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Modulation of adenylyl cyclase by GABAB receptors in vitro and in vivo

Knight, Antony R.; (1995) Modulation of adenylyl cyclase by GABAB receptors in vitro and in vivo. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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In previous studies it has been shown that forskolin and noradrenaline stimulate cAMP production in in vitro preparations of rat brain tissue, and that baclofen can inhibit the forskolin stimulation, and augment the noradrenaline stimulation. The purpose of the present study was to determine the pharmacological profile of this GABAb receptor mediated response, and investigate its physiological relevance by using in vivo microdialysis in awake freely moving rats, and by determining the class of cells in which the response occurs. Thus, in a cross-chopped rat cortical slice preparation, it was confirmed that (-)Baclofen inhibited forskolin stimulated adenylyl cyclase activity and augmented noradrenaline stimulated adenylyl cyclase activity. The potency of five further agonists was investigated (SKF97541, CGP47656, CG44533, 3-APA and CGP44532). Of these agonists two compounds were significantly more potent as inhibitors of forskolin stimulated adenylyl cyclase than as augmenters of noradrenaline stimulated adenylyl cyclase activity, these were (-)baclofen (pEC50 of 6.07±0.29 and 5.04±0.17 respectively (p<0.01)), and CGP47656 (pEC50=6.44±0.05 and 4.08±0.26 respectively (p<0.01)). The specificity of (-)baclofen and CGP47656 may be due to the low intrinsic activity of these compounds, and a relatively high receptor reserve for the GABAB receptor mediated inhibition of forskolin stimulated adenylyl cyclase activity. Six antagonists (CGP49311A, CGP46381, CGP45024, CGP35348, CGP45397, CGP36742) were also tested for their ability to antagonize 30 μM (-)baclofen. These ranged in potency in the forskolin assay from CGP49311A (pEC50 5.4±0.30) to CGP36742 (pEC50 3.87±0.16). Each compound had similar potency in the two assays. In vivo microdialysis was used to investigate the ability of forskolin, noradrenaline and (-)baclofen to modulate adenylyl cyclase activity in the frontal cortex of rats. Forskolin (100 μM) administered through the dialysis probe (flow rate 2 μl.min-l, 30 min fractions), caused a significant (p<0.01) elevation in cAMP in dialysates. A two pulse protocol gave a control S2/S1 ratio of 7.3±0.25. (-)Baclofen (10 μM, 100 μM, 1 mM and 10 mM) was administered through the probe during the second forskolin pulse, however, the S2/S1 ratios in the presence of (-)baclofen did not differ significantly from control (1.38±0.37, 1.27±0.52, 1.14±0.13 and 3.66±1,92 respectively). Using different conditions (flow rate 1.2 μ1 min-1, 10 min fractions) noradrenaline administered through the probe also caused a significant increase in cAMP concentration in dialysates (p<0.05), however a second pulse was ineffective. The inclusion of (-)baclofen in the perfusion medium during the first pulse increased the concentration of cAMP in dialysates, but the difference was not significant. Experiments were also conducted to determine the relative contribution made to forskolin and noradrenline stimulated adenylyl cyclase activity by glial cells and neurons. Flourocitrate, a glial cell specific metabolic inhibitor, reduced forskolin stimulated adenylyl cyclase activity by 78% and eliminated the stimulation caused by noradrenaline in cross chopped cortical slices. In cultured glial cells both forskolin and noradrenaline caused large increases in cAMP production (306 fold and 93 fold respectively). However, these responses were not modulated by 100 ?M (-)baclofen. These data suggest that most noradrenaline, and a proportion of forskolin and noradrenaline stimulated adenylyl cyclase activity may be associated with glial cells. In addition, the long term effects of GABAB receptor activation and antagonism were investigated. Thus rats were dosed with (-)baclofen and the GABAB antagonists CGP46381 and CGP36742 for three weeks ((-)baclofen, 10 mg.kg-1day-1; antagonists 100 mg.kg-1day-1, IP). No regulation of GABAB receptor function, as assayed by modulation of adenylyl cyclase activity in frontal cortex, was detected. Autoradiography detected an up regulation of radioligand receptor binding in the lateral thalamic nucleus (115% increase) in response to CGP46381, and CGP36742 caused an up regulation in the lateral dorsal thalamic nucleus (103 %), the substantia nigra (156%) and the CA3 pyramidal cell layer of the hippocampus (68%). (-)Baclofen caused a complex spectrum of up and down regulation in different brain regions which requires confirmation in a more detailed study. Genetic Absence Epilepsy Rats from Strasbourg are a strain of rats susceptible to absence seizures. An autoradiographic study of the brains of these animals revealed no differences in the levels of radioligand binding to GABAA and GABAB receptors between these animals and controls. This suggests that an alteration in these receptor populations might not underlie this genetic condition.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Modulation of adenylyl cyclase by GABAB receptors in vitro and in vivo
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Health and environmental sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10104881
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