The coupling of muscarinic receptors to hydrolysis of inositol lipids in human neuroblastoma SH-SY5Y cells

Carbachol (CCh)-stimulated hydrolysis of inositol lipids in human neuroblastoma SH-SY5Y cells was systematically characterized in parallel with the carbachol effects on cAMP formation. Carbachol concentration-dependently induced the hydrolysis of inositol lipids and formation of [3H]IP3, [3H]IP2 and [3H]IP1 in these cells labeled with [3H]inositol. The maximal amount of [3H]IP1 accumulated in the presence of 10 mM LiCl was about 50-fold above the basal level. The EC50 value of CCh was 14 microM. The muscarinic antagonists atropine, pirenzepine and 11-[[2-(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido [2,3-b] (1,4)-benzodiazepine-6-one (AF-DX 116) competitively inhibited CCh-induced [3H]IP1 accumulation. The functional inhibition constants (converted from the pA2 values) were 0.24, 8.1 and 470 nM, respectively. These values are in good agreement with the inhibition constants of these drugs from antagonist/[3H]pirenzepine studies using intact cells. Forskolin, adenosine and PGE1 stimulated cAMP formation in this cell line. Morphine decreased PGE1-induced cAMP formation as well as the basal cAMP formation. However, CCh did not stimulate or inhibit the basal cAMP formation. Also, CCh did not have any effects on the adenosine and PGE1-induced cAMP formation in these cells. These data suggest that muscarinic M1 receptors are coupled to the hydrolysis of inositol lipids and not to the adenylate cyclase system in human neuroblastoma SH-SY5Y cells.     

Influence of the acute stress on agonist-stimulated phosphoinositide hydrolysis in the rat cerebral cortex.

1. The present study was carried out in order to elucidate the influence of the acute stress on alpha 1-adrenergic, serotonin-2 (5-HT2) and muscarinic cholinergic (M-Ach) receptors-mediated phosphoinositide (PI) hydrolysis in rat cerebral cortex slices. 2. In rat cerebral cortex slices, noradrenaline (NA), serotonin (5-HT) and carbachol stimulated [3H]inositol-monophosphate (IP1) accumulation in a concentration-dependent manner. 3. The forced swimming test (FST) for 15 min induced a significant reduction of 5-HT-stimulated [3H]IP1 accumulation, but this stress situation did not produce a significant alteration of NA- and carbachol-stimulated [3H]IP1 accumulation. 4. The FST for 15 min did not affect the density and affinity of alpha 1-adrenergic, 5-HT2 and M-Ach receptors. 5. In a mild acute stress situation, the intracellular signal transduction mediated by 5-HT was promptly inhibited as compared to the signal transduction mediated by NA or carbachol. This inhibition may be induced by an acute uncoupling of 5-HT2 receptor-mediated intracellular signal transduction.     

Changes in pre- and postsynaptic components of noradrenergic transmission in hippocampal kindling in rats.

We investigated whether modifications in noradrenergic neurotransmission occurred during the development of hippocampal kindling in rats. We measured the release of [3H]norepinephrine (NE) induced by field-electrical stimulation, NE-stimulation of inositol phosphates [( 3H]IP) accumulation in the presence of LiCl and isoproterenol-induced accumulation of cAMP in hippocampal slices taken from rats electrically kindled at stages 2 and 5 in the dorsal hippocampus. One week after the last of at least 3 consecutive stage 5 seizures or 48 h after the last stage 2 stimulation, 2 min electrical stimulation of stratum pyramidale CA1-CA3 or dentate gyrus (DG) slices from kindled and contralateral hippocampi induced frequency-dependent NE release (respectively 2, 4 and 8 times spontaneous release measured at 2, 5 and 10 Hz) which did not significantly differ from that observed in shams (implanted with electrodes but not stimulated). Basal release of NE from kindled and sham-treated rats did not differ either. Isoproterenol induced a dose-dependent increase above basal cAMP concentration ranging from 40% at 0.01 microM to 180% at 10 microM (P less than 0.01, Dunnett's test) which did not differ between stages 2 and 5 and sham-hippocampi. NE (1-1000 microM) induced a dose-dependent, prazosin-sensitive increase in [3H]IP accumulation in the hippocampal slices. A significantly higher increase was found at stages 2 (P less than 0.05, Tukey's test) and 5 (P less than 0.05 and P less than 0.01, Tukey's test) compared to shams at all doses studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suramin disrupts insulin-like growth factor-II (IGF-II) mediated autocrine growth in human SH-SY5Y neuroblastoma cells

Suramin, traditionally used in the treatment of trypanosomiasis, is under investigation in the treatment of cancer. One side effect that limits its use is the onset of a sensorimotor polyneuropathy. In order to investigate the mechanism by which suramin induces polyneuropathy, we examined its effects on SH-SY5Y human neuroblastoma cells, an in vitro model of neuronal growth and differentiation. Addition of 50–400 μg/ml suramin to SH-SY5Y cells grown in 0.6% CS inhibited [³H]thymidine ([³H]TdR) incorporation and cell growth. Upon removal of suramin, [³H]TdR incorporation increased, demonstrating that levels of suramin used were cytostatic and not cytotoxic. Analysis of suramin-treated SH-SY5Y cells by flow cytometry revealed growth arrest in the G₁/G₀ phase of the cell cycle. IGF-II-induced SH-SY5Y growth is mediated by the type I IGF receptor (IGF-IR). Therefore, we examined its effect on IGF-IR tyrosine phosphorylation. Suramin prevented IGF-II-stimulated IGF-IR tyrosine phosphorylation. These results indicate that in SH-SY5Y cells, suramin acts as a cytostatic agent and can block IGF-II-dependent cell growth by preventing IGF-IR activation. Thus, suramin toxicity in the peripheral nervous system may be due, in part, to preventing IGFs and other growth factors from activating their receptors.     

Interferon-γ inhibits DNA synthesis and insulin-like growth factor-II expression in human neuroblastoma cells

Interferon-γ (IFN-γ) is known to be an antiproliferative, differentiation agent in many cell types, including neuroblastoma. In this study, we determined the effects of IFN-γ on cellular growth and expression of insulin-like growth factor II (IGF-II) and IGF receptors in the human neuroblastoma cell line SH-SY5Y. Incubation of SH-SY5Y cells in IFN-γ (20–100 U/ml) induced the formation of long neuritic processes. IFN-γ treatment also induced decreases in [³H]TdR incorporation, as well as serum-dependent changes in cell number. Treatment with IFN-γ reduced cell number 33% in the presence of serum but had no effect on cell number in the absence of serum. IGF-II mRNA content was 60% inhibited by IFN-γ, and was not serum dependent. The concentration of immunoreactive IGF-II in SH-SY5Y conditioned medium was also reduced in the presence of IFN-γ, to less than half of control levels. In contrast, type I IGF receptor mRNA content was increased more than three-fold after treatment with IFN-γ and serum. Co-incubation in IFN-γ (20–100 U/ml) and IGF-II on (3–10 nM) prevented the inhibitory effects of IFN-γ on [³H]TdR ncorporation in serum-free media. Our results suggest that IFN-γ may inhibit DNA synthesis and cell growth by interfering with an IGF-II/type I IGF receptor autocrine growth or survival mechanism.     

Insulin stimulates membrane phospholipid metabolism by enhancing endogenous alpha 1-adrenergic activity in the rat hippocampus.

Insulin stimulates membrane phospholipid metabolism and activates protein kinase C (PKC) in its peripheral target tissues. Additionally, insulin can stimulate PKC activity in cultured fetal chick neurons. In the present study, we tested whether insulin can stimulate membrane phospholipid metabolism in the rat hippocampus, a CNS region in which insulin has been reported to stimulate the phosphorylation of a PKC substrate protein and to suppress spontaneous electrical activity of pyramidal cells. Concentrations of 1, 10 and 100 nM insulin significantly stimulated the accumulation of [3H]inositol phosphate ([3H]IP1) and [3H]IP2 in hippocampal slices labelled with [3H]myoinositol. Significant (P less than 0.05) increases of hippocampal diacylglycerol (a product of phosphoinositol hydrolysis) content were observed at 1, 5 or 10 min of incubation with 50 or 100 nM insulin. Addition of tetrodotoxin resulted in a suppression of insulin stimulation of [3H]IP1 release, suggesting that insulin effects may be indirect and mediated via release of an endogenous neuronal transmitter within the hippocampus. Norepinephrine has been shown to both stimulate PI turnover and suppress the spontaneous electrical activity of pyramidal cells via alpha 1-adrenergic receptors. Therefore, we tested whether the effects of insulin were mediated by norepinephrine. We measured [3H]IP1 release in the presence or absence of the alpha 1-adrenergic antagonists prazobind and prazosin. These compounds blocked insulin stimulation of IP1 accumulation, suggesting that the action of insulin to stimulate PI turnover is secondary to enhancement of endogenous noradrenergic activity within the hippocampus.     

Clostridium botulinum neurotoxins act with a wide range of potencies on SH-SY5Y human neuroblastoma cells

We have described, in undifferentiated SH-SY5Y human neuroblastoma cells, the relative potency of Clostridium botulinum neurotoxin (BoNT) serotypes A-F Sensitivity of stimulated [3H]-noradrenaline ([3H]-NA) release to the toxins had a rank order of potency of: C > D > A > B > F after 3 days exposure. The difference between the most potent (BoNT/C: IC50 0.54 nM) and the least (BoNT/F: IC50 > 300 nM) was approximately 1,000-fold. Though fluid phase endocytosis may have been the mechanism of entry for low potency toxins the far higher potency of BoNT/C would suggest receptor-driven entry. Potency was not a reflection of the dependence of the release mechanism on a particular SNARE since the substrate specificities were mixed throughout the potency order. This indicated that the toxins differed in their efficiency of binding/endocytosis or enzymatic activity inside the cell. The serotypes that cleaved vesicle-associated membrane protein (VAMP) isoforms (BoNT/B, D and F) did not fully inhibit [3H]-NA release. Cleavage of the appropriate substrate proteins was observed for all serotypes. SNAP-25 cleavage by BoNT/A was shown to be a dose-dependent and correlated closely with reduction of release, supporting proteolysis as the mechanism by which toxin inhibited secretion. Comparison of the SH-SY5Y cell line sensitivity to BoNT/A with glycine releasing rat primary spinal cord neuron cultures, revealed a massive difference in potency; the primary cultures being approximately 200,000-fold more sensitive. The demonstration, using BoNTs, of the crucial role of SNAP-25, VAMP and syntaxin in SH-SY5Y cells suggests the use of this neuroblastoma as a model in the study of these proteins in neurotransmitter release.     

Muscarinic receptors, phosphoinositide metabolism and intracellular calcium in neuronal cells.

1. We have utilised SH-SY5Y human neuroblastoma cells and primary cultures of rat neonatal cerebellar granule cells, both expressing M3 muscarinic receptors, to examine agonist driven polyphosphoinositide hydrolysis and alterations in intracellular calcium. 2. Stimulation of SH-SY5Y cells leads to a biphasic increase in intracellular calcium, the initial peak being due to the release of calcium from an intracellular store and the second maintained phase being due to calcium entry across the plasma membrane. The channel involved does not appear to be voltage sensitive, to involve a pertussis toxin sensitive G protein, or be opened by inositol polyphosphates. 3. Muscarinic receptor stimulation also leads to increased inositol polyphosphate formation in SH-SY5Y cells. Ins(1,4,5)P3 mass formation was biphasic in profile whereas Ins(1,3,4,5)P4 mass formation was slower and monophasic in profile. These data are consistent with substantial activity of 5-phosphatase (dephosphorylating Ins(1,4,5)P3 to Ins(1,4)P2) and 3-kinase (phosphorylating Ins(1,4,5)P3 to Ins(1,3,4,5)P4) in SH-SY5Y cells. 4. In order to better understand the role of Ins(1,4,5)P3 and its metabolites in calcium homeostasis we have examined the ability of a variety of natural and synthetic analogues to release intracellular sequestered calcium. The Ins(1,4,5)P3 calcium mobilizing receptor displays a remarkable degree of stereo- and positional selectivity with the most potent agonist to date being Ins(1,4,5)P3 (EC50 = 0.09 microM). 5. As an alternative to the continuous SH-SY5Y neuroblastoma (tumour derived) cell line we have used the primary cultured cerebellar granule cell. These cells also display a biphasic increase in Ins(1,4,5)P3 mass and a subsequent release of intracellular stored calcium. In our hands carbachol appears to increase calcium influx, a response which is only visible in the absence of magnesium.     

Binding of [3H]prazosin and [3H]p-aminoclonidine to alpha-adrenoceptors in rat spinal cord

alpha-Adrenoceptors in spinal cord appear to play a role in a number of physiologic processes including the control of blood pressure, pain and motor function. In order to evaluate more clearly these potential roles, the characteristics of binding of [3H]prazosin ([3H]PRZ) to spinal alpha 1 adrenoceptors and [3H]p-aminoclonidine ([3H]PAC) to spinal alpha 2 adrenoceptors were determined. Binding of each ligand to their respective adrenoceptors was saturable and Scatchard analysis revealed binding of each to a single class of adrenoceptors with characteristics of [3H]PRZ binding of Bmax = 78 fmol/mg protein and Kd = 0.75 nM and [3H]PAC binding Bmax = 70 fmol/mg protein and Kd = 1.39 nM. Whereas [3H]PRZ specific binding (Bmax) was unaltered by guanine nucleotides. [3H]PAC binding was increased with addition of 10 microM guanosine triphosphate (GTP) (P less than 0.05) and decreased with either 50 microM GTP or guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] (P less than 0.01). Competition for specific [3H]PRZ and [3H]PAC binding by various alpha 1 and alpha 2 adrenoceptor agonists and antagonists of known pharmacologic activity revealed that [3H]PRZ defines alpha 1 adrenoceptors (Ki = 2.1 nM for prazosin vs 4300 nM for yohimbine) and [3H]PAC defines alpha 2 adrenoceptors (Ki = 1.06 nM for yohimbine vs 15480 nM for prazosin). Regional spinal cord studies demonstrated that dorsal spinal cord in the lumbar region contains the highest density of both [3H]PRZ (Bmax = 93 +/- 14 fmol/mg protein) and [3H]PAC (Bmax = 101 +/- 6 fmol/mg protein) binding. In contrast, lowest binding was evident in thoracic cord with equal levels in both dorsal and ventral regions (Bmax = 44-48 fmol/mg protein). The regional distribution of both alpha 1 and alpha 2 adrenoceptors in spinal cord compares to the localization previously classified functionally utilizing various pharmacological agonists and antagonists at norepinephrine receptors.     

Pleiotropic effects of lysophosphatidic acid on striatal astrocytes.

Lysophosphatidic acid (LPA) is a potent lipid mediator that is likely involved in diverse functions in the brain. Several recent studies have suggested that astrocytes are important target cells for LPA. In the present study, we have identified the signal transduction pathways activated following LPA stimulation in mouse striatal astrocytes in primary culture. In cells prelabeled with myo-[3H]inositol, LPA stimulated the formation of [3H]inositol phosphates (EC50 = 0.7 microM). This effect was reproduced neither by other lysophospholipids nor by phosphatidic acid. Astrocyte pretreatment with pertussis toxin partially abolished this LPA response indicating the involvement of a Gi/Go protein. In [3H]adenine-prelabeled cells, LPA strongly inhibited the formation of [3H]cyclic AMP induced by forskolin (EC(50) = 0.3 microM) and by isoproterenol and PACAP-38. These inhibitory effects were strongly reduced by pertussis toxin treatment. Although with a lesser potency (EC50 = 5 microM), LPA also stimulated the release of [3H]arachidonic acid from [3H]arachidonic acid-prelabeled astrocytes. This latter effect was totally inhibited by mepacrine, did not involve a pertussis toxin-sensitive G protein, and was highly dependent on external calcium. LPA also stimulated the activity of both extracellular signal-regulated kinases (Erk) Erk1 and Erk2 by a mechanism involving a Gi/Go protein. Surprisingly, in contrast to that observed in fibroblasts, LPA was totally ineffective in stimulating DNA synthesis. These results provide additional evidence in favor of an important physiological role of LPA in the astrocytic functions.     

Nicotinic acetylcholine receptor expression on B-lymphoblasts of healthy versus schizophrenic subjects stratified for smoking: [<Superscript>3</Superscript>H]-nicotine binding is decreased in schizophrenia and correlates with negative symptoms

Heavy smoking and schizophrenia are diversely associated with nicotinic acetylcholine receptor expression, as was shown for brain and lymphocytes. Most studies so far have not systematically differentiated between schizophrenia smokers and non-smokers and were confined either to in vivo or post-mortem study approaches. In order to avoid variable in vivo influences or post-mortem bias, we used stably transformed B-lymphoblast cultures derived from healthy and schizophrenia subjects stratified for smoking versus non-smoking in order to differentiate these clinical conditions with regard to nicotinic acetylcholine receptor expression and regulation. Receptor quantities were measured using [(3)H]-nicotine and [(3)H]-epibatidine binding. At baseline, [(3)H]-nicotine binding was not statistically different between healthy smokers and never-smokers (1.59 ± 0.73 vs. 1.26 ± 0.91 fmol/10(6) cells), while it was reduced in schizophrenia smokers compared to healthy smokers (1.05 ± 0.69 fmol vs. 1.44 ± 0.84/10(6) cells, P = 0.01). In schizophrenia, baseline [(3)H]-nicotine correlated inversely with higher PANSS negative subscale scores. After long-term nicotine incubation (1 μM), [3H]-nicotine binding increased in the group of schizophrenia smokers only (from 1.05 ± 0.69 to 1.54 ± 0.77 fmol/106 cells, P = 0.013), while [(3)H]-epibatidine binding decreased in this group (4.52 ± 1.52 to 3.82 ± 1.38 fmol/10(6) cells, P = 0.038). Our data are in further support of a decrease of nicotinic acetylcholine receptor expression in schizophrenia linked to negative psychotic symptoms, which may be counter-regulated by nicotine exposure.     

Tacrine interacts with different sites on nicotinic receptor subtypes in SH-SY5Y neuroblastoma and M10 cells

The effect of chronic treatment with the cholinesterase inhibitor tacrine on nicotinic receptor subtypes was investigated in human SH-SY5Y neuroblastoma cells and in a fibroblast cell line (M10 cells) stably transfected with alpha4beta2 nicotinic receptors. Tacrine significantly increased the number of nicotinic receptors in SH-SY5Y cells, in a concentration dependent manner (10(-9) to 10(-4) M), when using [3H]epibatidine as labelled ligand. Chronic tacrine treatment of M10 cells significantly increased and decreased the number of alpha4beta2 nicotinic receptors in a concentration dependent manner (10(-9) to 5 x 10(-6) M and 2 x 10(-5) to 10(-4) M, respectively). The tacrine induced increase of nicotinic receptors in SH-SY5Y cells, was not blocked in the presence of the nicotinic antagonists tubocurarine or mecamylamine. A further increase in the number of nicotinic receptors was, however, observed in the presence of mecamylamine. This study demonstrates that the effect of tacrine on the number of nicotinic receptor subtypes is different in human SH-SY5Y neuroblastoma and M10 cells. The up-regulation of different nicotinic receptor subtypes obtained with tacrine might be achieved through interaction via different binding sites on the receptor, i.e. the acetylcholine binding site as well as an allosteric site.     

Oligodendrocytes express functional A1 adenosine receptors that stimulate cellular migration

A1 adenosine receptors (A1ARs) exert important effects in the central nervous system. However, the expression and function of A1ARs in oligodendrocyte precursor cells (OPCs) and oligodendrocytes (OLGs) is unclear. To address this issue, we examined A1AR expression during different stages of oligodendrocyte development. Radioreceptor studies showed that membranes prepared from OPCs and OLGs expressed high-affinity A1ARs with Kd values of 1.35 +/- 0.33 and 1.2 +/- 0.27 nM for [3H]CCPA, 1.17 +/- 0.24 and 1.4 +/- 0.34 nM for [3H]DPCPX, respectively. Bmax values were 64.31 +/- 6.14 and 75 +/- 6 fmol/mg protein for [3H]CCPA, and 153 +/- 12 and 205 +/- 17.8 fmol/mg protein for [3H]DPCPX, respectively. Activation of A1ARs using N6-cyclopentyladenosine (CPA) reduced both forskolin- and N-ethylcarboxyamidoadenosine (NECA)-stimulated cAMP accumulation, but did not affect basal cAMP levels. Activation of A1ARs by CPA stimulated OPC migration, but did not affect cell viability, proliferation, or differentiation. These results show that OPCs and OLGs express functional A1ARs that can stimulate the migration of OPCs.     

β-淀粉样肽对人SH-SY5Y细胞突触素、发动蛋白Ⅰ及衔接蛋白180表达的影响

目的 探讨β-淀粉样肽(Aβ)对人SH-SY5Y神经母细胞瘤细胞突触素(Syn)、发动蛋白Ⅰ (Dyn Ⅰ)及衔接蛋白180(AP180)表达的影响。 方法 分别应用0.01、0.1、1、2及5μmol/LAβ1-42处理SH-SY5Y细胞,对照组细胞不加任何处理。MTT法检测细胞增殖情况,Western blotting 检测0.5、1μmol/L Aβ1-42处理组及对照组细胞Syn、DynⅠ和AP180蛋白表达,RT-PCR检测1 μmol/L Aβ1-42处理组及对照组细胞Syn、DynⅠ和AP180 mRNA表达。 结果 0.1 μmol/L Aβ1-42处理细胞即可出现细胞增殖率下降,并呈剂量依赖性负性相关关系,与对照组比较差异均有统计学意义(P＜0.05); 0.5 μmol/L Aβ1-42处理细胞可见DynⅠ蛋白表达降低,与对照组比较差异有统计学意义(P＜0.05);1 μmol/L Aβ1-42处理细胞可见Syn、DynⅠ蛋白及mRNA表达均降低,与对照组比较差异均有统计学意义(P＜0.05);但未见AP180蛋白及mRNA表达水平发生改变。 结论 Aβ1-42可引起人SH-SY5Y细胞Syn和DynⅠ表达降低,该改变可能与AD发病中学习记忆能力减退有关。     

Phosphoinositide hydrolysis in vivo with group I metabotropic glutamate receptor agonists

The present report describes the effect of mGluR agonists and antagonists administration on phospholipase C activation by measuring accumulation of [3H] inositol monophosphates (IP) in rats pre-labeled with [3H]myo-inositol (i.c.v. 24 h pre-treatment). The levels of accumulated [3H]IP were then determined from clarified tissue homogenates using ion-exchange chromotography. Following lithium chloride treatment (10 mg/kg, s.c.), (R/S)-3, 5-dihydroxyphenylglycine (DHPG), a selective group I mGluR agonist was found to dose-dependently cause a maximal increase in the levels of [3H]IP at 0.3 to 3 micromol/8 microliter i.c.v. with lower doses resulting in less efficacious or no responses. This effect was temporal-dependent reaching a plateau at 2 h. The DHPG-induced increases in [3H]IP were most pronounced in the hippocampus where a 3- to 5-fold increase above vehicle was consistently found, but significant approximately 2-fold increases were also seen in the cerebellum, striatum and frontal cortex. The mixed group I and II agonist, (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (1S, 3R-t-ACPD), similarly resulted in dose-dependent increases in [3H]IP levels with doses of 1 to 3 micromol i.c.v. Furthermore, this effect was enantiomer specific since the less active 1R,3S-t-ACPD failed to alter phosphoinositol hydrolysis. Administration of the selective mGluR5 agonist (R/S)-2-chloro-5-hydroxyphenyl-glycine (CHPG) resulted in a dose-dependent increase in hippocampal but not cerebellar levels of [3H]IP, consistent with the receptor distribution of the two group I mGluRs. The Group II agonist LY354740 (1S,2S,5R,6S-2-aminobicycl[3.1.0]hexane-2,6-dicarboxylate monohydrate) and the group III agonist L-AP4 (L-(+)-2-amino-4-phosphonobutyric acid) failed to alter the levels of [3H]IP. LY341495 (2S-2-amino-2-(1S, 2S-2-carboxycycloprop-1-yl)-3-(xanth-9-yl)propanoic acid) is a nM potent Group II antagonist. However, LY341495 has also been found to have microM potency in inhibiting mGluR1 and 5. The stimulation of [3H]PI hydrolysis by 1 micromol DHPG was dose-dependently blocked by co-administration of the mGluR antagonists, LY341495 at doses that are constant with an interaction at Group I mGluR's. Taken together these results suggest that stimulation of group I mGluRs results in measurable increases in PI hydrolysis in vivo. This method could be quite useful in determining the doses and routes of administration of agonists and antagonists that are required to interact with group I mGluRs.     

SH-SY5Y neuroblastoma cells: a model system for studying biosynthesis of NAAG

N-Acetylaspartylglutamate (NAAG) is a neuropeptide that is thought to modulate neurotransmitter release through pre-synaptic mGluR3 receptors. Despite years of research into NAAG biochemistry, almost nothing is known about NAAG biosynthesis. To date, NAAG biosynthesis has only been demonstrated conclusively in explanted animal neural tissues, including frog retina, rat dorsal root ganglia and crayfish nerve cord, but not in human cells or tissues. We show here that a human neuroblastoma cell line, SH-SY5Y, provides a good model system for the study of NAAG biosynthesis. Radiolabled NAAG synthesis occurred using both L-[3H]glutamic acid and L-[3H]glutamine as precursors, with glutamine being the preferred substrate. Differentiation of SH-SY5Y cells with retinoic acid resulted in decreased radiolabel incorporation into NAAG, whereas differentiation with nerve growth factor did not affect radiolabel incorporation.     

Beta-estradiol attenuate amyloid beta-peptide toxicity via nicotinic receptors

A number of epidemiological studies suggest that estrogen therapy is linked to a reduced risk of developing Alzheimer's disease (AD). The present study was conducted to evaluate the effect of 17beta-estradiol on beta-amyloid (Abeta)-induced toxicity and was performed in rat pheochromocytoma PC 12 cells by measuring the mitochondrial activity. 17Beta-estradiol (10(-5), 10(-6) and 10(-8) M) attenuated Abeta(25-35)-induced toxicity in PC 12 cells. The neuroprotective effect of 17beta-estradiol (10(-5) M) was prevented in the presence of the nicotinic antagonists methyllycaconitine (MLA) and mecamylamine, suggesting an interaction probably via the alpha7 nicotinic receptor subtype. Chronic treatment with 17beta-estradiol (10(-10)-10(-5) M) alone did not change the number of [3H]epibatidine binding sites in human neuroblastoma SH-SY5Y cells and rat PC 12 cells, but significantly prevented the enhanced [3H]epibatidine binding in nicotine-treated PC 12 cells. This study demonstrates that 17beta-estradiol exerts neuroprotective effects which might involve interaction with the alpha7 nicotinic receptor subtype.     

Failure of FK506 (tacrolimus) to alleviate apomorphine-induced circling in rat Parkinson model in spite of some cytoprotective effects in SH-SY5Y dopaminergic cells

The mechanism of action of the neurotoxin 6-hydroxydopamine (6-OHDA) is thought to involve the generation of free radicals and subsequent apoptotic processes. We have demonstrated in vitro that the neuroimmunophilin, FK506 (10-100 nM), dose dependently and significantly restored the ROS production to the control level, increased the Bcl-2 protein level, partly inhibited the cytochrome C release from mitochondria and reduced the caspase-3 activation in SH-SY5Y cells. On the other hand, there was no significant restoration of the ATP level by FK506 and the toxin activated proteins, p53 and Bax, were not normalized by FK506. In support of these latter results, daily administration of FK506 for 7 days to rats (0.5, 1 and 3 mg/kg i.p.) did not significantly prevent the apomorphine-induced contralateral circling, measured 2 weeks after unilateral nigral lesioning. Moreover, FK506 pretreatment did not significantly lower the toxin elevated lipid peroxidation levels, indicating that oxidative stress was present even after the FK506 treatment in the lesioned striatum. Taken together, our results with FK506 are inconsistent. We confirm the antioxidant nature of FK506, that is, it blocks ROS production in SH-SY5Y cells. However, there were no significant protective effects in any apoptotic analyses in SH-SY5Y cells and in animal studies, a 7-day FK506 pre-treatment was not able to reverse the toxic effect of 6-OHDA in a rat model of Parkinson's disease.     

Effects of insulin, insulin-like growth factor-II and nerve growth factor on neurite outgrowth in cultured human neuroblastoma cells

The identification of biologically important and chemically well-defined substances that can promote axon and dendrite formation would improve present understanding of the development of the nervous system. Physiological concentrations of insulin and insulin-like growth factor-II (IGF-II) reversibly enhanced neurite outgrowth (NTO) in human neuroblastoma SH-SY5Y cells cultured in media with and without serum. Nerve growth factor (NGF), in contrast, did not enhance NTO in serum-free media. Furthermore, anti-NGF antiserum inhibited NGF but not insulin-enhanced NTO. Insulin increased [3H]leucine and [3H]uridine uptake. These increases, together with increased NTO, were inhibited by cycloheximide and actinomycin D, respectively. The inhibition of NTO by cycloheximide was reversible. Human neuroblastoma cell lines that were responsive by NTO to NGF were also responsive to insulin, with the exception of line CHP-270. Moreover, cell lines unresponsive by NTO to NGF, and to tumor promoters, were uniformly unresponsive to insulin. These findings suggest that there are common defects in distal sites, because specific NGF and tumor promotor receptors are present in these lines. Insulin increased [3H]thymidine uptake in SH-SY5Y and CHP-100 cells. However, the enhancement of NTO by insulin and IGF-II in SH-SY5Y cells was independent of the cellular proliferation rate. Our results, together with the observations of others, suggest that insulin and IGF-II may modulate NTO in the nervous system.     

Potentiation of [3H]inositol phosphate formation by receptor activation and membrane depolarization in brain cortical slices (I)

Potentiation of [3H]inositol phosphate ([3H]IP) accumulation by receptor agonists combined with depolarizing agents was studied in rat brain cortical slices, prelabeled with [3H]inositol. Muscarinic agonists, alpha 1-adrenergic, histaminergic and serotonergic agonists remarkably enhanced (2-7-fold) the accumulation of [3H]IP in the presence of KCl (30 mM). The potentiated levels of [3H]IP were strongly dependent on K+ concentration and displayed a dose-response relationship with the agonist. Other depolarizing agents such as veratridine and ouabain induce potentiation of [3H]IP formation similarly to that observed by KCl, but to a lesser extent. The production of elevated levels of [3H]IP is Ca2+-dependent with maximal effect at 0.6 mM which is similar to the Ca2+ dependency observed for the agonist and the depolarizing agent alone. Enhanced [3H]IP levels induced by agonists in the presence of depolarizing agents affect Vmax values only, since the apparent half maximal effective concentration of carbachol (CCh)-induced-IP-formation (1.2 x 10(-4) M) and of the phenylephrine-induced IP-formation (8 x 10(-6) M), were not affected in the presence of either K+ or veratridine. In addition the efficacy of various muscarinic agonists as inducers of IP-accumulation was conserved under depolarizing conditions as compared to IP accumulation under normal conditions. In the presence of KCl (30 mM) the maximal degree of potentiation was at a range of 5-7-fold, with order efficacy of ACh greater than CCh greater than Oxo M greater than arecoline much greater than pilocarpine.(ABSTRACT TRUNCATED AT 250 WORDS)