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Cholera toxin impairment of opioid-mediated inhibition of adenylate cyclase in neuroblastoma x glioma hybrid cells is
1Department of Biochemistry, University of Glasgow, Scotland, U.K.
Abstract:
Cholera toxin treatment (up to 1 microgram/ml, 16 h) of neuroblastoma x glioma hybrid NG108-15 cells produced a decrease of some 35% in both delta opioid receptor-mediated stimulation of high-affinity GTPase activity and inhibition of forskolin-amplified adenylate cyclase. Coincident with these decreases was a down-regulation of some 35% in the delta opioid receptor population. A similar pattern of a decrease in signalling capacity was noted for the alpha 2B-adrenergic receptor in these cells after cholera toxin treatment. Half-maximal effects of cholera toxin on all of the parameters assayed were noted at concentrations between 2 and 5 ng/ml. Neither levels of Gi2, as assessed by immunoblotting with specific antisera, nor the intrinsic activity of the alpha subunit of the guanine-nucleotide-binding protein which acts as the inhibitory G-protein of the adenylate cyclase in these cells, as assessed by guanosine 5'-[beta gamma-imido]triphosphate (Gpp[NH]p)-mediated inhibition of adenylate cyclase, was lowered by cholera toxin treatment. Furthermore, levels of another pertussis toxin-sensitive G-protein (Go) expressed by these cells was also not lowered by cholera toxin treatment. However, as previously noted in other cells [Milligan, Unson & Wakelam (1989) Biochem. J. 262, 643-649], marked down-regulation of the alpha subunit of the stimulatory G-protein (Gs) of the adenylate cyclase cascade was observed in response to cholera toxin treatment. Previous studies [Klee, Milligan, Simonds & Tocque (1985) Mol. Aspects Cell Regul. 4, 117-129] have shown that cholera toxin treatment can result in a decrease in the maximal effectiveness of agonists which function to inhibit adenylate cyclase. These data have been used as evidence to suggest a functional interaction between Gs and 'Gi'. The results provided herein demonstrate that such effects of the toxin can be explained adequately by a decrease in the number of receptors that function to produce inhibition of adenylate cyclase.
Insights
Cholera toxin treatment reduces delta opioid and alpha 2B-adrenergic receptor signaling by down-regulating receptor numbers. This explains the toxin's effect on adenylate cyclase inhibition, not by altering G-protein levels.
Area of Science:
- Cellular and Molecular Pharmacology
- Signal Transduction Pathways
- G Protein-Coupled Receptors
Background:
- Cholera toxin is known to affect G protein-coupled receptor (GPCR) signaling.
- Previous studies suggested a functional interaction between stimulatory G proteins (Gs) and inhibitory G proteins (Gi) based on cholera toxin's effects.
- Neuroblastoma x glioma hybrid NG108-15 cells express relevant receptors and G proteins for studying signal transduction.
Purpose of the Study:
- To investigate the precise mechanism by which cholera toxin treatment impacts delta opioid and alpha 2B-adrenergic receptor signaling.
- To determine if cholera toxin affects G protein levels or receptor populations.
- To clarify the role of receptor down-regulation versus G protein alterations in mediating the toxin's effects on adenylate cyclase activity.
Main Methods:
- Treatment of NG108-15 cells with varying concentrations of cholera toxin.
- Assay of delta opioid receptor-mediated high-affinity GTPase activity.
- Measurement of forskolin-amplified adenylate cyclase inhibition.
- Quantification of receptor populations via down-regulation.
- Immunoblotting to assess levels of Gi2 and Go proteins.
- Assessment of Gs alpha subunit levels and intrinsic activity.
Main Results:
- Cholera toxin treatment (2-5 ng/ml) decreased delta opioid receptor signaling and alpha 2B-adrenergic receptor signaling by approximately 35%.
- A corresponding 35% down-regulation of the delta opioid receptor population was observed.
- Cholera toxin did not alter levels of Gi2 or Go proteins, nor the intrinsic activity of the inhibitory G protein alpha subunit.
- Marked down-regulation of the stimulatory G-protein (Gs) alpha subunit was observed.
Conclusions:
- The observed decrease in delta opioid and alpha 2B-adrenergic receptor signaling capacity following cholera toxin treatment is adequately explained by a reduction in the number of these receptors.
- Cholera toxin's effects on inhibiting adenylate cyclase are primarily due to receptor down-regulation, not alterations in the levels or activity of inhibitory G proteins (Gi/Go).
- The data challenge previous suggestions of a direct functional interaction between Gs and Gi mediated by cholera toxin, attributing the effects to receptor number changes.