Alessandra Nejar Bruno

Ontogenetic profile of ectonucleotidase activities from brain synaptosomes of pilocarpine-treated rats

Adenosine, a well‐known neuromodulator, can act as an endogenous anticonvulsant via the activation of adenosine A1 receptors. This adenine nucleoside can be produced in the synaptic cleft by the ectonucleotidase cascade, which includes the nucleoside triphosphate diphosphohydrolase (NTPDase) family and ecto‐5′‐nucleotidase. It has been previously reported that ectonucleotidase activities are increased in female adult rats submitted to the pilocarpine model of epilepsy. Several studies have suggested that the immature brain is less vulnerable to morphologic and physiologic alterations after status epilepticus (SE). Here, we evaluate the ectonucleotidase activities of synaptosomes from the hippocampus and cerebral cortex of male and female rats at different ages (7–9, 14–16 and 27–30‐day old) submitted to the pilocarpine model of epilepsy. Our results show that ATP and ADP hydrolysis in the hippocampus and cerebral cortex were not altered by the pilocarpine treatment in female and male rats at 7–9, 14–16 and 27–30 days. There were no changes in AMP hydrolysis in female and male rats submitted to the model at different ages, but a significant increase in AMP hydrolysis (71%) was observed in synaptosomes from the cerebral cortex of male rats at 27–30 days. Pilocarpine‐treated male rats (60–70‐day old) presented an enhancement in ectonucleotidase activities in the synaptosomes of the cerebral cortex (33, 40 and 64% for ATP, ADP and AMP hydrolysis, respectively) and hippocampus (55, 98 and 101% for ATP, ADP and AMP hydrolysis, respectively). These findings highlight differences between the purinergic system of young and adult rats submitted to the pilocarpine model of epilepsy.

Increase of nucleotidase activities in rat blood serum after a single convulsive injection of pentylenetetrazol

Adenosine has been shown to be a major regulator in convulsive disorders exerting its anticonvulsant effects on various seizure models. The ectonucleotidase pathway is an important metabolic source of extracellular adenosine. In this study, we evaluated ATP, ADP and AMP hydrolysis in rat serum after a single convulsive injection of pentylenetetrazol (PTZ). The animals were sacrificed at 5 and 30 min, 1, 5, 12, 24 and 48 h after an intraperitoneal injection of PTZ (60 mg/kg). ATP, ADP and AMP hydrolysis by rat blood serum were significantly increased (40-50%) until 24 h after PTZ injection. There were no significant differences in the nucleotide hydrolysis when the in vitro effect of different concentrations of PTZ was analyzed. Changes in nucleotide hydrolysis observed after acute administration of PTZ could not be attributed to phosphodiesterase activity since PTZ-treated rats did not demonstrate significant differences in the hydrolysis of the substrate marker of this enzyme when compared with control rats. These results suggest that the stimulation of the nucleotidase pathway may play an important role in attenuating seizure activity.

Conditioned medium from mesenchymal stem cells induces cell death in organotypic cultures of rat hippocampus and aggravates lesion in a model of oxygen and glucose deprivation.

Cell therapy using bone marrow-derived mesenchymal stem cells (MSC) seems to be a new alternative for the treatment of neurological diseases, including stroke. In order to investigate the response of hippocampal tissue to factors secreted by MSC and if these factors are neuroprotective in a model of oxygen and glucose deprivation (OGD), we used organotypic hippocampal cultures exposed to conditioned medium from bone marrow-derived MSC. Our results suggest that the conditioned medium obtained from these cells aggravates lesion caused by OGD. In addition, the presence of the conditioned medium alone was toxic mainly to cells in the CA1, CA2 and CA3 areas of the hippocampal organotypic culture even in basal conditions. GABA stimulation and NMDA and AMPA receptors antagonists were able to reduce propidium iodide staining, suggesting that the cell death induced by the toxic factors secreted by MSC could involve these receptors.

Hypothyroidism changes adenine nucleotide hydrolysis in synaptosomes from hippocampus and cerebral cortex of rats in different phases of development

The influence of the thyroid hormones on the normal function of the mammalian central nervous system depends on the brain region and on the developmental stage. Adenine nucleotides and their products also affect the brain function; ATP is an excitatory neurotransmitter, and adenosine has inhibitory effects on neurotransmission. Thus, this study aimed to evaluate the effects of hypothyroidism on the hydrolysis of ATP to adenosine in hippocampal and cortical synaptosomes and blood serum of rats during different phases of development. Rats aged 60 and 420 days old were divided into three groups: control, sham‐operated and hypothyroid. Hypothyroidism was induced in these rats by thyroidectomy and methimazole (0.05%) added to their drinking water for 14 days. Neonatal hypothyroidism was induced by adding 0.02% methimazole in the drinking water from day 9 of gestation, and continually until 14 days old. Hypothyroidism increased the AMP hydrolysis in both hippocampus and cerebral cortex synaptosomes of rats in all aged tested. In blood serum, thyroid hormones deficiency increased the AMP hydrolysis in 14‐day‐old rats and the hydrolysis of ATP, ADP and AMP in 60‐day‐old rats; however, no alteration was observed in 420‐day‐old rats. Thus, our results suggest the involvement of the 5′‐nucleotidase in synaptic function control in hypothyroidism throughout brain development.

Hyperthyroidism modifies ecto-nucleotidase activities in synaptosomes from hippocampus and cerebral cortex of rats in different phases of development

Here we investigate the possible effects of the hyperthyroidism on the hydrolysis of the ATP to adenosine in the synaptosomes of hippocampus, cerebral cortex and blood serum of rats in different developmental phases. Manifestations of hyperthyroidism include anxiety, nervousness, tachycardia, physical hyperactivity and weight loss amongst others. The thyroid hormones modulate a number of physiological functions in central nervous system, including development, function, expression of adenosine A1 receptors and transport of neuromodulator adenosine. Thus, hyperthyroidism was induced in male Wistar rats (5‐, 60‐, 150‐ and 330‐day old) by daily injections of l‐thyroxine (T4) for 14 days. Nucleotide hydrolysis was decreased by about 14–52% in both hippocampus and cerebral cortex in 5 to 60‐day‐old rats. These changes were also observed in rat blood serum. In addition, in 11‐month‐old rats, inhibition of ADP and AMP hydrolysis persisted in the hippocampus, whereas, in cerebral cortex, an increase in AMP hydrolysis was detected. Thus, hyperthyroidism affects the extracellular nucleotides balance and adenosine production, interfering in neurotransmitter release, development and others physiological processes in different systems.

Adenosine Uptake is the Major Effector of Extracellular ATP Toxicity in Human Cervical Cancer Cells

Cervical cancer cells respond to high extracellular ATP. There is cooperation between ATP and its metabolites with regard to cytotoxicity, with adenosine necessary, but not sufficient, to induce cell death in the whole population of cells, which is significant in the context of cancer therapeutics.

Increase of adenine nucleotide hydrolysis in rat hippocampal slices after seizures induced by quinolinic acid.

Quinolinic acid (QUIN), an endogenous convulsant compound, overstimulates the glutamatergic system stimulating N-methyl-D-aspartate receptors, enhancing glutamate release and inhibiting glutamate uptake. Glutamate releases the neuroprotector adenosine, which in turn reduces glutamate release and depresses the neuronal activity. Additionally, adenine nucleotides are an important source of adenosine, by action of ecto-nucleotidases. Here we evaluated the adenine nucleotide hydrolysis in hippocampal slices of adult rats in different times after seizures induced by QUIN. After 45 min, there was an increase of ATP and ADP hydrolysis. After 5 h, there was an increase of ATP, ADP and AMP hydrolysis. After 12 h, there was an increase only of ATP hydrolysis. After 24 h, all hydrolysis returned to control levels. As slice preparations maintain tissue integrity, this study indicates, more than previously observed with synaptosomal preparations, that the extracellular production of the neuroprotector adenosine may be involved in brain responses to seizures.

The Inhibitory Effects of Phenolic and Terpenoid Compounds from Baccharis trimera in Siha Cells: Differences in Their Activity and Mechanism of Action

Baccharis trimera is used in folk medicine as a tea for digestive and liver diseases. It possesses anti-inflammatory and antioxidant properties that are related to the presence of phenolic compounds. The aim of this work was to investigate the anti-proliferative properties of phenolic (PHE) and terpenoid (SAP) compounds from B. trimera on human cervical cancer. The treatment of SiHa cells with PHE for 24 h suppressed colony formation in a dose-dependent manner, inhibited proliferation and inhibited cell motility. Although SAP inhibited the proliferation of SiHa cells in a dose-dependent manner, it increased colony formation and did not inhibit cell motility. PHE and SAP also promoted a significant increase in lactate dehydrogenase levels in the culture medium in a dose-dependent manner, indicating a loss of cell membrane integrity. Moreover, PHE promoted necrotic cell death, whereas SAP induced apoptosis. These compounds are new anticancer prototypes due their significant anticancer activity demonstrated herein.

Nociceptive Response and Adenine Nucleotide Hydrolysis in Synaptosomes Isolated from Spinal Cord of Hypothyroid Rats

Purinergic system exerts a significant influence on the modulation of pain pathways at the spinal site. Adenosine has antinociceptive properties in experimental and clinical situations, while ATP exerts pronociceptive actions in different pain models. In this study we investigated the hydrolysis of ATP to adenosine in synaptosomes from spinal cord in parallel with the nociceptive response of rats at different ages after hypothyroidism induction. Hypothyroidism elicited a significant increase in AMP hydrolysis to adenosine in synaptosomes from spinal cord of rats subjected to neonatal hypothyroidism and in 420-day-old rats submitted to thyroidectomy. Accordingly, these rats presented an analgesic response as a consequence of hypothyroidism. In contrast, the ATP hydrolysis was decreased in the spinal cord of 60-day-old hypothyroid rats in parallel with a significant increase in nociceptive response. These results indicate the involvement of adenine nucleotides in the control of the hypothyroidism-induced nociceptive response during development.

Acute caffeine treatment increases extracellular nucleotide hydrolysis from rat striatal and hippocampal synaptosomes

The psychostimulant caffeine promotes behavioral effects such as hyperlocomotion, anxiety, and disruption of sleep by blockade of adenosine receptors. The availability of extracellular adenosine depends on its release by transporters or by the extracellular ATP catabolism performed by the ecto-nucleotidase pathway. This study verified the effect of caffeine on NTP-Dase 1 (ATP diphosphohydrolase) and 5′-nucleotidase of synaptosomes from hippocampus and striatum of rats. Caffeine and theophylline tested in vitro were unable to modify nucleotide hydrolysis. Caffeine chronically administered in the drinking water at 0.3 g/L or 1 g/L for 14 days failed to affect nucleotide hydrolysis. However, acute administration of caffeine (30 mg/kg, ip) produced an enhancement of ATP (50%) and ADP (32%) hydrolysis in synaptosomes of hippocampus and striatum, respectively. This activation of ATP and ADP hydrolysis after acute treatment suggests a compensatory effect to increase adenosine levels and counteract the antagonist action of caffeine.