RESUMEN
Proteinase-activated receptors (PARs) are a family of G-protein-coupled receptors that are activated by endogenous serine proteinases that cleave the N-terminal domain of the receptor unmasking a "tethered ligand" sequence. Trypsin and other agonists at PAR(2) act on peripheral nerves to augment the transfer of nociceptive information. We tested whether PAR(2) agonists also exert a spinal pronociceptive effect by i.t. administering the selective ligand, Ser-Leu-Ile-Gly-Arg-Leu-NH(2) (SLI-GRL). This produced thermal and mechanical hyperalgesia in rats and mice and augmented mechanical and thermal hyperalgesia seen in the formalin inflammatory pain test. Effects of SLIGRL were abrogated in PAR(2)-deficient mice and were not seen with the inactive control peptide, Leu-Arg-Gly-Ile-Leu-Ser-NH(2). Surprisingly, electrophysiological studies, using whole-cell recording from rat substantia gelatinosa neurons, failed to demonstrate an increase in excitatory transmission or neuronal excitability following treatment with SLIGRL or trypsin. In fact, the actions of trypsin were consistent with a decrease in dorsal horn excitability. SLIGRL and trypsin did, however, depolarize and increase the excitability of large, medium and small primary afferent, dorsal root ganglion neurons. The effects were associated with an increase in conductance at hyperpolarized potentials and a decrease in conductance at depolarized potentials. PAR(2)-like immunoreactivity was found in DRG but not in spinal dorsal horn. These results suggest that activation of DRG neuron cell bodies may account for the pronociceptive actions of i.t. applied PAR(2) agonists. They also imply that pathophysiological release of PAR(2)-activating proteases in the vicinity of DRG neurons may produce profound effects on nociceptive processing in vivo.
Asunto(s)
Ganglios Espinales/citología , Hiperalgesia/fisiopatología , Neuronas Aferentes/efectos de los fármacos , Oligopéptidos/farmacología , Receptor PAR-2/agonistas , Animales , Formaldehído , Ganglios Espinales/fisiología , Calor , Hiperalgesia/inducido químicamente , Inyecciones Espinales , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Noqueados , Neuronas Aferentes/fisiología , Dolor/inducido químicamente , Dolor/fisiopatología , Ratas , Ratas Wistar , Receptor PAR-2/deficiencia , Receptor PAR-2/genética , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiopatología , Tripsina/farmacologíaRESUMEN
Galanin modulates spinal nociceptive processing by interacting with two receptors, GalR1 and GalR2. The underlying neurophysiological mechanisms were examined by whole-cell recording from identified neurons in the substantia gelatinosa of young adult rats. GalR1 was activated with a 'cocktail' containing the GalR1/2 agonist, AR-M 961 (0.5 microM), in the presence of the GalR2 antagonist, M871 (1.0-2.5 microM). GalR2 was activated with the selective agonist, AR-M 1896 (0.5-1.0 microM). Application of the 'GalR1 agonist cocktail' often activated an inwardly-rectifying conductance in delay firing (excitatory) and tonically firing (inhibitory) neurons. This conductance was not activated by AR-M 1896 which instead decreased or increased an outwardly-rectifying conductance at voltages positive to -70 mV. Despite this variability in its actions on current-voltage relationships, AR-M 1896 very consistently decreased membrane excitability, as measured by cumulative action potential latency in response to a depolarizing current ramp. This strong GalR2-mediated effect was seen in neurons where membrane conductance was decreased, and where membrane excitability might be predicted to increase. GalR2 was also located presynaptically, as AR-M 1896 increased the interevent interval of spontaneous EPSCs in both delay and tonic cells. By contrast, the 'GalR1 agonist cocktail' had little effect on spontaneous EPSCs, suggesting that presynaptic terminals do not express GalR1. These diverse actions of GalR1 and GalR2 activation on both inhibitory and excitatory neurons are discussed in relation to the known spinal antinociceptive and pro-nociceptive actions of galanin, to the possible association of GalR1 with the inhibitory G-protein, G(i/o) and to report that GalR2 activation suppresses Ca2+ channel currents.
Asunto(s)
Analgésicos/farmacología , Galanina/fisiología , Receptor de Galanina Tipo 1/fisiología , Receptor de Galanina Tipo 2/fisiología , Sustancia Gelatinosa/fisiología , Animales , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Ratas , Ratas Sprague-Dawley , Receptor de Galanina Tipo 1/agonistas , Receptor de Galanina Tipo 2/agonistas , Sustancia Gelatinosa/efectos de los fármacosRESUMEN
Peripheral nerve injury promotes an enduring increase in the excitability of the spinal dorsal horn. This change, that likely underlies the development of chronic pain, may be a consequence of prolonged exposure of dorsal horn neurons to mediators such as neurotrophins, cytokines, and neurotransmitters. The long-term effects of such mediators can be analyzed by applying them to spinal neurons in organotypic slice culture. To assess the validity of this approach, we established serum-free, defined-medium organotypic cultures (DMOTC) from E13-14 prenatal rats. Whole-cell recordings were made from neurons maintained in DMOTC for up to 42 days. These were compared with recordings from neurons of similar age in acute spinal cord slices from 15- to 45-day-old rats. Five cell types were defined in acute slices as 'Tonic', 'Irregular', 'Delay', 'Transient' or 'Phasic' according to their discharge patterns in response to depolarizing current. Although fewer 'Phasic' cells were found in cultures, the proportions of 'Tonic', 'Irregular', 'Delay', and 'Transient' were similar to those found in acute slices. GABAergic, glycinergic, and 'mixed' inhibition were observed in neurons in acute slices and DMOTC. Pure glycinergic inhibition was absent in 7d cultures but became more pronounced as cultures aged. This parallels the development of glycinergic inhibition in vivo. These and other findings suggest that fundamental developmental processes related to neurotransmitter phenotype and neuronal firing properties are preserved in DMOTC. This validates their use in evaluating the cellular mechanisms that may contribute to the development of chronic pain.