This article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.21/issuetoc.
BACKGROUND AND PURPOSEAgonists 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 1A -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 Ca2+ 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 RESULTSNoradrenaline, phenylephrine, methoxamine and A61603 increased Ca 2+ mobilization, cAMP accumulation and ERK1/2 phosphorylation. However, oxymetazoline showed low efficacy for Ca +2 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 offtarget effects at 5-HT 1B receptors endogenously expressed in CHO-K1 cells. Phenylephrine and methoxamine showed genuine bias towards ERK1/2 phosphorylation compared to Ca 2+ and cAMP pathways, whereas A61603 displayed bias towards cAMP accumulation compared to ERK1/2 phosphorylation.
CONCLUSION AND IMPLICATIONSWe have shown that while adrenergic agonists display bias at human α 1A -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.
AbbreviationsECAR, extracellular acidification rate; HEAT, 2-(β-4-hydroxyphenyl)ethylaminomethyltetralone; PTX, pertussis toxin
Diabetes augments the risk of hypertension. Although several factors have been implicated in the development of such hypertensive state, we designed this study to investigate blood pressure development, the activity of angiotensinconverting enzyme (ACE) in blood as well as sympathetic neurotransmission in the vas deferens of diabetic rats. We used streptozotocin (STZ)-induced diabetic rats (60 mg/kg) in order to evaluate the systolic blood pressure (SBP), ACE activity and peripheral sympathetic neurotransmission. We observed the following changes of parameters: increase of SBP, decrease of heart rate, augmentation of plasma ACE activity, enhancement of phasic and tonic vas deferens contractions elicited by electrical stimulation at 5 Hz, increase of maximal response to noradrenaline (NA) and decrease of adenosine triphosphate (ATP)-elicited contraction of vasa deferentia. The results reveal that in the development of hypertension in diabetic rats, augmentation of circulating ACE activity precedes the sympathetic dysfunction. Additionally, it seems that the purinergic and noradrenergic neurotransmission is compromised.
The testicular capsule contracts in response to noradrenaline and adrenaline, but the effects of adrenoceptor agonists, as for instance clonidine, had not yet been thoroughly evaluated. The testicular capsule from adult male Wistar rats was isolated and mounted in organ bath and cumulative concentration curves were performed for clonidine and other adrenergic agonists in the absence or presence of α-adrenoceptors antagonists. The order of potency for agonists (pD2) was clonidine=adrenaline>UK 14,304>noradrenaline>phenylephrine>methoxamine. The consecutive curves for clonidine showed desensitization with 3-fold rightward shift and Emax reduction of 40%. The noradrenaline curves were 4.5, 19 and 190-fold less potent after clonidine pretreatment at 10−5, 10−4 or 10−3 M for 10 min, respectively, added to Emax decrease by about 20%. Clonidine (10−5 M for 10 min) was unable to alter the noradrenaline curves if the treatment was made in the presence of idazoxan (α2-adrenoceptor antagonist) whereas prazosin (α1-adrenoceptor antagonist) was ineffective. The effect of idazoxan 3×10−7 M on noradrenaline curves was decreased by 50% after clonidine pretreatment, as reflected by the concentration ratio of 5.2±1.2 (treated tissue) and 10.1±1.0 (untreated tissue). However, the concentration ratio for prazosin 3×10−8 M was unchanged. After phenoxybenzamine (irreversible antagonist of α1-adrenoceptor) pretreatment, the residual noradrenaline contraction was antagonized by idazoxan or prazosin with pKB values of 7.8 and 5.1, respectively. The results indicate the presence of α2-adrenoceptors in testicular capsule. Furthermore, these receptors may be desensitized by clonidine, causing a decreased potency of noradrenaline.
Autonomic nerves release ATP, which is processed into adenosine in the synaptic cleft. Adenosine and ATP exert a negative chronotropic effect in the heart. This study aims to evaluate adenosine and P2 receptors and cellular signalling in cardiac arrest produced by purines in the heart. Right atria of adult Wistar rats were used to evaluate the effects of adenosine, ATP and CPA (an adenosine A1 receptor agonist), in the presence and absence of DPCPX, an adenosine A1 receptor antagonist. Effects of adenosine A2 and A3 receptors agonists and antagonists were also investigated. Finally, involvement of calcium and potassium channels in these responses was assessed using BayK 8644 and 4-Aminopyridine. Cumulative concentration-effect curves of adenosine and CPA resulted in a negative chronotropic effect culminating in cardiac arrest at 1000μM (adenosine) and 1µM (CPA). Furthermore, ATP produced a negative chronotropic effect at 1-300µM and cardiac arrest at 1000μM in the right atrium. ATPγS (a non-hydrolysable analogue of ATP) reduced chronotropism only. The effects of adenosine, CPA and ATP were inhibited by DPCPX, a selective adenosine A1 receptor antagonist. The selective adenosine A2 and A3 receptors antagonists did not alter the chronotropic response of adenosine. 4-Aminopyridine, a blocker of potassium channels at 10mM, prevented the cardiac arrest produced by adenosine and ATP, while BayK 8644, activator of calcium channels, did not prevent cardiac arrest. Adenosine A1 receptor activation by adenosine and ATP produces cardiac arrest in the right atrium of Wistar rats predominantly through activation of potassium channels.
In the normotensive rat atrium, adenosine-5'-triphosphate and uridine-5'-triphosphate exert a biphasic effect consisting of an initial negative inotropic effect (NIE) followed by a subsequent positive inotropic effect (PIE). We comparatively studied these responses in normotensive Wistar rats (NWRs) and spontaneously hypertensive rats (SHRs). Compared with NWRs, the NIE responses in the atria were lower and the PIE responses were higher in SHRs. The P1 purinoceptor antagonist, D 8-cyclopentyl-1,3-dipropylxanthine, partially blocked the NIE responses of both ATP and UTP and mildly enhanced the PIE responses in both NWRs and SHRs. Furthermore, the P2 purinoceptor blockers suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium salt induced a pronounced block of the PIE responses in both atria types. The PIE responses to ATP were inhibited more efficiently by nifedipine. These responses were depressed by ryanodine and, to a lesser extent, carbonyl cyanide 3-chlorophenylhydrazone in SHR atria compared with NWR atria. The higher responses in SHR rats suggest the existence of an augmented endoplasmic reticulum Ca(2+) store and faster mitochondrial Ca(2+) cycling in SHR atria compared with NWR atria. These data support the hypothesis that a dysfunction of purinergic neurotransmission and enhanced sympathetic activity are contributing factors in the pathogenesis of hypertension.
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