Extracellular UTP Research Articles

P2 receptor expression profiles in human vascular smooth muscle and endothelial cells.

P2 receptors mediate the actions of the extracellular nucleotides ATP, ADP, UTP, and UDP, regulating several physiologic responses including cardiac function, vascular tone, smooth muscle cell (SMC) proliferation, platelet aggregation, and the release of endothelial factors. P2 receptor characterization has been hampered by the lack of selective antagonists. The aim of the current study was to investigate the mRNA and protein expression of P2X and P2Y receptors in human SMC and in endothelial cells (EC). Smooth muscle cells were obtained from human mammary artery and EC from human umbilical vein. Using real-time PCR, the authors established quantitative mRNA assays. Protein expression was studied using Western blotting with recently developed antibodies. The P2X1 receptor was highly specific for human SMC, while the P2X4 was the highest expressed receptor in EC. The P2Y2 receptor was present in both SMC and EC. UTP-mediated effects in these cells are likely to be mediated by P2Y2 and not P2Y4 receptors since the latter had considerably lower expression. The P2Y6 receptor was expressed in both SMC and EC. The P2Y1 and surprisingly the P2Y11 receptors were the most abundantly expressed P2Y receptors in the endothelium. Overall, Western blotting confirmed the mRNA findings in most aspects, and most interestingly, indicated oligomerization of the P2Y1 receptor that may be important for its function. In conclusion, P2X1, P2Y2, and P2Y6 are the most expressed P2 receptors in SMC and are thus probably mediating the contractile and mitogenic actions of extracellular nucleotides. The P2X4, P2Y11, P2Y1, and P2Y2 are the most expressed P2 receptors in EC, and are most likely mediating release of nitric oxide, endothelium-dependent hyperpolarizing factor (EDHF), and t-PA induced by extracellular nucleotides. These findings will help to direct future cardiovascular drug development against the large P2 receptor family.

Effects of extracellular purines on ion transport across the integument of Hirudo medicinalis.

Little is known about the long-term regulation of epithelial ion transport in invertebrates and the specific mediators involved. For some years, we have been investigating the short-term regulation of transepithelial ion transport across the dorsal integument of the leech Hirudo medicinalis, and we have established a model of Na+ uptake. In the present study, we investigated the effect of long-term acclimation on transintegumental ion transport by adapting leeches to high-salinity conditions. We dissected segments of dorsal integument and measured ion currents in Ussing chamber experiments. Electrophysiological variables, such as transepithelial potential (V(T)) and short-circuit-current (I(sc)), were profoundly affected by adaptation to high-salinity conditions. The total transepithelial Na+ current (I(Na)) decreased from 7.66+/-0.82 to 4.6+/-0.54 microA cm(-2) in preparations adapted to high salinity. The involvement of epithelial Na+ channels was determined as current inhibition (I(ami)) by apical application of amiloride; Na+ channels were equally active in control epithelia and epithelia from leeches adapted to high salinity. Removal of Ca2+ from the apical solutions, which is believed to reduce intracellular Ca2+ concentrations, equalized transepithelial variables between high-salt-adapted integuments and control integuments. Extracellular purines regulate transepithelial Cl- secretion and Na+ absorption. In a variety of tissues we tested ATP and adenosine for their effects on epithelial transport. Examination of integuments from pondwater- and high-salinity-adapted leeches revealed different sensitivities for these purines. Apical and basolateral application of ATP both stimulated transepithelial Na+ uptake and I(ami). Adenosine upregulated non-Na+ currents and acted from the basolateral side only. Apical Ca2+-free conditions attenuated these effects of purines on transepithelial currents. Extracellular UTP had no effect on ion transport.

Uridine nucleotide-induced stimulation of gluconeogenesis in isolated rat proximal tubules.

Uridine nucleotides, released into the extracellular environment, influence a variety of metabolic and other cellular activities in a wide range of target tissues. Here we have studied the effects of uridine nucleotides on gluconeogenesis in isolated rat proximal tubules. Gluconeogenesis, from a range of precursors, was stimulated following exposure of isolated proximal tubules to either UTP or UTPgammaS, but not when exposed to other uridine-containing nucleotides. UTP- and UTPgammaS-induced gluconeogenesis was diminished in the presence of purinoceptor antagonists (e.g. suramin, PPADS) indicative of a role for P2Y(2)-like purinoceptors in these effects. Likewise, agents that interfere with either phospholipase C activation or intracellular Ca2+ mobilization decreased UTP- and UTPgammaS-induced stimulation of gluconeogenesis consistent with a role for these secondary messenger systems in the mechanism of action of extracellular UTP and UTPgammaS on proximal tubule metabolism.

UDP acts as a growth factor for vascular smooth muscle cells by activation of P2Y(6) receptors.

Mitogenic effects of the extracellular nucleotides ATP and UTP are mediated by P2Y(1), P2Y(2), and P2Y(4) receptors. However, it has not been possible to examine the highly expressed UDP-sensitive P2Y(6) receptor because of the lack of stable, selective agonists. In rat aorta smooth muscle cells (vascular smooth muscle cells; VSMC), UDP and UTP stimulated (3)H-labeled thymidine incorporation with similar pEC(50) values (5.96 and 5.69). Addition of hexokinase did not reduce the mitogenic effect of UDP. In cells transfected with P2Y receptors the stable pyrimidine agonist uridine 5'-O-(2-thiodiphosphate) (UDPbetaS) was specific for P2Y(6) with no effect on P2Y(1), P2Y(2), or P2Y(4) receptors. UDPbetaS stimulated [(3)H]thymidine and [(3)H]leucine incorporation and increased cell number in VSMC. Flow cytometry demonstrated that UDP stimulated cell cycle progression to both the S and G(2) phases. The intracellular signal pathways were dependent on phospholipase C, possibly protein kinase C-delta, and a tyrosine kinase pathway but independent of G(i) proteins, eicosanoids, and protein kinase A. The half-life of P2Y(6) receptor mRNA was <1 h by competitive RT-PCR. The mitogen-activated protein kinase kinase inhibitor PD-098059 significantly suppressed, whereas ATP and interleukin-1beta upregulated, expression of P2Y(6) receptor mRNA. The results demonstrate that UDP stimulates mitogenesis through activation of P2Y(6) receptors and that the receptor is regulated by factors important in the development of vascular disease.

Extracellular nucleotides induce arterial smooth muscle cell migration via osteopontin.

Migration and proliferation of arterial smooth muscle cells (SMCs) play a prominent role in the development of atherosclerotic plaques and restenosis lesions. Most of the growth-regulatory molecules potentially involved in these pathological conditions also demonstrate chemotactic properties. Extracellular purine and pyrimidine nucleotides have been shown to induce cell cycle progression and to elicit growth of cultured vascular SMCs. Moreover, the P2Y(2) ATP/UTP receptor was overexpressed in intimal thickening, suggesting a role of these nucleotides in vascular remodeling. Using the Transwell system migration assay, we demonstrate that extracellular ATP, UTP, and UDP exhibit a concentration-dependent chemotactic effect on cultured rat aortic SMCs. UTP, the most powerful nucleotide inducer of migration, elicited significant responses from 10 nmol/L. In parallel, UTP increased osteopontin expression dose-dependently. The blockade of osteopontin or its integrin receptors alpha(v)beta(3)/beta(5) by specific antibodies or antagonists inhibited UTP-induced migration. Moreover, the blockade of ERK-1/ERK-2 MAP kinase or rho protein pathways led to the inhibition of both UTP-induced osteopontin increase and migration, demonstrating the central role of osteopontin in this process. Taken together, these results suggest that extracellular nucleotides, and particularly UTP, can induce arterial SMC migration via the action of osteopontin.

Physiological evidence for a P2Y receptor responsive to diadenosine polyphosphates in human lung via Ca 2+ release studies in bronchial epithelial cells

P2Y 2 receptors that are activated by the extracellular nucleotides ATP or UTP mediate Cl − secretion via an increase in [Ca 2+] i (intracellular calcium concentration). Therefore, in the lung of patients suffering from cystic fibrosis, inhalation of aerosolized UTP offers a way to circumvent the defect in Cl − secretion by the cystic fibrosis transmembrane conductance regulator. A possible alternative for the relatively unstable UTP in inhalation therapy is the more resistant diadenosine tetraphosphate (Ap 4A). In human and rat lung membranes, Ap 4A binds to P2 receptor sites coupled to G proteins. Here, we showed that Ap 4A caused an increase in [Ca 2+] i with an ec 50 of 17 μM in human bronchial epithelial cells (HBE1). The [Ca 2+] i rise evoked by ATP and UTP was completely, but that induced by Ap 4A only partially, caused by release of Ca 2+ from internal stores. Moreover, the potency of Ap 4A to mobilize Ca 2+ was lower than that of ATP and UTP ( ec 50 1.5 and 1.8 μM, respectively), and the maximal increase in [Ca 2+] i was considerably smaller than that after ATP or UTP. In accordance with our previous results providing evidence for a common binding site for various diadenosine polyphosphates in lung membranes, all Ap nA analogues tested (n = 3 to 6) caused a comparable [Ca 2+] i increase. Homologous or heterologous prestimulation largely diminished the increase in [Ca 2+] i found after a second pulse of either UTP or Ap 4A. Although specific binding characteristics and functional responses of Ap 4A on lung cells are in favor of a distinct receptor for Ap 4A, the cross-talk between UTP and Ap 4A in HBE1 cells and the only slight differences in Ca 2+ mobilization by ATP or UTP and Ap 4A render it impossible at this point to state unequivocally whether there exists a distinct P2Y receptor specific for diadenosine polyphosphates in lung epithelia or whether Ap 4A activates one of the nucleotide receptors already described.

Extracellular ATP and UTP stimulate release of tissue-type plasminogen activator from vascular endothelium in human forearm David Erlinge, Th�rd�s Hrafnkelsd�ttir, Sverker Jern. Dept of Cardiology, Lund, Sweden, Dept of Clin. Exp. Research, Gothenburg, Sweden

Extracellular ATP and UTP stimulate release of tissue-type plasminogen activator from vascular endothelium in human forearm David Erlinge, Th�rd�s Hrafnkelsd�ttir, Sverker Jern. Dept of Cardiology, Lund, Sweden, Dept of Clin. Exp. Research, Gothenburg, Sweden

Activation of calcium entry in human carcinoma A431 cells by store depletion and phospholipase C- dependent mechanisms converge on ICRAC-like calcium channels.

Activation of phospholipase C in nonexcitable cells causes the release of calcium (Ca2+) from intracellular stores and activation of Ca2+ influx by means of Ca2+ release-activated channels (ICRAC) in the plasma membrane. The molecular identity and the mechanism of ICRAC channel activation are poorly understood. Using the patch-clamp technique, here we describe the plasma membrane Ca2+ channels in human carcinoma A431 cells, which can be activated by extracellular UTP, by depletion of intracellular Ca2+ stores after exposure to the Ca2+-pump inhibitor thapsigargin, or by loading the cells with Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate. The observed channels display the same conductance and gating properties as previously described I(min) channels, but have significantly lower conductance for monovalent cations than the ICRAC channels. Thus, we concluded that the depletion-activated Ca2+ current in A431 cells is supported by I(CRAC)-like (ICRACL) channels, identical to I(min). We further demonstrated synergism in activation of ICRACL Ca2+ channels by extracellular UTP and intracellular inositol (1,4,5)-triphosphate (IP3), apparently because of reduction in phosphatidylinositol 4,5-bisphosphate (PIP2) levels in the patch. Prolonged exposure of patches to thapsigargin renders ICRACL Ca2+ channels unresponsive to IP3 but still available to activation by the combined action of IP3 and anti-PIP2 antibody. Based on these data, we concluded that phospholipase C-mediated and store-operated Ca2+ influx pathways in A431 cells converge on the same I(CRACL) Ca2+ channel, which can be modulated by PIP2.

Extracellular Nucleotides ATP and UTP Induce a Marked Acute Release of Tissue-type Plasminogen Activator In Vivo in Man

Extracellular nucleotides such as ATP and UTP are released by activation of platelets and ischemic tissue injury. The aim of the present study was to investigate whether ATP and UTP can induce acute tPA release from the vascular endothelium in vivo. Nine healthy subjects were studied in a perfused-forearm model during stepwise intraarterial infusions of ATP and UTP (10-200 nmol/min), and UTP during inhibition of prostanoid and NO synthesis by indomethacin and L-NMMA. ATP and UTP induced a similar and marked stimulation of forearm tPA release which increased 11- and 18-fold above baseline (p < or =0.01 for both) in conjunction with pronounced vasodilation. Neither the acute tPA release nor the vasodilation could be abrogated by NO and prostanoid synthesis inhibition. The similar effect of ATP and UTP suggests that P2Y rather than adenosine receptors mediate the response. Release of extracellular nucleotides in ischemic tissue may induce a pronounced activation of the endogenous fibrinolytic system.

Characterization of intracellular calcium oscillations induced by extracellular nucleotides in HEp-2 cells

The effect of extracellular nucleotides on the cytosolic calcium concentration of fluo-3-loaded HEp-2 cells was examined using confocal microscopy. Extracellular ATP and UTP at micromolar concentration induced cytosolic calcium oscillations in 42–66% of the cells. Oscillations were usually sinusoid and their frequency depended only slightly on agonist concentration. Oscillations developed in calcium-free medium but were diminished by depletion of intracellular calcium stores with thapsigargin, indicating periodic calcium release from internal stores. Inhibition of phospholipase C with U73122 prevented the development of oscillations, while ryanodine did not abolish the response to extracellular nucleotides. Activation of protein kinase C with 4β-phorbol 12-myristate 13-acetate also prevented the development of oscillations. These results indicate that extracellular nucleotides induce periodic calcium release from inositol 1,4,5-trisphosphate-sensitive pools in HEp-2 cells and that the inhibitory effect of protein kinase C on the phosphatidylinositol signaling pathway can contribute to the development of intracellular calcium oscillations.

Extracellular ATP and UTP stimulate cartilage proteoglycan and collagen accumulation in bovine articular chondrocyte pellet cultures

Bovine articular chondrocytes were maintained in high density pellet cultures with and without serum and nucleotide triphosphates for different periods of time. Despite half-lives in culture of about 3 h, adenosine triphosphate and uridine triphosphate in the presence of serum increased sulphated glycosaminoglycan and collagen deposition above control levels. In the presence of serum a single dose of uridine triphosphate on the first day of culture was sufficient to induce significant increases in subsequent proteoglycan and collagen deposition. We conclude that both adenine triphosphate and uridine triphosphate are anabolic for articular chondrocytes, and that this effect on the chondrocyte is long-term.

Cytokines induce upregulation of vascular P2Y(2) receptors and increased mitogenic responses to UTP and ATP.

P2Y(2) receptors, which mediate contractile and mitogenic effects of extracellular nucleotides in vascular smooth muscle cells (VSMCs), are upregulated in the synthetic phenotype of VSMCs and in the neointima after balloon angioplasty, suggesting a role in the development of atherosclerosis. Because released cytokines in atherosclerotic lesions mediate multiple effects on gene transcription in VSMCs, we speculated that cytokines could be involved in the regulation of P2Y(2) receptor expression. Using a competitive reverse transcription-polymerase chain reaction, we detected that interleukin (IL)-1beta induced a time- and dose-dependent upregulation of P2Y(2) receptor mRNA, which was dramatically enhanced when combined with interferon-gamma or tumor necrosis factor-alpha. Lipopolysaccharide also significantly increased the expression of P2Y(2) receptor mRNA. The upregulation of P2Y(2) receptor mRNA was paralleled at the functional level because IL-1beta significantly increased the UTP-stimulated DNA synthesis and the release of intracellular Ca(2+). Actinomycin D completely blocked the upregulation of P2Y(2) receptor mRNA expression by IL-1beta, indicating de novo mRNA synthesis. There was no cAMP accumulation in the cells stimulated with IL-1beta. The cyclooxygenase inhibitor indomethacin and the protein kinase C inhibitor RO-31-8220 inhibited IL-1beta-induced upregulation of P2Y(2) receptor mRNA expression, whereas rapamycin and PD098059 had no effects. Furthermore, neither P38 mitogen-activated protein kinase inhibitor SB20358 alone nor its combination with PD098059 blocked the effect of IL-1beta on the expression of P2Y(2) receptor mRNA. Our results demonstrate that inflammatory mediators upregulate vascular P2Y(2) receptors at the transcriptional and at the functional level through protein kinase C and cyclooxygenase but not cAMP, extracellular signal-regulated kinases 1 and 2, or P38-dependent pathways. This may result in increased growth-stimulatory or contractile effects of extracellular UTP and ATP, which may be of importance in the development of vascular disease.

Extracellular UTP stimulates electrogenic bicarbonate secretion across CFTR knockout gallbladder epithelium.

The loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial HCO(3)(-) secretion contributes to the pathogenesis of pancreatic and biliary disease in cystic fibrosis (CF) patients. Recent studies have investigated P2Y(2) nucleotide receptor agonists, e.g., UTP, as a means to bypass the CFTR defect by stimulating Ca(2+)-activated Cl(-) secretion. However, the value of this treatment in facilitating transepithelial HCO(3)(-) secretion is unknown. Gallbladder mucosae from CFTR knockout mice were used to isolate the Ca(2+)-dependent anion conductance during activation of luminal P2Y(2) receptors. In Ussing chamber studies, UTP stimulated a transient peak in short-circuit current (I(sc)) that declined to a stable plateau phase lasting 30-60 min. The plateau I(sc) after UTP was Cl(-) independent, HCO(3)(-) dependent, insensitive to bumetanide, and blocked by luminal DIDS. In pH stat studies, luminal UTP increased both I(sc) and serosal-to-mucosal HCO(3)(-) flux (J(s-->m)) during a 30-min period. Substitution of Cl(-) with gluconate in the luminal bath to inhibit Cl(-)/HCO(3)(-) exchange did not prevent the increase in J(s-->m) and I(sc) during UTP. In contrast, luminal DIDS completely inhibited UTP-stimulated increases in J(s-->m) and I(sc). We conclude that P2Y(2) receptor activation results in a sustained (30-60 min) increase in electrogenic HCO(3)(-) secretion that is mediated via an intracellular Ca(2+)-dependent anion conductance in CF gallbladder.

Release of osmolytes induced by phagocytosis and hormones in rat liver.

Betaine, taurine, and inositol participate as osmolytes in liver cell volume homeostasis and interfere with cell function. In this study we investigated whether osmolytes are also released from the intact liver independent of osmolarity changes. In the perfused rat liver, phagocytosis of carbon particles led to a four- to fivefold stimulation of taurine efflux into the effluent perfusate above basal release rates. This taurine release was inhibited by 70-80% by the anion exchange inhibitor DIDS or by pretreatment of the rats with gadolinium chloride. Administration of vasopressin, cAMP, extracellular ATP, and glucagon also increased release of betaine and/or taurine, whereas insulin, extracellular UTP, and adenosine were without effect. In isolated liver cells, it was shown that parenchymal cells and sinusoidal endothelial cells, but not Kupffer cells and hepatic stellate cells, release osmolytes upon hormone stimulation. This may be caused by a lack of hormone receptor expression in these cells, because single-cell fluorescence measurements revealed an increase of intracellular calcium concentration in response to vasopressin and glucagon in parenchymal cells and sinusoidal endothelial cells but not in Kupffer cells and hepatic stellate cells. The data show that Kupffer cells release osmolytes during phagocytosis via DIDS-sensitive anion channels. This mechanism may be used to compensate for the increase in cell volume induced by the ingestion of phagocytosable material. The physiological significance of hormone-induced osmolyte release remains to be evaluated.

Extracellular nucleotides activate the p38-stress-activated protein kinase cascade in glomerular mesangial cells.

1. Extracellular ATP and UTP have been reported to activate a nucleotide receptor (P2Y2-receptor) that mediates arachidonic acid release with subsequent prostaglandin formation, a reaction critically depending on the activity of a cytosolic phospholipase A2. In addition, extracellular nucleotides trigger activation of the classical mitogen-activated protein kinase (MAPK) cascade and cell proliferation as well as of the stress-activated protein kinase (SAPK) cascade. 2. In this study, we report that ATP and UTP are also able to activate the p38-MAPK pathway as measured by phosphorylation of the p38-MAPK and its upstream activators MKK3/6, as well as phosphorylation of the transcription factor ATF2 in a immunocomplex-kinase assay. 3. Time courses reveal that ATP and UTP induce a rapid and transient activation of the p38-MAPK activity with a maximal activation after 5 min of stimulation which declined to control levels over the next 20 min. 4. A series of ATP and UPT analogues were tested for their ability to stimulate p38-MAPK activity. UTP and ATP were very effective analogues to activate p38-MAPK, whereas ADP and gamma-thio-ATP had only moderate activating effects. 2-Methyl-thio-ATP, beta gamma-imido-ATP, AMP, adenosine and UDP had no significant effects of p38-MAPK activity. In addition, the extracellular nucleotide-mediated effect on p38-MAPK was almost completely blocked by 1 mM of suramin, a putative P2-purinoceptor antagonist. 5. In summary, these results demonstrate for the first time that extracellular nucleotides are able to activate the MKK3/6- p38-MAPK cascade most likely via the P2Y2-receptor. Moreover, this finding implies that all three MAPK subtypes are signalling candidates for extracellular nucleotide-stimulated cell responses.

M-type K+ currents in rat cultured thoracolumbar sympathetic neurones and their role in uracil nucleotide-evoked noradrenaline release.

Cultured sympathetic neurones are depolarized and release noradrenaline in response to extracellular ATP, UDP and UTP. We examined the possibility that, in neurones cultured from rat thoracolumbar sympathetic ganglia, inhibition of the M-type potassium current might underlie the effects of UDP and UTP. Reverse transcriptase-polymerase chain reaction indicated that the cultured cells contained mRNA for P2Y(2)-, P2Y(4)- and P2Y(6)-receptors as well as for the KCNQ2- and KCNQ3-subunits which have been suggested to assemble into M-channels. In cultures of neurones taken from newborn as well as from 10 day-old rats, oxotremorine, the M-channel blocker Ba(2+) and UDP all released previously stored [(3)H]-noradrenaline. The neurones possessed M-currents, the kinetic properties of which were similar in neurones from newborn and 9 - 12 day-old rats. UDP, UTP and ATP had no effect on M-currents in neurones prepared from newborn rats. Oxotremorine and Ba(2+) substantially inhibited the current. ATP also had no effect on the M-current in neurones prepared from 9 - 12 day-old rats. Oxotremorine and Ba(2+) again caused marked inhibition. In contrast to cultures from newborn animals, UDP and UTP attenuated the M-current in neurones from 9 - 12 day-old rats; however, the maximal inhibition was less than 30%. The results indicate that inhibition of the M-current is not involved in uracil nucleotide-induced transmitter release from rat cultured sympathetic neurones during early development. M-current inhibition may contribute to release at later stages, but only to a minor extent. The mechanism leading to noradrenaline release by UDP and UTP remains unknown.

Nucleotide-induced PMN adhesion to cultured epithelial cells: possible role of MUC1 mucin.

Accumulation of intraluminal polymorphonuclear leukocytes (PMN) is a hallmark of inflammatory diseases of the airways. Extracellular nucleotides stimulate PMN adhesion to human main pulmonary artery endothelial cells (HPAEC) by a purinoceptor-mediated mechanism. We investigated the effects of nucleotides on adhesion of freshly isolated human PMN to cultured human tracheobronchial epithelial cells (HBEC). We found that extracellular ATP and UTP were much less effective in stimulating PMN adhesion to HBEC compared with HPAEC, whereas the bacterial chemotactic peptide N-formyl-Met-Leu-Phe stimulated PMN adhesion to both cell types to an equal degree. We investigated several mechanisms that might account for decreased nucleotide-induced PMN adhesion to HBEC. The ectonucleotidase-resistant ATP analog adenosine 5'-O-(3-thiotriphosphate) was also ineffective in stimulating PMN adhesion to HBEC, indicating that degradation of ATP by ectonucleotidase(s) was not responsible for altered PMN adhesion. HBEC responded to ATP and UTP with increased intracellular calcium, indicating that these cells are capable of purinoceptor-mediated responses. We found that ATP and UTP also did not stimulate PMN adhesion to Chinese hamster ovary (CHO) cells, which had been stably transfected with the gene for hamster Muc1, a cell-associated mucin. However, ATP and UTP did stimulate adhesion of PMN to nontransfected CHO cells. These results suggested that MUC1 mucin modulates PMN adhesion to epithelium. We found that cultured HBEC expressed more mRNA and protein for MUC1 mucin than did HPAEC. We conclude that extracellular nucleotides are less effective in stimulating PMN adhesion to epithelial cells than to endothelial cells and that overexpression of hamster Muc1 mucin inhibits nucleotide-induced PMN adhesion to CHO cells.

Quantitation of extracellular UTP using a sensitive enzymatic assay.

The wide distribution of the uridine nucleotide-activated P2Y2, P2Y4 and P2Y6 receptors suggests a role for UTP as an important extracellular signalling molecule. However, direct evidence for UTP release and extracellular accumulation has been addressed only recently due to the lack of a sensitive assay for UTP mass. In the present study, we describe a method that is based on the uridinylation of [14C]-glucose-1P by the enzyme UDP-glucose pyrophosphorylase which allows quantification of UTP in the sub-nanomolar concentration range. The UTP-dependent conversion of [14C]-glucose-1P to [14C]-UDP-glucose was made irreversible by including the pyrophosphate scavenger inorganic pyrophosphatase in the reaction medium and [14C]-glucose-1P and [14C]-UDP-glucose were separated and quantified by HPLC. Formation of [14C]-UDP-glucose was linearly observed between 1 and 300 nM UTP. The reaction was highly specific for UTP and was unaffected by a 1000 fold molar excess of ATP over UTP. Release of UTP was measured with a variety of cells including platelets and leukocytes, primary airway epithelial cells, rat astrocytes and several cell lines. In most resting attached cultures, extracellular UTP concentrations were found in the low nanomolar range (1-10 nM in 0.5 ml medium bathing 2.5 cm2 dish). Up to a 20 fold increase in extracellular UTP levels was observed in cells subjected to a medium change. Extracellular UTP levels were 10-30% of the ATP levels in both resting and mechanically-stimulated cultured cells. In unstirred platelets, a 1:100 ratio UTP/ ATP was observed. Extracellular UTP and ATP increased 10 fold in thrombin-stimulated platelets. Detection of UTP in nanomolar concentrations in the medium bathing resting cultures suggests that constitutive release of UTP may provide a mechanism of regulation of the basal activity of uridine nucleotide sensitive receptors.

Intracellular calcium oscillations induced by ATP in airway epithelial cells.

In airway epithelial cells, extracellular ATP (ATP(o)) stimulates an initial transient increase in intracellular Ca(2+) concentration that is followed by periodic increases in intracellular Ca(2+) concentration (Ca(2+) oscillations). The characteristics and mechanism of these ATP-induced Ca(2+) responses were studied in primary cultures of rabbit tracheal cells with digital video fluorescence microscopy and the Ca(2+)-indicator dye fura 2. The continual presence of ATP(o) at concentrations of 0.1-100 microM stimulated Ca(2+) oscillations that persisted for 20 min. The frequency of the Ca(2+) oscillations was found to be dependent on both ATP(o) concentration and intrinsic sensitivity of each cell to ATP(o). Cells exhibited similar Ca(2+) oscillations to extracellular UTP (UTP(o)), but the oscillations typically occurred at lower UTP(o) concentrations. The ATP-induced Ca(2+) oscillations were abolished by the phospholipase C inhibitor U-73122 and by the endoplasmic reticulum Ca(2+)-pump inhibitor thapsigargin but were maintained in Ca(2+)-free medium. These results are consistent with the hypothesis that in airway epithelial cells ATP(o) and UTP(o) act via P2U purinoceptors to stimulate Ca(2+) oscillations by the continuous production of inositol 1,4,5-trisphosphate and the oscillatory release of Ca(2+) from internal stores. ATP-induced Ca(2+) oscillations of adjacent individual cells occurred independently of each other. By contrast, a mechanically induced intercellular Ca(2+) wave propagated through a field of Ca(2+)-oscillating cells. Thus Ca(2+) oscillations and propagating Ca(2+) waves are two fundamental modes of Ca(2+) signaling that exist and operate simultaneously in airway epithelial cells.

Interplay between the NO pathway and elevated [Ca2+]i enhances ciliary activity in rabbit trachea.

1. Average intracellular calcium concentration ([Ca2+]i) and ciliary beat frequency (CBF) were simultaneously measured in rabbit airway ciliated cells in order to elucidate the molecular events that lead to ciliary activation by purinergic stimulation. 2. Extracellular ATP and extracellular UTP caused a rapid increase in both [Ca2+]i and CBF. These effects were practically abolished by a phospholipase C inhibitor (U-73122) or by suramin. 3. The effects of extracellular ATP were not altered: when protein kinase C (PKC) was inhibited by either GF 109203X or chelerythrine chloride, or when protein kinase A (PKA) was inhibited by RP-adenosine 3', 5'-cyclic monophosphothioate triethylamine (Rp-cAMPS). 4. Activation of PKC by phorbol 12-myristate, 13-acetate (TPA) had little effect on CBF or on [Ca2+]i, while activation of PKA by forskolin or by dibutyryl-cAMP led to a small rise in CBF without affecting [Ca2+]i. 5. Direct activation of protein kinase G (PKG) with dibutyryl-cGMP had a negligible effect on CBF when [Ca2+]i was at basal level. However, dibutyryl-cGMP strongly elevated CBF when [Ca2+]i was elevated either by extracellular ATP or by ionomycin. 6. The findings suggest that the initial rise in [Ca2+]i induced by extracellular ATP activates the NO pathway, thus leading to PKG activation. In the continuous presence of elevated [Ca2+]i the stimulated PKG then induces a robust enhancement in CBF. In parallel, activated PKG plays a central role in Ca2+ influx via a still unidentified mechanism, and thus, through positive feedback, maintains CBF close to its maximal level in the continuous presence of ATP.