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.

Gossypol toxicosis in the rat associated with protein malnutrition and experimental infection with Trypanosoma brucei.

The toxicity of gossypol, a compound occurring naturally in the cotton plant, was investigated in Trypanosoma brucei-infected, gossypol-treated rats, with and without protein malnutrition. The liver, heart, lungs, spleen and adrenal glands were enlarged in all gossypol-treated rats. Gossypol treatment or trypanosome infection, either alone or together, invariably caused significant reductions in the serum activity of creatine phosphokinase and amylase and in the serum concentration of cortisol. The serum biochemical changes, together with histopathological findings in various organs, indicated that the toxicity of gossypol and pathology of trypanosome infection, either alone or in concert, could be exacerbated by protein malnutrition. This finding suggests that the previously reported antiparasitic properties of gossypol may be of little ultimate benefit due to these serious side effects. The spleen in the protein-malnourished, trypanosome-infected and gossypol-treated animals exhibited only a slight decrease in the number of lymphatic nodules, but a marked cellular depletion, especially of cortical tissue, was observed in the thymus. These observations would seem to justify further study of the immune status of trypanosome-infected, gossypol-treated animals.

Identification of a stimulator of steroid hormone synthesis isolated from testis.

Gonadal steroidogenesis is regulated by pituitary gonadotropins and a locally produced, unidentified factor. A 70-kilodalton (kD) protein complex secreted from rat Sertoli cells was isolated. The complex, composed of 28- and 38-kD proteins, stimulated steroidogenesis by Leydig cells and ovarian granulosa cells in a dose-dependent and adenosine 3',5'-monophosphate-independent manner. The follicle-stimulating hormone-induced 28-kD protein appeared to be responsible for the bioactivity, but the 38-kD protein was indispensable for maximal activity. The 28- and 38-kD proteins were shown to be identical to the tissue inhibitor of metalloproteinase-1 (TIMP-1) and the proenzyme form of cathepsin L, respectively. Thus, a TIMP-1-procathepsin L complex is a potent activator of steroidogenesis and may regulate steroid concentrations and, thus, germ cell development in both males and females.

The effects of protein malnutrition and experimental infection with Trypanosoma brucei on gossypol treatment in the rat: haematological and serum biochemical changes.

This study was designed to evaluate the interaction between protein malnutrition, gossypol treatment and blood parasitosis (Trypanosoma brucei) in the Wistar rat. Haematological and serum biochemical changes were evaluated in the rats, which were placed on two planes of nutrition--low protein (LP) and normal protein (NP)--and either treated with gossypol or infected with Trypanosoma brucei, or both. Higher parasitaemia occurred in gossypol-treated NP rats than in the corresponding LP group. Gossypol treatment and trypanosomal infection, either alone or in concert, caused an anaemia that was both macrocytic and hypochromic. Both treatments together also caused increases in serum alkaline phosphatase and alanine aminotransferase activities, which were accompanied by depressed serum albumin concentrations, suggestive of hepatic dysfunction in affected rats. These results suggest that, with adequate protein intake, the growth and infectivity of trypanosomes is not inhibited by gossypol but that protein malnutrition has a beneficial effect of reduced parasitaemia. Unfortunately, this beneficial effect is counteracted by gossypol enhancement of hepatic dysfunction caused by trypanosomes.

The polypeptide diazepam-binding inhibitor and a higher affinity mitochondrial peripheral-type benzodiazepine receptor sustain constitutive steroidogenesis in the R2C Leydig tumor cell line.

The polypeptide diazepam binding inhibitor (DBI) and drug ligands for the mitochondrial peripheral-type benzodiazepine receptor (PBR) have been shown to regulate cholesterol transport, the rate-determining step in steroidogenesis, in hormone-responsive steroidogenic cells including the MA-10 Leydig tumor cells. The present study was designed to characterize the role of DBI and PBR in the R2C rat Leydig tumor constitutive steroid-producing cell model. Both DBI and PBR were present in R2C cells. R2C cell treatment with a cholesterol-linked phosphorothioate oligodeoxynucleotide antisense to DBI, but not sense, resulted in the reduction of DBI levels and a concomitant dramatic decrease of the amount of progesterone produced. These observations strongly suggested that DBI was important in maintaining constitutive steroidogenesis in R2C cells. Radioligand binding assays revealed the presence of a single class of PBR binding sites with an affinity 10 times higher (Kd approximately 0.5 nM) than that displayed by the MA-10 PBR (Kd approximately 5 nM). Photolabeling of R2C and MA-10 cell mitochondria with the photoactivatable PBR ligand [3H]1-(2-fluoro-5-nitrophenyl)-N-methyl-N-(1-methyl-propyl)-3- isoquinolinecarboxamide showed that the M(r) 18,000 PBR protein was specifically labeled. This indicates that the R2C cells express a PBR protein which has properties similar to the MA-10 PBR. Chemical crosslinking studies of purified metabolically radiolabeled DBI to mitochondria provided direct evidence that DBI specifically binds to the M(r) 18,000 PBR protein. Moreover, DBI and a PBR synthetic ligand were able to increase steroid production in isolated R2C cell mitochondria which express the 5 nM affinity receptor. However, mitochondrial PBR binding was increased by 6-fold upon addition of the post-mitochondrial fraction, suggesting that a cytosolic factor modulates the binding properties of PBR in R2C cells and is responsible for the 0.5 nM affinity receptor seen in intact cells. In conclusion, these data demonstrate that DBI plays a key role in maintaining R2C constitutive steroidogenesis by binding to the mitochondrial higher affinity PBR which promotes a continuous supply of cholesterol to the inner mitochondrial side chain cleavage cytochrome P450.

Effect of vasoactive intestinal peptide on the release of growth hormone in perifused pituitary and hypothalamus of the goat.

The effect of vasoactive intestinal peptide (VIP) on the release of growth hormone (GH) from the adenohypophysis of the goat was studied in vitro using the perifusion system. Two perifusion systems were employed to differentiate potential direct effects of VIP on the pituitary from indirect effects mediated through the hypothalamus. The first perifusion system used single chambers housing only pituitary fragments. The second system used two chambers in tandem, one containing hypothalamus and the second the pituitary fragments, the flow passing through the hypothalamic chamber first. VIP (10(-6), 10(-7), 10(-8)M) stimulated significant GH release in both perifusion systems (P less than 0.05, P less than 0.01) in a concentration related fashion. The quantity of GH release induced by the 10(-9)M and 10(-10)M VIP groups were not significant. Further, the GH released from the system that perifused the pituitary alone (10(-8)M VIP) was significantly higher than that containing both the hypothalamus and the pituitary fragments (P less than 0.05). The relative lower GH secretory response to the same dose of VIP applied to the hypothalamus-pituitary suggests that the perifused caprine hypothalamus may release an inhibitory factor, such as somatostatin in vitro. These results suggest that VIP stimulates GH release by acting directly on the adenohypophysis of the goat.

Egg-laying pattern of the semi-domesticated helmeted guinea fowl (Numida meleagris galeata).

1. Studies on the egg laying pattern of the semi-domesticated helmeted guinea fowl showed that the birds laid eggs between 06.00 and 20.00 h local time (05.00 to 19.00 h GMT). 2. More (67.9%) were laid in the evenings (15.00 to 20.00 h) than at any other period. 3. There were two distinct periods in the reproductive cycle: a breeding season which began in April and ended in September, and a resting or non-breeding period between October and March. 4. Sequence length was predominantly of 4 eggs and July was the month of peak egg production.

Effects of kola-nut extract administration on the liver, kidney, brain, testis and some serum constituents of the rat.

Kola-nut extract induced a number of overt neurotoxicological signs in male albino rats. A decrease in the total body weight and an increase in the absolute weights of the liver, kidney, brain and testis were observed after 18 weeks oral administration of kola-nut extract to the rats. Total protein, RNA and DNA of these organs were significantly depressed. The activity of beta-glucuronidase and beta-galactosidase was induced only in the kidney, brain and testis of treated animals. While the levels of serum phosphomonoesterases and total cholesterol were significantly enhanced due to kola-nut intake, serum total and conjugated bilirubin levels were significantly decreased.

Effects of dimethylnitrosamine on some actions of testosterone.

Using homogenates of mouse kidney and testes, the activities of the enzymes, beta-glucuronidase and beta-galactosidase, were studied as markers of androgen action. The results obtained differed between testes and kidney homogenates. Dimethylnitrosamine (DMN) may cause a competitive inhibition of the anabolic action of testosterone in kidney homogenates but this was not evident from the results obtained with testes homogenates.