RESUMO
PURPOSE: Nitric oxide (NO) donors and cholinergic agents decrease intraocular pressure, in part because they induce a decrease in aqueous humor production. Because Na,K-adenosine triphosphatase (ATPase) is involved in aqueous humor formation, this study was conducted to investigate the hypothesis that NO and cholinomimetics regulate its activity in bovine ciliary processes. METHODS: Bovine tissue slices were incubated with agonists and antagonists in a physiological buffer in vitro. Na,K-ATPase activity was determined by assaying hydrolysis of adenosine triphosphate (ATP) in suspended permeabilized tissue slices. RESULTS: Carbachol-induced inhibition of Na,K-ATPase activity correlated with increases in cGMP. This inhibition was abolished by the muscarinic blocker atropine, the NO inhibitor N(w)-nitro-L-arginine (L-NAME) and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Sodium nitroprusside (SNP) mimicked the actions of carbachol. The SNP-induced decrease in Na,K-ATPase activity correlated with an increase in cGMP and was also abolished by ODQ. Both 8-bromo (Br)-cGMP and okadaic acid also inhibited Na,K-ATPase activity. CONCLUSIONS: Carbachol-induced inhibition of Na,K-ATPase activity involves muscarinic receptor activation. That SNP mimics and L-NAME reverses carbachol's effect on Na,K-ATPase activity suggests that the actions of carbachol are mediated by NO. Carbachol's and SNP's effects on Na,K-ATPase activity involved soluble guanylate cyclase and cGMP. Inhibition of Na,K-ATPase activity by 8-Br-cGMP and okadaic acid indicates that protein phosphorylation events may mediate SNP-induced inhibition of Na,K-ATPase activity.
Assuntos
Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Corpo Ciliar/efeitos dos fármacos , Óxido Nítrico/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Atropina/farmacologia , Bovinos , Corpo Ciliar/enzimologia , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Inibidores Enzimáticos/farmacologia , NG-Nitroarginina Metil Éster/farmacologiaRESUMO
BACKGROUND: Inhibition of the Na,K-ATPase by ouabain results in decreased intraocular pressure (IOP), by mechanisms that are not completely understood. Cellular mechanisms that regulate aqueous humour outflow through the trabecular meshwork (TM) include changes in the TM cytoskeleton. Because inhibition of the Na,K-ATPase by ouabain alters the cell's cytoskeleton, the role of the ouabain in regulating aqueous humour outflow and TM cytoskeleton was investigated. METHOD: Porcine anterior eye segment and low-passage porcine TM cells were used. Outflow facility was measured using perfused anterior eye segment organ culture. Changes in TM cytoskeleton were assessed by rhodamine-phalloidin and anti-paxillin antibody staining and imaged with confocal microscopy. RESULTS: Ouabain (30 nM to 300 microM) increased outflow facility in perfused eye anterior segments. The time course for ouabain-induced changes in outflow facility correlated with morphological changes in the cytoskeleton of TM cells exposed to ouabain (30 nM to 300 microM). Morphological changes in TM cells include changes in actin filaments and focal adhesions. CONCLUSIONS: These data suggest that ouabain, at non-toxic concentrations, increases aqueous humour outflow facility possibly by altering the TM cell cytoskeleton.
Assuntos
Humor Aquoso/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ouabaína/farmacologia , Malha Trabecular/efeitos dos fármacos , Animais , Humor Aquoso/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Pressão Intraocular/fisiologia , Suínos , Malha Trabecular/citologia , Malha Trabecular/metabolismoRESUMO
Calcium (Ca2+) plays an integral role in the light response of the photoreceptors in both vertebrate and invertebrate organisms. In the ventral eye of the horseshoe crab, Limulus polyphemus, a flash of light delivered to a dark-adapted photoreceptor stimulates a rapid rise in intracellular free calcium concentration ([Ca2+]i), which in turn mediates light adaptation. It has previously been demonstrated that in Limulus photoreceptors light, via Ca2+, activates a calcium/calmodulin (Ca2+/CaM)-dependent protein kinase which increases the phosphorylation of arrestin. We now have identified biochemically, a calcium/calmodulin-dependent protein phosphatase (Ca2+/CaM PP) in homogenates of the Limulus lateral and ventral eye, brain, and lateral optic nerve using as a substrate, a 32P-labeled peptide fragment of the regulatory subunit of cAMP-dependent protein kinase (RII). This protein phosphatase shares biochemical properties with calcineurin, a Ca2+/CaM-dependent protein phosphatase (type-2B). Its activity is enhanced by Ca2+, calmodulin and Mn2+; and is inhibited by mastoparan, a calmodulin antagonist, and a synthetic peptide corresponding to the autoinhibitory domain of mammalian calcineurin. Most importantly, light regulates the Ca2+/CaM PP activity in the lateral eye. While there is no difference in basal activity in long-term dark- or light-adapted preparations, Ca2+ enhances Ca2+/CaM PP activity only in long-term light-adapted eyes.
Assuntos
Encéfalo/enzimologia , Cálcio/farmacologia , Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/farmacologia , Fosfoproteínas Fosfatases/metabolismo , Células Fotorreceptoras de Invertebrados/enzimologia , Sequência de Aminoácidos , Animais , Encéfalo/efeitos dos fármacos , Calcineurina , Proteínas de Ligação a Calmodulina/antagonistas & inibidores , Adaptação à Escuridão , Caranguejos Ferradura , Peptídeos e Proteínas de Sinalização Intercelular , Luz , Dados de Sequência Molecular , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/enzimologia , Fragmentos de Peptídeos , Peptídeos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Células Fotorreceptoras de Invertebrados/efeitos dos fármacos , Transdução de Sinais/fisiologia , Especificidade por Substrato , Venenos de Vespas/farmacologiaRESUMO
Secretion of cerebrospinal fluid by the choroid plexus can be inhibited by its cholinergic innervation. We demonstrated that carbachol inhibits the Na(+)-K(+)-ATPase in bovine choroid tissue slices and investigated the mechanism. Many of the actions of cholinergic agents are mediated by nitric oxide (NO), which plays important roles in fluid homeostasis. The inhibition of Na(+)-K(+)-ATPase was blocked by the NO synthase inhibitor [N(omega)-nitro-L-arginine methyl ester] and was quantitatively mimicked by the NO agonists sodium nitroprusside (SNP) and diethylenetriamine NO. Inhibition by SNP correlated with an increase in tissue cGMP and was abolished by 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase. Inhibition was mimicked by the protein kinase G activator 8-bromo-cGMP and by okadaic acid, an inhibitor of protein phosphatases 1 and 2A. cGMP-dependent protein kinase inhibitors Rp-8-pCPT-cGMP (0.5-5 microM) and KT-5823 (2.0 microM) did not block the effects of SNP, but higher concentrations of the more selective inhibitor (Rp-8-pCPT-cGMP) had a pharmacological inhibitory effect on Na(+)-K(+)-ATPase. The data suggest that cholinergic regulation of the Na(+)-K(+)-ATPase is mediated by NO and involves activation of guanylate cyclase and elevation of cGMP.
Assuntos
Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Plexo Corióideo/enzimologia , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Óxido Nítrico/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Bovinos , Plexo Corióideo/efeitos dos fármacos , GMP Cíclico/farmacologia , Inibidores Enzimáticos/farmacologia , Hidrólise , Técnicas In Vitro , NG-Nitroarginina Metil Éster/farmacologia , Nitroprussiato/farmacologia , Ouabaína , Oxidiazóis/farmacologia , Fosforilação , Quinoxalinas/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Solubilidade , Tionucleotídeos/farmacologia , Triazenos/farmacologia , Vasodilatadores/farmacologiaRESUMO
Changes in intracellular calcium are involved in phototransduction processes in both vertebrate and invertebrate photoreceptors. During this phototransduction process in the Limulus ventral eye, there is a biochemical change in the protein phosphatase, calcineurin, such that it becomes capable of activation by calcium and calmodulin. Here we show that the calcium/calmodulin-dependent calcineurin-like activity in light-adapted ventral eye was completely inhibited by the CaN autoinhibitory peptide, CaN A457-482 and the Merck analog of the membrane-permeable, immunosuppressant drug, FK 506, L-683, 590, but not an inactive analogue, L-685, 818. Whole-cell, voltage-clamp recordings of spontaneous quantal bump activity present in dark-adapted photoreceptors injected with either CaN A457-482 (500 microM) or superfused with L-683, 590 (20 microM) or L-685, 818 revealed that both CaN A457-482 and L-683, 590, but not L-685, 818, caused rapid decreases in quantal bump amplitude, rise time and fall time, resulting in smaller, sharper bumps. This was correlated with enhanced phosphorylation of arrestin in light-adapted ventral eye photoreceptors exposed to L-683, 590 or less reliably okadaic acid. Both CaN A457-482 and L-683, 590 markedly affected the light-stimulated inward currents recorded from light-adapted ventral photoreceptors, causing a "terracing" of the inward current, and an intensity-dependent delay in the time required to reach peak amplitude. Consequently, inhibition of calcineurin markedly affects two major rhodopsin-dependent electrophysiological processes, and implicates CaN as an integral component in the phototransduction cascade.