Context-Specific Switch from Anti- to Pro-epileptogenic Function of the P2Y1 Receptor in Experimental Epilepsy.

Extracellular ATP activates inflammatory responses to tissue injury. It is also implicated in establishing lasting network hyperexcitability in the brain by acting upon independent receptor systems. Whereas the fast-acting P2X channels have well-established roles driving neuroinflammation and increasing hyperexcitability, the slower-acting metabotropic P2Y receptors have received much less attention. Recent studies of P2Y1 receptor function in seizures and epilepsy have produced contradictory results, suggesting that the role of this receptor during seizure pathology may be highly sensitive to context. Here, by using male mice, we demonstrate that the metabotropic P2Y1 receptor mediates either proconvulsive or anticonvulsive responses, dependent on the time point of activation in relation to the induction of status epilepticus. P2Y1 deficiency or a P2Y1 antagonist (MRS2500) administered before a chemoconvulsant, exacerbates epileptiform activity, whereas a P2Y1 agonist (MRS2365) administered at this time point is anticonvulsant. When these drugs are administered after the onset of status epilepticus, however, their effect on seizure severity is reversed, with the antagonist now anticonvulsant and the agonist proconvulsant. This result was consistent across two different mouse models of status epilepticus (intra-amygdala kainic acid and intraperitoneal pilocarpine). Pharmacologic P2Y1 blockade during status epilepticus reduces also associated brain damage, delays the development of epilepsy and, when applied during epilepsy, suppresses spontaneous seizures, in mice. Our data show a context-specific role for P2Y1 during seizure pathology and demonstrate that blocking P2Y1 after status epilepticus and during epilepsy has potent anticonvulsive effects, suggesting that P2Y1 may be a novel candidate for the treatment of drug-refractory status epilepticus and epilepsy.SIGNIFICANCE STATEMENT This is the first study to fully characterize the contribution of a metabotropic purinergic P2Y receptor during acute seizures and epilepsy. The findings suggest that targeting P2Y1 may offer a potential novel treatment strategy for drug-refractory status epilepticus and epilepsy. Our data demonstrate a context-specific role of P2Y1 activation during seizures, switching from a proconvulsive to an anticonvulsive role depending on physiopathological context. Thus, our study provides a possible explanation for seemingly conflicting results obtained between studies of different brain diseases where P2Y1 targeting has been proposed as a potential treatment strategy and highlights that the timing of pharmacological interventions is of critical importance to the understanding of how receptors contribute to the generation of seizures and the development of epilepsy.

Fibrinogen γ-Chain Peptide-Coated Adenosine 5' Diphosphate-Encapsulated Liposomes Rescue Mice From Lethal Blast Lung Injury via Adenosine Signaling.

OBJECTIVES: Fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes can accumulate via dodecapeptide HHLGGAKQAGDV interactions at bleeding sites where they release adenosine 5'-diphosphate that is rapidly metabolized to adenosine, which has tissue-protective effects. We investigated the efficacy of fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes to treat blast lung injury, with a focus on adenosine signaling. DESIGN: Controlled animal study. SETTING: University research laboratory. SUBJECTS: Adult male C57BL/6 mice. INTERVENTIONS: Mice were pretreated with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes, dodecapeptide HHLGGAKQAGDV-(phosphate-buffered saline)-liposomes, adenosine 5' diphosphateliposomes, or phosphate-buffered saline-liposomes. Five minutes after treatment the mice received a single laser-induced shock wave (1.8 J/cm) that caused lethal blast lung injury, and their survival times and lung injuries were then assessed. We also evaluated the therapeutic effect of posttreatment with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes or H12-(phosphate-buffered saline)-liposomes 1 minute after laser-induced shock wave exposure. To examine the effect of adenosine signaling, adenosine A2A receptor (ZM241385) or adenosine A2B receptor (PSB 1115) antagonists were administered to the mice 1 hour before the pretreatment with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes that was followed by laser-induced shock wave exposure. MEASUREMENTS AND MAIN RESULTS: Pre- and posttreatment with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes significantly increased mouse survival [fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes: 58% survival vs H12-(phosphate-buffered saline)-liposomes: 8%; p < 0.05 (posttreatment)] and mitigated pulmonary tissue damage/hemorrhage and neutrophil accumulation after laser-induced shock wave exposure. fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes accumulated at pulmonary vessel injury sites after laser-induced shock wave exposure with both pre- and posttreatment. Furthermore, pretreatment with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes reduced albumin and macrophage inflammatory protein-2 levels in bronchoalveolar lavage fluid. Although fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes pretreatment did not affect blood coagulation activity in the injured mice, its beneficial effect on blast lung injury was significantly abrogated by A2A or A2B adenosine receptor antagonists (A2A antagonist: 17% survival; A2B antagonist: 33% vs dimethyl sulfoxide control: 80%; p < 0.05, respectively). CONCLUSIONS: Fibrinogen γ-chain (dodecapeptide HHLGGAKQA GDV)-coated adenosine 5'-diphosphate-encapsulated liposomes may be effective against blast lung injury by promoting tissue-protective adenosine signaling and could represent a novel controlled-release drug delivery system.

[The variability of response of thrombocytes to ADP: from theory of thrombogenesis to practical application of plasma rich in thrombocytes].

The study was carried out to evaluate variability of morpho-genetic potential of plasma rich in thrombocytes. The analysis of ADP-induced aggregation of thrombocytes separated from blood samples of 32 male volunteers aged 18-42 (22±3) was applied. The bimodal character of reaction of thrombocytes to ADP with peaks of aggregation in range of 30-50% and 60-80%. Besides individual differences in range of aggregation differences in time of reaching peak of aggregation and entering plateau were established. In some of volunteers the reversible character of aggregation curve reflecting absence of phase of thrombogenesis stabilization was established. The characteristics of aggregation curve, associated with activation of signal system, launching of degranulation of thrombocytes and exposition of GPIIaIIIb can reflect velocity and effectiveness of secretion of granules of thrombocytes at application of standard doses of stimulants of aggregation. These specifics substantiate necessity of preliminary evaluation of aggregation response of thrombocytes for prognosis of effectiveness of degranulation and release out of granules of thrombocytes biologically active molecules determining morpho-genetic potential of plasma rich with thrombocytes.

The Marine-Derived Kinase Inhibitor Fascaplysin Exerts Anti-Thrombotic Activity.

BACKGROUND: The marine-derived kinase inhibitor fascaplysin down-regulates the PI3K pathway in cancer cells. Since this pathway also plays an essential role in platelet signaling, we herein investigated the effect of fascaplysin on thrombosis. METHODS: Fascaplysin effects on platelet activation, platelet aggregation and platelet-leukocyte aggregates (PLA) formation were analyzed by flow cytometry. Mouse dorsal skinfold chambers were used to determine in vivo the effect of fascaplysin on photochemically induced thrombus formation and tail-vein bleeding time. RESULTS: Pre-treatment of platelets with fascaplysin reduced the activation of glycoprotein (GP)IIb/IIIa after protease-activated receptor-1-activating peptide (PAR-1-AP), adenosine diphosphate (ADP) and phorbol-12-myristate-13-acetate (PMA) stimulation, but did not markedly affect the expression of P-selectin. This was associated with a decreased platelet aggregation. Fascaplysin also decreased PLA formation after PMA but not PAR-1-AP and ADP stimulation. This may be explained by an increased expression of CD11b on leukocytes in PAR-1-AP- and ADP-treated whole blood. In the dorsal skinfold chamber model of photochemically induced thrombus formation, fascaplysin-treated mice revealed a significantly extended complete vessel occlusion time when compared to controls. Furthermore, fascaplysin increased the tail-vein bleeding time. CONCLUSION: Fascaplysin exerts anti-thrombotic activity, which represents a novel mode of action in the pleiotropic activity spectrum of this compound.

Pharmacokinetic study of the structural components of adenosine diphosphate-encapsulated liposomes coated with fibrinogen γ-chain dodecapeptide as a synthetic platelet substitute.

Fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV, H12)-coated, ADP-encapsulated liposomes [H12-(ADP)-liposomes] were developed as a synthetic platelet alternative that specifically accumulates at bleeding sites as the result of interactions with activated platelets via glycoprotein IIb/IIIa and augments platelet aggregation by releasing ADP. The aim of this study is to characterize the pharmacokinetic properties of H12-(ADP)-liposomes and structural components in rats, and to predict the blood retention of H12-(ADP)-liposomes in humans. With use of H12-(ADP)-liposomes in which the encapsulated ADP and liposomal membrane cholesterol were radiolabeled with (14)C and (3)H, respectively, it was found that the time courses for the plasma concentration curves of (14)C and (3)H radioactivity showed that the H12-(ADP)-liposomes remained intact in the blood circulation for up to 24 hours after injection, and were mainly distributed to the liver and spleen. However, the (14)C and (3)H radioactivity of H12-(ADP)-liposomes disappeared from organs within 7 days after injection. The encapsulated ADP was metabolized to allantoin, which is the final metabolite of ADP in rodents, and was mainly eliminated in the urine, whereas the cholesterol was mainly eliminated in feces. In addition, the half-life of the H12-(ADP)-liposomes in humans was predicted to be approximately 96 hours from pharmacokinetic data obtained for mice, rats, and rabbits using an allometric equation. These results suggest that the H12-(ADP)-liposome has potential with proper pharmacokinetic and acceptable biodegradable properties as a synthetic platelet substitute.

Sampling conditions influence multiple electrode platelet aggregometry in cardiac surgery patients.

OBJECTIVES: To investigate the importance of blood sampling conditions for multiple electrode platelet aggregometry (MEA) in cardiac surgery patients. DESIGN: Eighty-one patients undergoing first time CABG surgery were included in three prospective, observational studies. MEA was used to analyze platelet aggregability after addition of adenosine-diphosphate (ADP) or thrombin activating peptide 6 (TRAP). In substudy 1, hirudin and citrate tubes were compared. In substudy 2, samples from peripheral vein, central venous catheter, and radial artery were compared and in substudy 3, the effect of surgery was investigated by analyzing pre- and postoperative samples. RESULTS: Platelet aggregability values were 30% higher in hirudin tubes than in citrate tubes. There was a significant correlation between hirudin and citrate tubes in TRAP-induced aggregability (r = 0.84, p < 0.001) but not in ADP-induced aggregability (r = 0.25, p = 0.13). The blood sampling site did not influence platelet aggregability. Surgery reduced ADP-induced aggregability by 31% (p < 0.001) and TRAP-induced aggregability by 30% (p < 0.001) with large intraindividual variations. CONCLUSIONS: MEA results in cardiac surgery patients should not be compared between samples collected in test tubes with different anticoagulants. The choice of blood sampling site does not affect the results. The operation in itself reduces markedly mean platelet aggregability.

P2Y1 purinoceptor inhibition reduces extracellular signal-regulated protein kinase 1/2 phosphorylation in spinal cord and dorsal root ganglia: implications for cancer-induced bone pain.

It remains unclear as to whether P2Y1 purinergic receptor (P2Y1R) and the molecules that act downstream, such as extracellular signal-regulated protein kinase 1/2 (ERK1/2), are involved in the development of cancer-induced bone pain (CIBP) in vivo. Here, we investigated the role of the P2Y1R in the modulation of CIBP-associated nociception in spinal cord and dorsal root ganglia (DRG). A CIBP model was established by inoculating Walker 256 gland carcinoma cells into the tibia of female rats. Tactile allodynia and spontaneous pain were assessed using von Frey filaments and ambulatory scores. The results showed that both the paw withdrawal latency to tactile allodynia and the ambulatory score to spontaneous pain were significantly different between the CIBP group and the sham group on days 7-9 post-inoculation (P< 0.01). Furthermore, rats in the CIBP group also showed a progressive increase in ambulatory score, which is different from the sham group (P< 0.01). Furthermore, P2Y1R mRNA and phosphorylated ERK1/2 (p-ERK1/2) protein expression levels were increased in the spinal dorsal horn and DRG of the CIBP group relative to the sham group. However, intrathecal injection of the P2Y1R antagonist MRS2179 decreased P2Y1R mRNA and p-ERK1/2 protein expression in the spinal dorsal horn and DRG (P< 0.01). These results provide evidence that the inhibition of P2Y1R-mediated ERK1/2 phosphorylation in the spinal dorsal horn and DRG can attenuate nociception transmission.

Comparison of original and generic clopidogrel 600 mg loading dose in the patients who planned undergoing coronary angiography.

OBJECTIVE: To compare the efficacy and safety of original (Plavix) and generic (Apolets) clopidogrel 600 mg loading in patients planning to undergo coronary angiography. MATERIAL AND METHOD: This is an experimental design, parallel, randomized-controlled study. Coronary artery disease patients planned for cardiac catheterization were recruited Patients were randomized to receive either original or generic clopidogrel 600 mg loading dose. Platelet aggregation induced by 5 micromol/L and 20 micromol/L adenosine diphosphate (ADP) was measured by light transmission aggregometry (LTA) at baseline and 6 hours after clopidogrel 600 mg administration. RESULTS: Forty-nine patients were enrolled, 24 patients received original clopidogrel, and 25 patients received generic clopidogrel. After six hours of loading, there was significantly reduction in platelet aggregation induced by adenosine 5 micromol/L from 41.08 +/- 3.04% to 19.50 +/- 1.68% (p < 0.001) in original group compared to 36.76 +/- 2.66% to 21.32 +/- 2.60% (p < 0.001) in generic group. When induced by 20 micromol/L, the platelet aggregation was reduced from 58.50 +/- 2.09% to 32.25 +/- 2.30% (p < 0.001) in original group and from 61.12 +/- 2.54% to 30.04 +/- 3.14% (p < 0.001) in generic group. There was no significant difference between original and generic clopidogrel in reducing platelet aggregation induced by both adenosine 5 and 20 micromol/L. Groin hematoma was found in one case (4.2%) in the original clopidogrel group. CONCLUSION: Generic clopidogrel (Apolets) 600 mg loading dose is as effective as original clopidogrel (Plavix) in term of platelet aggregation inhibition.

P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells.

Extracellular ATP and ADP have been shown to exhibit potent angiogenic effects on pulmonary artery adventitial vasa vasorum endothelial cells (VVEC). However, the molecular signaling mechanisms of extracellular nucleotide-mediated angiogenesis remain not fully elucidated. Since elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is required for cell proliferation and occurs in response to extracellular nucleotides, this study was undertaken to delineate the purinergic receptor subtypes involved in Ca(2+) signaling and extracellular nucleotide-mediated mitogenic responses in VVEC. Our data indicate that stimulation of VVEC with extracellular ATP resulted in the elevation of [Ca(2+)](i) via Ca(2+) influx through plasma membrane channels as well as Ca(2+) mobilization from intracellular stores. Moreover, extracellular ATP induced simultaneous Ca(2+) responses in both cytosolic and nuclear compartments. An increase in [Ca(2+)](i) was observed in response to a wide range of purinergic receptor agonists, including ATP, ADP, ATPγS, ADPßS, UTP, UDP, 2-methylthio-ATP (MeSATP), 2-methylthio-ADP (MeSADP), and BzATP, but not adenosine, AMP, diadenosine tetraphosphate, αßMeATP, and ßγMeATP. Using RT-PCR, we identified mRNA for the P2Y1, P2Y2, P2Y4, P2Y13, P2Y14, P2X2, P2X5, P2X7, A1, A2b, and A3 purinergic receptors in VVEC. Preincubation of VVEC with the P2Y1 selective antagonist MRS2179 and the P2Y13 selective antagonist MRS2211, as well as with pertussis toxin, attenuated at varying degrees agonist-induced intracellular Ca(2+) responses and activation of ERK1/2, Akt, and S6 ribosomal protein, indicating that P2Y1 and P2Y13 receptors play a major role in VVEC growth responses. Considering the broad physiological implications of purinergic signaling in the regulation of angiogenesis and vascular homeostasis, our findings suggest that P2Y1 and P2Y13 receptors may represent novel and specific targets for treatment of pathological vascular remodeling involving vasa vasorum expansion.

Rutin present in Alibertia edulis extract acts on human platelet aggregation through inhibition of cyclooxygenase/thromboxane.

Alibertia edulis leaf extract is commonly used in folk medicine, with rutin caffeic and vanillic acids being its major compounds. The Alibertia edulis leaf extract was investigated for its pharmacological effects via platelet aggregation, calcium mobilization, cyclic nucleotides levels, vasodilator-stimulated phosphoprotein Ser157 and Ser239 and protein kinase Cß2 phosphorylation, thromboxane B2, cyclooxygenases 1 and 2, docking and molecular dynamics. Alibertia edulis leaf extract significantly inhibited (100-1000 µg mL-1) platelet aggregation induced by different agonists. Arachidonic acid increased levels of calcium and thromboxane B2, phosphorylation of vasodilator-stimulated phosphoprotein Ser157 and Ser239, and protein kinase Cß, which were significantly reduced by Alibertia edulis leaf extract, rutin, and caffeic acid as well mixtures of rutin/caffeic acid. Cyclooxygenase 1 activity was inhibited for Alibertia edulis leaf extract, rutin and caffeic acid. These inhibitions were firsrtly explored by specific stabilization of rutin and caffeic acid compared to diclofenac at the catalytic site from docking score and free-energy dissociation profiles. Then, simulations detailed the rutin interactions close to the heme group and Tyr385, responsible for catalyzing the conversion of arachidonic acid to its products. Our results reveal the antiplatelet aggregation properties of Alibertia edulis leaf extract, rutin and caffeic acid providing pharmacological information about its origin from cyclooxygenase 1 inhibition and its downstream pathway.

Platelet adhesion and intracellular calcium levels in antigen-challenged rats.

There is considerable evidence that platelet activation occurs in allergic airways diseases. In this study we aimed to investigate platelet adhesion to immobilized fibrinogen and intracellular calcium levels in a rat model of allergic inflammation. Male Wistar rats were challenged with ovalbumin (OVA). At 30 min to 24h after OVA-challenge, assays of platelet adhesion to immobilized fibrinogen and intracellular calcium levels using fura 2-AM loaded platelets were performed. The serum levels of IgE were approximately 5-fold greater in OVA-sensitized rats. A marked eosinophil influx in bronchoalveolar lavage (BAL) fluid of OVA-challenged rats at 24h after OVA-challenge was also seen. OVA-challenge resulted in a marked thrombocytopenia, as observed within 12h after OVA-challenge. The agonists ADP (0.5-50 microM) and thrombin (30-100 mU/ml) concentration-dependently increased platelet adhesion to immobilized fibrinogen. At an early time after OVA-challenge (30 min), platelets exhibited greater platelet adhesion compared with the non-sensitized group, whereas at a late time (24h) they exhibited lower platelet adhesion to both agonists. Moreover, at 30 min after OVA-challenge, intracellular calcium levels to ADP (20 microM) and thrombin (100 mU/ml)-activated platelets were greater compared with non-challenged rats. As opposed, at 24h after OVA challenge, a lower intracellular calcium level to ADP- and thrombin-activated platelets was observed. In conclusion, OVA-challenge in rats promotes a biphasic response in platelet adhesion consisting of an increased adhesion and intracellular calcium levels at an early phase (30 min), which progress to a reduction in adhesion and intracellular calcium levels at a late time (24h) after antigen challenge.

High sensitivity of intestinal CD8+ T cells to nucleotides indicates P2X7 as a regulator for intestinal T cell responses.

The purinoreceptor P2X7 is expressed on subsets of T cells and mediates responses of these cells to extracellular nucleotides such as ATP or NAD(+). We identified P2X7 as a molecule highly up-regulated on conventional CD8alphabeta(+) and unconventional CD8alphaalpha(+) T cells of the intestinal epithelium of mice. In contrast, CD8(+) T cells derived from spleen, mesenteric lymph nodes, and liver expressed only marginal levels of P2X7. However, P2X7 was highly up-regulated on CD8(+) T cells from spleen and lymph nodes when T cells were activated in the presence of retinoic acid. High P2X7 expression on intestinal CD8(+) T cells as well as on CD8(+) T cells incubated with retinoic acid resulted in enhanced sensitivity of cells to extracellular nucleotides. Both cell populations showed a high level of apoptosis following incubation with NAD(+) and the ATP derivative 2',3'-O-(benzoyl-4-benzoyl)-ATP, and injection of NAD(+) caused selective in vivo depletion of intestinal CD8(+) T cells. Following oral infection with Listeria monocytogenes, P2X7-deficient mice showed similar CD8(+) T cell responses in the spleen, but enhanced responses in the intestinal mucosa, when compared with similarly treated wild-type control mice. Overall, our observations define P2X7 as a new regulatory element in the control of CD8(+) T cell responses in the intestinal mucosa.

Time course of mitochondrial metabolism alterations to repeated injections of bupivacaine in rat muscle.

PURPOSE: Bupivacaine-induced myotoxicity is associated with mitochondrial bioenergetic alterations. The impact of the duration of bupivacaine treatment on mitochondrial energy production remains undetermined. Here, we assessed, in vivo, the alteration of mitochondrial metabolism following different durations of bupivacaine exposure (40, 56, or 112 hr) that correspond to 5, 7, or 14 repeated injections of 0.25% bupivacaine, respectively. METHODS: Rats were divided randomly into seven different groups: one control group (no catheter); three groups with normal saline injections (1 mL x kg(-1)) every eight hours via a femoral nerve catheter for 40, 56, and 112 hr, respectively; and three groups with 0.25% bupivacaine injections (1 mL x kg(-1)) every eight hours via a femoral nerve catheter for 40, 56, and 112 hr. Psoas and gracilis muscle samples located within the bupivacaine infusion-diffusion space were investigated. To estimate mitochondrial respiratory capacity, the protein content of the mitochondrial respiratory chain apparatus was evaluated by measuring citrate synthase activity. To measure mitochondrial respiratory function, adenosine diphosphate-stimulated oxygen consumption was measured by polarography in saponin-skinned muscle fibres using glutamate-malate or succinate as energy substrates. RESULTS: In psoas and gracilis muscles, saline solution had no effect on the two mitochondrial parameters. Bupivacaine induced a significant decrease in the citrate synthase activity in psoas (r(2) = 0.74; P < 0.001) and gracilis muscle (r(2) = 0.52; P < 0.001), and there was a significant decrease in the adenosine diphosphate-stimulated oxygen consumption using glutamate or succinate as substrates in both muscles (P < 0.001). CONCLUSIONS: The severity of bupivacaine-induced myotoxicity is closely linked to the duration of bupivacaine exposure in the muscle fibres located close to the catheter tip.

Adenine nucleotide-induced contraction on the inner mitochondrial membrane. II. Effect of bongkrekic acid.

In bovine heart mitochondria bongkrekic acid at concentrations as low as about 4 nmol/mg protein (a) completely inhibits phosphorylation of exogenous adenosine diphosphate (ADP) and dephosphorylation of exogenous adenosine triphosphate (ATP), (b) completely reverses atractyloside inhibition of inner membrane contraction induced by exogenous adenine nucleotides, and (c) decreases the amount of adenine nucleotide required to elicit maximal exogenous adenine nucleotide-induced inner membrane contraction to a level which appears to correspond closely with the concentration of contractile, exogenous adenine nucleotide binding sites Bongkrekic acid at concentrations greater than 4 nmol/mg protein induces inner membrane contraction which seems to depend on the presence of endogenous ADP and/or ATP. The findings appear to be consistent with the interpretations (a) that the inner mitochondrial membrane contains two types of contractile, adenine nucleotide binding sites, (b) that the two sites differ markedly with regard to adenine nucleotide affinity, (c) that the high affinity site is identical with the adenine nucleotide exchange carrier, (d) that the low affinity site is accessible exclusively to endogenous adenine nucleotides and is largely unoccupied in the absence of bongkrekic acid, and (e) that bongkrekic acid increases the affinity of both sites in proportion to the amount of the antibiotic bound to the inner membrane.

Ticagrelor yields consistent dose-dependent inhibition of ADP-induced platelet aggregation in patients with atherosclerotic disease regardless of genotypic variations in P2RY12, P2RY1, and ITGB3.

The platelet P2Y(12) receptor is the target of clopidogrel therapy, which has been shown to reduce thromboembolic complications of atherosclerotic disease but has limitations in terms of variability of response and irreversibility of effect. This receptor is also a target for ticagrelor (AZD6140), the first reversibly binding oral P2Y(12) receptor antagonist that does not require metabolic activation and yields more consistent inhibition of platelet aggregation than clopidogrel therapy. Single nucleotide polymorphisms (SNPs) have been described in the gene for this receptor (P2RY12), some of which have been associated with variability in platelet reactivity. SNPs in P2RY1 and ITGB3 have also been reported by some groups to affect platelet reactivity to adenosine diphosphate (ADP). We assessed whether SNPs in these genes influenced the pharmacodynamic response to ticagrelor in patients enrolled in both the DISPERSE study (stable atherosclerotic disease) and the DISPERSE2 study (non-ST-segment elevation acute coronary syndromes). Platelet aggregation data (at baseline and 4 weeks) and DNA samples from clopidogrel-naive Caucasian patients treated with ticagrelor were available for 151 patients. Seventy four SNPs within three genes were genotyped. After adjustment for multiple comparisons, none of these SNPs were found to significantly influence inhibition of ADP-induced platelet aggregation by ticagrelor.

Insights into the mechanism of ADP action on flagellar motility derived from studies on bull sperm.

Adenosine diphosphate (ADP) is known to have interesting effects on flagellar motility. Permeabilized and reactivated bull sperm exhibit a marked reduction in beating frequency and a greatly increased beat amplitude in the presence of 1-4 mM ADP. In this study we examined the force production of sperm reactivated with 0.1 mM ATP with and without 1 mM ADP and found that there is little or no resulting change in the stalling force produced by a bull sperm flagella in response to ADP. Because bull sperm bend to a higher curvature after ADP treatment we explored the possibility that ADP-treated sperm flagella are more flexible. We measured the stiffness of 50 muM sodium vanadate treated bull sperm in the presence of 4 mM ADP, but found no change in the passive flagellar stiffness. When we analyzed the torque that develops in ADP-treated sperm at the point of beat reversal we found that the torque developed by the flagellum is significantly increased. Our torque estimates also allow us to calculate the transverse force (t-force) acting on the flagellum at the point of beat direction reversal. We find that the t-force at the switch-point of the beat is increased significantly in the ADP treated condition, averaging 0.7 +/- 0.29 nN/microm in 0.1 mM ATP and increasing to 2.9 +/- 1.2 nN/microm in 0.1 mM ATP plus 4 mM ADP. This suggests that ADP is exerting its effect on the beat by increasing the tenacity of dynein attachment at the B-subtubule. This could be a direct result of a regulatory effect of ADP on the binding affinity of dynein for the B-subtubule of the outer doublets. This result could also help to explain a number of previous experimental observations, as discussed.

Real-time measurement of non-lethal platelet thromboembolic responses in the anaesthetized mouse.

Identifying and evaluating new therapeutic targets in platelets requires advanced animal models in which platelet responses can be measured directly and in situ. This is important because platelet function is strongly influenced by external factors such as those originating from the vascular endothelium. Our objectives were to record graded, non-lethal thromboembolic platelet responses to platelet agonists in situ in the mouse and to demonstrate an inhibitory effect of aspirin in our model. Radiolabelled platelets were infused into anaesthetized mice and responses to ADP, collagen and thrombin measured as changes in platelet associated counts in miniaturized detection probes placed over the thoracic region. All agonists induced dose-dependent changes in platelet counts due to accumulation of thrombi in the pulmonary vasculature. We confirmed a specific platelet effect by comparing platelet and erythrocyte responses and showing platelet aggregates in the lung vasculature histologically. Simultaneous injection of collagen and adrenaline induced increased and protracted synergistic platelet responses compared with individual injection of these agents and aspirin inhibited collagen-induced responses. We confirmed the clinical relevance of our model by showing that platelet thromboembolism in the mouse, like pulmonary embolism in humans, impaired cardiovascular performance. We present a refined method for measuring platelet agonist dose-responses and thromboembolism in real-time without inducing mortality in the mouse. Our technique will be useful in investigating the molecular determinants of physiological and pathophysiological platelet function in an in-vivo context and will enable investigations of both platelet and non-platelets mediators of thrombus formation.

Inhibition of ATP-induced glutamate release by MRS2179 in cultured dorsal spinal cord astrocytes.

It was reported that ATP, an excitatory chemical mediator, exerts its effects by activation of the P2X (ligand-gated cationic channels) and P2Y (G protein-coupled receptors) purinoceptors in the nervous system. In the present work, we used confocal laser scanning microscopy and high-performance liquid chromatography to assess the role of the P2Y1 receptor in ATP-evoked Ca2+ mobilization and glutamate release from cultured dorsal spinal cord astrocytes. ATP (0.01-100 micromol/l) produces a dose-dependent rise in the Ca2+ relative fluorescence intensity in cultured astrocytes. N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179, 0.01-100 micromol/l), a P2Y1-specific antagonist, could dose-dependently inhibit ATP-evoked Ca2+ mobilization. In addition, 100 micromol/l ATP caused glutamate efflux from cultured dorsal spinal cord astrocytes in a time-dependent manner. 100 micromol/l MRS2179 significantly inhibited the glutamate efflux induced by ATP, which suggests that P2Y1 receptor activation is responsible for the ATP-induced glutamate efflux from astrocytes. Taken together, our results demonstrate that P2Y1 receptor plays an important role in modulating the function of astrocytes, which raises the possibility that MRS2179, a potent P2Y1-specific antagonist, may become a potential drug in treating many chronic neurological diseases characterized by astrocytic activation in the nervous system.

The metabolism of ecto-5'-nucleotidase (CD73) inhibitor-α,ß-methylene adenosine diphosphate in BALB/c mice.

CD73 inhibitors are considered to be used in the therapies of melanomas, gliomas or breast cancer. However, little is known about their pharmacology and kinetics in mouse experimental models. Thus, this study is aimed to define a metabolic stability and elimination of the adenosine diphosphate (ADP) analog - α,ß-Methylene-ADP also known as AOPCP in BALB/c mice. The process starts with an intravenous injection of AOPCP, next blood and serum samples are collected. Urine samples are possessed by a bladder puncture. Mice aortas are dissected for the e5NT activity evaluation. In order to assess the AOPCP degradation, the incubation of AOPCP in mice blood and plasma is performed. The AOPCP concentration as well as the activity of e5NT were analyzed with the reverse phase-high pressure liquid chromatography (RP-HPLC). The study shows that after 60 minutes of the 20 mg/kg intravenous injection of AOPCP (body weight dose), the concentration of AOPCP in blood diminished rapidly from 38.6 ± 5.0 µM (measured 5 minutes after the injection) to 6.4 ± 1.4 µM. Interestingly, it is also noted that 60 minutes after the incubation of mice blood samples the AOPCP concentration decreases from 50 µM to 30.0 ± 0.3 µM. This study demonstrates a significant and quick decrease of AOPCP concentration in BALB/c mice blood after the intravenous injection and in isolated blood sample incubation. These findings emphasize the quick elimination of AOPCP as well as its instability and suggest that the AOPCP concentration have to be accurately and frequently monitored in all the studies that address its clinical application.