RESUMO
Phototoxicity-induced pain is a major clinical problem triggered by light acting on photosensitising drugs or endogenous porphyrins, notably protoporphyrin IX (PpIX), an intermediary in heme biosynthesis. Protoporphyrin IX accumulates in individuals with erythropoietic protoporphyria and is elevated during photodynamic therapy subsequent to application of 5-aminolevulinic acid (ALA). Pain occurs during irradiation of PpIX and responds poorly to conventional analgesics. Our objective was to develop a model of PpIX phototoxicity pain and investigate the potential of menthol as an analgesic. Application of ALA to the tails of C57 black and SWISS white mice caused PpIX accumulation and nociception during irradiation (630 nm at 3.7 J/cm). Despite similar PpIX accumulation, C57 mice exhibited less pain behavior compared with SWISS mice because of light absorption by pigmentation. Irradiation of ALA-treated dorsal root ganglion neurons caused phototoxicity-evoked action potentials (APs) in both mouse strains. The antioxidant L-tryptophan increased the light dose required to elicit such APs. By contrast, the addition of keratinocytes to neuronal cultures decreased the threshold for APs, suggesting a requirement for proliferating cells. Inhibition of fatty acid amide hydrolase, selective antagonism of TRPV1 or the application of lidocaine or its quaternary derivative QX-314, reduced AP frequency, whereas antagonism of TRPA1 had no effect. These results suggest that products of singlet oxygen-mediated lipid peroxidation trigger nociceptor activation via TRPV1. Menthol inhibited phototoxicity-evoked APs and reduced pain behavior when applied topically to mice. These findings suggest that menthol might provide pain relief in patients experiencing PpIX-phototoxicity pain caused by photodynamic therapy or erythropoietic protoporphyria.
Assuntos
Antipruriginosos/uso terapêutico , Dermatite Fototóxica/complicações , Mentol/uso terapêutico , Limiar da Dor/efeitos dos fármacos , Dor/tratamento farmacológico , Dor/etiologia , Fotoquimioterapia/efeitos adversos , Acrilamidas/farmacologia , Animais , Animais Recém-Nascidos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Modelos Animais de Doenças , Potenciais Evocados/efeitos dos fármacos , Células HEK293 , Humanos , Hiperalgesia/etiologia , Lidocaína/uso terapêutico , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
Picrotoxin and t-butylbicyclophosphorothionate (TBPS) are GABA(A) receptor (GABA(A)R) open channel blockers. However, picrotoxin displaceable [(35)S]TBPS binding to α1ß2γ2 GABA(A)Rs occurs in the absence of GABA, suggesting that access to the binding site is independent of activation. Alternatively, spontaneous gating may provide access to the channel. In the absence of episodic GABA application, picrotoxin and TBPS blocked (by 91 ± 3% and 85 ± 5%, respectively) GABA-evoked currents mediated by α1ß2γ2 receptors. We used two approaches to inhibit spontaneous GABA(A)R gating, bicuculline, which inhibits spontaneous current in the absence of exogenous agonist and the α1(K278M) mutant subunit. Whole-cell patch-clamp recordings demonstrated that α1(K278M)ß2γ2 receptors have negligible spontaneous gating. Application of bicuculline to α1ß2γ2 receptors in the absence of exogenous GABA caused a 35% reduction of current blockade by TBPS and reduced [(35)S]TBPS binding by 25%. Consistent with this, in the absence of exogenous GABA, α1(K278M)ß2γ2 receptors exhibited reduced blockade by TBPS current compared to wild-type receptors. These data suggest that a decrease in spontaneous gating reduces accessibility of TBPS to its binding site. GABA application during picrotoxin or TBPS administration enhanced α1ß2γ2 receptor blockade (to 98% in both cases). The GABA-dependent component of TBPS blockade accounts for the stimulation of [(35)S]TBPS binding to α1ß2γ2 receptors seen with GABA (1 µm) application. Moreover, application of GABA at concentrations that cause significant steady-state desensitization reduced [(35)S]TBPS binding. The α1(K278M) subunit slowed desensitization kinetics and increased the rate of deactivation of GABA-evoked currents. Furthermore, there was a marked increase in the GABA EC(50) for desensitization of α1(K278M)ß2γ2 receptors associated with a large increase in the GABA-dependent stimulation of [(35)S]TBPS binding. These data establish a relationship between GABA(A)R function and the three phases of [(35)S]TBPS binding seen in the absence and the presence of GABA.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Bicuculina/farmacologia , Sítios de Ligação , Linhagem Celular , Glicina/metabolismo , Células HEK293 , Humanos , Mutação , Técnicas de Patch-Clamp/métodos , Picrotoxina/farmacologia , Receptores 5-HT3 de Serotonina/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Analysis of membrane currents recorded from hormone-deprived H441 cells showed that the membrane potential (V(m)) in single cells (approximately -80 mV) was unaffected by lowering [Na+]o or [Cl(-)]o, indicating that cellular Na+ and Cl(-) conductances (GNa and GCl, respectively) are negligible. Although insulin (20 nM, approximately 24 h) and dexamethasone (0.2 microM, approximately 24 h) both depolarized Vm by approximately 20 mV, the response to insulin reflected a rise in GCl mediated via phosphatidylinositol 3-kinase (PI3K) whereas dexamethasone acted by inducing a serum- and glucocorticoid-regulated kinase 1 (SGK1)-dependent rise in GNa. Although insulin stimulation/PI3K-P110 alpha expression did not directly increase GNa, these maneuvers augmented the dexamethasone-induced conductance. The glucocorticoid/SGK1-induced GNa in single cells discriminated poorly between Na+ and K+ (PNa/PK approximately 0.6), was insensitive to amiloride (1 mM), but was partially blocked by LaCl3 (La3+; 1 mM, approximately 80%), pimozide (0.1 mM, approximately 40%), and dichlorobenzamil (15 microM, approximately 15%). Cells growing as small groups, on the other hand, expressed an amiloride-sensitive (10 microM), selective GNa that displayed the same pattern of hormonal regulation as the nonselective conductance in single cells. These data therefore 1) confirm that H441 cells can express selective or nonselective GNa (14, 48), 2) show that these conductances are both induced by glucocorticoids/SGK1 and subject to PI3K-dependent regulation, and 3) establish that cell-cell contact is vitally important to the development of Na+ selectivity and amiloride sensitivity.