Original article
Phospholipase C and cAMP-dependent positive inotropic effects of ATP in mouse cardiomyocytes via P2Y11-like receptors

https://doi.org/10.1016/j.yjmcc.2005.03.007Get rights and content

Abstract

ATP is released as a cotransmitter together with catecholamines from sympathetic nerves. In the heart ATP has been shown to cause a pronounced positive inotropic effect and may also act in synergy with β-adrenergic agonists to augment cardiomyocyte contractility. The aim of the present study was to investigate the inotropic effects mediated by purinergic P2 receptors using isolated mouse cardiomyocytes.

Stable adenine nucleotide analogs were used and the agonist rank order for adenine nucleotide stimulation of the mouse cardiomyocytes was AR-C67085 > ATPγS > 2-MeSATP >>> 2-MeSADP = 0, that fits the agonist profile of the P2Y11 receptor. ATPγS induced a positive inotropic response in single mouse cardiomyocytes. The response was similar to that for the β1 receptor agonist isoproterenol. The most potent response was obtained using AR-C67085, a P2Y11 receptor agonist. This agonist also potentiated contractions in isolated trabecular preparations. The adenylyl cyclase blocker (SQ22563) and phospholipase C (PLC) blocker (U73122) demonstrated that both pathways were required for the inotropic response of AR-C67085. A cAMP enzyme immunoassay confirmed that AR-C67085 increased cAMP in the cardiomyocytes. These findings are in agreement with the P2Y11 receptor, coupled both to activation of IP3 and cAMP, being a major receptor for ATP induced inotropy. Analyzing cardiomyocytes from desmin deficient mice, Des–/–, with a congenital cardiomyopathy, we found a lower sensitivity to AR-C67085, suggesting a down-regulation of P2Y11 receptor function in heart failure.

The prominent action of the P2Y11 receptor in controling cardiomyocyte contractility and possible alterations in its function during cardiomyopathy may suggest this receptor as a potential therapeutic target. It is possible that agonists for the P2Y11 receptor could be used to improve cardiac output in patients with circulatory shock and that P2Y11 receptor antagonist could be beneficial in patients with congestive heart failure (CHF).

Introduction

Extracellular adenosine 5′-triphosphate (ATP) exerts various potent actions in the cardiovascular system, e.g. regulation of vascular tone and platelet aggregation [1], [2], [3], [4], [5]. In the cardiomyocyte, ATP has been shown to cause a pronounced positive inotropic effect and may also act in synergy with β-adrenergic agonists to augment myocyte contractility [3], [6], [7], [8], [9].

ATP is released as a cotransmitter together with catecholamines from sympathetic nerves but it may also be released from other sources in the heart such as endothelium, platelets, red blood cells and ischemic myocardium [3], [10], [11]. Using microdialysis, ATP in the interstitial space has been estimated to be 40 nM but the levels may increase markedly during electrical stimulation, ischemia, challenge with cardiotonic agents, increase in blood flow, mechanical stretch and increased work load (for review see Vassort, 2001). At the place of release the concentration of ATP is high, but within seconds it is rapidly degraded into ADP, AMP and adenosine by ectonucleotidases.

Extracellular ATP mediates its effects via membrane bound P2 receptors [12]. P2 receptors can be divided into two classes: ligand-gated intrinsic ion channels, P2X receptors (P2X1-P2X7), and G-protein-coupled P2Y receptors [13], [14]. P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11 receptors are coupled to Gq, promoting phospholipase C (PLC) catalyzed generation of inositol phosphates (IP3) and subsequent mobilization of intracellular calcium. P2Y12, P2Y13 and P2Y14, are coupled to Gi inhibiting adenylyl cyclase.

In heart, ATP has been shown to stimulate an increase in cytosolic calcium and evidence for the involvement of IP3 coupled P2Y2 receptors and ion-channel coupled P2X receptors have been presented [7], [8], [15], [16], [17]. Increase of cAMP mediates the inotropic effects of catecholamines acting on β-receptors and antagonists of these receptors are important drugs for blood pressure lowering and reduce mortality in congestive heart failure (CHF). ATP stimulates increase in cAMP in cardiac myocytes and may act in synergy with the β1-adrenergic agonist isoproterenol by differential activation of adenylyl cyclase isoforms [3], [18], [19]. However, the ATP receptor mediating this increase in cAMP has not been related to any particular P2 receptor subtype in cardiac cells (Vassort, 2001). A candidate that has been suggested (Vassort, 2001), is the P2Y11 receptor that is additionally coupled to Gs and activates adenylyl cyclase [20], [21], [22]. The P2Y11 receptor has previously not been shown to be involved in heart physiology. It seems to play a role in the immune system in granulocytic differentiation [23] and dendritic cell maturation [24].

mRNA for the P2Y11 receptor has been detected in both atria and ventricles of human hearts [25]. Furthermore, 2-propylthio-β,γ-dichloromethylene-d-ATP (AR-C67085) has been shown to be a specific agonist for the P2Y11 receptor [21], giving us a tool to discriminate between the different ATP receptors. The purpose of the present study was therefore to examine a possible role for the P2Y11 receptor in mediating the positive inotropic effects of ATP. We also examined the response to the P2Y11 receptor agonist on cardiomyocytes from transgenic (desmin deficient) mice with cardiomyopathy [26], [27], [28] to further explore if alterations in P2Y11 receptor responses occur in the heart in pathophysiological conditions.

Section snippets

Animals

The major part of this study was performed using adult female NMRI mice (B & K AB Sollentuna). We also examined genetically modified desmin deficient mice (Des–/–) and their wild type controls (Des+/+), obtained of the strain C57BL/6J in the laboratory of Dr. Li et al. [26] at University Paris VII. The animals were kept in the university animal facilities with free access to food and water according to regulations of the local animal ethics committee. The investigation conforms with the Guide

Cardiomyocyte cell shortening

The amplitude of cardiomyocyte shortening was increased following addition of inotropic substances, such as the selective β1-agonist isoproterenol. The amplitude of cardiomyocyte shortening due to electrical stimulation in the absence of drug was 9.3 ± 0.6 μm. The mean relaxed cardiomyocyte length was 257 ± 12 μm. Isoproterenol (1 μM) caused 65 ± 17% (n = 24) increase in the cardiomyocyte shortening amplitude, i.e. 14.9 ± 2.1. An increase in shortening was also observed after addition of the stable

Discussion

In this study inotropic effects of adenine nucleotides were studied in cardiomyocytes from mouse. Different ATP analogs were examined and the most potent response was obtained using AR-C67085, a P2Y11 receptor agonist. The response to AR-C67085 recruited a P2Y11 receptor like signaling pathway associated with changes in both cAMP and IP3. Analyzing the cardiomyocytes from the desmin deficient mice, Des–/–, with cardiomyopathy, we found a lower sensitivity to AR-C67085.

To limit the contribution

Conclusion

We present data suggesting that P2Y11 is expressed in the heart myocytes mediating ATP stimulated positive inotropic effects. The signaling pathway includes activations of adenylyl cyclase with an increase of cAMP and was dependent on IP3 generation. Both pathways seem to be required for an inotropic response. Analyzing cardiomyocytes from desmin deficient mice which have a cardiomyopathy, we found a decreased response to AR-C67085. Several similarities with the sympathetic nervous system and

Acknowledgements

We are very grateful for the desmin deficient mice obtained from the laboratory of Dr. Z. Li and Dr. D. Paulin, Université Paris VII, France.

This study received support from the Swedish Research Council (DE: 04 × 13130; AA:04 × 8268), the Swedish Heart Lung Foundation and the Medical Faculty at Lund University, Franke and Margareta Bergqvist Foundation, the Wiberg Foundation, the Crafoord Foundation and the Zoegas Foundation.

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