Analysis of GABA(A)- and GABA(B)-receptor mediated effects on intracellular Ca(2+) 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 intracellular Ca(2+) concentration ([Ca(2+)]i) of hybrid neurones, called MD3 cells. The cell line was produced by fusion between a mouse neuroblastoma cell 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 applications of capsaicin and alpha, beta-methylene adenosine triphosphate reversibly increased [Ca(2+)]i. However, repeated applications of capsaicin were much less effective. 3. Pressure applications of GABA (100 microM), (Z)-3-[(aminoiminomethyl) thio] prop-2-enoic acid sulphate (ZAPA; 100 microM), an agonist at low affinity GABA(A)-receptors, or KCl (25 mM), transiently increased [Ca(2+)]i. 4. Bath application of bicuculline (100 nM - 100 microM), but not picrotoxinin (10 - 25 microM), antagonized GABA-induced increases in [Ca(2+)]i in a concentration-dependent manner (IC(50)=9.3 microM). 5. Ca(2+)-free perfusion reversibly abolished GABA-evoked increases in [Ca(2+)]i. Nifedipine and nimodipine eliminated GABA-evoked increases in [Ca(2+)]i. These results imply GABA response dependence on extracellular Ca(2+). 6. Baclofen (500 nM - 100 microM) activation of GABA(B)-receptors reversibly attenuated KCl-induced increases in [Ca(2+)]i in a concentration-dependent manner (EC(50)=1.8 microM). 2-hydroxy-saclofen (1 - 20 microM) antagonized the baclofen-depression of the KCl-induced increase in [Ca(2+)]i. 7. In conclusion, GABA(A)-receptor activation had effects similar to depolarization by high external K(+), initiating Ca(2+) influx through high voltage-activated channels, thereby transiently elevating [Ca(2+)]i. GABA(B)-receptor activation reduced Ca(2+) influx evoked by depolarization, possibly at Ca(2+)-channel sites in MD3 cells.
Yokogawa
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Division of Neurology, Department of Medicine, The University of British Columbia, Vancouver, BC, Canada.
Kim
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SU
Krieger
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Puil
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D000179:Acrylates / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001640:Bicuculline / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002211:Capsaicin / Q000494:pharmacology; D002460:Cell Line; D003975:Diazepam / Q000494:pharmacology; D004095:Dihydropyridines / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018755:GABA Agonists / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006822:Hybrid Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011963:Receptors, GABA-A / Q000502:physiology*; D018080:Receptors, GABA-B / Q000502:physiology*; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D000179:Acrylates; D004095:Dihydropyridines; D018755:GABA Agonists; D018756:GABA Antagonists; D011963:Receptors, GABA-A; D018080:Receptors, GABA-B; D002110:Caffeine; D005680:gamma-Aminobutyric Acid; D011189:Potassium Chloride; D019284:Thapsigargin; D000255:Adenosine Triphosphate; C058552:3-((aminoiminomethyl)thio)-2-propenoic acid; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D003975:Diazepam; D002211:Capsaicin; D002118:Calcium; D001640:Bicuculline