RESUMEN
Spontaneous action potentials precede phasic contractile activity in human collecting lymphatic vessels. In this study, we investigated the expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in human collecting lymphatics and by pharmacological inhibition ex vivo tested their potential role in controlling contractile function. Spontaneous and agonist-evoked tension changes of isolated thoracic duct and mesenteric lymphatic vessels-obtained from surgical patients with informed consent-were investigated by isometric myography, and ivabradine, ZD7288 or cesium were used to inhibit HCN. Analysis of HCN isoforms by RT-PCR and immunofluorescence revealed HCN2 to be the predominantly expressed mRNA isoform in human thoracic duct and mesenteric lymphatic vessels and HCN2-immunoreactivity confirmed protein expression in both vessel types. However, in functional experiments ex vivo the HCN inhibitors ivabradine, ZD7288, and cesium failed to lower contraction frequency: conversely, all three antagonists induced a positive chronotropic effect with concurrent negative inotropic action, though these effects first occurred at concentrations regarded as supramaximal for HCN inhibition. Based on these results, we conclude that human collecting vessels express HCN channel proteins but under the ex vivo experimental conditions described here HCN channels have little involvement in regulating contraction frequency in human collecting lymphatic vessels. Furthermore, HCN antagonists can produce concentration-dependent positive chronotropic and negative inotropic effects, which are apparently unrelated to HCN antagonism.
Asunto(s)
Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Vasos Linfáticos , Cesio/metabolismo , Cesio/farmacología , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Ivabradina , Contracción MuscularRESUMEN
Lymph vessels counteract edema by transporting interstitial fluid from peripheral tissues to the large veins and serve as conduits for immune cells, cancer cells, and pathogens. Because edema during inflammation and malignancies is frequently associated with acidosis, we tested the hypothesis that acid-base disturbances affect human thoracic duct contractions. We studied, by isometric and isobaric myography, the contractile function of human thoracic duct segments harvested with written informed consent from patients undergoing esophageal cancer surgery. Human thoracic ducts produce complex contractile patterns consisting of tonic rises in tension (isometric myography) or decreases in diameter (isobaric myography) with superimposed phasic contractions. Active tone development decreases substantially (~90% at 30 vs. 7 mmHg) at elevated transmural pressure. Acidosis inhibits spontaneous as well as noradrenaline- and serotonin-induced phasic contractions of human thoracic ducts by 70-90% at extracellular pH 6.8 compared to 7.4 with less pronounced effects observed at pH 7.1. Mean tension responses to noradrenaline and serotonin - averaged over the entire period of agonist exposure - decrease by ~50% at extracellular pH 6.8. Elevating extracellular [K+ ] from the normal resting level around 4 mmol/L increases overall tension development but reduces phasic activity to a level that is no different between human thoracic duct segments investigated at normal and low extracellular pH. In conclusion, we show that extracellular acidosis inhibits human thoracic duct contractions with more pronounced effects on phasic than tonic contractions. We propose that reduced phasic activity of lymph vessels at low pH attenuates lymph propulsion and increases the risk of edema formation.
Asunto(s)
Acidosis/fisiopatología , Edema/fisiopatología , Vasos Linfáticos/fisiopatología , Contracción Muscular , Músculo Liso/fisiopatología , Tórax/fisiopatología , Agonistas Adrenérgicos/farmacología , Anciano , Epinefrina/farmacología , Femenino , Humanos , Vasos Linfáticos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Tono Muscular , Músculo Liso/efectos de los fármacos , Periodicidad , Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Tórax/patologíaRESUMEN
Cl(-) and HCO3 (-) are the most prevalent membrane-permeable anions in the intra- and extracellular spaces of the vascular wall. Outwardly directed electrochemical gradients for Cl(-) and HCO3 (-) permit anion channel opening to depolarize vascular smooth muscle and endothelial cells. Transporters and channels for Cl(-) and HCO3 (-) also modify vascular contractility and structure independently of membrane potential. Transport of HCO3 (-) regulates intracellular pH and thereby modifies the activity of enzymes, ion channels, and receptors. There is also evidence that Cl(-) and HCO3 (-) transport proteins affect gene expression and protein trafficking. Considering the extensive implications of Cl(-) and HCO3 (-) in the vascular wall, it is critical to understand how these ions are transported under physiological conditions and how disturbances in their transport can contribute to disease development. Recently, sensing mechanisms for Cl(-) and HCO3 (-) have been identified in the vascular wall where they modify ion transport and vasomotor function, for instance, during metabolic disturbances. This review discusses current evidence that transport (e.g., via NKCC1, NBCn1, Ca(2+)-activated Cl(-) channels, volume-regulated anion channels, and CFTR) and sensing (e.g., via WNK and RPTPγ) of Cl(-) and HCO3 (-) influence cardiovascular health and disease.
Asunto(s)
Bicarbonatos/metabolismo , Cloruros/metabolismo , Transporte Iónico , Proteínas de Transporte de Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Animales , Canales de Cloruro/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ratones , Músculo Liso Vascular/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
We investigate sensing and signaling mechanisms for H(+), [Formula: see text] and CO2 in basilar arteries using out-of-equilibrium solutions. Selectively varying pHo, [[Formula: see text]]o, or pCO2, we find: (a) lowering pHo attenuates vasoconstriction and vascular smooth muscle cell (VSMC) Ca(2+)-responses whereas raising pHo augments vasoconstriction independently of VSMC [Ca(2+)]i, (b) lowering [[Formula: see text]]o increases arterial agonist-sensitivity of tone development without affecting VSMC [Ca(2+)]i but c) no evidence that CO2 has direct net vasomotor effects. Receptor protein tyrosine phosphatase (RPTP)γ is transcribed in endothelial cells, and direct vasomotor effects of [Formula: see text] are absent in arteries from RPTPγ-knockout mice. At pHo 7.4, selective changes in [[Formula: see text]]o or pCO2 have little effect on pHi At pHo 7.1, decreased [[Formula: see text]]o or increased pCO2 causes intracellular acidification, which attenuates vasoconstriction. Under equilibrated conditions, anti-contractile effects of CO2/[Formula: see text] are endothelium-dependent and absent in arteries from RPTPγ-knockout mice. With CO2/[Formula: see text] present, contractile responses to agonist-stimulation are potentiated in arteries from RPTPγ-knockout compared to wild-type mice, and this difference is larger for respiratory than metabolic acidosis. In conclusion, decreased pHo and pHi inhibit vasoconstriction, whereas decreased [[Formula: see text]]o promotes vasoconstriction through RPTPγ-dependent changes in VSMC Ca(2+)-sensitivity. [Formula: see text] serves dual roles, providing substrate for pHi-regulating membrane transporters and modulating arterial responses to acid-base disturbances.
Asunto(s)
Bicarbonatos , Arterias Cerebrales/fisiología , Músculo Liso Vascular/fisiología , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/fisiología , Animales , Calcio/metabolismo , Dióxido de Carbono/sangre , Células Endoteliales , Concentración de Iones de Hidrógeno , Ratones , Ratones Noqueados , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/genética , VasoconstricciónRESUMEN
The presence of Ca(2+)-activated Cl() currents (I(Cl(Ca))) in vascular smooth muscle cells (VSMCs) is well established. ICl(Ca) are supposedly important for arterial contraction by linking changes in [Ca(2+)]i and membrane depolarization. Bestrophins and some members of the TMEM16 protein family were recently associated with I(Cl(Ca)). Two distinct I(Cl(Ca)) are characterized in VSMCs; the cGMP-dependent I(Cl(Ca)) dependent upon bestrophin expression and the 'classical' Ca(2+)-activated Cl() current, which is bestrophin-independent. Interestingly, TMEM16A is essential for both the cGMP-dependent and the classical I(Cl(Ca)). Furthermore, TMEM16A has a role in arterial contraction while bestrophins do not. TMEM16A's role in the contractile response cannot be explained however only by a simple suppression of the depolarization by Cl() channels. It is suggested that TMEM16A expression modulates voltage-gated Ca(2+) influx in a voltage-independent manner and recent studies also demonstrate a complex role of TMEM16A in modulating other membrane proteins.
Asunto(s)
Canales de Cloruro/metabolismo , Proteínas del Ojo/metabolismo , Músculo Liso Vascular/metabolismo , Animales , Humanos , Activación del Canal Iónico , Unión Proteica , Relación Estructura-ActividadRESUMEN
OBJECTIVE: Cardiovascular diseases have high comorbidity with major depression. Endothelial dysfunction may explain the adverse cardiovascular outcome in depression; therefore, we analyzed it in vitro. In the chronic mild stress model, some rats develop depression-like symptoms (including "anhedonia"), whereas others are stress resilient. METHODS: After 8 weeks of chronic mild stress, anhedonic rats reduced their sucrose intake by 55% (7%), whereas resilient rats did not. Acetylcholine-induced endothelium-dependent relaxation of norepinephrine-preconstricted mesenteric arteries was analyzed in nonstressed, anhedonic, and resilient rat groups. RESULTS: Small resistance arteries from anhedonic rats were less sensitive to acetylcholine than those of the nonstressed and resilient groups (p = .029). Pathways of endothelium-dependent relaxation were altered in arteries from anhedonic rats. Nitric oxide (NO)-dependent relaxation and endothelial NO synthase expression were increased in arteries from anhedonic rats (0.235 [0.039] arbitrary units and 155.7% [8.15%]) compared with the nonstressed (0.135 [0.012] arbitrary units and 100.0% [8.08%]) and resilient (0.152 [0.018] arbitrary units and 108.1% [11.65%]) groups (p < .001 and p = .002, respectively). Inhibition of cyclooxygenase (COX) activity revealed increased COX-2-dependent relaxation in the anhedonic group. In contrast, endothelial NO synthase- and COX-independent relaxation to acetylcholine (endothelium-dependent hyperpolarization-like response) was reduced in anhedonic rats (p < .001). This was associated with decreased transcription of intermediate-conductance Ca-activated K channels. CONCLUSIONS: Our findings demonstrate that depression-like symptoms are associated with reduced endothelium-dependent relaxation due to suppressed endothelium-dependent hyperpolarization-like relaxation despite up-regulation of the NO and COX-2-dependent pathways in rat mesenteric arteries. These changes could affect peripheral resistance and organ perfusion in major depression.
Asunto(s)
Depresión/fisiopatología , Endotelio Vascular/fisiopatología , Arterias Mesentéricas/fisiopatología , Estrés Psicológico/fisiopatología , Vasodilatación/fisiología , Acetilcolina/farmacología , Anhedonia/fisiología , Animales , Factores Biológicos/fisiología , Enfermedad Crónica , Constricción Patológica , Depresión/metabolismo , Modelos Animales de Enfermedad , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa/fisiología , Norepinefrina/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandina-Endoperóxido Sintasas/fisiología , Ratas , Ratas Wistar , Resiliencia Psicológica , Estrés Psicológico/metabolismo , Sacarosa/administración & dosificación , Resistencia Vascular/fisiología , Vasodilatación/efectos de los fármacosRESUMEN
The presence of Ca(2+)-activated Cl(-) channels (CaCCs) in vascular smooth muscle cells (SMCs) is well established. Their molecular identity is, however, elusive. Two distinct Ca(2+)-activated Cl(-) currents (I Cl(Ca)) were previously characterized in SMCs. We have shown that the cGMP-dependent I Cl(Ca) depends on bestrophin expression, while the "classical" I Cl(Ca) is not. Downregulation of bestrophins did not affect arterial contraction but inhibited the rhythmic contractions, vasomotion. In this study, we have used in vivo siRNA transfection of rat mesenteric small arteries to investigate the role of a putative CaCC, TMEM16A. Isometric force, [Ca(2+)]i, and SMC membrane potential were measured in isolated arterial segments. I Cl(Ca) and GTPγS-induced nonselective cation current were measured in isolated SMCs. Downregulation of TMEM16A resulted in inhibition of both the cGMP-dependent I Cl(Ca) and the "classical" I Cl(Ca) in SMCs. TMEM16A downregulation also reduced expression of bestrophins. TMEM16A downregulation suppressed vasomotion both in vivo and in vitro. Downregulation of TMEM16A reduced agonist (noradrenaline and vasopressin) and K(+)-induced contractions. In accordance with the depolarizing role of CaCCs, TMEM16A downregulation suppressed agonist-induced depolarization and elevation in [Ca(2+)]i. Surprisingly, K(+)-induced depolarization was unchanged but Ca(2+) entry was reduced. We suggested that this is due to reduced expression of the L-type Ca(2+) channels, as observed at the mRNA level. Thus, the importance of TMEM16A for contraction is, at least in part, independent from membrane potential. This study demonstrates the significance of TMEM16A for two SMCs I Cl(Ca) and vascular function and suggests an interaction between TMEM16A and L-type Ca(2+) channels.