ABSTRACT
We developed a non-radioisotopic quantitative competitive RT-PCR method for the measurement of gamma-aminobutyric acid (GABA) type A receptor subunit mRNA levels. The specificity of the method was optimized by the use of four subunit-specific oligonucleotides in the sequential steps: reverse transcription, polymerase chain reaction (PCR), and detection. The biotinylated PCR products were bound on streptavidin-coated microtiter plates allowing detection of the products using dinitrophenyl (DNP)-labeled probes and anti-DNP alkaline phosphatase conjugate. The method was set up for the six major cerebellar GABA(A) receptor subunits: alpha1; alpha6; beta2; beta3; gamma2 and delta. The method is quantitative and rapid. With a large dynamic range from 10 fg to 1 ng of subunit mRNA, the accuracy was 12 and 19% (intra- and interassay coefficients of variation, respectively), which might be improved by using a smaller range of standards. The use of a double logarithmic standard curve [log (standard to competitor signal) vs. log (standard mRNA originally present)] requires only one reaction from each sample, allowing the analysis of a large number of samples in one experiment.
Subject(s)
RNA, Messenger/metabolism , Receptors, GABA-A/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Animals , Cerebellum/metabolism , Cerebral Cortex/metabolism , Male , Mice , Rats , Reproducibility of ResultsABSTRACT
Benzodiazepine- and alcohol-induced ataxias in rodents have been proposed to be affected by the gamma-aminobutyric acid type A (GABAA) receptor alpha 6 subunit, which contributes to receptors specifically expressed in cerebellar granule cells. We have studied an alpha 6 -/- mouse line for motor performance and drug sensitivity. These mice, as a result of a specific genetic lesion, carry a precise impairment at their Golgi-granule cell synapses. On motor performance tests (rotarod, horizontal wire, pole descending, staircase and swimming tests) there were no robust baseline differences in motor function or motor learning between alpha 6 -/- and alpha 6 +/+ mice. On the rotarod test, however, the mutant mice were significantly more impaired by diazepam (5-20 mg/kg, i.p.), when compared with alpha 6 +/+ control and background C57BL/6J and 129/SvJ mouse lines. Ethanol (2.0-2.5 g/kg, i.p.) produced similar impairment in the alpha 6 -/- and alpha +/+ mice. Diazepam-induced ataxia in alpha 6 -/- mice could be reversed by the benzodiazepine site antagonist flumazenil, indicating the involvement of the remaining alpha 1 beta 2/3 gamma 2 GABAA receptors of the granule cells. The level of activity in this synapse is crucial in regulating the execution of motor tasks. We conclude that GABAA receptor alpha 6 subunit-dependent actions in the cerebellar cortex can be compensated by other receptor subtypes; but if not for the alpha 6 subunit, patients on benzodiazepine medication would suffer considerably from ataxic side-effects.
Subject(s)
Ataxia/physiopathology , Benzodiazepines/pharmacology , Purkinje Cells/chemistry , Purkinje Cells/physiology , Receptors, GABA-A/genetics , Animals , Ataxia/chemically induced , Central Nervous System Depressants/pharmacology , Diazepam/pharmacology , Ethanol/pharmacology , GABA Modulators/pharmacology , GABA-A Receptor Agonists , Glutamic Acid/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Motor Activity/physiology , Nerve Fibers/chemistry , Nerve Fibers/physiology , Psychomotor Performance/drug effects , Receptors, GABA-A/metabolism , Synapses/chemistry , Synapses/physiologyABSTRACT
Inhibitory GABA(A) receptors are regulated by numerous allosteric modulators, the most receptor-subtype specific of which is furosemide. It recognises receptors of the subunit composition alpha6beta2/3gamma2, restricted to cerebellar granule cells. To locate furosemide's site of action we constructed chimeras of the furosemide-sensitive alpha6 and the furosemide-insensitive alpha1 subunit, and expressed and studied them together with the beta3 and gamma2 subunits in Xenopus oocytes by the two-electrode voltage clamp technique. The inhibition of GABA-induced currents by furosemide mainly depended on a short domain proximal to the first transmembrane region of the alpha6 subunit.
Subject(s)
Cell Membrane/physiology , Furosemide/pharmacology , GABA Antagonists/pharmacology , GABA-A Receptor Antagonists , Allosteric Regulation , Animals , Base Sequence , In Vitro Techniques , Molecular Sequence Data , Oocytes/physiology , XenopusABSTRACT
The NO synthases (NOS) generate NO from L-arginine. High concentrations of NO have been shown to be responsible for tissue injury and cell death, while low concentrations of NO induce vasodilatation and other signaling effects. We have investigated the involvement of NO in contact hypersensitivity (CHS) reactions. CHS induced by treatment of BALB/c mice with the contact allergen 2,4-dinitrofluorobenzene (DNFB) was significantly reduced by the NOS inhibitor N-methyl-L-arginine (L-NMA), but not by the stereoisomer D-NMA, as shown by reduced ear swelling responses and evaluation of ear tissue sections. The CHS response was also reduced by aminoguanidine, which is known to preferentially inhibit the inducible isoform of the enzyme (iNOS), suggesting that iNOS contributed to the inflammatory response. We therefore investigated whether iNOS was expressed by epidermal cells. Epidermal Langerhans cells produced low but significant amounts of iNOS mRNA at the single-cell level as indicated by RT-PCR. Likewise, keratinocytes expressed basic iNOS mRNA levels. Elicitation of a CHS response by DNFB in vivo resulted in enhanced iNOS mRNA expression in Langerhans cells and keratinocytes, with higher levels of expression in Langerhans cells. The enhanced mRNA expression in Langerhans cells correlated with iNOS protein production as shown by immunofluorescence staining of epidermal sheets performing double staining with anti-iNOS and anti-MHC class II antibodies. Our data suggest that epidermal cell-derived NO contributes to the ear swelling reaction in CHS.
