David A. Kendall

Isolation rearing in rats: pre- and postsynaptic changes in striatal dopaminergic systems.

Isolation rearing of rats produces a behavioral syndrome indicative of altered dopamine (DA) function in the nucleus accumbens (NAC). The present experiments extend these findings by investigating: (a) interactions between isolation rearing and repeated handling/testing on presynaptic DA function in the NAC using in vivo microdialysis: (b) the dose-response curve for the effects of d-amphetamine, and the responses elicited by high potassium, using in vivo microdialysis, and (c) postsynaptic function in isolates as indexed by DA receptor-linked cAMP production. Experiment 1 showed that both isolation rearing and repeated handling/testing had effects on monoamine function in the NAC. However, while both manipulations enhanced DA release evoked by d-amphetamine, only isolated rats had elevated basal DA levels. Opposite neurochemical changes were observed with respect to the serotonin metabolite 5-HIAA, isolates having lower, and repeatedly handled/tested animals having higher, extracellular levels. Experiment 2 provided evidence for enhanced d-amphetamine-evoked DA release in isolated animals, while potassium-evoked DA release was reduced. Experiment 3 provided evidence that the isolation rearing induced changes in presynaptic DA function were accompanied by postsynaptic changes. Specifically, the inhibitory influence of the D2 receptor on D1 receptor-stimulated cAMP production was attenuated in ventral striatal slices taken from isolates, suggesting a functional downregulation of D2 receptors.

Cannabinoid activation of peroxisome proliferator-activated receptors: Potential for modulation of inflammatory disease

Cannabinoids act via cell surface G protein-coupled receptors (CB(1) and CB(2)) and the ion channel receptor TRPV1. Evidence has now emerged suggesting that an additional target is the peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors. There are three PPAR subtypes alpha, delta (also known as beta) and gamma, which regulate cell differentiation, metabolism and immune function. The major endocannabinoids, anandamide and 2-arachidonoylglycerol, and ajulemic acid, a structural analogue of the phytocannabinoid Delta(9)-tetrahydrocannabinol (THC), have anti-inflammatory properties mediated by PPARgamma. Other cannabinoids which activate PPARgamma include N-arachidonoyl-dopamine, THC, cannabidiol, HU210, WIN55212-2 and CP55940. The endogenous acylethanolamines, oleoylethanolamide and palmitoylethanolamide regulate feeding and body weight, stimulate fat utilization and have neuroprotective effects mediated through PPARalpha. Other endocannabinoids that activate PPARalpha include anandamide, virodhamine and noladin ether. There is, as yet, little direct evidence for interactions of cannabinoids with PPARdelta. There is a convergence of effects of cannabinoids, acting via cell surface and nuclear receptors, on immune cell function which provides promise for the targeted therapy of a variety of immune, particularly neuroinflammatory, diseases.

Analgesic Effects of Fatty Acid Amide Hydrolase Inhibition in a Rat Model of Neuropathic Pain

Cannabinoid-based medicines have therapeutic potential for the treatment of pain. Augmentation of levels of endocannabinoids with inhibitors of fatty acid amide hydrolase (FAAH) is analgesic in models of acute and inflammatory pain states. The aim of this study was to determine whether local inhibition of FAAH alters nociceptive responses of spinal neurons in the spinal nerve ligation model of neuropathic pain. Electrophysiological studies were performed 14–18 d after spinal nerve ligation or sham surgery, and the effects of the FAAH inhibitor cyclohexylcarbamic acid 3-carbamoyl biphenyl-3-yl ester (URB597) on mechanically evoked responses of spinal neurons and levels of endocannabinoids were determined. Intraplantar URB597 (25 μg in 50 μl) significantly (p < 0.01) attenuated mechanically evoked responses of spinal neurons in sham-operated rats. Effects of URB597 were blocked by the cannabinoid 1 receptor (CB1) antagonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide] (30 μg in 50 μl) and the opioid receptor antagonist naloxone. URB597 treatment increased levels of anandamide, 2-arachidonyl glycerol, and oleoyl ethanolamide in the ipsilateral hindpaw of sham-operated rats. Intraplantar URB597 (25 μg in 50 μl) did not, however, alter mechanically evoked responses of spinal neurons in spinal nerve ligated (SNL) rats or hindpaw levels of endocannabinoids. Intraplantar injection of a higher dose of URB597 (100 μg in 50 μl) significantly (p < 0.05) attenuated evoked responses of spinal neurons in SNL rats but did not alter hindpaw levels of endocannabinoids. Spinal administration of URB597 attenuated evoked responses of spinal neurons and elevated levels of endocannabinoids in sham-operated and SNL rats. These data suggest that peripheral FAAH activity may be altered or that alternative pathways of metabolism have greater importance in SNL rats.

The complexities of the cardiovascular actions of cannabinoids

The cardiovascular actions of cannbinoids are complex. In general they cause vasorelaxation in isolated blood vessels, while in anaesthetised animals they cause multiphasic responses which involve an early bradycardia and long‐lasting hypotension. However, in conscious animals, the picture is one of bradycardia followed by pressor responses. Clearly, the responses to cannabinoids are dependent on the experimental conditions and synthetic cannabinoids and endocannabinoids exhibit different pharmacologies. In terms of mechanisms involved in the vascular responses to cannabinoids, the following have been implicated: the involvement of ‘classical’ cannabinoid receptors, the involvement of a novel endothelial cannabinoid receptor, the release of nitric oxide, the release of endothelium‐derived hyperpolarising factor (EDHF), the activation of vanilloid receptors, metabolism of endocannabinoids to vasoactive molecules, and both peripheral inhibition and central excitation of the sympathetic nervous system.

Cannabinoid CB2 receptor activation inhibits mechanically evoked responses of wide dynamic range dorsal horn neurons in naïve rats and in rat models of inflammatory and neuropathic pain

Peripheral cannabinoid 2 receptors (CB2 receptors) modulate immune responses and attenuate nociceptive behaviour in models of acute and persistent pain. The aim of the present study was to investigate whether peripheral CB2 receptors modulate spinal processing of innocuous and noxious responses and to determine whether there are altered roles of CB2 receptors in models of persistent pain. Effects of local administration of the CB2 receptor agonist JWH‐133 (5 and 15 µg/50 µL) on mechanically evoked responses of spinal wide dynamic range (WDR) neurons in noninflamed rats, rats with carrageenan‐induced hindpaw inflammation, sham operated rats and spinal nerve‐ligated (SNL) rats were determined in anaesthetized rats in vivo. Mechanical stimulation (von Frey filaments, 6–80 g) of the peripheral receptive field evoked firing of WDR neurons. Mechanically evoked responses of WDR neurons were similar in noninflamed, carrageenan‐inflamed, sham‐operated and SNL rats. Intraplantar injection of JWH‐133 (15 µg), but not vehicle, significantly (P < 0.05) inhibited innocuous and noxious mechanically evoked responses of WDR neurons in all four groups of rats. In many cases the selective CB2 receptor antagonist, SR144528 (10 µg/50 µL), attenuated the inhibitory effects of JWH‐133 (15 µg) on mechanically evoked WDR neuronal responses. The CB1 receptor antagonist, SR141716A, did not attenuate the inhibitory effects of JWH‐133 on these responses. Intraplantar preadministration of JWH‐133 also inhibited (P < 0.05) carrageenan‐induced expansion of peripheral receptive fields of WDR dorsal horn neurons. This study demonstrates that activation of peripheral CB2 receptors attenuates both innocuous‐ and noxious‐evoked responses of WDR neurons in models of acute, inflammatory and neuropathic pain.

Inhibitory effects of CB1 and CB2 receptor agonists on responses of DRG neurons and dorsal horn neurons in neuropathic rats

Cannabinoid 2 (CB2) receptor mediated antinociception and increased levels of spinal CB2 receptor mRNA are reported in neuropathic Sprague–Dawley rats. The aim of this study was to provide functional evidence for a role of peripheral, vs. spinal, CB2 and cannabinoid 1 (CB1) receptors in neuropathic rats. Effects of the CB2 receptor agonist, JWH‐133, and the CB1 receptor agonist, arachidonyl‐2‐chloroethylamide (ACEA), on primary afferent fibres were determined by calcium imaging studies of adult dorsal root ganglion (DRG) neurons taken from neuropathic and sham‐operated rats. Capsaicin (100 nm) increased [Ca2+]i in DRG neurons from sham and neuropathic rats. JWH‐133 (3 µm) or ACEA (1 µm) significantly (P < 0.001) attenuated capsaicin‐evoked calcium responses in DRG neurons in neuropathic and sham‐operated rats. The CB2 receptor antagonist, SR144528, (1 µm) significantly inhibited the effects of JWH‐133. Effects of ACEA were significantly inhibited by the CB1 receptor antagonist SR141716A (1 µm). In vivo experiments evaluated the effects of spinal administration of JWH‐133 (8–486 ng/50 µL) and ACEA (0.005–500 ng/50 µL) on mechanically evoked responses of neuropathic and sham‐operated rats. Spinal JWH‐133 attenuated mechanically evoked responses of spinal neurons in neuropathic, but not sham‐operated rats. These inhibitory effects were blocked by SR144528 (0.001 µg/50 µL). Spinal ACEA inhibited mechanically evoked responses of neuropathic and sham‐operated rats, these effects were blocked by SR141716A (0.01 µg/50 µL). Our data provide evidence for a functional role of CB2, as well as CB1 receptors on DRG neurons in sham and neuropathic rats. At the level of the spinal cord, CB2 receptors have inhibitory effects in neuropathic, but not sham‐operated rats suggesting that spinal CB2 may be an important analgesic target.

Cardiovascular effects of cannabinoids

The prototypic endocannabinoid, anandamide, and synthetic analogues have been shown to elicit pressor and depressor effects, bradycardia, vasorelaxation, and inhibition of neurotransmission in the central and peripheral nervous systems. Cannabinoid-mediated inhibition of neurotransmission is mediated by inhibition of voltage-gated Ca(2+) channels and adenylyl cyclase and activation of inwardly rectifying K(+) channels. The precise mechanisms underlying the vasorelaxant actions of cannabinoids are currently unclear, but might involve both receptor-dependent and -independent and endothelium-dependent and -independent pathways. Mechanisms proposed have included the release of endothelial autacoids, activation of myoendothelial gap junctions, activation of the Na(+) pump, activation of K(+) channels, inhibition of Ca(2+) channels, and activation of vanilloid receptors, leading to the release of sensory neurotransmitters. Pathophysiologically, the vasodilator actions of endocannabinoids have been implicated in the hypotension associated with both septic and haemorrhagic shock, but their physiological significance remains to be determined.

Endocannabinoids: a new class of vasoactive substances

Endogenous cannabinoids (endocannabinoids) have recently been identified in the CNS and attention has now turned to their cardiovascular actions. The prototypic endocannabinoid, anandamide, derived from arachidonic acid, has been shown to be a vasorelaxant, particularly in the resistance vasculature. This vasorelaxation has been shown to be both endothelium-independent and -dependent, depending on the vascular bed. It has been proposed that an endocannabinoid may mediate the nitric oxide- and prostanoid-independent component of endothelium-dependent relaxations, as these responses are sensitive to a cannabinoid receptor antagonist and show similarities to anandamide-induced relaxations. This hypothesis has generated much controversy and the emerging conflicts in the literature are discussed in this article by Michael Randall and David Kendall. Despite this controversy, it has recently been shown that anandamide is produced by endothelial cells. Clearly, much work is required to adequately define the physiological significance of endocannabinoids in the cardiovascular system.

Testosterone-induced vasorelaxation in the rat mesenteric arterial bed is mediated predominantly via potassium channels

We have investigated the involvement of nitric oxide and K+ channels in the vasorelaxant responses to physiologically‐relevant concentrations of testosterone in the rat isolated mesenteric arterial bed. Testosterone (100 pM – 10 μM) elicited concentration‐dependent relaxations in the isolated mesenteric arterial bed (pEC50=9.47 (9.22 – 9.73, 95% CI), maximal relaxation, Rmax=62.8±2.0%, n=6). A nitric oxide synthase (NOS) inhibitor, NG‐nitro‐L‐arginine methyl ester (L‐NAME, 300 μM) or removal of the endothelium significantly inhibited maximal relaxations to testosterone (L‐NAME: Rmax=51.4±1.1%, P<0.01, n=6; endothelium‐denuded: Rmax=46.9±2.8%, P<0.001, n=5). Raising the extracellular K+ concentration to 30 and 60 mM, or pre‐treatment with 300 μM tetrabutylammonium chloride (TBA), a calcium‐activated K+ channel inhibitor, abolished vasorelaxations induced by testosterone. A selective inhibitor of ATP‐sensitive K+ (KATP) channels, glibenclamide (10 μM) and an inhibitor of voltage‐sensitive K+ (KV) channels, 4‐aminopyridine (4‐AP, 1 mM) did not affect testosterone‐induced responses. Vasorelaxation to 1 μM testosterone was significantly (P<0.05) inhibited by 100 nM charybdotoxin (ChTx), an inhibitor of large conductance calcium‐activated K+ (BKCa) channels (control: 63.3±9.9%, n=6; ChTx: 11.9±12.7%, n=3). Neither the testosterone receptor antagonist, flutamide (10 μM) nor an aromatase inhibitor, aminoglutethimide (10 μM) inhibited testosterone‐induced responses. In conclusion, the present findings demonstrate, in the rat isolated mesenteric arterial bed, that testosterone causes acute vasorelaxations at physiologically relevant concentrations which are, in part, mediated via NO‐ and endothelium‐dependent pathways. However, the activation of BKCa channels plays a substantial role in testosterone‐induced vasorelaxation.

Influence of cannabinoids on electrically evoked dopamine release and cyclic AMP generation in the rat striatum.

Abstract: Using the endogenous cannabinoid receptor agonist anandamide, the synthetic agonist CP 55940 {[1α,2β(R)5α]‐(−)‐5‐(1,1‐dimethylheptyl)‐2‐[5‐hydroxy‐2‐(3‐hydroxypropyl)cyclohexyl]phenol}, and the specific antagonist SR 141716 [N‐(piperidin‐1‐yl)‐5‐(4‐chlorophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide hydrochloride], second messenger activation of the central cannabinoid receptor (CB1) was examined in rat striatal and cortical slices. The effects of these cannabinoid ligands on electrically evoked dopamine (DA) release from [3H]dopamine‐prelabelled striatal slices were also investigated. CP 55940 (1 µM) and anandamide (10 µM) caused significant reductions in forskolin‐stimulated cyclic AMP accumulation in rat striatal slices, which were reversed in the presence of SR 141716 (1 µM). CP 55940 (1 µM) had no effect on either KCl‐ or neurotransmitter‐stimulated 3H‐inositol phosphate accumulation in rat cortical slices. CP 55940 and anandamide caused significant reductions in the release of dopamine after electrical stimulation of [3H]dopamine‐prelabelled striatal slices, which were antagonised by SR 141716. SR 141716 alone had no effect on electrically evoked dopamine release from rat striatal slices. These data indicate that the CB1 receptors in rat striatum are negatively linked to adenylyl cyclase and dopamine release. That the CB1 receptor may influence dopamine release in the striatum suggests that cannabinoids play a modulatory role in dopaminergic neuronal pathways.