Regulation of Muscarinic Agonist-Induced Activation of Phosphoinositidase C in Electrically Permeabilized SH-SY5Y Human Neuroblastoma Cells by Guanine Nucleotides

myo-[3H]Inositol-labelled SH-SY5Y cells were permeabilized with electrical discharges. 3H-Inositol phosphate formation in cells shown to be fully permeable was stimulated by the muscarinic agonist carbachol, by guanosine 5'-(gamma-thio)triphosphate [GTP(S)], and by guanosine 5'-(beta gamma-imido)diphosphate (GppNHp). Synergism was observed on coincubation of these GTP analogues with carbachol. GTP was also stimulatory and guanosine 5'-(beta-thio)diphosphate was inhibitory in the presence of agonist. Atropine blocked the effects of carbachol. Stimulation by GTP(S) (0.1 mM) occurred after a 1-2-min lag, whereas Ca2+ (0.5 mM), carbachol (1 mM), and carbachol plus GTP(S) stimulated without delay. The effects of carbachol plus GTP(S) but not those of Ca2+ were inhibited by spermine (4 mM). Accumulation of 3H-inositol phosphates was enhanced by Li+ (4 mM) only in intact cells. In intact or permeabilized cells, the "partial" agonist arecoline was maximally 40-50% as efficacious as carbachol. In permeabilized cells, the maximal effects of carbachol and arecoline were enhanced 2.8- and 5.3-fold, respectively, by 0.1 mM GTP(S), but only the EC50 for carbachol was substantially reduced. The binding affinity of carbachol but not that of arecoline in permeabilized cells was significantly reduced by 0.1 mM GppNHp. These data indicate that a guanine nucleotide-binding regulatory protein is involved in coupling muscarinic receptors to phosphoinositidase C in SH-SY5Y cells and that the activity of this protein influences the relationship between receptor occupation and phosphoinositide response.     

Chronic muscarinic stimulation of SH-SY5Y neuroblastoma cells suppresses inositol 1,4,5-trisphosphate action. Parallel inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ mobilization and inositol 1,4,5-trisphosphate binding.

The possibility that chronic activation of the phosphoinositide-mediated signaling pathway modifies the Ca(2+)-mobilizing action of inositol 1,4,5-trisphosphate (InsP3) was examined. SH-SY5Y human neuroblastoma cells were exposed to carbachol, permeabilized electrically, loaded with 45Ca2+, and 45Ca2+ mobilization in response to exogenous InsP3 was assessed. In control permeabilized cells, InsP3 released 65 +/- 2% of sequestered 45Ca2+ (EC50 = 0.32 +/- 0.05 microM). Pre-treatment with carbachol reduced both maximal InsP3-induced 45Ca2+ release (to 34 +/- 3%, with half-maximal and maximal inhibition at approximately 3 and 6 h, respectively) and the potency of InsP3 (EC50 = 0.92 +/- 0.13 microM). This inhibitory effect of carbachol was half-maximal at approximately 5 microM, was mediated by muscarinic receptors, and was reversible following withdrawal of agonist. Pretreatment with phorbol 12,13-dibutyrate did not alter the maximal effect of InsP3 but doubled its EC50. Evidence suggesting that the inhibitory effects of carbachol pretreatment resulted from altered Ca2+ homeostasis was not forthcoming; both 45Ca2+ uptake and release induced by ionomycin and thapsigargin were identical in control and pretreated permeabilized cells, as were the characteristics of reuptake of released Ca2+. In contrast, carbachol pretreatment, without altering the affinity of InsP3 (Kd = 64 +/- 7 nM), reduced the density of [32P]InsP3-binding sites from 2.0 +/- 0.1 to 1.0 +/- 0.1 pmol/mg protein with a time course essentially identical to that for the reduction in responsiveness to InsP3. This effect was not mimicked by pretreatment of cells with phorbol 12,13-dibutyrate. These data indicate that chronic activation of phosphoinositide hydrolysis can reduce the abundance of InsP3 receptors and that this causes a reduction in size of the InsP3-sensitive Ca2+ store. This modification, possibly in conjunction with a protein kinase C-mediated event, appears to account for the carbachol-induced suppression of InsP3 action. As intracellular InsP3 mass remained elevated above basal for at least 24 h after addition of carbachol, suppression of the Ca(2+)-mobilizing activity of InsP3 represents an important adaptive response to cell stimulation that can limit the extent to which intracellular Ca2+ is mobilized.     

Forskolin alters sensitivity of the cAMP-generating system to stimulatory as well as to inhibitory agonists. A study with intact human platelets and guinea pig myometrium

1. In both the intact guinea pig myometrium and human platelets, cAMP accumulation was enhanced by prostaglandin I2 (prostacyclin, PGI2) and forskolin with potentiated responses in the simultaneous presence of both effectors. Under basal conditions, forskolin caused rises in platelet cAMP concentration through a single low-affinity interaction (Kapp = 90 microM) while in myometrium, activation involved both a low-affinity (Kapp = 10 microM) and a high-affinity (Kapp = 0.8 microM) component. The contribution of the high-affinity component could be reduced when endogenous PGI2 was decreased. In both tissues, the synergistic effect of forskolin in the presence of PGI2 was mediated by a single high-affinity interaction (Kapp = 0.3 microM). The data were consistent with a low-affinity interaction of the diterpene with the cyclase catalytic unit C generating the C...forskolin state and with a high-affinity interaction of the diterpene with the activated complex (stimulatory regulatory protein) and C generating the potentiated Gs-C...forskolin state. 2. Both norepinephrine in platelets and carbachol in the myometrium (via Gi, the inhibitory regulatory protein) inhibited PGI2-mediated cAMP accumulation (EC50 = 100 nM and 8 nM respectively). The persistently activated cAMP-generating system induced by cholera toxin in the myometrium was also susceptible to inhibition but the EC50 for carbachol was increased to 50 nM and the extent of inhibition was decreased. Forskolin-mediated effect in platelets was inhibited by norepinephrine as was the PGI2 response. By contrast, the synergistic state of the cyclase resisted the inhibitory action of norepinephrine and carbachol in platelets and myometrium respectively. In the myometrium, where the cAMP response due to forskolin alone partially involved some synergistic Gs-C ... forskolin species, carbachol at 50 microM elicited no more than 30% inhibition. Inhibition was partly improved (60% inhibition at 1 microM carbachol) when the contribution of the Gs-C species was decreased by lowering the concentration of local PGI2. Partial inhibition by norepinephrine was similarly observed in platelets under partial synergistic conditions. The data suggest that receptor-mediated inhibition of cAMP generation could be differentially expressed depending on the nature of the active species of the cyclase involved in the stimulatory responses.     

Coincident signalling between the Gi/Go-coupled delta-opioid receptor and the Gq-coupled m3 muscarinic receptor at the level of intracellular free calcium in SH-SY5Y cells

In SH-SY5Y cells, activation of delta-opioid receptors with [D-Pen(2,5)]-enkephalin (DPDPE; 1 microM) did not alter the intracellular free Ca(2+) concentration [Ca(2+)](i). However, when DPDPE was applied during concomitant Gq-coupled m3 muscarinic receptor stimulation by carbachol or oxotremorine-M, it produced an elevation of [Ca(2+)](i). The DPDPE-evoked increase in [Ca(2+)](i) was abolished when the carbachol-sensitive intracellular Ca(2+) store was emptied. There was a marked difference between the concentration-response relationship for the elevation of [Ca(2+)](i) by carbachol (EC(50) 13 microM, Hill slope 1) and the concentration-response relationship for carbachol's permissive action in revealing the delta-opioid receptor-mediated elevation of [Ca(2+)] (EC(50) 0.7 mM; Hill slope 1.8). Sequestration of free G protein beta gamma dimers by transient transfection of cells with a beta gamma binding protein (residues 495-689 of the C terminal tail of G protein-coupled receptor kinase 2) reduced the ability of delta opioid receptor activation to elevate [Ca(2+)](i). However, DPDPE did not elevate either basal or oxotremorine-M-evoked inositol phosphate production indicating that delta-opioid receptor activation did not stimulate phospholipase C. Furthermore, delta-opioid receptor activation did not result in the reversal of muscarinic receptor desensitization, membrane hyperpolarization or stimulation of sphingosine kinase. There was no coincident signalling between the delta-opioid receptor and the lysophosphatidic acid receptor which couples to elevation of [Ca(2+)](i) in SH-SY5Y cells by a PLC-independent mechanism. In SH-SY5Y cells the coincident signalling between the endogenously expressed delta-opioid and m3 muscarinic receptors appears to occur in the receptor activation-Ca(2+) release signalling pathway at a step after the activation of phospholipase C.     

Cilostazol, a cyclic AMP phosphodiesterase inhibitor, stimulates nitric oxide production and sodium potassium adenosine triphosphatase activity in SH-SY5Y human neuroblastoma cells.

Deficiencies in cellular cyclic AMP (cAMP) and nitric oxide (NO) production are thought to be involved in the pathogenesis of diabetic neuropathy. We used a human neuroblastoma cell line, SH-SY5Y, to investigate the effect of cilostazol, a specific cAMP phosphodiesterase inhibitor, on NO production and Na+, K+-ATPase activity. SH-SY5Y cells were cultured under 5 or 50 mM glucose for 5-6 days, the cells were then exposed to cilostazol or other chemicals and nitrite, cAMP and Na+, K+-ATPase activity were measured. In cells grown in 50 mM glucose, cilostazol was observed to increase significantly both NO production and cellular cAMP accumulation in a time- and dose-dependent manner. Cilostazol also significantly recovered reduced levels of protein kinase A activity (PKA) in 50 mM glucose. Furthermore, a PKA inhibitor, H-89 significantly suppressed the increase in NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production by activating PKA. Cilostazol did not affect either sorbitol or myo-inositol concentrations. Dexamethasone, which is known to induce inducible NO synthase, had no effect on NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production catalyzed by neuronal constitutive NO synthase (ncNOS) in SH-SY5Y cells. L-arginine, which is an NO agonist enhanced Na+, K+-ATPase activity in cells grown in 50 mM glucose, NG-nitro-L-arginine methyl ester (L-NAME), which is an NOS inhibitor inhibited basal Na+, K+-ATPase activity in 5 mM glucose and suppressed the increased enzyme activity induced by cilostazol in 50 mM glucose. The above results confirmed our previous observation that NO regulates Na+, K+-ATPase activity in SH-SY5Y cells and suggest that cilostazol increases Na+, K+-ATPase activity, at least in part, by stimulating NO production. The present results also suggest that cilostazol has a beneficial effect on diabetic neuropathy by improving Na+, K+-ATPase activity via directly increasing cAMP and NO production in nerves.     

Carbachol-induced reverse transformation of Chinese hamster ovary cells transfected with and expressing the m5 muscarinic acetylcholine receptor

Reverse transformation was induced in Chinese hamster ovary (CHO) cells transfected with and stably expressing the m5 subtype of the muscarinic acetylcholine receptor when stimulated with the muscarinic agonist, carbachol. Atropine, a muscarinic antagonist, blocked the carbachol-stimulated reverse transformation. CHO cells not transfected with the muscarinic receptor did not change with added carbachol. PMA induced reverse transformation without increasing cAMP accumulation in CHO cells. Carbachol, prostaglandin E2, and cholecystokinin increased cAMP accumulation but only carbachol caused reverse transformation. Carbachol-stimulated cAMP accumulation occurred at a higher concentration (EC50 10 microM) than did carbachol-stimulated reverse transformation (EC50 63 nM). Muscarinic m5 acetylcholine receptor transfected into CHO cells can induce reverse transformation which may be independent of cAMP.     

Carbachol Inhibits Insulin-Stimulated Phosphatidylinositol 3-Kinase Activity in SH-SY5Y Neuroblastoma Cells

Abstract: SH-SY5Y human neuroblastoma cells express muscarinic M₃ receptors as well as insulin receptors, thus offering the opportunity to investigate possible cross-talk following activation of two distinct intracellular signal transduction pathways that convert the precursor phosphatidylinositol (PI) to its 3′ phosphate or its 4′ phosphate, respectively. In this study, the effect of carbachol on insulin-stimulated PI 3-kinase (PI3K) activity was examined in SH-SY5Y cells. Insulin addition to the cell medium induced a 10–26-fold increase in anti-phosphotyrosine-immunoprecipitable PI3K activity. Preincubation with 1 mM carbachol inhibited the insulin-stimulated PI3K activity in a time-dependent manner, with half-maximal and maximal inhibition times of 4 and 15 min, respectively. Atropine blocked the inhibitory effect of carbachol. Although carbachol did not change the amount of 85-kDa subunit protein regulatory unit associated with tyrosine-phosphorylated proteins, either in control or in insulin-stimulated cells, it appears to decrease the amount of associated 110-kDa catalytic subunit protein in the latter instance. Because PI3K activity from SH-SY5Y cells has been shown to be inhibited in vitro in the presence of cytidine diphosphodiacylglycerol (CDP-DAG) or phosphatidate (PA), we examined the presence of these lipids in SH-SY5Y cells that had been treated with carbachol. Formation of both lipids was increased in a time-dependent manner following carbachol addition, and their increased levels are proposed to account for the observed in vivo inhibition of PI3K. Addition of the cell-permeable homologue didecanoyl-CDP-DAG to intact cells inhibited insulin-stimulated PI3K activity up to 75%, with an IC₅₀ of 0.5 µM, a result that further supports a proposed lipid-mediated inhibition of PI3K. Exogenously added didecanoyl-PA, however, did not affect PI3K activity. The possibility that stimulation of the PI 4-kinase-mediated signal transduction pathway leads to down-regulation of the PI3K-mediated signal transduction pathway in vivo, via inhibition of PI3K by CDP-DAG or by other consequences of phosphoinositidase C-linked receptor activation, is discussed.     

Muscarinic receptor-linked elevation of cAMP in SH-SY5Y neuroblastoma cells is mediated by Ca2+ and protein kinase C.

The mechanisms of muscarinic receptor-linked increase in cAMP accumulation in SH-SY5Y human neuroblastoma cells has been investigated. The dose-response relations of carbachol-induced cAMP synthesis and carbachol-induced rise in intracellular free Ca2+ were similar. The stimulated cAMP synthesis was inhibited by about 50% when cells were entrapped with the Ca2+ chelator BAPTA or in the presence of the protein kinase C (PKC) inhibitor staurosporine. Production of cAMP could be induced also by the Ca2+ ionophore, ionomycin and by TPA, an activator of PKC. When added together TPA and ionomycin had a synergistic effect. When cAMP synthesis was activated with cholera toxin, PGE1 or PGE1 + pertussis toxin carbachol stimulated cAMP production to the same extent as in control cells. Ca2+ and protein kinase C thus seem to be the mediators of muscarinic-receptor linked cAMP synthesis by a direct action on adenylate cyclase.     

Opioid agonists binding and responses in SH-SY5Y cells.

SH-SY5Y (human neuroblastoma) cultured cells, known to have mu-opioid receptors, have been used to assess and compare the ability of eight representative mu-selective compounds from diverse opioid families to recognize and activate these receptors. A wide range of receptor affinities spanning a factor of 10,000 was found between the highest affinity fentanyl analogs (Ki = 0.1nM) and the lowest affinity analog, meperidine (Ki = 1 microM). A similar range was found for inhibition of PGE1-stimulated cAMP accumulation with a rank order of activities that closely paralleled binding affinities. Maximum inhibition of cAMP accumulation by each compound was about 80%. Maximum stimulation of GTPase activity (approximately 50%) was also similar for all compounds except the lowest affinity meperidine. Both effects were naloxone reversible. These results provide further evidence that mu-receptors are coupled to inhibition of adenylate cyclase and that the SH-SY5Y cell line is a good system for assessment of mu-agonists functional responses.     

Inositol 1,4,5-Trisphosphate Receptor Immunoreactivity in SH-SY5Y Human Neuroblastoma Cells Is Reduced by Chronic Muscarinic Receptor Activation

Inositol 1,4,5-trisphosphate (InsP3) receptor immunoreactivity in SH-SY5Y human neuroblastoma cells was monitored with a monoclonal antibody raised against the mouse cerebellar InsP3 receptor. Recognition of a protein corresponding to the InsP3 receptor (molecular mass, approximately 275 kDa) was inhibited markedly following exposure of cells to 0.1 mM carbachol. This effect was half-maximal and maximal at approximately 2 and approximately 6 h, respectively; was blocked by atropine; but was not mimicked by thapsigargin, K+, or phorbol 12-myristate 13-acetate. However, the decrease in immunoreactivity following exposure of cells to carbachol for 5 h was blocked if the extracellular Ca2+ concentration was reduced from 1.3 mM to 200 nM. This manipulation also reduced markedly carbachol-induced increases in InsP3 concentration at 5 h. These data indicate that chronic muscarinic stimulation of phosphoinositide hydrolysis reduces InsP3 receptor concentration in SH-SY5Y cells, perhaps via a mechanism that involves prolonged elevation of InsP3 levels.     

Retinoic acid enhances VIP receptor expression and responsiveness in human neuroblastoma cell, SH-SY5Y

Retinoic acid (RA) induces partial differentiation of neuroblastoma (NB) cells in vitro. In the human NB line, SH-SY5Y (a neuroblastic subclone of SK-N-SH), RA was previously shown to enhance the stimulatory (PGE1) and inhibitory (opioid) regulation of adenylyl cyclase. Since these cells are also sensitive to cAMP stimulation by vasoactive intestinal peptide (VIP), we have tested the effects of RA on VIP receptor expression and function. Pretreatment of SH-SY5Y cells with 10 microM RA over 6 days dramatically increased VIP receptor number from approximately 3,000 to approximately 70,000 sites per cell and enhanced threefold the cAMP accumulation after external VIP addition, while VIP immunoreactive content in the cells increased 2-3-fold. In the light of the recently proposed autocrine function of VIP in this cell lineage, the strong enhancement of the VIP system may contribute to the differentiation effects of RA.     

Cholinergic Stimulation of Early Growth Response-1 DNA Binding Activity Requires Protein Kinase C and Mitogen-Activated Protein KInase Kinase Activation and Is Inhibited by Sodium Valporate in SH-SY5y Cells

Activation of muscarinic receptors in human neuroblastoma SH-SY5Y cells with carbachol stimulated a rapid and large increase in early growth response-1 (Egr-1, also called zif268 and NGF1-A) protein levels and DNA binding activity. Egr-1 DNA binding activity was stimulated within 15 min of treatment with carbachol and maintained a maximum 20-fold increase over basal between 1 and 2 h after treatment, and the EC50 was approximately 1 microM carbachol. Carbachol-stimulated Egr-1 DNA binding activity was dependent on protein kinase C, as it was potently inhibited by GF109203X (IC50 approximately 0.1 microM) and was reduced by 85 +/- 5% by down-regulation of protein kinase C. Inhibitors of increases in intracellular calcium levels reduced carbachol-induced Egr-1 DNA binding activity by 25-35%. Carbachol-stimulated activation of Egr-1 was reduced 35% by genistein, a tyrosine kinase inhibitor, and 60% by PD098059, an inhibitor of mitogen-activated protein kinase kinases 1/2 (MEK1/2) that activates extracellular-regulated kinases 1/2 (ERK1/2). A novel inhibitory action was caused by chronic (7-day) administration of sodium valproate but not by two other bipolar disorder therapeutic agents, lithium and carbamazepine. Valproate treatment reduced carbachol-stimulated Egr-1 DNA binding activity by 60% but did not alter carbachol-induced activation of ERK1/2 or p38 or increases in Egr-1 protein levels. These results reveal that muscarinic receptors activate Egr-1 through a signaling cascade primarily encompassing protein kinase C, MEK1/2, and ERK1/2 and that valproate substantially inhibits Egr-1 DNA binding activity stimulated by carbachol or protein kinase C.     

Interaction of synthetic D-6-deoxy-myo-inositol 1,4,5-trisphosphate with the Ca2(+)-releasing D-myo-inositol 1,4,5-trisphosphate receptor, and the metabolic enzymes 5-phosphatase and 3-kinase.

The ability of D-6-deoxy-myo-inositol 1,4,5-trisphosphate [6-deoxy-Ins(1,4,5)P3], a synthetic analogue of the second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], to mobilise intracellular Ca2+ stores in permeabilised SH-SY5Y neuroblastoma cells was investigated. 6-Deoxy-Ins(1,4,5)P3 was a full agonist (EC50 = 6.4 microM), but was some 70-fold less potent than Ins (1,4,5)P3 (EC50 = 0.09 microM), indicating that the 6-hydroxyl group of Ins(1,4,5)P3 is important for receptor binding and stimulation of Ca2+ release, but is not an essential structural feature. 6-Deoxy-Ins(1,4,5)P3 was not a substrate for Ins (1,4,5)P3 5-phosphatase, but inhibited both the hydrolysis of 5-[32P]+ Ins (1,4,5)P3 (Ki 76 microM) and the phosphorylation of [3H]Ins(1,4,5)P3 (apparent Ki 5.7 microM). 6-Deoxy-Ins (1,4,5)P3 mobilized Ca2+ with different kinetics to Ins(1,4,5)P3, indicating that it is probably a substrate for Ins (1,4,5)P3 3-kinase.     

Studies of Inositol Phosphate Export from Neuronal Tissue In Vitro

Abstract: Recent in vivo microdialysis studies have demonstrated the presence of extracellular levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] that can be increased in a concentration-dependent manner by muscarinic receptor activation. The aim of the present study was to determine whether extracellular levels of Ins(1,4,5)P₃ could be measured in vitro. Despite rapid increases in internal Ins(1,4,5)P₃ levels after stimulation with 1 m M carbachol, there was no change in external levels in both rat brain cortical slices and human neuroblastoma SH-SY5Y cells. Suprafusion of myo -[³H]inositol-prelabelled hippocampal slices with 1 m M carbachol caused an increase in ³H-inositol phosphates over basal levels in the perfusate after 10 min, reaching a peak (223 ± 56% of basal) 20 min after suprafusion with carbachol was started. This response to carbachol was potentiated in the presence of 30 m M K⁺. Analysis of the individual ³H-inositol phosphates in the perfusate revealed that levels of [³H]inositol monophosphate, [³H]inositol bisphosphate, [³H]inositol trisphosphate, and [³H]inositol tetrakisphosphate were all significantly increased. A similar increase in extracellular ³H-inositol phosphates was demonstrated in SH-SY5Y cells incubated with 1 m M carbachol for 30 min. This response was again enhanced by 30 m M K⁺, although the intracellular response was not potentiated. Possible roles for extracellular inositol phosphates are discussed.     

Cyclic AMP stimulates luteinizing-hormone (lutropin) exocytosis in permeabilized sheep anterior-pituitary cells. Synergism with protein kinase C and calcium.

Sheep anterior-pituitary cells permeabilized with Staphylococcus aureus alpha-toxin were used to investigate the role of cyclic AMP (cAMP) in exocytosis of luteinizing hormone (lutropin, LH) under conditions where the intracellular free Ca2+ concentration ([Ca2+]free) is clamped by Ca2+ buffers. At resting [Ca2+]free (pCa 7), cAMP rapidly stimulated LH exocytosis (within 5 min) and continued to stimulate exocytosis for at least 30 min. When cAMP breakdown was inhibited by 3-isobutyl-1-methylxanthine (IBMX), the concentration giving half-maximal response (EC50) for cAMP-stimulated exocytosis was 10 microM. cAMP-stimulated exocytosis required millimolar concentrations of MgATP, as has been found with Ca2(+)- and phorbol-ester-stimulated LH exocytosis. cAMP caused a modest enhancement of Ca2(+)-stimulated LH exocytosis by decreasing in the EC50 for Ca2+ from pCa 5.6 to pCa 5.9, but had little effect on the maximal LH response to Ca2+. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) dramatically enhanced cAMP-stimulated LH exocytosis by both increasing the maximal effect 5-7-fold and decreasing the EC50 for cAMP to 3 microM. This synergism between cAMP and PMA was further augmented by increasing the [Ca2+]free. Gonadotropin-releasing hormone (gonadoliberin, GnRH) stimulated cAMP production in intact pituitary cells. Since GnRH stimulation is reported to activate PKC and increase the intracellular [Ca2+]free, our results suggest that a synergistic interaction of the cAMP, PKC and Ca2+ second-messenger systems is of importance in the mechanism of GnRH-stimulated LH exocytosis.     

Regulation of Cyclic AMP by the μ-Opioid Receptor in Human Neuroblastoma SH-SY5Y Cells

The human neuroblastoma clonal cell line SH-SY5Y expresses both mu- and delta-opioid receptors (ratio approximately 4.5:1). Differentiation with retinoic acid (RA) was previously shown to enhance the inhibition of adenylyl cyclase (AC) by mu-opioid agonists. We tested here the inhibition of cyclic AMP (cAMP) accumulation by morphine under a variety of conditions: after stimulation with prostaglandin E1 (PGE1), forskolin, and vasoactive intestinal peptide (VIP), both in the presence and in the absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Morphine inhibition of the forskolin cAMP response (approximately 65%) was largely unaffected by the presence of IBMX. In contrast, deletion of IBMX enhanced morphine's inhibition of the PGE1 and VIP cAMP response from approximately 50 to approximately 80%. The use of highly mu- and delta-selective agents confirmed previous results that inhibition of cAMP accumulation by opioids is mostly mu, and not delta, receptor mediated in SH-SY5Y cells, regardless of the presence or absence of IBMX. Because of the large morphine inhibition and the high cAMP levels even in the absence of IBMX, PGE1-stimulated, RA-differentiated SH-SY5Y cells were subsequently used to study narcotic analgesic tolerance and dependence in vitro. Upon pretreatment with morphine over greater than or equal to 12 h, a fourfold shift of the PGE1-morphine dose-response curve was observed, whether or not IBMX was added. However, mu-opioid receptor number and affinity to the mu-selective [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin were largely unaffected, and Na(+)- and guanyl nucleotide-induced shifts of morphine-[3H]naloxone competition curves were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthetic D- and L-enantiomers of 2,2-difluoro-2-deoxy-myo-inositol 1,4,5-trisphosphate interact differently with myo-inositol 1,4,5-trisphosphate binding proteins: identification of a potent small molecule 3-kinase inhibitor.

The ability of two enantiomeric fluoro-analogues of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to mobilize intracellular Ca2+ stores in SH-SY5Y neuroblastoma cells has been investigated. (-)-D-2,2-difluoro-2-deoxy-myo-Ins(1,4,5)P3 [D-2,2-F2-Ins(1,4,5)P3] was a full agonist [EC50 0.21 microM] and slightly less potent than D-Ins(1,4,5)P3 [EC50 0.13 microM]. (+)-L-2,2-F2Ins(1,4,5)P3 was a very poor agonist, confirming the stereospecificity of the Ins(1,4,5)P3 receptor. D-2,2-F2-Ins(1,4,5)P3 mobilized Ca2+ with broadly similar kinetics to Ins(1,4,5)P3 and was a substrate for Ins(1,4,5)P3 3-kinase inhibiting Ins(1,4,5)P3 phosphorylation (apparent Ki = 10.2 microM) but was recognised less well than Ins(1,4,5)P3. L-2,2-F2-Ins(1,4,5)P3 was a potent competitive inhibitor of 3-kinase (Ki = 11.9 microM). Whereas D-2,2-F2-Ins(1,4,5)P3 was a good substrate for Ins(1,4,5)P3 5-phosphatase, L-2,2-F2Ins(1,4,5)P3 was a relatively potent inhibitor (Ki = 19.0 microM).     

PXR激活在拮抗SH-SY5Y细胞凋亡中的作用

人神经母细胞瘤细胞SH-SY5Y细胞可以表达神经元特异性的酪氨酸羟化酶、多巴胺-β-羟化酶以及多巴胺转运体等,因此可用于建立帕金森病的体外模型。虽然帕金森综合症发病的确切机制至今尚不清楚,但众多的病理学资料证实该病患者存在中脑黑质多巴胺能神经元的凋亡。自由基、兴奋性     

Turnover of bis-diphosphoinositol tetrakisphosphate in a smooth muscle cell line is regulated by beta2-adrenergic receptors through a cAMP-mediated, A-kinase-independent mechanism.

Bis-diphosphoinositol tetrakisphosphate ([PP]2-InsP4 or 'InsP8') is a 'high-energy' inositol phosphate; we report that its metabolism is receptor-regulated in DDT1 MF-2 smooth muscle cells. This conclusion arose by pursuing the mechanism by which F- decreased cellular levels of [PP]2-InsP4 up to 70%. A similar effect was induced by elevating cyclic nucleotide levels, either with IBMX or by application of either Bt2cAMP (EC50 = 14.7 microM), Bt2cGMP (EC50 = 7.9 microM) or isoproterenol (EC50 = 0.4 nM). Isoproterenol (1 microM) decreased [PP]2-InsP4 levels 25% by 5 min, and 71% by 60 min. This novel, agonist-mediated regulation of [PP]2-InsP4 turnover was very specific; isoproterenol did not decrease the cellular levels of either inositol pentakisphosphate, inositol hexakisphosphate or other diphosphorylated inositol polyphosphates. Bradykinin, which activated phospholipase C, did not affect [PP]2-InsP4 levels. Regulation of [PP]2-InsP4 turnover by both isoproterenol and cell-permeant cyclic nucleotides was unaffected by inhibitors of protein kinases A and G. The effectiveness of the kinase inhibitors was confirmed by their ability to block phosphorylation of the cAMP response element-binding protein. Our results indicate a new signaling action of cAMP, and furnish an important focus for future research into the roles of diphosphorylated inositol phosphates in signal transduction.