RESUMEN
BACKGROUND AND PURPOSE: Inflammasomes are multimeric complexes that facilitate caspase-1-mediated processing of the pro-inflammatory cytokines IL-1ß and IL-18. Clinical hypertension is associated with renal inflammation and elevated circulating levels of IL-1ß and IL-18. Therefore, we investigated whether hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and if inhibition of inflammasome activity reduces BP, markers of renal inflammation and fibrosis. EXPERIMENTAL APPROACH: Wild-type and inflammasome-deficient ASC(-/-) mice were uninephrectomized and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt). Control mice were uninephrectomized but received a placebo pellet and water. BP was measured by tail cuff; renal expression of inflammasome subunits and inflammatory markers was measured by real-time PCR and immunoblotting; macrophage and collagen accumulation was assessed by immunohistochemistry. KEY RESULTS: 1K/DOCA/salt-induced hypertension in mice was associated with increased renal mRNA expression of inflammasome subunits NLRP3, ASC and pro-caspase-1, and the cytokine, pro-IL-1ß, as well as protein levels of active caspase-1 and mature IL-1ß. Following treatment with 1K/DOCA/salt, ASC(-/-) mice displayed blunted pressor responses and were also protected from increases in renal expression of IL-6, IL-17A, CCL2, ICAM-1 and VCAM-1, and accumulation of macrophages and collagen. Finally, treatment with a novel inflammasome inhibitor, MCC950, reversed hypertension in 1K/DOCA/salt-treated mice. CONCLUSIONS AND IMPLICATIONS: Renal inflammation, fibrosis and elevated BP induced by 1K/DOCA/salt treatment are dependent on inflammasome activity, highlighting the inflammasome/IL-1ß pathway as a potential therapeutic target in hypertension.
Asunto(s)
Hipertensión/metabolismo , Inflamasomas/metabolismo , Enfermedades Renales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Adaptadoras de Señalización CARD , Desoxicorticosterona/administración & dosificación , Hipertensión/inducido químicamente , Inflamasomas/antagonistas & inhibidores , Enfermedades Renales/inducido químicamente , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Sales (Química)/administración & dosificaciónRESUMEN
Nicotinamide adenine dinucleotide phosphate oxidases (NOX) are enzymes that generate reactive oxygen species (ROS). NOX2 activity in the vascular wall is elevated in hypercholesterolemia, and contributes to oxidative stress and atherogenesis. Here we examined the role of another NOX isoform, NOX1, in atherogenesis in apolipoprotein E-knockout (APOE(-/-)) mice fed a Western diet for 14 weeks. Although NOX1 mRNA expression was unchanged in aortas from APOE(-/-) versus wild-type mice, expression of the NOX1-specific organizer, NOXO1, was diminished, consistent with an overall reduction in NOX1 activity in APOE(-/-) mice. To examine the impact of a further reduction in NOX1 activity, APOE(-/-) mice were crossed with NOX1(-/y) mice to generate NOX1(-/y)/APOE(-/-) double-knockouts. NOX1 deficiency in APOE(-/-) mice was associated with 30-50% higher plasma very-low-density lipoprotein (VLDL)/LDL and triglyceride levels (P < 0.01). Vascular ROS levels were also elevated by twofold in NOX1(-/y)/APOE(-/-) versus APOE(-/-) mice (P < 0.05), despite no changes in expression of other NOX subunits. Although en face analysis of the descending aorta revealed no differences in plaque area between NOX1(-/y)/APOE(-/-) and APOE(-/-) mice, intimal thickening in the aortic sinus was increased by 40% (P < 0.05) in the double-knockouts. Moreover, NOX1 deficiency was associated with a less stable plaque phenotype; aortic sinus lesions contained 60% less collagen (P < 0.01), 40% less smooth muscle (P < 0.01), and 2.5-fold higher levels of matrix metalloproteinase-9 (P < 0.001) than lesions in APOE(-/-) mice. Thus, these data, which suggest a protective role for NOX1 against hyperlipidemia and atherosclerosis in APOE(-/-) mice, highlight the complex and contrasting roles of different NOX isoforms (e.g., NOX2 versus NOX1) in vascular pathology.
Asunto(s)
Apolipoproteínas E/genética , Aterosclerosis/genética , Lipoproteínas LDL/sangre , Lipoproteínas VLDL/sangre , NADH NADPH Oxidorreductasas/genética , Triglicéridos/sangre , Animales , Aterosclerosis/sangre , Lipoproteínas LDL/genética , Lipoproteínas VLDL/genética , Ratones , Ratones Noqueados , NADPH Oxidasa 1 , Triglicéridos/genéticaRESUMEN
Chronic inflammation in the kidneys and vascular wall is a major contributor to hypertension. However, the stimuli and cellular mechanisms responsible for such inflammatory responses remain poorly defined. Inflammasomes are crucial initiators of sterile inflammation in other diseases such as rheumatoid arthritis and gout. These pattern recognition receptors detect host-derived danger-associated molecular patterns (DAMPs), such as microcrystals and reactive oxygen species, and respond by inducing activation of caspase-1. Caspase-1 then processes the cytokines pro-IL-1ß and pro-IL-18 into their active forms thus triggering inflammation. While IL-1ß and IL-18 are known to be elevated in hypertensive patients, no studies have examined whether this occurs downstream of inflammasome activation or whether inhibition of inflammasome and/or IL-1ß/IL-18 signalling prevents hypertension. In this review, we will discuss some known actions of IL-1ß and IL-18 on leukocyte and vessel wall function that could potentially underlie a prohypertensive role for these cytokines. We will describe the major classes of inflammasome-activating DAMPs and present evidence that at least some of these are elevated in the setting of hypertension. Finally, we will provide information on drugs that are currently used to inhibit inflammasome/IL-1ß/IL-18 signalling and how these might ultimately be used as therapeutic agents for the clinical management of hypertension.
Asunto(s)
Hipertensión/inmunología , Mediadores de Inflamación/inmunología , Interleucina-18/inmunología , Interleucina-1beta/inmunología , Antiinflamatorios/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Biomarcadores/metabolismo , Vasos Sanguíneos/inmunología , Caspasa 1/inmunología , Caspasa 1/metabolismo , Inhibidores de Caspasas/uso terapéutico , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Hipertensión/tratamiento farmacológico , Hipertensión/metabolismo , Inflamasomas/inmunología , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Interleucina-18/metabolismo , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/metabolismo , Riñón/inmunología , Antagonistas del Receptor Purinérgico P2X/uso terapéutico , Transducción de Señal/inmunologíaRESUMEN
Multi-protein complexes called inflammasomes have recently been identified and shown to contribute to cell death in tissue injury. Intravenous immunoglobulin (IVIg) is an FDA-approved therapeutic modality used for various inflammatory diseases. The objective of this study is to investigate dynamic responses of the NLRP1 and NLRP3 inflammasomes in stroke and to determine whether the NLRP1 and NLRP3 inflammasomes can be targeted with IVIg for therapeutic intervention. Primary cortical neurons were subjected to glucose deprivation (GD), oxygen-glucose deprivation (OGD) or simulated ischemia-reperfusion (I/R). Ischemic stroke was induced in C57BL/6J mice by middle cerebral artery occlusion, followed by reperfusion. Neurological assessment was performed, brain tissue damage was quantified, and NLRP1 and NLRP3 inflammasome protein levels were evaluated. NLRP1 and NLRP3 inflammasome components were also analyzed in postmortem brain tissue samples from stroke patients. Ischemia-like conditions increased the levels of NLRP1 and NLRP3 inflammasome proteins, and IL-1ß and IL-18, in primary cortical neurons. Similarly, levels of NLRP1 and NLRP3 inflammasome proteins, IL-1ß and IL-18 were elevated in ipsilateral brain tissues of cerebral I/R mice and stroke patients. Caspase-1 inhibitor treatment protected cultured cortical neurons and brain cells in vivo in experimental stroke models. IVIg treatment protected neurons in experimental stroke models by a mechanism involving suppression of NLRP1 and NLRP3 inflammasome activity. Our findings provide evidence that the NLRP1 and NLRP3 inflammasomes have a major role in neuronal cell death and behavioral deficits in stroke. We also identified NLRP1 and NLRP3 inflammasome inhibition as a novel mechanism by which IVIg can protect brain cells against ischemic damage, suggesting a potential clinical benefit of therapeutic interventions that target inflammasome assembly and activity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Portadoras/metabolismo , Inmunoglobulinas Intravenosas/farmacología , Inflamasomas/metabolismo , Neuronas/metabolismo , Accidente Cerebrovascular/patología , Animales , Isquemia Encefálica/complicaciones , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Caspasa 1/metabolismo , Inhibidores de Caspasas/farmacología , Muerte Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/patología , Citoprotección/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas NLR , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/patología , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/metabolismo , Resultado del TratamientoRESUMEN
BACKGROUND AND PURPOSE: Reactive oxygen species (ROS) derived from Nox2-containing reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity is reportedly detrimental in cerebrovascular disease. However, ROS generation by other Nox isoforms may have a physiological role. No Nox2-selective inhibitors have yet been identified, and thus it is unclear whether isoform non-selective Nox inhibitors would necessarily improve outcome after stroke. We assessed the effect of apocynin on cerebrovascular ROS production and also on outcome following cerebral ischaemia when administered either before ischaemia or after cerebral reperfusion. The involvement of Nox2-containing NADPH oxidase in the effects of apocynin was assessed using Nox2(-/-) mice. EXPERIMENTAL APPROACH: Transient cerebral ischaemia was induced by 0.5 h middle cerebral artery occlusion followed by 23.5 h reperfusion. Mice received apocynin (2.5 mg.kg(-1), i.p.) either 0.5 h before ischaemia or 1 h after reperfusion. In situ superoxide production after cerebral ischaemia-reperfusion was measured in brain sections of wild-type mice at 24 h using dihydroethidium fluorescence. KEY RESULTS: Treatment with apocynin 0.5 h before ischaemia reduced total infarct volume, neurological impairment and mortality in wild-type but not Nox2(-/-) mice. Conversely, treatment with apocynin 1 h after initiation of reperfusion had no protective effect. Cerebral ischaemia and reperfusion increased superoxide production in the brain at 24 h, and pretreatment but not posttreatment with apocynin reduced superoxide levels. CONCLUSIONS AND IMPLICATIONS: Apocynin improves outcome following stroke when administered before ischaemia in wild-type but not Nox2(-/-) mice.
Asunto(s)
Acetofenonas/uso terapéutico , Infarto de la Arteria Cerebral Media/prevención & control , Ataque Isquémico Transitorio/prevención & control , Glicoproteínas de Membrana/deficiencia , NADPH Oxidasas/deficiencia , Acetofenonas/administración & dosificación , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Esquema de Medicación , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/metabolismo , Ataque Isquémico Transitorio/complicaciones , Ataque Isquémico Transitorio/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND AND PURPOSE: To test whether development of enhanced vasoconstriction to 5-hydroxytryptamine (5-HT; serotonin) in SHR was temporally related to hypertension, elevated vascular superoxide (O(2)(-)) levels, decreased NO bioavailability, or increased contractile effects of cyclooxygenase or rho-kinase and/or PKC. EXPERIMENTAL APPROACH: We examined systolic blood pressure (SBP), vascular O(2)(-), and 5-HT-induced contractile responses of aortic segments from 4- and 8-week-old WKY and SHR. KEY RESULTS: SBP was 35% higher in SHR than WKY at 4 weeks and 60% higher at 8 weeks. Contractile responses to 5-HT were similar in WKY and SHR at 4 weeks, but were markedly augmented in SHR at 8 weeks. The NO synthase inhibitor, L-NAME, enhanced contractile responses to 5-HT markedly in both strains at 4 weeks and in WKY at 8 weeks, but only very modestly in SHR at 8 weeks. These functional differences were associated with higher O(2)(-) levels in SHR versus WKY at 8 weeks, but not at 4 weeks. The rho-kinase inhibitor, Y-27632, and the PKC inhibitor, Ro 31-8220, each only modestly attenuated contractions in WKY and SHR in each age group, and their effects in each strain were more pronounced at 8 weeks. The cyclooxygenase inhibitor, indomethacin, had no effect on contractile responses. CONCLUSIONS AND IMPLICATIONS: Development of augmented vascular contractile responses to 5-HT in SHR is preceded by hypertension. It is associated with increased vascular O(2)(-) levels and reduced modulatory effects of NO, and is unlikely to be due to enhanced activity of rho-kinase, PKC or cyclooxygenase.
Asunto(s)
Hipertensión/inducido químicamente , Prostaglandina-Endoperóxido Sintasas/metabolismo , Serotonina/farmacología , Vasoconstricción/efectos de los fármacos , Animales , Arterias , Presión Sanguínea/efectos de los fármacos , Hipertensión/enzimología , Hipertensión/metabolismo , Oxígeno/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismoRESUMEN
Chronic hypertension is a major risk factor for numerous cardiovascular disorders and is strongly associated with stroke. Hypertension alters cerebral vascular structure and may have profound deleterious effects on cerebral vascular function, the underlying mechanisms of which are still not well understood. Recent findings have led to important developments in our understanding of novel areas of cerebral vascular biology. This review briefly examines new evidence for physiologic and pathologic roles of K(+) channels, the renin-angiotensin system and reactive oxygen species, and Rho and Rho-kinase in regulation of cerebral vascular tone.
Asunto(s)
Circulación Cerebrovascular/fisiología , Hipertensión/complicaciones , Sistema Renina-Angiotensina/fisiología , Presión Sanguínea/fisiología , Enfermedad Crónica , Humanos , Hipertensión/enzimología , Hipertensión/fisiopatología , Péptidos y Proteínas de Señalización Intracelular , Canales de Potasio/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Especies Reactivas de Oxígeno/farmacología , Quinasas Asociadas a rhoRESUMEN
1. When a cerebral aneurysm ruptures, bleeding and clot formation occur around the surface of the brain, including several major blood vessels. The resulting condition, known as subarachnoid haemorrhage (SAH), often results in death or severe disability and is a significant cause of stroke. Delayed cerebral vasospasm and impaired vasodilatation are critical clinical complications that occur after SAH. Mechanisms contributing to the development of vasospasm and abnormal reactivity of cerebral arteries after SAH have been intensively investigated in recent years. The present short review briefly decribes recent advances in our knowledge of two relatively novel aspects of the mechanism(s) underlying the vascular abnormalities following SAH. 2. Cerebral arteries are depolarized after SAH, possibly due to decreased activity of potassium channels in vascular muscle. Decreased basal activation of potassium channels may be due to several mechanisms, including impaired activity of nitric oxide (NO). Vasodilator drugs that produce hyperpolarization, such as potassium channel openers, appear to be particularly effective for dilating cerebral arteries after experimental SAH. 3. Subarachnoid haemorrhage often involves decreased responsiveness of cerebral arteries to NO. This could be due to impaired activity of soluble guanylate cyclase, resulting in reduced basal levels of cGMP in cerebral vessels. However, an alternative explanation is that there may be an increased rate of cGMP hydrolysis by phosphodiesterase (PDE)-V in the cerebral vascular wall and that this abnormality contributes substantially to the impairment of NO-mediated cerebral vasodilatation after SAH. In support of this proposal, vasodilator responses to NO are reported to be normalized when coadministered with a PDE-V inhibitor following experimental SAH. 4. Thus, in cerebral vascular muscle after SAH, abnormalities of vasodilator mechanisms involving potassium channel function and also NO/cGMP activity may contribute to cerebral vascular dysfunction. These mechanisms may also represent useful and novel therapeutic targets for the treatment of vasospasm.
Asunto(s)
Trastornos Cerebrovasculares/etiología , Hemorragia Subaracnoidea/complicaciones , 3',5'-GMP Cíclico Fosfodiesterasas , Animales , Trastornos Cerebrovasculares/tratamiento farmacológico , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5 , Humanos , Donantes de Óxido Nítrico/uso terapéutico , Inhibidores de Fosfodiesterasa/uso terapéutico , Hidrolasas Diéster Fosfóricas/efectos de los fármacos , Hidrolasas Diéster Fosfóricas/metabolismo , Canales de Potasio/metabolismo , Hemorragia Subaracnoidea/tratamiento farmacológicoRESUMEN
Dilatation of cerebral arterioles in response to arachidonic acid is dependent on activity of cyclooxygenase. In this study, we examined mechanisms that mediate dilatation of the basilar artery in response to arachidonate. Diameter of the basilar artery (baseline diameter = 216 +/- 7 micrometer) (means +/- SE) was measured using a cranial window in anesthetized rats. Arachidonic acid (10 and 100 microM) produced concentration-dependent vasodilatation that was not inhibited by indomethacin (10 mg/kg iv) or N(G)-nitro-L-arginine (100 microM) but was inhibited markedly by baicalein (10 micrometerM) or nordihydroguaiaretic acid (NDGA; 10 microM), inhibitors of the lipoxygenase pathway. Dilatation of the basilar artery was also inhibited markedly by tetraethylammonium ion (TEA; 1 mM) or iberiotoxin (50 nM), inhibitors of calcium-dependent potassium channels. For example, 10 microM arachidonate dilated the basilar artery by 19 +/- 7 and 1 +/- 1% in the absence and presence of iberiotoxin, respectively. Measurements of membrane potential indicated that arachidonate produced hyperpolarization of the basilar artery that was blocked completely by TEA. Incubation with [(3)H]arachidonic acid followed by reverse-phase and chiral HPLC indicated that the basilar artery produces relatively small quantities of prostanoids but large quantities of 12(S)-hydroxyeicosatetraenoic acid (12-S-HETE), a lipoxygenase product. Moreover, the production of 12-HETE was inhibited by baicalein or NDGA. These findings suggest that dilatation of the basilar artery in response to arachidonate is mediated by a product(s) of the lipoxygenase pathway, with activation of calcium-dependent potassium channels and hyperpolarization of vascular muscle.
Asunto(s)
Ácido Araquidónico/farmacología , Arteria Basilar/fisiología , Flavanonas , Lipooxigenasa/metabolismo , Canales de Potasio/metabolismo , Vasodilatación/fisiología , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Animales , Arteria Basilar/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Indometacina/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Músculo Liso Vascular/enzimología , Óxido Nítrico Sintasa/metabolismo , Nitroarginina/farmacología , Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Tetraetilamonio/farmacología , Tritio , Vasodilatación/efectos de los fármacosRESUMEN
The small G protein Rho and its target Rho-kinase may participate in the mechanisms underlying vascular contractile tone via inhibition of myosin light chain phosphatase. The present study has tested the hypothesis that Rho-kinase activity normally contributes to cerebral vascular tone in vivo, and that this effect is augmented during chronic hypertension. Comparative studies also examined the role of protein kinase C (PKC) in regulation of cerebral artery tone. Two Rho-kinase inhibitors, Y-27632 (0.1 to 100 micromol/L) and HA1077 (1 to 10 micromol/L), caused marked concentration-dependent increases in basilar artery diameter of anesthetized normotensive rats (Sprague-Dawley and Wistar-Kyoto [WKY] strains), as measured using a cranial window approach. By comparison, the selective PKC inhibitors calphostin C (0.01 to 0.5 micromol/L) and Ro 31-8220 (5 micromol/L) had little or no effect on basilar artery diameter. Vasodilator responses to Y-27632 were unaffected by PKC inhibition or activation. In two models of chronic hypertension (spontaneously hypertensive rats and WKY rats treated with N-nitro-L-arginine methyl ester for 4 weeks), Y-27632 elicited cerebral vasodilator responses that were significantly greater than in control WKY rats (P<0.05), indicating that the chronically hypertensive state and not genetic factors contributed to the increased responses to Rho-kinase inhibition. PKC inhibition had no significant effect on basilar artery diameter in chronically hypertensive rats. These data suggest that Rho-kinase, but not PKC, activity contributes substantially to cerebral artery tone in vivo, and this effect is augmented in the cerebral circulation during chronic hypertension.
Asunto(s)
Arterias Cerebrales/enzimología , Hipertensión/enzimología , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Sistema Vasomotor/enzimología , Amidas/farmacología , Animales , Arteria Basilar/efectos de los fármacos , Arteria Basilar/enzimología , Arteria Basilar/fisiopatología , Presión Sanguínea/efectos de los fármacos , Arterias Cerebrales/efectos de los fármacos , Arterias Cerebrales/fisiopatología , Enfermedad Crónica , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Hipertensión/inducido químicamente , Hipertensión/fisiopatología , Péptidos y Proteínas de Señalización Intracelular , Masculino , NG-Nitroarginina Metil Éster , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridinas/farmacología , Ratas , Ratas Endogámicas WKY , Ratas Sprague-Dawley , Especificidad de la Especie , Vasodilatadores/farmacología , Sistema Vasomotor/efectos de los fármacos , Sistema Vasomotor/fisiopatología , Quinasas Asociadas a rhoRESUMEN
Potassium ion (K(+)) channel activity is a major regulator of vascular muscle cell membrane potential (E(m)) and is therefore an important determinant of vascular tone. There is growing evidence that the function of several types of vascular K(+) channels is altered during major cardiovascular diseases, such as chronic hypertension, diabetes, and atherosclerosis. Vasoconstriction and the compromised ability of an artery to dilate are likely consequences of defective K(+) channel function in blood vessels during these disease states. In some instances, increased K(+) channel function may help to compensate for increased vascular tone. Endothelial cell dysfunction is commonly associated with cardiovascular disease, and altered activity of nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor could also contribute to changes in resting K(+) channel activity, E(m), and K(+) channel-mediated vasodilatation. Our current knowledge of the effects of disease on vascular K(+) channel function almost exclusively relies on interpretation of data obtained by using pharmacological modulators of K(+) channels. As further progress is made in the development of more selective drugs and through molecular approaches such as gene targeting technology in mice, specific K(+) channel abnormalities and their causes in particular diseases should be more readily identified, providing novel directions for vascular therapy.
Asunto(s)
Canales de Potasio/fisiología , Enfermedades Vasculares/metabolismo , Animales , Endotelio Vascular/metabolismo , Humanos , Potenciales de la Membrana/fisiología , Enfermedades Vasculares/fisiopatologíaRESUMEN
We tested whether activation of inwardly rectifying K(+) (Kir) channels, Na(+)-K(+)-ATPase, or nitric oxide synthase (NOS) play a role in K(+)-induced dilatation of the rat basilar artery in vivo. When cerebrospinal fluid [K(+)] was elevated from 3 to 5, 10, 15, 20, and 30 mM, a reproducible concentration-dependent vasodilator response was elicited (change in diameter = 9 +/- 1, 27 +/- 4, 35 +/- 4, 43 +/- 12, and 47 +/- 16%, respectively). Responses to K(+) were inhibited by approximately 50% by the Kir channel inhibitor BaCl(2) (30 and 100 microM). In contrast, neither ouabain (1-100 microM, a Na(+)-K(+)-ATPase inhibitor) nor N(G)-nitro-L-arginine (30 microM, a NOS inhibitor) had any effect on K(+)-induced vasodilatation. These concentrations of K(+) also hyperpolarized smooth muscle in isolated segments of basilar artery, and these hyperpolarizations were virtually abolished by 30 microM BaCl(2). RT-PCR experiments confirmed the presence of mRNA for Kir2.1 in the basilar artery. Thus K(+)-induced dilatation of the basilar artery in vivo appears to partly involve hyperpolarization mediated by Kir channel activity and possibly another mechanism that does not involve hyperpolarization, activation of Na(+)-K(+)-ATPase, or NOS.
Asunto(s)
Arteria Basilar/fisiología , Circulación Cerebrovascular/fisiología , Canales de Potasio de Rectificación Interna , Canales de Potasio/metabolismo , Vasodilatación/fisiología , Acetilcolina/farmacología , Animales , Compuestos de Bario/farmacología , Arteria Basilar/efectos de los fármacos , Cloruros/farmacología , Cromakalim/farmacología , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Expresión Génica/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Nitroarginina/farmacología , Nitroprusiato/farmacología , Ouabaína/farmacología , Picolinas/farmacología , Potasio/farmacología , Canales de Potasio/genética , Piranos/farmacología , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: We postulated that some abnormalities in cerebrovascular function after subarachnoid hemorrhage (SAH) may involve underlying alterations in K(+) channel function. Thus, using pharmacological inhibitors, we assessed the influence of SAH on function of 2 types of K(+) channel in regulation of basilar artery diameter in vivo and membrane potential (E(m)) in vitro. METHODS: Rats were injected with saline (control) or autologous blood (SAH) into the cisterna magna. Two days later, effects of vasoactive drugs on the basilar artery were examined with a cranial window preparation. Vascular responses to 4-aminopyridine (4-AP), 3-aminopyridine (3-AP), tetraethylammonium (TEA), serotonin, acetylcholine, and adenosine were compared in control and SAH rats. Additional studies using intracellular microelectrodes evaluated the effects of 4-AP and serotonin on E(m) of basilar arteries isolated from control and SAH rats. RESULTS: Baseline artery diameter was 236+/-5 micrometer in control rats and 220+/-7 micrometer in SAH rats (P:<0. 05). 4-AP, but not 3-AP, constricted the basilar artery in control rats, and responses to 4-AP were reduced in SAH rats. Constrictor responses to TEA or serotonin were unaffected by SAH. Vasodilator responses to acetylcholine were impaired in SAH rats, whereas responses to adenosine were not different. Resting E(m) was -81+/-3 mV in control arteries and -79+/-3 mV in SAH arteries. Both 4-AP and serotonin depolarized the basilar artery, but only 4-AP-induced depolarization was impaired in SAH arteries. CONCLUSIONS: These data suggest that 4-AP induces cerebral vasoconstriction in vivo through smooth muscle depolarization due to inhibition of voltage-dependent K(+) channels. Furthermore, function of these K(+) channels may be selectively reduced in the basilar artery after SAH and thus could contribute to cerebral vascular dysfunction.
Asunto(s)
4-Aminopiridina/farmacología , Encéfalo/irrigación sanguínea , Bloqueadores de los Canales de Potasio , Hemorragia Subaracnoidea/fisiopatología , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Acetilcolina/metabolismo , Acetilcolina/farmacología , Adenosina/metabolismo , Adenosina/farmacología , Aminopiridinas/farmacología , Animales , Arteria Basilar/efectos de los fármacos , Arteria Basilar/metabolismo , Presión Sanguínea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Canales de Potasio/metabolismo , Ratas , Ratas Sprague-Dawley , Serotonina/metabolismo , Serotonina/farmacología , Tetraetilamonio/farmacología , Vasodilatadores/farmacologíaRESUMEN
1. We compared the effects of inhibiting nitric oxide synthase (NOS), soluble guanylate cyclase (sGC) and K+ channel activation on dilator responses to acetylcholine (ACh) in rat resistance (hindquarters) and conduit arteries (thoracic aorta). 2. In rat perfused hindquarters, the NO synthase inhibitor N omega-nitro-L-arginine (L-NNA; 1 mmol/L) partially inhibited the ACh-induced dilatation and the combination of L-NNA + haemoglobin (Hb; 20 mumol/L), a NO scavenger, did not further affect the response. Exposure to high K+ (30 mmol/L) also inhibited the response to ACh and this response was further reduced by L-NNA + high K+. Surprisingly, when applied alone 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of sGC, did not affect responses to ACh, whereas treatment with ODQ + high K+ markedly impaired dilatation. 3. In aortic rings precontracted with phenylephrine (PE; 0.01-1 mumol/L), the maximum relaxation to ACh was significantly reduced by L-NNA (0.1 mmol/L) and further inhibited by L-NNA + Hb (20 mumol/L). At 10 mumol/L, ODQ alone inhibited the maximum relaxation to ACh, which was further reduced by ODQ + high K+ (30 mmol/L). High K+ caused a smaller but significant inhibition of ACh-induced relaxation. 4. These results suggest that NO and cGMP play a relatively greater role in ACh-induced dilatation of the aorta compared with the hindquarters resistance vasculature and are consistent with the hypothesis that a non-NO endothelium-derived hyperpolarizing factor (endothelium-derived hyperpolarizing factor; EDHF) makes a relatively greater contribution to dilatation of resistance vessels than in conduit arteries. The data suggest that when sGC is inhibited, a compensatory mechanism involving K+ channel opening by NO can largely maintain ACh-induced vasodilator responses of resistance vessels. Furthermore, when NO synthesis is blocked, a non-NO EDHF may play a role in ACh-induced dilatation of the resistance vasculature.
Asunto(s)
GMP Cíclico/fisiología , Endotelio Vascular/fisiología , Óxido Nítrico/fisiología , Canales de Potasio/fisiología , Acetilcolina/antagonistas & inhibidores , Acetilcolina/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/fisiología , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Miembro Posterior , Nitroarginina/farmacología , Nitroprusiato/farmacología , Oxadiazoles/farmacología , Potasio/farmacología , Canales de Potasio/efectos de los fármacos , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiología , Vasodilatadores/antagonistas & inhibidores , Vasodilatadores/farmacologíaRESUMEN
Subarachnoid hemorrhage (SAH) is associated with impaired nitric oxide (NO)-mediated cerebral vasodilatation. We tested the hypothesis that SAH causes alterations in the production of, hydrolysis of, or responsiveness to cGMP in the rat basilar artery in vivo. Rats were injected with saline or autologous blood into the cisterna magna. Two days later, effects of vasoactive drugs on basilar artery diameter were examined using a cranial window preparation. Vasodilator responses to ACh, sodium nitroprusside (SNP), and low concentrations (=10(-5) M) of zaprinast, an inhibitor of phosphodiesterase V (PDE V), were impaired in SAH rats (P < 0.05). In contrast, vasodilator responses to adenosine and 8-BrcGMP were similar in control and SAH rats. Vasoconstrictor responses to 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase, were unaffected by SAH. In the presence of zaprinast (10(-5)-10(-4) M), responses to ACh and SNP were equivalent in control and SAH rats. Thus an increased rate of cGMP hydrolysis by PDE V may be a major factor contributing to the impairment of NO-mediated cerebral vasodilatation after SAH.
Asunto(s)
Circulación Cerebrovascular/efectos de los fármacos , Óxido Nítrico/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Hemorragia Subaracnoidea/fisiopatología , Vasodilatación , 3',5'-GMP Cíclico Fosfodiesterasas , Acetilcolina/farmacología , Animales , Arteria Basilar/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5 , Masculino , Nitroprusiato/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Purinonas/farmacología , Ratas , Ratas Sprague-Dawley , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: Protease-activated receptor-2 (PAR-2) can be activated after proteolysis of the amino terminal of the receptor by trypsin or by synthetic peptides with a sequence corresponding to the endogenous tethered ligand exposed by trypsin (eg, SLIGRL-NH(2)). PAR-2 mediates nitric oxide (NO)-dependent dilatation in cerebral arteries, but it is unknown whether PAR-2 function is altered in cardiovascular diseases. Since hypertension selectively impairs NO-mediated cerebral vasodilatation in response to acetylcholine and bradykinin, we sought to determine whether PAR-2-mediated vasodilatation is similarly adversely affected by this disease state. METHODS: We studied basilar artery responses in Wistar-Kyoto rats (WKY) (normotensive) and spontaneously hypertensive rats (SHR) in vivo (cranial window preparation) and in vitro (isolated arterial rings). The vasodilator effects of acetylcholine, sodium nitroprusside, and activators of PAR-2 and protease-activated receptor-1 (PAR-1) were compared in WKY versus SHR. Immunohistochemical localization of PAR-2 was also assessed in the basilar artery. RESULTS: Increases in basilar artery diameter in response to acetylcholine were 65% to 85% smaller in SHR versus WKY, whereas responses to sodium nitroprusside were not different. In contrast to acetylcholine, vasodilatation in vivo to SLIGRL-NH(2) was largely preserved in SHR, and SLIGRL-NH(2) was approximately 3-fold more potent in causing vasorelaxation in SHR versus WKY in vitro. In both strains, responses to SLIGRL-NH(2) were abolished by N(G)-nitro-L-arginine, an inhibitor of NO synthesis. Activators of PAR-1 had little or no effect on the rat basilar artery. PAR-2-like immunoreactivity was observed in both the endothelial and smooth muscle cells of the basilar artery in both strains of rat. CONCLUSIONS: These data indicate that NO-mediated vasodilatation to PAR-2 activation is selectively preserved or augmented in SHR and may suggest protective roles for PAR-2 in the cerebral circulation during chronic hypertension.
Asunto(s)
Arterias Cerebrales/fisiopatología , Hipertensión/fisiopatología , Fragmentos de Péptidos/farmacología , Receptores de Trombina/fisiología , Vasodilatación , Acetilcolina/farmacología , Animales , Arterias Cerebrales/efectos de los fármacos , Enfermedad Crónica , Inhibidores Enzimáticos/farmacología , Masculino , Nitroarginina/farmacología , Nitroprusiato/farmacología , Oligopéptidos/farmacología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptor PAR-1 , Receptor PAR-2 , Receptores de Trombina/agonistas , Vasodilatación/efectos de los fármacosRESUMEN
1. Voltage-dependent K+ channels are present in cerebral arteries and may modulate vascular tone. We used 200 microM 4-aminopyridine (4-AP), thought to be a relatively selective inhibitor of voltage-dependent K+ channels at this concentration, to test whether activation of these channels may influence baseline diameter of the basilar artery and dilator responses to nitric oxide (NO) and cyclic GMP in vivo. 2. Using a cranial window in anaesthetized rats, topical application of 4-AP to the basilar artery (baseline diameter = 240+/-5 microm, mean +/- s.e.mean) produced 10+/-1% constriction. Sodium nitroprusside (a NO donor), acetylcholine (which stimulates endothelial release of NO), 8-bromo cyclic GMP (a cyclic GMP analogue), cromakalim (an activator of ATP-sensitive K+ channels) and papaverine (a non-NO, non-K+ channel-related vasodilator) produced concentration-dependent vasodilator responses that were reproducible. 3. Responses to 10 and 100 nM nitroprusside were inhibited by 4-AP (20+/-4 vs 8+/-2% and 51+/-5 vs 33+/-5%, respectively, n=10; P<0.05). Responses to acetylcholine and 8-bromo cyclic GMP were also partially inhibited by 4-AP. In contrast, 4-AP had no effect on vasodilator responses to cromakalim or papaverine. These findings suggest that NO/cyclic GMP-induced dilator responses of the basilar artery are selectively inhibited by 4-aminopyridine. 4. Responses to nitroprusside were also markedly inhibited by 10 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (an inhibitor of soluble guanylate cyclase; 16+/-4 vs 1+/-1% and 44+/-7 vs 7+/-1%; n=10; P<0.05). 5. Thus, dilator responses of the rat basilar artery to NO appear to be mediated by activation of soluble guanylate cyclase and partially by activation of a 4-aminopyridine-sensitive mechanism. The most likely mechanism would appear to be activation of voltage-dependent K+ channels by NO/cyclic GMP.
Asunto(s)
4-Aminopiridina/farmacología , Arteria Basilar/efectos de los fármacos , Óxido Nítrico/farmacología , Acetilcolina/farmacología , Adenosina Trifosfato/fisiología , Animales , Arteria Basilar/fisiología , Cromakalim/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Gliburida/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Masculino , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Oxadiazoles/farmacología , Papaverina/farmacología , Bloqueadores de los Canales de Potasio , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
Responses of cerebral blood vessels to nitric oxide (NO) are mediated by soluble guanylate cyclase (sGC)-dependent and potentially by sGC-independent mechanisms. One sGC-independent mechanism by which NO may produce vasodilatation is inhibition of formation of a vasoconstrictor metabolite produced through the cytochrome P450 pathway. In these experiments, we examined the hypothesis that dilatation of cerebral microvessels in response to NO is dependent on activation of sGC. Diameters of cerebral arterioles (baseline diameter=94+/-5 micrometers, mean+/-S.E.) were measured using a closed cranial window in anesthetized rabbits. Under control conditions, YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole], an NO-independent activator of sGC, produced vasodilation that was blocked by ODQ (1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one)(10 microM), an inhibitor of sGC. These findings indicate that sGC is functionally important in cerebral arterioles. In addition, acetylcholine (which stimulates endogenous production of NO by endothelium) produced dilatation of cerebral arterioles that was inhibited by ODQ. For example, 1 microM acetylcholine dilated cerebral arterioles by 34+/-7 and 5+/-1% in the absence and presence of ODQ (10 microM), respectively. Increases in arteriolar diameter in response to sodium nitroprusside (1 microM, an NO donor) were inhibited by approximately 80% by ODQ, but were not affected by 17-ODYA (10 microM) or clotrimazole (10 microM), inhibitors of the cytochrome P450 pathway. Thus, dilatation of the cerebral microcirculation in response to exogenously applied and endogenously produced NO is dependent, in large part, on activation of sGC.
Asunto(s)
Encéfalo/irrigación sanguínea , Encéfalo/enzimología , Circulación Cerebrovascular/fisiología , Guanilato Ciclasa/metabolismo , Vasodilatación/fisiología , Acetilcolina/fisiología , Adenosina/farmacología , Animales , Arterias Cerebrales , Clotrimazol/farmacología , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/fisiología , Inhibidores Enzimáticos/farmacología , Ácidos Grasos Insaturados/farmacología , Inhibidores de Crecimiento/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Ácidos Hidroxieicosatetraenoicos/farmacología , Indazoles/farmacología , Microcirculación/fisiología , Óxido Nítrico/metabolismo , Nitroprusiato/farmacología , Oxadiazoles/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Quinoxalinas/farmacología , Conejos , Solubilidad , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
1. Subarachnoid haemorrhage (SAH) is a unique disorder and a major clinical problem that most commonly occurs when an aneurysm in a cerebral artery ruptures, leading to bleeding and clot formation. Subarachnoid haemorrhage results in death or severe disability of 50-70% of victims and is the cause of up to 10% of all strokes. Delayed cerebral vasospasm, which is the most critical clinical complication that occurs after SAH, seems to be associated with both impaired dilator and increased constrictor mechanisms in cerebral arteries. Mechanisms contributing to development of vasospasm and abnormal reactivity of cerebral arteries after SAH have been intensively investigated in recent years. In the present review we focus on recent advances in our knowledge of the roles of nitric oxide (NO) and cGMP, endothelin (ET), protein kinase C (PKC) and potassium channels as they relate to SAH. 2. Nitric oxide is produced by the endothelium and is an important regulator of cerebral vascular tone by tonically maintaining the vasculature in a dilated state. Endothelial injury after SAH may interfere with NO production and lead to vasoconstriction and impaired responses to endothelium-dependent vasodilators. Inactivation of NO by oxyhaemoglobin or superoxide from erythrocytes may also occur in the subarachnoid space after SAH. 3. Nitric oxide stimulates activity of soluble guanylate cyclase in vascular muscle, leading to intracellular generation of cGMP and relaxation. Subarachnoid haemorrhage appears to cause impaired activity of soluble guanylate cyclase, resulting in reduced basal levels of cGMP in cerebral vessels and often decreased responsiveness of cerebral arteries to NO. 4. Endothelin is a potent, long-lasting vasoconstrictor that may contribute to the spasm of cerebral arteries after SAH. Endothelin is present in increased levels in the cerebrospinal fluid of SAH patients. Pharmacological inhibition of ET synthesis or of ET receptors has been reported to attenuate cerebral vasospasm. Production of and vasoconstriction by ET may be due, in part, to the decreased activity of NO and formation of cGMP. 5. Protein kinase C is an important enzyme involved in the contraction of vascular muscle in response to several agonists, including ET. Activity of PKC appears to be increased in cerebral arteries after SAH, indicating that PKC may be critical in the development of cerebral vasospasm. Recent evidence suggests that PKC activation may occur in cerebral arteries after SAH as a result of decreased negative feedback influence of NO/cGMP. 6. Cerebral arteries are depolarized after SAH, possibly due to decreased activity of potassium channels in vascular muscle. Decreased basal activation of potassium channels may be due to several mechanisms, including impaired activity of NO (and/or cGMP) or increased activity of PKC. Vasodilator drugs that produce hyperpolarization, such as potassium channel openers, appear to be unusually effective in cerebral arteries after SAH. 7. Thus, endothelial damage and reduced activity of NO may contribute to cerebral vascular dysfunction after SAH. Potassium channels may represent an important therapeutic target for the treatment of cerebral vasospasm after SAH.