Beta-methylene ATP Research Articles

Dysfunctional voiding behavior and impaired muscle contractility in a rat model of detrusor underactivity.

Detrusor underactivity (DU) is an understudied health concern with inadequate clinical management. The pathophysiology of DU is unclear, and current therapies fail to improve symptoms. The current studies characterized voiding function and contractility of bladder and urethral tissues in a novel rat model of DU. Female obese prone (OP) and obese resistant (OR) rats were fed a 60 kcal% fat diet at 8 weeks old. A subset of rats (n = 4/strain) underwent uroflowmetry biweekly for 18 weeks in metabolic cages. At 40-56 weeks old, rats (n = 9-10/strain) underwent instrumented cystometry under urethane anesthesia. Following cystometry, bladder and urethral tissues (n = 8-9/strain) were harvested for in vitro assessments of contractility in response to carbachol, electric field stimulation, atropine, alpha, beta-methylene ATP, and caffeine. OP rats exhibited increased urinary frequency (p = 0.0031), decreased voided volume (p = 0.0093), and urine flow rate (p = 0.0064) compared to OR rats during uroflowmetry. Bethanechol (10 mg/kg) did not alter uroflowmetry parameters. During cystometry, OP rats exhibited decreased bladder emptying efficiency (p < 0.0001), decreased pressure to generate a void (p < 0.0001), and increased EUS activity during filling (p = 0.0011). Bladder contractility was decreased in OP rats when exposed to carbachol (p < 0.0003) and ATP (p = 0.0004), whereas middle urethral contractility was increased when exposed to carbachol (p = 0.0014), EFS (p = 0.0289), and caffeine (p = 0.0031). Impaired cholinergic and purinergic signaling in the bladder may contribute to poor voiding function in OP rats. In addition, increased urethral activity may engage a guarding reflex to augment continence and exacerbate incomplete emptying.

AB294. SPR-21 Sex differences in bladder dysfunction in response to enteric neuronal NFκB overactivation and experimentally induced colitis in mice

ObjectiveIn experimental models, animals with induced colitis have increased urinary frequency and abnormalities in bladder contraction to cholinergic and electric field stimulation (EFS). Nuclear factor kappa B (NFκB) is a major mediator of the inflammatory response in inflammatory bowel disease and syndrome. The aim of the present study was to determine the effect of increased NFκB signalling in enteric neurons on bladder contraction with and without experimentally induced colitis.MethodsCalretinin-Cre-ERT2-IKK2CA/tdTomato transgenic (TG) mice were generated and compared to littermate wild-type (WT) controls. At two months of age, the mice were treated for three days with tamoxifen causing continuous NFκB activation in enteric neuronal cells. One month later, the mice were treated with vehicle or 2.5% dextran sulfate sodium (DSS) for 7 days to induce colitis. Full thickness urinary bladder strips were stimulated with potassium and then EFS to induce nerve mediated contraction. The effect of atropine to inhibit muscarinic receptors and alpha, beta methylene ATP (ATP) to desensitize purinergic receptors was determined.ResultsEven though the bladder contractile response to high potassium stimulation was similar between sexes in both WT and TG mice, the EFS induced maximal contraction was greater in WT males than in WT females. Bladder strips from WT female mice had a significantly greater atropine resistant (purinergic) component of EFS induced contraction, compared to WT male mice, but this was not found in TG mice. The cholinergic component of EFS induced contraction was not different between male and female mice (WT or TG). Acute colitis significantly reduced KCl stimulated and nerve mediated contraction in bladder strips from WT female and male mice, and from TG female mice. Constitutive activation of neuronal NFκB preserved the nerve mediated and KCL induced contractile response in male mice. Acute colitis increased the purinergic component of nerve mediated contractions in male WT mice only.ConclusionsExperimentally induced colitis causes decreased bladder contraction. Neuronal NFκB signalling rescued the decreased bladder contraction due to colitis, perhaps through purinergic regulation in males. Neurogenic inflammation in the bladder may cause a larger degree of bladder dysfunction in female than in male mice perhaps because females have a greater purinergic component of nerve mediated bladder contraction that does not further increase due to colitisFunding Source(s)RO1DK075684; P2ODK097819

AB314. SPR-41 Localization of neuromuscular nicotinic receptors in the functionally reinnervated canine bladder after prolonged decentralization

ObjectiveWe previously found that intravenous succinylcholine, a depolarizing neuromuscular nicotinic receptors blocker, prevents bladder contractions induced by new neuronal pathways established by nerve transfer in decentralized dogs. We studied the detrusor pressure response in vivo and contractile response of bladder smooth muscle strips in vitro from sham, decentralized and reinnervated animals to localize the neuromuscular nicotinic receptors involved.MethodsThree groups of female mongrel hound dogs were used: sham (N=4), 12-month decentralized (N=3) and 6-month reinnervated (N=3). Decentralization was created by bilateral transection of all spinal roots caudal to L7, including the dorsal roots of L7 and the hypogastric nerves. Reinnervation was created by bilateral transfer of the obturator nerve to anterior vesical branches of the pelvic nerve. Two-way ANOVAs and Sidak post-hoc tests were used to determine group differences.ResultsIn reinnervated dogs, blockade of neuromuscular nicotinic receptor with intravenous injections of the competitive antagonist atracurium besylate significantly reduced the increase in detrusor pressure induced by electrical stimulation of the transferred obturator nerve (L1 or L2). Atracurium did not block the increase in pressure induced by stimulation of sacral nerve roots in sham-operated controls. In vitro, neither the competitive neuromuscular nicotinic receptor antagonist d-tubocurarine nor the ganglionic antagonist hexamethonium inhibited electric field stimulation (EFS)-induced contractions of reinnervated or sham-operated control bladder strips. No contractile response was elicited in the presence of 1 µM tetrodotoxin (TTX) across groups. Similarly, EFS-evoked contractions were strongly reduced by 10 µM alpha, beta-methylene ATP (α,β-mATP) and 1 µM atropine in all groups relative to the vehicle (water).ConclusionsIn vivo blockade of nerve-evoked bladder pressure by atracurium in the reinnervated, but not sham operated controls, suggests that neuromuscular nicotinic receptors become involved in bladder contractions induced by the new neuronal pathway. Because d-tubocurarine did not block in vitro contractions induced by EFS in the reinnervated bladders, the neuromuscular nicotinic receptors involved in the new neuronal pathway must not be located in the bladder muscle or intramural ganglia and therefore, are likely in preganglionic neurons. TTX blockade validates that EFS-induced contractions at all frequencies were nerve-evoked. Both muscarinic and purinergic components contributed similarly to neurotransmission based on response to blockade of nerve evoked muscle strip contractions with a combination of atropine and α,β-mATP.Funding Source(s)NIH-NINDS NS070267

AB318. SPR-45 Decentralization reduces nicotinic receptor-mediated canine bladder contractions in vitro

ObjectiveBladder function depends upon several complex signaling pathways that induce either contraction or relaxation. We performed nerve re-routing surgery on bladder of decentralized dogs (reinnervated) as a model with the goal of restoring bladder function in spinal cord injured patients. The neuromuscular nicotinic receptor blocker, succinylcholine blocks spinal root-stimulated bladder contraction in vivo in reinnervated dogs, but not in sham-operated dogs. Our lab explored the function and location of nicotinic receptors involved in bladder contraction, with emphasis on their possible role in the release of other neurotransmitters such as acetylcholine or ATP in sham-operated, 12-month-decentralized and immediate-reinnervated bladders.MethodsSmooth muscle strips (mucosa denuded) were isolated from the region just rostral to the trigone and suspended in muscle baths. Strips were ranked based on their contractile response to a 3-min exposure to 120 mM KCl and sorted so that the average response in each group was equal. Strips were incubated with either 1 µM atropine (ATR), 1 µM tetrodotoxin (TTX), 10 µM alpha, beta-methylene ATP (α,β-ATP) or vehicle (water) and then induced to contract with the nicotinic receptor agonists 1,1 dimethyl-4-phenyl-piperazinium iodide (DMPP, 100 µM), TC2559 (100 µM) or epibatidine (10 µM) or 1 mM nicotine itself.ResultsThe DMPP-induced contraction was not different between sham, reinnervated or decentralized bladders (11%, 3.3%, and 3.6% of KCl contraction respectively). While the epibatidine-induced contraction in shams was not different relative to that in the reinnervated (41% vs. 27% KCl respectively), it was significantly greater than that in decentralized bladders (13% of KCl). TC2559 did not induce bladder contractions. Nicotine-induced contractions in sham-operated controls were 16% of KCl. ATR completely blocked nicotine-induced contraction while α,β-ATP had no statistically significant effect in shams. TTX had no significant inhibitory effect on DMPP, epibatidine or nicotine-induced contraction in any group.ConclusionsNicotinic receptors mediate contraction in sham, reinnervated and decentralized bladders. This nicotinic receptor-mediated contraction is decreased after decentralization. TTX does not block nicotinic receptor-mediated contractions, indicating that action potentials are not required to induce contraction. In sham-operated dog bladders, the nicotine-induced contraction is blocked by ATR, suggesting that these nicotinic receptors are located on cholinergic nerve terminals and induce the release of acetylcholine, which activates muscarinic receptors on the smooth muscle.Funding Source(s)NIH-NINDS NS070267

Functional ligand-gated purinergic receptors (P2X) in rat vestibular ganglion neurons

The expression of purinergic receptors (P2X) on rat vestibular ganglion neurons (VGNs) was examined using whole-cell patch-clamp recordings. An application of adenosine 5′-triphosphate (ATP; 100μM) evoked inward currents in VGNs at a holding potential of −60mV. The decay time constant of the ATP-evoked currents was 2–4s, which is in between the values for rapidly desensitizing subgroups (P2X1 and P2X3) and slowly desensitizing subgroups (P2X2, P2X4, etc.), suggesting the heterogeneous expression of P2X receptors. A dose–response experiment showed an EC50 of 11.0μM and a Hill’s coefficient of 0.82. Suramin (100μM) reversibly inhibited the ATP-evoked inward currents. Alpha, beta-methylene ATP (100μM), a P2X-specific agonist, also evoked inward currents but less extensively than ATP. An application of adenosine 5′-dihosphate (ADP; 100μM) evoked similar, but much smaller, currents. The current–voltage relationship of the ATP-evoked conductance showed pronounced inward rectification with a reversal potential more positive than 0mV, suggesting non-selective cation conductance. However, the channel was not permeable to a large cation (N-methyl-d-glucamine) and acidification (pH 6.3) had little effect on the ATP-evoked conductance. RT-PCR confirmed the expression of five subtypes (P2X2–P2X6) in VGNs. The physiological role of P2X receptors includes the modulation of excitability at the synapses between hair cells and dendrites and/or trophic support (or also neuromodulation) from supporting cells surrounding the VGNs.

Modification of rat detrusor muscle contraction by ascorbic acid and citric acid involving enhanced neurotransmitter release and Ca2+ influx

Consumption of carbonated soft drinks is independently associated with the development of overactive bladder (OR 1.41, 95% Cl 1.02-1.95). We have shown previously that artificial sweeteners, present in carbonated soft drinks, enhanced detrusor muscle contraction. Other constituents of soft drinks are preservatives and antioxidants, we evaluated the effects of two of these, ascorbic acid and citric acid, on the contractile response of isolated rat bladder muscle strips. Detrusor muscle strips were suspended in a perfusion organ bath. We determined the effect of ascorbic acid and citric acid on the contractile responses to electrical field stimulation (EFS) in the absence and presence of atropine, carbachol, alpha, beta methylene ATP, potassium and calcium. Ascorbic acid and citric acid (10(-7) M to 10(-3) M) enhanced the contractile response to 10 Hz EFS compared to control (P < 0.01). The frequency and amplitude of spontaneous bladder contractions were enhanced in the presence of ascorbic acid and citric acid by 14%, 21%, 21%, and 11% respectively. Ascorbic acid 10(-4) M significantly increased the atropine resistant response to EFS 5 Hz by 37% (P < 0.01) and inhibited contraction in response to carbachol 10(-4) M by 24%, (P < 0.05). Both ascorbic acid 10(-4) M and citric acid 10(-5) M significantly enhanced maximum contractile responses to alpha, beta methylene ATP, KCI and calcium compared to control. Ascorbic acid and citric acid augmented bladder muscle contraction possibly by enhanced Ca(2+) influx. Presynaptic neurotransmitter release was enhanced by ascorbic acid. Carbonated beverages containing preservatives may aggravate symptoms of OAB.

Rho-kinase inhibition reduces pressure-mediated autoregulatory adjustments in afferent arteriolar diameter.

Preglomerular resistance is regulated by calcium influx- and mobilization-dependent mechanisms; however, the role of Rho-kinase in calcium sensitization in the intact kidney has not been carefully examined. Experiments were performed to test the hypothesis that Rho-kinase inhibition blunts pressure-mediated afferent arteriolar autoregulatory behavior and vasoconstrictor responses evoked by angiotensin II and P2X1 receptor activation. Rat kidneys were studied in vitro using the blood-perfused juxtamedullary nephron technique. Autoregulatory behavior was assessed before and during Rho-kinase inhibition with Y-27632 (1.0 microM; n = 5). Control diameter averaged 14.3 +/- 0.8 microm and increased to 18.1 +/- 0.9 microm (P < 0.05) during Y-27632 treatment. In the continued presence of Y-27632, reducing perfusion pressure to 65 mmHg slightly increased diameter to 18.7 +/- 1.0 microm. Subsequent pressure increases to 130 and 160 mmHg yielded afferent arteriolar diameters of 17.5 +/- 0.8 and 16.6 +/- 0.6 microm (P < 0.05). This 11% decline in diameter is significantly smaller than the 40% decrease obtained in untreated kidneys. The inhibitory effects of Y-27632 on autoregulatory behavior were concentration dependent. Angiotensin II responses were blunted by Y-27632. Angiotensin II (1.0 nM) reduced afferent diameter by 17 +/- 1% in untreated arterioles and by 6 +/- 2% during exposure to Y-27632. The P2X1 receptor agonist, alpha, beta-methylene ATP, reduced afferent arteriolar diameter by 8 +/- 1% but this response was eliminated during exposure to Y-27632. Western blot analysis confirms expression of the Rho-kinase signaling pathway. Thus, Rho-kinase may be important in pressure-mediated autoregulatory adjustments in preglomerular resistance and responsiveness to angiotensin II and autoregulatory P2X1 receptor agonists.

Dual Effect of ATP on Glucose-Induced Insulin Secretion in HIT-T15 Cells

: The study investigated the dual effect of purinergic nucleotides on the secretion of insulin from pancreatic beta cells. : The level of insulin secretion in HIT-T15 cells of static incubation was measured using a radioimmunoassay. : The adenine nucleotides reduced the level of glucose-induced insulin secretion in a concentration-dependent manner, and the relative potency order (IC50; muM) was BzATP (6.9) > ATP (20.4) >/= alpha, beta-methylene ATP (23.3) >/= 2-methylthio-ATP (24.9). Suramin and PPADS (200 muM), which are blockers of the purinergic receptors, had a little influence on the activity of ATP. However, the inhibitory effect of ATP was reversed by preincubation with oxidized ATP (200 muM), which is a P2X7 antagonist. The level of insulin secretion in these preincubated cells exposed to the purinergic nucleotides increased in the following order: ATP > alpha, beta-methylene ATP >/= 2-methylthio-ATP. A pretreatment with foskolin and PDBu (100 nM) potentiated the increasing effect of ATP on insulin secretion. The Western blotting showed the expression of P2X7 and P2Y11 receptors. : Purinergic stimulation has inhibitory activity on glucose-dependent insulin secretion through the activation of the P2X7 receptor, whereas it has enhancing effect through the activation of the P2Y11 receptor in HIT-T15 cells.

Electrophysiological determination of antagonist mechanism of action at a P2X2‐3 chimeric receptor

P2X3 receptors are ligand gated ion channels activated by ATP. In the periphery, these receptors are located on nociceptive neurons which convey inflammatory pain information to the central nervous system. Thus, these receptors are likely targets for analgesic drug development. Here, we devise an electrophysiological method to determine the mechanism of action (competitive or non-competitive inhibition) by which compounds exert their antagonistic effects on a P2X2-3 chimeric receptor. By applying a fixed concentration of the antagonist in the presence of the P2X3 receptor agonist alpha, beta-methylene ATP, we can compare the rate of inhibition at different concentrations of the agonist. We show that the rate of inhibition of the P2X3 antagonist TNP-ATP is linearly dependent on the concentration of agonist with which it is co-applied, consistent with the pharmacological profile of TNP-ATP acting as a competitive antagonist at the receptor's ligand binding site. However, the rate of inhibition of Compound A is independent of the agonist concentration in which it is co-applied, suggesting that the site of inhibition is not competitive with that of the agonist. These results are consistent with radioligand binding data of Compound A on a homomeric P2X3 receptor. These data therefore provide an electrophysiological kinetic determination of the competitivity of a compound at a ligand gated ion channel.

P2X1 and P2X4 receptor currents in mouse macrophages

Activation of P2X receptors on macrophages is an important stimulus for cytokine release. This study seeks evidence for functional expression of P2X receptors in macrophages that had been only minimally activated. Whole-cell recordings were made from macrophages isolated 2-6 h before by lavage from mouse peritoneum, without further experimental activation. ATP (1-1000 muM) elicited inward currents in all cells (holding potential -60 mV). The properties of this current were compared among cells from wild type, P2X1 (-/-) and P2X4 (-/-) mice. Immunoreactivity for P2X1 and P2X4 receptors was observed in wild type macrophages but was absent from the respective knock-out mice. In cells from wild type mice, ATP and alpha beta methyleneATP (alpha beta meATP) evoked inward currents rising in 10-30 ms and declining in 100-300 ms: these were blocked by pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM). ATP also elicited a second, smaller ( approximately 10% peak amplitude), more slowly decaying (1-3 s) at concentrations > or =10 microM: this was resistant to PPADS and prolonged by ivermectin. Macrophages from P2X1 (-/-) mice responded to ATP (>100 microM) but not alpha beta meATP: these small currents were prolonged by ivermectin. Macrophages from P2X4 (-/-) mice responded to ATP and alpha beta meATP as cells from wild type mice, except that ATP did not evoke the small, slowly decaying component: these currents were blocked by PPADS. Mouse peritoneal macrophages that are minimally activated demonstrate membrane currents in response to ATP and alpha beta meATP that have the predominate features of P2X1 receptors.

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.

The diverse series of recombinant P2Y purinoceptors.

A cDNA encoding a P2Y purinoceptor was originally cloned from chick brain and the bovine and human homologues have recently been obtained. These are seven-transmembrane-domain polypetides, i.e. G protein-coupled receptors. When activated by agonists, this P2Y receptor mobilizes intracellular Ca2+ and has been shown to be coupled to inositol-1,4,5-trisphosphate formation. Its pharmacology has been established in several expression systems, using both ligand binding and functional responses: 2-methylthioATP has the highest potency of nucleotides and derivatives tested, while UTP and alpha, beta-methylene ATP are inactive. This was hence assigned as a new subtype of the pharmacologically defined P2Y receptors, P2Y1. P2Y1 receptors are exceptionally abundant in the brain. A P2U receptor reported by others can be designated P2Y2. Another P2 receptor subtype, P2Y3, now cloned as a cDNA from the brain and expressed in oocytes and in transfected cells, shows a quite different ligand potency profile to the first two. A fourth subtype is expressed primarily in certain haemopoietic cells and in cardiac muscle. A putative fifth subtype is expressed only in T lymphocytes, upon activation. Yet other P2Y subtypes are indicated by recent cloning studies. The amino acid sequences of all of these P2 receptors, while displaying some homology, are strikingly diverse: they form a separate and unusual new family in the G protein-coupled receptor main superfamily.

Melatonin inhibits nitric oxide production by microvascular endothelial cells in vivo and in vitro

We have previously shown that melatonin inhibits bradykinin-induced NO production by endothelial cells in vitro. The purpose of this investigation was to extend this observation to an in vivo condition and to explore the mechanism of action of melatonin. RT-PCR assays were performed with rat cultured endothelial cells. The putative effect of melatonin upon arteriolar tone was investigated by intravital microscopy while NO production by endothelial cells in vitro was assayed by fluorimetry, and intracellular Ca(2+) measurements were assayed by confocal microscopy. No expression of the mRNA for the melatonin synthesizing enzymes, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase, or for the melatonin MT(2) receptor was detected in microvascular endothelial cells. Melatonin fully inhibited L-NAME-sensitive bradykinin-induced vasodilation and also inhibited NO production induced by histamine, carbachol and 2-methylthio ATP, but did not inhibit NO production induced by ATP or alpha, beta-methylene ATP. None of its inhibitory effects was prevented by the melatonin receptor antagonist, luzindole. In nominally Ca(2+)-free solution, melatonin reduced intracellular Ca(2+) mobilization induced by bradykinin (40%) and 2-methylthio ATP (62%) but not Ca(2+) mobilization induced by ATP. We have confirmed that melatonin inhibited NO production both in vivo and in vitro. In addition, the melatonin effect was selective for some G protein-coupled receptors and most probably reflects an inhibition of Ca(2+) mobilization from intracellular stores.

Enhancement of rat bladder contraction by artificial sweeteners via increased extracellular Ca2+ influx

Consumption of carbonated soft drinks has been shown to be independently associated with the development of overactive bladder symptoms (OR 1.62, 95% CI 1.18, 2.22) [Dallosso, H.M., McGrother, C.W., Matthews, R.J., Donaldson, M.M.K., 2003. The association of diet and other lifestyle factors with overactive bladder and stress incontinence: a longitudinal study in women. BJU Int. 92, 69-77]. We evaluated the effects of three artificial sweeteners, acesulfame K, aspartame and sodium saccharin, on the contractile response of isolated rat detrusor muscle strips. Strips of detrusor muscle were placed in an organ bath and stimulated with electrical field stimulation (EFS) in the absence and presence of atropine, and with alpha,beta methylene ATP, potassium, calcium and carbachol. Sweeteners 10(-7) M to 10(-2) M enhanced the contractile response to 10 Hz EFS compared to control (p<0.01). The atropine-resistant response to EFS was marginally increased by acesulfame K 10(-6) M, aspartame 10(-7) M and sodium saccharin 10(-7) M. Acesulfame K 10(-6) M increased the maximum contractile response to alpha,beta methylene ATP by 35% (+/-9.6%) (p<0.05) and to KCl by 12% (+/-3.1%) (p<0.01). Sodium saccharin also increased the response to KCl by 37% (+/-15.2%) (p<0.05). These sweeteners shifted the calcium concentration-response curves to the left. Acesulfame K 10(-6) M increased the log EC(50) from -2.79 (+/-0.037) to -3.03 (+/-0.048, p<0.01) and sodium saccharin 10(-7) M from -2.74 (+/-0.03) to 2.86 (+/-0.031, p<0.05). The sweeteners had no significant effect on the contractile response to carbachol but they did increase the amplitude of spontaneous bladder contractions. These results suggest that low concentrations of artificial sweeteners enhanced detrusor muscle contraction via modulation of L-type Ca(+2) channels.

ATP inhibits the hypoxia response in type I cells of rat carotid bodies

Summary During hypoxia, ATP was released from type I (glomus) cells in the carotid bodies. We studied the action of ATP on the intracellular Ca(2+) concentration ([Ca(2+)](i)) of type I cells dissociated from rat carotid bodies using a Ca(2+) imaging technique. ATP did not affect the resting [Ca(2+)](i) but strongly suppressed the hypoxia-induced [Ca(2+)](i) elevations in type I cells. The order of purinoreceptor agonist potency in inhibiting the hypoxia response was 2-methylthioATP > ATP > ADP >> alpha, beta-methylene ATP > UTP, implicating the involvement of P2Y(1) receptors. Simultaneous measurements of membrane potential and [Ca(2+)](i) show that ATP inhibited the hypoxia-induced Ca(2+) signal by reversing the hypoxia-triggered depolarization. However, ATP did not oppose the hypoxia-mediated inhibition of the oxygen-sensitive TASK-like K(+) background current. Neither the inhibition of the large-conductance Ca(2+)-activated K(+) (maxi-K) channels nor the removal of extracellular Na(+) could affect the inhibitory action of ATP. Under normoxic condition, ATP caused hyperpolarization and increase in cell input resistance. These results suggest that the inhibitory action of ATP is mediated via the closure of background conductance(s) other than the TASK-like K(+), maxi-K or Na(+) channels. In summary, ATP exerts strong negative feedback regulation on hypoxia signaling in rat carotid type I cells.

Novel role for P2X receptor activation in endothelium-dependent vasodilation.

1 ATP is an important vasoactive mediator, which acts via two receptor classes: P2X and P2Y. Activation of P2X receptors has traditionally been associated with the well-characterised vasoconstrictor properties of ATP. 2 In the current study, we have shown that the P2X(1 & 3) receptor ligand, alpha, beta methylene ATP, induces vasodilation of rat isolated mesenteric arteries and that P2X1 receptors are abundantly expressed in the endothelium of these vessels. 3 Second-order rat mesenteric arteries were mounted in myographs and vasomotor responses recorded. Both ATP and alpha, beta methylene ATP induced a constriction followed by a vasodilation. The dilator effects of either ATP or alpha, beta methylene ATP were slower in onset than those induced by acetylcholine. By contrast, the traditional vasodilator P2Y ligand, ADP, induced vasodilation without contraction. 4 Vasodilation induced by alpha, beta methylene ATP was endothelial dependent, but was not affected by treatment of the vessels with L-NAME plus indomethacin alone. Dilation was, however, partially inhibited by the combination of apamin plus charybdotoxin and blocked by treating vessels with all four drugs. 5 Using confocal microscopy, P2X1 receptor immunoreactivity was localised to the endothelial, smooth muscle and adventitial layers of mesenteric vessels. P2X1 protein migrated as a primary band at around 50-60 kDa in vascular tissue. 6 These results show for the first time that P2X1 receptors are expressed on the endothelium and that a selective ligand of this receptor results in vasoconstriction followed by vasodilation. These observations have important implications for our understanding of the role of purines in biological responses.

Contribution of sensitized P2X receptors in inflamed tissue to the mechanical hypersensitivity revealed by phosphorylated ERK in DRG neurons

The mechanism of mechanical hyperalgesia in inflammation might involve a ‘mechanochemical’ process whereby stretch evokes the release of adenosine 5′-triphosphate (ATP) from the damaged tissue that then excites nearby primary sensory nerve terminals. In the present study, phosphorylated extracellular signal-regulated protein kinase (pERK) immunoreactivity was used as a marker indicating functional activation of primary afferent neurons to examine the P2X receptor-mediated noxious response in DRG neurons in a rat model of peripheral inflammation. We found that very few pERK-labeled DRG neurons were detected in normal rats after alpha, beta methylene-ATP (αβme-ATP) intraplantar injection. However, a number of DRG neurons were labeled for pERK after αβme-ATP injection to the complete Freund's adjuvant (CFA) induced inflamed paw. Seventy-three percent of pERK-labeled DRG neurons co-expressed the P2X3 receptor. After mechanical noxious stimulation to the hind paw of CFA-inflamed rats, we found many more pERK-labeled neurons compared to those in the normal rats. Administration of the P2X 3 receptor antagonists, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid or 2′- (or 3′)- O-(trinitrophenyl)adenosine 5′-triphosphate (TNP-ATP), significantly decreased the mechanical stimulation-evoked pERK labeling in CFA-inflamed rats, but not in normal rats. We also found the recruitment of neurons with myelinated A fibers labeled for pERK in CFA-inflamed rats, which was reversed by P2X 3 receptor antagonists. Moreover, TNP-ATP dose dependently reduced the mechanical hypersensitivity of CFA rats. These data suggest that the P2X receptors in primary afferent neurons increase their activity with enhanced sensitivity of the intracellular ERK signaling pathway during inflammation and then contribute to the hypersensitivity to mechanical noxious stimulation in the inflammatory state.

Expression and function of P2X purinoceptors in rat histaminergic neurons

(1) The pharmacology of ATP responses and the expression pattern of seven known subunits of the P2X receptor were investigated in individual histaminergic neurons of the tuberomamillary nucleus (TM). (2) ATP (3-1000 micro M) evoked fast non-desensitizing inward currents in TM neurons. 2-methylthioATP (2MeSATP) displayed the same efficacy but a lower potency, EC(50)s 84 micro M versus 48 micro M, when compared with ATP. Adenosine-diphosphate (ADP), uridine-triphosphate (UTP) and alpha beta methylene-ATP (alphabeta-meATP) were inactive. (3) ATP-mediated whole cell currents were potentiated by acidification of the recording solution (pH 7.5 and 6.6 were compared). (4) Single-cell RT-PCR (scRT-PCR) analysis revealed that the P2X(2) receptor is expressed in all PCR-positive neurons. Each of the P2X(1), P2X(3), P2X(4), P2X(5) and P2X(6) mRNAs were detected in less than 35% of the cells. (5) Suramin antagonized ATP responses with an IC(50) of 4.2 micro M and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 1 micro M) reduced ATP responses to 43% of control, when antagonists were pre-applied 90s before the agonist. Cibacron blue (3 micro M) given together with ATP potentiated control responses by 67%, but inhibited it to 10% after pre-application. (6) 2',3'-O-(2,4,6-Trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) antagonized ATP responses with an IC(50) of 7 micro M. (7) Pharmacological properties of ATP responses together with scRT-PCR data suggest that P2X(2) is the major purinoceptor on the soma of TM neurons, however the presence of heteromeric P2X(2/5) receptors in some neurons cannot be excluded.

ATP-induced [Ca2+]i changes in the human corneal epithelium.

ATP, when leaked from damaged cells, is capable of eliciting responses in neighboring cells. A better understanding of the mechanism of this response is essential for designing therapeutic strategies for disease, there have been only a limited number of studies on the effect of ATP on the human cornea. We examined ATP-induced intracellular Ca2+ ([Ca2+]i) changes in the human corneal epithelia, cultured to near confluence. Cells were loaded with the Ca2+ indicators, Indo-1 or Fluo-4, and [Ca2+]i was monitored. ATP was found to induce an increase in [Ca2+]i, which was initiated from the perinuclear region and the nuclear envelope per se, and then propagated gradually towards the periphery. Intranuclear Ca2+ was momentarily increased. UTP elicited an identical response, but adenosine and alpha, beta-methylene ATP had no effect. Pretreatment with U73122 or thapsigargin inhibited the ATP-induced increase in [Ca2+]i. When a cell was topically stimulated with ATP, the [Ca2+]i increase spread beyond the cell boundary. The intercellular communications that accompanied the [Ca2+]i changes were inhibited by octanol. We conclude that extracellular ATP in the human cornea caused the mobilization of Ca2+ from intracellular Ca2+ stores (e.g. the endoplasmic reticulum and nuclear envelope) via P2Y purinoceptors of the epithelial cell. The response to ATP appears to spread to neighboring regions through gap junctions in the epithelium.

Inhibitory interactions between 5-HT3 and P2X channels in submucosal neurons.

Inhibitory interactions between 5-HT subtype 3 (5-HT(3)) and P2X receptors were characterized using whole cell recording techniques. Currents induced by 5-HT (I(5-HT)) and ATP (I(ATP)) were blocked by tropisetron (or ondansetron) and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, respectively. Currents induced by 5-HT + ATP (I(5-HT+ATP)) were only as large as the current induced by the most effective transmitter, revealing current occlusion. Occlusion was observed at membrane potentials of -60 and 0 mV (for inward currents), but it was not present at +40 mV (for outward currents). Kinetic and pharmacological properties of I(5-HT+ATP) indicate that they are carried through 5-HT(3) and P2X channels. Current occlusion occurred as fast as activation of I(5-HT) and I(ATP), was still present in the absence of Ca(2+) or Mg(2+), after adding staurosporine, genistein, K-252a, or N-ethylmaleimide to the pipette solution, after substituting ATP with proportional to, beta-methylene ATP or GTP with GTP-gamma-S in the pipette, and was observed at 35 degrees C, 23 degrees C, and 8 degrees C. These results are in agreement with a model that considers that 5-HT(3) and P2X channels are in functional clusters and that these channels might directly inhibit each other.