Effects of chronic ligand exposure on nicotinic acetylcholine receptors

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ProQuest Information and Learning 300 North Zeeb Road, Ann Art)or. Ml 48106-1346 USA 800-521-0600

EFFECTS OF CHRONIC UGAND EXPOSURE ON NICOTINIC ACETYLCHOLINE RECEPTORS

by

Cynthia Lee Gentry

A Dissertation Submitted to the Faculty of the

GRADUATE INTERDISCIPLINARY PROGRAM IN NEUROSCIENCE

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY

In the Graduate College

THE UNIVERSITY OF ARIZONA

2002 UMI Number; 3061001

® UMI

UMI Microform 3061001 Copyright 2002 by ProQuest Information and Leaming Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.

ProQuest Information and Leaming Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor. Ml 48106-1346 0

THE UNIVERSITY OF ARIZONA iS GRADUATE COLLEGE

As members of the Final Examination Committee, we certify that we have read the dissertation prepared by Cynthia Lee Gentry entitled Effects of Chronic Ligand Exposure on Nicotinic;

Acetylcholine Receptors.

and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy

Rpna^ld/Tlukas, Ph.D. Date Li =f-1^11 b Leslie Tolbert, Ph.D. Date 7> -] U \ hs-L Gai^Burd, Ph.0. / ^ce

He^ 3ate~i/ P '?-' "^/-o 2- Ed French, Ph.D. / Date

Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

7- 5(-0- Dissercati^ directorRonald Lukas, Ph.D. Date 3

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. 4

ACKNOWLEDGEMENTS

This work was supported by the University of Arizona Committee on Neuroscience, by scholarship support from the FORD Foundation, the International Chapter P.E.O. Sisterhood and from the graduate college of the University of Arizona, by grant support from National Institute of Health training grant (AG007434) and the Arizona Disease Control Research Commission (100II), by endowment and/or capitalization funds from the Men's and Women's Boards of the Barrow Neurological Foundation and the Robert and Gloria Wallace Foundation, and was conducted in part in the Charlotte and Harold Simensky Neurochemistry of Alzheimer's Disease Laboratory. The contents of this report are solely the responsibility of the author and do not necessarily represent the views of the aforementioned rewarding agencies. The author wishes to thank Teri Lamour, Claire Dellarciprete, Jennifer Lawrence, Ruth Daniels, Ken Stynen, Jan Carey, Eleanor Swislow, and Behrooz Kousari for administrative support. The author acknowledges contributions of scientific training from the following persons and/or organizations: the American Psychological Association-sponsored Summer Program in Neuroscience Ethics and Survival (SPINES), the Society for Neuroscience Minority Educational Training and Professional Advancement Committee (METPAC), the University of Arizona's Biology Research Program (UBRP), the laboratories of Ronald J. Lukas, Ph.D., Thomas P. Davis, Ph.D., Paul A. St. John, Ph.D., Gail D. Burd, Ph.D., Dick van Der Horst, Ph.D., Rik van Antwerpen, PhX)., and John A. Law, Ph.D. Special thanks is extended to members of the dissertation conunittee: Leslie Tolbert, Ph.D., Henry Yamamura, Ph.D., Gail Burd, Ph.D., Ed French, Ph.D., and Ronald Lukas, Ph.D. The work presented in this document was performed under the guidance of Dr. Ronald Lukas at the Barrow Neurological Institute/St. Joseph's Hospital, Phoenix, AZ. 5

DEDICATION

This body of work is dedicated to my loving family, friends, and colleagues who have been a rich source of encouragement throughout my educational training and during the preparation of this dissertation. The love of my family and friends has been and will always be my greatest asset. If noble prizes were awarded to homemakers, my mother,

Lois Gentry would certainly earn one. My father. Dr. Michael Gentry, has preceeded me in earning a Ph.D. and is responsible for teaching me among many things, the value of an education. He was the first to teach me what a Ph.D. is and instill in me the desire to get one. My brother and friend, Christopher Gentry, has readily offered support both financially and otherwise at every opportunity, and I am sincerely grateful. Gratitude is also extended to my dear friend Jerod Carr, who has tolerated me through good times and more challenging times over the past four years, making himself available often when I needed a shoulder on which to cry. I also dedicate this work to my dissertation director,

Dr. Ronald Lukas, who has been and continues to be an exemplary role model for me and others.

Earning a Ph.D. is a team effort, and this dream could not have been accomplished without assistance and lessons-learned from many wonderful teachers and mentors in addition to the unwaivering support I received from my family and friends.

As I complete the requirements for this degree, it is with heartfelt thanks that I proudly announce to all of these invaluable people, "We did it!" 6

TABLE OF CONTENTS

UST OF ILLUSTRATIONS 9

UST OF TABLES 13

ABSTRACT 14 I. GENERAL INTRODUCTION Overview of nicotinic acetylcholine receptors (nAChR)...... 16 Roles of nAChR 17 nAChR Structure 18 Several nAChR subtypes exist 26 Properties of nAChR 31 nAChR are targets of nicotine...... 33 Nicotine acting on nAChR elicits drug seeking behavior 33 Therapeutic potential of drugs targeting nAChR...... 34 Tobacco related disease and nicotine dependence 34 Nicotine self-medication hypothesis 36 Parkinson's Disease 37 Alzheimer's Disease 38 Tourette's Syndrome 39 Attention-Deficit Hyperactivity Disorder 40 Autosomal Dominant Nocturnal Frontal Lobe Epilepsy 40 Mood elevation 41 Subtype selective ligands 41 Review of chronic nicotine exposure effects on nAChR...... 45 Nicotine Pharmacokinetics 45 nAChR upregulation following chronic nicotine exposure 46 nAChR functional desensitization 51 nAChR functional persistent inactivation 52 n. PRESENT STUDY: PROJECT DESIGN AND TECHNICAL CONSIDERATIONS 56 Hypothesis...... 57 Specific Aims...... 58 Aim 1: Evaluate a4^2- and a4^nAChR functional responses following chronic nicotine, carbamylcholine or mecamylamine exposure 59 Aim 2: Compare nicotinic ligand abilites to induce changes in a4^2- and a4P4-nAChR function 59 7

TABLE OF CONTENTS - Continued

Aim 3: Examine local anesthetics' acute functional potency at four distinct nAChR subtypes 60 Aim 4: Examine the chronic effects of local anesthetics on CNS-type nAChR 60 Technical considerations...... 61 in. SPECIFIC AIM 1; PROLONGED LIGAND EXPOSURE DEPRESSES FUNCTION OF HUMAN a4p2- AND a4P4-NICOTINIC ACETYLCHOLINE RECEPTORS (nAChR) HETEROLOGOUSLY EXPRESSED IN HUMAN EPITHELLVL CELL LINES 84 Abstract 85 Introduction 87 Materials and Methods 91 Results 96 Discussion 119

IV. SPECIFIC AIM 2; MIMICRY OR BLOCKADE OF CHRONIC NICOTINE- INDUCED LOSSES IN a4P2. AND a4P4-nAChR FUNCTION 128 Abstract 129 Introduction 131 Materials and Methods 133 Results 139 Discussion 161

V. SPECIFIC AIM 3; LOCAL ANESTHETICS NONCOMPETITIVELY INHIBIT FUNCTION OF FOUR DISTINCT NICOTINIC ACETYLCHOLINE RECEPTOR SUBTYPES 167 Abstract 168 Introduction 170 Materials and Methods 173 Results 177 Discussion 202

VI. SPECIFIC AIM4t FUNCTIONAL EFFECTS AT 04^2- AND a4P4-NAChR FOLLOWING PROLONGED LOCAL ANESTHETIC EXPOSURE 210 Abstract 211 Introduction 213 Materials and Methods 216 Results 222 8

TABLE OF CONTENTS - Continued

Discussion 238 vn. GENERAL DISCUSSION 245 Summary of major findings 246 Conclusions of the present research 252

APPENDIX A: REGULATION OF NUMBERS AND FUNCTION OF nAChR BY NICOTINE 256

REFERENCES 286 9

UST OF ILLUSTRATIONS

FIGURE l.l, nAChR are ligand gated ion channels 19

FIGURE 1.2A, Three-dimensional map of extracellular entrance of nAChR channel 22

FIGURE 1.2B, Electron micrograph cross-section of crystallized nAChR 22

FIGURE 1.2C, Fourier transform of digitized electron micrograph image showing nAChR channel profile 22

FIGURE 1.3, Pentameric structure of acetylcholine binding protein (ACBP): ball-and-stick representation 25 nGURE 1.4, Evolutionary relationship between nAChR subtypes 27

FIGURE 1.5, Structure of nAChR subunits 28

FIGURE 1.6, Examples of pentameric subunit combinations that yield functional nAChR 30 nGURE 1.7, nAChR kinetics model 55

FIGURE 2.1A, Operation of the Na^/lC-ATPase pump - mechanism for loading cells with ^^Rb"^ 63

FIGURE 2.IB, ^'^b^ loading is maximal withing 4 hours 64

FIGURE 2.2, Diagram of efflux assay protocol 67

FIGURE 2.3, Flow chart of experimental approach 69

FIGURE 2.4, Comparisons of pretreatment administration methods 72

FIGURE 2.5, How the data were normalized 74

FIGURE 2.6, Effectiveness of Rinses Following Chronic Nicotine Pretreatments .. 78

FIGURE 2.7, Nicotine pretreatment-induced persistent inactivation of a4P2-nAChR regardless of challenge drug and dose used to assess nAChR function 82 10

UST OF ILLUSTRATIONS - Continued

FIGURE 2.8, Observed efflux responses are mediated by nAChR 83

RGURE 3.1, Time- and dose-dependence of a4P2-nAChR functional effects following chronic nicotine exposure 107

FIGURE 3.2, Time- and dose-dependence of a4P4-nAChR functional effects following chronic nicotine exposure 109

FIGURE 3.3, Time- and dose-dependence of a4p2-nAChR functional effects following chronic carbamylcholine exposure Ill

FIGURE 3.4, Time- and dose-dependence of a4P4-nAChR functional effects following chronic carbamylcholine exposure 113

FIGURE 3.5, Time- and dose-dependence of a4P2-nAChR functional effects following chronic mecamylamine exposure 115

FIGURE 3.6, Time- and dose-dependence of a4P4-nAChR functional effects following chronic mecamylamine exposure 117

RGURE 4.1, SH-EPl-ha4P2-nAChR functional response to 1 mM carbamylcholine challenge following 1 hour nicotinic agonist exposure 145

FIGURE 4.2, SH-EPI-ha4p4-nAChR functional response to I mM carbamylcholine challenge following 1 hour nicotinic agonist exposure 147

HGURE 4.3, Correlations between agonist potency for inducing persistent functional inactivation (ICso) and agonist functional potency for acute activation (ECso) 148

FIGURE 4.4, Acetylcholine conenctration-response curves at a4P2- and a4p4-nAChR following 1 hour or 8-10 hours pretreatment with fixed concentrations of nicotine 150

FIGURE 4.5, SH-EPl-ha4P2-nAChR functional response to I mM carbamylcholine challenge following 1 hour nicotinic antagonist exposure 152 II

UST OF ILLUSTRATIONS - Continued

FIGURE 4.6, SH-EPI-ha4P4-nAChR functional response to 1 niM carbamylcholine challenge following 1 hour nicotinic antagonist exposure 154

FIGURE 4.7, Pancuronium block of nicotine, carbamylcholine, and mecamylamine induced nAChR functional inactivation at SH-EPl-ha4P2-nAChR 156

FIGURE 4.8, Pancuronium block of nicotine, carbamylcholine, and mecamylamine induced nAChR functional inactivation at SH-EPl-ha4P4-nAChR 158

FIGURE 5.1, Molecular structures of LAs used in this study 182

RGURE 5.2, LA dose dependence for functional blockade of al*-nAChR in TE671/RD cells 184

FIGURE 5.3, Dose-response profiles for carb-stimulated ®'^Rb^ efflux through al*-nAChR at different concentrations of LA 186

FIGURE 5.4, LA dose dependence for functional blockade of a3P4*-nAChR in SH-SY5Y cells 188

FIGURE 5.5, Dose-response profiles for carb-stimulated ^^b^ efflux through a3P4*-nAChR at different concentrations of LA 190

FIGURE 5.6, LA dose dependence for functional blockade of a4P2-nAChR in SH-EPl-ha4P2 cells 192

FIGURE 5.7, LA dose dependence for functional blockade of a4P4-nAChR in SH-EPl-ha4P4 cells 194

FIGURE 5.8, Dose-response profiles for carb-stimulated ®^b^ efflux through a4P2-nAChR at different concentrations of LA 196

FIGURE 5.9, Dose-response profiles for carb-stimulated ®^b^ efflux through a4P4-nAChR at different concentrations of LA 199 12

UST OF ILLUSTRATIONS - Continued

HGURE 6.1, SH-EPl-ha4p2-nAChR and SH-EPl-ha4p4-nAChR functional responses to 1 mM carbamylcholine challenge following 1 hour lidocaine, adiphenine, or dimethisoquin exposure 230

FIGURE 6.2, Pancuronium block of lidocaine-, adiphenine-, or dimethisoquin- induced nAChR functional inactivation at SH-EPl-ha4P2-nAChR ..232

FIGURE 6.3, Pancuronium block of lidocaine-, adiphenine-, or dimethisoquin- induced nAChR functional inactivation at SH-EPl-ha4P2-nAChR. .234

FIGURE 6.4, Effects of fixed concentrations of lidocaine, adiphenine, or dimethisoquin on nicotine-induced nAChR functional inactivation at CNS-type nAChR 237 13

UST OF TABLES

TABLE LIA, Table of subtype selective agonists 43

TABLE I.IB, Table of subtype selective antagonists 44

TABLE 3.1, Comparisons between nicotinic ligand functional inactivation and acute activation/inhibition potencies 118

TABLE 4.1, Parameters for persistent inactivation of oA^l- and a4P4-nAChR induced by nicotinic ligands 160

TABLE 5.1, Parameters for functional nAChR block by LA 201 14

ABSTRACT

To illuminate effects of chronic nicotinic iigand treatment on nicotinic acetylcholine receptor (nAChR) function, responses to acute agonist challenge after nicotinic Iigand pre-exposure were studied using ion efflux assays. Losses in function of heterologous!y-expressed, human a4P2- and a4P4-nAChR were observed following exposure to concentrations of nicotine at or below levels of nicotine present in the brain and serum of human smokers and showed both time- and concentration-dependence. a432- and a4P4-nAChR functional responsiveness to acute agonist challenge following prolonged exposure to various nicotinic receptor agonists or antagonists also was evaluated to determine whether these agents block or mimic nicotine effects and to gain insight as to mechanisms underlying nAChR functional changes. For all agonists tested, prolonged exposure induced functional losses at both a4^2- and a4P4-nAChR that persisted beyond removal of the drug. A strong positive correlation between acute functional potency of agonists and the amount of functional loss induced by pre-exposure to the agonists was found. Some but not all antagonists tested were able to induce persistent functional inactivation of a4P2- and a4P4-nAChR, but more weakly than agonists. However, sensitivity to antagonist pre-exposure allowed discrimination between closely related nAChR subtypes. Pre-exposure to pancuronium, a competitive antagonist of nAChR, had no lasting effect on nAChR function after its removal, but instead protected nAChR from nicotine-induced persistent inactivation. In studies to explore the 15 idea that open channel block is involved in nAChR persistent inactivation, local anesthetics (LAs) were found to act mostly as non-competitive antagonists at al-, (x3P4*-

, a4P2- and a4P4-nAChR subtypes. Prolonged exposure to some LAs also induced persistent functional losses at a4P2- and a4P4-nAChR, and some evidence was obtained suggesting that other LAs may protect against nicotine-induced persistent inactivation of a4P2- or a4P4-nAChR. Collectively, these studies provide insight into changes in nAChR functional state induced by prolonged nicotinic ligand exposure for diverse nAChR subtypes including those found in the brain. These findings have implications for nicotine dependence and for anticipated nicotinic therapies of potential clinical

Importance in treatment of a variety of neurological and psychiatric conditions. 16

CHAPTER I

General introduction

Nicotinic acetylcholine receptors, receptor desensitization and inactivation: introduction 17

CHAPTER I

The role of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChR) are among the best characterized members of the ligand-gated ion channel super family. NAChR are found throughout the peripheral and central nervous system (CNS) where they play critical roles in numerous physiological functions. Excitatory neurotransmission at the vertebrate neuromuscular

Junction and in autonomic ganglia is an important role of peripheral nAChR (reviewed in

Lukas and Bencherif, 1992). Other nAChR present at selected synapses of the CNS are believed to participate not only in classical exicitatory neurotransmission but also in the modulation of neurotransmitter release and neurotrophism (Lukas et al., 1999). Several nAChR subtypes with different regional distributions have been described in brain

(Sargent, 1993). Physiological roles for nAChR in the brain are less well defined. Still, these receptors have been implicated in both Alzheimer's and Parkinson's Diseases as well as anxiety and mood disorders (reviewed in Court et al., 2000; Mihailescu and

Drucker-Colin, 2000b). Nicotinic receptors in the brain are also believed to play an important role in specific cognitive functions, including attention and memory processes

(Newhouse et al., 1997; Levin and Simon, 1998; Levin et al., 1999). Some believe that nAChR play a more general role in synaptic plasticity and use of nicotine products could be rewiring the brain (Changeux et al., 1998). Because nAChR participate in many vital physiological and perhaps also pathological roles, a more in-depth invesitgation of the nature of nicotine and nicotine analogs interacting with particular nAChR subtypes is needed.

nAChR Structure

As ligand-gated ion channels, nAChR interact with selective agonists (activating ligands), such as the endogenous neurotransmitter, acetylcholine, or exogenously applied nicotine to induce a conformational change in the receptor that allows select ions to flow through the internal pore of the receptor (see fig. l.l). Ions traversing the cell membrane via open nAChR channels can initiate electrical signaling responsible for critical physiological events. The structure of nAChR is an important factor in determining how nAChR function. The nAChR was initially identified in Torpedo electric organ as a protein heteropentamer of about 3(K),(X)0 MW [reviewed in Karlin, 1993 #349].

Biochemical anaylsis revealed that the muscle-type nAChR has a subunit stoichiometry of

(al)2PlY/£S and contains two ACh binding sites located at interfaces between subunits a

+ y/E a + 5. Binding sites for other ligands and on other nAChR subtypes can differ. 19

Figure 1.1 iiAChR are ligand-gated ion channels

channel closed

channel opens

Fig. 1.1. Figure illustrates ligands, such as nicotine or acetylcholine, first binding to extracellular binding sites on a ligand-gated ion channel and then causing the channel to undergo a conformational change that opens the channel pore. Membrane impermeant ions are selectively allowed to flow into or out of a cell when the channel is open. The structure of muscie-type nAChR isolated from electric organ of the Torpedo ray has been studied using a cryo-equipped electron microscope (Miyazawa et al., 1999).

Closed-channel features were described from an average of 500,000 molecules at a resolution of 4.6 A (Miyazawa et al., 1999). Extracellular portions of a-subunits appear to have binding pockets for ACh -30 A away from the membrane. It was estimated that acetylcholine molecules (ACh) travel along with other cations through a -65 A long water-filled vestibule of the channel that is --20 A in diameter at the extracellular entrance then narrows sharply at the membrane surface (Miyazawa et al., 1999). Structural characteristics led to speculation that ACh is selectively guided through narrow tunnels

-10-15 A long which lead to cavities approximately the correct size and shape to accommodate an ACh molecule. The guidance mechanism was speculated to involve a combination of electrostatic effects and hydrophobic interactions. Closed-channel nAChR are gated by a narrow density close to the nuddle of the membrane. A total barrier to ions is formed -15 A away from the cytoplasmic membrane surface (Unwin,

1996). The observed location of the channel gate differs, however, from the location proposed based on cysteine-substitution experiments involving amino acids in the loop between transmembrane segments Ml and M2 (Wilson and Karlin, 1998). Precise physical aspects of the channel-gate are still in question. The <6 A width, however, led to a suggestion that the gate could be formed by conserved leucine side-chains projecting inwards from the middle of the M2 segments of each subunit (Unwin, 1993). 21

Interestingly the high-resolution electron microscopic study also revealed passages through the cytoplasmic wall wide enough for ions to pass through (Miyazawa et al.,

1999).

Cryo-electron microscopy of membranes expressing nAChR frozen either before or after being sprayed with saturating concentrations of acetylcholine revealed structural differences in open versus closed nAChR. No significant changes appeared near the rim.

Helices, however, switched to a new configuration in which bends formerly pointing toward the axis of the pore had rotated over to the side (Unwin, 1998). Upon binding neurotransmitter, nAChR protein undergoes a conformational change opening the channel and allowing ions to flow at a rate of - lO' sec"' (10,000 ions/ms) through each channel pore (Sansom, 1995; Unwin, 1998). Estimated open channel pore diameter was -10 A or greater and is consistent with measurements made using organic cations of various sizes

(Cohen et al., 1992). Three-dimensional images compiled from available data indicate that acetylcholine binding leads to channel opening via a series of propagated rotations from extracellular regions downward toward membrane spanning regions of the nAChR

(Sansom, 1995; Sankararamakrishnan et al., 1996; Mongan et al., 1998; Adcock et al.,

2000; Law et al., 2000). 22 Figure 1.2 uAChR mapping using electron microscopy and diffraction patterns.

(Unwin, 1998) (Unwin, 1998)

synapse

Fig. 1.2. A) Three-dimensional shape of nAChR based on superimposed two-dimensional images obtained by electron microscopic analysis of crystalline Torpedo electric ray postsynaptic membrane. Subunit assignments based on localization made with subunit-specific reagents. Scale bar. 20 A. B) Cross-section showing protein components of postsynaptic membrane protruding from the lipid bilayer. C) nAChR in profrle obtained by fourier transform of electron microscope data with estimated limits of the lipid bilary superimposed (dotted line, 30 A apart).

Cytoplasm (Unwin, 1993) 23

Crystal structure of a recently identified soluble acetylcholine binding protein

(ACBP) has been examined at 2.7 A resolution and provides improved structural information relevant to N-terminal domains of a-subunits of nAChR (Brejc et al., 2001).

The AChBP aligns with N-terminai domains of pentameric ligand gated ion channels and is closely related to a-subunits of nAChR. Nearly all residues conserved within the nAChR family are present in AChBP. including those relevant for ligand binding.

However, AChBP lacks the transmsmembrane domains and C-terminus that nAChR subunits possess. The AChBP isolated from the snail Lymnaea stagnalis is produced, stored and released in an ACh-dependent manner from glial cells (Brejc et al., 2001).

AChBP is 210 residues long and forms a stable homopentamer whose crystal structure, when viewed along the five-fold axis (top down view facing cell membrane), resembles a

windmill toy with petal-like protomers (Brejc et al., 2001). A pore diameter of ~18 A and cylinder height extending from the membrane surface of -62 A determined by crystallography are in close agreement with muscle-type nAChR electron microscopical data (Brejc et al., 2001). Each AChBP protomer is an asymmetrically shaped single

domain protein consisting of an N-terminal a-helix, two short 3io helices and a core of

ten P-strands forming a ^-sandwich (Brejc et al., 2001). The central pore of the pentamer

is very hydrophilic, lined with charged residues. Each AChBP protomer has a large

pocket accessible from the central pore that is uncharged and mainly hydrophobic. There 24 is a different cavity at each interface beween the subunits. These cavities, located close to the outside of the pentameric ring, have been shown biochemically to be involved in ligand binding in nAChR and are putative ligand-binding sites (Brejc et al., 2001).

Crystal structure of the AChBP further tells that although the size of the binding site is roughly similar in AChBP and acetylcholinesterase (AChE), observed arrangement of aromatic residues is quite different. Among pemtameric ligand gated ion channel receptors, the ligand-binding site is conserved, but ligand-binding residues vary creating specificity for different ligands. At present, it is uncertain whether AChBP performs the allosteric and desensitization movements critical for nAChR function. Lack of a transmembrane domain necessitates that ligand binding to the AChBP does not open a membrane pore. Some movements, however, could be conserved between AChBP and nAChR. Further studies are required to determine this and whether non-competitive antagonist-binding sites are present in AChBP (Brejc et al., 2001). Figure 1.3 Pentameric Structure of Acetylcholine Binding Protein.

Fig. 1.3. Bail-and-stick representation of pentameric structure of AChBP. AChBP viewed perpendicular to the five­ fold axis. (Brejc et ai., 2001) 26

Several nAChR subtypes exist

Several pharmacologically distinct subtypes of nAChR exist. To date 17 AChR subunit genes have been cloned from vertebrates (Lukas, 1998). Multiple combinations of subunits (al-lO, pi-4, y,e,S) can form a variety of nAChR pentamers, and certain combinations produce functional nAChR subtypes (Fig. 1.6). Sequence anaylsis, combined with observations from biochemical and pharmacological studies, suggests that the genes for all of the subunits share a common origin with the a9 subunit being the most primitive (Changeux et al., 1998). The nAChR subunits could have evolved from the serotonin receptor. There are many caveats in determining evolutionary relationships between nAChR subunits because the outcome of a phylogenetic classification may differ depending on factors such as, what subunit is used as the reference point and what evidence is used for comparison (i.e. sequence information, secondary structure, ion permeability, functional properties, etc.). One depiction of evolutionary relationship (in million years) between nAChR subunits based on sequence information is represented in

Fig. 1.4: 27 Figure 1.4 Phylogenetic tree illustrating evolutionary relationship of nAChR subunits. a2 a4

a3

a6

P3 a5

p4

al pi

S

Y e on a8 ] a9 ]

5-HT3

—r- —I— 1 800 600 400 200 0 (Changeux et al., 1998) Fig. 1.4. Phylogenetic tree diagram representing evolutionary relationship between nAChR subunits based on sequence similarites. 28

Figure 1.5 Structure of the nAChR subunit

NHa CC30H

Intracellular

Figure taken from: Fundamental Neuroscience 1999 by MJ. Zigmond, F.E. Bloom, S.C. Landis, J.L. Roberts L.R. Squire. Academic Press, San Diego CA, USA. ISBN: 0-12-780870-1

Fig. 1.5. Structure of the nAChR subunit including four transmembrane domains, a long extracellular N-terminus, and a large intracellular loop between membrane spanning domains M3 and M4. Five nAChR subunits assemble to form a functional nAChR ligand gated ion channel. Each subunit has a large extracellular amino-terminus, four membrane spanning domains (M1-M4) and an extracellular C-terminus. Subunits can be distinguished by sequence difference occurring in the substantial M3-M4 cytoplasmic loop.

Each subunit may assemble with other like or unlike subunits to form a pentameric nAChR. Thus both differences in individual nAChR subunit gene sequences and various combinations of subunits assembling into functional pentameric nAChR contribute to a diverse array of nAChR subtypes. Each nAChR subtype possesses a unique pharmacological and physiological profile (reviewed in Lukas, 1989b; Lukas and

Bencherif, 1992; Lukas, 1995). 30

Figure 1.6 Examples of subunit combinations that form functional nAChR:

Alpha 7 homomer Muscle-type nAChR

a7 a7 al

a7 a7

a7 al "CNS"-type nAChR ^ Alpha 4, beta 2 nAChR; ((x4)202)

a4

Alpha 4, beta 4 nAChR Gangllonlc-type nAChR (a4),(P4)3 a3* or (*3^4* a4

a4

a4 ol3

Fig. 1.6. Examples of nAChR subunit pentameric combinations that are known to form functional nicotinic receptors. 31

Properties of nAChR subtypes

Each nAChR subtype may respond differently upon exposure to a given iigand.

Ligand affinities vary across nAChR subtypes. Additionally, some ligands may act as an agonist or partial agonist at one subtype while acting as an antagonist at a different nAChR subtype. Certain nAChR subtypes may exhibit differences in levels of cooperativity upon ligand binding or differences in voltage dependence. A particular nAChR subtype may also exhibit unique ion selectivity when activated. Individual nAChR subtypes also have a unique kinetic profiles. Single channel open times, and whole cell current responses are often used to characterize nAChR subtypes. It is also thought that desensitization/inactivation properties vary across nAChR subtypes, a hypothesis that is the focus of this dissertation (reviewed in Gentry and Lukas, 2002a).

Additionally, whether or not a particular nAChR subtype responds to a ligand and the type of response may depend on its subcellular localization (axon, dendrite, soma; pre- or post-synaptic) and its regional distribution throughout tissues and organs. nAChR are generally found wherever there are cholinergic projections, cholinergic intemeurons, cholineacetyltransferase, or acetylcholinesterase. For example, evidence from a variety of species increasingly supports that a4- and a7- nAChR subunits are prominently expressed and widely distributed throughout the central nervous system. nAChR functional roles may differ according to their anatomical localization. For example, a7- nAChR found in hippocampal tissue is believed to play critical roles in learning and 32 memory, while a4P2-nAChR found in amygdala are thought to play a role in mood

(Decker et al., 1995a; Court et al., 2000). 33

Nicotine acting on nAChR elicits drug seeiung behavior.

Nicotine, a primary ingredient of tobacco products, is likely to exert its powerful and diverse physiological effects by acting on nAChR. Thus, effects of nicotine consumption from tobacco products have provided clues as to physiological roles for nAChR. nAChR have been implicated in mediating a wide range of physical and behavioral changes in humans and animal. For example, there is substantial evidence that nicotine elicits drug seeking behavior (Corrigali, 1991; Stolerman and Shoaib, 1991;

Stolerman and Jarvis, 1995). Habitual users of tobacco products experience craving, tolerance, physical and psychological (mild euphoriant) dependence, relapse during abstinence and withdrawal symptoms (Benowitz et al., 1989; Benowitz, 1996). Nicotine is thought to be responsible for smokers' continued use of cigarettes despite harmful effects generated by other substances contained in cigarettes (Benowitz, 1996). In the

United States, over 52 million adults and nearly 4 million adolescents use tobacco products (Smith and Fiore, 1999). This use of tobacco products results in more than

400,000 deaths in the United States each year (www.cdc.gov/tobacco/issue). This costly social issue has brought nAChR to the forefront in research aimed at understanding addictive processes and developing effective smoking cessation therapies. 34

Therapeutic potential of drugs targeting nAChR and importance of studying ciironic drug effects.

In its pure form, nicotine, aside from being addictive, is also recognized for many positive attributes. An important focus of future nicotinic receptor research is understanding the role of nAChR in disease pathology and capitalizing on the potential of cholinergic ligands for treating diseases. In addition to nicotine dependence, nAChR have been implicated in several disease pathologies: Alzheimer's Disease, Parkinson's

Disease, Tourette's Syndrome, Attention-Deficit Hyperactivity Disorder, Schizophrenia,

Pain, Depression, and Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (reviewed in Dursun and Kutcher, 1999). Many cholinergic ligands are being considered for alleviating symptoms associated with these diseases, making it essential that regulation of each nAChR subtype following chronic ligand exposure be well understood.

NAChR renulation implications for Tobacco-related disease and nicotine dependence

Habitual users of tobacco products are considered "nicotine dependent." By definition, a person experiences "tolerance" if she or he obtains a diminished behavioral response after being exposed to the drug for a defined period of time (Ochoa et al., 1990).

Thus, higher intake is required in order to achieve the same behavioral effect as that obtained at the initial stages of the drug use. Tolerance may lead to "dependence" in which a person feels she or he needs the drug in order to continue functioning normally in civilized life (Ochoa et al., 1990). Dependence is less severe than "drug abuse", in which drug usage leads to dangerously deranged health and behavior problems affecting interpersonal relationships and often associated with criminal actions (Ochoa et al.,

1990). While nicotine is not legally considered a drug of abuse, proven addictive properties of tobacco products, of which nicotine is the primary ingredient, are still a substantial concern. Nationwide, tobacco product usage leads to an estimated 50 billion dollars per year in direct health care costs and an additional 50 billion dollars per year in indirect health care costs (www.cdc.gov). Understanding the effects of chronic nicotine exposure on the regulation of nAChR may lead to improved smoking cessation therapies.

New therapies could reduce the use of tobacco products. In turn, this could reduce health care costs and loss of life/quality-of-life associated with use of tobacco products. Some nicotinic ligands are already in use as smoking cessation aids while others are still in development for similar purposes.

Early experiments evaluating chronic nicotine effects revealed a quantitative increase in nicotine binding sites (reviewed in Gentry and Lukas, 2002a). This nAChR

"upregulation" did not agree with dogma that receptors down-regulate in response to chronic agonist exposure (Wonnacott, 1990). Later, when it was discovered that nAChR

undergo functional desensitization and inactivation, this phenomenon became a suspected

mechanism for tolerance development. Several hypotheses began to arise as to how

nAChR upregulation and functional changes relate to smokers' behavior. Some

investigators speculated that nAChR functional potentiation after treatment with low

concentrations of nicotine could mediate some of the beneficial neurochemical and behavioral effects of nicotine, whereas the down-regulation of function observed after treatment with high-concentrations of nicotine could underlie the development of tolerance. Other ideas about the relationship between nAChR functional changes following nicotine exposure and the development of tolerance have also been discussed in the literature (Collins et al., 1990b). One hypothesis is that rather than acute activation of nAChR providing beneficial physiological enhancement, receptor inactivation may parallel these effects while the recovery of nAChR from inactivation prompts the desire for more nicotine (Lukas, 1991; Ke et al., 1998). These ideas and the processes of upregulation, desensitization, and inactivation are discussed more thoroughly in upcoming sections and in a review article by Gentry and Lukas, 2002 (see appendix).

Nicotine self-medication hypothesis

Nicotine in its purest form has promising therapeutic potential. The incidence of smoking is greater among patients suffering from depression and schizophrenia than in the normal population, suggesting that smoking may represent a form of self-medication

(Masterson and O'Shea, 1984; Hughes, 1986; Goff et al., 1992). Self-medicating with nicotine products may also be occurring among patients experiencing anxiety and pain. nAChR regulation by chronic nicotine at the molecular level could explain self- medicating behavior of smokers (Lukas, 1991; Ke et al., 1998). One hypothesis proposes that craving a cigarette is a means for keeping nAChR in a persistently inactivated state to prevent an overactive cholinergic system from generating anxiety (Ke et al., 1998). The 37 role of nAChR function under such conditions and its relationship to smoking behavior warrants additional research. It should be noted that physiological effects of nicotine at nAChR, including nAChR upregulation and functional inactivation, have been demonstrated for concentrations of nicotine even lower than what is observed in serum and brain of smokers, making the hypothesis relating nAChR regulation to smoking behavior reasonable (Gentry and Lukas, 2002a).

Prospects for the use of chronic nicotinic lieand treatment for psychiatric and neurolosical disorders

Parkinson's Disease (PD). Deterioration of substantia nigra dopamine-producing neurons leads to severe muscular rigidity, tremor and bradykinesia characteristic of PD.

Cholinergic striatal neurons are thought to be released from dopaminergic inhibition upon depletion of dopamine in the basal ganglia. Evidence that high-affinity nAChR density is reduced in humans with PD, combined with evidence that PD is 20-70% less frequent in smokers than in non-smokers, suggests that nAChR may play a role in PD (Baron, 1986).

Differences in acute and chronic nicotine effects on degenerating nigrostriatal neurons in mouse have been observed (Janson et al., 1992b). Chronic intermittent nicotine treatment demonstrated protective effects in several studies of male rat's with lesioned nigrostriatal dopamine neurons (Janson et al., 1992a). Human subjects also exhibited improvements in motor control, cognitive performance and emotional status following nicotine administration (Fagerstrom et al., 1994). Thus appropriate regulation of nAChR by 38 chronic nicotinic ligand administration could be beneficial in treating Parkinson's patients

and in gaining an improved understanding of the complex mechanisms underlying

symptoms of PD.

Alzheimer's Disease (AD). Alzheimer's dementia results in part from degeneration of

basal forebrain cholinergic neurons which project to various cortical areas and memory

centers such as the hippocampus. A decline in [^H]nicotine binding but not a-

bungarotoxin binding in post-morten human brain tissue of Alzheimer's patients suggests

that high affinity/slow-desensitizing nicotine receptors are selectively reduced (Sugaya et

al., 1990). Fast-desensitizing, a-bungarotoxin sensitive nAChR have also been

implicated in AD. p-amyloid peptide (APi_t2), material composing neuritic plaques

present in brains of patients with AD, was recently reported to bind with femtomolar

affinity and inhibit a7-nAChR expressed in human SK-N-MC neuroblastoma cells

(Wang et al., 2000). Follow-up studies found AP1.42 could elicit functional blockade of

a7-nAChR native in rat hippocampal slices, but binding studies have not been replicated

(Pettit et al., 2001). Nevertheless, nicotine administered subcutanteously or intravenously

improves sustained visual attention, reaction time and perception among Alzheimer's

patients (Newhouse et al., 1988; Sahakian et al., 1989). Thus, it has been established that

nicotinic ligands have therapeutic potential in treating AD. New links between nAChR

and AD are still surfacing, and the precise role of nAChR regulation remains unclear. 39

Tourette's Syndrome (TS). Altered levels of nAChR functional activity may be key in controlling sudden, rapid, brief motor and vocal tics associated with TS. Nicotine has been shown to reduce tics when combined with neuroleptic treatment. Nicorette gum containing 2 mg of nicotine administered together with haloperidol decreased TS- associated tic frequency and severity relative to haloperidol administered alone (Sanberg et al., 1988; Sanberg et al., 1989; McConville et al., 1991; McConville et al., 1992).

Similar results were obtained using nicotine patches (7 mg/24 h), and improved patient compliance was expected (Silver and Sanberg, 1993). Interestingly, evidence of neuroadaptation was shown in a study of Tourette's patients taking haloperidol simultaneously treated with transdermal nicotine patches (7 nig/24 hr). A single administration of nicotine reduced tics for a 1-2 week period, while a second administration reduced tics for an even longer period (Silver et al., 1996). Nicotine was also capable of improving tics in the absence of neuroleptics. Nausea and vomitting, however, were common side effects of this treatment (Shytle et al., 2000; Newman et al.,

2001). Shytle and colleagues hypothesize that beneficial effects of nicotine therapy in

Tourette's patients may be the result of presynaptic nAChR desensitization ultimately limiting dopamine release. Silver and colleagues proposed that low doses of chronically administered nicotine would mimic antagonist's action by inactivating nAChR (Silver et al., 2001a). A follow-up clinical trial using nAChR antagonist, mecamylamine, as

therapy for Tourette's patients met with very limited success (Silver et al., 2001a). While

mecamylamine may have led to moderate reduction of sudden mood changes and 40 depression in severly affected individuals, mecamylamine did not significantly improve

motor tics relative to placebo control (Silver et al., 2001b). Cholinergic ligand therapy

has not been discarded as a potential treatment for Tourette's patients, but a better

understanding of nAChR regulation and response to chronic ligand exposure including

neuroadaptive capabilities is needed.

Attention-deficit Hweractivitv Disorder (ADHD). ADHD is another target for nicotine

therapy. Impaired attentiveness, increased impulsivity and hyperactivity are characteristic

symptoms of ADHD. Cigarette smoking or nicotine administration has been found to

improve symptoms, and withdrawal from cigarettes worsens symptoms. 40% of adults

with ADHD are smokers compared with 26% of the general population (Pomerleau et al.,

1995). The extent of nAChR participation in alleviating ADHD symptoms is not

known.

Autosomal Dominant Nocturnal Frontal Lobe Epilepsv (ADNFLE). nAChR

desensitization plays an important role in the pathogenesis of ADNFLE. In this epilepsy,

characterized by brief seizures occurring during light sleep, there are specific mutations

located in the a4 subunit of nAChR: I) phenylalanine is substituted for serine at position

247 (S247F), and/or 2) leucine insertion (776ins3) occurs near the extracellular end of

M2. Either mutation will lead to an increased rate of desensitization and hence, reduced

calcium permeability. Taking into consideration current knowledge of nAChR,

functional responses to chronic nicotinic ligands may assist in the development of a

successful treatment regime for patients with ADNFLE (reviewed in Mihailescu, 2000). Mood elevation. While nAChR regulation via chronic ligand effects at particular nAChR subtypes is likely to be important for development of improved drug therapies, not all effects of smoking can be entirely attributed to nicotine acting directly on nAChR to regulate nicotinic receptor function. For example, chronic smoking has been shown to inhibit monoamine oxidase B, an enzyme involved in the breakdown of dopamine, and of monoamine oxidase A which may explain some of the mood enhancing and other beneficial effects of smoking (Fowler et al., 1996b; Fowler et al., 1996a). Nicotine has also been shown to increase firing of serotonergic neurons from dorsal raphe nucleus of rat midbrain slices (Mihailescu et al., 1998; Mihailescu and Drucker-Colin, 2000a). Still, subtype selctive ligands are highly sought for their potential to advantageously regulate nAChR in such a way that avoids many of the harmful side effects commonly associated with smoking.

Subtype Selective Ligands

One approach to developing drug therapies for treating the above-mentioned diseases is finding subtype-selective ligands. Such ligand may allow the formulation of a drug treatment that targets some but not all subtypes of nAChR in a combination that

yields a desirable outcome with minimal side effects. Of the nAChR suptype selective

ligands in development, there is almost no information available about

pharmacodynamics of these drugs following prolonged exposures because very few

studies have evaluated their chronic effects on nAChR function or general physiology. The following table outlines site of action and proposed mechanisms of action for the subtype-selective ligands currently available (see table 1.1A,B), (reviewed in Decker et al., 2001). 43 Table I.IA nAChR Agonist subtype Characteristic Reference

GTS-21 rat a? Potent partial agonist at (Hunter et al., 0(7; weak partial agonist 1994) at a432 AR.R 17779 on Potent full agonist at a7 (Levin et al., 1999) IVIG624 and F3 ot7/(x8 (Gotti et al., 2000) Choline an Weak full agonist? (Papke et al., 1996) DMXB a7 (Meyer et al., 1997) A-85380 04^2 (Sullivan et al., 1996) ABT-418 oe4p2 Potent full agonist at 04^2 (Americ et al., 1994) ABT.594 a4p2 Full agonist at neuronal (Bannon et al., oe432, a7, and a3Sy 1998) ABT-089 oe4p2 Weak partial agonist at (Brioni et al., 04^2; complex weak 1997; Sullivan et agonist, partial agonist al., 1997) and inhibitory interaction with other subtypes. DBO-83 a4P2 Full agonist at oc4P2 and (Decker et al., ganglionic nAChR 2001) SIB 1508Y a4P2 (Menzaghi et al., 1997; Schneider et al., 1998; Schneider et al., 1999) SIB 1765F a4p2 Full agonist (Sacaan et al., 1997) SIB 1553A P4 (Bontempi et al., 2001) Lobeline ot4fi4 Potent partial agonist Buhlman, unpublished UB-165 a4/a3 Intermediate potency (Sharpies et al., 2000)

Table 1.1A. Agonists recognized for selectivity activating particular nAChR subtypes while being much less potent at other nAChR subtypes. 44

Table I.IB Antagonist nAChR subtype Characteristics Reference Lophotoxin a432, al^, a* competitive (Luetje et al., 1990) Neosurugatoxin all except al*, competitive (Luetje et al., 1990) neuronal nAChR II-BRT al*, a7*, a8*, a9* competitive Luetje, 1990 #725] DHBE 0402 competitive (Grottick et al., 2000) Erysodine neuronal nAChR ? competitive (Decker et al., 1995b) MLA an competitive (Grottick et al., 2000) a-conotoxin-mll oc3/(x6 competitive (Harvey et al., 1997) a>conotoxin-inI al/y interface, al"** competitive (Luetje et al., 1990; muscle nAChR Martinez et al., 1995) a-contoxin-GlA al*, muscle nAChR competitive (Luetje et al., 1990) a-conotoxin-lml a7, a9 competitive (Johnson et al., 1995) a-conotoxin-EI al/S interface competitive (Park et al., 2001); Martinez, 1995 #733] a-contoxin-lpl a3p2, Gc3p4, ? competitive (Loughnan et al., 1998) a>conotoxin-AuIB a3B4,? competitive (Cho et al., 2(M)0) omega-conotoxin a3B4,? via Ca*^ block (Herrero et al., 1999)

Table I. IB. Antagonists recognized for selectivity activating particular nAChR subtypes while being much less potent at other nAChR subtypes. Nicotine Pharmacokinetics

Nicotine is absorbed rapidly from cigarette smoke. Within 10-19 seconds nicotine is distributed to body tissues, including brain via arterial circulation. Levels of nicotine present in the serum of two pack per day smokers is 0.22 (Russell et al., 1980). Brain nicotine levels were found to be 3.1 fold greater than plasma levels in a study of rats subjected to constant nicotine infusion (Rowell and Li, 1997). Physiological effects of nicotine at nAChR have been demonstrated for concentrations of nicotine even lower than what is observed in serum and brain of smokers (reviewed in Gentry and Lukas,

2002a).

Nicotine is subject to extensive metabolism in the liver. Metabolism of nicotine can vary from individual to individual. Typically 80% of nicotine is metabolized to cotinine and about 4% to nicotine-oxide (Benowitz, 1996). The elimination of nicotine occurs in two phases. The half-life relevant to the accumulation of nicotine during use of nicotine or tobacco products averages 2-3 hours (Benowitz, 1996). Slow release of nicotine from body tissues is presumed to be responsible for a very long terminal half-life of 20 or more hours (Benowitz, 1996). Nicotine conversion to cotinine is the rate- limiting step in nicotine metabolism and occurs via a two step processs in which a

CYP450 enzyme (thought to be CYP2A6) convens nicotine to the intermediary metabolite, nicotine iminium ion, and aldehyde oxidase converts the intermediary to cotinine (Benowitz, 1996). Cotinine is further metabolized to 3'-hydroxycotinine, the most abundant metabolite found in urine (Benowitz, 1996). Glucuronidation leads to additional metabolites of nicotine, cotinine, and 3'-hydroxycolinine (Benowitz, 1996).

Evidence suggests that persons of certain races or ethnicity may be different with regard to their CYP2A6 expression and thus be more or less capable of metabolizing nicotine (Pianezza et al., 1998; Tyndale et al., 1999; Sellers et al., 2000; Sellers and

Tyndale, 2000; Tyndale and Sellers, 2002). One individual identified to be deficient in

C-oxidation converted only 8% of nicotine to cotinine and consequently had an unusually long half-life of nicotine (Benowitz et al., 1995). Some have hypothesized that smoking may itself reduce nicotine metabolic activity (Benowitz and Jacob, 2000).

nAChR upregulation following prolonged llgand exposure

Several in vitro and in vivo studies have shown chronic nicotine affects numbers of nicotine binding sites present. Benwell and colleagues reported human smokers to have elevated levels of brain nicotinic receptor binding when compared with nonsmokers

(Benwell et al., 1988). In vivo investigations using mice and rats allowed for the elucidation of specific brain regions affected as well as effects of withdrawal on nicotinic receptor binding sites to be examined. Studies using the DBA/2 mouse strain demonstrated L-[^H]-nicotine binding increases in a time- and dose-dependent manner, yet numbers of binding sites return to control level after a sufHcient withdrawal period

(Marks et al.. 1985; Marks et al., 1986b; Marks et al., 1986a; Marks et al., 1986c).

Collins and colleagues also examined nicotine and bungarotoxin binding in rat brain 47 during withdrawal following 7 days of gradually increasing concentrations of chronic nicotine infusion (0.2 mg/kg/hr gradually increasing to 0.8 mgAcg/hr) (Collins et al.,

1990a). The chronic nicotine treatment led to a significant increase in both nicotine and bungarotoxin binding for all brain regions examined: cortex, midbrain, hindbrain, hippocampus, striatum, and hypothalamus. The greatest increase in nicotine binding was observed in cortex. The increase in nicotine binding returned to control levels within 8 days for most brain regions, however, the cortex still exhibited a significantly greater amount of nicotine binding with respect to control at 8 days of withdrawal. Increased levels of nicotine binding also persisted in the hippocampus. After 21 days of withdrawal, nicotine binding in the cortex and hippocampus were able to return to control levels. In comparison to bungarotoxin binding, nicotine binding exhibited slower returns to control levels. Bungarotoxin binding persisted the longest in hippocampus and hindbrain regions. For all brain regions examined, the observed increases in bungarotoxin binding following the seven day chronic nicotine infusion were reversed within 8 days of withdrawal. Thus, chronic nicotine infusion elicited increases in nicotine and bungarotoxin binding in ail brain regions examined. Scatchard analysis demonstrated that Kd values were unaffected by chronic nicotine, and B max values were increased (Collins et al., 1990b). This result suggests that chronic nicotine induces an upregulation in the number of nicotine binding sites. A detailed account of brain regions affected by chronic nicotine in terms of nAChR upregulation found statistically signiHcant increases in L-^-nicotine binding to occur in every major brain area 48 examined following chronic nicotine infusion in mice (Pauly et al., 1991; Marks et al.,

1992).

The phenomenon of chronic nicotine induced upreguiation of receptor binding has been observed in in vitro systems as well. Investigations of several cell lines each containing different nAChR subtypes, have demonstrated increased binding to various degrees in response to chronic nicotine treatment. The use of in vitro systems has allowed for individual responses of nAChR subtypes to be examined. Fenster and colleagues reported chronic nicotine exposure initiates an upreguiation of surface a4^2- nAChRs expressed in Xenopus oocytes (Fenster et al., 1999b). Another study of the a4p2-nAChR in HEK 293 cells found up-regulation of [^H]cyiisine binding following 7 day chronic nicotine treatment. The magnitude of upreguiation observed depended on the concentration of chronic nicotine used (Gopalakrishnan et al., 1996).

Investigators first speculated that changes in nAChR binding would parallel the onset of nicotine tolerance, however, data soon indicated that there is no correlation between receptor upreguiation and nicotine sensitivity. Studies showed that mice which have been subjected to chronic nicotine infusion exhibit reduced sensitivity to acute nicotine stimulus with respect to their locomotor, respiration, heart rate, body temperature, and startle responses (Marks et al., 1983; Marks et al., 1985; Marks et al.,

1986b; Marks et al., 1986a). Chronic nicotine injection (1.6 mg/kg, twice daily for 1-8 days) of rats revealed increased nicotine binding in various brain regions which returned to control levels within 7 days of treatment initiation. In contrast, the rat's tolerance to nicotine as measured by behavioral effects of nicotine did not subside during the 7 day withdrawal period (Collins et al., 1988). Thus, the time course for reversal of nicotine binding during withdrawal did not correlate with that of tolerance to nicotine's effects on body temperature and locomotor activity (Collins et al., 1990a). Additional studies demonstrated that up-regulation of brain nicotinic receptors can occur prior to development of tolerance (Collins and Marks, 1991) and tolerance can occur without receptor upregulation (Pauly et al., 1992). Marks (1993), found that mouse in vivo behavioral responsiveness to chronic nicotine decreased linearly with increasing concentrations of nicotine. In contrast, binding site densities showed maximal increases following treatment with low dosage (0.5 mg/kg/hr) (Marks et al., 1993a). Collectively these results suggest that receptor upregulation and nicotine tolerance are independent.

The mechanism underlying the nAChR upregulation response to chronic ligand stimulation is not known. However, recent evidence suggests that modification of nAChR binding site numbers that results from chronic ligand exposure does not involve an increase in receptor subunit mRNA production. In vivo studies of DBA mice chronically infused for 10 days with nicotine (4 mg/kg/hr) found increased L-[^H1- nicotine binding primarily in cortex and midbrain. In contrast, the mRNA for a4 and ^2 nAChR subunits as well as less prominent a2, a3, and a5 subunits measured in these

brain regions of chronically treated animals showed no significant increases in mRNA

levels (Marks et al., 1992). These findings suggest that nAChR up-regulation upon chronic nicotine exposure results from a postranslational modiHcation. In addition to nicotine, chronic treatment with nicotinic agonists cytisine

(Schwartz and Kellar, 1985), (+)-anatoxin-a (Wonnacott, 1990), anabasine (Bhat et al.,

1991), and methylcarbamylcholine (MCC) (Yang and Buccafusco, 1994) has been shown to induce upregulation of [^H]nicotine binding in rodent brain. Warpman and colleagues tested the effects of chronic treatment with six additional nicotinic agonists using MIO cells stably transfected with chick the a4P2-nAChR and SH-SY5Y cells expressing the a3P4-nAChR (Warpman et al., 1998). Most nicotinic agonists up-regulated nicotine binding. Two cases in which chronic ureatment with nicotinic agonists did not significantly up-regulate high-affinity nAChR binding are lobeline (Bhat et al., 1991) and l-acetyl-4-methylpiperazine methiodide (AMP) (Warpman et al., 1998). Each of the nicotinic agonists raise nicotinic receptor binding site numbers to varying degrees with some having little or no affect at all. It is speculated that individual pharmacological properties of agonists play a role in determining the magnitude of up-regulation that results from chronic agonist exposure.

Chronic treatment with nicotinic receptor antagonists has also been investigated.

Chronic infusiong of DBA/2 mice with nicotinic antagonists, mecamylamine, led to an increase in [^H] nicotine binding. Chronic infusion of mecamylamine together with nicotine resulted in an additive increase in nicotine binding (Collins et al., 1994).

These results, however, were not in complete agreement with those of Schwartz and

Kellar (1985) who were unable to detect differences in nicotinic receptor binding in rat brain following chronic injection with mecamylamine. Both studies similarly found that chronic mecamylamine was unable to protect against upreguiation in nicotinic receptor binding that occurs with chronic nicotine injection. Zhang and colleagues reported that competitive antagonists dihydro-P-erythroidine and tubocurare as well as the noncompetitive antagonist hexamethonium do not lead to increased nicotinic receptor binding (Zhang et al., 1995). Furthermore, nicotine evoked up-regulation was partially inhibited by curare and hexamethonium in MIO cells (Peng et al., 1994; Zhang et al.,

1995) and dihydro-B-erythroidine in rat brain (Yang and Buccafusco, 1994).

nAChR desensUization

Agonist binding to nAChR results in a rapid and transient opening of a transmembrane ion channel. It has been accepted for many years that nAChR

"desensitize" following a brief exposure to nicotine. Receptor desensitization is the process by which the receptor becomes no longer responsive to repeated or prolonged stimuli and is thought to be a mechanism by which receptors protect the target organ from overstimulation. The term "desensitization" by classical definition applies to an instance of reversible functional loss. Desensitization of nAChRs was originally thought to occur by a single-exponential process. Feltz and Trautman (1982) first proposed that desensitization is a more complex phenomenon and a wealth of evidence since has given strength to that proposal (Feltz and Trautmann, 1982). 52 nAChR persistent inactivation

In 1987, Boyd demonstrated with Na^ influx assays that nAChRs of the PC 12 sympathetic cell line exhibited more than one kinetic phase of functional inactivation.

Boyd found that most receptors were in a rapidly recovering state after short agonist

incubation periods, however, with increasing duration of agonist exposure, many

receptors were converted to a distinctly different kinetic state which recovered function

more slowly (Boyd, 1987). The later, more deeply desensitized state is now termed

"persistent inactivation" (Ke et al., 1998). Boyd also found that exposure to the agonist, carbamylcholine, for 30 minutes at 22 and 37 °C was capable of inducing an irreversible

decrease in the nAChR function (Boyd, 1987). An in vivo rat study contlrmed the

functional inactivation response to chronic nicotine infusion (10 days) in midbrain

synaptosomes (Marks et al., 1993b; Marks et al., 1993a).

Investigations in which nAChR subunit cRNAs were injected into Xenopus

oocytes to express functional nAChRs support the hypothesis that persistent inactivation

occurs to varying degrees in different nAChR subtypes. Hsu and colleagues found the

a4P2-nAChR exhibited a decrease in functional response to agonist following 24 hour

incubation with nanomolar concentrations of nicotine and the functional loss occurred in

a concentration dependent manner (Hsu et al., 1996). A total loss of agonist ability to

elicit function was achieved at between 100 and 500 nM concentrations of nicotine. In

contrast, loss in responsiveness of the a3P2-nAChR occurred only at micromolar 53 concentrations, and total loss of responsiveness could not be achieved even at high concentrations of nicotine.

An independent study by Fenster and colleagues, measured function of the aA^l- nAChR expressed in Xenopus oocytes by two-electrode voltage clamp. Peak currents were measured before and immediately after removal from 24 hour nicotine incubation.

The magnitude of nicotine-induced desensitization was defmed as the ratio of the fractional response remaining after nicotine exposure relative to the fractional response remaining over the same period of time in the absence of nicotine. Consistent with the

Hsu (1996) study, a dose-dependent decrease in a4P2-nAChR function was observed following the chronic nicotine treatment. A small increase in peak currents was observed after 1-2 hours recovery period in which oocytes were removed from nicotine incubation.

Almost no additional recovery could be achieved with extended washout times (Fenster et al., 1999b).

Gopalakrishnan and colleagues provided the first chronic nicotine study of the human a432-nAChR expressed in a human cell line (Gopalakrishnan et al., 1997).

Interestingly, this receptor type stably expressed in HEK-293 cells chronically exposed to nicotine for 24 hours at concentrations less than 1 ^M, followed by 30 minute recovery period, exhibited enhanced ion flux. Functional inactivation consistent with previous studies of other receptor subtypes occurred at concentrations greater than 1 ^M

(Gopalakrishnan et al., 1997). 54

Several models for nAChR kinetics have been introduced into the literature. A current model, such as the model illustrated in Fig. 1.7), would have to incorporate recent developments in understanding nAChR regulation. Thus, collectively evidence gathered to date requires that a model for nAChR kinetics include a basal- ready state (B) for the receptor, an activated open channel state (A), a rapid-initial desensitized state (D), and a more persistently inactivated state or states (1). The model should take into account that a given nAChR may exist in a B state with one molecule bound but not enter the A state unless two molecules are bound. The one molecule bound condition may represent an indepent B state with affinity that is distinct from B with no molecules bound. The model needs to include a pathway for "silent" recovery where the receptor can return to B after ligand removal without passing through the A state. The model may/may not depict nAChR returning from I to B after ligand removal in a gradual or step-wise fashion.

Most data indicate that ligand dissociation and removal is a prerequiste for entering the pathway from I to B although this is not yet supported with conclusive evidence.

Numerous reports of nAChR desensitization/inactivation inducable by pretreatment concentrations of agonist that are subthreshold for provoking channel opening, combined with data suggesting nAChR antagonist pretreatment may also induce desensitization/inactivation except to a lesser extent, indicate that a model of nAChR kinetics should include a pathway from B to D or I without passing through A. A model of this design is the basis of the hypothesis tested in this dissertation (Rg. 1.7). 55 Figure 1.7 nAChR Kinetics Model nicotine B A* nicotine -D +t If h I.

Fig. 1.7. One proposed model for nAChR kinetic states following nicotinic ligand exposure. 56

CHAPTER n

Present Study: Project Design and Technical Considerations 57

CHAPTER n

Hypothesis: Based on previous investigations demonstrating that prolonged nicotine exposure renders peripheral nAChR incapable of responding to future agonist stimulation, it was hypothesized that other nAChR subtypes, including CNS-type nAChR, may become functionally inactivated by prolonged nicotine exposure. It was further hypothesized that the extent of functional inactivation (i.e. percentage of functional loss relative to unexposed nAChR and length of time necessary for functionally inactivated nAChR to regain their ability to respond to agonist challenge) could depend on the type of ligand, concentration of ligand, and duration of ligand exposure that nAChR received.

Objective: The primary goal of this project was to improve upon current understanding of how central nervous svstem fCNS)-tvpe nicotinic acetylcholine receptors (nAChRs) are regulated upon chronic ligand exposure. The study characterizes changes in a4P2- and a4P4-nAChR function induced by prolonged nicotinic ligand exposure. aA^2- nAChR is putatively the highest-affinity binding nAChR subtype present in the CNS. The a4P4-nAChR is gaining recognition as a potentially major subtype of nAChR present in

human CNS, and ^4 subunit mRNA has been anatomically localized to brain regions

important for regulating human emotions and memory. This research is intended to 58 provide insight as to the molecular events underlying addiction and thus provide a necessary knowledge base for more direct approaches to smoking cessation therapv.

Nicotine dependence and addiction to other drugs of abuse, such as cocaine, morphine and amphetamines, may have common underlying mechanisms. The impact of this study may reach beyond nicotine dependence to serve as a model for other addictive processes and as a predictor of how nicotinic lieand administered chronically to patients as neuropharmaceuticals may affect brain chemistry.

Specific aims:

1. a4P2- and a4P4-nAChR function is affected by prolonged exposure to nicotine,

carbamylcholine, or mecamylamine in a time- and concentration- dependent manner.

Time- and concentration-dependence varies for each nAChR subtype.

2. Nicotinic ligands will have different abilities to induce functional changes of a4P2-

and a4P4-nAChR.

3. Local anesthetics will be functional non-competitive antagonists of nAChR. LA

functional potency will differ across nAChR subtypes and local anesthetic structural

categories.

4. Prolonged local anesthetic application will induce changes in a4^2- and a4^

nAChR functional response to subsequent acute agonist challenge. 59

The hypothesis that chronic nicotine treatment leads to persistent inactivation of both peripherally- and centrally-acting nAChR subtypes, but that the induction of persistent inactivation requires different doses and lengths of nicotine exposure is addressed in chapter QI. It was further hypothesized that high-affinity receptors such as the a4P2-nAChR may be subject to greater functional inactivation than lower-affinity counterparts. To test the hypothesis of specifc aim 1 (chapter QI), conditions necessary for inducing changes in functional activity of the aA^l- and a4P4-nAChR following chronic nicotine exposure were characterized using ion flux assays. Results were compared with data from previous studies of peripheral nAChR subtypes. Studying a4P4- and a4P2-nAChR expressed in the same cellular environment and subject to equivalent handling conditions, made it possible to draw conclusions about whether different subtypes of CNS-nAChR are affected by chronic nicotine similarly. The direct comparison with only a single variable altered also allowed for examining the role of the

P subunit in nAChR desensitization and persistent inactivation.

Specific aim 2 is discussed in chapter IV. Pharmacological tools were used to gain a better understanding of mechanisms underlying the nAChR functional inactivation that is observed following chronic ligand exposure. It was hypothesized that channel opening precedes nicotinic receptor entrance to a desensitized and persistently inactivated receptor conformation. Experiments using nAChR agonists other than nicotine tested whether the ability to induce functional inactivation of the receptor is isolated to a specific nicotine binding site. Chronic antagonist studies provided insight as to whether functional activation of the receptor is a necessary event for induction of persistent inactivation. The relative ability of several agonists and antagonists to downregulate a4P2- or a4P4-nAChR function was tested using ion efflux assays.

The ability of select noncompetitive antagonists to induce nAChR persistent inactivation prompted additional studies of other ligands also thought to induce channel block. Specific aim 3 hypothesized that local anesthetics are functional non-competitive antagonists of nAChR that block open channels and could be useful tools for further investigating the nature of persitent changes in receptor function upon chronic drug exposure. Chapter V discusses the results of ion efflux assays used to generate concentration-response profiles determining local anesthetics' (LA) acute functional potency at four distinct nAChR subtypes and to examine their mechanism of inhibition.

Comparisons were made across nAChR subtypes and local anesthetic structural categories.

Specific aim 4 addresses the chronic effects of LAs acting on CNS-type nAChR.

The effects of pretreating a4^2- or a4P4-nAChR with one LA from each of three structural categories were examined. Aside from the hypothesis that LA acting as nAChR non-competitive antagonists would mimic function of nicotine in inducing persistent inactivation, it was further hypothesized that a relationship may exist between acute functional potency of the LAs and their ability to induce functional inactivation or that a relationship may exist between the structural category of the LAs and LA ability to induce 61 functional inactivation of the nAChR. Additionally, chronic effects of LA were hypothesized to affect particular nAChR subtypes selectively, possibly even differentiating between a4^2- and a4P4-nAChR. Finally, hypotheses relating to non­ competitive antagonists abilities to induce or protect nAChR from functional loss were tested. Ion efflux assays were used to assess the function of CNS-type nAChR following chronic LA treatment.

Technical considerations:

This project relied heavily on ion efflux assays to evaluate receptor function during and after nAChR were subjected to various pharmacological manipulations. A standard ion efflux assay protocol is described here and outlined in table 2.2. A discussion of modifications necessary to accommodate pretreating cells expressing nAChR with various nicotinic ligands follows, including explanations for several experiments completed to characterize the modified ion efflux assay.

Standard ^^b^ Ion Rux Assay.

Both ion influx and ion efflux assays were evaluated for potential use in these studies, ^^b^ efflux assays clearly exhibited the highest sensitivity and resolution of any flux assay of nAChR function tested, yielding consistent data on a regular basis. For standard efflux assays performed in our laboratory, cells expressing the receptor of interest are harvested from master plates via trypsinization and reseeded evenly onto 24- well Falcon culture plates, 500 |il per well. Once cells are adherent to the plate surface

(minimum 4 hours, usually overnight) they are loaded with (approx. 25,000 cpm per 25 ^l of media) via Na*-K*-ATPase activity occurring at the cell membrane (see Fig.

2.1a). The radioactively labeled rubidium as a analog is actively transported against the K* concentration gradient into the cell. It was determined that the cells are fully loaded after 4 hours (i.e. ^'^b^'s electrochemical gradient has reached equilibrium and the amount of ®®Rb* that can enter the cell has reached its maximal level) (Fig. 2.1b). 63

Figure 2.1A

Cells are loaded with via active transport using the Na'^/K'^ ATPase pump.

E^otein Phosphoiylation and confonaadonal change MM Release of Binding of Na ion mmmm»• t-ia luu U/r ^ ^^*Ia1 ^MA Jii|^oTAof thetne cefl.ceu.

Na,K/8«Rb+• •/ pump ,

Release ( K\o &e v|^ Dephosphoiyiation and inside of M||5« struccoial change in protein. theCell ' • The Top is die Outter membrane. • The Bottom is the inner membrane (inside of the CeU) Modified from: http7/www.biochein.arizona.edu/classes/bioc462/462a/NOTES/LIPIDS/tninsport.html by M.Ziegler. 64

Figure 2.1B loading is maximal within 4 hours. i

E CL SH-EP wt O iS IS o

3000 > I -o 0) 2000 >1

CO 1000 SH-EP ha4p2 O • + JD flC

SH-EP ha4P4

7.5 10.0 12.5 15.0 17.5 20.0 Loading time (hours) 65

Rinses are necessary to remove extracellular that has not been internalized by the cells. The "flip plate method" refers to a drug application or rinse method developed in our laboratory previously (Lukas et al., 2002). By forming a tight seal between the cell plate placed on top of another 24 well Falcon plate that contains the appropriate solution, in this case rinse buffer, the solution to be applied can be gently

"flipped" into culture wells containing the cells. Using this method, all wells of the cell plate receive rinse buffer or drug exposure efficiently and simultaneously. Typically, 2 rinses of 2 ml per well efflux buffer remove the extracellular ®^Rb^. Following sufficient

rinsing away of extracellular ^^Rb*, cells are subjected to an acute (2 minute) ligand exposure. Each well in the 24-well plate pre-set for use in the acute (2 minute) ligand exposure will typically contain either I mM carbamylcholine for positive control to define total ion flux, buffer alone as a negative control to define non-specific ion flux,

agonist together with antagonist as another negative control to also assess any spontaneous nAChR-mediated flux, and/or various concentrations of drug being evaluated in the presence of agonist to test for drug antagonist activity or in the presence of drug alone to test for its intrinsic agonist activity. Because removal of extracellular

^^b^ during rinses before nicotinic drug application establishes an infinite gradient for

the ^®Rb^ rapidly leaves the cell upon nAChR stimulation, ^^b^ that is released

from cells into extracellular buffer during the 2 minute assay period is counted using a

state-of-the-art Wallac Trilux 1450 microbeta liquid scintillation and luminescence plate 66 counter and serves as a quantitative indicator of receptor function. Experiments of the present study were performed at room temperature (20° C), and efflux buffer was pH 7.4.

Presentation of standard efflux assay data involves subtracting the radioactive counts representing ^^b^ released under negative control condition (i.e. ^'^Rb* release due to spontaneous channel opening or leak) and then expressing the specific efflux from each sample as a percentage of the positive control (i.e. sample exposed to 1 niM carbamylcholine represents 100% efflux).

The use of a clonal ceil line for these studies minimizes pharmacokinetic complications and allows for comparison of function and levels of expression in parallel studies of nAChR. Preliniinary experiments tested instrumentation for reproducibility and to determine essential procedures for obtaining optimal and accurate results. 67

Table 2.2 Standard Efflux Assay - Flip Plate Method

1. Plate cells into 24-well tissue culture plates and allow to adhere (typically about 3-6 hours or overnight).

2. Aspirate medium with a Pasteur pipette that has a P-200 pipette tip on the end and add ®1lb^ medium (see note below) to each well. Use a minimum 250 |iL per well for a 24-well plate.

3. Allow cells to load by allowing them to incubate in the medium for a minimum of 4 hours (can be done overnight).

4. Before the start of the assay, fill 2 (or 3 if you expect high background signal) clean multi-well plates with efflux buffer. These will be the "rinse plates." For 24-well plates use 2 mL in each well. This high volume allows the extracellular '^b'^ to be completely washed away.

5. In addition to 2 "rinse plates", you will need a "test plate" containing the hots (agonist alone) and colds (agonist + antagonist or efflux buffer alone) and dilutions of drug teing tested. Use the same volumes as in Step 4, with one exception. If the cells are going to be counted in 24-well plates, both the "test plate" and the cell plate should be filled with I mL of efflux solution or 0.1% SDS/O.OIN NaOH, respectively. Higher volumes in the cell plate tend to cause overflow when the plate counter manipulates the plates.

6. To start the assay, aspirate the *^b^ medium with a Pasteur pipette that has a P-200 pipette tip on the end and hold inverted over the first "rinse plate." Align the edges of the plate so that all the wells line up. It is critical to feel around the edges of the plate to ensure proper alignment. Any misalignment will allow buffer to spill into adjacent wells or spaces in between wells. Holding the plates firmly together, slowly and gently rotate the plates over and allow the rinse buffer to bathe the cells. Rotate plates back over and remove the used "rinse plate" and set aside. ("Flip-plate" method)

7. Without aspirating, hold the "cell plate" over the second "rinse plate" and rinse as in Step 6.

8. There is a small, but uniform, residual volume that remains in the wells of the "cell plate" which must be aspirated so that applied test concentrations remain exact when the "test plate" is applied. Aspirate the wells as quickly as possible (should take no more than 10 seconds for a 24-well plate).

9. Place the "cell plate" over the "test plate" and carefully align. Rotate the plates to initiate the efflux and set on the benchtop. 68

Table 2.2 con't.

10. After appropriate efflux time (usually 2-5 min), carefully realign plates and flip the plates back over so that the test concentrations are flipped back into the "test plate."

11. For 24-well plates, place clean, dry inserts into each well. Cover the plate and inserts with a Top-Seal film.

12. Place test plate onto a loading rack for the Trilux plate counter. Use appropriate protocol 1-5 minutes per well counting.

13. Perform appropriate analysis of data.

Notes: Cells should be plated at high density, usually 2 confluent 10 cm' dishes are used for each multi-well plate. SH-SY5Y cells are not very adherent and therefore the plate must be pre-coated with poly-d-lysine. TE cells are fairly adherent and poly-d- lysine is usually not required. SH-EPl cells are very adherent so poly-d-lysine is not required, unless pretreating cells with ligand as in chronic ligand experiment. Chronic ligand experiments require additional rinses and thus it is important to precoat dishes with poly-d-lysine to help cells adhere to plates.

Prep of ^®Rb'^ medium: Determine the minimum amount of medium necessary to load plates for the assay (add about 15% to ensure that you have enough). Place completed medium (plus antibiotics if necessary for that cell line) into a 50 mL conical tube and place the tube in a ^-blocking container. Under the isotope hood, pipet the *^b^ from the manufacturer's stock into the medium. Newly received stocks are very hot, so a 15 mL volume of medium might require as linle as 1 to 2 of the stock. Swirl the medium thoroughly to mix and count ®^b* medium in scintillation counter, ^^b"^ medium should contain approx 250,000 to 350,000 cpm/250 ^L. However, for some cell lines and polyclonal pools (especially the SHEP ca7-V274T), as much as 500,000 cpm/250 fxL must be used to ensure adequate signal. 69

Figure 2.3 Exnerimental Design for Studies of Chronic T Jgand EffFecte;

efflux pretreatment drug free recovery assay defined length of defined length of time^ with drug of intei 2 minutes ImMcarb

Step 1 Step 2 Step 3

Fig. 23. Experimental design for studies of chronic ligand effects. Cells expresssing nAChR of interest were first treated with drug of interest for an experimentally defined length of time. Next the cells were allowed to recover in drug-free media for an experimentally defined length of time. Finally, nAChR functional response to 1 mM carbamylcholine acute (2 min) challenge was measured. The use of ion efflux assays for evaluating nAChR function haS been established for sometime. However, to evaluate nAChR function following chronic treatment with various nicotinic ligands, the standard ®®Rb* efflux assay protocol required some modification and further characterization. The overlap between loading cells with ®®Rb^ for the functional assay and pretreating cells with nicotinic ligand/allowing cells to

recover from ligand pretreatment raised a few issues of concern. First, nicotinic ligand could interfere with the loading of ®®Rb* into the cells. It was necessary to perform

preliminary experiments to confirm that ^^Rb^ could still be incorporated into cells allowing for sufficient ®®Rb^ to be present intracellularly at the time of measuring

receptor function. Second, any initial stimulation of nAChR by pretreatment ligand could

trigger the release of ^^Rb^ prior to the subsequent functional assay. The amount of

released ®^b^ could then vary across wells on a cell plate exposed to different concentrations of pretreatment ligand leaving the amount of ^^Rb^ present intracellularly

at the start of the functional assay unequal for various samples, perhaps even fully

depleting the intracellularly loaded ^^b^ in some samples before beginning the functional

assay. If the amount of intracellular ^^b^ present in cells for different samples is unequal

at the start of the assay, counting the amount of ^*^b^ released by cells and comparing

that with ®^b^ released from control cells on the same assay plate would not accurately

depict the relative function of nAChR on those cells. Correction for differences in loaded

was made via a normalizing process in the analysis of our data (explained under

heading "data analysis", and Table 2.S). A third concern introduced by chronic treatment 71 was ensuring that our rinsing method was effective in removing not only extracellular

but also the ligand used for pretreating the cells. Residual pretreatment ligand would certainly affect the function of receptors being subjected to a subsequent acute agonist challenge and artifactually skew the data. Experiments were done to evaluate the amount of residual nicotine present after our rinses to ensure that a sufficient rinse method was being used. Finally, whether the choice of agonist used in the functional assay involving an acute agonist challenge could change the outcome of the experiment was also tested.

Conditions for loading. To address the first two of the above-mentioned concerns, a control study was completed prior to initiating experiments in which effects of three different methods for 5 min ligand pretreatment were compared: (1) simply adding a concentrated aliquot of ligand in media to cells maintained in ^^Rb^ loading media, (2) using the "flip plate" method to replace ^^b* loading media with ^"^Rb^-free media containing ligand, and (3) using the "flip plate" method to apply ligand in ^^b^ loading medium. Regardless of the method used, double-normalized results obtained (see

Data Analysis below) were nearly identical (see Fig. 2.4). This indicated that final results were not influenced by differences in loading during ligand pretreatment. 72

Figure 2.4 SH-EP1-a4p2-nAChR

125 o ^ log IC50 Inactivation » o mec Spike -4.156 U w mec Rb pretreat •4.265 O "S mec fresn flip •3 998 100 Cam spike •4.146 m cam Rb pretreat •4.350 5" 3 cart) fresf) flip -4.568 nic spike -5.808 nic Rb pretreat -5.964 nic (resn flip -6.082 ISs"! >9 pretreatment m 75 ^ drug and method: ^nlc spike 11o s -•-nic w/®®Rb"flip z ^ Tk-nic fresh flip ^ lU i> ^ carb spike n n carb w/ ^^Rb" flip -ak- carb fresh flip o3

-8 -7 -6 -5 -4 -3 -2 log [drug] (M) 5 min pretreatment, 3 min recovery

Fig. 2.4. Various methods of pretreatment lead to the same outcome. Applying

pretreatment drug by either spiking the cell culture media (best for long pretreatment

times in the range of days), using the flip plate method (best for short pretreatment times

in the range of less than one hour), or using the flip plate method together with ®^b^ for

loading cells with radioactivity (necessary for pretreatments in the 1-4 hour range),

gives the same result for any given time and dose of pretreatment provided the data are

properly normalized. 73

Data Analysis. To control for possible influences of ligand pretreatment and recovery conditions on ®^b^ loading, data were subjected to a double normalization process. Background radioactivity was subtracted from all samples. Amount of ®^b^ loaded and present intracelluiarly at the start of the "acute agonist challenge" functional efflux assay was determined as the sum for each sample of released (extracellular) and remaining (intracellular) "^b"^ present at the conclusion of the functional assay. Specific

®'^Rb^ efflux was defined as total ^^b^ efflux minus nonspecific ^^Rb"*" efflux.

Normalized specific ^"^b^ efflux was then expressed as a percentage of specific ^^Rb* efflux divided by ^^b^ loaded. Double-normalized specific ^'^Rb^ efflux was then expressed as a percentage of normalized specific ®^b* efflux for experimental samples subjected to ligand pretreatment divided by normalized specific ^^b* efflux for control samples not subjected to ligand pretreatment (see Table 2.5). Data was not reported if

ligand pretreatment caused ^^b^ loaded into cells to be more than 20% lower than in control cells not pre-exposed to ligand because independent experiments (Eaton and

Lukas, unpublished) show that double normalization cannot correct for the large

difference. Only one such case was encountered. Figure 2.5 How the data were normalized Definitions; Nonspecific Efflux = released from cells not exposed to drug pretreatment and no exposed to acute agonist challenge. Control Efflux (used to define maximum "total" efflux under control conditions) released from cells during 2 minute 1 mM carbamylcholine "acute" challenge following no exposure to pretreatment drug. Experimental Sample Effluj^ ^"Rb* released from cells during 2 minute 1 mM carbamylcholine "acute" challenge following exposure to nicotinic ligand for experimentally defmed length of time. Sum of radioactivit^total radioactivity prpent intracellularly at start of functional assay) = intracellular Rb^ -t- extracellular^^Rb'^ for that particular sample at completion of assay % Nonspecific Efflux= nonspecific efflux expressed as a percentage of total radioactivity for that particular sample. % Specific Control Efflux= (equal to 100% ) % hot control ion flux - nonspecific ion flux Specific experimental ion flu)^ % experimental ion flux - nonspecific ion flux Specific''Rb* Efflux (% of control)= value quantitatively representing the level of nAChR function expressed as a percentage of efflux observed relative to specific contro efflux, where specific control efflux = 100%.

Nnnspecifir efflux Sum of radioacitivity X 100 = % nonspecific efflux

Control efflux Sum of radioacitivity X 100 = % control efflux

Experimental efflux Sum of radioacitivity X 100 = % experimental efflux

% control % nonspecific ___ % specific efflux efflux control efflux

% experimental % nonspecific efflux — % specific efflux experimental efflux

% specific e»pffrimenta! efflii X 1(K) =SpecificEfflux (% of Control) % specific control efflux 75

For experiments presented throughout this dissertation that involve pretreating cells with nicotinic ligand, the methods for ligand application are described in the

Materials and Methods section of Chapter ID. The present chapter addresses, in pan, how we arrived at and confirmed the validity of those methods.

Rinse Effectiveness: Once the ligand pretreatment exposure time is complete, media containing the pretreatment ligand is aspirated and cells are rinsed with fresh efflux buffer three times, then allowed to "recover" in a drug-free environment for an experimentally defined length of "recovery" time. It was important to confirm that pretreatment ligand concentrations were being reduced sufficiently so that residual pretreatment ligand would not contaminate the recovery process or the subsequent functional assay of nAChR. To address this concern, additional control experiments were done to verify that the rinse method was adequate for removing the pretreatment drug and terminating the chronic pretreatment period. Rinses of 30-60 sec duration were found to be adequate to allow equilibration in nicotine distribution intra- and extra-cellularly

(Fryer and Lukas, unpublished observations). Independent experiments were completed in which cells were pretreated with nicotine for 0, 1 or 48 hr before samples from each successive rinse were tested for their ability to elicit ion efflux from a separate set of untreated cells preloaded with ^^b^ (Figure 2.6). These functional assays demonstrated that nicotine levels in first-rinse samples were >IOO-fold lower than the initial nicotine concentration (i.e., there was a more than two log unit leftward shift in the dose-response profile for acute functional potency of drug in first-rinse samples). Residual nicotine present in second-rinse samples elicited ion flux only if initial nicotine concentrations were 1 mM, but with efficacy equivalent to 0.3-1 ^iM nicotine, indicating a 1000-3000- fold removal of drug. Levels of residual nicotine present in third-rinse samples had been reduced to below those capable of eliciting any ^^Rb"*" efflux; i.e., below 100 nM, a greater than 10,000-fold reduction. The efficiency of removal of nicotine in the second rinse appeared to be slightly lower following longer pretreatment times of I hr or 48 hrs relative to 20 sec of nicotine pretreatment, but no pretreatment time-dependent difference in nicotine removal was observed after the third rinse (see Figure 2.6). Thus, three rinses were routinely used to process samples. 77

Fig. 2.6. SH-EPl ceils heterologously expressing ha4P2 were pre-exposed to nicotine

(abscissa, concentration) for 20 seconds (top), 1 hr (center) or 48 hr (bottom). Pre­ exposure media was aspirated and the pre-exposed cells were subjected to 4 successive rinses of 2 ml drug-free assay buffer. The first rinse was one min and each subsequent rinse was 2 min. Activity of nicotine collected in the rinse buffer was tested for its ability to elicit function from SH-EPl cells heterologously expressing ha4p2 that had not been previously exposed to nicotine. Specific ^®Rb^ efflux (ordinate, percentage of control) measured as described in Materials and Methods was determined by eliciting nAChR function using acute nicotine (O) or nicotine collected in each of 4 successive rinses: l^' rinse (A), 2"'' rinse (V), 3"* rinse (•), and 4'*' rinse (•). Smooth curves drawn through data points are means ± S.E.M. from at least three separate experiments. Concentration- response profiles indicating the percentage of ha4P2-nAChR function measured relative to control where 100% equals untreated control cells response to I mM carb. 78

Figure 2.6 Rinse Effectiveness: SH-EP1 a4P2 nAChR 100| 20 second pretreatment acuta nicotina

1*' rinaa

O 100 1 hour pretreatment acuta nicotina

1" rinaa

3'<' rinaa •"rinaa

48 hour pretreatment acuta nicotina

2™'rin

zW a"*411. rinrinaa -8 -7 -6 -5 -4 -3 -2 -1 log [nicotine] (M) pretreatment 79

Choice ofasonist for acute aeonist challense

Additional control experiments were completed to evaluate the choice of agonist used for testing function of the receptors after they underwent pretreatment with nicotinic ligand and an experimentally defined recovery period.

Various concentrations of the nAChR agonists, carbamylcholine, nicotine and acetylcholine, have been used to measure function of receptors in other studies of chronic nicotine exposure effects on nAChR function. While most studies of nAChR in various expression systems have found nAChR to become functionally inactivated following chronic nicotine exposure, hypersensitivity of nAChR following nicotine pretreatment was reported in at least two cases (see discussion chapter QI). It was hypothesized that perhaps differences in the type or concentration of agonist used in the efflux assays following nicotine pre-exposure could account for the difference in observed receptor function. Thus, carbamylcholine, nicotine and acetylcholine used in the functional assay were compared. Regardless of the agoinst used to evaluate nAChR function at the conclusion of the experiment involving 1 hour nicotine exposure and 5 min recovery

(drug-washout), 1 |xM nicotine pretreatment led to approximately 50% reduction in nAChR function, while pretreatment with I mM nicotine led to nearly 75% nAChR functional decline. Regardless of the efflux assay agonist (acute challenge) concentration, hypersensitivity of nAChR was not observed. Carbamylcholine is a charged molecule that acutely is less likely to encounter intracellular trapping or otherwise interfere with nAChR regulation than would be nicotine. Thus, the readily available agonist 80 carbamyicholine was chosen to challenge nAChR acutely following chronic ligand exposure.

Efflux elicited by nicotine, carbamyicholine or acetylcholine is modulated by nAChR. It was necessary to establish that the observed efflux responses to nicotine, carbamyicholine or acetylcholine are mediated by nAChR rather than other receptors natively expressed by the SH-EPl cell line. SH-EP wild-type, SH-EPl ha4P2, or SH-

EPl ha434 cells were subjected to an acute challenge of either nicotine, carbamyicholine or acetylcholine (Fig 2.8). Results indicate that wildtype SH-EPi cells (not expressing nAChR) do not exhibit any ^^Rb* efflux response to nicotinic agonist challenge, while cells heterologously expressing either a4P2- or a4P4-nAChR do release ®^Rb^ upon nicotinic ligand challenge (Fig 2.8). Additionally, the efflux response elicited by nicotinic agonists from SH-EPl ha4P2 or SH-EPl ha4P4 cells is antagonized by I mM d-tubocurare (d-TC) or by 100 (iM mecamylamine, two nAChR antagonists (Fig 2.8).

Effects of the sodium channel antagonist, tetrodotoxin (TTX), were not statistically significant (Fig. 2.8).

The remaining chapters utilize procedures described or modifications of the

procedures described to address the hypotheses relating to chronic ligand effects on

nAChR function. 81

Figure 2.7. Nicotine pretreatment-induced persistent inactivation of a4P2-nACIiR is evident regardless of challenge drug and dose used to assess nAChR function. SH-

EPI cells heterologously expressing ha4P2-nAChR following 1 hour pretreatment with

0 M (•), I (V), or I mM nicotine (A) in cell culture media (see Materials and

Methods chapter m). Pretreatment media was aspirated, and the pre-exposed cells were subjected to 3 successive rinses (1 min, 1 min, and 3 min) of 2 ml drug-free assay buffer.

Specific ®^Rb* efflux (ordinate; percentage of control) measured as described in Materials and Methods was determined by eliciting nAChR function using acute (2 min) exposure to either carbamylcholine (top panel), nicotine (middle panel), or ACh (bottom panel) at indicated concentrations ranging from 10 nM up to 1 mM (abscissa). 100% response was defined by untreated control cells response to 1 mM carb. Lines drawn through data

points (means ± S£.M. from at least three separate experiments) are best fits to the Hill equation.. Carb ECsos following 0 M, 1 ^iM, and 1 mM pretreatment were 5.18 x/-?- 1.07

HM, 22.49 X/-5-1.27 ^M. and 14.45 x/^ n/a fxM, respectively. Nicotine EC50S following 0

M, 1 nM, and 1 mM pretreatment were 0.75 x/^ 1.12 |aM, 6.19 x/-^- 1.23 (iM, and 3.90

x/^ 1.46 nM, respectively. ACh EC50S following 0 M, 1 ^iM, and I mM pretreatment

were 0.63 x/-i-1.10 ^lM, 8.36 x/-5- n/a nM, and 4.76 x/^ 3.16 |4M, respectively. 82

Figure 2.7 SH-EP1-ha4p2-nAChR

1 hour nieotlna 100- pretreatment: • OM 75- VI ^M A 1 mM 50-

25

c 0- o -9-8-7-6-5-4-3-2 O acute challenge log [ Carb ] ( M)

100- 1 hour nleotina pratraatmanti 75- • OM VI ^M A 1 mM 50- ..V- —"7

UJ 25 — A +

cc -9-8-7-6-5-4-3-2 8 acute challenge log [ Nic ] (M)

o 100- 1 hour nieotina 0) pratraatmant: a 75- • OM (/) VI )iM A 1 mM 50-

25-

0-

•8-7-6-5-4-3-2 acute challenge log [ ACh ] (M) 83

Figure 2.8 efflux responses to nicotine, carbamylcholine, or acetylcholine are mediated by nAChR.

T 1 1 i T n j h 1 1 : ! ! s i 1 usyy ^ ? "" 1 "^1 ii ii •&! Q. " 0 - X - 3 - 1 - ••!_ li: Pffl 1•• « ^ J flc „

! ~ 1 1 . iraa . 1H ^ SH-EP1 wild-type SH-EP1 haA^2 SH-EP1

Fig. 2.8. Observed efflux reponses are nAChR specific, efflux (q)m) from SH-EP wild-type (left column), SH-EPl ha4P2 (middle column), and SH-EP ha4^ (right column) cells elicited by acute (2 min) challenge with nicotine (top row), carbamylcholine (middle row), or acetylcholine (bottom row). Each bar represents a different acute challenge stimulus as indicated. 84

CHAPTER m

Prolonged Ligand Exposure Depresses Function of Human a4p2-and a4P4-nAChR Heterologously Expressed in Human Epithelial Cell Lines 85

CHAPTER m

ABSTRACT

Effects of chronic nicotinic ligand exposure on the function of human aA^l- and a4P4-nicotinic acetylcholine receptor (nAChR) subtypes were studied using receptors heterologously expressed in SH-EPl human epithelial cells. Magnitudes of acute, nAChR-mediated, specific ®®Rb*-efflux responses to I mM carbamylcholine were reduced after pretreatment with specific nAChR ligands in effects that depended on pretreatment drug dose, duration of drug pretreatment, and duration of drug-free recovery.

Fifty percent inhibition of a4P2-nAChR function following 5 minutes of recovery occurred after 1 minute pretreatment with 1 mM nicotine but also after I hour pretreatment at 10 nM nicotine. Seventy-five percent loss in function persisted one hour after drug removal following 15 minutes or more of exposure to 1 mM nicotine.

However, functional recovery was nearly complete after I hour in drug-free medium following I minute-24 hour pretreatment with 0.1-1 ^iM nicotine, i.e., in the range of smoker plasma nicotine levels. a4p4-nAChR were similarly sensitive to functional 86 inactivation by prolonged nicotine exposure. Carbamyicholine exhibited slightly lower functional inactivation potency than nicotine at both a4P2- and a4P4-nAChR. The nAChR antagonist, mecamylamine, exhibited functional inactivation potency and efficacy similar to nicotine at a4P2-nAChR but had a much reduced effect on a4P4-nAChR.

These studies illustrate functional inactivation of human a4P2- or a4P4-nAChR isa induced by prolonged exposure to nicotine and show that other ligands induce nAChR functional inactivation in a subtype-specific manner. 87

INTRODUCTION

Nicotinic acetylcholine receptors (nAChR) are found throughout the peripheral and central nervous systems (CNS) where they play numerous, critical physiological roles. In vertebrates, multiple pharmacologically-distinct nAChR subtypes exist, each of which is composed of a unique combination of subunits encoded from 17 different nAChR subunit genes (reviewed in Lukas, 1995, 1998; Elgoyhen et al., 2001). Each nAChR subtype has unique channel gating and ion permeability characteristics, and some can have both pre- and post-synaptic dispositions, allowing them to modulate or mediate, respectively, synaptic transmission (Dani, 2001). Moreover, each nAChR subtype has a unique regional distribution in the brain (reviewed in Lukas, 1998).

nAChR in the CNS are suggested to play roles in attention and memory, in mood and emotion, and in sensation and perception (reviewed in Paterson and Nordberg, 2000).

CNS nAChR also are likely to be involved in synaptic plasticity and are most certainly involved in nicotine dependence (Balfour, 1994). Moreover, nAChR have been implicated in the etiology of Alzheimer's and Parkinson's diseases and as targets for treatment of neurodegenerative and mood disorders (reviewed in Mihailescu and

Drucker-Colin, 2000b). Thus, nicotinic ligands may be developed as therapeutic agents for the treatment of a number of neurological disorders.

Either through therapeutic use of nicotinic ligands to treat neuropsychiatric disorders or via habitual use of tobacco products, there will be or is sustained exposure of the brain and body to nicotinic ligands. Any effects of such exposure are likely mediated 88 by effects on numbers and function of nAChR, and an improved understanding of such effects is of high significance. Historically, much attention has focused on the phenomenon of quantitative increases in nAChR-like radioligand binding sites

("upregulation") following chronic nicotine exposure (Gentry and Lukas, 2002a).

However, upregulation is predominantly attributable to changes in internal rather than cell surface pools of nAChR-like radioligand binding sites (Ke et al., 1998; Whiteaker et al.,

1998), and its physiological relevance remains open to question (Gentry and Lukas,

2002a). By contrast, effects on nAChR function are of obvious, primary importance in determining physiological and behavioral effects of sustained exposure to either nicotine or therapeutic agents targeting CNS nAChR (Gentry and Lukas, 2002a).

The literature predominantly indicates that sustained exposure to nicotinic agonists induces losses in nAChR function. nAChR "desensitization," defmed as "a rapid in onset and quickly reversible loss of function induced on brief exposure to nicotinic agonists" (Ke et al., 1998), was first discovered and described at the nerve-muscle

junction by Katz and Thesleff (1957). Subsequently, distinct processes involving longer

lasting loss of nAChR function following longer-term nicotine exposure became

recognized for nAChR subtypes found in the periphery (Boyd, 1987; Lukas, 1991; Ke et

al., 1998; Reitstetter et al., 1999). These processes have been called "specific chronic

desensitization" (Ochoa et al., 1990; Collins et al., 1994), "stable desensitization"

(Mihailescu and E)rucker-Colin, 2000a), "functional down-regulation" (Marks et al.,

1993a), "long-lasting inactivation" (Kawai and Berg, 2001), or "persistent inactivation" 89

(Lukas, 1991; Lukas et al., 1996; Ke et al., 1998). A clear, operational definition provided by Ke and colleagues distinguished desensitization from "persistent inactivation," which was defined as "a loss of nAChR function that is not reversed during a 5 min period of recovery from agonist exposure" (Ke et al., 1998).

Sustained nicotinic agonist exposure also has been found in most studies to induce functional loss for the predominant, high-affinity nicotine-binding nAChR in the brain, composed of a4 and ^2 subunits (Flores et al., 1992), whether expressed naturally or heterologously in the oocyte system (Collins et al., 1990b; Marks et al., 1993a; Hsu et al.,

1996; Fenster et al., 1997; Gopalakrishnan et al., 1997; Olale et al., 1997; Gentry and

Lukas, 2002a). However, a recent electrophysiological study of human a432-nAChR heterologously expressed in a mammalian HEK-derived cell line reported an enhancement of function under some experimental conditions following chronic exposure to nicotine (Buisson and Bertrand, 2001). This finding opens questions about whether and how different expression systems and details of experimental design influence functional responsiveness of individual nAChR subtypes to chronic nicotinic ligand exposure.

This study examines functional responses of human aA^l- and a4P4-nAChR heterologously expressed in the SH-EPl human epithelial line following pretreatment with nicotinic ligands. Whereas the a4P2-nAChR subtype is of obvious importance, having been recognized for years as the most abundant, high affinity nicotine binding site in rodent brain (Flores et al., 1992). nAChR ^4 subunit messages as detected by in situ hybridization in non-human primate brain also are abundant and colocalize with a4 subunit messages (Quik et al., 2000). Moreover, a4 and P4 subunits combine to form a functional nAChR ion channel with high affinity for nicotine (Eaton et al., 2000). The current studies examined and contrasted effects of nicotine as a membrane-permeant agonist, carbamylcholine (carb) as an ionic nAChR agonist, and mecamylamine (mec) as a prototypical nAChR antagonist with potential as an aid to smoking cessation (Rose et al., 1998) and a therapeutic agent (Sanberg et al., 1997). A preliminary report of these findings has appeared (Gentry and Lukas, 2001a). 91

MATERIALS AND METHODS

Drug Dilutions. All drugs were prepared fresh on the day of the assay as 10 mM stock solutions in cell culture medium (see cell culture) and diluted appropriately for each experiment. Efflux assay buffer consisted of 130 mM NaCl, 5.4 mM KCl, 2 mM CaCb,

5 mM glucose, and 50 mM HEPES, pH 7.4. Ringer's buffer consists of 115 mM NaCl, 5 mM KCl, 1.8 mM CaCb, 1.3 mM MgS04, and 33 mM Tris supplemented with 1.5 mM

NaNs. Buffer components as well as (-)-nicotine bitartrate, carb CI, and mec HCl were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Model Cell Lines and Cell Culture. The present study used low passage (less than 50) SH-EPl human epithelial cell clones stably and heterologously expressing human a4p2- or a4P4-nAChR to examine functional responsiveness of receptors following pretreatment with nicotine or other nicotinic ligands. These clonal cell lines were generated using techniques that have been previously reported (Peng et al., 1999b;

Eaton et al., 2000; Lukas et al., 2002). Briefly, transfection with pcDNA3.1-zeo-human a4 and pcDNA3.l-hygro-human P2 constructs created in our lab (subcloned using human a4(S452) and P2 subunit cDNAs kindly provided by Dr. Ortrud Steinlein, Rheinishce-

Friedrich-Wilhelms-Universitaet, Bonn, Germany), followed by isolation of a stable, high-expressing clonal line, was performed to create SH-EPl-ha4P2 cells. Transfection with pCEP4-hygro-human a4 and pcDNA3.1-zeo-human P4 constructs created in our lab

(the latter was subcloned using a human ^4 subunit cDNA kindly provided by Dr. Jon 92

Lindstrom, University of Pennsylvania, Philadelphia, PA) also followed by isolation of a high-expressing clone was used to generate SH-EPl-ha4P4 cells. Cells were maintained at 37°C, under 95% O2 / 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum (Hyclone, Logan, UT), 10% horse serum (Life

Technologies/Gibco BRL Grand Island, NY), 1% sodium pyruvate (Cellgro AK,

Mediatech Inc., Hemdon, VA), 2% glutamine penicillin-streptomycin (Irvine Scientific,

Santa Ana, CA) and 0.02% amphotericin B (Sigma). Cell culture medium was also supplemented with 0.01% hygromycin (Calbiochem, San Diego, CA) and 0.03% zeocin

(Invitrogen, Carlsbad, CA) to maintain stable expression of a4 and P2 or P4 subunits.

Assays of nACIiR Function. ^"^Rb^ efflux assays (Lukas and Cullen, 1988;

Lukas et al., 2002) were performed using SH-EPI-ha4P2 or SH-EPl-ha4P4 cells with some modifications to account for the nicotinic ligand pretreatment protocol. Cells were cultured (~2 x 10^ cells per 15.5 mm diameter well; -150 ng total cell protein per well) on Falcon 24-well culture plates (BD Biosciences, Bedford, MA) pre-coated with poly-D- lysine M.W. 70,000-150,000 (Sigma). Cells were grown overnight to confluence.

^®RbCl (3.8 mCi/mg or 0.2 cpm/fmol at 40% counting efficiency) was obtained from

New England Nuclear (Boston, MA).

In all cases, cells were loaded with 0.5 ml of serum- and antibiotic-supplemented cell culture medium also supplemented with ®^b^ (-2 (iCi per well) for 4 hr or longer to ensure maximal ^^b^ uptake by the cells. Control samples were not subjected to ligand 93 pretreatment but were otherwise processed identically. For I, 15, or 60 niin drug pretreatments, medium containing only was removed and replaced at specific times with medium containing ®^b^ plus the appropriate concentration of drug. For 24 hr drug pretreatments, medium containing drug only was removed after 20 hr and replaced for the final 4 hr of pretreatment with medium containing ^^Rb"^ plus the appropriate concentration of drug. Cells were kept in incubators in 5% CO2 at 37°C for drug pretreatments of more than 5 min and for ®^Rb* loading except during medium changes.

At the end of any drug pretreatment and *^b^ loading period, medium was removed by aspiration to mark the beginning of the recovery period, and the "flip plate"

(Lukas et al., 2002) method was employed to administer three rinses with 2 ml per well of drug- and ®^b^-free efflux assay buffer (22°C for recovery times of 5 min or less, 37°C for recovery times greater than 5 min) to remove extracellular ^®Rb^ and pretreatment drug. For rinse times of 3 min or more, the first rinse solution was applied for 1 min, the second rinse solution was applied for 1 min, and the third rinse solution was applied for the remainder of the experimentally defined length of lime (1 min for 3-min recovery, 3 min for 5-min recovery, etc.). Each of the three rinses was for 20 sec for 1-min recovery samples.

After the experimentally defined periods of recovery from ligand exposure, efflux assays were used to evaluate nAChR function. Some wells of cells on each 24-well cell culture plate were reserved for controls. Total ®^b^ efflux (or positive control condition) was defined by cells not exposed to ligand pretreatment and only exposed for 2 min to 1 94 ml of 1 mM carb alone in efflux buffer ("acute agonist challenge"). Non-specific ^®Rb^ efflux was defined using other control samples exposed to efflux buffer alone or to efflux buffer containing both I mM carb and 100 ^M d-tubocurarine (either negative control approach gave comparable results). Simultaneously, wells of cells that had received ligand pretreatment were exposed to I ml of 1 mM carb alone in efflux buffer for the 2 min "acute agonist challenge" period.

At the end of the efflux period, buffer samples were collected, and amounts of

®^Rb^ released into extracellular fluids were quantified by Cerenkov counting using a

Wallac Trilux system (40% efficiency) (Perkin-Elmer Life Sciences, Boston, MA). One ml per well of 0.1% SDS/O.IM NaOH was then added to lyse the cells and to prepare samples for determination of the amounts of remaining, intracellular ®^Rb^ (see Data

Analysis below). Specific nAChR function was defined as total minus non-specific ^®Rb^ efflux. Typical values for specific and non-specific ®®Rb^ efflux in control cells not subjected to ligand pretreatment were 2500 cpm and 500 cpm, respectively, for SH-EPl- ha4P2 cells (loaded with 3000 cpm out of 350,000 cpm of applied ^^b*) and 35(X) cpm and 400 cpm, respectively, for SH-EPl-ha4P4 cells (loaded with 5000 cpm out of

350,000 cpm of applied ®^b^).

Data Analysis. To control for possible influences of ligand pretreatment and recovery conditions on ®^b^ loading, data were subjected to a double normalization process. Background radioactivity was subtracted from all samples. Amounts of ^^b^ 95 loaded and present intracelluiarly at the start of the "acute agonist challenge" to initiate the functional efflux assay were determined as the sum for each sample of released

(extracellular) and remaining (intracellular) present at the conclusion of the functional assay. Specific efflux was defined as total efflux minus non­ specific ^^b"*" efflux (described above). Normalized specific ^^Rb"^ efflux was then expressed as a percentage of specific ^^b^ efflux divided by ®^Rb^ loaded. Double normalized specific ®®Rb^ efflux was then expressed as a percentage of normalized specific ®®Rb^ efflux for experimental samples subjected to ligand pretreatment divided by normalized specific ®^b* effiux for control samples not subjected to ligand pretreatment. Data were plotted as means ± S.E.M using GraphPad Prism 3.0 (San

Diego, CA) software. Results from 3 or more independent experiments are shown.

Multiple dependent variables made comparisons by statistical analysis inappropriate due to lack of sufficient power for determining significance, however, the reproducability of the data allowed for obvious discrimination of major effects. 96

RESULTS

Effects of nicotine pretreatment on SH-EPl cell human (x4P2-nAChR. Pre­ exposure of SH-EPl-ha4p2 cells for one min to nicotine at concentrations as low as 1

|xM produced an ~20% loss of heterologously expressed, human a4P2-nAChR function when tested with an acute challenge dose of carb after I min of recovery from nicotine pre-exposure (Fig. 3.1: top-left). Losses of function for 1 min pretreatment with I or 10 fxM nicotine reversed (i.e., function elicited by an acute challenge dose of carb returned to control levels) after I hr of recovery. However, after 1 hr (or less) of recovery, losses of function were still evident in cells pre-exposed to 100 |iM (-20% loss for 1 hr recovery) or 1 mM (-30% loss for 1 hr recovery) nicotine. Using the 5 min recovery period previously used to operationally define "persistent inactivation" (Ke et al., 1998), losses of function were -10% for pre-treatment with 1 |aM nicotine and -55% for pretreatment

with 1 mM nicotine.

Extension of the nicotine pretreatment period led to progressively greater and

longer-lasting inactivation of a4P2-nAChR, such that doses of nicotine as low as 10 tiM

(compare to smoker plasma/brain levels of 100 nM-1 nM) induced losses of nAChR

function (Fig. 3.1). Thus, a4P2-nAChR recovery from functional inactivation was both

time- and concentration-dependent.

For example, following nicotine exposure for 15 min, a4P2-nAChR became

inactivated at doses as low as 100 nM, 75% of a4P2-nAChR function was lost following 97 exposure to 1 nicotine, and pretreatment with nicotine at 10 ^iM or greater caused nearly total loss of receptor function (Fig. 3.1: top-right). While the functional loss resulting from pretreatment with 10 nM nicotine recovered almost completely within 1 hr, nAChR exposed to I mM nicotine regained only 20% of function following I hr in drug-free buffer. After 5 min of recovery, the extent of "persistent inactivation" varied between -15% for 100 nM nicotine pretreatment to -90% for pre-exposure to 1 mM nicotine.

Following a 1 hr pretreatment (compared to 1 or 15 min pretreatment) with

nicotine, stronger functional inactivation was observed throughout the dose range examined (Fig. 3.1: bottom-left). The most remarkable effects were those at lower concentrations of nicotine. Pre-exposure to 10 nM nicotine caused -60% loss of a432-

nAChR function at one min of recovery, -20% functional loss that persisted after 30 min

of recovery, and -10% functional loss evident after I hr of recovery. Losses in function

at higher doses of nicotine pretreatment were even higher. Overall, for I hr pretreatment,

"persistent inactivation" remaining after 5 min of recovery ranged from -50% at 10-100

nM nicotine to full inactivation at 1 mM nicotine.

Functional profiles for a4P2-nAChR pretreated with nicotine for 24 hr and

allowed to recover for times ranging from I min to I hr were similar to profiles for I hr

pretreatment (Fig. 3.1: bottom-right), although recovery of function tended to be slightly

faster than for 1 hr nicotine pretreatment. Still, substantial loss of receptor function even 98 for low concentrations of nicotine pretreatment was again observed. One hr recovery was sufficient to bring receptors exposed to 10 or less nicotine back to levels of function matching that of untreated control cells. However, for the 5 min recovery period, losses of function ranged between -45% for 10 nM nicotine pretreatment and -100% for pre­ exposure to 1 mM nicotine.

Effects of nicotine pretreatment on SH-EPl cell human (x4P4-nAChR. Upon

prolonged nicotine exposure, heterologously expressed, human a4P4-nAChR in

transfected SH-EPl cells became less responsive to subsequent activation by carb (Fig.

3.2), and patterns of functional inactivation matched closely with that for a4P2-nAChR.

Following I min of exposure to I mM nicotine, an -75% reduction in efflux from a4P4-

nAChR was observed (Fig. 3.2: top-left). Function did not return to normal (-50%

inactivation) when cells were allowed to recover for one hr in drug-free buffer. A dose of

100 nM nicotine led to -50% reduction in a4P4-nAChR function that did recover

substantially (to -20% loss) following one hr in drug-free buffer. One min pretreatment

with nicotine at 1 fiM or less did not induce persistent inactivation. Five min recovery

allowed a4P4-nAChR function to return to -80%, 70%, or 40% of control, respectively,

after 10 ^M, 100 |AM, or 1 mM nicotine pre-exposure.

a4P4-nAChR exposed to nicotine for 15 min showed slightly greater functional

inactivation than was observed following only one min of nicotine exposure and slightly

less inactivation than was observed for a4P2-nAChR following an equivalent nicotine 99 exposure (Fig. 3.2: top-right). Only 75% functional loss of a4P4-nAChR occurred following 15 min exposure to nicotine concentrations of 10 nM or greater compared with complete loss of function observed for a4P2-nAChR. a434-nAChR function did not return to normal following one hr of recovery in drug-free buffer except when pretreatment nicotine concentration was less than 100 nM. There was significant

"persistent inactivation" operationally defined for 5 min recovery samples at all doses tested (10 nM-l mM).

Increasing the pretreatment time to 1 or 24 hr led to greater loss of function for a4P4-nAChR (Figs. 3.2: bottom-left; bottom-right). Effects at 10 or 100 nM nicotine became increasingly profound. Losses of function were -35-80% after I min recovery and remained at -10-25% after I hr recovery. Pretreatment with I nicotine for one hr followed by one min of recovery produced -75% loss of receptor function, and increasing the pretreatment time to 24 hr completely eliminated responses to subsequent carb challenge. Function was entirely lost for 1 or 24 hr pretreatment with 10 |xM-l mM nicotine after I min of recovery, although function returned to between 30 and 89% of control after 1 hr in drug-free medium. Using the "persistent inactivation" criteria, losses in function after 5 min recovery ranged between -30% at 10 nM nicotine and -90% at 1 mM nicotine.

To sunmiarize the results of nicotine pre-exposure on a4P2- and a4P4-nAChR, the extent of functional loss for each subtype depended on duration of nicotine 100 pretreatment, duration of recovery in nicotine-free buffer, and concentration of nicotine during pretreatment. Inactivation of nAChR function occured at low (10-100 nM) concentrations of nicotine, but only for pretreatment times of 15 min or more. Overall, a4p2- and a4P4-nAChR exhibited similar responses to nicotine pretreatment.

Effects of carbamylcholine pretreatment on SH-EPl cell human 04^2' nAChR. One min pretreatments with 0.1 and 1 mM carb caused a reduction in heterologously expressed, human a4P2-nAChR function of 15% and 25%, respectively, when measured with a subsequent, acute carb challenge (Fig. 5: top-left). In both cases, receptor function fully recovered within 30 min. One nun pretreatment with carb concentrations of 10 nM or less had no effect. Only 100 (aM or 1 mM pretreatments

produced "persistent inactivation" measured after 5 min of recovery.

Fifteen min pretreatment with I mM carb reduced a4P2-nAChR function by more

than 80% (Fig. 3.3: top-right). Following a 1 hr recovery period, a4P2-nAChR function

had returned to 75% of untreated control. With a 15 min pretreatment, functional effects

were seen at concentrations as low as 10 |iM. a4P2-nAChR function reduced by 30%

under these conditions recovered fully within 15 min. However, 15 min pre-exposure to

carb at concentrations between 10 nM and 1 |xM produced no functional loss, although

a4P2-nAChR "persistent inactivation" of -25%, -50%, and -70% was observed for pre­

exposure to 10 |iM, 100 |xM and 1 mM carb, respectively. 101

An additional decline in receptor function was observed following one hr pretreatment with carb (Fig. 3.3: bottom-left) when compared with 15 min of pretreatment. Most notable differences included some inactivation of receptors at lower concentrations (100 nM - 1 ^M carb and an increase in the degree of inactivation at higher concentrations, e.g., to 100% for I hr pretreatment with I mM carb after 1 min

recovery. However, "persistent inactivation" was detectable at a dose as low as 100 nM.

Functional dose-response profiles for a4P2-nAChR inactivation following 24 hr

pretreatment with carb appeared to be similar to those for 1 hr pretreatment (Fig. 3.3:

bottom-right).

Effects of carbamylcholine pretreatment on SH-EPl cell human a4p4-

nAChR. Following one min of carb exposure, heterologously expressed, human a4P4-

nAChR functioned normally except when pretreated with concentrations of carb greater

than 100 ^M (Fig. 6: top-left). One min exposure to 100 ^iM or 1 mM carb led to

approximately 8% or 20% reductions of efflux responses after one min of recovery.

Receptor function returned to normal after 30 min in drug-free buffer. Slightly greater

a4P4-nAChR inactivation resulted from 15 min of exposure to carb (Fig. 3.4: top-right).

Pretreatment concentrations of carb as low as 10 fiM began to have an effect on a4p4-

nAChR function after one hr of incubation (Fig. 3.4; bottom-left), although function

recovered fully within 1 hr of drug-free recovery. Functional losses of -60-70% occurred

for 1 hr pretreatment with 0.1-1 mM carb, although function was at -80% of control level 102 after 1 hr of recovery. Little change in a4P4-nAChR functional inactivation was observed by increasing the carb exposure time to 24 hr (Fig. 3.4: bottom-right), although the range for functional loss remaining at I hr of recovery extended to 100 nM carb

(~10% loss; -25% at 0.01-1 mM carb). "Persistent inactivation" first became evident after 5 min recovery for 1 min pretreatment with I mM carb, for 15 min-1 hr pretreatment

with 10 |iM carb, or for 24 hr pretreatment with 100 nM carb.

To summarize, these data suggested that a4P2-nAChR and a4P4-nAChR exhibited subtle differences in their response to chronic carb exposure. a4P2-nAChR

were more sensitive to carb pretreatment at shorter times of pre-exposure or at lower

doses for a given pre-exposure time. Moreover, the data indicated that carb inactivated

a4P2-nAChR to a lesser extent and with lower potency than does nicotine, and such a

difference was even more evident for a4P4-nAChR.

Effects of mecamylamine pretreatment on SH-EPl cell human a4P2-nAChR.

Pretreatment with the nAChR antagonist, mec, was capable of inducing heterologously

expressed, human a4^2-nAChR inactivation that persisted after removal of the drug from

media (Fig. 3.5). Following a one min pretreatment with I mM mec, a4P2-nAChR

underwent complete functional loss, and levels of a4P2-nAChR function remained at

25% of normal following a drug-free recovery period of 1 hr (Fig. 3.5; top-left). a4P2-

nAChR were functioning at 25% of normal following 100 fiM mec pre-exposure for one

min and one min of recovery, but 5-60 min of recovery allowed function to return to 103

-85% of control levels. Pretreatment for one min with mec at concentrations less than 1

HM had no effect on a4P2-nAChR function. "Persistent inactivation" evident after 5 min of recovery was about 15% and 75% for one min pretreatment with O.I and I mM mec, respectively.

Fifteen min pre-exposure to 100 jiM-1 mM mec caused a4P2-nAChR to lose nearly all of their function (Fig. 3.5: top-right). Recovery of function was slower after 15

min pre-exposure than for I min of pretreatment. After 1 hr of recovery from pre­ exposure to O.L or 1 mM mec, function was -60% or -10% of control, respectively.

Effects were evident for pretreatment with 10 ^iM mec; there was an -25% loss of

function after one min of drug-free recovery, but one hr after drug removal, function was at control levels. Functional losses of -15%, -65%, and -95% were seen at 5 min of

recovery from pretreatment with 10, 100, or 1000 ^M mec, respectively.

Pretreatment with mec for I hr led to receptor inactivation that required longer

recovery time (Fig. 3.5: bottom-left). Additionally, pretreatment with mec at

concentrations as low as 10 nM led to a4P2-nAChR functional loss of 25%, although this

effect was fully reversed within 30 min of recovery in drug-free media. Incomplete

recovery from functional loss after pretreatment with mec occurred at pretreatment

concentrations of I ^M or higher. Defined as the loss of function 5 min after drug

removal, "persistent inactivation" of a4P2-nAChR ranged from -10% at 10 nM mec to

-90% at 100 |iM mec. 104

Increasing the mec pretreatment time to 24 hr did not further inactivate a4^2- nAChR function (Fig. 3.5: bottom-right). Indeed, 24 hr of mec pre-exposure at concentrations of 1 or less caused less inactivation than did pre-exposure at the same concentrations for I hr. "Persistent inactivation" ranged from 20% at 100 nM mec to

-100% at 10 |aM mec or higher.

Effects of mecamylamine pretreatment on SH-EPl cell human (x4p4>nACliR.

One min of pretreatment with as much as 10 mec had no effect on subsequently assessed function of heterologously expressed, human a4P4-nAChR (Fig.

3.6: top-left). One min of 100 mec pre-exposure caused a small desensitization of a4P4-nAChR function that quickly recovered to normal. a4P4-nAChR exposed to 1 mM mec for one min lost -50% of function assessed after I min of recovery, lost -20% of function measured after 5 min of recovery, and lost -5% of function after 0.5-1 hr of recovery. Little-to-no increase in the extent of a4p4-nAChR inactivation was observed if mec pretreatment times were extended to 15 min (Fig. 3.6: top-right). One or 24 hr of pretreatment with 1 mM mec gave an only modest increase in the extent of functional loss

(Fig. 3.6: bottom-left; bottom-right). However, pre-exposure to 100 ^M mec for 1 hr induced an -30% loss in function, although recovery occurred within 5-60 min of drug- free incubation.

Thus, substantial differences were observed between 04^2- and a4P4-nAChR functional responses to prolonged mec pre-exposure. a4P2-nAChR exhibited much 105 greater functional inactivation following mec pretreatments whereas a4P4-nAChR were less vulnerable to persistent inactivation caused by mec exposure. 106

Fig. 3.1. Time and concentration dependent effects of nicotine exposure on function of nAChR measured using efflux assays. SH-EPl cells heterologously expressing ha4p2 were pre-exposed to nicotine (abscissa, log scale concentration) for 1 min (top-left), 15 min (top-right), 1 hr (bottom-left) or 24 hr (bottom-right). Pre-exposure media was aspirated and the pre-exposed cells were subjected to 3 successive rinses of 2 ml drug-free assay buffer. The first and second rinses were one min, and the third rinse consumed the remainder of the experimentally defined recovery time. Recovery times were (O) I min, (•) 3 min, (V) 5 min, (•) 15 min, (•) 30 min, or (•) 60 min.

Specific ^^b^ efflux (ordinate, percentage of control) measured as described in Materials and Methods was determined by eliciting nAChR function using acute (2 min) exposure to I mM carb. 100% response was defined by untreated control cells response to 1 mM carb. Linear curves are drawn through data points (means ± S.E.M. from at least three separate experiments). 107

Figure 3.1 Time- and Dose-Dependence: Nicotine Pretreatment: SIH-EPI a4P2-nAChR

1 minute pretreatment 15 minute pretreatment

recovery time 25|.01 min -#-15 min •^3min 030 min •V-5 min •BO min o> 125 • minute pretreatment 24 hour pretreatment h DC S i- !• I (0

-8-7-6-5-4-3 -8-7-6-5-4-3 log [nicotine]{M) pretreatment 108

Fig. 3.2. Time and concentration dependent effects of nicotine exposure on function of nAChR measured using efflux assays. SH-EPl cells heterologously expressing ha4P4 were pre-exposed to nicotine (abscissa, concentration) for I min (top- left), 15 min (top-right), I hr (bottom-left) or 24 hr (bottom-right). Pre-exposure media was aspirated and the pre-exposed cells were subjected to 3 successive rinses of 2 ml drug-free assay buffer. The first and second rinses were one min, and the third rinse consumed the remainder of the experimentally defmed recovery time. Recovery times were (O) 1 min, (•) 3 min, (V) 5 min, (•) 15 min, (•) 30 min, or (•) 60 min.

Specific ^^Rb"^ efflux (ordinate, percentage of control) measured as described in Materials and Methods was determined by eliciting nAChR function using acute (2 min) exposure to 1 mM carb. 100% response was defined by untreated control cells response to 1 mM carb. Linear curves are drawn through data points (means ± S.E.M. from at least three separate experiments). 109

Figure 3.2 Time-and Dose-De|3endence: Nicotine Pretreatment: SH-EP1 a4p4-nAChR

1 minute pretreatment. 15 minute pretreatment .

^ recovery time: ^ 251-0-1 min-•-ISmin •A-3 min 030 min X QL -^5 niin -B-l hour

UJ * 125- 60 minute pretreatment 24 liour pretreatment

S 100

•8 -7 -6 -5 -4 -3 -8 -7-6-5-4 log [ nicotine ]{M) pretreatment 110

Fig. 3.3. Time and concentration dependent effects of carb exposure on function of nAChR measured using efflux assays. SH-EPl cells heterologously expressing ha4^2 were pre-exposed to carb (abscissa, concentration) for I min (top-left), 15 min

(top-right), I hr (bottom-left) or 24 hr (bottom-right). Pre-exposure media was aspirated and the pre-exposed cells were subjected to 3 successive rinses of 2 ml drug-free assay buffer. The first and second rinses were one min, and the third rinse consumed the remainder of the experimentally defined recovery time. Recovery times were (O) 1 min,

(A) 3 min, (V) 5 min, (•) 15 min, (•) 30 min, or (•) 60 min. Specific '^'^Rb^ efflux

(ordinate, percentage of control) measured as described in Materials and Methods was determined by eliciting nAChR function using acute (2 min) exposure to I mM carb.

100% response was defined by untreated control cells response to I mM carb. Linear curves are drawn through data points (means ± S.E.M. from at least three separate experiments). Ill

Figure 3.3 Time- and Dose- Dependence: Carbamylchoiine Pretreatment: SH-EP1 a4P2-nAChR

^ 1 minute pretreatment 15 minute pretreatment.

recovery time; 0-1 min -«-15 min •a-3 min O30 min •V-5 min •SO min 2 0-

60 minute pretreatment '24 liour pretreatment. A. :i

-8 -7-6-5-4 -3 -8 -7-6-5-4 log [ cartMimylcholine ] (M) pretreatment 112

Fig. 3.4. Time and concentration dependent effects of carb exposure on function of nAChR measured using efHux assays. SH-EPI cells heterologously expressing ha4^ were pre-exposed to carb (abscissa, concentration) for 1 min (top-left), 15 min

(top-right), 1 hr (bottom-left) or 24 hr (bottom-right). Pre-exposure media was aspirated and the pre-exposed cells were subjected to 3 successive rinses of 2 ml drug-free assay buffer. The first and second rinses were one min, and the third rinse consumed the remainder of the experimentally defmed recovery time. Recovery times were (O) 1 min,

(A) 3 min, (V) 5 min, (•) 15 min, (•) 30 min, or (•) 60 min. Specific ^^Rb" efflux

(ordinate, percentage of control) measured as described in Materials and Methods was determined by eliciting nAChR function using acute (2 min) exposure to 1 mM carb.

100% response was defined by untreated control ceils response to 1 mM carb. Linear curves are drawn through data points (means ± S.E.M. from at least three separate experiments). 113

Figure 3.4 Time-and Dose-Dependence: Carbamylcholine Pretreatment: SH-EP1 a4P4 nAChR

1 minute pretreatment 15 minute pretreatment.

S loo o recovery time: •s O"! min -•-15 min -A-3 min 030 min •V-5 min -B-l tiour X 3 UJ + 125 • 60 minute pretreatment 24 hour pretreatment S lOG- I

-8 -7 -6 -5 -4 -3 •8 -7 -6 -5 -4 -3

log [ carbamylcholine ] (M) pretreatment 114

Fig. 3.5. Time and concentration dependent effects of mec exposure on function of nAChR measured using efflux assays. SH-EPl cells heterologously expressing ha4P2 were pre-exposed to mec (abscissa, concentration) for 1 min (top-left), 15 min

(top-right), 1 hr (bottom-left) or 24 hr (bottom-right). Pre-exposure media was aspirated and the pre-exposed cells were subjected to 3 successive rinses of 2 ml drug-free assay buffer. The first and second rinses were one min, and the third rinse consumed the remainder of the experimentally defined recovery time. Recovery times were (O) 1 min,

(A) 3 min, (V) 5 min, (•) 15 min, (•) 30 min, or (•) 60 min. Specific ®®Rb^ efflux

(ordinate, percentage of control) measured as described in Materials and Methods was determined by eliciting nAChR function using acute (2 min) exposure to I mM carb.

100% response was defined by untreated control cells response to 1 mM carb. Linear curves are drawn through data points (means ± S.E.M. from at least three separate experiments). 115

Figure 3.5 Time-and Dose-Dependence: {Mecamylamine Pretreatment: SH-EP1 a4p2-nAChR

I I I I I 1 minute pretreatment 15 minute pretreatment .

o racovary Hmea; \ > o 0>1 min -•-ISmin -A-3 min -QSO min -^5 min -B-eO min

60 minute pretreatment 24 hour pretreatment

log [ mecamylamine ] (M) pretreatment 116

Fig. 3.6. Time and concentration dependent effects of nicotine exposure on function of nAChR measured using efflux assays. SH-EPl ceils heterologously expressing ha4P4 were pre-exposed to mec (abscissa, concentration) for I min (top-ieft),

15 min (top-right), I iir (bottom-left) or 24 hr (bottom-right). E*re-exposure media was aspirated and the pre-exposed cells were subjected to 3 successive rinses of 2 ml drug- free assay buffer. The first and second rinses were one min, and the third rinse consumed the remainder of the experimentally defined recovery time. Recovery times were (O) I min, (•) 3 min, (V) 5 min, (•) 15 min, (•) 30 min, or (•) 60 min. Specific ^^Rb" efflux (ordinate, percentage of control) measured as described in Materials and Methods was determined by eliciting nAChR function using acute (2 min) exposure to 1 mM carb.

100% response was defined by untreated control cells response to I mM carb. Linear curves are drawn through data points (means ± S.E.M. from at least three separate experiments). Figure 3.6 Time- and Dose-Dependence: l\/lecamylamine Pretreatment: SH-EP1 a4p4-nAChR

I I I I I I 125 1 minute pretreatment 15 minute pretreatment 100 75 50- recQvcry time; 25 O-l min -•-IS min -A-3 min 030 min -V-S min •I hour I I I I

i I I I I I 125 60 minute pretreatment 24 hour pretreatment 100 75 50- 25

-8-7-6-5-4-3 -8-7-6-5-4-3

log [ mecamylamine ] (M) pretreatment lis

Table 3.1 Comparisons between nicotinic ligand functional inactivation and acute activation/inhibition potencies.

Persistent Acute Functional Ligand and Inactivation IC50 EC50 or IC50 PI-1C«. nAChR Subtype (FI-ICsoXuM) [A-ECdOso] (uM) A-ECaC)5« Nicotine a4^2 O.Ol-O.l 0.63 0.016-0.16 Nicotine a434 0.5 0.68 0.74 Carbamylcholine a432 160 17 9.4 Carbamylcholine a434 >1,000 4.1 >200 Mecamylamine a432 2 0.47 4.3 Mecamylamine a434 >1,000 18 >56

Table 3.1. Concentrations of the indicated ligands acting at the specified nAChR subtypes (column I) needed to induce persistent inactivation (50% loss of function after I hour of drug pretreatment and 5 min of drug-free recovery; PI-IC50 in nM; column 2) were compared to concentrations needed to acutely induce 50% of maximal functional activation (for agonists nicotine and carbamylcholine; A-ECso in ^M: column 3) or to acutely inhibit agonist-induced function by 50% (for the antagonist mecamylamine; A-

ICso in ^M; column 3). Persistent inhibition potency normalized to acute ligand potency was then calculated as the dimensionless ratio between persistent inactivation ICso and either acute EC50 or IC50 values [PI-IC5o/A-EC(IC)5o; column 4]. nAChR function was determined using ^^b* efflux assays (see Materials and Methods). Acute functional

EC50 or IC50 values are from Peng et al. (1999) or Eaton et al. (2000), respectively, for a4P2- or a4p4-nAChR, and persistent inactivation IC50 values are from the present study

(Peng et al., 1999b; Eaton et al., 2000). 119

DISCUSSION

Major findings. One of the major findings of this study is that pre-exposure to nicotine induces losses in function of heterologously expressed, human a4P2- or a4P4- nAChR. These losses in function, measured at maximally efficacious challenge doses of acute agonist, are significant after pretreatment with concentrations of nicotine (-100 nM-

1 i^M) found in the plasma of human smokers. These losses in function are even more substantial after pretreatment at higher doses of nicotine, i.e., at concentrations that have acute functional efficacy equivalent to acetylcholine when acting at active synapses.

Using the operational defmition of "persistent inactivation" as the loss of function after 5 min of drug-free incubation (Ke et al., 1998), the present study found that one hr of nicotine pretreatment at "smoker doses" of 100 nM-l (aM induces 60-80% loss of human a4*-nAChR function, and recovery to 90% of control levels requires ~1 hr after drug removal.

Another major finding is that human a4P2- and a4P4-nAChR subtypes differ in their sensitivity to functional inactivation after carb or mec pretreatment. Carb is less effective than nicotine at inducing functional loss of either a4P2- or a4P4-nAChR as assessed by functional inactivation potency (dose dependence), rate of functional inactivation (time dependence), and extent of functional inactivation (percentage of functional loss for a given dose and time of drug exposure). Pretreatment with mec powerfully induces functional inactivation of a4P2-nAChR but has no lasting effect on 120 a4P4-nAChR function except when mec concentrations during pretreatment are greater than 100 ^M. These observations provide insight into molecular bases for functional inactivation.

Ligand and nAChR subtype selectivity of functional inactivation. As an ionic species, carb does not freely traverse the membrane lipid bilayer and gain access to the intracellular space as readily as nicotine does. The fmding that carb induces a4*-nAChR receptor desensitization and inactivation suggests that ligand access to nAChR cytoplasmic or other buried domains is not required for functional inactivation.

Nevertheless, whether expressed in absolute terms or normalized to agonist acute functional efficacy and potency, nicotine has more persistent inactivation potency (I hr pretreatment, 5 min recovery) than carb at either a4P2- or a4P4-nAChR (Table 3.1).

This suggests that nicotine's ability to access the cell interior may indeed play a role in its strong induction of persistent inactivation. Whether expressed absolutely or after normalization to agonist acute potency, persistent inactivation potency of carb is higher

(>6->20-fold) at a4P2- than at a4P4-nAChR (Table 3.1), suggesting an influence of nAChR P subunit sequence on this effect.

Absolute or normalized persistent inactivation potency for mec is lower than that for nicotine but higher than that for carb at either a4P2- or a4P4-nAChR (Table 3.1).

The higher sensitivity of a4P2-nAChR than a4P4-nAChR to mec-induced persistent inactivation (Table 3.1) implicates P2-P4 subunit sequence differences in this effect. 121

Mec acts as an open channel blocker of nAChR (Varanda et al., 1985) and is not a steric inhibitor of nicotinic agonist binding to a432- or a4P4-nAChR (Peng et al., 1999b;

Eaton et al., 2000). Although it is possible that mec induces persistent inactivation of spontaneously opening nAChR or activates transient channel openings not detected using contemporary ion flux or electrophysiological measurements, our data with mec suggests that channel opening is not required for conversion of nAChR to an inactivated state(s).

Some non-competitive antagonists as well as agonists can induce desensitization and/or persistent inactivation of other nAChR subtypes (Lukas, 1991). As previously discussed, open channel blocking ability may be a common feature for agonists and antagonists that produce functional inactivation, although other possibilities include mutual interaction at novel regulatory sites (Lukas, 1991) or stabilization of distinct, closed-channel, nAChR conformations (Changeux et al., 1990). Interestingly the highest concentration of mec used in the pretreatment exhibited less functional loss than lower concentrations. This result is contrary to the trend observed at lower concentrations of mec and for all concentrations of the other ligands tested when applied chronically. Typically greater functional loss was observed with increasing concentration of ligand pretreatment. One possible explanation for the contradictory 1 mM mec data is that the mec pretreatment may have induced increased nonspecific ®^b^ efflux.

Also of signiHcance is the finding that concentrations of nicotine below those needed to acutely activate a4*-nAChR induce nAChR inactivation. This supports the hypothesis that channel activation is not required for nAChR functional inactivation. It 122 also is consistent with potential mechanisms involving open channel block (but of

infrequently opening channels), allosteric interactions at distinct ligand binding sites, or stabilization of nAChR in closed-channel states.

Comparisons to other in vitro findings. Findings in the present study using the

SH-EPl human epithelial cell expression system are consistent with numerous reports of

functional inactivation of a4P2-nAChR in diverse experimental systems following

chronic nicotine exposure. Treatment with 10 ^M nicotine produces functional

inactivation of chicken a4p2-nAChR heterologously expressed in Xenopus oocytes or in

a transfected murine fibroblast cell line (Peng et al., 1994). Pretreatment for 24 hr with

nicotine produces dose-dependent (from nM concentrations) and sometimes total (at

concentrations) functional loss of rat a4P2-nAChR heterologously expressed in Xenopus

oocytes (Hsu et al., 1996). Human a4P2-nAChR heterologously expressed in Xenopus

oocytes and exposed to 0.2 ^iM nicotine for 2.8 hr lose essentially all of their

responsiveness to 100 |iM ACh through a process that requires more than one hr for

recovery, and longer (48 hr) pretreatment produces even more rapid functional losses

(Olale et al., 1997). Functional responses of rat a4P2- or a4P4-nAChR expressed in

Xenopus oocytes become desensitized following nicotine pretreatment at concentrations

fnjm 0.1 nM-lOO nM (Fenster et al., 1997).

Other nAChR subtypes that undergo functional inactivation following prolonged

nicotine exposure include human muscle-type al*-nAChR naturally expressed in 123

TE671/RD cells, a7-nAChR and a3*-nAChR expressed in Xenopus oocytes, and ganglionic a3*-nAChR naturally expressed in rat PC 12 or human SH-SY5Y cells (see references cited in (Lukas, 1991; Peng et al., 1994; Hsu et al., 1996; Ke et al., 1998)).

Contrary to these observations are reports that human a4P2-nAChR function can be upregulated in response to appropriate ligand pretreatment (Gopalakrishnan et al.,

1997; Buisson and Bertrand, 2001). Functional "sensitization" measured as enhanced ion flux responses was found for human a4p2-nAChR heterologously expressed in the K-177 cell variant of the HEK 293 human embryonic kidney fibroblast cell line following 7 days of nicotine pretreatment at 0.1 - 1 nM and 4 hr of subsequent recovery in drug free media

(Gopalakrishnan et al., 1997). In the same study, if pretreatment was with 10 |iM or higher nicotine, there was functional inactivation of human a4P2-nAChR. An independent investigation found that whole cell current responses recorded from K-177- expressed, human a4P2-nAChR exposed to 0.1 |iM nicotine for 8 hr followed by 2 min of drug removal showed a large increase in acute responsiveness to ACh that was attributed to an increase in the percentage of functional a4P2-nAChR with high affinity for agonist (Buisson and Bertrand, 2001). Common to these studies is potentiated human a4P2-nAChR function, especially assessed at lower agonist concentrations, following

0.1-10 nicotine pretreatment of transfected K-177 cells. Thus, the experimental model (host cell environment) used may be an important factor influencing effects of nicotinic ligand pretreatment. 124

As suggested by Buisson and Bertrand, use of oocyte or mammalian cell expression systems, studies of native or heterologously-expressed nAChR, differences in temperature for cell maintenance, culture medium composition (presence of serum), and presence of Idnases, phosphatases, or other means for post-translational modification, also may influence effects of chronic ligand exposure on nAChR function (Buisson and

Bertrand, 2001). Additionally, species differences, variations in pH, assay temperature, passage number (if cell lines are used) and confluence of cells (amount of cell-to-cell contact) (Boyd, 1987), and subunit sequence variations may play a role as well. Our ion flux-based studies (but also those of Gopalakrishnan et al.) assess function of an ensemble of nAChR across hundreds of thousands of cells integrated over 2 min, whereas whole cell current studies typically measure only peak currents during the first few msec- sec of drug application. More work is needed to reconcile whether there truly are discrepant observations and/or diverse responses of a4P2-nAChR to chronic nicotine exposure and, if so, to elucidate the reasons for these differences.

Comparisons to in vivo studies. Studies using animal models allow evaluation of effects of nicotine pretreatment and recovery times on the order of days and on physiological parameters beyond acute functional responsiveness of nAChR. Some behavioral studies involving chronic nicotine pretreatment reveal sensitization of responses to acute nicotine but other studies indicate attenuation of responses and development of tolerance (Collins et al., 1990a; Marks et al., 1993a; Benwell et al.,

199S). In a rat model, 10 days of twice daily subcutaneous injections of 4.8 ^Mol/kg or 125

0.8 mg/kg of nicotine free-base equivalent abolished prolactin release responses to acute intraveneous injection of nicotine, consistent with the hypothesis that chronic nicotine inactivates brain nAChR (Hulihan-Giblin et al., 1990). In vivo microdialysis studies in rats found that norepinephrine responses to a second nicotine injection (0.5 mg/^g, ip) were reduced by ~50% compared to responses following initial injections, leading to the conclusion that nAChR become rapidly desensitized (Sharp and Matta, 1993). Nicotine infusion in mice for 10 days revealed dose-dependent (0.25-4.0 mg/kg/hr) decreases in nicotine-stimulated [^Hldopamine release from striatal synaptosomes, again suggesting that the involved nAChR become functionally down-regulated (Marks et al., 1993a).

Relationships between chronic nicotine-induced functional inactivation and nAChR numerical upregulation. Several factors can account for changes in the level of measurable nAChR function. Among these are enhanced/reduced conductance of each channel, enhanced/reduced frequency of channel opening, and longer/shorter channel open time. Among these possibilities, changes brought about by nicotinic ligands may be influenced by membrane lipid composition effecting conformational equilibria of nAChR

(Baenziger et al., 2000), electrostatic interactions (Song and Pedersen, 2000), enzymatic carboxymethylation (Kloog et al., 1980), conformation changes (Baenziger and Chew,

1997), phosphorylation (Hsu et al., 1997; Fenster et al., 1999b), endocytosis/exocytosis

(Peng et al., 1994), and number of nAChR on the cell surface (Ke et al., 1998).

Regarding numbers of nAChR expressed at the cell surface, substantial evidence indicates that chronic nicotine exposure leads to an increase (upregulation) of nAChR 126 binding sites, reviewed in (Wonnacott, 1990). Nicotine pretreatment does not appear to affect nAChR subunit mRNA levels, but rather seems to increase nAChR numbers via post-translational effects (Marks et al., 1992; Bencherif et al., 1994; Peng et al., 1994;

Bencherif et al., 1995). The proposition that nAChR desensitization initiates upregulation (Marks et al., 1983; Schwartz and Kellar, 1985; Fenster et al., 1999a) seems untenable given that the processes occur with widely different time- and concentration- dependencies and because any upregulated nAChR appearing on the cell surface would also quickly become inactivated in the presence of pretreatment ligand (Lapchak et al..

1989; Collins et al., 1990a; Lukas, 1991). Perhaps a different issue is the rate of recovery

from functional inactivation. Although not statistically significant, we found that human

a4P2-nAChR responses on SH-EPl cells exposed to nicotine for 24 hr recovered somewhat more quickly than nAChR on cells exposed to nicotine for only I hr. One could speculate that this reflects the presence of more surface nAChR or nAChR

precursors due to an increase in numbers of nAChR-like radioligand binding sites as

would occur over the longer period of nicotine exposure.

Conclusions. The current characterization of effects of prolonged nicotinic

ligand exposure on function of human a4^2- and a4P4-nAChR adds to findings

indicating that all nAChR subtypes are vulnerable to persistent inactivation. Knowledge

of nAChR subtype-selectivity with regard to rates, extents, and nicotine concentration

dependence of persistent inactivation is critical toward development of rational

approaches for nicotinic drug therapies and successful approaches to smoking cessation. 127

Acknowledgements:

Kind gifts of human nicotinic acetylcholine receptor subunit DNA from Dr. Ortrud

Steinlein of the Rheinische-Friedrich-Wilhelms-Universitaet and from Dr. Jon M.

Lindstrom of the University of Pennsylvania made this project possible. I also appreciate the statistical analysis consult of Drs. Kris Horn and Curtis Bay. 128

CHAPTER IV

Mimicry or Blockade of Chronic Nicotine-Induced Losses in a4p2- and a4P4-nAChR Function. 129

CHAPTER IV

ABSTRACT

Effects of chronic nicotinic ligand exposure on function of human a4P2- and a4P4-nicotinic acetylcholine receptor (nAChR) subtypes were studied using receptors heterologously expressed in SH-EPl human epithelial cells. Magnitudes of acute, nAChR-mediated, specific ®^Rb^ efflux responses to I mM carbamylcholine were reduced after 1 hour pretreatment with specific nicotinic ligands in effects that depended on pretreatment drug dose. All agonists tested induced persistent inactivation (loss of function following 5 minutes of recovery from drug pretreatment) of and a4P4- nAChR. Rank orders for both acute agonist potency and chronic agonist exposure inactivation potency at both a4P2-nAChR and a4P4-nAChR were positively correlated and were: cytisine > nicotine > DMPP > acetylcholine (ACh) + eserine > carbamylcholine chloride (carb) > ACh. Losses of a4p2-nAChR function were evident throughout the dose range for acute responses to carbamylcholine, nicotine, or ACh regardless of time for recovery from agonist pretreatment. With the exception of mecamylamine, antagonists did not induce persistent changes in a4P2- or a4p4-function upon chronic exposure. However, the nAChR antagonist, pancuronium, was able to partially protect a4P2- and a4P4'nAChR from chronic agonist-induced, but not chronic mecamylamine- 130 induced, functional inactivation. These studies define profiles for agonist-induced persistent inactivation of a4P2- and a4P4-nAChR and demonstrate the ability of mecamylamine to mimic such effects, possibly through a shared mechanism. These findings also indicate that selected nicotinic antagonists can block agonist-induced persistent inactivation of a4P2- and a434-nAChR. 131

INTRODUCTION

Multiple subtypes of nicotinic acetylcholine receptors (nAChR) are expressed in various tissues and cell types throughout the brain and body. To date, 17 genetically distinct nAChR subunits (al-alO, Pl-P4, 5, 8, and y) have been identified (Lukas et al.,

1999; Elgoyhen et al., 2001). These subunits assemble as pentamers in different combinations to form diverse nAChR subtypes that function as ion channels gated by acute actions of the endogenous neurotransmitter, acetylcholine (ACh), or of the tobacco alkaloid, nicotine, and have distinctive ligand recognition and functional properties.

Among several nAChR subtypes in the mammalian brain are those that contain a4 subunits (collectively termed a4*-nAChR according to accepted nomenclature (Lukas et al., 1999)). a432-nAChR are the predominant form of high affinity nicotinic binding site in rodent brain (Flores et al., 1992). ^ subunit messages also are abundant in non- human primate brain and colocalize with a4 subunits (Quik et al., 2000), with which they can combine to create a4P4-nAChR that also exhibit high nicotine binding affinity

(Eaton et al., 2000).

Both in vitro and in vivo evidence has established that chronic and continual nicotine presence elicits an increase in nicotine binding site density (Gentry and Lukas,

2002a). Generally (but see Buisson and Bertrand, 2001), chronic nicotine treatment produces loss of nicotine-sensitive nAChR function that persists well beyond drug removal for ganglionic a3P4*-nAChR, muscle-type al*-nAChR, or high affinity 132 nicotine-binding a4P2- or a4P4-nAChR, expressed heterologously in either oocytes or mammalian cell lines or naturally (Gentry and Lukas, 2002a). This persistent inactivation of nAChR is a possible mechanism underlying the development of nicotine tolerance.

Persistent inactivation is thought to be a process distinct from more reversible nAChR "desensitization" described for nAChR first by Katz and Thesleff 1957.

"Desensitization" has been operationally defined as nAChR functional loss that is at least partially reversible following 5 min of drug washout, and "persistent inactivation" describes functional loss that has a longer half-time for recovery that can be on the order of hours or days (Lukas, 1991; Lukas et al., 1996). Evidence thus far suggests that the function of each nAChR subtype is affected by chronic nicotine exposure; however, responses vary across nAChR subtypes (Lukas, 1991; Lukas et al., 1993)

An improved understanding about effects of chronic nicotinic ligand exposure on function of a4*-nAChR is important because (i) a4*-nAChR are thought to play critical roles in brain function, (ii) such effects are relevant to nicotine dependence and use of tobacco products, and (iii) chronic nicotinic ligand exposure for therapeutic purposes in treatment of neuropsychiatric disorders is anticipated. The present study, previously presented in preliminary form (Gentry and Lukas, 2001a), evaluates functional responses of 04^2- and a4^-nAChR heterologously expressed in an SH-EPl human epithelial cell line following chronic exposure to different nicotinic ligands. 133

MATERIALS AND METHODS

Drug Dilutions. All drugs were prepared fresh on the day of the assay as 10 mM stock solutions in cell culture medium (see cell culture) and diluted appropriately for each experiment. Efflux assay buffer consisted of 130 mM NaCl, 5.4 mM KCl, 2 mM CaCh,

5 mM glucose, and 50 mM HEPES, pH 7.4. Buffer components as well as S(-)-nicotine

(free base), carbamylcholine CI (carb), cytisine, acetylcholine (ACh), d-tubocurarine CI

(d-TC), pancuronium Br, decamethonium Br, hexamethonium Br, dihydro-P-erythroidine

(DHBE), and eserine were purchased from Sigma Chemical Co. (St. Louis, MO, USA). l,l-Dimethyl-4-phenyl-piperazinium (DMPP) and epibatidine (EBDN) were purchased from Research Biochemicals International (RBI) (Natick, MA), and mecamylamine HCl was purchased from Merck Sharp & Dohme Research Lab (Rahway, NJ).

Model Cell Lines and Cell Culture. The present study used low passage (less than 50) SH-EPl human epithelial cell clones stably and heterologously expressing human a4P2- or a4P4-nAChR to examine functional responsiveness of receptors following pretreatment with nicotine or other nicotinic ligands. These clonal cell lines were generated using techniques that have been previously reported (Peng et al., 1999a;

Eaton et al., 2000; Pacheco et al., 2001; Lukas et al., 2002). Briefly, transfection with pcDNA3.1-zeo-human a4 (S452) and pcDNA3.l-hygro-human P2 constructs created in our laboratory (subcloned using human a4 and P2 subunit cDNAs kindly provided by Dr.

Ortrud Steinlein, Rheinishce-Friedrich-Wilhelms-Universitaet, Bonn, Germany) followed 134 by isolation of a stable, high-expressing clonal line was accomplished to create SH-EPl- ha4P2 cells. Transfection with pCEP4-hygro-human a4 and pcDNA3.1-zeo-human P4 constructs created in our laboratory (the latter was subcloned using a human ^4 subunit cDNA kindly provided by Dr. Jon Lindstrom, University of Pennsylvania, Philadelphia,

PA) also followed by isolation of a high-expressing clone was used to generate SH-EPl- ha4P4 cells. Cells were maintained at 37°C, under 95% O2 / 5% COt in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum (Hyclone, Logan,

UT), 10% horse serum (Life Technologies/Gibco BRL Grand Island, NY), 1% sodium pyruvate (Cellgro AK, Mediatech Inc., Hemdon, VA), 2% glutamine penicillin- streptomycin (Irvine Scientific, Santa Ana, CA) and 0.02% amphotericin B (Sigma). Cell culture medium was also supplemented with 0.01% hygromycin (Calbiochem, San Diego,

CA) and 0.03% zeocin (Invitrogen, Carlsbad, CA) to maintain stable expression of a4 and 32 or ^ subunits.

Assays of iiAChR Function. ^^Rb^ efflux assays were performed using SH-EPl-

ha4P2, or SH-EPl-ha4P4 cells according to the procedure of (Lukas and Cullen, 1988;

Gentry et al., 2002; Lukas et al., 2002) with some modifications to account for

pretreatment with with nicotinic ligand. Modifications are described here. Cells were cultured (-2 x 10^ cells per 15.5 mm diameter well; ~I50 |ig total cell protein per well)

on Falcon 24-well culture plates (ED Biosciences, Bedford, MA) precoated with poly-D-

lysine m.w. 70,000-150,(XX) (Sigma). Cells were grown overnight to confluence verified 135 using light microscopy. ®®Rubidium-Cl 3.8 mCi/mg or 0.4 cpm/fmol at 40% counting efficiency) was obtained from Perkin-Elmer Life Sciences (New England

Nuclear) (Boston, MA).

In all cases, cells were loaded with 0.5 ml of serum- and antibiotic-supplemented cell culture medium also supplemented with ®^b^ (~2 nCi per well) for 4 hr or longer to ensure maximal ^'^b* uptake by the cells. Control samples were not subjected to ligand pretreatment but were otherwise processed identically. Medium containing "^^b" only was removed and replaced at specific times with medium containing ^^Rb^ plus the appropriate concentration of drug so as to allow cells to incubate in ligand-containing media for 1 hr prior to drug washout for nAChR recovery period and subsequent assay of nAChR function. Cells were kept in incubators in 5% CO2 at 37°C for drug pretreatments and for ^^b^ loading except during medium changes.

At the end of any drug pretreatment and "^"^Rb^ loading period, medium was removed by aspiration to mark the beginning of the recovery period, and the "flip plate"

(Lukas et al., 2002) method was employed to administer 3 rinses with 2 ml per well of drug- and ®^b"*^-free efflux assay buffer at 22°C to remove extracellular ®^b* and pretreatment drug. A 5 min recovery period consisted of the first rinse solution applied for I min, the second rinse solution applied for 1 min, and the third rinse solution was applied for 3 min. However, for some studies employing a 2 min recovery period, the 136 first and second rinse solutions were applied for 30 sec each, and the third rinse solution was applied for the remaining 1 min.

Most typically, after the 5 min period of recovery from ligand exposure, efflux assays were used to evaluate nAChR function. Some wells of cells on each 24-well cell culture plate were reserved for controls. Total efflux (or positive control condition) was deflned by cells not exposed to ligand pretreatment, only exposed for 2 min to 1 ml of I mM carb alone in efflux buffer ("acute agonist challenge"). Non-specific ®^b* efflux was defined using other control samples exposed to efflux buffer alone or to efflux buffer containing both 1 mM carb and 100 |aM d-TC (either negative control approach gave comparable results). Simultaneously, wells of cells that had received ligand pretreatment were exposed to 1 ml of I mM carb alone in efflux buffer for the 2 min

"acute agonist challenge" period. In other studies, samples pretreated with nicotinic ligands and washed free of pretreatment drug (and suitable control samples not subjected to drug pretreatment) were assayed for function in response to acute challenge with nicotinic agonists at several concentrations to allow generation of agonist dose-response profiles following control or nicotinic ligand pretreatment.

At the end of the efflux period, buffer samples were collected, and amounts of

^^b^ released into extracellular fluids were quantified by Cerenkov counting using a

Wallac Trilux system (40% efficiency). One ml per well of 0.1% SDS/0.1 M NaOH was then added to lyse the cells and to prepare samples for determination of the amounts of remaining, intracellular ^b^ (see Data Analysis below). Specific nAChR function was 137 deHned as total minus nonspecific efflux. Typical values for specific and nonspecific efflux in control cells not subjected to ligand pretreatment were 2500 cpm and 500 cpm for SH-EPl-ha4P2 cells, and 3500 cpm and 400 cpm for SH-EPl- ha4P4 cells (loaded with 350,(XX) cpm of applied ®'^Rb*).

Data Analysis. To control for possible influences of ligand pretreatment and recovery conditions on ®'^Rb^ loading, data were subjected to a double normalization process. Background radioactivity was subtracted from all samples. Amount of ^^Rb^ loaded and present intracellularly at the start of the "acute agonist challenge" functional efflux assay was determined as the sum for each sample of released (extracellular) and remaining (intracellular) *^b^ present at the conclusion of the functional assay. Specific

®®Rb* efflux was defined as total efflux minus non-specific ^"^b* efflux (described above). Normalized specific ®®Rb^ efflux was then expressed as a percentage of specific

®'^b^ efflux divided by ^^Rb^ loaded. Double normalized specific ^"^b* efflux was then expressed as a percentage of normalized specific ^^Rb^ efflux for experimental samples subjected to ligand pretreatment divided by normalized specific ®®Rb^ efflux for control samples not subjected to ligand pretreatment. Data were plotted as mean ± S.E.M using

GraphPad Prism 3.0 (San Diego, CA) software. Results from 3 or more independent experiments are shown. Statistical analysis were performed using SPSS software (SPSS,

Inc., Chicago, IL). 138

Linear regression analysis was applied to log ECso values for acute functional potency as a function of the log IC50 for functional persistent inactivation observed following pretreatment of cells expressing a4P2- or a4P4-nAChR with media containing ligand of interest for I hr and subsequent 5 min recovery period in drug-free efflux buffer.

Data were analyzed and plotted using GraphPad Prism 3.0 (San Diego, CA) software. 139

RESULTS

Mimicry by agonist pretreatment of losses in oAfiil- or a4P4-nAChR function induced by nicotine pretreatment. Although acute nicotine exposure activates nAChR ion channel function, evidence from many previous investigations indicates that more sustained nicotine exposure induces multiple phases of nAChR functional loss. The first phase of rapid-in-onset, rapidly reversible functional loss called "desensitization" occurs following a brief exposure to nicotine, but nAChR enter a persistently inactivated state(s) following more prolonged nicotine exposure. "Persistent inactivation" has previously been operationally defined as the loss of nAChR function that is not reversed following a

5 min period of recovery from agonist exposure (Ke et al., 1998). Experiments were conducted to investigate the potential for nicotinic ligands besides nicotine to persistently inactivate aA^l- or a4p4-nAChR. Following 1 hr pretreatment with drug and 5 min of recovery in drug-free medium, nAChR function was assessed using ®^b* efflux assay involving an acute, 2 min challenge with 1 mM carb.

One hr pre-exposure to every nicotinic agonist tested produced persistent inactivation of a4P2-nAChR (Fig. 4.1) and a4P4-nAChR (Fig. 4.2). The extent of persistent inactivation was dependent on the concentration of drug used in pretreatment; i.e., effects were pretreatment drug dose-dependent. ACh alone was a weak functional inactivator, but this was evidently due to enzymatic hydrolysis of this ligand during the 1

hr pretreatment period, because ACh persistent inactivation potency was increased when 140

I mM eserine, an ACh-esterase inhibitor, was included in pretreatment medium.

Nicotine at doses like those found in the plasma of human tobacco users (i.e., ~l(X) nM) produced -50% persistent inactivation of a4p2-nAChR and -25% persistent inactivation of a4P4-nAChR. At ICX) nM, cytisine produced -50% inactivation of both a4P2- and a4P4-nAChR and was maximally effective exhibiting a steeper dose-persistent inactivation profile than any of the other agonists. Membrane-permeant agonists, nicotine and cytisine, had higher persistent inactivation potency than ionized agonists DMPP, carb, and ACh, but there was a positive correlation between agonist acute functional potency and agonist persistent inactivation potency (Fig. 4.3; r^ = 0.83 for agonists acting at a4P2-nAChR; r^ = 0.99 for agonists acting at a4P4-nAChR). The rank order for persistent inactivation potency at a4P2-nAChR (approximate IC50 value for persistent inactivation precedes the drug's name) was: 23 nM cytisine > 0.84 |iM nicotine >12 |iM

DMPP > 13 nM ACh + eserine >71 ^iM Carb > 230 nM ACh (Table 4.1). The rank

order for persistent inactivation potency at a4P4-nAChR (approximate IC50 value for persistent inactivation precedes the drug's name) was: 2.4 nM cytisine >0.11 |xM nicotine > 2.2 |iM DMPP > 4.5 ^iM ACh + eserine >1.4 mM carb (Table 4.1).

Human a4p2-iiADChR function in SH-EPl-ha4P2 cells is not potentiated following nicotine pretreatment and 2 min recovery. To further clarify conditions leading to changes in nAChR function that persist after nicotine removal, functional responses of a4P2- and a4p4-nAChR were tested using acute ACh challenges following 141

1 hr or 8-10 hr exposure to 0.1 or 1 ^iM nicotine and 2 min of drug-free recovery. These conditions match those used in a study of effects of chronic nicotine exposure on function of a4P2-nAChR heterologously expressed in an HEK-293-derived cell line in which

"potentiation" rather than persistent inactivation of nAChR function was reported following 8-10 hr of chronic nicotine exposure (Buisson and Bertrand, 2001). ACh concentration-response curves for a4P2- and a4P4-nAChR heterologously expressed in human SH-EPl celts treated with 1 nicotine for 1 hr are shifted rightward indicating a reduction in efflux response relative to untreated control cells (Fig. 4.4 top-left and top-right). Dose-response profiles for acute ACh challenge for a4P2- and a4P4-nAChR in transfected SH-EPl cells treated with 0.1 or 1 iaM nicotine for 8-10 hr are more markedly shifted downward and to the right indicating a more marked persistent inactivation of nAChR function (Fig. 4.4 bottom-left and bottom-right). These results indicate that there is no potentiation of a4P2- or a4p4-nAChR function in transfected

SH-EPl cells measured using ®^Rb^ efflux assays under conditions reported to induce potentiation of a4P2-nAChR function in transfected K-117 cells.

EfTects of chronic antagonist exposure on nAChR function. Additional experiments were conducted to test whether nAChR persistent inactivation could be induced upon chronic antagonist exposure. These included competitive (alcuronium, pancuronium, DHBE) and non-competitive (hexamethonium, mecamylamine (Peng et al.,

1999a; Eaton et al., 2000) antagonists. Most antagonists tested did not induce persistent 142 inactivation of a4^2- or a4P4-nAChR (Figs. 4.5-4.6), and lack of such effect, could not be explained by lack of acute functional antagonist potency (Table 4.1). However, mecamyiamine exposure did produce substantial persistent inactivation of a4P2-nAChR, although it produced modest persistent inactivation of a4P4-nAChR only at 1 mM.

Antagonist block of persistent inactivation. Previous studies indicated that the competitive nicotinic antagonist, pancuronium, was capable of protecting muscle-type al-nAChR from nicotine-induced persistent inactivation (Lukas, 1991). The present study investigated pancuronium's ability to protect a4P2- and a4P4-nAChR from persistent inactivation induced by nicotine, carb or mecamyiamine. Combined nicotinic ligand and l mM pancuronium treatment for 1 hour followed by either 5 or 15 min recovery in drug-free efflux buffer revealed that pancuronium, which did not produce functional inactivation of nAChR alone (Figs. 4.5-4.6), was capable of blocking the persistent inactivation induced by nicotine or carb at a4P2-nAChR (Fig. 4.7) or a4P4- nAChR (Fig. 4.8). At a4P2-nAChR, nicotine-induced persistent inactivation was completely blocked by pancuronium except at nicotine concentrations of 100 nM or greater where persistent inactivation was reduced by -50% (Fig. 4.7). Pancuronium also completely blocked carb-induced persistent inactivation except in the presence of 1 mM carb where the extent of persistent inactivation was reduced by pancuronium from -60% to only -25%. Mecamylamine-induced persistent inactivation of a4P2-nAChR was not affected by the co-application of 1 mM pancuronium during pretreatment (Fig. 4.7). At 143 a4P4-nAChR, nicotine-induced persistent inactivation was completely blocked by pancuronium except at nicotine concentrations of 100 fiM or greater, where persistent inactivation was reduced by more than 50%. Pancuronium also completely blocked carb- induced persistent inactivation of a4P4-nAChR. However, pancuronium co-exposure did not change the modest ability of mecamylamine to induce persistent inactivation of oA^A- nAChR. 144

Fig. 4.1 Persistent inactivation of a4P2-iiAChR function following 1 hour nicotinic agonist exposure. SH-EPl-ha4p2 cells were pre-exposed to acetylcholine

(ACh) alone (O) or in the presence of 1 mM eserine (A), nicotine (•) (upper panel), carbamylcholine (•), DMPP (V), or cytisine (•) (lower panel) at the indicated concentrations (abscissa, log M) for 1 hr. Cells were rinsed free of pretreatment drug for

5 min and subjected to specific ®^Rb* efflux assays (ordinate, percentage of control efflux in response to 2 min challenge with I mM carbamylcholine) as described in Materials and Methods. Lines drawn through data points (means ± S.E.M. from at least three separate experiments) are best fits to the Hill equation. Concentration by drug interaction was significant using Greenhouse-Geisser correction based on repeated measures analysis of variance (ANOVA): F( 16.56, 86.11) = 1.89, p = 0.03. 145

Figure 4.1 SH-EP1 •ha4p2-nAChR 1 hour pretreatment followed by 5 min recovery period

ACh O 25 Eserine ^ Nicotine i

Cytisine

-12-11 -10 -9-8-7-6-5-4 -3 -2 -1 0 log [ agonist ] pretreatment {M) 143

Fig. 4.2 Persistent inactivation of a4P4-nAChR function following 1 hour nicotinic agonist exposure. SH-EPl-ha4P4 cells were pre-exposed to acetylcholine (ACh) alone

(O) or in the presence of I mM eserine (T), nicotine (•) (upper panel), carbamylcholine

(•), DMPP (A), or cytisine (•) (lower panel) at the indicated concentrations (abscissa, log M) for 1 hr. Cells were processed for specific ^^b* efflux assays (ordinate, percentage of control) as described in Materials and Methods and in the legend to Fig. I.

Lines drawn through data points (means ± S.E.M. from at least three separate experiments) are best fits to the Hill equation. Concentration by drug interaction was significant using Greenhouse-Geisser correction based on repeated measures analysis of variance (ANOVA): F(l 1.28,60.93) = 6.70, p <0.001. 147

Figure 4.2 SH-EP1-ha4p4-nAChR 1 hour pretreatment followed by 5 min recovery period I I I I

P—~—Q—O—Q—

ACh-i-

Nicotine

Cytisinc

-12 -11 -10 -9-8-7-6-5-4 -3 -2 -1 0 log [ agonist ] pretreatment (M) 148

Figure 4.3

- SH-EP1-ha4p2-nAChR SH-EP1-ha4P4-nAChR r^ = 0.99

DMPP^Carb

Cytisifw Nlcotlna^ ACh (w/aaarina) ACh (w^Mcriiw) Cytisina

-7 < -5 -4 -3 -7-6-5-4-3 Log ICso ( M ) persistent inactivation Log ICso (M) persistent inactivation (nAChR function mMSurad following 1 hour (nAChR furKtion maaaurad following 1 hour protroatmant and 5 minuta racovary) pratraatmant and 5 minuta racovary)

Fig. 4.3 Positive correlations between agonist persistent inactivation potency and agonist acute functional potency in actions at a4*-nACliR. Agonist acute functional potency (EC30; ordinate, log molar scale) for the indicated agonists acting at a4P2- nAChR (left panel) or a4P4-nAChR (right panel) as determined in previous studies (Peng et al., 1999a; Eaton et al., 2000) is plotted against persistent inactivation potency (PI-IC50; abscissa; log molar scale) as derived from studies shown in Figs. 4.1-4.2. Linear regression gives correlation coefficients of 0.88 for a4P2-nAChR and 0.99 for aA^A- nAChR. 149

Fig. 4.4 Persistent inactivation of a4*-nAChR occurs after 1 or 8-10 hr of nicotine pretreatment. Specific efflux (ordinate, percentage of control) in response to 2 min challenge with acetylcholine at the indicated concentrations (abscissa; log molar scale) was measured as described in Materials and Methods using SH-EPl-ha4P2 cells

(left panels) or SH-EPl-ha4P4 cells (right panels) following treatment with nicotine at 0

^iM (•), O.I nM (A), or I |iM (•) for I hr (top panels) or 8-10 hr (bottom panels) and 2 min of drug-free recovery. Results are means (+ S.E.M.) from at least three separate experiments. 150

Figure 4.4

SH-EP1- SH-EP1- ha4p2-nAChR ha434-nAChR

1 hr nicotine 1 hr nicotine c 2 min recovery 2 min recovery o o •••OmM 1^0.1 pM -*-1 nM

X

^ 1! yj •I- 12 8-10 hr nicotine XI 8-10 hr nicotine flC 1C 2 min recovery 2 min recovery «(O -!

(0

^ -8 -7 -6 -5 -4 -3 -2-9 -8 -7 -6 -5 -4 -3 -2 log acute [ ACh ] (M ) 2 min challenge 151

Fig. 4.5 a4P2-nACliR function following 1 hour nicotinic antagonist exposure. SH-

EPl-ha4P2 cells were pre-exposed to mecamylamine (•), hexamethonium (A), dihyrdo-

P-eryhthroidine (V), d-tubocurarine (T) (upper panel), decamthonium (•), pancuronium (•), or alcuronium (•) (lower panel) at the indicated concentrations

(abscissa, log molar scale) for 1 hr. Cells were processed for specific ^^b^ efflux assays

(ordinate, percentage of control) as described in Materials and Methods and in the legend to Fig. 1. Lines drawn through data points (means ± S.E.M. from at least three separate experiments) are best fits to the Hill equation. 152

Figure 4.5

SH-EP1-ha4p2-nAChR 1 hour pretreatment followed by 5 m in recovery period

O o 50- • Mecamylamine A Hexamethonium 25 VDHBE • d-TC

5 0*—^ lU + A 100- scc o 75-

A Alcuronium SCL SOI- (0 • Decamethonium • Pancuronium 251-

-8 -7 -6 -5 -4 -3 log [ antagonist ] pretreatment (M) 153

Fig. 4.6 a4P4-iiAChR function following 1 hour nicotinic antagonist exposure. SH-

EPl-ha4P4 cells were pre-exposed to d-tubocurarine (•), decamethonium (•), alcuronium (•) (upper panel), mecamylamine (•), hexamethonium (A), pancuronium

(•), or dihyrdo-3-eryhthroidine (V) (lower panel) at the indicated concentrations

(abscissa, log molar scale) for i hr. Cells were processed for specific ®^b^ efflux assays

(ordinate, percentage of control) as described in Materials and Methods and in the legend

to Fig. 1. Lines drawn through data points (means ± S.E.M. from at least three separate experiments) are best fits to the Hill equation. 154 Figure 4.6 SH-EP1-ha4P4-nAChR 1 hour pretreatment followed by 5 min recovery period

100-

75 O o 50- o • d-TC p • Alcuronium or 25 • Decamethonkim X 0 LU + 100- oc S 75 m^mo m^m O 50 • Mecamylamine 0) A Hexamethonium Q. • Pancuronium 0) 25 V DHBE

-8-7-6-5-4-3 log [ antagonist ] pretreatment (M) 155

Fig. 4.7 Pancuronium block of nicotine- or carbamylcholine-induced persistent inactivation of (x4P2-nACliR. SH-EPl-ha4p2 cells were exposed for 1 hour to nicotine

(upper panel), carbamylcholine (middle panel) or mecamylamine (lower panel) at the indicated concentrations (abscissa, log molar scale) in the absence (#, A; solid lines) or presence (O, •; dashed lines) of 1 mM pancuronium before being rinsed free of pretreatment drug(s) for 5 min (•, •) or for 15 min (•, O). Specific ^^b^ efflux

(ordinate, percentage of control) was then measured as described in Materials and

Methods and in the legend to Fig. I. Results are means (± S.E.M.) from 3 or more separate experiments. 156 Figure 4.7 SH-EP1-ha4p2-nAChR 125

Nicotine

Recovary 5 min 15 min O-f pane O-f pane

a, Carb

1 •

Rgcovery 5 min 15 min 0-»>panc O-fpanc

lUiecamylamine

Rgcovery 5 min 15 min O+panc O-fpanc

-8-7-6-5-4-3 log [ pretreatment drug ] (M) 157

Fig. 4.8 Pancuronium block of nicotine- or carbamylcholine-induced persistent inactivation of (x4P4-nACliR. SH-EPl-ha4P4 cells were exposed for 1 hour to nicotine

(upper panel), carbamylcholine (middle panel) or mecamylamine (lower panel) at the indicated concentrations (abscissa, log molar scale) in the absence (#, A; solid lines) or presence (O, •; dashed lines) of I mM pancuronium before being rinsed free of pretreatment drug(s) for 5 min (•, •) or for 15 min (•, O). Specific ®*^Rb* efflux

(ordinate, percentage of control) was then measured as described in Materials and

Methods and in the legend to Fig. I. Results are means (± S.E.M.) from 3 or more separate experiments. 158

Figure 4.8 SH-EP1-ha4p4-nAChR

125i Nicotine 100 >

75-

21 SO- Recovery o Smin 15 min 25- •ih o 0+ pane '^+ pane

2£ I Carb 100-

;t= 75- UJ + 50- n tc, Recovery 5 min 15 min S 25- O+panc O+panc mmmo 9^m 0< U 125 0) a Mecamylamine (0 100-

75-

50- Recovery Smin 15 min 25- pane O-^panc

log [ pretreatment drug ] (M) 159

Table 4.1 Parameters for agonist-induced persistent inactivation of a4'''-nACIiR.

Data from studies illustrated in Figs. 4.1-4.2 were fit to the Hill equation to derive PI-IC50 values (i.e., drug concentration producing half-maximal loss of nAChR function) for I hr pretreatment with the indicated drugs (first column) and 5 min recovery for a4P2-nAChR

(second column) or a4P4-nAChR (third column). Results in bold face type are mean PI-

ICso values (nM), and the S.E.M. for those values (as a multiple of the PI-IC50 value) follows in parentheses. In smaller scale font below PI-ICso values are the Hill coefficient

(± S.E.M.) followed by the r^ values for goodness of fit of the Hill equation with the indicated parameters to the data. 160

Table 4.1 IC50 (mM) persistent inactivation (x/-i-S.E.M.) nAChR Coefficient ± S.E.M., Msaad a4B2 a4B4

Cytisine 0.0024 (0.70) 0.023 (0.27)

-0.24 ± 0.08, 0.69 -0.35 ±0.07,0.83

Nicotine 0.11 (0.43) 0.84 (0.37)

-0.36 ±0.13,0.70 -0.29 ±0.08,0.85

DMPP 2.2 (0.60) 12.1 (0.39)

-0.25 ± 0.08,0.58 -0.38 ±0.08,0.89

ACh + Eserine 4.5 (2.6) 12.8 (3.4)

-0.15 ±0.06,0.61 -0.17 ±0.10,0.36

Carb 1370 (3.0) 71.3 (0.66)

-0.25 ±0.11,0.53 -0.47 ±0.19, 0.73

ACh N/A 227.5 (3.4)

-1.87 ± 13.04,0.54 161

DISCUSSION

Major flndings. This study shows (I) that nAChR agonists induce persistent inactivation of a4P2- or a4P4-nAChR function; (2) that there is a positive correlation between acute functional potency and chronic exposure-induced persistent inactivation potency for agonists acting on a4P2- and a4P4-nAChR; (3) that the nAChR non­ competitive antagonist mecamylamine, but not other non-competitive or competitive antagonists tested, mimics the ability of nicotine and other agonists to induce persistent inactivation of a4P2-nAChR while only weakly doing so at a4P4-nAChr; (4) that the

nAChR competitive antagonist, pancuronium, is able to protect a4P2- or a4P4-nAChR

from chronic agonist-induced persistent inactivation, but; (5) that pancuronium is unable

to protect a432- or a4P4-nAChR from mecamylamine-induced persistent inactivation.

Central to these findings is the clear demonstration that a4*-nAChR function is lost and

not potentiated by chronic nicotine exposure when a4P2- or a4P4-nAChR are

heterologously expressed in the SH-EPl human cell line.

Mimicry of chronic nicotine effects by other nicotinic agonists. Whereas

chronic nicotine exposure has repeatedly been shown to induce long-lasting loss of

nAChR function, few studies have examined the ability of other compounds with various

characteristics (i.e. differing nAChR ligand binding affinity, acute functional potency, and

lipophilicity, etc.) to mimic these effects. The findings presented here demonstrate such

mimicry and also show striking, positive correlations between agonist acute functional 162 potency and persistent inactivation potency at human a4^2- and a4P4-nAChR. This suggests that agonist-induced nAChR channel opening and agonist-induced persistent inactivation are closely related, perhaps causally and mechanistically. By contrast, and in keeping with questions about the relationships between nicotine-induced nAChR persistent inactivation and nicotine-induced nAChR radioligand binding site numerical upregulation, let alone about the physiological relevance of the latter phenomenon

(Gentry and Lukas, 2002a), in vivo studies making comparisons across nicotinic agonists found no clear relationship between affinity for the nicotine binding site and agonist- induced nAChR upregulation (Collins et al., 1990b).

Elucidation of the dose dependencies for persistent inactivation effects among agonists is also informative and enlightening. For example, at equal doses, cytisine is more effective than nicotine in inducing persistent inactivation of a4*-nAChR. Perhaps this observation helps to explain why cytisine patch therapies to treat nicotine dependence marketed as Tabex in Europe have reportedly met with greater success than nicotine- based therapies (www.Tabex.com). Thus, treatment of nicotine dependence might exploit the new findings by employing progressively less potent compounds to wean nicotine-dependent individuals from nicotinic agonist replacement therapies and to help identify nAChR subtypes involved in nicotine dependence and its treatment.

Nicotine pretreatment induction of a4*-nAChR functional hypersensitivity or persistent inactivation? The vast majority of evidence to date indicates that nAChR 163 subtypes endure functional loss following chronic nicotine exposure (reviewed in Gentry and Lukas, 2002a). The current study shows chronic agonist-induced persistent inactivation of a4*-nAChR function as assessed in several ways. However, some findings are in disagreement, reporting enhanced ion flux- or electrophysiologically- measured responses for human a4P2-nAChR heterologously expressed in the K-177 cell variant of the HEK-293 human embryonic kidney fibroblast cell line following a 7 day pretreatment with 0.1-1 uM nicotine and 4 hour recovery (Gopalakrishnan et al., 1997) or following 0.1 or 10 nM nicotine exposure for 1 or 8-10 hr (Buisson and Bertrand,

2001), although persistent inactivation following pretreatment at higher nicotine doses was confirmed in both of these studies. The current findings rule out a difference between the current or previous studies and those examining K-l77-expressed a4P2- nAChR due to pretreatment or drug-free recovery conditions or the type or dose of agonist used for acute challenge to assess nAChR function following ligand pretreatment.

Thus, we hypothesize that the experimental model (host cell environment) used may be an important factor influencing effects of nicotinic ligand pretreatment. Differences between cell types in kinases, phosphatases, other means for post-translational modification, and/or other cell-specific features could exist and influence effects of chronic ligand exposure on nAChR function (Buisson and Bertrand, 2001).

Alternatively, differences in temperature for cell maintenance, culture medium composition (presence of serum), variations in pH, assay temperature, passage number (if 164 cell lines are used), confluence of ceils (amount of cell-to-cell contact) (Boyd, 1987), and subunit sequence variations may play a role as well. Important for future investigation is whether a4*-nAChR function in neurons is potentiated or persistently inactivated by chronic nicotine exposure.

Antagonist mimicry or blocicade of nicotinic agonist-induced, a4*-nAChR persistent inactivation. The fmding that chronic pretreatment with mecamylamine, which acts acutely as a non-competitive antagonist perhaps by engaging in open channel block, was able to induce operationally-defined persistent inactivation of a4P2-nAChR, was consistent with suggestions chat open channel block might be a mechanism explaining nicotinic agonist-induced persistent inactivation and argued against a requirement for nAChR activation as a precondition for persistent inactivation. The confound of the relatively reduced potency of mecamylamine in persistent inactivation of a4P4-nAChR could be explained by mecamylamine's lower acute functional antagonist potency at a4P4-nAChR. However, the inability of hexamethonium, another non­ competitive antagonist, to induce a4*-nAChR persistent inactivation, even when corrected for its acute functional inhibition potency, argues against a common, open channel block mechanism to explain persistent inactivation. Perhaps more compelling is the finding that pancuronium fails to protect against mecamylamine pretreatment-induced persistent inactivation of a4P2-nAChR while being protective against nicotine- or carb- induced persistent inactivation of 04^2- or a4p4-nAChR. Collectively, these findings 165 suggest that mecamylamine and nicotinic agonists induce persistent inactivation by different mechanisms.

Nevertheless, blockade by pancuronium of agonist-induced persistent inactivation remains consistent with a requirement for nAChR activation as a precondition for the more delimited phenomena of agonist-induced persistent inactivation. Pancuronium block of agonist-induced persistent inactivation applies not only to a4*-nAChR as revealed in this study, but also to muscle-type al*-nAChR as expressed by TE671/RD cells and ganglionic (x3P4*-nAChR as expressed by PC 12 cells (Lukas, 1991). Thus, a testable hypothesis is that any competitive nicotinic antagonist would provide protection against persistent inactivation by preventing acute agonist activation of nAChR function and the subsequent cascade of events leading to persistent inactivation.

Although mecamylamine-induced persistent inactivation appears to have a different mechanism from nicotine-induced persistent inactivation, the fact that either drug induces a lasting loss of a4p2-nAChR function helps explain the utility of either mecamylamine or nicotine as a smoking cessation aid. Treatment with either drug, if they also induce persistent inactivation of a4P2-nAChR on brain neurons, would help reduce nicotinic cholinergic hyperactivity postulated to underlie cognitive and emotional disorders expressed in some individuals who resort to tobacco use in an effort to achieve self medication for those disorders (Gentry and Lukas, 2002a). 166

Acknowledgements:

This work could not have been accomplished without the kind gifts of human nicotinic acetylcholine receptor subunit DNA from Dr. Ortrud Steinlein of the Rheinische-

Friedrich-Wilhelms-Universitaet and from Dr. Jon M. Lindstrom of the University of

Pennsylvania. Special thanks is extended to Dr. Curtis Bay for consultation regarding statistical analysis. 167

CHAPTER V

Local Anesthetics Noncompetitively Inhibit Function of Four Distinct nAChR Subtypes 168

CHAPTER V

ABSTRACT

Local anesthetics (LAs) are considered to act primarily by inhibiting voltage-gated

Na^ channels. However, LAs also are pharmacologically active at other ion channels including nicotinic acetylcholine receptors (nAChR). nAChR exist as a family of diverse subtypes, each of which has a unique pharmacological profile. The current studies established effects of LAs on function of four human nAChR subtypes: naturally expressed muscle-type (al*-nAChR) or autonomic (a3P4*-nAChR) nAChR, or heterologously expressed nAChR containing a4 with either P2 or P4 subunits (a4P2- or a4P4-nAChR). Of the LAs tested, those with structures containing two separated aromatic rings (e.g. proadifen and adiphenine) had the greatest inhibition potency (IC50 values between 0.34 and 6.3 |aM) but lowest selectivity (~4-fold) across the four nAChR subtypes examined. From the fused, two-ring (isoquinoline backbone) class of LAs, dimethisoquin had comparatively moderate inhibition potency (ICso values between 2.4 and 61 jiM) and ~30-fold selectivity across nAChR subtypes. Lidocaine, a commonly used LA from the single ring category of LAs, blocked nAChR function with IC50 values of between 52 and 250 |iM and had only ~5-fold selectivity across nAChR subtypes. Its quaternary triethyl ammonium analogue, QX-314, had greater inhibition potency, but the 169 trimethyl ammonium derivative, QX-222, was the least potent LA at all but the a4P2- nAChR subtype. With only a few exceptions, LA effects were consistent with noncompetitive inhibition of nAChR function and occurred at therapeutic doses. These studies suggest structural determinants for LA action at diverse nAChR subtypes and that nAChR likely are clinically relevant targets of LAs. 170

INTRODUCTION

LAs block nerve conduction in the peripheral nervous system (Arias, 1999). They also have a wide range of behavioral effects implicating actions in the central nervous system (CNS). Among these effects are restlessness, euphoria, muscle twitching, and tremor, which have been attributed to selective depression of inhibitory neurons by LAs.

Other effects of LAs suggesting CNS actions include drowsiness, disorientation, slurred speech, respiratory depression, tinnitus, and sedation. At high concentrations, LAs may cause loss of consciousness or even death (Naguib et al., 1998; Hodgson et al., 2000).

LAs also differ from one another in several ways, and different bases have been used to classify LAs. LAs have been classified into three categories by Arias (Arias,

1999). Agents from one category of LAs (Group I; see Fig. 5.1), typified by tetracaine, procaine and lidocaine, possess only one aromatic ring. An amide (lidocaine) or an ester

(tetracaine, procaine) linkage couples the ring to one aliphatic chain that typically ends in a ternary amino group (tetracaine, procaine, lidocaine) or a quaternary ammonium ion

(the charged lidocaine analogue QX-314 or the dimethylanunonio variant QX-222). The

Group I esters also have a second amino (procaine) or alkylamino (butylamino for tetracaine) chain para to the ester-linked alkylamino chain. The Group I amides include compounds (lidocaine, QX-314, QX-222) having two methyl groups ortho to the alkyiamino or alkylammonio chain. Thus, Group I compounds might be subdivided into two subgroups representing para-(alkyl)amino-phenyl-alkylesters and ortho-dimethyl- phenyl-alkylamides. A second category of LAs (Group 11; see Fig. 5.1) includes 171 molecules with two aromatic rings separated and linked by a single a-carbon chain and is typified by proadifen and adiphenine. Group II LAs also have an ester linking the ring region through the a-carbon to an aliphatic chain that ends in a ternary amino group

(proadifen, adephinine) or in a quaternary ammonium ion (charged proadifen derivative meproadifen; not shown). In some cases. Group II LAs have an additional alkyl chain

(propyl for proadifen) linked to the a-carbon bridging the two aromatic rings. A third category (Group HI; see Fig. 5.1), represented by dimethisoquin, contains drugs having a fused, two-ring structure (i.e., an isoquinoline backbone). An aliphatic chain ending again in either a ternary amino group (dimethisoquin) or in a quaternary ammonium ion

(charged dimethisoquin analogue trimethisoquin; not shown) is coupled via an ester linkage to the isoquinoline ring, which is further derivatized with an alkly chain (butyl for dimethisoquin) meta to the ester link.

Whereas peripheral effects of LAs are attributed to actions on voltage-sensitive

Na^ channels, mechanisms of LA action on presumably CNS-mediated behaviors are not well understood. lonotropic neurotransmitter receptors such as cholinergic and serotonergic receptors have been investigated as potential targets of LA action in the brain (Katz and Miledi, 1975; Neher and Steinbach, 1978; Lukas and Bennett, 1979;

Horn et al., 1980; Forman and Miller, 1989; Chamet et al., 1990; Revah et al., 1991;

Barann et al., 1993; Dilger and Vidal, 1994; Fan and Weight, 1994). 172

Nicotinic acetylcholine receptors (nAChR) are neurotransmitter-gated ion channels. At least 16 distinct genes encode nAChR subunits that combine in a variety of

ways to form diverse, pentameric nAChR ion channels (reviewed in Lukas, 1998). Some

physiological roles of diverse nAChR subtypes are known whereas others remain

incompletely defined (Albuquerque et al., 2000). However, each nAChR subtype has a

unique pharmacological profile, and this may also translate into differences in sensitivity

to LAs (Dani, 1993; Eterovic et al., 1993; Cuevas and Adams, 1994). Conversely,

nAChR may prove to contribute to clinically relevant therapeutic actions and/or side

effects of LAs. Additionally, the understanding the extent and mechanism of LA action

at nAChR provides a foundation for their use in studying regulation of nAChR by chronic

ligand exposure.

This study assessed acute effects of several members from the three categories of

LAs on function of four different, human nAChR subtypes. A preliminary report of these

fmdings has appeared (Gentry et al., 2000). 173

MATERIALS AND METHODS

Drug Dilutions. All drugs were prepared fresh the day of the assay as stock solution in as much as 50% ethanol depending on the drug and then diluted so that final concentrations of ethanol were no higher than 5% in assay buffer (130 mM NaCl, 5.4 mM

KCl, 2 mM CaCl2 • 2 H2O, 5 mM Glucose, 50 mM HEPES) at the highest concentrations of LA used in each experiment. Ethanol alone up to 50% in assay buffer had no effects on function of any of the examined nAChR subtypes during a 3 min exposure (data not shown). Ethanol was obtained from Sigma Chemical Co. a-phenylbenzeneacetic acid-2-

(diethylamino)ethyl ester HCl (adiphenine), carbamylcholine chloride (carb), 2- diethyIamino-N-(2,6-dimethylphenyl)acetamide HCl (lidocaine), a-phenyl-a- propyibenzeneacetic acid-2-(diethylamino)ethyl ester HCl (proadifen; SKF-525A), 4- aminobenzoic acid-2-(diethylamino)ethyl ester HCl (procaine), and 4-

(butylamino)benzoic acid 2-(dimethylamino)ethyl ester HCl (tetracaine) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2-(trimethylanmionio)-N-(2,6- dimethylphenyOacetamide chloride (QX-222) and 2-(triethylanmionio)-N-(2,6- dimethylphenyl)acetamide Br or CI' were obtained from Alomone Labs (Jerusalem,

Israel). 3-butyl-l-[2-(dimethylamino)ethoxyl isoquinoline HCl (dimethisoquin) was purchased from ICN Biomedicals, Inc. (Plainview, NY) (discontinued - now available from Research Diagnostics, Inc, Flanders, NJ). 174

Model Cell Lines and Cell Culture. The present study used low passage (less than SO) human cell lines naturally or heterologously expressing different nAChR subtypes to examine inhibition potencies of LAs. The TE671/RD human cell line naturally expresses muscle-type nAChR (al*-nAChR according to suggested nomenclature); (Lukas et al.,

1999) as an assembly of two al subunits and one each of pi, 5 and y subunits (Lukas,

1986; Schoepferet al., 1988; Lukas, 1989a; Luther et al., 1989). Human neuroblastoma- derived SH-SY5Y cells naturally express two nAChR subtypes found in autonomic neurons. However, the subtype that contains a3 and P4 subunits (a3P4*-nAChR) dominates ion flux assay measures of nAChR function in these cells (Lukas et al., 1993;

Conroy and Berg, 1995). nAChR containing a4 and P2 subunits (a4P2-nAChR) are a major form of high affinity nicotine binding site in mammalian brain, and nAChR containing a4 and ^4 subunits (a4P4-nAChR) may also be substantially expressed in the

CNS. Human a4P2- and a4P4-nAChR were stably and heterologously expressed in native nAChR-null SH-EPl human epithelial cells to create SH-EPl-ha4P2 and SH-EPl- ha4P4 cell lines, respectively, using techniques that have been previously reported (Peng et al., 1999b; Eaton et al., 2000; Ferchmin et al., 2001). Cells were maintained at 37°C, under 95% O2 / 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum (Hyclone, Logan, UT), 10% horse serum (Life Technologies/Gibco

BRL Grand Island, NY), 1% sodium pyruvate (Cellgro, AK by Mediatech Inc., Hemdon, 175

VA), 2% glutamine penicillin-streptomycin (Irvine Scientific, Santa Ana, CA and 0.02% amphotericin B (Sigma Chemical Co.).

Assays of nAChR Function. efflux assays were performed using TE67 l/RD, SH-

SY5Y, SH-EPl-ha4P2, or SH-EPl-ha4P4 cells according to the procedure of (Lukas and

Cullen, 1988). Cells were cultured (-2 x 10^ cells per 15.5 mm diameter well; -150 ng total cell protein per well) on Falcon 24-well culture plates (BD Biosciences, Bedford,

MA) precoated with poly-D-lysine m.w. 70,000-150,000 (Sigma Chemical Co., St. Louis,

MO, USA) according to Bencherif et al. (1995). Cells were allowed to grow (overnight) until a confluent monolayer was formed and cells adhered to culture plate. Confluence was monitored using light microscopy. Rubidium-86 (^®Rb*) was obtained from New

England Nuclear (Boston, MA). In all cases, cells were loaded with ^^Rb^ for no less than 4 hours and subsequently rinsed twice with 2 ml per well of drug-free assay buffer.

Cells expressing each nAChR subtype of interest were exposed to efflux buffer alone or experimental concentrations of LA and/or carbamylcholine for three minutes.

Radioactivity released into the extracellular medium was quantified by Cerenkov counting using a Wallac Trilux system (40% efficiency). Levels of nonspecific ion flux from each cell line were comparable whether detined with samples containing antagonist,

100 fxM d-tubocurarine and I mM carb, or with control samples containing buffer alone

without agonist. Specific nAChR function was defined as total, experimentally

determined ion flux evoked by agonist, carb, in the absence or presence of LA, minus 176 nonspecific ion flux. Typical values for specific and nonspecific efflux for cell samples are 9000 cpm and 1200 cpm for TE671/RD cells, 2900 cpm and 900 cpm for

SH-SY5Y cells, 3500 cpm and 600 cpm for SH-EPl-ha4P2 cells, and 9000 cpm and 800 cpm for SH-EPl-ha4P4 cells.

Data Analysis. Dose-response curves were fit to data points by the general equation Y =

b + [(a-byi+lO"*^'^^"*] where Y is the observed specific efflux response (% of control), X is the experimental concentration of LA, b is nonspecific flux, a is total ion flux (1 mM carb control), c is the log IC50 value for antagonist dose-response profiles at

fixed agonist concenU'ation or the log EC50 value for agonist dose-response profiles at

fixed antagonist concentration, and n is the Hill coefficient (<0 for antagonist dose-

response profiles; >0 for agonist dose-response profiles). Best fit, nonlinear regression

variable slope curves were determined by an iterative process using GraphPad Prism (San

Diego, CA) software, from which values of a, b, c, and n were derived for each

experiment. Values of a and b parameters were normalized to percentage of control flux.

Because LA acted as noncompetitive inhibitors in nearly all cases, there was no

dependence on concentration of agonist used to stimulate nAChR function for LA ICso

values, and there was no need to apply modifications, such as the Cheng-Prusoff

correction, to the data. Results from 3-16 independent measurements were fit to the

logistic equation and plotted as mean ± S£.M. 177

RESULTS

Acute Effects of LAs on al*-nAChR Function, efflux assays with the

TE671/RD ceil line expressing muscle-type al*-nAChR were used to evaluate receptor function during acute exposure to selected LAs. Simultaneous exposure of cells to increasing concentrations of LA and 1 mM carb indicated that all LAs inhibited al*- nAChR function in a concentration-dependent manner (Fig. 5.2). Concentrations at which half maximal block of TE671/RD cell al*-nAChR function were achieved rank ordered from high to low inhibition potency were (IC50 value precedes drug's name):

0.34 (aM proadifen > 1.9 |iM adiphenine > 2.4 |aM dimethisoquin > 13 ^iM tetracaine >

19 |iM QX-314 > 52 (aM lidocaine > 230 |xM procaine > > 3.4 mM QX-222 (Table 5.1).

Thus, representative Group Q LAs exhibited greatest inhibitory potency at al*-nAChR followed by Group III LAs, with Group I LAs exhibiting the lowest inhibitory potency

(Fig. 5.2). Hill coefficients for antagonist concentration-response profiles ranged between -0.74 proadifen to -1.6 for lidocaine (Table 5.1).

Subsequent experiments were completed in which carbamylcholine concentration- response profiles were obtained at fixed concentrations of LAs near to and encompassing their ICso values for inhibition of 1 mM carb response. Representative LAs from each of the structural categories were used for these studies: Group I was represented by tetracaine and procaine; Group n was represented by adiphenine; and Group QI was represented by dimethisoquin. For each of the LAs tested, functional block produced by 178 near ICso concentrations was insurmountable by increasing the concentration of carb, suggesting a noncompetitive mechanism for functional inhibition of al*-nAChR (Fig.

5.3).

Acute Effects of LAs on (x3P4*-iiAChR Function. ^^Rb^ efflux assays using SH-

SY5Y cells expressing a3P4*-nAChR showed concentration-dependent functional blockade by all LAs tested (Fig. 5.4). Concentrations at which half maximal block (IC50) of SH-SY5Y cell 0t3P4*-nAChR function was achieved were (by rank order: high to low inhibition potency): 0.6 joM proadifen > 1.8 (iM adiphenine > 4.7 |iM dimethisoquin >

8.3 |xM tetracaine = 9.2 jiM QX-314 > 63 nM lidocaine > 87 nM procaine > 150 |aM QX-

222 (Table 5.1). Thus, the general rank order inhibition potency of LA's observed in the

TE67I/RD cell line (Group II > Group HI > Group I) held for a334*-nAChR expressed by the SH-SY5Y cell line (Fig. 5.3,5.4; Table 5.1). Hill slopes ranged between -0.78 for proadifen and -1.23 for lidocaine.

Carb dose-response profiles at increasing concentrations of LAs revealed that functional block of a3P4*-nAChR produced by each of the representative LAs was consistent with a noncompetitive mechanism of functional inhibition (Fig. 5.5).

Acute Effects of Drags on 04^2- or a4P4- nAChR Function. Acute effects of selected LAs on function of nAChR containing a4 subunits were also evaluated by ®^b^ efflux assay. SH-EPl cells in which a4P2- or a4P4- nAChR were heterologously expressed were simultaneously exposed to test concentrations of LA and 1 mM carb. All 179

LAs inhibited a4*-nAChR function in a concentration-dependent manner with one exception (Figs. 5.6-5.7). The quartemary LA, QX-222, at a concentration of 2.5 mM only weakly inhibited I mM carb-evoked response mediated by a4P4-nAChR (Fig. 5.7).

The rank order inhibition potency for LAs tested at a4P4-nAChR was identical to that for al*- or a3P4*- nAChR and was (IC50 value precedes drug name): 1.5 ^iM proadifen >

6.3 ^M adiphenine = 7.2 ^iM dimethisoquin > 30 ^M tetracaine > 64 |iM QX-314 > 250

HM lidocaine >1.0 mM procaine > > 2.5 mM QX-222 (Table 1). Interestingly, the rank order inhibition potency for LAs at a4P2-nAChR was somewhat unique. Rank order and

IC50 values for LA inhibition of a4P2-nAChR function were: 2.0 ^M proadifen > 3.1 |iM adiphenine > 27 ^M tetracaine >61 ^M dimethisoquin = 68 |iM QX-314 > 190 ^M lidocaine > 390 ^M QX-222 > 2.1 mM procaine (Fig. 5.6 and Table 5.1). Thus, in general. Group Q LAs (adiphenine and proadifen) exhibited the greatest ability to inhibit carb-evoked a4*-nAChR function, while Group I compounds exhibited weaker ability to inhibit. However, the Group 01 compound has an inhibition potency for a4P2-nAChR within the range of potencies tested for Group I compounds. Moreover, the diethylamino group I ligand, lidocaine, its triethylammonium derivative, QX-314, and the trimethylammonium analogue, QX-222, had the most similar inhibitory capability for a4P2-nAChR compared to other nAChR subtypes (Figs. 5.1 and 5.6; Table 5.1). Hill slopes were shallowest for proadifen (-0.73 to -0.94) and steepest for tetracaine (-1.24 to

-1.61) at a4*-nAChR (Table 1). 180

Concentration-response profiles for carb-evoked efflux exhibited both right- and downward shifts with increasing LA concentration suggesting that LAs tested inhibit a4P2-nAChR mediated responses via a combination of noncompetitive and competitive mechanisms (Fig. 5.8). Carb concentration-response profiles at antagonist concentrations fixed near their respective IC50 value suggested that most of the representative LAs produce functional block of a4P4-nAChR via a noncompetitive mechanism (Fig. 5.9).

The exception is for procaine, for which a rightward shift in the agonist dose-response profiles suggests that procaine may have some steric effects at the carb binding site(s) on a4p4-nAChR. 181

Fig. 5.1. Molecular structures of LAs used in this study. Names for each LA and their position according to the classification scheme of Arias, 1999, in Groups I, II, or HI, are shown for each structure (note however that dimethisoquin, proadifen and adiphenine are ternary amino ligands rather than quaternary ammonium ions as figure 2 in Arias, 1999, might implicate). Structures are listed left top-to-bottom and then right top-to-bottom in the rank order, with some minor exceptions, observed for actions at nAChR. See the text.

Figs 5.2-5.9, and Table 5.1 for experimental details. 182 Figure 5.1 Molecular Structures of LA tested n. proadifen L QX-314

N Ot

CHj

II. adiphenine I. lidocaine .ot

in. dimethisoquin I. procame r

1. tetracaine L QX-222 Q ^ ^ Hac

Oi 1 CHJ 183

Fig. 5.2. LA dose dependence for functional blockade of al*-nAChR in TE671/RD ceils. Specific efflux (ordinate, percentage of control) measured as described in

Materials and Methods was determined in the presence of the indicated concentrations

(abscissa, log molar scale) of: A - proadifen (•), dimethisioquin (•), QX-314 (A), or procaine (V), or B - adiphenine (O), tetracaine (•), lidocaine (A), or QX-222 (•).

Smooth curves drawn through data points (means ± S.E.M. from at least three separate experiments) are from iterative fits to the logistic function setting a = 100% (see

Materials and Methods). Results yield IC50 values. Hill coefficients, and r^ values indicated in the text and/or in Table 5.1. Values for the parameter, b, as a percentage of control ^"^b^ efflux are 3.0 ± 2.9% for proadifen, 11.2 ± 5.4% for dimethisoquin, 13.8 ±

7.0% for QX-314, -1.3 ± 13.5% for procaine 2.4 ± 2.0% for adiphenine, 1.6 ± 2.9% for tetracaine, and 2.4 ± 3.6% for lidocaine. Figure 5.2 TE671/RD cells: Inhibition of Carb Response

100-

75 4 O 50- c o O 25 • proadifen • dimethisoquin • QX-314 V procaine i i I

U 100I- +

S 75

u SO- 0) O) 25 O adiphenine • tetracaine A iidocaine • QX-222 -9 -8 -7-6-5-4 -3 -2 log [ drug ] (M ) 185

Fig. 53. Dose-response profiles for carbamylcholine-stimulated efflux through al*-nAChR at different concentrations of LA. Measurements of specific efflux

(ordinate, percentage of 1 mM carbamylcholine control) were made with TE67 l/RD cells assayed in the presence of carb alone (O) at the indicated doses (abscissa, molar log scale) or in the presence of carb with adiphenine, dimethisoquin, tetracaine or procaine at indicated concentrations. Data points are from 3 or more independent experiments (mean

± S.E.M.). Smooth curves drawn through the data points are from iterative fits to the logistic function setting b = 0% {see Materials and Methods). Values for the parameter, a

(as a percentage of specific ^^Rb"" efflux), and r^ values (in parentheses) are 103.1 ± 4.0%

(0.90) for untreated control, 76.3 ± 2.9% (0.97) for 1.3 (aM adiphenine, 63.2 ± 3.3%

(0.95) for 2.5 jiM adiphenine, 41.6 ± 3.3% (0.90) for 5 nM adiphenine, 72.4 ± 0.9%

(0.99) for I nM dimethisoquin, 48.7 ± 1.5% (0.99) for 3 ^iM dimethisioquin, 29.5 ± 1.2%

(0.97) for 10 ^iM dimethisoquin, 78.4 ± 2.2% (0.99) for 3 ^iM tetracaine, 47.5 ± 2.2%

(0.98) for 10 nM tetracaine, 21.8 ± 1.8% (0.94) for 32 |iM tetracaine, 84.6 ± 0.9% (0.99) for 32 jiM procaine, 64.5 t i.4% (0.99) for 100 nM procaine, and 36.1 ± 1.4% (0.98) for

316 jiM procaine. None of the carb dose-response profiles displayed any significant shift from EC50 value of control (39 nM x/-5- 0.05). Figure 5.3 TE671/RD cells

100 adiphenine •Q 0,iM , dimethisoquin

O 75 1 tilM - c O 50 O S|iM

o 25

H 3 T T T T T T T T T T T

100 tetracaine procaine •Q 0 |iM . ^ 32(ilM SF 75 •b 5 50 O 316 (iM 6 25 32(ilM'

-8 -7 •6 -5 -4 3 -2 log [carb] (M) 00 187

Fig. 5.4. LA dose dependence for functional blockade of 0t3P4*-nAChR in SH-SY5Y cells. Specific efflux (ordinate, percentage of control) measured as described in

Materials and Methods was determined in the presence of the indicated concentrations

(abscissa, log molar scale) of: A - proadifen (•), dimethisioquin (•), QX-314 (A), or procaine (V), or B - adiphenine (O), tetracaine (•), lidocaine (A), or QX-222 (•).

Smooth curves drawn through data points (means ± S.E.M. from at least three separate experiments) are from iterative fits to the logistic function setting a = 100% (see

Materials and Methods). Results yield IC50 values. Hill coefficients, and r^ values indicated in the text and/or in Table 5.1. Values for the parameter, b, as a percentage of control ^^b"*" efflux are 5.7 ± 3.8% for proadifen, 3.5 ± 2.5% for dimethisoquin, l.l ±

3.6% for QX-314, 1.4 ± 5.1% for procaine, 1.9 ± 4.2% for adiphenine, 5.6 ± 4.0% for tetracaine, 1.0 ± 10.0% for lidocaine, and 7.0 ± 9.6% for QX-222. Figi 5.4 SH-SY5Y cells: Inhibition of Carb Response

100

75

50

• proadifen 25 • dimettiisoquin A QX-314 V procaine 0

100

75

50

O adiphenine I 25 • tetracaine A lidocaine 0 • QX-222 J -8 -7 -6 -5 -4 -3 log [ drug ] (M ) 189

Fig. 5.5. Dose-response profiles for carb-stimulated efflux through a3P4*-nAChR at different concentrations of LA. Measurements of specific efflux (ordinate, percentage of 1 mM carb control) were made with SH-SY5Y ceils assayed in the presence of carb alone (O) at the indicated doses (abscissa, molar log scale) or in the presence of carb with adiphenine, dimethisoquin, tetracaine or procaine at indicated concentrations. Data points are from 3 or more independent experiments (mean ±

S.E.M.). Smooth curves drawn through the data points are from iterative fits to the logistic function setting b = 0% (see Materials and Methods). Values for the parameter, a

(as a percentage of specific ^^b^ efflux), and r^ values (in parentheses) are 113.3 ± 9.5%

(0.85) for untreated control, 68.2 ± 6.0% (0.72) for 0.5 |iM adiphenine, 48.6 ± 6.0%

(0.61) for 1 |aM adiphenine, 40.9 ± 4.8% (0.60) for 2 ^iM adiphenine, 75.6 ± 5.5% (0.94) for 1 nM dimethisoquin, 41.6 ± 6.4% (0.82) for 3 jiM dimethisoquin, 25.6 ± 3.6% (0.75) for 10 ^iM dimethisoquin, 74.2 ± 4.2% (0.96) for 3 tetracaine, 37.4 ± 2.6% (0.92) for

10 |iM tetracaine, 17.9 ± 6.1% (0.66) for 32 ^iM tetracaine, 72.3 ± 4.2% (0.89) for 32 nM procaine, 48.4 ± 4.0% (0.81) for 100 nM procaine, and 24.4 ± 4.2% ( ) for 320 procaine. None of the carb dose-response profiles displayed any significant shift in EC50 value (155 ^iM x/^0.12). Figure 5.5 SH-SY5Y cells

0\if* 100- adiphenine dimethisoquin A .

— 75 1|iM ' O 0.5 ^Nl i JL C 50 o o >^'"5 10»iM -

X 3 £ lU A 0|iM ^ 1001- tetracaine procaine A . flC i S 75 3|ilM o 50 100 |iM 10 MM l 25 320 uM- (0 32 ^IM

-7 -5 -3 -1 -7 -2 -1 log [carb] (M) 191

Fig. 5.6. LA dose dependence for functional blockade of a4P2-nAChR in transfected

SH-EPl-ha4P2 cells. Specific ®^Rb^ efflux (ordinate, percentage of control) measured as

described in Materials and Methods was determined in the presence of the indicated concentrations (abscissa, log molar scale) of: A - proadifen (•), dimethisioquin (•), QX-

314 (A), or procaine (V), or B - adiphenine (O), tetracaine {•), lidocaine (A), or QX-

222 (T). Smooth curves drawn through data points (means ± S.E.M. from at least three

separate experiments) are from iterative fits to the logistic function setting a = 100% (see

Materials and Methods). Results yield IC50 values. Hill coefficients, and r^ values

indicated in the text and/or in Table 5.1. Values for the parameter, b, as a percentage of

control ^^b* efflux are 5.3 ± 6.8% for proadifen, 16.5 ±5.1% for dimethisoquin, -4.1 ±

7.7% for QX-314, n/a for procaine, -1.2 ± 4.1% for adiphenine, 2.3 ± 2.5% for tetracaine,

-3.1 ± 20% for lidocaine. and n/a for QX-222. 192 Figure 5.6 SH-EP a4P2 cells: Inhibition of Carb Response

100

75

50 o o • proadifen 25 • dimethisoquin • QX-314 V procaine ^ 0 X 3 + + r 111 100 (C (O 00 75

O & 50 (O O adiphenine 25 • tetracaine A lidocaine • QX-222

-9 -7-6-5-4 -2 log [ drug ] (M ) 193

Fig. 5.7. LA dose dependence for functional blockade of a4^nAChR in transfected

SH-EPl-ha4P4 cells. Specific efflux (ordinate, percentage of control) measured as described in Materials and Methods was determined in the presence of the indicated concentrations (abscissa, log molar scale) of: A - proadifen (•), dimethisioquin (•), QX-

314 (A), or procaine (V), or B - adiphenine (O), tetracaine (•), lidocaine (A), or QX-

222 (T). Smooth curves drawn through data points (means ± S.E.M. from at least three separate experiments) are from iterative fits to the logistic function setting a = 100% (see

Materials and Methods). Results yield IC50 values. Hill coefficients, and r^ values indicated in the text and/or in Table 1. Values for the parameter, b, as a percentage of control efflux are -5.2 ± 2.3% for proadifen, 6.4 ± 2.8% for dimethisoquin, 7.0 ±

2.9% for QX-314, 12.3 ± 46% for procaine, 0.64 ± 4.4% for adiphenine, 1.4 ± 2.1% for tetracaine, -8.6 ± 7.6% for lidocaine, and n/a for QX-222. 194

Figure 5.7 SH-EP a4P4 cells: Inhibition of Carb Response

100

75

50

o • proadifen o 25 • dimethisoquin • QX-314 0 V procaine + + +

91 UJ 100 B +

(O 00 75

U 50 & 0) 25 O adiphenine • tetracaine A iidocaine 0 • QX-222

-9 -7-6-5-4 -2 log [ drug ] (M ) 195

Fig. 5.8. Dose-response profiles for carb-stimulated efflux through a4P2-nAChR at different concentrations of LA. Measurements of specific efflux (ordinate, percentage of 1 mM carb control) were made with SH-EPI-ha4P2 cells assayed in the presence of carb alone (O) at the indicated doses (abscissa, molar log scale) or in the presence of carb with adiphenine, dimethisoquin, tetracaine or procaine at indicated concentrations. Data points are from 3 or more independent experiments (mean ±

S.E.M.). Smooth curves drawn through the data points are from iterative fits to the logistic function setting b = 0% (see Materials and Methods). Values for the parameter, a

(as a percentage of specific ^^b^ efflux), and r^ values (in parentheses) are 100.8 ± 1.4%

(0.98) for untreated control, 47.2 ± 3.0% (0.81) for 1 adiphenine, 36.9 ± 2.5% (0.83)

for 3 (aM adiphenine, 22.1 ± 2.6% (0.70) for 6 nM adiphenine, 54.1 ± 0.7% (0.99) for 32

HM dimethisoquin, 40.0 ± 3.2% (0.82) for 100 jiM dimethisoquin, 13.2 ± 1.6% (0.95) for

320 dimethisoquin, 65.4 ± 1.5% (0.97) for 10 nM tetracaine, 44.0 ±1.1% (0.98) for

32 nM tetracaine, 6.9 ± 0.3% (0.92) for 100 tetracaine, 85.4 ± 1.9% (0.98) for 100

|aM procaine, 71.6 ± 2.9% (0.95) for 320 |IM procaine, and 27.4 ± 1.3% (0.94) for 1000

HM procaine. All of the carb dose-response profiles produced a shift in EC50 value from

(19 nM X/-5- 0.03) for control to higher values (up to ~400 nM x/^ 0.08) in the presence of

LAs. Figure 5.8 SH-EP a4p2 cells

100 • adiphenine jy-O 0|iM dimethisoquin _Q.-0.-Q 0|tM-

32|iM Io o ^100 )1M "B 320 |iM n X 3 S Ul 100- tetracaine Q-X)—2 OjiM. procaine J3,.0"g OMW h

75- §320 |iM

& 50- O x> 32 & 1000 uM » 25- 100 tiM o..o-^-Sr:^

-7 -5 -3 -2 -1 log [carb] (M) 197

Fig. 5.9. Dose-response profiles for carb-stimulated efflux through a4P4-nAChR at different concentrations of LA. Measurements of specific ®^Rb^ efflux (ordinate, percentage of I mM carb control) were made with SH-EPl-ha4P4 cells assayed in the presence of carbamylcholine alone (O) at the indicated doses (abscissa, molar log scale) or in the presence of carb with adiphenine, dimethisoquin, tetracaine or procaine at indicated concentrations. Data points are from 3 or more independent experiments (mean

± S.E.M.). Smooth curves drawn through the data points are from iterative fits to the

logistic function setting b = 0% (see Materials and Methods). Values for the parameter, a

(as a percentage of specific ®^b^ efflux), and r values (in parentheses) are 97.9 ± 2.6%

(0.99) for untreated control, 70.0 ± 3.1% (0.93) for 2 jiM adiphenine, 48.7 ± 2.4% (0.91)

for 4 ^iM adiphenine, 25.8 ± 3.0% (0.66) for 8 nM adiphenine, 68.9 ± 3.5% (0.97) for 4

|xM dimethisoquin, 46.2 ± 2.9% (0.96) for 8 dimethisoquin, 52.0 ± 51.6% (0.89) for

16 ^iM dimethisoquin, 81.2 ± 1.2% (0.99) for 8 |aM tetracaine, 72.2 ± 1.8% (0.97) for 16

tetracaine, 51.9 ± 1.6% (0.98) for 32 nM tetracaine, 85.0 ± 4.2% (0.95) for 250 ^iM

procaine, 73.5 ± 8.6 (0.86) for 500 nM procaine, and 39.3 ± 7.4% (0.89) for 1000 nM

procaine. The dose response profile for procaine produced a rightward shift in ECso value

(from 50 nM x/^ 0.03 control to 316 |iM x/-i- 0.25 in the presence of 1000 nM procaine)

indicating that procaine may partially compete with carb for a binding site on the a4P4-

nAChR. Adiphenine, dimethisoquin, tetracaine and procaine dose-response profiles all 198 produced functional blockade of the a4p2-nAChR insurmountable with increasing concentrations of carb suggesting noncompetitive binding properties. Figure 5.9 SH-EP a4p4 cells I I I

lOOl- adiphenine .0..0 OfilM • dimethisoquin ,.0 0)ilM - /<5' « 75 9d ^X 9 J. 4tilM *t -rX^ C 50- • .--a 8|iM ' O JX !w * ./J. 16^iM • -V- * ^A 2 25 wV */ ^ o Jr ol- ».Q»g»0"8

tetracaine o 0)iM procaine ,a-o 0|iM

250 uM 500 |iM i 1000 iiN

-7 -5 -3 log [carb] (M) 200

Table 5.1. ^*^5^ efflux (3 min) was stimulated by 1 mM carb alone (to deflne total efflux), 1 mM carb in the presence of LA (to define experimental LA dose-efflux response curves), or in the presence of carb- and LA- free buffer (to define nonspecific

^"^Rb^ efflux). Data as illustrated in Rgs. 5.2, 5.4, 5.6, and 5.7 were analyzed to determine IC50 values for LA inhibition of function of the nAChR subtype indicated.

Mean IC50 values in bold face (x/-^ S.E.M.) are reported on the top line of each entry and represent the concentration at which the LA reduces the maximal agonist induced response by one-half. LLill coefficients (± S.E.M.) and r^ values are reported in the lower line of each entry. 201

Table 5.1

Parameters for Functional nAChR Block by LA

IC50 [iM (x/-j- S.E.M.) Hill coefficient ± S.E.M. R" al*- a3B4*- a4B2- proadifen 0.34(1.3) 0.60(1.3) 2.0(1.6) 1.5(1.2) -0.74 ±0.13, 0.93 -0.78 ±0.12.0.77 -0.73 ± 0.24. 0.86 -0.94 ± 0.16.0.92 adiphenine 1.9(1.1) 1.8(1.3) 3.7(1.3) 6.3(1.2) -1.05 ±0.12, 0.97 -0.93 ±0.11,0.88 -0.91 ± 0.19.0.96 -1.12 ±0.24,0.94 dimethisoquin 2.4(1.5) 4.7(1.1) 61 *(1.2) 7.2(1.1) -0.77 ± 0.23, 0.82 -1.09 ± 0.05, 0.94 -1.10 ± 0.20, 0.88 -1.33 ± 0.24,0.96 tetracaine 13(1.1) 8.3(1.2) 27 *(1.1) 30(1.1) -1.21 ±0.17.0.95 -1.19 ± 0.08. 0.88 -1.23 ±0.12, 0.96 -1.61 ±0.18.0.97

QX-314 19(1.5) 9.2(1.2) 68(1.3) 64(1.1) -1.43 ± 0.67,0.83 -0.95 ± 0.16, 0.88 -1.03 ± 0.23, 0.94 -1.40 ±0.16.0.94

lidocaine 52(1.1) 63(1.4) 190(1.8) 250(1.2) -1.60 ± 0.24. 0.95 -1.23 ±032.0.64 -1.10 ± 0.44.0.76 -1.22 ± 0.20,0.92

procaine 230(1.4) 87(1.2) 2090 *(5.2) 1030(4.6) -1.10 ± 0.24,0.88 -1.06 ±0.19.0.94 -0.84 ± 0.20.0.92 -0.82 ± 0.40.0.55

QX-222 >3400 150(1.4) 390 *(1.4) >2500 N/A -1.14 ± 0.34,0.85 -1.41 ±0.42,0.86 N/A

* Rank order inhibition potency not in agreement with muscle -type nAChR results 202

DISCUSSION

Major findings. The primary findings of this study are: (i) that LAs have reasonably strong ability to inhibit diverse, human nAChR subtypes (al*-, (x3P4*-, a4P2- and a434- nAChR), and (ii) that, with a few exceptions, LAs act at these nAChR subtypes as noncompetitive functional inhibitors. Two exceptions to the noncompetitive inhibition rule are for LAs acting at a4P2-nAChR and for procaine acting at a4P4- nAChR. Although LAs can be classified in several ways, our data regarding LA function at nAChR are well segregated based on the structural classification for LAs as put forth by (Arias, 1999) (represented in Fig. 5.1). Although the sample size of LAs representing each structural category is limited, the data serve as a foundation for studies of LA-

nAChR structure-activity relationships.

Group n LAs, proadifen and adiphenine, were the most potent functional

inhibitors at all nAChR tested, with proadifen having 2- to 6-fold greater inhibition

potency across nAChR subtypes than adiphenine (see Table 5.1). The only structural difference that might account for the greater inhibition potency of nAChR for proadifen is

its additional propyl moiety linked to the a-carbon (see Fig. 5.1).

In general, the group ED ligand built on the butyi-isoquinoline backbone, dimethisoquin, had the next greatest ability to inhibit each nAChR subtype tested (see

Table 5.1). Dimethisoquin, like the Group Q LAs, has a two carbon chain separating

amino firom ester moieties on one aliphatic chain (see Fig. 1). However, dimethisoquin is 203 a dimethyiamino rather than a diethylamino compound, suggesting that presence of the more compact amino group might contribute to lower inhibition potency for n AChR.

Group I drugs generally have the lowest inhibition potencies of the four nAChR subtypes examined. Tetracaine, which has a dimethyiamino residue ending its ester- linked aliphatic chain but also has a markedly longer para-aminobutyl chain, consistently has > 10-fold greater potency for the nAChR subtypes tested than the diethylamino, para- amino ligand, procaine (see Table 5.1 and Fig. 5.1). Tetracaine's ability to inhibit at all nAChR subtypes tested is only 4- to 8-fold greater than that of the diethylamino amide, lidocaine. The quaternary, triethylammonium amide, QX-314, generally has greater (up to 7-fold) inhibition potency for nAChR than does the ternary, diethylamino analogue, lidocaine, but QX-314 has much greater inhibition potency than does the more compact quaternary, trimethylammonium amide, QX-222. Thus for Group I esters, presence of a diethylamino (procaine) moiety rather than a more compact dimethylanuno (tetracaine) group, and perhaps the shortened length of the para-(alkyl)amino aliphatic chain, correlates with diminished potency for nAChR (see Fig. 5.1). However, for Group I amides, the diethylammonium (QX-314) has greater inhibition potency than does the more compact dimethyiammonium (QX-222). The diethylanmionium (QX-314) also has greater inhibition potency than does the diethylamino (lidocaine) compound. These fmdings indicate that structural determinants of LA ability to inhibit nAChR include bulk at the amino/ammonio moiety. However, these findings also indicate that these structural 204 determinants differ between Group I esters and amides, suggesting that ligands in Group I should be further subclassified.

Of the nAChR subtypes tested, muscle-type al*- and ganglionic 03^4*- nAChR naturally expressed in the periphery each were generally more sensitive to block by LAs than were CNS-type a4^2- or a4P4-nAChR. al*- and cx3P4*-nAChR had similar sensitivity to block by most of the LA tested. As an exception, a3P4*-nAChR showed nearly 20-fold higher sensitivity than al*-nAChR to QX-222. Another notable exception was for a4P2-nAChR, which was ~9-fold more sensitive than al •-nAChR to QX-222 block. Thus, across nAChR subtypes, the only rank order inhibition potency profile that deviated from proadifen > adiphenine > dimethisoquin > tetracaine > QX-314 > lidocaine

> procaine » QX-222 was for a4P2-nAChR, which had the profile tetracaine > dimethisoquin ~ QX-314 > lidocaine > QX-222 » procaine. Comparisons between a4P2- and a4P4-nAChR indicated comparable inhibition potency or slightly higher potency at a4P2-nAChR for most of the LAs tested except for dimethisoquin (~9-fold greater inhibition potency at a4P4-nAChR) and QX-222 (> 6-fold greater inhibition potency at a4P2-nAChR). Therefore, the small differences between P2 and P4 subunits influence potency for only a select few LAs. Curiously, a4P2-nAChR showed the highest level of discrimination between Group I esters (tetracaine/procaine) but by far the lowest level of discrimination between Group I amides (QX compounds and lidocaine). 205

There is substantial evidence that LAs bind to and inhibit nAChR via noncompetitive mechanisms implicating open channel block (reviewed by Arias, 1999).

Our findings are largely consistent with results and conclusions from these studies, but our studies extend to broader comparisons across LAs and across nAChR subtypes. For example, consistent with our results, QX-314 was found in electrophysiological studies to be a more effective blocker than QX-222 at frog extrajunctional (fetal or y subunit- containing) al*-nAChR (Neher and Steinbach, 1978). The comparatively weak effects of these charged ligands, which we also observed, were attributed to inability to gain access to the cell's interior except via open channels. In another electrophysiological study, QX-314 and QX-222 were found to produce half-maximal inhibition of mouse muscle al*-nAChR heterologously expressed in Xenopus oocytes at concentrations of 78 jiM and 2780 (aM, respectively (Pascual and Karlin, 1998), which compare to ICso values of 19 nM and 34(X) ^M, respectively, in the current studies. Electrophysiological analyses of effects on peak currents evoked by presumed a3P4*-nAChR naturally expressed in cultured rat parasympathetic cardiac neurons yielded ICso values of 28 ^ for QX-222 and 2.8 fxM for procaine (Cuevas and Adams, 1994), which have the same rank order but show considerably higher activity when compared with 150 and 87

^lM ICso values for QX-222 and procaine, respectively, acting in the current study at himian a3P4*-nAChR natively expressed in ganglionic SH-SY5Y cells. Another electrophysiological study reported voltage-dependent ICso values of 1 or 40 ^M, 206 respectively, for tetracaine acting at mouse muscle or Torpedo electroplax al*-nAChR heterologously expressed in Xenopus oocytes (Eterovic et al., 1993); these values bracket the IC50 value of 13 ^iM found in the current study for actions of tetracaine at human al*- nAChR. Mouse al*-nAChR expressed in Xenopus oocytes were blocked by procaine with an IC50 of 66 |iM (Yost and Dodson, 1993), showing higher sensitivity than human al*-nAChR natively expressed in the TE671/RD cell line (IC50 value of 230 ^M).

Differences in species, expression systems (cell environment), experimental approach

(electrophysiological or ion flux assays), or perhaps degrees of nAChR desensitization

(Ryan and Baenziger, 1999) could account for differences in IC50 values observed in different studies.

Rank order inhibition potency for LA action at Torpedo marmorata electric organ al*-nAChR measured using electron spin resonance and fluorescence techniques was procaine > tetracaine > QX-222 (Arias et al., 1990) and contrasts to our findings. This probably is because the biophysical measures taken may not relate to functional effects of

LA at nAChR.

With regard to clinical relevance of LA inhibition of nAChR function, pharmacokinetic findings and ease of LA access to nAChR are of primary importance.

Actions of charged anmionium LAs are likely restricted to the periphery. Also, peripheral routes of administration are less likely to yield LA concentrations high enough to induce neurotoxicity (Ritchie and Greengard, 1966). Roles played by blood-brain barrier- 207 permeable LAs in neurological effects depend on perineural concentrations achieved rather than the dose of administered LA (Johnson, 2000), and plasma, cerebrospinal fluid

(CSF), and brain concentrations of LAs are influenced by rates and routes of LA administration (Robinson et al., 1994; Xuecheng et al., 1997; Youngs, 1999).

For example, lidocaine is widely used for clinical procedures that include topical ointment application, epidural anesthesia, spinal administration, and systemic bolus administration. Peak plasma concentrations of lidocaine following topical application of

5% ointment to intact skin of healthy volunteers was 9 ng/ml (33.2 nM) and occurred 24 hours following the treatment. Thus, absorption of lidocaine from intact skin is poor, and even if applied to damaged skin, effects of topical lidocaine at nAChR (IC50 values of 52-

250 nM) would be unlikely to occur. However, plasma and CSF concentrations of lidocaine following systemic bolus administration in humans peak at -1.7 ng/ml (~6 nM) within 5-15 minutes rapidly declining thereafter (Tsai et al., 1998). Intra-venous (i.v.) infusion of rabbits with lidocaine at a rate of 4 mg«kg''«min'' produces whole brain = cortical brain > plasma > CSF concentrations of 373 nM, 295 |iM, 166 nM, and 77.5 nM, respectively, at the time of lidocaine-induced seizure onset (718 seconds) (Momota et al.,

2000). These concentrations, especially those in cortical or whole brain, in which lidocaine accumulates, are higher than IC50 values for blockade of nAChR. An independent investigation of adiphenine metabolism showed that concentration of ethyl-

['^Cladiphenine 5 minutes after i.v. administration is 106.7 ± 17.2 ng/g brain tissue 208 versus 7.1 ± 0.4 ng/g in blood, a difference of nearly fifteen times more concentrated in brain tissue (Michelot et al., 1981). Spinal administration, despite dilution of drug in the subarachnoid space, of anesthetics provides perhaps the greatest opportunity for LA to access CNS receptors (Rigler et al., 1991). Spinally administered LAs are distributed

non-homogenously and can briefly reach very high local concentrations (Drasner et al.,

1994; Robinson et al., 1994). For example, five minutes following routine spinal administration of a 5% solution of lidocaine, the mean CSF concentration can reach 14

mM (Van Zundert et al., 1996), which clearly would have effects at nAChR.

Conclusions. Collectively, these considerations and our results suggest

therapeutic application of LA may produce peak plasma and/or brain concentrations capable of inducing significant functional inhibition of nAChR in the periphery or centrally. Enhanced deposition of LAs in the brain may actually make their concentrations there higher than in the periphery, meaning that inhibition of nAChR in

the brain (e.g., a4*-nAChR and perhaps central a3*-nAChR) would occur even if their

acute sensitivity to LAs is lower than LA sensitivity of nAChR in the periphery. Thus,

nAChR need to be considered as potential targets for therapeutic actions of LAs and in

mediation of side effects of LA action. Moreover, LAs present a broad and interesting

family of pharmacophores, action of which at nAChR can be used to help define

structural determinants on drugs and receptors that could be exploited to develop novel

nAChR antagonists as well as safer and more effective LAs. 209

Acknowledgements

Thank you Alomone Labs for generous donation of lidocaine derivatives QX-314Cr and

QX-314Br. 210

CHAPTER VI

Functional Effects of Prolonged Local Anesthetic Exposure on and a4^-Nicotinic Acetylcholine Receptors (nAChR) 211

CHAPTER VI

ABSTRACT

Previous investigations suggested that nicotinic ligands acting acutely as open channel blockers might have abilities to discriminately induce lasting functional

inhibition of a4^2- and/or a4P4-nAChR subtypes when applied chronically (Gentry and

Lukas, 2001a). Lidocaine, adiphenine, and dimethisoquin, representative compounds

from each of three structural categories of local anesthetics (LA) previously shown to act

acutely as non-competitive antagonists of nAChR function, were tested for their ability to

induce losses in a4p2- and a4P4-nAChR function that persist beyond drug removal. In

this study, only dimethisoquin was capable of inducing persistent inactivation of a4P2-

and a4P4-nAChR function at micromolar concentrations, but not discriminately. In

pharmacological studies to examine mechanisms involved with persistent inactivation of

nAChR function, pancuronium, a competitive nAChR antagonist previously shown to

protect a4P2- and a4p4-nAChR from persistent inactivation induced by nicotine or

carbamylcholine, did not protect those nAChR from persistent inactivation induced by 1

hr exposure to dimethisoquin. On the other hand, two of the LAs tested were able to

produce some protection of a4P2-nAChR, but not of a4P4-nAChR, from persistent

inactivation induced by I hr exposure to nicotine at I mM concentrations. The results

indicate that select LA are capable of inducing or protecting against persistent

inactivation of nAChR containing a4 subunits and thought to be the principal targets of 212 exogenous nicotine action. These findings illuminate mechanisms involved in long lasting losses of nAChR function on chronic nicotinic ligand exposure and suggest possible exploitation of LA in potential therapies for treatment of nicotine dependence and use of tobacco products. 213

INTRODUCTION

Nicotinic acetylcholine receptors (nAChR) are diverse ligand-gated ion channels and are expressed in various tissue- and cell- types throughout the brain and body.

Multiple subtypes of nAChR exist, each composed of various pentameric combinations of nAChR subunits. To date, 17 nAChR subunit genes have been identified including (al- aiO, Pl-P4, 5, e, y) (Lukas et al., 1999; Elgoyhen et al., 2001). nAChR are capable of controlling flux of ions such as Na^, and Ca^ across cell membranes in response to stimulation by the endogenous neurotransmitter, acetylcholine, or upon acute exposure to other, exogenously-applied nicotinic agonists. For example, nAChR are targets of

nicotine, a major component of tobacco, which acutely acts to mimic acute actions of acetylcholine at nAChR.

nAChR have been carefully studied for many years, in part, because of their

important physiological role in regulating synaptic transmission. nAChR are subject to

sustained nicotinic ligand exposure via habitual use of tobacco products or when nicotinic

ligands are used therapeutically to treat neuropsychiatric disorders. As a result, a large

effort has been underway to understand nAChR responses to chronic nicotinic ligand

exposure (reviewed in Gentry and Lukas, 2002a). It has been consistently shown in both

in vitro and in vivo systems that chronic nicotine exposure induces an upregulation in

nAChR-like radioligand binding sites. In an apparent paradox, in vitro and in vivo studies

have found all nAChR subtypes evaluated to date are subject to functional down-

regulation following chronic nicotine exposure. However, the paradox is partially 214 remedied with evidence that quantitative increases in nAChR-like radioligand binding sites occur largely in internal rather than cell surface pools (Ke et al., 1998; Whiteaker et al., 1998). The physiological relevance of nAChR upregulation remains open to question although surface nAChR function is undoubtedly important in determining physiological and behavioral effects of sustained exposure to either nicotine or therapeutic agents targeting nAChR in the brain (Gentry and Lukas, 2002a).

E^evious investigations in vitro of nAChR functional responses after chronic nicotinic agonist or antagonist exposure revealed that most nAChR agonists, but only a select few antagonists, induce losses in nAChR function that persist well after drug has been removed (Ke et al.. 1998; Gentry and Lukas, 2001a). The phenomenon is arguably distinct from rapidly reversible receptor "desensitization" first described by Katz and

Thesleff in 1957, and it has been operationally defined as "persistent inactivation," a loss of nAChR function that is not reversed during a 5 min period of recovery from agonist exposure" (Katz and Thesleff, 1957; Ke et al., 1998). Chronic exposure to the competitive antagonist, pancuronium, does not induce persistent inactivation of nAChR;

however, it can offer protection of nAChR from nicotine-induced persistent inactivation

(Lukas, 1991; Ke et al., 1998; Gentry and Lukas, 2001a). Interestingly, persistent

inactivation of a4P2- or a4P4-nAChR function following chronic exposure to the

noncompetitive antagonist, mecamylamine, suggested that noncompetitive antagonists

may have some ability to discriminate between nAChR subtypes, perhaps by sharing with

nicotine and other agonists the ability to engage in long-lasting open channel block 215

(Gentry and Lukas, 2001a). Additional noncompetitive antagonists would have to be tested to address unanswered questions: Do other noncompetitive antagonists induce persistent inactivation of nAChR? Can noncompetitive antagonists selectively inhibit function of particular nAChR subtypes when administered chronically? Are noncompetitive antagonists able to mimic the protective capabilities of the competitive antagonist, pancuronium, against chronic agonist exposure-induced persistent inactivation? Can pancuronium protect against any persistent inactivation mediated by noncompetitive antagonists?

Local anesthetics (LA), recognized for acting primarily by inhibiting voltage- gated Na" channels, also noncompetitively inhibit function of nAChR (Gentry and Lukas,

2001b). LA are useful tools for identifying noncompetitive inhibitor binding sites on the nAChR and for studying conformational states of nAChR (McCarthy and Stroud, 1989;

Charaet et al., 1990; Arias, 1996; Pascual and Karlin, 1998; Ryan and Baenziger, 1999).

However, effects of sustained LA exposure on nAChR function are not known.

Understanding functional responses of CNS-type nAChR to chronic LA exposure may help elucidate the mechanisms of CNS-type nAChR regulation. The present study evaluates functional responses of a4p2- or a4P4-nAChR, two putative nAChR subtypes with known or potential functional importance in the brain, expressed in a SH-EPl human epithelial cell line, following chronic exposure to the non-competitive antagonists, lidocaine, adiphenine, and dimethisoquin. 216

MATERIALS AND METHODS

Drug Dilutions. All drugs were prepared fresh on the day of the assay as 10 mM stock solutions in cell culture medium (see cell culture) and diluted appropriately for each experiment. Efflux assay buffer consisted of 130 mM NaCl, 5.4 mM KCl, 2 mM CaCU,

5 mM glucose, and 50 mM HEPES, pH 7.4. Buffer components as well as carbamylcholine (carb), S(-)-nicotine (free base), 2-diethyIamino-N-(2,6- dimethylphenyl)acetamide HCl (lidocaine), a-phenylbenzeneacetic acid-2-

(diethylamino)ethyl ester HCl (adiphenine), and pancuronium Br were purchased from

Sigma Chemical Co. (St. Louis, MO, USA). 3-butyl-l-[2-(dimethylamino)ethoxy] isoquinoline HCl (dimethisoquin) was purchased from ICN Biomedicals, Inc. (Plainview,

NY) (discontinued - now available from Research Diagnostics, Inc, Flanders, NJ).

Model Cell Lines and Cell Culture. The present study used low passage (less

than 50) SH-EPl human epithelial cell clones stably and heterologousiy expressing

human a4P2- or a4P4-nAChR to examine functional responsiveness of receptors

following various drug treatments. These clonal cell lines were generated using

techniques that have been previously reponed (Peng et al., 1999a; Eaton et al., 2000;

Pacheco et al., 2001; Lukas et al., 2002). Briefly, transfection with pcDNA3.1-zeo-

human a4(S452) and pcDNA3.1-hygro-human P2 constructs created in our lab

(subcloned using human a4 and ^2 subunit cDNAs kindly provided by Dr. Ortrud

Steinlein, Rheinishce-Friedrich-Wilhelms-Universitaet, Bonn, Germany) followed by 217 isolation of a stable, high-expressing clonal line was accomplished to create SH-EPl- ha4P2 cells. Transfection with pCEP4-hygro-human a4(S452) and pcDNA3.1-zeo- human ^4 constructs created in our lab (the latter was subcloned using a human ^4 subunit cDNA kindly provided by Dr. Jon Lindstrom, University of Pennsylvania,

Philadelphia, PA) also followed by isolation of a high-expressing clone was used to generate SH-EPl-ha4P4 cells. Cells were maintained at 37°C, under 95% O2 / 5% CO2

in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum

(Hyclone, Logan, UT), 10% horse serum (Life Technologies/Gibco BRL Grand Island,

NY), 1% sodium pyruvate (Cellgro AK, Mediatech Inc., Hemdon, VA), 2% glutamine

penicillin-streptomycin (Irvine Scientific, Santa Ana, CA) and 0.02% amphotericin B

(Sigma). Cell culture medium was also supplemented with 0.01% hygromycin

(Calbiochem, San Diego, CA) and 0.03% zeocin (Invitrogen, Carlsbad, CA) to maintain

stable expression of a4 and P2 or ^4 subunits.

Assays of nAChR Function, ^^b^ efflux assays were performed using SH-EPl-

ha4p2, or SH-EPl-ha4P4 cells according to the procedure of (Lukas and Cullen, 1988;

Gentry et al., 2002; Lukas et al., 2(X)2) with some modifications to account for

pretreatments with nicotinic ligand. Modifications are described here. Cells were

cultured (~2 x 10^ cells per 15.5 mm diameter well; -150 ng total cell protein per well)

on Falcon 24-well culture plates (BD Biosciences, Bedford, MA) precoated with poly-D-

lysine m.w. 70,000-150,000 (Sigma). Cells were grown overnight to confluence verified 218 using light microscopy. ^^ubidium-Cl 3.8 mCi/mg or 0.4 cpm/fmol at 40% counting efficiency) was obtained from Perkin-Elmer Life Sciences (New England

Nuclear) (Boston, MA).

In all cases, cells were loaded with 0.5 ml of serum- and antibiotic-supplemented cell culture medium also supplemented with ®^b^ (-2 nCi per well) for 4 hr or longer to ensure maximal ^^Rb^ uptake by the cells. Control samples were not subjected to ligand pretreatment but were otherwise processed identically. Medium containing ^"^b* only was removed and replaced at specific times with medium containing plus the appropriate concentration of drug so as to allow cells to incubate in ligand containing media for I hr prior to drug washout for nAChR recovery period and subsequent assay of nAChR function. Cells were kept in Incubators in 5% CO2 at 37°C for drug pretreatments and for ®^b^ loading except during medium changes.

At the end of any drug pretreatment and ^^b^ loading period, medium was removed by aspiration to mark the beginning of the recovery period, and the "flip plate"

(Lukas et al., 2002) method was employed to administer three rinses with 2 ml per well of drug- and ®®Rb^ -free efflux assay buffer (22°C for recovery times of 5 min or less, 37°C for recovery times greater than 5 min) to remove extracellular and pretreatment drug. For rinse times of 3 min or more, the first rinse solution was applied for 1 min, the second rinse solution was applied for 1 min, and the third rinse solution was applied for the remainder of the experimentally defined length of time (1 min for 3 min recovery, 3 219 tnin for 5 min recovery, etc.). For I min recovery samples, each of the three rinses was for 20 sec.

After the defmed period of recovery from ligand exposure, efflux assays were used to evaluate nAChR function. Some wells of cells on each 24-well cell culture plate were reserved for controls. Total efflux (or positive control condition) was defined by cells not exposed to ligand pretreatment, only exposed for 2 min to 1 ml of 1 mM carb alone in efflux buffer ("acute agonist challenge"). Non-specific ^"^b* efflux was defined using other control samples exposed to efflux buffer alone or to efflux buffer containing both I mM carb and 100 |iM d-TC (either negative control approach gave comparable results). Simultaneously, wells of cells that had received ligand pretreatment were exposed to 1 ml of I mM carb alone in efflux buffer for the 2 min "acute agonist challenge" period.

At the end of the efflux period, buffer samples were collected, and amounts of

®*^b^ released into extracellular fluids were quantified by Cerenkov counting using a

Wallac Trilux system (40% efficiency). One ml per well of 0.1% SDS/O.l M NaOH was then added to lyse the cells and to prepare samples for determination of the amounts of remaining, intracellular ^®Rb^ (see Data Analysis below). Specific nAChR function was defined as total minus nonspecific ®^b^ efflux. Typical values for specific and nonspecific ^"^b^ efflux in control cells not subjected to ligand pretreatment were 2500 220 cpm and 500 cpm for SH-EPl-ha4P2 cells, and 3500 cpm and 400 cpm for SH-EPl- ha4P4 cells (loaded with 350,000 cpm of applied

Data Analysis. To control for possible influences of ligand pretreatment and recovery conditions on ^^b^ loading, data were subjected to a double normalization process. Background radioactivity was subtracted from all samples. Amount of ^®Rb^ loaded and present intracellularly at the start of the "acute agonist challenge" functional efflux assay was determined as the sum for each sample of released (extracellular) and

remaining (intracellular) present at the conclusion of the functional assay. Specific

®^Rb^ efflux was defined as total ®'^b^ efflux minus non-specific ^^Rb" efflux (described above). Normalized specific ^®Rb* efflux was then expressed as a percentage of specific

^^Rb"^ efflux divided by ®^Rb^ loaded. Double normalized specific ^®Rb^ efflux was then expressed as a percentage of normalized specific ^^b"" efflux for experimental samples

subjected to ligand pretreatment divided by normalized specific ^^b* efflux for control

samples not subjected to ligand pretreatment. Data were plotted as mean ± S.E.M using

GraphPad Prism 3.0 (San Diego, CA) software. Results from 3 or more independent

experiments are shown. Statistical analysis was performed using GraphPad Prism 3.0

(San Diego, CA) software. Parameters were compared to control using analysis of

variance (ANOVA) followed by Dunnett's post-test, and multiple comparisons between

data set were made using ANOVA followed by Bonferroni post-test. The extent of 221 nAChR functional loss following 5 min of drug washout was operationally defined as

"persistent inactivation." 222

RESULTS

efflux assays with SH-EPl human epithelial cells transfected to express either human a4P2-nAChR or human a4P4-nAChR were used to evaluate receptor function following 1 hr-long exposures to various LAs or combinations of nicotinic ligands. LAs differ from one another in several ways and have previously been classified into three categories based on structural characteristics and acute functional potencies

(Arias, 1999; Gentry and Lukas, 2001b). The LAs chosen for this study each represent a different category of L\. Lidocaine exhibits features characteristic of the first category of

LA, group I, which possess only one aromatic ring. Other examples of group I compounds include tetracaine, procaine, and QX-222 and QX-314, two lidocaine derivatives. Some of these molecules have ester linkages coupling the ring to an aliphatic chain, while others such as lidocaine have an amide linkage. For lidocaine, the aliphatic chain terminates with a ternary amino group. Adiphenine represents a second category, group Q LAs, which includes molecules with two aromatic rings separated and linked by a single a-carbon chain. Adiphenine has an ester linking the ring region through the a- carbon to an aliphatic chain that ends in a ternary anuno group. The third category of LA, group m, is represented in this study by dimethisoquin. Group m contains compounds having a fused, two-ring soiicture (i.e. an isoquinoline backbone) and in the case of dimethisoquin, an aliphatic chain ending in a ternary amino group.

Select noncompetitive antagonists induce persistent inactivation of CNS«type nAChR. 223

Effects of 1 hr lidocaine pre-exposure on function of human aA^l- and a4^4- nicotinic acetylcholine receptor (nAChR) subtypes were studied using receptors heterologously expressed in SH-EPl human epithelial cells. For a4P2-nAChR, magnitudes of acute, specific efflux responses to 1 mM carbamylcholine following high concentration (I mM) lidocaine exposure and 5 min recovery in drug free buffer were reduced -30% relative to untreated controls (i.e., there was -30% "persistent inactivation of a4P2-nAChR function; p < 0.05, one-way ANOVA followed by Dunnett's post test) (Fig. 6.1, top-left). Lower concentrations of lidocaine did not induce significant persistent inactivation of a4P2-nAChR. Only after pretreatment with 1 mM lidocaine was there a modest -20% loss of a434-nAChR function, and there was little recovery beyond that after 5 min of drug-free rinsing, but no significant, long-lasting effects of lidocaine at lower concentrations were observed at a4P4-nAChR (Fig.6.1, top-right).

Chronic treatment of SH-EPl-ha4P2 cells with adiphenine, a group II LA, did not result in any significant, long-lasting functional effects except at a concentration of i mM

(p < 0.01, one-way ANOVA folowed by Dunnett's post test) (Fig. 6.1, middle-left).

There was -35% persistent inactivation of a4P2-nAChR function after 5 min of recovery from I mM adiphenine exposure. a4P4-nAChR did become persistently inactivated following 1 hr of exposure to adiphenine at concentrations of 100 ^M or greater (Fig. 6.1, middle-right). Functional loss was -40% and -73% for cells treated with 100 ^M or 1 mM adiphenine, respectively, following 1 min of drug-free recovery, and there was about 224

25-35% persistent inactivation after 5 min of drug removal, a4P4-nAChR function returned to normal within 1 hr after adiphenine removal.

Dimethisoquin induced significant and substantial persistent functional losses relative to untreated controls for both a4P2- and a4P4-nAChR (p < 0.01, one-way

ANOVA followed by Dunett's post test; Fig. 6.1, bottom-left and bottom-right). a4P2- nAChR function was reduced by -75-80% following 1 hr of 10 ^iM or 100 |xM dimethisoquin exposure (Fig. 6.1, bottom-left). There was 50-60% persistent inactivation after 5 min of drug-free recovery for samples treated with 10-100 nM dimethisoquin.

Although function had not completely recovered within 1 hr, a4p2-nAChR had regained more than 50% of their normal efflux response. a4P4-nAChR function was completely eliminated following 1 hr of 10 nM or 100 jiM dimethisoquin pre-exposure, and function recovered more slowly than was observed for a4P2-nAChR (Fig. 6.1, bottom-right).

After 5 min in drug-free medium, a4P4-nAChR function was persistently inactivated to only ~10% of control values. At the end of a 1 hr recovery period, a4P4-nAChR that had been pretreated for i hr with 10 or 100 dimethisoquin were functioning at

-25% and -50% of normal, respectively.

Pancuronium shows mixed ability to block LA-induced persistent Inactivation of a4*.nAChR. 225

The competitive antagonist, pancuronium, was previously shown to protect al*- nAChR naturally expressed in TE-67 l/RD cells and a3P4*-nAChR naturally expressed in PC-12 cells from nicotine-induced persistent inactivation (Lukas, 1991). Pancuronium pretreatment by itself had no lasting functional effects at any of the nAChR subtypes tested (Lukas, 1991). Pancuronium was used in the present study to determine whether exposure to it together with a LA noncompetitive antagonist would have additive effects in induction of nAChR persistent inactivation or whether it would protect against any such effects of LA. Pancuronium was able to inhibit the modest persistent inactivation of a4P2-nAChR function induced by 1-hr treatment at high (I mM) concentrations with lidocaine (Fig. 6.2, top) or adiphenine (Fig. 6.2, middle). Pancuronium also was able to protect against persistent inactivation induced by 10 dimethisoquin pretreatment, although pancuronium was ineffective in altering persistent inactivation induced at higher or lower doses of this more potently-acting LA (Fig. 6.2, bottom). Although lidocaine alone induced only modest persistent inactivation of a4P4-nAChR and only for I mM pretreatment, that effect was attenuated in combined pancuronium/lidocaine treatment

(Fig. 6.3, top). Unlike for a4P2-nAChR, pancuronium was not able to prevent the persistent inactivation of a4P4-nAChR induced by high (I mM) concentrations of adiphenine pretreatment (although there was some protection against the effect of pretreatment with 100 |aM adiphenine; Fig. 6.3, middle). Effects of pancuronium on dimethisoquin pretreatment of a4P4-nAChR were more complex. When I mM 226 pancuronium was added to the pretreatment media containing 10 ^M dimethisoquin, which alone produced nearly complete persistent inactivation of a4^4-nAChR, functional losses were reduced to only -40% (Fig. 6.3, bottom). However, pancuronium did not significantly reduce the amount of persistent inactivation of a4P4-nAChR following 1

HM or 100 ^iM dimethisoquin pretreatments (Fig. 6.3, bottom).

Noncompetitive antagonist block of nicotine-induced persistent inactivation of CNS- type nAChR.

Tests were also conducted of the ability of LA noncompetitive antagonists to protect nAChR from nicotine-induced persistent inactivation. Cells were pretreated for 1 hr with nicotine at the indicated concentrations alone or in the presence of LA at concentrations selected because they show some effect alone on a4*-nAChR persistent inactivation. Following 5 min of drug-free recovery, functional assays were done to assess levels of nAChR persistent inactivation. Profiles for nicotine pretreatment alone showed typical, pronounced persistent inactivation of a4P2-nAChR (Fig. 6.4, top) or a4P4-nAChR (Fig. 6.4, bottom). Lidocaine pretreatment at I mM by itself produced

-30% persistent inactivation of a4P2-nAChR function and -20% persistent inactivation of a4P4-nAChR function (Fig. 6.1, top). However, segments in nicotine pretreatment dose-persistent inactivation profiles for both a4P2-nAChR (Fig. 6.4, top) and a434- nAChR (Fig. 6.4, bottom) were shifted upward in the presence of 1 mM lidocaine, suggesting that lidocaine rendered some protection against nicotine pretreatment-induced 227 functional loss. Note that doses of nicotine where lidocaine produced statistically significant (p < 0.05 one-way ANOVA, Bonferroni post-test) protection against persistent inactivation were slightly different between a4P2-nAChR (100 ^iM and I mM nicotine) and a4P4-nAChR (1-10 (aM nicotine). However, for both a4*-nAChR subtypes, the fact that lidocaine and nicotine effects were not additive, which would have produced downward shifts in nicotine dose-response profiles in the presence of LA, is also notable.

At a4P2-nAChR, 100 ^M adiphenine pretreatment alone produced modest,

-20% persistent inactivation of a4P2-nAChR and -25% persistent inactivation of a4P4- nAChR (Fig. 6.1, middle), i.e., levels of persistent inactivation comparable to those produced by I mM lidocaine alone. Interestingly, dose-response curves for nicotine- induced persistent inactivation of a4P2-nAChR (Fig. 6.4, top) or a4P4-nAChR (Fig. 6.4, bottom) function were shifted upward to the same degree in the presence of 100 ^iM adiphenine as they were with concurrent nicotine plus 1 mM lidocaine pretreatment.

Thus, effects of nicotine and adephenine were not additive, but adephenine provided some degree of protection against nicotine-induce persistent inactivation.

Following 1 hr pretreatment with 10 ^M dimethisoquin alone and 5 min recovery, a4P2-nAChR were persistently inactivated by -50% (Fig. 6.1, bottom-left), and a4P4- nAChR were persistently inactivated by -35% after pretreatment with I nM dimethisoquin (Fig. 6.1, bottom-right). The concentration proHle for nicotine-induced

persistent inactivation of a4P2-nAChR was not significantly different from samples 228 subjected to 1 hr pretreatment with nicotine plus 10 |iM dimethisoquin (Fig. 6.4, top).

Similarly, addition of dimethisoquin to the nicotine pretreatment media did not have additive functional effects on a4P4-nAChR, nor was dimethisoquin able to protect a4P4- nAChR from nicotine-induced persistent inactivation (Fig. 6.4, bottom). 229

Fig. 6.1 SH-EPl-ha4P2-iiACIiR and SH-EPl-ha4P4-iiAChR functional responses to 1 mM carbamylcholine challenge following 1 hour lidocaine, adiphenine, or dimethisoquin exposure. SH-EPl cells heterologously expressing ha4P2-nAChR (left panels) or ha4P4-nAChR (right panels) were pre-exposed to non-competitive antagonists, lidocaine (top panels), adiphenine (middle panels), dimethisoquin (bottom panels)

(abscissa, concentration) for 1 hour. E^re-exposure media was aspirated and the pre- exposed cells were subjected to 3 successive rinses of 2 ml drug-free assay buffer. The first and second rinses were one min, and the third rinse consumed the remainder of the experimentally defined recovery time. Recovery times were (O) I min, (•) 3 min, (V) 5 min, (•) 15 min. (•) 30 min, or (•) 60 min. Specific ^®Rb* efflux (ordinate, percentage of control) measured as described in Materials and Methods was determined by eliciting nAChR function using acute (2 min) exposure to I mM carb. 100% response was defined by untreated control cells response to 1 mM carb. Linear curves are drawn through data points (means ± S.E.M. from at least three separate experiments). 230

Figure 6.1

ha4p2-nAChR ha4p4-nAChR

100 75- racBwry tima: o 50- 01 min -••ismin -*-3min 030 min •9-S min -a-l hour c 25- o lidocaine Hdocain* o 0 o 100 75- X 3 50-

0) 25 + adiphenine adiphanin* ja 0- cc (O 00 100- O 75 o o 50- Q. (0 25- diin«thi«oqutn 0 dimathisoquin -7 log [ pretreatment local anesthetic ] (M ) 231

Fig. 6.2 Pancuronium block of lidocaine-, adiphenine-, or dimetiiisoquin-induced nAChR functional inactivation at SH-EPl-ha4P2-nAChR. I mM pancuronium block of lidocaine (top panel), adiphenine (middle panel), or dimethisoquin (bottom panel) induced nAChR functional inactivation was evaluated. SH-EPl-ha4P2-nAChR were exposed for 1 hour to lidocaine, adiphenine or dimethisoquin treatment (abscissa, concenu-ation) in the absence of pancuronium (solid lines) or in the presence of I mM

pancuronium (dashed tines) and subsequently allowed 3 min to recover in drug free

media before receptor function was assessed. Specific efflux (ordinate, percentage of control) measured as described in Materials and Methods was determined by eliciting

nAChR function using acute (2 min) exposure to I mM carb. Pancuronium did not significantly reduce the amount of functional loss observed following pretreatment with

dimethisoquin. Pancuronium did not significantly change lidocaine or adiphenine's lack

of long-lasting functional effects. 232

Fieure 6.2 ha4p2-nAChR

100- f-

75

O

c O lidocaine o *5 p 125-

100-

75

pane

(O 25 00 adiphenine

O 100- Q. 75-

50-

25- dimethisoquin

log [ pretreatment LA ] (M ) 233

Fig. 6.3 Pancuronium block of lidocaine-, adiphenine-, or dimethisoquin-induced nAChR functional inactivation at SH-EPl-ha4P4-nAChR. 1 mM pancuronium block of lidocaine (top panel), adiphenine (middle panel), or dimethisoquin (bottom panel) induced nAChR functional inactivation was evaluated. SH-EPl-ha4P4-nAChR were exposed for 1 hour to lidocaine, adiphenine or dimethisoquin treatment (abscissa, concentration) in the absence of pancuronium (solid lines) or in the presence of 1 mM pancuronium (dashed lines) and subsequently allowed 5 min to recover in drug free media before receptor function was assessed. Specific ^^b^ efflux (ordinate, percentage of control) measured as described in Materials and Methods was determined by eliciting nAChR function using acute (2 min) exposure to I mM carb. Pancuronium did not significantly change lidocaine or adiphenine's lack of long-lasting functional effects.

Pancuronium reduced the amount of 10 ^M dimethisoquin-induced functional loss observed at SH-EPl-ha4P4-nAChR, but did not completely eliminate dimethisoquin's long-lasting functional effects (p < 0.05, unpaired t-test). 234 Figure 6.3 ha4p4-nAChR

IOC •

75-

50 •

25' lidocaine

IOC-

75-

ifc 50 • % Ol-A't-I mM pane 25' adiphenine

50 •

25 • dimethisoquin

log [ pretreatment LA ] (M ) 235

Fig. 6.4 Effects of fixed concentratioiis of lidocaine, adiphenine, and dimethisoquin on nicotine-induced nAChR functional inactivation at CNS-type nAChR. SH-EPl- ha4p2-nAChR (top panel) were treated for I hour with nicotine (abscissa, concentration) and simultaneously with either 0 M anesthetic (•), I mM lidocaine (A), 100 (aM adiphenine (V), or 10 ^iM dimethisoquin (•). Following pretreatment, nAChR were rinsed free of drug (see materials and methods) and allowed to recover in drug-free efflux buffer for 5 minutes prior to evaluating nAChR function using ^^b"*" efflux assay and 2 min challenge with I mM carb. The presence of either lidocaine or adiphenine in nicotine pretreatment media mildy reduced the amount of persistent inactivation observed relative to high concentrations of nicotine alone. Dimethisoquin co-application with nicotine did not significantly change the concentration-response profile of a432-nAChR function from that expected for either drug alone. Thus, dimethisoquin does not protect a4P2-nAChR from nicotine-induced persistent inactivation. SH-EPI-ha4P4-nAChR

(bottom panel) were treated for 1 hour with nicotine (abscissa, concentration) and simultaneously with either 0 M anesthetic (#), I mM lidocaine (A), 100 ^M adiphenine

(V), or 1 nM dimethisoquin (•). After 5 min recovery, functional responsiveness of a4P4-nAChR remained less than that of untreated control in all cases. No significant differences in concentration-response profiles were observed relative to nicotine alone, except for adiphenine treated a4P2-nAChR (* represents p < 0.05, one-way ANOVA 236 followed by Bonferroni post test) which offered incomplete protection of a4p2-nAChR from 1 mM nicotine-induced persistent inactivation. 237 Figure 6.4 Combined Effects of Prolonged Exposure to Nicotine and Local Anesthetic 100 0 M anesthetic ha4P2-nAChR ^ * 1 mM lidocaine 100 adiph ^ 10 fiM dimeth

8 50

O P 25

S&100 -f 0 M anesthetic + ha4P4-nAChR ^ 1 mM lidocaine n -f 100 adiph oc CO 75 * 1 ^M dimeth 00 O = 50 0) Q. ^ 25

-8 -7 -6 -5 -3 log [ pretreatment nicotine ] (M) 238

DISCUSSION

Major findings. The present study was based on the relatively simple and logical notion that LA would prove useful as noncompetitive nAChR antagonists with likely channel blocking ability that could help elucidate mechanisms involved in the persistent loss of a4*-nAChR function induced by pretreatment with nicotine, other nAChR agonists, or select nAChR antagonists. The current findings show modest induction of persistent inactivation of a4P2- and a4P4-nAChR only after pretreatment with high doses of the representative class I and class Q LA, lidocaine and adephinine, but more potent and effective persistent inactivation of a4*-nAChR by pretreatment with dimethisoquin, a group HI, fused ring, isoquinoline-based LA. The degree of a4*- nAChR functional loss is higher at longer times of LA pretreatment, at higher doses of

LA pretreatment, and for shorter times of drug-free recovery following LA pretreatment as was seen for effects of nicotine and other nAChR agonists or select antagonists inactivation (chapter IV). There is a slightly (but not significant), greater ability of the

LA tested to induce persistent inactivation of a4p4-nAChR than of a4P2-nAChR, unlike the substantial differences in ability of the noncompetitive inhibitor mecamylamine to produce greater persistent inactivation of a4P2-nAChR than of a4P4-nAChR (Gentry et al., 2(X)2). The competitive nAChR antagonist, pancuronium, produces some degree of

protection against LA-induced persistent inactivation of oA^l- or a4P4-nAChR for some doses of LA, but not as effectively as it protects against agonist-induced persistent 239 inactivation (chapter IV). The LAs tested did not protect a4*-nAChR from nicotine- induced persistent inactivation with the exception of adiphenine providing limited protection of a4P2-nAChR from high (I mM) concentrations of nicotine-induced persistent inactivation. Interestingly LAs tested were previously shown to exhibit both competitive and noncompetitive pharmacological activity at a4P2-nAChR, while acting purely via noncompetitive mechanisms at a4P4-nAChR (Chapter V).

Insights into structure-activity relationsiiips for ligands inducing persistent inactivation. By evaluating three ligands representative of three structurally diverse classes of LA, we hoped to add to a structure-activity-based foundation to illuminate mechanisms involved in nAChR persistent inactivation and sites of ligand interaction that could influence that phenomenon. Some degree of persistent functional loss for a4*- nAChR is induced by lidocaine and adephinine at high concentrations, but dimethisoquin is clearly the most potent and effective LA, especially when its persistent inactivation potency is normalized to its acute functional inhibitory potency at a4P2- or a4P4- nAChR, showing that it actually has higher persistent inactivation potency than acute functional inhibitory potency. Previous studies indicated that group II LA, such as adiphenine, are the most potent acute functional inhibitors of nAChR, group III LA, typified by dimethisoquin, have intermediate acute functional inhibitory potency, and group I LA, represented here by lidocaine, are least potent acutely. Studies comparing

nAChR agonists found a strong, positive correlation between agonist acute functional 240 potency and chronically induced persistent inactivation potency at both a4P2- and a4P4- nAChR (Gentry and Lukas, 2002b). Such a relationship, however, did not exist for either competitive or noncompetitive, classical nAChR antagonist, nor does it exist for the LAs tested. This suggests that structural features important for LA-mediated acute block of a4*-nAChR function are not the same as those important for persistent inactivation. One caveat with this line of interpretation is that we do not know whether all ligands that induce operationally-defined "persistent inactivation" have or must have a common mechanism of action. One approach yet to be executed to investigate this, but perhaps limited until prototypical ligands acting at different nAChR sites are identified that have high enough affinity for those sites to be useful as radioligands, is to ascertain whether there are correlations between ligand binding affinities to those sites and persistent inactivation potencies. Nevertheless, it is clear that not all noncompetitive antagonists,

including the LA tested here as well as more classical nAChR ligands such as

mecamylamine and hexamethonium (Gentry et al., 2002) (chapter IV), are able to induce

persistent inactivation of nAChR, either in absolute terms or with potencies normalized to

their acute functional inhibitory potencies. Thus, support for the notions that actions as open channel blockers defines agents acting to produce persistent inactivation and that

persistent inactivation involves long-lasting open channel block is not derived from the current studies. Furthermore, because LA are poor inhibitors of radiolabeled agonist

binding to nAChR, it seems unlikely that those agonist binding sites are the exclusive loci

for induction of nAChR persistent inactivation. However, of potential utility in further 241 investigation of these issues are the observations made here that dimethisoquin induces persisting loss of a4*-nAChR function with higher potency at a4P4-nAChR and the previous finding that mecamyiamine is another noncompetitive antagonist that induces persistent inactivation of a4*-nAChR but with higher potency at a4P2-nAChR (Gentry et al., 2002).

Another possible caveat is that any ligand that induces persistent inactivation of nAChR does so because it is inefficiently removed from the medium bathing cells, in the immediate vicinity of nAChR under study, or from the cells themselves if the cells act as

"sinks" for ligand accumulation. Our previous study demonstrated that nicotine applied to nAChR expressed in SH-EPl cells under similar conditions is reduced by more than

10,000-fold using the same rinse method as was used in the present study. Nevertheless, antagonists with high affinity for nAChR may be more resistant to wash-out than nicotine, and residual antagonist could produce persisting block of nAChR function.

However, dimethisoquin is more potent in persistent inactivation than it is in acute block of a4P32- or a4P4-nAChR function, which can not be explained even if all dimethisoquin was retained. Furthermore, adephinine is more potent acutely than dimethisoquin, and assuming that both LA would be removed with about the same efficiency, given that the effect of pretreatment with 1 mM adephinine is equivalent to the block produced by 1 adephenine acutely, an --1000-fold reduction in LA concentration can be expected when using the rinse protocol applied in this study. Thus, 242 these observations support the hypothesis that some ligands indeed interact with nAChR to induce persistent inactivation long after bulk removal of the drugs (Lukas, 1991; Ke et al., 1998; Gentry and Lukas, 2001a; Gentry et al., 2002).

Pancuronium does not protect nAChR from persistent inactivation induced by prolonged noncompetitive antagonist exposure. The competitive antagonist, pancuronium, found previously to protect nAChR from nicotine-induced persistent inactivation, did not significantly alter concentration-response profiles of a4P2-nAChR following exposure to LA alone (Fig. 6.2). Similarly, pancuronium did not change the concentration-response profiles of a4P4-nAChR following prolonged exposure to either lidocaine or adiphenine (Fig 6.3, top and middle panels). This was not surprising in light of the weak persistent functional effects of lidocaine and adiphenine to begin. However, pancuronium did offer some protection of nAChR from dimethisoquin effects.

Interestingly, while pancuronium did not significantly protect a4P2-nAChR from the functional effects of prolonged dimethisoquin exposure, pancuronium did significantly block effects of i nM dimethisoquin 1 hour exposure at a4P4-nAChR (p < 0.05) (Fig. 6.2 and 6.3, bottom panels). Pancuronium may interact with dimethisoquin's binding site to interrupt the mechanism by which dimethisoquin induces persistent inactivation of oA^A- nAChR. Differences between pancuronium's ability to block dimethisoquin-induced persistent inactivation at a4^2- and a4P4-nAChR leads to speculation that the ^ subunit 243 may be involved with binding interaction of dimethisoquin and pancuronium that determines whether the receptor will transition into an inactivated conformational state.

Noncompetitive antagonists do not protect nAChR from nicotine-induced persistent inactivation. Results of the present study suggest that noncompetitive antagonists are less effective than competitive antagonists at blocking nicotine-induced persistent inactivation of nAChR. Previous studies found both pancuronium and alcuronium, two competitive nAChR antagonists, to protect effectively muscle-type al*- nAChR natively expressed in TE671/RD cells and ganglionic a3P4*-nAChR native in

SHSY-5Y cells from persistent inactivation induced by nicotine (Lukas, 1991).

Pancuronium was also shown to reduce functional effects observed at a4P2- and a4P4-

nAChR heterologously expressed in SH-EPl human epithelial cell line following chronic

nicotine exposure (Gentry and Lukas, 2001a). In the present study, these same nAChR

were not well protected from nicotine induced persistent inactivation by the

noncompetitive antagonists lidocaine, adiphenine, or dimethisioquin. This evidence suggests that the mechanism by which noncompetitive antagonists induce persistent

inactivation of nAChR may differ from that of agonists and/or competitive antagonists.

Noncompetitive antagonists may be acting at a unique site on nAChR to induce persistent

inactivation or stablize a close conformation of the receptor without the receptor first

being activated. 244

Conclusions. Pharmacology is unveiling some of the secrets of nAChR functional desensitization and persistent inactivation, however, it will require a large assembly of data to draw appropriate conclusions regarding mechanisms underlying this phenomenon. More evidence should be gathered to discern properties that allow some but not all antagonists to induce persistent inactivation at select nAChR subtypes. 245

CHAPTER vn

General Discussion 246

CHAPTER Vn

Summary of major findings:

The overall objective of this dissertation was to characterize functional responsiveness of CNS-type nAChR following prolonged exposure to nicotinic ligands and explore pharmacologically regulation of CNS-type nAChR by nicotinic ligands. The following outlines major fmdings from each chapter and summarizes the overall conclusions of this study:

I. Chapter Q established that ^^Rb"^ Efflux assays provide an effective means for

evaluating nicotinic receptor function following prolonged ligand exposure. It was

determined that applying nicotinic ligand by spiking cell culture media or using the

"flip plate" method yielded similar results. It was further concluded that double

normalizing the data sufficiently corrects for any reduction in loaded ^^b^ that may

occur in samples for which ligand pretreatment overlapped with ®®Rb^ loading or

recovery in drug/®'^b* free media may have depleted the amount of ^^b"^ inside the

cells prior to the functional assay. It was further shown that a rinse method

consisting of removing drug-containing media from samples via aspiration and

subsequently applying three successive rinses of 2 ml drug-free buffer per well for

one or more minutes each reduces nicotine content by greater than 10-fold. 247

Cummulatively the rinse method used reduced nicotine presence greater than

10,000-fold before acute functional assays were performed to measure function of

the nAChR. Finally, receptor function following ligand pretreatment can be

measured using an acute agonist challenge of either carbamylcholine, nicotine, or

acetylcholine.

2. Chapter HI characterized functional responses of two CNS-type nAChR subtypes

following prolonged exposure to nicotine. Pre-exposure to nicotine induces losses

in function of heterologously expressed, human a4p2- or a4p4-nAChR. Loss in

receptor function occurs following nAChR exposure to concentrations of nicotine at

or below the level of nicotine present in the brain and serum of human smokers.

Functional losses and recovery from those losses are both concentration- and time-

dependent with greater nicotine exposure causing a greater % reduction in nAChR

function which in turn requires longer time in a drug-free environment to regain

function. The expression system may play an important role in regulating nAChR

functional response following sustained nicotine exposure, because an independent

investigation of a4p2-nAChR heterologously expressed in a different cell line found

hypersensitivity of a4P2-nAChR following nicotine exposure. Persistent

inactivation of nAChR resulting from prolonged ligand exposure found in the

present study and elsewhere is speculated to be a possible mechanism of tolerance. 248

The work presented here is a strong foundation for additional experimentation and

analysis to define nAChR inactivation kinetics.

3. Chapter HI also sought to determine whether other nicotinic ligands could mimic

nicotine's effects on nAChR function. Human a4^2- and a4P4-nAChR subtypes

functional responses to prolonged carbamylcholine or mecamyiamine exposure

differ from responses following nicotine exposure. Carbamylcholine exposure

induced long lasting decline in functional responsiveness of both cell lines, however

the percentage change was less dramatic than was observed to occur following

equivalent nicotine exposure. Responsiveness of each cell line following

mecamyiamine exposure were substantially different. Mecamyiamine pretreatment

only weakly affected a4P4-nAChR yet induced persistent functional losses ranging

from 20-100% at a4p2-nAChR depending on the concentration and duration of

exposure. Data suggested chronic exposure to noncompetitve antagonists may affect

particular nAChR subtypes selectively. This finding was motivation for further

studying noncompetitive inhibitor effects on nAChR function (chapters V and VI).

4. In chapter IV, a comparison of nicotinic ligand abilities for inducing persistent

inactivation at and a4^nAChR found nAChR agonists are more likely to

induce persistent functional inactivation of the two CNS-type-nAChR than are 249

nAChR antagonists. Rank order of ligand potency for inducing persistent

inactivation was the same for both nAChR subtypes tested. Furthermore, there is a

strong positive correlation between acute functional potency and chronically

induced functional inactivation potency of agonists acting on CNS-type nAChR.

5. Mechanisms of nAChR inactivation were further explored in chapter IV using

pharmacological tools. The nAChR competitive antagonist, pancuronium, exhibits

limited ability to protect a4P2- and a4P4-nAChR from chronic nicotine or

carbamylcholine induced functional inactivation. Pancuronium did not protect

receptors from mecamylamine effects. This evidence supports that nAChR channel

opening is required for nicotine or carbamylcholine to induce persistent

inactivation, while mecamylamine is likely to induce persistent inactivation of

a4P2-nAChR via a different mechanism. Alternatively, mecamylamine has greater

affinity for a4P4-nAChR and residual mecamylamine that is not effectively

removed during post-exposure rinses could be responsible for the functional

antagonism observed.

6. In light of recent Hndings from another laboratory reporting hypersensitivity of

nAChR function following nicotine exposure, chapter IV provided a different

means for measuring chronic nicotine effects on nAChR function and clarified 250

conditions under wiiich persistent inactivation of nAChR is observed. Regardless

of acute agonist challenge used to elicit function from nAChR following nicotine

exposure, the a4P2- and a4P4-nAChR heterologously expressed in the human SH-

EPl cell line consistently exhibit a decline in functional responsiveness to agonist

challenge following prolonged nicotine exposure. Experiments analogous to those

of Buisson and Bertrand performed using SH-EPl human epithelial cell line rather

than HEK human kidney derived cell line as host to a4P2-nAChR and ^'^b^ efflux

assays to measure function rather than electrophysiology, found I hour or 8 - 10 hr

nicotine exposure led to decline in receptor function.

7. Chapter V found local anesthetics have reasonably strong abilities to inhibit diverse,

human nAChR subtypes (ai*-, 03^4*-, a4P2-, and a4P4- nAChR). With a few

exceptions, LAs act at nAChR subtypes as noncompetitive functional inhibitors. LA

function at nAChR may be well segregated based on the structural classification for

LAs proposed by Arias 1999. Group II LAs defined by a separated aromatic ring

structure were the most potent functional inhibitors at nAChR subtypes examined,

while the Group DI compound dimethisoquin, classified based on its fused aromatic

ring structure, was the next most potent functional inhibitor of nAChR. Group I

LAs, characterized by a single aromatic ring, were the least potent functional

inhibitors at nAChR. 251

8. Chapter VI measured functional responses of aA^2- and a4^-nAChR following

chronic exposure to LAs. Sustained exposure to some but not all LAs induces

lasting effects on function at a4^2- and a4P4-nAChR. Dimethisoquin, a group in

LA representative, is a potent functional inactivator of both CNS-type nAChR

examined, while LAs representative of group I and group Q exhibited little or no

ability to induce persistent functional inactivation of the aA^2- or a4P4-nAChR.

9. Chapter VI used pharmacological tools to examine mechanisms by which

noncompetitive antagonists, such as the LAs tested, produce long lasting functional

effects at nAChR. Whereas nAChR antagonist, pancuronium, was able to provide

some protection of a4P2- and a4P4-nAChR from persistent functional inactivation

induced by nicotine or carbamylcholine, additional experimentation showed that

persistent inactivation induced by group m LA dimethisoquin, characterized by a

fused ring structure and best able to induce persistent inactivation among LAs tested,

is relatively resistant to pancuronium effects.

10. Chapter VI also evaluated the abilities of noncompetitive antagonists to prevent

persistent inactivation. Combined nicotine and LA pretreatments showed that LAs,

which acutely act as noncompetitive inhibitors of nAChR, offer no protection of 252

a4P2- or a4P4-nAChR from nicotine induced persistent inactivation. Only minimal

protection from nicotine-induced persistent inactivation was observed when LAs

with mixed competitive and noncompetitive mechanisms of action were used to

block nicotine effects. These observations taken together with the observations from

chapter V regarding pancuronium effects, suggest that competitive inhibitors are

better able to protect nAChR from agonist induced persistent inactivation than are

noncompetitive inhibitors..

Conclusions of the present research. The present studies have made important advances in understanding the phenomenon of nAChR persistent inactivation. Evidence presented here supports the hypothesis that all nAChR subtypes, including CNS-type nAChR, are vulnerable to persistent inactivation induced by nicotine. A model of nAChR kinetics with multiple functionally inactivated conformational states is supported by the data presented here. Results suggest that nicotine is not alone in causing persistent inactivation of nAChR as most agonists induce long lasting functional changes at nAChR to varying degrees (chapters QI and IV). It was found that a striking postive correlation exists between acute potency of agonists and their efficacy of persistent inactivation following chronic exposure (chapter IV). This finding implies that extent of persistent inactivation that occurs following chronic agonist exposure is irrespective of factors aside from acute potency of the agonist, such as charge, lipophilicity, structural characteristics, etc. Furthermore, the observation that competitive antagonists, such as pancuronium, are 253 capable of blocking the persistent inactivation induced by agonists (chapter IV) and lack of open-channel blocking antagonists preventing persistent inactivation induced by agonists (chapter VI), suggests that agonist-induced persistent inactivation requires channel opening via agonist binding at specific sites on cell surface exposed portions of the nAChR.

While not all noncompetitive inhibitors of nAChR induced persistent inactivation, observations that mecamylamine and dimethisoquin induced persistent inactivation of nAChR implies open-channel block could be involved (chapter in and VT). Among the open-channel blockers that induce persistent inactivation, their effects were not prevented by addition of the competitive antagonist, pancuronium, suggesting these drugs induce persistent inactivation in a different way than agonists (chapter VT). Interestingly, mecamylamine has a different efficacy for inducing persistent inactivation at aA^2- nAChR than a4P4-nAChR, whereas chronic effects of dimethisoquin do not discriminate between the two receptor subtypes (chapters in and VI).

The present study focused entirely on functional changes occuring at nAChR following chronic nicotinic ligand exposure without addressing other known effects of chronic nicotine exposure such as a quantitive upregulation of nAChR binding sites

(reviewed in Gentry and Lukas, 2002a). Previous studies have supplied evidence to support the argument that the two phenomena, nAChR functional changes following chronic nicotine and nAChR quantitative changes following chronic nicotine, could be mechanistically unrelated (see review Gentry and Lukas, 2002a). The dynamic nature of 254 synapses suggests that numbers of nAChR present, alteration of nAChR function, as well as other factors are collectively responsible for behavioral effects associated with chronic nicotinic ligand exposure. Nevertheless, an increase in the number of nAChR does not increase the level of cholinergic signalling if the nAChR are functionally inactivated.

Additional work paralleling the present study in scope and detail for purposes of thoroughly evaluating nAChR upregulation and its relationship or lack of relationship to functional changes occuring after chronic nicotine exposure will be worthwhile in the future.

This work illuminated roles of the 3 subunit in persistent inactivation of nAChR.

Studying two nAChR subtypes that differ by only one subunit and all other conditions being equal revealed differences between a4P2- and a4P4-nAChR that can be attributed to the P subunit. Collectively the data suggested that antagonists may act via a different

mechanism than agonists to induce persistent inactivation. It can also be speculated that the P subunit may play an important role in regulating the functional response of nAChR

to chronic antagonist exposure. a4P2- and a4P4-nAChR subtypes responded similarly

to chronic agonist exposure, but differences were observed following chronic antagonist exposure. Data indicate that P subunit is involved with at least one mechanism by which

the nAChR studied become persistently inactivated.

Results of this study are clinically relevant as indicated. For example, it was

shown that chronic exposure to concentations of nicotine at or below the level of nicotine 255 present in the brain or serum of a smoker will have long-lasting functional effects on brain nAChR. Thus, conclusions drawn from this study may explain some behavioral effects of nicotinic ligands observed in humans and may have clinically relevant applications. Understanding CNS-type nAChR response to chronic ligand stimulation is important because CNS-type nAChR response to chronic ligand stimulation may relate to nicotine dependence and withdrawal observed in cigarette smokers and other tobacco product users. For example, the evidence found here is consistent with the hypothesis that nicotine dependent individuals crave additional nicotine as a means for stablizing the inactivate conformation of nAChR rather than stimulating nAChR. Applications of such knowledge include the development of improved smoking cessation therapies. Because long-term effects of drugs may differ from their acute effects as seen here, nAChR regulation following sustained exposure to nicotinic ligands should be considered in the development of therapeutics that may be chronically administered to patients. 256

APPENDIX A Volume 1, Number 4, August. 2002 ISSN; 1568 - 007X Current Drug Targets

CNS cind Neurological Disorders

BENTHAM SCIENCE PUBUSHERS 258

On July 1, 2002, Bentham Science Publishers' manager of publications, Saima

Rao, granted permission to reprint the following review article:

Gentry, CL and RJ Lukas. 2002 "Regulation of nicotinic acetylcholine numbers and junction by chronic nicotine exposure." Current Drug Targets - CNS & Neurological Disorders. Bentham Publishers Ltd.

Bentham Science Publishers have consented to the inclusion of the above named journal article in the appendix of Cynthia Gentry's dissertation entitled, "Effects of

Chronic Ligand Exposure on Nicotinic Acetylcholine Receptors." The dissertation partially fulfills requirements for the degree of Doctor of Philosophy at the University of

Arizona. The permission extends to publication and/or microfilming of the above named journal article within the dissertation by the university-affiliated publisher for scholarly use. University-affliated publishers will be granted the non-exclusive right to reproduce and distribute the dissertation in whole or in part, in and from microform and may supply copies on demand. One copy of the dissertation will be deposited with the Library of

Congress. These rights in no way restrict republication of the material by Bentham

Publishers Ltd. Permission granted acicnowledges that Bentham Publishers, Ltd. owns the copyright to the above-described journal article.

PERMISSION GRANTED FOR THE USE REQUESTED ABOVE.

Company Name: Bentham Science Publishers

By: Saima Rao

Title: Manager of Publications

Date: July 1, 2002 259

Cmmml Dnf Trngta - QVJ A StwUglctl DbsHtn.imo. /. 35»-3M 3S9 Regulation of Nicotinic Acetylclioline Receptor Numbers and Function by Chronic Nicotine Exposure

C.L. Gentry and R.J. Lukas*

Division of Neurobiology, Barrow Neurological Insiituie. Phoenix. Arizona, USA

Abilract: Rccenl tdvanccs concerning cflects of chronic nicotine exposure on nicotinic acetylcholine receptor (nAChR) expression are reviewed. Implications are assessed of thw findings for roles of nAChR in health and disease and for design of drugs for treatment of neurological and psychiatric disorders. Most studies continue to show that chronic nicotine exposure induces increases in numbers of nAChR-like binding or antigenic sites C^ipregulalion') across all nAChR subtypes investigated, but with time- and dose- dependencies and magnitudes for these effects that are unique to subsets of nAChR subtypes. These elTects appear to be post-transcriptionally based, but mechanisms involved remain obscure. With notable exceptions, most studies also show that chronic nicotine exposure induces several phases of nAChR functional loss ("desensitization" and longer-lasting "persistent inactivation'*) assessed in response to acute nicotinic agonist challenges. Times for onset and recovery and dose^dependencies for nicotine-induced functional loss also are nAChR subtype-specific. Some findings suggest that upregulation and functional' loss are not causally- or meclianistically-relatcd. It is suggested that uptegutatkm is luM as physiologically significant m vivo as functional effects of chronic nicotine exposure. By contrast, brain levels of nicotine iri tobacco users. and perhaps levels of acetylcholine in the extracellular space, clearly are in the range that would alter the balance between nAChR in functionally ready or inactivated states. Further work is warranted to illuminate how effects of chronic nicotinic ligand exposure are integrated across nAChR subtypes and the neuronal circuits and chemical signaling pathways that they service to produce nicotine dependence and/or therapeutic benefit. I. INTRODUCTION AND BACKGROUND brain homogenates Cupregulation'^ after chronic nicotine exposure in vivo or with smoking [12]. Convenlional Many advances in knowledge of diversity, structure and wisdom at the time said that chronic agonist exposure function of the Tamily or nicotinic acetylcholine recepiois should induce downregulation in numbers of fimctional (nAChR) have occurred during the past decade [t-4|. receptors, whereas chronic exposure lo antagonists would Insights have been gained into local izaiions of diverse indim upregulation, perhaps through a process mimicking iiAQtR subtypes, their associations with nervous system the phenomenon of ''dentation supersensitivity." The fuiKtion in health and disease, and design of nAChR conventional effects of chronic agonist or antagonist subcypc-selcctive ilnig9 [S-9). Chronic' exposure to nicotiite exposure clearly applied to metabotropic receptors, such as occufs during tobacco use. and the iiKidencc of chronic muscarinic acetylcholine receptors. However, chronic exposure to mcotinic ligands is expected lo increase as they exposure to nicotine or other nicotinic agonists iiKluding are applied to treat neurological and psychiatric disorders anabasine, (-f)analoxin-a, and cytisine produced increases in [10, II]. Consequently, interest in efTects of chronic nAChR radioligand binding Bums levels without altering nicotinic ligand exposure on numbers and function of radioligand binding affinities (Kp values), and these effects diverse nAChR subtypes has grown. Here, relying on were specific (e.g., muscarinic receptor numbers were not rererence to previous reviews for more detailed discussion altered following chronic nicotinic agonist exposures) [12]. and compfchcnsive listings of the earlier literattire and on a Effects of long-term antagonist treatment on nAChR distillation of selected, reccnt findittgs presented here in numbers had nol been well si^ied at the time. tabular form', we provide an update on major, recent findings concerning effects of ciirotiic nicotine expostve on At the time of Wonnacott's review [12], some studies of nAChR and assess the implications for roles of nAChR and chronic nicotine exposure in vrvo already had shown losses ligands targeting them in health and disease. of fimctional responses to acute nicotinic agimist challenges that persisted for sometimes extended periods after the end Wonnacott's timely review confronted the apparent of chronic nicotine exposm (reviewed and refaenced in [13, paradox of the iiKrease in numbers of itAChR-like 14]). Nicotine "tolerance" (the need to increase drug dose to radioligand binding sites in rodent or post-mortem human achieve tlie same acute effect after chronic tieatment with that drug) based on a variety of behavioral snd physiological meastces was a long-recognized phenomenon consistent with *A4drm cattopaadcncc lo ddt Mdnr *. the Divisioa of Ncurobiokity. chronic nicotine-induced loss of nAChR fimction. Some in Baimit Neuralogicai ImiMie. 150 West Thomas Roail. Phocaix. Arizoia vitro studies also suggested that prolonged nicotine exposure ISOtJ USA; Voicc - 602-406-)399: Fix: »|.«02-406-4172; induced persisting losses (lasting minutes-hours) in nAChR E-Mak [email protected] fimction beyond Ifutse associated with the process of nAChR I9ia4t7XA2S35.M4-.M O MU •ealtkaa Scinct PaMUtn LM. 260

3M CmnmlttnmTmt^-CNS*MimnlitkalD^m*n 2tn.Vttl,l 4 Cnrntmrndlatm "deseasilizaiioii," whicli ia > tnosicnl (revenible within 2. EFFECTS OF CHRONIC NICOTINIC LICAND seconds-minutcs) loss of nAChR function during brief EXPOSURE ON NUMBERS OF aAChR (seeonds-minutes) nicotinic agonist exposure (findings reviewed and referenced in [13, 14]). This compounded the 2.1 Changes ia aAChR Nambers in Respoasc to apparent parados, in that both nAOiR radioli^nd binding Chraaic Nlcatiae Eipasart im VUro site upregiilaliaa and functional inactivalioQ seemed to occur with chronic nkotine exposure. Kellar and associates' [IS] Studies using Xenopus oocyte or mammalian cell introduction of the conc^ of nicotine as a "time-averaged heterologous expression systems or using mammalian antagonist" provided a possible solution to the conundrum primaiy or clonal cells naturally expressing different nAChR of nicotine-induced nAChR binding site upregulation. subtypes continue to demonstrate upregulation of nAChR to However, the dissociation between chronic nicotine effects varying degrees in response to chmic nicotine treatment on "numbeis" and fimctian was in sharp contrast to parallel (results fnm selected studies are summarized in Table I). chronic effects of drugs on numben and fimction of Generalizations an difficult to make in part because of the metabotropic receptors. Debate began whether short- or wide variety of model systems, nAChR subtypes, assays, longer-term nAChR functional inactivation and/or and nicotine exposure conditions used. Nevertheless, there upregulation was a mechanism for the productioa of nicotine are no clear differences in effects across species or across toletance. Furthermore, the question arose as to whether heterologous or natural expressim systems for a given nAChR binding site upreguMon could be a response to nAChR subtype. Half-times for iqxegulation or for recoveiy nAChR functianal inactivatian: a call to the cell (or to the from it are usually on the order of one day. Nicotine nucleus) that "we no longer have respcmses to nicotine or concentrations needed for half-maximal upregulation and acetylcholine, so please make more recepton.** magnitudes of upregulation are not always precisely determined because levels of nAC^hR as measured sometimes A later review [14] summarized progress canfirming that continue lo climb up to the highest dose tested. In general, chronic nicotinic exposure induced increases in numbers of however, upregulation EC50 values for nicoline are in the nAChR radioligand binding sites not only ia brain, but high |iM range for at*-, a3*-. and a7-nAChR. but are in also, for example, in cells naturally (N) or heterologously the high nM range for a4*-nAChR. Increases in al*- (H) expressing a4P2-. a3P4*-, a I*-, or a7-nAChR (* nAChR are 2-S-fold and in a4*- or a7*-nAChR are about nomenclature indicates nAChR subtypes lcna%m to contain 2-fold, whereas increases in a3*-nAChR vary quite widely the indicated plus other subtwitt [7]). Based on work done perhaps dependent on the proportion of those containing fi2 to that time, the following proposals wen made: (highly seiuitive lo upregulation) or P4 (resistant to upregulation) subunits. In studies assessing such effects, • Chronic nicotine exposure induces an increase in there is a transient downregulation of surface, naturally- nicotimc radioligand binding sites (upregulation) for expressed al*- or a7-nAChR shortly afler exposure to 1 all nAChR subtypes. mM nicotine, and there is only a modest increase at longer • Upregulation of nicotinic radioligand binding sites limes of nicoline exposure, consistent with a dominant eflMt involves post-lranscriptional mechanisms, because on intracellular pools of assayable al*- or a7-nAChR. subunit mRNA levels are not significantly changed However, surfm levels of a3P2-nAChR (but not 0304- by chronic nicoline exposufc [16-19). nAChR) or a4P2-nAChR more closely parallel levels of total pools following chronic nicotine ncalment. • Doses and durations of nicotine exposure lequired to induce nAChR binding site upregulation and the magnitude of upregulation aro different across native 2.2 Changes ia aAChR Numbers ia Response ta or heterologpusly expressed nAChR subtypes. Chraaie Nieadae Expaaare Ai Khw • Chronic nicotine exposure also induces a decrease in Consistett with and extending knowledge at the time of nAChR firactional responsiveness to subsequent Wonnacott's review [12], studies of effects of chronic nicotinic agonist application. nicoline exposure in vivo oa numbers of nAChR-like radioligand binding or antigenic sites typically show • Agonist-induced nAChR functional loss occurs by increases typically of about SOK in rodent (results &om two distinct (operationally-defined) processes: a selected studies are summarized in Table 2). Rales for onset comparatively npid ia onset and rapiiUy reversible and recovery (nxn upregulation are not frequently reported, 'desensitizalioa'' that recovers afler S min of diug but ihey seem lo be on the order of a week in rodent studies removal, and a more slow to develop and slow to (7 days is the shoitest exposure lime listed in Table 2). recover "persistent inactivation" functional loss Higher magnitude changes tave been reported in some of thie requiring more than S min for recovety. hunuui studies (nAChR sites in brain or blood cells; see • Kinetics and doses of nicotiiic required to inducc Table 2), perhaps reflecting a longer-lerm exposure thM in peisislem inactivatian and magnitudes of those effects rodent studies. Few dose studies have been done, but these differ across nAChR subtypes. suggest that maximal effects have not been reached at ihe highest nicotine doses used in rodent studies [20]. • Chronic nicotinic agonist-induced persistent Microdissection studies are consistent with more fimctional inactivation nd numerical upregulation of comprehensive receptor binding autoradiopaphic analyses nAChR Ac mrrh«ntnir«lly- mnA lhal show brain region variations in levels of upregulation 261

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0402-7 Mik SyniM-OtwIty iM ('H1ACk.Ctai I]0-I40 10 H|aico M drnigf 39di,0.4|A«i.cAii 117] a4|l2-7 Fmule Spitfoe-Dtwity iM ('Hhiico . T 9 4i,0.Serl.«awnif2/ita (•1) a7-7 I-Br no chMgc a4(l2-7 Ral ('HlACKHCmonti 140 14 lii 0.45ni^( •.€. 2/4i (Ml MB 150 a4|l2-7 Mtic SpraiiK-DiwIcy nl ('HKiieo 130 lldi.0.45ntAi|2/ib(l5kr) (521 a4p2-7 Mil* Sfngut-Diwlty iM (>H)iitc«.STntiiib MO 1 di 1J •«. iahiin (0 nk) (46] 210 S di 1J ifitAf^ditx. kflnioa (0 ak) 120 14 di I.S mi/ki/di ic. kllMiM (0 mk) 0402-7 M«l* Sprague-Onrky m I'hjucc IM) lOdi, 0J9iii(/kf (3,6|Unol/k()i.c. 2/di (19) (241.) IM (4di) 0402-7 Mih uid Fcmile Win* nl I'HJACh 120 l< di. 0.45 nf/kf i.c. 2/di (12 hr) (901 I'mieo l» FCIMIC Spnguc-Dntlcy nl ['H)nko,CR -220 7 di 0.1 ni/kflir com kfiuion (0 di) 7.7 di ("1 MB >160 4.6 di HB -190 7.1 di HC -230 10 di ST -170 4.0 di HY -210 4.1 dt 07-7 1-Bft.Cei -ISO 2.2 di MB -120 1.9 di HB -150 5.5 di HC -IM 4.0 di ST -420 2.2 di HY -IM <2di 11402- Mil* SpntM-Dawtoy m 130-ISO I0di.2.0nt/l«2/di(lllv) (911 Uiiva4«d02AM | MCM wVVHHi ••• Amtf SafCMtral* CaadMeM! Eipaaatv (Ricave^) ECH Oatctt Reeavify t Her. SOTiypf»-• 04{l2-7 Hit |'H)nico, Cti marib ISO 14 da, 4 aig/hg^di coal laflnhNi (921 a7-? Cti mmb no chMfc a4p]-7 Mile Spnguc-Dtwlty m (>H)iiico,Cli,IIC.MB. ISO II da. 0.4S mg kg Le. 2/da (II hr) (931 STmmk (>H)aieo.CBiM(nb ae change a4{U-T Ral ['H]ako, CH acuroM 110 {onioflMfiHc agplic. (941 a4p2-7 Milt Spfffue-Ontlcy iM ['H)iiica, Cn incmb 140 13 «4a, noae ei|NMUR w matniutam t'JI ST IW ciganite moke CB 130 TH no cbaage HC no cbange 04P2-7 Rai ['H]aico Clx imnh IIO-IM IOda,2m^2/dB (Ml HCaionb 100-140 a4-7 Mate Spn|it(-D*«>l(y nt |'ll)cyt, memb 110-100 IS da, 0.( mglii i.cyda (-) <20 di (97) (J mo) a4-7 Funic Spnint-Diwlty ncmb no change IS da, 0.< Bg^g txMt (-) (97J a4-7 Mile Spnfue-Dniiy rd ('H]cyioc('H)cpi.Cei. 100-150 IOd8,0.7g/lr|2/da(l2M (91) a4(>2-? a4.7 Mile SpcifM-Dntlcy m f'HJcyt 140 10 da, 2.4 aifltg/da 2/da (20) 120 10 da, 2.4 ing.h|Mi coal iaIlMiaa a4|l2-7 Mile mt6 Finitlt Spngw- (>H)iiee.C«x 120 1 da, 0.1 n^i i.e. 2/di H|ACh.Cli as dua|f l< di, 0.45 mf/kg ic. 2/di (12 If) (90) ('H)iko.Clii asekanie a4(l2-7 Mile Papio hiiuAyM ('"l]Mi>-A.I5]WSPEa -l«0 15 di, 2 mg/kg/krcoflt filWon (7di) rioi} baboon (ValdiNr.),CB,CR,TH a4P2-7 Hiimin poilBiortciii I'HInicoHC 202 •moken 7-20 cig/da, >]0 yean (102) HCCR 215 CB IM median laplie 149 nedutta 104 a4{l2-7 Human [mliiioiHin [%)epi, Cei manb 250-)00 niioken >20 cig/da (0 hr) (lOJ) aipi-l ('HJeyt CH memb 250-JOO a402-7 ('H]epi. CS mMond 340 1 8

a4(l2-7 ['H)epi, HC Miond Is a4P2-7 Mil* md Female huaim (>H|aieoHC 210 l.<2 pk/da nnokcn fiw) po0tfnoftcfii TH tM ('H]aieo. HC ao chmie 0.17 pk/da iiBokefi (>2 montfi) TH no chiBie a4P2-7 Hinnaa (polymeipho- |'H]nica 220 20 cig/di imaken lyr (105) nuelw celU) MMHd in Wm n^ilimul iiKnici nMiif Is chM|n ki MMkai of aACkK-Uk* MaMi| • Miink lim MMaf dtmk akariM iiiiuwi h wMliiMA CmifiritMt cm kt mda mcmi pmmmi impM ikupii (NL— 1). nlml NWM (caliiMi 2X wiir lypt iMd Is witnn nnptw ckMfn mi knki MLW>—>LI piipiniluii (IUIHMI I), ikmi ki kliilag tilt miMkin npiiinM M H of cmml (MkMM 4, ain«iiiii) kmm akMiat afaun mi m-ir (uhmt S). co«c«a»tila« af afcalkw piiliiaaaial taidiat M kalf-aiaakMl Hten tim colama ) tDallHiai (calann «). Mf-tkm Ite OMM af aACkl(-«kf kki4k« tim |iiilll) dMHa tl*« »)«<• > caiMaat (catami T). mi kalf^kaa fm nm af aACkK-llka bki4iat iMa aaa*m lo po-akoiiM nyeoon Imli (colaan I). Coloiaa * cooMh cwiupwrfkn nftitata kiftnaMlna by mw for du ll«o4. 268

3M CmnmDntTmgm'CNSANtimhtkalDlitt^m Itl2,VaL l,Ni.4 Cttm/amJLiiMm (with some showing no change; [16, 21)). lUdiolabeled requirementi for nAChR functional stimulation to induce nicotine, acetylcholine (ACh). cylisine, or epibatidine upreguiation.i However, perhaps the mechanism involved is binding assays employed are thought to predominantly differenti for agonists or antagonists, and shuttling of identify a4p2-iiAChR in studies of rodent or human brain, nAChRi to a fimctionally inactivated state by antagonists or and radiolabeled a-bungarotoxin binding seems to identify chronici agonist exposure (desensitization) could still be a a7-nAChR, but more work is needed to assess eflects on commoni outcome for agonist or antagonist treatment. a3*- or a6*-nAChR. There is no consensus about gender Similarly, blockade of upreguiation by antagonists could be diflerences in sensitivity to upreguiation, but older animals takeni as evidence for a requirement for nAChR activation in seem to be more resistant than younger ones to upregulatory induction of upreguiation. However, it could be argued that efTects of chronic nicotine exposure, which when initiated in conversion of nAChR to a functionally inactive slate also younger animals promotes preservation in some brain would be prevented by antagonists. We suggest that much regions of nAChR that otheiwise undergo age-dependent more work is needed with attention to permeability of decline [22]. ligands and acute mechanism of action of antagonists (competitive, non-competitive) and especially to expression system and experimental details before agonist and Z.3 luighta int* Mcchaaiaas lavolved in Nlcoliae- antagonist studies present a coherent view of how they aflect ladMccd aAChR Upregiilalioa nAChR numbers. Mimicry of nicotine-induced upreguiation of nAChR The consensus that nAChR radioligand binding site after chronic treatment in vitro is reported for a number of upreguiation in vivo or in vitro occurs without significant or nicotinic agonists whether they carry a charge or not substantial changes in nAChR subunit mRNA levels (earlier (selected findings are summarized in Table 3). The work reviewed in [14] has been extended [19, 28, 29] magnitude of upreguiation in some studies is reported not to although increases on the order or S0% or less would be very be as high as for nicotine exposure, but not always difficult to discrimirute becaose of limits in resolution of consistently so for a specific drug at a specific nAChR Northern, in situ hybridization, or even reverse transcription- subtype. Agreement across studies is even less consistent polymerase chain reaction analyses. It was postulated that with regard to abilities of antagonists to mimic or block the post-ttanscriptional pmess contributing to upreguiation nicotine-induced upreguiation (Table 3). It remains to be of ['H]nicoiine binding sites in MIO mouse fibroblast cells determined whether these inconsistencies in effects in vitro hcterologously expressing chick a492-nAChR is a decline reflect diflisences in expression system, experimental design, in receptor turnover [17]. However, no difTerence in turnover or perhaps species of origin for nAChR subtypes. was found in other studies evaluating MIO cell a4P2- nAChR nonover from control and fhm nicotine-upregulMed Mimicry of nicotine-induced upreguiation after in vivo levels, and. depending on the nicotine dose, results could be treatment is reported for nicolinic agonists cytisine [23], (-•-)- obtained showing that the rate of nAChR degradation firom anatoxin-a [I2|, anabasine [24], or methylcarbamylcholine control levels of expression could be partially or fully (MCQ [25], but not for lobeline [24], Whereas chronic balanced or overcome by the rate of nicotine-induced infiision with the nicotinic antagonist mecamylamine upreguiation of nAChR-like binding sites [IS]. Thus, induces increases in [^H]nicotine binding sites in mouse p^cularly given the evidence for a dominant effect of brain [19,26], chronic injections of mecamylamine in the rat nicotine exposure on an increase in internal pools of do not alter numbers of brain [^H]ACh binding sites [23]. nAChR-like radioligand binding sites (Table I), it is Neither of these studies found that chronic mecamylamine hypothesized that nicotine induces upreguiation because it could prevent nicotine-induced upreguiation, but additive enters the cell and facilitates assembly of nAChR subunits effects of mecamylamine and nicotine were observed [26, to the point where they can engage in radioligand binding. 27]. Such a postulate may not hold for all nAChR subtypes, however, because upreguiation in rat cortical neurons of a7- Because a consensus is yet to emerge about effects of nAChR does not occur if there is inhibition of protein agonists and antagonists on numbers of nAChR-like sites, synthesis or glycosylation [30]. caution is warramed in making extrapolations from these studies. However, at least in principle, clues as to mechanisms involvol in nicotine-induced upreguiation can 2.4 Perspectives M Vprcgiilation be drawn ftom these studies based on logics arguments, if one assumes that mechanisms of nAChR regulation are the Several factors complicate interpretation of effects of same for exposure to these diverse ligands. For example, it chronic nicotine exposure in vivo and in vitro on nAChR can be argwed that the abilify of membrane-impeimeable, numbers. The descriptive, qualitative, term "upreguiation" quaternary ammonium agonists, such as carbamylcholine characterizes all of these effects, but magnitudes of and DMPP, to induce upreguiation of nAChR suggests that upreguiation differ across nAChR subtypes. Doses of ligand access to the intracellular space is not required for nicotine (or other nicotinic ligands) needed to induce upreguiation. However, more close attention to the upreguiation differ across subtypes. The kinetics of expression system and experimental conditions used as well upreguiation also seem to be unique for each subtype. as to majpiitvde and potencies of agonists in inducing upreguiation seems warranted. It also can be argued that if Comparisons of findings obtained in studies of antagonists induce upreguiation. then there would be no heterologously-expressed tiAChR in oocytes or in transfwted TaMt 3. CirMtt af Ckraalc Nkattalc Lliiad TrMlacat ImVltnm Nmbcn araACbll-Uk* lla4Mif*a4 Madlai ar Aali|calc SKcf

•ACkR Eapnalia EifnalH A|mMI AgnlitiWct AatagnlHi Aa

o3*. Nboviac b idnMl * d-TC DHPE (IM) cknmctlb mcci he» Nhunuii h SH-SY5Y d-TC d-TC I"1 DHPE OH0E mcca mica NhuaiM h SH-SY5Y epibttidiM (29) NhwiiM hsH-sysY GTS-21 l"l Ii432- Hckidi mMIO cirb mcci d-TC d-TC (•7) cytifine DMrr H chick mMiO d.TC hcxa (107) DtipE heia H human h K-177 (HEK 'cpibctidiM d-TC mcci d-TC (JJl 293) cytiiinc DHPE mcci DMTT MLA ABT-4II A-R5)(0 H chick mMIO cpibtlidiac AMP (29J DMCC -ABT-4II -MCC Hcbicfc mMIO •cpibMidine •OHPE d-TC d-TC DHpe (7«I cytifiiM •MLA meca MLA •aatmlii-A dcca mcci ABT-4II hcM dcei TMA bcxi •MCC a4P2-7 N nl rCuncwaat DHPE DHPE 177) mcca mcei ••niiilm ikMtoUf nWt ninttlntii). Cahmm10hiBriiii »»«ini)i afI* KfMi •pacifM a7- II tt&m mmimiMl mi4 S^NIB ttfnmhm N human H human N human mt> iiMc Nitl nACkll nt^pu (nUm akatiM (caKaiiM nUtact nMiif»rtn|W itmti hr4m rHCnnmm hSH-SVSy hSH.SV5V hHEK29) Kipmlaa Syitaa I). lACk*ipniu tm4 1,mfofiotly),•MafoaM*ikM 'AMiriiki I*cataani4ml» 'cpibilidiac laitaiin-A MhiricUH NkaMaa* •OTS-21 h ii—iiiiat«ACMI-NtciWigH|«id OTS-}l AfanMi cytliine •DMFT OMIT m4 npKHiMi Afnlia W»l MInrfridnt lyini (cahNiiM NItattot MkaM *««iAai mthn aM MaMif maMifaiic I ciftM* afMacUai AniHMiMi MfankUaC Nkadaa* •MLA fntmea i^ifilaiteakalwM •4-TC MLA i|i«lm 4m< aittt bllnttalckraife akaXac lafcctln iolta MloiMcakatlmMwW AMafaaMiNat NkaHat DHPE d-TC mfci a a » utmaiially lamtHktcyitaaakatlai. • af aACkR-likt npamt Antafaakto Nkariaa MMltiiis M wiMi mcca d-TC HikMm lilt tffima (cohmn RkotMc Iipii4.ConiyirimiCM AntafaaMiNal apRfalMlM (calaiMU NIcatiac MLA 4 nrf S, riif«cll»tly(. I Mil, 175) IW) ("I f3Jl Raf. k« 271

ttgidrnkH afSkMUc Att^Uftrnt tttfmr Ci MDrmTmgtm-CNSANnnlitinlDimrJm 2tU.yUI,N^4 371 mammaliin celb and of native nAChR in cultured neurons levels at active brain synapses are like those at the active seem reasonable given the relatively slow kinetics of neuromuscular jimction, thm doses of ACh on the order of I upregulatory effects and the wide range of manipulations of mM are achieved subsyiuptically during cholinergic fiber drug dose and freatment time that can be applied in such stimulation, which ensures activation of most nAChR studies. Moieover, whether a particular nAChR sid)type is of subtypes given that their EC50 values for ACh stimulation avian, rodent, or primate derivation seems not to matter. of fiiMtion is 10-100 |iM. It has been accepted for many However, there is a gap in relating findings obtained using years that nAChR "desensitize" following a brief exposure to cell systems in vitro to effects of nicotinic (igand treatment nicotine [31]. nAChR desensitization is thought to be a in vivo on nAChR numbers. Studies of brain nAChR in vivo mechanism for protecting systems &am overstimulatioa that or in primary cultures concern post-mitotic cells expressing a renders the receptor no longer responsive to subse«|uent clearly neuronal environment, whereas oocyte or mammalian sthnuli and is thought to involve conversion of nAChR to a fibroblast, epithelial, or other cell hosts for recombinant state with higher aflinity binding for agonist (<-l |iM for nAChR expression present a diverse range in terms of ACh). The fU-safe mechanism of nAChR desensitization is mitotic activity and environmental context. not thought to be utilized often in the exogenous drug-free brain, because the enzyme that degrades ACh Also confounding comparisons is the practical limitation (acetylcholinesterase; AChE) has a very high catalytic rate in the dose of nicotinic ligand exposure that is feasible for and is found in high concentrations in extracellular space at studies in vivo. Compounding this problem are cholinergic nerve endings. Pesticides and bioloycal warlive pharmacokinetic uncertainties about the concentrations of agents that inactivate AChE lead to desensitization of total, free, and protein-bound or intracellularly-sequestered nAChR and, for example, respiratory compromise. On the nicotine in cerebrospinal fluid and brain parenchyma. This other hand, neurologi^ disorders that involve deficits in uncertainty still applies to levels of brain nicotine in human ACh release and/or nAChR numbers can be treated with tobacco users. reversible AChE blockers so th^ ACh at the synapse can There also is a lingering uncertainty about just what is increase to effective levels. However, despite the actions of being measured by nAChR radioligand binding assays. AChE, levels of ACh in the rat brain extracellular space There is a need for many more protein chemistry and based on microdialysis measurements are about S nM and in ioununoassay studies to monitor efTects of nicotinic ligands human cerebrospinal fluid or hippocampal microdialysale on nAChR assembly states and processing to the cell range between 33 and 300 nM [32]. Thm levels are well surface. None of the assays conducted to date have below those needed to activate nAChR but could produce determined how internal pools of partially- or fully- conversion of at least a proportion of functional nAChR to assembled nAChR and cell surface i\AChR ate affected by "desensitized" sutes. nicotine action in vivo. Studies using cell lines and membrane-permeable radioprobes such as nicotine and A somewhat different situation occurs in the presence of epibatidine need to be designed very carefully because of nicotine, which is not degraded by enzymes present in access of the probes to internal as well as cell surface pools extracellular space. Rates of nicotine metabolism and of nAChR. behavioral doses of nicotine (1-3 mg/~48 kg human * -100- 300 nM; OJ-1.2 mg/kg for --0.4 kg rat » -2-7 |iM; l-S It seems evident that levels of upregulation of nicotinic mg/kg for -80 g mouse • -6-30 )iM) arc (|uite different radioligand binding sites in brain aflCT drug exposure in vivo across species. However, brain concentrations rather than arc smaller dian many measures of upregulation magnitude m^g d^ or even plasma leveb probably are most relevant in vim, but to wliat extent is this due to the inability to for behavioral responses to smoked, infused, or injected achieve the rather high concentrations of ligand needed to nicotine. Brain levels in rats are -3-foht higher than plasma induce full upregulation? On the other hMd, are effects levels (9-86 nM in plasma, 26-189 nM in brain for constant obtained in vitro afler treatment with high concentrations of infusion at 0.6-4.8 m^^^y; peak plasma level of 3 J )iM nicotinic liganb of physiological significance, and how well and peak brain level of 7 |iM for twice-daily injections of do diese effects at the exuene model eflects and mechanisms 1.2 mg/kg. but typical brain/plasma ratio of 3.1; half-times of action operant in vivo? Given the lower magnitude of in brain or plasma of just under I hr) [20]; Rowell and Li. upregulatoiy effects in vivo, are small changes in nAChR 1997). Plasma levels of about 4 |iM are achieved in mice subunit mRNA levels tliat cannot accoimt for effects following chronic nicotine mfimon at 4 m^k^hr, and peak obsen^ in vitro now in range to explain upregulation as a levels after hourly injections of 4 mg/kg are -6 |iM [32a]. tivisGriptianal process after all? What percoitage changie in TOIMCCO users typically have steady-state plasma nicotine nAChR subunit message, protein, binding site, and cell levels of 100-300 nM, and their brain levels would be OJ-1 surftce levels after chranic ligand exposure is neaM to have |iM at steady state if brain/plasma ratios are like those in physiolagiGal significance? Is upiegulation of nAChR radio­ rats. Thus, a proportion of some nAChR with nicotine ECso ligand binding sites or protein of fimctiaaal relevance at all? values of-1-3 |iM (e-g.. a4P2-nAChR) might be constantly stimulated at steaiiy-state nicotine levels in a smoker 3. EFFECTS OF CHRONIC NICOTINIC LIGAND whereas a3*-. a7- and al*-nAChR with nicotine ECso EXPOSURE ON NACHR FUNCTION values of -30 |iM. -10 |iM, and -100 |iM. respectively, would not However, given that nicotine binding affinities 3.1 General Backgraud for nAChR in desensitized states are estimated to lie between 10 nM and I |iM. a prediction is that a much higher Agonist binding to nAChR results in a rapid and proportion of each nAChR subtype would lie converted to transient opening of a transmembrane ion channel. If ACh 272

371 CmmuDngTmttu-CNStfftmaltgkalDiur^tn UIJ,VU. I,N»^4 CnMVMtflata desensitized states in the presence of smoker leveSs of nAChRi [30]), and the 2-6-fold increase in numbers of nicotine. membrane-toundi a7-nAChR-like l-Bgt binding sites in nicotine-treatedI cells (surface a7-nAChR were not In Wonnacott's review, the statement was made, "it is determined)i is equal to or higher than the -2-fold increase in not at all obvious whether chronic treatment with a nicotinic a7-nAChRi fiinction observed under the same conditions cholinergic agonist will produce enhanced, diminished or (33|.j Thus, there is a major difference between these studies unchanged respansiveness* [12]. Today, despite a substantial showing no or only a modest change in a7-nAChR function increase in the volume of evidence indicating that nAChR with chronic nicotine exposure and studies using the oocyte become functionally inactivated following prolonged expression system (Table 4) showing profound loss of a7- exposure to nicotinic ligand, some recent evideiice has te- nAChR function. Studies using HEK 293-derived K-177 ignited debate as to whether long-term administration of cells heterologously expressing human a4P2-nAChR also nicotinic agonists leads to hypersensitivity or stand out because they show potentiation of fiinction upon desensitization/persistent inactivation of all nAChR chronic exposure to 100 nM-l )iM nicotine [34-36], again subtypes. contrasting with studies done using the oocyte expression system or another mammalian cell host (Table 4). 3.2 Changes in nAChR Function Following Chronic Ligand Exposure in Kfro 3J Changes in nAChR Function Following Chronie Ugaad Esposnrc /it yivo Seminal studies suggested that muscle-type and ganglionic nAChR subtypes undergo more than one phase of Effects of acute or more chronic nicotine exposure in vivo functional inactivation upon sustained exposure to nicotinic on animal behavioral or physiological measures (e.g., heart agonists (reviewed in [U]). Most of the more itceni studies and respiration rates, locomotor activity, cognitive also indicate that chronic nicotine exposure induces perfonnance) and how mote chronic nicotine exposure aflixts functional losses for nAChR heterologously- or naturally- subsequent responses to actde nicotine challenge are reported expressed in cells or naturally expressed in brain tissue widely in the literature and will not be discussed in detail samples pretreated and challenged with nicotine in vitro here (see [37, 38]). Briefly, evidence can be found for (selected findings are summarized in Table 4). When sensitization (suggesting nAChR potentiation?) or for evaluated at a auflicient level of detail, magnitudes of attenuation (suggesting nAChR inactivation?) of responses nAChR functional loss show complex interdependence on to acute nicotine after chronic nicotine treatment. Among the lime of nicotine exposure, pretreatment concentration, and measures showing reduced sensitivity to acute nicotine are time for recovery l^m nicotine pretreatment. Rates of onset mouse (or, in some cases, rat) locomotor, respiration, heart of functional loss also show dependence on pretreatment rate, bo^ temperature, and startle responses (37,39-44], and concentration, and rates of recovery vary for diffin'ent these effects show nicotine dose-dependeitce and take days to pretreatment conditions yet often are described by single dissipate. By contrast, other studies of rat locomotor activity exponentials, suggesting that nAChR are converted to a [38, 45-47] found evideiKe that chronic nicotine exposure series of inactivated states recovery from which does not causes a sensitization to acute nicotine challenge. require transit through inlnmediates. For extended exposuie to nicotine in the 100-300 nM (human smoker plasma level) Other studies have been done on effects of chronic range with no substantial recovery time in drug-free nicotine exposure in vivo on biochemical measures of acute medium, losses of function based on most studies would be responses to nicotine challenge in rat, mouse or guinea pig < 10% for al*-nAChR, -50% for a3fl2-nAChR. -10-50% brain (see Table 5 for findings from selected studies). Just as for a334-nAChR. -50-100% for a402-nAChR. -20-50% for investigations involving behavioral and physiological for a4^nAChR, and -50-100% for a7-nAChR (for oocyte measures, there are many variables in these studies, expression of a7 subunits). Half-times for recoveiy in drug- including age, gender, species and strain, mechanism of frw media after exposure lo 100-300 nM nicotine are not chronic nicotine delivery in vivo (constant infusion, periodic reported for every nAChR subtype, but are a few minutes for injection, drinking water), dose (plasma and brain) and time a3*-nAChR and ate on the ordw of hours for a4*-nAChR of nicotine pretreatment. time of dnig-lm recovery (both in expressed in oocytes but on the order of min for a4*- vivo and ex vcvo, as, for example, in processing of tissues to nAChR expressed in mammalian cells. Magnitudes of create syiuptosomes or slices), and particulars of the inactivation would be higher and rates of tecovery vrauld be measure. There is additional uncertainty in terms of the slower for exposure to -1 fiM nicotine as might be found in identity of the nAChR subtype(s) and the extent of the the brains of human smokets. neuronal circuitry and chemic^ signals involved in effects of chronic nicotine exposure and in responses to acute Nevettheless, a subset of studies has found that absolute challenge. Nevertheless, most studies indicate that chronic measures of a7-nAChR function show potentiation with nicotine exposure leads to a reduction in a subsequent acute chronic nicotine exposure (heterologous expression in HEK response to nicotine when compared to animals pretreated 293 ceils [33]; natural expression in rat cortical neurons with vehicle (Table 5). For example, infusion of [30]). However, in the latter study, after correction of behaviorally-active doses of nicotine can produce losses in absolute potentiation of function for nicotine-induced acute nicotine-provoked ion flux from synaptosomes. upregulation of surface nAChR, there was no change in nAChR in the hypothalamic-pituitafy-adrenal axis appear to function (i.e., when expressed per expressed surface a7- be particularly sensitive to nicotine exposure-induced TaM(4. MfcebelCW«*Nfce

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MOntTmtm-CNSASnnlitktlDiMr^in Jtn. Vtl I, 4 37> nAChR fimctioiial inactivation measured by honnone levels functional inactivation observed using in vitro expression in lespoose to acute nicotiiie challenge, and recovery fn>m systems can be on the order of minuto-days depending on such eflwts can be very slow. Acute nicotine-evalced ACh or the nAChR subtype being studied. At first glance, norepinephrine release also is markedly attenuated after magnitudes of inactivation after chronic nicotine exposure in chro^ nicotine exposure. vivo seem to be less and functional recovery seems to be more complete when assessed using measures of liAChR function in synaptosome or slice preparations. Diflerences in 3.4 iMights iito Mechanisms Underlying Nicotinic cell mitotic activity and environment are factors (hat could Ligand-lndnced nAChR Fnnctional Changes contribute to these apparent differences. However, pharmacokinetics of nicotine exposure and details in There is evidence that phosphorylation modulates experimental design appear to play more central roles. For desensitization of muscle-t;^ nAChR function [48] example, the practical limitation in doses of nicotinic ligand (reviewed in [49]). Roles of phosphorylation in conversion exposure in vivo make it impossible to mimic the higher of nAChR to deq^ states of inactivation are suggested by magnitudes of ftmctional loss observed after treatment of studies showing that calcium-mediated, PKC-dependent cells in vitro with higher doses (over 1 |iM) of nicotine. phosphoiylation of a4 subunits affects the rate of recovery Nevertheless, ctinently available data for sub-|iM dosing of oocyte-expressed a4P2-nAChR &om desensitization [SO]. controlled for time of expostve and time of recovery show However, another study using an HEK 293-based comparable levels of nAChR inactivation for cells in vitro heteiokig^ expcession system found evidence to exclude and for synaptosomes or slice work. a4P2-nAChR-mediated calcium ion influx in nicotine- induced functional faiactivatian of those receptois [SI]. Neither the in vitro studies dqne to date that allow rapid changes in nicotine concentrations pre- and post-exposure Another emerging idea is that factors including nicotine nor the in vivo studies that involve nicotine removal often concentration and length of exposure time may control the for an extended period prior to and during processing of relative balance of receptors in interconvertable basal or tissue for slice or synaptosome studies precisely mimic the desensitized f\mctional states that differ in nicotine binding nicotinic ligand exposure profile that occurs in tobacco users aflinities [36, S2-S4]. There is single channel evidence or that would occur with chronic drug therapy. The supporting the existence of a heterogeneous population of complication of possible acid trapping of nicotine in large a4^-nAChR (100 nM ACh induces channel openings with and poorly perfiised cells such as oocytes muddles the conductance levels of 38-43 pS; 30 (iM ACh induces interpretation of that segment of the data. However, in a channel openings with lower conductance levels of 16- 23 way, the oocyte system might actually be quite a good pS [36]). This may reflect activation of different pools of model for the profile of nicotine exposure in a b^n (with its nAChR (with low or high agonist affinity) or effects on a hydrophobic sinks for nicotine during loading acting as single population of nAChR that convert to different sources for sustained release of nicotine) bathed in nicotine conducting states dependent on agonist concentration [36]. in a tobacco user whose waking hours involve titration of plasma nicotine levels, perhaps to maintain a constant level of drug in the CSF and brain tissue. Probably more accurate 3.5 Perspcedvcs on Nicolinic Ligand-indMced nACkR evaluation of functional effects of nAChR inactivation Functional Changes and Whether they Relate t* would come from behavioral and general physiological Upregnlation measures of nicotine tolerance during sustained exposure. Although the nAChR subtypes involved are not always There has been stunning agreement, across heterologous known in these studies, and mechanisms are complicated in or natural expression experimental systems, means of these un-reduced experimental models, recovery of nAChR nAChR functional measurement (electrophysiological, Ca^'^ function after cessation of chronic nicotine administration in signaling, ion flux assays), and across species of derivation vivo (relief of tolerance) when assessed using behavioral or for specific nAChR subqrpes, that chronic nicotine exposure general physiological measures in intact animals is quite induM a series of steps involving nAChR functional loss. slow, requiring days-weeks to return to control levels. One exception would he for non-oocyte-expressed a7- nAChR, md another would be for the cluster of studies It does seem more and more clear that there are several using HEK 293 cells heterologously expressing human phases of nAChR fimctiorml loss as time and or dose of a4^-nAChR, function of which seems to te potentiated by nicotine exposure increases. When the kinetics of fimctiotMl sustained nicotine exposure. Perhaps the HEK cell loss and recovery are examined in suflicient detail, it also is environment is unique, potentially offering clues to clear that there must be many conformational states of mechanisms involved in responses to chronic nicotine, or nAChR. Thus, there are more nAChR states than can be perhaps there are sequence diflerences in subunits making up accounted for by the simpler sequential or cyclical models these a4P2-nAChR compared to others that have been for conversion of nAChR from basal to activated to studied. Nevertheless, in the many cases when functional desensitized arul back again to basal states. The allosterk (4] loss has been observed and participating nAChR subtypes or "fishing net" models of nAChR state transitioiis thus are known, diversity of those subtypes translates into seem to be earning greater appeal. Any applicable model diversity in magnitudes, extents, and dose- and time- would have to incorporate recent findings about nAChR dependence of onset of nAChR functional loss and in inactivation, arul would require a basal (reidy) state (B), an diCTerences in kinetics and extents of nAChR functional activated (open-charmel) sute (A), perhaps a partially- recovery after drug removal. The rate of recovery fhim inactivated state that maintaiiu some open channel 280 im cmttmlonttft*»-cnsantmalatftaldismhm 2t02.vtli.no.4I CtmUf mm^ Lukmt condiictaiice (I), a rapidly desensiiized slate (D), and a more activation or any phase of functional inactivation, it seems pcnislenily inactivated slate or slates (P or Pg). Aside from clear( that upregulation and fimctional effects are not causally allowing the sequential conversion of these slates in the and mechanistically linked. Even if upregulation could order presented, silent (cyclical) conversions back to B from conceivably( provide more nAChR assembly intermediates to I. D or P suies should be allowed, perhaps in a step-wise allow faster recovery from chronic nicotine exposure, given manner. Whether ligand remains bound in P stales and ithe high permeability of nicotine and its consequent access whether ligand dissociation is a prerequiste for conversion toI assembled nAChR before they are inserted into the from P to B stales is not yet supported with conclusive |plasma membrane, would not these "new recruits'* be evidence. Reports of nAChR desensitization/inactivallon similarly inactivated before they had a chance to respond to induced by agonists at concentrations that are below those endogenous< acetylcholine? Thus, even if upregulation is a needed to induce channel opening or by antagonists suggest response to functional inactivation, it is diflicult to imagine a need for conversion from B to I, D, or P without passing how such a response would be anything but futile. through A. However, the possibility remains that even a slow frequency of channel opening not detectable using 4. THERAPEUTIC AND TOBACCO CONTROL contemporary techniques could trigger conversion to P stales IMPLICATIONS OF EFFECTS OF CHRONIC and, if recovery from P states is sufficiently slow, depletion NICOTINIC LIGAND EXPOSURE of B. Obviously, the allosieric model, in which specific states of nAChR are siabili2ed by specific ligands, could 4.1 Nicotine Self-Medieatioa and Nicotine-Induced accommodate conversions from/to B to/from I, D or P and nAChR Inactivation Hypotheses: Implications for is free of constraints of the sequential and cyclical models. Tobacco-Related Disease and Nicotine Dependence It is argued here that any level of nAChR functional At the time of World War 11, the percentage of American alteration with chronic nicotine exposure could have adults who used tobacco products was over 70%. This is physiological significance. What remains unclear is how comparable to the adult smoking prevalence rate found magnitudes and kinetics of these effects integrate across currently in many Pacific rim countries and in eastern nAChR subtypes (i) as a function of absolute nicotine Europe. The percentage of American adults who currently concentration, and (ii) with changes in nicotine levels during use tobacco products has fallen to about 25%, thanks in part tobacco use (or with changes in nicotinic ligand levels to the heightened awareness in the United States over the during drug therapy). For example, assume a brain nAChR past SO years about the ill health consequences of tobacco subtype that is persistently inactivated by SOS during use. However, there has been no change in this rate in continuous exposure to 100 nM nicotine but not if nicotine America over the last 10-IS years. We hypothesize that levels are 10 nM or lower. Would such a nAChR's activity within this group of American nicotine-dependent recover only during overnight tobacco abstinence (half-time individuals is a subpopulation prone to clinical or for recovery fhim inactivation of a few hours and > 10% subclinical mood imbalance (anxiety, depression) and clearance of brain nicotine within thai period)? If so, then attentional and cognitive difnculties (perhaps in part due to would chronic nicotine self-administration suggest that the mood imbalance) due to hyperactive nicotinic cholinergic user was subconsciously seeking to quell nAChR fiwction? signaling. We further hypothesize that as these individuals Or would such a nAChR's activity recover between engage in tobacco use, chronic nicotine exposure produces cigarettes (half-time for recovery from inactivation of a few inhibition of nAChR fimction and attenuates nicotinic minutes and > lOK clearance of brain nicotine within that cholinergic hyperactivity. As a consequence, there is period)? If so, then would tobacco use by that iiuiividual normalization of mood and improved attention and cognitive indicate subconscious effort to stimulate nAChR recurrently performance. Upon nicotine withdrawal, irritability, mood and intermittently? What would happen to nAChR with swings, and attentional and cognitive difliculties would these characteristics in individuals with 3-fold or 10-fold return. Some tobacco users with good introspective differences in rates of nicotine clearance? Would iheir rates capabilities would be consciously aware of these effects on (and volume) of tobacco consumption differ? How would thdr state of mind, whereas others might only have a vague, things change if these nAChR were SOK inactivated at I |iM subconscious feeling about how tobacco use affects them, nicotine and only 10% inactivated at 100 nM nicotine? their sense of being, and their productivity. There is major Would I OK inactivation of such a nAChR produce a discrepancy between reports by the vast majority of tobacco detectable change in behavior or general physiological state? users that they want to stop smoking and the low rates of What would hapj^ if brain nicotine levels were lO-times or smoking cessation initiation and success. We propose that 3-tiraes higher than plasma levels, meaning that they this is in large part because tobacco users have conscious or appRMch I |iM or more? Could an individual titrate nicotine subconscious awareness of psychophysiological effects of to persistently inactivale oneclass of nAChR and recurrently nicotine on them and have not developed a successful activate another class of nAChR? strategy to subsist in a tobacco-firee existence. Thus, smoking cessation may be more effective if subjects are Because upregulatioa occurs at higher doses (perhaps out educated not only about the ill health consequences of of the physiologically-relevam range) and for longer times of tobacco use and nicotine dependence, but also about how to nicotine treatment than those need^ to induce fimctioiul bring to consdous awareness the psychophysiological efTccts activation, acute desensitization, or even "longer-term" of nicotine on that individual. This would allow the user inactivalion, and because upregulation can occur in the and counselor to formulate a reasonable plan for smoking presence of non-competitive antagonists and cannot be cessation that involves coping with psychophysiological blacked by competitive antagonists that prevent functional consequences. 281

MDnitTmtia-CNStHnnligkalOlmnkn JtU,VaLI.Nm.4 Ml irchronic, coalinuoiis nicotine exposuie indeed induces attention enhancing, and pro-cognitive effects and coiuisteni behavionlly-relevnt lunctional inactivation of nAChR that with evidence that tobacro use as a form of nicotine self- is cenml to nicotine dependence, then one prediction about medication (S8, 63-67]. nAChR have been implicated in smoking cessation efTorts is that a similar level nAChR several neurological or psychiatric disorders including iiuctivation would be required, at least in initial stages of Alzheimer's Disease, Parkinson's Disease, Tourette's cessation, to make those efforts effective. Nicotine Syndrome, Attention-Deficit Hyperactivity Disorder, replacemeM in smoking cessation therapy has had sigiiificant schizophrenia, pain, depression, and Autosomal Dominant but only limited success. However, we would argue that the Nocturnal Frontal Lobis Epilepsy (reviewed in [8, 68)). limited success is because use of nicotine gum or the 22 mg Nicotinic ligands are being explored for their therapeutic nicotine patch is insuflicient to bring plasma nicotine to potential in treatment of these disorders. Such treatment is levels achieved in smokers through tobacco use. Thus, an likely to involve long-term therapy, meaning that subjects inadequate level of nAChR inactivatian would occur, taking will be subjected to chronic nicotinic ligand exposure. Thus the edge off nicotine dependence, but not fiilly it will be important to understand how actions of these accommodating it. Indeed, case reports by Hurt and ligands are integrated across nAChR subtypes, treatment colleagues indicate improved smoking cessation success by time, and drug dose at sites of nAChR expression. In providing through nicotine patches enough nicotine so that nicotinic therapeutic drug design, it will be necessary to serum cotinine levels rise to levels achieved during smoking consider acute and more chronic effixts of ligand exposure at [SS-S7]. Given a high likelihood that a subpopulation of specific nAChR subtypes, whether activation or inactivation American adults who use tobacco would othe^se show of nAChR is a consequence, and whether those effects are nicotine dependence for a good proportion of their lives, it modulating excitatory or inhibitory circuits. In addition, probably would b« wise to extoid any period of nicotine consideration should be given to design of nicotinic replacement therapy for longer than the six weeks-six antagonists as therapeutics if nAChR inactivation indeed is a months customarily employed in smoking cessation consequence of chronic nicotinic agonist exposure and a programs. therapeutic objective. On the other hand, if modulation of the balance between active and inactive nAChR is the Another prediction based on the hypothesis that chronic, therapeutic objective, and it could be argued that this is the continuous nicotine exposure induces fimctional inactivation most critical factor in either exposure to exogenously-applied of nAChR is that nicotinic antagonists would show utility nicotine through tobacco use or even in natural actions in smoking cessation. This idea is supported by the reported modulating ACh levels around doses that will alter the success relative to placebo and relative to limited nicotine balance between nAChR activation and inactivation. then replacement therapy of treatment with mecamylamine or perhaps design of nicotinic partial agonists would have bupropion, which now is known to have non-competitive greater therapieutic potential. Given evidence that at least ftinctional inhibitory potencies at several itAChR subtypes some drugs thought to target other systems, such as similar to its potency for interaction with dopamine neurotransmitter transporters or other kinds of ion chamels, tnnspoitcfs. also have effects on nAChR at comparable and/or clinically- acbieved doses, it could be argu^ that rational, target- For years, it has been recognized that the incidence of directed drug design aiming for sdective actions at a specific tobacco use in alcoholics and schizophrenics is as great as nAChR subtype might not yield the best clinical drug. 90% [S8-6I]. Now that the percentage of American adults Instead, use of "soA-pharmacophores" blending elements of who use lobKco has fallen, phenotypes of those susceptible diverse targets to discover and design drugs that have effects to nicotine dependence have become more clear. balanced across a range of targets may prove, accidentally or Cooiemporaiy epidemiological studies now indicate that 40- intentionally, to yield superior therapeutics. SOS or more of the mentally ill are tobacco users [60,62|, meaning that these often life-long disorders should be recognized and treated in as many as one-half of those ACKNOWLEDGEMENTS entering smoking cessation programs. Aside from depression and anxiety, subclinical manifistalions of these a^ other The audion are grateful to Drs. Jon Lindstiom and Allan fonns of mood imbalance, such as oppositional and/or C. Collins for insights and perspectives. Work in the aggressive behavior, may occur with higher frequency in authors'laboratory is supported by the National Institules of nicotine dependent indi^uals. Attention and cognitive Health, the Arizona Disew Control Research Commission, diHiculties including sensory processing and other leaming and the Barrow Ndinilogical Foundatian. disabilities also may cosegregate with susceptibility to nicotine dependence. Thus, successful treatment of a potentially Ufe-long nicotine depoidence probably might NONSTANDARD ABBREVUTIONS also require treatment of life-long clinical or subclinical mood and^or cognitive diflfeulties. ACh - acetykholine AMP • l-«ceyl < iiKlhylpipciaiinf 4.2 PrMpecls for (fee u« af Ckranie Nirattaic Ligaad Trantft Ibr FiycMalric sad Wenralaifcil DiwrJcri Bgt ~ a-bungarotoxin Nicotine in its purest form has promising therapeutic BrSt = brainstem potential, given its anxiolytic, antidepressant, analgesic. 282

3a omtmonmtftm'oadnimmnkwio^*!* hu,ytl gtimyam^lm = CA - inlnceUular calcium (Fufa-2) fhiofcscence nieinb membranes = cab carbanykhoiine min minutes = Caud - caudate MLA methyllycaconitine

CB - cerebdhan N S naturally expressed = CU m cartex nAChR nicotinic acetylcholine receptor

DA - [^Hldopamine ideaae NE - noiepiiiephtine da - days nico - nicotine deca decamethonium OC - occipital cortex

DHflE - dihydro'^-erytivodine r m lat

DMPP - dimethylphenylpiperazmum s.c. ' subcutaneous d-TC 8 d-tubocurarine SCol ' superior colliculus

EP S elecirophysiology sec seconds epi - epibaiidine sol • solubilized

f m frog SpCd - spinal cord

FCtx s fionial cortex ST - striatum = GTS-21 - dimethoxybenzylidine anabaseine surf siafice

H m heterologously expressed synapto St synaptosomes

h « human TH - thalamus

HB at bindbrain TMA SM tetramethylammonium HC hippo&Mwpus FOOTNOTES hen hexamdhanium 'Chronic exposure here refers to long-term continuous hr exposure or perhaps to intermittent spikes of higher exposure above a long-term continuous back^ound level of exposure HY hypothalamus and is not necessarily comparable to repeated, interminent l-Bgl '^l-tabeled a-bungarotoMn exposure, cecovety from which may occur between drug administrations. IF ioa flux ^Selected findings presented in the data tables and/or l-MAb i^t-labeled raonoclonal antibody mentioned in the narrative are certain to have excluded, unintentionally, pertinent studies. Nevertheless, the authors i.p. intnpefilMieal intend to maintain the tables as a database and invite correspondence from readers to correct errors or omissions. I-PH i^l-laheled epibaiidine analog

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