Deep brain stimulation of the anterior thalamus attenuates PTZ kindling with concomitant reduction of adenosine kinase expression in rats.

BACKGROUND: Deep brain stimulation (DBS) of the anterior nucleus of the thalamus (ANT) is an emerging therapy to provide seizure control in patients with refractory epilepsy, although its therapeutic mechanisms remain elusive. OBJECTIVE: We tested the hypothesis that ANT-DBS might interfere with the kindling process using three experimental groups: PTZ, DBS-ON and DBS-OFF. METHODS: 79 male rats were used in two experiments and exposed to chemical kindling with pentylenetetrazole (PTZ, 30 mg/kg i.p.), delivered three times a week for a total of 18 kindling days (KD). These animals were divided into two sets of three groups: PTZ (n = 26), DBS-ON (n = 28) and DBS-OFF (n = 25). ANT-DBS (130 Hz, 90 µs, and 200 µA) was paired with PTZ injections, while DBS-OFF group, although implanted remained unstimulated. After KD 18, the first set of PTZ-treated animals and an additional group of 11 naïve rats were euthanized for brain extraction to study adenosine kinase (ADK) expression. To observe possible long-lasting effects of ANT stimulation, the second set of animals underwent a 1-week treatment and stimulation-free period after KD 18 before a final PTZ challenge. RESULTS: ANT-DBS markedly attenuated kindling progression in the DBS-ON group, which developed seizure scores of 2.4 on KD 13, whereas equivalent seizure scores were reached in the DBS-OFF and PTZ groups as early as KD5 and KD6, respectively. The incidence of animals with generalized seizures following 3 consecutive PTZ injections was 94%, 74% and 21% in PTZ, DBS-OFF and DBS-ON groups, respectively. Seizure scores triggered by a PTZ challenge one week after cessation of stimulation revealed lasting suppression of seizure scores in the DBS-ON group (2.7 ± 0.2) compared to scores of 4.5 ± 0.1 for the PTZ group and 4.3 ± 0.1 for the DBS-OFF group (P = 0.0001). While ANT-DBS protected hippocampal cells, the expression of ADK was decreased in the DBS-ON group compared to both PTZ (P < 0.01) and naïve animals (P < 0.01). CONCLUSIONS: Our study demonstrates that ANT-DBS interferes with the kindling process and reduced seizure activity was maintained after a stimulation free period of one week. Our findings suggest that ANT-DBS might have additional therapeutic benefits to attenuate seizure progression in epilepsy.

AICAR promotes endothelium-independent vasorelaxation by activating AMP-activated protein kinase via increased ZMP and decreased ATP/ADP ratio in aortic smooth muscle.

OBJECTIVES: AICAR, an adenosine analog, has been shown to exhibit vascular protective effects through activation of AMP-activated protein kinase (AMPK). However, it remains unclear as to whether adenosine kinase-mediated ZMP formation or adenosine receptor activation contributes to AICAR-mediated AMPK activation and/or vasorelaxant response in vascular smooth muscle. METHODS AND RESULTS: In the present study using endothelium-denuded rat aortic ring preparations, isometric tension measurements revealed that exposure to 1 mM AICAR for 30 min resulted in inhibition of phenylephrine (1 µM)-induced smooth muscle contractility by ∼35%. Importantly, this vasorelaxant response by AICAR was prevented after pretreatment of aortic rings with an AMPK inhibitor (compound C, 40 µM) and adenosine kinase inhibitor (5-iodotubercidin, 1 µM), but not with an adenosine receptor blocker (8-sulfophenyltheophylline, 100 µM). Immunoblot analysis of respective aortic tissues showed that AMPK activation seen during vasorelaxant response by AICAR was abolished by compound C and 5-iodotubercidin, but not by 8-sulfophenyltheophylline, suggesting ZMP involvement in AMPK activation. Furthermore, LC-MS/MS MRM analysis revealed that exposure of aortic smooth muscle cells to 1 mM AICAR for 30 min enhanced ZMP level to 2014.9 ± 179.4 picomoles/mg protein (vs. control value of 8.5 ± 0.6; p<0.01), which was accompanied by a significant decrease in ATP/ADP ratio (1.08 ± 0.02 vs. 2.08 ± 0.06; p<0.01). CONCLUSIONS: Together, the present findings demonstrate that AICAR-mediated ZMP elevation and the resultant AMPK activation in vascular smooth muscle contribute to vasorelaxation.

Adenosine kinase inhibition selectively promotes rodent and porcine islet ß-cell replication.

Diabetes is a pathological condition characterized by relative insulin deficiency, persistent hyperglycemia, and, consequently, diffuse micro- and macrovascular disease. One therapeutic strategy is to amplify insulin-secretion capacity by increasing the number of the insulin-producing ß cells without triggering a generalized proliferative response. Here, we present the development of a small-molecule screening platform for the identification of molecules that increase ß-cell replication. Using this platform, we identify a class of compounds [adenosine kinase inhibitors (ADK-Is)] that promote replication of primary ß cells in three species (mouse, rat, and pig). Furthermore, the replication effect of ADK-Is is cell type-selective: treatment of islet cell cultures with ADK-Is increases replication of ß cells but not that of α cells, PP cells, or fibroblasts. Short-term in vivo treatment with an ADK-I also increases ß-cell replication but not exocrine cell or hepatocyte replication. Therefore, we propose ADK inhibition as a strategy for the treatment of diabetes.

Differential effect of insulin and elevated glucose level on adenosine handling in rat T lymphocytes.

Reduced proliferation potential is among other T cell functional defects long known feature of diabetes. However, the mechanism responsible for this impairment is still unknown. Our study was undertaken to investigate the effect of changes in glucose and insulin concentrations on adenosine metabolism, transport and receptor-mediated action in rat T lymphocytes. Presented results indicate that vulnerability of T cells to metabolic stress is determined by insulin but not by glucose concentration. However, glucose and insulin differentially affected the activities of adenosine metabolizing enzymes in resting and proliferating T cells. The Con A-induced proliferation of cultured T lymphocytes did not depended on expression level and functional state of nucleoside transporters. Inhibition of adenosine kinase (AK) with 5-iodotubercidin lowers the proliferation potential of T cells to the level observed for insulin-deprived cells. Moreover, insulin-deprived T lymphocytes but not cells cultured in the presence of insulin released significant quantities of adenosine. Under resting conditions, the cAMP level was fivefold higher in cells deprived of insulin comparing to cells cultured in the presence of insulin. Exposition of insulin-deprived T lymphocytes to specific antagonist (ZM241385) of A2a receptor but not to specific antagonist (Alloxazine) of A2b receptor suppressed cAMP elevation and completely restored the proliferation potential of T cells. Concluding, adenosine released by insulin-deprived T cells due to suppressed AK activity by acting on A2a receptors leads to increases in cAMP level and suppression of T cell proliferation. We assume that this mechanism may significantly contribute to immune impairment observed in diabetes.

Enzyme activities controlling adenosine levels in normal and neoplastic tissues.

Adenosine is known to be associated with effects such as inhibition of immune response, coronary vasodilation, stimulation of angiogenesis, and inhibition of inflammatory reactions. Some authors suggest that adenosine may also have similar functions in tumor tissues. Tissue levels of adenosine are under close regulation by different enzymes acting at different levels. Adenosine is produced from AMP by the action of 5'-nucleotidase (5'-NT) and is converted back into AMP by adenosine kinase (AK) or into inosine by adenosine deaminase (ADA). Inosine is converted into purine catabolites by purine nucleoside phosphorylase (PNP), whereas AMP is converted into ADP and ATP by adenylate kinase (MK). The aim of this study was to analyze the activities of the above enzymes in fragments of neoplastic and apparently normal mucosa, obtained less than 5 cm and at least 10 cm from tumors, in 40 patients with colorectal cancer. The results showed much higher activities of ADA, AK, 5'-NT, and PNP in tumor tissue than in neighboring mucosa (p > 0.01 for ADA, AK, and PNP; p > 0.05 for 5'-NT), suggesting that the activities of purine metabolizing enzymes increase to cope with accelerated purine metabolism in cancerous tissue. The simultaneous increase in ADA and 5'-NT activities might be a physiological attempt by cancer cells to provide more substrate to accelerate salvage pathway activity.

Effect of adenosine kinase, adenosine deaminase and transport inhibitors on striatal dopamine and stereotypy after methamphetamine administration.

The effect of adenosine kinase (AKA), adenosine deaminase (ADA) and transport inhibitors on the release of dopamine (DA) induced by methamphetamine (MTH) in rat striatum was assessed using in vivo microdialysis in freely moving rats. MTH injected in a dose of 3 x 5 mg/kg i.p. at 2-hour intervals produced a massive release of DA. This excessive release of DA was inhibited by the ADA inhibitor 2'-deoxycoformycin (DCF), the AKA inhibitor 5'-iodotubercidin (IOT) and the adenosine uptake inhibitor dilazep (DIL), each of them given locally to the striatum via a microdialysis probe at a concentration of 100 microM. Perfusion with the same concentrations of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and 5'-amino-5'-deoxyadenosine (NH(2)dAD), ADA and AKA inhibitors, respectively, induced a considerably weaker effect on DA release. The non-selective antagonist of adenosine A(1)/A(2A) receptor caffeine (75 microM) significantly prevented the inhibitory effect of DCF, IOT and DIL on the MTH-induced DA release. Intrastriatal administration of DCF, IOT and DIL (5 nmol/microl before each injection of MTH) inhibited the stereotypy induced by MTH. The striatal content of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), decreased by MTH administration and measured 5 days after treatment with the toxin, was reversed by all the inhibitors at the order of potency as follows: IOT>DCF>DIL. Direct agonists of adenosine A(1) and A(1)/A(2A) receptors, N(6)-cyclopentyladenosine (CPA) and 5'-N-ethylcarboxamidoadenosine (NECA), respectively, given intrastriatally (5 nmol/microl) completely abolished the MTH-induced stereotypy and the fall in the striatal content of DA, DOPAC and HVA. The above results show that augmentation of endogenous adenosine in rat striatum by inhibition of its metabolism or uptake-despite the differences in the efficacy of various inhibitors-may provide neuroprotection against a toxic action of MTH.

Inhibition of phosphoribosylpyrophosphate synthetase by 4-methoxy-(MRPP) and 4-amino-8-(D-ribofuranosylamino) pyrimido[5,4-d]pyrimidine (ARPP).

The basis for the antitumor activities of the exocyclic amino nucleosides 4-amino-(ARPP) and 4-methoxy-8-(D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (MRPP) was investigated. The primary target of these nucleosides appeared to be 5-phospho-alpha-D-ribofuranose-1-pyrophosphate (PRPP) synthetase. MRPP-5'-monophosphate was a competitive inhibitor (Ki = 40 microM) of the activation of this enzyme by the cofactor inorganic phosphate (K alpha = 2.2 mM). Consequently, ARPP and MRPP treatment of WI-L2 cultures rapidly inhibited both de novo pyrimidine and purine synthesis as well as the nucleotide salvage reactions dependent on PRPP, ARPP or MRPP treatment completely prevented [14C]bicarbonate incorporation into acid-soluble pyrimidine and purine nucleotides. The rate of salvage of [8-14C]hypoxanthine to form IMP was decreased by 85%. Treatment of cells with these agents caused a 50% reduction in the steady-state level of PRPP. When the capacity of the treated cells for sustained synthesis of PRPP was examined by adenine incorporation, the rate of adenine uptake was inhibited by greater than 50%. In vivo treatment of BDF1 mice with a single dose of ARPP (173 mg/kg) or MRPP (62 mg/kg) extended the mean life span of the mice, which had been inoculated intraperitoneally 1 day earlier with 1 x 10(6) L1210 murine leukemia cells, by 62 and 82% respectively. These studies indicate that MRPP and ARPP inhibit PRPP synthetase, and that PRPP synthetase may be a viable target in the development of certain antitumor agents.

Metabolism and action of neplanocin A in Chinese hamster ovary cells.

Neplanocin A is a naturally occurring carbocyclic analog of adenosine which contains a cyclopentene moiety in place of ribose and has demonstrated antitumor and antimicrobial activity. This compound was highly toxic to Chinese hamster ovary (CHO) cells; the approximate minimum inhibitory concentration of neplanocin A for inhibition of clone formation was 0.1 microM. The toxicity of the agent was greatly reduced by prior treatment with adenosine deaminase. [3H]Uridine incorporation into perchloric acid insoluble material in growing cells was inhibited by neplanocin A more dramatically than that of [3H]thymidine or [3H]leucine. Treatment with the drug resulted in a marked depression of ATP pool levels. High pressure liquid chromatographic analysis of cellular nucleotide pools from cells treated with neplanocin A revealed the formation of an apparent drug metabolite (NpcTP) that eluted in the triphosphate region of the chromatographic profile. Treatment of NpcTP with alkaline phosphatase produced a nucleoside with properties similar to neplanocin A. An adenosine-kinase-deficient cell line formed little, if any, NpcTP but demonstrated only slight resistance to the agent. These observations suggest that neplanocin A was efficiently metabolized to the triphosphate level but that this metabolite was responsible for only a fraction of the observed toxicity.