RESUMO
Kinin B1 receptors are known to be highly induced after inflammatory stimuli in several biological systems. We report that incubation of pig iris sphincter with lipopolysaccharide from Escherichia coli caused a marked and time-related up-regulation of B1, accompanied by a reduction of B2 receptor-mediated contractile responses. The up-regulation of B1 receptors by lipopolysaccharide stimulation was decreased by the inhibitors of protein synthesis, cycloheximide and actinomycin D, and by dexamethasone and the nuclear factor-kappaB (NF-kappaB) inhibitor pyrrolidinedithiocarbamate (PDTC). In addition, lipopolysaccharide-induced up-regulation of B1 receptors in the pig iris sphincter was significantly reduced by the p38 inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) and to a lesser extent by the extracellular signal-regulated kinases 1 and 2 (ERK1/2) blocker 2'-amino-3'-methoxyflavone (PD98059). Molecular biology experiments demonstrated that in vitro incubation with lipopolysaccharide resulted in a time-dependent and remarkable activation of NF-kappaB and of p38 and ERK1/2 mitogen-activated protein (MAP) kinases, in pig iris sphincter preparations. While attempting to verify how MAP kinases are part of the B1 receptor-activated signaling transduction pathways, we observed that PD98059 was able to markedly reduce the contraction induced by B1 receptor activation in lipopolysaccharide-pretreated pig iris sphincter muscle but that this response was only partially decreased by SB203580. Our results extend the previous evidence on the mechanisms underlying the B1 receptor upregulation processes and demonstrate for the first time how this takes place in an ocular tissue, the pig iris sphincter. It is therefore possible to define B1 receptors as therapeutic targets for the treatment of infectious and inflammatory alterations of the eye.
Assuntos
Iris/fisiologia , Lipopolissacarídeos/farmacologia , Receptor B1 da Bradicinina/genética , Animais , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Cicloeximida/farmacologia , Iris/efeitos dos fármacos , Cinética , Receptor B1 da Bradicinina/efeitos dos fármacos , Receptor B1 da Bradicinina/fisiologia , Suínos , Regulação para CimaRESUMO
We have reported previously that bradykinin (BK) induces potent and reproducible concentration-dependent contractions of the pig iris sphincter (PIS) muscle in vitro through the activation of BK B(2) receptors. Here we attempted to investigate additional mechanisms by which BK induces contraction of the PIS in vitro. BK-mediated contraction of the PIS relied largely on the external Ca2+ influx by a mechanism sensitive to the L-, N- and P-type of Ca2+ channel selective blockers. Likewise, BK-induced contraction of the PIS was greatly inhibited by the CGRP-(8-37), NK(2) or NK(3) receptor antagonists (SR 48968, SR 142801), and to a lesser extent by the NK(1) antagonist (FK 888). Capsaicin desensitization of PIS or capsazepine pre-incubation also significantly reduced BK-mediated contraction in the PIS. Furthermore, KT 5720 or GF 109203X (the protein kinase A and C inhibitors, respectively) also significantly inhibited BK-mediated contraction. Taken together, these results indicate that BK-mediated contraction of the PIS seems to be mediated primarily by the release of CGRP and tachykinins from sensory nerve fibers, and relies largely on extracellular Ca2+ influx via activation of L-, N- and P-type of Ca2+ channels. Finally, these responses are mediated by activation of both protein kinase A- and C-dependent mechanisms.
Assuntos
Bradicinina/farmacologia , Capsaicina/análogos & derivados , Iris/fisiologia , Contração Muscular/efeitos dos fármacos , Animais , Benzamidas/farmacologia , Bradicinina/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Cálcio/metabolismo , Cálcio/farmacologia , Canais de Cálcio/fisiologia , Capsaicina/farmacologia , Carbazóis/farmacologia , Conotoxinas/farmacologia , Dipeptídeos/farmacologia , Relação Dose-Resposta a Droga , Ácido Egtázico/farmacologia , Técnicas In Vitro , Indóis/farmacologia , Iris/efeitos dos fármacos , Maleimidas/farmacologia , Nicardipino/farmacologia , Fragmentos de Peptídeos/farmacologia , Piperidinas/farmacologia , Inibidores de Proteínas Quinases , Proteínas Quinases/metabolismo , Pirróis/farmacologia , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/fisiologia , Receptores de Droga/antagonistas & inibidores , Receptores de Droga/fisiologia , Receptores da Neurocinina-2/antagonistas & inibidores , Receptores da Neurocinina-2/fisiologia , Receptores da Neurocinina-3/antagonistas & inibidores , Receptores da Neurocinina-3/fisiologia , Suínos , Taquicininas/fisiologia , ômega-Agatoxina IVA/farmacologiaRESUMO
This study was designed to investigate the mechanisms by which bradykinin induces contraction of the pig iris sphincter muscle in vitro. Addition of bradykinin, Lys-bradykinin and Met-Lys-bradykinin to the pig iris sphincter resulted in a graded contraction with a mean EC(50s) of 21, 11 and 5 nM, respectively. The bradykinin B(1) receptor agonist des-Arg(9)-bradykinin only caused a slight contraction, measured 6 h after the tissue was set up. The B(2) receptor antagonists FR 173657 ((E)-3-(6-acetamido-3-pyridyl)-N [N-2-4-dichloro-3-[(2-methyl-8-quinolinyl) oxymethyl] phenyl]-N-methylamino-carbonyl-ethyl] acrylamide) and Hoe 140 (D-Arg(0)-[Hyp(3), Thi(5), D-Tic(7), Oic(8)]-bradykinin produced a graded shift to the right associated with marked inhibition of the bradykinin-induced contraction. Atropine, guanethidine or tetrodotoxin significantly reduced the bradykinin-induced contraction. Dazoxiben, an inhibitor of thromboxane A(2), and MK-571 (3-(3-(2-(7-chloro-2-quinolinyl) ethenyl) phenyl ((3-dimethyl amino-3oxo-propyl) thio) methyl) propanoic acid, a leukotriene D(4) receptor-selective antagonist, also caused inhibition of the bradykinin-mediated contraction. Cyclooxygenase-1 and -2 inhibitors, indomethacin, ibuprofen, valeryl salicylate and NS 398 (N-[2-(cyclohexyloxy)-4-nitrophenyl]methanosulfonamide) all significantly inhibited the bradykinin-mediated contraction without affecting the carbachol-induced contraction of the pig iris sphincter. Taken together, these results indicate that the bradykinin-mediated contraction of the pig iris sphincter muscle seems to be mediated primarily by the activation of the B(2) receptor release of acetylcholine, noradrenaline and both cyclooxygenase-1 and -2 metabolites besides the release of leukotriene D(4) and tromboxane A(2) from the arachidonic acid pathway.