The CB₂-preferring agonist JWH015 also potently and efficaciously activates CB₁ in autaptic hippocampal neurons

Abstract

The G protein coupled receptors CB₁ and CB₂ are targets for the psychoactive constituents of cannabis, chief among them Δ⁹-THC. They are also key components of the multifunctional endogenous cannabinoid signaling system. CB₁ and CB₂ receptors modulate a wide variety of physiological systems including analgesia, memory, mood, reward, appetite and immunity. Identification and characterization of selective CB₁ and CB₂ receptor agonists and antagonists will facilitate understanding the precise physiological and pathophysiological roles of cannabinoid receptors in these systems. This is particularly necessary in the case of CB₂ because these receptors are sparsely expressed and problematic to detect using traditional immunocytochemical approaches.

1-Propyl-2-methyl-3-(1-naphthoyl)indole (JWH015) is an aminoalkylindole that has been employed as a “CB₂-selective” agonist in more than 40 published papers. However, we have found that JWH015 potently and efficaciously activates CB₁ receptors in neurons. Using murine autaptic hippocampal neurons, which express CB₁, but not CB₂ receptors, we find that JWH015 inhibits excitatory postsynaptic currents with an EC50 of 216 nM. JWH015 inhibition is absent in neurons from CB₁^−/− cultures and is reversed by the CB₁ antagonist, SR141716 [200 nM]. Furthermore, JWH015 partially occludes CB₁-mediated DSE (∼35% remaining), an action reversed by the CB₂ antagonist, AM630 [1 and 3 μM], suggesting that high concentrations of AM630 also antagonize CB₁ receptors.

We conclude that while JWH015 is a CB₂-preferring agonist, it also activates CB₁ receptors at experimentally encountered concentrations. Thus, CB₁ agonism of JWH015 needs to be considered in the design and interpretation of experiments that use JWH015 to probe CB₂-signaling.

Introduction

The endocannabinoid system has many roles within the body. Its functions are mediated via endogenous cannabinoids, including anandamide (AEA [1]) and 2-arachidonoylglycerol (2-AG [2]), binding to the well-characterized metabotropic cannabinoid receptors, CB₁ and CB₂ [3], [4]. Cannabinoid receptors are best known as the endogenous targets of the psychoactive ingredients of marijuana and hashish, chief among them Δ⁹-THC [5]. These G protein-coupled receptors are widely distributed throughout the body and have been found to modulate diverse physiological systems including analgesia, memory, mood, reward, appetite, and immunity [6].

CB₁ is richly expressed in the CNS [7], [8] and is the chief mediator of the psychoactive effects of marijuana and hashish. Because of its prominent role in the psychoactive effects of THC, CB₁ has received more attention than CB₂. However, CB₂ has been the object of growing interest as a potential therapeutic target, particularly for pain, inflammation, and osteoporosis [9]. CB₂ is widely expressed in the immune system and is known to modulate some inflammatory responses [10], [11], [12], [13], [14]. As compared to CB₁, CB₂ is expressed at low levels in the healthy brain and has been proposed as a promising pharmacological target, insofar as CB₂ activation is hypothesized to be less likely to cause adverse psychoactivity. However, to fully characterize the therapeutic potential of CB₂ receptors it is essential to employ appropriately selective CB₂ agonists and antagonists.

Many synthetic cannabinoids have been developed, with varying degrees of selectivity for the two cannabinoid receptors [15], [16]. However the pharmacology of cannabinoid receptor ligands—endogenous, exogenous (e.g. derived from cannabis), and synthetic—has proved complex. Some nominally CB₂-selective agonists have come into widespread use without a full consideration of their selectivity in a functional context. One of these is 1-propyl-2-methyl-3-(1-naphthoyl)indole (JWH015, Fig. 1A), an aminoalkylindole that has been reported to be 12–24 times more selective for CB₂ than for CB₁ [17], [18], [19].

Since first described, JWH015 has been used as a CB₂-selective agonist in more than 40 published articles. Initial characterization reported a K_i of 13.8 nM at the CB₂ receptor, and a K_i of 336 nM at the CB₁ receptor [17]. This offers a ∼25-fold selectivity for CB₂ over CB₁, though a subsequent study reported only a 12-fold selectivity [19]. Regardless, a 12- to 24-fold selectivity is relatively slender margin, especially when CB₁ is found at very high levels and may efficaciously signal at low occupancy. For example, CB₁-signaling can be observed at receptor occupancy ranging from 4 to 14% [20]. This narrow selectivity range raises the possibility that some reported effects of JWH015 have in fact occurred via CB₁, especially when employing higher concentrations or doses of the drug. But how efficacious and potent is JWH015 in an endogenous neuronal CB₁ signaling system? In autaptic hippocampal neurons, CB₁ activation is coupled to inhibition of calcium channels and neurotransmitter release [21], [22], [23]. These neurons express a robust CB₁-dependent endogenous cannabinoid signaling system [22], [24], [25] including depolarization-induced suppression of excitation (DSE) [26], [27]. DSE is a well-described 2-AG/CB₁ receptor-dependent signaling mechanism characterized by a transient decrease in excitatory post-synaptic current (EPSC) size, with subsequent recovery back to baseline over tens of seconds.

Using autaptic hippocampal cultures we explored the action of JWH015 at CB₁. Neurons in these cultures express CB₁ receptors, but lack CB₂ receptors, and express robust DSE [22]. Thus, they serve as a useful model for the study of the selectivity of CB₁ signaling in a controlled neuronal environment. Using this system we found that JWH015 is an efficacious and relatively potent CB₁ receptor agonist, similarly, the CB₂-preferring antagonist, AM630, has appreciable antagonistic activity at CB₁ receptors. Thus, both compounds should be used with caution as “CB₂-selective” agents.