RESUMO
Episodic ataxia type 1 (EA1) is a rare human neurological syndrome characterized by continuous myokymia and attacks of generalized ataxia that can be triggered by abrupt movements, emotional stress and fatigue. An Italian family has been identified where related members displayed continuous myokymia, episodes of ataxia, attacks characterized by myokymia only, and neuromyotonia. A novel missense mutation (F414C), in the C-terminal region of the K(+) channel Kv1.1, was identified in the affected individuals. The mutant homotetrameric channels were non-functional in Xenopus laevis oocytes. In addition, heteromeric channels resulting from the co-expression of wild-type Kv1.1 and Kv1.1(F414C), or wild-type Kv1.2 and Kv1.1(F414C) subunits displayed reduced current amplitudes and altered gating properties. This indicates that the pathogenic effect of this KCNA1 mutation is likely to be related to the defective functional properties we have identified.
Assuntos
Ataxia/genética , Saúde da Família , Canal de Potássio Kv1.1/genética , Mutação de Sentido Incorreto/genética , Mioquimia/genética , Adulto , Animais , Ataxia/complicações , Fenômenos Biofísicos , Cromossomos Humanos Par 12/genética , Cisteína/genética , Análise Mutacional de DNA , Estimulação Elétrica , Proteínas de Fluorescência Verde/genética , Humanos , Itália , Canal de Potássio Kv1.2/genética , Masculino , Potenciais da Membrana/genética , Microinjeções/métodos , Modelos Moleculares , Mioquimia/complicações , Oócitos , Técnicas de Patch-Clamp/métodos , Fenilalanina/genética , Proteínas de Xenopus/genética , Xenopus laevis , Adulto JovemRESUMO
Kir 5.1 is a member of the inward rectifier potassium channel superfamily which does not form functional channels when expressed by itself in Xenopus laevis oocytes. rt-PCR reveals high levels of Kir 5.1 mRNA expression in testis but the function of this channel remains unknown. To determine the cell-specific expression of this channel in the testis we raised a polyclonal antibody against an external epitope of Kir 5.1 and tested its specificity in Xenopus oocytes expressing several cloned Kir subunits. Strong immunoreactivity for Kir 5.1 was found in seminiferous tubules of rat testis and, particularly, in spermatogonia, primary and secondary spermatocytes, spermatids and in the head and body of spermatozoa. The intensity of Kir 5.1 immunofluorescence, quantified using laser scanning microscopy, increased with age at every stage in the development of sperm from spermatogonia and reached a peak in 60-day-old rats. In contrast, the immunofluorescence decreased in 90-day-old animals and was detected mostly in spermatozoa. The results demonstrate that Kir 5.1 expression in the testis is localised to cells involved in spermatogenesis, showing a temporal pattern of expression during sexual maturity.
Assuntos
Envelhecimento/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/biossíntese , Testículo/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , Masculino , Dados de Sequência Molecular , Coelhos , Ratos , Testículo/citologia , Xenopus laevisRESUMO
Adrenergic stimulation induces an acute fibrinolytic response in the vascular wall and can play an important role in limiting thrombus growth. The incidence of thrombotic disease increases with age. As many adrenergic functions are affected by aging, the vascular response to adrenergic stimulation was studied in young (3 +/- 1 months) and aged (18 +/- 3 months) rats, using an experimental model of vascular perfusion. Five min epinephrine (EPI) infusion (0.15-25 microM) induced a dose-dependent increase in tissue-type plasminogen activator (t-PA) release and in perfusion pressure, in both young and aged rats. In the latter, however, the basal activity of t-PA was significantly higher than in young animals (0.22 +/- 0.02 vs 0.13 +/- 0.01 U/ml; means +/- SE; p < 0.01, n = 40) and EPI infusion induced a significantly higher increase in t-PA activity (0.78 +/- 0.06 vs 0.47 +/- 0.08 U/ml, p < 0.01, for 6.25 microM EPI). The response was characterized by a delayed onset. In contrast, the increase in the perfusion pressure by EPI was significantly lower in aged that in young rats, its basal levels being normal. To evaluate whether the increased vascular response of t-PA was peculiar for adrenergic stimulation, we infused platelet activating factor (PAF), an inducer of t-PA release, not related to catecholamines. PAF, during five-minute infusion induced a rapid and dose-dependent (100-1250 pM) increase in t-PA release, to the same extent in both young and aged rats. In conclusion, basal t-PA secretion was increased in aged rats probably as a response of vascular endothelium to chronic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Envelhecimento/fisiologia , Endotélio Vascular/efeitos dos fármacos , Epinefrina/farmacologia , Fibrinólise/efeitos dos fármacos , Trombose/fisiopatologia , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Suscetibilidade a Doenças/fisiopatologia , Membro Posterior/irrigação sanguínea , Masculino , Fator de Ativação de Plaquetas/farmacologia , Ratos , Ratos EndogâmicosRESUMO
Activated neutrophils may promote thrombus formation by releasing proteases which may activate platelets, impair the fibrinolytic balance and injure the endothelial monolayer. We have investigated the morphological correlates of damage induced by activated neutrophils on the vascular wall, in particular the vascular injury induced by released cathepsin G in both static and dynamic conditions. Human umbilical vein endothelial cells were studied both in a cell culture system and in a model of perfused umbilical veins. At scanning electron microscopy, progressive alterations of the cell monolayer resulted in cell contraction, disruption of the intercellular contacts, formation of gaps and cell detachment. Contraction was associated with shape change of the endothelial cells, that appeared star-like, while the underlying extracellular matrix, a potentially thrombogenic surface, was exposed. Comparable cellular response was observed in an "in vivo" model of perfused rat arterial segment. Interestingly, cathepsin G was active at lower concentrations in perfused vessels than in culture systems. Restoration of blood flow in the arterial segment previously damaged by cathepsin G caused adhesion and spreading of platelets on the surface of the exposed extracellular matrix. The subsequent deposition of a fibrin network among adherent platelets, could be at least partially ascribed to the inhibition by cathepsin G of the vascular fibrinolytic potential. This study supports the suggestion that the release of cathepsin G by activated neutrophils, f.i. during inflammation, may contribute to thrombus formation by inducing extensive vascular damage.
Assuntos
Catepsinas/sangue , Endotélio Vascular/metabolismo , Neutrófilos/metabolismo , Trombose/metabolismo , Animais , Aorta/citologia , Aorta/metabolismo , Catepsina G , Células Cultivadas , Endotélio Vascular/citologia , Humanos , Técnicas In Vitro , Masculino , Microscopia Eletrônica de Varredura , Perfusão , Ratos , Serina Endopeptidases , Veias Umbilicais/citologia , Veias Umbilicais/metabolismoRESUMO
To investigate the relationship among lipids, coagulation and thrombosis in the absence of atherosclerosis, spontaneous or dietary-induced hyperlipidemic (FHL) rats were studied. FHL showed higher levels of coagulation factors VII, IX, X, VIII and XII and a shortening of the occlusion time (OT) of an artificial arterial prosthesis as compared with normolipidemic (FNL) animals. Damage of abdominal aorta of FHL was followed by increased fibrin deposition in the vascular intima as compared to FNL. After 5 months of cholesterol-rich diet FNL showed increased cholesterol, triglycerides and factor II, VII, IX, X, XII levels. A significant shortening of the OT and increased fibrin deposition was also observed. Two-month diet withdrawal restored the initial condition. Warfarin treatment, at a dose decreasing vitamin K-dependent factor to levels found in FNL, prolonged the OT and reduced fibrin deposition, without modifying F XII or changing lipid profile. An increase in the activated form of F VII was observed. In contrast, no difference was found in F VII clearance. High lipid levels favour the process of thrombus formation by increasing the activation of vitamin K-dependent coagulation factors. Low-dose warfarin treatment reverts the prothrombotic effect of hyperlipidemia.
Assuntos
Anticoagulantes/uso terapêutico , Fatores de Coagulação Sanguínea/análise , Modelos Animais de Doenças , Hiperlipidemias/complicações , Trombofilia/etiologia , Trombose/etiologia , Varfarina/uso terapêutico , Administração Oral , Animais , Anticoagulantes/administração & dosagem , Aorta Abdominal/patologia , Aorta Abdominal/cirurgia , Doenças da Aorta/sangue , Doenças da Aorta/etiologia , Doenças da Aorta/patologia , Doenças da Aorta/prevenção & controle , Fatores de Coagulação Sanguínea/fisiologia , Prótese Vascular , Colesterol na Dieta/administração & dosagem , Dieta Aterogênica , Ativação Enzimática , Fator VII/metabolismo , Hipercolesterolemia/complicações , Hipercolesterolemia/genética , Hiperlipidemias/etiologia , Hiperlipidemias/genética , Hipertrigliceridemia/complicações , Hipertrigliceridemia/genética , Complicações Pós-Operatórias/sangue , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/patologia , Complicações Pós-Operatórias/prevenção & controle , Ratos , Ratos Endogâmicos , Ratos Sprague-Dawley , Trombofilia/sangue , Trombofilia/tratamento farmacológico , Trombose/sangue , Trombose/patologia , Trombose/prevenção & controle , Vitamina K/fisiologia , Varfarina/administração & dosagemRESUMO
Dermatan sulphates have been shown to inhibit thrombus formation and thrombus growth in different experimental model of venous thrombosis. At variance with heparins, they show a remarkably low haemorrhagic potential. On the other hand, very few data are available on the effect of dermatan sulphates on arterial thrombus formation. We evaluated the effects of a low molecular weight (LMW)-dermatan sulphate, a high molecular weight (HMW)-dermatan sulphate and sulodexide (a mixture of fast-moving heparin fraction and dermatan sulphate) in comparison with LMW- and HMW-heparin, in a model of arterial thrombosis in rats. The insertion of an artificial prosthesis into the abdominal aorta of the animals induced the formation of an occluding thrombus within 2-5 days. The time in which 50% of the loops became occluded (OT50) was also calculated and used to compare the efficacy of the different drug treatments. This was 2.84 days for control animals and 4.25 and 5.80 days for HMW- and LMW-dermatan sulphate, respectively. Neither drug changed the "template" bleeding time, even at higher doses. In contrast, HMW-heparin at doses (8 mg/kg, sc, twice a day) inducing an antithrombotic activity comparable to that of dermatan sulphates, dramatically prolonged the bleeding time. LMW-heparin at the same doses was ineffective. Sulodexide (10 mg/Kg, sc, twice a day) prolonged the occlusion time to the same extent as HMW-heparin (OT50 5.10 vs. 4.14 days), with less an effect on the bleeding time (144 +/- 6 s vs. > 300 s, respectively). Histological examination confirms that the prolongation of occlusion time induced by the drugs is really related to thrombus formation inhibition at the site of arterial wall injury. Acetyl salicylic acid (ASA) (100 mg/kg/day in drinking water as lysine acetylsalicylate) did not modify the effect of Desmin 370 and Sulodexide on both occlusion and bleeding time. However, while it did not increase the antithrombotic activity of HMW-heparin, it significantly prolonged its haemorrhagic effect. In conclusion, dermatan sulphates are effective inhibitors of arterial thrombosis in rats, without inducing bleeding complications.
Assuntos
Anticoagulantes/uso terapêutico , Dermatan Sulfato/uso terapêutico , Glicosaminoglicanos/uso terapêutico , Heparina/uso terapêutico , Trombose/tratamento farmacológico , Animais , Modelos Animais de Doenças , Quimioterapia Combinada , Masculino , Ratos , Trombose/patologiaRESUMO
Prion diseases are fatal neurodegenerative disorders of the CNS of men and animals, characterized by spongiform degeneration of the CNS, astrogliosis and deposition of amyloid into the brain. The conversion of a cellular glycoprotein (the prion protein, PrP(C)) into an altered isoform (the prion scrapie, PrP(Sc)), which accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism, is currently believed to represent the etiologic agent responsible for these diseases. Synthetic or recombinant polypeptides are commonly used to elucidate the mechanism of proteins involved in neurodegenerative diseases. Here we describe a procedure, which allows the synthesis and purification in its native folding, of the human prion protein fragment 90-231, corresponding to the protease resistant core of PrP(Sc). We synthesized the polypeptides 90-231 of both the wild type and the E200K mutant isoforms of PrP. Using a gluthatione S-transferase (GST) fusion protein approach, milligram amounts of polypeptides were obtained after expression in E. coli. The recovery of the purified fusion protein was monitored following the evaluation of the GST activity. The PrP fragment was released from the fusion protein immobilized on a glutathione-coupled agarose resin by direct cleavage with thrombin. The recombinant protein was identified by comassie stained acrylamide gel and by immunoblotting employing a monoclonal anti-PrP antibody. The peptide purified by gel filtration chromatography showed mainly an alpha-helix structure, as analysed by circular dichroism (CD) and an intact disulfide bridge. The same procedure was also successfully employed to synthesize and purify the E200K mutant PrP fragment.
Assuntos
Escherichia coli/genética , Príons/genética , Sequência de Bases , Western Blotting , Cromatografia Líquida , Dicroísmo Circular , Primers do DNA , Humanos , Espectrometria de Massas , Príons/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Espectrometria de FluorescênciaRESUMO
The role of aspirin on tissue plasminogen activator (t-PA) release was studied in rats after experimental venous occlusion. For this purpose, we developed a new experimental model which combines a vascular perfusion system (isolated rat hindquarters) with vascular stimulation, namely the application of venous stasis. Application of venous stasis for 30 min induced the release of t-PA from the vascular endothelium into the perfusate (from 0.19 +/- 0.05 to 0.39 +/- 0.05 UI/ml), reaching a peak 90 s after reperfusion. Aspirin administered to rats 60 min before the experiments (100 mg/kg i.v.), or dissolved in Tyrode solution (100 microM), suppressed 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) synthesis (0.38 +/- 0.09 in control and < 0.01 and 0.15 +/- 0.09 ng/ml, respectively, in aspirin-treated groups) but did not prevent the increase in fibrinolytic activity after venous occlusion (from 0.20 +/- 0.04 to 0.38 +/- 0.06 and from 0.07 +/- 0.03 to 0.27 +/- 0.03 IU/ml, respectively, in the aspirin-treated group). Our results suggest that the increase in fibrinolytic activity after experimental venous occlusion in isolated rat hindlegs is modulated by mechanism(s) other than the cyclooxygenase pathway in the vascular wall.
Assuntos
Aspirina/farmacologia , Fibrinólise/efeitos dos fármacos , 6-Cetoprostaglandina F1 alfa/metabolismo , Animais , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Membro Posterior/irrigação sanguínea , Ligadura , Masculino , Perfusão , Ratos , Ativador de Plasminogênio Tecidual/metabolismo , Veias , Insuficiência Venosa/sangueRESUMO
We evaluated the changes over time in hemostatic factors during ongoing arterial thrombosis in rats, as induced by a loop-shaped aortic prosthesis. Moreover, we investigated this condition by inspecting in parallel local thrombus growth, systemic vascular prostacyclin and t-PA production. One minute after loop insertion, activated platelets spread on the internal surface of the prosthesis and 24 hrs later numerous platelet aggregates supported by a fibrin network could be observed. However, no evidence for platelet activation could be concomitantly found in peripheral blood. A sustained increased in PGI2 formation was detected together with a progressive increase in plasma fibrinolytic activity during thrombus growth. The levels of fibrinogen as well as antithrombin III (ATIII) and heparin cofactor II (HCII) activities were steadily increased in loop-bearing animals. In conclusion, the dynamic phases of thrombus formation, in an aortic prosthesis, produce changes in vascular function and in hemostatic factors at the level of systemic blood.
Assuntos
Hemostasia , Trombose/patologia , 6-Cetoprostaglandina F1 alfa/sangue , Animais , Aorta , Fatores de Coagulação Sanguínea/metabolismo , Prótese Vascular , Fibrina/metabolismo , Fibrinólise , Contagem de Leucócitos , Masculino , Agregação Plaquetária , Contagem de Plaquetas , Próteses e Implantes , Ratos , Fatores de TempoAssuntos
Amilorida/farmacologia , Diuréticos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Endotélio Vascular/metabolismo , Epinefrina/farmacologia , Fibrinólise/efeitos dos fármacos , Perfusão , Ratos , Taxa Secretória/efeitos dos fármacos , Veia Cava InferiorRESUMO
1. The inwardly rectifying potassium channel Kir5.1 appears to form functional channels only by coexpression with either Kir4.1 or Kir4.2. Kir4.1-Kir5.1 heteromeric channels have been shown to exist in vivo in renal tubular epithelia. However, Kir5.1 is expressed in many other tissues where Kir4.1 is not found. Using Kir5.1-specific antibodies we have localised Kir5.1 expression in the pancreas, a tissue where Kir4.2 is also highly expressed. 2. Heteromeric Kir5.1-Kir4.1 channels are significantly more sensitive to intracellular acidification than Kir4.1 currents. We demonstrate that this increased sensitivity is primarily due to modulation of the intrinsic Kir4.1 pH sensitivity by Kir5.1. 3. Kir4.2 was found to be significantly more pH sensitive (pK(a) = 7.1) than Kir4.1 (pK(a) = 5.99) due to an additional pH-sensing mechanism involving the C-terminus. As a result, coexpression with Kir5.1 does not cause a major shift in the pH sensitivity of the heteromeric Kir4.2-Kir5.1 channel. 4. Cell-attached single channel analysis of Kir4.2 revealed a channel with a high open probability (P(o) > 0.9) and single channel conductance of approximately 25 pS, whilst coexpression with Kir5.1 produced novel bursting channels (P(o) < 0.3) and a principal conductance of approximately 54 pS with several subconductance states. 5. These results indicate that Kir5.1 may form heteromeric channels with Kir4.2 in tissues where Kir4.1 is not expressed (e.g. pancreas) and that these novel channels are likely to be regulated by changes in intracellular pH. In addition, the extreme pH sensitivity of Kir4.2 has implications for the role of this subunit as a homotetrameric channel.
Assuntos
Polímeros/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/fisiologia , Animais , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Membranas Intracelulares/fisiologia , Masculino , Pâncreas/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Xenopus laevisRESUMO
The activity of voltage-gated potassium (Kv) channels can be dynamically modulated by several events, including neurotransmitter-stimulated biochemical cascades mediated by G-protein-coupled receptors. By using a heterologous expression system, we show that activating the 5-HT2C receptor inhibits both Kv1.1 and Kv1.2 channels through a tyrosine phosphorylation mechanism. The major molecular determinants of channel inhibition were identified as two tyrosine residues located in the N-terminal region of the Kv channel subunit. Furthermore, we demonstrate that receptor protein tyrosine phosphatase alpha (RPTPalpha), a receptor protein tyrosine phosphatase, co-ordinates the inhibition process mediated via 5-HT2C receptors. We therefore propose that the serotonergic regulation of human Kv1.1 and Kv1.2 channel activity by the 5-HT2C receptor involves the dual coordination of both RPTPalpha and specific tyrosine kinases coupled to this receptor.
Assuntos
Fosfotirosina/metabolismo , Bloqueadores dos Canais de Potássio , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Proteínas Tirosina Fosfatases/fisiologia , Receptores de Superfície Celular , Serotonina/fisiologia , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Expressão Gênica , Genisteína/farmacologia , Humanos , Cinética , Canal de Potássio Kv1.1 , Canal de Potássio Kv1.2 , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Fosforilação , Canais de Potássio/genética , Canais de Potássio/fisiologia , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores , Receptores de Serotonina/genética , Receptores de Serotonina/fisiologia , Transfecção , Vanadatos/farmacologia , Xenopus laevisRESUMO
The physiological role of the inwardly rectifying potassium channel, Kir5.1, is poorly understood, as is the molecular identity of many renal potassium channels. In this study we have used Kir5.1-specific antibodies to reveal abundant expression of Kir5.1 in renal tubular epithelial cells, where Kir4.1 is also expressed. Moreover, we also show that Kir5.1/Kir4.1 heteromeric channel activity is extremely sensitive to inhibition by intracellular acidification and that this novel property is conferred predominantly by the Kir5.1 subunit. These findings suggest that Kir5.1/Kir4.1 heteromeric channels are likely to exist in vivo and implicate an important and novel functional role for the Kir5.1 subunit.
Assuntos
Concentração de Íons de Hidrogênio , Túbulos Renais/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Animais , Sequência de Bases , Primers do DNA , Epitélio/metabolismo , Imuno-Histoquímica , Dados de Sequência Molecular , RatosRESUMO
Episodic ataxia type-1 syndrome (EA-1) is an autosomal dominant neurological disorder that manifests itself during infancy and results from point mutations in the voltage-gated potassium channel gene hKv1.1. The hallmark of the disease is continuous myokymia and episodic attacks of spastic contractions of the skeletal muscles, which cause permanent disability. Coexpression of hKv1.1 and hKv1.2 subunits produces heteromeric potassium channels with biophysical and pharmacological properties intermediate between the respective homomers. By using tandemly linked subunits, we demonstrate that hKv1.1 subunits bearing the EA-1 mutations V408A and E325D combine with hKv1.2 to produce channels with altered kinetics of activation, deactivation, C-type inactivation, and voltage dependence. Moreover, hKv1.1V408A single-channel analysis reveals a approximately threefold reduction of the mean open duration of the channel compared with the wild-type, and this mutation alters the open-state stability of both homomeric and heteromeric channels. The results demonstrate that human Kv1.2 and Kv1.1 subunits coassemble to form a novel channel with distinct gating properties that are altered profoundly by EA-1 mutations, thus uncovering novel physiopathogenetic mechanisms of episodic ataxia type-1 myokymia syndrome.
Assuntos
Ataxia Cerebelar/genética , Mutação , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/fisiologia , Animais , Ataxia Cerebelar/fisiopatologia , Humanos , Ativação do Canal Iônico/fisiologia , Canal de Potássio Kv1.1 , Técnicas de Patch-Clamp , Síndrome , XenopusRESUMO
Episodic ataxia type-1 is a rare human neurological syndrome which occurs during childhood and persists through the whole life of affected patients. Several heterozygous point mutations have been found in the coding sequence of the voltage-gated potassium channel gene hKv1.1 of different affected families. V408A and E325D mutations are located in the cytoplasmic putative pore region of hKv1.1 channels and profoundly alter their gating properties. V408A channels showed increased kinetic rates of activation, deactivation and C-type inactivation. Expression of E325D channels in Xenopus oocytes led to an approximately 13-fold current amplitude reduction and to a 52.4 mV positive shift in the voltage dependence of activation. Moreover, the E325D mutation altered the kinetics of activation, deactivation, C-type inactivation and channel open probability. Heteromeric channels composed of two wild-type and two mutated subunits, linked as dimers, showed gating properties intermediate between channels formed from four normal or four mutated subunits. The results demonstrate that the highly conserved residues Val408 and Glu325 play a pivotal role in several gating processes of a human potassium channel, and suggest a pathogenetic mechanism by which the impairment of the delayed-rectifier function of affected neurons is related to the type and number of mutated subunits which make up the hKv1.1 channels.
Assuntos
Ataxia/fisiopatologia , Ativação do Canal Iônico/genética , Mutação/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/genética , Canais de Potássio/fisiologia , Animais , Citoplasma , DNA Complementar , Dimerização , Potenciais Evocados , Humanos , Cinética , Canal de Potássio Kv1.1 , Oócitos , Técnicas de Patch-Clamp , Canais de Potássio/química , Conformação Proteica , RNA Mensageiro , Xenopus laevisRESUMO
The role of vascular cyclooxygenase pathway on tissue-type plasminogen activator (t-PA) release after venous occlusion was studied in anesthetized rats. After the inferior vena cava was clamped for 30 min, fibrinolytic activity increased from 143.7 +/- 14.5 to 209.5 +/- 10.3 mm2 (mean +/- SE, P < 0.002). This increase was prevented by aspirin at high (100 mg/kg i.v.) but not at low doses (1 mg/kg i.v.). Dazoxiben (10 mg/kg i.v.), an inhibitor of thromboxane synthase, was ineffective on the fibrinolytic response. Both the basal levels of 6-ketoprostaglandin F1 alpha and its increase after venous occlusion were suppressed by 100 mg/kg aspirin administration (from 0.64 +/- 0.2 to 0.05 +/- 0.002 ng/ml before occlusion, P < 0.001; and from 1.08 +/- 0.2 to 0.06 +/- 0.002 ng/kg after occlusion, P < 0.001), whereas they were both unaffected by aspirin at low doses (from 0.53 +/- 0.06 before to 1.20 +/- 0.08 ng/ml after stasis). Moreover, iloprost, a stable analogue of prostacyclin, reversed the aspirin inhibitory effects on fibrinolytic activity by restoring t-PA vascular release after venous stasis. Our results provide experimental evidence that an intact cyclooxygenase pathway in vascular wall is required for the fibrinolytic activity increase after venous occlusion in rats.