δ‐ and μ‐opioid receptor mobilization of intracellular calcium in SH‐SY5Y human neuroblastoma cells

Connor, Mark; Henderson, Graeme

doi:10.1111/j.1476-5381.1996.tb15195.x

Cited by 62 publications

(54 citation statements)

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“…In SH-SY5Y cells, DOR agonists couple to G␣ i/o proteins to inhibit Ca 2ϩ -sensitive AC enzymes 1 and 8 (30) and muscarinic agonists such as carbachol act at M 3 R to activate G␣ q and increase [Ca 2ϩ ] i , with no observable effect on AC (34,43,44). Alteration of RGS4 levels in SH-SY5Y cells by shRNA knockdown or MG132 protection against degradation modulates sig- naling of the DOR agonist SNC80 to AC and the MAPK pathway (Ref.…”

Section: Discussionmentioning

confidence: 99%

Opioid-induced Down-Regulation of RGS4

Wang¹,

Traynor

2011

Journal of Biological Chemistry

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Regulator of G protein signaling protein 4 (RGS4) acts as a GTPase accelerating protein to modulate -and ␦-opioid receptor (MOR and DOR, respectively) signaling. In turn, exposure to MOR agonists leads to changes in RGS4 at the mRNA and/or protein level. Here we have used human neuroblastoma SH-SY5Y cells that endogenously express MOR, DOR, and RGS4 to study opioid-mediated down-regulation of RGS4. Overnight treatment of SH-SY5Y cells with the MOR agonist DAMGO or the DOR agonist DPDPE decreased RGS4 protein by ϳ60% accompanied by a profound loss of opioid receptors but with no change in RGS4 mRNA. The decrease in RGS4 protein was prevented by the pretreatment with pertussis toxin or the opioid antagonist naloxone. The agonist-induced down-regulation of RGS4 proteins was completely blocked by treatment with the proteasome inhibitors MG132 or lactacystin or high concentrations of leupeptin, indicating involvement of ubiquitin-proteasome and lysosomal degradation. Polyubiquitinated RGS4 protein was observed in the presence of MG132 or the specific proteasome inhibitor lactacystin and promoted by opioid agonist. The loss of opioid receptors was not prevented by MG132, demonstrating a different degradation pathway. RGS4 is a GTPase accelerating protein for both G␣ i/o and G␣ q proteins. After overnight treatment with DAMGO to reduce RGS4 protein, signaling at the G␣ i/o -coupled DOR and the G␣ q -coupled M 3 muscarinic receptor (M 3 R) was increased but not signaling of the ␣ 2 adrenergic receptor or bradykinin BK 2 receptor, suggesting the development of cross-talk between the DOR and M 3 R involving RGS4. Regulator of G-protein signaling (RGS)2 proteins are a family of more than 30 molecules that control the duration of G protein-coupled receptor (GPCR)-mediated G protein signaling by acting as GTPase accelerating proteins (GAPs) (1-3). RGS4 is a small RGS protein (ϳ28 kDa) and a member of the R4 subfamily of RGS proteins with a structure consisting of the RGS homology domain (RH domain) and a small N terminus that may be important for defining specificity of action at GPCRs (1). However, RGS4 is a promiscuous GAP and shares activity at both G␣ q (4, 5) and G␣ i/o proteins (6, 7). The protein is unstable and subject to degradation by the N-end rule pathway (8 -12). RGS4 is also regulated by transcription and RNA stabilization (13). This means that levels of this protein can be readily regulated. Indeed, stress and several drugs of abuse have been shown to alter RGS4 levels (13-15).RGS4 has a wide distribution in brain, including regions important for the action of analgesic drugs acting at -opioid receptors (MOR) such as the cortex, thalamus, caudate putamen, nucleus accumbens, amygdala, locus ceruleus, periaqueductal gray, and dorsal horn of the spinal cord (16). Consequently, RGS4 has been linked to the regulation of MOR signaling in several in vitro systems (17-19), and a RGS4 knockout mouse shows phenotypic behaviors in response to MOR opioid agonist (20). In support of a role for RGS4 in the pharmacology of M...

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Section: Discussionmentioning

confidence: 99%

Opioid-induced Down-Regulation of RGS4

Wang¹,

Traynor

2011

Journal of Biological Chemistry

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“…Intracellular calcium mobilization was measured in con¯uent monolayers of NG108-15 cells using the¯uorescent Ca 2+ dye fura-2 (Connor & Henderson, 1996). Brie¯y, cells were seeded onto poly-L-lysine-treated (0.1 mg ml 71 ) plastic slides and cultured in Leighton tubes.…”

Section: Intracellular Calcium Measurementsmentioning

confidence: 99%

Second messenger‐dependent protein kinases and protein synthesis regulate endogenous secretin receptor responsiveness

Ghadessy

Kelly

2002

British J Pharmacology

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1 The present study investigated the role of second messenger-dependent protein kinase A (PKA) and C (PKC) in the regulation of endogenous secretin receptor responsiveness in NG108-15 mouse neuroblastoma6rat glioma hybrid cells. 2 In whole cell cyclic AMP accumulation studies, activation of PKC either by phorbol 12-myristate 13-acetate (PMA) or by purinoceptor stimulation using uridine 5'-triphosphate (UTP) decreased secretin receptor responsiveness. PKC activation also inhibited forskolin-stimulated cyclic AMP accumulation but did not a ect cyclic AMP responses mediated by the prostanoid-IP receptor agonist iloprost, or the A 2 adenosine receptor agonist 5'-(N-ethylcarboxamido) adenosine (NECA). 3 In additivity experiments, saturating concentrations of secretin and iloprost were found to be additive in terms of cyclic AMP accumulation, whereas saturating concentrations of NECA and iloprost together were not. This suggests compartmentalization of G s -coupling components in NG108-15 cells and possible heterologous regulation of secretin receptor responsiveness at the level of adenylyl cyclase activation. 4 Cells exposed to the PKA inhibitor H-89, exhibited a time-dependent increase in secretin receptor responsiveness compared to control cells. This e ect was selective since cyclic AMP responses to forskolin, iloprost and NECA were not a ected by H-89 treatment. Furthermore, treatment with the protein synthesis inhibitor cycloheximide produced a time-dependent increase in secretin receptor responsiveness. 5 Together these results indicate that endogenous secretin receptor responsiveness is regulated by PKC, PKA and protein neosynthesis in NG108-15 cells.

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“…Opioid receptor activation has been shown to evoke numerous cellular responses via G i /G o proteins, for example, inhibition of N-type Ca 2+ channels, inhibition of adenylyl cyclase, activation of K + currents (for review, see [12]) and intracellular Ca 2+ mobilization [2,8]. Mu-opioid receptor activation in cultured bovine adrenal medullary chromaffin cells has also been shown to potentiate Ca 2+ -dependent K + channel activation [17].…”

Section: +mentioning

confidence: 99%

“…Mu-opioid receptor activation in cultured bovine adrenal medullary chromaffin cells has also been shown to potentiate Ca 2+ -dependent K + channel activation [17]. Opioid receptor-mediated increases in intracellular Ca 2+ has been reported to arise from two mechanisms, namely, Ca 2+ influx across the plasmalemma [4,16] and Ca 2+ release from inositol 1,4,5-trisphosphate (IP 3 )-sensitive Ca 2+ stores [2,8]. In rat intracardiac neurones, activation of μ-opioid receptors has been shown to inhibit N-type Ca 2+ channels via a Pertussis toxin-sensitive G-protein [1].…”

Section: +mentioning

confidence: 99%

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Met-enkephalin-induced mobilization of intracellular Ca2+ in rat intracardiac ganglion neurones

Smith

Adams

1999

Neuroscience Letters

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The effects of Met-enkephalin on Ca 2+-dependent K + channel activity were investigated using the cell-attached patch recording technique on isolated parasympathetic neurones of rat intracardiac ganglia. Large-conductance, Ca 2+ -dependent K + channels (BK Ca ) were examined as an assay of agonist-induced changes in the intracellular free calcium ion concentration ([Ca 2+ ] i ). These BK Ca channels had a conductance of ˜200 pS and were charybdotoxin-and voltage-sensitive. Caffeine (5 mM), used as a control, evoked a large increase in BK Ca channel activity, which was inhibited by 10 μM ryanodine. Met-enkephalin (10 μM) evoked a similar increase in BK Ca channel activity, which was dependent on the presence of extracellular Ca 2+ and inhibited by either ryanodine (10 μM) or naloxone (1 μM). In Fura-2-loaded intracardiac neurones, Met-enkephalin evoked a transient increase in [Ca 2+ ] i . Metenkephalin-induced mobilization of intracellular Ca 2+ may play a role in neuronal excitability and firing behaviour in mammalian intracardiac ganglia.Keywords: Parasympathetic ganglia; Intracellular Ca 2+ ; Caffeine; Ryanodine; Opioid receptor; CaIndex Terms: heart innervation Endogenous opioid peptides have been found in the extrinsic and intrinsic innervation of the rat heart [7] and vagal transmission to the heart has been shown to be inhibited by exogenous opioid substances [11,18] channels via a Pertussis toxin-sensitive G-protein [1]. However, the action potential after-hyperpolarization due to a Ca 2+ -dependent K + conductance(s) in these neurones was unaltered in the presence of Met-enkephalin. In the present study, the actions of Met-enkephalin and caffeine, a known intracellular Ca 2+ mobilizing agent, on intracellular Ca 2+ mobilization and activation of the large conductance Ca 2+ -dependent K + channels (BK Ca ) were investigated.Parasympathetic neurones from neonatal rat intracardiac ganglia were isolated as previously described [19]. The dissociated neurones were plated onto glass coverslips coated with laminin, incubated at 37°C in 95% O 2 -5% CO 2 atmosphere and used for experiments within 36-72 h. At the time of experiments, the neuroneplated glass coverslips were transferred to a recording chamber (0.5 ml volume) and mounted on the stage of an inverted phase contrast microscope. Unitary current recordings were obtained using the cell-attached patch recording configuration of the patch clamp technique [6]. In the cell-attached configuration, K + currents in the membrane patch follow the relationship i K =γ(V m −V h −E K ). When the cells were bathed in a depolarizing external solution (V m ˜0 mV) and V h was held at 0 mV, the membrane potential in the patch was close to 0 mV. Unitary currents were recorded using an Axopatch 200B patch clamp amplifier (Axon Instruments, Foster City, CA), filtered at 5 kHz, and stored on digital tape using a DAT digital recorder (DTR-1204, Biologic Science Instruments, Claix, France). Records were transferred to a Pentium computer using an analogue-to-digit...

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δ‐ and μ‐opioid receptor mobilization of intracellular calcium in SH‐SY5Y human neuroblastoma cells

Cited by 62 publications

References 32 publications

Opioid-induced Down-Regulation of RGS4

Opioid-induced Down-Regulation of RGS4

Second messenger‐dependent protein kinases and protein synthesis regulate endogenous secretin receptor responsiveness

Met-enkephalin-induced mobilization of intracellular Ca2+ in rat intracardiac ganglion neurones

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