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Purine nucleosides (adenosine) and nucleotides such as adenosine mono/di/triphosphate (AMP/ADP/ATP) may produce complex cardiovascular responses. For example, adenosine-5′-(β-thio)-diphosphate (ADPβS; a stable synthetic analogue of ADP) can induce vasodilatation/vasodepressor responses by endothelium-dependent and independent mechanisms involving purinergic P2Y receptors; however, the specific subtypes participating in these responses remain unknown. Therefore, this study investigated the receptor subtypes mediating the blood pressure changes induced by intravenous bolus of ADPβS in male Wistar rats in the absence and presence of central mechanisms with the antagonists MRS2500 (P2Y1), PSB0739 (P2Y12), and MRS2211 (P2Y13). For this purpose, 120 rats were divided into 60 anaesthetised rats and 60 pithed rats, and further subdivided into four groups (n = 30 each), namely: (a) anaesthetised rats, (b) anaesthetised rats with bilateral vagotomy, (c) pithed rats, and (d) pithed rats continuously infused (intravenously) with methoxamine (an α1-adrenergic agonist that restores systemic vascular tone). We observed, in all four groups, that the immediate decreases in diastolic blood pressure produced by ADPβS were exclusively mediated by peripheral activation of P2Y1 receptors. Nevertheless, the subsequent increases in systolic blood pressure elicited by ADPβS in pithed rats infused with methoxamine probably involved peripheral activation of P2Y1, P2Y12, and P2Y13 receptors. [ABSTRACT FROM AUTHOR]

Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

The aim: To evaluate the developmental dynamics of the hemostatic properties of platelets in rats with a low amount of antioxidants in their diet. The study took 116 healthy male Wistar rats, which during their lives contained on a standard diet, not rich in antioxidants, under normal vivarium conditions, including 23 rats of 3 months of age, 22 rats aged 6 months, 24 rats aged 12 months, 22 rats 18 months of age and 25 animals at the age of 24 months. In rats, their general state and parameters of biochemical and hematological blood parameters were determined. The results were processed by student's criterion. In the rats examined, the biochemical parameters of platelets and their activity remained stable between 3 and 6 months of life. The intensity of lipid peroxidation in the platelets of the observed rats remained normal until 6 months of age, and subsequently increased. In the blood plates of animals over the age of 6 months, the processes of self-assembly of the actin-myosin complex intensified and the content of platelets increased and the secretion of adenosine phosphates from them increased. Platelet activity in rats that are on the course of ontogenesis on the diet of not rich with antioxidants, grew older than 6 months and increased until the end of the observation-24 months. [ABSTRACT FROM AUTHOR]

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The clinical use of antineoplastic agent doxorubicin (DOX) is limited due to its cardiotoxic action. [βAla14, His15]-galanine (2–15) (G) is a novel synthetic agonist of galanin receptors GalR1-3 having cardioprotective properties in animal models in vivo. The aim of the present study was to explore effects of G on DOX-induced cardiotoxicity. Wistar rats were divided into four groups and treated with DOX (D group), DOX and G (D + G group), G (G group), and saline (control). Before treatment and at the end of the study, concentration of thiobarbituric acid reactive substances (TBARS) and activity of creatine kinase-MB (CK-MB) were determined in blood plasma, the animals were weighed, and cardiac function was evaluated by echocardiography. At the end of experiments, the hearts were used to determine energy metabolites and mitochondrial respiration in permeabilized fibers. After an 8-week study, D group exhibited a pronounced cardiac failure, the absence of weight gain, an increased plasma TBARS concentration, and CK-MB activity. These disorders were accompanied by a reduced myocardial content of high-energy phosphates and mitochondrial respiratory parameters. Co-administration of G with DOX significantly decreased plasma TBARS level and prevented an increase in plasma CK-MB activity. In D + G group, myocardial contents of ATP, PCr, total adenine nucleotides, and total creatine as well as myocardial PCr/ATP ratio and the respiratory control index were higher than in D group at the end of the experiments. Peptide G significantly improved parameters of left ventricular (LV) function and caused weight gain in animals of D + G group. These results suggest that peptide G may be a potential pharmacological agent that attenuates the cardiotoxic effects of DOX. [ABSTRACT FROM AUTHOR]

The molecular mechanisms underlying stress-induced depression have not been fully outlined. Hence, the current study aimed at testing the link between behavioral changes in chronic mild stress (CMS) model and changes in hippocampal energy metabolism and the role of paroxetine (PAROX) in ameliorating these changes. Male Wistar rats were divided into three groups: vehicle control, CMS-exposed rats, and CMS-exposed rats receiving PAROX (10 mg/kg/day intraperitoneally). Sucrose preference, open-field, and forced swimming tests were carried out. Corticosterone (CORT) was measured in serum, while adenosine triphosphate and its metabolites, cytosolic cytochrome-c (Cyt-c), caspase-3 (Casp-3), as well as nitric oxide metabolites (NOx) were measured in hippocampal tissue homogenates. CMS-exposed rats showed a decrease in sucrose preference as well as body weight compared to control, which was reversed by PAROX. The latter further ameliorated the CMS-induced elevation of CORT in serum (91.71±1.77 ng/mL vs 124.5±4.44 ng/mL, P<0.001) as well as the changes in adenosine triphosphate/adenosine diphosphate (3.76±0.02 nmol/mg protein vs 1.07±0.01 nmol/mg protein, P<0.001). Furthermore, PAROX reduced the expression of Cyt-c and Casp-3, as well as restoring NOx levels. This study highlights the role of PAROX in reversing depressive behavior associated with stress-induced apoptosis and changes in hippocampal energy metabolism in the CMS model of depression. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is the deadliest primary brain tumor in adults due to the high rate of relapse with current treatment. Therefore, the search for therapeutic alternatives is urgent. Gallic acid (GA), a potent natural antioxidant, has antitumor and modulatory actions on purinergic signaling. In this study, we investigated the cytotoxic effects of GA on the rat GBM (C6) cell line and on astrocyte culture and analyzed its role in regulating oxidative stress and purinergic enzymes involved in GBM proliferation. Cells were exposed to GA from 50 to 400 µM for 24 and/or 48 h. Next, the effect of GA was evaluated in the preclinical model of GBM. Wistar rats were treated with 50 or 100 mg/kg of GA for 15 days, and cerebral and systemic redox status and degradation of adenine nucleotides and nucleosides in circulating platelets, lymphocytes, and serum were evaluated. Our results demonstrated that GA has selective anti-glioma activity in vitro, without inducing cytotoxicity in astrocyte. Furthermore, GA prevented oxidative stress and changes in the hydrolysis of nucleotides in GBM cells. The anti-glioma effect was also observed in vivo, as GA reduced tumor volume by 90%. Interestingly, GA decreased the oxidative damage induced by a tumor in the brain, serum, and platelets, and, also prevented changes in the degradation of nucleotides and nucleosides in lymphocytes, platelets, and serum. These results indicate, for the first time, the therapeutic potential of GA in a preclinical model of GBM, whose effects may be related to its role in redox and purinergic modulation. [ABSTRACT FROM AUTHOR]

Three aspects of cardioprotection are discussed in this article. The first is myocyte death as a function of the duration and severity of ischemia in experimental acute myocardial infarction in the dog heart. The short period of time during which reperfusion with arterial blood will salvage myocytes is demonstrated along with data showing that this period diminishes significantly if collateral flow is very low or absent. The second topic is a discussion of potential mechanisms underlying postconditioning. It begins with a review of the changes that lead to irreversible injury during acute ischemia in the dog heart along with a discussion of the genesis of contraction band necrosis and no reflow when myocardium is salvaged by unrestricted reperfusion with arterial blood in order to provide a basis to discuss the potential mechanisms underlying postconditioning, a situation in which reflow is intermittent and restricted. Postconditioning is reported to achieve greater myocyte salvage than unrestricted reflow. Potential explanations for this beneficial effect include: first, sufficient sarcolemmal repair occurring during the intermittent reflow (reoxygenation) to prevent cell death by explosive cell swelling, and second, prevention of the opening of the mitochondrial permeability transition pore, thereby preventing mitochondrial failure and cell death in the reperfused tissue. Since there is no way available to identify and specifically study the myocytes that would have died if not protected by postconditioning, direct demonstration of mechanisms is difficult or impossible. Finally, the third topic in this commentary is an analysis of the obstacles faced by investigators using small rodent hearts to establish cardioprotective mechanisms. Such studies provide valid data but the relationship of the changes and the proposed mechanisms underlying these changes are not necessarily directly transferable to ischemic large animal hearts including the heart of man. [ABSTRACT FROM PUBLISHER]

Circadian rhythms represent an important mechanism to prepare the organism for environmental variations. ATP, ADP, AMP, and adenosine can act as extracellular messengers in a range of biological processes and are metabolized by a number of enzymes, including NTPDases and 5′-nucleotidase. In the present study the authors report that ATPase and ADPase activities present 24-h temporal variations that peak during dark (activity) span. These findings suggest that this enzymatic temporal pattern in blood serum might be important for the normal physiology and function of the organism through the maintenance of extracellular nucleotides at physiological levels. (Author correspondence: ) [ABSTRACT FROM AUTHOR]

Several stress conditions are characterized by activation of 5-AMP-activated protein kinase (AMPK) and the development of leucine resistance in skeletal muscle. In the present study, we determined whether direct activation of the AMPK by 5-aminoimidazole-4-carboxamide-1 -β-D-ribonucleoside (AICAR) prevents the characteristic leucine-induced increase in protein synthesis by altering mammalian target of rapamycin (mTOR) signal transduction. Rats were injected with AICAR or saline (Sal) and 1 h thereafter received an oral gavage of leucine (or Sal). Efficacy of AICAR was verified by increased AMPK phosphorylation. AICAR decreased basal in vivo muscle (gastrocnemius) protein synthesis and completely prevented the leucine-induced increase, independent of a change in muscle adenine nucleotide concentration. AICAR also prevented the hyperphosphorylation of eukaryotic initiation factor (elF) 4E binding protein (4E-BP1), ribosomal protein S6 kinase (56K1), S6, and elF4G in response to leucine, suggesting a decrease in mTOR activity. Moreover, AICAR prevented the leucine- induced redistribution of elF4E from the inactive el F4E.4E-BP1 to the active eIF4EeIF4G complex. This ability of AICAR to produce muscle leucine resistance could not be attributed to a change in phosphorylation of tuberous sclerosis complex (TSC)2, the formation of a TSC1TSC2 complex, the binding of raptor with mTOR, or the phosphorylation of eukaryotic elongation factor-2. However, the inhibitory actions of AICAR were associated with reduced phosphorylation of praline-rich Akt substrate-40 and increased phosphorylation of raptor, which represent potential mechanisms by which AICAR might be expected to inhibit leucine-induced increases in mTOR activity and protein synthesis under in viva conditions. [ABSTRACT FROM AUTHOR]

Abstract: The inhibitory effects of nucleotides and related substances on rat hepatic UDP-glucuronosyltransferase (UGT) were studied. ATP and NADP+ markedly reduced 4-methylumbelliferone (4-MU) UGT activity only when detergent-treated rat liver microsomes were used as the enzyme source. The IC50 values of adenine, ATP, NAD+ and NADP+ were estimated to be below 20 μM, whereas AMP had no inhibitory effect. From the kinetic behavior observed, these adenine-related compounds were assumed to inhibit UGT activity non-competitively without competing with either 4-MU or UDP-glucuronic acid. Among guanine, cytosine and their related nucleotides, only triphosphate nucleotides (CTP and GTP) exhibited potent UGT inhibition, although the effect of GTP was weak. Estradiol 3- and 17-glucuronidation were also inhibited by the inhibitors of 4-MU UGT. The only exception was that estradiol 17-glucuronidation activity was inhibited by AMP (IC50 =31 μM). In addition, AMP antagonized the inhibitory effects of adenine, ATP, and NADP+ on 4-MU and estradiol 3- glucuronidation activities. These results suggest that (1) a number of cellular nucleotides present within the endoplasmic reticulum regulate UGT function; and (2) these substances bind to a common allosteric site on UGT to reduce catalytic function. [Copyright &y& Elsevier]

During ischemic heart diseases and when heart failure progresses depletion of myocardial energy stores occurs. D-Ribose (R) has been shown to improve cardiac function and energy status after ischemia. Folic acid (FA) is an essential cofactor in the formation of adenine nucleotides. Therefore, we assessed whether chronic R-FA administration during the development of hypertrophy resulted in an improved cardiac function and energy status. In Wistar rats (n = 40) compensatory right ventricular (RV) hypertrophy was induced by monocrotaline (30 mg/kg; MCT), whereas saline served as control. Both groups received a daily oral dose of either 150 mg · kg-1 day dextrose (placebo) or R-FA (150 and 40 mg · kg-1 · day-1, respectively). In Langendorff-perfused hearts, RV and left ventricular (LV) pressure development and collagen content as well as total RV adenine nucleotides (TAN), creatine content, and RV and LV collagen content were determined. In the control group R-FA had no effect. In the MCT-placebo group, TAN and creatine content were reduced, RV and LV diastolic pressure-volume relations were steeper, RV systolic pressures were elevated, RV and LV collagen content was increased, and RV-LV diastolic interaction was altered compared with controls. In the MCT- R-FA group, TAN, RV and LV diastolic stiffness, RV and LV collagen content, and RV-LV diastolic interaction were normalized to the values in the control group while creatine content remained depressed and RV systolic function remained elevated. In conclusion, the depression of energy status in compensated hypertrophic myocardium observed was partly prevented by chronic R-FA administration and accompanied by a preservation of diastolic function and collagen deposition. [ABSTRACT FROM AUTHOR]

Nicotinic acid adenine dinucleotide phosphate (NAADP) is capable of inducing global Ca2+ increases via a lysosome-associated mechanism, but the mechanism mediating NAADP-induced intracellular Ca2+ release remains unclear. The present study reconstituted and characterized a lysosomal NAADP-sensitive Ca2+ release channel using purified lysosomes from rat liver. Furthermore, the identity of lysosomal NAADP-sensitive Ca2+ release channels was also investigated. It was found that NAADP activates lysosomal Ca2+ release channels at concentrations of 1 nM to 1 μM, but this activating effect of NAADP was significantly reduced when the concentrations used increased to 10 or 100 μM. Either activators or blockers of Ca2+ release channels on the sarcoplasmic reticulum (SR) had no effect on the activity of these NAADP-activated Ca2+ release channels. Interestingly, the activity of this lysosomal NAADP-sensitive Ca2+ release channel increased when the pH in cis solution decreased, but it could not be inhibited by a lysosomal H+-ATPase antagonist, bafilomycin A1. However, the activity of this channel was significantly inhibited by plasma membrane L-type Ca2+ channel blockers such as verapamil, diltiazem, and nifedipine, or the nonselective Ca2+, Na+ channel blocker, amiloride. In addition, blockade of TRP-ML1 (transient receptor potential-mucolipin 1) protein by anti-TRP-ML1 antibody markedly attenuated NAADP-induced activation of these lysosomal Ca2+ channels. These results for the first time provide direct evidence that a NAADP-sensitive Ca2+ release channel is present in the lysosome of native liver cells and that this channel is associated with TRP-ML1, which is different from ER/SR Ca2+ release channels. [ABSTRACT FROM AUTHOR]

Background and Purpose: The ATP-gated P2X(7) receptor has been shown to play a role in several inflammatory processes, making it an attractive target for anti-inflammatory drug discovery. We have recently identified a novel set of cyclic imide compounds that inhibited P2X(7) receptor-mediated dye uptake in human macrophage THP-1 cells. In this study the actions and selectivity of one of these compounds, AZ11645373, were characterized.Experimental Approach: We measured membrane currents, calcium influx, and YOPRO-1 uptake from HEK cells expressing individual P2X receptors, and YOPRO1 uptake and interleukin-1beta release from THP-1 cells in response to ATP and the ATP analogue benzoylbenzoyl ATP (BzATP).Key Results: AZ11645373 up to 10 microM, had no agonist or antagonist actions on membrane currents due to P2X receptor activation at human P2X(1), rat P2X(2), human P2X(3), rat P2X(2/3), human P2X(4), or human P2X(5) receptors expressed in HEK cells. AZ11645373 inhibited human P2X(7) receptor responses in HEK cells in a non-surmountable manner with K (B) values ranging from 5 - 20 nM, with mean values not significantly different between assays. K (B) values were not altered by removing extracellular calcium and magnesium. ATP-evoked IL-1beta release from lipopolysaccharide-activated THP-1 cells was inhibited by AZ11645373, IC(50) = 90 nM. AZ11645373 was > 500-fold less effective at inhibiting rat P2X(7) receptor-mediated currents with less than 50% inhibition occurring at 10 microM.Conclusions and Implications: AZ11645373 is a highly selective and potent antagonist at human but not rat P2X(7) receptors and will have much practical value in studies of human cells. [ABSTRACT FROM AUTHOR]

The protective effect of ischemic preconditioning (IPC) has been reported on improvement of survival, reduction of liver necrosis and enhancement of the regenerative capacity of hepatocytes after partial hepatectomy. This study was undertaken to confirm that IPC has a significant impact on regeneration of hepatocytes after partial hepatectomy in ischemically damaged liver. In addition, we sought to examine the role of adenine nucleotides in this process. Methods: Wistar rats were subjected to 60 min of total hepatic ischemia, followed by 70% hepatectomy. The animals were subdivided into an IPC (10/15 min) group and a non-IPC (control) group. Liver function tests and arginase activity were analyzed. Hepatic adenosine triphosphate (ATP), adenosine diphosphate and adenosine monophosphate were measured using gradient high-performance liquid chromatography. The liver regeneration was identified using relative liver weight and proliferating cell nuclear antigen (PCNA) labeling index. Results: IPC treatment improved serum liver enzymes and tissue arginase activity ( P<0.05) when compared with the control group. The preconditioned livers were associated with upregulation of ATP expression and also increased tissue energy charge. Regenerated liver weight in the IPC group was significantly higher than in the control group ( P<0.05). The PCNA labeling index in the remnant livers in the IPC group was also significantly increased at 24 and 48 h after partial hepatectomy ( P<0.05). Conclusion: These results suggest that IPC-augmented liver regeneration after hepatectomy, probably due to the stabilization of energy metabolism in rats. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We exposed cultured neurons prelabeled with 14C-adenine to H2O2 with or without the poly(ADP-ribose) polymerase (PARP) inhibitor 3,4-Dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ) to quantify its effects on acute ATP depletion, later ATP synthesis, cellular and nuclear morphology, extent of DNA fragmentation, and PARP cleavage. According to the extent of the acute ATP depletion, the exposures were classified as ‘mild’ (50 μM H2O2), ‘moderate’ (100–250 μM H2O2), or ‘severe’ (500 μM–1 mM H2O2) insults. Mild exposure had no significant effects on the parameters studied. In the ‘moderately’ exposed neurons, ATP depletion to 59±6% of control was associated with a decrease in the cell counts, apoptotic morphology, and cleavage of PARP. In this group, DPQ prevented the acute ATP (to 95±15% of control), preserved cell morphology, and improved cell survival. In the ‘severe’ group, ATP depletion to 18±4% was associated with necrosis and intact PARP. DPQ elevated ATP levels (to 44±12% of control) and post-insult ATP synthesis, improved cell counts, and altered cell morphology towards apoptosis rather than necrosis. Post-insult application of DPQ was less effective. Our results show that the extent of oxidant-induced ATP depletion and cell fate can be modified by PARP inhibition, to some extent also after the insult. [Copyright &y& Elsevier]

In this study, we investigated the influence of intracellular cyclic adenosine monophosphate (cAMP) changes on the rat mast cell hyporesponsiveness following immunological and non-immunological stimuli. Compared with mast cells from normal rats, those recovered from 21-day diabetic animals showed a significant augmentation in the intracellular levels of cAMP, in directly correlated with secretion of lower amounts of histamine after stimulation with antigen, bradykinin and compound 48/80 in vitro. Incubation of normal mast cells with selective inhibitors of phosphodiesterase type 4 (PDE 4) rolipram, NCS 613 and RP 73401, or the cell permeable analogue N6-2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate (db cAMP), led to a decrease of histamine secretion in vitro. However, the effectiveness of either NCS 613 or db cAMP in inhibiting antigen-induced degranulation is comparable in both normal and diabetic mast cells. We suggest that (a) there is a close correlation between higher levels of intracellular cAMP and hyporesponsiveness of diabetic mast cells, phenomena probably associated with a reduction in the expression and/or activity of PDE 4 and that (b) the mechanism of cAMP-mediated down-regulation of mast cell function is saturated in diabetic rats. [Copyright &y& Elsevier]

The hypothesis that an increase in intracellular calcium [Ca2+]c in carotid body (CB) glomus cells will cause enhanced afferent carotid sinus nerve (CSN) activities was tested in the rat CB in-vitro with the use of extracellular ATP. ATP caused a dose dependent [Ca2+]c increase in identified glomus cells. A major part of total [Ca2+]c increase (2/3) was due to the [Ca2+] influx. The rest of [Ca2+]c increase (1/3) was due to the release of [Ca2+] from the endoplasmic reticulum (ER) [Ca2+] stores, and it was inhibited by the pretreatment of cells with cyclopiazonic acid (CPA), an intracellular Ca2+-ATPase blocker. Suramin, a purinergic P2 receptor membrane blocker, blocked [Ca2+] influx due to ATP in the presence of extracellular [Ca2+]. Perfusion with 5 and 10 μM ATP stimulated CSN activities in both normoxia (Nx) and hypoxia (Hx). Above that level, 100 μM ATP induced slight initial stimulation in CSN activities which were subsided subsequently in Nx and partly diminished in Hx, while 500 μM ATP completely inhibited CSN activities in Nx and Hx after a slight initial stimulation. Electrophysiological measurements of the glomus cell membrane potential in the presence of ATP (100 μM) during Nx indicated cellular enhanced outward K+ current and hyperpolarization, suggesting potential mechanism for the inhibition of CSN activities. Thus, ATP dependent linear increases in [Ca2+]c did not give rise to a corresponding increase in CSN activities, contravening the normally expected increase in CSN activities following [Ca2+]c rise. [Copyright &y& Elsevier]

The antiestrogen tamoxifen (TAM) inhibits the growth of different estrogen receptor (ER)-negative cells. Recently, multiple effects of TAM on mitochondrial bioenergetic functions have been pointed to explain its ER-independent cell death mechanisms. We have shown that TAM and its major active metabolite 4-hydroxytamoxifen (OHTAM) induce depolarization of the mitochondrial membrane potential (ΔΨ) and uncouple the mitochondrial respiration, depressing the oxidative phosphorylation efficiency. To clarify the biochemical mechanisms underlying the changes in the regulation of ATP synthesis and yield, in this work we evaluated the alterations of mitochondrial adenine nucleotides induced by both drugs and ascertained whether such changes could reflect a specific inhibition of either the adenine nucleotide translocase (ANT) or the phosphate carrier, as well as the activation of ATP hydrolysis due to ΔΨ depolarization. We found that both antiestrogens caused a concentration-dependent decrease in mitochondrial ATP levels. Mitochondrial ADP and AMP were concomitantly increased with a subsequent decrease in the ATP/ADP or ATP/AMP ratios. The total concentration of adenine nucleotides also changed. Additionally, both drugs decreased the ANT content of mitochondria, inhibited the phosphate carrier and induced ATP hydrolysis. However, the effects of TAM were more drastic than those induced by OHTAM. Therefore, the depletion of ATP might result from an activation of ATP catabolism, as well as from a decrease in the mitochondrial content of ANT and partial inhibition of the phosphate carrier. Our data may explain the ER-independent effects and cytotoxicity of both drugs and, in agreement with other previous studies, suggest that OHTAM is much less toxic to mitochondria than TAM. [Copyright &y& Elsevier]

Studies the structures of beta-methylenethiazole-4-carboxamide adenine dinucleotide, NAD[sup +] and NADH as bound to ecto-ADP-ribosyltransferase 2.2 from rat and to its mutants E189I and E189A respectively. Positions and conformations of NAD[sup +] and its analogues that agree in general with those in other ADP-ribosyltransferases; Analysis of pertinent topics and relevant issues; Implications on biochemistry.

We have previously observed that adenosine 5′-diphosphate (ADP) hydrolysis was decreased 25% in spinal cord synaptosomes of chronically stressed male rats, while no changes were observed in ATPase activity. In the present study, we investigated the effect of chronic stress on the hydrolysis of adenine nucleotides in two cerebral structures (frontal cortex and hypothalamus) and in the blood serum of male rats. Adult male Wistar rats were submitted to 1-h restraint stress/day for 45 days (chronic) and were sacrificed 24 h after the last session of stress. Adenosine 5′-triphosphate (ATP) or ADP hydrolysis was assayed in the synaptosomal fraction obtained from the frontal cortex and hypothalamus of control and chronically stressed animals. No effects on ADP or ATP hydrolysis were observed in any of the cerebral structures analyzed after chronic stress. On the other hand, reduced ADP hydrolysis was observed in the blood serum of chronic stressed rats. It is possible that the effects observed in the blood serum may represent an adaptation to chronic stress and may reflect different functions of nucleotides and/or enzymes in these tissues. It is possible that altered levels of ADPase activity in the serum may be a biochemical marker for chronic stress situations. [Copyright &y& Elsevier]

In the brain, the levels of adenosine increase up to 100-fold during cerebral ischernia; however, the roles of specific cell types, enzymatic pathways and membrane transport processes in regulating intra- and extracellular concentrations of adenosine are poorly characterized. Rat primary cortical neurons and astrocytes were incubated with [3H]adenine for 30 min to radiolabel intracellular ATP. Cells were then treated with buffer, glucose deprivation (GD), oxygen–glucose deprivation (OGD), 100 μM sodium cyanide (NaCN) or 500 μM iodoacetate (IAA) for 1 h to stimulate the metabolism of ATP and cellular release of [3H]purines. The nucleoside transport inhibitor dipyridamole (DPR) (10 μM), the adenosine kinase inhibitor iodotubercidin (ITU) (1 μM), the adenosine deaminase inhibitor EHNA (1 μM) and the purine nucleoside phosphorylase inhibitor BCX-34 (10 μM) were tested to investigate the contribution of specific enzymes and transporters in the metabolism and release of purines from each cell type. Our results indicate that (a) under basal conditions astrocytes released significantly more [3H]adenine nucleotides and [3H]adenosine than neurons, (b) OGD, NaCN and IAA conditions produced significant increases in [3H]adenosine release from neurons but not astrocytes, and (c) DPR blocked [3H]inosine release from both astrocytes and neurons but only blocked [3H]adenosine release from neurons. These data suggest that, in these experimental conditions, adenosine was formed by an intracellular pathway in neurons and then released via a nucleoside transporter. In contrast, adenine nucleotide release and extracellular metabolism to adenosine appeared to predominate in astrocytes. [Copyright &y& Elsevier]

The article investigates: the cytotoxic effects of &b.beta;-thujaplicin on rat immature thymocytes and the preventive effects of &b.beta;-thujaplicin on tributyltin-induced thymocyte damage. The incubation of thymocytes with &b.beta; -thujaplicin caused a concentration- and time-dependent cell killing accompanied by the loss of intracellular adenosine triphosphate (ATP). The pretreatment of the thymocyte suspension with EDTA, a hydrophilic chelator, enhanced the cytotoxicity of &b.beta; -thujapiicin at concentrations of 1 and 2 mM, accompanied by the depletion of intracellular levels of ATP.

The interstitial accumulation of adenine nucleotide breakdown products (ANBP) in the myocardium during ischemia has been shown to provide a useful index of the ischemic injury, whereas reperfusion ANBP washout rate has been regarded as an index of reperfusion damage. The purpose of this study was, using cardiac microdialysis, to examine in the rat model of regional ischemia/reperfusion the relationship between the duration of ischemia and these indices and to assess the profile of interstitial ATP concentrations and the beneficial effects of ischemic preconditioning (IP). The rats underwent 10, 20, 30 or 40 min of coronary artery occlusion and 50 min of reperfusion. Regional ischemia, with its duration, provoked a progressive increase in dialysate ANBP in the ischemic zone. The rate of purine washout during reperfusion exponentially declined with an increase in duration of the ischemic period. IP, induced by three 5-min episodes of ischemia, each separated by 5 min of reperfusion, significantly reduced the accumulation of ANBP during the 30-min period of sustained ischemia and resulted in a marked acceleration of reperfusion ANBP washout, indicating the improvement of postischemic microcirculation. These effects were suggested to be, at least in part, responsible for the infarct size limitation observed. Using the relationship between the duration of ischemia and ANBP washout rate, it could be demonstrated that IP produced similar facilitation of purine washout as shortening of the ischemic period in nonpreconditioned rats from 30 to approximately 7 min. Regional 20-min ischemia induced an early peak increase in interstitial fluid ATP which correlated with the maximal incidence of ventricular arrhythmias, whereas IP abolished both ATP release and arrhythmias during the sustained ischemia. These findings suggest that ATP may be an important mediator of ischemia-induced ventricular arrhythmias. [ABSTRACT FROM AUTHOR]

Presents a study in which a cardiac mycrodialysis technique was used to examine adenine nucleotide breakdown in in situ rat hearts. How cardiac mycrodialysis was performed in the rats; Technique used for dialysate adenosinetriphosphatase (ATP) measurement; Analysis of the dialysate samples; In-depth look at the study.

Examines the temporal relationships between eating behavior and liver adenine nucleotides in rats treated with 2,5-AM. Information on the eating response to the 2,5-AM.

The control exerted by the adenine nucleotide translocator and the ADP-regenerating system on oxidative phosphorylation was studied in isolated rat-liver mitochondria respiring with succinate. At intermediate rates of respiration the flux control coefficient (control strength) of the adenine nucleotide translocator on respiration was much higher with creatine/creatine kinase than with glucose/hexokinase as the ADP-regenerating system. In contrast, at the same rate of respiration the flux control coefficient of creatine kinase was much lower than that of hexokinase. On addition of a small amount of carboxyatractyloside to mitochondria respiring in the presence of glucose/hexokinase or creatine/creatine kinase, the rate of respiration decreased abruptly and then increased again to a new steady state which was lower with creatine/creatine kinase than with glucose/hexokinase. In the new steady state, the extramitochondrial ATP/ADP ratio was lower with glucose/hexokinase than with creatine/creatine kinase. At the same rate of respiration, the elasticity coefficient of creatine kinase towards the extramitochondrial ATP/ADP ratio was much higher than that of hexokinase. The connectivity theorem [Kacser, H. and Burns, J. A. (1973) in Rate Control of Biological Processes (Davies, D. D., ed.) pp. 65–104, Cambridge University Press, London] which relates the flux control coefficients of two adjacent enzymes to their elasticity coefficients towards the common intermediate, provides an explanation for the difference in flux control coefficient of the adenine nucleotide translocator with the two ADP-regenerating systems. Using principles developed by R. Heinrich and T. A. Rapoport [BioSystems 7, 130–136 (1975)], the flux control coefficients of the adenine nucleotide translocator and hexokinase on phosphorylation were also calculated from the elasticity coefficients of these enzymes towards the extramitochondrial ATP/ADP ratio and the controls exerted by these enzymes on the extramitochondrial ATP/ADP ratio. The calculated values were approximately 30% lower than the values measured directly. The elasticity coefficient of the adenine nucleotide translocator towards the extramitochondrial ATP/ADP ratio was determined, using either the connectivity theorem of Kacser and Burns (loc. cit.) or a new connectivity theorem developed by Westerhoff( following paper in this journal). Good agreement was obtained using the different methods of calculation. It is concluded that flux through the adenine nucleotide translocator, although in principle dependent on ([ATP]/[ADPDout, ([ATP/[ADP])in and Δψ, is regulated primarily by ([ATP]/[ADP])out, under the conditions used in this study. [ABSTRACT FROM AUTHOR]

Rat mast cell granules were obtained by sonication of highly purified rat mast cells and isolated in a Percoll gradient. Phosphorylation of endogenous phosphatidylinositol in rat mast cell granules, which is catalysed by phosphatidylinositol kinase in the granules, was assayed by measuring the incorporation of sap from [γ32P] ATP into phosphatidylinositol 4-phosphate. Lipids were isolated with methanol/chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. Phosphatidylino- sitol 4-phosphate areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The phosphorylation reaction was inhibited by 50–500 μM adenosine, ADP and 500 μM AMP in a concentration-dependent manner. Among several anti- allergic drugs investigated, 100–1000 μM theophylline and 10–100 μM azelastine inhibited the phosphorylation reaction, but disodium cromoglycate and ketotifen had little effect. [ABSTRACT FROM AUTHOR]