Abstract
N-arachidonoyl serine (ARA-S) is one of a number of acyl amino acids recently identified in mammalian tissues. It has been referred to as an endocannabinoid-like lipid largely based on its structural similarities with the endocannabinoid, N-arachidonoyl ethanolamide (anandamide). However, little is known about its potential physiological functions and receptor targets. In this issue of the British Journal of Pharmacology, Zhang and colleagues show that ARA-S is a potent inducer of endothelial cell proliferation and migration, and angiogenesis in vitro. Furthermore, this pro-angiogenic action is mediated, at least partly, by activation of the poorly characterized, G-protein-coupled GPR55 receptor. ARA-S, via GPR55, increases phosphorylation of extracellular signal-regulated kinases and Akt, and vascular endothelial growth factor signalling. These exciting findings highlight the endothelium as an endogenous target for ARA-S and GPR55.
Keywords: acyl amino acids, angiogenesis, endocannabinoid, endothelium, extracellular signal-regulated kinases, GPR55, migration, proliferation, vascular endothelial growth factor
Endocannabinoids, including N-arachidonoyl ethanolamide (anandamide), are novel lipid signalling molecules that activate cannabinoid CB1 and/or CB2 receptors, and, in some cases, the transient receptor potential vanilloid type1 (TRPV1) receptor (see Howlett et al., 2002). They are believed to play a role, for example, in fine tuning neuronal activation, cardiovascular functions and inflammatory responses (Howlett et al., 2002). While the endocannabinoid system continues to be a focus of much research effort, technological advances in detecting traces of cellular lipids have led to the identification of an increasing number of novel lipids that share structural similarities with endocannabinoids (Bradshaw and Walker, 2005). However, little information on their synthetic and degradation pathways, as well as biological activities, is currently available. One of these endogenous lipids, N-arachidonoyl serine (ARA-S) is an example of an acyl amino acid detected in mammalian tissues (Huang et al., 2001). ARA-S induces arterial relaxation (EC50∼1–10 µM; Milman et al., 2006; Godlewski et al., 2009; Parmar and Ho, 2010), and thus might play a role in the regulation of blood flow. Unlike anandamide, ARA-S has negligible activity at CB1, CB2 and TRPV1 receptors (Milman et al., 2006). Instead, vasorelaxation may partly be mediated by a novel, Gi/o-coupled receptor in the endothelium, and partly through direct activation of large-conductance Ca2+-activated K+ channels in the vascular smooth muscle (Milman et al., 2006; Godlewski et al., 2009).
In this issue of the British Journal of Pharmacology, Zhang et al. (2010) provide convincing evidence that ARA-S acts as a pro-angiogenic factor, in addition to being a vasorelaxant. The authors show that ARA-S promotes proliferation and migration of primary human dermal microvascular endothelial cells, with maximal effect achieved at 1 µM. Furthermore, ARA-S also increases endothelial tube formation (by 50%) and neovascularization (by 100%) in vitro, suggesting a potent pro-angiogenic action. ARA-S appears to act by increasing the release of vascular endothelial growth factor C (VEGF-C), which is an important regulator of angiogenesis, and expression of VEGF-2 and VEGF-3 receptors. Interestingly, in contrast, cannabinoids have been reported to reduce endothelial cell survival and migration (Blázquez et al., 2003). Anandamide, via CB1 receptors, also suppresses VEGF and VEGF-1 receptor expression in tumour cells in vivo (Portella et al., 2003), indicating that ARA-S and anandamide exert opposing effects on angiogenesis. To better understand the role of ARA-S as a pro-angiogenic factor, investigation into the synthetic and degradation pathways for ARA-S, and perhaps endogenous regulators of tissue contents of ARA-S (and anandamide) especially in cases of endothelial damage, is much needed. Notably, the relevant cellular source and (patho)physiological level of ARA-S remain unknown. In vivo effects of ARA-S on angiogenesis in wound healing and/or tumour growth should also be explored.
In an effort to elucidate the mechanisms of actions of ARA-S, Zhang et al. (2010) also reveal a new physiological function for the poorly characterized G-protein-coupled receptor, GPR55 (Ross, 2009). At present, the pharmacology of GPR55 is highly controversial; l-α-lysophosphatidylinositol is thought to be the endogenous agonist, whereas cannabinoid receptor ligands might also activate the receptor depending on the cellular system and functional assay used (see Ross, 2009). GPR55 has been implicated as a novel, Gi/o-coupled endothelial cannabinoid (CBx) receptor which modulates vascular tone. This CBx receptor is distinct from CB1 and CB2 receptors, but has been suggested to contribute to vasorelaxation to anandamide (Howlett et al., 2002) and cannabinoid receptor-inactive arachidonoyl amino acids (ARA-S, Milman et al., 2006; N-arachidonoyl glycine, Parmar and Ho, 2010). Unfortunately, research in this area has been hampered by the limited pharmacological tools available; cannabidiol and O-1918 are often used as GPR55 and CBx antagonists, respectively, but their specificity is questionable (Lauckner et al., 2008; Godlewski et al., 2009; Parmar and Ho, 2010). Here, the authors overcome this hurdle by using siRNA technique to specifically knock down GPR55 expression in endothelial cells. They confirm the expression of GPR55 in the primary human endothelial cells used, and, more importantly, demonstrate that GPR55 knock-down partially inhibits the pro-angiogenic effects of ARA-S. Interestingly, knock-down of GPR55 alone also reduces basal endothelial tube formation in the absence of ARA-S, substantiating the role of GPR55 in the regulation of endothelial function and angiogenesis. In theory, GRP55 could therefore serves as a novel target for improving circulation in persistent wounds and ischaemic heart disease. It would also be beneficial to explore if GPR55 knock-out animals are more resistant to tumour growth and metastasis.
Previous studies suggest that GPR55 is coupled to G12/13 (and probably Gq) proteins, and thus activates the small GTPase, RhoA, and causes Ca2+ mobilization (Ross, 2009). In contrast, ARA-S appears to act via Gi/o proteins, and downstream phosphorylation of extracellular signal-regulated kinases and Akt, which have long been associated with cell proliferation and migration, supporting the proposal that GPR55 displays agonist-dependent signalling pathways (Ross, 2009). This is the first indication that ARA-S can activate GPR55. Importantly, however, a direct agonist action by ARA-S on GPR55 was not verified. Further studies will be needed to test if other intermediates are involved. It should also be borne in mind that signalling mechanisms independent of GPR55 might also be deployed by ARA-S.
Beyond the role of GPR55 and ARA-S in angiogenesis, Zhang et al. (2010) provide tantalizing evidence for GPR55 in vascular reactivity responses to ARA-S. The apparent coupling of endothelial GPR55 to Gi/o, instead of G12/13, is consistent with the ability of Pertussis toxin, a Gi/o uncoupler, to inhibit vasorelaxation attributed to CBx receptor (e.g. ARA-S, Milman et al., 2006). In addition, O-1918, which has unproven action on GPR55, antagonizes both the vasorelaxation (Milman et al., 2006) and endothelial tube formation (Zhang et al., 2010) induced by ARA-S. This mirrors the observation that abnormal cannabidiol, a putative agonist for the vascular CBx receptor, also enhances endothelial cell migration through Gi/o and Akt activation (Mo et al., 2004). However, as Zhang and colleagues suggest, it still remains to identify definitively the GPR55 as the long sought-after CBx receptor involved in vasorelaxation. Given the conflicting results obtained from studies using GPR55 over-expressed in cultured cells and the lack of established selective antagonists, molecular approaches that inhibit the function of native receptors, similar to those used in the current study, would seem more desirable. Of particular interest would be to examine the effects of siRNA knock-down of GPR55, or whole animal knock-out of GPR55, on the vasorelaxant responses to ARA-S and anandamide.
Zhang et al. (2010) have clearly shown that ARA-S and GPR55 regulate endothelial function and promote angiogenesis. This paper represents an exciting step forward in improving our understanding of the physiological roles of both the endogenous lipid and the receptor. It also raises intriguing questions that will undoubtedly be addressed in future studies.
Glossary
Abbreviations:
- ARA-S
N-arachidonoyl serine
- VEGF
vascular endothelial growth factor
Conflict of interest
The author states no conflict of interest.
References
- Blázquez C, Casanova ML, Planas A, Gómez Del Pulgar T, Villanueva C, Fernández-Aceñero MJ, et al. Inhibition of tumor angiogenesis by cannabinoids. FASEB J. 2003;17:529–531. doi: 10.1096/fj.02-0795fje. [DOI] [PubMed] [Google Scholar]
- Bradshaw HB, Walker JM. The expanding field of cannabimimetic and related lipid mediators. Br J Pharmacol. 2005;144:459–465. doi: 10.1038/sj.bjp.0706093. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Godlewski G, Offertáler L, Osei-Hyiaman D, Mo FM, Harvey-White J, Liu J, et al. The endogenous brain constituent N-arachidonoyl l-serine is an activator of large conductance Ca2+-activated K+ channels. J Pharmacol Exp Ther. 2009;328:351–356. doi: 10.1124/jpet.108.144717. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, Devane WA, et al. International Union of Pharmacology. XXVII. Classification of cannabinoid receptors. Pharmacol Rev. 2002;54:161–202. doi: 10.1124/pr.54.2.161. [DOI] [PubMed] [Google Scholar]
- Huang SM, Bisogno T, Petros TJ, Chang SY, Zavitsanos PA, Zipkin RE, et al. Identification of a new class of molecules, the arachidonyl amino acids, and characterization of one member that inhibits pain. J Biol Chem. 2001;276:42639–42644. doi: 10.1074/jbc.M107351200. [DOI] [PubMed] [Google Scholar]
- Lauckner JE, Jensen JB, Chen HY, Lu HC, Hille B, Mackie K. GPR55 is a cannabinoid receptor that increases intracellular calcium and inhibits M current. Proc Natl Acad Sci USA. 2008;105:2699–2704. doi: 10.1073/pnas.0711278105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Milman G, Maor Y, Abu-Lafi S, Horowitz M, Gallily R, Batkai S, et al. N-arachidonoyl l-serine, an endocannabinoid-like brain constituent with vasodilatory properties. Proc Natl Acad Sci USA. 2006;103:2428–2433. doi: 10.1073/pnas.0510676103. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mo FM, Offertáler L, Kunos G. Atypical cannabinoid stimulates endothelial cell migration via a Gi/Go-coupled receptor distinct from CB1, CB2 or EDG-1. Eur J Pharmacol. 2004;489:21–27. doi: 10.1016/j.ejphar.2004.02.034. [DOI] [PubMed] [Google Scholar]
- Parmar N, Ho WS. N-arachidonoyl glycine, an endogenous lipid that acts as a vasorelaxant via nitric oxide and large conductance calcium-activated potassium channels. Br J Pharmacol. 2010 doi: 10.1111/j.1476-5381.2009.00622.x. DOI: 10.1111/j.1476-5381.2009.00622 [Epub ahead of print] [DOI] [PMC free article] [PubMed] [Google Scholar]
- Portella G, Laezza C, Laccetti P, De Petrocellis L, Di Marzo V, Bifulco M. Inhibitory effects of cannabinoid CB1 receptor stimulation on tumor growth and metastatic spreading: actions on signals involved in angiogenesis and metastasis. FASEB J. 2003;17:1771–1773. doi: 10.1096/fj.02-1129fje. [DOI] [PubMed] [Google Scholar]
- Ross RA. The enigmatic pharmacology of GPR55. Trends Pharmacol Sci. 2009;30:156–163. doi: 10.1016/j.tips.2008.12.004. [DOI] [PubMed] [Google Scholar]
- Zhang X, Maor Y, Wang JF, Kunos G, Groopman JE. Endocannabinoid-like N-arachidonoyl serine is a novel proangiogenic mediator. Br J Pharmacol. 2010;160:1583–1594. doi: 10.1111/j.1476-5381.2010.00841.x. [DOI] [PMC free article] [PubMed] [Google Scholar]