Crucial role of carbon monoxide as a regulator of diarrhea caused by cholera toxin: Evidence of direct interaction with toxin.

The present study evaluated the role of heme oxygenase 1 (HO-1)/carbon monoxide (CO) pathway in the cholera toxin-induced diarrhea and its possible action mechanism. The pharmacological modulation with CORM-2 (a CO donor) or Hemin (a HO-1 inducer) decreased the intestinal fluid secretion and Cl- efflux, altered by cholera toxin. In contrast, ZnPP (a HO-1 inhibitor) reversed the antisecretory effect of Hemin and potentiated cholera toxin-induced intestinal secretion. Moreover, CORM-2 also prevented the alteration of intestinal epithelial architecture and local vascular permeability promoted by cholera toxin. The intestinal absorption was not altered by any of the pharmacological modulators. Cholera toxin inoculation also increased HO-1 immunoreactivity and bilirubin levels, a possible protective physiological response. Finally, using fluorometric technique, ELISA assay and molecular docking simulations, we show evidence that CO directly interacts with cholera toxin, forming a complex that affects its binding to GM1 receptor, which help explain the antisecretory effect. Thus, CO is an essential molecule for protection against choleric diarrhea and suggests its use as a possible therapeutic tool.

(R)-ketamine attenuates neurodevelopmental disease-related phenotypes in a mouse model of maternal immune activation.

Infections during pregnancy are associated with an increased risk of neuropsychiatric disorders with developmental etiologies, such as schizophrenia and autism spectrum disorders (ASD). Studies have shown that the animal model of maternal immune activation (MIA) reproduces a wide range of phenotypes relevant to the study of neurodevelopmental disorders. Emerging evidence shows that (R)-ketamine attenuates behavioral, cellular, and molecular changes observed in animal models of neuropsychiatric disorders. Here, we investigate whether (R)-ketamine administration during adolescence attenuates some of the phenotypes related to neurodevelopmental disorders in an animal model of MIA. For MIA, pregnant Swiss mice received intraperitoneally (i.p.) lipopolysaccharide (LPS; 100 µg/kg/day) or saline on gestational days 15 and 16. The two MIA-based groups of male offspring received (R)-ketamine (20 mg/kg/day; i.p.) or saline from postnatal day (PND) 36 to 50. At PND 62, the animals were examined for anxiety-like behavior and locomotor activity in the open-field test (OFT), as well as in the social interaction test (SIT). At PND 63, the prefrontal cortex (PFC) was collected for analysis of oxidative balance and gene expression of the cytokines IL-1ß, IL-6, and TGF-ß1. We show that (R)-ketamine abolishes anxiety-related behavior and social interaction deficits induced by MIA. Additionally, (R)-ketamine attenuated the increase in lipid peroxidation and the cytokines in the PFC of the offspring exposed to MIA. The present work suggests that (R)-ketamine administration may have a long-lasting attenuation in deficits in emotional behavior induced by MIA, and that these effects may be attributed to its antioxidant and anti-inflammatory activity in the PFC.

Acute and sub-acute toxicity study of ethanol extract from Nectandra leucantha Nees & Mart. (Lauraceae) barks.

Nectandra leucantha has been used in traditional medicine. Several metabolites isolated from N. leucantha extracts displayed immunomodulatory, antileishmanial properties, but the determination of the toxicological profile in mammals has not previously been performed. In this study, the ethanol extract from N. leucantha barks (EENl) was characterized by HPLC/HRESIMS. To study acute toxicity, female mice received EENl in a single dose of 100, 300, 1000, or 2000 mg/kg bw. Later, sub-acute toxicity was introduced in female and male mice by oral gavage at 100, 500 or 1000 mg/kg bw for 28 consecutive days. Hematological and biochemical profiles from the blood as well as histological analysis from the liver and kidney were performed. The HPLC/HRESIMS analysis of the EENl revealed the presence of six neolignans chemically related to dehydrodieugenol B. In the oral acute and sub-chronic studies, EENl did not produce in all doses evaluated any alteration in behavior, biochemical, hematological, body weight gain and food intake or sudden death in Swiss mice. In addition, histopathological data did not reveal any disturbance in liver and kidney morphology after 28 days of EENl treatment. Our results indicate that EENl at dosage levels up to 2000 mg/kg bw is non-toxic and can be considered safe for mammals.

Cytokines and Immune Cells Profile in Different Tissues of Rodents Induced by Environmental Enrichment: Systematic Review.

Environmental Enrichment (EE) is based on the promotion of socio-environmental stimuli, which mimic favorable environmental conditions for the practice of physical activity and health. The objective of the present systematic review was to evaluate the influence of EE on pro-and anti-inflammatory immune parameters, but also in cell activation related to the innate and acquired immune responses in the brain and peripheral tissues in rodents. Three databases [PubMed (2209 articles), Scopus (1154 articles), and Science Direct (1040 articles)] were researched. After applying the eligibility criteria, articles were selected for peer review, independently, as they were identified by September 2021. The protocol for this systematic review was registered in the PROSPERO. Of the 4417 articles found, 16 were selected for this systematic review. In the brain, EE promoted a reduction in proinflammatory cytokines and chemokines. In the blood, EE promoted a higher percentage of leukocytes, an increase in CD19+ B lymphocytes, and the proliferation of Natura Killer (NK cells). In the bone marrow, there was an increase in the number of CD27- and CD11b+ mature NK cells and a reduction in CD27- and CD11b+ immature Natural Killer cells. In conclusion, EE can be an immune modulation approach and plays a key role in the prevention of numerous chronic diseases, including cancer, that have a pro-inflammatory response and immunosuppressive condition as part of their pathophysiology.

Safety assessment of Bauhinia cheilantha Bong. Steud leaves extract: acute, sub-acute toxicity, antioxidant, and antihemolytic evaluations.

Bauhinia cheilantha (Fabaceae), known popularly as pata-de-vaca and mororó has been largely recommended treating several diseases in folk medicine. However, information on safe doses and use is still scarce. The goal was to evaluate in-vitro antioxidant and antihemolytic and also acute and sub-acute toxicity effects of hydroalcoholic extract from B. cheilantha leaves (HaEBcl). The identification of the compounds in the HaEBcl was performed by ultra-performance liquid chromatography coupled with a diode array detector and quadrupole time-of-flight mass spectrometry. Antioxidant and hemolytic activity of HaEBcl was evaluated in vitro. To study acute toxicity, female mice received HaEBcl in a single dose of 300 and 2.000 mg/kg. Later, sub-acute toxicity was introduced in both female and male mice by oral gavage at 300, 1000, or 2000 mg/kg for 28 consecutive days. Hematological and biochemical profiles were created from the blood as well as from histological analysis of the liver. HaEBcl is rich in flavonoids (quercitrin and afzelin), has no hemolytic effects and moderate antioxidant effects in vitro. Acute toxicity evaluation showed that lethal dose (LD50) of HaEBcl was over 2000 mg/kg. Sub-acute toxicity testing elicited no clinical signs of toxicity, morbidity, or mortality. The hematological and biochemical parameters discounted any chance of hepatic or kidney toxicity. Furthermore, histopathological data did not reveal any disturbance in liver morphology in treated mice. Results indicate that HaEBcl has no hemolytic and moderate antioxidant effects in vitro. In addition, HaEBcl dosage levels up to 2000 mg/kg are nontoxic and can be considered safe for mammals.

Sepsis impairs the propagation of cortical spreading depression in rats and this effect is prevented by antioxidant extract.

Sepsis is a clinical syndrome with high morbidity and mortality. It is characterized by acute inflammatory response and oxidative stress, which is implicated in cerebral dysfunction. Murici (Byrsonimacrassifolia (L.) Kunth) is a fruit rich in antioxidant compounds, which could be an alternative to prevent damage to tissues induced by sepsis . Here, we evaluated the effects of sepsis on the propagation of cortical spreading depression (CSD) and oxidative stress, and tested the action of murici antioxidant extract in prevention against the effect of sepsis. Male Wistar rats (90-210 days, n = 40) were previously supplemented, orogastrically, with murici extract (150 mg/kg/day or 300 mg/kg/day), or an equivalent volume of the vehicle solution, for fifteen days. Then the animals were subjected to experimental sepsis through cecal ligation and perforation (CLP). Subsequently, CSD recordings were obtained and brain oxidative stress was evaluated. Sepsis decelerated CSD and increased the malondialdehyde (MDA) levels in the brain cortex of the animals. In contrast, septic rats that had been previously supplemented with murici antioxidant extract in doses of 150 and 300 mg/kg/day showed an increase in CSD propagation velocity, low levels of MDA and GSH/GSSG ratio and an increase of superoxide dismutase (SOD) activity, regardless of the dose tested. Our results demonstrate that sepsis affects brain excitability and that this effect can be prevented by murici antioxidant extract. The effects of sepsis and/or murici extract on CSD may be due to the oxidative state of the brain.

Hexane fraction from Brazilian Morus nigra leaves improved oral carbohydrate tolerance and inhibits α-amylase and α-glucosidase activities in diabetic mice.

Morus nigra, popularly known as mulberry, has been traditionally used as anti-diabetic herbal medication. This study focused on hexane fraction from Brazilian M. nigra leaves (Hex-Mn) effects on digestion and absorption of carbohydrate in diabetic mice. The high-performance liquid chromatography (HPLC) analysis was performed, and showed the presence of flavonoids isoquercetin and kaempferol-3-O-rhamnoside. Hex-Mn did not alter oral glucose tolerance test; however, it prevented hyperglycemia in oral sucrose and starch tolerance test in diabetic mice. Also, Hex-Mn was more efficient to inhibit the α-glucosidase, showing lower inhibitory effect on α-amylase activity in vitro. The results suggest that Hex-Mn may delay the carbohydrate digestion, but not glucose transport through brush border membrane of the intestine, which contribute with reduction in postprandial hyperglycemia in mice. Hex-Mn has antihyperglycemic effect by attenuating the carbohydrate digestion in diabetic mice, which could be explained, at least in part, by the presence of isoquercetin and kaempferol-3-O-rhamnoside.

Vasopressin and copeptin release during sepsis and septic shock.

Sepsis is defined as a potentially fatal organ dysfunction caused by a dysregulated host response to infection. Despite tremendous progress in the medical sciences, sepsis remains one of the leading causes of morbidity and mortality worldwide. The host response to sepsis and septic shock involves changes in the immune, autonomic, and neuroendocrine systems. Regarding neuroendocrine changes, studies show an increase in plasma vasopressin (AVP) concentrations followed by a decline, which may be correlated with septic shock. AVP is a peptide hormone derived from a larger precursor (preprohormone), along with two peptides, neurophysin II and copeptin. AVP is synthesized in the hypothalamus, stored and released from the neurohypophysis into the bloodstream by a wide range of stimuli. The measurement of AVP has limitations due to its plasma instability and short half-life. Copeptin is a more stable peptide than AVP, and its immunoassay is feasible. The blood concentrations of copeptin mirror those of AVP in many physiological states; paradoxically, during sepsis-related organ dysfunction, an uncoupling between copeptin and AVP blood levels appears to happen. In this review, we focus on clinical and experimental studies that analyzed AVP and copeptin blood concentrations over time in sepsis. The findings suggest that AVP and copeptin behave similarly in the early stages of sepsis; however, we did not find a proportional decrease in copeptin concentrations as seen with AVP during septic shock. Copeptin levels were higher in nonsurvivors than in survivors, suggesting that copeptin may work as a marker of severity or sepsis-related organ dysfunction.

Effects of Physical Exercise on Neuroplasticity and Brain Function: A Systematic Review in Human and Animal Studies.

Background: Physical exercise (PE) has been associated with increase neuroplasticity, neurotrophic factors, and improvements in brain function. Objective: To evaluate the effects of different PE protocols on neuroplasticity components and brain function in a human and animal model. Methods: We conducted a systematic review process from November 2019 to January 2020 of the following databases: PubMed, ScienceDirect, SciELO, LILACS, and Scopus. A keyword combination referring to PE and neuroplasticity was included as part of a more thorough search process. From an initial number of 20,782 original articles, after reading the titles and abstracts, twenty-one original articles were included. Two investigators evaluated the abstract, the data of the study, the design, the sample size, the participant characteristics, and the PE protocol. Results: PE increases neuroplasticity via neurotrophic factors (BDNF, GDNF, and NGF) and receptor (TrkB and P75NTR) production providing improvements in neuroplasticity, and cognitive function (learning and memory) in human and animal models. Conclusion: PE was effective for increasing the production of neurotrophic factors, cell growth, and proliferation, as well as for improving brain functionality.

Maternal Protein Restriction in Two Successive Generations Impairs Mitochondrial Electron Coupling in the Progeny's Brainstem of Wistar Rats From Both Sexes.

Maternal protein deficiency during the critical development period of the progeny disturbs mitochondrial metabolism in the brainstem, which increases the risk of developing cardiovascular diseases in the first-generation (F1) offspring, but is unknown if this effect persists in the second-generation (F2) offspring. The study tested whether mitochondrial health and oxidative balance will be restored in F2 rats. Male and female rats were divided into six groups according to the diet fed to their mothers throughout gestation and lactation periods. These groups were: (1) normoprotein (NP) and (2) low-protein (LP) rats of the first filial generation (F1-NP and F1-LP, respectively) and (3) NP and (4) LP rats of the second filial generation (F2-NP and F2-LP, respectively). After weaning, all groups received commercial chow and a portion of each group was sacrificed on the 30th day of life for determination of mitochondrial and oxidative parameters. The remaining portion of the F1 group was mated at adulthood and fed an NP or LP diet during the periods of gestation and lactation, to produce progeny belonging to (5) F2R-NP and (6) F2R-LP group, respectively. Our results demonstrated that male F1-LP rats suffered mitochondrial impairment associated with an 89% higher production of reactive species (RS) and 137% higher oxidative stress biomarkers, but that the oxidative stress was blunted in female F1-LP animals despite the antioxidant impairment. In the second generation following F0 malnutrition, brainstem antioxidant defenses were restored in the F2-LP group of both sexes. However, F2R-LP offspring, exposed to LP in the diets of the two preceding generations displayed a RS overproduction with a concomitant decrease in mitochondrial bioenergetics. Our findings demonstrate that nutritional stress during the reproductive life of the mother can negatively affect mitochondrial metabolism and oxidative balance in the brainstem of F1 progeny, but that restoration of a normal diet during the reproductive life of those individuals leads toward a mitochondrial recovery in their own (F2) progeny. Otherwise, if protein deprivation is continued from the F0 generation and into the F1 generation, the F2 progeny will exhibit no recovery, but instead will remain vulnerable to further oxidative damage.

Spondias tuberosa inner bark extract exert antidiabetic effects in streptozotocin-induced diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Diabetes mellitus (DM) is one of the most important medical emergencies of the 21st century. However, commercially available oral drugs with antidiabetic properties have been limited because of potential side effects, such as: hypoglycemia, weight gain, hepatic dysfunction and abdominal discomfort. As well as antidiabetic drugs, many types of medicinal herbal supplements are utilized as alternative treatments for DM and related comorbidities. Spondias tuberosa Arruda (Anacardiaceae), popularly known as "umbu", has been used in traditional medicine to treat a vast range of diseases, including DM, infections, digestive disorders, diarrhea and menstrual abnormalities. AIM OF THE STUDY: This study evaluated the effect of the hydroethanolic extract of the inner stem bark of Spondias tuberosa (EEStb) in streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Diabetes was induced in rats by a single injection of STZ (40 mg/kg i.p.). Diabetic rats were treated with 250 mg/kg or 500 mg/kg of the EEStb for 21 days. Water intake, urinary volume, body weight, as well as biochemical parameters, such as cholesterol total (TC), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), hepatic and muscle glycogen urea, alanine and aspartate aminotransferase, total protein, albumin, and glucose blood levels, were analyzed. We also determined the hepatic antioxidant state, as well as both of insulin and glucose tolerance. RESULTS: The extract was evaluated by HPLC, and the major components of EESTb were identified (i.e. gallic acid and quercetin). The 500 mg/kg dosage of EEStb significantly decreased fasting blood glucose and post-prandial glucose. The EEStb also reduced urinary volume, food and water intake, as well as decreased body weight gain. Diabetic rats that received EEStb had a lower loss of muscle mass and white adipose tissue. Additionally, EEStb improved the urinary excretion of urea and glucose. The extract significantly decreased triglycerides, total cholesterol and VLDL in diabetic rats. However, no significant effect was observed on the levels of total and HDL cholesterol. EEStb treatment prevented hepatotoxic diabetic-induced, improved GSH:GSSG ratio, SOD and CAT activity as well as reduced nitrite and TBARs levels. CONCLUSIONS: Our results demonstrate that EEStb has antioxidant and hepatoprotective effects as well as improves insulin sensibility in diabetic rats. This indicates that S. tuberosa could be a potential resource for alternative therapies in the treatment of hyperglycemic conditions. These results also support the use of EEStb in ethnomedicine for the management of diabetes.

Cardioprotective effect of thyroid hormone is mediated by AT2 receptor and involves nitric oxide production via Akt activation in mice.

Studies have demonstrated that thyroid hormone (T3) can precondition the heart against ischaemic injury and improve post-ischaemic recovery. This study investigated whether the AT2 receptor (AT2R) is involved in cardioprotection and the potential molecular mechanism responsible for this effect. Hyperthyroidism was induced in male wild-type (WT) and AT2R knockout (KO) mice by administering daily intraperitoneal injections of T3 (7 µg/100 g body weight) for 14 days. The mouse hearts were harvested and perfused with a Krebs-Henseleit solution at a constant flow in a Langendorff set-up. After 30 min of stabilization, the hearts were subjected to global ischaemia for 20 min and reperfused for 45 min. Baseline cardiac function was assessed by measuring four parameters: LVDP (mmHg), heart rate (bpm), + dP/dt and - dP/dt (mmHg/s). After reperfusion, the total protein from cardiac ventricles was obtained, and the Akt signalling pathway and NO production were evaluated. Post-ischaemic functional recovery was significantly greater (p < 0.05) in the T3-treated WT mice compared to the control, demonstrating the cardioprotective effect of T3. This effect was abolished in T3-treated KO mice, demonstrating the physiological relevance of AT2R to the cardioprotective phenotype induced by T3. Akt activation, iNOS expression and NO production increased in cardiac tissue after T3 treatment in the WT animals, but no difference was observed after treatment in the KO mice. This study indicates that AT2R acts as a cardioprotector in the case of hyperthyroidism. Strategies targeting AT2R agonists might improve cardiac function through NO production and suggest potential therapeutic targets for heart diseases.

Angiotensin II type 2 receptor (AT2R) is associated with increased tolerance of the hyperthyroid heart to ischemia-reperfusion.

BACKGROUND: Thyroid hormone induces cardiac hypertrophy and preconditions the myocardium against Ischemia/Reperfusion (I/R) injury. Type 2 Angiotensin II receptors (AT2R) are shown to be upregulated in cardiac hypertrophy observed in hyperthyroidism and this receptor has been reported to mediate cardioprotection against ischemic injury. METHODS: The aim of the present study was to evaluate the role of AT2R in the recovery of myocardium after I/R in isolated hearts from T3 treated rats. Male Wistar rats were treated with triiodothyronine (T3; 7 µg/100 g BW/day, i.p.) in the presence or not of a specific AT2R blocker (PD123,319; 10 mg/Kg) for 14 days, while normal rats served as control. After treatment, isolated hearts were perfused in Langendorff mode; after 30 min of stabilization, hearts were subjected to 20 min of zero-flow global ischemia followed by 25 min, 35 min and 45 min of reperfusion. RESULTS: T3 treatment induced cardiac hypertrophy, which was not changed by PD treatment. Post-ischemic recovery of cardiac function was increased in T3-treated hearts after 35 min and 45 min of reperfusion as compared to control and the ischemic contracture was accelerated and intensified. AT2R blockade was able to return the evaluated functional parameters of cardiac performance (LVDP, +dP/dt(máx) and -dP/dt(min)) to the control condition. Furthermore, AT2R blockade prevented the increase in AMPK expression levels induced by T3, suggesting its possible involvement in this process. CONCLUSION: AT2R plays a significant role in T3-induced cardioprotection.

Carbon monoxide and nitric oxide modulate hyperosmolality-induced oxytocin secretion by the hypothalamus in vitro.

OT (oxytocin) is secreted from the posterior pituitary gland, and its secretion has been shown to be modulated by NO (nitric oxide). In rats, OT secretion is also stimulated by hyperosmolarity of the extracellular fluid. Furthermore, NOS (nitric oxide synthase) is located in hypothalamic areas involved in fluid balance control. In the present study, we evaluated the role of the NOS/NO and HO (haem oxygenase)/CO (carbon monoxide) systems in the osmotic regulation of OT release from rat hypothalamus in vitro. We conducted experiments on hypothalamic fragments to determine the following: (i) whether NO donors and NOS inhibitors modulate OT release and (ii) whether the changes in OT response occur concurrently with changes in NOS or HO activity in the hypothalamus. Hyperosmotic stimulation induced a significant increase in OT release that was associated with a reduction in nitrite production. Osmotic stimulation of OT release was inhibited by NO donors. NOS inhibitors did not affect either basal or osmotically stimulated OT release. Blockade of HO inhibited both basal and osmotically stimulated OT release, and induced a marked increase in NOS activity. These results indicate the involvement of CO in the regulation of NOS activity. The present data demonstrate that hypothalamic OT release induced by osmotic stimuli is modulated, at least in part, by interactions between NO and CO.

Central nitric oxide blocks vasopressin, oxytocin and atrial natriuretic peptide release and antidiuretic and natriuretic responses induced by central angiotensin II in conscious rats.

The presence of nitric oxide synthase (NOS), the enzyme that catalyses the formation of nitric oxide (NO), in the circumventricular organs and magnocellular neurones suggests an important role of NO in the modulation of vasopressin (AVP) and oxytocin (OT) release. Intracerebroventricular (I.C.V.) injection of angiotensin II (Ang II) stimulates the release of AVP, OT and atrial natriuretic peptide (ANP), with the resultant antidiuretic and natriuretic effects. This study investigated the interaction between nitrergic and angiotensinergic pathways on the release of AVP, OT and ANP and on urinary volume and sodium excretion in water-loaded rats. Unanaesthetized, freely moving, male Wistar rats received two water loads followed by an injection into the lateral ventricle of an inhibitor of NOS (L-NAME), a NO donor [3-morpholinylsydnoneimine chloride (SIN-1) or S-nitroso-N-acetyl penicillamine (SNAP)] or vehicle (isotonic saline) and, 20 min after, they received a second I.C.V. injection of Ang II or vehicle. Injections of L-NAME or Ang II produced an increase in plasma levels of AVP, OT and ANP, a reduction in urinary volume and an increase in sodium excretion. Pretreatment with L-NAME enhanced the Ang II-induced increase in AVP, OT and ANP release, as well as the antidiuresis and natriuresis. Injection of SIN-1 or SNAP did not modify hormonal plasma levels and urinary parameters. In contrast SNAP blocked the AVP, OT and ANP release, as well as antidiuretic and natriuretic responses induced by ANG-II. Thus, the central nitrergic system can act to inhibit AVP, OT and ANP secretion and the antidiuretic and natriuretic effects in response to Ang II.

Neuronal nitric oxide synthase inhibition differentially affects oxytocin and vasopressin secretion in salt loaded rats.

Nitric oxide, an endogenous gas produced by nitric oxide synthase (NOS), has been described as a neuromodulator of hormone secretion, including the neurohypophysial peptides oxytocin (OT) and vasopressin (AVP), hormones involved in the sodium and water homeostasis. The presence of NOS in the hypothalamic nuclei as well as in the circumventricular organs suggests a nitrergic regulation of OT and AVP secretion. Thus, the aim of this study was to evaluate the effect of 7-nitroindazole (7-NI), a selective inhibitor of neuronal NOS, in the plasma OT and AVP levels in rats submitted to a short and long-term salt loading. We also evaluated the NOS activity in the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. Our data showed an increase of plasma OT and AVP levels in both short and long-term salt loading. The augment of plasma OT and AVP levels was accompanied by an increase of NOS activity in the SON and PVN. The injection of 7-NI potentiated the increase of plasma OT induced by salt loading, but inhibited the increase of plasma AVP in the same experimental conditions. These results indicate that, under short and prolonged osmotic stimulation, nitric oxide may differentially control the neurohypophysial secretion.

The role of carbon monoxide and nitric oxide in hyperosmolality-induced atrial natriuretic peptide release by hypothalamus in vitro.

We evaluated the participation of the nitrergic and carbon monoxide (CO) systems in the atrial natriuretic peptide (ANP) release induced by osmotic stimulation of the rat anterior and medial basal hypothalamus (BH) fragments in vitro. The increase in the medium osmolality (NaCl, 340 mOsm/kg H2O) induced an elevated ANP release, which was associated with a decrease in nitric oxide synthase (NOS) activity (p<0.001), nitric oxide (NO) production and nitrate (p<0.001) release into the medium. The NO donors sodium nitroprusside (SNP, 300 microM), S-nitroso-N-acetylpenicillamine (SNAP, 300 microM) and 3-morpholinylsydnoneimine chloride (SIN-1, 300 microM) promoted a significant decrease in ANP release in response to hyperosmolality (p<0.001). ANP release observed in the present study did not result from injury to the BH caused by the increase in medium osmolality nor a toxic effect of the NO donors as demonstrated by the ANP release after incubation with KCl (56 mM). Furthermore, hyperosmolality or NO donors did not increase the LDH content in the medium. The hyperosmotic-induced ANP release and reduction of NOS activity were prevented by the heme oxygenase inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG). In conclusion, these results suggest that NO, the production of which is dependent on CO, modulates the osmolality-induced ANP release by BH fragments.