Diphenyl Diselenide Attenuates Mitochondrial Damage During Initial Hypoxia and Enhances Resistance to Recurrent Hypoxia.

Hypoxia plays a significant role in the development of various cerebral diseases, many of which are associated with the potential risk of recurrence due to mitochondrial damage. Conventional drug treatments are not always effective for hypoxia-related brain diseases, necessitating the exploration of alternative compounds. In this study, we investigated the potential of diphenyl diselenide [(PhSe)2] to ameliorate locomotor impairments and mitigate brain mitochondrial dysfunction in zebrafish subjected to hypoxia. Additionally, we explored whether these improvements could confer resistance to recurrent hypoxia. Through a screening process, an appropriate dose of (PhSe)2 was determined, and animals exposed to hypoxia received a single intraperitoneal injection of 100 mg/kg of the compound or vehicle. After 1 h from the injection, evaluations were conducted on locomotor deficits, (PhSe)2 content, mitochondrial electron transport system, and mitochondrial viability in the brain. The animals were subsequently exposed to recurrent hypoxia to assess the latency time to hypoxia symptoms. The findings revealed that (PhSe)2 effectively crossed the blood-brain barrier, attenuated locomotor deficits induced by hypoxia, and improved brain mitochondrial respiration by modulating complex III. Furthermore, it enhanced mitochondrial viability in the telencephalon, contributing to greater resistance to recurrent hypoxia. These results demonstrate the beneficial effects of (PhSe)2 on both hypoxia and recurrent hypoxia, with cerebral mitochondria being a critical target of its action. Considering the involvement of brain hypoxia in numerous pathologies, (PhSe)2 should be further tested to determine its effectiveness as a potential treatment for hypoxia-related brain diseases.

Ibuprofen during gestation prevents some changes in physical and reflex development in offspring in a model of hyperleucinemia and maternal inflammation.

Maple Syrup Urine Disease (MSUD) is caused by a severe deficiency in the branched-chain ketoacid dehydrogenase complex activity. Patients MSUD accumulate the branched-chain amino acids leucine (Leu), isoleucine, valine in blood, and other tissues. Leu and/or their branched-chain α-keto acids are linked to neurological damage in MSUD. When immediately diagnosed and treated, patients develop normally. Inflammation in MSUD can elicit a metabolic decompensation crisis. There are few cases of pregnancy in MSUD women, and little is known about the effect of maternal hyperleucinemia on the neurodevelopment of their babies. During pregnancy, some intercurrences like maternal infection or inflammation may affect fetal development and are linked to neurologic diseases. Lipopolysaccharide is widely accepted as a model of maternal inflammation. We analyzed the effects of maternal hyperleucinemia and inflammation and the possible positive impact the use of ibuprofen in Wistar rats on a battery of physics (ear unfolding, hair growing, incisors eruption, eye-opening, and auditive channel opening) and neurological reflexes (palmar grasp, surface righting, negative geotaxis, air-righting, and auditory-startle response) maturation parameters in the offspring. Maternal hyperleucinemia and inflammation delayed some physical parameters and neurological reflexes, indicating that both situations may be harmful to fetuses, and ibuprofen reversed some settings.

Antioxidant, hepatoprotective, genoprotective, and cytoprotective effects of quercetin in a murine model of arthritis.

Rheumatoid arthritis is a highly debilitating inflammatory autoimmune disease which is characterized by joint destruction. The present study sought to investigate the effect of quercetin in rats with complete Freund's adjuvant-induced arthritis. Animals were divided into control/saline, control/quercetin (5 mg/kg, 25 mg/kg, and 50 mg/kg) arthritis/saline, and arthritis/quercetin (5 mg/kg, 25 mg/kg, and 50 mg/kg); the treatments were administered for 45 days. Biochemical, oxidative stress, genotoxicity, and cytotoxicity parameters were evaluated. All doses of quercetin reduced the levels of aspartate aminotransferase, thiobarbituric acid-reactive substances, and reactive oxygen species; however, only treatment with 25 or 50 mg/kg increased catalase activity. Total thiol and reduced glutathione levels were not significantly affected by the induction nor by the treatments. Genotoxicity assessed by DNA damage, and cytotoxicity through picogreen assay, decreased after treatments with quercetin. Our results present evidence of the antioxidant, cytoprotective, genoprotective and hepatoprotective, and effects of quercetin, demonstrating its potential as a candidate for coadjuvant therapy.

Effect of quercetin on E-NTPDase/E-ADA activities and cytokine secretion of complete Freund adjuvant-induced arthritic rats.

The effect of quercetin was assessed in rats induced with complete Freund adjuvant (CFA). Arthritis scores, paw oedema, latency, activities of myeloperoxidase (MPO), ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), and ectoadenosine deaminase (E-ADA) in lymphocytes were determined. Furthermore, nucleotide and nucleoside levels as well as the secretion of pro- and anti-inflammatory cytokines were evaluated. Animals were treated with saline and quercetin in doses of 5, 25, and 50 mg/kg for 45 days. The result revealed that quercetin (50 mg/kg) reduced arthritis score and paw oedema, and increased the latency in the thermal hyperalgesia test. Histopathological analysis showed that all the doses of quercetin reduced infiltration of inflammatory cells. MPO activity was increased in the arthritis group; however, quercetin reduced this activity. E-NTPDase activity was increased in lymphocytes of arthritis rats, and treatment with quercetin reversed this increase. However, E-ADA activity was reduced in the arthritis group, and treatment with quercetin modulated the activity of this enzyme in arthritis rat groups. Serum adenosine levels were increased in arthritis, and the levels were lowered with quercetin treatment. Quercetin treatment in arthritis groups decreased the elevated levels of cytokines in the arthritis control group. Thus, quercetin demonstrated an anti-inflammatory effect, and this flavonoid may be a promising natural compound for the treatment of arthritis. SIGNIFICANCE OF THE STUDY: Quercetin may represent a potential therapeutic compound in the treatment of rheumatoid arthritis. Findings from this study indicate that quercetin suppresses swelling and attenuates the underlying inflammatory responses. This is the first report where quercetin was shown to modulate the immune response to arthritis via attenuation of the purinergic system (E-NTPDase and E-ADA activities) and the levels of IFN-gamma and IL-4. Thus, this work is relevant to basic research and may be translated into clinical practice.

Activity and expression of E-NTPDase is altered in peripheral lymphocytes of systemic lupus erythematosus patients.

BACKGROUND: Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease, where there is irreversible breakdown of immunological self-tolerance. Extracellular adenosine triphosphate (ATP) and adenosine are signaling molecules that play an important part in the immune response. During inflammation and the immune response, a group of enzymes control these molecules, including ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), E-5'-nucleotidase, and ecto-adenosine deaminase (E-ADA). We determined the activity and expression of E-NTPDase, the expression of E-5'-nucleotidase, the activity of E-ADA in lymphocytes and serum of SLE patients. METHODS: This study involved 35 patients with SLE and 30 healthy subjects as a control group. E-NTPDase activity and expression were increased in lymphocytes from SLE patients (31% and 37% for activity and expression, respectively) compared with the control group. RESULTS: An approximately 42% increase in E-ADA activity in lymphocytes was observed in SLE patients compared with the control group, in serum the ADA activity was decreased by 57% in SLE patients. Expression of E-5'-nucleotidase was not changed in SLE patients. CONCLUSIONS: E-NTPDase and E-ADA perform key functions in the modulation of the immune and inflammatory response in SLE.

Altered extracellular ATP, ADP, and AMP hydrolysis in blood serum of sedentary individuals after an acute, aerobic, moderate exercise session.

Nucleotidases participate in the regulation of physiological and pathological events, such as inflammation and coagulation. Exercise promotes distinct adaptations, and can influence purinergic signaling. In the present study, we investigated soluble nucleotidase activities in the blood serum of sedentary young male adults at pre- and post-acute moderate aerobic exercise. In addition, we evaluated how this kind of exercise could influence adenine nucleotide concentrations in the blood serum. Sedentary individuals were submitted to moderate aerobic exercise on a treadmill; blood samples were collected pre- and post-exercise, and serum was separated for analysis. Results showed increases in ATP, ADP, and AMP hydrolysis post-exercise, compared to pre-exercise values. The ecto-nucleotide pyrophosphatase/phosphodiesterase was also evaluated, showing an increased activity post-exercise, compared to pre-exercise. Purine levels were analyzed by HPLC in the blood serum, pre- and post-exercise. Decreased levels of ATP and ADP were found post-exercise, in contrast with pre-exercise values. Conversely, post-exercise levels of adenosine and inosine increased compared to pre-exercise levels. Our results indicate an influence of acute exercise on ATP metabolism, modifying enzymatic behavior to promote a protective biological environment.

Involvement of purinergic system in the release of cytokines by macrophages exposed to glioma-conditioned medium.

Macrophages are involved in cancer progression. M1 macrophages have an antitumor effect, whereas M2 phenotype are associated with tumor growth. The progression of gliomas involves the participation of an inflammatory microenvironment. Adenosine triphosphate (ATP) can act as pro-inflammatory signal, whereas adenosine has opposite properties. The biological effects of extracellular nucleotides/nucleosides mediated by purinergic receptors are controlled by ectonucleotidases. In the present work, we evaluated whether glioma-conditioned medium (GL-CM) modulates macrophage differentiation and the participation of ATP and adenosine in the release of pro-and anti-inflammatory cytokines by these cells. The results show that macrophages exposed to GL-CM were modulated to an M2-like phenotype. HPLC analysis of GL-CM demonstrated the presence of significant amounts of ATP and its metabolites. Macrophages exposed to GL-CM presented decreased ATP and AMP hydrolysis and increased IL-10 and MCP-1 secretion, effects that were diminished by P1 or P2 antagonists. GL-CM did not alter the release of IL-6 by macrophages, although treatment with ATP promoted an increase in the release of IL-6, which was prevented by a P2X7 antagonist. In summary, we found that A2A and P2X7 activation is necessary for IL-10, MCP-1, and IL-6 release by macrophages exposed to GL-CM, which, in turn, modulates the macrophages to M2-phenotype. The present study establishes a relationship between M2-like polarization, cytokine release and purinergic receptor activation in macrophages exposed to GL-CM. Therefore, the data presented herein contributes to advancing in the field of cancer-related inflammation and point specific purinergic receptors as targets for modulation of the phenotype of glioma-associated macrophages.

Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells.

In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2 × 7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2 × 7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2 × 7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling-p53 increase, AMPK activation, and PARP cleavage-as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells.

Ectonucleotidase expression profile and activity in human cervical cancer cell lines.

Cervical cancer is the third most frequent cancer in women worldwide. Adenine nucleotide signaling is modulated by the ectonucleotidases that act in sequence, forming an enzymatic cascade. Considering the relationship between the purinergic signaling and cancer, we studied the E-NTPDases, ecto-5'-nucleotidase, and E-NPPs in human cervical cancer cell lines and keratinocytes. We evaluated the expression profiles of these enzymes using RT-PCR and quantitative real-time PCR analysis. The activities of these enzymes were examined using ATP, ADP, AMP, and p-nitrophenyl-5'-thymidine monophosphate (p-Nph-5'-TMP) as substrate, in a colorimetric assay. The extracellular adenine nucleotide hydrolysis was estimated by HPLC analysis. The hydrolysis of all substrates exhibited a linear pattern and these activities were cation-dependent. An interesting difference in the degradation rate was observed between cervical cancer cell lines SiHa, HeLa, and C33A and normal imortalized keratinocytes, HaCaT cells. The mRNA of ecto-5'-nucleotidase, E-NTPDases 5 and 6 were detectable in all cell lines, and the dominant gene expressed was the Entpd 5 enzyme, in SiHa cell line (HPV16 positive). In accordance with this result, a higher hydrolysis activity for UDP and GDP nucleotides was observed in the supernatant of the SiHa cells. Both normal and cancer cells presented activity and mRNAs of members of the NPP family. Considering that these enzymes exert an important catalytic activity, controlling purinergic nucleotide concentrations in tumors, the presence of ectonucleotidases in cervical cancer cells can be important to regulate the levels of extracellular adenine nucleotides, limiting their effects.

E-ADA activity in lymphocytes of an experimental model of pythiosis treated with immunotherapy.

Pythiosis is a life-threatening disease caused by the oomycete Pythium insidiosum. Some authors have suggested the involvement of a Th2-like immune response in the infected host, which leads to extensive tissue damage. The switch from a Th2 to a Th1 response pattern is one hypothesis to explain the curative properties of immunotherapy. Taking into account the importance of immunotherapy for pythiosis treatment and the contribution of adenine nucleotides in the immunoregulation of the host, we evaluated the ecto-adenosine deaminase (E-ADA; EC 3·5.4·4) activity in lymphocytes from rabbits inoculated with P. insidiosum. Rabbits were inoculated with 1 milliliter of zoospores subcutaneously injected into the lateral thorax; after developing lesions, the rabbits received eight doses of immunotherapy. E-ADA activity was measured in lymphocytes and the adenine nucleotides and adenosine levels were quantitatively determined in serum. Rabbits with characteristic lesions of pythiosis showed a decreased E-ADA activity (82·36%), a decreased adenosine triphosphate concentration (54·04%) and a higher adenosine concentration (2·51 fold), when compared with controls, after 28 days of inoculation. However, after the immunotherapy, the rabbits showed an increase in the E-ADA activity when compared with control (78·62%), contributing for the change in the immune response. Our results reinforce the hypothesis that the change from a Th2 to a Th1 immune response with the participation of the purinergic system could be responsible for the curative properties of immunotherapy.

E-NTPDase and E-ADA activities are altered in lymphocytes of patients with indeterminate form of Chagas' disease.

Trypanosoma cruzi infection triggers a chronic inflammatory process in human host and purinergic system ecto-enzymes play an important role in modulating the inflammatory and immune responses. In this study, it was investigated ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5; CD39) and ecto-adenosine deaminase (E-ADA; EC 3.5.4.4) activities in lymphocytes from patients with indeterminate form of Chagas' disease (IFCD). Twenty-five IFCD patients and 25 healthy subjects (control group) were selected. The peripheral lymphocytes were isolated and E-NTPDase and E-ADA activities were determined. Adenine nucleotides and adenosine levels were determined in serum by HPLC and the E-NTPDase1 expression in lymphocytes by Western blot analysis. E-NTPDase (ATP and ADP as substrates) and E-ADA (adenosine as substrate) activities were decreased in lymphocytes from IFCD patients (P<0.05 and P<0.01, respectively), while the E-NTPDase1 expression presented no changes in these patients. Serum ATP levels showed to be decreased (P<0.05) and both AMP (P<0.01) and adenosine (P<0.001) levels were increased in the IFCD group. The enzymatic alterations observed are in agreement with the immune response against T. cruzi infection in IFCD patients, since the decreased extracellular ATP and the increased adenosine levels trigger a Th2 anti-inflammatory response, which it is associated to adaptation of host to parasite, preventing clinical progress of disease.

Physical training normalizes nucleotide hydrolysis and biochemical parameters in blood serum from streptozotocin-diabetic rats.

Ectonucleotidases and the nucleotide metabolism have been implicated as important regulators in diabetes disease. We evaluated the ectonucleotidase activities and biochemical parameters in blood serum of streptozotocin (STZ)-induced diabetic rats submitted a physical training protocol. We observed a raise in ATP, ADP, AMP and p-Nph-5'-TMP hydrolysis rate and in the levels of cholesterol and triglycerides in rat blood serum, after 30 days of diabetes induction. However, in serum of rats submitted a physical training protocol by forced swimming, both the nucleotide hydrolysis rate and the lipids levels returned to the control values. Considering that diabetes leads to multiple pathophysiological alterations, the modulations observed in ectonucleotidase activities may be part of the events involved in these alterations. Then the physical training is a very important way to control the vascular alterations developed in diabetes.

L-NAME-treatment alters ectonucleotidase activities in kidney membranes of rats.

AIMS: To investigate the effect of N(omega)-Nitro-L-arginine methyl ester (l-NAME) treatment, known to induce a sustained elevation of blood pressure, on ectonucleotidase activities in kidney membranes of rats. MAIN METHODS: L-NAME (30 mg/kg/day) was administered to Wistar rats for 14 days in the drinking water. Enzyme activities were determined colorimetrically and their gene expression patterns were analyzed by semi-quantitative RT-PCR. The metabolism of ATP and the accumulation of adenosine were evaluated by HPLC in kidney membranes from control and hypertensive rats. PKC phosphorylation state was investigated by Western blot. KEY FINDINGS: We observed an increase in systolic blood pressure from 115+/-12 mmHg (control group) to 152+/-18 mmHg (l-NAME-treated group). Furthermore, the hydrolysis of ATP, ADP, AMP, and p-Nph-5'TMP was also increased (17%, 35%, 27%, 20%, respectively) as was the gene expression of NTPDase2, NTPDase3 and NPP3 in kidneys of hypertensive animals. Phospho-PKC was increased in hypertensive rats. SIGNIFICANCE: The general increase in ATP hydrolysis and in ecto-5'-nucleotidase activity suggests a rise in renal adenosine levels and in renal autoregulatory responses in order to protect the kidney against the threat presented by hypertension.

Ectonucleotidase activities are altered in serum and platelets of L-NAME-treated rats.

It is well known that hypertension is closely associated to the development of vascular diseases and that the inhibition of nitric oxide biosynthesis by administration of Nomega-Nitro-L-arginine methyl ester hydrochloride(L-NAME) leads to arterial hypertension. In the vascular system, extracellular purines mediate several effects;thus, ADP is the most important platelet agonist and recruiting ag ent, while adenosine, an end product of nucleotide metabolism, is a vasodilator and inhibitor of platelet activation and recruitment. Members of several families of enzymes, known as ectonucleotidases, including E-NTPDases (ecto-nucleoside triphosphate diphosphohydrolase), E-NPP (ecto-nucleotide pyrophosphatase/phosphodiesterase) and 5'-nucleotidase are able to hydrolyze extracellular nucleotides until their respective nucleosides. We investigated the ectonucleotidase activities of serum and platelets from rats made hypertensive by oral administration of L-NAME (30 mg/kg/day for 14 days or 30 mg/kg/day for 14 days plus 7 days of L-NAME washout, in the drinking water) in comparison to normotensive control rats. L-NAME promoted a significant rise in systolic blood pressure from 112 +/- 9.8 to 158 +/- 23 mmHg. The left ventricle weight index (LVWI) was increased in rats treated with L-NAME for 14 days when compared to control animals. In serum samples, ATP, ADP and AMP hydrolysis were reduced by about 27%, 36% and 27%, respectively. In platelets, the decrease in ATP, ADP and AMP hydrolysis was approximately 27%, 24% and 32%, respectively. All parameters recovered after 7 days of L-NAME washout. HPLC demonstrated a reduction in ADP, AMP and hypoxanthine levels by about 64%, 69% and 87%,respectively. In this study, we showed that ectonucleotidase activities are decreased in serum and platelets from L-NAME-treated rats, which should represent an additional risk for the development of hypertension. The modulation of ectonucleotidase activities may represent an approach to antihypertensive therapy via inhibition of spontaneous platelet activation and recruitment, as well as thrombus formation.

Genomic-independent action of thyroid hormones on NTPDase activities in Sertoli cell cultures from congenital hypothyroid rats.

The Sertoli cells play an essential role in the maintenance and control of spermatogenesis. The ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) and 5'-nucleotidase activities can modulate the extracellular adenine nucleotide levels, controlling nucleotide-mediated signaling events in Sertoli cells. Since thyroid hormones (TH) and adenine nucleotides and nucleosides play important modulatory roles in Sertoli cell proliferation and differentiation, the aim of our study was to investigate the effect of hypothyroidism upon the NTPDase and 5'-nucleotidase activities in Sertoli cell cultures, as well as to verify whether these effects may be reversed by short and long-term supplementation with TH. Congenital hypothyroidism was induced by adding 0.02% methimazole in the drinking water from day 9 of gestation and continually until 18 days of age. Hypothyroidism significantly decreased the extracellular ATP and ADP hydrolysis and this effect was significantly reversed when cell cultures were supplemented with 1 microM T3 or 0.1 microM T4 for 30 min. In contrast, AMP hydrolysis was not altered by hypothyroidism, but was increased by T4 supplementation for 24 h. The presence of the enzymes NTPDase 1, 2 and 3 was detected by RT-PCR in Sertoli cell cultures, however, hypothyroidism was not able to alter the expression of these enzymes. These findings demonstrate that TH modify NTPDase activities in hypothyroid Sertoli cells, probably via nongenomic mechanisms and, consequently, may influence the reproductive function throughout development.

Kinetic characterization of ATP diphosphohydrolase and 5'-nucleotidase activities in cells cultured from submandibular salivary glands of rats.

The participation of ecto-ATP diphosphohydrolase (CD39; ecto-NTPDase) and ecto-5'-nucleotidase (CD73) activities in the nucleotide hydrolysis by salivary gland cells from rats was evaluated. We investigated the biochemical characteristics of these ectoenzymes in cells cultured from submandibular salivary glands of rats. The V(max) for the hydrolysis of ATP, ADP and AMP were 2275+/-153 (mean+/-SEM, n = 4), 941+/-96 (mean+/-SEM, n = 5) and 175+/-5 (mean+/-SEM, n = 5) nmol Pi liberated per min per mg of protein, respectively. The K(m) values for ATP, ADP and AMP were 224+/-8 microM (mean+/-SEM, n = 4), 163+/-15 microM (mean+/-SEM, n = 5) and 117+/-5 microM (mean+/-SEM, n = 5), respectively. The competition plot showed that ATP and ADP were hydrolyzed at the same active site on the enzyme. It may be postulated that the physiological role for this ecto-enzyme cascade is to terminate the action of the co-transmitter ATP, generating adenosine.

Effects of follicle-stimulating hormone and vitamin A upon purinergic secretion by rat Sertoli cells.

Follicle-stimulating hormone (FSH) and vitamin A (retinol) are two of the main regulators of the male reproductive system. Recently, it has been described that extracellular purines can affect some important reproductive-related functions in Sertoli cells and germinative cells, by activating specific purinergic receptors. In this work, we report that both FSH and retinol are able to induce changes in the levels of extracellular purines of cultured rat Sertoli cells. FSH induced an increase in adenosine, mainly caused by enhanced ecto-ATPase activity, while retinol increased xanthine and hypoxanthine levels, and decreased uric acid concentration by an unknown mechanism. These data indicate that purinergic signaling may be involved in the control and/or regulation of some of the reproductive-related actions of these hormones.

Biochemical brain markers and purinergic parameters in rat CSF after seizure induced by pentylenetetrazol.

Cellular and molecular mechanisms involved in the generation of seizures and the magnitude of neural cells injury are not fully understood. We evaluated astrocyte and/or neuronal injury in rats in the pentylenetetrazol model of acute seizures by measuring S100B and NSE levels in cerebrospinal fluid. Additionally, we determined ADP and GDP hydrolysis by soluble nucleoside triphosphate diphosphohydrolase in the cerebrospinal fluid, and the concentration of nucleosides adenosine, inosine and guanosine as putative markers of brain injury. After pentylenetetrazol-induced seizures: (i) S100B values increased from 10 to 30 min, returning to control levels at 24 h; NSE levels presented a biphasic increase: an increase at 10 to 30 min returning to control levels, and again at 240 min followed by a decline at 24 h; (ii) nucleotidase activities increased from 10 min, returning to control levels at 240 min; (iii) guanosine and inosine levels increased exclusively after 30 min. In summary, this study showed biochemical changes in the cerebrospinal fluid occurring after seizures induced by pentylenetetrazol. Such events may have a modulating effect upon seizure expression, particularly nucleoside triphosphate diphosphohydrolase activities and nucleoside concentrations, but are nevertheless followed by neural death as evidenced by the increase in NSE and S100B levels.

Changes in ectonucleotidase activities in rat Sertoli cells during sexual maturation.

Sertoli cell maturation is a complex process involving both morphological and biochemical changes. These cells have previously been shown to be targets for extracellular purine structures such as ATP and adenosine. These compounds evoke responses in rat Sertoli cells through the purinoceptor families, P2X and P2Y and PA1. The signals to purinoceptors are usually terminated by the action of ectonucleotidases. In a previous work, we demonstrated that rat Sertoli cells have ecto-ATPdiphosphohydrolase (EC 3.6.1.5), ecto-5'-nucleotidase (EC 3.1.3.5) and ecto-adenosine deaminase (ecto-ADA) (EC 3.5.4.4) activities. Here we investigated whether some changes occur during rat Sertoli cell maturation in these activities. Rat Sertoli cells obtained from rats of different ages representing the pre-pubertal, mid-pubertal and 'young adult' (10-, 18- and 35-day-old, respectively) were cultured and used for different assays. The nucleotide hydrolysis was estimated by measuring the Pi released using a colorimetric method and by HPLC analysis. ATP and ADP hydrolysis was increased 3-fold during sexual maturation. AMP hydrolysis increased 4-fold in 10- to 35-day-old Sertoli cells. Similar results were obtained when we used other substrates to measure the extracellular hydrolysis of nucleotides (GTP, GDP, GMP and IMP). The ecto-ADA activity showed a 2-fold increase in the specific activity (18- to 35-day-old Sertoli cells). The termination of the purine cascade by adenosine degradation was faster in the 35- than in 18-day-old Sertoli cells. Follicle Stimulating Hormone (FSH) influences on the ectonucleotidase activities were investigated in 10- and 18-day-old Sertoli cells and a significant increase in the ATP and ADP hydrolysis was observed. Our results show an increase in the extracellular purine cascade during the Sertoli cell development, indicating a rise in the purine communication inside the seminiferous tubules with rat sexual maturation.