RESUMO
Aging and its related disorders are important issues nowadays and the first cause of this physio-pathological condition is the overproduction of ROS. Ascorbic acid is an antioxidant mediator and its anti-aging proprieties are well known. Our previous data demonstrated that Voghera sweet pepper (VP), a distinctive type of pepper cultivated in Italy, is particularly rich in ascorbic acid. Based on these data, the anti-aging effect mediated by extracts of the edible part of VP was evaluated on an in vitro model of both young and old Normal Human Diploid Fibroblasts (NHDF). Using phase contrast microscopy, we observed that VP may help cells in the maintenance of physiological morphology during aging. Cytofluorimetric analyses revealed that VP extracts led to an increase in DNA synthesis and percentage of living cells, linked to a consequent increase in mitotic events. This hypothesis is supported by the enhancement of PCNA expression levels observed in old, treated fibroblasts, corroborating the idea that this extract could recover a young phenotype in adult fibroblasts, confirmed by the study of p16 and p53 expression levels and TEM analyses. Based on these results, we may suppose that VP can lead to the partial recovery of "young-like" phenotypes in old fibroblasts.
Assuntos
Ácido Ascórbico , Capsicum , Proliferação de Células , Senescência Celular , Fibroblastos , Extratos Vegetais , Proteína Supressora de Tumor p53 , Humanos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Capsicum/química , Senescência Celular/efeitos dos fármacos , Extratos Vegetais/farmacologia , Proliferação de Células/efeitos dos fármacos , Ácido Ascórbico/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Envelhecimento/fisiologia , Antioxidantes/farmacologia , Diploide , Células Cultivadas , ItáliaRESUMO
In the Medicago genus, saponins are complex mixtures of triterpene pentacyclic glycosides extensively studied for their different and economically relevant biological and pharmaceutical properties. This research is aimed at determining for the first time the tissue and cellular localization of triterpene saponins in vegetative organs of Medicago truncatula, a model plant species for legumes, by histochemistry and transmission electron microscopy. The results showed that saponins are present mainly in the palisade mesophyll layer of leaves, whereas in stems they are mostly located in the primary phloem and the subepidermal cells of cortical parenchyma. In root tissue, saponins occur in the secondary phloem region. Transmission electron microscopy revealed prominent saponin accumulation within the leaf and stem chloroplasts, while in the roots the saponins are found in the vesicular structures. Our results demonstrate the feasibility of using histochemistry and transmission electron microscopy to localize M. truncatula saponins at tissue and cellular levels and provide important information for further studies on biosynthesis and regulation of valuable bioactive saponins on agronomic relevant Medicago spp., such as alfalfa (Medicago sativa L.). RESEARCH HIGHLIGHTS: The Medicago genus represents a valuable rich source of saponins, one of the most interesting groups of secondary plant metabolites, which possess relevant biological and pharmacological properties. Plant tissue and cellular localization of saponins is of great importance to better understand their biological functions, biosynthetic pathway, and regulatory mechanisms. We elucidate the localization of saponins in Medicago truncatula with histochemical and transmission electron microscopy studies.
Assuntos
Medicago truncatula , Microscopia Eletrônica de Transmissão , Folhas de Planta , Raízes de Plantas , Saponinas , Triterpenos , Medicago truncatula/ultraestrutura , Medicago truncatula/metabolismo , Medicago truncatula/química , Saponinas/metabolismo , Triterpenos/metabolismo , Triterpenos/química , Raízes de Plantas/química , Raízes de Plantas/ultraestrutura , Folhas de Planta/química , Folhas de Planta/ultraestrutura , Caules de Planta/química , Caules de Planta/ultraestrutura , Floema/ultraestrutura , Floema/química , Floema/metabolismo , Histocitoquímica , Cloroplastos/ultraestrutura , Cloroplastos/metabolismo , Cloroplastos/químicaRESUMO
Photodynamic therapy (PDT) is a promising anticancer strategy based on the light energy stimulation of photosensitizers (PS) molecules within a malignant cell. Among a multitude of recently challenged PS, Rose bengal (RB) has been already reported as an inducer of cytotoxicity in different tumor cells. However, RB displays a low penetration capability across cell membranes. We have therefore developed a short-term amino acids starvation protocol that significantly increases RB uptake in human astrocytoma cells compared to normal rat astrocytes. Following induced starvation uptake, RB is released outside cells by the exocytosis of extracellular vesicles (EVs). Thus, we have introduced a specific pharmacological treatment, based on the GW4869 exosomes inhibitor, to interfere with RB extracellular release. These combined treatments allow significantly reduced nanomolar amounts of administered RB and a decrease in the time interval required for PDT stimulation. The overall conditions affected astrocytoma viability through the activation of apoptotic pathways. In conclusion, we have developed for the first time a combined scheme to simultaneously increase the RB uptake in human astrocytoma cells, reduce the extracellular release of the drug by EVs, and improve the effectiveness of PDT-based treatments. Importantly, this strategy might be a valuable approach to efficiently deliver other PS or chemotherapeutic drugs in tumor cells.
Assuntos
Astrocitoma , Exossomos , Fotoquimioterapia , Aminoácidos , Animais , Astrocitoma/tratamento farmacológico , Humanos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Ratos , Rosa Bengala/química , Rosa Bengala/farmacologiaRESUMO
Photodynamic therapy (PDT) has recently attracted interest as an innovative and adjuvant treatment for different cancers including malignant gliomas. Among these, Glioblastoma (GBM) is the most prevalent neoplasm in the central nervous system. Despite conventional therapeutic approaches that include surgical removal, radiation, and chemotherapy, GBM is characterized by an extremely poor prognosis and a high rate of recurrence. PDT is a physical process that induces tumor cell death through the genesis and accumulation of reactive oxygen species (ROS) produced by light energy interaction with a photosensitizing agent. In this contribution, we explored the potentiality of the plant alkaloid berberine (BBR) as a photosensitizing and cytotoxic agent coupled with a PDT scheme using a blue light source in human established astrocytoma cell lines. Our data mainly indicated for the combined BBR-PDT scheme a potent activation of the apoptosis pathway, through a massive ROS production, a great extent of mitochondria depolarization, and the sub-sequent activation of caspases. Altogether, these results demonstrated that BBR is an efficient photosensitizer agent and that its association with PDT may be a potential anticancer strategy for high malignant gliomas.
RESUMO
The identification of new biomarkers allowing an early and more accurate characterization of patients with ST-segment elevation myocardial infarction (STEMI) is still needed, and exosomes represent an attractive diagnostic tool in this context. However, the characterization of their protein cargo in relation to cardiovascular clinical manifestation is still lacking. To this end, 35 STEMI patients (17 experiencing resuscitated out-of-hospital cardiac arrest (OHCA-STEMI) and 18 uncomplicated) and 32 patients with chronic coronary syndrome (CCS) were enrolled. Plasma exosomes were characterized by the nanoparticle tracking analysis and Western blotting. Exosomes from STEMI patients displayed a higher concentration and size and a greater expression of platelet (GPIIb) and vascular endothelial (VE-cadherin) markers, but a similar amount of cardiac troponin compared to CCS. In addition, a difference in exosome expression of acute-phase proteins (ceruloplasmin, transthyretin and fibronectin) between STEMI and CCS patients was found. GPIIb and brain-associated marker PLP1 accurately discriminated between OHCA and uncomplicated STEMI. In conclusion, the exosome profile of STEMI patients has peculiar features that differentiate it from that of CCS patients, reflecting the pathophysiological mechanisms involved in STEMI. Additionally, the exosome expression of brain- and platelet-specific markers might allow the identification of patients experiencing ischemic brain injury in STEMI.
Assuntos
Exossomos/metabolismo , Parada Cardíaca Extra-Hospitalar/sangue , Infarto do Miocárdio com Supradesnível do Segmento ST/sangue , Idoso , Biomarcadores/sangue , Ceruloplasmina/análise , Exossomos/química , Fibronectinas/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Pré-Albumina/análise , Infarto do Miocárdio com Supradesnível do Segmento ST/complicações , Troponina/sangueRESUMO
BACKGROUND: Waiting lists that continue to grow and the lack of organs available for transplantation necessitate the use of marginal livers, such as fatty livers. Since steatotic livers are more susceptible to damage from ischemia and reperfusion, it was investigated whether fatty livers with different lipidomic profiles show a different outcome when subjected to long-term cold storage preservation. METHODS: Eight-week-old male Wistar rats fed for 2 weeks by a methionine-choline-deficient (MCD) diet or control diet were employed in this study. Livers were preserved in a University of Wisconsin (UW) solution at 4 °C for 6, 12 or 24 h and, after washout, reperfused for 2 h with a Krebs-Henseleit buffer at 37 °C. Hepatic enzyme release, bile production, O2-uptake, and portal venous pressure (PVP) were evaluated. The liver fatty acid profile was evaluated by a gas chromatography-mass spectrometry (GC/MS). RESULTS: MCD rats showed higher LDH and AST levels with respect to the control group. When comparing MCD livers preserved for 6, 12 or 24 h, no differences in enzyme release were found during both the washout or the reperfusion period. The same trend occurred for O2-uptake, PVP, and bile flow. A general decrease in SFA and MUFA, except for oleic acid, and a decrease in PUFA, except for arachidonic, eicosadienoic, and docosahexanaeoic acids, were found in MCD rats when compared with control rats. Moreover, the ratio between SFA and the various types of unsaturated fatty acids (UFA) was significantly lower in MCD rats. CONCLUSIONS: Although prolonged cold ischemia negatively affects the graft outcome, our data suggest that the quality of lipid constituents could influence liver injury during cold storage: the lack of an increased hepatic injury in MCD may be justified by low SFA, which likely reduces the deleterious tendency toward lipid crystallization occurring under cold ischemia.
Assuntos
Deficiência de Colina/complicações , Fígado Gorduroso/patologia , Metionina/deficiência , Preservação de Tecido , Animais , Colina/administração & dosagem , Deficiência de Colina/patologia , Dieta , Fígado/patologia , Masculino , Ratos , Ratos Wistar , Preservação de Tecido/métodosRESUMO
Nonalcoholic fatty liver disease (NAFLD) is a serious health problem in developed countries. We documented the effects of feeding with a NAFLD-inducing, methionine- and choline-deficient (MCD) diet, for 1-4 weeks on rat liver oxidative stress, with respect to a control diet. Glycogen, neutral lipids, ROS, peroxidated proteins, and SOD2 were investigated using histochemical procedures; ATP, GSH, and TBARS concentrations were investigated by biochemical dosages, and SOD2 expression was investigated by Western Blotting. In the 4-week-diet period, glycogen stores decreased whereas lipid droplets, ROS, and peroxidated proteins expression (especially around lipid droplets of hepatocytes) increased. SOD2 immunostaining decreased in poorly steatotic hepatocytes but increased in the thin cytoplasm of macrosteatotic cells; a trend towards a quantitative decrease of SOD expression in homogenates occurred after 3 weeks. ATP and GSH values were significantly lower for rats fed with the MCD diet with respect to the controls. An increase of TBARS in the last period of the diet is in keeping with the high ROS production and low antioxidant defense; these TBARS may promote protein peroxidation around lipid droplets. Since these proteins play key roles in lipid mobilization, storage, and metabolism, this last information appears significant, as it points towards a previously misconsidered target of NAFLD-associated oxidative stress that might be responsible for lipid dysfunction.
Assuntos
Colina/metabolismo , Dieta , Fígado Gorduroso/patologia , Metionina/deficiência , Estresse Oxidativo , Trifosfato de Adenosina/metabolismo , Animais , Western Blotting , Fígado Gorduroso/metabolismo , Glutationa/metabolismo , Glicogênio/metabolismo , Hidrazinas , Imuno-Histoquímica , Fígado/metabolismo , Fígado/patologia , Masculino , Carbonilação Proteica , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
BACKGROUND/AIM: A new platinum compound, (Pt(O,O'-acac)(γ-acac)(DMS)) (PtAcacDMS), has been shown to possess higher cytotoxic activity than cisplatin on several cancer and chemoresistant cell lines. The aim of the present study was to compare the nephrotoxic effects - particularly renal fibrogenesis- of PtAcacDMS and cisplatin in rats after the subcutaneous administration of a single dose (5 mg/Kg b.w., s.c.) of either compound to ten-day-old rats. MATERIALS AND METHODS: Control and treated rats were killed 1 day (PD11), 7 days (PD17), 21 days (PD31) and 40 days (PD50) after the injection. Kidneys were processed for light and electron microscopy, and platinum determination. Antibodies against E-cadherin (E-cad), vimentin (VIM) and α-smooth muscle actin (αSMA) were used to identify epithelial and mesenchymal cells. RESULTS AND CONCLUSION: Cisplatin produced progressive cortical fibrotic lesions displaying a variable number of VIM-positive tubules and interstitial αSMA-positive cells around. By contrast, PtAcacDMS induced a minimal number of histopathological changes, which declined in the adult samples, while the renal platinum content was generally higher after PtAcacDMS than after cisplatin. The present experimental model was proven suitable to investigate the occurrence of epithelial-mesenchymal transition (EMT) in renal fibrogenesis induced by the platinum-based compounds.
Assuntos
Cisplatino/toxicidade , Modelos Animais de Doenças , Fibrose/induzido quimicamente , Rim/efeitos dos fármacos , Micelas , Compostos de Platina/toxicidade , Animais , Peso Corporal/efeitos dos fármacos , Feminino , Rim/patologia , Ratos , Ratos WistarRESUMO
Oxidative stress in fatty livers is mainly generated by impaired mitochondrial ß-oxidation, inducing tissue damages and disease progression. Under suitable excitation, light liver endogenous fluorophores can give rise to autofluorescence (AF) emission, the properties of which depend on the organ morphofunctional state. In this work, we characterized the AF properties of a rat liver model of lipid accumulation and oxidative stress, induced by a 1-9-week hypercaloric methionine-choline deficient (MCD) diet administration. The AF analysis (excitation at 366 nm) was performed in vivo, via fiber optic probe, or ex vivo. The contribution of endogenous fluorophores involved in redox reactions and in tissue organization was estimated through spectral curve fitting analysis, and AF results were validated by means of different histochemical and biochemical assays (lipids, collagen, vitamin A, ROS, peroxidised proteins, and lipid peroxidation -TBARS-, GSH, and ATP). In comparison with the control, AF spectra changes found already at 1 week of MCD diet reflect alterations both in tissue composition and organization (proteins, lipopigments, and vitamin A) and in oxidoreductive pathway engagement (NAD(P)H, flavins), with a subsequent attempt to recover redox homeostasis. These data confirm the AF analysis potential to provide a comprehensive diagnostic information on negative effects of oxidative metabolism alteration.
Assuntos
Dieta , Metabolismo dos Lipídeos , Fígado/metabolismo , Fígado/patologia , Estresse Oxidativo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Modelos Animais de Doenças , Glutationa/metabolismo , Peroxidação de Lipídeos , Masculino , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Fluorescência , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Vitamina A/metabolismoRESUMO
Tumor interstitial fluid (TIF) is a watery phase that accumulates inside the tumor interstitium. Its genesis and fate depend on various factors, namely tumor type, metabolic state of the tumor, expression of vascular endothelial growth factor, and absence of lymphatic system. For almost 30 years TIF remained a neglected entity until it was demonstrated that TIF, and in particular its high pressure, constitutes an important obstacle to drug delivery and immunotherapy. The present review not only summarizes the abundant literature on the processes of TIF genesis and on its effects on therapy but it also presents data that, in our opinion, point towards what is perhaps the real physiological purpose of TIF: a primitive means of providing nourishment, oxygen, cytokines and matrikines to tumor cells that furthermore promotes the invasion of the normal surrounding tissue and passive metastatization through lymphatics. It is also an inducer of inflammation through increased osmolarity due to albumin loss. Recently, a role for TIF as a possible source of biomarkers has also been suggested.
Assuntos
Líquido Extracelular/metabolismo , Neoplasias/metabolismo , Animais , Líquido Extracelular/imunologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Neoplasias/irrigação sanguínea , Neoplasias/imunologia , Neoplasias/patologia , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Trombose/imunologia , Trombose/metabolismo , Trombose/patologiaRESUMO
Widely used in the past against termites and soil insects, the chlorinated insecticide heptachlor (H) is a toxic contaminant which represents a risk for both terrestrial and aquatic organisms. Like many organochlorine pesticides, heptachlor and heptachlor epoxide (HE), with oxidation products synthesized by many plant and animal species, degrade slowly since many of the derived compounds are persistent. This increases the status of heptachlor as a hazardous pollutant. In the present experimental study we exposed specimens of Rana kl. esculenta, from the tadpole stage through to their complete metamorphosis, to three different concentrations of heptachlor (4, 40 and 400 ppb). Mortality and HE bioaccumulation were evaluated on all the experimental groups. Since amphibian integument directly interacts with the environmental constituents (water, air and soil), we investigated the toxic effects on the ventral epidermis of both tadpole and adult samples by employing such histo-cytopathological biomarkers as ultrastructural morphology, certain enzyme activities (acid and alkaline phosphatases, AcPase, and AlkPase; succinic dehydrogenase, SDH; alpha-naphtyl butyrate esterase, ANBE; nitric oxide synthase/NADPH diaphorase, NOS/NADPHd). Also, the levels of reactive oxygen species (ROS) in the different conditions were evaluated. The results obtained were of ecological relevance, in particular as regards the effects of this environmental toxicant on the samples of tadpole epidermis. Severe morphological alterations were observed in the larval epidermal cells (apical and skein cells), whereas the cell epidermis (keratinocytes and mitochondria-rich cells) of the adult survivors showed changes in enzyme activities, particularly those involved in the protective response to xenobiotic injury. In general, morpho-histochemical studies, analysis of HE bioaccumulation and mortality showed a relation to the H doses employed.
Assuntos
Epiderme/efeitos dos fármacos , Heptacloro/toxicidade , Rana esculenta/fisiologia , Poluentes Químicos da Água/toxicidade , Animais , Enzimas/metabolismo , Epiderme/enzimologia , Epiderme/ultraestrutura , Larva/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Análise de SobrevidaRESUMO
This study compares the effects of machine perfusion (MP) at different temperatures with simple cold storage. In addition, the role of Ca(2+) levels in the MP medium was evaluated. For MP, rat livers were perfused for 6 hours with Krebs-Henseleit (KH) solution (with 1.25 or 2.5 mM CaCl(2)) at 4 degrees C, 10 degrees C, 20 degrees C, 25 degrees C, 30 degrees C, or 37 degrees C. For cold storage, livers were perfused in situ and preserved with Celsior solution at 4 degrees C for 6 hours. The reperfusion period (2 hours at 37 degrees C) was performed under the same conditions used for MP-preserved and cold storage-preserved livers. Hepatic enzyme release, bile production, adenosine triphosphate (ATP) levels, and morphology were evaluated during MP and reperfusion. MP at 37 degrees C caused marked enzyme release; the same findings were obtained during reperfusion. By contrast, MP temperature lowering induced a significant decrease in liver damage. High levels of biliary gamma-glutamyltransferase and lactate dehydrogenase were found with MP at 4 degrees C and 10 degrees C but not with MP at 20 degrees C. When a KH-1.25 mM CaCl(2) solution was used during MP at 20 degrees C, very low enzyme release was observed and significantly lower hepatic damage was present at the end of the reperfusion period in comparison with cold storage. The same results were obtained when ruthenium red, a calcium uniporter blocker, was added to KH-2.5 mM CaCl(2). ATP levels were higher and morphology was better in liver preserved with KH-1.25 mM CaCl(2). MP at 20 degrees C with KH-1.25 mM CaCl(2) resulted in better quality liver preservation, improving hepatocyte and endothelial biliary cell survival, in comparison with cold storage. This raises the need to reconsider the temperature and calcium levels to be used during liver MP.
Assuntos
Cálcio/farmacologia , Fígado/fisiologia , Traumatismo por Reperfusão/fisiopatologia , Reperfusão/métodos , Temperatura , Trifosfato de Adenosina/metabolismo , Animais , Aspartato Aminotransferases/análise , Automação , Cloreto de Cálcio/farmacologia , Bombas de Infusão , L-Lactato Desidrogenase/análise , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Circulação Hepática/fisiologia , Masculino , Modelos Animais , Ratos , Ratos WistarRESUMO
In vertebrates, the biotransformation processes of xenobiotics are performed mainly by the liver which involves both hepatocytes and Kupffer-melanomacrophagic cells through enzymatic and nonenzymatic mechanisms. In this study, we investigated the liver of Rana esculenta adult frogs collected at two sample rice fields, one heavily polluted and one relatively unpolluted. Water pollution was determined by chemical analysis on tadpoles. The specific activities of some enzymes (glucose-6-phosphate dehydrogenase (G6PDH), acid and alkaline phosphatases (AcPase and AlkPase), succinic dehydrogenase (SDH), and catalase) were studied in the liver of adult frogs to identify the possible changes induced by contamination in the metabolic processes which depend on the function of the liver. The production of reactive oxygen species (ROS) were also evaluated through histochemical techniques. In the polluted samples, hepatocytes showed variations in the activity of G6PDH, AlkPase, and SDH and a moderate to intense ROS expression. Prominent changes were observed in Kupffer cells (KCs) and melanomacrophages (MMPs), both showing intense reactivity for AcPase and catalase and variations in melanin content and distribution. Results thus indicate a general adaptive response of liver parenchyma to environmental pollution. The possible role of both KCs and MMPs as scavengers of foreign substances is discussed.
Assuntos
Exposição Ambiental , Hepatócitos/efeitos dos fármacos , Células de Kupffer/efeitos dos fármacos , Fígado/efeitos dos fármacos , Rana esculenta , Poluição da Água , Fosfatase Ácida/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Catalase/metabolismo , Monitoramento Ambiental , Glucose-6-Fosfatase/metabolismo , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Histocitoquímica , Células de Kupffer/metabolismo , Células de Kupffer/ultraestrutura , Larva/química , Larva/efeitos dos fármacos , Fígado/citologia , Fígado/enzimologia , Melaninas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Succinato Desidrogenase/metabolismo , Oligoelementos/análise , Poluição da Água/análiseRESUMO
Natriuretic peptides (NPs), a family of structurally related hormones and nitric oxide (NO), generated by nitric oxide synthase (NOS), are believed to be involved in the regulation of fluid balance and sodium homeostasis. Differential expression and regulation of these factors depend on both physiological and pathological conditions. Both NPs and NO act in target organs through the activation of guanylate cyclase (GC) and the generation of guanosine 3',5'-cyclic monophosphate (cGMP), which is considered a common messenger for the action of these factors. The present study was designed to investigate--by histochemical methods--the expression of some NPs (proANP and ANP) and isoforms of NOS (neuronal NOS, nNOS, and inducible NOS, iNOS) in the mesonephros of Rana esculenta in different periods of the year including hibernation, to evaluate possible seasonal changes in their expression. We also studied the enzyme activity of NOS-related nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) and of GC. The experiments were performed on pieces of kidney of R. esculenta collected in their natural environment during active and hibernating life. The study was carried out using immunohistochemical techniques to demonstrate proANP, ANP, and some NOS isoforms. Antigen capture by enzyme linked immunosorbent assay (ELISA) was also performed to determine the presence of NPs in the frog kidney extract. Enzyme histochemistry was used to demonstrate the NOS-related NADPHd activity at light microscopy; GC activity was visualized at the electron microscope, using cerium as capture agent. The application of the immunohistochemical techniques demonstrated that frog mesonephros tubules express different patterns of distribution and/or expression of ANP and NOS during the annual cycle. Comparing the results obtained on active and hibernating frogs has provided interesting data; the NOS/NADPHd and GC activities showed some variations as well. Furthermore, the presence of NPs in the frog kidney extract was evidenced by dose-dependent response in the ELISA. The data suggest that both ANP and NO are intra-renal paracrine and/or autocrine factors which may modulate the adaptations of frog renal functions to seasonal changes through the action of the cGMP generated from GC activity.