Role of Purinergic Signaling in Acupuncture Therapeutics.

Acupuncture is a therapeutic treatment that is well recognized in many countries. However, the initiation mechanisms of acupuncture are not well understood. Purinergic signaling has been considered a key signaling pathway in acupuncture in recent years. Acupuncture-induced ATP is mainly produced by mast cells and fibroblasts, and ATP is gradually hydrolyzed into adenosine. ATP and adenosine further participate in the process of acupuncture information transmission to the nervous and immune systems through specific purine receptors. Acupuncture initiates analgesia via the down-regulation of the expression of P2 receptors or up-regulation of the expression of adenosine A1 receptors on nerve fibers. ATP also promotes the proliferation of immune cells through P2 receptors and A3 receptors, causing inflammation. In contrast, adenosine activates A2 receptors, promotes the production and infiltration of immunosuppressive cells, and causes an anti-inflammatory response. In summary, we described the role of purinergic signaling as a general signaling pathway in the initiation of acupuncture and the influence of purinergic signaling on the neuroimmune network to lay the foundation for future systematic research on the mechanisms of acupuncture therapeutics.

Complementary Role of P2 and Adenosine Receptors in ATP Induced-Anti-Apoptotic Effects Against Hypoxic Injury of HUVECs.

BACKGROUND: Vascular endothelial injury during ischemia generates apoptotic cell death and precedes apoptosis of underlying tissues. We aimed at studying the role of extracellular adenosine triphosphate (ATP) on endothelial cells protection against hypoxia injury. METHODS: In a hypoxic model on endothelial cells, we quantified the extracellular concentration of ATP and adenosine. The expression of mRNA (ectonucleotidases, adenosine, and P2 receptors) was measured. Apoptosis was evaluated by the expression of cleaved caspase 3. The involvement of P2 and adenosine receptors and signaling pathways was investigated using selective inhibitors. RESULTS: Hypoxic stress induced a significant increase in extracellular ATP and adenosine. After a 2-h hypoxic injury, an increase of cleaved caspase 3 was observed. ATP anti-apoptotic effect was prevented by suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and CGS15943, as well as by selective A2A, A2B, and A3 receptor antagonists. P2 receptor-mediated anti-apoptotic effect of ATP involved phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinases (ERK1/2), mitoKATP, and nitric oxide synthase (NOS) pathways whereas adenosine receptor-mediated anti-apoptotic effect involved ERK1/2, protein kinase A (PKA), and NOS. CONCLUSIONS: These results suggest a complementary role of P2 and adenosine receptors in ATP-induced protective effects against hypoxia injury of endothelial. This could be considered therapeutic targets to limit the development of ischemic injury of organs such as heart, brain, and kidney.

Coffee, caffeine, chlorogenic acid, and the purinergic system.

Coffee is a drink prepared from roasted coffee beans and is lauded for its aroma and flavour. It is the third most popular beverage in the world. This beverage is known by its stimulant effect associated with the presence of methylxanthines. Caffeine, a purine-like molecule (1,3,7 trymetylxantine), is the most important bioactive compound in coffee, among others such as chlorogenic acid (CGA), diterpenes, and trigonelline. CGA is a phenolic acid with biological properties as antioxidant, anti-inflammatory, neuroprotector, hypolipidemic, and hypoglicemic. Purinergic system plays a key role inneuromodulation and homeostasis. Extracellular ATP, other nucleotides and adenosine are signalling molecules that act through their specific receptors, namely purinoceptors, P1 for nucleosides and P2 for nucleotides. They regulate many pathological processes, since adenosine, for instance, can limit the damage caused by ATP in the excitotoxicity from the neuronal cells. The primary purpose of this review is to discuss the effects of coffee, caffeine, and CGA on the purinergic system. This review focuses on the relationship/interplay between coffee, caffeine, CGA, and adenosine, and their effects on ectonucleotidases activities as well as on the modulation of P1 and P2 receptors from central nervous system and also in peripheral tissue.

Comparison of common platelet receptors between the chacma baboon (Papio ursinus) and human for use in pre-clinical human-targeted anti-platelet studies.

Anti-platelet agents play a central part in the treatment and prevention of acute thrombotic events. Discriminating animal models are needed for the development of novel agents. The chacma baboon has been extensively used as a model to evaluate anti-platelet agents. However, limited data exist to prove the translatability of this species to humans. We aimed to determine the suitability of the chacma baboon in preclinical human targeted GPIIb/IIIa, GPIbα and P2Y12 studies. Light-transmission platelet aggregometry (LTA), whole blood impedance aggregometry, receptor number quantification and genomic DNA sequencing were performed. Baboon ADP and arachidonic acid-induced LTA aggregation results differed significantly from human values, even at increased concentrations. LTA ristocetin-induced agglutination was comparable between species, but baboon platelets needed twice the concentration of ristocetin to elicit a similar response. Citrated baboon blood had significantly less aggregation than humans when evaluated with impedance aggregometry. However, hirudinised baboon whole blood gave similar aggregation as humans at the same agonist concentrations. GPIIb, GPIIIa and GPIbα numbers were significantly more on the baboon platelets. None of the amino acids deemed vital for receptor function, ligand binding or receptor inhibition, were radically different between the species. However, a conservative change in a calcium-binding region of GPIIb may render the baboon platelets more sensitive to calcium-binding agents. The chacma baboon may be used for the evaluation of human-targeted GPIIb/IIIa-, GPIbα- and P2Y12-inhibiting agents. However, the best anticoagulant, optimal agonist concentrations, increase in receptor number and sequence differences must be considered for any future studies.

Platelet P2Y12 receptors are involved in the haemostatic effect of notoginsenoside Ft1, a saponin isolated from Panax notoginseng.

BACKGROUND AND PURPOSE: Saponins isolated from Panax notoginseng (Burk.) F.H. Chen have been shown to relieve thrombogenesis and facilitate haemostasis. However, it is not known which saponin accounts for this haemostatic effect. Hence, in the present study we aimed to identify which saponins contribute to its haemostatic activity and to elucidate the possible underlying mechanisms. EXPERIMENTAL APPROACH: Platelet aggregation was analysed using a platelet aggregometer. Prothrombin time, activated partial thromboplastin time and thrombin time were measured using a blood coagulation analyser, which was further corroborated with bleeding time and thrombotic assays. The interaction of notoginsenoside Ft1 with the platelet P2Y12 receptor was determined by molecular docking analysis, cytosolic Ca(2+) and cAMP measurements, and phosphorylation of PI3K and Akt assays. KEY RESULTS: Among the saponins examined, Ft1 was the most potent procoagulant and induced dose-dependent platelet aggregation. Ft1 reduced plasma coagulation indexes, decreased tail bleeding time and increased thrombogenesis. Moreover, it potentiated ADP-induced platelet aggregation and increased cytosolic Ca(2+) accumulation, effects that were attenuated by clopidogrel. Molecular docking analysis suggested that Ft1 binds to platelet P2Y12 receptors. The increase in intracellular Ca(2+) evoked by Ft1 in HEK293 cells overexpressing P2Y12 receptors could be blocked by ticagrelor. Ft1 also affected the production of cAMP and increased phosphorylation of PI3K and Akt downstream of P2Y12 signalling pathways. CONCLUSION AND IMPLICATIONS: Ft1 enhanced platelet aggregation by activating a signalling network mediated through P2Y12 receptors. These novel findings may contribute to the effective utilization of this compound in the therapy of haematological disorders.

Discovery of novel P2Y14 agonist and antagonist using conventional and nonconventional methods.

P2Y14 is a member of the pyrimidinergic GPCR family. UDP-Glc has been previously shown to activate human P2Y14, whereas UDP was unable to activate the receptor. In this study, the authors used conventional and nonconventional methods to further characterize P2Y14 and its ligands. Conventional calcium mobilization and nonconventional cellular impedance functional assays revealed that UMP and UDP selectively activated HEK cells coexpressing P2Y14 and Gα(qi5). In the impedance assays, the presence of exogenous Gα(qi5) resulted in agonist-induced Gq signaling, whereas in the absence of exogenous Gα(qi5), the signal was indicative of Gi. The authors established the first P2Y14 membrane filtration binding assay using a novel optimized expression vector and [(3)H]UDP as radioligand. UDP-Glc, UMP, and UDP dose dependently inhibited [(3)H]UDP binding in the binding assay, and saturation analysis revealed that UDP bound P2Y14 with a K(D) = 10 nM and a B(max) = 110 pmol/mg. The authors screened a phosphonate library and identified compound A, which inhibited UDP-Glc-mediated calcium signaling in the fluorometric imaging plate reader assay (IC(50) = 2.3 µM) and competed for [(3)H]UDP binding in the novel binding assay with a K(i) = 1280 nM.

Gi- and Gq-coupled ADP (P2Y) receptors act in opposition to modulate nociceptive signaling and inflammatory pain behavior.

BACKGROUND: Investigations of nucleotide signaling in nociception to date have focused on actions of adenosine triphosphate (ATP). Both ATP-gated ion channels (P2X receptors) and G protein-coupled (P2Y) receptors contribute to nociceptive signaling in peripheral sensory neurons. In addition, several studies have implicated the Gq-coupled adenosine diphosphate (ADP) receptor P2Y1 in sensory transduction. In this study, we examined the expression and function of P2Y1 and the Gi-coupled receptors P2Y12, P2Y13 and P2Y14 in sensory neurons to determine their contribution to nociception. RESULTS: We detected mRNA and protein for ADP receptors P2Y12 and P2Y13 in mouse dorsal root ganglia (DRG). P2Y14, a homologous Gi-coupled nucleotide receptor, is also expressed in DRG. Immunohistochemical analysis of receptor distribution indicated that these receptors are widely expressed in nociceptive neurons. Using ratiometric calcium imaging, we found that ADP evokes increases in intracellular calcium in isolated DRG neurons and also produces a pertussis toxin-sensitive inhibition of depolarization-evoked calcium transients. The inhibitory effect of ADP was unaltered in the presence of the selective P2Y1 antagonist MRS2179 and in neurons isolated from P2Y1 knockout mice, whereas ADP-evoked calcium transients were greatly reduced. Analysis of behavioral responses to noxious heat before and after inflammatory injury (injection of complete Freund's adjuvant into the hindpaw) revealed that P2Y1 is required for the full expression of inflammatory hyperalgesia, whereas local injection of agonists for Gi-coupled P2Y receptors reduced hyperalgesia. CONCLUSIONS: We report that Gi-coupled P2Y receptors are widely expressed in peripheral sensory neurons. Agonists for these receptors inhibit nociceptive signaling in isolated neurons and reduce behavioral hyperalgesia in vivo. Anti-nociceptive actions of these receptors appear to be antagonized by the Gq-coupled ADP receptor, P2Y1, which is required for the full expression of inflammatory hyperalgesia. We propose that nociceptor sensitivity is modulated by the integration of nucleotide signaling through Gq- and Gi-coupled P2Y receptors, and this balance is altered in response to inflammatory injury. Taken together, our data suggest that Gi-coupled P2Y receptors are broadly expressed in nociceptors, inhibit nociceptive signaling in vivo, and represent potential targets for the development of novel analgesic drugs.

Role of midbrain periaqueductal gray P2X3 receptors in electroacupuncture-mediated endogenous pain modulatory systems.

Extracellular ATP facilitates pain transmission at peripheral and spinal sites via the P2X receptors and the P2X3 subtype is an important candidate for this effect. Electroacupuncture (EA) has been clinically utilized to manage chronic pain. In this study, with neuropathic pain model of Sprague-Dawley (SD) rats, the P2X3 receptor protein level and expression location in the midbrain periaqueductal gray (PAG), a crucial site in endogenous pain modulatory system, were evaluated by Western blotting and immunohistochemistry. The results showed (1) pain thresholds were decreased while P2X3 receptor expression was up-regulated in the lateral PAG (lPAG) when neuropathic pain occurred. When the lPAG was pretreated with P2X3 receptors, antagonist A-317491 attenuated the antinociceptive effect produced by intra-lPAG injection of alpha,beta-methylene-ATP (alpha, beta-meATP), an agonist for P2X3 receptor. (2) Multiple EA treatments begot enhanced pain thresholds and increased P2X3 receptor immunoreactivity in the lPAG in neuropathic pain rats. Conversely, the down-regulated P2X3 receptor expression in the lPAG with antisense oligodeoxynucleotide (ODN) for P2X3 gene significantly attenuated the antinociceptive effect of EA treatment. These results suggest that P2X3 receptors in the lPAG play an inhibitory role in pain modulation and EA exerts a marked therapeutic effect in relieving neuropathic pain in CCI rats, which may be related to its regulative effect on the expression of P2X3 receptors in the lPAG. In conclusion, P2X3 receptors in the lPAG are involved in the supraspinal antiociception effect of EA treatment.

An extract (THC-002) of Ba-Wei-Die-Huang-Wan inhibits expression of tachykinins, and P2X3 and TRPV1 receptors, and inhibits ATP-induced detrusor overactivity in spontaneously hypertensive rats.

AIMS: To investigate possible mechanisms of action of THC-002 (HARNCARE), a galenical produced from the traditional Chinese herbal mixture Ba-Wei-Die-Huang-Wan, which has been reported to improve lower urinary tract symptoms (LUTS) in patients. METHODS: Forty-five female SHRs were randomly separated into three groups. Two groups were given 20 ml physiological saline solution (PSS) per kg-body weight orally daily for 1 week. An hour after the administration of PSS, one of the groups received 20 mg THC-002 per kg body weight, and the other a similar volume of THC-002-free saline. The third group received no treatments. The bladders were analyzed by real time RT-PCR (n = 6) and immunohistochemistry (n = 3) for the expression of tachykinins and P2X3 and TRPV1 receptors. Cystometric investigation (n = 6) was conducted after intravesical instillation of saline followed by 5 mg/ml ATP solution. RESULTS: Treatment with PSS caused and upregulation of tachykinins and P2X3 and TRPV1 receptors, which was prevented in the group treated with THC-002. In the normal (non-treated) and non-THC-002-treated SHRs, instillation of the ATP solution decreased voiding interval, micturition volume, and bladder capacity compared to the instillation of saline. However, in the THC-002-treated SHRs, ATP instillation had no effect. CONCLUSIONS: In SHRs, THC-002 reduced the bladder expression of tachykinins and P2X3 and TRPV1 receptors, and inhibited ATP-induced detrusor overactivity. These effects may explain part of its beneficial effects on LUTS.

Role of sodium ferulate in the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X(3) receptor.

Neuropathic pain usually is persistent and no effective treatment. ATP plays an important role in the initiation of pain. P2X(3) receptors are localized in the dorsal root ganglion (DRG) neurons and activated by extracellular ATP. Sodium ferulate (SF) is an active principle from Chinese herbal medicine and has anti-inflammatory activities. This study observed the effects of SF on the nociceptive facilitation of the primary sensory afferent after chronic constriction injury (CCI) mediated by P2X(3) receptor. In this study, the content of ATP in DRG neurons was measured by high-performance liquid chromatography (HPLC). P2X(3) agonist-activated currents in DRG neurons was recorded by the whole-cell patch-clamp skill. The expression of P2X(3) mRNA in DRG neurons was analyzed by in situ hybridization. The ATP content of DRG was increased after CCI. In CCI rats treated with SF, the content of ATP in DRG neurons was reduced. SF decreased the increment of P2X(3) agonist-activated currents and P2X(3) mRNA expression in DRG neurons during CCI. SF may inhibit the initiation of pain and primary afferent sensitization mediated by P2X(3) receptor during CCI.

Gosha-jinki-gan reduces transmitter proteins and sensory receptors associated with C fiber activation induced by acetic acid in rat urinary bladder.

AIM: We determined if Gosha-jinki-gan, a traditional Chinese herbal mixture, reduced the presence of the tachykinins neurokinin A, neurokinin B, and substance P, as well as the transient receptor potential vanilloid 1 (TRPV1) and P2X3 purine receptors that are functionally associated with C fibers in the urinary bladder. METHODS: Thirty-six female rats were fed with either a standard diet or one supplemented with 1.08% Gosha-jinki-gan. After 4 weeks, the urinary bladders were instilled with either saline or 0.1% acetic acid. After 30 min, the bladders were removed and expression of the tachykinins and the TRPV1 and P2X3 receptors was determined by immunohistochemistry and mRNA expression. RESULTS: In rats fed with the standard diet, the tachykinins and the TRPV1 and P2X3 receptors expressed nearby or within urothelium of the acetic acid-treated rats increased compared with the saline-instilled rats. In rats pretreated with Gosha-jinki-gan, the tachykinins and the TRPV1 and P2X3 receptors in the acetic acid-treated rats also increased compared with the saline-instilled rats. However, with the instillation of acetic acid, the tachykinins and the TRPV1 and P2X3 receptors of Gosha-jinki-gan pretreated rats decreased compared with standard diet fed rats. The mRNA expression levels of neurokinin A, substance P, and the TRPV1 receptor in acetic acid-treated Gosha-jinki-gan pretreated rats were lower than that in acetic acid-treated standard diet fed rats. Gosha-jinki-gan did not destroy nerve fibers within the bladders. CONCLUSIONS: Gosha-jinki-gan partially reduced the tachykinins and TRPV1 and P2X3 purine receptors without destroying the nerve fibers.

Thermal nociception and TRPV1 function are attenuated in mice lacking the nucleotide receptor P2Y2.

Recent studies indicate that ATP and UTP act at G protein-coupled (P2Y) nucleotide receptors to excite nociceptive sensory neurons; nucleotides also potentiate signaling through the pro-nociceptive capsaicin receptor, TRPV1. We demonstrate here that P2Y(2) is the principal UTP receptor in somatosensory neurons: P2Y(2) is highly expressed in dorsal root ganglia and P2Y(2)-/- mice showed profound deficits in UTP-evoked calcium transients and potentiation of capsaicin responses. P2Y(2)-/- mice were also deficient in the detection of painful heat: baseline thermal response latencies were increased and mutant mice failed to develop thermal hypersensitivity in response to inflammatory injury (injection of complete Freund's adjuvant into the hindpaw). P2Y(2) was the only Gq-coupled P2Y receptor examined that showed an increase in DRG mRNA levels in response to inflammation. Surprisingly, TRPV1 function was also attenuated in P2Y(2)-/- mice, as measured by the frequency and magnitude of capsaicin responses in vitro and behavioral responses to capsaicin administration in vivo. However, TRPV1 mRNA levels and immunoreactivity were not reduced, and behavioral sensitivity to capsaicin could be largely restored in P2Y(2)-/- mice by pretreatment with bradykinin, suggesting that normal function of TRPV1 requires ongoing modulation by G protein-coupled receptors. These results indicate that nucleotide signaling through P2Y(2) plays a key role in thermal nociception.

In vitro and in vivo evidence for a role of the P2X7 receptor in the release of IL-1 beta in the murine brain.

The P2X(7) receptor (P2X(7)R) is a purinoceptor expressed predominantly by cells of immune origin, including microglial cells. P2X(7)R has a role in the release of biologically active proinflammatory cytokines such as IL-1 beta, IL-6 and TNFalpha. Here we demonstrate that when incubated with lipopolysaccharide (LPS), glial cells cultured from brain of P2X(7)R(-/-) mice produce less IL-1 beta compared to glial cells from brains of wild-type mice. This is not the case for TNFalpha and IL-6. Our results indicate a selective effect of the P2X7R gene deletion on release of IL-1 beta release but not of IL-6 and TNFalpha. In addition, we confirm that only microglial cells produce IL-1beta, and this release is dependent on P2X(7)R and ABC1 transporter. Because IL-1 beta is a key regulator of the brain cytokine network and P2X(7)R is an absolute requirement for IL-1 beta release, we further investigated whether response of brain cytokines to LPS in vivo was altered in P2X(7)R(-/-) mice compared to wild-type mice. IL-1 beta and TNFalpha mRNAs were less elevated in the brain of P2X(7)R(-/-) than in the brain of wild-type mice in response to systemic LPS. These results show that P2X7R plays a key role in the brain cytokine response to immune stimuli, which certainly applies also to cytokine-dependent alterations in brain functions including sickness behavior.

Dasatinib: a tyrosine kinase inhibitor for the treatment of chronic myelogenous leukemia and philadelphia chromosome-positive acute lymphoblastic leukemia.

BACKGROUND: The Philadelphia chromosome is formed from a translocation of genetic material involving human chromosomes 9 and 22. The resulting gene product, BCR-ABL, encodes for an abnormal tyrosine kinase (TK) that is a factor in the pathology of chronic myelogenous leukemia (CML). Use of targeted therapy that inhibits BCR-ABL kinase activity may lead to hematologic and cytogenetic responses in affected individuals. The oral TK inhibitor dasatinib was approved in 2006 for use in patients with CML or Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL) who are unable to tolerate or have not responded to other treatments. OBJECTIVE: This paper reviews the available data on dasatinib, including its pharmacokinetic and pharmacodynamic properties, findings of in vitro and in vivo studies, adverse effects, and potential place in therapy. METHODS: Pertinent information was identified through searches of MEDLINE (1966-May 2007), EMBASE (1980-first quarter 2007), and International Pharmaceutical Abstracts (1970-May 2007) using the terms dasatinib, BMS-354825, chronic myelogenous leukemia, Sprycel, Philadelphia chromosome, and acute lymphoblastic leukemia. All clinical studies and case reports published at the time of the search were included in this review. RESULTS: Observed mutations in the amino acid sequence of BCR-ABL cause the failure of treatment with existing TK inhibitors. Dasatinib has shown in vitro and in vivo activity against BCR-ABL, including mutations that are resistant to other available TK inhibitors. Preliminary results are available from several noncomparative studies of dasatinib in patients who were unable to tolerate or were resistant to previous therapies. The 5 phases of START (SRC/ABL Tyrosine kinase inhibition Activity Research Trials of dasatinib) represent the largest and most comprehensive evaluation of dasatinib in the treatment of patients in all stages of CML or Philadelphia chromosome-positive ALL who had undergone previous treatment for leukemia. Dasatanib had the greatest benefit in patients in the chronic phase of CML, with complete hematologic responses in 90% of patients, 52% of whom achieved a major hematologic response. Compared with those in the chronic phase, patients in the accelerated phase or blast crisis of CML, or with Philadelphia chromosome-positive ALL had lower responses. In the START-R trial, which compared the response to dasatinib and high-dose imatinib (800 mg/d), both regimens had comparable ability to induce a complete hematologic response (95% and 93%, respectively), although more patients achieved a major cytogenetic response with dasatinib (32% vs 7%). Adverse effects include significant myelosuppression. Dasatinib may have the potential for use in the management of nonleukemic malignancies. CONCLUSIONS: Dasatinib has a wider spectrum of activity against a broader range of BCR-ABL forms than existing TK inhibitors. It has shown clinical benefit and tolerability in patients in all phases of CML, as well as in those with Philadelphia chromosome-positive ALL. Dasatinib illustrates the potential for targeted drug development based on an understanding of the genetic alterations leading to CML and the development of resistance to treatment.

Effect of hyperhomocysteinemia on cardiovascular risk factors and initiation of atherosclerosis in Wistar rats.

Hyperhomocysteinemia is considered an independent risk factor for atherosclerosis. The present study was designed to assess the effect of high level of serum homocysteine on other cardiovascular risk factors and markers in rats and to study its mode of action in initiating atherosclerosis. To address this issue, four different doses of methionine (0.1 g/kg, 0.25 g/kg, 0.5 g/kg, 1 g/kg) were orally administered to four groups (Group II, III, IV, V respectively) of rats (6 rats in each group) for a period of 8 weeks to get different level of homocysteine in serum. Group I was administered with saline and served as control. Our results revealed that the level of Total cholesterol, Triglyceride, and Oxidized low-density lipoproteins increased significantly with the increase in the level of serum homocysteine. The levels of Resistin, C-reactive protein and cysteinyl-leukotrienes were found to be significantly high in Group IV (P<0.001 vs Group I) and Group V (P<0.001 vs Group I) at 8 weeks. Total antioxidant capacity and nitrite/nitrate level in serum showed negative correlation with the increased dose of methionine. The mRNA expression and the enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase significantly increased only in livers of rats of Group V. Furthermore, high mRNA expression of P2 receptors and caveolin were found in aorta of rats administered with high dose of methionine (Group IV and V at 8 weeks). Data obtained from in-vitro effect of homocysteine on isolated aortic arch also showed induction in P2 receptors and caveolin with the increase in the concentration of homocysteine. These findings collectively suggest that hyperhomocysteinemia initiates atherosclerosis by modulating the cholesterol biosynthesis and by significantly inducing the level of other cardiovascular risk factors and markers, which play important role in initiating atherosclerosis.

Immobilized P2X2 purinergic receptor stationary phase for chromatographic determination of pharmacological properties and drug screening.

The purinergic receptor signaling system plays an important role in communication between cells in the nervous system and opens new opportunities for screening of potential drugs. Our objective was to explore the pharmacological properties and establish a new methodology for ligand screening for the P2X2 receptor, which has been developed by the combinatorial library approach Systematic Evolution of Ligands by Exponential enrichment (SELEX). To this end, membranes of 1321N1 cells stably transfected with rat P2X2 receptors were resuspended in 2% cholate detergent and subsequently coupled onto an immobilized artificial membrane (IAM). The IAM-cholate-P2X2 mixture was then dialyzed, centrifuged and packed into a FPLC column. Equilibrium binding to the receptor and competition between ATP and the purinergic antagonists suramin and 2'3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) were analyzed by a chromatographic assay using 32P alpha ATP as a radioligand. Our data indicate that suramin does not compete with ATP for the ligand binding site and TNP-ATP is a competitive antagonist, confirming previous studies [C.A. Trujillo, A.A. Nery, A.H. Martins, P. Majumder, F.A. Gonzalez, H. Ulrich, Biochemistry 45 (2006) 224-233]. In addition, we demonstrate that this assay can be used in in vitro selection procedures for RNA aptamers binding to P2X2 receptors. The results demonstrate that the receptor can be immobilized in a stable format and reused over an extended period of time, facilitating the exploration of ligand-receptor interactions and screening of combinatorial pools for possible ligands.

P2X2R purinergic receptor subunit mRNA and protein are expressed by all hypothalamic hypocretin/orexin neurons.

Neurophysiologic data suggest that orexin neurons are directly excited by ATP through purinergic receptors (P2XR). Anatomical studies, though reporting P2XR in the hypothalamus, did not describe it in the perifornical hypothalamic area, where orexinergic neurons are located. Here we report the presence of the P2X(2)R subunit in the rat perifornical hypothalamus and demonstrate that hypothalamic orexin neurons express the P2X(2)R. Double immunohistochemistry showed that virtually all orexin-immunoreactive neurons are also P2X(2)R immunoreactive, whereas 80% of P2X(2)R-immunoreactive neurons are also orexin positive. Triple-labeling experiments, combining fluorescence in situ hybridization for P2X(2)R mRNA and P2X(2)R/orexin double immunofluorescence, confirmed these findings. In addition, in situ hybridization demonstrated that P2X(2)R mRNA is localized in cellular processes of orexinergic neurons. The present data support neurophysiologic findings on ATP modulation of orexinergic function and provide direct evidence that the entire population of orexin neurons expresses a P2XR subtype, namely, P2X(2)R. Thus, purinergic transmission might intervene in modulating key functions known to be controlled by the orexinergic system, such as feeding behavior and arousal.

P2X5 receptors are expressed on neurons containing arginine vasopressin and nitric oxide synthase in the rat hypothalamus.

In this study, the P2X(5) receptor was found to be distributed widely in the rat hypothalamus using single and double labeling immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR) methods. The regions of the hypothalamus with the highest expression of P2X(5) receptors in neurons are the paraventricular and supraoptic nuclei. The intensity of P2X(5) immunofluorescence in neurons of the ventromedial nucleus was low. 70-90% of the neurons in the paraventricular nucleus and 46-58% of neurons in the supraoptic and accessory neurosecretory nuclei show colocalization of P2X(5) receptors and arginine vasopressin (AVP). None of the neurons expressing P2X(5) receptors shows colocalization with AVP in the suprachiasmatic and ventromedial nuclei. 87-90% of the neurons in the lateral and ventral paraventricular nucleus and 42-56% of the neurons in the accessory neurosecretory, supraoptic and ventromedial nuclei show colocalization of P2X(5) receptors with neuronal nitric oxide synthase (nNOS). None of the neurons expressing P2X(5) receptors in the suprachiasmatic nucleus shows colocalization with nNOS. These findings provide a morphological basis for possible functional interactions between the purinergic and nitrergic or vasopressinergic neurotransmitter systems.

Expression of purinergic receptors in the hypothalamus of the rat is modified by reduced food availability.

ATP-sensitive P2 receptors are suggested to play an important role in the cerebral signal transduction. We examined the expression of the P2Y1 receptor and the possibly downstream-related neuronal nitric oxide synthase (nNOS) in the hypothalamus of rats food-restricted for 3 or 10 days and rats refed after a restriction of 10 days. The restriction caused a reduction of the body weight and plasma triacylglyceride, an increase of non-esterified fatty acid levels correlating with a decrease of leptin levels and an enhancement of plasma corticosterone. All changes returned to basal levels after refeeding. The restriction induced an enhanced intake within 30 min after food presentation and a reduction in the latency. Interestingly, the latter was not abolished by refeeding. The daily food intake induced by refeeding was enhanced at the first day only. The expression of hypothalamic P2Y1 receptor/nNOS mRNA and protein and of leptin receptor mRNA were enhanced after restricted feeding. These changes were abolished after 3 days of refeeding. Immunofluorescence studies indicated that P2Y1 receptor and nNOS immunoreactivities are present in the dorsomedial, ventromedial and lateral hypothalamus and in the nucleus arcuatus. P2Y1 receptor-positive cells were partially also nNOS-positive. The P2Y1 receptor labeling was restricted to cell bodies of obviously non-glial cells, whereas nNOS labeling could be detected also at cellular processes of these cells. In the nucleus arcuatus, astrocytes were identified, expressing P2Y1 receptors at cell bodies and cellular processes. The data suggest that restricted feeding may enhance the sensitivity of the hypothalamus to extracellular ADP/ATP by regulation of the expression of P2Y1 receptors and possibly of their signal transduction pathway via nitric oxide production.