Francis V. DeFeudis

Ginkgo biloba extracts and cancer: a research area in its infancy

Recent studies conducted with various molecular, cellular and whole animal models have revealed that leaf extracts of Ginkgo biloba may have anticancer (chemopreventive) properties that are related to their antioxidant, anti‐angiogenic and gene‐regulatory actions. The antioxidant and associated anti‐lipoperoxidative effects of Ginkgo extracts appear to involve both their flavonoid and terpenoid constituents. The anti‐angiogenic activity of the extracts may involve their antioxidant activity and their ability to inhibit both inducible and endothelial forms of nitric oxide synthase. With regard to gene expression, a Ginkgo extract and one of its terpenoid constituents, ginkgolide B, inhibited the proliferation of a highly aggressive human breast cancer cell line and xenografts of this cell line in nude mice. cDNA microarray analyses have shown that exposure of human breast cancer cells to a Ginkgo extract altered the expression of genes that are involved in the regulation of cell proliferation, cell differentiation or apoptosis, and that exposure of human bladder cancer cells to a Ginkgo extract produced an adaptive transcriptional response that augments antioxidant status and inhibits DNA damage. In humans, Ginkgo extracts inhibit the formation of radiation‐induced (chromosome‐damaging) clastogenic factors and ultraviolet light‐induced oxidative stress – effects that may also be associated with anticancer activity. Flavonoid and terpenoid constituents of Ginkgo extracts may act in a complementary manner to inhibit several carcinogenesis‐related processes, and therefore the total extracts may be required for producing optimal effects.

Contents of β-alanine and γ-aminobutyric acid in regions of rat CNS

[3-Alanine, l ike y a m i n o b u t y r i c acid ( G A B A ) , exer ts a p o t e n t dep re s san t effect on v e r t e b r a t e cen t ra l neu rones (e.g. , Cur t is and Watk ins , 1960; Biscoe et al. , 1972). This, a long with the p r e s e n c e in CNS tissues of smal l amoun t s of ~ -a lan ine (e.g., Ta l l an et al., 1954; Yosh ino et al., 1970) and its poss ib le p r e cursors (d ihyd ro -u rac i l and a spa r t a t e ) and synthesiz ing and deg rad ing enzymes (Komi t i an i , 1959; Meis te r , 1965; B e a r t and Johns ton , i 9 7 3 ) , ind ica te tha t it m a y be a cent ra l neu ro t r ansmi t t e r . A n o t h e r c r i t e r ion c ons ide r e d useful for estab l i sh ing a t r ansmi t t e r ro le for a pa r t i cu la r subs tance is the exis tence of a special r eg iona l d i s t r ibu t ion . Using a rapid , sensi t ive m e t h o d for de t e rmin ing ~a lan ine (Mar t in de l Rio , 1977), its concen t r a t ions in severa l s t ruc tures of the rat CNS have been e s t ima ted and c o m p a r e d with those of G A B A , a l ike ly CNS inh ib i to ry t r ansmi t t e r (see e.g., Cur t is and Johns ton , 1974; Krnjevi6 , 1974; D e F e u d i s , 1975, 1977).

Action of N-methyl-bicuculline on the binding of γ-aminobutyric acid to a synaptosomal fraction of rat cerebral cortex ☆

Abstract The interaction of N -methyl-bicuculline and γ-aminobutyric acid (GABA) on a synaptosomal fraction of rat cerebral cortex was examined at 0 C in a balanced bicarbonate-buffered medium using differential centrifugation and double-isotope scintillation spectrometry. The drug, at 10 −4 m , produced a significant decrease in the binding of [ 3 H]GABA (10 −8 , 10 −7 m ) to the particles. Further experiments revealed that N -methyl-bicuculline, at concentrations of 10 −7 –10 −3 m , significantly decreased the “binding” of GABA (4.8 × 10 −8 m ) to the particles. This bicuculline-induced reduction in GABA binding exhibited linear characteristics over a 10 −10 –10 −3 m concentration range of the drug, and when present at 10 −3 m , the drug decreased GABA binding by about 23%. This demonstration of a bicuculline-sensitive component of GABA binding to synaptosomal particles adds support to neurophysiological studies which have indicated that bicuculline may serve as an antagonist to the GABA receptor in vivo .

High-affinity glycine binding sites in rat CNS: regional variation and strychnine sensitivity.

Abstract 1. Specific high-affinity binding of [3H]glycine (KB ≅ 1−1.8 × 10−7 M; Bmax ≅ 0.8−2.1 nmoles/g pellet) occurred to synaptosome-enriched fractions of 6 regions of rat CNS. 2. Strychnine-sensitive and strychnine-insensitive glycine binding sites appeared to exist. Strychninesensitive sites had KB ≅ 0.2−0.8 × 10−7 M and Bmax ≅ 0.2−0.55 nmole/g pellet. 3. About 565–1370 pmoles/g, original weight of tissue, of specific glycine sites and about 109–358 pmoles/g of strychnine-sensitive glycine sites were present in various CNS regions. 4. These findings provide a direct approach for examining the properties of glycine-receptors.

Further characterization of the behavioral despair test in mice: Positive effects of convulsants

Abstract The behavioral despair test in mice has been further characterized using three convulsant agents (at non-convulsant doses), pilocarpine, and several other substances. False positive responses (reductions in immobility) produced by orally-administered strychnine-SO 4 , bicuculline, picrotoxin and pilocarpine further limit the use of this test as a method of screening for antidepressants, unless it is considered that these substances have potential value as antidepressants.

Comparison of the analgesic actions of THIP and morphine

The antinociceptive actions of intraperitoneally-administered 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and morphine were compared using three strains of mice. With the hot-plate assay, ED50 values for the action of THIP were about 4 mg/kg in OF1, CD1 and NMRI strains, whereas ED50 values for morphine varied among strains, being 6.8 mg/kg for OF1, 16.9 mg/kg for CD1, and about 29 mg/kg for NMRI mice; thus, the genetic control of the analgesic action of THIP appears to differ from that of morphine. The analgesic action of THIP in the hot-plate test was not blocked by naloxone, bicuculline, phentolamine or methysergide, but was partially reversed by a high dose of atropine, indicating that classic opiate-receptors, bicuculline-sensitive GABA-receptors, alpha-adrenoceptors and serotonin-receptors do not appear to mediate the action of THIP but that cholinergic receptors might be indirectly involved. THIP was about equipotent or more potent than morphine in the phenylbenzoquinone writhing test, evasion test, and traction test. Since the ED50 values for THIP in OF1 mice were similar for hot-plate, evasion and traction tests, the analgesic action of THIP might not be readily dissociated from its sedative or myorelaxant action.

Density of GABA-receptors in rat cerebral cortex measured with bicuculline-methiodide in the presence of Na+ and other inorganic ions☆

Abstract 1. 1. In the presence of physiological concentrations of Na+ and other ions, GABA was bound to a “synaptosomal-mitochondrial” (P2) fraction of rat cerebral cortex by ‘double-’ or ‘triple-affinity’ mechanisms. 2. 2. ‘High-affinity’ GABA binding had a K B ≜ 0.06 μ M and a B max ≜ 1–3 nmole GABA/g P 2 ; bicuculline-methiodide (BMI) inhibited this binding ( ED 50 ≜ 0.07 μ M ). 3. 3. BMI (10−3 M) displaced about 1.2 pmole GABA/mg protein (about 60 pmole GABA/g original weight of tissue), a value which agreed with estimates of GABA-receptor density made on synaptic membrane fractions in the absence of Na+. 4. 4. BMI-sensitive, high-affinity GABA binding represents about 1/40,000th the total GABA content of cerebral cortex, or about 1/1065th the total GABA bound to this P2 fraction in the presence of Na+. 5. 5. This BMI-sensitive component of GABA binding may represent the synaptic GABA-receptor.

“Binding” of γ-aminobutyric acid and glycine to synaptic particles of the brains of differentially-housed mice; Evidence for morphological changes

Abstract Using subcellular fractionation and a double-isotope method, it was shown that differential housing of male Swiss albino mice for 6 to 7 weeks caused pronounced neurochemical changes in a region of discontinuous sucrose gradient fractions which contains synaptosomes. The “binding” of the possible inhibitory neurotransmitters, [14C]GABA (2.4 × 10−5 m ) and [3H] glycine (9 × 10−8 m ), and the total protein content of these fractions were significantly lower for the brains of aggressive “isolated” mice than for those of their “aggregated” counterparts. These differences in protein content and in amino acid binding (when expressed in mole amino acid/fraction) occurred in particles that were localized just above the 1.2:1.6 m sucrose interface of the gradients. However, when the “binding” data were expressed in mole amino acid/mg protein, no differences existed between gradient subfractions of the brains of isolated and aggregated mice. Because the protein content and total amino acid binding capacities of synaptosomal fractions were lower for the brains of isolated mice than for those of their aggregated counterparts, while no differences existed in amino acid binding on a protein basis, it seems apparent than an environmentally-induced morphological change had been produced in the brain—the brains of isolated mice contained a lesser number of subcellular binding sites for GABA and glycine. These environmentally-sensitive subcellular structures may reside on “heavy” synaptosomes, as they were localized just above the 1.2:1.6 m sucrose (“mitochondrial”) interface. This demonstration of environmentally-sensitive constituents of cerebral nerve-ending particles further validates the view that the brain exhibits structural plasticity when an animal is adapting to conditions which produce dramatic changes in behavior.

Effects of Ginkgo biloba on arterial smooth muscle responses to vasoactive stimuli.

1. An extract of Ginkgo biloba (Gb), at a concentration (100 microgram/ml) that did not provoke isometrically-recorded contractions of rabbit aorta, was tested on the contractile effects of norepinephrine (NE), serotonin (5-HT) and dopamine (DA). 2. Gb potentiated the contractile effect of NE (reduced the EC50 from 75 to 36 nM), but had no obvious action on the contractile effects of 5-HT or DA. 3. These results indicate that low concentrations of Gb might influence catecholaminergic systems by an indirect mechanism.

Physiological and behavioral studies with muscimol.

Muscimol has been used to increase our knowledge of central GABAergic systems, CNS physiology, and behavior. Some studies concerning the neurophysiological and behavioral effects of muscimol and its analogs have been reviewed and analyzed. In vivo iontophoretic studies have greatly increased our knowledge of the active conformation(s) adopted by GABA during its interaction with neuronal synaptic (or extrasynaptic) receptors, and behaviors. studies have supported the notion that central GABAergic systems might be involved in convulsions, extrapyramidal functions, and other behaviors. However, behavioral studies with muscimol remain difficult to interpret in terms of central GABAergic systems, especially since muscimol is extensively metabolized and since it appears to interact with membrane sites other than GABA receptors. Muscimol does not appear to be useful for reversing human neurologic-psychiatric disorders.