Neurochemical regulation of Aedes aegypti salivary gland function.

The salivary gland of hematophagous arthropods is critical for blood meal acquisition, blood vessel localization, and secretion of digestive enzymes. Thus, there is significant interest in the regulation of salivary gland function and mechanisms driving the secretion of saliva and digestive proteins. We aimed to gain a broader understanding of the regulatory role of aminergic, cholinergic, and octopaminergic neuromodulators to saliva and protein secretion from the female A. aegypti salivary gland. Quantification of saliva after injection with neuromodulators showed that dopamine, serotonin, and pilocarpine increased the secretory activity of the salivary gland with potency rankings dopamine = serotonin > pilocarpine. No change in saliva secretion was observed with octopamine or ergonovine, which indicates the A. aegypti salivary gland may be regulated by dopaminergic, serotonergic, and cholinergic systems, but are not likely regulated by octopaminergic or tryptaminergic systems. Next, we studied the regulatory control of dopamine-mediated salivation. Data indicate extracellular calcium flux, but not neural function, is critical for dopamine-mediated salivation, which suggests epithelial transport of ions and not neuronal control is responsible for dopamine-mediated salivation. For regulation of protein secretion, data indicate dopamine or serotonin exposure facilitates amylase secretion, whereas serotonin but not dopamine exposure increased apyrase concentrations in the secreted saliva. General immunoreactivity to anti-rat D1-dopamine receptor antibody was observed, yet immunoreactivity to the anti-rat D2-receptor antibody was identified in the proximal regions of the lateral lobes and slight immunoreactivity in the distal portion of the lateral lobe, with no expression in the medial lobe.

On the mechanism of anti-CD39 immune checkpoint therapy.

With the coming of age of cancer immunotherapy, the search for new therapeutic targets has led to the identification of immunosuppressive adenosine as an important regulator of antitumor immunity. This resulted in the development of selective inhibitors targeting various components of the adenosinergic pathway, including small molecules antagonists targeting the high affinity A2A adenosine receptor and low affinity A2B receptor, therapeutic monoclonal antibodies (mAbs) and small molecules targeting CD73 and therapeutic mAbs targeting CD39. As each regulator of the adenosinergic pathway present non-overlapping biologic functions, a better understanding of the mechanisms of action of each targeted approach should accelerate clinical translation and improve rational design of combination treatments. In this review, we discuss the potential mechanisms-of-action of anti-CD39 cancer therapy and potential toxicities that may emerge from sustained CD39 inhibition. Caution should be taken, however, in extrapolating data from gene-targeted mice to patients treated with blocking anti-CD39 agents. As phase I clinical trials are now underway, further insights into the mechanism of action and potential adverse events associated with anti-CD39 therapy are anticipated in coming years.

Regulation of Treg-associated CD39 in multiple sclerosis and effects of corticotherapy during relapse.

BACKGROUND: Accumulating data highlight proinflammatory processes leading to MS relapses. Whether anti-inflammatory mechanisms are concomitantly activated is unclear. The ectonucleotidase CD39 has been described as a novel T regulatory cell (Treg) marker. The purpose of this study was to explore whether regulatory mechanisms are activated during MS relapses and reinforced by intravenous methylprednisolone (ivMP). METHODS: Blood samples were collected from stable and relapsing MS patients and healthy controls. We used FOXP3 methylation-specific qPCR and CD4(+)CD25(high)FOXP3(+) analysis to quantify Tregs. Cytokine mRNA expression levels were measured in peripheral blood mononuclear cells (PBMCs) and in CD4(+) T cells. CD39 expression was determined by flow cytometry in monocytes, NK, T and B cells. CD39 enzymatic activity was assessed by ATP luminometry. RESULTS: The proportion of Tregs was similar in relapsing MS patients and healthy controls. CD39 mRNA level was higher in PBMCs of relapsing MS patients than in controls. The proportion of CD39-expressing Tregs was higher in MS patients. IvMP decreased the overall proportion of Tregs while it increased CD39 mRNA levels, the proportions of CD39-expressing Tregs and monocytes as well as CD39 ectonucleotidase activity. CONCLUSIONS: Our data suggest that immunoregulatory mechanisms are ongoing in MS patients, particularly during relapses, and strengthened by ivMP.

P2X(1) receptor inhibition and soluble CD39 administration as novel approaches to widen the cardiovascular therapeutic window.

Thrombus formation at sites of disrupted atherosclerotic plaques is a leading cause of death and disability worldwide. Although the platelet is now recognized to be a central regulator of thrombus formation, development of antiplatelet reagents that selectively target thrombosis over hemostasis represents a challenge. Existing prophylactic antiplatelet therapies are centered on the use of aspirin, an irreversible cyclooxygenase inhibitor, and a thienopyridine such as clopidogrel, which inactivates the adenosine diphosphate-stimulated P2Y(12) receptor. Although these compounds are widely used and have beneficial effects for patients, their antithrombotic benefit is complicated by an elevated bleeding risk and substantial or partial "resistance." Moreover, combination therapy with these two drugs increases the hemorrhagic risk even further. This review explores the possibility of inhibiting the platelet-surface ionotropic P2X(1) receptor and/or elevating CD39/NTPDase1 activity as new therapeutic approaches to reduce overall platelet reactivity and recruitment of surrounding platelets at prothrombotic locations. Because both proteins affect platelet activation at an early stage in the events leading to thrombosis but are less crucial in hemostasis, they provide new strategies to widen the cardiovascular therapeutic window without compromising safety.

The effect of aluminium on NTPDase and 5'-nucleotidase activities from rat synaptosomes and platelets.

Aluminium (Al), a neurotoxic compound, has been investigated in a large number of studies both in vivo and in vitro. In this study, we investigated the effect in vivo of long-term exposure to Al on NTPDase (nucleoside triphosphate diphosphohydrolase) and 5'-nucleotidase activities in the synaptosomes (obtained from the cerebral cortex and hippocampus) and platelets of rats. Here, we investigated a possible role of platelets as peripheral markers in rats. Rats were loaded by gavage with AlCl(3) 50 mg/(kg day), 5 days per week, totalizing 60 administrations. The animals were divided into four groups: (1) control (C), (2) 50 mg/kg of citrate solution (Ci), (3) 50 mg/kg of Al plus citrate (Al+Ci) solution and (4) 50 mg/kg of Al (Al). ATP hydrolysis was increased in the synaptosomes from the cerebral cortex by 42.9% for Al+Ci and 39.39% for Al, when compared to their respective control (p<0.05). ADP hydrolysis was increased by 13.15% for both Al and Al+Ci, and AMP hydrolysis increased by 32.7% for Al and 27.25% for Al+Ci (p<0.05). In hippocampal synaptosomes, the hydrolysis of ATP, ADP and AMP, was increased by 58.5%, 28.5% and 25.92%, respectively, for Al (p<0.05) and 36.7%, 22.5% and 37.64% for Al+Ci, both when compared to their respective controls. ATP, ADP and AMP hydrolysis, in platelets, was increased by 172.3%, 188.52% and 92.1%, respectively in Al+Ci, and 317.9%, 342.8% and 177.9%, respectively, for Al, when compared to their respective controls (p<0.05). Together, these results indicate that Al increases NTPDase and 5'-nucleotidase activities, in synaptosomal fractions and platelets. Thus, we suggest that platelets could be sensitive peripheral markers of Al toxicity of the central nervous system.

Oral L-arginine administration does not inhibit thrombosis on an experimental model of arterial thrombosis: the effect on the apyrasic activity of the arterial wall.

Despite the extensive research on the pharmacology of L-arginine, there are only few data on its antithrombotic properties. We studied the effect of oral L-arginine administration in a model of arterial thrombosis in rabbits divided into three groups: group 1, group without intervention; group 2, control group, treated with normal diet and submitted to the thrombosis-triggering protocol; group 3, treated for 2 weeks with L-arginine (2.25%) prior the protocol. L-Arginine did not alter platelet aggregation nor coagulation parameters but reduced vascular activities of both ADPase (49.1 +/- 8.5 versus 28.9 +/- 8.3 versus 18.8 +/- 10.3 nmoles inorganic phosphate/min per mg protein; mean +/- SD; group 1 versus group 2 versus group 3, respectively; ANOVA F = 19.21; P < 0.0001) and ATPase (97.8 +/- 15.8 versus 52.1 +/- 11.6 versus 31.9 +/- 16.3 nmoles inorganic phosphate/min per mg protein; mean +/- SD; group 1 versus group 2 versus group 3, respectively; ANOVA, F = 34.65; P < 0.0001). L-Arginine did not reduce the thrombi area (17.1 mm, 9.02 and 48.07, versus 27.04 mm, 25.4 and 70.39, median, percentile 25 and 75 respectively, P = 0.079; group 2 versus group 3, respectively). In conclusion, oral L-arginine administration did not inhibit thrombosis, and, conversely, it significantly reduced the arterial wall ADPase and ATPase activities. This effect may limit its antithrombotic properties.

Trypanosoma rangeli: characterization of a Mg-dependent ecto ATP-diphosphohydrolase activity.

In this work we describe the ability of living Trypanosoma rangeli to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by Trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (1.53+/-0.12 nmol P(i)/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 5.24+/-0.64 nmol P(i)/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. This stimulatory effect on the ATP hydrolysis was also observed when MgCl(2) was replaced by MnCl(2), but not by CaCl(2), SrCl(2), and ZnCl(2). The apparent K(m) for Mg-ATP2- was 0.53+/-0.11 mM. The optimum pH for the T. rangeli Mg-dependent ecto-ATPase activity lies in the alkaline range. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase activity was stimulated by carbohydrates involved in the attachment/invasion of salivary glands of Rhodnius prolixus and by lipophorin, an insect lipoprotein circulating in the hemolymph.

Changes in nucleotide hydrolysis in rat blood serum induced by pentylenetetrazol-kindling.

There is growing pharmacological evidence from several animal models of seizure disorders that adenosine possesses endogenous anticonvulsant activity. Apart from being released from cells, adenosine can be produced by the degradation of adenine nucleotides by ectoenzymes or soluble nucleotidases. These enzymes constitute an important mechanism in synaptic modulation, as they hydrolyze ATP, an excitatory neurotransmitter, to adenosine, a neuroprotective compound. We recently demonstrated an increase in ectoenzyme activity in rat brain synaptosomes after pentylenetetrazol-kindling in rats resistant to kindling, suggesting a role for ectonucleotidases in the seizure control. The present work investigates the effect of seizures induced by pentylenetetrazol kindling on the enzymes that could be playing a role in ATP, ADP and AMP hydrolysis to adenosine in rat blood serum. Animals received injections of PTZ (30 mg/kg, i.p., dissolved in 0.9% saline) once every 48 h, totaling 10 stimulations and the controls animals were injected with saline. The hydrolysis of ATP, ADP and AMP were significantly increased (42, 40, and 45%, respectively), while phosphodiesterase activity was unchanged. These results suggest once more that an increase in the ATP diphosphohydrolase and 5'-nucleotidase activities and, possibly, in adenosine levels, could represent an important compensatory mechanism in the development of chronic epilepsy. Moreover, the fact that this increase can also be measured in serum could mean that these enzymes might be useful as plasma markers of seizures in epilepsy.

Long-read pyrosequencing using pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate) Sp-isomer.

Pyrosequencing, a nonelectrophoretic DNA sequencing method that uses a luciferase-based enzymatic system to monitor DNA synthesis in real time, has so far been limited to sequencing of short stretches of DNA. To increase the signal-to-noise ratio in pyrosequencing the natural dATP was replaced by dATPalphaS (M. Ronaghi et al., 1996, Anal. Biochem. 242, 84-89). The applied dATPalphaS was a mixture of two isomers (Sp and Rp). We show here that by the introduction of pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate) Sp-isomer in pyrosequencing substantial longer reads could be obtained. The pure Sp-isomer allowed lower nucleotide concentration to be used and improved the possibility to read through poly(T) regions. In general, a doubling of the read length could be obtained by the use of pure Sp-isomer. Pyrosequencing data for 50 to 100 bases could be generated on different types of template. The longer read will enable numerous new applications, such as identification and typing of medically important microorganisms as well as resequencing of DNA fragments for mutation screening and clone checking.

Expression and characterization of human ecto-ATPase and chimeras with CD39 ecto-apyrase.

Human ecto-ATPase (ecto-nucleoside triphosphate diphosphohydrolase 2 [eNTPDase2], also known as CD39L1) has been expressed and characterized in COS cells. It exhibits some unusual enzymology that is similar to a few members of this class of proteins but different from the majority of the family members. Hydrolysis of ATP by human ecto-ATPase is nonlinear with time, and its activity is stimulated/stabilized by both the lectin concanavalin A and the chemical cross-linking agent disuccinimidyl suberate. Like other members of the eNTPDase family, the human ecto-ATPase is a tetramer, the activity of which depends on its glycosylation. Chimeras of this protein with human CD39 (eNTPDasel) were constructed to test the hypothesis that the N-terminal half of these proteins regulates nucleotide specificity. The two chimeras generated demonstrated that the N-terminal half of these proteins is crucial for determining the relative activities of the nucleoside di- and triphosphatases. Chemical cross-linking of the two chimeras suggests that disuccinimidyl suberate interacts with the C-terminal half of ecto-ATPase in a manner that results in an increase of activity for both the ecto-ATPase and the ecto-apyrase/ecto-ATPase chimera.

Transforming growth factor-beta(1) restores antiplatelet function of endothelial cells exposed to anoxia-reoxygenation injury.

Transforming growth factor-beta(1) released from platelet alpha-granules may preserve endothelial functions in injured vessels. However, direct evidence is lacking regarding how this cytokine modifies the antithrombotic function of injured endothelial cells. We performed an in vitro study to investigate the effects of transforming growth factor-beta(1) on platelet functions in the presence of cultured endothelial cells exposed to anoxia-reoxygenation injury. Cultured bovine aorta endothelial cells were placed in an anoxic chamber (0.5% O(2), 5% CO(2)) for 60 minutes followed by a 90-minute reoxygenation. Collagen (2 microg/mL)-induced platelet aggregation (10(8) platelets/mL), as determined by impedance aggregometry, was potently inhibited in the presence of control endothelial cells (17.4+/-3.3 Omega) at a concentration of 5x10(4) cells/mL, as compared to their absence (68. 2+/-2.2 Omega). Inhibition of platelet aggregation was attenuated in endothelial cells exposed to anoxia-reoxygenation (54.6+/-2.5 Omega). However, preincubation of endothelial cells with transforming growth factor-beta(1) (1.0 ng/mL) for 16 hours partially recovered the inhibitory capability of platelet aggregation by injured endothelial cells (40.6+/-3.8 Omega). Cell viability, confirmed by a trypan blue dye exclusion test, was similar (93-96%), including control, 1.0 ng/mL transforming growth factor-beta(1)- and/or anoxia-reoxygenation-pretreated cells. The capability of platelet inhibition was restored when the endothelial cells were preincubated for 4 hours or more. Restoration of antiplatelet capacity in endothelial cells by transforming growth factor-beta(1) was suggested to be due to several mechanisms, including an increase in nitric oxide synthase activity, up-regulation of prostacyclin release, and restoration of adenosine triphosphate diphosphohydrolase activity, which was attenuated by anoxia-reoxygenation pretreatment. In summary, transforming growth factor-beta(1) released from activated platelets may play a compensatory role in the preservation of endothelial functions to inhibit platelet activation.

An in vitro method for evaluating vascular endothelial ADPase activity.

Some xenobiotics, known to promote the development of thrombotic phenomena, affect vascular endothelium ADPase, a regulatory enzyme that inactivates vaso- and platelet-active adenine nucleotides. This proposed new experimental approach represents an improved method of evaluation of vascular endothelial ADPase activity which is assessed by measuring, at pre-established times, the degradation rate of exogenous ADP incubated with aortic bovine patches. The ADP dosage was performed by using a spectrophotometric enzymatic assay. Statistical analyses showed that the method is capable of highlighting the linearity of the ADPase activity time-course, thus indicating that the slopes of time-degradation curves of ADP are a valid index for this endothelial ectoenzyme activity. Results obtained with ADPase inhibiting or stimulating agent confirm that this in vitro method is an efficient tool for estimating the ability of xenobiotics or drugs to modify the nonthrombogenic properties of vascular endothelium.

The increased endotoxin-sensitivity of pregnant rats, as reflected by glomerular ecto-ADP-ase activity, is not dependent on the presence of decidual cells.

In the present study the possible role of decidual cells in the pregnancy-associated increased sensitivity of glomerular ecto-ADP-ase to endotoxin was investigated. Early (day 5) pregnant (E-Pr; n = 10), pseudopregnant (E-PSP; n = 10), (day 5), pseudopregnant rats with a decidualized uterus (E-DEC; n - 10), as well as late (day 14) pregnant (L-Pr; n = 10), pseudopregnant (L-PSP: n = 10) (day 14), and pseudopregnant rats with a decidualized uterus (E-DEC; n = 10) were infused with either endotoxin (1.0 mg/kg bw) or saline. Three days later rats were killed and specimens of the left kidney were snap-frozen. Cryostat kidney sections (4 microns) were stained for ecto-ADP-ase activity and quantitatively evaluated. The results show that only glomerular ecto-ADP-ase activity of both groups of pregnant rats (E-Pr and L-Pr) was significantly decreased after endotoxin infusion as compared to saline infusion. In the other groups of rats, no significant differences in ecto-ADP-ase activity were observed between saline and endotoxin infusion. It is concluded that decidual cells do not play a role in the increased sensitivity of ecto-ADP-ase to endotoxin during pregnancy.

In vitro and in vivo effects of HgCl2 on synaptosomal ATP diphosphohydrolase (EC 3.6.1.5) from cerebral cortex of developing rats.

The aim of the present investigation was to evaluate the in vitro (10-500 microM) and in vivo (1-21 subcutaneous injections of 2.5 mg/kg each) effects of HgCl2 on the ATP diphosphohydrolase activity (EC 3.6.1.5; apyrase) of synaptosomes from cerebral cortex of rats at different ages (5, 11, 18 and 25 days of life). The in vitro results showed that HgCl2 (from 10 to 500 microM) inhibited the hydrolysis of both substrates by the synaptosomal enzyme at all ages studied. In contrast, HgCl2 injected in vivo did no affected the normal ontogeny of ATP and ADP hydrolysis. The hydrolysis of both nucleotides increased at the same rate as a function of age in control and HgCl2-treated rats (the specific activity of enzyme increased about 5-fold from the first week of postnatal life of weaning). The results of the present study demonstrated that in vitro HgCl2 inhibited the enzyme, but was ineffective when tested in vivo. Probably the absence of an in vivo effect is due to the low permeability of blood-brain barrier to inorganic forms of mercury.

Effect of phenylalanine and its metabolites on ATP diphosphohydrolase activity in synaptosomes from rat cerebral cortex.

The in vitro effects of phenylalanine and some of its metabolites on ATP diphosphohydrolase (apyrase, EC 3.6.1.5) activity in synaptosomes from rat cerebral cortex were investigated. The enzyme activity in synaptosomes from rats subjected to experimental hyperphenylalaninemia (alpha-methylphenylalanine plus phenylalanine) was also studied. In the in vitro studies, a biphasic effect of phenylalanine on both enzyme substrates (ATP and ADP) was observed, with maximal inhibition at 2.0 mM and maximal activation at 5.0 mM. Inhibition of the enzyme activity was not due to calcium chelation. Moreover, phenylpyruvate, when compared with phenylalanine showed opposite effects on the enzyme activity, suggesting that phenylalanine and phenylpyruvate bind to two different sites on the enzyme. The other tested phenylalanine metabolites phenyllactate, phenylacetate and phenylethylamine) had no effect on ATP diphosphohydrolase activity. In addition, we found that ATP diphosphohydrolase activity in synaptosomes from cerebral cortex of rats with chemically induced hyperphenylalaninemia was significantly enhanced by acute or chronic treatment. Since it is conceivable that ATPase-ADPase activities play an important role in neurotransmitter (ATP) metabolism, it is tempting to speculate that our results on the deleterious effects of phenylalanine and phenylpyruvate on ATP diphosphohydrolase activity may be related to the neurological dysfunction characteristics of naturally and chemically induced hyperphenylalaninemia.