Human recombinant follicle stimulating hormone (rFSH) compared to urinary human menopausal gonadotropin (HMG) for ovarian stimulation in assisted reproduction: a literature review and cost evaluation.

BACKGROUND: Gonadotropins are protein hormones which are central to the complex endocrine system that regulates normal growth, sexual development, and reproductive function. There is still a lively debate on which type of gonadotropin medication should be used, either human menopausal gonadotropin or recombinant follicle-stimulating hormone. The objective of the study was to perform a systematic review of the recent literature to compare recombinant follicle-stimulating hormone to human menopausal gonadotropin with the aim to assess any differences in terms of efficacy and to provide a cost evaluation based on findings of this systematic review. METHODS: The review was conducted selecting prospective, randomized, controlled trials comparing the two gonadotropin medications from a literature search of several databases. The outcome measure used to evaluate efficacy was the number of oocytes retrieved per cycle. In addition, a cost evaluation was performed based on retrieved efficacy data. RESULTS: The number of oocytes retrieved appeared to be higher for human menopausal gonadotropin in only 2 studies while 10 out of 13 studies showed a higher mean number of oocytes retrieved per cycle for recombinant follicle-stimulating hormone. The results of the cost evaluation provided a similar cost per oocyte for both hormones. CONCLUSIONS: Recombinant follicle-stimulating hormone treatment resulted in a higher oocytes yield per cycle than human menopausal gonadotropin at similar cost per oocyte.

Functional polymorphisms in COMT and SLC6A4 genes influence the prognosis of patients with medication overuse headache after withdrawal therapy.

BACKGROUND AND PURPOSE: It is currently unknown if common genetic variants influence the prognosis of patients with medication overuse headache (MOH). Here the role of two common single nucleotide polymorphisms in the COMT gene (rs4680 and rs6269), as well as the STin2 variable number tandem repeat (VNTR) polymorphism in the SLC6A4 gene, were evaluated as predictors for long-term outcomes of MOH patients after withdrawal therapy. METHODS: Genotyping was conducted by polymerase chain reaction (PCR), PCR restriction fragment length polymorphism analysis or real-time PCR allelic discrimination assay on genomic DNA extracted from peripheral blood. Gene variants association was evaluated by logistic regression analysis adjusted for clinical confounding factors, and the threshold of statistical significance for multiple testing was set at P < 0.012. RESULTS: Sixty-five MOH patients with unsuccessful detoxification and 83 MOH patients with effective drug withdrawal therapy were available for the analysis. rs4680G allele carriers or the COMT rs6269G-rs4680G haplotype were found to be associated with a lower risk of relapse within the first year after successful detoxification therapy, in comparison with homozygous rs4680A allele carriers [odds ratio (OR) 0.17, 95% confidence interval (CI) 0.05-0.61, P = 0.007] or with the COMT rs6269A-rs4680A haplotype (OR 0.19, 95% CI 0.06-0.54, P = 0.003), respectively. In addition, carriers of the STin2 VNTR short allele were found at higher odds for the composite poor outcome including unsuccessful withdrawal therapy and relapse within 12 months of follow-up after successful detoxification (OR 2.81, 95%CI 1.26-6.25, P = 0.009). CONCLUSIONS: Our results indicate that genotyping for COMT rs4680 and SLC6A4 STin2 VNTR could be useful for the identification of MOH patients at higher risk of poor prognosis after drug withdrawal.

Association of haplotype combination of serotonin transporter gene polymorphisms with monthly headache days in MOH patients.

OBJECTIVES: To evaluate the role of 5-HTTLPR, STin2 VNTR, and rs1042173T>G polymorphisms of the serotonin transporter gene (SLC6A4) as susceptibility factors for medication overuse headache (MOH) and to assess their value as predictors of the number of headache days per month, a potential marker of disease severity. METHODS: Genotyping was performed by PCR and PCR-RFLP on genomic DNA extracted from peripheral blood of 227 MOH patients and 312 control subjects. Logistic regression analysis was used to evaluate the association between the SL6A4 gene polymorphisms and MOH risk. The association between polymorphic variants and monthly headache days was evaluated by linear regression analysis. RESULTS: Logistic regression analysis, adjusted for age and gender, revealed a nominal association between rs1042173T>G and MOH risk (TT vs. TG + GG, OR: 1.58 95% CI: 1.05-2.37, P = 0.028). In the linear regression analysis adjusted for age, gender, primary headache diagnosis, acute drug overused and monthly drug number, STin2 VNTR was found nominally associated with monthly headache days (12/12 vs. others, difference: 1.55 days, 95% CI: 0.01-3.08, P = 0.050). When STin2 VNTR and rs1042173T>G were analyzed in haplotypic combination, a global haplotype association emerged with monthly headache days which remained significant after Bonferroni correction for multiple comparisons (global haplotype association P = 0.0056). CONCLUSION: Although a minor contribution of SLC6A4 variants in the genetic liability of MOH cannot be excluded, haplotype-based analysis of STin2 VNTR and rs1042173T>G polymorphisms allowed to identify a subgroup of MOH patients with a higher number of monthly headache and, possibly, with a more severe disease.

Calcineurin primes immature gonadotropin-releasing hormone-secreting neuroendocrine cells for migration.

During development, many neurons display calcium-dependent migration, but the role of this messenger in regulating gene expression leading to this event has not yet been elucidated. Among the decoders of calcium signals is calcineurin, a Ca(2+)/calmodulin serine/threonine phosphatase that has been involved in both short-term and long-term cellular changes. By using immortalized GnRH-secreting neurons, we now show that, in vitro, Ca(2+)-dependent gene expression, proceeding via calcineurin and the transcription factor nuclear factor of activated T cells, is a key player controlling the chemomigratory potential of developing GnRH-secreting neurons. Furthermore, our data highlight the switch nature of this phosphatase, whose activation or inactivation guides cells to proceed from one genetic program to the next.

Gene regulation in the frontal cortex of rats exposed to the chronic mild stress paradigm, an animal model of human depression.

In the present study, we have coupled the chronic mild stress (CMS) protocol with Affymetrix microarray technology to screen the rat genome for gene changes in the frontal cortex. The aim of our work was to assess whether the CMS protocol could be a useful experimental model to provide insights into the molecular basis of depression. Under our experimental conditions, 59 transcripts changed by more than +/-1.5-fold between naïve and anhedonic rats and showed significantly altered expression levels (P < 0.05). Among these, 18 were upregulated (fold change range +1.509 to +3.161) and 41 were downregulated (fold change range -1.505 to -2.659). To confirm the data obtained with microarrays, we used real-time reverse transcription polymerase chain reaction (RT-PCR). The results confirmed the downregulation of Itga6, Camk2a, Plcb1, Cart, Gad1, Homer1 and Th and the upregulation of Egr2 and Ptgs2 observed in the DNA microarray analysis. Moreover, the fold change data of the nine validated transcripts from microarray analysis and real-time polymerase chain reaction showed a good correlation (r = 0.863, 7 d.f., P < 0.01; slope = 0.976). It is of great interest that prostaglandin-endoperoxide synthase 2, tyrosine hydroxylase, Cart, Homer1 and glutamate decarboxylase have already been implicated in affective disorders by different approaches in previous reports. In conclusion, our findings indicate that the CMS paradigm is a useful preclinical model with which to investigate the molecular basis of anhedonia and to help in the discovery of novel targets for antidepressant drugs.

Inhibitors of histone deacetylase (HDAC) restore the p53 pathway in neuroblastoma cells.

BACKGROUND AND PURPOSE: Inhibitors of histone deacetylase (HDAC) are emerging as a promising class of anti-cancer drugs, but a generic deregulation of transcription in neoplastic cells cannot fully explain their therapeutic effects. In this study we evaluated alternative molecular mechanisms by which HDAC inhibitors could affect neuroblastoma viability. EXPERIMENTAL APPROACH: Effects of HDAC inhibitors on survival of the I-type SK-N-BE and the N-type NB SH-SY5Y neuroblastoma cell lines were assessed by the MTT assay. Molecular pathways leading to this were examined by western blot, confocal microscopy and cytofluorometry. The mRNA levels of apoptotic mediators were assessed semi-quantitatively by RT-PCR. Tumour-suppressor p53 trans activity was assessed in EMSA experiments. HDAC inhibitors were also studied in cells subjected to plasmid-based p53 interference (p53i). KEY RESULTS: HDAC inhibitors induced cell death via the mitochondrial pathway of apoptosis with recruitment of Bcl-2 family members. Bcl-2 overexpression rendered neuroblastoma cells resistant to HDAC inhibitor treatment. Low concentrations of HDAC inhibitors (0.9 mM) caused a G(2) cell-cycle arrest and a marked upregulation of the p21/Waf1/Cip1 protein. HDAC inhibitors also activate the p53 protein via hyper-acetylation and nuclear re-localization, without affecting its protein expression. Accordingly, HDAC inhibitor-induced cell-killing and p21/Waf1/Cip1 upregulation is impaired in p53i-cells. CONCLUSIONS AND IMPLICATIONS: In neuroblastoma cells, HDAC inhibitors may overcome the resistance to classical chemotherapeutic drugs by restoring the p53 tumour-repressor function via its hyper-acetylation and nuclear migration, events usually impaired in such tumours. In neuroblastoma cells, HDAC inhibitors are not able to induce p21/Waf1/Cip1 in the absence of a functional p53.

Transcriptional Remodeling in Primary Hippocampal Astrocytes from an Alzheimer's Disease Mouse Model.

BACKGROUND: It is well known that alterations in astrocytes occur in Alzheimer's disease and reactive astrogliosis is one of the hallmarks of the disease. Recently, data has emerged that suggests that alterations in astrocytes may also occur early in the pathogenesis of the disease. OBJECTIVE: The aim of present work was to characterize the transcriptional alterations occurring in cultured astrocytes from 3xTg-AD mouse pups compared to control non-transgenic mice. Furthermore, we also compared these changes to those reported by others in astrocytes from symptomatic AD mice. METHOD: We conducted a whole-genome microarray study on primary cultured astrocytes from the hippocampus of 3xTg-AD and non-transgenic mouse newborn pups. We used cross-platform normalization and an unsupervised hierarchical clustering algorithm to compare our results with other datasets of cultured or freshly isolated astrocytes, including those isolated from plaque-stage APPswe/PS1dE9 AD mice. RESULTS: We found a set of 993 genes differentially expressed in 3xTg-AD as compared with non-Tg astrocytes. Over-represented gene ontology terms were related to calcium, cell-cell communication, mitochondria, transcription, nucleotide binding and phosphorylation. Of note, no genes related to inflammation were found in cultured 3xTg-AD astrocytes. Comparison with astrocytes isolated from plaque stage APPswe/PS1dE9 showed that 882 out of 993 genes were selectively changed in primary 3xTg-AD astrocytes while 50 genes were co-regulated and 61 were anti-regulated (regulated in the opposite direction in the datasets). CONCLUSION: Our data show that in cultured astrocytes from an AD mouse model, transcriptional changes occur and are different from those reported in models mimicking later stages of the disease.

Common European Mitochondrial Haplogroups in the Risk for Radiation-induced Subcutaneous Fibrosis in Breast Cancer Patients.

AIMS: The contribution of mitochondrial DNA (mtDNA) variations to clinical radiosensitivity is largely unknown. In the present study, we evaluated the association between mtDNA haplogroups and the risk of radiation-induced subcutaneous fibrosis after postoperative radiotherapy in breast cancer patients. MATERIALS AND METHODS: Subcutaneous fibrosis was scored according to the Late Effects of Normal Tissue-Subjective Objective Management Analytical (LENT-SOMA) scale in 286 Italian breast cancer patients who received radiotherapy after breast-conserving surgery. Eight mtDNA single nucleotide polymorphisms that define the nine major haplogroups in the European population were determined by polymerase chain reaction restriction fragment length polymorphism analysis on genomic DNA extracted from peripheral blood. RESULTS: In a Kaplan-Meier analysis evaluated by the Log-rank test, carriers of haplogroup H were found to be at lower risk of grade ≥2 subcutaneous fibrosis (P = 0.018) compared with all other haplotypes combined. In the multivariate Cox regression analysis adjusted for clinical factors (body mass index, breast diameter, adjuvant treatment, dose per fraction, radiation type and acute skin toxicity), haplogroup H emerged as a protective factor for moderate to severe radiation-induced fibrosis at a nominal significance level (hazard ratio: 0.50, 95% confidence interval 0.27-0.92, P = 0.027), which did not survive correction for multiple testing. CONCLUSIONS: Our results suggest a protective effect of the mitochondrial haplogroup H in the development of radiation-induced fibrosis in breast cancer patients. However, the loss of statistical significance after correction for multiple comparisons and the lack of an independent validation cohort make our findings preliminary, requiring further confirmation in large-scale prospective studies.

Mitogenic effect of nerve growth factor (NGF) in LNCaP prostate adenocarcinoma cells: role of the high- and low-affinity NGF receptors.

We have investigated the effect of nerve growth factor (NGF) in the androgen-dependent, prostate adenocarcinoma LNCaP cell line. Exposure of LNCaP cells to NGF resulted in a significant increase of cell proliferation. The effect was concentration dependent and equally present in serum- or charcoal-stripped serum-supplemented and serum-deprived conditions. The mitogenic action of NGF was accompanied by an enhanced expression of prostate-specific antigen (PSA) and resulted additive to the proliferative effect of dihydrotestosterone. The proliferative effect of NGF appeared to be mediated by the high-affinity NGF receptor, p140trka. Only p140trka, but not the low-affinity NGF receptor, p75LNGFR, was expressed in LNCaP cells; both the proliferative response and the phosphorylation of p140trka upon NGF treatment were prevented by the tyrosine kinase inhibitor K252a. LNCaP cells transiently transfected with the cDNA encoding for p75LNGFR appeared more sensitive to NGF, as demonstrated by the increased number of p75LNGFR-transfected LNCaP cells exposed for 72 h to NGF compared with wild LNCaP cultures. However, p75LNGFR-transfected LNCaP cells rapidly underwent apoptotic death when deprived of NGF. Our study demonstrates the physiological relevance of NGF in the regulation of prostate cell proliferation and the relative contribution of the high- and low-affinity NGF receptors in this control.

D-2 dopamine receptor activation reduces free [3H]arachidonate release induced by hypophysiotropic peptides in anterior pituitary cells.

Dopamine reduces the stimulation of intracellular [3H]arachidonate release produced by the two PRL-stimulating peptides angiotensin-II and TRH. This effect is concentration dependent and is mediated by stimulation of D-2 dopamine receptors. D-2 receptor agonists (bromocriptine, dihydroergocryptine, and dihydroergocristine) inhibit the release of fatty acid induced by angiotensin-II with a potency that parallels their ability to inhibit PRL release in vitro. Conversely, the selective D-2 receptor antagonist L-sulpiride completely prevents dopamine's effect, whereas SCH 23390 (a D-1 receptor antagonist) is ineffective. The inhibitory action of dopamine does not seem to be consequent to an action on the adenylate cyclase-cAMP system, as 8-bromo-cAMP (1 mM) does not affect either basal or dopamine-inhibited [3H]arachidonate release. However, a 24-h pertussis toxin pretreatment significantly reduces the action of dopamine on fatty acid release. Collectively, these results suggest that D-2 dopamine receptor-mediated inhibition of intracellular [3H]arachidonate release requires the action of a GTP-binding protein, but is not a consequence of an inhibitory action on cAMP levels.

Angiotensin peptides stimulate phosphoinositide breakdown and prolactin release in anterior pituitary cells in culture.

We investigated the effects of angiotensin peptides on the breakdown of specific membrane phospholipids, the inositol lipids, in anterior pituitary cells in culture, measuring the water-soluble products (inositol phosphates) produced during the cleavage of phosphoinositides by phospholipase C. Both angiotensin II and angiotensin I in the presence of 10 mM LiCl potently increased, in a concentration-dependent manner, total [3H]inositol phosphate and PRL release in cultured rat anterior pituitary cells. The release of LH, TSH, or GH was not significantly enhanced by the peptides. The effect on inositol phosphate accumulation was significant at 0.01 nM, and maximal stimulation (approximately 5-fold increase) occurred at 10 nM, with an ED50 of about 0.3 nM. The stimulatory effects of both angiotensin II and angiotensin I were antagonized by the receptor antagonists saralasin and Sar1,Ile8-angiotensin II. Moreover, 1 microM captopril, an inhibitor of angiotensin-converting enzyme, antagonized the effects of 0.1 and 1 nM angiotensin I, suggesting that the effect of angiotensin I on phosphoinositide breakdown and PRL release is dependent on prior conversion of angiotensin I to angiotensin II. The effect of angiotensin II was very rapid. Fractionation of the water-soluble inositol phosphates showed that angiotensin II significantly increased inositol bisphosphate and inositol triphosphate at 10 sec, whereas inositol monophosphate was increased only after 40 sec. These data indicate that in the pituitary, and presumably in the lactotroph, the binding of angiotensin II to specific membrane receptors provokes increased polyphosphoinositide hydrolysis, leading to increased production of intracellular messengers, i.e. inositol triphosphate and 1,2-diacylglycerol, responsible for the stimulation of PRL release.

Neuroprotective effect of insulin-like growth factor I in immortalized hypothalamic cells.

The neuroprotective action of insulin-like growth factor I (IGF-I) was tested in immortalized hypothalamic GT1-7 cells exposed to reduced glutathione depleting agents, which cause oxidative stress and cell death. The extent of cell survival was assessed by either using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide cytotoxicity assay or counting at the fluorescence microscope GT1-7 cells prelabeled with fluorescent dyes selective for viable and dead cells. Treatments with buthionine sulfoximine (500 microns), diethylmaleate (1 mM), and ethacrynic acid (200 microns) caused diffuse GT1-7 cell death (40-60%). Exposure of the same cells to IGF-I (either before or concomitant to the toxic agent, depending on the drug used) significantly prevented neuronal death. This effect was rapid, concentration-dependent, maximal at concentrations of 25-50 ng/ml, and mimicked by IGF-II, fibroblast growth factor, and the potent antioxidant idebenone. In contrast, IGF-I, as well as idebenone, were completely ineffective in antagonizing the toxic effect produced by different concentrations of menadione. In conclusion, the present data demonstrate a protective role for IGF-I against glutathione depleting agents-induced damage in GT1-7 cells suggesting an antioxidant action of this growth factor in hypothalamic neurons.

Tumor necrosis factor-alpha induces apoptosis in immortalized hypothalamic neurons: involvement of ceramide-generating pathways.

To investigate possible effects that may contribute, together with a direct action on neurohormone secretion, to the impairment of gonadal axis function during inflammation, we evaluated the effect of TNF alpha on the growth and viability of GT1-7 hypothalamic neurons and the intracellular transduction pathways involved in these effects. TNF alpha caused a reduction of cell number and an induction of apoptotic death. These effects were mimicked by cell-permeable analogs of ceramide and by neutral or acidic sphingomyelinase. Exposure to acidic sphingomyelinase induced a persistent (up to 48 h) reduction of cell growth and apoptosis, whereas the effect of neutral sphingomyelinase was time limited. The involvement of acidic sphingomyelinase in TNF alpha action was demonstrated by the partial prevention of ceramide generation, apoptosis, and reduced cell growth by the inhibitor of the acidic sphingomyelinase-generating pathway, D609, whereas the involvement of ceramide was proved by complete prevention of TNF alpha-induced effects by treatment with okadaic acid at concentrations inhibiting ceramide-dependent protein phosphatase. The present data indicate that TNF alpha, through activation of ceramide-generating pathways, is able to affect GT1-7 cell viability, suggesting an additional effect that may contribute to the global action of this cytokine on neuroendocrine activities.

Involvement of nitric oxide in the regulation of gonadotropin-releasing hormone release from the GT1-1 neuronal cell line.

A role for nitric oxide (NO) in the regulation of hypothalamic neurohormone secretion has been suggested. The aim of the present study was to establish a direct involvement of this novel intracellular regulatory molecule in the control of GnRH release. For this purpose, the GT1-1 GnRH-secreting continuous cell line was treated with various agents that can modify the endogenous NO synthase activity or, alternatively, with substances that can liberate NO, mimicking an increased concentration of this molecule in the cell. Treatment of GT1-1 cells with increasing concentrations of L-arginine, the direct precursor of NO, produced a marked reduction of norepinephrine-stimulated GnRH release despite a lack of effect on basal secretion. Similarly, the NO donors SIN-1 and acidified NaNO2 potently reduced basal as well as KCl-stimulated GnRH secretion. Conversely, sodium nitroprusside caused a significant inhibition of KCl-stimulated, but not basal, GnRH secretion. Addition of these agents to GT1-1 cells resulted in a marked increase in intracellular cGMP accumulation. Addition of the NO synthase inhibitors N-nitro-L-arginine and N-nitro-L-arginine methyl ester stimulated basal GnRH secretion without modifying norepinephrine- or KCl-stimulated release. In addition, treatment of GT1-1 cells with both L-arginine analogs produced a significant inhibition of the basal cGMP concentration. Together, these data suggest an inhibitory role for NO in the control of GnRH secretion from GT1-1 cells.

The inhibition of phosphatidylinositol turnover: a possible postreceptor mechanism for the prolactin secretion-inhibiting effect of dopamine.

We studied the association between the inhibition of phosphatidylinositol (PI) turnover and the inhibition of PRL secretion in the presence of dopamine. The incorporation of radiolabeled phosphate into anterior pituitary gland PI as well as serum PRL levels were significantly (P less than 0.01) greater in female than in male rats. No significant sex-related difference was found in the incorporation by pituitary tissue of 32P into phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Dopamine decreased the incorporation of 32P into PI, but not into PC or PE, by female rat pituitary glands; this effect was reversed by two dopamine receptor-blocking agents, haloperidol and pimozide. After dopamine was removed from the incubation medium, basal 32P incorporation into PI was restored within 10 min. The administration of bromocriptine (500 micrograms/kg, ip, 4 h earlier) significantly reduced pituitary PI turnover. Conversely, in vivo injection of alpha-methyl-p-tyrosine (alpha MpT; 200 mg, ip, 2.5 h before death), an inhibitor of catecholamine biosynthesis, dramatically increased serum PRL levels. In vitro incorporation of 32P into PI, but not into PC or PE, increased (+130%) when these glands were incubated for 30 min with radiolabeled phosphate. The in vitro addition of 0.5 microM dopamine to glands from alpha MpT-treated rats counteracted the stimulation of 32P incorporation into PI produced by alph MpT treatment. In rats bearing the transplantable PRL-secreting tumor MtTW15, the hyperprolactinemia produced by the tumor stimulates hypothalamic turnover of dopamine, with a consequent inhibition of pituitary gland PRL secretion. 32P incorporation into PI, but not into PC or PE, was significantly (P less than 0.01) inhibited (-41%) in pituitary glands from these rats. The injection of alpha MpT (200 mg/kg, ip) or haloperidol (2 mg/kg, ip) 12 and 3 h before death into MtTW15 tumor-bearing rats abolished the inhibition of 32P incorporation into pituitary PI. Dopamine also decreased PI turnover in the 7315a PRL-secreting pituitary tumor. Our data indicate that the PI cycle may be an intracellular mechanism controlling PRL release in the rat and that the changes in its cleavage and turnover may be an early postreceptor event responsible for the inhibition of PRL secretion produced by factors such as dopamine.

Dopamine inhibits 32P incorporation into phosphatidylinositol in the anterior pituitary gland of the rat.

The in vitro effect of dopamine, haloperidol and pimozide on 32Pi incorporation into phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine by anterior pituitary glands was studied. Dopamine decreased the incorporation of 32Pi into phosphatidylinositol in a dose-related manner without affecting phosphatidylcholine and phosphatidylethanolamine. Haloperidol and pimozide did not modify 32Pi incorporation into these phospholipids by themselves but completely reversed the inhibitory effect of dopamine on phosphatidylinositol. The inhibition of 32Pi incorporation into phosphatidylinositol synthesis in the presence of 500 nM dopamine was significant at 20 min of incubation and maximal at 30 min. The possibility that a decrease in phosphatidylinositol cleavage and turnover may be involved in the inhibitory regulation of prolactin secretion by DA is suggested.

Arachidonate stimulates prolactin release in vitro: a role for the fatty acid and its metabolites as intracellular regulator(s) in mammotrophs.

We investigated the involvement of arachidonate in the PRL secretory process using three experimental systems: hemipituitary glands incubated in vitro, cultured pituitary cells, and dispersed anterior pituitary cells perifused in columns. Arachidonate (100 microM) significantly (P less than 0.05) stimulated PRL release in the former system and stimulated PRL secretion in a dose-related manner in cultured cells. In hemipituitary glands, indomethacin, a cyclooxygenase inhibitor, potentiated the arachidonate-mediated stimulation, whereas nordihydroguaiaretic acid or BW755c abolished it. The latter two agents, but not indomethacin, abolished the effect of phospholipase A2 on PRL release in vitro. BW755c also inhibited the stimulatory effect of TRH on PRL release in both experimental systems. Conversely, the stimulation of PRL release by phorbol myristate acetate (PMA), although significantly reduced, was not abolished by either nordihydroguaiaretic acid or BW755c. Quinacrine, a phospholipase A2 inhibitor, also abolished the stimulatory effect of phospholipase A2 or TRH on PRL release. In cultured cells, quinacrine inhibits basal PRL release, but does not affect PRL release induced by arachidonate or (Bu)2 cAMP. These results more firmly establish a role for arachidonate as an intracellular mediator of PRL release and suggest the involvement of an arachidonate metabolic pathway(s) (lipoxygenase and epoxygenase) other than prostaglandin or thromboxane formation. The effect of PMA on PRL release may be attributable only in part to an increase in the production of arachidonate metabolites, and most of PMA's effect on PRL release may relate to its activation of protein kinase C.

Glia mediates the neuroprotective action of estradiol on beta-amyloid-induced neuronal death.

17beta-Estradiol (17beta-E(2)) is known to exert neuroprotective activity against beta-amyloid, but its exact target and mechanism of action in this effect have not been elucidated. The involvement of astroglia in neuroprotection of 17beta-E(2) against the beta-amyloid fragment [betaAP((25-35))] has been evaluated using an experimental paradigm in which medium conditioned from rat astroglia pretreated with 17beta-E2 was transferred to pure rat cortical neurons challenged with 25 microm betaAP((25-35)) for 24 h. The toxicity of betaAP((25-35)) was assessed by flow cytometry, evaluating the ability of the peptide to induce an aberrant mitotic cell cycle in neurons. The results obtained indicate that conditioned medium from astrocytes preexposed to 17beta-E(2) for 4 h increased the viability of cortical neurons treated with betaAP((25-35)). This effect was not modified by treatment with the estrogen receptor antagonist ICI 182,780, added directly to neurons, nor was it mimicked by direct addition of 17beta-E(2) to neuronal cultures during exposure to betaAP((25-35)). A soluble factor stimulated by 17beta-E(2) seemed to be involved, and accordingly, the intracellular and released levels of TGF-beta1 were increased by 17beta-E(2) treatment, as established by Western blot analysis. In addition, the intracellular content of TGF-beta1 in immunopositive cells, as detected by flow cytometry, was reduced, suggesting that 17beta-E(2) stimulated mainly the release of the cytokine. In support of a role for TGF-beta1 in astrocyte-mediated 17beta-E(2) neuroprotective activity, incubation with a neutralizing anti-TGF-beta1 antibody significantly modified the reduction of neuronal death induced by 17beta-E(2)-treated astrocyte-conditioned medium.

Ubiquinone protects cultured neurons against spontaneous and excitotoxin-induced degeneration.

Ubiquinone is an endogenous quinone with pharmacological actions mainly related to its antioxidant properties. Here we report that ubiquinone protects cultured cerebellar granule cells against glutamate-induced neurotoxicity. In control cultures at 9 days of maturation in vitro (DIV), a 30-min exposure to 100 microM glutamate induced neuronal degeneration, as reflected by the great percentage (greater than 90%) of cells labeled with propidium iodide 24 h after the exposure. Glutamate-induced neuronal death was dramatically reduced in cultures treated daily with ubiquinone since the second DIV. In these cultures, glutamate failed to induce a "delayed" increase in the influx of 45Ca2+, an established parameter of excitotoxicity. Similarly, repeated addition of ubiquinone attenuated in a concentration-dependent manner the age-dependent degeneration of granule cells that is due to the toxic action of the endogenous glutamate progressively released into the medium. These results suggest that ubiquinone may be a useful drug in the therapy of acute and chronic neurodegenerative diseases related to hyperactivity of excitatory amino acid neurotransmission.

Behavioral effects of phosphatidylserine in aged rats.

The behavioral effects of 5 days of administration of phosphatidylserine (PS) was studied in aged rats. The intraperitoneal (5, 10, and 20 mg/kg) or intracerebroventricular (5, 10, and 20 micrograms/2 microliters) injection of PS liposomes caused a facilitated acquisition of active avoidance behavior as studied in shuttle-box and pole jumping test situations. The retention of active and passive avoidance responses was also improved. No substantial difference between PS-treated and control animals was apparent either in the responsiveness to electrical footshock or in the motor activity tested in an open field. Grooming behavior appeared to be enhanced in rats treated with the highest dose of the substance. Since PS affects both central catecholaminergic and cholinergic transmission, which is known to be impaired in old animals, the possibility that the behavioral effects of PS involve brain dopamine and/or acetylcholine systems is discussed.