Synthesis of a new allelopathic agent from the biotransformation of ent-15α-hydroxy-16-kauren-19-oic acid with Fusarium proliferatum.

The use of kaurane diterpenes as substrates in fungal biotransformation to achieve bioactive compounds has been widely reported. In this work, the natural product kaurenoic acid, a diterpene widely distributed in the plant Kingdom, was chemically converted into ent-15α-hydroxy-kaur-16-en-19-oic acid (1). Substrate 1 was subjected to biotransformation by the fungus Fusarium proliferatum, furnishing a new derivative, ent-2α,15α-dihydroxy-kaur-16-en-19-oic acid (2). The structure of metabolite 2 was deduced on the basis of spectroscopy and MS data. Derivative 2 showed allelopathic activity on germination and growth of root and stem of lettuce (Lactuca sativa), inhibiting 100% of germination and growth of roots and stem, at higher concentration assayed (10-4 mol/L).

Constituents from stem barks of Luehea ochrophylla Mart and evaluation of their antiparasitic, antimicrobial, and antioxidant activities.

Luehea species are found in almost all Central and South American countries. The present work describes the phytochemical study, isolation, and structural characterisation of friedelin, ß-friedelinol, lupeol, pseudotaraxasterol, ß-sitosterol, betulinic acid, taraxasterol, (-)-epicatechin, ß-sitosterol-3-O-ß-d-glucopyranoside, and (+)-epicatechin-(4ß→8)-epicatechin from stem barks of Luehea ochrophylla Mart. The structural identification of the isolated compounds was mainly performed by NMR analyses and comparison with the data from literature. These compounds were isolated for the first time in the genus Luehea, except ß-sitosterol glucopyranoside, (-)-epicatechin, and lupeol. Hexane extract (HE) and dichloromethane (DF) and ethyl acetate (AF) fractions exhibited antiparasitic activity against amastigote (intracellular) and trypomastigote culture forms of Trypanosoma cruzi. The ethanol extract (EE), DF, and ethanol fraction (EF) exhibited considerable antifungal activity against Candida albicans. Moreover, extracts and fractions exhibited significant percentage of capture free radicals of 2,2-diphenyl-picrylhydrazyl (DPPH) when compared to the standard of ascorbic acid.

New jacaranone glucoside from Jacaranda oxyphylla leaves.

Jacaranda oxyphylla Cham. is popularly known as 'caroba-de-São-Paulo' and it is used in traditional medicine for microbial infections. A new phytoquinoid (α/ß-glucoside-4-phenylacetate-6-(1-hydroxy-4-oxo-2,5-cyclohexadiene-1-acetate) (1) was isolated from J. oxyphylla leaves, together with three known compounds: quercetin-3-O-ß-d-galactoside (2), verbascoside (3) and polystyrene (4). Their chemical structures were elucidated using spectroscopic techniques and by comparison with the related known compounds. In addition, it was found a pronounced acetylcholinesterase inhibitory activity for the quinoid 1 (100.0 ± 0.8%) and phenolic compounds 2 and 3 (99.9 ± 0.7 and 99.3 ± 0.5%, respectively), if compared to the standard eserine (92.7 ± 0.4%), that was analysed by a microplate spectrophotometer.

Antioedematogenic activity, acetylcholinesterase inhibition and antimicrobial properties of Jacaranda oxyphylla.

Jacaranda oxyphylla Cham. (Bignoniaceae) is a shrub found in the Brazilian cerrado and used in folk medicine to treat microbial infections. The aim of this study was to carry out a phytochemical screening and evaluate antioedematogenic, antimicrobial and antiacetylcholinesterase properties of J. oxyphylla crude extracts. All extracts analysed showed presence of terpenoids, which are potentially active chemical substances. A high AChE inhibitory activity for hexane extract from leaves and for the extracts from twigs was found. Ethanol extract from leaves of J. oxyphylla showed activity against Gram-positive (Staphylococcus aureus and Bacillus cereus) and Gram-negative (Escherichia coli) bacteria. This extract was also effective in inhibiting the stages of inflammation evaluated. Biological investigation and phytochemical screening of J. oxyphylla extracts provided additional evidence of its traditional medicinal value.

MDGC-MS analysis of essential oils from Protium heptaphyllum (Aubl. ) and their antifungal activity against Candida specie / Análise MDGC-MS de óleos essenciais de Protium heptaphyllum (Aubl. ) e sua atividade antifúngica contra espécies de Candida

ABSTRACT Protium heptaphyllum is found in the Amazon region, and in various Brazilian states and South American countries. Also Known as almecega, it produces an oil resin used in traditional medicine as analgesic, anti-inflammatory, cicatrizant and expectorant, it is rich in pentacyclic triterpenes and essential oil. The main objective of this study was to analyze the chemical composition of P. heptaphyllumresin (OEPh) over different extraction times and to evaluate their antifungal activity against Candida species, obtained from gardeners with onychomycosis, using the disk diffusion method. The OEPh was obtained by hydrodistillation and analyzed by Multidimensional Gas Chromatography coupled with Mass Spectrometry (MDGC / MS). Candida species were obtained from lesions on the nails of horticulturist from a community garden in the city of Teresina, Piauí, Brazil. The antifungal activity in concentrations of 1000 µg/L, 500 µg/L and 250 µg/L, PROTOCOL M44-A2 (CLSI 2009) OEPh was tested. The main constituents identified were: l-limonene, α-terpineol, p-cineol, o-cymene and α-phellandrene, however, its composition varies significantly with extraction time. All species, except C. rugosa, were inhibited with halo (≥ 14 mm) at 1000 μg / L. C. krusei is naturally resistant to the drug fluconazole, but when tested with OEPh the clinical species (case 9) demonstrated sensitivity in three dilutions (halo ≤ 10 ≥ 14) and the standard strain was inhibited at concentration of 1000 μg/Lg / L (halo 14mm). A similar situation also occurred with the standard strain of C. parapsilosis (halo ≥ 11mm). OEPh has considerable antifungal activity, which merits further investigation for alternative clinical applications, since this species is widely distributed in our community, and it presents good yields, and also has important therapeutic applications.

RESUMO Protium heptaphyllum é encontrada na região amazônica, em vários estados do Brasil e países da América do Sul. Conhecida como almecega produz uma resina oleosa usada na medicina popular como analgésica, antiinflamatória, cicatrizante e expectorante, é rica em triterpenos pentaciclicos e óleo essencial. O objetivo principal do presente trabalho foi analisar a composição química do óleo essencial da resina P. heptaphyllum (OEPh) em diferentes tempo de extração e avaliarsuaatividade antifúngica contra espécies de Candida, isoladas de horticultores com onicomicoses, por método de disco-difusão. O OEPh foi obtido por hidrodestilação, analisado por Cromatografia Gasosa Multidimensinal Acoplada a Espectrometria de Massas (MDGC/MS). As espécies de Candida foram obtidas de lesões nas unhas de horticultores de uma horta comunitária na cidade de Teresina, Piauí, Brasil. Testou-se a atividade antifúngica do OEPhnas concentrações de 1000 μg/L, 500 μg/L e 250 μg/L, protocolo M44-A2 (CLSI 2009). Os principais constituintes identificados foram l- limoneno, α-terpineol, p-cineol, o-cimeno e α-felandreno, entretanto, sua composição varia significativamente em decorrência do tempo de extração. Todas as espécies, exceto a C. rugosa, foram inibidas com halo ( Χ ≥ 14 mm) na concentração de 1000 μg/L. C. krusei é naturalmente resistente ao fármaco fluconazol, mas quando testado com OEPh,a espécie clínico (caso 9) demonstrou sensibilidade nas três diluições (halo Χ ≤ 10 ≥ 14) e a cepa padrão foi inibida na concentração de 1000 μg/L (halo Χ 14mm). Fato semelhante também ocorreu com a cepa padrão de C. parapsilosis (halo Χ ≥ 11mm). O OEPh possui atividade antifúngica considerável, merecendo uma investigação mais aprofundada para aplicações clínicas alternativas, uma vez que esta espécie é amplamente distribuída em nossa comunidade, apresenta bom rendimento e, ainda, aplicações terapêuticas importantes.

The phytochemistry and biological aspects of Caryocaraceae family / Aspectos fitoquímicos e biológicos da família Caryocaraceae

The Caryocaraceae family is constituted of 25 species distributed in two genera (Caryocar and Anthodiscus). Plants of this family have been used in several phytochemical studies for isolation and characterization of chemical compounds. Some of these studies evaluated in vitro and in vivo biological activities of extracts and pure substances isolated from plants of this family. Nine species of Anthodiscus genus have been described, while no phytochemical study related to them has been reported. On the other hand, Caryocar genus presents 16 species with several medicinal uses like for the treatment of colds and bronchitis, in the prevention of tumours, as a regulating agent of the menstrual flow, to treat ophthalmological problems and for the cure of hematomas and bruises. Some species of this genus were targeted by phytochemical studies and presented, in their composition, the following classes of secondary metabolites: triterpenes, fatty acids, tannins, carotenoids, triterpenic saponins, phenolic coumarins, phenolic glycosides, and others. The fruits of Caryocar species are very nutritive, containing in their composition fibers, proteins, carbohydrates and minerals. Seeds have been widely used as oil source with nutritional and cosmetic value. The biological evaluation of some species was carried out by using relevant biological assays such as: antioxidant, allelopathic and antifungal activities against Biomphalaria glabrata and toxicity on Artemia salina.

A família Caryocaraceae é constituída por 25 espécies distribuídas em dois gêneros (Caryocar e Anthodiscus). Plantas desta família têm sido utilizadas em diversos estudos fitoquímicos para isolamento e caracterização de constituintes químicos. Alguns destes estudos avaliaram atividades biológicas in vitro e in vivo de extratos e substâncias puras isoladas a partir de plantas desta família. São descritas nove espécies dentro do gênero Anthodiscus e nenhum estudo fitoquímico relacionado a elas foi relatado. Por outro lado, o gênero Caryocar apresenta 16 espécies com diversos usos medicinais, tais como: para o tratamento de resfriados, bronquites, na prevenção de tumores, como reguladores do fluxo menstrual, em problemas oftalmológicos, e na cura de hematomas e contusões. Algumas espécies deste gênero foram submetidas a estudos fitoquímicos e apresentaram, em sua composição, as seguintes classes de metabólitos secundários: triterpenos, ácidos graxos, taninos, carotenoides, saponinas triterpênicas, cumarinas, glicosídeos fenólicos, entre outros. Os frutos de espécies Caryocar são muito nutritivos contendo em sua composição, fibras, proteínas, carboidratos e minerais. As sementes têm sido amplamente usadas como fonte de óleo com valor nutricional e cosmético. A avaliação biológica de algumas espécies foi realizada utilizando-se testes biológicos relevantes, entre eles: a atividade antioxidante, alelopática, antifúngica, contra Biomphalaria glabrata, e toxicidade sobre Artemia salina.

Screening filamentous tropical fungi for their nutritional potential as sources of crude proteins, lipids and minerals.

The use of fungal and yeast biomass in foodstuff, either as supplements or as major ingredients in formulations is an area of growing interest for the modern food industry. The aim of this study was to evaluate the nutritional potential of biomasses obtained from filamentous fungi Penicillium sclerotiorum, Penicillium janthinellum, Rhizopus stolonifer and Syncephalastrum racemosum. Biomasses presented 26-37% of total proteins, 1.7-3.5% of lipids and 4.6-9.1% of ashes. The humidity level reached 75-83%. Ashes were screened for minerals contents with a special outcome for S. racemosum biomass that presented 3438 mg/100 g (dw) of magnesium. Fatty acids present in the biomasses were screened and the palmitic (C16:0), estearic (C18:0), elaidic (18:1n9-t), oleic (18:1n9-c), linolelaidic (C18:2n6-t), linoleic (C18:2n6-c) and γ-linolenic (C18:3n6) acids were found to be the most abundant, from a total of 88-90% of identified fatty acids. Overall data indicate that the filamentous fungi studied have good nutritional properties, possessing a combination of good level of proteins, low level of fat, and presence of essential fatty acids, including omega-3 derivatives, along with the presence of Ca, Mg, Zn and Fe.

Comparison of oral falecalcitriol and intravenous calcitriol in hemodialysis patients with secondary hyperparathyroidism: a randomized, crossover trial.

BACKGROUND: Falecalcitriol is a novel vitamin D analog, which has a greater potential to suppress parathyroid hormone (PTH) and a longer half-life. There are few studies to compare clinical effects of oral falecalcitriol treatment with those of intravenous calcitriol treatment. METHODS: Twenty-one patients with moderate to severe SHPT were included in a random 2 x 2 crossover trial with the two vitamin D analogs (12 weeks for each treatment). The primary endpoint measure was a decrease in serum intact PTH (iPTH) level, and the secondary outcome measures included changes in serum calcium (Ca), phosphate (P), and metabolic bone marker levels. RESULTS: Both treatments decreased iPTH and whole PTH (wPTH) levels by similar degrees (iPTH, -200.1 +/- 107.0 with falecalcitriol vs. -200.8 +/- 114.9 pg/ml with calcitriol, p = 0.9895; wPTH, -137.1 +/- 73.1 with falecalcitriol vs. -120.4 +/- 81.1 pg/ml with calcitriol, p = 0.5603). Serum Ca, P, and Ca x P product levels at the end of each treatment were comparable and the frequencies of hypercalcemia and hyperphosphatemia were also similar during each treatment period. Although intravenous calcitriol treatment significantly changed intact osteocalcin and cross-linked N-telopeptide of type I collagen after 12 weeks, oral falecalcitriol treatment did not change any bone metabolic marker level. CONCLUSION: The present study showed that oral falecalcitriol treatment is effective for PTH suppression, and Ca and P metabolism in hemodialysis patients with moderate to severe SHPT, as well as intravenous calcitriol administration.

Synergic interaction between pomegranate extract and antibiotics against Staphylococcus aureus.

We evaluated the interaction between Punica granatum (pomegranate) methanolic extract (PGME) and antibiotics against 30 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA). Susceptibility testing of the isolates to PGME and antibiotics was performed by the broth dilution method. Synergic activity was detected between PGME and the 5 antibiotics tested, chloramphenicol, gentamicin, ampicillin, tetracycline, and oxacillin, ranging from 38% to 73%. For some isolates, PGME did not interfere with the action of any of the antibiotics tested. The bactericidal activity of PGME (0.1 x MIC) in combination with ampicillin (0.5 x MIC) was assessed using chosen isolates by time-kill assays, and they confirmed the synergic activity. Using this combination, cell viability was reduced by 99.9% and 72.5% in MSSA and MRSA populations, respectively. PGME increased the post-antibiotic effect (PAE) of ampicillin from 3 to 7 h. In addition, PGME demonstrated the potential to either inhibit the efflux pump NorA or to enhance the influx of the drug. The detection of in vitro variant colonies of S. aureus resistant to PGME was low and they did not survive. In conclusion, PGME dramatically enhanced the activity of all antibiotics tested, and thus, offers an alternative for the extension of the useful lifetime of these antibiotics.

Radical resection for intrinsic midbrain pilocytic astrocytoma: report of two cases.

BACKGROUND: Intrinsic midbrain glioma has been one of the most challenging therapeutic tasks in neurosurgery due to its prognosis and risks associated with surgical procedures. It is known that the prognosis of pilocytic astrocytoma is relatively good if radical resection can be achieved without severe complications. In order to remove pilocytic astrocytoma within the midbrain radically, we used microsurgical techniques. METHOD: Two patients with intrinsic pilocytic astrocytomas located at the midbrain were operated on. The subtemporal approach was used with a point of entry on the lateral surface of the midbrain just behind the cerebral peduncle. Major vessels were preserved, followed by resection of the intrinsic tumor making the cleavage between tumour and midbrain. FINDINGS: In both patients, intrinsic pilocytic astrocytoma was grossly totally removed with minimal permanent morbidity. They have been able to maintain independent activities in their daily lives without tumor recurrance. CONCLUSIONS: Surgical cure can be accomplished in some cases of midbrain pilocytic astrocytoma, even if the lesions are intrinsic to the midbrain. To remove the tumor totally without further neurological deficits, it is necessary to select a safe access or entrance point to the tumor, and to demarcate the gliotic plane between tumour and midbrain. A long-term follow up with a larger number of patients is needed to establish the significance of radical resection for intrinsic midbrain pilocytic astrocytoma.

Pomegranate extract inhibits Staphylococcus aureus growth and subsequent enterotoxin production.

In Brazil, pomegranate (Punica granatum L. (Punicaceae)) is widely used as a phytotherapeutic agent. This study evaluates the effect of pomegranate extract on Staphylococcus aureus FRI 722 growth and subsequent enterotoxin production. Bacterial susceptibility was determined by tube dilution method and production of enterotoxin was assessed using membrane-over-agar (MOA) plates. At a low extract concentration (0.01% v/v) bacterial growth was delayed, while a higher concentration (1% v/v) eliminated bacterial growth. Most interestingly, a 0.05% (v/v) concentration of extract was found to inhibit Staphylococcal enterotoxin (SE) A production. These data further implicate pomegranate extracts as potential antibacterial therapeutics with the added ability to inhibit enterotoxin production.

Studies on osteogenic differentiation of rat bone marrow stromal cells cultured in type I collagen gel by RT-PCR analysis.

The purpose of this investigation was to examine by reverse-transcriptase polymerase chain reaction analysis the osteogenic differentiation of twice-passaged Sprague-Dawley rat bone marrow stromal cells in type I collagen gel cultured for 3 weeks. Two culture media were used here, namely Dulbecco's modified Eagle (DME) medium supplemented with vitamin C [Dex (-)] and those with vitamin C, dexamethasone and beta-glycerophosphate [Dex (+)]. Culture with Dex (-) medium in collagen gel for 3 weeks brought about the well-developed cell network and middle-stage osteogenic phenotype expression characterized by mRNA for alkaline phosphatase, osteonectin and osteopontin while those for bone sialo protein and osteocalcin were not detected. On the contrary, culture with Dex (+) medium in collagen gel for 3 weeks lead to necrosis of the cells. These results indicate that culture in collagen gel with Dex (-) DME medium containing vitamin C was useful for three-dimensional culture and middle-stage osteogenic differentiation of twice-passaged bone marrow stromal cells. This study might contribute to tissue engineering therapy to fix bone and periodontal defects in the future.

Biological screening of Annonaceous Brazilian Medicinal Plants using Artemia salina (brine shrimp test).

Eighteen different extracts from five Annona species collected in Minas Gerais state, Brazil, were submitted to the brine shrimp lethality test in order to detect potential sources of novel cytotoxic, antitumor, pesticidal and anti-Trypanosoma cruzi compounds. All of the Annonaceous species tested showed good larvicidal activity as compared to a reference compounds and literature data.

Inhibitory effect of losartan, an AT1 angiotensin II receptor antagonist, on increased leucocyte entrapment in retinal microcirculation of diabetic rats.

BACKGROUND: The effectiveness of losartan for the treatment of leucocyte entrapment in the retinal microcirculation of diabetic rats was evaluated quantitatively. METHODS: After diabetes was induced by injection of streptozotocin (STZ), the rats were divided into two subgroups. The first subgroup (n = 6), received no medications; the second subgroup (n = 6) was given fresh drinking water supplemented with losartan (5 mg/kg/day) for 4 weeks. Six rats that were not injected with STZ or given medications served as controls. 4 weeks after intervention, leucocyte dynamics in the retina were observed using acridine orange digital fluorography. Leucocyte entrapment in the retina was compared among the three groups. RESULTS: In the untreated diabetic rats, the number of trapped leucocytes (6.1 (SD 1.4) cells/mm(2)) increased significantly compared with control rats (2.8 (1.2) cells/mm(2); p = 0.005) and diabetic rats treated with losartan (3.1 (0.9) cells/mm(2); p = 0.0002). CONCLUSIONS: Losartan, an AT1 angiotensin II receptor antagonist, inhibited increased leucocyte entrapment in the diabetic retina. The authors demonstrated that losartan may have therapeutic efficacy in preventing development of diabetic retinopathy. Further clinical studies of the effect of the angiotensin receptor antagonist on preventing development of diabetic retinopathy are needed.

Constituents from aerial parts of Wedelia paludosa.

Tetrachyrin (1), 3alpha-tigloyloxykaur-16-en-19-oic acid (2) and 3alpha-cinnamoyloxykaur-16-en-19-oic acid (3) have been isolated from the ethanol extract of Wedelia paludosa aerial parts.

Visual sensitivities of nur77 (NGFI-B) and zif268 (NGFI-A) induction in the suprachiasmatic nucleus are dissociated from c-fos induction and behavioral phase-shifting responses.

Mammalian circadian rhythms are regulated by a pacemaker in the suprachiasmatic nucleus of the hypothalamus. Recent work from several laboratories has shown that light induces the IEGs, c-fos and jun-B, in the rodent suprachiasmatic nucleus. In hamsters, there is a strong correlation between circadian entrainment and the induction of c-fos and jun-B in the suprachiasmatic nucleus by light. Previous work has shown that the IEGs, nur77 and zif268, both of which encode transcription factors, are also light-inducible in the rat suprachiasmatic nucleus [Rusak, B., McNaughton, L., Robertson, H.A. and Hunt, S.P., Circadian variation in photic regulation of IEG mRNAs in rat suprachiasmatic nucleus cells, Mol. Brain Res., 14 (1992) 124-130.; Sutin, E.L. and Kilduff, T.S., Circadian and light-induced expression of IEG mRNAs in the rat suprachiasmatic nucleus, Mol. Brain Res., 15 (1992) 281-290.]. To characterize the photic-regulation of these genes in the suprachiasmatic nucleus of golden hamsters, we used in situ hybridization to measure nur77 and zif268 mRNA levels with 33P-labeled complementary RNA probes. 5-min monochromatic light pulses at CT19 induced a dramatic increase in both nur77 and zif268 mRNA levels. Peak mRNA levels occurred 45-60 min after light onset for both nur77 and zif268. In addition, the induction of both nur77 and zif268 mRNA levels was gated by the circadian pacemaker. Light pulses during subjective day (CT3 and CT9), which do not cause behavioral phase-shifts, did not significantly alter mRNA levels of either nur77 or zif268; whereas light pulses during the subjective night (CT14 and CT19), which induce phase-shifts, dramatically increased both nur77 and zif268 mRNA levels. In contrast to c-fos induction, which has a photic threshold indistinguishable from that of the behavioral phase-shifting response, nur77 and zif268 mRNA induction were found to have visual sensitivities greater than the phase-shifting response by 1-2 log units (10-100-fold). Although light and circadian phase regulate nur77 and zif268 expression in the SCN, these results demonstrate that their induction is not rate-limiting for photic entrainment of the hamster circadian system.

Apoptosis induced by selenium in human glioma cell lines.

Several studies have shown that selenium can inhibit tumorigenesis in tissues. However, little is known about the mechanism and the effect of selenium on DNA, especially in brain tumor cells. In this study we examined the biological effect of selenium on human glioma cell lines (A172 and T98G). Selenium exhibited an antiproliferative effect on these cell lines (and induced the typical ladder pattern of DNA fragmentation commonly found in apoptosis), which were prevented by catalase. Few effects of selenium on NT14 fibroblasts were found. These findings demonstrate that selenium may induce, by apoptosis, cell death of human glioma cell lines, which are resulting from free radical oxygen forming.

Pharmacological and genetic approaches for the study of circadian rhythms in mammals.

Two different approaches have been utilized to study the controlling mechanisms that underlie the generation and entrainment of circadian rhythms in mammals. The use of specific drugs to alter the period and/or the phase of circadian rhythms has provided new insights into both the pathways by which environmental information reaches the mammalian circadian pacemaker in the suprachiasmatic nuclei (SCN) and the cellular and neurochemical events within the SCN itself which are involved in circadian rhythmicity. A second approach, which seeks to exploit genetic differences in the properties of the circadian system, holds the promise of eventually defining the cellular and molecular events that are part of the clock itself, the events that underlie the entrainment of the circadian clock by environmental factors, and the expression of overt rhythms driven by the clock. It is anticipated that the pharmacological and genetic approaches to the study of circadian rhythms will complement each other as the underlying physiological mechanisms of the circadian clock system become defined.

Neuropeptide Y stimulates luteinizing hormone-releasing hormone release from superfused hypothalamic GT1-7 cells.

The effects of neuropeptide Y (NPY) on LHRH release from an immortalized cell line were investigated using a flow-through cell culture superfusion system. Immortalized hypothalamic GT1-7 cells were cultured for 72 h and superfused for a total of 180 min. In initial experiments, discrete 5-min pulses of NPY (10(-12)-10(-5) M) were administered to the cells. A clear dose-dependent stimulatory effect on NPY on LHRH release from the cells was observed with a calculated 50% effectiveness concentration of 33 nM. The stimulatory effects of brief NPY exposure were rapid and robust, e.g. reaching and maintaining levels of 173% over baseline for 20 min at the 10(-7) dose. The lowest dose of NPY that showed a significant effect was 10(-10) M; maximal responses were observed at 10(-6) M and reached a plateau thereafter. Control pulses of Dulbecco's modified Eagle's medium (DMEM) and 10(-6) M substance P or arg-vasopressin were also presented to the cells to serve as controls for our pulse protocol, and these challenges produced no significant LHRH responses. The NPY receptor antagonists, PYX1 and PYX2, at 10(-8) M, completely blocked the observed NPY responses in these cells. To assess the NPY receptor subtypes that mediate the NPY effects pharmacologically, GT1-7 cells were challenged with a Y1 receptor agonist, (Leu31Pro34)NPY, a Y2 receptor agonist, NPY(13-36), or peptide YY, at doses 10(-12)-10(-5) M. All four peptides stimulated LHRH release from GT1-7 cells with a rank-ordered potency of NPY = peptide YY > Y1 agonist = Y2 agonist. To examine possible signal transduction mechanism(s) involved in mediating this effect, pertussis toxin, RpcAMPs (cyclic adenosine-3'5'-monophosphothioate Rp diastereomer), Ca(2+)-free DMEM and TMB-8 (3, 4, 5-trimethoxybenzoic acid 8-(diethylamino) octylester) were used to treat the cells before and during superfusion with NPY. Treatment with pertussis toxin, RpcAMPs, and Ca(2+)-free DMEM did not significantly alter NPY-stimulated LHRH release responses to 10(-7) M NPY. However, the addition of 100 microM and 250 microM TMB-8 to Ca(2+)-free DMEM almost completely blocked this NPY effect, as did 10 microM ryanodine. Finally, the locus of action for this NPY effect was examined using tetrodotoxin to reduce action potential propagation in the GT1-7 cells. Tetrodotoxin treatment blocked the LHRH response to NPY by more than 50%.(ABSTRACT TRUNCATED AT 400 WORDS)