Purinoceptor Antagonist Research Articles

Functional expression of muscarinic and purinoceptors in the urinary bladder of male and female rats and guinea pigs

The objectives of this study were to compare the functional expression of muscarinic and purinergic receptors in the urinary bladder of 2 species, rat and guinea pig under comparable experimental conditions; and to test whether the receptors in males and females differ. Reverse transcription-polymerase chain reaction (RT-PCR) techniques were used to identify gene expression profiles in bladder smooth muscle (total n=8 rats, 7 guinea pigs) and mechanical responses to nerve stimulation and applied acetylcholine (ACh) in the presence of specific antagonists were used to identify functional receptor sub-types (total n=12 rats, 16 guinea pigs). RT-PCR indicated that M2 and M3 were the predominant muscarinic receptor genes in both the male and female rat and guinea pig bladders. The phasic component of the nerve-induced contraction was greater in guinea pigs vs. rats. The tonic component and the ACh response were inhibited by the M3 receptor antagonist, darifenacin (10(-6) M, P≤0.05), but not by the M2 receptor antagonist, methoctramine (10(-5) M). The antipurinergic drug α, β-methylene ATP (5 × (-5) M) caused a significant reduction in the amplitude of the phasic response to nerve stimulation in all groups, and this effect was significantly greater in male vs. female rats. mRNA for the purinergic P2X1, P2X2, P2X4, P2X5 and P2X7 receptors was detected in both male and female rats, whereas P2X3 and P2X6 were inconsistently detected in male rats. The P2X1 purinoceptor antagonist pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4', 8'-disulphonate) (PPNDS), only inhibited nerve induced contractions at high concentrations (up to 10(-4) M). While only minor functional differences were documented in cholinergic and purinergic bladder contractile responses between male and female animals, and between rats and guinea pigs, data such as presented in this study are critical in determining how relative functional contributions may change in the diseased state, providing valuable information towards new treatment options.

Comparison of the peripheral mediator background of heat injury- and plantar incision-induced drop of the noxious heat threshold in the rat

Aims Previously we described the drop of the noxious heat threshold in response to mild heat injury or plantar incision. While mild heat injury elicits an immediate and short-lasting thermal hyperalgesia, surgical incision leads to a delayed and sustained heat hyperalgesia. Only very few peripheral mediators of these phenomena have been identified. Therefore the present study aimed at comparing the peripheral mediator background of heat hyperalgesia evoked by mild heat injury or surgical incision. Main methods Heat hyperalgesia was assessed by measuring the behavioural noxious heat threshold in conscious rats employing an increasing-temperature water bath. Key findings The heat threshold drop evoked by a mild heat injury and measured 10 min afterwards was reduced by intraplantarly applied HOE 140, a bradykinin B 2 receptor antagonist, NDGA, a non-selective lipoxygenase inhibitor, L-NOARG, a non-selective nitric oxide synthase inhibitor, TNP-ATP, a P2X purinoceptor antagonist and AMG9810, an antagonist of the transient receptor potential vanilloid type 1 (TRPV1) receptor. The heat threshold drop evoked by plantar incision and measured 18 h later was reduced by intraplantarly applied HOE 140, [des-Arg 10]-HOE 140, a bradykinin B 1 receptor antagonist, L-NOARG, TNP-ATP and the TRPV1 receptor antagonist SB-366791. Significance Only small differences have been revealed between the examined peripheral mediators of the acute heat hyperalgesia evoked by mild heat injury and the sustained increase in heat responsiveness induced by surgical incision. The B 2 and B 1 bradykinin receptor, P2X purinoceptors, TRPV1 receptor, nitric oxide synthase and lipoxygenase(s) are involved in at least one of these hyperalgesia models.

P2X purinoceptors mediate an endothelium‐dependent hyperpolarization in longitudinal smooth muscle of anterior mesenteric artery in young chickens

The chicken anterior mesenteric artery contains an outer longitudinal smooth muscle layer, whose neural regulation remains to be elucidated. ATP evokes a depolarization in the smooth muscle through P2Y purinoceptors. However, there may be an additional inhibitory regulation because blockade of P2Y purinoceptors converts the depolarization to hyperpolarization. The objective of the present study was to examine the mechanism underlying this hyperpolarization. Membrane potentials of longitudinal smooth muscle of the chicken mesenteric artery were recorded with a microelectrode technique. Perivascular nerves were stimulated by applying electrical field stimulation (EFS). EFS induced a hyperpolarization in preparations obtained from 5-week-old chickens, whereas it evoked a depolarization in those from 12-week-old chickens. The EFS-evoked hyperpolarization in 5-week-old chickens was blocked by a non-specific purinoceptor antagonist, suramin, and by a specific P2X purinoceptor antagonist, pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid. Desensitization of the P2X purinoceptor with its agonist alpha,beta-MeATP significantly suppressed EFS-evoked hyperpolarization. Blockade of the P2Y purinoceptor did not affect EFS-evoked hyperpolarization. The application of the NOS inhibitor Nomega-nitro-L-arginine methyl ester or the removal of the endothelium inhibited the hyperpolarization. The application of the nitric oxide (NO) donor sodium nitroprusside mimicked the hyperpolarization. Reverse transcriptase-PCR showed that P2X purinoceptors are expressed in the endothelium of the anterior mesenteric artery. Hyperpolarization in the longitudinal smooth muscle of the chicken anterior mesenteric artery was induced by ATP. ATP released from perivascular nerves may act on P2X purinoceptors in the endothelium and thereby stimulate NO production.

Functional Interaction Between Purinergic System and Opioid Withdrawal: In Vitro Evidence

The effects exerted by P1 and P2 as well as by A1 and A2 agonists and antagonists purinoceptor on the acute opiate withdrawal induced by morphine were investigated in vitro. Following a 4 min in vitro exposure to morphine, the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone. The P1 purinoceptor agonist, adenosine, was able dose-dependently to reduce morphine withdrawal whereas alpha,beta-methylene ATP (APCPP), a P2 purinoceptor agonist, increased morphine withdrawal. Caffeine, a P1 purinoceptor antagonist, was able significantly and in a concentration dependent manner to increase morphine withdrawal whereas quinidine, a P2 receptor antagonist, reduced it. Also, the adenosine A1 receptor agonist, N6-Cyclopentyladenosine (CPA) was able to reduce dose-dependently naloxone-precipitated withdrawal whereas the selective adenosine A(2A) receptor agonist CGS 21680 increased the naloxone-precipitated withdrawal phenomenon. Dipyridamole, a blocker of adenosine reuptake, induced a significant reduction of morphine dependence. Caffeine, an adenosine receptor antagonist, significantly increased the naloxone-precipitated withdrawal effect in a concentration dependent manner. The same effect was observed with 8-phenyltheophylline (8PT), an A1 adenosine receptor antagonist, whereas 3,7-dimethyl-1-propargylxanthine (DMPX), an A2 adenosine receptor antagonist, reduced the naloxone-precipitated withdrawal phenomenon. The results of our experiments indicate that P1 and P2 as well as A1 and A2 purinoceptor agonists and antagonists are able to influence opiate withdrawal in vitro, suggesting an important functional interaction between the purinergic system and opioid withdrawal.

The NANC relaxation of the human ileal longitudinal and circular muscles is inhibited by MRS 2179, a P 2 purinoceptor antagonist

Aims Functional innervation of the human small intestine may be different from that of experimental animals. These experiments set out to assess the mediating roles of P 2 purinoceptors in the non-adrenergic, non-cholinergic (NANC) relaxation of the human ileum longitudinal and circular muscles. Main methods In organ bath experiments NANC relaxations were evoked by electrical field stimulation (EFS). The relaxant effects of ATP and sodium nitroprusside were also examined. Key findings In the longitudinal muscle, relaxation in response to EFS (2 or 10 Hz for 30 s) or the relaxant effect of exogenous ATP were strongly inhibited or abolished, respectively, by the P 2 purinoceptor antagonist MRS 2179 (10 μM). MRS 2179 had a smaller effect at 3 μM. The NANC relaxation was also inhibited by apamin (3 μM), but not by N G-nitro-L-arginine (100 μM), an inhibitor of nitric oxide synthesis. Both apamin (3 μM) and MRS 2179 (3 μM, a concentration below the effective range if administered alone) strongly inhibited the NANC response in preparations pretreated with the nitric oxide synthase inhibitor. NANC relaxations of the circular muscle were also inhibited by MRS 2179 (3−10 μM). Significance MRS 2179-sensitive P 2 purinoceptors play a mediating role the NANC relaxation in the ileal longitudinal and circular muscle. There seems to be a supra-additive relationship between the purinergic and nitrergic mechanisms in the longitudinal muscle.

Antiplatelet effects of piplartine, an alkamide isolated from <I>Piper tuberculatum</I>: possible involvement of cyclooxygenase blockade and antioxidant activity

Piplartine (piperlongumine; 5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl]-2(1H) pyridinone) is an alkaloid amide isolated from Piper species (Piperaceae). It has been reported to show multiple pharmacological activities in vitro and in vivo. We evaluated the in-vitro antiplatelet effect of piplartine isolated from the roots of P. tuberculatum, on human platelet aggregation induced in platelet-rich plasma by the agonists collagen, adenosine 5'-diphosphate (ADP), arachidonic acid (AA) and thrombin. Piplartine (100 mug/ml) caused a 30% inhibition in platelet aggregation when collagen was the agonist. At 200 mug/ml, piplartine significantly inhibited the aggregation induced by arachidonic acid (100%), collagen (59%) or ADP (52%) but not that induced by thrombin. The highest concentration of piplartine (300 mug/ml) inhibited thrombin- (37%), ADP- (71%) and collagen- (98%) induced aggregation. The inhibitory effect of piplartine on ADP-induced platelet aggregation was not modified by pretreatment with pentoxifylline (a phosphodiesterase inhibitor), L-arginine (a substrate for nitric oxide synthase) or ticlopidine (a P2Y(12) purinoceptor antagonist). However, aspirin, a well-known inhibitor of cyclooxygenase, greatly increased the inhibitory effect of piplartine on arachidonic-acid-induced platelet aggregation. The mechanism underlying the piplartine antiplatelet action is not totally clarified. It could be related to the inhibition of cyclooxgenase activity and a decrease in thromboxane A(2) formation, similar to that occurring with aspirin. This and other possible mechanisms require further study.

Antiplatelet effects of piplartine, an alkamide isolated from Piper tuberculatum: possible involvement of cyclooxygenase blockade and antioxidant activity

Abstract Objectives Piplartine (piperlongumine; 5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl]-2(1H) pyridinone) is an alkaloid amide isolated from Piper species (Piperaceae). It has been reported to show multiple pharmacological activities in vitro and in vivo. Methods We evaluated the in-vitro antiplatelet effect of piplartine isolated from the roots of P. tuberculatum, on human platelet aggregation induced in platelet-rich plasma by the agonists collagen, adenosine 5′-diphosphate (ADP), arachidonic acid (AA) and thrombin. Key findings Piplartine (100μg/ml) caused a 30% inhibition in platelet aggregation when collagen was the agonist. At 200 μg/ml, piplartine significantly inhibited the aggregation induced by arachidonic acid (100%), collagen (59%) or ADP (52%) but not that induced by thrombin. The highest concentration of piplartine (300 μg/ml) inhibited thrombin- (37%), ADP- (71%) and collagen- (98%) induced aggregation. The inhibitory effect of piplartine on ADP-induced platelet aggregation was not modified by pretreatment with pentoxifylline (a phosphodiesterase inhibitor), l-arginine (a substrate for nitric oxide synthase) or ticlopidine (a P2Y12 purinoceptor antagonist). However, aspirin, a well-known inhibitor of cyclooxygenase, greatly increased the inhibitory effect of piplartine on arachidonic-acid-induced platelet aggregation. Conclusions The mechanism underlying the piplartine antiplatelet action is not totally clarified. It could be related to the inhibition of cyclooxgenase activity and a decrease in thromboxane A2 formation, similar to that occurring with aspirin. This and other possible mechanisms require further study.

Reciprocal regulation of ATPγS‐induced monocyte chemoattractant protein‐1 production by ERK and p38 MAP kinases in rat corticostriatal slice cultures

Monocyte chemoattractant protein-1 (MCP-1, CCL2) is a well-defined chemokine implicated in the pathology of various neurodegenerative diseases and brain injuries, such as Alzheimer's disease, multiple sclerosis, stroke, and traumatic injury. We investigated the effect of the activation of P2 purinoceptors on MCP-1 production in rat corticostriatal slice cultures. Treatment with adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), a hydrolysis-resistant adenosine triphosphate (ATP) analog, induced MCP-1 production in astrocytes. The induction was in a concentration-dependent manner and was antagonized by a P2 purinoceptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid. The inhibition of an extracellular signal-regulated kinase (ERK) pathway by PD98059 and U0126 significantly suppressed ATPgammaS-induced MCP-1 mRNA expression and protein production, while inhibition of c-Jun N-terminal kinase by SP600125 resulted in the partial suppression. Conversely, SB203580, a p38 mitogen-activated protein (MAP) kinase inhibitor, significantly enhanced ATPgammaS-induced MCP-1 production. Similar effects of ERK and p38 MAP kinase inhibitors on MCP-1 production were observed in the slices stimulated by ATP and BzATP. These results demonstrate that astrocytic MCP-1 production induced by P2 purinoceptor stimulation is reciprocally regulated by ERK and p38 MAP kinases in the organotypic slice cultures.

P02-202 Autoimmune-induced pain alters affective behaviors in a model of neuropsychiatric lupus?

As commonly seen in patients with neuropsychiatric systemic lupus erythematosus (NP-SLE), spontaneous disease onset in the MRL/MpJ-Faslpr/J (MRL-lpr) mouse model of NP-SLE is accompanied by increased autoantibodies, pro-inflammatory cytokines and behavioral dysfunction which precede neuroinflammation and structural brain lesions. Given that P2X purine receptors are important in peripheral "pain" signaling, the possibility that purinoception is involved in the development of aberrant affective behaviors was explored in the present study. Suramin, a P2X purinoceptor antagonist, was administered to lupus-prone mice from 5 to 14 weeks of age (60 mg/kg, i.p.). Novel object and sucrose preference tests were performed to examine behavior, and enzyme-linked immunosorbant assays for autoantibodies and pro-inflammatory cytokines were employed in immunopathological analyses. Suramin was found to attenuate markers of autoimmunity and inflammation, but was not immunosuppressive. Treatment did, however, prevent neophobic- and anhedonic-like deficits in MRL-lpr mice. More specifically, animals receiving the drug showed a profound 3-fold increase in their responsiveness to palatable solution (i.e. a reversal of anhedonia) and were less "distressed" by novelty. The present data supports the hypothesis that sustained activation of the peripheral immune system induces nociceptive-related behavioral symptomatology in lupus animals which is attenuated by the analgesic effects of suramin. It is proposed that anti-purine or cross-reactive autoantibodies cause neuronal hyperexcitability within "pain" pathways early in the disease process, which manifests as affective deficits. At a later stage of chronic lupus-like disease (following breakdown of the blood-brain barrier), fixed structural lesions in dopaminergic pathways likely account for and maintain impairments in emotionality.

Coronary response to diadenosine pentaphosphate after ischaemia-reperfusion in the isolated rat heart

Diadenosine polyphosphates are vasoactive mediators that may be released from platelet granules and which may be present at higher concentrations during coronary ischaemia-reperfusion. The objective of this study was to analyse their effects in such conditions. Rat hearts were perfused in a Langendorff preparation and the response to diadenosine pentaphosphate (Ap5A, 10(-7)-10(-5) M) was recorded. In control hearts, Ap5A produced a small, transient coronary vasoconstriction followed by marked vasodilatation, as well as a reduction in the left ventricular developed pressure dP/dt and heart rate, both at the basal coronary resting tone or after pre-contracting coronary arteries with 9,11-dideoxy-11alpha, 9alpha-epoxymethanoprostaglandin F2alpha (U46619). After ischaemia-reperfusion, the vasoconstriction in response to Ap5A was augmented and vasodilatation diminished, both in hearts with basal or increased vascular tone. The pyridoxal derivative P(2) purinoceptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 3 x 10(-6) M), inhibited this vasoconstriction, while the antagonist of purinergic P(2Y) receptors, Reactive Blue 2 (2 x 10(-6) M), inhibited the vasodilatation, both before and after ischaemia-reperfusion. The antagonist of nitric oxide synthesis N-omega-nitro-L- arginine methyl ester (L-NAME, 10(-4) M) did not modify the response to Ap5A, whereas the cyclooxygenase inhibitor, meclofenamate (2 x 10(-6) M), reduced contraction and increased the relaxation in response to Ap5A after ischaemia-reperfusion but not under control conditions. Ischaemia-reperfusion reduces the vasodilatory response to Ap5A and increases the vasoconstriction provoked due to a reduced influence of purinergic P(2Y) receptors and/or to the production of vasoconstrictor prostanoids.

PARTICIPATION OF EXTRACELLULAR NUCLEOTIDES IN THE WOUND RESPONSE OF DASYCLADUS VERMICULARIS AND ACETABULARIA ACETABULUM (DASYCLADALES, CHLOROPHYTA)(1).

As assayed by fluorescent reporter dyes, nitric oxide (NO) and H2 O2 , two downstream signaling agents induced by wounding in the alga Dasycladus vermicularis (Scop.) Krasser, can also be induced in unwounded Dasycladus cells by μM Adenosine 5'[γ-thio]triphosphate (ATPγS) and Adenosine 5'-[β-thio]diphosphate (ADPβS), but not by Adenosine 5'-O-thiomonophosphate (AMPS). These nucleotide-induced responses are blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), an antagonist of animal purinoceptors, and by adenosine, a feed-back inhibitor of extracellular nucleotide responses in animals. Similar nucleotide- and nucleotide-antagonist responses were observed in Acetabularia acetabulum (L.) P. C. Silva. Significant levels of ATP released from Dasycladus cells were measured at wound sites by a sensitive luciferin-luciferase assay. Additionally, the normal wound-induced production of NO and H2 O2 in Dasycladus can be blocked by pretreating the cells with PPADS. Our results indicate that nucleotides released from wounds can serve as a signal to trigger wound responses in algae, and that coordinated signaling between extracellular nucleotides and the NO pathway may have been established early during the evolution of plants.

Alteration of cholinergic, purinergic and sensory neurotransmission in the mouse colon of food allergy model

It is well known that intestinal anaphylaxis results in a disturbed intestinal motility. It is hypothesized that the chronic intestinal anaphylaxis-induced changes in the enteric neuronal circuitry cause intestinal motor malfunctions. However, detailed mechanisms largely remain unclear. The aim of this study was to investigate the pathophysiological role of ATP, which acts as a non-cholinergic neurotransmitter and a neuroimmune modulator, in a disturbed intestinal motility of food allergy (FA). The FA mice developed allergic diarrhea accompanied with chronic inflammation and mast cell hyperplasia in the colon. The excised proximal colons (PCs) were suspended in the longitudinal direction in organ baths. In the PCs precontracted by KCl (50 mM), contractile responses to exogenous ATP (1 mM) were significantly ( P < 0.01) higher in FA mice (34.2% of KCl-induced precontractions) as compared to control mice (17.2%). Pretreatment with P2 purinoceptor antagonists [suramin and PPADs] significantly ( P < 0.01) reduced the ATP-evoked contractions to 7.7% and 1.5% in FA and control PCs, respectively. Furthermore, in the presence of inhibitors of cholinergic nerves and capsaicin-sensitive sensory nerves the electrical field stimulation (EFS; 10 Hz)-evoked contractions were significantly ( P < 0.05) higher in FA mice (65.8% of EFS-evoked maximum contractions, n = 6) than those in control mice (47.9%, n = 6). In addition, cumulative application of suramin and PPADs further inhibited EFS-induced contractions by 21.7% in FA mice ( n = 6, P < 0.01) and 8.7% in control mice ( n = 6, P < 0.05). Thus, the present study suggests that the sustained alteration in cholinergic, purinergic and sensory neurotransmission contribute to the disturbed motility during the chronic intestinal anaphylaxis.

Purine receptor antagonist modulates serology and affective behaviors in lupus-prone mice: Evidence of autoimmune-induced pain?

Neurologic and psychiatric (NP) manifestations are severe complications of systemic lupus erythematosus (SLE). As commonly seen in patients, spontaneous disease onset in the MRL/MpJ-Fas lpr/J (MRL-lpr) mouse model of NP–SLE is accompanied by increased autoantibodies, pro-inflammatory cytokines and behavioral dysfunction which precede neuroinflammation and structural brain lesions. The role of purinergic receptors in the regulation of immunity and behavior remains largely unexplored in the field of neuropsychiatry. To examine the possibility that purinoception is involved in the development of affective behaviors, the P2X purinoceptor antagonist, suramin, was administered to lupus-prone mice from 5 to 14 weeks of age. In addition to food and water measures, novel object and sucrose preference tests were performed to assess neophobic anxiety- and anhedonic-like behaviors. Enzyme-linked immunosorbant assays for anti-nuclear antibodies (ANA) and pro-inflammatory cytokines were employed in immunopathological analyses. Changes in dendritic morphology in the hippocampal CA1 region were examined by a Golgi impregnation method. Suramin significantly lowered serum ANA and prevented behavioral deficits, but did not prevent neuronal atrophy in MRL-lpr animals. In a new batch of asymptomatic mice, systemic administration of corticosterone was found to induce aberrations in CA1 dendrites, comparable to the “stress” of chronic disease. The precise mechanism(s) through which purine receptor inhibition exerted beneficial effects is not known. The present data supports the hypothesis that activation of the peripheral immune system induces nociceptive-related behavioral symptomatology which is attenuated by the analgesic effects of suramin. Hypercortisolemia may also initiate neuronal damage, and metabolic perturbations may underlie neuro-immuno-endocrine imbalances in MRL-lpr mice.

ZP120 causes relaxation by pre‐junctional inhibition of noradrenergic neurotransmission in rat mesenteric resistance arteries

ZP120 (Ac-RYYRWKKKKKKK-NH(2)), is a new partial nociceptin/orphanin FQ (NOP) receptor agonist with sodium-potassium sparing aquaretic effects. The mechanisms of vasodilatation of ZP120 were examined in rat mesenteric resistance arteries. Arterial segments (internal diameters 206+/-4 microm, n=224) were mounted in microvascular myographs for isometric tension recordings and electrical field stimulation (EFS). ZP120 and the endogenous NOP receptor ligand, N/OFQ, did not relax arteries contracted with noradrenaline or adenosine-triphosphate. EFS-evoked contractions were inhibited by a purinoceptor antagonist, suramin, and the alpha(1)-adrenoceptor antagonist prazosin. N/OFQ inhibited, concentration-dependently, EFS-evoked contractions with a maximal effect of 52+/-3% (n=8) at 1 microM. The maximal effect of 1 microM ZP120 was lower (27+/-5%, P<0.05, n=9) than for N/OFQ. Endothelial removal or pretreatment with capsaicin did not influence the vasodilator effects of ZP120 and N/OFQ. ZP120 and N/OFQ responses were preserved in the presence of suramin. The alpha(2)-adrenoceptor antagonist, rauwolscine, antagonized the effect of clonidine and brimonidine, but ZP120 and N/OFQ inhibition of EFS-evoked contraction was unaltered. The competitive NOP receptor antagonist, UFP-101 (10 microM), prevented the inhibitory effect of N/OFQ, but not ZP120 suggesting that N/OFQ and ZP120 have distinct modes of interaction with the NOP receptor. Our findings suggest that the vasodilator effect of ZP120 and N/OFQ in rat mesenteric resistance arteries is mediated by prejunctional inhibition of adrenergic neurotransmission. These properties, that promote diuresis and attenuate the cardiovascular consequences of increased sympathetic nerve activity, make ZP120 a promising drug candidate.

Emerging Mucus Regulating Drugs in Inflammatory and Allergic Lung Disease

Mucus hypersecretion is common in inflammatory and allergic lung disease. Excessive mucus production leads to obstruction of airways and favours bacterial colonization. Advances in understanding the signalling and transduction pathways of mucin gene expression as well as mechanisms of mucin protein production and secretion have defined new therapeutic targets. Conventional therapies include anticholinergics, beta2-adrenoceptor agonists, glucocorticosteroids, mucolytics and macrolide antibiotics. Novel therapeutic approaches are inhibitors of cholinergic nerve activity, tachykinin receptor antagonists, epoxygenase inducers, inhibitors of mucin exocytosis, inhibitors of mucin synthesis and goblet cell hyperplasia, inducers of goblet cell apoptosis and P2Y2 purinoceptor antagonists to inhibit mucin secretion. After providing a short overview on conventional therapies this review will focus on new therapeutic targets.

Role of P2X receptors in positive inotropic effect of rat myocardium during ontogeny

Experiments with selective agonists and antagonists of purinoceptors allowed us to evaluate the subtype of P2X receptors. We showed that the myocardium of 14- 100-day-old rats contains functionally active P2X1 receptors. These receptors are involved in the realization of the positive inotropic effect of the atria and ventricles. Selective P2X1 receptor agonist beta,gamma-methylene-ATP induced a dose-dependent increase in the strength of atrial and ventricular contractions. P2X1 receptor antagonist TNP-ATP abolished the effect of the agonist in rats of all age groups.

Purinergic junctional transmission and propagation of calcium waves in cultured spinal cord microglial networks

In order to elucidate the mechanisms of purinergic transmission of calcium (Ca2 + ) waves between microglial cells, we have employed micro-photolithographic methods to form discrete patterns of microglia that allow quantitative measurements of Ca2 + wave propagation. Microglia were confined to lanes 20–100 mu mbox{m} wide and Ca2 + waves propagated from a point of mechanical stimulation, with a diminution in amplitude, for about 120 mu mbox{m}. The number of cells participating in propagation also decreased over this distance. Ca2 + waves could propagate across a cell-free lane from one microglia lane to another if this distance of separation was less than about 60 mu mbox{m}, indicating that propagation involved diffusion of a chemical transmitter. This transmitter was identified as ATP since all Ca2 + wave propagation was blocked by the purinoceptor antagonist suramin, which blocks P2Y2 and P2Y12 at relatively low concentrations. Antibodies to P2Y12 showed these at very high density compared with P2Y2, indicating a role for P2Y12 receptors. These observations were quantitatively accounted for by a model in which the main determinants are the diffusion of ATP released from a stimulated microglial cell and differences in the dissociation constant of the purinoceptors on the microglial cells.

Inhibitory purinergic transmission in mouse caecum: Role for P2Y1 receptors as prejunctional modulators of ATP release

Using conventional microelectrode recording techniques, we investigated, in the circular muscle of the mouse caecum, the neurotransmitter(s) involved in the neurally-evoked inhibitory junction potentials (IJPs) and the existence of possible prejunctional mechanisms controlling neurotransmitter release. Electrical field stimulation with single pulses elicited IJPs, consisting only of a "fast" hyperpolarization, while using train stimuli (30-50 Hz) the initial fast hyperpolarization was followed by a slower hyperpolarization. The fast and the slow component were selectively antagonized by apamin, a blocker of calcium-activated potassium channels, and N(omega)-nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor, respectively. Fast IJPs were antagonized also by P2 purinoceptor antagonists, suramin or 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid tetrasodium salt (PPADS), P2Y purinoceptor desensitization by adenosine 5'-O-2-thiodiphosphate (ADPbetaS). 2'-Deoxy-N(6)-methyl ADP diammonium salt (MRS 2179), P2Y1 purinoceptor antagonist, at the concentration of 1 microM increased the amplitude of the fast IJP, while at the concentration of 10 microM induced a reduction. 8,8'-[Carbonylbis[imino-3,1-phenylenecarbonylimino (4-fluoro-3,1-phenylene) carbonylimino]] bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt (NF 157) and 2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyl-oxymethyl)-propyl ester (MRS 2395), P2Y11 and P2Y12 purinoceptor antagonist, were without any effect. ATP-induced hyperpolarization was affected by apamin and by P2Y purinoceptor desensitization, but not by MRS 2179. 2-(Methylthio)ATP tetrasodium salt hydrate (2-MeSATP), P2Y1 purinoceptor agonist, at a concentration which did not cause changes in the membrane potential, reduced the amplitude of the fast IJPs. This effect was prevented by MRS 2179. Paired nerve stimulation, either using single pulses or train stimuli, did not cause any alteration of the second-evoked IJP. In conclusion, in the circular muscle of the mouse caecum, ATP is responsible for the fast IJP while nitric oxide is responsible for the slow IJP. ATP-mediated response is dependent on ADPbetaS-sensitive P2Y receptors, which are in part P2Y1, but not P2Y11 or P2Y12 receptor subtypes. In addition, the most substantial finding of this study is the functional demonstration that ATP released by nerve stimulation activates P2Y1 receptors, located prejunctionally, limiting its release by motoneurons.

ATP release and its prejunctional modulation.

We studied some properties of the release of noradrenaline and ATP in isolated sympathetically innervated tissues. Release was elicited by electric stimulation and assessed as overflow of tritiated compounds (after labelling with [3H]noradrenaline) and enzymically measured ATP, respectively. Evans blue, which inhibits ectonucleotidases, greatly increased the evoked overflow of ATP, indicating that a major part of the ATP was metabolized after release. Much of the ATP was postjunctional in origin. The neural fraction was isolated when postjunctional release was suppressed by prazosin (alpha 1-adrenoceptor antagonist) and suramin (P2 purinoceptor antagonist). Comparison of neural ATP and [3H]-noradrenaline release showed that prostaglandin E2 reduced the release of both co-transmitters to a similar extent. Activation of prejunctional alpha 2-adrenoceptors, however, preferentially reduced the release of [3H]noradrenaline, and activation of prejunctional A1 purinoceptors reduced preferentially the release of ATP. Nucleotides such as ATP depressed the release of [3H]noradrenaline through two receptors: the well-known prejunctional A1 receptors and a separate group of prejunctional P2 purinoceptors. P2 antagonists increased the release of [3H]-noradrenaline. Overall, the results indicate differential storage, release and modulation of release of the two sympathetic co-transmitters. They also indicate that postganglionic sympathetic axons possess receptors for both co-transmitters: alpha 2 and P2 autoreceptors.

ATP as a co-transmitter with noradrenaline in sympathetic nerves--function and fate.

ATP and noradrenaline are co-stored in synaptic vesicles in sympathetic nerves and when co-released act postjunctionally to evoke contraction of visceral and vascular smooth muscle. In the original purinergic nerve hypothesis it was proposed that ATP would then be sequentially broken down to ADP, AMP and adenosine. Although such breakdown can be measured, it is not clear how the time-scale of breakdown compares with the time-course of the postjunctional actions of ATP. We have investigated the role of ectoATPase in modulating purinergic neurotransmission in the guinea-pig vas deferens using ARL67156 (formerly FPL67516), a recently developed inhibitor of ectoATPase. ARL67156 (1-100 microM) potentiated neurogenic contractions in a concentration-dependent manner. Onset of potentiation was rapid and the effect reversed rapidly on washout of the drug. The effect was also frequency dependent, being greater at lower frequencies. The purinergic component of the neurogenic contraction was isolated using the alpha 1 antagonist prazosin (100 nM) and ARL67156 caused a similar potentiation. ARL67156 also potentiated contractions evoked by exogenous ATP (100 microM), but had no effect on those of the stable analogue alpha, beta-methylene ATP (500 nM). In the presence of the P2 purinoceptor antagonist PPADS (100 microM), ARL67156 also had no effect on contractions evoked by noradrenaline (10 microM) or KCI (40 mM). These results are consistent with an inhibitory action of ARL67156 on ectoATPase and suggest that ectoATPase modulates purinergic transmission in the guinea-pig vas deferens. When released from sympathetic nerves, ATP acts at the P2X purinoceptor, a ligand-gated cation channel, to evoke depolarization and contraction. In single acutely dissociated smooth muscle cells of the rat tail artery, studied under voltage-clamp conditions, ATP and its analogues evoke an inward current, with a rank order potency of 2-methylthioATP = ATP > alpha, beta-methylene ATP. This is very different from the order of potency for evoking contraction in whole vessel rings, which is alpha, beta-methylene ATP > > 2-methylthioATP > or = ATP. This discrepancy can be explained by a previously unrecognized attenuation of the action of ATP and 2-methylthioATP, but not alpha, beta-methylene ATP, by ectoATPase in whole tissues.