RESUMEN
Activation of endothelin receptors expressed in DRG neurons is functionally coupled to translocation of PKCε from cytoplasm to the plasma membrane. Using immunocytochemistry we show that in DRG cultured neurons PKCε translocation induced by endothelin-1 was prominently seen in a peptidergic subpopulation of cultured DRG neurons largely negative for isolectin B4 staining, indicating that in basal conditions functional expression of endothelin receptors does not occur in non-peptidergic, RET-expressing nociceptors. Translocation was blocked by the specific ETA-R antagonist BQ-123 while it was unaffected by the ETB-R antagonist BQ-788. No calcium response in response to endothelin-1 was observed in sensory neurons, while large and long-lasting responses were observed in the majority of non-neuronal cells present in DRG cultures, which are ensheathing Schwann cells and satellite cells, identified with the glial marker S-100. Calcium responses in non-neuronal cells were abolished by BQ-788. The fraction of peptidergic PKCε-translocated neurons was significantly increased by nerve growth factor, while in the presence of neurturin or glia-derived neurotropic factor (GDNF), an IB4-positive subpopulation of small- and medium-sized neurons showed PKCε translocation induced by endothelin-1 which could be blocked by BQ-123 but not by BQ-788. Our in vitro results show that the level of expression of functional endothelin receptors coupled to PKCε is different in peptidergic and non-peptidergic nociceptors and is modulated with different mechanisms in distinct neuronal subpopulations.
Asunto(s)
Ganglios Espinales/metabolismo , Glicoproteínas/metabolismo , Lectinas/metabolismo , Receptores de Endotelina/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Endotelina-1/farmacología , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Inmunohistoquímica , Factor de Crecimiento Nervioso/farmacología , Neurturina/farmacología , Proteína Quinasa C-epsilon/metabolismo , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , VersicanosRESUMEN
The plant cannabinoids (phytocannabinoids), cannabidiol (CBD), and Delta(9)-tetrahydrocannabinol (THC) were previously shown to activate transient receptor potential channels of both vanilloid type 1 (TRPV1) and ankyrin type 1 (TRPA1), respectively. Furthermore, the endocannabinoid anandamide is known to activate TRPV1 and was recently found to antagonize the menthol- and icilin-sensitive transient receptor potential channels of melastatin type 8 (TRPM8). In this study, we investigated the effects of six phytocannabinoids [i.e., CBD, THC, CBD acid, THC acid, cannabichromene (CBC), and cannabigerol (CBG)] on TRPA1- and TRPM8-mediated increase in intracellular Ca2+ in either HEK-293 cells overexpressing the two channels or rat dorsal root ganglia (DRG) sensory neurons. All of the compounds tested induced TRPA1-mediated Ca2+ elevation in HEK-293 cells with efficacy comparable with that of mustard oil isothiocyanates (MO), the most potent being CBC (EC(50) = 60 nM) and the least potent being CBG and CBD acid (EC(50) = 3.4-12.0 microM). CBC also activated MO-sensitive DRG neurons, although with lower potency (EC(50) = 34.3 microM). Furthermore, although none of the compounds tested activated TRPM8-mediated Ca2+ elevation in HEK-293 cells, they all, with the exception of CBC, antagonized this response when it was induced by either menthol or icilin. CBD, CBG, THC, and THC acid were equipotent (IC(50) = 70-160 nM), whereas CBD acid was the least potent compound (IC(50) = 0.9-1.6 microM). CBG inhibited Ca2+ elevation also in icilin-sensitive DRG neurons with potency (IC(50) = 4.5 microM) similar to that of anandamide (IC(50) = 10 microM). Our findings suggest that phytocannabinoids and cannabis extracts exert some of their pharmacological actions also by interacting with TRPA1 and TRPM8 channels, with potential implications for the treatment of pain and cancer.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Cannabinoides/farmacología , Ganglios Espinales/metabolismo , Neuronas/efectos de los fármacos , Canales Catiónicos TRPM/metabolismo , Animales , Ancirinas , Canales de Calcio/genética , Cannabis/química , Línea Celular , Células Cultivadas , Ganglios Espinales/citología , Humanos , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Canal Catiónico TRPA1 , Canales Catiónicos TRPC , Canales Catiónicos TRPM/genética , TransfecciónRESUMEN
N-arachidonoylethanolamine (anandamide, AEA), is a full agonist at both cannabinoid CB(1) receptors and "transient receptor potential vanilloid" type 1 (TRPV1) channels, and N-palmitoylethanolamine (PEA) potentiates these effects. In neurons of the rat dorsal root ganglia (DRG), TRPV1 is activated and/or sensitised by AEA as well as upon activation of protein kinases C (PKC) and A (PKA). We investigated here the effect on AEA levels of PKC and PKA activators in DRG neurons. AEA levels were significantly enhanced by both phorbol-miristoyl-acetate (PMA), a typical PKC activator, and forskolin (FSK), an adenylate cyclase stimulant, as well as by thrombin, which also activates PKC by stimulating protease-activated receptors (PARs). The levels of the other endocannabinoid and TRPV1-inactive compound, 2-arachidonoylglycerol (2-AG), were enhanced only by thrombin and to a lesser extent than AEA, whereas PEA was not affected by any of the treatments. Importantly, FSK- and PMA-induced elevation of AEA levels was not sensitive to intracellular Ca2+ chelation with BAPTA-acetoxymethyl (AM) ester. In human embryonic kidney (HEK-293) cells, which constitutively express PARs, thrombin, PMA and FSK elevated AEA levels, and the effects of the two former compounds were counteracted by the PKC inhibitor, RO318220, whereas the effect of FSK was reduced by the PKA inhibitor RpcAMPs. In conclusion, we report that AEA levels are stimulated by both PKC, either directly or after thrombin receptor activation, and PKA, possibly in a way independent from intracellular calcium. Since AEA activates TRPV1, these findings may suggest the existence of an amplificatory cascades on this receptor in sensory neurons.
Asunto(s)
Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteína Quinasa C/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Trombina/farmacología , Animales , Animales Recién Nacidos , Ácidos Araquidónicos/metabolismo , Moduladores de Receptores de Cannabinoides , Células Cultivadas , Colforsina/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Endocannabinoides , Ganglios Espinales/citología , Glicéridos/metabolismo , Humanos , Ratas , Ratas Sprague-Dawley , Receptores Proteinasa-Activados/metabolismo , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
No direct evidence has been found for expression of functional AMPA receptors by dorsal root ganglion neurons despite immunocytochemical evidence suggesting they are present. Here we report evidence for expression of functional AMPA receptors by a subpopulation of dorsal root ganglion neurons. The AMPA receptors are most prominently located near central terminals of primary afferent fibers. AMPA and kainate receptors were detected by recording receptor-mediated depolarization of the central terminals under selective pharmacological conditions. We demonstrate that activation of presynaptic AMPA receptors by exogenous agonists causes inhibition of glutamate release from the terminals, possibly via primary afferent depolarization (PAD). These results challenge the traditional view that GABA and GABA(A) receptors exclusively mediate PAD, and indicate that PAD is also mediated by glutamate acting on presynaptically localized AMPA and kainate receptors.