion and protein phosphorylation of permeabilized platelets from stroke-prone spontaneously hypertensive rats

Aggregation, secretion and 47kDa protein (P47) phosphorylation by various agonists such as thrombin, ADP and ionophore A23187 were markedly reduced in platelets from stroke-prone spontaneously hypertensive rats (SHRSP) compared with those of age-matched Wistar Kyoto rat (WKY) platelets, suggesting defective functions of intracellular Ca²⁺ in SHRSP platelets (Tomita et al. Hypertension 1989: 14: 304–315). To clarify the mechanism of the platelet hypofunctions, saponin permeabilized platelets were prepared to compare the responses of platelets from both rats in varying concentrations of extracellular Ca²⁺. The leakage of lactate dehydrogenase from saponin (15 μg/ml)-treated platelets was approx. 5 % of total activity; the degree of the leakage in both platelets did not differ. In saponin-treated platelets, extracellular Ca²⁺ alone did not induce either aggregation or secretion in both strains. However, in the presence of 1-oleoyl-2-acetylglycerol (10 μg/ml), Ca²⁺ dose dependently stimulated both aggregation and secretion. Under this condition, Ca²⁺ sensitivity of aggregation, secretion and P47 phosphorylation in SHRSP platelets were significantly reduced compared with those in WKY platelets. These results strongly suggest that intracellular Ca²⁺ functions are impaired in SHRSP platelets.     

Brain cell membrane Na,K-ATPase activity following severe hypoxic injury in the newborn piglet

This study tests the hypothesis that severe brain hypoxia causes decreased Na⁺,K⁺-ATPase activity, resulting in permanent alterations in the neuronal cell membranes. Seventeen anesthetized piglets (normoxic control (NC), no recovery after hypoxia (Group 1), 6 h normoxic recovery (Group 2), and 48 h normoxic recovery (Group 3) were studied. Hypoxia was induced by lowering the FiO₂ to maintain PCr/P_i ratio at 25% of baseline for 1 h as monitored by ³¹P-NMR spectroscopy. PCr/P_i returned to 57% of baseline by 6 h and was normal by 48 h. At termination, cortical tissue Na⁺,K⁺-ATPase activity was determined. Na⁺,K⁺-ATPase activity was measured in cortical membrane preparations by determining the rate of ATP hydrolysis. NC membranes had Na⁺,K⁺-ATPase activity of 58.3 ± 1.3 μM P_i/mg protein/h (mean ± S.E.M.). Na⁺,K⁺-ATPase activity was reduced in Groups 1, 2, and 3 (45.8 ± 1.3, 47.4 ± 3.6, 48.7 ± 2.9 μM P_i/mg protein/h) (P < 0.05 compared to NC). There was no differene in enzyme activity among Groups 1, 2, or 3. The data show that in spite of recovery of neuronal oxiditive phosphorylation (PCr/P_i) by 48 h, there is a permanent decrease in Na⁺,K⁺-ATPase activity in cells that have undergone severe hypoxic injury. The persistent decrease in Na⁺,K⁺-ATPase activity indicates ongoing cell injury following severe cerebral hypoxia, and that recovery of oxidative phosphorylation as indicated by PCr/P_i values cannot be used as an index of recovery of cell function.     

Divalent transition-metal ions (Cu2+ and Zn2+) in the brains of epileptogenic and normal mice

The concentrations of Cu²⁺ and Zn²⁺ in 3 strains of mice were determined spectrophotometrically. The brain of the inborn audiogenic mouse (DBA/2J) contains higher levels of Zn²⁺ and Cu²⁺ than those found in the normal mouse (CBA/Ca or Parkes). Small differences in the metallic content in the whole brains of audiogenic and normal mice are accentuated in the hippocampus and the colliculus.     

Potassium and sodium channels in human malignant glioma cells

Human malignant glioma cells from 5 different cell lines were voltage clamped and examined for the presence of depolarization-activated ion channels. Outward K-currents were elicited at membrane potentials > 40 mV, which had two main components, one which was delayed and blocked by externally applied tetraethylammonium (TEA, 10 mM), and another which was instantaneous and insensitive to TEA in the outside solution. The proportion of the two K-current components varied between cell lines. An increase in [Ca²⁺] in the range 0–4 mM, decreased the leak conductance and shifted the activation of the instantaneous outward K-current towards more positive potenttials. Mg²⁺, Zn²⁺ and Co²⁺ had qualitatively similar effects. Patch recordings with 150–160 mM K⁺-solution on both sides of the membrane revealed that the delayed outward K-current was carried through large conductance (250–300 pS) channels. Changes in free [Ca²⁺]_i from 0 to 2 × 10⁻⁸ M increased the activation of the large conductance K-channel. Small Na-currents were identified in cells from one cell line (Tp-378MG). The Na-conductance rangedfrom 0.5 to 7.5 nS in 25% of the cells, and was less than 0.5 nS in 75%. The Na-channels were activated and inactivated at 30–40 mV more positive potentials than in the mammalian peripheral nerve. Tetrodotoxin (100 mM) blocked g_Na almost completely.     

Procoagulant and defibrinating potency of the venom gland extract of Thelotornis kirtlandi

The coagulant activity of the venom gland extract of Thelotornis kirtlandi (TK) was studied. Citrated plasma is coagulated independently of Ca⁺⁺ ions, platelets and phospholipids. The extract has no thrombin-like activity. It converts prothrombin directly to thrombin without other plasmatic cofactors of the intrinsic thromboplastin being necessary. The ensuing thrombin is far less inhibited by heparin or plasmatic antithrombins. Factor X is not activated by this coagulase. A dose of 10 μg/kg of the extracted protein causes (in rats) a rapidly onsetting and prolonged defibrination accompanied in the first 12 hours by haemoglobinaemia. Coagulant activity in vitro and defibrinating potency in vivo of TK are compared with other prothrombin converting and defibrinating snake venoms.     

Interaction of fibrinogen with mercury

The interaction of bovine fibrinogen with mercuric chloride was studied. Gel filtration on Sephadex G-25 revealed that fibrinogen bound twice the amount of mercury such as fibrin or fibrin monomers (8.8, 4.5, and 3.4 μg Hg²⁺ ions/ mg protein, respectively). Fibrinogen complexed with mercury or in the presence of Hg²⁺ ions at concentration above 10⁻⁶ M was clotted by thrombin more effectively than in the control system which was devoid of this metal. Reaggregation of the purified fibrin monomers was not affected by mercury.     

Interaction of human blood platelet aggregation inhibitors with phospholipid films

A series of carbamoylpiperidines and related entities, a class of compounds only recently identified by the authors as potent human blood platelet aggregation inhibitors, showed correspondingly strong levels of “specific” interaction, in monomolecular film systems, with some phospholipids viewed as prototypes of actual platelet plasma membrane constituents. The data strongly corroborate our previously articulated view that our compounds possess appropriate hydrophobic character to penetrate the lipid bilayer of the platelet plasma membrane, subsequently are capable of generating sufficient quantities of their cationic species to counteract massively stimulus-induced mobilization of Ca⁺⁺ ions, and thereby restrain or void Ca⁺⁺-dependent phospholipase activity.     

Purification and characterization of a serine protease with fibrinolytic activity from the dung beetles, Catharsius molossus

Catharsius protease-1 (CPM-1) was isolated from the whole body of the dung beetles, Catharsius molossus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel Blue gel). The purified CPM-1 that has a molecular weight of 27 kDa was assessed homogeneous by SDS-polyacrylamide gel electrophoresis and an isoelectric point of 4.4 was determined by isoelectric focusing. N-terminal amino acid sequence of the protease was composed of Ile-Val-Gly-Gly-Gln-Ala-Val-Glu-Ile-Gly-Asp-Tyr-Pro-Ala-Gln. The enzyme was inactivated by Cu²⁺ and Zn²⁺ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine and -antitrypsin. However, EDTA, EGTA, cysteine, β-mercaptoethanol, E64, chymostatin, elastatinal and TPCK did not/less affect activity. Also, antiplasmin and antithrombin III were not sensitive to CPM-1. On the basis of amidolytic activity test, CPM-1 preferably hydrolysed chromogenic protease substrates containing Arg or Lys residues of the P1 position at pH 7.0 and 37 °C. CPM-1 preferentially cleaved the oxidized B-chain of insulin between Arg²² and Gly²³. CPM-1 readily digested A- and γ-chains and more slowly Bβ-chain of fibrinogen. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. D-dimer concentration increased on incubation of cross-linked fibrin with CPM-1, indicating that the enzyme has a significant fibrinolytic activity.     

The co-expression of VR1 and VRL-1 in the rat vagal sensory ganglia

Immunohistochemistry for two nociceptive transducers, the vanilloid receptor 1 (VR1) and vanilloid receptor 1-like receptor (VRL-1), was performed on the vagal sensory ganglia. In the jugular ganglion, VR1-immunoreactive (IR) neurons were small to medium-sized (range 49.7–1125.6 μm², mean±S.D. 407.7±219.7 μm²), whereas VRL-1-IR neurons were medium-sized to large (range 223.6–1341.1 μm², mean±S.D. 584.3±253.5 μm²). In the nodose ganglion, VR1- and VRL-1-IR neurons were mostly small to medium-sized (VR1: range 148.5–1464.4 μm², mean±S.D. 554.3±207.4 μm²; VRL-1: range 161.7–1166.2 μm², mean±S.D. 541.9±186.2 μm²). The double immunofluorescence method revealed that co-expression of VR1-immunoreactivity among VRL-1-IR neurons was more abundant in the nodose ganglion (63%) than in the jugular ganglion (4%). The present study suggests that co-expression of VR1 and VRL-1 may be more common in visceral sensory neurons than in somatic sensory neurons.     

Affinity crosslinking of I-labeled human ß-endorphin to cell lines possessing either μ- or δ-type opioid binding sites

The specific labeling of opioid receptor-related polypeptides was compared in two cell lines which differ in their opioid receptor population: SK-N-SH which contains predominantly μ-type opioid receptors, and NG-108-15, which contains exclusively δ-type opioid receptors. Labeling of opioid receptors was achieved by affinity cross-linking of membranes, using ¹²⁵I-labeled human ß-endorphin, followed by solubilization in sodium dodecyl sulphate (SDS), SDS-gel electrophoresis and autoradiography. Different labeling patterns were obtained from these two cell lines. In SK-N-SH cells, 3 major proteins were labeled, corresponding to molecular weights of 92, 65 and 25 kDa, while in the NG-108-15 cells, 53-kDa and 25-kDa polypeptides were the major ones labeled. The radioactivity incorporated into the 92- and 65-kDa peptide bands derived from SK-N-SH cells was displayed by the μ-selective ligand Tyr- -Ala-Gly-MePhe-Gly-ol (DAGO) but not by the δ-selective ligand [ -Pen², -Pen⁵]enkephalin (DPDPE). The radioactivity incorporated into the NG-108-15-derived peptide bands was displaced by the δ-selective ligand, but not by the μ-selective ligand. This confirms our previous finding in mammalian brain which demonstrated that μ- and δ-opioid binding sites can be identified as distinct proteins which differe in molecular size.     

Analysis of potential shifts associated with recurrent spreading depression and prolonged unstable spreading depression induced by microdialysis of elevated K in hippocampus of anesthetized rats

The potential shifts (ΔV_o) associated with spreading depression (SD) were analysed with the help of multiple extracellular recording and ion-selective microelectrodes in the CA1 region of the dorsal hippocampus of anesthetized rats. Recurrent waves of SD were induced by perfusing high K⁺ solution through microdialysis probes. SD-related ΔV_o had a composite wave shape, consisting of an early, rapidly shifting part (phase I) followed by a slower shift to a second negative maximum (phase II). ΔV_o shifts in stratum radiatum usually started earlier, always lasted longer and had lartger amplitude than those recorded in stratum pyramidale. The ΔV_o responses in stratum radiatum had an inverted saddle shape created by a transient relatively positive “hump” interposed between phases I and II. During this “hump”, the potentials in the two layers transiently approached one another. During continuous high K⁺ dialysis, successive ΔV_o waves episodes evolved according to a consistent pattern: while phase I remained unchanged, phase II increased in amplitude and duration with each episode. Eventually, a depressed state developed which lasted for many minutes, termed here prolonged unstable spreading depression. During phase I, ΔV_o and extracellular K ([K⁺]_o) changes were correlated. During phase II, [K⁺]_o decreased even as ΔV_o continued to increase. During SD, [Ca²⁺]_o decreased to <0.01 mM. During phases I and II, both [Ca²⁺]_o and [Na⁺]_o remained low. the recoverries of [Ca²⁺]_o and [Na⁺]_o had an initial fast and a later much slower phase and took several minutes longer than the recoveries of [K⁺]_o and ΔV_o. Depth profiles of ΔV_o and Δ[K⁺]_o revealed strikingly steep gradients early and late during a wave; but voltage and ion gradients were not precisely correlated either in time or in space. We conclude that ΔV_o of phases I and II are generated by different processes. Membrane ion currents cannot fully explain the ΔV_o responses. The possible contributions by ion diffusion and by active ion transport are discussed. The extremely low level to which [Ca²⁺]_o sinks during SD, and its two-phase recovery, indicate intracellular sequestration or binding of substantial amounts of Ca²⁺ ions. The residual deficit of [Ca²⁺_o following recovery of SP shifts may account for the persistent depression of synaptic transmission after repolarization of neurons.     

IL-1beta 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.