Purinoceptor Antagonist Research Articles

Hexamethonium-induced augmentation of the electrical twitch response in the guinea-pig ileum longitudinal muscle–myenteric plexus strip

Longitudinal muscle–myenteric plexus strips of the guinea-pig ileum were used to investigate the nature of the hexamethonium-induced augmentation of the twitch response. All preparations were set up in Tyrode solution and intermittent longitudinal twitch contractions were evoked by single pulse electrical field stimulation. Hexamethonium, a blocker of nicotinic ganglionic transmission, at 300μmol/l and 1mmol/l augmented the twitch contractions by 21% and 35%, respectively. First we tested for a possible nicotinic drive onto an inhibitory neuronal component to the longitudinal smooth muscle cells. However, guanethidine (5μmol/l), naloxone (1μmol/l), or l-NAME (300μmol/l) were without effect on the hexamethonium-induced augmentation. The P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS), 25–100μmol/l, without altering the control twitch responses, dose-dependently reduced the hexamethonium-induced augmentation; at 100μmol/l a statistically significantly inhibition was observed. Based on these functional experiments we found no evidence that blocking nicotinic transmission removed a tonic adrenergic, opioidergic or nitrergic inhibitory input to the longitudinal muscle. However, we provide evidence for a hexamethonium-induced augmentation of the P2 purinergic input to cholinergic motoneurons of the guinea-pig ileum longitudinal muscle. The P2-nicotinic receptor interaction presents a novel modulatory mechanism to cholinergic myenteric motor neurons.

A positive inotropic effect of UTP in the human cardiac atrium

In the cardio-vascular system extracellular UTP can induce receptor-mediated vasoconstriction via smooth muscle cells and vasodilatation via endothelial cells. We evaluated inotropic effects of UTP in preparations from human heart. Contractile effects were studied in atrial preparations from patients undergoing cardiac bypass surgery. For comparison, contractility in isolated spontaneously beating right atrial and paced left atrial preparations from mice was investigated. UTP and UTPγS concentration-dependently exerted a positive inotropic effect with a maximum at 100µM UTP that amounted to 156% of pre-drug value (n=13) without changing time parameters of contraction. UTP was able to partially attenuate the positive inotropic effect of β-adrenoceptor stimulation. UTP did not change the beating rate in right atrial mouse preparations. The positive inotropic effect of UTP could not be blocked by the P2 purinoceptor antagonists suramin (100µM and 500µM), PPADS (50µM) and reactive blue (100µM). Likewise inhibitors of PLC activity (U73122) and of adenylyl cyclase activity (SQ22563; 10µM each) failed to affect the effects of UTP. In summary, we describe a novel positive inotropic effect of UTP on force contraction in the isolated human atrium. We tentatively suggest that UTP might act via P2Y2- or P2Y4-like receptors.

Differential regulation of atrial contraction by P1 and P2 purinoceptors in normotensive and spontaneously hypertensive rats

In the normotensive rat atrium, adenosine-5'-triphosphate and uridine-5'-triphosphate exert a biphasic effect consisting of an initial negative inotropic effect (NIE) followed by a subsequent positive inotropic effect (PIE). We comparatively studied these responses in normotensive Wistar rats (NWRs) and spontaneously hypertensive rats (SHRs). Compared with NWRs, the NIE responses in the atria were lower and the PIE responses were higher in SHRs. The P1 purinoceptor antagonist, D 8-cyclopentyl-1,3-dipropylxanthine, partially blocked the NIE responses of both ATP and UTP and mildly enhanced the PIE responses in both NWRs and SHRs. Furthermore, the P2 purinoceptor blockers suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium salt induced a pronounced block of the PIE responses in both atria types. The PIE responses to ATP were inhibited more efficiently by nifedipine. These responses were depressed by ryanodine and, to a lesser extent, carbonyl cyanide 3-chlorophenylhydrazone in SHR atria compared with NWR atria. The higher responses in SHR rats suggest the existence of an augmented endoplasmic reticulum Ca(2+) store and faster mitochondrial Ca(2+) cycling in SHR atria compared with NWR atria. These data support the hypothesis that a dysfunction of purinergic neurotransmission and enhanced sympathetic activity are contributing factors in the pathogenesis of hypertension.

The Operculina macrocarpa (l.) urb. (jalapa) tincture modulates human blood platelet aggregation

Ethnopharmacological relevanceOperculina macrocarpa is an ornamental climbing plant of the Northeastern Brazil extensively used in traditional medicine as depurative of the blood and for the treatment of thrombosis. To investigate the antiplatelet and anticoagulant potential of Operculina macrocarpa and to determine the possible mechanisms of action. Material and methodsThe Operculina macrocarpa tincture (OMT) was characterized by the polyphenol content and chromatographic profile established by HPLC with detection and quantification of three phenol acids (caffeic, clorogenic and gallic acids). The human platelet aggregation was induced in vitro by the agonists ADP, collagen, thrombin, epinephrine or arachidonic acid, and the antiplatelet effect of OMT was evaluated in the presence or absence of aspirin (a nonselective inhibitor of cyclooxygenase), pentoxifylline (a phosphodiesterase inhibitor), ticlopidine (a P2Y12 purinoceptor antagonist) or ODQ (a selective inhibitor of guanilate cyclase). The effect of OMT on the partial thromboplastin time, prothrombin time and bleeding time were investigated on human or rat plasma. ResultsThe strongest antiplatelet effect of OMT (50–400µg/mL) was observed on the ADP- induced aggregation with inhibitions up to 55%, while among others agonists (epinephrine, collagen, thrombin and arachidonic acid) maximal inhibitions reached by OMT (200µg/mL) were on platelet aggregation induced by collagen (18%) or epinephrine (20%). The antiplatelet effect of OMT (400µg/mL) was comparable to aspirin, a nonspecific inhibitor of cyclooxygenase. The ticlopidine and pentoxifylline increased 5.1 and 3.8 fold the inhibitory effect of OMT on ADP-induced platelet aggregation, respectively. On the other hand, l-arginine, ODQ and aspirin showed a slightly or no effect on antiplatelet effect of OMT. The bleeding time in rats was significantly increased by OMT, but the tincture did not interfere on the activated partial thromboplastin or prothrombin time in human plasma. ConclusionsThis study showed that the tincture of Operculina macrocarpa has antiplatelet effect that cannot be attributed to a single biochemical mechanism and at least part of it cannot be related to the OMT inhibition of P2Y12 purinergic receptors.

Purinoceptor-mediated, Capsaicin-resistant Excitatory Effect of Allyl Isothiocyanate on Neurons of the Guinea-Pig Small Intestine

Allyl isothiocyanate (AITC; 200μM) caused atropine- and tetrodotoxin-sensitive longitudinal muscle contraction on the guinea-pig small intestine. The response was not influenced by hexamethonium, a functional blockade of capsaicin-sensitive neurons or by antagonists acting at TRPV1 or TRPA1, but was abolished by the P2 purinoceptor antagonist PPADS (50μM). It is concluded that cholinergic motoneurons are activated by a purinergic mechanism in the course of the AITC response, independently of capsaicin-sensitive processes or even TRPA1.

Influence of pressure on adenosine triphosphate function as a sympathetic neurotransmitter in small mesenteric arteries from the spontaneously hypertensive rat

Enhanced sympathetic neurotransmission contributes to hypertension in the spontaneously hypertensive rat (SHR). We recently reported a method for studying sympathetic neurotransmission in pressurized small arteries, demonstrating a major role of adenosine triphosphate (ATP) as a sympathetic neurotransmitter under these physiological conditions. We have now used this methodology to assess the role of ATP as a sympathetic neurotransmitter in small mesenteric arteries isolated from SHRs. Small arteries were mounted in a suction electrode, cannulated and pressurized to either 30 or 90 mmHg. Nerve-evoked alterations in membrane potential were assessed using sharp microelectrodes. Neurally evoked vasoconstrictor responses were measured in the absence and presence of the α1-adrenoceptor antagonist, tamsulosin (0.1 μmol/l), or the P2 purinoceptor antagonist suramin (0.1 mmol/l). At 30 mmHg the P2X-receptor-mediated excitatory junctional potential (EJP) was larger in arteries from SHRs (7.9 ± 0.9 mV) than Wistar-Kyoto (WKY) rats (3.2 ± 0.4 mV, P < 0.05). Increasing pressure increased the amplitude of the EJP, which again, was larger in SHRs. At 90 mmHg, activation of the perivascular nerves produced a larger vasoconstriction in arteries isolated from SHRs compared with WKY rats. The vasoconstrictor response in SHRs was abolished by either suramin or tamsulosin. These data provide electrophysiological evidence for enhanced purinergic function in the SHR and show that ATP is fundamentally important in contributing to the vasoconstriction produced after activation of the perivascular nerves in pressurized arteries from the SHR. This involves a synergistic interaction with noradrenaline to causes enhanced mesenteric arterial vasoconstriction, which may contribute to the hypertension in this model.

RNA‐seq analysis identifies potential modulators of gravity response in spores of Ceratopteris (Parkeriaceae): Evidence for modulation by calcium pumps and apyrase activity

Gravity regulates the magnitude and direction of a trans-cell calcium current in germinating spores of Ceratopteris richardii. Blocking this current with nifedipine blocks the spore's downward polarity alignment, a polarization that is fixed by gravity ∼10 h after light induces the spores to germinate. RNA-seq analysis at 10 h was used to identify genes potentially important for the gravity response. The data set will be valuable for other developmental and phylogenetic studies. De novo Newbler assembly of 958 527 reads from Roche 454 sequencing was executed. The sequences were identified and analyzed using in silico methods. The roles of endomembrane Ca(2+)-ATPase pumps and apyrases in the gravity response were further tested using pharmacological agents. Transcripts related to calcium signaling and ethylene biosynthesis were identified as notable constituents of the transcriptome. Inhibiting the activity of endomembrane Ca(2+)-ATPase pumps with 2,5-di-(t-butyl)-1,4-hydroquinone diminished the trans-cell current, but increased the orientation of the polar axis to gravity. The effects of applied nucleotides and purinoceptor antagonists gave novel evidence implicating extracellular nucleotides as regulators of the gravity response in these fern spores. In addition to revealing general features of the transcriptome of germinating spores, the results highlight a number of calcium-responsive and light-receptive transcripts. Pharmacologic assays indicate endomembrane Ca(2+)-ATPases and extracellular nucleotides may play regulatory roles in the gravity response of Ceratopteris spores.

N-cadherin expression is regulated by UTP in schwannoma cells

Schwann cells (SCs) are peripheral myelinating glial cells that express the neuronal Ca(2+)-dependent cell adhesion molecule, neural cadherin (N-cadherin). N-cadherin is involved in glia-glia and axon-glia interactions and participates in many key events, which range from the control of axonal growth and guidance to synapse formation and plasticity. Extracellular UTP activates P2Y purinergic receptors and exerts short- and long-term effects on several tissues to promote wound healing. Nevertheless, the contribution of P2Y receptors in peripheral nervous system functions is not completely understood. The current study demonstrated that UTP induced a dose- and time-dependent increase in N-cadherin expression in SCs. Furthermore, N-cadherin expression was blocked by the P2 purinoceptor antagonist suramin. The increased N-cadherin expression induced by UTP was mediated by phosphorylation of mitogen-activated protein kinases (MAPKs), such as Jun N-terminal kinase, extracellular-regulated kinase and p38 kinase. Moreover, the Rho kinase inhibitor Y27632, the phospholipase C inhibitor U73122 and the protein kinase C inhibitor calphostin C attenuated the UTP-induced activation of MAPKs significantly. Extracellular UTP also modulated increased in the expression of the early transcription factors c-Fos and c-Jun. We also demonstrated that the region of the N-cadherin promoter between nucleotide positions -3698 and -2620, which contained one activator protein-1-binding site, was necessary for UTP-induced gene expression. These results suggest a novel role for P2Y purinergic receptors in the regulation of N-cadherin expression in SCs.

Adenosine receptor antagonism suppresses functional and histological inflammatory changes in the rat urinary bladder

Cyclophosphamide (CYP) induces an interstitial cystitis-like inflammation. The resulting bladder dysfunction has been associated with increased release of adenosine-5'-triphosphate (ATP), structural bladder wall changes and contractile impairment. Due to the inflammatory modulatory effects of purines it was presently wondered if pre-treatment with P1 and P2 purinoceptor antagonists affect the CYP-induced alterations. Rats were pre-treated with saline or antagonists for five days, and 60 h before the in vitro functional examination the rats were administered either saline or CYP. Histological examination revealed CYP-induced bladder wall thickening largely depending on submucosal enlargement, mast cell invasion of the detrusor muscle, increase in muscarinic M5 receptor expression and macrophage migration inhibitory factor (MIF) occurrence in large parts of the urothelium. Functionally, methacholine- and ATP-evoked contractions were smaller in urinary bladders from CYP-treated rats. Pre-treatment with the P2 purinoceptor antagonist suramin and the P1A2B antagonist PSB1115 did not to any great extent affect the CYP-induced changes. The P1A1 antagonist DPCPX, however, abolished the difference of methacholine-evoked contractions between saline- and CYP-treated rats. ATP-evoked contractions were reduced in control after the DPCPX pre-treatment, but not in cystitis. The functional observations for DPCPX were supported by its suppression of CYP-induced submucosal thickening, muscarinic M5 receptor expression and, possibly, detrusor mast cell infiltration and the spread of urothelial MIF occurrence. Thus, P1A1 is an important pro-inflammatory receptor in the acute CYP-induced cystitis and a P1A1 blockade during the initial phase may suppress CYP-induced cystitis. P1A1 purinoceptors seem to regulate contractility in healthy and in inflamed rat urinary bladders.

A role for ATP in bronchoconstriction‐induced activation of guinea pig vagal intrapulmonary C‐fibres

Activation of vagal afferent sensory C-fibres in the lungs leads to reflex responses that produce many of the symptoms associated with airway allergy. There are two subtypes of respiratory C-fibres whose cell bodies reside within two distinct ganglia, the nodose and jugular, and whose properties allow for differing responses to stimuli. We here used extracellular recording of action potentials in an ex vivo isolated, perfused lung-nerve preparation to study the electrical activity of nodose C-fibres in response to bronchoconstriction. We found that treatment with both histamine and methacholine caused strong increases in tracheal perfusion pressure that were accompanied by action potential discharge in nodose, but not in jugular C-fibres. Both the increase in tracheal perfusion pressure and action potential discharge in response to histamine were significantly reduced by functionally antagonizing the smooth muscle contraction with isoproterenol, or by blocking myosin light chain kinase with ML-7. We further found that pretreatment with AF-353 or 2',3'-O-(2,4,6-Trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP), structurally distinct P2X3 and P2X2/3 purinoceptor antagonists, blocked the bronchoconstriction-induced nodose C-fibre discharge. Likewise, treatment with the ATPase apyrase, in the presence of the adenosine A1 and A2 receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and SCH 58261, blocked the C-fibre response to histamine, without inhibiting the bronchoconstriction. These results suggest that ATP released within the tissues in response to bronchoconstriction plays a pivotal role in the mechanical activation of nodose C-fibres.

Inhibitory effect of schisandrin on spontaneous contraction of isolated rat colon

This study examined the effect of schisandrin, one of the major lignans isolated from Schisandra chinensis, on spontaneous contraction in rat colon and its possible mechanisms. Schisandrin produced a concentration-dependent inhibition (EC₅₀=1.66 μM) on the colonic spontaneous contraction. The relaxant effect of schisandrin could be abolished by the neuronal Na+ channel blocker tetrodotoxin (1 μM) but not affected by propranolol (1 μM), phentolamine (1 μM), atropine (1 μM) or nicotine desensitization, suggesting possible involvement of non-adrenergic non-cholinergic (NANC) transmitters released from enteric nerves. N(ω)-nitro-l-arginine methyl ester (100-300 μM), a nitric oxide synthase inhibitor, attenuated the schisandrin response. The role of nitric oxide (NO) was confirmed by an increase in colonic NO production after schisandrin incubation, and the inhibition on the schisandrin responses by soluble guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-α]-quinoxalin-1-one (1-30 μM). Non-nitrergic NANC components may also be involved in the action of schisandrin, as suggested by the significant inhibition of apamin on the schisandrin-induced responses. Pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate (100 μM), a selective P2 purinoceptor antagonist, markedly attenuated the responses to schisandrin. In contrast, neither 8-cyclopentyl-1,3-dipropylxanthine, an antagonist for adenosine A₁ receptors, nor chymotrypsin, a serine endopeptidase, affected the responses. All available results have demonstrated that schisandrin produced NANC relaxation on the rat colon, with the involvement of NO and acting via cGMP-dependent pathways. ATP, but not adenosine and VIP, likely plays a role in the non-nitrergic, apamin-sensitive component of the response.

Postnatal development of excitatory innervations in longitudinal smooth muscle of the chicken anterior mesenteric artery

Aims The anterior mesenteric artery of chickens contains a well-developed outer longitudinal smooth muscle layer in addition to an inner circular layer. Cholinergic and purinergic neurons play crucial roles in excitatory transmission at the longitudinal smooth muscle. The aim of this study was to clarify postnatal development of excitatory neurotransmission of the longitudinal smooth muscle. Main methods Membrane potentials of smooth muscle were recorded with a microelectrode technique. Perivascular nerves were stimulated by applying electrical field stimulation (EFS). Key findings Histological examination showed that longitudinal smooth muscles exist in the artery at birth. EFS failed to evoke membrane response in 1-day-old chickens, though it caused depolarization (excitatory junction potential; EJP) in 12-week-old chickens. However, exogenous application of acetylcholine (ACh) or ATP produced depolarization in longitudinal smooth muscle of 1-day-old chickens, suggesting that responsiveness of smooth muscle to excitatory neurotransmitters is already established at birth. In preparations isolated from 10-day-old chickens, EFS caused EJP, which was totally blocked by atropine but not by a non-specific purinoceptor antagonist, suramin. Several purinoceptor subtypes including P2Y1, which may be related to depolarizing response in smooth muscle of adult chickens, were expressed in the anterior mesenteric artery of 10-day-old chickens. Significance Excitatory innervation in longitudinal smooth muscle of the chicken anterior mesenteric artery is not established at birth but develops during the early postnatal period. Moreover, development of cholinergic excitatory innervation precedes that of purinergic excitatory innervation, although receptors that mediate purinergic control are already expressed in smooth muscle.

A nicardipine-sensitive Ca2+ entry contributes to the hypotonicity-induced increase in [Ca2+]i of principal cells in rat cortical collecting duct

We examined the mechanisms involved in the [Ca2+]i response to the extracellular hypotonicity in the principal cells of freshly isolated rat cortical collecting duct (CCD), using Fura-2/AM fluorescence imaging.Reduction of extracellular osmolality from 305 (control) to 195mosmol/kgH2O (hypotonic) evoked transient increase in [Ca2+]i of principal cells of rat CCDs. The [Ca2+]i increase was markedly attenuated by the removal of extracellular Ca2+. The application of a P2 purinoceptor antagonist, suramin failed to inhibit the hypotonicity-induced [Ca2+]i increase. The [Ca2+]i increase in response to extracellular hypotonicity was not influenced by application of Gd3+ and ruthenium red. On the other hand, a voltage-gated Ca2+ channel inhibitor, nicardipine, significantly reduced the peak amplitude of [Ca2+]i increase in the principal cells. In order to assess Ca2+ entry during the hypotonic stimulation, we measured the quenching of Fura-2 fluorescence intensity by Mn2+. The hypotonic stimulation enhanced quenching of Fura-2 fluorescence by Mn2+, indicating that a Ca2+-permeable pathway was activated by the hypotonicity. The hypotonicity-mediated enhancement of Mn2+ quenching was significantly inhibited by nicardipine.These results strongly suggested that a nicardipine-sensitive Ca2+ entry pathway would contribute to the mechanisms underlying the hypotonicity-induced [Ca2+]i elevation of principal cells in rat CCD.

Purinergic signalling is involved in the malaria parasite Plasmodium falciparum invasion to red blood cells

Plasmodium falciparum, the most important etiological agent of human malaria, is endowed with a highly complex cell cycle that is essential for its successful replication within the host. A number of evidence suggest that changes in parasite Ca(2+) levels occur during the intracellular cycle of the parasites and play a role in modulating its functions within the RBC. However, the molecular identification of Plasmodium receptors linked with calcium signalling and the causal relationship between Ca(2+) increases and parasite functions are still largely mysterious. We here describe that increases in P. falciparum Ca(2+) levels, induced by extracellular ATP, modulate parasite invasion. In particular, we show that addition of ATP leads to an increase of cytosolic Ca(2+) in trophozoites and segmented schizonts. Addition of the compounds KN62 and Ip5I on parasites blocked the ATP-induced rise in [Ca(2+)](c). Besides, the compounds or hydrolysis of ATP with apyrase added in culture drastically reduce RBC infection by parasites, suggesting strongly a role of extracellular ATP during RBC invasion. The use of purinoceptor antagonists Ip5I and KN62 in this study suggests the presence of putative purinoceptor in P. falciparum. In conclusion, we have demonstrated that increases in [Ca(2+)](c) in the malarial parasite P. falciparum by ATP leads to the modulation of its invasion of red blood cells. The online version of this article (doi:10.1007/s11302-010-9202-y) contains supplementary material, which is available to authorized users.

Can guanine-based purines be considered modulators of intestinal motility in rodents?

Adenine-based purines play a pivotal role in the control of gastrointestinal motility in rodents. Recently, guanine-based purines have been also shown to exert extracellular effects in the central nervous system raising the possibility of the existence of distinct receptors for guanine-based purines. Thus, it seems likely to speculate that also guanine-based purines may play a role in the modulation of the intestinal contractility. Spontaneous and neurally-evoked mechanical activity was recorded in vitro as changes in isometric tension in circular muscle strips from mouse distal colon. Guanosine up to 3 mM or guanine up to 1 mM failed to affect the spontaneous mechanical activity, but reduced the amplitude of the electrical field stimulation (EFS)-induced cholinergic contractions, without affecting the early nitrergic relaxation. Both compounds failed to affect the direct contractile responses evoked by carbachol. No desensitization of the response was observed. Guanine-based purine effects were not altered by theophylline, P1 purinoceptor antagonist, by PPADS or suramin, P2 purinoceptor antagonists, by ODQ, guanilyl cyclase inhibitor, or by DDA, adenylyl cyclase inhibitor. Nucleoside uptake inhibitors, dipyridamole or 6-[(4-Nitrobenzyl)thio]-9-β-D-ribofuranosylpurine (NBTI), antagonized the inhibitory effects induced by guanosine without interfering with guanine. On the contrary, adenine, a competitive inhibitor of nucleobase uptake, antagonized guanine-induced effects. In conclusion, our data indicate that guanosine and guanine are able to modulate negatively the excitatory cholinergic neurotransmission in the circular muscle layer of mouse colon. Guanine-based purines appear to interfere with prejunctional acethylcoline release. Their effects are dependent by their cellular uptake, and independent by adenine-based purine receptors.

Pharmacological modulation of the micturition pattern in normal and cyclophosphamide pre-treated conscious rats

In the current study, we wanted to assess the influence of muscarinic receptors, nitric oxide and purinoceptors on the micturition pattern of conscious normal and cyclophosphamide (CYP) pre-treated rats. The micturition parameters were assessed using a metabolic cage. Rats were pre-treated with either saline or CYP, to induce cystitis, followed by treatment with either the muscarinic M1/M3/M5 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), the nitric oxide synthase blocker N ω-nitro-L-arginine methyl (L-NAME), the P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) or a combination of 4-DAMP with PPADS or L-NAME. Voiding volumes per micturition event were significantly lower in CYP pre-treated than in saline pre-treated rats. Neither 4-DAMP nor L-NAME had any effect in the normal rats, whereas PPADS reduced the micturition volume per event. In CYP pre-treated rats, 4-DAMP and L-NAME significantly increased voiding volumes per event and micturition frequency, respectively. 4-DAMP dose-dependently reduced the differences in micturition activity between saline and CYP pre-treated rats. We show that cystitis changes the urodynamics in conscious rats and that this change seems to depend on the production of NO and on altered muscarinic receptor effects. The altered muscarinic receptor responses are likely to per se involve NO-mediated mechanisms.

Both the stimulation and inhibition of root hair growth induced by extracellular nucleotides in Arabidopsis are mediated by nitric oxide and reactive oxygen species

Root hairs secrete ATP as they grow, and extracellular ATP and ADP can trigger signaling pathways that regulate plant cell growth. In several plant tissues the level of extracellular nucleotides is limited in part by ectoapyrases (ecto-NTPDases), and the growth of these tissues is strongly influenced by their level of ectoapyrase expression. Both chemical inhibition of ectoapyrase activity and suppression of the expression of two ectoapyrase enzymes by RNAi in Arabidopsis resulted in inhibition of root hair growth. As assayed by a dose-response curve, different concentrations of the poorly hydrolysable nucleotides, ATPγS and ADPβS, could either stimulate (at 7.5-25μM) or inhibit (at≥150μM) the growth rate of root hairs in less than an hour. Equal amounts of AMPS, used as a control, had no effect on root hair growth. Root hairs of nia1nia2 mutants, which are suppressed in nitric oxide (NO) production, and of atrbohD/F mutants, which are suppressed in the production of H(2)O(2), did not show growth responses to applied nucleotides, indicating that the growth changes induced by these nucleotides in wild-type plants were likely transduced via NO and H(2)O(2) signals. Consistent with this interpretation, treatment of root hairs with different concentrations of ATPγS induced different accumulations of NO and H(2)O(2) in root hair tips. Two mammalian purinoceptor antagonists also blocked the growth responses induced by extracellular nucleotides, suggesting that they were initiated by a receptor-based mechanism.

Involvement of peripheral adenosine 5′‐triphosphate and P2X purinoceptor in pain‐related behavior produced by orthotopic melanoma inoculation in mice

Adenosine 5'-triphosphate (ATP) plays an important role in nociceptive processing. We used a mouse model of skin cancer pain to investigate the role of ATP in cancer pain. Orthotopic inoculation of B16-BL6 melanoma cells into the hind paw produced spontaneous licking of the tumor-bearing paw. Intraperitoneal injection of the P2 purinoceptor antagonist suramin suppressed spontaneous licking dose-dependently. Two P2X purinoceptor antagonists also suppressed spontaneous licking. An intraplantar injection of ATP, which did not induce licking in the healthy paw, increased licking of the tumor-bearing paw. Spontaneous firing of the tibial nerve was significantly increased in tumor-bearing mice and was inhibited by suramin. Extracellular concentration of ATP was significantly increased in the tumor-bearing paw than in the normal paw. ATP is concentrated in the culture medium of melanoma, lung cancer and breast cancer cells, but not fibroblasts. The P2X(3) receptor was expressed in about 40% of peripherin-positive small and medium-sized neurons in the dorsal root ganglia. P2X(3)-positive neurons were significantly increased in melanoma-bearing mice. These results suggest that ATP and P2X, especially P2X(3), receptors are involved in skin cancer pain, due to the increased release of ATP and increased expression of P2X(3) receptors in the sensory neurons.

Assessment and Characterization of Purinergic Contractions and Relaxations in the Rat Urinary Bladder

The aim of the present study was to assess the purinoceptor functional responses of the urinary bladder by using isolated rat urinary bladder strip preparations. ATP elicited a transient bladder contraction followed by a sustained relaxation and ADP, UDP and UTP generated predominantly potent relaxations (relaxatory potencies: ADP = ATP > UDP = UTP). The ATP contractions were desensitized with the P2X(1/3) purinoceptor agonist/desensitizer alpha,beta-meATP and reduced by the P2 purinoceptor antagonist PPADS but unaffected by the P2 purinoceptor antagonist suramin. Electrical field stimulation (1-60 Hz) evoked frequency-dependent bladder contractions that were decreased by incubation with alpha,beta-meATP but not further decreased by PPADS. Suramin antagonized relaxations generated by UDP but not those by ADP, ATP or UTP. PPADS antagonized and tended to antagonize UTP and UDP relaxations, respectively, but did neither affect ADP nor ATP relaxations. ADP relaxations were insensitive to the P2Y(1) purinoceptor antagonist MRS 2179 and the ATP-sensitive potassium channel antagonist glibenclamide. The ATP relaxations were inhibited by the P1 purinoceptor antagonist 8-p-sulfophenyltheophylline but unaffected by the A2A adenosine receptor antagonist 8-(3-chlorostyryl)caffeine and glibenclamide. Adenosine evoked relaxations that were antagonized by the A2B adenosine receptor antagonist PSB 1115. Thus, in the rat urinary bladder purinergic contractions are elicited predominantly by stimulation of the P2X(1) purinoceptors, while UDP/UTP-sensitive P2Y purinoceptor(s) and P1 purinoceptors of the A2B adenosine receptor subtype are involved in bladder relaxation.

P2Y receptors and atherosclerosis in apolipoprotein E‐deficient mice

P2Y nucleotide receptors are involved in the regulation of vascular tone, smooth muscle cell (SMC) proliferation and inflammatory responses. The present study investigated whether they are involved in atherosclerosis. mRNA of P2Y receptors was quantified (RT-PCR) in atherosclerotic and plaque-free aorta segments of apolipoprotein E-deficient (apoE(-/-)) mice. Macrophage activation was assessed in J774 macrophages, and effects of non-selective purinoceptor antagonists on atherosclerosis were evaluated in cholesterol-fed apoE(-/-) mice. P2Y(6) receptor mRNA was consistently elevated in segments with atherosclerosis, whereas P2Y(2) receptor expression remained unchanged. Expression of P2Y(1) or P2Y(4) receptor mRNA was low or undetectable, and not influenced by atherosclerosis. P2Y(6) mRNA expression was higher in cultured J774 macrophages than in cultured aortic SMCs. Furthermore, immunohistochemical staining of plaques demonstrated P2Y(6)-positive macrophages, but few SMCs, suggesting that macrophage recruitment accounted for the increase in P2Y(6) receptor mRNA during atherosclerosis. In contrast to ATP, the P2Y(6)-selective agonist UDP increased mRNA expression and activity of inducible nitric oxide synthase and interleukin-6 in J774 macrophages; this effect was blocked by suramin (100-300 microM) or pyridoxal-phosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS, 10-30 microM). Finally, 4-week treatment of cholesterol-fed apoE(-/-) mice with suramin or PPADS (50 and 25 mg.kg(-1).day(-1) respectively) reduced plaque size, without changing plaque composition (relative SMC and macrophage content) or cell replication. These results suggest involvement of nucleotide receptors, particularly P2Y(6) receptors, during atherosclerosis, and warrant further research with selective purinoceptor antagonists or P2Y(6) receptor-deficient mice.