Original article
Endocannabinoids and cannabinoid analogues block human cardiac Kv4.3 channels in a receptor-independent manner

https://doi.org/10.1016/j.yjmcc.2009.07.011Get rights and content

Abstract

Endocannabinoids are amides and esters of long chain fatty acids that can modulate ion channels through both receptor-dependent and receptor-independent effects. Nowadays, their effects on cardiac K+ channels are unknown even when they can be synthesized within the heart. We have analyzed the direct effects of endocannabinoids, such as anandamide (AEA), 2-arachidonoylglycerol (2-AG), the endogenous lipid lysophosphatidylinositol, and cannabinoid analogues such as palmitoylethanolamide (PEA), and oleoylethanolamide, as well as the fatty acids from which they are endogenously synthesized, on human cardiac Kv4.3 channels, which generate the transient outward K+ current (Ito1). Currents were recorded in Chinese hamster ovary cells, which do not express cannabinoid receptors, by using the whole-cell patch-clamp. All these compounds inhibited IKv4.3 in a concentration-dependent manner, AEA and 2-AG being the most potent (IC50  0.3–0.4 µM), while PEA was the least potent. The potency of block increased as the complexity and the number of C atoms in the fatty acyl chain increased. The effects were not mediated by modifications in the lipid order and microviscosity of the membrane and were independent of the presence of MiRP2 or DPP6 subunits in the channel complex. Indeed, effects produced by AEA were reproduced in human atrial Ito1 recorded in isolated myocytes. Moreover, AEA effects were exclusively apparent when it was applied to the external surface of the cell membrane. These results indicate that at low micromolar concentrations the endocannabinoids AEA and 2-AG directly block human cardiac Kv4.3 channels, which represent a novel molecular target for these compounds.

Introduction

Endogenous cannabinoids (endocannabinoids), which are synthesized from lipid precursors in plasma membranes, are signaling lipids consisting of amides and esters of long chain fatty acids [1]. Some of these endocannabinoids are derivatives of arachidonic acid (AA), namely anandamide (N-arachidonoylethanolamide, AEA) and 2-arachidonoylglycerol (2-AG). Others, such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), are synthesized from the palmitic (PA) and oleic (OA) fatty acids, respectively [1]. PEA and OEA are classified on the basis of their mode of action as endocannabinoids or cannabinoid analogues since they have been shown to potentiate AEA responses [1]. Endocannabinoids can be generated by virtually all cell types, including the cardiac muscle, and it is considered that they exert their broad range of biological effects mainly through their interaction with the G-protein-coupled receptors CB1, CB2, and GPR55, as well as additional, as yet unidentified, receptors [1], [2].

It has been extensively demonstrated that endocannabinoids can produce biological effects which are not mediated by the interaction with receptors (i.e., the receptor-independent or direct effects of endocannabinoids) [1], [3], [4]. Indeed, endocannabinoids and cannabinoid analogues can modulate Na+ [5], K+ [6], [7], [8], and Ca2+ [9] channels in a receptor-independent manner (for review see [3], [4]). However, almost all these studies analyzed their effects on neuronal or smooth muscle tissues, whereas data on the putative direct effects of endocannabinoids on human cardiac channels are scarce or absent.

It has been described that AEA accelerated the inactivation of neuronal rapidly inactivating A-type (Kv3.4) channels, an effect that was independent of the endocannabinoid receptor activation [8]. In the human myocardium the most important rapidly inactivating K+ current is the Ca2+-independent component of the transient outward K+ current (Ito1), which is critical in determining the height and the duration of the plateau phase of the action potential (AP). Ito1 is predominantly carried by Kv4.3 α-subunits, which assemble with KChIP2, DPP6, and MiRP ancillary subunits [10], [11], [12], [13].

In the present paper we analyzed the direct effects of endocannabinoids and cannabinoid analogues such as AEA, its metabolically stable analogue, (R)-(+)-arachidonoyl-1′-hydroxy-2′-propylamide (MetAEA), 2-AG, PEA, OEA, and the endogenous lipid lysophosphatidylinositol (LPI) on human cardiac Kv4.3 + KChIP2 current (IKv4.3). Moreover, the effects of the fatty acids from which some of them are endogenously synthesized have also been examined. The results demonstrated, for the first time, that among endocannabinoids and cannabinoid analogues, AEA and 2-AG are the more potent agents for inhibiting IKv4.3, an effect that was not mediated by either their interaction with cannabinoid receptors or the modification of the lipid order and microviscosity of the cell membrane.

Section snippets

Material and methods

Chinese hamster ovary (CHO) cells stably transfected with hKv4.3-L/hKChIP2, that do not endogenously express any of the known cannabinoid receptors [2], [9], [14], were cultured as described [15], [16]. In some experiments, CHO cells were transiently transfected with the cDNA encoding KCNE3 (1.6 μg) or DPP6 (1 μg) proteins together with the cDNA encoding the CD8 antigen (0.5 μg) by using FUGENE6 [13], [16]. Ito1 was recorded on myocytes isolated from right atrial appendages of patients

Results

Fig. 1 shows the chemical structure of the endocannabinoids and cannabinoid analogues tested in the present study. Since most of them are ethanolamides of fatty acids, we also studied the effects of their corresponding fatty acids. Moreover, to gain more insight into the putative structure–effect relationship we also studied the effects of SA, an 18:0 fatty acid, and its ethanolamide (SEA). Fig. 2 shows IKv4.3 traces recorded by applying 250 ms-pulses to + 50 mV from a holding potential of − 

Discussion

In the present paper the effects produced by endocannabinoids (AEA, 2-AG, and LPI) and cannabinoid analogues (PEA and OEA) on human cardiac Kv4.3 channels have been systematically analyzed for the first time. The results demonstrated that these lipid mediators, which are synthesized within the heart, inhibit IKv4.3 and Ito1 currents by specifically interacting with the channel, AEA and 2-AG being the most potent for this effect. Furthermore, the effects produced by AEA and 2-AG on Kv4.3

Acknowledgments

We thank Prof. Manuel Guzmán for his helpful suggestions. Supported by Fondo de Investigación Sanitaria (PI080665), Ministerio de Educación y Ciencia (SAF2008-04903), Instituto de Salud Carlos III (Red HERACLES RD06/0009), Fundación LILLY, Centro Nacional de Investigaciones Cardiovasculares (CNIC-13), Universidad Complutense de Madrid (UCM-4195), BFU2006-03905 (MPL) and Sociedad Española de Cardiología. Ricardo Gómez is a fellow of Comunidad Autónoma de Madrid.

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    Both authors contributed equally.

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