Dual effects of muscarinic M2 receptors on the synthesis of cyclic AMP in CHO cells: Background and model

doi:10.1016/S0024-3205(01)01045-1

Life Sciences

Volume 68, Issues 22–23, 27 April 2001, Pages 2501-2510

https://doi.org/10.1016/S0024-3205(01)01045-1 Get rights and content

Abstract

It has been observed in several laboratories that muscarinic agonists have dual effects on the synthesis of cyclic AMP in cell lines expressing muscarinic M₂ or M₄ receptors, producing strong inhibition at low agonist concentrations and lesser inhibition or stimulation at high agonist concentrations. Data obtained on CHO cells (known to express adenylyl cyclases VI and VII) are best interpreted on the assumption that the upward phase of the concentration-response curves reflects simultaneous inhibition of adenylyl cyclase VI via the Gi proteins, with which the M₂ and M₄ receptors communicate with high affinity, and stimulation of adenyly cyclases VI and VII via the Gs proteins, with which the M₂ and M₄ receptors communicate with low affinity. A simplified model is described which permits one to predict how the shapes of the concentration-response curves will be affected by changes in the concentration of receptors, the affinities of activated receptors for Gi or Gs proteins, and other parameters.

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Although the present data, together with the above-mentioned literature, point to high receptor expression levels as being a key factor in detection of bell-shape responses, the exact mechanisms underlying such responses are a matter of discussion. One group [27,28] suggested that U-shaped concentration-response in forskolin-stimulated cAMP production was due to a progressive saturation of muscarinic M2 receptor coupling to a “preferred Gα protein” (Gαi), followed by progressive receptor coupling to a “second preferred Gα protein” (Gαs), thus resulting in neutralization of the inhibitory response mediated by Gαi by the opposite effect of stimulatory Gαs. A similar idea was described for adenosine A1-induced cAMP accumulation in CHO cells.
A previous study observed bell-shaped concentration-response isotherms for activation of Gα_i3 G-protein subunits by high efficacy 5-HT_1A receptor agonists in a Chinese hamster ovary (CHO) cell line expressing high levels of these receptors. This suggested that a signaling switch took place in that cell line (from Gα_i3 to activation of other G-proteins) but it was unclear if such effects are observed for 5-HT_1A receptors in other cellular environments.
Here, using an antibody capture-based [³⁵S]GTPγS binding assay for Gα_i3 activation, we investigated whether efficacious 5-HT_1A receptor agonists (5-HT, F13714, befiradol, NLX-101), prototypical agonists ((+) and (−)8-OH-DPAT), and partial agonist, antagonists, inverse agonists (pindolol, WAY100635, spiperone) produced similar effects on 5 cell lines expressing different levels of human 5-HT_1A receptors.
In membranes from cell lines (HeLa, C6-glia and CHO-low) expressing moderate receptor levels (between 1 and 4 pmol/mg of protein), 5-HT, F13714, befiradol and NLX-101 elicited classical sigmoid concentration-response isotherms. In contrast, in cell lines (CHO-high, HEK-293F) expressing high receptor levels (>9 pmol/mg) these agonists elicited bell-shaped concentration-response isotherms that peaked at nanomolar-range concentrations and then returned to baseline or below. Spiperone elicited inverse agonist inhibitory sigmoid isotherms in all membrane preparations while WAY100635 was mostly ‘silent’ for Gα_i3 activation. The other compounds elicited diverse responses in the different cell lines suggesting that other factors, in addition to receptor expression levels, could be influencing Gα_i3 activation.
These data indicate that Gα_i3 G-protein activation by 5-HT_1A receptor ligands is highly dependent on receptor expression levels and on cellular background. Moreover, the induction of bell-shape concentration-response isotherms by 5-HT and other high-efficacy agonists is consistent with a switch in signaling to other G-protein-mediated signaling cascades, possibly elicited by receptor conformational changes.
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Thus, at porcine α2A-adrenoceptors, the agonist UK14304 can both inhibit adenylyl cyclase via Gi/o proteins and stimulate it via Gs proteins for higher concentrations [26]. Several mechanisms may give rise to bell-shaped isotherms in pharmacological systems [27]. Bell-shaped drug concentration response isotherms in in vitro models have been attributed to the presence of cross-reacting receptors [28,29] or promiscuous coupling to two or more signal transduction systems [30].
Following agonist action, G-protein-coupled receptors may exhibit differential coupling to G-proteins or second messenger pathways, supporting the notion of agonist-directed trafficking. To explore these mechanisms, we have designed and transfected synthetic siRNA duplexes to knockdown different G_α subunits in Chinese hamster ovary (CHO) cells expressing human (h)5-hydroxytryptamine 1A receptors (CHO-h5-HT_1A). siRNAs against G_αi2 and G_αi3 transfected alone or in combination caused a large decrease in the corresponding mRNA level (64–80%) and also at the protein level for G_αi3 (60–70%), whereas a non-specific siRNA showed no effect. In membranes of CHO-h5-HT_1A, 5-HT stimulated guanosine-5′-O-(3-[³⁵S]thio)-triphosphate ([³⁵S]GTPγS) binding was differentially affected by transfection of siRNAs against G_αi protein, siRNAs against G_αi2 inducing a more important decrease in the efficacy of 5-HT than transfection of siRNAs against G_αi3. The high potency component was abolished after transfection of siRNAs against G_αi3 and the lower potency component was suppressed after transfection of siRNAs against G_αi2. To directly investigate G_αi3 activation we used an antibody-capture/scintillation proximity assay. (+)8-OH-DPAT yielded bell-shaped curves for G_αi3 activation, a response that was abolished after transfection of siRNAs against G_αi3 protein. Interestingly, (+)8-OH-DPAT yielded a sigmoidal response when only G_αi3 protein was expressed. These data suggest that when efficacious agonists attain a high level of occupation of h5-HT_1A receptors, a change occurs that induces coupling to G_αi2 protein and suppresses signalling through G_αi3 subunits.
Section I. The Cholinergic System
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Signaling to a new form of MAPK—ERK5—involves the G‐proteins Gα(q) and Gα(12/13) and not Gα(i), Gα(s), or Gβγ subunits (Fukuhara et al., 2000). This variability is marked in pancreatic cells, where M2Rs and M4Rs if bound to G(i) inhibit adenylyl cyclase VI, whereas, if they are bound to G(s), they stimulate adenylyl cyclases VI and VII (Tucek et al., 2001). Alfonzo et al. (1998) show that membrane‐bound guanylyl cyclase (GC) is regulated by muscarinic agents through two opposing signaling pathways.
This chapter explains the cholinergic system that consists of the muscarinic system and the nicotinic system. Muscarinic receptors occur in all layers in the neocortex and are also distributed in the rest of the brain. The brain contains several potassium channels that are modulated by the cholinergic system. Acetylcholine (ACh) regulates two aspects of calcium activity in neurons: (1) receptor-mediated entry of calcium into the cell, and (2) the redistribution of intracellular calcium in different compartments of the neuron. Cholinergic receptor activation evokes complex changes in the distribution of intracellular calcium. Muscarinic receptors also modulate calcium waves in neurons. The muscarinic receptors activate postsynaptic cascades, including the mitogen-activated protein ser/thr kinase (MAPK) pathway that induces the phosphorylation of key protein molecules in the neuron by a second protein kinase leading to changes inter alia in ion channel activity. The ACh nicotinic receptor (NR) molecule forms a transmembrane channel that conducts Na⁺, K⁺, and Ca²⁺ ions. Stimulation of the receptor leads to depolarization of the membrane and activation of the neuron. NRs are active at the presynaptic and postsynaptic sites. The nicotinic system plays an important role in long-term potentiation (LTP) and learning.
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Properties, regulation and function of muscarinic receptorDual effects of muscarinic M2 receptors on the synthesis of cyclic AMP in CHO cells: Background and model

Abstract

Properties, regulation and function of muscarinic receptor
Dual effects of muscarinic M₂ receptors on the synthesis of cyclic AMP in CHO cells: Background and model