P A Calkin

Baylor College of Medicine, Houston, Texas, United States

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Publications (2)6.65 Total impact

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    ABSTRACT: Chick embryos with an undeveloped blood-brain barrier were used to examine the down-regulation of GABAA receptors in vivo. The GABAA receptor agonist isoguvacine (5 mumol) was applied to the vascularized chorioallantoic membrane of 8 day embryos. This treatment was repeated on embryonic days 11, 14, and 17, and the embryos were sacrificed on day 18 (stage 42). Isoguvacine administration reduced the clonazepam-displaceable binding of [3H]flunitrazepam to washed cerebellar membranes by 34.0 +/- 3.0% compared to vehicle-treated controls. Binding reductions of lower magnitude were found in membranes from the cerebrum and optic lobes. Administration of isoguvacine had no significant effect on the wet weights of whole embryos or cerebella, the yield of cerebellar membranes, or the binding of [3H]N-methylscopolamine. The reduction of [3H]flunitrazepam binding to cerebellar membranes was dose-dependent, allowing a half saturation value of 8 microM isoguvacine to be estimated. Scatchard analysis showed that the Bmax for [3H]flunitrazepam binding was reduced by 28.3 +/- 6.7% compared to controls, without a change in the Kd. Embryonic exposure to isoguvacine also caused a reduction of 43.6 +/- 6.0% in the binding of the GABAA receptor channel ligand [35S]t-butylbicyclophosphorothionate to washed cerebellar membranes. Taken together, these results indicate that isoguvacine induces a down-regulation of the receptor subunits in vivo. However, measurements of cerebellar GABAA receptor mRNAs for the alpha 1, beta 2L, beta 2S, beta 4, gamma 1, gamma 2L, and gamma 2S subunits by reverse transcriptase-polymerase chain reaction (RT-PCR) revealed no significant alterations by isoguvacine administration. The data suggest that translational or post-translational mechanisms, rather than those modulating the synthesis or stability of subunit mRNAs, take precedence in establishing GABAA receptor down-regulation.
    Molecular Brain Research 11/1994; 26(1-2):18-25. · 2.00 Impact Factor
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    P A Calkin, E M Barnes
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    ABSTRACT: The impermeant SH-cleavable reagent, 125I-labeled 3,3'-dithiopropionyl 1-sulfosuccinimidyl 1'-glycyltyrosine (DPSgt) and specific immunoprecipitation were used to quantitate gamma-aminobutyric acid-A (GABAA) receptor polypeptides with domains exposed on the surface of chick cortical neurons. The 125I label incorporated into 50- and 53-kDa receptor subunits was completely removed by washing the cells with glutathione (GSH) buffer. A single dose of 100 microns GABA or GABA agonist was added to the neurons, and after 5 days in culture the cells were washed and labeled with 125I-DPSgt. This treatment with GABA or isoguvacine reduced the level of the receptor 125I-labeled subunits by 50-60%, while the reduction by 4,5,6,7-tetrahydroisoazolo[5,4-c]pyridin-3-ol was less than 17%. The subunit down-regulation by agonist was prevented by the GABAA antagonist 3 alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (R5135). Direct iodination of membranes isolated from treated cells revealed a similar loss of subunits, indicating that the down-regulated polypeptides are not retained intracellularly. Furthermore, the level of intracellular [3H]flunitrazepam binding (per mg of protein) did not change significantly during chronic GABA treatment, while the fraction of intracellular binding rose from 7% to 15% of the total, owing to a decline in surface binding. The fate of the surface subunits during acute agonist exposure was examined by labeling intact neurons with 125I-DPSgt at 0 degrees C, incubating with GABA for 2-4 h at 37 degrees C and then washing with GSH buffer. Of the 50-53-kDa receptor peptides originally on the surface, 16 +/- 2% became protected from GSH during the 2-h GABA treatment. This sequestration was not found in cells incubated without GABA, with GABA + R5135, or with GABA at 0 degree C. A consistently lower level of sequestered subunits was recovered after a 4- versus 2-h GABA treatment at 37 degrees C, suggesting polypeptide degradation. Overall, the results indicate that GABAA receptor sequestration and subsequent degradation play an important role in agonist-dependent down-regulation.
    Journal of Biological Chemistry 02/1994; 269(2):1548-53. · 4.65 Impact Factor

Publication Stats

34 Citations
6.65 Total Impact Points

Institutions

  • 1994
    • Baylor College of Medicine
      Houston, Texas, United States