Further characterisation of the [35S]-TBPS binding site of the GABA receptor complex in locust (Schistocerca gregaria) ganglia membranes

1. Using homogenates of supraoesophageal ganglia from locust we observed specific binding of [35S]-TBPS which was linear with protein concentration up to 7 mg/ml, showed a pH optimum at pH 9.0 and was linear with NaCl concentration up to 350 mM. 2. Kinetic analysis of the binding showed positive cooperativity as a result of changes in on and off-rates with occupation of the binding site by the ligand. The apparent KD = 417 nM and Bmax = 1083 fmol/mg of membrane protein were calculated using a computer program for dose-response curve fitting. 3. The binding was enhanced by GABA, pentobarbital and benzodiazepines. Picrotoxinin had no effect on the binding at 0.1 mM. Only the cage convulsants TBPS and IBP were able to displace the binding. 4. Whilst the characteristics of the binding are similar to those reported for house fly thorax and abdomen preparations they are significantly different from those reported for house fly head, cockroach nerve cord and rat brain.     

Stereoselective Nicotine-Induced Release of Dopamine from Striatal Synaptosomes: Concentration Dependence and Repetitive Stimulation

Using a sensitive perfusion system we have studied the nicotine-induced release of [3H]dopamine ([( 3H]DA) from striatal synaptosomes. Nicotine-evoked release was concentration dependent with an EC50 of 3.8 microM. The response to 1 microM nicotine was comparable to that to 16 mM K+; 10 microM veratridine evoked a larger response. All three stimuli were Ca2+ dependent but only the response to veratridine was blocked by tetrodotoxin. Repetitive stimulations by 1 microM (-)-nicotine (100 microliters) at 30-min intervals resulted in similar levels of [3H]DA release; higher concentrations of (-)-nicotine resulted in an attenuation of the response particularly following the third stimulation. This may reflect desensitisation or tachyphylaxis of the presynaptic nicotinic receptor. The action of nicotine was markedly stereoselective: a 100-fold higher concentration of (+)-nicotine was necessary to evoke the same level of response as 1 microM (-)-nicotine. It is proposed that these presynaptic nicotinic receptors on striatal terminals are equivalent to high-affinity nicotine binding sites described in mammalian brain.     

γ-Aminobutyric Acid Receptor Complex of Insect CNS: Characterization of a Benzodiazepine Binding Site

The specific binding of [N-methyl-3H]flunitrazepam ([3H]FNZP) to a membrane fraction from the supraoesophageal ganglion of the locust (Schistocerca gregaria) has been measured. The ligand binds reversibly with a KD of 47 nM. The binding is Ca2+-dependent, a property not found for the equivalent binding site in vertebrate brain. The pharmacological characteristics of the locust binding site show similarities to both central and peripheral benzodiazepine receptors in mammals. Thus binding is enhanced by gamma-aminobutyric acid (GABA), a feature of mammalian central receptors, whereas the ligand Ro 5-4864 was more effective in displacing [3H]FNZP than was clonazepam, which is the pattern seen in mammalian peripheral receptors. The locust benzodiazepine binding site was photoaffinity-labelled by [3H]FNZP, and two major proteins of Mr 45K and 59K were specifically labelled. In parallel experiments with rat brain membranes a single major protein of Mr 49K was labelled, a finding in keeping with many reports in the literature. We suggest that the FNZP binding site described here is part of the GABA receptor complex of locust ganglia. The insect receptor appears to have the same general organization as its mammalian counterpart but differs significantly in its detailed properties.     

INCREASED INCORPORATION OF [G-3H]LEUCINE INTO A POSSIBLE ''RECEPTOR'' PROTEOLIPID IN DENERVATED MUSCLE IN VIVO

Abstract— The incorporation of radioactive leucine into the total proteins and the proteolipids of normal and denervated rat diaphragm has been studied in vivo. Denervation increased the incorporation of isotopically labelled leucine into each of the isolated proteolipids and the effect was particularly marked in a single proteolipid which has been designated a 'receptor' proteolipid. In normal muscle this particular proteolipid was found to have a higher incorporation of isotopically labelled leucine in the area of the muscle rich in endplates compared with an area devoid of endplates. However the stimulatory effect of denervation on the incorporation of radioactive leucine into this proteolipid was considerably more marked in the latter region. An attempt has been made to correlate these findings with the development of the hypersensitivity to ACh characteristic of denervated muscle.     

Interrelationship of carbohydrate and the alpha-toxin binding site on the acetylcholine receptor from Torpedo marmorata

The relationship of the carbohydrate components of purified acetylcholine receptor (AChR) to its acetylcholine binding site was investigated by two approaches. In the first, the effect of periodate or glycosidase treatment of AChR on its ability to bind α-bungarotoxin was assessed. Although loss of binding capacity was observed, this could be attributed to increased temperature, acid pH or high salt concentrations of the incubation conditions rather than to the specific action of periodate or glycosidases, indicating that the α-toxin binding site does not directly involve carbohydrate.The second approach involved the use of concanavalin A to block the binding of α-toxin to AChR, when a maximum inhibition of approximately 40% was obtained. The results are interpreted in terms of heterogeneity of AChR molecules, of which some 40% have sterically interacting sites binding concanavalin A and α-toxin respectively.     

Cervical Cytology Specimen Stability in Surepath Preservative and Analytical Sensitivity for HPV Testing with the cobas and Hybrid Capture 2 Tests

None of the commercial HPV tests are U.S. FDA-approved for testing of cervical cytology specimens in SurePath preservative. Still, ~30% of HPV testing is performed on specimens in this formalin-containing preservative. Formalin-induced DNA fragmentation and cross-linking may interfere with HPV detection. We evaluated analytical sensitivity and specimen stability of the cobas 4800 HPV (Roche) and Hybrid Capture 2 HPV (HC2, Qiagen) tests with residual cervical cytology samples in SurePath preservative available within 1 week of collection. Cobas testing was performed with and without heating samples at 120°C for 20 min diluted 1:1 in an alkaline environment (pretreatment) to revert DNA crosslinking. Stability was tested after 2 weeks of storage at ambient temperature followed by ≤10 weeks at 4°C. Analytical sensitivity and positivity rates (HC2, 18%; cobas pretreated, 46%; cobas untreated, 47%) were greater for cobas than HC2 (n = 682). After 6 weeks of storage, mean HC2 ratios were lower (mean 0.9, SD 6.3) but high variability limited statistical power to detect trends. Cobas threshold cycles (Ct’s) increased in untreated (mean 2.1) but not pretreated samples (mean 0.3; n = 110; p≤0.0001). Overall, cobas had greater analytical sensitivity for samples in SurePath preservative. Although pretreatment introduced a manual sample transfer step and 30 min of incubation times, it improved stability without negatively affecting analytical sensitivity. While awaiting results of large trials to evaluate the clinical performance of cobas, the addition of the pretreatment step may improve the detection of HPV, especially after prolonged sample storage.