Our hypothesis explaining this behavior is that this elevated [Ca2+]i has reduced the Ca2+ sensitivity of the SMCs to almost 0 because of an increase in myosin light chain phosphatase (MLCP) activity. Airway relaxation induced by NOC-5 was accompanied by a decrease in the frequency of these Ca2+ oscillations. The cGMP analogues and selective PKG activators 8Br-cGMP and 8pCPT-cGMP also induced airway relaxation and decreased the frequency of the Ca2+ oscillations. NOC-5 inhibited the increase of [Ca2+]i and contraction induced by the photolytic release of inositol 1,4,5-trisphosphate (IP3) in airway SMCs. The effect of NO around the Ca2+ sensitivity of the airway SMCs was examined in lung slices permeabilized to Ca2+ by treatment with caffeine and ryanodine. Neither NOC-5 nor 8pCPT-cGMP induced relaxation in agonist-contracted Ca2+-permeabilized airways. Consequently, we conclude that NO, acting via the cGMPCPKG pathway, induced airway SMC relaxation by predominately inhibiting the release of Ca2+ via the IP3 receptor to decrease the frequency of agonist-induced Ca2+ oscillations. INTRODUCTION In the airways and lungs, nitric oxide (NO) is usually produced by epithelial ciliated cells, type II alveolar cells, and neural fibers that innervate the airway clean muscle cells (SMCs) (Ricciardolo et al., 2004). It has been suggested that this NO released by these cells decreases airway resistance and that NO, released by neural fibers, is a major nonadrenergic, noncholinergic neurotransmitter responsible for airway SMC relaxation (Belvisi et al., 1995). In addition, airway inflammation is usually associated with a considerable increase in NO synthesis by inflammatory cells, including macrophages, mast cells, and neutrophils. However, in asthma, airway inflammation is accompanied by airway hyperresponsiveness, a behavior characterized by increased airway contraction in response to a variety of stimuli that suggests an impediment of the airways to relax in response to NO and other natural or pharmacological bronchodilators (Ricciardolo et al., 2004). The mechanisms responsible for these asthma-associated changes are still not completely comprehended. A first step toward understanding how asthma and other obstructive lung diseases alter airway responsiveness is usually to elucidate the cellular mechanisms regulating changes in airway resistance induced by agonists and NO in healthy individuals. The small intrapulmonary airways are considered a major site for this regulation; however, the contractile responses of the SMCs of these airways are relatively unexplored because they are difficult to isolate and study with conventional methods used to measure cell signaling and/or pressure development. Consequently, we have adopted a novel approach that combines the use of mouse lung slices and confocal microscopy to simultaneously evaluate Ca2+ signaling in SMCs and airway contraction. With this approach, we previously emphasized that agonist-induced airway contraction is usually regulated by two intracellular processes or signals (Sanderson et al., 2008). These are (a) the frequency of agonist-induced oscillations in [Ca2+]i, referred to as Ca2+ oscillations (Perez and Sanderson, 2005) (a Ca2+-dependent mechanism), and (b) the magnitude of an agonist-induced increase in the sensitivity of the contractile machinery to [Ca2+]i, referred to as Ca2+ sensitivity (Bai and Sanderson, 2006b) (a Ca2+-impartial mechanism). In addition, the relaxation of airway SMCs induced by the activation of 2 adrenergic receptors with isoproterenol (ISO), albuterol, or formoterol was shown to be accompanied by both a cAMP-dependent decrease in the frequency of Ca2+ oscillations and a decrease in Ca2+ sensitivity (Bai and Sanderson, 2006a; Delmotte and Sanderson, 2008, 2009; Ressmeyer et al., 2009). Here, we extended these studies to investigate the mechanisms responsible for airway relaxation induced by NO. The signaling cascade by which NO induces SMC relaxation has been mainly studied in vascular SMCs of the systemic circulation. In these blood vessels, NO is usually synthesized in the endothelial cells and diffuses to the adjacent SMCs, where it activates soluble guanylate cyclase (sGC) to synthesize cGMP. This cGMP acts as a second messenger to activate cGMP-dependent PKG and/or other effector proteins, including ion channels, ion pumps, and phosphodiesterases (PDEs) (Carvajal et al., 2000). The phosphorylation of one or more target molecules by PKG and/or a direct activation/inhibition of ion channels by cGMP are believed to lead to SMC relaxation. However, the details of the mechanism by which an increase in cGMP results in SMC relaxation are often controversial and can vary considerably, and the precise mechanism occurring in the small pulmonary airways is usually unknown. For example, the contribution of Ca2+-dependent and Ca2+-impartial pathways to SMC relaxation can differ with the location of the blood vessels (Chitaley and Webb, 2002; Kitazawa et al., 2009). Furthermore, many of the regulatory proteins participating in these pathways have been shown to be affected by cGMP or PKG..(F) Summary of the effect of cGMP analogues around the frequency of Ca2+ oscillations measured before (control) and after the addition of cGMP analogues (= 5 SMCs from different slices from three mice; *, P 0.05). In keeping with the reduction of the Ca2+ oscillation frequency by NOC-5, these cGMP analogues gradually reduced the frequency of the 5-HTCinduced Ca2+ oscillations until they almost completely stopped (with 8pCPT-cGMP) or attained a new slower constant frequency (with 8Br-cGMP) (Fig. contraction induced by 5-HT correlated with the occurrence of Ca2+ oscillations in the airway SMCs. Airway relaxation induced by NOC-5 was accompanied by a decrease in the frequency of these Ca2+ oscillations. The cGMP analogues and selective PKG activators 8Br-cGMP and 8pCPT-cGMP also induced L-701324 airway relaxation and decreased the frequency of the Ca2+ oscillations. L-701324 NOC-5 inhibited the increase of [Ca2+]i and contraction induced by the photolytic release of inositol 1,4,5-trisphosphate (IP3) in airway SMCs. The effect of NO around the Ca2+ sensitivity of the airway SMCs was examined in lung slices permeabilized to Ca2+ by L-701324 treatment with caffeine and ryanodine. Neither NOC-5 nor 8pCPT-cGMP induced relaxation in agonist-contracted Ca2+-permeabilized airways. Consequently, we conclude that NO, acting via the cGMPCPKG pathway, induced airway SMC relaxation by predominately inhibiting the release of Ca2+ via the IP3 receptor to decrease the frequency of agonist-induced Ca2+ oscillations. INTRODUCTION In the airways and lungs, nitric oxide (NO) is usually produced by epithelial ciliated cells, type II alveolar cells, and neural fibers Rabbit Polyclonal to DNA-PK that innervate the airway clean muscle cells (SMCs) (Ricciardolo et al., 2004). It has been suggested that this NO released by these cells decreases airway resistance and that NO, released by neural fibers, is a major nonadrenergic, noncholinergic neurotransmitter responsible for airway SMC relaxation (Belvisi et al., 1995). In addition, airway inflammation is usually associated with a considerable increase in NO synthesis by inflammatory cells, including macrophages, mast cells, and neutrophils. However, in asthma, airway inflammation is accompanied by airway hyperresponsiveness, a behavior characterized by increased airway contraction in response to a variety of stimuli that suggests an impediment of the airways to relax in response to NO and other natural or pharmacological bronchodilators (Ricciardolo et al., 2004). The mechanisms responsible for these asthma-associated changes are still not completely understood. A first step toward understanding how asthma and other obstructive lung diseases alter airway responsiveness is to elucidate the cellular mechanisms regulating changes in airway resistance induced by agonists and NO in healthy individuals. The small intrapulmonary airways are considered a major site for this regulation; however, the contractile responses of the SMCs of these airways are relatively unexplored because they are difficult to isolate and study with conventional methods used to measure cell signaling and/or force development. Consequently, we have adopted a novel approach that combines the use of mouse lung slices and confocal microscopy to simultaneously evaluate Ca2+ signaling in SMCs and airway contraction. With this approach, we previously emphasized that agonist-induced airway contraction is regulated by two intracellular processes or signals (Sanderson et al., 2008). These are (a) the frequency of agonist-induced oscillations in [Ca2+]i, referred to as Ca2+ oscillations (Perez and Sanderson, 2005) (a Ca2+-dependent mechanism), and (b) the magnitude of an agonist-induced increase in the sensitivity of the contractile machinery to [Ca2+]i, referred to as Ca2+ sensitivity (Bai and Sanderson, 2006b) (a Ca2+-independent mechanism). In addition, the relaxation of airway SMCs induced by the activation of 2 adrenergic receptors with isoproterenol (ISO), albuterol, or formoterol was shown to be accompanied by both a cAMP-dependent decrease in the frequency of Ca2+ oscillations and a decrease in Ca2+ sensitivity (Bai and Sanderson, 2006a; Delmotte and Sanderson, 2008, 2009; L-701324 Ressmeyer et al., 2009). Here, we extended these studies to investigate the mechanisms responsible for airway relaxation induced by NO. The signaling cascade by which NO induces SMC relaxation has been mainly studied in vascular SMCs of the systemic circulation. In these blood vessels, NO is synthesized in the endothelial cells and diffuses to the adjacent SMCs, where it activates soluble guanylate cyclase (sGC) to synthesize cGMP. This cGMP acts as a second messenger to activate cGMP-dependent PKG and/or other effector proteins, including ion channels, ion pumps, and phosphodiesterases (PDEs) (Carvajal et al., 2000). The phosphorylation of one or more target molecules by PKG and/or a direct activation/inhibition of ion channels by cGMP are believed to lead to SMC relaxation. However, the details of the mechanism by which an increase in cGMP results in SMC relaxation are often controversial and can vary considerably, and the precise mechanism occurring in the small pulmonary airways is unknown. For example, the contribution of Ca2+-dependent and Ca2+-independent pathways to SMC relaxation can differ with the location of the blood vessels (Chitaley and Webb, 2002; Kitazawa et al., 2009). Furthermore, many of the regulatory proteins participating in these pathways have been shown to be affected.