Original articlePhospholipase C and cAMP-dependent positive inotropic effects of ATP in mouse cardiomyocytes via P2Y11-like receptors
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.
References (43)
- et al.
Extracellular ATP has a potent effect to enhance cytosolic calcium and contractility in single ventricular myocytes
Cell. Calcium
(1988) - et al.
Purinoceptors: are there families of P2X and P2Y purinoceptors?
Pharmacol. Ther.
(1994) - et al.
Specific activation of adenylyl cyclase V by a purinergic agonist
FEBS Lett.
(1998) - et al.
Cloning of a human purinergic P2Y receptor coupled to phospholipase C and adenylyl cyclase
J. Biol. Chem.
(1997) - et al.
Rapid up-regulation of P2Y messengers during granulocytic differentiation of HL-60 cells
FEBS Lett.
(2000) - et al.
Increase in cardiac P2X1-and P2Y2-receptor mRNA levels in congestive heart failure
Life Sci.
(1999) - et al.
Cardiovascular lesions and skeletal myopathy in mice lacking desmin
Dev. Biol.
(1996) - et al.
The absence of desmin leads to cardiomyocyte hypertrophy and cardiac dilation with compromised systolic function
J. Mol. Cell. Cardiol.
(1999) - et al.
Endothelial P2Y receptors induce hyperpolarisation of vascular smooth muscle by release of endothelium-derived hyperpolarising factor
Eur. J. Pharmacol.
(1999) - et al.
Null mutation in the desmin gene gives rise to a cardiomyopathy
J. Mol. Cell. Cardiol.
(1997)
Generation of tension by skinned fibers and intact skeletal muscles from desmin-deficient mice
Biochem. Biophys. Res. Commun.
Involvement of purinergic signaling in cardiovascular diseases
Drug News Perspect.
Purinergic signaling and vascular cell proliferation and death
Arterioscler. Thromb. Vasc. Biol.
Adenosine 5′-triphosphate: a P2-purinergic agonist in the myocardium
Physiol. Rev.
P2 receptors: new potential players in atherosclerosis
Br. J. Pharmacol.
Roles of P2-purinoceptors in the cardiovascular system
Circulation
Extracellular ATP inhibits adrenergic agonist-induced hypertrophy of neonatal cardiac myocytes
Circ. Res.
A novel phospholipase C- and cAMP-independent positive inotropic mechanism via a P2 purinoceptor
Am. J. Physiol.
P2 purinergic receptor activation enhances cardiac contractility in isolated rat and mouse hearts
Am. J. Physiol. Heart Circ. Physiol.
Extracellular ATP: effects, sources and fate
Biochem. J.
Appearance of adenosine triphosphate in the coronary sinus effluent from isolated working rat heart in response to hypoxia
J. Physiol.
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Authors contributed equally.