NMDA induces NO release from primary cell cultures of human fetal cerebral cortex

In order to investigate the role of insulin-like growth factors (IGFs) in human ovulation, we evaluated the concentrations of IGF-binding proteins (IGFBPs) in human follicular fluid (FF). The concentrations of IGFBP-1 in the FFs of 15 women undergoing in vitro fertilization and embryo transfer were measured and related to those of 17beta-estradiol (E2), progesterone and androstenedione in the FFs. IGFBP-1 levels in the FFs were positively correlated with those of E2 and progesterone. No correlation was found between the IGFBP-1 and androstenedione levels in FFs. The concentrations of IGFBP-1 were significantly increased in the FFs which contained mature oocytes compared with those of immature oocytes, whereas IGFBP-3 in FFs tended to decrease as oocytes matured. It is suggested that IGFs may play important roles in human preovulatory processes, and that IGFBP-1 may be a valuable biochemical marker in the evaluation of oocytes.     

Acetylcholine release from the frontal cortex during exploratory activity

The activation of the cortical cholinergic system was investigated in 3- and 25-month-old male Wistar rats, by measuring by transversal microdialysis the changes in cortical extracellular acetylcholine (ACh) levels during the performance of simple spontaneous tasks involving exploratory activity and working memory. Two days after implantation of the microdialysis probe in the frontal cortex, object recognition was investigated by either moving the rats from the home cage to the arena containing the objects or keeping the rats in the arena and introducing the objects. Spontaneous alternation was investigated in a Y runway. Young rats discriminated between familiar and novel objects and alternated in the Y runway, while aged rats were unable to discriminate. Whenever rats were moved from the home cage to the arena, ACh release increased (+70-80%) during the exploratory activity. Handling per se had no effect on extracellular ACh levels. When young rats were left in the arena, introduction of the objects caused some exploratory activity and object recognition but no increase in ACh release. ACh release increased by about 300% during spontaneous alternation. In aging rats basal extracellular ACh levels and their increase after placement in the arena were less than half that in young rats. Our work demonstrates that a novel environment activates the cortical cholinergic system, which presumably is associated with arousal mechanisms and selective attentional functions. It also demonstrates that in aging rats the cortical cholinergic hypofunction is associated with a loss of non-spatial working memory.     

Effect of transient cerebral ischaemia on acetylcholine release in the gerbil hippocampus

To clarify the relationship between presynaptic cholinergic dysfunction and postsynaptic cell death in the hippocampus, extracellular levels of acetylcholine (ACh) were assayed and CA1 pyramidal cells were histologically investigated in gerbils which had undergone 2, 5 and 10 min ischaemia. It was found that the KCl- and atropine-induced release of ACh, an index of the functioning cholinergic system at the presynaptic terminals, was significantly lower in the ischaemic groups than in control groups. The hippocampal CA1 pyramidal cell area of the 5 and 10 min ischaemic animals was also significantly decreased, but the 2 min ischaemia caused no cell damage. These findings indicate that the presynaptic terminals of the cholinergic neurone are vulnerable to ischaemic insult and that cholinergic dysfunction precedes postsynaptic CA1 pyramidal cell death in the hippocampus.     

Muscarinic M1 receptor mediated inhibition of GABA release from rat cerebral cortex

Presynaptic modulation of [3H]GABA release was examined using rat cerebral cortical slices. In vitro addition of carbachol, a muscarinic receptor agonist, resulted in a significant suppression of the release of [3H]GABA evoked by high potassium (50 mM) stimulation in a dose dependent manner, while noradrenaline, isoproterenol, dopamine, 5-hydroxytryptamine, histamine and glutamic acid had no significant effect on the evoked release of [3H]GABA. This suppressive effect of carbachol was antagonized invariably by atropine. Furthermore, it was found that the suppressive action of carbachol could be antagonized by pirenzepine, a selective M1 muscarinic receptor antagonist, but not by AF-DX 116 and 4-DAMP, M2 and M3 receptor antagonists, respectively. These results suggest that the release of GABA from cerebral cortical GABA neurons may be modulated by presynaptic M1 muscarinic receptor.     

Exocytotic and nonexocytotic modes of glutamate release from cultured cerebellar granule cells during chemical ischaemia

We previously reported the generation of a library of hydrophobic oxazole-based small molecules designed as inhibitors of phosphatases involved in cellular signaling and cell cycle control. One member of the targeted array library, 4-(benzyl-(2-[(2, 5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylami no butyric acid (SC-alphaalphadelta9), inhibited cell growth in the G0/G1 phase of the cell cycle. To investigate potential mechanisms for SC-alphaalphadelta9 antiproliferative activity, we have used mouse embryonic fibroblasts transformed with simian virus 40 large T antigen mouse embryonic fibroblasts as a model system for a malignant phenotype that depends on overexpression of cell cycle regulators and autocrine stimulation by insulin-like growth factor-1. Structure-activity relationship studies with SC-alphaalphadelta9 and four library congeners demonstrated that antiproliferative activity was not a result of overall hydrophobicity. Rather, SC-alphaalphadelta9 decreased insulin-like growth factor-1 receptor tyrosine phosphorylation, receptor expression, mitogen-activated protein kinase activation and levels of the cyclin-dependent kinase Cdc2. Less toxic congeners only partially affected receptor expression, receptor tyrosine phosphorylation and Cdc2 levels. Thus SC-alphaalphadelta9, which is structurally distinct from other known small molecules that decrease intracellular Cdc2 levels, has profound effects on intracellular signaling. Furthermore, SC-alphaalphadelta9, but not vanadate or okadaic acid, selectively inhibited the growth of simian virus 40 large T antigen mouse embryonic fibroblasts compared to the parental cells. These results suggest that overexpression of Cdc2 and increased dependence on insulin-like growth factor-1 autocrine stimulation are responsible for the increased sensitivity of simian virus 40 large T antigen mouse embryonic fibroblasts to SC-alphaalphadelta9. The SC-alphaalphadelta9 pharmacophore could be a useful platform for the development of novel antisignaling agents.     

Microsphere embolism-induced changes in noradrenaline release in the cerebral cortex in rats

A human cell line LMS6-93 has been established from a leiomyosarcoma (LMS). Characteristics for ultrastructure, growth characteristics, cell cycle distribution, karyotype, protein expression detected by immunohistochemistry (IHC), p53 mutational status and liposomal transfection behaviour were studied and determined. The primary tumor was clearly positive for á-smooth muscle type actin and desmin in moderately differentiated areas and indicated a loss of myogenic differentiation in other regions and therefore was classified as a poorly differentiated LMS. The cell line LMS6-93 contains mainly polymorphic spindle shaped or polygonal tumor cells which possess the characteristics of primitive mesenchymal cells, based on their morphology and positive reaction with an antibody to vimentin. IHC staining for S100, synaptophysin A, NSE, neurofilament proteins and cytokeratins were negative. Cytogenetic analysis revealed in the cell line diploid karyotypes comparatively close to several structural and numerical aberrations for chromosomes 2, 5, 6, 9, 10, 12, 14, 17, 18, 20, 22, and Y. IHC positivity was found for the tumor suppressor protein Rb and the oncogene product MDM2. In a p53 mutational analysis a 1 bp insertional mutation in exon 6 (G insertion in codon 215) was detected and confirmed in the original primary tumor. The other p53 allele appears to be wild-type as indicated in Western hybridization. Using different cationic lipid formulations complexed with a reporter expression vector (GFP) successful transfection into LMS6-93 cells was observed. The highest transfection rates (20-30% GFP expression in the viable cell population) were obtained with lipofectin. These results suggest that LMS6-93 functions as a good in vitro model for transfection studies on an LMS cell line carrying a heterozygous p53-frameshift mutation.     

Lithium regulation of protein phosphorylation in rat cerebral cortex slices in vitro

Histamine type 2 receptor antagonists (H2RAs) have been found to alter gastric motility. The aims of this study were to determine if H2RAs affect antral contractility in vitro and the mechanism of this effect. Guinea pig antral muscle strips were pinned in an organ bath after removing the mucosa, and circular muscle tension was measured using an isometric force transducer. Gastric myocytes were isolated from guinea pig stomach using collagenase digestion, and cell lengths were measured using an image analysis system. In muscle strips, ranitidine and nizatidine increased the amplitude of spontaneous phasic antral contractions in a concentration-dependent fashion with threshold concentrations of 5 microM. The order of potency for the H2RAs was ranitidine = nizatidine > cimetidine > famotidine. The contractile effects of ranitidine and nizatidine were reduced, but not abolished, by tetrodotoxin and omega-conotoxin GVIA and nearly abolished by atropine. In isolated cells, ranitidine and nizatidine, but not famotidine or cimetidine, induced concentration-dependent cell shortening, with maximal shortening at 10 microM. These contractile effects of ranitidine and nizatidine in isolated cells were inhibited by atropine. Ranitidine and nizatidine increase antral contractility; this effect appears to be mediated by an interaction between ranitidine and nizatidine on cholinergic pathways with both direct effects on smooth muscle cholinergic receptors and indirect effects by increasing cholinergic neurotransmission.     

Glutamate release in human cerebral cortex and its modulation by 5-hydroxytryptamine acting at h 5-HT1D receptors

The action of amiloride on two different components of airway electrical potential difference, one stable (PD) and the other transiently hyperpolarizable after gentle mechanical stimulation (dPD), was studied by means of isolated tracheal wall mounted in a modified Ussing apparatus. The immediate effect of amiloride, when added to the bathing and stimulating medium, was a partial depolarization, and a diminution or elimination of dPD. After at least 60 min incubation of the tracheal wall with amiloride in the presence of the drug in the bathing and stimulating fluid, both the PD, and dPD were no different from the control. This difference between the immediate and the sustained action of amiloride on airway PD suggests that there is an efficient regulatory system in the airway walls which stabilizes the transepithelial PD. The usefulness of amiloride as a drug for cystic fibrosis patients is discussed in the light of these findings.     

Measurement of nitric oxide in the rat cerebral cortex during hypercapnoea

Changes in nitric oxide (NO) concentration and cerebral blood flow (CBF) in the parietal cortex during hypercapnoea were investigated in anaesthetized rats, using a NO-selective electrode and laser Doppler flowmetry. When hypercapnoea was induced by inhalation of 5% CO2 for 10 min, both the NO concentration and CBF increased. After administration of 7-nitroindazole, a neuronal NO synthase (nNOS) inhibitor, both the basal NO and CBF decreased, and responses to hypercapnoea were also significantly suppressed by 70.1% and 73.2%, respectively, compared with the control state. These results suggest that NO derived from nNOS is involved not only in maintaining resting cerebral circulation but also in regulating CBF response during hypercapnoea.     

Effects of ropivacaine on eicosanoid release from human granulocytes and endothelial cells in vitro

OBJECTIVE: To examine the effects of ropivacaine, currently being investigated for treatment of ulcerative colitis, on the release of arachidonic acid metabolites. MATERIAL: Human granulocytes and endothelial cells. TREATMENT: Ropivacaine, lidocaine, hydrocortisone, 5-aminosalicylic acid or acetylsalicylic acid (10-1000 microM). METHODS: Leukotriene B4, 5-hydroxyeicosatetraenoic acid, 6-keto PGF1 alpha and 15-hydroxyeicosatetraenoic acid were measured using immuno assays. Wilcoxon signed rank test was used for statistical calculations. RESULTS: Ropivacaine dose-dependently inhibited zymosan-induced release of leukotriene B4 and 5-hydroxyeicosatetraenoic acid whereas the release after ionophore stimulation was not affected. Ropivacaine was more potent than 5-aminosalicylic acid but less potent compared to hydrocortisone. Ropivacaine had only a weak inhibitory effect on the release of 15-hydroxyeicosatetraenoic acid from zymosan- or ionophore-stimulated cells. In contrast to hydrocortisone and 5-aminosalicylic acid, ropivacaine only weakly affected the release of 6-keto PGF1 alpha after stimulation with thrombin. CONCLUSIONS: The inhibited release of 5-lipoxygenase products may account for some of the anti-inflammatory effects of ropivacaine seen in the treatment of ulcerative colitis.     

Action of L-acetylcarnitine on different cerebral mitochondrial populations from cerebral cortex

The maximum rate (Vmax) of some mitochondrial enzymatic activities related to the energy transduction (citrate synthase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, cytochrome oxidase) and amino acid metabolism (glutamate dehydrogenase, glutamate-pyruvate-transaminase, glutamate-oxaloacetate-transaminase) was evaluated in non-synaptic (free) and intra-synaptic mitochondria from rat brain cerebral cortex. Three types of mitochondria were isolated from rats subjected to i.p. treatment with L-acetylcarnitine at two different doses (30 and 60 mg.kg-1, 28 days, 5 days/week). In control (vehicle-treated) animals, enzyme activities are differently expressed in non-synaptic mitochondria respect to intra-synaptic "light" and "heavy" ones. In fact, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glutamate-pyruvate-transaminase and glutamate-oxaloacetate-transaminase are lower, while citrate synthase, cytochrome oxidase and glutamate dehydrogenase are higher in intra-synaptic mitochondria than in non-synaptic ones. This confirms that in various types of brain mitochondria a different metabolic machinery exists, due to their location in vivo. Treatment with L-acetylcarnitine decreased citrate synthase and glutamate dehydrogenase activities, while increased cytochrome oxidase and alpha-ketoglutarate dehydrogenase activities only in intra-synaptic mitochondria. Therefore in vivo administration of L-acetylcarnitine mainly affects some specific enzyme activities, suggesting a specific molecular trigger mode of action and only of the intra-synaptic mitochondria, suggesting a specific subcellular trigger site of action.     

Induction of tyrosine hydroxylase gene expression in human foetal cerebral cortex

INTRODUCTION: The poisoning severity score is a four-point severity-classification scale, developed by the International Programme on Chemical Safety, the Commission of the European Union, and the European Association of Poison Centres and Clinical Toxicologists (IPCS/EC/EAPCCT), for the retrospective assessment of cases of poisoning reported to poisons information centers. OBJECTIVES: The aims of this study were to test the validity of using the poisoning severity score obtained at initial referral to assess clinical severity and the likelihood of subsequent deterioration, to select cases for follow-up, and also to determine the need for referral to a clinical toxicologist. METHODS: The poisoning severity score was determined at the time of initial inquiry. Follow-up was then undertaken until either the patient was discharged from medical attention or died. A second poisoning severity score was then calculated taking note of the most severe features present after the initial inquiry. RESULTS: Of 718 consecutive telephone inquiries, 397 were given an initial poisoning severity score of 0 (no signs and symptoms), 225 a score of 1 (mild symptoms), 71 a score of 2 (moderate symptoms), and 25 a score of 3 (severe symptoms). Follow-up data are available only in 638 cases because the patient or referring doctor could not be traced in 80 instances. Of the 638 cases, 41 deteriorated; 31 of these were graded initially as poisoning severity score 0, four as 1, and six as 2. Five patients died (two with an initial score of 2 and three with an initial score of 3). CONCLUSIONS: This study demonstrates that it is useful to score telephone inquiries to a poisons information service at initial referral with the poisoning severity score. First, the poisoning severity score is helpful in assessing accurately the clinical severity and the likelihood of further deterioration. Second, the poisoning severity score is useful in determining the need for referral of the inquiry to a clinical toxicologist, thus ensuring that more serious and complicated cases of poisoning receive expert medical advice on management. Third, the poisoning severity score is helpful in selecting those cases which warrant follow-up for medical and epidemiological reasons.     

Nitric oxide regulation of corticotropin-releasing hormone release from the human perfused placenta in vitro

We have investigated the regulatory role of nitric oxide (NO) in corticotropin-releasing hormone (CRH) release from the human perfused placental lobule in vitro. The effects of the NO donor sodium nitroprusside, the NO synthase inhibitor N omega-nitro-L-arginine, and the NO substrate L-arginine on human (h) placental CRH secretion have been studied. Single lobules of term placentae were bilaterally perfused with Krebs solution (5 mL/min; 95% O2-5% CO2; 37 C; pH 7.3). Fetal and maternal perfusates were collected at 4 C every 30 min for 3 h. CRH immunoreactivity (CRH-IR) in perfusates was measured by RIA using the 41-residue synthetic CRH as standard, 125I-labeled Tyr-hCRH as tracer, and a rabbit anti-CRH antibody Y2BO. The sensitivity of the assay was 0.13 pmol/L. Under basal conditions, human perfused placentae in vitro continuously secreted CRH-IR, which diluted in parallel to a synthetic hCRH-(1-41) standard curve. Size-exclusion chromatography of placental perfusates using a Sephadex G-50 column indicated that placental CRH-IR predominately coeluted with hCRH-(1-41) standard. Basal maternal perfusate CRH-IR levels (27 +/- 4 pmol/L) released from perfused placental lobules were nearly 10-fold greater than fetal perfusate CRH-IR levels (3.4 +/- 0.7 pmol/L; P < 0.05). Infusion of sodium nitroprusside (30-100 mumol/L) into the maternal and fetal placental circulations inhibited CRH-IR release into maternal perfusate in a concentration-dependent manner, but did not inhibit CRH-IR release into the fetal perfusate. N omega-nitro-L-arginine (100 mumol/L) increased placental CRH-IR secretion into fetal perfusate, and this effect was reversed by the infusion of L-arginine (100 mumol/L), which also reduced release below basal levels. In contrast, maternal perfusate CRH-IR levels were not affected by N omega-nitro-L-arginine or L-arginine. These results indicate that the human perfused placenta in vitro releases a substance of similar mol wt and hCRH-IR. Moreover, modulators of the NO signaling pathway differentially affect placental secretion of CRH-IR into the maternal and fetal perfusates. These data are consistent with the involvement of NO in the regulation of placental CRH release during pregnancy.     

Inhibition by anion channel blockers of ischemia-evoked release of excitotoxic and other amino acids from rat cerebral cortex

In febrile neutropenic patients with high-grade hematologic malignancies, empirical antimicrobial intervention is mandatory. Large randomized clinical trials have elucidated the benefit of broad-spectrum beta lactam antibiotics used as single drugs or in combination with aminoglycosides in order to provide activity against gram-negative aerobes as well as against streptococci and Staphylococcus aureus. As a result, infection-related mortality was reduced to less than 10% also in patients undergoing intensified remission induction or consolidation therapy for acute leukemias. Distinct subgroups of patients have been identified who need an empirical modification of antimicrobial treatment i.e., patients with catheter-related infections, patients with pulmonary infiltrates, and patients with unexplained fever not responding to first-line antibiotics. In two consecutive, prospectively randomized trials conducted by the Paul Ehrlich Society it was demonstrated that empirical antifungal therapy is beneficial for second-line treatment in patients with persistent unexplained fever and should be part of the first-line approach in patients with lung infiltrates. The empirical addition of glycopeptides, however, should be restricted to patients with catheter-related infections due to coagulase-negative staphylococci.     

Developmental changes in intracellular calcium regulation in rat cerebral cortex during hypoxia

During the first weeks of life, injury to the central nervous system caused by brief periods of oxygen deprivation greatly increases. To investigate possible causes for this change, the effects of hypoxia or application of the excitatory neurotransmitter glutamate on intracellular calcium ([Ca2+]i) and ATP were studied in rat cerebrocortical brain slices. [Ca2+]i was measured fluorometrically with the indicator Fura-2. Hypoxia (95% N2/5% CO2) or 100 microM sodium cyanide produced gradual elevations in [Ca2+]i and ATP depletion in slices from rats < 2 weeks old, but rapid changes in older rats. After 20 min, [Ca2+]i in adult slices exposed to cyanide was 1,980 +/- 310 nM; in day 1-14 animals, it was 796 +/- 181 nM (p < 0.05). Combination of cyanide and a glycolytic inhibitor (iodoacetate) rapidly elevated [Ca2+]i and depleted ATP in all age groups. Energy utilization during anoxia, assessed by measuring ATP fall in cyanide/iodoacetate-treated brain slices, increased with age. Elevations in [Ca2+]i caused by application of 500 microM glutamate increased 240% from days 1-2 to day 28, but ATP loss caused by glutamate did not change with age. The N-methyl-D-aspartate antagonist MK-801 delayed calcium entry during the initial 5-7 min of hypoxia or cyanide in rats < 2 weeks old. We conclude that anaerobic ATP production, conservation of energy by reduced ATP consumption, and reduced sensitivity to glutamate contribute to delaying elevation in [Ca2+]i in neonatal rat brain during hypoxia.     

Effect of adenosine receptor agonists on spontaneous and K(+)-evoked acetylcholine release from the in vivo rat cerebral cortex

Repeated applications of elevated K+ (100 mM) in artificial cerebrospinal fluid (CSF) were used to evoke an efflux of acetylcholine (ACh) from the in vivo rat cerebral cortex using a cortical cup technique. Elevated K+ reproducibly increased the levels of ACh in cup superfusates by a factor of 3-5-fold above basal levels (27.2 +/- 9.7 nM). The adenosine A1 receptor agonist N6-cyclopentyl adenosine (CPA), at a concentration of 10(-8) M, depressed basal, but not K(+)-evoked ACh efflux. 10(-6) M CPA increased basal, but did not alter K(+)-evoked, ACh efflux. The A2 selective agonist CGS 21680 did not alter either basal, or K(+)-evoked, ACh efflux. The inhibitory effects of 10(-8) M CPA on ACh efflux would be consistent with the presence of adenosine A1 receptors on cholinergic nerve terminals in the cerebral cortex. At a higher concentration (10(-6) M) CPA elevated basal release, possibly by activating low affinity A2 receptors. The failure of CGS 21680 (10(-8) M) to alter basal ACh release suggests an absence of high affinity A2 receptors in these terminals. Whereas elevated K+ in cup superfusates consistently enhanced ACh efflux from the cerebral cortex, this increase was not affected by either CPA or CGS 21680. High K(+)-evoked release of cerebral cortical ACh may be an inappropriate model for the study of adenosine's actions on neurotransmitter release.