Mechanisms underlying epithelium-dependent relaxation in rat bronchioles: analogy to EDHF-type relaxation in rat pulmonary arteries

Abstract

Kroigaard C, Dalsgaard T, Simonsen U. Mechanisms underlying epithelium-dependent relaxation in rat bronchioles: analogy to EDHFtype relaxation in rat pulmonary arteries. Am J Physiol Lung Cell Mol Physiol 298: L531-L542, 2010. First published January 29, 2010 doi:10.1152/ajplung.00220.2009.-This study investigated the mechanisms underlying epithelium-derived hyperpolarizing factor (EpDHF)-type relaxation in rat bronchioles. Immunohistochemistry was performed, and rat bronchioles and pulmonary arteries were mounted in microvascular myographs for functional studies. An opener of small (SKCa) and intermediate (IKCa)-conductance calcium-activated potassium channels, NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime) was used to induce EpDHF-type relaxation. IK Ca and SKCa3 positive immunoreactions were observed mainly in the epithelium and endothelium of bronchioles and arteries, respectively. In 5-hydroxytryptamine (1 M)-contracted bronchioles (828 Ϯ 20 m, n ϭ 84) and U46619 (0.03 M)-contracted arteries (720 Ϯ 24 m, n ϭ 68), NS309 (0.001-10 M) induced concentration-dependent relaxations that were reduced by epithelium/endothelium removal and by blocking IK Ca channels with charybdotoxin and in bronchioles also by blocking SK Ca channels with apamin. Inhibition of cyclooxygenase, nitric oxide synthase, and cytochrome 2C isoenzymes, or blockade of large (BK Ca)-conductance calciumactivated potassium channels with iberiotoxin, failed to reduce NS309 relaxation. In contrast to the pulmonary arteries, relaxations to a ␤ 2-adrenoceptor agonist, salbutamol, were reduced in bronchioles by removing the epithelium or blocking IK Ca and/or SK Ca channels. Extracellular K ϩ (2-20 mM) induced relaxation in both bronchioles and arteries. An inhibitor of Na ϩ -K ϩ -ATPase, ouabain, abolished relaxations to NS309, salbutamol, and K ϩ . These results suggest that IK Ca and SKCa3 channels are located in the epithelium of bronchioles and endothelium of pulmonary arteries. Analog to the endotheliumderived hyperpolarizing factor (EDHF)-type relaxation in pulmonary arteries, these channels may be involved in EpDHF-type relaxation of bronchioles caused by epithelial K ϩ efflux followed by activation of Na ϩ -K ϩ -ATPase in the underlying smooth muscle layer.NS309; salbutamol; potassium; endothelium; GEA 3175 THE RESPIRATORY EPITHELIUM releases factors that regulate bronchial smooth muscle relaxations (20), and removal of the bronchial epithelium in larger airways has been shown to enhance the effect of contractile agonists and reduce the relaxing response in bronchioles (50, 51). Nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) are mediators of human bronchiole smooth muscle epithelium-dependent relaxations (22). In addition, a non-NO nonprostanoid epithelium-dependent hyperpolarizing factor (EpDHF) is also involved in epitheliumdependent relaxations. Epoxyeicosatrienoic acids (EETs) have been suggested to account for EpDHF-mediated relaxation in guinea pig airways (3). In comparison, opening of intermediate (IK Ca ) and endothelial subt...

Estrogen Regulates the Expression of Small-Conductance Ca²⁺-Activated K⁺ Channels in Colonic Smooth Muscle Cells

“…The existence of functional SK2 in human and mouse cardiac myocytes has been reported, and genetic knockout of SK2 results in a delay in cardiac repolarization and atrial arrhythmias [32]. Functional SK3 channels are associated with a variety of smooth muscle tissues, such as the smooth muscle of blood vessels [33], bronchus [34], bladder [35], and uterus [12]. Moreover, SK3 channels participate in the purine-induced inhibition of colonic smooth muscle contraction [36].…”

Pulmonary Hypertension in Wild Type Mice and Animals with Genetic Deficit in KCa2.3 and KCa3.1 Channels

“…In pulmonary arteries relaxation sensitive to blockers of the K Ca 2.3 and K Ca 3.1 channels has been observed in several studies [26], [28]–[30]. Moreover, the potent K Ca 2.3- and K Ca 3.1 channel activator NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime) has been shown to induce relaxation being sensitive to K Ca 2.3- and K Ca 3.1 blockade in rat and human pulmonary arteries [26], [27], rat mesenteric arteries [31], and in porcine retinal arterioles [32]. In addition, K Ca 3.1 expression has also been found in proliferating vascular smooth muscle cells [33], [34], and it may also be increased in the smooth muscle of the hypertrophied arteries in pulmonary hypertension.…”

“…In previous studies we have performed immunoblotting of lungs from rat and man [26], [27], but unfortunately we were unable to validate the gene expression results in this study with protein quantification in mouse lung using western blotting, as we experienced a series of difficulties (e.g. lack of specificity and multiple bands) with the mouse antibodies for the K Ca 2.3 and K Ca 3.1 channels, despite trying several types (K Ca 2.3: SC-28621 (Santa Cruz Biotechnology, Santa Cruz, CA); APC-025 (Alamone Labs, Jerusalem, Israel); H00003782-A01 (Abnova, Taiwan).…”

“…Thus far, K Ca 2.3 and K Ca 3.1 expression have been found in human, porcine, and rat pulmonary artery endothelial cells [24]–[27], as well as in the bronchial epithelium [26], [27]. In pulmonary arteries relaxation sensitive to blockers of the K Ca 2.3 and K Ca 3.1 channels has been observed in several studies [26], [28]–[30].…”

Tracheal relaxation of five Ivorian anti-asthmatic plants: Role of epithelium and K+ channels in the effect of the aqueous-alcoholic extract of Dichrostachys cinerea root bark

“…The fact that the relaxation was only reduced but not abolished might be explained either by an incomplete epithelium denudation or by the existence of a concomitant epithelium-independent relaxing mechanism. Recently, it has been shown that K + channels participate to epithelium-dependent relaxation in guinea-pig trachea and rat bronchioles (Benoît et al, 2001;Kroigaard et al, 2010). This mechanism might be the underlying explanation of the epithelium-and K + channels-dependency of the Dichrostachys cinerea EtOH/H 2 O extract relaxing effect.…”