RESUMO
The aim of the present study was to determine the effect of pertussis toxin (PTX) on inflammatory hypernociception measured by the rat paw pressure test and to elucidate the mechanism involved in this effect. In this test, prostaglandin E(2) (PGE(2)) administered subcutaneously induces hypernociception via a mechanism associated with neuronal cAMP increase. Local intraplantar pre-treatment (30 min before), and post-treatment (5 min after) with PTX (600 ng/paw1, in 100 microL) reduced hypernociception induced by prostaglandin E(2) (100 ng/paw, in 100 microL, intraplantar). Furthermore, local intraplantar pre-treatment (30 min before) with PTX (600 ng/paw, in 100 microL) reduced hypernociception induced by DbcAMP, a stable analogue of cAMP (100 microg/paw, in 100 microL, intraplantar), which indicates that PTX may have an effect other than just G(i)/G(0) inhibition. PTX-induced analgesia was blocked by selective inhibitors of nitric oxide synthase (L-NMMA), guanylyl cyclase (ODQ), protein kinase G (KT5823) and ATP-sensitive K(+) channel (Kir6) blockers (glybenclamide and tolbutamide). In addition, PTX was shown to induce nitric oxide (NO) production in cultured neurons of the dorsal root ganglia. In conclusion, this study shows a peripheral antinociceptive effect of pertussis toxin, resulting from the activation of the arginine/NO/cGMP/PKG/ATP-sensitive K(+) channel pathway.
Assuntos
Analgésicos/metabolismo , Arginina/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Óxido Nítrico/metabolismo , Toxina Pertussis/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Trifosfato de Adenosina/metabolismo , Analgesia , Animais , Bucladesina/metabolismo , Carbazóis/metabolismo , Células Cultivadas , Dinoprostona/administração & dosagem , Dinoprostona/imunologia , Inibidores Enzimáticos/metabolismo , Gânglios Espinais/citologia , Glibureto/metabolismo , Indóis/metabolismo , Canais KATP , Masculino , Neurônios/citologia , Neurônios/metabolismo , Oxidiazóis/metabolismo , Dor/metabolismo , Medição da Dor , Quinoxalinas/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Tolbutamida/metabolismo , ômega-N-Metilarginina/metabolismoRESUMO
Mammalian sperm must undergo a series of physiological changes after leaving the testis to become competent for fertilization. These changes, collectively known as capacitation, occur in the female reproductive tract where the sperm plasma membrane is modified in terms of its components and ionic permeability. Among other events, mouse sperm capacitation leads to an increase in the intracellular Ca(2+) and pH as well as to a hyperpolarization of the membrane potential. It is well known that ion channels play a crucial role in these events, though the molecular identity of the particular channels involved in capacitation is poorly defined. In the present work, we report the identification and potential functional role of K(ATP) channels in mouse spermatogenic cells and sperm. By using whole-cell patch clamp recordings in mouse spermatogenic cells, we found K(+) inwardly rectifying (K(ir)) currents that are sensitive to Ba(2+), glucose and the sulfonylureas (tolbutamide and glibenclamide) that block K(ATP) channels. The presence of these channels was confirmed using inhibitors of the ATP synthesis and K(ATP) channel activators. Furthermore, RT-PCR assays allowed us to detect transcripts for the K(ATP) subunits SUR1, SUR2, K(ir)6.1 and K(ir)6.2 in total RNA from elongated spermatids. In addition, immunoconfocal microscopy revealed the presence of these K(ATP) subunits in mouse spermatogenic cells and sperm. Notably, incubation of sperm with tolbutamide during capacitation abolished hyperpolarization and significantly decreased the percentage of AR in a dose-dependent fashion. Together, our results provide evidence for the presence of K(ATP) channels in mouse spermatogenic cells and sperm and disclose the contribution of these channels to the capacitation-associated hyperpolarization.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Capacitação Espermática/fisiologia , Espermatogênese/fisiologia , Espermatozoides/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bário/metabolismo , Bário/farmacologia , Diazóxido/metabolismo , Diazóxido/farmacologia , Relação Dose-Resposta a Droga , Glibureto/metabolismo , Glibureto/farmacologia , Canais KATP , Masculino , Potenciais da Membrana , Camundongos , Microscopia Confocal , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Pinacidil/metabolismo , Pinacidil/farmacologia , RNA Mensageiro/metabolismo , Receptores de Droga , Espermatozoides/citologia , Receptores de Sulfonilureias , Fatores de Tempo , Tolbutamida/metabolismo , Tolbutamida/farmacologiaRESUMO
In order to investigate to the contribution of K+ channels on the peripheral antinociception induced by diclofenac, we evaluated the effect of several K+ channel blockers, using the rat paw pressure test, in which sensitivity is increased by intraplantar injection (2 microg) of prostaglandin E2. Diclofenac administered locally into the right hindpaw (25, 50, 100 and 200 microg) elicited a dose-dependent antinociceptive effect which was demonstrated to be local, since only higher doses produced an effect when injected in the contralateral paw. This blockade of PGE2 mechanical hyperalgesia induced by diclofenac (100 microg/paw) was antagonized in a dose-dependent manner by intraplantar administration of the sulphonylureas glibenclamide (40, 80 and 160 microg) and tolbutamide (80, 160 and 320 microg), specific blockers of ATP-sensitive K+ channels, and it was observed even when the hyperalgesic agent used was carrageenin, while the antinociceptive action of indomethacin (200 microg/paw), a typical cyclo-oxygenase inhibitor, over carrageenin-induced hyperalgesia was not affected by this treatment. Charybdotoxin (2 microg/paw), a blocker of large conductance Ca2+-activated K+ channels and dequalinium (50 microg/paw), a selective blocker of small conductance Ca2+-activated K+ channels, did not modify the effect of diclofenac. This effect was also unaffected by intraplantar administration of non-specific voltage-dependent K+ channel blockers tetraethylammonium (1700 microg) and 4-aminopyridine (100 microg) or cesium (500 microg), a non-specific K+ channel blocker. The peripheral antinociceptive effect induced by diclofenac was antagonized by NG-Nitro L-arginine (NOarg, 50 microg/paw), a NO synthase inhibitor and methylene blue (MB, 500 microg/paw), a guanylate cyclase inhibitor, and this antagonism was reversed by diazoxide (300 microg/paw), an ATP-sensitive K+ channel opener. We also suggest that an endogenous opioid system may not be involved since naloxone (50 microg/paw) did not affect diclofenac-induced antinociception in the PGE2-induced hyperalgesia model. This study provides evidence that the peripheral antinociceptive effect of diclofenac may result from activation of ATP-sensitive K+ channels, possible involving stimulation of L-arginine/NO/cGMP pathway, while Ca2+-activated K+ channels, voltage-dependent K+ channels as well as endogenous opioids appear not to be involved in the process.
Assuntos
Analgesia , Anti-Inflamatórios não Esteroides/metabolismo , Diclofenaco/metabolismo , Canais de Potássio/metabolismo , Animais , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/metabolismo , Glibureto/metabolismo , Hiperalgesia/induzido quimicamente , Indometacina/metabolismo , Masculino , Naloxona/metabolismo , Antagonistas de Entorpecentes/metabolismo , Medição da Dor , Bloqueadores dos Canais de Potássio/metabolismo , Ratos , Ratos Wistar , Tolbutamida/metabolismoRESUMO
Se hace un estudio experimental sobre la absorción de Vit. Apor el intestino delgado de la rata y su variación por efecto de la Tolbutamida que se mostró ineficaz para estimular dicha absorción