Analysis of GABA(A)- and GABA(B)-receptor mediated effects on intracellular Ca2+ in DRG hybrid neurones

1 Using pharmacological analysis and fura-2 spectrofluorimetry, we examined the effects of gamma -aminobutyric acid (GABA) and related substances on i ntracellular Ca2+ concentration ([Ca2+](i)) of hybrid neurones, called MD3 cells. The cell line was produced by fusion between a mouse neuroblastoma c ell and a mouse dorsal root ganglion (DRG) neurone. 2 MD3 cells exhibited DRG neurone-like properties, such as immunoreactivity to microtubule-associated protein-2 and neurofilament proteins. Bath appli cations of capsaicin and alpha, beta -methylene adenosine triphosphate reve rsibly increased [Ca2+](i). However, repeated applications of capsaicin wer e much less effective. 3 Pressure applications of GABA (100 muM), (Z)-3-[(aminoiminomethyl) thio] prop-2-enoic acid sulphate (ZAPA, 100 muM), an agonist at low affinity GABA (A)-receptors, or KCl (25 mM), transiently increased [Ca2+](i). 4 Bath application of bicuculline (100 nM-100 muM), but not picrotoxinin (1 0-25 muM), antagonized GABA-induced increases in [Ca2+](i) in a concentrati on-dependent manner (IC50=9.3 muM). 5 Ca2+-free perfusion reversibly abolished GABA-evoked increases in [Ca2+]( i). Nifedipine and nimodipine eliminated GABA-evoked increases in [Ca2+](i) . These results imply GABA response dependence on extracellular Ca2+. 6 Baclofen (500 nM-100 muM) activation of GABA(B)-receptors reversibly atte nuated KCl-induced increases in [Ca2+](i) in a concentration-dependent mann er (EC50 = 1.8 muM). 2-hydroxy-saclofen (1-20 muM) antagonized the baclofen -depression of the KCl-induced increase in [Ca2+](i). 7 In conclusion, GABA(A)-receptor activation had effects similar to depolar ization by high external K+, initiating Ca2+ influx through high volta ge-a ctivated channels, thereby transiently elevating [Ca2+](i). GABA(B)-recepto r activation reduced Ca2+ influx evoked by depolarization, possibly at Ca2 channel sites in MD3 cells.