A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes

Cyclic adenosine monophosphate (cAMP) has been implicated as an important regulator of meiotic maturation in mammalian oocytes. A decrease in cAMP, brought about by the action of cAMP phosphodiesterase (PDE), is thought to initiate germinal vesicle breakdown (GVB) by the inactivation of cAMP-dependent protein kinase. However, the product of PDE activity, 5'-AMP, is a potent activator of an important regulatory enzyme, AMP-activated protein kinase (AMPK). The aim of this study was to evaluate a possible role for AMPK in meiotic induction, using oocytes obtained from eCG-primed, immature mice. Alpha-1 and -2 isoforms of the catalytic subunit of AMPK were detected in both oocytes and cumulus cells. When 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICA riboside), an activator of AMPK, was tested on denuded oocytes (DO) and cumulus cell-enclosed oocytes (CEO) maintained in meiotic arrest by dbcAMP or hypoxanthine, GVB was dose-dependently induced. Meiotic induction by AICA riboside in dbcAMP-supplemented medium was initiated within 3 h in DO and 4 h in CEO and was accompanied by increased AMPK activity in the oocyte. AICA riboside also triggered GVB when meiotic arrest was maintained with hypoxanthine, 8-AHA-cAMP, guanosine, or milrinone, but was ineffective in olomoucine- or roscovitine-arrested oocytes, indicating that it acts upstream of maturation-promoting factor. Adenosine monophosphate dose-dependently stimulated GVB in DO when meiotic arrest was maintained with dbcAMP or hypoxanthine. This effect was not mimicked by other monophosphate or adenosine nucleotides and was not affected by inhibitors of ectophosphatases. Combined treatment with adenosine and deoxycoformycin, an adenosine deaminase inhibitor, stimulated GVB in dbcAMP-arrested CEO, suggesting AMPK activation due to AMP accumulation. It is concluded that phosphodiesterase-generated AMP may serve as a transducer of the meiotic induction process through activation of AMPK.     

AMP-activated protein kinase-independent inhibition of hepatic mitochondrial oxidative phosphorylation by AICA riboside

AICA riboside (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside) has been extensively used in cells to activate the AMPK (AMP-activated protein kinase), a metabolic sensor involved in cell energy homoeostasis. In the present study, we investigated the effects of AICA riboside on mitochondrial oxidative; phosphorylation. AICA riboside was found to dose-dependently inhibit the oligomycin-sensitive JO2 (oxygen consumption rate) of isolated rat hepatocytes. A decrease in P(i) (inorganic phosphate), ATP, AMP and total adenine nucleotide contents was also observed with AICA riboside concentrations >0.1 mM. Interestingly, in hepatocytes from mice lacking both alpha1 and alpha2 AMPK catalytic subunits, basal JO2 and expression of several mitochondrial proteins were significantly reduced compared with wild-type mice, suggesting that mitochondrial biogenesis was perturbed. However, inhibition of JO2 by AICA riboside was still present in the mutant mice and thus was clearly not mediated by AMPK. In permeabilized hepatocytes, this inhibition was no longer evident, suggesting that it could be due to intracellular accumulation of Z nucleotides and/or loss of adenine nucleotides and P(i). ZMP did indeed inhibit respiration in isolated rat mitochondria through a direct effect on the respiratory-chain complex I. In addition, inhibition of JO2 by AICA riboside was also potentiated in cells incubated with fructose to deplete adenine nucleotides and P(i). We conclude that AICA riboside inhibits cellular respiration by an AMPK-independent mechanism that likely results from the combined intracellular P(i) depletion and ZMP accumulation. Our data also demonstrate that the cellular effects of AICA riboside are not necessarily caused by AMPK activation and that their interpretation should be taken with caution.     

Protein kinase inhibitors block the stimulation of the AMP-activated protein kinase by 5-amino-4-imidazolecarboxamide riboside

The AMP-activated protein kinase (AMPK) is the central component of a protein kinase cascade that plays a major role in energy sensing. AMPK is activated pharmacologically by 5-amino-4-imidazolecarboxamide (AICA) riboside monophosphate (ZMP), which mimics the effects of AMP on the AMPK cascade. Here we show that uptake of AICA riboside into cells, mediated by the adenosine transport system, is blocked by a number of protein kinase inhibitors. Under these conditions, ZMP does not accumulate to sufficient levels to stimulate AMPK. Our results demonstrate that careful interpretation is required when using AICA riboside in conjunction with protein kinase inhibitors to investigate the physiological role of AMPK.     

5 '-Amino-4-imidazolecarboxamide riboside induces apoptosis in human neuroblastoma cells via the mitochondrial pathway

5'-Amino-4-imidazolecarboxamide (AICA) riboside induces apoptosis in neuronal cell models. In order to exert its effect, AICA riboside must enter the cell and be phosphorylated to the ribotide. In the present work, we have further studied the mechanism of apoptosis induced by AICA riboside. The results demonstrate that AICA riboside activates AMP-dependent protein kinase (AMPK), induces release of cytochrome c from mitochondria and activation of caspase 9. The role of AMPK in determining cell fate is controversial. In fact, AICA riboside has been reported to be neuroprotective or to induce apoptosis depending on its concentration, cell type or apoptotic stimuli used. In order to clarify whether the activation of AMPK is related to apoptosis in our model, we have used another AMPK stimulator, metformin, and we have analysed its effects on cell viability, nuclear morphology and AMPK activity. Five mM metformin increased AMPK activity, inhibited viability, and increased the number of apoptotic nuclei. AICA riboside, which can be generated from the ribotide (an intermediate of the purine de novo synthesis) by the action of the ubiquitous cytosolic 5'-nucleotidase (cN-II), may accumulate in those individuals in which an inborn error of purine metabolism causes both a building up of intermediates and/or an increase of the rate of de novo synthesis, and/or an overexpression of cN-II. Therefore, our results suggest that the toxic effect of AICA riboside on some types of neurons may participate in the neurological manifestations of syndromes related to purine dismetabolisms.     

5 ′-Amino-4-Imidazolecarboxamide Riboside Induces Apoptosis in Human Neuroblastoma Cells Via the Mitochondrial Pathway

5′-Amino-4-imidazolecarboxamide (AICA) riboside induces apoptosis in neuronal cell models. In order to exert its effect, AICA riboside must enter the cell and be phosphorylated to the ribotide. In the present work, we have further studied the mechanism of apoptosis induced by AICA riboside. The results demonstrate that AICA riboside activates AMP-dependent protein kinase (AMPK), induces release of cytochrome c from mitochondria and activation of caspase 9. The role of AMPK in determining cell fate is controversial. In fact, AICA riboside has been reported to be neuroprotective or to induce apoptosis depending on its concentration, cell type or apoptotic stimuli used. In order to clarify whether the activation of AMPK is related to apoptosis in our model, we have used another AMPK stimulator, metformin, and we have analysed its effects on cell viability, nuclear morphology and AMPK activity. Five mM metformin increased AMPK activity, inhibited viability, and increased the number of apoptotic nuclei. AICA riboside, which can be generated from the ribotide (an intermediate of the purine de novo synthesis) by the action of the ubiquitous cytosolic 5′-nucleotidase (cN-II), may accumulate in those individuals in which an inborn error of purine metabolism causes both a building up of intermediates and/or an increase of the rate of de novo synthesis, and/or an overexpression of cN-II. Therefore, our results suggest that the toxic effect of AICA riboside on some types of neurons may participate in the neurological manifestations of syndromes related to purine dismetabolisms.     

Purine uptake and release in rat C6 glioma cells: nucleoside transport and purine metabolism under ATP-depleting conditions

Adenosine, through activation of membrane-bound receptors, has been reported to have neuroprotective properties during strokes or seizures. The role of astrocytes in regulating brain interstitial adenosine levels has not been clearly defined. We have determined the nucleoside transporters present in rat C6 glioma cells. RT-PCR analysis, (3)H-nucleoside uptake experiments, and [(3)H]nitrobenzylthioinosine ([(3)H]NBMPR) binding assays indicated that the primary functional nucleoside transporter in C6 cells was rENT2, an equilibrative nucleoside transporter (ENT) that is relatively insensitive to inhibition by NBMPR. [(3)H]Formycin B, a poorly metabolized nucleoside analogue, was used to investigate nucleoside release processes, and rENT2 transporters mediated [(3)H]formycin B release from these cells. Adenosine release was investigated by first loading cells with [(3)H]adenine to label adenine nucleotide pools. Tritium release was initiated by inhibiting glycolytic and oxidative ATP generation and thus depleting ATP levels. Our results indicate that during ATP-depleting conditions, AMP catabolism progressed via the reactions AMP --> IMP --> inosine --> hypoxanthine, which accounted for >90% of the evoked tritium release. It was surprising that adenosine was not released during ATP-depleting conditions unless AMP deaminase and adenosine deaminase were inhibited. Inosine release was enhanced by inhibition of purine nucleoside phosphorylase; ENT2 transporters mediated the release of adenosine or inosine. However, inhibition of AMP deaminase/adenosine deaminase or purine nucleoside phosphorylase during ATP depletion produced release of adenosine or inosine, respectively, via the rENT2 transporter. This indicates that C6 glioma cells possess primarily rENT2 nucleoside transporters that function in adenosine uptake but that intracellular metabolism prevents the release of adenosine from these cells even during ATP-depleting conditions.     

Activation of AMP-activated protein kinase leads to the phosphorylation of elongation factor 2 and an inhibition of protein synthesis

Protein synthesis, in particular peptide-chain elongation, consumes cellular energy. Anoxia activates AMP-activated protein kinase (AMPK, see ), resulting in the inhibition of biosynthetic pathways to conserve ATP. In anoxic rat hepatocytes or in hepatocytes treated with 5-aminoimidazole-4-carboxamide (AICA) riboside, AMPK was activated and protein synthesis was inhibited. The inhibition of protein synthesis could not be explained by changes in the phosphorylation states of initiation factor 4E binding protein-1 (4E-BP1) or eukaryotic initiation factor 2alpha (eIF2alpha). However, the phosphorylation state of eukaryotic elongation factor 2 (eEF2) was increased in anoxic and AICA riboside-treated hepatocytes and in AICA riboside-treated CHO-K1 cells, and eEF2 phosphorylation is known to inhibit its activity. Incubation of CHO-K1 cells with increasing concentrations of 2-deoxyglucose suggested that the mammalian target of the rapamycin (mTOR) signaling pathway did not play a major role in controlling the level of eEF2 phosphorylation in response to mild ATP depletion. In HEK293 cells, transfection of a dominant-negative AMPK construct abolished the oligomycin-induced inhibition of protein synthesis and eEF2 phosphorylation. Lastly, eEF2 kinase, the kinase that phosphorylates eEF2, was activated in anoxic or AICA riboside-treated hepatocytes. Therefore, the activation of eEF2 kinase by AMPK, resulting in the phosphorylation and inactivation of eEF2, provides a novel mechanism for the inhibition of protein synthesis.     

Beyond AICA riboside: in search of new specific AMP-activated protein kinase activators

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICA riboside) has been extensively used in vitro and in vivo to activate the AMP-activated protein kinase (AMPK), a metabolic sensor involved in both cellular and whole body energy homeostasis. However, it has been recently highlighted that AICA riboside also exerts AMPK-independent effects, mainly on AMP-regulated enzymes and mitochondrial oxidative phosphorylation (OXPHOS), leading to the conclusion that new compounds with reduced off target effects are needed to specifically activate AMPK. Here, we review recent findings on newly discovered AMPK activators, notably on A-769662, a nonnucleoside compound from the thienopyridone family. We also report that A-769662 is able to activate AMPK and stimulate glucose uptake in both L6 cells and primary myotubes derived from human satellite cells. In addition, A-769662 increases AMPK activity and phosphorylation of its main downstream targets in primary cultured rat hepatocytes but, by contrast with AICA riboside, does neither affect mitochondrial OXPHOS nor change cellular AMP:ATP ratio. We conclude that A-769662 could be one of the new promising chemical agents to activate AMPK with limited AMPK-independent side effects.     

Activation of adenosine monophosphate activated protein kinase inhibits growth of multiple myeloma cells

The role of adenosine monophosphate activated protein kinase (AMPK) in regulating multiple myeloma (MM) cell growth is not yet clear. In this study, we show that the AMPK activators 5-aminoimidazole-4-carboxamide riboside (AICAr) and D942 inhibit cell growth in MM cell lines. AICAr also induced an S-phase cell cycle arrest in all four tested cell lines and led to phosphorylation and thus activation of AMPK. Furthermore, the inhibition of a nucleoside transporter by nitrobenzyl-thio-9-beta-d-ribofuranosylpurine (NBTI), inhibition of the adenosine kinase by iodotubericidine and inhibition of AMPK by AMPKI Compound C reversed AICAr effects, indicating that the cellular effects of AICAr were mediated by AMPK. Activation of AMPK inhibited basal extracellular signal-regulated kinase (ERK), mammalian target of rapamycin (mTOR) and P70S6 kinase (P70S6K) as well as AKT phosphorylation, and blocked IL-6, IGF-1, and HS-5 stromal cell conditioned medium-induced increase of cell growth. Troglitazone, which has previously been shown to activate AMPK, similarly inhibited MM cell growth, activated AMPK, and decreased ERK and P70S6K phosphorylation. Our results suggest that activation of AMPK inhibits MM cell growth despite stimulation with IL-6, IGF-1, or HS-5 stromal cell conditioned medium and represents a potential new target in the therapy of MM.     

AMP-activated protein kinase phosphorylates and inactivates liver glycogen synthase

Recombinant muscle GYS1 (glycogen synthase 1) and recombinant liver GYS2 were phosphorylated by recombinant AMPK (AMP-activated protein kinase) in a time-dependent manner and to a similar stoichiometry. The phosphorylation site in GYS2 was identified as Ser7, which lies in a favourable consensus for phosphorylation by AMPK. Phosphorylation of GYS1 or GYS2 by AMPK led to enzyme inactivation by decreasing the affinity for both UDP-Glc (UDP-glucose) [assayed in the absence of Glc-6-P (glucose-6-phosphate)] and Glc-6-P (assayed at low UDP-Glc concentrations). Incubation of freshly isolated rat hepatocytes with the pharmacological AMPK activators AICA riboside (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) or A769662 led to persistent GYS inactivation and Ser7 phosphorylation, whereas inactivation by glucagon treatment was transient. In hepatocytes from mice harbouring a liver-specific deletion of the AMPK catalytic α1/α2 subunits, GYS2 inactivation by AICA riboside and A769662 was blunted, whereas inactivation by glucagon was unaffected. The results suggest that GYS inactivation by AMPK activators in hepatocytes is due to GYS2 Ser7 phosphorylation.     

Role of hypothalamic adenosinergic systems in regulating states of wakefulness in the rat

Immunohistochemical localization of adenosine deaminase (ADA), marker for the putative neurotransmitter/neuromodulator adenosine, has revealed a population of ADA-positive neurons in the ventrolateral hypothalamus in the rat brain. These posterior neurons possess adenosine uptake sites. We have studied the effects of local injections of adenosinergic drugs on the sleep-wake cycle in the rat. Microinjection of erythro-9-(hydroxy-2, nonyl-3) adenine (EHNA), a specific inhibitor of adenosine deaminase, resulted in a significant decrease in wakefulness (W) and an increase in deep slow wave sleep (SWS, or S2) and paradoxical sleep (SP). On the other hand, microinjections of soluflazine, a nucleoside transport inhibitor, increased W and decreased total sleep. These opposite modifications may reflect opposite variations in the extracellular concentrations of Ado and consequently different responses of A1/A2 adenosine receptors.     

Purine catabolism in rat brain (author's transl)

In soluble rat brain fraction, the specific activities of purine nucleoside phosphorylase, guanine deaminase, 5'Nucleotidase and adenosine deaminase, decrease in their mentioned order. A kinetic parameter comparison between these enzymes shows that 5'Nucleotidase with AMP has the lowest KM and the greatest Vmax values, while purine nucleoside phosphorylase has its lowest KM and its greatest Vmax values with guanosine and with inosine, respectively. The enzymes activity is not modified by the metabolic intermediates differently from their own reaction products which behave as competitive inhibitors.     

The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia

Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis – a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA.KEY WORDS: Ribosomal protein deficiency, Rps19, Rpl11, p53, ATR, RNR, Chk1, ATP, AMPK, Exogenous nucleosides     

Kinetics of the 5′-nucleotidase and the adenosine deaminase in subcellular fractions of rat brain

Suspensions of rat brain microsomes, synaptosomes, and synaptic vesicles were able to convert adenosine to inosine by means of adenosine deaminase. Isosbestic points of this transformation, at 222, 250 and 281 nm, remained unchanged with time-course. This fact suggests that adenosine deaminase (ADA, E.C. 3.5.4.4) is located on the surface of the vesicles whereas purine nucleoside phosphorylase (PNP, E.C. 2.1.2.4) is located inside the vesicles. Kinetic parameters of the particulate 5-nucleotidase (5N, E.C. 3.1.3.5) and adenosine deaminase were analogous to those of the cytosolic enzymes. These results suggest that soluble and particulate enzymes represent different pools of the same molecular species.     

Presynaptic Adenosine A2 and N-Methyl-D-Aspartate Receptors Regulate Dopamine Synthesis in Rat Striatal Synaptosomes

Dopamine synthesis rate and cyclic AMP concentration were measured in synaptosomes prepared from rat striatum. Dopamine synthesis rate was decreased by the addition of either adenosine deaminase or 8-phenyltheophylline, an adenosine receptor blocker, and was increased by the addition of 2-chloroadenosine. The addition of L-glutamate in the absence of adenosine deaminase decreased both dopamine synthesis rate and cyclic AMP concentration; in the presence of adenosine deaminase, glutamate had no effect on basal dopamine synthesis, but enhanced K(+)-stimulated synthesis. Both these effects of glutamate were abolished in Ca2(+)-free medium or in the presence of 2-amino-5-phosphonovalerate, an N-methyl-D-aspartate (NMDA) receptor blocker. In Mg2(+)-free medium with adenosine deaminase, glutamate enhanced both basal and K(+)-stimulated synthesis. These results suggest that dopaminergic terminals have A2 adenosine receptors, whose activation can stimulate dopamine synthesis by a cyclic AMP-dependent mechanism, and NMDA receptors, which modulate dopamine synthesis by a Ca2(+)-dependent mechanism.     

Evidence for extracellular deamination of adenosine in the rat heart

• 1.1. In rat heart perfused with adenosine (10⁻⁶M), dilazep (10⁻⁴M) inhibited incorporation of adenosine into nucleotides (an index of nucleoside transport and phosphorylation) to a greater extent (70%) than metabolism to inosine and uric acid (40%) and actually increased the recovery of inosine to 30% of the adenosine infused.
• 2.2. Extrapolating for complete inhibition of transport suggested that 60% of adenosine metabolism was intracellular and 40% extracellular.
• 3.3. Static incubations of atria also gave an estimate for extracellular metabolism of 40%.
• 4.4. Adenosine deaminase was localised by immunocytochemistry to the extracellular surface of endothelial cells of small coronary arteries.
• 5.5. Extracellular deamination may explain the lack of effect of nucleoside transport inhibitors on responses to adenosine in rat heart.

     

Adenosine and deoxyadenosine induces apoptosis in oestrogen receptor-positive and -negative human breast cancer cells via the intrinsic pathway

In this study we have examined the cytotoxic effects of different concentrations of adenosine (Ado) and deoxyadenosine (dAdo) on human breast cancer cell lines. Ado and dAdo alone had little effect on cell cytotoxicity. However, in the presence of adenosine deaminase (ADA) inhibitor, EHNA, adenosine and deoxyadenosine led to significant growth inhibition of cells of the lines tested. Ado/EHNA and dAdo/EHNA-induced cell death was significantly inhibited by NBTI, an inhibitor of nucleoside transport, and 5'-amino-5'-deoxyadenosine, an inhibitor of adenosine kinase, but the effects were not affected by 8-phenyltheophylline, a broad inhibitor of adenosine receptors. The Ado/EHNA combination brought about morphological changes consistent with apoptosis. Caspase-9 activation was observed in MCF-7 and MDA-MB468 human breast cancer cell lines on treatment with Ado/EHNA or dAdo/EHNA, but, as expected, caspase-3 activation was only observed in MDA-MB468 cells. The results of the study, thus, suggest that extracellular adenosine and deoxyadenosine induce apoptosis in both oestrogen receptor-positive (MCF-7) and also oestrogen receptor-negative (MDA-MB468) human breast cancer cells by its uptake into the cells and conversion to AMP (dAMP) followed by activation of nucleoside kinase, and finally by the activation of the mitochondrial/intrinsic apoptotic pathway.     

Differential Distribution of Purine Metabolizing Enzymes Between Glia and Neurons

Abstract: Previous studies showed that in cultured chick ciliary ganglion neurons and CNS glia, adenosine can be synthesized by hydrolysis of 5'-AMP and that the accumulation of the adenosine degradative products inosine and hypoxanthine was significantly greater in glial than in neuronal cultures. Furthermore, previous immunochemical and histochemical studies in brain showed that adenosine deaminase and nucleoside phosphorylase are localized in endothelial and glial cells but are absent in neurons; however, adenosine deaminase may be found in a few neurons in discrete brain regions. These results suggested that adenosine degradative pathways may be more active in glia. Thus, we have determined if there is a differential distribution of adenosine deaminase, nucleoside phosphorylase, and xanthlne oxidase enzyme fluxes in glia, comparing primary cultures of central and ciliary ganglion neurons and glial cells from chick embryos. Hypoxanthine-guanine phosphoribosyltransferase and production of adenosine by S-adenosylhomocysteine hydrolase activity were also examined. Our results show that there is a distinct profile of purine metabolizing enzymes for glia and neurons in culture. Both cell types have an S-adenosylhomocysteine hydrolase, but it was more active in neurons than in glia. In contrast, in glia the enzymatic activities of xanthine oxidase (443 ± 61 pmol/min/10⁷ cells), nucleoside phosphorylase (187 ± B pmol/min/10⁷ cells), and adenosine deaminase (233 ± 32 pmol/min/10⁷ cells) were more active at least 100, 20, and five times, respectively, than in ciliary ganglion neurons and 100, 100, and nine times, respectively, than in central neurons.     

alpha2 But not alpha1 AMP-activated protein kinase mediates oxidative stress-induced inhibition of retinal pigment epithelium cell phagocytosis of photoreceptor outer segments

Oxidative stress causes retinal pigment epithelium (RPE) cell dysfunction and is a major risk factor leading to the development of dry-type age-related macular degeneration. Taking pharmacological and genetic approaches, we address the mechanisms by which sublethal oxidative stress inhibits RPE cell phagocytosis. Sublethal oxidative stress dose-dependently inhibited RPE cell phagocytosis of photoreceptor outer segments (POS) and activated AMP-activated protein kinase (AMPK) as determined by increased Thr172 and Ser79 phosphorylation of AMPKalpha and its substrate acetyl-CoA carboxylase, respectively. Similar to oxidative stress, 5-aminoimidazole-4-carboxamide riboside (AICAR), a pharmacological activator of AMPK, inhibited RPE cell phagocytosis of POS in a dose-dependent manner. Inhibition of RPE cell phagocytosis by AICAR was fully reversed by blockade of AICAR translocation into cells by dipyridamole or inhibition of AICAR conversion to ZMP by adenosine kinase inhibitor 5-iodotubercidin. In agreement, AICAR-induced activation of AMPK was abolished by preincubation with dipyridamole or 5-iodotubercidin. Knock-out experiments further revealed that alpha2 but not alpha1 AMPK was involved in RPE cell phagocytosis and that activation of alpha2 AMPK contributed to the inhibition of RPE cell phagocytosis by oxidative stress. Inhibition of RPE cell phagocytosis by activation of alpha2 AMPK was associated with a dramatic increase in acetyl-CoA carboxylase phosphorylation. In comparison, AMPK had no role in oxidative stress-induced breakdown of RPE barrier function. Taken together, reduction in POS load under oxidative stress might direct RPE cells to a self-protected status. Thus, activating AMPK could have therapeutic potential in treating dry macular degeneration.     

Heterogeneous localization of some purine enzymes in subcellular fractions of rat brain and cerebellum

The activity of guanine deaminase (GAH, E.C. 3.5.4.3) was lower in rat cerebellum soluble and microsomal fractions than in rat brain subfractions. Adenosine deaminase (ADA, E.C. 3.5.4.4) activity was released in higher proportion than guanine deaminase, purine nucleoside phosphorylase (PNP, E.C. 2.1.2.4), 5-nucleotidase (5N, E.C. 3.1.3.5), and lactate (LDH, E.C. 1.1.1.27) and malate (MDH, E.C. 1.1.1.37) dehydrogenase in press-juices of rat brain. Furthermore, nerve ending-derived fractions (synaptosomes and synaptic vesicles) showed an enrichment of adenosine deaminase and also of 5-nucleotidase. The action of deoxycholate over the subfractions did not increase the activity of either enzyme. The contrary occurred with the remaining enzymes studied. Thus, it is possible that one set of enzymes are located on the surface of the particulate vesicles, whereas another set are located inside these vesicles, suggesting a compartmentation of purine catabolic enzymes in different areas of the central nervous system.