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The generation of acetylcholine-induced asynchronous Ca²⁺ waves and their role in airway smooth muscle Dai, Jiazhen Minnie 2006

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THE GENERATION OF ACETYLCHOLINE-INDUCED ASYNCHRONOUS Ca 2 + WAVES AND THEIR ROLE IN AIRWAY SMOOTH MUSCLE by Jiazhen Minnie Dai B.Sc., The University of Calgary, 2002 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Pharmacology & Therapeutics) THE.UNIVERSITY OF BRITISH COLUMBIA August 2006 © Jiazhen Minnie Dai, 2006 Abstract Agonist-stimulated repetitive asynchronous C a 2 + waves (ACW) have emerged as ubiquitous C a 2 + signals in airway smooth muscle cells. Even though the role of this type of C a 2 + signal in airway smooth muscle (ASM) has yet to be defined, it is likely that A C W are involved in the regulation of airway constriction due to the significance of C a 2 + in A S M contraction. This thesis focuses on: 1) the primary function of A C W and 2) the signaling pathway(s) underlying agonist-induced A C W in intact ASM. Employing confocal imaging of Ca2+-sensitive dyes, we found that ACh elicits recurring intracellular C a 2 + waves in cells of the intact porcine tracheal and more importantly human bronchial muscle bundle. These C a 2 + waves were not synchronized between neighboring cells. Simultaneous measurement of intracellular C a 2 + concentration ([Ca2+]i) and isometric contraction indicates that induction of these A C W was temporally associated with development of force by the muscle bundles. By comparing the concentration dependence of force generation and the parameters characterizing ACW, we found that the concentration-dependent increase in ACh-induced force development by the A S M bundle is achieved by differential recruitment of cells to initiate C a 2 + waves followed by enhancement in the frequency of A C W and elevation of interspike [Ca2+]i once the cells are recruited. Furthermore, pharmacological characterization of the mechanism of ACh-induced A C W revealed that they are a result of repetitive cycles of sarcoplasmic reticulum (SR) C a 2 + release via ryanodine-sensitive C a 2 + release channels followed by refilling of the SR via sarco(endo)plasmic reticulum C a 2 + ATPase. Plasmalemmal C a 2 + entry via the reverse-mode Na + /Ca 2 + exchange coupled with the non-selective cation permeable receptor-operated channels/store-operated channels, and to a lesser extent via the L-type voltage-gated C a 2 + channels is involved in replenishing the SR and supporting the ongoing ACW. Given ii the significance of A S M in the pathogenesis of airway diseases such as asthma, these findings provide insights into the regulation of A S M contraction and potential therapeutic targets for the management of these diseases. T A B L E O F C O N T E N T S A B S T R A C T ii T A B L E O F C O N T E N T S iv LIST O F T A B L E S vii LIST O F FIGURES viii ABBREVIATIONS x P R E F A C E xi A C K N O W L E D G E M E N T S xii C H A P T E R 1: INTRODUCTION 1 1.1 Pathophysiological role of airway smooth muscle 1 1.2 The role of Ca2+ in contractile activation of airway smooth muscle 2 1.3 Ca2+ regulation in airway smooth muscle 4 1.4 Classic model of agonist-induced Ca2+ signal in airway smooth muscle 8 1.5 Disadvantage of single airway smooth muscle cells 9 1.6 Repetitive asynchronous Ca2+ waves in intact airway smooth muscle 11 1.7 Hypothesis 12 1.8 Bibliography 16 C H A P T E R 2: T H E RELATIONSHIP B E T W E E N A S Y N C H R O N O U S C a 2 + W A V E S A N D F O R C E D E V E L O P M E N T IN INTACT S M O O T H M U S C L E BUNDLES O F T H E PORCINE T R A C H E A 21 2.1 Introduction 21 2.2 Methods 22 2.2.1 Tissue preparations 22 2.2.2 Histology and electron microscopy study 23 2.2.3 Simultaneous isometric force measurement and confocal microscopy of Ca2+ induced fluorescence 23 2.2.4 Data analysis 25 2.2.5 Solutions^ and chemicals 26 iv 2.3 Results 27 2.3.1 Organization of tracheal smooth muscle 28 2.3.2 ACh-induced Ca2+ signals in ASMC of the intact porcine tracheal muscle bundle 28 2.3.3 Relationship between repetitive asynchronous Ca2+ wave and force generation 31 2.3.4 Role ofL-type voltage-gated Ca2+ channel in ACh-induced repetitive asynchronous Ca2+ wave 35 2.4 Discussion 37 2.5 Bibliography 45 C H P A T E R 3: T H E M E C H A N I S M O F A C E T Y L C H O L I N E - I N D U C E D A S Y N C H R O N O U S C A L C I U M W A V E S IN T H E PORCINE T R A C H E A L M U S C L E B U N D L E 48 3.1 Introduction 48 3.2 Material and Methods 50 3.2.1 Tissue Preparations 50 3.2.2 Cell Permeabilization 50 3.2.3 Isometric Force Measurement 51 3.2.4 Confocal Imaging 51 3.2.5 Data Analysis 52 3.2.6 Solutions And Chemicals 53 3.3 Results 54 3.3.1 Dependence of ACW on plasmalemmal Ca2+ influx 54 3.3.2 Dependence of ACW on SR Ca2+ release 62 3.4 Discussion 73 3.5 Bibliography 83 C H A P T E R 4: A C E T Y L C H O L I N E - I N D U C E D A S Y N C H R O N O U S C A L C I U M W A V E S IN I N T A C T HU M A N B R O N C H I A L M U S C L E B U N D L E 90 4.1 Introduction 90 4.2 Materials and methods 91 4.2.1 Tissue preparation 91 4.2.2 Solutions and Chemicals 92 v 4.2.3 Isometric force measurement 92 4.2.4 Simultaneous isometric force measurement and confocal microscopy of Ca2+-induced fluorescence 93 4.2.5 Data analysis 93 4.3 Results 94 4.3.1 Patient background 94 4.3.2 Characterization of ACh-induced Ca2+ signal in human bronchial smooth muscle 95 4.3.3 Relationship between repetitive asynchronous Ca2+ waves and force development 98 4.3.4 Dependence of repetitive asynchronous Ca2* waves on plasmalemmal Ca2+ influx 101 4.3.5 Dependence of repetitive asynchronous Ca2+ waves on SR Ca2+ release 105 4.4 Discussion 108 4.5 Bibliography 113 C H A P T E R 5: C O N C L U S I O N AND F U T U R E DIRECTIONS 116 5.1 Overview of repetitive asynchronous Ca2+ waves 116 5.2 Repetitive asynchronous Ca2+ waves and constriction of the airway 118 5.3 Mechanism of repetitive asynchronous Ca2* waves 119 5.4 Functional advantage of repetitive asynchronous Ca2+ waves 123 5.5 Summary 125 5.6 Bibliography.... 128 vi LIST O F T A B L E S Table 4.1: Demographic information of the patients LIST O F FIGURES Page Figure 1.1: Ca2+-mediated contraction of A S M 3 Figure 1.2: Molecules involve in transporting C a 2 + in A S M C 4 Figure 1.3: Biphasic C a 2 + signaling and activation of contractile machinery 8 Figure 2.1: Structure of A S M C in an intact porcine tracheal muscle strip 27 Figure 2.2: ACh induces A C W in the A S M C of intact porcine tracheal muscle bundles 30 Figure 2.3: Temporal association between ACh-induced force generation and Ca 2 +signal 31 Figure 2.4: ACh induces concentration-dependent contraction of the tracheal smooth muscle 32 Figure 2.5: Concentration-response relationships of ACh-induced force generation and A C W are depicted 33 Figure 2.6: Effects of nifedipine (NIF) on ongoing ACh-mediated A C W and force generation 37 Figure 3.1: Effect of removal of extracellular C a 2 + on ACh-induced as A C W 54 Figure 3.2: Effect of 10 uM nifedipine and 50 uM SKF-96365 on ACh-induced A C W and tonic contraction 55 Figure 3.3: Effect of 10 uM 2',4'-DCB, 20 uM KB-R7943, and zero-Na+ PSS on the nifedipine-resistant component of ACh-induced A C W and tonic contraction 59 Figure 3.4: Effect of 10 u.M nifedipine and 50 uM SKF96365 on the initiation of ACh-induced A C W 62 Figure 3.5: Effect of 10 uM CPA on ACh-induced A C W 64 Figure 3.6: Effect of 75 uM 2-APB on ACh-induced A C W and tonic contraction and on IP3-induced contraction in smooth muscle cell from intact porcine tracheal muscle 65 Figure 3.7: Effect of 10 uM xestospongin C (Xe-C) on ACh-induced A C W and tonic contraction 68 viii Figure 3.8: Effect of 12.5 mM caffeine and 25 uM ryanodine on ACh-induced A C W in smooth muscle cells from intact porcine tracheal muscle 69 Figure 3.9: Effect of 2 mM procaine on ACh-induced A C W and tonic contraction in smooth muscle cells from intact porcine tracheal muscle 71 Figure 3.10: Effect of 100 uM tetracaine on ACh-mediated A C W and tonic contraction in porcine tracheal smooth muscle cells 72 Figure 4.1: ACh-induced A C W in the A S M C of intact human bronchial muscle bundles 97 Figure 4.2: Temporal association between ACh-induced force generation and C a 2 + signal 98 Figure 4.3: Concentration-response relationships of ACh-induced force generation and A C W 100 Figure 4.4: Effect of 10 uM nifedipine and 50 uM SKF96365 on ACh-induced A C W and tonic contraction 102 Figure 4.5: Effect of zero Na + PSS and 10 uM KB-R7943 on ACh-induced A C W and tonic contraction 104 Figure 4.6: Effect of 10 uM CPA on ACh-induced A C W 106 Figure 4.7: Effects of 100 uM tetracaine and 2 mM procaine on the ACh-induced A C W and tonic contraction in human bronchial smooth muscle strips 107 Figure 5.1: Histamine- and endothelin-induced A C W in A S M C of intact porcine tracheal muscle strips 117 Figure 5.2: Model for ACh-induced A C W in A S M C of intact porcine tracheal smooth muscle 120 ix ABBREVIATIONS 2-APB: 2-aminoethoxydiphenyl borate [Ca2+]j: intracellular C a 2 + concentration cADPr: cyclic-ADP ribose CICR: Ca2+rinduced C a 2 + release CPA: cyclopiazonic acid DMSO: dimethyl sulfoxide E-C coupling: excitation-contraction coupling E M : electron miscroscopy ER: endoplasmic reticulum IP3: inositol- 1,4,5-trisphosphate IP3R: inositol-1,4,5-trisphosphate-sensitive SR C a 2 + release M L C K : myosin light chain kinase NCX: Na + /Ca 2 + exchanger PM: plasma membrane PMCA: plasma membrane C a 2 + ATPase PSS: physiological saline solution ROC: receptor-operated channels RyR: ryanodine-sensitive SR C a 2 + release channel SERCA: sarco(endo)plasmic/ reticulum C a 2 + ATPase SOC: store-operated channels SR: sarcoplasmic reticulum TRP: transient receptor potential V G C C : voltage-gated C a 2 + channels ASMC: airway smooth muscle cells ASM: airway smooth muscle NIF: nifedipine [Ca2 +]: C a 2 + concentration Xe-C: xestospongin C ACh: acetylcholine ACW: repetitive asynchronous Ca waves VSMC: vascular smooth muscle cells TSMC: tracheal smooth muscle cells 2',4'-DCB: 2',4'-dichlorobenzamil N M D G + : N-methyl-D-glucamine ATP: adenosine triphosphate NSCC: non-selective cation channel P R E F A C E Material from this dissertation has been published or accepted in the following journals: *- Dai J M , Kuo KH, Leo JM, van Breemen C, Lee CH. The mechanism of ACh-induced asynchronous calcum waves and tonic contraction in the porcine tracheal muscle bundle. Am J Physiol Lung Cell Mol Physiol. 2006; 290(3):L459-69. •* Kuo K H * , Dai J * , Seow CY, Lee CH, van Breemen C. The relationship between asynchronous Ca waves and force development in intact smooth muscle bundles of the porcine trachea. Am J Physiol Lung Cell Mol Physiol. 2003 Dec;285(6):L1345-53. (* co-first author) Material from this dissertation has been submitted to the following journals: * Dai J M , Kuo KH, Leo JM, Pare P, van Breemen C, Lee CH. Acetylcholine-induced asynchronous calcium waves in intact human bronchial muscle bundle. Am J Respir Cell Mol Biol. 2006 x i A C K N O W L E G E M E N T S First of all, I would like to thank my supervisor Dr. Casey van Breemen for his guidance throughout my pursuit of a PhD degree. Over the past four years, Casey has always been there for me during difficult times and was very supportive of my work. He was not only my supervisor but also a dear friend who make my graduate research an enjoyable and memorable experience. To my supervisory committee, I am very grateful for the valuable advice that Dr. Peter Pare, Dr. Xiaodong Wang and Dr.Chun Seow have provided. I would like to thank the individuals in the laboratory, especially Dr. Cheng-Han Lee and Dr. Kuo-Hsing Kuo for introducing me to confocal microscopy and helping me during my earlier days in the laboratory. I would also like to thank Drs. Elena Okon and Mark Elliott for their help and support at St. Paul's hospital. Finally, and more importantly, I appreciate the understanding and support from my parents, my sister, and my fiance. I am particularly grateful for all the sacrifice my parents made to provide me the opportunity to educate in Canada. To my beloved grandpas, I thank them for teaching me the value and importance of education when 1 was little. xii C H A P T E R 1 - INTRODUCTION 1.1 Pathophysiological role of airway smooth muscle The obstructive airway diseases - asthma and chronic obstructive pulmonary disease (COPD) are common in our society and are responsible for a large burden of decreased length and quality of life. Airway obstruction in these disorders is related to acute and chronic inflammation and is caused by structural remodeling, mucus hyper-secretion and airway smooth muscle (ASM) contraction. There is substantial evidence that the contraction of A S M is excessive in asthma and COPD (as well as other inflammatory airway diseases), but the exact mechanism for this abnormality is unknown. The exaggerated bronchoconstriction can be quantified as non-specific airway or bronchial hyperresponsiveness (NSBH). NSBH is the exaggerated airway narrowing that occurs in response to challenging the airways with a wide variety of pharmacological agonists and non-specific irritants such as cold, dry air and oxidant gases. The stimuli that elicit the exaggerated response have in common the ability to directly or indirectly stimulate A S M to contract. NSBH is characterized both by an increase in the sensitivity of the airways (i.e., they narrow more easily than the airways of normal subjects at lower concentrations of pharmacologic agonists or lower levels of irritants) and by greater maximal airway narrowing than can be achieved in normal airways (Woolcock et al , 1984; Sterk et al , 1985). Although it 1 is generally agreed that the narrowing is caused by contraction of the ASM, it is still unclear if the phenomenon is due to fundamental changes in the phenotype (structure and/or function) of the smooth muscle itself or is caused by structural and/or mechanical changes in the non-contractile elements of the airway wall. With respect to the phenotype of A S M , considerable gaps still remain in our current understanding of contractile activation in healthy ASM, particularly with regard to the cellular C a 2 + signal. Without a thorough understanding of the mechanism of excitation-contraction coupling (E-C coupling) of normal airways, it is difficult to appreciate the potential phenotypic changes in A S M that may underlie NSBH in diseased airways. 1.2 The role of Ca2+ in contractile activation of airway smooth muscle The ability of A S M to contract is attributed to the interaction among contractile proteins and regulatory enzymes, including myosin thick filaments, actin thin filaments, and myosin light chain kinase (MLCK). In its activated form, M L C K phosphorylates myosin light chain which then allows actin to activate the myosin ATPase activity required for cross-bridge cycling. Coincident with myosin phosphorylation, myosin light chain phosphatase (MLCP) opposes the action of M L C K and de-phosphorylates myosin, thus promoting relaxation of the ASM. During cross-bridge cycling, binding of myosin filament to actin filaments and their subsequent 2 sliding motion toward each other generates force (Gerthoffer, 1991; Gunst and Tang, 2000). 4Ca2+ + Calmodulin 4Ca2+-Calm6dulin + MLCK Mypsin ATP Ca2+-Calmodulin-MLCK ADP Myosin phosphatase n-P; + Actin Actin-Myosin-Pj Contraction Figure 1.1. Ca2+-mediated contraction of A S M As it is the case in other types of smooth muscle, activation of M L C K and cross-bridge cycling in A S M is primarily triggered by an elevation of intracellular C a 2 + concentration ([Ca2+];) and the formation of the Ca2+-calmodulin complex. The induction of A S M contraction by an elevation in [Ca2+]i is illustrated in Fig. 1.1. Numerous studies have shown that in addition agonist stimulation induces C a 2 + sensitization of the myofilament by inhibiting the activity of MLCP (Croxton et al , 1998; Yoshii et al., 1999; Yoshimura et al., 2001; Bai and Sanderson, 2006). This phenomenon leads to enhancement of the overall contractile response as a result of an increased number of phosphorylated myofilaments without further elevation in the [Ca2+]; of 3 the A S M C . Figure 1.2. Molecules involve in transporting Ca in A S M C 1.3 Ca regulation in airway smooth muscle In view o f the dependence of the contractile filaments to changes in [Ca 2 + ] i (de Lanerolle et al., 1982; Gerthoffer, 1991), airway smooth muscle cells ( A S M C ) actively modulate their [Ca 2 +]j to regulate their contractile activity. This is achieved through the concerted action of various C a 2 + translocating molecules as shown in Fig. 1.2. 4 Upon stimulation, [Ca ]j is elevated up to about 1 \iM to produce force (Sims et al., 1996; Barnes, 1998). This rise in [Ca2+]j can result from regulated C a 2 + entry from extracellular space, where the C a 2 + concentration ([Ca2+]) is approximately 1 mM (Barnes, 1998; Janssen, 1998). This involves the activity of a number of C a 2 + permeable proteins on the sarcolemma, including L-type voltage-gated C a 2 + channels (VGCC) (Liu and Farley, 1996; Janssen, 1998, 2002), non-selective cation permeable receptor-operated channels/store-operated channels (ROC/SOC) (Fleischmann et al., 1997; Janssen, 1998; Ay et al., 2004; Marthan, 2004), and Na + /Ca 2 + exchangers (NCX) operating in the reverse mode (Chideckel et al., 1987; Pitt and Knox, 1996; Mejia-Elizondo et al., 2002; Cortijo et al., 2003). Alternatively, release of C a 2 + from the intracellular store, sarcoplasmic reticulum (SR), which has a free luminal [Ca2+] of up to 400 | l M (Corbett and Michalak, 2000), can also lead to elevation of [Ca2+]i in ASMC. Calcium within the SR may be released via inositol 1,4,5-trisphosphate sensitive SR C a 2 + release channels (IP3R) (Coburn and Baron, 1990; Marmy et al., 1993; Kannan et al., 1997; Iizuka et al., 1998) and/or ryanodine sensitive SR C a 2 + release channels (RyR) (Kannan et al , 1997; Du et al., 2005). The open probability of IR3R and RyR is determined by the local [Ca ]. Inositol 1,4,5-trisphosphate (IP3) and cyclic adenosine diphosphate ribose enhance the sensitivity of IP3R and RyR respectively to the surrounding C a 2 + and promote the activation of these two channels (Prakash et al., 1998; Amrani et al, 2004; Deshpande et al., 2005). To reduce the [Ca 2 + ] i of A S M C to the basal level of approximately 100 n M (Sims et al., 1996; Roux et al., 1997; Barnes, 1998; Roux and Marhl , 2004), cytoplasmic C a 2 + is removed by C a 2 + pumps and exchangers located on the plasma membrane and membrane of the SR. On the sarcolemma, the plasma membrane C a 2 + ATPase ( P M C A ) and the N C X operating in the forward mode extrude cytoplasmic C a 2 + to the extracellular space (Janssen, 1998). On the SR membrane, sarco(endo)plasmic reticulum C a 2 + ATPase ( S E R C A ) actively sequester cytosolic C a 2 + into the SR (Janssen, 1998; Roux and Marhl , 2004). Both P M C A and S E R C A are pumps with high C a 2 + affinity and utilize adenosine triphosphate (ATP) as their energy source. N C X is a bidirectional ion transporter whose mode of operation ( C a 2 + influx and C a 2 + exit) is determined by the trans-membrane electrochemical gradients for N a + and C a 2 + ions. A t physiological N a + and Ca concentrations, N C X has a reversal potential of about -15 mV. This can also be estimated by the following equation: where [Ca 2 + ] 0 and [Na + ] 0 are the extracellular concentration of C a i + and N a + respectively, [Ca z + ] i and [Na + ] i are the intracellular concentration of C a 2 + and N a + respectively, n is the exchange ratio between N a + and C a 2 + , E r e v i s the reversal potential of N C X , F is the Faraday's constant, R is the Gas constant, and T is the absolute temperature (Blaustein and Lederer, 1999). Since the resting membrane potential of A S M C is within the range of -40 m V to -65 mV, N C X generally 6 operates in the C a i + extrusion mode when cells are un-stimulated (Thirstrup, 2000; Montano and Bazan-Perkins, 2005). Regardless of its mode of operation, the coupling ratio for the exchanger is 3 N a + : 1 C a 2 + , i.e. for every three N a + that is carried in one direction, one C a 2 + is delivered in the opposite path. In the non-stimulated A S M C , there are constantly active C a 2 + fluxes that maintain the basal [Ca 2 +]j in the cell. Resting C a 2 + entry across the P M through primarily R O C and S O C have been reported to be balanced by the basal C a 2 + entrusion by P M C A and forward-mode N C X (Montano and Bazan-Perkins, 2005). Owing to its non-selective nature, R O C and S O C are permeable to N a as well as Ca . 7 C a 2 + C a 2 + C a 2 t • • • Figure 1.3. Biphasic Ca signaling and activation of contractile machinery. A. Agonist stimulation leads to IP3-mediated SR C a 2 + release. This represents the initial C a 2 + transient which contributes solely to the potentiation of contractile response. B. Subsequent activation of the plasma membrane C a 2 + channels results in sustained influx of extracellular C a 2 + . This represents the second plateau phase of the agonist-mediated C a 2 + signal which is responsible for the maintenance of tonic contraction. SR, sarcoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; Gq, receptor coupled G protein. 1.4 Classic model of agonist-induced Ca signal in airway smooth muscle Early studies measuring average changes in [Ca / +] of an entire muscle strip proposed a simple two-phase model of agonist-induced C a 2 + signal, which underlies E-C coupling in A S M (Fig. 1.3) (Shieh et al., 1991). In this model, stimulation of A S M C with agonist first elicits SR 8 C a 2 + release to cause a large transient increase in [Ca2+]j, which is responsible only for the initiation of contraction. Immediately following the [Ca2+]j transient, the [Ca2+]j declines to a steady-state level that stays above the basal value for as long as the agonist is present. This second phase was thought to be the result of sustained influx of extracellular C a 2 + across the sarcolemma and responsible for maintaining the steady state portion of the agonist-induced tonic contraction. However, as will be discussed later, with the development of more advanced imaging techniques, for example, the fast laser scanning confocal microscope in combination 2+ 2+ with Ca -sensitive dyes, more complex types of Ca signals were found to be elicited by various contractile agonists in ASMC. This diversity of C a 2 + signaling in ASMC suggests that agonist-mediated C a 2 + signal is not a simple event and the average change in [Ca2+] of an entire muscle strip may not reflect the change within each individual cell in the intact muscle strip. 7.5 Disadvantage of single airway smooth muscle cells Unlike many of the second messengers involved in intracellular signaling, the C a 2 + signal is unique in the sense that the message is not solely conveyed by a rise in its concentration, but also by the spatial and temporal pattern of the C a 2 + signal (Lee et al., 2002a; Lee et al., 2002b). Because of this complexity the study of C a 2 + signaling requires imaging of sub-cellular [Ca2 +] with high spatio-temporal resolution. In single cell preparations, which were first examined by 9 confocal microscopy, agonist stimulation of ASMC produced varying patterns of changes in the [Ca 2 + ] b including the biphasic model discussed above (Sims et al., 1996), and recurrent wave-like C a 2 + transients that propagates along the length of the ASMC (Colliard-Rouiller and Durand, 1997; Kannan et al , 1997; Prakash et al., 1997; Roux et al., 1997; Prakash et al., 2000). Although these studies provided a great deal of information regarding the C a 2 + regulation of ASMC, there are several problems associated with the use of freshly isolated and cultured cells. First, within their intact gap junctions and tissue matrix in situ ASMC are electrically and functionally interconnected with each other (Daniel et al., 1986). The gap junctions allow changes in membrane potential and [Ca2+]; in one cell to influence the respective parameters in neighboring cells. In isolated cell studies, the disruption of gap junctions is unfortunately unavoidable. This not only removes signals coming from neighbouring cells, but also potentially alters the primary mode of smooth muscle Ca signaling. Second, the relatively non-selective enzymatic proteolysis employed during isolation of ASMC will undoubtedly damage many of the surface proteins, including channels, pumps and receptors (Hall and Kotlikoff, 1995). This may alter cellular physiology and affect normal C a 2 + signaling. Indeed, as demonstrated previously and later in this thesis, single cell preparations of vascular smooth muscle (Ruehlmann et al., 2000; Lee et al , 2001) and A S M tend to behave very differently from the more physiological intact tissue preparation. Given that the tissue is significantly perturbed 10 during enzymatic digestion, standardization of the quality of the digested cells is difficult and this can make quantitative comparison between sensitized and normal airways challenging. Third, many single cell preparations involve cell culture. However, during the process of culturing, smooth muscle cells are known to undergo phenotypic changes that include changes in structure and protein expression (Halayko et al., 1996; Hirst, 1996; Worth et al., 2001). In cultured canine tracheal smooth muscle cells, the level of contractile proteins and enzymes such as smooth muscle myosin heavy chain, smooth muscle a-actin, and M L C K were greatly decreased, (Halayko et al , 1996) which may lead to the loss of contractility. In cultured human airway smooth muscle cells, the expression of muscarinic receptors is different from that in intact muscle, which may be the cause of marked attenuation or lack of Ca response to stimulation (Panettieri et al., 1989; Widdop et al, 1993; Hall and Kotlikoff, 1995). In summary the data collected from single cultured or freshly isolated cells do not reliably reflect the pharmacological function of airway smooth muscle. 1.6 Repetitive asynchronous Ca2+ waves in intact airway smooth muscle The phenomenon of agonist-mediated repetitive C a 2 + waves was first observed in single A S M C (Prakash et al., 1997; Prakash et al., 2000). However, as discussed earlier, due to possible damage of the cell surface protein from non-specific isolation procedure and the phenotypic alteration that can occur d u r i n g ce l l culture, the response observed m a y not accurately represent the true p h y s i o l o g y o f the A S M C . Recently , Bergner and Sanderson e x a m i n e d agonist- induced [ C a 2 + ] i changes o f intact A S M that reside i n the bronchiolar w a l l o f m i c e l u n g slices (Bergner and Sanderson, 2002). T h e y observed that appropriate concentrations o f acetylchol ine ( A C h ) stimulate recurr ing C a 2 + waves i n the intact A S M C . A n interesting observation o f their study is that, i n addit ion to the regenerative and propagating nature, these C a 2 + waves observed in lung slice are not s y n c h r o n i z e d between ne ighbor ing cells. 1.7 Hypothesis T h e observation o f repetitive asynchronous C a 2 + waves ( A C W ) raised a series o f questions regarding our knowledge i n C a 2 + s ignal ing and contraction o f A S M : C o u l d these A C W observed i n b r o n c h i occur i n smooth muscle cells o f other parts o f the tracheobronchial tree, or more importantly , i n intact h u m a n A S M ? W h a t is the role o f these A C W ? H o w are these A C W generated? T o address these questions, I examined the c o m p l e x C a signal i n relation to force development i n A S M based on the f o l l o w i n g hypothesis: A S M C respond to agonist s t imulat ion w i t h the generation o f A C W w h i c h underl ie E - C c o u p l i n g o f intact A S M bundle. These A C W are a result o f the repetitive release o f C a 2 + f rom the S R , and their maintenance requires r e f i l l i n g o f the S R v i a p l a s m a l e m m a l C a 2 + entry. 12 The specific aims are: Aim #1: Examination of ACh-mediated Ca2+ signaling in ASMC of the intact porcine tracheal muscle bundle. Using confocal microscopy of Ca2+-sensitive fluorescent molecules, I will examine at high spatio-temporal resolution the C a 2 + signals elicited by ACh in smooth muscle cells within the porcine tracheal muscle bundle. Aim #2: Correlation of ACh-mediated ACW to contractile function in porcine tracheal muscle bundle. I will examine the C a 2 + signal and tonic contraction induced by various concentrations of ACh. The concentration-dependence of selected properties of the agonist-induced C a 2 + signal will be compared to the concentration-dependence of force generation. The selected properties of the ACh-induced ACW are: the recruitment of the ASMC to initiate the C a 2 + signal, the amplitude of the ACW, the frequency of the ACW, the baseline [Ca 2 +]i, and the average [Ca2+]j. Aim #3: Examination of the mechanism of ACh-mediated ACW in the cells of the intact porcine tracheal muscle bundle. 13 Using various pharmacological agents, I w i l l systematically examine the mechanism of ACh-mediated A C W and tonic contraction in intact porcine tracheal muscle bundle. This involves use of selective inhibitors of the known C a 2 + handling molecules to examine their role in mediating the A C W . The proteins in question include L-type V G C C , non-selective cation permeable R O C / S O C type channel, N C X , S E R C A , and IP 3 R/RyR. Aim #4: Examination of the ACh-induced Ca2+ signaling in intact adult human bronchial smooth muscle. I w d l examine the C a signal elicited by A C h in the A S M C within the intact human bronchial muscle bundle. Aim #5: Correlation of ACh-mediated ACW to tonic contraction in intact human bronchial smooth muscle. 1 w i l l examine the C a 2 + signal and tonic contraction induced by various concentrations of A C h . The concentration-dependence of selected properties of the agonist-induced C a 2 + signal w i l l be compared to the concentration-dependence of force generation. Aim #6: Examination of the mechanism of ACh-mediated ACW in the ASMC of the intact human bronchial muscle bundle 14 I will pharmacologically characterize the signaling pathways for ACh-mediated ACW which induce tonic contraction in intact human bronchial smooth muscle bundle. 15 1.8 Bibliography Amrani, Y., O. Tliba, D.A. Deshpande, T.F. Walseth, M.S. Kannan, and R.A. Panettieri, Jr. 2004. Bronchial hyperresponsiveness: insights into new signaling molecules. Curr Opin Pharmacol. 4:230-234. Ay, B., Y.S. Prakash, C M . Pabelick, and G.C. Sieck. 2004. Store-operated C a 2 + entry in porcine airway smooth muscle. Am J Physiol Lung Cell Mol Physiol. 286:L909-917. Bai, Y , and M.J. Sanderson. 2006. Modulation of the C a 2 + sensitivity of airway smooth muscle cells in murine lung slices. Am J Physiol Lung Cell Mol Physiol. 7:34. Barnes, P.J. 1998. Pharmacology of airway smooth muscle. Am J Respir Crit Care Med. 158:S123-132. Bergner, A., and M.J. Sanderson. 2002. 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InsP.i, but not novel C a 2 + releasers, contributes to agonist-initiated contraction in rabbit airway smooth muscle. J Physiol. 511 ( Pt 3):915-933. 17 Janssen, L.J. 1998. Calcium handling in airway smooth muscle: mechanisms and therapeutic implications. Can Respir J. 5:491-498. Janssen, L.J. 2002. Ionic mechanisms and C a 2 + regulation in airway smooth muscle contraction: do the data contradict dogma? Am J Physiol Lung Cell Mol Physiol. 282:L1161-1178. Kannan, M.S., Y.S. Prakash, T. Brenner, J.R. Mickelson, and G C . Sieck. 1997. Role of ryanodine receptor channels in C a 2 + oscillations of porcine tracheal smooth muscle. Am J Physiol. 272:L659-664. Lee, C.H., D. Poburko, K.H. Kuo, C. Seow, and C. van Breemen. 2002a. Relationship between the sarcoplasmic reticulum and the plasma membrane. Novartis FoundSymp. 246:26-41; discussion 41-27, 48-51. Lee, C.H., D. Poburko, K.H. Kuo, C.Y. Seow, and C. van Breemen. 2002b. C a 2 + oscillations, gradients, and homeostasis in vascular smooth muscle. Am J Physiol Heart Ore Physiol. 282:H1571-1583. Lee, C.H., D. Poburko, P. Sahota, J. Sandhu, D.O. Ruehlmann, and C. van Breemen. 2001. The mechanism of phenylephrine-mediated [Ca 2 + ]i oscillations underlying tonic contraction in the rabbit inferior vena cava. J Physiol. 534:641-650. Liu, X., and J.M. Farley. 1996. Acetylcholine-induced chloride current oscillations in swine tracheal smooth muscle cells. J Pharmacol Exp Ther. 276:178-186. Marmy, N . , J. Mottas, and J. Durand. 1993. Signal transduction in smooth muscle cells from human airways. Respir Physiol. 91:295-306. Marthan, R. 2004. Store-operated calcium entry and intracellular calcium release channels in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol. 286:L907-908. Mejia-Elizondo, R., R. Espinosa-Tanguma, and V.M. Saavedra-Alanis. 2002. Molecular identification of the N C X isoform expressed in tracheal smooth muscle of guinea pig. Ann N YAcad Sci. 976:73-76. Montano, L .M. , and B. Bazan-Perkins. 2005. Resting calcium influx in airway smooth muscle. Can J Physiol Pharmacol. 83:717-723. Panettieri, R.A., R.K. Murray, L.R. DePalo, RA. Yadvish, and M.I. Kotlikoff. 1989. A human airway smooth muscle cell line that retains physiological responsiveness. Am J Physiol. 256:C329-335. Pitt, A., and A.J. Knox. 1996. Molecular characterization of the human airway smooth muscle Na + /Ca 2 + exchanger. Am J Respir Cell Mol Biol. 15:726-730. Prakash, Y.S., M.S. Kannan, and G C . Sieck. 1997. Regulation of intracellular calcium oscillations in porcine tracheal smooth muscle cells. Am J Physiol Cell Physiol. 272:C966-975. Prakash, Y.S., M.S. Kannan, T.F. Walseth, and G C . Sieck. 1998. Role of cyclic ADP-ribose in the regulation of [Ca2+]i in porcine tracheal smooth muscle. Am J Physiol. 274:C1653-1660. Prakash, Y.S., C M . Pabelick, M.S. Kannan, and G C . Sieck. 2000. Spatial and temporal aspects of ACh-induced [Ca2+]j oscillations in porcine tracheal smooth muscle. Cell Calcium. 27:153-162. Roux, E. , C. Guibert, J.P. Savineau, and R. Marthan. 1997. [Ca2+]i oscillations induced by muscarinic stimulation in airway smooth muscle cells: receptor subtypes and correlation with the mechanical activity. Br J Pharmacol. 120:1294-1301. Roux, E. , and M . Marhl. 2004. Role of sarcoplasmic reticulum and mitochondria in Ca removal in airway myocytes. Biophys J. 86:2583-2595. 2_|_ Ruehlmann, D.O., C H . Lee, D. Poburko, and C. van Breemen. 2000. Asynchronous Ca waves in intact venous smooth muscle. Circ Res. 86:E72-79. Shieh, C.C., M.F. Petrini, T.M. Dwyer, and J.M. Farley. 1991. Concentration-dependence of acetylcholine-induced changes in calcium and tension in swine trachealis. J Pharmacol ExpTher. 256:141-148. Sims, S.M., Y. Jiao, and Z .G Zheng. 1996. Intracellular calcium stores in isolated tracheal smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 271 :L300-309. Sterk, P.J., E.E. Daniel, N. Zamel, and E E . Hargreave. 1985. Limited maximal airway narrowing in nonasthmatic subjects. Role of neural control and prostaglandin release. Am Rev Respir Dis. 132:865-870. Thirstrup, S. 2000. Control of airway smooth muscle tone. I—electrophysiology and contractile mediators. Respir Med. 94:328-336. Widdop, S., K. Daykin, and LP. Hall. 1993. Expression of muscarinic M 2 receptors in cultured human airway smooth muscle cells. Am J Respir Cell Mol Biol. 9:541-546. 19 Woolcock, A.J., C M . Salome, and K. Yan. 1 9 8 4 . The shape of the dose-response curve to histamine in asthmatic and normal subjects. Am Rev Respir Dis. 1 3 0 : 7 1 - 7 5 . Worth, N.F., B.E. Rolfe, J. Song, and GR. Campbell. 2 0 0 1 . Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins. Cell Motil Cy to skeleton. 4 9 : 1 3 0 - 1 4 5 . Yoshii, A., K. lizuka, K. Dobashi, T. Horie, T. Harada, T. Nakazawa, and M. Mori. 1 9 9 9 . Relaxation of contracted rabbit tracheal and human bronchial smooth muscle by Y - 2 7 6 3 2 through inhibition of C a 2 + sensitization. Am J Respir Cell Mol Biol. 2 0 : 1 1 9 0 - 1 2 0 0 . Yoshimura, H., K.A. Jones, W.J. Perkins, T. Kai, and D.O. Warner. 2 0 0 1 . Calcium sensitization produced by G protein activation in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol. 2 8 1 : L 6 3 1 - 6 3 8 . 20 CHAPTER 2 - THE RELATIONSHIP BETWEEN ASYNCHRONOUS Caz+ WAVES AND FORCE DEVELOPMENT IN INTACT SMOOTH MUSCLE BUNDLES OF THE PORCINE TRACHEA 1 2.1 Introduction As mentioned earlier, repetitive asynchronous C a 2 + waves (ACW) have been reported in airway smooth muscle cells (ASMC) of the murine bronchi stimulated by acetylcholine (ACh) (Bergner and Sanderson, 2 0 0 2 ) . Coincidentally, concomitant narrowing of the bronchiolar lumen was observed with ACh stimulation in these murine ASMC. Even though these authors attempted to relate the frequency of the A C W with force generation, the data were inconclusive due to considerable variation and the paucity of data points. Thus the relationship between ACW occurring in individual intact A S M C and the overall airway smooth muscle (ASM) tissue contraction remains poorly defined. For the purpose of this study, we have developed a novel technique employing confocal imaging of intact A S M bundles from the porcine trachea attached to a tension transducer. In ' A version of this chapter has been published as: Kuo K H , Dai J, Seow CY, Lee C H , van Breemen C. 2003. Relationship between asynchronous C a 2 + waves and force development in intact smooth muscle bundles of the porcine trachea. Am J Physiol Lung Cell Mol Physiol. 285(6):L1345-53. 21 this case, the ASMC are examined at the single-cell level while they reside within their native extracellular matrix with preserved intercellular adhesion or communication. There is minimal perturbation of the muscle bundle during preparation. This novel method of investigating single-cell C a 2 + signaling in the intact airway muscle bundle offers a consistent, stable and physiological preparation for examining ASMC C a 2 + signaling while simultaneously measuring overall force generation by the muscle bundle. The aim of this section is to examine whether agonist-induced A C W occur in ASMC within other segments of the airway by characterizing the ACh-induced C a 2 + signaling in intact ASMC of the tracheal muscle bundles in relation to their force generation. 2.2 Methods 2.2.1 Tissue preparations. Porcine ASM obtained from a local abattoir was used for the experiments. Immediately after removal of the trachea from the pig, it was placed in physiological saline solution (PSS) at 4°C. After the epithelium was removed, smooth muscle strips (~6 x 1.5 x 0.3 mm in dimension) were dissected from the trachea. Each muscle strip contained multiple muscle bundles. The strips were subsequently attached at both ends to aluminum foil clips designed for mounting onto the custom-built setup. 22 2.2.2 Histology and electron microscopy study. Tissues were rinsed in 0.1 M PBS. The tissues were then embedded in Tissue-Tek optimum cutting temperature compound (an embedding medium for frozen tissue specimens), frozen quickly in liquid nitrogen, and stored at -70°C. Eight-micrometer thick sections were cut at -20°C, collected on slides, and fixed with liquid nitrogen-cooled acetone. The sections were then stained with hematoxylin and eosin. Details of the electron microscopy study have been presented previously, and the procedures, reagents, and chemicals used were identical to what has been previously described (Herrera et al., 2002) Images of the cross sections of the muscle cells were obtained with a Phillips 300 electron microscope. 2.2.3 Simultaneous isometric force measurement and confocal microscopy of Ca2+-induced fluorescence. The clipped muscle strips were loaded with Fluo-4 A M (5 uM with 5 uM Pluronic F-127) for 90 min at 25°C and then left to equilibrate for 10 min in normal PSS. Fluo-4 A M is a cell permeable C a 2 + indicator generally used to monitor cytosolic C a 2 + concentration ([Ca2+]j). The affinity of Fluo-4 for C a 2 + is high with a Ka(Ca2+) value of approximately 345 nM (Gee et al , 2000). The Fluo-4 loaded muscle strips were then immediately mounted onto the custom-made stiff force transducer setup for simultaneous isometric force and [Ca2+]i measurements. The employment of a stiff force transducer, the application of firm clipping to secure the tissue, and the use of small-sized muscle strips are all 23 important for minimizing tissue movements during confocal Ca imaging. Inside the organ bath, one end of the tissue was placed over a stiff metal bar mounted on a micromanipulator for adjustment of muscle strip length, and the other end was connected to the lever arm of a servo-controlled force transducer. The lengths of the mounted muscle strips were set approximately to the intact length. Details of the force measurements employed in this study are similar to what has been described previously (Opazo Saez et al., 2000; Herrera et al., 2002). Briefly, the servo-controlled force lever system had a force resolution of 10 u\N. The analog signals were converted to digital signals via a National Instrument analog-to-digital converter and were recorded by a computer. The details of confocal C a 2 + imaging are also similar to what has been described by us previously (Ruehlmann et al., 2000; Lee et al., 2001; Lee et al , 2002b). Briefly, once the muscle strips were isometrically mounted, the changes in [Ca ]i were measured using a Noran Oz laser scanning confocal microscope through either an air x60 (numerical aperture 0.7) or an air x20 (numerical aperture 0.45) lens on an inverted Nikon microscope. The tissue was illuminated using the 488-nm line of an argon-krypton laser, and a high-gain photomultiplier tube collected the emission after it passed through a 525/52 band-pass filter. The measured Fluo-4 fluorescence level (F525) indicates relative [Ca 2 +]j, and thus changes in [Ca 2 +]i are directly reflected by proportional changes in F525. All parameters (laser intensity, gain, etc.) were left unchanged during the experiment. Generally, the acquisition speed was set at 66 ms/frame with two-frame integration resulting in an effective frame rate of 24 133 ms/frame. Comparisons between recordings made at 66 ms/frame and 133 ms/frame were made when necessary to exclude possible sampling artifacts in the case that the sampling speed was insufficient. Initially, all mounted muscle strips were equilibrated in PSS at 37°C and stimulated twice with high-K+ PSS for 5 min each time. Once the muscle strips were fully relaxed after the second high-K+ stimulation, the experimental protocols were applied. 2.2.4 Data analysis. All confocal image analyses were performed in ImageProPlus using customized routines written in Visual Basic. The muscle strips with recordings that showed significant horizontal and/or vertical movement artifacts were excluded from the study. To obtain data on recruitment of cells during ACh stimulation, a fixed field of view under the x20 lens was chosen, and the number of cells responding with C a 2 + wave(s) was recorded at each concentration of ACh. Recruitment of cells was calculated as a fraction of the number of cells that responded to the highest ACh concentration (100 uM). Further analysis of wave parameters was performed using a three-pixel-wide line along the longitudinal axis of a single cell. The resulting x-t plot revealed the point of origin as well as the progression of the apparent"Ca2+ wave". The frequency of the A C W was determined by counting the number of waves occurring during a period of 40 s. The amplitude of the A C W reflected the difference between the peak F525 of individual C a 2 + spikes in the waves and the pre-stimulation baseline F525. The representative fluorescence traces shown in this report reflect the averaged 25 fluorescence signals from a 3x3-pixel region (1.36 |im2) of the ribbon-shaped ASMC. The fluorescence level (F525) derived in each region is linearly proportional to the [Ca2 +]i in that region in such a fashion that any change in [Ca2 +]i would be proportionally reflected in the change in F525 level. Such a linear relationship between F525 and [Ca2+]j is not necessarily an absolute since it can be skewed if parameters such as intracellular temperature or pH level change significantly during the course of the experiment. Attempts were made in the experiment to minimize the change of these intracellular temperatures and pH with the use of a precision bath temperature control device and extracellular pH buffer, respectively. All summarized data are presented as means ± SE. For numerical analysis, all data were analyzed in Excel or Sigma Plot using the appropriate statistical tests. A paired Student's t-test was used for comparisons. A value of P < 0.05 was considered significant. The n values indicated for force development experiments represent the number of tracheal muscle strips from the same number of animals used, and the n values indicated for the C a 2 + studies represent the number of ASMC analyzed from the specified numbers of tracheal muscle strips. 2.2.5 Solutions and chemicals. Normal PSS containing (in mM) 140 NaCl, 5 KC1, 1.5 CaCl 2 , 1 MgCl 2 , 10 glucose, and 5 HEPES, pH 7.4, at 37°C was used for all studies. High-K+ (80 mM extracellular K + ) PSS was identical in composition to normal PSS with the exception of 26 (in mM) 65 NaCl and 80 KC1. Fluo-4 A M and Pluronic F-127, purchased from Molecular Probes, were dissolved in DMSO. Stocks of ACh (Sigma) were prepared in normal PSS, and stocks of nifedipine (Sigma) were prepared in ethanol. 2.3 Results A C Figure 2.1. Structure of A S M C in an intact porcine tracheal muscle strip. A. Histological (hematoxylin and eosin stain) section of a tracheal muscle strip (courtesy of Dr. Kuo-Hsing Kuo). 27 The muscle strip is free of pseudostratified columnar epithelial layer and contains multiple muscle bundles. The scale bar indicates 10 um. B. Electron microscopic image shows cross section of the tracheal muscle strip. It is at the junction between two clusters of ASMC (2 muscle bundles) that are separated by connective tissues. The scale bar indicates 2 urn. C. These ASMC contain intact intercellular gap junctions (arrow). The scale bar indicates 1 um. 2.3.1 Organization of tracheal smooth muscle. Structural aspects of the porcine tracheal smooth muscle strips used in this study are shown in Fig. 2.1. These images reveal that the tracheal muscle strip is devoid of prominent pseudostratified columnar epithelium (Fig. 2.1 A) and contains several smooth muscle bundles (Fig. 2.1 A and B). In addition, as shown in the electron microscopic image in Fig. 2.1C, these intact ASMC possess intact intercellular gap junctions. 2.3.2 ACh-induced Ca2* signals in intact ASMC of porcine trachea. As revealed by the basal fluorescence level (F525) under confocal microscopy, the intact A S M C of the porcine trachea displays the expected long, ribbon-shaped appearance with a width of ~3 um (Fig. 2.2A). When stimulated with 5 uM ACh, all the visualized intact ASMC initially responded synchronously with a large C a 2 + wave (Fig. 2.2A). This initial large C a 2 + wave resulted in a local [Ca 2 +]i elevation that was transient and dissipated within -10 -15 s (Fig. 2.3). After this initial large C a 2 + wave, repetitive intracellular C a 2 + waves were observed traveling along the longitudinal axis of the ribbon-shaped cell. In contrast to the first C a 2 + wave, the subsequent 28 repetitive Ca2+waves, as depicted in Fig. 2.3, produced [Ca 2 +]i elevations that were smaller in amplitude and shorter in duration. As shown in the x-t plot in Fig. 2.2B, which displays the change in F525 (oc [Ca 2 +]i) over a longitudinal section of the cell over time, a rapid rise in [Ca2+]; was first seen on the left side of this cellular segment and subsequently propagated to the right side of the cellular segment in an apparent wave-like fashion. TTiese apparent C a 2 + waves continue to recur in the same cell for as long as the agonist is present. Figure 2.2A shows that with the exception of the initial C a 2 + wave, the subsequent oscillatory C a 2 + signals did not occur in a synchronized fashion between neighboring cells. Thus ACh (5 J I M ) induces A C W in intact A S M C of porcine trachea. 29 (A) Os 1s 10s 11s 12s 13s 5uM acetylcholine x-tplot Figure 2.2. A C h induces A C W in the A S M C of intact porcine tracheal muscle bundles. A. These time series images depict the ACh-induced changes in [Ca 2 +]i over time (as revealed by the F525 fluorescence level) in the ASMC within this selected field of view. At 1-s post-ACh application, the A S M C responded with elevation in [Ca 2 +]j that was synchronized between different cells. At the 10-s time mark when the initial [Ca 2 +]j elevation had subsided, the cells began to initiate oscillatory C a 2 + signals (recurring C a 2 + waves) in a nonsynchronized fashion since the rise in F525 level was not simultaneous in all cells in the latter 4 time series images. B . The x-t plot is a 3-pixel (1.36-um)-wide line scan that depicts the changes in F525 ([Ca 2 +]0 over time in this longitudinal line section of the ribbon-shaped A S M C stimulated with 5 uM ACh. The still frame image (left) delineates the placement of the line, and the x-t plot derived from the line is shown (right). The x-t plot shows recurring C a 2 + waves that are initiated on one end (Xo) of the scanned cellular segment and subsequently propagated to the other end (Xi). C. F525 changes in a two 3><3-pixel intracellular region (1.36 um2) from two neighboring A S M C are depicted in the traces (right). The ACh-induced A C W in the two neighboring cells occur at different frequencies. 30 2+ Figure 2.3. Temporal association between ACh-induced force generation and Ca signal. The representative experimental traces (n = 6 muscle strips) shown depict simultaneous force generation by the muscle strip and [Ca2+]; changes in ASMC of the same intact muscle strip. The ACh-mediated induction of A C W at the cellular level is temporally associated with the induction of tonic contraction of the tracheal smooth muscle. 2.3.3 Relationship between repetitive asynchronous Ca2+ wave and force generation. Simultaneous measurements of force and [Ca2+]j showed that 5 uM ACh nearly simultaneously initiated the development of force (tonic contraction) and the generation of A C W in individual intact A S M C residing within the same muscle strip (Fig. 2.3). In other words, the induction of the cellular A C W is temporally closely associated with the induction of tissue force generation, which typically lags ~0.5 - 1.0 s behind the initial generation of the C a 2 + signal (n = 6 muscle strips). It is important to note that synchronized non-wavelike oscillation in [Ca 2 +]i usually 31 result in oscillatory force generation, whereas the asynchronous nature of the A C W observed in the intact A S M C accounts for the tonic nature of the force generation by the entire muscle strip. 100 t Time (s) Figure 2.4. ACh induces concentration-dependent contraction of the tracheal smooth muscle. In the representative trace (n = 6 muscle strips), application of increasing concentrations of ACh over the selected concentration range (~1 nM - 100 uM) results in graded increases in the amplitude of the tonic contraction, which eventually reaches a maximum. 32 7 0 ? 6 0 S S O g 4 0 O 3 0 2 0 1 0 0 D. c .2 _ 9 • S s , > c 7 ID m 5 §>K! 3 p ~~ 1 - 8 - 7 - 6 Log [Ach] { - 1 0 9 0 . 3 0 . 2 S C 0 . 1 5 5 • 0 . 1 tT £ o.os L L 0 E. 6 0 a I S 2 0 TO v P C L 0 - 8 - 7 - 6 - 6 Log [Ach] i < f — 100 '—' 8 0 +J C «j 6 0 Si g 40 O 20 a: 0 3 0 - 8 - 7 - 6 - 5 - 4 Log [Ach] o C 2 5 3 m 2 0 C N K> 1 5 CD Q) < 5 5; s - 8 - 7 - 6 - 6 Log [Ach] - 1 0 Log [Ach] - 8 - 7 - 6 - 5 - 4 - 3 Log [Ach] G. 80 -70 -60 -| » -M 40 -o 30 -LL 20 10 ft ; i * 0.15 16 0.3 Frequency (Hz) 32 Average [Ca2*], (F525 unit) 50 100 Recruitment (%) Figure 2.5. Concentration-response relationships of ACh-induced force generation and A C W are depicted. These concentration-response curves are generated from simultaneous force and [Ca 2 + ]i measurements of intact tracheal muscle bundles (110 cells in 6 tracheal muscle strips from 6 animals). A. Concentration dependence of the magnitude of ACh-induced tonic contraction (n = 6 muscle strips). B. Concentration dependence of the frequency of ACh-induced A C W (n = 110 cells). The determination of the frequency parameter is described in METHODS. C. Concentration dependence of the degree of cell recruitment by ACh to initiate C a 2 + wave(s) (n = 110 cells). The quantification of the degree of cell recruitment is described in METHODS. D. Trough elevation refers to the interspike baseline [Ca 2 + ]i elevation (see RESULTS for details) (n = 110). The baseline interspike [Ca 2 +]t elevation as a function of ACh concentration is depicted here. E. Concentration dependence of the amplitudes of the ACh-mediated A C W (n = 110 cells). F. Concentration dependence of the average [Ca2+]j level 33 (time average of the oscillatory Ca signal) of ACh-mediated A C W (n = 110 cells). G. Dependence of force development on the frequency of A C W (circular marker), percent recruitment of cells (square marker), and average [Ca2+]; level (triangular marker). Furthermore, it was found that increasing concentrations of ACh ranging from 0.01 uM to 100 uM elicited tonic contraction of increasing amplitude (Figs. 2.4 and 2.5A). To determine the relationship between the C a 2 + waves and force development, concentration dependences of selected aspects of the A C W were compared with the concentration dependence of force generation in ACh-stimulated tissues. As shown in the concentration-response curves in Fig. 2.5B, increasing concentrations of ACh over the concentration range of 0.01 uM to 100 uM are correlated with increasing frequency of ACW, reaching 0.28 ± 0.01 Hz at 100 uM ACh (n = 110 cells from 6 muscle strips). This observation suggests that the message involved in regulating force development is encoded in the frequency domain of the ACW. Increasing the concentration of ACh over the lower concentration range from 0.01 uM to 0.1 uM resulted in increased recruitment of cells that display C a 2 + waves (Fig. 2.5C). It thus appears that incremental recruitment of cells is involved in the modulation of force generation at low-stimulation intensity. A third parameter that was assessed is baseline [Ca2+]j elevation. As shown in Fig. 2.3, with higher concentrations of ACh and consequently higher frequency of ACW, the interspike trough [Ca2+]; frequently did not return to the prestimulation baseline level. The baseline [Ca2+]j elevation is defined as the average difference between the trough F525 and 34 the prestimulation resting F525. As shown in Fig. 2.5D, baseline [Ca ]i elevations were significant and showed concentration-dependent increases only over the higher concentration range of ACh (-10-100 uM). It is important to note that the baseline elevation of [Ca2+]i at its highest level was only a quarter of the averaged amplitude of ACW. In contrast to the other assessed parameters, the amplitude of ACh-mediated A C W did not display a statistically significant concentration dependency (Fig. 2.5E), indicating that amplitude is not involved in the modulation of the tonic contraction. To obtain the average [Ca 2 + ]i elevation at different ACh concentrations, the oscillatory C a 2 + signal was averaged over time (the entire recording interval). Figure 2.5F shows that increasing ACh concentration results also in increasing average [Ca ] i elevation over time in a concentration-dependent manner. Finally, force development was plotted against frequency of ACW, average [Ca2+];, and percentage of cell recruitment (Figure 2.5G). 2.3.4 Role of L-type voltage-gated Ca2+ channels in ACh-induced repetitive asynchronous Ca2+ waves. C a 2 + influx through the L-type voltage-gated C a 2 + channels (VGCC) is known to play an important role in Ca2+signaling in A S M (Bourreau et al., 1993; Darby et al., 2000). More specifically, it was reported that ongoing ACh-mediated recurring C a 2 + waves observed in enzymatically dissociated ASMC from the porcine trachea could be abolished with a high dose of nifedipine (Prakash et al., 1997). To assess the contribution by L-type V G C C , nifedipine 35 was added to ACh-stimulated tissues exhibiting ongoing cellular A C W and tonic contraction. As shown in Fig. 2.6A, in contrast to what was reported before in enzymatically isolated cells, nifedipine (10 uM) reduced the frequency of ACh (5 u.M)-induced A C W from 0.24 ± 0.02 Hz to 0.17 ± 0.01 Hz (P < 0.002, n = 54 cells from 4 muscle strips) but did not abolish the ongoing A C W in intact ASMC (even after 15 min of drug treatment). In terms of the average [Ca2+]i levels, nifedipine reduced ACh (5 (xM)-induced average [Ca2+]i elevation from 17.6 ± 2.9 F525 units to 11.2 ± 2.1 F525 units (P < 0.02, n = 54 cells from 4 tissues), which is a 36% decrease in average [Ca2+]; elevation. Simultaneously recorded force data revealed that 10 uM nifedipine caused a 32.8 ± 2.9% (P < 0.01, n = 4 tissues) inhibition of the tonic contraction elicited with 5 u,M ACh (Fig. 2.6B). It is important to note that at 10 uM, nifedipine was able to completely abolish the contraction induced by 80 mM K + PSS in the tracheal muscle bundle. To examine the possibility that the high-dose nifedipine may produce non-specific inhibition beyond the blockade of the L-type V G C C , we used 100 nM nifedipine, and it produced a similar degree of inhibition (33.0 ± 3.3%, P = 0.0096, n = 3 animals) of ACh-induced tonic contraction as produced by 10 uM nifedipine. These findings indicate that C a 2 + influx through the L-type V G C C does contribute to, but is nonetheless not essential for, the generation of ACh-induced A C W and ACh-induced tonic contraction of intact trachea smooth muscle bundles. The 32% reduction in force was similar to the 29% decrease in frequency. The role of the L-type V G C C is ascertained when both low-dose (100 nM) and high-dose (10 uM) nifedipine produced 36 comparable partial inhibitory effects on ACh-induced A C W and tonic contraction. Figure 2.6. Effects of nifedipine (NIF) on ongoing ACh-mediated A C W and force generation. A. In the displayed representative [Ca2+]i trace (n = 54 cells), application of 10 uM NIF reduced the frequency of ongoing ACh-mediated A C W but did not abolish them. B. representative trace shown (n = 4 muscle strips) reveals that application of 10 fiM NIF partially inhibited ACh-mediated force generation. 2.4 Discussion The tracheal smooth muscle strip appears to be an ideal preparation for simultaneous 37 confocal Ca imaging and recording of force generation in ASM. This preparation contains an abundance of intact ASMC organized in bundles. To enhance the purity of the preparations, the epithelium has been physically removed, ensuring that there is no active epithelial influence on A S M function during the course of the experiment. Conceivably, there are intact nerve endings from the parasympathetic nervous system present in the tracheal smooth muscle strips. However, aside from the use of high-K+ PSS, which can stimulate release of ACh from the nerve endings, our experiments did not involve conditions that would lead to nerve stimulation. For these reasons, we assume that our experimental data were the consequence of activation of muscarinic receptors located on the tracheal smooth muscle. The findings presented in this study reveal that ACh induces A C W in the intact A S M C of the porcine trachea. This pattern of C a 2 + signaling has also been recently described in intact A S M C of the terminal bronchiole in a murine lung slice (Bergner and Sanderson, 2002). In murine ASMC, ACh initially induced a large wave-like C a 2 + transient, which was followed immediately by the appearance of ACW. The appearance of the C a 2 + signal coincided with the development of tonic constriction of the terminal bronchiole airway. The frequency of the A C W reached 0.33 Hz with 100 fiM ACh in the murine bronchiole, which is comparable with the frequency of 0.28 Hz attained with 100 fiM ACh in ASMC from porcine trachea obtained in the present study. Together, these observations demonstrate several important aspects regarding C a 2 + signaling in 38 A S M C at both the tracheal and bronchiolar levels. First, they confirm the ACh-induced recurring C a 2 + waves reported in enzymatically dissociated ASMC, although as discussed later, the ability of the A C W in the intact A S M C to persist in the presence of high-dose nifedipine is a characteristic not shared by its enzymatically isolated counterpart (Prakash et al., 1997). Interestingly, A C W are seen in these intact ASMC despite the presence of intact gap junctions (Fig. 2.1). Second, despite the oscillatory nature of the C a 2 + signal at the cellular level, the stimulated constriction of the airway at the tissue level is tonic. This can be explained by the observation that the A C W do not propagate from cell to cell and are not synchronized between neighboring cells. This property allows for tonic force generation as a result of intermittent cellular C a 2 + signals. Third, it is important to note that similar A C W have been described in intact vascular smooth muscle cells (VSMC) from a variety of blood vessels (lino et al., 1994; Ruehlmann et al., 2000; Lee et al., 2002a). It therefore appears that A C W represent a fundamental mechanism for stimulation of tonic smooth muscle contraction. The main function of ACh-induced A C W is to signal contractile activation in the ASMC. Our findings provide strong evidence that this, indeed, is the case. The observation that the two processes, A C W and tonic contraction, are closely associated temporally, with the Ca signals preceding the force generation by 0.5 s, supports this speculation. To further examine how the ACh-induced A C W at the cellular level can regulate force generation at the A S M tissue level, the 39 concentration dependences of selected aspects of the A C W were studied and correlated with the concentration dependence of force generation. It was found that the amplitude of the A C W remains constant regardless of the ACh concentration. This is similar to what was reported in both intact bronchial smooth muscle cells (Bergner and Sanderson, 2002) and enzymatically dissociated tracheal smooth muscle cells (Prakash et al , 2000). It is, therefore, unlikely that the amplitude domain of the A C W is involved in regulating concentration-dependent contraction. The observed elevation of interspike baseline [Ca2 +]i at the high concentrations of ACh (10 and 100 U.M) is similar to what has been previously observed by Sieck and coworkers (Sieck et al., 1997) in isolated ASMC. However, in contrast to the isolated ASMC, the average elevation in baseline [Ca2+], is small (approximately one-quarter) compared with the amplitude of the C a 2 + spikes that constitute the A C W in intact ASMC. Therefore, it is likely that even over the high concentration range of ACh, the degree of contractile activation is more dependent on the frequency C a 2 + waves than the much smaller interspike [Ca2 +]i elevations. Furthermore, as shown in Fig. 2.5, significant interspike (trough) [Ca2+]; elevation occurs only above the 1 uM level of ACh. However, without the input of interspike [Ca2+]j level, 74% of maximal force modulation has already been completed at 1 | i M ACh stimulation, apparently achieved through 100% of cell recruitment and 70% of maximal frequency modulation. We thus speculate that the baseline [Ca2+]; elevation observed only at very high ACh concentrations plays little or no role in the physiological regulation of A S M contractility. As mentioned, the recruitment of 40 A S M C initiating Ca waves was observed over the lower concentration range of ACh. This implies that the intact A S M C may have differential sensitivity of the muscarinic receptors to ACh and/or differential effectiveness in the process of initiating C a 2 + waves. Interestingly, a similar observation of differential cell recruitment has been made in V S M C of intact rabbit inferior vena cava (Ruehlmann et al., 2000). In the A S M , it appears that differential recruitment of the intact A S M C initiating C a 2 + waves may be important in regulating A S M contractility over the lower concentration range of ACh stimulation. As for the frequency of the ACW, they exhibited a concentration dependence that was parallel with force generation. Together, our findings provide strong evidence that the A C W underlie ACh-mediated tonic contraction of the tracheal smooth muscle. More specifically, with increasing intensity of ACh stimulation, the graded A S M contraction of the intact A S M tissue is achieved first by differential recruitment of the intact A S M C to initiate C a 2 + signals and second by enhancement of the frequency of the A C W and elevation of interspike [Ca2+]; once the cells are recruited. This, however, does not mean that force development in these ASMC is a frequency-sensitive process, although it remains a possibility, since enzymes that are sensitive to the frequency of recurring C a 2 + waves have been described (De Koninck and Schulman, 1998). Alternatively, as shown in Fig. 2.5F, it is plausible that the graded force generation is achieved on the basis of the average amount of Ca2+exposed to a cell. In this instance, the average [Ca2+]; over time is determined by the frequency of A C W and the interspike [Ca 2 +]i elevation. Therefore, by increasing the 41 frequency of the A C W and elevating the interspike (trough) Ca level, we expose individual ASMC to higher average [Ca2+]i over time. The spatiotemporal pattern of the C a 2 + signal also provides mechanistic insight into the C a 2 + signal at A S M C (Berridge and Dupont, 1994; Lee et al., 2001). In a variety of excitable cells, intracellular C a 2 + waves are initiated by an increase in local [Ca2+]i as a result of C a 2 + influx and/or localized endoplasmic/sarcoplasmic reticulum (ER/SR) C a 2 + release that triggers regenerative C a 2 + release from ER/SR (Lee et al., 2002a). The wave-like pattern is a reflection of the SR-mediated C a 2 + release in one cellular region triggering Ca2+-induced C a 2 + release in an adjacent cellular region. Thus the A C W observed in A S M C of the porcine trachea appear to be the result of repetitive "waves" of SR-mediated C a 2 + release as described in VSMC. This is consistent with the findings by Bergner and Sanderson that the A C W in the bronchiole can be abolished by either pre-depletion of the SR C a 2 + store or blockade of sarco(endo)plasmic reticulum Ca2+-ATPase (Bergner and Sanderson, 2002). However, with repetitive SR C a 2 + release, loss of C a 2 + to the extracellular space is inevitable, and C a 2 + influx must occur to replenish the SR C a 2 + store to maintain ongoing recurring C a 2 + waves (Prakash et al., 1997; Lee et al , 2001). In enzymatically dissociated ASMC from porcine trachea, it was found that ongoing ACh-mediated recurring C a 2 + wave could not persist when L-type V G C C were blocked by 100 nM nifedipine (Prakash et al., 1997). This would suggest that C a 2 + influx through the 42 L-type V G C C is crucial in replenishing the loss of intracellular Ca and in maintaining the SR C a 2 + store. However, in our intact A S M C of the intact porcine tracheal muscle bundle, it was found that a high concentration (10 uM) of nifedipine, maximally effective in blocking V G C C , only partially inhibited the frequency of the ongoing A C W and reduced the tonic contraction by 32.8%. This relatively small inhibitory effect is comparable with what was described previously (Janssen, 2002). The attenuation in frequency may be the consequence of a slightly reduced rate of SR C a 2 + store refilling in the absence of stimulated C a 2 + entry through L-type V G C C . Given that the apparent A C W likely represents recurring release of SR C a 2 + and the free luminal SR [Ca2+] can modulate the open probability of SR C a 2 + release channels (Meldolesi and Pozzan, 1998; Verkhratsky and Toescu, 1998), a decrease in the rate of SR Ca refilling may lead to reduced frequency of SR C a 2 + release, and thus reduced frequency of the ACW. Because removal of external C a 2 + abolished maintained ACh-induced contraction (Nouailhetas et al , 1988), C a 2 + entry through other pathway(s) is important for replenishing the SR C a 2 + level in ASM. This nifedipine-resistant C a 2 + entry pathway(s) is capable of supporting ongoing A C W and -67% of the tonic contraction elicited by ACh. Possible nifedipine-resistant C a 2 + entry pathways include receptor-operated channels (ROC, a type of non-selective cation channels whose activation required ligand binding to the receptor), store-operated channels (SOC, a type of non-selective cation channels that are activated as a direct result of SR Ca store depletion), reverse-mode Na + /Ca 2 + exchange, and other subtypes of V G C C such as the 43 T-type V G C C (Fleischmann et al., 1997; Janssen et al., 1997; Wang et al., 1997; Barritt, 1999; Wang and Kotlikoff, 2000; Lee et al., 2001; Sweeney et al., 2002). The observe difference between isolated myocytes and intact A S M could be due to enzymatic alteration of both V G C C and ROC/SOC or loss of cell contact. The findings presented in this chapter clearly implicate A C W in the regulation of contractile activity of tracheal smooth muscle. In relation to pulmonary disease, such as asthma, aberrations in C a 2 + signaling of A S M have been somewhat overlooked in the past because of the failure of V G C C blockers to attenuate airway hyperresponsiveness clinically (Patel and Al-Shamma, 1982; Walters et al., 1984; Fish and Norman, 1986; Henderson and Costello, 1988). However, the unveiling of A C W as a fundamental mode of C a 2 + signaling in A S M and our finding that these A C W can persist under maximal blockade of L-type V G C C have shed new light on this matter. Further studies on the mechanism of the A C W in porcine tracheal smooth muscle cells as described in the next chapter together with an examination on how C a 2 + signaling in diseased airways differ from the typical A C W observed in healthy airways may reveal novel insights into the pathophysiology of common airway diseases such as asthma. 2.5 Bibliography Barritt, G.J. 1999. Receptor-activated Ca inflow in animal cells: a variety of pathways tailored to meet different intracellular C a 2 + signalling requirements. Biochem J. 337 ( Pt 2):153-169. Bergner, A., and M.J. Sanderson. 2002. Acetylcholine-induced Calcium Signaling and Contraction of Airway Smooth Muscle Cells in Lung Slices. J. Gen. Physiol. 119:187-198. Berridge, M.J., and G Dupont. 1994. Spatial and temporal signalling by calcium. Curr Opin Cell Biol. 6:267-274. Bourreau, J.P., C.Y. Kwan, and E.E. Daniel. 1993. Distinct pathways to refdl ACh-sensitive internal C a 2 + stores in canine airway smooth muscle. 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C a 2 + oscillations, gradients, and homeostasis in vascular smooth muscle. Am J Physiol Heart Circ Physiol. 282:H1571-1583. Lee, C.H., D. Poburko, P. Sahota, J. Sandhu, D.O. Ruehlmann, and C. van Breemen. 2001. Trie mechanism of phenylephrine-mediated [Ca2+]i oscillations underlying tonic contraction in the rabbit inferior vena cava. J Physiol. 534:641-650. Lee, C.H., R. Rahimian, T. Szado, J. Sandhu, D. Poburko, T. Behra, L. Chan, and C. van Breemen. 2002b. Sequential opening of IP3-sensitive C a 2 + channels and SOC during alpha-adrenergic activation of rabbit vena cava. Am J Physiol Heart Circ Physiol. 282:H1768-1777. Meldolesi, J., and T. Pozzan. 1998. The endoplasmic reticulum Ca2+ store: a view from the lumen. Trends Biochem Sci. 23:10-14. Nouailhetas, V.L., N.J. Lodge, C H . Twort, and C. Van Breemen. 1988. The intracellular calcium stores in the rabbit trachealis. Eur J Pharmacol. 157:165-172. Opazo Saez, A . M . , C.Y. Seow, and RD. Pare. 2000. Peripheral airway smooth muscle mechanics in obstructive airways disease. Am J Respir Crit Care Med. 161:910-917. Patel, K.R., and M . Al-Shamma. 1982. Effect of nifedipine on histamine reactivity in asthma. Br Med J (Clin Res Ed). 284:1916. Prakash, Y.S., M.S. Kannan, and G.C. Sieck. 1997. Regulation of intracellular calcium oscillations in porcine tracheal smooth muscle cells. Am J Physiol Cell Physiol. 272:C966-975. 46 Prakash, Y.S., C M . Pabelick, M.S. Kannan, and G C . Sieck. 2000. Spatial and temporal aspects of ACh-induced [Ca2+]; oscillations in porcine tracheal smooth muscle. Cell Calcium. 27:153-162. Ruehlmann, D.O., C H . Lee, D. Poburko, and C. van Breemen. 2000. Asynchronous Ca waves in intact venous smooth muscle. Circ Res. 86:E72-79. Sieck, G.C., M.S. Kannan, and Y.S. Prakash. 1997. Heterogeneity in dynamic regulation of intracellular calcium in airway smooth muscle cells. Can J Physiol Pharmacol. 75:878-888. Sweeney, M . , S.S. McDaniel, O. Platoshyn, S. Zhang, Y. Yu, B.R. Lapp, Y. Zhao, RA. Thistlethwaite, and J.X. Yuan. 2002. Role of capacitative C a 2 + entry in bronchial contraction and remodeling. JAppl Physiol. 92:1594-1602. Verkhratsky, A., and E. Toescu. 1998. Integrative Aspects of C a 2 + signalling. Plenum Press, New York. 408 pp. Walters, E.H., J. Banks, A. Fennerty, and B.H. Davies. 1984. Effects of calcium channel blockade on histamine induced bronchoconstriction in mild asthma. Thorax. 39:572-575. Wang, Y.X., B.K. Fleischmann, and M.I. Kotlikoff. 1997. M2 receptor activation of nonselective cation channels in smooth muscle cells: calcium and G;/G 0 requirements. Am J Physiol. 273:C500-508. Wang, Y.X., and M.I. Kotlikoff. 2000. Signalling pathway for histamine activation of non-selective cation channels in equine tracheal myocytes. J Physiol. 523 Pt 1:131-138. 47 CHAPTER 3 - THE MECHANISM OF ACH-INDUCED ASYCHRONOUS CALCIUM WAVES IN THE PORCINE TRACHEAL MUSCLE BUNDLE 2 3.1 Introduction In the previous chapter, I have demonstrated that smooth muscle cells of the intact porcine trachea respond to cholinergic stimulation with repetitive asynchronous C a 2 + waves (ACW). Functionally, these A C W were found to be responsible for the development of force in the tracheal smooth muscle cells (TSMC). However, the detailed mechanism for the generation of ACh-induced A C W and tonic contraction has not been elucidated in the smooth muscle cells of the intact tracheal muscle bundle. In enzymatically-isolated porcine TSMC, ACh-induced repetitive C a 2 + waves have been described and their mechanism has been extensively studied. It was found that these agonist-induced repetitive C a 2 + waves are produced by sarcoplasmic reticulum (SR)-mediated C a 2 + release (Chopra et al., 1991; Liu and Farley, 1996; Kannan et al., 1997; Prakash et al., 1997; Sieck et al., 1997; Prakash et al., 2000). More specifically, C a 2 + release through the 2 A version of this chapter has been published as: Dai JM, Kuo KH, Leo JM, van Breemen C, Lee CH. 2006. Mechanism of ACh-induced asynchronous calcium waves and tonic contraction in porcine tracheal muscle bundle. Am J Physiol Lung Cell Mol Physiol. 290(3):L459-69. 48 inositol-l,4,5-trisphosphate sensitive SR Ca release channels (IP3R) is important in initiating the C a 2 + waves while C a 2 + release through the ryanodine-sensitive SR C a 2 + release channels (RyR) is responsible for the recurrent wave generation (Chopra et al., 1991; Liu and Farley, 1996; Kannan et al., 1997). Furthermore, extracellular C a 2 + entry through the L-type V G C C is the main pathway utilized for refilling the SR C a 2 + store and the maintenance of the repetitive cycles of SR C a 2 + release in these cells (Prakash et al., 1997). However, our initial investigation into the mechanism of the A C W of the intact porcine TSMC revealed a crucial difference from the isolated cell preparation. As shown in chapter 2, unlike the enzymatically-isolated TSMC, Ca influx through the L-type V G C C is not obligatory for maintaining the A C W of the TSMC of the intact tissue as blockade of these channels did not abolish the ongoing A C W or the tonic contraction stimulated with ACh. Such a significant difference in the phenotypic characteristics between freshly dissociated cells and the intact tissue may be the result of the disruption of crucial intercellular communication or may reflect damage of important surface proteins on the TSMC due to non-specific enzymatic digestion (Ives et al., 1978). In any case, this is a crucial observation indicating that essential elements of the airway smooth muscle (ASM) Ca waves have been missed by previous studies. Therefore, the detailed mechanism for the generation of A C W in intact tracheal muscle bundle requires further investigation. In this part of the thesis, I examine the mechanism for the generation of ACh-induced A C W 49 in the TSMC of the intact porcine tracheal muscle bundle. The focus of this study is to identify the Ca2+-transport molecules involved in the generation and the maintenance of ACh-mediated ACW. 3.2 Materials and Methods 3.2.1 Tissue preparations. Porcine trachea obtained from a local abattoir was placed in physiological saline solution (PSS) at 4°C. Tracheal smooth muscle strips (~6 x 1.5 x 0.3 mm in dimension) free of epithelium and connective tissue were isolated from the trachea. The tracheal muscle strips were subsequently attached at both ends to aluminum foil clips designed for mounting onto the custom-built setup. 3.2.2 Cell permeabilization. Tracheal muscle strips were permeabilized using 10 U.M digitonin in an intracellular substitution solution (refer to Solutions and Chemicals) for 10 min. The experiments were performed in intracellular substitution solution for permeabilized tracheal muscle strips. Successful permeabilization of tracheal muscle bundle was verified by the observation of force generation following either the application of inositol 1,4,5-trisphosphate (IP3) or increased extracellular C a 2 + concentration at the end of each experiment. 50 3.2.3 Isometric force measurement. The porcine tracheal muscle strips were attached to an isometric force transducer and equilibrated in PSS at 37°C for 1 hour. During this time, the resting tension was maintained at 0.3 g. Exchange of bathing solutions was accomplished by simultaneously draining and refilling the tissue bath. The muscle strips were stimulated twice with 80 mM K + PSS for 5 min each time. The experiment protocols were applied following complete relaxation of the tissue from the second dose of 80 mM K + stimulation. Chart v3.4.5 (ADIinstruments) was employed for data acquisition and analysis. 3.2.4 Confocal Imaging. The details of the confocal C a 2 + imaging method have been described previously in chapter 2. Briefly, the clipped tracheal smooth muscle strips were loaded with Fluo-4 A M (5 uM, with 5 p M pluronic F-127) for 90 min at 25°C and then left to equilibrate for 10 min in normal PSS. They were then isometrically mounted onto the custom-made stiff force transducer setup for intracellular C a 2 + concentration ([Ca2+]i) measurements. The changes in [Ca2+]j were measured using an inverted Leica TCS SP2 AOBS, fast laser scanning confocal microscope with an air x 10 (numerical aperture 0.3) lens. Unlike the imaging system used in the previous study, this system incorporates the acusto-optical beam splitter (AOBS) for reflecting excitation light and transmitting emitted fluorescence light, and the SP prism spectrophotometer for detection of fluorescence emission. These two elements allow me to maximize signal collection from the porcine tracheal muscle by electronically 51 program the range of wavelengths of interest instead of using the standard fixed optical filter. The tissue was illuminated using the 488 nm line of an argon-krypton laser and a high-gain photomultiplier tube collected the emission at wavelengths between 505 nm and 550 nm. The acquisition rate was 3 frame/s. The measured changes in Fluo-4 fluorescence level are proportional to the relative changes in [Ca2+];. All parameters (laser intensity, gain etc.) were maintained during the experiment. 3.2.5 Data analysis. All confocal image analysis was performed in ImageProPlus using customized routines written in Visual Basic. Analysis of frequency of the A C W was performed using a 3 pixel-wide line along the longitudinal axis of a single cell. The frequency of the A C W was determined by counting the number of waves occurring during a period of 50 s. The amplitude of the A C W reflects the difference between the peak fluorescence of individual C a 2 + spikes in the A C W and the pre-stimulation baseline level. The fluorescence level derived in each region is linearly proportional to the [Ca2+]i in that region in such a fashion that any fluctuation in [Ca2+]j would be proportionally reflected in the changes in fluorescence. All summarized data are presented as mean ± SEM. For numerical analysis, all data were analyzed in Excel or Sigma Plot using the appropriate statistical tests. Paired student's t test was used for comparisons. A value of P < 0.05 was considered significant. The n-values 52 indicated for contraction experiments represent the number of animals studied and the n-values indicated for the C a 2 + studies represent the number of TSMC studied from the specified numbers of animals. For each study protocol, only one muscle strip from each animal is used and the number of tissues indicated therefore reflects the number of animals. For the analysis of the C a 2 + signals, when the data obtained from individual TSMC are pooled together from different animals, ANOVA is performed and variance is reported when significant. In our current study, no significant variance was found. 3.2.6 Solutions and chemicals. Normal PSS containing (in mM): 140 NaCl, 5 KC1, 1.5 CaCl 2 , 1 MgCl 2 , 10 glucose, 5 HEPES, (pH 7.4 at 37°C) was used for all the studies. High K + (80 mM extracellular K + ) PSS was identical in composition to normal PSS with the exception of (in mM) 65 NaCl and 80 KC1. Zero-Ca 2 + PSS and zero-Na+ PSS were prepared in the same way as normal PSS but CaCl2 was replaced with 1 mM EGTA, and Na + was replaced with equimolar of A^-methyl-D-glucamine (NMDG +). The intracellular substitution solution used contained (in mM): 130 KC1, 10 NaCl, 5 K 2 H P 0 4 , 5.6 glucose, 1 MgS0 4 , 5 Tris-succinate, 1 ATP, 75 EGTA uM, 20 HEPES (pH 7.0 at 37°C). Fluo-4 A M , pluronic F-127, and 2',4'-dichlorobenzamil (2',4'-DCB) were purchased from Molecular Probes and were dissolved in dimethyl sulfoxide (DMSO). Stocks of ACh, caffeine, digitonin, and IP3 (Sigma) were prepared in normal PSS and stocks of 2-aminoethoxydiphenyl borate (2-APB), xestospongin C, 53 nifedipine, cyclopiazonic acid (CPA), KB-R7943, SKF96365, procaine, and tetracaine (Sigma) were prepared in 100% ethanol. 3.3 Results 3.3.1 Dependence of ACW on plasmalemmal CaJ+ influx. There are two potential sources of C a 2 + that can contribute to the generation of ACh-mediated ACW: C a 2 + release from intracellular store and C a 2 + influx from the extracellular space. In order to determine the significance of plasmalemmal C a 2 + entry in ACh-mediated ACW, I studied the effect of extracellular C a 2 + removal on the ACh-induced ACW. As shown in Fig. 3.1, ACh-mediated A C W were completely abolished within 5 min of removal of extracellular C a 2 + (n = 30 cells from 5 animals). This indicates that C a 2 + entry from the extracellular space is required for maintaining ACh-induced ACW. Subcellular [Ca2*], 5s Normal PSS 0Ca 2 +PSS v :f.:: 3 uM ACh 54 Figure 3.1. Effect of removal of extracellular Ca on ACh-induced ACW. Application of 3 [iM ACh elicited A C W in porcine TSMC placed in normal PSS with 1.5 mM extracellular Ca 2 + . Replacement of the normal PSS with zero-Ca2+ PSS resulted in the cessation of the ACW. Experimental [Ca2+]j trace is representative of results in 30 cells from 5 different animals. A Subcellular [Ca2*], 10 units | : ' • • S s ' : . 50 uM SKF96365 50 uM SKF96365 55 3 uM A C h Figure 3.2. Effect of 10 uM nifedipine and 50 uM SKF-96365 on ACh-induced ACW and tonic contraction. A. L-type V G C C blockade by nifedipine did not abolish ACh-induced A C W but reduced the frequency of the ACW. Additional application of SK-F96365 abolished ACh-induced A C W completely. Experimental [Ca2+]i trace is representative of results in 30 cells from 4 different animals. B. Application of nifedipine partially reduced the ACh-induced contraction, whereas SKF-96365 nearly abolished the remaining contraction. Experimental tissue contraction trace is representative of results from 5 different animals. C. Time-control traces of ACh-mediated A C W at 30 s and 45 min post stimulation. To further define the C a 2 + entry pathways that are responsible for maintaining the ACW, we used SKF96365, an inhibitor of receptor-operated channels (ROC) and store-operated channels (SOC) together with nifedipine, a selective inhibitor of L-type V G C C . Results from the previous study (chapter 2) indicate that C a 2 + influx via L-type V G C C plays only a minor role in the maintenance of ACh-induced A C W and tonic contraction in the porcine TSMC. Therefore, additional pathway(s) for C a 2 + entry must be responsible for supporting the nifedipine-resistant component of the ACh-mediated A C W and tonic contraction. In the isolated airway smooth muscle cells, non-selective cation permeable channels have been implicated in C a 2 + signaling. While some studies suggested that these channels are predominantly ROC, others favor a 56 combination of SOC and ROC (Murray and Kotlikoff, 1991; Murray et al., 1993; Ay et al., 2004; Marthan, 2004). As shown in Fig. 3.2, 50 uM SKF96365 abolished the nifedipine-resistant A C W within 2 min of its application (n - 30 cells from 4 animals). The corresponding time control trace of the ACh-mediated A C W in Fig. 3.2C shows the persistent generation of A C W up to 45 min post ACh stimulation and the frequency of the A C W at 30 s post-stimulation and 45 min post-stimulation were 0.36 ± 0.02 Hz and 0.33 ± 0.02 Hz respectively (n - 40 cells from 5 animals, P - 0.168). In parallel contraction studies (Fig. 3.2), a combination of 10 uM nifedipine and 50 uM SKF96365 abolished 93.7 ± 1% of ACh-induced tonic contraction in comparison to the 32.8 ± 2.9% reduction in ACh-induced tonic contraction seen with nifedipine alone (P < 0.0001, n = 5 animals). SKF96365 was used after the addition of nifedipine but not in the reverse sequence because it is known to inhibit the L-type V G C C as well. These findings suggest that C a 2 + entry via a ROC/SOC dependent mechanism is essential for maintaining ACh-induced A C W as well as tonic contraction in the tracheal muscle bundle. In addition to the conventional plasmalemmal C a 2 + permeable channels, the Na + /Ca 2 + exchanger (NCX) operating in the reverse mode can be an important pathway for Ca entry into smooth muscle cells (Blaustein and Lederer, 1999; Arnon et al , 2000). The role of N C X in airway smooth muscle (ASM) is controversial. While some studies reported little contribution by N C X (Fleischmann et al., 1996; Janssen et al., 1997), others have implicated N C X in the C a 2 + 57 and contractile regulation of A S M (Chideckel et al., 1987; Raeburn, 1990; Pitt and Knox, 1996; Mustafa et al., 1999; Cortijo et al., 2003; Espinosa-Tanguma et al., 2003). To examine whether the reverse-mode Na + /Ca 2 + exchange is involved in supporting the nifedipine-resistant A C W in the porcine TSMC, we used an inhibitor of both forward- and reverse-mode Na + /Ca 2 + exchange, 2',4'-DCB and a selective reverse-mode inhibitor of NCX, KB-R7943 (Slaughter et al., 1988; Iwamoto et al., 1996; Blaustein and Lederer, 1999; Lee et al., 2001; Shigekawa and Iwamoto, 2001; Takai et al., 2004; Zhang et al., 2005). As shown in Fig. 3.3, the application of 10 uM 2',4'-DCB abolished nifedipine-resistant A C W induced by ACh (n = 45 cells from 7 animals) and inhibited nifedipine-resistant tonic contraction by 91.0 ± 1.4% (P < 0.001, n = 8 animals). Similarly, the application of 20 uM KB-R7943 abolished nifedipine-resistant A C W induced by ACh (n = 45 cells from 7 animals) and inhibited the corresponding tonic contraction by 86.9 ± 4.3%). (P < 0.0001, n = 8 animals). In addition to the use of the above-mentioned pharmacological inhibitors, we also examined the effect of removal of extracellular Na + on ACh-mediated A C W and tonic contraction. As shown in Fig. 3.3C, the removal of extracellular Na + using zero-Na+ PSS - a procedure which will deplete smooth muscle of cellular Na + abolished ACh-mediated A C W within 10 min (n = 40 cells from 5 animals). In a parallel contraction study (Fig. 3.3C), the removal of extracellular Na + reduced ACh-mediated tonic contraction by 72.7 ± 2.2% (P < 0.001, n = 5 animals). These data indicate that reverse-mode Na + /Ca 2 + exchange is involved in maintaining ACh-induced A C W and tonic contraction. As 58 will be discussed later, the sensitivity of the nidefipine-resistant A C W and tonic contraction to both SKF96365 and 2',4'-DCB or KB-R7943 would suggest that the ROC/SOC are likely operating in series with the N C X to allow C a 2 + entry into the cytoplasm in exchange for Na + as proposed previously (Arnon et al., 2000; Lee et al., 2001). Jis >sue con tractio . . . i '9 150 s 10 uM 2',4'-DCB 3 uM ACh 10 u.M Nifedipine 10uNI 2,4,-DCB 59 Subcellular [Ca2*]; 20 uM KB-R7943 60 Subcellular [Ca2 +] zero Na + PSS Figure 3.3. Effect of 10 uM 2',4'-DCB, 20 uM KB-R7943, and zero-Na+ PSS on the nifedipine-resistant component of ACh-induced ACW and tonic contraction. A. Application of 10 u,M nifedipine reduced the frequency of ACh-induced ACW, whereas additional application of 2',4'-DCB resulted in inhibition of the nifedipine-resistant component of the ACh-induced A C W and tonic contraction. Experimental [Ca2+]i trace is representative of results in 45 cells from 7 different animals, and tissue contraction trace is representative of results from 8 different animals. B. The nifedipine-insensitive portion of the ACh-induced A C W and tonic contraction were inhibited by KB-R7943. Experimental [Ca ]; trace is 61 representative of results in 45 cells from 7 different animals, and tissue contraction trace is representative of results from 8 different animals. C. Replacement of the bathing solution with zero-Na+ PSS resulted in cessation of ACh-induced A C W and significant inhibition of the tonic contraction. Experimental [Ca2+]j trace is representative of results in 40 cells in 5 different animals, and tissue contraction trace is representative of results from 5 different animals. Subcellular [Ca2+], , 10 units I • 3 uM ACh Figure 3.4. Effect of 10 uM nifedipine and 50 uM SKF96365 on the initiation of ACh-induced A C W . Application of ACh elicited transient repetitive C a 2 + waves in tissues pretreated with nifedipine (L-type V G C C blocker) and SKF96365 [receptor-operated channels/store-operated channels (ROC/SOC) blocker] in contrast to the sustained repetitive C a 2 + waves in the control tissue. Experimental [Ca2+]; trace is representative of results in 20 cells from 4 different animals. 3.3.2 Dependence of ACW on SR Ca release. As described previously, ACh induces A C W in the TSMC of the intact porcine tracheal muscle bundle. The wave-like nature of the C a 2 + signal implies that SR C a 2 + release is most likely responsible for raising the [Ca2+]j because the C a 2 + signal produced by extracellular C a 2 + entry would typically result in a spatially more 62 uniform elevation of [Ca2+]j (Ruehlmann et al., 2000). To examine the role of plasmalemmal C a 2 + entry in the generation of the ACW, the tracheal muscle bundle was pretreated with 10 uM nifedipine and 50 uM SKF96365 for 5 min prior to ACh-stimulation to block all the C a 2 + entry pathways that were found to be important in supporting the A C W (Fig. 3.2). As indicated in Fig. 3.4, blockade of the L-type V G C C and ROC/SOC did not prevent the induction of A C W as ACh was able to induce C a 2 + waves initially (n = 20 cells from 4 animals). However, the A C W did not persist as they did in the absence of nifedipine and SKF96365 (Fig. 3.4). More interestingly, caffeine (12.5 mM) induced no significant rise in [Ca2 +]i after the cessation of the A C W as the [Ca2+]i prior to and immediately after the addition of caffeine were 100.9 ± 0.8% and 100.6 ± 1.0% of baseline level pre-ACh stimulation respectively (n = 20 cells from 4 animals, P = 0.862), indicating that the SR C a 2 + stores had been emptied when refilling was prevented by SKF96365. It is important to note that the pretreatment of the muscle bundle with only nifedipine and SKF96365 at the same concentration did not affect the amplitude of the caffeine (12.5 mM) induced C a 2 + transient as compared to the control amplitude prior to the addition of nifedipine and SKF96365 (n = 28 cells from 4 animals, P - 0.896). This suggests that the A C W are the result of SR C a 2 + release and that the SR C a 2 + release can be initiated in the absence of extracellular C a 2 + entry, thereby excluding plasmalemmal C a 2 + entry induced SR C a 2 + release by the process of Ca2+-induced C a 2 + release (CICR) as the primary activation mechanism. Furthermore, if SR C a 2 + release is responsible for the generation of the ACW, blockade of the 63 sarco(endo)plasmic reticulum C a 2 + ATPase (SERCA) should completely inhibit the A C W as the SR Ca Z T store can no longer be replenished. As shown in Fig. 3.5, the application of CPA (10 uM), a selective inhibitor of SERCA, resulted in a brief broadening of the C a 2 + waves followed by complete abolition of the ACh-mediated A C W within 10 s and a small but significant elevation in baseline [Ca2+]i (n = 30 cells from 4 animals, p < 0.0001) that corresponds to 26 ± 2% of the peak [Ca2+]j of the ACW. In a parallel contraction study, the application of 10 uM CPA produced a 83.4 ± 2.8% inhibition of the tonic contraction induced by ACh (n = 4 animals, p < 0.001). These findings collectively indicate that A C W are produced by repetitive cycles of SR C a 2 + release followed by C a 2 + reuptake and that C a 2 + entry through the L-type V G C C , ROC/SOC and N C X pathway is necessary to ensure the continual refilling of the SR C a 2 + store to sustain the ongoing ACW. Subcellular [Ca2+]i 10 uM CPA Figure 3.5. Effect of 10 u M CPA on ACh-induced A C W . Application of CPA to ACh-stimulated porcine TSMC completely abolished the ongoing ACW. Experimental [Ca2+]i trace is representative of results in 30 cells from 4 different animals. A Subcellular [Ca2+], B Subcellular [Ca2+]( — Control — 2-APB 3uM ACh 10 units |_ 5s 75uM 2-APB C Tissue contraction — Control — 2-APB 19 150 s D Tissue contraction l _ 150 s 3uM ACh 3uM ACh 75uM 2-APB E Tissue contraction F Tissue contraction Control 2-APB pretreated 0 2 g | _ 0.2 g | _ 50 s 2-APB pretreated 50 s 10 uM IP3 12.5 mM caffeine Figure 3.6. Effect of 75 u M 2-APB on ACh-induced A C W and tonic contraction and D?3-induced contraction in smooth muscle cell from intact porcine tracheal muscle. A. Applications of ACh initiated comparable A C W in cells from the control group and the 2-APB pretreated (30 min) group. Experimental [Ca2+]i traces are representative of results in 20 cells from 4 different animals. B. Application of 2-APB did not affect the ongoing A C W mediated by ACh. Experimental [Ca2+]i trace is representative of results in 20 cells from 4 different animals. C. ACh stimulated tension development in both the control smooth muscle bundle and the 2-APB-pretreated (30 min) muscle bundle. Experimental tissue contraction traces are representative of results from 5 different animals. D. Application of 2-APB did not affect the ongoing tonic contraction mediated by ACh. Experimental tissue contraction trace is representative of results from 5 different animals. E. Pretreatment with 75 uM 2-APB prevented the generation of force transient induced by 10 uM IP3 shown in control trace in digitonin (10 uM)-permeabilized tracheal muscle bundle. Experimental tissue contraction traces are representative of results in 4 animals. F. Pretreatment with 75 uM 2-APB did not affect the generation of force transient induced by 12.5 mM caffeine in permeabilized tracheal muscle bundle. Experimental tissue contraction traces are representative of results in 4 animals. Given that SR Ca release produces the ACW, we proceeded to identify the type(s) of SR C a 2 + release channels involved in the generation of ACh-induced ACW. As mentioned earlier, in isolated TSMC, SR C a 2 + release through the IP3R is required for the initiation of ACh-induced repetitive C a 2 + waves (Kannan et al., 1997). In order to determine whether SR C a 2 + release through the IP3R is responsible for producing ACh-induced A C W in the intact porcine tracheal muscle bundle, we used 2-APB and xestospongin C, both well-established cell-permeable inhibitors of IP3R that have been found to block IP3R-mediated C a 2 + release in a variety of tissues that include the A S M as well as non-smooth muscle cell type (Garni et al., 1997; Maruyama et al., 1997; Ascher-Landsberg et al., 1999; Mitchell et al., 2000; Wu et al., 2000; Missiaen et al., 2001; Ozaki et al., 2002; Perez and Sanderson, 2005b; Perez and Sanderson, 66 2005a). As shown in Fig. 3.6, after pretreatment of the tissue with 75 uM 2-APB for 30 min, ACh (3 uM) was still able to induce A C W and tonic contraction in a manner similar to that observed in the absence of 2-APB in the same tissue. Furthermore, the addition of 2-APB to ongoing ACh-mediated A C W and tonic contraction produced no measurable effect on the A C W and the tonic contraction (Fig. 3.6). It is important to note that the 30 min pretreatment of the tissue with the same batch of 2-APB at 75 uM was able to inhibit phenylephrine (5 uM) induced tonic contraction of the porcine aorta by 94.4 ± 5.4% (n = 4 animals, P < 0.0001). More importantly, in a separate experiment where we permeabilized the tracheal muscle bundle with 10 uM digitonin (Somlyo et al., 1985; Saito et al., 1993; Yang et al., 1994; Nassar and Simpson, 2000), the addition of IP3 (10 uM) produced a transient contraction that was completely prevented by 2-APB (75uM) pretreatment (n = 4 animals) (Fig. 3.6E). In contrast, as shown in Figure 3.6F, 2-APB pretreatment did not prevent or attenuate caffeine-induced contraction (n = 4 animals). These positive control studies show that 75 jiM 2-APB is able to effectively inhibit porcine A S M IP3R and porcine vascular smooth muscle IP3R as the vascular smooth muscle cells (VSMC) in the aorta is known to exhibit A C W and that the phenylephrine-induced A C W in V S M C depend on SR C a 2 + release through the IP3R (Lee et al., 2002c). Similar to the results with 2-APB, pre-treatment of the tissue for 45 min with 10 jxM xestospongin C produced no significant effect on ACh-induced A C W and tonic contraction in the intact porcine muscle bundle while the addition of 10 uM xestospongin C to tissues already stimulated with ACh 67 produced no measurable effect on the ongoing A C W and tonic contraction (Fig. 3.7). The lack of effect seen with these two structurally distinct inhibitors o f the IP3R suggests that Ca release via the IP3R is not required for the generation or the maintenance o f ACh-induced A C W and tonic contraction. A Subcellular [Ca2+] B Subcellular [Ca2*], — Control — Xe-C 3 uM ACh C Tissue contraction — Control — Xe-C 10 uM Xe-C D Tissue contraction 150 s 150 s 3 \M ACh 3 uM ACh 10 uM Xe-C F i g u r e 3.7. E f f e c t o f 10 u,M xes tospong in C ( X e - C ) o n A C h - i n d u c e d A C W a n d ton ic c o n t r a c t i o n . A . Pre-exposure o f porcine tracheal muscle strip with X e - C (10 u M ) for 45 min 68 did not prevent the initiation of ACh-mediated A C W . Experimental [Ca ]; traces are representative of results in 30 cells from 5 different animals. B . Application of X e - C did not affect the ongoing A C W induced by A C h . Experimental [Ca 2 + ] ; trace is representative of results in 30 cells from 5 different animals. C. Pre-exposure of porcine tracheal muscle strip with X e - C for 45 min did not prevent the generation of ACh-induced tissue contraction. Experimental tissue contraction traces are representative of results in 5 different animals. D . Application of X e - C did not affect the ACh-induced tonic contraction. Experimental tissue contraction trace is representative of results in 5 different animals. A Subcellular [Ca2*], • 12.5 mM Caffeine • 3 LIM ACh B Subcellular [Ca2*], 3 u.M ACh Figure 3.8. Effect of 12.5 mM caffeine and 25 u M ryanodine on ACh-induced ACW in smooth muscle cells from intact porcine tracheal muscle. A . Application of A C h initiated A C W in cells from the control group, whereas it resulted in no measurable C a signal in cells 69 from the caffeine-pretreated group. Experimental [Ca2+]j traces are representative of results in 20 cells from 4 different animals. B. Application of ACh stimulated A C W in control smooth muscle bundle but produced no response in ryanodine-pretreated muscle bundles. Experimental [Ca2 +]i traces are representative of results in 20 cells from 4 different animals. In addition to the IP3R, another type of SR C a 2 + release channel that is known to be functionally important in the TSMC is the ryanodine-sensitive SR C a 2 + release channels (RyR). As shown in Fig. 3.8, pretreatment of the tracheal muscle bundle for approximately 20 s with either 12.5 mM caffeine or 25 uM ryanodine to empty C a 2 + from the RyR-sensitive SR C a 2 + store completely prevented the generation of ACh-induced ACW. These findings support the notion that SR C a 2 + release from the RyR-dependent SR store is responsible for the generation of A C W but nonetheless does not prove the involvement of RyR in the C a 2 + release. We therefore employed procaine and tetracaine, both known membrane-permeable inhibitors of RyR-mediated SR C a 2 + release to block the RyR channels (Coronado et al., 1994; Gyorke et al., 1997; Cheranov and Jaggar, 2002). As demonstrated in Fig. 3.9, pretreatment of tracheal muscle bundle with 2 mM procaine for 30 min completely prevented ACh-induced A C W and tonic contraction. When 2 mM procaine was applied to ACh-stimulated tracheal muscle bundles exhibiting ongoing A C W and tonic contraction, it immediately abolished ongoing A C W and reduced the ongoing tonic contraction to 5.3 ± 0.7% of the original level (P < 0.0001, n = 6 animals, Figure. 9). Similarly, as shown in Fig. 3.10, pretreatment of the tracheal muscle bundle with 100 uM tetracaine for 30 min completely prevented ACh-induced A C W and tonic contraction as well. 70 The application of 100 uM tetracaine to ACh-stimulated tissues resulted in the immediate cessation of the ongoing A C W and near-complete inhibition of the ongoing tonic contraction to 13.3 ± 5.3% of the original level (P = 0.0038, n = 5 animals). These findings suggest that SR C a 2 + release via the R y R channels is responsible for both the initiation and the maintenance of ACh-mediated A C W and tonic contraction in the porcine tracheal smooth muscle. A Subcellular [Ca2+]; — — Control 10 units • procaine B Subcellular [Ca 2! 10 units 5s 3 uM ACh 3 uM ACh 2 mM procaine C Tissue contraction D Tissue contraction —— Control 1 g — procaine 150S L 150 S 3uM ACh 3uM ACh 2mM procaine Figure 3.9. Effect of 2 m M procaine on ACh-induced A C W and tonic contraction in smooth 71 muscle cells from intact porcine tracheal muscle. A. Applications of ACh initiated ACW in cells from the control group, whereas it resulted in no measurable C a 2 + signals in cells from the procaine-pretreated (30 min) group. Experimental [Ca 2 +]j traces are representative of results in 20 cells from 4 different animals. B . Application of procaine immediately abolished the ongoing A C W mediated by ACh. Experimental [Ca ]j trace is representative of results in 20 cells from 4 different animals. C. Application of ACh stimulated tension development in control smooth muscle bundle but elicited no response in procaine-pretreated (30 min) muscle bundle. Experimental tissue contraction traces are representative of results from 6 different animals. D . Application of procaine abolished the ongoing tonic contraction mediated by ACh. Experimental tissue contraction trace is representative of results from 6 different animals. A Subcellular [Ca2+]j — Control 10 units | _ — tetracaine 5 s B Subcellular [Ca2*], 10 units |^ 5 s 3 LIM ACh 3 JLIM ACh 100 LIM tetracaine C Tissue Contraction D Tissue Contraction —- Control — tetracaine L 150 s ig L 150 s I J L • • 3 uM ACh 3 uM ACh 100 LIM tetracaine 72 Figure 3.10. Effect of 100 u M tetracaine on ACh-mediated ACW and tonic contraction in porcine tracheal smooth muscle cells. A. ACh-mediated A C W were observed in control porcine tracheal muscle cells but were absent in porcine tracheal muscle strip pre-incubated with tetracaine for 30 min. Experimental [Ca 2 + ] i traces are representative of results in 30 cells from 5 different animals. B. Application of tetracaine immediately abolished the ongoing ACh-induced ACW. Experimental [Ca 2 + ] i trace is representative of results in 30 cells from 5 animals. C. ACh induced tonic contraction in control muscle bundle, whereas it produced no response in tetracaine-pretreated (30 min) muscle bundle. Experimental tissue contraction traces are representative of results in 5 different animals. D. Application of tetracaine immediately attenuated the ongoing ACh-induced tonic contraction. Experimental tissue contraction trace is representative of results in 5 different animals. 3.4 Discussion The aim of this study was to examine the mechanism of the A C W in the porcine tracheal smooth muscle. Our findings show that extracellular C a 2 + entry is not directly responsible for the generation of the C a 2 + waves (Fig. 3.4) and that the A C W are produced by recurring cycles of SR C a 2 + release and SR C a 2 + reuptake (Fig. 3.5). We also characterized the type of SR C a 2 + release channels involved. A major finding of the current study is the lack of involvement of IP3R in mediating the ACh-induced A C W in porcine TSMC. Activation of G-protein-coupled receptors leading to IP^R-mediated intracellular C a 2 + release is a well established signaling pathway in many cell types, including airway smooth muscle cells (Somlyo and Somlyo, 1994). Given that phenylephrine-induced A C W in V S M C is caused by repetitive release of SR C a 2 + via IP3R, it could be expected that IP3R mediated SR C a 2 + release also contributes to generation of 73 ACh-induced A C W and tonic contraction in porcine TSMC. However, as shown in Fig. 3.6 and 3.7, the application of IP3R inhibitors 2-APB and xestospongin C produced no measurable effect on the initial generation and the maintenance of ACh-induced A C W and tonic contraction. It is unlikely that two structurally unrelated inhibitors of IP3R with well-demonstrated efficacy in various smooth muscle and non smooth muscle cell types would be completely ineffective in inhibiting the IP3R in the porcine tracheal smooth muscle (Garni et al., 1997; Maruyama et al., 1997; Ascher-Landsberg et al., 1999; Mitchell et al., 2000; Wu et al , 2000; Missiaen et al., 2001; Ozaki et al., 2002; Perez and Sanderson, 2005b; Perez and Sanderson, 2005a). In addition, our control studies demonstrate that 2-APB is an effective inhibitor of porcine airway IP3R because 2-APB pretreatment completely inhibited IP3-induced force transient in permeabilized tracheal muscle bundle. 2-APB also produced significant inhibition of phenylephrine-induced tonic contraction of the non-permeabilized porcine aorta, a process that is driven by IP3R-mediated A C W in the vascular smooth muscle (Lee et al., 2002b). Furthermore, a similar concentration of xestospongin C has been shown to be effective in preventing ACh- and adenosine triphosphate-induced A C W in the mouse bronchiolar smooth muscle cells (Bergner and Sanderson, 2002; Perez and Sanderson, 2005b), suggesting that xestospongin C is able to inhibit IP3R in the A S M as well. The observed difference in the sensitivity to xestospongin C between ACh-induced A C W in the porcine tracheal smooth muscle and ACh-induced A C W in the mouse bronchial smooth muscle may reflect inter-specie or inter-airway segment heterogeneity in the 74 mechanism of ACW. The lack of effect with 2-APB and xestospongin C observed in our study, however, indicates that C a 2 + release through the IP3R is not involved in the generation of the A C W in ACh-stimulated intact porcine tracheal smooth muscle. Apart from SR C a 2 + release, plasmalemmal C a 2 + entry inducing SR C a 2 + release by the process of CICR could potentially initiate the generation of ACW. In smooth muscle cells of the rat cerebral resistance vessel, recurring C a 2 + release is triggered by C a 2 + entry via L-type V G C C (Lee et al., 2002b). The increased surrounding [Ca2+]j activates the RyR to initiate generation of the C a 2 + waves. In this case, obstruction of the L-type V G C C was sufficient to inhibit the generation of recurring C a 2 + waves. On the contrary, in my study, pretreatment of porcine tracheal muscle strip with nifedipine and SKF96365 to block all known C a 2 + entry pathways did not prevent the generation of ACh-induced ACW, which rule out CICR as the primary mode of activation for ACW. In contrast to the effect of IP3R and plasmalemmal Ca channel blockers, pretreatment of the tracheal muscle bundle with caffeine or ryanodine prevented the generation of the A C W by ACh. More importantly, application of procaine or tetracaine prevented the generation of the A C W and produced immediate and complete inhibition of ongoing A C W induced by ACh. This suggests that A C W is initiated by SR C a 2 + release via a channel that is resistant to 2-APB 75 and xestospongin C but sensitive to procaine and tetracaine. Procaine and tetracaine are potent inhibitors of RyR in various types of tissue (Coronado et al., 1994; Gyorke et al., 1997; Hyvelin et al., 2000; Cheranov and Jaggar, 2002). Therefore, all of the above findings together support that ACh-induced A C W in the intact porcine tracheal smooth muscle are the result of repetitive waves of SR C a 2 + release through the RyR. At present, the identity of the RyR activator has not been revealed, although cyclic adenosine diphosphate ribose is an obvious candidate. Even though extracellular C a 2 + entry is not immediately required for the generation of the ACW, it is necessary to support ongoing A C W over time as shown in Fig. 3.4. This is likely due to the fact that a proportion of the C a 2 + released by the SR to produce the C a 2 + waves is inevitably extruded to the extracellular space possibly via the plasma membrane Ca2+-ATPase and N C X operating in the C a 2 + extrusion mode. In the absence of sufficient extracellular C a 2 + entry, the SR is unable to continually replenish itself after repetitive waves of C a 2 + release and the A C W cease after a period of time because the SR C a 2 + store is depleted (Fig. 3.4). Therefore, in order to maintain the ongoing ACW, it is important to replenish the SR C a 2 + store with additional C a 2 + from the extracellular space. Our pharmacological characterizations in this study have implicated the ROC/SOC-type channel and the N C X operating in the reverse-mode. 76 The exact nature of the ROC/SOC-type channel is not known at this point. However, our findings have revealed a few characteristics of this ROC/SOC-type channel in porcine tracheal smooth muscle. First, it is sensitive to SKF96365 but resistant to nifedipine, 2-APB and xestospongin C. Second, this channel is likely a nonselective cation (e.g. permeable to both Na + and Ca 2 +) channel, given the involvement of the reverse-mode Na + /Ca 2 + exchange in supporting the ACW. This ROC/SOC type channel may predominantly allow Na + to enter the cells since the concentration gradients across the sarcolemma for both Na + and C a 2 + ions are about the same while in the extracellular space there are approximately 150 Na + ions to compete with every C a 2 + ion for entry into the ROC/SOC type channel. There may be some channels that are activated via ligand binding to receptors (i.e. ROC) leading to release of second messengers (e.g. G-protein, IP3, and diacylglycerol) that signal channel opening (Fasolato et al., 1994; Barritt, 1999), or other channels activated via depletion of SR Ca store. Alternatively, ROC/SOC type channel may represent a common channel type that can be activated by either a ligand or store depletion (Wang and van Breemen, 1997). The latter scenario may apply to what I observed in porcine TSMC as the C a 2 + entry through the ROC/SOC type channel was stimulated by ACh binding to cholinergic receptors, which typically results in activation of G q proteins, synthesis of various second messengers, and release of SR Ca 2 + , thus leading to activation of both ROC and SOC. Recently, the transient receptor potential proteins (TRP) have been identified as molecular candidates for ROC/SOC type channels (Beech et al., 2004). 77 While TRPC1, TRPC4, and TPvPC5 are suggested to be components of the SOC, TRPC3 and TRPC6 are implicated in ROC (Lee et al., 2002c; Beech et al., 2004; Thebault et al , 2005). Furthermore, diacylglycerol has been shown to regulate ROC activity in V S M C (Albert and Large, 2006). Given that airway smooth muscle cells also expressed TRPC (Ong et al., 2002; Sweeney et al., 2002; Ong et al., 2003; Corteling et al., 2004), these proteins may constitute the ROC/SOC type channel presented in porcine tracheal smooth muscle. For the N C X to contribute to C a 2 + entry into the cell, it would need to operate in its reverse mode. To activate reverse-mode Na + /Ca 2 + exchange, ACh-stimulated TSMC must allow for an influx of Na + into the cell, and such influx of Na + in smooth muscle cells typically occurs through a non-selective cation channel (NSCC) (Blaustein and Lederer, 1999; Arnon et al., 2000). Na + entering via the NSCC is assumed to accumulate in a sub-plasmalemmal restricted space. In smooth muscle, replenishment of the SR C a 2 + store has been shown to occur in part across a cytoplasmic microdomain formed between part of the plasma membrane (PM) and the peripheral SR (van Breemen et al., 1995). These PM-SR junctions, have been demonstrated in smooth muscle cells of the vasculature as well as the airway (Lee et al., 2002a; Lee et al., 2002b; Poburko et al., 2004; Dai et al., 2005; Lee et al., 2005b). The significance of PM-SR junction on the generation of A C W has been demonstrated in V S M C when disruption of the PM-SR junction prevented the generation of phenylephrine-induced A C W (Lee et al., 2005a). If the 78 NSCC is localized to the PM-SR junction Na + entry would rapidly elevate [Na+] in the junctional space as well as depolarize the membrane to reverse the N C X driving force. In this model, the nonselective cation permeable ROC/SOC type channel is coupled in series to the N C X operating in the C a 2 + influx mode within the PM-SR junction to deliver C a 2 + to SERCA. This would explain our findings that the nifedipine-resistant A C W and tonic contraction are similarly sensitive to both inhibitor of the ROC/SOC, SKF96365 and inhibitors of the NCX, 2',4'-DCB and KB-R7943. It is important to note that while the phenomenon of reverse-mode Na + /Ca 2 + exchange coupled with the nonselective cation channel has never been reported previously in ASM, it has been well described in vascular smooth muscle (Arnon et al., 2000; Lee et al., 2001). In both animal and human A S M , it is known that N C X is present and is capable of operating in the reverse mode (Chideckel et al., 1987; Raeburn, 1990; Pitt and Knox, 1996). The observation that N C X and other ion transport proteins such as the a2- and ct3-isoforms of the Na+/K+-ATPase (a plasmalemmal Na + pump which regulates cytoplasmic Na + and K + concentration by extruding 3 Na + out of and introducing 2 K + into the cell) are co-localized with superficial SR and SERCA in smooth muscle further supports the postulated functional association between ROC/SOC, NCX, and SERCA (Moore et al., 1993; Juhaszova et al., 1994; Juhaszova and Blaustein, 1997; Arnon et al., 2000). Depolarization of the PM by Na + influx through the ROC/SOC type channel may result in 79 activation of the L-type V G C C to allow influx of more extracellular Ca for repletion of the SR. However, in contrast to the ROC/SOC type channel and NCX, L-type V G C C may be located outside of the PM-SR junction. In ASM, depolarization of membrane potential alone is capable of inducing contraction of smooth muscle by sustained influx of C a 2 + through V G C C and may not be located within the PM-SR junction. Our study of the mechanism of A C W in ACh-stimulated TSMC of the intact tissue has revealed some crucial differences from the enzymatically dissociated porcine TSMC. First, in isolated porcine TSMC challenged with ACh, it was shown previously that C a 2 + release through IP3R is important in initiating the repetitive C a 2 + waves (Kannan et al., 1997). However, in the porcine TSMC of the intact tissue, two structurally unrelated inhibitors of IP3R did not affect either the initial generation or the maintenance of ACh-induced A C W and tonic contraction. Second, Prakash et al. (Prakash et al., 1997) showed that the repetitive C a 2 + waves could be abolished by 100 nM nifedipine, which suggested that C a 2 + entry through the L-type V G C C is the main pathway utilized for refilling the SR C a 2 + store and maintenance of the repetitive cycles of SR C a 2 + release. In contrast, our previous study showed that the inhibition of the L-type V G C C by high-dose nifedipine attenuated the frequency of the A C W but did not abolish the ongoing A C W or the tonic contraction stimulated with ACh. These findings indicate that C a 2 + entry through the L-type V G C C is not obligatory for maintaining the A C W in intact tracheal 80 smooth muscle and as shown in this report, C a 2 + entry dependent on ROC/SOC and the N C X operating in the reverse mode is able to sustain the A C W in the absence of Ca entry through the L-type V G C C . Third, results from earlier single-cell studies also showed that N C X plays only a minor role in the regulation of C a 2 + signaling in the tracheal smooth muscle (Janssen et al., 1997). However, the significant inhibitory effects produced by KB-R7943 and 2',4'-DCB, as well as extracellular Na + removal, on ACh-induced A C W and tonic contraction indicate that the N C X plays an important role in agonist-induced C a 2 + signaling in the porcine TSMC. These discrepancies in the characteristics between single-cell preparations and intact tissue show that enzymatically isolated TSMC are phenotypically different from TSMC of the intact tissue. It would appear that the process of enzymatic dissociation may have altered the phenotype of these TSMC, possibly as a result of the nonspecific proteolysis and the disruption of intercellular communication as suggested previously (Ives et al., 1978). 2"b In summary, the data presented in this chapter show that multiple Ca translocating proteins are involved in the generation of the A C W observed in ACh-stimulated smooth muscle cells of the intact porcine tracheal muscle bundle. The ACW appear to be produced by repetitive cycles of RyR-mediated SR C a 2 + release followed by SERCA-mediated SR C a 2 + reuptake. 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Am J Physiol Cell Physiol. 288:C245-252. 89 C H A P T E R 4 - A C E T Y L C H O L I N E - I N D U C E D A S Y C H R O N O U S C A L C I U M WAVES IN INTACT H U M A N B R O N C H I A L M U S C L E B U N D L E 3 4.1 Introduction In previous chapters, I have described the development of repetitive asynchronous C a 2 + waves (ACW) in the in situ porcine tracheal smooth muscle cells stimulated by acetylcholine (ACh), which are responsible for regulating agonist-induced tonic contraction. Most significantly, I found that high-dose nifedipine produced only partial attenuation of the A C W and tonic contraction in the ACh-stimulated porcine airway smooth muscle (ASM) (data presented in chapter 2). It is therefore likely that the poor bronchodilatory effect observed with nifedipine in the treatment of asthmatic attacks (Patel and Al-Shamma, 1982; Walters et al., 1984; Fish and Norman, 1986; Henderson and Costello, 1988) is due to the fact that nifedipine is unable to abolish the A C W responsible for A S M contractile activation. Furthermore, if the same type of C a 2 + signal (i.e. ACW) found in porcine tracheal muscle is also utilized for the regulation of bronchoconstriction in human ASM, it is likely that airway hyperresponsiveness may be due to aberration in agonist-mediated A C W (Parameswaran et al., 2002). Recent findings by Tao et al. 3 A version of this chapter has been submitted for publication as: Dai JM, Kuo KH, Leo JM., Pare PD, van Breemen C, Lee CH. 2006. Acetylcholine-induced asychronous calcium waves in intact human bronchial muscle bundle. Am J Respir Cell Mol Biol. 90 (Tao et al., 2000) demonstrating abnormal SR C a 2 + release in A S M of hyperresponsive rats lends support to this hypothesis. However, despite the advances in our understanding of A S M C a 2 + signaling in the animal airway, very little is known about contractile regulation and C a 2 + signaling in the intact human ASM. As it was previously demonstrated, the observation of A C W in animal tissues does not necessarily correlate with what occurs in the adult human tissues (Lee et al., 2001; Crowley et al., 2002). This likely reflects inter-specie differences or the effect of aging and relevant disease processes on smooth muscle physiology. It is therefore prudent to determine whether A C W are elicited in response to agonist-stimulation in smooth muscle cells of the human airway and, more importantly, underlie excitation-contraction (E-C) coupling in adult human ASM. In this study I examined the ACh-induced C a 2 + signal in the intact human bronchial muscle bundle in relation to tissue contraction. I also explored the mechanism of the ACh-induced C a 2 + signal with emphasis on the mode of C a 2 + entry. 4.2 Materials and methods 4.2.1 Tissue preparation. Lung tissues were obtained from patients who required surgical resection of a lobe for bronchogenic carcinoma at St. Paul's Hospital (Vancouver, BC, Canada). Subjects were studied with the approval of the University of British Columbia-St. Paul's Hospital Ethics Committee and after obtaining informed consent from the subjects. The third-91 and fourth-branch bronchi were carefully dissected from a tumor-free part of the lobe, and immediately transferred to ice-cold sterile physiological salt solution (PSS). From these bronchi, strips of smooth muscles (~ 5x1.5x0.3 mm in dimension) were isolated free of epithelium and cartilage. The strips were subsequently attached at both ends to aluminum foil clips designed for mounting onto the Confocal Wire Myograph system (J.P. Trading). 4.2.2 Solutions and chemicals. Normal PSS containing (in mM): NaCl 140, KC1 5, CaCb, 1.5, MgCl 2 1, glucose 10, HEPES 5, (pH 7.4 at 37°C) was used for all the studies. Zero Na + PSS was identical in composition to normal PSS except Na + was replaced with equimolar of N-methyl-D-glucamine (NMDG +). Fluo-4 A M and pluronic F-127 were purchased from Molecular Probes (Burlington, Ontario, Canada) and were dissolved in dimethyl sulfoxide (DMSO). Stocks of ACh, procaine, and tetracaine were prepared in normal PSS, and stocks of nifedipine, cyclopiazonic acid (CPA), KB-R7943, SKF96365 were prepared in 100% ethanol. These chemicals were purchased from Sigma-Aldrich (Oakville, Ontario, Canada). 4.2.3 Isometric force measurement. Force measurement was performed using Wire Myograph (J.P. Trading). The bronchial muscle strips were attached to isometric force transducer with 1 mN baseline tension and equilibrated in PSS at 37°C for 1 hour. Isometric 92 tension was recorded on-line via a serial connection to a computer hard drive at a rate of 1 Hz. Myodaq 2.01/Myodata 2.02 (J.P. Trading) was employed for data acquisition and analysis. 4.2.4 Simultaneous isometric force measurement and confocal microscopy of Ca2+-induced fluorescence. The clipped muscle strips were loaded with Fluo-4 A M (5 uM with 5 uM Pluronic F-127) for 120 min at 25°C and then left to equilibrate for 10 min in normal PSS. They were then immediately mounted onto the Confocal Wire Myograph (J. P. Trading) for simultaneous isometric force and [Ca2 +]; measurements. Details of the force measurements have been described above (section 4.2.3). Please refer to chapter 3 for details of confocal C a 2 + imaging. 4.2.5 Data analysis. Confocal image analysis was performed with ImageProPlus software using a customized routine written in Visual Basic as described previously in chapter 2. Please refer to 2.2.5 Data analysis for details on data acquisition of recruitment of cells to display ACW, frequency of the ACW, amplitude of the ACW, and baseline [Ca 2 +]i elevation. The representative fluorescence traces shown reflect the averaged fluorescence signals from a 3><3 pixels region (1.36 um2) of a bronchial smooth muscle cell. 93 All numerical data are presented as mean ± SEM. The data were analyzed in Excel or Prism. Paired Student's t tests were used and a value of P<0.05 was considered significant. The n-values indicated for the contraction studies represent the number of bronchial muscle 2+ * * strips used from the specified numbers of patients. The n-values indicated for the Ca imagmg studies represent the number of cells analyzed from the specified numbers of bronchial muscle strips. For C a 2 + imaging studies ANOVA was performed to assess for inter-tissue variance within the same study group wherever appropriate and no significant variance was found. 4.3 Results 4.3.1 Patient background. The demographic, clinical and physiologic data for the patients from whom the tissues were obtained are shown in Table 4.1 with the exception of patients number eight and twelve who received their pulmonary function tests outside of St. Paul's Hospital, and patient number eleven who did not receive a pulmonary function test post surgery. Fresh bronchial tissues from eighteen patients with an average age of 66 ± 2 were used in this study. Because of the small size of the specimens, we were able to isolate only two to four viable bronchial muscle strips from each specimen. As shown in Table 4.1, twelve of the eighteen patients had a significant smoking history. Nine patients exhibited mild to moderate 94 degree of airway obstruction on pulmonary function test with no significant reversible component. Table 4.1. Demographic information of the patients. PATIENT AGE GENDER FEV, (%PRED) PRE FEVj (%PRED) POST FEVj/FVC PRE SMOKE EXPOSURE (pack-years) 1 76 F 85.8 84.9 0.753 0 2 69 M 96.5 79.2 0.798 17 3 69 M 65.5 71.7 0.620 39 4 78 M 90.3 76.4 0.496 39 5 60 F 81.0 90.1 0.693 39 6 75 F 85.4 69.2 0.605 15 7 78 M 58.0 61.0 0.397 35 8 63 M N/A N/A N/A N/A 9 70 M 69.6 52.8 0.502 62 10 45 M 108.6 102.5 0.775 0 11 59 M 66.0 N/A 0.660 40 12 74 M N/A N/A N/A N/A 13 62 M 66.4 67.0 0.692 45 14 51 F 97.7 103.7 0.728 3 15 64 M 82.6 84.3 0.722 125 16 47 M 109.1 112.3 0.791 27 17 67 F 93 92 0.786 N/A 18 60 F 56 66 0.476 69 4.3.2 Characterization of ACh-induced Ca + signal in human bronchial smooth muscle. 95 Confocal microscopy was performed on Fluo-4 loaded human bronchial muscle strip to visualize the changes in [Ca2+]i that occur in individual smooth muscle cells. As shown in Fig. 4.1 A, the cells of the intact bronchial smooth muscle strip appear to be long ribbon-shaped. Application of 3 uM ACh to the intact muscle strip first induced a synchronized C a 2 + wave in the individual bronchial smooth muscle cells (BSMC) (n = 238 cells in 31 muscle strips from 15 patients) (Fig. 1A). This C a 2 + wave gave rise to a transient [Ca2+]i elevation which subsided within 10 s. Subsequently, recurrent intracellular C a 2 + waves developed and traveled along the longitudinal axis of the ribbon-shaped cell. The x-t plot in Fig. 4.IB displays the change in fluorescence level ( o c [Ca2+],) over a longitudinal section of the cell over time. As shown in this figure, a rapid rise in [Ca2+]j was first seen on the left side of this region of interest and subsequently propagated to the right side of the region in an apparent wave-like fashion. These C a 2 + waves continue to recur in the same cell for as long as the agonist is present. According to Fig. 4.1 A, with the exception of the initial C a 2 + wave, the subsequent oscillatory C a 2 + signals did not occur in a synchronized fashion between neighboring cells. Thus ACh induces asynchronous repetitive C a 2 + waves (ACW) in cells of the intact human bronchial muscle bundle. (Please visit http://www.mrl.ubc.ca/pare/ppare.html for a video clip of the A C W in ASMC). Apart from the asynchronous nature, the ensuing repetitive C a 2 + waves were smaller in amplitude than the initial large C a 2 + transient. Interestingly, the A C W induced by ACh observed in the human bronchial smooth muscle cells are qualitatively indistinguishable from the ACh-induced A C W observed in 96 the porcine tracheal smooth muscle cells. 0000 10 s 11 s 12 s 13s B 3u.M Acetylcholine X 0 X, 5s 5 Lim x-t plot 3LIM Acetylcholine Figure 4.1. ACh-induced A C W in A S M C of the intact human bronchial muscle bundles. A. These time series images depict the ACh-induced changes in [Ca 2 +]j over time (as revealed by the fluorescence level) in the BSMC within this selected field of view. At 1-s post-ACh application, the BSMC responded with elevation in [Ca2+]; that was synchronized between different cells. At the 10-s time mark when the initial [Ca 2 + ] i elevation had subsided, the cells began to initiate oscillatory C a 2 + signals (recurring C a 2 + waves) in a nonsynchronized fashion since the rise in fluorescence level was not simultaneous in all cells in the latter 4 time series images. The white scale bar shown indicates a distance of 1 0 urn. B. The x-t plot is a 3-pixel (1.36-um)-wide line scan that depicts the changes in fluorescence ([Ca 2 +]0 over time in this longitudinal line section of the ribbon-shaped BSMC stimulated with 3 uM ACh. The still frame image (left) delineates the placement of the line, and the x-t plot derived from the line is shown (right). The x-t plot shows recurring C a 2 + waves that are initiated on one end (Xo) of the scanned cellular segment and subsequently propagated to the other end (Xi). C. Fluorescence 9 7 changes in two 3*3-pixel intracellular regions (1.36 um2) from two neighboring BSMC are depicted in the traces (right). The ACh-induced A C W in the two neighboring cells occur at different frequencies. 70 -1 2 4 6 8 10 12 14 16 18 20 Time (s) Figure 4.2. Temporal association between ACh-induced force generation and C a 2 + signal. The representative experimental traces (n = 8 strips from 8 patients) shown depict simultaneous measurement of the force generation by a muscle strip (indicated by a black trace) and [Ca + ] i changes in a cell residing in the muscle strip (indicated by a grey trace) that occur in the same muscle strip. Following the application of 3 uM ACh, the appearance of A C W at the cellular level preceded the onset of force generation in the human bronchial muscle strip. 4.3.3 Relationship between repetitive asynchronous Ca2+ waves and force development. We performed simultaneous measurements of both force and [Ca ]j from the same muscle strip stimulated with ACh to study the relationship between the A C W and force generation. We found that the appearance of the A C W consistently precedes the onset of force generation by the muscle strips (Fig. 4.2). This suggests that the A C W is the signal responsible for contractile 98 activation in human bronchial smooth muscle. As shown in Fig. 4.3A, increasing concentrations of ACh produce tonic contraction of increasing amplitude (n = 8 muscle trips from 5 patients). It is interesting to note that the ACh concentration-response relationship of the human bronchial smooth muscle (as shown in Fig. 4.3A) is similar to that described earlier for the porcine tracheal smooth muscle (as shown in Fig. 2.5A). To examine how these C a 2 + waves modulate contraction, concentration dependences of selected quantitative parameters of the A C W were compared with the concentration dependence of force generation in ACh-stimulated tissues. As shown in the concentration-response curves in Fig. 4.3B, increases in the concentration of ACh over the lower concentration range from 0.01 uM to 1 fiM results in increasing recruitment of cells to initiate C a 2 + waves, while increases in concentrations of ACh over the concentration range of 0.01 | i M to 100 uM are correlated with rising frequency of A C W (Fig. 4.3C), reaching 0.47 ± 0.02 Hz at 100 uM ACh (n = 65 cells of 8 muscle strips from 5 patients). In contrast, baseline [Ca 2 +]i elevation and amplitude of the A C W show no significant concentration dependence (Fig. 2D-E). These results indicate that cell recruitment and frequency of the A C W are two main parameters involved in determining the degree of force generation. 0) u o u. E 3 E "S ra 2 120 100 80 60 :40 20 0 -10 -9 Log [ACh] B o fl> 120 r 100 -80 , 60 '•-40 -20 0 - • -10 -8 -7 -6 -5 log [ACh] a -0) : 0.6 0.5 0.4 0.3 0.2 0.1 0 -10 -9 -8 -7 -6 -5 log [ACh] 30 S 25 •a = 20 = § 15 Q. oo E £ 10 "I 5 I -10 -9 -8 log [ACh] I f '>•'•*-•• ( D C " i 2 I 30 25 20 15 10 5 0 3-: * * * -10 -8 -5 log [ACh] Figure 4.3. Concentration-response relationships of ACh-induced force generation and ACW. These concentration-response curves are generated from simultaneous force and [Ca2+]j measurements of intact human bronchial muscle bundles (65 cells in 8 muscle strips from 5 patients). A. Concentration dependence of the magnitude of ACh-induced tonic contraction. B. Concentration dependence of the percentage cell recruitment by ACh to initiate C a 2 + wave(s). C. Concentration dependence of the frequency of ACh-induced ACW. D. Concentration dependence of amplitude of ACh-induced ACW. E. Concentration dependence of trough [Ca 2 + ]i elevation (interspike baseline [Ca 2 + ] i elevation). 100 4.3.4 Dependence of repetitive asynchronous Ca + waves on plasmalemmal Ca influx. Various pharmacological inhibitors were used to characterize the plasmalemmal Ca entry pathway(s) important in ACh-mediated A C W and tonic contraction. Both L-type V G C C and receptor-operated channels/store-operated channels (ROC/SOC) have been implicated in the process of C a 2 + signaling in freshly isolated and cultured human BSMC (Murray and Kotlikoff, 1991; Murray et al., 1993; Prakash et al, 1997; Sweeney et al., 2002). As shown in Fig. 4.4, application of 10 | i M nifedipine partially inhibited ACh-induced tonic contraction and attenuated frequency of the ACW, but did not abolish the ongoing ACW. The reduction in frequency of the A C W and tonic contraction induced by ACh was 35 ± 5% (n - 30 cells from 4 muscle strips from 4 patients, P < 0.001) and 35 ± 4% (n = 6 muscle strips from 4 patients, P = 0.0004) respectively. Additional application of SKF96365 (an inhibitor of the ROC/SOC) completely abolished the nifedipine-resistant component of ACh-induced A C W (n = 30 cells from 4 muscle strips from 4 patients) and reduced the nifedipine-resistant tonic contraction by 94 ± 1% (n = 6 muscle strips from 4 patients, P < 0.0001) of its peak level (Fig. 4.4). These results suggest that ACh-induced tonic contraction in the human bronchial smooth muscle is maintained by plasmalemmal C a 2 + entry involving the L-type V G C C and the ROC/SOC. This is similar both qualitatively and quantitatively to our previous observations in porcine tracheal smooth muscle (data presented in chapter 3). 101 A Subcellular [Ca2+]r C Figure 4.4. Effect of 10 uM nifedipine and 50 uM SKF96365 on ACh-induced ACW and tonic contraction. A. L-type V G C C blockade by nifedipine did not abolish ACh-induced A C W but reduced the frequency of the A C W Additional application of SKF96365 abolished ACh-induced A C W completely. Experimental [Ca2+]j trace is representative of results in 30 cells in 4 muscle strips from 4 patients. B. Application of nifedipine partially reduced the ACh-induced contraction, whereas SKF96365 nearly abolished the remaining contraction. Experimental tissue contraction trace is representative of results in 6 muscle strips from 4 patients. C. Percentage reduction in frequency of A C W and tonic contraction by nifedipine and SKF96365 in ACh-stimulated human bronchial smooth muscle. In vascular smooth muscle cells, reverse-mode Na + /Ca 2 + exchange is implicated in the generation of agonist-induced A C W (Blaustein and Lederer, 1999; Lee et al., 2001). Intriguingly, in porcine tracheal smooth muscle cells, I have provided evidence that reverse-mode N C X play an important role in the maintenance of ACh-induced A C W and tonic contraction (Dai et al., 2006). Previously, it was proposed that an influx of Na + through a non-selective cation permeable ROC/SOC across the plasma membrane can lead to a large subplasmalemmal rise in Na + concentration near NCX, which then drives the N C X into its reverse-mode of operation, bringing C a 2 + into the cell (Lee et al., 2001; Dai et al., 2006). As described in section 3.3.1, if C a 2 + entry via reverse-mode Na + /Ca 2 + exchange contributes to the maintenance of ACh-mediated ACW, removal of extracellular Na + should result in the inhibition of the A C W and the consequent force generation. Indeed, as shown in Fig. 4.5, removal of extracellular Na + abolished the ongoing ACh-mediated A C W (n = 35 cells in 5 muscle strips from 5 patients) and reduced ongoing tonic contraction by 65 ± 11% (n = 5 muscle strips from 4 103 patients, P = 0.0039). In addition, we also employed a selective inhibitor of reverse-mode N a 7 C a 2 + exchange, KB-R7943 (Blaustein and Lederer, 1999; Lee et al., 2001; Shigekawa and Iwamoto, 2001). As shown in Fig. 4.5, application of 10 uM KB-R7943 abolished nifedipine-resistant A C W (n = 32 cells of 4 muscle strips from 4 patients) and inhibited nifedipine-resistant tonic contraction by 78 ± 8% (n = 4 muscle strips from 4 patients, P -0.0024). These results indicate that extracellular C a 2 + entry via reverse-mode Na + /Ca 2 + exchange is required for maintaining ACh-induced A C W and tonic contraction. 3uM ACh 3uM ACh '. Zero Na+ PSS 10uM'nifedipine-V: 10uM KB-R7943 ^ 104 Figure 4.5. Effect of zero Na + PSS and 10 uM KB-R7943 on ACh-induced ACW and tonic contraction. A. Removal of extracellular Na + resulted in the cessation of ACh-induced ACW. Experimental [Ca 2 +]i trace shown is representative of the result of 35 cells in 5 muscle strips from 5 patients. B. Removal of extracellular Na + inhibited ACh-induced tonic contraction. Experimental tissue contraction trace shown is representative of the result in 5 muscle strips from 4 patients. C. Application of 10 uM KB-R7943 inhibited nifedipine-resistant ACW. Experimental [Ca 2 +]j trace shown is representative of the results in 32 cells in 4 muscle strips from 4 patients. D. Application of KB-R7943 resulted in the inhibition of ACh-induced nifedipine-resistant tonic contraction. Experimental tissue contraction trace shown is representative of the results in 4 muscle strips from 4 patients. 4.3.5 Dependence of repetitive asynchronous Ca2+ waves on SR CaJ+ release. In intact porcine tracheal smooth muscle, ACh-induced A C W are the result of repetitive cycles of SR C a 2 + release followed by SR C a 2 + reuptake (Dai et al., 2006). As demonstrated previously, the wave-like nature of the C a 2 + signal seen in ACh-induced A C W (Fig. 4.1) indicates that they are likely the result of SR C a 2 + release rather than plasmalemmal C a 2 + entry (Lee et al., 2001). In addition, blockade of sarco(endo)plasmic reticulum ATPase (SERCA), with 10 uM CPA rapidly and completely abolished ongoing A C W induced by ACh (n = 28 cells from 4 muscle strips from 3 patient, Fig. 4.6). This observation indicates that SR C a 2 + release is required for ACh-induced ACW in human BSMC. I thus proceeded to determine the type of SR C a 2 + release channels involved in generating the ACW. In the previous chapter I observed that ryanodine-sensitive SR Ca release channels (RyR) mediate bronchoconstriction in animal A S M (Du et al., 2005; Dai et al., 2006). To examine whether C a 2 + release via RyR is important in 105 mediating ACh-induced A C W and tonic contraction in human bronchial muscle strips, we used two inhibitors of RyR, procaine and tetracaine (Coronado et al., 1994; Gyorke et al., 1997; Cheranov and Jaggar, 2002). In the present study, the application of 2 mM procaine (n = 24 cells in 3 muscle strips from 3 patients) or 100 | i M tetracaine (n = 24 cells in 3 muscle strips from 3 patients) caused a complete inhibition of ACh-induced A C W (Fig. 4.7). Similarly, the application of the same dose of procaine (n = 5 muscle strips from 4 patients) and tetracaine (n = 5 muscle strips from 4 patients) resulted in 97 ± 1% (P < 0.0001) and 94 ± 4% (P < 0.0001) inhibition of ACh-induced tonic contraction respectively (Fig. 4.7). These results indicate that SR C a 2 + release via the RyR is crucial to the generation of A C W and development of force induced by ACh. Subcellular [Ca2*], fOuMCPA Figure 4.6. Effect of 10 uM CPA on ACh-induced A C W . Application of CPA to ACh-stimulated human BSMC completely abolished the ongoing ACW. Experimental [Ca2+]j trace shown is representative of the results in 28 cells in 4 muscle strips from 3 patients. 106 Figure 4.7. Effects of 100 uM tetracaine and 2 mM procaine on the ACh-induced ACW and tonic contraction in human bronchial smooth muscle strips. A. Application of tetracaine (100 uM) immediately abolished the ongoing ACh-induced ACW. Experimental [Ca2+]; trace shown is representative of 24 cells in 3 muscle strips from 3 patients. B. Application of tetracaine (100 uM) nearly abolished the ongoing ACh-induced tonic contraction. Experimental tissue contraction trace shown is representative of the results in 5 muscle strips from 4 patients. C. Application of procaine (2 mM) immediately abolished ongoing A C W mediated by ACh. Experimental [Ca2+]j trace shown is representative of the results in 24 cells in 3 muscle strips from 3 patients. D. Application of procaine (2 mM) abolished ongoing tonic contraction mediated by ACh. Experimental tissue contraction trace shown is representative of the results in 5 muscle strips from 4 patients. 107 4.4 Discussion This chapter presents the first study of C a 2 + mediated E-C coupling in human bronchial smooth muscle and reveals several previously unexplored pharmacological targets for the treatment of broncho-spasm. In this study, ACh consistently and reproducibly induced A C W and tonic contraction in the human bronchial muscle strips from successive surgical specimens despite the potential variability that may exist between different patients. I have previously shown that the A C W is the signal responsible for E-C coupling in ACh-stimulated porcine tracheal smooth muscle. In the case of human BSMC, the present study supports that the purpose of the A C W appears to be the same because the A C W precede force generation and inhibition of A C W with various pharmacologic agents consistently reduces contraction. Comparison of the concentration dependence of the properties of the A C W to that of the force development indicates that differential cell recruitment and modulation of the frequency of the A C W determine the magnitude of ACh-induced force generation. Interestingly, in contrast to what was found in intact porcine tracheal muscle bundle, trough [Ca 2 +]i elevation shows no significant concentration dependence (Fig. 4.3). These findings indicate that 1) ACh induces A C W in cells of the intact human A S M and 2) A C W represent the fundamental signaling mechanism for E-C coupling in the human ASM. 108 A C W induced by ACh in the human BSMC exhibit a similar spatiotemporal pattern as A C W observed in the animal ASM. There is however one noteworthy difference. Even though the dose-response relationships are similar between porcine tracheal smooth muscle and human bronchial smooth muscle, ACh-induced A C W at the highest concentration examined (100 |iM) recurred at higher peak frequency of 0.47 Hz in human tissue than the frequency of 0.28 Hz observed in the porcine tissue. It is unclear at this time whether this difference in the frequency of A C W represents a physiologically significant inter-species difference or is the result of pathological alterations in the human bronchial smooth muscle related to the factors such as age and smoke exposure. With regard to the mechanism for the generation of ACW, the pharmacological characterization in this study indicates that the A C W of human and animal A S M likely share similar mechanisms. Using the same maximally effective dose of nifedipine to inhibit the L-type V G C C , we observed a 35% reduction in ACh-induced tonic contraction in human ASM, a result that is similar to the 33% reduction in ACh-induced tonic contraction seen in porcine tracheal smooth muscle. Correspondingly, nifedipine reduced the frequency of the A C W in the human and the porcine tissue by 35% and 29% respectively. When SKF96365 is added in the presence of nifedipine, the nifedipine-resistant A C W were abolished in both human and porcine tissue, while the corresponding nifedipine-resistant tonic contraction was nearly completely inhibited as well in both human and porcine tissues. In addition, blockade of the reverse-mode Na + /Ca 2 + exchange with either zero Na + PSS or KB-R7943 resulted in significant 109 inhibition of the A C W and tonic contraction induced by ACh in both human bronchial and porcine tracheal muscle. These findings directly implicate the N C X in A S M C a 2 + signaling and E-C coupling in both human and porcine airway. Furthermore, SR C a 2 + release via the RyR and C a 2 + refilling via the SERCA are crucial in maintaining ACh-induced A C W and tonic contraction in the human airway as shown by the abolition of the A C W by inhibitor of SERCA -CPA and inhibitors of RyR - procaine and tetracaine. The new findings resulting from the present series of pharmacologic characterizations indicate the need to revise our knowledge of human A S M physiology. The concept that A S M contraction is brought on by stimulated C a 2 + influx via the L-type V G C C is oversimplified and no longer tenable. In contrast, the generation of A C W requires a coordinated sequence of C a 2 + fluxes inside the cell that occurs on a repetitive basis (Lee et al., 2001; Lee et al., 2002b). Multiple C a 2 + transporting molecules on the sarcolemma and the SR are required for this recurring sequence of C a 2 + fluxes. The L-type V G C C , the NCX, the SERCA, the RyR and a putative non-selective cation permeable ROC/SOC type channel have been implicated thus far in the generation of A C W in the human BSMC. The ROC and SOC include a wide range of plasmalemmal channels that are activated secondary to receptor activation and SR C a 2 + depletion respectively (Barritt, 1999; Lee et al., 2002a). The exact molecular identities of the various types of ROC and SOC are not known at this time, but are likely related to transient receptor 110 potential molecules (TRP). Identification of the various subtypes of TRP and N C X involved in the pathophysiology of human airway may lead to new therapeutic approaches. Clinically, our appreciation of the importance of A C W in agonist-induced contractile activation of human A S M introduces many exciting therapeutic possibilities. Though it remains to be verified in future studies, it is plausible that the hyperresponsive airway smooth muscle cells found in asthmatic airways still signal for tonic contraction via ACW. Accordingly, anomalies in A C W related to altered expression of TRPs, NCX, or RyR may actually be responsible for the altered A S M contractility (Opazo Saez et al , 2000). Therefore, it is likely that pharmacological agents targeting the various C a 2 + translocators and effectively modulate or abolish the A C W may prove to be efficacious inhaled and/or systemic bronchodilators in the clinical setting. Furthermore, the development of novel inhalational therapeutic agents that target C a 2 + signaling in the A S M will certainly be a welcome addition to the p-agonists that are the first line bronchodilator used clinically at the present. This is especially true in cases where p-agonists are poorly tolerated by the patients and in cases where other beneficial drugs such as P-blockers used in the treatment of acute coronary syndrome are withheld because of the potential adverse clinical interaction with the p-agonists. In summary, I have demonstrated for the first time the presence of ACh-induced A C W in i l l the smooth muscle cells of intact human bronchial muscle strips. The A C W are the primary-signal used by the agonist to stimulate cellular contraction. Furthermore, we have identified a number of C a 2 + transport molecules that are involve in mediating ACh-induced A C W and tonic contraction, including the L-type V G C C , the NCX, the SERCA, the RyR and a putative non-selective cation permeable ROC/SOC type channel. 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Thorax. 39:572-575. 115 CHAPTER 5 - CONCLUSION AND FUTURE DIRECTIONS 5.1 Overview of repetitive asynchronous Ca2+ waves Following the first documentation by Bergner and Sanderson, repetitive asynchronous C a 2 + waves (ACW) have been found in agonist-stimulated airway smooth muscle cells (ASMC) in various locations of the tracheobronchial tree of different species (Bergner and Sanderson, 2002a). In this thesis, I presented evidence that acetylcholine (ACh) stimulation elicits A C W at specific regions of the porcine ASMC. These A C W subsequently propagate along the longitudinal axis of the ribbon-shaped cells. In addition to ACh, other bronchoconstricting agents such as 5-hydroxytrypamine, adenosine triphosphate (ATP), histamine, and endothelin can also induce A C W in ASMC of the murine lung slices and intact porcine tracheal muscle strips (Bergner and Sanderson, 2002b; Perez and Sanderson, 2005b). The intracellular C a 2 + concentration ([Ca2+]i) traces shown in Fig. 5.1 represent the A C W in A S M C of the histamine-and endothelin-stimulated porcine tracheal smooth muscle bundle. Aside from airway smooth muscle (ASM), A C W have been found in other types of smooth muscle cells (Ruehlmann et al., 2000; Lee et al., 2002; Lee et al., 2005). Consequently, dysfunction of molecules that are found in the signaling cascade of A C W have been postulated to underlie various vascular and pulmonary diseases such as hypertension and asthma (Parameswaran et al., 2002; Gudermann et 116 al., 2004; Ong and Barritt, 2004). However, due to the lack of evidence for the occurrence of A C W in intact human tissue, their significance in human physiology and pathophysiology was controversial. Nonetheless, using intact smooth muscle strips isolated from the 3 r d and 4 t h generation of adult human bronchi, I have demonstrated that ACh induces A C W in human A S M C , which provides support for the important role that A C W may play in human tissue. The ACh-induced A C W in adult human bronchial smooth muscle are comparable to those found in porcine tracheal smooth muscle. Collectively, these findings confirm that A C W are ubiquitous C a 2 + signals initiated by a variety of contractile agonists to induce contraction of A S M C in animal and human airway. A. Subcellular [Ca 2 ^ 30u.M Histamine 117 Figure 5.1. Histamine- and endothelin-induced ACW in ASMC of intact porcine tracheal muscle strips. A. 30 uM histamine stimulated A C W in two different ASMC on the same porcine tracheal muscle strip. Experimental [Ca 2 +]i traces are representative of results from 5 muscle strips from 5 animals. B. 10 nM endothelin elicited A C W in two different ASMC on the same porcine tracheal muscle strip. Experimental [Ca 2 +]i traces are representative of results from 5 muscle strips from 5 animals. 5.2 Repetitive asynchronous Ca + waves and constriction of the airway As discussed in chapter 1, contraction of the A S M was thought to be maintained by sustained 118 elevation of [Ca2+];. However, results presented in this thesis and other recent studies (Perez and Sanderson, 2005b; Dai et al., 2006) have challenged the accepted views of A S M contractile regulation, which were deduced from studies of single cells either freshly isolated or derived from tissue culture. In our studies, employment of fast scanning confocal microscopy of intracellular Ca2+-sensitive dyes to examine ASMC of the intact muscle strips suggests that the apparent sustained elevation of C a 2 + concentration ([Ca2+]) at the whole tissue level may be, in fact, due to summation of agonist-induced A C W with constant amplitude in individual cells. Therefore, ASMC of murine lung slices, intact porcine tracheal bundle, and human bronchial smooth muscle bundle utilize A C W to modulate whole tissue force development (Perez and Sanderson, 2005b). Furthermore, the degree of contractile activation is determined by the number of cells that are recruited to elicit A C W at lower agonist concentration and the frequency of the A C W at higher agonist concentration. 5.3 Mechanism of repetitive asynchronous Ca2+ waves The observation of agonist-induced A C W in ASMC of the intact tissue preparation is relatively recent, and our understanding of the mechanism of this type of C a 2 + signal is rather limited. Currently, the cellular events involved in ACh-induced A C W in relation to force development have been characterized to certain extend for porcine tracheal smooth muscle cells. 119 Essential ly , the A C h - m e d i a t e d A C W result f rom repetit ive cycles o f sarcoplasmic r e t i c u l u m ( S R ) C a 2 + release f o l l o w e d b y S R C a 2 + reuptake and r e f i l l o f the S R w i t h extracel lular C a 2 + is required to m a i n t a i n the recurr ing C a 2 + waves. • • • Figure 5.2. Model for ACh-induced A C W in A S M C of intact porcine tracheal smooth muscle. Please refer to text b e l o w for detail descr ipt ion. L - t y p e V G C C , L-type voltage gated C a 2 + channels; R O C / S O C , receptor-operated channels/store-operated channels; P M C A , p l a s m a membrane C a 2 + A T P a s e ; R y R , ryanodine sensit ive S R C a 2 + release channels; IP3R, IP3 sensitive S R C a 2 + release channels; S E R C A , sarco(endo)plasmic r e t i c u l u m C a 2 + A T P a s e ; S R , sarcoplasmic r e t i c u l u m ; N K A a 2 , a2 insoform o f N a + , K + - A T P a s e . 120 The postulated detailed signaling cascade is illustrated in Figure 5.2. Stimulation of cholinergic receptors by ACh results in activation of ryanodine-sensitive SR C a 2 + release channel (RyR). SR C a 2 + released in the form of C a 2 + wave binds to calmodulin tethered to the contractile filament, and increase the open probability of the nearby SR C a 2 + release channel on the neighboring SR via the process of Ca2+-induced C a 2 + release (CICR). Propagation of the C a 2 + wave may be result from a train of CICR events progressing along the ribbon-shaped ASMC. The receptor-operated channels/store-operated channels (ROC/SOC) open in response to receptor binding or reduction in SR C a 2 + content allowing entry of predominantly Na + into the restricted space sandwiched between plasma membrane (PM) and SR membrane (i.e. PM-SR junction). Influx of Na + across the sarcolemma causes depolarization of the membrane potential which activates L-type voltage-gated C a 2 + channel (VGCC) leading to C a 2 + influx into the bulk cytosol. Coincidentally, accumulation of Na + ion within the PM-SR junction creates a large Na + gradient that drives the neighboring Na + /Ca 2 + exchanger (NCX) into the reverse mode to allow C a 2 + enter into the PM-SR junction. As the local [Ca2 +]i near the cytoplasmic side of the RyR increases, RyR is inactivated. Sarco(endo)plasmic recticulum ATPase (SERCA) starts to refill the SR by sequestering intracellular C a 2 + released from the SR, and C a 2 + entering via the N C X and L-type V G C C . When the SR luminal C a 2 + content refills and reaches the threshold for activation of RyR, another wave of SR C a 2 + begins. Some studies suggest that the low Na + affinity oc2 and a3 isoforms of Na+/K+-ATPase are also present in the PM-SR junctions and 121 indirectly facilitate reversal of N C X (Juhaszova and Blaustein, 1997; Arnon et al., 2000). The molecular identity and mode of activation of The ROC/SOC type channel are yet to be defined. Furthermore, the cellular events coupling ACh binding of muscarinic receptor to activation of RyR remain elusive. Recently, cyclic adenosine diphosphate ribose (cADPr), a nucleotide metabolite synthesized by a membrane-bound glycoprotein, has emerged as a potential endogenous second messenger that mediates SR C a 2 + release via RyR in various types of smooth muscle cells (Prakash et al., 1998; Li et al., 2001; Barone et al , 2002; Deshpande et al , 2005). As proposed by others, cADPr may activate RyR directly by binding to cADPr receptors on the RyR, or indirectly through binding to other proteins, such as FKB12.6 which forms complex with RyR and calmodulin kinase II which phosphorylates RyR (Takasawa et al., 1995; Tang et al., 2002). In cultured ASMC, FKB12.6 has been found to associate with type 2 RyR and contribute to cADPr mediated SR C a 2 + release (Wang et al., 2004). Given the importance of RyR in ACh-induced A C W and contractile activation in intact ASM, future examination of the involvement of cADPr and its interaction with proteins associated with RyR are necessary. Consistent with the findings in porcine tracheal smooth muscle, results from studies using human bronchial smooth muscle suggest that ACh-induced A C W in this tissue are also are the 122 consequence of Ca cycling in and out of the SR, involving RyR and SERCA. As in the porcine trachea, refilling of the SR in human bronchial smooth muscle requires Ca entry via reverse mode N C X driven by Na + in flux through non-selective cation channel. As the role of A C W in agonist-induced contractile activation in human A S M in health and disease become more clearly established, our identification and characterization of the C a 2 + transporting proteins, namely, non-selective cation permeable ROC/SOC type channel, NCX, SERCA, and RyR, involving in mediating A C W is expected to lead to the development of agents that target the activation of these proteins as prophylactics and therapeutic for acute broncho-spasm. 5.4 Functional advantage of repetitive asynchronous Ca2+ waves Perhaps the most intriguing question regarding the A C W is why A S M C adopt this complex form of C a 2 + signaling for activating its contractile filaments. The answer to this question remains elusive at this point. However, there are a number of important considerations that may shed some light on this issue. First, C a 2 + as an intracellular signaling molecule is capable of activating a large number of effector molecules and processes in the cell. A C W may be a mechanism to achieve high localized [Ca2+] allowing effective activation of specific effector functions without a generalized activation of Ca2+-sensitive pathways. A sustained local increase in C a 2 + could lead to C a 2 + diffusion and more generalized activation unless C a 2 + is 123 rapidly re-compartmentalized as is achieved with ACW. The SR network in smooth muscle cells permeates through the myosin-rich deep cytoplasm and provides a conduit for C a 2 + delivery to the myoplasm where the target molecule myosin light chain kinase is tethered on the myosin filament (Lee et al., 2002; Wilson et al., 2002). Therefore, utilizing repetitive waves ofSR C a 2 + release to deliver C a 2 + to the myosin light chain kinase in comparison to introducing C a 2 + across the PM that is typically about 100 nm away from the myosin-rich cytoplasm may represent a more efficient mechanism to activate the contractile machinery (Lee et al., 2002; Poburko et al., 2004). If this is true, it would indicate that a state of C a 2 + overload in the SR may be an important determinant of smooth muscle hypercontractility. Intriguingly, an increasing degree of SR C a 2 + release has been correlated with enhanced intraparenchymal airway narrowing in the rat A S M (Tao et al., 1999). Second, if the kinetics for calmodulin activation by C a 2 + is fast while inactivation is slow, the C a 2 + level at peak of the A C W would be sufficient to signal for contractile activation. In this case, the average [Ca2+]; achieved is lower than when a sustained rise in [Ca2+]j is used to stimulate contraction. Third, one cannot exclude the possibility that there may be frequency-sensitive enzymes that are activated to a greater extent by the ACW. An enzyme such as calmoduline kinase II and transcription factors such as NF-AT that are selectively activated by the frequency-domain of oscillatory [Ca2+] rise have previously been demonstrated in mammalian cells (De Koninck and Schulman, 1998; Dupont and Goldbeter, 1998; Hu et al., 1999). Fourth, repetitive SR C a 2 + release also appears to be important for 124 mitochondrial Ca signaling and regulation of energy metabolism. In intact A S M , it has been shown that SR is tightly associated with mitochondria such that their apposing membranes form a narrow restricted space referred to as mito-SR junction (Dai et al., 2005). Similar to the PM-SR junction, this junctional space limits the movement of ions, thus allow generation of sufficiently high local [Ca2+] to activate the C a 2 + uptake system on the mitochondrial membrane. The resulting rise in mitochondrial [Ca2+] can activate Ca2+-sensitive dehydrogenases and leads to increased ATP synthesis in order to match the increase energy demand for contraction of A S M (Hajnoczky et al., 1995; Robb-Gaspers et al., 1998). Finally, A C W represent a signaling mechanism that is widely utilized by a variety of smooth muscle cells to stimulate tonic cellular contraction (Ruehlmann et al., 2000; Lee et al., 2002; Lee et al., 2005; Perez and Sanderson, 2005b; Perez and Sanderson, 2005a). It is unlikely that nature has adopted and preserved this more complex form of C a 2 + signaling across different tissue types and species serendipitously without any underlying advantage. 5.5 Summary The primary objective of my research was to investigate the generation of agonist-induced ACW, their functional role, and their mechanism in porcine tracheal and human bronchial smooth muscle. Findings presented in this thesis demonstrate that ACh stimulation elicits 125 A C W which underlie excitation-contraction (E-C) coupling in intact porcine tracheal smooth muscle and, most importantly, human bronchial smooth muscle. Furthermore, variation in the frequency of the A C W regulates the level of force development. Characterization of the mechanism of the A C W revealed a coordinated sequence of C a 2 + transporting events involving RyR, ROC/SOC type channel, NCX, and SERCA (Fig. 5.1). This study enhances our understanding of C a 2 + signaling in healthy animal and human ASM. As mentioned earlier, Parameswaran et al. postulated that airway hyperresponsiveness may be due to aberrant C a 2 + signaling (Parameswaran et al., 2002). To clarify this hypothesis, results presented in this thesis can be used to compare with future studies using A S M from animal models of airway diseases or asthmatic and COPD patients. This will enable us to comprehensively investigate abnormalities of C a 2 + signaling in A S M of asthma and COPD patients, and may thus finally unveil the mystery of NSBH in these inflammatory airway diseases. While my work provides in some detail the molecular basis of agonist-induced ACW, the mechanism of the RyR activation resulting from receptor binding remains unclear. Due to the central role RyR play in mediating A C W and tonic contraction, it is important to determine the second messenger that activates it. In this regard, a popular candidate is cADPr. In addition 126 to contractile regulation, it is quite possible that A C W may involve in the regulation of other processes in ASMC. On this note, transcription factors that are sensitive to frequency of A C W have been identified. If A C W is also employed to signal for transcription and gene expression, abnormality in A C W may lead to hypertrophy and hyperplasia of A S M , which in addition to smooth muscle hypercontractility are characteristics of pulmonary diseases such as asthma. Further studies are required to address these issues. 127 5.6 Bibliography Anion, A., J.M. Hamlyn, and M.P. Blaustein. 2000. Na + entry via store-operated channels modulates C a 2 + signaling in arterial myocytes. Am J Physiol Cell Physiol. 278:C163-173. Barone, R, A. Genazzani, A. Conti, G Churchill, R Palombi, E. Ziparo, V. Sorrentino, A. Galione, and A. Filippini. 2002. A pivotal role for cADPR-mediated C a 2 + signaling: regulation of endothelin-induced contraction in peritubular smooth muscle cells. FASEB J. 16:697-705. Bergner, A., and M.J. Sanderson. 2002a. Acetylcholine-induced Calcium Signaling and Contraction of Airway Smooth Muscle Cells in Lung Slices. J. Gen. Physiol. 119:187-198. Bergner, A., and M.J. 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