Phosphorylation-Dependent Desensitization by Anandamide of Vanilloid Receptor-1 (TRPV1) Function in Rat Skeletal Muscle Arterioles and in Chinese Hamster Ovary Cells Expressing TRPV1

Abstract

It has been proposed that activation of vanilloid receptor-1 (TRPV1) affects the vasotone of resistance arteries. One of the endogenous activators of TRPV1 is anandamide. The effects of anandamide on TRPV1 responsiveness were tested on isolated, pressurized (80 mm Hg) skeletal muscle (m. gracilis) arterioles (179 Ϯ 33 m in diameter). We found that the TRPV1 agonist capsaicin (1 M) elicited a substantial constriction in isolated arterioles (51 Ϯ 12%). In contrast, anandamide (0 -100 M) did not affect arteriolar diameter significantly (3 Ϯ 5%). Isolated vessels were also preincubated with anandamide (30 M for 20 min). This anandamide pretreatment completely blocked capsaicin-induced arteriolar constriction (response decreased to 1 Ϯ 0.6%), and this inhibition was reversed by a protein phosphatase-2B inhibitor (cyclosporin-A; 100 nM, 5 min) treatment (constriction, 31 Ϯ 1%). An exogenous TRPV1-expressing cell line [Chinese hamster ovary (CHO)-TRPV1] was used to specifically evaluate TRPV1-mediated effects of anandamide. The efficacy of anandamide in this system, as determined by 45 Ca 2ϩ uptake, was 65 Ϯ 8% of that of capsaicin. Upon treatment of the cells with cyclosporin-A or the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), anandamide was transformed to a full agonist. Anandamide treatment caused an acute desensitization in these cells as measured by intracellular Ca 2ϩ imaging. Application of cyclosporin-A or PMA reversed this desensitization. Our data suggest that anandamide may cause a complete (albeit phosphorylation-dependent) desensitization of TRPV1 in skeletal muscle arterioles and in CHO-TRPV1 cells, which apparently transforms the ligand-gated TRPV1 into a phosphorylationgated channel. This property of anandamide may provide a new therapeutic strategy to manipulate TRPV1 activity.The vanilloid receptor 1 (TRPV1) is a nonselective cation channel, the structure of which places it in the transient receptor potential family of ion channels. In the periphery, TRPV1 is primarily expressed in sensory C and A-␦ fibers (Caterina et al., 1997) and acts as a ligand-, proton-, and heat-activated molecular integrator of nociceptive stimuli Di Marzo et al., 2002a,b;Ross, 2003). Activation of TRPV1 leads to central (pain) and to local "sensory-efferent" effects (Szolcsanyi, 2000). These include the release of vasoactive agents (such as calcitonin gene-related peptide) and subsequent vasorelaxation (Zygmunt et al., 1999).Compared with the well characterized effects of TRPV1 upon stimulation by exogenous substances , relatively little is known about its endogenous ligands. So far, anandamide (N-arachidonoyl-ethanolamide) (Zygmunt et al., 1999), N-arachidonoyl-dopamine (NADA) (Huang et al., 2002), and different lipoxygenase products (Hwang et al., 2000) have been identified as endogenous agonists of TRPV1. Among them, anandamide was originally identified as an endocannabinoid activating the

TRPV1 activation improves exercise endurance and energy metabolism through PGC-1α upregulation in mice

“…Anandamide is the most abundant endogenous endocannabinoid and regulates systemic energy metabolism [43]. Anandamide in skeletal muscles participates in the regulation of TRPV1 function [44,45]. We showed that anandamide treatment significantly increased TRPV1 expression (Supplementary information, Figure S6).…”

Structure‐activity relationships of vanilloid receptor agonists for arteriolar TRPV1

“…Vasoconstriction in response to TRPV1 stimulation was reported decades ago (Molnar and Gyorgy, 1967;Toda et al, 1972;Donnerer and Lembeck, 1982;Duckles, 1986;Edvinsson et al, 1990) and this effect was confirmed later (Szolcsanyi et al, 2001;Dux et al, 2003;Scotland et al, 2004;Keeble and Brain, 2006;Lizanecz et al, 2006). Nonetheless, these responses were not thought to be of interest.…”

“…We have recently shown that stimulation of TRPV1 in skeletal muscle arterioles results in a substantial vasoconstriction (Lizanecz et al, 2006). Moreover, intra-arterial injection of capsaicin into the hind limb evoked a dose-dependent increase in blood flow in the skin (probably representing vasodilatation in this organ) and simultaneously, a decrease of blood flow in skeletal muscle (representing vasoconstriction) (Kark et al, 2008).…”

“…The isolation of skeletal muscle (m. gracilis) arterioles of the rat and measurement of the diameter of arterioles were performed as described previously (Lizanecz et al, 2006). Briefly, arterioles were kept in a physiological saline solution (PSS; composition in mM: 110 NaCl, 5.0 KCl, 2.5 CaCl2, 1.0 MgSO4, 1.0 KH2PO4, 5.0 glucose and 24.0 NaHCO3 equilibrated with a gas mixture of 10% O2, 5% CO2 and 85% N2, at pH 7.4.)…”

Cannabinoids, the Heart of the Matter

“…TRPV1 ion channels have important functions as cellular sensors, and are involved in nociception, taste perception, thermosensation, mechano‐ and osmolarity sensing, and regulation of signal transmission 15, 55, 56. In addition to ECS and physicochemical activators,15, 33, 55, 56, 57, 58 TRPV1 is activated by tetrahydrocannabinol, cannabinol, cannabigerol and some propyl homologs of THC and cannabigerol 59, 60, 61, 62, 63, 64, 65, 66, 67. Cannabichromene (CBC), cannabidiol, and cannabinol are strong TRPA1 agonists and desensitizers, and THCV (from a botanical extract) is a potent regulator of TRPA1 62…”