Silica-collagen nanoformulations with extended human growth hormone release.

Growth hormone deficiency has been treated by the daily administration of recombinant human growth hormone (hGH) for decades. Patient compliance to this treatment is generally incomplete due to challenges including dose frequency and lack of perceived benefits. This stimulates the research on new formulations to reduce the number of periodic administrations. In this study silica nanoparticles and silica-collagen nanocomposites were evaluated for hGH loading and release. Bare nanoparticles showed higher hGH adsorption capacity than thiol- and isobutyl-bearing particles of similar diameters. Monitoring of bound protein conformation changes indicated hGH structure retention when adsorbed on bare silica nanoparticles and suggested no alterations on protein activity. Protein-loaded particles incorporated into collagen matrices (silica-collagen nanocomposites) showed a progressive protein release profile different from the observed for hGH-loaded silica nanoparticles and hGH-loaded collagen matrices. While both the collagen and the silica nanoparticle systems reached a 100 % release after 4 and 7 days respectively, silica-collagen nanocomposites showed a bi-phasic prolonged hGH release reaching approximately an 80 % after 15 days. These findings suggest that biocompatible silica-collagen nanocomposites could be used as vehicles for the prolonged delivery of hGH which could lead to a potential reduction in the number of periodic administrations.

Determination of Antibacterial Activity of Film Coatings Against Four Clinically Relevant Bacterial Strains.

Antibacterial coatings have currently gained great importance in biomedical technology investigations. Because of the spatial arrangement of the film coatings, evaluation of antibacterial activity presents a new challenge regarding traditional bacterial counting methods. In this protocol, four clinically relevant pathogens, Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were incubated on titania mesostructured thin film coatings for 24 h. Then, cell viability was studied considering three methods: counting of the number of colony forming units (CFU), live/dead staining, and quantification of extracellular DNA in suspension. Firstly, bacterial count was determined by the standard plate-count technique. Secondly, bacteria membrane integrity was evaluated by utilization of two fluorescent dyes, which allow distinction between live (membrane intact) and dead (membrane disrupted) bacteria. Lastly, extracellular DNA was quantified by spectrophotometry. In this manner, the three aforementioned techniques enabled the study of bacterial viability by qualitative and quantitative analyses.

A Survey on Analytical Methods for the Characterization of Green Synthesized Nanomaterials.

Nowadays, nanotechnologies are well established and the uses of a great variety of nanomaterials show exponential growth. The development of green synthesis procedures experienced a great development thanks to the contribution of researchers of diverse origins. The versatility of green chemistry allows producing a wide range of organic and inorganic nanomaterials with numerous promising applications. In all cases, it is of paramount importance to carefully characterize the resulting nanomaterials because their properties will determine their correct performance to accomplish the function to which they were synthesized or even their detrimental effects like nanotoxicological behavior. This review provides an overview of frequently employed characterization methods and their applications for green synthesized nanomaterials. However, while several different nanoscale materials and their associated green construction methodology are being developed, other important techniques would be extensively incorporated into this field soon. The aim is to encourage researchers in the field to employ a variety of these techniques for achieving an exhaustive characterization of new nanomaterials and for contributing to the development of validated green synthesis procedures.

Antibiofilm effect of mesoporous titania coatings on Pseudomonas aeruginosa biofilms.

Activation of photocatalytic titania by ultraviolet-A (UVA) radiation has been proposed as a good approach for combating bacteria. Titania powder, in solution or immobilized on a surface, has excellent UVA-assisted killing properties on several microorganisms. However, these properties could not be demonstrated in biofilms of Pseudomonas aeruginosa, a resistant opportunistic human pathogen that can cause severe complications in patients who are immunocompromised or have burn wounds or cystic fibrosis. P. aeruginosa biofilms have detrimental effects on health and industry, causing serious economic damage. In this study, the effect of titania photocatalysis for controlling P. aeruginosa biofilms was investigated by employing different coatings obtained through sol-gel and evaporation-induced self-assembly. Biofilms were grown on non-mesoporous and mesoporous titania surfaces with different pore sizes, which were achieved based on the use of surfactants Brij-58 and Pluronics-F127. In addition, two structural forms of titania were assayed: amorphous and anatase. As well as inhibiting biofilm formation, these coatings significantly enhanced the bactericidal effect of UVA on P. aeruginosa biofilms. The most efficient surface with regard to total antibacterial effect was the mesoporous Brij-58-templated anatase film, which, compared to control biofilms, decreased the number of viable bacteria by about 5 orders, demonstrating the efficacy of this methodology as a disinfection system.

Transforming an inert nanopolymer into broad-spectrum bactericidal by superstructure tuning.

Poloxamer block copolymers (also known as Pluronic®) are particularly useful for drug delivery and self-assembly techniques. These nanopolymers are generally considered to be biologically inert and they were used to generate only bacteria repellent surfaces but keeps bacteria alive and as a latent threat. However, the inherent capabilities of these nanopolymers to kill bacteria have been largely overlooked. Here, we report that Pluronic shaped as superstructures (self-organized array of micelles) in fact possess a broad-spectrum bactericidal activity (capability of killing bacteria) similar to that shown for some antibiotics. This further represents the first report that shows that appropriate control of superstructured mesophase architecture is a key parameter for bactericidal efficacy. Based on this finding, we have developed a highly bactericidal coating (>99.9% kill) against all tested Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Salmonella typhimurium LT2, Escherichia coli K12 and Pseudomonas aeruginosa PAO1) bacteria which moreover allows the adhesion and proliferation of mammalian cells. The inexpensiveness and ease of production make these versatile nanopolymer structures a powerful tool for the development of a new generation of highly effective antimicrobial coatings.

Antibiofilm effect of supramolecularly templated mesoporous silica coatings.

Bacteria attached to solid surfaces and encased in a self-synthesized matrix, so-called biofilms, are highly difficult to eradicate and present negative impact on industry and human health. The ability of supramolecularly templated mesoporous silica coatings to inhibit biofilm formation in Pseudomonas aeruginosa is shown here. Assays employing submerged and air-liquid interface biofilms demonstrated that mesoporous coatings with tuned pore size significantly reduce the number of attached bacteria and matrix production. Given its versatility, scalability, robustness and low cost, our proposal is attractive for the production of transparent, inert and permanent antibiofilm coatings that could be applied on multiple surfaces.

Innovative Immobilization Matrices.

We present a brief survey of some of the recent work of Professor Luis E. Díaz, performed together with his students and collaborators at the University of Buenos Aires. Dr Luis E. Díaz has been involved in research on biochemical and pharmaceutical sciences solving scientific and industry problems for over 40 years until he passed away. Prof. Díaz scientific interests included various topics from NMR spectroscopy to biomedicine but fundamentally he focused in various aspects of chemistry (analytical, organic, inorganic and environmental). This is not a complete survey but a sampling of prominent projects related to sol-gel chemistry with a focus on some of his recent publications.

Wired enzymes in mesoporous materials: A benchmark for fabricating biofuel cells.

Evolution of fuel cells using metallic inorganic catalysts has led to the development of biofuel cells with potential applications in implantable devices. However, the main disadvantages in real world applications of enzymatic biofuel cells are short lifetime and low power density. Many efforts have been devoted to immobilize redox enzymes on surfaces to allow efficient electrical communication with electrodes and to provide an adequate habitat for biochemical activity. In this context, nanocavities of mesoporous materials offer a tailored environment for protein immobilization. Mesostructured platforms with high surface area and stability have been developed to enhance mass transport, charge transfer from biocatalysts to electrodes and enzyme stability, leading to biofuel cells with improved power density (up to 602 µW cm(-2) at physiological conditions) and overall performance (high stability after 30 h of continuous operation and after 10 days of storage). This review discusses recent developments using mesoporous materials as novel platforms for effective electronic charge transfer in the context of current and emerging technologies in enzymatic fuel cell research, emphasizing their practical implications and potential improvements leading to a major impact on medical science and portable electronics.

The redox-active nanomaterial toolbox for cancer therapy.

Advances in nanomaterials science contributed in recent years to develop new devices and systems in the micro and nanoscale for improving the diagnosis and treatment of cancer. Substantial evidences associate cancer cells and tumor microenvironment with reactive oxygen species (ROS), while conventional cancer treatments and particularly radiotherapy, are often mediated by ROS increase. However, the poor selectivity and the toxicity of these therapies encourage researchers to focus efforts in order to enhance delivery and to decrease side effects. Thus, the development of redox-active nanomaterials is an interesting approach to improve selectivity and outcome of cancer treatments. Herein, we describe an overview of recent advances in redox nanomaterials in the context of current and emerging strategies for cancer therapy based on ROS modulation.

Lack of functional GABAB receptors alters Kiss1 , Gnrh1 and Gad1 mRNA expression in the medial basal hypothalamus at postnatal day 4.

BACKGROUND/AIMS: Adult mice lacking functional GABAB receptors (GABAB1KO) show altered Gnrh1 and Gad1 expressions in the preoptic area-anterior hypothalamus (POA-AH) and females display disruption of cyclicity and fertility. Here we addressed whether sexual differentiation of the brain and the proper wiring of the GnRH and kisspeptin systems were already disturbed in postnatal day 4 (PND4) GABAB1KO mice. METHODS: PND4 wild-type (WT) and GABAB1KO mice of both sexes were sacrificed; tissues were collected to determine mRNA expression (qPCR), amino acids (HPLC), and hormones (RIA and/or IHC). RESULTS: GnRH neuron number (IHC) did not differ among groups in olfactory bulbs or OVLT-POA. Gnrh1 mRNA (qPCR) in POA-AH was similar among groups. Gnrh1 mRNA in medial basal hypothalamus (MBH) was similar in WTs but was increased in GABAB1KO females compared to GABAB1KO males. Hypothalamic GnRH (RIA) was sexually different in WTs (males > females), but this sex difference was lost in GABAB1KOs; the same pattern was observed when analyzing only the MBH, but not in the POA-AH. Arcuate nucleus Kiss1 mRNA (micropunch-qPCR) was higher in WT females than in WT males and GABAB1KO females. Gad1 mRNA in MBH was increased in GABAB1KO females compared to GABAB1KO males. Serum LH and gonadal estradiol content were also increased in GABAB1KOs. CONCLUSION: We demonstrate that GABABRs participate in the sexual differentiation of the ARC/MBH, because sex differences in several reproductive genes, such as Gad1, Kiss1 and Gnrh1, are critically disturbed in GABAB1KO mice at PND4, probably altering the organization and development of neural circuits governing the reproductive axis.

Exhaustion of racing sperm in nature-mimicking microfluidic channels during sorting.

Fertilization is central to the survival and propagation of a species, however, the precise mechanisms that regulate the sperm's journey to the egg are not well understood. In nature, the sperm has to swim through the cervical mucus, akin to a microfluidic channel. Inspired by this, a simple, cost-effective microfluidic channel is designed on the same scale. The experimental results are supported by a computational model incorporating the exhaustion time of sperm.

Emerging technologies in medical applications of minimum volume vitrification.

Cell/tissue biopreservation has broad public health and socio-economic impact affecting millions of lives. Cryopreservation technologies provide an efficient way to preserve cells and tissues targeting the clinic for applications including reproductive medicine and organ transplantation. Among these technologies, vitrification has displayed significant improvement in post-thaw cell viability and function by eliminating harmful effects of ice crystal formation compared to the traditional slow freezing methods. However, high cryoprotectant agent concentrations are required, which induces toxicity and osmotic stress to cells and tissues. It has been shown that vitrification using small sample volumes (i.e., <1 µl) significantly increases cooling rates and hence reduces the required cryoprotectant agent levels. Recently, emerging nano- and micro-scale technologies have shown potential to manipulate picoliter to nanoliter sample sizes. Therefore, the synergistic integration of nanoscale technologies with cryogenics has the potential to improve biopreservation methods.

Lack of functional GABA(B) receptors alters GnRH physiology and sexual dimorphic expression of GnRH and GAD-67 in the brain.

GABA, the main inhibitory neurotransmitter, acts through GABA(A/C) and GABA(B) receptors (GABA(B)Rs); it is critical for gonadotropin regulation. We studied whether the lack of functional GABA(B)Rs in GABA(B1) knockout (GABA(B1)KO) mice affected the gonadotropin axis physiology. Adult male and female GABA(B1)KO and wild-type (WT) mice were killed to collect blood and tissue samples. Gonadotropin-releasing hormone (GnRH) content in whole hypothalami (HT), olfactory bulbs (OB), and frontoparietal cortexes (CT) were determined (RIA). GnRH expression by quantitative real-time PCR (qRT-PCR) was evaluated in preoptic area-anterior hypothalamus (POA-AH), medial basal-posterior hypothalamus (MBH-PH), OB, and CT. Pulsatile GnRH secretion from hypothalamic explants was measured by RIA. GABA, glutamate, and taurine contents in HT and CT were determined by HPLC. Glutamic acid decarboxylase-67 (GAD-67) mRNA was measured by qRT-PCR in POA-AH, MBH-PH, and CT. Gonadotropin content, serum levels, and secretion from adenohypophyseal cell cultures (ACC) were measured by RIA. GnRH mRNA expression was increased in POA-AH of WT males compared with females; this pattern of expression was inversed in GABA(B1)KO mice. MBH-PH, OB, and CT did not follow this pattern. In GABA(B1)KO females, GnRH pulse frequency was increased and GABA and glutamate contents were augmented. POA-AH GAD-67 mRNA showed the same expression pattern as GnRH mRNA in this area. Gonadotropin pituitary contents and serum levels showed no differences between genotypes. Increased basal LH secretion and decreased GnRH-stimulated gonadotropin response were observed in GABA(B1)KO female ACCs. These results support the hypothesis that the absence of functional GABA(B)Rs alters GnRH physiology and critically affects sexual dimorphic expression of GnRH and GAD-67 in POA-AH.

Participación de los receptores GABAB en la regulación del eje gonadotrófico: evaluación en ratones GABA / Participation of GABAB receptors on neuroendrocrine regulation in the regulation of the hypothalamic-pituitary-gonadal axis: evaluation in GABA mouse

Hasta el momento, los estudios realizados sobre la participación de los receptores GABAB (REGABAB) en la regulación neuroendocrina habían sido llevados a cabo a través de abordajes farmacológicos, mediante la utilización de agonistas y antagonistas específicos. En el presente trabajo utilizamos el modelo de ratón GABA para analizar las consecuencias endocrinas de la falta constitutiva de los RGABAB en la unidad hipotálamo-hipófiso-gonadal. No observamos diferencias en los contenidos hipofisarios ni en los niveles séricos de LH y FSH entre los genotipos en ningún sexo. Sin embargo, nuestros estudios in vitro, demostraron la existencia de alteraciones de la fisiología de los gonadotropos provenientes de hembras GABA, con una secreción basal aumentada de gonadotropinas y una menor respuesta el estímulo con GnRH. Al analizar más específicamente la funcionalidad del eje en esos ratones, encontramos alteraciones en el aumento de LH postcastración en las hembras, confirmando la participación de los RGABAB en este fenómeno. Por otro lado, en la hemras GABA adultas demostramos la presencia de alteraciones en el contenido hipotalámico de GnRH, el cual estaba francamente disminuido, y su secreción pulsátil, en la que se observa un aumento significativo de la frecuencia de los pulsos de GnRH. También observamos un aumento en los contenidos hipotalámicos de neurotransmisores aminoacídicos que podrían afectar la liberación de GnRH... (AU)

Participación de los receptores GABAB en la regulación del eje gonadotrófico: evaluación en ratones GABA / Participation of GABAB receptors on neuroendrocrine regulation in the regulation of the hypothalamic-pituitary-gonadal axis: evaluation in GABA mouse

Hasta el momento, los estudios realizados sobre la participación de los receptores GABAB (REGABAB) en la regulación neuroendocrina habían sido llevados a cabo a través de abordajes farmacológicos, mediante la utilización de agonistas y antagonistas específicos. En el presente trabajo utilizamos el modelo de ratón GABA para analizar las consecuencias endocrinas de la falta constitutiva de los RGABAB en la unidad hipotálamo-hipófiso-gonadal. No observamos diferencias en los contenidos hipofisarios ni en los niveles séricos de LH y FSH entre los genotipos en ningún sexo. Sin embargo, nuestros estudios in vitro, demostraron la existencia de alteraciones de la fisiología de los gonadotropos provenientes de hembras GABA, con una secreción basal aumentada de gonadotropinas y una menor respuesta el estímulo con GnRH. Al analizar más específicamente la funcionalidad del eje en esos ratones, encontramos alteraciones en el aumento de LH postcastración en las hembras, confirmando la participación de los RGABAB en este fenómeno. Por otro lado, en la hemras GABA adultas demostramos la presencia de alteraciones en el contenido hipotalámico de GnRH, el cual estaba francamente disminuido, y su secreción pulsátil, en la que se observa un aumento significativo de la frecuencia de los pulsos de GnRH. También observamos un aumento en los contenidos hipotalámicos de neurotransmisores aminoacídicos que podrían afectar la liberación de GnRH...

GABA(B) receptors in neuroendocrine regulation.

Gamma-amino butyric acid (GABA), in addition to being a metabolic intermediate and the main inhibitory neurotransmitter in the synaptic cleft, is postulated as a neurohormone, a paracrine signaling molecule, and a trophic factor. It acts through pre- and post-synaptic receptors, named GABA(A) and GABA(C) (ionotropic receptors) and GABA(B) (metabotropic receptor). Here we reviewed the participation of GABA(B) receptors in the regulation of the hypothalamic-pituitary-gonadal axis, using physiological, biochemical, and pharmacological approaches in rats, as well as in GABA(B1) knock-out mice, that lack functional GABA(B) receptors. Our general conclusion indicates that GABA(B )receptors participate in the regulation of pituitary hormone secretion acting both in the central nervous system and directly on the gland. PRL and gonadotropin axes are affected by GABA(B) receptor activation, as demonstrated in the rat and also in the GABA(B1) knock-out mouse. In addition, hypothalamic and pituitary GABA(B) receptor expression is modulated by steroid hormones. GABA participation in the brain control of pituitary secretion through GABA(B) receptors depends on physiological conditions, being age and sex critical factors.These results indicate that patients receiving GABA(B) agonists/antagonists should be monitored for possible endocrine side effects.

Impact of proestrous milieu on expression of orexin receptors and prepro-orexin in rat hypothalamus and hypophysis: actions of Cetrorelix and Nembutal.

Orexins and their receptors OX1 and OX2 regulate energy balance and the sleep-wake cycle. We studied the expression of prepro-orexin (PPO), OX1, and OX2 in brain and pituitary under the influence of the hormonal status in adult rats. Primarily, PPO, OX1, and OX2 expression was determined in Sprague-Dawley female cycling rats during proestrus and in males. Animals were killed at 2-h intervals. Anterior (AH) and mediobasal (MBH) hypothalamus, anterior pituitary (P), and frontoparietal cortex (CC) were homogenized in TRIzol, and mRNAs were obtained for screening of PPO, OX1, OX2 expression by semiquantitative RT-PCR. Main findings were confirmed and extended to all days of the cycle by quantitative real-time RT-PCR. Hormones and food consumption were determined. Finally, OX1, OX2, and PPO were measured by real-time RT-PCR in tissues collected at 1900 of proestrus after treatments at 1400 with ovulation-blocking agents Cetrorelix or pentobarbital. OX1 and OX2 expression increased at least threefold in AH, MBH, and P, but not in CC, between 1700 and 2300 of proestrus, without variations in estrus, diestrus, or in males. PPO in AH and MBH showed a fourfold or higher increase only during proestrus afternoon. Cetrorelix or pentobarbital prevented increases of OX1 and OX2 only in the pituitary and blunted gonadotropin surges, but left OX1, OX2, and PPO brain expression unchanged. Reproduction, energy balance, and sleep-wake cycle are integrated. Here, we demonstrate that, in the physiological neuroendocrine condition leading to ovulation, information to the orexinergic system acts in hypothalamus and pituitary by different mechanisms.

GABA(B1) knockout mice reveal alterations in prolactin levels, gonadotropic axis, and reproductive function.

gamma-Aminobutyric acid (GABA) has been implicated in the control of hypophyseal functions. We evaluated whether the constitutive loss of functional GABA(B) receptors in GABA(B1) knockout (GABA(B1)(-/-)) mice alters hormonal levels, under basal and stimulated conditions, and reproductive function. The serum hormone levels were measured by radioimmunoassay, the estrous cyclicity was evaluated by vaginal lavages, and the mating behavior was determined by the presence of vaginal plugs. A moderate hyperprolactinemic condition was observed, in which prolactin increase and thyroid-stimulating hormone decrease were similar between genotypes. Basal luteinizing hormone (LH), follicle-stimulating hormone, thyroid-stimulating hormone, and growth hormone levels were similar between genotypes in each sex. Analysis of the gonadotropin axis revealed no differences in puberty onset between female genotypes. In con trast, the estrous cyclicity was significantly disrupted in GABA(B1)(-/-) female mice, showing significantly extended periods in estrus and shortened periods in proestrus. Reproduction was significantly compromised in GABA(B1)(-/-) females, with a significantly lower proportion of mice (37.5%) getting pregnant during the first 30 days of mating as compared with wild-type controls (87.5%). Moreover, only 14% of vaginal plug positive GABA(B1)(-/-) females had successful pregnancies as compared with 75% in the controls. In addition, the postovariectomy LH rise was significantly advanced in GABA(B1)(-/-) mice, while the response to estradiol feedback was similar in both genotypes. In conclusion, our endocrine analysis of GABA(B1)(-/-) mice reveals that GABA(B) receptors are involved in the regulation of basal prolactin titers. Moreover, the hypothalamic-hypophyseal-ovarian axis is seriously disturbed, with alterations in cyclicity, postcastration LH increase, and fertility indexes. The molecular mechanism underlying these hormonal disturbances remains to be addressed.

Effect of androgens on sexual differentiation of pituitary gamma-aminobutyric acid receptor subunit GABA(B) expression.

Previous work demonstrated a sexually dimorphic ontogenic expression of gamma-aminobutyric acid receptors (GABA(B)R) in rat pituitary. As sex steroids determine sex-specific expression patterns, we now studied the effect of sex hormones on pituitary GABA(B)R expression. GABA(B)R subunits, measured by Western blot and by semi-quantitative RT-PCR and luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone measured by RIA were determined in two experimental designs: First experimental design: 8- and 15-day-old females (8F, 15F); 8F and 15F treated with 100 mug testosterone propionate (TP) on day 1 of life (8F100TP, 15F100TP), 8- and 15-day-old males (8M, 15M) and 8M and 15M castrated on day 1 (8MC, 15MC). Second experimental design: 8-day-old female and male animals: 8F, 8F100TP, 8F treated with 1 mug/day TP on days 1-4 (8F1TP), 8F treated with the androgen antagonist Flutamide (Flut: 2.5 mg/100 g BW of pregnant mother on days E17-E23) (8F-Flut), 8M, 8MC, 8M treated with Flut as above (8M-Flut) and 8MC-Flut. In these animals, in addition, GABA, glutamate, aspartate and taurine were measured by HPLC in hypothalami and cortex. In the first set of experiments, GABA(B1)R mRNA/protein expression was higher in 8F than in 15F, 8M or 15M. In 8F100TP, GABA(B1)R mRNA/protein decreased to male levels. TP treatment did not alter GABA(B1)R expression in 15F. There was no difference in GABA(B1)R expression between 8M and 15M and neonatal castration did not modify its expression. In the second set of experiments, TP (1 mug) or Flut did not modify GABA(B1)R in 8F, while 100 microg TP continued to decrease GABA(B1)R expression. In 8M, Flut, alone or with castration, increased GABA(B1)R mRNA/protein expression to 8F. Hypothalamic GABA content followed the same pattern as pituitary GABA(B)R expression in 8-day-old animals, suggesting a cross-regulation. With regard to hormonal levels, 100 microg, but not 1 microg TP altered gonadotropins at 8 days, although both treatments effectively androgenized females as evidenced by lack of cycling. We conclude that androgens, acting pre- and postnatally, decrease pituitary GABA(B)R subunit expression.

Gonadotropin-releasing hormone signaling pathways in an experimental ovarian tumor.

Previous results showed that GnRH signaling is altered in cells from rat luteinized ovarian tumors (tumor group) because it did not activate the phospholipase C pathway, in contrast to control ovarian cells from superovulated prepubertal rats (SPO). In the present work, alternate GnRH-induced second messengers such as phospholipase A(2) and phospholipase D activation, cAMP production, ERK1/2 phosphorylation, and the presence of G proteins were evaluated to determine GnRH mechanism of action in tumor cells. G proteins examined were present in both cell types. Buserelin, a GnRH agonist, (1, 10, and 100 ng/ml) increased phosphatidylethanol in SPO, indicating phospholipase D activation. Only 100 ng/ml buserelin induced a significant response in the tumor group. Buserelin (100 ng/ml) increased (3)H-arachidonic acid in culture media in SPO, indicating phospholipase A(2) activation; no effect was observed in the tumor group. Buserelin (100 and 1000 ng/ml) induced pertussis toxin-insensitive cAMP increases in both cell types, with similar potencies. In the tumor group, buserelin (100 ng/ml) inhibited human chorionic gonadotropin-induced cAMP and progesterone; this effect was protein kinase C (PKC) dependent (inhibited by GF109203X, a PKC inhibitor). Buserelin (100 and 1000 ng/ml) induced ERK1/2 phosphorylation in both cell kinds. Buserelin-induced ERK1/2 activation was G(i/0) independent and PKC dependent. Only in the tumor group, buserelin-induced ERK1/2 activation was cAMP dependent (abolished by SQ 22536, the adenylyl cyclase inhibitor). Furthermore, dibutyryl cAMP-induced ERK1/2 activation in the tumor group was PKC dependent (inhibited by GF109203X). In conclusion, activation of phospholipases in tumor cells does not seem to mediate GnRH effects. GnRH signaling seems to involve adenylyl cyclase activation, PKC stimulation, and ERK1/2 phosphorylation.