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Intracellular calcium changes in a hybrid mouse dorsel root ganglion (DRG) neuronal cell line MD3 following [gamma]-aminobutyric acid (GABA) receptor activation

University of British Columbia

Application of y-aminobutyric acid (GABA) to dorsal root ganglion (DRG) neurons in vivo increases their membrane conductance for Cl⁻. I studied a mouse DRG 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 GABA on MD3 cells in vitro. I used fura-2 to monitor GABA-evoked depolarizations in terms of intracellular Ca²⁺, [Ca²⁺][sub i]. Application of GABA to hybrid cells caused [Ca²⁺i increases that implied GABA[sub A] receptor (GABA[sub A]R) activation. Applications of GABA[sub A]R antagonists bicuculline (BIC) and picrotoxinin (PTX) confirmed the GABA[sub A]R involvement in the intracellular response. BIC, but not PTX, blocked the GAB A-mediated elevations in [Ca²⁺i. ZAP A, a potent agonist of low affinity GABAARs, also increased [Ca²⁺i in hybrid cells. The GABA-evoked increases in [Ca²⁺i were not apparent during the absence of Ca²⁺ in the extracellular environment and exposure to dihydropyridines (DHP) nimodipine and nifedipine. The results imply that depolarization-activated Ca²⁺ influx increased [Ca²⁺i during GABA application. I also studied the effects of GABA[sub B] receptor (GABA[sub B]R) agonist, p-parachlorophenyl-y-aminobutyric acid (baclofen), on the hybrid cells. Detectable changes in the [Ca2+]j were not apparent during GABA[sub B]R activation alone. 2-hydroxy sac lo fen blocked the inhibitory effect of baclofen and antagonized the GABA[sub B]R. The MD3 hybrid cell line possesses GABA[sub A] and GABA[sub B]R[sub S]. The augmentation in [Ca²⁺j occurred when GABA activated the GABA[sub A]R, increasing Cl⁻ conductance and depolarizing the membrane potential, thus resulting Ca²⁺ influx through voltage-dependent Ca²⁺ channels that increase intracellular Ca²⁺. The source of the elevations in [Ca²⁺][sub i] was the extracellular environment. Nifedipine and nimodipine inhibited the GABA-induced transmembrane influx of Ca²⁺ through VGCCs. Activation of GABA[sub B]Rs with baclofen attenuated the depolarization-induced increases in [Ca²⁺][sub i]. In addition, baclofen influenced L - type channels since DHPs inhibited the K⁺-mediated [Ca²⁺][sub i] elevations. The MD3 cells exhibited similarities to its sensory parent. Nevertheless, it is a useful model system for the examination of GABA and other drug effects.

Thesis/Dissertation

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2009-07-20

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http://hdl.handle.net/2429/11036

Experimental Medicine

University of British Columbia

UBCV

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