Regional differences in the modulation of [3H] muscimol binding to GABAA receptor complexes by kavapyrones, compounds of the rhizome of the plant Piper methysticum which possess sedative activity, were demonstrated using membrane fractions obtained from target brain centers of kavapyrone action: hippocampus (HIP), amygdala (AMY) and medulla oblongata (MED), and from brain centers outside the main kavapyrone effects as frontal cortex (FC) and cerebellum (CER). The kava extract enhanced the binding of [3H] muscimol in a concentration-dependent manner with maximal potentiation of 358% over control in HIP followed by AMY and MED (main target brain centers). Minimal stimulation was observed in CER followed by FC. In contrast, apart from CER, the potency of kavapyrones was similar in the brain areas investigated with EC50 values ranging between 200 and 300 microM kavapyrones. Scatchard analysis revealed that the observed effects of kavapyrones were due to an increase in the number of binding sites (Bmax), rather than to a change in affinity. At a kavapyrone concentration of 500 microM the order of enhancement in Bmax was HIP = AMY > MED > FC > CER. When kavapyrones are included together with pentobarbital or HPO the two classes of compounds produced a more than additive, i.e., synergetic effect on [3H] muscimol binding. Our findings suggest that one way kavapyrones might mediate sedative effects in vivo is through effects on GABAA receptor binding.

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The aim of the present investigation was to answer the question if there exists a relation between the equipment of human gliomas with GABA binding sites and the degree of malignancy of these tumours diagnostically characterized according to WHO classification. The following parameters were assessed: the density, the affinity and the sensitivity to the modulating steroids 3 alpha-hydroxy-5 alpha-pregnane-20-one (3 alpha OH-DHP) and 3 alpha,21 dihydroxy-5 alpha-pregnane-20-one (THDOC). Scatchard analysis and non linear computerization revealed that the occurrence of GABA sites was directly related to the degree of tumour malignancy: GABA sites were only detectable in lower malignant gliomas of WHO grade II but not in the very malignant glioblastomas. However, irrespective of the individual density to be detected all glioma GABA sites were sensitive to 3 alpha-hydroxy-5 alpha-pregnane-20-one (3 alpha OH-DHP) and 3 alpha,21 dihydroxy-5 alpha-pregnane-20-one (THDOC) without exception. The effects of THDOC were due to increases in the number of binding sites whereas in the presence of 3 alpha OH-DHP a decrease in affinity was noted, additionally. These findings support the view of a normal functional integrity of GABA receptors in gliomas.

ATPase was shown to be present on the cytoplasmic membrane of the methanogenic bacterium strain Gol. The enzyme was identified by an immunoelectron microscopic technique by using polyclonal antiserum directed against the j subunit of Escherichia coli FOF,-ATPase. Negatively stained membrane vesicles exhibited a dense population of stalked particles similar in dimensions and fine structure to typical FOF,-ATPase particles.The presence of ATPases has been demonstrated in a variety of methanogenic bacteria (4,6,10,16). Based on results obtained with Methanosarcina barkeri (1-3), it is assumed that the terminal step in methanogenesis, the 2-(methylthio)ethanesulfonic acid (methyl coenzyme M) methylreductase reaction (14,17), is coupled to the generation of an electrochemical proton gradient across the cytoplasmic membrane. This gradient is then taken advantage of for ATP synthesis (3).Recently, a methylotrophic methanogenic bacterium, strain Gol, has been isolated from which protoplasts can easily be obtained (8). Membrane vesicles and fragments derived from these protoplasts could be used in an electron microscopic approach aimed at the physical demonstration of the presence of ATPase. The enzyme has not been purified from this microorganism, and its identity was expected to be proven by an immunoelectron microscopic technique with polyclonal antiserum directed against the ,B subunit of the F1-ATPase particle of Escherichia coli. This serum has recently been shown to cross-react with membrane vesicle preparations obtained from strain Gol (unpublished data).Strain Gol, obtained from the Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany, was grown with methanol as the energy and carbon source, as described previously (8). Protoplasts were prepared by pronase treatment of whole cells (8). Inside-out and right side-out membrane vesicles and fragments were produced from protoplasts by disruption in a French pressure cell (11) operated at 65 MPa, using buffer (pH 7.0) containing 0.2 M sodium maleinate, 0.05 M N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate (TES), 0.25 M sucrose, 5 mM MgSO4, titanium citrate (19), resazurin, and DNase I, by repeated freezing and thawing (12) or by osmotic lysis (9).Experiments aimed at the identification of ATPase particles assumed to be attached to membrane vesicles and fragments were performed as follows. Samples (0.5 ,ul) of the vesicle suspension were applied to Formvar-coated 300-mesh nickel grids and kept at room temperature for 1 h. The * Corresponding author. samples were incubated overnight at 4°C on drops containing rabbit antibodies specific for the 1B subunit of the E. coli ATPase (1.4 mg/ml). The grids were then rinsed with phosphate-buffered saline (0.05 M phosphate buffer, 0.9% sodium chloride [pH 6.9]) before incubation with anti-rabbit antibody-gold complexes for 2 h at room temperature. Subsequently, the samples were rinsed with phosphate-buffered saline and distilled water. Colloidal gold was prepared by the method of Slot and Geuze (15) b...

The methanogenic bacterium strain G61 harbors a high-molecular-weight enzyme complex containing methyl coenzyme M methylreductase as revealed by immunoelectron microscopy. This Methanogenic bacteria utilize substrates such as H2-CO2, methanol, monomethylamine, dimethylamine, trimethylamine, and acetate (2,12,18,22) which are converted to a common intermediate, (2-methylthio)ethanesulfonic acid (methyl-CoM) (34). This compound is then reduced to methane and 2-mercaptoethanesulfonic acid in a reaction catalyzed by the enzyme methyl-CoM methylreductase (11,28,30). Studies of the effects of N,N'-dicyclohexylcarbodiimide and 3,5,4',5'-tetrachlorosalicylanilide [3,5-dichloro-N-(4,5-dichlorophenyl)-2-hydroxybenzamide] on methanogenesis, the transmembrane electrical gradient, and the cellular ATP level strongly indicate that ATP is synthesized by a chemiosmotic mechanism and that the methylreductase system is involved in proton translocation (3,4,6,7,22).Various models have been proposed for the generation of an electrochemical transmembrane gradient of protons in methanogenic bacteria (5, 10); one assumes that methylreductase might be membrane associated; one includes the view that a redox carrier might mediate between membrane and (cytoplasmic) methylreductase. Indications have accumulated that the methylreductase system is a multienzyme complex (3,17,23).