Differential responses to ATPgammaS in the mesenteric and hindlimb vascular bed of the cat.

The mechanism by which the purinergic agonist adenosine 5'-O-(3 thiotriphosphate) (ATPgammaS) decreases vascular resistance was investigated in the mesenteric and hindlimb vascular beds of the cat. Injections of ATPgammaS into the hindlimb perfusion circuit elicited dose-dependent decreases in perfusion pressure while injections into the mesenteric circuit produced a biphasic response with an initial vasopressor response followed by a vasodepressor response. In the mesenteric vascular bed the pressor response to ATPgammaS was blocked by a P2X1 receptor antagonist. Also an inhibitor of nitric oxide synthase enhanced the vasoconstrictive responses to ATPgammaS. However, the vasodepressor response in the mesenteric bed was not altered by the adminstration of an alpha adrenergic receptor antagonist, a cyclooxygenase inhibitor, a P2Y1 receptor antagonist, or a K+ATP channel blocking agent. These data suggest that the vasopressor response to ATPgammaS in the mesenteric vascular bed of the cat is mediated via P2X1 receptor activation. The differential responses to ATPgammaS in the hindlimb and mesentery suggest differences in purinergic receptor distribution in the vascular system of the cat. In addition, the results suggest that prostaglandin synthesis, P2Y1 receptor activation, alpha receptor inhibition, and K+ATP channels activation play little to no role in mediating the vascular response to ATPgammaS in the mesentery of the cat.     

Dopamine induces inhibitory effects on the circular muscle contractility of mouse distal colon via D1- and D2-like receptors

Dopamine (DA) acts as gut motility modulator, via D1- and D2-like receptors, but its effective role is far from being clear. Since alterations of the dopaminergic system could lead to gastrointestinal dysfunctions, a characterization of the enteric dopaminergic system is mandatory. In this study, we investigated the role of DA and D1- and D2-like receptors in the contractility of the circular muscle of mouse distal colon by organ-bath technique. DA caused relaxation in carbachol-precontracted circular muscle strips, sensitive to domperidone, D2-like receptor antagonist, and mimicked by bromocriptine, D2-like receptor agonist. 7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH-23390), D1-like receptor antagonist, neural toxins, L-NAME (nitric oxide (NO) synthase inhibitor), 2′-deoxy-N₆-methyl adenosine 3′,5′-diphosphate diammonium salt (MRS 2179), purinergic P2Y1 antagonist, or adrenergic antagonists were ineffective. DA also reduced the amplitude of neurally evoked cholinergic contractions. The effect was mimicked by (±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrobromide (SKF-38393), D1-like receptor agonist and antagonized by SCH-23390, MRS 2179, or L-NAME. Western blotting analysis determined the expression of DA receptor proteins in mouse distal colon. Notably, SCH-23390 per se induced an increase in amplitude of spontaneous and neurally evoked cholinergic contractions, unaffected by neural blockers, L-NAME, MRS 2179, muscarinic, adrenergic, or D2-like receptor antagonists. Indeed, SCH-23390-induced effects were antagonized by an adenylyl cyclase blocker. In conclusion, DA inhibits colonic motility in mice via D2- and D1-like receptors, the latter reducing acetylcholine release from enteric neurons, involving nitrergic and purinergic systems. Whether constitutively active D1-like receptors, linked to adenylyl cyclase pathway, are involved in a tonic inhibitory control of colonic contractility is questioned.     

Aggravation of chemically-induced injury in perfused rat liver by extracellular ATP

The effects of purinergic receptor agonists on acute liver damage and hemodynamics were studied using chemically-induced liver injury. Rat livers were perfused in situ 24 h after treatment with D-galactosamine (800 mg/kg, i.p.). In these livers, infusion of ATP (50 microM) into the portal vein caused a rapid increase in the leakage of LDH and AST from perfused liver in a dose dependent manner, accompanied with flow reduction. The similar but less effective responses were also observed by the infusion of ADP. Infusion of adenosine, a P1-receptor agonist, induced only minimal changes of liver damage and flow rate. The ATP-induced changes were almost completely suppressed by P2-receptor antagonist, suramin, but not affected by P1-receptor antagonist, 8-phenyltheophylline. Pretreatment of rats with gadolinium chloride, which depletes Kupffer cells, did not inhibit the potentiation of liver damage caused by ATP, whereas hemodynamic effects of ATP were significantly attenuated by gadolinium. These results indicate that extracellular ATP aggravates acute liver injury mediated by P2-type purinergic receptors.     

Nerve growth factor-stimulated neuronal differentiation induces changes in P2 receptor expression and nucleotide-stimulated catecholamine release

Extracellular nucleotides modulate synaptic transmission and neuronal communication by activating purinergic 2 (P2) (nucleotide) receptors. Here, we assessed changes in the regulation by nucleotides and their receptors of an important physiological response - release and uptake of catecholamines - that accompanies sympathoadrenal neuronal differentiation. Nerve growth factor (NGF)-promoted differentiation of pheochromocytoma 12 (PC12) cells enhanced the ability of the non-hydrolyzable ATP analog, ATPgammaS, to stimulate catecholamine (norepinephrine, NE) release and this enhancement occurred without a significant alteration in NE uptake. In addition to ATPgammaS, 2-MeSATP and alphabetaMeATP, P2X receptor-selective agonists, caused greater NE release from NGF-differentiated than from undifferentiated PC12 cells. NGF-differentiated PC12 cells had altered mRNA expression of several P2Y and P2X receptors but protein expression was only increased for P2X, in particular P2X(1-4,) receptors and P2X, but not P2Y, receptor inhibitors blunted the NGF-promoted enhancement in nucleotide-regulated catecholamine release. Surprisingly, siRNA directed against P2X(2), the receptor with the highest expression, failed to alter NE release by ATPgammaS. These findings indicate that sympathetic neuronal differentiation by NGF increases both the expression of P2X receptor sub-types and their regulation of catecholamine release. NGF-promoted increased expression of P2X receptors thus appears to be a physiologically important response that characterizes sympathetic neuronal differentiation.     

Physiological role of neuropeptide Y in the regulation of the ascending phase of the peristaltic reflex

The physiological role of neuropeptide Y (NPY) and of specific NPY receptors in regulating the intestinal peristaltic reflex was examined in three-compartment flat-sheet preparations of rat colon. Graded muscle stretch or mucosal stimulation applied to the central compartment inhibited NPY release in the orad compartment where ascending contraction was measured. NPY and the Y1-receptor agonist [Leu31, Pro34]NPY inhibited, whereas the selective Y1-receptor antagonist BIBP 3226 augmented ascending contraction and substance P (SP) release in the orad compartment induced by muscle stretch or mucosal stimulation. Neither agonist nor antagonist had any effect on descending relaxation or VIP release in the caudad compartment. The Y2-receptor agonist NPY13-36 and antagonist BIIE 0246 had no effect on peptide release or mechanical response. The results indicate that suppression of a tonic inhibitory influence of NPY neurons on excitatory neurotransmitter release contributes substantially to the orad contractile phase of the peristaltic reflex. The effect of NPY on neurotransmitter release is mediated by Y1 receptors.     

Use of biological methods in the analysis of the purinergic activity of etimizol

To analyse etimizol purinergic activity, methods of biological analysis were applied using isolated smooth muscle strips of the guinea-pig caecum and developing embryo cultures of the sea urchin (Strongylocentrotus intermedius). Stimulating etimizol action in 10(-8)-10(-12) M concentration was observed on embryonal development in the sea urchin to be eliminated by co-action of etimizol and P1-purinoceptor antagonist theophylline. The analogous action of the preparations was established using isolated smooth muscle strips of the guinea-pig caecum.     

Balance of purines may determine life or death of retinal ganglion cells as A3 adenosine receptors prevent loss following P2X7 receptor stimulation

The purines ATP and adenosine can act as a coordinated team of transmitters. As extracellular adenosine is frequently derived from the enzymatic dephosphorylation of released ATP, the distinct actions of the two purines can be synchronized. In retinal ganglion cells (RGCs), stimulation of the P2X7 receptor for ATP leads to increased intracellular Ca2+ and death. Here we define the contrasting effects of adenosine and identify protective actions mediated by the A3 receptor. Adenosine attenuated the rise in Ca2+ produced by the P2X7 agonist 3'-O-(4-benzoylbenzoyl)ATP (BzATP). Adenosine was also neuroprotective, increasing the survival of ganglion cells exposed to BzATP. The A3 adenosine receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronimide (Cl-IB-MECA) mimicked the inhibition of the Ca2+ rise, whereas the A3 antagonist 3-Ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS-1191) reduced the protective effects of adenosine. Both Cl-IB-MECA and a second A3 receptor agonist IB-MECA reduced the cell loss triggered by BzATP. The actions of BzATP were mimicked by ATPgammaS, but not by ATP. In summary, adenosine can stop the rise in Ca2+ and cell death resulting from stimulation of the P2X7 receptor on RGCs, with the A3 adenosine receptor contributing to this protection. Hydrolysis of ATP into adenosine and perhaps inosine shifts the balance of purinergic action from that of death to the preservation of life.     

Possible Agonistic Interaction of Defibrotide,A DNA Derivative,with Adenosine Receptors “In Vitro”

Abstract

Defibrotide (DFT), a single-stranded DNA, dose-dependently displaced only 3H-CHA and 3H-NECA (markers of adenosine receptors), among 37 radioligands tested. On isolated gastro-intestinal smooth muscle, DFT caused a dose-dependent relaxation, that was antagonized by 8-phenyltheophylline, a mixed Al- and A2-receptor antagonist.     

Modulating influence of adenosine triphosphate on the oscillating properties of hippocampal neurons during early ontogenesis

Extracellular ATP was used to test the involvement of purinergic P2 receptors in the modulation of GAB Aergic giant depolarizing potentials (GDPs) in rat neonatal hippocampus. Bath application of ATP reduced the GDP frequency in a concentration-dependent manner. The inhibitory effect of ATP persisted in the presence of adenosine P1 receptor antagonist DPCPX indicating the action of ATP on GDPs. It was suggested that, during development, ATP can modulate network-driven activity in the neonatal hippocampus through the activation of purinergic P2 receptors.     

Relaxation of guinea pig trachealis during electrical field stimulation increases with age.

Our laboratory has previously shown that maturation of airway smooth muscle (ASM) contractility may play a role in the airway hyperresponsiveness displayed by juveniles of many species, including humans (Chitano P, Wang J, Cox CM, Stephens NL, and Murphy TM. J Appl Physiol 88: 1338-1345, 2000). ASM relaxation, which could also contribute to airway hyperresponsiveness, has neither been described nor quantified during maturation. Therefore, we studied ASM relaxation during and after electrical field stimulation (EFS) in tracheal strips from 1-wk-old, 3-wk-old, and 3-mo-old guinea pigs. Strips were stimulated (60 Hz, 18 V) at their optimal length for 15, 20, and 25 s, with and without the cyclooxygenase inhibitor indomethacin. To evaluate the role of the epithelium, deepithelialized strips from adult animals were also studied. New indexes were developed to quantify relaxation during EFS. We measured the time course of tension relaxation and its maximum rate (RTR) during the EFS, as well as the residual tension at the end of the EFS (TCT(end)). After EFS, we measured the maximum RTR and the time needed to reduce to half the TCT(end). Relaxation during the EFS significantly increased with age. Indomethacin reduced this age difference by increasing relaxation in strips from younger animals. By contrast, removal of the epithelium in adult strips decreased relaxation. Relaxation after EFS decreased with age and was not affected by indomethacin. In adult strips, it was further reduced by epithelium removal. Our results show that during EFS 1) airway smooth muscle relaxation increases with age, 2) cyclooxygenase metabolites oppose relaxation in younger animals, and 3) epithelium removal inhibits relaxation. We suggest that a reduced ASM relaxing ability during stimulation may be involved in juvenile airway hyperresponsiveness.     

Expression and Characterization of Purinergic Receptors in Rat Middle Meningeal Artery–Potential Role in Migraine

The dura mater and its vasculature have for decades been central in the hypothesis of migraine and headache pathophysiology. Although recent studies have questioned the role of the vasculature as the primary cause, dural vessel physiology is still relevant in understanding the complex pathophysiology of migraine. The aim of the present study was to isolate the middle meningeal artery (MMA) from rodents and characterize their purinergic receptors using a sensitive wire myograph method and RT-PCR. The data presented herein suggest that blood flow through the MMA is, at least in part, regulated by purinergic receptors. P2X1 and P2Y6 receptors are the strongest contractile receptors and, surprisingly, ADPβS caused contraction most likely via P2Y1 or P2Y13 receptors, which is not observed in other arteries. Adenosine addition, however, caused relaxation of the MMA. The adenosine relaxation could be inhibited by {"type":"entrez-protein","attrs":{"text":"SCH58261","term_id":"1052882304","term_text":"SCH58261"}}SCH58261 (A2A receptor antagonist) and caffeine (adenosine receptor antagonist). This gives one putative molecular mechanism for the effect of caffeine, often used as an adjuvant remedy of cranial pain. Semi-quantitative RT-PCR expression data for the receptors correlate well with the functional findings. Together these observations could be used as targets for future understanding of the in vivo role of purinergic receptors in the MMA.     

嘌呤拟似物对豚鼠胃窦环行肌自发性收缩及电活动的影响

为探讨非肾上腺素能非胆碱能神经递质对胃窦环行肌功能的调节作用,在离体胃平滑肌上观察了嘌呤拟似物对胃窦环行肌自发性收缩活动和电活动的影响。电活动用传统的细胞内微电极记录,并和收缩活动同步描记于多道生理记录仪。结果表明,嘌呤能P2Y受体激动剂,三磷酸腺苷(ATP)和2-methylthio ATP(2-MeSATP)均增强胃窦平滑肌的收缩活动,但不影响电活动,而且ATP和2-MeSATP对胃平滑肌收缩活动的增强作用可被嘌呤能P2Y受体阻断剂,reactive blue-2和苏拉明(suramin)所阻断。用100μmol／L α,β-MeATP引起的脱敏感使P2X受体被阻断,ATP增强胃窦平滑肌收缩活动的效应不受影响。嘌呤能P2X受体激动剂,α,β-MeATP明显抑制胃窦环行肌自发性收缩活动,同时使膜电位明显超极化。ATP对胃窦平滑肌的收缩作用不被L型钙通道阻断剂尼卡地平(nicardipine)阻断,但细胞外用无钙液灌流时这种效应则完全被阻断。用前列腺素合成抑制剂消炎痛预先处理20min后,ATP和2-MeSATP仍能增强胃窦平滑肌的自发性收缩活动。以上结果提示：(1)ATP和2-MeSATP通过嘌呤能P2Y受体增强胃窦平滑肌的自发性收缩活动,而α,β-MeATP或β,γ-MeATP通过嘌呤能P2X受体使膜电位超极化,引起胃窦平滑肌的舒张;(2)ATP和2-MeSATP增强胃窦平滑肌自发性收缩活动的效应依赖于细胞外钙,但钙离子进入细胞的途径并不是电压依赖性钙通道;(3)ATP和2-MeSATP增强胃窦平滑肌自发性收缩活动的效应不通过前列腺素介导。     

Functional characterization of P2Y and P2X receptors in human eosinophils

Activation of purinoceptor by ATP induces in eosinophils various cell responses including calcium transients, actin polymerization, production of reactive oxygen metabolites, CD11b-expression, and chemotaxis. Here, the effect of ion channel-gated P2X and/or G protein-coupled P2Y receptor agonists ATP, ATPgammaS, alpha,beta-meATP, 2-MeSATP, BzATP, ADP, CTP, and UTP on the intracellular Ca(2+)-mobilization, actin polymerization, production of reactive oxygen metabolites, CD11b expression and chemotaxis of human eosinophils were measured and the biological activity was analyzed. Although all tested nucleotides were able to induce all these cell responses, the biological activity of the analyzed nucleotides were distinct. Agonists of the G protein-coupled P2Y receptors such as 2-MeSATP, UTP, and ADP have a higher biological activity for production of reactive oxygen metabolites, actin polymerization and chemotaxis in comparison to the ion channel-gated P2X agonists alphabeta-meATP, BzATP, and CTP. In contrast, P2Y and P2X agonist showed similar potencies in respect to intracellular calcium transient and CD11b up-regulation. This conclusion was further supported by experiments with receptor iso-type antagonist KN62, EGTA or with the G(i) protein-inactivating pertussis toxin. These findings indicate participation of different purinorecptors in the regulation of cell responses in eosinophils.     

Species variations in the relaxation of tail arteries to electrical stimulation

Relaxation in response to electrical stimulation has been studied in isolated tail arteries from rats, cats and pigs. Electrical stimulation elicited contractile responses in unstimulated strips, but caused frequency-dependent relaxations in tail arteries from all species studied, following contractile responses by various agents. The order of susceptibility to the relaxant effect of electrical stimulation in arteries from all three species was pig greater than cat greater than rat. Relaxations to electrical stimulation were unaffected by prior treatment of arteries with atropine, propranolol, tetrodotoxin, indomethacin, ouabain, pyrilamine and chemical denervation with 6-hydroxy-dopamine, but were prevented, dose-dependently, by cimetidine and aminophylline (antagonists of histamine H2-receptor and adenosine P1-receptor, respectively). The results suggest that relaxation of tail arteries from the rat, cat and pig in response to electrical stimulation is modulated by histamine and adenosine.     

Histological changes in the pituitary-thyroid axis during spontaneous and artificially-induced metamorphosis of larvae of the flounder Paralichthys olivaceus

Summary Histological changes in the pituitary TSH cells and in the thyroid gland of flounder (Paralichthys olivaceus) larvae during spontaneous or artificially induced metamorphosis were studied. Activity of the immunoreactive TSH cells (IrTSH cells) gradually increased during premetamorphosis, reaching the highest level in prometamorphic larvae, and the cells were degranulated in metamorphic climax. The IrTSH cells were most inactive at the post-climax stage. The thyroid gland was morphologically the most active in metamorphic climax when the degranulation occurred in the pituitary IrTSH cells, and appeared inactive at post-climax. A few weeks after metamorphosis, both the IrTSH cells and the thyroid gland appeared to be activated again in the benthic, juvenile flounder. Administration of thyroxine or thiourea revealed negative feedback regulation of the pituitary-thyroid axis in flounder larvae. These results indicate that activation of the pituitary-thyroid axis induces metamorphosis in the flounder.     

Functional evidence for purinergic inhibitory neuromuscular transmission in the mouse internal anal sphincter

The neurotransmitter(s) underlying nitric oxide synthase (NOS)-independent neural inhibition in the internal anal sphincter (IAS) is still uncertain. The present study investigated the role of purinergic transmission. Contractile and electrical responses to electrical field stimulation of nerves (0.1-5 Hz for 10-60 s) were recorded in strips of mouse IAS. A single stimulus generated a 28-mV fast inhibitory junction potential (IJP) and relaxation. The NOS inhibitor N(omega)-nitro-l-arginine (l-NNA) reduced the fast IJP duration by 20%. Repetitive stimulation at 2.5-5 Hz caused a more sustained IJP and sustained relaxation. l-NNA reduced relaxation at 1 Hz and the sustained IJP at 2.5-5 Hz. All other experiments were carried out in the presence of NOS blockade. IJPs and relaxation were significantly reduced by the P2 receptor antagonists 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid (PPADS) (100 microM), by desensitization of P2Y receptors with adenosine 5'-[beta-thio]diphosphate (ADP-betaS) (10 microM), and by the selective P2Y1 receptor blocker 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate (MRS2179) (10 microM). Relaxation and IJPs were also significantly reduced by the K(+) channel blocker apamin (1 microM). Removal of extracellular potassium (K(o)) increased IJP amplitude to 205% of control, whereas return of K(o) 30 min later hyperpolarized cells by 19 mV and reduced IJP amplitude to 50% of control. Exogenous ATP (3 mM) relaxed muscles in the presence of TTX (1 microM) and hyperpolarized cells by 15 mV. In conclusion, these data suggest that purinergic transmission significantly contributes to NOS-independent neural inhibition in the mouse IAS. P2Y1 receptors, as well as at least one other P2 receptor subtype, contribute to this pathway. Purinergic receptors activate apamin-sensitive K(+) channels as well as other apamin-insensitive conductances leading to hyperpolarization and relaxation.     

Domain-specific purinergic signaling in polarized rat cholangiocytes

In cholangiocytes, adenine nucleotides function as autocrine/paracrine signals that modulate ductular ion transport by activation of purinergic receptors. The purpose of these studies was to identify cellular signals that modulate ATP release and nucleotide processing in polarized normal rat cholangiocytes. In Ussing chamber studies, selective exposure of the apical and basolateral membranes to ATP or adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) stimulated increases in short-circuit current. Apical purinergic receptor agonist preference was consistent with the P2Y(2) subtype. In contrast, basolateral ADP was more potent in stimulating transepithelial currents, consistent with the expression of different basolateral P2 receptor(s). Luminometric analysis revealed that both membranes exhibited constitutive ATP efflux. Hypotonic exposure enhanced ATP release in both compartments, whereas decreases in ATP efflux during hypertonicity were more prominent at the apical membrane. Increases in intracellular cAMP, cGMP, and Ca(2+) also increased ATP permeability, but selective effects on apical and basolateral ATP release differed. Finally, the kinetics of ATP degradation in apical and basolateral compartments were distinct. These findings suggest that there are domain-specific signaling pathways that contribute to purinergic responses in polarized cholangiocytes.     

Effect of arylazido aminopropionyl ATP (ANAPP3), a putative ATP antagonist,on ATP responses of isolated guinea pig smooth muscle

Arylazido aminopropionyl ATP (ANAPP₃), a photoaffinity analogue of adenosine 5′-triphosphate, photoactivated with visible light (+hv), specifically and irreversibly antagonized ATP contractions of the guinea pig vas deferens. ANAPP₃ (30 μM) antagonized responses to exogenously added ATP in untreated, and in tissues pretreated with indomethacin (2.9 μM) and 6-(2-hydroxy-5-nitrobenzyl)-thio guanosine (10 μM). It was of interest to see if this pharmacological antagonist of ATP could be used to assess the validity of the purinergic nerve hypothesis by allowing a differentiation between, or proof of the identity of, responses to ATP and the non-adrenergic inhibitory transmitter in guinea pig stomach fundus. After photoactivation (+hv) in the organ bath and subsequent washout, ANAPP₃ (30 and 100 μM) failed to antagonize relaxant responses to ATP (1.0 – 1000 μM) in fundic strips. In addition ANAPP₃ failed to antagonize ATP-induced inhibition of the twitch response in electrically stimulated guinea pig ileum longitudinal muscle strips. We conclude that ANAPP₃ does not antagonize all actions of ATP, which may limit its usefulness in assessing the above hypothesis. Results with this compound suggest that ATP excitatory receptors may differ from those mediating relaxation and other ATP actions.