Peripheral nucleotide hydrolysis in rats submitted to a model of electroconvulsive therapy.

Electroconvulsive therapy (ECT) is an efficacious and safe method for the treatment of mood disorders. Its utilization is accompanied by a myriad of biochemical and cellular changes, which are far from fully understood. The present work investigates in rat serum the effects of seizures induced by electroconvulsive shocks (ECS), an animal model of ECT, on enzymes that hydrolyze ATP, ADP and AMP to adenosine. Two different models of ECS were used, consisting in the application of one or eight ECS sessions, and respectively named acute or chronic. Serum samples were collected at several time points after the single shock in the acute and after the eighth and last shock in the chronic model. A single shock produced a sudden and short-lived inhibition of enzymatic activity (P<0.01 for ADP and AMP), whereas in the chronic model significant increases were noticed starting as early as 12 h after the last shock, remaining significantly elevated until the last measurement 7 days later for ATP and ADP. Analysis of hydrolysis was assessed at the selected time point of 7 days in cerebrospinal fluid samples, also demonstrating a significant activation in the chronic model (P<0.0001 for ATP and ADP). These results support the idea that adenosine nucleotides may be involved in the biochemical mechanisms underlying longer lasting therapeutic effects associated with ECT, and suggest that peripheral markers can possibly contribute to the evaluation of activity in the central nervous system.

Pentylenetetrazol kindling alters adenine and guanine nucleotide catabolism in rat hippocampal slices and cerebrospinal fluid.

Pentylenetetrazol (PTZ) is commonly used as a convulsant drug. The enhanced seizure susceptibility induced by kindling is probably attributable to plastic changes in the synaptic efficacy. Adenosine and guanosine act both as important neuromodulators and neuroprotectors with mostly inhibitory effects on neuronal activity. Adenosine and guanosine can be released per se or generated from released nucleotides (ATP, ADP, AMP, GTP, GDP, and GMP) that are metabolized and rapidly converted to adenosine and guanosine. The aim of this study was to evaluate nucleotide hydrolysis by ecto- and soluble nucleotidases (hippocampal slices and CSF, respectively) after PTZ-kindling (stages 3, 4, or 5 seizures) or saline treatment in rats. Additionally, the levels of purines in rat cerebrospinal fluid (CSF), as well as ecto-NTPDases (1, 2, 3, 5, 6 and 8) and ecto- 5'-nucleotidase expression were determined. Ecto-enzyme assays demonstrated that ATP, AMP, GDP, and GMP hydrolysis enhanced when compared with controls. In addition, there was an increase of ADP, GDP, and GMP hydrolysis by soluble nucleotidases in PTZ-kindling rats compared to control group. The HPLC analysis showed a marked increase in PTZ-kindled CSF concentrations of GTP, ADP, and uric acid, but GDP, AMP, and hypoxanthine concentrations were decreased. Such alterations indicate that the modulatory role of purines in CNS could be affected by PTZ-kindling. However, the physiological significance of these findings remains to be elucidated.

Guanine and adenine nucleotidase activities in rat cerebrospinal fluid.

Adenine and guanine nucleotides have been shown to exert multiple roles in central and peripheral nervous systems, and the sequential breakdown of these nucleotides by enzymatic systems is an important step in the modulation of their extracellular effects. The aim of this study was to investigate whether nucleotide hydrolysis also occurs in the cerebrospinal fluid (CSF) of rats. CSF was able to hydrolyze all guanine and adenine nucleotides investigated (2.0 mM): GDPz.Gt;ADP=ATP=GTPz.Gt;AMP=GMP. More detailed studies with the diphosphate nucleotides showed that the hydrolysis of ADP and GDP was linear with incubation time and protein concentration. The apparent K(M) (Henry-Michaelis-Menten constant) and V (maximal velocity) values for ADP and GDP were 164.3+/-54.7 microM and 12.2+/-3.8 nmol P(i)/min per mg protein, and 841.0+/-90.2 microM and 22.8+/-8.0 nmol P(i)/min per mg protein. The sum of ADP, GDP and UDP hydrolysis (2.0 mM) upon individual incubations with CSF was similar to the hydrolysis observed when all three nucleotides were incubated together. This pattern of hydrolysis strongly suggests the involvement of more than one enzyme activity. The higher maximum activity for GDP and UDP compared to ADP is compatible with presence of a soluble NTDPase5.

Guanine nucleotides are present in human CSF.

Several studies have shown that experimentally added guanine nucleotides (GN) may extracellularly modulate the glutamatergic system. However, there is no previous report of the extracellular occurrence of GN in the CNS. This study used HPLC to investigate the presence of GN in cerebrospinal fluid (CSF) samples of 26 patients. We demonstrated the extracellular presence of GN in the CNS. In human CSF, GMP was detected in a remarkably high concentration (236.20 microM). This evidence stimulates further investigation of extracellular GN modulation of neurotransmission, physiological mechanisms of action(s), and therapeutic potential(s) in the CNS.

ATP, ADP and AMP in plasma from peripheral venous blood.

ATP, ADP and AMP in concentrations at least 1 mumol/l have been found by high pressure liquid chromatography (HPLC) in plasma from peripheral venous blood. Total adenine nucleotide concentrations of about 15-20 mumol/l can be found in some conventional clinical samples of blood using EDTA as an anticoagulant. EDTA prevented adenine nucleotide conversion to inosine in plasma. In order to estimate concentrations in vivo, the contribution derived from the cell breakage inherent even in careful venous blood sampling has been estimated by extrapolation to zero 'haemoglobin' concentration in plasma and minimum values in samples of small volume. Available results appear to be consistent with the release of small amounts of ATP in or near the peripheral circulation at the time of venepuncture. In CSF, ATP, ADP and AMP concentrations were less than 0.05 mumol/l suggesting that membrane activity in the central nervous system is not associated with non-specific leakage. The high Km variant of lymphocyte ecto-5'-nucleotidase was not associated with a significantly higher concentration of its substrate AMP in plasma. However, this enzyme may function on the lymphocyte in the thymus and spleen.