Activation of M 3 cholinoceptors attenuates vascular injury after ischaemia/reperfusion by inhibiting the Ca 2+/calmodulin‐dependent protein kinase II pathway

Background and Purpose

The activation of M ₃ cholinoceptors (M ₃ receptors) by choline reduces cardiovascular risk, but it is unclear whether these receptors can regulate ischaemia/reperfusion (I/R)‐induced vascular injury. Thus, the primary goal of the present study was to explore the effects of choline on the function of mesenteric arteries following I/R, with a major focus on Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) regulation.

Experimental Approach

Rats were given choline (10 mg·kg⁻¹, i.v.) and then the superior mesenteric artery was occluded for 60 min (ischaemia), followed by 90 min of reperfusion. The M ₃ receptor antagonist, 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP), was injected (0.12 μg·kg⁻¹, i.v.) 5 min prior to choline treatment. Vascular function was examined in rings of mesenteric arteries isolated after the reperfusion procedure. Vascular superoxide anion production, CaMKII and the levels of Ca²⁺‐cycling proteins were also assessed.

Key Results

Choline treatment attenuated I/R‐induced vascular dysfunction, blocked elevations in the levels of reactive oxygen species (ROS) and decreased the up‐regulated expression of oxidised CaMKII and phosphorylated CaMKII. In addition, choline reversed the abnormal expression of Ca²⁺‐cycling proteins, including Na⁺/Ca²⁺ exchanger, inositol 1,4,5‐trisphosphate receptor, sarcoplasmic reticulum Ca²⁺‐ATPase and phospholamban. All of these cholinergic effects of choline were abolished by 4‐DAMP.

Conclusions and Implications

Our data suggest that inhibition of the ROS‐mediated CaMKII pathway and modulation of Ca²⁺‐cycling proteins may be novel mechanisms underlying choline‐induced vascular protection. These results represent a significant addition to the understanding of the pharmacological roles of M ₃ receptors in the vasculature, providing a new therapeutic strategy for I/R‐induced vascular injury.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

4‐DAMP

4‐diphenylacetoxy‐N‐methylpiperidine methiodide

CaMKII

Ca²⁺/calmodulin‐dependent protein kinase II

DHE

dihydroethidium

I/R

ischaemia/reperfusion

IP₃R

inositol 1,4,5‐trisphosphate receptor

NAC

N‐acetyl‐L‐cysteine

NCX

Na⁺/Ca²⁺ exchanger

PLB

phospholamban

ROS

reactive oxygen species

SERCA

sarcoplasmic reticulum Ca²⁺‐ATPase

SNP

sodium nitroprusside

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Biased β2‐adrenoceptor signalling in heart failure: pathophysiology and drug discovery

The body is constantly faced with a dynamic requirement for blood flow. The heart is able to respond to these changing needs by adjusting cardiac output based on cues emitted by circulating catecholamine levels. Cardiac β‐adrenoceptors transduce the signal produced by catecholamine stimulation via G_s proteins to their downstream effectors to increase heart contractility. During heart failure, cardiac output is insufficient to meet the needs of the body; catecholamine levels are high and β‐adrenoceptors become hyperstimulated. The hyperstimulated β₁‐adrenoceptors induce a cardiotoxic effect, which could be counteracted by the cardioprotective effect of β₂‐adrenoceptor‐mediated G_i signalling. However, β₂‐adrenoceptor‐G_i signalling negates the stimulatory effect of the G_s signalling on cardiomyocyte contraction and further exacerbates cardiodepression. Here, further to the localization of β₁‐ and β₂‐adrenoceptors and β₂‐adrenoceptor‐mediated β‐arrestin signalling in cardiomyocytes, we discuss features of the dysregulation of β‐adrenoceptor subtype signalling in the failing heart, and conclude that G_i‐biased β₂‐adrenoceptor signalling is a pathogenic pathway in heart failure that plays a crucial role in cardiac remodelling. In contrast, β₂‐adrenoceptor‐G_s signalling increases cardiomyocyte contractility without causing cardiotoxicity. Finally, we discuss a novel therapeutic approach for heart failure using a G_s‐biased β₂‐adrenoceptor agonist and a β₁‐adrenoceptor antagonist in combination. This combination treatment normalizes the β‐adrenoceptor subtype signalling in the failing heart and produces therapeutic effects that outperform traditional heart failure therapies in animal models. The present review illustrates how the concept of biased signalling can be applied to increase our understanding of the pathophysiology of diseases and in the development of novel therapies.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

ACEI

ACE inhibitors

CaMKII

Ca²⁺/calmodulin‐dependent kinase II

ct

carboxy terminus

EGFR

epidermal growth receptor

Epac

exchange protein directly activated by cAMP

G_i

inhibitory G protein

GRK

GPCR kinase

G_s

stimulatory G protein

HF

heart failure

PKA

cAMP‐dependent protein kinase

SNS

sympathetic nervous system

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Regulator of G‐protein signalling 5 protects against atherosclerosis in apolipoprotein E‐deficient mice

Background and Purpose

Atherosclerosis is a chronic inflammatory disease, in which ‘vulnerable plaques’ have been recognized as the underlying risk factor for coronary disease. Regulator of G‐protein signalling (RGS) 5 controls endothelial cell function and inflammation. In this study, we explored the effect of RGS5 on atherosclerosis and the potential underlying mechanisms.

Experimental Approach

RGS5^−/− apolipoprotein E (ApoE)^−/− and ApoE ^−/− littermates were fed a high‐fat diet for 28 weeks. Total aorta surface and lipid accumulation were measured by Oil Red O staining and haematoxylin–eosin staining was used to analyse the morphology of atherosclerotic lesions. Inflammatory cell infiltration and general inflammatory mediators were examined by immunofluorescence staining. Apoptotic endothelial cells and macrophages were assayed with TUNEL. Expression of RGS5and adhesion molecules, and ERK1/2 phosphorylation were evaluated by co‐staining with CD31. Expression of mRNA and protein were determined by quantitative real‐time PCR and Western blotting respectively.

Key Results

Atherosclerotic phenotypes were significantly accelerated in RGS5^−/− ApoE ^−/− mice, as indicated by increased inflammatory mediator expression and apoptosis of endothelial cells and macrophages. RGS5 deficiency enhanced instability of vulnerable plaques by increasing infiltration of macrophages in parallel with the accumulation of lipids, and decreased smooth muscle cell and collagen content. Mechanistically, increased activation of NF‐κB and MAPK/ERK 1/2 could be responsible for the accelerated development of atherosclerosis in RGS5‐deficient mice.

Conclusions and Implications

RGS5 deletion accelerated development of atherosclerosis and decreased the stability of atherosclerotic plaques partly through activating NF‐κB and the MEK‐ERK1/2 signalling pathways.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

ApoE

apolipoprotein E

CHD

coronary heart disease

H&E

haematoxylin‐eosin

ICAM‐1

intercellular adhesion molecue‐1

LDL

low‐density lipoprotein

MEK

MAPK/ERK kinase

RGS

regulator of G‐protein signalling

SMC

smooth muscle cell

VCAM‐1

vascular cell adhesion molecule‐1

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Role for engagement of β‐arrestin2 by the transactivated EGFR in agonist‐specific regulation of δ receptor activation of ERK1/2

Background and Purpose

β‐Arrestins function as signal transducers linking GPCRs to ERK1/2 signalling either by scaffolding members of ERK1/2s cascades or by transactivating receptor tyrosine kinases through Src‐mediated release of transactivating factor. Recruitment of β‐arrestins to the activated GPCRs is required for ERK1/2 activation. Our previous studies showed that δ receptors activate ERK1/2 through a β‐arrestin‐dependent mechanism without inducing β‐arrestin binding to the δ receptors. However, the precise mechanisms involved remain to be established.

Experimental Approach

ERK1/2 activation by δ receptor ligands was assessed using HEK293 cells in vitro and male Sprague Dawley rats in vivo. Immunoprecipitation, immunoblotting, siRNA transfection, intracerebroventricular injection and immunohistochemistry were used to elucidate the underlying mechanism.

Key Results

We identified a new signalling pathway in which recruitment of β‐arrestin2 to the EGFR rather than δ receptor was required for its role in δ receptor‐mediated ERK1/2 activation in response to H‐Tyr–Tic–Phe–Phe–OH (TIPP) or morphine stimulation. Stimulation of the δ receptor with ligands leads to the phosphorylation of PKCδ, which acts upstream of EGFR transactivation and is needed for the release of the EGFR‐activating factor, whereas β‐arrestin2 was found to act downstream of the EGFR transactivation. Moreover, we demonstrated that coupling of the PKCδ/EGFR/β‐arrestin2 transactivation pathway to δ receptor‐mediated ERK1/2 activation was ligand‐specific and the Ser³⁶³ of δ receptors was crucial for ligand‐specific implementation of this ERK1/2 activation pathway.

Conclusions and Implications

The δ receptor‐mediated activation of ERK1/2 is via ligand‐specific transactivation of EGFR. This study adds new insights into the mechanism by which δ receptors activate ERK1/2.

Abbreviations

DPDPE

[D‐Pen2, D‐Pen5] enkephalin

HB‐EGF

heparin‐binding EGF‐like growth factor

IGFR

insulin‐like growth factor receptor

NG108‐15

cell mouse neuroblastoma x rat glioma hybrid cell

RTK

receptor tyrosine kinase

TIPP

H‐Tyr‐Tic‐Phe‐Phe‐OH