Publications by authors named "Jon Merlin"

14 Publications

  • Page 1 of 1

Pharmacological Insights Into Safety and Efficacy Determinants for the Development of Adenosine Receptor Biased Agonists in the Treatment of Heart Failure.

Front Pharmacol 2021 11;12:628060. Epub 2021 Mar 11.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

Adenosine A receptors (AR) are a potential target for cardiac injury treatment due to their cardioprotective/antihypertrophic actions, but drug development has been hampered by on-target side effects such as bradycardia and altered renal hemodynamics. Biased agonism has emerged as an attractive mechanism for AR-mediated cardioprotection that is haemodynamically safe. Here we investigate the pre-clinical pharmacology, efficacy and side-effect profile of the AR agonist neladenoson, shown to be safe but ineffective in phase IIb trials for the treatment of heart failure. We compare this agent with the well-characterized, pan-adenosine receptor (AR) agonist NECA, capadenoson, and the AR biased agonist VCP746, previously shown to be safe and cardioprotective in pre-clinical models of heart failure. We show that like VCP746, neladenoson is biased away from Ca influx relative to NECA and the cAMP pathway at the AR, a profile predictive of a lack of adenosine-like side effects. Additionally, neladenoson was also biased away from the MAPK pathway at the AR. In contrast to VCP746, which displays more 'adenosine-like' signaling at the AR, neladenoson was a highly selective AR agonist, with biased, weak agonism at the AR. Together these results show that unwanted hemodynamic effects of AR agonists can be avoided by compounds biased away from Ca influx relative to cAMP, relative to NECA. The failure of neladenoson to reach primary endpoints in clinical trials suggests that AR-mediated cAMP inhibition may be a poor indicator of effectiveness in chronic heart failure. This study provides additional information that can aid future screening and/or design of improved AR agonists that are safe and efficacious in treating heart failure in patients.
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http://dx.doi.org/10.3389/fphar.2021.628060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7991592PMC
March 2021

The metabolic effects of mirabegron are mediated primarily by β -adrenoceptors.

Pharmacol Res Perspect 2020 10;8(5):e00643

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic., Australia.

The β -adrenoceptor agonist mirabegron is approved for use for overactive bladder and has been purported to be useful in the treatment of obesity-related metabolic diseases in humans, including those involving disturbances of glucose homeostasis. We investigated the effect of mirabegron on glucose homeostasis with in vitro and in vivo models, focusing on its selectivity at β-adrenoceptors, ability to cause browning of white adipocytes, and the role of UCP1 in glucose homeostasis. In mouse brown, white, and brite adipocytes, mirabegron-mediated effects were examined on cyclic AMP, UCP1 mRNA, [ H]-2-deoxyglucose uptake, cellular glycolysis, and O consumption. Mirabegron increased cyclic AMP levels, UCP1 mRNA content, glucose uptake, and cellular glycolysis in brown adipocytes, and these effects were either absent or reduced in white adipocytes. In brite adipocytes, mirabegron increased cyclic AMP levels and UCP1 mRNA content resulting in increased UCP1-mediated oxygen consumption, glucose uptake, and cellular glycolysis. The metabolic effects of mirabegron in both brown and brite adipocytes were primarily due to actions at β -adrenoceptors as they were largely absent in adipocytes derived from β -adrenoceptor knockout mice. In vivo, mirabegron increased whole body oxygen consumption, glucose uptake into brown and inguinal white adipose tissue, and improved glucose tolerance, all effects that required the presence of the β -adrenoceptor. Furthermore, in UCP1 knockout mice, the effects of mirabegron on glucose tolerance were attenuated. Thus, mirabegron had effects on cellular metabolism in adipocytes that improved glucose handling in vivo, and were primarily due to actions at the β -adrenoceptor.
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http://dx.doi.org/10.1002/prp2.643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437350PMC
October 2020

BRL37344 stimulates GLUT4 translocation and glucose uptake in skeletal muscle via β-adrenoceptors without causing classical receptor desensitization.

Am J Physiol Regul Integr Comp Physiol 2019 05 20;316(5):R666-R677. Epub 2019 Mar 20.

Department of Molecular Biosciences, The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University , Stockholm , Sweden.

The type 2 diabetes epidemic makes it important to find insulin-independent ways to improve glucose homeostasis. This study examines the mechanisms activated by a dual β-/β-adrenoceptor agonist, BRL37344, to increase glucose uptake in skeletal muscle and its effects on glucose homeostasis in vivo. We measured the effect of BRL37344 on glucose uptake, glucose transporter 4 (GLUT4) translocation, cAMP levels, β-adrenoceptor desensitization, β-arrestin recruitment, Akt, AMPK, and mammalian target of rapamycin (mTOR) phosphorylation using L6 skeletal muscle cells as a model. We further tested the ability of BRL37344 to modulate skeletal muscle glucose metabolism in animal models (glucose tolerance tests and in vivo and ex vivo skeletal muscle glucose uptake). In L6 cells, BRL37344 increased GLUT4 translocation and glucose uptake only by activation of β-adrenoceptors, with a similar potency and efficacy to that of the nonselective β-adrenoceptor agonist isoprenaline, despite being a partial agonist with respect to cAMP generation. GLUT4 translocation occurred independently of Akt and AMPK phosphorylation but was dependent on mTORC2. Furthermore, in contrast to isoprenaline, BRL37344 did not promote agonist-mediated desensitization and failed to recruit β-arrestin1/2 to the β-adrenoceptor. In conclusion, BRL37344 improved glucose tolerance and increased glucose uptake into skeletal muscle in vivo and ex vivo through a β-adrenoceptor-mediated mechanism independently of Akt. BRL37344 was a partial agonist with respect to cAMP, but a full agonist for glucose uptake, and importantly did not cause classical receptor desensitization or internalization of the receptor.
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http://dx.doi.org/10.1152/ajpregu.00285.2018DOI Listing
May 2019

Adrenoceptors in white, brown, and brite adipocytes.

Br J Pharmacol 2019 07 7;176(14):2416-2432. Epub 2019 Apr 7.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

Adrenoceptors play an important role in adipose tissue biology and physiology that includes regulating the synthesis and storage of triglycerides (lipogenesis), the breakdown of stored triglycerides (lipolysis), thermogenesis (heat production), glucose metabolism, and the secretion of adipocyte-derived hormones that can control whole-body energy homeostasis. These processes are regulated by the sympathetic nervous system through actions at different adrenoceptor subtypes expressed in adipose tissue depots. In this review, we have highlighted the role of adrenoceptor subtypes in white, brown, and brite adipocytes in both rodents and humans and have included detailed analysis of adrenoceptor expression in human adipose tissue and clonally derived adipocytes. We discuss important considerations when investigating adrenoceptor function in adipose tissue or adipocytes. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.
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http://dx.doi.org/10.1111/bph.14631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592855PMC
July 2019

Effects of hypoxia-ischemia and inotropes on expression of cardiac adrenoceptors in the preterm fetal sheep.

J Appl Physiol (1985) 2018 11 23;125(5):1368-1377. Epub 2018 Aug 23.

The Ritchie Centre, The Hudson Institute of Medical Research , Melbourne , Australia.

Preterm infants frequently suffer cardiovascular compromise, with hypotension and/or low systemic blood flow, leading to tissue hypoxia-ischemia (HI). Many preterm infants respond inadequately to inotropic treatments using adrenergic agonists such as dobutamine (DB) or dopamine (DA). This may be because of altered cardiac adrenoceptor expression because of tissue HI or prolonged exposure to adrenergic agonists. We assessed the effects of severe HI with and without DB/DA treatment on cardiac adrenoceptor expression in preterm fetal sheep. Fetal sheep (93-95 days) exposed to sham surgery or severe HI induced by umbilical cord occlusion received intravenous DB or saline for 74 h (HI + DB, HI, Sham + DB, Sham). The HI groups were also compared with fetal sheep exposed to HI and DA. Fetal hearts were collected to determine β-adrenoceptor numbers using [I]-cyanopindolol binding and mRNA expression of β-, β-, α-, α-, or α-adrenoceptors. The HI group had increased β-adrenoceptor numbers compared with all other groups in all four heart chambers ( P < 0.05). This increase in β-adrenoceptor numbers in the HI group was significantly reduced by DB infusion in all four heart chambers, but DA infusion in the HI group only reduced β-adrenoceptor numbers in the left atria and ventricle. DB alone did not affect β-adrenoceptor numbers in the sham animals. Changes in β-adrenoceptor mRNA levels trended to parallel the binding results. We conclude that HI upregulates preterm fetal cardiac β-adrenoceptors, but prolonged exposure to adrenergic agonists downregulates adrenoceptors in the preterm heart exposed to HI and may underpin the frequent failure of inotropic therapy in preterm infants. NEW & NOTEWORTHY This is the first study, to our knowledge, on the effects of hypoxia-ischemia and adrenergic agonists on adrenoceptors in the preterm heart. In fetal sheep, we demonstrate that hypoxia-ischemia increases cardiac β-adrenoceptor numbers. However, exposure to both hypoxia-ischemia and adrenergic agonists (dobutamine or dopamine) reduces the increase in β-adrenoceptor numbers, which may underpin the inadequate response in human preterm infants to inotropic therapy using adrenergic agonists. Dobutamine alone does not affect the cardiac adrenoceptors in the sham animals.
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http://dx.doi.org/10.1152/japplphysiol.00472.2018DOI Listing
November 2018

Rosiglitazone and a β-Adrenoceptor Agonist Are Both Required for Functional Browning of White Adipocytes in Culture.

Front Endocrinol (Lausanne) 2018 30;9:249. Epub 2018 May 30.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

The recruitment of brite (or beige) adipocytes has been advocated as a means to combat obesity, due to their ability to phenotypically resemble brown adipocytes (BA). Lineage studies indicate that brite adipocytes are formed by differentiation of precursor cells or by direct conversion of existing white adipocytes, depending on the adipose depot examined. We have systematically compared the gene expression profile and a functional output (oxygen consumption) in mouse adipocytes cultured from two contrasting depots, namely interscapular brown adipose tissue, and inguinal white adipose tissue (iWAT), following treatment with a known browning agent, the peroxisome proliferator-activated receptor (PPARγ) activator rosiglitazone. Prototypical BA readily express uncoupling protein (UCP)1, and upstream regulators including the β-adrenoceptor and transcription factors involved in energy homeostasis. Adipocytes from inguinal WAT display maximal UCP1 expression and mitochondrial uncoupling only when treated with a combination of the PPARγ activator rosiglitazone and a β-adrenoceptor agonist. In conclusion, brite adipocytes are fully activated only when a browning agent (rosiglitazone) and a thermogenic agent (β-adrenoceptor agonist) are added in combination. The presence of rosiglitazone throughout the 7-day culture period partially masks the effects of β-adrenoceptor signaling in inguinal white adipocyte cultures, whereas including rosiglitazone only for the first 3 days promotes robust β-adrenoceptor expression and provides an improved window for detection of β-adrenoceptor responses.
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http://dx.doi.org/10.3389/fendo.2018.00249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992408PMC
May 2018

The PPARγ agonist rosiglitazone promotes the induction of brite adipocytes, increasing β-adrenoceptor-mediated mitochondrial function and glucose uptake.

Cell Signal 2018 Jan 29;42:54-66. Epub 2017 Sep 29.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Monash University, Parkville, Victoria 3052, Australia; Department of Pharmacology, 9 Ancora Imparo Way, Monash University, Clayton, Victoria 3800, Australia. Electronic address:

Recruitment and activation of brite (or beige) adipocytes has been advocated as a potential avenue for manipulating whole-body energy expenditure. Despite numerous studies illustrating the differences in gene and protein markers between brown, brite and white adipocytes, there is very little information on the adrenergic regulation and function of these brite adipocytes. We have compared the functional (cyclic AMP accumulation, oxygen consumption rates, mitochondrial function, glucose uptake, extracellular acidification rates, calcium influx) profiles of mouse adipocytes cultured from three contrasting depots, namely interscapular brown adipose tissue, and inguinal or epididymal white adipose tissues, following chronic treatment with the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone. Prototypical brown adipocytes readily express β-adrenoceptors, and β-adrenoceptor stimulation increases cyclic AMP accumulation, oxygen consumption rates, mitochondrial function, glucose uptake, and extracellular acidification rates. Treatment of brown adipocytes with rosiglitazone increases uncoupling protein 1 (UCP1) levels, and increases β-adrenoceptor mitochondrial function but does not affect glucose uptake responses. In contrast, inguinal white adipocytes only express UCP1 and β-adrenoceptors following rosiglitazone treatment, which results in an increase in all β-adrenoceptor-mediated functions. The effect of rosiglitazone in epididymal white adipocytes, was much lower compared to inguinal white adipocytes. Rosiglitazone also increased α-adrenoceptor mediated increases in calcium influx and glucose uptake (but not mitochondrial function) in inguinal and epididymal white adipocytes. In conclusion, the PPARγ agonist rosiglitazone promotes the induction and function of brite adipocytes cultured from inguinal and epididymal white adipose depots.
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http://dx.doi.org/10.1016/j.cellsig.2017.09.023DOI Listing
January 2018

Factors influencing biased agonism in recombinant cells expressing the human α -adrenoceptor.

Br J Pharmacol 2017 Jul 10;174(14):2318-2333. Epub 2017 Jun 10.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

Background And Purpose: Agonists acting at GPCRs promote biased signalling via Gα or Gβγ subunits, GPCR kinases and β-arrestins. Since the demonstration of biased agonism has implications for drug discovery, it is essential to consider confounding factors contributing to bias. We have examined bias at human α -adrenoceptors stably expressed at low levels in CHO-K1 cells, identifying off-target effects at endogenous receptors that contribute to ERK1/2 phosphorylation in response to the agonist oxymetazoline.

Experimental Approach: Intracellular Ca mobilization was monitored in a Flexstation® using Fluo 4-AM. The accumulation of cAMP and ERK1/2 phosphorylation were measured using AlphaScreen® proximity assays, and mRNA expression was measured by RT-qPCR. Ligand bias was determined using the operational model of agonism.

Key Results: Noradrenaline, phenylephrine, methoxamine and A61603 increased Ca mobilization, cAMP accumulation and ERK1/2 phosphorylation. However, oxymetazoline showed low efficacy for Ca mobilization, no effect on cAMP generation and high efficacy for ERK1/2 phosphorylation. The apparent functional selectivity of oxymetazoline towards ERK1/2 was related to off-target effects at 5-HT receptors endogenously expressed in CHO-K1 cells. Phenylephrine and methoxamine showed genuine bias towards ERK1/2 phosphorylation compared to Ca and cAMP pathways, whereas A61603 displayed bias towards cAMP accumulation compared to ERK1/2 phosphorylation.

Conclusion And Implications: We have shown that while adrenergic agonists display bias at human α -adrenoceptors, the marked bias of oxymetazoline for ERK1/2 phosphorylation originates from off-target effects. Commonly used cell lines express a repertoire of endogenous GPCRs that may confound studies on biased agonism at recombinant receptors.
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http://dx.doi.org/10.1111/bph.13837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481649PMC
July 2017

Could burning fat start with a brite spark? Pharmacological and nutritional ways to promote thermogenesis.

Mol Nutr Food Res 2016 Jan 27;60(1):18-42. Epub 2015 Aug 27.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia.

There are two types of adipose tissue with distinct functions-white adipose tissue stores chemical energy as triglycerides, whereas brown adipose tissue consumes energy and releases heat (thermogenesis) in response to sympathetic nerve activity. In humans, treatments that promote greater brown adipose tissue deposition and/or activity would be highly beneficial in regimes aimed at reducing obesity. Adult humans have restricted populations of prototypical brown adipocytes in the neck and chest areas, but recent advances have established that adipocytes with similar properties, termed "brite" adipocytes, can be recruited in subcutaneous depots thought to be primarily white adipose tissue. These brite adipocytes express the protein machinery required for thermogenesis, but to assess brite adipocytes as viable therapeutic targets we need to understand how to promote conversion of white adipocytes to brite adipocytes and ways to increase optimal energy consumption and thermogenesis in these brite adipocytes. This can be accomplished by pharmacological and nutritional therapies to differing degrees, as reviewed in detail here.
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http://dx.doi.org/10.1002/mnfr.201500251DOI Listing
January 2016

Orthosteric binding of ρ-Da1a, a natural peptide of snake venom interacting selectively with the α1A-adrenoceptor.

PLoS One 2013 25;8(7):e68841. Epub 2013 Jul 25.

Commissariat à l'Énergie Atomique Et Aux Énergies Alternatives, iBiTec-S, Service d'Ingénierie Moléculaire des Protéines, Gif sur Yvette, France.

ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of (3)H-prazosin or (125)I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of (3)H-prazosin or (125)I-HEAT, and the IC50 of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca(2+) release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α1A-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D106(3.32)A and the S188(5.42)A/S192(5.46)A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86(2.64)A, F288(6.51)A and F312(7.39)A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86(2.64) was identified as a key interaction point for (125)I-HEAT, as the variant F86(2.64)A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86(2.64), F288(6.51) and F312(7.39)) and agonist (F288(6.51) and F312(7.39)) ligands. Its selectivity for the α1A-adrenoceptor may result, at least partly, from its interaction with the residue F86(2.64), which appears to be important also for HEAT binding.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0068841PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3723878PMC
March 2014

β(2)-Adrenoceptors increase translocation of GLUT4 via GPCR kinase sites in the receptor C-terminal tail.

Br J Pharmacol 2012 Mar;165(5):1442-56

Department of Physiology, The Wenner-Gren Institute, Arrhenius Laboratories F3, Stockholm University, Stockholm, Sweden.

Background And Purpose: β-Adrenoceptor stimulation induces glucose uptake in several insulin-sensitive tissues by poorly understood mechanisms.

Experimental Approach: We used a model system in CHO-K1 cells expressing the human β(2)-adrenoceptor and glucose transporter 4 (GLUT4) to investigate the signalling mechanisms involved.

Key Results: In CHO-K1 cells, there was no response to β-adrenoceptor agonists. The introduction of β(2)-adrenoceptors and GLUT4 into these cells caused increased glucose uptake in response to β-adrenoceptor agonists. GLUT4 translocation occurred in response to insulin and β(2)-adrenoceptor stimulation, although the key insulin signalling intermediate PKB was not phosphorylated in response to β(2)-adrenoceptor stimulation. Truncation of the C-terminus of the β(2)-adrenoceptor at position 349 to remove known phosphorylation sites for GPCR kinases (GRKs) or at position 344 to remove an additional PKA site together with the GRK phosphorylation sites did not significantly affect cAMP accumulation but decreased β(2)-adrenoceptor-stimulated glucose uptake. Furthermore, inhibition of GRK by transfection of the βARKct construct inhibited β(2)-adrenoceptor-mediated glucose uptake and GLUT4 translocation, and overexpression of a kinase-dead GRK2 mutant (GRK2 K220R) also inhibited GLUT4 translocation. Introducing β(2)-adrenoceptors lacking phosphorylation sites for GRK or PKA demonstrated that the GRK sites, but not the PKA sites, were necessary for GLUT4 translocation.

Conclusions And Implications: Glucose uptake in response to activation of β(2)-adrenoceptors involves translocation of GLUT4 in this model system. The mechanism is dependent on the C-terminus of the β(2)-adrenoceptor, requires GRK phosphorylation sites, and involves a signalling pathway distinct from that stimulated by insulin.
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http://dx.doi.org/10.1111/j.1476-5381.2011.01647.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3372728PMC
March 2012

α₂-Adrenoceptors activate noradrenaline-mediated glycogen turnover in chick astrocytes.

J Neurochem 2011 Jun 26;117(5):915-26. Epub 2011 Apr 26.

Department of Pharmacology, Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 399 Royal Parade, Parkville, Victoria 3052, Australia.

In the brain, glycogen is primarily stored in astrocytes where it is regulated by several hormones/neurotransmitters, including noradrenaline that controls glycogen breakdown (in the short term) and synthesis. Here, we have examined the adrenoceptor (AR) subtype that mediates the glycogenic effect of noradrenaline in chick primary astrocytes by the measurement of glycogen turnover (total (14) C incorporation of glucose into glycogen) following noradrenergic activation. Noradrenaline and insulin increased glycogen turnover in a concentration-dependent manner. The effect of noradrenaline was mimicked by stimulation of α(2) -ARs (and to a lesser degree by β(3) -ARs), but not by stimulation of α(1) -, β(1) -, or β(2) -ARs, and occurred only in astrocytes and not neurons. In chick astrocytes, studies using RT-PCR and radioligand binding showed that α(2A) - and α(2C) -AR mRNA and protein were present. α(2) -AR- or insulin-mediated glycogen turnover was inhibited by phosphatidylinositol-3 kinase inhibitors, and both insulin and clonidine caused phosphorylation of Akt and glycogen synthase kinase-3 in chick astrocytes. α(2) -AR but not insulin-mediated glycogen turnover was inhibited by pertussis toxin pre-treatment indicating involvement of Gi/o proteins. These results show that the increase in glycogen turnover caused by noradrenaline is because of activation of α(2) -ARs that increase glycogen turnover in astrocytes utilizing a Gi/o-PI3K pathway.
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http://dx.doi.org/10.1111/j.1471-4159.2011.07261.xDOI Listing
June 2011

Quantification of functional selectivity at the human α(1A)-adrenoceptor.

Mol Pharmacol 2011 Feb 26;79(2):298-307. Epub 2010 Oct 26.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, 399 Royal Parade, Parkville, Victoria 3052, Australia.

Although G protein-coupled receptors are often categorized in terms of their primary coupling to a given type of Gα protein subunit, it is now well established that many show promiscuous coupling and activate multiple signaling pathways. Furthermore, some agonists selectively activate signaling pathways by promoting interaction between distinct receptor conformational states and particular Gα subunits or alternative signaling proteins. We have tested the capacity of agonists to stimulate Ca(2+) release, cAMP accumulation, and changes in extracellular acidification rate (ECAR) at the human α(1A)-adrenoceptor. Signaling bias factors were determined by novel application of an operational model of agonism and compared with the reference endogenous agonist norepinephrine; values significantly different from 1.0 indicated an agonist that promoted receptor conformations distinct from that favored by norepinephrine. Oxymetazoline was a full agonist for ECAR and a partial agonist for Ca(2+) release (bias factor 8.2) but failed to stimulate cAMP production. Phenylephrine showed substantial bias toward ECAR versus Ca(2+) release or cAMP accumulation (bias factors 21 and 33, respectively) but did not display bias between Ca(2+) and cAMP pathways. Cirazoline and N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide (A61603) displayed bias toward cAMP relative to Ca(2+) release (bias factors of 7.4 and 8.6). It is noteworthy that epinephrine, a second endogenous adrenoceptor agonist, did not display bias relative to norepinephrine. Our finding that phenylephrine displayed significant signaling bias, despite being highly similar in structure to epinephrine, indicates that subtle differences in agonist-receptor interaction can affect conformational changes in cytoplasmic domains and thereby modulate the repertoire of effector proteins that are activated.
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http://dx.doi.org/10.1124/mol.110.067454DOI Listing
February 2011

The M3-muscarinic acetylcholine receptor stimulates glucose uptake in L6 skeletal muscle cells by a CaMKK-AMPK-dependent mechanism.

Cell Signal 2010 Jul 4;22(7):1104-13. Epub 2010 Mar 4.

Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Victoria, 3800, Australia.

The role of muscarinic acetylcholine receptors (mAChRs) in regulating glucose uptake in L6 skeletal muscle cells was investigated. [(3)H]-2-Deoxyglucose uptake was increased in differentiated L6 cells by insulin, acetylcholine, oxotremorine-M and carbachol. mAChR-mediated glucose uptake was inhibited by the AMPK inhibitor Compound C. Whole cell radioligand binding using [(3)H]-N-methyl scopolamine chloride identified mAChRs in differentiated but not undifferentiated L6 cells and M(3) mAChR mRNA was detected only in differentiated cells. M(3) mAChRs are Gq-coupled, and cholinergic stimulation by the mAChR agonists acetylcholine, oxotremorine-M and carbachol increased Ca(2+) in differentiated but not undifferentiated L6 cells. This was due to muscarinic but not nicotinic activation as responses were antagonised by the muscarinic antagonist atropine but not the nicotinic antagonist tubocurarine. Western blotting showed that both carbachol and the AMPK activator AICAR increased phosphorylation of the AMPKalpha subunit at Thr172, with responses to carbachol blocked by Compound C and the CaMKK inhibitor STO609 but not by the PI3K inhibitor wortmannin. AICAR-stimulated AMPK phosphorylation was not sensitive to STO-609, confirming that this compound inhibits CaMKK but not the classical AMPK kinase LKB1. The TAK1 inhibitor (5Z)-7-oxozeaenol and the G(i) inhibitor pertussis toxin both failed to block AMPK phosphorylation in response to carbachol. Using CHO-K1 cells stably expressing each of the mAChR subtypes (M(1)-M(4)), it was determined that only the M(1) and M(3) mAChRs phosphorylate AMPK, confirming a G(q)-dependent mechanism. This study demonstrates that activation of M(3) mAChRs in L6 skeletal muscle cells stimulates glucose uptake via a CaMKK-AMPK-dependent mechanism, independent of the insulin-stimulated pathway.
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http://dx.doi.org/10.1016/j.cellsig.2010.03.004DOI Listing
July 2010