Reduced rather than enhanced cholinergic airway constriction in mice with ablation of the large conductance Ca2+‐activated K+channel

Sausbier, Matthias; Zhou, Xiao‐Bo; Beier, Caroline; Sausbier, Ulrike; Wolpers, Daniela; Maget, Sylvi; Martin, Christian; Dietrich, Alexander; Ressmeyer, Anna-Rebekka; Renz, Harald; Schlossmann, Jens; Hofmann, Franz; Neuhuber, Winfried; Gudermann, Thomas; Uhlig, Stefan; Korth, Michael; Ruth, Peter

doi:10.1096/fj.06-7167com

Cited by 39 publications

(40 citation statements)

References 48 publications

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“…The optimal resting tension was determined empirically and we have found that passive tension of~5 -10 mN results in an equivalent, maximal response to high potassium stimulation. This is consistent with a number of other studies that utilize a passive tension in this range 16,17,18 . Over the first 5-10 minutes, trachea passive tension tends to decline somewhat (stress-relaxation phenomenon) and the micrometer is used to adjust the passive tension to~10 mN during equilibration.…”

Section: Representative Resultssupporting

confidence: 91%

“…Some examples include analysis of gene knockouts of ion channels 17,20,25,26 , metabotropic receptors 27,28,29,30 , and downstream signaling cascades 31 . In addition, the antigen challenged mouse is frequently used for asthma studies 32 and the in vitro trachea preparation provides a useful assay for changes in contractility that ensues following development of asthma.…”

Section: Discussionmentioning

confidence: 99%

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In vitro Measurements of Tracheal Constriction Using Mice

Semenov

Herlihy

Brenner

2012

JoVE

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Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways 1,2 . In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM ( Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively 3,4,5 . M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum 3,6,7 . M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels 5,8,9,10 . These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle 11 . In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling") 4,7 . More detailed reviews on signaling pathways controlling airway constriction can be found 4,12 . The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine 13,14 , both well-known ASM modulators in humans and other species 5,15 .Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K + , Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction. Video LinkThe video component of this article can be found at http://www.jove.com/video/3703/ Protocol EquipmentThe major components of a contraction measurement device are shown ...

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Section: Representative Resultssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

In vitro Measurements of Tracheal Constriction Using Mice

Semenov

Herlihy

Brenner

2012

JoVE

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“…Ruth's group also observed that their BK-KO mouse model had an enhanced sensitivity to isoproterenol and that airway smooth muscle cells from BK-KO mice were depolarized with about 10 mV compared with cells from WT mice (21). However, it should be mentioned that in the absence of ␤-AR/PKA stimulation, IBTX and genetic BK channel deletion had equivalent effects on UBSM contractility (14,22,24).…”

Section: Discussionmentioning

confidence: 98%

β-Adrenergic relaxation of mouse urinary bladder smooth muscle in the absence of large-conductance Ca²⁺-activated K⁺ channel

Brown¹,

Bentcheva-Petkova²,

Liu³

et al. 2008

American Journal of Physiology-Renal Physiology

110

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GV. ␤-Adrenergic relaxation of mouse urinary bladder smooth muscle in the absence of large-conductance Ca 2ϩ -activated K ϩ channel. Am J Physiol Renal Physiol 295: F1149 -F1157, 2008. First published August 13, 2008; doi:10.1152/ajprenal.00440.2007.-In urinary bladder smooth muscle (UBSM), stimulation of ␤-adrenergic receptors (␤-ARs) leads to activation of the large-conductance Ca 2ϩ -activated K ϩ (BK) channel currents (Petkov GV and Nelson MT. Am J Physiol Cell Physiol 288: C1255-C1263, 2005. In this study we tested the hypothesis that the BK channel mediates UBSM relaxation in response to ␤-AR stimulation using the highly specific BK channel inhibitor iberiotoxin (IBTX) and a BK channel knockout (BK-KO) mouse model in which the gene for the pore-forming subunit was deleted. UBSM strips isolated from wild-type (WT) and BK-KO mice were stimulated with 20 mM K ϩ or 1 M carbachol to induce phasic and tonic contractions. BK-KO and WT UBSM strips pretreated with IBTX had increased overall contractility, and UBSM BK-KO cells were depolarized with ϳ12 mV. Isoproterenol, a nonspecific ␤-AR agonist, and forskolin, an adenylate cyclase activator, decreased phasic and tonic contractions of WT UBSM strips in a concentrationdependent manner. In the presence of IBTX, the concentrationresponse curves to isoproterenol and forskolin were shifted to the right in WT UBSM strips. Isoproterenol-and forskolin-mediated relaxations were enhanced in BK-KO UBSM strips, and a leftward shift in the concentration-response curves was observed. The leftward shift was eliminated upon PKA inhibition with H-89, suggesting upregulation of the ␤-AR-cAMP pathway in BK-KO mice. These results indicate that the BK channel is a key modulator in ␤-AR-mediated relaxation of UBSM and further suggest that alterations in BK channel expression or function could contribute to some pathophysiological conditions such as overactive bladder and urinary incontinence.BK channel knockout mouse; isoproterenol; forskolin; iberiotoxin DURING VOIDING, the urinary bladder smooth muscle (UBSM) contracts forcefully to expel urine. UBSM exhibits spontaneous action potentials that are associated with the phasic nature of the contractions in this tissue (2,6,17). Ca 2ϩ entry through dihydropyridine-sensitive L-type voltage-gated Ca 2ϩ (Ca V ) channels is responsible for the upstroke of the action potential and leads to an increase in global intracellular Ca 2ϩ , which activates the UBSM phasic contractions. Blocking L-type Ca V channels eliminates the spontaneous action potentials and contractions in UBSM (2, 6). The repolarization phase of the UBSM action potential is mediated by the activity of the large-conductance Ca 2ϩ -activated K ϩ (BK) channels (6) and perhaps the voltage-gated K ϩ (K V ) channels (23). Pharmacological inhibition of UBSM BK channels with a highly specific inhibitor, iberiotoxin (IBTX), increases the action potential duration and frequency, causes membrane potential depolarization (6, 19), and increases the amplitude of phasic contractions (8, 17)...

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“…However, knockout mice of the accessory calcium-sensitive ␤ 1 -subunit were not hypertensive (96). Interestingly, the BK Ca ␣-subunit deletion enhanced the expression of cGMP signaling proteins (PKG1 and IRAG) in tracheal smooth muscle and led to cholinergic bronchoconstriction in the airways (71). cGMP enhanced BK Ca activity via PKG1 in vascular smooth muscle and thereby mediated smooth muscle hyperpolarization (70).…”

Section: Other Targets For Pkgmentioning

confidence: 99%

IRAG and novel PKG targeting in the cardiovascular system

Schlossmann

Desch

2011

American Journal of Physiology-Heart and Circulatory Physiology

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Schlossmann J, Desch M. IRAG and novel PKG targeting in the cardiovascular system. Am J Physiol Heart Circ Physiol 301: H672-H682, 2011. First published June 10, 2011; doi:10.1152/ajpheart.00198.2011.-Signaling by nitric oxide (NO) determines several cardiovascular functions including blood pressure regulation, cardiac and smooth muscle hypertrophy, and platelet function. NO stimulates the synthesis of cGMP by soluble guanylyl cyclases and thereby activates cGMP-dependent protein kinases (PKGs), mediating most of the cGMP functions. Hence, an elucidation of the PKG signaling cascade is essential for the understanding of the (patho)physiological aspects of NO. Several PKG signaling pathways were identified, meanwhile regulating the intracellular calcium concentration, mediating calcium desensitization or cytoskeletal rearrangement. During the last decade it emerged that the inositol trisphosphate receptor-associated cGMP-kinase substrate (IRAG), an endoplasmic reticulum-anchored 125-kDa membrane protein, is a main signal transducer of PKG activity in the cardiovascular system. IRAG interacts specifically in a trimeric complex with the PKG1␤ isoform and the inositol 1,4,5-trisphosphate receptor I and, upon phosphorylation, reduces the intracellular calcium release from the intracellular stores. IRAG motifs for phosphorylation and for targeting to PKG1␤ and 1,4,5-trisphosphate receptor I were identified by several approaches. The (patho)physiological functions for the regulation of smooth muscle contractility and the inhibition of platelet activation were perceived. In this review, the IRAG recognition, targeting, and function are summarized compared with PKG and several PKG substrates in the cardiovascular system. inositol trisphosphate receptor-associated guanosine 3=,5=-cyclic monophosphatekinase substrate; nitric oxide; guanosine 3=,5=-cyclic monophosphate-dependent protein kinase THIS ARTICLE is part of a collection on Hypertension and Novel Modulators of Vascular Tone. Other articles appearing in this collection, as well as a full archive of all collections, can be found online at http://ajpheart.physiology.org/.Nitric oxide (NO) leads to cGMP synthesis and thereby activates cGMP-dependent protein kinase (PKG), mediating various cardiovascular functions. The identification of PKG substrates including the inositol trisphosphate receptor-associated cGMP-kinase substrate (IRAG) has lead to a new view of PKG1 isozyme PKG1␤ and PKG1␣ function. Intracellular targeting and function of these isozymes are modulated by different substrates. The recognition of these substrates is mediated by the NH 2 -terminal leucine/isoleucine zipper (LZ) domains of PKG1. In this review the current view of the molecular interaction and function of IRAG compared with other substrates regulated by PKG are described, mainly focusing on the cardiovascular system. Furthermore, the subtle pathways for cGMP/PKG signaling are discussed. Function of NO/cGMP/PKG Signaling CascadeNO/cGMP signaling regulates several tasks in the cardiovascular sy...

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Reduced rather than enhanced cholinergic airway constriction in mice with ablation of the large conductance Ca²⁺‐activated K⁺channel

Cited by 39 publications

References 48 publications

<em>In vitro</em> Measurements of Tracheal Constriction Using Mice

<em>In vitro</em> Measurements of Tracheal Constriction Using Mice

β-Adrenergic relaxation of mouse urinary bladder smooth muscle in the absence of large-conductance Ca²⁺-activated K⁺ channel

IRAG and novel PKG targeting in the cardiovascular system

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Reduced rather than enhanced cholinergic airway constriction in mice with ablation of the large conductance Ca2+‐activated K+channel

Cited by 39 publications

References 48 publications

<em>In vitro</em> Measurements of Tracheal Constriction Using Mice

<em>In vitro</em> Measurements of Tracheal Constriction Using Mice

β-Adrenergic relaxation of mouse urinary bladder smooth muscle in the absence of large-conductance Ca2+-activated K+ channel

IRAG and novel PKG targeting in the cardiovascular system

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Resources

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Reduced rather than enhanced cholinergic airway constriction in mice with ablation of the large conductance Ca²⁺‐activated K⁺channel

β-Adrenergic relaxation of mouse urinary bladder smooth muscle in the absence of large-conductance Ca²⁺-activated K⁺ channel