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The role of intracellular free calcium as a second messenger in human airway epithelia Harris, Robert Arthur 1992

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THE ROLE OF INTRACELLULAR FREE CALCIUM AS A SECOND MESSENGER IN HUMAN AIRWAY EPITHELIA by ROBERT ARTHUR HARRIS B.Sc., University of Alberta, 1981 M.Sc, University of Alberta, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ZOOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April, 1992 © Robert A. Harris, 1992 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. (Signature) Department of Zoology The University of British Columbia Vancouver, Canada Date April 23, 1992 DE-6 (2/88) ii Abstract The role of [Ca2+]i as a second messenger controlling electrolyte transport in human airway epithelia was investigated using cultured human nasal epithelial cells as a model. Human nasal epithelial tissue was obtained as a by-product of surgery and put into monolayer cell culture. After 2-5 days in culture, cells were loaded with fura-2 and intracellular free Ca2+ measured using wavelength emission ratio cytofluorometry. Initially, bradykinin increased Ca2+]; in all cells tested, whereas isoproterenol increased Ca2+]i; in only half the cells tested. Epinephrine, norepinephrine, and prostaglandin E2 had no stimulatory effect on Ca2+]i. However, nucleotide receptor agonists, such as adenosine 5'-triphosphate, uridine 5'-triphosphate, and adenosine 5'-diphosphate, stimulated increases in Ca2+]i in a manner similar to bradykinin. Other related compounds such as guanidine 5'-triphosphate and cyclic 3'-5'-adenosine monophosphate had no detectable stimulatory effect on [Ca2+]i. Verapamil, a voltage-sensitive Ca2+ channel blocker, had no effect on bradykinin-induced increases in Ca2+]i. Removal of extracellular Ca2+ for periods as long as 30 minutes only slightly attenuated the increase in Ca2+]i which resulted from both bradykinin stimulation and stimulation with a variety of exogenous nucleotides. Histamine also increased Ca2+]i in a dose dependent manner; however, the kinetics of theCa2+]i response to histamine differed markedly from stimulation with either bradykinin or exogenous nucleotides. In order to elicit a change in Ca2+]i, cells required at least a 30 second exposure to histamine. Bathing cells in Ca2+-free saline for 5 minutes had no effect on the histamine-induced change in Ca2+]i. However, bathing the cells in Ca2+-free saline for 30 minutes completely abolished the histamine-induced change in Ca2+]i, yet subsequent stimulation with bradykinin or exogenous nucleotides resulted in a dramatic increase in Ca2+]i. iii Over the course of this study a change in the responsiveness of the cultured cells to bradykinin-mediated increases in Ca2+]i was observed. All aspects of the cell culture procedure were investigated and virtually ruled out as the agent which caused the observed change in physiological response to bradykinin. During the critical time frame (February-March 1990), new topical steroid treatments consisting of budesonide became popular with the local ENT surgeons from whom I obtained tissue samples. These steroid treatments may be good candidates for the agent which changed the cellular physiology. Subsequent experiments demonstrated that, after introduction of this steroid treatment, human nasal epithelial cells retained a Ca2+]i response to bradykinin and exogenous nucleotides for the first 2 days in cell culture only. After 2 days in cell culture, the number of responsive cells decreased rapidly such that after 6 days in culture only about 15% of the cells responded to bradykinin. A model for the second messenger pathways mediating CI" secretion in human airway epithelial cells is proposed. Briefly, 8-adrenergic agonists activate the cAMP second messenger pathway independent of Ca2+]i. Adenosine 5'-triphosphate and bradykinin stimulate the production of D-myo-inositol 1,4,5-trisphosphate, which in turn releases sequestered calcium from intracellular stores. Histamine also releases sequestered calcium; however, the calcium released by histamine comes from a pool which is separate from that released by D-myo-inositol 1,4,5-trisphosphate. In addition, histamine may act directly as the second messenger which releases calcium from this hitherto undescribed intracellular store. iv Table of contents Abstract ii List of Tables vii List of Figures viii List of Abbreviations xi Acknowledgements xiii CHAPTER ONE: General Introduction 1 Mucociliary Transport in the Airway and the Role of the Periciliary Fluid Layer 1 Mechanisms of Epithelial Ion Transport 2 Ion Transport Events in Airway Epithelia 5 Intracellular Regulation of Ion Transport 8 Signal Transduction at the Plasma Membrane 8 Rationale for the Present Study 12 CHAPTER TWO: Effects of Bradykinin, PGE2 and Adrenergic Agonists on [Ca2+], 17 Materials and Methods 18 Cell Culture 18 V Cell Preparation for [Ca2Jr]i Determination 19 Fluorescence measurements 20 Calculation of cytosolic calcium concentration 21 Drug treatment 22 Results 22 Discussion 36 CHAPTER THREE: Observed Changes in Secretagogue Response of Cultured Cells Over Time 41 Materials and Methods 41 Pre-operative Patient Treatment 41 Cell Culture and [Ca2+]t Determination 42 Results and Discussion 42 CHAPTER FOUR: Effects of Nucleotide Stimulation on [Ca2+], in Cultured Human Nasal Epithelial Cells 46 Materials and Methods 47 Cell Culture 47 Drug treatment 49 Results 49 Discussion 60 CHAPTER 5: Effects of Histaminergic Stimulation on [Ca2+], in Cultured Human Nasal Epithelial Cells 62 Materials and Methods 63 vi Cell Culture 63 Calculation of cytosolic free calcium concentration 63 Drug treatment 64 Results 64 Discussion 81 CHAPTER SIX: General Discussion 86 The Role of CFTR and cAMP 86 Role of [Ca2+]i 90 Source of the Increased [Ca2+]; 95 Organization of Second Messengers Mediating CI" Secretion in Airway Epithelia 97 Future Studies 99 REFERENCES 100 Appendix 1 112 Important Diseases Which Affect Electrolyte Transport Across Airway Epithelia 112 Cystic Fibrosis 112 Asthma 113 Appendix 2 115 Ratio Fluorometric Determination of [Ca2+]; 115 vii List of Tables Table 5.1. Effect of Various Receptor-Antagonists on [Ca2+]i Response to Histamine Stimulation 80 viii List of Figures Figure 1.1. The Two Common Arrangements of Transport Across Epithelial Cells 4 Figure 1.2. Initial Model for NaCl Transport Across Airway Epithelia 6 Figure 1.3. Interactions between hormone receptor, G-protein, and effector enzyme 9 Figure 2.1. Change in [Ca2+]j in Response to Bradykinin 24 Figure 2.2. Effect of Increasing Bradykinin Concentration on the [Ca2+]i Response 25 Figure 2.3. Inconsistent [Ca2+]i Response to Isoproterenol 26 Figure 2.4. Effect of Epinephrine and Norepinephrine on [Ca2+]i in Single Cells 30 Figure 2.5. Effect of 8-bromo-cAMP and Forskolin on [Ca2+]j 31 Figure 2.6. Effect of Prostaglandin E2 on [Ca2+]; in a Single Cell 32 Figure 2.7. Effect of Verapamil on the Bradykinin-induced [Ca2+]; Response in a Single Cell 33 Figure 2.8. Effect of High Extracellular K+ on [Ca2+]j in a Single Cell 34 Figure 2.9. Effect of Nominally Ca2+-free Medium on the Bradykinin-induced [Ca2+]; Response in a Single Cell 35 Figure 3.1. Effect of Cell Culture Duration on Cell Responsiveness 43 Figure 4.1. Effect of Increasing Adenosine Triphosphate Concentration on [Ca2+]i 50 Figure 4.2. Change in [Ca2+]j During Sequential Exposures to Adenosine Triphosphate and Uridine Triphosphate 51 Figure 4.3. Effect of Increasing Uridine Triphosphate Concentration on [Ca2+]i 52 Figure 4.4. Effect of Increasing Adenosine Diphosphate Concentration on [Ca2+]j 54 Figure 4.5. Effect of Increasing Adenosine Monophosphate Concentration on [Ca2+];. . . . 55 Figure 4.6. Effect of Increasing Adenosine Concentration on [Ca2+]i 56 Figure 4.7. Effect of Increasing Guanosine Triphosphate Concentration on [Ca2+]; 57 ix Figure 4.8. Effect of Guanosine Triphosphate and Adenosine 3'-5' Cyclic Monophosphate on [Ca2+]i 58 Figure 4.9. Effect of Nominally Ca2+-free Saline on the UTP Stimulated Increase in [Ca2+]j in a Single Cell 59 Figure 5.1. Effect of a 15 Second Exposure to 100 uM Histamine on a Single Cell 65 Figure 5.2. Effect of a 45 Second Exposure to 100 uM Histamine on a Single Cell 66 Figure 5.3. Effect of Repeated Stimulations with Histamine, 8 Minutes Apart, in a Single Cell 67 Figure 5.4. Effect of Repeated Stimulations with Histamine, 30 Minutes Apart, in a Single Cell 68 Figure 5.5. Effect of Increasing Histamine Concentration on [Ca2+]j 69 Figure 5.6. Effect of a 30 minute Exposure to Ca2+-free Saline on the Histamine-stimulated Increase of [Ca2+]; in a Single Cell 71 Figure 5.7. Removal of Extracellular Ca2+ for 30 minutes Without Histamine Pre-stimulation Also Abolishes the Histamine-stimulated Increase in [Ca2+]i in a Single Cell 72 Figure 5.8. Removal of Extracellular Ca2+ for 5 minutes Does Not Attenuate the Histamine-stimulated [Ca2+]i Response 73 Figure 5.9. Effect of Restoring Extracellular Ca2+ on the [Ca2+]j Response to Histamine-stimulation 74 Figure 5.10. UTP-stimulated [Ca2+]j Increase After Abolition of the Histamine-stimulated [Ca2+]i Increase by Incubation in Ca2+-free Saline 75 Figure 5.11. Effect of the Hrreceptor Antagonist Pyrilamine on the Histamine-stimulated Increase in [Ca2+]j 76 X Figure 5.12. Variable Effect of the H2-receptor Antagonist Cimetidine on the Histamine -stimulated Increase in [Ca2+]; 77 Figure 5.13. Effect of the H3-receptor Antagonist Thioperamide on the Histamine-stimulated Increase in [Ca2+]i 79 Figure 6.1. Current Model of cAMP Mediated CI" Transport 89 Figure 6.2. Proposed Organization of Intracellular Second Messengers in Human Airway Epithelia 98 Figure app. 1. Shifting Excitation Peak of Fura-2 with Changes in [Ca2+] 116 xi List of Abbreviations ADP AMP ATP BK Br-A23187 Ca2+ t e a 2 ! [Ca2+]0 cAMP CF CFTR C02 DPPE EGTA Epi F12-5 supp. F12-6 supp. FBS FEV, fura-2 AM GnRH - adenosine 5'-diphosphate - adenosine 5'-monophosphate - adenosine 5'-triphosphate - bradykinin - calcium ionophore - free ionic calcium - intracellular free ionic calcium - extracellular free ionic calcium - cyclic adenosine 3':5'-monophosphate - cystic fibrosis - cystic fibrosis transmembrane regulator - carbon dioxide - Af,A -^diethly-2-[4-(phenlymethyl)phenoxy]ethanamine HCl - ethyleneglycol-bis-(6-aminoethyl ether) N,N,N',N'-tetraacetic acid - epinephrine - Ham's F12 culture medium (with 5 media supplements) - Ham's F12-5 supp. with cholera toxin - fetal bovine serum - 1 second forced expiratory volume - fura 2-acetoxy-methyl ester - gonadotropic hormone-releasing hormone GTP HBS Hm IP3 Iso MEM Na+-K+ ATPase NE PAF PGE2 Quin-2 UTP xn guanidine 5' triphosphate Hank's balanced saline histamine D-myo-inositol 1,4,5-trisphosphate isoproterenol minimum essential medium sodium, potassium pump norepinephrine platelet activating factor prostaglandin E2 2 -[(2-bis-[carboxymethyl] am in o-5 -methyl p h e n o x y ) m e t h y l ] - 6 - m e t h o x y - 8 bis [carboxymethyl] aminoquinoline uridine 5' triphosphate xiii Acknowledgements I wish to thank Dr. John Phillips for the support, guidance and generosity which he extended throughout this study. I also wish to thank Dr. Sidney Katz for extending a helping hand when I needed it the most. I thank Dr. Kenneth Baimbridge for the timely use of his equipment as well as the time, effort, and enthusiasm he invested in this project. I thank Dr. John Hanrahan for allowing me the opportunity to work in his laboratory, as well as for his kind tutelage in the art of patch-clamping. I thank Drs. N. Auersperg, D. Randall, D. Applegarth and J. Steves for their advice and comments concerning this study and this manuscript. I thank Maryette Mar for her expert technical assistance as well as for helping me keep everything in perspective. I thank Joan Martin for helping me make the original transition into the laboratory. I would also like to thank the local otolaryngology surgeons, especially Drs. A. Blokmanis and H. Stevens for supplying the human tissue used in these experiments. I would like to thank the Canadian Cystic Fibrosis Foundation and the Natural Sciences and Engineering Research Council of Canada for their financial support during my graduate studies. I acknowledge the assistance of Dr. M. Bridges with tissue culture techniques during the initial period of this study. I would like to thank my parents, and particularly my father for their support and assistance in the preparation of this manuscript. I especially thank my wife Marianne, for her support and tolerance during my doctoral quest as well as for her critical review of this manuscript, and my sons Patrick and Robert, without whom all this would be meaningless. 1 CHAPTER ONE: General Introduction Mucociliary Transport in the Airway and the Role of the Periciliary Fluid Layer The primary pulmonary defense against inhaled particles, such as dust or smoke, is mucociliary clearance. The surface of the airway is covered with a layer of mucus which is propelled up the respiratory tree into the esophagus. The mucus floats on a thin watery layer, referred to as the periciliary fluid layer. Cilia, located on the apical surface of the epithelial cells, beat in a coordinated fashion, which propels the mucus over the airway surface. The depth of the periciliary fluid layer is crucial to the efficient movement of the mucus blanket (Welsh, 1987). The periciliary fluid layer is maintained at a thickness of about 6 urn, approximately the same as the length of the cilia. Although the relationship between the periciliary fluid layer, ciliary beating, and mucus movement is not clearly understood, it is reasonable to postulate that if the periciliary fluid layer is too thick, then ciliary beating will fail to impart adequate movement to the mucus blanket. Alternatively, if the periciliary fluid layer is too thin, the rhythmic beating of the cilia would be impaired by direct contact with the viscous mucus, also resulting in inefficient clearance of mucus from the respiratory tree. The elimination of the periciliary fluid layer would likely result in insufficient hydration of the mucus blanket. This may interfere with bulk mucus clearance during coughing. Electrolyte transport plays a critical role in regulating the composition and depth of the periciliary fluid layer in the respiratory tract (Welsh, 1987), yet the precise relationship between electrolyte transport and mucociliary clearance is not well understood. One hypothesis in the literature suggests that mucus secretion by the submucosal glands and goblet cells is followed by electrolyte and fluid transport (Nadel et al, 1979). This is certainly true in the upper airways of larger mammals, where evaporative water loss is significant (Man, 1979). However, this is 2 probably not the case in the airways of smaller animals, as well as the smaller diameter airways in larger organisms, such as man. An alternative hypothesis is that a surfeit of fluid produced in the lungs migrates up the respiratory tree (Kilburn, 1968). The progressively smaller surface area of the airways encountered by the fluid as it moves up the respiratory tree necessitates the active absorption of electrolytes and fluid to prevent an obstructive build-up in the airway (Welsh, 1987). Dehydration of the airway surface becomes problematic in upper airways due to evaporative water loss. In these airways fluid secretion would function to replenish the diminished periciliary fluid layer. Mechanisms of Epithelial Ion Transport Primary active ion transport across epithelia always occurs as two steps. The energy consuming step consists of translocation of an ion across the cell membrane, against its electrochemical gradient. The active step can occur at either the basolateral membrane or the apical membrane and can function to increase or decrease the intracellular levels of the ion being transported. Because active epithelial transport involves the movement of ions across two membranes (apical and basolateral), carrier mechanisms such as ion channels or ion exchangers must be located on the membrane which is contralateral to the active translocating step. There are two possible arrangements for these components: The ion translocating enzyme can increase the intracellular electrochemical potential of an ion, which will result in the ions exiting the cell by diffusion through passive transport mechanisms (such as ion selective channels, ion exchangers or ion cotransporters) in the opposite membrane. However, if the active step is to pump ions out of the cytoplasm into the extracellular fluid, the ions will enter the cell through passive transport mechanisms located in the opposite membrane. In both cases, the functions of both membranes must be tightly coordinated. 3 Ions (and other substrates) can also be translocated across epithelia via the indirect action of an ATPase. For example, amino acids are transported across the gut via a Na+-amino acid cotransport mechanism which utilizes the Na+ concentration gradient established by the actions of the Na+-K+ ATPase to raise intracellular amino acid levels above thermodynamic equilibrium. The amino acids then move downhill out of the gut epithelial cell and into the blood. Thus, amino acids are actively transported, but in an indirect manner. This type of transport is referred to as secondary active transport. CI" ions are transported in many tissues via a Na+-K+-2C1" cotransport mechanism which also utilizes the Na+ gradient established by the actions of Na+-K+-ATPase. As a result, intracellular CI" levels are raised above thermodynamic equilibrium with the external fluid. CI" then diffuses out of the cells through anion selective channels (Al-Bazzaz et al, 1981; Frizzell et al, 1976; Welsh, 1983; Welsh and Widdicombe, 1980). Thus, CI" is actively transported through secondary active transport. Commonly, two patterns of transport are seen in epithelia (Frizzel et al, 1979; Fig. 1.1). Absorbing epithelia (e.g. kidney, gall bladder, ileum and toad urinary bladder) have apically located Na+-coupled cotransport mechanisms which import into the cell the desired substrates (CI', amino acids, sugars). Na+, which enters the cell in this manner, is pumped out basolaterally by the Na+-K+ ATPase (DeWeer, 1983). Basolaterally located transport systems (usually channels or cotransport mechanisms) allow the transported substrate to exit the cell. In contrast, the other major pattern of ion transport is seen in secretory epithelia {e.g. trachea, colon, cornea, and stomach). In this instance, the Na+-K+-2 CI" cotransport mechanism is located on the basolateral membrane. CI" enters the cell from the blood side against a ^ Apical 7TT Na J Basal ^ Na ^ . tr X CI r t£ j ^ B Figure 1.1. The Two Common Arrangements of Transport Across Epithelial Cells. A) Actions of the Na+-K+-ATPase results in a decrease in intracellular Na+ levels. Uptake of substrate X (such as amino acids, sugars, and ions) is coupled to the influx of Na+ down its concentration gradient, or Na+ simply enters the cells through apically located cation specific channels. Substrate X then leaves the cell usually through ion selective channels or carriers located on the opposite membrane. The Na+ which entered apically, is then pumped out basolaterally. B) CI* enters the cell through a basolaterally located Na+-K+-2C1' cotransport mechanism, which is driven by the Na+ gradient. CI" exits the cell apically through anion-specific channels. (Figure adapted from Frizzell et ah, 1979). 5 concentration gradient. Apical CI" channels allow CI" to exit the cell. K+ brought in with the CI" exits the cell through basolateral K+ channels (Welsh and McCann, 1985). Generally, Ca2+ activates the basolateral K+-specific channel, while cAMP activates the apical CI" channels. Na+ is recycled through the basolateral membrane. The increased [Na+]j caused by the cotransport mechanism stimulates the Na+-K+-ATPase, which in turn ejects excess Na+ (see review by Frizzell et al, 1979). Ion Transport Events in Airway Epithelia As previously mentioned, mucus clearance by the airway depends on the maintenance of an aqueous layer between the apical membrane and the mucus layer. This layer is maintained by the ion transporting cells of the airway epithelia. The fluid movements which occur in the upper airway epithelia appear to be the result of the movement of two ions in opposite directions, active absorption of Na+ (with CI" following passively) and secondary active secretion of CI" (with Na+ following passively: Frizzell et al, 1982; Jarnigan et al, 1983; Oliver et al, 1975; Shorofsky et al, 1982; Widdicombe et al, 1979; Widdicombe and Welsh, 1980). It is generally accepted that both transport events occur in only a single cell type in airway epithelia (Widdicombe et al, 1979). Figure 1.2 illustrates the model for ion transport at the inception of this study. Uphill CI' entry into the cell across the basolateral membrane is coupled to the entry of Na+ through a bumetanide-sensitive Na+-K+-2C1 -cotransport mechanism. CI" exits the cell passively through apically located channels. The Na+ which enters the cell with the CI' is pumped out of the cell by the basolaterally located Na+-K+-ATPase which is inhibited by ouabain. Excess intracellular K+ exits the cell through basolaterally located Ca2+-activated K+-specific channels (Frizzell, 1977; Welsh and McCann, 1985; Welsh and Leidtke, 1986). At the apical membrane, Na+ enters the cell through an amiloride- sensitive cation channel and is pumped out by the basolateral Na+-K+-6 Figure 1.2. Initial Model for NaCl Transport Across Airway Epithelia. Under unstimulated conditions, Na+ enters the cell through cation channels located in the apical membrane. The Na+ diffuses across the cell and is pumped out by the basolaterally located Na+-K+ ATPase. CI" ions follow passively through the paracellular shunt, down the electrical gradient created by the movement of Na+. Upon stimulation with a variety of secretagogues, the apical Na+ channels close and apical CI" channels open, allowing CI" to exit the cell on the luminal surface. CI" enters the cell across the basolateral membrane via the Na+-K+-2 CI" cotransport mechanism which is driven by the Na+ concentration gradient. Excess K+ exits through the basolateral membrane via a Ca2+- activated K+ channel (based on data presented at the First Annual North American Cystic Fibrosis Conference, Oct. 1987). 7 ATPase. The net result is generation of a lumen negative transepithelial potential difference which draws Na+ and K+ passively along via the paracellular pathway (Welsh et al, 1982a; Welsh et al, 1982b; Welsh et al, 1983). The relative contributions of the Na+ uptake (fluid absorption) versus CI" secretion (fluid secretion) to the total current flow in short-circuited airway epithelia depends, at least in part, upon the degree of 6-adrenergic stimulation (epinephrine). Under unstimulated conditions, ~33%of the IJC in human trachea can be accounted for by the bumetanide-sensitive CI' flux to the mucosal side (Widdicombe et al, 1985a; Widdicombe et al, 1985b). The amiloride-sensitive uptake of Na+ accounts for approximately 38% of 1^ . The addition of ouabain to the adventitial side results in a total collapse of \. in canine trachea in vitro (Widdicombe et al, 1979), demonstrating that all ion transport across this epithelium is driven by the actions of the Na+-K+ ATPase. 6-adrenergic stimulation results in a dramatic increase in the amount of CI" secretion (Welsh et al, 1983; Widdicombe and Welsh, 1980). At first appearance, the mode of ion transport exhibited by these epithelia seems energetically wasteful with active transport of NaCl in both directions at once (Fig. 1.2). However, if one considers the tight constraints applied to the depth of the periciliary fluid layer, the functioning of the ion transporting mechanisms in the tissue becomes clearer. Under conditions where the aqueous layer is too thick, active Na+ absorption takes place to reduce the depth of the fluid layer. However, during enviromental conditions under which water is being lost from the periciliary fluid through evaporation , the more potent system (active CI" secretion) is stimulated resulting in the replenishing of the periciliary fluid. 8 Intracellular Regulation of Ion Transport Signal Transduction at the Plasma Membrane The involvement of second messengers in hormonal signal transduction has been well reviewed (Berridge and Irvine, 1984; Rasmussen et al., 1985; Casey and Gilman, 1988; Casey et al., 1988). Several second messengers have been established, including cAMP, Ca2+, inositol 1,4,5-trisphosphate, diacylglycerol, cGMP, and histamine. The most studied second messenger system involves the production of cAMP. Briefly, in the case of a stimulatory hormone, an agonist binds with a specific receptor in the cell membrane. The hormone-receptor complex (H-R) then binds to a membrane-bound GTP-binding protein (Gs), which is composed of 3 subunits, an alpha unit Ga, a beta unit G6, and a gamma unit G r The binding of the hormone-receptor complex to the GS-GDP complex results in the release of GDP from the hormone-receptor-Gs complex after which GTP is taken up in its place. The hormone-receptor complex dissociates from the activated GS-GTP complex. The Gey dissociates from the Ga-GTP complex which binds to and activates adenylate cyclase (E) on the internal membrane surface (Casey and Gilman, 1988; Casey et al., 1988). Endogenous GTPase activity in the Ga subunit eventually converts the GTP to GDP, rendering the adenylate cyclase-Ga complex inactive. After inactivation, the effector enzyme dissociates from the Ga-GDP complex, which then combines with the Gfir thus completing the cycle. The initial stages of inhibitory hormone action are similar to the initial steps already outlined in the previous paragraph. Binding of the hormone to the receptor results in the formation of a G^-GTP complex which dissociates into Ga-GTP and G6. However, in this case GDP G-GTP H-R G-GDP H-R G-GTP Figure 1.3. Interactions between hormone receptor, G-protein, and effector enzyme. (*) Denotes the initiation of the cycle which begins with the binding of a hormone to the plasma membrane-bound receptor. The reaction then proceeds in the direction of the arrows (see text for a complete description). 10 Ga-GTP does not bind to an effector enzyme. Rather, the excess production of the GBr complex, which is interchangeable with the Gay-complex from the GS-GTP, drives the equilibrium away from the adenylate cyclase-Ga-GTP complex resulting in less cAMP production (see reviews by Casey and Gilman, 1988; Casey et al, 1988). The Ga-activated adenylate cyclase converts ATP to cAMP, thus increasing the cytosolic concentration of this cyclic nucleotide (Gilman, 1987; Gilman, 1990). Cyclic-AMP binds to a cAMP-dependent protein kinase (A-kinase) which in turn phosphorylates target proteins in the cell. In the case of ion transporting epithelia, these proteins can be either the ion channels or ion carrier proteins themselves, or they can be secondary kinases which will subsequently phosphorylate the carrier or channel proteins. The net result of the phosphorylation of the carrier or channel protein is a change in the rate of ion transport. Intracellular levels of cAMP are returned to normal by phosphodiesterases, which break down cAMP to 5'AMP (Rasmussen, 1970). Activation of cAMP mediated channel or carrier proteins is ended by their dephosphorylation. Ca2+ has now been widely implicated as a second messenger (Borle and Uchikawa, 1979; Berridge and Irvine, 1984; Rasmussen 1970; Rasmussen etal, 1985; Berridge etal, 1988). As with the cAMP-activated pathway, the initial binding of hormone to receptor results in activation of a membrane GTP-binding protein (different from that involved in the cAMP-activated pathway). The activated GTP-binding protein activates phospholipase-C, which cleaves phosphatidylinositol 4,5-bisphosphate to D-myo-inositol 1,4,5-trisphosphate (IP3), which is released into the cytoplasm, and 57?-l,2-diacylglycerol (DG), which remains within the membrane. IP3 through conversion into inositol 1,3,4,5-tetrakisphosphate, may open Ca2+ channels in the plasma membrane (Irvine, 1989, Luckhoff and Clapham, 1992). IP3 may also initiate an inositide 11 shuttle (which transports Ca2+ across membranes through rapid phosphorylation-dephosphorylation of phosphatidylinositol; Brockeroff, 1986). More commonly, IP3 triggers release of Ca2+ sequestered in the endoplasmic reticulum (Joseph et al, 1984; Gill et al, 1986; Spat et al, 1986), which results in the elevated cytoplasmic Ca2+ concentration ([Ca2+]j). Ca2+ may act in several ways to stimulate a response. Sometimes Ca2+ first binds to calmodulin. Many enzymes (such as adenylate cyclase, some nucleotide phosphodiesterases, and Ca2+-ATPase) are activated upon binding to the Ca2+-calmodulin complex (or other Ca2+-binding proteins; Rasmussen, 1983). The Ca2+-calmodulin complex can activate one of several protein kinases (Cheung, 1980) resulting in the phosphorylation of target enzymes (Marme and Maltzenauer, 1985) analogous to the adenylate cyclase-mediated pathway (Alberts et al, 1983; Walaas and Nairn, 1985). Ca2+ can also act without the involvement of a calcium binding protein. In these instances, Ca2+ binds directly to the target enzyme or ion channel {e.g. the Ca2+-activated K+ channels) resulting in a conformational change which gives rise to a change in the enzyme activity or transport rate (Berridge and Irvine, 1984). A significant modulator of ion channel activity is protein kinase-C (PKC), which is activated by DG and Ca2+. PKC has been shown to activate many ion channels (Shearman et al, 1989). Recently, it has become apparent that [Ca2+]j and cAMP often interact as synarchic (interdependent) second messengers within cells (Rasmussen et al, 1985). In many instances, an increase in cytosolic cAMP results in an increase in [Ca2+]; (Reuter, 1983; Luini et al, 1985; Rasmussen et al, 1985; Piascik et al, 1986). Thus, cAMP may activate Ca2+ channels within the endoplasmic reticulum or the plasma membrane. Evidence suggests that the Ca2+-specific channels are activated through phosphorylation by a cAMP-dependent protein kinase (A-kinase; Reuter, 1983). 12 A second possible mechanism by which cAMP and Ca2+ may act as synarchic messengers involves a cAMP-mediated change in Ca2+ sensitivity of one or more of the Ca2+dependent response elements (Rasmussen et al. 1985). A common mechanism by which this so-called sensitivity modulation is accomplished involves phosphorylation of a Ca2+ response element by a cAMP-dependent protein kinase (Rasmussen et al, 1985; Reuter, 1983). Thus, although no change can be detected in [Ca2+];, it is still intimately involved in controlling the cellular response to a hormone. Many ion channels have been shown to be under second messenger control (Camardo and Sieglebaum, 1983; Kolb etal, 1986; Petersen and Maruyama, 1983; Reuter,1983; Sauve et al, 1986). Because Ca2+ often acts on different channels than does cAMP {e.g. cAMP activates apical CI" channels and Ca2+ activates basolateral K+ channels in many CI" secreting epithelia), Ca2+ has been suggested as a second messenger which coordinates ion movement across apical and basolateral membranes of transporting epithelia (Taylor and Windhager, 1979; Chase, 1984; Cuthbert, 1985). [Ca2+]i has also been shown to stimulate epithelial ion secretion in CI* secreting epithelia (Frizzel, 1976; Bolton and Field, 1977). Rationale for the Present Study Two major diseases, cystic fibrosis (CF) and asthma both affect fluid secretion by airway epithelia.1 Cystic fibrosis causes inadequate fluid secretion, whereas asthma is characterized, in part, by elevated secretion of fluid and mucus by airway epithelia. In order to devise more effective treatments for these diseases, a better understanding of the processes which control fluid 1 For a detailed description of these diseases refer to appendix 1. 13 secretion and absorption in airway epithelia is essential. This thesis examines the role of several secretagogues and inflammatory mediators in stimulating increased [Ca2+]j, and thus initiating CI" and fluid secretion. The effects of these inflammatory mediators and secretagogues is discussed in the context of the two major medically important disease processes in which abnormal CI" and fluid secretion are involved. At the inception of this study, the precise nature of the cause of CF was unknown. Quinton (1983) first suggested that CF was the result of an abnormally low CI" permeability, on the basis of ion replacement studies performed on sweat gland ducts. Similarly, in monolayer cultures of human tracheal cells, cultures established from patients with CF exhibit a decreased apical CI" permeability (Widdicombe et al, 1985). In situ potential difference measurements demonstrate that CF airway epithelia generate a greater potential difference than do normal airway epithelia. This has also been ascribed to a lack of CI" transport (Knowles et al, 1981; Knowles et al, 1982; Knowles et al, 1983). Using the patch-clamp technique (Hamil et al, 1981), Welsh (1986) described an apical CI" channel in human tracheal cells. This channel has a conductance of -25 pico-Siemens and shows strong inward rectification (Shoemaker et al, 1986). In the cell-attached mode, this channel is activated by 13-adrenergic agonists which are known to stimulate CI" secretion (Frizzel et al, 1986; Shoemaker et al, 1986). As well, this channel can be activated by an increase in [Ca2+]j (Frizzel et al, 1986; Shoemaker et al, 1986; Welsh, 1986). The CF defect had been localized to the second messenger pathway which was activated by adrenergic stimulation (Sato and Sato, 1984; Fig. 1.2). Several groups reported that the above mentioned channel could be directly activated by ATP and the catalytic subunit of cAMP-dependent protein kinase, in excised inside-out membrane patches, from cells of normal individuals, but not of patients with CF (Shoumacher et al, 1987; Li et al, 1988; Hwang et al, 1989). However, it now appears that this channel is not the one involved in CF (as will be 14 discussed in chapter 6) but rather a channel involved in cell-volume regulation (Worrel et al, 1989). One of the characteristics of asthma is a hypersecretion of airway luminal fluid. It is reasonable to suggest, on the basis of this observation, that some of the inflammatory mediators which are involved in the allergic response to inhaled allergens could stimulate increased airway fluid secretion. Histamine (Hra) is clearly involved in the development of the asthmatic response, although the exact mechanism is poorly understood (Howarth, 1990). However, Hm has been shown to work through increasing [Ca2+]j levels in a variety of target cells (Nakahata and Harden, 1987). Abnormal fluid secretion is at the heart of both CF and asthma, and both diseases exhibit their major, clinically important, symptoms in the respiratory tree (asthma exclusively so). Thus, it becomes important to understand exactly what role [Ca2*]; plays in mediating airway fluid secretion. Specifically, manipulation of [Ca2+]i levels may offer at least symptomatic relief of CF respiratory problems. Conversely, understanding what role [Ca2+]i plays in mediating the hyper-secretion observed in the asthmatic response may open new avenues of treatment for this widespread disease. In this thesis, I address the following general questions: 1) Do inflammatory mediators which stimulate CI" and fluid secretion across airway epithelia, operate through [Ca2+]i as a second messenger? 2) Are there interactions between the second messengers cAMP and [Ca2+]j? 3) What is (are) the source(s) of the [Ca2+]j second messenger signal? 15 Human nasal epithelial tissue was used in this study for three reasons: First, this tissue exhibits CI" secretion both in vivo (Knowles et al, 1981), and in vitro (Knowles et al, 1983), and it is affected by CF (Knowles et al, 1983; Yankaskas et al, 1985). Second, excised human nasal tissue is readily available as a by-product of corrective surgery for nasal obstruction (removal of nasal polyps and correction of congenital airway obstruction). And finally, nasal tissue probably exhibits some of the characteristic of asthma (George and Owens, 1991). I investigated the role of the inflammatory mediator bradykinin (BK) on [Ca2+]j in cultured human nasal epithelial cells. BK has been shown to stimulate the release of prostaglandins (including PGE2) by airway epithelial cells (Leikauf et al, 1985), so the effects of PGEj on [Ca2+]j were also tested. In addition, I investigated the effects on [Ca2+]i of the 8-adrenergic agonists epinephrine, norepinephrine, and isoproterenol. These G-adrenergic agonists have been shown to stimulate airway CI" and fluid secretion. These results are presented in chapter 2. I then discuss potential interactions between Ca2+ and cAMP, as well as potential sources for the increased [Ca2+]j which was detected after BK stimulation. During the course of this study, the physiological responses of the cultured nasal epithelial cells changed radically. Specifically, cells which were cultured before February 1990, responded to BK and ATP stimulation even after 7 days in cell culture. Cells which were cultured after February 1990 significantly lost receptivity to ATP and BK after as little as 3 days in cell culture. In chapter 3, I discuss the observed changes in cell physiology, as well as a potential pre-operative cause of the change. Recently, it has been reported that many cell types release ATP (Burnstock, 1990). ATP is released either as a product included in granules of secretory cells, or is simply extruded 16 across the cell membrane through a mechanism which is unknown at the present time. ATP has been shown to stimulate degranulation in several cells of the immune system (McDonald, 1988; Huang et al, 1989). In chapter 4, I investigated whether extracellular ATP, which is clearly involved in the inflammatory response, can influence [Ca2*];. In addition, I provide evidence that ATP stimulation releases [Ca2+]i from intracellular stores in a manner similar to BK. Histamine has been shown to stimulate CI" secretion across canine tracheal epithelium (Marin et al, 1978), and has been shown to stimulate increased [Ca2+]j in many cell types which are Hm responsive (see review by Hill, 1990). Operating on the general assumption that inflammatory mediators operate through the [Ca2+]i second messenger pathway, I tested whether Hm could stimulate increased [Ca2+]i. These experiments are discussed in chapter 5. I also investigated the source of the increased [Ca2+]j which was observed upon Hm stimulation, and present data which demonstrate that Hm stimulation releases [Ca2+]; from an intracellular pool which is distinct from the pool which is mobilized by stimulation with BK or ATP. In chapter 6,1 discuss the results obtained from these investigations, and present a model for the second messenger pathways which are involved in the control of CI" and fluid secretion across airway epithelial cells. 17 CHAPTER TWO: Effects of Bradykinin, PGE2 and Adrenergic Agonists on [Ca2+], Research on cystic fibrosis has been hampered by the diverse clinical symptoms which present in the face of a simple, single autosomal recessive gene inheritance pattern. Indeed, the defective gene has been identified in cystic fibrosis (Rommens et al, 1989), yet the actual mechanism of the dysfunction is still unknown. Defective electrolyte transport across exocrine epithelia is thought to precipitate the many symptoms of CF. Extensive studies have been conducted on the ion transport processes across mammalian airway epithelia (Quinton, 1990). In particular, much experimentation has been conducted using canine tracheal tissue as a model for human airway epithelia (see Welsh, 1987 for review). Normally, under unstimulated conditions, Na+ enters airway cells through ion channels located in the apical membrane, eventually to be extruded basolaterally by the Na+-K+ ATPase. CI" and water follow passively, resulting in dehydration of the luminal surface of the respiratory mucosa (Welsh, 1990). Upon stimulation with a variety of secretagogues (primarily adrenergic agonists), apical Cl"-specific channels open. A basolaterally located Na+-K+-2C1" co-transporter, driven by the Na+ gradient, imports CI" against its thermodynamic equilibrium. Attention has been focussed on the intracellular second messenger pathways which control the apical ion channels in airway epithelia. Increased intracellular levels of cAMP and Ca2+ have both been implicated in stimulating CI' secretion across airway epithelia. It has been well documented that increased intracellular levels of cAMP can stimulate CI" secretion (Welsh, 1987). However, our knowledge of the role that intracellular free ionic Ca2+ ([Ca2+]j) plays in controlling electrolyte and water transport across both canine and human upper airway epithelia 18 is incomplete. Accordingly, in the present study I have investigated secretagogue stimulation of the intracellular Ca2+-based second messenger systems in primary cultures of human airway epithelia. McCann et al. (1989) reported that 13-adrenergic agonists and bradykinin (BK) stimulated increased [Ca2+]j in canine tracheal epithelial cells. I investigated whether these secretagogues would have similar effects on human upper airway epithelia. Materials and Methods Cell Culture Excised human airway epithelial tissue specimens were obtained as by-products of nasal surgeries (either polypectomies or turbinate resections) from non-CF patients. All experimental procedures had the approval of the University of British Columbia ethics committee (certificate # C90-222). Epithelial cells were dissociated from tissues by treatment with pronase E (0.1% [w/v]; Type XIV, Sigma Chemical Co., St. Louis, MO) and DNAase (0.1 mg/mL; Type 1 from bovine pancreas, Sigma Chemical Co., St. Louis, MO) in Joklik's modified Ca2+-free minimum essential medium (MEM) for 48 hours at 4° C. Fetal bovine serum (FBS) was added (to a final concentration of 10% v/v) to neutralize the enzymatic reactions, and the dispersed cells were filtered through a sterile Nitex nylon mesh (60 um). Two hundred-fifty microliter aliquots of the cell suspension were taken to determine cell numbers (using a stage micrometer), and cell viability (using trypan blue exclusion). The cell suspension was centrifuged at 150 x g for 6 minutes, resuspended in MEM containing 10% FBS, and recentrifuged as before. Cell pellets were resuspended at a density of 1.5 x 105 cells/mL in Ham's F 12 medium (Sigma Chemical Co., St. Louis, MO) supplemented with insulin (2 |ig/mL), hydrocortisone (100 nM), T3 (3 nM), endothelial cell growth supplement (4 ug/mL), epidermal growth factor (12.5 ng/mL), cholera 19 toxin (10 ng/mL) and antibiotics (penicillin 60 units/mL, streptomycin 60 units/mL and gentamicin 50 ng/mL; Wu et al.., 1985). All supplements were obtained from Collaborative Research (Bedford, MA), except cholera toxin which was purchased from Calbiochem Biochemicals (San Diego, CA). Cells were then plated onto poly-L-lysine-treated 18 mm round glass coverslips and incubated at 37° C for 24 hours, to facilitate attachment. Cells were washed and fed every two days with the above mentioned supplemented Ham's F 12 medium without cholera toxin. Cholera toxin in the initial plating medium was required for cell attachment to the coverslips. After attachment, the cholera toxin was withdrawn so as not to influence intracellular cAMP metabolism during experiments. Cell Preparation for [Ca2+]i Determination After 3-5 days of incubation at 37°C in a humidified (100% relative humidity) atmosphere of 5% C02 in air, sub-confluent cell cultures were loaded with fura-2 AM (Molecular Probes, Inc., Eugene, OR) as described previously (Grynkiewicz et al., 1985). Briefly, 40 ug of fura-2 AM was dissolved in 25 uL dimethylsulfoxide (DMSO), to which 12.5 uL of a 20% pluronic F-127 (a non-ionic detergent) solution (in DMSO) was then added. This mixture was added, under intense agitation, to 2 mL of Hank's balanced saline (HBS, Sigma Chemical Co., St. Louis, MO) containing 0.1% (w/v) bovine serum albumin. Slow addition under intense agitation was necessary to prevent the formation of droplets of the fura-2 AM solution described above. The above mixture was diluted 1:1 with HBS. The 18 mm round coverslips containing the nasal cells were immersed in the loading medium for 1.5 hours at 21°C, after which they were rinsed once with HBS to wash out excess fura-2 AM. 20 Fluorescence measurements Fluorescence measurements were performed as previously reported (Wang et al., 1989). Briefly, individual coverslips were mounted, with the cellular monolayer facing downwards, onto a laminar flow-through chamber (volume, -350 uL). Silicone rubber cement was used to complete a watertight seal. The chamber was inserted into a stainless steel holder, and the entire assembly was mounted onto a temperature-controlled stage of a Zeiss Jenalumar microscope equipped for epifluorescence (Zeiss-Jena, New York, NY). The perfusion chamber was maintained at 37°C. The light source was a 200-watt mercury arc lamp powered by a DC power supply. The light was first passed through one of two differential interference filters (350 or 380 nm; bandwidth 10 nm) mounted in a turret which could be rotated by a computer-controlled stepping motor. The light was then passed through a 410-nm dichroic mirror and a xlOO apochromat oil immersion lens with a numerical aperture of 1.4 and an adjustable diaphragm to reduce the light intensity. A field diaphragm in the light path before the dichroic mirror was used to reduce the area of illumination to cover the cytosol of, at most, 2-3 epithelial cells at their junction, with care taken to exclude the nuclei of the cells (wherever possible, fluorescence from the cytosol of single cells was collected). All fluorescent light passed through a 450-nm bandpass filter to reduce background fluorescence. The emitted fluorescence was deflected to either the eyepieces or a camera port in which was mounted a photomultiplier tube, used to convert the fluorescence into a DC voltage. The voltage was converted to digital form, and measurements of fluorescent ratios, corrected for background, were obtained on a 1.8 second time base (measurements of fluorescence at each excitation wavelength were taken 0.35 seconds apart) with HBS constantly flowing at a rate of 3 mL/min through the chamber during the experiment. 21 Calculation of cytosolic calcium concentration The cytosolic calcium concentration was calculated using the following formula, as described by Grynkiewicz et al. (1985)2: [1] tCa2! = K, x 6 x (R - R^VCR^ - R) Where Kd is the equilibrium dissociation constant for the association of fura-2 with cytosolic free calcium {i.e. 224 uM), 6 is the ratio 380 nm with zero [Ca2+]/380 nm with infinite [Ca2+], R is the ratio at 350 nm/380 nm, R ^ is the ratio of 350 nm/380 nm at zero [Ca2+], R ^ is the ratio of 350 nm/380 nm at infinite [Ca2+]. Background fluorescence was determined by obtaining fluorescence samples for areas which were devoid of cells on each coverslip . For the present study, 8 = 4.28, R ^ = 0.52 and R ^ = 4.21. These values were determined using the same epithelial cell cultures exposed to Br-A23187 (HSC Research Development Corp., Toronto, Ontario, Canada), first in the presence of 1.8 mM Ca2+ in the perfusion medium, and then in a medium nominally free of Ca2+ and containing 5 mM EGTA. Because cellular components quench fura-2 fluorescence the calibration was conducted on permeabilized cells rather than on standardized Ca2+ solutions. 2 For a detailed description of the ratio fluorometric determination of [Ca2+]j refer to appendix 2. 22 Drug treatment All drugs were obtained from Sigma Chemical Co. (St. Louis, MO), dissolved in HBS and diluted to the desired concentration with the same medium. Five hundred uL bolus injections were made into the flow-through chamber. At a chamber perfusion rate of 3 mL/min, it was estimated that the drug solution would be in contact with the cultured cells for approximately 10 seconds. Cells were perfused with HBS for a minimum of 8 minutes between drug exposures to allow for the recovery of receptors and intracellular Ca2+ pools. Statistics Mean [Ca2+]j values before and after stimulation were compared using Student's t-test (Sokal and Rohlf, 1973). Differences were considered significant at the p < 0.05 (*) and p < 0.01 (**) levels. Results The observed morphology of the cultured cells agreed well with reports in the literature (Wu et al, 1985). Three cell types were readily distinguishable morphologically. The bulk of the cells were approximately 40-50 jam across, and had few processes which, when present, were very short. These cells were usually ciliated for the first 12-18 hours after plating, after which time the cilia were lost. These cells were considered to be the transporting cells. Larger cells (70-90 urn across) were also seen. These cells were never observed with cilia, and were assumed to be goblet cells. The third cell type observed possessed a completely different morphology. 23 These cells generally had a spindle shaped cell body (50-60 um in length and 10-15 um across) and possessed multiple, long cytoplasmic processes. These cells were assumed to be fibroblasts. [Ca2+]j was measured in only the first cell type. In preliminary experiments, I established that optimal fura-2 loading was obtained by incubating the cells in the fura-2 AM loading medium for 1.5-2 hr. at 21°C. Such an incubation minimized the incorporation of fura-2 into perinuclear granules (determined observationally). Figure 2.1 shows a typical [Ca2+]j response to bradykinin (BK). The duration of exposure was approximately 10 seconds (see Materials and Methods). [Ca2+]j begins to increase 15-20 seconds after the introduction of BK into the outer well of the perfusion chamber. This lag period mainly reflects the time requires for the agonists to actually reach the cells. [Ca2+]i increased sharply to a peak which was typically 3-10 times the normal resting [Ca2"1"]^  Often, [Ca2+]i levels oscillated around a peak value for 10-15 seconds. A moderately fast decrease in [Ca2+Ji followed, resulting in restoration of normal resting [Ca2+]; levels within 1-1.5 minutes after initial exposure to BK. Bradykinin stimulated an increase in [Ca2+]j in a dose-dependent fashion (Fig. 2.2). Although it is not apparent from the dose-response curve, individual cells behaved as if they had an all-or-none threshold for BK stimulation. Once the threshold had been reached, bradykinin pulses of increasing concentration would not further increase [Ca2+]i. Normal resting [Ca2+]; levels for this experiment were 149 ± 35 nM (n=10). Treatment with isoproterenol (Iso) had a somewhat paradoxical effect. In 4 out of 8 cells Iso (as high as 1 uM) had no effect (pre-stimulation [Ca2+]; was 124 ± 17 nM, peak stimulated 24 600 J 5 0 0 --1 .0 - 0 . 5 0.0 0.5 1.0 1.5 2.0 Time (min) Figure 2.1. Change in [Ca2+]i in Response to Bradykinin. At time 0, a single cell from a monolayer primary culture of human nasal epithelium was exposed to a 10 second (approx.) pulse of 100 nM BK. [Ca2+]i was calculated based on fura-2 fluorescence (see Materials and Methods). [Ca2+]j rose sharply to nearly 10 fold that of the resting [Ca2"1"]^  25 id + o 10 100 1000 BK Concentrat ion (nM) Figure 2.2. Effect of Increasing Bradykinin Concentration on the [Ca2+], Response. Increasing doses of BK increased [Ca2% The filled circle denotes normal unstimulated cellular [Ca2+]; levels for this study. Mean, S.E.M. (vertical bars) and N are indicated for each point. Significant increases in [Ca2+]i are indicated (* p < 0.05; ** p , 0.01). 26 Figure 2.3. Inconsistent [Ca2+], Response to Isoproterenol. (A) [Ca2+]i insensitivity of a single cell exposed to 0.1 and 1.0 uM isoproterenol, although the cell was responsive to BK (100 nM). (B) Isoproterenol induced change in [Ca2+]i followed by a standard dose of BK, in a single cell from a different primary culture than A (identical culture conditions). The cell had responded to 0.1 JUM BK prior to exposure to Iso (data not shown). 27 300 T 225 r-r 150 5 0 0 -4 0 0 -3 0 0 -2 0 0 -100-0-• 1 • • I •1 1 • • 1 1/xM 1 ^m Iso — H Time (min) 1 B ^ \ \ V ? 100 n 1 1 0 10 15 20 25 30 Time (min) 28 [Ca2+]j was 122 ± 29 nM; Fig. 2.3A, response of a single cell). However, in the other 4 cells, Iso (1 uM) had a profound stimulatory effect (p < 0.01) on [Ca2+]j release (pre-stimulation [Ca2+]j was 128 ± 21 nM, peak stimulated [Ca2+]i was 428 ± 53 nM; Fig. 2.3B, single cell). On at least two occasions, Iso exhibited this paradoxical effect on [Ca2+]j in different cells from the same primary culture. All these cells responded to BK (peak stimulated [Ca2+]i was 376 ± 62 nM; p < 0.01). Treatment with epinephrine (Epi; 1 uM, n=6; Fig. 2.4), norepinephrine (NE; 1 pM, n=5; Fig. 2.4) or prostaglandin E2 (PGF^ ;1 pM, n=5; Fig. 2.5) did not result in any detectable change in [Ca2+]j. The pre-stimulation [Ca2+]i levels for Epi, NE, and PGE2 were 102 ± 19 nM, 118 ± 24 nM, and 121 ±26 nM respectively. The peak [Ca2+]i levels reached after stimulation with Epi, Ne, and PGE2 were 112 ± 23 nM, 123 ± 21 nM, and 121 ± 19 nM respectively. The minor fluctuations of [Ca2+]; seen in figures 2.4 and 2.5 simply reflect the normal fluctuations in basal [Ca2+]( levels within these cells. Studies conducted on canine tracheal cells (McCann et al, 1989) have suggested that intracellular cAMP may elicit a [Ca2+]i response; however, as previously mentioned, experiments conducted on human nasal epithelial cells were not consistent with this hypothesis. I therefore further examined whether intracellular cAMP could initiate a [Ca2+]i response by testing the effects of both 8-bromo-cAMP (a membrane permeant cAMP analogue) and forskolin (Fig. 2.5). Perfusion of the cells with both 8-bromo-cAMP (500 uM, 2 minutes, n=5) and forskolin (100 uM, 10 minutes, n=5) had no effect on [Ca2+];. The pre-stimulation [Ca2+]j levels for cells exposed to 8-bromo-cAMP and forskolin was 98 ± 12 nM. The peak [Ca2+]i level during exposure to 8-bromo cAMP was 103 ± 14 nM, and the peak [Ca2+]i level during exposure to 29 forskolin was 100 ± 13 nM. The [Ca2+]; level in these cells increased to 248 ± 43 nM when exposed to a 1 uM ATP pulse (p < 0.01; see chapter 4). To determine the source of the increased [Ca2+]j, the effect of the voltage-gated Ca2+ channel blocker verapamil was tested, in an attempt to block any influx of extracellular Ca2+ ([Ca2+]0). Figure 2.7 depicts a typical [Ca2+]i response of a single cell to 100 uM BK, with and without 50 uM verapamil present in the bathing medium. Bathing the cells in HBS containing 50 uM verapamil for 10 minutes had no effect on the [Ca2+]i response to 100 u.M BK (n=4). The peak stimulated [Ca2+]i levels for these cells were 378 ± 54 nM and 386 ± 44 nM respectively (increased from a pre-stimulation [Ca2+]j level of 132 ± 24 nM). Furthermore, stimulation with a 100 nM BK pulse after verapamil had been washed out for 10 minutes resulted in a peak [Ca2+]j level of 401 ± 56 nM. In addition, depolarizing the cells by increasing extracellular K+ (50 mM) did not stimulate any increase in [Ca2+]j (n=4; Fig. 2.8). The pre-exposure [Ca2+]j level for cells exposed to 50 mM K+ was 99 ± 21 nM, while the peak [Ca2+]; level reached during K+ exposure was 116 ± 31 nM. To further test the hypothesis that [Ca2+]0 is not directly involved in the generation of the BK-induced [Ca2+]i response, cells were bathed for 15 minutes in nominally Ca2+-free saline (HBS, from which all calcium had been omitted). Such treatment decreased resting [Ca2+]j from 125 ± 28 nM, observed prior to perfusion of the "Ca2+-free" saline, to 70 ± 22 nM after removal of extracellular Ca2+ (n=4). As can been seen in figure 2.9, bradykinin (100 nM) was able to stimulate an increase in [Ca2+]i, although the peak Ca2+ levels observed (322 ± 43 nM) were much lower than the levels obtained by BK (100 nM) stimulation in saline containing 1.8 mM Ca2+ (511 ± 63 nM). [Ca2^ in cells bathed in "Ca2+-free" saline dropped from 138 ± 31 nM to 18 ± 6 nM when treated with A-23187 (n=3). 30 + u 200 150 100 50 0 1 1 [JM Epi 200 a 150 + i 10° 06 O 50 + 0 0 0.5 \ / -W v. 1 MM NE 1.5 Time (min) 2 2.5 Figure 2.4. Effect of Epinephrine and Norepinephrine on [Ca2+], in Single Cells. Exposure of different cells to 1 uM doses of Epi and NE failed to elicit an increase in [Ca2+]j (Epi, n=6; NE, n=5 for these treatments). 31 + O 200 150 1 0 0 -50 0 0 0.5 A v ^ -\ 1 fiM PGE, 1 1.5 Time (min) 2.5 Figure 2.5. Effect of Prostaglandin E2 on [Ca2+], in a Single Cell. Exposure of a single cell to 1 |iM prostaglandin E2 failed to stimulate an increase in [Ca2+]i (n=5 for this treatment). 32 S C + 300 2 5 0 -2 0 0 -150 100 50 0 0 Forskolin 8-Br-cAMP » Vv*- -i 1 fuM ATP 10 15 20 Time ( m i n ) 25 30 Figure 2.6. Effect of 8-bromo-cAMP and Forskolin on [Ca2+],. Exposing cells to 8-bromo-cAMP (500 uM, 2 min, n=5) and forskolin (100 uM, 10 min, n=5) had no effect on [Ca2+]i nor did such treatment inhibit the [Ca2+]i response to a 1 uM pulse of ATP (as compared to the response to an identical dose of ATP shown in figure 4.2). 33 + CQ O 500 T 400 300 2 0 0 -100 0 50 /JM Verapamil 4 \ 0 —'" ~| 10 15 Time (min) 20 25 Figure 2.7. Effect of Verapamil on the Bradykinin-induced [Ca2+], Response in a Single Cell. Bathing cells in saline containing 50 uM verapamil for 10 minutes did not attenuate the [Ca2+]j response to BK (n=4). Ten second pulses of 100 nM BK are denoted by the arrows. 34 + 03 O 600T 500 400 300 200 + 100 0 0 50 mM K + 3 Time (min) 6 7 Figure 2.8. Effect of High Extracellular K+ on [Ca2+], in a Single Cell. Exposure of the cell to high extracellular K+ causes depolarization of the plasma membrane which would trigger the opening of any voltage-gated Ca2+-specific channels. No increase in [Ca2"1"]; was observed during such treatment, suggesting that influx of extracellular Ca2+ through voltage-gated channels is not responsible for the BK-induced increase in [Ca2+]j (n=4). 35 + o ouu -600-400-200-0-_ / i i ' i AN I 75 nM BK 1 1— p_i_ Ca free saline i - - - , — j | • 1 100 nM BK 1 1 1 0 10 20 30 Time (min) 40 50 Figure 2.9. Effect of Nominally Ca2+-free Medium on the Bradykinin-Induced [Ca2*], Response in a Single Cell. Removal of all external Ca2+ for a period of 15 minutes attenuated, but did not abolish the [Ca2+]; response to BK (n=4). 36 Discussion Research into the physiological dysfunction underlying CF has been hampered by the lack of a suitable model tissue on which to conduct experiments. For this reason, much attention has been directed toward the development of immortal cell lines and the use of animal tissues. Some success has been achieved in transforming human airway epithelial cells using retroviral vectors (Sholte et al, 1989; Jetten et al, 1989; Buchanan et al, 1990). However, such cell lines may be viewed as undesirable for a variety of reasons. Transformed airway cell lines are still in their infancy and much more characterization of their physiology is needed. In addition, many subtle physiological perturbations associated with transformation may affect the cellular systems being studied. Furthermore, some researchers may have reservations about using potentially biohazardous material. Animal models are attractive due to the availability of tissue. However, CF appears to be restricted to humans, thus the use of animal models is limited to the study of normal ion transport events. Also, interspecies variations must be anticipated when using animal tissues to model human physiology. These considerations notwithstanding, electrolyte transport across canine airway epithelia has been very well studied (see review by Welsh, 1987) and much of what has been learned has been applied to our understanding of electrolyte transport processes across the human airway epithelium. Several factors affected the choice of tissue for this investigation. Nasal airway epithelium is a relatively simple tissue consisting of only 2-3 cell types, with ion transporting cells making up the majority of the cell population (Borysenko, 1984), making it a favorable experimental tissue for studies involving ion transporting cells in primary cell culture. Nasal 37 epithelium has been well studied and cells cultured from CF patients have been shown to exhibit the CF defect (Knowles et al, 1981; Knowles et al, 1982; Knowles et al, 1983). My results are consistent with McCann et al (1989), who observed that BK was able to increase [Ca2+]j in monolayer cultures of canine tracheal cells. Similarities between the magnitude and time course of the transient [Ca2+]; increase observed in both the canine tracheal cells and the human nasal and tracheal epithelial cells suggest that the cell types from both species respond to bradykinin in the same manner. However, my observations on the [Ca2+]j response to isoproterenol were less consistent than the observations of either McCann et al or Murphy et al (1988). McCann et al observed that isoproterenol stimulated an increase in [Ca2+]; in all the canine tracheal cells which they tested. However, Murphy et al found that in human nasal cell suspensions, Iso (luM) was ineffective at stimulating changes in [Ca2+]j. In my hands, Iso was successful at increasing [Ca2+]j in half the attempts, although all the cells in question responded to BK. In addition, morphologically similar cells from the same primary tissue sample behaved in both fashions on exposure to isoproterenol. These results suggest that in humans there may be a subpopulation of epithelial cells that respond to a different set of secretagogues. In addition, the experimental cells in my study remained attached to the substrate whereas the cells used by Murphy et al were put into suspension after monolayer culture for approximately 25 days under media conditions which were significantly different from those used for my study. It is therefore not surprising that the [Ca2+]; response to isoproterenol observed in my cultured cells was different from that observed by Murphy et al My results with epinephrine suggests significant differences between canine and human airway epithelial cells. Welsh and McCann (1985) reported that stimulation of suspended canine tracheal cells with epinephrine increased [Ca2+]j, as determined by Quin-2 fluorescence. 38 I was unable to induce a change in [Ca2+]i with either Epi, NE or PGE2 (BK stimulation of canine tracheal cells has been shown to cause the release of PGE2; Leikauf et al, 1985; Widdecombe et al, 1989). These results also suggest differences in the mechanism by which Iso stimulates increased [Ca2+]i between canine and human airway epithelial cells. McCann et al. (1989) proposed that isoproterenol acted through cAMP to increase [Ca2+]i. However, Epi, which has been shown to increase intracellular cAMP levels (Boucher et al, 1989) in human nasal epithelial tissue, did not increase [Ca2+]; in my cultured human nasal epithelial cells. Further, treatments which elevate intracellular cAMP levels (membrane permeant forms of cAMP and forskolin) did not affect [Ca2"1"];. Thus, unless the epinephrine-cAMP second messenger pathway had been somehow incapacitated distal to cAMP production under my culture conditions, my observations indicate that increased cAMP does not necessarily result in increased [Ca2+]i in human nasal epithelium. Recently, Feldman et al. (1990) demonstrated that 8-adrenergic-stimulated CI" secretion across canine tracheal epithelia had at least some component that is independent of cAMP formation, supporting the hypothesis that the increase in [Ca2+]i observed after 6-adrenergic stimulation with Iso was not due to increased cAMP. The inability of verapamil, a known Ca2+ channel blocker, to block or even attenuate the [Ca2"1"]; response elicited by exposure to BK, coupled with a failure to increase [Ca2+]j levels in the face of membrane depolarization by high external K+, strongly suggests that an influx of external Ca2+ through voltage-gated Ca2+ channels is not involved in the increase in [Ca2+]i. Bathing the cells in nominally Ca2+-free saline during stimulation with BK had interesting effects. [Ca2+]i in cells which were exposed to A-23187 while bathed in nominally Ca2+-free saline (this saline did not contain EGTA) dropped significantly (p < 0.01). Although [Ca2+]0 was not measured directly, this result suggests that under nominally Ca2+-free conditions the prevailing 39 electrochemical gradient for Ca2+ would drive Ca2+ out of the cell. Resting [Ca2+]j levels dropped by over 50% during the 15 minute exposure to "Ca2+-free" medium, yet stimulation with BK still resulted in a 3.5-fold increase in [Ca2+]j. These results are consistent with a simple 3 compartment model in dynamic equilibrium, with the extracellular fluid as one compartment, the cytosol as the second compartment, and the intracellular Ca2+ reserve (presumably the endoplasmic reticulum; Streb et al, 1983; Joseph et al, 1984; Prentki et al, 1984; Gill et al, 1986) as the final compartment. Removal of extracellular Ca2+ would force a repartitioning of the available Ca2+ in the intracellular compartments until a new equilibrium is reached. Thus, one would expect Ca2+ levels in both the cytosol and the intracellular Ca2+ reservoir(s) to drop. Indeed, the directly measurable Ca2+ in the cytosolic compartment does drop to a new level. If the Ca2+ in the separate intracellular compartments is in dynamic equilibrium, then a drop in the Ca2+ in the intracellular reservoir would result in an attenuation of the BK- induced [Ca2+]i response. Such an attenuation was observed. A similar depletion of intracellular Ca2+ stores has been observed in rat granulosa cells (Wang et al, 1989). In that study, it was observed that the increase in [Ca2+]j in response to stimulation with gonadotropic hormone-releasing hormone (GnRH) was attenuated during exposure to a bathing saline which contained no added Ca2+. Repeated stimulation of the granulosa cells with LHRH depleted the intracellular Ca2+ stores more rapidly than simple exposure to Ca2+-free saline, implying that, although Ca2+ is actively sequestered in the intracellular reservoir, such a reservoir is reasonably labile. My observations on [Ca2+]j dynamics during exposure to low [Ca2+]0 agree with this conclusion. Further support for the hypothesis that the cytosolic Ca2+ is in a dynamic equilibrium with the intracellular Ca2+ reservoir comes from studies conducted on Ca2+-dependent contraction of guinea pig myometrium. Coleman et al (1988) observed that oxytocin-induced smooth muscle contractions were augmented when lanthanum was present in the bathing saline. Under these 40 conditions, lanthanum inhibits extrusion of Ca2+ across the plasma membrane, causing cytosolic Ca2+ levels to rise. They reasoned that an increase in cytosolic Ca2+ would force a repartitioning of Ca2+ between the cytosol and the intracellular Ca2+ reservoir. This would cause an increase in [Ca2+]i upon stimulation with oxytocin, resulting in augmentation of contraction, which they observed. In summary, although individual nasal epithelial cells did not exhibit a clear dose-dependent relationship between BK and [Ca2+]i, different cells had different thresholds which, when summed, resulted in a clear dose-dependent response for the epithelial sheet as a whole. These results are similar to those already reported for canine tracheal epithelial cells. Significant differences exist, however, between the action of isoproterenol on [Ca2+]j levels in canine and human airway cells. My results indicate that the inter-relationship between cAMP and [Ca2+]j may be less complex than that reported in canine trachea. I am, as yet, uncertain as to whether or not this reflects interspecies variability, or whether differences in the physiology of electrolyte transport control mechanisms exist between nasal and tracheal epithelia. Finally, influx of extracellular Ca2+ through voltage-gated Ca2+-specific channels does not appear to be involved in generating the transient Ca2+ signal stimulated by bradykinin. 41 CHAPTER THREE: Observed Changes in Secretogogue Response of Cultured Cells Over Time. The results presented in chapter 2 were obtained on cells which had been maintained in tissue culture for a minimum of 5 days. The tissue culture protocol consisted, in part, of treating the dispersed cells with cholera toxin. Cholera toxin stimulates the synthesis of cAMP. Cell attachment and viability was significantly improved with this treatment. However, in order to rule out any influence of excess cAMP on the physiological responses tested, the cholera toxin was withdrawn from the culture medium at least 48 hours before any experiments were conducted. During the period of February-March 1990, virtually all response to BK was lost in cells which had been cultured for 5 days. No changes in the tissue culture protocol were found which could account for the observed loss of tissue sensitivity to BK. Therefore I investigated whether sensitivity to BK was lost with time in tissue culture or whether changes in tissue culture media and supplements, or tissue source, patient pretreatment, or time of year might be the cause. Materials and Methods Pre-operative Patient Treatment The majority of tissue utilized in this study was nasal polyp tissue (>65%). The pre-operative treatment of patients varied considerably according to the course of their condition and response to previous treatment. However, patients typically presented to the Otolaryngologist, suffering from allergic rhinitis and complaining of nasal obstruction and sinus pain. The routine course of action included treatment with topical corticosteroid sprays in an attempt to decrease 42 or eliminate the nasal polyps. Airway obstruction due to chronic inflammation of the nasal mucosa, usually including the nasal turbinates, was also routinely treated with topical corticosteroids in an attempt to ameliorate the mucosal edema. If this failed, patients were referred for surgery, and the nasal polyps and / or obstructing tissues were excised. Cell Culture and [Ca2*]i Determination The cells used in this study were cultured as already mentioned in chapter 2. However, in some experiments, the tissue culture medium was supplemented with 10% FBS. The cells were loaded with fura-2 in the same manner as previously described in chapter 2. For the present study, 6 = 4.81, R ^ = 0.35 and R ^ = 3.51. BK and ATP were used to stimulate the cells at concentrations which were known to give a maximal response (BK, 1 uM; ATP, 10 uM). Results and Discussion The onset of the loss of sensitivity to BK in the cultured nasal epithelial cells was relatively abrupt. In experiments conducted during January 1990, 100% of the cells ( which had been in cell culture for a minimum of 5 days) exposed to 100 uM pulses of BK responded with an increase of [Ca2+]; (n=56; pre-stimulation [Ca2+]i was 128 ± 18 nM, peak stimulated [Ca2+]; was 418 ± 63 nM; /? < 0.01). However, primary cultures (after 5 days in cell culture) which had been prepared after March, 1990 (using the protocol described in chapter 2), did not respond to BK (n=43). Initially, I thought that some change had occurred in the tissue culture protocol, which adversely affected the response of the tissue to BK. During this time, the cells were being cultured in the laboratory of Dr. M.A. Bridges (Department of Pathology, Faculty of Medicine, University of British Columbia). Consultations with Dr. Bridges failed to reveal any change in 43 m o •i—I fi O O H W QJ m 'a; O • H o 100 7 5 -50--2 5 -0 10 0 6 7 8 Days in Culture Figure 3.1. Effect of Cell Culture Duration on Cell Responsiveness. After 2 days in culture the percentage of cells which respond to BK stimulation declined dramatically. A similar decline in response to isoproterenol was observed with 7 days in culture, (o) Stimulated with 100 nM BK. (A) Stimulated with 1.0 uM Iso. 44 the procedures used to prepare the primary cultures used in the experiments. More detailed investigations required setting up an independent tissue culture facility, which was done with the assistance of Dr. S. Katz and Dr. G. Brown (Faculty of Pharmaceutical Sciences, University of British Columbia). Several lots of each tissue culture supplement were tested (a minimum of three each from at least two different sources), as well as inclusion of 10% FBS, with no detectable improvement in cellular responses. Water from several different purification systems was tested, again with no detectable improvement in the cellular response to BK. Consultations with the Greater Vancouver Regional Water District revealed that no changes in water treatment had occurred during the time period in question. I then performed a time course experiment in an attempt to determine if the loss of response to BK represented a total lack of response or a progressive loss of response. Figure 3.1 illustrates the results obtained (the values plotted reflect the percentage of all the cells tested which responded). Initially, when the primary cultures were established, the cells respond "normally" to BK stimulation ("normal" meaning that the [Ca2+]i responses obtained were similar those presented in chapter 2, peak stimulated value 436 ± 48 nM). However, after 2 days in culture, an increasing percentage of cells failed to respond to BK stimulation. After 5 days in culture, only about 10-15% of the cells tested responded to BK. Although, as demonstrated in chapter 2, only 50% of cells tested responded to Iso, a similar trend in the loss of sensitivity was observed (Fig. 3.2). The observed loss of response to Iso was less pronounced than to BK, with approximately 38.5% of cells responding on the fifth day in culture (5 out of 13). However, by day 7 of culture only 10% of the cells tested responded (Fig. 3.1). As will be shown in chapter 4, stimulation with adenosine triphosphate (ATP) also increased [Ca2+]j. The cultured nasal epithelial cells lost their responsiveness to ATP in a manner which was indistinguishable from the loss of responsiveness to BK (15% and 10% of all the cells tested responded to ATP 45 stimulation on days 5 and 7, respectively). In addition, after this period the resting [Ca2+]j observed in the cells dropped from 149 ± 35 nM (as reported in chapter 2) to 90 ± 15 nM (see chapter 4). The loss of secretagogue sensitivity in early 1990 can be accounted for in two ways: First, there may have been some fortuitous contamination which prevented the loss of sensitivity to BK and ATP, which was consistently present in one of the tissue culture supplements prior to February-March 1990. I consider this explanation to be unlikely for the following reason: The results presented in chapter 2 were obtained from cultures which covered a minimum of five different lots of each supplement. As well, cultures which were maintained in the presence of fetal bovine serum, which presumably would contain any potential contaminant, should have retained sensitivity to BK and ATP. Alternatively, since no explanation is readily discernible from the tissue culture procedures used in this study, it is reasonable to assume that some change in pre-operative patient treatment could account for the loss of BK and ATP sensitivity. During the course of this study, a fundamental shift in the pre-operative corticosteroid treatment regime occurred. Patients with nasal polyps are routinely treated with topical corticosteroids. However, in the spring of 1990 (Feb.-March), a new topical corticosteroid treatment, containing the corticosteroid budesonide, became popular amongst the local otolaryngologists. Budesonide has superior efficacy in reducing or eliminating nasal polyps, as well as ameliorating the symptoms of allergic rhinitis. Limited resources, financial and human, did not allow this line of investigation to be pursued further. 46 CHAPTER FOUR: Effects of Nucleotide Stimulation on [Ca2+], in Cultured Human Nasal Epithelial Cells. Cyclic AMP and Ca2+ have both been implicated as second messengers in regulating CI" secretion by airway epithelia. Agents which raise intracellular cAMP stimulate CI" secretion (Smith et al, 1982). Clancey et al (1990) have demonstrated that inhibition of phosphorylation interferes with cAMP stimulation of apical CI" permeability in canine tracheal cells. The role of intracellular ionic Ca2+ concentration in epithelial CI" secretion is less well understood. Bradykinin increases [Ca2+]j in canine tracheal cells (McCann et al, 1989) and human nasal epithelial cells (Murphy et al, 1988; chapter 2). Purinergic stimulation of many endothelial and epithelial cells has been demonstrated (Burnstock, 1990). Purinergic receptors have been divided into two major classes, Pj and P2 (Burnstock, 1990) on the basis of their general sensitivity to exogenous nucleotides as well as which second messenger system is activated. Prreceptors are more sensitive to adenosine monophosphate than to ATP. In addition, Prreceptors stimulate the production of intracellular cAMP through the actions of adenylate cyclase (Burnstock, 1990). P2-receptors are more sensitive to ATP than to adenosine monophosphate and trigger cellular responses independently of intracellular cAMP (Burnstock 1990). Purinergic stimulation has been shown to affect ion and water transport in another exocrine epithelium, namely the rat epididymis (Wong, 1988) and the function of this tissue is believed to be compromised in CF patients (Seale et al, 1985; Wen and Wong, 1988). Indeed, in the rat epididymis, adenosine 3'-5' triphosphate (ATP) directly stimulates CI" transport into the lumen, a transport pattern which is also observed in the upper airway. Several groups have 47 recently investigated the possible role of nucleotide stimulation of [Ca2+]i in human airway epithelia. In cystic fibrosis, cAMP-mediated CI" secretion in exocrine epithelia is defective (see review by Quinton, 1990), Ca2+-mediated CI" secretion is still intact (Boucher et al, 1989), and Ca2+ mediated calmodulin-dependent protein kinase activates CI" channels (Wagner et al, 1991). Thus, a more complete understanding of the role [Ca2+]i plays in mediating CI" secretion could lead to more effective treatments of the symptoms of the disease. The goal of this study was to determine if purinergic stimulation influences the intracellular free calcium concentration in upper airway epithelial cells, and if so, what type of purinergic receptor was involved and what was the source of the increased [Ca2+]j was. Materials and Methods Cell Culture Primary cultures of human nasal epithelial cells were prepared as described in chapter 2. Based on the results presented in chapter 3, 18 mm round coverslips containing 1-3 day old cultures (rather than 5-7 day old cultures) were immersed in the loading medium for 1.5 hours at 21° C, after which they were rinsed once with HBS to wash out excess fura-2 AM. Calculation of cytosolic free calcium concentration Cells were loaded with fura-2 and the cytosolic calcium concentration was calculated as described in chapter 2. For the present study, 6 = 4.28, R ^ = 0.29 and Rmax = 3.50. These values were determined using the same epithelial cell cultures exposed to Br-A23187 (HSC 48 Research Development Corp., Toronto, Ont., Canada), first in the presence of 1.8 mM Ca2+ in the perfusion medium, and then in a medium nominally free of Ca2+ and containing 5 mM EGTA. Statistical differences were determined as described in chapter 2. 49 Drug treatment All drugs were dissolved in HBS and diluted to the desired concentration with the same medium as described in chapter 2. Five hundred uL bolus injections were made into the flow-through chamber. At a chamber perfusion rate of 3 mL/min, it is estimated that the drug solution would be in contact with the cultured cells for approximately 10 seconds. In some experiments in "Ca2+-free" saline, extracellular Ca2+ was transiently replaced, and this was facilitated by the flow rate through the perfusion chamber being reduced to 1 mL/min in order to expose the cells to Ca2+ containing medium for 30 seconds. Cells were perfused with HBS for a minimum of 8 minutes between drug exposures to allow for a full recovery of the [Ca2+]; response to ATP (or related nucleotide) stimulation. Results [Ca2+]i levels in cultured human nasal epithelial cells increased in a dose-dependent manner in response to addition of ATP (Fig. 4.1). Maximal [Ca2+]j levels were achieved by a 10 second pulse of 50 uM ATP. A minimum ATP concentration of -1 uM ATP was required to elicit a significant change in [Ca2+]j. UTP also stimulated increased [Ca2+]i levels in a dose-dependent manner (Fig. 4.3). Lower concentrations of UTP (0.5 uM) than ATP (1.0 uM) were required to cause minimally significant increases in [Ca2+]j. Maximal stimulation of [Ca2+]i by UTP appeared to be reached with a concentration of 5.0 uM, ten times the threshold concentration. ATP, however, required a 50-fold increase to reach a maximal response (Fig. 4.1). 50 S a + o 600 500 400 300 200 100 04 0.01 6 0.1 1.0 10 ATP concentra t ion (/uM) * * * * 100 Figure 4.1. Effect of Increasing Adenosine Triphosphate Concentration on [Ca2+],. ATP increases [Ca2+]j in a dose-dependent manner. Maximal [Ca2+]i levels were stimulated by a 10-15 second exposure to 50 uM ATP (see Materials and Methods). A threshold dose of ~1 (aM ATP was required to elicit a detectable rise in [Ca2"1"];. Maximum responses were obtained with 50 uM ATP. Mean, S.E.M. (vertical bars) and n are indicated for each point. A 50-fold increase in [ATP] was required to reach maximum stimulation from the threshold dose (1 pM ATP). Significant increases in [Ca2+]j are indicated (* p < 0.05; ** p < 0.01). 51 + cv O 400 300 2 0 0 -100 0 0 ^ A T P 1 yU,M + 10 Time (min) t UTP 1 fjM 15 20 Figure 4.2. Change in [Ca2+], During Sequential Exposures to Adenosine Triphosphate and Uridine Triphosphate. The arrows denote the time at which a single cell from a monolayer primary culture of human nasal epithelium was exposed to a pulse of 1 \JM ATP and UTP. In each case, [Ca2+]i rose sharply after a delay of approximately 10-12 seconds. UTP was a more potent stimulator of [Ca2+]i in all cells tested. 52 + O 600 j 5 0 0 -400 300 200 100 0 0.01 0.1 1.0 * * 10 UTP concent ra t ion (/zM) 100 Figure 4.3. Effect of Increasing Uridine Triphosphate Concentration on [Ca2+],. UTP elevates [Ca2+]j in a dose-dependent manner and was more potent than ATP: the threshold concentration for UTP was 0.5 uM and the maximal [Ca2+]i response was obtained with 5 \iM. Mean, S.E.M. (vertical bars) and n are indicated for each point. Significant increases in [Ca2+]j are indicated (* p < 0.05; ** p < 0.01). 53 Figure 4.2 illustrates the [Ca2+]j response of a single cell to luM doses of both ATP and UTP. In both cases, the cellular [Ca2+]j response was similar to that previously described for stimulation with BK in these cells. After a delay of 15-20 seconds, [Ca2+]j rose sharply to a peak which was typically 5-10 times the normal resting [Ca2+]i (90 ±15 nM, n=36). Frequently, [Ca2+]j levels oscillated around the peak value for 10-15 seconds before a moderately rapid decline back to pre-stimulation levels, usually within 1.5 minutes of the initial stimulation. To delineate further the nature of the purinergic receptor, I tested other related compounds. Dose-response curves were obtained for ADP (Fig.4.4), AMP (Fig 4.5), adenosine (Fig. 4.6), and GTP (Fig.4.7). A selectivity sequence of UTP > ATP > ADP > AMP = adenosine was obtained. Exogenous GTP (n=7) and cAMP, which is membrane impermeant (n=7), had no effect on [Ca2+]i at concentrations up to 100 uM (Fig. 4.8). In an attempt to determine the source of the purinergically-stimulated increase in [Ca2+]i; I used the following protocol: Cells (n=7) were bathed in HBS containing 1.8 mM Ca2+ and stimulated with a luM dose of UTP. This treatment stimulated a [Ca2+]; response of 397 ± 48 nM. After allowing the cells to recover for 8 minutes, the cells were then bathed for 15 minutes in HBS which contained no added Ca2+ ("Ca2+-free" medium). The cells were then stimulated every 8 minutes with 1 uM UTP (in "Ca2+-free" medium). Similar to the results reported for stimulation of the cells with BK, bathing the cells in "Ca2+-free" saline (Fig. 4.9) attenuated the peak response to UTP (luM, peak stimulated [Ca2+]j level for this treatment was 306 ± 28 nM). Subsequent exposures to UTP (1 uM) resulted in progressively diminished [Ca2+]; responses so that, typically, after the third exposure to UTP (1 uM) the response was abolished (peak stimulated [Ca2+]j level of 102 ± 18 nM). In order to determine if an acute influx of Ca2+ through Ca2+ channels was the source of the increased [Ca2+]i; the cells were then stimulated with 1 ^M 54 s Pi + o 3 5 0 -I 300-250-200-150-100-50-0-7 T o 1 1 1.0 10 ioo ADP concentra t ion (/JM) Figure 4.4. Effect of Increasing Adenosine Diphosphate Concentration on [Ca ],. ADP elevated [Ca2+]i in a dose-dependent manner. ADP was clearly less effective than either ATP or UTP. A concentration of 100 uM was required to stimulate a significant increase in [Ca2+]i (* p=0.05). Mean, S.E.M. and n are indicated for each point. 55 + o 350 300 250 200 150 100 50 0 1.0 10 100 AMP concent ra t ion {fjM) — I 1000 Figure 4.5. Effect of Increasing Adenosine Monophosphate Concentration on [Ca2+],. AMP did not elevate [Ca2+]i at any concentration tested. Mean and n are given for each point. The S.E.M. fell within the symbol for each point shown. 56 S £ + CV2 O 3 5 0 T 300-250-200-150 100 50 0 1.0 10 100 1000 Adenosine concent ra t ion (/xM) Figure 4.6. Effect of Increasing Adenosine Concentration on [Ca2+],. Adenosine did not elevate [Ca2+]j at any concentration tested. Mean and n are given for each point. The S.E.M. fell within the symbol for each point shown. 57 s fi • . — 1 + 02 O 350 300 250 200 150 100 50 0 1.0 10 100 GTP concent ra t ion (/U.M) Figure 4.7. Effect of Increasing Guanosine Triphosphate Concentration on [Ca2+],. GTP did not elevate [Ca2+]j at the 2 concentrations tested. Mean and n are given for each point. The S.E.M. fell within the symbol for each point shown. 58 + cv cd o uuu -4 0 0 -3 0 0 -2 0 0 -100-0 -1 GTP lfM 1 1 1 1 1 1 cAMP 1/i.M • H 1 1 1 0 2 3 Time (min) Figure 4.8. Effect of Guanosine Triphosphate and Adenosine 3'-5' Cyclic Monophosphate on [Ca2+],. The arrows denote 500 uL bolus injections of 1 ^M GTP and cAMP into the perfusion chamber. Neither GTP (n=7) nor cAMP (n=7) had any effect on [Ca2!. 59 9T 3 o D U U -500-400-300-200-100-0 -• ^ t Ca2 + • V • -i 1 1 1 free saline 1 1 —H H * I H • J 1 H 1 0 10 20 30 40 Time (min) 50 60 70 Figure 4.9. Effect of Nominally Ca2+-free Saline on the UTP Stimulated Increase in [Ca2+], in a Single Cell. Cells were perfused with nominally Ca2+-free saline and repeatedly stimulated with 1 uM UTP. A progressive diminution of the [Ca2+]i response was observed. Brief exposure, during stimulation, to 5.4 mM external Ca2+ (*) failed to restore the [Ca2+]i response. Stimulation with 1 uM UTP after exposure to normal medium (1.8 mM Ca2+) for 8 minutes caused only partial recovery of the [Ca2+]i response. 60 UTP in saline which contained 5.4 mM Ca2+ (3x normal). Inclusion of high [Ca2+] in the bathing saline only during the course of UTP stimulation had no effect (maximum [Ca2+]j stimulated by this treatment was 112 ± 25 nM) on the diminished [Ca2+]j response (Fig. 4.9). The cells were then returned to HBS containing 1.8 mM Ca2+ for 8 minutes before a final exposure to 1 uM UTP. Only a partial recovery (p < 0.05) of the [Ca2+]j response was observed after this time (Fig. 4.9, peak stimulated [Ca2+]; was 214 ± 46 nM). The unstimulated [Ca2+]j concentration in these cells was 96 ± 24 nM. Discussion My results, as well as those of Mason et al. (1991)3 have clearly demonstrated that purinergic agents can initiate a transient rise in [Ca2+]i in cultured airway cells in a dose-dependent manner. A growing body of reports in the literature (see Table 4.1) all suggest the existence of an, as yet, uncharacterized ATP receptor, similar to a purinergic P2y-receptor, yet having a UTP sensitivity greater than or equal to its ATP sensitivity (O'Connor et al, 1990; Mason et al, 1991). The selectivity sequence, UTP > ATP > ADP » AMP = adenosine = GTP = cAMP, combined with a similar selectivity sequence recently reported by Mason et al (1991), strongly suggest that the nucleotide stimulation is mediated through this so-called "Nucleotide" receptor (receptor nomenclature as suggested by Davidson et al, 1990). There is confusion as to whether the receptor described here, and elsewhere (Mason et al, 1991), is the recently proposed pyrimidial receptor (O'Connor et al, 1990). A clearer picture of the relationship between those two receptor types will have to await a more definitive characterization of the pyrimidial receptor. 3 This paper appeared after my studies were completed and submitted for publication. 61 As previously demonstrated (chapter 2), the source of the increase in [Ca2+]j caused by BK was intracellular in nature, presumably through the actions of inositol 1,4,5-trisphosphate on endoplasmic reticulum (Joseph et al, 1984). As in that study, I found that bathing the cells in nominally Ca2+-free saline for 15 minutes attenuated, but did not abolish the [Ca2+]; signal. However, repeated purinergic stimulation in nominally Ca2+-free saline virtually abolished the [Ca2+]i response. These data suggest that the rise in intracellular free Ca2+ is caused by release from intracellular stores. Responses in "Ca2+-free" saline were probably not due to residual extracellular Ca2+ because the chamber was flushed with 120 volume equivalents before any stimulation and because the transient inclusion of 1.8 mM extracellular Ca2+ during nucleotide exposure failed to restore [Ca2+]j responses. Thus, repeated stimulation in "Ca2+-free" saline probably depletes intracellular Ca2+ stores, which are replenished only slowly (>8 min) when extracellular Ca2+ is permanently restored. Nucleotide-stimulated release of Ca2+ from an intracellular reservoir has been shown to operate through the IP3-mediated pathway, in several other systems studied (Dubyak, 1991). Endogenous purinergic stimulation probably consists of exposure to ATP rather than to UTP. ATP has been shown to be secreted at the same time as granule secretion in at least two secretory cell types (Rojas et al, 1986; DeLisle and Hopfer, 1986). In addition, ATP can be released by cells which mediate the inflammatory response (McDonald, 1988; Huang et al, 1989). In summary, purinergic stimulation transiently increased [Ca2+]j in cultured human nasal epithelial cells by mobilizing calcium from intracellular stores. This stimulation was shown to be dose-dependent and had a selectivity sequence of UTP > ATP > ADT » AMP = adenosine = cAMP = GTP, consistent with a nucleotide receptor (Davidson et al, 1990). Unlike canine tracheal cells, increased intracellular cAMP had no effect on [Ca2+]j in cultured human nasal epithelial cells. 62 CHAPTER 5: Effects of Histaminergic Stimulation on [Ca2+], in Cultured Human Nasal Epithelial Cells. Asthma is one of the most common respiratory ailments, afflicting over 5% of individuals in the industrialized world (George and Owens, 1991). Although primarily precipitated by environmental factors, a genetic predisposition to the disease has been reported (Hopp et al, 1990). Physiologically, the asthmatic response consists, in part, of bronchospasm and airway epithelial inflammation and hypersecretion of mucus and fluid (George and Owens, 1991). A potential role for histamine (Hm) in mediating at least some of the asthmatic reaction was proposed as early as 1919 (Dale et al). Administration of intravenous Hm was shown to trigger bronchospasm in individuals who suffered from asthma (Weiss et al, 1929), supporting the hypothesis that Hm was involved in the generation of the asthmatic reaction. More recently, the role of Hm in asthmatic bronchoconstriction has been more thoroughly investigated. Plasma Hm levels are elevated during the acute phase of the asthmatic response. This elevation is probably due to increased mast cell degranulation (Bruce et al, 1976; Simon et al, 1977; George and Owens, 1991). Increased Hm is also present in fluid retrieved during bronchoalveolar lavage from patients suffering from allergic asthma (Jarjour et al, 1991). Three types of Hm receptors ( denoted as H1? H2, and H3) have been identified on the basis of agonist and antagonist binding studies (see review by Schwartz et al, 1985). Hm evokes asthmatic symptoms through both Hr and H2-receptor mediated pathways. H^receptors mediate vasodilation, elevated vascular permeability, smooth muscle contraction, and sensory nerve stimulation. H2-receptors mediate vasodilation, increased mucus secretion, immunoregulation, and neutrophil chemotaxis (see review by Howarth, 1990). 63 Less clear is the potential role of Hm in mediating the elevated airway fluid secretion which is characteristic of the asthmatic attack. Hm has been shown to elevate [Ca2+]i levels in several different cell types, including at least two CI" secreting cell lines derived from human colonic epithelia (Wasserman et al, 1988; Pickles and Cuthbert, 1991). As well, Hm has been shown to be a weak CI" secretagogue in the canine trachea (Marin et al., 1977). I therefore tested whether Hm would stimulate elevated [Ca2+]; in cultured human nasal epithelial cells. Materials and Methods Cell Culture Primary cultures of human nasal epithelial cells were prepared as described in chapter 2. Based on the results presented in chapter 3, 18 mm round coverslips containing 1-3 day old cultures were immersed in the loading medium for 1.5 hours at 21° C, after which they were rinsed once with HBS to wash out excess fura-2 AM. This protocol has been shown to minimize the incorporation of fura-2 into perinuclear granules (chapter 2). Calculation of cytosolic free calcium concentration Cells were loaded with fura-2 and the cytosolic calcium concentration was calculated as described in chapter 2. For the present study, 6 = 4.30, R ^ = 0.27 and R ^ = 3.54. These values were determined using the same epithelial cell cultures exposed to Br-A23187 (HSC Research Development Corp., Toronto, Ont., Canada), first the presence of 1.8 mM Ca2+ in the perfusion medium, and then in medium nominally free of Ca2+ and containing 5 mM EGTA. Statistical differences were calculated as described in chapter 2. 64 Drug treatment All drugs were dissolved in HBS and diluted to the desired concentration with the same medium as described in chapter 2. One mL aliquots of HBS containing Hm were perfused through the chamber at a flow rate of 1.3 mL/min, resulting in a 45 second exposure to Hm. During exposure to "Ca2+-free" saline, the chamber was perfused at a rate of 10 mL/min for the first 30 seconds in order to flush any residual Ca2+ out of the chamber. A period of thirty minutes between Hm exposures was maintained in order to allow for maximal recovery of the [Ca2+]; response to histamine. Results Compared to BK or nucleotide stimulation, a longer exposure of cells to Hm was required to elicit a maximal response (n=5, approx. 30 seconds for Hm vs. 10 seconds for both BK and ATP). Figure 5.1 shows the effect of a 15 second exposure to 100 uM Hm (pre-stimulation [Ca2+]i was 94 ± 21 nM, peak stimulated [Ca2+]i was 99 ± 22 nM). This response can be compared to figure 5.2 which shows the [Ca2+]; response elicited by a 45 second exposure to 100 uM Hm (n=12, pre-stimulation [Ca2+]i was 89 ± 24 nM, peak stimulated [Ca2"^ was 324 ± 56 nM). In addition, the period of time required for recovery of the [Ca2+]j response between Hm exposures (30 minutes, n=6) was significantly longer than that required for either BK or nucleotide stimulation (8 minutes). Figure 5.3 demonstrates that two 100 uM Hm doses given 8 minutes apart (as was sufficient for BK and ATP) do not give consistent results (n=4, pre-stimulation [Ca2+]s was 90 ± 27 nM, [Ca2+]j elicited by the first Hm pulse was 329 ± 53 nM whereas [Ca2+]j elicited by a Hm pulse 8 minutes later was 153 ± 38 nM). As well, unlike stimulation with BK or nucleotides, the [Ca2"1"^  response to repeated Hm stimulation demonstrated 65 300 T S a 200 + CV2 CO O 100 0 0 0.25 0.5 Time (min) 0.75 1.0 Figure 5.1. Effect of a 15 Second Exposure to 100 uM Histamine on a Single Cell. A single cell was stimulated with 100 uM Hm (shown to be a maximal dose, see Fig. 5.4), in a manner identical to BK and ATP (Figures 2.1 and 4.2 respectively). [Ca2+]; did not increase detectably in response to this treatment in any cell tested (n=5). 66 + cd o 600 500 400 300 200 + 100 0 0 \A •\ 4 6 Time (min) Figure 5.2. Effect of a 45 Second Exposure to 100 uM Histamine on a Single Cell. The arrow denotes the initiation of a 45 second perfusion to 100 |iM Hm (n=12). [Ca2+]j rose sharply after a slight delay. 67 + O 300 T 200 100 0 m \ 0 6 8 Time (min) 10 Figure 5.3. Effect of Repeated Stimulations with Histamine, 8 Minutes Apart, in a Single Cell. A single cell was stimulated twice (45 second pulses, 8 minutes apart) with 100 uM Hm. The second pulse of 100 \M Hm resulted in a greatly attenuated [Ca2+]j response (n=4). With similar treatment with BK or ATP, the second [Ca2+]j response would have been of equal magnitude as the first [Ca2+]j response. 68 + cv? o 600 500 400 + 300 200 + 100 0 •ijj • 0 u V I -25 50 Time (min) 75 8 100 Figure 5.4. Effect of Repeated Stimulations with Histamine, 30 Minutes Apart, in a Single Cell. Repeated stimulation of cultured human nasal epithelial cells resulted in a progressive diminution of the [Ca2+]i response even when the tissue was allowed to recover for 30 minutes between exposures to Hm (n=4). 69 500 T 0.1 1.0 10 100 1000 10000 Histamine Concentrat ion (,uM) Figure 5.5. Effect of Increasing Histamine Concentration on [Ca2+],. Histamine triggers an increase in [Ca2+]j in a dose-dependent fashion. A maximal [Ca2+]i level (-340 nM) was obtained with a Hm concentration of 100 uM. The threshold Hm concentration was > 10 uM. The maximal [Ca2+]j level obtained was less than that observed for stimulation with either BK or nucleotides (520 nM and 480 nM respectively). Mean ± SEM (vertical bars) and n are indicated for each point. Significant increases in [Ca2+]j are indicated (* p < 0.05; ** p < 0.01). 4 0 0 -1 3 0 °" ' 'i—l -T"1 ^ 200 --CO o 1 0 0 -0 0.01 70 significant diminution regardless of the time allowed for recovery (n=4, Fig. 5.4). The peak stimulated [Ca2+]i levels for 100 uM Hm pulses given at 5, 35, and 95 minutes (approximately) after initiation of saline perfusion resulted in peak [Ca2+]i values of 368 ± 65 nM, 302 ± 53 nM, and 178 ± 49 nM respectively (the pre-stimulation [Ca2+]i was 87 ± 29). Hm stimulates [Ca2+]j in a dose-dependent manner (Fig. 5.5). A maximal [Ca2+]j response was obtained with a concentration of 100 uM Hm while a threshold Hm concentration of > 10 \iM was required to stimulate a significant change in [Ca2+];. Maximum [Ca2+]i levels stimulated with Hm (350 nM) were significantly (p<0.05) smaller than those observed for either BK stimulation or nucleotide stimulation (500 nM). Several experiments were performed in order to determine the source of the [Ca2+]j response to Hm. As demonstrated in figure 5.6, bathing the cells (n=5) in Ca2+-free saline for 30 minutes totally abolished the [Ca2+]i response to Hm stimulation (peak stimulated value was 81 ± 26 nM, pre-stimulation [Ca2+]j was 88 ± 24 nM) and returning extracellular Ca2+ to normal (1.8 mM) restored (partially) the [Ca2+]i response to Hm (peak stimulated [Ca2"1"]; level was 218 ± 63 nM). Eliminating the initial Hm stimulation before removal of the extracellular Ca2+ for 30 minutes did not restore the [Ca2+]j response to subsequent Hm stimulation (n=6, Fig. 5.7). Pre-stimulation and peak stimulated [Ca2+]j levels for cells exposed to this treatment were 92 ± 32 nM and 88 ± 29 nM respectively. However, bathing cells in Ca2+-free saline for only 5 minutes before Hm stimulation did not attenuate the [Ca2+]j response (n=5, Fig. 5.8). Pre-stimulation and peak stimulated [Ca2+]j for cells exposed to the preceding treatment were 82 ± 23 nM and 352 ± 43 nM. Bathing the cells in Ca2+-free saline for 30 minutes and then including 3.6 mM Ca2+ with the Hm stimulation failed to restore the [Ca2+]i response to Hm (n=5, Fig. 5.9). 71 Ca —free saline 500 j 4 0 0 -a 3 0 0 -0 10 20 30 40 50 60 70 80 Time (min) Figure 5.6. Effect of a 30 minute Exposure to Ca2+-free Saline on the Histamine-stimulated Increase of [Ca2+], in a Single Cell. The arrows denote addition of 100 uM Hm to the perfusion medium. Duration of the Hm perfusion was -45 seconds. Cells (n=5) were stimulated with Hm, then bathed in Ca2+-free saline for the time indicated by the bar. Before Ca2+ was restored to the bathing medium, the cells were again stimulated with 100 uM Hm. Ca2+ was then restored to the bathing saline and the cells were allowed to recover for a further 30 minutes. A final stimulation with 100 uM Hm was performed in order to determine whether the cells were still responsive to Hm. Note that exposure to Ca2+-free saline for 30 minutes completely abolished the [Ca2+]j response to Hm. After Ca2+ was restored to the bathing saline for 30 minutes, the cells responded normally to stimulation with 100 uM Hm (i.e. the decrease in the [Ca2+]j response is consistent with the effect of repeated exposures to Hm [Fig. 5.1]). 72 S 3 cC O 600 500 400 300 200 1 0 0 -0 2 + Ca free saline 25 50 Time (min) 75 Figure 5.7. Removal of Extracellular Ca2+ for 30 minutes Without Histamine Pre-stimulation Also Abolishes the Histamine-stimulated Increase in [Ca2+], in a Single Cell. In order to demonstrate that the lack of response observed in Fig. 5.3 was due to a true depletion of an intracellular pool, rather than due to incomplete filling of the intracellular pool before removal of the extracellular Ca2+, cells (n=6) were incubated in Ca2+-free saline without prior stimulation with Hm. Note that after a 30 minute exposure to Ca2+-free saline the cell did not respond to Hm stimulation (first arrow), even though the Hm-releasable pool had not been depleted by exposure to Hm. A strong response to Hm (second arrow) was observed after the cell had been allowed to recover in saline containing normal extracellular Ca2+ (1.8 mM). 73 + cc! U 600 j 5 0 0 -400 300 200--1 0 0 -0 0 Ca Free saline - % 1 25 50 Time (mirt) 75 Figure 5.8. Removal of Extracellular Ca2+ for 5 minutes Does Not Attenuate the Histamine-stiniulated [Ca2+], Response. Ca2+-free saline was perfused through the chamber during the time indicated. Stimulation of cells (n=5) with 100 jiM Hm (first arrow) after 5 minutes resulted in a full [Ca2+]i response. However, a similar stimulation (second arrow) after 30 minutes in Ca2+-free saline did not result in any change in [Ca2+]j. The cell did respond to 100 |iM Hm after the extracellular Ca2+ was restored for 30 minutes. 74 + CO o 600 500 400 300 2 0 0 -1 0 0 -0 C a 2 + Free Saline U !-•-A 0 1 * 10 Time (min) u 15 Figure 5.9. Effect of Restoring Extracellular Ca2+ on the [Ca2+], Response to Histamine-stimulation. After stimulation with a 45 second pulses of 100 |iM Hm (first arrow), cells (n=5) were bathed in Ca2+-free saline for 30 minutes before a second stimulation with 100 uM Hm (second arrow). (*) Normal extracellular Ca2+ (1.8 mM) was re-introduced in the perfusion medium simultaneously with the second stimulation with 100 uM Hm. Note that the cells did not respond to Hm stimulation under these conditions. If Hm stimulation resulted in opening of Ca2+ channels in the plasma membrane, then an increase in [Ca2+]j should have been observed under these conditions. A normal response to Hm (third arrow) was observed after the cells had been allowed to recover for 30 minutes in saline containing normal Ca2+ (1.8 mM). 75 Ca free saline . T" | f 1 MM UTP 20 30 40 50 Time (min) Figure 5.10. UTP-stimulated [Ca2+], Increase After Abolition of the Histamine-stimulated [Ca2+], Increase by Incubation in Ca2+-free Saline. After stimulation with 100 uM Hm (first arrow), cells (n=4) were bathed for 30 minutes in Ca2+-free saline in order to completely abolish the [Ca2+]j response to Hm. Immediately after the ineffective stimulation with 100 uM Hm (second arrow) and while still under Ca2+-free conditions, the cell was stimulated with 1 |iM UTP (third arrow). Note that a full [Ca2+]j response to stimulation with UTP was observed. Other cells which were tested with BK rather than UTP also exhibited a full [Ca2+]j response (data not shown). + o 500 4 0 0 -3 0 0 -2 0 0 -1 0 0 -0 .1 A 0 10 76 s £ • ( — 1 cd O DUU -500-400-300-200-100-0 -• — 0 »— 1 1 0.1 fuM Pyrilamine # - • — • - . • • 1 1 1 2 3 4 Time (min) 1 1 5 • 1 6 Figure 5.11. Effect of the Hrreceptor Antagonist Pyrilamine on the Histamine-stimulated Increase in [Ca2+],. Incubation of the cells (n=5) with 0.1 \JM pyrilamine (indicated by the bar) totally abolished the [Ca2+]j increase stimulated by Hm. The arrow indicates the onset of a 45 second exposure to 100 uM Hm. 77 Figure 5.12. Variable Effect of the H2-receptor Antagonist Cimetidine on the Histamine-stimulated Increase in [Ca2+],. A) Incubation of a cell with 0.1 uM cimetidine (indicated by the bar) greatly attenuated the [Ca2+]i response to a 45 second exposure to 100 uM Hm (as denoted by the arrow). B) A different cell was treated in an identical manner as in panel A. In this instance, cimetidine had no apparent effect on the Hm-stimulated increase in [Ca2+]; (as compared to figure 5.3). [Ca2+]i responses, all greatly attenuated, were observed in 3 of the 7 cells tested (no [Ca2+]; responses were observed in the other 4 cells). 78 600 T 5 0 0 -400 300 200 100 0-0.1 fjM C imet id ine -V • V o 6 7 8 9 600 500 + 400 300 200 + 100 0 0 B 0.1 JJM C imet id ine I .J 1, 4 7 8 Time (min) 79 + 600 T 500 4 0 0 -3 0 0 -2 0 0 -100 0 0 0.1 yU-M Thioperamide ft Time (min) 6 8 Figure 5.13. Effect of the H3-receptor Antagonist Thioperamide on the Histamine-stimulated Increase in [Ca2+],. Incubation of the cells (n=5) with 0.1 uM thioperamide (indicated by the bar) had no apparent effect on the Hm-stimulated (45 sec, 100 uM; arrow) [Ca2+]j response as compared to Fig. 5.3. 80 Table 5.1. Effect of Various Receptor-Antagonists on [Ca2+], Response to Histamine Stimulation. Receptor Antagonist (Receptor Type) Pyrilamine Cimetidine (H2) Thioperamide (H3) Pre-stimulated [Ca2+]j level mean ± S.E. (nM) 82 ± 8 74 ± 5 77 ± 11 Peak stimulated [Ca2+]j mean ± S.E. (nM) 86 ± 11 178 ± 93 326 ± 38 81 In order to determine whether Hm stimulation released Ca2+ from the same intracellular pool as BK or nucleotides, cells were bathed in Ca2+-free saline in order to abolish the Hm response. Immediately following a 100 uM Hm stimulation, which resulted in a [Ca2+]i level of 99 ± 34 nM, the cells were stimulated with either 1 uM UTP or 1 uM BK. Figure 5.10 illustrates a typical trace of [Ca2+]j obtained under such conditions. No change in [Ca2+]j was observed in response to stimulation with Hm; however, stimulation with UTP resulted in a normal [Ca2+]; response (452 ± 28 nM; n=4). Results obtained with BK (468 ± 31 nM; n=3) were not statistically different from results obtained with UTP. The basal [Ca2+]i level before removal of extracellular Ca2+ was 89 ± 12 nM (n=7, basal [Ca2+]i levels for the cells exposed to UTP and cells exposed to BK were combined) while the basal [Ca2+]; level after removal of extracellular Ca2+ for 30 minutes was 78 ± 9 nM (not statistically different). Table 5.1 summarizes the results obtained using Hx, H2 and H3 specific Hm-receptor antagonists. Cells were bathed in saline containing 0.1 uM of the antagonist being tested for 3 minutes before stimulation with 100 uM Hm (maximal dose). The Hi-specific receptor blocker, pyrilamine maleate, blocked the [Ca2+]j response in all cells tested (n=5; Fig 5.11 shows a typical response of a single cell to pyrilamine). Cimetidine (response of a single cell shown in Fig. 5.12), an H2-specific receptor antagonist, abolished the [Ca2+]i response in 4 of 7 cells tested. Thioperamide maleate, an H3- specific antagonist, had no effect on the [Ca2+]i response to Hm (n=5). The response of a single cell to thioperamide is shown in Fig. 5.13. Discussion Changes in [Ca2+]i have been shown to affect electrolyte and fluid secretion by airway epithelia (see review by Welsh, 1990). One of the characteristics of asthma is a hyper-secretion 82 of fluid and mucus by airway epithelia (Tamaoki et al, 1991), and histamine has certainly been implicated in mediating at least some of the symptoms of asthma (Howarth, 1990; George and Owens, 1991). Because Hm has been shown to stimulate increased [Ca2+]i in canine tracheal epithelia (Marin et al, 1977), I attempted to determine if Hm influenced [Ca2+]; levels in human nasal epithelial cells. As demonstrated in Fig. 5.1, Hm does stimulate increased [Ca2"1"^  levels in airway epithelial cells. However, the dynamics of the [Ca2+]i response to Hm stimulation are unusual compared to other Hm-mediated systems studied. Although Hm binding studies were not conducted, the affinity of the Hm-receptor in airway epithelial cells is apparently relatively low as evidenced by the high Hm concentrations required to initiate a [Ca2+]j response (a threshold Hm concentration of >10 uM). Another unusual characteristic of Hm stimulation of cultured human nasal epithelial cells is the length of exposure required to initiate a [Ca2+]j response. Unlike BK or nucleotides, which initiate a full [Ca2+]j response with exposure durations of 10-15 seconds, Hm must be in contact with the cells for at least 20 seconds before any [Ca2+]j response is observed, and a maximal [C&2% response is not obtained until the cells have been exposed to Hm for at least 30 seconds. The results obtained with the Hm-receptor antagonists suggest that Hm is operating through an H, receptor; however, the classically defined Hj receptor is responsive to Hm in the sub-micromolar range. Recently, an intracellular Hm-receptor has been described in rat brain membranes (Brandes et al, 1987) and rat liver microsome preparations (Brandes et al., 1990). Indeed, Hm has been demonstrated to satisfy the criteria that define a second messenger in human platelets (Saxena et al., 1990). In that system, Hm is formed as a second messenger in response to stimulation by platelet activating factor (PAF). Interestingly, PAF has been shown to be 83 generated by lung epithelial tissues, and has been shown to stimulate electrolyte transport across monolayers of cultured airway epithelial cells (Tamaoki et al, 1991). The intracellular Hm-receptor has an Hm affinity in the micro-molar range. In addition, pyrilamine was a more potent antagonist than cimetidine, prompting those investigators to propose an Hlc receptor nomenclature. In that system, N,A^diethly-2-[4-(phenlymethyl)phenoxy]ethanamine (DPPE) was the most potent antagonist tested. The unusual response of human nasal epithelial cells to Hm could be explained in a similar fashion. The unusually long exposure time required to stimulate an increase in [Ca2+]j may reflect the time required for the Hm to penetrate the cells and come in contact with any intracellular receptors. In addition, the effective Hm dose is in agreement with the known Hlc receptor affinity, as both are approximately in the micro-molar range. The fact that 10-100 times the concentration was required for the human nasal epithelial cells could be explained by the fact that intact cells were used in this study, whereas the platelet Hlc receptor binding was directly studied in a microsomal preparation. The Hrreceptor antagonist pyrilamine was found to be more potent than the H2-receptor antagonist cimetidine. This potency ranking supports both the hypothesis that Hm operates as a second messenger in this tissue, as well as the hypothesis that Hm actions on airway epithelia are mediated by an Hj-receptor (Howarth, 1990). The effects of DPPE should be tested on the cultured human nasal epithelial cells in order to help to resolve this question. DPPE is not commercially available and thus was not tested in this study. Although bathing the cells in Ca2+-free saline for 30 minutes abolished the [Ca2+]j response to Hm, the source of the increased [Ca2+]j appears to be intracellular for the following reasons. 84 Bathing the cells in Ca2+-free saline for only 5 minutes did not attenuate the response to Hm, even though the perfusion rate had been increased such that the perfusion chamber had been flushed with at least 50 volume equivalents, effectively removing all the residual Ca2+-containing saline. As well, when normal [Ca2+]0 was restored simultaneously with Hm stimulation, [Ca2+]j did not change. If the [Ca2+]i response was due to an influx of extracellular Ca2+, then inclusion of normal [Ca2+]0 with Hm stimulation would have resulted in a normal [Ca2+]; response. Although the source of the [Ca2+]i response to Hm is an intracellular reservoir, it is separate from the IP3-releasable pool which is tapped by stimulation with BK or nucleotides. Bathing the cells in Ca2+-free saline for 30 minutes abolished the [Ca2+]i response to Hm stimulation, suggesting that the Hm-releasable Ca2+ reservoir is very labile. However, stimulation with BK or nucleotides, while still under Ca2+-free conditions, results in a strong [Ca2+]i response. Thus, Hm stimulation accesses a Ca2+ reservoir which is distinct from that accessed by BK or nucleotides, both of which have been shown to operate through IP3 in airway epithelial cells (BK in canine tracheal cells, McCann et al, 1986; ATP in cultured human nasal epithelial cells, Mason et al, 1991). Again, these results are consistent with the hypothesis that Hm is operating as a second messenger in this tissue. Hm would be produced in response to some extracellular stimulus (presumably PAF) and would trigger the release in [Ca2+]i from a store distinct from the IPj-releasable pool. In summary, Hm stimulation increases [Ca2"1"]; in cultured human nasal epithelial cells in a dose-dependent fashion. Results obtained from experiments conducted with Hu H2, and H3 specific Hm-receptor antagonists are consistent with either an H r or Hlc-receptor. However, the relatively high concentration of Hm which is required in order to stimulate a significant change in [Ca2+]j (>10 uM) suggests an Hic receptor type, rather than an Hj receptor. Hm stimulation 85 appears to release Ca2+ from an intracellular store, and this reservoir is probably distinct from the IP3-releasable pool. It has been suggested that Hm is functioning as a second messenger in this tissue, probably mediating stimulation with PAF. 86 CHAPTER SIX: General Discussion Asthma and CF are both important diseases which affect fluid secretion in airway epithelia. CF is the most common lethal genetic disorder in people of western European descent. Although modern treatments have dramatically increased the average life span for individuals afflicted with the disease, CF is still regarded as being ultimately fatal under current treatment regimes. Recently, the cystic fibrosis transmembrane conductance regulator (CFTR; Riordan et al, 1989) gene has been successfully transfected into CF cells (Drumm et al, 1990; Rich et al, 1990); however, genetic manipulation is not yet a viable treatment alternative. One possible route for new treatment alternatives is to explore the mechanisms controlling fluid secretion which are unaffected by the CF defect. It has long been known that pharmacologic factors which increase [Ca2+]i are capable of stimulating electrolyte and fluid secretion in airway epithelia (Welsh, 1990). However, endogenous factors which raise [Ca2+]i in airway epithelia have not been studied in any detail. As discussed earlier (Fig. 1.2), at the outset of this study very little was known about the mechanisms controlling fluid secretion in airway epithelia. With the identification of the gene which is responsible for CF came many new avenues of investigation which have already yielded much information about cAMP-mediated CI" secretion in airway tissue. The Role of CFTR and cAMP The projected structure of CFTR implies that it is a trans-membrane protein, and has much structure in common with known ion channels. Recently, expression of CFTR in non-mammalian cells has demonstrated that CFTR is in fact a low conductance, Ci"-specific channel 87 (Kartner et al, 1990). The channel kinetics of CFTR from several tissues and cell lines were described before the channel nature of CFTR was known (pancreatic ductal epithelial cells, Gray et al, 1988, Gray et al, 1989; porcine thyroid cells, Champigny et al, 1990; T^ colon carcinoma cells, Tabcharani et al, 1990). The low-conductance CI" channel (presumably CFTR) has a conductance 6-9 of pS (Tabcharani et al, 1990) and is more highly permeable to nitrate and bromide than to chloride, and less permeable to iodide, fluoride, and bicarbonate when studied in the cell-attached configuration (Grey et al, 1990). The presumed structure of CFTR consists of 12 membrane-spanning regions, 2 nucleotide binding folds, and a highly charged regulatory (R) domain. CFTR has a multitude of phosphorylation sites for both A-kinase and C-kinase, primarily located on the R-domain (Riordan et al, 1989). Activation of the low-conductance CI channel in excised membrane patches by cAMP-dependent protein kinase (A-kinase) and/or Ca2+-dependent protein kinase (C-kinase) yielded unusual results. Activation of membrane patches with the catalytic subunit of C-kinase alone resulted in a very weak increase of the channel open probability (P0). Activation of excised membrane patches with the catalytic subunit of A-kinase, in the presence of ATP, resulted in a strong increase in the P0 of the channel. However, activation of excised membrane patches with A-kinase after activation with C-kinase resulted in a dramatic increase in P0 as compared to activation with A-kinase alone. This activation potentiation was greater than the response to C-kinase and A-kinase added together (Tabcharani et al, 1991). These results underscore the interaction of second messenger systems in controlling cellular responses. Mutations in the first and sixth membrane-spanning regions suggest that these amino acids interact with the permeating anions (Anderson et al, 1991a). CFTR anion channel function requires ATP even after the protein has been activated by phosphorylation, suggesting a critical regulatory role for the nucleotide-binding folds in the protein (Anderson et al, 1991b). This 88 conclusion is further supported by the effect that the most common CF mutation (a deletion of a phenlyalanine from position 508 in the first nucleotide binding fold) has on the channel function of CFTR. This mutation increases the mean closed time of the channel by a factor of four (Dalemans et al, 1991). Regulation of channel activity by the nucleotide binding folds and the R-domain probably overlap because a large CI" conductance can be evoked in oocytes expressing the A508 mutation when the concentrations of phosphodiesterase inhibitor (and thus presumably cytoplasmic cAMP) is sufficiently high (Drumm et al, 1991). Figure 6.1 illustrates our current knowledge of cAMP-mediated CI" secretion and the CF defect. It has been clearly demonstrated that in CF, cAMP but not [Ca2+]rmediated CI" secretion is impaired (Boucher et al, 1989; Willumsen and Boucher, 1989). 6- adrenergic stimulation triggers an increase in cAMP through activation of adenylate cyclase. The increased cAMP triggers A-kinase-mediated phosphorylation of CFTR. Such phosphorylation results in opening of the apical CI" conductance (through CFTR). Intracellular CI" levels decrease with an efflux of CI' through the apical CI" channels. The resultant decrease in [Cl'Ji stimulates the basolaterally located Na+-K+-2Cl"-cotransporter, which imports CI" against its electrical gradient. It is unclear, as yet, how Na+ movement is affected by the activation of CI" secretion. Clearly, Na+ influx through the apical membrane must be curtailed in some way, yet no evidence has been reported which would indicate a direct connection between CFTR and the apical Na+ conductance. In CF, basal Na+ absorption is abnormal (Boucher et al, 1986). This abnormal Na+ absorption is probably due to the deranged CI" handling since the CF mutation has been shown to be localized to the CFTR protein, which is not suspected to directly regulate the apical cation channels (believed to account for the apical Na+ conductance; Duszyk et al, 1989). 89 BASAL K Na \ Y\ JK JA-\ v-—/N—-v \ 2C1 I I I K rffl ' ^ -A-kinase (> /CFTR . ? W (channel) a i-H Epi / cAMP Na APICAL Figure 6.1. Current Model of cAMP Mediated CI" Transport. Stimulation of the basolateral B-adrenergic receptor stimulates the production of cAMP. cAMP then stimulates A-kinase (a cAMP-dependent protein kinase) which, in turn phosphorylates CFTR. CFTR has been shown to be a low conductance (5-8 pS) CI" specific channel. Several phosphorylation sites for both A-kinase and C-kinase have been identified on CFTR (see Riordan et al., 1989; for proposed structure of CFTR). Phosphorylation of CFTR increases the open probability of the channel from 0.0 to 0.34 (Tabcharani et al, 1991). Increasing the apical CI" conductance likely lowers the [CI]; which in turn stimulates the Na+-K+-2Cl"-cotransporter. It is unknown at this time how, or indeed if, the apical Na+ channels are inhibited. It is logical to assume some type of regulation because if the apical Na+ channels remained open, the driving force for CI" transport would be dissipated. 90 Role of [Ca2+], As previously mentioned, the role of [Ca2+]rmediated CI" secretion is less well understood than that of cAMP-mediated CI' secretion. Only recently have there been any investigations of secretagogues which raise [Ca2+]i (this study; Welsh and McCann, 1986; McCann etal., 1989; and Mason et al., 1991). BK increased [Ca2+]j in a dose-dependent fashion in cultured human nasal epithelial cells (chapter 2). My results parallel those obtained by McCann et al. (1989) using cultured canine tracheal epithelial cells. BK elevated [Ca2+]; levels in cultured human nasal epithelial cells at comparable levels to those reported by McCann et al. (1989) for canine tracheal epithelial cells, suggesting that BK is working in a similar manner in both tissues. However, based on results obtained on cultured canine tracheal cells, McCann et a/.(1989) suggested that increased intracellular cAMP could trigger an increase in [Ca2+]j. This hypothesis was based primarily on the [Ca2+]; response to stimulation with Iso. When canine tracheal epithelial cells were stimulated with Iso, a 6-adrenergic agonist, cAMP and [Ca2+]i increased significantly. This response was unaffected by the a-adrenergic antagonist, prazosin, yet was significantly inhibited by the 6-adrenergic antagonist nadolol. These observations, coupled with the observations of Welsh and McCann (1986), where stimulation with Epi increased [Ca2+]; in a cell suspension of canine tracheal epithelial cells, led the authors to conclude that cAMP could directly stimulate increased [Ca2+];. However, in their own study, not all of the cells tested responded to CPT-cAMP (a membrane permeant form of cAMP), suggesting the possibility that the link between cAMP and [Ca2+]; is not as direct as they hypothesized. My experience with cultured human nasal epithelial cells suggests that fundamental differences exist in the way cAMP and [Ca2+]; interact in epithelial cells from canine airway and human airway. Treatment of human nasal epithelial cells with forskolin, a compound which 91 directly stimulates adenylate cyclase to produce cAMP, as well as exposure to a membrane-permeant form of cAMP (8-Bromo-cAMP) did not increase [Ca2+]i (chapter 4). Iso, Epi, and NE had significantly different effects on human nasal epithelial cells as compared to canine tracheal cells. Some secretagogues which stimulate CI" secretion through the cAMP-mediated pathway, such as Epi and NE, had no effect on [Ca2+]j levels in cultured nasal epithelial cells. Iso did stimulate elevated [Ca2+]j, but in only half the cells studied, a result which is at variance with the study published by Murphy et a/.(1988), in which no effect of Iso on human nasal epithelial cells was observed. The difference between the present study and that of Murphy et al. could be due to the vastly different tissue culture conditions employed in the two studies. Murphy et al. used human nasal epithelial cells which had been put into suspension after they had been cultured for 25 days. The cells utilized in the present study were cultured for a considerably shorter period of time (5 days) and remained attached to the substrate. As seen in chapter 3, the response of the cells to several stimulants is clearly related to time in culture. The lack of response to Iso in the study by Murphy et al. must therefore be interpreted cautiously. I believe that the differences between my results and those of McCann et al. (1989) reflect differences between species rather than differences between nasal and tracheal cell types. Fundamentally, nasal epithelial cells and tracheal epithelial cells perform the same physiological tasks (mucus secretion and regulation of the periciliary fluid layer) and are derived from the same embryological source (Moore, 1977)4. In cultured human nasal epithelial cells, increased intracellular cAMP does not mediate an increase in [Ca2+]j. The results that I obtained with Iso suggest that there is a sub-population 4 Initially, the nasal cavities originate as invaginations just above the oral cavity, whereas the trachea originates as an invagination of the primitive pharynx. However, the epithelial lining of both structures consists of endothelial cells originating from the lining of the laryngotracheal tube. 92 of cells in the airway which respond to Iso through [Ca2+]j as a second messenger. At this time it is not known whether Iso also stimulates cAMP production in those cells in which [Ca2+]; levels were increased. The indication is that in those cells which respond to Iso (approximately 50% of the cell population), cAMP does not play a role in the stimulation of [Ca2+]i levels. If cAMP were involved in the Iso-mediated increase in [Ca2+]i, then 50% of the cells stimulated with Epi, NE, forskolin, or 8-bromo-cAMP should also have demonstrated an increase in [Ca2+]j. [Ca2+]i did not increase in any of the cells tested with any of these compounds. Thus, an alternative mechanism independent of cAMP must be invoked to explain the increase in [Ca2"1"]; stimulated by Iso. Presumably, there is a G-adrenergic-like receptor, which responds to Iso but not Epi, that is coupled to IP3 production. This conclusion is based on two assumptions. First, the receptor types which are found on canine tracheal cells are identical to the receptor types found on human nasal cells, and second, the distribution of these receptors varies between species. Thus, the canine tracheal cells all possess this unknown 6-like receptor, while it is found on only about 50% of the human nasal cells. My results, as well as other studies conducted concurrently (Mason et al, 1991), have demonstrated the involvement of ATP and related compounds in stimulating increased [Ca2+]j. In their study, when the agonists were added apically, Mason et al. (1991) obtained an agonist selectivity sequence of UTP > ATP > 2MeSATP when measuring nucleotide-stimulated CI' secretion (as reflected by 1^ ). In that study, a similar selectivity sequence was obtained when [Ca2+]j was measured. This selectivity sequence is indicative of the so-called "nucleotide" receptor (Davidson et al, 1990; O'Connor et al, 1991). My receptor characterization studies followed a slightly different approach. O'Connor et a/.(1991) suggested that the relative potencies of ATP and ADP could also be used to distinguish between P2y receptors and nucleotide receptors. The selectivity sequence I obtained, UTP > ATP > ADP, also supports the 93 hypothesis that a nucleotide receptor is mediating increased [Ca2+]i stimulated by ATP. Interestingly, in their 1^  experiments, Mason et al. (1991) found an agonist selectivity sequence characteristic of a mixed receptor population when the agonists were added basolaterally. It is technically difficult to gain access exclusively to the basolateral membrane when measuring [Ca2+]i using fura-2, thus neither I nor Mason et al. were able to determine any sidedness to the [Ca2+]i response to nucleotide stimulation. Histamine has been shown to be a weak mediator of CI" secretion in canine tracheal epithelium (Marin et al, 1977). This observation, coupled with the demonstrated involvement of histamine in producing the symptoms of asthma, led me to investigate whether Hm influenced [Ca2+]i in airway epithelial cells. Clearly, Hm does influence [Ca2+]i levels in a dose-dependent manner (Fig. 5.2). However, the concentrations required to initiate an increase in [Ca2+]i appear, at first observation, to be unusually high as compared to other known [Ca2+]rmediated secretagogues (100 uM vs. 1.0 uM). Attempts to block the response to Hm, using antagonists for the three classical Hm-receptor types, suggest an Hj receptor. At a concentration of 0.1 uM, cimetidine (an H2-receptor antagonist) did not completely block the response to Hm, whereas pyrilamine (an Hj-receptor agonist) completely abolished the [Ca2+]j response to Hm. Thioperamide, an H3-receptor antagonist (0.1 uM), had no effect on the [Ca2+]j response to Hm. These results suggest an Hrreceptor type. It is difficult to reconcile the data compiled using the Hm-receptor blockers with the dose response curve obtained for the cultured human nasal epithelial cells. In other systems studied, Hrreceptors have a much higher affinity (i.e. a lower threshold) than that observed in this study. Recently, a novel Hm-receptor system has been described in human platelets (Brandes et al, 1989). In that system, PAF stimulates intracellular Hm production (Saxena et al, 1989a; Saxena 94 et al, 1989b). A novel Hm-receptor has been described in rat brain membranes (Brandes et al., 1987) and rat liver microsomes (Brandes et al., 1991), which has a low (uM) affinity and is different from the classically described Hu H2, or H3-receptor types. iV,iV-diethly-2-[4-(phenlymethyl)phenoxy]ethanamine HC1 (DPPE) is the most potent antagonist known for this receptor. Of the classical Hm-receptor agonists, pyrilamine is the most potent, followed by cimetidine (Brandes et al., 1991). This ranking of receptor blocker potency has prompted the proposal of an Hlc-receptor classification by those investigators. It is reasonable to propose that an Hlc-receptor may account for the results I obtained with Hm in the cultured human nasal epithelial cells. Certainly, the minimum effective Hm dose for increasing [Ca2+]j is in the same relative range as that described for the Hlc-receptor. The fact that the cultured human nasal epithelial cells required 10-100 times the concentration described for platelets may simply reflect the fact that I was using intact cells, not membrane fractions. Presumably, the rate at which Hm penetrates the cells necessitated higher Hm concentrations. Further support for this hypothesis comes from the relatively long perfusion time required to stimulate an increase in [Ca2+]i. Hm had to be in contact with the cells for a minimum of -30 seconds before any change in [Ca2+]j could be detected (as opposed to the 10-15 seconds required to initiate a [Ca2+]; response with BK or ATP). Again, this could possibly reflect the time required for the Hm to penetrate the cells. Certainly, if the Hm-receptors were on the cell surface as with BK or ATP, similar perfusion times should have been required. Unfortunately, the effects of DPPE were not tested since DPPE is not commercially available, and our laboratory was not equipped to synthesize that compound. Interestingly, PAF has been shown to stimulate CI" secretion of canine tracheal epithelia. Thus PAF, which has already been shown to work through Hm as a second messenger in human platelets (Brandes et al, 1991), may work through Hm as a second messenger in airway epithelia as well. 95 If Hm is not acting as a second messenger in airway epithelia, then an as yet unknown second messenger must be involved in the release of [Ca2^. The data presented in figure 5.7 suggest that Hm stimulates release from an intracellular pool that is separate from the IP3-releasable pool. Hm apparently does not operate through IP3 since secretagogues, which have been shown to stimulate an increase in cellular IP3 levels, were effective in increasing [Ca2+]i even after the Hm stimulated [Ca2+]j response was abolished by incubation in Ca2+-free saline (discussed later). Hm apparently does not operate through the second messenger cAMP either, since treatment with forskolin or membrane permeant forms of cAMP (8-bromo-cAMP) would have resulted in an increase of [Ca2+]j. Source of the Increased [Ca2+], None of the secretagogues tested appeared to stimulate an influx of extracellular Ca2+. This conclusion is supported by the insensitivity of the [Ca2+]j response to treatment with verapamil. Verapamil has been shown to block voltage-sensitive Ca2+ channels in the plasma membrane. Certainly, bathing the cells in Ca2+-free saline for periods even as long as 30 minutes did not strongly inhibit the [Ca2"1"]; response to either BK or exogenous nucleotides, suggesting that influx of extracellular Ca2+ through any channels, voltage-dependent or not, does not directly contribute to the increased [Ca2+]j observed. Thus, since influx of extracellular Ca2+ is not involved in the generation of the [Ca2+]i signal, intracellular Ca2+ reserves must be the source of the increased [Ca2+]i. BK has been shown to increase IP3 production in cultured canine tracheal cells, and that is almost certainly the case for cultured human nasal epithelial cells as well. Mason et al. (1991) demonstrated that exogenous nucleotides stimulated IP3 production in their cultured human nasal epithelial cells. Thus nucleotide stimulation and BK stimulation activate the classical IP3-Ca2+ second messenger pathway. 96 Stimulation with Hm also increases [Ca2+]; but through an entirely different mechanism. As already mentioned, short-term exposure to Ca2+-free saline (-5 minutes) does not adversely affect the change in [Ca2+]i in response to stimulation with Hm. Thus, Hm stimulation probably also triggers the release of Ca2+ from an intracellular reservoir. However, this reservoir is distinct from the IP3-releasable Ca2+ reservoir for two reasons. First, the Hm-releasable Ca2+ reservoir is much more labile than the IPj-releasable pool. Second, bathing the cells in Ca2+-free saline for 30 minutes did not seriously affect the [Ca2+]i response to BK or nucleotide stimulation, yet the same treatment completely abolished the [Ca2+]; response to Hm stimulation. Indeed, when cells were stimulated with ATP or BK immediately after an ineffectual histamine stimulation while under Ca2+-free conditions, a normal [Ca2+]i response was observed. It appears that the Hm-releasable intracellular Ca2+ pool was depleted by incubation of the cells in Ca2+-free saline, whereas the IP3-releasable intracellular Ca2+ pool was virtually unaffected. These observations necessitate only a slight modification of the three compartment model which was presented in chapter 2. Under Ca2+-free conditions, [Ca2+]; levels tend to decline. Such a diminution is probably counteracted by a slow loss of sequestered Ca2+ into the cytosolic compartment. My results indicate that more than one cytosolic compartment is present in the cultured human nasal epithelial cells. The different cytosolic Ca2+ reservoirs have different susceptibilities to decreased [Ca2+];. The Hm-releasable pool is clearly more labile than the IP3-releasable pool and thus is the first pool to be sacrificed as the cell attempts to maintain a constant [Ca2+]; level. 97 Organization of Second Messengers Mediating CI" Secretion in Airway Epithelia Control of CI" secretion is clearly more complicated than the model presented in fig 1.2. In the past 3-4 years several advances have taken place which have improved our understanding of the systems involved in the control of CI" transport across airway epithelia, primarily due to research into the cause of cystic fibrosis. Figure 6.2 incorporates our current knowledge of stimulators and second messengers involved in mediating CI" secretion across airway epithelia (information about which side a secretagogue acts on was taken from Welsh, 1987). Stimulation with 6-adrenergic agonists (Epi, Iso) increases cytoplasmic cAMP, which activates A-kinase, which in turn phosphorylates CFTR, a low-conductance CI" channel. This phosphorylation results in an increased P0 of CFTR. A second type of 8-adrenergic receptor apparently is present in the plasma membrane of some of the airway epithelial cells. Iso, but not Epi (or NE) stimulates a receptor which can be blocked by 8-adrenergic antagonists, and which stimulates an increase in [Ca2+]i. This response is totally independent of cAMP, suggesting the existence of a different receptor which can be activated by some, but not all, 6-adrenergic agonists and which is coupled to [Ca2+]j in some way. This hypothesis is further supported by data obtained with canine tracheal cells treated with pindolol (Feldman et al, 1990). Pindolol is a 6-adrenergic receptor antagonist which possesses weak intrinsic sympathomimetic activity. Pindolol stimulated CI" secretion by primary culture monolayers of canine tracheal epithelial cells. However, cAMP levels in those monolayers did not change significantly. The authors were prompted to suggest a non-cAMP-dependent pathway of activation for pindolol. My results suggest that such a pathway is operating through [Ca2+]j. 98 APICAL ATP, UTP BASOLATERAL PAF Figure 6.2. Proposed Organization of Intracellular Second Messengers in Human Airway Epithelia. Epinephrine stimulates cAMP production via 6-adrenergic receptors on the basolateral surface. Isoproterenol also stimulates these receptors, resulting in cAMP production. However, Iso also stimulates another receptor, which can be blocked with 6-adrenergic antagonists, which stimulates an increase in [Ca2+]j. Bradykinin stimulates receptors on both the apical and basolateral membranes, which activate the IP3-[Ca2+]i second messenger system. Nucleotide receptors which also activate the rP3-[Ca2+]i second messenger system are present on both the apical and basolateral membranes. PAF, operating through apical receptors, probably stimulates production of intracellular Hm, which in turn releases Ca2+ from a different intracellular pool than the one which is stimulated by IP3. 99 Stimulation of airway epithelial cells with BK and exogenous nucleotides, such as ATP and UTP, results in the liberation of sequestered intracellular Ca2+. The release of Ca2+ from intracellular reservoirs, stimulated by BK and exogenous nucleotides, has been shown to be mediated by the production of IP3 (McCann et al., 1989; Mason et ah, 1991). Future Studies Two major lines of investigation are suggested by the results of this study. First, and most important, is the suggestion that Hm is operating as a second messenger in airway epithelia. IP3 levels during stimulation with BK, ATP, and Hm must be conducted in order to determine whether these secretagogues are working through the IP3-[Ca2+]i second messenger axis. The effects of DPPE must be tested on these cells. 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Wu, R., Yankaskas, J., Cheng, E., Knowles, M.R., and Boucher, R. (1985). Growth and differentiation of human nasal epithelial cells in culture. Am. Rev. Respir. Dis. 132: 311-320. Yankaskas, J.R., Knowles, M.R., Gatzy, J.T., and Boucher, R.C. (1985). Persistance of abnormal chloride ion permiability in cystic fibrosis nasal epithelial cells in heterologous culture. Lancet 1: 954-956. 112 Appendix 1 Important Diseases Which Affect Electrolyte Transport Across Airway Epithelia Cystic Fibrosis Cystic fibrosis (CF) is a major cause of mortality in Caucasians of western European origin. With an incidence of approximately 1 in every 1600-2000 births, CF is the most common genetic disease with lethal consequences in North America and western Europe. It is estimated that 5% of these populations are carriers of the CF gene (reviewed by Nadler, 1984). The medically important symptoms of CF are manifest in the respiratory and digestive tracts. Respiratory effects can usually be found within the first year of life and are characterized by repeated bouts of pneumonia. Pronounced continual coughing (productive) and wheezing are typical, with increased chest diameter and clubbing of the fingers and toes in later stages which is probably due to insufficient oxygen uptake (Schwartz and Milner, 1984). Death is most often caused by pneumonia or respiratory insufficiency due to bronchial and peribronchial damage caused by bacterial infection (Taussig, 1984). Involvement of the digestive tract is manifest in several ways. Blockage of the pancreatic ducts results in pancreatic enzyme deficiency. In addition, bicarbonate secretion appears to be decreased (Mangos, 1978). Obstruction of the intestine often presents at birth (meconium ileus) or can develop at a later age. As well, most patients are found to have at least focal biliary 113 cirrhosis at autopsy (Mangos, 1978). This has been attributed to obstruction of the bile canaliculi by inspissated mucous secretions. As can be seen by the symptoms just mentioned (by no means a complete description), CF most seriously afflicts exocrine systems which also secrete mucus or proteins. No definitive changes in mucus secretions have yet been noted in CF (Boat, 1984). However, the symptoms are consistent with a decrease in water transport in these systems which results in inspissation (Applegarth and Bridges, 1983). Other water transporting systems are also affected. Patients with CF are often subject to shock during profuse sweating, a fact that led directly to the discovery of high CI" levels in the sweat of CF patients (diSant'Agnese et ah, 1953). Indeed, tantalizing hints of CF can be found in medieval literature. Several references can be found stating that children who tasted salty when kissed were bewitched, and would soon die. This could possibly reflect the elevated salt content in the sweat of the victims of this disease (see review by Taussig, 1984). Asthma Although not generally fatal, asthma can develop into a life-threatening disorder. Asthma is one of the most prevalent respiratory disorders in the industrialized world, with an incidence in excess of 5% of the total population (George and Owens, 1991). Like CF, asthma presents a plethora of variations. Symptoms vary from mild bronchospasm triggered by allergens, to chronic airway obstruction, with life-threatening consequences. Asthma attacks can be precipitated by a bewildering variety of factors and encompass several mechanisms. Asthma has been defined by the American Thoracic Society as a clinical syndrome characterized by a hyper-responsiveness of the tracheobronchial tree to a variety of stimuli. The major symptoms include 114 bouts of dyspnea, wheezing, and cough varying from very mild to severe and unremitting (American Thoracic Society, 1987). A genetic predisposition to asthma has been demonstrated (Hopp et al, 1990). George and Owens (1991) have defined 5 distinct types of asthma: 1. Allergic asthma, which is characterized by early onset, intermittent symptoms, rapid response to therapy, and possibly a reduction of symptoms with age. 2. Asthmatic bronchitis, which is characterized by onset after the age of thirty, progressive decrease in FEV! (1 second forced expiratory volume), and partial response to therapy. 3. Occupational asthma, in which symptoms are work-related and only partially respond to therapy (symptoms may persist after changing jobs). 4. Drug-induced asthma, in which the causative agent is usually apparent (agents such as aspirin) and there is a partial or complete remission after removal of the drug. 5. And finally, allergic bronchopulmonary aspergillosis, which is characterized by proximal bronchiectasis, respiratory infiltrates, chronic symptoms, response to steroids, and Aspergillus precipitates. Typically, an acute asthma attack can be resolved into two episodes: The early response, which usually lasts from 30 to 60 minutes, consists primarily of bronchospasm. This response is followed several hours later by a late response which is characteristically a hyperresponsiveness of the airway and can last several weeks or months (George and Owens, 1991). The asthmatic response includes edema and inflammation of the airway epithelia, hypersecretion of mucus and fluid by the airway epithelia, and constriction of the smooth muscles underlying the airway mucosa. Mediators which trigger the inflammatory responses involved in asthma include histamine, platelet-activating factor, and leukotrienes (George and Owens, 1991). 115 Appendix 2 Ratio Fluorometric Determination of [Ca2+], Initially, the first generation of Ca2+ indicators such as aequorin, had distinct disadvantages when used to determine [Ca2+];. Aequorin is a protein which changes in fluorescence when exposed to changes in Ca2+ ion concentrations (McNeil and Taylor, 1985). Because aequorin is such a large molecule, loading it into cells posed a considerable obstacle. Usually, cells were microinjected with aequorin , limiting the size of the cells which could be tested. Smaller cells could be loaded with aequorin through mechanically disrupting (by scraping cultured cells off the culture substrate) the cells in the presence of aequorin and allowing the cells to re-seal, a procedure which at best could be described as invasive. Further, aequorin tends to mildly buffer the [Ca2+]j during measurement. Rapid advances were made in the study of [Ca2+]; when it was found that the acetoxymethyl esters of tetracarboxylate chelators were easily loaded into cells. These acetoxymethyl esters (the so-called AM forms) are membrane permeant and can be loaded into cells non-disruptively by diffusion from the extracellular milieu (Tsien, 1981). Intracellular acetylesterases cleave the acetylester linkage, regenerating the original membrane impermeant chelator which is then trapped within the cells. The first of these compounds to be widely used was quin-2 (see review by Rink and Pozzan, 1985). The major drawbacks to quin-2 determination of [Ca2+]; are that the fluorescence is relatively weak, it has a relatively high dissociation constant, and absolute fluorescence is used to estimate [Ca2^. Quin-2 responds to Ca2+ mainly by a change in fluorescence emission under excitation at 339 nm. Using absolute fluorescence is difficult since it is directly dependent on dye concentration in the cytoplasm, specimen thickness, and the intensity of the excitation illumination. More recently, the second generation Ca2+ indicator fura-2 has become widely 116 w CD CD O CD O OT CD S-i o 2 0 300 1 mM 350 400 Excitation wavelength (r)m) Figure app. 1. Shifting Excitation Peak of Fura-2 with Changes in [Ca2+]. Increasing the [Ca2+] results in the shifting of the excitation peak for fura-2 from -380 nm down to ~ 340 nm. (graph adapted from Grynkiewicz et al, 1985; exact Ca2+ titration curves can be found in Grynkiewicz et al, 1985 and Tsien et al, 1985) 117 used. Fura-2 has three major advantages over quin-2: Thirty t imes less fura-2 is required to produce a given fluorescence intensity, resulting in less buffering of the [Ca2"1"]; signal. Fura-2 has a Kd of approximately 220 nM, resulting in increased sensitivity to Ca2 + in the physiological range. Finally, and probably most importantly, on binding Ca2+, fura-2 shifts its excitation peak from 380 to 340 nm (Fig. 1.4). This shift in the excitation peak facilitates the use of fluorescence ratios for determination of [Ca2+]i. Four proportionality coefficients can be derived from the two dye species and two wavelengths (k) measured. As described by Grynkiewicz et al (1985), these are S n for free dye at Xv Sa for free dye at ^ Sb l and Sb2 for bound dye at Xx and A^ respectively. The total fluorescence intensities, for a mixture of bound and free dye at concentrations cf and cb, are given by the following. 1.2J Fj=Sflcf+c>blCb L-3J F 2 = S f 2 C f + i b 2 C b Cf and Cb are related to the Ca2+ concentration by the equation, [4] Cb=Cf[Ca2+]/Kd Kd is the effective dissociation constant for fura-2. By substitution with equations 1,2, and 3, fluorescence ratio R (F/F^ can also be expressed as [5] R=(Sn+Sbl[Ca2+]/Kd)/(Sf2+Sb2[Ca2+]/Kd) Rearrangement of equation 4 yields [1] [Ca2+]=Kd(R-Rmin/Rmax-R)(Sf2/Sb2) 118 Where R^^Sf/S^ and Rmax=Sbl/Sb2. These equations are based on the assumption that the dye forms a 1:1 complex with Ca2+ (equation 3). Grynkiewicz et al. (1985) made a second assumption, namely that the dye behaves the same whether in cells or in the calibration medium which they used. However, by estimating R ^ and Rmax by permeabilizing the cells to Ca2+ and then measuring the fluorescence ratios at maximal [Ca2+] (1.8 mM) and at minimal [Ca2+] (0 mM + 5 mM EGTA), that assumption need not be made. By rapidly switching from 340 nm to 380 nm excitation illumination and measuring fluorescence at -510 nm, a reasonably accurate fluorescence ratio can be obtained from which [Ca2+] can be calculated (for a detailed description of the epifluorescence microscope and photometer arrangement, see chapter 2). 

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