Inhibitory effects of jackbean (Canavalia ensiformis L.) leaf residues on germination and vigour of crops and weeds

The effects of jackbean leaf residues incorporated in the soil on germination and seedlings growth of cucumber, radish and some weeds was examined. Trials were carried out under greenhouse conditions to (a) determine the amount of incorporated residue that is inhibitory to two test plants, (b) to determine if decomposition time changes the inhibitory levels of jackbean residues on test plants and (c) to determine the amount of residue that is inhibitory to the weed species. Jackbean leaf residues incorporated in soil at concentration of 2% or higher and allowed to decompose for a period of 0 to 2 weeks before sowing, reduced the initial growth of cucumber and radish and at different concentrations, reduced germination and growth of three weed species. These results suggest the presence of allelopathic components in Jackbean leaves that could affect seed germination and seedling development.

Inhibitory effect of deferoxamine on Paracoccidioides brasiliensis survival in human monocytes: Reversal by holotransferrin not by apotransferrin

The mechanisms used by Paracoccidioides brasiliensis to survive into phagocytic cells are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Thus, the objective of this paper was to investigate the role of intracellular iron in regulating the capacity of P. brasiliensis yeast cells to survive within human monocytes. Treatment of monocytes with deferoxamine, an iron chelator, suppressed the survival of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by nonsaturated transferrin (apotransferrin). These results strongly suggest that P. brasiliensis survival in human monocytes is iron dependent.

Fluoride concentration in public water supply: 72 months of analysis

Known as one of the ten most important advances on Public Health in the 20th century, fluoridation of public water supply is a measure of wide population coverage, which is effective on caries control. The city of Araçatuba, in the Northwest region of the São Paulo state, Brazil, started public water supply fluoridation in 1972 and, based on the average annual highest temperature, has kept the fluoride concentration between 0.6 to 0.8 mgF/L. The purpose of this study was to analyze monthly the fluoride concentration in public water supply in the city of Araçatuba during 72 months. Water samples were collected monthly on weekdays, directly from the water distribution network, on pre-established locations and analyzed in duplicate between November 2004 and October 2010 at the Research Laboratory of the Nucleus for Public Health (NEPESCO) of the Public Health Graduate Program from Araçatuba Dental School/UNESP, Brazil, using an fluoride-specific electrode connected to an ion analyzer. From the total of samples (n=591), 67.2% (n=397) presented fluoride concentration between 0.6 and 0.8 mgF/L; 20.6% (n=122) below 0.6 mgF/L; 11.5% (n=68) between 0.8 and 1.2 mgF/L and 0.7% (n=4) above 1.2 mgF/L. Most samples showed fluoride levels within the recommended parameters. Minimal variation was observed among the analyzed collection locations, showing that the city has been able to control the fluoride levels in the public water supply and reinforcing the importance of surveillance and constant monitoring to assure the quality of the water delivered to the population.

Sugarcane bagasse ozonolysis pretreatment: Effect on enzymatic digestibility and inhibitory compound formation

Sugarcane bagasse was pretreated with ozone to increase lignocellulosic material digestibility. Bagasse was ozonated in a fixed bed reactor at room temperature, and the effect of the two major parameters, ozone concentration and sample moisture, was studied. Acid insoluble and total lignin decreased whereas acid soluble lignin increased in all experiments. Pretreatment barely attacked carbohydrates, with cellulose and xylan recovery rates being >92%. Ozonolysis increased fermentable carbohydrate release considerably during enzymatic hydrolysis. Glucose and xylose yields increased from 6.64% and 2.05%, for raw bagasse, to 41.79% and 52.44% under the best experimental conditions. Only xylitol, lactic, formic and acetic acid degradation compounds were found, with neither furfural nor HMF (5-hydroxymethylfurfural) being detected. Washing detoxification provided inhibitor removal percentages above 85%, increasing glucose hydrolysis, but decreasing xylose yield by xylan solubilization. SEM analysis showed structural changes after ozonization and washing. © 2013 Elsevier Ltd.

Inhibitory mechanism of the nucleus of the solitary tract involved in the control of cardiovascular, dipsogenic, hormonal, and renal responses to hyperosmolality

The nucleus of the solitary tract (NTS) is the primary site of visceral afferents to the central nervous system. In the present study, we investigated the effects of lesions in the commissural portion of the NTS (commNTS) on the activity of vasopressinergic neurons in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, plasma vasopressin, arterial pressure, water intake, and sodium excretion in rats with plasma hyperosmolality produced by intragastric 2 M NaCl (2 ml/rat). Male Holtzman rats with 15-20 days of sham or electrolytic lesion (1 mA; 10 s) of the commNTS were used. CommNTS lesions enhanced a 2 M NaCl intragastrically induced increase in the number of vasopressinergic neurons expressing c-Fos in the PVN (28 ± 1, vs. sham: 22 ± 2 c-Fos/AVP cells) and SON (26 ± 4, vs. sham: 11 ± 1 c-Fos/AVP cells), plasma vasopressin levels (21 ± 8, vs. sham: 6.6 ± 1.3 pg/ml), pressor responses (25 ± 7 mmHg, vs. sham: 7 ± 2 mmHg), water intake (17.5 ± 0.8, vs. sham: 11.2 ± 1.8 ml/2 h), and natriuresis (4.9 ± 0.8, vs. sham: 1.4 ± 0.3 meq/1 h). The pretreatment with vasopressin antagonist abolished the pressor response to intragastric 2 M NaCl in commNTS-lesioned rats (8 ± 2.4 mmHg at 10 min), suggesting that this response is dependent on vasopressin secretion. The results suggest that inhibitory mechanisms dependent on commNTS act to limit or counterbalance behavioral, hormonal, cardiovascular, and renal responses to an acute increase in plasma osmolality. © 2013 the American Physiological Society.

Virulência de linhagens de Rhodococcus equi isoladas de linfonodo de suínos e javalis (Sus scrofa) de abatedouros

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factors

The organization of the nervous and immune systems is characterized by obvious differences and striking parallels. Both systems need to relay information across very short and very long distances. The nervous system communicates over both long and short ranges primarily by means of more or less hardwired intercellular connections, consisting of axons, dendrites, and synapses. Longrange communication in the immune system occurs mainly via the ordered and guided migration of immune cells and systemically acting soluble factors such as antibodies, cytokines, and chemokines. Its short-range communication either is mediated by locally acting soluble factors or transpires during direct cell–cell contact across specialized areas called “immunological synapses” (Kirschensteiner et al., 2003). These parallels in intercellular communication are complemented by a complex array of factors that induce cell growth and differentiation: these factors in the immune system are called cytokines; in the nervous system, they are called neurotrophic factors. Neither the cytokines nor the neurotrophic factors appear to be completely exclusive to either system (Neumann et al., 2002). In particular, mounting evidence indicates that some of the most potent members of the neurotrophin family, for example, nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF), act on or are produced by immune cells (Kerschensteiner et al., 1999) There are, however, other neurotrophic factors, for example the insulin-like growth factor-1 (IGF-1), that can behave similarly (Kermer et al., 2000). These factors may allow the two systems to “cross-talk” and eventually may provide a molecular explanation for the reports that inflammation after central nervous system (CNS) injury has beneficial effects (Moalem et al., 1999). In order to shed some more light on such a cross-talk, therefore, transcription factors modulating mu-opioid receptor (MOPr) expression in neurons and immune cells are here investigated. More precisely, I focused my attention on IGF-I modulation of MOPr in neurons and T-cell receptor induction of MOPr expression in T-lymphocytes. Three different opioid receptors [mu (MOPr), delta (DOPr), and kappa (KOPr)] belonging to the G-protein coupled receptor super-family have been cloned. They are activated by structurallyrelated exogenous opioids or endogenous opioid peptides, and contribute to the regulation of several functions including pain transmission, respiration, cardiac and gastrointestinal functions, and immune response (Zollner and Stein 2007). MOPr is expressed mainly in the central nervous system where it regulates morphine-induced analgesia, tolerance and dependence (Mayer and Hollt 2006). Recently, induction of MOPr expression in different immune cells induced by cytokines has been reported (Kraus et al., 2001; Kraus et al., 2003). The human mu-opioid receptor gene (OPRM1) promoter is of the TATA-less type and has clusters of potential binding sites for different transcription factors (Law et al. 2004). Several studies, primarily focused on the upstream region of the OPRM1 promoter, have investigated transcriptional regulation of MOPr expression. Presently, however, it is still not completely clear how positive and negative transcription regulators cooperatively coordinate cellor tissue-specific transcription of the OPRM1 gene, and how specific growth factors influence its expression. IGF-I and its receptors are widely distributed throughout the nervous system during development, and their involvement in neurogenesis has been extensively investigated (Arsenijevic et al. 1998; van Golen and Feldman 2000). As previously mentioned, such neurotrophic factors can be also produced and/or act on immune cells (Kerschenseteiner et al., 2003). Most of the physiologic effects of IGF-I are mediated by the type I IGF surface receptor which, after ligand binding-induced autophosphorylation, associates with specific adaptor proteins and activates different second messengers (Bondy and Cheng 2004). These include: phosphatidylinositol 3-kinase, mitogen-activated protein kinase (Vincent and Feldman 2002; Di Toro et al. 2005) and members of the Janus kinase (JAK)/STAT3 signalling pathway (Zong et al. 2000; Yadav et al. 2005). REST plays a complex role in neuronal cells by differentially repressing target gene expression (Lunyak et al. 2004; Coulson 2005; Ballas and Mandel 2005). REST expression decreases during neurogenesis, but has been detected in the adult rat brain (Palm et al. 1998) and is up-regulated in response to global ischemia (Calderone et al. 2003) and induction of epilepsy (Spencer et al. 2006). Thus, the REST concentration seems to influence its function and the expression of neuronal genes, and may have different effects in embryonic and differentiated neurons (Su et al. 2004; Sun et al. 2005). In a previous study, REST was elevated during the early stages of neural induction by IGF-I in neuroblastoma cells. REST may contribute to the down-regulation of genes not yet required by the differentiation program, but its expression decreases after five days of treatment to allow for the acquisition of neural phenotypes. Di Toro et al. proposed a model in which the extent of neurite outgrowth in differentiating neuroblastoma cells was affected by the disappearance of REST (Di Toro et al. 2005). The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. Therefore, in the fist part of this thesis, I investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I upregulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 (STAT3) signaling pathway and this transcription factor, binding to the STAT1/3 DNA element located in the promoter, increases OPRM1 transcription. T-cell receptor (TCR) recognizes peptide antigens displayed in the context of the major histocompatibility complex (MHC) and gives rise to a potent as well as branched intracellular signalling that convert naïve T-cells in mature effectors, thus significantly contributing to the genesis of a specific immune response. In the second part of my work I exposed wild type Jurkat CD4+ T-cells to a mixture of CD3 and CD28 antigens in order to fully activate TCR and study whether its signalling influence OPRM1 expression. Results were that TCR engagement determined a significant induction of OPRM1 expression through the activation of transcription factors AP-1, NF-kB and NFAT. Eventually, I investigated MOPr turnover once it has been expressed on T-cells outer membrane. It turned out that DAMGO induced MOPr internalisation and recycling, whereas morphine did not. Overall, from the data collected in this thesis we can conclude that that a reduction in REST is a critical switch enabling IGF-I to up-regulate human MOPr, helping these findings clarify how human MOPr expression is regulated in neuronal cells, and that TCR engagement up-regulates OPRM1 transcription in T-cells. My results that neurotrophic factors a and TCR engagement, as well as it is reported for cytokines, seem to up-regulate OPRM1 in both neurons and immune cells suggest an important role for MOPr as a molecular bridge between neurons and immune cells; therefore, MOPr could play a key role in the cross-talk between immune system and nervous system and in particular in the balance between pro-inflammatory and pro-nociceptive stimuli and analgesic and neuroprotective effects.

Transport of nanoparticles into polymersomes : a minimal model system of particles passage through biological membranes

This thesis focuses on the design and characterization of a novel, artificial minimal model membrane system with chosen physical parameters to mimic a nanoparticle uptake process driven exclusively by adhesion and softness of the bilayer. The realization is based on polymersomes composed of poly(dimethylsiloxane)-b-poly(2-methyloxazoline) (PMDS-b-PMOXA) and nanoscopic colloidal particles (polystyrene, silica), and the utilization of powerful characterization techniques. rnPDMS-b-PMOXA polymersomes with a radius, Rh ~100 nm, a size polydispersity, PD = 1.1 and a membrane thickness, h = 16 nm, were prepared using the film rehydratation method. Due to the suitable mechanical properties (Young’s modulus of ~17 MPa and a bending modulus of ~7⋅10-8 J) along with the long-term stability and the modifiability, these kind of polymersomes can be used as model membranes to study physical and physicochemical aspects of transmembrane transport of nanoparticles. A combination of photon (PCS) and fluorescence (FCS) correlation spectroscopies optimizes species selectivity, necessary for a unique internalization study encompassing two main efforts. rnFor the proof of concepts, the first effort focused on the interaction of nanoparticles (Rh NP SiO2 = 14 nm, Rh NP PS = 16 nm; cNP = 0.1 gL-1) and polymersomes (Rh P = 112 nm; cP = 0.045 gL-1) with fixed size and concentration. Identification of a modified form factor of the polymersome entities, selectively seen in the PCS experiment, enabled a precise monitor and quantitative description of the incorporation process. Combining PCS and FCS led to the estimation of the incorporated particles per polymersome (about 8 in the examined system) and the development of an appropriate methodology for the kinetics and dynamics of the internalization process. rnThe second effort aimed at the establishment of the necessary phenomenology to facilitate comparison with theories. The size and concentration of the nanoparticles were chosen as the most important system variables (Rh NP = 14 - 57 nm; cNP = 0.05 - 0.2 gL-1). It was revealed that the incorporation process could be controlled to a significant extent by changing the nanoparticles size and concentration. Average number of 7 up to 11 NPs with Rh NP = 14 nm and 3 up to 6 NPs with Rh NP = 25 nm can be internalized into the present polymersomes by changing initial nanoparticles concentration in the range 0.1- 0.2 gL-1. Rapid internalization of the particles by polymersomes is observed only above a critical threshold particles concentration, dependent on the nanoparticle size. rnWith regard possible pathways for the particle uptake, cryogenic transmission electron microscopy (cryo-TEM) has revealed two different incorporation mechanisms depending on the size of the involved nanoparticles: cooperative incorporation of nanoparticles groups or single nanoparticles incorporation. Conditions for nanoparticle uptake and controlled filling of polymersomes were presented. rnIn the framework of this thesis, the experimental observation of transmembrane transport of spherical PS and SiO2 NPs into polymersomes via an internalization process was reported and examined quantitatively for the first time. rnIn a summary the work performed in frames of this thesis might have significant impact on cell model systems’ development and thus improved understanding of transmembrane transport processes. The present experimental findings help create the missing phenomenology necessary for a detailed understanding of a phenomenon with great relevance in transmembrane transport. The fact that transmembrane transport of nanoparticles can be performed by artificial model system without any additional stimuli has a fundamental impact on the understanding, not only of the nanoparticle invagination process but also of the interaction of nanoparticles with biological as well as polymeric membranes. rn

Effect of Acute Psychological Stress on Prefrontal GABA Concentration Determined by Proton Magnetic Resonance Spectroscopy

Objective Impaired function of the central gamma-aminobutyric acid (GABA) system, which provides the brain’s major inhibitory pathways, is thought to play an important role in the pathophysiology of anxiety disorders. The effect of acute psychological stress on the human GABA-ergic system is still unknown, however. The purpose of this study was to determine the effect of acute stress on prefrontal GABA levels. Method A recently developed noninvasive magnetic resonance spectroscopy method was used to measure changes in the GABA concentration of the prefrontal cortex in 10 healthy human subjects during a threat-of-shock condition and during a safe condition (two sessions on different days). The main outcome measure was the mean GABA concentration within a 3×3×2-cm3 voxel selected from the medial prefrontal cortex. Results Prefrontal GABA decreased by approximately 18% in the threat-of-shock condition relative to the safe condition. This reduction was specific to GABA, since the concentrations of N-acetyl-aspartate, choline-containing compounds, and glutamate/glutamine levels obtained in the same spectra did not change significantly. Conclusions This result appeared compatible with evidence from preclinical studies in rodents, which showed rapid presynaptic down-regulation of GABA-ergic neurotransmission in response to acute psychological stress. The molecular mechanism and functional significance of this reduced inhibitory effect of acute psychological stress in relation to impaired GABA-ergic function in anxiety disorders merit further investigation.

Blood concentration curve of cyclosporine: impact of itraconazole in lung transplant recipients

Although the influence of cytochrome P450 inhibitory drugs on the area under the curve (AUC) of cyclosporine (CsA) has been described, data concerning the impact of these substances on the shape of the blood concentration curve are scarce. By assessment of CsA blood levels before and 1, 2, and 4 hr after oral intake (C0, C1, C2, and C4, respectively) CsA profiling examinations were performed in 20 lung transplant recipients taking 400 mg, 200 mg, and no itraconazole, respectively. The three groups showed comparable results for C0, C2, and AUC(0-12). Greater values were found for Cmax, Cmax-C0, peak-trough fluctuation and rise to Cmax in favor of the non-itraconazole group. Additionally, tmax was shorter in the non-itraconazole group. Comedication with the metabolic inhibitor itraconazole is associated with a flattening of the CsA blood concentration profile in lung transplant recipients. These changes cannot be assessed by isolated C0, C2, or AUC(0-12) values alone.

Investigation of the inhibitory effects of the benzodiazepine derivative, 5-BDBD on P2X4 purinergic receptors by two complementary methods

BACKGROUND/AIMS ATP-gated P2X4 purinergic receptors (P2X4Rs) are cation channels with important roles in diverse cell types. To date, lack of specific inhibitors has hampered investigations on P2X4Rs. Recently, the benzodiazepine derivative, 5-BDBD has been proposed to selectively inhibit P2X4Rs. However, limited evidences are currently available on its inhibitory properties. Thus, we aimed to characterize the inhibitory effects of 5-BDBD on recombinant human P2X4Rs. METHODS We investigated ATP-induced intracellular Ca(2+) signals and whole cell ion currents in HEK 293 cells that were either transiently or stably transfected with hP2X4Rs. RESULTS Our data show that ATP (< 1 μM) stimulates P2X4R-mediated Ca(2+) influx while endogenously expressed P2Y receptors are not activated to any significant extent. Both 5-BDBD and TNP-ATP inhibit ATP-induced Ca(2+) signals and inward ion currents in a concentration-dependent manner. Application of two different concentrations of 5-BDBD causes a rightward shift in ATP dose-response curve. Since the magnitude of maximal stimulation does not change, these data suggest that 5-BDBD may competitively inhibit the P2X4Rs. CONCLUSIONS Our results demonstrate that application of submicromolar ATP concentrations allows reliable assessment of recombinant P2XR functions in HEK 293 cells. Furthermore, 5-BDBD and TNP-ATP have similar inhibitory potencies on the P2X4Rs although their mechanisms of actions are different.

Time Course of Antibiotic and Antifungal Concentrations in Corneal Organ Culture.

PURPOSE Contamination with bacteria and/or fungi is a serious complication in organ-cultured corneas. Hence, antibiotic and antifungal agents are added to the culture medium. The concentration of different antimicrobial and antifungal additives to the media over time has so far not been investigated in detail and is the aim of this study. METHODS Nine human fresh corneoscleral discs were stored in corneal culture medium consisting of 2% fetal bovine serum and minimal essential medium. In addition, the culture medium contained 1200 μg/mL penicillin G, 25 μg/mL amphotericin B, 120 μg/mL streptomycin, and 100 μg/mL voriconazole. The concentration of amphotericin B used was 10 times higher than in clinical routine to facilitate its detection. The cultures were kept at 37°C for 28 days. At days 0, 7, 14, 21, and 28, samples of the culture medium were harvested for analysis of antimicrobial concentrations by liquid chromatography and electrospray ionization tandem mass spectrometry. RESULTS During corneal storage, the concentration of all antibiotics and antifungal agents declined significantly. By day 28, penicillin G was reduced to 14% of the original concentration. Amphotericin B and streptomycin retained approximately 60% of the original concentration to the end of the experiment and voriconazole maintained stable concentrations after an initial decline to approximately 80% at 7 days. CONCLUSIONS Throughout the entire storage period, the concentrations of penicillin G, streptomycin, and voriconazole exceeded the minimum inhibitory concentrations of all common contaminants, obviating the need for a change of the medium for antimicrobial reasons. Based on the minimum inhibitory concentrations and our findings, the initial concentration of amphotericin B should be raised to 5 μg/mL.

Concentration of the rare-earth elements in metalliferous sediments from DSDP Leg 92 holes

DSDP Leg 92 drilled at four sites along an east-west transect at 19°S on the western flank of the East Pacific Rise (EPR), in an area where sediments are essentially a mixture of hydrothermal and biogenic components, with only a minimal contribution of clastic material. Rare-earth element (REE) data on the metalliferous (non-carbonate) fraction of samples ranging in age from ~2 to ~27 Ma indicate the existence of two distinct groups of patterns corresponding to two broad age groups, one <=8 Ma, the other >=10 Ma. Within each group, REE patterns have characteristics which are near-uniform, despite large variations in total REE abundances. Sediments of the younger group are enriched in light REE (LREE) relative to deep bottom waters influenced by the hydrothermal plume extending west from the EPR at 19°S. Sediments of the older groups show further relative LREE enrichment and/or heavy REE (HREE) depletion. Surficial sediments deposited beneath the lysocline have high Sum REE concentrations resulting from slow accumulation rates, and patterns resembling older sediments due to early diagenetic effects. A correlation between the mass accumulation rates (MAR) of Sum REE and Fe + Mn suggests that ferromanganese particulate matter supplied by the hydrothermal plume scavenges REE; during this process the LREE are preferentially removed from plume seawater. The MAR of Fe + Mn shows a general decrease with age above basement, whereas Sum REE concentrations in the metalliferous component increase with age above basement. This supports the Ruhlin and Owen model wherein limited scavenging of REE, due to rapid burial of sediment near the palaeo-axis, leads to low concentrations (but high MAR-values) for the REE. Following deposition and burial of the hydrothermal component, further relative flattening of the REE pattern takes place, probably the result of diagenetic reactions over several million years. Phase partitioning data indicate that the proportion of REE residing in more poorly crystalline phases tends to increase with age (from ~45% to 90% of Sum REE). This suggests that as initial ferromanganese precipitates undergo diagenetic recrystallization, REE are transferred to the poorly crystalline phases, and/or are scavenged from pore waters by these phases. Because of the various modifications to REE patterns apparently produced both in the water column and post-depositional settings, the REE patterns of metalliferous sediments will not reflect fine-scale REE variations in associated oceanic water masses.

Ascorbic acid acts as an inhibitory transmitter in the hypothalamus to inhibit stimulated luteinizing hormone-releasing hormone release by scavenging nitric oxide

Because ascorbic acid (AA) is concentrated in synaptic vesicles containing glutamic acid, we hypothesized that AA might act as a neurotransmitter. Because AA is an antioxidant, it might therefore inhibit nitric oxidergic (NOergic) activation of luteinizing hormone-releasing hormone (LH-RH) release from medial basal hypothalamic explants by chemically reducing NO. Cell membrane depolarization induced by increased potassium concentration [K+] increased medium concentrations of both AA and LH-RH. An inhibitor of NO synthase (NOS), NG-monomethyl-l-arginine (NMMA), prevented the increase in medium concentrations of AA and LH-RH induced by high [K+], suggesting that NO mediates release of both AA and LH-RH. Calcium-free medium blocked not only the increase in AA in the medium but also the release of LH-RH. Sodium nitroprusside, which releases NO, stimulated LH-RH release and decreased the concentration of AA in the incubation medium, presumably because the NO released oxidized AA to dehydro-AA. AA (10−5 to 10−3 M) had no effect on basal LH-RH release but completely blocked high [K+]- and nitroprusside-induced LH-RH release. N-Methyl-d-aspartic acid (NMDA), which mimics the action of the excitatory amino acid neurotransmitter glutamic acid, releases LH-RH by releasing NO. AA (10−5 to 10−3 M) inhibited the LH-RH-releasing action of NMDA. AA may be an inhibitory neurotransmitter that blocks NOergic stimulation of LH-RH release by chemically reducing the NO released by the NOergic neurons.

Luteinizing hormone-releasing hormone (LH-RH) antagonist Cetrorelix down-regulates the mRNA expression of pituitary receptors for LH-RH by counteracting the stimulatory effect of endogenous LH-RH

The mechanisms through which LH-RH antagonists suppress gonadotroph functions and LH-RH receptor (LH-RH-R) production are incompletely understood. To elucidate these mechanisms, we investigated the effects of Cetrorelix on the mRNA expression of pituitary LH-RH-R and luteinizing hormone (LH) secretion in three experimental systems with different pituitary LH-RH environments. Ovariectomy induced 3.61-fold and 6.34-fold increases in the mRNA expression of pituitary LH-RH-R in rats after 11 and 21 days, respectively. After (5 h) a single injection of 100 μg Cetrorelix, no significant decrease occurred in the mRNA levels of pituitary LH-RH-R in ovariectomized (OVX) rats with high pituitary exposure to LH-RH, but there was a significant 23.2% reduction in cycling rats with normal hypophysial LH-RH environment. Prolonged treatment for 10 days with a Cetrorelix depot formulation releasing 100 μg/day decreased the concentration of mRNA for pituitary LH-RH-R by 72.6% in OVX rats, but only by 32.9% in normal rats. The decline in serum LH was 98.7% in OVX rats and 63.2% in normal rats, resulting in a minimal 0.1–0.2 ng/ml LH concentration in both groups. A continuous exposure of pituitary cells to 100 nM Cetrorelix in the superfusion system, which is devoid of LH-RH, did not cause any significant changes in LH-RH-R mRNA level. These studies demonstrate that prolonged exposure to Cetrorelix in vivo, but not in vitro, down-regulates the mRNA expression of the pituitary receptors for LH-RH. Our findings indicate that LH-RH antagonists exert their inhibitory effects on the gene expression of pituitary LH-RH-R by counteracting the stimulatory effect of endogenous LH-RH.