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Intracellular calcium changes in a hybrid mouse dorsel root ganglion (DRG) neuronal cell line MD3 following… Yokogawa, Tomonori 2000

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I N T R A C E L L U L A R C A L C I U M C H A N G E S IN A HYBRID M O U S E D O R S A L R O O T G A N G L I O N (DRG) N E U R O N A L C E L L LINE M D 3 F O L L O W I N G y -AMINOBUTYRIC ACID (GABA) R E C E P T O R A C T I V A T I O N by Tomonori Yokogawa B . S c , The University of British Columbia, 1996 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E in F A C U L T Y OF G R A D U A T E S T U D I E S (Department of Medicine/Division of Neurology; Experimental Medicine Programme) We accept this thesis as conforming to the required standard T H E UNIVERSITY O F BRITISH C O L U M B I A O C T O B E R 2000 © Tomonori Yokogawa, 2000 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada Date Od. 12 , 2ooo DE-6 (2/88) Abstract Application of y-aminobutyric acid (GABA) to dorsal root ganglion (DRG) neurons in vivo increases their membrane conductance for CF. I studied a mouse D R G neuronal cell line MD3, produced from hybridization between mouse (Balb/C) DRG neurons and N18TG2 neuroblastoma cells, for sensory and pharmacological properties. Immunostaining with markers for neurofilaments demonstrated the cells had neuronal characteristics. In addition, the hybrid cells reacted to CAP and ATP, similar to sensory neurons. Then, I investigated the mechanism and effects of externally applied agonists and antagonists of G A B A on MD3 cells in vitro. I used fura-2 to monitor GABA-evoked depolarizations in terms of intracellular C a 2 + , [Ca2+]j. Application of G A B A to hybrid cells caused [Ca 2 +]i increases that implied G A B A A receptor (GABA A R) activation. Applications of G A B A A R antagonists bicuculline (BIC) and picrotoxinin (PTX) confirmed the G A B A A R involvement in the intracellular response. B I C , but not PTX, blocked the G A B A-mediated elevations in [Ca2+]i. ZAP A, a potent agonist of low affinity G A B A A R s , also increased [Ca2+]i in hybrid cells. The GABA-evoked increases in [Ca2+]i were not apparent during the absence of C a 2 + in the extracellular environment and exposure to dihydropyridines (DHP) nimodipine and nifedipine. The results imply that depolarization-activated C a 2 + influx increased [Ca 2 +]i during G A B A application. I also studied the effects of G A B A B receptor ( G A B A B R ) agonist, p-parachlorophenyl-y-aminobutyric acid (baclofen), on the hybrid cells. Detectable changes in the [Ca2+]j were not apparent during G A B A B R activation alone. 2-hydroxy sac lo fen blocked the inhibitory effect of baclofen and antagonized the G A B A B R . The MD3 hybrid cell line possesses G A B A A and G A B A B R S . The augmentation in [Ca2+]j occurred when G A B A activated the G A B A A R , increasing C l " conductance and depolarizing the membrane potential, thus resulting C a 2 + influx through voltage-dependent C a 2 + iii channels that increase intracellular Ca 2 + . The source of the elevations in [Ca 2 + ]i was the extracellular environment. Nifedipine and nimodipine inhibited the GABA-induced transmembrane influx of C a 2 + through VGCCs. Activation of G A B A B R S with baclofen attenuated the depolarization-induced increases in [Ca 2 + ]\. In addition, baclofen influenced L -type channels since DHPs inhibited the K+-mediated [Ca 2 + ]i elevations. The M D 3 cells exhibited similarities to its sensory parent. Nevertheless, it is a useful model system for the examination of G A B A and other drug effects. Table of Contents Abstract ii-Table of Contents iv. List of Figures vi. List of Tables vii. List of Abbreviations viii. Acknowledgements ix. Chapter I. Introduction 1 1.1 General Introduction 1 1.2. Synopsis of the Central and Peripheral Nervous Systems 1 1.2.1. Anatomical Organization of the PNS 2 1.2.2. Classification of Nerve Fibres 3 1.3. Anatomical Organization of Dorsal Root Ganglion (DRG) Neurons 4 1.3.1. Embryology and Morphology of Neurons in Sensory Ganglia 5 1.3.2. Processes of DRG Sensory Neurons 6 1.3.3. Electrophysiological Properties of D R G Neurons 6 1.3.4. Presynaptic Inhibition 7 1.4. Pharmacological Responses 8 1.4.1. y-Amino butyric Acid Receptor (GAB AR) 8 1.4.2. y-Aminobutyric Acid Subtype A Receptor (GABA A R) 9 1.4.3. y-Aminobutyric Acid Subtype B Receptor (GABA B R) 11 1.4.4. y-Aminobutyric Acid Subtype C Receptor (GABAcR) 12 1.5. GAB A in D R G Neurons 13 1.5.1. G A B A A R S of D R G Neurons 13 1.5.2. G A B A B R s of D R G Neurons 14 1.6. Objectives 15 Chapter II. Methods 17 2.1. Tissue Cultures 17 2.2. Immunocytochemistry 18 2.3. Prepartation of Test Solution 18 2.4. Application of Drugs 19 2.5. Chemicals and Drugs 20 2.6. Measurement of Intracellular Free C a 2 + Concentration, [Ca 2 +]j 21 2.6.1. Preparation of Cells for Calcium Recording 21 2.6.2. Calcium Digital Imaging 21 Chapter HI. Results 26 3.1. Identifying the M D 3 cell line 26 3.1.1. Immunocytochemistry 26 3.1.2. Capsaicin-evoked Response 26 3.1.3. a,P-methylene ATP-induced Response : 28 3.2. M D 3 Cells Express G A B A Receptors 30 3.2.1. GABA-evoked [Ca 2 ' ] ; Elevation 32 3.2.2. Effect of Bicuculline on G A B A Response 32 3.2.3. Effect of Picrotoxinin on M D 3 Neuronal Cells 35 3.3. Mechanism o f Increase in [Ca 2 +]j 39 3.3.1. Zero C a 2 + in the Extracellular Environment 39 3.3.2. GABA-induced C a 2 + Transient Inhibition with D H P s 41 3.4. Ionic Mechanism of Depolarization 44 3.4.1. Depolarization-induced [Ca 2 + ] i Increase 44 3.4.2. Inhibition of KCl-evoked increase in [Ca 2 ]j 46 3.5. Activation of G A B A B Receptor 46 3.5.1. G A B A B Receptor Agonism 46 3.5.2. G A B A B Receptor Antagonism 49 Chapter IV. Discussion 55 4.1 Identifying the M D 3 Cel l Line 55 4.1.1. Immunocytochemistry 55 4.1.2. Capsaicin-evoked Response 55 4.1.3. ATP-induced [Ca 2 + ] ; Increase 56 4.2. G A B A Experiments 57 4.2.1. GABA-induced Intracellular C a 2 + Increase 58 4.2.2. GABA-evoked Depolarization Mechanism 58 4.2.3. Depolarization-evoked C a 2 + Influx 60 4.2.4. Bicuculline Antagonism of G A B A Response 61 4.2.5. Effect of Picrotoxinin and Diazepam 61 4.3. Mechanism o f G A B A - e v o k e d Response 62 4.3.1. Absence of C a 2 + in the Extracellular Environment 63 4.3.2. GABA-response Inhibition with D H P s 63 4.4. Potassium-induced Elevation in [Ca 2 + ] , 65 4.5. G A B A B Receptor Activation 66 4.6. Ca 2 +Sequestration and Efflux Mechanism 68 4.7. Sensory Ganglion Soma 69 Chapter V. Summary and Conclusions 70 References 72 vi List of Figures Fig. 1 Experimental Setup o f Fura-2 System 23 Fig. 2 Immunostaining o f MD3 Cells 27 Fig. 3A,B Capsaicin-mediated Response 29 Fig. 4 Effect o f A T P on Hybrid Cells 31 Fig. 5A,B GABA-mediated [Ca 2 + ] i Increases 33 Fig. 6 ZAPA-evoked Elevations in [Ca 2 +]j 34 Fig. 7 Dose-response Curve for BIC-mediated Inhibition 36 Fig. 8 Picrotoxinin Co-application with G A B A 38 Fig. 9 G A B A-evoked Response in the Absence o f C a 2 + 40 Fig. 10 Effect o f Nifedipine on GABA- induced Responses 42 Fig. 11 Effect o f Nimodipine on GABA- induced Responses 43 Fig. 12 KCl-evoked [Ca 2 +]j Increases 45 Fig. 13 Effect o f Nifedipine on KCl-induced [Ca 2 + ] i Elevations 47 Fig. 14 Effect o f Nimodipine on Depolarization-evoked [Ca 2 +]j Elevations 48 Fig. 15,4,1? G A B A B Receptor Activation and Antagonism 51 Fig. 16 Dose-response Curve for Baclofen-mediated Inhibition 52 Fig. 17 Pretreatment with 2-hydroxy-saclofen 54 vii List of Tables Table 1 Effects of BIC on GABA-induced [Ca 2 +]i Increase. 37 Table 2 Effects of Baclofen on K+-mediated [Ca 2 +]i Increase 52 viii List of Abbreviations A P action potential A T P adenosine triphosphate Baclofen P-parachlorophenyl-y-aminobutyric acid B I C bicuculline B S A bovine serum albumin C A P capsaicin [Ca 2 + ] i intracellular free calcium concentration C N S central nervous system D A B 3,3'-diaminobenzidine D H P dihydropyridine D M E M Dulbecco's Modified Eagle Medium D M S O dimethyl sulfoxide D R G dorsal root ganglion E G T A ethylene glycol-bis((3-aminoethyl ether)- N,N,N',N'-tetraacetic acid F B S fetal bovine serum Fura -2 /AM fura-2 acetoxymethyl ester G A B A y-aminobutyric acid G A B A R y-aminobutyric acid receptor G A B A A R y-aminobutyric acid subtype A receptor G A B A B R y-aminobutyric acid subtype B receptor GABAfcR y-aminobutyric acid subtype C receptor G D P guanine diphoshate H E P E S N-2-hydroxyethylpiperazine-N'-2-etheansulfonic acid H H S S HEPES-buffered Hank's balanced salt solution H P R T hypoxanthine phosphoribosyltransferase H S horse serum K d dissociation constant M A b monoclonal antibody min minute(s) msec millisecond(s) nm nanometre P A D primary afferent depolarization P B S phosphate-buffered saline P N S peripheral nervous system P T X picrotoxinin Rmin minimum fluorescence intensity ratio Rmax maximum fluorescence intensity ratio rpm revolutions per minute sec second(s) SIT silicone intensified target V G C C voltage-gated calcium channel Z A P A (Z)-3-[aminoiminomethul) thio] prop-2-enoic acid Acknowledgements I would like to express my gratitude to Drs. Kim, Puil, and Krieger without whose guidance, encouragement, and support this work would not have been possible. Also, I would like to thank the many people in Drs. Kim and Puil's laboratories for their kindness and support. Finally, I am appreciative of my family and friends for making life interesting and meaningful. 1 C H A P T E R I. INTRODUCTION 1.1 General Introduction Certain aspects of the peripheral nervous system are relevant to the study of the dorsal root ganglion (DRG) neuron. For example, the general organization, electrophysiological properties, processes and structure of the sensory ganglia. In this thesis, I focused on G A B A effects of the intracellular C a 2 + concentration ([Ca2+]0 increase in a mouse hybrid sensory neuronal cell line, MD3. Since primary cell cultures of neurons are heterogeneous and labour intensive to prepare, I used clonal cells that exhibit neuronal features and immortalization. Every experimental approach has its limitations; however, cell lines represent an unlimited source of material for analyses. I will review the anatomical organization of the central and peripheral nervous systems, especially with a focus on the D R G neuron, and the pharmacological aspects of y-aminobutyric acid (GABA). 1.2. Synopsis of the Central and Peripheral Nervous Systems The nervous system depends on input from the external world and the body to maintain homeostasis in the organism. Two major subdivisions of the nervous system include: the central (CNS) and peripheral (PNS) nervous systems. Both systems function synergistically to accomplish necessary actions and have similar anatomical organization and neurochemistry (Sterini, 1997). The CNS consists of the brain and spinal cord while the PNS includes peripheral nerves (spinal and cranial), the autonomic nervous system, and the sensory ganglia. The PNS mediates communication between the CNS, the body of the organism, and the external environment. The CNS monitors the external environment via sight, sound, taste, smell, and touch. It also regulates the internal homeostasis of the organism through other modalities such as pH level, O2 concentration, distention of internal vessels and viscera, and osmotic pressure of fluids. The CNS interacts with the PNS through the primary afferent fibers in the dorsal root 2 and the outgoing efferent motor fibers in the ventral horns of the spinal cord (Terzis & Smith, 1990). 1.2.1. Anatomical Organization of the PNS The PNS is a complex entity composed of cell bodies motor, sensory and autonomic neurons as well as their supportive connective tissue, cellular elements, and associated end-organs. The microscopic structural elements of the PNS include the axons, sensory receptors, neuromuscular junctions, synapses, and ganglia. Situated near the ventrolateral surface of the spinal cord, the ventral root joins with the dorsal root to form the spinal root. Located inside the ventral root are the somatic motor axons and visceral motor axons. The dorsal root extends from the dorsolateral surface of the spinal cord and has both central and peripheral processes. At the vertebral level enclosed within the spinal canal, the majority of dorsal roots have both autonomic (visceral) and somatic sensory axons. The autonomic nervous system innervates the periphery using two neurons. Located in the CNS, the preganglionic neuron sends its axon peripherally to synapse on the postganglionic neuron in the wall of a target tissue or in a peripheral ganglion. The sympathetic system influences activities that expend energy such as increasing the heart rate and the force of each beat, raising arterial pressure, elevating the blood sugar level, and directing blood flow to the skeletal muscles (Barr & Kiernan, 1988). Sympathetic responses occur also during times of stress and emergency (flight or fight response). The parasympathetic system conserves and restores energy by decreasing the rate and force of the heart, lowering blood pressure, and increasing the activity of the digestive system (Barr & Kiernan, 1988). Peripheral ganglia are collections of sensory, sympathetic or parasympathetic neurons outside the CNS. Both the D R G and cranial ganglia are sensory ganglia. The location of DRGs, also known as posterior root or spinal ganglia, is on the posterior roots of the spinal 3 nerves. Cranial sensory ganglia such as the trigeminal root ganglia have cell bodies located near the attachment of the nerve roots to the brainstem. The trigeminal root ganglia have functional equivalence to the DRG, innervating the head, neck, and oral structures. Motor ganglia are part of the autonomic nervous system, terminating in the ear, glands, or smooth muscle. The D R G contains the soma of a variable number of ganglia cells including sensory neurons, proximal portions of axons, associated satellite and Schwann cell sheaths, and connective and vascular tissue elements of the PNS (Lieberman, 1976). 1.2.2. Classification of Nerve Fibers Connective tissue supports afferent and efferent nerve fibers that travel in bundles called fascicles. Spinal nerves have both a dorsal root and a ventral root. The three major categories of afferent fibers, depending upon where the impulse originates, are somatic, visceral, and proprioceptive. Nerve fibers of the dorsal roots are important for sensory information to reach the spinal cord and the rest of the CNS. A whole range of nerve fibers constitutes the peripheral nerves and spinal roots. Erlanger and Gasser originally defined the classification of fibres in a mixed peripheral nerve trunk as one of three groups: A, B, or C (Perl, 1992). The A fibers, the myelinated somatic afferent and efferent nerve fibers, transmit impulses from afferent endings for pain, temperature, touch, pressure, and vibration and send impulses to motor end plates. The four subgroups of the A group are a , P, 8, and y neurons, a neurons have the most heavily myelinated axons and the highest conduction speeds while the 5 neurons have lightly myelinated fibers with the slowest conduction rates of the A group (Bessou & Perl, 1969; Lynn & Carpenter, 1982). The B fibers consist of myelinated efferent preganglionic fibers of the autonomic system. Their diameter reaches approximately 3 um in diameter with impulse rates ranging from 3-15 m/s (Bessou & Perl, 1964). The C fibers are unmyelinated fibers from the postganglionic sympathetic axons of the autonomic nervous system, and the unmyelinated 4 afferent fibers of the peripheral nerves and dorsal roots. As with ^ 48 fibres, C fibres have an entire range of sensory modalities such as pain, touch, and temperature (Fitzgerald, 1979; Lynn & Carpenter, 1982). The postganglionic sympathetic C fibers have diameters ranging from 0.3 to 1.3 pm with impulse speeds from 0.7 to 2.3 m/s. The afferent C fibers range from 0.4 to 1.2 pm with conduction speeds ranging from 0.6 to 2.0 m/s (Daniel & Terzis, 1977). Nerve fibres of the Ad and C classes transmit pain information (Burgess & Perl, 1967; Kumazawa & Perl, 1977; Fyffe, 1983). 1.3. Anatomical Organization of D R G Neurons DRG neurons are sensory neurons, located within the dorsal root ganglion of the PNS. They and their cranial nerve equivalents are the primary sensory cells for general somatic sensation. The DRG neuron is a functional unit of the nervous system, capable of receiving, conducting, processing, transmitting stimuli, and making functional contact with other neurons. The vertebrate D R G neuron is a mononucleate cell that possesses a cell body, also known as the soma or perikaryon, and a population of fine cytoplasmic processes (neurites). These extensions of the soma surround tissues, possessing an excitable plasma membrane. The peripheral dendrites of DRG neurons convey pain, pressure, temperature, and stretch impulses from either free endings in skin or a number of specialized end-organs in skin, muscle, tendons, joints, connective tissue, and bone to the somata (Lieberman, 1976). The DRG neuron is spheroidal with round or oval profiles (Bunge et al., 1967). They vary enormously in size, ranging from diameters of 10 pm for the smallest cells of small vertebrates to 115 pm for the largest spinal ganglion cells of man (Ohta et al., 1974). The D R G neurons are pseudounipolar with a stem process or the initial tract of the axon. On leaving the cell body, the stem process often pursues a complex course near the cell body. The central 5 process enters the spinal cord through the dorsal root while the peripheral process extends to the periphery in a peripheral nerve, to receive sensation and terminate in a sensory ending (Taylor & Pierau, 1991). The T- or Y-shaped bifurcation occurs at a variable distance from the cell body, commonly close to or within the axial fibre region (Lieberman, 1976). 1.3.1. Embryology and Morphology of Neurons in Sensory Ganglia The nervous system develops from ectoderm, the outermost of the three germ layers in the embryo. The derivation of the CNS is primarily from this germ layer; in the PNS, the ectodermal and mesodermal structures combine to produce the final structures. During the third week of gestation, when the neural groove appears on the dorsal surface of the embryo, the longitudinal groove deepens where the lateral margins meet to close the neural groove to form the neural tube. The neural crest is the portion of the ectodermal tissue that separates from the tube, forming a pair of elongated tissue masses along the dorsolateral margins of the neural tube and eventually developing into sensory ganglia (Pannese, 1974). The cells are first bipolar, but the two processes fuse to form the single process of this pseudounipolar type of neuron (Tennyson, 1965, 1970). The stem process that arises from smaller cell bodies is short and unconvoluted, whereas the stem process of larger neurons often at first wind around the parent cell body (Barr & Kiernan, 1988). During development, D R G neuronal processes begin to associate with the surrounding tissues that are forming in the periphery (Pannese, 1974). Two morphological subtypes of D R G neurons are: "large light" ("light" or L) and "small dark" (SD) neurons (Jacobs et al., 1975, Rambourg et al., 1983). From the statistical analysis of mice and rats, both L and SD neurons have a normal distribution of cell size and overlap each other. The L neuron distribution extends over the entire size range of the ganglion while SD neurons are limited to the lower end of the distribution (Lawson, 1979). 6 1.3.2. Processes of D R G Sensory Neurons Situated on structures near the surface of the body and in deeper tissues, somatic sensory axons are the peripheral part of D R G neurons in spinal nerves, and cranial sensory ganglia in cranial nerves. Somatic afferents carry information concerning changes in the external environment. Receptors detect three types of sensations: pain; temperature, warm and cold; and touch, light and deep. Light touch requires only surface stimulation while deep touch requires firmer stimulation that provokes the receptors that lie deeper in the tissues. DRG neurons are afferent units with dendritic processes, its peripheral afferent fiber and receptive terminals in peripheral tissue, and its central axon's branches and synaptic contacts in central tissue (Perl, 1992). The primary function of the spinal dorsal roots and their cranial equivalents is to serve as afferent conduits to the CNS (Perl, 1992). The axons vary in diameter from a maximum of 20 p.m to a minimum of 0.2 jam depending on the destination of the nerve and the sensory modality conveyed (Iggo, 1974). Both the peripherally and centrally directed processes, and stem process that connects them with the cell body, are structurally axons and all three may be myelinated. At its origin, the stem process is small and generally less than 3 um in diameter in small mammals and leaves the perikaryon rather abruptly, commonly at an acute angle. The diameter of the stem process may increase at some distance from the cell body and the myelinated part of the stem process is usually considerably larger than unmyelinated axons of peripheral nervous tissue. 1.3.3. Electrophysiological Properties of D R G Neurons Under physiological conditions, the initiation of action potentials (AP) in sensory ganglion neurons develops at, or close to the receptor terminals. Although the position of the soma is off-stream in relation to the impulse traffic 'through-route1, ganglion cell perikarya are believed to play a far less dominant role in the electrophysiological activities than is the case in 7 multi-polar neurons. The terminal portions of the peripheral process are vital in the sensory ganglion cell; however, one should not consider the soma and stem process as silent partners in the process of electrical signalling. The anatomy and the physiology of the spinal cord and the brain stem nuclei, which the sensory ganglion cells terminate, also play a major role because these areas generate activity and control reflex activity. In neurons, the observed resting membrane potential is typically -50 to -60 mV, meaning that the internal environment is more negative than the extracellular component (Gallego & Eyzaguirre, 1978; Mayer & Westbrook, 1983). The calculation of the potential difference (PD) developed across a membrane using the Nernst equation is: E v = (RT/zF) loglO [K + ]o / [K + ] i Determining membrane potentials requires R, the universal gas constant (8.31 J/mol), T, the absolute temperature, F, the Faraday constant (96500 C/mol), z, the charge constant, [K + ] 0 , the extracellular potassium ion concentration, and [K + ] j , the intracellular potassium ion concentration. Although initiation is not normally at the level of the cell body, action potentials always invade the soma passing along its processes (Scott et al., 1969; Varon & Raiborn, 1971). Conduction along the initial axon is slow, however, the central terminals of cells with large diameter myelinated axons are depolarized before the soma (Darian-Smith, 1973). 1.3.4. Presynaptic Inhibition Many hypotheses exist concerning presynaptic inhibition mechanisms. The C l " equilibrium potential relative to the membrane potential influences the C f conductance that leads to hyperpolarization, depolarization, or no potential change depending on the C F gradient across the membrane. An increased C F conductance shunts the presynaptic spike, reducing transmitter output (Eccles, 1964; Takeuchi & Takeuchi, 1966). A high extracellular K + 8 environment near the presynaptic terminals may induce depolarization (Rudomin et al, 1981). In addition, the decrease in presynaptic C a 2 + current may reduce the transmitter release from the presynaptic cell (Dunlap & Fischbach, 1978; Kretz et al, 1986). Previous research provided evidence for primary afferent depolarizations (PAD) produced from GABAergic pathways (Eccles et al, 1963a,b; Curtis et al., 1977). The most accepted hypothesis is the shunting effect of the chloride conductance, lowering the amplitude of the action potential in the nerve terminal (Dunlap & Fischbach, 1978; Kretz et al, 1986), reducing C a 2 + influx through voltage-activated channels and transmitter release. However, the exact ionic mechanisms involved in presynaptic inhibition in primary afferents by G A B A agonists remain unknown because the nerve terminals are not readily accessible to investigation. 1.4. Pharmacological Responses 1.4.1. y-Aminobutyric Acid (GABA) Receptors G A B A has many functions that include inhibitory transmitter, seizures, and trophic factor in synaptogenesis (Wolff*?/ al., 1978; Roberts, 1991). It has also been implicated in the pathogenesis of Huntington's disease, Parkinsonism, epilepsy, schizophrenia, tardive dyskinesia, and senile dementia, as well as other behavioral disorders (Mountjoy et al, 1984; Gunne & Andren, 1993; Reynolds et al, 1997; Volpi et al, 1997; Wassef et al, 1999; Brailowsky et al, 1999; Casamenti et al, 1999). The term G A B A receptor (GABAR) refers to a G A B A recognition site on pre-and postsynaptic membranes. When an appropriate agonist binds to this receptor, a change in membrane permeability permits inorganic ions to cross. For example, a change in permeability to C F , for example, results in hyperpolarization of the receptive neuron for postsynaptic inhibition or depolarization in the case of presynaptic inhibition. In vertebrates, three major types of GABARs are: type A (GABA A R ) , B (GABA B R ) , and C (GABAcR). The GABARs have differences based on their physiology, pharmacology, and anatomical location as well as their second messenger mechanisms. The subunit combination influenced the pharmacological properties of a particular G A B A R (MacDonald & Olsen, 1994; Costa, 1998). 1.4.2. G A B A A Receptors G A B A and structural analogues of G A B A like muscimol, a substance from the hallucinogenic mushroom Amanita muscaria, and synthetic analogues such as 4, 5, 6, 7-tetrahydroisoxazolopyridin-3-ol (THIP), 3-aminopropanesulfonate, peiperidin-4-sulfonate, and isoguvacine activate G A B A A R s , controlling the opening and closing of a C l " channel conductance (Macdonald & Olsen, 1994). Astroglia, central and peripheral neurons have G A B A A R S (Grobin et ah, 1999). This major inhibitory neurotransmitter receptor is a multi-subunit receptor-channel complex that belongs to a large superfamily of ligand-gated ion channels that includes the nicotinic-cholinergic, ionotropic glutamate, and glycine receptors. The G A B A A R - i on channel complex is a heteropentameric glycoprotein of approximately 250 kD, combining multiple polypeptide subunits (Delaney & Sah, 1999). The five distinct classes of polypeptide subunits (a, p\ 5, y, and p) have multiple isoforms. Therefore, a structural diversity exists for the G A B A A R with a family of at least 17 genes coding for diverse subunits (a 1-6, pi-4, yl-3, 8, pi-2, n, and s) (Schofield et al., 1987; Olsen & Tobin, 1990; Sieghart, 1995; Hedblom & Kirkness, 1997). Many pharmacological agents, including anesthetics (for review, see Mody et al., 1991; Weight et al, 1992), ethanol (Sudzak et ah, 1986) and endogenous neurosteroids (Majewska et al, 1986), affect GABA-mediated C l " conductance. Evidence for the biochemical and structural relationships between G A B A and benzodiazepine showed that they share the same receptor 10 complex and G A B A enhances the binding of benzodiazepines (Sigel et al, 1983; Schofield et al, 1987; Stauber et al, 1987). While G A B A agonists activate the G A B A A R , benzodiazepines increase the frequency of the channel opening without altering the channel conductance or duration of opening (Choi et al, 1977; Macdonald & Barker, 1978a; Macdonald & Barker, 1979). However, barbiturates slightly decrease the opening frequency but prolong the duration of opening (Study and Barker, 1981; Olsen, 1981, 1987; Twyman et al, 1989a,b; Macdonald and Twyman, 1992). The steroids that may include anesthetic steroids and progesterone metabolites also augment G A B A R current (Majewska et al, 1986; Callachan et al, 1987a,b). G A B A antagonists bicuculline (BIC), picrotoxinin (PTX), methyl 6,7-dimethoxy-4-ethyl~P-carboline-3-carboxylate (DMCM), and penicillin (Raichle et al, 1971; Macdonald and Barker, 1978b) diminish GABA-mediated Cl~ conductance. Therefore, distinct compounds affect the G A B A A R function through several different sites: G A B A , P T X / barbiturate, benzodiazepine, and steroid (Olsen, 1981; Sigel et al, 1983; Stauber et al, 1987). Compounds that increase protein phosphorylation may also influence G A B A A R channel function (Whiting et al, 1990). However, the significance of phosphorylation of G A B A A R S remains uncertain but may have an important role for the regulation of the receptor function and expression (Browning et al, 1990, 1993). G A B A antagonists compete with G A B A directly at the G A B A A R or non-competitively through modification of the receptor or inhibition of the GABA-activated ionophore. Two classic G A B A antagonists are plant convulsants BIC and PTX, each acting differently on the G A B A A R with BIC competitively antagonizing G A B A at the receptor level whereas PTX non-competitively blocking GABA-activated ionophores (Johnston, 1991). Other antagonists include petrazepin, the amidine steroid RU5135, and a series of pyridazinyl-GABA derivatives like SR-95531 (Johnston, 1991). 11 G A B A is also an important neurodevelopmental factor and signal molecule during early ontogenesis (Meier et al, 1991; Belhage et ah, 1998). The elucidation of the exact mechanism will involve G A B A A R activation since studies discovered that G A B A agonists replicate G A B A effects and antagonists inhibit neurodifferentiation (Meier et al., 1985; Belhage et al., 1986). 1.4.3. G A B A B Receptors In mammals, G A B A B R binding sites occur inside and outside the CNS, and are present in lower species. For example, at the neuromuscular synpase of lobsters, both inhibitory and excitatory axon terminals possess G A B A B R S (Miwa et al., 1990). G A B A B R S are BIC- and PTX-insensitive, activated by P-para-chlorophenyl-GABA (baclofen), antagonized by 2-hydroxy-saclofen, and unassociated with CF conductance (Sivilotti & Nistri, 1991; Bowery, 1993). Coupling of G A B A B R s to G-proteins activates intracellular second-messenger pathways. G A B A B R activation also mediates the release of amines, excitatory amino acids, neuropeptides, and hormones. While the G A B A A R is linked to C P channels, the G A B A B R is associated with C a 2 + or K + channels via second messenger systems (Newberry & Nicoll, 1984; Asano et al., 1985; Inoue et al, 1985; Holz et al., 1986; Ogata et al, 1987; Dolphin & Scott, 1987; Dutar & Nicoll, 1988). Therefore, G A B A B R s activate an inhibitory effect associated with a K + conductance increase, a C a 2 + conductance decrease, or an adenylyl cyclase functional decrease. Metabotropic G A B A B R require coupling between two distinct gene products: G A B A B R I and G A B A B R 2 (Kaupmann et al, 1997,1998; Martin et al, 1999; Bowery & Enna, 2000). In terms of pharmacology, the G A B A B R is different from the G A B A A R by its selective affinity for baclofen and lack of affinity for BIC and muscimol (Bowery et al, 1980,1981; Bowery, 1993). Only a limited number of G A B A B R agonists are active, presumably because they cannot differ significantly in structure from GABA. Baclofen activation of the G A B A B presynaptic receptors decreases Ca conductance and hence, transmitter release (Scholz & 12 Miller, 1991; Diverse-Pierluissi & Dunlap, 1995). Baclofen, (-)-baclofen being the more active form, is virtually inactive at G A B A A sites but is stereospecifically active at G A B A B sites. The most potent agonists are the phosphonic analogues, 3-aminopropyl phosphinic acid and its methyl analogue (Pratt et al, 1989). Unlike the G A B A A R , the G A B A B R does not respond to benzodiazepines or barbiturates. The first selective but relatively weak G A B A B antagonist was phaclofen (Kerr et al., 1987). In other brain regions, Lambert et al (1989) observed similar results using the more potent derivatives, saclofen and 2-hydroxy-saclofen. The antagonistic compounds that affect the G A B A A R have no effect on the G A B A B R mechanism. G A B A B R antagonists include CGP 35348, the first phosphinic derivative to be reported (Olpe et al., 1990; Seabrook et ah, 1990), and CGP 36742, a brain-penetrating antagonist, competitively antagonizes responses to baclofen when applied to rat cerebrocortical neurons, (Bowery, 1993). Many thioether analogues of saclofen and phaclofen are pharmacologically less active than the original compounds (Allan et al., 1990). 1.4.4. G A B A c Receptors Recent pharmacological and molecular biological experiments discovered a third class of GABARs, named GABAcRs. In the retina, many types of species, bipolar and horizontal cells had GABAcRs (Qian & Dowling, 1994; Bormann & Feigenspan, 1995; Enz et al, 1995). Coupled to BIC-insensitive, PTX-sensitive C f channels, GABAcRs are activated by C A C A and T A C A (Shimada et al, 1992; Qian & Dowling, 1993). Although GABAcRs also gate a C l " conductance, they are not blocked by BIC or SR95531 and are markedly less sensitive to PTX. They are also insensitive to modulation by benzodiazepines and barbiturates (Qian & Dowling, 1993; Bormann & Feigenspan, 1995; Johnston, 1996). Assembled from p subunits (p i , p2, p3), GABAcRs share some homology with G A B A A R subunits, but do not appear to coassemble with them (Cutting et al, 1991; 13 Shimada et al, 1992). However, the significance of these receptors outside of the retina is unknown. 1.5. G A B A in D R G Neurons Past experiments demonstrated that G A B A depolarizes the somata and terminals of primary afferent neurons, causing presynaptic inhibition in the spinal cord (Eccles et al., 1963a,b; Schmidt, 1963; Tebecis & Phillis, 1969; Davidson & Southwick, 1971; Barker & Nicoll, 1973; Krnjevic, 1974; Feltz and Rasminsky, 1974; Nishi et al., 1974; Deschenes et al, 1976; Deschenes & Feltz, 1976; Gallagher et al, 1978). Nishi and colleagues (1974) observed in bullfrog D R G neurons that G A B A was the most potent depolarizing substance among its analogues, resulting in increased CF conductance. G A B A is a major neurotransmitter that generates presynaptic inhibition at central terminals of primary afferents in the dorsal horn, where released G A B A simultaneously activates both G A B A A and G A B A B R S . GABARs in human DRG neurons behaves differently compared to other vertebrate neurons that hyperpolarize during G A B A application. Valeyev et al. (1996) determined that BIC and PTX did not influence G A B A currents in human D R G neurons. However, in rat D R G neurons, both antagonists inhibited GABA-mediated responses (Nowak et al, 1982; Twyman et al, 1989b). These differences may be due to the particular receptor subunit composition in human D R G cells or the experimental design. In the membrane of DRG cells, G A B A A R S and G A B A B R S may exist together or separately. A recent study found 50 % of each kind of receptor in the primary sensory terminals (Si et al, 1997). 1.5.1. G A B A a Receptors of D R G Neurons In mature DRG neurons, BIC competitively and PTX non-competitively inhibit G A B A A R subunit proteins that have C l " conductance (De Groat, 1972; Nishi et al, 1974; 14 Gallagher et al, 1978; Choi & Fischbach, 1981; Dunlap, 1984; Inoue & Akaike, 1988). As mentioned above, activation of G A B A A R channel with an appropriate agonist conducts CF ions predominantly. Since the CP equilibrium potential is near resting potential in most neurons, increasing the CF conductance depresses excitability by causing a shunt of the depolarizing effects of an excitatory input. In DRG neurons, the concentration of CY is higher inside compared to the extracellular environment (Feltz & Rasminsky, 1974; Nishi et al, 1974; Gallagher et al., 1978). The activation of GABAARs results in the opening of CF channels and a concomitant CF efflux out of DRG neurons, depolarizing the membrane (Nishi et al, 1974; 2+ Gallagher et al., 1978; Bormann, 1988). The depolarization activates voltage-gated Ca channels (VGCC), allowing Ca from the external environment to enter the cells. GABARs affect a variety of VGCCs in DRG neurons. 1.5.2. GABAB Receptors of DRG Neurons Initial investigations concerning G A B A B R in DRG neurons found that stimulation or activation of the receptor decreased membrane Ca 2 + conductance. This depression was attributed to the decrease in the duration of APs in C and A S neurons (Dunlap, 1981; Desarmenien et al., 1984) and in DRG neurons of cat, mouse, and rat from voltage clamp studies (Robertson & Taylor, 1986; Green & Cottrell, 1988; Formenti & Sansone, 1991). The action of baclofen on the G A B A B R also produces an inhibition of VGCCs, decreasing the amount of Ca 2 + influx or shortening the A P duration in the membrane of DRG neurons (Dunlap and Fischbach, 1978; Llinas et al., 1981; Desarmenien et al., 1984; Deisz & Lux, 1985; Dolphin & Scott, 1986,1987). A diminution of Ca 2 + influx through VGCCs may account for the ability of baclofen and G A B A to reduce the evoked-release of neurotransmitters (Bowery et al., 1980,1981; Price etal, 1984). 15 1.6. Objectives Previously, our laboratory created the MD3 cell line according to the protocol described by Hammond and colleagues (1986). Immortal cell lines have advantages over primary cultures, owing to ease of maintenance of cells in simple media and acquisition of an infinite lifetime. Neuronal cell lines are also an unlimited source of a pure neuron population, allowing investigations of receptor properties. Fusion of mouse DRG sensory neurons with a mouse neuroblastoma N18TG2, deficient in hypoxanthine phosphoribosyltransferase (HPRT), generated the clonal cell line MD3. The reasons for choosing the neuroblastoma parent include its embryological similarity to neurons (Augusti-Tocco & Sato, 1969); therefore, the chances of ehrninating some neuronal gene expression are reduced (Killary & Fournier, 1984). In Platika's study (1985), the hybrid sensory neuronal cells expressed a neuron-like morphology, cell-surface gangliosides and excitable membranes. Successful fusion occurs when these neuronal properties represent the expression of neuronal genes. Suburo and colleagues (1992) also found specific markers and neuropeptides, and cell depolarization in response to capsaicin and bradykinin in their hybrid sensory neuronal cells. This study investigated the GABA pharmacology of the mouse hybrid sensory neuronal cell line created from hybridization termed MD3. The MD3 cell line may possess GABARs and be a model of GABAR interactions. Therefore, a central hypothesis of this thesis is that GABA binds to the GABA subtype A receptor, opening the Cl~ chloride channels that depolarizes the cell; this activates voltage-gated Ca 2 + channels, increasing [Ca 2 +]i as a consequence of Ca 2 + influx. GABA subtype B receptor activation mav attenuate the evoked elevations in [Ca 2 +]i. Various GABAR antagonists and Ca 2 + channel-blockers should inhibit both types of responses. Fura-2 was used to measure the [Ca2+]i of MD3 cells to determine the presence of GABAR and its responses to different compounds. To determine the mechanisms and effects of GABA-evoked depolarizations, bath and local applications of GABA-related compounds influenced [Ca 2 +]j, exhibiting G A B A R pharmacology. Determining the G A B A R subtype involved in the generation of intracellular transients involved applying antagonists that distinguished between G A B A A and G A B A B R subtypes. A principal mechanism proposed to explain GABA-evoked depolarizations in the mammalian system is that G A B A depolarizes the cell through activation of the G A B A A R coupled to anion-selective channels, resulting in efflux of Cl". 17 Chapter II. METHODS 2.1. Cell Culture We previously produced the hybrid sensory neuronal cell line MD3 by somatic fusion of hypoxanthine phophoribosyl transferase (HPRT)-deficient mouse N18TG2 neuroblastoma cells and Balb/c mouse DRG sensory neurons using methods described by Hammond et al. (1986). In brief, DRGs were removed from 3 week old Balb/C mice and incubated in phosphate-buffered saline (PBS) containing 0.25 % collagenase (type I; Worthington Biochemical, Freehold, NJ) and 20 ug/ml DNase (Sigma, St. Louis, MO) for 40 min at 37 °C and dissociated into single cells by repeated pipetting. Single D R G neurons were re-suspended in Dulbecco's modified Eagle's medium (DMEM) containing phytohenmgglutinin (40 ug/ml) that facilitates the adherence of DRG neurons to the N18TG2 cells. Then, the N18TG2 neuroblastoma cells were added to the DRG neurons. After a 15 min incubation, the medium was aspirated and the cells were fused with 50 % (v/v) polyethylene glycol (PEG 4000, Sigma). The fusion products were plated in D M E M containing 5 % fetal calf serum, 5 % horse serum (HS), 100 uM hypoxanthine, 0.4 uM aminopterin, and 16 uM thymidine (HAT medium) which selects against HPRT-deficient cells. After 7 days of treatment in HAT medium, cultures were fed with D M E M containing 5 % fetal bovine serum (FBS) and 5 % HS. Individual colonies were isolated and expanded further in D M E M containing 5 % fetal bovine serum (FBS), 5 % HS, 20 ug/ml gentamicin, and 2.5 ug/ml fungizone. One of the expanded colonies was named MD3 to be used in the present study. Cells frozen in a nitrogen tank were quickly thawed, washed, and resuspended in media. Stock cultures of mouse hybrid sensory neuronal cell line MD3 were grown in tissue culture flasks with growth medium consisting of D M E M with 5 % FBS, 5 % HS, 20 ug/ml gentamicin, 18 and 2.5 ug/ml fungizone. MD3 cells were maintained in a humidified atmosphere of 5 % CO2 and 95 % air at 37 °C and fed twice a week. Experiments were performed on MD3 cells, plated onto poly-D-lysine-cbated 22 mm glass coverslips (#1 thickness, Carolina Biological Supply, Burlington, NC). 2.2. Immunocytochemistry MD3 cells on aclar plastic coverslips (9 mm, round) were cultured for 2-4 days. The cells were rinsed thoroughly in PBS, fixed in methanol at -20 °C for 10 min, and incubated with a mouse monoclonal antibody (Ab) against microtubule-associated protein-2 (MAP-2, 1: 100; Sigma Chemical) and a rat monoclonal Ab against high molecular weight neurofilament protein (NF-H, 1:10, Ta51; gift from Dr. V . Lee) at 4 °C for 48 h. Cells treated with the primary antibodies were washed 3 X 2 min in PBS. Appropriate biotinylated anti-mouse or anti-rat IgG Ab was diluted in PBS (1: 200) and applied to the coverslips for 1 h at room temperature (RT). Hybrid sensory neuronal cells were extensively washed in PBS and then incubated in adivin-biotin complex solution (ABC, Vector Laboratories, Mississauga, ON) that binds to the biotinylated secondary antibody for 1 h at RT. The chromogen diaminobenzadine (DAB) revealed this antibody staining. The cells were washed extensively in PBS and incubated for 2 min in 0.1 M Tris-HCl buffer containing 0.5 m M 3,3'-DAB and 0.02 % hydrogen peroxide. Coverslips were dehydrated in 70-100 % ethanol, cleaned in xylene, and embedded on microscopic slides with Permount. 2.3. Preparation of Test Solutions HEPES-buffered Hank's balanced salt solution (HHBSS, adjusted to pH 7.4 with NaOH) consisted of (in mM): 145 NaCI, 2.5 KC1, 1.0 M g C l 2 , 20 HEPES, 10 glucose, and 1.8 CaCl 2 . In Ca2 +-free HHBSS buffer, 50 p M ethylene glycol bis (P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) replaced the CaCb. to chelate any excess C a 2 + ions present. G A B A , ap-methylene ATP, capsaicin (CAP), (±)-baclofen, diazepam, 2-hydroxy-saclofen, nifedipine, nimodipine, (Z)-3-[ammoiminomethyl) thio] prop-2-enoic acid (ZAPA), BIC methobromide, and PTX were prepared from frozen aliquots of stock solution (1-100 mM) and diluted in HHBSS buffer. Since CAP, diazepam, nimodipine, and nifedipine are water-insoluble, they were first dissolved in 100 % alcohol and diluted in HHBSS with a final alcohol concentration of 0.01 %. 2.4. Application of Drugs The experimental protocol involved recording the changes in [Ca2+]j of the MD3 cells in a series of different solutions (for example, normal saline, Ca2 +-free saline, and normal saline with agonist or antagonist, recovery in normal saline). MD3 cells were placed in a recording chamber, with a bath volume of 0.4 ml, on the stage of a Nikon inverted microscope (Nikon Diaphot TMD). This chamber was superfused with the different types of HHBSS buffer using a system of multiple rubber tubes connected to reservoirs containing the various solutions. A three-way solenoid valve allowed switching between the different types of recording solutions. Trials with a marker dye showed that complete replacement of the solution occurred within 30 sec at a flow rate of 2 ml/min. This relatively slow speed of solution exchange does not affect the results because the kinetics of the responses were not measured. An ejection technique with a Picospritzer was used where pressurized nitrogen gas ejected drugs from pipettes. This "puffer" technique allowed rapid and repeated applications of drugs of known concentrations (Fischbach & Choi, 1981). Small quantities of ct,p-methylene ATP (100 pM), CAP (30 pM), G A B A (10-100 pM), Z A P A (100 pM), or KC1 (25-100 mM) were applied to individual cells by pressure microejection (5-20 psi, 10-150 ms) from a two-channel Picospritzer (General Valve, 20 Fairfield, NJ). A rmcromanipulator was used to position the single-barrel micropipette over the cells near the recording site. At the start of an experiment, G A B A agonists or KC1 were applied by pressure to initiate a response. If no response resulted, then the ejection time was increased, or the pipette was moved closer to the cells. Care was taken to optimize the pipette tip-to-cell distance, such that a maximal response was obtained at a given pressure. Using a double pull protocol, thin-wall glass tubings were drawn under low heat on using an instrument (Narashigi Instrument, Tokyo, Japan) that produced a sharp tip from thin-walled glass (diameter of 1.0 mm o.d., World Precision Instruments). The tips were broken to give a tip diameter of 10-20 um. The micropipettes were backfilled with a long, thin needle. If the solution could not be expressed from the tip, the electrode was assumed clogged and discarded. Experiments with trypan-blue dye solutions showed the ejection pulses covered the cell area during drug application. In addition, trypan blue allowed visualization of the perfusion flow inside the chamber. BIC (100 nM-100 uM), baclofen (50 nM-100 uM), nimodipine (1-20 uM), nifedipine (1-20 uM), 2-hydroxy-saclofen (500 nM-10 uM), PTX (10-25 uM), and diazepam (1-20 uM) were mixed directly to HHBSS buffer and applied to cells in the recording chamber. At the end of each experimental day, the recording chamber, apparatus and rubber tubings were washed first with 10 % alcohol, followed by distilled water. 2.5. Chemicals and Drugs GABA, N-2-hydroxyethylpiperazine-Nl-2-etheansulfonic acid (HEPES), nifedipine, nimodipine, diazepam, potassium chloride (KC1), bovine serum albumin (BSA), CAP (8-methyl-N-vanillyl-6-nonenamide), EGTA were all purchased from Sigma; Z A P A sulfate from Tocris Cookson (Ballwin, MO); (±)-baclofen, (-)-BIC methobromide, PTX and 2-hydroxy-saclofen from Precision Biochemicals (Vancouver, BC); fura-2/AM, pentapotassium salt ((2-[6-21 (bis(carboxymethyl)-ammo)-5-methylph^ carboxylic acid), was purchased from Molecular Probes (Eugene, OR). 2.6. Measurement of Intracellular Free [Ca 2 +] 2.6.1. Preparation of Cells for C a 2 + Recording Cell cultures grew on poly-D-lysine-coated 22 mm glass coverslips for at least 12 h before initiation of any experiments. Intracellular C a 2 + levels were measured using the C a 2 + -sensitive fluorescent chelator fura-2 (Grynkiewicz et al., 1985). An ampule containing 50 u,g of fura-2/AM (acetoxy methyl ester) was mixed with 50 uL of dimethyl sulphoxide (DMSO) to make a 1 uM stock solution. The stock solution was aliquoted into Eppendorf tubes at 3 uL each and stored in -20 °C. Fura-2/AM was mixed with 0.2 % pluronic F-127 acid (Molecular Probes) and 0.02 % B S A in the recording buffer (HHBSS). The addition of pluronic acid increases the solubility of the highly hydrophobic dye, facilitating the fura-2 loading of the cells. From earlier work (Yoo et al., 1999), adding glucose to HHBSS sustained cell viability during loading, washing, and testing. Without glucose, the cells would also lose their fura-2 fluorescence signal at a much faster rate. 2.6.2. Calcium Imaging Al l cells examined were incubated with fura-2/AM (5 uM) in HHBSS for 45 min at 37 °C. Fura-2/AM permeates cell membranes into the cytosol where it is hydrolyzed into fura-2, a non-esteric form that is membrane-impermeable, remaining trapped inside the cell. After loading, the cells were washed twice in recording buffer to remove any excess fura-2 that may be present on the exterior. The cells were incubated a further 15 min at 37 °C to completely de-esterify fura-2/AM to fura-2. Finally, the glass coverslip with the fura-2 loaded cells was mounted on a perfusion chamber and placed onto the stage of the Nikon inverted microscope. 22 Figure 1 illustrates the overall experimental setup. Cells on glass coverslips were used within a week after plating and passaged between 2-20 times before taking a new vial of cells from the liquid nitrogen storage. Excitation light from a 100 W xenon arc lamp (Osram, Germany) passed through 340 or 380 nm filters mounted in a plate, reflected by a dichroic mirror and focused onto the cells via a 40X-fluorite epifluorescence objective. The fura-2 fluorescence signal was detected by a silicone intensified target (SIT) video camera (Hamamatsu C2400-08). The ratios of the fluorescence intensity at 340 and 380nm wavelengths were stored on a desktop computer. Before each experiment, cells regions were marked and chosen based on their appearance with an even distribution of fura-2. Regions of high cell density were avoided because of the difficulty in discriminating signals between cells. Ratios were obtained from 8-frame averages of pixel intensities (ranging from 0 to 255) at each of the excitation frequencies. An appropriate software program, Image-1/FL Quantitative Fluorescence System (Universal Imaging, West Chester, PA), was used on the desktop computer to control optic equipment and the parameters of fluorescence data acquisition (background subtraction, filter wheel/ shutter switch, sampling rate and camera sensitivity). Using ratios should, to a first approximation, normalize for differences in preparation geometry, as well as for the effects of light scattering, illumination nonuniformity, and indicator concentration differences (Grynkiewicz et al, 1985). Then, the ratios were converted to a direct measurement of [Ca 2 + ] j using the following equation: [Ca 2 +]i (in nM) = K D p(R-R m i n ) / (R m a x -R ) j Glass Pipette Dichroic Miiror(410 Shutter & Filter Selector Control Logic I Main Computer Analog-to-Digital Converter o^mp Data Storage Drive Computer Monitor Video Monitor Figure 1 Experimental Setup o f Fura-2 Imaging System. Fura-2 loaded cells were mounted in a perfusion chamber and viewed through an inverted microscope. Excitation light passed through 340 or 380 nm filters and reflected off a dichroic mirror. A video camera recorded the fluorescence signal from fura-2 loaded cells and the signals were converted from analog to digital and analyzed by a computer. A picosprizter that was connected to a N 2 pressure source locally applied drugs to the MD3 cells and ejected known drug concentrations rapidly and repeatedly from a single-barrel glass micropipette. 24 Converting the ratio values to [Ca 2 + ]i required the values Rmax = 0.12, Rmm = 5.5, KD - 221 (constant), p = 5.4 nM. R m i n is the minimum value of the fluorescence ratio (340 nm/380 nm) observed with 0 m M C a 2 + HHBSS buffer with 50 p M EGTA. R ^ x is the maximum ratio value when 4-bromo-A23582 (10 uM, Molecular Probes), a C a 2 + ionophore, was added to increase the [Ca 2 + ] , The imaging system was calibrated using fura-2 pentapotassium salt (fura-2/K5, 5 p M , Molecular Probes) with a series of buffers containing 100 mM KC1, 10 mM MOPS, and C a 2 + -EGTA/EGTA in ratios that yielded C a 2 + concentrations between 0 and 39.8 p M (pH 7.2) on the current system previously (Yoo et al., 1999). The dissociation constant for fura-2 binding (Kd) and the ratio of fluorescence intensities at 380 nm under zero and saturating C a 2 + concentrations (P) were determined to be 221 and 5.4 nM, respectively. The value for K<j in our fura-2 imaging system was similar to values reported by Grynkiewicz et al. (224 nM, 1985). Experiments were completed in a dark room at RT (22-24 °C) where full-field images of fura-2 fluorescence at 340 nm and 380 nm were recorded every 10 s. Temperature was previously shown not to influence the K D value (Uto etal., 1991). Although the individual levels of fluorescence were different in normal saline, the ratio signal was nearly uniform over the population of cells. The resting fluorescence ratio corresponded to C a 2 + level of approximately ranging from 38 to 142 nM. Baseline level of cytoplasmic free C a 2 + in individual cells was constant during perfusion of HHBSS buffer for up to 2 h. The isobestic point is the wavelength at which the excitation spectra for the C a 2 + bound and unbound forms of the dye intersect and not influenced by ion binding. Therefore, in the present system, the fluorescence measured at the excitation wavelength of 340 nm will rise while excitation from 380 nm will produce a fluorescence signal that will decrease when fura-2 and C a 2 + form a complex (Thayer et ah, 1988). The ratio of these two fluorescent intensities is related to C a 2 + concentration in a manner that is independent of dye concentration, optical path length, or dye bleaching (Thayer et al., 1988). Data are presented as mean ± S E M unless otherwise stated. Most of the data were graphed as measurements of [Ca 2 + ]i over time. Statistical comparison between control and experimental groups was performed by Student's t-test. The data were analyzed using StatView version 4.02 (Abacus Concepts, Berkeley, CA), Prism version 2.0 software (GraphPad, San Diego, CA), and Microsoft Excel version 8.0 software (Microsoft Corporation, Redmond, WA). 26 Chapter III. RESULTS 3.1. Identifying the MD3 cell line From cultures of MD3 cells plated on glass coverslips, I quantified the [Ca 2 + ]i using microfluorimetry imaging. Recording the fura-2 fluorescent ratio values of mouse hybrid sensory neuronal cells enabled the calculation of [Ca 2 +], from the methods described by Grynkiewicz et al. (1985). 3.1.1. Immunocytochemistry First, the immunocytochemistry technique showed that the MD3 cell line possesses neuronal characteristics. Anti-MAP-2 and anti-NF-H (clone Ta51) Abs labelled the hybrid sensory neuronal cells for neuronal properties (Lee et al., 1982). After growing in culture for at least 24 h, cells were fixed and irnmunostained with cell type-specific Abs, staining all the cells in culture (Figure 2). The cells appeared to be a morphologically heterogeneous population containing cells of different sizes and different number of processes with cell body diameters ranging between 10 and 25 pm. The majority of the cells had two or more short- or medium-sized processes (89.6 %; n = 1012). Positive immunoreactivity to MAP-2 and NF-H Abs indicate that the MD3 cells express neuronal phenotypes. 3.1.2. Capsaicin-evoked Response After determining cytoskeletal immunoreactivity, I examined the mouse hybrid sensory neuronal cells for sensitivity to capsaicin (CAP), a pungent tasting ingredient in peppers of the Capsicum family. Sensitivity to CAP helps to identify nociceptive sensory neurons (Bevan & Szolcsanyi, 1990) because of its ability to elicit pain and burning sensations (Simone et al., 1987,1989). It selectively damages sensory neurons, depleting them of neurotransmitter substances like substance P and other neuropeptides (for review see Jancso et al., 1977; Nagy & Hunt, 1983; Holzer, 1988, 1991). Fura-2 imaging showed the effects of CAP on the MD3 27 Figure 2A,B Immunocytochemistry. A. Antibodies against N F - H (Ta51) and B. M A P - 2 specifically labelled the M D 3 cells. Diaminobenzidine revealed the antibody staining. The selected antibodies showed that the hybrid sensory neuronal cells possess neuronal characteristics. Scale bars = 10 pm. 28 cells. The average baseline level of [Ca2 +]i was 89.6 + 7.4 nM (n = 27). The concentration for CAP was chosen on the results of a previous report that found CAP had an EC50 =1.1 pM on rat D R G neurons (Oh et al, 1996). After observing a stable baseline [Ca2+]j, I applied 3 pM CAP for less than 15 sec increased [Ca 2 +]i (14 independent experiments), as illustrated in Figure 3A. The rise in cytoplasmic C a 2 + from a second application of CAP was -80 % smaller than the original exposure, or disappeared completely, presumably due to acute desensitization with rapid onset. Subsequent CAP applications were similarly ineffective for observation periods of up to 30 min. In summary, the CAP-evoked response exhibited pronounced acute desensitization and tachyphylaxis when bath applied. However, to circumvent desensitization and evoke repeatable responses, I applied CAP (30 pM) onto MD3 cells with a Picospritzer, resulting in reproducible responses with increased [Ca2 +]i responses with repeated agonist exposures. Consecutive applications of CAP resulted in increases of [Ca 2 + ]„ ranging from 191 to 630 nM (mean increase in [Ca2+]j = 404 + 56 nM; n = 14; Figure 3B). In 5 separate experiments, I observed little or no increase in [Ca2 +]i on applying capsaicin after a 10 min pretreatment with capsazepine (5 pM), a CAP receptor antagonist (Walpole et al., 1994). The responsiveness of the MD3 cells to CAP is consistent with the characteristics of sensory neurons in vitro and in vivo (Bevan & Szolcsanyi, 1990; Simone et al., 1991). 3.1.3. Effects of a,p-methylene adenosine triphosphate I examined the MD3 cells for responsiveness to a,(3-methylene adenosine triphosphate (ATP) because sensory neurons express an ATP-gated cation conductance (Krishtal et al., 1988a,b; Bouvier etal., 1991). The MD3 cells responded to applications of 100 pM a,P-methylene ATP, increasing [Ca2+]i(mean increase in [Ca 2 +]; = 317 ± 20 nM; n = 58; Figure 4). After a 5 min wash with normal HHBSS, a second response to 100 pM a,p-methylene ATP was observed. The responses were similar in reproducibility in terms of their amplitude and time-A. 3 |LIM C A P 200 + u 100 10 20 B. 600 3 0 J J M C A P - 400 + 200 20 time (min) Figure 3/4,2? Capsaicin (CAP)-mediated responses in the MD3 cells. A Application of 3 u M CAP increased [Ca 2 +] f; however, repeated bath applications led to acute desensitization and tachyphylaxis. B. Local applications of 30 u M CAP increased [Ca 2 +] ; while reducing desensitization. Squares indicate bath applications while arrowheads indicate local applications of CAP. 30 course. I examined the effects of varying the extracellular C a 2 + on the ct,P-methylene ATP-mediated response. Application of 100 uM a,P-methylene ATP did not increase [Ca 2 + ] i inMD3 cells bathing in Ca2 +-free recording solution containing E G T A (Figure 4). This figure shows first two responses to a,P-methylene ATP application in C a 2 + containing HHBSS buffer, then a no response to a,p-methylene ATP during perfusion of Ca2 +-free HHBSS, and finally another response to a,p-methylene ATP in a 1.8 mM Ca2 +recording solution. None of the MD3 cells bathed in this manner showed any C a 2 + elevation in response to ct,p-methylene ATP application during 0 mM C a 2 + HHBSS perfusion. Therefore, the rise in [Ca2 +]i depended on extracellular C a 2 + . Extracellular E G T A had no effect on the indicator and did not produce obvious morphological deterioration in the cells. Cells contain either purinergic receptors P2X1 or P2X3 that react to a,P-methylene ATP (Collo et al, 1996). P2X3 receptors are present in only CAP-sensitive small-diameter sensory neurons (Chen et al., 1995). Therefore, the sensitivity of MD3 neurons to ct,P-methylene ATP is consistent with the presence of purinergic receptors. 3.2. M D 3 Cells Express G A B A Receptors I recorded the [Ca2 +]i to determine the mechanisms and effects of GABA-evoked responses in the mouse neuronal MD3 cells. The [Ca2 +]i for MD3 neurons was recorded during and following application of G A B A alone and in the presence of various drugs. I monitored the intracellular C a 2 + levels to establish baseline levels, then studied [Ca 2 +]i following G A B A application, or G A B A together with G A B A antagonists, followed by washout, and finally G A B A re-stimulation. The response was biphasic at times, with a rapid, transient rise, declining to a plateau, and finally a gradual decrease to basal levels. Construction of a dose-response curve was not possible because of desensitization to longer applications of GABA. MD3 cells soo. 100 uM ATP Figure 4 Effect of ATP on hybrid sensory neuronal cells. 100 u M a,P-methylene ATP applications increased [Ca 2 +] i 5 implying the presence of purinergic receptors in the MD3 cells. The lack of extracellular C a 2 + prevented increases in [Ca2 +]j, implying that this rise in [Ca 2 +] ; depended on the presence of extracellular C a 2 + . Filled squares indicate applications of a,P-methylene ATP. 32 presumably had a high internal [CF] resulting in an efflux of CF on opening of G A B A A R -activated channels. i 3.2.1. GABA-evoked [Ca 2 +]i Elevation In this study, I identified G A B A chemosensitivity with brief pulses of G A B A using a HFfBSS perfusion system. Applications of G A B A (100 pM) evoked repeatable, transient elevations in [Ca2+]jthat recovered to the pre-drug baseline (mean increase in [Ca2+]j = 347 ± 42 nM; n = 54; Figure 5A). The observed GABA-induced C a 2 + increases have similar amplitudes and time courses. MD3 neurons recovered from locally applied G A B A within a few minutes, minimizing desensitization. The GABA-evoked changes in intracellular C a 2 + had either a simple or complex recovery phase. The complex recovery had an initial fast phase of [Ca 2 +]; decline that ended at a level to form a plateau from which [Ca 2 +] i recovered at a much slower rate. I, then, investigated the effects of specific G A B A A R agonists to clarify the role of G A B A A R S in the transient increase of [Ca 2 +] f in MD3 cells. Similar to G A B A , Z A P A (100 pM), a selective and potent agonist of low affinity G A B A A R S , also increased [Ca2+]j of the mouse hybrid DRG neuronal cells (mean increase in [Ca2 +]i= 393 ± 44 nM; n = 24; Figure 6). From these results, I suggest that the MD3 cell line possesses GABARs, more specifically G A B A A R S that were activated, thus increasing [Ca2+]j caused by the application of G A B A or a related agonist. 3.2.2. Effect of Bicuculline on GABA-mediated Response Application of antagonists of GABAergic transmission were used to examine the nature of the receptors responsible for mediating the response in MD3 cells to G A B A stimulation. Applying different compounds that were selective between the G A B A A and G A B A B R , I determined the G A B A R subtype responsible for the production of the [Ca2+]j transients. As mentioned in the Introduction, the G A B A A R is part of a superfamily of ligand-gated ion 33 Figure 5A,B GABA-induced [Ca ]{increases and antagonism of G A B A A receptor. A. Repeated, control responses to 100 uM G A B A in MD3 cells led to rises in intracellular free C a 2 + , [Ca2 +] j. B. 25 u M bicuculline antagonized the G A B A A receptor and decreased in the rise of intracellular C a 2 + . The arrowheads represent local application of G A B A . The solid, black bar represents the bath application of bicuculline for 5 min. Figure 6 Effects of ZAP A. Local applications of ZAP A (100 pM), a G A B A agonist, increased the cytoplasmic free C a 2 + , similar to G A B A -evoked responses. Arrowheads indicate applications of ZAP A. 35 channels that on activation increases CP conductance. Applications of G A B A A R antagonists confirmed the involvement of G A B A A R s in increasing internal C a 2 + for the hybrid D R G neuronal cells. In the recording chamber, bath application of BIC to the MD3 cells, at 2+ concentrations ranging from 100 nM-100 uM, reversibly blocked the increase in [Ca resulting from local applications of GABA. BIC reversibly reduced the amplitude of the GABA-mediated response in MD3 neurons but did not alter the time course of the responses (n = 13). Figure 5B illustrates the time courses of GABA-activated responses under control conditions and during bath application of BIC. This figure shows first GABA-mediated control responses, followed by pre-treatment of BIC for a period of 4 min, followed by a G A B A stimulation of the neurons with BIC present. The concentration-response curve for BIC-mediated inhibition of GABA-induced increase in [Ca2 +]i showed an E D 5 0 = 9.3 u M (Figure 7, cf. with Table 1). I suggest from the BIC and ZAP A experiments provide evidence that G A B A A R S influence increases in [Ca2 +];. With BIC present, the amplitude of GABA-evoked rise in intracellular C a 2 + was reversibly depressed. However, BIC did not affect the baseline level of [Ca2 +]i during pre-treatment of cells prior to co-application with G A B A at the concentrations studied. 3.2.3. Effect of Picrotoxinin and Diazepam on MD3 Neuronal Cells As with BIC, PTX application did not affect the resting [Ca2+]j levels in the MD3 cells. When PTX was bath applied for 5 min, as shown in Figure 8, it did not reduce the amplitude of [Ca2 +]i transients evoked by 100 uM G A B A (n = 9). Simultaneous application of G A B A with 10 u M PTX potentiated the GABA-activated response by an average of 18 ± 4 % over control values. PTX is known to depress GABA-stimulated responses in a non- competitive manner (Gallagher et al., 1978; Akaike et al, 1985); however, in this experiment, PTX did not inhibit the GABA-mediated response. In addition, I applied diazepam to determine if the Figure 7 Concentration-response relationship for bicuculline-induced blockade of G A B A-mediated increase in [Ca 2 +] f, (cf. Table 1). G A B A was locally applied to MD3 cells to elicit the control responses. Then, bath application of bicuculline (100 nM-100 pM) blocked the GABA-evoked increase in [Ca 2 +]j. The inhibition was calculated as a percentage of the averaged controls. The E D 5 0 for bicuculline was 9.3 pM. The relationship between the mean % inhibition and the logarithm of the bicuculline concentration was fitted with a sigmoidal function using GraphPad software. Data represent mean ± S E M (%) (n = 3-5). 37 Table 1 Effects of bicuculline on GABA-induced increase in [Ca 2 +] i . The control responses were averaged elevation in [Ca 2 +]; due to two local applications of G A B A (100 pM), Bath application of bicuculline (100 nM-100 pM) blocked the responses due to local application of GABA. The percent inhibition of the GABA-evoked response was calculated as a fraction of the averaged control values. The values are expressed as mean ± S E M (%). The p values indicate statistical differences from control values (Student's t-test). [Bicuculline]" [ C a 2 ! P value n (pM) (% of control)6 0.1 99.3 ± 7.0 0.6845 4 1 91.8 ± 5.9 0.1541 4 5 80.0 ± 10.8 0.0731 4 10 45.2 ± 8.9 0.0005 5 20 18.3 ± 11.7 0.0001 5 25 13.6 ± 7.3 < 0.0001 3 100 3.8 ± 2.4 < 0.0001 4 a Bicuculline was applied for 5 min. * Average of 2 responses to G A B A applied from micropipette 800 600 + ( N U 400 200 G A B A 10 uM picrotoxinin 10 20 30 time (min) Figure 8 Effects of picrotoxinin. PTX (10 uM) was bath applied to MD3 cells for 5 min prior to G A B A (100 uM) stimulation. PTX did not inhibit the G A B A response. The arrowheads indicate local applications of G A B A . The black, solid bar represents applications of PTX. 39 benzodiazepine receptor is an integral part of the G A B A A R S in MD3 neurons, as described in D R G neurons (Akaike et al, 1989). Bath applications of diazepam (1-50 pM) for 20 min did not significantly affect the [Ca2+]j responses evoked by local applications of G A B A (n = 11; data not shown). The failure of diazepam applications to potentiate the GABA-stimulated response implies that some differences in G A B A A R s may exist between MD3 cells and D R G neurons (see Discussion). 3.3. Mechanism of Increase in [Ca2 +]j 3.3.1. Absence of C a 2 + in the Extracellular Environment I examined the possible ionic mechanisms involved in the GABA-induced response leading to an increase in [Ca2+]j. To determine the route involved in raising internal C a 2 + subsequent to G A B A A R activation, I observed the effect of changes in extracellular C a 2 + on the GABA-evoked response. During the perfusion of Ca2 +-free extracellular solution (0 mM Ca 2 + ) , G A B A (100 pM) did not raise the [Ca 2 +] f, as illustrated in Figure 9A. First, control GABA-evoked responses were established in regular recording buffer with 1.8 mM C a 2 + , and then the hybrid neurons were perfused in Ca2 +-free media with local applications of GABA. The MD3 cells, in the absence of extracellular C a 2 + , failed to respond to GABA. Previous studies have reported that incubation of cells in Ca2 +-free buffer depletes internal C a 2 + stores (Matsuda et al, 1997; Kerper & Hinkle, 1997). The perfusion time was about one minute to avoid depleting intracellular C a 2 + stores while exposing MD3 cells to Ca2 +-free media. The rate of perfusion enabled complete solution exchange within 30 s. During the replacement of Ca2 +-free solution back to normal buffer, applications of G A B A were effective again in increasing [Ca2+]j. Also, during the absence of extracellular C a 2 + , I applied thapsigargin (1-10 pM) and caffeine (1-10 mM) in the bath for 5 min prior to local application of G A B A in 15 cells. Pre-treatment with G A B A 0mMCa 2 + buffer 400-5 200-_ 7 10 20 time (min) Figure 9 GABA-mediated response with changes in extracellular C a 2 + . G A B A (100 pM) application during perfusion of Ca2 +-free buffer resulted in the disappearance of the increase in [Ca 2 +]j. The presence of extracellular C a 2 + was required to initiate the GABA-mediated response. The arrowheads represent G A B A stimulation of MD3 cells. The solid bar represents a 1 min bath application of Ca2 +-free HHBSS buffer. 41 either thapsigargin or caffeine produced little or no change in the baseline [Ca2+]j. The application of either thapsigargin or caffeine also produced no difference in the amplitude of the GABA-induced increase in [Ca 2 +]; (data not shown). The inability of the hybrid sensory neuronal cells to respond to G A B A in Ca2 +-free solution and the return of the GABA-mediated response following the return of Ca2+-containing buffer imply that the increase in [Ca2 +]i following G A B A application was a result of transmembrane influx of C a 2 + , requiring a C a 2 + environment to observe intracellular Ca2+transients. The rise in [Ca 2 +]i resulted from a C a 2 + influx and not release from internal stores. No indicator signal decrease or cell deterioration occurred from the E G T A in the Ca2 +-free HHBSS. 3.3.2 GABA-induced C a 2 + Transient Inhibition with Dihydropyridines My hypothesis was that G A B A stimulation of receptors in MD3 cells increased intracellular free C a 2 + through VGCCs. Therefore, to determine the consistency of this hypothesis, I assessed the effects of C a 2 + channel blockers on VGCC-mediated increase in [Ca 2 + ]i . Neuronal L-type C a 2 + channels are sensitive to DHP antagonists (Fox et al., 1987a,b; Aosaki & Kasai, 1989). G A B A was applied to the hybrid DRG neuronal cells in the presence of nimodipine and nifedipine. I studied the changes in [Ca 2 +]i in MD3 cells during and after applications of G A B A alone as well as in the presence of a dihydropyridine (DHP). Intracellular Ca levels were first monitored for at least 2 min to establish basal levels, then during G A B A application, followed by application of G A B A and a DHP, and finally G A B A alone after washout of the C a 2 + channel blocker. Nifedipine and nimodipine, at concentrations of 1-20 uM, attenuated elevations in [Ca 2 + ]i (n = 27; Figures 10,11). The V G C C blockers were both bath applied, inhibiting the GABA-induced increase in cytoplasmic free Ca in the hybrid neuronal cells. Therefore, I suggest the GABA-induced response results from a depolarization because of a transmembrane influx of C a 2 + through, voltage-gated L-type C a 2 + channels. 60(h GABA 400 KS u 200 10 uM nifedipine 10 20 time (min) 30 Figure 10 Nifedipine-induced inhibition of GABA-evoked response. Nifedipine (10 pM), an L-type C a 2 + channel blocker, inhibited the transient increases in [Ca2+]j. Nifedipine was bath applied for 5 min while G A B A was locally applied simultaneously. The arrowheads represent local applications of G A B A (100 pM). The solid, black bar represents bath application of nifedipine. GABA 300-200-10 uM nimodipine 100^ 10 time (min) 20 Figure 11 Nimodipine-induced inhibition of GABA-mediated response. Nimodipine (10 pM), an L-type C a 2 + channel blocker, attenuated the increase in [Ca2 +] { from G A B A application. Nimodipine was bath applied for 5 min, followed by a picospritzer application of G A B A . The arrowheads represent local applications of G A B A (100 pM). The solid, black bar represents bath application of nimodipine. 44 Extended washing of 10-15 min failed to reverse the effects of nifedipine and nimodipine completely in 12 out of 27 neurons. Both L-type C a 2 + channel blockers were first dissolved in alcohol and then diluted in HHBSS to a final concentration of 0.01 % alcohol. However, the vehicle, alcohol, had no effect on the basal [Ca 2 + ]i when cells were applied with G A B A in the presence of 0.01 % alcohol-containing HHBSS (data not shown). 3.4. Ionic Mechanism of Depolarization 3.4.1. Depolarization-induced [Ca 2 +]i Increase I also studied K+-evoked C a 2 + entry through voltage-sensitive C a 2 + channels in the sensory neuron-derived hybrid cells. High external K + depolarizes excitable cells and, consequently, induces C a 2 + influx via VGCCs (Benham etal., 1992). Increasing K + from its normal level of 2.5 mM to 25 mM elevated [Ca 2 + ]i in MD3 cells (mean increase in [Ca 2 +]i=538 + 60 nM; n = 58). Figure 12 illustrates the effect of local applications of K + (25 mM) and the subsequent depolarization-induced elevations in [Ca2+]j. When the extracellular C a 2 + was replaced with EGTA, no change in [Ca2+]j resulted from the depolarization with high K + . Similar to the GABA-mediated response in the absence of external C a 2 + , I did not observe an elevation of [Ca 2 +]i following K+-evoked depolarizations when the external solution contained no C a 2 + . The response returned following a 3-5 min wash in Ca2+-containing HHBSS buffer. I suggest that the MD3 neurons possess VGCCs that mediate C a 2 + when depolarized with K + . As with GABA-mediated responses, one type of recovery was smooth while the other pattern, observed at times, was more complicated. The initial fast decline of [Ca 2 +]; decline ended at a level from which [Ca2+]j recovered at a much slower rate. Adverse effects of high K + solutions KC1 Figure 12 K+-evoked [Ca2+]j increase. Local applications of K + (100 pM) depolarized hybrid cells and increased their cytoplasmic free C a 2 + . When C a 2 + was removed from the external solution, no increase in [Ca2"1"]! occurred with high K + application. However, on return of extracellular C a 2 + perfusion, the depolarization-evoked [Ca] ; increases returned. Bar indicates perfusion of zero C a 2 + buffer. Arrowheads represent applications of KC1. were not apparent on the cells because the increase in [Ca 2 +]i returned to baseline levels. 3.4.2. Inhibition of K+-evoked Increase in [Ca2+]j As with GABA-induced responses, I investigated the effects of C a 2 + channel blockers on the K+-stimulated C a 2 + increase. I observed the effects of nifedipine and nimodipine on voltage-dependent C a 2 + influx in mouse hybrid D R G neuronal cells in culture. K + evoked depolarizations that activate VGCCs, resulting in [Ca2+]j increases as a consequence of C a 2 + influx. To confirm this mechanism, I applied nifedipine and nimodipine to inhibit C a 2 + influx through VGCCs. When 25 mM K + stimulated the MD3 cells, control responses elevated cytosolic C a 2 + . However, pre-treatment of hybrid sensory neuronal cells with nimodipine (1-20 pM) and nifedipine (1-20 pM) inhibited the increase in [Ca 2 +]i due to K + application (n = 20; Figures 13,14). Full recovery from dihydropyridine application occurred in 10 out of 20 MD3 cells. In some cells (n = 10), however, extended washing for about 10-15 min only partially reversed the total blockade produced by either nimodipine or nifedipine. From the above data, I suggest that similar to G A B A , K+-mediated responses involved C a 2 + influx through L-type VGCCs in the MD3 cells. 3.5. Activation of G A B A B Receptor 3.5.1. G A B A B Receptor Agon ism Using baclofen, an agonist at G A B A B R S , I investigated the presence of G A B A B R S on the MD3 cells. Therefore, in order to observe the effects of G A B A B R stimulation, I first used K + to depolarize the cells while applying baclofen. Baclofen application (50 nM-100 pM) for 5 KCl 400 300 + & 200 100 10 uM nifedipine 10 time (min) 20 Figure 13 Nifedipine-induced inhibition of K+-mediated [Ca2+]j increases. Nifedipine (10 uM), an L-type C a 2 + channel blocker, inhibited the transient increases in [Ca2+]j. Nifedipine was bath applied for 5 min while K C l (25 mM) was locally applied intermittently. The arrowheads represent local applications of K C l . The solid, black bar represents bath application of nifedipine. KCl 300 10 U.M nimodipine + U 200 time (min) Figure 14 Nimodipine-induced inhibition of K+-mediated depolarization. Nimodipine (10 uM), an L-type C a 2 + channel blocker, attenuated the transient increases in [Ca2+]j. Nimodipine was bath applied for 5 min while K + was applied locally. The arrowheads represent local applications of K C l (25 mM). The solid, black bar represents bath application of nifedipine. 49 rnin did not produce significant effects on the baseline values of [Ca2 +]i (n = 87). However, during the application of K + (25 mM), (±)-baclofen (500 nM-100 pM) significantly reduced the amplitude of the K+-induced [Ca 2 +]; transients by 18-100 % (n = 51; Figure 15,4). Baclofen (50 nM-100 M-M) did not cause any perturbations in baseline [Ca2 +]i, implying that there was no constitutive activity involving G A B A B R S . The graph in Figure 16 shows the concentration-response curve for baclofen-induced inhibition of K+-evoked increase in [Ca2+]j which revealed an ED50 =1.8 p M (cf. with Table 2). In these experiments, the K + applications presumably depolarized the MD3 cells, activating VGCCs that permitted C a 2 + influx that increased [Ca2+]j. 3.5.2. G A B A B Receptor Antagonism Once I determined that the MD3 cells possessed G A B A B R S , I investigated the ability of 2-hydroxy-saclofen (1-20 pM) to antagonize the inhibitory effect of baclofen (500 nM-10 pM) on the K+-induced increase in [Ca 2 + ] i (n = 18). 2-hydroxy-saclofen (1 pM) was bath applied for the first 4 min, followed by a 4 min co-application with baclofen (1 pM). Firstly, bath application of 2-hydroxy-saclofen for 4-8 min had little effect on the baseline [Ca2+]j. However, I observed that 2-hydroxy-saclofen antagonized the effects of baclofen on the K+-mediated increase in [Ca2+]j. Before 2-hydroxy-saclofen application, baclofen application decreased the rise in [Ca2 +]iby 25-85 % but 2-hydroxy-saclofen prevented any further effects of baclofen-mediated blockade of the K+-evoked increase in [Ca 2 +]; (n = 11; Figure 15B). Application of 2-hydroxy-saclofen (1-20 pM) also antagonized the subsequent responses to baclofen. 2-hydroxy-saclofen blocked the effects of baclofen-mediated inhibition of KCl-evoked elevation in [Ca ] i (n = 7 ; Figure 17). Therefore, the results show that 2-hydroxy-saclofen antagonized baclofen and blocked the inhibitory effect of baclofen on K+-mediated increase in [Ca 2 +], through VGCCs. When K + presumably depolarized the MD3 cells during co-applications of 2-hydroxy-saclofen and baclofen, the response to K + did not decrease as observed during baclofen 50 application alone (see Figure 15,4). The above results with 2-hydroxy-saclofen imply that MD3 cells have functional G A B A B R s in addition to G A B A A R S . Figure 15,4,1? G A B A B receptor activation and antagonism. A. Using K C l to depolarize cells, baclofen (1 uM) decreased the rise in [Ca2+]j. B. 2-hydroxy saclofen (1 uM) antagonized the baclofen-induced inhibitory response. The arrowheads represent local applications of K C l (25 mM). The solid, black and white lines represent bath applications of baclofen (10 min) and 2-hydroxy saclofen (4 min), respectively. 52 [Baclofen] (M) Figure 16 Concentration-response relationship for baclofen-mediated inhibition of the KCl-induced increase in [Ca 2 +] f, (cf. Table 2). First, K C l was locally applied to MD3 cells for control responses. Then, during bath application of baclofen (50 nM-100 uM) blocked the KCl-evoked increases in [Ca 2 + ] j . The inhibition was calculated as a percentage of two averaged controls. The E D 5 0 for baclofen was 1.8 uM. The relationship between the mean % inhibition and the logarithm of the baclofen concentration was fitted with a sigmoidal function using GraphPad software. Data represent mean + S E M (%) (n = 4-19). 53 Table 2 Effects of baclofen on the KCl-induced increase in [Ca2+]j. The control responses were an averaged elevation in [Ca2 +]i due to two local applications of K C l (25 mM). Then, bath application of baclofen (50 nM-100 uM) blocked the increase in [Ca2+]j due to KC l . The percent inhibition of the KCl-mediated response was calculated as a fraction of the averaged control values and expressed as mean ± S E M (%). The p values indicate statistical differences from control values (Student's t-test). / [Baclofen]" [Ca 2 +] ; P value n (uM) (% of control)6 0.05 94.0 ± 2.8 0.1975 19 0.1 96.7 ± 2.8 0.4595 18 0.5 74.3 ± 5.2 0.0080 15 1 59.6 ± 8.8 0.0107 14 10 28.8 ±7.6 < 0.0001 13 25 4.6 ±3.0 < 0.0001 6 100 0.8 ± 0.5 < 0.0001 4 a Baclofen was applied for 4 min h Average of 2 responses to K C l applied from micropipette Figure 17 Reverse application of 2-hydroxy-saclofen and baclofen. The hybrid cells were first treated with 2-hydroxy-saclofen, followed by baclofen. 2-hydroxy-saclofen prevented subsequent application of baclofen from influencing the K+-mediated increase in [Ca2 +] j. Arrowheads indicate K + (25 mM) application. The solid, black and white bars represent bath applications of baclofen (1 pM) and 2-hydroxy saclofen (1 pM), respectively. 55 Chapter IV. DISCUSSION I studied the [Ca2+]j changes in a mouse hybrid sensory neuronal cell line MD3, generated from mouse D R G sensory neurons and N18TG2 neuroblastoma cells in culture, using C a 2 + imaging with fluorescent Ca2+-chelator fura-2. Fura-2 was trapped inside the cells and visualized using digital imaging of the fluorescence (Williams et al, 1985; Connor, 1985, 1986). Fura-2 was found to be effective in competing with the endogenous C a 2 + buffers, to capture virtually all mcoming C a 2 + (Bollman et al., 1998). Upon binding C a 2 + ions, the excitation maxima of fura-2 are shifted (Thayer et al., 1988). The indicator method does not give a quantitative measure of the C a 2 + influx. However, this procedure directly provides a measure of the [Ca2+]j perturbations. The technique is very powerful, because C a 2 + currents or channel activity is extremely sensitive to the internal dialysis that accompanies methods such as suction-electrode or whole-cell patch recording (Fedulova et al., 1981; Byerly & Hagiwara, 1982; Fenwick etal, 1982). 4.1. Identifying the MD3 Cell Line 4.1.1. Immunocytochemistry The immunocytochemical findings show that the MD3 cells were neuronal in nature. Immunocytochemical detection of cytoskeletal proteins indicates that MD3 cells have a neuronal phenotype (Lee et al., 1982). Reactivity to monoclonal Abs NF-H and MAP-2 provided an identification of MD3 cells as shown in Figure 2. MAP-2 and neurofilament immunoreactivity also exists in D R G neurons (Naves et al, 1996). 4.1.2. Capsaicin-evoked Response CAP causes intense burning pain because of its excitatory action on vanilloid receptors of sensory neurons (Geppetti et al, 1988; Simone et al, 1989). The CAP-activated channels are permeable to cations, including C a 2 + (Cholewinski et al, 1993; Koplas et al, 1997). 56 Applications of CAP to the MD3 cells provided a tentative identification that they possessed nociceptive properties similar to sensory neurons in vivo. From Figure 3 A, for example, the hybrid cells responded to 3 JJ.M CAP, raising the [Ca2+]j. The cells showed acute desensitization, a diminished response during constant CAP application, as well as tachyphylaxis, a decreased response to successive applications to CAP. In Figure 3A, consecutive bath applications of CAP decreased the increases in cytoplasmic C a 2 + with successive stimulation, illustrating both acute desensitization and tachyphylaxis. Acute desensitization was observed as an inactivation of the response during prolonged application of CAP whereas tachyphylaxis is a decrease in responsiveness of the receptor during successive applications (Koplas et al., 1997). CAP stimulates sensory neurons associated with the C- and A8-fibres (Bevan & Szolcsanyi, 1990). Therefore, the similar sensitivity to CAP as D R G sensory neurons (Gschossman et ai, 2000) implies the hybrid sensory neuronal cells have vanilloid receptors and nociceptive properties. 4.1.3. a,p-methylene ATP-induced [Ca2 +]j Increase Sensory neurons are known to have an ATP-gated ion conductances (Krishtal et al., 1983, 1988a,b). Once ATP binds to its receptor, a depolarization results from N a + influx. With fura-2 microfluorometry, I measured the accumulation of cytoplasmic C a 2 + , most likely through C a 2 + influx through ATP-gated cation channels. In general, cells that express P2X1 or P2X3 receptors respond to ap-methylene ATP (Collo et al., 1996). While P2X1 receptors are expressed in sensory ganglion neurons and spinal cord motoneurons (Valera et al., 1994), the P2X3 receptors are found only in CAP-sensitive sensory neurons (Chen et al., 1995). From Figure 4, 100 uM ct,P-methylene ATP applications to MD3 cells elevated [Ca2 +]i. However, during perfusion of zero C a 2 + HHBSS buffer, ct,P-methylene ATP had no effect. Therefore, I suggest that the rise in [Ca2+]j depended on extracellular C a 2 + , resulting from C a 2 + influx through the ATP-gated channels as reported in other studies (Krishtal et ah, 1988a,b; Bean et al., 1990). The results imply that these channels allow sufficient C a 2 + entry in a normal C a 2 + gradient to elevate somatic [Ca 2 +]j. The MD3 cells express receptors that respond to a,p-methylene ATP as well as CAP, corresponding to a nociceptive phenotype. The CAP- and a,P-methylene ATP-mediated responses imply that the hybrid sensory neuronal cell line has nociceptive properties. Also, combining the results from immunocytochemistry and the responses to a,P-methylene ATP and CAP imply the hybrid sensory neuronal cells possess neuronal characteristics. 4.2. G A B A Experiments I investigated the GABA-evoked responses as well as agonists and antagonists in MD3 cells. In the present investigation, G A B A produced a transient rise in [Ca 2 +]j, presumably a consequence of changes in membrane potential. Previous research has revealed that a G A B A -induced depolarization in many types of excitable tissue including crustacean stretch receptors of crayfish muscle (McGeer et al., 1961) and frog spinal ganglion (Nishi et al., 1974). G A B A enhance [Ca 2 +]j in explanted, early postnatal cerebellar granule neurons, mammalian dorsal horn neurons in culture, and early postnatal neocortical neurons of slices (Connor et al., 1987; Yuste & Katz, 1991; Reichling et al, 1994). The data from the current study imply that the [Ca 2 +] ; transients in this cell line may be due to the V G C C s activation from Cl~ conductance-induced membrane depolarization, resulting from an unusually high internal [CP] and an efflux of Cl " . In the present study, the time courses of GABA- , KC1-, and ZAPA-evoked increases in [Ca 2 +]i varied from one experiment to another. The differences in time course of these drugs may arise from the distance between the ejection pipette and the mouse hybrid sensory neuronal cells. 58 4.2.1. GABA-induced [Ca2+]i Increase After having identified the responses of sensory neurons to CAP and a,(3-methylene-ATP sensitivity, and performed immunocytochemistry, I examined the hybrid sensory neuronal cells for GABARs because G A B A modulates transmission in primary afferent neurons (Rudomin and Schmidt, 1999). When stimulated with G A B A , fura-2 fluorescent imaging methods revealed [Ca2 +]i perturbations in the MD3 cells. G A B A evoked reproducible, transient increases in [Ca2+]j in the MD3 cells, implying that they have receptors for GABA. The data from the experiments are consistent with the hypothesis that G A B A activates its receptor, resulting in a Cl~ conductance-induced membrane depolarization that produces C a 2 + influx through nimodipine- and nifedipine-sensitive VGCCs, thereby elevating [Ca2+]j. This mechanism has been observed in acute preparations of dorsal spinal cord and the trigeminal complex of the brain stem from 1- and 2-week old rat pups that have both high- and low-threshold C a 2 + channels (Huang, 1989; Mintz et al, 1992). Of interest is the fact that G A B A A R s and DHP-sensitive VGCCs form tight spatial coupling in neuronal somata (Hansen etal, 1992). 4.2.2. GABA-evoked Depolarization Mechanisms Several different mechanisms may explain the role that G A B A has in evoking an increase in [Ca2 +]i in the MD3 cells. Current through the Cr-permeable channel may depolarize the cell when the sustained concentration for the internal C F environment is greater than that in the extracellular space. This process of GABA-induced depolarization occurs in peripheral ganglion neurons (Nishi et al, 1974; Adams & Brown, 1975). However, in other neuronal preparations such as hippocampal pyramidal cells, this mechanism is insufficient to explain both hyperpolarizing and depolarizing effects within a single neuron (Obata et al, 1978; Alger & Nicoll, 1979; Lambert et al, 1991). Nevertheless, the sensory neuron-derived cells presumably 59 contain a high CT internal environment as demonstrated by their responses to G A B A agonists and K + . A DRG neuron may generate a high CF internal environment through a variety of mechanisms. To maintain a stable depolarizing CT reversal potential, a neuron may employ an inward pump such as a Cr-HCC>3-exchanger with the production of CO2/HCO3 as the driving force or a CF cation transporter driven by the Na + gradient (Kopito, 1990). Evidence for this comes from research on mouse oligodendrocytes and guinea-pig hippocampal neurons where the Cl~ transporter inhibitor, furosemide, reduces GABA-evoked depolarizations (Misgeld et al, 1986; Hoppe & Kettleman, 1989). Depolarization and subsequent activation of VGCCs would result when the intracellular Cl~ concentration is sufficiently high for a net efflux of CF during activation of GABARs. The electrochemical C F diffusion gradient across the afferent neuron membrane is directed outward, the result of an inwardly directed CFpump (Nishi et al., 1974; Gallagher et al., 1978). Also, a major difference has been substantiated when comparing neurons in terms of their recovery time following intracellular Crinjection. Motoneurons recovered in about 30 seconds (Eccles, 1964), 90 seconds for cortical neurons (Kelly et al., 1969), but 30 minutes for the spinal ganglion. One reason for the slow recovery in DRG neurons may be the lower resting CT conductance. In addition, the primary afferent neuron may have effective pumps that prevent outward leakage of CF , not found in motor and cortical neurons (Gallagher et al., 1978). In previous experiments, removal of CF from the external solution was possible by replacing NaCl with an equimolar solution of sodium isethionate. However, there are conflicting reports. Barker & Nicoll (1973) observed in the amphibian that Cr-free media produced a small increase in the membrane potential while others found a decrease in the G A B A potential (Nishi et al, 1974; Kudo et al, 1975) or no change (Constanti & Nistri, 1976) 60 in GABA 's depolarizing capacity following C l " removal. A removal of external CF failed to increase the CP gradient across the nerve terminal membrane during a drug-induced reduction in activity of concentration-dependent CP pump that maintains the gradient. 4.2.3. GABA-evoked C a 2 + Influx As explained earlier, GABA-induced a transient increase in [Ca2+]j through membrane depolarization, presumably due to an increased CP conductance. Previous studies also have demonstrated that stimulation of G A B A A R S results in C a 2 + influx in cultured dorsal horn neurons, as well as neocortical and hippocampal neurons (Reichling et al., 1994; Owens et al., 1996; Canepari et al., 2000). What happens to the hybrid sensory neuron following depolarization? The data show that the GABA-evoked depolarization activated VGCCs since DHPs inhibited the C a 2 + transients. The depolarization would activate other voltage-gated channels such as voltage-gated N a + and K + channels. However, depolarization from GABA-induced CP conductance may inhibit neuronal activity such as APs because it could act as a current shunt, or indirectly activate Ca2+-dependent K + currents (Staley & Mody, 1992). The afferent activity along the sensory nerves would then depend on both the excitatory and inhibitory influences as well as their electrophysiological propertiesof the somata. The GABA-mediated [Ca2+]j increases may also serve a developmental role, since small tonic elevations of [Ca 2 + ] i promote neuronal survival and development in embryonic rat spinal cord cultures (Franklin & Johnson, 1992). Also, G A B A stimulates early stages of synaptogenesis in cultured neuroblastoma cells, influences the differentiation of cultured cerebellar granule neurons, and disrupts developing cerebellum in vivo (Belhage et al., 1998). One study observed the responses to G A B A in the cultures diminished over time; hence, the depolarizing response to G A B A may result in developmental regulation in vivo (Reichling et al., 1994). 61 The regulation of cytoplasmic free C a 2 + concentration is vital to controlling neuronal function (Mironov et al., 1993). The time course of [Ca2+]j accumulation and the rate of [Ca2+]j recovery following Ca influx may be important factors in the activation of different messenger systems. Many studies have observed transient rises of [Ca 2 + ] i induced from membrane depolarization in many neuronal types (Tsien & Tsien, 1990; Miller, 1991; Henzi & MacDermott, 1992). 4.2.4. Bicuculline Antagonism of GABA-response I examined further the GABA-mediated response in the DRG neuronal cell line MD3 to determine the nature of the receptors implicated in enhancing the [Ca2+]j. The results from this section provide evidence that BIC selectively and reversibly blocked the effects of GABA. As shown in Figure 5B, BIC antagonized the GABA-evoked response in the mouse hybrid D R G neuronal cells in culture. In invertebrate muscle (Smith & Costanti, 1981) and cat D R G neurons (Gallagher et al., 1978), BIC was a noncompetitive or 'mixed' noncompetitive antagonist. However, on most vertebrate central and peripheral neurons, BIC was a competitive inhibitor of the GABA-evoked response (Curtis et al., 1971; Bowery & Brown, 1974; Levy, 1977; Simmonds, 1980). The BIC effect on the GABA-mediated response in MD3 cells was either by direct competition with G A B A at its receptor (competitive inhibition) or decreased Cl" channel conductance (non-competitive inhibition). However, deterrnining the exact mechanism of antagonism was not possible using the fura-2 microfluorometry system. Nevertheless, ZAP A applications that activate G A B A A R S increased [Ca2+]j, consistent with the presence of G A B A A R s in MD3 cells (Allan et al, 1997). The observed antagonism by BIC with an ED5o = 9.3 p M is consistent with G A B A and G A B A R interactions (MacDonald & Olsen, 1994). 4.2.5. Effects of Picrotoxinin and Diazepam The mechanism of PTX inhibition of GABA-activated CF conductance is different in a variety of tissues. PTX inhibits the GABA-evoked response competitively and noncompetitively, or a mixture of both effects on lobster muscle (Takeuchi & Takeuchi, 1969; Shank et al, 1974; Constanti, 1978). PTX had similar effects on neuronal membranes (Gallagher et al, 1978; Homma & Rovainen, 1978; Hori et al, 1978; Akaike et al, 1985). However, no PTX inhibition of the GABA-stimulated response was observed in the MD3 cells, as in hippocampal (Segal, 1993) and human dorsal root ganglion neurons (Valeyev et al, 1996). This inconsistency may be due to G A B A R subtypes with different channel properties. Differential manifestation of individual G A B A R subunits and the combination of various distinct subunits influences channel properties (Shivers et al, 1989; Verdoorn et al, 1990; Zhang et al, 1991). Picotoxinin-insensitivity may result from unique subunit combinations, such as mutations in the y2 subunit of the G A B A A R (Shen et al, 1999). In MD3 cells, analogous variations in the benzodiazepine receptor may result in lack of the potentiation of GABA-mediated action by diazepam (Macdonald & Olsen, 1994; Knoflack et al, 1996; Malgrange et al, 1997; Williamson et al, 1998). 4.3. Mechanism of GABA-mediated Increase in [Ca2+]j I investigated the mechanisms concerning responses in MD3 cells to G A B A applications. G A B A binds to its receptor, opening the chloride channel to increase CP conductance and depolarizing the cell. However, what is the process that produced the heightened [Ca2+]i in the hybrid neurons? 63 4.3.1. Absence of C a 2 + in the Extracellular Environment The treatment of the MD3 cells with 0 m M C a 2 + recording buffer during G A B A stimulation established that the [Ca2+]j increase depended on extracellular C a 2 + . Perfusion of Ca2 +-free buffer through the recording chamber eliminated the GABA-induced response, resulting in no change in the in [Ca2+]j (Figure 9). This removed the possibility of the C a 2 + concentration increase occurring through release of internal stores such as the endoplasmic reticulum. Therefore, [Ca 2 +], increased because of C a 2 + influx from the exterior to the interior of the MD3 cell. To rninimize depletion of the internal stores during eradication of extracellular C a 2 + , the exposure time to EGTA-containing Ca2 +-free HHBSS buffer was about a minute. The [Ca2 +]i elevations following G A B A applications returned with the perfusion of Ca2+-containing HHBSS buffer. Also, I did not detect a change in baseline [Ca 2 +] ; on thapsigargin or caffeine application, observed in some cells (cf. Guerini & Carafoli, 1999). However, thapsigargin and caffeine had no effects on GABA-evoked increase in [Ca 2 +] i , implying that the GABA-induced response did not involve C a 2 + release from internal stores. Therefore, C a 2 + influx, not C a 2 + release from internal stores, and extracellular C a 2 + influenced the augmentation in [Ca 2 +] i in MD3 neurons. 4.3.2. GABA-response Inhibition with DHPs The results imply that the GABA- induced response involves G A B A A R activation and extracellular C a 2 + influx. The application of DHP antagonists inhibited GABA-mediated rise in cytoplasmic C a 2 + , verifying that the transmembrane influx of C a 2 + occurred through VGCCs, as in cultured dorsal horn neurons (Reichling et al., 1994) and gonadotropes (Williams et al., 2000). The present study used nimodipine and nifedipine to define the functional presence of L-type channels. From Figures 10 and 11, the application of DHPs inhibited the rise in cytoplasmic C a 2 + from G A B A induction. This provides evidence that the GABA-mediated response initiates entry of Ca2 +through L-type channels that DHPs potently inhibit (Fox et al, 64 1987a; Aosaki & Kasai, 1989; Plumrner et al, 1989). These channels exhibited a slowly inactivating, voltage-dependent C a 2 + current in primary sensory neurons as in chick DRG (Nowycky et al., 1985). In addition, L-type C a 2 + channels controlled the release of substance P from D R G neurons in chick and rat (Pernery et al., 1986; Holz et al., 1988). C a 2 + channels play a significant functional role, though poorly defined, in neurons (Nowycky, 1992). In addition, direct electrical excitability as well as simultaneous conversion of electrical activity into other functional activities such as transmitter release, regulation of gene expression, or further control of membrane excitability involve C a 2 + channels (Nowycky, 1992). VGCCs have an important role in a number of neuronal functions that depend on the intracellular C a 2 + concentration. Fox et al. (1987a) discovered the co-existence of three types of C a 2 + channels in D R G neurons. In addition, various neurotransmitters modulate C a 2 + channel currents in many neuronal cell types (Menon-Johansson et al., 1993), representing one of the mechanisms whereby presynaptic inhibition may occur in both the central and peripheral nervous systems (Dunlap & Fischbach, 1978). In mammalian neurons, cadmium and manganese block Ca2 +currents; however, these divalent ions were unsatisfactory as blockers in experiments with fura-2 (Connor et al., 1987). Conner and associates (1987) discovered that cadmium produced a steady increase in the fluorescence ratio of fura-2 while manganese rapidly decreased the fluorescence ratio values at about 10 % of initial values. Therefore, I applied DHPs to investigate its effects on GABA-stimulated the cytoplasmic C a 2 + increase. Nimodipine and nifedipine, at concentrations ranging from 1-20 uM, inhibited the GABA-induced depolarization that increase [Ca2+]j (Figures 10 and 11). Therefore, G A B A A R activation may have depolarized MD3 cells through a Cl" efflux, initiating C a 2 + influx through DHP-sensitive C a 2 + channels, leading to an increase in [Ca2+]j. 65 4.4. K +-induced Increase in [Ca2 +]j K+-induced [Ca 2 + ] i transients produced through VGCCs increased [Ca 2 +]; (Figure 12). Nimodipine and nifedipine blocked the rapid depolarization from K+-applications and mimicked the DHP inhibition of the GABA-activated response (Figures 10 and 11). Thus, these results demonstrate the influx of extracellular C a 2 + to be important in the depolarization-induced changes in [Ca ] i . Previous studies on neurons and neuronal cell lines showed DFfP inhibition of C a 2 + influx and transmitter release in response to K + depolarization (Toll, 1982; Takahashi & Ogura, 1983; Freedman et al, 1984; Sasakawa et al., 1984; Carboni et ah, 1985; Enyeart et al, 1985; Perney et al., 1986). DHP C a 2 + channel antagonists bind to Wgh-affinity sites and block the depolarization-induced entry of C a 2 + (Miller & Freedman, 1984). The inhibition of C a 2 + influx is due to the selective interaction of the DHPs with VGCCs (Rane et all, 1987). Mnay investigations have reported that DHPs do not affect C a 2 + influx or C a 2 + -dependent transmitter release in intact neurons or synaptosomes (Nachshen & Blaustein, 1979; Ebstein & Daly, 1982; Daniell et al, 1983; Rampe et al, 1984; Ogura & Takahashi, 1984; Shalaby et al, 1984; Boll & Lux, 1985; Perney et al, 1986; Suszkiw et al, 1986). However, other investigations observed the involvement of DHP-sensitive C a 2 + channels for both C a 2 + entry into and transmitter release from synaptosomes, neurons or neuronal cell lines (Toll, 1982; Takahashi & Ogura, 1983; Freedman et al, 1984; Sasakawa et al, 1984; Turner & Goldin, 1985; Enyeart et al, 1985; Middlemiss & Spedding, 1985; Carboni et al, 1985; Perney et al, 1986). In the present study, the C a 2 + channels of the MD3 cell line accounted for C a 2 + entry and were sensitive to nimodipine and nifedipine during K + - and GABA-induced responses. 66 4.5. G A B A B Receptor Activation. The results presented in this section show evidence for G A B A B R S in MD3 cells. Baclofen inhibited neurotransmission at peripheral and central synapses such as primary afferent terminals within the spinal cord (Fox et al., 1978). Without affecting the baseline [Ca2+]j, baclofen reduced the K+-evoked rises in [Ca 2 +]i in MD3 cells (Figure 15,4), agreeing with the results from other studies on rat and chick D R G neurons. G A B A B R s mediated this effect because 2-hydroxy-saclofen did not significantly change the baseline [Ca 2 +]i and completely blocked the baclofen-inhibition of KCl-induced responses. Baclofen directly depressed C a 2 + currents and modulated GABA B R's influence on neuronal excitability (Dunlap, 1984; Deisz & Lux, 1985). Another study showed that bath application of baclofen for 5-10 min suppressed the inward current produced by DRG neurons from exposure to G A B A (Xi et al., 1997). Baclofen-induced inhibition in the MD3 neurons may involve G proteins. Stimulation of G A B A B R S with baclofen or G A B A at the binding site activated G proteins and proceeded through different biochemical pathways (Holz et al., 1986), resulting in the closure of CL channels and a decrease in CF efflux in D R G neurons by phosphorylation of G A B A A R s . In chick D R G cultures, pertussis toxin reduced the effect of G A B A on A P duration, revealing a G -protein involvement in the process (Holz et al., 1986). G A B A B R activation may mediate a decrease in Ca2+conductance through a direct link with G-proteins. Holz et al. (1986) discovered that by incubating DRG neurons with either GDP -P -S , a non-hydrolyzeable analogue of GDP, or pertussis toxin, removed GABA-induced inhibition of Ca2+currents, implying the participation of a Gj-like protein membrane protein. G A B A B R S of embryonic chick sensory neurons in vitro decreased Ca2+-dependent APs with no change in the resting conductance. Activation of G A B A B R during an A P inhibits VGCCs in the membrane, decreases extracellular C a 2 + flux, and results in low neurotransmitter release from presynaptic terminals. Activation of 67 G A B A B R S in the MD3 neurons did not lead to an increase in cytoplasmic C a 2 + . The inhibitory effects of baclofen persisted for the duration of its presence. The ability of the G A B A B R to modulate second-messenger systems may explain the effect of baclofen in depressing [Ca2+]j elevations. For example, the G A B A B R decreased cAMP production (Desrues et al, 1995; Hashimoto & Kuriyama, 1997). In the previous section, nimodipine and nifedipine inhibited the KCl-mediated increase in [Ca2+]j. Since high K + depolarized the MD3 neurons during baclofen application, then the G A B A B R activation that decreased the influx of Ca2+presumably affected L-type Ca2+channels. The observed effects of baclofen at G A B A B R , with an ED50 =1.8 pM, may have resulted from a decrease in voltage-activated C a 2 + currents, as observed in spinal neurons (Batueva et al, 1999). Given that MD3 cells are similar to D R G neurons, the effects of baclofen and the antagonism by a G A B A B R antagonist imply that G A B A B R activation can influence the voltage-dependent activities in sensory ganglia. The co-administration of 2-hydroxy-saclofen with (-)-baclofen provided evidence that the effects of baclofen-mediated inhibition of K+-induced Ca2+transients were specific to the G A B A B R (Curtis et al, 1988; Lambert et al, 1989; Harrison et al, 1990). The results show that bath application of 2-hydroxy-saclofen antagonizes the depressant actions of baclofen in the MD3 neurons. As illustrated from Figure 17, 2-hydroxy-saclofen blocked the inhibitory effects of baclofen during K+-induced enhancement of [Ca2 +];. The effective treatment with 2-hydroxy-saclofen abolished the effect of (-)-baclofen. From these results, I suggest that the mouse hybrid sensory neuronal cells posses G A B A B R s that respond to baclofen and 2-hydroxy-saclofen, influencing K+-evoked Ca2+transients. 68 4.6. C a 2 + Sequestration and Efflux Mechanisms The regulation of cytoplasmic free C a 2 + is central to the control of neuronal function. When Ca2+enters through VGCCs, the heightened [Ca 2 +]j integrates with and influences other intracellular signals, controlling neurotransmitter release, activation of Ca2+-dependent enzymes, and modulation of membrane ion channels (Benham et al., 1992). In fact, extended increases in cytosolic Ca 2 +were acutely cytotoxic (Schanne et al., 1979; Choi, 1988, 1995). This present study showed heightened internal C a 2 + levels in hybrid D R G neuronal cells from G A B A R activation and K+-evoked depolarizations. Then, how do these cells recover from such acute mcreases m Ca 2 + load? One source of help is the mitochondria. In addition to their ubiquitous role as the major synthesizer of ATP in aerobic cells (Nicholls, 1985; Thayer & Miller, 1990), mitochondria have the ability to transport and store Ca 2 +(Nicholls & Akerman, 1982; Akerman & Nicholls, 1983). The mitochondria have a high rate of C a 2 + accumulation. When containing high free C a 2 + of 10 uM or more, mitochondria concentrated on the accumulation of the cation rather than ATP synthesis (Nicholls & Akerman, 1982). Two papers identified a role for mitochondrial uptake of C a 2 + following large C a 2 + loads in DRG neurons (Thayer & Miller, 1990; Duchen et al., 1990). In neuronal tissue, the plasma membrane, inner mitochondrial membrane, and endoplasmic reticulum have a role in the regulation of the cytosolic free C a 2 + concentration (Nicholls & Akerman, 1983). From this current study, G A B A may contributes to excitotoxicity as observed in cortical neuron cultures from heightened [Ca 2 +]i (Erdo et al., 1991). However, once C a 2 + enters the cell through C a 2 + permeable channels, C a 2 + encounters many obstacles that prevent it from influencing its physiochemical properties. C a 2 + binding proteins buffered most of the C a 2 + (> 99 %) that enters the cytoplasm (McBurney & Neering, 1987; Neher & Augustine, 1992; Zhou & 69 Neher, 1993) and taken up into intracellular organelles. However, the two main extrusion mechanisms, sodium-Ca2 + exchange and the ATP-driven Ca 2 +pump, send all the Ca 2 +back across the plasmalemma. 4.7. Sensory Ganglion Soma The arbitrary assumption that the effects of G A B A on the cell body membranes giving rise to terminals should be similar to the terminals comes from the following evidence: G A B A depolarized D R G neurons grown in culture (Nishi et al, 1974; Feltz & Rasminsky, 1974; Deschenes et al., 1976; Obata et al, 1978; Gallagher et al., 1978). Studies observed evidence for synapses on the soma of D R G in chick embryo tissue cultures (Miller et al., 1970; Lodin et ah, 1973). Kayahara and colleagues (1981,1984,1986) discovered synapses between the ganglion cell soma and the terminals of cell processes of unknown origin in their ultrastructural study of the cat spinal ganglion in vivo. The D R G has been viewed as a model for G A B A interactions at presynaptic sites within the spinal cord (Feltz & Rasminsky, 1974; Valeyev et al, 1999; Rudomin & Schmidt, 1999). The effects of transmitters at sensory ganglion somata may have significance for the mechanisms of action of drugs for potential therapy of sensory nerve injury and allodynia (Gordon et al, 1995; Baccei & Kocsis, 2000; van Hilten et al, 2000). The MD3 cells have many properties of D R G neurons in vitro and in vivo. They possess functional G A B A A and G A B A B R S , providing a simple system for studying drug effects of D R G neurons. 70 Chapter V . S U M M A R Y AND CONCLUSIONS 1.1 studied the properties of a cell line (MD3) produced from hybridization between mouse D R G sensory neurons and N l 8TG2 neuroblastoma cells. Immunostaining with MAP-2 and NF-H Abs demonstrated that MD3 cells had neuronal characteristics. In addition, the hybrid sensory neuronal cells reacted to CAP and ct,P-mefhylene ATP, similar to sensory neurons. 2.1 investigated the mechanisms and effects of GABA-evoked depolarizations on the mouse hybrid sensory neuronal cells in culture using fura-2 imaging of [Ca2+]j. 3. The MD3 cell line possessed G A B A A and GABA B Rs . G A B A A R activation increased the [Ca 2 +]i of these sensory neuronal cells. This augmentation of [Ca2+]j may have resulted from GABA-mediated increase in Cl~ conductance that depolarized the membrane potential, resulting in C a 2 + influx through voltage-dependent C a 2 + channel activation. BIC, but not PTX, prevented transient rises in [Ca2+]j. 4. Nifedipine and nimodipine inhibited the GABA- and K+-mediated transmembrane influx of C a 2 + through VGCCs, presumably through blockade of C a 2 + entry through L-type channels. 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