Αβ-methylene ATP Research Articles

Pharmacology of P2X3 and P2X2/3 receptors: An automated patch clamp study.

P2X receptors are ligand-gated ion channels activated by extracellular ATP. They are permeable to small monovalent cations, some having significant divalent or anion permeability. The P2X2 and P2X3 subunits are predominantly expressed in primary sensory neurons and have been proposed to play a role in thermal sensation, taste and pain. They form functional hetero- or homotrimers which are activated by αβ-methylene ATP (αβmeATP). The stoichiometry of P2X2/3 heteromers appears to be dependent on the relative abundance of the two subunits. A mixture of P2X2 and P2X3 homomers as well as P2X2/3 heteromers are likely to coexist but can be distinguished through their biophysical and pharmacological properties. The receptors open in response to an increase in extracellular ATP occurring under pathological conditions. The resulting depolarization leads to propagation of the pain signal. Due to its role in nociception and pain signaling, these receptors are important targets for pain management. Here we present data collected on automated patch clamp (APC) systems with ranging throughput from 1 up to 384 cells simultaneously. We show activation and inhibition of P2X2/3 and P2X3 receptors expressed in CHO cells with rapid and brief application of ligand. ATP was applied using the perfusion system of the Port-a-Patch or the pipette(s) of the Patchliner or SyncroPatch 384. The EC50 values for αβmeATP or ATP activation of P2X2/3 receptors were similar across the three devices. The kinetics of currents mediated by homomeric P2X3 receptors were distinctly faster than those of heteromeric receptors. P2X3-mediated currents activated by αβmeATP could be repetitively activated at least 6 times in the same cell with similar peak amplitudes. The P2X3-selective compound A-317491 blocked P2X3-mediated currents activated by αβmeATP with an IC50 of approximately 90 nM and was similar on all three devices.

Aqueous extracts of Urtica dioica (stinging nettle) leaf contain a P2-purinoceptor antagonist-Implications for male fertility.

Stinging nettle root and leaf extracts were tested for their effect on prostatic smooth muscle contractility. Root extract did not affect electrical field stimulation induced-nerve mediated contractions of isolated rat prostates. On the other hand, leaf extract attenuated electrical field stimulation-induced contractions at all frequencies. Similarly, contractions elicited by exogenous administration of ATP and αβ-methylene ATP were inhibited by leaf extract, whereas contractions elicited by exogenous administration of noradrenaline or acetylcholine were unaffected. The active component was present within the aqueous phase of the leaf extract. In mouse mating studies, stinging nettle leaf extract (50 mg p.o. daily) reduced male fertility by 53% compared to vehicle-treated male mice. Cardiovascular parameters were unaffected by administration of stinging nettle leaf extract (p ≥ 0.057). Treated mice exhibited normal mating behaviour. Bladder and testes weighed less in stinging nettle leaf extract treated mice. All other organs and total body weight were unaffected. It is concluded that stinging nettle leaf extract reduces contractility of genitourinary smooth muscle by acting as an antagonist at postjunctional P2X1-purinoceptors. These data indicates that blocking sperm transport through pharmacological blockade of P2X1-purinoceptors via oral administration is consistent with an effective and convenient biological strategy male contraception.

P2X receptor agonist enhances tumor-specific CTL responses through CD70+ DC-mediated Th17 induction.

Extracellular ATP is known to promote Th17 cell differentiation in the intestinal lamina propria by stimulating CD70+CD11clow dendritic cells (DCs) via P2X receptors (P2XRs). Recent studies have also shown that Th17 cells enhance antitumor immunity by directly promoting proliferation of cytotoxic T lymphocytes (CTLs). These finding led us to test a P2XR agonist, αβ-methylene ATP (αβ-ATP), as a mucosal vaccine adjuvant to promote CTL responses through Th17 induction. We demonstrated that (i) CD70+CD11clow DCs were present in the nasal lamina propria and expressed P2X1R, P2X2R and P2X4R; (ii) CD70+CD11clow DCs isolated from the nasal lamina propria enhanced Th17 cell differentiation of cocultured splenic CD4+ T cells upon stimulation with αβ-ATP; (iii) mice intranasally immunized with ovalbumin (OVA) and αβ-ATP had increased OVA-specific Th17 cells and CTLs in the nasal lamina propria and regional lymph nodes; (iv) mice intranasally immunized with OVA and αβ-ATP also had elevated resistance to E.G7-OVA tumor growth compared with those intranasally immunized with OVA alone; (v) suramin, a broad-range inhibitor of P2 receptors, suppressed the increases of OVA-specific Th17 cells and CTLs in mice intranasally immunized with OVA and αβ-ATP; and (vi) suramin also abrogated the enhanced antitumor immunity of mice intranasally immunized with OVA and αβ-ATP against E.G7-OVA. Collectively, αβ-ATP may be a promising mucosal adjuvant that promotes antigen-specific CTL responses via CD70+CD11clow DC-mediated Th17 induction.

One-Time Acute Heat Treatment Is Effective for Attenuation of the Exaggerated Exercise Pressor Reflex in Rats With Femoral Artery Occlusion.

The purpose of this study was to determine the effects of one-time acute heat treatment (HT) on the exaggerated exercise pressor reflex in a model of peripheral arterial insufficiency induced by ligation of the femoral artery and was to further examine the underlying mechanism of ATP-P2X3 signal activity during this process. The blood pressure (BP) response to static muscle contraction and muscle tendon stretch was recorded to determine the exercise pressor reflex. Also, αβ-methylene ATP (αβ-me ATP) was injected into the arterial blood supply of the hindlimb muscles to stimulate P2X3 receptors in the muscle afferent nerves. To process one-time acute HT, a heating pad was placed locally on the hindlimb and the muscle temperature (Tm) was increased by ~1.5°C and maintained for 5 min. Compared with control rats, a greater mean arterial pressure (MAP) response to muscle contraction was observed in rats with femoral occlusion in a pre-heat control session (28 ± 2 mmHg in occluded rats/n = 12 vs. 18 ± 2 mmHg in control rats/n = 9; p < 0.05). The one-time acute HT attenuated the amplification of the BP response in rats with femoral artery occlusion (MAP response: 19 ± 8 mmHg in occluded rats + HT/n = 11; p < 0.05 vs. occluded rats). In contrast, HT did not significantly attenuate amplification of MAP response to muscle stretch and αβ-me ATP injection in rats with femoral artery occlusion and controls (all p > 0.05). Our data suggest that one-time acute HT selectively attenuates the amplified pressor response induced by activation of the metabolic and mechanical components of the reflex in rats after femoral artery occlusion. The suppressing effects of acute HT on the exaggerated exercise pressor reflex are likely mediated through a reduction in metabolites (e.g., ATP) stimulating the muscle afferent nerves in contracting muscle, but unlikely through direct alteration of P2X receptors per se.

Kinetic and Pharmacological Properties of P2X3 and P2X2/3 Receptors

P2X receptors are ligand-gated ion channels activated by extracellular ATP. They are permeable to small monovalent cations, some having significant divalent or anion permeability. The P2X2 and P2X3 subunits are predominantly expressed in primary sensory neurons and have been proposed to play a role in thermal sensation, taste and pain. They form functional hetero- or homotrimers which are activated by αβ-methylene ATP (αβmeATP). The stoichiometry of P2X2/3 heteromers appears to be dependent on the relative abundance of the two subunits. A mixture of P2X2 and P2X3 homomers as well as P2X2/3 heteromers are likely to exist, which can be distinguished through their biophysical and pharmacological properties. The receptors open in response to an increase in extracellular ATP which occurs under pathological conditions such as tissue damage. The resulting depolarization leads to propagation of the pain signal. Due to its role in nociception and pain signaling, these receptors are important targets for pain management. Here we present data collected on automated patch clamp (APC) systems showing activation and inhibition of P2X2/3 and P2X3 receptors expressed in CHO cells with rapid and brief application of ligand. αβmeATP or ATP activated P2X2/3 receptors in a concentration-dependent manner with an EC50 of 8.3 µM (n = 190) and 1.1 µM (n = 191), respectively. The kinetics of currents mediated by homomeric P2X3 receptors were distinctly faster than those of heteromeric P2X2/3 receptors. Currents mediated by P2X3 receptors were activated by ATP and αβmeATP in a concentration-dependent manner with an EC50 of 2.0 µM (n = 372) and 1.4 µM (n = 311). Suramin blocked homomeric P2X2 or heteromeric P2X2/3 receptors with an IC50 of approximately 17 µM. The compound A-317491 also blocked P2X3-mediated currents activated by αβmeATP with an IC50 of 115 nM (n = 318).

Characterization of muscarinic and P2X receptors in the urothelium and detrusor muscle of the rat bladder

Muscarinic and purinergic (P2X) receptors play critical roles in bladder urothelium under physiological and pathological conditions. Aim of present study was to characterize these receptors in rat bladder urothelium and detrusor muscle using selective radioligands of [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) and αβ-methylene ATP [2,8-3H]tetrasodium salt ([3H]αβ-MeATP). Similar binding parameters for each radioligand were observed in urothelium and detrusor muscle. Pretreatment with N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) mustard revealed co-existence of M2 and M3 receptors, with the number of M2 receptors being larger in the urothelium and detrusor muscle. Intravesical administration of imidafenacin and Dpr-P-4 (N → O) (active metabolite of propiverine) displayed significant binding of muscarinic receptors in the urothelium and detrusor muscle. The treatment with cyclophosphamide (CYP) or resiniferatoxin (RTX) resulted in a significant decrease in maximal number of binding sites (Bmax) for [3H]NMS and/or [3H]αβ-MeATP in the urothelium and detrusor muscle. These results demonstrated that 1) pharmacological characteristics of muscarinic and P2X receptors in rat bladder urothelium were similar to those in the detrusor muscle, 2) that densities of these receptors were significantly altered by pretreatments with CYP and RTX, and 3) that these receptors may be pharmacologically affected by imidafenacin and Dpr-P-4 (N → O) which are excreted in the urine.

MP28-20 MUCOSA AFFECTS THE BLADDER DYSFUNCTION IN DIABETES WITH HIGH FAT DIET

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I1 Apr 2016MP28-20 MUCOSA AFFECTS THE BLADDER DYSFUNCTION IN DIABETES WITH HIGH FAT DIET Zongwei Wang, Vivian Cristofaro, Tongxiang Liu, Rongbin Ge, Maryrose Sullivian, and Aria Olumi Zongwei WangZongwei Wang More articles by this author , Vivian CristofaroVivian Cristofaro More articles by this author , Tongxiang LiuTongxiang Liu More articles by this author , Rongbin GeRongbin Ge More articles by this author , Maryrose SullivianMaryrose Sullivian More articles by this author , and Aria OlumiAria Olumi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1070AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Studies have demonstrated that obesity, diabetes and other metabolic syndromes are more vulnerable to develop bladder dysfunction. Previously we identified a diabetic bladder dysfunction (DBD) mouse model, in which hepatic specific double knockout insulin receptor substrate (IRS) 1 and 2 (DKO) causes insulin resistance and hyperglycemia but without development of obesity. These mice develop a temporal pattern of DBD that parallels the symptoms observed in humans. In this study we show the impact of high fat diet (HFD) on bladder function in diabetic mice and non-diabetic mice, and how the mucosa contributes to the bladder smooth muscle contractility. METHODS Female DKO mice and genetic control (CTR) were fed a standard chow (DKO, CTR) or a HFD (DKO+HFD, CTR+HFD) for 10 weeks. Urinary bladders were harvested from each animal for functional evaluation by ex vivo muscle tension studies. The mucosa was removed from half of each bladder. The amplitude of contractile responses induced by αβmethylene ATP (meATP), carbachol, KCl and electrical field stimulation (EFS) were measured. The contribution of the mucosa to the smooth muscle contractility was tested in bladder strips with and without the mucosal layer. RESULTS In bladder strips with intact mucosa, HFD did not affect contractions in response to CCh stimulation either in CTR or DKO. However, mucosa-denuded strips from DKO+HFD had significantly higher responses than that from normal diet animal, although HFD had little effect on CTR tissue without mucosa. Upon neurally-mediated responses evoked by EFS and membrane depolarization with KCl, bladder strip contractions in DKO+HFD with intact mucosa were not different from DKO fed standard chow, but significantly higher in mucosa-denuded tissue. Contrarily, the contractile responses to EFS in CTR+HFD were significantly higher than CTR, but little changed in mucosa-denuded tissue. Finally, HFD had little effect on aßmeATP elicited contractions either in CTR or DKO bladders with or without mucosa, although the contractions were higher in DKO bladder strips than CTR in normal chow. CONCLUSIONS Our data suggest that the effect of HFD appeared to be more pronounced in diabetic bladders than in control through cholinergic and neurogenic components but not purinergic component. More importantly, this augment of contractile responses was only observed in the absence of mucosa, which suggests that mucosa might have the protective effect in the condition of diabetes challenged with HFD. The data also suggest that diabetes patient might be more sensitive to HFD and vulnerable to develop bladder dysfunction. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e379 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Zongwei Wang More articles by this author Vivian Cristofaro More articles by this author Tongxiang Liu More articles by this author Rongbin Ge More articles by this author Maryrose Sullivian More articles by this author Aria Olumi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Interaction of αβ-Methylene ATP with the Cholinergic Twitch Response in the Guinea-Pig Ileum

In this study, direct effects of the P2X purinoceptor agonist αβ-methylene ATP (αβ-meATP) and effects on the cholinergic twitch response of the electrically stimulated longitudinal muscle-myenteric plexus (LMMP) strip of the guinea-pig ileum, were investigated. αβ-meATP (1, 3, and 10 µmol/l) induced short-lasting contractions on its own, followed by an inhibition of the twitch response during its presence in the organ bath. The inhibitor of small conductance Ca²⁺-activated K⁺ (SK) channels, apamin (100 nmol/l), prevented the inhibitory effect of αβ-meATP on the twitch response, whereas tetraethylammonium (300 µmol/l), a blocker of voltage-gated K⁺ channels and an inhibitor at nicotinic acetylcholine receptors, augmented the inhibitory effect of αβ-meATP on the twitch response. It is concluded, that there is a functional interaction between P2X receptors and nicotinic receptors in the LMMP strip, and that a major part of the excitatory input to the cholinergic motor neuron evoking the twitch response is purinergic and not nicotinergic.

Nicotine-Induced Modulation of the Cholinergic Twitch Response in the Ileum of Guinea Pig

In the present study, the direct drug effects of nicotine and its effects on the cholinergic twitch responses of the electrically stimulated longitudinal muscle-myenteric plexus strip from the ileum of guinea pig were investigated. Nicotine dose-dependently (0.3-10 µmol/l) evoked the well-known contractile responses on its own. Whereas the interposed twitch responses remained present without a change in height at 1 µmol/l nicotine, a nicotine concentration of 3 µmol/l slightly and a concentration of 10 µmol/l markedly diminished the twitch during their presence. After the washout of 1-10 µmol/l nicotine, the height of the twitch response was also temporarily and significantly reduced by 30-77%. The P2X purinoceptor agonist αβ-methylene ATP (1-10 µmol/l) dose-dependently induced contractions on its own and reduced the twitch response during its presence in the organ bath; however, it did not diminish the twitch responses after washout of the drug as nicotine did. The P2X antagonist pyridoxalphosphate-6-azophenyl-2′-4′-disulphonic acid, the NMDA channel blocker MK-801 and the inhibitor of small conductance Ca²⁺-activated K⁺ (SK) channels apamin reduced the contractile effect of 1 µmol/l nicotine. Apamin also significantly prevented the ‘post-nicotine inhibition of the twitch' following the washout of 1-3 µmol/l nicotine. As a conclusion, we provide evidence for a functional interaction between nicotinic receptors and the P2X receptors in the ileum of the guinea pig. The ‘post-nicotine inhibition of the twitch' is not due to nicotinic acetylcholine receptor desensitization or transmitter depletion, but most probably the secondary effects of nicotine on SK channels determine the reduced cholinergic motor neuron excitability.

Pain-enhancing mechanism through interaction between TRPV1 and anoctamin 1 in sensory neurons

The capsaicin receptor transient receptor potential cation channel vanilloid 1 (TRPV1) is activated by various noxious stimuli, and the stimuli are converted into electrical signals in primary sensory neurons. It is believed that cation influx through TRPV1 causes depolarization, leading to the activation of voltage-gated sodium channels, followed by the generation of action potential. Here we report that the capsaicin-evoked action potential could be induced by two components: a cation influx-mediated depolarization caused by TRPV1 activation and a subsequent anion efflux-mediated depolarization via activation of anoctamin 1 (ANO1), a calcium-activated chloride channel, resulting from the entry of calcium through TRPV1. The interaction between TRPV1 and ANO1 is based on their physical binding. Capsaicin activated the chloride currents in an extracellular calcium-dependent manner in HEK293T cells expressing TRPV1 and ANO1. Similarly, in mouse dorsal root ganglion neurons, capsaicin-activated inward currents were inhibited significantly by a specific ANO1 antagonist, T16Ainh-A01 (A01), in the presence of a high concentration of EGTA but not in the presence of BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid]. The generation of a capsaicin-evoked action potential also was inhibited by A01. Furthermore, pain-related behaviors in mice treated with capsaicin, but not with αβ-methylene ATP, were reduced significantly by the concomitant administration of A01. These results indicate that TRPV1-ANO1 interaction is a significant pain-enhancing mechanism in the peripheral nervous system.

Characterization of synapses in the rat subnucleus centralis of the nucleus tractus solitarius.

The nucleus tractus solitarius (NTS) receives subdiaphragmatic visceral sensory information via vagal A- or C-fibers. We have recently shown that, in contrast to cardiovascular NTS medialis neurons, which respond to either purinergic or vanilloid agonists, the majority of esophageal NTS centralis (cNTS) neurons respond to vanilloid agonists, whereas a smaller subset responds to both vanilloid and purinerigic agonists. The present study aimed to further investigate the neurochemical and synaptic characteristics of cNTS neurons using whole cell patch-clamp, single cell RT-PCR and immunohistochemistry. Excitatory postsynaptic currents (EPSCs) were evoked in cNTS by tractus solitarius stimulation, and in 19 of 64 neurons perfusion with the purinergic agonist αβ-methylene ATP (αβMeATP) increased the evoked EPSC amplitude significantly. Furthermore, neurons with αβMeATP-responsive synaptic inputs had different probabilities of release compared with nonresponsive neurons. Single cell RT-PCR revealed that 8 of 13 αβMeATP-responsive neurons expressed metabotropic glutamate receptor 8 (mGluR8) mRNA, which our previous studies have suggested is a marker of glutamatergic neurons, whereas only 3 of 13 expressed glutamic acid dehydroxylase, a marker of GABAergic neurons. A significantly lower proportion of αβMeATP-nonresponsive neurons expressed mGluR8 (2 of 30 neurons), whereas a greater proportion expressed glutamic acid dehydroxylase (12 of 30 neurons). Esophageal distension significantly increased the number of colocalized mGluR8- and c-Fos-immunoreactive neurons in the cNTS from 8.0 ± 4% to 20 ± 2.5%. These data indicate that cNTS comprises distinct neuronal subpopulations that can be distinguished based on their responses to purinergic agonists and that these subpopulations have distinct neurochemical and synaptic characteristics, suggesting that integration of sensory inputs from the esophagus relies on a discrete organization of synapses between vagal afferent fibers and cNTS neurons.

P2X7 receptor activation mediates organic cation uptake into human myeloid leukaemic KG-1 cells

The P2X7 purinergic receptor is an ATP-gated cation channel with an emerging role in neoplasia. In this study we demonstrate that the human KG-1 cell line, a model of acute myelogenous leukaemia, expresses functional P2X7. RT-PCR and immunochemical techniques demonstrated the presence of P2X7 mRNA and protein respectively in KG-l cells, as well as in positive control multiple myeloma RPMI 8226 cells. Flow cytometric measurements demonstrated that ATP induced ethidium(+) uptake into KG-l cells suspended in sucrose medium (EC(50) of ≈ 3 μM), but not into cells in NaCl medium. In contrast, ATP induced ethidium(+) uptake into RPMI 8226 cells suspended in either sucrose or NaCl medium (EC(50) of ≈ 3 or ≈ 99 μM, respectively), as well as into RPMI 8226 cells in KCl medium (EC(50) of ≈ 18 μM). BzATP and to a lesser extent ATPγS and αβ-methylene ATP, but not ADP or UTP, also induced ethidium(+) uptake into KG-1 cells. ATP-induced ethidium(+) uptake was completely impaired by the P2X7 antagonists, AZ10606120 and A-438079. ATP-induced ethidium(+) uptake was also impaired by probenecid but not by carbenoxolone, both pannexin-1 antagonists. ATP induced YO-PRO-1(2+) and propidium(2+) uptake into KG-1 cells. Finally, sequencing of full-length P2X7 cDNA identified several single nucleotide polymorphisms (SNPs) in KG-1 cells including H155Y, A348T, T357S and Q460R. RPMI 8226 cells contained A348T, A433V and H521Q SNPs. In conclusion, the KG-1 cell line expresses functional P2X7. This cell line may help elucidate the signalling pathways involved in P2X7-induced survival and invasiveness of myeloid leukaemic cells.

Intramuscular ketorolac inhibits activation of rat peripheral NMDA receptors

The nonsteroidal anti-inflammatory drug (NSAID) diclofenac has local anesthetic-like and peripheral N-methyl-d-aspartate (NMDA) receptor antagonist characteristics when administered at higher concentrations to masticatory muscle. It is not known if the ability to inhibit NMDA receptors is unique to diclofenac or shared by other NSAIDs. This study was undertaken to determine whether intramuscular injection of ketorolac or naproxen at concentrations that do not induce local anesthetic-like effects could attenuate jaw-closer muscle nociceptor discharge in anesthetized Sprague-Dawley rats. It was found that ketorolac (5 mM) inhibited hypertonic saline-evoked nociceptor discharge, which suggests that at this concentration, ketorolac has local anesthetic-like properties. A lower concentration of ketorolac (0.5 mM), which did not affect hypertonic saline-evoked discharge, did inhibit nociceptor discharge evoked by NMDA. In contrast, naproxen (5 mM) did not alter hypertonic saline- or NMDA-evoked nociceptor discharge. Subsequent experiments revealed that ketorolac (0.5 mM) had no effect on nociceptor discharge evoked by αβ-methylene ATP, 5-hydroxytryptamine, or AMPA. The inhibitory effect of ketorolac did not appear to be related to cyclooxygenase inhibition, because the concentration of prostaglandin E(2) in the masticatory muscles 10 min after injection of either NSAID was not significantly decreased. The present study indicates that in vivo, ketorolac, but not naproxen, can antagonize NMDA-evoked nociceptor discharge similarly to diclofenac. We speculate that structural similarities between ketorolac and diclofenac could account for the ability of these NSAIDs to inhibit NMDA-evoked nociceptor discharge. These properties may partly explain the analgesic effect of intramuscularly injected ketorolac in the clinic.

Sympathetic overdrive in obesity involves purinergic hyperactivity in the resistance vasculature

While a close correlation exists in obese humans between sympathetic, adrenergic hyperactivity and structural and functional organ damage, a role for the co-transmitter, ATP, in vascular function remains unexplored. We therefore studied sympathetic nerve-mediated responses of pressurised small mesenteric arteries from control and obese rats. Diet-induced obesity significantly increased the amplitude of vasoconstriction to transmural nerve stimulation (1-10 Hz; P <0.05). At 1 and 5 Hz, both adrenergic and purinergic responses were significantly augmented, while only the purinergic component was increased at 10 Hz (P <0.05). Nerve stimulation at 1 Hz evoked contractions and underlying excitatory junction potentials (EJPs), which were both significantly increased in amplitude during obesity (P <0.05) and abolished by αβ-methylene ATP (1 μM; desensitises purinergic receptors). The rise time and rate of decay of these EJPs were significant decreased (P <0.05), without change in resting membrane potential. Amplitude and frequency of spontaneous EJPs and the density of perivascular sympathetic nerves were also significantly increased (P <0.05). Inhibition of sensory neurotransmitter release (capsaicin; 10 μM) significantly increased the amplitude of nerve-mediated contraction (P <0.05), with a greater effect in control than obese animals, although the density of sensory nerves was unaffected by obesity. We demonstrate that sympathetic nerve-mediated vasoconstriction is enhanced by diet-induced obesity due to upregulation of purinergic, in addition to adrenergic, neurotransmission. Changes result from increased perivascular sympathetic innervation and release of ATP. We conclude that augmented sympathetic control of vasoconstriction induced by obesity could contribute directly to hypertension and global organ damage. A decrease in sensitivity to sensory vasodilatory neurotransmitters may also affect these processes.

Ca2+ entry following P2X receptor activation induces IP3 receptor-mediated Ca2+ release in myocytes from small renal arteries.

P2X receptors mediate sympathetic control and autoregulation of the renal circulation triggering contraction of renal vascular smooth muscle cells (RVSMCs) via an elevation of intracellular Ca(2+) concentration ([Ca(2+) ](i) ). Although it is well-appreciated that the myocyte Ca(2+) signalling system is composed of microdomains, little is known about the structure of the [Ca(2+) ](i) responses induced by P2X receptor stimulation in vascular myocytes. Using confocal microscopy, perforated-patch electrical recordings, immuno-/organelle-specific staining, flash photolysis and RT-PCR analysis we explored, at the subcellular level, the Ca(2+) signalling system engaged in RVSMCs on stimulation of P2X receptors with the selective agonist αβ-methylene ATP (αβ-meATP). RT-PCR analysis of single RVSMCs showed the presence of genes encoding inositol 1,4,5-trisphosphate receptor type 1(IP(3) R1) and ryanodine receptor type 2 (RyR2). The amplitude of the [Ca(2+) ](i) transients depended on αβ-meATP concentration. Depolarization induced by 10 µmol·L(-1) αβ-meATP triggered an abrupt Ca(2+) release from sub-plasmalemmal ('junctional') sarcoplasmic reticulum enriched with IP(3) Rs but poor in RyRs. Depletion of calcium stores, block of voltage-gated Ca(2+) channels (VGCCs) or IP(3) Rs suppressed the sub-plasmalemmal [Ca(2+) ](i) upstroke significantly more than block of RyRs. The effect of calcium store depletion or IP(3) R inhibition on the sub-plasmalemmal [Ca(2+) ](i) upstroke was attenuated following block of VGCCs. Depolarization of RVSMCs following P2X receptor activation induces IP(3) R-mediated Ca(2+) release from sub-plasmalemmal ('junctional') sarcoplasmic reticulum, which is activated mainly by Ca(2+) influx through VGCCs. This mechanism provides convergence of signalling pathways engaged in electromechanical and pharmacomechanical coupling in renal vascular myocytes.

Noradrenaline enhances ATP P2X3 receptor expression in dorsal root ganglion neurons of rats

Increasing evidence indicates that both the ATP P2X receptors and adrenergic systems play a very important role in the development of nociception. However, there is little information concerning the interactions between these two systems in the dorsal root ganglia (DRG). In the present study, we examined the effects of noradrenaline (NA) on the P2X3 receptor expression in the DRG of Sprague–Dawley rats. The direct effect of NA on the P2X3 mRNA and protein expression was determined in cultured DRG neurons. Treatment of neuronal cultures with NA (50 nM, 5 h) induced a two-fold increase in P2X3 expression, as detected with real-time RT-PCR and Western blots, but had no effect on P2X2 expression. In electrophysiological experiments, perfusion of neuronal cultures with the P2X3 agonist (αβ-methylene ATP) increased neuronal firing rate by 139% and 273% in neurons treated with either PBS (control) or NA, respectively, indicating that chronic NA treatment significantly enhanced the neuronal response to P2X3 activation. In behavior studies, combination of NA (2 or 20 nmol) with αβ-methylene ATP (10 nmol) produced a significant and long lasting augmentation of thermal hyperalgesia. These results indicate that NA stimulates P2X3 expression in DRG neurons, and this could contribute to the development of pain sensitization.

P2X1 Ion Channels Promote Neutrophil Chemotaxis through Rho Kinase Activation

ATP, released at the leading edge of migrating neutrophils, amplifies chemotactic signals. The aim of our study was to investigate whether neutrophils express ATP-gated P2X(1) ion channels and whether these channels could play a role in chemotaxis. Whole-cell patch clamp experiments showed rapidly desensitizing currents in both human and mouse neutrophils stimulated with P2X(1) agonists, alphabeta-methylene ATP (alphabetaMeATP) and betagammaMeATP. These currents were strongly impaired or absent in neutrophils from P2X(1)(-/-) mice. In Boyden chamber assays, alphabetaMeATP provoked chemokinesis and enhanced formylated peptide- and IL-8-induced chemotaxis of human neutrophils. This agonist similarly increased W-peptide-induced chemotaxis of wild-type mouse neutrophils, whereas it had no effect on P2X(1)(-/-) neutrophils. In human as in mouse neutrophils, alphabetaMeATP selectively activated the small RhoGTPase RhoA that caused reversible myosin L chain phosphorylation. Moreover, the alphabetaMeATP-elicited neutrophil movements were prevented by the two Rho kinase inhibitors, Y27632 and H1152. In a gradient of W-peptide, P2X(1)(-/-) neutrophils migrated with reduced speed and displayed impaired trailing edge retraction. Finally, neutrophil recruitment in mouse peritoneum upon Escherichia coli injection was enhanced in wild-type mice treated with alphabetaMeATP, whereas it was significantly impaired in the P2X(1)(-/-) mice. Thus, activation of P2X(1) ion channels by ATP promotes neutrophil chemotaxis, a process involving Rho kinase-dependent actomyosin-mediated contraction at the cell rear. These ion channels may therefore play a significant role in host defense and inflammation.

Involvement of prostaglandin F2α receptor in ATP-induced mechanical allodynia

Nociceptive primary afferents have the capacity to induce a state of increased excitability in the dorsal horn neurons of the spinal cord. It is well accepted that capsaicin-sensitive C-fibers transduce noxious stimulation and acute pain and that capsaicin-insensitive Aβ-fibers are responsible for touch and innocuous sensation. It has been reported that the intrathecal (i.t.) administration of prostaglandin F2α (PGF2α) and ATP induces mechanical allodynia via the capsaicin-insensitive primary afferent pathway. In the present study, we investigated the interaction of purinoceptor P2X and the PGF2α receptor (FP) in the induction of allodynia by use of mice lacking FP (FP−/−). Both PGF2α and the P2X receptor agonist αβ-methylene ATP administered i.t. strongly induced allodynia for 50 min by tactile stimuli to the flank of mice. The allodynia induced by αβ-methylene ATP, but not that by PGF2α, was suppressed by simultaneous i.t. administration of P2X receptor antagonists pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid and A-317491. In contrast, the allodynia induced by αβ-methylene ATP as well as that by PGF2α was not observed in FP−/− mice. Immunostaining of β-galactosidase, a reporter knocked into the endogenous FP locus in FP−/− mice, showed that the FP receptor was co-localized with P2X2 and P2X3 receptors in neurons of the spinal cord. αβ-Methylene ATP evoked a transient or sustained [Ca2+]i increase in most of the PGF2α-responsive cells in the deeper layer of the spinal cord, and the αβ-methylene ATP-evoked increase was blocked by the FP receptor antagonist AL-8810 in two-thirds of the cells. Neither PGF2α nor αβ-methylene ATP induced the activation of spinal microglia. The present study demonstrates that the αβ-methylene ATP-evoked allodynia is mediated by the FP receptor, possibly via the functional coupling between the activation of P2X2/3 receptors on the central terminal of capsaicin-insensitive fibers and FP receptors on spinal neurons.

Age‐related changes in vasoconstrictor response to heat in skeletal muscle arteries

Aging reduces skeletal muscle blood flow, but the mechanism remains unclear. The hypothesis was that aging results in less heat‐induced attenuation of vasoconstriction and this is attributable to decreased sensitivity of P2X receptors to heat. Femoral arteries from 28 young (2–3 months old) and 23 old (24 months old) rats were prepared for wire myography. Dose response curves for the P2X agonist, αβ‐methylene ATP (mATP: 10−7 to 10−3M), the α1‐agonist, phenylephrine (PE: 10−7 to 10−4M), the smooth muscle constrictor, potassium chloride (KCl: 20 to 80mM), and field stimulation (2–60Hz for 144 impulses) were performed at bath temperatures of 37°C and 41°C. The response to mATP was attenuated with heat in young (37°C: 0.95±0.14; 41°C: 1.48±0.12 g/log M mATP; p&lt;0.05) and old (37°C: 1.05±0.16; 41°C: 1.67±0.20 g/log M mATP; p&lt;0.05) femoral arteries. Phenylephrine dose response curves were not heat sensitive in young arteries, but were potentiated with 41°C in old arteries (p&lt;0.05). KCl dose response curves were potentiated by 41°C in the young (37°C: 2.10±0.25; 41°C: 2.67±0.19g peak tension; p&lt;0.05) and old arteries (37°C: 1.95±0.22; 41°C: 2.53±0.10g peak tension; p&lt;0.05). The tension produced by field stimulation was attenuated by a greater amount in old (−78±10%) than in young (−43±12%). Heat‐induced attenuation of vasoconstriction via P2X receptors is preserved in old femoral arteries. Unexpectedly, old femoral arteries exhibit heat potentiation of α1‐receptor mediated vasoconstriction.

Calcium oscillations induced by ATP in human umbilical cord smooth muscle cells

Arterial smooth muscle cells exhibit vasomotion, related to oscillations in intracellular Ca(2+) concentration, but the origin and function of these has not yet been fully determined. We measured intracellular Ca(2+) using conventional fluorescent methods in primary cultured, human umbilical cord artery smooth muscle cells (HUCASMC). Spontaneous oscillations in Ca(2+) was found in only 1% of all cells but exogenous, micromolar concentrations of ATP could induce Ca(2+) oscillations in 70% of cells with the most common pattern being one of regular amplitude and frequency with a return to basal levels between each peak. The P2Y agonist, UTP, but not the P2X agonist alphabeta-methylene ATP, could also induce Ca(2+) oscillations. Once induced, these oscillations could not be blocked by G-protein, PLC, VGCC or TRP channel antagonists applied individually, but could be prevented when antagonists were applied together. In the presence of EGTA, micromolar concentrations of ATP induced an elevation in intracellular Ca(2+) but did not induce Ca(2+) oscillations. The oscillation frequency induced by ATP was affected by bath Ca(2+) concentration. Taken together, these data suggest that external Ca(2+) entry maintains the Ca(2+) oscillation induced by activation of P2Y receptors. Once induced, multiple mechanisms are involved to maintain the oscillation and the oscillation frequency is determined by the speed of Ca(2+) refilling. Chronic hypoxia enhanced the Ca(2+) response and altered the oscillation frequency. We suggest that these oscillations may play a role in the maintenance of umbilical blood flow during situations in which GPCR are activated.