Direct inhibiting effects of [D-Trp6]gonadotropin-releasing hormone on the estrogen-sensitive progression of polyoma virus-induced mammary tumors in athymic mice.

The direct antitumoral effects of gonadotropin-releasing hormone (GnRH) analogues on breast tumors have been surmised from clinical observations and in vitro studies. The present study aimed to determine the effects of the GnRH agonist [D-Trp6]GnRH (Decapeptyl) on steps of experimental mammary carcinogenesis, and the mechanisms, other than the chemical castration, involved. We chose a recent model, i.e., mammary tumors induced by wild-type A2 polyoma (Py) virus in BALB/c female nu/nu mice, which displays the following characteristics. Tumors are mammary adenocarcinomas similar to well differentiated breast carcinomas. Tumor promotion period ends 20 days after Py virus inoculation and is estradiol dependent. The first palpable tumors occur 60 days after Py virus inoculation, and tumor growth is ovarian hormone independent. The effects of Decapeptyl treatment on tumor induction and tumor growth were studied in normal or ovariectomized 6-week-old nude mice inoculated with 10(7) plaque-forming units Py virus (day 0 of experiments). Normal mice and ovariectomized mice percutaneously supplemented with 0.6 micrograms 17 beta-estradiol every other day until day 30 (OvE2 mice) were treated with monthly s.c. injections of the sustained release form of Decapeptyl (5 mg/kg) until the end of 180-day experiments. Overall values for latency periods were included within a day 60 to day 130 time interval. Hormone-independent outgrowth was not affected. We focused on tumor progression before the outgrowth. Incidences on tumor appearance kinetics account for effects at this stage. 17 beta-Estradiol repletion strongly antagonized (P less than 0.001) the slowing effect of ovariectomy on the tumor appearance kinetics, indicating that tumor progression is estradiol sensitive in its early stages. [D-Trp6]GnRH treatment antagonized tumor appearance profiles, inducing similar kinetics in both normal and OvE2 mice. In normal mice, the antagonism (P less than 0.01) was concomitant with significant decreases (P less than 0.05) in serum levels of estradiol and prolactin, which are critical hormones for mammary tumor development in mice, suggesting a pituitary-mediated effect. In OvE2 mice, the antagonism (P less than 0.01) occurs independently of estradiol and prolactin, suggesting a direct effect at the mammary cell level. Because of alterations in kinetics, this effect is exerted at the early stages of tumor progression on Py virus-transformed, ovarian hormone-sensitive cells in the mammary tissue. This new animal model of breast cancer is shown to be useful in characterizing direct antitumoral effects of GnRH analogues and studying the basic mechanisms of mammary carcinogenesis.

Ex vivo regulation of adrenal cortical cell steroid and protein synthesis, in response to adrenocorticotropic hormone stimulation, by the Ginkgo biloba extract EGb 761 and isolated ginkgolide B.

We previously demonstrated that repeated treatment of rats with the standardized extract of Ginkgo biloba leaves, EGb 761, and its bioactive component ginkgolide B (GKB), specifically reduces the ligand binding, and protein and messenger RNA expression of the adrenal mitochondrial peripheral benzodiazepine receptor (PBR), a key element in the regulation of cholesterol transport, resulting in decreased circulating corticosterone levels. Adrenocortical cells were isolated from rats treated with EGb 761 or GKB and cultured for 2 and 12 days. The effect of ACTH on normal and metabolically labeled cells was examined. Corticosterone levels were measured by RIA, and protein synthesis was analyzed by two-dimensional gel electrophoresis. Ex vivo treatment with EGb 761 and GKB resulted, respectively, in 50% and 80% reductions of ACTH-stimulated corticosterone production by adrenocortical cells cultured for 2 days compared with that by cells isolated from saline-treated rats. Two-dimensional gel electrophoresis analysis revealed that in cells from both control and drug-treated animals, ACTH induced the synthesis, at the same level, of a 29-kDa and pI 6.4-6.7 protein identified as the adrenal steroidogenic acute regulatory protein (StAR). In addition, treatment with EGb 761 and GKB specifically altered the synthesis of seven proteins, including inhibition of synthesis of a 17-kDa, identified as PBR. After 12 days in culture, ACTH-stimulated adrenocortical cell steroid synthesis was maintained, and it was identical among the cells isolated from animals treated with GKB or saline. Under the same conditions, the expression of PBR was recovered, whereas no effect of ACTH on the 29-kDa and 6.4-6.7 pI protein (StAR) or other protein synthesis could be seen. A comparative analysis of the effects of GKB and EGb 761 on adrenocortical steroidogenesis and protein synthesis identified, in addition to the 17-kDa PBR, target proteins of 32 kDa (pI 6.7) and 40 kDa (pI 5.7-6.0) as potential mediators of the effect of EGb 761 and GKB on ACTH-stimulated glucocorticoid synthesis. In conclusion, these results 1) validate and extend our previous in vivo findings on the effect of EGb 761 and GKB on ACTH-stimulated adrenocortical steroidogenesis, 2) demonstrate the specificity and reversibility of EGb 761 and GKB treatment, 3) question the role of the 29-kDa, 6.4-6.7 pI protein (mature StAR) as the sole mediator of ACTH-stimulated steroid production, and 4) demonstrate the obligatory role of PBR in hormone-regulated steroidogenesis.

In vivo regulation of peripheral-type benzodiazepine receptor and glucocorticoid synthesis by Ginkgo biloba extract EGb 761 and isolated ginkgolides.

Glucocorticoid excess has broad pathogenic potential including neurotoxicity, neuroendangerment, and immunosuppression. Glucocorticoid synthesis is regulated by ACTH, which acts by accelerating the transport of the precursor cholesterol to the mitochondria where steroidogenesis begins. Ginkgo biloba is one of the most ancient trees, and extracts from its leaves have been used in traditional medicine. A standardized extract of Ginkgo biloba leaves, termed EGb 761 (EGb), has been shown to have neuroprotective and antistress effects. In vivo treatment of rats with EGb, and its bioactive components ginkgolide A and B, specifically reduces the ligand binding capacity, protein, and messenger RNA expression of the adrenocortical mitochondrial peripheral-type benzodiazepine receptor (PBR), a key element in the regulation of cholesterol transport, resulting in decreased corticosteroid synthesis. As expected, the ginkgolide-induced decrease in glucocorticoid levels resulted in increased ACTH release, which in turn induced the expression of the steroidogenic acute regulatory protein. Because ginkgolides reduced the adrenal PBR expression and corticosterone synthesis despite the presence of high levels of steroidogenic acute regulatory protein, these data demonstrate that PBR is indispensable for normal adrenal function. In addition, these results suggest that manipulation of PBR expression could control circulating glucocorticoid levels, and that the antistress and neuroprotective effects of EGb are caused by to its effect on glucocorticoid biosynthesis.

Triton X-100 eliminates plasma proteins interference in a radioimmunoassay for luteinizing hormone-releasing hormone (LHRH) and LHRH analogues.

A method is described for the radioimmunoassay of native LHRH and DTrp6-LHRH, an LHRH analogue which does not require extraction of plasma samples. Interference by binding proteins normally present in plasma is removed by addition of Triton X-100 to the binding buffer at a concentration of 1% for LHRH and 0.15% for the LHRH analogue. This approach permits a direct estimation of the peptide level in unextracted plasma with quantitative recoveries for concentrations ranging from 0.015 to 10 ng/ml. Although the antiserum titre is reduced, the affinity of the antibody does not change at the detergent concentrations used in this study. This procedure is recommended for peptide assays in which the non-specific effects of plasma prevent a direct assay.

Ginkgo biloba extract (EGb 761) and CNS functions: basic studies and clinical applications.

The effects of EGb 761 on the CNS underlie one of its major therapeutic indications; i.e., individuals suffering from deteriorating cerebral mechanisms related to age-associated impairments of memory, attention and other cognitive functions. EGb 761 is currently used as symptomatic treatment for cerebral insufficiency that occurs during normal ageing or which may be due to degenerative dementia, vascular dementia or mixed forms of both, and for neurosensory disturbances. Depressive symptoms of patients with Alzheimer's disease (AD) and aged non-Alzheimer patients may also respond to treatment with EGb 761 since this extract has an "anti-stress" effect. Basic and clinical studies, conducted both in vitro and in vivo, support these beneficial neuroprotective effects of EGb 761. EGb 761 has several major actions; it enhances cognition, improves blood rheology and tissue metabolism, and opposes the detrimental effects of ischaemia. Several mechanisms of action are useful in explaining how EGb 761 benefits patients with AD and other age-related, neurodegenerative disorders. In animals, EGb 761 possesses antioxidant and free radical-scavenging activities, it reverses age-related losses in brain alpha 1-adrenergic, 5-HT1A and muscarinic receptors, protects against ischaemic neuronal death, preserves the function of the hippocampal mossy fiber system, increases hippocampal high-affinity choline uptake, inhibits the down-regulation of hippocampal glucocorticoid receptors, enhances neuronal plasticity, and counteracts the cognitive deficits that follow stress or traumatic brain injury. Identified chemical constituents of EGb 761 have been associated with certain actions. Both flavonoid and ginkgolide constituents are involved in the free radical-scavenging and antioxidant effects of EGb 761 which decrease tissue levels of reactive oxygen species (ROS) and inhibit membrane lipid peroxidation. Regarding EGb 761-induced regulation of cerebral glucose utilization, bilobalide increases the respiratory control ratio of mitochondria by protecting against uncoupling of oxidative phosphorylation, thereby increasing ATP levels, a result that is supported by the finding that bilobalide increases the expression of the mitochondrial DNA-encoded COX III subunit of cytochrome oxidase. With regard to its "anti-stress" effect, EGb 761 acts via its ginkgolide constituents to decrease the expression of the peripheral benzodiazepine receptor (PBR) of the adrenal cortex.

Protection of mitochondrial respiration activity by bilobalide.

Mitochondria alteration is an early event in ischemia-induced damage, and its prevention improves tissue survival upon reperfusion. Adenine translocase and complex I activities are rapidly affected by ischemia. Ginkgo biloba extract demonstrates anti-ischemic properties attributable to the terpenoid fraction, mainly due to the presence of bilobalide. The mechanism of the protection afforded by bilobalide is not yet known. In this work, the effects of bilobalide on mitochondrial respiration were investigated. Mitochondria isolated from rats treated with bilobalide (2 to 8 mg/kg) showed a dose-dependent increase in the respiratory control ratio, due to a lower oxygen consumption during state 4. Bilobalide also decreased the sensitivity of oxygen consumption to inhibition of complex I by Amytal or to inhibition of complex III by antimycin A or myxothiazol. There was no protection of complexes IV and V. It also increased the activity of complex I but not of adenine translocase. Similar effects were also obtained in vitro when control mitochondria were preincubated for 1 hr with 0.8 microg/mL bilobalide. Treatment of the rats with 8 mg/kg bilobalide also prevented the ischemia-induced decrease in state 3 of the mitochondrial respiration and thus the decrease in RCR. The protective effect of bilobalide on cellular ATP content observed under ischemic conditions can be correlated with the above observations. By protecting complex I and III activities, bilobalide allows mitochondria to maintain their respiratory activity under ischemic conditions as long as some oxygen is present, thus delaying the onset of ischemia-induced damage. This mechanism provides a possible explanation for the anti-ischemic properties of bilobalide and of Ginkgo biloba extract in therapeutic interventions.

Protection of hypoxia-induced ATP decrease in endothelial cells by ginkgo biloba extract and bilobalide.

Due to their localization at the interface between blood and tissue, endothelial cells are the first target of any change occurring within the blood, and alterations of their functions can seriously impair organs. During hypoxia, which mimics in vivo ischemia, a cascade of events occurs in the endothelial cells, starting with a decrease in ATP content and leading to their activation and release of inflammatory mediators. EGb 761 and one of its constituents, bilobalide, were shown to inhibit the hypoxia-induced decrease in ATP content in endothelial cells in vitro. Under these conditions, glycolysis was activated, as evidenced by increased glucose transport, as well as increased lactate production. Bilobalide was found to increase glucose transport under normoxic but not hypoxic conditions. In addition, EGb and bilobalide prevented the increase in total lactate production observed after 60 min of hypoxia. However, after 120 min of hypoxia, the total lactate production was similar under normoxic and hypoxic conditions, and both compounds increased this production. These results indicate that glycolysis slowed down between the 60th and 120th minute of hypoxia, while EGb and bilobalide delayed the onset of glycolysis activation. In another experimental model, both compounds were shown to increase the respiratory control ratio of mitochondria isolated from liver of rats treated orally. Since ischemia is known to uncouple mitochondria, the protection of ATP content and the delay in glycolysis activation observed during hypoxia in the presence of EGb 761 or bilobalide is best explained by a protection of mitochondrial respiratory activity, at least during the first 60 min of hypoxia incubation. Both products retain the ability to form ATP, thereby reducing the cell's need to induce glycolysis, probably by preserving ATP regeneration by mitochondria as long as oxygen is available.

GnRH receptors in rat brain, pituitary and testis; modulation following surgical and gonadotropin-releasing hormone agonist-induced castration.

The regulation of rat gonadotropin-releasing hormone (GnRH) receptors in male rat pituitary, hippocampus and testis was studied, in vivo, under steady-state conditions during treatment with D-Trp6 GnRH (triptorelin, slow-release form, at 300 micrograms/kg/month). GnRH receptors were characterized on tissue sections by quantitative autoradiography using 125I-GnRHa as a tracer. Castrating doses of triptorelin strongly down-regulated pituitary GnRH receptors (50% of reduction after 8 h, 80% on days 1-30); in contrast, only a transient decrease (20% at 8 h) was observed in the hippocampus with a rapid return to control levels. Triptorelin induced a marked (2-fold) increase in GnRH receptors in testicular interstitial tissue during 5 days with a return to control value by day 20. Administration of a GnRH antagonist (BIM 21009, 1 mg/kg/24 h) induced a rapid reduction of pituitary and testicular receptors to undetectable levels at 24 h, while hippocampal receptors were strongly reduced only. This indicates that GnRH receptors with similar pharmacology are differently controlled in various tissues and that brain receptors are likely to be also regulated by GnRH agonists and antagonists.

Radioimmunoassay of [D-Trp6]-luteinizing hormone-releasing hormone: its application to animal pharmacokinetic studies after single injection and long-acting formulation administration.

A sensitive radioimmunoassay (RIA) for [D-Trp6]-luteinizing hormone-releasing hormone (LHRH) has been developed. This assay allowed measurement of the LHRH analog in unextracted plasma with a minimum detectable concentration of 10 pg/ml. Validation of plasma assays was performed through Sep-Pak and HPLC purification. The in vivo fate of the peptide was investigated in dogs after subcutaneous or intravenous injections. In both cases, the LHRH analog showed longer plasma half-life than native LHRH with an elimination half-life superior to 80 min. Long-acting formulations were tested in dogs and rats: the day following administration, [D-Trp6]-LHRH plasma level rose to 2.9-4.6 ng/ml in dogs and 0.8-3.8 ng/ml in rats. From day 4 to day 30, [D-Trp6]-LHRH plasma level followed a plateau with concentrations of 0.3-0.8 ng/ml in dogs and 0.2-0.4 ng/ml in rats. In parallel, testosterone plasma concentration was reduced to castrate level between day 4 and day 7 in dogs and was significantly lowered in rats. This sensitive [D-Trp6]-LHRH RIA will be particularly useful for the evaluation of long-acting formulations in patients with advanced prostate cancer.

Effect of the extract of Ginkgo biloba (EGb 761) on the circulating and cellular profiles of polyunsaturated fatty acids: correlation with the anti-oxidant properties of the extract.

Ginkgo biloba extract (EGb 761) has beneficial effects on cognitive functions in aging patients, and on various pathologies, including cardiovascular diseases. Although the extract is known to have antioxidant properties and improve membrane fluidity, the cellular mechanisms underlying these effects have not been determined. Here, we examined the in vivo effects of EGb 761 on circulating and cellular lipids. EGb 761 treatment induced significant increases in the levels of circulating polyunsaturated fatty acids (PUFAs), and a decrease in the saturation index SI (saturated/polyunsaturated species). Plasma triglycerides and cholesterol were not affected, while phospholipids were slightly increased at the higher dose of EGb 761. EGb 761 treatment also induced a significant increase in the levels of PUFAs in erythrocyte membranes, especially for the eicosapentaenoic acid (EPA omega 3), and a decrease in the saturation index. Moreover, the response of erythrocytes to oxidative stress was improved in EGb 761-treated animals (H(2)O(2)-induced cell lysis decreased by 50%). Considering that PUFAs are known to improve membrane fluidity and response to oxidative damage, and are precursors of signaling molecules such as prostaglandins, the effects of EGb 761 on circulating and cellular PUFAs may explain some of the pharmacological properties of Ginkgo biloba.

The Ginkgo biloba extract EGb 761 rescues the PC12 neuronal cells from beta-amyloid-induced cell death by inhibiting the formation of beta-amyloid-derived diffusible neurotoxic ligands.

beta Amyloid (Abeta) treatment induced free radical production and increased glucose uptake, apoptosis and cell death in PC12 nerve cells. Addition of the standardized extract of Ginkgo biloba leaves, EGb 761 together with the Abeta protein prevented, in a dose-dependent manner, the Abeta-induced free radical production, increased glucose uptake, apoptosis and cell death. However, pretreatment of the cells with EGb 761 did not rescue the cells from the Abeta-induced toxicity although it prevented the Abeta-induced reactive oxygen species generation. Moreover, the terpene and flavonoid-free EGb 761 extract, HE 208, although inhibited the Abeta-induced increased glucose uptake, it failed to protect the cells from apoptosis and cytotoxicity induced by Abeta. In conclusion, these results indicate that the terpenoid and flavonoid constituents of EGb 761, acting probably in combination with components present in HE 208, are responsible for rescuing the neuronal cells from Abeta-induced apoptosis and cell death; their mechanism of action being distinct of their antioxidant properties. Because pre- and post-treatment with EGb 761 did not protect the cells from Abeta-induced neurotoxicity, we examined whether EGb 761 interacts directly with Abeta. Indeed, in vitro reconstitution studies demonstrated that EGb 761 inhibits, in a dose-dependent manner, the formation of beta-amyloid-derived diffusible neurotoxic soluble ligands (ADDLs), suggested to be involved in the pathogenesis of Alzheimer's disease.

Ginkgo biloba extract EGb761 reduces the development of amphetamine-induced behavioral sensitization: effects on hippocampal type II corticosteroid receptors.

Pretreatment of rats with the extract of Ginkgo biloba termed EGb761 reduced the behavioral sensitization induced by successive D-amphetamine administrations (0.5 mg/kg) as estimated by increasing values of locomotor activity. EGb761 pretreatment also prevented the reduced density of [3H]dexamethasone binding sites in the dentate gyrus and the CA1 hippocampal regions of D-amphetamine treated animals. These observations suggest that EGb761, by reducing glucocorticoid levels, could modulate the activity of the neuronal systems involved in the expression of the behavioral sensitization.

Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia.

The neuroprotective effect of Ginkgo biloba extract (EGb 761) against ischemic injury has been demonstrated in animal models. In this study, we compared the protective effect of bilobalide, a purified terpene lactone from EGb 761, and EGb 761 against ischemic injury. We measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global forebrain ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In addition, both bilobalide and EGb 761 protected against ischemia-induced reductions in COX III mRNA in CA1 neurons prior to their death, at 1 day of reperfusion. These results suggest that oral administration of bilobalide and EGb 761 protect against ischemia-induced neuron death and reductions in mitochondrial gene expression.

Free radicals and lipid peroxidation do not mediate beta-amyloid-induced neuronal cell death.

"beta Amyloid (Abeta)-induced free radical-mediated neurotoxicity" is a leading hypothesis as a cause of Alzheimer's disease (AD). Abeta increased free radical production and lipid peroxidation in PC12 nerve cells, leading to increased 4-hydroxy-2-nonenal (HNE) production and modification of specific mitochondrial target proteins, apoptosis and cell death. Pretreatment of the cells with isolated ginkgolides, the anti-oxidant component of Ginkgo biloba leaves, or vitamin E, prevented the Abeta-induced increase of reactive oxygen species (ROS). Ginkgolides, but not vitamin E, inhibited the Abeta-induced HNE modification of mitochondrial proteins. However, treatment with these anti-oxidants did not rescue the cells from Abeta-induced apoptosis and cell death. These results indicate that free radicals and lipid peroxidation may not mediate Abeta-induced neurotoxicity.

Effects of stress on the functional properties of pre- and postsynaptic 5-HT1B receptors in the rat brain.

Numerous studies have clearly shown that the turnover and release of serotonin (5-hydroxytryptamine, 5-HT) are increased under acute stressful conditions. Inasmuch as this latter process is under the control of a feedback mechanism involving the stimulation of presynaptic 5-HT1B autoreceptors, we have investigated the possible effects of acute restraint (40 min) on the functional properties of 5-HT1B receptors. The efficacy of the selective 5-HT1B receptor agonist 3-[1,2,5,6-tetrahydropyrid-4-yl]pyrrolo-[3,2-b]pyrid-5-one (CP-93,129) in inhibiting in vitro the K+-evoked release of [3H]5-HT, was significantly reduced in stressed rats as compared to naive animals. Similarly, the responsiveness of 5-HT1B receptors inhibiting the release of [3H]acetylcholine (presynaptic 5-HT1B heteroreceptors), was reduced by restraint. These effects were observed in the hippocampus, but using the inhibitory effect of CP-93,129 on forskolin-stimulated adenylyl cyclase activity as an index of 5-HT1B receptor function, it could be shown that the 5-HT1B receptors located in the substantia nigra are also desensitized by stress. The number as well as the apparent affinity constant of 5-HT1B binding sites labelled by [125I]iodocyanopindolol, as measured by quantitative autoradiography and membrane binding, were similar in naive and restraint-stressed rats suggesting that the stress-induced desensitization of 5-HT1B receptors is not due to a reduced number of 5-HT1B binding sites. As stress is thought to be a causal factor for the etiology of anxiety and depression, these results support the potential involvement of 5-HT1B receptor dysfunction in the development of these neurological disorders.

Sub-chronic cold stress reduces 5-HT1A receptor responsiveness in the old but not in the young rat.

The inhibitory effect of the prototypical 5-hydroxytryptamine (5-HT)1A receptor agonist 8-hydroxy-2-(di-n-propyl amino)tetraline (8-OH-DPAT) on forskolin-stimulated adenylyl cyclase activity, has been examined as an index of the functional activity of 5-HT1A receptors in the hippocampus of young (3 months) and old (18 months) rats exposed during 24 h or 5 days to cold. In both young and old rats exposed to cold stress during 24 h, there was a reduction in the potency (EC50) and/or the maximal inhibitory effect (Emax) of 8-OH-DPAT in reducing forskolin-induced cAMP accumulation. The properties of the hippocampal 5-HT1A sites labelled by [3H]8-OH-DPAT were not affected by these stressful conditions. Moreover, while the sensitivity of 5-HT1A receptors to 8-OH-DPAT in young rats returned to control values after 5 days of cold exposure, old rats still exhibited a significant desensitization of 5-HT1A receptors as compared to naive animals. These results point out the capacity of young but not of old rats to adapt to the aversive effects of a subchronic stressor.

Effect of chronic administration of Ginkgo biloba extract or Ginkgolide on the hypothalamic-pituitary-adrenal axis in the rat.

The hypersecretion of glucocorticoids during exposure to various stressors may induce or worsen pathological states in predisposed subjects. Therefore it is of interest to evaluate drugs able to reduce glucocorticoid secretion. It has recently been shown that chronic administration of a Ginkgo biloba extract (EGb 761) inhibits stress-induced corticosterone hypersecretion through a reduction in the number of adrenal peripheral benzodiazepine receptors. The present study was designed to analyze the effect of EGb 761 and one of its components, Ginkgolide B on the biosynthesis and secretion of CRH and AVP, the hypothalamic neurohormones that regulate the pituitary-adrenal axis. Chronic administration of EGb 761 (50 or 100 mg/kg p.o. daily for 14 days) reduced basal corticosterone secretion and the subsequent increase in CRH and AVP gene expression. Under the same conditions, surgically-induced increase in CRH secretion was attenuated while the activation of CRH gene expression, ACTH and corticosterone secretion following insulin-induced hypoglycemia remained unchanged. Chronic i.p. injection of Ginkgolide B reduced basal corticosterone secretion without alteration in the subsequent CRH and AVP increase. However, the stimulation of CRH gene expression by insulin-induced hypoglycemia was attenuated by Ginkgolide B. These data confirm that the administration of EGb 761 and Ginkgolide B reduces corticosterone secretion. In addition, these substances act also at the hypothalamic level and are able to reduce CRH expression and secretion. However the latter effect appears to be complex and may depend upon both the nature of stress and substance (Ginkgolide B or other compounds of EGb 761).

Stress-induced 5-HT1A receptor desensitization: protective effects of Ginkgo biloba extract (EGb 761).

The effects of sub-chronic cold stress on the functioning of hippocampal 5-HT1A receptors in old isolated rats and the possible protective effects of Ginkgo biloba extract (EGb 761) were investigated. Cold exposure during five days, produced a significant reduction of the inhibitory effect of 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT) on forskolin-stimulated adenylyl cyclase activity. In contrast, neither the affinity nor the density of hippocampal [3H]8-OH-DPAT binding sites were affected indicating that the reduced sensitivity of 5-HT1A receptors induced by stress is probably due to a modification of their coupling mechanisms to adenylyl cyclase. The stress-induced desensitization of 5-HT1A receptors was prevented by the administration of EGb 761 (50 mg/kg per os/14 days). These results clearly indicate that 5-HT1A receptors are desensitized by stress and point out the reduced capacity of old rats to cope with the adverse effects of a chronic stressor. EGb 761 appears to restore the age-related decreased capacity to adapt to a chronic stressor.

Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis.

Steroidogenesis begins with the metabolism of cholesterol to pregnenolone by the inner mitochondrial membrane cytochrome P450 side-chain cleavage (P450scc) enzyme. The rate of steroid formation, however, depends on the rate of cholesterol transport from intracellular stores to the inner mitochondrial membrane and loading of P450scc with cholesterol. In previous in vitro studies, we demonstrated that a key element in the regulation of cholesterol transport is the mitochondrial peripheral-type benzodiazepine receptor (PBR). We also showed that the polypeptide diazepam binding inhibitor (DBI), an endogenous PBR ligand, stimulates cholesterol transport and promotes loading of cholesterol to P450scc in vitro, and that its presence is vital for hCG-induced steroidogenesis by Leydig cells. Based on these data and the observations that i) the mitochondrial PBR binding and topography are regulated by hormones; ii) the 18-kDa PBR protein is functionally coupled to the mitochondrial contact site voltage-dependent anion channel protein; iii) the 18-kDa PBR protein is a channel for cholesterol, as shown by molecular modeling and in vitro reconstitution studies; iv) targeted disruption of the PBR gene in steroidogenic cells dramatically reduces the ability of the cells to transport cholesterol in the mitochondria and produce steroids; v) endocrine disruptors, with known anisteroidogenic effect, inhibit PBR ligand binding; and vi) in vivo reduction of adrenal PBR expression results in reduced circulating glucocorticoid levels, we conclude that PBR is an indispensable element of the steroidogenic machinery.

Drug-induced inhibition of the peripheral-type benzodiazepine receptor expression and cell proliferation in human breast cancer cells.

The peripheral-type benzodiazepine receptor (PBR) expression and localization correlate with human breast cancer cell proliferation and aggressive phenotype expression. The standardized extract of Ginkgo biloba leaves (EGb 761) and isolated ginkgolide B (GKB) were shown to decrease PBR mRNA expression in adrenal cells. We examined the effect of EGb 761 and GKB on PBR expression and cell proliferation in human breast cancer cells. EGb 761 and GKB decreased in a time- and dose-dependent manner PBR expression and cell proliferation in the highly aggressive, rich in PBR, human breast cancer cell line MDA-231 whereas they did not affect the proliferation of the non-aggressive human breast cancer cell line MCF-7, which contains very low PBR levels. This effect was reversible and not due to the antioxidant properties of the compounds tested. Using a human cDNA expression array we determined that EGb 761 treatment altered, in addition to PBR, the expression of 36 gene products involved in various pathways regulating cell proliferation. These in vitro data were further validated in an in vivo model where EGb 761 and GKB significantly inhibited the nuclear PBR expression and growth of MDA-231 cell xenografts in nude mice. Taken together, these data suggest that the manipulation of PBR expression could be used to control tumor growth and that EGb 761 and GKB, under the conditions used, exert cytostatic properties.