Human Airway Contraction and Formoterol-Induced Relaxation Is Determined by Ca²⁺ Oscillations and Ca²⁺ Sensitivity

Abstract

The etiology of airway hyperresponsiveness associated with asthma requires an understanding of the regulatory mechanisms mediating human airway smooth muscle cell (SMC) contraction. The objective of this study was to determine how human airway SMC contraction (induced by histamine) and relaxation (induced by formoterol) are regulated by Ca 21 oscillations and Ca 21 sensitivity. The responses of human small airways and their associated SMCs were studied in human lung slices cut from agarose-inflated lungs. Airway contraction was measured with phase-contrast video microscopy. Ca 21 signaling and Ca 21 sensitivity of airway SMCs were measured with two-photon fluorescence microscopy and Ca 21 -permeabilized lung slices. The agonist histamine induced contraction of human small airways by stimulating both an increase in intracellular Ca 21 concentration in the SMCs in the form of oscillatory Ca 21 waves and an increase in Ca 21 sensitivity. The frequency of the Ca 21 oscillations increased with histamine concentration, and correlated with increased contraction. Formoterol induced airway relaxation at low concentrations by initially decreasing SMC Ca 21 sensitivity. At higher concentrations, formoterol additionally slowed or inhibited the Ca 21 oscillations of the SMCs to relax the airways. The action of formoterol was only slowly reversed. Human lung slices provide a powerful experimental assay for the investigation of small airway physiology and pharmacology. Histamine induces contraction by simultaneously increasing SMC Ca 21 signaling and Ca 21 sensitivity. Formoterol induces long-lasting relaxation by initially reducing the Ca 21 sensitivity and, subsequently, the frequency of the Ca 21 oscillations of the airway SMCs.Keywords: lung slice; smooth muscle cell; two-photon microscopy; hyperresponsiveness; histamine Because increased airway smooth muscle cell (SMC) contraction is a major characteristic of airway hyperresponsiveness (AHR) associated with asthma (1), a prerequisite to address AHR is an understanding of the mechanisms that regulate airway SMC contraction. It is well established that airway SMC contraction is regulated by the phosphorylation of the regulatory myosin light chain (rMLC) (2, 3) to induce its interaction with adjacent actin filaments via the classical mechanism of cross-bridge cycling to generate force (4). (5)(6)(7)(8). Although the frequency of these Ca 21 oscillations has been found to increase with agonist concentration, and that this correlates with increased contraction, the frequency of the Ca 21 oscillations generated by similar agonist concentrations differs considerably between species (9).To induce SMC relaxation, the increase in [Ca 21 ] i is generally reversed to inactivate MLCK, but the cessation of the cross-bridge cycle requires that the rMLC is dephosphorylated; this reaction is mediated by MLC phosphatase (MLCP). With the exception of mice (10), the activity of MLCP appears to be Ca 21 independent, but is regulated by the same agonists that stimulate increases in [Ca 21 ] ...

Cryopreserved Human Precision-Cut Lung Slices as a Bioassay for Live Tissue Banking. A Viability Study of Bronchodilation with Bitter-Taste Receptor Agonists

“…Airways in hPCLSs were first contracted with 500 nM His, the subsequent exposure to formoterol induced dose-dependent airway relaxation (half maximal effective concentration [EC 50 ] = 0.6 nM; Figure 2D). This response was similar to that observed in airways of never-frozen hPCLSs, as previously published (24). These observations provide strong evidence that ASMCs in cryopreserved hPCLSs are highly viable and functional.…”

Dilatation of the Constricted Human Airway by Tidal Expansion of Lung Parenchyma

“…We therefore developed a new experimental system in which human airways within thin lung slices can be studied under conditions of dynamic load that simulate breathing, while precisely monitoring airway size and its tidal fluctuation (Figure 1). Our approach builds on prior work by Wohlsen and others (40,41,(53)(54)(55)(56)(57), who assessed airway contraction in the absence of simulated breathing, and on that of Dassow and colleagues (58) and Sanderson (59), who have also developed systems to stretch lung slices.…”

“…Limited medical history was available for most donors; none were reported to have asthma or other pulmonary disease, although eight were known to have smoked tobacco products. The right lung was removed and a portion of its middle lobe was infused with approximately 120 to 180 ml of 1.5% low melting temperature agarose (Type IX; Sigma, St. Louis, MO) in Hanks balanced salt solution (pH ¼ 7.4; Invitrogen, Carlsbad, CA) (40,41). After solidifying at 4 8 C for 1 hour, the infused lung was cubed (z1 cm) and 250-mm precision-cut lung slices (PCLS) were cut using a VT1200S vibrating blade microtome (Leica Microsystems, Bannockburn, IL).…”

Formoterol and salmeterol induce a similar degree of β2‐adrenoceptor tolerance in human small airways but via different mechanisms

“…Although the exact mechanisms of salmeterol-induced relaxation remain unknown, low concentrations of formoterol induce airway relaxation by decreasing airway smooth muscle Ca 2+ sensitivity. At higher concentrations, formoterol additionally slows or inhibits Ca 2+ oscillations within smooth muscle cells to relax the airways (Ressmeyer et al, 2010). Whether these mechanisms explain the differential effects of salmeterol and formoterol on bronchoprotection requires further study.…”