Carbachol-stimulated calcium entry in SH-SY5Y human neuroblastoma cells: Which route?

doi:10.1016/S0928-4257(05)80010-5

Journal of Physiology-Paris

Volume 86, Issues 1–3, 1992, Pages 77-82

https://doi.org/10.1016/S0928-4257(05)80010-5 Get rights and content

Summary

M3 muscarinic receptors expressed on SH-SY5Y human neuroblastoma cells are linked to phosphoinositide turnover and rises in [Ca²⁺]_i. The rise in [Ca²⁺]_i is biphasic with the peak phase being due to release from an intracellular lns(1,4,5)P₃-sensitive site and the plateau phase being due to Ca²⁺ entry across the plasma membrane. Ca²⁺ entry does not appear to involve voltage sensitive Ca²⁺ channels, a pertussis toxin sensitive G-protein-operated Ca²⁺ channel or lns(1,4,5)P₃/lns(1,3,4,5)P₄-operated Ca²⁺ channel. We suggest that carbachol-stimulated Ca²⁺ entry in SH-SY5Y human neuroblastoma cells occurs via receptor operated Ca²⁺ channels and through capacitive refilling.

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Involvement of store-operated Ca2+ entry in activation of AMP-activated protein kinase and stimulation of glucose uptake by M<inf>3</inf> muscarinic acetylcholine receptors in human neuroblastoma cells
2014, Biochimica et Biophysica Acta - Molecular Cell Research
Citation Excerpt :
Collectively, these data indicate that the sustained AMPK phosphorylation induced by CCh required the influx of extracellular Ca2 +. Previous studies have shown that in SH-SY5Y cells the mAChR-induced [Ca2 +]i plateau requires the continued presence of the agonist [26,28]. Accordingly, addition of atropine (100 nM) to cells pre-treated with CCh (10 μM) induced a drop of [Ca2 +]i plateau to resting levels (Fig. 3D).
G_q/11-coupled muscarinic acetylcholine receptors (mAChRs) belonging to M₁, M₃ and M₅ subtypes have been shown to activate the metabolic sensor AMP-activated protein kinase (AMPK) through Ca^2 +/calmodulin-dependent protein kinase kinase-β (CaMKKβ)-mediated phosphorylation at Thr172. However, the source of Ca^2 + required for this response has not been yet elucidated. Here, we investigated the involvement of store-operated Ca^2 + entry (SOCE) in AMPK activation by pharmacologically defined M₃ mAChRs in human SH-SY5Y neuroblastoma cells. In Ca^2 +-free medium the cholinergic agonist carbachol (CCh) caused a transient increase of phospho-Thr172 AMPK that rapidly ceased within 2 min. Conversely, in the presence of extracellular Ca^2 + CCh-induced AMPK phosphorylation lasted for at least 180 min. The SOCE modulator 2-aminoethoxydiphephenyl borate (2-APB), at a concentration (50 μM) that suppressed CCh-induced intracellular Ca^2 + ([Ca^2 +]_i) plateau, inhibited CCh-induced AMPK phosphorylation. CCh triggered the activation of the endoplasmic reticulum Ca^2 + sensor stromal interaction molecule (STIM) 1, as indicated by redistribution of STIM1 immunofluorescence into puncta, and promoted the association of STIM1 with the SOCE channel component Orai1. Cell depletion of STIM1 by siRNA treatment reduced both CCh-induced [Ca^2 +]_i plateau and AMPK activation. M₃ mAChRs increased glucose uptake and this response required extracellular Ca^2 + and was inhibited by 2-APB, STIM1 knockdown, CaMKKβ and AMPK inhibitors, and adenovirus infection with dominant negative AMPK. Thus, the study provides evidence that SOCE is required for sustained activation of AMPK and stimulation of downstream glucose uptake by M₃ mAChRs and suggests that SOCE is a critical process connecting M₃ mAChRs to the control of neuronal energy metabolism.
Transcriptional response to muscarinic acetylcholine receptor stimulation: Regulation of Egr-1 biosynthesis by ERK, Elk-1, MKP-1, and calcineurin in carbachol-stimulated human neuroblastoma cells
2008, Archives of Biochemistry and Biophysics
Citation Excerpt :
An anti-phospho-Elk-1 antibody (Santa Cruz, Heidelberg, Germany, #sc-8406) was used for immunoprecipitation. SH-SY5Y neuroblastoma cells were used for this study because these cells express a relatively high density of pharmacologically homogenous M3 muscarinic acetylcholine receptors [1–7]. The expression of type M3 muscarinic acetylcholine receptor in SH-SY5Y cells was confirmed via RNase protection mapping.
Carbachol-mediated activation of type M₃ muscarinic acetylcholine receptors induces the biosynthesis of the transcription factor Egr-1 in human SH-SY5Y neuroblastoma cells involving an activation of extracellular signal-regulated protein kinase. Carbachol triggered the phosphorylation of the ternary complex factor Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, and strikingly enhanced the transcriptional activation potential of Elk-1. Chromatin immunoprecipitation experiments revealed that Elk-1 binds in vivo to the 5′-upstream region of the Egr-1 gene in carbachol-stimulated neuroblastoma cells. Together, these data indicate that Elk-1 connects the intracellular signaling cascade elicited by activation of M₃ muscarinic acetylcholine receptors with the transcription of the Egr-1 gene. Lentiviral-mediated expression of either MAP kinase phosphatase-1 (MKP-1) or a constitutively active mutant of calcineurin A inhibited Egr-1 biosynthesis following carbachol stimulation, indicating that these phosphatases function as shut-off devices of muscarinic acetylcholine receptor signaling. Additionally, carbachol stimulation increased transcription of a chromatin-embedded collagenase promoter/reporter gene, showing that AP-1 activity is enhanced in carbachol-stimulated neuroblastoma. Expression experiments revealed that both MKP-1 and a constitutively active mutant of calcineurin A impaired carbachol-induced upregulation of AP-1 activity. The fact that carbachol stimulation of neuroblastoma cells activates the transcription factors Egr-1 and AP-1 suggests that changes in the gene expression pattern are an integral part of muscarinic acetylcholine receptor signaling.
Store-operated Ca2+-channels are sensitive to changes in extracellular pH
2005, Biochemical and Biophysical Research Communications
Citation Excerpt :
Agonist-induced entry of Ca2+ into cells often exhibits a biphasic pattern: binding of the agonist to its cell surface receptor initiates a sharp rise in intracellular Ca2+ concentration ([Ca2+]i) followed by a recovery to a level which is higher than that of the original level and is therefore referred to as a “plateau.” This pattern of Ca2+-signal has been observed in a variety of different cell types (both excitable and non-excitable cells) following stimulation with a variety of agents, e.g., endothelin-1 in aortic smooth muscle cells [18], thrombin in platelets [27], or activation of muscarinic acetylcholine receptors in neuroblastoma cells with carbachol [20,28]. It has been established that the sharp rise is attributed to the opening of inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+-channels on the endoplasmic reticulum [24] membrane, whereas the plateau is the result of the opening of SOCs present in the plasma membrane [29–31].
The sensitivity of store-operated Ca²⁺-entry to changes in the extra- and intracellular pH (pH_o and pH_i, respectively) was investigated in SH-SY5Y human neuroblastoma cells. The intracellular Ca²⁺-stores were depleted either with 1 mM carbachol (CCH) or with 2 μM thapsigargin (TG). Extracellular acidification suppressed both the CCH- and TG-mediated Ca²⁺-entry while external alkalinization augmented both the CCH- and the TG-induced Ca²⁺-influx. Mn²⁺-quenching experiments revealed that the rates of Ca²⁺-entry at the thapsigargin- or carbachol-induced plateau were both accelerated at pH_o 8.2 and slowed down at pH_o 6.8 with respect to the control at pH_o 7.4. Alteration of pH_o between 6.8 and 8.2 did not have any significant prompt effect on pH_i and changes in pH_i left the CCH-induced Ca²⁺-entry unaffected. These findings demonstrate that physiologically relevant changes in pH_o affect the store-operated Ca²⁺-entry in SH-SY5Y cells and suggest that endogenous pH_o shifts may regulate cell activity in situ via modulating the store-operated Ca²⁺-entry.
The cooperation between the influx of extracellular calcium and α<inf>1</inf>-adrenoceptor-induced translocation of protein kinase C
1997, Pharmacological Research
Calcium ionophore A23187 or phenylephrine injected i.p. in doses of 0.05–0 .25 mg kg⁻¹and 0.1–1 mg kg⁻¹, respectively, induced translocation of protein kinase C (PKC) from the cytosol to the membrane fraction of the rat frontal cortex and hippocampus. The action of A23187 was blocked in a dose-dependent manner by nifedipine and verapamil. The phenylephrine induced translocation of PKC was blocked by prazosin and in a dose-dependent manner by nifedipine and verapamil. In contrast, pre-treatment with a small, ineffective by itself, dose of A23187 (0.02 mg kg⁻¹) potentiated the α₁-adrenoceptor induced translocation of PKC. Thus, it seems that the influx of calcium ions through an L-type calcium channel is probably necessary for a full α₁-adrenoceptor mediated activation and translocation of PKC.
Effect of halothane on K+ and carbachol stimulated [3H]noradrenaline release and increased [Ca2+](i) in SH-SY5Y human neuroblastoma cells
1997, British Journal of Anaesthesia
We have examined the effects of the volatile general anaesthetic agent, halothane, on K+ and carbachol stimulated [3H]noradrenaline release and associated increases in intracellular Ca2+ in a cultured human neuroblastoma cell line, SH-SY5Y. K+ (but not carbachol) stimulated [3H]noradrenaline release, and the increase in intracellular Ca2+ concentration was entirely extracellular Ca2+ dependent. Halothane produced a dose-dependent reduction in K+ evoked release of [3H]noradrenaline with significant inhibition (17%) occurring from 1.26 atm%. Basal and carbachol evoked release were unaffected. Halothane also produced a dose-dependent reduction in K+ evoked increases (measured at the peak) in intracellular Ca2+ with significant inhibition (29%) occurring from 0.88 atm%. K+ plateau, basal and carbachol evoked increases in intracellular Ca2+ were unaffected. These data suggest that halothane reduced Ca2+ entry through voltage-sensitive Ca2+ channels and implicate this important class of ion channel in the mechanism of anaesthesia.
Inhibition of M<inf>3</inf> muscarinic acetylcholine receptor-mediated Ca2+ influx and intracellular Ca2+ mobilization in neuroblastoma cells by the Ca2+/calmodulin-dependent protein kinase inhibitor 1-[N,O-bis(5- isoquinolinesulfonyl)-N-methyl-L-trosyl]-4-phenylpiperazine (KN-62)
1997, Biochemical Pharmacology
The role of $Ca^{2+} calmodulin - dependent$ protein kinase (CaM kinase; EC 2.7.1.123) in the generation of Ca²⁺ signals by muscarinic acetylcholine receptors (mAChR) was studied. Changes in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) induced by mAChR activation were monitored in SK-N-SH human neuroblastoma cells using the dye Fura-2. SK-N-SH cells express M₃ mAChR, as well as CaM kinase types II and IV, which are specifically inhibited by the CaM kinase antagonist KN-62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine). Carbamylcholine (100 μM) elicited an initial transient peak in [Ca²⁺]_i due to mobilization of Ca²⁺ from internal stores, followed by a sustained elevation in [Ca²⁺]_i that depended on the influx of extracellular Ca²⁺ and which was inhibited by EGTA and Ni²⁺. These mAChR-induced Ca²⁺ signals were diminished to an equal extent by preincubating the cells with 0.01 to 100 μM KN-62. KN-62 inhibited mAChR-induced Ca²⁺ influx and mobilization from internal stores by about 25–30%, producing a half-maximal effect at ≈ lμM. In contrast, KN-62 (25 μM) almost completely abolished carbamylcholinestimulated entry of divalent cations through Mn²⁺-permeant channels, as revealed by Mn²⁺ quenching of Fura-2 fluorescence. KN-62 also almost completely abolished Ca²⁺ influx induced by depolarization of the cells with 25 mM K⁺ (lC₅₀ ≈ 3 μM). These results suggest that CaM kinases regulate both the mobilization of intracellular Ca²⁺ and the stimulation of Ca²⁺ influx that are induced by mAChR activation, and indicate that the mAChR-induced influx of Ca²⁺ occurs through Ca²⁺ channels other than, or in addition to, the voltage-gated calcium channels or Mn²⁺-permeant channels which are inhibited by KN-62.

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Carbachol-stimulated calcium entry in SH-SY5Y human neuroblastoma cells: Which route?

Summary

Dev Brain Res

Trends Neurosci

Biochem Biophys Res Commun

Eur J Pharmacol

Trends Pharmacol Sci

Eur J Pharmacol

Biochem Pharmacol

Mol Brain Res

Trends Pharmacol Sci

Exp Cell Res

Trends Pharmacol Sci

Cell Calcium

FEBS Lett

FEBS Lett

Trends Neurosci

Morphology and growth, tumorigenicity and cytogenetics of human neuroblastoma cells in continuous culture

Cancer Res

Inositol trisphosphate and diacylglycerol: two interacting second messengers

Annu Rev Biochem

Inositol phosphates and cell signalling

Nature (Lond)