P2 Purinoceptors Research Articles

Modulation of dopamine-mediated facilitation at the neuromuscular junction of Wistar rats: A role for adenosine A1/A2A receptors and P2 purinoceptors

This study aims to understand how dopamine and the neuromodulators, adenosine and adenosine triphosphate (ATP) modulate neuromuscular transmission. Adenosine and ATP are well-recognized for their regulatory effects on dopamine in the central nervous system. However, if similar interactions occur at the neuromuscular junction is unknown. We hypothesize that the activation of adenosine A1/A2A and/or P2 purinoceptors may influence the action of dopamine on neuromuscular transmission. Using the rat phrenic nerve hemi-diaphragm, we assessed the influence of dopamine, adenosine and ATP on the height of nerve-evoked muscle twitches. We investigated how the selective blockade of adenosine A1 receptors (2.5nM DPCPX), adenosine A2A receptors (50nM CSC) and P2 purinoceptors (100μM suramin) modified the effects of dopamine. Dopamine alone increased indirect muscle contractions while adenosine and ATP either enhanced or depressed nerve-evoked muscle twitches in a concentration-dependent manner. The facilitatory effects of 256μM dopamine were significantly reduced to 29.62±2.79% or 53.69±5.45% in the presence of DPCPX or CSC, respectively, relative to 70.03±1.57% with dopamine alone. Alternatively, the action of 256μM dopamine was potentiated from 70.03±1.57, in the absence of suramin, to 86.83±4.36%, in the presence of suramin. It can be concluded that the activation of adenosine A1 and A2A receptors and P2 purinoceptors potentially play a central role in the regulation of dopamine effects at the neuromuscular junction. Clinically this study offers new insights for the indirect manipulation of neuromuscular transmission for the treatment of disorders characterized by motor dysfunction.

Mesenchymal Stem Cells Ageing: Targeting the "Purinome" to Promote Osteogenic Differentiation and Bone Repair.

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming cells. Such ability is compromised in elderly individuals resulting in bone disorders such as osteoporosis, also limiting their clinical usage for cell transplantation and bone tissue engineering strategies. In bone marrow niches, adenine and uracil nucleotides are important local regulators of osteogenic differentiation of MSCs. Nucleotides can be released to the extracellular milieu under both physiological and pathological conditions via (1) membrane cell damage, (2) vesicle exocytosis, (3) ATP-binding cassette transporters, and/or (4) facilitated diffusion through maxi-anion channels, hemichannels or ligand-gated receptor pores. Nucleotides and their derivatives act via adenosine P1 (A1 , A2A , A2B , and A3 ) and nucleotide-sensitive P2 purinoceptors comprising ionotropic P2X and G-protein-coupled P2Y receptors. Purinoceptors activation is terminated by membrane-bound ecto-nucleotidases and other ecto-phosphatases, which rapidly hydrolyse extracellular nucleotides to their respective nucleoside 5'-di- and mono-phosphates, nucleosides and free phosphates, or pyrophosphates. Current knowledge suggests that different players of the "purinome" cascade, namely nucleotide release sites, ecto-nucleotidases and purinoceptors, orchestrate to fine-tuning regulate the activity of MSCs in the bone microenvironment. Increasing studies, using osteoprogenitor cell lines, animal models and, more recently, non-modified MSCs from postmenopausal women, raised the possibility to target chief components of the purinergic signaling pathway to regenerate the ability of aged MSCs to differentiate into functional osteoblasts. This review summarizes the main findings of those studies, prompting for novel therapeutic strategies to control ageing disorders where bone destruction exceeds bone formation, like osteoporosis, rheumatoid arthritis, and fracture mal-union. J. Cell. Physiol. 231: 1852-1861, 2016. © 2016 Wiley Periodicals, Inc.

Shear stress induces a longitudinal Ca(2+) wave via autocrine activation of P2Y1 purinergic signalling in rat atrial myocytes.

Atrial myocytes are exposed to shear stress during the cardiac cycle and haemodynamic disturbance. In response, they generate a longitudinally propagating global Ca(2+) wave. Here, we investigated the cellular mechanisms underlying the shear stress-mediated Ca(2+) wave, using two-dimensional confocal Ca(2+) imaging combined with a pressurized microflow system in single rat atrial myocytes. Shear stress of ∼16 dyn cm(-2) for 8 s induced ∼1.2 aperiodic longitudinal Ca(2+) waves (∼79 μm s(-1)) with a delay of 0.2-3 s. Pharmacological blockade of ryanodine receptors (RyRs) or inositol 1,4,5-trisphosphate receptors (IP3 Rs) abolished shear stress-induced Ca(2+) wave generation. Furthermore, in atrial myocytes from type 2 IP3R (IP3R2) knock-out mice, shear stress failed to induce longitudinal Ca(2+) waves. The phospholipase C (PLC) inhibitor U73122, but not its inactive analogue U73343, abolished the shear-induced longitudinal Ca(2+) wave. However, pretreating atrial cells with blockers for stretch-activated channels, Na(+)-Ca(2+) exchanger, transient receptor potential melastatin subfamily 4, or nicotinamide adenine dinucleotide phosphate oxidase did not suppress wave generation under shear stress. The P2 purinoceptor inhibitor suramin, and the potent P2Y1 receptor antagonist MRS 2179, both suppressed the Ca(2+) wave, whereas the P2X receptor antagonist, iso-PPADS, did not alter it. Suppression of gap junction hemichannels permeable to ATP or extracellular application of ATP-metabolizing apyrase inhibited the wave. Removal of external Ca(2+) to enhance hemichannel opening facilitated the wave generation. Our data suggest that longitudinally propagating, regenerative Ca(2+) release through RyRs is triggered by P2Y1-PLC-IP3R2 signalling that is activated by gap junction hemichannel-mediated ATP release in atrial myocytes under shear stress.

Calcium signaling and the novel anti-proliferative effect of the UTP-sensitive P2Y11 receptor in rat cardiac myofibroblasts

During myocardial ischemia and reperfusion both purines and pyrimidines are released into the extracellular milieu, thus creating a signaling wave that propagates to neighboring cells via membrane-bound P2 purinoceptors activation. Cardiac fibroblasts (CF) are important players in heart remodeling, electrophysiological changes and hemodynamic alterations following myocardial infarction. Here, we investigated the role UTP on calcium signaling and proliferation of CF cultured from ventricles of adult rats.Co-expression of discoidin domain receptor 2 and α-smooth muscle actin indicate that cultured CF are activated myofibroblasts. Intracellular calcium ([Ca2+]i) signals were monitored in cells loaded with Fluo-4 NW. CF proliferation was evaluated by the MTT assay.UTP and the selective P2Y4 agonist, MRS4062, caused a fast desensitizing [Ca2+]i rise originated from thapsigargin-sensitive internal stores, which partially declined to a plateau providing the existence of Ca2+ in the extracellular fluid. The biphasic [Ca2+]i response to UTP was attenuated respectively by P2Y4 blockers, like reactive blue-2 and suramin, and by the P2Y11 antagonist, NF340. UTP and the P2Y2 receptor agonist MRS2768 increased, whereas the selective P2Y11 agonist NF546 decreased, CF growth; MRS4062 was ineffective. Blockage of the P2Y11 receptor or its coupling to adenylate cyclase boosted UTP-induced CF proliferation. Confocal microscopy and Western blot analysis confirmed the presence of P2Y2, P2Y4 and P2Y11 receptors.Data indicate that besides P2Y4 and P2Y2 receptors which are responsible for UTP-induced [Ca2+]i transients and growth of CF, respectively, synchronous activation of the previously unrecognized P2Y11 receptor may represent an important target for anti-fibrotic intervention in cardiac remodeling.

Effect of Exogenous Extracellular Nicotinamide Adenine Dinucleotide (NAD⁺) on Bioelectric Activity of the Pacemaker and Conduction System of the Heart.

In rat sinoatrial node, NAD(+) (10 μM) reduced the rate of spontaneous action potentials, duration of action potentials, and the velocity of slow diastolic depolarization, but the rate of action potential front propagation increases. In passed rabbit Purkinje fibers, NAD(+) (10 μM) reduced the duration of action potentials. Under conditions of spontaneous activity of Purkinje fibers, NAD(+) reduced the fi ring rate and the rate of slow diastolic depolarization. The effects of extracellular NAD(+) on bioelectric activity of the pacemaker (sinoatrial node) and conduction system of the heart (Purkinje fibers) are probably related to activation of P1 and P2 purinoceptors.

Impact of ectonucleotidases in autonomic nervous functions.

Adenine and uracil nucleotides play key functions in the autonomic nervous system (ANS). For instance, ATP acts as a neurotransmitter, co-transmitter and neuromodulator in the ANS. The purinergic system encompasses (1) receptors that respond to extracellular purines, which are designated as P1 and P2 purinoceptors, (2) purine release and uptake, and (3) a cascade of enzymes that regulate the concentration of purines near the cell surface. Ectonucleotidases and adenosine deaminase (ADA) are enzymes responsible for the hydrolysis of ATP (and other nucleotides such as ADP, UTP, UDP, AMP) and adenosine, respectively. Accordingly, these enzymes are expected to play an important role in the control of neuro-effector transmission in tissues innervated by both the sympathetic and parasympathetic divisions of the ANS. Indeed, ectonucleotidases have the ability to either terminate P2 receptor responses initiated by nucleoside triphosphates (ATP and UTP), and/or to favor the activation of ADP (e.g. P2Y1,12,13) and UDP (e.g. P2Y6) and/or adenosine (P1) specific receptors. In addition, ectonucleotidases can also importantly protect some P2 receptors from desensitization (e.g. P2X1, P2Y1). In this review, we present the (putative) roles of ectonucleotidases and ADA in the ANS with a focus on their regulatory activity at neuro-effector junctions in the following tissues: heart, vas deferens, urinary bladder, salivary glands, blood vessels and the intestine. We also present their implication in nociceptive transmission.

P2 Receptors in Renal Autoregulation

Autoregulation of renal blood flow and glomerular filtration rate is an essential function of the renal microcirculation. While the existence of this phenomenon has been known for many years, the exact mechanisms that underlie this regulatory system remain poorly understood. The work of many investigators has provided insights into many aspects of the autoregulatory mechanism, but many critical components remain elusive. This review is intended to update the reader on the role of P2 purinoceptors as a postulated mechanism responsible for renal autoregulatory resistance adjustments. It will summarize recent advances in normal function and it will touch on more recent ideas regarding autoregulatory insufficiency in hypertension and inflammation. Current thoughts on the nature of the mechanosensor responsible for myogenic behavior will be also be discussed as well as current thoughts on the mechanisms involved in ATP release to the extracellular fluid space.

Purinergic receptor activation facilitates astrocytic GABAB receptor calcium signalling

Gamma-aminobutyric acid B receptors (GABABRs) are heterodimeric G-protein coupled receptors, which mediate slow synaptic inhibition in the brain. Emerging evidence suggests astrocytes also express GABABRs, although their physiological significance remains unknown. To begin addressing this issue, we have used imaging and biochemical analysis to examine the role GABABRs play in regulating astrocytic Ca2+ signalling. Using live imaging of cultured cortical astrocytes loaded with calcium indicator Fluo-4/AM, we found that astrocytic GABABRs are able to induce astrocytic calcium transients only if they are pre-activated by P2 purinoceptors (P2YRs). The GABABR-mediated calcium transients were attenuated by the removal of extracellular calcium. Furthermore, P2YRs enhance the phosphorylation of astrocytic GABABR R2 subunits on both serine 783 (S783) and serine 892 (S892), two phosphorylation sites that are well known to regulate the activity and the cell surface stability of GABABRs. Collectively these results suggest that P2YR mediated signalling is an important determinant of GABABR activity and phosphorylation in astrocytes.This article is part of the Special Issue entitled ‘GABAergic Signaling in Health and Disease’.

Functional properties of submucosal venules in the rat stomach.

Venules in the stomach may have intrinsic properties for maintaining active microcirculation drainage even during gastric filling. Properties of spontaneous and nerve-mediated activity of submucosal venules in the rat stomach were investigated. Changes in vasodiameter and intracellular Ca(2+) in venular smooth muscle cells (SMCs) were monitored by video tracking and Fluo-8 Ca(2+) imaging, respectively. Venular SMCs developed synchronous spontaneous Ca(2+) transients and corresponding rhythmic constrictions of the venules. Nominally Ca(2+)-free solution or an L-type Ca(2+) channel blocker (1 μM nifedipine) disrupted the Ca(2+) transient synchrony and abolished spontaneous constrictions. Spontaneous constrictions were also prevented by inhibitors of sarcoplasmic reticulum Ca(2+)-ATPase (10 μM cyclopiazonic acid (CPA)), IP3 receptors (100 μM 2-APB) or Ca(2+)-activated Cl(-) channels (100 μM niflumic acid). Transmural nerve stimulation (TNS) induced a long-lasting venular constriction that was abolished by α-adrenoceptor antagonist (1 μM phentolamine), while TNS evoked a sympathetic transient constriction of arterioles that was abolished by a combination of phentolamine and a P2 purinoceptor antagonist (10 μM pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS)). Consistently, P2X1 purinoceptor immunoreactivity was detected in arteriolar but not venular SMCs. Primary afferent nerve stimulation (300 nM capsaicin) caused a venular dilatation by releasing calcitonin gene-related peptide. Thus, Ca(2+) release from the sarcoplasmic reticulum may play a fundamental role in the generation of spontaneous Ca(2+) transients, while electrical coupling amongst venular SMCs via L-type Ca(2+) channel activation appears to be critical for Ca(2+) transient synchrony as well as spontaneous contractions. Sympathetic venular constrictions appear to be exclusively mediated by noradrenaline due to the lack of P2X1 receptor in venular SMCs.

Suppression of cell membrane permeability by suramin: involvement of its inhibitory actions on connexin 43 hemichannels

Suramin is a clinically prescribed drug for treatment of human African trypanosomiasis, cancer and infection. It is also a well-known pharmacological antagonist of P2 purinoceptors. Despite its clinical use and use in research, the biological actions of this molecule are still incompletely understood. Here, we investigated the effects of suramin on membrane channels, as exemplified by its actions on non-junctional connexin43 (Cx43) hemichannels, pore-forming α-haemolysin and channels involved in ATP release under hypotonic conditions. Hemichannels were activated by removing extracellular Ca(2+) . The influences of suramin on hemichannel activities were evaluated by its effects on influx of fluorescent dyes and efflux of ATP. The membrane permeability and integrity were assessed through cellular retention of preloaded calcein and LDH release. Suramin blocked Cx43 hemichannel permeability induced by removal of extracellular Ca(2+) without much effect on Cx43 expression and gap junctional intercellular communication. This action of suramin was mimicked by its analogue NF023 and NF449 but not by another P2 purinoceptor antagonist PPADS. Besides hemichannels, suramin also significantly blocked intracellular and extracellular exchanges of small molecules caused by α-haemolysin from Staphylococcus aureus and by exposure of cells to hypotonic solution. Furthermore, it prevented α-haemolysin- and hypotonic stress-elicited cell injury. Suramin blocked membrane channels and protected cells against toxin- and hypotonic stress-elicited injury. Our finding provides novel mechanistic insights into the pharmacological actions of suramin. Suramin might be therapeutically exploited to protect membrane integrity under certain pathological situations.

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.

Purinergic control of AMPK activation by ATP released through connexin 43 hemichannels: pivotal roles in hemichannel-mediated cell injury

Connexin hemichannels regulate many cell functions. However, the molecular mechanisms involved remain elusive. Hemichannel opening causes loss of ATP, we therefore speculated a potential role for AMPK in the biological actions of hemichannels. Activation of hemichannels by removal of extracellular Ca(2+) led to an efflux of ATP and a weak activation of AMPK. Unexpectedly, dysfunction of hemichannels markedly potentiated AMPK activation, which was reproduced by promotion of extracellular ATP degradation or inhibition of P2 purinoceptors but counteracted by exogenous ATP. Further analysis revealed that ATP induced a purinoceptor-dependent activation of Akt and mTOR. Suppression of Akt or mTOR augmented AMPK activation, whereas activation of Akt by transfection of cells with myristoylated Akt, a constitutively active form of Akt, abolished AMPK activation. In a pathological model of hemichannel opening triggered by Cd(2+), disclosure of hemichannels similarly enhanced AMPK activity, which protected cells from Cd(2+)-induced cell injury through suppression of mTOR. In summary, our data point to a channel-mediated mechanism for the regulation of AMPK through a purinergic signaling pathway. Furthermore, we define AMPK as a pivotal molecule that underlies the regulatory effects of hemichannels on cell survival.

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.

P2X receptor signaling in skeletal muscle health and disease

Skeletal muscle (SM) is a heterogeneous and dynamic tissue that changes significantly in its form and function in response to external and internal stimuli and throughout life, from development right through to aging. The fully differentiated SM fiber is a highly specialized, complex and metabolically active cell containing finely tuned assemblies of contractile force‐generating proteins, while its growth and repair are maintained by a resident stem cell population. There is increasing evidence that extracellular ATP (ATPe) released during physiological activity and acting on P2 purinoceptors is involved in a number of muscle functions. Furthermore, very high levels of ATPe released from injured muscles can trigger further damage either by altered activation of P2 purinoceptors on muscle cells or by promoting inflammatory cell infiltration. Therefore, the effects of activation of specific P2 purinoceptors in SM can vary from physiologically beneficial to pathologically catastrophic. The most studied in SM so far have been the P2X purinoceptors, which are a family of homo/heterotrimeric ATP‐gated ion channels comprised of seven subtypes. Of these P2X1, P2X2, P2X4, P2X5, P2X6, and P2X7 have been identified, so far, predominantly in mouse SM and shown to influence cell growth, differentiation, death, and regeneration in health and disease. There is, however, a considerable diversity in expression of these receptors between different muscle groups and fiber types, which is not fully recognized yet. Understanding the roles of specific P2X purinoceptors in the physiology and pathology of different muscle groups might offer new opportunities for targeted pharmacological intervention in SM diseases. WIREs Membr Transp Signal 2013. doi: 10.1002/wmts.96Conflict of interest: The authors have declared no conflicts of interest for this article.For further resources related to this article, please visit the WIREs website.

Characterization of P1 (Adenosine) Purinoceptors

The purine nucleoside adenosine (ADO) is an important modulator of cellular function in mammalian tissues, modulating cellular function and neuronal excitability via interactions with different cell surface receptor subtypes that are heterogeneously distributed in both the mammalian CNS and peripheral tissues. Four ADO receptor subtypes have been cloned and characterized. Described in this unit are three radioligand binding assays for pharmacological characterization of the high-affinity ADO receptor subtypes A1, A2A, and A3 receptors. Pharmacological characterization of the low-affinity A2B receptor has been enabled by the use of tritiated xanthine PSB-603. Because receptor localization is an important criterion for differentiation of receptor subtypes, a support protocol that describes the methodology for the localization of ADO receptors in rat brain tissue using autoradiography is also included.

Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation

BackgroundChronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts.ResultsBradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2-octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor.ConclusionsBradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors.

Lung inflammation caused by adenosine-5′-triphosphate is mediated via Ca2+/PKCs-dependent COX-2/PGE2 induction

Up-regulation of cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) are implicated in lung inflammation. Adenosine 5′-triphosphate (ATP) has been shown to act via activation of P2 purinoceptors, leading to COX-2 expression in various inflammatory diseases. The mechanisms of ATP-induced COX-2 expression and PGE2 release remain unclear. We showed that pretreatment with the inhibitors of P2 receptors (PPADS and Suramin), Gq protein (GPA2A), phosphatidylcholine-phospholipase C (PC-PLC; D609), phosphoinositide-phospholipase C (PI-PLC; ET-18-OCH3), Ca2+/calmodulin-dependent protein kinase II (CaMKII; KN62), protein kinase C (PKC; Gö6976, Ro-318220, GF109203X, and rottlerin), MEK1/2 (PD98059), p38 MAPK (SB202190), and nuclear factor-kappaB (NF-κB; Bay11-7082) and the intracellular calcium chelator (BAPTA/AM) or transfection with siRNAs of these molecules and cPLA2 reduced ATPγS-induced COX-2 expression or PGE2 production in A549 cells. In addition, ATPγS-induced elevation of intracellular Ca2+ concentration was attenuated by PPADS, Suramin, D609, or ET-18-OCH3. ATPγS-induced p38 MAPK, p42/p44 MAPK, and NF-κB p65 activation were inhibited by Gö6976, Ro-318220, GF109203X, or rottlerin. ATPγS also induced cPLA2 phosphorylation and activity, which were reduced via inhibition of P2 receptors, PKCs, p38 MAPK, and p42/p44 MAPK. ATPγS-induced cPLA2 expression was inhibited by SB202190, PD98059, or Bay11-7082. In the in vitro study, we established that ATPγS induced PGE2 generation via a cPLA2/COX-2-dependent pathway. In the in vivo study, we found that ATPγS induced COX-2 mRNA expression in the lungs and leukocyte (mainly eosinophils and neutrophils) count in bronchoalveolar lavage (BAL) fluid in mice via a P2 receptors-dependent signaling pathway. We concluded that ATPγS may induce lung inflammation via a cPLA2/COX-2/PGE2-dependent pathway.

Purinergic modulation of smooth muscle intracellular Ca2+ concentration

Basal, steady‐state Ca2+ levels are determined by the balance between Ca2+ release/influx and uptake/extrusion mechanisms. Extrinsic factors can alter this balance to affect smooth muscle excitability. Extracellular ATP acts as an inhibitory neurotransmitter in gastrointestinal and vascular (portal vein) smooth muscle. This study examined the effect of extracellular ATP on the steady‐state intracellular Ca2+ concentration ([Ca2+]c). Extracellular ATP (1 mM; applied via picopritzer) reversibly decreased steady‐state [Ca2+]c (measured using fluo 4). To assess whether ATP functioned by altering influx or extrusion [Ca2+]c and inward currents were measured simultaneously in voltage‐clamped myocytes during a depolarizing pulse. ATP decreased the peak amplitude without altering the voltage‐dependence of the inward current. The P2 purinoceptor inhibitor, suramin, and the P2Y1 specific inhibitor, MRS2179, each prevented ATP inhibition of steady‐state [Ca2+]c and inward current. Interestingly, suramin and MRS2179 each also increased the magnitude of the inward current per se, indicating tonic ATP inhibition of Ca2+ channels by endogenous ATP. ATP did not alter Ca2+ uptake/extrusion mechanisms. ATP appears to influence Ca2+ influx mechanisms in smooth muscle via P2Y receptors to exert a negative regulation of excitability.

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.