Monophosphate Hydrolysis Research Articles

Phosphodiesterases 3 and 5 express activity in the trigeminal ganglion and co-localize with calcitonin gene-related peptide

Understanding of the neuropathology leading to migraine pain has centered on either a vascular or neuronal origin. Sildenafil, a specific inhibitor of phosphodiesterase 5 (PDE5), induces migraine-like headache in a human headache model without concomitant artery dilation. The presence and activity of PDE3 and PDE5 is known in cerebral arteries. However, the presence in the neuronal part of the trigeminovascular pathway, i.e. the trigeminal ganglion and the possible co-localization with calcitonin gene-related peptide (CGRP), is not known. Rat trigeminal ganglia were isolated and immunohistochemistry and in situ hybridization was applied. Evaluations of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) hydrolysis were performed using scintillation proximity assays. PDE3 and PDE5 were present and active in the trigeminal ganglia. A subset of PDE3- and PDE5-positive neurons contained CGRP. In contrast to cGMP, both sildenafil and cilostazol influenced cAMP hydrolysis. Sildenafil may exert its effect on the neuronal part of the migraine pain pathway. In addition to the effects on cGMP signaling, sildenafil may indirectly affect cAMP signaling in the trigeminal ganglion. This result may suggest a common cAMP-related pathway for sildenafil, cilostazol, and CGRP in eliciting migraine pain.

Combining Neurotrophin-Transduced Schwann Cells and Rolipram to Promote Functional Recovery from Subacute Spinal Cord Injury

Following spinal cord injury (SCI), both an inhibitory environment and lack of intrinsic growth capacity impede axonal regeneration. In a previous study, prevention of cyclic adenosine monophosphate (AMP) hydrolysis by the phosphodiesterase-4 inhibitor rolipram, in combination with Schwann cell (SC) grafts, promoted significant supraspinal and proprioceptive fiber growth and/or sparing and improved locomotion. In another study, transplanted SCs transduced to generate a bifunctional neurotrophin (D15A) led to significant increases in graft SCs and axons, including supraspinal and myelinated axons. Here we studied the growth and myelination of local and supraspinal axons and functional outcome following the combination of rolipram administration and neurotrophin-transduced SC implantation after SCI. Rolipram was administered subcutaneously for 4 weeks immediately after contusion at vertebral T8 (25.0-mm weight drop, MASCIS impactor). GFP or GFP-D15A-transduced SCs were injected into the injury epicenter 1 week after SCI. GFP-D15A SC grafts and GFP SC grafts with rolipram contained significantly more serotonergic fibers compared to GFP SCs. SC myelinated axons were increased significantly in GFP SC with rolipram-treated animals compared to animals receiving SCI alone. Rolipram administered with either GFP or GFP-D15A SCs significantly increased numbers of brain stem-derived axons below the lesion/implant area and improved hindlimb function. Compared to the single treatments, the combination led to the largest SC grafts, the highest numbers of serotonergic fibers in the grafts, and increased numbers of axons from the reticular formation below the lesion/implant area and provided the greatest improvement in hindlimb function. These findings demonstrate the therapeutic potential for a combination therapy involving the maintenance of cyclic AMP levels and neurotrophin-transduced SCs to repair the subacutely injured spinal cord.

Targeting CD73 Enhances the Antitumor Activity of Anti-PD-1 and Anti-CTLA-4 mAbs

Monoclonal antibodies (mAb) that block programmed death (PD)-1 or cytotoxic T lymphocyte antigen (CTLA-4) receptors have been associated with durable clinical responses against a variety of cancer types and hold great potential as novel cancer therapeutics. Recent evidence suggest that targeted blockade of multiple immunosuppressive pathways can induce synergistic antitumor responses. In this study, we investigated whether targeted blockade of CD73, an ectonucleotidase that catabolizes the hydrolysis of extracellular adenosine monophosphate (AMP) to adenosine, can enhance the antitumor activity of anti-CTLA-4 and anti-PD-1 mAbs against transplanted and chemically induced mouse tumors. Anti-CD73 mAb significantly enhanced the activity of both anti-CTLA-4 and anti-PD-1 mAbs against MC38-OVA (colon) and RM-1 (prostate) subcutaneous tumors, and established metastatic 4T1.2 breast cancer. Anti-CD73 mAb also significantly enhanced the activity of anti-PD-1 mAb against 3-methylcholanthrene (MCA)-induced fibrosarcomas. Gene-targeted mice revealed that single-agent therapies and combinatorial treatments were dependent on host IFN-γ and CD8(+) T cells, but independent of perforin. Interestingly, anti-CD73 mAb preferentially synergized with anti-PD-1 mAb. We investigated the effect of extracellular adenosine on tumor-infiltrating T cells and showed that activation of A2A adenosine receptor enhances PD-1 expression, but not CTLA-4 expression, on tumor-specific CD8+ T cells and CD4+ Foxp3+ T regulatory cells. Taken together, our study revealed that targeted blockade of CD73 can enhance the therapeutic activity of anti-PD-1 and anti-CTLA-4 mAbs and may thus potentiate therapeutic strategies targeting immune checkpoint inhibitors in general.

A cross-kingdom Nudix enzyme that pre-empts damage in thiamin metabolism

Genes specifying the thiamin monophosphate phosphatase and adenylated thiazole diphosphatase steps in fungal and plant thiamin biosynthesis remain unknown, as do genes for ThDP (thiamin diphosphate) hydrolysis in thiamin metabolism. A distinctive Nudix domain fused to Tnr3 (thiamin diphosphokinase) in Schizosaccharomyces pombe was evaluated as a candidate for these functions. Comparative genomic analysis predicted a role in thiamin metabolism, not biosynthesis, because free-standing homologues of this Nudix domain occur not only in fungi and plants, but also in proteobacteria (whose thiamin biosynthesis pathway has no adenylated thiazole or thiamin monophosphate hydrolysis steps) and animals (which do not make thiamin). Supporting this prediction, recombinant Tnr3 and its Saccharomyces cerevisiae, Arabidopsis and maize Nudix homologues lacked thiamin monophosphate phosphatase activity, but were active against ThDP, and up to 60-fold more active against diphosphates of the toxic thiamin degradation products oxy- and oxo-thiamin. Deleting the S. cerevisiae Nudix gene (YJR142W) lowered oxythiamin resistance, overexpressing it raised resistance, and expressing its plant or bacterial counterparts restored resistance to the YJR142W deletant. By converting the diphosphates of damaged forms of thiamin into monophosphates, the Tnr3 Nudix domain and its homologues can pre-empt the misincorporation of damaged diphosphates into ThDP-dependent enzymes, and the resulting toxicity.

Phosphodiesterase-2 inhibitor reverses corticosterone-induced neurotoxicity and related behavioural changes via cGMP/PKG dependent pathway

Phosphodiesterase 2 (PDE2) is an enzyme responsible for hydrolysis of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) to restrict intracellular signalling of these second messenger molecules. This study investigated how PDE2 inhibitor Bay 60-7550 affects the dysregulated glucocorticoid signalling in neuronal cells and regulates depressive behaviours after chronic stress in mice. We found that exposure of hippocampal neurons to corticosterone resulted in time- and concentration-dependent increases in PDE2 expression. These intriguing findings were confirmed in the hippocampal cell line HT-22. After corticosterone exposure for 24 h, HT-22 cells showed a concentration-dependent increase in mRNA levels for PDE2 subtypes, PDE2A1 and 2A3, as well as for the total PDE2A protein expression. Bay 60-7550 was found to reverse the cell lesion induced by corticosterone (50 μm). This neuroprotective effect was blocked by pretreatment with protein kinase G inhibitor KT5823, but not protein kinase A inhibitor H89, suggesting the involvement of cGMP-dependent signalling. Although Bay 60-7550 treatment for 24 h did not change the levels of phosphorylated mitogen-activated protein kinases ERK1/2 (pERK) and phosphorylated cAMP response element-binding protein (pCREB), it down-regulated pERK at 2 h and up-regulated a CREB co-activator, CREB-binding protein, at 24 h. Both of these effects were blocked by KT 5823. Furthermore, Bay 60-7550 reversed corticosterone-induced down-regulation of brain-derived neurotrophic factor protein levels 24 h after corticosterone exposure. In behavioural testing, Bay 60-7550 produced antidepressant-like effects and reduced corticosterone levels in stressed mice, further supporting the involvement of a PDE2-dependent pathway in mediating Bay 60-7550's effect during stress hormone insults.

Modulating the Cyclic Guanosine Monophosphate Substrate Selectivity of the Phosphodiesterase 3 Inhibitors by Pyridine, Pyrido[2,3-d]pyrimidine Derivatives and Their Effects upon the Growth of HT-29 Cancer Cell Line

Analogues with the scaffolds of 3-cyano-4-alkoxyphenyl-6-bromoaryl-2-pyridone and 2-amino-3-cyano-4-alkoxyphenyl-6-bromoarylpyridine were synthesized. Cyclization of the 2-amino derivatives with formic acid and formamide gave the corresponding pyrido[2,3-d]pyrimidin-4(3H)-one and the pyrido[2,3-d]-pyrimidin-4-amine derivatives, respectively. Active phosphodiesterase 3 (PDE3) inhibitors were identified from each of the four aforementioned scaffolds. This is the first report that pyrido[2,3-d]pyrimidin-4(3H)-one and pyrido[2,3-d]pyrimidin-4-amine derivatives can inhibit PDE3. The analogues with the pyridone and pyrido[2,3-d]pyrimidin-4(3H)-one scaffolds inhibited both cAMP and cyclic guanosine monophosphate (cGMP) hydrolysis by PDE3, while the amine containing scaffolds were more selective for cGMP hydrolysis. This observation may set the base for substrate-selective pharmacological modulation of this important class of drug targets and with less side effects, particularly tachcardia. The dual inhibitors of PDE3 were more potent inhibitor towards the growth of HT-29 cancer cell lines.

Morphine treatment alters nucleotidase activities in rat blood serum.

Morphine has been widely used in neonatal pain management. However, this treatment may produce adaptive changes in several physiologic systems. Our laboratory has demonstrated that morphine treatment in neonate rats alters nucleoside triphosphate diphosphohydrolase (NTPDase) activity and gene expression in central nervous system structures. Considering the relationship between the opioid and purinergic systems, our aim was to verify whether treatment with morphine from postnatal days 8 (P8) through 14 (P14) at a dose of 5 μg per day alters NTPDase and 5′-nucleotidase activities in rat serum over the short, medium, and long terms. After the in vivo assay, the morphine group showed increased hydrolysis of all nucleotides at P30, and a decrease in adenosine 5′-diphosphate hydrolysis at P60. Moreover, we found that nucleotidase activities change with age; adenosine 5′-triphosphate hydrolysis activity was lower at P16, and adenosine 5′-monophosphate hydrolysis activity was higher at P60. These changes are very important because these enzymes are the main regulators of blood nucleotide levels and, consequently, nucleotide signaling. Our findings showed that in vivo morphine treatment alters nucleotide hydrolysis in rat blood serum, suggesting that purine homeostasis can be influenced by opioid treatment during the neonatal period.

Microcystin-LR acute exposure does not alter in vitro and in vivo ATP, ADP and AMP hydrolysis in adult zebrafish (Danio rerio) brain membranes

Microcystins (MCs) are toxins produced by cyanobacteria during the blooms that could accumulate in aquatic animals and be relocated to higher trophic levels. Adenosine triphosphate (ATP) acts as an excitatory neurotransmitter and/or a neuromodulator in the extracellular space playing important roles in physiological and pathological conditions. The aim of this study was, therefore, to evaluate the acute effects of different concentrations of MC-LR on nucleoside triphosphate diphosphohydrolases and 5’-nucleotidade in adult zebrafish (<em>Danio rerio</em>) brain membranes. The results have shown no significant changes in ATP, adenosine diphosphate (ADP) and adenosine monophosphate (AMP) hydrolysis in zebrafish brain membranes. MC-LR <em>in vitro</em> also did not alter ATP, ADP and AMP hydrolysis in the concentrations tested. These findings show that acute exposure to MC-LR did not modulate ectonucleotidase activity in the conditions tested. However, additional studies including chronic exposure should be performed in order to achieve a better understanding about MC-LR toxicity mechanisms in the central nervous system.

Cloning, expression, purification, crystallization and X-ray analysis of inositol monophosphatase from Mus musculus and Homo sapiens.

Inositol monophosphatase (IMPase) catalyses the hydrolysis of inositol monophosphate to inositol and is crucial in the phosphatidylinositol (PI) signalling pathway. Lithium, which is the drug of choice for bipolar disorder, inhibits IMPase at therapeutically relevant plasma concentrations. Both mouse IMPase 1 (MmIMPase 1) and human IMPase 1 (HsIMPase 1) were cloned into pRSET5a, expressed in Escherichia coli, purified and crystallized using the sitting-drop method. The structures were solved at resolutions of 2.4 and 1.7 Å, respectively. Comparison of MmIMPase 1 and HsIMPase 1 revealed a core r.m.s. deviation of 0.516 Å.

Insights into the Role of Magnesium Triad in myo-Inositol Monophosphatase: Metal Mechanism, Substrate Binding, and Lithium Therapy

myo-Inositol monophosphatase (IMPase) plays a pivotal role in the intracellular phosphatidylinositol cell signaling pathway. It has attracted considerable attention as a putative therapeutic target for lithium therapy in the treatment of bipolar disorder. A trio of activated cofactor Mg²⁺ ions is required for inositol monophosphate hydrolysis by IMPase. In the present study, computational studies, including two-layered ONIOM-based quantum mechanics/mechanical mechanics (QM/MM) calculations, molecular modeling, and molecular dynamics (MD) simulations, were performed to ascertain the role of the Mg²⁺ triad in the IMPase active site. The QM/MM calculations show that the structural identity of the nucleophilic water molecule W1 shared by Mg²⁺-1 and Mg²⁺-3, activated by Thr95/Asp47 dyad, is a hydroxide ion. Moreover, Mg²⁺-3 needs to be conjugated with Mg²⁺-1 in the binding site to create the activated nucleophilic hydroxide ion in accordance with the three-metal ion catalytic mechanism. The MD simulation of the IMPase-substrate-Mg²⁺ complex shows that the three Mg²⁺ ions promote substrate binding and help fix the phosphate moiety of the substrate for nucleophilic attack by the hydroxide ion. When Mg²⁺-2 is displaced with Li⁺, the MD simulations of the postreaction complex indicate that the conformation of the catalytic loop (residues 33 to 44) is disrupted and water molecule W2 does not coordinate with Li⁺. This disruption traps the inorganic phosphate and inositolate in the active site, which lead to IMPase inhibition. By contrast, in the native Mg²⁺ system, the W2 ligated by Mg²⁺-2 and Asp200 aids in protonation of the leaving inositolate moiety.

Determination of the Lithium Binding Site in Inositol Monophosphatase, the Putative Target for Lithium Therapy, by Magic-Angle-Spinning Solid-State NMR

Inositol monophosphatase (IMPase) catalyzes the hydrolysis of inositol monophosphate to inorganic phosphate and inositol. For this catalytic process to occur, Mg(2+) cations must exist in the active site. According to the inositol depletion hypothesis, IMPase activity is assumed to be higher than normal in patients suffering from bipolar disorder. Treatment with Li(+), an inhibitor of IMPase, reduces its activity, but the mechanism by which lithium exerts its therapeutic effects is still at a stage of conjecture. The Escherichia coli SuhB gene product possesses IMPase activity, which is also strongly inhibited by Li(+). It has significant sequence similarity to human IMPase and has most of its key active-site residues. Here we show that by using (7)Li magic-angle-spinning solid-state NMR spectroscopy, including {(13)C}(7)Li dipolar recoupling experiments, the bound form of lithium in the active site of wild-type E. coli SuhB can be unambiguously detected, and on the basis of our data and other biochemical data, lithium binds to site II, coupled to aspartate residues 84, 87, and 212.

Apremilast mechanism of action and application to psoriasis and psoriatic arthritis

Psoriasis and psoriatic arthritis are common clinical conditions that negatively impact health-related quality of life and are linked to serious medical comorbidities. Disease mechanisms involve local and systemic chronic inflammatory processes. Available biologic therapies specifically target single inflammatory mediators, such as tumor necrosis factor-α (TNF-α), in the context of a larger inflammatory signaling cascade. To interrupt this pathological cascade earlier in the response or further upstream, and return pro-inflammatory and anti-inflammatory signaling to a homeostatic balance, the use of a phosphodiesterase4 (PDE4) inhibitor has been explored. PDE4 is the major enzyme class responsible for the hydrolysis of cyclic adenosine monophosphate (cAMP), an intracellular second messenger that controls a network of pro-inflammatory and anti-inflammatory mediators. With PDE4 inhibition, and the resulting increases in cAMP levels in immune and non-immune cell types, expression of a network of pro-inflammatory and anti-inflammatory mediators can be modulated. Apremilast is an orally available targeted PDE4 inhibitor that modulates a wide array of inflammatory mediators involved in psoriasis and psoriatic arthritis, including decreases in the expression of inducible nitric oxide synthase, TNF-α, and interleukin (IL)-23 and increases IL-10. In phase II studies of subjects with psoriasis and psoriatic arthritis, apremilast reversed features of the inflammatory pathophysiology in skin and joints and significantly reduces clinical symptoms. The use of an oral targeted PDE4 inhibitor for chronic inflammatory diseases, like psoriasis and psoriatic arthritis, represents a novel treatment approach that does not target any single mediator, but rather focuses on restoring a balance of pro-inflammatory and anti-inflammatory signals.

α‐Tocopherol regulates ectonucleotidase activities in synaptosomes from rats fed a high‐fat diet

α-Tocopherol (α-Toc) is involved in various physiologic processes, which present antioxidant and neuroprotective properties. High-fat diets have an important role in neurodegenerative diseases and neurological disturbances. This study aimed to investigate the effects of treatment with α-Toc and the consumption of high-fat diets on ectonucleotidase activities in synaptosomes of cerebral cortex, hippocampus and striatum of rats. Animals were divided into four different groups, which received standard diet (control), high-fat saturated diet (HF), α-Toc and high-fat saturated diet plus α-Toc (α-Toc + HF). High-fat saturated diet was administered ad libitum and α-Toc by gavage using a dose of 50 mg·kg(-1). After 3 months of treatment, animals were submitted to euthanasia, and cerebral cortex, hippocampus and striatum were collected for biochemical assays. Results showed that adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) hydrolysis in the cerebral cortex, hippocampus and striatum were decreased in HF in comparison to the other groups (P < 0·05). When rats that received HF were treated with α-Toc, the activity of the ectonucleotidases was similar to the control. ATP, ADP and AMP hydrolysis in the cerebral cortex, hippocampus and striatum were increased in the α-Toc group when compared with the other groups (P < 0·05). These findings demonstrated that the HF alters the purinergic signaling in the nervous system and that the treatment with α-Toc was capable of modulating the adenine nucleotide hydrolysis in this experimental condition.

Activation of PDE10 and PDE11 Phosphodiesterases

The most recently identified cyclic nucleotide phosphodiesterases, PDE10 and PDE11, contain a tandem of so-called GAF domains in their N-terminal regulatory regions. In PDE2 and PDE5, the GAF domains mediate cGMP stimulation; however, their function in PDE10 and PDE11 remains controversial. Although the GAF domains of PDE10 mediate cAMP-induced stimulation of chimeric adenylyl cyclases, cAMP binding did not stimulate the PDE10 holoenzyme. Comparable data about cGMP and the PDE11 GAF domains exist. Here, we identified synthetic ligands for the GAF domains of PDE10 and PDE11 to reduce interference of the GAF ligand with the catalytic reaction of PDE. With these ligands, GAF-mediated stimulation of the PDE10 and PDE11 holoenzymes is demonstrated for the first time. Furthermore, PDE10 is shown to be activated by cAMP, which paradoxically results in potent competitive inhibition of cGMP turnover by cAMP. PDE11, albeit susceptible to GAF-dependent stimulation, is not activated by the native cyclic nucleotides cAMP and cGMP. In summary, PDE11 can be stimulated by GAF domain ligands, but its native ligand remains to be identified, and PDE10 is the only PDE activated by cAMP.

Increased NTPDase Activity in Lymphocytes during Experimental Sepsis

We investigated in rats induced to sepsis the activity of ectonucleoside triphosphate diphosphohydrolase (NTPDase; CD39; E.C. 3.6.1.5), an enzyme involved in the modulation of immune responses. After 12 hours of surgery, lymphocytes were isolated from blood and NTPDase activity was determined. It was also performed the histology of kidney, liver, and lung. The results demonstrated an increase in the hydrolysis of adenosine-5′-triphosphate (ATP) (P < 0.01), but no changes regarding adenosine-5′-monophosphate (ADP) hydrolysis (P > 0.05). Histological analysis showed several morphological changes in the septic group, such as vascular congestion, necrosis, and infiltration of mononuclear cells. It is known that the intracellular milieu contains much more ATP nucleotides than the extracellular. In this context, the increased ATPasic activity was probably induced as a dynamic response to clean up the elevated ATP levels resulting from cellular death.

Lung cancer alters the hydrolysis of nucleotides and nucleosides in platelets

BackgroundThe nucleotides and nucleosides of adenine are signaling molecules related to thromboregulation and modulation of immune responses in patients with malignancies. Thus, this study aims to determine NTPDase, 5′-nucleotidase, ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) and adenosine deaminase (ADA) activities in the platelets of patients with lung cancer. MethodsWe collected blood samples from patients (n=33) previously treated for lung cancer with chemotherapy. Patients were classified as stage IIIb and IV according to the Union for International Cancer Control (UICC). ResultsPatients showed a significant decrease in the hydrolysis of adenosine diphosphate (ADP) and adenosine, whereas the adenosine monophosphate (AMP) hydrolysis and platelet aggregation were significantly increased in this group. Adenosine triphosphate (ATP) hydrolysis did not show significant results between the group of patients and the control group. ConclusionsWe may suggest that ectonucleotidases as well as ADA are enzymes involved in thromboembolic events but especially here we may see that they are also directly involved in the generation of adenosine formation in the cancer patient circulation.

Endotoxin-induced effects on nucleotide catabolism in mouse kidney

Extracellular adenosine 5′-triphosphate (ATP) acts as a proinflammatory mediator. Adenosine, the final product of ATP breakdown, is an anti-inflammatory compound, acting mainly on adenosine A2A receptors. Considering that the kidney is an organ strongly affected during systemic inflammatory responses and that ectonucleotidases are responsible for the control of extracellular nucleotide and nucleoside levels, we examined the endotoxin-induced effects on ectonucleotidases in kidney membranes of mice, and whether CGS-21680 hydrochloride (3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino]ethyl]phenyl]propanoic acid), a selective adenosine A2A receptor agonist, antagonizes the lipopolysaccharide (LPS)-induced effects on nucleotide catabolism in kidney. Animals were injected intraperitoneally with 12mg/kg LPS and/or 0.5mg/kg CGS-21680 or saline. Nucleotidase activities were determined in kidney membrane preparations and ATP metabolism was measured by high performance liquid chromatography (HPLC) assay. Analysis of ectonucleotidase expression was carried out by semi-quantitative semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Exposure to endotoxemia promoted an increase in ATP and p-Nitrophenyl thymidine 5′-monophosphate (p-Nph-5′-TMP) hydrolysis, and a decrease in adenosine 5′-monophosphate (AMP) hydrolysis. CGS-21680 treatment failed to reverse these changes. HPLC analysis indicated a decrease in extracellular ATP and adenosine levels in groups treated with LPS and LPS plus CGS-21680. The expression pattern of ectonucleotidases revealed an increase in Entpd3, Enpp2, and Enpp3 mRNA levels after LPS injection. These findings indicate that nucleotide and nucleoside availability in mouse kidney is altered at different stages of endotoxemia, in order to protect the integrity of this organ when exposed to systemic inflammation.

Hormonal control of mammalian oocyte meiosis at diplotene stage.

Mammalian oocytes grow and undergo meiosis within ovarian follicles. Fully grown oocytes are arrested at the first meiotic prophase by a mural granulosa origin "arrester" until a surge of luteinizing hormone (LH) from the pituitary at the mid-cycle stimulates the immature oocyte to resume meiosis. Recent evidence indicates that natriuretic peptide precursor type C (NPPC) produced by mural granulosa cells stimulates the generation of cyclic guanosine 3',5'-monophosphate (cGMP) by cumulus cell natriuretic peptide receptor 2 (NPR2), which diffuses into oocyte via gap junctions and inhibits oocyte phosphodiesterase 3A (PDE3A) activity and cyclic adenosine 3',5'-monophosphate (cAMP) hydrolysis and maintains meiotic arrest with a high intraoocyte cAMP level. This cAMP is generated through the activity of the Gs G-protein by the G-protein-coupled receptor, GPR3 and GPR12, and adenylyl cyclases (ADCY) endogenous to the oocyte. Further studies suggest that endocrine hormones, such as follicle-stimulating hormone (FSH), LH, 17β-estradiol (E2) and oocyte-derived paracrine factors (ODPFs), participate in oocyte meiosis possibly by the regulation of NPPC and/or NPR2. A detailed investigation of NPPC and NPR2 expression in follicle cells will elucidate the precise molecular mechanisms of gonadotropins, and control the arrest as well as resumption of meiosis.

Melatonin and IP3-induced Ca2+ Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.

Is prenatal myo‐inositol deficiency a mechanism of CNS injury in galactosemia?

Classic Galactosemia due to galactose-1-phosphate uridyltransferase (GALT) deficiency is associated with apparent diet-independent complications including cognitive impairment, learning problems and speech defects. As both galactose-1-phosphate and galactitol may be elevated in cord blood erythrocytes and amniotic fluid despite a maternal lactose-free diet, endogenous production of galactose may be responsible for the elevated fetal galactose metabolites, as well as postnatal CNS complications. A prenatal deficiency of myo-inositol due to an accumulation of both galactose-1- phosphate and galactitol may play a role in the production of the postnatal CNS dysfunction. Two independent mechanisms may result in fetal myo-inositol deficiency: competitive inhibition of the inositol monophosphatase1 (IMPA1)-mediated hydrolysis of inositol monophosphate by high galactose-1- phosphate levels leading to a sequestration of cellular myo-inositol as inositol monophosphate and galactitol-induced reduction in SMIT1-mediated myo-inositol transport. The subsequent reduction of myo-inositol within fetal brain cells could lead to inositide deficiencies with resultant perturbations in calcium and protein kinase C signaling, the AKT/mTOR/ cell growth and development pathway, cell migration, insulin sensitivity, vescular trafficking, endocytosis and exocytosis, actin cytoskeletal remodeling, nuclear metabolism, mRNA export and nuclear pore complex regulation, phosphatidylinositol-anchored proteins, protein phosphorylation and/or endogenous iron "chelation". Using a knockout animal model we have shown that a marked deficiency of myo-inositol in utero is lethal but the phenotype can be rescued by supplementing the drinking water of the pregnant mouse. If myo-inositol deficiency is found to exist in the GALT-deficient fetal brain, then the use of myo-inositol to treat the fetus via oral supplementation of the pregnant female may warrant consideration.