RESUMEN
The opioid-related receptor, ORL1, is activated by the neuropeptide nociceptin/orphanin FQ (N/OFQ) and inhibits high-voltage-activated (HVA) calcium channel currents (I(Ca)) via a G-protein-coupled mechanism. Endocytosis of ORL1 receptor during prolonged N/OFQ exposure was proposed to cause N-type voltage-gated calcium channel (VGCC) internalization via physical interaction between ORL1 and the N-type channel. However, there is no direct electrophysiological evidence for this mechanism in dorsal root ganglion (DRG) neurons or their central nerve terminals. The present study tested this using whole-cell patch-clamp recordings of HVA I(Ca) in rat DRG neurons and primary afferent excitatory synaptic currents (eEPSCs) in spinal cord slices. DRG neurons were classified on the basis of diameter, isolectin-B4 (IB4) binding and responses to capsaicin, N/OFQ and a µ-opioid agonist, DAMGO. IB4-negative neurons less than 20 µm diameter were selectively responsive to N/OFQ as well as DAMGO. In these neurons, ORL1 desensitization by a supramaximal concentration of N/OFQ was not followed by a decrease in HVA I(Ca) current density or proportion of whole-cell HVA I(Ca) contributed by N-type VGCC as determined using the N-type channel selective blocker, ω-conotoxin CVID. There was also no decrease in the proportion of N-type I(Ca) when neurons were incubated at 37°C with N/OFQ for 30 min prior to recording. In spinal cord slices, N/OFQ consistently inhibited eEPSCs onto dorsal horn neurons. As observed in DRG neurons, preincubation of slices in N/OFQ for 30 min produced no decrease in the proportion of eEPSCs inhibited by CVID. In conclusion, no internalization of the N-type VGCC occurs in either the soma or central nerve terminals of DRG neurons following prolonged exposure to high, desensitizing concentrations of N/OFQ.
Asunto(s)
Canales de Calcio Tipo N/fisiología , Ganglios Espinales/fisiología , Receptores Opioides/fisiología , Células Receptoras Sensoriales/fisiología , Médula Espinal/fisiología , Analgésicos Opioides/farmacología , Animales , Capsaicina/farmacología , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Ganglios Espinales/efectos de los fármacos , Técnicas In Vitro , Masculino , Péptidos Opioides/farmacología , Lectinas de Plantas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Opioides/agonistas , Médula Espinal/efectos de los fármacos , Receptor de Nociceptina , NociceptinaRESUMEN
Glutamate receptor response properties of nociceptive synapses on neurokinin 1 receptor positive (NK1R+) lamina I neurons were determined 3 days after induction of chronic peripheral inflammation with Freund's Complete Adjuvant (CFA). A significant increase in the AMPAR/NMDAR ratio was found during inflammation, which was associated with a significant reduction in the quantal amplitude of NMDAR-mediated synaptic currents. A significant shortening of the quantal AMPA current decay, a greater inward rectification of the AMPAR-mediated eEPSC amplitude and an increased sensitivity to the Ca2+-permeable AMPAR channel blocker 1-naphthylacetyl spermine (NAS) was also observed, indicating an increase in the contribution of Ca2+-permeable AMPARs at this synapse during inflammation. Furthermore the reduced effectiveness of the NR2B-specific antagonist CP-101,606 on NMDAR-mediated eEPSCs together with a decrease in Mg2+ sensitivity suggests a down regulation of the highly Mg2+-sensitive and high conductance NR2B subunit at this synapse. These changes in glutamatergic receptor function during inflammation support the selective effectiveness of Ca2+-permeable AMPAR antagonists in inflammatory pain models and may underlie the reported ineffectiveness of NR2B antagonists in spinal antinociception.
Asunto(s)
Calcio/metabolismo , Conducción Nerviosa/fisiología , Dolor/metabolismo , Células del Asta Posterior/metabolismo , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Animales , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Adyuvante de Freund , Inflamación/inducido químicamente , Inflamación/complicaciones , Inflamación/metabolismo , Masculino , Dolor/etiología , Ratas , Ratas Sprague-Dawley , Receptores de Neuroquinina-1/metabolismo , Receptores Opioides/metabolismo , Receptor de NociceptinaRESUMEN
N-type calcium channels contribute to the release of glutamate from primary afferent terminals synapsing onto nocisponsive neurons in the dorsal horn of the spinal cord, but little is known of functional adaptations to these channels in persistent pain states. Subtype-selective conotoxins and other drugs were used to determine the role of different calcium channel types in a rat model of inflammatory pain. Electrically evoked primary afferent synapses onto lumber dorsal horn neurons were examined three days after induction of inflammation with intraplantar complete Freund's adjuvant. The maximal inhibitory effect of the N-type calcium channel blockers, omega-conotoxins CVID and MVIIA, were attenuated in NK1 receptor-positive lamina I neurons after inflammation, but the potency of CVID was unchanged. This was associated with reduced inhibition of the frequency of asynchronous-evoked synaptic events by CVID studied in the presence of extracellular strontium, suggesting reduced N-type channel contribution to primary afferent synapses after inflammation. After application of CVID, the relative contributions of P/Q and L channels to primary afferent transmission and the residual current were unchanged by inflammation, suggesting the adaptation was specific to N-type channels. Blocking T-type channels did not affect synaptic amplitude under control or inflamed conditions. Reduction of N-type channel contribution to primary afferent transmission was selective for NK1 receptor-positive neurons identified by post hoc immunohistochemistry and did not occur at synapses in laminae II(o) or II(i), or inhibitory synapses. These results suggest that inflammation selectively downregulates N-type channels in the terminals of primary afferents synapsing onto (presumed) nociceptive lamina I NK1 receptor-positive neurons.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Inflamación/fisiopatología , Células del Asta Posterior/metabolismo , Receptores de Neuroquinina-1/metabolismo , Transmisión Sináptica , Vías Aferentes/fisiopatología , Animales , Canales de Calcio Tipo N/efectos de los fármacos , Enfermedad Crónica , Regulación hacia Abajo , Estimulación Eléctrica , Potenciales Evocados/efectos de los fármacos , Femenino , Adyuvante de Freund , Miembro Posterior , Inflamación/inducido químicamente , Inflamación/metabolismo , Masculino , Nociceptores , Dolor/metabolismo , Dolor/fisiopatología , Ratas , Ratas Sprague-Dawley , Médula Espinal/fisiopatología , Sinapsis , Ponzoñas/farmacología , omega-ConotoxinasRESUMEN
Calcineurin, protein phosphatase 2B, is a calcium-binding protein that has been shown to modulate NMDA receptor activity (Regulation of NMDA channel function by endogenous Ca(2+)-dependent phosphatase. Nature 369 (1994) 235; Regulation of glycine-insensitive desensitisation of the NMDA receptor in outside-out patches. J. Neurophysiol. 71 (1994) 754; Calcineurin acts via the C-terminus of NR2A to modulate desensitization of NMDA receptors. Neuropharmacology 42 (2002) 593) and synaptic transmission (Synaptic desensitization of NMDA receptors by calcineurin. Science 267 (1995) 1510; beta-adrenergic regulation of synaptic NMDA receptors by cAMP-dependent protein kinase. Neuron 16 (1996) 415). Calmodulin, a necessary co-factor for calcineurin (Calmodulin binding by calcineurin. J. Biol. Chem. 262 (1987) 15062), has also been shown to inhibit NMDA receptor activity (Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit. Cell 84 (1996) 745; Direct effects of calmodulin on NMDA receptor single-channel gating in rat hippocampal granule cells. J. Neurosci. 22 (2002) 8860) in a calcium dependent manner (Calmodulin mediates calcium-dependent inactivation of N-methyl-d-aspartate receptors. Neuron 21 (1998) 443; Interactions of calmodulin and alpha-actinin with the NR1 subunit modulate calcium-dependent inactivation of NMDA receptors. J. Neurosci. 19 (1999) 1165). In order to gain insight into the likely actions and interactions of calcineurin and calmodulin at excitatory synapses, we have investigated the effects of these two proteins on single NMDA receptor channel activity. Calcineurin and calmodulin are both known to reduce channel open time (Regulation of NMDA channel function by endogenous Ca(2+)-dependent phosphatase. Nature 369 (1994) 235; Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit. Cell 84 (1996) 745), and the duration of receptor activations or superclusters. They are, therefore, predicted to shorten the synaptic current decay (Regulation of NMDA channel function by endogenous Ca(2+)-dependent phosphatase. Nature 369 (1994) 235; Direct effects of calmodulin on NMDA receptor single-channel gating in rat hippocampal granule cells. J. Neurosci. 22 (2002) 8860). In agreement with Lieberman and Mody (Regulation of NMDA channel function by endogenous Ca(2+)-dependent phosphatase. Nature 369 (1994) 235), the results of this study indicate calcineurin plus calmodulin reduces channel open time. However, this effect is not as pronounced as that observed in the presence of calmodulin alone. Calcineurin plus calmodulin was also found to increase single channel shut time. We conclude that in addition to its direct effects on single channel activity, calcineurin regulates the effects of calmodulin on NMDA receptor activity.
Asunto(s)
Calcineurina/farmacología , Calmodulina/farmacología , Hipocampo/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Interacciones Farmacológicas/fisiología , Hipocampo/fisiología , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/fisiologíaRESUMEN
The NMDA receptor is modulated by changes in the intracellular calcium concentration, through activation of various intracellular calcium-dependent proteins. We have investigated regulation of single NMDA receptor channel activity by the calcium-sensing proteins alpha-actinin and calmodulin. Both of these proteins bind to the NMDA receptor NR1 subunit C-terminus at the C0 region where they compete for occupation of the C0 site and contribute to calcium-dependent inactivation of NMDA receptor-mediated whole-cell currents. Calmodulin has also been shown to bind to the neighbouring C1 region where it has been shown to reduce single channel open time. To investigate regulation of single NMDA channel activity by alpha-actinin and calmodulin, we selected concentrations of these two proteins that would result in maximal binding to the C0 region and/or the C1 region in the case of calmodulin. Alpha-actinin binding was found to predominantly decrease single channel shut time, resulting in an increased open probability (P(open)), whereas calmodulin binding reduced single channel mean open time, resulting in an overall reduction in P(open). The physiological implications of these findings are discussed.
Asunto(s)
Actinina/farmacología , Calmodulina/farmacología , Hipocampo/metabolismo , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Animales , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/fisiología , Estimulación Eléctrica , Electrofisiología , Regulación de la Expresión Génica/fisiología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/metabolismo , Técnicas de Placa-Clamp , Conejos , Ratas , Ratas Sprague-Dawley , Superóxidos/metabolismo , Sinapsis/fisiologíaRESUMEN
NMDA receptors are glutamate-sensitive ion channel receptors that mediate excitatory synaptic transmission and are widely implicated in synaptic plasticity and integration of synaptic activity in the CNS. This is in part attributable to the high calcium permeability of the ion channel, which allows receptor activation to influence the intracellular calcium concentration and also the slow time course of NMDA receptor-mediated synaptic currents. NMDA receptor activity is also regulated by the intracellular calcium concentration through activation of various calcium-dependent proteins, including calmodulin, calcineurin, protein kinase C, and alpha-actinin-2. Here, we have shown that calmodulin reduces the duration of native NMDA receptor single-channel openings from 3.5 +/- 0.6 msec to 1.71 +/- 0.2 msec in agreement with previous studies on recombinant NMDA receptors (Ehlers et al., 1996). NMDA receptor single-channel amplitudes and shut times were not affected. However, calmodulin reduced the duration of groups of channel openings called superclusters, which determine the slow time course of synaptic currents, from 121 +/- 25.4 msec to 60.4 +/- 11.6 msec. In addition, total open time, number of channel openings, and charge transfer per supercluster were all reduced by calmodulin. A 68% decrease in charge transfer per supercluster suggests that calmodulin activation will significantly reduce calcium influx during synaptic transmission. These results suggest that calmodulin-dependent inhibition of NMDA receptors will reduce the amplitude and time course of excitatory synaptic currents and thus affect synaptic plasticity and integration of synaptic activity in the CNS.