Receptors In SH-SY5Y Cells Research Articles

Guanosine modulates K+ membrane currents in SH-SY5Y cells: involvement of adenosine receptors

Guanosine (GUO), widely considered a key signaling mediator, is implicated in the regulation of several cellular processes. While its interaction with neural membranes has been described, GUO still is an orphan neuromodulator. It has been postulated that GUO may eventually interact with potassium channels and adenosine (ADO) receptors (ARs), both particularly important for the control of cellular excitability. Accordingly, here, we investigated the effects of GUO on the bioelectric activity of human neuroblastoma SH-SY5Y cells by whole-cell patch-clamp recordings. We first explored the contribution of voltage-dependent K+ channels and, besides this, the role of ARs in the regulation of GUO-dependent cellular electrophysiology. Our data support that GUO is able to specifically modulate K+-dependent outward currents over cell membranes. Importantly, administering ADO along with GUO potentiates its effects. Overall, these results suggested that K+ outward membrane channels may be targeted by GUO with an implication of ADO receptors in SH-SY5Y cells, but also support the hypothesis of a functional interaction of the two ligands. The present research runs through the leitmotif of the deorphanization of GUO, adding insight on the interplay with adenosinergic signaling and suggesting GUO as a powerful modulator of SH-SY5Y excitability.

Effects of Ropivacaine Hydrochloride on the Expression of Type I Interferon and Its Receptor in SH-SY5Y Cells

Nerve injury caused by local anesthetics is a hot issue that people pay close attention to, and its mechanism has not been fully clarified. Type I interferon (I-IFN) is an important factor in regulating inflammatory response. In this study, SH-SY5Y cells were injured by ropivacaine hydrochloride in vitro. The cell viability, apoptosis rate, mRNA and protein expression of I-IFN and its receptor IFNAR, as well as the contents of inflammatory cytokines TNF-α, IL-6 and IL-10 were detected to explore the correlation between I-IFN and neurotoxicity induced by ropivacaine hydrochloride. The results showed that after treated with ropivacaine hydrochloride, the cell viability was decreased, the apoptosis rate was increased, the mRNA and protein expressions of IFN-α, IFN-β, IFNAR1 and IFNAR2 were up-regulated, and the contents of inflammatory factors TNF - α, IL-6 and IL-10 were increased. These results suggest that type I interference and its receptor are associated with neurotoxicity of local anesthetics.

HMGB1 Receptors as Potential Novel Targets for Neuroblastoma

Background and Hypothesis: The pathogenesis of neuroblastoma remains elusive. In order to further understand the pathogenesis, it is necessary to identify molecular and cellular mechanisms of the cancer. One protein of interest is high-mobility group box 1 protein (HMGB1). HMGB1 has been implicated in cancer including tumor growth, invasion, angiogenesis, metastasis, relapse and therapeutic resistance. HMGB1 isoforms signals via different receptors including the chemokine receptor, CXCR4, receptor for advanced glycation end products (RAGE), and the prominent inflammation-associated receptor, toll-like receptor 4 (TLR4). To gain insight into the possible impact of HMGB1 on the SH-SY5Y neuroblastoma cell line, we determined the degree to which CXCR4, TLR4 and RAGE are present on SH-SY5Y cells. We hypothesize that the presence of these receptors in SH-SY5Y cells may mediate proliferation of neuroblastoma cells and other types of cancer.
 Experimental Design or Project Methods: The SH-SY5Y cell line (ATCC® CRL-2266™) was derived from a metastatic bone tumor in a 4 year old female. Cells were lysed in lysis buffer, electrophoresed on a 10% SDS-PAGE, and blotted onto PVDF membrane. After blocking, the membranes were incubated with primary antibodies against the receptor protein overnight at 4 °C, and then with HRP-conjugated secondary antibodies for 1h. Protein bands were visualized with a SuperSignal West Pico Chemiluminescent Substrate.
 Results: To be finalized 
 Conclusion and Potential Impact: Investigating SH-SY5Y cell line for the presence of these inflammation-associated receptors could potentially serve as a model to better understand the role of these receptors in cancer research.

Cocaine potently blocks neuronal α3β4 nicotinic acetylcholine receptors in SH-SY5Y cells.

Cocaine is one of the most abused illicit drugs worldwide. It is well known that the dopamine (DA) transporter is its major target; but cocaine also acts on other targets including nicotinic acetylcholine receptors (nAChRs). In this study, we investigated the effects of cocaine on a special subtype of neuronal nAChR, α3β4-nAChR expressed in native SH-SY5Y cells. α3β4-nAChR-mediated currents were recorded using whole-cell recordings. Drugs were applied using a computer-controlled U-tube drug perfusion system. We showed that bath application of nicotine induced inward currents in a concentration-dependent manner with an EC50 value of 20 µM. Pre-treatment with cocaine concentration-dependently inhibited nicotine-induced current with an IC50 of 1.5 μM. Kinetic analysis showed that cocaine accelerated α3β4-nAChR desensitization, which caused a reduction of the amplitude of nicotine-induced currents. Co-application of nicotine and cocaine (1.5 μM) depressed the maximum response on the nicotine concentration-response curve without changing the EC50 value, suggesting a non-competitive mechanism. The cocaine-induced inhibition of nicotine response exhibited both voltage- and use-dependence, suggesting an open-channel blocking mechanism. Furthermore, intracellular application of GDP-βS (via recording electrode) did not affect cocaine-induced inhibition, suggesting that cocaine did not alter receptor internalization. Moreover, intracellular application of cocaine (30 µM) failed to alter the nicotine response. Finally, cocaine (1.5 μM) was unable to inhibit the nicotine-induced inward current in heterologous expressed α6/α3β2β3-nAChRs and α4β2-nAChRs expressed in human SH-EP1 cells. Collectively, our results suggest that cocaine is a potent blocker for native α3β4-nAChRs expressed in SH-SY5Y cells.

PTPN11 induces endoplasmic stress and apoptosis in SH-SY5Y cells

PTPN11 is associated with regulation of growth factor signaling pathways in neuronal cells. Using SH-SY5Y neuroblastoma cells, we showed that adeno-associated virus (AAV)-mediated PTPN11 upregulation was associated with TrkB antagonism, reduced neuritogenesis and enhanced endoplasmic reticulum (ER) stress response leading to apoptotic changes. Genetic knock-down of PTPN11 on the other hand leads to increased TrkB phosphorylation in SH-SY5Y cells. ER stress response induced by PTPN11 upregulation was alleviated pharmacologically by a TrkB agonist. Conversely the enhanced ER stress response induced by TrkB receptor antagonism was ameliorated by PTPN11 suppression, providing evidence of cross-talk of PTPN11 effects with TrkB actions. BDNF treatment of neuronal cells with PTPN11 upregulation also resulted in reduced expression of ER stress protein markers. This study provides evidence of molecular interactions between PTPN11 and the TrkB receptor in SH-SY5Y cells. The results reinforce the role played by PTPN11 in regulating neurotrophin protective signaling in neuronal cells and highlight that PTPN11 dysregulation promotes apoptotic activation. Based on these findings we suggest that blocking PTPN11 could have potential beneficial effects to limit the progression of neuronal loss in neurodegenerative disorders.

Arrestins contribute to amyloid beta-induced cell death via modulation of autophagy and the \u03b17nAch receptor in SH-SY5Y cells

Amyloid β-protein (Aβ) is believed to contribute to the development of Alzheimer’s disease (AD). Here we showed that Aβ25-35 rapidly caused activation of autophagy, subsequently leading to reduction of autophagy associated with cellular apoptosis. Further investigation revealed that the accumulation of β-arrestin 1 (ARRB1) caused by Aβ25-35 contributed to the induction of autophagic flux. The depletion of ARRB1 led to decreases in the expression of LC3B, Atg7, and Beclin-1, which are essential for the initiation of autophagy. ARRB1 depletion also reduced downstream ERK activity and promoted Aβ25-35-induced cell death. As with ARRB1, transient upregulation of ARRB2 by Aβ25-35 was observed after short treatment durations, whereas genetic reduction of ARRB2 caused a marked increase in the expression of the α7nAch receptor at the cell surface, which resulted in partial reversal of Aβ25-35-induced cell death. Although expression of both ARRB1 and ARRB2 was reduced in serum from patients with AD, the levels of ARRB1 were much lower than those of ARRB2 in AD. Thus, our findings indicate that ARRB1/2 play different roles in Aβ25-35 cytotoxicity, which may provide additional support for exploring the underlying molecular mechanism of AD.

Beneficial Effects of Sulforaphane Treatment in Alzheimer's Disease May Be Mediated through Reduced HDAC1/3 and Increased P75NTR Expression.

Alzheimer's disease is an irreversible, progressive neurodegenerative disorder. The accumulation of Aβ in the brain is thought to play a causative role in the development of cognitive dysfunction in Alzheimer's disease. The p75 neurotrophin receptor is of great importance to protect against the Aβ burden and its expression is regulated by histone acetylation. This study investigated whether the phytochemical sulforaphane, a pan-histone deacetylase inhibitor, up-regulates the p75 neurotrophin receptor expression via affecting histone acetylation in protection against Alzheimer's disease. We found that sulforaphane ameliorated behavioral cognitive impairments and attenuated brain Aβ burden in Alzheimer's disease model mice. Additionally, sulforaphane reduced the expression of histone deacetylase1, 2, and 3, up-regulated p75 neurotrophin receptor, and increased levels of acetylated histone 3 lysine 9 and acetylated histone 4 lysine 12 in the cerebral cortex of Alzheimer's disease model mice as well as in Aβ-exposed SH-SY5Y cells. Furthermore, silencing of histone deacetylase1 and 3, but not histone deacetylase2, gene expression with small interfering RNA caused up-regulation of p75 neurotrophin receptor in SH-SY5Y cells. In conclusion, this study demonstrates that sulforaphane can ameliorate neurobehavioral deficits and reduce the Aβ burden in Alzheimer's disease model mice, and the mechanism underlying these effects may be associated with up-regulation of p75 neurotrophin receptor mediated, apparently at least in part, via reducing the expression of histone deacetylase1 and 3.

Propofol produces preventive analgesia via GluN2B-containing NMDA Receptor/ERK1/2 Signaling Pathway in a rat model of inflammatory pain.

Propofol, an intravenous anesthetic, has been shown to offer superior analgesic effect clinically. Whether propofol has preventive analgesic property remains unexplored. The present study investigated the antinociceptive effect of propofol and underlying molecular and cellular mechanisms via pre-emptive administration in a formalin-induced inflammatory pain model in rats. Male adult Sprague–Dawley rats were randomly allocated into four groups: naïve (Group Naïve), formalin injection only (Group Formalin), and formalin injection at 30 min (Group P-30 min) or 2 h (Group P-2 h) after intravenous infusion of propofol (0.6 mg kg−1 min−1) for 1 h. Nociceptive responses and protein expression of phosphorylated- or pan-GluN2B, ERK1/2, p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase in the spinal dorsal horn were evaluated. Alteration of intracellular Ca2+ concentration induced by N-methyl-D-aspartate (NMDA) receptor agonists with or without pre-treatment of propofol was measured using fluorometry in SH-SY5Y cells while neuronal activation in the spinal dorsal horn by immunofluorescence. Pre-emptive propofol reduced pain with a delayed response to formalin and a reduction in hypersensitivity that lasted at least for 2 h. The formalin-induced activation of spinal GluN2B and ERK1/2 but not p38 or c-Jun N-terminal kinase was also diminished by propofol treatment. Preconditioning treatment with 3 µM and 10 µM of propofol inhibited Ca2+ influx mediated through NMDA receptors in SH-SY5Y cells. Propofol also reduced the neuronal expression of c-Fos and p-ERK induced by formalin. This study shows that pre-emptive administration of propofol produces preventive analgesic effects on inflammatory pain through regulating neuronal GluN2B-containing NMDA receptor and ERK1/2 pathway in the spinal dorsal horn.

Heterologous Regulation of Mu-Opioid (MOP) Receptor Mobility in the Membrane of SH-SY5Y Cells

The dynamic organization of G protein-coupled receptors in the plasma membrane is suspected of playing a role in their function. The regulation of the diffusion mode of the mu-opioid (MOP) receptor was previously shown to be agonist-specific. Here we investigate the regulation of MOP receptor diffusion by heterologous activation of other G protein-coupled receptors and characterize the dynamic properties of the MOP receptor within the heterodimer MOP/neuropeptide FF (NPFF2) receptor. The data show that the dynamics and signaling of the MOP receptor in SH-SY5Y cells are modified by the activation of α2-adrenergic and NPFF2 receptors, but not by the activation of receptors not described to interact with the opioid receptor. By combining, for the first time, fluorescence recovery after photobleaching at variable radius experiments with bimolecular fluorescence complementation, we show that the MOP/NPFF2 heterodimer adopts a specific diffusion behavior that corresponds to a mix of the dynamic properties of both MOP and NPFF2 receptors. Altogether, the data suggest that heterologous regulation is accompanied by a specific organization of receptors in the membrane.

The influence of inhibiting or stimulating the expression of the α3 subunit of the nicotinic receptor in SH-SY5Y cells on levels of amyloid-β peptide and β-secretase

To examine the effects of the α3 subunit of the nicotinic acetylcholine receptor (nAChR) on the expression of β-secretase and the concomitant level of amyloid-β (Aβ), SH-SY5Y neuroblastoma cells were either transfected with small interference RNAs (siRNAs) specifically targeting this subunit or exposed to nicotine. The levels of α3 nAChR mRNA and protein, as well as the corresponding levels of BACE1 (which cleaves the β-site of APP) and BACE2 (cleaving in the Aβ domain) were determined by real-time PCR and Western blotting, respectively. The levels of Aβ1–42 in culture media were determined by an Elisa procedure.In SH-SY5Y cells transfected with siRNA, the levels of α3 nAChR mRNA and protein were reduced by 96% and 88%, respectively; the levels of BACE1 mRNA and protein were significantly enhanced, while those of BACE2 were reduced; and the level of Aβ in the culture medium was elevated. In contrast, when untransfected SH-SY5Y cells were exposed to nicotine, the levels of both α3 nAChR mRNA and protein were enhanced; while the levels of BACE1 mRNA and protein were diminished and the corresponding levels of BACE2 enhanced; and the level of Aβ in the culture medium was attenuated.These results indicate that the α3 subunit of nAChR inhibits the production of Aβ by reducing the expression of BACE1 and elevating the expression of BACE2, suggesting that this subunit might play an important neuroprotective role in connection with the pathogenesis of AD.

Agonist-selective Dynamic Compartmentalization of Human Mu Opioid Receptor as Revealed by Resolutive FRAP Analysis

Techniques for analyzing the membrane diffusion of molecules are the most promising methods for investigating the compartmentalization of G-protein-coupled receptors, particularly as relevant to receptor signaling processes. Here, we report fluorescence recovery after photobleaching (FRAP) measurements performed at variable spot radius for human mu opioid (hMOP) receptors on SH-SY5Y neuroblastoma cells in the presence of ligands. Although an antagonist did not affect the behavior of the receptors compared with the basal state, two different agonists, DAMGO and morphine, caused markedly different changes to receptor diffusion. Like receptors in the absence of ligand, receptors bound to morphine exhibited diffusion confined to joined semipermeable domains, but with smaller domain size and diffusion coefficient. This effect was inhibited by pertussis toxin, strongly suggesting that this dynamic behavior is associated with early steps of signaling. In the presence of DAMGO, half of the receptors displayed free long-range diffusion and the other half were confined to smaller isolated domains. Hypertonic sucrose buffer suppressed this effect, which we attribute to receptor entry into clathrin-coated pits. It is likely that the observation of distinct receptor dynamics in the presence of DAMGO and morphine involves the agonist-selective phosphorylation of the receptor.

Regulation of p53 expression, phosphorylation and subcellular localization by a G-protein-coupled receptor

G-protein coupled receptors (GPCRs) have been extremely successful drug targets for a multitude of diseases from heart failure to depression. This super-family of cell surface receptors have not, however, been widely considered as a viable target in cancer treatment. In the current study we demonstrate that a classical Gq/11-coupled GPCR, the M3-muscarinic receptor, was able to regulate apoptosis via receptors that are endogenously expressed in the human neuroblastoma cell line SH-SY5Y and when ectopically expressed in Chinese hamster ovary (CHO) cells. Stimulation of the M3-muscarinic receptor was shown to inhibit the ability of the DNA-damaging chemotherapeutic agent, etoposide, from mediating apoptosis. This protective response in CHO cells correlated with the ability of the receptor to regulate the expression levels of p53. In contrast, stimulation of endogenous muscarinic receptors in SH-SY5Y cells did not regulate p53 expression but rather was able to inhibit p53 translocation to the mitochondria and p53 phosphorylation at serine 15 and 37. This study suggests the possibility that a GPCR can regulate the apoptotic properties of a chemotherapeutic DNA-damaging agent by regulating the expression, sub-cellular trafficking and modification of p53 in a manner that is in part dependent on the cell type.

Hyoscine butylbromide potently blocks human nicotinic acetylcholine receptors in SH-SY5Y cells

Hyoscine butylbromide (HBB; tradenames: Buscopan ®/Buscapina ®) is an antispasmodic drug for the treatment of abdominal pain associated with gastrointestinal cramping. As a hyoscine derivative, this compound competitively inhibits muscarinic acetylcholine (ACh) receptors on smooth muscle cells in the gastrointestinal tract. Preliminary investigations suggested that it might also inhibit nicotinic ACh receptors. This study investigated the effect of HBB on nicotinic ACh receptor-mediated membrane currents in SH-SY5Y cells. ACh and nicotine application-induced comparable membrane currents with EC 50 values of 25.9 ± 0.6 and 40.1 ± 0.4 μM, respectively. When coapplied with 100 μM ACh, HBB concentration-dependently suppressed currents with an IC 50 value of 0.19 ± 0.04 μM, and was approximately seven-times more potent than the ganglionic blocker, hexamethonium (IC 50 = 1.3 ± 0.3 μM). Increasing the agonist concentration to 5 mM did not affect the amount of block by HBB, which suggests a non-competitive mode of action. These functional in vitro data demonstrate for the first time that HBB blocks neuronal nicotinic ACh receptors in the same concentration range as it inhibits muscarinic ACh receptors. If one hypothesizes that HBB might also affect nicotinic receptors in autonomic neurons in vivo (e. g. in the enteric nervous system), this effect could contribute to its spasmolytic activity.

Mutated recombinant human glucagon-like peptide-1 protects SH-SY5Y cells from apoptosis induced by amyloid-β peptide (1–42)

Accumulation and deposition of amyloid β peptide (Aβ) in the brain causes neuronal apoptosis and eventually leads to Alzheimer's disease (AD). A therapeutic target for AD is to block the cascade reaction induced by Aβ. It has been demonstrated that glucagon-like peptide-1 (GLP-1), which is an endogenous insulinotropic peptide secreted from the gut, binds to its receptor in the brain and possesses neuroprotective effects. Using site-directed mutagenesis and gene recombination techniques, we generated a mutated recombinant human glucagon-like peptide-1 (mGLP-1) which has longer half-life as compared with native GLP-1. This present work aims to examine whether mGLP-1 attenuates Aβ 1–42-mediated cytotoxicity in SH-SY5Y cells and to explore the possible mechanisms. Our data indicate that ≥0.02 ng/ml of mGLP-1 facilitated cell proliferation and 0.1 ng/ml and 0.5 ng/ml of mGLP-1 rescued SH-SY5Y cells from Aβ 1–42-induced apoptosis. Moreover, Aβ 1–42 treatment dramatically stimulated the release of Ca 2+ from internal calcium stores in SH-SY5Y cells, while mGLP-1 helped to maintain the intracellular Ca 2+ homeostasis. Aβ 1–42 also significantly increased the expression level of TP53 and Bax genes which are involved in apoptotic pathways, and mGLP-1 decreased Aβ 1–42-induced up-regulation of TP53 and Bax. Since mGLP-1 treatment elevated cytosolic cAMP concentration in SH-SY5Y cells, we postulate that mGLP-1 may exert its influence via binding to GLP-1 receptors in SH-SY5Y cells and stimulating the production of cAMP. These results suggest that mGLP-1 exhibited neuronal protection properties, and could potentially be a novel therapeutic agent for intervention in Alzheimer's disease.

Inhibiting gene expression of α3 nicotinic receptor in SH-SY5Y cells with the effects on APP metabolism and antioxidation in Alzheimer's disease

In order to examine the effects of α3 nicotinic acetylcholine receptor (nAChR) in connection with the pathogenesis of Alzheimer's disease (AD), neuroblastoma (SH-SY5Y) cells were transfected with small interference RNAs (siRNAs) that target specifically towards α3 nAChR. The expressions of α3 nAChR mRNA and protein were measured by real-time PCR and Western blotting, respectively. The levels of the α-form of secreted amyloid precursor protein (αAPPs) and total-APP were determined by Western blotting. SH-SY5Y cells transfected with siRNA were then treated with 1 μM β-amyloid peptide (Aβ) 1–42, following which the levels of lipid peroxidation, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the reduction rate of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were characterized by utilizing spectrophotometric procedures. As compared to controls, SH-SY5Y cells transfected with siRNA expressed the decreases in the levels of α3 nAChR mRNA and protein by 98% and 66% lower levels, respectively; exhibited reduced level of the αAPPs; and demonstrated enhanced lipid peroxidation, decreased rate of MTT reduction, and declined activities of SOD and GSH-Px. Inhibited gene expression of the α3 nAChR enhanced the toxicity exerted by Aβ. These results indicate that α3 nAChR may improve cleavage of APP by α-secretase, enhance antioxidation and inhibit the toxicity of Aβ, suggesting that the receptor might play an important role in AD.

Modulation of tyrosine hydroxylase expression by melatonin in human SH-SY5Y neuroblastoma cells

We have previously reported in vivo preservation of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, following treatment with physiological doses of melatonin, in a 6-hydroxydopamine model of Parkinson's disease. Based on these findings, we postulated that melatonin would similarly modulate the expression of TH in vitro. Therefore, using human SH-SY5Y neuroblastoma cells which can differentiate into dopaminergic neurons following treatment with retinoic acid, we first examined whether these cells express melatonin receptors. Subsequently, the physiological dose-dependent effects of melatonin on TH expression were examined in both undifferentiated and differentiated cells. The novel detection of the G protein-coupled melatonin MT(1) receptor in SH-SY5Y cells by RT-PCR was confirmed by sequencing and Western blotting. In addition, following treatment of SH-SY5Y cells with melatonin (0.1-100 nM) for 24h, Western analysis revealed a significant increase in TH protein levels. A biphasic response, with significant increases in TH protein at 0.5 and 1 nM melatonin and a reversal at higher doses was seen in undifferentiated cells; whereas in differentiated cells, melatonin was effective at doses of 1 and 100 nM. These findings suggest a physiological role for melatonin in modulating TH expression, possibly via the MT(1) receptor.

Nicotine-induced upregulation of human neuronal nicotinic α7-receptors is potentiated by modulation of cAMP and PKC in SH-EP1-hα7 cells

Chronic nicotine exposure induces upregulation of nicotinic receptors, but the mechanisms underlying this phenomenon are not well understood. The aim of this study was to examine the role of different second messenger systems in the nicotine-induced upregulation of α7-nicotinic receptors in SH-EP1-hα7 human epithelial cells. We show here that chronic exposure to nicotine results in accumulation of cAMP. Furthermore, an enhanced cAMP signalling potentiates nicotine-induced upregulation of α7-nicotinic receptors measured by [ 3H]methyllycaconitine ([ 3H]MLA) binding suggesting that cAMP is involved in the α7-nicotinic receptor upregulation. Down-regulation of protein kinase C (PKC) with a phorbol ester abolishes the nicotine-induced upregulation of α7-nicotinic receptors. Furthermore, overexpression of PKCα in SH-EP1-hα7 cells results in potentiation of nicotine-evoked upregulation indicating that PKC has a role in regulation of α7-nicotinic receptor number. The Ca 2+-calmodulin kinase II (CaMKII) and extracellular signal regulated kinase 1/2 (ERK1/2) appear not to participate in α7-nicotinic receptor upregulation since the specific inhibitors of these kinases did not have an effect on the nicotine-induced upregulation. Taken together this study provides evidence that nicotine induces accumulation of cAMP and that the upregulation mechanisms of α7-nicotinic receptors are potentiated both by cAMP and PKC. As nicotine-evoked upregulation of heteromeric nicotinic receptors in SH-SY5Y cells was unaffected by the treatment with drugs affecting cAMP signalling or PKC activity, our results suggest that the upregulation mechanisms of homomeric α7-nicotinic receptors and heteromeric nicotinic receptors differ from each other.

Internalization and down-regulation of mu opioid receptors by endomorphins and morphine in SH-SY5Y human neuroblastoma cells

The human neuroblastoma cell line, SH-SY5Y, was used to examine the effects of morphine and the endogenous opioid peptides, endomorphin-1 (EM-1) and endomorphin-2 (EM-2), on mu opioid receptor (MOR) internalization and down-regulation. Treatment for 24 h with EM-1, EM-2 or morphine at 100 nM, 1 μM and 10 μM resulted in a dose-dependent down-regulation of mu receptors. Exposure of cells to 10 μM EM-1 for 2.5, 5 and 24 h resulted in a time-dependent down-regulation of mu receptors. Down-regulation of mu receptors by morphine and EM-1 was blocked by treatment with hypertonic sucrose, consistent with an endocytosis-dependent mechanism. Sensitive cell-surface binding studies with a radiolabeled mu antagonist revealed that morphine was able to induce internalization of mu receptors naturally expressed in SH-SY5Y cells. EM-1 produced a more rapid internalization of mu receptors than morphine, but hypertonic sucrose blocked the internalization induced by each of these agonists. This study demonstrates that, like morphine, the endomorphins down-regulate mu opioid receptors in a dose- and time-dependent manner. This study also demonstrates that morphine, as well as EM-1, can induce rapid, endocytosis-dependent internalization of mu opioid receptors in SH-SY5Y cells. These results may help elucidate the ability of mu agonists to regulate the number and responsiveness of their receptors.

Characterisation of a sphingosine 1-phosphate-activated Ca2+ signalling pathway in human neuroblastoma cells.

Sphingosine 1-phosphate (S1P) has assumed great importance within neuroscience research because of putative links between S1P-sensitive Edg receptors and neuroregeneration, cell survival, and alterations in neurite outgrowth. In the present study, we examined the mechanisms by which the endogenous complement of S1P-sensitive human Edg receptors can elevate Ca(2+) in the human neuroblastoma cell line, SH-SY5Y. Reverse transcriptase-polymersase chain reaction (RT-PCR) confirmed the expression of mRNA for Edg 3, 5, and 8 subtypes of S1P-responsive Edg receptors in SH-SY5Y cells. Neither S1P nor the muscarinic agonist methacholine were able to cause a change in SH-SY5Y cell morphology, whereas retinoic acid caused a range of changes, including an increase in neurite outgrowth, under similar test conditions. Stimulation with S1P resulted in a slowly rising increase in cytosolic Ca(2+) levels. These responses were dependent upon inositol-1,4,5-trisphosphate receptors, thapsigargin-sensitive endoplasmic reticulum, and also intact functional mitochondria. S1P-evoked Ca(2+) responses were similar in mechanism to those of methacholine, which activated a much faster responding, larger amplitude Ca(2+) response. These studies indicate that in an endogenous human expression system, S1P appears to be an efficacious agonist of Edg receptors. Despite its slow time course of response, S1P appears to activate the same single Ca(2+) store in SH-SY5Y cells as is activated by methacholine and other G protein coupled receptors.

Induction of G protein-coupled receptor kinases 2 and 3 contributes to the cross-talk between μ and ORL1 receptors following prolonged agonist exposure

The molecular mechanism(s) underlying cross-tolerance between μ and opioid receptor-like 1 (ORL1) receptor agonists were investigated using two human neuroblastoma cell lines endogenously expressing these receptors and G protein-coupled receptor kinases (GRKs). Prolonged (24 h) activation of the μ receptor desensitized both μ and ORL1 receptor-mediated inhibition of forskolin-stimulated cAMP accumulation and upregulated GRK2 levels in SH-SY5Y and BE(2)-C cells. Prolonged ORL1 activation increased GRK2 levels and desensitized both receptors in SH-SY5Y cells. Upregulation of GRK2 correlated with increases in levels of transcription factors Sp1 or AP-2. PD98059, an upstream inhibitor of extracellular signal-regulated kinases 1 and 2 (ERK1/2), reversed all these events. Pretreatment with orphanin FQ/nociceptin (OFQ/N) also upregulated GRK3 levels in both cell lines, and desensitized both receptors in BE(2)-C cells. Protein kinase C (PKC), but not ERK1/2, inhibition blocked OFQ/N-mediated GRK3 induction and μ and ORL1 receptor desensitization in BE(2)-C cells. Antisense DNA treatment confirmed the involvement of GRK2/3 in μ and ORL1 desensitization. Here, we demonstrate for the first time a role for ERK1/2-mediated GRK2 induction in the development of tolerance to μ agonists, as well as cross-tolerance to OFQ/N. We also demonstrate that chronic OFQ/N-mediated desensitization of ORL1 and μ receptors occurs via cell-specific pathways, involving ERK1/2-dependent GRK2, or PKC-dependent and ERK1/2-independent GRK3 induction.